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JAIDS Journal of Acquired Immune Deficiency Syndromes:
doi: 10.1097/QAI.0b013e3182961cfc
Clinical Science

Plasma and Mucosal HIV Viral Loads Are Associated With Genital Tract Inflammation in HIV-Infected Women

Herold, Betsy C. MD*,†,‡; Keller, Marla J. MD†,§; Shi, Qiuhu PhD; Hoover, Donald R. PhD; Carpenter, Colleen A. BS*; Huber, Ashley BS*; Parikh, Urvi M. PhD#; Agnew, Kathy J. BS**; Minkoff, Howard MD††; Colie, Christine MD‡‡; Nowicki, Marek J. PhD§§; D'Souza, Gypsyamber PhD‖‖; Watts, D. Heather MD¶¶; Anastos, Kathryn MD†,§,##

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Author Information

Departments of *Pediatrics;

Obstetrics & Gynecology and Women's Health;

Microbiology and Immunology;

§Medicine, Albert Einstein College of Medicine, Bronx, NY;

New York Medical College, Valhalla, NY;

Institute for Health, Health Care Policy and Aging Research, Rutgers University, Piscataway, NJ;

#University of Pittsburgh, Pittsburgh, PA;

**University of Washington, Seattle, WA;

††Maimonides Medical Center, Brooklyn, NY;

‡‡Georgetown University Medical Center, Washington, DC;

§§University of Southern California, Los Angeles, CA;

‖‖Johns Hopkins Bloomberg School of Public Health, Baltimore, MD;

¶¶Pediatric, Adolescent and Maternal AIDS Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health, Bethesda, MD; and

##Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx NY.

Correspondence to: Betsy C. Herold, MD, Department of Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Forchheimer 702, Bronx, NY 10461 (betsy.herold@einstein.yu.edu).

The Women's Interagency HIV Study (WIHS) is supported by the National Institute of Allergy and Infectious Diseases Grants UO1-AI-35004, UO1-AI-31834, UO1-AI-34994, UO1-AI-34989, UO1-AI-34993, and UO1-AI-42590 and by the Eunice Kennedy Shriver National Institute of Child Health and Human Development Grant UO1-HD-32632. The study was also supported in part by Grants U19AI067980, R01AI065309, R33AI079763 and the Center for AIDS Research at the Albert Einstein College of Medicine and Montefiore Medical Center Grant NIH AI-51519. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.

The authors have no conflicts of interest to disclose.

Data in this manuscript were collected by the WIHS Collaborative Study Group with centers (Principal Investigators) at New York City/Bronx Consortium (K. Anastos); Brooklyn, NY (H. Minkoff); Washington DC Metropolitan Consortium (Mary Young); Los Angeles County/Southern California Consortium (Alexandra Levine); Data Coordinating Center (Stephen Gange).

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (www.jaids.com).

Received December 21, 2012

Accepted March 26, 2013

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Abstract

Background: Systemic and mucosal inflammation may play a role in HIV control. A cross-sectional comparison was conducted among women in the Women's Interagency HIV Study to explore the hypothesis that compared with HIV-uninfected participants, women with HIV, and, in particular, those with high plasma viral load (PVL) have increased levels of mucosal and systemic inflammatory mediators and impaired mucosal endogenous antimicrobial activity.

Methods: Nineteen HIV-uninfected, 40 HIV-infected on antiretroviral therapy (ART) with PVL ≤ 2600 copies/mL (low viral load) (HIV+-LVL), and 19 HIV-infected on or off ART with PVL >10,000 (high viral load) (HIV+-HVL) were evaluated. Immune mediators and viral RNA were quantified in plasma and cervicovaginal lavage (CVL). The CVL antimicrobial activity was also determined.

