Transcriptional Coactivator BOB1 (OBF1, OCA-B) Modulates the Specificity of DNA Recognition by the POU-Domain Factors OCT1 and OCT2 in a Monomeric Configuration.

BOB1, a mammalian lymphocyte-specific transcriptional coactivator of the transcription factors OCT1 and OCT2 (OCT1/2), plays important roles in normal immune responses, autoimmunity, and hematologic malignancies. The issue of a DNA sequence preference change imposed by BOB1 was raised more than two decades ago but remains unresolved. In this paper, using the EMSA-SELEX-Seq approach, we have reassessed the intrinsic ability of BOB1 to modulate the specificity of DNA recognition by OCT1 and OCT2. Our results have reaffirmed previous conclusions regarding BOB1 selectivity towards the dimer configuration of OCT1/2. However, they suggest that the monomeric configuration of these factors, assembled on the classical octamer ATGCAAAT and related motifs, are the primary targets of BOB1. Our data further specify the DNA sequence preference imposed by BOB1 and predict the probability of ternary complex formation. These results provide an additional insight into the action of BOB1-an essential immune regulator and a promising molecular target for the treatment of autoimmune diseases and hematologic malignancies.

Chronic Release of Tailless Phage Particles from Lactococcus lactis.

Lactococcus lactis strains residing in the microbial community of a complex dairy starter culture named "Ur" are hosts to prophages belonging to the family Siphoviridae. L. lactis strains (TIFN1 to TIFN7) showed detectable spontaneous phage production and release (109 to 1010 phage particles/ml) and up to 10-fold increases upon prophage induction, while in both cases we observed no obvious cell lysis typically described for the lytic life cycle of Siphoviridae phages. Intrigued by this phenomenon, we investigated the host-phage interaction using strain TIFN1 (harboring prophage proPhi1) as a representative. We confirmed that during the massive phage release, all bacterial cells remain viable. Further, by monitoring phage replication in vivo, using a green fluorescence protein reporter combined with flow cytometry, we demonstrated that the majority of the bacterial population (over 80%) is actively producing phage particles when induced with mitomycin C. The released tailless phage particles were found to be engulfed in lipid membranes, as evidenced by electron microscopy and lipid staining combined with chemical lipid analysis. Based on the collective observations, we propose a model of phage-host interaction in L. lactis TIFN1 where the phage particles are engulfed in membranes upon release, thereby leaving the producing host intact. Moreover, we discuss possible mechanisms of chronic, or nonlytic, release of LAB Siphoviridae phages and its impact on the bacterial host. IMPORTANCE In complex microbial consortia such as fermentation starters, bacteriophages can alter the dynamics and diversity of microbial communities. Bacteriophages infecting Lactococcus lactis are mostly studied for their detrimental impact on industrial dairy fermentation processes. In this study, we describe a novel form of phage-bacterium interaction in an L. lactis strain isolated from a complex dairy starter culture: when the prophages harbored in the L. lactis genome are activated, the phage particles are engulfed in lipid membranes upon release, leaving the producing host intact. Findings from this study provide additional insights into the diverse manners of phage-bacterium interactions and coevolution, which are essential for understanding the population dynamics in complex microbial communities like fermentation starters.

Out of the shadow of interleukin-17A: the role of interleukin-17F and other interleukin-17 family cytokines in spondyloarthritis.

PURPOSE OF REVIEW: The last decade has witnessed tremendous advances in revealing an important role for the interleukin (IL)-17 cytokine family in the pathogenesis of spondyloarthritis (SpA). Although most attention has been focused on IL-17A, a potential role of other IL-17 family members in inflammation and tissue remodelling is emerging. Herein, I review recent studies covering the role of IL-17B-F cytokines in the pathogenesis of SpA. RECENT FINDINGS: Several recent studies provided new insights into the cellular source, regulation and function of IL-17F. IL-17F/IL-17A expression ratio is higher in psoriatic skin compared to SpA synovitis. IL-17F-expressing T cells produce different proinflammatory mediators than IL-17A-expressing cells, and IL-17F and IL-17A signal through different receptor complex. Dual IL-17A and IL-17F neutralization resulted in greater suppression of downstream inflammatory and tissue remodelling responses. Furthermore, there is additional evidence of IL-23-independent IL-17 production. In contrast to IL-17A, IL-17F and IL-17C, which play proinflammatory roles in skin and joint inflammation, an anti-inflammatory function is proposed for IL-17D. An increase in IL-17E is associated with subclinical gut microbiome alterations after anti-IL-17A therapy in SpA patients. SUMMARY: IL-17 family cytokines may act as agonists or antagonists to IL-17A contributing in concert to local inflammatory responses. Understanding their function and identifying their cellular sources, and molecular mechanisms driving their expression will be the key to designing rational therapies in SpA.

