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In the gastro-intestinal tract, the complex network of multiple innate cell populations play critical roles not only as a first line of defense against invading pathogens and in driving adaptive immune responses but also in maintaining intestinal homeostasis. Here, we describe the roles of various innate immune cell populations in gut immunity and detail studies investigating the impact of acute and chronic HIV infection on these cell populations. Alterations in frequencies, phenotype and/or function of innate lymphoid cells, dendritic cells, macrophages, neutrophils, and innate-like T cells have been reported in people with HIV (PWH), with many of these features persisting despite anti-retroviral therapy and virological suppression. Dysregulated gut innate immunity in PWH is a feature of gut pathogenesis. A greater understanding of the mechanisms driving impairment in the multiple different gut innate immune cell populations and the downstream consequences of an altered innate immune response on host defense and gut homeostasis in PWH is needed to develop more effective HIV treatments and cure strategies.

Background The etiology of the low-level chronic inflammatory state associated with aging is likely multifactorial, but a number of animal and human studies have implicated a functional decline of the gastrointestinal immune system as a potential driver. Gut tissue-resident memory T cells play critical roles in mediating protective immunity and in maintaining gut homeostasis, yet few studies have investigated the effect of aging on human gut T cell immunity. To determine if aging impacted CD4 T cell immunity in the human large intestine, we utilized multi-color flow cytometry to measure colonic lamina propria (LP) CD4 T cell frequencies and immune-modulatory marker expression in younger (mean ± SEM: 38 ± 1.5 yrs) and older (77 ± 1.6 yrs) adults. To determine cellular specificity, we evaluated colon LP CD8 T cell frequency and phenotype in the same donors. To probe tissue specificity, we evaluated the same panel of markers in peripheral blood (PB) CD4 T cells in a separate cohort of similarly aged persons. Results Frequencies of colonic CD4 T cells as a fraction of total LP mononuclear cells were higher in older persons whereas absolute numbers of colonic LP CD4 T cells per gram of tissue were similar in both age groups. LP CD4 T cells from older versus younger persons exhibited reduced CTLA-4, PD-1 and Ki67 expression. Levels of Bcl-2, CD57, CD25 and percentages of activated CD38+HLA-DR+ CD4 T cells were similar in both age groups. In memory PB CD4 T cells, older age was only associated with increased CD57 expression. Significant age effects for LP CD8 T cells were only observed for CTLA-4 expression, with lower levels of expression observed on cells from older adults. Conclusions Greater age was associated with reduced expression of the co-inhibitory receptors CTLA-4 and PD-1 on LP CD4 T cells. Colonic LP CD8 T cells from older persons also displayed reduced CTLA-4 expression. These age-associated profiles were not observed in older PB memory CD4 T cells. The decline in co-inhibitory receptor expression on colonic LP T cells may contribute to local and systemic inflammation via a reduced ability to limit ongoing T cell responses to enteric microbial challenge.

Group 3 innate lymphoid cells (ILC3s) in the gut mucosa have long been thought to be noncytotoxic lymphocytes that are critical for homeostasis of intestinal epithelial cells through secretion of IL-22. Recent work using human tonsillar cells demonstrated that ILC3s exposed to exogenous inflammatory cytokines for a long period of time acquired expression of granzyme B, suggesting that under pathological conditions ILC3s may become cytotoxic. We hypothesized that inflammation associated with bacterial exposure might trigger granzyme B expression in gut ILC3s. To test this, we exposed human colon lamina propria mononuclear cells to a panel of enteric bacteria. We found that the Gram-negative commensal and pathogenic bacteria induced granzyme B expression in a subset of ILC3s that were distinct from IL-22–producing ILC3s. A fraction of granzyme B+ ILC3s coexpressed the cytolytic protein perforin. Granzyme B expression was mediated, in part, by IL-15 produced upon exposure to bacteria. ILC3s coexpressing all three IL-15R subunits (IL15Rα/β/γ) increased following bacterial stimulation, potentially allowing for cis presentation of IL-15 during bacterial exposure. Additionally, a large frequency of colonic myeloid dendritic cells expressed IL-15Rα, implicating myeloid dendritic cells in trans presentation of IL-15 to ILC3s. Tonsillar ILC3s minimally expressed granzyme B when exposed to the same bacteria or to rIL-15. Overall, these data establish the novel, to our knowledge, finding that human colonic ILC3s can express granzyme B in response to a subset of enteric bacteria through a process mediated by IL-15. These observations raise new questions about the multifunctional role of human gut ILC3s.

Background: Physiologic aging has been associated with gut dysbiosis. Although short exercise interventions have been linked to beneficial changes in gut microbiota in younger adults, limited data are available from older populations. We hypothesized that exercise would produce beneficial shifts in microbiota and short-chain fatty acid (SCFA) levels in older persons. Methods: Stool samples were collected before and at completion of a supervised 24-week cardiovascular and resistance exercise intervention among 50–75-year-old participants. SCFA levels were analyzed by gas chromatography and microbiome by 16S rRNA gene sequencing. Negative binomial regression models compared pre- and post-differences using false discovery rates for multiple comparison. Results: A total of 22 participants provided pre-intervention samples; 15 provided samples at study completion. At baseline, the majority of participants were men (95%), mean age 58.0 (8.8) years, mean body mass index 27.4 (6.4) kg/m2. After 24 weeks of exercise, at the genus level, exercise was associated with significant increases in Bifidobacterium (and other unidentified genera within Bifidobacteriaceae), Oscillospira, Anaerostipes, and decreased Prevotella and Oribacterium ( p Conclusion: Our pilot study suggested that an exercise intervention is associated with changes in the microbiome of older adults and a key bacterial metabolite, butyrate. Although some of these changes could potentially reverse age-related dysbiosis, future studies are required to determine the contribution of changes to the microbiome in the beneficial effect of exercise on overall health of older adults. Clinical Trials NCT02404792

BackgroundWhether human immunodeficiency virus (HIV) infection impacts gut microbial α-diversity is controversial. We reanalyzed raw 16S ribosomal RNA (rRNA) gene sequences and metadata from published studies to examine α-diversity measures between HIV-uninfected (HIV–) and HIV-infected (HIV+) individuals.MethodsWe conducted a systematic review and individual level meta-analysis by searching Embase, Medline, and Scopus for original research studies (inception to 31 December 2017). Included studies reported 16S rRNA gene sequences of fecal samples from HIV+ patients. Raw sequence reads and metadata were obtained from public databases or from study authors. Raw reads were processed through standardized pipelines with use of a high-resolution taxonomic classifier. The χ2 test, paired t tests, and generalized linear mixed models were used to relate α-diversity measures and clinical metadata.ResultsTwenty-two studies were identified with 17 datasets available for analysis, yielding 1032 samples (311 HIV–, 721 HIV+). HIV status was associated with a decrease in measures of α-diversity (P < .001). However, in stratified analysis, HIV status was associated with decreased α-diversity only in women and in men who have sex with women (MSW) but not in men who have sex with men (MSM). In analyses limited to women and MSW, controlling for HIV status, women displayed increased α-diversity compared with MSW.ConclusionsOur study suggests that HIV status, sexual risk category, and gender impact gut microbial community α-diversity. Future studies should consider MSM status in gut microbiome analyses.

Background HIV-1 infection and physiological aging are independently linked to elevated systemic inflammation and changes in enteric microbial communities (dysbiosis). However, knowledge of the direct effect of HIV infection on the aging microbiome and potential links to systemic inflammation is lacking. Methods In a cross-sectional study of older people living with HIV (PLWH) (median age 61.5 years, N = 14) and uninfected controls (median 58 years, n = 22) we compared stool microbiota, levels of microbial metabolites (short-chain fatty acid levels, SCFA) and systemic inflammatory biomarkers by HIV serostatus and age. Findings HIV and age were independently associated with distinct changes in the stool microbiome. For example, abundances of Enterobacter and Paraprevotella were higher and Eggerthella and Roseburia lower among PLWH compared to uninfected controls. Age-related microbiome changes also differed by HIV serostatus. Some bacteria with inflammatory potential (e.g. Escherichia) increased with age among PLWH, but not controls. Stool SCFA levels were similar between the two groups yet patterns of associations between individual microbial taxa and SCFA levels differed. Abundance of various genera including Escherichia and Bifidobacterium positively associated with inflammatory biomarkers (e.g. soluble Tumor Necrosis Factor Receptors) among PLWH, but not among controls. Interpretation The age effect on the gut microbiome and associations between microbiota and microbial metabolites or systemic inflammation differed based on HIV serostatus, raising important implications for the impact of therapeutic interventions, dependent on HIV serostatus or age.

