Objective. Glucocorticoid-induced leucine zipper (GILZ) has effects on inflammatory pathways that suggest it to be a key inhibitory regulator of the immune system, and its expression is exquisitely sensitive to induction by glucocorticoids. We undertook this study to test our hypothesis that GILZ deficiency would exacerbate experimental immune-mediated inflammation and impair the effects of glucocorticoids on inflammation and, correspondingly, that exogenous GILZ would inhibit these events.Methods. GILZ ؊/؊ mice were generated using the Cre/loxP system, and responses were studied in delayedtype hypersensitivity (DTH), antigen-induced arthritis (AIA), K/BxN serum-transfer arthritis, and lipopolysaccharide (LPS)-induced cytokinemia. Therapeutic expression of GILZ via administration of recombinant adeno-associated virus expressing the GILZ gene (GILZ-rAAV) was compared to the effects of glucocorticoid in collagen-induced arthritis (CIA).Results. Increased T cell proliferation and DTH were observed in GILZ ؊/؊ mice, but neither AIA nor K/BxN serum-transfer arthritis was affected, and GILZ deficiency did not affect LPS-induced cytokinemia. Deletion of GILZ did not impair the effects of exogenous glucocorticoids on CIA or cytokinemia. In contrast, overexpression of GILZ in joints significantly inhibited CIA, with an effect similar to that of dexamethasone. Conclusion. Despite effects on T cell activation, GILZ deficiency had no effect on effector pathways of arthritis and was unexpectedly redundant with effects of glucocorticoids. These findings do not support the hypothesis that GILZ is central to the actions of glucocorticoids, but the efficacy of exogenous GILZ in CIA suggests that further evaluation of GILZ in inflammatory disease is required.

Annexin A1 (AnxA1) is recognized as an endogenous anti-inflammatory molecule. However, its effects on the adaptive immune response and, in particular, on T cells remain unclear. In this study, we investigated the actions of AnxA1 in three distinct models of T cell–mediated inflammation. In contact hypersensitivity, collagen-induced arthritis, and inflammation induced by OT-II TCR transgenic T cells responding to OVA, AnxA1 deficiency significantly increased Ag-induced T cell proliferation and the resultant level of inflammation. In the contact hypersensitivity model, this was associated with increased adhesion of CD4+ T cells, CD8+ T cells, and neutrophils in the dermal microvasculature, as well as increased T cell expression of RORγt and IL-17A. In collagen-induced arthritis, deficiency of endogenous AnxA1 increased susceptibility to arthritis and Ag-specific T cell activation. Deficiency of AnxA1 also increased OVA-induced cutaneous delayed-type hypersensitivity and IFN-γ and IL-17 release. Transfer experiments using CD4+ T cells from AnxA1−/− mice demonstrated that the absence of AnxA1 solely in T cells resulted in increased inflammatory responses in wild-type recipients. Similarly, experiments using AnxA1−/− OT-II CD4+ T cells demonstrated that the absence of AnxA1 in T cells was sufficient to induce increased Ag-specific CD4+ T cell proliferation in vivo, augment T cell production of IFN-γ, IL-17, TNF, and IL-6, and increase Akt, ERK, and p38 activation. Together, these findings indicate that T cell–expressed AnxA1 functions to attenuate T cell–driven inflammatory responses via T cell–intrinsic effects on intracellular signaling, proliferation, and Th1/Th17 cytokine release.