Results: Compared to HIV-uninfected participants, HIV+-HVL women had higher levels of mucosal but not systemic proinflammatory cytokines and chemokines, higher Nugent scores, and lower Escherichia coli bactericidal activity. In contrast, there were no significant differences between HIV+-LVL and HIV-uninfected controls. After adjusting for PVL, HIV genital tract shedding was significantly associated with higher CVL concentrations of IL-6, IL-1β, MIP-1α, and CCL5 (RANTES) and higher plasma concentrations of MIP-1α. High PVL was associated with higher CVL levels of IL-1β and RANTES, as well as with higher Nugent scores, lower E. coli bactericidal activity, smoking, and lower CD4 counts; smoking and CD4 count retained statistical significance in a multivariate model.

Conclusions: Further study is needed to determine if the relationship between mucosal inflammation and PVL is causal and to determine if reducing mucosal inflammation is beneficial.

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INTRODUCTION

The genital tract is the most common site of HIV acquisition and transmission in women. Recent studies indicate that local and/or systemic inflammation facilitates HIV acquisition and may lead to a higher plasma viral load (PVL) set point.1–3 Inflammation may persist, even in those receiving antiretroviral therapy (ART), possibly reflecting chronic antigen exposure and immune activation. Increased levels of proinflammatory cytokines such as interleukin (IL)-1β and tumor necrosis factor-alpha (TNF-α) may disrupt the epithelial barrier and promote microbial translocation in the gut, further fueling the inflammatory response.4 Microbial translocation has been suggested to play an important role in HIV pathogenesis; however, recent studies have challenged this hypothesis. For example, no significant differences in microbial translocation biomarkers were observed in a cohort study of people with varying rates of HIV-1 disease progression in Uganda, and circulating cytokine levels either decreased or remained unchanged.5

The female genital tract is a reservoir for HIV and cell-free virus may reflect transudation of HIV from the blood and local viral replication; both may be influenced by systemic or local factors, including exposure to ART.6–8 Whether the genital tract is a site of chronic inflammation in HIV-infected women, as has been described for the gut, and how this might impact HIV replication is unknown.7,8 Prior studies suggest that increased levels of genital tract inflammatory cytokines may promote local viral replication and/or recruit immune target cells into the genital tract leading to increased HIV shedding.1–3,9 In vitro studies indicate that HIV may directly induce inflammatory cytokine expression through Toll-like receptors 7 and 8 activation.10 Cytokines and other inflammatory mediators may also be induced in response to sexually transmitted infections (STI), bacterial vaginosis (BV), and exogenous hormones. For example, in a recent prospective study of 57 HIV-infected women, higher concentrations of IL-1β and IL-8 in cervicovaginal lavage (CVL) samples were significantly associated with higher CVL HIV RNA.9 These associations persisted after adjusting for plasma HIV RNA but were attenuated after adjusting for STI and BV.

An important limitation of individual cytokine measures is that the biological impact of changes is difficult to predict as the same molecule may increase HIV replication while promoting host immune responses. A complementary approach that may facilitate interpretation of changes in cytokine levels is to incorporate functional assays, such as the ability of CVL to inhibit viral or bacterial pathogens. Several studies found that CVL samples provide variable inhibitory activity against HIV and herpes simplex virus (HSV)-2 and are bactericidal for Escherichia coli.11–17 Studies conducted among HIV-uninfected women indicate that CVL anti-HSV-2 activity correlates with inflammatory immune mediators but not with HSV serostatus, suggesting that this activity may provide a biomarker of the inflammatory state of the genital tract.11,12,18,20 The bactericidal activity of E. coli may reflect, at least in part, the vaginal microbiome. This notion is supported by prior studies in HIV-uninfected low-risk US women: E. coli bactericidal activity was reduced in the setting of BV,17 correlated inversely with E. coli vaginal colonization,21 and was associated with proteins expressed by healthy lactobacillus species in proteomic studies.22 Given the link between BV and HIV, including the observation of increased HIV transmission from women with BV to their male partners,23–25 we speculate that HIV-infected women and, in particular, those with high VLs or genital tract shedding, may have lower E. coli bactericidal activity compared with HIV-uninfected women.

