Complex Interactions Between Human Immunodeficiency Virus Type-1, Sex, and Osteopontin Influence Viral Replication and Leukocyte Proportions in Tissues

Evidence suggesting that HIV pathogenesis differs by sex, a variable known to influence the extent and breadth of immune responses in health and disease continues to accumulate. Host factors that promote or inhibit HIV replication may do so in a way that varies by sex. Prior studies using cultured human macrophages demonstrated that osteopontin (OPN)/secreted phosphoprotein-1 (SPP1) stimulates HIV replication. To test whether OPN has the same positive impact on virus replication at the level of tissues, we used a humanized mice model of low-level chronic HIV infection and in which OPN RNA and protein expression was inhibited with targeted aptamers. Interestingly, 4 months after infection when there were no significant differences in HIV viral load in plasma between groups however in contrast, in the spleen, lung, and liver the tissue burden of HIV RNA, as well as the proportion of leukocytes in female and male mice differed depending on whether OPN was expressed or not. The findings collectively demonstrate the potential for complex interactions between host factors like OPN and sex to influence different facets of HIV tissue-level pathogenesis.


INTRODUCTION
Human immunodeficiency virus type 1 (HIV-1) infection is managed with antiretroviral (ART) therapy while research toward a cure continues. HIV-1 disseminates rapidly into tissues forming sanctuaries and reservoirs which are harder to target (1)(2)(3). Replication-competent virus in such tissue reservoirs in tissues rebound into a full-fledged infection if ART is interrupted (4,5). One of the significant challenges in HIV eradication strategies has been the absolute suppression of replication from tissue reservoirs (1,4). Therefore, identifying and increasing our understanding of host cell genes that promote HIV replication in tissues would provide critical new knowledge.
Immune cells' ability to circulate and surveil tissues is potentially hijacked by HIV-1 to facilitate the formation of sanctuaries and reservoirs, suggesting a complex interaction between viral and host factors (6). Different anatomical and physiological properties of tissues also contribute to reservoir formation and add another confounding dimension to eradicating reservoirs (6). HIV-1 infects CD4 + T cells, monocytes, and tissue-resident macrophages throughout the body. The molecular mechanisms by which the virus establishes residency in these cells have pathways in common, while others are cell-autonomous (7). This reality highlights the heterogeneity of HIV reservoir formation and the challenge to eradication efforts.
There are also significant differences in HIV phenotypes and immune response between males and females, some of which have been attributed to socioeconomic and biological differences (8). Females account for almost half the population currently living with HIV, yet in early studies a consideration of sex differences in pathogenesis were rarely considered important in the design of effective treatment strategies (9). Plasma viral loads in females are lower than males, but females have a higher risk of acquisition (8,10). Estradiol negatively impacts HIV replication, while testosterone has a positive association with plasma viral loads (11). Despite similar plasma viral loads, the burden of HIV in female lymph nodes was reported to be higher compared with similar tissue from males (8). Recent HIV vaccine trials have shown that females have enhanced interferon-stimulated gene expression and interferon-α (IFNα) response upon TLR7/9 activation and higher titers of neutralizing antibody than males suggesting enhanced immune responses (8,11).
Moreover, these biological sequelae likely exacerbate the more robust activation of cytotoxic CD8 + and higher CD4 + loss seen in females vs. males (8,11). Increased immune activation linked with HIV infection drives co-morbid conditions such as cardiovascular disease, cancer, and neurocognitive complications, which are augmented risk factors for females than males (12)(13)(14). Persistent immune activation and inflammation contribute to the maintenance and enhancement of viral replication by activating and stimulating the proliferation of memory CD4 + T cells, which are highly susceptible to further infection (8,15).
Osteopontin (OPN) is a multi-functional protein that is elevated during HIV infection, having both pro-survival as well as pro-and anti-inflammatory roles (16)(17)(18). HIV can use OPN to enhance replication, as demonstrated in cultured monocytederived macrophages via cell-cell fusion and through NF-κB signaling, which activates the HIV promoter (19). OPN can also function as a chemoattractant for monocytes and macrophages (20)(21)(22). Therefore, OPN's chemotactic, pro-survival, and viral replication promoting potential suggests that it may contribute in vivo to HIV pathogenesis and the recruitment and survival of infected immune cells in tissues.
In this study, we sought to understand whether inhibition of OPN RNA and protein expression in a humanized mice model for low-level chronic HIV infection can without antiretrovirals, reduce HIV reservoirs in the spleen, liver, and lung. The chimeric mouse model where immunocompromised NOD.Cg-PrkdcscidIL2rγtm1Wjl/SzJ (NSG) mice engrafted with human hematopoietic stem cells that develop a human immune system was used (23). Human T cells, monocytes, and macrophages were infected after intravenous challenge with HIV-1 SF162 . Global inhibition of OPN expression at the RNA and protein level was achieved with oligonucleotide aptamers that block the interaction site of OPN with its receptors that, in turn, reduce signaling and gene expression through feedback suppression (24). At the study endpoint, viral loads in plasma were undetectable or very low and in the range of 2-3000 copies/ml. Interestingly, using in situ hybridization to detect HIV-1 viral RNA, we found numerous foci of productively infected cells in the spleen and moderate levels in the lung and liver. Moreover, in the liver of mice with reduced OPN expression, there were fewer numbers of infected foci in the tissues of female compared to male mice. Reduction of OPN expression did not affect CD4:CD8 ratios, an indicator of immune dysfunction, however in all tissues examined, HIV infection was an additional source of variation that interacted in the liver with the variable sex (25)(26)(27)(28). In the lungs, CD14 + monocyte counts were significantly influenced by interactions with OPN and HIV infection. Interestingly, in the spleen, lung, and liver, there were significant interactions with HIV infection and CD19 + B-cells counts, with an additional influence in the liver of male or female sex. Lastly, analyses of leukocyte proportions in each tissue revealed that OPN expression significantly influenced their relative levels.

