Abstract
Objectives:
Recent studies have identified vaginal bacterial taxa associated with increased HIV risk. A possible mechanism to explain these results is that individual taxa differentially promote cervicovaginal inflammation. This study aimed to explore relationships between concentrations of bacteria previously linked to HIV acquisition and vaginal concentrations of pro-inflammatory cytokines and chemokines.
Methods:
In this cross-sectional analysis, concentrations of 17 bacterial taxa and 4 pro-inflammatory cytokines (IL-1β, IL-6, IL-10, and TNFα) and 2 pro-inflammatory chemokines (IL-8, and IP-10) were measured in vaginal swabs collected from 80 HIV-uninfected women. Cytokine and chemokine concentrations were compared between women with bacterial concentrations above or below the lower limit of detection as determined by quantitative PCR for each taxon. Principal component analysis was used to create a summary score for closely correlated bacteria, and linear regression analysis was used to evaluate associations between this score and increasing concentrations of TNFα and IL-1β.
Results:
Detection of D. micraerophilus (p=0.01), Eggerthella species Type 1 (p=0.05) or M. hominis (p=0.03) was associated with higher TNFα concentrations, and detection of D. micraerophilus (p<0.01), Eggerthella species Type 1 (p=0.04), M. hominis (p=0.02), or Parvimonas species Type 2 (p=0.05) was associated with significantly higher IL-1β concentrations. Seven bacterial taxa (D. micraerophilus, Eggerthella species Type 1, G. asaccharolytica, Leptotrichia/Sneathia species, Megasphaera species, M. hominis, and Parvimonas species Type 2) were found to be highly correlated by principal component analysis (eigenvalue 5.24, explaining 74.92% of variability). Linear regression analysis demonstrated associations between this principal component and concentrations of TNFα (β=0.55, 95% CI 0.01, 1.08; p=0.048) and IL-1β (β=0.96, 95% CI 0.19, 1.74; p=0.016).
Conclusions:
This study provides evidence that several highly correlated vaginal bacterial taxa may influence vaginal cytokine and chemokine concentrations. These results suggest a mechanism whereby the presence of specific bacterial taxa could influence HIV susceptibility by increasing vaginal inflammation.
INTRODUCTION
The vaginal microbiota likely exists on a continuum between “optimal” states, characterized by Lactobacillus species predominance, and “suboptimal” states, distinguished by increased anaerobic bacteria, species richness, and species diversity [1-3]. Bacterial vaginosis (BV) is a clinical syndrome distinguished by increased detection of suboptimal bacterial taxa, and has been associated with elevated risk of HIV acquisition [2,4,5]. One possible explanation for this association is that BV causes a pro-inflammatory state, resulting in recruitment of activated immune cells and breakdown in the mucosal barriers of the cervix and vagina [5-7]. Assessment of vaginal inflammation in women has consistently demonstrated an association between BV and interleukin 1β (IL-1β), but associations between BV and other pro-inflammatory cytokines and chemokines have varied between studies [1].
Bacterial vaginosis does not represent a single microbiological entity, and the bacterial communities in BV are heterogeneous [8]. One plausible explanation for the variation in cytokine/chemokine patterns noted in prior reports is that cytokines and chemokines are influenced by individual bacterial taxa, which may differ between women with BV [8]. For example, a recent publication demonstrated that Gardnerella vaginalis and Atopobium vaginae were associated with decreased concentrations of IP-10 and increased concentrations of IL-8, IL-1α, IL-1β, and IL-12 [9]. A growing body of evidence supports the role of individual taxa in influencing clinical outcomes, such as HIV acquisition [10]. Recently, our group demonstrated an association between risk of HIV-1 acquisition in women and concentrations of seven bacterial taxa associated with suboptimal vaginal states, including Eggerthella species Type 1, Gemella asaccharolytica, Leptotrichia/Sneathia species, Megasphaera species, Mycoplasma hominis, Parvimonas species Type 1, and Parvimonas species Type 2 [10]. To better understand which bacterial taxa are associated with inflammation, concentrations of pro-inflammatory cytokines, chemokines, and select bacterial taxa were measured in vaginal fluid. In addition, principal component analysis (PCA) and linear regression were performed to assess patterns in bacterial concentrations and associations between those patterns and cytokine and chemokine levels. Together, these data provide insight into the potential mechanisms by which bacterial taxa may influence cervical inflammation and HIV risk.
