Tracking Cardiovascular Comorbidity in Models of Chronic Inflammatory Disease.

Immune-mediated inflammatory diseases (IMIDs) are commonly associated with complex coexisting conditions, and cardiovascular comorbidities are a common cause of mortality in systemic inflammation. Experimental models of disease provide an opportunity to dissect inflammatory mechanisms that promote damage to vascular tissues affected by comorbidity. Here, we describe methods to recover the thoracic aorta from mice during experimental inflammatory arthritis and assess vascular constriction responses by isometric tension myography. To complement the assessment of functional changes in the vasculature during inflammatory arthritis, we also outline a method to characterize vascular inflammation by immunohistochemistry.

AMPA/kainate glutamate receptor antagonists prevent posttraumatic osteoarthritis.

Musculoskeletal disorders represent the third greatest burden in terms of death and disability in the developed world. Osteoarthritis is the single greatest cause of chronic pain, has no cure, and affects 8.5 and 27 million people in the UK and US, respectively. Osteoarthritis is most prevalent in older people, but as it commonly occurs after joint injury, young people with such injuries are also susceptible. Painful joints are often treated with steroid or hyaluronic acid (HA) injections, but treatments to prevent subsequent joint degeneration remain elusive. In animals, joint injury increases glutamate release into the joint, acting on nerves to cause pain, and joint tissues to cause inflammation and degeneration. This study investigated synovial fluid glutamate concentrations and glutamate receptor (GluR) expression in injured human joints and compared the efficacy of GluR antagonists with current treatments in a mouse model of injury-induced osteoarthritis (ACL rupture). GluRs were expressed in the ligaments and meniscus after knee injury, and synovial fluid glutamate concentrations ranged from 19 to 129 µM. Intra-articular injection of NBQX (GluR antagonist) at the time of injury substantially reduced swelling and degeneration in the mouse ACL rupture model. HA had no effect, and Depo-Medrone reduced swelling for 1 day but increased degeneration by 50%. Intra-articular administration of NBQX modified both symptoms and disease to a greater extent than current treatments. There is an opportunity for repurposing related drugs, developed for CNS disorders and with proven safety in humans, to prevent injury-induced osteoarthritis. This could quickly reduce the substantial burden associated with osteoarthritis.

Oestrogen-deficiency induces bone loss by modulating CD14+ monocyte and CD4+ T cell DR3 expression and serum TL1A levels.

BACKGROUND: Oestrogen-deficiency induced by menopause is associated with reduced bone density and primary osteoporosis, resulting in an increased risk of fracture. While the exact etiology of menopause-induced primary osteoporotic bone loss is not fully known, members of the tumour necrosis factor super family (TNFSF) are known to play a role. Recent studies have revealed that the TNFSF members death receptor 3 (DR3) and one of its ligands, TNF-like protein 1A (TL1A) have a key role in secondary osteoporosis; enhancing CD14+ peripheral blood mononuclear cell (PBMC) osteoclast formation and bone resorption. Whether DR3 and TL1A contribute towards bone loss in menopause-induced primary osteoporosis however, remains unknown. METHODS: To investigate this we performed flow cytometry analysis of DR3 expression on CD14+ PBMCs isolated from pre- and early post-menopausal females and late post-menopausal osteoporotic patients. Serum levels of TL1A, CCL3 and total MMP-9 were measured by ELISA. In vitro osteoclast differentiation assays were performed to determine CD14+ monocyte osteoclastogenic potential. In addition, splenic CD4+ T cell DR3 expression was investigated 1 week and 8 weeks post-surgery, using the murine ovariectomy model. RESULTS: In contrast to pre-menopausal females, CD14+ monocytes isolated from post-menopausal females were unable to induce DR3 expression. Serum TL1A levels were decreased approx. 2-fold in early post-menopausal females compared to pre-menopausal controls and post-menopausal osteoporotic females; no difference was observed between pre-menopausal and late post-menopausal osteoporotic females. Analysis of in vitro CD14+ monocyte osteoclastogenic potential revealed no significant difference between the post-menopausal and post-menopausal osteoporotic cohorts. Interestingly, in the murine ovariectomy model splenic CD4+ T cell DR3 expression was significantly increased at 1 week but not 8 weeks post-surgery when compared to the sham control. CONCLUSION: Our results reveals for the first time that loss of oestrogen has a significant effect on DR3; decreasing expression on CD14+ monocytes and increasing expression on CD4+ T cells. These data suggest that while oestrogen-deficiency induced changes in DR3 expression do not affect late post-menopausal bone loss they could potentially have an indirect role in early menopausal bone loss through the modulation of T cell activity.

