Modeling the transcriptome of genital tract epithelial cells and macrophages in healthy mucosa versus mucosa inflamed by Chlamydia muridarum infection.

Chlamydia trachomatis urogenital serovars are intracellular bacteria that parasitize human reproductive tract epithelium. As the principal cell type supporting bacterial replication, epithelial cells are central to Chlamydia immunobiology initially as sentries and innate defenders, and subsequently as collaborators in adaptive immunity-mediated bacterial clearance. In asymptomatic individuals who do not seek medical care a decisive struggle between C. trachomatis and host defenses occurs at the epithelial interface. For this study, we modeled the immunobiology of epithelial cells and macrophages lining healthy genital mucosa and inflamed/infected mucosa during the transition from innate to adaptive immunity. Upper reproductive tract epithelial cell line responses were compared to bone marrow-derived macrophages utilizing gene expression microarray technology. Those comparisons showed minor differences in the intrinsic innate defenses of macrophages and epithelial cells. Major lineage-specific differences in immunobiology relate to epithelial collaboration with adaptive immunity including an epithelial requirement for inflammatory cytokines to express MHC class II molecules, and a paucity and imbalance between costimulatory and coinhibitory ligands on epithelial cells that potentially limits sterilizing immunity (replication termination) to Chlamydia-specific T cells activated with limited or unconventional second signals.

An atypical CD8 T-cell response to Chlamydia muridarum genital tract infections includes T cells that produce interleukin-13.

Chlamydia trachomatis urogenital serovars D-K are intracellular bacterial pathogens that replicate almost exclusively in human reproductive tract epithelium. In the C. muridarum mouse model for human Chlamydia genital tract infections CD4 T helper type 1 cell responses mediate protective immunity while CD8 T-cell responses have been associated with scarring and infertility. Scarring mediated by CD8 T cells requires production of tumour necrosis factor-α (TNF-α); however, TNF-α is associated with protective immunity mediated by CD4 T cells. The latter result suggests that TNF-α in-and-of itself may not be the sole determining factor in immunopathology. CD8 T cells mediating immunopathology presumably do something in addition to producing TNF-α that is detrimental during resolution of genital tract infections. To investigate the mechanism underlying CD8 immunopathology we attempted to isolate Chlamydia-specific CD8 T-cell clones from mice that self-cleared genital tract infections. They could not be derived with antigen-pulsed irradiated naive splenocytes; instead derivation required use of irradiated immune splenocyte antigen-presenting cells. The Chlamydia-specific CD8 T-cell clones had relatively low cell surface CD8 levels and the majority were not restricted by MHC class Ia molecules. They did not express Plac8, and had varying abilities to terminate Chlamydia replication in epithelial cells. Two of the five CD8 clones produced interleukin-13 (IL-13) in addition to IL-2, TNF-α, IL-10 and interferon-γ. IL-13-producing Chlamydia-specific CD8 T cells may contribute to immunopathology during C. muridarum genital tract infections based on known roles of TNF-α and IL-13 in scar formation.

Perforin is detrimental to controlling [corrected] C. muridarum replication in vitro, but not in vivo.

CD4 T cells are critical for clearing experimental Chlamydia muridarum genital tract infections. Two independent in vitro CD4 T cell mechanisms have been identified for terminating Chlamydia replication in epithelial cells. One mechanism, requiring IFN-γ and T cell-epithelial cell contact, terminates infection by triggering epithelial production of nitric oxide to chlamydiacidal levels; the second is dependent on T cell degranulation. We recently demonstrated that there are two independent in vivo clearance mechanisms singly sufficient for clearing genital tract infections within six weeks; one dependent on iNOS, the other on Plac8. Redundant genital tract clearance mechanisms bring into question negative results in single-gene knockout mice. Two groups have shown that perforin-knockout mice were not compromised in their ability to clear C. muridarum genital tract infections. Because cell lysis would be detrimental to epithelial nitric oxide production we hypothesized that perforin was not critical for iNOS-dependent clearance, but posited that perforin could play a role in Plac8-dependent clearance. We tested whether the Plac8-dependent clearance was perforin-dependent by pharmacologically inhibiting iNOS in perforin-knockout mice. In vitro we found that perforin was detrimental to iNOS-dependent CD4 T cell termination of Chlamydia replication in epithelial cells. In vivo, unexpectedly, clearance in perforin knockout mice was delayed to the end of week 7 regardless of iNOS status. The discordant in vitro/in vivo results suggest that the perforin's contribution to bacterial clearance in vivo is not though enhancing CD4 T cell termination of Chlamydia replication in epithelial cells, but likely via a mechanism independent of T cell-epithelial cell interactions.

