For monocyte differentiation assays on day six MDMs, iMDDC, dMonWC1+, and dMonWC1neg were infected. For MDDC maturation assays, γδ T lymphocyte subsets and bacterial suspensions of *Map* were added on day 6 to iMDDCs. Cell cultures were infected at a multiplicity of infection (MOI) of 10:1 for 48 h with an Ontario-derived clinical bovine *Map* strain (gc86). The *Map* strain was cultured in Middlebrook 7H9 broth supplemented with 10% OADC (oleic acid, albumin, dextrose, catalase) enrichment (BD Biosciences), 0.05% Tween 80 (Sigma Aldrich), and 2 mg/L of mycobactin J (Allied Monitor, Inc., Fayette, MO, USA) referred to below as 7H9-OADC-MJ-T. Optical density (OD) was measured with a spectrophotometer (Genesys 10S VIS, ThermoFisher Scientific, Waltham, MA, USA) at 540 nm wavelength and quantification of bacteria was performed using a standard growth curve. The bacterial suspension was briefly sonicated with a sonic dismembrator (Model 120, Fisher Scientific) at 60% amplitude during 2 s pulses to disperse bacterial clumps. Aliquots of *Map* with viability of 97.4% measured by fluorescein diacetate (Sigma Aldrich) as described previously (25) were kept at −80°C in saline to ensure that the same *Map* passage was used throughout this study.

#### Antibodies and Flow Cytometry

Antibodies used in this study are shown in **Table 1**. Cells were collected 48 h after *Map* infection into serum-free media before staining and assessment (FACSAria IIu, BD Biosciences). Viability was assessed using Zombie NIR fixable viability kit (Biolegend, CA, USA). The acquisition software used was FACS Diva II, and data were analyzed using FlowJo software (Treestar, Inc., San Carlos, CA, USA) (Figure S2 in Supplementary Material shows gating strategy).

# DQ-Ovalbumin (DQ-OVA) Endocytosis Assay

DQ-ovalbumin was added at a concentration of 10 µg/mL to 60 µL of cell suspension (2 × 105 cells) of monocytes, MDMs, iMDDCs, mMDDCs, dMonWC1+, dMonWC1neg, iMDDC + WC1+, or iMDDC + WC1neg. Cells were incubated at 37°C for 45 min. After incubation, cells were washed with cold PBS and immediately analyzed by flow cytometry (FACSAria IIu, BD Biosciences) to measure the bright green fluorescence exhibited by the ovalbumin labeled with the pH-insensitive fluorescent dye, boron-dipyrromethene, upon proteolytic degradation after phagocytosis.

### *Map* Viability

Forty-eight hours after *Map* infection of MDMs, iMDDCs, dMonWC1+, or dMonWC1neg, culture supernatants were collected and wells containing *Map*-infected cells were washed twice with

day of peripheral blood mononuclear cell isolation (day 0). After 6 days of differentiation, cultures of monocyte-derived macrophages (MDMs), monocyte-derived dendritic cells (MDDCs), monocytes differentiated in presence of WC1+ (dMonWC1+) or WC1neg (dMonWC1neg) γδ T lymphocytes were obtained. On day 6, live *Map* was added at a multiplicity of infection of 10:1 to evaluate how it affected phenotype of MDMs, dMonWC1+ and dMonWC1neg; and MDDC after 48 h.

Table 1 | Anti-bovine monoclonal antibodies used for peripheral blood mononuclear cell immunophenotyping and coculture experiments.

*a Washington State University, Monoclonal Antibody Center.*

*bThermoFisher Scientific.*

*c Jackson ImmunoResearch.*

*dZenon antibody labeling kit.*

warm PBS to remove free *Map*. Cells were then detached from the flasks using TrypLE Express (Gibco), centrifuged at 400 × *g* for 2 min, and resuspended in sterile saline solution. Cell suspensions were stored at −80°C until further analysis. After thawing, cell suspensions were vortexed vigorously for 10 s to lyse cells, centrifuged at 400 × *g* for 2 min, resuspended in 7H9-OADC-MJ-T, and incubated in 24-well plates for 24 h at 37°C in 5% CO2. Contents of each well were centrifuged at 400 × *g* for 10 min and pellets were resuspended in 100 µL of saline solution in 5 mL conical tubes. One microliter of fluorescein diacetate at a concentration of 2 mg/mL (Sigma Aldrich) was added to each tube. After 30 min of incubation at 37°C, samples were analyzed by flow cytometry (Accuri C6, BD Biosciences). Standardization of the procedure and determination of gates were performed using a standard curve generated from known proportions of live and heat-killed *Map* as described previously (25).

#### Statistical Analysis

Statistical comparisons were performed using analysis of variance with SAS 9.4 software (SAS Institute, Cary, NC, USA) and GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA). The mean and SEM were calculated in experiments containing multiple data points. A *P* value of ≤0.05 was considered statistically significant.

### RESULTS

#### Phenotypic and Functional Characterization of Bovine Cells from the MPS Show Clear Distinctions between MDMs and MDDCs

To determine the expression of MPS cell markers in this study, several cell types were defined: monocytes were freshly isolated by adherence; MDMs were collected from flasks after fresh adherent monocytes were cultured for 6 days; MDDCs were generated by adding IL-4 and GM-CSF to fresh adherent monocyte cultures (**Figure 1**). Monocytes display significantly lower expression (*p* < 0.0001) of CD14, CD11b, CD11c, and CD172a compared with MDMs or MDDCs (**Figure 2**). Fresh monocytes

lack expression of CD163, CD1b, and CD205. The markers that help to differentiate MDMs from MDDCs in our system are CD11b, CD163, and CD172a, which are each significantly higher on MDMs compared to MDDCs with *p* values of <0.0001, <0.0001, and 0.0333, respectively (**Figures 2B–D**). To establish the phagocytic capacity of MPS cells in our study, monocytes, MDMs, and MDDCs were assessed with the DQ-OVA endocytosis assay. As expected, our data show that MDMs had the highest phagocytic capacity followed by MDDC (*p* = 0.0418), while monocytes have minimal phagocytic activity compared with MDMs and MDDCs (*p* < 0.0001 and 0.0028, respectively) (**Figure 3A**). To compare the ability of MDMs and MDDCs to alter *Map* viability, the viability of *Map* recovered from MDMs and MDDCs 48 h after *in vitro* infection with live *Map* was evaluated by flow cytometry. No significant differences were found between the viability of *Map* harvested from MDMs and MDDCs (*p* = 0.2929) (**Figure 3B**).

### The Presence of **γδ** T Lymphocytes during Monocyte Differentiation Does Not Affect Phenotype or Phagocytic Capacity; However, Viability of *Map* Recovered from dMonWC1neg Was Significantly Lower

To analyze the effect of WC1+ or WC1neg γδ T lymphocyte subsets on monocytes during differentiation, sorted WC1+ or WC1neg γδ T lymphocytes were added to freshly isolated autologous monocytes, and the cells were left in direct contact for 6 days prior to assessment of MPS surface-marker expression (**Figure 1**). Our data show that the phenotype of monocytes differentiated after 6 days in presence of either WC1+ or WC1neg γδ T lymphocytes (dMonWC1+ and dMonWC1neg) was not significantly different from the phenotype of monocytes differentiated without γδ T lymphocytes (MDMs) in our system. The mean and SD median fluorescence intensity (MFI) of surface markers of MPS in this

study are shown in Table S1 in Supplementary Material. The phagocytic capacity of dMonWC1+ and dMonWC1neg was assessed with the DQ-OVA endocytosis assay. dMonWC1+ and dMonWC-1neg showed an intermediate phagocytic activity between MDMs (*p* = 0.5403 and 0.1582, respectively) and MDDC (*p* = 0.3165 and 0.8772, respectively) (**Figure 3A**). To determine the ability of dMonWC1+ and dMonWC1neg to alter *Map* viability*,* the viability of *Map* recovered from these cells 48 h after live *Map* infection was evaluated by flow cytometry. The viability of *Map* harvested from dMonWC1neg was significantly lower compared with the viability of *Map* harvested from either MDMs (*p* = 0.0492), MDDC (*p* = 0.0044), or dMonWC1+ (*p* = 0.0277) (**Figure 3B**).

#### Presence of Live *Map* Does Not Alter Phenotype or Functions of Cells from the MPS

To determine the effect of the presence of *Map* on the phenotype of MPS cells in this system, *Map* was added to cultures of MDMs, MDDC, dMonWC1+, and dMonWC1neg for 48 h (**Figure 1**). The effect of the presence of *Map* was examined by comparing the same cell type (i.e., uninfected MDMs vs. *Map-*infected MDMs). Our data show that infection of MDMs, MDDCs, dMonWC1+, or dMonWC1neg with *Map* did not significantly alter surface expression of CD163, CD1b, CD205, CD14, CD172a, CD11b, or CD11c at 48 h post infection (Table S1 in Supplementary Material).

### MDDC Maturation Is Characterized by Upregulation of Surface Expression of MHC-I, CD80, and CD86, and Significant Reduction in Phagocytic Capacity

To examine the DC maturation process, iMDDCs were generated by adding IL-4 and GM-CSF to fresh peripheral blood-derived adherent monocyte cultures and their maturation was then induced by adding LPS for 48 h (**Figure 4**). As expected, our data showed that expression of MHC-I, CD80, and CD86 were significantly higher on mMDDCs compared with iMMDCs (**Figure 5**, *p* = 0.0105, <0.0001, and 0.0002, respectively); however, expression of MHC-II on mMDDCs was not significantly different from iMDDCs (*p* = 0.0640) (**Figure 5**). To study the effect of maturation of MDDCs on their phagocytic capacity, a DQ-OVA phagocytosis assay was performed on iMDDCs and mMDDCs using flow cytometry. Our data showed that the phagocytic capacity of mMDDCs was significantly reduced compared with iMDDCs (*p* ≤ 0.0001, **Figure 6**).

#### WC1neg **γδ** T Lymphocytes Increase MHC-II Expression on iMDDCs and Both **γδ** T Lymphocyte Subsets Reduce Phagocytic Capacity of iMDDCs

To determine the effect of WC1+ and WC1neg γδ T lymphocytes on maturation of MDDCs, sorted γδ T lymphocyte subsets were added to iMDDCs for 48 h (**Figure 4**). iMDDC + WC1neg had significantly increased expression of MHC-II compared with both iMDDC (*p* = 0.0013) and mMDDC (*p* = 0.0046) (**Figure 7**). Presence of γδ T lymphocytes did not affect the expression of CD80, CD86, and MHC-I on mMDDCs at 48 h post *Map* infection (data not shown). An interesting finding was increased individual variation of expression of MHC-II on mMDDCs compared with all iMDDCs, iMDDC + WC1+, and iMDDC + WC1neg, which may suggest that the DC maturation process varies widely between animals. To study the effects of WC1+ and WC1neg γδ T lymphocytes on the maturation of MDDCs, γδ T lymphocytes were added to cultures of iMDDC for 48 h and then the DQ-OVA phagocytosis assay was performed using flow cytometry to measure changes in phagocytic ability. iMDDCs cultured in the presence of either WC1+ or WC1neg γδ T lymphocytes had significantly reduced phagocytic ability compared to iMDDCs cultured without γδ T lymphocytes (*p* = 0.0111 and 0.0174, respectively) (**Figure 6**). These findings indicate that both γδ T lymphocyte subsets reduce phagocytosis by iMDDCs in our model.

### Live *Map* Was Associated with Significantly Increased Expression of MHC-I on iMDDC **+** WC1neg

To determine the effect of *Map* on maturation of MDDCs, live *Map* was added to cultures of iMDDC, iMDDC + WC1+, and iMDDC + WC1neg for 48 h (**Figure 4**). The presence of live *Map* was associated with significantly increased expression of MHC-I on iMDDC + WC1neg compared to iMDDC + WC1neg unexposed to *Map* (*p* = 0.0090) (**Figure 8**). The presence of live *Map* did not affect the expression of CD80, CD86, and MHC-II on MDDCs at 48 h post *Map* infection (data not shown).

### DISCUSSION

During the development of host responses against pathogens, monocytes are recruited to the site of infection where they differentiate into effector cells amidst crosstalk with resident tissue immune cells (35, 36). After encountering an antigen, immature DCs begin their maturation process, migrate to local draining lymph nodes where they present antigen to naïve T lymphocytes to initiate, or perpetuate antigen-specific immune responses (37, 38). γδ T lymphocytes are resident sentinel cells in a variety of mucosal surfaces but especially in the ileum, where infection with *Map* is generally assumed to initially occur (39). In this study, we sought to first define and characterize MPS cells from young calves; using that information, we then set out to determine how WC1+ and WC1neg γδ T lymphocytes affect (1) monocyte differentiation and (2) DC maturation, both processes important to initiation and propagation of effective immune responses during infection by *Map* and other pathogens. For monocyte differentiation experiments, sorted peripheral blood derived WC1+ or WC1neg γδ T lymphocytes were cocultured with freshly isolated autologous monocytes for 6 days so that their phenotype and function could be compared with the experimental controls previously defined: monocytes, MDMs, and MDDCs. For DC maturation experiments, sorted peripheral blood WC1+ or WC1neg γδ T lymphocytes were cocultured for 48 h with iMDDCs and then compared with iMDDCs (without γδ T lymphocytes) and mMDDCs (obtained after stimulation of iMDDCs with LPS for 48 h).

