Editing the immune system in vivo in mice using CRISPR/Cas9 ribonucleoprotein (RNP)-mediated gene editing of transplanted hematopoietic stem cells.

CRISPR/Cas9-based genome editing has been widely used to evaluate target gene function in biomedical research. The CRISPR/Cas9 system can introduce gene knockout, knock-in and mutations with more ease than earlier generations of genome editing tools. Using CRISPR/Cas9-based genome editing, researchers have successfully modified the DNA of different immune components, including primary T cells, B cells, macrophages, and immune system progenitors, i.e. hematopoietic stem cells (HSCs), which are also known as Lin-Sca1 + Kit + cells (LSKs) in mice. We previously reported that the transplantation of HSCs with lentivirus-mediated CRISPR/Cas9-based genetic modifications into lethally irradiated congenic mice repopulated the ablated recipient immune system with the donor immune system. In this report, we efficiently manipulated CD40 expression in LSK cells using Cas9 RNP and demonstrated the functional impact in a colitis model. Compared to a virus-based strategy, the RNP approach has the potential to enable investigation of target gene biology in any mouse strain and eliminates the time and effort associated with virus production and infection. Therefore, in vivo RNP-based CRISPR/Cas9 gene editing of transplanted HSCs represents a promising new strategy for exploring gene function in the immune system of mice.

Molecular taxonomy of osteoarthritis for patient stratification, disease management and drug development: biochemical markers associated with emerging clinical phenotypes and molecular endotypes.

PURPOSE OF REVIEW: This review focuses on the molecular taxonomy of osteoarthritis from the perspective of molecular biomarkers. We discuss how wet biochemical markers may be used to understand disease pathogenesis and progression and define molecular endotypes of osteoarthritis and how these correspond to clinical phenotypes. RECENT FINDINGS: Emerging evidence suggests that osteoarthritis is a heterogeneous and multifaceted disease with multiple causes, molecular endotypes and corresponding clinical phenotypes. Biomarkers may be employed as tools for patient stratification in clinical trials, enhanced disease management in the primary care centres of the future and for directing more rational and targeted osteoarthritis drug development. Proximal molecular biomarkers (e.g synovial fluid) are more likely to distinguish between molecular endotypes because there is less interference from systemic sources of biomarker noise, including comorbidities. SUMMARY: In this review, we have focused on the molecular biomarkers of four distinct osteoarthritis subtypes including inflammatory, subchondral bone remodelling, metabolic syndrome and senescent age-related endotypes, which have corresponding phenotypes. Progress in the field of osteoarthritis endotype and phenotype research requires a better understanding of molecular biomarkers that may be used in conjunction with imaging, pain and functional assessments for the design of more effective, stratified and individualized osteoarthritis treatments.

Phase IIa, placebo-controlled, randomised study of lutikizumab, an anti-interleukin-1α and anti-interleukin-1ß dual variable domain immunoglobulin, in patients with erosive hand osteoarthritis.

OBJECTIVE: To assess the efficacy, safety, pharmacokinetics and pharmacodynamics of the anti-interleukin (IL)-1α/ß dual variable domain immunoglobulin lutikizumab (ABT-981) in erosive hand osteoarthritis (HOA). METHODS: Patients with ≥1 erosive and ≥3 tender and/or swollen hand joints were randomised to placebo or lutikizumab 200 mg subcutaneously every 2 weeks for 24 weeks. The primary endpoint was change in Australian/Canadian Osteoarthritis Hand Index (AUSCAN) pain subdomain score from baseline to 16 weeks. At baseline and week 26, subjects had bilateral hand radiographs and MRI of the hand with the greatest number of baseline tender and/or swollen joints. Continuous endpoints were assessed using analysis of covariance models, with treatment and country as main factors and baseline measurements as covariates. RESULTS: Of 132 randomised subjects, 1 received no study drug and 110 completed the study (placebo, 61/67 (91%); lutikizumab, 49/64 (77%)). AUSCAN pain was not different among subjects treated with lutikizumab versus placebo at week 16 (least squares mean difference, 1.5 (95% CI -1.9 to 5.0)). Other clinical and imaging endpoints were not different between lutikizumab and placebo. Lutikizumab significantly decreased serum high-sensitivity C reactive protein levels, IL-1α and IL-1ß levels, and blood neutrophils. Lutikizumab pharmacokinetics were consistent with phase I studies and not affected by antidrug antibodies. Injection site reactions and neutropaenia were more common in the lutikizumab group; discontinuations because of adverse events occurred more frequently with lutikizumab (4/64) versus placebo (1/67). CONCLUSION: Despite adequate blockade of IL-1, lutikizumab did not improve pain or imaging outcomes in erosive HOA compared with placebo.

