技术资料

Although activated inflammatory monocytes (IMCs) and inflammatory dendritic cells (IDCs) are potent T cell suppressors,nonactivated IMCs and IDCs promote T cell activation and Th1/Th17 cell differentiation. In this study,we investigated how to reduce the proinflammatory properties of IMCs and IDCs and further convert them into immune regulatory dendritic cells (DCs). We found that IL-4 and retinoic acid (RA) cotreatment of GM-CSF-differentiated IDCs synergistically induced the expression of aldehyde dehydrogenase family 1,subfamily A2,a rate-limiting enzyme for RA synthesis in DCs. IL-4 plus RA-treated IDCs upregulated CD103 expression and markedly reduced the production of proinflammatory cytokines upon activation. IL-4 plus RA-treated IDCs strongly induced CD4�?�Foxp3�?� regulatory T cell differentiation and suppressed Th1 and Th17 differentiation. Mechanistically,the transcription factors Stat6 and RA receptor $$ play important roles in aldehyde dehydrogenase family 1,subfamily A2,induction. In addition,IL-4 and RA signaling pathways interact closely to enhance the regulatory function of treated DCs. Adoptive transfer of IL-4 plus RA-treated DCs significantly increased regulatory T cell frequency in vivo. Direct treatment with IL-4 and RA also markedly suppressed actively induced experimental autoimmune encephalomyelitis. Our data demonstrate the synergistic effect of IL-4 and RA in inducing a regulatory phenotype in IDCs,providing a potential treatment strategy for autoimmune diseases. View Publication

Cancer stem cells (CSCs) are a small subset of cancer cells with indefinite potential for self-renewal and the capacity to drive tumorigenesis. Brefeldin A (BFA) is an antibiotic that is known to block protein transport and induce endoplasmic reticulum (ER) stress in eukaryotic cells,but its effects on colorectal CSCs are unknown. We investigated the inhibitory effect of BFA on human colorectal cancer Colo 205 cells. We found that BFA effectively reduced the survival of suspension Colo 205 cells (IC₅₀ = ˜15 ng/mL) by inducing apoptosis,and inhibited the clonogenic activity of Colo 205 CSCs in tumorsphere formation assay and soft agar colony formation assay in the same nanogram per milliliter range. We also discovered that at such low concentrations,BFA effectively induced endoplasmic reticulum (ER) stress response as indicated by the increased mRNA expression of ER stress-related genes,such as glucose-regulated protein 78 (GRP78),X-box binding protein 1 (XBP1),and C/EBP homologous protein (CHOP). Finally,we found that BFA reduced the activity of matrix metallopeptidase 9 (MMP-9). These findings suggest that BFA can effectively suppress the progression of colorectal cancer during the tumorigenesis and metastasis stages. These results may lead to the development of novel therapies for the treatment of colorectal cancer. View Publication

Although BMP9 is highly capable of promoting osteogenic differentiation of mesenchymal stem cell (MSCs),the molecular mechanism involved remains to be fully elucidated. Here,we explore the possible involvement and detail role of JNKs (c-Jun N-terminal kinases) in BMP9-induced osteogenic differentiation of MSCs. It was found that BMP9 stimulated the activation of JNKs in MSCs. BMP9-induced osteogenic differentiation of MSCs was dramatically inhibited by JNKs inhibitor SP600125. Moreover,BMP9-activated Smads signaling was decreased by SP600125 treatment in MSCs. The effects of inhibitor are reproduced with adenoviruses expressing siRNA targeted JNKs. Taken together,our results revealed that JNKs was activated in BMP9-induced osteogenic differentiation of MSCs. What is most noteworthy,however,is that inhibition of JNKs activity resulted in reduction of BMP9-induced osteogenic differentiation of MSCs,implying that activation of JNKs is essential for BMP9 osteoinductive activity. View Publication

We have examined a number of reagents for their ability to modulate activity of the Hh signaling pathway during embryonic development of Xenopus. In particular we have focused on regulation of events occurring during tailbud stages and later. Two inducible protein reagents based on the Gli1 and Gli3 transcription factors were generated and the activity of these proteins was compared to the Hh signaling pathway inhibitor,cyclopamine,and the activators,Smoothened agonist (SAG) and purmorphamine (PMA). Effectiveness of reagents was assayed using both molecular biological techniques and biological readouts. We found that the small molecule modulators of the Hh pathway were highly specific and effective and produced results generally superior to the more conventional protein reagents for examination of later stage developmental processes. View Publication

