Caractérisation de NKp44L, un ligand activateur des cellules NK, et implications dans la physiopathologie du VIH-1

Les Mauvais Répondeurs Immunologiques (MRI) représentent un groupe particulier de patients infectés par le VIH-1 qui ne parviennent pas à obtenir un taux de Lymphocytes T CD4+ (LT-CD4) suffisant pour éviter la détérioration progressive de leur état général, malgré une suppression efficace de la réplication virale. Nous avons émis l hypothèse que les cellules NK pourraient jouer un rôle délétère dans ce phénomène, via l expression de NKp44L sur les LT-CD4, un ligand cellulaire du récepteur activateur NKp44. Des travaux antérieurs avaient montré que NKp44L était induit sur les LT-CD4+ via un motif très conservé de la gp41 du VIH-1. Des études phénotypiques et fonctionnelles sur les LT-CD4 de ces patients ont confirmé notre hypothèse et ont mis en évidence que le statut MRI est associé à la présence de cellules très différenciées, multifonctionnelles et apoptotiques, exprimant NKp44L. Ces cellules très particulières pourraient ainsi participer à la mauvaise reconstitution immunitaire chez les patients MRI. La caractérisation de NKp44L a également été réalisée afin de comprendre son rôle en physiopathologie. Nous avons identifié ce ligand comme étant un isoforme de la protéine MLL5 qui est notamment impliquée dans la régulation de l hématopoïèse et du cycle cellulaire. Son expression est détectée à la surface de nombreux types cellulaires dans des situations pathologiques variées, mettent en avant le rôle que pourrait avoir des molécules du soi comme signal de stress. L ensemble de ces résultats permet d envisager des stratégies thérapeutiques ayant pour objectif de prévenir l expression de NKp44L chez les patients infectés par le VIH-1, afin de limiter la déplétion en LT-CD4Certain patients, defined as Immunological non Responders (InR), fail to significantly improve their CD4 count, despite an effective antiretroviral treatment. We hypothesized that NK cells could be involved in this phenomenon through the involvement of NKp44L, a cellular ligand of the activating receptor NKp44, on CD4+ T cells. Over the ensuing years, our team have demonstrated that NKp44L is translocated on LT-CD4 in the presence of the specific and highly conserved 3S motif of the HIV-1 gp41 protein, rendering them more sensitive to NK cell lysis, a phenomenon, which could be partially responsible of the CD4 depletion. Phenotypic and functional studies on InR s CD4+ T cells confirmed our hypothesis and showed that this status was associated with the presence of highly differentiated, multifunctional and apoptotic cells expressing NKp44L, which could be, at least partially, associated with the poor immune reconstitution in InR HIV-infected patients. In parallel, the characterization of NKp44L was conducted to better understand its role in the physiopathology. Thus, we have identified NKp44L, as a new isoform of the MLL5 protein, both involved in the hematopoiesis and the cell cycle regulation. Importantly, NKp44L is only expressed on cells in various pathological situations. Altogether, these data highlight the role of self-molecules, as a danger signal, to alert the NK innate immune system, and allow us to consider therapeutic strategies, to prevent NKp44L expression in HIV-1 infected patients, in order to limit CD4+T cells depletionPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

The Human Penis Is a Genuine Immunological Effector Site

The human penis is a main portal of entry for numerous pathogens, and vaccines able to control resulting infections locally are highly desirable. However, in contrast to the gastrointestinal or vaginal mucosa, the penile immune system and mechanisms inducing a penile immune response remain elusive. In this descriptive study, using multiparametric flow cytometry and immunohistochemistry, we characterized mucosal immune cells such as B, T, and natural killer (NK) cells from the urethra, fossa, and glans of human adult penile tissues. We show that memory B lymphocytes and CD138+ plasma cells are detected in all penile compartments. CD4+ and CD8+ T lymphocytes reside in the epithelium and lamina propria of the penile regions and have mostly a resting memory phenotype. All penile regions contain CD56dim NK cells surface expressing the natural cytotoxicity receptor NKp44 and the antibody-dependent cell cytotoxicity receptor CD16. These cells are also able to spontaneously secrete pro- and anti-inflammatory cytokines, such as IL-17 and IL-22. Finally, CCR10 is the main homing receptor detected in these penile cells although, together with CCR3, CCR6, and CCR9, their expression level differs between penile compartments. Unlike antigen-presenting cells which type differ between penile regions as we reported earlier, urethral, fossa, and glans content in immune B, T, and NK cells is comparable. However, median values per each analysis suggest that the glans, containing higher number and more activated NK cells together with higher number of terminally differentiate effector CD8+ T cells, is a superior effector site than the urethra and the fossa. Thus, the human penis is an immunologically active tissue containing the cellular machinery required to induce and produce a specific and effective response against mucosal pathogens. It can therefore be considered as a classic mucosal effector site, a feature that must be taken into account for the elaboration of efficient strategies, including vaccines, against sexually transmitted infections

