Virulence Protein Pgp3 Is Insufficient To Mediate Plasmid-Dependent Infectivity of Chlamydia trachomatis.

Chlamydia trachomatis is the most common cause of infectious blindness and sexually transmitted bacterial infection globally. C. trachomatis contains a conserved chlamydial plasmid with eight coding sequences. Plasmid-cured Chlamydia strains are attenuated and display reduced infectivity in cell culture and in vivo genital infection of female mice. Mutants that do not express the plasmid-encoded proteins Pgp3, a secreted protein with unknown function, or Pgp4, a putative regulator of pgp3 and other chromosomal loci, display an infectivity defect similar to plasmid-deficient strains. Our objective was to determine the combined and individual contributions of Pgp3 and Pgp4 to this phenotype. Deletion of pgp3 and pgp4 resulted in an infectivity defect detected by competition assay in cell culture and in mice. The pgp3 locus was placed under the control of an anhydrotetracycline-inducible promoter to examine the individual contributions of Pgp3 and Pgp4 to infectivity. Expression of pgp3 was induced 100- to 1,000-fold after anhydrotetracycline administration, regardless of the presence or absence of pgp4. However, secreted Pgp3 was not detected when pgp4 was deleted, confirming a role for Pgp4 in Pgp3 secretion. We discovered that expression of pgp3 or pgp4 alone was insufficient to restore normal infectivity, which required expression of both Pgp3 and Pgp4. These results suggest Pgp3 and Pgp4 are both required for infectivity during C. trachomatis infection. Future studies are required to determine the mechanism by which Pgp3 and Pgp4 influence chlamydial infectivity as well as the potential roles of Pgp4-regulated loci.

IL-1α Is Essential for Oviduct Pathology during Genital Chlamydial Infection in Mice.

Chlamydia trachomatis infection of the female genital tract can lead to irreversible fallopian tube scarring. In the mouse model of genital infection using Chlamydia muridarum, IL-1R signaling plays a critical role in oviduct tissue damage. In this study, we investigated the pathologic role of IL-1α, one of the two proinflammatory cytokines that bind to IL-1R. Il1a-/- mice infected with C. muridarum cleared infection at their cervix at the same rate as wild-type (WT) mice, but were significantly protected from end point oviduct damage and fibrosis. The contribution of IL-1α to oviduct pathology was more dramatic than observed in mice deficient for IL-1ß. Although chlamydial burden was similar in WT and Il1a-/- oviduct during peak days of infection, levels of IL-1ß, IL-6, CSF3, and CXCL2 were reduced in Il1a-/- oviduct lysates. During infection, Il1a-/- oviducts and uterine horns exhibited reduced neutrophil infiltration, and this reduction persisted after the infection resolved. The absence of IL-1α did not compromise CD4 T cell recruitment or function during primary or secondary chlamydial infection. IL-1α is expressed predominantly by luminal cells of the genital tract in response to infection, and low levels of expression persisted after the infection cleared. Ab-mediated depletion of IL-1α in WT mice prevented infection-induced oviduct damage, further supporting a key role for IL-1α in oviduct pathology.

Tumor Necrosis Factor Alpha-Induced Interleukin-1 Alpha Synthesis and Cell Death Is Increased in Mouse Epithelial Cells Infected With Chlamydia muridarum.

Chlamydia trachomatis-genital infection in women can be modeled in mice using Chlamydia muridarum. Using this model, it has been shown that the cytokines tumor necrosis factor (TNF)α and interleukin (IL)-1α lead to irreversible tissue damage in the oviducts. In this study, we investigated the contribution of TNFα on IL-1α synthesis in infected epithelial cells. We show that C muridarum infection enhanced TNFα-induced IL-1α expression and release in a mouse epithelial cell line. In addition to IL-1α, several TNFα-induced inflammatory genes were also highly induced, and infection enhanced TNF-induced cell death. In the mouse model of genital infection, oviducts from mice lacking the TNFα receptor displayed minimal staining for IL-1α compared with wild-type oviducts. Our results suggest TNFα and IL-1α enhance each other's downstream effects resulting in a hyperinflammatory response to chlamydial infection. We propose that biologics targeting TNF-induced IL-1α synthesis could be used to mitigate tissue damage during chlamydial infection.

