Immune cell activation by HIV-1 is essential to mount an effective host immune response. This process however also provides an immunological microenvironment that drives viral replication and disease progression in HIV-1–infected individuals.1–3
HIV-1 replication is regulated by a complex network of immune factors produced by a variety of hematopoietic and nonhematopoietic cells. Although immune factors with stimulatory and inhibitory effects on HIV-1 replication in peripheral blood mononuclear cells (PBMCs) are expressed at mucosal sites of HIV-1 exposure, for example, the female genital tract (FGT), knowledge about their contribution to HIV-1 replication is limited.4–7
The genital mucosa is populated with T cells, macrophages, and dendritic cells expressing the HIV-1 receptor CD4, and coreceptors CCR5 and CXCR4.8,9 Each of these cell populations is a potential target for infection; yet, recent findings identified CD4+ T cells as the main infected cell population in cervicovaginal tissues.10 Moreover, activated compared with resting CD4+ T cells are more susceptible to HIV-1.7,11–13 These findings suggest that HIV-1 induces inflammatory cytokine production in mucosal CD4+ T cells, leading to immune cell activation, proliferation, and HIV-1 receptor expression,2,14–16 thereby enhancing HIV-1 infection.7
The relevance of inflammatory cytokines in driving HIV-1 replication in the FGT is underscored by our previous work demonstrating that HIV-1 enhances interleukin (IL)-6 expression in cervical tissues (CTs).17 Higher IL-6 production is associated with increased HIV-1 transcription. Evaluating the impact of a woman's reproductive phase on HIV-1 replication, we demonstrated greater expression of pro-inflammatory cytokines and chemokines and enhanced HIV-1 replication in CTs from postmenopausal women compared with premenopausal women.7 Regarding the impact of sexually transmitted pathogens on HIV-1 infection, specifically herpes simplex virus type 2 (HSV-2), we reported enhanced viral replication in HIV-1/HSV-2 coinfected CTs compared with HIV-1–infected CTs.18 Greater HIV-1 replication was associated with higher CD4, CCR5, and CD38 expression and frequency of HIV-1–infected cells.18 These findings underscore the role of mucosal inflammation and sex hormones in regulating HIV-1 replication.19–22
The identification of factors driving immune cell activation and HIV-1 replication by HSV-2 is incomplete. Productive HSV-2 infection of cervical epithelial cells stimulates C-X-C motif chemokine 9 (CXCL9) expression, which controls viral infection.23–25 CXCL9 triggers CD4+ T-cell migration, activation, and proliferation26; yet, the extent to which CXCL9 impacts HIV-1 replication in the genital mucosa is unknown.
Increased CXCL9 expression has been reported in blood, semen, gut mucosa, and colostrum samples of HIV-infected individuals.27–29 Furthermore, low CXCL9 expression in the genital mucosa of HIV-exposed seronegative commercial sex workers was associated with HIV-1 protection,30 suggesting that CXCL9 may be a target to decrease HIV-1 replication.
We report on the scope and mechanism of CXCL9 modulation of HIV-1 replication. HIV-1 enhanced CXCL9 expression and blocking CXCL9 decreased HIV-1 replication in CTs. CXCL9 induction by HIV-1 and decreased HIV-1 replication by CXCL9 neutralization were also observed in PBMCs, suggesting that immune cells may contribute to CXCL9 expression and signaling in CTs. CXCL9 neutralization decreased mucosal immune cell activation, thereby lowering the number of activated CD4+ T cells.
CXCL9 neutralization enhanced the efficacy of suboptimal concentrations of tenofovir (TFV), the leading mucosal pre-exposure prophylactic microbicide, especially early during the infection. When used in combination with TFV at suboptimal concentrations possibly present in poor adherers, CXCL9 neutralization could further decrease HIV-1 replication in CTs, a main portal of viral entry.
