Cytokine Profiling in Vaginal Specimens from Asymptomatic Women with Human Papillomavirus and/or Sexually Transmitted Infections

1. Introduction

Human papillomavirus (HPV) and sexually transmitted diseases (STIs), including gonorrhea, chlamydia, trichomoniasis, syphilis, and herpes, are linked to a host of adverse health outcomes, including cancer, infertility, chronic infection, and an increased risk of acquiring other infections(1,2). The host immune system and these infections have a complex and heterogeneous interaction, and understanding the mechanisms that underline them is essential to develop targeted prevention and treatment strategies (3).

An effective response to infections depends on the balance and regulation of cytokines. Cytokine profiling has enabled the understanding of the immunological components of a variety of incurable and infectious diseases and is becoming increasingly relevant because of its central role in influencing the immune response (4). Thus, cytokine profiling in a biological specimens provides insights into the host’s inflammatory milieu and immune status (5). This process is especially important for STIs and hrHPV infections because both local and systemic immune responses may have a significant impact on the development, dissemination, and clinical outcomes of these infectious diseases(6). Individual differences in the cytokine milieu can be attributed to a variety of factors, including the type and the location of the disease, co-infections, and the host’s immune status(7).

A high-risk HPV infection exhibits a specific cytokine profile(8). Inflammation is not a strong enough immune response to hrHPV, which could let the virus spread and avoid immune detections(9). Research has demonstrated a correlation between hrHPV infection and altered cytokine profiles, including reduced interferon-gamma (IFN-γ), increased interleukin-10 (IL-10), and other immunosuppressive cytokines. This immunosuppressive environment may help to progress hrHPV infection, cervical dysplasia, and malignancy(8,9).

The appropriate response to sexually transmitted infections is typically demonstrated by a careful balancing act between pro- and anti-inflammatory cytokines. Increased levels of cytokines like TNF-α, IL-1, and IL-6 indicate bacterial pathogen infections(10), such as those brought on by Neisseria gonorrhoeae and Chlamydia trachomatis, almost always result in a strong pro-inflammatory response(6). These cytokines are fundamental in attracting and energizing immune cells to the site of the infections, aiding in the pathogen’s clearance. Conversely, excessive inflammation can lead to tissue damage and chronic problems like as infertility and pelvic inflammatory diseases (11,12).

Cytokine profiling provides a view into the intricate relationships that hrHPV and STIs have with host immune system. The immunological mechanisms that characterize human interactions with STI and hrHPV appear to be an exciting new research area that is revealing new cytokine profiles (13). Through evidence of distinct cytokine signatures associated with hrHPV and STIs infections, and clinical outcomes, we can get a significant understanding of the development of these diseases. Moreover, this data may help design antibodies and targeted treatment strategies that improve clinical outcomes, by modifying the immune response(6,13). With meticulous cytokine profiling, we might be possible to identify underutilized biomarkers for disease diagnosis, prognosis, and treatment. This kind of research is scarce in Ethiopia. This study aimed to determine and compare the vaginal profile of cytokines in asymptomatic women, with either hrHPV, STIs, hrHPV/STIs co-infections, and healthy controls among sexually active asymptomatic women in Ethiopia. We also explored changes in vaginal cytokine levels of women with cervical intraepithelial lesions and determined the value of cytokine detection in assessing cervical lesions.

2. Methods and Materials

2.1. Study Setting and Study Population

The samples used in the study were collected from vagina in women of reproductive age who were screened for high-risk human papillomavirus (hrHPV) and sexually transmitted infections (STIs) such as Trichomonas vaginalis, Neisseria gonorrhea, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum, Ureaplasma parvum, and Chlamydia trichomatis. Eighty women form attending cervical cancer screening in Adama cohort were included in this study. It comprised 16 women who tested positive for hrHPV, 16 women who tested positive for other STIs, 26 women who were co-infected, and 22 women who were negative controls. Among 42 hrHPV positive women, 33 had histology biopsy results, with 25 were negative for intraepithelial lesions or malignancy (NILM) or CIN1, and 8 had CIN2+ or invasive cervical cancer.