Reduced Uterine Tissue Damage during Chlamydia muridarum Infection in TREM-1,3-Deficient Mice.

Genital infections with Chlamydia trachomatis can lead to uterine and oviduct tissue damage in the female reproductive tract. Neutrophils are strongly associated with tissue damage during chlamydial infection, while an adaptive CD4 T cell response is necessary to combat infection. Activation of triggering receptor expressed on myeloid cells-1 (TREM-1) on neutrophils has previously been shown to induce and/or enhance degranulation synergistically with Toll-like receptor (TLR) signaling. Additionally, TREM-1 can promote neutrophil transepithelial migration. In this study, we sought to determine the contribution of TREM-1,3 to immunopathology in the female mouse genital tract during Chlamydia muridarum infection. Relative to control mice, trem1,3-/- mice had no difference in chlamydial burden or duration of lower-genital-tract infection. We also observed a similar incidence of hydrosalpinx 45 days postinfection in trem1,3-/- compared to wild-type (WT) mice. However, compared to WT mice, trem1,3-/- mice developed significantly fewer hydrometra in uterine horns. Early in infection, trem1,3-/- mice displayed a notable decrease in the number of uterine glands containing polymorphonuclear cells and uterine horn lumens had fewer neutrophils, with increased granulocyte colony-stimulating factor (G-CSF). trem1,3-/- mice also had reduced erosion of the luminal epithelium. These data indicate that TREM-1,3 contributes to transepithelial neutrophil migration in the uterus and uterine glands, promoting the occurrence of hydrometra in infected mice.

Human Fallopian Tube Epithelial Cell Culture Model To Study Host Responses to Chlamydia trachomatis Infection.

Chlamydia trachomatis infection of the human fallopian tubes can lead to damaging inflammation and scarring, ultimately resulting in infertility. To study the human cellular responses to chlamydial infection, researchers have frequently used transformed cell lines that can have limited translational relevance. We developed a primary human fallopian tube epithelial cell model based on a method previously established for culture of primary human bronchial epithelial cells. After protease digestion and physical dissociation of excised fallopian tubes, epithelial cell precursors were expanded in growth factor-containing medium. Expanded cells were cryopreserved to generate a biobank of cells from multiple donors and cultured at an air-liquid interface. Culture conditions stimulated cellular differentiation into polarized mucin-secreting and multiciliated cells, recapitulating the architecture of human fallopian tube epithelium. The polarized and differentiated cells were infected with a clinical isolate of C. trachomatis, and inclusions containing chlamydial developmental forms were visualized by fluorescence and electron microscopy. Apical secretions from infected cells contained increased amounts of proteins associated with chlamydial growth and replication, including transferrin receptor protein 1, the amino acid transporters SLC3A2 and SLC1A5, and the T-cell chemoattractants CXCL10, CXCL11, and RANTES. Flow cytometry revealed that chlamydial infection induced cell surface expression of T-cell homing and activation proteins, including ICAM-1, VCAM-1, HLA class I and II, and interferon gamma receptor. This human fallopian tube epithelial cell culture model is an important tool with translational potential for studying cellular responses to Chlamydia and other sexually transmitted pathogens.

Discovery of Blood Transcriptional Endotypes in Women with Pelvic Inflammatory Disease.

