Lyle R. McKinnon

The integrin α4β7 forms a complex with cell-surface CD4 and defines a T-cell subset that is highly susceptible to infection by HIV-1

Both activated and resting CD4+ T cells in mucosal tissues play important roles in the earliest phases of infection after sexual transmission of HIV-1, a process that is inefficient. HIV-1 gp120 binds to integrin α4β7 (α4β7), the gut mucosal homing receptor. We find that α4β7high CD4+ T cells are more susceptible to productive infection than are α4β7low-neg CD4+ T cells in part because this cellular subset is enriched with metabolically active CD4+ T cells. α4β7high CD4+ T cells are CCR5high and CXCR4low; on these cells, α4β7 appears in a complex with CD4. The specific affinity of gp120 for α4β7 provides a mechanism for HIV-1 to target activated cells that are critical for efficient virus propagation and dissemination following sexual transmission.

Toll-like receptor expression and responsiveness are increased in viraemic HIV-1 infection

Objectives:Toll-like receptors (TLR) are important in pathogen recognition and may play a role in HIV disease. We evaluated the effect of chronic untreated and treated HIV-1 infection on systemic TLR expression and TLR signalling. Methods:Two hundred HIV-infected and uninfected women from a Kenya cohort participated in the studies. TLR1 to TLR10 messenger RNA expression was determined by quantitative reverse transcriptase polymerase chain reaction in peripheral blood mononuclear cells (PBMC). TLR ligand responsiveness was determined in or using ex-vivo PBMC by cytokine production in culture supernatants. Results:Chronic, untreated HIV-1 infection was significantly associated with increased mRNA expression of TLR6, TLR7, and TLR8 and when analysis was limited to those with advanced disease (CD4 cell count < 200 cells/ml) TLR2, TLR3, and TLR4 were additionally elevated. TLR expression correlated with the plasma HIV-RNA load, which was significant for TLR6 and TLR7. In vitro HIV single-stranded RNA alone could enhance TLR mRNA expression. PBMC of HIV-infected subjects also demonstrated profoundly increased proinflammatory responsiveness to TLR ligands, suggesting sensitization of TLR signalling in HIV. Finally, viral suppression by HAART was associated with a normalization of TLR levels. Conclusion:Together, these data indicate that chronic viraemic HIV-1 is associated with increased TLR expression and responsiveness, which may perpetuate innate immune dysfunction and activation that underlies HIV pathogenesis, and thus reveal potential new targets for therapy.

Characterization of a Human Cervical CD4+ T Cell Subset Coexpressing Multiple Markers of HIV Susceptibility

The HIV pandemic disproportionately affects women, with most infections acquired through receptive vaginal sex. Although the target cells by which HIV establishes infection in the female genital tract remain poorly defined, it is known that immune activation results in CD4+ T cells with enhanced susceptibility, as does expression of the mucosal integrin α4β7 and the HIV coreceptor CCR5. Blood and cervical cytobrush specimens were collected from female sex workers (FSWs) in Nairobi, Kenya. Genital infection diagnostics were performed, T cell populations were defined by multiparameter flow cytometry based on their expression of surface receptors relevant to mucosal homing and/or HIV acquisition, and cytokine production was assayed by intracellular cytokine staining. The integrin α4β7 was expressed on 26.0% of cervical CD4+ T cells, and these cells were more likely to express both the HIV coreceptor CCR5 (p < 0.0001) and the early activation marker CD69 (p < 0.0001) but not CXCR4 (p = 0.34). Cervical Th17 frequencies were enhanced compared with blood (7.02 versus 1.24%; p < 0.0001), and cervical IL-17A+ CD4+ T cells preferentially coexpressed α4β7 and CCR5. Expression of IFN-γ and IL-22 was greater in cervical Th17 cells than in blood Th17 cells. In keeping with the hypothesis that these cells are preferential HIV targets, gp120 preferentially bound CCR5+ cervical T cells, and cervical Th17 cells were almost completely depleted in HIV+ FSWs compared with HIV− FSWs. In summary, a subset of Th17 CD4+ T cells in the cervical mucosa coexpresses multiple HIV susceptibility markers; their dramatic depletion after HIV infection suggests that these may serve as key target cells during HIV transmission.

Sustained virologic control in SIV+ macaques after antiretroviral and α4β7 antibody therapy

Antibodies sustain viral control For many infected individuals, antiretroviral therapy (ART) means that an HIV-1 diagnosis is no longer a death sentence. But the virus persists in treated individuals, and complying with the intense drug regimen to keep virus loads down can be challenging for patients. Seeking an alternative, Byrareddy et al. treated ART-suppressed monkeys with antibodies targeting α4β7 integrin. When ART was halted in the antibody-treated animals, viral loads stayed undetectable, and normal CD4 T cell counts were maintained for over 9 months—and persisted—even after stopping the antibody therapy. Science, this issue p. 197 Update: An Editorial Expression of Concern has been published here Combining short-term antiretroviral therapy with specific anti-integrin treatment sustains low viral loads in monkeys. Antiretroviral drug therapy (ART) effectively suppresses replication of both the immunodeficiency viruses, human (HIV) and simian (SIV); however, virus rebounds soon after ART is withdrawn. SIV-infected monkeys were treated with a 90-day course of ART initiated at 5 weeks post infection followed at 9 weeks post infection by infusions of a primatized monoclonal antibody against the α4β7 integrin administered every 3 weeks until week 32. These animals subsequently maintained low to undetectable viral loads and normal CD4+ T cell counts in plasma and gastrointestinal tissues for more than 9 months, even after all treatment was withdrawn. This combination therapy allows macaques to effectively control viremia and reconstitute their immune systems without a need for further therapy.

