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Hui Guan,1 Ming Zhong,1 Kongyang Ma,2 Chun Tang,1 Xiaohua Wang,1 Muzi Ouyang,3 Rencai Qin,2 Jiasi Chen,1 Enyi Zhu,1 Ting Zhu,1 Yongping Lu,1 Yu Liu,1 Chengzi Tian,4 Zhihua Zheng1 1Department of Nephrology, Center of Kidney and Urology, The Seventh Affiliated Hospital, Sun Yat-Sen University, Shenzhen, People’s Republic of China; 2Centre of Infection and Immunity Studies, School of Medicine, Sun Yat-Sen University, Shenzhen, People’s Republic of China; 3Department of Pharmacology, School of Medicine, Sun Yat-Sen University, Shenzhen, People’s Republic of China; 4Center of Reproductive Medical, The Seventh Affiliated Hospital, Sun Yat-Sen University, Shenzhen, People’s Republic of ChinaCorrespondence: Zhihua Zheng, Email zhzhihua@mail.sysu.edu.cnBackground: HMGB1 is a highly conserved nuclear protein widely expressed in mammalian cells. This study aimed to comprehensively investigate the roles and mechanisms of HMGB1 in different tumors.Methods: Original data on HMGB1 expression, localization, potential interacting proteins, genetics were obtained from The Cancer Genome Atlas, Genotype-Tissue Expression, Cancer Cell Line Encyclopedia, Human Protein Atlas, Compartmentalized Protein-Protein Interaction and cBioPortal databases. Then, correlation between HMGB1 expression levels and tumor stage, prognosis, potential pathways, tumor microenvironment, ESTIMATE score, immune-related genes, immune cell infiltration, microsatellite instability, tumor mutation burden, or anti-tumor drug resistance was investigated. The above results consistently indicated that high expression of HMGB1 protein may be related to clinical prognosis of HCC patients. Therefore, clinical tissues of HCC patients were selected to verify the differential expression of HMGB1 protein in HCC. The sensitivity of HMGB1-siRNA transfected HepG2 cells to sorafenib was assessed.Results: HMGB1 was found to be differentially expressed in many tumors and normal tissues. HMGB1 was mainly located in the nucleus and might interact with proteins such as TLR2 and TLR4. Furthermore, HMGB1 expression was closely related to tumor stage, prognosis, tumor microenvironment, immune-related genes, immune cell infiltration, microsatellite instability, tumor mutation burden, and anti-tumor drug resistance and might be involved in different pathways of various tumors. Immunohistochemistry results further verified the differential expression of HMGB1 in HCC and paracancerous tissues. HMGB1-siRNA transfected HepG2 cells had a tendency to be more insensitive to sorafenib treatment compared to the control group.Conclusions: HMGB1 was differentially expressed in most tumors and normal tissues, and was closely related to the clinical stage, prognosis, immune infiltration, tumor microenvironment, and drug resistance of tumors. Therefore, HMGB1 may serve as a novel biomarker for predicting tumor prognosis, efficacy of immune checkpoint inhibitors, and a potential target for anti-tumor therapy.Keywords: high mobility group box 1, pan-cancer analysis, tumor, bioinformatics, prognosis

Autoantibodies produced by B cells play a pivotal role in the pathogenesis of systemic lupus erythematosus (SLE). However, both the cellular source of anti-phospholipid antibodies and their contributions to the development of lupus nephritis (LN) remain largely unclear. Here, we report a pathogenic role ofanti-phosphatidylserine (PS) autoantibodies in the development of LN. In cohort study and mouse models, elevated serum PS-specific IgG levels were detected in SLE patients, especially in those with nephritis, and lupus mice. The deposition of PS-specific IgG was detected in kidney biopsied of lupus nephritis patients. Both transfer of SLE PS-specific IgG and PS immunization triggered lupus-like glomerular immune complex deposition in recipient mice. ELISPOT analysis identified B1a cells as the main cell type for secreting PS-specific IgG in both lupus mice and patients. Adoptive transfer of PS-specific B1a cells accelerated PS-specific autoimmune response and renal damage in recipient lupus mice whereas depletion of B1a cells attenuated lupus progression. In culture, PS-specific B1a cells were significantly expanded upon treatment with chromatin components while blockade of TLR signal cascades by DNase I digestion, inhibitory ODN 2088 or R406 treatment profoundly abrogated chromatin-induced PS-specific IgG secretion by lupus B1a cells. Thus, our study has demonstrated that the novel anti-PS autoantibodies produced by B1 cells contribute to lupus nephritis development. Our findings that blockade of TLR/Syk signaling cascade inhibits PS-specific B1 cell expansion may provide new insight in understanding lupus pathogenesis and may help develop novel therapeutic targets for the treatment of LN in SLE.

