Mucosal Immunity
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Mucosal Immunity
The largest immune tissue in the body
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Mucosal Immunity

is the most recent part of Immunology!

It appeared less than 40 years ago, while systemic immunity exploded 60  years ago.

It is still a minor part of Immunology teaching and research, while the mucosal immune system is at the frontline of encounters with germs, antigens... in other words the environment.

major keywords

IgA http://www.scoop.it/t/mucosal-immunity?q=IgA

tolerance http://www.scoop.it/t/mucosal-immunity?q=tolerance

microbiome http://www.scoop.it/t/mucosal-immunity?q=microbiome

 

july 2015: almost 2100 scoops, more than 1700 visitors, more than 3900 views

december 2015, more than 4700 views by more than 2000 visitors of more than 2300 scoops

november 2016, more than 7;2K views more than 2750 scoops

november 2017 >10K views of >3300 scoops

Gilbert C FAURE's insight:

This topic complements the more general Immunology topic.

 http://www.scoop.it/t/immunology

 

It includes also reproductive immunology searchable on

http://www.scoop.it/t/mucosal-immunity?q=reproductive

https://www.scoop.it/t/mucosal-immunity/?&tag=REPRODUCTION


and  also covers lung immunology

http://www.scoop.it/t/mucosal-immunity?q=lung

 

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Memory B Cells in the Lung May Be Important for More Effective Flu Vaccines

Memory B Cells in the Lung May Be Important for More Effective Flu Vaccines | Mucosal Immunity | Scoop.it
Seasonal influenza vaccines are typically less than 50 percent effective, according to Centers for Disease Control and Prevention studies.
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Frontiers | Tissue Adaptations of Memory and Tissue-Resident Gamma Delta T Cells | Immunology

Frontiers | Tissue Adaptations of Memory and Tissue-Resident Gamma Delta T Cells | Immunology | Mucosal Immunity | Scoop.it
Epithelial and mucosal barriers are critical interfaces physically separating the body from the outside environment and are the tissues most exposed to microorganisms and potential inflammatory agents. The integrity of these tissues requires fine tuning of the local immune system to enable the efficient elimination of invasive pathogens while simultaneously preserving a beneficial relationship with commensal organisms and preventing autoimmunity. Although they only represent a small fraction of circulating and lymphoid T cells,  T cells form a substantial population at barrier sites and even outnumber conventional  T cells in some tissues. After their egress from the thymus, several  T cell subsets naturally establish residency in predetermined mucosal and epithelial locations, as exemplified by the restricted location of murine V5+ and V3V1+ T cell subsets to the intestinal epithelium and epidermis, respectively. More recently, a growing body of studies have shown that  T cells form long-lived memory populations upon local inflammation or bacterial infection, some of which permanently populate the affected tissues after pathogen clearance or resolution of inflammation. Natural and induced resident  T cells have been implicated in many beneficial processes such as tissue homeostasis and pathogen control, but their presence may also exacerbate local inflammation under certain circumstances. Further understanding of the biology and role of these unconventiona
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Tipping the balance: inhibitory checkpoints in intestinal homeostasis

Tipping the balance: inhibitory checkpoints in intestinal homeostasis | Mucosal Immunity | Scoop.it
Review Article
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The role of immunotherapy in in vitro fertilization and recurrent pregnancy loss: a systematic review and meta-analysis

The role of immunotherapy in in vitro fertilization and recurrent pregnancy loss: a systematic review and meta-analysis | Mucosal Immunity | Scoop.it
To study the current evidence on the role of immunotherapy in IVF and in the management
of recurrent pregnancy loss (RPL).

Via Krishan Maggon
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Gut feelings

Gut feelings | Mucosal Immunity | Scoop.it
The same taste receptors found on the tongue are in the stomach, intestines and elsewhere, too. What are they doing there? Well, a lot.
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Non-invasive delivery strategies for biologics

Non-invasive delivery strategies for biologics | Mucosal Immunity | Scoop.it
The requirement for delivery by injection is currently a limitation for the use of biologic drugs such as antibodies. In this Review, Mitragotri and colleagues discuss advances made in non-invasive drug delivery for biologics, including the transdermal, oral and inhalation routes, highlighting...
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Role of eosinophils in airway inflammation of chronic obstructive pulm | COPD

Role of eosinophils in airway inflammation of chronic obstructive pulm | COPD | Mucosal Immunity | Scoop.it
Role of eosinophils in airway inflammation of chronic obstructive pulmonary disease Donald P Tashkin,1 Michael E Wechsler2 1Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA; 2Department of Medicine, National Jewish Health, Denver, CO, USA Abstract: COPD is a...
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Modulation of the microbiota by oral antibiotics treats immunoglobulin A nephropathy in humanized mice. - PubMed - NCBI

Modulation of the microbiota by oral antibiotics treats immunoglobulin A nephropathy in humanized mice. - PubMed - NCBI | Mucosal Immunity | Scoop.it
Nephrol Dial Transplant. 2018 Nov 20. doi: 10.1093/ndt/gfy323.[Epub ahead of print]...
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IL-12, IL-23 and IL-17 in IBD: immunobiology and therapeutic targeting

IL-12, IL-23 and IL-17 in IBD: immunobiology and therapeutic targeting | Mucosal Immunity | Scoop.it
This Review outlines the current understanding of IL-12 and IL-23 biology in IBD, as well as the roles of major downstream cytokines, including IL-17. The authors also discuss how emerging knowledge influences the development of therapeutic strategies in IBD.
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Frontiers | Origin, Differentiation, and Function of Intestinal Macrophages | Immunology