Transcriptional regulator BOB.1: Molecular mechanisms and emerging role in chronic inflammation and autoimmunity.

Lymphocytes constitute an essential and potent effector compartment of the immune system. Therefore, their development and functions must be strictly regulated to avoid inappropriate immune responses, such as autoimmune reactions. Several lines of evidence from genetics (e.g. association with multiple sclerosis and primary biliary cirrhosis), human expression studies (e.g. increased expression in target tissues and draining lymph nodes of patients with autoimmune diseases), animal models (e.g. loss of functional protein protects animals from the development of collagen-induced arthritis, experimental autoimmune encephalomyelitis, type 1 diabetes, bleomycin-induced fibrosis) strongly support a causal link between the aberrant expression of the lymphocyte-restricted transcriptional regulator BOB.1 and the development of autoimmune diseases. In this review, we summarize the current knowledge of unusual structural and functional plasticity of BOB.1, stringent regulation of its expression, and the pivotal role that BOB.1 plays in shaping B- and T-cell responses. We discuss recent developments highlighting the significant contribution of BOB.1 to the pathogenesis of autoimmune diseases and how to leverage our knowledge to target this regulator to treat autoimmune tissue inflammation.

IL-12p40/IL-23p40 Blockade With Ustekinumab Decreases the Synovial Inflammatory Infiltrate Through Modulation of Multiple Signaling Pathways Including MAPK-ERK and Wnt.

Background: Psoriatic arthritis (PsA) is a chronic inflammatory joint disease within the spondyloarthritis spectrum. IL-12p40/IL-23p40 blockade reduces PsA disease activity, but its impact on synovial inflammation remains unclear. Objectives: To investigate the cellular and molecular pathways affected by IL-12p40/IL-23p40 blockade with ustekinumab in the synovium of PsA patients. Methods: Eleven PsA patients with at least one inflamed knee or ankle joint were included in a 24-week single-center open-label study and received ustekinumab 45 mg/sc according to standard care at week 0, 4, and 16. Besides clinical outcomes, synovial tissue (ST) samples were obtained by needle arthroscopy from an inflamed knee or ankle joint at baseline, week 12 and 24 and analyzed by immunohistochemistry, RNA-sequencing and real-time quantitative polymerase chain reaction (qPCR). Results: We obtained paired baseline and week 12, and paired baseline, week 12 and 24 ST samples from nine and six patients, respectively. Eight patients completed 24 weeks of clinical follow-up. At 12 weeks 6/11 patients met ACR20, 2/11 met ACR50 and 1/11 met ACR70 improvement criteria, at 24 weeks this was 3/8, 2/8 and 1/8 patients, respectively. Clinical and serological markers improved significantly. No serious adverse events occurred. We observed numerical decreases of all infiltrating cell subtypes at week 12, reaching statistical significance for CD68+ sublining macrophages. For some cell types this was even more pronounced at week 24, but clearly synovial inflammation was incompletely resolved. IL-17A and F, TNF, IL-6, IL-8, and IL-12p40 were not significantly downregulated in qPCR analysis of W12 total biopsies, only MMP3 and IL-23p19 were significantly decreased. RNA-seq analysis revealed 178 significantly differentially expressed genes between baseline and 12 weeks (FDR 0.1). Gene Ontology and KEGG terms enrichment analyses identified overrepresentation of biological processes as response to reactive oxygen species, chemotaxis, migration and angiogenesis as well as MAPK-ERK and PI3K-Akt signaling pathways among the downregulated genes and of Wnt signaling pathway among the upregulated genes. Furthermore, ACR20 responders and non-responders differed strikingly in gene expression profiles in a post-hoc exploratory analysis. Conclusions: Ustekinumab suppresses PsA synovial inflammation through modulation of multiple signal transduction pathways, including MAPK-ERK, Wnt and potentially PI3K-Akt signaling rather than by directly impacting the IL-17 pathway.