Atherosclerosis, an inflammatory progressive vascular disease, causes heart disease and stroke worldwide. B cells with immune suppressive functions have been implicated in autoimmune, inflammatory, and cardiovascular diseases. However, the precise role of regulatory B cells and the interaction with macrophages in atherosclerosis remains undefined. In our study, eight-week-old female apolipoprotein E null (Apoe

Recent studies have demonstrated a central role for plasma cells in the development of autoimmune diseases, such as systemic lupus erythematosus (SLE). Currently, both the phenotypic features and functional regulation of autoreactive plasma cells during SLE pathogenesis remain largely unclear. In this study, we first found that a major subset of IL-17 receptor-expressing plasma cells potently produced anti-dsDNA IgG upon IL-17A (IL-17) stimulation in SLE patients and lupus mice. Using a humanized lupus mouse model, we showed that the transfer of Th17 cell-depleted PBMCs from lupus patients resulted in a significantly reduced plasma cell response and attenuated renal damage in recipient mice compared to the transfer of total SLE PBMCs. Moreover, long-term BrdU incorporation in lupus mice detected highly enriched long-lived BrdU(+) subsets among IL-17 receptor-expressing plasma cells. Lupus mice deficient in IL-17 or IL-17 receptor C (IL-17RC) exhibited a diminished plasma cell response and reduced autoantibody production with attenuated renal damage, while the adoptive transfer of Th17 cells triggered the plasma cell response and renal damage in IL-17-deficient lupus mice. In reconstituted chimeric mice, IL-17RC deficiency resulted in severely impaired plasma cell generation but showed no obvious effect on germinal center B cells. Further mechanistic studies revealed that IL-17 significantly promoted plasma cell survival via p38-mediated Bcl-xL transcript stabilization. Together, our findings identified a novel function of IL-17 in enhancing plasma cell survival for autoantibody production in lupus pathogenesis, which may provide new therapeutic strategies for the treatment of SLE.

Background Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies. Treatments such as surgical resection, radiation and chemotherapy have limited efficiency due to the dense stromal and immunosuppressive tumor microenvironment (TME). The interaction between the microenvironment and cancer cells remains to be further elucidated. In this study, we characterized the prognostic value of P311 in PDAC and aimed to examine the interaction between tumor-derived P311 and M2 macrophage and investigate the underlying mechanisms. Methods Expression datasets of PDAC patients were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. The immune score was calculated by applying the ESTIMATE algorithm to the matrix data. The deconvolution algorithm (CIBERSORT) was used to assess 22 tumor-infiltrating immune cells (TIICs) for their amounts in the PDAC microenvironment. Stable PANC-1 cells expressing P311 were generated from lentiviral particles. RNA sequencing (RNA-Seq) was conducted to investigate gene-expression profiling from P311 overexpressed PANC-1 cells. The association between P311 expression and M2 macrophage polarization was determined by flow cytometry. Results P311 was overexpressed in tissues of PDAC patients compared with the adjacent normal tissues and associated with poor overall survival (OS). Based on ESTIMATE algorithm, P311 positively correlated with ESTIMATE score and stromal score. CIBERSORT algorithm analysis showed that the two common TIICs in PDAC tissues were macrophages and T lymphocytes. The proportions of M2 macrophages were significantly higher in P311 high group compared with P311 low group(P Conclusions Collectively, our studies provide evidence for a novel role for P311 in M2 macrophage polarization and infiltration, uncovering the underlying oncogenic role of P311 in PDAC. Targeting P311 may serve as a novel potential therapeutic option to reset TAM polarization toward an antitumor state in PDAC.

