Mucosal Immunity

Abstract In addition to their critical role in allergic disorders, mast cells (MCs) are well recognized for their protective effector functions during bacteria and parasite infections. This review describes recent advancements of our understanding of the complex role of MCs in fungal infections. Specifically, we outline key features of the contribution of MCs to infections with six fungal pathogens, namely Sporothrix, Paracoccidioides, Aspergillus, Malassezia, Candida and Dermatophytes. Evidence from studies of these pathogens suggests that MCs can function as positive regulators that detect and contain fungi at the site of infection. However, it appears that the inflammation induced by MCs following fungal infections may not always and only be beneficial to the host. MC responses during fungal infections may primarily benefit the pathogen by facilitating its spreading and contributing to a greater severity of fungal infections. This review also highlights key drivers of MCs activation and effector mechanisms that have been identified for the multidimensional function of MCs in fungal diseases and in allergic diseases combined with fungal infection. 1 INTRODUCTION Fungi are a very diverse group of eukaryotic organisms that separate from plants and animals in their own kingdom. Today, fungal infections represent a serious public health problem around the world with more than 300 million people suffering from serious fungal infections and an estimated 1.6 million deaths annually.1 Fungi can cause a broad range of mycoses in humans: (a) Superficial fungal infections of the skin, nails and hair are commonly due to dermatophytes, for example Trichophyton rubrum or Trichophyton mentagrophytes, or yeasts, for example Candida or Malassezia furfur.2-5 (b) Subcutaneous fungal infections include sporotrichosis, chromoblastomycosis, mycetoma, phaeohyphomycosis, hyalohyphomycosis and lacaziosis.6, 7 (c) Paracoccidioidomycosis, coccidioidomycosis, histoplasmosis, mucormycosis and cryptococcosis are common systemic fungal infections.8 Subcutaneous and systemic mycoses occur mainly in immunosuppressed patients.7 Systemic fungal infections are linked to significant morbidity and mortality.8 In addition, fungal spores, for example, Aspergillus spores, are important allergenic agents and can cause persistent allergic inflammation of the airways.9 The immune mechanisms of defense against fungal infections are numerous, ranging from protective mechanisms that were present early in evolution (innate immunity) to sophisticated adaptive mechanisms that are induced specifically during infection and disease (adaptive immunity).10-12 Traditionally, mast cells (MCs) are seen as key effector cells in the elicitation of allergic signs and symptoms.13-15 However, there is an increasing number of studies that indicate that MCs are also essential players in innate immune responses.16-18 MCs are strategically located in tissues that are borders to the external environment such as the lung, the gut and the skin. Therefore, MCs are perfectly positioned to orchestrate protective immune responses against invading pathogens including fungi. Most studies of anti‐fungal immune responses are focused on the effects of epithelial cells, neutrophils, macrophages/monocytes and dendritic cells (DCs).19-23 As of now, only a few publications and reviews have addressed the role of MCs in the immune response to fungi infections. In the innate immune system, MCs act as sentinels for invading pathogens, as initiators of inflammatory responses and as regulatory cells in the coordination of raising and limiting innate immune responses.18, 24, 25 Recent findings suggest that, during innate immune responses against invading pathogens, MCs function as positive or negative immunoregulatory cells depending on the situation. In this review, we focus on the recent advancements of our understanding of the complex role of MCs in fungal infections—as positive regulators that detect and contain fungi at the site of infection and as negative regulators that promote a wider dissemination and a greater severity of fungal infections. Specifically, we highlight key features of the contribution of MCs to infections with six fungal pathogens, namely Sporothrix, Paracoccidioides, Aspergillus, Malassezia, Candida and Dermatophytes. 2 WHAT IS THE ROLE AND RELEVANCE OF MCs IN SPOROTHRIX SCHENCKII INFECTION? Sporothrix schenckii (S. schenckii) is an opportunistic dimorphic fungus with a global distribution.26, 27 S. schenckii is the cause of sporotrichosis, which usually affects the skin, less commonly the lungs, joints, bones, and brain of humans and other mammals.28 Veterinarians, animal technicians and caretakers, and owners of cats with sporotrichosis are regarded to be at increased risk for the acquisition of the disease.27 Sporotrichosis is transferred via the inoculation of S. schenckii (conidia or hyphae) from contaminated soil, plants or organic matter into sites of tissue injury such as wounds (traumatic inoculation).27 During interactions of S. schenckii with the host, the conidia‐phase of S. schenckii will transform into the yeast‐phase of S. schenckii in response to the host's temperature. Yeast is the dominant form of S. schenckii in sporotrichosis.29 In vitro studies showed that IgG‐seroreactive proteins from Sporothrix yeast cell extracts such as Gp 70 may be important for the pathogenesis and invasion of S. schenckii in human.30 In 2012, Romo‐Lozano et al. first reported that S. schenckii conidia can activate mouse peritoneal MCs to release early response cytokines such as TNF‐α and IL‐6 without degranulation.31, 32 By using an experimental murine model of systemic S. schenckii infection, which used intraperitoneal inoculation with S. schenckii conidia, they demonstrated that mice that had been depleted of peritoneal MCs presented with a decreased fungal load in organs and reduced severity of clinical manifestations. Their results suggest that mediators released by MCs may be implicated in promoting a wider dissemination of S. schenckii and a greater severity of the infection, indicating that MCs might act as negative immunoregulators in S. schenckii infection. However, this study used a functional MCs depletion model where MCs were depleted by injection of compound 48/80 into the peritoneal cavity. Moreover, their experimental model investigated systemic S. schenckii infection following intraperitoneal injection, whereas subcutaneous infection is the more common clinical manifestation of sporotrichosis. In summary, the in vivo evidence for a relevant role of MCs in S. schenckii infection is still limited. Based on the limited evidence available from mouse studies so far, MCs appear to promote S. schenkii, rather than protect from them. How MCs are involved in human sporotrichosis still needs to be clarified. 3 DO MCs PROMOTE PARACOCCIDIOIDES BRASILIENSIS INFECTION? The fungus Paracoccidioides brasiliensis (Pb) causes paracoccidioidomycosis (PCM), an autochthonous systemic mycosis found primarily in Central and South America.33 PCM is characterized by mucous membrane ulcerations of the mouth and nose with spreading through the lymphatic system. The route of infection is assumed to be inhalation. The lungs, lymph nodes and mucous membrane of the mouth are the most frequently infected tissues.34 That MCs interact with Pb has been shown by Valim et al., who demonstrated that co‐culturing of Pb yeast cells with MC lines rat basophilic leukaemia (RBL)‐2H3 MCs induced morphological changes of MCs and their degranulation.35 Furthermore, endogenous PbPga1, which is an immunoregulatory protein on the surface of Pb yeast cells, is able to activate rodent MCs through the NF‐κB pathway, resulting in the release of the inflammatory interleukin IL‐6. IL‐6 is involved in the induction of inflammatory responses that can lead to tissue destruction. Recently, a study of skin biopsies from patients with paracoccidioidomycosis suggested that MCs contribute to skin responses characterized by loose granuloma formation. This type of cutaneous response, which does not effectively contain or clear the infection, features markedly increased numbers of MCs and a Th2 cytokine profile.36 Interestingly, most MCs in these lesions were positive for IL‐10 suggesting that MCs are an important source of this immunosuppressive cytokine. Moreover, Batista et al. observed a downregulation of the inducible nitric oxide synthase (iNOS) in the tissues of oral paracoccidioidomycosis (OP). This results in reduced production of fungicidal nitric oxide (NO) in multinucleated giant cells and most mononuclear cells.37 MCs have been shown to be one of the major sources of NO in humans with fungus infections.38 These findings suggest that a low expression of iNOS in OP may represent a possible MC‐mediated mechanism that permits local fungal multiplication and maintenance of active oral lesions. Taken together, MCs appear to contribute to the morbidity of PCM, by several mechanisms. However, most of the above findings are from ex vivo or in vitro experiments, and proof that MCs play a harmful role in Pb infection needs to come from future in vivo studies. 4 MCs CONTRIBUTE TO THE MORBIDITY OF ASPERGILLUS FUMIGATUS INFECTION Aspergillus fumigatus (A. fumigatus) is a ubiquitous, spore‐forming fungus that has been associated with multiple pulmonary disorders. Inhaled conidia of A. fumigatus adhere to pulmonary epithelial cells, causing opportunistic infection followed by inflammatory responses to A. fumigatus antigens.39 Most studies on the role of MCs in chronic lung disease due to A. fumigatus focus on IgE‐mediated allergic bronchopulmonary aspergillosis (ABPA).40-42 ABPA is associated with early allergic and late‐phase inflammatory responses of the lungs to A. fumigatus antigens that occur in a minority (less than 1%) of patients with asthma and, more often (7%‐10%), in patients with cystic fibrosis.43 ABPA is characterized by elevated total and A. fumigatus‐specific immunoglobulin E (IgE) levels accompanied by eosinophilia, pulmonary infiltrates, bronchiectasis and fibrosis leading to asthmatic attacks and wheezing in affected patients. A. fumigatus‐specific IgE has been shown to activate MCs by cross‐linking of the high‐affinity receptor for IgE(FcεRI) leading to the immediate release of preformed mediators from intracellular granules and the de novo synthesis of lipid mediators and cytokines, all of which contribute to the subsequent development of inflammatory responses and obstruction of the airways.44-46 Proteases released from MCs can promote the release of growth factors from airway epithelial cells driving strong and irreversible remodelling reactions in airways of patients with ABPA, resulting in bronchiectasis lesions and fibrosis.46 Treatment with omalizumab, a humanized mAb that binds to IgE, has been shown to be an effective therapy for patients with severe allergic asthma and ABPA as shown by attenuated asthma symptoms and improved pulmonary function parameters in patients with ABPA.47 Recently, clinical trials demonstrated that omalizumab treatment not only provided a clinically important reduction in serum IgE, exacerbation rates and steroid requirement but also showed attenuated asthma symptoms and improved pulmonary function parameters in patients with ABPA.47 Interestingly, however, the majority of patients with airway colonization by A. fumigatus do not develop ABPA, yet they still exhibit a progressive decline in lung function.48 IgE‐independent MC degranulation in response to A. fumigates may be involved in this response. Urb and co‐workers found that direct contact with mature A. fumigatus hyphae can induce the degranulation of bone marrow‐derived mast cells (BMCMCs) in the absence of IgE, whereas no significant degranulation is induced by either conidia or immature hyphae.49 Although the exact mechanism of this is still unknown, the fungal proteins StuA and MedA, which control the composition of the cell surface molecules of mature A. fumigatus hyphae, were shown to induce BMCMC degranulation in the absence of IgE.49 Therefore, the authors speculated that A. fumigatus may directly worsen pulmonary disorders, by inducing degranulation in MCs that are in contact to hyphae, which may aggravate the inflammation and obstruction of airway rather than eliminate the fungus. Recent data shows that similar to ABPA in man, MCs may be implicated in recurrent airway obstruction (RAO), a type I hypersensitivity response to A. fumigatus in horses.49, 50 As in ABPA, specific IgE‐mediated cross‐linking of FcεRI results in MC degranulation and the release of histamine, serotonin, leukotrienes and eicosanoids such as thromboxane and 15‐hydroxyeicosatetraenoic acid. In addition, in experimental eosinophilic esophagitis induced by A. fumigatus, Niranjan et al. reported that MCs numbers are markedly increased and play a significant role in promoting oesophageal functional abnormalities, contributing to muscle cell hyperplasia and hypertrophy.51 Finally, a recent report suggests that MCs also contribute to A. fumigatus‐induced inflammation in acute invasive fungal rhinosinusitis (AIFR), an aggressive fungal infection with high morbidity and mortality rates in immunocompromised patients.52 Yan et al. created a rat model of AIFR based on A. fumigatus infection and investigated the role of MCs.53 Compared to control rats, the total number of MCs was unchanged, but MC degranulation was only found in the infected nasal cavities of AIFR rats. In summary, these findings suggest that MC responses to A. fumigatus appear to help the pathogen rather than the host. Further studies will be required to determine the underlying mechanisms of the contribution of MCs to A. fumigatus infection by valuable mice models.54 5 EXACERBATION OF MALASSEZIA‐INDUCED SKIN INFLAMMATION BY MCs IN PATIENTS WITH ATOPIC DERMATITIS Malassezia comprises a family of opportunistic pathogenic fungi, which includes 19 species found on the skin of humans and animals. These Malassezia species have been associated with dermatological diseases such as seborrhoeic dermatitis, pityriasis versicolor, atopic dermatitis (AD) and folliculitis.55 AD is one of the most common chronic inflammatory diseases of the skin.56 Malassezia sympodialis (M. sympodialis) is frequently isolated from AD patients and healthy individuals.57 Recently, it was demonstrated that mouse and human MCs respond to extracts from M. sympodialis.58-60 For example, M. sympodialis can enhance IgE‐mediated MC degranulation by signalling through the TLR2/MyD88 pathway. Interestingly, Ribbing et al. further observed that MCs derived from peripheral blood CD34+ progenitor cells of Malassezia‐infected patients contain more preformed granule mediators and exhibit enhanced IL‐6 secretion in response to M. sympodialis, as compared to MCs from healthy controls.60 In addition, Malassezia also can elicit specific IgE and T‐cell reactivity in AD patients.61, 62 Hiragun and colleagues identified the fungal protein, MGL_1304, derived from Malassezia globosa (M. globosa) in purified sweat, as an allergen in AD patients.63-65 They confirmed its ability to induce IgE‐dependent degranulation in human basophils and RBL cells expressing the human FcεRI receptor. Their results suggest that MGL_1304 is a major allergen in human sweat and could cause type I allergy in patients with AD. These findings indicate that M. sympodialis can activate MCs and thereby exacerbate inflammatory responses in AD patients. 6 DO MCs CONTRIBUTE TO THE PATHOGENIC EFFECTS OF CANDIDA ALBICANS? Candida albicans (C. albicans) is an opportunistic fungal pathogen, and they are normally grown as a harmless commensal on the skin and mucosal surfaces of healthy individuals.66 However, it can also cause superficial and systemic infections, especially in immunosuppression condition. C. albicans initially interacts with epithelial cells, resulting in fungal recognition and the formation of hyphae.67 The ability to undergo the morphological transition from yeast to hyphal growth is critical for its pathogenesis. Epithelial activation leads to the production of inflammatory mediators that recruit innate immune cells, which together work with epithelial cells to clear the fungal infection. The main immune cell in the defense against these candida infections is assigned to professional phagocytes, neutrophils and macrophages. In addition to the above cells, MCs also have been identified as the main cell types involved in the defense against this pathogen.68, 69 Yamaguchi et al. have shown that ovalbumin‐sensitized mice colonized with C. albicans show higher specific IgG and IgE titres than control mice, and colonized tissues promoted infiltration and degranulation of MCs.70 Their results suggest that gastrointestinal C. albicans colonization may promote sensitization against food antigens, at least partly due to MC‐mediated hyperpermeability in the gastrointestinal mucosa of mice. Furthermore, Takenaka and colleagues reported that increased levels of Candida‐specific IgE were linked to MC hyperplasia in lymph nodes in Kimura's disease.71 In addition, Segal and co‐workers observed that the injection of C. albicans together with Complete Freund's Adjuvant into the foot‐pads of guinea‐pigs provoked a state of hypersensitivity, characterized by direct degranulation of mouse peritoneal MCs when exposed in vitro to the antigen.72 Noverr and co‐workers reported that overgrowth of the yeast C. albicans following antibiotic treatment can promote the development of allergic airway disease characterized by increased levels of MCs.73 Taken together, these findings suggest that C. albicans may contribute to atopic diseases, at least in part by effects on MCs. Interestingly, MCs may also protect from C. albicans infection. Recent evidence shows that rat peritoneal MCs can kill C. albicans in the extracellular environment, very likely through secreted granular mediators.74 That C. albicans can degranulate rat peritoneal MCs to release mediators has been shown as early as in 1974 when Nosál et al. reported that glycoprotein from C. albicans can induce histamine release in isolated rat MCs, which was confirmed by later in vivo studies showing C. albicans‐induced MCs degranulation in the gut of mice.75 More recently, Nieto‐Patlán et al. have shown that both yeasts and hyphae of C. albicans can induce BMCMCs degranulation, production of TNF‐α, IL‐6, IL‐10, CCL3, CCL4, and ROS as well as NO involving TLR2 and dectin‐1.76 Moreover, MC seems to be able to phagocytose C. albicans. According to Lopes JP et al., the protective response