Inhibition of CCL3 abrogated precursor cell fusion and bone erosions in human osteoclast cultures and murine collagen-induced arthritis.

Objective: Macrophage inflammatory protein 1-alpha (CCL3) is a chemokine that regulates macrophage trafficking to the inflamed joint. The agonistic effect of CCL3 on osteolytic lesions in patients with multiple myeloma is recognized; however, its role in skeletal damage during inflammatory arthritis has not been established. The aim of the study was to explore the role of osteoclast-associated CCL3 upon bone resorption, and to test its pharmacological blockade for protecting against bone pathology during inflammatory arthritis. Methods: CCL3 production was studied during osteoclast differentiation from osteoclast precursor cells: human CD14-positive mononuclear cells. Mice with CIA were treated with an anti-CCL3 antibody. The effect of CCL3 blockade through mAb was studied through osteoclast number, cytokine production and bone resorption on ivory disks, and in vivo through CIA progression (clinical score, paw diameter, synovial inflammation and bone damage). Results: Over time, CCL3 increased in parallel with the number of osteoclasts in culture. Anti-CCL3 treatment achieved a concentration-dependent inhibition of osteoclast fusion and reduced pit formation on ivory disks (P ⩽ 0.05). In CIA, anti-CCL3 treatment reduced joint damage and significantly decreased multinucleated tartrate-resistant acid phosphatase-positive osteoclasts and erosions in the wrists (P < 0.05) and elbows (P < 0.05), while also reducing joint erosions in the hind (P < 0.01) and fore paws (P < 0.01) as confirmed by X-ray. Conclusion: Inhibition of osteoclast-associated CCL3 reduced osteoclast formation and function whilst attenuating arthritis-associated bone loss and controlling development of erosion in murine joints, thus uncoupling bone damage from inflammation. Our findings may help future innovations for the diagnosis and treatment of inflammatory arthritis.

In Vivo Models for Inflammatory Arthritis.

In vivo mouse models of inflammatory arthritis are extensively used to investigate pathogenic mechanisms governing inflammation-driven joint damage. Two commonly utilized models include collagen-induced arthritis (CIA) and methylated bovine serum albumin (mBSA) antigen-induced arthritis (AIA). These offer unique advantages for modeling different aspects of human disease. CIA involves breach of immunological tolerance resulting in systemic autoantibody-driven arthritis, while AIA results in local resolving inflammatory flares and articular T cell-mediated damage. Despite limitations that apply to all animal models of human disease, CIA and AIA have been instrumental in identifying pathogenic mediators, immune cell subsets and stromal cell responses that determine disease onset, progression, and severity. Moreover, these models have enabled investigation of disease phases not easily studied in patients and have served as testing beds for novel biological therapies, including cytokine blockers and small molecule inhibitors of intracellular signaling that have revolutionized rheumatoid arthritis treatment.

Alterations to adipose tissue morphology during inflammatory arthritis is indicative of vasculopathology in DBA/1 mice.

The physiologic function of adipose tissue is altered by the host's inflammatory response; the implications for maintaining human health and regulating inflammation-associated disease progression are ill defined. However, this cannot be investigated in humans, therefore the use of animal models is required. With the aim to determine morphological and molecular alterations to perivascular and organ-associated adipose tissues during inflammatory arthritis, collagen-induced arthritis (CIA) was established in male DBA/1 mice. Emerging evidence from this study signposts CIA in the DBA/1 mouse as a model that is relevant to study the development and treatment of early cardiovascular pathology associated with inflammatory arthritis. Here, we show global morphological changes in adipose tissue and the thoracic aorta in animals induced with CIA compared with the non-immunized controls. In CIA, we concluded that the increased cell count in PVAT was, at least in part, caused by an ingress and/or expansion of macrophages that had a mixed phenotype. A substantial increase of galectin-3 was expressed in PVAT from mice with CIA. Galectin-3 is elevated in the blood of patients with CVDs, however, it has never before been measured in PVAT in rodents or humans. Here, PVAT-associated galectin-3 is identified as a potential biomarker for detecting early vascular pathology in CIA and a promising candidate for translation to RA.