Transmembrane TNF drives osteoproliferative joint inflammation reminiscent of human spondyloarthritis.

TNF plays a key role in immune-mediated inflammatory diseases including rheumatoid arthritis (RA) and spondyloarthritis (SpA). It remains incompletely understood how TNF can lead to different disease phenotypes such as destructive peripheral polysynovitis in RA versus axial and peripheral osteoproliferative inflammation in SpA. We observed a marked increase of transmembrane (tm) versus soluble (s) TNF in SpA versus RA together with a decrease in the enzymatic activity of ADAM17. In contrast with the destructive polysynovitis observed in classical TNF overexpression models, mice overexpressing tmTNF developed axial and peripheral joint disease with synovitis, enthesitis, and osteitis. Histological and radiological assessment evidenced marked endochondral new bone formation leading to joint ankylosis over time. SpA-like inflammation, but not osteoproliferation, was dependent on TNF-receptor I and mediated by stromal tmTNF overexpression. Collectively, these data indicate that TNF can drive distinct inflammatory pathologies. We propose that tmTNF is responsible for the key pathological features of SpA.

Selective targeting of PI3Kδ suppresses human IL-17-producing T cells and innate-like lymphocytes and may be therapeutic for IL-17-mediated diseases.

The delta isoform of phosphoinositide 3-kinase (PI3Kδ) regulates various lymphocyte functions. Considering the key pro-inflammatory role of IL-17A and IL-17F cytokines in psoriasis and spondyloarthritis (SpA), we investigated the potential of PI3Kδ blockade to suppress IL-17A, IL-17F and associated pro-inflammatory cytokines that could synergize with IL-17A and IL-17F. Using in vitro studies with primary human cells and ex vivo studies with inflamed target tissues, we assessed if seletalisib, a selective PI3Kδ inhibitor, suppresses cytokine production by T cells and innate-like lymphocytes, and if seletalisib modulates the inflammatory responses in stromal cell populations in psoriasis (human dermal fibroblasts (HDF)) and SpA (fibroblast-like synoviocytes (FLS)). In vitro, seletalisib inhibited the production of pro-inflammatory cytokines, including IL-17A and IL-17F, from peripheral blood mononuclear cells (PBMCs), T helper 17 (Th17) cells as well as γδ-T cells and mucosal-associated invariant T cells. This inhibition resulted in decreased inflammatory activation of HDF in co-culture systems. Seletalisib was also efficacious in inhibiting SpA PBMCs and synovial fluid mononuclear cells (SFMCs) from producing pro-inflammatory cytokines. Furthermore, supernatant derived from cultured seletalisib-treated Th17 cells showed reduced potency for activating inflammatory responses from cultured SpA FLS and decreased their osteogenic differentiation capacity. Finally, analysis of inflamed SpA synovial tissue biopsies revealed activation of the PI3K-Akt-mTOR pathway. We observed that ex vivo seletalisib treatment of inflamed synovial tissue reduced IL-17A and IL-17F expression. Collectively, inhibition of PI3Kδ reduces the production of pro-inflammatory cytokines from IL-17-producing adaptive and innate-like lymphocytes and thereby inhibits downstream inflammatory and tissue remodeling responses. PI3Kδ-targeting may therefore represent a novel therapeutic avenue for the treatment of IL-17-mediated chronic inflammatory diseases such as psoriasis and SpA.

Interleukin 17A and IL-17F Expression and Functional Responses in Rheumatoid Arthritis and Peripheral Spondyloarthritis.

