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ObjectiveIL-17A plays a major role in the pathogenesis of spondyloarthritis (SpA). Here we assessed the impact of inhibition of RAR related orphan receptor-γ (RORC), the key transcription factor controlling IL-17 production, on experimental SpA in HLA-B27 transgenic (tg) rats.MethodsExperimental SpA was induced by immunization of HLA-B27 tg rats with heat-inactivated Mycobacterium tuberculosis. Splenocytes obtained at day 7, 14 and 21 after immunization were restimulated ex vivo to assess the induction of pro-inflammatory cytokines. Rats were then prophylactically treated with a RORC inhibitor versus vehicle control. The biologic effect of RORC inhibition was assessed by pro-inflammatory cytokine expression in draining lymph nodes. Arthritis and spondylitis were monitored clinically, and the degree of peripheral and axial inflammation, destruction and new bone formation was confirmed by histology.ResultsEx vivo mRNA and protein analyses revealed the rapid and selective induction of IL-17A and IL-22 production by a variety of lymphocyte subsets upon disease induction in HLA-B27 tg rats. Prophylactic RORC inhibition in vivo suppressed the expression of IL-17A, IL17F, and IL-22 without affecting the expression of other T helper cell subset related genes. This biological effect did not translate into clinical efficacy as RORC inhibition significantly accelerated the onset of arthritis and spondylitis, and aggravated the clinical severity of arthritis. This worsening of experimental SpA was confirmed by histopathological demonstration of increased inflammation, destruction, and new bone formation.ConclusionDespite a significant suppression of the IL-17 axis, RORC inhibitor treatment accelerates and aggravates experimental SpA in the HLA-B27 tg rat model.

Over the last 10 years considerable progress has been made in the application of small molecules to modulating protein-protein interactions (PPIs), and the navigation from “undruggable” to a host of candidate molecules in clinical trials has been well-charted in recent, comprehensive reviews. Structure-based design has played an important role in this scientific journey, with three dimensional structures guiding medicinal chemistry efforts. However, the importance of two additional dimensions: movement and time is only now being realised, as increasing computing power, closely aligned with wet lab validation, is applied to the challenge. Protein dynamics are fundamental to biology and disease, and application to PPI drug discovery has massively widened the scope for new chemical entities to influence function from allosteric, and previously unreported, sites. In this forward-looking perspective we highlight exciting, new opportunities for small molecules to modulate disease biology, by adjusting the frequency profile of natural conformational sampling, through the stabilisation of clinically desired conformers of target proteins.

Introduction: Spondyloarthritis (SpA) is characterized by inflammation, articular bone erosions and pathologic new bone formation. Targeting TNFα or IL-17A with current available therapies reduces inflammation in SpA, however, treatment of the bone pathology in SpA remains an unmet clinical need. Activation of the mammalian target Of rapamycin (mTOR) promotes IL-17A expression and osteogenesis. Therefore, the inhibition of mTOR (with rapamycin) could be a promising therapeutic avenue in SpA.Objectives: To investigate the effect of blocking mTOR on inflammation, bone erosions and new bone formation in SpA.Methods: Peripheral blood mononuclear cells (PBMCs) from patients with SpA were stimulated with anti-CD3/CD28 in the presence or absence of rapamycin and the resulting cytokine expression was assessed. Fibroblast-like synoviocytes (FLS) from SpA patients were assessed for osteogenic differentiation potential in conditions with TNFα, IL-17A, or TNFα plus IL-17A, in the presence or absence of rapamycin. HLA-B27/Huβ2m transgenic rats were immunized with low dose heat-inactivated Mycobacterium tuberculosis (M. tub), treated with 1.5 mg/kg rapamycin prophylactically or therapeutically and monitored for arthritis and spondylitis. Histology and mRNA analysis were performed after 5 weeks of treatment to assess inflammation and bone pathology.Results:In vitro TNFα and IL-17A protein production by SpA PBMCs was inhibited in the presence of rapamycin. Rapamycin also inhibited osteogenic differentiation of human SpA FLS. Ex vivo analysis of SpA synovial biopsies indicated activation of the mTOR pathway in the synovial tissue of SpA patients. In vivo, prophylactic treatment of HLA-B27/Huβ2m transgenic rats with rapamycin significantly inhibited the development and severity of inflammation in peripheral joints and spine (arthritis and spondylitis), with histological evidence of reduced bone erosions and new bone formation around peripheral joints. In addition, therapeutic treatment with rapamycin significantly decreased severity of arthritis and spondylitis, with peripheral joint histology showing reduced inflammation, bone erosions and new bone formation. IL-17A mRNA expression was decreased in the metacarpophalangeal joints after rapamycin treatment.Conclusion: mTOR blockade inhibits IL-17A and TNFα production by PBMCs, and osteogenic differentiation of FLS from patients with SpA in vitro. In the HLA-B27 transgenic rat model of SpA, rapamycin inhibits arthritis and spondylitis development and severity, reduces articular bone erosions, decreases pathologic new bone formation and suppresses IL-17A expression. These results may support efforts to evaluate the efficacy of targeting the mTOR pathway in SpA patients.