of primary human MCs in C. albicans infections has three stages: Initially, during the first 3 hours, MCs reduce C. albicans viability by degranulation. During the intermediate stage of the response, 3‐12 hours after infection, MC cytokine secretion and the recruitment of neutrophils by releasing chemoattractants such as CXCL1/CXCL2 help to contain the infections by phagocytosis, the release of proinflammatory mediators and by recruiting other leucocytes. Later responses, that is after 12 hours, are characterized by the release of anti‐inflammatory cytokines and the recruitment of effector cells of adaptive immunity.77 Taking together, these results suggest that MCs can help the infected host by killing C. albicans and by containing the infection by recruiting neutrophils and macrophages to the sites of infection. As for the role of MCs in C. albicans infection, there are still many questions to be answered, and further evidence from in vivo studies is needed to arrive at robust conclusions.78 7 WHAT IS THE RELEVANCE OF MCs IN DERMATOPHYTE INFECTIONS? Dermatophytes, a group of keratinophilic fungi with approximately 40 species, are grouped into three genera: Trichophyton, Microsporum and Epidermophyton.79 Dermatophytes can invade human skin, hair, and nails and cause dermatophytosis (tinea).80 The role and relevance of MCs in dermatophyte infections are ill characterized. MCs have been observed to accumulate at sites of dermatophyte infections. For example, in Microsporum canis‐infected cats, the histopathological features of lesional skin include MC infiltration of the superficial dermis.81 Moskalewski et al. reported that the number of skin MCs is higher in mice infected with dermatophytes.82 In a Trichophyton quinckeanum dermatophytosis mouse model,83 MCs also accumulated in the dermis at the site of infection. This may or may not be relevant for the course of dermatophyte infections, and we will need further studies to clarify this. 8 WHAT CAN SPOROTHRIX, PARACOCCIDIOIDES, ASPERGILLUS, MALASSEZIA, CANDIDA AND DERMATOPHYTES TEACH US ABOUT THE EFFECTS OF MCs IN FUNGAL INFECTIONS? It appears that the inflammation induced by MCs following fungal infections may not always and only be beneficial to the host, but may primarily benefit the fungal pathogen by facilitating spreading (Figure 1 and Table 1). Additional support for this comes from studies with the fungal cell wall product zymosan.84-86 Zymosan‐induced peritonitis was found to be suppressed in MC‐deficient mice (WBB6F1‐W/Wv), and this was restored by adoptively transferred BMCMCs.87 MCs also appear to be involved in the morphine‐mediated inhibition of zymosan‐induced peritonitis in mice.88, 89 Gelatinase B/MMP‐9 as an inflammatory marker enzyme in mouse zymosan peritonitis was constitutively present in unstimulated MCs,90 and zymosan‐activated peritoneal MCs reportedly contain and produce more MMP‐9 after zymosan administration. Fungal species (family) Main colonized tissues Study host Study type IgE/non–IgE‐mediated response Reference Sporothrix schenckii (Ophiostomataceae) Skin, lungs, joints, bones, brain Mouse In vivo Non–IgE‐mediated response 31, 32 Paracoccidioides brasiliensis (Ajellomycetaceae) Lymph nodes, mucous membrane of the mouth Mouse In vivo Non–IgE‐mediated response 35 Human In vitro Non–IgE‐mediated response 36, 37 Aspergillus fumigatus (Trichocomaceae) Lung Mouse In vivo IgE‐mediated response 40 Human In vivo IgE‐mediated response 41, 44, 47 Mouse In vivo Non–IgE‐mediated response 45 Mouse In vitro Non–IgE‐mediated response 49 Horse In vivo IgE‐mediated response 50 Mouse In vivo, In vitro Non–IgE‐mediated response 51 Malassezia (Malasseziaceae) Skin Human In vitro IgE‐mediated response 59-61 Candida albicans (Saccharomycetaceae) Skin, mucosal surfaces Mouse In vivo IgE‐mediated response 69, 72 Guinea‐pig In vitro IgE‐mediated response 71 Rat In vitro Non–IgE‐mediated response 73 Mouse In vivo Non–IgE‐mediated response 74 Mouse In vitro Non–IgE‐mediated response 75 Human In vitro Non–IgE‐mediated response 76 Dermatophyte Skin Mouse In vivo Unclear 87, 88 While the published findings from studies on MCs in fungal infections point to a role of MCs that is different from that in bacteria and parasite infection, they do not provide the underlying mechanisms. In Sporotrichosis, for example, MCs promote a wider dissemination of S. schenckii and a greater severity of the infection in mice, but it remains unclear how MCs can mediate these negative effects. Also, most of the investigations performed so far, as described above, have been conducted with mouse models, and it has to be stressed that care should be taken in the translation of findings from the murine to the human system. MCs in mice and humans share many characteristic features, but also differ significantly, for example, in the expression of certain mediators such as proteases.91 In addition, the characterization of the role of MCs in fungal infections, as of now, has not made use of novel models and insights recently introduced to MC research, a limitation that will need to be addressed by future studies. Further evidence from ongoing and forthcoming research will need to provide a better picture of the mechanisms and the relevance of MC responses in fungal infections in humans. Finally, more focus has to come to the clinical relevance of characterizing the role of MCs in fungal infections. Several MC inhibitors are currently under development for the treatment of MC‐driven diseases. These inhibitors should be explored for their effects on fungal diseases. They may be especially helpful (and instructive) in the treatment of patients with allergic diseases and fungal infections. On the other hand, MC inhibitors may carry a risk of exacerbating some fungal infections, candidiasis for example, as MCs can reportedly contribute significantly to the clearance of C. albicans. In conclusion, further studies are needed to characterize the complex function of MCs in fungal diseases and, especially, in allergic diseases exacerbated by fungal infections. In the long run, this will lead to better strategies for preventing and treating fungal infections. ACKNOWLEDGEMENTS This work was supported by grants from A Joint Sino‐German Research project (no. GZ901) and Jiangsu Provincial Medical Youth Talent (no. QNRC2016736). CONFLICT OF INTEREST The authors confirm that there are no conflicts of interest. REFERENCES

Abstract Despite advances in our understanding of immunoglobulin A nephropathy (IgAN) over the past decade, there are currently no specific therapies capable of targeting key pathways involved in the pathogenesis of the disease. Recent studies have, however, provided new insights into important molecular pathways that are likely to be amenable to therapeutic manipulation in the future. Specifically, a deeper understanding of the role of mucosal immunity, B‐cell activation and mesangial cell activation in IgAN has provided the impetus for a number of exciting phase II/III clinical trials in IgAN. In this review, we examine some of these on‐going studies, first examining studies that clarify the role of traditional immunosuppression in IgAN, then focusing on novel therapies in early clinical studies, looking closely at the rationale for these agents in relation to our current understanding of the pathogenesis of IgAN. Finally, we examine emerging pathways and therapeutic agents that have the potential to be developed as novel therapies in the coming years. It is hoped that as we continue to develop a greater understanding of IgAN, emerging therapies will soon become a reality in the day‐to‐day treatment of patients with IgAN. Immunoglobulin A nephropathy (IgAN) is the commonest pattern of glomerulonephritis in the world and an important cause of end‐stage renal disease (ESRD).1, 2 Since its first description more than 47 years ago,3 there have been significant advances in our understanding of the pathogenesis of IgAN, with numerous studies offering key insights into the molecular mechanisms of the disease.4 There is now convincing evidence that the production of poorly galactosylated IgA1 and glycan‐specific IgG and IgA autoantibodies leads to the formation of IgA‐containing immune complexes, and these immune complexes deposit within the mesangium, where their consequent effects on mesangial cells, podocytes, and proximal tubule cells are central to the development of IgAN. Despite gaining better insight into the pathogenesis of IgAN, current treatment strategies remain generic and common to all forms of chronic kidney disease, principally renin–angiotensin system (RAS) blockade, reduction of proteinuria and blood pressure control, or involve the use of non‐specific immunosuppression, such as corticosteroids.5 According to the recently published KDIGO guidelines (2012), RAS blockade with angiotensin‐converting enzyme inhibitors (ACEi) or angiotensinogen receptor blockers (ARB) when proteinuria is more than 1 g/day remains the mainstay of treatment in IgAN. Theoretically, more intense and complete RAS inhibition using more than one RAS blocker versus a single agent could offer more benefit in patients with proteinuric renal disease. However, following the retraction of the COOPERATE study, there are no studies looking specifically at dual RAS blockade in patients with IgAN.6 In addition, there is emerging evidence that dual blockade, in specific groups of patients, with kidney disease, does not provide additional benefit but actually increases the risk of adverse events.7-9 It has been suggested that it is premature to abandon combination RAS blockade on the basis of the current level of evidence and that an approach of individualizing therapy with careful dose titration according to proteinuria and tolerability in patients with kidney disease should be adopted,10 a practice that will require formal testing. Immunosuppression, in the form of a 6 month course of corticosteroid therapy, is only suggested in patients who have persistent proteinuria and preserved renal function (glomerular filtration rate (GFR) >50 mL/min per 1.73 m2) after a 3‐ to 6 month period of optimization of blood pressure control and RAS blockade (grade 2C evidence). At the time the KDIGO guidelines were written, there was insufficient evidence to support the use of any other form of immunosuppression (apart from in crescentic IgAN). With the exception of the recommendation on the use of RAS blockade when proteinuria exceeds 1 g/day, the remaining KDIGO suggestions for the treatment of IgAN were based on low levels of evidence (grade 2B–2D). The lack of high‐quality clinical trials has limited the robust evaluation of therapies in IgAN, particularly immunosuppressive regimens, and there remains a great deal of uncertainty over the optimum treatment of patients at high risk of progressive chronic kidney disease. In addition, current therapeutic options seek to ameliorate the damage occurring as a consequence of inflammation and scarring far downstream in the disease pathology, while disease‐specific therapies capable of targeting early events in the pathogenesis of IgAN, for example, preventing IgA immune‐complex formation or mesangial IgA deposition and mesangial cell activation, are currently lacking. In this context, there is a pressing need for a better understanding of the role of immune modulation in IgAN and in evaluation of novel treatment strategies that target specific pathogenic pathways beyond current traditional treatments. One of the challenges in the evaluation of treatment options is, like many other kidney diseases, IgAN is frequently slowly progressive, and high‐risk patients may develop ESRD only after many years. Significant loss of renal function (defined by the doubling of serum creatinine) has been accepted as a valid end point for clinical studies, but even this is a relatively long‐term outcome. Surrogate end points, such as proteinuria, thus become an important tool in the evaluation of clinical studies as these allow more rapid prediction of clinically important outcomes using smaller numbers of patients over a shorter period of time.11 In IgAN, proteinuria has been demonstrated to be an early marker of progressive disease, and treatment resulting in reduction of proteinuria predicts a favourable long‐term outcome.12 However, there is uncertainty over the use of proteinuria as a surrogate end point in clinical studies. First, while it has been demonstrated that proteinuria is a marker of kidney damage and predicts risk of progression in renal disease, it remains unclear if proteinuria causes or mediates progression of kidney disease. Furthermore, proteinuria is not always a necessary intermediate endpoint on the path to ESRD, implying that its relationship to progressive renal disease is not always direct. Intervention trials demonstrating association between effects of treatment on proteinuria and renal outcome are either limited to specific agents (e.g. ACEi) or specific diseases (e.g. diabetic nephropathy), and the results cannot necessarily be extrapolated to other kidney diseases or pharmacologic agents. This is especially so when considering that kidney disease is heterogeneous and the degree of proteinuria (and its consequent effects) varies widely. Finally, the size effect of proteinuria as a surrogate marker in predicting renal outcomes is not precisely defined, meaning clinicians cannot quantify reliably the effects on renal outcome based on the degree of reduction in proteinuria. For these reasons, regulatory agencies, such as the FDA, have been cautious when reviewing approvals based predominantly on a surrogate end point of proteinuria as evidence of treatment efficacy for drug approvals.13 Therefore, in the absence of a sensitive and specific mechanistic biomarker that mirrors disease activity or predicts response to treatment, the definition of the primary end point in many current clinical trials in IgAN remains contentious, balancing the pragmatic need to conduct a meaningful trial while appeasing regulators to allow early licensing of the drug under investigation. As you will see from Tables 1-7, most investigators have opted to use proteinuria reduction as a surrogate marker of reduced risk of renal function decline and ESRD. It remains to be seen how regulatory agencies will view relatively short‐term reductions in proteinuria as evidence of efficacy of emerging therapies in IgAN. Inclusion criteria All biopsy‐proven IgAN patients, between 18 and 70 years of age will be considered if: Proteinuria >0.75 g/day, and Hypertension (BP > 140/90 mm Hg or use of antihypertensive medications), or GFR < 90 mL/min Exclusion criteria GFR < 30 mL/min At the end of run‐in phase, patients will be excluded if: Proteinuria >3.5 g/day, or Decrease in GFR > 30% (from baseline) Trial design Intervention Follow‐up duration Run‐in phase All patients will receive supportive therapy: RAS blockade, BP control (target 125/75 mm Hg), statin therapy, dietary counselling for low salt diet and protein restriction (with verification of dietary restriction via measurement of 24‐hour urine sodium and urea excretion) and education/intervention programme to stop smoking 6 months Randomization phase Patients with persistent proteinuria exceeding 0.75 g/day at the end of the run‐in phase will be randomized to two groups: Supportive group Supportive group will continue supportive therapy as initiated in the run‐in phase Immunosuppressive group GFR ≥ 60 mL/min Six month course of corticosteroids, consisting of intravenous methylprednisolone 1 g per day for 3 days at the start of month 1, 3, 5 and oral prednisolone 0.5 mg/kg every other day for 6 months on the remaining days GFR between 30 and 60 mL/min Oral cyclophosphamide (1.5 mg/kg per day, adjusted down to the nearest 50 mg) for 3 months, together with oral prednisolone (40 mg/day, tapered to 10 mg at 3 months), after which patients will receive azathioprine (1.5 mg/kg per day), together with oral prednisolone (10 mg/day for months 4–6 and 7.5 mg/day after 6 months) for 3 years 3 years Primary end point Complete remission defined by proteinuria of less than 0.2 g/day with stable renal function (loss of GFR less than 5 mL/min from baseline) GFR loss of 15 mL/min or higher, at the end of 3 years BP, blood pressure; GFR, glomerular filtration rate; IgAN, IgA nephropathy. Inclusion criteria All biopsy‐proven IgAN patients, above 14 years of age will be considered if: Proteinuria >1 g/day, and GFR 20–90 mL/min Exclusion criteria Received immunosuppression in past 1 year Trial design Intervention Follow‐up duration Pre‐randomization phase Dose of ACEi or ARB and blood pressure will be optimized 4–8 weeks Randomization phase Patients with persistent proteinuria exceeding 1 g/day at the end of the pre‐randomization phase will be randomized to two groups: Oral methylprednisolone 0.8 mg/kg per day for 2 months (rounded to the nearest 4 mg and with a maximal dose of 48 mg/day) then tapered by 8 mg/day each month Matching placebo at the same dosage 6–8 months Primary end point Composite of: 50% decrease in GFR Development of ESRD, and Death due to kidney disease. The planned average follow‐up period is for a duration of 5 years, although the study is event driven and will be continued until 335 primary endpoints have occurred, so the final follow‐up duration may be longer or shorter depending on the event rate. ACEi, angiotensin‐converting enzyme inhibitor; ARB, angiotensinogen receptor blocker; BP, blood pressure; ESRD, end‐stage renal disease; GFR, glomerular filtration rate; IgAN, IgA nephropathy. Inclusion criteria All biopsy‐proven IgAN patients, above 18 years of age will be considered if: UPCR > 0.5 g/g or 24‐UTP > 0.75 g/day, and GFR > 45 mL/min Exclusion criteria Received immunosuppression in past 2 years At the end of run‐in phase, patients will be excluded if: Decrease in GFR > 30% (from baseline) Trial design Intervention Follow‐up duration Run phase ACEi and/or ARB will be dosed to target a blood pressure of 130/80 mm Hg and UPCR < 0.5 g/g 6 months Randomization phase Patients with persistent proteinuria exceeding UPCR > 0.5 g/g at the end of the run‐in phase will be randomized to three groups: Enteric budesonide 16 mg/day, Enteric budesonide 8 mg/day, or Matching placebo group Patients who received 16 mg/day dosing will taper the dose to 8 mg/day during the first 2 weeks of the follow on phase, with the placebo and 8 mg/day groups receiving placebo to maintain blinding. 