The role of BRD4 in the innate immune response to gastrointestinal infection

Modi, Nikita

Permalink

https://hdl.handle.net/2142/121236 Copy

Description

Title

The role of BRD4 in the innate immune response to gastrointestinal infection

Author(s)

Modi, Nikita

Issue Date

2023-07-14

Director of Research (if dissertation) or Advisor (if thesis)

Chen, Lin-Feng

Doctoral Committee Chair(s)

Chen, Lin-Feng

Committee Member(s)

• Prasanth, Kannanganattu V
• Kalsotra, Auinash
• Zhang, Kai

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• BRD4
• iNOS
• H. pylori
• Salmonella
• Gastrointestinal infections

Abstract

The innate immune response is the first line of defense in response to infection by various cellular responses regulated at different levels, including transcription and epigenetic modulation through chromatin remodeling. BRD4, a member of the bromodomain and extra terminal (BET) proteins, contains two highly conserved bromodomain subunits that bind to acetylated histone and non- histone proteins to promote gene transcription. Recent studies using conditional knockout models or BET inhibitors have demonstrated the critical role of BRD4 in innate immune responses by regulating the inflammatory gene expression in macrophages through transcription, enhancer, and super-enhancer formation. My thesis study focuses on elucidating the role of BRD4 in the innate immune response during gastrointestinal infection. H. pylori is a gram-negative bacterial pathogen with a unique ability to colonize in the harsh, acidic environment of the stomach. Approximately half of the world’s population is infected with H. pylori, a long-term infection significantly increases the risk of developing peptic ulcer disease and gastric adenocarcinomas. Both epithelial cells and macrophages contribute to the host innate immune response against infection for disease progression. Previous studies have demonstrated the role of BRD4 in gastric inflammation, gastric cancer progression, and metastasis by affecting the gastric epithelial cells. However, the role of BRD4 in macrophage-mediated innate immune response in response to H. pylori infection remains unclear. In this work, we found that H. pylori infection induced a shift to a BRD4-dependent pro-inflammatory (M1) phenotype in macrophages. The M1 macrophage phenotype included an increase in the expression of cytokines and production of nitric oxide and a shift to glycolysis. Transcriptome analysis revealed the central role of BRD4 in HIF-1a pathway gene expression. Glycolysis-related genes, Slc2a1 and Hk2, ii showed a defect in expression in Brd4-deficient BMDMs. We also found that HIF-1a and BRD4 colocalized at the promoter of Slc2a1 and Hk2, and inhibition of HIF-1a lead to reduced BRD4 recruitment, indicating a HIF-1a-dependent regulation of Slc2a1 and Hk2 expression by BRD4. Importantly, this BRD4-regulated HIF-1a-dependent glycolysis regulated the stability of Nos2 (gene for inducible nitric oxide synthase enzyme, iNOS) mRNA transcript and the production of NO, a free radical produced through the oxidation of L-arginine by iNOS. The NO-mediated killing of H. pylori in vitro was BRD4- and glycolysis-dependent. Consistently, The Brd4-CKO mice also showed a defect in its ability to kill H. pylori, with increased colonization of H. pylori in the stomachs of mice, indicating that iNOS is involved in the killing of H. pylori in vivo. Our results identified the crucial role of BRD4 in the innate immune response to H. pylori infection as a transcription regulator of glycolysis, which further post-transcriptionally regulates Nos2 transcript to combat H. pylori infection. Salmonella enterica serovar Typhimurium (S. Typhimurium), a gram-negative enteropathogen, is the leading cause of non-typhoidal salmonellosis (NTS) infections, such as gastroenteritis. Innate immune cells, including macrophages and innate lymphoid cells (ILCs), are essential for the immune response against infection as well as the pathogenesis of Salmonella infection. We recently showed that BRD4 is a critical regulator of IL-1b production in macrophages through the NLRC4 inflammasome activation in S. typhi infection. Recent studies also indicate that macrophages can activate innate lymphoid cells type 3 ILCs (ILC3s) via IL-1b in response to infection. In an effort to elucidate the role of macrophage BRD4 in ILC3 activation in response to S. Typhi infection, we found that WT mice showed an increase in colonization and inflammation in the cecum and colon post infection in comparison to Brd4-CKO mice in Streptomycin-pre-treated mice infected with Salmonella. Additionally, increased activation of iii IL22+ RORgt+ ILC3 cells were found in the colon of WT mice comparing to Brd4-CKO mice. Recent studies have shown that Salmonella can exploit ILC3-mediated production of IL-22 and production of antimicrobial peptides, such as RegIIIb and RegIIIg, for its colonization. Reg3b is known to affect the commensal microbiota to promote Salmonella colonization. Consistently, we found a BRD4-dependent increase in the RegIIIb and RegIIIg with a decrease in Bacteroides spp., which is responsible for pathogen clearance, in the colon of WT mice comparing to Brd4-CKO mice upon Salmonella infection. Another mechanism for Salmonella colonization is the exploitation of gut inflammation by utilizing nitrate released by inflammatory cells as electron acceptors for anaerobic respiration and growth in the lumen of intestine. We found a BRD4- dependent increase in iNOS expression in the cecum of WT mice promoted gut colonization. However, a nitrate respiration-deficient mutant of Salmonella, DnapA S. Typhi, failed to colonize in the gut of WT mice. Collectively, these data identify a critical role of Brd4 in the innate immune response upon Salmonella infection by regulating IL-1b-dependent recruitment and activation of ILC3s and the release of nitrate. These multi-layer regulations contribute to the severity of Salmonella-induced colitis in mice and can be explored as a target for future therapeutics. In addition to the pro-inflammatory cytokines, BRD4 can also regulate innate immune response via non-coding RNAs, such as miRNAs and enhancer RNAs. We found that BRD4, together with NF-kB, a key transcription factor in immune gene expression, regulated the expression of lincRNA-Cox2 upon H. pylori infection in macrophages. Knockout of lincRNA-Cox2 in macrophages decreased H. pylori-induced expression of Ptgs2, a gene involved in the production of cyclooxygenase 2 (COX-2), and prostaglandin (PGE2), a potent inflammatory mediator that is generated by COX2. Conversely, overexpression of lincRNA-Cox2 increased H. pylori-induced Ptgs2 and PGE2 expression in macrophages. These results indicate a potential novel iv mechanism by which BRD4 regulates inflammatory response via lincRNA-Cox2. Future study will focus on how BRD4-regulated lincRNA-Cox2 controls the expression of Ptgs2 and PGE2 in macrophages in response to H. pylori infection. In summary, BRD4 modulates innate immune response through transcription and post- transcriptional mechanisms. BRD4 binds to transcription factors, such as NF-kB and HIF-1a, via its bromodomains to activate immune gene transcription and macrophage functions. In response to invading pathogens, BRD4 promotes pro-inflammatory cytokine production, coupled with the activation of glycolysis and nitric oxide production in macrophages, allowing for the clearance of H. pylori infection. Interestingly, several pathogenic bacteria like Salmonella have developed mechanisms to exploit nitric oxide for colonization. Additionally, BRD4-mediated macrophage activation can recruit and activate ILC3s to produce antibacterial peptides like RegIIIb and RegIIIg and to disrupt the normal microbiome, prompting colonization of Salmonella. Therefore, the outcomes of BRD4-mediated innate immune response might vary in response to different pathogens.

Graduation Semester

2023-08

Type of Resource

Thesis

Copyright and License Information

Copyright 2023 Nikita Modi

Owning Collections