Cells from the MPS share precursors as well as several surface markers and functions which makes it difficult to clearly distinguish between them (6). We show that freshly isolated peripheral blood monocytes express low levels of CD14, CD11b, CD11c, and CD172a and lack expression of CD163, CD1b, and CD205. These data are consistent with a recent review showing that bovine monocytes express CD172a but lack expression of CD1b and CD205 [reviewed in Ref. (8)]. Other studies have defined three distinct phenotypic bovine monocyte subsets based on their variable surface expression of CD14 and CD16 among CD172a+ cells (40). Expression of CD11c and CD172a is considered constitutive in bovine monocyte subsets, while expression of CD11b is variable (41). We did not find significant expression of CD163 on monocytes in our study; however, expression of CD163 has been described by others in bovine monocytes (41). A possible explanation for these contradictory findings is the utilization of different clones of the CD163 monoclonal antibody. We used a murine anti-bovine clone (LND68A) while Corripio-Miyar et al. (41) used human clone (EDHu-1); potential concerns regarding interspecies cross-reactivity of monoclonal antibodies have been published (42).

In our model CD1b, CD11c, CD14, and CD205 were all upregulated following *in vitro* differentiation of monocytes; however, these particular markers do not reliably distinguish MDMs from MDDCs. Our data do suggest that MDMs can be phenotypically distinguished from MDDCs because of significantly higher expression of CD11b, CD163, and CD172a on MDMs compared to MDDCs. Bovine MDDCs have been described as CD172a+ while the expression of CD1b, CD11b, CD14, and CD205 vary depending on the subset of MDDC [reviewed in Ref. (8)]. MDMs have historically been classified by phenotype and function using surface-marker expression and cytokine-secretion profiles, respectively, but more recently classification of MDMs as either classically (M1) and alternatively activated (M2) macrophages using expression of CD163 has been described. M1 macrophages are CD163− and secrete pro-inflammatory cytokines while M2 macrophages are CD163+ and secrete low levels of pro-inflammatory cytokines and high levels of IL-10 (43). In our *in vitro* model, we neither identified CD163− populations of MDMs nor assessed cytokine concentration in supernatants. Thus, further research is required to characterize and classify cells of the bovine MPS under different isolation (a.k.a. magnetic beads, FACS, adherence), culture conditions *in vitro* and evaluating other relevant surface markers such as CD209 (DC-SIGN) (44), CD16 (41), CD68 (45), and CD11a (46). Regardless, our data support the basic hypothesis that MPS cells comprise a complex network of distinct cell subsets that though they share some overlapping phenotypic and functional characteristics, they polarize depending on the local microenvironment for specific functions (47).

After coculturing freshly isolated blood monocytes with sorted WC1+ and WC1neg γδ T lymphocytes (dMonWC1+ and dMonWC1neg, respectively) during the differentiation process, our data indicate that the presence of γδ T lymphocytes has no phenotype-altering effect on monocyte differentiation. Viability of *Map* recovered from dMonWC1neg, however, was significantly reduced suggesting that the presence of WC1neg γδ T lymphocytes improves the ability of differentiated monocytes (dMonWC1neg) to limit *Map* viability. In a previous study, we showed that the presence of either WC1+ or WC1neg γδ T lymphocytes cocultured with autologous *Map*-infected MDMs from 30- to 40-day-old calves was associated with reduced viability of *Map* recovered from MDMs (26). Taking into account that (1) in the previous study, MDMs were considered fully differentiated prior to the addition of γδ T lymphocytes into cocultures, (2) in the current study, significant differences were observed between the viability of *Map* recovered from dMonWC1+ and dMonWC1neg, and (3) when *Map* was introduced in the current study*,* WC1+ and WC1neg γδ T lymphocytes had been removed; we hypothesize that dMonWC1neg are distinct from classical MDMs, and that WC1neg γδ T lymphocytes alter the functional differentiation of peripheral blood monocytes. Based on these data, our hypothesis is that WC1neg γδ T lymphocytes have a direct effect on transcription factor expression during monocyte differentiation resulting in a monocyte-derived cell with increased ability to limit *Map* viability. Because we have observed that the number of γδ T lymphocyte subsets within tissues of mucosal surfaces is variable between calves (unpublished data), we hypothesize that inter-animal variability also influences the different immune responses and disease outcomes that can be commonly observed within groups of calves (i.e., a herd with endemic *Map* infection). This inter-animal variability might explain why some animals (i.e., those with more WC1neg γδ T lymphocytes or those that have more cognate γδ T lymphocyte/MPS cell interactions in the ileum) clear *Map* infection and do not progress to later stages of bovine paratuberculosis. Further studies are required to characterize the distribution and function of bovine γδ T lymphocytes in different mucosal and non-mucosal tissues.

To determine how γδ T lymphocyte subsets affect DC maturation, we first defined the characteristics of both iMDDC and mMDDC in our system by evaluating expression of specific maturation markers and co-stimulatory molecules. As expected and based on the current literature, our data confirm that MHC-I, CD80, and CD86 are useful markers to differentiate mMDDCs from iMDDCS because they are upregulated during the maturation process. Other bovine models have shown that *Salmonella typhimurium*-infected DCs had significantly increased expression of MHC-I, MHC-II, CD40, CD80, and CD86 (48). In our system, MHC-II was not upregulated on mMDDCs at 48 h post *Map* infection; however, the expression of MHC-II was highly variable between animals in our study. Inter-animal immune cell phenotypic variability has been well established in bovine studies of our lab and others (25, 26, 49), and the distinct pattern of high/low-effector functions and specific phenotypes in cattle is complex but probably explains an individual's unique ability to respond (or not) to infection. Timing may also have influenced our ability to detect early and transient changes in MHC-II expression. It is known that after stimulation of murine DC PAMPs receptors, MHC-II expression increases transiently but then decreases (50), though this phenomenon has not been shown in bovine DCs. Furthermore, it is known that MHC-II expression on bovine MDMs is downregulated between 24 and 48 h post *Map* infection (51) and because cells in this study were analyzed at only a single time point (48 h after *Map* infection), we may have thus been unable to detect early differences in the expression of MHC-II due to timing.

The presence of WC1neg γδ T lymphocytes in our coculture experiment was associated with significantly increased expression of MHC-II on iMDDC + WC1neg cells compared with iMDDCs and mMDDCs. Upregulation of MHC-II, along with CD86 and CD83 has also been induced by human Vγ9Vδ2 T lymphocytes on DCs, suggesting the ability of γδ T lymphocytes to specifically promote DC maturation (17). Furthermore, human iMDDCs induce Vγ9Vδ2 T lymphocytes to secrete pro-inflammatory cytokines required for their own maturation (52). This reciprocal effect has not been definitively demonstrated in cattle, and further research is required to determine if bovine DCs could induce this effect on either WC1+ and/or WC1neg γδ T lymphocytes.

A major functional change during MDDC maturation is reduced phagocytic capacity of MDDCs; this finding is supported by studies in adult cows (28). In our study, a reduced phagocytic capacity was observed in iMMDCs cocultured with either WC1+ or WC1neg γδ T lymphocyte subsets, which suggests that WC1+ or WC1neg γδ T lymphocytes induce "functional" MDDC maturation with respect to phagocytic capacity.

Our overall hypothesis was that early γδ T lymphocytes/ MPS/*Map* interactions influence the initiation of early local host immunity and potentially the induction of adaptive immunity and progression or eventual outcome of *Map* infection. To test our hypothesis, we first needed to define the phenotype and effector functions of MPS cells specifically under *in vitro* conditions in our laboratory. We have shown that cells from the MPS can be distinguished by collective examination of phenotype and function: (1) monocytes lack the expression of CD1b, CD205, and CD163; (2) MDMs express higher levels of CD11b, CD163, and CD172a compared to MDDCs; (3) mMDDCs express higher levels of CD11c, CD80, and CD86 compared to iMDDC; mMD-DCs have reduced phagocytic capacity compared to iMDDCs. In this study, the most significant findings related to the effect of γδ T lymphocytes include: (1) the presence of WC1neg γδ T lymphocytes contributes to differentiation of monocytes into cells with increased ability to limit *Map* viability and (2) both γδ T lymphocyte subsets induce functional MDDC maturation (reduced phagocytosis).

#### REFERENCES

#### ETHICS STATEMENT

All animal procedures in this study were approved by the Institutional Committee on Animal Care at the University of Guelph (Animal Utilization Protocol # 3373).

#### AUTHOR CONTRIBUTIONS

MMB and BLP performed the experiments; designed the experiments; interpreted the data; drafted the manuscript; reviewed and approved the final version of the manuscript; agreed to be accountable for the content of the work.

#### ACKNOWLEDGMENTS

We thank John F. Prescott, Lucy Mutharia, Stefan Keller and Shayan Sharif for their scientific advice and Laura Wright for her collaboration during the blood collection.

#### FUNDING

This work was funded by The National Science and Engineering Research Council (NSERC) of Canada. We acknowledge The Vanier Canada Graduate Scholarship and the Colombian Administrative Department of Science, Technology and Innovation (Colciencias) for their financial assistance.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at http://journal.frontiersin.org/article/10.3389/fimmu.2017.00534/ full#supplementary-material.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Baquero and Plattner. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Myeloid Cell interaction with Hiv: A Complex Relationship

*Vasco Rodrigues1 , Nicolas Ruffin1 , Mabel San-Roman2 and Philippe Benaroch1 \**

*1 Institut Curie, PSL Research University, INSERM U932, Paris, France, 2 Institut Curie, PSL Research University, UMR3216, Paris, France*

Cells of the myeloid lineage, particularly macrophages, serve as primary hosts for HIV *in vivo*, along with CD4 T lymphocytes. Macrophages are present in virtually every tissue of the organism, including locations with negligible T cell colonization, such as the brain, where HIV-mediated inflammation may lead to pathological sequelae. Moreover, infected macrophages are present in multiple other tissues. Recent evidence obtained in humanized mice and macaque models highlighted the capacity of macrophages to sustain HIV replication *in vivo* in the absence of T cells. Combined with the known resistance of the macrophage to the cytopathic effects of HIV infection, such data bring a renewed interest in this cell type both as a vehicle for viral spread as well as a viral reservoir. While our understanding of key processes of HIV infection of macrophages is far from complete, recent years have nevertheless brought important insight into the uniqueness of the macrophage infection. Productive infection of macrophages by HIV can occur by different routes including from phagocytosis of infected T cells. In macrophages, HIV assembles and buds into a peculiar plasma membrane-connected compartment that preexists to the infection. While the function of such compartment remains elusive, it supposedly allows for the persistence of infectious viral particles over extended periods of time and may play a role on viral transmission. As cells of the innate immune system, macrophages have the capacity to detect and respond to viral components. Recent data suggest that such sensing may occur at multiple steps of the viral cycle and impact subsequent viral spread. We aim to provide an overview of the HIV–macrophage interaction along the multiple stages of the viral life cycle, extending when pertinent such observations to additional myeloid cell types such as dendritic cells or blood monocytes.

Keywords: macrophages, sensing, viral assembly, antiretroviral therapy, reservoir, virus-containing compartment, restriction factors

# INTRODUCTION

The introduction of antiretroviral therapy (ART) to treat HIV infection in the mid 1990s was met with extraordinary success and dramatically improved the lives of patients, by turning a deadly infection into a manageable chronic disease. However, while able to prevent progression to AIDS, ART cannot eradicate HIV from the body, and a viral reservoir quickly rebounds after interruption of the therapy. In addition, HIV patients under suppressive therapy are at elevated risk of developing several non-AIDS related diseases, including cognitive impairment and cardiovascular problems.

#### *Edited by:*

*Christel Vérollet, Institut de Pharmacologie et de Biologie Structurale (IPBS), France*

#### *Reviewed by:*

*Florent Ginhoux, Singapore Immunology Network (A\*STAR), Singapore Quentin James Sattentau, University of Oxford, United Kingdom*

> *\*Correspondence: Philippe Benaroch<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology*

*Received: 05 October 2017 Accepted: 17 November 2017 Published: 30 November 2017*

#### *Citation:*

*Rodrigues V, Ruffin N, San-Roman M and Benaroch P (2017) Myeloid Cell Interaction with HIV: A Complex Relationship. Front. Immunol. 8:1698. doi: 10.3389/fimmu.2017.01698*

**445**

HIV mainly replicates in CD4 T cells and macrophages in the body. Loss of CD4 T cells has long been known as the major pathological event leading to AIDS. In macrophages, HIV infection does not induce immediate cell death and viral replication proceeds for extended periods of time.

Macrophages maintain tissue homeostasis by performing crucial housekeeping tasks. Their ubiquitous distribution in the body allows HIV to disseminate into organs and tissues and establish compartmentalized infection. Macrophages are also an important effector arm of the innate immune system. These cells detect HIV infection and express cellular factors that severely restrain the capacity of the virus to replicate.

Here, we discuss the interplay between HIV and macrophages. We review recent work highlighting the unique interaction between HIV and macrophages, at the cellular level. We further discuss evidence pointing to a role for macrophages as cellular reservoirs of HIV during ART and how they participate in the pathological morbidities that prevail in patients under therapy.

