Immune response against Chlamydia trachomatis via toll-like receptors is negatively regulated by SIGIRR

Abstract

Chlamydia trachomatis is the most common bacterial sexually-transmitted infection and the major cause of preventable blindness worldwide. The asymptomatic nature of many infections along with uncontrolled inflammation leads to irreversible damage in the upper genital tract and the tarsal conjunctivae, with the major complications of infertility and chronic pelvic pain, and blindness, respectively. Inflammation must, therefore, be tightly regulated to avoid an unrestrained immune response. The genetic factors that regulate inflammation through Toll-like receptor (TLR) signaling pathways during C. trachomatis infection have not been fully characterized. SIGIRR (also known as IL-1R8 or TIR8) can regulate inflammation in response to various pathogens and diseases. However, nothing is known about its role during C. trachomatis infection. Expression of the pro-inflammatory chemokine, IL-8, was measured in epithelial cells infected with C. trachomatis. The effect of SIGIRR was determined by depleting SIGIRR or over-expressing SIGIRR in the epithelial cells before infection. Our results indicate that, in the absence of SIGIRR, epithelial cells induce higher levels of the pro-inflammatory chemokine, IL-8, in response to C. trachomatis infection. In addition, SIGIRR associates with MyD88 in both infected and uninfected infected cells. Collectively, our data demonstrate that SIGIRR functions as a negative regulator of the immune response to C. trachomatis infection. This finding provides insights into the immuno-pathogenesis of C. trachomatis that can be used to treat and identify individuals at risk of uncontrolled inflammation during infection.

Fig 1. Expression of SIGIRR in cell lines.

PCR analysis of SIGIRR in various human cell lines. Cervical epithelial HeLa cells, HEK293 and THP-1 cells express SIGIRR, while HEK-TLR2, HEK-TLR3 and HEK-TLR4 do not.

Fig 2. Depletion of SIGIRR in HeLa cells induces higher levels of IL-8 mRNA during infection.

(A) HeLa cells were transfected with siRNA non-target control (siRNA CONT); SIGIRR-siRNA (sequence A or B); Lipofectamine treated (Lipo Cont) or non-transformed (N.T.). Relative expression level of SIGIRR compared with WT was reduced by 40 to 80% in SIGIRR-siRNA (panels A and B, respectively). (B) IL-8 expression in C. trachomatis serovar L2 infected HeLa cells, at MOI of 1 for 24 hours, showed higher levels of IL-8 expression in the cells that express lower levels of SIGIRR compared with WT infected cells. The increase of IL-8 expression inversely correlated with the expression levels of SIGIRR, as shown in panel A. Data were collected from 4 independent experiments. Error bars represent ±SD, and Student’s t test was conducted. ns. (not significant), (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig 3. SIGIRR associates with MyD88.

HeLa cells were transfected with 4.0 μg of MyD88-HA and either SIGIRR-FLAG construct or mock-transfected with 4.0 μg of pCDNA3.1+ empty vector. Forty-eight hours post transfection, cells were either left uninfected or infected with LGV/L2 as indicated. Seventy-two hours post transfection, cells were lysed and immunoprecipitated using FLAG Immunoprecipitation Kit (IP). Samples immunoblotted (IB) using anti-HA. Both L2 infected or uninfected HeLa cells showed an association of MyD88 with SIGIRR (top panel-right). Whole cell lysate indicated the presence of MyD88-HA in all the samples (bottom panel). Data shown is representative of 3 independent experiments.

Fig 4. Overexpression of SIGIRR slightly reduces expression of IL-8 mRNA after infection with C. trachomatis.

HEK-Blue-hTLR2 cells transiently transfected, or mock-transfected, with 0.1 μg or 1.0 μg of SIGIRR-FLAG construct or pCDNA3.1+ empty vector (A). The expression of SIGIRR in HEK-Blue hTLR2 cells was detected only in SIGIRR-FLAG transfected cells. The expression level of SIGIRR mRNA was proportional to the level of the plasmid DNA introduced into the cells. (B) Mock-cells and SIGIRR-FLAG infected with C. trachomatis at an MOI of 1 for 24 hrs. The mRNA expression levels of IL-8 were reduced in cells transfected with SIGIRR-FLAG construct compared with the empty vector. This reduction was statistically significant only at the lower concentration of SIGIRR (0.1 μg). Data were collected from 4 independent experiments. Error bars represent ±SD, and Student’s t test was conducted. N.D. (not detected), n.s. (not significant), *p < 0.05, **p < 0.01.