Sexually transmitted infections with Chlamydia trachomatis and/or Neisseria gonorrhoeae and rates of pelvic inflammatory disease (PID) in women continue to rise, with reinfection being common because of poor adaptive immunity. Diagnosis remains imprecise, and pathogenesis data are derived primarily from monoinfection of mice with C. trachomatis or N. gonorrhoeae By comparing blood mRNA responses of women with C. trachomatis- and/or N. gonorrhoeae-induced PID and histologic endometritis with those from women with C. trachomatis and/or N. gonorrhoeae infection limited to their cervix and asymptomatic uninfected women determined via microarray, we discovered important pathogenic mechanisms in PID and response differences that provide a pathway to biomarker discovery. Women with N. gonorrhoeae- and/or C. trachomatis-induced PID exhibit overexpression of myeloid cell genes and suppression of protein synthesis, mitochondrial oxidative phosphorylation, and T cell-specific genes. Coinfected women exhibited the greatest activation of cell death pathways and suppression of responses essential for adaptive immunity. Women solely infected with C. trachomatis expressed elevated levels of type I and type II IFN genes, and enhanced type I IFN-induced chemokines in cervical secretions were associated with ascension of C. trachomatis to the endometrium. Blood microarrays reveal discrete pathobiological endotypes in women with PID that are driven by pathogen invasion of the upper genital tract.

Caspase-11 Contributes to Oviduct Pathology during Genital Chlamydia Infection in Mice.

It has been shown that caspase-1, but not its upstream activator, ASC, contributes to oviduct pathology during mouse genital Chlamydia muridarum infection. We hypothesized that this dichotomy is due to the inadvertent absence of caspase-11 in previously used caspase-1-deficient mice. To address this, we studied the independent contributions of caspase-1 and -11 during genital Chlamydia infection. Our results show that caspase-11 deficiency was sufficient to recapitulate the effect of the combined absence of both caspase-1 and caspase-11 on oviduct pathology. Further, mice that were deficient for both caspase-1 and -11 but that expressed caspase-11 as a transgene (essentially, caspase-1-deficient mice) had no significant difference in oviduct pathology from control mice. Caspase-11-deficient mice showed reduced dilation in both the oviducts and uterus. To determine the mechanism by which caspase-11-deficient mice developed reduced pathology, the chlamydial burden and immune cell infiltration were determined in the oviducts. In the caspase-11-deficient mice, we observed increased chlamydial burdens in the upper genital tract, which correlated with increased CD4 T cell recruitment, suggesting a contribution of caspase-11 in infection control. Additionally, there were significantly fewer neutrophils in the oviducts of caspase-11-deficient mice, supporting the observed decrease in the incidence of oviduct pathology. Therefore, caspase-11 activation contributes to pathogen control and oviduct disease independently of caspase-1 activation.

A Chlamydia-Specific TCR-Transgenic Mouse Demonstrates Th1 Polyfunctionality with Enhanced Effector Function.

Chlamydia is responsible for millions of new infections annually, and current efforts focus on understanding cellular immunity for targeted vaccine development. The Chlamydia-specific CD4 T cell response is characterized by the production of IFN-γ, and polyfunctional Th1 responses are associated with enhanced protection. A major limitation in studying these responses is the paucity of tools available for detection, quantification, and characterization of polyfunctional Ag-specific T cells. We addressed this problem by developing a TCR-transgenic (Tg) mouse with CD4 T cells that respond to a common Ag in Chlamydia muridarum and Chlamydia trachomatis Using an adoptive-transfer approach, we show that naive Tg CD4 T cells become activated, proliferate, migrate to the infected tissue, and acquire a polyfunctional Th1 phenotype in infected mice. Polyfunctional Tg Th1 effectors demonstrated enhanced IFN-γ production compared with polyclonal cells, protected immune-deficient mice against lethality, mediated bacterial clearance, and orchestrated an anamnestic response. Adoptive transfer of Chlamydia-specific CD4 TCR-Tg T cells with polyfunctional capacity offers a powerful approach for analysis of protective effector and memory responses against chlamydial infection and demonstrates that an effective monoclonal CD4 T cell response may successfully guide subunit vaccination strategies.

T Cell-Independent Gamma Interferon and B Cells Cooperate To Prevent Mortality Associated with Disseminated Chlamydia muridarum Genital Tract Infection.