MATERIALS AND METHODS
Study Design and Participants
A secondary cross-sectional analysis of data collected from the Mombasa Cohort [11] and the Mama Salama Study [12] was performed. Detailed information on both cohorts and study procedures has been described [11,12]. In brief, the Mombasa Cohort is a longitudinal, open cohort study of female sex workers in Mombasa, Kenya, and the Mama Salama Study was a prospective study of HIV-negative, pregnant women presenting to the Ahero sub-District and Bondo District Hospitals in Kenya [11,12]. Country-specific and investigator-affiliated ethical review board approval was obtained for both studies. All participants provided written, informed consent.
After enrollment, women in both cohorts returned for follow-up visits every 1-3 months for collection of behavioral and demographic data, physical examination, and testing for sexually transmitted infections (STIs), as previously described [10-12]. At specific follow-up visits defined for each cohort, vaginal swabs were collected for quantitative polymerase chain reaction (qPCR), analysis of bacteria, and measurement of cytokines and chemokines. This cross-sectional analysis includes data from a single visit per woman at which both microbiota and cytokine/chemokine data were available. Among women who became infected with HIV, the visit selected was the last visit prior to HIV seroconversion, as previously described [10]. One woman who was HIV-positive and one woman with gonococcal cervicitis at the time of sampling were excluded, as these infections may alter the relationships between microbiota and cytokines [13,14]. Data on herpes simplex virus (HSV) serostatus or HSV shedding were not available for the majority of women and are not included in this analysis. Due to differences in the timing of vaginal swab collection and STI testing per study protocols, assessment for C. trachomatis (N=19) and genital ulcer disease (N=50) was only performed on a subset of women at the analysis visit.
Laboratory Procedures
STI Testing:
Testing for HIV was performed by enzyme linked immunosorbent assay (ELISA) in the Mombasa Cohort using the Pishtaz HIV 1.2 ELISA (Pishtaz Teb Diagnostics, Tehran, Iran) for HIV screening and the Vironostika HIV-1 Uni-Form II Ag/Ab (bioMérieux, Marcy I’Etoile, France) for confirmatory testing [10,11]. In the Mama Salama study, the first-generation Gen-Probe HIV viral load assay (Hologic/Gen-Probe, San Diego, CA, USA) was used for HIV testing [10,12]. The Gen-Probe APTIMA Combo-2 Assay (Hologic/Gen-Probe, San Diego, CA, USA) was used in both cohorts for diagnosis of infection with Neisseria gonorrhoeae and Chlamydia trachomatis [11,12]. For both cohorts, BV was diagnosed by Gram stain according to the method of Nugent and Hillier, and T. vaginalis infection was diagnosed by wet preparation [11,12,15].
Vaginal sample collection:
Vaginal samples were collected during speculum-assisted pelvic examination (Mombasa Cohort) or by self-collection (Mama Salama Study), using push-off Dacron swabs from FitzCo, Inc (Spring Park, MN, USA). Vaginal swabs were stored at −80°C in Kenya, shipped on dry ice to Seattle, then stored at −80°C at the Fred Hutchison Cancer Research Center in Seattle, WA, until use.
Quantitative PCR:
DNA extraction and bacterium specific qPCR from vaginal fluid samples were performed for the following bacterial taxa according to published protocols: Aerococcus christensenii, Atopobium vaginae, Bacterial vaginosis-associated bacterium 2 (BVAB2), Dialister micraerophilus, Dialister species Type 2, Eggerthella species Type 1, Gardnerella vaginalis, Gemella asaccharolytica, Lactobacillus crispatus, Leptotrichia/Sneathia species, Megasphaera species, Mycoplasma hominis, Parvimonas species Type 1, Parvimonas species Type 2, Porphyromonas asaccharolytica/uenonis, Porphyromonas species Type 1, Porphyromonas bennonis and Prevotella genus [8,10,16,17].
Measurement of vaginal cytokines and chemokines:
Levels of IL-1β, interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 10 (IL-10), tumor necrosis factor alpha (TNFα), and interferon gamma-induced protein 10 (IP-10) in vaginal samples were assessed using a V-Plex Custom Human Cytokine panel from Meso Scale Discovery (MSD; Rockville, MD, USA) following the manufacturer’s instructions. Cytokine and chemokine values for samples with levels below the lower limit of detection (LLD) were set to the midpoint between zero and the LLD for that cytokine.
