Defective apoptotic death of activated macrophages has been implicated in the pathogenesis of rheumatoid arthritis (RA). However, the molecular signatures defining apoptotic resistance of RA macrophages are not fully understood. Here, global transcriptome profiling of RA macrophages revealed that the osmoprotective transcription factor nuclear factor of activated T cells 5 (NFAT5) critically regulates diverse pathologic processes in synovial macrophages including the cell cycle, apoptosis, and proliferation. Transcriptomic analysis of NFAT5-deficient macrophages revealed the molecular networks defining cell survival and proliferation. Proinflammatory M1-polarizing stimuli and hypoxic conditions were responsible for enhanced NFAT5 expression in RA macrophages. An in vitro functional study demonstrated that NFAT5-deficient macrophages were more susceptible to apoptotic death. Specifically, CCL2 secretion in an NFAT5-dependent fashion bestowed apoptotic resistance to RA macrophages in vitro. Injection of recombinant CCL2 into one of the affected joints of Nfat5+/- mice increased joint destruction and macrophage infiltration, demonstrating the essential role of the NFAT5/CCL2 axis in arthritis progression in vivo. Moreover, after intra-articular injection, NFAT5-deficient macrophages were more susceptible to apoptosis and less efficient at promoting joint destruction than were NFAT5-sufficient macrophages. Thus, NFAT5 regulates macrophage survival by inducing CCL2 secretion. Our results provide evidence that NFAT5 expression in macrophages enhances chronic arthritis by conferring apoptotic resistance to activated macrophages.

Rheumatoid synoviocytes, which consist of fibroblast-like synoviocytes (FLSs) and synovial macrophages (SMs), are crucial for the progression of rheumatoid arthritis (RA). Particularly, FLSs of RA patients (RA-FLSs) exhibit invasive characteristics reminiscent of cancer cells, destroying cartilage and bone. RA-FLSs and SMs originate differently from mesenchymal and myeloid cells, respectively, but share many pathologic functions. However, the molecular signatures and biological networks representing the distinct and shared features of the two cell types are unknown. We performed global transcriptome profiling of FLSs and SMs obtained from RA and osteoarthritis patients. By comparing the transcriptomes, we identified distinct molecular signatures and cellular processes defining invasiveness of RA-FLSs and proinflammatory properties of RA-SMs, respectively. Interestingly, under the interleukin-1β (IL-1β)–stimulated condition, the RA-FLSs newly acquired proinflammatory signature dominant in RA-SMs without losing invasive properties. We next reconstructed a network model that delineates the shared, RA-FLS–dominant (invasive), and RA-SM–dominant (inflammatory) processes. From the network model, we selected 13 genes, including periostin, osteoblast-specific factor (POSTN) and twist basic helix–loop–helix transcription factor 1 (TWIST1), as key regulator candidates responsible for FLS invasiveness. Of note, POSTN and TWIST1 expressions were elevated in independent RA-FLSs and further instigated by IL-1β. Functional assays demonstrated the requirement of POSTN and TWIST1 for migration and invasion of RA-FLSs stimulated with IL-1β. Together, our systems approach to rheumatoid synovitis provides a basis for identifying key regulators responsible for pathological features of RA-FLSs and -SMs, demonstrating how a certain type of cells acquires functional redundancy under chronic inflammatory conditions.