OBJECTIVE: Targeting the interleukin 17 (IL-17) axis is efficacious in psoriasis and spondyloarthritis (SpA), but not in rheumatoid arthritis (RA). We investigated potential differences in tissue expression and function of IL-17A and IL-17F in these conditions. METHODS: mRNA expression of cytokines and their receptors was assessed by quantitative PCR in psoriasis skin samples, in SpA and RA synovial tissue (ST) samples and in fibroblast-like synoviocytes (FLS). Cytokines were measured in synovial fluid (SF) and FLS supernatants by ELISA. FLS were stimulated with IL-17A or IL-17F cytokines supplemented with tumor necrosis factor (TNF), or with pooled SF from patients with SpA or RA. RESULTS: Levels of IL-17A (P = 0.031) and IL-17F (P = 0.017) mRNA were lower in psoriatic arthritis ST compared to paired psoriasis skin samples. The level of IL-17A mRNA was 2.7-fold lower than that of IL-17F in skin (P = 0.0078), but 17.3-fold higher in ST (P < 0.0001). In SF, the level of IL-17A protein was 37.4-fold higher than that of IL-17F [median 292.4 (IQR 81.4-464.2) vs median 7.8 (IQR 7.7-8.7) pg/mL; P < 0.0001]. IL-17A and IL-17F mRNA and protein levels did not differ in SpA compared to RA synovitis samples, and neither were the IL-17 receptors IL-17RA and IL-17RC, or the TNF receptors TNFR1 and TNR2, differentially expressed between SpA and RA ST, nor between SpA and RA FLS. SpA and RA FLS produced similar amounts of IL-6 and IL-8 protein upon stimulation with IL-17A or IL-17F cytokines, supplemented with 1 ng/ml TNF. Pooled SpA or RA SF samples similarly enhanced the inflammatory response to IL-17A and IL-17F simulation in FLS. CONCLUSION: The IL-17A/IL-17F expression ratio is higher in SpA synovitis compared to psoriasis skin. Expression of IL-17A and IL-17F, and the functional response to these cytokines, appear to be similar in SpA and RA synovitis.

APRIL Induces a Novel Subset of IgA+ Regulatory B Cells That Suppress Inflammation via Expression of IL-10 and PD-L1.

Regulatory B cells (Bregs) are immunosuppressive cells that modulate immune responses through multiple mechanisms. The signals required for the differentiation and activation of these cells remain still poorly understood. We have already shown that overexpression of A PRoliferation-Inducing Ligand (APRIL) reduces the incidence and severity of collagen-induced arthritis (CIA) in mice. Furthermore, we have described that APRIL, but not BAFF, promoted IL-10 production and regulatory functions in human B cells. Therefore, we hypothesized that APRIL, but not BAFF, may be involved in the induction and/or activation of IL-10 producing Bregs that suppress inflammatory responses in vitro and in vivo. Here, we describe that APRIL promotes the differentiation of naïve human B cells to IL-10-producing IgA+ B cells. These APRIL-induced IgA+ B cells display a Breg phenotype and inhibit T cell and macrophage responses through IL-10 and PD-L1. Moreover, APRIL-induced IL-10 producing Bregs suppress inflammation in vivo in experimental autoimmune encephalitis (EAE) and contact hypersensitivity (CHS) models. Finally, we showed a strong correlation between APRIL and IL-10 in the inflamed synovial tissue of inflammatory arthritis patients. Collectively, these observations indicate the potential relevance of this novel APRIL-induced IgA+ Breg population for immune homeostasis and immunopathology.

BOB.1 controls memory B-cell fate in the germinal center reaction.

During T cell-dependent (TD) germinal center (GC) responses, naïve B cells are instructed to differentiate towards GC B cells (GCBC), high-affinity long-lived plasma cells (LLPC) or memory B cells (Bmem). Alterations in the B cell-fate choice could contribute to immune dysregulation leading to the loss of self-tolerance and the initiation of autoimmune disease. Here we show that mRNA levels of the transcription regulator BOB.1 are increased in the lymph node compartment of patients with rheumatoid arthritis (RA), a prototypical autoimmune disease caused by the loss of immunological tolerance. Investigating to what extent levels of BOB.1 impact B cells during TD immune responses we found that BOB.1 has a crucial role in determining the B cell-fate decision. High BOB.1 levels promote the generation of cells with phenotypic and functional characteristics of Bmem. Mechanistically, overexpression of BOB.1 drives ABF1 and suppresses BCL6, favouring Bmem over LLPC or recycling GCBC. Low levels of BOB.1 are sufficient for LLPC but not for Bmem differentiation. Our findings demonstrate a novel role for BOB.1 in B cells during TD GC responses and suggest that its dysregulation may contribute to the pathogenesis of RA by disturbing the B cell-fate determination.

