Blunted IL17/IL22 and pro-inflammatory cytokine responses in the genital tract and blood of HIV-exposed, seronegative female sex workers in Kenya.

Identifying the immune correlates of reduced susceptibility to HIV remains a key goal for the HIV vaccine field, and individuals who are HIV-exposed, seronegative (HESN) may offer important clues. Reduced systemic immune activation has been described in HESN individuals. Conversely, pro-inflammatory T cell subsets, particularly CD4+ T cells producing the cytokine IL17 (Th17 cells), may represent a highly susceptible target for HIV infection after sexual exposure. Therefore, we characterized the cellular pro-inflammatory and IL17/IL22 cytokine immune milieu in the genital mucosa and blood of HESN female sex workers (FSWs).Blinded lab personnel characterized basal and mitogen-induced gene and cytokine immune responses in the cervix and blood of HESN FSWs (n = 116) and non-FSW controls (n = 17) using qPCR and ELISA. IL17 and IL22 production was significantly reduced in both the cervix and blood of HESNs, both in resting cells and after mitogen stimulation. In addition, HESN participants demonstrated blunted production of both pro-inflammatory cytokines and β-chemokines.We conclude that HIV exposure without infection was associated with blunted IL17/IL22 and pro-inflammatory responses, both systemically and at the site of mucosal HIV exposure. It will be important for further studies to examine the causal nature of the association and to define the cell subsets responsible for these differences

Summary of cytokine production by blood lymphocytes from enrolled participants.

<p>A Mann-Whitney U test was performed to compare differences between groups and the ‘p’ statistic reported. Significant p values (p<0.05) are shown in bold. Staphylococcus enterotoxin B (SEB); HIV exposed seronegative (HESN), Low risk control (LRC); Enzyme linked immunoabsorbent assay (ELISA).</p

Decreased Th17 and pro-inflammatory cytokine production in HESN blood lymphocytes.

<p>Figure shows cytokine levels in supernatants of blood lymphocytes from study participants blood mononuclear cells (a) incubated in culture medium alone (unstimulated), and (b) after SEB mitogen stimulation. Cytokine concentrations (pg/ml; IL17, IL22, IFNg, IL8, SDF-1b, MIP1b, RANTES, IL10, MCP-1, MIP1a, and IL6) in culture supernatants were determined using an ELISA platform, and significant cytokine differences between groups are illustrated. Box and whisker plots are plotted with whiskers covering the 95-5 percentiles, dots representing outliers and horizontal lines representing the median.</p

Decreased Th17 and pro-inflammatory cytokine gene expression in the HESN cervix and blood.

<p>Figure shows immune gene expression in mononuclear cells from both blood (PBMC) and cervix (CMC) in study participants that were either (a) incubated in culture medium alone (unstimulated), or (b) after SEB mitogen stimulation. A qPCR assay was used to determine mRNA induction of IL17, IL22, IFNg, and IL6 genes. Box and whisker plots with whiskers covering the 95-5 percentiles, with horizontal lines representing the median.</p

Summary of immune gene induction in cervical cells from enrolled participants.

<p>A Mann-Whitney U test was performed to compare differences between groups and the ‘p’ statistic reported. Significant p values (p<0.05) are shown in bold. qPCR data is presented as a ratio of target gene expression over the housekeeping gene. Staphylococcus enterotoxin B (SEB); HIV exposed seronegative (HESN), Low risk control (LRC), qPCR (real time PCR).</p

Summary of condom use in enrolled female sex workers.

*<p>Data was not available for all 116 enrolled sex workers. Respondent numbers are shown for each variable.</p

Summary of immune gene induction in blood lymphocytes from enrolled participants.

<p>A Mann-Whitney U test was performed to compare differences between groups and the ‘p’ statistic reported. Significant p values (p<0.05) are shown in bold. qPCR data is presented as a ratio of target gene expression over the housekeeping gene. Staphylococcus enterotoxin B (SEB); HIV exposed seronegative (HESN), Low risk control (LRC), qPCR (real time PCR).</p

Primer sequences for SYBR green real-time PCR.

<p>Glyceraldehyde-3-phosphate dehydrogenase (GAPDH); Interferon Gamma (IFNγ); Interleukin 6 (IL6); Interleukin 17a (IL17); Interleukin 22 (IL22).</p