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

Abstract

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.

Summary

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.

Introduction

Human Immunodeficiency Virus (HIV) remains a global epidemic, with approximately 39 million people infected and 1.3 million new infections as of 2022 [1]. HIV is a retrovirus that targets immune cells and increasing evidence demonstrates that myeloid cells (monocytes/macrophages) are targets of infection and capable of establishing viral reservoirs [2]. Infected macrophages harbor latent virus and are resistant to the cytotoxic effects of infection and viral replication [3]. While antiretroviral treatment (ART) succeeds in blocking viral replication, latently infected cells are not targeted as they are transcriptionally silent. As long-lived innate immune cells, macrophages pose a significant barrier to HIV-1 cure strategies and there remains a gap in knowledge regarding the establishment and maintenance of myeloid reservoirs. Further, macrophages are composed of distinct cell types and distributed throughout the body, creating a challenge in targeting this cellular reservoir. Given this heterogeneity, it is important to study each subtype and the impact that each macrophage reservoir has on viral persistence within the body. The goal of this review is to compile studies that have provided evidence of monocyte and macrophage reservoirs in anatomic sites throughout the body and summarize their contribution to viral persistence and pathogenesis of HIV-1.

Myeloid Cells — Macrophages

Macrophages are a heterogenous population of innate immune cells that are broadly distributed in tissues [4, 5]. Most adult tissue macrophages are derived from yolk sac progenitors during early embryogenesis and seeded in the tissues as long-lived cells with the propensity for self-renewal [6–8]. These include microglia, brain macrophages (perivascular, meningeal, circumventricular, and choroid plexus macrophages), splenic macrophages, alveolar and interstitial macrophages in the lung, Kupffer cells in the liver, macrophages within the reproductive systems, and many others [6, 7]. Thus, macrophages populate a variety of lymphoid and non-lymphoid tissues [9]. The maintenance of tissue macrophage populations is typically independent of repopulation by monocyte-derived macrophages (MDMs); however, MDMs supplement tissue macrophage populations in circumstances of infection and inflammation [10]. These phagocytic cells are critical to the response against pathogens, participate in tissue repair, and modulate the immune response and inflammation [11–13]. Despite the development of in vitro and in vivo models used to study HIV-1 infection and reservoir dynamics in macrophages, critical aspects of macrophage biology remain challenging to study. Given the broad diversity of tissues that harbor macrophages, each type is highly specialized and requires the development of tissue-specific assays to properly study macrophages as cellular targets of HIV-1 infection, reservoir establishment, and reservoir maintenance.

HIV-1 Infection of Macrophages

Macrophages are targets for HIV-1 infection and contribute to viral reservoirs [2]. Early in the HIV epidemic, studies revealed that macrophages were productively infected by HIV-1 and produced replication-competent virus [3, 14, 15]. Macrophages can be infected through multiple mechanisms including, phagocytosis of infected CD4+ T cells [16, 17], direct infection with cell-free virus [18, 19], and cell-cell transmission [20••]. Similar to CD4+ T cells, macrophages also express CD4 and CCR5/CXCR4, though at lower levels compared to CD4+ T cells, resulting in inefficient infection with cell-free virus [18]. Cell-to-cell transmission has been demonstrated to be a more efficient manner of infection for macrophages. The precise mechanisms of cell-to-cell infection are being determined; however, the formation of virological and immune synapses is the likely route of transmission [20••]. Further, infected macrophages have been shown to assemble viral particles in intracellular vesicles, virus-containing compartments (VCCs), as opposed to viral assembly at the cell membrane as in CD4+ T cells. This facilitates viral escape from the host immune defense and from ART [21]. Additionally, macrophages employ methods that reduce their susceptibility to infection and infected macrophages withstand the cytotoxic effects of the virus for long periods of time [3]. Macrophage infection has been previously reviewed in detail [2, 3, 16, 17, 20••]. For the purposes of this review, we will focus on establishment and maintenance of macrophage reservoirs in tissues.