Summary: 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. : The role of ILC2s in the initiation phase of inflammatory arthritis has remained unclear. Omata et al. demonstrate that the ILC2-derived IL-4/13 decrease pro-inflammatory cytokine secretion by macrophages, thereby attenuating arthritis. Keywords: innate lymphoid cells, rheumatoid arthritis, immunoregulation

Abstract Background Histone deacetylase inhibitors (HDACi) suppress cytokine production in immune and stromal cells of patients with rheumatoid arthritis (RA). Here, we investigated the effects of the HDACi givinostat (ITF2357) on the transcriptional and post-transcriptional regulation of inflammatory markers in RA fibroblast-like synoviocytes (FLS). Methods The effects of ITF2357 on the expression and messenger RNA (mRNA) stability of IL-1β-inducible genes in FLS were analyzed using array-based qPCR and Luminex. The expression of primary and mature cytokine transcripts, the mRNA levels of tristetraprolin (TTP, or ZFP36) and other AU-rich element binding proteins (ARE-BP) and the cytokine profile of fibroblasts derived from ZFP36 +/+ and ZFP36 −/− mice was measured by qPCR. ARE-BP silencing was performed by small interfering RNA (siRNA)-mediated knockdown, and TTP post-translational modifications were analyzed by immunoblotting. Results ITF2357 reduced the expression of 85% of the analyzed IL-1β-inducible transcripts, including cytokines (IL6, IL8), chemokines (CXCL2, CXCL5, CXCL6, CXCL10), matrix-degrading enzymes (MMP1, ADAMTS1) and other inflammatory mediators. Analyses of mRNA stability demonstrated that ITF2357 accelerates IL6, IL8, PTGS2 and CXCL2 mRNA degradation, a phenomenon associated with the enhanced transcription of TTP, but not other ARE-BP, and the altered post-translational status of TTP protein. TTP knockdown potentiated cytokine production in RA FLS and murine fibroblasts, which in the latter case was insensitive to inhibition by ITF2357 treatment. Conclusions Our study identifies that regulation of cytokine mRNA stability is a predominant mechanism underlying ITF2357 anti-inflammatory properties, occurring via regulation of TTP. These results highlight the therapeutic potential of ITF2357 in the treatment of RA.

Abstract Background Anti-CD74 IgG antibodies are reported to be elevated in patients with axial spondyloarthritis (axSpA). This study assessed the diagnostic value of anti-CD74 antibodies in patients with early axSpA. Methods Anti-CD74 IgG and IgA antibodies were first measured in an exploratory cohort of patients with radiographic axSpA (138 patients with ankylosing spondyloarthritis (AS)) and 57 healthy controls and then were measured in patients with early axSpA (n = 274) and with non-SpA chronic back pain (CBP) (n = 319), participating in the spondyloarthritis caught early (SPACE) prospective cohort study of patients under 45 years old with early back pain (for ≥ 3 months, but ≤ 2 years). Results In the exploratory cohort, anti-CD74 IgG antibodies were present in 79.7% of patients with AS vs. 43.9% of healthy controls (p