PURPOSE OF REVIEW: Aging and HIV share features of intestinal damage and alterations in the communities of enteric bacteria, termed dysbiosis. The purpose of this review is to highlight the various features of the gut microbiome in 1) aging and in 2) people with HIV (PWH) and to discuss how aging and HIV converge to impact the gut microbiome. The term microbiome reflects the combined genetic material of micro-organisms present including bacteria, viruses, bacteriophages and fungi. To date, the majority of studies investigating the impact of aging and HIV on the gut microbiome have focused on bacteria and therefore, for the purposes of this review, the term “microbiome” is used to reflect enteric bacterial communities. RECENT FINDINGS: Aging is associated with alterations in the gut bacterial microbiome. Although changes vary by the age of the population, lifestyle (diet, physical activity) and geographic location, the age-associated dysbiosis is typically characterized by an increase in facultative anaerobes with inflammatory properties and a decrease in obligate anaerobes that play critical roles in maintaining intestinal homeostasis and in regulating host immunity. PWH also have dysbiotic gut microbiomes, many features of which reflect those observed in elderly persons. In one study, the age effect on the gut microbiome differed based on HIV serostatus in older adults. SUMMARY: HIV and age may interact to shape the gut microbiome. Future studies should investigate relationships between the gut microbiome and age-associated co-morbidities in older PWH populations. Identifying these links will provide new avenues for treatments and interventions to improve the healthspan and lifespan of older PWH.\

Objective(s) Type I interferon (IFN-I) responses confer both protective and pathogenic effects in persistent virus infections. IFN-I diversity, stage of infection and tissue compartment may account for this dichotomy. The gut is a major site of early HIV-1 replication and microbial translocation, but the nature of the IFN-I response in this compartment remains unclear. Design Samples were obtained from two IRB-approved cross-sectional studies. The first study included individuals with chronic, untreated HIV-1 infection (n = 24) and age/sex-balanced uninfected controls (n = 14). The second study included antiretroviral-treated, HIV-1-infected individuals (n = 15) and uninfected controls (n = 15). Methods The expression of 12 IFNα subtypes, IFNβ and antiviral IFN-stimulated genes (ISGs) were quantified in peripheral blood mononuclear cells (PBMCs) and colon biopsies using real-time PCR and next-generation sequencing. In untreated HIV-1-infected individuals, associations between IFN-I responses and gut HIV-1 RNA levels as well as previously established measures of colonic and systemic immunological indices were determined. Results IFNα1, IFNα2, IFNα4, IFNα5 and IFNα8 were upregulated in PBMCs during untreated chronic HIV-1 infection, but IFNβ was undetectable. By contrast, IFNβ was upregulated and all IFNα subtypes were downregulated in gut tissue. Gut ISG levels positively correlated with gut HIV-1 RNA and immune activation, microbial translocation and inflammation markers. Gut IFN-I responses were not significantly different between HIV-1-infected individuals on antiretroviral treatment and uninfected controls. Conclusion The IFN-I response is compartmentalized during chronic untreated HIV-1 infection, with IFNβ being more predominant in the gut. Gut IFN-I responses are associated with immunopathogenesis, and viral replication is likely a major driver of this response.

Group 1 Innate Lymphoid Cells (which include Natural Killer cells and ILC1s) aid in gut anti-bacterial defense through the production of IFNγ, which is critical for mobilizing protective responses against enteric pathogens. When intestinal epithelial barrier integrity is compromised, commensal bacteria are likely to translocate from the gut lumen into the lamina propria. Few studies have addressed the mechanisms by which commensal bacteria impact the function of gut Group 1 ILCs, especially ILC1s. Utilizing an in vitro human colonic lamina propria mononuclear cell (LPMC) model, we evaluated Group 1 ILC cytokine and cytolytic protein production in response to a panel of enteric Gram-positive and Gram-negative commensal and pathogenic bacteria. IFNγ-production by NK cells and ILC1s was significantly increased after LPMC exposure to Gram-negative commensal or pathogenic bacteria, but not after exposure to the Gram-positive bacteria commensals tested. Stimulation of IFNγ production from Group 1 ILCs was not through direct recognition of bacteria by NK cells or ILC1s, but rather required accessory cells within the LPMC population. Myeloid dendritic cells generated IL-12p70, IL-18, and IL-1β upon exposure to enteric bacteria and these cytokines contributed to Group 1 ILC production of IFNγ. Furthermore, Gram-negative commensal or pathogenic bacteria induced significant expression of Granzyme B in NK cells and ILC1s. Overall, these data demonstrate that some enteric commensal bacteria indirectly induce inflammatory cytokine production and cytolytic protein expression from human colonic Group 1 ILCs, a process which could contribute to inflammation in the setting of microbial translocation.

Commensal organisms appear to play significant roles in normal homeostasis as well as in the pathogenesis of HIV infection in a number of different organ systems. On November 17th and 18th, 2016, leading researchers from around the world met to discuss their insights on advances in our understanding of HIV and the microbiome at the National Institutes of Health (NIH) in Bethesda. Dr. Elhanan Borenstein of the University of Washington gave a keynote address where he discussed new developments in systems biology which hold the promise of illuminating the pathways by which these organisms interact with human physiology. He suggested that we need to get past correlations in microbiome research by using models and informatics which incorporate metagenomics to predict functional changes in the microbiome.

Innate lymphoid cells (ILCs) are a diverse family of cells that play critical roles in mucosal immunity. One subset of the ILC family, Group 3 ILCs (ILC3s), has been shown to aid in gut homeostasis through the production of IL-22. IL-22 promotes gut homeostasis through its functional effect on the epithelial barrier. When gut epithelial barrier integrity is compromised, such as in Human Immunodeficiency Virus (HIV) infection and inflammatory bowel disease (IBD), microbes from the gut lumen translocate into the lamina propria, inducing a multitude of potentially pathogenic immune responses. In murine models of bacterial infection, there is evidence that bacteria can induce pro-inflammatory IFNγ production in ILC3s. However, the impact of diverse translocating bacteria, particularly commensal bacteria, in dictating IFNγ versus IL-22 production by human gut ILC3s remains unclear. Here, we utilized an in vitro human lamina propria mononuclear cell (LPMC) model to evaluate ILC3 cytokine production in response to a panel of enteric Gram-positive and Gram-negative commensal and pathogenic bacteria and determined potential mechanisms by which these cytokine responses were induced. The percentages of IL-22-producing ILC3s, but not IFNγ-producing ILC3s, were significantly increased after LPMC exposure to both Gram-positive and Gram-negative commensal or pathogenic bacterial stimuli. Stimulation of IL-22 production from ILC3s was not through direct recognition of bacterial antigen by ILC3s, but rather required the help of accessory cells within the LPMC population. CD11c+ myeloid dendritic cells generated IL-23 and IL-1β in response to enteric bacteria and contributed to ILC3 production of IL-22. Furthermore, ligation of the natural cytotoxicity receptor NKp44 on ILC3s in response to bacteria stimulation also significantly increased the percentage of IL-22-producing ILC3s. Overall, these data demonstrate that human gut microbiota, including commensal bacteria, indirectly modulate colonic ILC3 function to induce IL-22, but additional signals are likely required to induce IFNγ production by colonic ILC3s in the setting of inflammation and microbial translocation.

The Type I Interferons (IFN-Is) are innate antiviral cytokines that include 12 different IFNα subtypes and IFNβ that signal through the IFN-I receptor (IFNAR), inducing hundreds of IFN-stimulated genes (ISGs) that comprise the ‘interferome’. Quantitative differences in IFNAR binding correlate with antiviral activity, but whether IFN-Is exhibit qualitative differences remains controversial. Moreover, the IFN-I response is protective during acute HIV-1 infection, but likely pathogenic during the chronic stages. To gain a deeper understanding of the IFN-I response, we compared the interferomes of IFNα subtypes dominantly-expressed in HIV-1-exposed plasmacytoid dendritic cells (1, 2, 5, 8 and 14) and IFNβ in the earliest cellular targets of HIV-1 infection. Primary gut CD4 T cells from 3 donors were treated for 18 hours ex vivo with individual IFN-Is normalized for IFNAR signaling strength. Of 1,969 IFN-regulated genes, 246 ‘core ISGs’ were induced by all IFN-Is tested. However, many IFN-regulated genes were not shared between the IFNα subtypes despite similar induction of canonical antiviral ISGs such as ISG15, RSAD2 and MX1, formally demonstrating qualitative differences between the IFNα subtypes. Notably, IFNβ induced a broader interferome than the individual IFNα subtypes. Since IFNβ, and not IFNα, is upregulated during chronic HIV-1 infection in the gut, we compared core ISGs and IFNβ-specific ISGs from colon pinch biopsies of HIV-1-uninfected (n = 13) versus age- and gender-matched, antiretroviral-therapy naïve persons with HIV-1 (PWH; n = 19). Core ISGs linked to inflammation, T cell activation and immune exhaustion were elevated in PWH, positively correlated with plasma lipopolysaccharide (LPS) levels and gut IFNβ levels, and negatively correlated with gut CD4 T cell frequencies. In sharp contrast, IFNβ-specific ISGs linked to protein translation and anti-inflammatory responses were significantly downregulated in PWH, negatively correlated with gut IFNβ and LPS, and positively correlated with plasma IL6 and gut CD4 T cell frequencies. Our findings reveal qualitative differences in interferome induction by diverse IFN-Is and suggest potential mechanisms for how IFNβ may drive HIV-1 pathogenesis in the gut., Author summary The Type I Interferons (IFN-Is) serve as the first line of defense against viral infections. IFN-Is are evolutionarily diverse, with 12 distinct IFNα subtypes and IFNβ in humans. All IFN-Is bind to the same receptor, but it remains unclear whether distinct IFN-Is will trigger the same set of IFN-stimulated genes. Here, we provide evidence that diverse IFN-Is altered gene expression in gut CD4 T cells in different ways. Specifically, IFNβ induced a broader gene expression profile than individual IFNα subtypes. Genes uniquely induced by IFNβ in gut CD4 T cells ex vivo were downregulated in the gut during chronic HIV-1 infection. This downregulation correlated with markers of inflammation and immune dysfunction. Our data unravel qualitative differences between the IFN-Is and suggest a complex picture of how IFNβ may be driving HIV-1 pathogenesis in the gastrointestinal tract.