Correction: HLA-C*07 in axial spondyloarthritis: data from the German Spondyloarthritis Inception Cohort and the Spondyloarthritis Caught Early cohort.

The original version of this Article contained an error in the spelling of the author Denis Poddubnyy, which was incorrectly given as Denis Podubbnyy. This has now been corrected in both the PDF and HTML versions of the Article.

HLA-C*07 in axial spondyloarthritis: data from the German Spondyloarthritis Inception Cohort and the Spondyloarthritis Caught Early cohort.

We aimed to assess the mRNA expression of MHC class 1-related molecules in ankylosing spondylitis (AS) patients vs healthy controls (HCs) and, subsequently, if the absence of HLA-C*07 is associated with genetic susceptibility to axial spondyloarthritis (axSpA). HLA-C*07 was assessed in (a) an exploratory cohort of 24 AS patients vs 40 HCs, (b) a confirmatory cohort of 113 AS patients and 83 non-radiographic axSpA patients from the GErman SPondyloarthritis Inception Cohort (GESPIC) vs 134,528 German potential stem cell donors, and (c) an early back pain cohort with 94 early axSpA patients vs 216 chronic back pain (CBP) patients from the SPondyloArthritis Caught Early (SPACE) cohort. In the exploratory cohort, 79% of the AS patients were HLA-C*07 negative compared to 35% of the HCs (p < 0.001). This difference was confirmed in GESPIC with 73% of AS patients being HLA-C*07 negative compared to 50% of the controls (p < 0.0001); 59% of the nr-axSpA patients were HLA-C*07 negative. In the SPACE cohort, 70% of the axSpA patients were HLA-C*07 negative compared to 44% of CBP patients (p < 0.0001); the association between HLA-C*07 negativity and a diagnosis of axSpA was independent from HLA-B*27. In conclusion, the absence of HLA-C*07 is associated with genetic susceptibility to axSpA.

Generation and Characterization of Anti-Citrullinated Protein Antibody-Producing B Cell Clones From Rheumatoid Arthritis Patients.

OBJECTIVE: Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA). Aside from autoantibody production, the function of autoantigen-specific B cells remains poorly understood in the context of this disease. This study set out to elucidate autoantigen-specific B cell functions through the isolation and immortalization of unique citrullinated protein/peptide (CP)-reactive B cell clones from RA patients. METHODS: B cell clones from either the blood or synovial fluid of cyclic citrullinated peptide 2 (CCP2) antibody-positive RA patients were immortalized by genetic reprogramming with Bcl-6 and Bcl-xL. Enzyme-linked immunosorbent assay and flow cytometry were used to identify CCP2-reactive clones and to further characterize surface marker and cytokine expression as well as B cell receptor signaling competence. Global gene expression profiles were interrogated by RNA sequencing. RESULTS: Three unique CP-reactive memory B cell clones were generated from the blood or synovial fluid of 2 RA patients. CP-reactive memory B cells did not appear to be broadly cross-reactive, but rather had a fairly restricted epitope recognition profile. These clones were able to secrete both pro- and antiinflammatory cytokines and had a unique surface profile of costimulatory molecules and receptors, including CD40 and C5a receptor type 1, when compared to non-CP-reactive clones from the same patient. In addition, CP-reactive clones bound citrullinated protein, but not native protein, and could mobilize calcium in response to antigen binding. CONCLUSION: CP-reactive memory B cells comprise a rare, seemingly oligoclonal population with restricted epitope specificity and distinct phenotypic and molecular characteristics suggestive of antigen-presenting cells. Cloning by genetic reprogramming opens new avenues to study the function of autoreactive memory B cells, especially in terms of antigen processing, presentation, and subsequent T cell polarization.

