Driss Ehirchiou

Immunosuppressive effects of streptozotocin-induced diabetes result in absolute lymphopenia and a relative increase of T regulatory cells

OBJECTIVE Streptozotocin (STZ) is the most widely used diabetogenic agent in animal models of islet transplantation. However, the immunomodifying effects of STZ and the ensuing hyperglycemia on lymphocyte subsets, particularly on T regulatory cells (Tregs), remain poorly understood. RESEARCH DESIGN AND METHODS This study evaluated how STZ-induced diabetes affects adaptive immunity and the consequences thereof on allograft rejection in murine models of islet and skin transplantation. The respective toxicity of STZ and hyperglycemia on lymphocyte subsets was tested in vitro. The effect of hyperglycemia was assessed independently of STZ in vivo by the removal of transplanted syngeneic islets, using an insulin pump, and with rat insulin promoter diphtheria toxin receptor transgenic mice. RESULTS Early lymphopenia in both blood and spleen was demonstrated after STZ administration. Direct toxicity of STZ on lymphocytes, particularly on CD8+ cells and B cells, was shown in vitro. Hyperglycemia also correlated with blood and spleen lymphopenia in vivo but was not lymphotoxic in vitro. Independently of hyperglycemia, STZ led to a relative increase of Tregs in vivo, with the latter retaining their suppressive capacity in vitro. The higher frequency of Tregs was associated with Treg proliferation in the blood, but not in the spleen, and higher blood levels of transforming growth factor-β. Finally, STZ administration delayed islet and skin allograft rejection compared with naive mice. CONCLUSIONS These data highlight the direct and indirect immunosuppressive effects of STZ and acute hyperglycemia, respectively. Thus, these results have important implications for the future development of tolerance-based protocols and their translation from the laboratory to the clinic.

Anti-CD154 mAb and Rapamycin Induce T Regulatory Cell Mediated Tolerance in Rat-to-Mouse Islet Transplantation

Background Anti-CD154 (MR1) monoclonal antibody (mAb) and rapamycin (RAPA) treatment both improve survival of rat-to-mouse islet xenograft. The present study investigated the effect of combined RAPA/MR1 treatment on rat-to-mouse islet xenograft survival and analyzed the role of CD4+CD25+Foxp3+ T regulatory cells (Treg) in the induction and maintenance of the ensuing tolerance. Methodology/Principal Findings C57BL/6 mice were treated with MR1/RAPA and received additional monoclonal anti-IL2 mAb or anti CD25 mAb either early (0–28 d) or late (100–128 d) post-transplantation. Treg were characterised in the blood, spleen, draining lymph nodes and within the graft of tolerant and rejecting mice by flow cytometry and immunohistochemistry. Fourteen days of RAPA/MR1 combination therapy allowed indefinite islet graft survival in >80% of the mice. Additional administration of anti-IL-2 mAb or depleting anti-CD25 mAb at the time of transplantation resulted in rejection (100% and 89% respectively), whereas administration at 100 days post transplantation lead to lower rejection rates (25% and 40% respectively). Tolerant mice showed an increase of Treg within the graft and in draining lymph nodes early post transplantation, whereas 100 days post transplantation no significant increase of Treg was observed. Rejecting mice showed a transient increase of Treg in the xenograft and secondary lymphoid organs, which disappeared within 7 days after rejection. Conclusions/Significances These results suggest a critical role for Treg in the induction phase of tolerance early after islet xenotransplantation. These encouraging data support the need of developing further Treg therapy for overcoming the species barrier in xenotransplantation.

T regulatory cells in xenotransplantation

Abstract:  The role of T regulatory cells (Treg) in the induction and maintenance of allograft tolerance is being studied to a great extent. In contrast, little is known on their potential to prevent graft rejection in the field of xenotransplantation, where acute vascular rejection mediated by cellular and humoral mechanisms and thrombotic microangiopathy still prevents long‐term graft survival. In this regard, the induction of donor‐specific tolerance through isolation and expansion of xenoantigen‐specific recipient Treg is currently becoming a focus of interest. This review will summarize the present knowledge concerning Treg and their potential use in xenotransplantation describing in particular CD4+CD25+Foxp3+ T cells, CD8+CD28− Treg, double negative CD4−CD8− T cells, and natural killer Treg. Although only studied in vitro so far, human CD4+CD25+Foxp3+ Treg is currently the best characterized subpopulation of regulatory cells in xenotransplantation. CD8+CD28− Treg and double negative CD4−CD8− Treg also seem to be implicated in tolerance maintenance of xenografts. Finally, one study revealing a role for natural killer CD4+Vα14+ Treg in the prolongation of xenograft survival needs further confirmation. To our opinion, CD4+CD25+Foxp3+ Treg are a promising candidate to protect xenografts. In contrast to cadaveric allotransplantation, the donor is known prior to xenotransplantation. This advantage allows the expansion of recipient Treg in a xenoantigen specific manner before transplantation.

