James D. Sikes

Early Local Cytokine Profiles in Strains of Mice with Different Outcomes from Chlamydial Genital Tract Infection

ABSTRACT In this study, we expand on the examination of genetically determined differences in host responses that correlate with clearance of Chlamydia trachomatis from the genital tract. We infected C57BL/6, BALB/c, and C3H/HeN mice with the mouse pneumonitis agent of C. trachomatis (MoPn). C57BL/6 mice had the shortest course of infection (22 days) and the lowest incidence of severe hydrosalpinx. BALB/c mice also had a short course of infection (25 days), but all developed hydrosalpinx. C3H/HeN mice had the longest course of infection (38 days), and all developed severe hydrosalpinx. Determination of local cytokine responses by enzyme-linked immunosorbent assay (ELISA) of genital tract secretions revealed that the levels of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β) were significantly increased in the C57BL/6 and BALB/c strains compared to those in the C3H/HeN strain whereas the level of IL-6 was not different. The level of the neutrophil chemokine macrophage inflammatory protein 2 (MIP-2) was increased during the first week of infection in all three strains but was significantly higher in the BALB/c strain, the strain with the most rapid influx of neutrophils into the genital tract. Prolonged detection of MIP-2 in C3H/HeN mice was associated with a protracted presence of neutrophils in the genital tract. Early increases in the levels of the proinflammatory cytokines TNF-α and IL-1β are associated with earlier eradication of infection in the C57BL/6 and BALB/c strains than in the C3H/HeN strain. Increased levels of MIP-2 and neutrophils in BALB/c and C3H/HeN mice relative to C57BL/6 mice suggest that these responses may contribute to pathology.

Type I Interferon Signaling Exacerbates Chlamydia muridarum Genital Infection in a Murine Model

ABSTRACT Type I interferons (IFNs) induced during in vitro chlamydial infection exert bactericidal and immunomodulatory functions. To determine the precise role of type I IFNs during in vivo chlamydial genital infection, we examined the course and outcome of Chlamydia muridarum genital infection in mice genetically deficient in the receptor for type I IFNs (IFNAR−/− mice). A significant reduction in chlamydial shedding and duration of lower genital tract infection was observed in IFNAR−/− mice in comparison to the level of chlamydial shedding and duration of infection in wild-type (WT) mice. Furthermore, IFNAR−/− mice developed less chronic oviduct pathology in comparison to that in WT mice. Compared to the WT, IFNAR−/− mice had a greater number of chlamydial-specific T cells in their iliac lymph nodes 21 days postinfection. IFNAR−/− mice also exhibited earlier and enhanced CD4 T-cell recruitment to the cervical tissues, which was associated with increased expression of CXCL9 in the genital secretions of IFNAR−/− mice, but not with expression of CXCL10, which was reduced in the genital secretions of IFNAR−/− mice. These data suggest that type I IFNs exacerbate C. muridarum genital infection through an inhibition of the chlamydial-specific CD4 T-cell response.

Critical Role for Interleukin-1β (IL-1β) during Chlamydia muridarum Genital Infection and Bacterial Replication-Independent Secretion of IL-1β in Mouse Macrophages

ABSTRACT Recent findings have implicated interleukin-1β (IL-1β) as an important mediator of the inflammatory response in the female genital tract during chlamydial infection. But how IL-1β is produced and its specific role in infection and pathology are unclear. Therefore, our goal was to determine the functional consequences and cellular sources of IL-1β expression during a chlamydial genital infection. In the present study, IL-1β−/− mice exhibited delayed chlamydial clearance and decreased frequency of hydrosalpinx compared to wild-type (WT) mice, implying an important role for IL-1β both in the clearance of infection and in the mediation of oviduct pathology. At the peak of IL-1β secretion in WT mice, the major producers of IL-1β in vivo are F4/80+ macrophages and GR-1+ neutrophils, but not CD45− epithelial cells. Although elicited mouse macrophages infected with Chlamydiamuridarum in vitro secrete minimal IL-1β, in vitro prestimulation of macrophages by Toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS) purified from Escherichiacoli or C. trachomatis L2 prior to infection greatly enhanced secretion of IL-1β from these cells. By using LPS-primed macrophages as a model system, it was determined that IL-1β secretion was dependent on caspase-1, potassium efflux, and the activity of serine proteases. Significantly, chlamydia-induced IL-1β secretion in macrophages required bacterial viability but not growth. Our findings demonstrate that IL-1β secreted by macrophages and neutrophils has important effects in vivo during chlamydial infection. Additionally, prestimulation of macrophages by chlamydial TLR ligands may account for the elevated levels of pro-IL-1β mRNA observed in vivo in this cell type.

