Brown adipose tissue-derived MaR2 contributes to cold-induced resolution of inflammation.

Obesity induces chronic inflammation resulting in insulin resistance and metabolic disorders. Cold exposure can improve insulin sensitivity in humans and rodents, but the mechanisms have not been fully elucidated. Here, we find that cold resolves obesity-induced inflammation and insulin resistance and improves glucose tolerance in diet-induced obese mice. The beneficial effects of cold exposure on improving obesity-induced inflammation and insulin resistance depend on brown adipose tissue (BAT) and liver. Using targeted liquid chromatography with tandem mass spectrometry, we discovered that cold and ß3-adrenergic stimulation promote BAT to produce maresin 2 (MaR2), a member of the specialized pro-resolving mediators of bioactive lipids that play a role in the resolution of inflammation. Notably, MaR2 reduces inflammation in obesity in part by targeting macrophages in the liver. Thus, BAT-derived MaR2 could contribute to the beneficial effects of BAT activation in resolving obesity-induced inflammation and may inform therapeutic approaches to combat obesity and its complications.

Placental superoxide dismutase 3 mediates benefits of maternal exercise on offspring health.

Poor maternal diet increases the risk of obesity and type 2 diabetes in offspring, adding to the ever-increasing prevalence of these diseases. In contrast, we find that maternal exercise improves the metabolic health of offspring, and here, we demonstrate that this occurs through a vitamin D receptor-mediated increase in placental superoxide dismutase 3 (SOD3) expression and secretion. SOD3 activates an AMPK/TET signaling axis in fetal offspring liver, resulting in DNA demethylation at the promoters of glucose metabolic genes, enhancing liver function, and improving glucose tolerance. In humans, SOD3 is upregulated in serum and placenta from physically active pregnant women. The discovery of maternal exercise-induced cross talk between placenta-derived SOD3 and offspring liver provides a central mechanism for improved offspring metabolic health. These findings may lead to novel therapeutic approaches to limit the transmission of metabolic disease to the next generation.

A biomimetic five-module chimeric antigen receptor (5MCAR) designed to target and eliminate antigen-specific T cells.

T cells express clonotypic T cell receptors (TCRs) that recognize peptide antigens in the context of class I or II MHC molecules (pMHCI/II). These receptor modules associate with three signaling modules (CD3γε, δε, and ζζ) and work in concert with a coreceptor module (either CD8 or CD4) to drive T cell activation in response to pMHCI/II. Here, we describe a first-generation biomimetic five-module chimeric antigen receptor (5MCAR). We show that 1) chimeric receptor modules built with the ectodomains of pMHCII assemble with CD3 signaling modules into complexes that redirect cytotoxic T lymphocyte (CTL) specificity and function in response to the clonotypic TCRs of pMHCII-specific CD4+ T cells, and 2) surrogate coreceptor modules enhance the function of these complexes. Furthermore, we demonstrate that adoptively transferred 5MCAR-CTLs can mitigate type I diabetes by targeting autoimmune CD4+ T cells in NOD mice. This work provides a framework for the construction of biomimetic 5MCARs that can be used as tools to study the impact of particular antigen-specific T cells in immune responses, and may hold potential for ameliorating diseases mediated by pathogenic T cells.

Human Sox4 facilitates the development of CXCL13-producing helper T cells in inflammatory environments.

In human inflammatory sites, PD-1hiCXCR5-CD4+ T cells are involved in the formation of ectopic lymphoid-like structures (ELSs) by the secretion of chemokine CXCL13, but how the transcription of CXCL13 is regulated in CD4+ T cells is still unclear. Here we show that Sox4 is a key transcription factor for CXCL13 production in human CD4+ T cells under inflammatory conditions. In vitro TGF-ß+, IL-2-neutralizing culture conditions give rise to PD-1hiCXCR5-CD4+ T cells that preferentially express CXCL13, and transcriptome analysis and lentiviral overexpression indicate Sox4 association with the CXCL13 transcription. In vivo, Sox4 is significantly upregulated in synovial CD4+ T cells, when compared with blood CD4+ T cells, from patients with rheumatoid arthritis (RA), and further correlates with ELS formation in RA synovium. Overall, our studies suggest that Sox4 contributes to CXCL13 production and ELS formation at inflammatory sites in humans.