CCL3 and MMP-9 are induced by TL1A during death receptor 3 (TNFRSF25)-dependent osteoclast function and systemic bone loss.

Reduced bone density and secondary osteoporosis, resulting in increased risk of fracture, is a significant complicating factor in the inflammatory arthritides. While the exact etiology of systemic bone loss is not fully elucidated, recent insights into the tumor necrosis factor super family (TNFSF) revealed a potential role for death receptor 3 (DR3/TNFRSF25) and one of its ligands, TNF-like protein 1A (TL1A/TNFSF15). The mechanisms by which DR3/TL1A signalling modulates bone loss are unclear. We investigated the effect of DR3/TL1A signalling upon osteoclast-dependent chemokine and MMP production to unravel novel mechanisms whereby this pathway regulates OC formation and OC-dependent bone resorption. Collagen induced arthritis (CIA) was established in DR3wt and DR3ko mice, joints were sectioned and analysed histologically for bone damage while systemic trabecular bone loss distal to the affected joints was compared by micro-CT. Ablation of DR3 protected DBA/1 mice against the development and progression of CIA. In DR3ko, joints of the ankle and mid-foot were almost free of bone erosions and long bones of mice with CIA were protected against systemic trabecular bone loss. In vitro, expression of DR3 was confirmed on primary human CD14+ osteoclast precursors by flow cytometry. These cells were treated with TL1A in osteoclast differentiation medium and TRAP+ osteoclasts, bone resorption, levels of osteoclast-associated chemokines (CCL3, CCL2 and CXCL8) and MMP-9 measured. TL1A intensified human osteoclast differentiation and bone resorption and increased osteoclast-associated production of CCL3 and MMP-9. Our data reveals the DR3 pathway as an attractive therapeutic target to combat adverse bone pathology associated with inflammatory arthritis. We demonstrate that DR3 is critical in the pathogenesis of murine CIA and associated secondary osteoporosis. Furthermore, we identify a novel mechanism by which the DR3/TL1A pathway directly enhances human OC formation and resorptive activity, controlling expression and activation of CCL3 and MMP-9.

Death Receptor 3 Promotes Chemokine-Directed Leukocyte Recruitment in Acute Resolving Inflammation and Is Essential for Pathological Development of Mesothelial Fibrosis in Chronic Disease.

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.

Characterization of death receptor 3-dependent aortic changes during inflammatory arthritis.

Murine collagen-induced arthritis (mCIA) is characterized by decreased vascular constriction responses and increased MMP-9. Here, we describe additional histological alterations within the aorta and surrounding perivascular adipose tissue (PVAT), study the role of PVAT in constriction response, and investigate the potential involvement of death receptor 3 (DR3). mCIA was induced in wild-type (WT) and DR3-/- mice with nonimmunized, age-matched controls. Vascular function was determined in isolated aortic rings ±PVAT, using isometric tension myography, in response to cumulative serotonin concentrations. Cellular expression of F4/80 (macrophages), Ly6G (neutrophils), DR3, and MMP-9 was determined using immunohistochemistry. In WTs, arthritis-induced vascular dysfunction was associated with increased F4/80+ macrophages and increased DR3 expression in the aorta and PVAT. MMP-9 was also up-regulated in PVAT, but did not correlate with alterations of PVAT intact constriction. DR3-/- mice inherently showed increased leukocyte numbers and MMP-9 expression in the PVAT, but retained the same nonarthritic constriction response as DR3WT mice ±PVAT. Arthritic DR3-/- mice had a worsened constriction response than DR3WT and showed an influx of neutrophils to the aorta and PVAT. Macrophage numbers were also up-regulated in DR3-/- PVAT. Despite this influx, PVAT intact DR3-/- constriction responses were restored to the same level as DR3WT. Impaired vascular constriction in inflammatory arthritis occurs independently of total MMP-9 levels, but correlates with macrophage and neutrophil ingress. Ablating DR3 worsens the associated vasculature dysfunction, however, DR3-/- PVAT is able to protect the aorta against aberrant vasoconstriction caused in this model.

