Gut microbiota from high-risk men who have sex with men drive immune activation in gnotobiotic mice and in vitro HIV infection.

Men who have sex with men (MSM) have differences in immune activation and gut microbiome composition compared with men who have sex with women (MSW), even in the absence of HIV infection. Gut microbiome differences associated with HIV itself when controlling for MSM, as assessed by 16S rRNA sequencing, are relatively subtle. Understanding whether gut microbiome composition impacts immune activation in HIV-negative and HIV-positive MSM has important implications since immune activation has been associated with HIV acquisition risk and disease progression. To investigate the effects of MSM and HIV-associated gut microbiota on immune activation, we transplanted feces from HIV-negative MSW, HIV-negative MSM, and HIV-positive untreated MSM to gnotobiotic mice. Following transplant, 16S rRNA gene sequencing determined that the microbiomes of MSM and MSW maintained distinct compositions in mice and that specific microbial differences between MSM and MSW were replicated. Immunologically, HIV-negative MSM donors had higher frequencies of blood CD38+ HLADR+ and CD103+ T cells and their fecal recipients had higher frequencies of gut CD69+ and CD103+ T cells, compared with HIV-negative MSW donors and recipients, respectively. Significant microbiome differences were not detected between HIV-negative and HIV-positive MSM in this small donor cohort, and immune differences between their recipients were trending but not statistically significant. A larger donor cohort may therefore be needed to detect immune-modulating microbes associated with HIV. To investigate whether our findings in mice could have implications for HIV replication, we infected primary human lamina propria cells stimulated with isolated fecal microbiota, and found that microbiota from MSM stimulated higher frequencies of HIV-infected cells than microbiota from MSW. Finally, we identified several microbes that correlated with immune readouts in both fecal recipients and donors, and with in vitro HIV infection, which suggests a role for gut microbiota in immune activation and potentially HIV acquisition in MSM.

Tetherin promotes the innate and adaptive cell-mediated immune response against retrovirus infection in vivo.

Tetherin/BST-2 is a host restriction factor that could directly inhibit retroviral particle release by tethering nascent virions to the plasma membrane. However, the immunological impact of Tetherin during retrovirus infection remains unknown. We now show that Tetherin influences antiretroviral cell-mediated immune responses. In contrast to the direct antiviral effects of Tetherin, which are dependent on cell surface expression, the immunomodulatory effects are linked to the endocytosis of the molecule. Mice encoding endocytosis-competent C57BL/6 Tetherin exhibited lower viremia and pathology at 7 d postinfection with Friend retrovirus (FV) compared with mice encoding endocytosis-defective NZW/LacJ Tetherin. Notably, antiretroviral protection correlated with stronger NK cell responses. In addition, Friend retrovirus infection levels were significantly lower in wild-type C57BL/6 mice than in Tetherin knockout mice at 2 wk postinfection, and antiretroviral protection correlated with stronger NK cell and virus-specific CD8+ T cell responses. The results demonstrate that Tetherin acts as a modulator of the cell-mediated immune response against retrovirus infection in vivo.

IFN-α treatment inhibits acute Friend retrovirus replication primarily through the antiviral effector molecule Apobec3.

Therapeutic administration of IFN-α in clinical trials significantly reduced HIV-1 plasma viral load and human T-lymphotropic virus type I proviral load in infected patients. The mechanism may involve the concerted action of multiple antiretroviral effectors collectively known as "restriction factors," which could vary in relative importance according to the magnitude of transcriptional induction. However, direct genetic approaches to identify the relevant IFN-α restriction factors will not be feasible in humans in vivo. Meanwhile, mice encode an analogous set of restriction factor genes and could be used to obtain insights on how IFN-α could inhibit retroviruses in vivo. As expected, IFN-α treatment of mice significantly upregulated the transcription of multiple restriction factors including Tetherin/BST2, SAMHD1, Viperin, ISG15, OAS1, and IFITM3. However, a dominant antiretroviral factor, Apobec3, was only minimally induced. To determine whether Apobec3 was necessary for direct IFN-α antiretroviral action in vivo, wild-type and Apobec3-deficient mice were infected with Friend retrovirus, then treated with IFN-α. Treatment of infected wild-type mice with IFN-α significantly reduced acute plasma viral load 28-fold, splenic proviral load 5-fold, bone marrow proviral load 14-fold, and infected bone marrow cells 7-fold, but no inhibition was observed in Apobec3-deficient mice. These findings reveal that IFN-α inhibits acute Friend retrovirus infection primarily through the antiviral effector Apobec3 in vivo, demonstrate that transcriptional induction levels did not predict the mechanism of IFN-α-mediated control, and highlight the potential of the human APOBEC3 proteins as therapeutic targets against pathogenic retrovirus infections.