Viral Reservoirs: Barriers to Cure

The major barrier to an HIV-1 cure is the latent HIV-1 reservoir which is established early in infection in immune cells and anatomical sites throughout the body. Three criteria define latent viral reservoirs: (1) viral genomes integrated into the host cells; (2) the reservoirs persist during transcriptional silencing; and (3) infected cells reactivate and produce infectious replication-competent virus after a period of latency [22, 23]. The establishment and maintenance of CD4+ T cell HIV-1 latent reservoirs have been extensively studied and continue to be a focus of research in the field [24, 25]. However, myeloid cells represent an additional cellular compartment that harbor latent virus. As latent HIV-1 cellular reservoirs in the blood and tissues, monocytes and macrophages play a critical role in the persistence of virus in the body despite ART. There is evidence in virologically suppressed people with HIV (vsPWH) and in non-human primate (NHP) simian immunodeficiency virus (SIV) models of HIV that HIV/SIV persists in monocytes and macrophages in blood and tissues. HIV-1/SIV DNA has been reported in highly purified monocytes [26–31, 32••] and tissue macrophages isolated from the brain [33, 34, 35••, 36••, 37, 38, 39••], lung [38, 39••, 40, 41••], spleen [38, 39••, 40], gut [42, 43], urethra [44, 45••, 46], and liver [47••]. However, there is ongoing discussion regarding the efficacy of ART in tissue macrophages and whether these cells are considered truly latent or if there is ongoing low-level replication despite ART [48, 49]. Therefore, assessment of ongoing replication, in addition to assessing DNA and inducibility, is essential to identify macrophage reservoirs within tissue.

While blood and cerebrospinal fluid (CSF) from PWH are regularly used in studies, tissues are more challenging to obtain. Animal models are a valuable resource to study HIV-1 infection, disease progression, reservoir dynamics, and cure strategies. NHP such as Asian macaques (rhesus, pigtail, and cynomolgus) are susceptible to SIV infection and develop AIDS-like disease similar to PWH [50]. Commonly used viruses in these macaque models include the SIVmac251, SIV/DeltaB670 and SIV/17E-Fr (dual inoculation), and SIVmac239 [38, 50, 51]. In addition, small animal models, such as “humanized mice” are commonly used to study viral replication and disease progression [50]. Animal studies point to the existence and persistence of monocyte and macrophage reservoirs in blood and tissues, including the central nervous system (CNS), throughout ART suppression [37, 38, 39••, 51, 52]. The main assays used to quantify the viral reservoir are the quantitative viral outgrowth assay (QVOA), which uses cells isolated from PWH or SIV-infected macaques and assesses replication-competent virus [40, 53]. As well as DNA-based assays that assess the total HIV-1 genomes and the quality of the genome via the intact proviral DNA assay (IPDA) [54, 55]. In this review, we explore key anatomic sites in which macrophages have been implicated as cellular viral reservoirs, their contribution to viral pathogenesis, and the models used to define them (Fig. 1, Table 1).

Fig. 1.

Graphical abstract depicting key anatomic sites in the body in which monocytes and/or macrophages are infected by HIV-1/SIV and evidence supporting the presence of viral reservoirs. Created with BioRender.com

Table 1.

Reports of HIV-1/SIV infection and reservoir characteristics in myeloid cells in blood and tissue