We conducted a cross-sectional comparison among participants in the Women's Interagency HIV Study (WIHS) to explore the hypothesis that compared with HIV-uninfected women; women chronically HIV-infected have higher levels of systemic and mucosal inflammatory immune mediators, higher CVL anti-HSV activity, and lower CVL E. coli bactericidal activity, which may be associated with mucosal HIV shedding. To evaluate whether the changes were linked to PVL, we compared HIV-infected women with high PVLs (HIV+-HVL) (on or off ART) and those on ART with low PVLs (HIV+-LVL) to HIV-uninfected controls.

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METHODS

Participants and Sample Collection

The WIHS is a prospective cohort study of HIV-infected and HIV-uninfected women; WIHS methods have been described previously.26 Briefly, at semiannual visits, interim history is obtained, blood is collected for CD4 cell count and quantitative HIV-1 RNA, a vaginal swab is collected, and CVL is performed with 10 mL of normal saline. For the present study, we analyzed plasma, CVL, and vaginal swabs (used to determine Nugent scores27) that were collected in parallel at Visit 33 (October 1, 2010 to March 31, 2011). Women were selected based on PVL from a previous WIHS visit but were analyzed based on the PVL results obtained for Visit 33 and divided into the following groups: (1) HIV-uninfected; (2) HIV-infected on ART with plasma HIV-1 RNA ≤2600 copies/mL (HIV+-LVL); and (3) HIV-infected with plasma HIV-1 RNA >10,000 copies/mL (HIV+-HVL). The latter included women on or off ART. The specimens were selected from the WIHS biorepository and included participants from Bronx, Brooklyn, Washington, DC, and Los Angeles WIHS sites. The Visit 33 plasma HIV RNA was quantified using a nucleic acid amplification test with a lower limit of detection (LLOD) of 48 copies/mL (COBAS AmpliPrep/COBAS Taqman HIV-1 test, Roche, Branchburg, NJ). The CD4 count was determined using standard flow cytometric methods (NIH/NIAID Flow Cytometry Quality Assessment Program). Plasma and CVL were stored at −80°C and shipped on dry ice to Albert Einstein College of Medicine.

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Measurement of Protein and Immune Mediators

The CVL protein concentration was measured by Micro BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). The HSV serostatus was determined in plasma for all subjects not previously identified as seropositive using HerpeSelect 1&2 Immunoblot IgG (Focus Diagnostics Cypress, CA). The concentration of IL-1α, IL-1 receptor antagonist (IL-1ra), IL-1β, IL-6, IL-8, IL-12p70, interferon alpha 2 (IFN-α2), macrophage inhibitory protein-1 α and β (MIP-1α and MIP-1β), and regulated on activation, normal T cell expressed and secreted (RANTES) in plasma, and CVL were determined by Luminex100 (Luminex Corp., Austin, TX) with beads from Chemicon International (Billerica, MA) and analyzed using StarStation (Applied Cytometry Systems, Sacramento, CA). Levels of secretory leukocyte protease inhibitor (SLPI) (R&D Systems, Minneapolis, MN) and human neutrophil peptides 1-3 (HNP1-3) (Hycult Biotech, Uden, the Netherlands) were determined in CVL by enzyme-linked immunosorbent assay (ELISA). Luminex assays were performed with undiluted samples, and concentrations below the LLOD were set at the midpoint between zero and the LLOD. The ELISA assays were conducted with diluted samples with those below the LLOD set at the midpoint between zero and LLOD and multiplied by the dilution factor if insufficient sample was available to repeat at lower dilutions. The LLOD were: IFN-α2, 12.25 pg/mL; IL-12p70, 0.4 pg/mL; IL-1ra, 2.9 pg/mL; IL-1α, 3.5 pg/mL; IL-1β, 0.4 pg/mL; IL-8, 0.2 pg/mL; MIP-1α, 3.5 pg/mL; MIP-1β, 4.5 pg/mL; RANTES, 1 pg/mL; HNP1-3, 156 pg/mL; and SLPI, 62.5 pg/mL.

