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ObjectivesMacrophage subsets, activated by T cells, are increasingly recognised to play a central role in rheumatoid arthritis (RA) pathogenesis. Janus kinase (JAK) inhibitors have proven beneficial clinical effects in RA. In this study, we investigated the effect of JAK inhibitors on the generation of cytokine-activated T (Tck) cells and the production of cytokines and chemokines induced by Tck cell/macrophage interactions.MethodsCD14+monocytes and CD4+T cells were purified from peripheral blood mononuclear cells from buffy coats of healthy donors. As representative JAK inhibitors, tofacitinib or ruxolitinib were added during Tck cell differentiation. Previously validated protocols were used to generate macrophages and Tck cells from monocytes and CD4+T cells, respectively. Cytokine and chemokine including TNF, IL-6, IL-15, IL-RA, IL-10, MIP1α, MIP1β and IP10 were measured by ELISA.ResultsJAK inhibitors prevented cytokine-induced maturation of Tck cells and decreased the production of proinflammatory cytokines TNF, IL-6, IL-15, IL-1RA and the chemokines IL-10, MIP1α, MIP1β, IP10 by Tck cell-activated macrophages in vitro (pConclusionsOur findings show that JAK inhibition disrupts T cell-induced macrophage activation and reduces downstream proinflammatory cytokine and chemokine responses, suggesting that suppressing the T cell-macrophage interaction contributes to the therapeutic effect of JAK inhibitors.

Kara et al. show that ACKR4 regulates early activated B cell differentiation fate. ACKR4 limits the early proliferation of activated B cells by restricting their initial access to the splenic interfollicular zone., Activated B cells can initially differentiate into three functionally distinct fates—early plasmablasts (PBs), germinal center (GC) B cells, or early memory B cells—by mechanisms that remain poorly understood. Here, we identify atypical chemokine receptor 4 (ACKR4), a decoy receptor that binds and degrades CCR7 ligands CCL19/CCL21, as a regulator of early activated B cell differentiation. By restricting initial access to splenic interfollicular zones (IFZs), ACKR4 limits the early proliferation of activated B cells, reducing the numbers available for subsequent differentiation. Consequently, ACKR4 deficiency enhanced early PB and GC B cell responses in a CCL19/CCL21-dependent and B cell–intrinsic manner. Conversely, aberrant localization of ACKR4-deficient activated B cells to the IFZ was associated with their preferential commitment to the early PB linage. Our results reveal a regulatory mechanism of B cell trafficking via an atypical chemokine receptor that shapes activated B cell fate.

B1 B cells produce natural IgM and play a critical role in the early defense against bacterial and viral infection. The polyreactive IgM also contributes to the clearance of apoptotic products and plays an important role in autoimmune pathogenesis. However, the mechanism of activation and proliferation of B1 cells remains obscure. In this study, we report that IL-33, a new member of IL-1 family, activates B1 cells, which express the IL-33 receptor α, ST2. IL-33 markedly activated B1 cell proliferation and enhanced IgM, IL-5, and IL-13 production in vitro and in vivo in a ST2-dependent manner. The IL-33–activated B1 cell functions could be largely abolished by IL-5 neutralization and partially reduced by T cell or mast cell deficiency in vivo. ST2-deficient mice developed less severe oxazolone-induced contact sensitivity (CS) than did wild-type (WT) mice. Furthermore, IL-33 treatment significantly exacerbated CS in WT mice with enhanced B1 cell proliferation and IgM and IL-5 production. Moreover, IL-33–activated B1 cells from WT mice could adoptively transfer enhanced CS in ST2−/− mice challenged with IL-33. Thus, we demonstrate, to the best of our knowledge, a hitherto unrecognized mechanism of B1 cell activation and IL-33 function, and suggest that IL-33 may play an important role in delayed-type hypersensitivity.

LPS comprises a major PAMP and is a key target of the immune system during bacterial infection. While LPS can be recognised by innate immune cells via the TLR4 complex, it is unknown whether T lymphocytes, especially CD8(+) T cells are also capable of doing so. We report here that naive human CD8(+) T cells, after activation by TCR stimulation, express surface TLR4 and CD14. These activated CD8(+) T cells can then secrete high concentrations of IFN-gamma, granzyme and perforin in response to LPS. These effects can be specifically inhibited using siRNA for TLR4. Furthermore, LPS can synergize with IL-12 to polarize the CD8(+) T cells into cytotoxic T-cell 1 (Tc1) that produce IFN-gamma but not IL-4, with or without TCR activation. Moreover, CD8(+)CD45RO(+) memory T cells constitutively expressed TLR4 and markedly enhanced IFN-gamma production when stimulated with LPS. In contrast, activated murine CD8(+) T cells lack TLR4 and CD14 expression and fail to respond to LPS for proliferation and cytokine production. Thus, human but not murine CD8(+) T cells are able to directly recognise bacterial LPS via LPS receptor complex and TLR4 provides a novel signal for the activation of effector and memory human CD8(+) T cells.