BACKGROUND Circular RNA of vimentin (circ-VIM) is a predictor for poor prognosis of acute myeloid leukemia,but we had little information on its function in esophageal cancer (EC). Here we examined the effects of circ-VIM together with sevoflurane on immune escape and multiple oncogenic activities of EC. METHODS Bioinformatic tools,luciferase assay,and RNA immunoprecipitation were used to examine regulations between circ-VIM,miR-124-3p (miR-124),and PD-L1. CCK-8,wound healing,and Transwell assays were used to measure cell proliferation,migration,and invasion,respectively. The impacts of EC cells on cytotoxicity,proliferation,and apoptosis of CD8+ T cells were examined using LDH assay,CFSE staining,and Annexin V/PI staining,respectively. The in vivo tumorigenesis and lung metastases were assessed using xenograft model and tail vein injection of EC cells. RESULTS Significant upregulation of circ-VIM and PD-L1 and downregulation of miR-124 were detected in EC tissues or cells. Circ-VIM sponged miR-124 and released its suppression on the downstream target PD-L1. Sevoflurane,independent of circ-VIM,also upregulated miR-124 to lower PD-L1 expression. By modulating miR-124/PD-L1 axis,silencing circ-VIM and applying sevoflurane both inhibited immune escape and multiple oncogenic activities of EC in vitro,and suppressed xenograft growth and lung metastases in vivo. The inactivation of Ras/ERK signaling pathway was involved in suppression of malignant phenotypes by silencing circ-VIM and sevoflurane treatment. CONCLUSIONS Silencing circ-VIM and applying sevoflurane,by separately regulating miR-124/PD-L1 axis,presented synergistic effects in inhibiting immune escape and multiple malignant phenotypes of EC cells. View Publication

AIMS Whereas intravenous administration of Toll-like receptor 4 ligand lipopolysaccharide (LPS) to human volunteers is frequently used in clinical pharmacology studies,systemic use of LPS has practical limitations. We aimed to characterize the intradermal LPS response in healthy volunteers,and as such qualify the method as local inflammation model for clinical pharmacology studies. METHODS Eighteen healthy male volunteers received 2 or 4 intradermal 5 ng LPS injections and 1 saline injection on the forearms. The LPS response was evaluated by noninvasive (perfusion,skin temperature and erythema) and invasive assessments (cellular and cytokine responses) in skin biopsy and blister exudate. RESULTS LPS elicited a visible response and returned to baseline at 48??hours. Erythema,perfusion and temperature were statistically significant (P  View Publication

Understanding human hematopoietic stem cell fate control is important for its improved therapeutic manipulation. Asymmetric cell division,the asymmetric inheritance of factors during division instructing future daughter cell fates,was recently described in mouse blood stem cells. In human blood stem cells,the possible existence of asymmetric cell division remained unclear because of technical challenges in its direct observation. Here,we use long-term quantitative single-cell imaging to show that lysosomes and active mitochondria are asymmetrically inherited in human blood stem cells and that their inheritance is a coordinated,nonrandom process. Furthermore,multiple additional organelles,including autophagosomes,mitophagosomes,autolysosomes,and recycling endosomes,show preferential asymmetric cosegregation with lysosomes. Importantly,asymmetric lysosomal inheritance predicts future asymmetric daughter cell-cycle length,differentiation,and stem cell marker expression,whereas asymmetric inheritance of active mitochondria correlates with daughter metabolic activity. Hence,human hematopoietic stem cell fates are regulated by asymmetric cell division,with both mechanistic evolutionary conservation and differences to the mouse system. View Publication

Resting CD4+ T cells are primary targets of early HIV infection events in vivo,but do not readily support HIV replication in vitro. This barrier to infection can be overcome by exposing resting CD4+ T cells to endothelial cells (ECs). ECs line blood vessels and direct T cell trafficking into inflamed tissues. Cell trafficking pathways have been shown to have overlapping roles in facilitating HIV replication,but their relevance to EC-mediated enhancement of HIV susceptibility in resting CD4+ T cells has not previously been examined. We characterized the phenotype of primary human resting CD4+ T cells that became productively infected with HIV when cocultured with primary human blood and lymphatic ECs. The infected CD4+ T cells were primarily central memory cells enriched for high expression of the integrins LFA-1 and VLA-4. ICAM-1 and VCAM-1,the cognate ligands for LFA-1 and VLA-4,respectively,were expressed by the ECs in the coculture. Blocking LFA-1 and VLA-4 on resting CD4+ T cells inhibited infection by 65.4%-96.9%,indicating that engagement of these integrins facilitates EC-mediated enhancement of productive HIV infection in resting CD4+ T cells. The demonstration that ECs influence cellular HIV susceptibility of resting memory CD4+ T cells through cell trafficking pathways engaged during the transmigration of T cells into tissues highlights the physiological relevance of these findings for HIV acquisition and opportunities for intervention. View Publication

Persistent inflammation is a major contributor to healing impairment in diabetic chronic wounds. Paradoxically,diabetic wound environment during the acute phase of healing is completely different because it exhibits a reduced macrophage response owing to inadequate expression of CCL2 proinflammatory cytokine. What causes a reduction in CCL2 expression in diabetic wounds early after injury remains unknown. In this study,we report that in contrast to prolonged exposure to high glucose,which makes monocytes proinflammatory,short-term exposure to high glucose causes a rapid monocyte reprogramming,manifested by increased expression and secretion of IL-10,which in an autocrine/paracrine fashion reduces glucose uptake and transforms monocytes into an anti-inflammatory phenotype by dampening signaling through toll-like receptors. We show that IL-10 expression is significantly increased in diabetic wounds during the acute phase of healing,causing significant reductions in toll-like receptor signaling and proinflammatory cytokine production,delaying macrophage and leukocyte responses,and underlying healing impairment in diabetic wounds. Importantly,blocking IL-10 signaling during the acute phase of healing improves toll-like receptor signaling,increases proinflammatory cytokine production,enhances macrophage and leukocyte responses,and stimulates healing in diabetic wounds. We posit that anti-IL-10 strategies have therapeutic potential if added topically after surgical debridement,which resets chronic wounds into acute fresh wounds. View Publication