Increased numbers of T follicular helper (Tfh) cells have been implicated in the development of autoimmune diseases including primary Sjögren’s syndrome (pSS), but how the Tfh cell response is regulated during autoimmune pathogenesis remains largely unclear. Here, we first found negative correlations between IL-10(+) regulatory B (Breg) cell numbers and Tfh cell responses and disease activity in patients with pSS and mice with experimental Sjögren’s syndrome (ESS). Moreover, we detected high expression of IL-10 receptor on Tfh cells and their precursors in both humans and mice. In culture, IL-10 suppressed human and murine Tfh cell differentiation by promoting STAT5 phosphorylation. By using an adoptive transfer approach and two-photon live imaging, we found significantly increased numbers of Tfh cells with enhanced T cell homing into B cell follicles in the draining cervical lymph nodes of RAG-2−/− mice transferred with IL-10-deficient B cells during ESS development compared with those of RAG-2−/− mice transferred with wild-type B cells. In ESS mice, CD19(+)CD1d(hi)CD5(+) Breg cells with decreased IL-10 production exhibited severely impaired suppressive effects on T cell proliferation. Consistently, CD19(+)CD24(+)CD38(hi) Breg cells from pSS patients showed significantly reduced IL-10 production with defective inhibitory function in the suppression of autologous Tfh cell expansion. Furthermore, the adoptive transfer of IL-10-producing Breg cells markedly suppressed the Tfh cell response and ameliorated ESS progression in ESS mice. Together, these findings demonstrate a critical role for IL-10-producing Breg cells in restraining the effector Tfh cell response during pSS development.

ObjectivesIn patients with systemic lupus erythematosus (SLE), immune tolerance breakdown leads to autoantibody production and immune-complex glomerulonephritis. This study aimed to identify pathogenic plasma cells (PC) in the development of lupus nephritis.MethodsPC subsets in peripheral blood and renal tissue of patients with SLE and lupus mice were examined by flow cytometry and confocal microscopy, respectively. Sorting-purified PCs from lupus mice were adoptively transferred into Rag2-deficient recipients, in which immune-complex deposition and renal pathology were investigated. In culture, PCs from lupus mice and patients with SLE were treated with a TLR4 inhibitor and examined for autoantibody secretion by enzyme-linked immunospot assay (ELISPOT). Moreover, lupus mice were treated with a TLR4 inhibitor, followed by the assessment of serum autoantibody levels and glomerulonephritis activity.ResultsThe frequencies of TLR4+CXCR4+ PCs in peripheral blood and renal tissue were found significantly increased with the potent production of anti-dsDNA IgG, which were associated with severe renal damages in patients with SLE and mice with experimental lupus. Adoptive transfer of TLR4+CXCR4+ PCs from lupus mice led to autoantibody production and glomerulonephritis development in Rag2-deficient recipients. In culture, TLR4+CXCR4+ PCs from both lupus mice and patients with SLE showed markedly reduced anti-dsDNA IgG secretion on TLR4 blockade. Moreover, in vivo treatment with TLR4 inhibitor significantly attenuated autoantibody production and renal damages in lupus mice.ConclusionsThese findings demonstrate a pathogenic role of TLR4+CXCR4+ PCs in the development of lupus nephritis and may provide new therapeutic strategies for the treatment of SLE.

