AUTOIMMUNITY

Abstract Between 50% and 86% of patients with autoimmune hepatitis (AIH) relapse after immunosuppression withdrawal; long‐term immunosuppression is associated with increased risk of neoplasias and infections. Chloroquine diphosphate (CQ) is an immunomodulatory drug that reduces the risk of flares in rheumatologic diseases. Our aims were to investigate the efficacy and safety of CQ for maintenance of biochemical remission of AIH in a double‐blind randomized trial and to define a subgroup that obtained a greater benefit from its use. A total of 61 patients with AIH in histologic remission (90.1% AIH type 1 [AIH‐1]) were randomized to receive CQ 250 mg/day or placebo for 36 months. Of the 61 patients, 31 received CQ and 30 placebo. At baseline, clinical, laboratory, histologic findings, and human leukocyte antigen (HLA) profile were similar between the two groups. Relapse‐free survival was significantly higher in the CQ group compared to the placebo group (59.3% and 19.9%, respectively P = 0.039). For those patients completing 3‐year treatment, relapse rates were 41.6% and 0% after CQ and placebo withdrawal, respectively. Factors associated with a higher risk of relapse in multiple Cox regression were placebo use (hazard ratio, 2.4; 95% confidence interval [CI], 1.055.5; P = 0.039) and anti‐soluble liver antigen/liver‐pancreas (anti‐SLA/LP) seropositivity (hazard ratio, 5.4; 95% CI, 1.91‐15.3; P = 0.002). Although it was not possible to define a subgroup that obtained a greater benefit from CQ according to anti‐SLA/LP reactivity or HLA profile, 100% of patients who were anti‐SLA/LP‐positive (+) relapsed with placebo compared to 50% with CQ (P = 0.055). In the CQ group, 54.8% had side effects and 19.3% interrupted the drug regimen. Conclusion: CQ safely reduced the risk of relapse of AIH, but it was not possible to define a subgroup that obtained a greater benefit with CQ use, probably because of sample size. Abbreviations – negative + positive AIH autoimmune hepatitis AIH‐1/2 AIH type 1/2 ALT alanine aminotransferase anti‐LKM1 anti‐liver/kidney microsomal antibody type 1 anti‐SLA/LP anti‐soluble liver antigen/liver‐pancreas CI confidence interval CQ chloroquine diphosphate HLA human leukocyte antigen HLA‐DQ human leukocyte antigen isotype DQ HLA‐DR human leukocyte antigen isotype DR IAIHG International Autoimmune Hepatitis Group IgG immunoglobulin G The goal of treatment in autoimmune hepatitis (AIH) is to achieve normalization of aminotransferase and immunoglobulin G (IgG) levels with absent or minimal histologic activity to prevent disease progression. A sustained remission of treatment after its withdrawal is the most desirable endpoint. However, most patients relapse within the first year and need reintroduction of treatment, and only approximately 20% achieve this goal.1-3 Patients who present multiple relapses evolve more frequently for liver cirrhosis, death due to liver disease, and the need for liver transplantation than those who remain in sustained remission.4 A significant advance in the treatment of AIH would be the identification of drugs with a good safety profile and few side effects that would allow the maintenance of AIH remission after withdrawal of the immunosuppressive treatment. Antimalarial drugs have been prescribed for almost 400 years and are used in the treatment of systemic lupus erythematosus with benefits in symptomatic control, general and disease‐free survival, and reduction of severity of disease exacerbations.5, 6 Chloroquine diphosphate (CQ) has multiple mechanisms of action in immune response; this could justify its use in the treatment of AIH. CQ 250 mg/day was used for maintenance of disease remission after immunosuppression withdrawal in a preliminary pilot study with 14 patients in histologic remission; in that study, the CQ‐treated group had a 6.49‐fold lower risk of disease relapse.7 In 2 patients who were anti‐soluble liver antigen/liver‐pancreas (anti‐SLA/LP)(+) and who relapsed after CQ withdrawal, its reintroduction as monotherapy (on request) resulted in normalization of aminotransferases and, in one case, histologic remission. The aims of this study were to evaluate whether CQ is safe and effective in preventing relapse of AIH in patients with histologic remission of the disease after immunosuppression withdrawal and whether there is a specific patient profile in which it could be more beneficial. Participants and Methods Trial Design and Participants This was an interventional, single‐center, randomized, double‐blind, controlled, parallel‐group trial of 36 months of CQ versus placebo in patients with AIH in histologic remission. The authors designed the protocol, monitored patients, acquired and maintained the data, and performed statistical analysis. The protocol was approved by the local ethics committees and registered in the ClinicalTrials.gov database (NCT01980745). All subjects signed a written informed consent before enrollment, and the study was performed according to good clinical practice and the Declaration of Helsinki. Patients 18 to 70 years of age with a probable/definite diagnosis of AIH according to International Autoimmune Hepatitis Group (IAIHG) criteria8 who had biochemical and histologic remission after immunosuppressive therapy were eligible for enrollment in this study. Exclusion criteria were overlapping syndromes with primary biliary cholangitis and primary sclerosing cholangitis, decompensated liver cirrhosis (presence of ascites, hepatic encephalopathy, spontaneous bacterial peritonitis, acute variceal bleeding, or Child‐Turcotte‐Pugh B and C classification), enrollment in a previous study with antimalarial drugs, and pregnancy. Interventions All patients received initial treatment with azathioprine and prednisone, most of them in doses of 50 mg/day and 30 mg/day, respectively. There were monthly reductions in prednisone dose and increases in dose of azathioprine if necessary (up to 2 mg/kg/day if tolerated) in order to maintain the levels of aminotransferases within the normal reference range with the lowest dose possible. Doses of prednisone ranged from 5 to 15 mg/day and azathioprine from 50 to 150 mg/day during maintenance treatment. After normalization of hepatic enzymes with stable doses of the drugs, there were no additional adjustments in the doses (except in the cases of adverse effects that justified some modification) until the liver biopsy was performed to evaluate histologic remission, which never occurred before 18 months of normal aminotransferases and 2 years of treatment. Histologic remission was defined as absence or presence of minimal interface hepatitis (hepatitis activity index <4) according to the IAIHG definition.2 The histologic evaluation was performed by experienced liver pathologists according to the Brazilian Society of Pathology.9 Following confirmation of histologic remission, patients were randomized to receive either a fixed dose of CQ (250 mg/day, drug A) or placebo (drug B). CQ was chosen because it was the only formulation available for dispensing by the public health system. In the first month after enrollment, patients received CQ or placebo in addition to the immunosuppressive regimen that induced histologic remission; this was azathioprine and prednisone in all cases. During the second month, azathioprine was discontinued immediately, with weekly weaning of 2.5 mg of the prednisone maintenance dose. Subsequently, the patients remained on drug A or drug B for up to 36 months, disease relapse, significant side effects, pregnancy, or willing withdrawal from the study. Follow‐up was for at least 1 year after the end of the protocol. Primary Outcomes The primary outcome was the maintenance of biochemical response following immunosuppression withdrawal, which was defined as aminotransferase levels within the normal reference range. Relapse of AIH was defined as elevation of aminotransferase levels above twice the normal upper reference value in at least two different measurements taken within a 15‐day interval during which there was no tendency for liver enzymes to fall or stabilize. Routine blood tests and clinical evaluation were performed monthly during the first 6 months of the study, every 2 months or earlier if necessary until the end of the first year, and every 3 months until the end of the study. Secondary Outcomes Secondary outcomes were to determine whether there was a specific group of patients who would benefit most from the ingestion of CQ and to assess the safety profile of the drug. Autoantibody Testing Anti‐nuclear, anti‐smooth muscle, anti‐liver/kidney microsomal antibody type 1 (anti‐LKM1), anti‐liver cytosol type 1, and anti‐mitochondrial antibodies were detected by indirect immunofluorescence; performed on frozen unfixed rodent substrate that included kidney, liver, and stomach; and were considered positive if the titers were greater than or equal to 1:40 according to IAIHG diagnosis criteria.10 The fluorescent patterns of anti‐nuclear and anti‐microfilament antibodies were detected by indirect immunofluorescence on human epithelial type 2 (HEp2) cells and human fibroblasts. Anti‐SLA/LP antibodies were detected by enzyme‐linked immunosorbent assay using the commercial kits Quanta Lite SLA (Inova Diagnostics Inc., San Diego, CA) and/or anti‐SLA/LP IgG (EUROIMMUN, Luebeck, Germany), according to the manufacturer’s instructions, which consider positive values as ≥25 units and ≥1, respectively. Human Leukocyte Antigen Typing Human leukocyte antigen (HLA) typing was performed with DNA samples previously extracted using the dodecyltrimethylammonium bromide (DTAB)/cetyltrimethylammonium bromide (CTAB) technique and stored at −20°C. For the amplification reaction of the HLA isotype DR (HLA‐DR) and HLA isotype DQ (HLA‐DQ) genes, specific primers were used to test the polymorphism in exon 2. The presence of the allele or allele group was based on the presence or absence of the amplified product. The determination of alleles or groups of class II alleles was performed by polymerase chain reaction (primer‐specific sequence) with Micro SSP HLA DNA typing trays (One Lambda). The reaction was performed following the manufacturer’s instructions. Treatment‐Related Side Effects The relationship between the drug and side effects was defined according to the Naranjo algorithm, which determines the probability that an adverse reaction to the drug is actually due to the drug and not the result of other factors.11 The severity of side effects was classified according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 4.03.12 All patients underwent ophthalmologic evaluation before inclusion in the study and at least one annual evaluation according to protocol thereafter for the detection of retinal antimalarial deposition signals. Sample Size Determination Our main hypothesis to be tested was whether there is a difference in AIH relapse rate between patients who use CQ and patients who use placebo. The rates of AIH relapse were estimated in a pilot study, which resulted in relapse of 23.5% in the CQ group and 72.2% in a historical group without medication.7 Assuming an accrual rate of 7 patients per year, a sample size of 31 patients was defined to allow the study to estimate a minimum detectable difference of 37.2% using a 2‐sided Fisher’s exact test with 80% power at a 5% level of significance. Randomization Eligible patients were randomly assigned to receive CQ or placebo after withdrawal of immunosuppression by simple randomization and were allocated to each group in a 1:1 ratio. A medical assistant unaware of the study generated the random allocation sequence by a draw with 62 numbered papers: 31 named drug A and 31 named drug B. One of the trial investigators enrolled and assigned participants to the proposed interventions after randomization. After patients agreed to participate in the study, the principal investigator checked each patient’s list number and determined which drug was destined for that number. The drugs were formulated at the hospital pharmacy as tablets with an identical external appearance. The research assistant and the patients were all blind to the type of medication used. After the study was completed, the pharmacist revealed the composition of the tablets initially named drugs A and B to the researchers. The secrecy of the formulation was maintained until the end of the study. Statistical Analysis For statistical analysis, all patients undergoing randomization (except those who refused medication immediately after enrollment) were included regardless of the time of follow‐up or withdrawal due to side effects or pregnancy. Relapse‐free survival was defined as the time between a patient’s enrollment and the end of treatment (36 months) or the occurrence of side effects leading to discontinuation of the drug and the subject being censored from the study. Time to relapse was defined as the period between the immunosuppression withdrawal and the biochemical relapse of the disease. Relapse‐free survival curves were estimated using the Kaplan‐Meier method and compared by the log‐rank test. The hazard ratios with their respective 95% confidence intervals (CIs) were estimated using the simple Cox regression. A multivariate regression model was fitted using clinically relevant covariates for AIH relapse defined in previous studies. The interactions between the drug and autoantibody reactivity and HLA profile were evaluated by multivariate Cox regression to investigate whether there would be any subgroup of patients with additional benefits with the use of CQ. The proportional hazard assumption was verified using the Schoenfeld residues.13 Baseline characteristics were compared with Fisher’s exact test for categorical variables and t test or Mann‐Whitney test for quantitative variables. The normality and homoscedasticity hypotheses were evaluated by the Anderson‐Darling14 and Levene15 tests, respectively. All hypothesis tests were 2‐sided and considered P < 0.05 as statistically significant. R, version 3.0.2 (R Core Team, 2013), was used for all calculations. Results Patients were enrolled from May 2002 to October 2011, and the follow‐up ended in 2015. All patients except 1 had a definite diagnosis of AIH before treatment according to IAIHG criteria. One patient who could not undergo a liver biopsy due to the presence of ascites had “probable diagnosis” with a score of 13 points. Clinical, laboratory, and histologic data at diagnosis are shown in Table 1. A total of 62 patients were randomized to receive CQ 250 mg/day (n = 31) or placebo (n = 31) for 36 months. Except for 1 patient in the placebo group who refused treatment immediately