Humanized Mice Generation
All animal protocols and procedures were reviewed and approved by Johns Hopkins University Animal Care and Use Committee and Institutional Review Board (Protocol # MO17M12). The humanization protocol has been described previously (29), and the schematic of the study is illustrated in Figure 1A. NOD.Cg-Prkdc scid IL2rγ tm1Wjl /SzJ (NSG) (5-8 weeks old) mice were purchased from Jackson Laboratory for breeding colonies. Mice were housed and handled in pathogen-free environments. Newborn pups (P1-3) from breeders were irradiated at 100 cGy using Gamma Cell-40 Cesium Extractor (Theratronics) and injected intrahepatically with 1-2 × 10 5 cells of human fetal liver-derived CD34+ hematopoietic stem cells while under 2.5% isoflurane anesthesia. In mice, successful engraftment was identified 8 weeks post-engraftment from their peripheral blood with flow cytometry by staining and detecting human CD45 cells.

Mouse Groups
The humanized mice were separated and stratified into four groups of females and males either inhibited for OPN (treated with inhibitory aptamers that block OPN receptor signaling decreasing RNA and protein levels) or not (scrambled aptamers) and injected with HIV or buffer control [ Figure 1A, (29)]. Mice were injected intravenously with HIV SF162 (1 × 10 5 tissue culture infectious doses/ml).