Although all 12 subtypes of human interferon alpha (IFN-α) bind the same receptor, recent results have demonstrated that they elicit unique host responses and display distinct efficacies in the control of different viral infections. The IFN-α2 subtype is currently in HIV-1 clinical trials, but it has not consistently reduced viral loads in HIV-1 patients and is not the most effective subtype against HIV-1 in vitro . We now demonstrate in humanized mice that, when delivered at the same high clinical dose, the human IFN-α14 subtype has very potent anti-HIV-1 activity whereas IFN-α2 does not. In both postexposure prophylaxis and treatment of acute infections, IFN-α14, but not IFN-α2, significantly suppressed HIV-1 replication and proviral loads. Furthermore, HIV-1-induced immune hyperactivation, which is a prognosticator of disease progression, was reduced by IFN-α14 but not IFN-α2. Whereas ineffective IFN-α2 therapy was associated with CD8 + T cell activation, successful IFN-α14 therapy was associated with increased intrinsic and innate immunity, including significantly higher induction of tetherin and MX2, increased APOBEC3G signature mutations in HIV-1 proviral DNA, and higher frequencies of TRAIL + NK cells. These results identify IFN-α14 as a potent new therapeutic that operates via mechanisms distinct from those of antiretroviral drugs. The ability of IFN-α14 to reduce both viremia and proviral loads in vivo suggests that it has strong potential as a component of a cure strategy for HIV-1 infections. The broad implication of these results is that the antiviral efficacy of each individual IFN-α subtype should be evaluated against the specific virus being treated. IMPORTANCE The naturally occurring antiviral protein IFN-α2 is used to treat hepatitis viruses but has proven rather ineffective against HIV in comparison to triple therapy with the antiretroviral (ARV) drugs. Although ARVs suppress the replication of HIV, they fail to completely clear infections. Since IFN-α acts by different mechanism than ARVs and has been shown to reduce HIV proviral loads, clinical trials are under way to test whether IFN-α2 combined with ARVs might eradicate HIV-1 infections. IFN-α is actually a family of 12 distinct proteins, and each IFN-α subtype has different efficacies toward different viruses. Here, we use mice that contain a human immune system, so they can be infected with HIV. With this model, we demonstrate that while IFN-α2 is only weakly effective against HIV, IFN-α14 is extremely potent. This discovery identifies IFN-α14 as a more powerful IFN-α subtype for use in combination therapy trials aimed toward an HIV cure.

HIV-1-associated disruption of intestinal homeostasis is a major factor contributing to chronic immune activation and inflammation. Dendritic cells (DCs) are crucial in maintaining intestinal homeostasis, but the impact of HIV-1 infection on intestinal DC number and function has not been extensively studied. We compared the frequency and activation/maturation status of colonic myeloid DC (mDC) subsets (CD1c(+) and CD1c(neg)) and plasmacytoid DCs in untreated HIV-1-infected subjects with uninfected controls. Colonic mDCs in HIV-1-infected subjects had increased CD40 but decreased CD83 expression, and CD40 expression on CD1c(+) mDCs positively correlated with mucosal HIV-1 viral load, with mucosal and systemic cytokine production, and with frequencies of activated colon and blood T cells. Percentage of CD83(+)CD1c(+) mDCs negatively correlated with frequencies of interferon-γ-producing colon CD4(+) and CD8(+) T cells. CD40 expression on CD1c(+) mDCs positively associated with abundance of high prevalence mucosal Prevotella copri and Prevotella stercorea but negatively associated with a number of low prevalence mucosal species, including Rumminococcus bromii. CD1c(+) mDC cytokine production was greater in response to in vitro stimulation with Prevotella species relative to R. bromii. These findings suggest that, during HIV infection, colonic mDCs become activated upon exposure to mucosal pathobiont bacteria leading to mucosal and systemic immune activation.

Global transcriptome studies can help pinpoint key cellular pathways exploited by viruses to replicate and cause pathogenesis. Previous data showed that laboratory-adapted HIV-1 triggers significant gene expression changes in CD4+ T cell lines and mitogen-activated CD4+ T cells from peripheral blood. However, HIV-1 primarily targets mucosal compartments during acute infection in vivo. Moreover, early HIV-1 infection causes extensive depletion of CD4+ T cells in the gastrointestinal tract that herald persistent inflammation due to the translocation of enteric microbes to the systemic circulation. Here, we profiled the transcriptome of primary intestinal CD4+ T cells infected ex vivo with transmitted/founder (TF) HIV-1. Infections were performed in the presence or absence of Prevotella stercorea, a gut microbe enriched in the mucosa of HIV-1-infected individuals that enhanced both TF HIV-1 replication and CD4+ T cell death ex vivo. In the absence of bacteria, HIV-1 triggered a cellular shutdown response involving the downregulation of HIV-1 reactome genes, while perturbing genes linked to OX40, PPAR and FOXO3 signaling. However, in the presence of bacteria, HIV-1 did not perturb these gene sets or pathways. Instead, HIV-1 enhanced granzyme expression and Th17 cell function, inhibited G1/S cell cycle checkpoint genes and triggered downstream cell death pathways in microbe-exposed gut CD4+ T cells. To gain insights on these differential effects, we profiled the gene expression landscape of HIV-1-uninfected gut CD4+ T cells exposed to bacteria. Microbial exposure upregulated genes involved in cellular proliferation, MAPK activation, Th17 cell differentiation and type I interferon signaling. Our findings reveal that microbial exposure influenced how HIV-1 altered the gut CD4+ T cell transcriptome, with potential consequences for HIV-1 susceptibility, cell survival and inflammation. The HIV-1- and microbe-altered pathways unraveled here may serve as a molecular blueprint to gain basic insights in mucosal HIV-1 pathogenesis., Author summary The gastrointestinal (GI) tract is a major site of early HIV-1 replication and death of CD4+ T cells. As HIV-1 replicates in the gut, the protective epithelial barrier gets disrupted, causing the entry of bacteria into the underlying tissue and the bloodstream, leading to inflammation and clinical complications even in HIV-1-infected patients taking antiviral drugs. Counteracting these pathogenic processes may require in-depth understanding of the molecular pathways that HIV-1 and microbes utilize to infect, functionally alter and/or kill CD4+ T cells. However, to date, the nature of the genes altered by transmitted/founder HIV-1 strains and gut bacteria in intestinal CD4+ T cells remains unclear. Here, we obtained the first gene expression profiles of primary gut CD4+ T cells infected in cell culture with HIV-1 in the context of microbes found in the GI tract of HIV-1 infected patients. Our findings reveal common and distinct signaling pathways altered by HIV-1 depending on the presence of microbes that may shed light on infection, inflammation and CD4+ T cell depletion in HIV-1 infected individuals. More detailed studies of these molecular programs may inform potential ways to counteract pathogenic outcomes initiated and/or sustained by HIV-1 infection in the GI tract.

The absence of immunoglobulin A (IgA) in the intestinal tract renders young infants highly susceptible to enteric infections. However, mediators of initial IgA induction in this population are undefined. We determined the temporal acquisition of plasma cells by isotype and expression of T cell-independent (TI) and -dependent (TD) IgA class switch factors in the human intestinal tract during early infancy. We found that IgA plasma cells were largely absent in the infant intestine until after 1 month of age, approaching adult densities later in infancy than both IgM and IgG. The restricted development of IgA plasma cells in the first month was accompanied by reduced expression of the TI factor a proliferation-inducing ligand (APRIL) and its receptors TACI (transmembrane activator and calcium-modulator and cyclophilin ligand interactor) and B cell maturation antigen (BCMA) within isolated lymphoid follicles (ILFs). Moreover, both APRIL and BCMA expression strongly correlated with increasing IgA plasma cell densities over time. Conversely, TD mediators (CD40 ligand (CD40L) and CD40) were expressed within ILFs before 1 month and were not associated with IgA plasma cell generation. In addition, preterm infants had lower densities of IgA plasma cells and reduced APRIL expression compared with full-term infants. Thus, blunted TI responses may contribute to the delayed induction of intestinal IgA during early human infancy.