Myeloid cell in blood/tissue	Evidence of HIV-1/SIV infection and reservoir
Blood  • Monocytes  • Monocyte-derived macrophages	▪ Bone marrow progenitor cells are potential sites of initial HIV-1 infection [27]. ▪ Detection of HIV-1 DNA in highly purified monocytes from vsPWH [26, 29–31]. ▪ The mean half-life of HIV-1 in CD14+ monocytes (41.3 months) was significantly lower compared with activated CD4+ T cells (19.8 months) in vsPWH [28]. ▪ Intact proviral DNA was measured in monocytes and infectious replication-competent virus was produced from monocyte-derived macrophages from vsPWH [32••].
Central nervous system  • Microglia  • Brain macrophages	▪ HIV-1 reservoirs in the brain, in microglia and brain macrophages, persist despite ART in PWH [33, 34]. ▪ Total intact HIV-1 DNA in the frontal lobe was similar between ART-suppressed and viremic PWH [35••]. ▪ HIV-1 V3 env sequences were measured from the nuclei of CD68+ macrophages isolated from the frontal lobe of vsPWH [35••]. ▪ Brain myeloid cells (BrMCs), isolated from NHP and rapid autopsy of vsPWH, contained infectious replication-competent virus and sequencing revealed homogeneity in BrMCs sequences which were also distinct from peripheral sequences [36••]. ▪ In both dual inoculated (SIV/DeltaB670 and SIV/17E-Fr) ART-suppressed pig-tailed macaques and SIVmac251-infected ART-suppressed macaques [37, 38, 39••]:  ⚬ SIV DNA was detectable in CD11b+ brain macrophages.   ⚬ Latently infected CD11b+ brain macrophages were reactivatable ex vivo to produce infectious virus.
Lung  • Alveolar macrophages  • Interstitial macrophages	▪ HIV-1 infection of alveolar macrophages requires cell-to-cell transmission from infected CD4+ T cells [41••]. ▪ In both dual inoculated (SIV/DeltaB670 and SIV/17E-Fr) ART-suppressed pig-tailed macaques and SIVmac251-infected ART-suppressed macaques [38, 39••, 40]:   ⚬ SIV DNA was detectable in CD11b+ lung macrophages.   ⚬ Latently infected CD11b+ lung macrophages were reactivatable ex vivo to produce infectious virus.
Gastrointestinal tract  • Intestinal macrophages	▪ HIV-1 proviral DNA and p24 were measured in intestinal macrophages from the duodenum of vsPWH on long-term ART [42]. ▪ Higher total HIV-1 DNA levels compared with CD4+ T cell HIV-1 DNA levels indicate that myeloid cells may compose part of the viral reservoir in the rectum [43].
Spleen  • Splenic macrophages	▪ HIV-1 env-nef single genome sequencing of spleen tissue from vsPWH revealed two distinct phylogenetic patterns which indicate that there may be infection two major cell types, myeloid cells and CD4+ T cells [56]. ▪ In both dual inoculated (SIV/DeltaB670 and SIV/17E-Fr) ART-suppressed pigtailed macaques and SIVmac251-infected ART-suppressed macaques [38, 39••, 40]:   ⚬ SIV DNA was detectable in CD11b+ splenic macrophages.   ⚬ Latently infected CD11b+ splenic macrophages were reactivatable ex vivo to produce infectious virus.
Liver  • Kupffer cells  • Liver macrophages	▪ Liver macrophages isolated from vsPWH contain HIV-1 DNA; however, they cannot be reactivated to produce replication competent virus [47••].
Genital tract  • Male urethral macrophages  • Female reproductive tract macrophages	▪ Penile urethral macrophages are initial targets of HIV-1 [44]. ▪ HIV-1 DNA is detectable in urethral macrophages from vsPWH through nested PCR for HIV-1 gp120 V loops and Alu-gag nested PCR [45••]. ▪ Urethral macrophages from vsPWH contain HIV-1 reservoirs that are inducible and produce infectious virus [46]. ▪ Using vaginal tissue explants and GFP-reporter R5 HIV-1 viral strains, viral uptake was demonstrated in HAM56+ vaginal macrophages post-inoculation of whole explant tissue and CD13+ selected vaginal macrophages produced p24 following infection in culture [57]. ▪ CD14+ selected endometrial and decidual macrophages produced p24 in culture following infection with HIV-1BAL [58].

HIV-1 Monocyte Reservoir in the Blood

While monocytes and macrophages are readily infected by HIV-1, there remain questions about the stage of development in which these cells become infected. Wong et al. theorize that myeloid progenitor cells, such as CD34+ hematopoietic stem cells (HSCs) and committed myeloid progenitors (CMPs), are infected within the highly vascularized bone marrow [27]. These infected cells differentiate into monocytes that are released into the blood and lymph and circulate before infiltrating tissues and differentiating into HIV+ macrophages [28, 59–61]. Alternatively, or conjointly, uninfected circulating monocytes migrate into the tissues, become infected, and re-enter circulation while retaining their monocyte phenotype which facilitates the spread of virus from one tissue site to another [27, 62]. In addition, infected tissue macrophages can infect other cells through cell-to-cell transmission or through the release of mature virions resulting in direct infection [63, 64]. A recent study reported that integrated HIV-1 DNA could be detected in highly purified monocytes from vsPWH who had been on long-term suppressive ART [26]. This study also demonstrated that HIV-1 DNA was persistent in monocytes despite ART, as integrated HIV-1 DNA could be detected in longitudinal monocytes samples. This data complements a previous study that reported that the mean viral decay rate in CD14+ monocytes was significantly slower compared to activated CD4+ T cells [28]. The mean half-life of HIV-1 DNA in monocytes was 41.3 months while in resting and activated CD4+ T cells it was 23.6 months and 19.8 months, respectively [28]. Persistent HIV-1 DNA in monocytes and a slower decay rate suggest that monocytes may persist as a cellular reservoir in vsPWH.