Galectin-3 is a β-galactoside-binding lectin that plays an important role in inflammatory diseases. It also interacts with the surface carbohydrates of many pathogens, including LPS. However, its role in infection is not fully understood. Data presented herein demonstrate for the first time that galectin-3 is a negative regulator of LPS-induced inflammation. Galectin-3 is constitutively produced by macrophages and directly binds to LPS. Galectin-3-deficient macrophages had markedly elevated LPS-induced signaling and inflammatory cytokine production compared with wild-type cells, which was specifically inhibited by the addition of recombinant galectin-3 protein. In contrast, blocking galectin-3 binding sites by using a neutralizing Ab or its ligand, β-lactose, enhanced LPS-induced inflammatory cytokine expression by wild-type macrophages. In vivo, mice lacking galectin-3 were more susceptible to LPS shock associated with excessive induction of inflammatory cytokines and NO production. However, these changes conferred greater resistance to Salmonella infection. Thus, galectin-3 is a previously unrecognized, naturally occurring, negative regulator of LPS function, which protects the host from endotoxin shock but, conversely, favors Salmonella survival.

Background Microparticles (MPs) have been described to function as extracellular vehicles that shuttle microRNAs (miR) from platelets to endothelial cells1 and regulate recipient cell gene expression. Crosstalk of synovial monocytes (MC) with synovial fibroblasts (SF) is a key step to the inflammatory process in rheumatoid arthritis (RA). Objectives Herein, we investigated whether delivery of specific miRs by MC-derived MPs affects RASF behaviour. Methods Blood samples of healthy volunteers and RA patients were collected. MPs were obtained by differential centrifugation. CD14+ cells were isolated from PBMCs by using positive selection and the cells were stimulated with 50 ng/ml recombinant human M-CSF for 6 days. Scanning electron microscopy, nanosight trafficking analysis and flow cytometry (FACS) have been applied to characterize MPs from human CD14+MCs and THP-1 cells. Total and smallRNA sequencing were performed on macrophages (n=4) and RASFs (n=4) as well as on macrophage-derived MPs (n=4). (Pre-)miR-223 expression and copy number was quantified in cells or tissues via TLDA, qPCR & in situ hybridisation with specific primers and probes. Targeted pathways were identified using prediction algorithms (TargetScanHuman6.2). Transfer of miR-223 from MCs to SFs was determined by fluorescence microscopy, direct cell co-culture and FACS, as well as transwell co-culture. RASF proliferation assays with FGF-2 as positive control, as well as THP-1 derived MPs, miR-223 mimic, miR-223 inhibitor, control mimic and inhibitor were carried out. Results MPs from MCs & THP-1 cells are about 250nm in size (range 50–800nm). SmallRNA sequencing revealed high levels of miR-223 in macrophages as opposed to a lack of its expression in RASF. If co-cultured with MCs for 3d, RASF acquire miR-223 expression, but not pre-miR-223 to a significant extent (n=6, Ct values Conclusions miR-223 transport from MCs to SFs by MPs represents a novel intercellular mechanism that could stimulate SF proliferation during initiation or chronicity of synovial inflammation in RA. References Laffont, B. et al. Blood (2013) Acknowledgement We wish to thank Derek Baxter, Diane Vaughan, Margaret Mullin, Pawel Herzyk, Julie Galbraith, Russka Shumnalieva for their support and technical assistance. Disclosure of Interest None declared

Multiple genetic studies in humans indicate a role for solute carrier family 11a member 1 [SLC11A1; formerly natural resistance-associated macrophage protein 1 (NRAMP1)] in autoimmune disease susceptibility, including ulcerative colitis. Murine Slc11a1 has many pleiotropic effects on macrophage activation and proinflammatory responses. To determine which of these are important in ulcerative colitis, we established a phenotype for oral dextran sulfate sodium (DSS)-induced acute colitis in congenic Slc11a1 wild-type (wt) and mutant (mt) mice on a B10 background. For over 7 days of treatment with 2% DSS in the drinking water, Slc11a1 wt mice showed enhanced acute ulcerative colitis, as demonstrated by significantly greater body weight loss and reduction in colon length, as well as a marked increase in monocyte/macrophage inflammatory infiltrates and histopathology changes in the colon. This was accompanied by a clear, inverse relationship between IFN-γ and IL-10 responses in Slc11a1 wt compared with mt mice, resulting in a significantly higher ratio of IFN-γ:IL-10 in wt compared with mt mice in lymph node and splenic T cells. RNase protection assays confirmed the presence of significantly higher IFN-γ at the RNA level in the colons of wt compared with mt mice at Day 7 of treatment. Interestingly this was accompanied by significantly enhanced RNA levels for the acute-phase protein IL-6, which is known to inhibit the generation of forkhead box P3+ regulatory T cells and help to drive the differentiation of Th17 from naive T cells and not by differences in RNA for IL-12p35 or IL-12p40 molecules that dimerize to form the Th1-inducing cytokine IL-12.

