HIV-1 Myeloid Reservoirs - Contributors to Viral Persistence and Pathogenesis.

PURPOSE OF REVIEW: HIV reservoirs are the main barrier to cure. CD4+ T cells have been extensively studied as the primary HIV-1 reservoir. However, there is substantial evidence that HIV-1-infected myeloid cells (monocytes/macrophages) also contribute to viral persistence and pathogenesis. RECENT FINDINGS: Recent studies in animal models and people with HIV-1 demonstrate that myeloid cells are cellular reservoirs of HIV-1. HIV-1 genomes and viral RNA have been reported in circulating monocytes and tissue-resident macrophages from the brain, urethra, gut, liver, and spleen. Importantly, viral outgrowth assays have quantified persistent infectious virus from monocyte-derived macrophages and tissue-resident macrophages. The myeloid cell compartment represents an important target of HIV-1 infection. While myeloid reservoirs may be more difficult to measure than CD4+ T cell reservoirs, they are long-lived, contribute to viral persistence, and, unless specifically targeted, will prevent an HIV-1 cure.

Monocyte-derived macrophages contain persistent latent HIV reservoirs.

The development of persistent cellular reservoirs of latent human immunodeficiency virus (HIV) is a critical obstacle to viral eradication since viral rebound takes place once anti-retroviral therapy (ART) is interrupted. Previous studies show that HIV persists in myeloid cells (monocytes and macrophages) in blood and tissues in virologically suppressed people with HIV (vsPWH). However, how myeloid cells contribute to the size of the HIV reservoir and what impact they have on rebound after treatment interruption remain unclear. Here we report the development of a human monocyte-derived macrophage quantitative viral outgrowth assay (MDM-QVOA) and highly sensitive T cell detection assays to confirm purity. We assess the frequency of latent HIV in monocytes using this assay in a longitudinal cohort of vsPWH (n = 10, 100% male, ART duration 5-14 yr) and find half of the participants showed latent HIV in monocytes. In some participants, these reservoirs could be detected over several years. Additionally, we assessed HIV genomes in monocytes from 30 vsPWH (27% male, ART duration 5-22 yr) utilizing a myeloid-adapted intact proviral DNA assay (IPDA) and demonstrate that intact genomes were present in 40% of the participants and higher total HIV DNA correlated with reactivatable latent reservoirs. The virus produced in the MDM-QVOA was capable of infecting bystander cells resulting in viral spread. These findings provide further evidence that myeloid cells meet the definition of a clinically relevant HIV reservoir and emphasize that myeloid reservoirs should be included in efforts towards an HIV cure.

Psychosocial Stress Alters the Immune Response and Results in Higher Viral Load During Acute Simian Immunodeficiency Virus Infection in a Pigtailed Macaque Model of Human Immunodeficiency Virus.

BACKGROUND: Although social distancing is a key public health response during viral pandemics, psychosocial stressors, such as social isolation, have been implicated in adverse health outcomes in general [1] and in the context of infectious disease, such as human immunodeficiency virus (HIV) [2, 3]. A comprehensive understanding of the direct pathophysiologic effects of psychosocial stress on viral pathogenesis is needed to provide strategic and comprehensive care to patients with viral infection. METHODS: To determine the effect of psychosocial stress on HIV pathogenesis during acute viral infection without sociobehavioral confounders inherent in human cohorts, we compared commonly measured parameters of HIV progression between singly (n = 35) and socially (n = 41) housed simian immunodeficiency virus (SIV)-infected pigtailed macaques (Macaca nemestrina). RESULTS: Singly housed macaques had a higher viral load in the plasma and cerebrospinal fluid and demonstrated greater CD4 T-cell declines and more CD4 and CD8 T-cell activation compared with socially housed macaques throughout acute SIV infection. CONCLUSIONS: These data demonstrate that psychosocial stress directly impacts the pathogenesis of acute SIV infection and imply that it may act as an integral variable in the progression of HIV infection and potentially of other viral infections.

HIV replication and latency in monocytes and macrophages.