CTs were obtained from HIV-1 seronegative women undergoing hysterectomy at Dartmouth–Hitchcock Medical Center for benign medical conditions after an institutional review board–approved protocol. Nonpolarized CTs were established as described.17,31–33 Using our culture conditions, tissues can be maintained for until 21 days without significant decrease in viability, as determined by lactate dehydrogenase viability assay (Cytotoxicity Detection Kit; Roche, Indianapolis, IN).
HIV-1 stocks were generated in human PBMCs. Tissues were infected with 104 cell-free R5-tropic HIV-1BaL, a 50% tissue culture infectious dose (TCID50)/mL. After overnight incubation at 37°C, tissues were washed to remove residual input virus (day 0) and cultured for 21 days in Leibovitz L-15 medium (Gibco, Grand Island, NY) as described.7,17,18 Day 11 and 21 supernatants were evaluated for HIV-1 p24 antigen by enzyme-linked immunosorbent assay (ELISA) (PerkinElmer, Boston, MA).
PBMC Isolation and Infection
PBMCs were isolated by Ficoll-Paque Plus (Amersham, Piscataway, NJ)34 and incubated overnight at 37°C in complete RPMI-1640 (GIBCO). Next day, cells were resuspended at 2 × 106 cells per milliliter and infected with one 50 TCID50/mL of HIV-1BaL. After overnight infection, PBMCs were washed, resuspended in medium, and cultured for 7 days.
Neutralizing Antibody and TFV Treatment
Of note, 1 μg/mL of either antigen affinity-purified polyclonal goat neutralizing CXCL9 or isotype control antibodies (abs; R&D systems, Minneapolis, MN) was added to CTs or PBMCs on day 0, replenished every 3 days, and maintained in the cultures throughout the experiment. No ab cytotoxic effects were observed. Tissues were treated with TFV at either 100 or 10 μg/mL for 6 hours before HIV-1 infection. When indicated, CXCL9 neutralizing or isotype control abs was added to designated wells on day 0. TFV was kindly provided by CONRAD (Arlington, VA).
Nucleic Acid Isolation
Genomic DNA and RNA were isolated as previously described.7,17,18,35
HIV-1 Reverse Transcription and Integration
HIV-1 total DNA and integration were detected by a 2-step quantitative real-time polymerase chain reaction amplification as described.35–37
HIV-1 RNA and HIV-1 receptor, coreceptor, and immune cell activation markers' expression was detected as described.18
Fluorescence–Activated Cell Sorting Experiments
CTs (8–10 pieces) were digested with collagenase D at 5 mg/mL in complete L15 for 1 and half hour at 37°C (Roche Diagnostics, Indianapolis, IN). Single cell suspensions were fixed and stained as described.38 Data were acquired on a BD FCASCanto II (BD Biosciences, San Jose, CA) using FACSDiva software (BD Biosciences) and analyzed with FlowJo version 10.0.7 (Ashland, OR). CD4+ T cells were defined as CD3+ and CD8−.10
Our experimental design fits a hierarchical model (dependent observations) to account for repeated measurements, each triplicated, within tissues. We included a random intercept for tissue and triplicates within tissue, in addition to fixed effects for the day and treatment. Analysis of datasets for HIV-1 reverse transcription, integration, and p24 containing 2 groups (Isotype and CXCL9 neutralizing ab)-treated tissues on days 11 and 21 after infection as well as CD4, CCR5, and CD38 RNA expression on days 3 and 5 after infection was performed by paired Student t test after logarithmic transformation to achieve normality. Nontransformed data of CXCL9 expression were expressed as arithmetic mean values and compared by paired Student t test. P values of <0.05 were considered significant.
