技术资料

The intradermal lipopolysaccharide (LPS) challenge in healthy volunteers has proven to be a valuable tool to study local inflammation in vivo. In the current study the inhibitory effects of oral and topical corticosteroid treatment on intradermal LPS responses were evaluated to benchmark the challenge for future investigational drugs. Twenty-four healthy male volunteers received a two-and-a-half-day twice daily (b.i.d.) pretreatment with topical clobetasol propionate 0.05% and six healthy volunteers received a two-and-a-half-day b.i.d. pretreatment with oral prednisolone at 0.25 mg/kg body weight per administration. Participants received one injection regimen of either 0,2,or 4 intradermal LPS injections (5 ng LPS in 50 µL 0.9% sodium chloride solution). The LPS response was evaluated by noninvasive (perfusion,skin temperature,and erythema) and invasive assessments (cellular and cytokine responses) in suction blister exudate. Both corticosteroids significantly suppressed the clinical inflammatory response (erythema P = 0.0001 for clobetasol and P = 0.0016 for prednisolone; heat P = 0.0245 for clobetasol,perfusion P < 0.0001 for clobetasol and P = 0.0036 for prednisolone). Clobetasol also significantly reduced the number of monocytes subsets,dendritic cells,natural killer cells,and T cells in blister exudate. A similar effect was observed for prednisolone. No relevant corticosteroid effects were observed on the cytokine response to LPS. We successfully demonstrated that the anti-inflammatory effects of corticosteroids can be detected using our intradermal LPS challenge model,validating it for evaluation of future investigational drugs,as an initial assessment of the anti-inflammatory effects of such compounds in a minimally invasive manner. View Publication

The atypical cannabinoid Abn-CBD improves the inflammatory status in preclinical models of several pathologies,including autoimmune diseases. However,its potential for modulating inflammation in autoimmune type 1 diabetes (T1D) is unknown. Herein we investigate whether Abn-CBD can modulate the inflammatory response during T1D onset using a mouse model of T1D (non-obese diabetic- (NOD)-mice) and of beta cell damage (streptozotocin (STZ)-injected mice). Six-week-old female NOD mice were treated with Abn-CBD (0.1-1 mg/kg) or vehicle during 12 weeks and then euthanized. Eight-to-ten-week-old male C57Bl6/J mice were pre-treated with Abn-CBD (1 mg/kg of body weight) or vehicle for 1 week,following STZ challenge,and euthanized 1 week later. Blood,pancreas,pancreatic lymph nodes (PLNs) and T cells were collected and processed for analysis. Glycemia was also monitored. In NOD mice,treatment with Abn-CBD significantly reduced the severity of insulitis and reduced the pro-inflammatory profile of CD4+ T cells compared to vehicle. Concomitantly,Abn-CBD significantly reduced islet cell apoptosis and improved glucose tolerance. In STZ-injected mice,Abn-CBD decreased circulating proinflammatory cytokines and ameliorated islet inflammation reducing intra-islet phospho-NF-$\kappa$B and TXNIP. Abn-CBD significantly reduced 2 folds intra-islet CD8+ T cells and reduced Th1/non-Th1 ratio in PLNs of STZ-injected mice. Islet cell apoptosis and intra-islet fibrosis were also significantly reduced in Abn-CBD pre-treated mice compared to vehicle. Altogether,Abn-CBD reduces circulating and intra-islet inflammation,preserving islets,thus delaying the progression of insulitis. Hence,Abn-CBD and related compounds emerge as new candidates to develop pharmacological strategies to treat the early stages of T1D. View Publication

Oxidative stress,caused by the imbalance between reactive species generation and the dysfunctional capacity of antioxidant defenses,is one of the characteristic features of cancer. Here,we quantified hydrogen peroxide in the tumor microenvironment (TME) and demonstrated that hydrogen peroxide concentrations are elevated in tumor interstitial fluid isolated from murine breast cancers in vivo,when compared with blood or normal subcutaneous fluid. Therefore,we investigated the effects of increased hydrogen peroxide concentration on immune cell functions. NK cells were more susceptible to hydrogen peroxide than T cells or B cells,and by comparing T,B,and NK cells' sensitivities to redox stress and their antioxidant capacities,we identified peroxiredoxin-1 (PRDX1) as a lacking element of NK cells' antioxidative defense. We observed that priming with IL15 protected NK cells' functions in the presence of high hydrogen peroxide and simultaneously upregulated PRDX1 expression. However,the effect of IL15 on PRDX1 expression was transient and strictly dependent on the presence of the cytokine. Therefore,we genetically modified NK cells to stably overexpress PRDX1,which led to increased survival and NK cell activity in redox stress conditions. Finally,we generated PD-L1-CAR NK cells overexpressing PRDX1 that displayed potent antitumor activity against breast cancer cells under oxidative stress. These results demonstrate that hydrogen peroxide,at concentrations detected in the TME,suppresses NK cell function and that genetic modification strategies can improve CAR NK cells' resistance and potency against solid tumors. View Publication