after randomization, all patients received their assigned treatment (Fig. 1). The mean follow‐up time was 3,526 ± 1,233 days. The median time of drug intake was 329 days (22‐1,275 days).The median age at diagnosis of AIH was 26.86 years (2.7‐63.4 years), and mean treatment duration time before immunosuppression withdrawal was 5.9 ± 4.08 years (median time, 4.56 years). Approximately 90% had AIH‐1; 23.3% of all patients had anti‐SLA/LP seropositivity. Out of 61 patients, 56 had an initial liver biopsy (91.8%), and hepatic cirrhosis was detected in 52.4%. At baseline, demographic, clinical, laboratory, and histologic data were similar between CQ and placebo groups (Table 2). When the HLA profile was analyzed, HLA‐DR3 (26/61 patients) and HLA‐DR13 (26/61 patients) were the most frequent, followed by HLA‐DR4 (17/61 patients) and HLA‐DR7 (13/61 patients). In the CQ group, there was a lower frequency of HLA‐DR3 (35.5% versus 50%), HLA‐DR4 (19.3% versus 36.7%), and HLA‐DR7 (16.1% versus 26.7%) compared to the placebo group and a higher frequency of DR13 (51.6% versus 33.4%). However, these differences did not reach statistical significance (Table 3). There was no specific HLA‐DR profile significantly related to anti‐SLA/LP seropositivity, although 50% harbored DR3. Data at Diagnosis n (%) Presentation of disease at onset Asymptomatic* 6 (9.8) Acute exacerbation of chronic AIH † 44 (72.2) Insidious onset ‡ 11 (18) Extrahepatic autoimmune diseases 8 (13.1) § AST ‖ (mean ± SD) 26.04 ± 20.1 ALT ‖ (mean ± SD) 22.83 ± 21.2 Alkaline phosphatase ‖ (mean ± SD) 1.73 ± 1.05 Gamma‐glutamyltransferase ‖ (mean ± SD) 4.91 ± 4.1 Albumin g/dL (mean ± SD) 3.37 ± 0.68 Total bilirubin mg/dL (mean ± SD) 9.13 ± 9.08 INR (mean ± SD) 1.5 ± 0.41 Gamma globulin ‖ (mean ± SD) 1.97 ± 1.06 Histologic findings ¶ n (%) Rosettes of hepatocytes 33 (58.9) Plasma cells inflammatory infiltrate 23 (41) Interface hepatitis 52 (92.8) Submassive necrosis 4 (7.14) * Diagnosis due to elevation of liver enzymes or ultrasonographic alterations during follow‐up of extrahepatic diseases (rheumatologic and endocrinologic diseases). † Presentation similar to viral hepatitis, with jaundice, coluria, and fecal acolia with or without signs of portal hypertension. ‡ Nonspecific symptoms of fatigue, general ill health, right upper quadrant pain, lethargy, malaise, anorexia, weight loss, nauseas, pruritus, polyarthargia. § Thyroid disease (n = 2), ulcerative colitis (n = 2), systemic erythematosus lupus (n = 2), Sjögren syndrome (n = 1), idiopathic thrombocytopenic purpura (n = 1). ‖ Values are shown as number of times above the upper normal limits. ¶ Percentage calculated considering 56 patients. Of these patients, 5 patients did not have liver biopsy at diagnosis due to presence of ascites and/or coagulopathy. Liver fibrosis staging according to Brazilian Society of Pathology Classification of chronic hepatitis.9 Abbreviations: AST, aspartate aminotransferase; INR, international normalized ratio. CQ (n = 31) Placebo (n = 30) P Value Female sex (%) 26 (83.9) 26 (86.7) 1 Age at diagnosis of AIH (mean ± SD) 29.4 ± 17.9 30.5 ± 17.1 0.98 Age at study inclusion (mean ± SD) 37.7 ± 16.1 39.1 ± 16.9 0.8 AIH classification (%) 0.27 Type 1 29 (93.5) 26 (86.7) Type 2 1 (3.2) 0 (0) Type 3 (isolated anti‐SLAP/LP) 1 (3.2) 1 (3.3) No serologic markers 0 (0) 3 (10) Anti‐SLA/LP seropositivity (%)* 6 (20) 8 (26.7) 0.76 Antimicrofilament seropositivity (%)† 11 (37.9) 16 (55.2) 0.29 Liver cirrhosis at diagnosis (%)‡ 14 (50) 18 (64.3) 0.42 Advanced fibrosis (F3/F4) at study inclusion (%)§ 18 (58) 16 (53.3) 0.8 Arterial hypertension (%) 3 (9.7) 5 (16.7) 0.47 Diabetes mellitus (%) 4 (12.9) 5 (16.7) 0.73 Normal gamma globulin/IgG levels|| at inclusion (%) 20 (64.5) 23 (76.7) 0.4 Immunosuppression at histologic remission (mg/day) (mean ± SD) Azathioprine 90.7 ± 26.6 97.5 ± 22.1 0.47 Prednisone 8.5 ± 2.8 8.9 ± 2.1 0.8 Treatment time before inclusion at study (years, mean ± SD) 6 ± 4.55 5.8 ± 3.53 0.44 *In 1 patient of the CQ group, it was not possible to assess anti‐SLA/LP seropositivity in initial screening. Regarding differences between patients who were anti‐SLA(+) or (–), patients who were anti‐SLA(+) had lower levels of alkaline phosphatase, gamma‐glutamyltranspeptidase, and bilirubin. There were no differences in histologic findings, IgG levels, albumin, INR, and aminotransferases (data not shown). †Information available in 29 patients in each group. ‡Two patients in each group did not have liver biopsy at diagnosis due to presence of ascites and/or coagulopathy. Liver fibrosis staging according to Brazilian Society of Pathology Classification of chronic hepatitis.9 §Liver biopsy was insufficient to stage liver fibrosis in 1 patient without impairing the degree of inflammatory activity. Classification of liver fibrosis according to METAVIR fibrosis score. ||Gamma globulin and/or IgG levels within the normal range (gamma globulin <1.5 g/dL and IgG <1,538 mg/dL). Abbreviation: INR, international normalized ratio. HLA Profile Chloroquine (n = 31) n (%) Placebo (n = 30) n (%) DR1 4 (12.9) 2 (6.7) DR3 11 (35.5) 15 (50)* DR4 6 (19.3) 11 (36.7)† DR7 5 (16.1) 8 (26.7)‡ DR8 0 (0) 1 (3.3) DR9 2 (6.4) 0 (0) DR10 1 (3.2) 0 (0) DR11 4 (12.9) 2 (6.7) DR13 16 (51.6) 10 (33.4)§ DR14 4 (12.9) 1 (3.3) DR15 3 (9.7) 4 (13.3) DR16 3 (9.7) 2 (6.7) DQ02 14 (45.1) 14 (46.7) DQ04 2 (6.4) 5 (16.7) DQ05 10 (32.2) 5 (16.7) DQ06 16 (51.6) 12 (40)|| DQ07 4 (12.9) 3 (10) DQ08 6 (19.3) 5 (16.7) There were no significant statistical differences in HLA‐DR and HLA‐DQ profiles between the CQ and placebo groups (P > 0.05). *P = 0.3 †P = 0.16 ‡P = 0.36 §P = 0.2 ||P = 0.44 Primary Outcomes Patients in the CQ group completed the study more frequently (38.7% versus 13.3%) and took the medication for a longer period compared to those in the placebo group (616 ± 484.8 versus 386 ± 377.4 days, respectively). The mean alanine aminotransferase (ALT) level at relapse was 271.1 ± 301.5 U/L (reference value <31 U/L). Patients who relapsed restarted immunosuppressive therapy without clinical or laboratory signs of liver dysfunction. Factors associated with higher risk of AIH relapse by univariate Cox regression were anti‐SLA/LP seropositivity, placebo randomization, and HLA‐DR3 profile (Fig. 2; Table 4). In the multivariate analysis, factors associated with AIH relapse were anti‐SLA/LP seropositivity and placebo randomization. Relapse‐free survival was significantly lower in the placebo group compared to the CQ group (CQ group, 59.3% [43.1%‐81.6%] versus placebo group, 19.9% [7.9%‐50.2%]; hazard ratio, 2.4; 95% CI, 1.05‐5.5; P = 0.039) and in patients with anti‐SLA/LP seropositivity (hazard ratio, 5.4; 95% CI, 1.91‐15.3; P = 0.002) (Table 5). After CQ/placebo withdrawal at the end of the study at 3 years, 5 of 12 patients (41.6%) in the CQ group relapsed compared to none of 4 patients in the placebo group. Relapse‐free survival 6 months after the study ended was 60% in the CQ group and 100% in the placebo group (P = 0.03) because no patient in the placebo group who maintained biochemical remission during the 3‐year follow‐up relapsed after the study’s end. Variable Hazard Ratio (95% CI) P Value Droga B (placebo) 2.51 (1.19‐5.25) 0.015 Anti‐SLA/LP seropositivity 2.84 (1.34‐5.98) 0.006 HLA‐DR3 2.04 (1‐4.16) 0.049 HLA‐DQ6 0.53 (0.25‐1.1) 0.089 HLA‐DR13 0.63 (0.3‐1.31) 0.21 HLA‐DR7 0.77 (0.32‐1.89) 0.57 HLA‐DR4 2.02 (0.96‐4.27) 0.066 Antimicrofilament seropositivity 1.24 (0.61‐2.54) 0.55 Classification of AIH* AIH‐2 0 (0‐infinity) 0.99 AIH‐3 0.26 (0.04‐1.93) 0.19 Liver cirrhosis at diagnosis 0.71 (0.34‐1.49) 0.36 Advanced fibrosis at study randomization (F3/F4) 1.09 (0.54‐2.24) 0.8 Normal gamma globulin level at randomization 1.14 (0.51‐2.56) 0.74 Diabetes mellitus 0.38 (0.09‐1.58) 0.18 Age at diagnosis 1 (0.98‐1.02) 0.71 *Considering type 1 AIH as the reference group; (AIH‐3 ‐ anti‐SLA/LP as sole marker). Variable Hazard Ratio (95% CI) P Value Placebo intake 2.4 (1.05‐5.5) 0.039 Anti‐SLA/LP seropositivity 5.4 (1.91‐15.3) 0.002 Age at diagnosis of AIH 1 (0.98‐1.03) 0.99 Normal gamma globulin levels at inclusion 0.54 (0.2‐1.45) 0.22 HLA‐DR3 1.84 (0.73‐4.63) 0.19 HLA‐DR13 0.9 (0.29‐2.79) 0.86 Secondary Outcomes We investigated whether a subgroup showed greater benefit with antimalarial use. Of the 14 patients who had anti‐SLA/LP reactivity, 8 were randomized for placebo and 6 for CQ. Of the 6 patients randomized to receive CQ, 3 relapsed compared to all patients in the placebo group (50% versus 100%, respectively; P = 0.055). The anti‐SLA/LP(+) patients had significantly higher levels of aminotransferases at AIH relapse compared to the anti‐SLA/LP‐negative (–) group (460 ± 429 U/L versus 165 ± 120 U/L; P = 0.048; normal reference value up to 31 U/L). The aminotransferase levels in the relapse of patients with anti‐SLA/LP(+) were higher in the placebo group compared to the CQ group (541 ± 475 U/L versus 243.3 ± 183 U/L), but this difference was not statistically significant (P = 0.33). It was not possible to establish an interaction between the drug and anti‐SLA/LP seropositivity (hazard ratio, 2.17; 95% CI, 0.57‐8.23; P = 0.254) by multivariable Cox analysis. It was also not possible to identify a subgroup achieving the greatest benefit according to HLA profile, AIH type, and antimicrofilament seropositivity (Table 6). Variable Hazard Ratio (95% CI) P Value Antimicrofilament seropositivity 3.82 (0.75‐19.38) 0.10 Anti‐SLA/LP 1.41 (0.28‐7.03) 0.67 HLA‐DR3 0.87 (0.2‐3.86) 0.85 HLA‐DR4 0.98 (0.17‐5.76) 0.98 HLA‐DR7 0.49 (0.07‐3.24) 0.46 HLA‐DR13 0.89 (0.19‐4.13) 0.88 Side Effects In the CQ group, 54.8% of the patients had some side effects compared with 16.7% in the placebo group. The discontinuation due to side effects occurred in 19.3% of patients in the CQ group and 10% in the placebo group. The majority of adverse events in the CQ group were classified as possible according to the Naranjo algorithm; none were classified as definite (Table 7). Adverse Event Chloroquine (n = 31) n (%) Placebo (n = 30) n (%) Any AE 17 (54.8) 5 (16.7) Discontinuation due to AE 6 (19.3) 3 (10) Classification according to Naranjo algorithm Definite (0) Definite (0) Probable (4)* Probable (0) Possible (12)† Possible (3) Doubtful (1)‡ Doubtful (2) Grade 3/4 0 0 Grade 2 Neuropathy 2 (6.4) 0 Dermatologic 6 (19.3) 2 (6.6) Seizure 1 (3.2) 0 Arthralgia 0 1 (3.3) Grade 1 Headache 2 (6.4) 1 (3.3) Worsening renal function 1 (3.2) 0 Dyspepsia 3 (9.7) 0 Myalgia 1 (3.2) 0 Dermatologic 3 (9.7) 0 Retinopathy 1 (3.2) 1 (3.3) *Probable causality between drug and adverse reaction: exfoliative dermatitis (1), neuropathy (1), pruritus (1), impairment of renal function (1). †Possible causality between drug and adverse reaction: headache (2), convulsion (1), nausea (1), cutaneous hyperpigmentation (3), pruritus (4), retinopathy (1), cutaneous papules (1), neuropathy (1), inappetence (1). ‡Doubtful causality between drug and adverse reaction: myalgia (1). Abbreviation: AE, adverse event. There were no grade 3 or 4 side effects. Most side effects in the CQ group were dermatologic and included pruritus (5 patients), skin darkening (3 patients), sporadic pruritic papules (1 patient), and skin lesions similar to pellagra (1 patient). Withdrawal of medications was necessary in only one case; the other symptoms were controlled with antihistamines, skin hydration, and vitamin B3 replacement. The most serious side effects were seizures (1 patient), worsening renal function (1 patient), and peripheral neuropathy (2 patients). Despite drug withdrawal improvement, there was no security that these adverse events were drug related. According to the Naranjo algorithm, none of the side effects were classified as definite in relation to the drug. The seizure was classified as possible (although the patient reported irregular use of anticonvulsants), the impaired renal function as probable (the patient had a further diagnosis of Gitelman’s syndrome), and the two cases of neuropathy as likely and possible (1 patient had the diagnosis of carpal tunnel syndrome). Two patients, 1 in the CQ group (after 19 months) and 1 in the placebo group (after 24 months), had suspicion of CQ deposition in the macula during ophthalmologic evaluations. Although none of these patients had visual loss and the suspicion was not confirmed during follow‐up, the drug was discontinued in both cases. Discussion This was a double‐blind randomized study of 61 patients with AIH in biochemical and histologic remission that demonstrated CQ was safe and effective to significantly reduce the risk of relapse 2.5 times in a period up to 3 years after immunosuppression withdrawal. Antimalarial drugs have immunomodulatory effects and have already been used in other autoimmune diseases. In systemic lupus erythematosus, for which they were better studied, they have beneficial effects on general and disease‐free survival, with a reduction in the risk and delay of quiescent disease reactivation, improvement of the metabolic profile, and protection against occurrence of serious infections.5, 6, 16 Their possible mechanisms of action in the pathophysiology of AIH include inhibition of the processing and presentation of antigens, inhibition of cytokine release (interleukins 1, 2, and 6; tumor necrosis factor α; and interferon‐γ), inhibition of the T helper 17 response (inhibition of the release of interleukins 6, 17, and 22), decreased natural killer cell activity, inhibition of polymorphonuclear chemotaxis, and inhibition of the activity of cytotoxic T lymphocytes and clusters of differentiation (CD)4+ T lymphocytes with autoreactivity.17, 18 In the current study, possible biases that could have interfered with the results were avoided. Patients were blindly randomized, the immunosuppressive treatment was maintained along with CQ for 1 month before withdrawal, and all subjects had at least 18 months of aminotransferase normalization and more than 2 years of immunosuppressive treatment before randomization, as recommended by IAIHG in relation to attempted withdrawal of medication. It appears that duration of treatment may influence relapse rates because patients treated continuously for more than 4 years had more sustained remission rates than those treated for less than 4 years.19 In the current study, the mean duration of treatment before immunosuppressive withdrawal was approximately 6 years. Liver biopsies were performed to confirm histologic remission because approximately 55% of the patients with liver enzymes and normal IgG levels still present interface hepatitis in the biopsy and disease relapse in case of treatment discontinuation.20 However, even normal histology does not completely rule out