Expansion of Interleukin-22- and Granulocyte-Macrophage Colony-Stimulating Factor-Expressing, but Not Interleukin-17A-Expressing, Group 3 Innate Lymphoid Cells in the Inflamed Joints of Patients With Spondyloarthritis.

OBJECTIVE: Clinical trials of the anti-interleukin-17A (anti-IL-17A) antibody secukinumab have demonstrated a crucial role of the cytokine IL-17A in the pathogenesis of spondyloarthritis (SpA); however, its cellular source in this condition remains a matter of controversy. Group 3 innate lymphoid cells (ILC3s) have been recently identified as potent producers of proinflammatory cytokines, including IL-17A and IL-22, in a number of different tissues. This study was undertaken to characterize the presence and composition of ILCs, and investigate whether these cells are an important source of IL-17A, in the synovial tissue (ST) of patients with SpA. METHODS: Matched ST, synovial fluid, and peripheral blood (PB) samples were obtained from SpA patients with actively inflamed knee joints. ILC subsets were characterized by flow cytometry. Gene expression analysis at the single-cell level was performed directly ex vivo and after in vitro activation. An IL-17A enzyme-linked immunospot assay was used to detect IL-17A-secreting cells. RESULTS: ILCs, and particularly NKp44+ ILC3s, were expanded in inflamed arthritic joints. Single-cell expression analysis demonstrated that ST ILCs were clearly distinguishable from ST T cells and from their PB counterparts. Expression of the Th17 signature transcripts RORC, AHR, and IL23R was detected in a large proportion of ST ILC3s. These cells were capable of inducing expression of IL22 and CSF2, but not IL17A, in response to in vitro restimulation. CONCLUSION: Our findings demonstrate that absolute and relative numbers of ILC3s are enriched in the synovial joints of patients with SpA. However, these cells are not a significant source of IL-17A in this disease.

Histologic evidence that mast cells contribute to local tissue inflammation in peripheral spondyloarthritis by regulating interleukin-17A content.

OBJECTIVES: Synovial mast cells contain IL-17A, a key driver of tissue inflammation in SpA. A recent in vitro study showed that tissue-derived mast cells can capture and release exogenous IL-17A. The present study aimed to investigate if this mechanism could contribute to tissue inflammation in SpA. METHODS: Potential activation of mast cells by IL-17A was assessed by gene expression analysis of the Laboratory of Allergic Diseases 2 (LAD2) mast cell line. The presence of IL-17A-positive mast cells was assessed by immunohistochemistry in synovial tissue obtained before and after secukinumab treatment, as well as in skin and gut tissues from SpA-related conditions. RESULTS: IL-17A did not induce a pro-inflammatory response in human LAD2 mast cells according to the canonical IL-17A signalling pathway. In SpA synovial tissue, the percentage of IL-17A-positive mast cells increased upon treatment with secukinumab. IL-17A-positive mast cells were also readily detectable in non-inflamed barrier tissues such as skin and gut. In non-inflamed dermis and gut submucosa, IL-17A-positive mast cells are the most prevalent IL-17A-positive cells in situ. Compared with non-inflamed tissues, both total mast cells and IL-17A-positive mast cells were increased in psoriatic skin dermis and in submucosa from inflammatory bowel disease gut. In contrast, the proportion of IL-17A-positive mast cells was strikingly lower in the inflamed compared with non-inflamed gut lamina propria. CONCLUSION: IL-17A-positive mast cells are present across SpA target tissues and correlate inversely with inflammation, indicating that their IL-17A content can be regulated. Tissue-resident mast cells may act as IL-17A-loaded sentinel cells, which release IL-17A to amplify tissue inflammation.

Group 2 Innate Lymphoid Cells Attenuate Inflammatory Arthritis and Protect from Bone Destruction in Mice.

