Bifidobacterium adolescentis-derived hypaphorine alleviates acetaminophen hepatotoxicity by promoting hepatic Cry1 expression.

Acetaminophen (APAP)-induced liver injury (AILI) is a pressing public health concern. Although evidence suggests that Bifidobacterium adolescentis (B. adolescentis) can be used to treat liver disease, it is unclear if it can prevent AILI. In this report, we prove that B. adolescentis significantly attenuated AILI in mice, as demonstrated through biochemical analysis, histopathology, and enzyme-linked immunosorbent assays. Based on untargeted metabolomics and in vitro cultures, we found that B. adolescentis generates microbial metabolite hypaphorine. Functionally, hypaphorine inhibits the inflammatory response and hepatic oxidative stress to alleviate AILI in mice. Transcriptomic analysis indicates that Cry1 expression is increased in APAP-treated mice after hypaphorine treatment. Overexpression of Cry1 by its stabilizer KL001 effectively mitigates liver damage arising from oxidative stress in APAP-treated mice. Using the gene expression omnibus (GEO) database, we verified that Cry1 gene expression was also decreased in patients with APAP-induced acute liver failure. In conclusion, this study demonstrates that B. adolescentis inhibits APAP-induced liver injury by generating hypaphorine, which subsequently upregulates Cry1 to decrease inflammation and oxidative stress.

Immune Cell-Related Genes in Juvenile Idiopathic Arthritis Identified Using Transcriptomic and Single-Cell Sequencing Data.

Juvenile idiopathic arthritis (JIA) is the most common chronic rheumatic disease in children. The heterogeneity of the disease can be investigated via single-cell RNA sequencing (scRNA-seq) for its gap in the literature. Firstly, five types of immune cells (plasma cells, naive CD4 T cells, memory-activated CD4 T cells, eosinophils, and neutrophils) were significantly different between normal control (NC) and JIA samples. WGCNA was performed to identify genes that exhibited the highest correlation to differential immune cells. Then, 168 differentially expressed immune cell-related genes (DE-ICRGs) were identified by overlapping 13,706 genes identified by WGCNA and 286 differentially expressed genes (DEGs) between JIA and NC specimens. Next, four key genes, namely SOCS3, JUN, CLEC4C, and NFKBIA, were identified by a protein-protein interaction (PPI) network and three machine learning algorithms. The results of functional enrichment revealed that SOCS3, JUN, and NFKBIA were all associated with hallmark TNF-α signaling via NF-κB. In addition, cells in JIA samples were clustered into four groups (B cell, monocyte, NK cell, and T cell groups) by single-cell data analysis. CLEC4C and JUN exhibited the highest level of expression in B cells; NFKBIA and SOCS3 exhibited the highest level of expression in monocytes. Finally, real-time quantitative PCR (RT-qPCR) revealed that the expression of three key genes was consistent with that determined by differential analysis. Our study revealed four key genes with prognostic value for JIA. Our findings could have potential implications for JIA treatment and investigation.

Blocking Matrix Metalloproteinase-9 Abrogates Collagen-Induced Arthritis via Inhibiting Dendritic Cell Migration.

Trafficking of dendritic cells (DCs) to lymph nodes (LNs) to present Ags is a crucial step in the pathogenesis of rheumatoid arthritis (RA). Matrix metalloproteinase-9 (MMP-9) is the key molecule for DC migration. Thus, blocking MMP-9 to inhibit DC migration may be a novel strategy to treat RA. In this study, we used anti-MMP-9 Ab to treat collagen-induced arthritis (CIA) in DBA/1J mice and demonstrated that anti-MMP-9 Ab treatment significantly suppressed the development of CIA via the modulation of DC trafficking. In anti-MMP-9 Ab-treated CIA mice, the number of DCs in draining LNs was obviously decreased. In vitro, anti-MMP-9 Ab and MMP-9 inhibitor restrained the migration of mature bone marrow-derived DCs in Matrigel in response to CCR7 ligand CCL21. In addition, blocking MMP-9 decreased T and B cell numbers in LNs of CIA mice but had no direct influence on the T cell response to collagen II by CD4+ T cells purified from LNs or spleen. Besides, anti-MMP-9 Ab did not impact on the expression of MHC class II, CD40, CD80, CD86, and chemokine receptors (CCR5 and CCR7) of DCs both in vivo and in vitro. Furthermore, we discovered the number of MMP-9-/- DCs trafficking from footpads to popliteal LNs was dramatically reduced as compared with wild type DCs in both MMP-9-/- mice and wild type mice. Taken together, these results indicated that DC-derived MMP-9 is the crucial factor for DC migration, and blocking MMP-9 to inhibit DC migration may constitute a novel strategy of future therapy for RA and other similar autoimmune diseases.