the possibility of disease relapse because it may occur even in patients with histologic remission.21 The option for the maintenance of immunosuppressive treatment that induced histologic remission during the first month of the study was justified because there is a delay in achieving effective plasma concentrations and stable serum concentrations of CQ are only achieved after 4 to 6 weeks.18 Another reason for this choice was to reduce the risk of confusing possible enzymatic alterations related to immunosuppression withdrawal with hepatotoxicity by the antimalarial, at least in the first month of introduction of a new medication.22 The beneficial effect of CQ on protection against disease relapse may be evidenced by several findings in this study. Patients who used the medication were more apt to complete the study and used the medication for a longer period than those who used the placebo. In addition, after cessation of the drug at the end of the study, relapse in the CQ group was significantly higher than in the placebo group, showing that the antimalarial was actually protecting patients against relapse. The relapse‐free survival was 59.3% in the CQ group and 19.9% in the placebo group during the 3 years of treatment, and this difference was also statistically significant. It can be argued that 59.3% is still a low relapse‐free survival when compared to maintenance of lifelong immunosuppressive therapy, with or without corticosteroids.1-3 These options are associated with the countless side effects of corticosteroids and the increased risk of neoplasias and bacterial and fungal infections secondary to thiopurine drugs, which justifies the search for new therapies.23 The use of immunomodulatory drugs without immunosuppressive properties to maintain AIH remission could add options to this scenario. Another aspect to consider is the adopted definition of disease relapse as the elevation of aminotransferase levels above twice the upper normal limit as proposed by the 1999 IAIHG criteria8 rather than 3 times as proposed by other guidelines.1, 2 This concept certainly increased the rates of relapse diagnosis. Another controversial point is the discontinuation of treatment because of side effects. In several cases, the treatment was interrupted because of the occurrence of clinical manifestations that were not clearly related to CQ. The withdrawal rate due to adverse effects was 19.3% in the CQ group, which may have influenced the final results because these patients did not relapse while taking the medication but after antimalarial withdrawal. In addition, this relapse rate occurred over a 3‐year period during which the patients remained without the side effects of corticosteroids and with the possibility of secondary gains in metabolic profile and bone density. With respect to autoantibody reactivity, anti‐SLA/LP and anti‐LKM1 are associated with an increased risk of relapse, and a meta‐analysis suggested never stopping therapy when the former is present.24 In the current study, anti‐SLA/LP(+) patients had higher levels of aminotransferases at disease relapse compared to anti‐SLA/LP(–) patients, and the relapse rates in this subgroup were 100% in the placebo group and 50% in the CQ group. However, when attempting to establish the relationship between anti‐SLA/LP reactivity and the drug used, the difference was not statistically significant; this may be related to sample size and should be confirmed in further studies. A study reporting a 20‐year experience with AIH treatment in childhood25 showed that all anti‐LKM1(+) patients relapsed after treatment discontinuation. In our series, there was one case of AIH‐2 in the CQ group. This patient completed the study without relapse and had biochemical relapse (aspartate aminotransferase, 197 U/L; ALT, 531 U/L [normal value, <31 U/L]) after approximately 90 days of its withdrawal. The HLA profile determines different clinical presentations and different responses to treatment, resulting in an autoimmune response of greater or lesser severity. Caucasian patients with HLA‐DR3 present earlier and more aggressive disease onset with inferior response to corticosteroid treatment compared to patients with HLA‐DR4.2, 26, 27 In Brazil, the susceptibility to AIH‐1 is primarily linked to HLA‐DR13, and there is a secondary association with DR3.28 In the current study, patients with AIH‐1 with HLA‐DR3 or HLA‐DR13 were the most common profiles. A study of AIH‐1 in North America evaluated 26 patients with non‐HLA‐DR3/DR4 and showed that patients in whom the only marker of susceptibility was HLA‐DR13 had lower rates of relapse after treatment withdrawal than those whose sole marker of susceptibility was HLA‐DR3.29 These findings are similar to those found in our study in which the presence of HLA‐DR3 was associated with a higher risk of relapse. Regarding HLA‐DQ, the two serotypes most frequently found in our series were DQ2 and DQ6. This finding is similar to findings in Brazilian and Argentinean patients with AIH‐1 in which susceptibility to the disease with the DR13‐DQ6 haplotype was observed.28, 30 The presence of HLA‐DQ6 resulted in a trend of lower risk of relapse (P = 0.089) with a risk ratio of 0.53, which should be evaluated in studies with a larger sample size. In a Brazilian study of HLA typing, a significant increase in DQ6 was observed in strong linkage disequilibrium with DR13.28 In the present study, 22 of the 28 patients with HLA‐DQ6 had at least one DR13 allele, which was associated with a lower risk of disease relapse compared to HLA‐DR3.29 Therefore, it remains an open question whether this protective effect is actually related to HLA‐DQ6 or to HLA‐DR13. Concerning the safety of antimalarial use, although hydroxychloroquine causes fewer side effects than CQ in patients with rheumatic diseases, CQ is 2‐3 times more potent than hydroxychloroquine.31 This probably influenced the rates of side effects. To minimize the risk of side effects, only patients with cirrhosis with preserved liver function were included because CQ binds moderately (60%) to plasma proteins and undergoes primarily hepatic metabolism by cytochrome P450 enzymes; in addition, impairments in liver function could affect drug metabolism.18, 32 Treatment suspension rates due to side effects were probably overestimated because of the inflexibility adopted in attributing a causal relationship between the symptoms presented during treatment and drug use. In addition, no higher incidence of side effects was observed when compared to published data, and there were no third‐ or fourth‐degree side effects in the current study. The most commonly reported side effects related to the use of CQ, occurring in up to 20% of patients, are dyspeptic and dermatologic symptoms, which were the most frequent in this series. Most of the time, dermatologic findings were reverted in this study with the use of antihistamines and hydration, without impairment of quality of life. The dyspeptic symptoms, which are more common in the first few weeks of treatment, usually improve over time and are minimized by dose reduction or fractionation and postprandial administration18, 33; this was not necessary in any of our patients. Neuromyopathy and seizures are rare events with few reported cases,18, 33, 34 which was also the case in the present study. One of the most feared side effects is retina damage by the antimalarial, which is usually irreversible and dose dependent and time dependent. It is present in less than 1% of patients after 5 years of use but reaches 20% after 20 years. In this series, retinopathy was detected in one case in the CQ group and one in the placebo group, without visual losses. Some risk factors associated with its occurrence are a cumulative total dose of 460 g or daily dose above 250 mg (or >3 mg/kg/day for individuals with low weight), age over 60 years, liver or kidney dysfunction, maculopathy, or retinal preexisting disease.35 In the case of a 50‐year‐old patient in the CQ group, injury suspicion occurred 19 months after starting the medication, the dose by weight was 2.84 mg/kg/day, and there was no renal or hepatic dysfunction. After drug discontinuation, the suspected lesion was completely reversed in less than 6 months of ophthalmologic follow‐up. It is worth remembering that, like antimalarials, long‐term use of corticosteroids is also associated with visual risk, such as glaucoma and cataracts, and a periodic ophthalmologic assessment is also recommended in patients who use long‐term medication. Some criticisms of this study could be made, such as the reduced sample size, the use of CQ instead of hydroxychloroquine, the early withdrawal of antimalarial drugs following the occurrence of some side effects even without a well‐documented causal relationship with the drug, and suspension in pregnancy. Despite the fact that hydroxychloroquine has a better safety profile in rheumatologic diseases, particularly with regard to retinopathy occurrence, a study with 940 patients with several rheumatic disorders revealed that 28% of patients had side effects with CQ compared to 15% with hydroxychloroquine. However, the interruption rate due to side effects was higher with CQ but due to clinical inefficacy was higher with hydroxychloroquine.36 Therefore, in the current study, the use of CQ was probably related to a higher rate of adverse events but maybe with a lower rate of disease relapse. CQ was the only formulation available for dispensing in our Public Health System, but the side effects most frequently reported in our patients were cutaneous and gastrointestinal; these were usually temporary and easy to alleviate. The placebo group also had a high rate of treatment discontinuation, probably reflecting the rigor of investigators in relation to the side effects due to the off‐label use of the drug in the treatment of AIH. On the other hand, the high rates of discontinuation were minimized by the evaluation of relapse‐free survival by the Kaplan‐Meier method because the patient could be observed until its censorship. Although the drug is considered relatively safe during pregnancy and already used in patients with antiphospholipid syndrome, there are no adequate and well‐controlled studies evaluating the safety and efficacy of CQ in pregnancy, justifying its withdrawal in the current study with more decreases in the sample size. The long enrollment period may have added some biases in this study, such as the changes in the AIH criteria for disease remission. The most recent AIH guidelines suggest that biochemical remission should include the normalization of aminotransferases and IgG levels. When the present study was planned and patient recruitment was started, the 1999 IAIHG recommendations, which are still valid, were the reference, and their considerations on complete remission, including histologic remission, were strictly followed. Liver biopsies at the end of the 3‐year CQ administration were not performed because the criteria for AIH relapse after full complete response were based only on biochemical results. However, this long period of time is justified by the low incidence/prevalence of the disease and the fact that the study is unicentric. This in turn also allowed better control of other confounding factors that could influence the results, such as inflammatory activity in liver biopsies before inclusion, standardization of the exams collected, criteria for suspension of the medication, and poststudy follow‐up. In conclusion, CQ safely and significantly reduces the risk of relapsing AIH in remission following discontinuation of immunosuppressive therapy; however, a subgroup with greater benefit from its use has yet to be defined. The side effects presented were mild and more frequently controlled with the use of symptomatic medication without affecting quality of life. The high rates of adverse effects and discontinuation of the drug were a concern in the study but should be interpreted with caution because of the stringent criteria for discontinuation of treatment in the case of side effects resulting from the “off‐label” treatment of AIH with CQ. Possibly the use of hydroxychloroquine instead of CQ could be a safer and better tolerated option to maintain AIH remission, but this should be investigated in further studies. The most outstanding finding of our study is the possibility of using a drug without immunosuppressive effect as part of the treatment of AIH. This creates the expectation that in the long term other drugs with immunomodulatory action can be formulated, allowing an increase of the scarce therapeutic options available for the treatment of the disease. In order to definitely include the drug as a possible therapeutic option, we should consider carrying out multicenter studies, including different countries and populations, to draw more solid conclusions regarding the role of antimalarial drugs in the treatment of AIH. Potential conflict of interest Nothing to report. References

Objective to appraise the role of epitope spreading in established human systemic lupus erythematosus (SLE). Methods IgG‐autoantibody reactivity to 398 distinct recombinant proteins was measured in 69 SLE patients over 6 years and compared to 45 controls. Changes in mean fluorescence intensity (MFI), number of autoantibodies to distinct antigens, and reactivity to distinct clones of selected established antigenic targets U1‐RNP, Sm, and ribosomal P representing epitope fine mapping were assessed. Linear mixed modelling adjusted for age and sex with Bonferroni correction was applied. Results The total number of autoantibodies, mean MFI, and number of autoantibodies in epitope fine mapping were higher in SLE compared to controls (p<0.0001). The total number of antibodies to distinct autoantigens remained stable, while the mean MFI decreased over time in SLE (p<0.021). SLE patients showed variable recognition of epitopes in fine mapping over time. In particular, SLE patients recognized more clones of the U1‐RNP complex at the time of new organ involvement (+0.65, p =0.007). Patients with lupus nephritis had a higher mean MFI (p=0.047). Time‐averaged MFI of 22 individual autoantibodies were higher in SLE after Bonferroni correction including dsDNA (p<0.0001). DsDNA and histone cluster 2 H3c MFIs were associated to the SLAM (p<0.0001). Conclusion Longitudinal surveillance of the IgG‐autoantibody repertoire in established SLE reveals evidence for sustained breadth of autoantibody repertoire without significant expansion. Associations of disease activity are confirmed for dsDNA and histone H3 autoantibodies. This article is protected by copyright. All rights reserved.