CD4 T cells and antibody are required for optimal acquired immunity to Chlamydia muridarum genital tract infection, and T cell-mediated gamma interferon (IFN-γ) production is necessary to clear infection in the absence of humoral immunity. However, the role of T cell-independent immune responses during primary infection remains unclear. We investigated this question by inoculating wild-type and immune-deficient mice with C. muridarum CM001, a clonal isolate capable of enhanced extragenital replication. Genital inoculation of wild-type mice resulted in transient dissemination to the lungs and spleen that then was rapidly cleared from these organs. However, CM001 genital infection proved lethal for STAT1-/- and IFNG-/- mice, in which IFN-γ signaling was absent, and for Rag1-/- mice, which lacked T and B cells and in which innate IFN-γ signaling was retained. In contrast, B cell-deficient muMT mice, which can generate a Th1 response, and T cell-deficient mice with intact B cell and innate IFN-γ signaling survived. These data collectively indicate that IFN-γ prevents lethal CM001 dissemination in the absence of T cells and suggests a B cell corequirement. Adoptive transfer of convalescent-phase immune serum but not naive IgM to Rag1-/- mice infected with CM001 significantly increased the survival time, while transfer of naive B cells completely rescued Rag1-/- mice from CM001 lethality. Protection was associated with a significant reduction in the lung chlamydial burden of genitally infected mice. These data reveal an important cooperation between T cell-independent B cell responses and innate IFN-γ in chlamydial host defense and suggest that interactions between T cell-independent antibody and IFN-γ are essential for limiting extragenital dissemination.

Differential signaling pathways are initiated in macrophages during infection depending on the intracellular fate of Chlamydia spp.

Chlamydia muridarum and Chlamydia caviae have equivalent growth rates in mouse epithelial cells but only C. muridarum replicates inside mouse macrophages, while C. caviae does not. Macrophages infected with C. muridarum or C. caviae were used to address the hypothesis that the early signaling pathways initiated during infection depend on the fate of chlamydiae in the host cell. Transmission electron microscopy of C. muridarum-infected macrophages showed intact chlamydial elementary bodies and reticulate bodies 2 h postinfection in compact vacuoles. Conversely, in macrophages infected with C. caviae, chlamydiae were observed in large phagocytic vacuoles. Furthermore, C. caviae infections failed to develop into inclusions or produce viable bacteria. Expression of proinflammatory cytokines TNFα, IL-1ß and MMP13 was similar in C. caviae- or C. muridarum-infected macrophages at 3 h postinfection, indicating that chlamydial survival is not required for initiation of these responses. IL-1ß secretion, dependent on inflammasome activation, occurred in C. caviae-infected macrophages despite no chlamydial growth. Conversely, IFNß mRNA was observed only in C. muridarum- but not in C. caviae-infected macrophages. These data demonstrate that differential signaling events are initiated during a productive versus nonproductive chlamydial infection in a macrophage.

The DNA sensor, cyclic GMP-AMP synthase, is essential for induction of IFN-ß during Chlamydia trachomatis infection.

IFN-ß has been implicated as an effector of oviduct pathology resulting from genital chlamydial infection in the mouse model. In this study, we investigated the role of cytosolic DNA and engagement of DNA sensors in IFN-ß expression during chlamydial infection. We determined that three-prime repair exonuclease-1, a host 3' to 5' exonuclease, reduced IFN-ß expression significantly during chlamydial infection using small interfering RNA and gene knockout fibroblasts, implicating cytosolic DNA as a ligand for this response. The DNA sensor cyclic GMP-AMP synthase (cGAS) has been shown to bind cytosolic DNA to generate cyclic GMP-AMP, which binds to the signaling adaptor stimulator of IFN genes (STING) to induce IFN-ß expression. We determined that cGAS is required for IFN-ß expression during chlamydial infection in multiple cell types. Interestingly, although infected cells deficient for STING or cGAS alone failed to induce IFN-ß, coculture of cells depleted for either STING or cGAS rescued IFN-ß expression. These data demonstrate that cyclic GMP-AMP produced in infected cGAS(+)STING(-) cells can migrate into adjacent cells via gap junctions to function in trans in cGAS(-)STING(+) cells. Furthermore, we observed cGAS localized in punctate regions on the cytosolic side of the chlamydial inclusion membrane in association with STING, indicating that chlamydial DNA is most likely recognized outside the inclusion as infection progresses. These novel findings provide evidence that cGAS-mediated DNA sensing directs IFN-ß expression during Chlamydia trachomatis infection and suggest that effectors from infected cells can directly upregulate IFN-ß expression in adjacent uninfected cells during in vivo infection, contributing to pathogenesis.