HIV-1 Enhances CXCL9 Expression and Blocking CXCL9 Decreases HIV-1 Replication in CTs
CXCL9 expression is enhanced in the peripheral blood, semen, and gut mucosa of HIV-1–infected individuals27,29; yet, modulation of CXCL9 expression by HIV-1 in the FGT remains to be evaluated. To address whether HIV-1 enhances CXCL9 expression at primary sites of viral exposure, we evaluated CXCL9 protein levels in uninfected and HIV-1–infected CTs. CXCL9 levels were expressed as fold increase in HIV-1–infected tissues compared with uninfected controls set to 1. CXCL9 expression was significantly enhanced by HIV-1 on day 7 (Fig. 1A, P = 0.04) compared with day 4 (P = 0.128) after infection.
To determine if there was a causal relationship between CXCL9 and HIV-1 replication, we blocked CXCL9 signaling with a specific neutralizing ab and infected CTs with HIV-1.
We detected a significant decrease in HIV-1 p24 levels in supernatants from HIV-1–infected CTs treated with CXCL9 neutralizing ab compared with isotype control–treated tissues on both days 11 (P = 0.009) and 21 (P = 0.027) after infection (Fig. 1B). Day 11 is one of the earliest time points where we consistently detect new viral release and HIV-1 DNA expression. Day 21 is the day when we terminated our experiments.7,18 To ascertain the step in the virus life cycle, we next tested whether CXCL9 neutralization decreased early events, that is, viral reverse transcription and integration. We detected no differences in HIV-1 reverse transcription between tissues treated with CXCL9 neutralizing or isotype control abs on either day 11 (P = 0.215) or 21 (P = 0.569) after infection (Fig. 1C). Likewise, CXCL9 neutralization did not alter HIV-1 integration at either time point (Fig. 1D; P = 0.824 and P = 0.698 on days 11 and 21 after infection, respectively).
Blocking CXCL9 Signaling Decreases HIV-1 Receptor and Immune Activation Markers in CTs
The lack of effect of CXCL9 neutralization on HIV-1 reverse transcription and integration suggests that CXCL9 decreases HIV-1 replication by targeting postintegration events. CXCL9 stimulates proliferation and activation of CD4+ T cells.26,39 Thus, CXCL9 may down-regulate HIV-1 replication by decreasing the activation phenotype of HIV-1 target cells. This hypothesis is supported by our published data demonstrating enhanced HIV-1 replication in CTs, which is associated with increased immune cell activation marker expression.18 Furthermore, decreasing mucosal inflammation by depressing NFκB signaling lowered HIV-1 replication.7
To test whether CXCL9 neutralization reduces mucosal immune cell activation, we evaluated the impact of blocking CXCL9 on CD4, CCR5, and CD38 RNA expression by reverse transcriptase polymerase chain reaction on days 3 and 5 after infection. Selection of these time points allowed evaluation of gene transcription after immune cell activation before T-cell depletion by HIV-1.18,40,41
On day 3, we detected a transient increase in CD4 (P = 0.0002), CCR5 (P = 0.0018), and CD38 (P = 0.03) RNA expression in CXCL9 neutralizing ab compared with isotype control–treated tissues (Figs. 2A–C), which was associated with similar levels of HIV-1 transcription (Fig. 2D). On day 5, CD4 (P = 0.0003), CCR5 (P = 0.0005), and CD38 (P = 0.006) RNA expression was decreased in HIV-1–infected tissues treated with CXCL9 neutralizing ab compared with isotype control–treated tissues (Figs. 2A–C). Down-regulation of receptor RNA expression correlated with decreased HIV-1 RNA production (P = 0.02, Fig. 2D).
HIV-1 Induces CXCL9 Expression, and CXCL9 Neutralization Decreases HIV-1 Replication in PBMCs
CD4+ immune cells are the primary HIV-1 targets in mucosal tissues.10 To assess the potential of immune cells as CXCL9 producers, we evaluated CXCL9 expression in supernatants from nonactivated HIV-1–infected PBMCs, which were used as a surrogate of mucosal immune cells. Supernatants were harvested on days 2 and 5 after infection and evaluated for CXCL9 levels by ELISA. CXCL9 expression was significantly enhanced in supernatants from HIV-1–infected compared with uninfected PBMCs. We detected a 5-fold and a 1.5-fold increase on days 2 (P = 0.001) and 5 (P = 0.02) after infection, respectively (Fig. 3A).