Summary Immune metabolism is a rapidly moving field. While most of the research has been conducted to define the metabolism of healthy immune cells in the mouse, it is recognized that the overactive immune system that drives autoimmune diseases presents metabolic abnormalities that provide therapeutic opportunities, as well as a means to understand the fundamental mechanisms of autoimmune activation more clearly. Here, we review recent publications that have reported how the major metabolic pathways are affected in autoimmune diseases, with a focus on rheumatic diseases. Introduction The connection between cellular metabolism and immune activation was first established when it was shown that activation of T cells through the engagement of the co‐stimulatory receptor CD28 triggered glycolysis through the phosphoinositide‐3‐kinase (PI3K) pathway 1 (Fig. 1). A few years later, it was shown that the ligation of the B cell receptor activated the same pathway, and that glycolysis was required for B cell activation and differentiation 2 (Fig. 2). The next decade has seen an explosive growth of research in immunometabolism that has defined nutrient utilization and cellular processes to sustain the unique energy biosynthetic needs of immune cells to achieve rapid growth and to perform effector functions upon challenge with pathogens 3. While most of the research has characterized the metabolism of healthy immune cells, it has been rapidly recognized that the overactive immune system that drives autoimmune diseases presents metabolic abnormalities that provide therapeutic opportunities 4, 5. Exploiting the intricately dysregulated metabolic networks in cancer is being considered to develop therapeutic approaches that would either starve tumor cells or boost the immune system 6, 7. Reviews have been recently published focusing on rheumatic diseases 8, rheumatoid arthritis (RA) 9 and systemic lupus erythematosus (SLE) 10, two autoimmune diseases for which the field of immunometabolism is the most advanced. Here, we review recent publications that have reported how the major metabolic pathways are affected in autoimmune diseases, with a focus on rheumatic diseases. The yin and yang regulation of autoimmunity by MTOR and AMPK Mammalian target of rapamycin (mTOR) and AMP‐activated protein kinase (AMPK) are metabolic sensors that play a major role in immune cell functions 11. Activated AMPK inhibits adenosine triphosphate (ATP)‐consuming processes, such as protein synthesis while enhancing ATP‐producing processes, such as fatty acid oxidation (FAO) and glucose uptake 12. Activated AMPK inhibits mTOR activation, which promotes energy‐consuming processes. mTOR signaling is mediated by two multimeric complexes, mTOR complex 1 (mTORC1) and mTORC2, which share the catalytic subunit mTOR but are distinguished by the scaffold proteins regulatory‐associated protein of mTOR (RAPTOR) and rapamycin‐insensitive companion of mammalian target of rapamycin (RICTOR), respectively. Activation of both complexes promote glucose metabolism, linking mTORC and glycolysis. Direct modulation of glucose metabolism through over‐expression of glucose transporter 1 (Glut1) enhances follicular helper T (Tfh) cell differentiation and leads to autoimmunity 13. mTOR activation promotes the differentiation of T helper type 1 (Th1), Th17 14 and Tfh T cells 13, three effector subsets that are expanded in lupus 15, 16. mTOR activation plays a more complex role in regulatory T cell (Treg) differentiation and function by preventing the generation of long‐lived central Tregs, but promoting the generation of effector Tregs 17. Treg‐specific deletion of mTOR reduced their frequency, leading to spontaneous effector T cell activation and inflammation 18. Deletion of Lkb1 in Tregs, the upstream kinase of AMPK and a well‐known sensor of metabolic stress, resulted in dysfunctional Tregs and the development of a Th2‐dominant severe autoimmune phenotype 19, 20. Deletion of PP2A in Tregs, a serine‐threonine phosphatase involved in the development SLE by regulating the production of interleukin (IL)‐2 and IL‐17 in CD4+ T cells 21, resulted in increased mTORC1 activation and a severe, multi‐organ autoimmune disorder 22. These results indicated that while mTOR activity is required for Treg development and function, its level of activation has to be kept in check by protein phosphatase 2 (PP2A), and possibly other mechanisms. The function and differentiation of follicular regulatory T cells (Tfr), a Treg subset that suppresses germinal center (GC) B cells and Tfh cells, is also mTORC1‐dependent 23. These results suggest that T cell differentiation of most T cell subsets is mTOR‐dependent and aberrant expression of mTOR might lead to autoimmunity. CD4+ T cells from lupus patients present a high level of mTOR activation that is directly implicated in the disease process 24. Indeed, treatment with sirolimus, an mTOR inhibitor, reduced disease activity in refractory lupus patients 25. Intriguingly, the therapeutic response in these patients was best associated with a reduced number of effector memory CD8+ T cells, a subset whose role in lupus pathogenesis is as yet undefined. Tfh cells in the B6.Sle1.Sle2.Sle3 (TC) model of lupus show a high level of mTORC1 activation, which was reduced by the inhibition of glucose metabolism 26. This reduction was associated with a decreased frequency of Tfh cells, GC B cells and autoantibody production. This effectively linked glycolysis, mTORC1 activation and Tfh expansion in lupus. mTOR also plays an essential role in B cell differentiation. In the Roquin mouse model of lupus, activation of AMPK and inhibition of mTOR limited B cell differentiation into GC B and plasma cells, which was associated with a reduced disease activity 27. In SLE patients, high mTOR activation in CD19+ B cells correlates with plasmablast numbers and disease activity 28 (Fig. 2). Conversely, treatment with metformin, which activates AMPK 29, has beneficial effects in lupus patients 30 and in mouse models of lupus 31, 32. Overall, these studies showed that mTOR plays a central role in lupus by affecting multiple cell types. However, these findings should not be generalized to other autoimmune diseases without further studies, in which the AMPK/mTOR pathway has not been explored in detail. Glycolysis Glycolysis refers to the metabolic pathway by which glucose is metabolized. The first common phase of glycolysis is the production of pyruvate. Pyruvate is then either oxidized in the Krebs cycle, leading to the production of up to 38 molecules of ATP per molecule of glucose, or reduced into lactate in either hypoxic conditions or when metabolite intermediates are needed over ATP production, which in this case is limited to two molecules. Glycolysis commonly refers to this lactate end‐point branch of glycolysis, while the other is referred to as glucose oxidative or mitochondrial metabolism. Activation of CD4+ T cells from lupus‐prone mice and SLE patients occurs with high levels of oxygen consumption and oxidation 31, 33. Lupus T cells also display a high level of glycolysis 31, with oxidation representing a major part of glucose utilization 32. Glucose transporters provide the ‘primary first step’ of glycolysis by importing glucose into the cell. The major glucose transporter expressed by T cells is Glut1, which is significantly up‐regulated upon T cell receptor and co‐stimulator CD28 signaling 34. Over‐expression of Glut1 in mice led to the accumulation of activated CD4+ T cells, the production of autoantibodies and a modest immune complex deposition in the glomeruli of aged mice 35. Furthermore, these mice showed increased Tfh and GC B cell numbers, with elevated IL‐21 and immunoglobulin (Ig)A production 13. The combination of 2‐deoxy‐D‐glucose (2DG), a glycolysis inhibitor, and metformin, which inhibits complex I of the mitochondrial electron transport chain 36, reversed lupus pathogenesis in mice 31. While treatment with either metformin or 2DG alone could prevent the development of the disease 32, these results indicate that targeting cellular metabolism could be a potential therapy for lupus and other autoimmune diseases 37. Among the subsets of T cells, Tfh cells from lupus mice are highly glycolytic (Fig. 1), and their expansion as well as that of GC B cells was abrogated by 2DG treatment 26. This glycolytic requirement is restricted to autoreactive Tfh cells, as Tfh cells induced by immunization with a nominal antigen or by infection with influenza virus were not affected by 2DG 26. This suggests that the metabolic requirements of autoreactive CD4+ T cells are unique, which may provide a window of opportunity for their selective elimination. The expansion of Tfh cells was also kept in check by 2DG in the K/BXN model of RA, which indicated that the high glucose requirement of autoreactive Tfh cells is not model‐dependent 38. 2DG‐treated K/BXN mice also showed a reduced disease severity, in association with a decreased T and B cell metabolism and a reduced activation of both adaptive and innate immune cells 38. In the same RA model, reducing glycolysis by targeting hexokinase (HK) showed beneficial effects by decreasing the activation of fibroblast‐like synoviocytes 39. Contrary to murine RA T cells and synoviocytes, glycolysis is reduced in the CD4+ T cells from RA patients, which develop a hyper‐reduced state due to an over‐active pentose phosphate pathway (PPP) 40. The pathogenicity of these T cells can be reduced by diverting the glucose flux away from PPP 41 or with oxidative agents 42. Lactate accumulation has been reported in the synovia of RA patients, which may be secondary to the hypoxic conditions in the inflamed joint. The excess is responsible for the ‘entrapment’ of CD4+ and CD8+ T cells. The expression of lactate transporters on these cells correlates with the clinical T cell score in the synovia of RA patients. Lactate directly inhibits CD4+ T cell motility by interfering with glycolysis activated upon engagement of the chemokine receptor C‐X‐C motif chemokine receptor 3 (CXCR3) with C‐X‐C motif chemokine ligand 10 (CXCL10) 43. These results suggest that blocking lactate production in the RA joint may decrease T cell infiltrates and present therapeutic benefits. Pyruvate dehydrogenase (PDH) promotes the oxidative phosphorylation of pyruvate over the lactate glycolytic pathway. Pyruvate dehydrogenase phosphatase catalytic subunit 2 (PDP2) converts the inactive PDH to its active form. PDP2 expression was decreased in memory Th17 cells from patients with SLE and forced expression of PDP2 in CD4+ T cells from lupus‐prone MRL/lpr mice and patients with SLE‐suppressed Th17 differentiation. This may be due at least partly to the direct control of energy production by the transcription factor‐inducible cAMP early repressor/cAMP response element modulator (ICER/CREM) at the PDH metabolism bifurcation level 44. These results are consistent with the glycolytic requirements of Th17 cells 45 and the expansion of Th17 cells in SLE patients 15. Finally, dimethyl fumarate, a derivative of the Krebs cycle intermediate fumarate that inactivates glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) and therefore inhibits both branches of glycolysis, altered the differentiation and function of Th1 and Th17 cells, attenuating disease in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis 46, stressing again the potentials of glucose metabolic inhibitors to target pathogenic autoreactive T cells. HIF1α, a transcription factor that controls the cellular response to hypoxia, activates the glycolytic pathway and, as such, promotes inflammation 47. HIF1α expression is required for the differentiation of Th17 cells 45, a T cell subset expanded in many autoimmune and inflammatory diseases. Mice with a B cell‐specific deletion of HIF1α have reduced numbers of IL‐10‐producing B cells, which exacerbate collagen‐induced arthritis and EAE 48. HIF1α‐inhibitor echinomycin reduced Th1 and Th17 responses, and attenuated a mouse model of acute graft‐versus‐host disease 49. Moreover, hypoxia induced HIF1α expression as well as signal transducer and activator of transcription (STAT)‐1 and STAT‐3 activation in human RA‐fibroblast‐like synoviocytes (FLS). STAT‐3 knock‐down inhibited HIF1α expression and the hypoxia‐induced cell invasion, migration and cytokine production, indicating a functional link between HIF1α and STAT‐3 in the regulation of proinflammatory mechanisms in RA 50. Prolyl hydroxylase domain (PHD) are enzymes that regulate HIFα levels by promoting its degradation. PHD‐2 is the major hydroxylase regulating HIF levels and the expression of angiogenic genes in RA‐FLS, illustrating the major role played by hypoxia in the RA joint 51. Finally, knocking down HIF1α in myeloid cells ameliorated induced colitis 52. Taken together, targeting glycolysis directly and indirectly may present promising therapeutic venues to treat autoimmune diseases; however, detailed disease‐specific analyses of preclinical models are necessary to understand the cellular targets and specific pathways in glucose flux that are responsible for pathogenesis. Oxidative phosphorylation and fatty acid oxidation In order to produce ATP, enzymes in the electron transport chain (ETC) transfer electrons from donor metabolites to electron acceptors. This is known as oxidative phosphorylation (OXPHOS) 53. In this process, complex I of the ETC, nicotinamide adenine dinucleotide hydrogenase (NADH), oxidizes NADH to nicotinamide adenine dinucleotide (NAD) 54. Succinate dehydrogenase, complex II, oxidizes succinate to fumarate, reducing ubiquinone 55. Ubiquinone is then oxidized by complex III, cytochrome c reductase, which reduces cytochrome c allowing for its oxidation by complex IV, cytochrome oxidase 56, 57. Thus, the design of OXPHOS is to produce ATP by the oxidation and reduction of key metabolites. A number of studies have demonstrated alterations in the OXPHOS and FAO metabolic pathways in autoimmune diseases. As mentioned earlier, T cells from RA patients function in a hyper‐reduced phenotype due to a hyperactive PPP, probably in response to the deficiency of necessary intermediates and the hypoxic environment 5, 9, 58. This is probably a result of oxidative stress from