PmpG303-311, a protective vaccine epitope that elicits persistent cellular immune responses in Chlamydia muridarum-immune mice.

Urogenital Chlamydia serovars replicating in reproductive epithelium pose a unique challenge to host immunity and vaccine development. Previous studies have shown that CD4 T cells are necessary and sufficient to clear primary Chlamydia muridarum genital tract infections in the mouse model, making a protective CD4 T cell response a logical endpoint for vaccine development. Our previous proteomics studies identified 13 candidate Chlamydia proteins for subunit vaccines. Of those, PmpG-1 is the most promising vaccine candidate. To further that work, we derived a PmpG(303-311)-specific multifunctional Th1 T cell clone, designated PmpG1.1, from an immune C57BL/6 mouse and used it to investigate the presentation of the PmpG(303-311) epitope by infected epithelial cells. Epithelial presentation of the PmpG(303-311) epitope required bacterial replication, occurred 15 to 18 h postinfection, and was unaffected by gamma interferon (IFN-γ) pretreatment. Unlike epitopes recognized by other Chlamydia-specific CD4 T cell clones, the PmpG(303-311) epitope persisted on splenic antigen-presenting cells (APC) of mice that cleared primary genital tract infections. PmpG1.1 was activated by unmanipulated irradiated splenocytes from immune mice without addition of exogenous Chlamydia antigen, and remarkably, activation of PmpG1.1 by unmanipulated immune splenocytes was stronger 6 months postinfection than it was 3 weeks postinfection. Enhanced presentation of PmpG(303-311) epitope on splenic APC 6 months postinfection reflects some type of "consolidation" of a protective immune response. Understanding the antigen-presenting cell populations responsible for presenting PmpG(303-311) early (3 weeks) and late (6 months) postinfection will likely provide important insights into stable protective immunity against Chlamydia infections of the genital tract.

Plac8-dependent and inducible NO synthase-dependent mechanisms clear Chlamydia muridarum infections from the genital tract.

Chlamydia trachomatis urogenital serovars replicate predominantly in genital tract epithelium. This tissue tropism poses a unique challenge for host defense and vaccine development. Studies utilizing the Chlamydia muridarum mouse model have shown that CD4 T cells are critical for clearing genital tract infections. In vitro studies have shown that CD4 T cells terminate infection by upregulating epithelial inducible NO synthase (iNOS) transcription and NO production. However, this mechanism is not critical, as iNOS-deficient mice clear infections normally. We recently showed that a subset of Chlamydia-specific CD4 T cell clones could terminate replication in epithelial cells using an iNOS-independent mechanism requiring T cell degranulation. We advance that work using microarrays to compare iNOS-dependent and iNOS-independent CD4 T cell clones. Plac8 was differentially expressed by clones having the iNOS-independent mechanism. Plac8-deficient mice had delayed clearance of infection, and Plac8-deficient mice treated with the iNOS inhibitor N-monomethyl-l-arginine were largely unable to resolve genital tract infections over 8 wk. These results demonstrate that there are two independent and redundant T cell mechanisms for clearing C. muridarum genital tract infections: one dependent on iNOS, and the other dependent on Plac8. Although T cell subsets are routinely defined by cytokine profiles, there may be important subdivisions by effector function, in this case CD4(Plac8).

Chlamydia muridarum-specific CD4 T-cell clones recognize infected reproductive tract epithelial cells in an interferon-dependent fashion.

During natural infections Chlamydia trachomatis urogenital serovars replicate predominantly in the epithelial cells lining the reproductive tract. This tissue tropism poses a unique challenge to host cellar immunity and future vaccine development. In the experimental mouse model, CD4 T cells are necessary and sufficient to clear Chlamydia muridarum genital tract infections. This implies that resolution of genital tract infection depends on CD4 T-cell interactions with infected epithelial cells. However, no laboratory has shown that Chlamydia-specific CD4 T cells can recognize Chlamydia antigens presented by major histocompatibility complex class II (MHC-I) molecules on epithelial cells. In this report we show that MHC-II-restricted Chlamydia-specific CD4 T-cell clones recognize infected upper reproductive tract epithelial cells as early as 12 h postinfection. The timing of recognition and degree of T-cell activation are dependent on the interferon (IFN) milieu. Beta IFN (IFN-beta) and IFN-gamma have different effects on T-cell activation, with IFN-beta blunting IFN-gamma-induced upregulation of epithelial cell surface MHC-II and T-cell activation. Individual CD4 T-cell clones differed in their degrees of dependence on IFN-gamma-regulated MHC-II for controlling Chlamydia replication in epithelial cells in vitro. We discuss our data as they relate to published studies with IFN knockout mice, proposing a straightforward interpretation of the existing literature based on CD4 T-cell interactions with the infected reproductive tract epithelium.

