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The cytokine interleukin-6 (IL-6) signals through three mechanisms called classic signaling, trans-signaling, and trans-presentation. IL-6 trans-signaling is distinctly mediated through a soluble form of its transmembrane receptor IL-6R (sIL-6R) and the coreceptor gp130 and is implicated in multiple autoimmune diseases. Although a soluble form of gp130 (sgp130) inhibits only IL-6 trans-signaling, it also blocks an analogous trans-signaling mechanism of IL-11 and its soluble receptor sIL-11R. Here, we report miniaturized chimeric soluble gp130 variants that efficiently trap IL-6:sIL-6R but not IL-11:sIL-11R complexes. We designed a novel IL-6 trans-signaling trap by fusing a miniaturized sgp130 variant to an IL-6:sIL-6R complex–binding nanobody and the Fc portion of immunoglobulin G (IgG). This trap, called cs-130Fc, exhibited improved inhibition of as well as increased selectivity for IL-6 trans-signaling compared to the conventional fusion protein sgp130Fc. We introduced affinity-enhancing mutations in cs-130Fc and sgp130Fc that further improved selectivity toward IL-6 trans-signaling. Moreover, cs-130Fc efficiently inhibited the expansion of T helper 17 (TH17) cells in cultures of mouse CD4+ T cells treated with IL-6:sIL-6R. Thus, these variants may provide or lead to the development of more precisely targeted therapeutics for inflammatory disorders associated with IL-6 trans-signaling.

The identification of elevated IL-6 levels in patients with severe COVID-19 led to the rapid development of clinical trials targeting this cytokine. Overall, these trials do not support the widespread use of IL-6 antagonists in hospitalized patients with mild-to-moderate disease, but IL-6 antagonists may be beneficial when rapidly deployed in patients with severe COVID-19, as we discuss here.

The classification of interleukin-6 (IL-6) as a pro-inflammatory cytokine undervalues the biological impact of this cytokine in health and disease. With broad activities affecting the immune system, tissue homeostasis and metabolic processes, IL-6 displays complex biology. The significance of these involvements has become increasingly important in clinical settings where IL-6 is identified as a prominent target for therapy. Here, clinical experience with IL-6 antagonists emphasises the need to understand the context-dependent properties of IL-6 within an inflammatory environment and the anticipated or unexpected consequences of IL-6 blockade. In this review, we will describe the immunobiology of IL-6 and explore the gamut of IL-6 bioactivity affecting the clinical response to biological drugs targeting this cytokine pathway.

Genome-wide association studies have identified PTPN2 as an important non-major histocompatibility complex gene for autoimmunity. Single nucleotide polymorphisms that reduce PTPN2 expression have been linked with the development of varied autoimmune disorders, including type 1 diabetes. The tyrosine-phosphatase PTPN2 attenuates T cell receptor and cytokine signalling in T cells to maintain peripheral tolerance, but the extent to which PTPN2-deficiency in T cells might influence type 1 diabetes onset remains unclear. Non-Obese Diabetic (NOD) mice develop spontaneous autoimmune type 1 diabetes, similar to that seen in humans. T cell PTPN2-deficiency in NOD mice markedly accelerated the onset and increased the incidence of type 1 diabetes, as well as that of other disorders, including colitis and Sjogren’s syndrome. Although PTPN2-deficiency in CD8+ T cells alone was able to drive the destruction of pancreatic β cells and onset of diabetes, T cell-specific PTPN2-deficiency was also accompanied by increased CD4+ T-helper type 1 differentiation and T follicular helper cell polarisation and an increased abundance of B cells in pancreatic islets as seen in human type 1 diabetes. These findings causally link PTPN2-deficiency in T cells with the development of type 1 diabetes and associated autoimmune co-morbidities.

The cytokine IL-6 controls the survival, proliferation and effector characteristics of lymphocytes through activation of the transcription factors STAT1 and STAT3. While STAT3 activity is an ever-present feature of IL-6 signaling in CD4 + T cells, prior activation via the T cell antigen receptor limits IL-6’s control of STAT1 in effector and memory populations. Here we found that phosphorylation of STAT1 in response to IL-6 was regulated by the tyrosine phosphatases PTPN2 and PTPN22 expressed in response to the activation of naïve CD4 + T cells. Transcriptomics and chromatin immunoprecipitation–sequencing (ChIP-seq) of IL-6 responses in naïve and effector memory CD4 + T cells showed how the suppression of STAT1 activation shaped the functional identity and effector characteristics of memory CD4 + T cells. Thus, tyrosine phosphatases induced by the activation of naïve T cells determine the way activated or memory CD4 + T cells sense and interpret cytokine signals.

No abstract available.

Acting in concert with TGF-β, interleukin-6 (IL-6) signaling induces T helper 17 (TH17) cell development by programming TH17-related genes via signal transducers and activators of transcription 3 (STAT3). A role for IL-6 signaling beyond the inductive phase of TH17 cell development has not been defined because IL-23 signaling downstream of TH17 cell induction also activates STAT3 and is thought responsible for TH17 cell maintenance. Here, we find that IL-6 signaling is required for both induction and maintenance of mouse TH17 cells; IL-6Rα-deficient TH17 cells rapidly lost their TH17 phenotype and did not cause disease in two models of colitis. Cotransfer of wild-type TH17 cells with IL-6Rα-deficient TH17 cells induced colitis but was unable to rescue phenotype loss of the latter. High IL-6 expression in the colon promoted classic, or cis, rather than transreceptor signaling that was required for maintenance of TH17 cells. Thus, ongoing classic IL-6 signaling underpins the TH17 program and is required for TH17 cell maintenance and function.

Emerging studies increasingly demonstrate the importance of the throat and salivary glands as sites of virus replication and transmission in early COVID-19 disease. SARS-CoV-2 is an enveloped virus, characterized by an outer lipid membrane derived from the host cell from which it buds. While it is highly sensitive to agents that disrupt lipid biomembranes, there has been no discussion about the potential role of oral rinsing in preventing transmission. Here, we review known mechanisms of viral lipid membrane disruption by widely available dental mouthwash components that include ethanol, chlorhexidine, cetylpyridinium chloride, hydrogen peroxide, and povidone-iodine. We also assess existing formulations for their potential ability to disrupt the SARS-CoV-2 lipid envelope, based on their concentrations of these agents, and conclude that several deserve clinical evaluation. We highlight that already published research on other enveloped viruses, including coronaviruses, directly supports the idea that oral rinsing should be considered as a potential way to reduce transmission of SARS-CoV-2. Research to test this could include evaluating existing or specifically tailored new formulations in well-designed viral inactivation assays, then in clinical trials. Population-based interventions could be undertaken with available mouthwashes, with active monitoring of outcome to determine efficacy. This is an under-researched area of major clinical need.

The cytokine interleukin (IL)−6 performs a diverse portfolio of functions in normal physiology and disease. These functions extend beyond the typical role for an inflammatory cytokine, and IL-6 often displays hormone-like properties that affect metabolic processes associated with lipid metabolism, insulin resistance, and the neuroendocrine system. Consequently, the biology of IL-6 is complex. Recent advances in the field have led to novel interpretations of how IL-6 delivers immune homeostasis in health and yet drives disease pathology during infection, autoimmunity, and cancer. Various biological drugs that target IL-6 are in clinical practice or emerging in clinical trials and pre-clinical development programmes. The challenge is knowing how and when to apply these therapies. In this review, we will explore the biology behind IL-6 directed therapies and identify some key hurdles for future investigation.