Frontiers | Origin, Differentiation, and Function of Intestinal Macrophages | Immunology | Mucosal Immunity | Scoop.it
Macrophages are increasingly recognised as essential players in the maintenance of intestinal homeostasis and as key sentinels of the intestinal immune system. However, somewhat paradoxically, they are also implicated in chronic pathologies of the gastrointestinal tract, such as inflammatory bowel disease (IBD) and are therefore considered potential targets for novel therapies. In this review, we will discuss recent advances in our understanding of intestinal macrophage heterogeneity, their ontogeny and the potential factors that regulate their origin. We will describe how the local environment of the intestine imprints the phenotypic and functional identity of the macrophage compartment, and how this changes during intestinal inflammation and infection. Finally, we highlight key outstanding questions that should be the focus of future research.
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Going with the flow

Going with the flow | Mucosal Immunity | Scoop.it
For scientists studying the microbiome, the immune system, and their intersection, flow cytometry was a breakthrough.
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Challenges with Point-Of-Care Tests (POCT) for Celiac Disease | IntechOpen

Current screening test for celiac disease involves blood test in centralized pathology laboratories, typically performing enzyme-linked immune-sorbent assays (ELISA) to detect specific celiac disease antibodies.
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Recent advances in understanding chronic rhinosinusitis endotypes - F1000Research

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.
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Viruses | Free Full-Text | Toll-Like Receptor 3 Is Involved in Detection of Enterovirus A71 Infection and Targeted by Viral 2A Protease

Viruses | Free Full-Text | Toll-Like Receptor 3 Is Involved in Detection of Enterovirus A71 Infection and Targeted by Viral 2A Protease | Mucosal Immunity | Scoop.it
Enterovirus A71 (EV-A71) has emerged as a major pathogen causing hand, foot, and mouth disease, as well as neurological disorders. The host immune response affects the outcomes of EV-A71 infection, leading to either resolution or disease progression. However, the mechanisms of how the mammalian innate immune system detects EV-A71 infection to elicit antiviral immunity remain elusive. Here, we report that the Toll-like receptor 3 (TLR3) is a key viral RNA sensor for sensing EV-A71 infection to trigger antiviral immunity. Expression of TLR3 in HEK293 cells enabled the cells to sense EV-A71 infection, leading to type I, IFN-mediated antiviral immunity. Viral double-stranded RNA derived from EV-A71 infection was a key ligand for TLR3 detection. Silencing of TLR3 in mouse and human primary immune cells impaired the activation of IFN-β upon EV-A71 infection, thus reinforcing the importance of the TLR3 pathway in defending against EV-A71 infection. Our results further demonstrated that TLR3 was a target of EV-A71 infection. EV-A71 protease 2A was implicated in the downregulation of TLR3. Together, our results not only demonstrate the importance of the TLR3 pathway in response to EV-A71 infection, but also reveal the involvement of EV-A71 protease 2A in subverting TLR3-mediated antiviral defenses.
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Microanatomical dissection of human intestinal T-cell immunity reveals site-specific changes in gut-associated lymphoid tissues over life

Microanatomical dissection of human intestinal T-cell immunity reveals site-specific changes in gut-associated lymphoid tissues over life | Mucosal Immunity | Scoop.it
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Paper: Loss of Microbiota Diversity after Autologous Stem Cell Transplant Is Comparable to Injury in Allogeneic Stem Cell Transplant

Paper: Loss of Microbiota Diversity after Autologous Stem Cell Transplant Is Comparable to Injury in Allogeneic Stem Cell Transplant | Mucosal Immunity | Scoop.it
Introduction: We have previously reported that clinically relevant, dramatic reductions occur in intestinal bacterial diversity during allogeneic hematopoietic stem cell transplant (allo-HSCT). These are likely attributable to antibiotic exposure, nutritional alterations, and intestinal mucosa injury from high-dose chemotherapy. Patients undergoing autologous hematopoietic stem cell transplantation (AHCT) also receive antibiotics and experience nutritional alterations due to mucositis and other gastrointestinal toxicities. We hypothesized that the pattern of dysbiosis seen in AHCT patients would reflect the changes in allo-HSCT patients. Here, we present a novel analysis of microbiota diversity in AHCT patients from two independent institutions. Methods: We retrospectively identified a cohort of 365 patients (median age 60 years) who underwent AHCT for treatment of hematologic malignancy between May 2009 to February 2018 at two large-volume transplant centers in the US. The population was diverse in terms of histology, conditioning regimens and remission status prior to transplant, with 179 (49%) patients diagnosed with multiple myeloma, 153 (42%) patients diagnosed with lymphoma, and 33 (9%) patients with other diseases. Stool samples from the selected patients were collected approximately weekly during inpatient hospitalization. Sequencing of the V4-V5 region of the bacterial 16S rRNA genes from all samples was performed on the Illumina platform at a central site. Microbial diversity was measured by the Simpson reciprocal a-diversity index (S). We defined the pre-AHCT period as days -10 to 0, and computed median values for patients with multiple samples within that period. We additionally defined monodomination of the microbiota as a single operational taxonomic unit comprising >30% of bacterial abundance. For comparison, we sequenced samples from 17 healthy volunteers and used a public dataset of sequences from 313 healthy volunteers from the NIH Human Microbiome Project (HMP). Median pre-transplant microbial diversity in the healthy patient and AHCT groups was compared by a pairwise Wilcox test to a retrospective cohort of allo-HSCT patients. Results: We evaluated 857 samples from 365 adult patients undergoing AHCT, with 316 patients from Memorial Sloan Kettering Cancer Center (MSKCC) and 49 patients from Duke University Medical Center (DUMC). Median pre-transplant diversity in AHCT patients from both centers was significantly lower than in normal controls (Fig 1A) (HMP vs MSKCC AHCT, S=12.05 vs. 9.19, p<0.005; HMP vs DUMC AHCT, S=12.05 vs 6.91, p<0.005) and reduced in both AHCT patients and allo-HSCT patients (MSKCC AHCT vs MSKCC allo-HSCT, S = 12.05 vs 8.74, p=0.53). In samples taken from days -10 to +30 after transplant, diversity decreased comparably after AHCT and allo-HSCT across both centers, while AHCT patients demonstrated a more rapid recovery at day +30 compared to allo-HSCT patients (Fig 1B). Finally, monodominance was observed in the samples (Fig 1C), with Streptococcus as the most common genus. The cumulative incidence of intestinal domination by any organism was >50% by day 0 and was >75% by day +14. Conclusion: Microbial diversity is reduced prior to transplant in both AHCT and allo-HSCT patients. Loss of diversity after AHCT occurs across centers and the degree of injury is comparable to the dysbiosis in allo-HSCT patients. Preliminary analysis suggests that lower diversity may correlate with worse progression-free survival (PFS) in myeloma patients in our diverse AHCT cohort. Given the known associations of alterations in microbiota composition with toxicities and overall survival in allo-HSCT patients, further evaluation of microbiota injury and its associations with toxicities, PFS, and overall survival (OS) in AHCT patients is warranted. Figure 1: A: The median Simpson reciprocal a-diversity index (S) of pre-transplant (days -10 to 0) samples of AHCT and allo-HSCT patients from two centers, as well as two cohorts of healthy volunteers, was plotted and a pairwise Wilcox test was performed, with p-values as indicated. B: (S) was plotted against time relative to allo-HSCT (on L) and AHCT (on R), for samples collected from day -10 to day +30. Larger values indicate greater diversity. C: Microbiota composition and changes in bacterial monodominance after transplant (days -14 to +28); the most common genus post-transplant is Streptococcus.