Statistical Analysis
For this cross-sectional analysis, data from the visit at which vaginal samples were collected were used to define the population characteristics. Demographic and behavioral data were reported using descriptive statistics. The primary exposure was dichotomized as detection of bacterial taxa above or below the LLD of qPCR. Bacterial taxa analyzed included: i) Group 1: seven bacterial taxa recently reported to have significant concentration-dependent associations with increased risk of HIV acquisition (Eggerthella species Type 1, G. asaccharolytica, Leptotrichia/Sneathia species, Megasphaera species, M. hominis, Parvimonas species Type 1, and Parvimonas species Type 2) [10]; ii) Group 2: ten additional taxa that demonstrated a statistical trend towards association with HIV acquisition by rank abundance in a prior analysis, including: Aerococcus christensenii, Atopobium vaginae, BVAB2, Dialister micraerophilus, Dialister species Type 2, Gardnerella vaginalis, Porphyromonas asaccharolytica/uenonis, Porphyromonas species Type 1, Porphyromonas bennonis and Prevotella genus [10]; and iii) L. crispatus, a well described marker of vaginal health [1-3]. Secondary analyses were conducted using the log10 concentration of bacterial taxa as the exposure. The primary outcome was the log2-transformed concentration of IL-1β, IL-6, IL-8, IL-10, TNFα, and IP-10. Transformation to the log2 scale was performed to normalize cytokine/chemokine concentrations and increase biological relevance, so that a one-unit change corresponds to a doubling (1 log2 increase) or halving (1 log2 decrease) of concentration.
For each taxon, cytokine/chemokine concentrations in participant samples with and without bacterial detection were compared using Wilcoxon rank-sum tests. All Group 1 bacterial taxa were carried forward for further analysis a priori. Each of the Group 2 bacterial taxa were carried forward only if associated with the cytokines or chemokines of interest at p<0.10. Of this set of exposure variables, only one Group 2 bacterial taxon, D. micraerophilus, met this criterion and was carried forward for PCA.
Due to significant correlations between the different bacterial taxa studied, and to reduce the dimensionality of the dataset, PCA was performed on log10-transformed concentrations of the bacterial taxa carried forward as described above. One factor had an eigenvalue of 5.39 and accounted for 67.4% of variability in the analysis; no other factors had eigenvalues >1. Parvimonas species Type 1 concentration had a uniqueness score of 82.62%, suggesting that this taxon did not share similar features with the other bacterial taxa in the model, and was subsequently removed. The seven remaining bacterial taxa (D. micraerophilus, Eggerthella species Type 1, G. asaccharolytica, Leptotrichia/Sneathia species, Megasphaera species, M. hominis, and Parvimonas species Type 2) underwent repeat PCA, confirming generation of a single factor of highly correlated bacterial species (eigenvalue 5.24, explaining 74.92% of variability). The high degree of variability explained by this factor suggests that these taxa are highly correlated, and are better analyzed as a single variable representing the degree of sub-optimal taxa present rather than as individual predictors assumed to be independent. Therefore, results of the PCA were used to generate a principal component score (labeled “suboptimal taxa score”) for each participant. This score is interpretable as a summary statistic generated for each woman based on the relative concentrations of the seven bacterial taxa included in the PCA.
Linear regression was performed to determine if the suboptimal taxa score and other components of the vaginal microbiota were independently associated with log2-tranformed concentrations of TNFα and IL-1β. Primary predictors included the suboptimal taxa score and Parvimonas species Type 1 concentration. Potential confounders included hormonal status (categorized as pregnant, no hormonal contraception, or use of hormonal contraception), vaginal washing (defined as washing beyond the introitus in the week prior to the analysis visit), age, number of sex partners, frequency of unprotected sex, frequency of vaginal sex, T. vaginalis infection, genital ulcer disease and pregnancy status (pregnant versus not pregnant). Hormonal status and vaginal washing were selected a priori for inclusion in multivariate analysis based on review of the literature suggesting that both may influence vaginal inflammation [18,19]. The remaining potential confounders were evaluated by linear regression for associations with TNFα or IL-1β, and included in multivariable modeling if associated with the cytokine of interest at p<0.10.
In addition to the above analyses, the relationship between diagnosis of BV (Nugent score ≥7) or abnormal microbiota (Nugent score ≥4) and cytokine, chemokine, or bacterial taxa concentration was evaluated using Wilcoxon rank-sum tests. Pearson’s correlation test was also performed to assess for correlations between the suboptimal taxa score, Nugent score, TNFα and IL-1β. Statistical analyses were conducted using Stata version 15.1 (College Station, Texas, USA).