Compounds that target interleukin (IL)−6 pathways include antibodies against the IL-6 receptor or ligand, and inhibitors of IL-6 signal transduction. The anti-IL-6 receptor (IL-6R) monoclonal antibody tocilizumab has been licensed for several years; data from multiple studies demonstrate its efficacy and tolerability in rheumatoid arthritis as monotherapy or in combination with methotrexate. In addition, another anti-IL-6R monoclonal antibody, sarilumab, has recently been approved in both the US and EU. Anti-IL-6 monoclonal antibodies include olokizumab and clazakizumab, which both have data from phase II studies, as well as sirukumab which has completed phase III trials but may not be brought to the market. Comparative data for olokizumab versus tocilizumab intervention in rheumatoid arthritis suggest no difference in efficacy between blocking the receptor or the ligand. Head-to-head studies are needed to determine whether inhibition of the Janus kinase pathway is similar in its overall efficacy to direct inhibition of IL-6 or its receptor. The IL-6 inhibitors appear to be more effective when combined with methotrexate. However, they have shown superiority to tumour necrosis factor inhibitors when used as monotherapy, and may have an advantage in patients who cannot use methotrexate or any other conventional synthetic disease modifying anti-rheumatic drug. Regarding disease activity assessment, CDAI is a more appropriate measure than DAS28 when looking at the effect of IL-6 inhibition, as these agents interfere with the acute phase response, which is heavily weighted in the formula of DAS28.

Interleukin (IL)−6 represents one of several possible targets for the treatment of rheumatoid arthritis. Drugs targeting IL-6 can be divided into monoclonal antibodies against IL-6 itself and monoclonal antibodies against the IL-6 receptor. Both types of agent inhibit both classical signalling through membrane­-bound IL-6 receptor, and trans-signalling via formation of a complex between IL-6 and soluble IL-6 receptor. The IL-6 receptor blockers tocilizumab and sarilumab inhibit the low affinity binding of IL-6 to its receptor. The anti-IL-6 agents clazakizumab and vobarilizumab also block binding of IL-6 to the receptor, while olokizumab blocks the higher affinity interaction of the IL-6-receptor complex with gp130. The doses and dosing intervals of the biologics targeting different elements vary, but no major differences in efficacy or safety have yet been seen between the two approaches, although more studies are needed in this area. In addition to the different blocking actions of monoclonal antibodies, we consider therapeutic strategies including the timing of IL-6 blockade and the use of monotherapy versus the addition of methotrexate.

Interleukin (IL)−6 inhibition has been approved for the treatment of rheumatoid arthritis, systemic juvenile arthritis, polyarticular juvenile idiopathic arthritis, giant cell arteritis, and, in some countries, Castleman’s disease. IL-6 has also been implicated in several non-rheumatoid arthritis inflammatory and immune conditions such as systemic sclerosis, vasculitides, systemic lupus erythematous, and psoriatic arthritis. In orphan diseases, such as systemic sclerosis, which are associated with significant morbidity and mortality and for which there are no approved treatments, IL-6 inhibition may offer a promising treatment strategy. It is also becoming clear that IL-6 may have an important role not only in inflammatory and immune diseases but also in non-immune mediated diseases such as endogenous depression and depression associated with chronic inflammatory conditions. Several studies have explored the effect of IL-6 pathway inhibition in Crohn’s disease and adult-onset Still’s disease, suggesting that IL-6 may be important in their pathogenesis.

Key Points Question Is genetic liability for rheumatoid arthritis associated with cognitive and psychiatric phenotypes in children prior to the clinical onset of disease? Findings In this cohort study of 7977 children and adolescents, genetic liability for rheumatoid arthritis was associated with lower total, performance, and verbal IQ at age 8 years and symptoms of hyperactivity and inattention from ages 4 to 16 years. However, there was little evidence of association with other domains of psychopathology. Meaning These findings support a primary etiological association between genetic risk for rheumatoid arthritis and cognitive phenotypes that is not simply secondary to disease-related processes or reverse causation., This cohort study investigates whether genomic risk for rheumatoid arthritis is associated with cognitive and psychiatric symptoms in children and adolescents., Importance The association of rheumatoid arthritis (RA) with cognitive and psychiatric phenotypes has been recognized. However, it is not known whether these phenotypes are a consequence of disease-related factors, such as pain, or reflect shared etiological factors. Objective To investigate whether genomic risk for RA is associated with cognitive and psychiatric symptoms in children and adolescents. Design, Setting, and Participants This cohort study analyzed data from 3296 to 5936 adolescents (depending on outcome) from the Avon Longitudinal Study of Parents and Children. Clinical and questionnaire data were collected periodically from September 6, 1990, with collection ongoing, and analyzed from August 21, 2017, to May 21, 2018. Exposures Polygenic risk scores (PRSs) for RA. Main Outcomes and Measures Measures of cognition (including IQ, working memory, verbal learning, processing speed, problem solving, selective attention, and attentional control) and psychopathology (including anxiety, depression, negative symptoms, psychotic experiences, attention-deficit/hyperactivity disorder, and hyperactive and inattentive symptoms) in childhood and adolescence. Results Polygenic risk scores for RA were generated for 7977 children and adolescents (3885 [48.7%] female). Of these 7977 participants, 9 (0.11%) had a known diagnosis of RA at age 22 years. Increased PRS for RA was associated with lower total IQ (β, −0.05; 95% CI, −0.07 to −0.02; P

ObjectivesMast cells (MCs) are involved in the pathogenesis of rheumatoid arthritis (RA). However, their contribution remains controversial. To establish their role in RA, we analysed their presence in the synovium of treatment-naïve patients with early RA and their association and functional relationship with histological features of synovitis.MethodsSynovial tissue was obtained by ultrasound-guided biopsy from treatment-naïve patients with early RA (n=99). Immune cells (CD3/CD20/CD138/CD68) and their relationship with CD117+MCs in synovial tissue were analysed by immunohistochemistry (IHC) and immunofluorescence (IF). The functional involvement of MCs in ectopic lymphoid structures (ELS) was investigated in vitro, by coculturing MCs with naïve B cells and anticitrullinated protein antibodies (ACPA)-producing B cell clones, and in vivo in interleukin-27 receptor alpha (IL27ra)-deficient and control mice during antigen-induced arthritis (AIA).ResultsHigh synovial MC counts are associated with local and systemic inflammation, autoantibody positivity and high disease activity. IHC/IF showed that MCs reside at the outer border of lymphoid aggregates. Furthermore, human MCs promote the activation and differentiation of naïve B cells and induce the production of ACPA, mainly via contact-dependent interactions. In AIA, synovial MC numbers increase in IL27ra deficient mice, in association with ELS and worse disease activity.ConclusionsSynovial MCs identify early RA patients with a severe clinical form of synovitis characterised by the presence of ELS.