TGF-ß induces the differentiation of human CXCL13-producing CD4(+) T cells.

In the ectopic lymphoid-like structures present in chronic inflammatory conditions such as rheumatoid arthritis, a subset of human effector memory CD4(+) T cells that lacks features of follicular helper T (Tfh) cells produces CXCL13. Here, we report that TGF-ß induces the differentiation of human CXCL13-producing CD4(+) T cells from naïve CD4(+) T cells. The TGF-ß-induced CXCL13-producing CD4(+) T cells do not express CXCR5, B-cell lymphoma 6 (BCL6), and other Tfh-cell markers. Furthermore, expression levels of CD25 (IL-2Rα) in CXCL13-producing CD4(+) T cells are significantly lower than those in FoxP3(+) in vitro induced Treg cells. Consistent with this, neutralization of IL-2 and knockdown of STAT5 clearly upregulate CXCL13 production by CD4(+) T cells, while downregulating the expression of FoxP3. Furthermore, overexpression of FoxP3 in naïve CD4(+) T cells downregulates CXCL13 production, and knockdown of FoxP3 fails to inhibit the differentiation of CXCL13-producing CD4(+) T cells. As reported in rheumatoid arthritis, proinflammatory cytokines enhance secondary CXCL13 production from reactivated CXCL13-producing CD4(+) T cells. Our findings demonstrate that CXCL13-producing CD4(+) T cells lacking Tfh-cell features differentiate via TGF-ß signaling but not via FoxP3, and exert their function in IL-2-limited but TGF-ß-rich and proinflammatory cytokine-rich inflammatory conditions.

A novel splice variant of human L-selectin encodes a soluble molecule that is elevated in serum of patients with rheumatic diseases.

L-selectin, a type I membrane protein, is a leukocyte adhesion molecule that mediates both lymphocyte homing to peripheral lymph nodes and leukocyte accumulation at sites of inflammation. L-selectin is rapidly shed from the cell surface after cellular activation, and the ectodomain thus released is thought to account for high levels of soluble L-selectin in serum. In this study, we report the identification of a novel, naturally occurring isoform of the human L-selectin gene. Sequence analysis revealed that this isoform is generated by an alternative splicing event: the 7th exon of the human L-selectin gene, which encodes the region containing the transmembrane domain, is excluded, predicting a soluble protein product. The mRNA for this splice variant was expressed in lymphoid organs, where conventional L-selectin mRNA was also expressed. Activating T cells increased the variant mRNA and its ratio to the membrane form. Soluble L-selectin translated from the variant mRNA was present in human serum, albeit at a much lower level than that arising from ectodomain shedding, and was markedly elevated in patients with various rheumatic diseases, including rheumatoid arthritis and systemic lupus erythematosus. These observations indicate that some of the soluble L-selectin present in human serum arises through alternative splicing, which may be upregulated during lymphocyte activation in patients with various clinical conditions.

TNFα, PDGF, and TGFß synergistically induce synovial lining hyperplasia via inducible PI3Kδ.

OBJECTIVES: To determine the mechanism underlying hypertrophic synovium in rheumatoid arthritis (RA). METHODS: We examined micromass cultures of fibroblast-like synoviocytes (FLSs) stimulated with tumor necrosis factor α (TNFα), platelet-derived growth factor (PDGF), and/or transforming growth factor ß (TGFß). The hypertrophic architecture of the micromasses, expression of phosphoinositide 3 kinase (PI3K) isoforms, and persistent activation of PI3K-Akt pathways were investigated. FLSs transfected with siRNA were also examined in the micromass cultures. RESULTS: The combination of TNFα, PDGF, and TGFß (TPT condition) induced obvious hypertrophic architecture of the intimal lining layer in FLSs in micromass cultures, and was accompanied by upregulated expression of matrix metalloproteinase-3 (MMP3), Cadherin-11, and PI3Kδ. In monolayer FLSs, the TPT condition enhanced the expression of PI3Kδ and persistent activation of the PI3K-Akt pathway. Knockdown of PI3Kδ significantly inhibited the formation of the hypertrophic synovial lining in the TPT condition. CONCLUSIONS: These results collectively indicate that inducible PI3Kδ plays a crucial role in persistent activation of PI3K-Akt in FLSs, and in the formation of a hypertrophic synovial lining. PI3Kδ may be an alternative treatment target for the regulation of proliferative synovium in RA.