### MACROPHAGE ONTOGENY AND FUNCTION

Macrophages populate virtually all tissues of the body, where they perform a multitude of functions that are essential for tissue homeostasis, architecture, and protection (1). This wide range of macrophage action was described more than a century ago by Elie Metchnikoff. In his pioneering work, Metchnikoff observed the swarming and subsequent clearance of foreign objects by phagocytic cells in starfish larvae and water fleas (1). He correctly foresaw the importance of macrophages in the removal of obsolete cells, pathogen elimination, or sterile inflammation (2, 3).

Tissue macrophages have classically been considered as originating exclusively and in a continuous manner from bone marrow-derived monocytes, as part of the mononuclear–phagocyte system, a concept put forward by Van Furth in the 1970s (4). However, fate-mapping studies over the past decade have drastically changed our views on macrophage ontogeny. It is now widely accepted that many tissues are seeded with macrophages derived from the yolk sac or the fetal liver, during embryonic development [reviewed in Ref. (5)]. Once at their site of residency, macrophages proliferate locally to maintain a population size able to meet the requirements of the developing tissue or organ (6). The ability to self-renew suggests the existence of a subpopulation of tissue-resident macrophages with stem cell properties and capable of asymmetric cell division, but no such cell has yet been described in the tissues (6), with the possible exception of a subpopulation of epidermal Langerhans cells (7). Alternatively, the whole population of macrophages residing in a given tissue may be endowed with self-renewal potential, as suggested in studies with microglial cells or peritoneal macrophages (8–10). In some tissues, such as the brain or the liver, the resident macrophage population appears to be exclusively derived from embryonic cells throughout all adulthood (6). While monocytes may infiltrate these tissues under inflammatory or pathologic conditions, and differentiate into macrophages, they do not become part of the stable resident population (11). In stark contrast, embryonic macrophages that seed the gut prenatally appear to be completely replaced by monocyte-derived cells after birth (12). The factors that dictate this differential capacity of embryonically or monocyte-derived cells to stably engraft different tissues are not well understood and are an area of active research (6).

In their tissues of residency, macrophages perform a wide range of tasks. Some of these functions, such as apoptotic cell removal or extracellular matrix (MA) remodeling, are required in all tissues to different extents, indicating that macrophages are engaged in cross talks with their local microenvironment. The capacity to perform such general functions appears to be imprinted in the whole macrophage lineage and possibly involves the role of master transcription regulators such as PU (13). Other functions, by contrast, are specific to certain tissues. For instance, alveolar macrophages are specialized in clearing excessive surfactant, while macrophages of the red pulp of the spleen recycle iron from senescent erythrocytes (6). These site-specific functions are presumably imprinted on macrophages by tissue-specific signals and will induce transcriptional programs that define macrophage populations in different tissues (13).

Tissue macrophages are further subjected to environmental cues that occur in non-homeostatic conditions such as inflammation. Evolution has shaped macrophages as primary tissue sentinels (14). These cells are equipped with a broad range of receptors capable of detecting molecular patterns from all classes of microbes and multiple types of tissue damage, as well as receptors for chemokines and cytokines produced by immune cells (1). Integration of these multiple signals leads to what is commonly known as macrophage polarization (15). For instance, in an infected/inflamed tissue, macrophages may encounter microbial products such as LPS or be exposed to T cell-derived IFN-γ, leading to a polarized state known as M1, that is highly efficient in killing intracellular or ingested pathogens (15). Importantly, the majority of the knowledge gathered on macrophage polarization derives from well-defined *in vitro* experiments (16). These studies led to the M1 versus M2 model of macrophage polarization, which is unlikely to capture the complexity and the diversity of signals that macrophages can integrate *in vivo*. Furthermore, these polarizing stimuli act upon macrophages with previously imprinted tissue-specific programs. As such, similar polarizing signals probably lead to distinct phenotypes in macrophages from different tissues (13).

A wealth of information on the ontogeny, differentiation, and function of macrophages, derived from multiple studies in recent years, has profound implications in how we perceive the role of the macrophage in pathological settings, such as cancer, metabolic disease, or infections like HIV.

#### MACROPHAGES DURING ACUTE AND CHRONIC HIV INFECTION

The first description that tissue macrophages were permissive to HIV infection and capable of replicating the virus came in 1986, from the lab of Robert Gallo (17), amid the fast-paced period that characterized the early years of HIV/AIDS research. That very same pioneering study further provided the initial evidence that macrophages produce HIV for extended periods of time, hence coping with viral-induced cytopathy (17). In addition, the study revealed that in macrophages, HIV accumulates in apparent intracellular compartments absent from T cells.

HIV can infect macrophages as these cells express both the viral entry receptor, CD4, and co-receptors, CCR5 and CXCR4, that bind the viral envelop protein, gp120. Macrophage infection by HIV requires initial adsorption of the virus to the cell surface, mediated by lectin-like receptors, integrins, and heparan sulfate proteoglycans (18). Entry then probably takes place following virion internalization into macropinosomes (19) or endosomes, where fusion between the viral envelope and the host cell appears to occur (20), as recently proposed by a study following the internalization of fluorescent quantum dots encapsulated by infectious HIV-1 particles in primary macrophages (21).

Classically, macrophage-tropic viruses (M-tropic) were thought to exclusively employ CCR5 for entry (R5 viruses), while CXCR4-using strains (X4 viruses) were viewed as unable to enter macrophages and establish productive infection (22). This simplified categorization of macrophage tropism based on co-receptor usage has been proven imperfect as many R5 viruses are unable to infect macrophages (23), whereas some X4 isolates can (24). While co-receptor usage may frequently predict macrophage tropism, categorizing a virus as M-tropic requires demonstration of its ability to replicate *in vitro* in macrophages, although, understandably, this may not be a practical approach to test every isolate (18).

Transmitted/founder (T/F) viruses are the viral variants that initiate infection in a new host, at genital or rectal mucosal surfaces. Their sequences can be inferred by the mathematical modeling of virus evolution after single-genome amplification analysis of the plasma viral population (25). These types of analyses, across multiple studies, support the idea that most infections are initiated by a single or a very limited number of founder viruses (26). Biological characterization of T/F viruses demonstrated that they are usually unable to replicate in macrophages (27, 28), possibly due to the lower densities of the CD4 molecule on the macrophage surface, as compared with CD4 T cells (29). This suggests that macrophages are not an important source of viral replication in the initial stages of infection, emerging only later, as the virus adapts to infect cells with a lower CD4 density at the surface. In agreement, studies with mucosal explants from the human reproductive tract (30–32), or in non-human primates (33) support the idea that CD4+ T cells are the crucial targets at very early time points of infection.

Following migration from mucosal entry points into regional lymph nodes, *via* yet poorly described mechanisms, HIV rapidly disseminates systemically in the host. In SIV-infected rhesus macaques, viral spread to distal tissues such as the gastrointestinal (GI) tract or the spleen can be detected as early as 1 day after intravaginal inoculation and systemic distribution of SIV was observed by day 7 (34). This rapid but clinically silent spread is followed by the acute phase of HIV infection characterized by unrestrained viral replication in multiple tissues (35–37).

Macrophages are likely targets of HIV during the acute phase of infection, as viral nucleic acids have been detected in tissue macrophages from multiple organs in infected patients. These include Kupffer cells in the liver (38), microglial cells in the brain (39), alveolar macrophages in the lung (40), and intestinal macrophages obtained from several segments of the GI tract (41, 42). Importantly, replication-competent virus can be recovered from cultures of macrophages purified from lymphoid tissues of acutely infected rhesus macaques (43), implying that productive infection is taking place. It remains unclear how HIV disseminates to establish infection in these cells and tissues.

Monocytes can seed many tissues and differentiate locally into macrophages, turning this cell type into a potential vehicle for HIV dissemination across the myeloid compartment. Several reports claim indeed that replication of HIV-1 can take place *in vivo* in monocytes, even in patients under ART (44–46). Infected monocytes have been proposed to play a key role in viral dissemination to the brain due to their capacity to cross the blood–brain barrier (47), see Ref. (48).

At their sites of residency, macrophages constitutively patrol the tissues for danger signals, while also performing several housekeeping tasks. Interestingly, through the action of the viral accessory protein Nef, HIV is capable of reprogramming the migration of macrophages and selectively promotes a mesenchymal type of migration, while inhibiting the amoeboid type (49). The mesenchymal mode of migration is characterized by extensive extracellular MA remodeling, thus allowing the invasion of dense microenvironments, which may further promote viral dissemination and persistence. The relevance of these findings is supported by the increased accumulation of macrophages in the tissues of mice engineered to express the HIV Nef protein (49).

Alternatively, migratory, infected CD4+ T cells may serve as vehicles for HIV systemic dissemination, as suggested by intravital microscopy in infected humanized mice (50), and possibly transmit the virus to tissue-resident macrophages. Interestingly, during the acute phase, SIV-DNA-positive myeloid cells present in lymphoid tissues also contain rearranged T cell receptor DNA (51). This suggests that phagocytosis of infected T cells allows macrophages to acquire viral DNA, which is presumably taken to the macrophage degradative compartments for destruction preventing potential infection of macrophages (51). However, at least *in vitro*, cultured macrophages become productively infected after ingesting infected T cells (52). This mode of direct T cell-to-macrophage HIV transmission results in more efficient macrophage infection than exposure to cell-free virus (52). While this mechanism has yet to be demonstrated *in vivo*, it seemingly represents a strategy employed by HIV to maximize its spread, by exploiting the extensive phagocytic capacity of macrophages (53).

Chronic untreated HIV infection leads to extensive depletion of the body's CD4+ T cell pool and progression to AIDS (54). Concurrently, the viral population evolves to become more M-tropic (55), presumably because extensive CD4+ T cell loss makes the macrophage the most abundant cell target in advanced disease. Rhesus macaques treated with an antibody depleting CD4+ T cells before SIV infection mimic this advanced stage AIDS (56). In these animals, macrophages represent about 80% of the SIV-RNA+ cells in the tissues with evidence of productive infection of macrophages from lymphoid tissues and the brain, frequently associated with activation markers. Remarkably, plasma viral loads were two logs higher in depleted animals as compared with CD4+ T cell-sufficient controls, which led to rapid disease progression (56). Thus, in the context of CD4+ T cell depletion that might reflect the advanced AIDS status, extensive activation and viral replication in macrophages drives a precipitous progression of clinical disease.

### MACROPHAGE SENSING OF HIV AND INTRINSIC RESTRICTIONS TO VIRAL REPLICATION

The macrophage paradox refers to the fact that macrophages represent both the first line of defense against many pathogens, including viruses, and yet are exploited by many of these pathogens as their favorite cellular niche for replication (57). Such is the case of HIV-1, which efficiently replicates in macrophages. However, as sentinel cells, macrophages are equipped with a range of sensors that detect ongoing infection at many steps of the viral cycle and trigger cellular responses that will activate antiviral immunity (58). A number of these induced antiviral effector genes, known as restrictions factors, will block infection at specific steps of the viral life cycle (59). Thus, from the viral perspective, the extent to which HIV-1 replicates in macrophages must be tightly regulated as to ensure viral transmission/dissemination, while avoiding significant antiviral responses. Such delicate balance is achieved by a combination of precise employment of viral accessory proteins and usurpation of the normal function of cellular factors. Complete reviews devoted to the various restriction factors are available (59), we will focus here on factors that play important roles in infected macrophages.

Studies examining the initial stages of the viral cycle, i.e., upon viral entry and retro-transcription (RT) of the viral RNA into cDNA in the cytosol, proposed that HIV-1 escapes early innate sensing in myeloid cells before viral DNA integration. This would result from a combination of shielding the newly synthesized cDNA by viral and cellular factors (60, 61), and maintenance of very low levels of cytosolic viral cDNA due to the action of the cellular nuclease TREX1 (62, 63). However, induction of a weak, yet detectable, interferon-stimulated gene (ISG) response after HIV-1 infection of macrophages has also been reported (64, 65), with type I IFNs levels remaining undetectable (64, 66).

Examining these apparent discrepancies, we recently confirmed this transient response of monocyte-derived macrophages (MDMs) to HIV-1 infection, detectable as soon as 6 h postexposure and peaking at 24 h (67). Such response induces an ISG signature and depends on the induction of low levels of type I IFN. This sensing step is macrophage specific as it does not occur in monocyte-derived dendritic cells (MDDCs) exposed to HIV-1 (Decalf et al., unpublished results). The signal inducing the early ISG wave preceded reverse transcription but required viral fusion. Virus-like particles devoid of their genome, but capable of fusing, elicited a similar ISG response, indicating that viral nucleic acids were not implicated is this sensing step (67). Importantly, this early and transient ISG induction alone conferred partial protection to macrophages against subsequent HIV-1 infection. Different viruses carrying different envelopes and thus entering MDM although different receptors exhibited similar capacities to induce this response (67). The actual sensor of viral entry involved in this process remains to be identified. Membrane perturbations, such as fusion events, can elicit antiviral responses in macrophages, *via* the stimulator of IFN genes (STING)/tank-binding kinase-1 (TBK-1)/interferon-responsive factor-3 (IRF-3) pathway (68, 69). Thus, it is tempting to consider that the plasma membrane of the macrophage represents its first line of defense, and that sensing membrane perturbations, like viral entry, as soon as it occurs would be advantageous for the rapidity of the establishment of the antiviral response (**Figure 1**).