CD8 T cell-mediated autoimmune diseases result from the breakdown of self-tolerance mechanisms in autoreactive CD8 T cells1. How autoimmune T cell populations arise and are sustained,and the molecular programmes defining the autoimmune T cell state,are unknown. In type 1 diabetes,$\beta$-cell-specific CD8 T cells destroy insulin-producing $\beta$-cells. Here we followed the fate of $\beta$-cell-specific CD8 T cells in non-obese diabetic mice throughout the course of type 1 diabetes. We identified a stem-like autoimmune progenitor population in the pancreatic draining lymph node (pLN),which self-renews and gives rise to pLN autoimmune mediators. pLN autoimmune mediators migrate to the pancreas,where they differentiate further and destroy $\beta$-cells. Whereas transplantation of as few as 20 ?»¿autoimmune progenitors induced type 1 diabetes,as many as 100,000 pancreatic autoimmune mediators did not. Pancreatic autoimmune mediators are short-lived,and stem-like ?»¿autoimmune progenitors must continuously seed the pancreas to sustain $\beta$-cell destruction. Single-cell RNA sequencing and clonal analysis revealed that autoimmune CD8 T cells represent unique T cell differentiation states and identified features driving the transition from autoimmune progenitor to autoimmune mediator. Strategies aimed at targeting the stem-like autoimmune progenitor pool could emerge as novel and powerful immunotherapeutic interventions for type 1 diabetes. View Publication

New approaches beyond PD-1/PD-L1 inhibition are required to target the immunologically diverse tumor microenvironment (TME) in high-grade serous ovarian cancer (HGSOC). In this study,we explored the immunosuppressive effect of B7-H3 (CD276) via the CCL2-CCR2-M2 macrophage axis and its potential as a therapeutic target. Transcriptome analysis revealed that B7-H3 is highly expressed in PD-L1-low,nonimmunoreactive HGSOC tumors,and its expression negatively correlated with an IFN$\gamma$ signature,which reflects the tumor immune reactivity. In syngeneic mouse models,B7-H3 (Cd276) knockout (KO) in tumor cells,but not in stromal cells,suppressed tumor progression,with a reduced number of M2 macrophages and an increased number of IFN$\gamma$+CD8+ T cells. CCL2 expression was downregulated in the B7-H3 KO tumor cell lines. Inhibition of the CCL2-CCR2 axis partly negated the effects of B7-H3 suppression on M2 macrophage migration and differentiation,and tumor progression. In patients with HGSOC,B7-H3 expression positively correlated with CCL2 expression and M2 macrophage abundance,and patients with B7-H3-high tumors had fewer tumoral IFN$\gamma$+CD8+ T cells and poorer prognosis than patients with B7-H3-low tumors. Thus,B7-H3 expression in tumor cells contributes to CCL2-CCR2-M2 macrophage axis-mediated immunosuppression and tumor progression. These findings provide new insights into the immunologic TME and could aid the development of new therapeutic approaches against the unfavorable HGSOC phenotype. View Publication

Successful implantation requires the coordinated migration and invasion of trophoblast cells from out of the blastocyst and into the endometrium. This process relies on signals produced by cells in the maternal endometrium. However,the relative contribution of stroma cells remains unclear. The study of human implantation has major technical limitations,therefore the need of in vitro models to elucidate the molecular mechanisms. Using a recently described 3D in vitro models we evaluated the interaction between trophoblasts and human endometrial stroma cells (hESC),we assessed the process of trophoblast migration and invasion in the presence of stroma derived factors. We demonstrate that hESC promotes trophoblast invasion through the generation of an inflammatory environment modulated by TNF-?. We also show the role of stromal derived IL-17 as a promoter of trophoblast migration through the induction of essential genes that confer invasive capacity to cells of the trophectoderm. In conclusion,we describe the characterization of a cellular inflammatory network that may be important for blastocyst implantation. Our findings provide a new insight into the complexity of the implantation process and reveal the importance of inflammation for embryo implantation. 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

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

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

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

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

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