The relevance of monocyte and macrophage reservoirs in virally suppressed people with HIV (vsPWH) has previously been debatable. Macrophages were assumed to have a moderate life span and lack self-renewing potential. However, recent studies have challenged this dogma and now suggest an important role of these cell as long-lived HIV reservoirs. Lentiviruses have a long-documented association with macrophages and abundant evidence exists that macrophages are important target cells for HIV in vivo. A critical understanding of HIV infection, replication, and latency in macrophages is needed in order to determine the appropriate method of measuring and eliminating this cellular reservoir. This review provides a brief discussion of the biology and acute and chronic infection of monocytes and macrophages, with a more substantial focus on replication, latency and measurement of the reservoir in cells of myeloid origin.

Brain macrophages harbor latent, infectious simian immunodeficiency virus.

: The current review examines the role of brain macrophages, that is perivascular macrophages and microglia, as a potential viral reservoir in antiretroviral therapy (ART) treated, simian immunodeficiency virus (SIV)-infected macaques. The role, if any, of latent viral reservoirs of HIV and SIV in the central nervous system during ART suppression is an unresolved issue. HIV and SIV infect both CD4 lymphocytes and myeloid cells in blood and tissues during acute and chronic infection. HIV spread to the brain occurs during acute infection by the infiltration of activated CD4 lymphocytes and monocytes from blood and is established in both embryonically derived resident microglia and monocyte-derived perivascular macrophages. ART controls viral replication in peripheral blood and cerebrospinal fluid in HIV-infected individuals but does not directly eliminate infected cells in blood, tissues or brain. Latently infected resting CD4 lymphocytes in blood and lymphoid tissues are a well recognized viral reservoir that can rebound once ART is withdrawn. In contrast, central nervous system resident microglia and perivascular macrophages in brain have not been examined as potential reservoirs for HIV during suppressive ART. Macrophages in tissues are long-lived cells that are HIV and SIV infected in tissues such as gut, lung, spleen, lymph node and brain and contribute to ongoing inflammation in tissues. However, their potential role in viral persistence and latency or their potential to rebound in the absence ART has not been examined. It has been shown that measurement of HIV latency by HIV DNA PCR in CD4 lymphocytes overestimates the size of the latent reservoirs of HIV that contribute to rebound that is cells containing the genomes of replicative viruses. Thus, the quantitative viral outgrowth assay has been used as a reliable measure of the number of latent cells that harbor infectious viral DNA and, may constitute a functional latent reservoir. Using quantitative viral outgrowth assays specifically designed to quantitate latently infected CD4 lymphocytes and myeloid cells in an SIV macaque model, we demonstrated that macrophages in brain harbor SIV genomes that reactivate and produce infectious virus in this assay, demonstrating that these cells have the potential to be a reservoir.

Myeloid and CD4 T Cells Comprise the Latent Reservoir in Antiretroviral Therapy-Suppressed SIVmac251-Infected Macaques.

Human immunodeficiency virus (HIV) eradication or long-term suppression in the absence of antiretroviral therapy (ART) requires an understanding of all viral reservoirs that could contribute to viral rebound after ART interruption. CD4 T cells (CD4s) are recognized as the predominant reservoir in HIV type 1 (HIV-1)-infected individuals. However, macrophages are also infected by HIV-1 and simian immunodeficiency virus (SIV) during acute infection and may persist throughout ART, contributing to the size of the latent reservoir. We sought to determine whether tissue macrophages contribute to the SIVmac251 reservoir in suppressed macaques. Using cell-specific quantitative viral outgrowth assays (CD4-QVOA and MΦ-QVOA), we measured functional latent reservoirs in CD4s and macrophages in ART-suppressed SIVmac251-infected macaques. Spleen, lung, and brain in all suppressed animals contained latently infected macrophages, undetectable or low-level SIV RNA, and detectable SIV DNA. Silent viral genomes with potential for reactivation and viral spread were also identified in blood monocytes, although these cells might not be considered reservoirs due to their short life span. Additionally, virus produced in the MΦ-QVOA was capable of infecting healthy activated CD4s. Our results strongly suggest that functional latent reservoirs in CD4s and macrophages can contribute to viral rebound and reestablishment of productive infection after ART interruption. These findings should be considered in the design and implementation of future HIV cure strategies.IMPORTANCE This study provides further evidence that the latent reservoir is comprised of both CD4+ T cells and myeloid cells. The data presented here suggest that CD4+ T cells and macrophages found throughout tissues in the body can contain replication-competent SIV and contribute to rebound of the virus after treatment interruption. Additionally, we have shown that monocytes in blood contain latent virus and, though not considered a reservoir themselves due to their short life span, could contribute to the size of the latent reservoir upon entering the tissue and differentiating into long-lived macrophages. These new insights into the size and location of the SIV reservoir using a model that is heavily studied in the HIV field could have great implications for HIV-infected individuals and should be taken into consideration with the development of future HIV cure strategies.