Scaling-down antibody radiolabeling reactions with zirconium-89.

The most widely cited procedures for radiolabeling antibodies with zirconium-89 for immuno-PET require multi-milligram amounts of antibody which can be cost-prohibitive, particularly during the research and development process. We therefore sought to develop a reliable (89)Zr-radiolabeling procedure that provides high radiochemical yields at the microgram scale.

Deletion of the membrane complement inhibitor CD59a drives age and gender-dependent alterations to bone phenotype in mice.

Degenerative joint diseases such as osteoarthritis are characterised by aberrant region-specific bone formation and abnormal bone mineral content. A recent study suggested a role for the complement membrane attack complex in experimental models of osteoarthritis. Since CD59a is the principal regulator of the membrane attack complex in mice, we evaluated the impact of CD59a gene deletion upon maintenance of bone architecture. In vivo bone morphology analysis revealed that male CD59a-deficient mice have increased femur length and cortical bone volume, albeit with reduced bone mineral density. However, this phenomenon was not observed in female mice. Histomorphometric analysis of the trabecular bone showed increased rates of bone homeostasis, with both increased bone resorption and mineral apposition rate in CD59a-deficient male mice. When bone cells were studied in isolation, in vitro osteoclastogenesis was significantly increased in male CD59a-deficient mice, although osteoblast formation was not altered. Our data reveal, for the first time, that CD59a is a regulator of bone growth and homeostasis. CD59a ablation in male mice results in longer and wider bones, but with less density, which is likely a major contributing factor for their susceptibility to osteoarthritis. These findings increase our understanding of the role of complement regulation in degenerative arthritis.

Novel Application of Behavioral Assays Allows Dissociation of Joint Pathology from Systemic Extra-Articular Alterations Induced by Inflammatory Arthritis.

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 antigen-induced 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 anxiety-related 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.

Interleukin-27 inhibits ectopic lymphoid-like structure development in early inflammatory arthritis.

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.

Death Receptor 3 (TNFRSF25) Increases Mineral Apposition by Osteoblasts and Region Specific New Bone Formation in the Axial Skeleton of Male DBA/1 Mice.

Objectives. Genome wide association studies identified TNFSF member TNF-like protein 1A (TL1A, TNFSF15) as a potential modulator of ankylosing spondylitis (AS). TL1A is the only confirmed TNFSF ligand of death receptor 3 (DR3, TNFRSF25); however, its role in disease pathology is not characterised. We evaluated DR3's role in controlling osteoblast- (OB-) dependent bone formation in vitro and in vivo. Methods. Osteoprogenitor cells and OB were cultured from male DR3-deficient (DR3(ko)) and wild-type (DR3(wt)) DBA/1 mice. DR3 and RANKL expression were tested by flow cytometry. Alkaline phosphatase and mineralization were quantified. Osteopontin, osteoprotegerin, and pro MMP-9 were measured by ELISA. A fluorescent probe (BoneTag) was used to measure in vivo mineralization in 10-month-old mice. Results. DR3 was expressed on osteoprogenitors and OB from DR3(wt) mice. Alkaline phosphatase, osteopontin, and mineral apposition were significantly elevated in DR3(wt) cultures. Levels of RANKL were comparable whilst osteoprotegerin was significantly increased in DR3(wt) cultures. In vivo incorporation of BoneTag was significantly lower in the thoracic vertebrae of 10-month-old DR3(ko) mice. Conclusions. These data identify new roles for DR3 in regulating OB-dependent bone mineral apposition. They potentially begin to explain the atypical pattern of new bone formation observed in the axial skeleton of grouped, aging DBA/1 mice.

AMPA/kainate glutamate receptors contribute to inflammation, degeneration and pain related behaviour in inflammatory stages of arthritis.