Autoantibody levels in myositis patients correlate with clinical response during B cell depletion with rituximab.

OBJECTIVES: To determine the longitudinal trends in serum levels of four myositis-associated autoantibodies: anti-Jo-1, -transcription intermediary factor 1 γ (TIF1-γ), -signal recognition particle (SRP) and -Mi-2, after B cell depletion with rituximab, and to determine the longitudinal association of these autoantibody levels with disease activity as measured by myositis core-set measures (CSMs). METHODS: Treatment-resistant adult and pediatric myositis subjects (n = 200) received rituximab in the 44-week Rituximab in Myositis Trial. CSMs [muscle enzymes, manual muscle testing (MMT), physician and patient global disease activity, HAQ, and extramuscular disease activity] were evaluated monthly and anti-Jo-1 (n = 28), -TIF1-γ (n = 23), -SRP (n = 25) and -Mi-2 (n = 26) serum levels were measured using validated quantitative ELISAs. Temporal trends and the longitudinal relationship between myositis-associated autoantibodies levels and CSM were estimated using linear mixed models. RESULTS: Following rituximab, anti-Jo-1 levels decreased over time (P < 0.001) and strongly correlated with all CSMs (P < 0.008). Anti-TIF1-γ levels also decreased over time (P < 0.001) and were only associated with HAQ, MMT and physician and patient global disease activity. Anti-SRP levels did not change significantly over time, but were significantly associated with serum muscle enzymes. Anti-Mi-2 levels significantly decreased over time and were associated with muscle enzymes, MMT and the physician global score. CONCLUSION: Anti-Jo-1, anti-TIF1-γ and anti-Mi-2 levels in myositis subjects decreased after B cell depletion and were correlated with changes in disease activity, whereas anti-SRP levels were only associated with longitudinal muscle enzyme levels. The strong association of anti-Jo-1 levels with clinical outcomes suggests that anti-Jo-1 autoantibodies may be a good biomarker for disease activity.

Complement component C5a permits the coexistence of pathogenic Th17 cells and type I IFN in lupus.

Systemic lupus erythematosus (SLE) is a type I IFN (IFN-I)-driven autoimmune disorder with exaggerated B and Th cell responses. Th17 cells, a recently identified Th cell subset, have been strongly implicated in the pathogenesis of SLE. Because IFN-I suppresses the generation and expansion of Th17 cells in an IL-27-dependent manner, it is unclear how pathogenic Th17 cells are generated in SLE in the presence of an environment characterized by high IFN-I levels. In this study, we showed that activation of c5aR on murine macrophages blocked IFN-I-mediated IL-27 production, thus permitting the development of Th17 cells. C5aR activation on IFN-I-responsive macrophages inhibits IRF-1-mediated transactivation of IL-27 gene expression via the PI3K/Akt pathway. Consistently, C5aR-deficient mice exhibited increased IL-27 expression and fewer Th17 cells and consequently developed reduced lupus nephritis in comparison with wild-type mice. In support of these findings in mice, we found that C5a inhibited IFN-I-induced IL-27 production from macrophages of lupus subjects. Moreover, the level of serum C5a correlated with Th17 frequency in peripheral blood. Collectively, these data indicate an essential role for C5a in the generation of pathogenic Th17 responses in SLE. Thus, therapeutic strategies to block C5aR activation may be beneficial for controlling pathogenic Th17-mediated inflammation in SLE.