9‐month treatment and 3‐month follow‐on Primary end point Change in UPCR at 9 months ACEi, angiotensin‐converting enzyme inhibitor; ARB, angiotensinogen receptor blocker; BP, blood pressure; GFR, glomerular filtration rate; IgAN, IgA nephropathy; UPCR, urine protein : creatinine ratio; UTP, urine total protein. Inclusion criteria All biopsy‐proven IgAN patients, between 18 and 65 years of age will be considered if: Proteinuria between 1 and 6 g/day GFR > 30 mL/min Stable dose of ACEi and/or ARB prior to screening Exclusion criteria Received immunosuppression within past 6 months Trial Design Intervention Follow‐up duration Randomization phase Patients will be randomized to two groups: Treatment group: Induction phase – subcutaneous blisibimod 100 mg three times weekly for 8 weeks, then Maintenance phase – subcutaneous blisibimod 200 mg weekly for 16 weeks Placebo group 24 weeks Primary end point Change in proteinuria at 24 weeks ACEi, angiotensin‐converting enzyme inhibitor; ARB, angiotensinogen receptor blocker; BP, blood pressure; GFR, glomerular filtration rate; IgAN, IgA nephropathy. Inclusion criteria All biopsy‐proven IgAN patients, between 18 and 70 years of age will be considered if: Proteinuria >0.5 g/day GFR > 30 mL/min Stable dose of ACEi and/or ARB for at least 90 days prior to randomization Exclusion criteria Prior use of immunosuppressant Trial design Intervention Follow‐up duration Randomization phase Patients will be randomized to three groups: Fostamatinib 150 mg twice daily, Fostamatinib 100 mg twice daily, or Placebo group 24 weeks Primary end point Change in proteinuria at 24 weeks ACEi, angiotensin‐converting enzyme inhibitor; ARB, angiotensinogen receptor blocker; GFR, glomerular filtration rate; IgAN, IgA nephropathy. Inclusion criteria All biopsy‐proven IgAN patients, between 18 and 70 years of age will be considered if: Proteinuria >1 g/day (or >0.5 g/day if on combination RAS blockers) GFR 30 to 90 mL/min Exclusion criteria Received >6 months of corticosteroids Trial design Intervention Follow‐up duration Randomization phase Patients will be randomized to two groups: Intravenous rituximab 1 g on day 1 and 15. The course will be repeated again at 6 months (i.e. day 168 and 182) after the first infusion Placebo All patients will receive, in addition to ACEi and/or ARBs, omega‐3 fatty acid fish oil supplements 3.6 g EPA/day 12 months Primary end point Change in proteinuria at 12 months Patients will be analysed according to response – complete response defined as proteinuria <300 mg/day and less than 10% decline in GFR from baseline, and partial response defined as reduction of proteinuria >50% from baseline and decline in GFR of less than 25% from baseline Patients with proteinuria reduction of less than 50%, unchanged or increasing proteinuria and/or decline in renal function will be classified as non‐responder. ACEi, angiotensin‐converting enzyme inhibitor; ARB, angiotensinogen receptor blocker; EPA, eicosapentaenoic acid; GFR, glomerular filtration rate; IgAN, IgA nephropathy; RAS, renin–angiotensin system. Inclusion criteria All biopsy‐proven IgAN patients, above 18 years of age, will be considered if: Proteinuria >1 g/day Stable dose of ACEi and/or ARB for at least 4 weeks prior to screening Trial design Intervention Follow‐up duration Open‐label study Patients will receive intravenous bortezomib 1.3 mg/m2 on days 1, 4, 8 and 11 A second cycle is to be given a month later for non‐responders 1 year Primary end point Reduction in proteinuria at 1 year ACEi, angiotensin‐converting enzyme inhibitor; ARB, angiotensinogen receptor blocker; IgAN, IgA nephropathy. In this article, we review the on‐going clinical trials in IgAN that examine both traditional treatments and emerging therapies, looking closely at the rationale, design and the progress of individual studies, and finally, we will examine some of the potential new therapies that hold promise but are not yet in clinical trials in IgAN. We have summarized our current understanding of the pathogenesis of IgAN in Figures 1 and 2 and have included the sites of action of the emerging therapies in relation to our understanding of key pathogenic pathways in IgAN. STOP‐IgAN (ClinicalTrials.gov Identifier: NCT00554502) Rationale The formation of IgA‐containing immune complexes is central to the pathogenesis of IgAN and suggests that modulation of B and T cell function with immunosuppressants may be beneficial in IgAN. A number of small clinical trials have assessed the value of immunosuppression (mostly involving corticosteroids) in IgAN; however, a major limitation of these older studies is the lack of conformity in the use of RAS inhibition and blood pressure targets with current best practice. This is likely to have led to the inclusion of patients who would have achieved a reduction in proteinuria and stabilization of GFR with RAS inhibition and blood pressure control alone. It is currently unclear, therefore, whether conventional immunosuppressive therapy adds benefit above current best practice supportive care. The STOP‐IgAN (Supportive Versus Immunosuppressive Therapy of Progressive IgA Nephropathy) trial was designed to specifically evaluate the additive benefit of traditional immunosuppressive therapy to current best practice standard care.14 Trial design, inclusion and exclusion criteria, and primary end point The STOP‐IgAN trial is a phase III prospective, randomized, multi‐centre, open‐label study conducted in Germany, comparing two parallel arms: immunosuppressive therapy added to comprehensive supportive therapy or comprehensive supportive therapy alone. Briefly, the trial encompasses a 6 month run‐in phase where patients receive best supportive therapy. Patients with persistent proteinuria exceeding 0.75 g/day at the end of the run‐in phase are randomized to either continue supportive therapy alone or to receive supportive therapy and immunosuppression. Patients randomized to the immunosuppression arm receive one of two‐treatment regimens, depending on their renal function (Table 1). Trial progress to date The trial began in 2008, and recruitment was completed in 2014 (148 patients – 74 in each arm). Interestingly, only slightly more than one‐third of the patients screened were eligible for randomization as there was a significant reduction of proteinuria during the run‐in phase with supportive treatment alone. This observation reinforces the fact that many IgAN patients with significant proteinuria can achieve a significant reduction in proteinuria (<0.75 g/d) with optimal supportive care only. Preliminary results from the study was announced at the 52nd ERA‐EDTA congress (Late Breaking Clinical Trials session) in London. The number of patients achieving complete remission was higher in the group receiving immunosuppression (odds ratio 5.33, P = 0.008, based on available case analysis); however, this was not accompanied by a difference in GFR decline between the two groups at three years (−1.6 versus −1.5 ml/min in supportive and immunosuppression group respectively, P = 0.362). Furthermore, treatment with immunosuppression was associated with higher rate of serious adverse events such as infections, diabetes and weight gain. The full report of the study is expected to be published soon. TESTING (ClinicalTrials.gov Identifier: NCT01560052) Rationale To date, there have been several small studies specifically investigating the use of corticosteroids in IgAN. Of these studies, three found that corticosteroids were associated with reduced progression of IgAN. In the largest published randomized controlled trial by Pozzi et al., treatment with corticosteroids was associated with a reduction in proteinuria and reduction in the risk of ESRD over a 10 year period.15 However, RAS blockade was used only in a minority of the patients, and blood pressure control was sub‐optimal by current standards. In two more recent trials, treatment with oral prednisolone for 6–8 months (a regimen less intense than that used by Pozzi et al.) resulted in a reduction in the proportion of patients progressing to doubling of serum creatinine or ESRD.16, 17 However, in both trials, patients stopped RAS blockade prior to entering the trial, and there was no run‐in phase before randomization to optimize the ACEi and/or ARB dose. It is therefore possible that the patients in these trials might have responded to ACEi/ARB alone (a hypothesis being tested by the STOP‐IgAN trial detailed above). In a recent systematic review, Lv et al. identified nine trials examining the effect of steroids in IgAN (536 IgAN patients with proteinuria >1 g/day and normal renal function at inclusion). The use of steroid therapy was associated with a lower risk of doubling of serum creatinine/halving of GFR or ESRD. In these studies, steroid therapy was, however, associated with a 55% higher risk of adverse events, including diabetes, weight gain and other features of steroid excess. Many of the included studies were single‐centre, comprised small numbers of patients and had only short‐term follow‐up data. Therefore, the balance of benefits and risks of corticosteroid treatment in IgAN remains uncertain, and it is not clear who should be treated with this therapy. The Therapeutic Evaluation of STeroids in IgA Nephropathy Global (TESTING) trial was designed to specifically answer this question. Trial design, inclusion and exclusion criteria and primary end point The TESTING study is an international, multi‐centre, double‐blind, randomized, placebo‐controlled trial to study the role of oral methylprednisolone in preventing kidney events in high‐risk IgAN patients. Unlike previous studies, this study includes patients with a GFR between 20 and 90 mL/min per 1.73 m2, and as with STOP‐IgAN, patients enrolled in the TESTING study enter a pre‐randomization period where the dose of ACEi or ARB and blood pressure is optimized over a 4‐ to 12 week period (depending on whether the patient was on stable dose of ACEi or ARB prior to the pre‐randomization period). Table 2 summarizes the design of the study. The target sample size for the study is 1300 patients (650 in each group) and will make this study the largest randomized controlled clinical trial ever performed in IgAN. Trial progress to date The study began recruitment in 2012, and randomization is expected to be complete in 2017. NEFIGAN (ClinicalTrials.gov Identifier: NCT01738035) Rationale One of the key observations in patients with IgAN is the presence of poorly galactosylated IgA1 in both the serum and glomerular immune deposits, a finding that is consistently reproduced in populations of different geographic origin. The precise origin of this IgA1 remains the subject of ongoing investigations.18-21 A number of observations have, however, led to the postulation that the mucosal immune system may be an important source of poorly galactosylated IgA1. It has been shown that the site of antigen encounter influences the phenotype of B lymphocytes and the IgA that they subsequently produce. In health, B cells that encounter antigen in the systemic compartment (e.g. following subcutaneous or intradermal immunization) synthesize heavily galactosylated IgA1 while mucosally programmed B cells, which encounter antigen across mucosal surfaces, synthesize poorly galactosylated IgA1.22 It is therefore postulated that the poorly galactosylated IgA1 in IgAN may originate from the mucosal immune system as an exaggerated mucosally primed B cell response to commonly encountered mucosal pathogens. Whether this response is limited to the mucosa‐associated lymphoid tissue or involves B cells that have mis‐trafficked to the bone marrow is currently not known.23, 24 A number of separate studies have reported high levels of IgA antibodies in the serum against a variety of mucosal antigens including Helicobacter pylori, oral polio vaccine, gliadin, ovalbumin and dextran B512 in patients with IgAN.25-29 Furthermore, the well‐recognized association of mucosal infection with episodes of visible haematuria suggests a close relationship between the mucosal immune system and nephritogenic IgA1 production in IgAN. Consistent with these observations, recently published genome‐wide association studies in IgAN have identified loci that are directly associated with intestinal mucosal immunity.30, 31 Therefore, given that the mucosal immune system may play a key role in the generation of nephritogenic IgA in IgAN, immunosuppression targeted to sites of mucosal B‐cell induction may provide an alternative to traditional regimens of systemic immunosuppression which are associated with significant side effects. In an open‐label, uncontrolled, proof‐of‐concept pilot study by Smerud et al., the potential treatment effects and safety profile of Nefecon® – a modified release formulation of budesonide, specifically designed to deliver budesonide to the ileocecal Peyer's patches, with minimal systemic exposure and side effects – were examined.32 Sixteen patients (62.5% males) with biopsy‐proven IgAN (albuminuria >500 mg/day and serum creatinine <200 μmol/L) were treated with Nefecon® 8 mg/day for 6 months and followed up for a further 3 months. During the 6 month treatment period, there was a median relative reduction in urinary albumin excretion of 23% (interquartile range −0.36 to −0.4, P = 0.04). This was accompanied by a minor reduction (6%) of serum creatinine and modest increase (8%) of GFR. Three out of 16 patients were withdrawn prematurely from the study due to abdominal pain relating to the study drug (n = 2) and sleep disturbances and increased micturition (n = 1). No major corticosteroid‐related side effects were reported. This study, coupled with our current understanding of the role of mucosal immunity in IgAN, formed the basis for the NEFIGAN trial. Trial design, inclusion and exclusion criteria, and primary end point The NEFIGAN trial is a multi‐centre, double‐blind, randomized, placebo‐controlled phase 2b study to evaluate the efficacy and safety of two different doses of Nefecon® in the treatment of patients with primary IgAN. The study is being conducted in 62 centres in 10 European countries. Consistent with other studies, there is an initial phase of rigorous blood pressure control and maximization of RAS blockade over a 6 month run‐in phase. Patients are eligible for randomization into the treatment phase of the trial (Table 3) if they have a UPCR of >0.5 g/g despite maximal supportive care. Trial progress to date The study began in 2012, and recruitment of patients was completed in January 2014. In contrast to the STOP‐IGAN trial, of the 299 patients who entered screening, 70% (208) were eligible for randomization suggesting that in the 4 years from commencement of STOP‐IgAN to 2012, there had been a significant improvement in the standard of care received by patients with IgAN across Europe. Results from this study are expected in mid‐2015. On the 14 April 2015, Pharmalink announced that the NEFIGAN trial had fully met its primary efficacy end point at a planned interim analysis and had been stopped early with respect to statistical analysis of the endpoint. BRIGHT‐SC (ClinicalTrials.gov Identifier: NCT02062684) Rationale Effective B cell maturation and survival is dependent on the presence of BAFF (B cell activating factor), also known as B‐lymphocyte stimulator (BlyS), and APRIL (a proliferation inducing ligand). BAFF and APRIL are expressed by T cells, dendritic cells, monocytes and macrophages but not by B cells. BAFF and APRIL bind to the TNF superfamily receptors B cell maturation antigen, TNFR homolog transmembrane activator and Ca2+ modulator and CAML interactor (TACI) and B‐cell‐activating factor receptor (does not bind APRIL). These receptors are expressed almost exclusively by immune cells in the B cell lineage. Expression is weak on immature bone marrow B cells but becomes progressively stronger during peripheral B cell development. Ligation of these receptors contributes to T‐cell‐independent responses of B cells, negative regulation of the size of the B‐cell compartment, class‐switch recombination and plasma cell survival.33-35 Serum levels of BAFF are elevated in patients with autoimmune disease, and levels of BAFF correlate with autoantibody levels.36-40 It has also been reported that elevated levels of BAFF are associated with an increased risk of antibody‐mediated renal allograft rejection.41 Evidence is also emerging for a role of BAFF in IgAN. Transgenic mice overexpressing BAFF display B‐cell hyperplasia, have elevated levels of IgA in the serum and intestinal lamina propia, and develop mesangial IgA deposition. Interestingly, mesangial IgA deposition only developed in those mice exposed to environmental antigens, presumably reflecting mucosal B cell activation.42, 43 In a separate study of tonsillar mononuclear cells (TMCs) exposed to deoxycytidyl‐deoxyguanosineoligodeoxynucleotides, to mimic the immunostimulatory activity of microbial DNA, Goto et al. demonstrated that high levels of BAFF were associated with an increased production of IgA by TMC in IgAN.44 Furthermore, the production of IgA by TMCs was inhibited by blockade of BAFF signalling. Serum levels of BAFF are elevated in IgAN, and BAFF levels correlate with a worse outcome in IgAN as measured by renal histology (increased mesangial hypercellularity, segmental glomerulosclerosis and tubular atrophy/interstitial fibrosis) and higher serum creatinine at time of renal biopsy.45 Interruption of BAFF and APRIL signalling with belimumab (Benlysta®, a human monoclonal antibody that inhibits BAFF), blisibimod (a selective peptibody antagonist of BAFF) and atacicept (a fusion protein containing the extracellular, ligand‐binding portion of TACI and the modified Fc portion of human IgG, blocks BAFF and APRIL) has proven effective in phase II/III trials in systemic lupus erythematosus (SLE).46 Given the success of treatment with BAFF inhibitors for patients with SLE,47, 48 there is considerable interest in examining the efficacy of BAFF inhibition in other B cell‐driven diseases, including IgAN. The first such study in IgAN is the BRIGHT‐SC study (Blisibimod Response in IgA Nephropathy Following At‐Home Treatment by Subcutaneous Administration). Trial design, inclusion and exclusion criteria, and primary end point BRIGHT‐SC is an international multi‐centre, randomized, double‐blind, placebo‐controlled phase II/III trial to examine the efficacy and safety of blisibimod in patients with IgAN (Table 4). Blisibimod (a fusion between the Fc portion of IgG and a peptide sequence selected for its ability to bind with high affinity to BAFF) is able to bind both soluble and membrane bound BAFF. Target recruitment for BRIGHT‐SC is 200 patients. Trial progress to date The study began in 2013 in Asia and is currently