2.2. Data Collection and Laboratory Procedures

Sample collection: Copan eNat^® swab collection and universal transport medium (Copan Italia SpA, Brescia, Italy) was used to collect and transport the vaginal samples for molecular investigations and cytokine profiling. The samples were collected from November 2021 to June 2022.

HPV detection and typing: HPV detection and typing was performed using a commercial kit, Anyplex II HPV HR (Seegene, Korea), which can detect 14 hr HPV types (i.e., 16, 18, 31, 33, 35, 39, 45, 51,52, 56, 58, 59, 66, and 68) in a single reaction tube by means of real-time PCR assays on the CFX96 real-time PCR instrument (Bio-Rad, Hercules, CA, USA).

STI detection: The STI detection was performed by using the Allplex™ STI-EA real-time multiplex kit with the CFX96™ thermal cycler (Bio-Rad, CA, USA).

Histology: Cervical biopsies were collected and handled routinely. The microscopic slides were examined and revealed the presence of benign alterations, low-grade squamous intraepithelial lesion (CIN1), high-grade squamous intraepithelial lesion (CIN2+), and invasive cervical cancer, as well as normal histology or no dysplasia (NILM).

Determination cytokine concentration: Invitrogen ProcartaPlex™ Multiplex Immunoassay (Lot number: 256805-001) was used for the determination of 45 cytokines, chemokines and growth factors including BDNF, EGF, Eotaxin, FGF-2, GM-CSF, GRO alpha, HGF, IFN alpha, IFN gamma, IL-1RA IL-1 alpha, IL-1 beta IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17A, IL-18, IL-21, IL-22, IL-23, IL-27, IL-31, IP-10, LIF, MCP-1, MIP-1 alpha, MIP-1 beta, NGF beta, RANTES, PDGF-BB, PIGF-1, SCF, SDF-1 alpha, TNF alpha, TNF beta, VEGF-A and VEGF-D.

ProcartaPlex™ Multiplex Immunoassay, a multiplex quantitative cytokine measurement technique, utilizes magnetic bead-based technology that allows for the simultaneous detection and quantitation of multiple cytokines from a single sample. Vaginal swab sample was used to measure cytokine, chemokine and growth factors using the manufacturer protocol. Luminex™ MAGPIX detection system from Invitrogen was used to assess the quantities cytokines. In a 96-well plate, all 45 analytes were detected at the same time following the manufacturers instruction (14).

Three distinct groups of beads were mixed and 50 µL of the mix were added per well, following the manufactories instructions. To keep the beads in place, a magnetic plate at the bottom of the plate was used for one wash. Addition of samples was done at 50 µL/well, and was kept at 4°C overnight.

In the next day, the plate was incubated for 30 minutes at room temperature with 500 rpm shaking. Following three washings, 25 µL of detection buffer per well was added, and the mixture was shaken at 500 rpm for 30 minutes at room temperature. Following three washings, 50 µL/well of the streptavidin–phycoerythrin (SAPE) solution was added, and the mixture was shaken at 500 rpm for 30 minutes at room temperature. Reading buffer (120 µL/well) was added after three washings. A MAGPIX Luminex equipment was used to read the plate following five minutes of 500 rpm shaking. The xPONENT^® software Version 4.3 was used to obtain and visualized the MAGPIX Luminex result(14).

Normalization and standard curve: The concentration of the samples was calculated by plotting the expected concentration of the standards against the median florescent intensity (MFI) generated by each standard. A 5PL algorithm is recommended for the best curve fit. The result was analyzed according to the operation manual for the Luminex™ instrument. Normalization and standard curve were determined after export the run data in.csv format and navigate to the ProcartaPlex™ Analysis App on ThermoFisher Connect website: https://apps.thermofisher.com/apps/procartaplex.

Measurements below detection limit were set to the lowest standard value, values above detection limit were set to the highest standard concentration(14).

