There is a strong link between integrins and interleukin-1 (IL-1), but the specifics of the role of integrins in IL-1 signaling are unclear. We describe that IL-1 specifically bound to integrins v3 and 51. The E128K mutation in the IL1R-binding site enhanced integrin binding. We studied whether direct integrin binding is involved in IL-1 signaling. We compared sequences of IL-1 and IL-1 receptor antagonist (IL1RN), which is an IL-1 homologue but has no agonistic activity. Several surface-exposed Lys residues are present in IL-1, but not in IL1RN. A disulfide linkage is present in IL1RN, but is not in IL-1 because of natural C117F mutation. Substitution of the Lys residues to Glu markedly reduced integrin binding of E128K IL-1, suggesting that the Lys residues mediate integrin binding. The Lys mutations reduced, but did not completely abrogate, agonistic action of IL-1. We studied whether the disulfide linkage plays a role in agonistic action of IL-1. Reintroduction of the disulfide linkage by the F117C mutation did not affect agonistic activity of WT IL-1, but effectively reduced the remaining agonistic activity of the Lys mutants. Also, deletion of the disulfide linkage in IL1RN by the C116F mutation did not make it agonistic. We propose that the direct binding to IL-1 to integrins is primarily important for agonistic IL-1 signaling, and that the disulfide linkage indirectly affects signaling by blocking conformational changes induced by weak integrin binding to the Lys mutants. The integrin-IL-1 interaction is a potential target for drug discovery.

Objective. SKG mice develop Interstitial lung disease (ILD) resembling rheumatoid arthritis-associated ILD In humans. The aim of this study was to clarify the mechanism underlying the lung pathology by analyzing lung-infiltrating cells In SKG mice with ILD.Methods. We assessed the severity of zymosan A (ZyA)-induced ILD in SKG mice histologically, and we examined lung-infiltrating cells by flow cytometry. Total lung cells and isolated monocytic myeloid-derived suppressor cells (MDSCs) were cultured In vitro with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4. The proliferation of 5,6-carboxyfluorescein diacetate N-succinimidyl ester-labeled naive T cells cocultured with Isolated CD11b+Gr-1(dim) cells and MDSCs was evaluated by flow cytometry. CD11b+Gr-1(dim) cells were adoptively transferred to ZyA-treated SKG mice.Results. MDSCs, Th17 cells, and group 1 and 3 Innate lymphoid cells (ILC1s and ILC3s) were Increased In the lungs; the proportion of these cells varied with ILD severity. In this process, we found that a unique cell population, CD11b+Gr-1(dim) cells, was expanded In the severely Inflamed lungs. Approximately half of the CD11b+Gr-1(dim) cells expressed CD11c. CD11b+Gr-1(dim) cells were induced from monocytic MDSCs with GM-CSF in vitro and were considered tolerogenic because they suppressed T cell proliferation. These CD11b+Gr-1(dim) cells have never been described previously, and we termed them CD11b+Gr-1(dim) tolerogenic dendritic cell (DC)-like cells. Th17 cells, ILC1s, and ILC3s in the Inflamed lung produced GM-CSF, which may have expanded CD11b+Gr-1(dim) tolerogenic DC-like cells in vivo. Furthermore, adoptive transfer of CD11b+Gr-1(dim) tolerogenic DC-like cells significantly suppressed progression of ILD In SKG mice.Conclusion. We Identified unique suppressive myeloid cells that were differentiated from monocytic MDSCs In SKG mice with ILD, and we termed them CD11b+Gr-1(dim) tolerogenic DC-like cells.

Recent studies have shown that cellular metabolism plays an important role in regulating immune cell functions. In immune cell differentiation, both interleukin-17-producing T (Th17) cells and dendritic cells (DCs) exhibit increased glycolysis through the upregulation of glycolytic enzymes, such as hexokinase-2 (HK2). Blocking glycolysis with 2-deoxyglucose was recently shown to inhibit Th17 cell differentiation while promoting regulatory T (Treg) cell generation. However, 2-DG inhibits all isoforms of HK. Thus, it is unclear which isoform has a critical role in Th17 cell differentiation and in rheumatoid arthritis (RA) pathogenesis. Here we demonstrated that 3-bromopyruvate (BrPA), a specific HK2 inhibitor, significantly decreased the arthritis scores and the histological scores in SKG mice, with a significant increase in Treg cells, decrease in Th17 cells, and decrease in activated DCs in the spleen. In vitro, BrPA facilitated the differentiation of Treg cells, suppressed Th17 cells, and inhibited the activation of DCs. These results suggested that BrPA may be a therapeutic target of murine arthritis. Although the role of IL-17 is not clarified in the treatment of RA, targeting cell metabolism to alter the immune cell functions might lead to a new therapeutic strategy for RA.