RESULTS
Paired vaginal bacterial qPCR and cytokine data were available for 80 eligible women, 50 from the Mama Salama Study, of whom 19 (23.75%) were pregnant, and 30 from the Mombasa Cohort. Median age at the time of sample collection used in this analysis was 24.5 years (IQR 19-35). Three women tested positive for T. vaginalis (3.8%) and 27 of 78 women (34.6%) for whom Gram stain results were available were diagnosed with BV (Nugent score ≥7). Additional demographic and health characteristics are reported in Table 1.
Table 1:
Characteristics at the Time of Sample Collection for 80 Participating Women
| Characteristic1 | All participants (N=80) |
Mombasa cohort (N=30) |
Mama Salama cohort (N=50) |
|---|---|---|---|
| Age (range, 15-57) | 24.0 (19, 35) | 37.5 (29, 48) | 20.0 (18, 23) |
| Married | 32 (40.0%) | 0 (0.0%) | 32 (64.0%) |
| Pregnancy and Contraception | |||
| Pregnant | 19 (23.75%) | 0 (0.0%) | 19 (38.0%) |
| Not pregnant, Implant | 5 (6.25%) | 4 (13.3%) | 1 (2.0%) |
| Not pregnant, DMPA | 7 (8.75%) | 4 (13.3%) | 3 (6.0%) |
| Not pregnant, oral contraceptive | 7 (8.75%) | 0 (0.0%) | 7 (14.0%) |
| Not pregnant, no hormonal contraception | 42 (52.5%) | 22 (73.3%) | 20 (40.0%) |
| Number of sexual partners (past month)2 | |||
| 0 | 34 (42.5%) | 10 (33.3%) | 24 (48.0%) |
| 1 | 37 (46.25%) | 11 (36.7%) | 26 (52.0%) |
| >1 | 9 (11.25%) | 9 (30.0%) | 0 (0.0%) |
| Sexual Practices | |||
| Frequency of vaginal sex (past month)2 | 1.5 (0,4) | 4 (0,8) | 1 (0,3) |
| Unprotected sex3 | 0 (0,1) | 0 (0,0) | 0 (0,1) |
| Number of sex partners3 | 1 (0,1) | 1 (0,2) | 0 (0,1) |
| Vaginal gram stain Nugent Score4 | |||
| Normal (0-3) | 36 (46.2%) | 15 (50.0%) | 21 (43.8%) |
| Intermediate (4-6) | 15 (19.2%) | 4 (13.3%) | 11 (22.9%) |
| Bacterial Vaginosis (7-10) | 27 (34.6%) | 11 (36.7%) | 16 (33.3%) |
| Sexually Transmitted Infections | |||
| Chlamydia trachomatis (N=19) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) |
| Trichomonas vaginalis | 3 (3.8%) | 0 (0.0%) | 3 (6.0%) |
| Genital ulcers on examination (N=50)5 | 1 (2.0%) | 0 (0.0%) | 1 (5.0%) |
| Vaginal Washing | |||
| Reports vaginal washing (past week)6 | 58 (72.5%) | 23 (76.7%) | 35 (70.0%) |
| Detection of bacterial taxa at the analysis visit | |||
| Dialister micraerophilus | 72 (90.0%) | 24 (80.0%) | 48 (96.0%) |
| Eggerthella species Type 1 | 48 (60.0%) | 22 (73.3%) | 26 (52.0%) |
| Gemella asaccharolytica | 45 (56.3%) | 22 (73.3%) | 23 (46.0%) |
| Lactobacillus crispatus | 19 (23.8%) | 7 (23.3%) | 12 (24.0%) |
| Leptotrichia/Sneathia species | 58 (72.5%) | 26 (86.7%) | 32 (64.0%) |
| Megasphaera species | 25 (31.3%) | 14 (46.7%) | 11 (22.0%) |
| Mycoplasma hominis | 38 (47.5%) | 15 (50.0%) | 23 (46.0%) |
| Parvimonas species Type 1 | 21 (26.3%) | 7 (23.3%) | 14 (28.0%) |
| Parvimonas species Type 2 | 34 (42.5%) | 17 (56.7%) | 17 (34.0%) |
Results are reported as N (%) or median (interquartile range).
Total sex acts or sex partners over the past month were imputed by multiplying the frequency of sexual acts or sex partners reported in the last week by four for the Mombasa cohort.
Past week for the Mombasa cohort and past month for the Mama Salama cohort.
Nugent score data were missing for two women in the Mama Salama cohort.
N=30 in the Mombasa cohort and N=20 in the Mama Salama cohort.
Vaginal washing was defined as insertion of cloth or finger to wash beyond the introitus.
