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

HIV-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 M4-macrophages 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.

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

Background: 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 NFκB/STAT4 signaling pathway that induces a variety of cooperative mechanisms. These include CGRP-mediated increase in the expression of the LC-specific 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 LCs-mediated 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 in LCs. In contrast, several N-terminal and N+C-terminal bivalent CGRP peptide fragments fail to increase 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.

Platelets from HIV-infected individuals on antiretroviral drug therapy with poor CD4+ T cell recovery can harbor replication-competent HIV despite viral suppression.

In addition to hemostasis, human platelets have several immune functions and interact with infectious pathogens including HIV in vitro. Here, we report that platelets from HIV-infected individuals on combined antiretroviral drug therapy (ART) with low blood CD4+ T cell counts (<350 cells/µl) contained replication-competent HIV despite viral suppression. In vitro, human platelets harboring HIV propagated the virus to macrophages, a process that could be prevented with the biologic abciximab, an anti-integrin αIIb/ß3 Fab. Furthermore, in our cohort, 88% of HIV-infected individuals on ART with viral suppression and with platelets containing HIV were poor immunological responders with CD4+ T cell counts remaining below <350 cells/µl for more than one year. Our study suggests that platelets may be transient carriers of HIV and may provide an alternative pathway for HIV dissemination in HIV-infected individuals on ART with viral suppression and poor CD4+ T cell recovery.

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

Human 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.

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

During sexual intercourse, HIV-1 crosses epithelial barriers composing the genital mucosa, a poorly understood feature that requires an HIV-1-infected cell vectoring efficient mucosal HIV-1 entry. Therefore, urethral mucosa comprising a polarized epithelium and a stroma composed of fibroblasts and macrophages were reconstructed in vitro. Using this system, we demonstrate by live imaging that efficient HIV-1 transmission to stromal macrophages depends on cell-mediated transfer of the virus through virological synapses formed between HIV-1-infected CD4+ T cells and the epithelial cell mucosal surface. We visualized HIV-1 translocation through mucosal epithelial cells via transcytosis in regions where virological synapses occurred. In turn, interleukin-13 is secreted and HIV-1 targets macrophages, which develop a latent state of infection reversed by lipopolysaccharide (LPS) activation. The live observation of virological synapse formation reported herein is key in the design of vaccines and antiretroviral therapies aimed at blocking HIV-1 access to cellular reservoirs in genital mucosa.

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.

NKp44L expression on CD4+ T cells is associated with impaired immunological recovery in HIV-infected patients under highly active antiretroviral therapy.

OBJECTIVE: HIV-infected immunological nonresponders (InRs) patients fail to show satisfactory CD4+ T-cell recovery despite virologically effective HAART. We propose that NKp44L, the cellular ligand of an activating natural killer (NK) receptor, expressed only on uninfected bystander CD4+ T cells from HIV-1 infected patients, could play a major role in this phenomenon by sensitizing these cells to NK killing. DESIGN: Phenotype and multifunctional status of CD4+ T cells, especially the subsets expressing and not expressing NKp44L, were characterized for HIV-infected patients receiving HAART for at least 2 years, during which their viral load remained less than 40 copies/ml; 53 were InRs (CD4 cell count always <350 cells/µl), and 82 immunological responders (CD4 cell count always ≥350 cells/µl). Flow cytometry determined NKp44L expression in association with specific markers of proliferation, maturation, activation, homeostasis, and intracellular cytokine production. Degranulation of NKp44+ determined the functional capacity of NK cells. RESULTS: InRs exhibited high levels of NKp44L+CD4+ T cells. Compared with NKp44L negative cells, the frequency of naive CD45RA+CCR7+ T cells expressing NKp44L fell (P < 0.001) and their proliferative capacity grew. Moreover, apoptosis and a unique ability to produce multiple cytokines (IL-2, IFN-γ, and TNF-α) without or after phytohemagglutinin or anti-CD3/CD28 stimulation distinguished NKp44L+ T cells. CONCLUSION: InR status is associated to a significant expansion of highly differentiated, multifunctional and apoptotic CD4+ T cells expressing NKp44L. This could explain a rapid CD4+ T-cell turnover in InR preventing immune recovery. These data suggest a new target for developing therapeutic strategies to prevent NKp44L expression and then stimulating immune recovery in InRs.