macrophages and other reactive oxygen species (ROS) releasing cells in the symptomatic tissue 59. Contrary to the RA phenotype, SLE T cells are hyper‐oxidative 33. These changes in oxidative profiles may be due in part to genetic predisposition. This hypothesis is supported by the finding that the murine lupus risk allele Esrrg encodes for a nuclear receptor, estrogen‐related receptor gamma (ERRγ), which regulates mitochondrial function and OXPHOS 60. Directly relevant to lupus, type 1 interferon (IFN) induces OXPHOS and FAO in plasmacytoid dendritic cells (pDCs), and this metabolic reprogramming is critical for their activation and amplification of IFN production 61. B cells and other myeloid cells are less well metabolically characterized, and it is not understood if these cell types also have altered OXPHOS or FAO metabolism in autoimmune disease 62. Notably, long‐lived plasma cells, which are a critical component of many antibody‐mediated autoimmune diseases, require mitochondrial import of pyruvate 63. A recent study utilizing several mice models of lupus nephritis demonstrated that kidney‐infiltrating T cells (KITs) present an exhausted phenotype similar to that observed in tumor‐infiltrating lymphocytes 64. Both CD4+ and CD8+ KITs demonstrated substantial loss of their spare respiratory capacity and little to no oxidative phosphorylation. The significance of this finding remains unclear, as CD4+ T cell infiltrates have been associated with lupus nephritis in mouse models 65, 66 and patients 67, 68. Another unexpected finding was the identification of C1q, a member of the complement pathway involved in efferocytosis, as a modulator of mitochondrial metabolism in CD8+ T cells in the chronic graft‐versus‐host‐disease (cGVHD)‐induced model of SLE 69. C1q‐deficiency reduced cGVHD induction and, mechanistically, resulted in the reduction of mitochondrial function in effector memory CD8+ T cells (Fig. 1). These results indicated a novel link between C1q, mitochondrial metabolism and CD8+ T cell effector functions in autoimmunity. OXPHOS may also play an important role in the pathogenesis of RA, according to a recent study showing that the Krebs cycle metabolite succinate is under‐utilized in the adjuvant‐induced arthritis (AIA) model of RA, possibly leading to increased joint inflammation 70. Suncr1, the cognate receptor succinate receptor 1, is an active participant in the innate immune response 71-73, and increased amounts of succinate correlate with disease in AIA 70, 72. Accordingly, Suncr1 deficiency reduced the expansion of pathogenic Th17 cells and AIA severity 70. Succinate is largely used by OXPHOS, which has a reduced activity in the T cells of RA patients and in other autoimmune diseases 74. This reduced OXPHOS probably results in an under‐utilization of succinate which would, in turn, create a microenvironment that increases disease activity. A recent study found, in a small subset of CD4+CXCR3+CXCR5–PD‐1hi, T cells in the blood of SLE patients who have increased ROS due to reverse ETC regulated by succinate 75. These cells provide help to B cells via IL‐10 and succinate, independently of IL‐21. These studies emphasize the importance of the metabolite succinate in rheumatic diseases. Itaconate inhibits succinate dehydrogenase, regulating succinate oxidation to fumarate. Itaconate is one of the most highly produced metabolites in lipopolysaccharide (LPS)‐activated macrophages, in which it has anti‐inflammatory effects 76. Mechanistically, itaconate derivatives can regulate nuclear factor kappa B inhibitor zeta (IκBζ) via activating transcription factor 3 (ATF3), ameliorating IL‐17 IκBζ‐driven inflammation 76. Another study identified that activation of nuclear factor‐like 2 (Nrf2) via Kelch‐like ECH‐associated protein 1 (KEAP1) alkylation could explain the anti‐inflammatory effects exhibited by itaconate 77. Further, cell‐permeable itaconate derivatives decreased cytokine production in macrophages and limited the type I IFN response, indicating a possible therapeutic use for itaconate and its derivatives in IFN‐driven diseases such as SLE 77, 78. FAO represents a major energy source for Treg and memory T cells 79, although a genetic rather than a pharmacological approach has recently challenged this tenet 80. The contribution to FAO to autoimmune diseases has not been well characterized. Elevated levels of adipocyte‐fatty acid binding protein (FABP), which is responsible for facilitating the transport of fatty acids to specific organelles for oxidation, correlate with disease activity in patients with multiple sclerosis (MS) and clinical isolated syndrome 81. Inhibition of FABP prevented EAE in mice 82, providing a possible treatment option to correct this specific metabolic defect that is contributing to immune cell dysfunction in MS‐related diseases. Finally, D‐mannose was found to induce Treg differentiation and decrease the production of inflammatory cytokines, indicating that D‐mannose has the potential to improve autoimmune pathology 83. Mechanistically, D‐mannose promotes transforming growth factor (TGF)‐β production, which is mediated at least in part through ROS produced by increased FAO, an indirect consequence of the inhibitory effect of D‐mannose on glycolysis 83. This study illustrates the complex relationships that exist between metabolic networks and their consequences on immune activation. Finally, glycolysis is required by Treg cells to migrate to the site of inflammation 84. Mechanistically, PI3K‐mTORC2 activation in Treg cells induces the expression of glycokinase, which binds actin and promotes the cytoskeletal remodeling that is necessary for cell migration. This latter finding emphasizes that specific metabolic pathways sustain different aspects of immune function. Branch chain amino acid and glutamine metabolism Amino acids are metabolites used for the biosynthesis of lipids, nucleotides, glutathione, glucosamine and polyamines, as well as in the anaplerosis of the Krebs cycle 85. Moreover, branch chain amino acids (BCAA) and glutamine function as signaling molecules of the cellular metabolic state to activate mTORC1 86. Here, we will focus on BCAAs, especially leucine and glutamine metabolism. The potential contribution of leucine metabolism to autoimmune diseases is largely attributed to its role as a critical checkpoint of mTORC1 signaling and glycolysis, which then control the function of T cells and monocytes/macrophages 87-89. Leucine influx is mediated by solute carrier family 7 member 5 (SLC7A5). Intracellular leucine undergoes reversible transamination by branched‐chain aminotransferase (BCAT) to form α‐ketoisocaproate, which is further metabolized into acetoacetate, then acetyl‐co‐enzyme A (CoA), to be subsequently oxidized in the Krebs cycle. BCAT exists in two isoforms, mitochondrial BCAT2 and cytosolic BCAT1. In human monocyte‐derived macrophages, BCAT1 is the most abundantly expressed BCAT isoform 90. Circulating monocytes from RA patients express higher SLC7A5 levels than cells from healthy controls, and these levels correlate with clinical parameters. Furthermore, blockade of SLC7A5 reduced IL‐1β production in these monocytes 87. Treatment with a selective inhibitor of BCAT1 reduced inflammation and macrophage infiltration in target organs in murine models of RA and crescentic glomerulonephritis 90. Glutamine (Gln) uptake is mediated by ASCT2/SLC1A5. It is converted to glutamate by glutaminase (GLS), followed by the conversion to α‐ketoglutaric acid (αKG) by transaminases or glutamate dehydrogenase 1 (GLUD1), after which αKG enters the Krebs cycle. Isocitrate dehydrogenase (IDH1 and IDH2) produce (D)‐2‐hydroxyglutarate (2HG) from αKG. In the SKG zymosan‐induced model of RA, the proliferation of RA‐FLS was decreased by glutamine but not glucose deprivation 91. Furthermore, Gls1 expression was higher in RA‐FLS than in osteoarthritis‐derived FLS. Accordingly, the administration of a Gls1 inhibitor ameliorated disease symptoms by suppressing FLS proliferation. Similarly, blocking glutaminolysis with the Gls1 inhibitor BPTES [bis‐2‐(5‐phenylactamido‐1,2,4‐thiadiazol‐2‐yl)ethyl sulfide] 92 or with the transaminase inhibitor aminooxyacetic acid (AOA) 93 prevented disease in the EAE model. The regulation of the balance between Th17 and Treg differentiation may underline the role of glutaminolysis in the pathogenesis of autoimmune diseases. Indeed, glutaminolysis is a major source of energy for the generation of Th17 cells 6. Gln deprivation led to the differentiation of activated naïve CD4+ T cells 6 and Th1‐polarized cells 94 into Treg cells. Gln transporter deficiency or depletion of Gln in culture media inhibited both Th1 and Th17 differentiation 95, but blocking the conversion of Gln to glutamate preferentially suppressed Th17 over other T cell subsets 6, 92. Blocking glutamate oxaloacetate transaminase 1 (Got1) also altered the balance between Th17 and Treg cells without affecting Th1 cells 93. However, Gls1 inhibition did not alter the number of Th17 cells and decreased the number of Treg cells in SKG mice 91. The reason for these discrepancies is as yet unclear, but it may include differences between healthy versus inflammatory or autoimmune strains. It may also be due to the role of some metabolic enzymes or metabolites in non‐metabolic pathways. The latter includes a greater accumulation of 2‐hydroxyglutarate (2‐HG) in differentiating Th17 than Treg cells, which promotes the methylation of the Foxp3 locus and silences its expression 91. Limiting glutamate production reversed this process and ameliorated EAE disease by expanding Treg and reducing Th17 cell numbers 92. This highlights the role of glutamine metabolism in epigenetic regulation in addition to energy production. Finally, the role of glutamine metabolism has been recently examined in Tfh cells in the TC model of SLE. Slc1a5 expression was lower in TC Tfh than in congenic non‐autoimmune B6 Tfh cells 26. Contrary to glycolysis, glutaminolysis inhibition greatly reduced immunization‐induced as well as autoimmune humoral responses. This diminished Tfh function, in both lupus‐prone and non‐autoimmune mice 26, indicating that it required for the development of germinal centers. Lipid synthesis De‐novo FA synthesis starts from the generation of malonyl‐CoA from acetyl‐CoA by the rate‐limiting enzyme acetyl‐CoA carboxylase (ACC1) 96. Subsequent steps and FA elongation require fatty acid synthase (FAS), stearoyl‐CoA desaturase (SCD) and the FA‐co‐enzyme A ligase family to generate diacetyl‐ and triacetyl‐glycerols and long‐chained FA 97. Most of the de‐novo synthesized FA are incorporated into phospholipids for membrane biogenesis and localize to lipid rafts to participate in crucial membrane‐based processes 98, while some form lipid droplets 99. Pathogenic T cells in RA patients are characterized by increased FA synthesis leading to the formation of cytoplasmic lipid droplets. The inhibition of FA synthesis corrected the pro‐arthritogenic tissue‐invasiveness of these T cells 100. FA, in the form of triglycerides, phosphoglycerides or sphingolipids, are directly involved in T cell activation and proliferation as key components of cell membranes, signaling molecules and energy‐yielding substrates. T cell‐specific deletion of ACC1 severely impaired the accumulation of antigen‐specific CD8+ T cell due to activation‐induced cell death. Furthermore, exogenous FA rescued defective blasting and survival of ACC1‐deficient CD8+ T cells in vitro 101. These results may provide insights for therapeutic targets to either increase or decrease CD8+ T cell activity. Th17 cell induction results in increased levels of mRNA and phosphorylated ACC1 and ACC2 proteins. Pharmacological inhibition or T cell‐specific deletion of ACC1 restrained the formation of human and mouse Th17 cells and promoted the induction of Treg cells, which attenuated Th17 cell‐mediated EAE 102. Inhibition of fatty acid synthase (FASN) also reduced EAE severity, with direct evidence from adaptive transfer that this effect is Th17‐dependent. However, FASN inhibition also promoted IFN‐γ production by Th1 and Th1‐like Th17 cells, which is different from the effect of ACC inhibition 103. The specific mechanisms by which FA synthesis regulates CD8+ T cell survival or inflammatory CD4+ T cell polarization have not yet been elucidated. Glycophingolipids (GSLs) are a key constituent of lipid rafts that have been implicated in lupus pathogenesis. CD4+ T cells from SLE patients present elevated GSL levels due to increased synthesis and altered trafficking 104. Normalization of GSL metabolism in these T cells normalized their function. In addition, activated T cells from SLE patients show a reduced induction of BTLA, an inhibitory receptor similar to cytotoxic T lymphocyte antigen (CTLA)‐4 and programmed death 1 (PD1) 105. The capacity of BTLA to restrain T cell activation in SLE is impaired, which could be due to poor BTLA recruitment to the immunological synapse. In support of this hypothesis, a glucosylceramide synthase inhibitor that normalized lipid metabolism restored BTLA inhibition. The dissociation of preclustered T cell receptor (TCR) molecules in the presence of this inhibitor allowed BTLA recruitment to TCR clusters directly linking GSL homeostasis to intracellular trafficking of components of the TCR 105. Cholesterol homeostasis Cholesterol homeostasis is maintained through the sophisticated balance between its synthesis, import and elimination of excessive cholesterol from the cells. Cholesterol is synthesized by endoplasmic reticulum‐bound 3‐hydroxy‐3‐methylglutaryl‐CoA reductase (HMGCR), which is controlled by the transcription factor sterol regulatory element‐binding protein (SREBP) 106. Apolipoprotein E (ApoE) and low‐density lipoprotein receptor (LDLR) contribute to the recycling and intercellular transport of cholesterol. ApoA1 and high‐density lipoproteins (HDL) mediate the efflux of cholesterol from immune cells via liver X receptor (LXR)‐regulated genes, such as ABCA1 and ABCG1. Cholesterol plays a key role in the regulation of immune responses through at least three mechanisms. First, cholesterol is required for membrane synthesis during cell expansion. Secondly, cholesterol is a key constituent of lipid