The role of STAT1 in Chlamydia-induced type I interferon responses in oviduct epithelium

Indiana University-Purdue University Indianapolis (IUPUI)Progression of Chlamydia into upper reproductive tract epithelium and the induction of subsequent immune responses to infection are major contributors to Chlamydia-induced pathogenesis of the genital tract. We reported that C. muridarum infection of the oviduct epithelial cells (OEs) secrete IFN-β in a TLR3 dependent manner. However, we showed that the C. muridarum infected TLR3-deficient OEs were still able to secrete minimal amounts of IFN-β into the supernatants, which is suggestive that there are other signaling pathways that contribute to Chlamydia-induced IFN-β synthesis in these cells. Previous studies describing the activation of the JAK/STAT signaling pathway during Chlamydia infection of cervical epithelial cells proposes a putative role for STAT1 in the synthesis of type I IFNs during Chlamydia infection. The present study investigated the role of STAT1 in Chlamydia-induced IFN-β production in OEs. OEs were infected with Chlamydia muridarum and analyzed at 24 hours by RT-PCR and western blot to determine STAT1 expression. STAT (-/-) OEs were infected and IFN-β production measured by ELISA. Quantitative real-time PCR analyses were performed at 6 and 16 hour post-infection to elucidate the mechanisms involved in IFN-β production during infection. Fluorescent microscopy was used to observe changes in Chlamydia replication. STAT1 activation and expression were significantly increased in wild-type (WT) OEs upon infection. TLR3 (-/-) OEs showed diminished STAT1 protein activation and expression. Augmented STAT1 protein expression corresponded to STAT1 mRNA levels. ELISA analyses revealed significantly less IFN-β production in infected STAT1 (-/-) OEs compared to WT OEs. Quantitative real-time PCR data showed that gene expression of IFN-β and of type I IFN signaling components were significantly increased during late stage Chlamydia infection, dependent on STAT1. Temporal regulation and increases in expression of IFN-α subtypes during infection were STAT1-dependent. Our results implicate STAT1-mediated signaling as a contributor to the C. muridarum-induced synthesis of IFN-β and other type I IFNs in OEs. We previously described a major role for TLR3 in the early-stage Chlamydia-induced synthesis of IFN-β in OEs; the results from this study suggest a role for STAT1 in the synthesis of type I IFNs that occurs during early and late stages of infection

Analyses of the pathways involved in early- and late-phase induction of IFN-beta during C. muridarum infection of oviduct epithelial cells.

We previously reported that the IFN-β secreted by Chlamydia muridarum-infected murine oviduct epithelial cells (OE cells) was mostly dependent on the TLR3 signaling pathway. To further characterize the mechanisms of IFN-β synthesis during Chlamydia infection of OE cells in vitro, we utilized specific inhibitory drugs to clarify the roles of IRF3 and NF-κB on both early- and late-phase C. muridarum infections. Our results showed that the pathways involved in the early-phase of IFN-β production were distinct from that in the late-phase of IFN-β production. Disruption of IRF3 activation using an inhibitor of TBK-1 at early-phase Chlamydia infection had a significant impact on the overall synthesis of IFN-β; however, disruption of IRF3 activation at late times during infection had no effect. Interestingly, inhibition of NF-κB early during Chlamydia infection also had a negative effect on IFN-β production; however, its impact was not significant. Our data show that the transcription factor IRF7 was induced late during Chlamydia infection, which is indicative of a positive feedback mechanism of IFN-β synthesis late during infection. In contrast, IRF7 appears to play little or no role in the early synthesis of IFN-β during Chlamydia infection. Finally, we demonstrate that antibiotics that target chlamydial DNA replication are much more effective at reducing IFN-β synthesis during infection versus antibiotics that target chlamydial transcription. These results provide evidence that early- and late-phase IFN-β production have distinct signaling pathways in Chlamydia-infected OE cells, and suggest that Chlamydia DNA replication might provide a link to the currently unknown chlamydial PAMP for TLR3

<i>C</i>. <i>muridarum</i>-induced IFN-β affects the gene expression levels of components found in the type-1 IFN signaling pathway.

<p>Bm1.1 cells were infected with 10 IFU/ cell <i>C</i>. <i>muridarum</i>, and the gene expression levels of IFN-β, TLR3, IRF3, IRF7, and Stat1 were measured by RT-qPCR after total cell mRNA was harvested at either early (12h) or late (24h) times post infection. <b>(A)</b> Transcription results of Bm1.11 OE cells harvested at 12h PI to measure the impact on transcription of candidate genes after cells were incubated from 4-to-12h PI in culture medium containing 1μg/ml of either IFN-β neutralizing antibody (α-IFNβ) or isotype control antibody (α-IgG). <b>(B)</b> Transcription results of these genes after cells were incubated in culture medium containing either antibody from 12-to-24h PI. <i>The results shown are representative of three independent experiments; Fold change is compared to Mock-infected controls</i>.</p

Gene expression levels of IFN-β and TLR3 during the course of <i>C</i>. <i>muridarum</i> infection.

<p>Bm1.11 cells were infected with 10 IFU/ cell <i>C</i>. <i>muridarum</i>, and the gene expression levels of: <b>(A)</b> IFN-β and <b>(B)</b> TLR3 were measured by RT-qPCR after total cell mRNA was harvested at each time-point indicated. <i>The results shown are representative of three independent experiments; Fold change is compared to Mock-infected controls</i>.</p

Kinetics of IFN-β production in <i>C</i>. <i>muridarum</i>-infected Bm1.1 OE cells.