OBJECTIVES: Synovial fluid glutamate concentrations increase in arthritis. Activation of kainate (KA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors (GluRs) increase interleukin-6 (IL-6) release and cause arthritic pain, respectively. We hypothesised that AMPA and KA GluRs are expressed in human arthritis, and that intra-articular NBQX (AMPA/KA GluR antagonist) prevents pain and pathology in antigen-induced arthritis (AIA). METHODS: GluR immunohistochemistry was related to synovial inflammation and degradation in osteoarthritis (OA) and rheumatoid arthritis (RA). A single intra-articular NBQX injection was given at induction, and knee swelling and gait of AIA and AIA+NBQX rats compared over 21 days, before imaging, RT-qPCR, histology and immunohistochemistry of joints. Effects of NBQX on human primary osteoblast (HOB) activity were determined. RESULTS: AMPAR2 and KA1 immunolocalised to remodelling bone, cartilage and synovial cells in human OA and RA, and rat AIA. All arthritic tissues showed degradation and synovial inflammation. NBQX reduced GluR abundance, knee swelling (p<0.001, days 1-21), gait abnormalities (days 1-2), end-stage joint destruction (p<0.001), synovial inflammation (p<0.001), and messenger RNA expression of meniscal IL-6 (p<0.05) and whole joint cathepsin K (p<0.01). X-ray and MRI revealed fewer cartilage and bone erosions, and less inflammation after NBQX treatment. NBQX reduced HOB number and prevented mineralisation. CONCLUSIONS: AMPA/KA GluRs are expressed in human OA and RA, and in AIA, where a single intra-articular injection of NBQX reduced swelling by 33%, and inflammation and degeneration scores by 34% and 27%, respectively, exceeding the efficacy of approved drugs in the same model. AMPA/KA GluR antagonists represent a potential treatment for arthritis.

Interleukin-10 regulates the inflammasome-driven augmentation of inflammatory arthritis and joint destruction.

INTRODUCTION: Activation of the inflammasome has been implicated in the pathology of various autoinflammatory and autoimmune diseases. While the NLRP3 inflammasome has been linked to arthritis progression, little is known about its synovial regulation or contribution to joint histopathology. Regulators of inflammation activation, such as interleukin (IL)-10, may have the potential to limit the inflammasome-driven arthritic disease course and associated structural damage. Hence, we used IL-10-deficient (IL-10KO) mice to assess NLRP3 inflammasome-driven arthritic pathology. METHODS: Antigen-induced arthritis (AIA) was established in IL-10KO mice and wild-type controls. Using histological and radiographic approaches together with quantitative real-time PCR of synovial mRNA studies, we explored the regulation of inflammasome components. These were combined with selective blocking agents and ex vivo investigative studies in osteoclast differentiation assays. RESULTS: In AIA, IL-10KO mice display severe disease with increased histological and radiographic joint scores. Here, focal bone erosions were associated with increased tartrate-resistant acid phosphatase (TRAP)-positive cells and a localized expression of IL-1ß. When compared to controls, IL-10KO synovium showed increased expression of Il1b, Il33 and NLRP3 inflammasome components. Synovial Nlrp3 and Casp1 expression further correlated with Acp5 (encoding TRAP), while neutralization of IL-10 receptor signaling in control mice caused increased expression of Nlrp3 and Casp1. In ex vivo osteoclast differentiation assays, addition of exogenous IL-10 or selective blockade of the NLRP3 inflammasome inhibited osteoclastogenesis. CONCLUSIONS: These data provide a link between IL-10, synovial regulation of the NLRP3 inflammasome and the degree of bone erosions observed in inflammatory arthritis.

Regulation of early cartilage destruction in inflammatory arthritis by death receptor 3.