Mucosal Th17 Cell Function Is Altered during HIV Infection and Is an Independent Predictor of Systemic Immune Activation

Mucosal Th17 cells maintain the gut epithelial barrier and prevent invasion by luminal bacteria through a delicate balance of immunosuppressive and proinflammatory functions. HIV infection is characterized by mucosal Th17 depletion, microbial translocation, and immune activation. Therefore, we assessed the function of blood and sigmoid Th17 cells during both early and chronic HIV infection, as well as the impact of short- and long-term antiretroviral therapy. Th17 cells were defined as IL-17a+ CD4 T cells, and their functional capacity was assessed by the coproduction of the inflammatory cytokines IL-22, TNF-α, and IFN-γ, as well as the immunoregulatory cytokine IL-10. Gut Th17 cells had a much greater capacity to produce proinflammatory cytokines than did those from the blood, but this capacity was dramatically reduced from the earliest stages of HIV infection. Immunoregulatory skewing of mucosal Th17 cell function, characterized by an increased IL-10/TNF-α ratio, was uniquely seen during early HIV infection and was independently associated with reduced systemic immune activation. Antiretroviral therapy rapidly restored mucosal Th17 cell numbers; however, normalization of mucosal Th17 function, microbial translocation, and mucosal/systemic immune activation was much delayed. These findings emphasize that strategies to preserve or to more rapidly restore mucosal Th17 function may have important therapeutic benefit.

Defining genital tract cytokine signatures of sexually transmitted infections and bacterial vaginosis in women at high risk of HIV infection: a cross-sectional study

Objectives Sexually transmitted infections (STI) and bacterial vaginosis (BV) cause female genital tract inflammation. This inflammation, which is often present in the absence of symptoms, is associated with increased susceptibility to HIV infection. We aimed to evaluate genital cytokine profiles and the degree of inflammation associated with common STIs and BV. Methods HIV-uninfected women (n=227) were screened for BV, Chlamydia trachomatis, Neisseria gonorrhoeae, Herpes simplex virus type 2 (HSV-2), and Trichomonas vaginalis. Concentrations of 42 cytokines in cervicovaginal lavages and 13 cytokines in plasma were measured using Luminex. Changes in cytokine profiles were evaluated using Mann–Whitney U test, logistic regression and factor analysis. p Values were adjusted for multiple comparisons using a false discovery rate step-down procedure. Results Women with chlamydia or gonorrhoea had the highest genital cytokine concentrations, with 17/42 and 14/42 cytokines upregulated compared with women with no infection, respectively. BV was associated with elevated proinflammatory cytokine concentrations, but lower chemokine and haematopoietic cytokine concentrations. HSV-2 reactivation was associated with lower levels of inflammation, while trichomoniasis did not cause significant differences in genital cytokine concentrations. Genital infections did not influence plasma cytokine concentrations. Although certain STIs, in particular chlamydia and gonorrhoea, were associated with high genital cytokine concentrations, only 19% of women with an STI/BV had clinical signs. Conclusions Chlamydia was associated with the highest genital cytokine levels, followed by gonorrhoea, HSV-2, trichomoniasis, and BV. In regions where HIV is prevalent and STIs are managed syndromically, better STI/BV screening is urgently needed, as certain infections were found to be highly inflammatory.

Vaginal bacteria modify HIV tenofovir microbicide efficacy in African women

Analysis of HIV clinical trials shows that the vaginal microbiome underlies varied responses to microbicide-based preexposure prophylaxis. Vaginal microbiome influences HIV acquisition Tenofovir is a preexposure drug used to prevent HIV infection. In clinical trials, tenofovir was effective for men, but not women. Klatt et al. now show that tenofovir efficacy in women depends on the composition of the vaginal microbiome (see the Perspective by Tuddenham and Ghanem). In a clinical trial of 688 women, tenofovir was three times as effective among those with a Lactobacillus-dominant vaginal microbiome as it was among other women. Gardnerella vaginalis tended to predominate in the women for whom tenofovir was less effective, and the authors found that the organism could rapidly metabolize and thereby inactivate the drug. Science, this issue p. 938; see also p. 907 Antiretroviral-based strategies for HIV prevention have shown inconsistent results in women. We investigated whether vaginal microbiota modulated tenofovir gel microbicide efficacy in the CAPRISA (Centre for the AIDS Program of Research in South Africa) 004 trial. Two major vaginal bacterial community types—one dominated by Lactobacillus (59.2%) and the other where Gardnerella vaginalis predominated with other anaerobic bacteria (40.8%)—were identified in 688 women profiled. Tenofovir reduced HIV incidence by 61% (P = 0.013) in Lactobacillus-dominant women but only 18% (P = 0.644) in women with non-Lactobacillus bacteria, a threefold difference in efficacy. Detectible mucosal tenofovir was lower in non-Lactobacillus women, negatively correlating with G. vaginalis and other anaerobic bacteria, which depleted tenofovir by metabolism more rapidly than target cells convert to pharmacologically active drug. This study provides evidence linking vaginal bacteria to microbicide efficacy through tenofovir depletion via bacterial metabolism.