Infectious Virus Persists in CD4+ T Cells and Macrophages in Antiretroviral Therapy-Suppressed Simian Immunodeficiency Virus-Infected Macaques.

Understanding the cellular and anatomical sites of latent virus that contribute to human immunodeficiency virus (HIV) rebound is essential for eradication. In HIV-positive patients, CD4+ T lymphocytes comprise a well-defined functional latent reservoir, defined as cells containing transcriptionally silent genomes able to produce infectious virus once reactivated. However, the persistence of infectious latent virus in CD4+ T cells in compartments other than blood and lymph nodes is unclear. Macrophages (Mϕ) are infected by HIV/simian immunodeficiency virus (SIV) and are likely to carry latent viral genomes during antiretroviral therapy (ART), contributing to the reservoir. Currently, the gold standard assay used to measure reservoirs containing replication-competent virus is the quantitative viral outgrowth assay (QVOA). Using an SIV-macaque model, the CD4+ T cell and Mϕ functional latent reservoirs were measured in various tissues using cell-specific QVOAs. Our results showed that blood, spleen, and lung in the majority of suppressed animals contain latently infected Mϕs. Surprisingly, the numbers of CD4+ T cells, monocytes, and Mϕs carrying infectious genomes in blood and spleen were at comparable frequencies (∼1 infected cell per million). We also demonstrate that ex vivo viruses produced in the Mϕ QVOA are capable of infecting activated CD4+ T cells. These results strongly suggest that latently infected tissue Mϕs can reestablish productive infection upon treatment interruption. This study provides the first comparison of CD4+ T cell and Mϕ functional reservoirs in a macaque model. It is the first confirmation of the persistence of latent genomes in monocytes in blood and Mϕs in the spleen and lung of SIV-infected ART-suppressed macaques. Our results demonstrate that transcriptionally silent genomes in Mϕs can contribute to viral rebound after ART interruption and should be considered in future HIV cure strategies.IMPORTANCE This study suggests that CD4+ T cells found throughout tissues in the body can contain replication-competent SIV and contribute to rebound of the virus after treatment interruption. In addition, this study demonstrates that macrophages in tissues are another cellular reservoir for SIV and may contribute to viral rebound after treatment interruption. This new insight into the size and location of the SIV reservoir could have great implications for HIV-infected individuals and should be taken into consideration for the development of future HIV cure strategies.

HIV Eradication Strategies: Implications for the Central Nervous System.

PURPOSE OF REVIEW: In addition to preventive protocols and antiretroviral therapy, HIV-1 eradication has been considered as an additional strategy to help fight the AIDS epidemic. With the support of multiple funding agencies, research groups worldwide have been developing protocols to achieve either a sterilizing or a functional cure for HIV-infection. RECENT FINDINGS: Most of the studies focus on the elimination or suppression of circulating CD4+ T cells, the best characterized HIV-1 latent reservoir. The role of the central nervous system (CNS) as a latent reservoir is still controversial. Although brain macrophages and astrocytes are susceptible to HIV-1 infection, it has not been ascertained whether the CNS carries latent HIV-1 during cART and, if so, whether the virus can be reactivated and spread to other compartments after ART interruption. Here, we examine the implications of HIV-1 eradication strategies on the CNS, regardless of whether it is a true latent reservoir and, if so, whether it is present in all patients.