Potential of T-regulatory cells to protect xenografts.

Purpose of reviewImmunological barriers still preclude clinical xenotransplantation. The protective role of CD4+CD25+Foxp3+ T-regulatory cells (Treg) in allotransplantation is well described and, therefore, could represent a promising therapeutical tool for xenotransplantation. This review addresses the latest findings on Treg in xenotransplantation research. Recent findingsIn vivo, costimulation blockade-based strategies including anti-CD154 monoclonal antibodies (mAbs) in combination with rapamycin or anti-LFA-1 mAb prolonged both concordant and discordant islets xenografts survival in a Treg-dependent manner. In vitro, IL-10 secretion was shown to be critical for the suppression of xenogeneic responses mediated by Treg. Moreover, transgenic expression of inducible costimulator-immunoglobulin or PD-L1 on porcine endothelial cells inhibited human T-cell proliferation in vitro and was associated with the induction of Treg and IL-10 secretion. CXCR3 mediated the recruitment of Treg to pig endothelium. Finally, the recruitment of human Treg was enhanced by the immobilization of human CCL17 on pig endothelium. SummaryThere is increasing evidence for the potential of CD4+CD25+Foxp3+ Treg to protect xenografts. Induction of Treg in recipients and/or recruitment of human Treg to pig endothelium may represent novel strategies to prevent cell-mediated rejection in pig-to-human xenotransplantation.

CD11b regulates the Treg/Th17 balance in murine arthritis via IL-6

Th17 cells are often associated with autoimmunity and been shown to be increased in CD11b−/− mice. Here, we examined the role of CD11b in murine collagen‐induced arthritis (CIA). C57BL/6 and CD11b−/− resistant mice were immunized with type II collagen. CD11b−/− mice developed arthritis with early onset, high incidence, and sustained severity compared with C57BL/6 mice. We observed a marked leukocyte infiltration, and histological examinations of the arthritic paws from CD11b−/− mice revealed that the cartilage was destroyed in association with strong lymphocytic infiltration. The CD11b deficiency led to enhanced Th17‐cell differentiation. CD11b−/− dendritic cells (DCs) induced much stronger IL‐6 production and hence Th17‐cell differentiation than wild‐type DCs. Treatment of CD11b−/− mice after establishment of the Treg/Th17 balance with an anti‐IL‐6 receptor mAb significantly suppressed the induction of Th17 cells and reduced arthritis severity. Finally, the severe phenotype of arthritis in CD11b−/− mice was rescued by adoptive transfer of CD11b+ DCs. Taken together, our results indicate that the resistance to CIA in C57BL/6 mice is regulated by CD11b via suppression of IL‐6 production leading to reduced Th17‐cell differentiation. Therefore, CD11b may represent a susceptibility factor for autoimmunity and could be a target for future therapy.

Chemoattractant Signals and Adhesion Molecules Promoting Human Regulatory T Cell Recruitment to Porcine Endothelium

Background Human CD4+CD25+Foxp3+ T regulatory cells (huTreg) suppress CD4+ T cell–mediated antipig xenogeneic responses in vitro and might therefore be used to induce xenograft tolerance. The present study investigated the role of the adhesion molecules, their porcine ligands, and the chemoattractant factors that may promote the recruitment of huTreg to porcine aortic endothelial cells (PAEC) and their capacity to regulate antiporcine natural killer (NK) cell responses. Methods Interactions between ex vivo expanded huTreg and PAEC were studied by static chemotaxis assays and flow-based adhesion and transmigration assays. In addition, the suppressive function of huTreg on human antiporcine NK cell responses was analyzed. Results The TNF&agr;-activated PAEC released factors that induce huTreg chemotaxis, partially inhibited by antihuman CXCR3 blocking antibodies. Coating of PAEC with human CCL17 significantly increased the transmigration of CCR4+ huTreg under physiological shear stress. Under static conditions, transendothelial Treg migration was inhibited by blocking integrin sub-units (CD18, CD49d) on huTreg, or their respective porcine ligands intercellular adhesion molecule 2 (CD102) and vascular cell adhesion molecule 1 (CD106). Finally, huTreg partially suppressed xenogeneic human NK cell adhesion, NK cytotoxicity and degranulation (CD107 expression) against PAEC; however, this inhibition was modest, and there was no significant change in the production of IFN&ggr;. Conclusions Recruitment of huTreg to porcine endothelium depends on particular chemokine receptors (CXCR3, CCR4) and integrins (CD18 and CD49d) and was increased by CCL17 coating. These results will help to develop new strategies to enhance the recruitment of host huTreg to xenogeneic grafts to regulate cell-mediated xenograft rejection including NK cell responses.