Not only anti-inflammation, etanercept abrogates collagen-induced arthritis by inhibiting dendritic cell migration and maturation.

The application of tumor necrosis factor inhibitors (TNFi) is a major breakthrough in the treatment of rheumatoid arthritis (RA). While the anti-inflammatory nature of TNFi is thought to contribute to the therapeutic effects, recent data show that the pharmacology of TNF-α blockade is probably more complex than previously thought. This study investigates whether etanercept (ETN), one of the TNF antagonists, suppresses arthritis development through modulation of dendritic cell (DC) functions. Bone marrow-derived DCs (BMDCs) were stimulated with lipopolysaccharide (LPS) and treated with ETN for 24 hrs. DC functions, including maturation and migration, were determined. DCs from the lymph nodes (LNs) of ETN-treated collagen-induced arthritis (CIA) mice were analyzed for phenotypes and subsets. ETN efficiently inhibited the phenotypic maturation both in vitro and in vivo. ETN treatment delayed the onset and reduced the severity of arthritis in CIA mice. Moreover, ETN treatment strongly down regulated the number of both myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in LNs, possibly due to the depressive effect on the expression of CXCR4 on DCs in peripheral blood. The impaired DC migration to local LNs by ETN down regulated the number of T cells and B cells, and changed the LN cellular composition. The data show that TNF-α blockade has profound effects on DC maturation and migration, which may contribute to its immune regulatory effects in RA patients.

FTY720 Abrogates Collagen-Induced Arthritis by Hindering Dendritic Cell Migration to Local Lymph Nodes.

Because dendritic cells (DCs) play critical roles in the pathogenesis of rheumatoid arthritis, modulation of their functions could serve as a novel therapy. In this study, we demonstrated that FTY720 treatment significantly suppressed the incidence and severity of collagen-induced arthritis (CIA) in DBA/1J mice via the modulation of DC functions. In FTY720-treated CIA mice, a decrease in the number of DCs in local draining lymph nodes (LNs) was observed. In vitro, FTY720 inhibited the trafficking of LPS-stimulated bone marrow-derived DCs (BMDCs). Decreased secretion of CCL19 and downregulation of CCR7 on DCs may explain the mechanisms underlying the impairment of DC migration induced by FTY720. In a DC-induced mouse arthritis model, FTY720 treatment also suppressed the incidence and severity of arthritis, which was correlated with a decrease in the migration of injected BMDCs to draining LNs. Although lower levels of costimulatory molecules (CD40, CD80, and CD86) and I-A(q) expressed on LN DCs were observed in FTY720-treated mice, in vitro analysis showed no effect of FTY720 on LPS-stimulated BMDC maturation. Furthermore, LN cells from FTY720-treated CIA mice displayed diminished production of proinflammatory cytokines in response to collagen II and Con A stimulation. In addition, the ratio of Th1/Th2 in the draining LNs of mice with DC-induced arthritis was decreased upon FTY720 treatment. This finding was consistent with the fact that FTY720 suppressed IL-12p70 production in cultured BMDCs. Taken together, these results indicate that inhibition of DC migration by FTY720 may provide a novel approach in treating autoimmune diseases such as rheumatoid arthritis.

Apigenin, a potent suppressor of dendritic cell maturation and migration, protects against collagen-induced arthritis.

