Richard M. Dunham

Dysbiosis of the gut microbiota is associated with HIV disease progression and tryptophan catabolism.

Gut-resident microbial populations may influence intestinal homeostasis during HIV disease. Gut Reaction to HIV Despite the value placed on individuality in the western world, no human is an island. Indeed, every person carries trillions of microorganisms in their intestine, and it’s becoming increasingly clear that the composition and interaction of these microorganisms can directly affect human health. Vujkovic-Cvijin et al. now demonstrate that this is the case for individuals infected with HIV, even those where viral load is controlled by therapy. The authors screened the intestinal microbiome for microbial drivers of HIV-associated immunopathology. They found that a dysbiotic mucosal microbial community associated with mucosal immune disruption, T cell activation, and chronic inflammation in HIV-infected subjects, even in patients controlled with antiviral therapy. They then looked further into the mechanism of this association and found that the extent of dysbiosis correlated with tryptophan catabolism and plasma concentrations of the inflammatory cytokine interleukin-6 (IL-6), two established markers of disease progression. Indeed, gut-resident bacteria with the capacity to metabolize tryptophan were found to be enriched in HIV-infected subjects. These data link gut-resident microbial populations with the immunopathogenesis of HIV and suggest that modulating this community could provide a new therapeutic strategy for managing HIV disease progression. Progressive HIV infection is characterized by dysregulation of the intestinal immune barrier, translocation of immunostimulatory microbial products, and chronic systemic inflammation that is thought to drive progression of disease to AIDS. Elements of this pathologic process persist despite viral suppression during highly active antiretroviral therapy (HAART), and drivers of these phenomena remain poorly understood. Disrupted intestinal immunity can precipitate dysbiosis that induces chronic inflammation in the mucosa and periphery of mice. However, putative microbial drivers of HIV-associated immunopathology versus recovery have not been identified in humans. Using high-resolution bacterial community profiling, we identified a dysbiotic mucosal-adherent community enriched in Proteobacteria and depleted of Bacteroidia members that was associated with markers of mucosal immune disruption, T cell activation, and chronic inflammation in HIV-infected subjects. Furthermore, this dysbiosis was evident among HIV-infected subjects undergoing HAART, and the extent of dysbiosis correlated with activity of the kynurenine pathway of tryptophan catabolism and plasma concentrations of the inflammatory cytokine interleukin-6 (IL-6), two established markers of disease progression. Gut-resident bacteria with capacity to catabolize tryptophan through the kynurenine pathway were found to be enriched in HIV-infected subjects, strongly correlated with kynurenine levels in HIV-infected subjects, and capable of kynurenine production in vitro. These observations demonstrate a link between mucosal-adherent colonic bacteria and immunopathogenesis during progressive HIV infection that is apparent even in the setting of viral suppression during HAART. This link suggests that gut-resident microbial populations may influence intestinal homeostasis during HIV disease.

Glucagon-like peptide-1 receptor is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway.

Glucagon‐like peptide 1 (GLP‐1) is a naturally occurring peptide secreted by the L cells of the small intestine. GLP‐1 functions as an incretin and stimulates glucose‐mediated insulin production by pancreatic β cells. In this study, we demonstrate that exendin‐4/GLP‐1 has a cognate receptor on human hepatocytes and that exendin‐4 has a direct effect on the reduction of hepatic steatosis in the absence of insulin. Both glucagon‐like peptide 1 receptor (GLP/R) messenger RNA and protein were detected on primary human hepatocytes, and receptor was internalized in the presence of GLP‐1. Exendin‐4 increased the phosphorylation of 3‐phosphoinositide‐dependent kinase‐1 (PDK‐1), AKT, and protein kinase C ζ (PKC‐ζ) in HepG2 and Huh7 cells. Small interfering RNA against GLP‐1R abolished the effects on PDK‐1 and PKC‐ζ. Treatment with exendin‐4 quantitatively reduced triglyceride stores compared with control‐treated cells. Conclusion: This is the first report that the G protein–coupled receptor GLP‐1R is present on human hepatocytes. Furthermore, it appears that exendin‐4 has the same beneficial effects in vitro as those seen in our previously published in vivo study in ob/ob mice, directly reducing hepatocyte steatosis. Future use for human nonalcoholic fatty liver disease, either in combination with dietary manipulation or other pharmacotherapy, may be a significant advance in treatment of this common form of liver disease. (HEPATOLOGY 2010)