Live Imaging of HIV-1 Transfer across T Cell Virological Synapse to Epithelial Cells that Promotes Stromal Macrophage Infection

International audienceGraphical Abstract Highlights d HIV-1-infected T cells form viral synapse with the epithelium in reconstructed mucosa d These viral synapse dynamics were recorded by live imaging for the first time d HIV-1 formed at the viral synapse crosses the epithelium by transcytosis d Transcytosed HIV-1 targets stromal macrophages, resulting in their latent infection In Brief Real et al. established by live imaging the dynamics of virological synapses formed between HIV-1-infected T cells and the epithelium at the surface of a human reconstructed mucosa. HIV-1 virions formed at the viral synapse cross the epithelium and reach the mucosal stroma, where the virus establishes a latent infection in macrophages

Multiple reprobing of Western blots after inactivation of peroxidase activity by its substrate, hydrogen peroxide

International audienceSequential detections of different proteins on Western blot save time and precious samples. The main problem concerning reprobing is that stripping buffers can unbind both the antibody and the tested antigen. An original reprobing method has been set up based on horseradish peroxidase (HRP) inhibition after enhanced chemiluminescence detection. Instead of removing previously fixed antibodies as common stripping buffers do, the HRP activity linked to the secondary antibody is irreversibly inhibited by excess of hydrogen peroxide. A 15-min incubation allows one to perform at least five different sequential detections without losing significant amounts of blotted proteins

The Human Penis Is a Genuine Immunological Effector Site

International audienceThe human penis is a main portal of entry for numerous pathogens, and vaccines able to control resulting infections locally are highly desirable. However, in contrast to the gastrointestinal or vaginal mucosa, the penile immune system and mechanisms inducing a penile immune response remain elusive. In this descriptive study, using multiparametric flow cytometry and immunohistochemistry, we characterized mucosal immune cells such as B, T, and natural killer (NK) cells from the urethra, fossa, and glans of human adult penile tissues. We show that memory B lymphocytes and CD138 + plasma cells are detected in all penile compartments. CD4 + and CD8 + T lymphocytes reside in the epithelium and lamina propria of the penile regions and have mostly a resting memory phenotype. All penile regions contain CD56 dim NK cells surface expressing the natural cytotoxicity receptor NKp44 and the antibody-dependent cell cytotoxicity receptor CD16. These cells are also able to spontaneously secrete pro-and anti-inflammatory cytokines, such as IL-17 and IL-22. Finally, CCR10 is the main homing receptor detected in these penile cells although, together with CCR3, CCR6, and CCR9, their expression level differs between penile compartments. Unlike antigen-presenting cells which type differ between penile regions as we reported earlier, urethral, fossa, and glans content in immune B, T, and NK cells is comparable. However, median values per each analysis suggest that the glans, containing higher number and more activated NK cells together with higher number of terminally differentiate effector CD8 + T cells, is a superior effector site than the urethra and the fossa. Thus, the human penis is an immunologically active tissue containing the cellular machinery required to induce and produce a specific and effective response against mucosal pathogens. It can therefore be considered as a classic mucosal effector site, a feature that must be taken into account for the elaboration of efficient strategies, including vaccines, against sexually transmitted infections

Native CGRP Neuropeptide and Its Stable Analogue SAX, But Not CGRP Peptide Fragments, Inhibit Mucosal HIV-1 Transmission