A Quantitative Approach to SIV Functional Latency in Brain Macrophages.

Lentiviruses are retroviruses that primarily infect myeloid cells, leading to acute inflammatory infections in many tissues particularly, lung, joints and the central nervous system (CNS). Acute infection by lentiviruses is followed by persistent/latent infections that are not cleared by the host immune system. HIV and SIV are lentiviruses that also infect CD4+ lymphocytes as well as myeloid cells in blood and multiple tissues. HIV infection of myeloid cells in brain, lung and heart cause tissue specific diseases as well as infect cells in gut, lymph nodes and spleen. AIDS dementia and other tissue specific disease are observed when infected individuals are immunosuppressed and the number of circulating CD4+ T cells declines to low levels. Antiretroviral therapy (ART) controls viral spread and dramatically changes the course of immunodeficiency and AIDS dementia. However, ART does not eliminate virus-infected cells. Brain macrophages contain HIV DNA and may represent a latent reservoir that persists. HIV latency in CD4+ lymphocytes is the main focus of current research and concern in efforts to eradicate HIV. However, a number of studies have demonstrated that myeloid cells in blood and tissues of ART suppressed individuals harbor HIV DNA. The resident macrophages in tissues such as brain (microglia), spleen (red pulp macrophages) and alveolar macrophages in lung are derived from the yolk sac and can self renew. The question of the latent myeloid reservoir in HIV has not been rigorously examined and its potential as a barrier to eradication been considered. Using a well characterized SIV ART suppressed, non-human primate (NHP) model, our laboratory developed the first quantitative viral outgrowth assay (QVOA) designed to evaluate latently infected CD4+ lymphocytes and more recently developed a similar protocol for the assessment of latently infected myeloid cells in blood and brain. Using an SIV ART model, it was demonstrated that myeloid cells in blood and brain harbor latent SIV that can be reactivated and produce infectious virus in vitro. These studies demonstrate for the first time that myeloid cells have the potential to be a latent reservoir of HIV that produces infectious virus that can be reactivated in the absence of ART and during HIV eradication strategies. Graphical Abstract.

CCR2 Signaling Selectively Regulates IFN-α: Role of ß-Arrestin 2 in IFNAR1 Internalization.

An integral component of the antiviral response, type I IFNs require regulation to modulate immune activation. We identify ß-arrestin 2 as a key modulator of type I IFN in primary human macrophages, an essential component of the innate immune response. ß-Arrestin 2 was selectively activated by CCL2/CCR2 signaling, which induced a decrease in IFN-α, but not IFN-ß expression. Small interfering RNA knockdown of ß-arrestin 2 demonstrated its role in IFNAR1 internalization, as well as STAT1 and IRF3 activation. As a result, cytokine responses were not propagated following HIV infection and TLR3 activation. However, remnants of IFN signaling remained intact, despite ß-arrestin 2 activation, as IFN-ß, IFN-γ, IFN-λ1, IRF7, TRAIL, and MxA expression were sustained. Similar effects of ß-arrestin 2 on IFN signaling occurred in hepatocytes, suggesting that arrestins may broadly modulate IFN responses in multiple cell types. In summary, we identify a novel role of ß-arrestin 2 as an integral regulator of type I IFN through its internalization of IFNAR1 and a subsequent selective loss of downstream IFN signaling.

SIV Latency in Macrophages in the CNS.

Lentiviruses infect myeloid cells, leading to acute infection followed by persistent/latent infections not cleared by the host immune system. HIV and SIV are lentiviruses that infect CD4+ lymphocytes in addition to myeloid cells in blood and tissues. HIV infection of myeloid cells in brain, lung, and heart causes tissue-specific diseases that are mostly observed during severe immunosuppression, when the number of circulating CD4+ T cells declines to exceeding low levels. Antiretroviral therapy (ART) controls viral replication but does not successfully eliminate latent virus, which leads to viral rebound once ART is interrupted. HIV latency in CD4+ lymphocytes is the main focus of research and concern when HIV eradication efforts are considered. However, myeloid cells in tissues are long-lived and have not been routinely examined as a potential reservoir. Based on a quantitative viral outgrowth assay (QVOA) designed to evaluate latently infected CD4+ lymphocytes, a similar protocol was developed for the assessment of latently infected myeloid cells in blood and tissues. Using an SIV ART model, it was demonstrated that myeloid cells in blood and brain harbor latent SIV that can be reactivated and produce infectious virus in vitro, demonstrating that myeloid cells have the potential to be an additional latent reservoir of HIV that should be considered during HIV eradication strategies.