Mouse Strain-Dependent Chemokine Regulation of the Genital Tract T Helper Cell Type 1 Immune Response

ABSTRACT Vaginal infection with the mouse pneumonitis agent ofChlamydia trachomatis (MoPn) produces shorter courses of infection in C57BL/6 and BALB/c mice than in C3H/HeN mice, while C57BL/6 mice are more resistant to oviduct pathology. A robust Th1 response is extremely important in host defense against chlamydia. In this study we examined gamma interferon (IFN-γ), interleukin 10 (IL-10), and the T-cell-regulatory chemokines macrophage inflammatory protein-1α (MIP-1α) and monocyte chemoattractant protein-1 (MCP-1) to determine if differences in these responses were associated with the differential courses of infection seen in these three strains of mice. Increased and prolonged IFN-γ responses and lower IL-10 responses were observed in the C57BL/6 strain compared to BALB/c and C3H. Examination of genital tract chemokines revealed a marked predominance of MIP-1α over MCP-1 only in the C57 strain. Thus, a pattern of high MIP-1α and low MCP-1 levels during the first week of infection is associated with an increased Th1 response and a shorter, more benign chlamydial infection. Inhibition of the MCP-1 response in C3H mice increased their later T-cell production of IFN-γ but decreased their early IFN-γ response and had no effect on the course or outcome of infection. Inhibition of MCP-1 is not beneficial in chlamydial infection because of its pleiotropic effects.

Significant Role of IL-1 Signaling, but Limited Role of Inflammasome Activation, in Oviduct Pathology during Chlamydia muridarum Genital Infection

IL-1β 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-1β 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-1β 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-1β 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.

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

ABSTRACT 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 Chemokine and Cytokine Response in the Endocervix within the First Developmental Cycle of Chlamydia muridarum

ABSTRACT The initial host response in a primary chlamydial infection is the onset of acute inflammation. However, we still know very little about the early temporal events in the induction of the acute inflammatory response and how these events relate to the initial chlamydial developmental cycle in an actual genital infection. Because it was critical to initiate a synchronous infection in the endocervix in the first 24 h to evaluate the sequential expression of the host response, we developed the surgical methodology of depositing Chlamydia muridarum directly on the endocervix. Cervical tissue was collected at 3, 12, and 24 h after inoculation and the expression array of chemokines, cytokines, and receptors was assessed to characterize the response during the initial developmental cycle. Polymorphonuclear leukocyte (PMN) infiltration was first observed at 12 h after inoculation, and a few PMNs could be seen in the epithelium at 24 h. Electron microscopic analysis at 24 h showed that virtually all inclusions were at the same stage of development, indicating a synchronous infection. Several chemokine and cytokine genes were expressed as early as 3 h after infection, but by 12 h, 41 genes were expressed. Thus, activation of the host response occurs both with the introduction of elementary bodies into the host and early replication of reticulate bodies. No significant response was observed when UV-inactivated organisms were inoculated into the cervix at any time interval. This model provides an ideal opportunity to investigate the mechanisms by which the early inflammatory response is induced in vivo.

Interferon Regulatory Transcription Factor 3 Protects Mice from Uterine Horn Pathology during Chlamydia muridarum Genital Infection

ABSTRACT Mice with the type I interferon (IFN) receptor gene knocked out (IFNAR KO mice) or deficient for alpha/beta IFN (IFN-α/β) signaling clear chlamydial infection earlier than control mice and develop less oviduct pathology. Initiation of host IFN-β 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-β 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-β 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-β versus IRF3, neutralizing IFN-β antibody was administered to wild-type (WT) mice during chlamydial infection. IFN-β 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-β expression.

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-1β, 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-1β, 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.

THE RESOLUTION OF CHLAMYDIAL GENITAL TRACT INFECTION IS INDEPENDENT OF TLR2 AND TLR4 BUT PARTIALLY DEPENDENT ON MYD88.: 336

Chlamydia trachomatis ligands are known to stimulate both TLR2 and TLR4. Our prior studies of chlamydial genital tract infection in the mouse model have revealed that the development of pathology is dependent on intact TLR2-mediated responses; however, in the absence of TLR2, normal resolution of infection occurred. Normal resolution of infection was also seen in mice deficient for TLR4. This suggests that the generation of a normal adaptive immune response to chlamydial infection may occur via multiple alternative or complementary pathogen recognition receptor (PRR) pathways. Purpose Our current studies were undertaken to examine the potential redundancy in the TLR2 and TLR4 pathways for the generation of adaptive immunity to chlamydial infection. Methods We examined the course of primary chlamydial infection in wild-type, MyD88 KO, TLR2/TLR4 double-knockout (DKO) and TLR4/MyD88 DKO mice after intravaginal inoculation. We evaluated cytokines, T-cell responses, and antibody responses after infection. Summary of Results The course of infection in TLR2/TLR4 DKO was similar to that of wild-type mice, with a moderate reduction in cytokine responses. In contrast, in both MyD88 KO and TLR4/MyD88 DKO mice, resolution of infection was significantly delayed with delayed or absent proinflammatory cytokine responses. In vitro proliferation of antigen-stimulated iliac node CD4 T cells was equal in wild-type and MyD88 KO mice but absent in TLR4/MyD88 DKOs. Chlamydia-specific antibody responses in MyD88 KO, TLR2/TLR4, and TLR4/MyD88 DKO mice were equal to wild-type responses. Conclusions Normal resolution infection in TLR2/TLR4 DKOs suggests that alternative PRR pathways drive the response that promotes clearance of infection. Our findings also demonstrate that there is a TLR4-dependent/MyD88-independent pathway, likely the TLR4-Trif pathway, that can drive CD4 T-cell proliferation in response to chlamydial infection. However, this response is not necessary for infection clearance, given the ability of TLR4/MyD88 DKO to resolve infection. An intact antibody response, shown here to be independent of TLR4, TLR2, and MyD88 pathways, is adequate to resolve chlamydial infection in the presence of innate immune cells despite an absence of CD4 T-cell activation.