Human T cells expressing BEND3 on their surface represent a novel subpopulation that preferentially produces IL-6 and IL-8.

BEN domain-containing protein 3 (BEND3) has no transmembrane region, is localized in the cytoplasm, and is involved in chromatin function and transcription. We here identified a novel subpopulation of human T cells that expressed BEND3 on their cell surface (BEND3(+) T cells). BEND3(+) T cells consisted of approximately 3% of T cells in the peripheral blood, were present in both CD4(+) and CD8(+) T cells, and were also observed in cord blood. The stimulation of BEND3(+) T cells through the TCR/CD3 complex led to the production of various kinds of cytokines; however, the levels of IL-6 and IL-8 produced by BEND3(+) T cells were higher than those by BEND3(-) T cells. The proportion of BEND3(+) T cells was also increased in some patients with inflammatory diseases. Taken together, these results indicate that BEND3(+) T cells are a new subpopulation of T cells in terms of their cytokine profile. Further analyses on BEND3(+) T cells may be of importance and useful in understanding human T cell immunology.

Transcriptional responses of a bicarbonate-tolerant monocot, Puccinellia tenuiflora, and a related bicarbonate-sensitive species, Poa annua, to NaHCO3 stress.

Puccinellia tenuiflora is an alkaline salt-tolerant monocot found in saline-alkali soil in China. To identify the genes which are determining the higher tolerance of P. tenuiflora compared to bicarbonate sensitive species, we examined the responses of P. tenuiflora and a related bicarbonate-sensitive Poeae plant, Poa annua, to two days of 20 mM NaHCO3 stress by RNA-seq analysis. We obtained 28 and 38 million reads for P. tenuiflora and P. annua, respectively. For each species, the reads of both unstressed and stressed samples were combined for de novo assembly of contigs. We obtained 77,329 contigs for P. tenuiflora and 115,335 contigs for P. annua. NaHCO3 stress resulted in greater than two-fold absolute expression value changes in 157 of the P. tenuiflora contigs and 1090 of P. annua contigs. Homologs of the genes involved in Fe acquisition, which are important for the survival of plants under alkaline stress, were up-regulated in P. tenuiflora and down-regulated in P. annua. The smaller number of the genes differentially regulated in P. tenuiflora suggests that the genes regulating bicarbonate tolerance are constitutively expressed in P. tenuiflora.

A distinct human CD4+ T cell subset that secretes CXCL13 in rheumatoid synovium.

OBJECTIVE: A subset of CD4+ T cells in the synovium of patients with rheumatoid arthritis (RA) produce CXCL13, a chemokine that is crucial for the formation of germinal centers. This study was undertaken to determine the relevance of this population to known subsets of T helper cells and to proinflammatory cytokines, and how these cells are generated. METHODS: The expression of Th markers and CXCL13 by CD4+ T cells in RA synovium and the involvement of proinflammatory cytokines in CXCL13 production were assessed. We also investigated whether CXCL13+CD4+ T cells could be newly induced. RESULTS: CXCL13+CD4+ T cells in RA synovium were negative for interferon-γ (IFNγ), interleukin-4 (IL-4), IL-17, FoxP3, and CXCR5 and expressed low levels of inducible T cell costimulator, indicating that this population is a distinct human CD4 subset. T cell receptor (TCR) stimulation of CD4+ T cells, obtained from RA synovium with low expression of CXCL13, promptly induced CXCL13 production and addition of proinflammatory cytokines supported the long-term production of CXCL13. These findings indicate that CXCL13-producing CD4+ T cells can be in a memory state ready to be reactivated upon TCR stimulation and that proinflammatory cytokines are involved in persistent CXCL13 production. TCR stimulation of CD4+ T cells from the blood of healthy volunteers, together with proinflammatory cytokine supplementation, induced a population that produced CXCL13, but not IFNγ. Synovial T cells recruited CXCR5+ cells in a CXCL13-dependent manner. CONCLUSION: CXCL13-producing CD4+ T cells induced in RA synovium may play a role in the recruitment of CXCR5+ cells, such as B cells and circulating follicular helper T cells, and in ectopic lymphoid neogenesis at sites of inflammation.

