The mucosal immune system of the human female reproductive tract (FRT) is unique. It is the only immune site that must balance the demands of protection from pathogen invasion with the requirements of fetal implantation and procreation. This task is accomplished by microbial recognition mediated by PRRs, which include members of the TLR and NOD families (reviewed in (Mitchell et al., 2007
). In this study, we report that TLRs 7–9 are constitutively expressed in FRT tissues including Fallopian tubes, uterine endometrium, cervix and ectocervix, while expression of TLR10 is restricted to the Fallopian tubes. NOD1 and NOD2 as well as the signal transducer RICK are detectable in all FRT tissues. Moreover, these receptors are functional, as treatment of FRT tissue cells with ligands for TLR and NOD induces production of pro-inflammatory CXCL8.
Immune surveillance in the FRT is critically influenced by the interplay of many factors including hormones, resident leukocyte populations, and the distinct microenvironment of each component of the reproductive tract. The non-sterile lower portions of the FRT harbor a variety of commensal bacteria, and are exposed to a wide diversity of sexually transmitted microbes (Quayle, 2002
). While the upper regions of the tract, notably the Fallopian tubes and uterine endometrium, have been considered sterile sites, emerging evidence demonstrates that these regions of the FRT are subject to challenge by ascending pathogens, such as Neisseria gonorrhoeae
and Chlamydia trachomatis
). Given the diversity of microbes to which the tract is exposed, it is imperative that tissues of the FRT are responsive to pathogenic challenge. In this regard, PRRs play a critical role in mediating microbial recognition. Previous studies indicate that TLRs 1–6 are present in tissues of the human FRT (Fazeli et al., 2005
, Hirata et al., 2005
, Pioli et al., 2004
, Young et al., 2004
). We now extend these findings to demonstrate constitutive expression of TLRs 7–9 and NOD1 and 2 in these tissues as well. Thus, cells of the human FRT are capable of mediating responses to a broad range of microbes.
This is particularly important with respect to NOD receptor expression, as these PRRs recognize intracellular cytoplasmic bacterial PAMPs. Because Chlamydia trachomatis
is an intracellular pathogen that can infect both the lower and upper FRT (den Hartog et al., 2006
), NODs may play a pivotal role in mediating immune responses to this microbe. Indeed, recent work has shown that functional NOD1 expression is important for the activation of NFκB during chlamydial infection (Welter-Stahl et al., 2006
). Clearance of C. trachomatis
infections is contingent on recognition of this microbe and stimulation of the inflammatory immune response. The consequences of persistent chlamydial infection include endosalpingeal tissue damage and tubal factor subfertility (den Hartog et al., 2006
). Moreover, increasing evidence suggests that chlamydial infections may be causally linked to pregnancy complications, particularly preeclampsia and preterm labor (Hossain et al., 1990
, Jain et al., 1991
). In this regard, studies conducted by Costello et al
have demonstrated functional NOD expression in first trimester trophoblasts (Costello et al., 2007
). Here we are now show for the first time the presence of functional NOD receptors in non-pregnant tissues of the human FRT.
It is intriguing that TLR10 was only detected in the Fallopian tubes. Our determination that human uterine endometrium lacks expression of TLR10 is consistent with studies conducted by Young et al
and Schaefer et al
(Schaefer et al., 2004
, Young et al., 2004
). Moreover, it is not surprising that TLR10 expression is restricted to the Fallopian tubes, as TLR10 is mainly expressed in B lymphocytes (Hornung et al., 2002
). Previous work has shown that human FRT tissues in general contain low numbers of B cells, with the exception of the Fallopian tubes (Givan et al., 1997
). However, our data are discordant with the findings of Aflatoonian et al
, in which human endometrium was shown to express TLR10 mRNA (Aflatoonian et al., 2007
). To attempt to reconcile this disparity, we performed PCR using the primers used to identify TLR10 in the study above, but failed to observe expression of TLR10 (data not shown). As we utilized appropriate positive and negative controls, the explanation for the variance in these results is unclear, but clearly merits further investigation. It will be important to determine if this receptor is functional in FRT tissues after a ligand has been identified for this receptor.
In addition to the characterization of NOD receptors, we also found that human FRT tissues express the signaling adapter RICK. Analysis of the signal transduction pathway initiated by NOD-ligand engagement demonstrates that RICK mediates NFκB activation through the polyubiquitylation of the inhibitor of NFκB (IκB)-kinase-γ (Abbott et al., 2004
). In this work, we have identified expression of RICK throughout the human FRT. Therefore, FRT tissues contain the necessary signaling machinery for inducing inflammation and immune responses.
Signaling through NODs has also been shown to activate caspase-1 and MAPK pathways and TLR engagement also triggers activation of MAPK signaling cascades and NFκB (Akira and Takeda, 2004
, Strober et al., 2006
). Stimulation of these signaling pathways leads to the generation of an inflammatory response that is characterized by the synthesis and secretion of pro-inflammatory cytokines. CXCL8 is a key mediator of inflammation that is rapidly synthesized by a broad variety of innate effector cells, including epithelial cells, uNK cells, monocytes and macrophages (Akahoshi et al., 1994
). For this reason, we utilized CXCL8 as an indicator of TLR and NOD activation. However, signaling through any TLR or NOD would be expected to result in production and secretion of CXCL8, and a recent report has shown that many TLR agonists are contaminated with the TLR4 agonist lipopolysaccharide (LPS) (Weaver et al., 2006
), To ensure the specificity of PRR activation, we tested all TLR and NOD agonists for LPS contamination using the Limulus
ameobocyte lysate (LAL) assay (data not shown). This assay is capable of detecting as little as 10 pg/ml LPS. Using this measure, all reagents were determined to be free of LPS contamination. As demonstrated in this study, stimulation of FRT tissue cells with specific TLR and NOD agonists leads to secretion of CXCL8. These data demonstrate that TLRs and NODs are functionally expressed in FRT tissues.
In summary, we have demonstrated for the first time functional expression of TLRs 7–9 and NOD1 and NOD2 in human Fallopian tubes, uterine endometrium, cervix and ectocervix. Expression of TLR10 was restricted to Fallopian tubes. In addition, we have also shown that each of the four FRT tissues expresses the NOD signaling adapter protein RICK. Significantly, treatment of human FRT tissue cells with TLR and NOD agonists induced secretion of the chemokine CXCL8, indicating these receptors are functional. Collectively, these data establish TLR and NOD expression in the female genital tract and implicate TLR and NOD-regulated immune surveillance as an important defense mechanism in the human FRT.