Agreement of Physicians and Nurses Performing Tender and Swollen Joint Counts in Rheumatoid Arthritis.

OBJECTIVE: The aims of this study were to assess the agreement of physicians and nurses performing tender and swollen joint counts (TJCs/SJCs) in rheumatoid arthritis (RA) and identify factors that might influence their examinations including patient age, sex, race, RA disease duration, body mass index, RA disease activity level, comorbid fibromyalgia, comorbid osteoarthritis, and levels of acute-phase reactants. METHODS: Seventy-two RA participants underwent TJCs/SJCs of 28 joints using a standardized protocol by 2 nurses and 2 rheumatologists. Demographic, laboratory, radiographic, and clinical data were obtained to assess the influence of these factors on TJCs/SJCs. Intraclass correlations (ICCs) among examiners were determined for TJCs/SJCs. Nurse-physician differences and agreement of individual joints were evaluated using Cohen κ. Analysis of variance was performed to detect differences in means between examiners for TJCs/SJCs. Intraclass correlation and Fisher Z tests were used to identify factors influencing TJCs/SJCs. RESULTS: Agreement was strong among these nurses and physicians for total TJCs/SJCs (ICC = 0.84/ICC = 0.79, respectively). κ was best for hand joint tenderness and poorest for shoulder swelling. Some significant differences in mean TJCs/SJCs were found between examiners. Fibromyalgia significantly reduced agreement of both TJCs and SJCs. Agreement of TJC was significantly reduced when patients had lower disease activity, greater work impairment, lower mental health quality of life, and elevated erythrocyte sedimentation rate, whereas female sex, assessor's perception of but not radiographic hand osteoarthritis, and elevated C-reactive protein significantly reduced agreement for SJC. CONCLUSIONS: Strong agreement was found among nurses and physicians for total 28-joint counts, with agreement at individual joints being stronger for tenderness than swelling. Fibromyalgia significantly reduced ICCs of TJCs/SJCs.

Anti-signal recognition particle autoantibody ELISA validation and clinical associations.

OBJECTIVE: The aim of this study was to develop and validate a quantitative anti-signal recognition particle (SRP) autoantibody serum ELISA in patients with myositis and longitudinal association with myositis disease activity. METHODS: We developed a serum ELISA using recombinant purified full-length human SRP coated on ELISA plates and a secondary antibody that bound human IgG to detect anti-SRP binding. Protein immunoprecipitation was used as the gold standard for the presence of anti-SRP. Serum samples from three groups were analysed: SRP(+) myositis subjects by immunoprecipitation, SRP(-) myositis subjects by immunoprecipitation and non-myositis controls. The ELISA's sensitivity, specificity, positive predictive value and negative predictive value were evaluated. Percentage agreement and test-retest reliability were assessed. Serial samples from seven SRP immunoprecipitation-positive subjects were also tested, along with serum muscle enzymes and manual muscle testing. RESULTS: Using immunoprecipitation, we identified 26 SRP(+) myositis patients and 77 SRP(-) controls (including 38 patients with necrotizing myopathy). Non-myositis control patients included SLE (n = 4) and SSc (n = 7) patients. Anti-SRP positivity by ELISA showed strong agreement (97.1%) with immunoprecipitation (κ = 0.94). The sensitivity, specificity, positive predictive value, and negative predictive value of the anti-SRP ELISA were 88, 100, 100 and 96, respectively. The area under the curve was 0.94, and test-retest reliability was strong (r = 0.91, P < 0.001). Serial samples showed that anti-SRP levels paralleled changes in muscle enzymes and manual muscle testing. CONCLUSION: We developed a quantitative ELISA for detecting serum anti-SRP autoantibodies and validated the assay in myositis. Longitudinal assessment of SRP levels by ELISA may be a useful biomarker for disease activity.