Via Krishan Maggon
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Regulating the immune system's 'regulator'

Regulating the immune system's 'regulator' | Mucosal Immunity | Scoop.it
A research team at the Academy of Immunology and Microbiology, within the Institute for Basic Science (IBS) has discovered a possible therapeutic target that pulls the reins of immunity. In Nature Communications, the scientists reported that mice lacking Foxp1 protein in some specific immune cells are more susceptible to immune-induced inflammation.
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Basophils are dispensable for the establishment of protective adaptive immunity against primary and challenge infection with the intestinal helminth parasite Strongyloides ratti

Basophils are dispensable for the establishment of protective adaptive immunity against primary and challenge infection with the intestinal helminth parasite Strongyloides ratti | Mucosal Immunity | Scoop.it
Author summary Helminths are large multicellular parasites that infect approximately every third person. Infections are controlled by a concerted action of innate and adaptive immune responses. Basophils and mast cells are innate effector cells with overlapping functions that have recently been...
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Birth mode is associated with earliest strain-conferred gut microbiome functions and immunostimulatory potential

Birth mode is associated with earliest strain-conferred gut microbiome functions and immunostimulatory potential | Mucosal Immunity | Scoop.it
The effects of caesarean section delivery on mother-to-neonate transmission of microbiota are unclear. Here the authors show that caesarean section delivery can affect the transmission of specific microbial strains and the immunomodulatory potential of the microbiota.
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Differential clustering of faecal and mucosa-associated microbiota in healthy individuals. - PubMed - NCBI

Differential clustering of faecal and mucosa-associated microbiota in healthy individuals. - PubMed - NCBI | Mucosal Immunity | Scoop.it
J Dig Dis. 2018 Nov 22. doi: 10.1111/1751-2980.12688.[Epub ahead of print]...
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A built-in adjuvant-engineered mucosal vaccine against dysbiotic periodontal diseases

A built-in adjuvant-engineered mucosal vaccine against dysbiotic periodontal diseases | Mucosal Immunity | Scoop.it
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Optimized procedures for generating an enhanced, near physiological 2D culture system from porcine intestinal organoids - ScienceDirect

Optimized procedures for generating an enhanced, near physiological 2D culture system from porcine intestinal organoids - ScienceDirect | Mucosal Immunity | Scoop.it
An important practical limitation of the three-dimensional geometry of stem-cell derived intestinal organoids is that it prevents easy access to the a…
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Tissue‐resident MAIT cell populations in human oral mucosa exhibit an activated profile and produce IL‐17 - Sobkowiak - - European Journal of Immunology - Wiley Online Library