Guanylate binding proteins enable rapid activation of canonical and noncanonical inflammasomes in Chlamydia-infected macrophages.

Interferon (IFN)-inducible guanylate binding proteins (GBPs) mediate cell-autonomous host resistance to bacterial pathogens and promote inflammasome activation. The prevailing model postulates that these two GBP-controlled activities are directly linked through GBP-dependent vacuolar lysis. It was proposed that the rupture of pathogen-containing vacuoles (PVs) by GBPs destroyed the microbial refuge and simultaneously contaminated the host cell cytosol with microbial activators of inflammasomes. Here, we demonstrate that GBP-mediated host resistance and GBP-mediated inflammatory responses can be uncoupled. We show that PVs formed by the rodent pathogen Chlamydia muridarum, so-called inclusions, remain free of GBPs and that C. muridarum is impervious to GBP-mediated restrictions on bacterial growth. Although GBPs neither bind to C. muridarum inclusions nor restrict C. muridarum growth, we find that GBPs promote inflammasome activation in C. muridarum-infected macrophages. We demonstrate that C. muridarum infections induce GBP-dependent pyroptosis through both caspase-11-dependent noncanonical and caspase-1-dependent canonical inflammasomes. Among canonical inflammasomes, we find that C. muridarum and the human pathogen Chlamydia trachomatis activate not only NLRP3 but also AIM2. Our data show that GBPs support fast-kinetics processing and secretion of interleukin-1ß (IL-1ß) and IL-18 by the NLRP3 inflammasome but are dispensable for the secretion of the same cytokines at later times postinfection. Because IFN-γ fails to induce IL-1ß transcription, GBP-dependent fast-kinetics inflammasome activation can drive the preferential processing of constitutively expressed IL-18 in IFN-γ-primed macrophages in the absence of prior Toll-like receptor stimulation. Together, our results reveal that GBPs control the kinetics of inflammasome activation and thereby shape macrophage responses to Chlamydia infections.

CD4+ T cell expression of MyD88 is essential for normal resolution of Chlamydia muridarum genital tract infection.

Resolution of Chlamydia genital tract infection is delayed in the absence of MyD88. In these studies, we first used bone marrow chimeras to demonstrate a requirement for MyD88 expression by hematopoietic cells in the presence of a wild-type epithelium. Using mixed bone marrow chimeras we then determined that MyD88 expression was specifically required in the adaptive immune compartment. Furthermore, adoptive transfer experiments revealed that CD4(+) T cell expression of MyD88 was necessary for normal resolution of genital tract infection. This requirement was associated with a reduced ability of MyD88(-/-)CD4(+) T cells to accumulate in the draining lymph nodes and genital tract when exposed to the same inflammatory milieu as wild-type CD4(+) T cells. We also demonstrated that the impaired infection control we observed in the absence of MyD88 could not be recapitulated by deficiencies in TLR or IL-1R signaling. In vitro, we detected an increased frequency of apoptotic MyD88(-/-)CD4(+) T cells upon activation in the absence of exogenous ligands for receptors upstream of MyD88. These data reveal an intrinsic requirement for MyD88 in CD4(+) T cells during Chlamydia infection and indicate that the importance of MyD88 extends beyond innate immune responses by directly influencing adaptive immunity.

IL-12 secretion by Langerhans cells stimulated with Candida skin test reagent is mediated by dectin-1 in some healthy individuals.