CXCL9 neutralization decreased HIV-1 p24 levels on both days 5 (P = 0.05) and 7 (P = 0.006) after infection in PBMCs (Fig. 3B). Blocking CXCL9 signaling had no direct effect on either HIV-1 reverse transcription (Fig. 3C) or viral integration (Fig. 3D).
Blocking CXCL9 Signaling Decreased the Number of Activated Cervical CD4+ T Cells
Our data suggest that blocking CXCL9 signaling reduces HIV-1 replication in both CTs and PBMCs (Figs. 1B, 3B). Given that our transcription experiments in CTs indicated a correlation between decreased HIV-1 transcription and immune cell activation marker expression (Fig. 2), we defined the impact of blocking CXCL9 on the activation phenotype of cervical CD4+ T cells.
Consistent with the results from our transcription experiments (Figs. 2A–C above), CXCL9 neutralization decreased the number of CD4+ T cells expressing CCR5 and CD38 on day 7 after infection (P = 0.019; Fig. 4B). On day 5, we detected a transient yet not significant increase in CCR5+ and CD38+ expression on CD4+ T cells in CXCL9 neutralizing compared with isotype control–treated tissues (P = 0.17; Fig. 4A).
Blocking CXCL9 Signaling is an Effective Target to Reduce HIV-1 Replication and Improve the Anti-HIV-1 Activity of TFV in CTs
To explore the translational/clinical relevance of our findings, we tested the effect of CXCL9 neutralization as a means to enhance the efficacy of TFV at suboptimal concentrations. In these experiments, CTs were left untreated or treated with TFV at either suboptimal concentrations (10 μg/mL) possibly found in women with incomplete/inadequate drug use42 or optimal prophylactic concentrations (100 μg/mL) in our experimental system,18 before infection with HIV-1. Compared with untreated controls, tissues treated with TFV at 100 µg/mL decreased HIV-1 p24 levels by 53% (P = 0.003) and 77% (P = 0.00003) on days 11 and 21 after infection, respectively (Fig. 5A). As expected, tissues treated with this TFV concentration displayed higher reduction in HIV-1 p24 levels than tissues treated with TFV at 10 µg/mL or those exposed to CXCL9 neutralizing ab (Figs. 5B, C). By comparing tissues treated with suboptimal concentrations of TFV and those subjected to CXCL9 neutralization, we found that TFV at 10 µg/mL lowered HIV-1 p24 levels by 17% (P = 0.01) on day 11 (Fig. 5B), whereas tissues treated with CXCL9 neutralizing ab reduced HIV-1 p24 levels by 42% (Fig. 5C; P = 0.009). On day 21, both treatments decreased HIV-1 p24 release by 52% (Figs. 5B, C; P = 0.0007 and P = 0.027 for TFV and CXCL9 neutralization, respectively).
Given that on day 11, CXCL9 neutralization decreased HIV-1 replication to a greater extent than TFV at 10 µg/mL, we next tested whether CXCL9 neutralization improved the anti–HIV-1 activity of suboptimal concentrations of TFV. On day 11, tissues treated with TFV in combination with CXCL9 neutralizing ab displayed a significant decrease (23%, P = 0.02) in HIV-1 replication compared with those treated with TFV alone (Fig. 5D). This effect was lost on day 21 (P = 0.309), given that treatment with either TFV at 10 μg/mL or CXCL9 neutralizing ab displayed similar levels of protection at this time point (Figs. 5B, C).
