HPV Induces Changes in Innate Immune and Adhesion Molecule Markers in Cervical Mucosa With Potential Impact on HIV Infection

Abstract

While most HPV infections are asymptomatic and clear spontaneously, persistent infection with high-risk HPVs is associated with cervical cancer and with increased risk of HIV acquisition. Although several hypotheses have been proposed to explain this phenomenon, none has been confirmed. Our aim was to investigate the expression of host factors involved in the susceptibility to HIV infection among HPV-infected women. Cervical samples were collected to characterize the expression levels of HIV susceptibility markers in the mucosa of HPV-infected compared with HPV-uninfected women. No differences in the frequency of CCR5+, integrin α4β7+, activated and memory CD4+ T-cell were detected between the groups. We additionally evaluated the expression levels of genes involved in innate immune responses and in cell adhesion. HPV infected patients expressed higher levels of TLR9 and lower levels of pattern recognition receptors that recognize RNA (TLR3, TLR7, and MDA5/IFIH1). We also detected an impaired IFN pathway, with an increased Type I IFN and a decreased IFNα2 receptor expression. HPV+ samples displayed reduced expression of genes for adherens and tight junctions. Taken together, these results suggest that although HPV infection does not result in the recruitment/activation of susceptible CD4+ T-cell in the female genital tract, it leads to changes in the innate antiviral immune responses and in cell adhesion that are likely to favor HIV infection.

Keywords: HIV; HPV; cell junctions; innate immunity; interferon type I; toll-like receptors.

Figure 1

Analysis of CD4+ T-cells subsets in cervical and blood samples from HPV- and HPV+ patients. (A,B) Cervical cells from pap smears were stained for CD4 and CCR5 and analyzed using the BD Accuri C6 cytometer. Women were grouped into HPV- (n = 7) or HPV+ (n = 6). (A) Proportion of CD4+ T-cells and (B) CD4+CCR5+ T-cells in cervix taking into account all cells obtained from the smears (the majority are squamous and columnar epithelial cells). (C,D) Cervical cells were stained and analyzed using FACS Canto II. Women were grouped into HPV- (n = 8; no leukocytes were obtained from three of the 11 samples) and HPV+ (n = 16). (C) Frequency of CD4+ T-cells expressing either CCR5+, CD38+, CD45RA-, α4β7+, or αEβ7+ in the cervix. (D) CD38 MFI on memory CD45RA- CD4+ T-cells in cervical samples. (E) β7 MFI on memory (CD45RA-) and activated (CD38+) blood CD4+ T-cells. Blood leucocytes from HPV- (n = 11) and HSIL (n = 7) patients were analyzed using FACS Canto II. The horizontal bar represents the median value. Statistical analysis: Mann-Whitney U-test; *p ≤ 0.05.

Figure 2

PRR expression levels in cervical cells. qPCR on HPV- and HPV+ cervical sample lysates was performed to assess the expression levels of (A) TLR3; (B) TRL7; (C) TLR4; (D) TRL9; (E) RIG-I/DDX58; (F) MDA5/IFIH1; (G) TRIM25. Relative expression of target genes was normalized to GAPDH (2^−ΔCt). Box plots represent 10–90 percentile; dots represent outlier samples; “+” represents the mean and the horizontal bar, the median. HPV- = 15 and HPV+ = 11 women. Mann-Whitney U-test; *p ≤ 0.05; ***p ≤ 0.001.

Figure 3

Cytokine expression in cervical cells. Expression levels of (A) IFNα2; (B) IFNβ1; (C) IFNαR2; (D) TNFα, and (E) IFNγ relative to GAPDH expression. Box plots represent 10–90 percentiles; dots represent outlier sample; “+” represents the mean and the horizontal bar, the median. In (F) the IFN-I/IFNαR2 ratios are presented in a histogram depicting medians and with interquartile range. HPV- = 15 and HPV+ = 11 women (A,B,C,F); HPV- = 8 and HPV+ = 4 women (D); HPV- = 10 and HPV+ = 10 women (E). Mann-Whitney U-test; *p ≤ 0.05.

Figure 4

Adhesion molecule expression in cervical cells. Expression levels of (A) E-caderin; (B) Claudin-1; (C) Claudin-4; (D) Ocludin and (E) ZO-1 relative to GAPDH expression. Box plots represent 10–90 percentiles; dots represent outlier samples; “+” represents the mean and the horizontal bar, the median. HPV- = 12 and HPV+ = 9 women. Statistical test: Mann-Whitney U-test.

Figure 5

Measured innate immune and adhesion gene expression patterns and gene interactions. (A) Protein network of analyzed genes showing action types and effects of proteins (created using string-db.org). (B) Heatmap of gene expression patterns comparing HPV- women with HPV+ counterparts. We used median to calculate the “relative expression activity” (n_α), except for TLR3, TLR7, E-caderin, Claudin-1, Occludin, and ZO-1, in that the HPV+ group was zero and we used the mean. In this graph, coordinates (X- and Y-axis) represent normalized values of the input network topology. Color gradient (Z-axis) represents the n_α value in the color scale. Red signal represents high expression and blue, low expression in HPV+ compared with HPV- group.