rafts; thus, any changes in cholesterol content modify raft‐dependent signaling of major immune pathways such as Toll‐like receptors (TLRs), major histocompatibility complex (MHC), TCRs and B cell receptors (BCRs) 107-109. Finally, type I IFN reprograms lipid metabolism, including a decreased synthetic and increased import of cholesterol 110. Interestingly, ApoE is bound not only to plasma lipoproteins but is also present on immune cell membranes 111. ApoE deficiency in DCs impaired the removal of cholesterol from the membrane, resulting in lipid raft accumulation, enhanced MHC‐II clustering on the membrane and increased antigen presentation. This, in turn, expanded proinflammatory CD4+ T cells and enhanced skin allograft rejection, independently of dyslipidemia 112. Atherogenic dyslipidemia caused by a Western diet in ApoE or LDLR‐deficient mice increased the production of autoantibodies and the severity of lupus by expanding the number of Tfh cells 113. A novel mechanism was identified by which dyslipidemia induces IL‐27 production by DCs, which in turn expands Tfh cell responses and GC reactions to lupus‐associated self‐antigens 113. The levels of IL‐27 were increased in patients with hypercholesterolemia, while blocking IL‐27 in atherogenic mice reduced autoantibodies and Tfh cells to levels similar to those of control mice. An almost complete absence of HDL due to ApoA1 deletion induced autoimmune phenotypes, characterized by cholesterol‐engorged, enlarged lymph nodes, anti‐dsDNA autoantibodies and increased T cell activation 114, suggesting a link between cellular cholesterol accumulation and autoimmunity. Mice lacking LXRs exhibit an age‐dependent systemic autoimmune disease, and pharmacological activation of LXRs attenuated disease progression in a mouse model of lupus 115. Transcriptional induction of Abca1 expression by LXRs promotes cholesterol efflux and alters plasma membrane cholesterol distribution. Mice with Abca1/g1 deficiency presented lymphadenopathy and glomerulonephritis 116, 117. Interestingly, autoimmune activation in these mice was due to the specific deficiency of these transporters in DCs (Fig. 1). Abca1/g1‐deficient DCs showed an activated inflammasome, increased proliferation and cytokine secretion. These led to the expansion of Th1, Th17 and Tfh cells, GC B cells and plasma cells, all changes which have been implicated in the pathogenesis of SLE 117. Macrophages lacking the ABC transporters Abca1 and Abcg1 showed an increased TLR cell surface expression and enhanced inflammatory responses to LPS 118, while Abcg1‐deficient T cells show enhanced TCR signaling and proliferation 119. None of these studies in macrophages and T cells have reported autoimmune phenotypes. The mechanisms responsible for cell‐specific consequences of cholesterol efflux are not clear at present. Conclusions Immune metabolism is a rapidly moving field. Earlier studies have proposed a simple model in which activated effector immune cells switched from mitochondrial respiration to aerobic glycolysis relying on an increased glucose import 120. More recent studies have demonstrated that glutamine and fatty acid utilization provided additional layers of metabolic reprogramming with functional consequences in immune cells. Additional levels of complexity have been uncovered. Altered metabolic pathways in one immune‐mediated disease cannot be automatically expanded to another immune‐mediated disease, as outlined in this review, with CD4+ T cells in SLE and RA at opposite poles of the oxidation spectrum. Metabolic reprogramming is cell type‐specific; for example, pDC activation by type I IFN relying on FAO instead of glycolysis 61, a process that has not been found in other types of DCs. Some metabolic enzymes may regulate immune function through mechanisms only indirectly linked to metabolism, such as GAPDH regulating IFN‐γ production 121. In addition, some metabolites such as succinate or itaconate show previously unsuspected anti‐inflammatory effects 122. Finally, mitochondria emerge as central players in immune responses, not only through the generation of these metabolite intermediates, but also by regulating ATP and ROS production through the fusion and fission processes, providing a platform for retinoic acid‐inducible gene I protein (RIG‐I) and nucleotide‐binding oligomerization domain‐like receptor family, pyrin domain‐containing‐3 (NRLP‐3) inflammasome signaling. Directly related to autoimmunity, mitochondria are a source of highly immunogenic mitochondrial DNA 123. The majority of these ground‐breaking studies have been conducted in normal mice, with only a few in models of autoimmune diseases, and even fewer in patients with autoimmune diseases as reported in this review. There are, however, reasons to believe that research in immunometabolism will have a significant long‐lasting impact in the field of autoimmunity. First, a clearer understanding of the specific nature of metabolic dysregulation in autoimmunity may provide much‐needed novel mechanistic insights into the nature of autoimmune activation. Secondly, these studies may lead to additional therapeutic tools, either alone or in combination with standard of care approaches. The metabolism of immune cells is already targeted by a drug commonly used by rheumatologists, methotrexate, which interferes with 1‐Carbon metabolism 124. Furthermore, mTOR inhibitors and metformin show promising results in SLE 25, 30. It would also be of great interest to explore the metabolic consequences of current treatments used or tested by rheumatologists to have a clearer understanding of the mechanisms by which drugs or biologics modulate the immune system effectively or not through altered metabolism. Preclinical studies guided by the results obtained in normal mice, as well as in‐depth characterization on the metabolic signatures of effector cells in patients with autoimmune diseases, are urgently needed to make significant strides in this direction. Acknowledgements This publication is supported by grants from the NIH (R01AI045050 and R01 AI128901) and from the Alliance for Lupus Research (TIL‐416522) to L. M. Disclosures None. References

Systemic Lupus Erythematosus (SLE) is a complex autoimmune disorder whose pathology involves multiple immune cell types, including B and T lymphocytes as well as myeloid cells. While it is clear that autoantibody producing B cells as well as CD4+ T cell help are key contributors to disease, little is known regarding the role of innate lymphoid cells such as Natural Killer (NK) cells in the pathogenesis of SLE. We have characterized the phenotype of NK cells by multicolor flow cytometry in a large cohort of SLE patients. While the overall percentage of NK cells was similar or slightly decreased compared to healthy controls, a subset of patients displayed a high frequency of NK cells expressing the proliferation marker, Ki‐67, which was not found in healthy donors. Although expression of Ki67 on NK cells correlated with Ki67 on other immune cell subsets, the frequency of Ki67 on NK cells was considerably higher. Increased frequencies of Ki67+ NK cells correlated strongly with clinical severity and active nephritis and was also related to low NK cell numbers but not overall leukopenia. Proteomic and functional data indicate that the cytokine IL‐15 promotes the induction of Ki67 on NK cells. These results suggest a role for NK cells in regulating the immune‐mediated pathology of SLE as well as reveal a possible target for therapeutic intervention. This article is protected by copyright. All rights reserved.

Summary Systemic sclerosis (SSc) is an idiopathic systemic autoimmune disease. It is characterized by a triad of hallmarks: immune dysfunction, fibrosis and vasculopathy. Immune dysfunction in SSc is characterized by the activation and recruitment of immune cells and the production of autoantibodies and cytokines. How immune abnormalities link the fibrosis and vasculopathy in SSc is poorly understood. A plethora of immune cell types are implicated in the immunopathogenesis of SSc, including T cells, B cells, dendritic cells, mast cells and macrophages. How these different cell types interact to contribute to SSc is complicated, and can involve cell‐to‐cell interactions and communication via cytokines, including transforming growth factor (TGF)‐β, interleukin (IL)‐6 and IL‐4. We will attempt to review significant and recent research demonstrating the importance of immune cell regulation in the immunopathogenesis of SSc with a particular focus on fibrosis. Introduction Systemic sclerosis (SSc) is an idiopathic systemic autoimmune disease. The pathogenesis of SSc is mostly unknown. Immunological abnormality, which includes autoimmunity and infiltration and activation of immune cells, is one of three key features of the disease. The other two hallmarks of SSc are fibrosis and vasculopathy. How autoimmunity is linked to fibrosis and vascular damage remains poorly understood, with no single hypothesis or mechanism to explain the links. It is therefore likely that a combination of mechanisms is involved in the pathogenesis of SSc, and picking apart these mechanisms to establish how the triad of fibrosis, vasculopathy and immune cell activation progress in SSc is a mammoth task. Many studies have linked immune abnormality as a cause of or at least a contributor to fibrosis in SSc, while fibrosis may also contribute to immune cell activation. Fibrosis in SSc occurs mainly in the skin but may progress to visceral organs, including heart and lungs. Fibrosis is the result of activation of fibroblasts and excessive extracellular matrix (ECM) deposition, both of which are hallmarks of SSc. As well as fibrosis, immune abnormalities are also linked to vasculopathy in SSc, with most research implicating vascular damage as an activator of immune cells. However, this review will focus mainly on the role of immunological abnormalities in the development of fibrosis in SSc; we recommend the paper by Asano and Sato for a comprehensive review of vasculopathy in SSc 1. Multiple different cell types of the immune system have been implicated in SSc, including T cells, B cells, dendritic cells, mast cells and macrophages. It appears that both innate and adaptive immunity is critical in the disease. We will attempt to review the old and emerging literature to examine the potential roles of these individual cell types in the immunopathogenesis of SSc. First, we will summarize major cytokines implicated in the immunopathogenesis of SSc, because these major players reappear in many studies, linking immune cell activation with other phenotypes in SSc, especially fibrosis. Cytokines in SSc Many cytokines are elevated in SSc, but interleukin (IL)‐4, transforming growth factor (TGF)‐β and IL‐6 are considered to be the major fibrogenic cytokines, or are at least the most studied. Genetic deletion of both IL‐4 and TGF‐β prevents skin fibrosis in a mouse model of SSc 2. Many other in‐vitro studies support the idea that IL‐4 promotes fibrosis through its ability to enhance the production of collagen 3, 4 and other ECM proteins 5, 6 while antibodies against IL‐4 prevent dermal fibrosis in the tight skin (Tsk) mouse model 7 targeted deletion of IL‐4 receptor in the Tsk mouse also reduces fibrosis 2. Skin and lung in SSc have high levels of IL‐4 8 and increased levels of IL‐4 in the blood are a common feature in patients with SSc 9-11 suggesting systemic release. TGF‐β is a well‐known potent inducer of fibrosis, with TGF‐β‐stimulated fibroblasts resembling those from SSc patients 12. Activation of the TGF‐β receptor following the binding of TGF‐β results in the phosphorylation and activation of SMAD proteins in the cytoplasm 13. TGF‐β also activates the three mitogen‐activated protein kinase (MAPK) signalling branches, c‐Jun N‐terminal kinase (JNK), p38 and extracellular signal‐regulated kinases 1 and 2 (ERK1 and 2) 12 all of which can promote inflammatory signalling. TGF‐β‐induced collagen production from both healthy and SSc dermal fibroblasts was found to be dependent on p38 14. JNK activation has also been implicated in fibrosis 15. However, in one study ERK activation inhibited skin fibroblast collagens I and III production while, p38 activation up‐regulated collagen I 16. IL‐6 is a classic proinflammatory cytokine and is also considered to be an important protein in the immunopathogenesis of SSc. For example, IL‐6 levels are increased in SSc patient sera 9 and skin 17. IL‐6 levels also correlate with SSc disease severity 18. A mouse model with development of autoimmune disease with SSc‐like skin thickening and lung fibrosis was found to be mediated by IL‐6 signalling 19. Bleomycin‐induced lung inflammation with collagen deposition was significantly attenuated in IL‐6‐deficient mice 20. IL‐6 signalling through trans‐signalling appears to be important, and we found that IL‐6 and the soluble form of the IL‐6 receptor are necessary for collagen production 21. We further showed in the same study that this was critical, dependent on the downstream signalling molecule signal transducer and activator of transcription (STAT)‐3. A crucial early step hypothesized to trigger the immune abnormalities and fibrosis in SSc is vasculopathy, including the damage and apoptosis of endothelial cells, resulting in the release of internal damage‐associated molecular patterns (DAMPs), which go on to activate and recruit immune cells 22. IL‐6 was found to mediate endothelial activation and apoptosis caused by the serum of patients with SSc 23, suggesting that it may play a major role in the very early stages of SSc. However, IL‐6 was found to be up‐regulated at the late stage of the disease using immunohistological analysis of skin biopsies from SSc patients 17. In both IL‐6 knock‐out (KO) mice and mice exposed to an IL‐6 blocking antibody, bleomycin‐induced dermal fibrosis was greatly induced by supressing fibroblast activation 24. The anti‐IL‐6 receptor antibody tocilizumab has had promising results with softening of the skin in two patients with SSc in one study 25 while a Phase II trial provided SSc patients with improvement in fibrosis of the skin 26 although statistically this was not significant. Thus, IL‐6 antibody therapy could be the first biological