2.3. Statistical Analysis

All cytokine levels underwent log-transformation to ensure raw values were homogeneous and normal. The different cytokine levels were compared with the Kruskal–Wallis’s test. Kruskal–Wallis’s test was also used to compare cytokine levels of HIV negative, HIV positive, and HIV/HPV co-infected groups. Dunn’s test used to correct for multiple comparison groups of Kruskal–Wallis’s test results. Mann–Whitney U statistical tests used to compare differences between the two histological grades (≤CIN1 and ≥CIN2), and HIV positive and negative groups.

3. Result

Receiver operating characteristic (ROC) and area under the curve (AUC) analysis was used to identify optimal the minimum threshold values of cytokines levels to identify with maximum sensitivity and specificity for detection of ≥CIN2+ histological grades. All comparisons were two-sided, and a 5% level of significance was used. Statistical analysis (median, mean, standard error (SE), confidence interval (CI), AUC, ROC) was carried out with GraphPad Prism v10.3 was used (GraphPad Software Inc. USA).

3.1. Characteristics of the Study Population

The study included 80 women diagnosed with hrHPV, other sexually transmitted infections, co-infections, and healthy controls (Table 1). Of the participants, 10(13%) women lived with HIV. Thirty-three hrHPV positive women had biopsy results. The median age of the participants was with 32, with a range of 24-44 years. All participants were married.

3.2. Expression Levels of Cytokines

Out of the 45 cytokines analyzed, 42 (93%) were expressed, with at least one of the swab samples with cytokine showing detectable levels. Eighteen cytokines (40%), were expressed in more than half of the vaginal swabs. Eight (10%) cytokines, including Eotaxin, hepatocyte growth factor (HGF), Tumor Necrosis Factor-alpha (TNF-α), Leukemia Inhibitory Factor (LIF), Interleukin-12p70 (IL-12p70), IL-17 receptor A (IL-17RA), IL-4, and IL-10 were expressed in all samples (100%). Placenta Growth Factor 1 (PIGF-1), TNF-beta, and VEGF-D expression were not detected at any level in either the patients or control groups (Table 2).

Eighteen cytokines, including epidermal growth factor (EGF), Eotaxin, HGF, Interleukin-17 Receptor A (IL-1RA), Interleukin 2(IL-2), Interleukin-4 (IL-4), Interleukin-10 (IL-10), Interleukin-12 p70(IL-12p70), IL-13, IL-15, IL-17A (CTLA-8), IL-27, Interferon Gamma-Induced Protein 10 (IP-10 (CXCL10)), LIF, Monocyte Chemoattractant Protein 1 (MCP-1 (CCL2)), Stem Cell Factor (SCF), TNF alpha, and Vascular Endothelial Growth Factor A (VEGF-A) expression were higher in the swabs tested(Table 2).

3.3. Cytokine Expression in Women Live with HIV, and HIV+HPV

Women live with HIV (WLHIV) present a distinctive pattern of cytokines compared to HIV-negative women. WLHIV had higher levels of IL-10 than HIV-negative women (p<0.0001) (Figure 1). There has been no observed difference in cervical cytokine expression between HIV positive and HIV negative women for cytokines like EGF, Eotaxin, IL-2, IL-4, TNF-α, IP-10, INF-gamma, TGF-beta and so on.

Vaginal swab samples from women with HIV/hrHPV co-infection showed a positive correlation with IL-10 levels.

3.4. Cytokine Concentration in hrHPV, STI, hrHPV and STI Coinfection, and Controls

Women having STIs and coinfection with hrHPV had increased concentration of EGF, Eotaxin (CCL11), GM-CSF, HGF, IL-2, IL-4, IL-10, IP-10, and VEGF-A compared with controls. All of the chemokines tested, Eotaxin, HGF, TNF alpha, LIF, IL-12p70, IL-17RA, IL-4, and IL-10 were detected in all of the groups analyzed. HrHPV and STI co-infected participants showed significantly higher levels of IP-10, IL-10, IL-2, and Eotaxin (Table 3).

In this study, we analysis cytokines expression levels hrHPV + STI against controls (without hrHPV and STI), The median expression levels of the targeted cytokines were not statically different from controls.