Interferon-regulated chemokine score associated with improvement in disease activity in refractory myositis patients treated with rituximab.

OBJECTIVES: The purpose of this study was to investigate whether serum interferon (IFN)-regulated chemokine and distinct cytokine response profiles are associated with clinical improvement in patients with refractory inflammatory myopathy treated with rituximab. METHODS: In a randomised, placebo-phase trial Rituximab in Myositis Trial (RIM), 200 refractory adult and paediatric myositis subjects received rituximab. Following rituximab, clinical response and disease activity were assessed. Serum samples and clinical data were collected at baseline and several time-points after rituximab treatment. Multiplexed sandwich immunoassays quantified serum levels of IFN-regulated chemokines and other pro-inflammatory cytokines. Composite IFN-regulated chemokine and Th1, Th2, Th17 and regulatory cytokine scores were computed. RESULTS: Baseline IFN-regulated chemokine, Th1, Th2, Th17 and regulatory cytokine scores correlated with baseline physician global VAS, whereas the baseline Th1, Th2 and Th17 cytokine scores correlated with baseline muscle VAS. We also found baseline IFN-regulated chemokine scores correlated with specific non-muscular targets such as baseline cutaneous (r=0.29; p=0.002) and pulmonary (r=0.18; p=0.02) VAS scores. Among all cytokine/chemokines examined, the baseline score of IFN-regulated chemokines demonstrated the best correlation with changes in muscle VAS at 8 (r=-0.19; p=0.01) and 16 weeks (r=-0.17; p=0.03) following rituximab and physician global VAS at 16 weeks (r=-0.16; p=0.04). In vitro experiments showed increased levels of IL-8 (p=0.04), MCP-1 (p=0.04), IL-6 (p=0.03), IL-1ß (p=0.04), IL-13 (p=0.04), IL-10 (p=0.02), IL-2 (p=0.04) and IFN-γ (p=0.02) in supernatants of TLR-3 stimulated PBMCs from non-responder compared to patients responders to rituximab. CONCLUSIONS: IFN-regulated chemokines before treatment is associated with improvement in disease activity measures in refractory myositis patients treated with rituximab.

Plasma B lymphocyte stimulator and B cell differentiation in idiopathic pulmonary fibrosis patients.

We hypothesized B cells are involved in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a progressive, restrictive lung disease that is refractory to glucocorticoids and other nonspecific therapies, and almost invariably lethal. Accordingly, we sought to identify clinically associated B cell-related abnormalities in these patients. Phenotypes of circulating B cells were characterized by flow cytometry. Intrapulmonary processes were evaluated by immunohistochemistry. Plasma B lymphocyte stimulating factor (BLyS) was assayed by ELISA. Circulating B cells of IPF subjects were more Ag differentiated, with greater plasmablast proportions (3.1 ± 0.8%) than in normal controls (1.3 ± 0.3%) (p < 0.03), and the extent of this differentiation correlated with IPF patient lung volumes (r = 0.44, p < 0.03). CD20(+) B cell aggregates, diffuse parenchymal and perivascular immune complexes, and complement depositions were all prevalent in IPF lungs, but much less prominent or absent in normal lungs. Plasma concentrations of BLyS, an obligate factor for B cell survival and differentiation, were significantly greater (p < 0.0001) in 110 IPF (2.05 ± 0.05 ng/ml) than among 53 normal (1.40 ± 0.04 ng/ml) and 90 chronic obstructive pulmonary disease subjects (1.59 ± 0.05 ng/ml). BLyS levels were uniquely correlated among IPF patients with pulmonary artery pressures (r = 0.58, p < 0.0001). The 25% of IPF subjects with the greatest BLyS values also had diminished 1-y survival (46 ± 11%), compared with those with lesser BLyS concentrations (81 ± 5%) (hazard ratio = 4.0, 95% confidence interval = 1.8-8.7, p = 0.0002). Abnormalities of B cells and BLyS are common in IPF patients, and highly associated with disease manifestations and patient outcomes. These findings have implications regarding IPF pathogenesis and illuminate the potential for novel treatment regimens that specifically target B cells in patients with this lung disease.