recruiting patients in Asia and Europe. Recruitment is expected to be complete by 2016. SIGN (ClinicalTrials.gov Identifier: NCT02112838) Rationale Effective B cell maturation and survival are not only dependent on BAFF and APRIL but also signalling through the B cell receptor (BCR). Activation through the BCR is required for normal antibody production, and defects in BCR signal transduction may lead to immunodeficiency, autoimmunity and B‐cell malignancy. One of the key intracellular signal transduction pathways activated on ligation of the BCR is the spleen tyrosine kinase (Syk)–Bruton's tyrosine kinase (BTK) axis, where BTK acts as an essential downstream effector of Syk in regulating both the maturation and survival of the B‐cell lineage. Given the central role of Syk in transmission of activating signals within B‐cells, it has been proposed that Syk plays a key role in autoantibody production and the pathogenesis of autoimmune disorders. In the only published clinical trial to date, administration of fostamatinib, an orally active relatively selective small molecule Syk inhibitor, resulted in a dose‐dependent improvement in disease severity in a 6 month double‐blind, placebo‐controlled trial in rheumatoid arthritis. While Syk is primarily expressed in haematopoietic tissues, there is expression of Syk in a variety of other tissues where it mediates diverse biological functions including cellular adhesion, innate immune recognition, osteoclast maturation, platelet activation and vascular development. This wide tissue distribution may explain the side effects seen with fostamatanib in the rheumatoid arthritis trial. Side effects included diarrhoea, upper respiratory tract infections, neutropenia and hypertension. Interestingly, Syk expression has also recently been reported in the kidney where it appears to regulate the kidney's response to injury.49 In an animal model of nephrotoxic nephritis Syk inhibition reduced the severity of the inflammatory cell infiltrate and proinflammatory cytokine levels in the kidney, reduced the extent of histological damage and reduced the level of proteinuria, even when treatment was delayed until disease was well established.50 In vitro experiments by the same authors demonstrated a down‐regulation of MCP‐1 production when mesangial cells and macrophages were stimulated with aggregated IgG in the presence of Syk inhibitors. Separately, Syk inhibition was shown to significantly reduce autoantibody production in a rodent model of experimental autoimmune glomerulonephritis.51 Similar to the nephrotoxic nephritis model, Syk inhibition decreased the production of proinflammatory cytokines in ex vivo nephritic glomeruli and in vitro bone marrow‐derived macrophages suggesting a therapeutic effect of Syk inhibition independent of its effect on autoantibody production. There is also now evidence that Syk plays a role in determining the mesangial cell response to IgA in IgAN. Kim et al. demonstrated that inhibition of Syk in mesangial cells was capable of blocking the proliferative and pro‐inflammatory effects of IgA immune complexes in IgAN.52 Furthermore, when renal biopsies were stained for total and phosphorylated Syk, there was a clear upregulation of Syk in glomeruli of patients with IgAN. As a result of these experiments and the crucial role of Syk in BCR signalling, the SIGN (Syk Inhibition for Glomerulonephritis) study was developed to evaluate the efficacy of Fostamatinib disodium, the orally bioavailable prodrug of the active compound R406, in the treatment of IgAN.53 Trial design, inclusion and exclusion criteria, and primary end point SIGN is an international, multi‐centre, phase 2, double‐blind, placebo‐controlled, randomized trial on the safety and efficacy of fostamatinib in the treatment of IgAN (Table 6). The study aims to recruit 75 patients, and notably, patients will undergo a repeat renal biopsy after treatment to evaluate the effects of Syk inhibition on histopathology. Trial progress to date The first patient was randomized in late 2014, and recruitment is expected to be complete by 2016. Rituximab in IgAN (ClinicalTrials.gov Identifier: NCT00498368) Rationale Rituximab (Rituxan®, MabThera® and Zytux®), a chimeric monoclonal antibody directed against the CD20 antigen of B cells, causes B‐cell depletion and has proven efficacy in treating B cell lymphomas, leukaemias, transplant rejection and autoimmune disorders including SLE, rheumatoid arthritis, dermatomyositis and ANCA‐associated vasculitis.54 B cell depletion is clearly an attractive therapeutic approach in IgAN as this should switch off production of poorly galactosylated IgA1 and glycan‐specific IgG/IgA autoantibodies and limit the formation of circulating immune complexes. There have, however, been few studies of the use of rituximab in IgAN. In a case series reported by Sugiura et al., the effects of a single‐dose of rituximab (375 mg/m2) was evaluated in 24 patients with primary glomerular diseases, of which five patients had IgAN.55 In this small observational study, there was no benefit from rituximab over conventional treatment in patients with IgAN with regard to proteinuria reduction at 6 month follow‐up, despite a significant reduction in B‐cell numbers. Trial design, inclusion and exclusion criteria, and primary end point A phase IV multi‐centre, randomized, prospective, open‐label trial for the treatment of progressive IgAN with rituximab is currently underway in the United States, with a recruitment target of 54 patients (Table 6). Similar to the SIGN study, patients will undergo a repeat renal biopsy after treatment to examine the effects of rituximab on histopathology. Trial progress to date The trial started in February 2009 and has completed recruitment. The study is expected to report in late 2015. Bortezomib in IgAN (ClinicalTrials.gov Identifier: NCT01103778) Rationale In normal cells, proteasomes regulate protein expression and function by degradation of ubiquitylated proteins. A specific form of proteasome, the immunoproteasome, found exclusively in antigen presenting cells (APC), is capable of generating peptides which are of optimal size and composition for MHC binding. Expression of the immunoproteasome is induced by IFN gamma during an immune response, and excessive immunoproteasomal activity has been linked to inflammatory and autoimmune diseases including SLE and rheumatoid arthritis. Increased immunoproteasome activity in APC results in upregulation of the activated form of NF‐κB, an anti‐apoptotic and pro‐inflammatory regulator of cytokine expression. There is also an emerging evidence for a role of increased immunoproteasome activity in IgAN.56 Excessive switching from proteasome to immunoproteasome in peripheral blood mononuclear cells has been observed in patients with IgAN with high levels of proteinuria.57 The trigger for this switch is not clear, but it has been postulated that an aberrant response to mucosal immune challenge, such as to viral infections that trigger IFN gamma release, may be involved. Bortezomib (Velcade®) is the most widely used proteasome inhibitor in current clinical practice and is licensed for the treatment of multiple myeloma and relapsed mantle cell lymphoma. Multiple mechanisms are likely to be involved in the B cell/plasma cell killing, but ultimately, it is thought bortezomib prevents degradation of pro‐apoptotic factors, permitting activation of programmed cell death in targeted cells. The selectivity of bortezomib for B lineage cells makes it an attractive proposition as a novel treatment in IgAN, although the side effect profile, which includes peripheral neuropathy, myelosuppression causing neutropenia and thrombocytopenia and gastrointestinal effects, may limit its utility to only those patients at greatest risk of progressive renal failure. Trial design, inclusion and exclusion criteria, and primary end point A single‐centre, open‐label, exploratory study examining the effects of bortezomib in IgAN is currently underway in the United States. The details of the study are listed in Table 7. Trial progress to date Recruitment commenced in July 2010 and is currently ongoing; the study is expected to report in late 2016. Potential New Pathways With an increasing understanding of mucosal immunology and the progressive translation of these findings to IgAN, potential new therapeutic targets are continually emerging. At the same time, investigators are looking at whether it might be possible to use both existing and novel therapies developed for other autoimmune diseases or those targeting B cell malignancies to treat IgAN. Manipulation of toll‐like receptor activation in IgAN Toll‐like receptors (TLRs) play a critical role in the early innate immune response to invading pathogens by sensing microbial pathogens and endogenous danger signals via recognition of a diverse range of pathogen‐associated molecular patterns (PAMPs), such as bacterial lipopolysaccharide, RNAs, and DNAs and danger‐associated molecular patterns (DAMPs). TLRs are found on a diverse range of cells including macrophages and dendritic cells, and stimulation of TLRs by the corresponding PAMPs or DAMPs initiates signalling cascades that result in a variety of cellular responses including the production of interferons (IFNs), pro‐inflammatory cytokines and effector cytokines that direct the adaptive immune response. There is increasing interest in the role of TLRs in both maintenance of normal mucosal immunity and the aberrant mucosal responses observed in IgAN. In a murine model of IgAN, B cell expression of TLR‐9 and the signalling molecule, myeloid differentiation factor 88 (MyD88) was significantly greater in mice that were conventionally housed (and therefore exposed to environmental antigens) than in mice housed in pathogen‐free conditions.58 This increased expression of TLR‐9 and MyD88 was associated with more severe IgA‐mediated renal injury. Furthermore, nasal challenge with TLR‐9 ligands aggravated the renal injury and resulted in strong Th1 polarization and increases in serum and mucosal IgA levels. An association between polymorphisms in the TLR‐9 gene and disease progression in two cohorts of patients with IgAN has also been reported. Importantly, TLR‐9 is expressed on mucosal B cells, and it has been suggested that B cell activation through TLR‐9 may be pivotal in the generation of the mucosal IgA response and therefore may be implicated in the generation of poorly galactosylated IgA1. TLR‐9 expression by dendritic cells has also been reported, and activation of dendritic cells through TLR‐9 ligation has been proposed as a potent stimulator of autoantibody production in IgAN.59 Targeting B‐cell TLR activation, therefore, may offer an alternative strategy for modulating mucosal B‐cell activation, poorly galactosylated IgA production and IgA immune complex formation in IgAN. In SLE, it has been shown that signalling through TLR‐7 and TLR‐9 directly antagonizes the immunosuppressive effect of corticosteroids. Furthermore, dual TLR‐7 and TLR‐9 inhibitors alleviate this antagonism and allow lower doses of corticosteroids to be used in SLE without loss of efficacy.60 Hydroxychloroquine (Plaquenil®), a drug commonly used in SLE, is a potent inhibitor of TLR‐9 and, to a lesser extent, TLR‐7 and TLR‐8. In addition, hydroxychloroquine interferes with functioning of the immunoproteasome and antigen presentation by APC. While not a new drug, perhaps it is time to consider a trial of hydroxychloroquine, either alone, or in combination with Nefecon®, to specifically target the mucosal IgA immune system in IgAN. Dissolution of mesangial IgA deposits with IgA1 proteases In order to circumvent the mucosal IgA immune system, several species of pathogenic bacteria including Haemophilus influenzae, Streptococcus pneumonia, Neisseria gonorrhoeae and Neisseria meningitides have evolved the ability to synthesize potent IgA1 proteases that are designed to proteolytically degrade human mucosally secreted IgA1, specifically targeting the O‐glycosylated IgA1 hinge region. These bacterial IgA1 proteases offer an interesting proposition. If it is not possible to completely turn off IgA immune complex formation in IgAN, perhaps it might be possible to use these IgA1 proteases to directly target the IgA1 molecule itself and degrade circulating IgA immune complexes and dissolve away mesangial IgA deposits.61 In a proof‐of‐concept study, Lamm et al. demonstrated that the H. influenzae IgA1 protease could cleave human IgA1 and IgA1‐containing immune complexes in vitro. When systemically administered in a passive mouse model of IgAN, the IgA1 protease significantly reduced the extent of mesangial IgA immune complexes.62 How these observations will translate into clinical trials in humans is currently unclear, but interestingly, in 2012, Shire acquired a worldwide exclusive license from IGAN Biosciences to develop and commercialize protease‐based therapeutics for the treatment of IgAN.61 Conclusion In the relatively short history of clinical trials in IgAN, there has not been a more exciting time. There is worldwide recruitment of IgAN patients to a number of well designed, appropriately sized and powered randomized controlled clinical trials evaluating the effectiveness of both traditional and novel immunomodulatory agents in IgAN. Furthermore, advances in our understanding of the pathogenesis of IgAN are identifying more potential biochemical pathways that might be amenable to therapeutic manipulation. Many of the trials discussed in this review will not report for a number of years, but it is hoped that some of these will provide sufficient evidence to translate into meaningful new treatments for our patients with IgAN in the next 3–5 years. References Notes : BP, blood pressure; GFR, glomerular filtration rate; IgAN, IgA nephropathy. ACEi, angiotensin‐converting enzyme inhibitor; ARB, angiotensinogen receptor blocker; BP, blood pressure; ESRD, end‐stage renal disease; GFR, glomerular filtration rate; IgAN, IgA nephropathy. ACEi, angiotensin‐converting enzyme inhibitor; ARB, angiotensinogen receptor blocker; BP, blood pressure; GFR, glomerular filtration rate; IgAN, IgA nephropathy; UPCR, urine protein : creatinine ratio; UTP, urine total protein. ACEi, angiotensin‐converting enzyme inhibitor; ARB, angiotensinogen receptor blocker; BP, blood pressure; GFR, glomerular filtration rate; IgAN, IgA nephropathy. ACEi, angiotensin‐converting enzyme inhibitor; ARB, angiotensinogen receptor blocker; GFR, glomerular filtration rate; IgAN, IgA nephropathy. ACEi, angiotensin‐converting enzyme inhibitor; ARB, angiotensinogen receptor blocker; EPA, eicosapentaenoic acid; GFR, glomerular filtration rate; IgAN, IgA nephropathy; RAS, renin–angiotensin system. ACEi, angiotensin‐converting enzyme inhibitor; ARB, angiotensinogen receptor blocker; IgAN, IgA nephropathy. Citing Literature Number of times cited: 6 Bengt C Fellström, Jonathan Barratt, Heather Cook, Rosanna Coppo, John Feehally, Johan W de Fijter, Jürgen Floege, Gerd Hetzel, Alan G Jardine, Francesco Locatelli, Bart D Maes, Alex Mercer, Fernanda Ortiz, Manuel Praga, Søren S Sørensen, Vladimir Tesar and Lucia Del Vecchio, Targeted-release budesonide versus placebo in patients with IgA nephropathy (NEFIGAN): a double-blind, randomised, placebo-controlled phase 2b trial, The Lancet, 389, 10084, (2117), (2017). Crossref Robert J Wyatt, Are we ready for targeted therapy for IgA nephropathy?, The Lancet, 389, 10084, (2083), (2017). Crossref See Cheng Yeo, Chee Kay Cheung and Jonathan Barratt, New insights into the pathogenesis of IgA nephropathy, Pediatric Nephrology, (2017). Crossref Magdalena Krochmal, Katryna Cisek, Szymon Filip, Katerina Markoska, Clare Orange, Jerome Zoidakis, Chara Gakiopoulou, Goce Spasovski, Harald Mischak, Christian Delles, Antonia Vlahou and Joachim Jankowski, Identification of novel molecular signatures of IgA nephropathy through an integrative -omics analysis, Scientific Reports, 10.1038/s41598-017-09393-w, 7, 1, (2017). Crossref Bing Du, Ye Jia, Wenhua Zhou, Xu Min, Lining Miao and Wenpeng Cui, Efficacy and safety of mycophenolate mofetil in patients with IgA nephropathy: an update meta-analysis, BMC Nephrology, 18, 1, (2017). Crossref Sigrid Lundberg, Emelie Westergren, Jessica Smolander and Annette Bruchfeld, B cell–depleting therapy with rituximab or ofatumumab in immunoglobulin A nephropathy or vasculitis with nephritis, Clinical Kidney Journal, 10.1093/ckj/sfw106, (sfw106), (2016). Crossref

