In this cohort of African American women with mild to moderate PID, TLR1 and TLR4 variants were associated with C. trachomatis infection. These variants also displayed trends toward increased odds of upper genital tract infection. TLR1 variants were also associated with decreased pregnancy rates among a subset of women with C. trachomatis infection. This study provides novel insight into the pathogenesis of C. trachomatis infection.
Toll-like receptors initiate microbial elimination through the production of inflammatory cytokines and chemokines via activation of nuclear factor κ-B (NF-κB) [10
]. Although this initially results in a healthy immune response, variations in these genes may alter TLR signaling, possibly playing a role in disease progression. Variations in these genes may also explain the variability seen in the course and outcome of C. trachomatis
infection. Our results show that TLR1 and TLR4 variants may play a role in the development of chlamydial PID. Chlamydia
infect epithelial cells, leading to the secretion of proinflammatory cytokines [7
]. This inflammatory response may be responsible for long-term damage of the reproductive tract following C. trachomatis
]. We found that compared with chlamydial-negative women, chlamydial-positive women were more likely to have elevated CRP. C-reactive protein is an acute-phase protein and is an indicator of inflammation, suggesting that the women with PID due to chlamydial infection had an increased systemic inflammatory response compared with women with PID without chlamydial infection. Chlamydial-positive women were also more likely to have evidence of histologic endometritis. It is possible that chlamydial infection more frequently causes detectable endometrial inflammation in women with PID symptoms. However, the diagnosis of PID was likely more specific in women with PID symptoms and documented chlamydial infection.
We found that among women with PID, the CC genotype of TLR4 SNP rs1927911 was associated with increased odds of C. trachomatis
infection. Studies among Dutch white populations have not found significant associations between TLR4 variants and C. trachomatis
]. However, our patient populations differed, and the authors did not include rs1927911 in their study. It is possible that TLR4 plays a role in C. trachomatis
pathogenesis. TLR4 is expressed in the female reproductive tract and has been reported to be present in the endocervix, endometrium, and Fallopian tubes [12
]. In addition, TLR4 can recognize both chlamydial LPS and cHSP60 [26
]. Several retrospective studies have found cHSP60 to be linked with chlamydia-associated tubal infertility and PID [27
], whereas others have determined immune responses to cHSP60 correlate with protection from infection or disease [31
]. However, little is known about the functional or clinical relevance of TLR4 rs1927911. In a white population consisting of 110 lung transplant recipients and 422 healthy controls, the TT genotype was found to increase the odds of bronchiolitis obliterans (OR, 4.20; 95% CI, 1.43–12.35; P
]. Functional analyses are needed to determine the relevance of rs1927911. Because this SNP is located in the intron, it may be in LD with another functional SNP. However, using Haploview, we were unable to find any reported SNPs in strong LD with this variant in African American populations.
TLR2 has been recognized to play a role in chlamydial infections [13
]. However, we did not find any significant associations between TLR2 variants and C. trachomatis
, although we only had 35% gene coverage for TLR2. Other TLR2 variants that were not tagged by our SNPs should be further examined. TLR2 can form a dimer with TLR1 to recognize a wide range of pathogens, and TLR1 can affect the level of signaling through TLR2 [34
]. We found an association between the TLR1 rs5743618 TT genotype and increased C. trachomatis
infection. TLR1 variants were also associated with decreased pregnancy rates among women with C. trachomatis
infection. In addition, rs5743618 was associated with upper genital tract infection after adjusting for age. The association between the TT genotype and chlamydia did not reach statistical significance after permutations. However, our sample size limited our power. TLR1 rs5743618 is a nonsynonymous mutation and results in an amino acid change. The G allele has been reported to be associated with deficient TLR signaling in comparison with the T allele [35
] and has been reported to reduce leprosy [35
]. Hawn et al [35
] reported that the T allele expressed significantly greater NF-κB signaling in transfected HEK293 cells compared with the G allele. Because rs5743618 has possible functional relevance, it should be further explored in C. trachomatis
Interactions between the responses elicited by chlamydial stimulation of multiple TLRs and other innate immune receptors may also be involved in determining the balance of cytokine production and thus the ultimate outcome [40
]. Functional interactions between TLR2 and TLR1 or TLR6 have been demonstrated in vitro, with TLR6 and TLR1 enhancing or impeding, respectively, the TLR2-dependent response to phenol-soluble modulin, a factor secreted by Staphylococcus epidermidis
]. Thus, the ratio of different TLRs within a cell may modify the response to a given ligand. It is clear that interactions between TLR2 and other TLRs following C. trachomatis
infection need to be further explored. Due to our sample size, we did not examine any interactions, but we acknowledge that our results may be masked by gene–gene or gene–environment interactions.
Our study has several strengths. First, data were obtained from a large, multicenter, prospective, randomized clinical trial with comprehensive demographic, clinical, and obstetric measurements. These findings are generalizable to patients treated for clinically suspected PID. Not all women in the PEACH study had blood samples available for analyses. However, important demographic and clinical characteristics between women with and without blood samples did not differ. This is the first study to examine the role of several TLR and adaptor-molecule SNPs in chlamydial PID. However, our sample size limited our power. We also had low SNP coverage for our genes. Other TLR variants and variants in other PRRs that recognize Chlamydiae [40
] should be explored in chlamydial pathogenesis. We also relied on an internal control group for comparison. Therefore, women in the control groups all had clinically suspected PID. This may have biased comparisons between chlamydial-positive and chlamydial-negative women toward the null. For comparisons of postchlamydial PID sequelae, this was not a limitation.
Studies in the mouse model of chlamydial genital tract infection have revealed TLR2 activation is critical for the development of oviduct pathology [13
]. TLR2 activation is the result of engagement of TLR1/2 or TLR2/6 heterodimers [34
]. The association of the TLR1 rs5743618 TT genotype with increased C. trachomatis
as a cause of PID and a trend for increased upper genital tract infection and decreased pregnancy raises the possibility that we have discovered a genetic risk predictor of enhanced disease due to chlamydia. This is supported by studies documenting that the T allele leads to significantly greater NF-κB signaling and inflammatory cytokine production [35
]. Although the CC genotype of TLR4 SNP rs1927911 was associated with increased odds for chlamydial infection as a cause of PID, this allele was not associated with decreased pregnancy. This may not be surprising because TLR4-deficient mice demonstrate levels of pathology similar to wild-type mice after chlamydial infection [13
Further exploration of the role of innate immune receptors in the course and outcome of C. trachomatis is needed to delineate its pathogenesis. This should include comparisons between women with chlamydial PID and women with uncomplicated lower genital C. trachomatis infection. Such research may lead to better management and control of C. trachomatis, possibly by identification of biomarkers that predict patients with increased risk that require increased screening and would benefit most from a vaccine.