Abstract Mucosa‐associated invariant T (MAIT) cells are unconventional T lymphocytes defined by their innate‐like characteristics and broad antimicrobial responsiveness. Whether MAIT cells are part of the tissue‐resident defense in the oral mucosal barrier is unknown. Here, we found MAIT cells present in the buccal mucosa, with a tendency to cluster near the basement membrane, and located in both epithelium and the underlying connective tissue. Overall MAIT cell levels were similar in the mucosa compared to peripheral blood, in contrast to conventional T cells that showed an altered representation of CD4+ and CD8+ subsets. The major mucosal MAIT cell subset displayed a tissue‐resident and activated profile with high expression of CD69, CD103, HLA‐DR, and PD‐1, as well as a skewed subset distribution with higher representation of CD4–/CD8– double‐negative cells and CD8αα+ cells. Interestingly, tissue‐resident MAIT cells had a specialized polyfunctional response profile with higher IL‐17 levels, as assessed by polyclonal stimulus and compared to tissue nonresident and circulating populations. Furthermore, resident buccal MAIT cells were low in perforin. Together, these data indicate that MAIT cells form a part of the oral mucosal T cell compartment, where they exhibit a tissue‐resident‐activated profile biased toward IL‐17 production. Introduction Mucosa‐associated invariant T (MAIT) cells are nonclassical innate‐like T cells that recognize microbial antigens presented by the MHC‐Ib‐related protein 1 (MR1) 1, 2. MR1 displays an extraordinary level of evolutionary conservation among mammals 2-4, and was shown to present microbial vitamin B2 (riboflavin) metabolites from a wide range of microbes that carry the riboflavin biosynthesis pathway 5, 6. MAIT cells express a semi‐invariant T cell receptor (TCR), including the Vα7.2 segment coupled with Jα33, Jα12, or Jα20, and limited TCR β‐chain diversity 7, 8. This TCR repertoire endows MAIT cells with the capacity to respond to the MR1‐restricted riboflavin derivatives produced by diverse microbes 9, 10. MAIT cells respond to antigenic stimulus with an innate‐like speed and produce proinflammatory cytokines including TNF, IFN‐γ, and IL‐17 9, 11-14. They can furthermore kill cells infected by microbes expressing the riboflavin pathway 15, 16 and inhibit intracellular microbial growth 17. Human MAIT cells are also defined by their high expression of CD161, the IL‐18 receptor α subunit (IL‐18Rα), and the transcription factor ZBTB16 18, also known as promyelocytic leukemia zinc finger protein (PLZF) 9, 19. The majority of MAIT cells are CD8+, with some being CD4–/CD8– double‐negative (DN), and a minor CD4+ population 9, 11, 12, 19. Human MAIT cells acquire innate‐like antimicrobial activity in the fetal intestinal mucosa prenatally, prior to the establishment of the commensal microflora 14. In adults, MAIT cells are highly abundant in mucosal tissues, liver, and peripheral blood 1, 2, 20-22. Studies of the murine oral immune system revealed diverse populations of DCs 23, 24, and provided insight into oral T‐cell function 25, but the immunology of the human oral mucosa remains relatively little studied. However, Dutzan et al. recently performed a broad characterization of the immune cell network at the gingival interface and identified T cells as the dominant immune cell population in both the buccal and gingival mucosa 26. CD4+ T cells, mainly of the CD45RO+ "memory" phenotype, were found to be the largest subset at around half of CD3+ cells, whereas smaller subsets of CD8+ and γδ T cells were also present 26. The same study also identified tissue‐resident CD4+ T cells as major producers of IL‐17 in periodontitis. IL‐17 is a cytokine central to the regulation of immune activity at mucosal surfaces 27, 28. Although IL‐17 is implicated in a variety of functions, its role in the regulation of mucosal immunity hinges on three core effects: maintenance of mucosal integrity via regulation of tight junction proteins, induction of antimicrobial molecule production by epithelial cells, and recruitment of neutrophils to sites of infection. Given the capacity of MAIT cells to recognize and respond to several commensal and disease causing microbes occurring in the oral cavity, and the ability of these cells to locate to mucosal sites, one can hypothesize a role for MAIT cells in the oral mucosal barrier. However, the immunobiology of MAIT cells at this mucosal site has to date not been explored. In the present study, we therefore investigated the presence, location, characteristics, and function of MAIT cells in the human buccal mucosa. The findings demonstrate that MAIT cells are part of the healthy buccal mucosal immune defense with specialized tissue‐resident and nonresident subpopulations exhibiting distinct functional profiles, where in particular, the CD69+CD103+ MAIT cell population show strong IL‐17 production. We discuss these findings in the context of contemporary knowledge of immune homeostasis and defense at this important barrier site. Results Identification of MAIT cells in the buccal mucosa Given the broad antimicrobial reactivity of MAIT cells, we were interested in investigating the potential role of these cells in oral mucosa, a site continuously exposed to a broad array of microbial stimuli. Buccal biopsies and matched peripheral blood samples were collected from healthy donors (Supporting Information Table 1), and cells isolated after tissue processing were analyzed by flow cytometry. All subjects were examined for their oral health and showed no oral mucosal disease, active dental caries, infection, or periodontitis. Using antibody combinations allowing identification of MAIT cells in blood, buccal MAIT cells were defined as CD45+ CD3+ CD161hi Vα7.2+ cells (Fig. 1A). To ensure that cells defined by this gating strategy were bona fide MAIT cells, their MR1 restriction was confirmed by staining with the MR1 5‐(2‐oxopropylideneamino)‐6‐D‐ribitylaminouracil (5‐OP‐RU) tetramer (Fig. 1B). The percentage of MAIT cells in the mucosa ranged between 0.1 and 7% of total T cells (Fig. 1C). Overall, these levels were not significantly different from those in matched blood, although in any given individual, the levels seen at the two sites were often rather divergent. Interestingly, major conventional T‐cell subsets were present in the buccal mucosa at frequencies different from those in the