The IL-6 family of cytokines consists of IL-6, IL-11, IL-27, IL-31, oncostatin M (OSM), leukaemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), cardiotrophin 1 (CT-1) and cardiotrophin-like cytokine factor 1 (CLCF1). Membership of this cytokine family is defined by usage of common β-receptor signalling subunits, which activate various intracellular signalling pathways. Each IL-6 family member elicits responses essential to the physiological control of immune homeostasis, haematopoiesis, inflammation, development and metabolism. Accordingly, distortion of these cytokine activities often promotes chronic disease and cancer; the pathological importance of this is exemplified by the successful treatment of certain autoimmune conditions with drugs that target the IL-6 pathway. Here, we discuss the emerging roles for IL-6 family members in infection, chronic inflammation, autoimmunity and cancer and review therapeutic strategies designed to manipulate these cytokines in disease.

Summary The cytokine interleukin (IL)-6 is a major therapeutic target for the treatment of various inflammatory and autoimmune diseases. While IL-6 receives considerable attention in studies of innate and adaptive immunity, the IL-6-related family member IL-27 is recognized increasingly for its effects on cellular proliferation, differentiation and leucocyte effector functions. Both cytokines activate responses in myeloid and stromal tissue cells, where they direct the transition from innate to adaptive immunity. However, they are identified frequently as lymphokines that control responses in T cells and B cells. In this regard, IL-27 often opposes the action of IL-6. Here, we will review the role of IL-6 and IL-27 in inflammation, with a particular focus on inflammatory arthritis, and discuss their importance in the diagnosis, stratification and treatment of autoimmune disease.

Anti-microbial host defence is dependent on the rapid recruitment of inflammatory cells to the site of infection, the elimination of invading pathogens, and the efficient resolution of inflammation so as to minimise damage to the host. The peritoneal cavity provides an easily accessible and physiologically relevant system where the delicate balance of these processes may be studied. Here, we describe murine models of peritoneal inflammation that enable studies of both competent anti-microbial immunity and inflammation associated tissue damage as a consequence of recurrent bacterial challenge. The inflammatory hallmarks of these models reflect the clinical and molecular features of peritonitis episodes seen in renal failure patients on peritoneal dialysis. Development of these models relies on the preparation of a cell-free supernatant derived from an isolate of Staphylococcus epidermidis (termed SES). Intraperitoneal administration of SES induces a TLR2-driven acute inflammatory response that is characterised by an initial transient influx of neutrophils that are replaced by a more sustained recruitment of mononuclear cells and lymphocytes. Adaptation of this model using a repeated administration of SES allows investigations into the development of adaptive immunity and memory responses, and the hallmarks associated with tissue remodelling and fibrosis. These models are therefore clinically relevant and provide exciting opportunities to study both innate and adaptive immune responses in the control of bacterial infection and pathogenesis.

The IL-6 signaling complex is described as a hexamer, formed by the association of two IL-6·IL-6 receptor (IL-6R)·gp130 trimers, with gp130 being the signal transducer inducing cis- and trans-mediated signaling via a membrane-bound or soluble form of the IL-6R, respectively. 25F10 is an anti-mouse IL-6R mAb that binds to both membrane-bound IL-6R and soluble IL-6R with the unique property of specifically inhibiting trans-mediated signaling events. In this study, epitope mapping revealed that 25F10 interacts at site IIb of IL-6R but allows the binding of IL-6 to the IL-6R and the recruitment of gp130, forming a trimer complex. Binding of 25F10 to IL-6R prevented the formation of the hexameric complex obligate for trans-mediated signaling, suggesting that the cis- and trans-modes of IL-6 signaling adopt different mechanisms for receptor complex assembly. To study this phenomenon also in the human system, we developed NI-1201, a mAb that targets, in the human IL-6R sequence, the epitope recognized by 25F10 for mice. Interestingly, NI-1201, however, did not selectively inhibit human IL-6 trans-signaling, although both mAbs produced beneficial outcomes in conditions of exacerbated IL-6 as compared with a site I-directed mAb. These findings shed light on the complexity of IL-6 signaling. First, triggering cis- versus trans-mediated IL-6 signaling occurs via distinctive mechanisms for receptor complex assembly in mice. Second, the formation of the receptor complex leading to cis- and trans-signaling biology in mice and humans is different, and this should be taken into account when developing strategies to inhibit IL-6 clinically.

Interleukin 6 (IL-6) has a broad effect on cells of the immune system and those not of the immune system and often displays hormone-like characteristics that affect homeostatic processes. IL-6 has context-dependent pro- and anti-inflammatory properties and is now regarded as a prominent target for clinical intervention. However, the signaling cassette that controls the activity of IL-6 is complicated, and distinct intervention strategies can inhibit this pathway. Clinical experience with antagonists of IL-6 has raised new questions about how and when to block this cytokine to improve disease outcome and patient wellbeing. Here we discuss the effect of IL-6 on innate and adaptive immunity and the possible advantages of various antagonists of IL-6 and consider how the immunobiology of IL-6 may inform clinical decisions.

Item does not contain fulltext Blood coagulation functions as part of the innate immune system by preventing bacterial invasion, and it is critical to stopping blood loss (hemostasis). Coagulation involves the external membrane surface of activated platelets and leukocytes. Using lipidomic, genetic, biochemical, and mathematical modeling approaches, we found that enzymatically oxidized phospholipids (eoxPLs) generated by the activity of leukocyte or platelet lipoxygenases (LOXs) were required for normal hemostasis and promoted coagulation factor activities in a Ca(2+)- and phosphatidylserine (PS)-dependent manner. In wild-type mice, hydroxyeicosatetraenoic acid-phospholipids (HETE-PLs) enhanced coagulation and restored normal hemostasis in clotting-deficient animals genetically lacking p12-LOX or 12/15-LOX activity. Murine platelets generated 22 eoxPL species, all of which were missing in the absence of p12-LOX. Humans with the thrombotic disorder antiphospholipid syndrome (APS) had statistically significantly increased HETE-PLs in platelets and leukocytes, as well as greater HETE-PL immunoreactivity, than healthy controls. HETE-PLs enhanced membrane binding of the serum protein beta2GP1 (beta2-glycoprotein 1), an event considered central to the autoimmune reactivity responsible for APS symptoms. Correlation network analysis of 47 platelet eoxPL species in platelets from APS and control subjects identified their enzymatic origin and revealed a complex network of regulation, with the abundance of 31 p12-LOX-derived eoxPL molecules substantially increased in APS. In summary, circulating blood cells generate networks of eoxPL molecules, including HETE-PLs, which change membrane properties to enhance blood coagulation and contribute to the excessive clotting and immunoreactivity of patients with APS.