Mucosal immune responses modulate HIV-1 replication. Understanding mechanisms of HIV-1 regulation of mucosal immune responses and identifying novel targets that control HIV-1 replication in the FGT, the main portal of viral entry in women after vaginal intercourse43 is critical in the development of approaches to decrease viral replication and prevent systemic HIV-1 dissemination. We provide novel insights into the scope and mechanism of CXCL9 regulation of HIV-1 replication in CTs, a relevant model of mucosal HIV-1 transmission. HIV-1 enhanced CXCL9 expression, and blocking either constitutive or HIV-1–induced CXCL9 levels decreased HIV-1 replication in CTs. Reduced HIV-1 p24 release was the result of lower immune cell activation, that is, CCR5 and CD38 expression on cervical CD4+ T cells. Data consistency between CTs and PBMCs underscores the role of immune cells on CXCL9 production in CTs and highlights the relevance of CXCL9 signaling on enhancing HIV-1 replication. When evaluating the anti–HIV-1 activity of CXCL9 neutralization in combination with suboptimal concentrations of TFV possibly present in poor adherers in clinical trials,44,45 we detected a greater decrease in HIV-1 replication in tissues cotreated with TFV and CXCL9 neutralizing ab compared with those treated with TFV alone at earlier stages of infection. Thus, targeting mucosal innate immune responses by blocking CXCL9 signaling may be a relevant therapeutic approach to down-regulate HIV-1 replication and potentially reduce viral genital shedding or dissemination to the periphery when suboptimal concentrations of microbicides such as TFV are present in the cervicovaginal mucosa.
HIV-1 Infection Enhances CXCL9 Expression
CXCL9 is produced in a wide range of inflammatory conditions.46,47 In line with epidemiological studies describing increased CXCL9 levels in the blood, semen, and gut mucosa of HIV-1–infected treatment-naive patients,27,29 we demonstrated enhanced CXCL9 expression by HIV-1 in CTs (Fig. 1A) and PBMCs (Fig. 3A). Although we acknowledge variations in phenotype between PBMCs and cervical leukocytes, our findings suggest that immune cells may contribute to CXCL9 expression in CTs.
Blocking CXCL9 Signaling Decreases HIV-1 Replication
Blocking CXCL9 signaling significantly decreased HIV-1 p24 release in CTs (Fig. 1B) and PBMCs (Fig. 3B), supporting the hypothesis that CXCL9 promotes HIV-1 replication in immune cells of the FGT. This conclusion is consistent with studies demonstrating greater genital mucosa CXCL9 levels in HIV-1–infected compared with uninfected female sex workers (FSWs) or uninfected control women, suggesting that CXCL9 expression induces a mucosal microenvironment that favors HIV-1 replication.48 Furthermore, HIV-1-exposed seronegative FSWs displayed significant lower CXCL9 expression compared with HIV-1-seropositive FSWs,30 implying a potential role of CXCL9 in the establishment of the infection.
CXCL9 Signaling Decreases the Activation Phenotype of Mucosal CD4+ T Cells
Knowing that each infected cell harbors 1 copy of integrated viral DNA,49 and using HIV-1 integration as a surrogate of the number of infected cells, we saw no effect of CXCL9 neutralization on HIV-1 integration in either CTs (Fig. 1D) or PBMCs (Fig. 3D). No difference in the frequency of HIV-1–infected cells, however, was associated with decreased HIV-1 p24 release from tissues or cells treated with CXCL9 neutralizing compared with isotype control abs (Figs. 1B, 3B), suggesting that, under the settings of CXCL9 neutralization, HIV-1–infected cells produced fewer viral particles. HIV-1 replication depends on immune cell activation,50–52 and CXCL9 induces activation and proliferation of CD4+ T cells.53 Thus, we evaluated CD4 and immune activation markers' CCR5 and CD38 RNA expression in CTs. Early during the infection, CXCL9 neutralization enhanced CD4, CCR5, and CD38 RNA levels (Figs. 2A–C), suggesting an increase in immune cell activation marker expression likely by additional chemokines such as CXCL10 or CXCL11. This transient increase in receptor expression was associated with no differences in HIV-1 transcription (Fig. 2D). In contrast, at later time points, we detected a significant decrease in CD4, CCR5, and CD38 transcription in tissues treated with a CXCL9 neutralizing ab compared with isotype control–treated tissues (Figs. 2A–C) that was associated with decreased HIV-1 RNA expression. These findings imply that CXCL9 neutralization down-regulates HIV-1 replication in CTs by reducing CD4+ T-cell activation. Supporting our results, it has been demonstrated that compared with resting T lymphocytes activated CD4+ T cells are more susceptible to HIV-1 infection.11–13 Findings from our fluorescence–activated cell sorting experiments demonstrated a lower frequency of cervical CCR5+ CD38+ CD4+ T cells in tissues treated with CXCL9 neutralizing compared with isotype control abs on day 7 after infection (Fig. 4B), indicating consistency yet different kinetics of RNA and protein expression of HIV-1 receptor and immune activation marker expression in CTs.