OPN Knockdown by Aptamer Treatments
HPLC purified Opn-R3 PTO (targets Opn) and Scrambled Opn-R3 aptamers were purchased from IBA Solutions for Life Sciences. Modifications to block endogenous nucleases were added to extend the half-life in blood circulation (ref 29). The sequence for Opn-R3-PTO was: 5 ′ GGGAGGACGAUGCGG AUCAGCCAUGUUUACGUCACUCCU-3 ′ and for scrambled FIGURE 1 | Experimental design and correlation of viral loads with engraftment levels. (A) Illustrates the experimental design for the generation of hCD34+ humanized mice, HIV infection, groups implemented and the tissues harvested for the study. The time point for the tissue harvest reflects chronic infection. Representative flow cytometric dot plot of mouse blood-stained for human CD45-PerCP and mouse CD45-FITC used to identify successfully engrafted animals. The 2 × 2 design for female and male mice is indicated. (B) Correlation analyses between measured variables %hCD45 + cells (at time of engraftment) and viral loads (at study endpoint) for each mouse in the four HIV-infected groups. The Spearman coefficients (rs) indicated, while positive, did not reach significance (P < 0.05) in any of the groups. Opn-R3 was 5 ′ -CGGCCACAGAAUGAAUCAUCGAUGUUG CAUAGUUG-3 ′ . Aptamers were refolded into their tertiary structure before injection by heating them in folding buffer (1× PBS and 1 mM MgCl 2 ) at 93 • C for 5 min. Seventy microliter of aptamers were intraperitoneally injected every week at a concentration of 1 mg/kg. Successful knockdown of Opn was confirmed by RT-PCR and IHC and reported previously (29).

RNA in situ Hybridization (RNAscope)
Chromogenic RNA in situ hybridization on HIV SF162 infected animals have been described previously (29). RNAscope kits and protocol were purchased from ACD Biosciences. HIV SF162 target probes were custom-designed to target Env-Nef sequences obtained from the accession number M65024.1 (2621-3701). Slides containing 10 µM sections of spleens, livers, and lungs were pretreated to retrieve their antigens according to the manufacturer's instructions. Custom probes were hybridized for 2 h in the HybEZ oven at 40 • C. Amplification of target probe signal was performed the following day according to RNAscope 2.5 HD detection protocol. The fast red reagent was applied following amplification and developed for 10 min at room temperature. The nuclear stain was achieved with 30% Gill's Hematoxylin I (MilliporeSigma GHS132). Slides were mounted with Cytoseal 60 (#8310-4 Richard-Allan Scientific, ThermoFisher) and imaged on an ECHO Revolve microscope at 40× magnification.

RNAscope Images
Area covering the red staining (which depicts viral RNA) from the chromogenic images were quantified per image with FiJi Image J (NIH). The average area of a single RNA was quantified, and the total area per image was divided by this number to determine the number of viral RNA foci. The group means and standard deviations were combined using the average and square root of the variance approach. Particle analyses were performed with ordinary one-way ANOVA with Tukey's post-hoc test for multiple comparisons was performed to determine any differences between groups. Correlation analysis was performed to determine the relationship between independently measured values with reporting of the Spearman coefficient for nonparametric data and P-value <0.05 considered as significant (Graphpad Prism v8).

Flow Cytometry
FCS files from flow cytometry acquisition were analyzed with Flowjo v10. The data scanned for any anomalies using the flowAI plugin (30). Singlets were selected, followed by gating of cells from side-scatter vs. forward scatter. Human-specific leukocytes were selected by gating on hCD45 cells. Total T cells from cohort 2-4 were determined by gating on hCD3, and this was further used to characterize CD4 and CD8 populations. Monocytes and macrophages were evaluated using the CD14 marker, and absolute B cells were depicted using the CD19 marker. Dot plots were used when cells were abundant (T cells and B cells), and density plots were used to identify cell populations when their counts were lower (monocytes and macrophages). Three-way ANOVA analyses with a post-hoc test for normally distributed data by Tukey's and Kruskal-Wallis for non-parametric continuous data were employed to determine the variation in the immune cells due to Opn status, sex differences, and infection (Graphpad Prism v8). The mean and standard deviation (SD) is reported on all the figures with * P < 0.05, * * P < 0.01, * * * P < 0.001, and * * * * P < 0.0001.