An SIV/macaque model targeted to study HIV-associated neurocognitive disorders.

Simian immunodeficiency virus (SIV) infection of pigtailed macaques is a highly representative and well-characterized animal model for HIV neuropathogenesis studies that provides an excellent opportunity to study and develop prognostic markers of HIV-associated neurocognitive disorders (HAND) for HIV-infected individuals. SIV studies can be performed in a controlled setting that enhances reproducibility and offers high-translational value. Similar to observations in HIV-infected patients receiving antiretroviral therapy (ART), ongoing neurodegeneration and inflammation are present in SIV-infected pigtailed macaques treated with suppressive ART. By developing quantitative viral outgrowth assays that measure both CD4+ T cells and macrophages harboring replication competent SIV as well as a highly sensitive mouse-based viral outgrowth assay, we have positioned the SIV/pigtailed macaque model to advance our understanding of latent cellular reservoirs, including potential CNS reservoirs, to promote HIV cure. In addition to contributing to our understanding of the pathogenesis of HAND, the SIV/pigtailed macaque model also provides an excellent opportunity to test innovative approaches to eliminate the latent HIV reservoir in the brain.

Brain Macrophages in Simian Immunodeficiency Virus-Infected, Antiretroviral-Suppressed Macaques: a Functional Latent Reservoir.

A human immunodeficiency virus (HIV) infection cure requires an understanding of the cellular and anatomical sites harboring virus that contribute to viral rebound upon treatment interruption. Despite antiretroviral therapy (ART), HIV-associated neurocognitive disorders (HAND) are reported in HIV-infected individuals on ART. Biomarkers for macrophage activation and neuronal damage in cerebrospinal fluid (CSF) of HIV-infected individuals demonstrate continued effects of HIV in brain and suggest that the central nervous system (CNS) may serve as a viral reservoir. Using a simian immunodeficiency virus (SIV)/macaque model for HIV encephalitis and AIDS, we evaluated whether infected cells persist in brain despite ART. Eight SIV-infected pig-tailed macaques were virally suppressed with ART, and plasma and CSF viremia levels were analyzed longitudinally. To assess whether virus persisted in brain macrophages (BrMΦ) in these macaques, we used a macrophage quantitative viral outgrowth assay (MΦ-QVOA), PCR, and in situ hybridization (ISH) to measure the frequency of infected cells and the levels of viral RNA and DNA in brain. Viral RNA in brain tissue of suppressed macaques was undetectable, although viral DNA was detected in all animals. The MΦ-QVOA demonstrated that the majority of suppressed animals contained latently infected BrMΦ. We also showed that virus produced in the MΦ-QVOAs was replication competent, suggesting that latently infected BrMΦ are capable of reestablishing productive infection upon treatment interruption. This report provides the first confirmation of the presence of replication-competent SIV in BrMΦ of ART-suppressed macaques and suggests that the highly debated issue of viral latency in macrophages, at least in brain, has been addressed in SIV-infected macaques treated with ART.IMPORTANCE Resting CD4+ T cells are currently the only cells that fit the definition of a latent reservoir. However, recent evidence suggests that HIV/SIV-infected macrophages persist despite ART. Markers of macrophage activation and neuronal damage are observed in the CSF of HIV-infected individuals and of SIV-infected macaques on suppressive ART regimens, suggesting that the CNS has continued virus infection and latent infection. A controversy exists as to whether brain macrophages represent a latent source of replication-competent virus capable of reestablishing infection upon treatment interruption. In this study, we demonstrated the presence of the latent macrophage reservoir in brains of SIV-infected ART-treated macaques and analyzed the reservoir using our established outgrowth assay to quantitate macrophages harboring replication-competent SIV genomes. Our results support the idea of the existence of other latent reservoirs in addition to resting CD4+ T cells and underscore the importance of macrophages in developing strategies to eradicate HIV.