Objective Natural killer (NK) cells are important in innate immune responses to bacterial and viral pathogens. HIV-1 infection is associated with opportunistic bacterial infections and with microbial translocation, but the nature of the NK cell response to bacteria during HIV-1 infection has not been studied extensively. The objective of this study was to compare NK cell responses to bacteria in HIV-1-infected versus that in uninfected individuals. Methods Multicolor flow cytometry was used to evaluate the ability of blood NK cell subsets (CD56CD16, CD56CD16, and CD56CD16) from treated, virally suppressed, and untreated viremic subjects with chronic HIV-1 infection and uninfected controls, to secrete interferon gamma (IFN-γ) in response to the in vitro stimulation of peripheral blood mononuclear cells with heat-killed commensal Escherichia coli or pathogenic Salmonella typhimurium. Results All 3 NK cell subsets produced IFN-γ in response to bacteria, but CD56CD16 NK cells were least responsive. Untreated HIV-1-infected donors had increased frequencies of CD56CD16 NK cells and lower overall frequencies of IFN-γ-producing NK cells responding to E. coli and S. typhimurium than did NK cells from uninfected donors. These NK cell defects were not fully restored in antiretroviral therapy-treated donors. Monocytes were necessary for NK cells to respond to bacteria, but the HIV-associated defect was intrinsic to NK cells because the addition of normal monocytes did not restore IFN-γ production in response to bacteria. Conclusions Functional defects and numeric alterations of NK cell subsets lead to decreased frequencies of bacteria-reactive, IFN-γ-producing NK cells in HIV-1-infected subjects, even those on antiretroviral therapy.

HIV-1 infection is associated with substantial damage to the gastrointestinal (GI) tract resulting in structural impairment of the epithelial barrier and a disruption of intestinal homeostasis. The accompanying translocation of microbial products and potentially microbes themselves from the lumen into systemic circulation has been linked to immune activation, inflammation, and HIV-1 disease progression. The importance of microbial translocation in the setting of HIV-1 infection has led to a recent focus on understanding how the communities of microbes that make up the intestinal microbiome are altered during HIV-1 infection and how they interact with mucosal immune cells to contribute to inflammation. This review details the dysbiotic intestinal communities associated with HIV-1 infection and their potential link to HIV-1 pathogenesis. We detail studies that begin to address the mechanisms driving microbiota-associated immune activation and inflammation and the various treatment strategies aimed at correcting dysbiosis and improving the overall health of HIV-1 infected individuals. Finally, we discuss how this relatively new field of research can advance to provide a more comprehensive understanding of the contribution of the gut microbiome to HIV-1 pathogenesis.

The components of the human gut microbiome have been found to influence a broad array of pathologic conditions ranging from heart disease to diabetes and even to cancer. HIV infection upsets the delicate balance in the normal host-microbe interaction both through alterations in the taxonomic composition of gut microbial communities as well as through disruption of the normal host response mechanisms. In this article we review the current methods of gut microbiome analysis and the resulting data regarding how HIV infection might change the balance of commensal bacteria in the gut. Additionally, we cover the various effects gut microbes have on host immune homeostasis and the preliminary but intriguing data on how HIV disrupts those mechanisms. Finally, we briefly describe some of the important biomolecules produced by gut microbiota and the role that they may play in maintaining host immune homeostasis with and without HIV infection.

Increased chronic immune activation and inflammation are hallmarks of HIV/SIV infection and are highly correlated with progression to AIDS and development of non-AIDS comorbidities, such as hypercoagulability and cardiovascular disease. Intestinal dysfunction resulting in microbial translocation has been proposed as a lead cause of systemic immune activation and hypercoagulability in HIV/SIV infection. Our goal was to assess the biological and clinical impact of a therapeutic strategy designed to reduce microbial translocation through reduction of the microbial content of the intestine (Rifaximin-RFX) and of gut inflammation (Sulfasalazine-SFZ). RFX is an intraluminal antibiotic that was successfully used in patients with hepatic encephalopathy. SFZ is an antiinflammatory drug successfully used in patients with mild to moderate inflammatory bowel disease. Both these clinical conditions are associated with increased microbial translocation, similar to HIV-infected patients. Treatment was administered for 90 days to five acutely SIV-infected pigtailed macaques (PTMs) starting at the time of infection; seven untreated SIVsab-infected PTMs were used as controls. RFX+SFZ were also administered for 90 days to three chronically SIVsab-infected PTMs. RFX+SFZ administration during acute SIVsab infection of PTMs resulted in: significantly lower microbial translocation, lower systemic immune activation, lower viral replication, better preservation of mucosal CD4+ T cells and significantly lower levels of hypercoagulation biomarkers. This effect was clear during the first 40 days of treatment and was lost during the last stages of treatment. Administration of RFX+SFZ to chronically SIVsab–infected PTMs had no discernible effect on infection. Our data thus indicate that early RFX+SFZ administration transiently improves the natural history of acute and postacute SIV infection, but has no effect during chronic infection., Author Summary We report that administration of the intraluminal antibiotic Rifaximin and the gut-focused anti-inflammatory drug Sulfasalazine to acutely SIV-infected pigtailed macaques is associated with a transient disruption of the vicious circle of inflammation-microbial translocation-immune activation which is pathognomonic to pathogenic HIV/SIV infection and drives HIV disease progression and non-AIDS comorbidities in HIV-infected patients. This therapeutic approach resulted in transient lower microbial translocation, lower systemic immune activation, lower viral replication, better preservation of mucosal CD4+ T cells and lower levels of hypercoagulation biomarkers throughout acute SIV infection. Our results thus support the use of therapeutic approaches to reduce microbial translocation, improve the clinical outcome of HIV-infected patients receiving antiretroviral therapy and prevent non-AIDS comorbidities. Our results also reinforce the importance of early therapeutic management of HIV infection.

HIV infection is associated with increased risk of cardiovascular complications, the underlying mechanism of which remains unclear. Plasma levels of the coagulation biomarker D-dimer (DD) correlate with increased mortality and cardiovascular events in HIV-infected patients. We compared the incidence of cardiovascular lesions and the levels of the coagulation markers DD and thrombin antithrombin in pathogenic SIV infections of rhesus and pigtailed macaques (PTMs) and in nonpathogenic SIV infection of African green monkeys (AGMs) and sooty mangabeys. Hypercoagulability and cardiovascular pathology were only observed in pathogenic SIV infections. In PTMs infected with SIV from AGMs (SIVagm), DD levels were highly indicative of AIDS progression and increased mortality and were associated with cardiovascular lesions, pointing to SIVagm-infected PTMs as an ideal animal model for the study of HIV-associated cardiovascular disease. In pathogenic SIV infection, DD increased early after infection, was strongly correlated with markers of immune activation/inflammation and microbial translocation (MT), and was only peripherally associated with viral loads. Endotoxin administration to SIVagm-infected AGMs (which lack chronic SIV-induced MT and immune activation) resulted in significant increases of DD. Our results demonstrate that hypercoagulation and cardiovascular pathology are at least in part a consequence of excessive immune activation and MT in SIV infection.

Microbial translocation has been linked to systemic immune activation in HIV-1 disease, yet mechanisms by which microbes may contribute to HIV-associated intestinal pathogenesis are poorly understood. Importantly, our understanding of the impact of translocating commensal intestinal bacteria on mucosal-associated T cell responses in the context of ongoing viral replication that occurs early in HIV-1 infection is limited. We previously identified commensal Escherichia coli-reactive Th1 and Th17 cells in normal human intestinal lamina propria (LP). In this article, we established an ex vivo assay to investigate the interactions between Th cell subsets in primary human LP mononuclear cells (LPMCs), commensal E. coli, and CCR5-tropic HIV-1Bal. Addition of heat-killed E. coli to HIV-1–exposed LPMCs resulted in increases in HIV-1 replication, CD4 T cell activation and infection, and IL-17 and IFN-γ production. Conversely, purified LPS derived from commensal E. coli did not enhance CD4 T cell infection. E. coli exposure induced greater proliferation of LPMC Th17 than Th1 cells. Th17 cells were more permissive to infection than Th1 cells in HIV-1–exposed LPMC cultures, and Th17 cell infection frequencies significantly increased in the presence of E. coli. The E. coli-associated enhancement of infection was dependent on the presence of CD11c+ LP dendritic cells and, in part, on MHC class II-restricted Ag presentation. These results highlight a potential role for translocating microbes in impacting mucosal HIV-1 pathogenesis during early infection by increasing HIV-1 replication and infection of intestinal Th1 and Th17 cells.