Identification of a cellular ligand for the natural cytotoxicity receptor NKp44.

With an array of activating and inhibitory receptors, natural killer (NK) cells are involved in the eradication of infected, transformed, and tumor cells. NKp44 is a member of the natural cytotoxicity receptor family, which is exclusively expressed on activated NK cells. Here, we identify natural cytotoxicity receptor NKp44 (NKp44L), a novel isoform of the mixed-lineage leukemia-5 protein, as a cellular ligand for NKp44. Unlike the other MLL family members, NKp44L is excluded from the nucleus, but expressed at the cell-surface level; its subcellular localization is being associated with the presence of a specific C-terminal motif. Strikingly, NKp44L has not been detected on circulating cells isolated from healthy individuals, but it is expressed on a large panel of the tumor and transformed cells. The sharply decreased NK lysis activity induced by anti-NKp44L antibodies directly demonstrates the role of NKp44L in cytotoxicity. Taken together, these results show that NKp44L could be critical for NK cell-mediated innate immunity. The identification and cellular distribution of NKp44L highlight the role of this self-molecule as a danger signal to alert the NK cell network.

A single amino-acid change in a highly conserved motif of gp41 elicits HIV-1 neutralization and protects against CD4 depletion.

BACKGROUND: The induction of neutralizing antibodies against conserved regions of the human immunodeficiency virus type 1 (HIV-1) envelope protein is a major goal of vaccine strategies. We previously identified 3S, a critical conserved motif of gp41 that induces the NKp44L ligand of an activating NK receptor. In vivo, anti-3S antibodies protect against the natural killer (NK) cell-mediated CD4 depletion that occurs without efficient viral neutralization. METHODS: Specific substitutions within the 3S peptide motif were prepared by directed mutagenesis. Virus production was monitored by measuring the p24 production. Neutralization assays were performed with immune-purified antibodies from immunized mice and a cohort of HIV-infected patients. Expression of NKp44L on CD4(+) T cells and degranulation assay on activating NK cells were both performed by flow cytometry. RESULTS: Here, we show that specific substitutions in the 3S motif reduce viral infection without affecting gp41 production, while decreasing both its capacity to induce NKp44L expression on CD4(+) T cells and its sensitivity to autologous NK cells. Generation of antibodies in mice against the W614 specific position in the 3S motif elicited a capacity to neutralize cross-clade viruses, notable in its magnitude, breadth, and durability. Antibodies against this 3S variant were also detected in sera from some HIV-1-infected patients, demonstrating both neutralization activity and protection against CD4 depletion. CONCLUSIONS: These findings suggest that a specific substitution in a 3S-based immunogen might allow the generation of specific antibodies, providing a foundation for a rational vaccine that combine a capacity to neutralize HIV-1 and to protect CD4(+) T cells.

Specific anti-gp41 antibodies predict HIV-1 disease progression.

The immunodeficiency-defining AIDS results from a progressive decline of CD4 cell count. We previously showed that 3S, a unique motif of the HIV-1 gp41 envelop protein, is highly conserved in HIV-1 strains and induces expression of NKp44L, rendering CD4 cells sensitive to NK killing. Here we found from a well-characterized cohort of 244 untreated HIV-1 seroconverters that high levels of anti-3S antibodies significantly delay spontaneous disease progression in the first years after seroconversion; this effect was not mediated through baseline viral load or CD4. These results could have important implications both for clinical care and better understanding of pathogenicity.

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

Sequential 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.