OBJECTIVE: To investigate the role of death receptor 3 (DR-3) and its ligand tumor necrosis factor-like molecule 1A (TL1A) in the early stages of inflammatory arthritis. METHODS: Antigen-induced arthritis (AIA) was generated in C57BL/6 mice deficient in the DR-3 gene (DR3(-/-) ) and their DR3(+/+) (wild-type) littermates by priming and intraarticular injection of methylated bovine serum albumin. The joints were sectioned and analyzed histochemically for damage to cartilage and expression of DR3, TL1A, Ly-6G (a marker for neutrophils), the gelatinase matrix metalloproteinase 9 (MMP-9), the aggrecanase ADAMTS-5, and the neutrophil chemoattractant CXCL1. In vitro production of MMP-9 was measured in cultures from fibroblasts, macrophages, and neutrophils following the addition of TL1A and other proinflammatory stimuli. RESULTS: DR3 expression was up-regulated in the joints of wild-type mice following generation of AIA. DR3(-/-) mice were protected against cartilage damage compared with wild-type mice, even at early time points prior to the main accumulation of Teff cells in the joint. Early protection against AIA in vivo correlated with reduced levels of MMP-9. In vitro, neutrophils were major producers of MMP-9, while neutrophil numbers were reduced in the joints of DR3(-/-) mice. However, TL1A neither induced MMP-9 release nor affected the survival of neutrophils. Instead, reduced levels of CXCL1 were observed in the joints of DR3(-/-) mice. CONCLUSION: DR-3 drives early cartilage destruction in the AIA model of inflammatory arthritis through the release of CXCL1, maximizing neutrophil recruitment to the joint and leading to enhanced local production of cartilage-destroying enzymes.

Interleukin-6 signaling drives fibrosis in unresolved inflammation.

Fibrosis in response to tissue damage or persistent inflammation is a pathological hallmark of many chronic degenerative diseases. By using a model of acute peritoneal inflammation, we have examined how repeated inflammatory activation promotes fibrotic tissue injury. In this context, fibrosis was strictly dependent on interleukin-6 (IL-6). Repeat inflammation induced IL-6-mediated T helper 1 (Th1) cell effector commitment and the emergence of STAT1 (signal transducer and activator of transcription-1) activity within the peritoneal membrane. Fibrosis was not observed in mice lacking interferon-γ (IFN-γ), STAT1, or RAG-1. Here, IFN-γ and STAT1 signaling disrupted the turnover of extracellular matrix by metalloproteases. Whereas IL-6-deficient mice resisted fibrosis, transfer of polarized Th1 cells or inhibition of MMP activity reversed this outcome. Thus, IL-6 causes compromised tissue repair by shifting acute inflammation into a more chronic profibrotic state through induction of Th1 cell responses as a consequence of recurrent inflammation.

Interleukin-6 limits influenza-induced inflammation and protects against fatal lung pathology.

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.

Suppression of leukocyte infiltration and cartilage degradation by selective inhibition of pre-B cell colony-enhancing factor/visfatin/nicotinamide phosphoribosyltransferase: Apo866-mediated therapy in human fibroblasts and murine collagen-induced arthritis.

OBJECTIVE: To assess the ability of pre-B cell colony-enhancing factor (PBEF) to regulate inflammation and degradative processes in inflammatory arthritis, using the small molecule inhibitor APO866 in human fibroblasts in vitro and in murine collagen-induced arthritis (CIA). METHODS: Enzyme-linked immunosorbent assays were used to examine regulation of expression of metalloproteinases and chemokines in human fibroblasts. The role of PBEF was further examined using APO866 in mice with CIA, with effects on disease activity assessed using radiography, histology, in vivo imaging, and quantitative polymerase chain reaction (qPCR). RESULTS: In vitro activation of human fibroblasts with PBEF promoted expression of matrix metalloproteinase 3 (MMP-3), CCL2, and CXCL8, an effect inhibited by APO866. In mice with CIA, early intervention with APO866 inhibited synovial inflammation, including chemokine-directed leukocyte infiltration, and reduced a systemic marker of inflammation, serum hyaluronic acid. APO866 blockade led to reduced expression of MMP-3 and MMP-13 in joint extracts and to a reduction in a systemic marker of cartilage erosion, serum cartilage oligomeric matrix protein. Radiologic images revealed that APO866 protected against bone erosion, while qPCR demonstrated inhibition of RANKL expression. In mice with established disease, APO866 reduced synovial inflammation and cartilage destruction, and halted bone erosion. In addition, APO866 reduced the activity of MMP-3, CCL2, and RANKL in vivo, and inhibited production of CCL2 and RANKL in synovial explants from arthritic mice, a result that was reversed with nicotinamide mononucleotide. CONCLUSION: These findings confirm PBEF to be an important regulator of inflammation, cartilage catabolism, and bone erosion, and highlight APO866 as a promising therapeutic agent for targeting PBEF activity in inflammatory arthritis.