Reactivation of simian immunodeficiency virus reservoirs in the brain of virally suppressed macaques.

OBJECTIVE: Resting CD4 T cells have been recognized as the major cell reservoir of latent HIV-1 during antiretroviral therapy (ART). Using an simian immunodeficiency virus (SIV)/macaque model for AIDS and HIV-related neurocognitive disorders we assessed the contribution of the brain to viral latency and reactivation. DESIGN: Pigtailed macaques were dual inoculated with SIVDeltaB670 and SIV17E-Fr and treated with an efficacious central nervous system-penetrant ART. After 500 days of viral suppression animals were treated with two cycles of latency reversing agents and increases in viral transcripts were examined. METHODS: Longitudinal plasma and cerebrospinal fluid (CSF) viral loads were analyzed by quantitative and digital droplet PCR. After necropsy, viral transcripts in organs were analyzed by PCR, in-situ hybridization, and phylogenetic genotyping based on env V1 loop sequences. Markers for neuronal damage and CSF activation were measured by ELISA. RESULTS: Increases in activation markers and plasma and CSF viral loads were observed in one animal treated with latency reversing agents, despite ongoing ART. SIV transcripts were identified in occipital cortex macrophages by in-situ hybridization and CD68 staining. The most abundant SIV genotype in CSF was unique and expanded independent from viruses found in the periphery. CONCLUSION: The central nervous system harbors latent SIV genomes after long-term viral suppression by ART, indicating that the brain represents a potential viral reservoir and should be seriously considered during AIDS cure strategies.

HIV Suppression Restores the Lung Mucosal CD4+ T-Cell Viral Immune Response and Resolves CD8+ T-Cell Alveolitis in Patients at Risk for HIV-Associated Chronic Obstructive Pulmonary Disease.

BACKGROUND: Lung CD4+ T-cell depletion and dysfunction, CD8+ T-cell alveolitis, smoking, and poor control of human immunodeficiency virus (HIV) are features of HIV-associated chronic obstructive pulmonary disease (COPD), but these changes have not been evaluated in smokers at risk for COPD. We evaluated the impact of viral suppression following initiation of antiretroviral therapy (ART) on HIV-specific immunity and the balance of the CD4+ T-cell to CD8+ T-cell ratio in the lung. METHODS: Using flow cytometry, we assessed the T-cell immune response in lung and blood specimens obtained from 12 actively smoking HIV-positive patients before ART initiation and after ART-associated viral suppression. RESULTS: HIV suppression resulted in enhanced lung and systemic HIV-specific CD4+ T-cell immune responses without significant changes in CD8+ T-cell responses. We observed an increase in lung ratios of CD4+ T cells to CD8+ T cells and CD4+ T-cell frequencies, decreased CD8+ T-cell numbers, and resolution of CD8+ T-cell alveolitis after ART in 9 of 12 individuals. Viral suppression reduced Fas receptor and programmed death 1 expression in lung CD4+ T cells, correlating with enhanced effector function and reduced susceptibility to apoptosis. HIV suppression rescued peripheral but not lung HIV-specific CD4+ T-cell proliferation, resulting in augmented effector multifunction. DISCUSSION: Together, our results demonstrate that HIV suppression restores lung mucosal HIV-specific CD4+ T-cell multifunctional immunity and balance in the ratio of CD4+ T cells to CD8+ T cells, often resolving CD8+ T-cell alveolitis in active smokers. Peripheral expansion and redistribution of CD4+ T cells and increased resistance to apoptosis are 2 mechanisms contributing to immunologic improvement following viral suppression in patients at risk for HIV-associated COPD.

Splenic Damage during SIV Infection: Role of T-Cell Depletion and Macrophage Polarization and Infection.