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Antimicrobial Activity of CVL

The CVL antimicrobial activity against HSV-2 and E. coli was measured within 6 months of collection, as previously described.16 Vero cells were infected with approximately 50–200 plaque forming units (pfu) of HSV-2 (G) mixed 1:1 with each CVL or control buffer, and plaques were counted after 48 hours. All samples were tested in duplicate in 2 independent experiments. E. coli (ATCC strain 4382627) was grown overnight to stationary phase and then approximately 109 cfu/mL were mixed with CVL or control buffer (20 mmol/L of potassium phosphate, 60 mmol/L of sodium chloride, 0.2 mg/mL of albumin; pH 4.5) and incubated at 37°C for 2 hours. The mixtures were further diluted in buffer to yield 800–1000 colonies on control plates and added in duplicate to agar enriched with trypticase soy broth. Colonies were counted after an overnight incubation at 37°C. Results are presented as the mean percentage inhibition relative to colonies formed on control plates.

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CVL Viral Loads

The HIV RNA levels were determined by centrifuging CVL at 700g and quantifying HIV RNA in the supernatants, using the Abbott m2000 HIV-1 RealTime System (Abbott Molecular, Des Plaines, IL) with a LLOD of 40 copies/mL.

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Statistical Analyses

Concentrations of mediators were log10 transformed to reduce skewness in the data. Because >25% of samples were below the LLOD for CVL IL-12p70 and IFN-α2 and plasma IL-1α, IL-1β, IL-1ra, IL-12p70, IFN-α2, and MIP-1α, these measures were dichotomized at the LLOD. Categorical variables were compared between groups by Fisher exact test. Continuous variables (including Nugent score) were compared by Mann–Whitney U test. Spearman rank-order correlation coefficients were calculated to assess within-person associations between mediators and bactericidal activity; a rho (r) value = 0.30 was considered indicating a correlation of modest clinical significance. Two-sided P values ≤0.05 were considered to be statistically significant. Sign and sign rank tests compared within-person differences of each immune mediator between the plasma and genital tract compartments. Logistic regression analyses (restricted to HIV+ women) were performed to identify variables associated with genital tract shedding (yes vs. no) or with high PVL (>10,000 vs. ≤2600). As PVL is strongly associated with genital tract HIV shedding, the univariate logistic regression analyses for shedding were repeated adjusting for PVL >10,000 together with each of the other predictor variables. Odds ratios in logistic regression models for log10 transformed laboratory measures are reported per 0.3 log10 units, which corresponds to a doubling of the amount on the original scale. All statistical analyses were performed using SAS version 9.2 (Cary, NC).

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RESULTS

Participant Characteristics

Nineteen HIV-uninfected, 40 HIV+-LVL, and 19 HIV+-HVL women who met the study criteria were included in this cross-sectional comparison (Table 1). The HIV+-LVL cohort included 29 women with plasma HIV RNA levels below the LLOD and 11 with low but detectable PVL (51, 59, 115, 133, 146, 146, 443, 545, 1180, 1610, and 2600 copies/mL). These 11 subjects had a PVL below the LLOD at the preceding WIHS visit from which eligible participants were selected. The median interquartile range [IQR] plasma log10 HIV RNA in the HIV+-HVL cohort was 5.0 (4.5–5.4); this included 9 women on ART. The 3 groups did not differ by age (including the proportion who were postmenopausal), race, education, HSV serostatus, or other demographic characteristics (Table 1). None reported taking hormonal contraception. Fewer women in the HIV+-LVL (but not the HIV+-HVL) group reported current smoking compared with HIV-uninfected controls (P = 0.054) (Table 1). The groups also differed in that all HIV-uninfected women were from the Bronx site, whereas HIV+ women were from all 4 participating WIHS sites (P < 0.001).