Biologic predictors of clinical improvement in rituximab-treated refractory myositis.

BACKGROUND: To examine the longitudinal utility of a biomarker signature in conjunction with myositis autoantibodies (autoAbs) as predictors of disease improvement in refractory myositis patients treated with rituximab. METHODS: In the RIM Trial, all subjects received rituximab on 2 consecutive weeks. Using start of treatment as baseline, serum samples (n = 177) were analyzed at baseline and after rituximab with multiplexed sandwich immunoassays to quantify type-1 IFN-regulated and other pro-inflammatory chemokines and cytokines. Biomarker scores were generated for the following pathways: type-1 IFN-inducible (IFNCK), innate, Th1, Th2, Th17 and regulatory cytokines. Myositis autoAbs (anti-synthetase n = 28, TIF-γ n = 19, Mi-2 n = 25, SRP n = 21, MJ n = 18, non-MAA n = 24, unidentified autoantibody n = 9, and no autoantibodies n = 33) determined by immunoprecipitation at baseline, were correlated with outcome measures. Kruskal-Wallis rank sum tests were used for comparisons. RESULTS: The mean (SD) values for muscle disease and physician global disease activity VAS scores (0-100 mm) were 46 (22) and 49 (19). IFNCK scores (median values) were higher at baseline in subjects with anti-synthetase (43), TIF1-γ (31) and Mi-2 (30) compared with other autoAb groups (p < 0.001). At 16 weeks after rituximab, anti-synthetase and Mi-2 autoAb positive subjects and non-MAA had a greater improvement in IFNCK scores (- 6.7, - 6.1 and -7.2, p < .001). Both IFNCK high scores (>30) and autoAb group (Mi-2, non-MAA, and undefined autoantibody) demonstrated the greatest clinical improvement based on muscle VAS (muscle-interaction p = 0.075). CONCLUSION: Biomarker signatures in conjunction with autoAbs help predict response to rituximab in refractory myositis. Biomarker and clinical responses are greatest at 16 weeks after rituximab.

Anti-transcription intermediary factor 1-gamma autoantibody ELISA development and validation.

OBJECTIVES: A quantitative anti-transcription intermediary factor 1-gamma (anti-TIF1-γ) ELISA may improve the detection of cancer-associated myositis (CAM). The aims of this study were the development and validation of a quantitative anti-TIF1-γ autoantibody ELISA in patients with myositis. METHODS: We developed an ELISA using recombinant purified full-length human TIF1-γ. Patient serum was incubated with TIF1-γ-coated ELISA plates, and secondary antibody that bound human IgG was used to detect anti-TIF1-γ binding. Protein immunoprecipitation (IP) was used as the gold standard for the presence of anti-TIF1-γ. Serum samples from myositis patients with positive and negative anti-TIF1-γ by IP, from non-myositis autoimmune patients (SSc, SLE and RA) and from healthy controls were analysed. The ELISA's sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were evaluated. Agreement between the ELISA and IP results was determined using chi-squared and kappa tests. Test-retest reliability of the ELISA was assessed. RESULTS: We identified 55 myositis patients with and 111 controls without anti-TIF1-γ by IP. Anti-TIF1-γ positivity by ELISA showed strong agreement (93.9%) with IP results (κ = 0.87). The sensitivity, specificity, PPV, NPV and overall accuracy of the anti-TIF1-γ ELISA were 91%, 96%, 93%, 95% and 94%, respectively. The area under the curve (AUC) of a receiver operating characteristic (ROC) curve was 0.938. Test-retest reliability was strong (Pearson r = 0.913, P < 0.001). CONCLUSION: We developed a quantitative ELISA for detecting serum anti-TIF1-γ autoantibodies and validated the assay in myositis and other connective tissue disease patients. The availability of a validated, quantitative ELISA should improve the detection of anti-TIF1-γ autoantibodies and may improve the detection of CAM.