This study aimed to investigate whether apigenin (API) suppresses arthritis development through the modulation of dendritic cell functions. Bone marrow-derived dendritic cells (BMDCs) were stimulated in vitro with lipopolysaccharide (LPS) and treated with API for 24 hrs; DC functions, including phenotype expressions, cytokine secretion, phagocytosis and chemotaxis, were then investigated. The effects of API on collagen-induced arthritis (CIA) were examined in vivo, and purified DCs from the lymph nodes (LNs) of API-treated CIA mice were analysed for phenotypes and subsets. In in vitro, API efficiently restrained the phenotypic and functional maturation of LPS-stimulated BMDCs while maintaining phagocytotic capabilities. Moreover, API inhibited the chemotactic responses of LPS-stimulated BMDCs, which may be related to the depressive effect on chemokine receptor 4 (CXCR4). In in vivo, API treatment delayed the onset and reduced the severity of arthritis in CIA mice, and diminished secretion of pro-inflammatory cytokines in the serum and supernatants from the LN cells of the CIA mice. Similar to the in vitro findings, the API-treated mice exhibited reduced expression of co-stimulatory molecules and major histocompatibility complex II on DCs. Furthermore, API treatment strongly down-regulated the number of Langerhans cells, but not plasmacytoid DCs (pDCs) in LNs, which may be related to the depressive effect of API on the expression of CXCR4 on DCs of peripheral blood. These data provide new insight into the mechanism of action of API on arthritis and indicate that the inhibition of maturation and migration of DCs by API may contribute to its immunosuppressive effects.

Blocking GSDME-mediated pyroptosis in renal tubular epithelial cells alleviates disease activity in lupus mice.

An increase in apoptosis and/or defects in the clearance of apoptotic cells resulting in massive secondary necrosis have been recognized as the main causes of systemic lupus erythematosus (SLE). Recent findings have revealed that gasdermin E (GSDME)-mediated pyroptosis is a mechanism associated with secondary necrosis. We aimed to investigate the effects of GSDME-mediated pyroptosis on disease activity in lupus mice. In vivo, high levels of GSDME expression were observed in the renal tubules of pristane-induced lupus (PIL) mice and SLE patients. In lupus mice, GSDME knockout or SP600125 administration effectively ameliorated lupus-like features by inhibiting GSDME-mediated renal tubular epithelial cell pyroptosis. In vitro, treatment with tumour necrosis factor-α (TNF-α) plus cycloheximide (CHX) or SLE sera induced HK2 cells to undergo pyroptosis in a caspase-3- and GSDME-dependent manner. Likewise, SP600125 significantly reduced GSDME expression and decreased pyroptosis in HK2 cells. GSDME-mediated pyroptosis may be associated with SLE pathogenesis, and targeting GSDME may be a potential strategy for treating SLE.

Attenuation of Rheumatoid Arthritis Through the Inhibition of Tumor Necrosis Factor-Induced Caspase 3/Gasdermin E-Mediated Pyroptosis.

OBJECTIVE: To determine the role of gasdermin E (GSDME)-mediated pyroptosis in the pathogenesis and progression of rheumatoid arthritis (RA), and to explore the potential of GSDME as a therapeutic target in RA. METHODS: The expression and activation of caspase 3 and GSDME in the synovium, macrophages, and monocytes of RA patients were determined by immunohistochemistry, immunofluorescence, and Western blot analysis. The correlation of activated GSDME with RA disease activity was evaluated. The pyroptotic ability of monocytes from RA patients was tested, and the effect of tumor necrosis factor (TNF) on caspase 3/GSDME-mediated pyroptosis of monocytes and macrophages was investigated. In addition, collagen-induced arthritis (CIA) was induced in mice lacking Gsdme, and the incidence and severity of arthritis were assessed. RESULTS: Compared to cells from healthy controls, monocytes and synovial macrophages from RA patients showed increased expression of activated caspase 3, GSDME, and the N-terminal fragment of GSDME (GSDME-N). The expression of GSDME-N in monocytes from RA patients correlated positively with disease activity. Monocytes from RA patients with higher GSDME levels were more susceptible to pyroptosis. Furthermore, TNF induced pyroptosis in monocytes and macrophages by activating the caspase 3/GSDME pathway. The use of a caspase 3 inhibitor and silencing of GSDME significantly blocked TNF-induced pyroptosis. Gsdme deficiency effectively alleviated arthritis in a mouse model of CIA. CONCLUSION: These results support the notion of a pathogenic role of GSDME in RA and provide an alternative mechanism for RA pathogenesis involving TNF, which activates GSDME-mediated pyroptosis of monocytes and macrophages in RA. In addition, targeting GSDME might be a potential therapeutic approach for RA.