A U-Box E3 ubiquitin ligase, PUB20, interacts with the Arabidopsis G-protein ß subunit, AGB1.

An Arabidopsis U-box E3 ubiquitin ligase Plant U-box 20 (PUB20; alternatively called AtCMPG1) was identified as a possible interactor of the Arabidopsis G-protein ß subunit, AGB1, by yeast two-hybrid screening. A bimolecular fluorescence complementation (BiFC) assay showed that PUB20 interacted with AGB1 in the nuclei and the cytosol. The expression levels of PUB20 and its closest homolog, PUB21 were stable under many conditions. GUS driven by the PUB20 promoter was active in anthers, pollen, premature seeds and receptacles and GUS driven by the PUB21 promoter was active in anthers and funiculi. PUB20 was found to have autoubiquitination activity in vitro.

Molecular cloning and characterization of plasma membrane- and vacuolar-type Na⁺/H⁺ antiporters of an alkaline-salt-tolerant monocot, Puccinellia tenuiflora.

A better understanding of salt tolerance in plants might lead to the genetic engineering of crops that can grow in saline soils. Here we cloned and characterized plasma membrane and vacuolar Na⁺/H⁺ antiporters of a monocotyledonous alkaline-tolerant halophyte, Puccinellia tenuiflora. The predicted amino acid sequence of the transporters were very similar to those of orthologs in monocotyledonous crops. Expression analysis showed that (1) NHA was more strongly induced by NaCl in the roots of P. tenuiflora while in rice it was rather induced in the shoots, suggesting that the role of NHA in salt excretion from the roots partly accounts for the difference in the tolerance of the two species, and that (2) NHXs were specifically induced by NaHCO3 but not by NaCl in the roots of both species, suggesting that vacuolar-type Na⁺/H⁺ antiporters play roles in pH regulation under alkaline salt conditions. Overexpression of the antiporters resulted in increased tolerance of shoots to NaCl and roots to NaHCO3. Overexpression lines exhibited a lower Na⁺ content and a higher K⁺ content in shoots under NaCl treatments, leading to a higher Na⁺/H⁺ ratio.

Overexpression of a minimal domain of calpastatin suppresses IL-6 production and Th17 development via reduced NF-κB and increased STAT5 signals.

Calpain, a calcium-dependent cysteine protease, is reportedly involved in the pathophysiology of autoimmune diseases such as rheumatoid arthritis (RA). In addition, autoantibodies against calpastatin, a natural and specific inhibitor of calpain, are widely observed in RA. We previously reported that E-64-d, a membrane-permeable cysteine protease inhibitor, is effective in treating experimental arthritis. However, the exact role of the calpastatin-calpain balance in primary inflammatory cells remains unclear. Here we investigated the effect of calpain-specific inhibition by overexpressing a minimal functional domain of calpastatin in primary helper T (Th) cells, primary fibroblasts from RA patients, and fibroblast cell lines. We found that the calpastatin-calpain balance varied during Th1, Th2, and Th17 development, and that overexpression of a minimal domain of calpastatin (by retroviral gene transduction) or the inhibition of calpain by E-64-d suppressed the production of IL-6 and IL-17 by Th cells and the production of IL-6 by fibroblasts. These suppressions were associated with reductions in RORγt expression and STAT3 phosphorylation. Furthermore, inhibiting calpain by silencing its small regulatory subunit (CPNS) suppressed Th17 development. We also confirmed that overexpressing a minimal domain of calpastatin suppressed IL-6 by reducing NF-κB signaling via the stabilization of IκBα, without affecting the upstream signal. Moreover, our findings indicated that calpastatin overexpression suppressed IL-17 production by Th cells by up-regulating the STAT5 signal. Finally, overexpression of a minimal domain of calpastatin suppressed IL-6 production efficiently in primary fibroblasts derived from the RA synovium. These findings suggest that inhibiting calpain by overexpressing a minimal domain of calpastatin could coordinately suppress proinflammatory activities, not only those of Th cells but also of synovial fibroblasts. Thus, this strategy may prove viable as a candidate treatment for inflammatory diseases such as RA.