Background: The myeloid enzyme 12/15-lipoxygenase (LOX), which generates bioactive oxidized lipids, has been implicated in numerous inflammatory diseases, with several studies demonstrating an improvement in pathology in mice lacking the enzyme. However, the ability of 12/15-LOX to directly regulate B cell function has not been studied. Methods: The influence of 12/15-LOX on B cell phenotype and function, and IgM generation, was compared using wildtype (WT) and 12/15-LOX (Alox15-/-) deficient mice. The proliferative and functional capacity of splenic CD19+ B cells was measured in vitro in response to various toll-like receptor agonists. Results: WT and Alox15-/- displayed comparable responses. However in vivo, splenic B cell numbers were significantly elevated in Alox15-/- mice with a corresponding elevation in titres of total IgM in lung, gut and serum, and lower serum IgM directed against the 12/15-LOX product, 12-hydroxyeicosatetraenoic acid-phosphatidylethanolamine (HETE-PE). Discussion: Myeloid 12/15-LOX can regulate B cell numbers and innate immune antibody levels in vivo, potentially contributing to its ability to regulate inflammatory disease. Furthermore, the alterations seen in 12/15-LOX deficiency likely result from changes in the equilibrium of the immune system that develop from birth. Further studies in disease models are warranted to elucidate the contribution of 12/15-LOX mediated alterations in B cell numbers and innate immune antibody generation to driving inflammation in vivo.

Balancing the generation of immune responses capable of controlling virus replication with those causing immunopathology is critical for the survival of the host and resolution of influenza-induced inflammation. Based on the capacity of interleukin-6 (IL-6) to govern both optimal T-cell responses and inflammatory resolution, we hypothesised that IL-6 plays an important role in maintaining this balance. Comparison of innate and adaptive immune responses in influenza-infected wild-type control and IL-6-deficient mice revealed striking differences in virus clearance, lung immunopathology and generation of heterosubtypic immunity. Mice lacking IL-6 displayed a profound defect in their ability to mount an anti-viral T-cell response. Failure to adequately control virus was further associated with an enhanced infiltration of inflammatory monocytes into the lung and an elevated production of the pro-inflammatory cytokines, IFN-α and TNF-α. These events were associated with severe lung damage, characterised by profound vascular leakage and death. Our data highlight an essential role for IL-6 in orchestrating anti-viral immunity through an ability to limit inflammation, promote protective adaptive immune responses and prevent fatal immunopathology.

Background: Interleukin-6 (IL-6) signalling involves the interplay between IL-6, soluble IL-6 receptor (sIL-6R) and soluble gp130 (sgp130). IL-6 activity is modulated by the soluble receptors to produce both pro- and anti-inflammatory effects in human diseases and animal models. The expression and functional activity of these molecules in lungs of preterm ventilated infants is unknown. Objectives: We investigated this pathway in preterm infants who were at risk of developing chronic lung disease of prematurity (CLD). Methods: Cytokines and soluble receptors were measured in bronchoalveolar lavage fluid (BALF) from ventilated preterm infants ≤32 weeks of gestation who did or did not develop CLD. B9 cells, which specifically proliferate to IL-6, were used to assess BALF IL-6 functional activity. Results: Inflammatory cells, IL-8 and monocyte chemotactic protein-1 were increased in BALF from the CLD group when compared to the No CLD group (p < 0.05). BALF IL-6 and sIL-6R were similar in both groups. In contrast, BALF sgp130 and sgp130/sIL-6R were greater in the CLD group when compared to the No CLD group (p = 0.01 and p = 0.02, respectively). However, the increased BALF sgp130 did not appear to modulate the BALF IL-6 functional activity. Conclusion: Lung inflammation was observed in the CLD group. Increased BALF sgp130 was noted in the CLD group but it did not appear to modulate the pulmonary IL-6 bioactivity. Further research is needed to investigate the potential modulatory activity of sgp130 in the preterm lung.

Background: Mortality is increased in RA patients, mainly due to cardiovascular (CV) disease; however, the biologic mechanisms are unknown. Increased CV risk in RA is attributed to both traditional risk factors and systemic inflammation. CV risk scores, even after modification as recommended by EULAR, underestimate risk in RA and thus there is a need to develop a better means of risk stratification. Murine collagen-induced arthritis (mCIA) is associated with vascular dysfunction, characterized by reduced vascular constriction to 5- hydroxytryptamine (5-HT). This study was undertaken to characterize the relationship between vascular cell adhesion molecule 1 (VCAM-1), which is induced in pro-atherosclerotic conditions in the general population and vascular dysfunction in mCIA and CV risk in RA patients. Methods: mCIA was induced in DBA/1 mice. The severity of arthritis was assessed by the arthritis index score. Constriction responses to 5- HT were used to assess vascular function in isolated sections of thoracic aorta. Serum VCAM-1 was measured with ELISA. Serum VCAM-1, IL-6, ESR and CRP were measured with ELISA in RA patients [182 patients, 4 female:1 male, mean age 60 years (range 21–89), mean disease duration 11.4 years (S.D. 11)]. CV risk was calculated using the Framingham risk calculator and the QRISK2 algorithm. The latter includes RA as an independent CV risk factor. Results: In mCIA, VCAM-1 levels [mean 1500 mg/ml range (929–2528)] correlated with the arthritis index score (r¼0.43, P¼0.03) and negatively correlated with maximal aortic contraction (r¼�0.35, P10% over 10 years [1200 ng/ ml (S.D. 547)] compared with those with

Introduction: Although rheumatoid arthritis (RA) is a disease of articular joints, patients often suffer from co-morbid neuropsychiatric changes, such as anxiety, that may reflect links between heightened systemic inflammation and abnormal regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Here, we apply behavioral neuroscience methods to assess the impact of antigeninduced arthritis (AIA) on behavioral performance in wild type (WT) and interleukin-10 deficient (Il10-/-) mice. Our aim was to identify limb-specific motor impairments, as well as neuropsychological responses to inflammatory arthritis. Methods: Behavioral testing was performed longitudinally in WT and Il10-/- mice before and after the induction of arthritic joint pathology. Footprint analysis, beam walking and open field assessment determined a range of motor, exploratory and anxietyrelated parameters. Specific gene changes in HPA axis tissues were analyzed using qPCR. Results: Behavioral assessment revealed transient motor and exploratory impairments in mice receiving AIA, coinciding with joint swelling. Hind limb coordination deficits were independent of joint pathology. Behavioral impairments returned to baseline by 10 days post-AIA in WT mice. Il10-/- mice demonstrated comparable levels of swelling and joint pathology as WT mice up to 15 days post-AIA, but systemic differences were evident in mRNA expression in HPA axis tissues from Il10-/- mice post-AIA. Interestingly, the behavioral profile of Il10-/- mice revealed a significantly longer time post-AIA for activity and anxiety-related behaviors to recover. Conclusions: The novel application of sensitive behavioral tasks has enabled dissociation between behaviors that occur due to transient joint-specific pathology and those generated by more subtle systemic alterations that manifest post-AIA.

Purpose of review: Biological drugs that target the inflammatory cytokine interleukin-6 (IL-6) are increasingly considered as therapies for chronic disease and cancer. The purpose of this review is to place the biology of IL-6 in context. Here, we provide information on the biology behind IL-6 and consider mechanisms that are relevant to the application of IL-6 targeted therapies. Recent findings: The clinical blockade of IL-6 activity with tocilizumab has fuelled considerable interest in the biology behind this inflammatory cytokine. Although IL-6 impacts both innate and adaptive immunity, and the control of tissue homeostasis, the signalling mechanisms that control IL-6 responsiveness are complex. Several alternative IL-6-directed interventions with unique modes of action are now approaching the clinic. However, various questions still remain about how and when to block IL-6. Owing to the complexity of IL-6 biology, this is not trivial. In this review, we introduce the immunobiology of IL-6 and explore the different therapeutic strategies in development that inhibit IL-6 activity. Summary: Various inhibitors of IL-6 bioactivity are now in development of routine clinical practice. The key is to understand how best to apply these drugs. This review provides useful insight into the workings of IL-6 in health and disease.