Rituximab therapy for primary Sjögren's syndrome: an open-label clinical trial and mechanistic analysis.

OBJECTIVE: To study the safety and clinical efficacy of rituximab therapy for primary Sjögren's syndrome, as well as to investigate its mechanisms. METHODS: Patients with primary Sjögren's syndrome were enrolled in an open-label trial, were given rituximab (1 gm) infusions on days 1 and 15, and were monitored through week 52. The primary end point was safety, with secondary end points evaluating clinical and biologic efficacy. Blood was obtained for enumeration of lymphocyte subsets, measurement of serum autoantibody and BAFF levels, and analysis of gene expression. RESULTS: Twelve female patients with primary Sjögren's syndrome were administered rituximab. They had a median age of 51 years (range 34-69 years) and a median disease duration of 8.0 years (range 2-18 years). We observed no unexpected toxicities from the rituximab therapy. Modest improvements were observed at week 26 in patient-reported symptoms of fatigue and oral dryness, with no significant improvement in the objective measures of lacrimal and salivary gland function. The recovery of blood B cells following the nadir from rituximab therapy was characterized by a predominance of transitional B cells and a lack of memory B cells. While blood B cell depletion was associated with an increase in serum BAFF levels, no significant changes were observed in the levels of serum anti-Ro/SSA, anti-La/SSB, and anti-type 3 muscarinic acetylcholine receptor autoantibodies or in the blood interferon signature. CONCLUSION: In patients with primary Sjögren's syndrome, a single treatment course of rituximab was not associated with any unexpected toxicities and led to only modest clinical benefits despite effective depletion of blood B cells.

Rituximab in the treatment of refractory adult and juvenile dermatomyositis and adult polymyositis: a randomized, placebo-phase trial.

OBJECTIVE: To assess the safety and efficacy of rituximab in a randomized, double-blind, placebo-phase trial in adult and pediatric myositis patients. METHODS: Adults with refractory polymyositis (PM) and adults and children with refractory dermatomyositis (DM) were enrolled. Entry criteria included muscle weakness and ≥2 additional abnormal values on core set measures (CSMs) for adults. Juvenile DM patients required ≥3 abnormal CSMs, with or without muscle weakness. Patients were randomized to receive either rituximab early or rituximab late, and glucocorticoid or immunosuppressive therapy was allowed at study entry. The primary end point compared the time to achieve the International Myositis Assessment and Clinical Studies Group preliminary definition of improvement (DOI) between the 2 groups. The secondary end points were the time to achieve ≥20% improvement in muscle strength and the proportions of patients in the early and late rituximab groups achieving the DOI at week 8. RESULTS: Among 200 randomized patients (76 with PM, 76 with DM, and 48 with juvenile DM), 195 showed no difference in the time to achieving the DOI between the rituximab late (n = 102) and rituximab early (n = 93) groups (P = 0.74 by log rank test), with a median time to achieving a DOI of 20.2 weeks and 20.0 weeks, respectively. The secondary end points also did not significantly differ between the 2 treatment groups. However, 161 (83%) of the randomized patients met the DOI, and individual CSMs improved in both groups throughout the 44-week trial. CONCLUSION: Although there were no significant differences in the 2 treatment arms for the primary and secondary end points, 83% of adult and juvenile myositis patients with refractory disease met the DOI. The role of B cell-depleting therapies in myositis warrants further study, with consideration for a different trial design.

Patients with idiopathic pulmonary fibrosis with antibodies to heat shock protein 70 have poor prognoses.