Reduced frequencies of myeloid and plasmacytoid dendritic cell (DC) subsets (mDCs and pDCs, respectively) have been observed in the peripheral blood of HIV-1-infected individuals throughout the course of disease. Accumulation of DCs in lymph nodes (LNs) may partly account for the decreased numbers observed in blood, but increased DC death may also be a contributing factor. We used multiparameter flow cytometry to evaluate pro- and antiapoptotic markers in blood mDCs and pDCs from untreated HIV-1-infected donors, from a subset of infected donors before and after receiving antiretroviral therapy (ART), and from uninfected control donors. Blood mDCs, but not pDCs, from untreated HIV-1-infected donors expressed lower levels of antiapoptotic Bcl-2 than DCs from uninfected donors. A subset of HIV-1-infected donors had elevated frequencies of proapoptotic caspase-3 + blood mDCs, and positive correlations were observed between caspase-3 + mDC frequencies and plasma viral load and CD8 + T-cell activation levels. Caspase-3 + mDC frequencies, but not mDC Bcl-2 expression, were reduced with viral suppression on ART. Apoptosis markers on DCs in blood and LN samples from a cohort of untreated, HIV-1-infected donors with chronic disease were also evaluated. LN mDCs displayed higher levels of Bcl-2 and lower caspase-3 + frequencies than did matched blood mDCs. Conversely, LN pDCs expressed lower Bcl-2 levels than their blood counterparts. In summary, blood mDCs from untreated HIV-1-infected subjects displayed a proapoptotic profile that was partially reversed with viral suppression, suggesting that DC death may be a factor contributing to blood DC depletion in the setting of chronic, untreated HIV disease.

Elevated expression of inhibitory receptors on virus-specific T cells has been implicated as a mechanism by which viruses evade host immune surveillance. Blockade of these pathways during chronic infection leads to increased T cell function and improved immune control of viral replication. To explore the association between costimulatory receptors and HIV replication, we examined the expression of programmed death 1 (PD-1), CTLA-4, T cell Ig domain and mucin domain 3 (TIM-3), and CD28 on HIV-specific CD4+ T cells from HIV-infected subjects. Greater than 30% of HIV-specific CD4+ T cells from untreated subjects coexpressed PD-1, CTLA-4, and TIM-3, whereas

Intestinal dendritic cells (DCs) play key roles in mediating tolerance to commensal flora and inflammatory responses against mucosal pathogens. The mechanisms by which intestinal “conditioning” influences human DC responses to microbial stimuli remain poorly understood. Infections with viruses, such as HIV-1, that target mucosal tissue result in intestinal epithelial barrier breakdown and increased translocation of commensal bacteria into the lamina propria (LP). It is unclear whether innate LP DC responses to concurrent viral and bacterial stimuli influence mucosal HIV-1 pathogenesis. In this study, direct ex vivo phenotype and in vitro constitutive cytokine production of CD1c+ DCs in human intestinal LP were compared with those in peripheral blood (PB). To evaluate innate responses to viral and bacterial stimuli, intracellular cytokine production by LP and PB DCs following stimulation with ligands for TLRs 2, 4, 5, and 7/8 was evaluated. At steady state, LP CD1c+ DCs expressed higher levels of activation markers (CD40, CD83, CD86, HLA-DR, and CCR7) than did PB CD1c+ DCs, and higher frequencies of LP CD1c+ DCs constitutively produced IL-6 and -10 and TNF-α. LP DCs had blunted cytokine responses to TLR4 ligand and TLR5 ligand stimulation relative to PB DCs, yet similarly produced IL-10 in response to TLR2 ligand. Only synthetic TLR7/8 ligand, a mimic of viral ssRNA, induced IL-23 production by LP CD1c+ DCs, and this proinflammatory cytokine response was synergistically enhanced following combined TLR7/8 and TLR4 stimulation. These findings highlight a potential mechanism by which viruses like HIV-1 may subvert homeostatic mechanisms and induce inflammation in the intestinal mucosa.

Background Melanoma often elicits a profound immune response, and this response has been exploited by various immune therapies. These immunotherapies ultimately fail, however, and advanced melanoma is uniformly fatal, suggesting the development of an immune escape mechanism. In this study, markers of immune escape including regulatory T cells (Tregs), dendritic cells (DCs), and TGF-β were evaluated in 14 Stage IV melanoma patients and correlated with survival. Materials and methods Peripheral blood mononuclear cells were isolated from Stage IV melanoma patients and analyzed for Tregs and DCs by flow cytometry using fluorescent CD3, CD4, CD25, Lin, HLA-DR, CD11c, and CD123 antibodies. Serum TGF-β levels were evaluated by ELISA from these patients. Clinical data were extracted from the patients' medical records. Results Stage IV melanoma patients with shorter survival (less than 24 mo) had a significantly higher proportion of Tregs than those with longer survival (15% versus 8%, respectively, P = 0.004). The numbers of DCs and the serum TGF-β levels were not significantly different in these two groups. There was an inverse relationship between the percentage of Tregs and survival, although this did not reach statistical significance (r = −0.35, P = 0.22). There was also an inverse relationship between peripheral Tregs and DCs. When patients were divided into groups of greater than or less than 7% Tregs, the number of total DCs was higher in the patients with fewer Tregs than in those with more Tregs, but this did not reach statistical significance (16,535 versus 12,126 total DCs/mL, P = 0.52). Conclusions In Stage IV melanoma patients, a high percentage of Tregs appears to be associated with shorter survival. The inverse relationship of the number of DCs and Tregs in these patients may provide an insight to the origin of this observation.

Reactivity of lamina propria (LP) T cells to commensal bacteria has been demonstrated in animal models of inflammatory bowel disease (IBD) and in humans with IBD, but few studies have evaluated the function of such cells in normal individuals. LP mononuclear cells (LPMC) were disaggregated from healthy human intestinal tissue and cultured with heat-killed commensal and pathogenic bacteria. CD3(+)CD4(+) IFN-gamma-producing (Th1) cells reactive to commensal bacteria were demonstrated at frequencies ranging from 0.05 to 2.28% in LPMC. Bacteria-specific Th1 responses were inhibited by anti-HLA-DR antibodies and chloroquine exposure, were enriched in LP relative to peripheral blood, and expressed effector memory cell markers. Bacteria-specific CD4(+) T cell proliferation in vitro was dependent on the presence of LP dendritic cells (DCs), which produced pro-inflammatory cytokines upon bacterial exposure. These results suggest that bacteria-reactive DCs and CD4(+) T cells in normal LP have substantial pro-inflammatory potential that is revealed upon disaggregation in vitro.

Objective: CD4 + CD8 + double-positive (DP) T cells represent a poorly characterized population of effector T cells found at low frequencies in the peripheral blood. Virus-specific DP T cells have been identified in HIV-1-infected patients but their origin, relationship to conventional CD4 + and CD8 + single-positive (SP) T cells, and role in disease pathogenesis are unclear. Methods: In this study, peripheral blood T cells were analyzed for cytokine production, maturation, and cytolytic marker expression by polychromatic flow cytometry in subjects with both early (n = 27) and chronic (n = 21) HIV-1 infection. Results and Conclusions: HIV-1-specific interferon gamma (IFN-γ)-producing DP T cells were identified at a median frequency of 0.48% compared with 1.08% and 0.02% for CD8 and CD4 SP cells, respectively, in response to pooled HIV-1 peptides. HIV-1-specific DP T cells exhibited polyfunctionality with characteristics of both CD4 and CD8 SP T cells, including coproduction of IFN-γ and IL-2 and expression of cytolytic-associated lysosomal-associated membrane protein. No differences in frequencies of unstimulated DP T cells were observed in early compared with chronic infection. However, chronic infection was associated with higher frequencies of HIV-specific, IFN-γ-producing DP T cells and higher fractions of effector memory and lysosomal-associated membrane protein expression among these cells, suggesting an effect of cumulative viral antigen burden on DP T-cell function.

Background: Dendritic cell (DC) defects may contribute to chronicity in hepatitis C virus (HCV) infection and determine response to PEG–interferon and ribavirin therapy via poor T cell stimulation. Studies to date have produced inconsistent results regarding DC maturation and function: no large study has examined DCs before and after therapy. Aims: We examined if DC defects in maturation and chemotaxis are present by comparing therapeutic responders to non-responders. Methods: We analysed peripheral DCs of 64 HCV genotype 1-infected patients from the Virahep-C study 2 weeks before and 24 weeks after therapy. We used flow cytometry to enumerate plasmacytoid DC (pDC) and myeloid DCs (mDC) and quantify expression of chemokine receptors and maturation markers. Chemotaxis was measured with an in vitro assay. Results: Pre-treatment frequencies of pDCs and mDCs were significantly lower in HCV patients than controls and successful therapy normalised pDCs. Levels of CXCR3 and CXCR4 on pDCs were higher at baseline compared to normal controls and decreased with therapy. Pre-therapy levels of co-stimulatory marker CD40 and the maturation marker CD83 were higher in pDCs of patients chronically infected with HCV compared to normal patients, and levels of both markers dropped significantly with therapy in the SVR+ group only. Other maturation markers (CD86 and CCR7) were not elevated suggesting a partially activated phenotype. Baseline chemotaxis of pDCs to CXCL12 and CXCL10 predicted failure of antiviral response and correlated with the histological activity index inflammation score. Conclusions: Plasmacytoid DC defects exist in chronic HCV and successful antiviral therapy normalises many phenotypic and functional abnormalities.