In vivo Mouse Modeling of Low-Level Chronic HIV-1 Infection
Having identified OPN as an HIV-induced host cell protein that stimulates NF-κb mediated transcriptional activation, we utilized a model of low-level chronic HIV infection to assess OPN's role in vivo on HIV replication in plasma, spleen, lung, and liver of male and female NSG-hCD34+ humanized mice ( Figure 1A). This model reflects many, but not all, aspects of viral pathogenesis and immunologic dysfunction observed in HIV infection (23,31). The animals were stratified into four groups with varying degrees of human CD45 engraftment ( Table 1). Successful inhibition of OPN was confirmed by RT-PCR and IHC and reported previously (29). For female HIV-infected mice irrespective of OPN levels, while a positive trend in the relationship between the percentage of hCD45 + cells at initial infection and viral load at the study endpoint was observed [ Figure 1B, Spearman coefficients (rs)], the correlations were not significant ( Figure 1B, NS). These results suggest that the overall number of hCD45 + cells at the time of engraftment did not drive plasma HIV copy number at the study endpoint. It is important here to highlight that the relationship between hCD45 + cells and viral copy number is also expected to be influenced by the genetic background and susceptibility of the three human donors used for humanization (32,33).

Variation in Levels of HIV RNA in the Spleen and Lungs in a Manner Dependent on Sex and OPN
More than 4 months after HIV infection, plasma viral load reached levels below 1,000 copies/ml in 25 out of 28 mice ( Table 1). Tissues were harvested at a time point after HIV infection that represented 48-55 human years of chronic infection. This provided an opportunity to determine whether the spleen, a secondary lymphoid tissue, and the lung harbored productively infected cells and whether the formation of such sanctuaries differed in animals with reduced levels of OPN. Recent studies using models of humanized mice similar to that described herein have studied the establishment and kinetics of HIV sanctuaries, examining alterations in immune cell subsets, activation markers, and viral nucleic acids, but did not quantify vRNA at the single-cell level in tissues (31,34,35). RNA in situ hybridization was used to detect and enumerate HIV-1 load in the spleen, lung, and liver. Viral RNA was detected in the spleens of all the groups as strong and defined fast-red staining showing clusters of RNA coalesced together (Figures 2A,B, arrowheads, 40× magnification). Dense staining was detected in the white pulp, while sparse puncta were also observed in the red pulp, confirming the presence of HIV-infected lymphocytes and macrophages (Figure 2A, black and red arrowheads). Positive immunoreactivity with antisera against hCD45 to detect human leukocytes (black arrowheads) and hCD68 (red arrowheads, a marker for resident macrophages) further confirmed the presence of a subset of infected immune cells in these regions (Figure 2A). T-lymphocyte subsets were identified in the spleen by flow cytometry, suggesting the high likelihood that these cells harbor the majority of HIV RNA (Figures 5D,E). Some M-Opn+ spleens displayed viral RNA with a bursting pattern denoting possible cell lysis and spread ( Figure 2B, third panel, arrows). The area covered by the fast red stain was measured and divided by the area of a single, representing one viral particle to determine the approximate total number of HIV viral particles per image ( Figure 2C). Analyses of viral particle means and variation within groups followed by ordinary one-way ANOVA showed that the reduction of OPN expression significantly decreased HIV RNA burdens in the spleen, but only in female mice ( Figure 2D, P < 0.0001). Significantly more HIV RNA was seen in the spleen of females expressing OPN compared to either male group ( Figure 2D, P < 0.0001). A perfect positive correlation between plasma and tissue HIV RNA was found although it did not reach statistical significance ( Figure 2D, Table rs = 1.0, P = 0.0833). At the study endpoint in mice with plasma loads below the limit of detection (Table 1), viral RNA puncta in tissues were found (Figure 2C, arrows). These findings suggest in the spleen a discordance between plasma viral load and the burden of splenic lymphocytes and macrophages persistently replicating HIV.
In the lungs, sparse HIV RNA puncta were detected in all infected groups ( Figure 3B). H&E stains showed an influx of hCD45 + cells from blood vessels into the lung parenchyma ( Figure 3A, black arrows). Viral RNA was present in these recent migrating cells ( Figure 3A, black arrowheads) and cells near (interstitial macrophages) or lining lung alveoli (alveolar macrophages) ( Figure 3B, black arrowheads). A small number of cells were also found to be hCD68 positive and HIV-infected ( Figure 3A, red arrowheads).
Overall, HIV RNA levels were 10-fold lower in the lung than in the spleen (Figure 2D vs. Figure 3D). In the lung, HIV RNA levels were 2-fold higher in females with reduced OPN than those with regular OPN expression (Figure 3D, P < 0.0001). The reverse relationship was seen in male lungs, where the number of HIV RNA particles was significantly higher with OPN normal expression ( Figure 3D, P < 0.002). We also noted viral RNA puncta in the lungs of mice with undetectable viral RNA in their plasma at the study endpoint (Table 1, Figure 3C, arrows).

