Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Genome-wide CRISPR knockout screen in Mycobacterium tuberculosis infected macrophages to identify regulators of TNF and iNOS expression

ABSTRACT: To elucidate the early regulators of inflammation during Mycobacterium tuberculosis infection, we performed a genome-wide CRISPR knockout screen in macrophages to identify genes that influence the induction of TNF and iNOS upon infection. A genome-wide knockout library in murine macrophages was infected with Mycobacterium tuberculosis and 24 hours post infection cells were FACS sorted based on TNF and iNOS expression. Samples were collected from the unsorted library, as well as two sorted populations: TNF positive and iNOS negative, and TNF positive and iNOS positive.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Mus musculus

SUBMITTER: Allison Roberts

PROVIDER: E-MTAB-15739 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

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Genome-wide CRISPR knockout screen in Mycobacterium tuberculosis infected macrophages to identify regulators of TNF and iNOS expression

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