FOXP3 Genetic Variants Do Not Impact Circulating and Cervical Interleukin-10 Levels in Human Papillomavirus Infection in Women

Forkhead box P3 (FOXP3) transcription factor is the master gene that regulates CD4⁺ CD25⁺ FOXP3⁺ regulatory T cell (Treg) development and function, and frameshift mutations in this gene were reported to cause several diseases associated with an over-reactive immune response (13). This T cell subset is known to display an immunosuppressive phenotype, characterized by high production of immunoregulatory cytokines, mainly TGF-β, interleukin-35 (IL-35), and IL-10 (4,14). IL-10 is a cytokine that may exert a range of inhibitor activities on several cell types that express IL-10 receptors, and the generation of IL-10-producing Tregs is one of the first types of regulators linked to the outcome of viral infections (15).

IL-10 is strongly involved in the persistence of the human papillomavirus (HPV) in the squamous metaplastic tissue of the cervix, inducing HPV immune escape and creating an immunosuppressive microenvironment permissive to tumorigenesis (1). In IL-10-deficient mice, Treg cells are unable to sustain FOXP3 expression and fail to exert immunoregulatory activities (8). In contrast with naturally occurring FOXP3⁺ CD25⁺ Treg cells, FOXP3 seems not to be required for IL-10–producing CD4⁺ Tregs development (10). However, Treg cells expressing IL-10 may develop from Foxp3⁺ and Foxp3⁻ precursor cells in mice (7). FOXP3 activates the promoters of IL-10, TGF-β1, and IL-35 genes (9).

Occurrence of single nucleotide variants (SNVs) in FOXP3 regulatory regions as introns may alter Treg activity by regulating FOXP3 gene transcription, decreasing FOXP3 mRNA expression and FOXP3 tissue immunostaining (11,16). Moreover, a report showed that multiple sclerosis patients carrying the FOXP3 intronic SNV rs3761548 present lower circulating serum levels of IL-35 (6). It indicates that FOXP3 SNVs may indirectly affect cytokine production since they change FOXP3 transcription factor levels. Therefore, we hypothesized that the FOXP3 SNVs rs3761548 and rs2232365 in the intron -1 region influence on IL-10 plasmatic and cervical levels in an HPV infection context.

This study was in accordance with the ethical guidelines established by the 2013 revised Helsinki Declaration and obtained the approval of the Institutional Ethics Committee Involving Humans of the Londrina State University, Londrina-PR, Brazil, CEP/UEL 133/2012, number 05505912.0.0000.5231. In this research protocol, we assessed 308 women between March 2015 and December 2016 attended by an ambulatory colposcopy facility that performs the cytological examination and belongs to the Intermunicipal Consortium of Health of the Middle Paranapanema, at the University Hospital and Clinic Center of the State University of Londrina and two Basic Healthcare Units in Londrina-PR, Brazil.

Technical procedures and aim of the study were carefully explained to all patients and the written and signed informed consent was obtained before blood and cervical samples collection. Cervical cytobrushes used in the sample collection for cytological analysis were stored in 2 mL of TE buffer (10 mM Tris-HCl, 1 mM EDTA pH 8.0) at −20°C. Peripheral blood was drawn into vacutainer tubes with EDTA as anticoagulant and stored at −20°C. Cervical samples and blood samples were used for HPV molecular testing that was performed by polymerase chain reaction (PCR) technique and FOXP3 SNVs genotyping, respectively, according to the Cezar-dos-Santos et al. methods (3).

In brief, PCR-restriction fragment length polymorphism (RFLP) was carried out using peripheral blood genomic DNA to detect the FOXP3 SNVs. For rs2232365 genotyping, the following primers were used: 5′-AGGAGAAGGAGTGGGCATTT-3′ (forward) and 5′-TGTGAGTGGAGGAGCTGAGG-3′ (reverse) (12). The rs3761548 genotyping was performed with the following primers: 5′-GGCAGAGTTGAAATCCAAGC-3′ (forward) and 5′-CAACGTGTGAGAAGGCAGAA-3′ (reverse) (5). Reactions and cycling protocol specificities are minutely described in Cezar-dos-Santos et al. (3), as well as the enzymatic digestion step, which was carried out for rs2232365 and rs3761548 SNVs employing the BsmBI and PstI restriction endonucleases (New England Biolabs, Ipswich, MA), respectively.

For primers specificity confirmation, some amplicons of FOXP3 DNA were purified using PureLinkTM PCR Purification Kit (Invitrogen), following the manufacturer instructions. Sequencing reaction was performed using BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems™, Foster City, CA). Amplicons were sequenced in a 24-capillary 3500xl Genetic Analyzer (Applied Biosystems). Percentage of identity was determined through BLAST program, and the obtained sequences demonstrated 100% of identity with known FOXP3 nucleotide sequences in the GenBank databases. Both TE buffer from cytobrushes and plasma were used for IL-10 levels measurement. Sampling (n) for IL-10 plasmatic levels analysis was 146 HPV-infected and 162 uninfected women.

Collected samples of peripheral blood in EDTA vacutainer tubes were centrifuged to obtain plasma. Cervical epithelial scrappings of HPV-infected (n = 30) and uninfected (n = 70) women were collected at the time of diagnosis during the colposcopy procedure and collection for cytological analysis into falcon tubes (2 mL TE buffer) and then centrifuged to obtain the supernatants.