blood. Levels of CD4+ T cells were lower than in blood, whereas CD8+ T cells and CD4–CD8– T cells were enriched in buccal mucosa as compared to blood (Fig. 1C). These data demonstrate that MAIT cells are part of the buccal mucosa T‐cell compartment. Detection of buccal mucosal MAIT cells in situ To determine MAIT cell location within the buccal mucosa, we performed in situ staining for Vα7.2 and IL‐18Rα in oral mucosal sections (Fig. 1D). In mucosa, staining for IL‐18Rα can be used in lieu of CD161, as we have previously shown for genital mucosa 22. Using the Vα7.2+ IL‐18Rα+ definition, MAIT cells were readily identified in buccal mucosal sections, and they tended to locate close to the basement membrane (Fig. 1D). MAIT cells were present in the epithelial layer, just above the basal membrane (distance from basal membrane: median = 14.6 μm; range = 1.0–75.9 μm), as well as in the underlying connective tissue, just beneath the basal membrane (median = 20.7 μm; range = 2.8–292.2 μm). Non‐MAIT T cells, as well as MR1‐expressing HLA‐DR+ antigen‐presenting cells, were also found in buccal mucosa as determined by immunofluorescence staining (Fig. 1E and F). MAIT cell location around the basement membrane is consistent with a possible role as innate‐like T cell sentinels to detect microbial epithelial barrier breach. Altered subset distribution and Jα‐TCR usage in mucosal MAIT cells In peripheral blood, MAIT cells are predominantly CD8+, with some CD4–CD8– cells and a very small CD4+ subpopulation. In the buccal mucosa, MAIT cells were enriched in CD4–CD8– and CD4+ subsets, and had significantly lower levels of the CD8+ subset (Fig. 2A). Within the CD8+ subset, similar to blood MAIT cells, the mucosal MAIT cells exhibited an increased frequency of CD8αα+ cells compared to non‐MAIT T cells (Fig. 2B and C). Notably, MAIT cells represented almost half of all CD8αα+ T cells in the buccal mucosa, with significant donor variability (Fig. 2D). The distribution of Jα‐TCR usage within the MAIT cell populations in the buccal mucosa and blood was compared using quantitative real‐time PCR (qRT‐PCR; Fig. 2E). We quantified the relative expression of three Jα previously shown to be represented within the MAIT cell TCR repertoire, the Jα12, Jα20, and Jα33 8, 29, 30. As expected, blood MAIT cells displayed a consistent pattern with dominant Jα33 usage, with smaller Jα20 and Jα12 usage. However, in matched mucosa, the pattern was more diverse, with different Jα segments dominating in different donors. Together, these data support a model of compartmentalization where the subset representation and TCR‐Jα usage in buccal MAIT cell population are partly different that of the circulating MAIT cells found in blood. MAIT cells in buccal mucosa express an activated perforinlow phenotype To assess the phenotypic characteristics of the buccal MAIT cells in more detail, we next stained isolated mucosal cells with markers of activation, CD38 and HLA‐DR (Fig. 3A). The staining pattern revealed the presence of an activated HLA‐DR+CD38+ MAIT cell population in buccal mucosa (Fig. 3B). Compared to their blood counterparts, the mucosal MAIT cells expressed higher levels of HLA‐DR, whereas CD38 levels appear to be slightly lower (Fig. 3C). In addition, expression of PD‐1 was common in mucosal MAIT cells, whereas PD‐1 was less expressed in peripheral blood MAIT cells (Fig. 3C). To investigate the phenotype of MAIT cells in more detail, paired blood and mucosal cell samples were stained intracellularly for the expression of the master transcription factor PLZF, as well as the cytolytic effector molecules perforin and granulysin (Fig. 3D). Perforin levels were mostly lower in the buccal MAIT cells than in their blood counterparts (Fig. 3E), whereas expression levels of PLZF and granulysin were similar in both compartments. These findings suggest that MAIT cells in the buccal mucosa are highly activated cells with significant expression of the checkpoint inhibition receptor PD‐1, and display a reduced perforin content as compared to circulating MAIT cells. Characterization of resident and nonresident MAIT cells in the buccal mucosa We next investigated the MAIT cell expression of receptors associated with tissue residency in the mucosa. Resident T cells can be distinguished by coexpression of CD69 and CD103 (αE integrin) in a range of tissues 31-35, including intestinal mucosa 36. Flow cytometric assessment of CD69 and CD103 expression revealed the existence of a major CD69+CD103+ tissue‐resident MAIT cell population in the buccal mucosa (Fig. 4A). However, a minority of MAIT cells were negative for CD69 and CD103, consistent with a tissue nonresident or "passing through" phenotype (Fig. 4A). Compared to peripheral blood MAIT cells, the buccal mucosal MAIT cell population was highly enriched in CD69 and CD103 expression, and CD69 expression was mostly seen in the CD103+ population (Fig. 4B). As the CD69 expression formed a continuum including dim cells of both CD103+ and CD103– character, we compared the four MAIT cell populations defined by CD69 and CD103 with regard to CD4+ and CD8+ expression. CD103+ MAIT cells were mainly CD8+ with a minority CD4–CD8– cells, whereas CD103– MAIT cells had a distinct opposite pattern with a majority CD4–CD8– character (Fig. 4C and D). CD69 expression did not significantly influence this pattern (Fig. 4C). In addition, CD103+ MAIT cells were more activated than their CD103– counterparts, as evidenced by higher expression of HLA‐DR and CD38 (Fig. 4E). In contrast, the CD103+ subpopulation expressed lower levels of the cytolytic effector molecule perforin, whereas PLZF did not vary depending on CD103 (Fig. 4F). These results indicate the existence of two distinct buccal MAIT cell subpopulations where one express the CD103 marker of tissue residency and one lacks CD103 consistent with a nonresident character. Distinct polyfunctional responses of mucosal resident, nonresident, and circulating MAIT cells To investigate the functional profile of MAIT cells in buccal mucosa in comparison with peripheral blood, isolated lymphocytes from the two tissues were activated with PMA/ionomycin and their repertoire of functions evaluated by flow cytometry staining for TNF, IFN‐γ, IL‐2, IL‐17, and granzyme B (Fig. 5A). Overall, both blood and mucosal MAIT cells displayed detectable production of all these effector functions, with some notable tissue‐dependent