Browsing by Author "Derbigny, Wilbert A."

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    Analyses of the pathways involved in early- and late-phase induction of IFN-beta during C. muridarum infection of oviduct epithelial cells
    (PLoS, 2015-03-23) Hu, Sishun; Hosey, Kristen L.; Derbigny, Wilbert A.; Department of Microbiology and Immunology, IU School of Medicine
    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.
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    TLR3 deficiency exacerbates the loss of epithelial barrier function during genital tract Chlamydia muridarum infection
    (PLOS, 2019-01-09) Kumar, Ramesh; Gong, Haoli; Liu, Luyao; Ramos-Solis, Nicole; Seye, Cheikh I.; Derbigny, Wilbert A.; Microbiology and Immunology, School of Medicine
    PROBLEM: Chlamydia trachomatis infections are often associated with acute syndromes including cervicitis, urethritis, and endometritis, which can lead to chronic sequelae such as pelvic inflammatory disease (PID), chronic pelvic pain, ectopic pregnancy, and tubal infertility. As epithelial cells are the primary cell type productively infected during genital tract Chlamydia infections, we investigated whether Chlamydia has any impact on the integrity of the host epithelial barrier as a possible mechanism to facilitate the dissemination of infection, and examined whether TLR3 function modulates its impact. METHOD OF STUDY: We used wild-type and TLR3-deficient murine oviduct epithelial (OE) cells to ascertain whether C. muridarum infection had any effect on the epithelial barrier integrity of these cells as measured by transepithelial resistance (TER) and cell permeability assays. We next assessed whether infection impacted the transcription and protein function of the cellular tight-junction (TJ) genes for claudins1-4, ZO-1, JAM1 and occludin via quantitative real-time PCR (qPCR) and western blot. RESULTS: qPCR, immunoblotting, transwell permeability assays, and TER studies show that Chlamydia compromises cellular TJ function throughout infection in murine OE cells and that TLR3 deficiency significantly exacerbates this effect. CONCLUSION: Our data show that TLR3 plays a role in modulating epithelial barrier function during Chlamydia infection of epithelial cells lining the genital tract. These findings propose a role for TLR3 signaling in maintaining the integrity of epithelial barrier function during genital tract Chlamydia infection, a function that we hypothesize is important in helping limit the chlamydial spread and subsequent genital tract pathology.
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    Toll-like receptor 3 (TLR3) promotes the resolution of Chlamydia muridarum genital tract infection in congenic C57BL/6N mice
    (Public Library of Science, 2018-04-06) Carrasco, Sebastian E.; Hu, Sishun; Imai, Denise M.; Kumar, Ramesh; Sandusky, George E.; Yang, X. Frank; Derbigny, Wilbert A.; Microbiology and Immunology, School of Medicine
    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.
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    Toll-Like Receptor 3 Deficiency Leads to Altered Immune Responses to Chlamydia trachomatis Infection in Human Oviduct Epithelial Cells
    (American Society for Microbiology, 2019-09) Xu, Jerry Z.; Kumar, Ramesh; Gong, Haoli; Liu, Luyao; Ramos-Solis, Nicole; Li, Yujing; Derbigny, Wilbert A.; Microbiology and Immunology, School of Medicine
    Reproductive tract pathology caused by Chlamydia trachomatis infection is an important global cause of human infertility. To better understand the mechanisms associated with Chlamydia-induced genital tract pathogenesis in humans, we used CRISPR genome editing to disrupt Toll-like receptor 3 (TLR3) function in the human oviduct epithelial (hOE) cell line OE-E6/E7 in order to investigate the possible role(s) of TLR3 signaling in the immune response to Chlamydia. Disruption of TLR3 function in these cells significantly diminished the Chlamydia-induced synthesis of several inflammation biomarkers, including interferon beta (IFN-β), interleukin-6 (IL-6), interleukin-6 receptor alpha (IL-6Rα), soluble interleukin-6 receptor beta (sIL-6Rβ, or gp130), IL-8, IL-20, IL-26, IL-34, soluble tumor necrosis factor receptor 1 (sTNF-R1), tumor necrosis factor ligand superfamily member 13B (TNFSF13B), matrix metalloproteinase 1 (MMP-1), MMP-2, and MMP-3. In contrast, the Chlamydia-induced synthesis of CCL5, IL-29 (IFN-λ1), and IL-28A (IFN-λ2) was significantly increased in TLR3-deficient hOE cells compared to their wild-type counterparts. Our results indicate a role for TLR3 signaling in limiting the genital tract fibrosis, scarring, and chronic inflammation often associated with human chlamydial disease. Interestingly, we saw that Chlamydia infection induced the production of biomarkers associated with persistence, tumor metastasis, and autoimmunity, such as soluble CD163 (sCD163), chitinase-3-like protein 1, osteopontin, and pentraxin-3, in hOE cells; however, their expression levels were significantly dysregulated in TLR3-deficient hOE cells. Finally, we demonstrate using hOE cells that TLR3 deficiency resulted in an increased amount of chlamydial lipopolysaccharide (LPS) within Chlamydia inclusions, which is suggestive that TLR3 deficiency leads to enhanced chlamydial replication and possibly increased genital tract pathogenesis during human infection.
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    XPC DNA REPAIR PROTEIN REGULATION IN THE CONTEXT OF THE G1/S CELL CYCLE CHECKPOINT
    (2010-10-15) Hardy, Tabitha M.; Smith, Martin L.; Derbigny, Wilbert A.; Herbert, Brittney-Shea; Roman, Ann
    DNA is subject to various types of damage that can impair cellular function or cause cell death. DNA damage blocks normal cellular processes such as replication and transcription and can have catastrophic consequences for the cell and for the organism. It has long been thought that the G1/S cell cycle checkpoint allows time for DNA repair by delaying S-phase entry. The p53 tumor suppressor pathway regulates the G1/S checkpoint by regulating the cyclin-dependent kinase inhibitor p21Waf1/Cip1, but p53 also regulates the nucleotide excision DNA repair protein XPC. Here, using p53-null cell lines we show that additional mechanisms stabilize XPC protein and promote NER in concert with the G1/S checkpoint. At least one mechanism to stabilize and destabilize XPC involves ubiquitin-mediated degradation of XPC, as the ubiquitin ligase inhibitor MG-132 blocked XPC degradation. The retinoblastoma protein, RB, in its unphosphorylated form actually stabilized XPC and promoted NER as measured by host-cell reactivation experiments. The data suggest that XPC protein and XPC-mediated NER is tightly linked to the G1/S checkpoint even in cells lacking functional p53.