Table 1
Table 1
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Mucosal and Plasma Immune Mediators

There were higher CVL concentrations of IL-1α, IL-1β, MIP-1β, and RANTES in HIV+-HVL compared with HIV-uninfected and/or HIV+-LVL participants (Table 2; Figs. 1A–D). In contrast, there were no statistically significant differences in concentrations of cytokines/chemokines between HIV+-LVL and HIV-uninfected. Conversely, plasma levels of MIP-1β and RANTES were significantly lower in HIV+-HVL compared with HIV-uninfected subjects (Table 2; Figs. 1E–F). IL-1α and IL-1β were below the LLOD in the majority of all plasma samples (Table 2). Within the HIV+-HVL group, there were no significant differences in immune mediator levels between participants who reported taking (open symbols) or not taking (closed symbols) ART (see Figure S1a, Supplemental Digital Content, http://links.lww.com/QAI/A423) or within the HIV+-LVL group, between those with detectable (open symbols) compared with those with undetectable (closed symbols) PVL.

Table 2
Table 2
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Figure 1
Figure 1
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We measured concentrations of HNP1-3 and SLPI in CVL as these antimicrobial peptides have anti-HIV activity in vitro18,28–33 and have been associated with protection against HIV in the genital tract or oral mucosa in small clinical studies.34–36 Moreover, lower levels of antimicrobial peptides have been observed in women with BV, a risk factor for HIV acquisition and shedding.17,25 However, the relationship between inhibitory activity and protection is complex as increases in HNP1-3 and LL-37, another defensin-like molecule, have also been associated with ongoing genital infections and, thus, indirectly associated with increased risk of HIV acquisition.37 There were no significant group differences in HNP1-3 or SLPI (Table 2).

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Relationship Between Cytokine/Chemokine Levels in the Plasma and Genital Tract

The IL-1α, IL-1β, IL-1ra, and MIP-1α were below the LLOD in plasma but were detectable in most CVL samples (P < 0.001 for equality between compartments for each by sign test) (Table 2). Conversely, RANTES was more abundant in plasma than CVL (P < 0.001, sign test). The IL-6 and IL-8 were detected at low levels in both compartments but did not correlate with one another (r = 0.11 and 0.04, respectively). Overall, there was no significant (r ≥ 0.30) within-person correlation between the concentrations of individual mediators in plasma compared with CVL.

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Differences in Endogenous Antimicrobial Activity

There were small statistically nonsignificant higher levels of anti-HSV activity in CVL in HIV+-HVL compared with HIV-uninfected [median (IQR) % inhibition 84.3% (76.4%, 87.7%) vs 64.0% (28.9%, 89.1%)], and compared with HIV+-LVL [78.8% (29.3%–86.8%)] (Fig. 2A). There was also less variability in the anti-HSV activity in the HIV+-HVL group. This could reflect the increased concentrations of mucosal proinflammatory mediators in the HIV+-HVL subjects, as prior studies of HIV-uninfected women found a significant correlation between inflammatory proteins and anti-HSV activity11,12,19,38 or differences in HSV-2–specific antibodies (IgA and/or IgG) in CVL, although there are no validated assays to quantify these antibodies in genital tract secretions. To assess the association between inflammatory proteins and activity, Spearman correlation coefficients were calculated within the combined HIV-infected groups. Anti-HSV activity modestly correlated with CVL concentrations of IL-β (r = 0.36), IL-8 (r = 0.41), MIP-1α (r = 0.36), MIP-1β (r = 0.36), RANTES (r = 0.44), and HNP1-3 (r = 0.43), each P < 0.01, but did not statistically correlate with any of the plasma cytokines or chemokines (not shown). Notably, there were no statistically significant differences in CVL anti-HSV activity in the HVL group between those on (open symbols) or not on (closed symbols) ART or within the LVL between those with detectable (open symbols) versus those with undetectable (closed symbols) PVL.