differences. Peripheral blood MAIT cells produced TNF and IFN‐γ more strongly than their mucosal counterparts, whereas IL‐2 and granzyme B were similar (Fig. 5A and B). In contrast, IL‐17 production was very low in blood MAIT cells, with the exception of one outlier, while this cytokine was clearly more highly expressed in the buccal MAIT cells (Fig. 5A and B). In the mucosal tissue, the resident CD103+ subset and the CD103– nonresident subset were largely similar in their response patterns with two notable exceptions. IL‐17 in particular, and to some extent granzyme B expression, were significantly higher in the CD103+ MAIT cell subset. To understand the functional specialization of mucosal resident, mucosal nonresident, and circulating MAIT cells in more detail, we analyzed the polyfunctional response profiles of these subsets. First, we determined the percentages of MAIT cells in the three locations that responded with at least one of the five functions measured (Fig. 5C). For all three sites, the vast majority of MAIT cells expressed at least one function. However, the mucosal MAIT cells displayed somewhat lower responsiveness, where in particular, the CD103– MAIT cells were less responsive. When the overall polyfunctionality was analyzed, the ability to express 1, 2, 3, 4, or 5 functions was fairly similar between the different tissue locations, although the CD103+ resident MAIT cells tended to be more 4‐ or 5‐functional (Fig. 5D). Finally, we analyzed the data by Boolean gating to assess which specific polyfunctional profiles were expressed by peripheral blood, CD103+ mucosal resident, and CD103– nonresident MAIT cells (Fig. 5E; Supporting Information Table 2). This analysis brought forward the pattern that peripheral blood MAIT cells were focused primarily on TNF production, either alone or in combinations with IFN‐γ and IL‐2. The mucosal CD103+ resident MAIT cells instead primarily focused on IL‐17 production either alone or in combinations with TNF or IL‐2. The CD103– nonresident MAIT cells had a functional profile intermediate between the circulating and resident populations, and were mostly expressing TNF with or without IL‐2, and less IL‐17. Altogether, these findings indicate that the mucosal resident, the mucosal nonresident, and the circulating MAIT cells deploy different effector cytokine response profiles. Discussion Tissue‐resident immune cell populations are increasingly recognized for their importance in defense against invading pathogens. The oral mucosa is continuously exposed to a range of antigens derived from food, microbiota, and pathogens. To maintain barrier immune defenses at this site, while not reacting against nonpathogenic encounters, is a considerable challenge. Recent studies have started to address this research question 37, 38, but no studies to date have addressed the potential role of the MR1‐restricted MAIT cell population in oral mucosa. Here, we observe that MAIT cells are dispersed throughout the buccal mucosa with a preferential location close to the basement membrane. The buccal MAIT cells are biased toward a CD4–/CD8– DN profile, and the CD8+ subset is primarily CD8αα and make up a significant proportion of all CD8αα T cells in the mucosa. The MAIT cell population in this mucosal site is composed of distinct CD69+CD103+ intraepithelial resident and CD69–CD103– nonresident subsets. The CD103+ subset is enriched in CD8+ MAIT cells, is more activated, and has lower cytolytic potential. Finally, the buccal MAIT cells show a distinct functional profile with enhanced IL‐17 production and less TNF and IFN‐γ, as compared to their peripheral blood counterparts, and this pattern was particularly pronounced for the CD103+ resident MAIT cells. The MR1‐presented riboflavin metabolite antigens recognized by MAIT cells are conserved among many prokaryotic and eukaryotic microbes relevant for mucosal health. Thus, MAIT cells may play a unique role in the oral mucosa with broad innate‐like recognition of such microbes in a manner that does not require the kind of priming which conventional adaptive T‐cell responses do. It is interesting that the MAIT cells to at least some extent locate around the basement membrane, where they may function as innate‐like T‐cell sentinels to detect microbial epithelial barrier breach. Around the similar location, MR1+HLA–DR+ cells are residing and those may be able to present riboflavin metabolite antigens from invading microbes. Together, MAIT cells and MR1+ antigen presenting cells may form a rapid innate response immune unit at the mucosal barrier, capable of responding to pathogens to which the host has no preexisting adaptive immunity. The CD103+CD69+ mucosal‐resident MAIT cell population is fairly strongly biased toward IL‐17 production, with less TNF and IFN‐γ expression, as compared to the CD103– MAIT cell subpopulation. Resident MAIT cell recognition of an invading microbial pathogen may thus setoff an IL‐17‐mediated inflammatory response with induction of antimicrobial peptides in epithelium and recruitment of neutrophils. Such a pattern would be reminiscent to the situation described for chronic severe periodontitis by Dutzan et al. 26, where IL‐17 produced by presumably adaptive Th17 cells was associated with increased gingival tissue infiltration of neutrophils. Given their innate‐like response characteristics, MAIT cells may be among the first immune cells to detect microbial infiltration and respond with IL‐17 release. Here, it is interesting to note that IL‐17 has emerged as an important mediator and amplifier of immunity against Candida albicans 38. The dimorphic fungus C. albicans expresses the riboflavin biosynthesis pathway and is recognized by MAIT cells in an MR1‐restricted manner 10. Many cells respond to IL‐17 by upregulation of proinflammatory cytokines, such as IL‐6, and chemokines for recruitment of neutrophils including CXCL1, CXCL2, and CXCL5 39. In addition, IL‐17 stimulates production of β‐defensins in epithelial cells 40. MAIT cells are thus well located to initiate mucosal immune responses in oropharyngeal candidiasis. Peripheral blood MAIT cells are predominantly CD8+ with a minority CD4–CD8– subset. This pattern is reversed in buccal mucosa, such that the CD4–CD8– MAIT cells are more numerous. It is however interesting to note that the CD8+ MAIT subset in buccal mucosa is primarily CD8αα, and that this subset makeup almost half