Abstract United airways disease (UAD) is the concept that the upper and lower airways, which are anatomically and immunologically related, form a single organ. According to this concept, upper and lower airway diseases are frequently comorbid because they reflect manifestations of a single underlying disease at different sites of the respiratory tract. Allergic asthma‐allergic rhinitis is the archetypal UAD, but emerging data indicate that UAD is a heterogeneous condition and consists of multiple phenotypes (observable clinical characteristics) and endotypes (pathobiologic mechanisms). The UAD paradigm also extends to myriad sinonasal diseases (eg, chronic rhinosinusitis with or without nasal polyps) and lower airway diseases (eg, bronchiectasis, chronic obstructive pulmonary disease). Here, we review currently known phenoendotypes of UAD and propose a “treatable traits” approach for the classification and management of UAD, wherein pathophysiological mechanisms and factors contributing to disease are identified and targeted for treatment. Treatable traits in UAD can be analyzed according to a framework comprising airway inflammation (eosinophilic, neutrophilic), impaired airway mucosal defense (impaired mucociliary clearance, antibody deficiency), and exogenous cofactors (allergic sensitizers, tobacco smoke, microbes). Appreciation of treatable traits is necessary in advancing the effort to deliver precise treatments and achieve better outcomes in patients with UAD. 1 INTRODUCTION The respiratory tract is divided into the upper and lower airways at the level of the vocal cords. Despite this artificial distinction, they are anatomically contiguous and immunologically related. United airways disease (UAD) is the concept that upper and lower airways form a single organ, with upper and lower airway diseases co‐occurring frequently because they reflect different manifestations of a single underlying disease process.1 Possible mechanisms linking the upper and lower airways include aspiration of nasal inflammatory cells and mediators into the tracheobronchial tree, hematogenous propagation of inflammatory mediators from one part of the airway to another, stimulation of bone marrow progenitor cells, or the nasobronchial reflex.2 The archetypal UAD is allergic asthma‐rhinitis, but emerging data indicate that UAD is complex and heterogenous, and may present with other clinical phenotypes of sinonasal disease (eg, chronic rhinosinusitis with or without nasal polyps) and lower airway disease (eg, bronchiectasis, chronic obstructive pulmonary disease [COPD]). Even within a given clinical phenotype, there is further variability based on the underlying pathobiologic mechanisms, known as endotypes. Current guideline‐based management approaches (summarized in Table 1) stratify patients according to symptoms, disease control, future risk, and occasionally broad clinical phenotypes, but do not sufficiently consider complexity and variability in other important dimensions such as airway inflammation, airway mucosal defense, and exogenous cofactors. Failure to recognize this complexity or treat both upper and lower compartments of the airways inevitably leads to suboptimal outcomes for patients. Site Disease Approach to stratification of patients and selection of therapies Patient stratification groups Recommended treatments according to patient stratification Other possible treatments Upper airway Allergic rhinitis Depends on symptoms, time course, severity, control, patient preferences, availability of treatment, and cost. One approach is a step‐up/step‐down approach based on assessment of control Step 1: mild symptoms Intranasal or oral non‐sedating H1‐antihistamines Leukotriene antagonists Allergen and irritant avoidance Allergen immunotherapy Nasal filters or saline Step 2: moderate‐to‐severe symptoms or persistent AR Intranasal corticosteroids Step 3: uncontrolled at step 2 (current or historical) Combination intranasal corticosteroids and intranasal H1‐antihistamines Step 4: uncontrolled at step 3 Short course oral steroids Chronic rhinosinusitis Based on clinical phenotype CRSsNP Intranasal corticosteroids Antibiotics (second line) Consider short course of oral steroids Saline irrigation Sinus surgery for medically refractory disease CRSwNP Intranasal corticosteroids Short course of oral steroids Antibiotic if suspicion of infection Consider leukotriene receptor for acetylsalicylic acid sensitivity Lower airway Asthma Assessment of symptoms, exacerbations, and lung function followed by stepwise escalation/de‐escalation of therapy Step 1 Consider low‐dose ICS Leukotriene receptor antagonists Theophylline Allergen avoidance Allergen immunotherapy for patients with allergic rhinitis and sensitized to house dust mites, frequent exacerbations, and FEV1 > 70% Vaccinations Weight reduction for obese asthma patients Step 2 Low‐dose ICS Step 3 Low‐dose ICS + LABA Step 4 Medium‐/high‐dose ICS + LABA Step 5 Low‐dose oral corticosteroids Add‐on treatments: tiotropium, anti‐IgE, anti‐IL5, bronchial thermoplasty Patients may benefit from phenotyping and endotyping at Step 5 Chronic obstructive pulmonary disease Assessment of symptoms (CAT, mMRC), exacerbations, airflow obstruction (FEV1) Group A (CAT < 10, mMRC < 2, infrequent exacerbations) Bronchodilator Smoking cessation (including pharmacologic treatment) Education, self‐management, and pulmonary rehabilitation Pneumococcal and influenza vaccinations Long‐term oxygen therapy in the presence of severe resting hypoxemia Long‐term noninvasive ventilation for patients with severe chronic hypercapnia and a history of hospitalization for acute respiratory failure Bronchoscopic or surgical lung volume reduction surgery for selected patients Group B (CAT ≥ 10, mMRC ≥ 2, infrequent exacerbations) LABA or LAMA Increase to LAMA + LABA if persistent symptoms Group C (CAT < 10, mMRC < 2, frequent exacerbations) LAMA Increase to LAMA + LABA or change to LABA + ICS if further exacerbations Group D (CAT ≥ 10, mMRC ≥ 2, frequent exacerbations) LAMA + LABA Increase to LAMA + LABA + ICS if persistent symptoms/further exacerbations Consider macrolides Consider roflumilast if FEV1 < 50% predicted and patient has chronic bronchitis Bronchiectasis Assessment of microbial infection, exacerbations and airway symptoms Pseudomonas aeruginosa infection with frequent exacerbations Long‐term inhaled antibiotic treatment Escalate to combined oral and inhaled antibiotic treatment if inadequate response Physiotherapy, pulmonary rehabilitation, and/or airway clearance Pneumococcal and influenza vaccinations Long‐acting bronchodilators for patients with significant breathlessness Non‐Pseudomonas aeruginosa infection with frequent exacerbations Long‐term macrolide treatment Escalate to combined oral and inhaled antibiotic treatment if inadequate response Multiple sequential airway treatments Short‐ or long‐acting bronchodilators → Mucolytic/physiotherapy adjuncts → airway clearance → inhaled antibiotics AR, allergic rhinitis; CAT, COPD Assessment Tool; CRSsNP, chronic rhinosinusitis without nasal polyps; CRSwNP, chronic rhinosinusitis with nasal polyps; FEV1, forced expiratory volume in one second; ICS, inhaled corticosteroids; ICS + LABA, combination inhaled corticosteroids and long‐acting beta agonist; LABA, long‐acting beta agonist; LAMA, long‐acting muscarinic antagonist; mMRC, modified Medical Research Council dyspnea score. The treatable traits approach has been described for lower airway diseases,3 and in this review, we extend its application to combined upper and lower airway disease. We summarize the current evidence for known clinicopathobiologic subtypes (“phenoendotypes”) of UAD. We have grouped the clinical manifestations of UAD according to the “treatable traits” of airway inflammation, airway mucosal defense dysfunction, and exogenous cofactors (Figure 1 and Table 2). These traits are not mutually exclusive and multiple traits may contribute to disease in an individual at any one point in time. Treatable traits may also vary over time. It is with the intention of drawing the readers’ attention to traits that are common to both airway compartments that we have artificially classified the upper and lower airway conditions as presented in this review. We believe that targeting common disease processes, when used in combination with current patient stratification approaches, is likely to lead to clinical improvements at both sites of the respiratory tract. Trait Endotype/pathophysiology Method of assessment/biomarker Clinical phenotype Targeted treatment Upper airway Lower airway Inflammation Eosinophilic, high type 2/TH2‐cytokines IL‐4, IL‐5, IL‐13 Blood periostin, high FeNO, sputum eosinophilia, blood eosinophilia CRSwNP Eosinophilic asthma Corticosteroids (systemic, inhaled, intranasal), anti‐IL‐5, IL‐4, IL‐13, anti‐TSLP, ?CRTh2 antagonist Allergic, systemic CD4+ TH2 cells Blood total IgE, blood specific IgEs, skin prick tests Allergic rhinitis Allergic asthma Allergen avoidance, corticosteroids, antileukotrienes, anti‐IgE, immunotherapy Allergic, local Local nasal and bronchial mucosa specific IgE production Blood total IgE not raised, negative blood specific IgEs, negative skin prick tests, local IgE and specific IgE production, nasal allergen provocation test Local allergic rhinitis Intrinsic asthma ?Omalizumab, ?immunotherapy Non‐allergic eosinophilic IL‐25, IL‐33, TSLP, group 2 innate lymphoid cells Blood periostin, high FeNO, sputum eosinophilia, blood eosinophilia, non‐reactive skin prick tests, absent specific IgE in blood Non‐allergic rhinitis with eosinophilia syndrome (NARES) Adult onset eosinophilic asthma Anti‐TSLP, anti‐IL‐4, IL‐5, IL‐13, ?CRTh2 antagonist Neutrophilic TH17 cells, IL‐17, CXCL1, CXCL8 IL‐8, sputum neutrophilia CRSsNP COPD, neutrophilic asthma Smoking cessation, macrolides Impaired airway mucosal defense Cystic fibrosis Autosomal recessive disorder in CFTR gene leading to improper salt balance and viscous airway surface liquid Genetic testing, sweat chloride test CRSwNP (acid mucin) Bronchiectasis CFTR modulators Primary ciliary dyskinesia Autosomal recessive mutations causing dynein and radial spoke defects Low nasal nitric oxide, ciliary motion and ultrastructure CRSsNP Bronchiectasis No validated PCD‐specific therapies Primary antibody deficiency Deficient immunoglobulins or functional antibody response Quantitative serum immunoglobulin levels, functional antibody responses CRS Bronchiectasis Immunoglobulins, vaccinations Exogenous cofactors Occupational exposures High molecular weight sensitizers: IgE‐mediated type 2 inflammation Low molecular weight sensitizers: haptens Irritant‐induced: no immunological reaction Total IgE, skin prick tests Peak flow monitoring Specific bronchoprovocation challenge Occupational rhinitis, reactive upper airways dysfunction syndrome Occupational asthma, reactive airways dysfunction syndrome Exposure avoidance, respiratory protection devices, ?anti‐IgE Aspirin/non‐steroidal anti‐inflammatories Constitutive overexpression of 5‐lipoxygenase and leukotriene C4 synthase Aspirin/NSAID challenge, urinary leukotriene E4 CRSwNP Severe asthma Avoidance, desensitization, antileukotrienes Staphylococcus aureus enterotoxin Superantigens elicit Th1, Th17 and Th2 cytokines, including IL‐4, IL‐5, IL‐13 Staph aureus enterotoxin IgE's CRSwNP Severe asthma ?anti‐IgE Aspergillus/fungal hypersensitivity TH2 cells, IL‐4, IL‐5, IL‐13, IgE, eosinophilia Aspergillus skin test positivity, Aspergillus specific IgE, IgE > 1000 IU/mL, Blood eosinophil >500/μL, precipitating antibodies to Aspergillus Allergic fungal rhinosinusitis Allergic bronchopulmonary aspergillosis (asthma, bronchiectasis) Steroids, antifungal, ?anti‐IgE Upper respiratory tract infection Variable depending on microbe and underlying disease Respiratory cultures and nucleic acid tests Exacerbation of rhinitis/rhinosinusitis Exacerbation of asthma, COPD or bronchiectasis Antivirals, antibiotics, vaccinations Typical upper and lower airway clinical phenotypes associated with underlying treatable traits are listed. Taxonomy based on clinical phenotypes has significant limitations: each phenotype may represent comprise treatable traits, for example, CRSwNP and asthma may be neutrophilic or eosinophilic inflammation depending on the population studied. Hence, a more precise approach is needed to identify contributory treatable trait(s) in each case, which can aid in selection of targeted treatments. Question mark (?) indicates targeted therapies under investigation. Treatable traits and targeted therapies may be used to complement the current approaches for patient stratification and treatment selection—including non‐targeted treatments—shown in Table 1. 2 AIRWAY INFLAMMATION Airway inflammation is important in the pathogenesis of many upper and lower airway diseases. It is a dynamic trait which varies over time and is modulated by factors such as treatment, infection, environmental exposures, and natural history of disease.4 Clinicians commonly assume that asthma and chronic rhinosinusitis with nasal polyps (CRSwNP) are stereotypically eosinophilic in nature, and that chronic rhinosinusitis without nasal polyps (CRSsNP) or COPD is stereotypically neutrophilic. As discussed below, while these generalizations may hold true for many cases there is substantial variability in the underlying pathophysiological mechanisms for each clinical phenotype or disease. Identification and quantification of airway inflammation can help to direct appropriate treatment. Current methods to assess upper and lower airway inflammation are summarized in Table 3. Method Comments Strengths Limitations Nasal lavage Luminal proteins, cytokines and cells Simple and safe Correlates with, but less invasive than induced sputum Variable fluid recovery and dilution with lavages No sampling of the epithelium Nasal brush or scraping Samples the nasal epithelium Simple and minimally invasive Does not sample deeper layers of the mucosa or the nasal lumen Nasal biopsy Identifies tissue inflammatory cellular subtypes of rhinitis or rhinosinusitis Samples the total nasal mucosa Technically difficult No information about nasal lumen Blood periostin Marker of Th2, eosinophilic airway inflammation Predicts severity of chronic rhinosinusitis Predicts chronic rhinosinusitis with nasal polyps phenotype Noninvasive Not widely available Conflicting evidence on whether periostin is associated with sputum eosinophilia Sputum induction Neutrophil count ≥65% or >500 × 104/mL identifies neutrophilic airway inflammation Eosinophil count ≥3% identifies eosinophilic airway inflammation Noninvasive Well‐validated Not widely available Logistically, technically and practically difficult High failure rate Risk of inducing bronchoconstriction Peripheral blood eosinophils Eosinophil count ≥2% identifies eosinophilic airway inflammation Noninvasive, minimal risk Widely available, cheap Predicts with important clinical outcomes such as exacerbation risk and response to treatment Wide diurnal variation of readings (40%) within the same subject May not be applicable when only local mucosal inflammation occurs Fractional exhaled nitric oxide (FeNO) FeNO < 25 ppb indicates low likelihood of airway eosinophilia FeNO > 47 ppb suggests eosinophilic airway inflammation and corticosteroid responsiveness Noninvasive Quick, easy, and safe to perform Readings may be affected by smoking, respiratory tract infections Sputum eosinophilia and FeNO are not always concordant Urinary leukotriene E4 (LTE4) Marker of cysteinyl leukotriene activity Higher levels observed in aspirin‐exacerbated respiratory disease Collection of urine is simple and of no risk Special equipment and training are required to measure LTE4 2.1 Allergic asthma‐rhinitis The link between allergic asthma and allergic rhinitis (AR) is well established. Up to 40% of AR patients have asthma and almost all patients with asthma have AR.5 Many patients with AR also exhibit nonspecific bronchial hyperresponsiveness.6 IgE‐mediated hypersensitivity to aeroallergens results in type 2/eosinophilic inflammation in the respiratory tract, manifesting as rhinitis in the upper airways and asthma in the lower airways. Upon allergen exposure, sensitization occurs whereby specific IgE (sIgE) are generated, circulate in the blood, and attach to the surface of mast cells and basophils, including those that reside in the airway mucosa. Subsequent exposure to allergen triggers mast cells degranulation, release of chemical mediators, and synthesis of leukotrienes and cytokines, resulting in an immediate allergic airway reaction. The late phase response involves Th2 lymphocyte recruitment with production of inflammatory cytokines such as IL‐4, IL‐5, and IL‐13, as well as activation of eosinophils. The central role of sIgE in allergic asthma‐rhinitis is highlighted by a population‐based birth cohort study showing that presence of peripheral blood sIgE directed against common allergens is associated with increased odds of asthma and rhinitis in childhood.7 In adults with AR, experimental nasal provocation with house dust mite allergens induces not only nasal eosinophilia but also bronchial eosinophilic inflammation, bronchoconstriction, and bronchial hyperresponsiveness,8 suggesting inflammatory crosstalk between upper and lower airways. The allergic asthma‐rhinitis phenoendotype usually begins in childhood and can be identified by measuring peripheral blood total IgE which is elevated, plus demonstration of atopy by the presence of raised sIgE levels in blood or skin prick test positivity. Omalizumab, an anti‐IgE monoclonal antibody, is efficacious for both severe allergic asthma9 and uncontrolled AR.10 Therapeutic response to omalizumab is predicted by higher baseline periostin levels and reduced free serum IgE levels during period of treatment.11 In some cases, specific causative allergens can be identified and targeted with allergen immunotherapy. In both AR12 and allergic asthma,13 immunotherapy improves symptoms and reduces the need for pharmacotherapy in the short term, albeit with limited evidence for longer‐term efficacy. Treatment of AR patients with grass pollen sublingual immunotherapy tablets was associated with slower AR progression, less frequent asthma onset, and slower asthma progression.14 Candidate biomarkers that can identify and/or predict response to allergen immunotherapy include the sIgE:total IgE ratio, specific IgG4 and IgE‐FAB.15 2.2 Local allergic rhinitis‐non‐allergic asthma: local mucosal IgE production A subset of asthma and rhinitis patients demonstrate type 2/eosinophilic allergic airway responses mediated by sIgE which is produced and sequestered in the airway mucosa, with skin prick test non‐reactivity and no detectable circulating sIgE in peripheral blood. In the upper airway, this phenoendotype is known as local allergic rhinitis (LAR), a distinct entity characterized by local production of sIgE in the nasal mucosa without evidence of systemic atopy. LAR does not appear to represent a forme fruste of AR,16 and is associated with increased risk of asthma compared to healthy individuals without LAR.17 The lower airway analog of LAR may correspond to a subtype of non‐allergic asthma which is currently not well defined, but whose existence is supported by studies (Supporting Information: References S1‐S4). In the aforementioned entity, intramucosal IgE synthesis is observed in the lower airways, and functional sIgE can be found in the sputum despite negative skin prick tests and blood sIgE levels. Bronchial inhalational challenge with allergen induces a clinical lower airway allergic response characterized by sputum eosinophilia and bronchial hyperresponsiveness. It is not known why individuals with allergic asthma‐rhinitis demonstrate positive skin prick tests and elevated sIgE in blood, whereas non‐allergic asthmatics and patients with LAR do not although local mucosal IgE synthesis occurs in all those conditions. One possibility is that local mucosal IgE synthesis may not reach sufficient quantities to spill over into peripheral blood. Alternatively, IgE may be sequestered within the bronchial mucosa. Regardless, efficacy of omalizumab has been shown in non‐atopic asthma with evidence of local mucosal IgE production. Pillai et al. (Supporting Information: References S5) randomized 18 such subjects and obtained bronchial biopsies from subjects before and after treatment with 12‐14 weeks of omalizumab. The patients subsequently underwent supervised dose reduction of their regular asthma therapy. Compared to placebo, omalizumab therapy significantly reduced total bronchial mucosal IgE+ cells. Lung function declined in the placebo‐treated group but improved in the omalizumab‐treated group. This small but important study serves as proof‐of‐concept that anti‐IgE can target disease associated with local mucosal IgE production. 