Objective MicroRNAs (miRNAs) are small endogenous, non-coding RNAs that act as post-transcriptional regulators. We analysed the in vivo effect of miRNA-124 (miR-124, the rat analogue of human miR-124a) on adjuvant-induced arthritis (AIA) in rats.Methods AIA was induced in Lewis rats by injecting incomplete Freund's adjuvant with heat-killed Mycobacterium tuberculosis. Precursor (pre)-miR-124 was injected into the right hind ankle on day 9. Morphological changes in the ankle joint were assessed by micro-CT and histopathology. Cytokine expression was examined by western blotting and realtime RT-PCR. The effect of miR-124 on predicted target messenger RNAs (mRNAs) was examined by luciferase reporter assays. The effect of pre-miR-124 or pre-miR-124a on the differentiation of human osteoclasts was examined by tartrate-resistant acid phosphatase staining.Results We found that miR-124 suppressed AIA in rats, as demonstrated by decreased synoviocyte proliferation, leucocyte infiltration and cartilage or bone destruction. Osteoclast counts and expression level of receptor activator of the nuclear factor kappa B ligand (RANKL), integrin beta 1 (ITGB1) and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) were reduced in AIA rats treated with pre-miR-124. Luciferase analysis showed that miR-124 directly targeted the 3'UTR of the rat NFATc1, ITGB1, specificity protein 1 and CCAAT/enhancer-binding protein a mRNAs. Pre-miR-124 also suppressed NFATc1 expression in RAW264.7 cells. Both miR-124 and miR-124a directly targeted the 3'-UTR of human NFATc1 mRNA, and both pre-miR-124 and pre-miR-124a suppressed the differentiation of human osteoclasts.Conclusions We found that miR-124 ameliorated AIA by suppressing critical prerequisites for arthritis development, such as RANKL and NFATc1. Thus, miR-124a is a candidate for therapeutic use for human rheumatoid arthritis.

The recent development of salivary proteomics has led to the identification of potential biomarkers for diagnosing patients with primary Sjogren's syndrome (pSS). Here we sought to identify differentially produced salivary metabolites from pSS patients and healthy controls (HCs) that might be used to characterize this disease. We obtained salivary samples from 12 female pSS patients (mean age 44.2 +/- 13.01) and 21 age-matched female HCs. The metabolite profiles of saliva were analysed by gas chromatography-mass spectrometry. The total metabolite levels in each of the samples were calculated and compared across the study participants. A total of 88 metabolites were detected across the study samples, 41 of which were observed at reduced levels in the samples frompSS patients. Principal component analysis (PCA) revealed a loss in salivary metabolite diversity in the pSS patient samples compared to the HC samples. The reduced presence of glycine, tyrosine, uric acid and fucose, which may reflect salivary gland destruction due to chronic sialoadenitis, contributed to the loss of diversity. Comparative PCA of the pSS patients revealed the presence of two subpopulations based on their metabolite profiles, and these two subpopulations showed a significant difference in the prevalence of major salivary glanditis (P=0014). In this study, we found that the salivary metabolite profile of pSS patients was less diverse than that of HCs and that the metabolite profiles in pSS patients were affected by the presence of major salivary glanditis.

Objective. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that have the ability to suppress T cell responses. The aim of this study was to evaluate the effects of the JAK inhibitor tofacitinib on MDSCs in a mouse model of rheumatoid arthritis.Methods. Arthritis was induced in SKG mice by zymosan A (ZyA) injection. MDSCs isolated from the bone marrow (BM) of donor SKG mice with arthritis were adoptively transferred to recipient mice with arthritis. In a separate experiment, tofacitinib was administered to arthritic SKG mice subcutaneously via osmotic pump, in some cases followed by injection of an anti-Gr-1 monoclonal antibody (mAb). BM cells from untreated mice were cultured for 5 days with granulocyte-macrophage colony-stimulating factor, with or without tofacitinib, and then analyzed by flow cytometry.Results. The numbers of MDSCs and polymorphonuclear MDSCs (PMN-MDSCs) were significantly increased in the spleens of SKG mice following ZyA injection. Adoptive transfer of MDSCs to recipient arthritic mice reduced the severity of arthritis compared to that in untreated control mice. Treatment with tofacitinib also ameliorated the progression of arthritis in SKG mice and induced significantly higher numbers of MDSCs and PMN-MDSCs in the BM of these animals. Furthermore, administration of an anti-Gr-1 mAb reduced the antiarthritic effect of tofacitinib in SKG mice. In vitro, tofacitinib facilitated the differentiation of BM cells to MDSCs, and inhibited their differentiation to dendritic cells.Conclusion. Tofacitinib facilitates the expansion of MDSCs and ameliorates arthritis in SKG mice.