OBJECTIVE: Our group and others have shown that serial intra-lesional injections of common warts with skin testing reagents such as Candida, mumps and Trichophyton are effective in regressing injected and non-injected warts. Anti-HPV T-cell responses appear to be induced. The goal of this study was to understand the mechanisms of how Candida skin testing reagent enhances immune responses. METHODS: The following immunological features were studied to understand how Candida induces immune responses in healthy subjects: (1) proliferative capacity of T-cells upon exposure to Candida through monocyte-derived human Langerhans cells (LCs) measured using alamarBlue, (2) cytokine (IL-1ß, IL-6, IL-8, IL-10, IL-12p40, IL-23Ap19, IFN-γ, and TNF- expression upon Candida stimulation of LCs by quantitative reverse transcription (qRT)-PCR and cytokine secretion by ELISA, (3) expression of pattern recognition receptors (PRRs) known to associate with Candida albicans (DC-SIGN, dectin-1, dectin-2, galectin-3, mincle, mannose receptor, Toll-like receptors 1, 2, 4, 6, and 9) on LCs by qRT-PCR, (4) role of dectin-1 in IL-12 production by antibody blocking, and (5) induction of Th1, Th2, and/or Th17 responses by intracellular cytokine staining of CD4 cells exposed to Candida pulsed LCs for IFN-γ, IL-4, and IL-17A. RESULTS: T-cell proliferation upon stimulation with Candida-pulsed LCs was significantly higher compared to proliferation in the absence of Candida (p=0.004). The most frequently expressed cytokine in stimulated LCs was IL-12p40 mRNA, and IL-12p40 and IL-12p70 were also detected at protein levels. All other cytokine mRNAs examined were detected in the following order of decreasing frequency: IL23Ap19, IFN-γ, IL-1ß, IL-6, IL-8, and IL-10. LCs expressed all PRRs examined. Anti-dectin-1 inhibited IL-12p40 mRNA production upon Candida stimulation of LCs from some healthy subjects. IFN-γ secretion was increased and IL-4 secretion was decreased in CD4 cells of a few healthy subjects, but IL-17A was essentially unchanged upon Candida treatment. CONCLUSIONS: Proliferation of T-cells in a substantial majority of healthy subjects can be demonstrated with Candida stimulation. We show Th1 promotion and dectin-1 stimulation of LCs as potential mechanisms in some healthy subjects.

Significant role of IL-1 signaling, but limited role of inflammasome activation, in oviduct pathology during Chlamydia muridarum genital infection.

IL-1ß has been implicated in the development of oviduct pathology during Chlamydia muridarum genital infection in the mouse model. The goal of this study was to characterize the role of IL-1 signaling and the inflammasome-activation pathways during genital chlamydial infection. Compared with control mice, IL-1R-deficient mice displayed delayed clearance and increased chlamydial colonization. Consistent with the role for IL-1 signaling in infection clearance, mice deficient for the IL-1R antagonist cleared infection at a faster rate. Despite increased infection, IL-1R-deficient mice had significantly reduced oviduct pathology, which was associated with decreased numbers of neutrophils, but more macrophages, in the genital tract. IL-1ß secretion is dependent on caspase-1 and apoptosis-associated speck-like protein containing caspase recruitment domain (ASC) inflammasome during in vitro infection of primed macrophages with C. muridarum. To investigate the role of inflammasome components during in vivo genital infection, mice lacking NLRP3, NLRC4, and ASC were tested and found to display no reduction in oviduct pathology compared with control mice. Mice deficient for ASC displayed a prolonged course of infection, which was associated with reduced T cell recruitment and proliferation. Further, ASC-deficient mice displayed normal levels of IL-1ß in genital secretions. However, a significant decrease in caspase-1-dependent IL-18 was observed in both ASC- and NLRP3-deficient mice. These data demonstrate a major role for IL-1 signaling, but a limited role for the inflammasome pathway, in IL-1ß secretion and development of oviduct pathology during genital chlamydial infection. The data also suggest an IL-1-independent role for ASC in adaptive immunity during genital chlamydial infection.

Stimulator of IFN gene is critical for induction of IFN-beta during Chlamydia muridarum infection.