Our group recently published that monocytes from vsPWH who were on long-term suppressive ART (5–20+ years) contain persistent and replication-competent HIV-1 reservoirs [32••]. Utilizing a myeloid-adapted IPDA we reported that 100% of participants had detectable HIV-1 in some form, while 40% of participants had detectable intact proviruses in monocytes [32••]. In addition, monocyte-derived macrophage quantitative viral outgrowth assays (MDM-QVOAs) from vsPWH demonstrated that the monocyte reservoir was replication-competent, as 50% of the participants had inducible proviruses with a median IUPM (infectious units per million cells) of 0.44, or a rate of 1 infectious monocyte in 2.5 million monocytes [32••]. The monocyte reservoir is stable and persistent in vsPWH as HIV-1 DNA and replication-competent virus could be detected at multiple visits, spanning 3 to 4 years [32••]. Together, these studies demonstrate that monocytes represent persistent cellular reservoirs of HIV-1 that need to be addressed as they have the potential to infiltrate the tissues and disseminate virus to anatomic sites throughout the body.

Central Nervous System (CNS) — Microglia and Brain Macrophages

The CNS harbors an HIV reservoir that is established in the brain within the first few days to weeks of infection and has been reported in both humans and NHP models [65]. Historically, the CNS was presumed to be an immune-privileged site; however, it is now recognized to be accessible to peripheral immune cells, such as T cells and monocytes, which play a crucial role in the persistence of virus within the brain [66]. Additionally, the CNS, protected by the blood brain barrier (BBB), is known to be a viral sanctuary. It is widely accepted that ART does not penetrate the CNS as efficiently as other tissues [67]. Drug concentrations within the brain tissue are highly variable and can exceed what is observed in CSF [68]. Additionally, CNS-targeted ART studies do not improve clinical outcomes [69]. During the immune response to infection within the brain, intermediate monocytes (CD14+CD16+) are preferentially recruited to the brain through the release of chemokines such as CCL2 [34, 70–72]. Infected intermediate monocyte recruitment has been linked to the infection of non-classical cells [65], glial cells such as microglia and astrocytes [73], as well as brain macrophages such as perivascular, meningeal, and choroid plexus macrophages [74].

HIV-1 infection in the brain results in cognitive impairment in approximately 50% of PWH [75]. The advent of ART has reduced the incidence of advanced forms of cognitive impairment; however, the percentage of individuals with impairment has remain unchanged [76, 77]. A study conducted on brain tissues from PWH, obtained from the National NeuroAIDS Tissues Consortium (NNTC), measured intact, defective, and total proviral genomes [78••]. Increased intact proviral genomes correlated with increased neuroinflammation as measured through NanoString analysis of genes related to inflammation and stress responses [78••]. Total HIV-1 DNA in intermediate monocytes (CD16+), that are likely to be infected in the periphery, have been correlated with poorer cognitive outcomes in vsPWH [79]. In virally suppressed women with HIV-1 (vsWWH), a higher proportion of intermediate monocytes in the blood correlated with poorer neuropsychiatric outcomes including lower global neuropsychological function, executive function, and processing speed [80••]. This suggests that HIV-1+ intermediate monocytes may be the primary drivers in the maintenance of the CNS reservoir and neuroinflammation and that sex may play a role in worsened cognitive outcomes seen in WWH.

In a recent study using brain tissue from PWH, donated to the Last Gift Cohort and National Disease Research Interchange (NDRI), and SIVmac239-infected ART-suppressed rhesus macaques, brain myeloid cells were isolated and cultured to assess the viral reservoir. There was successful measurement of viral DNA through gag ddPCR and integrated proviral DNA through Alu-gag PCR from brain myeloid cells from both PWH and macaques [36••]. Furthermore, sequencing of brain and peripheral viral genomes indicated that brain sequences cluster together, unlike peripheral sequences, which were more diverse. Additionally, brain sequences were distinct from the peripheral sequences [36••]. The most compelling evidence of an independent CNS reservoir is the example of symptomatic CNS viral escape in vsPWH. Several studies describe patients developing HIV-associated encephalitis (HIVE) [81] or elevated HIV-1 RNA in the cerebral spinal fluid (CSF) despite successful suppression of viremia in the plasma by long-term ART [82, 83]. These studies suggest that HIV-1 replicates independently in the CNS, despite ART-induced suppression of plasma viremia.