Foxp3+ T regulatory (Treg) cells can be induced to produce interleukin (IL)-17 by in vitro exposure to proinflammatory cytokines, drawing into question their functional stability at sites of inflammation. Unlike their splenic counterparts, Treg cells from the inflamed central nervous system (CNS-Treg cells) during EAE resisted conversion to IL-17 production when exposed to IL-6. We show that the highly activated phenotype of CNS-Treg cells includes elevated expression of the Th1-associated molecules CXCR3 and T-bet, but reduced expression of the IL-6 receptor α chain (CD126) and the signaling chain gp130. We found a lack of IL-6 receptor on all CNS CD4+ T cells, which was reflected by an absence of both classical and trans-IL-6 signaling in CNS CD4+ cells, compared with their splenic counterparts. We propose that extinguished responsiveness to IL-6 (via down-regulation of CD126 and gp130) stabilizes the regulatory phenotype of activated Treg cells at sites of autoimmune inflammation.

Death receptor 3 (DR3; TNFRSF25) and its tumor necrosis factor–like ligand TL1A (TNFSF15) control several processes in inflammatory diseases through the expansion of effector T cells and the induction of proinflammatory cytokines from myeloid and innate lymphoid cells. Using wild-type (DR3 +/+ ) and DR3-knockout (DR3 −/− ) mice, we show that the DR3/TL1A pathway triggers the release of multiple chemokines after acute peritoneal inflammation initiated by a single application of Staphylococcus epidermidis supernatant, correlating with the infiltration of multiple leukocyte subsets. In contrast, leukocyte infiltration was not DR3 dependent after viral challenge with murine cytomegalovirus. DR3 expression was recorded on connective tissue stroma, which provided DR3-dependent release of chemokine (C-C motif) ligand (CCL) 2, CCL7, CXCL1, and CXCL13. CCL3, CCL4, and CXCL10 production was also DR3 dependent, but quantitative RT-PCR showed that their derivation was not stromal. In vitro cultures identified resident macrophages as a DR3-dependent source of CCL3. Whether DR3 signaling could contribute to a related peritoneal pathology was then tested using multiple applications of S. epidermidis supernatant, the repetitive inflammatory episodes of which lead to peritoneal membrane thickening and collagen deposition. Unlike their DR3 +/+ counterparts, DR3 −/− mice did not develop fibrosis of the mesothelial layer. Thus, this work describes both a novel function and essential requirement for the DR3/TL1A pathway in acute, resolving, and chronic inflammation in the peritoneal cavity.

The successful treatment of certain autoimmune conditions with the humanized anti-IL-6 receptor (IL-6R) antibody tocilizumab has emphasized the clinical importance of cytokines that signal through the β-receptor subunit glycoprotein 130 (gp130). In this Review, we explore how gp130 signaling controls disease progression and examine why IL-6 has a special role among these cytokines as an inflammatory regulator. Attention will be given to the role of the soluble IL-6R, and we will provide a perspective into the clinical blockade of IL-6 activity in autoimmunity, inflammation, and cancer.

In this Viewpoint, we concentrate on the aspects of macrophage biology that we believe are fundamental for an appropriate contextual understanding of macrophage function during acute inflammation. These are the different origins of macrophage populations (and the implications of this for the renewal of these populations in the adult); and the impact of specific homeostatic or disease-associated microenvironments upon cellular heterogeneity, activation and effector functions.

Chronic activation of the gastric mucosal adaptive immune response is a characteristic trait of gastric cancer. It has recently emerged that a new class of T helper (Th) cells, defined by their ability to produce interleukin (IL)-17A (Th17), is associated with a host of inflammatory responses, including gastritis. However, the role of these Th17 cells in the pathogenesis of gastric cancer is less clear. To formally address this, we employed gp130F/F mice, which spontaneously develop gastric inflammation-associated tumours akin to human intestinal-type gastric cancer. At the molecular level, these tumours demonstrate hyper-activation of the latent transcription factor signal transducer and activator of transcription (STAT)3 via the IL-6 cytokine family member, IL-11. In gp130F/F mice, the generation of Th17 cells, as well as the gastric expression of IL-17a and other Th17-related factors (Rort, IL-23), were augmented compared to wild-type gp130+/+ mice. Consistent with a role for IL-6 and STAT3 in regulating IL-17A, increased Th17 generation and gastric expression of Th17-related factors in gp130F/F mice were reduced to wild-type levels in gp130F/F:Stat3−/+ mice displaying normalized STAT3 activity, and also in gp130F/F:IL-6−/− mice. Importantly, genetic ablation of IL-17A in gp130F/F:IL-17a−/− mice did not suppress the initiation and growth of gastric tumours. Furthermore, IL-17A and RORC gene expression was strongly increased in human gastric biopsies from patients with gastritis, but not gastric cancer. Collectively, our data suggest that increased expression of Th17-related factors does not correlate with the molecular pathogenesis of gastric tumourigenesis.

Background. Bacterial infection remains a major cause of morbidity and mortality in peritoneal dialysis (PD) patients worldwide. Previous studies have identified a key role for mesothelial cells, lining the peritoneal cavity, in coordinating inflammation and host defense. Toll-like receptor (TLR) involvement in early activation events within the mesothelium, however, remains poorly defined. To investigate the initiation of bacterial peritonitis, we characterized TLR activation by bacterial ligands in human peritoneal mesothelial cells (HPMC). Methods. Primary HPMC were isolated from omental biopsies and TLR expression detected by real-time polymerase chain reaction (PCR), reverse transcription (RT)–PCR and flow cytometry. The responsiveness of HPMC to specific bacterial TLR agonists was determined using chemokine production as a biological readout. The requirement for CD14 in HPMC responses to a clinically relevant Staphylococcus epidermidis cell-free supernatant (SES) was investigated using soluble CD14 or anti-CD14-blocking antibodies. Results. Real-time PCR detected TLR1-6 messenger RNA expression in HPMC and responses to TLR2/1 and TLR2/6 ligands and SES. No cell surface TLR4 expression or responses to lipopolysaccharide were detectable in HPMC, but they did respond to flagellin, a TLR5 ligand. SES-mediated responses were dependent on TLR2 but did not require CD14 in HPMC for optimal efficiency, unlike peripheral blood mononuclear cells. HPMC expression of TLR2 was also modulated by TLR2 ligands and inflammatory cytokines. Conclusions. These data suggest that mesothelial cell activation by TLR2/1, TLR2/6 and TLR5 contributes to bacterial recognition influencing the course of the infective process and has implications for improving treatment of infection in PD patients.