Group 2 innate lymphoid cells (ILC2s) were detected in the peripheral blood and the joints of rheumatoid arthritis (RA) patients, serum-induced arthritis (SIA), and collagen-induced arthritis (CIA) using flow cytometry. Circulating ILC2s were significantly increased in RA patients compared with healthy controls and inversely correlated with disease activity. Induction of arthritis in mice led to a fast increase in ILC2 number. To elucidate the role of ILC2 in arthritis, loss- and gain-of-function mouse models for ILC2 were subjected to arthritis. Reduction of ILC2 numbers in RORαcre/GATA3fl/fl and Tie2cre/RORαfl/fl mice significantly exacerbated arthritis. Increasing ILC2 numbers in mice by IL-25/IL-33 mini-circles or IL-2/IL-2 antibody complex and the adoptive transfer of wild-type (WT) ILC2s significantly attenuated arthritis by affecting the initiation phase. In addition, adoptive transfer of IL-4/13-competent WT but not IL-4/13-/- ILC2s and decreased cytokine secretion by macrophages. These data show that ILC2s have immune-regulatory functions in arthritis.

Brief Report: Interleukin-17 Blockade With Secukinumab in Peripheral Spondyloarthritis Impacts Synovial Immunopathology Without Compromising Systemic Immune Responses.

OBJECTIVE: Secukinumab (anti-interleukin-17A [anti-IL-17A]) is an effective therapy for ankylosing spondylitis and psoriatic arthritis, the prototypical forms of spondyloarthritis (SpA). We undertook this study to determine whether secukinumab modulates the immunopathology of target lesions without blunting systemic immune responses, using peripheral SpA as a model. METHODS: Twenty patients with active peripheral SpA were included in a 12-week open-label trial with secukinumab (300 mg once weekly from baseline to week 4 and then every 4 weeks thereafter). Outcomes included clinical response, cytokine production by peripheral blood cells using TruCulture technology, and histologic and real-time quantitative polymerase chain reaction analysis of synovial biopsy samples before and after treatment. RESULTS: All patients completed the 12-week study without severe adverse events (AEs) or severe treatment-related AEs. The efficacy end point, the number of patients meeting the American College of Rheumatology 20% improvement criteria (achieving an ACR20 response) at 12 weeks, was achieved by 13 of the 20 patients, of whom 8 achieved an ACR50 response and 5 achieved an ACR70 response, with rapid and significant improvements in all clinical disease activity measures. Clinical improvement in joint counts was associated with a histologic decrease in synovial sublining macrophages (P = 0.028) and neutrophils (P = 0.004), both of which are sensitive synovial biomarkers of inflammatory response in peripheral SpA, as well as with decreased synovial expression of IL-17A messenger RNA (mRNA) (P = 0.010) but not of tumor necrosis factor mRNA. Systemically, secukinumab treatment decreased the C-reactive protein level and the erythrocyte sedimentation rate (both P < 0.01), and also decreased matrix metalloproteinase 3 production in the TruCulture system (P < 0.05). However, with the exception of IL-17A itself, the capacity of peripheral blood cells to produce a broad panel of cytokines and chemokines upon stimulation with microbial antigens was not affected. CONCLUSION: This mechanism-of-action study in peripheral SpA indicates that clinical improvement with secukinumab treatment is paralleled by immunomodulation of inflamed target tissues without compromising systemic immune responses.

Altered cytokine expression by macrophages from HLA-B27-positive spondyloarthritis patients without evidence of endoplasmic reticulum stress.

OBJECTIVES: We investigated endoplasmic reticulum (ER) stress and cytokine expression in peripheral blood-derived macrophages and synovial tissue from HLA-B27+ SpA patients. METHODS: Macrophages from healthy donors, SpA and RA patients were polarized with IFN-γ or IL-10 and activated with lipopolysaccharide. Expression of ER stress markers (BiP, CHOP, ERdj4) and cytokines (IL-23, IL-12, TNF, IL-10) was measured by qRT-PCR. Expression of ER stress markers and cytokines in synovial tissue from SpA patients was evaluated by microarray analysis. RESULTS: Macrophages from HLA-B27+ SpA patients did not show elevated ER stress markers. However, the expression of IL-23 and IL-12 by peripheral blood-derived macrophages was higher in HLA-B27+ SpA in comparison with healthy donors. Synovial tissue from HLA-B27+ SpA patients showed higher expression of TNF compared with HLA-B27- SpA patients. CONCLUSION: HLA-B27+ SpA patients showed increased expression of IL-23, IL-12 and TNF without evidence of ER stress.