of all CD8αα T cells in the oral mucosa. These CD8αα MAIT cells bear resemblance to the intestinal mucosal CD8αα intraepithelial lymphocytes (IELs) that have been extensively characterized in murine models, but may be less frequent in humans 41, 42. The intestinal IELs of mice are a mix of TCRγδ T cells and TCRαβ T cells with diverse specificities, and the representation of MAIT cells among these IELs in different sites is to our knowledge largely unknown. Our data indicate that the CD103+ MAIT cell population is mostly, but not exclusively, CD8+ and composed of both CD8αα and CD8αβ cells. These findings together suggest that MAIT cells makeup a significant part of the human buccal IEL‐like population. The human oral mucosal barrier retains a commensal bacterial microbiota that is both varied and unique among other sites 43, 44, dominated by the genera Streptococcus, Haemophilus, Prevotella, and Veillonella. In addition to bacteria, the oral mucosal barrier is home to many species of fungi including C. albicans 45. The oral immune system thus has to manage and tolerate a diverse commensal microbiome, and at the same time guard against conditions arising either from dysfunction of normal oral homeostasis or caused by pathogens normally not present in the oral cavity. T cells are believed to play a role in multiple oral mucosa pathologies including aphthous stomatitis, oral leukoplakia, oral reticular or ulcerative lichen planus, celiac disease, and oral psoriasis 46, 47. Whether MAIT cells have a role in any such conditions in humans remains to be explored. In summary, we have shown that MAIT cell populations with resident and nonresident characteristics are part of the buccal mucosal immune system in healthy donors and that they have unique functional profiles. Future studies should aim to investigate how these populations respond to commensal and pathogenic microbes, and how they are affected in different disease conditions. Materials and methods Tissue donor recruitment and sample collection A total of 94 volunteers were recruited in two healthy donor groups A and B (Supporting Information Table 1). Inclusion criteria for both groups were: 20–50 years of age, HIV‐negative, non‐smoking, no antibiotics in the last 3 months, and not pregnant. Ethical permission was obtained from the Regional Ethical Review Board in Stockholm in accordance with the Declaration of Helsinki. All participants gave written informed consent. The oral health of all subjects was evaluated using standard dental examination procedures, including inspection of oral mucosa, teeth, and surrounding soft tissues, to ensure the donors had no visible mucosal lesions, no signs of gingivitis, active dental caries, or periodontitis. All donors were instructed to abstain from food or drink for 1 h before tissue collection. Oral mucosal tissue samples and matched peripheral blood samples were collected from two sets of healthy donors. Donor group A was recruited from the general population. A matched blood sample of 20–30 mL venous blood was taken from each participant on the day of the procedure, prior to the oral sample collection. The blood was stored in heparin tubes at room temperature until analysis. Local anesthesia with xylocain/adrenalin was applied to the left bucca and three punch biopsies (5 mm in diameter, 3–4 mm deep) were taken from the site. For donor group B, tissue samples were taken before wound closure on patients who underwent orthognatic surgery of the mandible. The patients underwent these corrective surgeries due to abnormal position of the mandible in relation to the base of skull, but no buccal disease or inflammation is associated with these conditions. The patients were treated under general anesthesia and local anesthesia was additionally applied. Tissue samples 20 × 10 mm in size were taken bilaterally from the mucosa. Matched blood samples from donor group B were collected at the time of the procedure. After collection, the biopsies were stored at 4°C in serum‐free RPMI (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 50 μg/mL gentamicin (ThermoFisher Scientific) and 100 μg/mL normocin (InvivoGen, San Diego, CA, USA). Biopsies intended for qPCR analysis were stored in RNAlater (QIAgen, Venlo, Netherlands) at −20°C. Biopsies intended for in situ microscopic analysis were submerged in OCT Cryomount (HistoLab, Askim, Sweden), snap‐frozen in liquid nitrogen, and then stored at −80°C. Sample processing for flow cytometry All collected samples were processed within 3 h of collection. Buccal biopsies were placed into serum‐free RPMI supplemented with 50 μg/mL gentamicin and 100 μg/mL normocin and then incubated with 1 mg/mL DNase I and collagenase A (both from Roche, Penzberg, Germany) at 37°C with shaking at 800 rpm for 1 h. After incubation, the reaction was stopped by addition of fetal calf serum (FCS; Sigma‐Aldrich, St Louis, MO, USA) to a final concentration of 10%. The biopsies were then pressed through a 100 μm cell strainer in order to detach loose cells. The cells were then washed in PBS and incubated in RBC lysis buffer for 10 min at RT to remove any erythrocyte contamination. The cells were washed again in PBS and stained for flow cytometry. Donor‐matched PBMCs were isolated from blood samples by Ficoll‐Hypaque density gradient centrifugation using Lymphoprep (Axis‐Shield, Dundee, Scotland). The PBMC layer was washed twice with PBS, and incubated in RBC lysis buffer for 10 minutes at room temperature to remove erythrocyte contamination. The cells were washed again in PBS and 106 cells were stained for flow cytometry. In situ staining In situ staining was performed on 8‐μm‐thick sections of frozen biopsies, as previously described 22. Sections were fixed in 2% formaldehyde and stained sequentially. Antibody reagents used for tissue staining are listed in Supporting Information Table 3. The tissue sections stained for Vα7.2 in combination with IL‐18Rα were scanned into digital images using a Pannoramic 250 Flash Slide Scanner (3DHistech, Budapest, Hungary). The distance measurement was performed using the digital microscope application CaseViewer (3DHistech), where the distance from the surface of the double‐positive cells to the basal membrane was measured. The tissue sections stained for HLA‐DR in combination with MR1, and Vα7.2 in combination with CD3 were visualized using DMR‐X microscope (Leica, Weitzlar, Germany) and images