Mature neutrophils are notoriously short-lived immune cells that cannot be genetically manipulated. Analysis of gene function therefore requires genetically modified animals, which is expensive, time-consuming, and costly in animal life. Analysis of gene function in neutrophils in a physiologically relevant context thus represents a significant problem in the field. We sought to overcome this obstruction in the field by developing a strategy for the analysis of gene function in neutrophils in a physiologically relevant context. Here, we demonstrate the functional relevance of in vitro conditional-Hoxb8 immortalized precursor-derived neutrophils. In vitro-derived neutrophils functionally resembled primary neutrophils, but critically, neutrophils generated in this way can be adoptively transferred into live animals and tracked during inflammatory responses using single-cell analysis to define functional attributes. We have validated this approach using CD11b-deficient neutrophils and replicated the key findings observed in gene-targeted animals and in naturally CD11b-deficient humans. Furthermore, we show that by retroviral transduction, one can generate stable alterations in the precursor cell lines and thus a continuous supply of functionally altered neutrophils. This novel technological advance offers for the first time the possibility of applying higher-throughput genetic modification and in vivo functional analysis to the neutrophil-lineage.

Background: Seasonal influenza A infection affects a significant cohort of the global population annually, resulting in considerable morbidity and mortality. Therapeutic strategies are of limited efficacy, and during a pandemic outbreak would only be available to a minority of the global population. Over-the-counter medicines are routinely taken by individuals suffering from influenza, but few studies have been conducted to determine their effectiveness in reducing pulmonary immunopathology or the influence they exert upon the generation of protective immunity. Methods: A mouse model of influenza infection was utilised to assess the efficacy of paracetamol (acetaminophen) in reducing influenza-induced pathology and to examine whether paracetamol affects generation of protective immunity. Results: Administration (intraperitoneal) of paracetamol significantly decreased the infiltration of inflammatory cells into the airway spaces, reduced pulmonary immunopathology associated with acute infection and improved the overall lung function of mice, without adversely affecting the induction of virus-specific adaptive responses. Mice treated with paracetamol exhibited an ability to resist a second infection with heterologous virus comparable with that of untreated mice. Conclusions: Our results demonstrate that paracetamol dramatically reduces the morbidity associated with influenza but does not compromise the development of adaptive immune responses. Overall, these data support the utility of paracetamol for reducing the clinical symptoms associated with influenza virus infection.

5-Lipoxygenase (5-LOX) plays key roles in infection and allergic responses. Herein, four 5-LOX–derived lipids comprising 5-hydroxyeicosatetraenoic acid (HETE) attached to phospholipids (PLs), either phosphatidylethanolamine (PE) or phosphatidylcholine (18:0p/5-HETE-PE, 18:1p/5-HETE-PE, 16:0p/5-HETE-PE, and 16:0a/5-HETE-PC), were identified in primary human neutrophils. They formed within 2 minutes in response to serum-opsonized Staphylococcus epidermidis or f-methionine-leucine-phenylalanine, with priming by lipopolysaccharide, granulocyte macrophage colony-stimulating factor, or cytochalasin D. Levels generated were similar to free 5-HETE (0.37 ± 0.14 ng vs 0.55 ± 0.18 ng/106 cells, esterified vs free 5-HETE, respectively). They remained cell associated, localizing to nuclear and extranuclear membrane, and were formed by fast esterification of newly synthesized free 5-HETE. Generation also required Ca2+, phospholipase C, cytosolic and secretory phospholipase A2, 5-LOX activating protein, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1. 5-HETE-PLs were detected in murine S epidermidis peritonitis, paralleling neutrophil influx, and in effluent from Gram-positive human bacterial peritonitis. Formation of neutrophil extracellular traps was significantly enhanced by 5-LOX inhibition but attenuated by HETE-PE, whereas 5-HETE-PE enhanced superoxide and interleukin-8 generation. Thus, new molecular species of oxidized PL formed by human neutrophils during bacterial infection are identified and characterized.

The heterodimeric cytokine interleukin 27 (IL-27) signals through the IL-27Rα subunit of its receptor, combined with gp130, a common receptor chain used by several cytokines, including IL-6. Notably, the IL-27 subunits p28 (IL-27p28) and EBI3 are not always expressed together, which suggests that they may have unique functions. Here we show that IL-27p28, independently of EBI3, antagonized cytokine signaling through gp130 and IL-6-mediated production of IL-17 and IL-10. Similarly, the ability to generate antibody responses was dependent on the activity of gp130-signaling cytokines. Mice transgenic for expression of IL-27p28 showed a substantial defect in the formation of germinal centers and antibody production. Thus, IL-27p28, as a natural antagonist of gp130-mediated signaling, may be useful as a therapeutic for managing inflammation mediated by cytokines that signal through gp130.

IL-6–mediated T cell-driven immune responses are associated with signaling occurring through the membrane-bound cognate receptor α-chain (mIL-6Rα). Once formed, IL-6–mIL-6Rα complexes induce the homodimerization and subsequent phosphorylation of the ubiquitously expressed signal-transducing protein, gp130. This signaling event is defined as classical IL-6 signaling. However, many inflammatory processes assigned to IL-6 may be mediated via binding a naturally occurring soluble IL-6Rα, which forms an agonistic complex (IL-6/soluble IL-6Rα) capable of evoking responses on a wide range of cell types that lack mIL-6Rα (IL-6 trans-signaling). To dissect the differential contribution of the two IL-6 signaling pathways in cell-mediated inflammatory processes, we pharmaceutically targeted each using two murine models of human arthritis. Whereas intra-articular neutralization of trans-signaling attenuated local inflammatory responses, the classical pathway was found to be obligate and sufficient to induce pathogenic T cells and humoral responses, leading to systemic disease. Our data illustrate that mechanisms occurring in the secondary lymphoid organs underlying arthropathies are mediated via the classical pathway of IL-6 signaling, whereas trans-signaling contributes only at the local site, that is, in the affected tissues.

Tumor necrosis factor (TNF)-like cytokine (TL1A) is a T-cell costimulator that bolsters cytokine-induced activation through death receptor 3 (DR3). To explore the relationship between T-cell activation and TL1A responsiveness, flow cytometry profiled DR3 expression in resting and activated T cells. In human CD4+ T cells, DR3 was induced rapidly following activation and expressed prominently by interleukin (IL)-17-secreting T cells (Th17). Splenic T cells from wild-type and DR3-deficient mice showed that TL1A activation of DR3 inhibits Th17 generation (81±2.6% at 100 ng/ml TL1A) from naive T cells. This response was not associated with suppression of T-cell proliferation. Using neutralizing antibodies or T cells derived from genetically modified mice, TL1A inhibition of Th17 development was found to be independent of IL-2, IL-27, γIFN, IFNAR1, and STAT1. Under suboptimal TCR activation, TL1A continued to block IL-17A secretion, however, the reduced threshold of TCR engagement was now linked with an increase in TL1A-driven proliferation. In contrast, fully committed Th17 cells displayed an altered TL1A responsiveness and in the absence of TCR costimulation supported the maintenance of T cell IL-17A expression. Consequently, TL1A orchestrates unique outcomes in naive and effector T-helper cells, which may affect the proliferation, differentiation and maintenance of Th17 cells in peripheral compartments and inflamed tissues.—Jones, G. W., Stumhofer, J. S., Foster, T., Twohig, J.P., Hertzog, P., Topley, N., Williams, A. S., Hunter, C. A., Jenkins, B. J., Wang, E. C. Y., Jones, S. A. Naive and activated T cells display differential responsiveness to TL1A that affects Th17 generation, maintenance, and proliferation.