Abbreviations: BVAB2, bacterial vaginosis-associated bacterium 2; DMPA, depo-medroxyprogesterone acetate; IQR, inter-quartile range; LLD, lower limit of detection.
Figure 1 shows the relationship between log2 cytokine or chemokine concentrations and detection of bacterial taxa selected for analysis. Detection of D. micraerophilus (p=0.01), Eggerthella species Type 1 (p=0.05), or M. hominis (p=0.03) was associated with higher concentrations of TNFα. Similarly, detection of D. micraerophilus (p<0.01), Eggerthella species Type 1 (p=0.04), M. hominis (p=0.02), or Parvimonas species Type 2 (p=0.05), was associated with higher concentrations of IL-1β. In contrast, detection of L. crispatus was associated with lower concentrations of TNFα (p=0.04) and IL-1β (p=0.04). Detection of Megasphaera species was associated with decreased concentrations of IP-10 (p=0.027). Because multiple bacterial taxa were associated with TNFα and IL-1β, and to restrict the total number of statistical comparisons, subsequent analysis focused on these two cytokines.
Figure 1: Box plots of log2 transformed cytokine level by detection of bacterial taxa.
The log2 of cytokine concentrations when bacterial taxa were below (dark bars) or above (white bars) the lower limit of detection. P-values were calculated using the Wilcoxon rank-sum test. The lower limits of detection for each cytokine are as follows: IL-1β, 0.048 pcg/mL; IL-6, 0.164 pcg/mL; IL-8, 0.090 pcg/mL; IL-10, 0.033 pcg/mL; TNFα 0.118 pcg/mL; IP-10, 0.109 pcg/mL.
Table 2 presents results of bivariable and multivariable linear regression using log2-transformed TNFα concentration as the outcome. Higher values of the suboptimal taxa score (β=0.56, 95% CI 0.06, 1.07; p=0.029) were associated with higher TNFα concentrations. In contrast, higher concentrations of Parvimonas species Type 1 were not associated with higher TNFα concentrations (β=0.34, 95% CI −0.12, 0.79; p=0.14). The association of the suboptimal taxa score (β=0.55, 95% CI 0.01, 1.08; p=0.048) with higher TNFα concentrations remained statistically significant in analyses adjusted for T. vaginalis, hormonal status, vaginal washing and age.
Table 2:
Cofactors for Log2 TNFα Concentration
| Characteristic | Unadjusted β Coefficient (95% CI) | P value | Adjusted β Coefficient (95% CI)9 | P value |
|---|---|---|---|---|
| Suboptimal taxa score1 | 0.56 (0.06, 1.07) | 0.029 | 0.55 (0.01, 1.08) | 0.048 |
| Parvimonas species Type 1 concentration2 | 0.34 (−0.12, 0.79) | 0.143 | 0.12 (−0.34, 0.58) | 0.608 |
| Trichomonas vaginalis infection3 | 3.35 (0.73, 5.97) | 0.013 | 2.61 (0.04, 5.18) | 0.047 |
| Hormonal status4 | 0.17 (−0.08, 0.42) | 0.176 | 0.11 (−0.14, 0.37) | 0.365 |
| Vaginal washing5 | 0.82 (−0.32, 1.97) | 0.156 | 0.65 (−0.43, 1.72) | 0.234 |
| Age | −0.05 (−0.10, 0.00) | 0.032 | −0.05 (−0.10, 0.00) | 0.059 |
| Number of sex partners6 | 0.11 (−0.51, 0.74) | 0.727 | ||
| Frequency of unprotected sex6 | 0.44 (−0.64, 1.52) | 0.421 | ||
| Frequency of vaginal sex in the past month7 | −0.03 (−0.09, 0.04) | 0.406 | ||
| Genital ulcer | 0.91 (−3.56, 5.39) | 0.683 | ||
| Pregnancy status8 | 0.84 (−0.37, 2.04) | 0.173 |
The suboptimal taxa score was generated by principal component analysis including the following bacterial taxa: Dialister micraerophilus, Eggerthella species Type 1, Gemella asaccharolytica, Leptotrichia/Sneathia species, Megasphaera species, Mycoplasma hominis, and Parvimonas species Type 2. This can be interpreted as a summary statistic based on the concentrations of the aforementioned bacterial taxa in each woman.
Log10 concentrations of bacterial taxa.
Diagnosed by wet preparation.
Hormonal status was categorized as: pregnant, no hormonal contraception, or use of hormonal contraception (depo-medroxyprogesterone acetate, oral contraceptive pills, or implant).