Background The cytokine interleukin 33 (IL-33), a member of the IL-1 family, is released in response to various types of endothelial or epithelial cell damage. IL-33 is constitutively expressed in epidermal keratinocytes, upregulated in inflamed skin and acts as an endogenous danger signal that mediates the recruitment of immune cells to sites of cellular damage. Our preliminary data have shown that psoriatic lesions express more nuclear IL-33 compared to perilesional biopsies from the same patients and mature IL-33 induces skin inflammation in a mouse model. Objectives To determine the role and function of IL-33 in skin inflammation, in particular to understand the mechanism how IL-33 contributes to the pathology and recruitment of inflammatory cells. Materials and Methods Transgenic mice with skin-specific expression of full length IL-33 were generated via pronuclear injection of K14-IL-33. This construct with IL-33 under keratin 14 promotor was tested for the functionality in vitro following the generation of the transgenic mice. For analysis control littermates with no transgene were used. K14-IL-33 mice were tested in various skin inflammation models including TLR7 agonistic stimulation with imiquimod 5%, ear injection model (rIL-23, 500 ng/ear for 14 days), and skin tape stripping. As readout we used caliper measurement of the ear thickness, skin histology H&E staining and immunohistochemistry as well as mRNA expression by quantitative RT-PCR. Results Pronuclear injection resulted in 16 pups with 2 transgenic mice expressing the IL-33 transgene. K14-IL-33 mice were born and developed normally with no obvious phenotype. Increased expression in the skin in K14-IL-33 compare to wildtype (WT) mice was detected using RT-PCR and immunohistology; as expected IL-33 was localised in the nuclei of the keratinocytes. No significant expression was measured in other organs. Due to the lack of a skin phenotype we performed different skin inflammation models in the K14-IL-33 mouse. Neither TLR7 stimulation via imiquimod, ear injection of IL-23, a pivotal cytokine for psoriasis, nor skin tape stripping as a model for chronic skin damage induced a clinical/histological change compared to littermates. Conclusions We demonstrate that local skin overexpression of the alarmin IL-33 does not induce spontaneous or triggered skin inflammation. In comparison to a published K14-IL-33 mouse with atopic inflammatory phenotype our data could not reproduce the described phenotype. These data suggest that different expression thresholds might lead to exacerbation of skin diseases.

Psoriasis is an inflammatory skin disease characterised by hyperproliferation of keratinocytes, infiltration of inflammatory cells, impaired barrier function and alteration of tight junction proteins. The interleukin 1 (IL1) family member IL33 has recently been described as an alarmin expressed in epithelial cells at body barriers, …

Background Tendon disorders comprise the commonest musculoskeletal clinical presentation and accordingly represent a significant unmet clinical need. Recent animal and human studies have highlighted a central role for inflammation and subsequent matrix remodelling in tendon pathophysiology. Interleukin-33 (IL-33) acting via its receptor ST2 is increasingly recognised as a critical endogenous tissue danger signal that can initiate inflammation. Objectives We investigated the role of the IL-33/ST2 signallingpathway on tendon pathology in animal and human models of tendinopathy. Results We show here that human and mouse tendons over express IL-33 when damaged, and drives tenocytes to undergo an early switch in collagen matrix production toward a collagen III phenotype. Moreover, administration of rh IL-33 in an in vivo model of tendinopathy results in reduced biomechanical tendon strength at early time points while neutralising antibodies to IL-33 attenuates these changes. Furthermore we highlight a key regulatory role for the microRNA29 family in IL-33 induced collagen matrix changes through direct targeting of the soluble ST2 receptor and Collagen III. Image/graph Conclusions For the first time we provide evidence that IL-33/miR-29 pathway orchestrates inflammatory and matrix responses following tissue injury, and thus may offer future strategies to treat tendon diseases diseases. Disclosure of Interest None Declared