Severe Depletion of Mucosal CD4+ T Cells in AIDS-Free Simian Immunodeficiency Virus-Infected Sooty Mangabeys

HIV-infected humans and SIV-infected rhesus macaques experience a rapid and dramatic loss of mucosal CD4+ T cells that is considered to be a key determinant of AIDS pathogenesis. In this study, we show that nonpathogenic SIV infection of sooty mangabeys (SMs), a natural host species for SIV, is also associated with an early, severe, and persistent depletion of memory CD4+ T cells from the intestinal and respiratory mucosa. Importantly, the kinetics of the loss of mucosal CD4+ T cells in SMs is similar to that of SIVmac239-infected rhesus macaques. Although the nonpathogenic SIV infection of SMs induces the same pattern of mucosal target cell depletion observed during pathogenic HIV/SIV infections, the depletion in SMs occurs in the context of limited local and systemic immune activation and can be reverted if virus replication is suppressed by antiretroviral treatment. These results indicate that a profound depletion of mucosal CD4+ T cells is not sufficient per se to induce loss of mucosal immunity and disease progression during a primate lentiviral infection. We propose that, in the disease-resistant SIV-infected SMs, evolutionary adaptation to both preserve immune function with fewer mucosal CD4+ T cells and attenuate the immune activation that follows acute viral infection protect these animals from progressing to AIDS.

IL-7 administration drives T cell-cycle entry and expansion in HIV-1 infection.

Interleukin 7 (IL-7) is a common gamma chain receptor cytokine implicated in thymopoiesis and in peripheral expansion and survival of T lymphocytes. The safety and activity of recombinant human IL-7 (rhIL-7) administration were therefore examined in HIV-infected persons. In this prospective randomized placebo-controlled study, a single subcutaneous dose of rhIL-7 was well tolerated with biologic activity demonstrable at 3 microg/kg and a maximum tolerated dose of 30 microg/kg. Injection site reactions and transient elevations of liver function tests were the most notable side effects. Transient increases in plasma HIV-RNA levels were observed in 6 of 11 IL-7-treated patients. Recombinant hIL-7 induced CD4 and CD8 T cells to enter cell cycle; cell-cycle entry was also confirmed in antigen-specific CD8 T cells. Administration of rhIL-7 led to transient down-regulation of the IL-7 receptor alpha chain (CD127) in both CD4(+) and CD8(+) T cells. Single-dose rhIL-7 increased the numbers of circulating CD4(+) and CD8(+) T cells, predominantly of central memory phenotype. The frequency of CD4(+) T cells with a regulatory T-cell phenotype (CD25(high) CD127(low)) did not change after rhIL-7 administration. Thus, rhIL-7 has a biologic and toxicity profile suggesting a potential for therapeutic trials in HIV infection and other settings of lymphopenia. This clinical trial has been registered at http://www.clinicaltrials.gov under NCT0099671.

Loss of CD127 expression defines an expansion of effector CD8+ T cells in HIV-infected individuals

The immunodeficiency that follows HIV infection is related to the virus-mediated killing of infected CD4+ T cells, the chronic activation of the immune system, and the impairment of T cell production. In this study we show that in HIV-infected individuals the loss of IL-7R (CD127) expression defines the expansion of a subset of CD8+ T cells, specific for HIV as well as other Ags, that show phenotypic (i.e., loss of CCR7 and CD62 ligand expression with enrichment in activated and/or proliferating cells) as well as functional (i.e., production of IFN-γ, but not IL-2, decreased ex vivo proliferative potential and increased susceptibility to apoptosis) features of effector T cells. Importantly, in HIV-infected individuals the levels of CD8+CD127− T cells are directly correlated with the main markers of disease progression (i.e., plasma viremia and CD4+ T cell depletion) as well as with the indices of overall T cell activation. In all, these results identify the expansion of CD8+CD127− effector-like T cells as a novel feature of the HIV-associated immune perturbation. Further studies are thus warranted to determine whether measurements of CD127 expression on CD8+ T cells may be useful in the clinical management of HIV-infected individuals.