Dendritic cells cause bone lesions in a new mouse model of histiocytosis

Langerhans cell histiocytosis (LCH) is a rare disease caused by the clonal accumulation of dendritic Langerhans cells, which is often accompanied by osteolytic lesions. It has been reported that osteoclast-like cells play a major role in the pathogenic bone destruction seen in patients with LCH and these cells are postulated to originate from the fusion of DCs. However, due to the lack of reliable animal models the pathogenesis of LCH is still poorly understood. In this study, we have established a mouse model of histiocytosis- recapitulating human disease for osteolytic lesions seen in LCH patients. At 12 weeks after birth, severe bone lesions were observed in our multisystem histiocytosis (Mushi) model, when CD8α conventional dendritic cells (DCs) are transformed (MuTuDC) and accumulate. Most importantly, our study demonstrates that bone loss in LCH can be accounted for the transdifferentiation of MuTuDCs into functional osteoclasts both in vivo and in vitro. Moreover, we have shown that injected MuTuDCs reverse the osteopetrotic phenotype of oc/oc mice in vivo. In conclusion, our results support a crucial role of DCs in bone lesions in histiocytosis patients. Furthermore, our new model of LCH based on adoptive transfer of MuTuDC lines, leading to bone lesions within 1–2 weeks, will be an important tool for investigating the pathophysiology of this disease and ultimately for evaluating the potential of anti-resorptive drugs for the treatment of bone lesions.

Hydrogen sulfide inhibits NLRP3 inflammasome activation and reduces cytokine production both : javax.xml.bind.JAXBElement@1792cb87 : and in a mouse model of inflammation.

A variety of stimuli, including monosodium urate (MSU) crystals, activate the NLRP3 inflammasome, and this activation involves several molecular mechanisms including xanthine oxidase (XO) up-regulation and mitochondrial dysfunction. Upon oligomerization of apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1 becomes active and cleaves the proinflammatory cytokine IL-1β into its active secreted form. Hydrogen sulfide (H2S), a gasotransmitter mainly produced by cystathionine γ-lyase (CSE) in macrophages, could modulate inflammation. Here, we sought to investigate the effects of exogenous and endogenous H2S on NLRP3 inflammasome activation in vitro and in vivo. Primed bone marrow–derived macrophages (BMDM) isolated from wildtype (wt) or CSE-deficient mice and human macrophages (THP1 cells and primary macrophages), were stimulated with MSU crystals in the presence or absence of a H2S donor, sodium thiosulfate (STS) or GYY4137 (GYY). In murine and human macrophages in vitro, both STS and GYY inhibited MSU crystal–induced IL-1β secretion in a dose-dependent manner. Moreover, the H2S donors inhibited MSU crystal–induced XO/caspase-1 activities, mitochondrial reactive oxygen species (ROS) generation, and ASC oligomerization. Accordingly, IL-1β secretion and XO/caspase-1 activities were higher in CSE-deficient BMDMs than in wt BMDMs. For in vivo studies, we experimentally induced peritonitis by intraperitoneal injection of MSU crystals into mice. GYY pretreatment ameliorated inflammation, evidenced by decreased IL-6/monocyte chemoattractant protein-1 (MCP-1) released into peritoneal lavages. Taken together, our results suggest that both exogenous (via H2S donors) and endogenous (via CSE) H2S production may represent approaches for managing, for example, acute gout or other inflammation conditions

Role of proapoptotic BH3-only proteins in Listeria monocytogenes infection

The ability of pathogens to influence host cell survival is a crucial virulence factor. Listeria monocytogenes (Lm) infection is known to be associated with severe apoptosis of hepatocytes and spleen cells. This impairs host defense mechanisms and thereby facilitates the spread of intracellular pathogens. The general mechanisms of apoptosis elicited by Lm infection are understood, however, the roles of BH3‐only proteins during primary Lm infection have not been examined. To explore the roles of BH3‐only proteins in Lm‐induced apoptosis, we studied Listeria infections in mice deficient in Bim, Bid, Noxa or double deficient in BimBid or BimNoxa. We found that BimNoxa double knockout mice were highly resistant to high‐dose challenge with Listeria. Decreased bacterial burden and decreased host cell apoptosis were found in the spleens of these mice. The ability of the BH3‐deficient mice to clear bacterial infection more efficiently than WT was correlated with increased concentrations of ROS, neutrophil extracellular DNA trap release and downregulation of TNF‐α. Our data show a novel pathway of infection‐induced apoptosis that enhances our understanding of the mechanism by which BH3‐only proteins control apoptotic host cell death during Listeria infection.