A novel HDAC inhibitor chidamide combined with imatinib synergistically targets tyrosine kinase inhibitor resistant chronic myeloid leukemia cells.

Chidamide is a novel selective histone deacetylase inhibitor (HDACi) with promising activity in hematological malignancies, but its role in chronic myeloid leukemia (CML) was marginally addressed. In this study, we firstly demonstrated that chidamide alone inhibited CML cells proliferation, induced apoptosis and cell cycle arrest. Further, chidamide combined with imatinib (IM) induced synergistic lethality in CML cell line KBM5, as well as IM-resistant CML cells KBM5T315I, associated with a marked reduction of Bcr-Abl kinase activity and acetyl-histone H3 expression. The combination treatment markedly inhibited constitutive activity of ß-catenin signaling in IM-resistant cells and abolished the protective effects of mesenchymal stromal cells (MSCs) to CML cells. In addition, the co-treatment significantly reduced Bcr-Abl and ß-catenin transcript levels and induced apoptosis of primary CD34+ stem/progenitor cells derived from blast crisis (BC)-CML patients, but exhibited minimal toxicity to normal CD34+ progenitors. Collectively, our data show that combination of chidamide and imatinib synergistically targets tyrosine kinase inhibitor (TKI) -resistant BC-CML cells via inhibition of Bcr-Abl and ß-catenin signaling, suggesting that this combination has the potential for treating TKI-resistant CML and improving clinical outcomes of BC-CML patients.

The mechanisms of hydroxychloroquine in rheumatoid arthritis treatment: Inhibition of dendritic cell functions via Toll like receptor 9 signaling.

Hydroxychloroquine (HCQ) is one of the most commonly prescribed immune-suppressants in treating rheumatoid arthritis (RA). Our previous research showed that HCQ suppressed RA development by inhibiting T follicular helper (Tfh) cells directly. Dendritic cells (DCs) serve as the link between innate and acquired immunity. Whether HCQ suppressed Tfh cell through DCs was not clear. In current study, we found that HCQ efficiently inhibited CD86, chemokine (C-X-C motif) receptor 4 (CXCR4) expression and interferon-α (IFN-α) secretion of healthy donor derived purified DCs stimulated by RA patient serum. To mimic RA, collagen-induced arthritis (CIA) mouse model was used and treated with HCQ daily for fifty-four days prior to sacrifice. We found HCQ inhibited DC maturation and migration to lymph nodes (LNs), manifested as down-regulated expression of CD40, CD80, CD86, MHCII (I-Aq) on LN DCs. In addition, HCQ reduced the level of chemokine receptor 7 (CCR7) and L-selectin on peripheral blood DCs and diminished percentage of LN DCs. Of note, HCQ only inhibited CpG ODN 1826-induced IL-12 secretion by bone marrow DCs (BMDCs) stimulated by various toll like receptor (TLR) agonists. Mechanistically, HCQ down-regulated the expression of TLR9 not only in healthy donor PBMC-derived DCs stimulated by RA patient serum, but also in LN DCs of CIA mice and CpG-activated BMDCs. Furthermore, arthritis scores in TLR9-/- mice were much lower than that in wild type mice with impaired maturity and migration capability of DCs. Collectively, activation of DCs contributes to the pathogenesis of RA and HCQ shows protective effects on RA by inhibition of DC activation via blocking TLR9.