Early Resolution of Acute Immune Activation and Induction of PD-1 in SIV-Infected Sooty Mangabeys Distinguishes Nonpathogenic from Pathogenic Infection in Rhesus Macaques

Primate lentiviruses are typically apathogenic in their evolutionarily coadapted host species but can be lethal when transferred to new host species. Why such infections are pathogenic in humans and rhesus macaques (RMs) but not in sooty mangabeys (SMs), a natural host, remains unclear. Studies of chronically infected animals point to the importance of diminished immune activation in response to the infection in SMs. In this study, we sought the causes and timing of the differences in immune activation in a comparative study of acute SIV infection in RMs and SMs. Surprisingly, we show that in acute infection immune activation is comparable in SMs and RMs but thereafter, SMs quickly resolve immune activation, whereas RMs did not. Early resolution of immune activation in SMs correlated with increased expression of PD-1 and with preservation of CD4+ T cell counts and lymphatic tissue architecture. These findings point to early control of immune activation by host immunoregulatory mechanisms as a major determinant of the different disease outcomes in SIV infection of natural vs non-natural hosts.

Correlates of Preserved CD4+ T Cell Homeostasis during Natural, Nonpathogenic Simian Immunodeficiency Virus Infection of Sooty Mangabeys: Implications for AIDS Pathogenesis

In contrast to HIV-infected humans, naturally SIV-infected sooty mangabeys (SMs) very rarely progress to AIDS. Although the mechanisms underlying this disease resistance are unknown, a consistent feature of natural SIV infection is the absence of the generalized immune activation associated with HIV infection. To define the correlates of preserved CD4+ T cell counts in SMs, we conducted a cross-sectional immunological study of 110 naturally SIV-infected SMs. The nonpathogenic nature of the infection was confirmed by an average CD4+ T cell count of 1,076 ± 589/mm3 despite chronic infection with a highly replicating virus. No correlation was found between CD4+ T cell counts and either age (used as a surrogate marker for length of infection) or viremia. The strongest correlates of preserved CD4+ T cell counts were a low percentage of circulating effector T cells (CD28−CD95+ and/or IL-7R/CD127−) and a high percentage of CD4+CD25+ T cells. These findings support the hypothesis that the level of immune activation is a key determinant of CD4+ T cell counts in SIV-infected SMs. Interestingly, we identified 14 animals with CD4+ T cell counts of <500/mm3, of which two show severe and persistent CD4+ T cell depletion (<50/mm3). Thus, significant CD4+ T cell depletion does occasionally follow SIV infection of SMs even in the context of generally low levels of immune activation, lending support to the hypothesis of multifactorial control of CD4+ T cell homeostasis in this model of infection. The absence of AIDS in these “CD4low” naturally SIV-infected SMs defines a protective role of the reduced immune activation even in the context of a significant CD4+ T cell depletion.

CD127 and CD25 Expression Defines CD4+ T Cell Subsets That Are Differentially Depleted during HIV Infection

