Inflammatory bowel diseases (IBD) are diseases of intestinal epithelial cell (IEC) death and altered barrier function. Bacteria have been extensively implicated in IBD through studies showing that the gut microbial community is altered in IBD and that bacteria are necessary for development of colitis in murine models. A role for bacteria in IBD is also supported by IBD-associated genetic polymorphisms in bacterial sensing and response genes. However, the specific mechanisms whereby bacteria contribute to IBD remain relatively unknown.Background:
High mobility group box 1 (HMGB1) is a multifunctional protein whose intracellular expression is decreased in IEC from active IBD lesions. During microbial stress, intracellular HMGB1 regulates the cellular autophagy/apoptosis checkpoint to promote cell survival. These factors suggest that altered regulation of HMGB1 during microbial stress could contribute to the pathophysiology of IBD. Therefore, the aims of this study were to identify HMGB1-dependent microbial stress response pathways that are altered in IBD and to develop novel therapeutic strategies targeting these pathways to improve IEC survival and promote mucosal healing.Methods:
This study utilized primary IEC (organoids) derived from wild type mice (HMGB1f/f) or mice conditionally deficient in IEC HMGB1 (HMGB1f/f, Vil-CRE) and primary IEC derived from human intestinal biopsies. Human IEC were used in an assay for HMGB1 production in response to bacterial ligands. Mouse IEC were used in standard assays of cellular response to microbial components and a high-throughput screen to identify approved drugs with the ability to promote survival of HMGB1-deficient cells during microbial stress.Results:
Intracellular HMGB1 was increased in mouse IEC in response to muramyl dipeptide (MDP), lipopolysaccharide (LPS), flagellin, lipoteichoic acid, and CpG-DNA. Intracellular HMGB1 was also increased in MDP-treated IEC isolated from human intestinal biopsies. HMGB1 deficient IEC underwent higher levels of cell death in response to MDP or LPS than wild type cells, as evidenced by caspase-3 activation on immunoblots. Likewise, ATP production in response to MDP treatment was lower in HMGB1-deficient cells versus controls. The Akt/PI3K signaling pathway is activated by MDP and involved in survival and energy production during cellular stress. Akt phosphorylation at Ser 473 in response to MDP was diminished in HMGB1 deficient cells versus controls. High-throughput screening utilizing HMGB1 deficient mouse organoids and the Prestwick chemical library of 1280 approved drugs was then performed. Drugs that increased ATP production by at least 25% over controls were considered “hits.” Sixteen drugs satisfied screening criteria and are considered potential novel therapeutics for IBD.Conclusions:
HMGB1 is a key regulator of cell survival pathways, including those required for energy production, during microbial stress. Decreased HMGB1 expression in IEC from active IBD lesions likely contributes to cell death and mucosal damage through loss of its regulatory effects on apoptosis and energy production. Therefore, identification of drugs that improve cell survival and ATP production during microbial stress in the absence of HMGB1 has the potential to uncover novel therapeutic agents with the ability to promote mucosal healing during IBD.