Macrophages are central to the viral reservoirs within the CNS. In addition to microglia, the primary innate immune cell within the CNS, brain macrophages such as perivascular macrophages, among others, are infected and produce replication-competent virus. Given the difficulty of obtaining brain tissue from PWH to conduct studies on microglia and brain macrophages, animal models such as the SIV-infected macaques are used to understand the viral reservoir dynamics within this tissue [23, 51]. In both dual inoculated and SIVmac251-infected ART-suppressed macaques, we demonstrated that SIV DNA was detectable in brain tissue and that CD11b+ brain macrophages could be reactivated ex vivo to produce replication-competent virus despite long-term ART-suppression [37, 38, 39••]. These studies demonstrate that despite long-term ART, brain macrophages remain a persistent cellular viral reservoir. Given the reduced bioavailability of ART in the brain due to the BBB [84], the release of viral proteins from infected brain macrophages [85], and the chronic activation of these cells, we can hypothesize that the brain macrophage reservoir likely contributes to persistent neuroinflammation and cognitive deficits in vsPWH [86]. Therefore, it remains important to understand the dynamics of CNS reservoirs in order to address the CNS-related clinical outcomes in vsPWH [87].

Lymphoid Organs and Mucosa-Associated Lymphoid Tissues

Alveolar and Interstitial Macrophages

Two main types of macrophages within the lung are the alveolar macrophages and interstitial macrophages. Alveolar macrophages reside in the airspaces of the lung and are critical first responders against airborne pathogens and respiratory infections. Interstitial macrophages reside in and maintain the lung connective tissue along with maintaining vasculature integrity [88]. However, both types serve as targets for HIV-1 and contribute to viral persistence and pathogenesis despite effective ART.

HIV-1 infection results in an increased risk for acute and chronic pulmonary diseases [89, 90]. It has been demonstrated that cell-to-cell contact with an infected CD4+ T cell is required for efficient infection of alveolar macrophages [41••]. Further, sequencing of HIV-1 env from both alveolar macrophages and plasma samples from ART-naïve PWH demonstrated that the alveolar macrophage and plasma env sequences clustered together indicating that plasma virus is likely disseminated to the lungs by infected CD4+ T cells [41••]. Infection of alveolar macrophages results in impaired function characterized by elevated phagocytic activity, increased pro-inflammatory cytokine production, and ultimately disruption of alveolar epithelium integrity. This further exacerbates pulmonary dysfunction and the increases the potential for respiratory diseases [91, 92]. Additionally, HIV-1 infection results in a decrease of anti-inflammatory CD163+ alveolar macrophages while pro-inflammatory CD163-CCR7+ alveolar macrophages were increased, shifting the immune profile in the lung. This promotes and prolongs an inflammatory response to infection in the lung [93]. While infected alveolar macrophages are long-lived, interstitial macrophages rapidly die post infection [94, 95]. It is proposed that the quick turnover and loss of interstitial macrophages is what is mainly responsible for lung tissue damage that is seen in PWH.

A study of HIV-1 infection in alveolar and interstitial macrophages in PWH is challenging as the collection of these cells is difficult. Alveolar macrophages can be assessed by bronchoalveolar lavage (BAL), whereas interstitial macrophages require lung biopsy or tissue collection post-mortem. However, the HIV macrophage reservoirs in the lungs have been assessed through NHP models. One study reported that SIV infects the lungs by targeting interstitial macrophages in the lung parenchyma [96]. SIV RNA was detected in lung macrophages as early as 6 days post infection, measured by combined immunohistochemistry (IHC) and in situ hybridization [96]. In untreated and treated dual inoculated pigtail macaques, we have shown that the number of infected interstitial macrophages correlated with the number of alveolar macrophages within each macaque, measured through MO-QVOAs using CD11b+ selected cells from lung tissue and BAL fluid, respectively [38, 40]. Further, in SIVmac251-infected ART-suppressed rhesus macaques, we demonstrated that the lung contained latently infected CD11b+ macrophages (both alveolar and interstitial), with detectable SIV gag DNA and reactivatable infectious virus [39••]. Additional studies are needed to determine the role lung macrophages play as an HIV-1 reservoir and their contribution to ongoing inflammatory issues in the lung in PWH.

