Exposure of the female reproductive tract (FRT) to seminal fluid provokes an inflammation-like response characterised by cytokine and chemokine induction and subsequent leukocyte recruitment. To date, several signalling mediators involved in eliciting FRT cytokine production have been identified in seminal plasma (SP). However, specific neutralisation of these factors does not completely abolish the observed response, implying additional factors account for the residual activity.
We aimed to identify receptors present on ectocervical epithelial (Ect1) cells, and ligands contained within SP that contribute to eliciting the female response. We performed ingenuity pathway analysis (IPA) on microarray data generated from Ect1 cells treated with either 10% SP or media alone (control) to identify upstream regulators of the cytokine response, and then used neutralising antibodies to inhibit receptor activation of the identified signalling pathways.
IPA identified several potential upstream regulators, including many members of the toll-like receptor family. TLR2 and TLR4 were selected for further investigation. qPCR confirmed the presence of genes encoding TLR2, TLR4, and co-receptor MD2, however, co-receptor CD14 was undetectable. Treatment of Ect1 cells with TLR2 and TLR4 agonists lipoteichoic acid and ultra-pure lipopolysaccharide did not induce cytokine production. When Ect1 cells were treated with 10% SP, significant cytokine production was observed. However, treatment with anti-hTLR4 antibody and TLR4-specific small-molecule inhibitor, TAK-242, failed to reduce cytokine induction. Interestingly, when Ect1 cells were treated with TLR2 and TLR4 agonists in the presence of exogenous soluble CD14 (sCD14), cytokine production was increased.
We conclude that despite Ect1 cells expressing both TLR2 and TLR4, these cells do not possess all the necessary machinery to respond to TLR-ligands and may depend on soluble factors released from other cell lineages. We are now investigating whether the addition of sCD14 can alter the Ect1 cell response to SP and whether inhibition of TLR4 reduces sCD14-enabled SP signalling.