Project description:Comparison of two Chlamydia-specific CD4 T cells that are dependent on iNOS to terminate Chlamydia replication in epithelial cells to two Chlamydia-specific CD4 T cells that are iNOS-independent: Chlamydia trachomatis urogenital serovars replicate predominately in epithelial cells lining the reproductive tract. This tissue tropism poses a unique challenge for the host immune system and vaccine development. Studies utilizing the Chlamydia muridarum mouse model have shown that CD4 T cells are critical and sufficient to clear primary genital tract infections. In vitro studies have shown that CD4 T cells terminate the infection in epithelial cells by up regulating epithelial iNOS transcription and nitric oxide production via IFN-gamma and T cell-epithelial cell interactions mediated by LFA-1-ICAM-1. This mechanism however is not critical as iNOS-deficient mice clear infections normally, and IFN-gamma deficient mice clear 99.9% of the infection with near normal kinetics. We recently showed that a subset of Chlamydia-specific CD4 T cell clones were able to terminate replication in epithelial cells using a mechanism that was independent of iNOS and IFN-gamma. That mechanism did not require physical lysis of infected cells, but instead required T cell degranulation. In this study we advanced that work using gene expression microarrays to compare CD4 T cell clones that are able to terminate epithelial replication via an iNOS-independent mechanism to iNOS-dependent CD4 T cell clones. Micro array experiments showed that Plac8 was differentially expressed by the T cell clones having the iNOS-independent mechanism. Plac8-deficient mice had significantly delayed clearance of C. muridarum genital tract infections, and that the large majority of Plac8-deficient mice treated with the iNOS-inhibitor N-monomethyl-L-arginine (MLA) were unable to resolve a C. muridarum genital tract infection over 8 weeks. These results demonstrate that there are two independent and redundant T cell mechanisms for clearing C. muridarum genital tract infections; one mechanism dependent on iNOS, the other mechanism dependent on Plac8. While T cells subsets have been defined by cytokine profiles, there are important subdivisions by effector functions, in this case CD4Plac8. Gene expression study using 4 experimental groups with 4 replicates each.

Project description:C57BL/6J mice were 105-fold more resistant to Chlamydia psittaci infection than DBA/2J mice by LD100 determinations. Linkage analysis using BXD recombinant inbred strains revealed a single effector locus at a 1.5 Mbp region on chromosome 11 encoding a cluster of three p47GTPases (Irgb10, Igtp, and Iigp2). Western blots of infected tissue showed that Irgb10 was elevated in resistant mice and one of the two possible Iigp2 protein isoforms was preferentially expressed in susceptible mice. The BXD39 strain, susceptible at Irgb10 and resistant at Iigp2, had an intermediate phenotype, implicating the non-redundant role of these p47GTPases. C57BL/6J and DBA/2J exhibited a difference in IFNg dependent chlamydial control, which was reversible by Iigp2 siRNA knockdown. Microarrays of infected peritoneal lavage revealed >10 fold up regulation of neutrophil recruiting chemokines in susceptible mice and >100 fold increase in macrophage differentiation genes in resistant mice, indicating that susceptibility pattern involves stimulation of different inflammatory cell recruiting pathways. Massive neutrophil recruitment was seen in susceptible mice by histology and flow cytometry, and neutrophil chemokine receptor (CXCR2) knockout mice on a susceptible background survived lethal challenge confirming that neutrophil recruitment was required for susceptibility. Congenic Igtp knockout mice also susceptible at Irgb10 and Iigp2 on a resistant background recruited neutrophils and succumbed to infection. We conclude that Irgb10 and Iigp2 act together to confer differential susceptibility against murine chlamydial infection. Results indicate that these p47GTPases have cell autonomous effects, which results in vastly different inflammatory stimulation leading to either recovery or death. Experiment Overall Design: C57BL/6J and DBA2J mice (4 mice each) were infected I.p. with 10E4 IFU of Chlamydia psittaci then peritoneal lavage was collected on day 3 post infection. Cells were centrifuged then treated with Trizol for total RNA extraction.

Project description:Comparison of two Chlamydia-specific CD4 T cells that are dependent on iNOS to terminate Chlamydia replication in epithelial cells to two Chlamydia-specific CD4 T cells that are iNOS-independent: Chlamydia trachomatis urogenital serovars replicate predominately in epithelial cells lining the reproductive tract. This tissue tropism poses a unique challenge for the host immune system and vaccine development. Studies utilizing the Chlamydia muridarum mouse model have shown that CD4 T cells are critical and sufficient to clear primary genital tract infections. In vitro studies have shown that CD4 T cells terminate the infection in epithelial cells by up regulating epithelial iNOS transcription and nitric oxide production via IFN-gamma and T cell-epithelial cell interactions mediated by LFA-1-ICAM-1. This mechanism however is not critical as iNOS-deficient mice clear infections normally, and IFN-gamma deficient mice clear 99.9% of the infection with near normal kinetics. We recently showed that a subset of Chlamydia-specific CD4 T cell clones were able to terminate replication in epithelial cells using a mechanism that was independent of iNOS and IFN-gamma. That mechanism did not require physical lysis of infected cells, but instead required T cell degranulation. In this study we advanced that work using gene expression microarrays to compare CD4 T cell clones that are able to terminate epithelial replication via an iNOS-independent mechanism to iNOS-dependent CD4 T cell clones. Micro array experiments showed that Plac8 was differentially expressed by the T cell clones having the iNOS-independent mechanism. Plac8-deficient mice had significantly delayed clearance of C. muridarum genital tract infections, and that the large majority of Plac8-deficient mice treated with the iNOS-inhibitor N-monomethyl-L-arginine (MLA) were unable to resolve a C. muridarum genital tract infection over 8 weeks. These results demonstrate that there are two independent and redundant T cell mechanisms for clearing C. muridarum genital tract infections; one mechanism dependent on iNOS, the other mechanism dependent on Plac8. While T cells subsets have been defined by cytokine profiles, there are important subdivisions by effector functions, in this case CD4Plac8.

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