Retro-transcription represents a very specific and mandatory step for retroviral replication and is the target of SAM domainand HD domain-containing protein 1 (SAMHD1), a major restriction factor for HIV-1 replication in macrophages and other cells of the myeloid lineage, such as DCs (71, 72). Upon fusion of the HIV envelope with the host cell membrane, the viral capsid (CA) is released into the cytoplasm and the viral reverse transcriptase initiates reverse transcription of the viral RNA genome. SAMHD1 restricts HIV infection by depleting the cytosolic pool of dNTPs available for reverse transcription, *via* its deoxynucleoside triphosphate triphosphohydrolase activity (73) and possibly also by directly attacking viral RNA *via* its ribonuclease activity (74). While HIV-1 has no known factor to counteract SAMHD1 restriction, HIV-2 and several SIV strains encode the accessory protein Vpx that targets SAMHD1 for degradation (71, 72). Yet, HIV-1 is capable of replicating in macrophages, suggesting alternative mechanisms to bypass restriction.

Cyclin-dependent kinases (CDKs) phosphorylate SAMHD1 in proliferating cells, halting its activity (75, 76). Interestingly, primary macrophages in culture spontaneously and temporarily enter a G1-like state, without progressing to actual cell division, leading to CDK1 induction that limits the levels of SAMHD1 in its active form (77). This provides HIV-1 with a window of opportunity, as the virus preferentially infects these G1-like phase macrophages (77). Importantly, microglial and peritoneal macrophages recovered from mouse tissues similarly exhibit spontaneous cycling between G0 and the G1 state, suggesting a relevance for this mechanism *in vivo* (77). Other recent studies reported elevated levels of different members of the cyclin family in macrophages, rendering them permissive to HIV-1 infection, further supporting an important role for cell cycle proteins in the mechanism through which HIV-1 bypasses SAMDH1 restriction (78, 79).

Packaging Vpx into HIV-1 virions leads to a strong increase in infection efficiency of macrophages and DCs (80). However, such increased infectivity comes at the cost of detection of the viral cDNA by the cytoplasmic DNA sensor cGAS and induction of antiviral type I IFN (63). This suggests that HIV-1, unlike HIV-2, adopts a strategy of co-habitation with SAMHD1 in myeloid cells to avoid triggering antiviral immunity (81).

There is, however, a second phase of ISG induction in macrophages, peaking around 96 h postinfection that requires retro-transcription (67) and viral integration (70). This response is induced by detection of newly transcribed viral RNA by the RNA sensor retinoic acid-inducible gene I, and it requires the activity of the trans-activating (tat) HIV-1 accessory protein, responsible for the elongation of HIV-1 transcripts (70). Together,

Figure 1 | Schematic view of HIV-1 sensing by macrophages. (A) Macrophages sense HIV-1 at two independent steps of the viral cycle. *Left panel*—Early sensing of HIV-1 by macrophages requires viral fusion with the plasma membrane but precedes retro-transcription (RT). This sensing step is detectable by 4 h after cell exposure to the virus and declines after 24 h when RT is inhibited. While the actual sensor involved remains to be identified, it activates the kinase tank-binding kinase-1 (TBK1), leading to production of type I IFN, signaling *via* IFNAR, and triggering of interferon-stimulated genes (ISGs). *Right panel*—The second wave on HIV-1 sensing is measurable only 48 h after cell exposure to the virus. It requires integration of the viral genome in the host DNA and transcription of viral RNAs, which appear to be the viral component triggering the late ISG response. Here also, the actual sensor remains to be identified, but retinoic acid-inducible gene I (RIG-I) is a likely candidate as the signaling cascade involves the adaptor MAVS and IRF-1 and IRF-7, leading to type I IFN production. (B) Schematic representation of the two ISG waves induced by the sensing steps described in panel (A). The full line represents the putative measurable ISG response, whereas the dashed lines indicate the contribution of the individual waves for the measurable response. (C) This table resumes the main characteristics associated with the two sensing mechanisms through which macrophages detect HIV-1 and was established based on Ref. (67, 70).

the two sensing steps confer an ISG signature in HIV-1-infected macrophages that may contribute to maintaining a low level of viral replication in this cell type (67, 70) (**Figure 1**).

Once viral proteins are produced, a key step in the assembly of new viral particles is the incorporation of the viral envelope. Two recently identified restriction factors, active in macrophages, target the viral envelope and thus reduce infectivity. Membraneassociated RING-CH 8 is highly expressed in myeloid cells where it retains the viral envelope glycoproteins intracellularly hence impairing their incorporation into the budding viral particles (82). The guanylate binding protein-5 (GBP5) is highly inducible by type I IFN and interferes with processing, trimming and incorporation of the HIV-1 envelope, rendering the produced virions less infectious (83). Interestingly, the viral genes encoding Env and the HIV-1 accessory protein Vpu are expressed from the same bicistronic RNA (84). Deletion of *Vpu* from the viral genome enhances Env expression and renders HIV-1 less susceptible to GBP5 restriction (83). These observations may explain the high frequencies of defective Vpu gene observed in M-tropic HIV-1 strains (85).

Not surprisingly, restriction also takes place during the late phase of HIV replication cycle in macrophages. Tetherin (or BST2) is an interferon-inducible transmembrane protein that restricts HIV-1 particle release by inserting its C-terminal end into the viral lipid bilayer (86). As a result, newly formed virions are unable to leave the surface of infected T cells; i.e., they stay tethered (87). Tetherin activity is counteracted by Vpu that mediates its surface downregulation and degradation (88). HIV-1 infection of macrophages upregulates tetherin in an apparently IFN-independent but Nef-dependent manner (89). However, despite the presence of Vpu, HIV-1-infected macrophages still express detectable levels of tetherin that appears to partially restrict viral particle release (89).

The interferon-induced transmembrane (IFITM) proteins belong to a small family of highly related proteins and act has as broad restriction factors able to interfere with the replication of many viruses. These relatively short proteins (around 130 aa) were initially characterized for their capacity to protect IFITM expressing cells from HIV-1 infection (90, 91). In addition, viral particles produced by IFITM expressing cells exhibit a reduced infectivity due to their incorporation of IFITM into the viral envelope (92). Whether present in the membrane of the target cell or the viral particles, IFITM proteins appear to impair viral fusion. Of note, endogenous IFITM3 also inhibits cell-to-cell transmission, and its inhibitory effect is stronger when it is present in viral particles than when part of the target cell membrane (92). The mechanism of action of IFITM is still unclear but probably relies on their capacity to reduce the fluidity of the membranes where they insert, thereby preventing viral fusion (or hemifusion). The precise topology of IFITM proteins is probably key to understand how they work, but this aspect is still debated. Mutagenesis studies combined with secondary structure predictions indicate that IFITM3 is a type 2 transmembrane protein that possesses an amphipathic helix adjacent to two palmitoylated cysteines (93). Silencing of IFITM1, 2, and 3 in HIV-1 producing cells resulted in increased infectivity of the viruses released (94). Importantly, the impact of such silencing was more striking in MDM than in any other cell type (94), raising the possibility that IFITM expression induced early in HIV-1-exposed MDM, as we documented (67), reduces the infectivity of the viral progeny.

In conclusion, low-level sensing of HIV-1 by macrophages results in the induction of a panoply of restriction factors that severely restricts infection. This antiviral state still allows for a certain degree of replication-competent viral production and may explain why HIV-1-infected macrophages are so resistant to the cytopathic effects that the virus exhibits in other cell types. The net effect, however, is that macrophages produce HIV-1 for extended periods of time which may favor long-term viral persistence.

### CELL BIOLOGY OF HIV ASSEMBLY IN MACROPHAGES: FROM GAG SYNTHESIS TO PARTICLE RELEASE

In CD4+ T cells and model cell lines, HIV assembly and budding take place at the plasma membrane. By contrast, in infected macrophages HIV buds into an apparently intracellular compartment, known as the virus-containing compartment (VCC). The VCC is a unique compartment with topological and biochemical properties distinct from late endosomes or multivesicular bodies, see Ref. (95). Indeed, the VCC (i) lacks classical markers of endosomal and lysosomal compartments (96, 97); (ii) possesses a near neutral pH (97); and (iii) is connected to the extracellular milieu making its lumen accessible to small membrane-impermeable dies (96, 98–100). The compartment consists of a complex membranous system of interconnected tubules and vesicles enclosing immature and mature viral particles in its lumen as well as budding virions in its limiting membrane (99) (**Figure 2**). The limiting membrane of the VCC possesses specific biophysical properties due in part to the particular membrane topology of the proteins that are inserted and the presence of high levels of cholesterol (101).

Evidence supports the notion that the VCC originates from intracellular sequestration of domains of the plasma membrane with a specific protein and lipid composition (102, 103). Cell surface proteins found in the limiting membrane of the VCC include the tetraspanins CD9, CD53, CD81, and CD82 (96, 104) that provide membrane rigidity; the scavenger receptor CD36 (105) that contains two cytoplasmic tails; CD44 (98), a receptor with promiscuous capacity to bind numerous ligands; and MHC II complexes (106) (**Figure 2**). The restriction factors IFITM, with their peculiar topology of membrane insertion, are incorporated into viral membranes, especially in viruses produced by MDM (94), and may therefore also associate with the limiting membrane of the VCC, potentially contributing to a decreased membrane fluidity. As in T cells, the ESCRT complex is in charge of promoting the fission of nascent particles (107) and ESCRT III proteins that are key players in the late abscission process have been found at the VCC limiting membrane (108). Further supporting a surface origin for the VCC, fluorescence recovery after photobleaching (FRAP) experiments demonstrated that the VCC limiting membrane and the plasma membrane are in rapid equilibrium (102).

Given that newly formed particles bud away from the cytosol toward the lumen of the VCC, they become wrapped with VCCderived membrane (**Figure 2**). The proteomic analysis of purified HIV-1 particles released by infected MDM published more than 10 years ago (109) thus reflects the protein composition of the VCC limiting membrane, and still represents a valuable source of information. Studies published thereafter indeed confirmed not only the presence of the given proteins at the VCC limiting membrane but also revealed their role in the viral assembly process [see, for instance, CD36 (105), IFITM (94), and CD18 or Filamin A (110)].

An issue debated since the initial characterization of the VCC is whether some degree of viral budding can also occur at the surface of the macrophage. Indeed, some studies claimed that budding at the surface occurs alongside budding at the VCC (99, 111). A recent study provided an exhaustive examination of the site of virion assembly in individual macrophages by using a viral mutant arrested at the budding stage (103). Although such arrested buds could potentially move toward the plasma membrane, the analysis indicated that only about 5–12% of budding events occur at the cell surface. Moreover, these events were concentrated in the region where the VCC connected with the surface (103). These observations confirm that the VCC is the primary site of HIV assembly in macrophages, while budding at the surface is, at best, rare.

Thus, the specific lipid and protein composition of the limiting membrane of the VCC seem to constitute an assembly platform (101, 112), and presumably possess specific properties required for assembly and budding. However, which cellular and viral determinants direct HIV assembly and budding in macrophages to the VCC, remain poorly elucidated. The Gag polyprotein precursor drives the multistep process of HIV assembly and coordinates the activity of the cellular players involved. Recent studies indicate that viral genomic RNA is an important player/cofactor in the viral assembly process. Using elegant cross-linking immunoprecipitation sequencing coupled to membrane flotations, Kutluay

Figure 2 | The virus-containing compartment (VCC) in macrophages. (A) Electron micrograph depicting HIV-1-infected monocyte-derived macrophages (MDMs). MDMs were differentiated from monocytes purified from the peripheral blood of healthy human donors, by culture over 7 days in the presence of M-cerebrospinal fluid. Cells were then infected with HIV-1 NL-AD8 and fixed and embedded in epon 5 days postinfection. Ultrathin sections were processed for electron microscopy and imaged using a Philips 120 keV. For clarity, VCC is pseudo-colored in green and mitochondria in blue. (B,C) Magnifications of VCC present in the MDM depicted in panel (A). Arrowheads indicate viral buds. A thick molecular coat, electron dense and often associated with the VCC limiting membrane of the VCC can be seen in panel (C), see arrowheads. (D) Schematic representation of the late phases of the HIV life cycle in macrophages. (1) Gag monomers initiate oligomerization in the cytoplasm, forming dimers with the viral genomic RNA and (2) subsequently bind the plasma membrane *via* interactions with acidic phospholipids. (3) In macrophages, high-order Gag multimerization and formation of a viral bud only occurs at the limiting membrane of the VCC. (4) Gag subsequently recruits the components of the ESCRT complex that ensure fission of the budding viral particle into the lumen of the VCC. (5) Immature viral particles accumulate inside the VCC and (6) convert into mature viral particles *via* the activity of the viral protease. The restriction factor BST2/tetherin and Siglec-1/CD169 may contribute to the retention of viral particles within the lumen of the VCC. The limiting membrane of the VCC is tightly associated with the microtubule network on which kinesins may drive the transport of the VCC toward the cell periphery. *Inset*: Magnification of a region associated with a molecular coat: the VCC limiting membrane constitutes a platform for viral assembly where Gag oligomerization will lead to virus production. Therefore, the viral and VCC membranes share similar composition. They are enriched in particular lipids such as cholesterol and transmembrane proteins. These include the tetraspanins CD9, CD53, CD81, and CD82, the scavenger receptor CD36, the integrins CD18/CD11, or the surface glycoprotein CD44 (see text for details and references). The electron micrograph depicted in this figure panel results from original work performed in our lab and has not been published elsewhere previously.

et al. established that assembly starts in the cytoplasm where monomers and dimers of newly synthesized Gag bound to viral genome accumulate (113, 114). Oligomerization of Gag requires interactions with membrane enriched in cholesterol (115), *via* its MA domain that directly binds to membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) (116) [see Ref. (112)]. The levels of PI(4,5)P2 in the VCC are similar to those at the surface (102) and hence should not be a determining factor that directs viral assembly to the VCC. Assembly of the viral particle at the membrane is coordinated *via* lateral interactions promoted by the CA and nucleocapsid (NC) domains of Gag, leading to a nascent virion that buds off the membrane (117). Gag possesses two RNA binding domains (NC and MA) with different specificities (115). In addition, the RNA binding specificities for the viral genome versus cellular RNA, i.e., mRNA and tRNA, present in the cytosol change during these processes finally promoting packaging of viral genomic RNA into the nascent viral particles (115).