A single nucleotide polymorphism in tetherin promotes retrovirus restriction in vivo.

Tetherin is a membrane protein of unusual topology expressed from rodents to humans that accumulates enveloped virus particles on the surface of infected cells. However, whether this 'tethering' activity promotes or restricts retroviral spread during acute retrovirus infection in vivo is controversial. We report here the identification of a single nucleotide polymorphism in the Tetherin gene of NZW/LacJ (NZW) mice that mutated the canonical ATG start site to GTG. Translation of NZW Tetherin from downstream ATGs deleted a conserved dual-tyrosine endosomal sorting motif, resulting in higher cell surface expression and more potent inhibition of Friend retrovirus release compared to C57BL/6 (B6) Tetherin in vitro. Analysis of (B6×NZW)F(1) hybrid mice revealed that increased Tetherin cell surface expression in NZW mice is a recessive trait in vivo. Using a classical genetic backcrossing approach, NZW Tetherin expression strongly correlated with decreased Friend retrovirus replication and pathogenesis. However, the protective effect of NZW Tetherin was not observed in the context of B6 Apobec3/Rfv3 resistance. These findings identify the first functional Tetherin polymorphism within a mammalian host, demonstrate that Tetherin cell surface expression is a key parameter for retroviral restriction, and suggest the existence of a restriction factor hierarchy to counteract pathogenic retrovirus infections in vivo.

Systems Analysis of Gut Microbiome Influence on Metabolic Disease in HIV-Positive and High-Risk Populations.

Poor metabolic health, characterized by insulin resistance and dyslipidemia, is higher in people living with HIV and has been linked with inflammation, antiretroviral therapy (ART) drugs, and ART-associated lipodystrophy (LD). Metabolic disease is associated with gut microbiome composition outside the context of HIV but has not been deeply explored in HIV infection or in high-risk men who have sex with men (HR-MSM), who have a highly altered gut microbiome composition. Furthermore, the contribution of increased bacterial translocation and associated systemic inflammation that has been described in HIV-positive and HR-MSM individuals has not been explored. We used a multiomic approach to explore relationships between impaired metabolic health, defined using fasting blood markers, gut microbes, immune phenotypes, and diet. Our cohort included ART-treated HIV-positive MSM with or without LD, untreated HIV-positive MSM, and HR-MSM. For HIV-positive MSM on ART, we further explored associations with the plasma metabolome. We found that elevated plasma lipopolysaccharide binding protein (LBP) was the most important predictor of impaired metabolic health and network analysis showed that LBP formed a hub joining correlated microbial and immune predictors of metabolic disease. Taken together, our results suggest the role of inflammatory processes linked with bacterial translocation and interaction with the gut microbiome in metabolic disease among HIV-positive and -negative MSM.IMPORTANCE The gut microbiome in people living with HIV (PLWH) is of interest since chronic infection often results in long-term comorbidities. Metabolic disease is prevalent in PLWH even in well-controlled infection and has been linked with the gut microbiome in previous studies, but little attention has been given to PLWH. Furthermore, integrated analyses that consider gut microbiome, together with diet, systemic immune activation, metabolites, and demographics, have been lacking. In a systems-level analysis of predictors of metabolic disease in PLWH and men who are at high risk of acquiring HIV, we found that increased lipopolysaccharide-binding protein, an inflammatory marker indicative of compromised intestinal barrier function, was associated with worse metabolic health. We also found impaired metabolic health associated with specific dietary components, gut microbes, and host and microbial metabolites. This study lays the framework for mechanistic studies aimed at targeting the microbiome to prevent or treat metabolic endotoxemia in HIV-infected individuals.