Inhibition of NET formation by polydatin protects against collagen-induced arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a systematic, inflammatory, autoimmune disease, associated with a high number of disabilities. Increasing evidence has demonstrated that neutrophil extracellular trap (NET) formation plays a significant role in the pathogenesis and progression of RA. In this study, we have aimed to investigate the effects of polydatin (PD) on NET formation and its effects on disease activity in a collagen-induced arthritis (CIA) mouse model. METHODS: In the presence of PD or vehicle, neutrophils isolated from RA patients and mice were treated with phorbol 12-myristate 13-acetate (PMA) for 4 h, and NET formation investigated. For in vivo experiments, PD was administered intraperitoneally (45 mg/kg per day) to collagen-induced arthritis (CIA) mice. The incidence and severity of collagen-induced arthritis were assessed and NET deposition tested. RESULTS: In vitro, PD significantly suppressed NET formation of neutrophils from RA patients. Consistently, decreased NETs were observed in PD treated bone marrow-derived neutrophils. In CIA mouse model, PD treatment delayed the onset of arthritis and attenuated arthritis severity. Compared with vehicle-treated CIA mice, the deposition of NETs in ankle joints was also reduced in PD-treated CIA mice. CONCLUSION: In this study, we found that PD treatment markedly inhibited NET formation and protected CIA mice from the development of arthritis. These findings suggest that inhibition of NET formation by PD may serve as a novel mechanism for the treatment of RA.

Isorhamnetin, the active constituent of a Chinese herb Hippophae rhamnoides L, is a potent suppressor of dendritic-cell maturation and trafficking.

Dendritic cells (DCs) have been recognized as major targets of immunosuppressive therapies for their significant roles in connecting innate and adaptive immunity. Isorhamnetin (Iso), one of the most common flavonoid compounds extracted from the Chinese herb Hippophae rhamnoides L, has been proved to have anti-inflammatory, anticarcinogenic, and antioxidant activities in many chronic inflammatory conditions, but the effects of Iso on DCs have rarely been reported before. Here we investigated the functions and the mechanisms of Iso on bone marrow-derived DCs (BMDCs) including maturation, phagocytosis, and trafficking. Our data showed that Iso effectively inhibited the maturation of lipopolysaccharide (LPS)-treated BMDCs by down regulation of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1ß and IL-12p70, up regulation of IL-10, and depression of costimulatory molecules CD40, CD80, and CD86, while had no effects on phagocytosis. Furthermore, Iso inhibited the migration of LPS-treated BMDCs, which may be due to its inhibition on chemokine receptor 7 (CCR7) expression. These findings strongly suggest that Iso is a potent immunosuppressive agent by inhibiting DC activation and trafficking, and may be used to prevent or treat chronic inflammation, autoimmune diseases, and graft rejections.

Novel function of hydroxychloroquine: Down regulation of T follicular helper cells in collagen-induced arthritis.

Hydroxychloroquine (HCQ) is an immunosuppressive agent widely used in rheumatoid arthritis (RA). T follicular helper (Tfh) cells play a vital role in the pathogenesis of RA. However, whether HCQ suppresses arthritis development through interfering with Tfh cells have never been reported. To address this issue, we investigated the percent of Tfh cells in newly diagnosed RA patients and found that they were up-regulated in peripheral blood. Importantly, in ex vivo experiments of peripheral blood mononuclear cells (PBMCs) from healthy volunteers, we proved that the percentage of Tfh cells in PBMCs and purified CD4+ T cells were decreased after HCQ treatment. In in vivo experiments of collagen-induced arthritis (CIA) model, we discovered that HCQ suppressed the incidence and score of arthritis, reduced the secretion of proinflammatory cytokines in serum. Similar to ex vivo study, the ratio of Tfh cells in HCQ treated CIA mice declined to the level of vehicle-treated group. Further research demonstrated that HCQ inhibited the generation of Tfh cells stimulated by IL-12 and IL-21. In conclusion, our study indicates a previously unrecognized mechanism of HCQ in RA, that HCQ directly suppresses the generation of Tfh cells by blocking IL-12 and IL-21 signaling pathways probably.

Mycophenolic Acid Synergizing with Lipopolysaccharide to Induce Interleukin-1ß Release via Activation of Caspase-1.