Higher Levels of HIV RNA Were Detected in the Liver of Male Mice Expressing OPN
A large influx of hCD45+ cells into the tissue parenchyma was observed in the liver (Figure 4A, arrows). Viral RNA was present in a subset of these cells (Figures 4A,B, black arrowheads). Human CD68 + macrophages were found within and adjacent  Table. to blood vessels with a subset showing positivity for viral RNA (Figure 4A, red arrowheads). Three males exhibiting very high plasma loads had giant clusters of viral RNA surrounded by many hematoxylin positive nuclei, a pattern not observed in any female group (Figure 4B, arrows). While no differences in HIV RNA burden in the two female mice groups were seen, males expressing normal levels of OPN had the highest levels of viral RNA particles (Figure 4D, P < 0.0001). Moreover, there was a significant positive correlation between the plasma and liver viral loads in infected males expressing OPN (Figure 4D, Table, rs = 0.893, P = 0.0417).

Variation in CD4 + Cell Levels and CD4:CD8 Ratios Are Not Modulated by OPN Expression
In addition to lowering viral loads, ART normalizes CD4:CD8 ratios and reverses some of the HIV-induced T cell dysfunction when administered during the acute phase of infection (36)(37)(38). OPN through a NF-κB signaling pathway enhances HIV replication in HIV target cells (19, 39). ART was not used in this study, but rather we tested the hypothesis that inhibiting OPN expression would serve to decrease HIV replication and thereby spare CD4 + T-cell depletion. Human CD4 and CD8 T cell (hCD4, hCD8) population levels found in the spleen, lung and liver where quantified by flow cytometric analyses on freshly prepared single-cell suspensions generated from PBS-perfused tissues. A gating strategy based on human CD45 (hCD45) lymphocytes was used to analyze human specific leukocytes in the spleen (Supplementary Figure 1). For all tissues there were no significant between group differences (Figures 5-7A-C, NS). However, three-way ANOVA analyses with post-test for normally distributed data by Tukey's and Kruskal-Wallis for nonparametric continuous data were used to enumerate potential variation and interaction between sex, OPN status and HIV infection. In the spleen, a decrease in total T cells (hCD3 + ) in both female infected groups was observed (Figure 5D   In the liver, there were no significant between-or intra-group differences in hCD3 and hCD8 cell numbers [ Figures 7A-C