Aiming to elucidate the role of the viral factors dictating the assembly site of HIV in macrophages, Inlora and colleagues evaluated the impact of targeted deletions or mutations in the different Gag domains, on virion assembly at the VCC (118). Viral mutants unable of high-order multimerization, due to NC substitutions, distributed equally between the VCC and the cell surface (118). This indicates that Gag initially binds the membrane arbitrarily at the surface membrane or the VCC and initiates low-order multimerization. However, in macrophages, high-order multimerization and complete virion assembly occur at the VCC but not at the surface.

Assembly of the polyprotein Gag precursor leads to the formation of a bud head and stalk. Gag further coordinates the recruitment of important cellular factors. These include the ESCRT proteins; key players critically required for proper abscission of the nascent viral particles. Gag recruits components of the ESCRT complex through its late p6 C-ter domain, thus promoting severance of the viral stalk and particle release in the VCC. The precise molecular mechanisms involved in membrane abscission have stimulated numerous studies and corresponding excellent reviews [see, for instance, Ref. (107, 119)], and thus will not be discussed here.

The molecular players implicated in the establishment and maintenance of the VCC's intricate architecture remain poorly characterized. The cytoskeleton appears to play a key role in maintaining the integrity of the VCC. Indeed, a meshwork of filamentous actin surrounds the VCC and treating MDMs with actin-depolymerizing agents causes dispersion of the VCC throughout the cell (102). Moreover, the VCC limiting membrane is often surrounded by an electron dense and thick molecular coat visible by EM and containing CD18, a β2 integrin and its associated α integrins CD11b and CD11c (**Figure 2**) (120). Proteins known to interact with integrins are also associated with the molecular coat, including actin and focal adhesion scaffold/ linker proteins such as talin, vinculin, and paxillin (120). These adherent complexes appear to be involved in the maintenance of the architecture of the VCC; however, CD18 silencing does not affect the amount of virus released nor its infectivity (120). Links between VCC and the autophagy machinery in HIV-1-infected macrophages have been investigated in a few studies that should be extended in the future (121).

The VCC limiting membrane appears tightly associated with the microtubule network and disruption of this network by nocodazole exposure leads to relocalization of the VCC into the perinuclear area (122). This suggested that kinesins, molecular motors associated with microtubules, could be involved in maintaining the correct positioning of the compartment. The kinesin II, KIF3A, is closely associated with VCCs in infected macrophages (122). Moreover, time-lapse microscopy in primary MDM showed paired movements of Gag and KIF3A. Importantly, KIF3A silencing in HIV-1-infected macrophages reduced viral particle release and increased intracellular Gag and the VCC volume. Overall, KIF3A is likely involved in the transport of the VCC along microtubules and toward the macrophage periphery or provides a force for particle release from the VCC (122). Other molecular motors are likely involved in the transport and positioning of the VCC but also in transport steps of viral components toward the assembly sites, i.e., the VCC (123). Future work will aim to establish at the molecular level how viral release from VCC is regulated.

While the VCC is defined by its viral content, the question of its induction by HIV or its existence in macrophages before infection was of interest. Analysis of primary MDMs by ultrastructural approaches suggested that indeed similar compartments are present in non-infected macrophages (96, 99, 120). Confocal microscopy confirmed the presence in non-infected MDMs of compartments sharing specific features with the VCC; i.e., containing CD36 and CD9, rapid accessibility to small dextran and therefore connected to the external medium (105). The direct proof of HIV capacity to highjack such compartments was obtained when transduced macrophages exhibiting CD36- GFP+ compartments were subjected to time-lapse epifluorescent microscopy after infection with HIV-1 Gag-iCherry allowing the visualization in real time of Gag recruitment and accumulation only to preexisting CD36+ compartments (105). Thus, in macrophages, HIV-1 hijacks these CD36+ compartments for viral assembly (105), and expands them (96).

Viral particles accumulate in the VCC over time (100). Infected MDMs initially contain sparse, barely filled VCCs, but as the time after infection progresses the compartments become crowded with virions and Brownian-like movements of particles within the VCC become highly limited as shown by FRAP experiments (100). In parallel, viral release into the extracellular media decreases over time, suggesting that viral particles remain within the VCC (100). The restriction factor tetherin is concentrated in the VCC where it may connect viral particles to each other or to the VCC limiting membrane, hence apparently tethering virions to the compartment (89, 124). Indeed, silencing tetherin expression increases viral release and reduces the size of the compartment (89).

Whether HIV-1 release from the VCC is inducible remains indeed elusive. The connections between the VCC and the membrane appear too narrow to allow passive viral diffusion to the extracellular media (99). The release of HIV-1 from infected MDMs can be induced by exposure to extracellular ATP, *via* activation of the P2X7 purinergic receptor that triggers a drastic remodeling of the cytoskeleton and the VCC, accompanied by sudden release of the viral particles packed within the VCC (125). However, we also propose that the highly dynamic nature of the plasma membrane of macrophages, which is subjected to a very active flux of exocytosis and endocytosis/phagocytosis, may promote the temporary widening of the VCC connections to the plasma membrane and allow the release of viral particles to the extracellular media. Supporting this hypothesis, antibodies specific for VCC components, such as tetraspanins (96), gp120 (126), or CD36 (105) had access to the VCC only after several hours of incubation at 37°C and not at 4°C. These observations suggest that the access of extracellular molecules to the VCC depends on the dynamics of the macrophage's plasma membrane, which possibly promotes a frequent opening of the connections between the lumen of the compartment and the surface. Future insight into the role of the cytoskeleton and its associated proteins in regulating the dynamics of the VCC and its impact on viral release may open new avenues to pharmacologically target the VCC (123, 127).

Whether tissue macrophages *in vivo* possess VCCs remains poorly investigated. Indeed, our current view of the VCC results almost exclusively from studies *in vitro* with MDMs. Of note, former ultrastructural analysis of tissue macrophages from patient's organs confirmed the presence of mature and immature virions in intracellular compartments (128, 129). Accumulation of viral particles within VCC in macrophages *in vivo* could be advantageous for the virus to be less accessible to (i) soluble immune mediators such as neutralizing antibodies as observed *in vitro* (126), (ii) to innate sensors that are cytoplasmic or endosomal, and (iii) to anti-pathogen effector mechanisms such as reactive oxygen species or acidic pH.

Direct cell-to-cell transfer is more efficient for spreading HIV infection than the cell-free route (130). This process involves the formation of a stable interface between an infected and an uninfected cell, known as viral synapse. Interaction between the viral envelope present at the membrane of the infected cell and CD4+ in the target cell initiates formation of the viral synapse, which is then maintained *via* additional interactions between cell adhesion molecules (131). Directed release leads to virion accumulation at the viral synapse and efficient infection of the target cell (132). Macrophages can transfer HIV directly to T cells (133), and the VCC appears to play a role in this process. Real-time imaging suggested a recruitment of the compartment to the proximity of viral synapses leading to subsequent T cell infection (134, 135), and the VCC markers CD9, CD18, and CD81 were found enriched at the macrophage to T cell interface (124). However, the precise mechanisms underlying viral transfer from infected macrophages to target cells (T cells or macrophages) remains incompletely understood as compared with T cell to T cell transfer and thus deserve further studies.

Despite their monocytic origin MDDCs are rather resistant to HIV-1 infection as compared with MDMs. Yet, MDDCs, when activated by LPS, can capture and retain vial particles in surface-connected compartments that bear some resemblance with the VCC. Although activated MDDCs are resistant to the infection and do not produce new viral progeny, they can efficiently transfer captured viruses to activated CD4+ T cells that get then productively infected. This transfer mode known as infection *in trans* [see accompanying review by Izquierdo-Useros or Izquierdo-Useros et al. (136)] appears to be highly related to the virological synapse established between HIV-1 infected T cells and target cells (137). This process is thought to play an important role in viral dissemination at the early stages of HIV-1 infection at mucosal entry sites (138). Intravital microscopy revealed that subcapsular sinus macrophages from the peripheral lymph nodes can capture and transfer HIV-1 to target cells without getting productively infected in the process (139). Viral capture is mediated by the sialoadhesin CD169/ SIGLEC1 that binds gangliosides embedded in the envelope glycoprotein (139). Interestingly, CD169-mediated capture of HIV-1 in macrophages leads to virion retention in the VCC and subsequent transfer to and productive infection of CD4+ T cells (140). Remarkably, viral particles captured *via* CD169 intermingled with virions endogenously produced by the macrophage in the same VCCs (140), suggesting that the VCC is not only the site of HIV budding and assembly in macrophages but also a compartment of retention of particles captured from the extracellular media. Indeed, compartments with topologies resembling the VCC have been described in the past after exposure of macrophages to various particulate matter, including latex, cholesterol, or low-density lipoprotein (95).

#### MACROPHAGES AND THE HIV RESERVOIR IN THE POST-ART ERA

While ART can efficiently prevent AIDS by restoring CD4+ T cell counts and suppressing viral load to undetectable levels, it fails to provide a sterilizing cure (141). A viral reservoir remains stable in HIV-infected patients under prolonged therapy (142, 143), and is responsible for the quick viral rebound observed within weeks after ART interruption (144). The cumulative toxicity and the cost associated with lifelong ART made it imperative to devise new strategies to eliminate or curb the viral reservoir (145). Unfortunately, such goal has remained elusive.

At the cellular level, the HIV reservoir during ART is mainly composed of resting memory CD4+ T cells that are latently infected; i.e., cells bearing integrated, transcriptionally silent, but replication-competent proviruses (146). The mechanism behind HIV latency is not fully understood but likely results from multiple factors acting together, such as sequestration of cellular transcription factors in the cytoplasm, epigenetic regulation, or the action of transcriptional repressors [reviewed in Ref. (147)]. T cells with central memory (TCM), transitional memory (TTM), and effector memory (TEM) phenotypes contain the highest levels of latent HIV-1 (148–150). A major breakthrough in the characterization of the latent CD4+ reservoir is the recent identification of CD32a as a surface marker highly enriched in circulating cells harboring replication-competent quiescent proviruses (151). The HIV reservoir is seeded very early after infection (within 2–3 days) (152, 153), and its size remains stable even after years of suppressive therapy (154, 155).

Whether residual viral replication under ART, the so-called active reservoir, contributes to HIV persistence is a rather contentious issue in the field (156). Low-level viremia ("Blips") can be detected in patients under therapy (157, 158). However, HIV shows little sign of genetic evolution during ART (159, 160), and the emergence of drug-resistant virus is remarkably low (158, 161), suggesting that ongoing residual replication does not significantly influence long-term viral persistence. However, a recent high-depth temporal analysis of the phylogeny of viral sequences from the blood and lymph node of patients under ART revealed a constant replenishment of the circulating reservoir as a result of low-level viral replication in sanctuary sites within lymphoid tissues (162). Experimental evidence for the existence of such lymphoid sanctuaries has emerged in the last years. B cell follicles and more specifically germinal centers have long been known as primary sites of HIV replication (163), possibly due to lower antiretroviral drug penetration (164), exclusion of cytotoxic CD8+ T cells (165, 166), and retention of infectious virions within immune complexes on the surface of follicular dendritic cells (167). In patients under treatment and with an aviremic status, CD4+ T cells with a T follicular helper phenotype that reside within germinal centers are the major source of residual infectious virus and contain the highest levels of HIV DNA (168–170).

These recent advances highlight the importance of accurately defining the HIV reservoir, with respect to its cellular and anatomic composition (171). However, studies evaluating the importance of cellular reservoirs other than CD4+ T lymphocytes have been scarce and, for the most part, non-conclusive. Analysis of the rebounding viral sequences in patients after therapy interruption revealed that they differ from proviral sequences integrated in resting CD4 T cells (172). Recovery of M-tropic sequences among the pool of rebounding virus has been recently reported (173). However, there is no readily available method to measure viral rebound from macrophages or other cellular reservoirs, equivalent to the viral outgrowth assay (VOA) typically used to quantify the latent CD4+ reservoir (171). Usually, VOAs require the culture of several million purified cells for accurate quantification of the CD4 reservoir, due to the rarity of latently infected cells (150), making an adaptation of the assay to tissue macrophages unfeasible. Alternatively, quantification of viral nucleic acids provides a more practical manner to evaluate the macrophage reservoir (174). In patients under ART, HIV DNA and/or RNA has been detected in alveolar (175) and duodenal (41) macrophages, microglia in the brain (39), as well as in liver Kupffer cells (176).

Evaluation of the HIV reservoir based on cell-associated DNA or RNA tends to largely overestimate the pool of replicationcompetent virus, as many defective viral genomes accumulate in patients (177, 178). Also, macrophages may acquire viral DNA *via* phagocytosis of infected T cells (51), as discussed previously. In the absence of a reliable outgrowth assay to measure the macrophage HIV reservoir from treated patients, animal models become a valuable alternative.