Correction to: An exploration of Prevotella-rich microbiomes in HIV and men who have sex with men.

Following publication of the original article [1], the authors reported an error in Fig. 2. The original Fig. 2 has been incorrectly replaced with the Supplementary Fig. 2. The correct Fig. 2 is presented here.

Can gut microbiota of men who have sex with men influence HIV transmission?

Gaining a complete understanding of transmission risk factors will assist in efforts to reduce new HIV infections, especially within the disproportionally affected population of men who have sex with men (MSM). We recently reported that the fecal microbiota of MSM elevates immune activation in gnotobiotic mice and enhances HIV infection in vitro over that of fecal microbiota from men who have sex with women. We also demonstrated elevation of the gut homing marker CD103 (integrin αE) on CD4+ T cells by MSM-microbiota. Here we provide additional evidence that the gut microbiota is a risk factor for HIV transmission in MSM by showing elevated frequencies of the HIV co-receptor CCR5 on CD4+ T cells in human rectosigmoid colon biopsies. We discuss our interest in specific MSM-associated bacteria and propose the influx of CD103+ and CCR5+ CD4+ T cells into the colon as a potential link between the MSM microbiota and HIV transmission.

An exploration of Prevotella-rich microbiomes in HIV and men who have sex with men.

BACKGROUND: Gut microbiome characteristics associated with HIV infection are of intense research interest but a deep understanding has been challenged by confounding factors across studied populations. Notably, a Prevotella-rich microbiome described in HIV-infected populations is now understood to be common in men who have sex with men (MSM) regardless of HIV status, but driving factors and potential health implications are unknown. RESULTS: Here, we further define the MSM-associated gut microbiome and describe compositional differences between the fecal microbiomes of Prevotella-rich MSM and non-MSM that may underlie observed pro-inflammatory properties. Furthermore, we show relatively subtle gut microbiome changes in HIV infection in MSM and women that include an increase in potential pathogens that is ameliorated with antiretroviral therapy (ART). Lastly, using a longitudinal cohort, we describe microbiome changes that happen after ART initiation. CONCLUSIONS: This study provides an in-depth characterization of microbiome differences that occur in a US population infected with HIV and demonstrates the degree to which these differences may be driven by lifestyle factors, ART, and HIV infection itself. Understanding microbiome compositions that occur with sexual behaviors that are high risk for acquiring HIV and untreated and ART-treated HIV infection will guide the investigation of immune and metabolic functional implications to ultimately target the microbiome therapeutically.

The HIV-Associated Enteric Microbiome Has Gone Viral.

HIV infection is associated with dramatic alterations in enteric bacteria, but little is known about other microbiome components. In this issue of Cell Host & Microbe, studies by Monaco et al. (2016) and Handley et al. (2016) reveal an under-appreciated role of the enteric virome in HIV-associated gastroenteritis and pathogenesis.

Tetherin/BST-2: Restriction Factor or Immunomodulator?