Vaginal washing was defined as insertion of cloth or finger to wash beyond the introitus.
Recall period was the past week for the Mombasa cohort and the past month for the Mama Salama cohort.
Total sex acts over the past month were imputed by multiplying the frequency of sexual acts reported in the last week by four for the Mombasa cohort.
Pregnant versus not pregnant at the time of sample collection.
Combined model with adjustment for all primary predictors together with potential confounders (age, vaginal washing, T. vaginalis, and hormonal status), which were selected as described in the materials and methods.
Table 3 presents results of bivariable and multivariable linear regression using log2-transformed IL-1β concentration as the outcome. Unadjusted analysis demonstrated that higher values of the suboptimal taxa score (β=0.97, 95% CI 0.25, 1.70; p=0.009) were associated with higher concentrations of IL-1β. Higher concentrations of Parvimonas species Type 1 were not associated with higher concentrations of IL-1β (β=0.43, 95% CI −0.23, 1.09; p=0.20). In multivariable analysis adjusting for T. vaginalis, hormonal status, vaginal washing and age, the suboptimal taxa score remained significantly associated with IL-1β concentration (β=0.96, 95% CI 0.19, 1.74; p=0.016).
Table 3:
Cofactors for Log2 IL-1β Concentration
| Characteristic | Unadjusted β Coefficient (95% CI) | P value | Adjusted β Coefficient (95% CI)9 | P value |
|---|---|---|---|---|
| Suboptimal taxa score1 | 0.97 (0.25, 1.70) | 0.009 | 0.96 (0.19, 1.74) | 0.016 |
| Parvimonas species Type 1 concentration2 | 0.43 ( −0.23, 1.09) | 0.201 | 0.04 (−0.62, 0.70) | 0.910 |
| Trichomonas vaginalis infection3 | 4.51 (0.69, 8.33) | 0.021 | 3.34 (−0.37, 7.04) | 0.077 |
| Hormonal status4 | 0.09 (−0.27, 0.46) | 0.609 | 0.01 (−0.35, 0.37) | 0.964 |
| Vaginal washing5 | 1.54 (−0.10, 3.19) | 0.065 | 1.32 (−0.23, 2.86) | 0.095 |
| Age | −0.06 (−0.13, 0.00) | 0.058 | −0.07 (−0.14, 0.00) | 0.042 |
| Number of sex partners6 | −0.20 (−1.11, 0.70) | 0.655 | ||
| Frequency of unprotected sex6 | 0.04 (−1.54, 1.61) | 0.962 | ||
| Frequency of vaginal sex in the past month7 | −0.06 (−0.16, 0.03) | 0.199 | ||
| Genital ulcer | 2.52 (−4.19, 9.23) | 0.454 | ||
| Pregnancy status8 | 0.39 (−1.38, 2.17) | 0.662 |
The suboptimal taxa score was generated by principal component analysis including the following bacterial taxa: Dialister micraerophilus, Eggerthella species Type 1, Gemella asaccharolytica, Leptotrichia/Sneathia species, Megasphaera species, Mycoplasma hominis, and Parvimonas species Type 2. This can be interpreted as a summary statistic based on the concentrations of the aforementioned bacterial taxa in each woman.
Log10 concentrations of bacterial taxa.
Diagnosed by wet preparation.
Hormonal status was categorized as: pregnant, no hormonal contraception, or use of hormonal contraception (depo-medroxyprogesterone acetate, oral contraceptive pills, or implant).
Vaginal washing was defined as insertion of cloth or finger to wash beyond the introitus.
Recall period was the past week for the Mombasa cohort and the past month for the Mama Salama cohort.
Total sex acts over the past month were imputed by multiplying the frequency of sexual acts reported in the last week by four for the Mombasa cohort.
Pregnant versus not pregnant at the time of sample collection.
Combined model with adjustment for all primary predictors together with potential confounders (age, vaginal washing, T. vaginalis, and hormonal status), which were selected as described in the materials and methods.
Pearson’s correlation test demonstrated statistically significant correlations between the suboptimal taxa score and Nugent score (p<0.001), TNFα (p=0.030) and IL-1β (p=0.009) (Supplemental Figure 1). No significant associations were found between diagnosis of BV and concentrations of any cytokines or chemokines tested, including TNFα and IL-1β (Supplemental Table 1); increasing concentrations of IL-1β were associated with the presence of abnormal microbiota (Nugent score ≥4) (Supplemental Table 2). However, diagnosis of BV was associated with the suboptimal taxa score (p<0.001). Diagnosis of BV was also associated with higher concentrations of all bacterial taxa associated with HIV acquisition except M. hominis, and with lower concentrations of L. crispatus (Supplemental Table 1). Sensitivity analysis excluding the 3 women with T. vaginalis infections, did not change the overall results (data not shown).