In a recent study with Asian macaques chronically infected with SIV, replication-competent virus could be recovered from macrophages purified from the spleen and mesenteric lymph nodes, using a modified version of the VOA (43). However, in macaques that had been under ART for 5 months, no replicationcompetent virus could be recovered after macrophage culture, from any of the animals under study and despite the presence of detectable SIV-DNA in macrophages in 40% of the animals (43). A possible limitation of this study is the small number of macrophages that could be purified for the viral outgrowth experiments. However, in parallel experiments, replicating virus could be recovered after culture of purified memory CD4+ T cells from all animals under therapy (43). This suggests that, in the SIV model, the macrophage reservoir, if existent, is clearly smaller than the memory CD4 reservoir.

T cell-deficient humanized mice can be generated by transferring CD34+ human hematopoietic cells into NOD/SCID mice. These mice are reconstituted with human myeloid cells and B cells, but completely devoid of T cells (179). As only macrophages can sustain HIV replication, these myeloid-only mice (MoM) provide a valuable model to test the HIV macrophage reservoir without the confounding effects conferred by the presence of the more abundant CD4+ T cell compartment. When infected with macrophage-tropic HIV-1, MoM present sustained viremia, and viral nucleic acids were detected in macrophages of the liver, bone marrow, spleen, lungs, and brain (179). Initiation of ART in infected MoM leads to undetectable viremia within 2 weeks, and a drastic reduction in the levels of HIV DNA and RNA in the tissues. Importantly, ART interruption led to viral rebound about 7 weeks later, although only in three out of nine animals under study (180). This represents a significantly longer period of viral remission after treatment interruption when compared with T cell-sufficient humanized mice, where rebound occurs within 1–2 weeks after ART interruption (181). Due to the short life span of the MoM mice, the authors could not extend the study and evaluate whether the non-rebounding group of mice would eventually show signs of reactivation of the infection (180).

The absence of T cells in the MoM model of HIV infection, certainly constitute a large deviation from the regular course of HIV infection in humans. Nevertheless, these observations provide the first direct evidence for HIV persistence in macrophages, in the setting of suppressive therapy. Whether such persistence is due to latent infection or ongoing residual replication remains unclear from the data available, as both viral DNA and RNA dropped to undetectable levels in most treated mice (180). Efforts to purge the CD4+ T cell reservoir have mostly employed the "shock and kill" approach (145); a latency-reversal agent (LRA) is initially administered to reactivate viral production in latently infected cells (shock), and followed by an immune-modulatory intervention that renders infected cells susceptible to destruction by the immune system (kill) (182). However, HIV latency in macrophages is not well understood (183), and LRAs that efficiently purge the CD4 reservoir may not have a similar effect in latently infected macrophages. Worse, if the macrophage reservoir is maintained mostly *via* residual ongoing replication or retention of infectious viral particles within VCCs then LRA therapy will be of negligible effect.

Definitive proof of the macrophage reservoir will require demonstration in human patients under therapy. This will likely require more sensitive methods of measuring the reservoir. A promising alternative is the recent report of an *in vivo* VOA, wherein cells from patients with undetectable viremia are transplanted into humanized mice (184). This method appears more sensitive than *in vitro* VOAs, although its widespread application will likely be hampered by the costs associated (185).

#### MACROPHAGES DURING CHRONIC DISEASE IN TREATED HIV-1 INFECTION

Introduction of ART effectively halted the AIDS pandemic, improved health, and prolonged the life of patients. However, a new group of problems, commonly known as "non-AIDS-related conditions," is emerging in HIV patients with long-term suppressed viremia (186). People living with HIV are at increased risk of developing, among others, cardiovascular and neurocognitive disease, osteoporosis, or cancer (187).

Persistent inflammation appears to lie at the origin of these pathologies, although its causes remain incompletely elucidated and may involve multiple factors. Microbial translocation across the gut mucosa is a well-established cause of systemic inflammation during HIV-1 (188, 189). Long-term suppressive therapy does not completely reconstitute the pool of CD4+ T cells in the gut mucosa, particularly those of the Th17 subset (190). This leads to loss of integrity of the epithelial mucosa and translocation of bacterial products through the lamina propria to mesenteric lymph nodes and extranodal sites (189). These microbial products engage pattern-recognition receptors in cells of the innate immune system, particularly monocytes, macrophages and DCs, leading to widespread production of inflammatory mediators (191). Indeed, myeloid cell-derived biomarkers of microbial translocation, such as IL-6, soluble CD14 (sCD14) or sCD163 are found elevated in ART-treated individuals, as compared with age-matched controls, and are strongly associated with premature mortality of HIV-infected individuals (191, 192). This persistent pro-inflammatory state appears to feedback on intestinal macrophages as they become unable to phagocyte microbial debris in the lamina propria and are thus unable to halt this inflammatory cycle (193). Importantly, persistent systemic inflammation drives the occurrence of non-AIDS comorbidities. For instance, inflammatory monocytes migrate to the heart and contribute to HIVassociated myocarditis (194).

Before the implementation of ART, more than half of HIVinfected patients exhibited HIV-1-associated dementia (HAD); a broad term used to described symptoms of cognitive impairment, including psychiatric disorders, loss of motor coordination, and in severe cases, HIV-1-associated encephalitis (195). The incidence of HAD has dramatically decreased with ART, but a set of milder cognitive problems have emerged and are collectively known as HIV-associated neurological disorders (HAND) that still affect about half of the HIV-1-infected population (196).

Residual viral replication in the CNS and neuronal death has been proposed has an explanation for the occurrence of HAND (197). This effect is probably indirect, as neurons and cells of the macroglia do not support productive HIV infection. Instead, residual viral replication in macrophages leads to the production of inflammatory mediators with neurotoxic action (196). While it is challenging to assess productive infection of brain-resident cells, macrophages are known to contain the highest levels of viral nucleic acids of all CNS cell populations (198). Both perivascular macrophages and microglial cells are targeted by HIV in the CNS (199) and HIV DNA has been detected in brain macrophages from patients under long-term therapy (39). HIV RNA persists in the cerebrospinal fluid (CSF) of ART-treated patients even after suppression of plasma viral RNA to undetectable levels (200), and genetic analysis revealed a significant compartmentalization between the CSF and plasma viral populations (201). Importantly, viruses isolated from the CSF are frequently M-tropic (202). Taken together, these studies suggest that the CNS is a tissue reservoir of HIV-1 during ART, likely maintained through low-level viral replication in resident macrophages (203).

Systemic inflammation is also likely to play a role in the progression of HAND. There is a strong association between circulating levels of sCD14 and the development of neurological disorders in HIV-1 infected individuals (204). It has been proposed that microbial products activate circulating monocytes, particularly those of the CD16+ subset that subsequently cross the blood–brain barrier and differentiate into a pro-inflammatory macrophage population in the brain by producing chemokines, cytokines, and neurotoxic factors such as nitric oxide (48). Supporting this model, CD14+CD16+ cells accumulate in the white matter and perivascular space of brains from non-treated patients (205). Also, these CD14+CD16+ monocytes are capable of transmigrating across *in vitro* models of the blood–brain barrier in response to the chemokine CCL2 (206).

These selected examples highlight the pathological role played by macrophages and other myeloid cells in non-AIDS conditions that afflict HIV-1-infected patients with suppressed viremia. It is thus crucial to devise new therapies able to complement ART and capable of targeting the persistent inflammation that drives these morbidities.

#### CONCLUSION

Because of their localization in many tissues, their long life span and the unique nature of their interaction with HIV-1, macrophages play a key role in HIV-1 pathogenesis. The last decade of research brought new and exciting insight into the ontogeny and functional specialization of tissue-resident macrophages. Whether these recently described macrophage properties, such as their proliferative potential, are explored HIV for dissemination and persistence remains unknown, but this subject should deserve increased interest in future studies. Macrophages are susceptible to HIV-1 infection but also sense the virus and thus participate in the general immune activation observed in infected patients. However, initiation of the antiviral immune response relies on the DC population whose capacity to perform antigen presentation and deal with infection is highly organized in space and time. This dual function appears to be ensured by a division of labor between DC subsets (207). While cDC1 (CD141+) are resistant to productive HIV-1 infection, they can cross-present viral antigens derived from cDC2 (CD1c+) that are susceptible to HIV-1. Thus, this dissociation of the viral infection and the antigen presentation function provides to DC populations the capacity to elicit antiviral immune responses and prime T cell responses (207). How macrophages cross talk with DCs and contribute to the antiviral response remains obscure. Reciprocally, how HIV succeeds to cope with these immune cells specialized in antiviral immunity is similarly incompletely understood. Future work addressing these questions should keep on producing exciting results enlightening our general comprehension of the virus–immune system relationships.

#### AUTHOR CONTRIBUTIONS

VR and PB wrote the review. MS-R performed the electron microscopy. NR, PB, and VR designed the cartoon. All the authors edited the review.

#### ACKNOWLEDGMENTS

The authors would like to thank Nicolas Manel at the Institut Curie for critical reading of the manuscript.

### FUNDING

This work was supported by grants from «Agence Nationale de Recherche contre le SIDA et les hépatites virales» (ANRS), «Ensemble contre le SIDA» (Sidaction), ANR-10-IDEX-0001-02 PSL and ANR-11-LABX-0043 to PB. VR and NR were supported by postdoctoral fellowships from ANRS.

### REFERENCES

which inhibits viral production. *Blood* (2012) 120:778–88. doi:10.1182/ blood-2012-01-407395

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Rodrigues, Ruffin, San-Roman and Benaroch. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Role of Monocyte/Macrophages during Hiv/Siv infection in Adult and Pediatric Acquired immune Deficiency Syndrome

#### *Kristen M. Merino1 , Carolina Allers1 , Elizabeth S. Didier 2 and Marcelo J. Kuroda1 \**

*1Division of Immunology, Tulane National Primate Research Center, Covington LA, United States, 2Division of Microbiology, Tulane National Primate Research Center, Covington LA, United States*

Monocytes/macrophages are a diverse group of cells that act as first responders in

#### *Edited by:*

*Geanncarlo Lugo-Villarino, UMR5089 Institut de Pharmacologie et de Biologie Structurale (IPBS), France*

#### *Reviewed by:*

*Masaaki Miyazawa, Kindai University, Japan Nina Derby, Population Council, United States Donald Sodora, Center for Infectious Disease Research, United States*

> *\*Correspondence: Marcelo J. Kuroda<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology*

*Received: 14 August 2017 Accepted: 16 November 2017 Published: 05 December 2017*

#### *Citation:*

*Merino KM, Allers C, Didier ES and Kuroda MJ (2017) Role of Monocyte/ Macrophages during HIV/SIV Infection in Adult and Pediatric Acquired Immune Deficiency Syndrome. Front. Immunol. 8:1693. doi: 10.3389/fimmu.2017.01693*

innate immunity and then as mediators for adaptive immunity to help clear infections. In performing these functions, however, the macrophage inflammatory responses can also contribute to pathogenesis. Various monocyte and tissue macrophage subsets have been associated with inflammatory disorders and tissue pathogeneses such as occur during HIV infection. Non-human primate research of simian immunodeficiency virus (SIV) has been invaluable in better understanding the pathogenesis of HIV infection. The question of HIV/SIV-infected macrophages serving as a viral reservoir has become significant for achieving a cure. In the rhesus macaque model, SIV-infected macrophages have been shown to promote pathogenesis in several tissues resulting in cardiovascular, metabolic, and neurological diseases. Results from human studies illustrated that alveolar macrophages could be an important HIV reservoir and humanized myeloid-only mice supported productive HIV infection and viral persistence in macrophages during ART treatment. Depletion of CD4+ T cells is considered the primary cause for terminal progression, but it was reported that increasing monocyte turnover was a significantly better predictor in SIV-infected adult macaques. Notably, pediatric cases of HIV/SIV exhibit faster and more severe disease progression than adults, yet neonates have fewer target T cells and generally lack the hallmark CD4+ T cell depletion typical of adult infections. Current data show that the baseline blood monocyte turnover rate was significantly higher in neonatal macaques compared to adults and this remained high with disease progression. In this review, we discuss recent data exploring the contribution of monocytes and macrophages to HIV/SIV infection and progression. Furthermore, we highlight the need to further investigate their role in pediatric cases of infection.

Keywords: HIV, SIV, macrophages, monocytes, pediatrics, Acquired Immune Deficiency Syndrome

# INTRODUCTION

A cure for HIV infection continues to be elusive, despite the use of antiretroviral therapy (ART). Studies using non-human primate (NHPs) models of simian immunodeficiency virus (SIV) have been invaluable in better understanding the pathogenesis of this infection. Declining numbers of CD4+ T cells during HIV/SIV infections have long been attributed as the primary cause of immune-deficiency, progression to acquired immune deficiency syndrome (AIDS), and sites of virus reservoirs established during ART. While it is likely that various other immune cells influence disease progression and viral seeding, the purpose of this review is to focus on the contribution of monocytes and macrophages to HIV/SIV infection. Although in adult macaques, monocyte levels in blood remain constant during infection, the monocyte turnover rate increases during the onset of simian AIDS (SAIDS) and reflects damage to short-lived tissue macrophages. Interestingly, physiological monocyte turnover rate in neonates (<1 month old) is higher than in adults and appears to be associated with the more rapid disease progression that occurs in pediatric HIV/SIV infections. As infants became older (<1 year old), they exhibited a biphasic pattern of monocyte turnover and disease progression, with some animals presenting with rapid disease such as seen in neonates, while others progressed similarly as adult macaques. We theorize that the magnitude of infected macrophages influences disease outcome, and susceptibility of macrophages to infection is impacted by age. Our data suggest shorter-lived macrophages are more easily destroyed by virus while longerlived macrophages appear more resistant to destruction and may contribute to the viral reservoir. This review discusses the possible contributions of macrophages to HIV/SIV infection and pathogenesis. It is important to consider that macrophages also may need to be targeted to achieve cure.