licensed for SSc. T cells T cells have been identified early in SSc progression before any evidence of fibrosis 27. SSc skin has a greater propensity to recruit/adhere T cells compared to healthy controls because of a greater expression of intercellular adhesion molecule (ICAM‐1), which is a ligand for the lymphocyte function‐associated antigen 1 (LFA1) receptor found on the surface of lymphocytes such as T cells 28. T cells from SSc skin biopsies have increased expression of the early T cell activation marker CD69 29. TGF‐β, which is elevated in SSc, was also found to be important for the recruitment of T cells to the skin in an SSc mouse model 30. A recent paper demonstrated that abatacept, which is an antibody that interferes with T cell activation, reduced fibrosis in not one, but two animal models of fibrosis 31. This was associated with reduced T cell activation and reduced levels of IL‐6, which may be mediated by blockade of cross‐talk between T cells and antigen‐presenting cells such as monocytes. Abatacept works by blocking the interaction of CD80/86 with cytotoxic T lymphocyte antigen (CTLA)‐4 on T cells, which is required for co‐stimulatory activation of T cells alongside major histocompatibility complex (MHC) and antigen presentation from antigen‐presenting cells such as dendritic cells, macrophages and B cells 32 Abatacept was initiated in three patients with morphea, and all three showed regression of the modified Rodnan skin score 33. T cells can be further classified into diverse subsets characterized by their distinct function, activators and the cytokines they subsequently release. Some of these T cell types have been found to play a role in SSc, which has been reviewed in more detail 34. However, many studies have identified the T helper type 2 (Th2) subtype as playing the largest role in SSc. Th2 cells are a major IL‐4‐producing cell type, but also secrete IL‐5, IL‐6, IL‐10 and IL‐13. Depletion of CD3+ T cells in a bleomycin mouse model reduced both fibrosis and IL‐4 secretion 35. As described above, IL‐4 can directly induce fibroblasts to promote fibrosis, but in addition IL‐4 can induce TGF‐β production in fibroblasts 36 and macrophages 37, which then leads to further signalling to fibroblasts to produce more collagen. Therefore, it is very likely that T cells have a major role to play in the onset of fibrosis in SSc through activation of macrophages and fibroblasts. An unusual population of CD4+CD8+ double‐positive T cells has been described in the skin that has very high levels of IL‐4 38. Gross et al. showed almost 30 years ago that IL‐4 is required to induce naive T cells to produce further IL‐4 39. If IL‐4 is required for T cells to produce IL‐4, this leads to the question: ‘what is the initial source of IL‐4 in SSc?’. This question remains unanswered, but potentially implicates other immune cells in the onset or progression of SSc. IL‐13 is produced predominantly by activated Th2 cells 34. IL‐13 was established as a major profibrotic agent in a model of hepatic fibrosis 40. IL‐13 inhibited IL‐1β‐induced matrix metalloproteinase (MMP)‐1 and MMP‐3 production and enhanced tissue inhibitor of metalloproteinase (TIMP)‐1 and collagen generation in fibroblasts 41. In the bleomycin model of SSc pulmonary fibrosis IL‐13 levels increased with pathogenesis, while neutralization of IL‐13 attenuated bleomycin‐induced pulmonary fibrosis 42. The potent fibrosis‐inducer, TGF‐β, may also contribute to an increase in Th2‐originating IL‐13 because TGF‐β up‐regulates GATA binding protein 3 (GATA‐3) expression in the T cells of patients with SSc, resulting in an increase in IL‐13 synthesis 43. As well as CD4+CD8+ double‐positive T cells, CD8+ single‐positive cells have been described that produce exuberant levels of IL‐13 44. These cells appear to be memory CD8+ T cells 45. We have shown that IL‐13 is directly profibrotic and is STAT‐6‐dependent 46. Thus, IL‐13 produced from T cells probably contributes to fibrosis in SSc and a monoclonal IL‐13 clinical‐grade antibody exists. Very recently, IL‐13 was shown to decrease MMP‐1 expression in both healthy and SSc fibroblasts and therefore may have an anti‐fibrotic role 47. However, this was only demonstrated in tumour necrosis factor (TNF)‐α‐induced fibroblasts, thus the effect of IL‐13 may be specific to the mechanism with which TNF‐α induces MMP‐1 expression. Another cytokine, IL‐17, prevalent in the serum of SSc patients in some studies, also implicates T cells 48. Th17 cells, which are characterized by their production of IL‐17A, IL‐17F, IL‐21 and IL‐22, are elevated in SSc skin compared to healthy controls 49, 50. Thus, Th17 cells and the IL‐17 they produce may play an important role in SSc. However, the roles of Th17 and IL‐17 in SSc are controversial, and have been recently reviewed in detail 51. Importantly, some studies have not detected differences in IL‐17 levels between SSc patients and healthy controls 52, 53 while IL‐17 has been shown to both increase 50 and decrease 54, 55 collagen production. The reasons for the differences in these studies between pro‐ or anti‐fibrotic effects of IL‐17 are not clear, and may reflect the source of the recombinant protein. A reduction in regulatory T cells has been recorded in the skin lesions of patients with SSc 56 suggesting that there is compromised capacity to regulate immune responses in SSc. A recent study identified an imbalance of regulatory T cells and Th17 cells, with a decrease in the former and an increase in the latter in the peripheral blood of SSc patients compared to healthy controls 57. In addition, a more recent study has confirmed that Th2 and Th17 cells are found in higher frequencies in SSc patients, and this was found to positively correlate with IL‐35 levels, although a causal link has not been established 58. Further evidence of disruption to T cell homeostasis comes from a recent study showing that SSc patients displaying severe peripheral vascular complications have an expansion of the recently discovered angiogenic T cells 59. This expansion in angiogenic T cells may also demonstrate a link between immune cell activation and vasculopathy in SSc. Macrophages More than 30 years ago circulating monocytes were found to be strongly activated in patients with SSc 60. Since then, many studies have found evidence of monocyte/macrophage activation in SSc. Higher numbers of macrophages have been observed in skin from SSc patients 27, 61 while cells positive for CD163, a putative marker for M2 macrophages 62, have been found to be increased in the serum of SSc patients 61, 63-65. M2 macrophages are therefore prominent in SSc, with a very recent study involving more than 200 SSc patients, not only confirming this but also demonstrating that soluble CD163 (sCD163) is significantly elevated in the serum of SSc patients and therefore may be of use as a biomarker 66. Microarray gene expression data have also supported a role for monocytes/macrophages in the immunopathogenesis of SSc, with studies demonstrating that peripheral blood mononuclear cells (PBMCs) from SSc patients have an increased expression of genes associated with monocyte/macrophages 67-69. Such large‐scale genomic studies have identified multiple innate immune regulators in SSc. Macrophages are also a major source of TGF‐β 70, which is a potent inducer of fibrosis 71. Many studies have implicated macrophages in the initiation or progression of fibrosis in SSc. Microarray data from SSc lung tissue confirm what has been observed in the blood with markers of macrophage activation 72 and emigration, which correlate with progressive lung fibrosis 73. In another recent study, a novel multi‐network approach to compare gene expression profiles has identified a gene expression signature indicative of profibrotic M2 macrophages in SSc tissues 74. Interestingly, the profibrotic macrophage gene expression profile differed between skin and lung, suggesting that although a role for macrophages in the immunopathogenesis of fibrosis in SSc seems likely in both skin and lung, there may still be subtle differences in what those roles are. Recently nintedanib, an inhibitor of the receptor tyrosine kinase platelet‐derived growth factor receptor, fibroblast growth factor receptor and vascular endothelial growth factor receptor, blocked myofibroblast differentiation and subsequent fibrosis in a mouse model of SSc. Interestingly, these nintedanib‐mediated anti‐fibrotic effects were associated with reduced numbers of M2 macrophages 75. Many chemokines which both recruit and can be primarily secreted by macrophages are up‐regulated in SSc skin 76-79 which is not surprising, given that infiltration of macrophages in SSc skin has been known since the early 1990s 80. However, the chemokine (C‐C motif) ligand 19 (CCL19) was up‐regulated in SSc skin and co‐localized with CD163‐positive macrophages, suggesting that it has a role in the recruitment of macrophages 76. The authors of the study also suggest that macrophages are the source of the CCL19, and in‐vivo experiments demonstrate that Toll‐like receptor (TLR)‐3, ‐4 or ‐9 activation is required for CCL19 expression in monocytes. TLR activation is considered a major event in the immunopathogenesis of SSc 81, 82. Cell types which highly express TLRs such as macrophages are thought to play a major role in SSc. However, many different cell types express TLRs, and thus TLR‐mediated signalling in the immunopathogenesis of SSc may not be limited to macrophages. It is possible that TLR activation in any cell type expressing TLRs is also a source of CCL19, and therefore a potential cause of macrophage recruitment in SSc. Indeed, Mathes et al. also observed that CCL19 expression was up‐regulated from TLR activation in T cells and other cell types from PBMCs, but monocytes gave the most robust increase 76. We have found that macrophages that respond to the TLR‐8 stimulus single‐stranded RNA in SSc result in up‐regulation of TIMP‐1 that is functional and leads to increased collagen deposition 83. Interestingly, these monocytes seemed to be perivascular. IL‐6 appears to play an important part in the role of macrophages in SSc because inhibition of phosphodiesterase‐4, which blocks M2 differentiation, also inhibits IL‐6 production, fibroblast activation and fibrosis in an SSc mouse model 84. IL‐6 may also be an important activator of M2 macrophages, as indicated by IL‐6 receptor blockage by tocilizumab 85 causing down‐regulation of genes associated with M2 macrophages in SSc skin. Oncostatin M is another IL‐6‐like cytokine that appears to be involved in fibrosis, as cells treated with OSM induced ECM accumulation in fibroblasts 86. SSc patients with higher expression of the M2 macrophage marker, MRC1, were also found to have elevated levels of IL‐13 in their plasma 69 suggesting that macrophage activation in SSc results in production of cytokines from macrophages themselves or indirectly through interaction with other immune cells, because macrophages can both secrete IL‐13 87 and, through being antigen‐presenting cells, can activate T cells which could lead to IL‐13 production. There have been more than 30 years of research implicating macrophages in the immunopathogenesis of SSc, with various cytokines produced by macrophages and other immune cells, internal molecules from damaged endothelial cells and chemokines all contributing to the recruitment and activation of macrophages, resulting in a profibrotic environment (Fig. 1). However, how macrophages are recruited to and activated in the tissues of SSc patients is not fully understood. What role is played by macrophages in the progression of fibrosis is also not fully known, but activation of TLRs and the subsequent profibrotic signalling remains an obvious choice. Targeting of TLRs may be a therapeutic option. B cells B cells are heavily implicated in the immunopathogenesis of SSc. B cells are known inducers of fibrosis generally and in SSc 88 and mounting evidence suggests that they are modified and activated in patients with SSc. SSc patients have abnormalities of B cell homeostasis in the blood, which includes an expansion of naive B cells and activated but diminished memory B cells 89. The cytokine B cell activating factor (BAFF), which belongs to the TNF ligand family, is a potent activator of B cells 90. Not only are BAFF levels elevated in SSc; levels also correlate with disease severity, while B cells isolated from SSc patients produce more IL‐6 when exposed to BAFF 91. A BAFF antagonist inhibited IL‐6 and IL‐10 expression in the skin of the Tsk mouse model of SSc, while stimulation of B cells with BAFF greatly increased IL‐6 92. IL‐6 can direct the differentiation of T cells into IL‐4‐producing Th2 cells 93, therefore B cell‐secreted IL‐6 could contribute to the Th2 phenotype detected in SSc. As well as being involved in IL‐6 cytokine production, B cells are also suggested to play an important role in fibrosis. Co‐cultures of B cells and fibroblasts from SSc patients induced a fibrotic response, including collagen, TGF‐β1 and IL‐6 secretion. Exposure of co‐cultures to BAFF (and anti‐IgM) further increased secretion of the profibrotic compounds 88. The majority of B cells are IL‐6‐producing, termed B effector cells (Beffs), and have a proinflammatory and autoimmunity role. However, a small subset of B cells termed regulatory B cells (Bregs) are potent negative regulators of inflammation and autoimmunity, in part through their ability to express IL‐10 94. These Bregs therefore have the potential to inhibit diseases with immune abnormalities such as SSc. Indeed, Bregs were able to inhibit the initiation of a mouse model of multiple sclerosis and depletion of Bregs increased symptom severity 95. Interestingly, the frequency of blood Bregs was found to be significantly lower in SSc patients compared to healthy controls 96 while in a very recent study by Matsushita et al., Bregs were found to have a similar role in a mouse model of SSc. Depletion of IL‐6‐producing Beffs reduced skin and lung fibrosis, while depletion of IL‐10‐producing Bregs caused more severe fibrosis 97 backing up findings from a previous study that Bregs can suppress skin fibrosis in a graft‐versus‐host disease mouse model of SSc 98. A tipping of the balance