Figure 2
Figure 2
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In contrast to the small increase in anti-HSV activity in the HIV+-HVL group, there was less E. coli bactericidal activity in HIV+-HVL compared with HIV-uninfected [% inhibition, 40.0% (21.0%–55.0%) versus 54.1% (43.0%–79.0%)] (P = 0.04) and compared with HIV+-LVL [56.1% (36.0%–88.0%)] (P = 0.01); there was no difference between the HIV+-LVL and the HIV-uninfected (P = 0.70) (Table 2; Fig. 2B). Within the HIV+-HVL group, there were no significant differences in CVL E. coli bactericidal activity between participants who did or did not report taking ART (see Figure S1b, Supplemental Digital Content, http://links.lww.com/QAI/A423). Conversely, the Nugent scores were significantly higher in HIV+-HVL compared to HIV-uninfected (P = 0.02) and HIV+-LVL (P = 0.01) but not between HIV+-LVL and HIV-uninfected (P = 0.24) (Table 1; Fig. 2C). The Nugent scores correlated negatively and modestly with E. coli bactericidal activity (r = −0.36, P = 0.003).

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Correlations Between HIV Shedding and Clinical and Laboratory Variables

Among the HIV+-HVL cohort, 9 of the 19 (47.4%) had HIV RNA detected in their CVL supernatants (3 on ART and 6 not on ART) with a median (IQR) of 147 HIV RNA copies/mL (40–1485). In contrast, only 2 (5%) of the 40 women in the HIV+-LVL cohort had HIV RNA detected in CVL (40 and 236 copies/mL); both had undetectable PVL (P < 0.001) (Table 1). Subjects with HIV RNA detected in their CVL are indicated by gray symbols in Figures 1 and 2. In a 1-variable logistic regression model, genital tract HIV shedding (yes vs. no) was positively associated with PVL >10,000 (P = 0.002) and, after adjusting for PVL >10,000, with CVL concentrations of IL-6, IL-1β, MIP-1α, and RANTES, all P < 0.05 (Table 3). In addition, genital tract HIV shedding was negatively associated with plasma MIP-1β after adjusting for PVL (P = 0.02, Table 3).

Table 3
Table 3
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In contrast, univariate analysis exploring factors associated with high PVL (>10,000 copies/mL) identified higher CVL IL-1β and RANTES levels, higher Nugent scores, less E. coli bactericidal activity, current smoking, and lower CD4 counts as statistically significant (P < 0.05, Table 3). Only smoking and CD4 count retained statistical significance in a multivariate model (model not shown).

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DISCUSSION

Compared with HIV-uninfected women, HIV+ women with PVL >10,000 copies/mL had higher concentrations of several mucosal (genital tract) but not systemic (plasma) inflammatory immune mediators, higher Nugent scores, and reduced CVL E. coli bactericidal activity. There were no differences within the HVL cohort when women were on or not on ART. In contrast, the women with low VLs were similar to HIV-uninfected women. Notably, lower levels of RANTES and MIP-1β were detected in the plasma of HIV+-HVL compared with HIV-uninfected or HIV+-LVL cohorts. This finding is consistent with the notion that these chemokines may contribute to the HIV-suppressive activity produced by CD8+ T cells.39 However, the converse findings of higher RANTES, MIP-1α, and MIP-1β in the genital tract, which were associated with a higher likelihood of genital tract HIV shedding, suggest differences in the source and/or role of these chemokines in the 2 compartments. Possibly, these chemokines are secreted by genital tract epithelial cells in response to HIV,40 microbiota,41 and/or subclinical STI.42,43 That microbiota may contribute to altered mucosal chemokine levels is suggested by the finding of significantly higher Nugent scores in the HIV+-HVL cohort. Higher levels of cytokines and chemokines in the genital tract have been observed among HIV-infected women in other small studies.9,44