DISCUSSION
In this exploratory cross-sectional analysis, detection of D. micraerophilus, Eggerthella species Type 1, or M. hominis was associated with higher concentrations of TNFα, while detection of G. asaccharolytica, Eggerthella species Type 1, M. hominis, or Parvimonas species Type 2 was associated with higher concentrations of IL-1β. Principal component analysis highlighted an association between a principal component reflecting correlated suboptimal bacterial taxa and higher concentrations of TNFα and IL-1β.
TNFα is a pro-inflammatory cytokine with multiple functions, including activation of neutrophils and macrophages [20]. Studies performed using in vitro co-culture models have consistently shown higher concentrations of TNFα in the presence of bacteria associated with BV [4,6]. On the other hand, results of clinical studies examining the association between BV and TNFα have been mixed [4,6,21-23]. In this analysis, some bacterial taxa were associated with higher concentrations of TNFα, while others were not. Interestingly, the principal component score of highly correlated bacterial taxa, several of which were individually associated with TNFα, was associated with higher TNFα concentrations, while a diagnosis of BV was not. Together, these data suggest that vaginal TNFα concentration may vary based on the specific composition of the vaginal microbiota rather than the presence or absence of clinical BV.
IL-1β is produced as an inactive precursor by multiple cell types and functions to activate CD4+ T cells and generate pro-inflammatory cytokines [24]. Numerous studies have shown an association between BV and IL-1β, and treatment of BV decreases levels of IL-1β [1,25]. This analysis demonstrates that detection of several individual bacterial taxa (D. micraerophilus, Eggerthella species Type 1, M. hominis, or Parvimonas species Type 2) is associated with higher IL-1β concentrations, and that a principal component comprised of highly correlated bacterial taxa was also associated with higher IL-1β concentrations after adjustment for potential confounders. As with TNFα, diagnosis of BV was not associated with IL-1β, suggesting that IL-1β expression may be highly dependent on the presence of specific bacteria, either alone or in combination.
The suboptimal vaginal bacterial taxa analyzed here have been associated with increased risk of HIV acquisition [10]. One hypothesis to explain this increased risk is that these bacteria recruit CD4+ T cells to the site of HIV entry by inducing a pro-inflammatory state [26]. In this study, these high-risk bacterial taxa were associated with elevated levels of TNFα and IL-1β, which may promote HIV acquisition via a number of pathways. For example, in vitro studies suggest that TNFα signaling disrupts vaginal mucin production, which may facilitate HIV entry by decreasing epithelial integrity [6,27]. Similarly, exposure to bacteria that increase levels of both cytokines has been associated with upregulation of NFκB and other pro-inflammatory pathways in vaginal tissue models [6]. However, given the difficulty of culturing some vaginal bacteria, there is an incomplete understanding of the bacterial antigens that trigger this signaling cascade. It is also possible that other mechanisms are involved. For example, individual bacterial taxa may secrete a factor that promotes HIV replication [26], or alter the vaginal mucosal barrier via other mechanisms, such as direct cytoskeletal disruption or alteration of proteolytic activity at the epithelial surface [28].
This study had a number of strengths. The use of taxon-specific qPCR allowed for examination of individual bacterial taxa concentrations and vaginal cytokines concentrations, which is becoming increasingly important as more studies report associations between individual bacterial taxa and adverse outcomes [5,10]. Generation of a principal component facilitated analysis of bacterial taxa that were shown to be highly correlated, and provided information about how highly correlated taxa influence cytokine production in concert. These results should also be interpreted in the setting of several limitations. As a secondary analysis, the data presented should be considered hypothesis generating, and a prospective study to address changes in cytokines before and after treatment of BV will be necessary to prove causality. Future studies of the relationship between individual bacterial taxa and chemokines involved in T cell recruitment (i.e. CCL5, MIP-1α) will also be critical for developing a mechanistic understanding of the role of specific taxa in HIV acquisition [29]. Furthermore, this study may not have had the power to detect small differences in cytokine concentrations. Additionally, data were not available for HSV-2, which is associated with both inflammation and HIV acquisition, and could act as a confounder of the association between vaginal bacteria and inflammation [30,31]. Similarly, only 19 women were tested for C. trachomatis at the visits included in this study. However, women in the Mombasa Cohort were tested multiple times during their participation, and C. trachomatis was treated when detected. Women in the Mama Salama Study were only tested and treated at the baseline visit for C. trachomatis, however the overall prevalence was only 6%, suggesting this is a relatively infrequent diagnosis in this population [12]. Therefore, the number of untreated cases of chlamydia should have been low.