Background Cytokines regulate a broad range of inflammatory pathways in the pathogenesis of Rheumatoid Arthritis (RA), and cytokine blockade against TNF-a and IL-6 has offered substantial advances in the treatment of articular inflammation. However, a large proportion of patients become unresponsive or exhibit only a partial response to treatment and new therapies are thus required. IL-21 is a member of the four-a-helix bundle family of cytokines that mediates pleiotropic effects through the IL-21 receptor (IL-21R)[1]. Since potency of IL-21 is mainly dependent on presence and abundance of its receptor on different cell types, the objective of this study was to characterize expression of IL-21R in the synovium and blood of patients with RA. Methods Immunohistochemistry for IL-21R was carried out on synovial tissue samples derived by arthroplasty from patients with RA (n=5) obtained from our synovial tissue bank. Peripheral blood mononuclear cells (PBMC) from 10 RA patients and 6 healthy subjects were isolated using Histopaque (Sigma). Cells were analyzed by flow cytometry for IL-21R expression on T cells (CD3/CD4/CD8), B cells (CD19/CD27) and NK cells (CD16/CD56). In parallel, RNA was purified from total PBMC using Qiagen RNeasy kit, converted into cDNA using Quantitect reverse transcription kit (Qiagen) and the absolute levels of IL-21R transcripts measured by quantitative Real-Time PCR. Samples were normalized against endogenous GAPDH control. Results Expression of IL-21R was detected in all synovial RA tissues tested. The IL-21R + cells were located in the synovial intimal and sub-lining layers and in lymphoid aggregates. Flow cytometric analysis of PBMC revealed IL-21R highly expressed on both CD4 + (12.58 mean fluorescent intensity (MFI)) and CD8 + (13.69 MFI) T cells, as well as on a subset of NK cells (19.15 MFI) in RA patients. Interestingly, the amount of IL-21R per CD8 + T cell is higher in patients with RA than in healthy subjects (13.69 MFI vs. 5.06 MFI, p=0.007, respectively). On B cells, IL-21R expression was higher on the CD27 - fraction of naive B cells (37.02 MFI), with lower expression on the CD27 + memory B cells (32.22 MFI). In this study cohort, Real-Time PCR revealed IL-21R to be at significantly higher transcript levels in total PBMC of healthy subjects compared to RA patients (2239 vs. 827.3 mean gene copy numbers, normalized to 10 4 copy numbers of GAPDH, p=0.001, respectively), suggesting discordant mRNA and protein expression indicating complex regulation of IL-21R biology. Conclusions Our results show abundant expression of IL-21R in synovial tissue as well as peripheral blood of RA patients. Diversity in the signature between RA patients and healthy volunteers requires further phenotyping at the protein as well as transcriptional level. Such data will be essential in translating to proof of concept clinical trials. References Habib T. et al., J Allergy Clin Immunol. 2003 Dec;112(6):1033-45. Disclosure of Interest M. Frleta: None Declared, V. King: None Declared, J. Reilly: None Declared, S. Kerr: None Declared, D. Gilchrist: None Declared, D. Tornehave Shareholder of: Minor stock holder at Novo Nordisk A/S, Employee of: Novo Nordisk A/S, A. Neisig Employee of: Novo Nordisk A/S, D. Lundsgaard Shareholder of: Minor stock holder at Novo Nordisk A/S, Employee of: Novo Nordisk A/S, A. Miller: None Declared, I. McInnes: None Declared

Background and objectives Cytokines regulate a broad range of inflammatory pathways in the pathogenesis of Rheumatoid Arthritis (RA), and cytokine blockade against tumour necrosis factor α and IL-6 has offered substantial advances in the treatment of articular inflammation. However, a large proportion of patients will not respond or exhibit only a partial response to treatment and new therapies are thus required. IL-21 is a member of the four-α-helix bundle family of cytokines that mediates pleiotropic effects through the IL-21 receptor (IL-21R). Since potency of IL-21 is mainly dependent on presence and abundance of its receptor on different cells types, the objective of this study was to characterise expression of IL-21R in the synovium and blood of patients with RA. Materials and methods Immunohistochemistry for IL-21R was carried out on synovial tissue samples derived by arthroplasty from patients with RA (n=5) obtained from the Institute of Infection, Immunity and Inflammation Research Tissue Bank. Mononuclear cells were separated out from peripheral blood (PBMC) of 10 RA patients or 3 healthy controls on a density gradient using Histopaque (Sigma) and analysed by flow cytometry for IL-21R expression on T cells (CD3/CD4/CD8), B cells (CD19/CD27) and NK cells (CD16/CD56). Results Expression of IL-21R was detected in 5/5 synovial RA tissues. The IL-21R + cells were located in the synovial intimal and sublining layers and in lymphoid aggregates. Flow cytometric analysis on blood PBMC revealed that IL-21R is highly expressed on both CD4 + (73.04%, 12.58 MFI) and CD8 + (50.88%, 13.69 MFI) T cells, as well as on a proportion of NK cells (73.83%, 19.15 MFI) in RA patients. On B cells, IL-21R expression was higher on the CD27 - fraction of naive B cells (95.19%, 37.02 MFI), with lower expression on the CD27 + memory B cells (15.2%, 32.22 MFI). Conclusions Our results show increased expression of IL-21R in established RA synovial tissue and peripheral blood, and indicate that targeting of the IL-21/IL-21R pathway may be a valid therapeutic strategy for the treatment of RA.