Rapid Dissemination of SIV Follows Multisite Entry after Rectal Inoculation

Receptive ano-rectal intercourse is a major cause of HIV infection in men having sex with men and in heterosexuals. Current knowledge of the mechanisms of entry and dissemination during HIV rectal transmission is scarce and does not allow the development of preventive strategies. We investigated the early steps of rectal infection in rhesus macaques inoculated with the pathogenic isolate SIVmac251 and necropsied four hours to nine days later. All macaques were positive for SIV. Control macaques inoculated with heat-inactivated virus were consistently negative for SIV. SIV DNA was detected in the rectum as early as four hours post infection by nested PCR for gag in many laser-microdissected samples of lymphoid aggregates and lamina propria but never in follicle-associated epithelium. Scarce SIV antigen positive cells were observed by immunohistofluorescence in the rectum, among intraepithelial and lamina propria cells as well as in clusters in lymphoid aggregates, four hours post infection and onwards. These cells were T cells and non-T cells that were not epithelial cells, CD68+ macrophages, DC-SIGN+ cells or fascin+ dendritic cells. DC-SIGN+ cells carried infectious virus. Detection of Env singly spliced mRNA in the mucosa by nested RT-PCR indicated ongoing viral replication. Strikingly, four hours post infection colic lymph nodes were also infected in all macaques as either SIV DNA or infectious virus was recovered. Rapid SIV entry and dissemination is consistent with trans-epithelial transport. Virions appear to cross the follicle-associated epithelium, and also the digestive epithelium. Viral replication could however be more efficient in lymphoid aggregates. The initial sequence of events differs from both vaginal and oral infections, which implies that prevention strategies for rectal transmission will have to be specific. Microbicides will need to protect both digestive and follicle-associated epithelia. Vaccines will need to induce immunity in lymph nodes as well as in the rectum

Entrée du virus de l'immunodéficience simienne et mouvements des cellules dendritiques au cours de l'infection rectale aiguë

PARIS7-Bibliothèque centrale (751132105) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Chemokines at mucosal barriers and their impact on HIV infection

International audienceAside from representing a physical barrier and providing an unfavorable chemical milieu to viral and bacterial infections, mucosae of gut and female genital tract also contain organized lymphoid structures that support the initiation of anti-microbial immune responses, and more diffuse lymphoid tissues that represent immune effector mucosal sites. Local expression of specific chemokines orchestrates lymphoid cell trafficking and positioning in the mucosa-associated lymphoid tissues, leading to their efficient priming during antigenic stimulations as well as their specific homing back where they were primed. This review examines productions and roles of mucosae-specific chemokines in healthy and pathological conditions, as well as their possible positive and deleterious effects during mucosal HIV infection

IL-7-dependent chemokine production by intestinal endothelial cells during acute HIV/SIV infection

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Mucosal IL-7 response in the gut during HIV/SIV acute infection

International audienceBackground: The very early events of the mucosal immune response to HIV/SIV infection still remain poorly understood. Our previous results demonstrate that IL-7 is expressed in the gut as part of the cytokine storm that occurs during initial virus dissemination, coincidently with viral spread and associated with an increased production of chemokines, leading to immune cell homing.The aim of this work was to identify mucosal cells responsible for this early IL-7 production as well as those responding to IL-7 by chemokine production in the gut.Methods: We thus analysed separately different cell types, known to be present in the gut mucosa to find out how much they can up-regulate their IL-7 production upon inflammatory conditions or how much they are able to secrete chemokines upon IL-7 stimulation. We used epithelial and endothelial cells isolated from gut mucosa of healthy macaques, as well as human endothelial cells differentiated from circulating endothelial cell precursors. These cells were cultured with the supernatant of SIV-infected or non-infected activated PBL or stimulated by pro-inflammatory cytokines with or without IL-7. IL-7 production was measured by RT-qPCR and ELISA in the supernatant of cultured cells. CD127 expression was analysed by RT-qPCR and FACS analysis. Chemokine production was measured by RT-qPCR and MSD.Results: We demonstrated that supernatants of infected activated PBLs boosted IL-7 production by epithelial cells, contrarily to the supernatants of non-infected PBLs. These cells also up-regulated their IL-7 production when stimulated by Interferons (IFNs). Endothelial cells showed an increased expression of CD127 when stimulated by TNF or IFNs, with higher expression when co-stimulated by IL-7, suggesting an increased responsiveness of cells to the IL-7 present in the gut environment during the SIV infection driven inflammatory status. In addition, IL-7 combined to inflammatory cytokines induced higher expression of chemokines by endothelial cells, such as IP-10, IL-8 and RANTES, which are important chemo-attractive molecules for immune cells.Conclusions: This work demonstrates that IL-7 production by intestinal epithelial cells is up regulated by inflammatory signals and identifies endothelial cells as one of the chemokine-expressing cell types under IL-7 stimulation