BACKGROUND & AIMS An invasive form of intestinal Entamoeba (E.) histolytica infection,which causes amoebic liver abscess,is more common in men than in women. Immunopathological mechanisms are responsible for the more severe outcome in males. Here,we used a mouse model of hepatic amoebiasis to investigate the contribution of hepatic hypoxia-inducible factor (HIF)-1$\alpha$ to T helper 17 (Th17)/regulatory T cell (Treg) responses in the context of the sex-specific outcome of liver damage. METHODS C57BL/6J mice were infected intrahepatically with E. histolytica trophozoites. HIF-1$\alpha$ expression was determined by qPCR,flow cytometry and immunohistochemistry. Tregs and Th17 cells were analysed by immunohistochemistry and flow cytometry. Finally,male and female hepatocyte-specific Hif1$\alpha$ knockout mice were generated,and the effect of HIF-1$\alpha$ on abscess development,the cytokine milieu,and Th17/Treg differentiation was examined. RESULTS E. histolytica infection increased hepatic HIF-1$\alpha$ levels,along with the elevated frequencies of hepatic Th17 and Treg cells. While the Th17 cell population was larger in male mice,Tregs characterised by increased expression of Foxp3 in female mice. Male mice displayed increased IL-6 expression,contributing to immunopathology; this increase in IL-6 expression declined upon deletion of hepatic HIF-1$\alpha$. In both sexes,hepatic deletion of HIF-1$\alpha$ reduced the Th17 cell frequency; however,the percentage of Tregs was reduced in female mice only. CONCLUSIONS Hepatic HIF-1$\alpha$ modulates the sex-specific outcome of murine E. histolytica infection. Our results suggest that in male mice,Th17 cells can be modulated by hepatic HIF-1$\alpha$ via IL-6,indicating marked involvement in the immunopathology underlying abscess development. Strong expression of Foxp3 by hepatic Tregs from female mice suggests a potent immunosuppressive function,leading to initiation of liver regeneration. LAY SUMMARY Infection with the parasite Entamoeba histolytica activates immunopathological mechanisms in male mice,which lead to liver abscesses that are larger than those in female mice. In the absence of the protein HIF-1$\alpha$ in hepatocytes,abscess formation is reduced; moreover,the sex difference in abscess size is abolished. These results suggest that HIF-1$\alpha$ modulates the immune response involved in the induction of immunopathology,resulting in differential disease susceptibility in males and females. View Publication

BACKGROUND During the COVID-19 pandemic,thousands of pregnant women have been infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The implications of maternal SARS-CoV-2 infection on fetal and childhood well-being need to be characterized. We aimed to characterize the fetal immune response to maternal SARS-CoV-2 infection. METHODS We performed single-cell RNA-sequencing and T cell receptor sequencing on cord blood mononuclear cells (CBMCs) from newborns of mothers infected with SARS-CoV-2 in the third trimester (cases) or without SARS-CoV-2 infection (controls). RESULTS We identified widespread gene expression changes in CBMCs from cases,including upregulation of interferon-stimulated genes and major histocompatibility complex genes in CD14+ monocytes,transcriptional changes suggestive of activation of plasmacytoid dendritic cells,and activation and exhaustion of natural killer cells. Lastly,we observed fetal T cell clonal expansion in cases compared to controls. CONCLUSIONS As none of the infants were infected with SARS-CoV-2,our results suggest that maternal SARS-CoV-2 infection might modulate the fetal immune system in the absence of vertical transmission. IMPACT The implications of maternal SARS-CoV-2 infection in the absence of vertical transmission on fetal and childhood well-being are poorly understood. Maternal SARS-CoV-2 infection might modulate the fetal immune system in the absence of vertical transmission. This study raises important questions about the untoward effects of maternal SARS-CoV-2 on the fetus,even in the absence of vertical transmission. View Publication

Peptidylarginine deiminase 4 (PAD4) is a key regulator of inflammation but its function in infections remains incompletely understood. We investigate PAD4 in the context of malaria and demonstrate a role in regulation of immune cell trafficking and chemokine production. PAD4 regulates liver immunopathology by promoting neutrophil trafficking in a Plasmodium chabaudi mouse malaria model. In human macrophages,PAD4 regulates expression of CXCL chemokines in response to stimulation with TLR ligands and P. falciparum. Using patient samples,we show that CXCL1 may be a biomarker for severe malaria. PAD4 inhibition promotes disease tolerance and may represent a therapeutic avenue in malaria. View Publication