#### MACROPHAGES IN HEALTH AND DISEASE

The immune system is essential for maintaining health and combatting infectious disease (1). Innate immune responses typically act immediately to produce inflammation and are performed directly by immune surveillance by effector cells such as neutrophils, natural killer (NK) cells, and macrophages. The innate immune cells work en masse to detect and clear pathogens and this is thought to limit the initial infection and thus buys time for adaptive immune responses to mount. Adaptive immune responses generally require weeks-to-months to develop and rely heavily on antigen-presenting cells (e.g., dendritic cells, macrophages, B cells) and T cells to clear infection and establish immunologic memory.

Macrophages are a diverse group of cells that play important roles in both innate and adaptive immune responses. During innate immune responses, macrophages sense and promote inflammation and recruit effector cells for clearance of pathogens. In adaptive immune responses, they function in antigen presentation and secretion of immune modulators for recruitment of lymphocytes (1). In addition, macrophages are imperative for tissue modeling during fetal development, maintaining homeostasis, and wound healing (2–4). Conversely, dysfunctional macrophages and unregulated inflammation are devastating to homeostasis and may instead contribute to pathogenesis in specific disease settings, such as pulmonary tissue injury during asthma (5), islet cell destruction in diabetes (6), and neurological damage, lymphoma and cardiovascular disease during human immunodeficiency virus (HIV) infection [reviewed in Ref. (7)].

Macrophages are derived from multiple sources during development, with earliest generation from the yolk sac during embryogenesis, and later generation from the fetal liver and bone marrow. In human and rhesus macaque adults, replenishment of tissue macrophages lost from death due to infection or injury occurs mainly from monocytes generated by hematopoietic stem cells in the bone marrow, which then circulate through the blood stream, deposit into the tissues, and differentiate into various types of macrophages (8–10). Macrophages in the mouse model have been shown to self-renew rather than being replaced with emigrating monocytes from the periphery (11–13). Based on published reports in murine models, tissue-resident macrophages can originate from two sources, with the self-renewing subset derived from the fetal liver during embryonic development and from the bone marrow after birth [reviewed in Ref. (10, 14)]. Studies have yet to identify self-renewing macrophages in primates, with possible exception of the brain (15). As summarized in **Figure 1**, there exist at least two types of macrophages in the tissues; the short-lived macrophages that are continuously replenished by circulating classical monocytes arising from the bone marrow and the long-lived macrophages. These two types of macrophages appear to differ in their phenotypes and functions, so it is critical to better understand their unique roles during development, immune responses, disease pathogenesis, and maintaining homeostasis.

Historically, subsets of macrophages have largely been classified as pro-inflammatory (M1) or anti-inflammatory (M2) on the basis of expressing various markers, secreting cytokines, and phagocytic and antigen-presenting functions. Results from more recent research has suggested that the polarization of macrophage phenotypes is more fluid and less dichotomous than originally thought, based on the ability of macrophages to change phenotypes in response to changes in the surrounding environment within the tissues and vasculature (16). Generally, tissue-resident macrophages appear more M2-like with functions supporting an anti-inflammatory environment such as phagocytosing debris and promoting healing and growth. These tissue-resident macrophages are considered longer-lived as illustrated in the lung with alveolar macrophages (17). Macrophages recruited from circulating monocytes, in contrast, largely constitute the interstitial macrophages (IM) in the lung and subsets of perivascular macrophages in the brain and are thought to be shorter-lived. These have been classified as both M1-like and M2-like depending on immune homeostasis, infection events, and expression of MAC387 and CD163 differentiation markers, respectively (16). CD163, a hemoglobin/haptoglobin scavenger receptor associated with anti-inflammatory responses, is expressed on subsets of tissue macrophages as well as on monocyte populations, which are thought to derive from CD14+CD16− monocytes (18). Soluble CD163 shed from monocyte populations exerts anti-inflammatory functions, and studies showed that increased levels are associated with infectious and autoimmune diseases [reviewed in Ref. (19)]. MAC387 recognizes the L1 or Calprotectin molecule, and has been used to identify recently infiltrated macrophages associated with acute inflammation (20, 21). During infections, particularly in the case of HIV/SIV disease progression, increased monocyte turnover occurs in association with an accumulation

of these various short-lived macrophage subsets in the tissues. This macrophage deposition seems to primarily occur in classical sites of AIDS pathology, including the brain, heart, lung, and gut tissues (15, 22–24). One study proposes that during acute infection M1-like macrophages are recruited into tissues to help fight infection, while M2-like macrophages accumulate later during chronic disease to dampen inflammation and repair tissues. The accumulation of both macrophage subsets seems to promote cardiovascular disease (16). Thus, the characterization of monocyte and macrophage subsets in relation to their number, phenotype, and function becomes increasingly important for predicting disease outcomes and subsequently attempting to ameliorate or reverse pathogenesis.

# HIV INFECTION

Human immunodeficiency virus is the etiological agent of AIDS and, since the beginning of this pandemic in the early 1980s, has caused the loss of over 40 million lives (25). HIV infection in humans follows a distinct pattern (26) starting with an eclipse phase during the first 1–2 weeks of infection, which constitutes a time when the virus replicates and disseminates while the immune response is developing and not yet effective at clearing the virus. HIV preferentially targets T cells for infection, using the CD4 receptor in combination with the CCR5 chemokine receptor to gain entry. Effector CD4+ T cells express the highest level of CCR5 and are depleted drastically early after infection (27) in what is known as the acute phase. This particular phase follows 2–4 weeks post-infection (pi) and is evident by high viral RNA loads in the plasma, and a reduction in peripheral CD4+ T cells. During the acute phase, there is a stark increase in detectable immune responses, most notably with higher levels of virus-specific effector CD8+ T cells and antibodies. The end of the acute phase and transition to the chronic stages of infection is characterized by a marked reduction in plasma viral load (VL) and establishment of a viral "set point" that appears to result from incomplete control of viral replication by adaptive immune responses with concurrent loss of target CD4+ T cells available for new infection. The chronic phase of HIV infection is accompanied by clinical latency that may last from 1 to 20 years in untreated individuals. Eventually CD4+ T cells decline to levels below 200 cells/μl in blood and infected individuals become susceptible to opportunistic infections such as *Pneumocystis jiroveci* (earlier named *P. carinii*), cytomegalovirus, *Cryptosporidium* spp., and *Mycobacterium tuberculosis,* and cancers such as Kaposi's sarcoma [reviewed in Ref. (28, 29)]. These late-stage infections and cancers associated with a loss of CD4+ T cells define the onset of AIDS that ultimately leads to death (30). With the development and application of combination ART (cART), survival in HIV-infected adults has been extended by suppression of virus to undetectable levels and restoration of CD4+ T cell numbers to normal levels, thus preventing onset of AIDS (31). Instead, longer-surviving HIV-infected individuals administered cART may experience inflammation-associated chronic diseases referred to as HIV-associated non-AIDS (HANA) conditions (32).

Despite the large success of ART treatment for lifespan and clinical symptoms, it does not completely eliminate the virus, which persists in reservoirs. In general terms, a viral reservoir is defined as a virus-infected cell that persists despite treatment. A reservoir can be described as active or latent. Active reservoirs continue to produce virus, such as HIV or SIV, and in this case the virus actively produces virons using the cells machinery for transcription and translation. This could result from an infected cell being refractory to ART treatment because the drugs may not penetrate the cell membrane or intracellular drugs may be ineffective at inhibiting the reverse transcriptase or integrase enzymes. In such cases, viral RNA or protein should be detected if expressed above threshold limits of the assay used. Alternatively latent virus reservoirs develop after insertion of viral DNA into a host cell genome. Here, virus is not actively being transcribed and effectively remains dormant. In this case, viral DNA should be detectable with sensitive assays. Cellular activation and some chemical compounds (latency reversal agents) can stimulate the active transcription and translation of the dormant virus, wherby viral RNA and protein will then be produced. Resting CD4+ T cells are thought to serve as latent reservoirs of HIV and SIV during ART treatment, and some latency reversal agents have produced successful reactivation of those reservoirs in attempt to eliminate the infection with continued long-term ART treatment. This is a fascinating and growing area of study that we will not review here but is described in depth in other reviews (33, 34).

Interestingly, in human and macaques, macrophages and dendritic cells (DC) are also capable of expressing the CD4 and CCR5 receptors required for HIV/SIV infection (35–39) and earlier work shows that macrophages can be targeted by HIV (40, 41) and SIV *in vivo* (42). Initially, virus tropism for the T cell and macrophage CCR5 receptor was used to delineate specific strains of these lentiviruses (43). It was largely accepted that while macrophages were capable of being infected by HIV, they were second to infection of CD4+ T cells, and possibly only targeted after significant loss of CD4+ T cells in the host (44). This evoked some persisting controversies regarding macrophage infection by HIV/SIV. *In vitro* studies showed that macrophages can be infected productively (45–47), and several strains of virus were considered macrophage-tropic yet *in vivo*, these same strains predominantly infected CD4+ T cells. *In vivo* data revealed that alveolar macrophages collected from bronchoalveolar lavage (BAL) in ART-treated human patients were positive for HIV DNA, indicating they are likely an important macrophage reservoir (48). Contradicting reports have indicated that while HIV DNA could be found in ART-treated human alveolar macrophages, subsequent outgrowth assays failed to detect any replication-competent virus, attributing positive DNA results to macrophage engulfment of infected CD4+ T cells (42). Work performed with rapidly progressing adult macaques (SAIDS progression occurring in months compared to years) reported detectable SIV RNA in macrophages of the lung and lymph nodes (LNs) (49). For example, Avalos and colleagues recently found that in virus-suppressed ART-treated macaques, macrophages isolated from the brain harbored replication-competent virus. These macrophages were designated as latently infected given that viral DNA was detectable, while *in situ* hybridization and qPCR of brain tissue showed no detectable viral RNA. These infected macrophages were found after 2 years of ART treatment, supporting the notion that they constituted a long-lived macrophage reservoir (50). Alternatively, Dinapoli and others detected SIV DNA and replication-competent virus in lymphoid macrophages isolated from ART-naïve macaques, but they were unable to detect viral outgrowth from similar macrophage populations in ART-treated animals. They suggested that infected macrophages were short-lived and were lost after longer treatment periods (42). In the humanized mouse model, results showed productive infection of macrophages as well as viral rebound after successful ART treatment in myeloid-only mice (MoM), which lacked human T cells (51). The viral rebound in the MoM study supports infection of long-lived tissue macrophage populations because short-lived macrophages have a half-life of 1 day and viral rebound occurred 7 weeks after withdrawal of ART (52). Additional studies in macaques have also reported infection of long-lived tissue macrophages and together these findings support the theory that macrophages can contribute to the viral reservoir (53, 54).

There is increasing attention on the characterization of macrophage phenotypes and functions during active HIV/SIV infections to better understand their roles in immune responses and tissue pathogenesis. For example, Burdo and colleagues found that an increase of MAC387+ macrophages in the central nervous system (CNS) was associated with more damage to the dorsal root ganglia, which is related to neurological complications in HIV infection (55). They also found that while MAC387+ macrophages were within brain lesions during acute SIV encephalitis (SIVE), higher numbers of CD163+ macrophages were associated with severe SIV encephalitic lesions during chronic infection. Additionally, an increase in CD163+CD16+ monocytes was found in HIV-infected patients with detectable viral loads despite ART treatment. The numbers of these cells were inversely correlated with numbers of CD4+ T cells among patients with <450 CD4 T cells/μl, suggesting that the CD163+CD16+ monocytes were contributing to viral replication (18). Another study reported increasing CD163 expression on monocytes in HIV-infected, ART-treated patients that inversely correlated with CD4 T cell number, however, this did not correlate with viral load (VL) and the use of protease inhibitors during treatment resulted in reduced shedding of CD163 on monocyte populations (56). In addition, increased monocyte turnover in the periphery was associated with increased trafficking of CD16+ macrophages into the gut tissue of chronic SIV-infected, progressing rhesus macaques (24). These gut macrophages were skewed functionally for anti-inflammatory responses and exhibited impaired phagocytic ability, which in turn was associated with increased disease of the gut. In *ex vivo* studies, monocytes expressing CD16 were preferentially infected by HIV over CD16-negative populations, and HIV DNA was detected in this cell subset sampled from ART-treated patients, also suggesting that these monocytes may serve as a viral reservoir (57).