The RIG-I-like receptor IFIH1/MDA5 is a dermatomyositis-specific autoantigen identified by the anti-CADM-140 antibody.

OBJECTIVES: Various autoantibodies are detected in the sera of PM/DM patients. Some of them are specific to PM/DM patients and closely associated with clinical manifestations of the diseases. Recently, the anti-CADM-140 antibody was reported to be found specifically in clinically amyopathic DM (C-ADM) patients and to be associated with acute interstitial lung disease (ILD). We assessed the clinical significance of the anti-CADM-140 antibody and then investigated the autoantigen recognized by the anti-CADM-140 antibody. METHODS: Autoantibodies were screened in 192 patients with various CTDs and 21 healthy controls using immunoprecipitation with [(35)S]methionine-labelled HeLa cells. Immunoabsorbent column chromatography was used to purify an autoantigen that was subsequently subjected to peptide mass fingerprinting. RESULTS: The anti-CADM-140 antibody was revealed to be specific to DM. Most of the anti-CADM-140-positive patients were C-ADM although some of them showed apparent myositis. The anti-CADM-140-positive patients frequently showed hyperferritinaemia and acute progressive ILD with poor prognosis. The anti-CADM-140 antibody was shown to recognize IFN induced with helicase C domain protein 1 (IFIH1), also known as the melanoma differentiation-associated gene 5 (MDA5), which is one of the RIG-I-like receptors and plays a role in innate immune responses. CONCLUSION: The anti-CADM-140 antibody was a marker of DM and intractable ILD and recognized IFIH1/MDA5, which is involved in innate immunity. These findings may give a new insight into the pathogenesis of DM.

Gamma/delta T cells are the predominant source of interleukin-17 in affected joints in collagen-induced arthritis, but not in rheumatoid arthritis.

OBJECTIVE: Although interleukin-17 (IL-17)-producing gamma/delta T cells were reported to play pathogenic roles in collagen-induced arthritis (CIA), their characteristics remain unknown. The aim of this study was to clarify whether gamma/delta T cells or CD4+ T cells are the predominant IL-17-producing cells, and to determine what stimulates gamma/delta T cells to secret IL-17 in mice with CIA. The involvement of IL-17-producing gamma/delta T cells in SKG mice with autoimmune arthritis and patients with rheumatoid arthritis (RA) was also investigated. METHODS: IL-17-producing cells in the affected joints of mice with CIA were counted by intracellular cytokine staining during 6 distinct disease phases, and these cells were stimulated with various combinations of cytokines or specific antigens to determine the signaling requirements. Similar studies were performed using SKG mice with arthritis and patients with RA. RESULTS: Gamma/delta T cells were the predominant population in IL-17-producing cells in the swollen joints of mice with CIA, and the absolute numbers of these cells increased in parallel with disease activity. IL-17-producing gamma/delta T cells expressed CC chemokine receptor 6, were maintained by IL-23 but not by type II collagen in vitro, and were induced antigen independently in vivo. Furthermore, IL-17 production by gamma/delta T cells was induced by IL-1beta plus IL-23 independently of T cell receptor. In contrast to what was observed in mice with CIA, IL-17-producing gamma/delta T cells were nearly absent in the affected joints of SKG mice and patients with RA, and Th1 cells were predominant in the joints of patients with RA. CONCLUSION: Gamma/delta T cells were antigen independently stimulated by inflammation at affected joints and produced enhanced amounts of IL-17 to exacerbate arthritis in mice with CIA but not in SKG mice with arthritis or patients with RA.