BACKGROUND: Cell-mediated immune (CMI) responses are critical for the control of HIV-1 infection and their importance was highlighted by the existence of viral proteins, particularly Vpu and Nef, that antagonize these responses. Pandemic HIV-1 Vpu counteracts Tetherin/BST-2, a host factor that could prevent the release of HIV-1 virions by tethering virions on the cell surface, but a link between Tetherin and HIV-1 CMI responses has not yet been demonstrated in vivo. In vitro, the virological and immunological impact of Tetherin-mediated accumulation of virions ranged from enhanced or diminished cell-to-cell spread to enhanced recognition by virus-specific antibodies for natural killer cellmediated lysis. However, Tetherin-restricted virions could be internalized through an endocytosis motif in the Tetherin cytoplasmic tail. METHODS: Given the uncertainties on which in vitro results manifest in vivo and the dearth of knowledge on how Tetherin influences retroviral immunity, in vivo retrovirus infections in mice encoding wild-type, null and endocytosis-defective Tetherin were performed. Here, we review and highlight the results from these in vivo studies. RESULTS: Current data suggests that endocytosis-defective Tetherin functions as a potent innate restriction factor. By contrast, endocytosis-competent Tetherin, the form found in most mammals including humans and the form counteracted by HIV-1 Vpu, was linked to stronger CMI responses in mice. CONCLUSION: We propose that the main role of endocytosis-competent Tetherin is not to directly restrict retroviral replication, but to promote a more effective CMI response against retroviruses.

Tetherin/BST-2 promotes dendritic cell activation and function during acute retrovirus infection.

Tetherin/BST-2 is a host restriction factor that inhibits retrovirus release from infected cells in vitro by tethering nascent virions to the plasma membrane. However, contradictory data exists on whether Tetherin inhibits acute retrovirus infection in vivo. Previously, we reported that Tetherin-mediated inhibition of Friend retrovirus (FV) replication at 2 weeks post-infection correlated with stronger natural killer, CD4+ T and CD8+ T cell responses. Here, we further investigated the role of Tetherin in counteracting retrovirus replication in vivo. FV infection levels were similar between wild-type (WT) and Tetherin KO mice at 3 to 7 days post-infection despite removal of a potent restriction factor, Apobec3/Rfv3. However, during this phase of acute infection, Tetherin enhanced myeloid dendritic cell (DC) function. DCs from infected, but not uninfected, WT mice expressed significantly higher MHC class II and the co-stimulatory molecule CD80 compared to Tetherin KO DCs. Tetherin-associated DC activation during acute FV infection correlated with stronger NK cell responses. Furthermore, Tetherin+ DCs from FV-infected mice more strongly stimulated FV-specific CD4+ T cells ex vivo compared to Tetherin KO DCs. The results link the antiretroviral and immunomodulatory activity of Tetherin in vivo to improved DC activation and MHC class II antigen presentation.

T Cell Production of IFNγ in Response to TLR7/IL-12 Stimulates Optimal B Cell Responses to Viruses.

Knowledge of the processes that underlie IgG subclass switching could inform strategies designed to counteract infections and autoimmunity. Here we show that TLR7 ligands induce subsets of memory CD4 and CD8 T cells to secrete interferon γ (IFNγ) in the absence of antigen receptor stimulation. In turn, TLR ligation and IFNγ cause B cells to express the transcription factor, T-bet, and to switch immunoglobulin production to IgG2a/c. Absence of TLR7 in T cells leads to the impaired T-bet expression in B cells and subsequent inefficient IgG2a isotype switching both in vitro and during the infection with Friend virus in vivo. Our results reveal a surprising mechanism of antiviral IgG subclass switching through T-cell intrinsic TLR7/IL-12 signaling.

Reassessment of murine APOBEC1 as a retrovirus restriction factor in vivo.

APOBEC1 is a cytidine deaminase involved in cholesterol metabolism that has been linked to retrovirus restriction, analogous to the evolutionarily-related APOBEC3 proteins. In particular, murine APOBEC1 was shown to inhibit Friend retrovirus (FV) in vitro, generating high levels of C-to-T and G-to-A mutations. These observations raised the possibility that FV infection might be altered in APOBEC1-null mice. To examine this question directly, we infected wild-type and APOBEC1-null mice with FV complex and evaluated acute infection levels. Surprisingly, APOBEC1-null mice exhibited similar cellular infection levels and plasma viremia relative to wild-type mice. Moreover, next-generation sequencing analyses revealed that in contrast to APOBEC3, APOBEC1 did not enhance retroviral C-to-T and G-to-A mutational frequencies in genomic DNA. Thus, APOBEC1 neither inhibited nor significantly drove the molecular evolution of FV in vivo. Our findings reinforce that not all retrovirus restriction factors characterized as potent in vitro may be functionally relevant in vivo.

