Determining the Major Genetic Regulators of Mycobacterium tuberculosis-induced Cytokine Expression in Monocytes and Macrophages with a Cellular GWAS

Abstract

Tuberculosis (TB), largely due to infection with the causative pathogen Mycobacterium tuberculosis (Mtb), has claimed over 1 billion lives in the past 200 years, including 1.3 million lives in 2022 alone. Efforts to eradicate TB have been complicated by heterogeneity in infection outcomes after Mtb exposure. Host genetics may contribute as much as half of an individual’s susceptibility to certain infection outcomes, however, the causal variants responsible remain unknown. 70,000 years of human infection has led to intricate pathogen and host adaptations that fight for control of crucial infection responses. In particular, the macrophage cytokine response is heavily modulated by both pathogen and host and requires a fine-tuned balance for successful control of infection. Prior studies attempting to define genetic regulators of Mtb infection have often focused on genome-wide association studies (GWAS) measuring clinical phenotypes. Acellular GWAS, which measures genetic regulation of the cytokine response to live Mtb infection, in vitro, may identify important biology, and has not been assessed previously. This dissertation aimed to assess if Mtb-induced human monocyte and macrophage cytokine responses are genetically controlled and if a cellular GWAS approach can be used to identify genetic regulators that are important in modulating immune and clinical TB response outcomes. Using Mtb-induced cytokine expression profiles from individuals in a Ugandan cohort, we performed WGCNA, heritability, and cellular GWAS analyses. These findings were further assessed in independent cohorts from South Africa and Seattle, and in subsequent in vitro validation and mechanistic investigation. Initial expression profiling identified 35 hub genes which were central to the Mtb-induced cytokine response, and determined a heritable component of Mtb-induced IL1B and IL6 expression. Our cellular GWAS in Ugandan individuals identified several SNPs surpassing suggestive significance and a significant enrichment of Mtb-induced TNF association in the genes of two pathways. Further investigation revealed that one of these pathways, alpha-linolenic acid metabolism, was validated in vitro using PLA2 inhibitors. We further identified multiple SNPs which showed population spanning effect when assessed in the Seattle population and validated cytokine effect for two of these SNPs which mapped to the genes SLIT3 and SLC1A1. SLIT3 was additionally found to enhance Mtb intracellular replication. Finally, SNPs from both of our in vitro validated loci, associated with SLC1A1 and SLIT3, were associated with the clinical TB phenotypes, susceptibility to tuberculous meningitis (TBM), and TBM survival, respectively. Taken together, this research identified multiple genetic regulators of the Mtb-induced myeloid cell cytokine response which were validated in vitro, and shows the promise of the cellular GWAS approach for identifying novel host response factors with clinical relevance.