Chlamydia trachomatis is the most common bacterial sexually transmitted disease in the United States and a major cause of female infertility due to infection-induced Fallopian tube scarring. Epithelial cells are likely central to host defense and pathophysiology as they are the principal cell type productively infected by C. trachomatis. We generated cloned murine oviduct epithelial cell lines without viral or chemical transformation to investigate the role of the TLRs and cytosolic nucleotide binding site/leucine-rich repeat proteins Nod1 and Nod2 in epithelial responses to Chlamydia muridarum infection. RT-PCR assays detected mRNA for TLR2 (TLRs 1 and 6), TLR3, and TLR5. No mRNA was detected for TLRs 4, 7, 8, and 9. Messenger RNAs for Nod1 and Nod2 were present in the epithelial cell lines. Oviduct epithelial cell lines infected with C. muridarum or exposed to the TLR2 agonist peptidoglycan secreted representative acute phase cytokines IL-6 and GM-CSF in a MyD88-dependent fashion. Infected epithelial cell lines secreted the immunomodulatory cytokine IFN-β, even though C. muridarum does not have a clear pathogen-associated molecular pattern (PAMP) for triggering IFN-β transcription. The oviduct epithelial lines did not secrete IFN-β in response to the TLR2 agonist peptidoglycan or to the TLR3 agonist poly(I:C). Our data identify TLR2 as the principal TLR responsible for secretion of acute phase cytokines by C. muridarum-infected oviduct epithelial cell lines. The pattern recognition molecule responsible for infection-induced IFN-β secretion by oviduct epithelial cells remains to be determined.

Because epithelial cells are the major cell type productively infected with Chlamydia during genital tract infections, the overall goal of our research was to understand the contribution of infected epithelial cells to the host defense. We previously showed that Toll-like receptor 3 (TLR3) is the critical pattern recognition receptor in oviduct epithelial (OE) cells that is stimulated during Chlamydia infection, resulting in the synthesis of beta interferon (IFN-␤). Here, we present data that implicates TLR3 in the expression of a multitude of other innate-inflammatory immune modulators including interleukin-6 (IL-6), CXCL10, CXCL16, and CCL5. We demonstrate that Chlamydia-induced expression of these cytokines is severely disrupted in TLR3-deficient OE cells, whereas Chlamydia replication in the TLR3-deficient cells is more efficient than in wild-type OE cells. Pretreatment of the TLR3-deficient OE cells with 50 U of IFN-␤/ml prior to infection diminished Chlamydia replication and restored the ability of Chlamydia infection to induce IL-6, CXCL10, and CCL5 expression in TLR3-deficient OE cells; however, CXCL16 induction was not restored by IFN-␤ preincubation. Our findings were corroborated in pathway-focused PCR arrays, which demonstrated a multitude of different inflammatory genes that were defectively regulated during Chlamydia infection of the TLR3-deficient OE cells, and we found that some of these genes were induced only when IFN-␤ was added prior to infection. Our OE cell data implicate TLR3 as an essential inducer of IFN-␤ and other inflammatory mediators by epithelial cells during Chlamydia infection and highlight the contribution of TLR3 to the inflammatory cytokine response.

Epithelial cells lining the murine genital tract act as sentinels for microbial infection, play a major role in the initiation of the early inflammatory response, and can secrete factors that modulate the adaptive immune response when infected with Chlamydia. C. muridarum-infected murine oviduct epithelial cells secrete the inflammatory cytokines IL-6 and GM-CSF in a TLR2-dependent manner. Further, C. muridarum infection induces IFN-β synthesis in the oviduct epithelial cells in a TRIF-dependent manner. Because murine oviduct epithelial cells express TLR3 but not TLRs 4, 7, 8, or 9, we hypothesized that TLR3 or an unknown TRIF-dependent pattern recognition receptor was the critical receptor for IFN-β production. To investigate the role of TLR3 in the Chlamydia-induced IFN-β response in oviduct epithelial cells, we used small interfering RNA, dominant-negative TLR3 mutants, and TLR3-deficient oviduct epithelial cells to show that the IFN-β secreted during C. muridarum infection requires a functional TLR3. Interestingly, we demonstrate that the TLR3 signaling pathway is not required for IFN-β synthesis in C. muridarum-infected macrophages, suggesting that there are alternate and redundant pathways to Chlamydia-induced IFN-β synthesis that seem to be dependent upon the cell type infected. Finally, because there is no obvious dsRNA molecule associated with Chlamydia infection, the requirement for TLR3 in Chlamydia-induced IFN-β synthesis in infected oviduct epithelial cells implicates a novel ligand that binds to and signals through TLR3.