RATIONALE: Diverse autoantibodies are present in most patients with idiopathic pulmonary fibrosis (IPF). We hypothesized that specific autoantibodies may associate with IPF manifestations. OBJECTIVES: To identify clinically relevant, antigen-specific immune responses in patients with IPF. METHODS: Autoantibodies were detected by immunoblots and ELISA. Intrapulmonary immune processes were evaluated by immunohistochemistry. Anti-heat shock protein 70 (HSP70) IgG was isolated from plasma by immunoaffinity. Flow cytometry was used for leukocyte functional studies. MEASUREMENTS AND MAIN RESULTS: HSP70 was identified as a potential IPF autoantigen in discovery assays. Anti-HSP70 IgG autoantibodies were detected by immunoblots in 3% of 60 control subjects versus 25% of a cross-sectional IPF cohort (n = 122) (P = 0.0004), one-half the patients with IPF who died (P = 0.008), and 70% of those with acute exacerbations (P = 0.0005). Anti-HSP70 autoantibodies in patients with IPF were significantly associated with HLA allele biases, greater subsequent FVC reductions (P = 0.0004), and lesser 1-year survival (40 ± 10% vs. 80 ± 5%; hazard ratio = 4.2; 95% confidence interval, 2.0-8.6; P < 0.0001). HSP70 protein, antigen-antibody complexes, and complement were prevalent in IPF lungs. HSP70 protein was an autoantigen for IPF CD4 T cells, inducing lymphocyte proliferation (P = 0.004) and IL-4 production (P = 0.01). IPF anti-HSP70 autoantibodies activated monocytes (P = 0.009) and increased monocyte IL-8 production (P = 0.049). ELISA confirmed the association between anti-HSP70 autoreactivity and IPF outcome. Anti-HSP70 autoantibodies were also found in patients with other interstitial lung diseases but were not associated with their clinical progression. CONCLUSIONS: Patients with IPF with anti-HSP70 autoantibodies have more near-term lung function deterioration and mortality. These findings suggest antigen-specific immunoassays could provide useful clinical information in individual patients with IPF and may have implications for understanding IPF progression.

IL11-mediated stromal cell activation may not be the master regulator of pro-fibrotic signaling downstream of TGFß.

Fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF) and systemic scleroderma (SSc), are commonly associated with high morbidity and mortality, thereby representing a significant unmet medical need. Interleukin 11 (IL11)-mediated cell activation has been identified as a central mechanism for promoting fibrosis downstream of TGFß. IL11 signaling has recently been reported to promote fibroblast-to-myofibroblast transition, thus leading to various pro-fibrotic phenotypic changes. We confirmed increased mRNA expression of IL11 and IL11Rα in fibrotic diseases by OMICs approaches and in situ hybridization. However, the vital role of IL11 as a driver for fibrosis was not recapitulated. While induction of IL11 secretion was observed downstream of TGFß signaling in human lung fibroblasts and epithelial cells, the cellular responses induced by IL11 was quantitatively and qualitatively inferior to that of TGFß at the transcriptional and translational levels. IL11 blocking antibodies inhibited IL11Rα-proximal STAT3 activation but failed to block TGFß-induced profibrotic signals. In summary, our results challenge the concept of IL11 blockade as a strategy for providing transformative treatment for fibrosis.

The Clinical Response of Upadacitinib and Risankizumab Is Associated With Reduced Inflammatory Bowel Disease Anti-TNF-α Inadequate Response Mechanisms.

BACKGROUND: Janus kinase (JAK) 1 inhibitor upadacitinib and IL-23 inhibitor risankizumab are efficacious in inflammatory bowel disease (IBD) patients who are antitumor necrosis factor (anti-TNF)-α inadequate responders (TNF-IRs). We aimed to understand the mechanisms mediating the response of upadacitinib and risankizumab. METHODS: Eight tissue transcriptomic data sets from IBD patients treated with anti-TNF-α therapies along with single-cell RNAseq data from ulcerative colitis were integrated to identify TNF-IR mechanisms. The RNAseq colon tissue data from clinical studies of TNF-IR Crohn's disease patients treated with upadacitinib or risankizumab were used to identify TNF-IR mechanisms that were favorably modified by upadacitinib and risankizumab. RESULTS: We found 7 TNF-IR upregulated modules related to innate/adaptive immune responses, interferon signaling, and tissue remodeling and 6 TNF-IR upregulated cell types related to inflammatory fibroblasts, postcapillary venules, inflammatory monocytes, macrophages, dendritic cells, and cycling B cells. Upadacitinib was associated with a significant decrease in the expression of most TNF-IR upregulated modules in JAK1 responders (JAK1-R); in contrast, there was no change in these modules among TNF-IR patients treated with a placebo or among JAK1 inadequate responders (JAK1-IR). In addition, 4 of the 6 TNF-IR upregulated cell types were significantly decreased after upadacitinib treatment in JAK1-R but not among subjects treated with a placebo or among JAK1-IR patients. We observed similar findings from colon biopsy samples from TNF-IR patients treated with risankizumab. CONCLUSIONS: Collectively, these data suggest that upadacitinib and risankizumab affect TNF-IR upregulated mechanisms, which may account for their clinical response among TNF-IR IBD patients.