The observation that plasma cytokines were not increased in either the HIV+-HVL or HIV+-LVL compared with HIV-uninfected women challenges the notion that HIV induces a state of chronic systemic inflammation in women. The latter hypothesis stems from observations that HIV-infected individuals, even those well controlled on ART, are at increased risk for complications linked to chronic inflammation, although males predominated in these studies,45 whereas our study is exclusively among women. Higher systemic levels of proinflammatory cytokines including IL-6 are among the immunologic changes that have been observed with HIV infection.46 However, we observed no significant increase in plasma IL-6 levels (or other cytokines/chemokines) in HIV-infected compared with HIV-uninfected women, although it should be noted that other markers of systemic inflammation such as C-reactive protein, D-dimer, cystatin C, and TNF-α were not evaluated here. A prior WIHS study found higher TNF-α levels in HIV-infected compared with uninfected women but did not differ between women with high and low viral burden.47 A second study that took advantage of both WIHS and Multicenter AIDS Cohort Study (MACS) found that the introduction of HAART was associated with reductions in D-dimer and IL-6 levels in MACS (male) participants and the decline correlated with HIV RNA reduction. However, in WIHS (female) participants only D-dimer levels, not IL-6, declined after the introduction of HAART. WIHS women had significantly lower IL-6 and CRP levels at the baseline visit compared with MACS men.48

Genital tract HIV shedding was associated with CVL IL-6, IL-1β, MIP-1α, and RANTES, and these associations retained significance when adjusting for high PVL. This could reflect either direct activation of local HIV replication by inflammatory molecules through activation of the HIV long terminal repeats and/or recruitment of immune target cells into the genital tract. Surprisingly, high PVL did not correlate with concentrations of any cytokines/chemokines in the plasma but with higher CVL IL-1β and RANTES, higher Nugent scores and lower E. coli bactericidal activity in univariate analyses, potentially linking mucosal immunity with PVL by as yet undetermined mechanisms. However, only cigarette smoking and lower CD4 cell counts retained statistical significance in a multivariate model.

Studies examining the association between smoking and PVL have yielded conflicting results. For example, 1 study (n = 36 HIV-infected subjects) found that smoking correlated with higher VL and lower CD4 cell count,49 whereas another found no correlation.50 Whether the association observed here reflects immunomodulatory effects of smoking or is a biomarker of behaviors including lower adherence as suggested in 1 study warrants further investigation.51

The association of decreased E. coli bactericidal activity with PVL and a similar trend with respect to genital tract HIV shedding (P = 0.14, Table 3) is novel and consistent with the link between E. coli bactericidal activity and BV, previously reported in HIV-uninfected women.17 This link may reflect findings from a recent proteomic study in HIV-uninfected women, in which proteins unique to Lactobacillus species (eg, S-layer protein) were found exclusively in CVL with high anti-E. coli activity but were absent from CVL with little or no anti-E. coli activity.22 BV has been associated with HIV acquisition and transmission and with higher genital tract HIV RNA.23,25,52 It is surprising that higher Nugent scores and lower E. coli activity were more strongly associated with PVL than with genital tract shedding; this may reflect the infrequent finding of viral shedding in CVL overall (which is a relatively dilute sample) and the small sample size and warrants further study.

There are several limitations to this cross-sectional study, including measurement of mediators at a single time point, the inclusion of women on and off ART in the HVL and those with undetectable and detectable PVL in the LVL cohorts, lack of data on immune cell populations, and vaginal microbiota or molecular testing for STI, HPV, or HSV shedding at the study visit. Despite these limitations, the findings demonstrate that higher HIV PVL is associated with higher levels of mucosal but not systemic inflammatory immune mediators and with less E. coli bactericidal activity. Larger studies are needed to determine whether bactericidal activity is associated with viral loads after adjusting for Nugent scores. Larger longitudinal studies are also needed to identify the factors that trigger mucosal inflammation and the loss in bactericidal activity and to determine how and whether the responses contribute to higher PVL. The findings suggest that strategies to reduce mucosal inflammation may play an important role in both local and systemic HIV control.

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Keywords:

HIV; HSV; mucosal immunity; inflammation; female genital tract; WIHS

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