NFAT5 (nuclear factor of activated T cells), a well-known osmoprotective factor, can be activated by isotonic stimuli such as Toll-like receptor (TLR) triggering. However, it is unclear how NFAT5 discriminates between isotonic and hypertonic stimuli to produce different functional and molecular outcomes. Here, we identified a novel XO-ROS-p38 MAPK-NFAT5 pathway (XO is xanthine oxidase, ROS is reactive oxygen species) that is activated in RAW 264.7 macrophages upon isotonic TLR stimulation. Unlike what is seen under hypertonic conditions, XO-derived ROS were selectively required for the TLR-induced NFAT5 activation and NFAT5 binding to the IL-6 promoter in RAW 264.7 macrophages under isotonic conditions. In mouse peritoneal macrophages and human macrophages, TLR ligation also induced NFAT5 activation, which was dependent on XO and p38 kinase. The involvement of XO in NFAT5 activation by TLR was confirmed in RAW 264.7 macrophages implanted in BALB/c mice. Moreover, allopurinol, an XO inhibitor, suppressed arthritis severity and decreased the expression of NFAT5 and IL-6 in splenic macrophages in C57BL/6 mice. Collectively, these data support a novel function of the XO-NFAT5 axis in macrophage activation and TLR-induced arthritis, and suggest that XO inhibitor(s) could serve as a therapeutic agent for chronic inflammatory arthritis.Keywords: Inflammatory arthritis r Innate immunity r NFAT5 r Reactive oxygen species r Xanthine oxidase Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionThe nuclear factor of activated T cells (NFAT) family is a group of five versatile transcription factors that are crucial to the develCorrespondence: Dr. Wan-Uk Kim e-mail: wan725@catholic.ac.kr opment and function of the immune system. Among the five members of the NFAT family (NFAT1-5), NFAT5 is unique in that it is the only protein that responds to hypertonicity [1]. NFAT5 is very similar to a Drosophila transcription factor involved * These authors contributed equally to this work. 2722 Nam-Hoon Kim et al. Eur. J. Immunol. 2014. 44: 2721-2736 in the defense against hypertonicity [2], indicating that it may represent an ancient prototype among members of the NFAT family. Indeed, the ability to guard cells against osmotic damage is commonly required in invertebrates as well as in vertebrates including humans. Accordingly, the role of NFAT5 is most pronounced in tissues constantly exposed to osmotic stress, such as kidney medulla epithelia, intestine epithelia, skin epidermis, and cornea [3]. Interestingly, NFAT5 is also activated in nonhypertonic conditions. Biologic processes that accompany NFAT5 activation include integrin-mediated carcinoma invasion [4], migration and differentiation of myoblasts [5], and T-cell proliferation [6]. Additionally, we have demonstrated for the first time that NFAT5 activity is increased by tumor necrosis factor alpha (TNF-α) and interleukin (IL) 1β stimulation under isotonic conditions [7]. NFAT5-deficient mice show a marked r...

Macrophage migration inhibitory factor (MIF) is involved in tumorigenesis by facilitating tumor proliferation and evasion of apoptosis; however, its role in tumor immunity is unclear. In this study, we investigated the effect of MIF on the progression of the syngenic, CT26 colon carcinoma and the generation of tumor regulatory T cells (Tregs). The results showed that the tumor growth rate was significantly lower in MIF knockout (MIF−/−) mice than in wild type (MIF+/+) mice. Flow cytometric analysis of both spleen and tumor cells revealed that MIF−/− mice had significantly lower levels of tumor-associated CD4+Tregs than MIF+/+ mice. The splenic cells of MIF−/− mice also showed a decrease in CD8+Tregs, which was accompanied by an increase in CD8-induced tumor cytotoxicity. Interestingly, the inducible Treg response in spleen cells to anti-CD3/CD28+IL-2+TGF-β was greater in MIF−/− mice than in MIF+/+ mice. Spleen cells of MIF−/− mice, stimulated with anti-CD3/CD28, produced lower levels of IL-2, but not TGF-β, than those of MIF+/+ mice, which was recovered by the addition of recombinant MIF. Conversely, a neutralizing anti-MIF Ab blocked anti-CD3-induced IL-2 production by splenocytes of MIF+/+ mice and suppressed the inducible Treg generation. Moreover, the administration of IL-2 into tumor-bearing MIF−/− mice restored the generation of Tregs and tumor growth. Taken together, our data suggest that MIF promotes tumor growth by increasing Tregs generation through the modulation of IL-2 production. Thus, anti-MIF treatment might be useful in enhancing the adaptive immune response to colon cancers.

Copy number variations (CNVs) have been implicated in human diseases. However, it remains unclear how they affect immune dysfunction and autoimmune diseases, including rheumatoid arthritis (RA). Here, we identified a novel leukocyte-specific protein 1 (LSP1) deletion variant for RA susceptibility located in 11p15.5. We replicated that the copy number of LSP1 gene is significantly lower in patients with RA, which correlates positively with LSP1 protein expression levels. Differentially expressed genes in Lsp1-deficient primary T cells represent cell motility and immune and cytokine responses. Functional assays demonstrated that LSP1, induced by T-cell receptor activation, negatively regulates T-cell migration by reducing ERK activation in vitro. In mice with T-cell–dependent chronic inflammation, loss of Lsp1 promotes migration of T cells into the target tissues as well as draining lymph nodes, exacerbating disease severity. Moreover, patients with RA show diminished expression of LSP1 in peripheral T cells with increased migratory capacity, suggesting that the defect in LSP1 signaling lowers the threshold for T-cell activation. To our knowledge, our work is the first to demonstrate how CNVs result in immune dysfunction and a disease phenotype. Particularly, our data highlight the importance of LSP1 CNVs and LSP1 insufficiency in the pathogenesis of RA and provide previously unidentified insights into the mechanisms underlying T-cell migration toward the inflamed synovium in RA.