The ability of IL-6 to signal via both membrane bound and soluble receptors is thought to explain the capacity of this cytokine to act in both the initiation and resolution of acute inflammatory responses. In cystic fibrosis (CF), poorly resolved neutrophillic inflammation of the lungs is a primary cause of morbidity and mortality. Expression of IL-6 has been reported to be low in CF lung secretions, despite ongoing inflammation, but the status of soluble IL-6 receptor (sIL-6R) in these patients is unknown. We hypothesised that sIL-6R may be an important potentiator of IL-6 activity in CF associated lung disease. IL-6, sIL-6R and sgp130 (a natural antagonist of responses mediated by the sIL-6R) were analysed by ELISA and Western blot in bronchoalveolar lavage fluid (BALF) from 28 paediatric CF patients and nine non-CF controls. Total cell counts in CF were four fold higher compared to controls (median: 1.4 × 106 cells/ml v. 0.35 × 106 cells/ml in controls) (p < 0.001) and the infiltrate was dominated by neutrophils which were elevated by 89 fold (0.62 × 106 cells/ml v. 0.007 × 106 cells/ml in controls) (p < 0.001). Other markers of inflammation such as IL-8 and MCP-1 were elevated 17.5 and 3.8 fold respectively (IL-8; median: 1122 pg/ml v. 64 pg/ml in controls, p < 0.01 and MCP-1; median: 692 pg/ml v. 182 pg/ml in controls, p < 0.05). IL-6, although present in 23/32 CF BALF specimens compared to 1/9 controls (p < 0.01), was weakly expressed (median: 50 pg/ml). Expression of sIL-6R and sgp130 in CF was no different to control patients. We tested whether weak expression of all three molecules was due to degradation by CF BALF. Degradative activity was observed in association with BALF elastase activity and could be specifically blocked by serine protease inhibitors. Degradation of sIL-6R by purified serine proteases (elastase, cathepsin G and proteinase 3) was also observed leading to a loss of trans-signalling activity. Interestingly, sIL-6R was protected from proteolysis by interaction with IL-6. Our data identify and define a novel protease mediated deficiency of IL-6 signalling in the CF lung.

As interleukin-6 (IL-6), its soluble receptor (sIL-6R), and the IL-6/sIL-6R complex is transiently elevated in response to prolonged moderate-intensity exercise, this study investigated how these levels would be modulated by an acute bout of high-intensity intermittent (HIIT) exercise in comparison to continuous moderate-intensity exercise (MOD). This study also investigated the expression of the differentially spliced sIL-6R (DS-sIL-6R) in response to exercise. Eleven healthy males completed two exercise trials matched for external work done (582 ± 82 kJ). During MOD, participants cycled at 61.8 (2.6)% VO2peak for 58.7 (1.9) min, while HIIT consisted of ten 4-min intervals cycling at 87.5 (3.4)% \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \dot{V}{{\hbox{O}}_{2{\rm{peak}}}} $$\end{document} separated by 2-min rest. Blood samples were collected pre-exercise, post-exercise, and 1.5, 6, and 23 h post-exercise. Plasma IL-6, sIL-6R, IL-6/sIL-6R complex, and DS-sIL-6R levels were measured by enzyme-linked immunosorbent assay. HIIT caused a significantly greater increase in IL-6 than MOD (P = 0.018). Both MOD and HIIT resulted in an increase in sIL-6R and IL-6/sIL-6R complex (P

TLR overactivation may lead to end organ damage and serious acute and chronic inflammatory conditions. TLR responses must therefore be tightly regulated to control disease outcomes. We show in this study the ability of the soluble form of TLR2 (sTLR2) to regulate proinflammatory responses, and demonstrate the mechanisms underlying sTLR2 regulatory capacity. Cells overexpressing sTLR2, or stimulated in the presence of the sTLR2 protein, are hyporesponsive to TLR2 ligands. Regulation was TLR2 specific, and affected NF-κB activation, phagocytosis, and superoxide production. Natural sTLR2-depleted serum rendered leukocytes hypersensitive to TLR2-mediated stimulation. Mice administered sTLR2 together with Gram-positive bacteria-derived components showed lower peritoneal levels of the neutrophil (PMN) chemoattractant, keratinocyte-derived chemokine; lower PMN numbers; and a reduction in late apoptotic PMN. Mononuclear cell recruitment remained unaffected, and endogenous peritoneal sTLR2 levels increased. Notably, the capacity of sTLR2 to modulate acute inflammatory parameters did not compromise the ability of mice to clear live Gram-positive bacteria-induced infection. Mechanistically, sTLR2 interfered with TLR2 mobilization to lipid rafts for signaling, acted as a decoy microbial receptor, and disrupted the interaction of TLR2 with its coreceptor, CD14, by associating with CD14. These findings establish sTLR2 as a regulator of TLR2-mediated inflammatory responses, capable of blunting immune responses without abrogating microbial recognition and may inform the design of novel therapeutics against acute and chronic inflammatory conditions.

The peritoneal macrophage (Mφ) is the site of greatest 12/15-lipoxygenase (12/15-LOX) expression in the mouse; however, its immunoregulatory role in this tissue has not been explored. Herein, we show that 12/15-LOX is expressed by 95% of resident peritoneal CD11bhigh cells, with the remaining 5% being 12/15-LOX−. 12/15-LOX+ cells are phenotypically defined by high F4/80, SR-A, and Siglec1 expression, and enhanced IL-10 and G-CSF generation. In contrast, 12/15-LOX− cells are a dendritic cell population. Resident peritoneal Mφ numbers were significantly increased in 12/15-LOX−/− mice, suggesting alterations in migratory trafficking or cell differentiation in vivo. In vitro, Mφ from 12/15-LOX−/− mice exhibit multiple abnormalities in the regulation of cytokine/growth factor production both basally and after stimulation with Staphylococcus epidermidis cell-free supernatant. Resident adherent cells from 12/15-LOX−/− mice generate more IL-1, IL-3, GM-CSF, and IL-17, but less CCL5/RANTES than do cells from wild-type mice, while Staphylococcus epidermidis cell-free supernatant-elicited 12/15-LOX−/− adherent cells release less IL-12p40, IL-12p70, and RANTES, but more GM-CSF. This indicates a selective effect of 12/15-LOX on peritoneal cell cytokine production. In acute sterile peritonitis, 12/15-LOX+ cells and LOX products were cleared, then reappeared during the resolution phase. The peritoneal lavage of 12/15-LOX−/− mice showed elevated TGF-β1, along with increased immigration of monocytes/Mφ, but decreases in several cytokines including RANTES/CCL5, MCP-1/CCL2, G-CSF, IL-12-p40, IL-17, and TNF-α. No changes in neutrophil or lymphocyte numbers were seen. In summary, endogenous 12/15-LOX defines the resident MΦ population and regulates both the recruitment of monocytes/Mφ and cytokine response to bacterial products in vivo.