PS1-034. Accelerated thymocyte maturation during the acute phase of SIV infection in rhesus macaques

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Acute Simian Immunodeficiency Virus Infection Triggers Early and Transient Interleukin-7 Production in the Gut, Leading to Enhanced Local Chemokine Expression and Intestinal Immune Cell Homing

The intestinal barrier, one of the first targets of HIV/simian immunodeficiency virus (SIV) is subjected to major physiological changes during acute infection. Having previously shown that pharmaceutical injection of interleukin-7 (IL-7) triggers chemokine expression in many organs leading to massive T-cell homing, in particular to the intestine, we here explored mucosal IL-7 expression as part of the cytokine storm occurring during the acute phase of SIV infection in rhesus macaques. Quantifying both mRNA and protein in tissues, we demonstrated a transient increase of IL-7 expression in the small intestine of SIV-infected rhesus macaques, starting with local detection of the virus by day 3 of infection. We also observed increased transcription levels of several chemokines in the small intestine. In infected macaques, ileal IL-7 expression correlated with the transcription of four of these chemokines. Among these chemokines, the macrophage and/or T-cell attractant chemokines CCL4, CCL25, and CCL28 also demonstrated increased transcription in uninfected IL-7-treated monkeys. Through immunohistofluorescence staining and image analysis, we observed increased CD8+ T-cell numbers and stable CD4+ T-cell counts in the infected lamina propria (LP) during hyperacute infection. Concomitantly, circulating CCR9+beta7+ CD4+ and CD8+ T-cells dropped during acute infection, suggesting augmented intestinal homing of gut-imprinted T-cells. Finally, CD4+ macrophages transiently decreased in the submucosa and concentrated in the LP during the first days of infection. Overall, our study identifies IL-7 as a danger signal in the small intestine of Chinese rhesus macaques in response to acute SIV infection. Through stimulation of local chemokine expressions, this overexpression of IL-7 triggers immune cell recruitment to the gut. These findings suggest a role for IL-7 in the initiation of early mucosal immune responses to SIV and HIV infections. However, IL-7 triggered CD4+ T-cells and macrophages localization at viral replication sites could also participate to viral spread and establishment of viral reservoirs

IL-7 as an adjuvant for mucosal vaccine development

International audienceBackground: Despite considerable research efforts, mucosal immunity remains particularly difficult to stimulate through vaccines. Systemic injection of IL-7 stimulates chemokines-induced recruitment of circulating T-cells into mucosae.Methods: The optimal dose of IL-7 to be sprayed on mucosal surface was defined on 14 rhesus macaques. On mucosal biopsies collected after IL-7 administration, we quantified local transcription of 19 chemokines by qRT-PCR and cell infiltration by immunochemistry plus image analysis. Six macaques were immunized with antigens (DT and the HIV-1 gp41-P1 peptide) applied directly on the vaginal mucosa, two days after either IL-7 or PBS administration. The immunizations were repeated thrice, four months apart, and the macaques were euthanized 2 weeks after the last immunization. Antigen-specific IgA and IgG productions were quantified in vaginal secretions by ELISA. Antigen-specific plasma cells were detected by reverse immunohistochemistry in tissue, and by B-cell ELISPOT in PBMCs.Results: A significant overexpression of twelve chemokines was observed 48 hours after mucosal administration of 10µg of IL-7. Subsequently, mDC, macrophage, NK, B- and T-cell numbers significantly raised in the IL-7-treated mucosae, suggesting massive chemokine-driven infiltration. Administration of antigens led to a stronger mucosal immune response in the IL-7-treated macaques as compared to animals immunized with antigens alone. Robust production of antigen-specific IgAs and IgGs was detected in vaginal secretions. The immunizations repeated thrice sustained mucosal specific immune responses. Antigen-specific-antibody secreting cells were recovered from PBMC and more DT-specific plasma cells were found in the vaginal mucosae (IgA isotype) and the draining lymph nodes (IgG isotype) of IL-7-treated macaques. Tertiary lymphoid organs were observed in vaginal mucosae from IL-7-treated macaques only.Conclusions: Pre-treatment by non-traumatic vaginal administration of IL-7 (10µg by spray), allows for the development of a strong mucosal immune response in macaques following subsequent mucosal vaccination, through local chemokine expression and the recruitment of immune cells in the vaginal mucosa. The mucosal localization of IgA-specific plasma cells argues for their main contribution in the high levels of specific-IgAs evidenced in the vaginal secretions. These data suggest that IL-7 could be used as a mucosal adjuvant to elicit vaginal antibody response, the most promising way to confer protection to numerous STD