We identified molecular and cellular mechanisms associated with and potentially mediating the response of upadacitinib and risankizumab for IBD patients that inadequately responded to anti-TNF-α treatment.

Synovial Inflammatory Pathways Characterize Anti-TNF-Responsive Rheumatoid Arthritis Patients.

OBJECTIVE: This study was undertaken to understand the mechanistic basis of response to anti-tumor necrosis factor (anti-TNF) therapies and to determine whether transcriptomic changes in the synovium are reflected in peripheral protein markers. METHODS: Synovial tissue from 46 rheumatoid arthritis (RA) patients was profiled with RNA sequencing before and 12 weeks after treatment with anti-TNF therapies. Pathway and gene signature analyses were performed on RNA expression profiles of synovial biopsies to identify mechanisms that could discriminate among patients with a good response, a moderate response, or no response, according to the American College of Rheumatology (ACR)/EULAR response criteria. Serum proteins encoded by synovial genes that were differentially expressed between ACR/EULAR response groups were measured in the same patients. RESULTS: Gene signatures predicted which patients would have good responses, and pathway analysis identified elevated immune pathways, including chemokine signaling, Th1/Th2 cell differentiation, and Toll-like receptor signaling, uniquely in good responders. These inflammatory pathways were correspondingly down-modulated by anti-TNF therapy only in good responders. Based on cell signature analysis, lymphocyte, myeloid, and fibroblast cell populations were elevated in good responders relative to nonresponders, consistent with the increased inflammatory pathways. Cell signatures that decreased following anti-TNF treatment were predominately associated with lymphocytes, and fewer were associated with myeloid and fibroblast populations. Following anti-TNF treatment, and only in good responders, several peripheral inflammatory proteins decreased in a manner that was consistent with corresponding synovial gene changes. CONCLUSION: Collectively, these data suggest that RA patients with robust responses to anti-TNF therapies are characterized at baseline by immune pathway activation, which decreases following anti-TNF treatment. Understanding mechanisms that define patient responsiveness to anti-TNF treatment may assist in development of predictive markers of patient response and earlier treatment options.

Epithelial dysfunction is prevented by IL-22 treatment in a Citrobacter rodentium-induced colitis model that shares similarities with inflammatory bowel disease.

Inflammatory bowel disease (IBD) is characterized by a dysregulated intestinal epithelial barrier leading to breach of barrier immunity. Here we identified similar protein expression changes between IBD and Citrobacter rodentium-infected FVB mice with respect to dysregulation of solute transporters as well as components critical for intestinal barrier integrity. We attribute the disease associated changes in the model to the emergence of undifferentiated intermediate intestinal epithelial cells. Prophylactic treatment with IL-22.Fc in C. rodentium-infected FVB mice reduced disease severity and rescued the mice from lethality. Multi-omics and solute analyses revealed that IL-22.Fc treatment prevented disease-associated changes including disruption of the solute transporter machinery and restored proper physiological functions of the intestine, respectively. Taken together, we established the disease relevance of the C. rodentium-induced colitis model to IBD, demonstrated the protective role of IL-22 in amelioration of epithelial dysfunction and elucidated the molecular mechanisms with IL-22's effect on intestinal epithelial cells.