Interleukin 6 (IL6) trans-signaling has emerged as a prominent regulator of immune responses during both innate and acquired immunity. Regulation of IL6 trans-signaling is reliant upon the release of soluble IL6 receptor (sIL6R), which binds IL6 to create an agonistic IL6/sIL6R complex capable of activating cell types that would not normally respond to IL6 itself. Here we show that intrinsic and extrinsic apoptotic stimulation by DNA damage, cytokine deprivation, and Fas stimulation promotes shedding of sIL6R. Apoptosis-induced shedding of the IL6R was caspase dependent but PKC independent, with inhibition of ADAM17 preventing IL6R shedding. Such insight is relevant to the control of acute inflammation, where transition from the initial neutrophil infiltration to a more sustained population of mononuclear cells is essential for the resolution of the inflammatory process. This transitional event is governed by IL6 trans-signaling. This study demonstrates that IL6R is shed from apoptotic human neutrophils. In vivo studies in a murine inflammation model showed that neutrophil depletion resulted in reduced local sIL6R levels and a concomitant decrease in mononuclear cells, suggesting that apoptosis-induced IL6R shedding from neutrophils promotes IL6 trans-signaling and regulates the attraction of monocytic cells involved in the clearance of apoptotic neutrophils.

OBJECTIVE: Local interaction between soluble mediators within the inflamed synovium is a key factor that governs the pathologic outcome of inflammatory arthritides. Our aim was to investigate the interplay between the Th1 lymphokine interferon-gamma (IFNgamma) and pivotal cytokines that drive rheumatoid arthritis (RA) pathology (interleukin-1beta [IL-1beta] and tumor necrosis factor alpha [TNFalpha]) in modulating inflammation and arthritis in vitro and in vivo. METHODS: Monarticular antigen-induced arthritis (AIA) was initiated in IFNgamma-deficient (IFNgamma(-/-)) mice and age-matched wild-type (IFNgamma(+/+)) mice. Joint swelling was measured and histologic analysis was performed in order to assess changes in both inflammatory and degenerative parameters in vivo. In vitro, the influence of IFNgamma in regulating IL-1beta- and TNFalpha-driven CXCL8 and CCL2 production was quantified by enzyme-linked immunosorbent assay. RESULTS: In murine AIA, both inflammatory and degenerative arthritis parameters were significantly exacerbated in the absence of IFNgamma. IFNgamma appeared to be a crucial factor in regulating CXCR2+ neutrophil influx in the joint. In in vitro studies using RA fibroblast-like synoviocytes, IFNgamma modulated both IL-1beta- and TNFalpha-driven chemokine synthesis, resulting in the down-regulation of CXCL8 production. CONCLUSION: IFNgamma exerts antiinflammatory, chondroprotective, and antiosteoclastogenic effects in murine AIA through a mechanism that involves the regulation of chemokine synthesis and local neutrophil recruitment. These studies suggest a potential therapeutic role of modulating IFNgamma signaling in the treatment of inflammatory arthritides.

Ectopic lymphoid-like structures (ELSs) reminiscent of secondary lymphoid organs often develop at sites of chronic inflammation where they contribute to immune-mediated pathology. Through evaluation of synovial tissues from rheumatoid arthritis (RA) patients, we now show that low interleukin-27 (IL-27) expression corresponds with an increased incidence of ELS and gene signatures associated with their development and activity. The presence of synovial ELS was also noted in mice deficient in the IL-27 receptor (IL-27R) after the onset of inflammatory arthritis. Here, pathology was associated with increased synovial expression of pro-inflammatory cytokines, homeostatic chemokines, and transcriptional regulators linked with lymphoid neogenesis. In both clinical and experimental RA, synovial ELS coincided with the heightened local expression of cytokines and transcription factors of the Th17 and T follicular helper (Tfh) cell lineages, and included podoplanin-expressing T cells within lymphoid aggregates. IL-27 inhibited the differentiation of podoplanin-expressing Th17 cells, and an increased number of these cells were observed in IL-27R–deficient mice with inflammatory arthritis. Thus, IL-27 appears to negatively regulate ELS development in RA through control of effector T cells. These studies open new opportunities for patient stratification and treatment.

ObjectivesTo investigate the role of liver X receptors (LXRs) in experimental skin fibrosis and evaluate their potential as novel antifibrotic targets.MethodsWe studied the role of LXRs in bleomycin-induced skin fibrosis, in the model of sclerodermatous graft-versus-host disease (sclGvHD) and in tight skin-1 (Tsk-1) mice, reflecting different subtypes of fibrotic disease. We examined both LXR isoforms using LXRα-, LXRβ- and LXR-α/β-double-knockout mice. Finally, we investigated the effects of LXRs on fibroblasts and macrophages to establish the antifibrotic mode of action of LXRs.ResultsLXR activation by the agonist T0901317 had antifibrotic effects in bleomycin-induced skin fibrosis, in the sclGvHD model and in Tsk-1 mice. The antifibrotic activity of LXRs was particularly prominent in the inflammation-driven bleomycin and sclGvHD models. LXRα-, LXRβ- and LXRα/β-double-knockout mice showed a similar response to bleomycin as wildtype animals. Low levels of the LXR target gene ABCA-1 in the skin of bleomycin-challenged and control mice suggested a low baseline activation of the antifibrotic LXR signalling, which, however, could be specifically activated by T0901317. Fibroblasts were not the direct target cells of LXRs agonists, but LXR activation inhibited fibrosis by interfering with infiltration of macrophages and their release of the pro-fibrotic interleukin-6.ConclusionsWe identified LXRs as novel targets for antifibrotic therapies, a yet unknown aspect of these nuclear receptors. Our data suggest that LXR activation might be particularly effective in patients with inflammatory disease subtypes. Activation of LXRs interfered with the release of interleukin-6 from macrophages and, thus, inhibited fibroblast activation and collagen release.

The linear glycosaminoglycan hyaluronan (HA) is synthesized at the plasma membrane by the HA synthase (HAS) enzymes HAS1, -2, and -3 and performs multiple functions as part of the vertebrate extracellular matrix. Up-regulation of HA synthesis in the renal corticointerstitium, and the resultant extracellular matrix expansion, is a common feature of renal fibrosis. However, the regulation of expression of these HAS isoforms at transcriptional and translational levels is poorly understood. We have recently described the genomic structures of the human HAS genes, thereby identifying putative promoter regions for each isoform. Further analysis of the HAS2 gene identified the transcription initiation site and showed that region F3, comprising the proximal 121 bp of promoter sequence, mediated full constitutive transcription. In the present study, we have analyzed this region in the human renal proximal tubular epithelial cell line HK-2. Electrophoretic mobility shift and promoter assay data demonstrated that transcription factors Sp1 and Sp3 bound to three sites immediately upstream of the HAS2 transcription initiation site and that mutation of the consensus recognition sequences within these sites ablated their transcriptional response. Furthermore, subsequent knockdown of Sp1 or Sp3 using small interfering RNAs decreased constitutive HAS2 mRNA synthesis. In contrast, significant binding of HK-2 nuclear proteins by putative upstream NF-Y, CCAAT, and NF-kappaB recognition sites was not observed. The identification of Sp1 and Sp3 as principal mediators of HAS2 constitutive transcription augments recent findings identifying upstream promoter elements and provides further insights into the mechanism of HAS2 transcriptional activation.