The IL-10 protein levels from cervical and blood samples were measured a posteriori employing the Human IL-10 ELISA Ready-SET-Go!™ (eBioscience, San Diego, CA), according to Berti et al. (2). The average of the readings of samples in duplicate was calculated, and results are expressed as picograms per milliliter (pg/mL).

Regarding the analysis of data, the Kolmogorov–Smirnov normality test was performed to analyze deviations from Gaussian distribution. Two-way analysis of variance (ANOVA) was carried out to verify interactions between the continuous variable IL-10 and FOXP3 inheritance models, considering HPV infection. Tukey's post hoc test for multiple comparisons was used to detect differences between models. All tests were two tailed with a significance level set at 0.05. Statistical analyses were performed using the GraphPad Prism 8.0 for Windows (GraphPad Software, Inc., La Jolla, CA).

None of the HPV uninfected women (n = 162) screened by PCR presented cervical lesions by the cytological examination. Of the 146 HPV-infected women, 72 (49.3%) presented no lesions, 20 (13.7%) presented low-grade squamous intraepithelial lesions, and 54 (37%) presented high-grade squamous intraepithelial lesions. The FOXP3 genotype distribution and HPV infection association analysis for the researched SNVs were described by our group recently and found to be independent biomarkers of the HPV-associated carcinogenesis (3). Considering the analysis for intragroup comparisons of FOXP3 genotypes and inheritance models, we were not able to detect any significant differences both in plasmatic and in cervical IL-10 levels of HPV-infected and uninfected women by the capture enzyme-linked immunosorbent assay (ELISA) method (Table 1).

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Table 1.

Interleukin-10 Plasmatic and Cervical Levels in Human Papillomavirus-Infected and Uninfected Women Stratified by Inheritance Models

FOXP3 models	IL-10 plasmatic levels a Mean ± SD		IL-10 cervical levels a Mean ± SD
	HPV uninfected (n = 162)	HPV infected (n = 146)	HPV uninfected (n = 30)	HPV infected (n = 70)
rs3761548
Codominant
C/C	3.53 ± 2.15	3.47 ± 1.69	3.57 ± 2.25	5.31 ± 3.42
C/A	3.74 ± 2.21	3.49 ± 1.91	3.93 ± 2.41	6.02 ± 2.90
A/A	3.76 ± 2.36	3.58 ± 1.86	3.90 ± 2.72	5.84 ± 3.23
Dominant
C/A+A/A	3.75 ± 2.25	3.51 ± 1.89	3.92 ± 2.52	5.95 ± 2.98
Recessive
C/C+C/A	3.65 ± 2.18	3.48 ± 1.82	3.80 ± 2.33	5.83 ± 2.99
Overdominant
C/C+A/A	3.62 ± 2.22	3.50 ± 1.73	3.76 ± 2.50	5.64 ± 3.22
rs2232365
Codominant
A/A	3.77 ± 2.48	3.97 ± 1.71	3.67 ± 1.84	4.67 ± 2.96
A/G	3.70 ± 2.12	3.61 ± 1.89	4.01 ± 2.79	5.70 ± 3.01
G/G	3.31 ± 1.95	2.87 ± 1.52	4.56 ± 3.40	6.40 ± 3.45
Dominant
A/G+G/G	3.60 ± 2.08	3.42 ± 1.83	4.13 ± 2.90	5.81 ± 3.04
Recessive
A/A+A/G	3.72 ± 2.22	3.69 ± 1.85	3.91 ± 2.54	5.53 ± 2.98
Overdominant
A/A+G/G	3.55 ± 2.24	3.36 ± 1.68	4.03 ± 2.55	5.53 ± 3.16

Comparisons of IL-10 levels between genetic models were performed by two-way ANOVA followed by Tukey's post hoc test. The variation of n among groups is due to availability of samples (i.e., adequate blood samples, TE availability).

a

In both groups (HPV uninfected and HPV infected), all intragroup analysis of IL-10 levels comparing all genotypes and genetic models showed no significant differences (p > 0.05).

ANOVA, analysis of variance; IL-10, interleukin-10; FOXP3, forkhead box P3; HPV, human papillomavirus; SD, standard deviation.

It is noteworthy that we found a significant difference in analysis for intergroup comparisons, that is, IL-10 cervical levels between HPV-infected versus uninfected women by two-way ANOVA followed by Bonferroni's correction, irrespective of the FOXP3 genotypes (data not shown). This predictable finding is related to HPV infection, reported earlier by our research group (2). Maynard et al. (7) showed a dual reporter mouse system of the genes encoding IL-10 and Foxp3 to track Treg subsets based on coordinate or differential expression of these genes. They found that a subset of Foxp3⁻-expressing Treg cells secrete IL-10.

We suggest that although IL-10 might be a putative binding site to FOXP3 transcription factor (17), IL-10 transcription may occur in a FOXP3-independent manner, which would explain why the FOXP3 SNVs (known to alter gene expression) do not interfere with IL-10 production. Finally, we conclude that the rs3761548 and rs2232365 FOXP3 SNVs do not change local and circulating levels of IL-10 in healthy and HPV-infected women. More studies are necessary to better explain whether and how FOXP3 and IL-10 physically interact with each other and might lead to alterations in IL-10 gene products in multiple molecular levels.