Electronic Thesis and Dissertation Repository

Degree

Master of Science

Program

Microbiology and Immunology

Supervisor

Dr. Sung O. Kim

Abstract

Anthrax is a lethal infectious disease caused by the bacterium Bacillus anthracis. B. anthracis secretes the virulence factor anthrax lethal toxin (LeTx), which causes rapid cell death known as pyroptosis and immune suppression in macrophages. Strikingly, RAW 264.7 macrophages pre-exposed to sub-lethal doses of LeTx become refractory to subsequent high cytolytic doses. The phenomenon is termed toxin-induced resistance (TIR). TIR is in part linked to the down-regulation of three mitochondrial death genes, BCL2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3), BNIP3-like (BNIP3L), and metastatic lymph node 64 (MLN64) protein, as well as the up-regulation of a gene-silencing epigenetic regulator, histone deacetylase (HDAC) 8. Interestingly, I found that inhibiting HDAC8 with the HDAC8-specific inhibitor PCI-34051 in RAW 264.7 TIR cells sensitized them to LeTx-induced pyroptosis. Furthermore, resistance to LeTx-induced pyroptosis is likely mediated by HDAC8-dependent H3K27Ac deacetylation in the regulatory regions of the mitochondrial death genes, BNIP3 and MLN64. Although, sub-lethal doses of LeTx induced resistance to pyroptosis in RAW 264.7 macrophages, LeTx still caused immune suppression. Here, I found that RAW 264.7 LeTx-treated macrophages and RAW 264.7 macrophages over-expressing HDAC8 failed to produce pro-interleukin (IL)-1β, a pro-inflammatory cytokine, in response to the Gram-negative bacterial cell wall component lipopolysaccharide (LPS), whereas inhibiting HDAC8 with PCI-34051 restored IL-1β and tumor necrosis factor (TNF)-α production in response to LPS. Furthermore, chromatin immunoprecipitation (ChIP)- quantitative real-time polymerase chain reaction (qPCR) analysis revealed that inhibiting HDAC8 in the presence of LeTx increased H3K27Ac association with the genomic regions of IL-1β in response to LPS. Collectively, these results suggest that HDAC8 plays a key role in resistance to LeTx-induced pyroptosis and LeTx-induced immunosuppression, through targeting the H3K27Ac-associated regions of BNIP3, MLN64 and IL-1β.


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