The effects of HIV infection on spleen and its cellular subsets have not been fully characterized, particularly for macrophages in which diverse populations exist. We used an accelerated SIV-infected macaque model to examine longitudinal effects on T-cell and macrophage populations and their susceptibilities to infection. Substantial lymphoid depletion occurred, characterized by follicular burn out and a loss of CD3 T lymphocytes, which was associated with cellular activation and transient dysregulations in CD4/CD8 ratios and memory effector populations. In contrast, the loss of CD68 and CD163(+)CD68(+) macrophages and increase in CD163 cells was irreversible, which began during acute infection and persisted until terminal disease. Mac387 macrophages and monocytes were transiently recruited into spleen, but were not sufficient to mitigate the changes in macrophage subsets. Type I interferon, M2 polarizing genes, and chemokine-chemokine receptor signaling were up-regulated in spleen and drove macrophage alterations. SIV-infected T cells were numerous within the white pulp during acute infection, but were rarely observed thereafter. CD68, CD163, and Mac387 macrophages were highly infected, which primarily occurred in the red pulp independent of T cells. Few macrophages underwent apoptosis, indicating that they are a long-lasting target for HIV/SIV. Our results identify macrophages as an important contributor to HIV/SIV infection in spleen and in promoting morphologic changes through the loss of specific macrophage subsets that mediate splenic organization.

Quantitation of Productively Infected Monocytes and Macrophages of Simian Immunodeficiency Virus-Infected Macaques.

UNLABELLED: Despite the success of combined antiretroviral therapy (ART), human immunodeficiency virus (HIV) infection remains a lifelong infection because of latent viral reservoirs in infected patients. The contribution of CD4(+) T cells to infection and disease progression has been extensively studied. However, during early HIV infection, macrophages in brain and other tissues are infected and contribute to tissue-specific diseases, such as encephalitis and dementia in brain and pneumonia in lung. The extent of infection of monocytes and macrophages has not been rigorously assessed with assays comparable to those used to study infection of CD4(+) T cells and to evaluate the number of CD4(+) T cells that harbor infectious viral genomes. To assess the contribution of productively infected monocytes and macrophages to HIV- and simian immunodeficiency virus (SIV)-infected cells in vivo, we developed a quantitative virus outgrowth assay (QVOA) based on similar assays used to quantitate CD4(+) T cell latent reservoirs in HIV- and SIV-infected individuals in whom the infection is suppressed by ART. Myeloid cells expressing CD11b were serially diluted and cocultured with susceptible cells to amplify virus. T cell receptor ß RNA was measured as a control to assess the potential contribution of CD4(+) T cells in the assay. Virus production in the supernatant was quantitated by quantitative reverse transcription-PCR. Productively infected myeloid cells were detected in blood, bronchoalveolar lavage fluid, lungs, spleen, and brain, demonstrating that these cells persist throughout SIV infection and have the potential to contribute to the viral reservoir during ART. IMPORTANCE: Infection of CD4(+) T cells and their role as latent reservoirs have been rigorously assessed; however, the frequency of productively infected monocytes and macrophages in vivo has not been similarly studied. Myeloid cells, unlike lymphocytes, are resistant to the cytopathic effects of HIV. Moreover, tissue-resident macrophages have the ability to self-renew and persist in the body for months to years. Thus, tissue macrophages, once infected, have the characteristics of a potentially stable viral reservoir. A better understanding of the number of productively infected macrophages is crucial to further evaluate the role of infected myeloid cells as a potential viral reservoir. In the study described here we compared the frequency of productively infected CD4(+) T cells and macrophages in an SIV-infected macaque model. We developed a critical assay that will allow us to quantitate myeloid cells containing viral genomes that lead to productive infection in SIV-infected macaques and assess the role of macrophages as potential reservoirs.

Activation-induced cell death drives profound lung CD4(+) T-cell depletion in HIV-associated chronic obstructive pulmonary disease.