Both HIV Infection and OPN Expression Influence the Number of CD14 + Monocytes in the Lungs, but Not in the Spleen or Liver
Monocyte trafficking into tissues is increased in HIV-infected individuals and plays a role in promoting inflammatory sequelae and enhancing the HIV reservoir (40). Monocyte numbers in the spleen, lung, and liver were assessed by FACS using antisera against hCD14, a marker for the lipoprotein receptor (LPS) responsible in concert with toll-like receptor signaling, for potentiating inflammatory responses (40,41). The gating strategy for spleen, lung and liver is shown in Supplementary Figure 4. Three-way ANOVA (non-parametric, spleen and lung; parametric, liver) of single-cell suspensions of the spleen demonstrated that there were no differences in the hCD14 + levels in the spleen between groups, but most of the females from every group had very low counts compared to males [Figures 8A-C . Similar analyses of cells in the liver did not reveal any differences due to infection, sex or OPN status ( Figure 8C).
These results indicate that irrespective of virus replication, OPN expression can impact monocyte numbers in the lungs. Chronic HIV infection independently reduces the number of monocytes in the lungs, while reducing OPN increases monocyte trafficking to this tissue in a manner independent of HIV infection. Individual points and mean ± SD are displayed. Three-way ANOVA, with Tukey's post-hoc for normally distributed data to determine the variation in the immune cells due to Opn status, sex differences, and infection; results are displayed below the bar charts with significant results highlighted. (D,E) CD4:CD8 ratio analyses were performed with three-way ANOVA for non-parametric continuous data and post-hoc test by Kruskal-Wallis to determine the variation in the immune cells due to Opn status, sex differences, and infection. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.  The absolute number of B cells in plasma usually declines in response to acute or chronic HIV replication (25,42). Therefore, we evaluated whether human B cell numbers in spleen, lung and liver followed a similar pattern in our HIV-infected humanized mice and whether OPN expression had any impact on this response. The FACS gating strategy for spleen, lung and liver is provided in Supplementary Figure 5 Proportional Differences of CD3 + T Cells, CD19 + B Cells, and CD14 + Monocytes in Mice With Low Levels of OPN Expression OPN can function as a chemoattractant facilitating the recruitment of leukocytes into tissues and promoting cell survival (17,18,20). In this scenario, knockdown of chemotactic and pro-survival signaling through OPN expression interference would be expected to reduce any increased immune cell influx stimulated by HIV infection. Therefore, we hypothesized that the percent frequency of each subpopulation of leukocytes might be differentially reduced in the absence of OPN expression. Twoway ANOVA between cell types (T cell, B cell, and monocytes) and OPN status revealed highly significant differences in the percent of leukocytes in tissues where OPN expression was unaffected or knockdown with aptamers ( Figures 10A,B, Opn status, P < 0.0001). Surprisingly, comparing the two HIVinfected female groups in the spleen (Figure 10A, F-Opn+ 11.2 ± 6.9, F-Opn-27.8 ± 13.8) and lung (Figure 10A (Figures 3B, 4B). There was a decrease in the percent frequency of splenic B cells (Figure 10A