HIV reservoir dynamics in HAART-treated poor immunological responder patients under IL-7 therapy

International audienceObjectives: Recombinant Human IL-7 (rhIL-7) therapy allows reconstituting systemic and tissue-associated CD4+ T-cell populations in HIV-infected poor immunological responder (PIR) patients. However, in-vitro studies suggest that the impact of rhIL-7 treatment on HIV-DNA loads in vivo remains questionable.Design: We assessed the dynamics of circulating HIV-DNA loads in IL-7-treated HIV-infected PIR individuals.Methods: Forty-one rhIL-7-treated and 16 control participants from the INSPIRE-3 clinical trial were included. Participants received three weekly subcutaneous injections of rhIL-7. HIV-DNA was quantified by nested quantitative PCR in white blood cells sampled at D0, D28 and M3 and expressed as per milliliters and per CD4+ T-cell. Changes in HIV-DNA loads in the CD4+ compartment at M3 were confirmed on sorted CD4+ cells.Results: Together with rhIL-7-induced T-cell expansion, we observed a significant raise in both infected cell frequencies and counts during the first 28 days of follow-up. During this period, HIV-DNA load per CD4+ T-cell also increased, to a lower extent. Three months post-therapy, both the frequencies and counts of infected cells diminished in blood as compared with D28 but remained significantly higher than before IL-7 therapy. In contrast, infection frequencies strongly diminished within CD4+ cells, reaching slightly but significantly lower levels than at baseline.Conclusion: rhIL-7 treatment initially drives an expansion of HIV reservoir in PIR patients by D28. This expansion is probably not only because of infected cell proliferation, but also to possible enhanced neoinfection, despite highly active antiretroviral therapy. In contrast, subsequent reduction in HIV-DNA load per CD4+ T-cell argues for partial elimination of infected cells between D28 and M3

IL-7 as a mucosal adjuvant in pulmonary immunization protocols

International audienceObjective: Mucosae are the gateway for many pathogens. However, few vaccines have been developed to specifically target mucosal immunity. Recent studies in the laboratory evidenced local expression of IL-7 in the acutely SIV-infected intestinal mucosa and IAV-infected lung in macaques or mice, respectively. This overexpression led to chemokine production and infiltration of immune cells into these mucosae. In addition, systemic injection of IL-7 to macaques rapidly stimulates both the production of chemokines and immune cells homing into the mucosa. These results prompted us to study if IL-7 could be used as a mucosal vaccine adjuvant.Methods: Mice were intratracheally treated with IL-7 or PBS, then immunized by the same route against diphtheria toxoid (DT) or inactivated influenza virus (IAVi) two days later. DT immunized-mice were sacrificed at day 14 while those immunized with IAVi were infected with virulent IAV at day 14 and sacrificed at day 29. We measured antigen-specific antibody responses (anti-DT & anti-IAV) in both bronchoalveolar lavages (BAL) and sera, as well as chemokine expressions in lung tissue, by ELISA. The immune cell infiltration into the pulmonary mucosa was evaluated by immunochemistry on lung sections. The effectiveness of IL-7-adjuvanted vaccine in providing protection against IAV pathology was assessed by daily monitoring of animal body weight.Results: Intratracheal administration of IL-7 stimulated the production of pro-inflammatory chemokines in the lung parenchyma, leading to massive infiltration of immune cells found to be mainly organized in lymphoid aggregates. Moreover, intratracheal administration of IL-7 before primo-immunization allowed antigens-specific IgAs and IgGs production in the pulmonary mucosa. These effects were not observed in control mice vaccinated without IL-7 administration. In addition, only IL-7-treated immunized mice were protected against influenza pathology. This protection seems to be related to a high level of IAV-specific antibodies in BAL and lymphoid aggregate appearance in the pulmonary mucosa.Conclusion: By attracting immune cells into mucosae, local IL-7 administration prepares the mucosal immune system, gathering conditions that result in enhanced antigen-specific pulmonary immune responses upon antigenic stimulation. Hence, IL-7 appears as a mucosal adjuvant able to increase mucosal antibody responses, an important immune arm implicated in the protection against most mucosal infections