Toll-like receptor 3 (TLR3) promotes the resolution of Chlamydia muridarum genital tract infection in congenic C57BL/6N mice

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

Direct Effects of HIV-1 Tat on Excitability and Survival of Primary Dorsal Root Ganglion Neurons: Possible Contribution to HIV-1-Associated Pain.

The vast majority of people living with human immunodeficiency virus type 1 (HIV-1) have pain syndrome, which has a significant impact on their quality of life. The underlying causes of HIV-1-associated pain are not likely attributable to direct viral infection of the nervous system due to the lack of evidence of neuronal infection by HIV-1. However, HIV-1 proteins are possibly involved as they have been implicated in neuronal damage and death. The current study assesses the direct effects of HIV-1 Tat, one of potent neurotoxic viral proteins released from HIV-1-infected cells, on the excitability and survival of rat primary dorsal root ganglion (DRG) neurons. We demonstrated that HIV-1 Tat triggered rapid and sustained enhancement of the excitability of small-diameter rat primary DRG neurons, which was accompanied by marked reductions in the rheobase and resting membrane potential (RMP), and an increase in the resistance at threshold (RTh). Such Tat-induced DRG hyperexcitability may be a consequence of the inhibition of cyclin-dependent kinase 5 (Cdk5) activity. Tat rapidly inhibited Cdk5 kinase activity and mRNA production, and roscovitine, a well-known Cdk5 inhibitor, induced a very similar pattern of DRG hyperexcitability. Indeed, pre-application of Tat prevented roscovitine from having additional effects on the RMP and action potentials (APs) of DRGs. However, Tat-mediated actions on the rheobase and RTh were accelerated by roscovitine. These results suggest that Tat-mediated changes in DRG excitability are partly facilitated by Cdk5 inhibition. In addition, Cdk5 is most abundant in DRG neurons and participates in the regulation of pain signaling. We also demonstrated that HIV-1 Tat markedly induced apoptosis of primary DRG neurons after exposure for longer than 48 h. Together, this work indicates that HIV-1 proteins are capable of producing pain signaling through direct actions on excitability and survival of sensory neurons

A Novel RAYM_RS09735/RAYM_RS09740 Two-Component Signaling System Regulates Gene Expression and Virulence in Riemerella anatipestifer

The Gram-negative bacterium Riemerella anatipestifer is an important waterfowl pathogen, causing major economic losses to the duck-producing industry. However, little is known of the virulence factors that mediate pathogenesis during R. anatipestifer infection. In this study, RAYM_RS09735 and RAYM_RS09740 were predicted to form a two-component signaling system (TCS) through bioinformatics analysis. This TCS was highly conserved across the Flavobacteriaceae. A mutant YMΔRS09735/RS09740 strain was constructed to investigate the role of the RAYM_RS09735/RAYM_RS09740 TCS in R. anatipestifer virulence and gene regulation. The median lethal dose (LD50) of YMΔRS09735/RS09740 was found to be >1011 CFU, equivalent to that of avirulent bacterial strains. The bacterial abundances of the YMΔRS09735/RS09740 strain in the heart, brain, liver, blood, and spleen were significantly lower than that of the wild-type R. anatipestifer YM strain. Pathological analysis using hematoxylin and eosin staining showed that, compared to the wild-type, the mutant YMΔRS09735/RS09740 strain caused significantly less virulence in infected ducklings. RNAseq and real-time PCR analysis indicated that the RAYM_RS09735/RAYM_RS09740 TCS is a PhoP/PhoR system. This is a novel type of TCS for Gram-negative bacteria. The TCS was also found to be a global regulator of expression in R. anatipestifer, with 112 genes up-regulated and 693 genes down-regulated in the YMΔRS09735/RS09740 strain (~33% genes demonstrated differential expression). In summary, we have reported the first PhoP/PhoR TCS identified in a Gram-negative bacterium and demonstrated that it is involved in virulence and gene regulation in R. anatipestifer

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

Role of STAT1 in Chlamydia-Induced Type-1 Interferon Production in Oviduct Epithelial Cells

We previously reported that Chlamydia muridarum-infected murine oviduct epithelial cells (OE cells) secrete interferon β (IFN-β) in a mostly TLR3-dependent manner. However, C. muridarum-infected TLR3-deficient OE cells were still able to secrete detectable levels of IFN-β into the supernatants, suggesting that other signaling pathways contribute to Chlamydia-induced IFN-β synthesis in these cells. We investigated the role of STAT1 as a possible contributor in the Chlamydia-induced type-1 IFN production in wild-type (WT) and TLR3-deficient OE cells to ascertain its putative role at early- and late-times during Chlamydia infection. Our data show that C. muridarum infection significantly increased STAT1 gene expression and protein activation in WT OE cells; however, TLR3-deficient OE cells showed diminished STAT1 protein activation and gene expression. There was significantly less IFN-β detected in the supernatants of C. muridarum-infected OE cells derived from mice deficient in STAT1 when compared with WT OE cells, which suggest that STAT1 is required for the optimal synthesis of IFN-β during infection. Real-time quantitative polymerase chain reaction analyses of signaling components of the type-1 IFN signaling pathway demonstrated equal upregulation in the expression of STAT2 and IRF7 genes in the WT and TLR3-deficient OE cells, but no upregulation in these genes in the STAT1-deficient OE cells. Finally, experiments in which INFAR1 was blocked with neutralizing antibody revealed that IFNAR1-mediated signaling was critical to the Chlamydia-induced upregulation in IFN-α gene transcription, but had no role in the Chlamydia-induced upregulation in IFN-β gene transcription

<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

Primers for RT-qPCR.

<p>Primers for RT-qPCR.</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

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