DISCUSSION
Replication-competent HIV-1 can persist within immune cells contained in secondary lymphoid tissues and other organs (43). Most recent estimates suggest that more than 70 years of uninterrupted ART is required to eliminate such reservoirs (4). Many factors affect the pharmacodynamics/kinetics of ART therapeutic efficacy (44-47). Therefore, understanding and identifying host proteins and associated cellular pathways that support viral persistence in tissues can inform therapeutic development aimed at halting HIV-1 spread and associated disease sequelae. This study shows evidence of osteopontin's (OPN) potential to affect the formation of HIV-infected tissue sanctuaries in female humanized mice. OPN expression is   (16,19,20). Therefore, the decline in tissue persistence seen in this study could be related to reducing HIV replication, decreased trafficking of infected cells into the tissues, or a combination of the two. An essential consideration of the NSG-hCD34 model is to understand that the phenotypes observed are also likely in direct relationship to the genetic background of the donor cells used for engraftment and could explain some of the FIGURE 10 | The proportion of T cells, B cells, and monocytes in the infected spleen, lung, and liver. (A) Shows stacked bar charts depicting the proportion of hCD3, hCD19, and hCD14 as a percentage of hCD45 in the different tissues assessed. Proportional differences were computed by two-way ANOVA, and the results are displayed in (B) with significant results highlighted. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. variation observed in this study (33). Nevertheless, through 3way ANOVA to further investigate the sources of variation and interaction, we found significant impacts of HIV, sex, and OPN.
Infected female mice in which OPN expression was blocked, had lower viral persistence in the spleen as evident by the significantly fewer number of viral RNA foci detected. The spleen, a secondary lymphoid organ, is a major HIV sanctuary with HIV infected T cells found in the white pulp and macrophages in the red pulp. In this study, most of the vRNA foci in the spleen were localized to the white pulp. In a humanized mouse model with ART beginning 4 weeks post-infection and continuing for a total time of 22 weeks, HIV viral DNA decreased 2-logs and remained stable highlighting the limitations of ART to hasten the turnover of infected cells in the spleen (48). Splenic T cell dysfunction has been previously associated with a decrease CD4 T cells and concomitant increase in CD8 cytotoxic lymphocytes (49). Our results indicated a similar trend of low CD4 and increased CD8, which was not recovered by inhibiting OPN expression. There were no significant differences in CD4 or CD8 levels with OPN expression, suggesting that OPN does not modulate these cells' recruitment into the spleen. Rather, reduced OPN expression results in a decline in HIV replication and lower vRNA persistence in tissues. Therefore, while infected T cells can extravasate into the spleen in animals with reduced OPN, HIV replication in these cells is limited. Female infected mice suffered a more significant loss in splenic CD4 than males. This likely reflects reported sex differences in HIV pathogenesis with infected females showing a faster rate of CD4 cell loss than males (11).
B-cell follicles found in lymph nodes and gut-associated lymphoid tissue are sites of dynamic interaction between specific T-cell subsets and NK cells involved in immunity against HIV (50,51). We also evaluated whether HIV-mediated B cell loss can be recovered by lowering viral persistence in the spleen. We found, however, that B cells declined in all infected animals irrespective of OPN levels. Interestingly, a role for OPN in Bcell differentiation in immune-mediated inflammatory disorders, including multiple sclerosis, alcoholic and non-alcoholic liver diseases, and others, has been reported (52,53). This could potentially explain why CD19 numbers did not increase in the HIV-infected groups with reduced OPN expression.
In HIV infection, monocyte recruitment is increased contributing to tissue macrophage viral reservoirs (49,54). However, in this low-level chronic infection model, we did not observe an increase in monocyte recruitment into the spleen. Rather in HIV-infected animals with or without OPN, CD14 cells were decreased. Further characterization of monocytic subsets with CD16, chemokine profiling, and sampling at an earlier time point would shed further light on their movement from blood to tissues.
Analysis of lungs also revealed a decrease in viral persistence in infected females with reduced OPN levels. Like the spleen, we did not observe CD4 or B cell recovery or CD8 decrease in the lungs despite the lower levels of HIV vRNA seen in this tissue. The prevalence of lung complications is increased with HIV infection and include respiratory infections like Mycobacterium tuberculosis and non-infectious dysfunctions such as emphysema and chronic obstructive pulmonary disease (55). These complications have been attributed to increased CD8 infiltrates in lung mucosa (55). Therefore, although reducing OPN expression lowered virus replication levels in the lungs, immune dysfunction was not reversed. With HIV infection monocyte levels were decreased in the lungs but interestingly, mice with reduced OPN expression showed a trend of an increased number of monocytes compared to animals with normal levels of OPN, however the sample sizes were small.
Sex differences in HIV persistence were observed in the liver. Males with and without OPN exhibited higher levels of viral RNA than females, and three out of seven males had giant hCD45 + clusters of cells that produced viral RNA. This pattern was not observed in females, even those with higher levels of virus in plasma. Reduction of OPN in females resulted in lower viral RNA in the liver but not in male mice, and T cell dysfunction and B cell decline again were not recovered in this context. Overall, in all tissues assessed, inhibition of OPN expression resulted in greater recruitment of CD3 + T cells and a lower influx of B cells.
Collectively, our findings demonstrate the potential for host factors to influence in a sex-dependent or -independent manner immune cell-HIV replication dynamics in tissues and warrant further investigation of the mechanisms involved.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding author/s.