BACKGROUND: The previous study showed that mycophenolic acid (MPA) synergizing with lipopolysaccharide (LPS) promoted interleukin (IL)-1ß release, but the mechanism is unclear. This study aimed to investigate the mechanism of MPA synergizing with LPS to induce IL-1ß release. METHODS: Undiluted human blood cells, THP-1 human myeloid leukemia mononuclear cells (THP-1) cells, or monocytes were stimulated with LPS and treated with or without MPA, and the supernatant IL-1ß was detected by enzyme-linked immunosorbent assay. The mRNA levels of IL-1ß were detected by real-time quantitative polymerase chain reaction. The intracellular protein levels of nuclear factor kappa B (NF-κB) phospho-p65 (p-p65), precursor interleukin-1ß (pro-IL-1ß), NOD-like receptor pyrin domain containing-3 (NLRP3), and cysteine aspartic acid-specific protease-1 (caspase-1) p20 in THP-1 cell were measured by Western blot. RESULTS: The MPA alone failed to induce IL-1ß, whereas MPA synergized with LPS to increase IL-1ß in a dose-dependent manner (685.00 ± 20.00 pg/ml in LPS + 5 µmol/L MPA group, P = 0.035; 742.00 ± 31.58 pg/ml in LPS + 25 µmol/L MPA group, P = 0.017; 1000.00 ± 65.59 pg/ml in LPS + 75 µmol/L MPA group, P = 0.024; versus 408.00 ± 35.50 pg/ml in LPS group). MPA alone has no effect on the IL-1ß mRNA expression, LPS induced the expression of IL-1ß mRNA 2761 fold, and LPS + MPA increased the IL-1ß expression 3018 fold, which had the same effect with LPS group (P = 0.834). MPA did not affect the intracellular NF-κB p-p65 and pro-IL-1ß protein levels but activated NLRP3 inflammasome. Ac-YVAD-cmk blocked the activation of caspase-1 and subsequently attenuated IL-1ß secretion (181.00 ± 45.24 pg/ml in LPS + MPA + YVAD group vs. 588.00 ± 41.99 pg/ml in LPS + MPA group, P = 0.014). CONCLUSIONS: Taken together, MPA synergized with LPS to induce IL-1ß release via the activation of caspase-1, rather than the enhanced production of pro-IL-1ß. These findings suggested that patients immunosuppressed with mycophenolate mofetil may have overly activated caspase-1 during infection, which might contribute to a more sensitive host defense response to invading germs.

Immunomodulatory effects of mesenchymal stromal cells-derived exosome.

The mechanisms underlying immunomodulatory ability of mesenchymal stromal cells (MSCs) remain unknown. Recently, studies suggested that the immunomodulatory activity of MSCs is largely mediated by paracrine factors. Among which, exosome is considered to play a major role in the communication between MSCs and target tissue. The aim of our study is to investigate the effect of MSCs-derived exosome on peripheral blood mononuclear cells (PBMCs), especially T cells. We find that the MSCs-derived exosome extracted from healthy donors' bone marrow suppressed the secretion of pro-inflammatory factor TNF-α and IL-1ß, but increased the concentration of anti-inflammatory factor TGF-ß during in vitro culture. In addition, exosome may induce conversion of T helper type 1 (Th1) into T helper type 2 (Th2) cells and reduced potential of T cells to differentiate into interleukin 17-producing effector T cells (Th17). Moreover, the level of regulatory T cells (Treg) and cytotoxic T lymphocyte-associated protein 4 were also increased. These results suggested that MSC-derived exosome possesses the immunomodulatory properties. However, it showed no effects on the proliferation of PBMCs or CD3+ T cells, but increases the apoptosis of them. In addition, indoleamine 2, 3-dioxygenase (IDO) was previously shown to mediate the immunoregulation of MSCs, which was increased in PBMCs co-cultured with MSCs. In our study, IDO showed no significant changes in PBMCs exposed to MSCs-derived exosome. We conclude that exosome and MSCs might differ in their immune-modulating activities and mechanisms.