Intestinal Macrophages

Macrophages play a critical role in immune surveillance and maintaining tissue integrity and function within the gastrointestinal (GI) tract [97]. Residing in the mucosal lining, the intestinal macrophages protect the body from microbes present in the intestinal lumen, maintain the microbiome, and provide antigens for the development of tolerance against commensal bacteria [97]. HIV-1 infection results in significant intestinal disease characterized by inflammation, increased permeability of the mucosa, malabsorption, and depletion of CD4+ T cells [98, 99]. In addition, studies have shown that HIV-1 infection results in an enrichment of macrophages within the GI tract of untreated PWH compared with vsPWH [100]. These findings were supported in SIVmac251-infected rhesus macaques in which the accumulation of intestinal macrophages correlated with progression to AIDS [101]. This is likely due to increased recruitment of monocytes in response to infection and contributes to the inflammatory environment in the gut.

While intestinal macrophages are susceptible to HIV-1 infection, their susceptibility is reduced when compared with monocyte-derived macrophages. This is due to decreased expression of CD4 and HIV-1 co-receptor CCR5 [57, 102]. There is detectable HIV-1 DNA and p24 in intestinal macrophages isolated from vsPWH [42, 43]. However, despite these findings, intestinal macrophages have been reported to have reduced or no capability to support viral replication [57, 103]. Questions remain about whether intestinal macrophages are productively infected with HIV-1 and capable of producing replication-competent virus. Additional studies are needed to determine the role intestinal macrophages play as an HIV-1 reservoir.

Splenic Macrophages

As the largest secondary lymphoid organ, the spleen has many functions, including housing immune cells. A variety of macrophages exist within in the spleen: marginal zone macrophages, metallophilic macrophages, red-pulp macrophages, and white pulp macrophages [104, 105]. Marginal zone macrophages bridge the innate and adaptive immune responses and are in a prime location to encounter pathogens from the blood, such as HIV [105]. HIV-1 infection in the spleen results in morphological and functional changes such as a reduction in the white pulp, recruitment of inflammatory cells, and cell and tissue death [106, 107]. While early studies have investigated the spleen as a sanctuary for HIV-1 and characterized CD4+ T cells as cellular reservoirs in vsPWH [56, 108, 109], little has been done to analyze macrophage reservoirs in this organ and their impact on viral persistence.

One study used HIV-1 env-nef single genome sequencing (SGS) and HIV-1 gag droplet digital PCR (ddPCR) in post-mortem tissues from vsPWH to analyze viral sequences [56]. SGS from the spleen demonstrated that env and nef sequences branched in two phylogenetically distinct patterns which suggest that there may be infection of multiple cell types, such as macrophages and clonal T cell populations [56]. Our group has demonstrated that splenic macrophages isolated from SIV-infected ART-suppressed rhesus macaques contain replication-competent virus [38, 39••]. In dual inoculated ART-suppressed pigtailed macaques, SIV DNA was detected in whole spleen tissue while SIV RNA was undetectable, demonstrating that ART is sufficient to block viral transcription while the reservoir persists [38]. MO-QVOAs performed using splenic CD11b+ macrophages showed consistent reactivation of replication-competent virus in culture [38]. We supported these findings in an additional NHP model, SIVmac251-infected ART-suppressed rhesus macaques, in which similar results were observed [39••]. In addition, our group has demonstrated that virus released by splenic CD11b+ macrophages from SIVmac251-infected ART-suppressed macaques was capable of de novo infection of activated CD4+ T cells from a healthy rhesus macaque [39••]. Further, in dual inoculated pigtail macaques, infection has been demonstrated to deplete CD68+ and fetal-derived CD163+CD68+ red pulp macrophage populations in the spleen while increasing CD163+ populations, likely causing a shift in the inflammatory profile in the tissue [107]. This change was observed in acute infection and was not resolved during chronic infection. One caveat to the work reported on splenic macrophages is that currently there is no marker that distinguishes tissue-resident macrophage populations from infiltrating monocytes in the spleen. As the spleen is a dense organ that is not easily perfused, the results reported in these studies are likely a mix of splenic resident macrophages and infiltrating blood monocytes. Together, these findings emphasize that splenic macrophages are persistently inflamed throughout infection and likely contain inducible viral reservoirs that may be supplemented by blood monocytes.