Fv1 restriction and retrovirus vaccine immunity in Apobec3-deficient 129P2 mice.

Understanding the host genetics of the immune response in retrovirus infection models could provide insights for basic HIV vaccine discovery. In Friend retrovirus (FV) infection of mice, Fv1 differentially inhibits N-tropic versus B-tropic FV infection by mediating a capsid-dependent post-entry block, Fv2 susceptibility governs splenomegaly induction, and Rfv3 resistance primes a stronger neutralizing antibody response due to more potent Apobec3 activity. Apobec3 polymorphisms in inbred mouse strains correlate with Rfv3 resistance and susceptibility, with one unresolved exception. The 129/OlaHsd (129P2) mouse strain is Fv2 and Rfv3 susceptible based on genotyping, but infection of 129P2 mice with B-tropic FV resulted in strong neutralizing antibody responses and no splenomegaly. Here we confirm that 129P2 mice are Fv1(nr/nr), explaining its resistance to B-tropic FV. Infection of 129P2 mice with NB-tropic FV, which can efficiently infect mice independent of Fv1 genotype, resulted in severe splenomegaly, high levels of viremia and weak neutralizing antibody responses regardless of Apobec3 status. Notably, high-dose B-tropic FV infection of 129P2 Apobec3-deficient mice induced significant adaptive immune responses and conferred high levels of protection following challenge with pathogenic NB-tropic FV. This immunological protection complemented previous studies that N-tropic FV can act as a live-attenuated vaccine in Fv1 (b/b) mice. Altogether, the results obtained in 129P2 mice strengthen the conclusion that Rfv3 is encoded by Apobec3, and highlight Fv1 incompatibility as a retroviral vaccine paradigm in mice. Due to its susceptibility to disease that allows for pathogenic challenge studies, B-tropic FV infection of 129P2 mice may be a useful model to study the immunological pathways induced by retroviral capsid restriction.

Humoral immunity in the Friend retrovirus infection model.

Major conceptual roadblocks impede the development of an HIV-1 vaccine that can stimulate a potent neutralizing antibody response. Animal models that support HIV-1 replication and allow for host genetic manipulation would be an ideal platform for testing various immunological hypotheses, but progress on this research front has been slow and disappointing. In contrast, many valuable concepts emerged from more than 50 years of studying the Friend retrovirus model. This was recently exemplified by the identification of an innate restriction gene, Apobec3, that could promote the retrovirus-specific neutralizing antibody response. Here we review both classical and recent data on humoral immunity against Friend retrovirus infection, and highlight the potential of this model for unraveling novel aspects of the retrovirus-specific antibody response that may guide HIV-1 vaccine development efforts.

Ribonuclease L is not critical for innate restriction and adaptive immunity against Friend retrovirus infection.

Ribonuclease L (RNase L) is a type I interferon regulated factor that can significantly inhibit retroviruses in vitro and may activate cytoplasmic sensing pathways to augment adaptive immunity. However, the antiretroviral activity of RNase L remains to be validated in vivo. We investigated the role of RNaseL in counteracting Friend retrovirus (FV) infection relative to a well-described restriction factor, Apobec3. C57BL/6 wild-type (WT) and RNaseL knock-out (KO) mice exhibited similar acute FV infection levels despite significant transcriptional induction of oligoadenylate synthetase 1, which produces activators of RNase L. Apobec3 KO mice showed higher FV infection levels relative to WT mice, but deletion of RNaseL in Apobec3 KO mice did not augment FV infection. Moreover, RNaseL did not influence FV-specific IgG responses and recovery from viremia by 28 days post-infection. The results suggest that RNase L is not an evolutionarily-conserved host defense mechanism to counteract retroviruses in vivo.