Type I IFN signaling has recently been shown to be detrimental to the host during infection with Chlamydia muridarum in both mouse lung and female genital tract. However, the pattern recognition receptor and the signaling pathways involved in chlamydial-induced IFN-beta are unclear. Previous studies have demonstrated no role for TLR4 and a partial role for MyD88 in chlamydial-induced IFN-beta. In this study, we demonstrate that mouse macrophages lacking TLR3, TRIF, TLR7, or TLR9 individually or both TLR4 and MyD88, still induce IFN-beta equivalent to wild type controls, leading to the hypothesis that TLR-independent cytosolic pathogen receptor pathways are crucial for this response. Silencing nucleotide-binding oligomerization domain 1 in HeLa cells partially decreased chlamydial-induced IFN-beta. Independently, small interfering RNA-mediated knockdown of the stimulator of IFN gene (STING) protein in HeLa cells and mouse oviduct epithelial cells significantly decreased IFN-beta mRNA expression, suggesting a critical role for STING in chlamydial-induced IFN-beta induction. Conversely, silencing of mitochondria-associated antiviral signaling proteins and the Rig-I-like receptors, RIG-I, and melanoma differentiation associated protein 5, had no effect. In addition, induction of IFN-beta depended on the downstream transcription IFN regulatory factor 3, and on activation of NF-kappaB and MAPK p38. Finally, STING, an endoplasmic reticulum-resident protein, was found to localize in close proximity to the chlamydial inclusion membrane during infection. These results indicate that C. muridarum induces IFN-beta via stimulation of nucleotide-binding oligomerization domain 1 pathway, and TLR- and Rig-I-like receptor-independent pathways that require STING, culminating in activation of IFN regulatory factor 3, NF-kappaB, and p38 MAPK.

Essential role for neutrophils in pathogenesis and adaptive immunity in Chlamydia caviae ocular infections.

Trachoma, the world's leading cause of preventable blindness, is produced by chronic ocular infection with Chlamydia trachomatis, an obligate intracellular bacterium. While many studies have focused on immune mechanisms for trachoma during chronic stages of infection, less research has targeted immune mechanisms in primary ocular infections, events that could impact chronic responses. The goal of this study was to investigate the function of neutrophils during primary chlamydial ocular infection by using the guinea pig model of Chlamydia caviae inclusion conjunctivitis. We hypothesized that neutrophils help modulate the adaptive response and promote host tissue damage. To test these hypotheses, guinea pigs with primary C. caviae ocular infections were depleted of neutrophils by using rabbit antineutrophil antiserum, and immune responses and immunopathology were evaluated during the first 7 days of infection. Results showed that neutrophil depletion dramatically decreased ocular pathology, both clinically and histologically. The adaptive response was also altered, with increased C. caviae-specific IgA titers in tears and serum and decreased numbers of CD4(+) and CD8(+) T cells in infected conjunctivae. Additionally, there were changes in conjunctival chemokines and cytokines, such as increased expression of IgA-promoting interleukin-5 and anti-inflammatory transforming growth factor ß, along with decreased expression of T cell-recruiting CCL5 (RANTES). This study, the first to investigate the role of neutrophils in primary chlamydial ocular infection, indicates a previously unappreciated role for neutrophils in modulating the adaptive response and suggests a prominent role for neutrophils in chlamydia-associated ocular pathology.

Interferon regulatory transcription factor 3 protects mice from uterine horn pathology during Chlamydia muridarum genital infection.

Mice with the type I interferon (IFN) receptor gene knocked out (IFNAR KO mice) or deficient for alpha/beta IFN (IFN-α/ß) signaling clear chlamydial infection earlier than control mice and develop less oviduct pathology. Initiation of host IFN-ß transcription during an in vitro chlamydial infection requires interferon regulatory transcription factor 3 (IRF3). The goal of the present study was to characterize the influence of IRF3 on chlamydial genital infection and its relationship to IFN-ß expression in the mouse model. IRF3 KO mice were able to resolve infection as well as control mice, overcoming increased chlamydial colonization and tissue burden early during infection. As previously observed for IFNAR KO mice, IRF3 KO mice generated a potent antigen-specific T cell response. However, in contrast to IFNAR KO mice, IRF3 KO mice exhibited unusually severe dilatation and pathology in the uterine horns but normal oviduct pathology after infection. Although IFN-ß expression in vivo was dependent on the presence of IRF3 early in infection (before day 4), the IFN-independent function of IRF3 was likely driving this phenotype. Specifically, early during infection, the number of apoptotic cells and the number of inflammatory cells were significantly less in uterine horns from IRF3 KO mice than in those from control mice, despite an increased chlamydial burden. To delineate the effects of IFN-ß versus IRF3, neutralizing IFN-ß antibody was administered to wild-type (WT) mice during chlamydial infection. IFN-ß depletion in WT mice mimicked that in IFNΑR KO mice but not that in IRF3 KO mice with respect to both chlamydial clearance and reduced oviduct pathology. These data suggest that IRF3 has a role in protection from uterine horn pathology that is independent of its function in IFN-ß expression.