Chlamydia trachomatis is the most common sexually transmitted bacterial infection in the United States. Utilizing cloned murine oviduct epithelial cell lines, we previously identified Toll-like receptor 2 (TLR2) as the principal epithelial pattern recognition receptor (PRR) for infection-triggered release of the acute inflammatory cytokines interleukin-6 and granulocyte-macrophage colony-stimulating factor. The infected oviduct epithelial cell lines also secreted the immunomodulatory cytokine beta interferon (IFN-␤) in a largely MyD88-independent manner. Although TLR3 was the only IFN-␤ production-capable TLR expressed by the oviduct cell lines, we were not able to determine whether TLR3 was responsible for IFN-␤ production because the epithelial cells were unresponsive to the TLR3 ligand poly(I-C), and small interfering RNA (siRNA) techniques were ineffective at knocking down TLR3 expression. To further investigate the potential role of TLR3 in the infected epithelial cell secretion of IFN-␤, we examined the roles of its downstream signaling molecules TRIF and IFN regulatory factor 3 (IRF-3) using a dominant-negative TRIF molecule and siRNA specific for TRIF and IRF-3. Antagonism of either IRF-3 or TRIF signaling significantly decreased IFN-␤ production. These data implicate TLR3, or an unknown PRR utilizing TRIF, as the source of IFN-␤ production by Chlamydia-infected oviduct epithelial cells.

Chlamydia trachomatis urogenital serovars primarily replicate in epithelial cells lining the reproductive tract. Epithelial cells recognize Chlamydia through cell surface and cytosolic receptors, and/or endosomal innate receptors such as Toll-like receptors (TLRs). Activation of these receptors triggers both innate and adaptive immune mechanisms that are required for chlamydial clearance, but are also responsible for the immunopathology in the reproductive tract. We previously demonstrated that Chlamydia muridarum (Cm) induces IFN-β in oviduct epithelial cells (OE) in a TLR3-dependent manner, and that the synthesis of several cytokines and chemokines are diminished in Cm-challenged OE derived from TLR3-/- 129S1 mice. Furthermore, our in vitro studies showed that Cm replication in TLR3-/- OE is more efficient than in wild-type OE. Because TLR3 modulates the release inflammatory mediators involved in host defense during Cm infection, we hypothesized that TLR3 plays a protective role against Cm-induced genital tract pathology in congenic C57BL/6N mice. Using the Cm mouse model for human Chlamydia genital tract infections, we demonstrated that TLR3-/- mice had increased Cm shedding during early and mid-stage genital infection. In early stage infection, TLR3-/- mice showed a diminished synthesis of IFN-β, IL-1β, and IL-6, but enhanced production of IL-10, TNF-α, and IFN-γ. In mid-stage infection, TLR3-/- mice exhibited significantly enhanced lymphocytic endometritis and salpingitis than wild-type mice. These lymphocytes were predominantly scattered along the endometrial stroma and the associated smooth muscle, and the lamina propria supporting the oviducts. Surprisingly, our data show that CD4+ T-cells are significantly enhanced in the genital tract TLR3-/- mice during mid-stage Chlamydial infection. In late-stage infections, both mouse strains developed hydrosalpinx; however, the extent of hydrosalpinx was more severe in TLR3-/- mice. Together, these data suggest that TLR3 promotes the clearance of Cm during early and mid-stages of genital tract infection, and that loss of TLR3 is detrimental in the development hydrosalpinx.

Among the eight equid herpesviruses identified to date (52), equine herpesvirus 1 (EHV-1) is one of the most pathogenic herpesviruses of horses, causing spontaneous abortions in pregnant mares, as well as respiratory tract infections and neurological disorders (1,12,45). The virus is a member of the subfamily Alphaherpesvirinae and serves as a model for the investigation of alphaherpesvirus gene regulation during both productive and persistent infections. The 77 EHV-1 genes are temporally and coordinately expressed at immediate-early (IE), early, and late (␥1 and ␥2) times of the lytic infection cycle (8, 18), analogous to that of herpes simplex virus type 1 (HSV-1) (11,33). In contrast to HSV-1, EHV-1 carries only one IE gene (also termed IR1 gene) that is expressed without prior viral protein synthesis due to the EHV-1 ␣-trans-inducing factor (ETIF), a homolog of the HSV-1 VP16 protein (14,41,47). The EHV-1 IE gene (i) is located within each invertedrepeat region and encodes a polypeptide of 1,487 amino acids (aa) with a predicted molecular mass of approximately 155 kDa (19,21,27), (ii) has a product with a high degree of homology with HSV-1 ICP4 and the varicella-zoster virus ORF62 gene products (21), and (iii) is transcribed as a 6.0-kb spliced mRNA (19,27,51) that gives rise to both structurally and antigenically related protein species ranging from 125 to 200 kDa (7,8,51). In transient-cotransfection assays, the IE protein is a bifunctional regulatory protein capable of (i) negatively autoregulating its own promoter (55), (ii) independently activating EHV-1 early and heterologous viral promoters (55, 56), (iii) cooperating synergistically with two early auxiliary regulatory proteins (EICP22 and EICP27) to activate EHV-1 early and ␥1 late promoters (32,44,55,57,64), and (iv) acting antagonistically with a third early major regulatory protein, EICP0, to selectively repress expression of certain promoters from all classes of EHV-1 promoters, including ␥2 late promoters (3,35).Sequence alignment of the EHV-1 IE protein and other homologs in the subfamily Alphaherpesvirinae defined five colinear regions that harbor specific functional domains. Region 1 contains an acidic transactivation domain (TAD; aa 3 to 89) (58) and a serine-rich tract (SRT; aa 181 to 220). Regions 2 and 3 harbor a helix-loop-helix motif that mediates a sequencespecific DNA-binding activity (aa 422 to 597) (38), while the nuclear localization signal (aa 963 to 970) lies within region 3 (56). Region 5 contains a transcriptional-enhancement domain that is required for the full transactivation activity of the IE protein (5, 56