Antisynthetase syndrome associated with sarcoidosis.

A 30-year-old man complained of polyarthralgia and fatigue. The clinical findings and laboratory data included myositis, polyarthritis, interstitial pneumonia, Raynaud's phenomenon, mechanic's hand, and anti PL-7 antibody (threonyl-tRNA synthetase antibody). All of these signs were consistent with antisynthetase syndrome. His chest radiograph revealed bilateral hilar lymphadenopathy. Biopsy specimens from his mediastinal lymph node and muscle showed noncaseating epithelioid cell granulomas. Lung histology revealed nonspecific interstitial pneumonia. Antisynthetase syndrome associated with sarcoidosis was diagnosed. Interstitial pneumonia in this patient responded well to high-dose corticosteroid therapy.

Anti-aminoacyl-tRNA synthetase antibodies in clinical course prediction of interstitial lung disease complicated with idiopathic inflammatory myopathies.

In the treatment of polymyositis and dermatomyositis (PM/DM), the complication of interstitial lung disease (ILD) is an important prognostic factor. It has been reported that autoantibodies against aminoacyl-tRNA synthetases (ARS) are strongly associated with ILD. The aim of this study is to examine the correlation between anti-ARS and the clinical course of ILD. We investigated 41 cases of PM/DM with ILD. The response of ILD to corticosteroids (CS) was determined according to the change in respiratory symptoms, image findings, and pulmonary function between, before and 2 months after the treatment. Anti-ARS (anti-Jo-1, PL-7, PL-12, EJ, OJ and KS) antibodies were screened with the RNA immunoprecipitation assay. In the stratification into ILD-preceding, simultaneous and myopathy-preceding types, anti-ARS antibodies were significantly frequent in the ILD-preceding type (p < 0.05). In the stratification into anti-ARS-positive and negative groups, the response of ILD to CS was significantly better in the positive group (p < 0.05). However, recurrence of ILD was significantly more frequent in the positive group (p < 0.01), and 2 year prognoses of pulmonary function (%VC and %DLCO) were not different between the two groups. In conclusion, screening of anti-ARS may be useful to predict late-onset myopathy in ILD-preceding patients and to predict the clinical course of ILD in PM/DM patients.

Immunogenetic features in 120 Japanese patients with idiopathic inflammatory myopathy.

OBJECTIVE: To examine the role of HLA-DRB1 and tumor necrosis factor (TNF) promoter genotypes in the development and the autoantibody profiles of idiopathic inflammatory myopathy (IIM) in Japanese patients. METHODS: HLA-DRB1 and TNF promoter genotypes were determined, and serum antinuclear autoantibodies were identified in 120 adult Japanese patients with IIM [72 with dermatomyositis (DM), 30 with polymyositis (PM), 18 with myositis overlapping with other collagen vascular diseases], as well as in 265 controls. RESULTS: Forty-two patients (35%) were positive for myositis-specific autoantibodies (MSA), including 37 (31%) for anti-aminoacyl-tRNA synthetase (ARS) autoantibodies. Allele carrier frequency of HLA-DRB1*0803 was increased in the patients with IIM [p = 0.02, corrected p (pc) NS, 23% vs 14%, odds ratio (OR) = 1.9 (95% confidence interval, CI = 1.1-3.2)], with PM [p = 0.006, pc NS, 33%, OR 3.1 (95% CI 1.3-7.1)], and with anti-ARS autoantibodies [27%, p = 0.04, OR 2.3 (95% CI 1.0-5.1)] compared with controls. DRB1*0405 was increased in patients with anti-ARS autoantibodies compared with controls [41% vs 25%, p = 0.04, pc NS, OR 2.1 (95% CI 1.0-4.3)]. TNF promoter genotype was associated with the presence of interstitial lung disease (ILD). The carriage of a TNF-a haplotype formed by -1031C, -863A, and -857C was increased in the patients with ILD versus those without ILD [33% vs 18%, p = 0.05, pc NS, OR 2.3 (95% CI 0.94-5.5)]. CONCLUSION: HLA-DRB1 alleles were associated with development of IIM and MSA in a Japanese population.