RATIONALE: As overall survival improves, individuals with HIV infection become susceptible to other chronic diseases, including accelerated chronic obstructive pulmonary disease (COPD). OBJECTIVES: To determine whether individuals with HIV-associated COPD exhibit dysregulated lung mucosal T-cell immunity compared with control subjects. METHODS: Using flow cytometry, we evaluated peripheral blood and lung mucosal T-cell immunity in 14 HIV(+)COPD(+), 13 HIV(+)COPD(-), and 7 HIV(-)COPD(+) individuals. MEASUREMENTS AND MAIN RESULTS: HIV(+)COPD(+) individuals demonstrated profound CD4(+) T-cell depletion with reduced CD4/CD8 T-cell ratios in bronchoalveolar lavage-derived lung mononuclear cells, not observed in peripheral blood mononuclear cells, and diminished CD4(+) T cell absolute numbers, compared with control subjects. Furthermore, HIV(+)COPD(+) individuals demonstrated decreased pulmonary HIV-specific and staphylococcal enterotoxin B-reactive CD4(+) memory responses, including loss of multifunctionality, compared with HIV(+)COPD(-) control subjects. In contrast, lung mucosal HIV-specific CD8(+) T-cell responses were preserved. Lung CD4(+) T cells from HIV(+)COPD(+) individuals expressed increased surface Fas death receptor (CD95) and programmed death-1, but similar bronchoalveolar lavage viral loads as control subjects. However, programmed death-1 expression inversely correlated with HIV-specific lung CD4(+)IFN-γ(+) T-cell responses, suggesting functional exhaustion. Moreover, lung CD4(+) T cells from HIV(+)COPD(+) patients demonstrated increased basal and HIV antigen-induced expression of the early apoptosis marker annexin V compared with control subjects, which was significantly attenuated with anti-Fas blockade. Lastly, lung mucosal, but not blood, CD4(+)/CD8(+) ratios from HIV(+) patients significantly correlated with the FEV1, but not in HIV(-)COPD(+) patients. CONCLUSIONS: Together, our results provide evidence for profound lung mucosal CD4(+) T-cell depletion via a Fas-dependent activation-induced cell death mechanism, along with impaired HIV-specific CD4(+) immunity as immunologic features of HIV-associated COPD.

Tristetraprolin expression and microRNA-mediated regulation during simian immunodeficiency virus infection of the central nervous system.

BACKGROUND: The RNA-binding protein tristetraprolin (TTP) participates in normal post-transcriptional control of cytokine and chemokine gene expression, dysregulation of which contributes to the HIV-associated neurocognitive disorders. Transcriptional and post-transcriptional regulation of TTP has been described, including regulation by microRNA-29a. In the simian immunodeficiency virus (SIV) model of HIV CNS disease, control of cytokine/chemokine expression coincides with the end of acute phase infection. This control is lost during progression to disease. In this study, we assessed TTP regulation and association with cytokine regulation in the brain during SIV infection. RESULTS: Quantitation of TTP expression over the course of SIV infection revealed downregulation of TTP during acute infection, maintenance of relatively low levels during asymptomatic phase, and increased expression only during late-stage CNS disease, particularly in association with severe disease. The ability of miR-29a to regulate TTP was confirmed, and evidence for additional miRNA targeters of TTP was found. However, increased miR-29a expression in brain was not found to be significantly negatively correlated with TTP. Similarly, increased TTP during late-stage disease was not associated with lower cytokine expression. CONCLUSIONS: TTP expression is regulated during SIV infection of the CNS. The lack of significant negative correlation of miR-29a and TTP expression levels suggests that while miR-29a may contribute to TTP regulation, additional factors are involved. Reduced TTP expression during acute infection is consistent with increased cytokine production during this phase of infection, but the increases in TTP observed during late-stage infection were insufficient to halt runaway cytokine levels. While antisense inhibitors of the post-transcriptional targeters of TTP identified here could conceivably be used further to augment TTP regulation of cytokines, it is possible that high levels of TTP are undesirable. Additional research is needed to characterize members of the miRNA/TTP/cytokine regulatory network and identify nodes that may be best targeted therapeutically to ameliorate the effects of chronic inflammation in retrovirus-associated CNS disease.