Peripheral Tissue

Liver Macrophages and Kupffer Cells

The liver functions to clear gut-derived products, including microbial products, from the blood before it enters systemic circulation. Kupffer cells, resident hepatic macrophages, are the first line of defense against bacterial and viral infections and maintain liver function by clearing microbes, microbial products, and dead or dying erythrocytes [110]. These cells make up about 80–90% of all tissue macrophages in the body. HIV-1 infection causes liver damage and disease through several pathways including cytopathic effects on hepatocytes through the induction of apoptosis due to exposure to the viral envelope glycoprotein, gp120 [111, 112]. Additionally, HIV-1 infection causes increased microbial translocation to the liver through the hepatic portal system due to the depletion of intestinal CD4+ T cells [99]. As the liver processes the increased gut-derived products in circulation, the Kupffer cells, and other immune cells, remain activated resulting in increased inflammation and immune activation in the tissue. Constant inflammation leads to the development of fibrosis as the Kupffer cells respond to the influx of pathogenic material [113].

Early studies demonstrate that Kupffer cells are susceptible to HIV-1 infection and produce viral products in viremic and vsPWH, as well as in animal models [114, 115]. However, limited studies exist that characterize the macrophage viral reservoirs in the liver. One study by Kandathil et al. indicated that liver macrophages from vsPWH contain integrated HIV-1 genomes [47••]. The liver tissue for this study came from vsPWH who required a liver transplant due to liver disease. Macrophages isolated from the liver were defined as liver macrophages as it is challenging to distinguish long-term tissue-resident macrophages from macrophages derived from infiltrating inflammatory monocytes. Despite this finding, it was determined that these latently infected cells did not contain replication-competent virus as exponential viral growth was not observed in culture [47••]. Viral propagation was only successfully measured from liver macrophages isolated from one patient, who was on ART for less than 12 months. The liver macrophages from the other patients, who were on ART for 15–113+ months, produced no infectious virus [47••]. Ultimately, this study demonstrates that while liver macrophages harbor HIV-1 DNA that persists despite ART, there is insufficient evidence of replication-competent HIV-1 in this cellular compartment. This highlights the issue of replication-competent HIV-1 reservoirs being extremely rare and difficult to measure. Further studies in liver macrophages, and Kupffer cells in particular, are required to determine if these macrophage subsets contribute to HIV persistence.

Genital Tract Tissue

Male Urethral Macrophages

One of the main modes of HIV-1 infection is sexual transmission [116, 117]. During sexual intercourse, micro injuries incurred on the male genital mucosa surface epithelia create a favorable environment for cell-free or cell-associated virus to breach through the submucosa to reach their target host cells [118]. This occurs through paracellular transport or transcytosis through the epithelial cells that comprise the urethral mucosa [116, 117, 119]. Penile urethral macrophages are the initial cells infected by HIV-1 [44]. Studies such as the work of Ganor et al. have highlighted that HIV-1 preferentially targets and infects resident urethral macrophages.

In penile tissue obtained from vsPWH, HIV-1 DNA was measured from isolated CD68+ macrophages using both nested PCR for HIV-1 gp120 V1/V2 and V3 loops and Alu-gag nested PCR [45••]. Infectious viral outgrowth was confirmed from urethral macrophages of vsPWH in a modified MO-QVOA using CD4+CCR5+CXCR4+ green fluorescent protein (GFP)-reporter GHOST cells [45••]. Urethral macrophages were transcriptionally active and produced HIV-1 RNA as shown through in situ hybridization of urethral tissue sections from vsPWH [45••]. In addition, both the gag protein precursor, p55, and its processed product, p24, were identified through immunofluorescence staining of VCC structures in CD68+ urethral macrophages [45••]. These findings suggest that urethral macrophages are persistent cellular reservoirs in vsPWH. In addition, infected and transcriptionally active urethral macrophages may contribute to the high viral loads measured in semen despite ART [120].

There has also been an examination of control of latency in infected urethral macrophages. A subset of latently infected inflammatory macrophages, IL-1R+S100A8+MMP7+ (M4), have been identified as transcriptionally active and enriched in the genital mucosa of vsPWH [121••]. Compared with other macrophages and CD4+ T cells, M4 macrophages in the urethra have increased expression of the alarmin S100A8 which was shown to reactivate latent reservoirs through shifting the metabolic profile of these cells and increasing glycolysis [121••]. S100A8 was shown to reactivate latent reservoirs in a glycolysis-dependent manner as inhibition of glycolysis in these cells blocked reactivation of replication-competent virus [121••]. This was further supported by the presence of whole viral particles observed in VCCs in M4 macrophages from vsPWH which additionally indicates that there is ongoing low-level viral production in this macrophage population [45••, 121••]. The macrophage reservoirs in the urethra pose a challenge for viral eradication and have raised questions about the potential for viral rebound and increased transmission risk during unprotected sexual intercourse.