of Bregs and Beffs in favour of fewer Bregs is therefore a potential important event in the initiation of fibrosis in SSc. However, the cause of this shift in B cell homeostasis remains unknown, and future studies to identify the trigger/s may provide useful early therapeutic targets. Various response regulators on the surface of B cells exist, with some increasing and others decreasing B cell receptor (BCR) signals. Of these, the positive B cell regulator, CD19, is the most established in SSc disease research. CD19 is an important regulatory molecule expressed by B cells. CD19+ B cells increase in SSc, with expression of CD19 on the surface of B cells significantly higher in SSc patients compared to healthy controls 99. CD19‐deficient mice have decreased sera levels of several autoantibodies 100 suggesting that B cells could be responsible for the autoantibodies detected in SSc patients. In agreement, a transgenic mouse model, with an elevation in CD19 expression similar to SSc human patients, shows an increase in the levels of autoantibodies 101, 102. It is also worth noting that CD19 over‐expression has not been observed in the Tsk mouse model, suggesting that a role for CD19 in B cell activation in SSc is not straightforward or that the animal models can only replicate the human disease phenotypes to a degree. However, the apparent lack of CD19 over‐expression may be explained partly through constitutive activation of CD19 and the downstream signalling pathway via up‐regulated CD19 tyrosine phosphorylation, which could negate the need for over‐expression 102. Autoantibody production is prevalent, and can be used diagnostically in SSc. Furthermore, autoantibodies against platelet‐derived growth factor receptor (PDGFR) were found in SSc patients, and caused collagen I‐increased expression and transition of normal human primary fibroblasts to a myofibroblast phenotype through activation of an intracellular loop that involves Ha‐Ras, ERK1/2 and reactive oxygen species (ROS) 103. However, it is still possible that B cell activation is the cause of both autoantibody production and fibrosis, as the two may be linked. Interestingly, B cell activation via CD19 specifically may also be a cause of other immune cell type infiltration in SSc. CD19‐deficient mice showed less infiltration of mast cells, macrophages, T cells and B cells after bleomycin treatment compared to wild‐type cells exposed to bleomycin 104. In the same study, it was discovered that B cells, via hyaluronan‐induced TLR‐4 activation, produced various cytokines which were dramatically reduced in CD19‐deficient cells. Therefore, B cells have the potential to contribute to fibrosis directly as well as indirectly through recruitment and activation of other immune cells to promote a cycle of cytokine and fibrogenic compound release. Mast cells Mast cell infiltration was detected from lip biopsy in very early stages of SSc and preceded the onset of skin changes 105 and more recently cutaneous mastocytosis was found to precede the onset of SSc in a 36‐year‐old woman 106 suggesting that mast cells may play an early role in the onset of SSc. Mast cell infiltration has also been detected late in the development of SSc 107 and in a subset of SSc patients with localized scleroderma‐like lesions 108. An increase in mast cell numbers has also been detected in SSc skin 109. Mast cells are able to produce TGF‐β and are found localized to fibroblasts in the skin of SSc patients 110. Interestingly, mast cells are not only a source of TGF‐β; they can also directly transfer TGF‐β to fibroblasts mediated by transgranulation via cell–cell contacts 111, which has also been observed in the dermis of SSc patients 112. The triggers of mast infiltration in SSc or other fibrotic diseases are not known, but a very recent study has identified Snail‐dependent production of plasminogen activator inhibitor‐1 (PAI1) in keratinocytes as a chemoattractant of mast cells, a mediator of mast cell–fibroblast adhesion and a promoter of fibrogenesis 113. Interestingly, PAI1 up‐regulated tenascin‐C (TENC) expression, which is the main activator of TLR‐4 to promote fibrosis and found to be elevated in SSc 114. Therefore, the role of PAI1 in SSc could involve mast cell recruitment and activation of fibroblasts to promote an inflammatory environment. Dendritic cells Two major types of dendritic cell are known: conventional (cDC) and plasmacytoid (pDC). pDC are a specialized cell type that when activated produce large amounts of interferon (IFN). A very recent study observed that pDCs infiltrate the skin of SSc patients and are chronically activated, producing IFN‐α and chemokine (C‐X‐C motif) ligand 4 (CXCL4). Fibrosis was reverted in a mouse model of SSc after pDCs depletion 115. In the same study, TLR‐8 signalling was found to be important for CXCL4 production and recruitment of pDCs to fibrotic skin. TLR involvement has also been found to be important for mediating the increased cytokine production in DCs in SSc, which has been comprehensively reviewed 116. pDCs isolated from SSc patients show up‐regulation of CXCL4 115, 117, which can also be detected at elevated levels and correlates with disease severity in SSc patient plasma 118 and has been suggested as a biomarker in SSc. In another recent study, pDC depletion in a bleomycin mouse model improved the clinical score, lung histopathology score, skin thickness and collagen content. The expression of genes involved in chemotaxis, dendritic cell differentiation, inflammation and fibrosis in the lungs of pDC‐depleted mice was also significantly reduced. Alongside this, B and T cells were also found to be reduced in the lungs, suggesting an important role for ongoing immune abnormalities induced by DCs 119. In another bleomycin‐induced pulmonary fibrosis mouse model, DC accumulation in the lung was reduced through blocking of TGF‐β with the use of inhibitor SB431542 120. TGF‐β was also found to be important for the recruitment of pDCs (and T cells) to the skin in an SSc mouse model 30 suggesting an important role for TGF‐β in DC location and activation. An important role for microRNAs in the pathogenesis SSc is emerging 121. Recently, microRNA expression profiling has identified miRNA‐618 (miR‐618) as being up‐regulated in pDCs from SSc patients, while over‐expression in healthy pDCs resulted in an SSc‐like pDC 122. Thus, microRNAs are disturbed, leading to altered functionality of immune cells. Alterations of microRNAs by restoration using microRNA therapeutics may be one way to reset dendritic cells to a ‘tolerogenic’ phenotype. Anti‐nuclear antibodies, which are a subset of autoantibodies, are found to be elevated in SSc patients 123. DNA topoisomerase I (TopoI) is an intracellular target of anti‐nuclear antibody anti‐TopoI. The presence of anti‐TopoI antibodies has been clinically associated with a more severe form of SSc 124. Immunization of mice with TopoI peptide‐loaded DCs induces anti‐TopoI autoantibody response and long‐term fibrosis of skin and lung, which is preceded by inflammation with increased IL‐17A and CXCL4 expression 125. TopoI peptide‐loaded DCs also induce proliferation of SSc and healthy‐derived T cells, but activation of T cells requires a fragmented form of TopoI and activation of full‐length TopoI is IL‐2‐dependent 126. Interestingly, antigen‐presenting cells within PBMCs were able to activate T cells more efficiently than dendritic cells, and even processed TopoI peptides differently, suggesting that there is a complex regulation of T cells by dendritic cells and other antigen‐presenting cells with regard to anti‐nuclear antibodies. Overall, experimental manipulation of DCS can mimic some major events in the pathogenesis of SSc, and therefore raises the intriguing prospect that DCs may be capable of initiating SSc. A broken loop that may perpetuate inflammation with cytokine‐mediated up‐regulation of co‐stimulatory molecules may also exist in SSc. Other immune cells Dysregulation and activation of immune cells is clearly a major hallmark of SSc involving many cell types. This complex network of signalling between different immune cells is only now beginning to become unravelled, with new evidence emerging regularly to provide new players in SSc immunopathogenesis. For example, only a few studies have highlighted the potential for platelets 127-129, neutrophils 130, 131, natural killer cells 132, 133 and innate lymphoid cells 134, 135 to be dysregulated and contribute to the pathogenesis of SSc. Type 2 innate lymphoid cells are found in higher numbers in SSc and correlate strongly with the Rodnan skin score and also the presence of lung fibrosis. Although not considered to be innate immune cells, fibroblasts play a crucial role in the fibrotic phenotype of SSc, but they also express TLRs and are therefore capable of responding to DAMPs. Expression of TLR‐2 was found to be increased in fibroblasts from SSc skin 136. Recently, fibroblasts exposed to TGF‐β and IL‐17A responded with a 100‐fold increase in the production of IL‐6 137, a known profibrotic mediator. Conclusion Various cell types of the immune system appear to be involved to some degree in the immunopathogenesis of SSc through promoting other immune cell activation, fibrosis or vascular damage. Very recent studies have highlighted the importance of this plethora of immune cell types in SSc (Table 1). However, research to date provides evidence for macrophages, T cells and B cells having the most important role in SSc. Whether this accurately represents their importance in SSc or is a result of research interests combined with the practicality of investigating specific cell types remains to be seen. Nevertheless, immune cell activation is a recurring observation in SSc research and is leading to the development and testing of therapeutic interventions. Although the over‐production of cytokines is a well‐accepted symptom of SSc, TGF‐β, IL‐6 and IL‐4 specifically seem to be integral to many of the immune abnormalities recorded. Thoroughly understanding how these and other cytokines fully regulate, and are regulated by, all the different immune cells and their subcategories in SSc will be a difficult task, but every study into immune cell activation, regardless of cell type, brings us closer to achieving it and as a result developing therapeutic interventions. Current trials such as the Fassinate trial in SSc blocking IL‐6 seem promising, with the Phase II trial positive, although the primary end‐point was not reached. Phosphodiesterase type 4 (PDE4) inhibitors appear to regulate macrophages leading to reduced fibroblast activation. There is an ongoing trial concerning morphea. Immune cell Recent findings Reference T cell Angiogenic T cells were elevated in SSc patients displaying digital ulcers, which is a severe peripheral vascular complication 59 Macrophage The soluble form of the M2 macrophage marker CD163 is elevated in the serum of patients with SSc, highlighting it as a potential biomarker 66 B cell Used a mouse model of SSc to demonstrate the importance of B cell homeostasis. Depletion of IL‐6‐producing B effector cells reduced fibrosis while depleting of IL‐10‐producing B regulatory cells had the opposite effect 97 Mast cell A subset of SSc patients with localized scleroderma‐like lesions were found to have an inflammatory phenotype leading to the activation of mast cells in the dermis of mechanically stressed skin 108 Dendritic cell After depletion of pDCs fibrosis was reverted in mice with established SSc‐like disease. pDC depletion prior to induction of disease also prevented fibrosis 115 Platelets Activated platelets induced the production of the profibrotic mediator thymic stromal lymphopoietin (TSLP) in human dermal endothelial cells 128 Neutrophils Neutrophil activation was induced by SSc microparticles. Microparticles were derived from platelets and expressed the damage‐associated molecular pattern HMGB1. An inhibitor of HMGB1 attenuated neutrophil activation 131 Natural killer A peculiar natural killer cell phenotype in SSc patients was identified characterized by decreased chemokine and activation receptors expression. These SSc‐derived natural killer cells were potent inducers of endothelial microparticle release suggesting that there may be a role for natural killer cells in the activation of endothelial cells in SSc 133 Innate lymphoid Type 2 innate lymphoid (ILC2) cells are elevated in patients with SSc. ILC2 counts correlated with skin fibrosis. This study highlights that there may be a profibrotic role for ILC2 cells in SSc 134 Fibroblast Although not an immune cell type, fibroblasts exposed to TGF‐β and IL‐17a responded with a 100‐fold increase in the production of IL‐6 137 DCs = dendritic cells; IL = interleukin; TGF = transforming growth factor; HMGB1 = high mobility group box 1. Disclosures None. References

Abstract
Introduction: Multiple pathways are involved in the pathogenesis of systemic lupus erythematosus (SLE). The Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway mediates the intracellular signals of more than 60 cytokines, growth factors and hormones from the type I/II cytokine receptors. Dysregulation of the cytokines is a hallmark of SLE; inhibition of downstream signaling mediated by the JAKs is an attractive therapeutic option.

Areas covered: This article reviews the preliminary data concerning the efficacy of the JAK inhibitors (Jakinibs) in SLE. JAK inhibition has shown promise in murine lupus dermatitis and nephritis. Ex-vivo studies of human SLE have demonstrated the effect of JAK1/2 inhibition on the activation of the STAT proteins and autoantibody production from B cells. A phase II trial reported modest efficacy of baricitinib in improving synovitis in SLE patients.

Expert opinions: Inhibition of the JAK-STAT pathway is an attractive therapeutic option. The convenience of oral administration and lower production cost of the Jakinibs could replace the biological agents in the treatment hierarchy of autoimmune inflammatory diseases. Additional clinical data are needed; results of ongoing studies of the newer Jakinibs in cutaneous and non-life-threatening lupus are eagerly awaited.

Keywords: JAK, Jakinib, lupus, targeted, therapeutic, systemic lupus, SLE, autoimmune disease