HIV-1 RNA Dysregulates the Natural TLR Response to Subclinical Endotoxemia in Kenyan Female Sex-Workers

Background Subclinical endotoxemia has been reported in HIV-1 infected persons and may drive systemic immune activation and pathogenesis. Proinflammatory responsiveness to endotoxin (LPS) is mediated by Toll-like receptor 4 (TLR4). We therefore examined the association between plasma LPS levels, HIV RNA, and TLR4 expression and cytokine responses in the blood of HIV infected and uninfected participants in a cohort of female sex-workers in Kenya. Methodology/Principal Findings Ex vivo plasma and peripheral blood mononuclear cells (PBMC) were assessed for LPS and TLR mRNA, respectively. The effects of HIV single stranded RNA, a TLR8 ligand, on TLR4 and LPS signaling were further assessed in short term PBMC culture. Both HIV uninfected and infected subjects frequently had low detectable LPS levels in their plasmas. Significantly increased LPS levels were associated with chronic HIV-1 infection, both treated and untreated, but not with other acute or semi-chronic conditions reported. In HIV-uninfected subjects, TLR4 mRNA expression levels correlated inversely with plasma LPS levels, suggesting chronic endotoxin ‘tolerance’ in vivo. A similar effect of reduced TLR4 mRNA was seen in short term PBMC culture after stimulation with LPS. Interestingly, the apparent in vivo tolerance effect was diminished in subjects with HIV infection. Additionally, pre-stimulation of PBMC with LPS lead to proinflammatory (TNF-α) tolerance to subsequent LPS stimulation; however, pre-treatment of PBMC with HIV single-stranded RNA40, could enhance TLR4-mediated LPS responsiveness in vitro. Conclusions/Significance Thus, dysregulation of endotoxin tolerance by HIV-1 RNA may exacerbate HIV chronic immune activation and pathogenesis.

Increased levels of inflammatory cytokines in the female reproductive tract are associated with altered expression of proteases, mucosal barrier proteins, and an influx of HIV-susceptible target cells

Elevated inflammatory cytokines (EMCs) at mucosal surfaces have been associated with HIV susceptibility, but the underlying mechanisms remain unclear. We characterized the soluble mucosal proteome associated with elevated cytokine expression in the female reproductive tract. A scoring system was devised based on the elevation (upper quartile) of at least three of seven inflammatory cytokines in cervicovaginal lavage. Using this score, HIV-uninfected Kenyan women were classified as either having EMC (n=28) or not (n=68). Of 455 proteins quantified in proteomic analyses, 53 were associated with EMC (5% false discovery rate threshold). EMCs were associated with proteases, cell motility, and actin cytoskeletal pathways, whereas protease inhibitor, epidermal cell differentiation, and cornified envelope pathways were decreased. Multivariate analysis identified an optimal signature of 16 proteins that distinguished the EMC group with 88% accuracy. Three proteins in this signature were neutrophil-associated proteases that correlated with many cytokines, especially GM-CSF (granulocyte-macrophage colony-stimulating factor), IL-1β (interleukin-1β), MIP-3α (macrophage inflammatory protein-3α), IL-17, and IL-8. Gene set enrichment analyses implicated activated immune cells; we verified experimentally that EMC women had an increased frequency of endocervical CD4(+) T cells. These data reveal strong linkages between mucosal cytokines, barrier function, proteases, and immune cell movement, and propose these as potential mechanisms that increase risk of HIV acquisition.

Quality And Quantity: Mucosal CD4+ T Cells And Hiv Susceptibility

PURPOSE OF REVIEW The HIV sexual transmission probability measured in the context of discordant couples appears too low to fuel the HIV pandemic, but these rates are substantially amplified by specific co-factors. The most consistent predictors of transmission are the HIV levels in the blood and genital tract of an infected individual, each of which increases the transmission probability in a dose-dependent manner. In an analogous fashion, we propose that both the quantity and quality of HIV-susceptible target cells in the exposed genital or rectal mucosa may be key predictors of HIV susceptibility. RECENT FINDINGS The absolute number of mucosal CD4+ T cells is increased in several situations that are associated with amplified HIV transmission, particularly during genital infections. In addition, qualitative mucosal T-cell parameters such as immune activation and the expression of the HIV binding molecules CCR5 and/or α4β7 are important determinants of gp120 binding and productive HIV infection. In particular, the Th17 and Th22 cell subsets are enhanced within mucosal compartments and appear to be highly HIV-susceptible. SUMMARY Blockade of specific HIV target cell subsets at the site of exposure, if done in a safe and effective manner, represents an opportunity for new HIV prevention tools.