In STI positive women, we found significantly higher levels of Eotaxin median concentration compared to controls (p=0.025) (Figure 2A). The median concentration level of IL-2 was significantly differed in STI (p=0.016) and coinfected women (p=0.045) compared to controls (Figure 2B). The median concentration levels of IP-10 (p=0.041) and IL-10 (p=0.021) differed significantly in coinfected women compared to controls, (Figure 2C,D).

3.5. Cytokine Concentration in NILM/CIN1 and CIN2+ and Cervical Cancer

We analyzed the relationships between cytokine levels (pg/ml) and the degree of cervical lesion (Table 4). The median vaginal swab concentration levels of Eotaxin, IL-1RA, IL-2, IL-4, IL-γ, IP-10, LIF, MCP-1, and VEGF were higher in the ≥CIN2+ and invasive cervical cancer groups than in the ≤CIN1 group. However, compared to the ≤CIN1 group, the median concentration levels of SCF and IL-27 was lower than in CIN2+ and cervical cancer groups. The median concentration levels of EGF, IL-1β, and TNF-α differed significantly among the two histological groups (≤CIN1 vs. ≥CIN2; P < 0.05). According to these findings, women with ≥CIN2 (CIN2+ and cervical cancer) had increased median concentrations of EGF (P = 0.01), IL-1β (P = 0.02), and TNF-α (p = 0.04) than those with ≤CIN1. No statistically significant differences were found in the median concentration levels of other cytokines with histological grades (P > 0.05).

3.6. Cytokine Score Effectiveness in Diagnosing Advanced Cervical Lesions

Table 5 displays the cytokine receiver operator characteristic (ROC) curve analysis used to distinguish between invasive cervical cancer and ≥CIN2+ from ≤CIN1. In the diagnosis of CIN2+ and invasive cervical cancer, the AUCs of EGF, TNF-α, and IL-1β, were 0.81 (95% CI: 0.57–1.0), 0.75(95% CI: 0.56–0.96), and 0.82 (95% CI: 0.56–1.0), respectively. When the three cytokines were pooled, the AUC was 0.89 (95% CI: 0.70-1.0) for differentiated CIN2+ and cervical cancer.

According to the ROC curve of the EGF, TNF-α, and IL-1β, sensitivities to detect CIN2+ and/or invasive cervical cancer at thresholds of >35.7, >16.8, and >8.0 were 0.67(95% CI:0.30-0.94), 1.0(95% CI:0.68-1.0), and 1.0 (95% CI:0.57-1.0), respectively, and specificities to detect CIN2+ and/or invasive cervical cancer at thresholds of >35.7, >16.8, and >8.0 were 0.93(95% CI:0.70-0.99), 0.14(95% CI:0.05-0.33), and 0.11(95% CI:0.01-0.44),respectively. The minimum threshold criterion was 8.0 pg/ml set to screen out CIN2+ and/or cervical cancer groups (Figure 3A–D).

4. Conclusion

The findings reveal distinct cytokine profiles associated with sexually transmitted infections (STIs), co-infections, and the progression of cervical lesions, underscoring the role of immune modulation in these conditions. Differences in Eotaxin, IL-2, IP-10, and IL-10 between controls and coinfected individuals indicate shifts in immune signaling pathways, with IL-10 suggesting an anti-inflammatory response and IP-10 reflecting enhanced immune cell recruitment during coinfections. The significant elevation of EGF, IL-1β, and TNF-α in women with ≥CIN2+ compared to ≤CIN1 highlights their potential role in lesion progression and cervical carcinogenesis. These findings suggest a targeted immunological response in STI-positive individuals and those with advanced cervical lesions, offering insights into potential biomarkers for disease monitoring and progression. According to our findings, IL-1β and EGF may be utilized as predictors for severe cervical lesions. As a result, cytokine testing may serve as indicators of the progression of lesions. Further research is needed to explore these cytokines’ mechanistic roles and their potential as therapeutic targets in STI-related cervical disease.