Dendritic cells (DCs) from HIV-1-infected individuals display numeric and functional defects, and recent evidence suggests that HIV-1 can directly and indirectly activate DCs in vitro. The in vivo activation state and compartmentalization of DC subsets during HIV-1 infection remain poorly understood, however. We evaluated phenotypic and functional characteristics of myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) directly ex vivo in peripheral blood and lymphoid tissue from HIV-1-infected and HIV-seronegative individuals. Analysis of a wide range of chemokine receptors and activation/maturation markers on circulating DCs from viremic HIV-1-infected donors revealed a phenotype indicative of partial activation. Yet, blood DCs from viremic subjects still achieved full maturation when stimulated in vitro. In addition, blood pDCs from viremic individuals had a reduced capacity to migrate to CXCL12 in vitro. Total numbers of both DC subsets were increased in lymph nodes of asymptomatic untreated HIV-1-infected subjects, consistent with DC accumulation in the lymphoid compartment. Lymph node DCs also expressed high levels of CD40 in the absence of increases of other typical activation/maturation markers. Activation and depletion of DCs in blood with accumulation in lymphoid tissue may contribute to HIV-associated chronic immune activation and T-cell dysfunction.

Background Early HIV-1 infection is characterized by high levels of HIV-1 replication and substantial CD4 T cell depletion in the intestinal mucosa, intestinal epithelial barrier breakdown, and microbial translocation. HIV-1-induced disruption of intestinal homeostasis has also been associated with changes in the intestinal microbiome that are linked to mucosal and systemic immune activation. In this study, we investigated the impact of representative bacterial species that were altered in the colonic mucosa of viremic HIV-1 infected individuals (HIV-altered mucosal bacteria; HAMB) on intestinal CD4 T cell function, infection by HIV-1, and survival in vitro. Lamina propria (LP) mononuclear cells were infected with CCR5-tropic HIV-1BaL or mock infected, exposed to high (3 gram-negative) or low (2 gram-positive) abundance HAMB or control gram-negative Escherichia coli and levels of productive HIV-1 infection and CD4 T cell depletion assessed. HAMB-associated changes in LP CD4 T cell activation, proliferation and HIV-1 co-receptor expression were also evaluated. Results The majority of HAMB increased HIV-1 infection and depletion of LP CD4 T cells, but gram-negative HAMB enhanced CD4 T cell infection to a greater degree than gram-positive HAMB. Most gram-negative HAMB enhanced T cell infection to levels similar to that induced by gram-negative E. coli despite lower induction of T cell activation and proliferation by HAMB. Both gram-negative HAMB and E. coli significantly increased expression of HIV-1 co-receptor CCR5 on LP CD4 T cells. Lipopolysaccharide, a gram-negative bacteria cell wall component, up-regulated CCR5 expression on LP CD4 T cells whereas gram-positive cell wall lipoteichoic acid did not. Upregulation of CCR5 by gram-negative HAMB was largely abrogated in CD4 T cell-enriched cultures suggesting an indirect mode of stimulation. Conclusions Gram-negative commensal bacteria that are altered in abundance in the colonic mucosa of HIV-1 infected individuals have the capacity to enhance CCR5-tropic HIV-1 productive infection and depletion of LP CD4 T cells in vitro. Enhanced infection appears to be primarily mediated indirectly through increased expression of CCR5 on LP CD4 T cells without concomitant large scale T cell activation. This represents a novel mechanism potentially linking intestinal dysbiosis to HIV-1 mucosal pathogenesis. Electronic supplementary material The online version of this article (doi:10.1186/s12977-016-0237-1) contains supplementary material, which is available to authorized users.

HIV-1 is transmitted primarily across mucosal surfaces and rapidly spreads within the intestinal mucosa during acute infection. The type I interferons (IFNs) likely serve as a first line of defense, but the relative expression and antiviral properties of the 12 IFNα subtypes against HIV-1 infection of mucosal tissues remain unknown. Here, we evaluated the expression of all IFNα subtypes in HIV-1-exposed plasmacytoid dendritic cells by next-generation sequencing. We then determined the relative antiviral potency of each IFNα subtype ex vivo using the human intestinal Lamina Propria Aggregate Culture model. IFNα subtype transcripts from the centromeric half of the IFNA gene complex were highly expressed in pDCs following HIV-1 exposure. There was an inverse relationship between IFNA subtype expression and potency. IFNα8, IFNα6 and IFNα14 were the most potent in restricting HIV-1 infection. IFNα2, the clinically-approved subtype, and IFNα1 were both highly expressed but exhibited relatively weak antiviral activity. The relative potencies correlated with binding affinity to the type I IFN receptor and the induction levels of HIV-1 restriction factors Mx2 and Tetherin/BST-2 but not APOBEC3G, F and D. However, despite the lack of APOBEC3 transcriptional induction, the higher relative potency of IFNα8 and IFNα14 correlated with stronger inhibition of virion infectivity, which is linked to deaminase-independent APOBEC3 restriction activity. By contrast, both potent (IFNα8) and weak (IFNα1) subtypes significantly induced HIV-1 GG-to-AG hypermutation. The results unravel non-redundant functions of the IFNα subtypes against HIV-1 infection, with strong implications for HIV-1 mucosal immunity, viral evolution and IFNα-based functional cure strategies., Author Summary The therapeutic potential of recombinant IFNα against HIV-1 infection has been explored for 25 years, but its effectiveness was inconsistent. However, these clinical trials administered IFNα2, which is only one member of a 12-protein family of IFNα subtypes. More recently, IFNα was found to activate ‘restriction factors’–proteins that can directly inhibit HIV-1. To date, it remains unknown which IFNα subtypes are produced by professional IFNα producing cells known as plasmacytoid dendritic cells and which IFNα subtypes are more effective in inhibiting HIV-1 infection in the gastrointestinal tract, the primary site of early HIV-1 replication. Here, we show that weaker IFNα subtypes were more highly expressed following HIV-1 infection. Using an infection platform that captures important characteristics of early HIV-1 infection in the gut, several IFNα subtypes were found to be more effective at inhibiting HIV-1 than IFNα2. In particular, IFNα8 and IFNα14 more potently reduced the infectivity of HIV-1 virions, an activity that can be attributed to the APOBEC3 proteins. Our findings strongly support the evaluation of potent IFNα subtypes in currently evolving HIV-1 curative strategies.

Objectives: To determine whether increased sequence variation occurs in regions of endogenous HIV-1 targeted by HIV-1-specific CD4 + T cells. The presence of increased variation would be suggestive of immune evasion by HIV-1. Design: We performed a cross-sectional study of untreated HIV-1-infected subjects measuring HIV-1-specific interferon (IFN)-γ-secreting CD4 + T-cell responses against epitopes in Gag p17 and p24 and concurrent endogenous plasma HIV-1 RNA epitope sequence variation. Methods: CD8- depleted IFNγ enzyme-linked immunospot assays were used to identify regions of HIV-1 Gag recognized by CD4 + T cells. Reverse transcriptase polymerase chain reaction and TA cloning were used to sequence endogenous plasma HIV-1 virus and identify variants. Results: CD4 + T-cell epitopes in Gag p17 and p24 were identified in 5 individuals, and concurrent sequence information on endogenous HIV-1 was obtained in 4 of these individuals. Endogenous plasma HIV-1 RNA sequencing revealed no intrapatient amino acid sequence variation through identified epitopes. Conclusions: In these chronically infected viremic subjects, circulating IFNγ-secreting CD4 + T-cell responses were directed against epitope sequences found in the predominant strain of endogenous circulating plasma HIV-1, suggesting that escape from CD4 + T-cell responses is not a common process in vivo.

HIV-1 replication is associated with reduced or absent HIV-1-specific CD4+ T cell proliferation and skewing of HIV-1-specific CD4+ T cells toward an IFN-γ-producing, CCR7− phenotype. The CCR7− T cell population is heterogeneous and can be subdivided based on the expression of CD57. Although CD57 expression on CD8+ T cells is associated with proliferation incompetence and replicative senescence, less is known about the function of CD57-expressing CD4+ T cells. In this study, the frequency, phenotype, and function of CD57+CD4+ T cells were evaluated in 25 HIV-1-infected subjects and 10 seronegative controls. CD57+CD4+ T cells were found to be proliferation incompetent, even after strong mitogen stimulation. Percentages of CD4+ T cells that expressed CD57 were significantly higher in untreated HIV-1-infected subjects than in HIV-1-seronegative donors, and CD57 expression did not normalize in subjects receiving at least 6 mo of effective antiretroviral therapy. CD57 was predominately expressed on the CCR7− fraction of the CD4+ T cell compartment and accounted for the majority of cells in the CCR7−CD45RA+ population from untreated HIV-1-infected subjects. HIV-1-specific CD4+ T cells producing only IFN-γ had the highest expression of CD57, whereas few cells producing IL-2 alone expressed CD57. These findings further define a novel population of proliferation-incompetent CD4+ T cells that are generated in the presence of chronic Ag exposure. A better understanding of the generation and persistence of CD57+ T cells in HIV-1 infection could provide important insights into the immunopathogenesis of this disease.