<p><b>(A)</b> Bm1.11 cells were infected with 10 IFU/ cell <i>C</i>. <i>muridarum</i> to measure the amount of IFN-β secreted into the supernatants for 24 hours. The supernatants were collected from the infected cells at each time point listed (denoted as 0-Xh), and replaced with fresh medium for the remainder of the 24 hour infection (denoted as X-24h). Total IFN-β secreted in the supernatants of the two phases was measured by ELISA. <b>(B)</b> Bm1.11 cells were infected with 10 IFU/cell <i>C</i>. <i>muridarum</i> to measure only the residual amount of IFN-β secreted into the supernatants <u>after</u> the media was replaced. Each time interval shows residual amounts of IFN-β synthesis up until 12hr post-infection. Horizontal bars represent mean ± SD of cytokine levels from experiments performed in triplicate. <i>Results are representative data from one of three independent experiments</i>. ** = <i>p< 0</i>.<i>01 compared to 24h C</i>. <i>muridarum infection control</i>.</p

Inhibition of IRF3 and NF-κB affects early synthesis of IFN-β induced during <i>Chlamydia</i> infection.

<p><b>(A)</b> Bm1.1 cells were infected with 10 IFU/ cell <i>C</i>. <i>muridarum</i> up until the indicated time-points when the media was supplemented with either the IRF3 inhibitor BX-795, the NF-κB inhibitor JSH-23, or the solubilizing agent DMSO as a negative control. Cells were allowed to incubate in the presence of each inhibitor from the time indicated until cell supernatants were harvested at 24h PI, and IFN-β secreted was measured by ELISA. <b>(B)</b><i>C</i>. <i>muridarum</i>-infected Bm1.11 cells were allowed to incubate in the presence of each inhibitor from the time indicated until cell supernatants were harvested at 24h PI, and IL-6 secreted was measured by ELISA. <b>(C)</b> Uninfected Bm1.11 cells were either DMSO-treated, transfected with 10, 25, or 50 μg/ml poly-IC, or transfected with poly-IC prior adding the inhibitors BX-795 and JSH-23 to the cells 1h post-transfection. IFN-β secreted into the supernatants 24 h post transfection was measured by ELISA. <i>The results shown are representative of three independent experiments</i>. * = <i>p<0</i>.<i>05; ** = p< 0</i>.<i>01; NS = not statistically significant compared to poly-IC alone (C)</i>, <i>or 24h C</i>. <i>muridarum infection control without inhibitor (A and B; denoted as MoPn)</i>.</p

Rifampicin does not affect host-cell mechanisms.

<p>Bm1.11 cells were either mock-treated, treated with 10 μg/ml of the purified <i>E</i>. <i>coli</i> peptidoglycan (PGN), or infected with 10 IFU/ cell <i>C</i>. <i>muridarum</i>, and cells were incubated in the presence of increasing concentrations of rifampicin starting at 2h post-treatment/ infection. After 24 h post-treatment/ infection, cell lysates and supernatants were harvested for: <b>(A)</b> western-blot analyses for the <i>Chlamydia</i>-specific major outer membrane protein (MOMP) and cellular β-actin. <b>(B)</b> ELISA analyses of PGN-induced versus <i>Chlamydia</i>-infection specific cytokine expression. <i>The results shown are representative of three independent experiments</i>. * = <i>p<0</i>.<i>05; ** = p< 0</i>.<i>01; NS = not statistically significant compared to control cells without rifampicin (0</i> μ<i>g/ml); MoPn denotes Chlamydia infection</i>.</p

Role of bacterial DNA replication and bacterial transcription in <i>Chlamydia</i> induced IFN-β synthesis.

<p><b>(A)</b> Bm1.11 cells were infected with 10 IFU/ cell <i>C</i>. <i>muridarum</i> and cells were incubated in the presence of increasing concentrations of either rifampicin or ofloxacin starting at 2h PI. The medium was replaced with antibiotic-free medium at 18h PI, cells were harvested at 30h PI, and <i>C</i>. <i>muridarum</i> titers (IFU/ml) were measured on McCoy cell monolayers as described in Materials and Methods. <b>(B)</b> Bm1.11 cells were infected with 10 IFU/ cell <i>C</i>. <i>muridarum</i> and cells were incubated in the presence of either 0.01 μg/ml rifampicin or 0.1 μg/ml ofloxacin for each 4h interval indicated, before cell supernatants were harvested at 24h PI and IFN-β secreted was measured by ELISA. <i>The results shown are representative of three independent experiments</i>. * = <i>p< 0</i>.<i>05; NS = not statistically significant compared to 24h C</i>. <i>muridarum infection control without antibiotic (denoted as MoPn)</i>.</p

Disruption of IFN-β autocrine-paracrine pathways with neutralizing antibody.

<p>Bm1.11 cells were infected with 10 IFU/ cell <i>C</i>. <i>muridarum</i> to measure the amount of IFN-β secreted into the supernatants for 24 hours by ELISA. The supernatants were supplemented at each time-point listed with either 0.1μg/ml of: <b>(A)</b> IFN-β neutralizing antibody, or <b>(B)</b> isotype control antibody. The amount of IFN-β secreted into the supernatants <u>after</u> the addition of antibody (ab/IgG-24h) was estimated by subtracting out the amount of IFN-β secreted in control experiments in which supernatants were instead collected and assayed for IFN-β synthesis at the same time-point (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119235#sec002" target="_blank">Materials and Methods</a>). <i>Results are representative data from one of three independent experiments</i>. * = <i>p</i>< 0.05 <i>compared to 24h C</i>. <i>muridarum infection control</i>; <u><i>0-ab/IgG</i></u><i>denotes “0hr PI until time antibody added”;</i><u><i>ab/IgG-24h</i></u><i>denotes “time antibody added until 24h PI”</i>.</p

Primers for RT-qPCR.

<p>Primers for RT-qPCR.</p