Decreased CD4+ T cell counts are the best marker of disease progression during HIV infection. However, CD4+ T cells are heterogeneous in phenotype and function, and it is unknown how preferential depletion of specific CD4+ T cell subsets influences disease severity. CD4+ T cells can be classified into three subsets by the expression of receptors for two T cell-tropic cytokines, IL-2 (CD25) and IL-7 (CD127). The CD127+CD25low/− subset includes IL-2-producing naive and central memory T cells; the CD127−CD25− subset includes mainly effector T cells expressing perforin and IFN-γ; and the CD127lowCD25high subset includes FoxP3-expressing regulatory T cells. Herein we investigated how the proportions of these T cell subsets are changed during HIV infection. When compared with healthy controls, HIV-infected patients show a relative increase in CD4+CD127−CD25− T cells that is related to an absolute decline of CD4+CD127+CD25low/− T cells. Interestingly, this expansion of CD4+CD127− T cells was not observed in naturally SIV-infected sooty mangabeys. The relative expansion of CD4+CD127−CD25− T cells correlated directly with the levels of total CD4+ T cell depletion and immune activation. CD4+CD127−CD25− T cells were not selectively resistant to HIV infection as levels of cell-associated virus were similar in all non-naive CD4+ T cell subsets. These data indicate that, during HIV infection, specific changes in the fraction of CD4+ T cells expressing CD25 and/or CD127 are associated with disease progression. Further studies will determine whether monitoring the three subsets of CD4+ T cells defined based on the expression of CD25 and CD127 should be used in the clinical management of HIV-infected individuals.

Short-Lived Infected Cells Support Virus Replication in Sooty Mangabeys Naturally Infected with Simian Immunodeficiency Virus: Implications for AIDS Pathogenesis

ABSTRACT Sooty mangabeys (SMs) naturally infected with simian immunodeficiency virus (SIV) do not develop AIDS despite high levels of virus replication. At present, the mechanisms underlying this disease resistance are poorly understood. Here we tested the hypothesis that SIV-infected SMs avoid immunodeficiency as a result of virus replication occurring in infected cells that live significantly longer than human immunodeficiency virus (HIV)-infected human cells. To this end, we treated six SIV-infected SMs with potent antiretroviral therapy (ART) and longitudinally measured the decline in plasma viremia. We applied the same mathematical models used in HIV-infected individuals and observed that SMs naturally infected with SIV also present a two-phase decay of viremia following ART, with the bulk (92 to 99%) of virus replication sustained by short-lived cells (average life span, 1.06 days), and only 1 to 8% occurring in longer-lived cells. In addition, we observed that ART had a limited impact on CD4+ T cells and the prevailing level of T-cell activation and proliferation in SIV-infected SMs. Collectively, these results suggest that in SIV-infected SMs, similar to HIV type 1-infected humans, short-lived activated CD4+ T cells, rather than macrophages, are the main source of virus production. These findings indicate that a short in vivo life span of infected cells is a common feature of both pathogenic and nonpathogenic primate lentivirus infections and support a model for AIDS pathogenesis whereby the direct killing of infected cells by HIV is not the main determinant of disease progression.

Availability of activated CD4+ T cells dictates the level of viremia in naturally SIV-infected sooty mangabeys

Naturally SIV-infected sooty mangabeys (SMs) remain asymptomatic despite high virus replication. Elucidating the mechanisms underlying AIDS resistance of SIV-infected SMs may provide crucial information to better understand AIDS pathogenesis. In this study, we assessed the determinants of set-point viremia in naturally SIV-infected SMs, i.e., immune control of SIV replication versus target cell limitation. We depleted CD4+ T cells in 6 naturally SIV-infected SMs by treating with humanized anti-CD4 mAb (Cdr-OKT4A-huIgG1). CD4+ T cells were depleted almost completely in blood and BM and at variable levels in mucosal tissues and LNs. No marked depletion of CD14+ monocytes was observed. Importantly, CD4+ T cell depletion was associated with a rapid, significant decline in viral load, which returned to baseline level at day 30-45, coincident with an increased fraction of proliferating and activated CD4+ T cells. Throughout the study, virus replication correlated with the level of proliferating CD4+ T cells. CD4+ T cell depletion did not induce any changes in the fraction of Tregs or the level of SIV-specific CD8+ T cells. Our results suggest that the availability of activated CD4+ T cells, rather than immune control of SIV replication, is the main determinant of set-point viral load during natural SIV infection of SMs.