International audienceBackground: The vasodilator neuropeptide calcitonin gene-related peptide (CGRP) plays both detrimental and protective roles in different pathologies. CGRP is also an essential component of the neuro-immune dialogue between nociceptors and mucosal immune cells. We previously discovered that CGRP is endowed with anti-viral activity and strongly inhibits human immunodeficiency virus type 1 (HIV-1) infection, by suppressing Langerhans cells (LCs)-mediated HIV-1 trans-infection in-vitro and mucosal HIV-1 transmission ex-vivo. This inhibition is mediated via activation of the CGRP receptor non-canonical NFkB/STAT4 signaling pathway that induces a variety of cooperative mechanisms. These include CGRP-mediated increase in the expression of the LCspecific pathogen recognition C-type lectin langerin and decrease in LC-T-cell conjugates formation. The clinical utility of CGRP and modalities of CGRP receptor activation, for inhibition of mucosal HIV-1 transmission, remain elusive.Methods: We tested the capacity of CGRP to inhibit HIV-1 infection in-vivo in humanized mice. We further compared the anti-HIV-1 activities of full-length native CGRP, its metabolically stable analogue SAX, and several CGRP peptide fragments containing its binding C-terminal and activating N-terminal regions. These agonists were evaluated for their capacity to inhibit LCs-mediated HIV-1 trans-infection in-vitro and mucosal HIV-1 transmission in human mucosal tissues ex-vivo.Results: A single CGRP intravaginal topical treatment of humanized mice, followed by HIV-1 vaginal challenge, transiently restricts the increase in HIV-1 plasma viral loads but maintains long-lasting higher CD4+ T-cell counts. Similarly to CGRP, SAX inhibits LCsmediated HIV-1 trans-infection in-vitro, but with lower potency. This inhibition is mediated via CGRP receptor activation, leading to increased expression of both langerin and STAT4 , and accordingly lack anti-HIV-1 activities. Finally, like CGRP, treatment of human inner foreskin tissue explants with SAX, followed by polarized inoculation with cell-associated HIV-1, completely blocks formation of LC-T-cell conjugates and HIV-1 infection of T-cells.Conclusion: Our results show that CGRP receptor activation by full-length CGRP or SAX is required for efficient inhibition of LCs-mediated mucosal HIV-1 transmission. These findings suggest that formulations containing CGRP, SAX and/or their optimized agonists/analogues could be harnessed for HIV-1 prevention

S100A8-mediated metabolic adaptation controls HIV-1 persistence in macrophages in vivo

International audienceHIV-1 eradication is hindered by viral persistence in cell reservoirs, established not only in circulatory CD4 + T-cells but also in tissue-resident macrophages. The nature of macrophage reservoirs and mechanisms of persistence despite combined anti-retroviral therapy (cART) remain unclear. Using genital mucosa from cART-suppressed HIV-1-infected individuals, we evaluated the implication of macrophage immunometabolic pathways in HIV-1 persistence. We demonstrate that ex vivo, macrophage tissue reservoirs contain transcriptionally active HIV-1 and viral particles accumulated in virus-containing compartments, and harbor an inflammatory IL-1R + S100A8 + MMP7 + M4-phenotype prone to glycolysis. Reactivation of infectious virus production and release from these reservoirs in vitro are induced by the alarmin S100A8, an endogenous factor produced by M4-macrophages and implicated in "sterile" inflammation. This process metabolically depends on glycolysis. Altogether, inflammatory M4macrophages form a major tissue reservoir of replication-competent HIV-1, which reactivate viral production upon autocrine/paracrine S100A8-mediated glycolytic stimulation. This HIV-1 persistence pathway needs to be targeted in future HIV eradication strategies

Platelets from cART-suppressed HIV-infected patients with poor CD4+T cell 1 recovery carry infectious HIV

International audienc

Platelets from cART-suppressed HIV-infected patients with poor CD4+T cell 1 recovery carry infectious HIV

International audienc

HIV-1 reservoirs in urethral macrophages of patients under suppressive antiretroviral therapy

International audienceHuman immunodeficiency virus type 1 (HIV-1) eradication is prevented by the establishment on infection of cellular HIV-1 reservoirs that are not fully characterized, especially in genital mucosal tissues (the main HIV-1 entry portal on sexual transmission). Here, we show, using penile tissues from HIV-1-infected individuals under suppressive combination antiretroviral therapy, that urethral macrophages contain integrated HIV-1 DNA, RNA, proteins and intact virions in virus-containing compartment-like structures, whereas viral components remain undetectable in urethral T cells. Moreover, urethral cells specifically release replication-competent infectious HIV-1 following reactivation with the macrophage activator lipopolysaccharide, while the T-cell activator phytohaemagglutinin is ineffective. HIV-1 urethral reservoirs localize preferentially in a subset of polarized macrophages that highly expresses the interleukin-1 receptor, CD206 and interleukin-4 receptor, but not CD163. To our knowledge, these results are the first evidence that human urethral tissue macrophages constitute a principal HIV-1 reservoir. Such findings are determinant for therapeutic strategies aimed at HIV-1 eradication