IL-17A is a central driver of spondyloarthritis (SpA), its production was originally proposed to be IL-23 dependent. Emerging preclinical and clinical evidence suggests, however, that IL-17A and IL-23 have a partially overlapping but distinct biology. We aimed to assess the extent to which IL-17A-driven pathology is IL-23 dependent in experimental SpA. Experimental SpA was induced in HLA-B27/Huβ2m transgenic rats, followed by prophylactic or therapeutic treatment with an anti-IL23R antibody or vehicle control. Spondylitis and arthritis were scored clinically and hind limb swelling was measured. Draining lymph node cytokine expression levels were analyzed directly ex vivo, and IL-17A protein was measured upon restimulation with PMA/ionomycin. Prophylactic treatment with anti-IL23R completely protected against the development of both spondylitis and arthritis, while vehicle-treated controls did develop spondylitis and arthritis. In a therapeutic study, anti-IL23R treatment failed to reduce the incidence or decrease the severity of experimental SpA. Mechanistically, expression of downstream effector cytokines, including IL-17A and IL-22, was significantly suppressed in anti-IL23R versus vehicle-treated rats in the prophylactic experiments. Accordingly, the production of IL-17A upon restimulation was reduced. In contrast, there was no difference in IL-17A and IL-22 expression after therapeutic anti-IL23R treatment. Targeting the IL-23 axis during the initiation phase of experimental SpA—but not in established disease—inhibits IL-17A expression and suppresses disease, suggesting the existence of IL-23-independent IL-17A production. Whether IL-17A can be produced independent of IL-23 in human SpA remains to be established.

Spondyloarthritis (SpA) does not display the typical features of auto-immune disease. Despite the strong association with MHC class I, CD8+ T cells are not required for disease induction in the HLA-B27/Huβ2m transgenic rats. We used Lewis HLA-B27/Huβ2m transgenic rats [21-3 × 283-2]F1, HLA-B7/Huβ2m transgenic rats [120-4 × 283-2]F1, and wild-type rats to test our hypothesis that SpA may be primarily driven by the innate immune response. In vitro, splenocytes were stimulated with heat-inactivated Mycobacterium tuberculosis and cytokine expression and production was measured. In vivo, male and female rats were immunized with 30, 60, or 90 µg of heat-inactivated M. tuberculosis and clinically monitored for spondylitis and arthritis development. After validation of the model, we tested whether prophylactic and therapeutic TNF targeting affected spondylitis and arthritis. In vitro stimulation with heat-inactivated M. tuberculosis strongly induced gene expression of pro-inflammatory cytokines such as TNF, IL-6, IL-1α, and IL-1β, in the HLA-B27 transgenic rats compared with controls. In vivo immunization induced an increased spondylitis and arthritis incidence and an accelerated and synchronized onset of spondylitis and arthritis in HLA-B27 transgenic males and females. Moreover, immunization overcame the protective effect of orchiectomy. Prophylactic TNF targeting resulted in delayed spondylitis and arthritis development and reduced arthritis severity, whereas therapeutic TNF blockade did not affect spondylitis and arthritis severity. Collectively, these data indicate that innate immune activation plays a role in the initiation of HLA-B27-associated disease and allowed to establish a useful in vivo model to study the cellular and molecular mechanisms of disease initiation and progression.

Objective Spondyloarthritis (SpA) can encompass axial, peripheral and extra-articular disease manifestations. Patients are classified as axial or peripheral SpA depending on the presence or absence of current back pain, independently of the other disease manifestations. Therefore, we aimed to assess the percentage of patients with axial SpA with peripheral disease and how this peripheral disease contributes to the overall disease activity.Methods Prevalence and disease activity of peripheral disease manifestations were assessed in a real-life observational cohort of 314 patients with the clinical diagnosis of SpA and fulfilling the Assessment of SpondyloArthritis international Society (ASAS) criteria.Results Of the 314 patients fulfilling the ASAS criteria, 230 fulfilled the axial and 84 the peripheral SpA criteria. Of the 230 patients with axial SpA, 49% had purely axial disease without peripheral disease manifestations whereas 51% had combined axial (back pain) and peripheral (arthritis, enthesitis and/or dactylitis) disease. The latter group had the highest disease activity in comparison with pure axial SpA as well as with peripheral SpA.Conclusion Half of the patients classified as axial SpA according to the ASAS criteria also have peripheral disease manifestations such as arthritis, enthesitis and/or dactylitis. These peripheral disease manifestations contribute significantly to overall disease activity.

Insulin like growth factor (IGF)-I can act on a variety of cells involved in cartilage and bone repair, yet IGF-I has not been studied extensively in the context of inflammatory arthritis. The objective of this study was to investigate whether IGF-I overexpression in the osteoblast lineage could lead to increased reparative or pathological bone formation in rheumatoid arthritis and/or spondyloarthritis respectively.Mice overexpressing IGF-I in the osteoblast lineage (Ob-IGF-I+/-) line 324-7 were studied during collagen induced arthritis and in the DBA/1 aging model for ankylosing enthesitis. Mice were scored clinically and peripheral joints were analysed histologically for the presence of hypertrophic chondrocytes and osteocalcin positive osteoblasts.90-100% of the mice developed CIA with no differences between the Ob-IGF-I+/- and non-transgenic littermates. Histological analysis revealed similar levels of hypertrophic chondrocytes and osteocalcin positive osteoblasts in the ankle joints. In the DBA/1 aging model for ankylosing enthesitis 60% of the mice in both groups had a clinical score 1