Our results suggest that CXCL9 down-regulates HIV-1 replication by decreasing immune cell activation in CTs. Supporting this hypothesis, we have previously demonstrated that HSV-2, a virus that also induces CXCL9 expression,23 enhances HIV-1 replication by up-regulating CCR5 and CD38 expression in CTs.18 These findings underscore a positive correlation between immune cell activation and HIV-1 production at the site of primary infection.
One limitation of the tissue explant model is that we cannot evaluate cell recruitment and influx of new cells.31,33 Because CXCL9 induces immune cell recruitment, we are potentially underestimating the effect of CXCL9 neutralization on HIV-1 replication in our model.
Blocking CXCL9 Signaling Improves the Anti-HIV-1 Activity of Suboptimal Concentrations of TFV in CTs
Pre-exposure prophylactic regimens containing nucleoside reverse transcriptase inhibitors have shown discrepant results in men who have sex with men, serodiscordant couples, and women.43,54–58 Although biological factors cannot be ruled out, these inconsistencies are most likely attributable to poor adherence resulting in inadequate antiretrovirals' tissue concentrations and protective effects. This concept has also been cited to explain the discrepant results of TFV 1% vaginal gel showing 39%, 14%, and 0% effectiveness under intent to treat analysis in CAPRISA 004, MTN-003, and FACTS 001 trials, respectively.59–61 Case–cohort analysis based on returned applicators and/or drug levels, however, demonstrated protection in 50%–60% of women who used the gel consistently per protocol.44 These results underscore the role of adherence in decreasing the efficacy of TFV.54 Additional data, however, suggest that partial protection may also have been due to biological factors such as subclinical inflammation triggered by vaginal infections known to enhance mucosal susceptibility to HIV-1.7,18 Thus, alternative and multi target treatments are likely to provide more efficient preexposure prophylaxis to women.
Given that blocking CXCL9 signaling significantly reduced HIV-1 replication, we explored the anti-HIV-1 efficacy of CXCL9 neutralization in combination with TFV at suboptimal concentrations. When TFV concentrations were suboptimal, CXCL9 neutralization decreased HIV-1 replication to a higher degree than TFV alone early during the infection (Figs. 5B, C). Indeed a combined multitarget treatment of TFV at suboptimal concentrations and CXCL9 neutralizing ab demonstrated an additional protection compared with the treatment with TFV alone (Fig. 5D). We have previously demonstrated that TFV at suboptimal concentrations enhances the frequency of CD4+ T cells early during the infection.38 Thus, CXCL9 reduced HIV-1 replication, likely by decreasing the immune activation of CD4+ T cells. This additive effect provides a proof of concept that understanding the pathogenesis of the initial HIV-1 mucosal infection and designing specific interventions may be a potential approach to enhance the activity of topical or systemic prophylactic antiretrovirals. Thus, by decreasing activation of mucosal CD4+ T cells, CXCL9 neutralization could delay viral spread to the periphery or reduce HIV-1 genital shedding complementing the antiviral effects of topical antiretrovirals.
The authors thank Anatomic Pathology, DHMC, Lebanon, NH, for tissue procurement.
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