Importantly, in contrast to T cells, macrophages and DC are described as more resistant to the cytopathic effects of the virus infection (47, 58, 59) and are not cleared by antigen-specific cytotoxic CD8+ T cells (60). Thus, in theory, infection of longlived macrophages would allow the virus to rapidly evade CD8+ T cell immunity and replicate to produce "escape" mutations that then preferentially infect CD4+ T cells. As CD4+ T cells are depleted, B cell responses for antibody production may become less effective. These events would then promote widespread activation and infection of macrophages that survive and enable continued virus replication and release for long periods of time. Macrophages are clearly capable of contributing to disease progression, treatment failure, and viral rebound. Thus, our studies are currently devoted to depleting macrophages during various stages of SIV infection to develop a clearer understanding about their role in disease, with and without concomitant T-cell depletion.

Despite recent advances in antiretroviral therapies for treatment of HIV, the question of an HIV-infected macrophage reservoir has become significant as efforts are aimed at developing a cure. It has been shown that nucleoside reverse transcriptase inhibitors and/or protease inhibitors were less effective at targeting infected macrophages while being more effective against virus-infected T lymphocytes in SIV-infected rhesus macaques (61, 62). Also, macrophages may be productively infected by mechanisms different from CD4+ T cells such as by phagocytosis of infected cells (63) or by direct spread of infection between monocyte-derived macrophages using nanotubes (64). Macrophages can also harbor cell-free virus and spread infection to lymphocytes *in trans* using lectin receptors (e.g., CD169) (65). In addition, the mean half-life of HIV DNA was found to be longer in monocyte populations compared to CD4+ T cells (66). Together, these findings highlight the need to characterize macrophages in relation to disease pathogenesis and for developing effective treatments to eliminate this population as a viral reservoir.

#### RHESUS MACAQUE MODEL FOR HIV

Non-human primates experimentally infected with SIV, primarily Indian and Chinese rhesus macaques, have been used to study the pathogenesis, immunology, and therapeutic interventions. They display a similar course of infection and disease progression pattern as seen in persons infected with HIV but at a faster rate. Rhesus macaques inoculated with SIV typically exhibit a high initial viral load, significant depletion of CCR5+CD4+T cells in the gut, and rapid seeding of virus in the tissues followed by establishment of the viral set point and gradual loss of CD4+ T cells in the periphery. After a few years, SIV-infected macaques develop SAIDS and succumb to opportunistic infections and wasting (67, 68).

Compared to HIV in humans, SIV in rhesus macaques uses the same cellular targets and anatomical sites, displays similar persistence, latency and viral loads, and is similarly responsive, though to a lesser degree, to the cART regimen used in human patients (69). The SIV-infected macaque model has enabled more detailed studies about cART therapy, viral seeding of reservoir cells, immune cell depletion effects, administration or generation of neutralizing antibodies, viral rebound, vaccine therapies and latency reversal agents [reviewed in Ref. (69)], much of which could not be examined to the same degree in human patients. Most importantly, animal studies have assisted in the development of effective drug treatments, advanced to producing undetectable viral loads and prolonged maintenance of normal levels of CD4+ T cells to thereby extend a long asymptomatic lifespan to HIV-infected individuals. However, even with strict adherence to cART therapy, drug treatment does not completely eliminate latent virus or virus production from long-lived host cells, and disruption of treatment consistently results in HIV viral rebound from a cellular reservoir [reviewed in Ref. (70)]. Viral seeding studies in macaques showed that even with ART initiation 3 days pi, prior to detectable VL or antigen-specific immune responses, viral rebound occurred when treatment was stopped. While this observed viral rebound was delayed, the post-rebound set points were similar to those observed in animals with ART initiation 2 weeks pi (71). Notably, studies performed in NHPs have found that viral reservoir seeding can be accomplished within 24 h of infection, and ART initiated as soon as 4 h pi, while beneficial in reduction of viral load, dissemination and reservoir seeding, was still not curative (72, 73). Taken together these studies highlight the difficulty and necessity of defining the viral reservoir and for eliminating long-lived or proliferating reservoirs to achieve cure. Much work has been devoted to investigating the role of memory CD4+T cell populations as a reservoir but other host cells, particularly long-lived macrophages, require attention as well due to their being refractory to ART and their potential to harbor virus for years.

### SIV INFECTION AND DISEASE PROGRESSION IN ADULT RHESUS MACAQUES

The typical course of SIV infection in rhesus macaques varies with age of the host as depicted in **Figure 2**. Early after infection, adult macaques produce a high initial viral load at ~108 RNA copies per ml followed by a set point at ~105 viral copies per ml that corresponds to a loss in CD4+ T cell numbers in the blood, all of which resemble HIV infection kinetics in adult human patients (74, 75). Disease progression and onset of AIDS/SAIDS is described typically using parameters such as CD4+ T cell count <200/μl with significant increase in viral load, and presentation with opportunistic infections, weight loss, anemia, pneumonia, fatigue, diarrhea, thymic atrophy, lymphoid atrophy, bone marrow hyperplasia, encephalitis, and colitis. Common reported opportunistic infections with SAIDS include cytomegalovirus, adenovirus, *Cryptosporidium, Pneumocystis*, *Mycobacterium, Shigella*, and *Campylobacter* (76, 77).

One of the many focal points in HIV/SIV research has been to characterize the physiological "flashpoint" and reliable prognostic markers that define onset of AIDS. Depletion of CD4+ T cells has been described as the primary cause for terminal progression to AIDS. Interestingly, however, CD4+ T cells can be depleted or remain at stable low levels for years before SAIDS onset occurs in rhesus macaques (78–80). In addition, CD4+ T-cell depletion or turnover has proven to not be the best predictive value for onset of terminal disease progression (80, 81). A recent working theory is that the depletion of CD4+ T cells occurs in three stages during the chronic stages of infection prior to AIDS onset such that initially effector memory (EM) T cells with high expression of CCR5 are depleted. This is

Figure 2 | Patterns observed for monocyte turnover, CD4 T cell counts and simian immunodeficiency virus (SIV) infection differ depending on age at infection. Results from Kuroda and colleagues showing monocyte turnover determined by quantification of bromodeoxyuridine (BrdU) incorporation by flow cytometry in monocytes at 24 h post-BrdU intravenous injection, CD4 T cell count obtained by peripheral blood cellular immunophenotyping *via* flow cytometry in conjunction with complete blood count (CBC), and SIV RNA plasma viremia as determined by quantitative PCR. Distinct patterns emerged for clinical progression of SIV in rhesus macaques illustrated in (A) adults, (B) newborns, and (C) as what we refer to as an "intermediate" phenotype in older infants infected at ~3–4 months of age.

followed by immune reconstitution of this population by CCR5 negative central memory (CM) cells and then ultimately leads to progressive loss of both effector and CM T cell pools over time with repeating cycles of reconstitution and depletion (27). It is thought that the loss of CM T cells leads to dysregulation of the immune network as a whole and immunodeficiency allowing for opportunistic infections that define the onset of AIDS (27).

During studies to further define biomarkers for the onset of SAIDS, we reported that increasing monocyte turnover with corresponding destruction of tissue macrophages was a significantly better predictor than declining CD4+ T cell counts or increasing plasma viral load (81), as diagrammed in **Figure 2A**. This suggested that in untreated adult rhesus macaques, macrophages play important roles in HIV/SIV pathogenesis and persistence. Additional studies show that they do so, in a tissue–specific manner as elaborated further in the next section.

### THE ROLE OF MACROPHAGES IN ADULT SAIDS

Increases in peripheral CD16+ monocyte subsets have been associated with inflammatory disorders such as cardiac disease (82), lesions within the kidney vasculature during systemic lupus erythematosus (83), rheumatoid arthritis (84) and intestinal pathology (85). A subset of CD14+ macrophages has been linked to greater inflammatory damage in intestinal tissues, impacted by Crohn's disease (86, 87). To also characterize the relationship between macrophages and SIV pathogenesis, we measured turnover rates of monocytes during steady-state homeostasis in uninfected macaques in comparison to SIV-infected rhesus macaques. Monocyte turnover was followed by measuring uptake of the thymidine analog, bromodeoxyuridine (BrdU), into dividing cells followed by immunofluorescent antibody staining and flow cytometry. The homeostatic baseline monocyte turnover in the blood of uninfected adult rhesus macaques was ~5% across several study groups, while the turnover of monocytes increased up to 50% prior to onset of terminal SAIDS (81). This agrees with data in a study where increased turnover of monocyte populations in the blood was predictive of more severe disease progression, in particular the development of SIVE (88). This was also related to an increase in macrophage populations deposited in the brain tissue, leading to localized pathology. Our study showed that an increase in monocyte turnover kinetics was associated with a loss of tissue macrophages by SIV infection in the LNs, and that this was not accompanied by changes in the absolute count of monocytes in peripheral blood (23, 54, 81). Interestingly, the shift in monocyte turnover was also not linked to numbers of CD4+ T cells or lymphocyte activation, similar to results reported by Burdo et al. (88), suggesting an independent mechanism of disease pathogenesis relating specifically to monocyte/macrophage infection (81). Further evaluations found that increased trafficking of monocytes from the bone marrow during SIV infection did occur with successive recruitment to the LN, CNS, and lung (23, 54, 88). In fact, increased rates of monocyte turnover and recruitment to become tissue macrophages were linked to rate of progression to SAIDS, severity of lung tissue pathology, and SIV encephalitis.

The lung, followed by the brain, appears to be among the organs most affected by HIV in the post-ART era (89, 90) with macrophages thought to play an important role in the chronic immune activation required for lung and brain lesions associated with chronic HIV/SIV infection. The accrual of CD163+ macrophages has also been observed in the hearts of rhesus macaques infected with SIV, which positively associated with cardiac disease (22). Given that respiratory infections such as *Pneumocystis* pneumonia and tuberculosis are among the most common AIDS-defining diseases and involve direct bacterial infections of macrophages, we focused further on characterizing macrophages of the lung. At least two different populations of macrophages were identified in the lung tissue of rhesus macaques; namely IM (23) and macrophages located in the alveolar spaces (AM) (91). BAL macrophages did not entirely represent macrophagemediated innate immune responses of the lung, so we examined macrophages in whole lung tissues and observed that IM and alveolar macrophages (AM) exhibited different turnover kinetics and phenotypes in adult rhesus macaque lung tissue. IM were smaller, located exclusively in the interstitium, and phenotypically resembled blood monocytes. *In vivo* BrdU incorporation by dividing cells demonstrated higher turnover rates for blood monocytes and lung IM compared to AM during steady state (91). The relatively higher TUNEL-staining IM also suggested a continuous transition of blood monocytes replacing apoptotic IM in an effort to maintain homeostasis in the lung. These data led us to believe that IMs are a short-lived population of macrophages in the lung. AM on the other hand, were larger, expressed CD206 (not expressed on IM), and were located in the alveolar spaces. AM turnover was negligible during steady state indicating that these are longer-lived cells. In addition, *in vivo* BrdU labeling suggested that IM can differentiate into AM when AMs are depleted (91). These findings suggest that AM and IM possess different functions.

Investigations of BAL collected from HIV-infected patients have produced some conflicting data and merited some interesting questions about whether AMS are productively infected and contribute to the viral reservoir. Results from several studies have found viral DNA and RNA in AMs (92–95) and some work has even defined a specific subset of HIV-specific CD8+ T cells whose function is thought to target HIV-infected alveolar macrophages (92, 96). There remains, however, a persisting argument that these results reflect macrophages engulfing infected CD4+T cells rather than true infection and replication within macrophages. In one study, viral DNA was found in AMS collected from a patient after 3 years of ART treatment, but the levels of rearranged TCR DNA supported their interpretation that viral DNA was present due to phagocytosis of infected CD4 T cells rather than macrophage infection (42). We thus investigated the effects of SIV infection on lung macrophage populations in ART-naïve macaques. Confocal imaging techniques were applied and lung macrophages were identified containing SIV RNA **Figure 3A**. This strongly supports the prospect that viral replication can occur in tissue macrophages. Furthermore, data showed that increasing blood monocyte turnover significantly correlated with turnover and apoptosis of IM but not with turnover of AM in SIV-infected macaques during terminal disease progression as shown in **Figure 3B** (54, 91). Interestingly, virus DNA copies increased in IM and AM after monocyte turnover increased in SIV-infected macaques but remained at the same levels in CD4+ T cells regardless of monocyte turnover (**Figure 3B**). The low turnover rate of AM despite their being infected with SIV strongly suggested that if infected with replication-competent virus, this macrophage subset could constitute an important long-lived virus reservoir (23, 91). Our interpretation is that the increased monocyte turnover was due to a compensatory mechanism to replace the short-lived macrophages (IM), which were destroyed by SIV in the tissues. This increase in turnover of both monocytes and IMs corresponds to greater infection of long-lived AMs, however mechanisms for this relationship are unclear. Based on these studies we proposed a working model as shown in **Figure 4** that (a) There exist at least two types of macrophages in the lung including the shorter-lived IM exhibiting relatively higher turnover during homeostasis suggesting a critical role for daily protection, and longer-lived AM that exhibit lower turnover rate; (b) Increased SIV infection of both IM and AM correlates with AIDS disease progression; (c) SIV-induced IM apoptosis promotes further increases in IM (and monocyte) turnover rate in contrast to AM that become infected with SIV but exhibit far lower rates of apoptosis and turnover. This further suggested that similarly longer-lived macrophage populations in other tissues/organs also may become infected, and because they appear to be resistant to apoptosis, are likely also to support viral infection for extended periods of time to serve as a virus reservoir and source of chronic inflammation. Together, these

studies illustrate the rationale for our proposed macrophage depletion strategies.