were acquired with a Retiga 2000 R camera (Qimaging, Surrey, Canada). Functional assay One million lymphocytes in suspension were cultured overnight in RPMI +10% FCS, supplemented with 100 μg/mL normocin and 50 μg/mL gentamicin. After overnight incubation, they were activated using Leukocyte Activation Cocktail (PMA/ionomycin) (BD Biosciences, San Jose, CA, USA) in the presence of GolgiPlug (monensin) for 6 h. At the end of the incubation, the cells were stained and analyzed by flow cytometry. Flow cytometry and antibodies Cells in suspension were stained for surface antigens in FACS buffer (PBS with 2 mM EDTA and 2% FCS) for 20 min at 4°C. Afterwards, they were washed in FACS buffer and permeabilized using the transcription factor fixation and permeabilization buffer (BD Biosciences) for 30 min at 4°C. Following permeabilization, cells were washed twice in transcription factor wash and permeabilization buffer (BD Biosciences), and then stained for intracellular antigens in the same buffer for 20 min at 4°C. PE‐conjugated human MR1:5‐OP‐RU tetramer was obtained from the NIH Tetramer Core Facility. For tetramer staining, cells in suspension were incubated with MR1:5‐OP‐RU tetramer for 40 min at room temperature and then washed with FACS buffer prior to surface antibody staining. Monoclonal antibody reagents used in the study are listed in Supporting Information Table 4. After staining, cells were washed and analyzed using an LSRFortessa flow cytometer (BD Biosciences) equipped with 355, 405, 488, 561, and 639 nm lasers. In addition to the antibodies, cells were stained with LIVE/DEAD Fixable Dead Cell Stain (Near‐Infrared or Aqua; ThermoFisher Scientific). Quantitative RT‐PCR qRT‐PCR was performed to determine the mRNA expression of TCRs Vα7.2‐Jα33, Vα7.2‐Jα20, and Vα7.2‐Jα12 as well as the constant Cα chain, in healthy oral mucosa and blood. Briefly, total RNA was extracted from buccal mucosa and PBMCs using TRIzol reagent (ThermoFisher Scientific). For reverse transcription, 250 ng of RNA was used in a 15 μL total reaction volume using the IScript cDNA Synthesis kit (BioRad, Hercules, CA, USA). For amplification, a 20 μL supermix reaction was prepared accordingly: 1 μL cDNA, 5 μL SsoAdvanced™ Universal® SYBR Green (BioRad), 1 μL of each primer at a final concentration of 500 nM, and nuclease free H2O. Primer sequences as well as annealing temperatures for each of the primer sets are listed in Supporting Information Table 5. qPCR was performed using 7500 Fast real time PCR system (Applied Biosystems, Waltham, CA, USA) with the following cycling conditions: 1 cycle at 95°C for 5 min, then 40 cycles at 94°C for 10 s, 58°C or 60°C depending on primer for 30 s, and 72°C for 27 s. The relative abundance of each Vα7.2‐Jα was determined as relative to Cα by the comparative ΔΔCT method. Software and statistical analysis Analysis of flow cytometric data was performed using FlowJo 10 (FlowJo LLC, Ashland, OR, USA), and microscopic images were processed using Image‐Pro Premier 9.3 (Media Cybernetics, Rockville, MD, USA). Statistical tests were performed using GraphPad Prism version 7.0c for Mac OS X (GraphPad Software, La Jolla, CA, USA). Statistical significance was assessed using Wilcoxon matched pairs test for paired data, and Mann–Whitney U‐test for unpaired data. For data presented in Fig. 5, analysis and presentation of distributions were performed using SPICE version 5.1, downloaded from http://exon.niaid.nih.gov 48. Comparison of distributions was performed using a Wilcoxon matched pairs test as described 48. For all statistical analysis, p‐values below 0.05 were regarded as significant. Acknowledgements The authors wish to acknowledge the contribution of Sam Chehrehgosha, Angelica Kroonder, Emelie Molin, and Beatrice Wiberg in recruiting study volunteers and gathering oral biopsies. This research was supported by grants to JKS from the Swedish Research Council (2016‐03052), the Swedish Cancer Society (CAN 2017/777), and the US National Institutes of Health (R01DK108350). Grants to M.S.C. were from the Foundation for Odontological Research (OF11211233), and the Swedish Cancer Society (CAN 2016/731). Further support to E.L. was from the Swedish Research Council (2015‐00174) and Marie Skłodowska Curie Actions, Cofund, Project INCA 600398. J.D. was supported by Fundação para a Ciência e a Tecnologia (SFRH/BD/85290/2012, doctoral fellowship), through program QREN‐POPH‐typology 4.1. The MR1 tetramer technology was developed jointly by Dr. James McCluskey, Dr. Jamie Rossjohn, and Dr. David Fairlie, and the material was produced by the NIH Tetramer Core Facility as permitted to be distributed by the University of Melbourne. The authors wish to thank the healthy volunteers that contributed time and effort to sample donation for this study. Author contributions J.K.S., E.L., and M.S.C. conceived the original research idea and study design. M.J.S., H.D., M.M., A.T., J.D., E.L., M.S.C., and J.K.S. designed experiments. M.J.S., H.D, A.G., and J.E. conducted experiments. M.J.S., H.D., and A.G. analyzed experimental data. R.H., S.A., and C.K.W. recruited study volunteers and collected essential human samples. M.J.S. and J.K.S. wrote the manuscript. All authors reviewed and commented on the manuscript. Conflict of interest The authors declare no commercial or financial conflict of interest. Supporting Information References Abbreviations IEL intraepithelial lymphocyte MAIT mucosal‐associated invariant T MR1 MHC‐Ib‐related protein 1 PLZF promyelocytic leukemia zinc finger protein
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Differential distribution of IgA-protease genotypes in mucosal and invasive isolates of Haemophilus influenzae in Sweden | BMC Infectious Diseases | Full Text

Differential distribution of IgA-protease genotypes in mucosal and invasive isolates of Haemophilus influenzae in Sweden | BMC Infectious Diseases | Full Text | Mucosal Immunity | Scoop.it
Several different IgA-proteases exist in Haemophilus influenzae. The variants have been suggested to play differential roles in pathogenesis, but there is limited information on their distribution in clinical isolates.
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Emerging therapies in immunoglobulin A nephropathy - Yeo - 2015 - Nephrology - Wiley Online Library

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
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