Dual IL-17A and IL-17F neutralisation by bimekizumab in psoriatic arthritis: evidence from preclinical experiments and a randomised placebo-controlled clinical trial that IL-17F contributes to human chronic tissue inflammation.

OBJECTIVE: Interleukin (IL)-17A has emerged as pivotal in driving tissue pathology in immune-mediated inflammatory diseases. The role of IL-17F, sharing 50% sequence homology and overlapping biological function, remains less clear. We hypothesised that IL-17F, together with IL-17A, contributes to chronic tissue inflammation, and that dual neutralisation may lead to more profound suppression of inflammation than inhibition of IL-17A alone. METHODS: Preclinical experiments assessed the role of IL-17A and IL-17F in tissue inflammation using disease-relevant human cells. A placebo-controlled proof-of-concept (PoC) clinical trial randomised patients with psoriatic arthritis (PsA) to bimekizumab (n=39) or placebo (n=14). Safety, pharmacokinetics and clinical efficacy of multiple doses (weeks 0, 3, 6 (240 mg/160 mg/160 mg; 80 mg/40 mg/40 mg; 160 mg/80 mg/80 mg and 560 mg/320 mg/320 mg)) of bimekizumab, a humanised monoclonal IgG1 antibody neutralising both IL-17A and IL-17F, were investigated. RESULTS: IL-17F induced qualitatively similar inflammatory responses to IL-17A in skin and joint cells. Neutralisation of IL-17A and IL-17F with bimekizumab more effectively suppressed in vitro cytokine responses and neutrophil chemotaxis than inhibition of IL-17A or IL-17F alone. The PoC trial met both prespecified efficacy success criteria and showed rapid, profound responses in both joint and skin (pooled top three doses vs placebo at week 8: American College of Rheumatology 20% response criteria 80.0% vs 16.7% (posterior probability >99%); Psoriasis Area and Severity Index 100% response criteria 86.7% vs 0%), sustained to week 20, without unexpected safety signals. CONCLUSIONS: These data support IL-17F as a key driver of human chronic tissue inflammation and the rationale for dual neutralisation of IL-17A and IL-17F in PsA and related conditions. TRIAL REGISTRATION NUMBER: NCT02141763; Results.

Endoplasmic reticulum stress cooperates with Toll-like receptor ligation in driving activation of rheumatoid arthritis fibroblast-like synoviocytes.

BACKGROUND: Endoplasmic reticulum (ER) stress has proinflammatory properties, and transgenic animal studies of rheumatoid arthritis (RA) indicate its relevance in the process of joint destruction. Because currently available studies are focused primarily on myeloid cells, we assessed how ER stress might affect the inflammatory responses of stromal cells in RA. METHODS: ER stress was induced in RA fibroblast-like synoviocytes (FLS), dermal fibroblasts, and macrophages with thapsigargin or tunicamycin alone or in combination with Toll-like receptor (TLR) ligands, and gene expression and messenger RNA (mRNA) stability was measured by quantitative polymerase chain reaction. Cellular viability was measured using cell death enzyme-linked immunosorbent assays and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, and signaling pathway activation was analyzed by immunoblotting. RESULTS: No cytotoxicity was observed in FLS exposed to thapsigargin, despite significant induction of ER stress markers. Screening of 84 proinflammatory genes revealed minor changes in their expression (fold change 90th percentile range 2.8-8.3) by thapsigargin alone, but the vast majority were hyperinduced during combined stimulation with thapsigargin and TLR ligands (35% greater than fivefold vs lipopolysaccharide alone). The synergistic response could not be explained by quantitative effects on nuclear factor-κB and mitogen-activated protein kinase pathways alone, but it was dependent on increased mRNA stability. mRNA stabilization was similarly enhanced by ER stress in dermal fibroblasts but not in macrophages, correlating with minimal cooperative effects on gene induction in macrophages. CONCLUSIONS: RA FLS are resistant to apoptosis induced by ER stress, but ER stress potentiates their activation by multiple TLR ligands. Interfering with downstream signaling pathway components of ER stress may be of therapeutic potential in the treatment of RA.