2.3 Non‐allergic rhinitis with eosinophilia syndrome‐non‐allergic eosinophilic asthma Type 2 (eosinophilic) inflammation can occur in UAD without evidence of IgE‐mediated hypersensitivity. In the upper airways, this endotype has been termed non‐allergic rhinitis with eosinophilia syndrome (NARES) and is characterized by persistent rhinitis and marked eosinophilia of the nasal mucosa in the absence of local or systemic IgE production, suggesting allergen‐independent induction of eosinophilic airway inflammation.18 Almost half of patients with NARES who do not report asthmatic symptoms nevertheless demonstrate bronchial hyperresponsiveness and sputum eosinophilia, supporting a link between NARES and asthma.19 The corresponding asthma phenoendotype is non‐allergic eosinophilic asthma, in which eosinophilic inflammation and type 2 cytokine production are not driven by allergen‐activated Th2 lymphocytes, but is instead postulated to be initiated by type 2 innate lymphoid cells (ILC2).20 ILC2s are a recently discovered population of lymphocytes capable of producing large amounts of type 2 cytokines (IL‐5 and IL‐13) and orchestrate eosinophilic inflammation in the absence of adaptive immunity, including Th2 cells, B cells, and IgE antibody.21 ILC2s are activated by epithelium‐derived cytokines IL‐25, IL‐33, and thymic stromal lymphopoietins (TSLP), which are released by epithelial cells in response to epithelial damage caused by a wide range of stimuli including microbiota and pollutants. ILC2s are increased in peripheral blood in eosinophilic asthmatics compared to non‐eosinophilic asthmatics and their numbers in sputum correlate with eosinophil counts.22 Increased ILC2s has also been observed in eosinophilic nasal polyps,23 but evidence for the involvement of ILC2 cells in NARES and non‐allergic eosinophilic asthma is lacking and should be the focus of future studies. Potential targeted treatments for this pathway include monoclonal antibodies targeting any of the type 2 cytokines, discussed in the section below. TSLP, targeted by the monoclonal antibody tezepelumab, was shown in a recent dose‐ranging proof‐of‐concept study to reduce exacerbations in patients with uncontrolled asthma, including in those with low IgE (<100 IU/mL).24 Antagonists of CRTH2, the receptor for prostaglandin D2 on ILC2 cells, are currently undergoing evaluation to assess clinical efficacy (Supporting Information: References S6, S7). 2.4 Asthma‐chronic rhinosinusitis Chronic rhinosinusitis (CRS) is broadly defined as an inflammatory disorder of the paranasal sinuses and mucosa of the nasal passages that lasts 12 weeks or longer. The condition is heterogeneous, with 2 broad clinical phenotypes, with nasal polyps (CRSwNP) and without nasal polyps (CRSsNP), as well as diverse endotypes.25 Epidemiologic studies have demonstrated higher prevalence of CRS in asthmatics compared to non‐asthmatics across different populations.26, 27 However, the inflammatory endotype of CRS varies according to the population being studied. In Caucasian subjects, CRSwNP is predominantly eosinophilic and CRSsNP is predominantly neutrophilic, whereas in Chinese subjects CRSwNP is mainly neutrophilic, with fewer Chinese subjects demonstrating eosinophilic nasal polyps.28 Various studies have elucidated the detailed phenoendotypes of either CRS or asthma separately, but there is a paucity of data about phenoendotypes of combined asthma‐CRS. Despite this, phenoendotypes of asthma and CRS are remarkably analogous to each other. Based on sputum inflammatory cells, asthma can be eosinophilic, neutrophilic, mixed eosinophilic and neutrophilic, or paucigranulocytic.29 Based on bronchoepithelial cell gene expression profiles, asthma inflammatory signatures can be partitioned into “Th2 high” (associated with elevated IL‐4, IL‐5, IL‐13, sputum, and blood eosinophilia) or “Th2 low”.30 CRS endotypes can be similarly classified into “IL‐5 high” and “IL‐5 low” clusters.31 Subjects with IL‐5 high CRS have high eosinophilic cationic protein and IgE from nasal specimens, and present clinically with CRSwNP and asthma. Low IL‐5 clusters clinically exhibit the CRSsNP phenotype. “IL‐5 high” CRS appears to be the upper airway analog of “Th2‐high” asthma. The inflammatory pattern of the upper and lower airways in patients with nasal polyps and comorbid asthma was evaluated in a recent study using nasal polyp biopsies and exhaled nitric oxide.32 Approximately, two‐thirds of patients were eosinophilic and one‐third was non‐eosinophilic, with concordance demonstrated between the upper and lower airways in individual subjects. More studies are needed to clarify concordance between upper and lower airway phenoendotypes across the spectrum of inflammatory endotypes in asthma and CRS. Systemic, inhaled, or intranasal corticosteroids are cost‐effective treatments for eosinophilic upper and lower airway inflammation. In cases that are refractory to corticosteroids or in patients who are unable to tolerate high doses of corticosteroids, several biologics targeting eosinophilic inflammation can be considered, such as anti‐IL‐13 (tralokinumab), anti‐IL‐5 (mepolizumab and reslizumab), antibodies against alpha chain of IL‐5 receptor (benralizumab), and alpha subunit of the IL‐4 receptor (dupilumab). Mepolizumab is efficacious in both eosinophilic nasal polyposis and severe eosinophilic asthma,33, 34 reducing polyp burden and need for surgery in the former and reducing exacerbations in the latter. Similarly, dupilumab was demonstrated in small phase 2 trials to reduce polyp burden in patients with chronic sinusitis and nasal polyposis,35 and was found to decrease exacerbations and improve forced expiratory volume in one second (FEV1) in patients with severe eosinophilic asthma.36 Patients with asthma and nasal polyps show greater improvement in Asthma Control Questionnaire score with reslizumab compared to asthmatics without nasal polyps.37 2.5 Chronic obstructive pulmonary disease‐chronic rhinosinusitis Chronic obstructive pulmonary disease is a disease characterized by chronic respiratory symptoms and persistent airflow limitation caused by significant exposure to noxious particles or gases, most commonly tobacco smoke. Although COPD has traditionally been thought to be a purely lower airway disease, there is increasing evidence to suggest frequent concomitant involvement of the upper airways. Multiple epidemiologic studies have shown a high prevalence of nasal symptoms among COPD patients. An early population‐based epidemiologic study conducted in the Swedish general population in 1992 found that 40.1% of individuals with self‐reported chronic bronchitis or emphysema had symptoms of rhinosinusitis, compared with 32.7% in the total study sample.38 In a follow‐up study in 2000, those who reported thick, yellow, nasal discharge and nasal blockage in 1992 had a higher incidence of COPD diagnosis over an 8‐year follow‐up period.39 Another cross‐sectional population‐based Canadian study also found an association between CRS and COPD, both self‐reported.40 Self‐report may lead to misclassification errors of COPD and sinonasal disease but are an inherent limitation of large‐scale epidemiologic studies. Medical record diagnoses, which are considered more reliable than self‐reported medical history, were used in one study from Taiwan which extracted International Classification of Diseases diagnostic codes from a national insurance database and found an increased risk of CRSsNP among COPD patients.41 Recent studies have utilized more stringent and objective criteria for classifying COPD and rhinosinusitis. Bergqvist et al.42 used spirometry for identifying COPD, defined as FEV1/forced vital capacity (FVC) ratio <0.7, and found that the 5‐year incidence of nasal symptoms without having a cold was higher in subjects who demonstrated obstruction on spirometry compared to subjects without baseline spirometric evidence of airflow limitation. Hansen et al43 evaluated the prevalence of paranasal sinus opacification at magnetic resonance imaging in COPD and asthma patients compared to a control group. COPD was defined using similar spirometric criteria as the Bergqvist et al study, whereas asthma was self‐reported. COPD and asthma patients were, respectively, found to be 6 and 2 times more likely than healthy controls to demonstrate paranasal sinus opacification. These findings support the link between upper and lower airway diseases in both asthma and COPD, but we cannot conclude from this study that the association with sinonasal disease is stronger for COPD than for asthma because misclassification bias may have occurred among asthma patients, who were diagnosed based on self‐report instead of objective evidence of variable airflow limitation. Similarly, uncontrolled COPD cohort studies have also found a high prevalence of up to 75% of sinonasal disease, when using spirometry for COPD diagnosis and CT paranasal sinuses for diagnosis of CRS.44, 45 An important limitation of all the aforementioned studies is the use of prebronchodilator, instead of postbronchodilator, FEV1/FVC ratio for identifying cases of COPD. Such a definition could result in other obstructive lung diseases (such as asthma or bronchiectasis) being misclassified as COPD. Only one small study thus far has evaluated COPD using postbronchodilator spirometry and found higher nasal symptoms scored on a visual analogue scale compared to healthy age‐matched controls.46 Although prevalence of sinonasal disease does not increase with declining FEV1,44, 45 the presence of sinonasal disease has profound effects on other important COPD‐related outcomes such as symptom morbidity and exacerbations, which are known to be relatively independent of FEV1. Upper airway symptoms worsen over the natural history of COPD,47 and are associated with lower airway symptoms44 and impaired quality of life.48 Sinonasal symptoms also feature prominently during COPD exacerbations, and are associated with frequent exacerbations47 as well as treatment failure of acute exacerbation of COPD.49 Tobacco smoke forms the common link between COPD and CRS,50 likely by inducing neutrophilic inflammation in the nasal and bronchial mucosa. Compared with non‐smoking healthy controls, smokers without COPD present with nasal inflammation characterized by increased neutrophils and CD8 T lymphocytes51 although this has not been consistently demonstrated.52 Nasal and bronchial IL‐8 levels, neutrophil counts, and CD8 Th1 cells are even higher in smoking COPD patients than in smoking “healthy” controls matched for pack‐year smoking history.51, 53 Nasal and sputum IL‐8 concentrations correlate in COPD patients,53 indicating concordance of neutrophilic inflammation between upper and lower airways in keeping with the UAD hypothesis. Release of neutrophil‐derived proteases such as elastase and matrix metalloproteinase‐9 causes alveolar wall destruction and mucous hypersecretion characteristic of COPD, and may contribute to tissue destruction and remodeling of the sinonasal mucosa seen in CRS. Neutrophilic inflammation results in airflow limitation in the lower airways in COPD, which is mirrored in the upper airways by reduced nasal patency54 and higher nasal resistance.55 Severity of lower airway obstruction, as measured by FEV1, correlates with severity of CRS symptoms.45 Smoking cessation alters the progressive clinical course of COPD by reducing lung function decline and mortality, hence remaining a cornerstone of COPD treatment in addition to other medical therapies. Additionally, in CRS, smoking cessation improves symptoms, quality of life and decreases CRS‐related antibiotic use.56Neutrophilic airway inflammation per se may potentially be targeted for treatment using macrolides, which exert a repertory of effects that suppress neutrophilic inflammation, including downregulation of IL‐8, elastase and matrix metalloproteinase‐9, and reduction of neutrophil chemotaxis and survival.57 Macrolides have proven efficacy in decreasing the frequency of COPD exacerbations,58 and in a small double‐blind randomized controlled trial was associated with improvement in symptoms, endoscopic scores, and nasal lavage IL‐8 levels specifically in Th1, neutrophilic CRS patients.59 Further studies are needed to confirm the efficacy of macrolides. In addition, long‐term macrolides should only be used in carefully selected patients after optimization of other standard medical therapies because of potential cardiac and otological adverse effects, as well as risk of inducing antibiotic resistance. 3 IMPAIRED AIRWAY MUCOSAL DEFENCE Major components of the airway mucosal defense mechanism include the mucociliary escalator, immunoglobulins, antimicrobial peptides, the epithelial barrier, and cells of the innate and adaptive immune system. Impaired mucosal airway defense has a causal role in several phenoendotypes of UAD. The predominant lower airway phenotype in UAD due to impaired mucosal defense is bronchiectasis, which is characterized by abnormal and irreversible dilatation of the lower airways. Frequent coexistence of bronchiectasis and sinonasal disease has long been recognized.60 Patients with bronchiectasis have a high prevalence of CRS when assessed using European Position Paper on Rhinosinusitis and Nasal Polyps criteria, that is, presence of symptoms and positive findings on either nasoendoscopy or CT of paranasal sinuses.61, 62 Conversely, patients with CRS more frequently demonstrate CT evidence of dilated airways compared to individuals without CRS.63 3.1 Cystic fibrosis: bronchiectasis‐chronic rhinosinusitis due to airway surface liquid viscosity Airway surface liquid (ASL) viscosity is regulated in part by the cystic fibrosis transmembrane regulator (CFTR) protein, which mediates ion transport at the airway epithelial surface. Pathological processes which impair CFTR activity can result in abnormal ion flux at the epithelial surface, increased sodium reabsorption, periciliary liquid depletion, and increased mucous viscosity, thereby impairing mucociliary clearance.64 Loss of CFTR‐mediated bicarbonate secretion in the airway epithelium also results in acidification of the ASL, which impairs the action of antimicrobial peptides in the ASL, rendering the airway more susceptible to colonization and infection by microbes.64 Cystic fibrosis (CF) is due to heterogeneous autosomal recessive mutations in the CFTR gene and affects mucociliary clearance at the sinoepithelial lining, lower airways, gastrointestinal tract, and exocrine organs. Sinonasal disease is ubiquitous in CF and characterized by neutrophilic polyps with acid mucin glands, mucoceles, or pseudomucoceles.65 Lower airway disease is characterized by intense neutrophilic inflammation resulting in bronchiectasis, which occurs in 70%‐75% of children by 5 years of age and represents the major cause of morbidity and death in this condition. Treatment of CF has been transformed by the discovery of novel CFTR modulators aimed at addressing the specific defects causing reduced CFTR activity. The most common gene mutation in CF is F508del which causes a processing defect that severely reduces CFTR levels at the epithelial surface and also disrupts channel opening. The latter gating defect is also seen in other mutations, such as the G551D mutation. Lumacaftor and tezacaftor are both CFTR correctors, which improve trafficking of CFTR to the epithelial cell surface. Ivacaftor is a CFTR potentiator and acts by increasing the probability of the channel remaining open. Randomized controlled trials have proven that treatment with these compounds is associated with improved lung function in CF,66-69 and improvement of CF‐associated CRS following treatment with CFTR modulators has been reported anecdotally.70, 71 There is emerging evidence that CFTR dysfunction is a trait in other UADs and may be a target for treatment. CF carrier mutations are implicated in CRS patients who do not have CF.72 Constituents in tobacco smoke such as cadmium, arsenic, and acrolein can cause CFTR dysfunction,73 either by reducing CFTR expression or by inducing gating defects. CFTR decrements are also seen in COPD, including reduced nasal and lower airway potential difference74 and elevated sweat chloride,75 and the degree of CFTR impairment correlates with severity of symptoms and airflow limitation.74, 75 Ivacaftor reverses the acute reduction of CFTR activity after exposure to cigarette smoke in human bronchoepithelial cells,76 and has been demonstrated to ameliorate chronic bronchitis symptoms and improve CFTR activity in a preliminary study.77 Roflumilast, a phosphodiesterase‐4 inhibitor, can stimulate CFTR by increasing cyclic adenosine monophosphate and reverses smoke‐induced inhibition of CFTR activity.78 Roflumilast is approved for reducing exacerbations in severe COPD associated with chronic bronchitis. 3.2 Primary ciliary dyskinesia (PCD): bronchiectasis‐chronic rhinosinusitis due to ciliary dysfunction Chronic rhinosinusitis and bronchiectasis are universal in adults with PCD. The underlying pathophysiology is absent or impaired ciliary motility due to heterogeneous recessive mutations affecting the biogenesis of cilia lining the upper and lower airways.79 In turn, this leads to impaired mucociliary clearance manifesting in the neonatal period as difficulty feeding due to nasal blockage/rhinitis, neonatal respiratory distress syndrome and later in life as recurrent sinopulmonary infections, and bronchiectasis. Nasal nitric oxide is an excellent screening test for patients with a history suggestive of PCD.80 Other tests to assess for ciliary dyskinesia include analysis of ciliary ultrastructure by electron microscopy, assessment of ciliary beat frequency and pattern by high resolution, high‐speed video microscopy, and immunofluorescent staining of ciliary proteins. Ciliary dysfunction occurs in other airway diseases but is usually acquired, for example, due to smoking or infection. In CRS, blunted ciliary response to environmental stimuli has been demonstrated, and this is reversed once the tissue is removed from infected/inflamed environment.81 In COPD, cigarette smoke is associated with ciliary abnormalities in both bronchial82 and nasal mucosae.83 Sputum from atopic and non‐atopic asthmatics have ciliary inhibitory effect on human bronchoepithelial cell cultures, which was reversible upon removal of the sputum.84 Bronchial biopsies from asthma patients demonstrate reduced ciliary number which recovers after treatment with inhaled corticosteroids.85 There are currently no validated effective therapies for primary ciliary dyskinesia and this should be a focus for future research. In acquired cases, removal of the causative factor may reverse ciliary impairment. 