B cells have a critical role in the initiation and acceleration of autoimmune diseases, especially those mediated by autoantibodies. In the peripheral lymphoid system, mature B cells are activated by self or/and foreign antigens and signals from helper T cells for differentiating into either memory B cells or antibody-producing plasma cells. Accumulating evidence has shown that epigenetic regulations modulate somatic hypermutation and class switch DNA recombination during B-cell activation and differentiation. Any abnormalities in these complex regulatory processes may contribute to aberrant antibody production, resulting in autoimmune pathogenesis such as systemic lupus erythematosus. Newly generated knowledge from advanced modern technologies such as next-generation sequencing, single-cell sequencing and DNA methylation sequencing has enabled us to better understand B-cell biology and its role in autoimmune development. Thus this review aims to summarize current research progress in epigenetic modifications contributing to B-cell activation and differentiation, especially under autoimmune conditions such as lupus, rheumatoid arthritis and type 1 diabetes.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by excessive autoantibody production and multi-organ involvement. Although the etiology of SLE still remains unclear, recent studies have characterized several pathogenic B cell subsets and regulatory B cell subsets involved in the pathogenesis of SLE. Among pathogenic B cell subsets, age-associated B cells (ABCs) are a newly identified subset of autoreactive B cells with T-bet-dependent transcriptional programs and unique functional features in SLE. Accumulation of T-bet+ CD11c+ ABCs has been observed in SLE patients and lupus mouse models. In addition, innate-like B cells with the autoreactive B cell receptor (BCR) expression and long-lived plasma cells with persistent autoantibody production contribute to the development of SLE. Moreover, several regulatory B cell subsets with immune suppressive functions have been identified, while the impaired inhibitory effects of regulatory B cells have been indicated in SLE. Thus, further elucidation on the functional features of B cell subsets will provide new insights in understanding lupus pathogenesis and lead to novel therapeutic interventions in the treatment of SLE.

In patients with systemic lupus erythematosus (SLE), immune tolerance breakdown leads to autoantibody production and immune-complex glomerulonephritis. This study aimed to identify pathogenic plasma cells (PC) in the development of lupus nephritis.PC subsets in peripheral blood and renal tissue of patients with SLE and lupus mice were examined by flow cytometry and confocal microscopy, respectively. Sorting-purified PCs from lupus mice were adoptively transferred intoThe frequencies of TLR4These findings demonstrate a pathogenic role of TLR4

B-1 cells play a critical role in early protection during influenza infections by producing natural IgM antibodies. However, the underlying mechanisms involved in regulating this process are largely unknown. Here we found that during influenza infection pleural cavity B-1a cells rapidly infiltrated lungs, where they underwent plasmacytic differentiation with enhanced IgM production. This process was promoted by IL-17A signaling via induction of Blimp-1 expression and NF-kB activation in B-1a cells. Deficiency of IL-17A led to severely impaired B-1a-derived antibody production in the respiratory tract, resulting in a deficiency in viral clearance. Transfer of B-1a-derived natural antibodies rescued Il17a(-/-) mice from otherwise lethal infections. Together, we identify a critical function of IL-17A in promoting the plasmacytic differentiation of B-1a cells. Our findings provide new insights into the mechanisms underlying the regulation of pulmonary B-1a cell response against influenza infection., published_or_final_version

We thank Alunno et al for their interest in our recently published work and their insightful comments regarding the roles of T helper (Th) 17 cell subsets during the pathogenesis of Sjogren's syndrome (SS) in mouse model and humans.1 In our study, we characterised the kinetic changes of IL-17-producing CD4 Th17 cells in salivary glands (SG) and draining cervical lymph nodes (CLN), and examined their correlations with clinical, histological and serological features of SS in mice immunised with SG proteins. Moreover, we revealed that IL-17 knockout (KO) mice were completely resistant for SS induction, while adoptive transfer of Th17 cells rapidly induced a full profile of SS in immunised IL-17 KO mice with markedly reduced saliva secretion, elevated autoantibody production and pronounced lymphocytic inflammation and tissue damage in SG. Together, our findings have defined a critical role of Th17 cells in the pathogenesis of SS in mice. Recently, there is increasing evidence indicating the involvement of several IL-17-producing T cell subsets in the development of SS in humans and mouse.2–4 In particular, an increased IL-17-producing T cell subset lacking CD4 and CD8 surface molecules (double negative, DN) was found in the peripheral blood …