Female Reproductive Tract (FRT) Macrophages

As one of the primary sites of viral entry and transmission in females, the reproductive tract plays a critical role in the context of HIV-1 infection. HIV-1 is transported to the FRT through semen during heterosexual transmission. The FRT consists of a lower (vagina and ectocervix) and upper (endocervix, endometrium, fallopian tubes, and ovaries) portion. In rhesus macaques, challenged vaginally with a single dose of a non-replicating SIV with a dual reporter system, it was shown that infection occurs in multiple sites throughout the FRT [122]. There appeared to be a preference for infection of both the vagina and the ovary indicating that viral migration towards the upper tract can occur [122].

Despite these findings, HIV-1 infection of macrophages in the human FRT remains relatively understudied. This may, in part, be due to difficulty in obtaining tissue. Macrophage distribution and phenotype varies throughout the FRT with an increased abundance found in the endometrium compared to other regions [123–125]. Vaginal macrophages have a monocyte-like phenotype as they express typical monocyte markers such as CD14, CD89, CD32, CD64 in addition to HIV CD4 receptor and co-receptors, CCR5 and CXCR4 [57]. In this study, vaginal tissue explants from healthy donors were inoculated with GFP-reporter R5 HIV-1 viral strains, and subsequent IHC of tissue sections revealed viral uptake in HAM56+ vaginal macrophages [57]. Additionally, CD13+ macrophages isolated from vaginal tissue support viral replication, as measured by p24, when inoculated with R5 viral strains in vitro [57]. In another study, macrophages in the upper portion of the FRT were implicated as the main target of HIV-1 infection [58]. Macrophages in the endometrium and decidua, the uterine mucosa during pregnancy, have an M2-like phenotype as they express CD68, CD163, CD206, and IL-10 [58]. Following infection with HIV-1_BAL, p24 antigen, measured through ELISA (enzyme-linked immunosorbent assay), could be detected in the supernatants of CD14+ selected endometrial and decidual macrophages [58]. In addition, both endometrial and decidual macrophages express high levels of SAMHD1 (sterile α motif domain HD domain-containing protein 1). SAMHD1 is an HIV restriction factor that hydrolyzes dNTPs thus reducing the capability of viruses to infect cells [126]. Protein expression of SAMHD1 was decreased in both endometrial and decidual macrophages that were treated with virus-like particles (VLPs) containing Vpx (virion-associated protein) compared to those treated with VPLs alone [58]. Vpx, encoded by HIV-2 and most SIV strains, is known to degrade SAMHD1 resulting in increased viral infection [127]. This was confirmed as HIV-1 infection was enhanced in both endometrial and decidual macrophages when the cells were infected with HIV-1 and treated with VPLs containing Vpx, and this effect was stronger in the decidua compared with the endometrium [58]. While further studies are required to measure the reservoir in FRT macrophages, these studies demonstrate that FRT macrophages are productively infected with HIV-1 and provide a great foundation to explore their contribution towards viral persistence and pathogenesis.

Conclusion

In this comprehensive review, we highlight the role of myeloid cells, particularly monocytes and macrophages, in the establishment and maintenance of HIV-1 reservoirs and their contributions to immune dysfunction. The complexity of HIV-1 myeloid reservoirs is compounded by specialized nature of each cell population and the tissue environment that harbors them. The evidence demonstrates the importance of these long-lived innate immune cells as contributors to viral persistence in vsPWH. Monocyte populations play an important role in dissemination of virus and reservoir maintenance in the tissues, including the brain. While the presence of integrated HIV-1 DNA is detectable in macrophage populations, the inability to reactivate latent reservoirs in some tissues raises questions of the contributions of these infected cells to viral persistence or whether the isolated cell populations, and assays performed, were sufficient to reach confident conclusions about reservoir characteristics. A deeper examination of the signaling pathways that lead to reactivation of HIV-1 myeloid reservoirs is required to understand the reservoir dynamics and to develop treatments and interventions for eliminating latent reservoirs. Ultimately, despite the challenges with obtaining tissue from PWH, various studies have utilized animal models and ex vivo methods to assess myeloid HIV-1 reservoirs. We demonstrate the need for a nuanced understanding of macrophage biology in the context of viral reservoir establishment and maintenance to determine how these reservoirs can be targeted for eradication.