Beryllium exposure can lead to the development of beryllium-specific CD4+ T cells and chronic beryllium disease (CBD), which is characterized by the presence of lung granulomas and a CD4+ T cell alveolitis. Studies have documented the presence of proliferating and cytokine-secreting CD4+ T cells in blood of CBD patients after beryllium stimulation. However, some patients were noted to have cytokine-secreting CD4 T cells in blood in the absence of beryllium-induced proliferation, and overall, the correlation between the 2 types of responses was poor. We hypothesized that the relative proportion of memory T cell subsets determined antigen-specific proliferation. In most CBD patients, the majority of beryllium-specific CD4+ T cells in blood expressed an effector memory T cell maturation phenotype. However, the ability of blood cells to proliferate in the presence of beryllium strongly correlated with the fraction expressing a central memory T cell phenotype. In addition, we found a direct correlation between the percentage of beryllium-specific CD4+ T(EM) cells in blood and T cell lymphocytosis in the lung. Together, these findings indicate that the functional capability of antigen-specific CD4+ T cells is determined by the relative proportion of memory T cell subsets, which may reflect internal organ involvement.

Diminished in vitro proliferation of human immunodeficiency virus type 1 (HIV-1)-specific CD4 + T cells has been associated with HIV-1 viremia and declining CD4 + T-cell counts during chronic infection. To better understand this phenomenon, we examined whether HIV-1 Gag p24 antigen-induced CD4 + T-cell proliferation might recover in vitro in a group of subjects with chronic HIV-1 viremia and no history of antiretroviral therapy (ART). We found that depletion of CD8 + cells from peripheral blood mononuclear cells (PBMC) before antigen stimulation was associated with a 6.5-fold increase in the median p24-induced CD4 + T-cell proliferative response and a 57% increase in the number of subjects with positive responses. These p24-induced CD4 + T-cell proliferative responses from CD8-depleted PBMC were associated with expansion of the numbers of p24-specific, gamma interferon (IFN-γ)-producing CD4 + T cells. Among the 20 viremic, treatment-naïve subjects studied, the only 5 subjects lacking proliferation-competent, p24-specific CD4 + T-cell responses from CD8-depleted PBMC showed plasma HIV-1 RNA levels > 100,000 copies/ml. Furthermore, both the magnitude of p24-induced CD4 + T-cell proliferative responses from CD8-depleted PBMC and the frequency of p24-specific, IFN-γ-producing CD4 + T cells expanded from CD8-depleted PBMC were associated inversely with plasma HIV-1 RNA levels. Therefore, proliferation-competent, HIV-1-specific CD4 + T cells that might help control HIV-1 disease may persist during chronic, progressive HIV-1 disease except at very high levels of in vivo HIV-1 replication.

Recognition by CD8+ T lymphocytes (CTL) of epitopes that are derived from conserved gene products, such as Gag and Pol, is well documented and conceptually supports the development of epitope-based vaccines for use against diverse HIV-1 subtypes. However, many CTL epitopes from highly conserved regions within the HIV-1 genome are highly variable, when assessed by comparison of amino acid sequences. The TCR is somewhat promiscuous with respect to peptide binding, and, as such, CTL can often recognize related epitopes. In these studies, we evaluated CTL recognition of five sets of variant HIV-1 epitopes restricted to HLA-A*0201 and HLA-A*1101 using HLA transgenic mice. We found that numerous different amino acid substitutions can be introduced into epitopes without abrogating their recognition by CTL. Based on our findings, we constructed an algorithm to predict those CTL epitopes capable of inducing responses in the HLA transgenic mice to the greatest numbers of variant epitopes. Similarity of CTL specificity for variant epitopes was demonstrated for humans using PBMC from HIV-1-infected individuals and CTL lines produced in vitro using PBMC from HIV-1-uninfected donors. We believe the ability to predict CTL epitope immunogenicity and recognition patterns of variant epitopes can be useful for designing vaccines against multiple subtypes and circulating recombinant forms of HIV-1.

Epitope-based vaccines designed to induce CTL responses specific for HIV-1 are being developed as a means for addressing vaccine potency and viral heterogeneity. We identified a set of 21 HLA-A2, HLA-A3, and HLA-B7 restricted supertype epitopes from conserved regions of HIV-1 to develop such a vaccine. Based on peptide-binding studies and phenotypic frequencies of HLA-A2, HLA-A3, and HLA-B7 allelic variants, these epitopes are predicted to be immunogenic in greater than 85% of individuals. Immunological recognition of all but one of the vaccine candidate epitopes was demonstrated by IFN-γ ELISPOT assays in PBMC from HIV-1-infected subjects. The HLA supertypes of the subjects was a very strong predictor of epitope-specific responses, but some subjects responded to epitopes outside of the predicted HLA type. A DNA plasmid vaccine, EP HIV-1090, was designed to express the 21 CTL epitopes as a single Ag and tested for immunogenicity using HLA transgenic mice. Immunization of HLA transgenic mice with this vaccine was sufficient to induce CTL responses to multiple HIV-1 epitopes, comparable in magnitude to those induced by immunization with peptides. The CTL induced by the vaccine recognized target cells pulsed with peptide or cells transfected with HIV-1 env or gag genes. There was no indication of immunodominance, as the vaccine induced CTL responses specific for multiple epitopes in individual mice. These data indicate that the EP HIV-1090 DNA vaccine may be suitable for inducing relevant HIV-1-specific CTL responses in humans.

HIV-1 Ag-specific CD4+ T cell proliferative responses in human subjects with advanced, untreated HIV-1 disease are often weak or undetectable. Conversely, HIV-1-specific CD4+ T cell proliferation is occasionally detected following suppression of HIV-1 replication with highly active antiretroviral therapy (HAART). These observations suggest that unchecked HIV-1 replication may lead to depletion or dysfunction of HIV-1-specific CD4+ T cells, and that these defects may be partially corrected by viral suppression and subsequent immune reconstitution. However, the impact of this immune reconstitution on the repertoire of HIV-1-specific CD4+ T cells has not been thoroughly evaluated. To examine the HIV-1-specific CD4+ T cell repertoire in this clinical setting, we established HIV-1 p24-specific CD4+ T cell clones from a successfully HAART-treated subject whose pretreatment peripheral CD4 count was 0 cells/μl. Eleven different p24-specific CD4+ T cell clonotypes were distinguished among 13 clones obtained. Most clones produced both IFN-γ and IL-4 upon Ag stimulation. Clones targeted eight distinct epitopes that varied in their conservancy among HIV-1 strains, and responses were restricted by one of three MHC II molecules. Clones showed a range of functional avidities for both protein and peptide Ags. Additional studies confirmed that multiple HIV-1 p24-derived epitopes were targeted by IFN-γ-producing CD4+ cells from subjects first treated with HAART during advanced HIV-1 disease (median, 4.5 peptides/subject; range, 3–6). These results suggest that in HAART-treated subjects whose peripheral CD4+ T cell pools were once severely depleted, the HIV-1-specific CD4+ T cell repertoire may include a diverse array of clonotypes targeting multiple HIV-1 epitopes.

Dendritic cells (DCs) are postulated to be involved in transmission of human immunodeficiency virus (HIV) type 1 to T cells and in stimulation of HIV-1-specific cell-mediated immunity. Blood DCs have been categorized as myeloid (mDC) and plasmacytoid (pDC) subsets, on the basis of differences in phenotype and function. Blood DC subset numbers and expression of costimulatory molecules and HIV-1 coreceptors on DCs were measured in the blood of treated and untreated HIV-1-infected subjects and uninfected control subjects. Absolute numbers of mDCs and pDCs were lower in HIV-1-infected subjects than in control subjects, most significantly in those with active HIV-1 replication. Increased surface expression of costimulatory molecules was observed on both DC subsets in subjects with HIV-1 viremia. Highly active antiretroviral therapy suppression of plasma viremia resulted in increases in blood DC numbers and decreases in DC costimulatory molecule expression. These findings further define the impact of HIV-1 replication on blood DC subsets in vivo.

The demonstration that highly active anti-retroviral therapy (HAART) can control human immunodeficiency virus type 1 (HIV-1) viral replication and the associated destruction of the immune system provides an opportunity to implement therapeutic vaccine strategies. The Epimmune approach is based on separate vaccine immunogens designed to induce, or augment, helper T-lymphocyte (HTL) or cytotoxic T-lymphocyte (CTL) responsiveness when administered in conjunction with HAART. The vaccines are composed of carefully selected, minimal HTL or CTL epitopes. Vaccines composed of multiple epitopes can be produced and delivered using different formats, including deoxyribonucleic acid (DNA) plasmid-based vaccines and recombinant proteins.