In summary, this analysis demonstrates associations between individual bacterial taxa and pro-inflammatory cytokines, suggesting that individual bacterial taxa may play an important role in determining the inflammatory state of the vagina. Future studies focusing on changes in inflammation if these bacteria are eliminated could help to strengthen the evidence for a causal relationship between vaginal bacteria and inflammation, as well as demonstrate the potential of vaginal health approaches for reducing HIV susceptibility.
Supplementary Material
Supplemental Figure 1: Correlation between principal component suboptimal taxa score and Nugent score, TNFα and IL-1β. Scatter plots comparing the suboptimal taxa score for each woman and Nugent score (A), log2 concentration of TNFα (B), and log2 concentration of IL-1β (C). The suboptimal taxa score represents a summary statistic generated for each woman based on the concentrations of bacterial taxa found to be highly associated in principal component analysis (D. micraerophilus, Eggerthella species Type 1, G. asaccharolytica, Leptotrichia/Sneathia, Megasphaera species, M. hominis, and Parvimonas species Type 2). Pearson correlation coefficients (“r”) and p-values are in the upper left corner of each graph. A line of best fit was generated in Stata/IC 15.1 using the command “lfitci.”
Supplemental Table 1: Association between concentrations of cytokines, chemokines, and bacterial taxa, and the presence of BV.
Supplemental Table 2: Association between concentrations of cytokines, chemokines, and bacterial taxa, and the presence of abnormal microbiota.
KEY MESSAGES.
Several bacterial taxa associated with HIV acquisition were associated with higher concentrations of TNFα and IL-1β.
A score derived from principal component analysis of these highly correlated bacterial taxa was associated with higher Nugent score and with higher concentrations of TNFα and IL-1β.
Bacterially mediated inflammation in the vagina is one mechanism that could account for increased HIV risk in women.
Acknowledgements:
We would like to thank all of the women who participated in the Mama Salama Study and Mombasa Cohort. We would also like to acknowledge the clinical, laboratory and administrative staff at each study site for their dedication and assistance with this project.
Funding: This study was supported by the National Institute of Child Health and Human Development of the National Institutes of Health (NIH P01-HD64915). Data and sample collection in the Mombasa Cohort were supported through the National Institute of Allergy and Infectious Diseases of the NIH (NIH R37 AI38518). The Mombasa research site receives infrastructure support from the University of Washington Center for AIDS Research (NIH P30-AI27757). RSM receives funding for mentoring through NIH K24 HD88229. ALD receives support from K01 AI116298. MCS is supported by the T32 Host Defense Training grant (NIH 5T32AI007044-43 PI, van Voorhis) as an infectious disease fellow. SMG was supported by the Robert W. Anderson Endowed Professorship in Medicine.
Footnotes
Conflicts of interest: RSM receives research funding, paid to the University of Washington, from Hologic Corporation. TLF has a patent, Molecular Diagnosis of Bacterial Vaginosis, licensed to Becton Dickinson. SS, MM, DAL and TLF report grants from the NIH during the conduct of the study. All other authors report nothing to disclose.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Supplemental Figure 1: Correlation between principal component suboptimal taxa score and Nugent score, TNFα and IL-1β. Scatter plots comparing the suboptimal taxa score for each woman and Nugent score (A), log2 concentration of TNFα (B), and log2 concentration of IL-1β (C). The suboptimal taxa score represents a summary statistic generated for each woman based on the concentrations of bacterial taxa found to be highly associated in principal component analysis (D. micraerophilus, Eggerthella species Type 1, G. asaccharolytica, Leptotrichia/Sneathia, Megasphaera species, M. hominis, and Parvimonas species Type 2). Pearson correlation coefficients (“r”) and p-values are in the upper left corner of each graph. A line of best fit was generated in Stata/IC 15.1 using the command “lfitci.”
Supplemental Table 1: Association between concentrations of cytokines, chemokines, and bacterial taxa, and the presence of BV.
Supplemental Table 2: Association between concentrations of cytokines, chemokines, and bacterial taxa, and the presence of abnormal microbiota.