Chlamydia muridarum infection elicits a beta interferon response in murine oviduct epithelial cells dependent on interferon regulatory factor 3 and TRIF.

Chlamydia trachomatis is the most common sexually transmitted bacterial infection in the United States. Utilizing cloned murine oviduct epithelial cell lines, we previously identified Toll-like receptor 2 (TLR2) as the principal epithelial pattern recognition receptor (PRR) for infection-triggered release of the acute inflammatory cytokines interleukin-6 and granulocyte-macrophage colony-stimulating factor. The infected oviduct epithelial cell lines also secreted the immunomodulatory cytokine beta interferon (IFN-beta) in a largely MyD88-independent manner. Although TLR3 was the only IFN-beta production-capable TLR expressed by the oviduct cell lines, we were not able to determine whether TLR3 was responsible for IFN-beta production because the epithelial cells were unresponsive to the TLR3 ligand poly(I-C), and small interfering RNA (siRNA) techniques were ineffective at knocking down TLR3 expression. To further investigate the potential role of TLR3 in the infected epithelial cell secretion of IFN-beta, we examined the roles of its downstream signaling molecules TRIF and IFN regulatory factor 3 (IRF-3) using a dominant-negative TRIF molecule and siRNA specific for TRIF and IRF-3. Antagonism of either IRF-3 or TRIF signaling significantly decreased IFN-beta production. These data implicate TLR3, or an unknown PRR utilizing TRIF, as the source of IFN-beta production by Chlamydia-infected oviduct epithelial cells.

Pattern recognition molecules activated by Chlamydia muridarum infection of cloned murine oviduct epithelial cell lines.

Chlamydia trachomatis is the most common bacterial sexually transmitted disease in the United States and a major cause of female infertility due to infection-induced Fallopian tube scarring. Epithelial cells are likely central to host defense and pathophysiology as they are the principal cell type productively infected by C. trachomatis. We generated cloned murine oviduct epithelial cell lines without viral or chemical transformation to investigate the role of the TLRs and cytosolic nucleotide binding site/leucine-rich repeat proteins Nod1 and Nod2 in epithelial responses to Chlamydia muridarum infection. RT-PCR assays detected mRNA for TLR2 (TLRs 1 and 6), TLR3, and TLR5. No mRNA was detected for TLRs 4, 7, 8, and 9. Messenger RNAs for Nod1 and Nod2 were present in the epithelial cell lines. Oviduct epithelial cell lines infected with C. muridarum or exposed to the TLR2 agonist peptidoglycan secreted representative acute phase cytokines IL-6 and GM-CSF in a MyD88-dependent fashion. Infected epithelial cell lines secreted the immunomodulatory cytokine IFN-beta, even though C. muridarum does not have a clear pathogen-associated molecular pattern (PAMP) for triggering IFN-beta transcription. The oviduct epithelial lines did not secrete IFN-beta in response to the TLR2 agonist peptidoglycan or to the TLR3 agonist poly(I:C). Our data identify TLR2 as the principal TLR responsible for secretion of acute phase cytokines by C. muridarum-infected oviduct epithelial cell lines. The pattern recognition molecule responsible for infection-induced IFN-beta secretion by oviduct epithelial cells remains to be determined.

Parameters associated with cloning in Actinobacillus actinomycetemcomitans.

Characterization of virulence traits in Actinobacillus actinomycetemcomitans requires the application of recombinant DNA techniques. To develop appropriate genetic tools it is necessary to identify suitable host-vector systems. The current study assessed cloning parameters in A. actinomycetemcomitans for two previously described vectors, pDMG4 and pMMB67. It was determined that the maximum size of recombinant molecules that could be transferred to A. actinomycetemcomitans strain ATCC29522 via electroporation was 33 kb. The size limit for transformation of the same strain with ligation mixtures (direct cloning), however, was limited to 23-24 kb. Additional experiments included electroporation of various A. actinomycetemcomitans strains with plasmid DNA isolated from Escherichia coli and different A. actinomycetemcomitans sources. Differences in transformation efficiencies suggested the presence of a restriction modification system for pDMG4 in some strains of A. actinomycetemcomitans. Cloning of portions of the enterococcal plasmid pJH1 into A. actinomycetemcomitans resulted in the insertion of the intact vector into the chromosome.