Background and PurposeAnnexin A1 (AnxA1) is an endogenous anti‐inflammatory protein and agonist of the formyl peptide receptor 2 (FPR2). However, the potential for therapeutic FPR ligands to modify immune‐mediated disease has been little explored. We investigated the effects of a synthetic FPR agonist on joint disease in the K/BxN model of rheumatoid arthritis (RA) and RA fibroblast‐like synoviocytes (FLS).Experimental ApproachArthritis was induced by injection of K/BxN serum at day 0 and 2 in wild‐type (WT) or AnxA1−/− mice and clinical and histopathological manifestations measured 8–11 days later. WT mice were given the FPR agonist compound 43 (Cpd43) (6 or 30 mg·kg−1 i.p.) for 4 days. Effects of AnxA1 and Cpd43 on RANKL‐induced osteoclastogenesis were assessed in RAW 264.7 cells and human RA FLS and macrophages.Key ResultsTreatment with Cpd43 before or after the onset of arthritis reduced clinical disease severity and attenuated synovial TNF‐α and osteoclast‐associated gene expression. Deletion of AnxA1 in mice exacerbated arthritis severity in the K/BxN model. In vitro, Cpd43 suppressed osteoclastogenesis and NFAT activity elicited by RANKL, and inhibited IL‐6 secretion by mouse macrophages. In human RA joint‐derived FLS and monocyte‐derived macrophages, Cpd43 treatment inhibited IL‐6 release, while blocking FPR2 or silencing AnxA1 increased this release.Conclusions and ImplicationsThe FPR agonist Cpd43 reduced osteoclastogenesis and inflammation in a mouse model of RA and exhibited anti‐inflammatory effects in relevant human cells. These data suggest that FPR ligands may represent novel therapeutic agents capable of ameliorating inflammation and bone damage in RA.

Objective. Myeloperoxidase (MPO) is implicated as a local mediator of tissue damage when released extracellularly in many chronic inflammatory diseases. The purpose of this study was to explore the role of endogenous MPO in experimental rheumatoid arthritis (RA).Methods. K/BxN serum-transfer arthritis was induced in C57BL/6 wild-type (WT) and MPO knockout (MPO ؊/؊ ) mice, and disease development was assessed. MPO activity was measured in joint tissues from mice with or without K/BxN arthritis. Collagen-induced arthritis (CIA) was induced in WT and MPO ؊/؊ mice, and disease development and immune responses were examined. MPO expression was assessed in synovial biopsy samples from patients with active RA, and the effect of MPO on synovial fibroblasts was tested in vitro.Results. MPO was up-regulated in the joints of mice with K/BxN arthritis, and MPO deficiency attenuated the severity of the disease without affecting circulating cytokine levels. In CIA, MPO ؊/؊ mice had enhanced CD4؉ T cell responses and reduced frequency of regulatory T cells in the lymph nodes and spleen, as well as augmented interleukin-17A and diminished interferon-␥ secretion by collagen-stimulated splenocytes, without an effect on circulating anticollagen antibody levels. Despite enhanced adaptive immunity in secondary lymphoid organs, CIA development was attenuated in MPO ؊/؊ mice. Intracellular and extracellular MPO was detected in the synovium of patients with active RA, and human MPO enhanced the proliferation and decreased the apoptosis of synovial fibroblasts in vitro.Conclusion. MPO contributes to the development of arthritis despite suppressing adaptive immunity in secondary lymphoid organs. This suggests distinct effects of local MPO on arthritogenic effector responses.

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Objectives Glucocorticoids remain a mainstay of the treatment of rheumatoid arthritis (RA). Dose-dependent adverse effects highlight the potential for therapies that regulate glucocorticoid sensitivity to enable glucocorticoid dose reduction. Macrophage migration inhibitory factor (MIF) is a pro-inflammatory protein implicated in the pathogenesis of RA, which also impairs glucocorticoid sensitivity via inhibition of the MAP kinase phosphatase, MKP-1. The intracellular protein glucocorticoid-induced leucine zipper (GILZ) mimics the effects of glucocorticoids in models of RA, but whether it represents a target for the modulation of glucocorticoid sensitivity remains unknown. We therefore investigated whether GILZ is implicated in regulation of glucocorticoid sensitivity by MIF. Methods GILZ expression was studied in the presence and absence of MIF, and the role of GILZ in the MIF-dependent regulation of the glucocorticoid sensitivity mediator MAP kinase phosphatase-1 (MKP-1) studied at the level of expression and function. Results GILZ expression was significantly inhibited by endogenous MIF, both basally and during responses to glucocorticoid treatment. The effects of MIF on GILZ were dependent on the expression and Akt-induced nuclear translocation of the transcription factor FoxO3a. GILZ was shown to regulate the expression of MKP-1, and consequent MAP kinase phosphorylation and cytokine release. Conclusions MIF exerts its effects on MKP-1 expression and MAP kinase activity through inhibitory effects on GILZ. These findings suggest a previously unsuspected intersection between these two molecules and identify GILZ as a potential target for the therapeutic regulation of glucocorticoid sensitivity.

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