MyD88 deficiency leads to decreased NK cell gamma interferon production and T cell recruitment during Chlamydia muridarum genital tract infection, but a predominant Th1 response and enhanced monocytic inflammation are associated with infection resolution.

We have previously shown that MyD88 knockout (KO) mice exhibit delayed clearance of Chlamydia muridarum genital infection compared to wild-type (WT) mice. A blunted Th1 response and ineffective suppression of the Th2 response were also observed in MyD88 KO mice. The goal of the present study was to investigate specific mechanisms whereby absence of MyD88 leads to these effects and address the compensatory mechanisms in the genital tract that ultimately clear infection in the absence of MyD88. It was observed that NK cells recruited to the genital tract in MyD88 KO mice failed to produce gamma interferon (IFN-γ) mRNA and protein. This defect was associated with decreased local production of interleukin-17 (IL-17), IL-18, and tumor necrosis factor alpha (TNF-α) but normal levels of IL-12p70. Additionally, recruitment of CD4 T cells to the genital tract was reduced in MyD88 KO mice compared to that in WT mice. Although chronic infection in MyD88 KO mice resulted in oviduct pathology comparable to that of WT mice, increased histiocytic inflammation was observed in the uterine horns. This was associated with increased CCL2 levels and recruitment of macrophages as a potential compensatory mechanism. Further deletion of TLR4-TRIF signaling in MyD88 KO mice, using TLR4/MyD88 double-KO mice, did not further compromise host defense against chlamydiae, suggesting that compensatory mechanisms are Toll-like receptor (TLR) independent. Despite some polarization toward a Th2 response, a Th1 response remained predominant in the absence of MyD88, and it provided equivalent protection against a secondary infection as observed in WT mice.

Host-Pathogen Interactions of Chlamydia trachomatis in Porcine Oviduct Epithelial Cells.

Chlamydia trachomatis (Ct) causes the most prevalent bacterial sexually transmitted disease leading to ectopic pregnancy and infertility. Swine not only have many similarities to humans, but they are also susceptible to Ct. Despite these benefits and the ease of access to primary tissue from this food animal, in vitro research in swine has been underutilized. This study will provide basic understanding of the Ct host-pathogen interactions in porcine oviduct epithelial cells (pOECs)-the counterparts of human Fallopian tube epithelial cells. Using NanoString technology, flow cytometry, and confocal and transmission-electron microscopy, we studied the Ct developmental cycle in pOECs, the cellular immune response, and the expression and location of the tight junction protein claudin-4. We show that Ct productively completes its developmental cycle in pOECs and induces an immune response to Ct similar to human cells: Ct mainly induced the upregulation of interferon regulated genes and T-cell attracting chemokines. Furthermore, Ct infection induced an accumulation of claudin-4 in the Ct inclusion with a coinciding reduction of membrane-bound claudin-4. Downstream effects of the reduced membrane-bound claudin-4 expression could potentially include a reduction in tight-junction expression, impaired epithelial barrier function as well as increased susceptibility to co-infections. Thereby, this study justifies the investigation of the effect of Ct on tight junctions and the mucosal epithelial barrier function. Taken together, this study demonstrates that primary pOECs represent an excellent in vitro model for research into Ct pathogenesis, cell biology and immunity.