3.3 Primary antibody deficiency The most common immunodeficiencies associated with CRS and bronchiectasis are primary antibody deficiencies,86 of which common variable immunodeficiency (CVID), characterized by reduced serum IgG concentrations, is the most common in adults. One‐third and two‐thirds of patients with CVID have bronchiectasis and recurrent sinusitis, respectively.87 Specific antibody deficiency, which is associated with normal immunoglobulin and IgG subclass levels but impaired antibody response to encapsulated respiratory pathogens such as Streptococcus pneumoniae and Hemophilus influenza, is also associated with a high prevalence of CRS and bronchiectasis, accounting for as many as 50% of idiopathic bronchiectasis.88 Patients with primary antibody deficiency most likely require lifelong immunoglobulin replacement, vaccinations, and prophylactic antibiotics. There is also some evidence that antibody deficiency may be a treatable underlying contributor in other airway diseases. For example, low circulating IgG levels are associated with higher rate of exacerbations and shorter time to next exacerbation in two COPD cohorts.89 Treatment with immunoglobulin replacement is associated with significant reduction in exacerbations and rescue antibiotic use in a small case series of 14 COPD patients (Supporting Information: References S8), warranting future prospective, randomized clinical trials to assess the efficacy of IVIG in preventing exacerbations in selected COPD patients with antibody deficiency. A potential contributing role of antibody deficiency in asthma has also been suggested by a small case‐control study which found that asthma patients are more likely to have a diagnosis of CVID or selective IgA deficiency than non‐asthmatic individuals, potentially accounting for the increased risk of bacterial infections in some individuals with asthma.90 A preliminary proof‐of‐concept study found that IVIG significantly reduces the frequency of asthma exacerbations and improved asthma control in adults with IgG subclass deficiency.91 4 EXOGENOUS COFACTORS The respiratory tract is continuously in contact with air from the environment which contains particulates such as microbes, pollutants, and other noxious agents that can cause or aggravate UAD. Aeroallergens such as house dust mites, pollen, and animal dander are the archetypal examples of how specific exposures can cause and worsen allergic asthma‐rhinitis. There are several other well‐delineated phenoendotypes of UAD in which specific inciting or aggravating cofactors are implicated. 4.1 Occupational asthma‐rhinitis More than 90% of individuals with occupational asthma (OA) also have occupational rhinitis (OR).92 Different occupational exposures can cause OA‐OR by different mechanisms. High molecular weight sensitizers, such as proteins and glycopeptides, often cause symptoms after a latency period via IgE‐mediated hypersensitivity.93 In contrast, the mechanism by which low molecular weight agents induces OA‐OR is poorly understood. Single or multiple exposures to high concentrations of irritants can lead to nonspecific inflammation in both upper and lower airways, clinically recognized as reactive upper airways dysfunction syndrome and reactive airways dysfunction syndrome, respectively. In addition to usual care for asthma and rhinitis, the management of OA‐OR is centered around exposure avoidance and use of appropriate respiratory protection equipment. The efficacy of omalizumab for sensitizing agents with IgE‐dependent reactions requires future studies. 4.2 Aspirin‐exacerbated respiratory disease The clinical phenotype of aspirin‐exacerbated respiratory disease (AERD) consists of a triad of asthma, CRSwNP, and exacerbation of upper and lower respiratory tract symptoms within 30 minutes to 3 hours after exposure to aspirin and other cyclooxygenase‐1‐inhibiting drugs. Nasal polyps in AERD can be distinguished from CRSwNP in individuals without AERD by the presence of enhanced eosinophilia, GM‐CSF, and decreased tissue plasminogen activator.94 The endotype of AERD is not fully understood but current available data suggest an underlying dysregulation of arachidonic acid metabolism, with constitutive overproduction of proinflammatory cysteinyl leukotrienes and decreased levels of anti‐inflammatory mediators such as prostaglandin E2.95 This dysregulation is aggravated by cyclooxygenase inhibition following exposure to aspirin and other non‐steroidal anti‐inflammatory drugs in susceptible individuals. The gold standard for diagnosing AERD is aspirin challenge, but challenge testing carries the risk of severe bronchospasm. Urinary leukotriene E4, the end‐product of arachidonic acid metabolism, is raised in AERD and may have a role in diagnosing AERD.96 Treatment options include avoidance of aspirin and NSAIDs, desensitization, and leukotriene receptor antagonists. Omalizumab and mepolizumab may be helpful in AERD as suggested by small trials involving subsets of patients with AERD (Supporting Information: References S9, S10). Whether the observed clinical efficacy of these biologics can be attributed to target AERD‐specific endotype above and beyond their effect on type‐2 eosinophilic inflammation is unclear. 4.3 Asthma‐CRS: hypersensitivity to Staphylococcus aureus enterotoxins Staphylococcus aureus is consistently found in nasal tissues of CRS patients, and evidence supports a role for S. aureus in the pathogenesis of CRS‐asthma. S. aureus colonizes nasal polyps and secretes proteins, including enterotoxins and serine protease‐like proteins in the nasal mucosa, which have the ability to elicit a polyclonal IgE response resulting in an intense type 2, IL‐5 high, eosinophilic inflammation which is likely responsible for chronic severe inflammation of the airways. Presence of sIgE to S. aureus enterotoxins in nasal polyp tissue is strongly associated with asthma in patients with CRSwNP,31, 97 providing an etiological link between asthma‐CRS. In a small placebo‐controlled, proof‐of‐concept study, omalizumab was found to ameliorate nasal polyps and airway symptoms in a small cohort of patients with asthma‐CRSwNP who demonstrated sIgE toward S. aureus enterotoxins.98 4.4 Allergic bronchopulmonary aspergillosis‐allergic aspergillus rhinosinusitis: fungal hypersensitivity IgE‐mediated hypersensitivity to Aspergillus hyphae is the underlying endotype for allergic bronchopulmonary aspergillosis (ABPA) and allergic aspergillus rhinosinusitis (AARS). Upper airway disease is characterized by pansinusitis, passage of nasal plugs, and recurrent nasal polyposis. Lower airway disease is characterized by mucus inspissation within the bronchi and severe asthma, central bronchiectasis, mucous plugging, and fleeting pulmonary infiltrates. In a case series, 22/95 patients of ABPA had also concomitant AARS. All these patients had positive skin tests, positive precipitins, and raised total and sIgE (Supporting Information: References S11). This combination of ABPA and AAS has been termed “sinobronchial allergic mycosis” to recognize their shared etiology and association. The optimal treatment of ABPA and AAS is not known but includes the use of steroids, antifungals, or omalizumab. 5 CONCLUSION The evidence for UAD is strongest for asthma and AR/CRS. However, close associations between upper airway diseases and other lower respiratory tract disorders can also be appreciated. UAD is complex and heterogeneous but a “treatable traits” paradigm, based on assessment of airway inflammation, mucosal defense dysfunction, and exogenous cofactors can help to identify pathophysiological mechanisms involved in the development of disease and potential targets for intervention in individuals presenting with combined upper and lower airway disease. A given individual with disease can demonstrate multiple traits which may vary over time. We have summarized the various treatable traits in UAD and their proven or notional targeted treatments in Table 2. Appreciation of these treatable traits is necessary toward advancing the precision medicine approach in UAD. CONFLICT OF INTEREST The authors declare that they have no conflicts of interest. Supporting Information REFERENCES Notes : AR, allergic rhinitis; CAT, COPD Assessment Tool; CRSsNP, chronic rhinosinusitis without nasal polyps; CRSwNP, chronic rhinosinusitis with nasal polyps; FEV1, forced expiratory volume in one second; ICS, inhaled corticosteroids; ICS + LABA, combination inhaled corticosteroids and long‐acting beta agonist; LABA, long‐acting beta agonist; LAMA, long‐acting muscarinic antagonist; mMRC, modified Medical Research Council dyspnea score. Typical upper and lower airway clinical phenotypes associated with underlying treatable traits are listed. Taxonomy based on clinical phenotypes has significant limitations: each phenotype may represent comprise treatable traits, for example, CRSwNP and asthma may be neutrophilic or eosinophilic inflammation depending on the population studied. Hence, a more precise approach is needed to identify contributory treatable trait(s) in each case, which can aid in selection of targeted treatments. Question mark (?) indicates targeted therapies under investigation. Treatable traits and targeted therapies may be used to complement the current approaches for patient stratification and treatment selection—including non‐targeted treatments—shown in Table 1. Citing Literature Number of times cited: 2 Ana Margarida Pereira, Cristina Jácome, Rute Almeida and João Almeida Fonseca, How the Smartphone Is Changing Allergy Diagnostics, Current Allergy and Asthma Reports, 10.1007/s11882-018-0824-4, 18, 12, (2018). Crossref Umut Can Kucuksezer, Cevdet Ozdemir, Mubeccel Akdis and Cezmi A. Akdis, Precision/Personalized Medicine in Allergic Diseases and Asthma, Archivum Immunologiae et Therapiae Experimentalis, 10.1007/s00005-018-0526-6, (2018). Crossref

National Academy of Sciences

Abstract Background Nonspecific induction of local innate immune responses by mucosally administered immunotherapy is a new approach to protection from upper respiratory tract infections. Therefore, a new liposome‐toll‐like receptor complex (LTC) immune stimulant was developed and investigated for its ability to activate innate immune responses in cats, both in vitro and in vivo, as part of an initial evaluation of LTC for use as an immunotherapeutic agent in cats. Objectives We hypothesized that LTC could activate innate immune responses in cats after topical application to nasal and oropharyngeal mucosal surfaces. Animals Mucosal immune responses to topical administration of LTC were assessed in 7 healthy, purpose‐bred cats, and in vitro responses were assessed using blood samples from healthy cats. Methods Cytokine and cellular immune responses to LTC were evaluated in blood samples, nasal lavage specimens, and pharyngeal swabs from cats, using reverse transcriptase polymerase chain reaction assays, ELISA assays, and flow cytometry. Results Liposome‐TLR complexes rapidly activated leukocytes in vitro, including upregulation of costimulatory molecule expression and cytokine production. Topical administration of LTC in healthy cats triggered rapid recruitment of monocytes to the nasal and oropharyngeal mucosa. Conclusions and Clinical Importance Liposome‐TLR complexes were found to effectively activate innate immune responses in cats after mucosal administration. These findings suggest that LTC have potential for use as a new mucosally administered immunotherapy for nonspecific protection from viral and bacterial respiratory tract infections. Abbreviations FBS fetal bovine serum IL interleukin INF interferon LTC liposome‐TLR complexes NALT nasopharyngeal‐associated lymphoid tissue NK natural killer OX40 tumor necrosis factor receptor superfamily, member 4 PBMC peripheral blood mononuclear cells PBS phosphate‐buffered saline RT‐PCR reverse transcriptase polymerase chain reaction TLR toll‐like receptor TNF tumor necrosis factor 1 INTRODUCTION Upper respiratory infection (URI) is a common and serious problem in cats in boarding facilities, animal hospitals, and shelters. Unfortunately, few quickly effective options are available for preventing these infections in newly exposed cats. Immunoprophylaxis may be most effective if focused at the likely portal of pathogen entry. The upper respiratory tract is the site of entry for several important respiratory pathogens of cats, consisting of a diverse array of viral and bacterial organisms, including feline herpesvirus 1 (FHV‐1), calicivirus, Mycoplasma sp., Bordetella bronchiseptica, and Chlamydia felis.1-6 Activation of host innate immune defenses, including airway epithelial cells and immune cells in oropharyngeal lymphoid tissues, results in production of antiviral and antibacterial cytokines, including type I (INF‐α) and II (INF‐γ) interferons, tumor necrosis factor alpha (TNF‐α), and interleukin 12 (IL‐12) and chemokines such as monocyte chemoattractant protein 1 and IL‐8 that recruit monocytes, T cells, and neutrophils. Local activation of innate immune responses may create an environment that is less susceptible to viral entry and replication or to bacterial invasion and colonization.7 Clinically, such an immune protective effect could be manifested as either partial or complete protection from infection or as a decrease in the severity and duration of clinical signs of illness, along with decreased viral or bacterial shedding. One potential solution to the problem of complex upper respiratory tract infections in cats would be an immunotherapeutic agent capable of inducing nonspecific immune activation at mucosal surfaces in the oropharynx, such as liposome‐toll‐like receptor complexes (LTC). These complexes have been shown previously to induce strong nonspecific protection from lethal bacterial and viral respiratory tract infections after intranasal administration in several different mouse infection models.8-12 In addition, we demonstrated induction of therapeutic immune responses in cats with chronic rhinitis after parenteral administration of LTC.13 Therefore, we reasoned that immune stimulatory complexes could be further optimized for mucosal delivery and generation of local mucosal immune responses in the upper respiratory tract of cats. Therefore, the original LTC formulation was modified to improve its innate immune stimulatory potency and to improve adhesion and contact time with mucosal surfaces. In the present study, the immune activating properties of modified LTC were evaluated in cats.12, 14 The modified LTC consisted of cationic liposomes formulated with 2 TLR agonists: polyinosinic‐polycytidylic acid (poly I:C), as a TLR3, retinoic acid‐inducible gene I, melanoma differentiation‐associated protein 5 agonist, and noncoding plasmid DNA (pDNA), as a TLR9 agonist rich in cytosine‐guanine. In addition, the modified LTC contained a mucosal adhesive agent (carboxymethylcellulose) to increase adhesion to mucosal cell surfaces. We also assessed cytokine and cell activation responses by feline leukocytes in vitro and also assessed the local mucosal immune responses to topical administration of LTC in healthy cats. 2 MATERIALS AND METHODS 2.1 Preparation of liposome‐TLR agonist complexes for mucosal immune stimulation Liposomes were prepared by dissolving the cationic lipid, 1,2‐dioleoyl‐3‐trimentylammonium‐propane (DOTAP) and cholesterol (Avanti Polar Lipids, Alabaster, AL) in chloroform, and the solution was dried overnight in a vacuum desiccator to a thin film. The lipids were rehydrated in 5% sterile dextrose in water at 50°C as described previously.12, 14 Poly I:C (InVivoGen, San Diego, California) and pDNA (noncoding commercial plasmid PCR2.1) Thermo Fisher Scientific, Waltham, MA were added to form liposome complexes, as reported previously.12, 14 The endotoxin content of the plasmid was between 0.04 and 0.25 EU/μg. Carboxymethylcellulose (Sigma‐Aldrich, St. Louis, Missouri) was added to the LTCs to increase mucosal adhesion. In studies in which fluorescent LTCs were delivered, the liposomes were formulated to contain 10% (vol/vol) Topfluor‐labeled cholesterol (Avanti Polar Lipids). 2.2 Animal studies Animal protocols were approved by the Institutional Animal Care and Use Committee of an independent contract research facility in Fort Collins, Colorado. Purpose‐bred cats for the studies were purchased from Liberty Research (Waverly, New York). All animals used in the studies were adopted to owners in the Fort Collins area after completion of the studies, and no animals were euthanized as part of these studies. For collection of nasal lavage samples, cats were restrained manually in a head‐down position, and 0.5 mL of prewarmed sterile phosphate‐buffered saline (PBS) solution was administered into each nostril, and the resulting fluid backflow was collected from the nostrils using a 15 mL conical tube. The procedure was repeated once for each nostril, and all of the fluid collected was pooled for later analysis and stored on ice. For oropharyngeal samples, alginate swabs were gently rubbed and rolled against the mucosa of the caudal oropharyngeal region and placed in tubes with 1 mL PBS and stored on ice before analysis. Nasal and oropharyngeal samples were collected 24 hours before LTC administration, 6 hours after LTC administration, and again 20 and 72 hours after LTC administration. Cats were treated with LTC administered on day 0. Control cats were treated with an equivalent volume of sterile PBS. Each cat was given 1 mL of LTC (0.2 mL intranasally in each nostril and 0.6 mL PO) while being gently restrained. In a separate experiment designed to track LTC uptake by mucosal cells in the nostril and oropharynx of cats, 1 mL of LTC labeled with TopFluor fluorescent dye was administered by instilling 0.2 mL intranasally in each nostril and 0.6 mL (equivalent to 60 μg of Poly I:C and PCR2.1) PO. Average weight of the cats was approximately 3 kg; thus, the intranasal dose of each TLR ligand was approximately 20 μg per kilogram body weight. No adverse effects, either acute or longer term, were observed after intranasal LTC administration (data not shown). 