Studies on the role of B lymphocytes in atherosclerosis development, have yielded contradictory results. Whereas B lymphocyte-deficiency aggravates atherosclerosis in mice; depletion of mature B lymphocytes reduces atherosclerosis. These observations led to the notion that distinct B lymphocyte subsets have different roles. B1a lymphocytes exert an atheroprotective effect, which has been attributed to secretion of IgM, which can be deposited in atherosclerotic lesions thereby reducing necrotic core formation. Tumor necrosis factor (TNF)-family member 'A Proliferation-Inducing Ligand' (APRIL, also known as TNFSF13) was previously shown to increase serum IgM levels in a murine model. In this study, we investigated the effect of APRIL overexpression on advanced lesion formation and composition, IgM production and B cell phenotype. We crossed APRIL transgenic (APRIL-Tg) mice with ApoE knockout (ApoE-/-) mice. After a 12-week Western Type Diet, ApoE-/-APRIL-Tg mice and ApoE-/- littermates showed similar increases in body weight and lipid levels. Histologic evaluation showed no differences in lesion size, stage or necrotic area. However, smooth muscle cell (α-actin stain) content was increased in ApoE-/-APRIL-Tg mice, implying more stable lesions. In addition, increases in both plaque IgM deposition and plasma IgM levels were found in ApoE-/-APRIL-Tg mice compared with ApoE-/- mice. Flow cytometry revealed a concomitant increase in peritoneal B1a lymphocytes in ApoE-/-APRIL-Tg mice. This study shows that ApoE-/-APRIL-Tg mice have increased oxLDL-specific serum IgM levels, potentially mediated via an increase in B1a lymphocytes. Although no differences in lesion size were found, transgenic ApoE-/-APRIL-Tg mice do show potential plaque stabilizing features in advanced atherosclerotic lesions.

Angiopoietin (Ang) -1 and -2 and their receptor Tie2 play critical roles in regulating angiogenic processes during development, homeostasis, tumorigenesis, inflammation and tissue repair. Tie2 signaling is best characterized in endothelial cells, but a subset of human and murine circulating monocytes/macrophages essential to solid tumor formation express Tie2 and display immunosuppressive properties consistent with M2 macrophage polarization. However, we have recently shown that Tie2 is strongly activated in pro-inflammatory macrophages present in rheumatoid arthritis patient synovial tissue. Here we examined the relationship between Tie2 expression and function during human macrophage polarization. Tie2 expression was observed under all polarization conditions, but was highest in IFN-γ and IL-10 -differentiated macrophages. While TNF enhanced expression of a common restricted set of genes involved in angiogenesis and inflammation in GM-CSF, IFN-γ and IL-10 -differentiated macrophages, expression of multiple chemokines and cytokines, including CXCL3, CXCL5, CXCL8, IL6, and IL12B was further augmented in the presence of Ang-1 and Ang-2, via Tie2 activation of JAK/STAT signaling. Conditioned medium from macrophages stimulated with Ang-1 or Ang-2 in combination with TNF, sustained monocyte recruitment. Our findings suggest a general role for Tie2 in cooperatively promoting the inflammatory activation of macrophages, independently of polarization conditions.

BACKGROUND: There is growing interest in soluble biomarkers that could be used on the group level for screening purposes in small proof of principle studies during early drug development. We investigated early changes in serum levels of several candidate biomarkers involved in cartilage and bone metabolism following the initiation of adalimumab as a prototypic active treatment in psoriatic arthritis (PsA) compared to placebo. MATERIALS AND METHODS: Twenty-four PsA patients were randomized to receive either adalimumab 40 mg s.c. every other week or placebo for 4 weeks, followed by an open label extension phase. Serum samples were obtained at baseline and after 4 and 12 weeks of treatment and analyzed for levels of CPII and PINP (synthesis of type II and type I procollagen), melanoma inhibitory activity (MIA) (chondrocyte anabolism), matrix metalloproteinase (MMP)-3, C2C and cartilage oligomeric matrix protein (COMP) (type II collagen degradation), osteocalcin (OC) (bone formation), NTX-I and ICTP (both type I collagen degradation). RESULTS: After 4 weeks, there was a significant decrease in serum MMP-3 levels in adalimumab-treated patients (P