One hallmark of uncontrolled, chronic human immunodeficiency virus type 1 (HIV-1) infection is the absence of strong HIV-1-specific, CD4+T-cell-proliferative responses, yet the mechanism underlying this T helper (Th)-cell defect remains controversial. To better understand the impact of HIV-1 replication on Th-cell function, we compared the frequency of CD4+Th-cell responses based on production of gamma interferon to lymphoproliferative responses directed against HIV-1 proteins in HIV-1-infected subjects with active in vivo viral replication versus those on suppressed highly active antiretroviral therapy (HAART). No statistically significant differences in the frequencies of cytokine-secreting, HIV-1-specific CD4+T cells between the donor groups were found, despite differences in viral load and treatment status. However, HIV-1-specific lymphoproliferative responses were significantly greater in the subjects with HAART suppression than in subjects with active viral replication. Similar levels of HIV-1 RNA were measured in T-cell cultures stimulated with HIV-1 antigens regardless of donor in vivo viral loads, but only HIV-1-specific CD4+T cells from subjects with HAART suppression proliferated in vitro, suggesting that HIV-1 replication in vitro does not preclude HIV-1-specific lymphoproliferation. This study demonstrates a discordance between the frequency and proliferative capacity of HIV-1-specific CD4+T cells in subjects with ongoing in vivo viral replication and suggests that in vivo HIV-1 replication contributes to the observed defect in HIV-1-specific CD4+T-cell proliferation.

AIDS-related complications and mortality have decreased dramatically in the combination antiretroviral therapy (cART) era, but the risk of cardiovascular disease and other morbidities linked to chronic immune dysfunction and inflammation remains high [1]. Increased CD8 T-cell activation and inflammatory markers are associated with impaired CD4 T-cell recovery and excess mortality in ART recipients [1, 2]. Systemic translocation of microbial products such as lipopolysaccharide (LPS) across a permeable gut mucosa may contribute to persistent inflammation [2]. Individuals with chronic human immunodeficiency virus (HIV) infection have increased circulating levels of LPS, LPS-binding protein (LBP), and soluble CD14 (sCD14), an LPS coreceptor [2]. ART decreases but does not normalize these levels [2]. Thus, an agent that decreases levels of LPS and its inflammatory consequences may improve clinical outcomes in HIV infection. Sevelamer carbonate, a phosphate-lowering drug, decreases circulating LPS levels in patients with renal insufficiency, possibly by binding chylomicron-LPS complexes and preventing their reabsorption; in this population, sevelamer also reduces levels of sCD14, interleukin 6 (IL-6), C-reactive protein (CRP), and total and low-density lipoprotein (LDL) cholesterol [3–6]. These effects may contribute to the decreased mortality risk observed in sevelamer-treated patients undergoing dialysis [7]. Given the possible role of microbial translocation in HIV-associated inflammation and the potential for its reversal by sevelamer, we performed a single-arm, open-label clinical trial (AIDS Clinical Trials Group [ACTG] A5296), to test the hypothesis that 8 weeks of sevelamer treatment would decrease plasma LPS and sCD14 levels. We studied its effects in subjects who were not receiving cART because (1) sevelamer-cART interactions are unknown, (2) sevelamer might exacerbate tenofovir's phosphate-lowering effect, and (3) microbial translocation is more consistently elevated in untreated disease, increasing the likelihood of seeing an effect.

Damage to the intestinal mucosa results in the translocation of microbes from the intestinal lumen into the circulation. Microbial translocation has been proposed to trigger immune activation, inflammation, and coagulopathy, all of which are key factors that drive HIV disease progression and non-HIV comorbidities; however, direct proof of a causal link is still lacking. Here, we have demonstrated that treatment of acutely SIV-infected pigtailed macaques with the drug sevelamer, which binds microbial lipopolysaccharide in the gut, dramatically reduces immune activation and inflammation and slightly reduces viral replication. Furthermore, sevelamer administration reduced coagulation biomarkers, confirming the contribution of microbial translocation in the development of cardiovascular comorbidities in SIV-infected nonhuman primates. Together, our data suggest that early control of microbial translocation may improve the outcome of HIV infection and limit noninfectious comorbidities associated with AIDS.

Background Many HIV-2 and SIV isolates, as well as some HIV-1 strains, can use the orphan 7-transmembrane receptor GPR15 as co-receptor for efficient entry into host cells. GPR15 is expressed on central memory and effector memory CD4+ T cells in healthy individuals and a subset of these cells is susceptible to HIV-1 and SIV infection. However, it has not been determined whether GPR15 expression is altered in the context of HIV-1 infection. Results Here, we show that GPR15 expression in CD4+ T cells is markedly up-regulated in some HIV-1 infected individuals compared to the rest of the infected patients and to healthy controls. Infection of the PM1 T cell line with primary HIV-1 isolates was found to up-regulate GPR15 expression on the infected cells, indicating that viral components can induce GPR15 expression. Up-regulation of GPR15 expression on CD4+ T cells was induced by activation of Toll-like receptor 3 signalling via TIR-domain-containing adapter-inducing interferon-β (TRIF) and was more prominent on gut-homing compared to lymph node-homing CD4+ T cells. Conclusion These results suggest that infection-induced up-regulation of GPR15 expression could increase susceptibility of CD4+ T cells to HIV infection and target cell availability in the gut in some infected individuals. peerReviewed

There is currently a need for vaccines that stimulate cell-mediated immunity—particularly that mediated by CD8+ cytotoxic T lymphocytes (CTLs)—against viral and tumor antigens. The optimal induction of cell-mediated immunity requires the presentation of antigens by specialized cells of the immune system called dendritic cells1 (DCs). DCs are unique in their ability to process exogenous antigens via the major histocompatibility complex (MHC) class I pathway2 as well as in their ability to activate naive, antigen-specific CD8+ and CD4+ T cells1,3. Vaccine strategies that target or activate DCs in order to elicit potent CTL-mediated immunity are the subject of intense research. We report here that whole recombinant Saccharomyces cerevisiae yeast expressing tumor or HIV-1 antigens potently induced antigen-specific, CTL responses, including those mediating tumor protection, in vaccinated animals. Interactions between yeast and DCs led to DC maturation, IL-12 production and the efficient priming of MHC class I- and class II-restricted, antigen-specific T-cell responses. Yeast exerted a strong adjuvant effect, augmenting DC presentation of exogenous whole-protein antigen to MHC class I- and class II-restricted T cells. Recombinant yeast represent a novel vaccine strategy for the induction of broad-based cellular immune responses.

The level of microbial translocation from the intestine is increased in HIV-1 infection. Proinflammatory cytokine production by peripheral antigen-presenting cells in response to translocated microbes or microbial products may contribute to systemic immune activation, a hallmark of HIV-1 infection. We investigated the cytokine responses of peripheral blood myeloid dendritic cells (mDCs) and monocytes to in vitro stimulation with commensal enteric Escherichia coli in peripheral blood mononuclear cells (PBMC) from untreated HIV-1-infected subjects and from uninfected controls. Levels of interleukin 23 (IL-23) produced by PBMC from HIV-1-infected subjects in response to E. coli stimulation were significantly higher than those produced by PBMC from uninfected subjects. IL-23 was produced primarily by CD16 + monocytes. This subset of monocytes was increased in frequency and expressed higher levels of Toll-like receptor 4 (TLR4) in HIV-1-infected individuals than in controls. Blocking TLR4 on total CD14 + monocytes reduced IL-23 production in response to E. coli stimulation. Levels of soluble CD27, an indicator of systemic immune activation, were elevated in HIV-1-infected subjects and were associated with the percentage of CD16 + monocytes and the induction of IL-23 by E. coli , providing a link between these parameters and systemic inflammation. Taken together, these results suggest that IL-23 produced by CD16 + monocytes in response to microbial stimulation may contribute to systemic immune activation in HIV-1-infected individuals.

Background Systemic inflammation is a characteristic of both HIV-1 infection and aging (“inflammaging”). Intestinal epithelial barrier damage (IEBD) and microbial translocation (MT) contribute to HIV-associated inflammation, but their impact on inflammaging remains unclear. Methods Plasma biomarkers for IEBD (iFABP), MT (LPS, sCD14), T-cell activation (sCD27), and inflammation (hsCRP, IL-6) were measured in 88 HIV-1 uninfected (HIVneg) and 83 treated, HIV-1-infected (HIVpos) adults from 20–100 years old. Results Age positively correlated with iFABP (r = 0.284, p = 0.008), sCD14 (r = 0.646, p =

Human immunodeficiency virus-1 (HIV-1) infection disrupts the intestinal immune system, leading to microbial translocation and systemic immune activation. We investigated the impact of HIV-1 infection on the intestinal microbiome and its association with mucosal T-cell and dendritic cell (DC) frequency and activation, as well as with levels of systemic T-cell activation, inflammation, and microbial translocation. Bacterial 16S ribosomal DNA sequencing was performed on colon biopsies and fecal samples from subjects with chronic, untreated HIV-1 infection and uninfected control subjects. Colon biopsies of HIV-1-infected subjects had increased abundances of Proteobacteria and decreased abundances of Firmicutes compared with uninfected donors. Furthermore at the genus level, a significant increase in Prevotella and decrease in Bacteroides was observed in HIV-1-infected subjects, indicating a disruption in the Bacteroidetes bacterial community structure. This HIV-1-associated increase in Prevotella abundance was associated with increased numbers of activated colonic T cells and myeloid DCs. Principal coordinates analysis demonstrated an HIV-1-related change in the microbiome that was associated with increased mucosal cellular immune activation, microbial translocation, and blood T-cell activation. These observations suggest that an important relationship exists between altered mucosal bacterial communities and intestinal inflammation during chronic HIV-1 infection.