2.3 Preparation of peripheral blood mononuclear cells Whole blood was obtained by jugular venipuncture from a separate group healthy young adult cats and collected into EDTA tubes. For separation of peripheral blood mononuclear cell (PBMC), blood was diluted 1:2 with sterile PBS, then layered over a Ficoll (GE Healthcare, Uppsala, Sweden) gradient and centrifuged. The PBMC were collected and washed twice in PBS and then resuspended in complete tissue culture medium before flow cytometric analysis. 2.4 Cell culture and PBMC activation with LTC Peripheral blood mononuclear cells were cultured in complete medium, which consisted of Dulbecco's modified eagle medium (Thermo Fisher Scientific, Watham, Massachusetts) containing 15% fetal bovine serum (FBS; Avanti Polar Lipids), essential and nonessential amino acids, penicillin, and streptomycin (Gibco‐Thermo Fisher Scientific, Pittsburgh, Pennsylvania). Cells were plated in 96‐well flat bottom plates (Celltreat, Pepperell, Massachusetts) at a density of 1 × 106 cells/well in 200 μL medium. For assays involving LTC activation of PBMC, LTC were added at 3 different dilutions (1 μL per well, 0.3 μL per well, and 0.1 μL per well) in triplicate wells of PBMC in 100 μL complete medium, with careful mixing, and then the cells were incubated for an additional 48 hours. Conditioned medium was collected for IFN‐γ assay, and cells were collected for flow cytometric analysis of activation markers (see below) and for analysis of cytokine gene expression by quantitative reverse transcriptase polymerase chain reaction (qRT‐PCR) (see below). Assays were repeated at least twice, using PBMC from different donor animals. 2.5 Quantitative RT‐PCR analysis of cytokine gene expression in vitro Ribonucleic acid was extracted from PBMC incubated for 48 hours with LTC, as noted above, and expression of IL‐1, IL‐12, IFN‐α, IFN‐γ, and TNF‐α genes was determined using quantitative RT‐PCR. Briefly, RNA was isolated and reverse transcribed using a commercial kit (Qiagen, Germantown, Maryland), and the complementary DNA then was amplified using Cyber green primers (Bio‐Rad, Hercules, California) using a qPCR MX3000p system instrument (Agilent, Santa Clara, California). Cytokine transcript copy numbers were determined by quantitative RT‐PCR, as noted previously.13 2.6 IFN‐γ ELISA Cell culture supernatants from PBMC cultures were analyzed for IFN‐γ concentrations, using a feline IFN‐γ specific ELISA (DuoSet Feline IFN‐γ kit; R&D systems, Minneapolis, Minnesota) according to the manufacturer's instructions. 2.7 Flow cytometry Cells were harvested after 48 hours of in vitro stimulation and immunostained with the following antibodies conjugated with fluorochromes: CD5‐PE (clone f43; Southern Biotech, Birmingham, Alabama), CD21‐APC (clone CA2.1Dg; Bio‐Rad), and OX40‐FITC (clone 7D6; Bio‐Rad), or CD14‐APC (clone TÜK4; Bio‐Rad) and MHCII‐FITC (clone TÜ39; BD Biosciences, San Jose, California). Cells were immunostained with primary antibodies for 20 minutes at 4°C in FACs buffer (PBS with 2% FBS and 0.05% sodium azide) after a 5‐minute incubation with normal cat serum (Jackson ImmunoResearch) to block nonspecific binding. Cells obtained by nasal lavage and oropharyngeal swabs were washed once with PBS, then immunostained in 96‐well plates with the directly conjugated antibodies noted above. Isotype‐matched antibodies were used as control for each primary antibody. Cells from in vitro activated PBMC cultures were collected 48 hours after stimulation and measured using the same panel of antibodies, with appropriate controls. Cells were analyzed using a Beckman Coulter Gallios flow cytometer (Beckman Coulter, Indianapolis, Indiana), and data were analyzed using FlowJo software (Tree Star, Ashland, Oregon). 2.8 Preparation and analysis of nasal and pharyngeal samples Swabs were collected from the distal oropharynx and immediately placed in 2 mL of complete tissue culture medium in 15 mL polypropylene tubes on ice. The samples were lightly vortexed to free cells from the swabs. Fluid was transferred to a new tube, and 2 mL of PBS was added to the empty tube containing the swab, vortexed again, and pooled with the first sample. The sample then was filtered using a 70 μm cell strainer (Celltreat, Pepperell, Massachusetts) to remove debris and mucus. Samples were centrifuged, the cell pellets were resuspended in 0.5 mL of PBS, and the cells were counted. Nasal lavage samples were filtered, washed, centrifuged, resuspended, and counted in the same fashion as for oropharyngeal swab samples. Cells were stained in the same manner as described for PBMC, and equal numbers were analyzed by flow cytometry to determine the cell type and activation. 2.9 Confocal microscopy Monocyte‐derived macrophages were generated from cat PBMC by incubation of plastic adherent monocytes with 10 ng/mL human recombinant macrophage colony stimulating factor for 5 days. The macrophages that were generated were then incubated with 0.1, 1, or 2 μL per well of TopFluor‐labeled LTC in complete medium at 37°C for 4 hours, then washed gently 3 times, fixed with 2% paraformaldehyde for 30 minutes, washed again with distilled water, cover‐slipped, and analyzed by confocal microscopy. Confocal images of feline monocyte‐derived macrophages (untreated control cells or cells incubated with labeled LTC) were obtained using an Olympus IX3 confocal microscope (Waltham, Massachusetts). Images were processed and analyzed using Olympus cellSens software. 2.10 Statistical methods Statistical comparisons between data sets with 2 treatment groups were performed using the nonparametric Mann‐Whitney test. Comparisons among 3 or more groups were performed using analysis of variance, followed by the Tukey multiple means post‐test. Analyses were performed using Prism6 software (GraphPad, La Jolla, California). For all analyses, statistical significance was set at P < .05. 3 RESULTS 3.1 B cells, T cells, and monocytes from feline PBMC activated by LTC in vitro Feline lymphoid tissues express TLRs such as TLR3 and TLR9, and feline immune effector cells (T cells and B cells) express TNF receptor superfamily, member 4 (OX40). When bound to its ligand OX40L, OX40 augments immune responses by prolonging survival of T cells, generation of immune memory, preventing emergence of T‐cell tolerance, and mediating decreased immunosuppressive activity of regulatory T cells. Thus, we chose to examine the effect of in vitro treatment of PBMC with LTC on OX40 expression in these cells.13, 15-17 Unfractionated PBMC were used because LTC may have both a direct effect on leukocytes as well as an indirect effect through crosstalk among various cell types in the mixture. B lymphocytes that were CD21+ were found to exhibit significant upregulation of expression of the costimulatory molecule OX40, in a dose‐dependent fashion with respect to the amount of added LTC (Figure 1, panel A). For T‐cell responses to LTC stimulation, the percentage of CD5+ T cells expressing OX40 also increased significantly as a function of LTC concentration (Figure 1, panel B). Finally, a significant increase in expression of MHCII by CD14+ monocytes was observed in cultures of PBMC treated with LTC (Figure 1, panel C). Similar results were observed in 3 separate studies. 3.2 LTC treatment activates cytokine gene expression by PBMC Quantitative RT‐PCR analysis was used to assess induction of cytokine gene expression by PBMC treated with LTC (Figure 2). Significant upregulation of expression of mRNA for INF‐α as well as upregulation of IL‐1β, IL‐12, and TNF‐α expression after LTC stimulation was identified in most cats. 3.3 LTC treatment triggers IFN‐γ secretion by PBMC Supernatants were collected from PBMC after 48 hours stimulation with LTC, for analysis of IFN‐γ production (Figure 3). Liposome‐TLR complexes treatment stimulated a dose‐dependent, significant secretion of IFN‐γ secretion, as determined by ELISA analysis of supernatants. 3.4 Uptake of labeled LTC by macrophages in vitro Monocyte‐derived macrophages were generated from PBMC of cats by plastic adherence, followed by culture for 4 days in complete medium, after which macrophage uptake of LTC was assessed. Extensive uptake of LTC complexes by macrophages was observed (Figure 4), consistent with the known targeting of liposome complexes to monocytes and macrophages and other phagocytic immune cells. 3.5 Retention of labeled LTC by cells in the oropharynx and nasal cavity Seven healthy adult cats were given 0.1 mL of labeled LTC bilaterally into the nostrils and 0.6 mL PO. Nasal lavage and throat swab samples were obtained before administration, and 6, 20, and 72 hours after LTC administration; cell populations were analyzed by flow cytometry, as described in the Materials and Methods section. Compared to pretreatment samples, samples obtained from the nose and oropharynx at 6 and 20 hours after LTC administration contained a readily identifiable population of cells containing fluorescently‐labeled LTC (Figure 5). The LTC‐containing cells were detected in both nasal lavage cells (Figure 5A) and in throat swab cells (oropharynx, Figure 5B). Peak uptake of LTC‐labeled cells in the nasal lavage samples occurred 6 hours after LTC administration, whereas in the oropharynx peak of LTC uptake occurred 20 hours after administration. 3.6 Intranasal and PO administration of LTC triggers influx of inflammatory leukocytes to mucosal surfaces Cells from the nasal cavity or oropharynx were obtained by nasal lavage or swabbing the back of the throat, respectively, as described above. Cells were immunostained with fluorescently conjugated antibodies against specific cell markers for T cells (CD5+) and monocytes (CD14+) and analyzed by flow cytometry. Neutrophils were identified by their characteristic forward versus side scatter profiles. Pretreatment samples from the nasal cavity of 7 healthy cats contained an average of 7% CD5+ T cells, whereas the average percentage of CD5+ T cells in the oropharynx of cats before treatment was 73%. The percentage of CD14+ monocytes in the nasal cavity before treatment was 2.5%, similar to the 3% of CD14+ monocytes found in the oropharynx (Figure 6). Percentages of neutrophils found in the nasal cavity were 10%, and 2% in the oropharynx of untreated animals. Six hours after topical administration of LTC, a marked increase in the percentage of CD14+ monocytes was observed in both the nasal mucosa (Figure 6A, increasing from an average of 2.5% to an average of 14%) and in the oropharynx (Figure 6B, increasing from an average of 3% to an average of 11%). The percentage of CD14+ monocytes from both nasal and oropharynx samples had decreased by 20 and 72 hours compared to the 6 hours samples but still remained higher than in pretreatment samples. Interestingly, the percentages of CD5+ T cells from the nasal mucosa decreased significantly 6 hours post‐LTC treatment (decreasing from 7% to 3%) and from 73% to 51% in the oropharynx. Nasal CD5+ T cells returned to pretreatment values by 72 hours after treatment. In the oropharynx, the decrease in CD5+ cells observed at 6 hours was also transient, with percentages of CD5+ T cells returning to nearly baseline values (67%) at 20 hours post‐LTC treatment. The neutrophil population was essentially unchanged throughout the study time course in both the nose and the oropharynx after LTC administration (Figure 6). 4 DISCUSSION An effective, broad‐spectrum immunotherapeutic agent capable of activating protective mucosal immune responses would provide a valuable means of inducing rapid protection from a variety of upper respiratory tract pathogens in cats. Therefore, we investigated innate immune responses to LTC, a new immune therapeutic agent designed for mucosal application. We found that LTC induced activation of feline leukocytes, including upregulation of costimulatory molecule (OX40 and MHCII) expression. In addition, LTC induced production of cytokines associated with innate immune responses, including INF‐α, INF‐γ, TNF‐α, and IL‐12. in vivo studies in healthy cats demonstrated uptake of LTC by immune cells and epithelial cells in the upper airways, as well as recruitment of monocytes to the upper airways. Our studies indicated that cats were capable of responding to mucosal immune activation by LTC. Significant upregulation of IFN‐γ along with increases in lymphocyte expression of OX40 and monocyte expression of MHCII all would be associated with innate immune system activation for better viral and bacterial pathogen clearance.7, 18, 19 These findings are consistent with LTC activation of mucosal innate immune responses in cats and induction of partial protection against inhalational challenge with FHV‐1.19 Nasopharyngeal‐associated lymphoid tissue (NALT) is a network of immune cells located in the oropharyngeal region that is primed for immune recognition of viral, bacterial, and fungal pathogens.20-23 When activated by infections, innate immune effector cells in the NALT (including monocytes, neutrophils, innate lymphoid cells, and natural killer [NK] cells) respond by producing cytokines and chemokines, and by activating other immune effector cells including tissue macrophages, epithelial cells, and T and B cells. In our study, several key antiviral and antibacterial cytokines were produced after LTC treatment, including significant increases in secretion of IFN‐γ protein, and increases in INF‐α and IL‐12 transcription, as assessed by qRT‐PCR. These results together suggest broad activation of innate immune responses in nasal and oropharyngeal tissues after administration of LTC in cats. Distinct cellular trafficking responses to LTC treatment were noted when monocyte and T‐cell responses were compared in the nose and oropharynx. For example, LTC administration triggered strong recruitment of CD14+ monocytes to both the nasal cavity and the oropharynx, while at the same time triggering significant, transient disappearance of T cells from these mucosal sites (see Figure 6). Interestingly, minimal neutrophil responses to LTC administration were observed at any time point evaluated, in either the nose or oropharynx. The recruitment of monocytes by LTC could be attributed to production of the chemokine CCL2, which has been noted previously after treatment with LTC.11 In contrast, the transient disappearance of T cells from mucosal sites after LTC administration most likely could be attributed to transient redistribution of T cells after adherence to capillary blood vessels or migration into tissues. A similar effect has been observed previously in dogs treated systemically with LTC.24 In support of transient redistribution, we observed that CD5+ T lymphocytes were rapidly restored in mucosal sites within 72 hours after LTC administration. We also observed distinct differences in resident immune cell populations in the nasal cavity and oropharynx, especially with respect to the density of mucosal T cells. For example, the oropharynx was found to be highly enriched in mucosal CD5+ T cells, whereas T cells were much less numerous in the nasal cavity (see Figure 6). These differences are considered real (and not simply a reflection of differences in sampling techniques), because the percentages of unmarked cells (assumed to represent primarily mucosal epithelial cells, see Figure 6) were similar in samples from the oropharynx, compared to nasal lavage samples. Thus, the oropharynx in cats appears to be enriched in superficially located T cells compared to the nasal cavity and thus well suited to induction of mucosal immune responses after PO administration of nonspecific immune stimuli. The ability of LTC to rapidly induce mucosal immune responses in the upper airway and oropharynx of cats suggests potential use as a nonspecific immune therapeutic agent for prevention of viral and bacterial infections in cats. Administration of LTC significantly decreased signs of FHV‐1 infection and viral shedding in experimentally inoculated cats.19 In addition, we have found that topical application of LTC in dogs can induce mucosal activation and cellular recruitment, similar to the responses seen in cats (Dow S, unpublished data). Thus, topical treatment in the upper airways with LTC may have utility for preventing or mitigating infections with common respiratory tract pathogens in both dogs and cats. The present study had several limitations. For example, the lack of available reagents for full phenotyping of feline leukocytes precluded a more complete assessment of cellular responses to topical LTC administration. In addition, the relatively small numbers of cells collected by nasal lavage or oral swabs precluded cytokine analysis after LTC administration. The in vitro studies using PBMC did not determine the cellular source of specific cytokine production after LTC activation. However, previous studies of LTC in mice indicated that dendritic cells were the primary cellular source of IL‐12 production after LTC administration, and that NK cells were the primary cellular source of IFN‐γ.25, 26 In summary, our studies describe the innate immune activation properties of a novel mucosally active immune stimulant in cats. Innate immune activation by LTC was associated with production of key antiviral and antibacterial cytokines, and with upregulation of important costimulatory molecules on immune effector cells. These findings suggest further evaluation of the LTC mucosal immune stimulant for prevention of URI in cats and for treatment of established URI refractory to treatment with antibiotics or other medications such as lysine. ACKNOWLEDGMENTS The authors acknowledge the assistance of Ms. Jade Kurihara with preparation of LTC used in the study, and Ms. Jennifer Hawley for performing RT‐PCR assays. Portions of this research were presented at the 2017 American College of Veterinary Internal Medicine Forum, National Harbor, Maryland. This study was supported by a grant from the state of Colorado Office of Economic Development and International Trade. CONFLICT OF INTEREST DECLARATION Drs. Lappin and Dow both hold stock options and corporate positions in Poudre Canyon Therapeutics, a Fort Collins company developing the LTC immunotherapy platform technology. OFF‐LABEL ANTIMICROBIAL DECLARATION Authors declare no off‐label use of antimicrobials. INSTITUTIONAL ANIMAL AND CARE AND USE COMMITTEE (IACUC) OR OTHER APPROVAL DECLARATION The cats were managed by a contract research facility in Fort Collins under an approved IACUC protocol. HUMAN ETHICS APPROVAL DECLARATION Authors declare human ethics approval was not needed for this study. REFERENCES

Chronic rhinosinusitis (CRS) is a heterogeneous inflammatory disease with an as-yet-undefined etiology. The management of CRS has historically been phenotypically driven, and the presence or absence of nasal polyps has frequently guided diagnosis, prognosis, and treatment algorithms.