Aims Cytotoxic T lymphocyte antigen 4 (CTLA4) exerts inhibitory effects on T-cell activation by competition with CD28. In this study, we investigated the effect of CTLA4-IgG on homocysteine (Hcy)-induced T-cell activation and potential signal pathways involved in atherosclerotic formation. Methods and results The CD28 signal was significantly amplified by Hcy treatment in splenic T cells and hyperhomocysteinaemia (HHcy)-accelerated plaques in apolipoprotein E-deficient (apoE–/–) mice. As a major competitor of CD28, CTLA4-IgG (abatacept) pretreatment, 100 μg/week, in apoE–/– mice could reverse 2- and 4-week HHcy-accelerated atherosclerosis. Furthermore, the membrane level of CTLA4 was decreased and the endocytosis level was increased by HHcy. Endocytosed CTLA4 molecules by Hcy were in large vesicles, colocalized with lysosomes and endosomes. Hcy-increased CTLA4 endocytosis and secretion of inflammatory cytokines in T cells were blocked by CTLA4-IgG and the PI3K inhibitor LY294002. Blocking the CD28 signal pathway in T cells significantly decreased Hcy-promoted macrophage migration. Conclusion These results illustrate a novel mechanism of CD28-dependent T-cell costimulation involved in HHcy-accelerated atherosclerosis, which extends the pharmacological application of CTLA4-IgG for atherosclerosis.

Rationale: A number of epidemiological studies have suggested an association of hyperhomocysteinemia (HHcy) and abdominal aortic aneurysm (AAA), but discrepancies exist. In addition, we lack direct evidence supporting a causal role. Objective: We determined the association and contribution of HHcy to AAA formation. Methods and Results: We first performed a meta-analysis of studies involving 1489 subjects and found a strong association of HHcy and AAA (odds ratio, 7.39). Next, we used angiotensin II–infused male apolipoprotein E–deficient mice and tested whether HHcy contributes to AAA pathogenesis. Homocysteine (Hcy) supplement (1.8 g/L) in drinking water resulted in mild HHcy. Intriguingly, HHcy greatly increased the incidence of angiotensin II–induced AAA and aortic dissection in apolipoprotein E–deficient mice (vehicle versus Hcy: 50% versus 100%; P ). Histology indicated HHcy markedly exaggerated aortic adventitial inflammation. Increased levels of proinflammatory interleukin-6 and monocyte chemoattractant protein-1 were preferentially colocalized within adventitial fibroblasts in HHcy plus angiotensin II mice, which suggested the importance of adventitial fibroblasts activation in Hcy-aggravated AAA. Hcy sequentially stimulated adventitial fibroblasts transformation into myofibroblasts, secretion of interleukin-6 and monocyte chemoattractant protein-1, and consequent recruitment of monocytes/macrophages to adventitial fibroblasts, which was abolished by the NADPH oxidase inhibitor diphenyliodonium. NADPH oxidase 4, but not other homologs of NADPH oxidase, was significantly upregulated by Hcy in adventitial fibroblasts, whereas NADPH oxidase 4 small interfering RNA silencing diminished Hcy-induced adventitial fibroblasts activation. Finally, folic acid supplement (0.071 μg/g per day) markedly reduced HHcy-aggravated angiotensin II–induced AAA formation in apolipoprotein E–deficient mice. Conclusions: HHcy may aggravate AAA formation at least partially via activating adventitial fibroblast NADPH oxidase 4.

Epoxyeicosatrienoic acids (EETs), synthesized from arachidonic acid by cytochrome P450 epoxygenases, are converted to dihydroxyeicosatrienoic acids by soluble epoxide hydrolase. EETs exert anti-inflammatory effects. However, the effect of EETs on humoral immunity is poorly understood. The present study is to investigate the potential role of EETs on B cell function and mechanisms. We examined the role of EETs on antibody production of splenic B cells from C57BL/6 and apolipoprotein E-deficient (ApoE-/-) mice by means of ELISA. Of the 4 EET regioisomers, 8,9-EET decreased basal and activation-induced B cell antibody secretion. As well, 8,9-EET significantly inhibited B-cell proliferation and survival, plasma cell differentiation and class-switch recombination. Western blot analysis revealed that lipopolysaccharide-induced nuclear translocation of NF-κB could be attenuated by 8,9-EET. Furthermore, germinal center formation was impaired by 8,9-EET in mice in vivo. 8,9-EET may inhibit B-cell function in vitro and in vivo, which suggests a new therapeutic strategy for diseases with excess B cell activation.