The equine herpesvirus 1 (EHV-1) immediate-early (IE) and EICP0 proteins are potent trans-activators of EHV-1 promoters; however, in transient-transfection assays, the IE protein inhibits the trans-activation function of the EICP0 protein. Assays with IE mutant proteins revealed that its DNA-binding domain, TFIIB-binding domain, and nuclear localization signal may be important for the antagonism between the IE and EICP0 proteins. In vitro interaction assays with the purified IE and EICP0 proteins indicated that these proteins interact directly. At late times postinfection, the IE and EICP0 proteins colocalized in the nuclei of infected equine cells. Transient-transfection assays showed that the EICP0 protein trans-activated EHV-1 promoters harboring only a minimal promoter region (TATA box and cap site), suggesting that the EICP0 protein trans-activates EHV-1 promoters by interactions with general transcription factor(s). In vitro interaction assays revealed that the EICP0 protein interacted directly with the basal transcription factors TFIIB and TBP and that the EICP0 protein (amino acids [aa] 143 to 278) mediated the interaction with aa 125 to 174 of TFIIB. Our unpublished data showed that the IE protein interacts with the same domain (aa 125 to 174) of TFIIB and with TBP. Taken together, these results suggested that interaction of the EICP0 protein with the IE protein, TFIIB, and TBP may mediate the antagonism between the IE and EICP0 proteins.The immediate-early (IE) gene of equine herpesvirus 1 (EHV-1) is essential for replication (16), lies within each of the two inverted repeats, and encodes a 1,487-amino-acid (aa) polypeptide (21). The IE protein trans-activates EHV-1 and heterologous viral promoters and trans-represses its own expression (56, 57). Residues 422 to 597 of the IE protein are sufficient for its sequence-specific DNA binding to the consensus binding sequence 5Ј-ATCGT-3Ј that overlaps the transcription initiation site of the IE promoter and to sequences in the E and L promoters that contain a degenerate version of this cognate cis element (36). A potent transcriptional activation domain lies within the first 89 aa residues of the IE protein (58), and aa 963 to 970 are necessary for nuclear localization of truncated IE polypeptides (57). The IE protein binds to the transcription initiation site of the glycoprotein K (gK) promoter sequences, thereby repressing transcription of this true late gene (35). The EICP0 protein is able to release the ␥2 L gK promoter from repression mediated by the IE protein (35). EHV-1 EICP22 (ICP22 homolog) (27), EICP27 (ICP27 homolog) (64), and EICP0 (2) are regulated as E genes, in contrast to the case for herpes simplex virus type 1 (HSV-1), in which the homologs of these three regulatory genes are members of the IE gene family (29,42). The EICP22 protein physically interacts with the IE protein (11, 12) and increases the in vitro DNA-binding activity of the IE protein for sequences in the IE, E, and L promoters (37).The EICP0 gene of the KyA virus encodes a prot...

Toll-Like Receptor (TLR) activation is important in immune responses and in differentiation of hematopoietic stem cells. We detected mRNA expression of TLR’s 1, 2, 3, 5, and 6, but not TLR’s 4,7,8, and 9 in murine (m)ESC line E14, and noted high cell surface protein expression of TLR-2, but not TLR-4, for mESC lines R1, CGR8, and E14. ESC lines were cultured in the presence of leukemia inhibitory factor (LIF). Pam3Cys, enhanced proliferation and survival of the three ESC lines. In contrast, LPS decreased proliferation and survival. Pam3Cys and LPS effects on proliferation and survival were blocked by antibody to TLR2, suggesting that effects of both Pam3Cys and LPS on these mESC lines were likely mediated through TLR2. E14 ESC line expressed MyD88. Pam3Cys stimulation of E14 ESCs was associated with induced NF-κB translocation, enhanced phosphorylation of IKKα/β, and enhanced mRNA, but not protein, expression of tumor necrosis factor-alpha, interferon-gamma, and IL-6. TLR-2 activation by Pam3Cys or inhibition by LPS was not associated with changes in morphology or expression of alkaline phosphatase, Oct4, SSEA1, KLF4, or Sox2, markers of undifferentiated mESCs. Our studies identify TLR-2 as present and functional in E14, R1, and CGR8 mESC lines.