Abstract
Background
GB004 is a small molecule that stabilizes hypoxia inducible factor (HIF-1α), a key transcription factor involved in the cellular responses at the intersection of hypoxia and inflammation. GB004 induction of HIF-1a target genes improves barrier integrity and reduces barrier permeability in rodent models of colitis. Here we investigated gene expression and secretion of soluble factors elicited by GB004 treatment on immune cells and epithelial cells, human monolayer assays and immune co-culture studies to further explore its protective mechanism.
Methods
In the BioMAP human co-culture assay, cells were treated with GB004 one hour prior to stimulation and incubated for 24–168 hours. Biomarkers, cell viability and proliferation were assessed. Repligut human stem-cell-derived monolayer platforms were used to assess GB004 effects under unstimulated or TNFa stimulation conditions. Barrier integrity was assessed by Transepithelial Endothelial Electric Resistance (TEER), HIF-1a target genes were assessed in cell lysates, and tight junction formation and epithelial monolayer viability were investigated by immunofluorescence staining.
Results
GB004 demonstrated activity in 11 of 12 systems in the BioMAP human co-culture assay, which covers a range of disease-relevant immune and non-immune mechanisms under stimulation conditions. Collectively, GB004 treatment led to changes in biomarkers associated with inflammation (sTNFa, IL-8, MCP-1, CXCL11), immunomodulation (sIL-17F, sIL-17A), and tissue remodeling (collagen I, collagen IV). GB004 treatment also reduced the proliferative activity in the in vitro lymphocyte assay systems. In the human Repligut intestinal epithelium assay, GB004 significantly reduced cell death and improved barrier integrity in response to pro-inflammatory cytokines. The protective role of GB004 on barrier integrity was further supported by immunostaining experiments demonstrating that GB004 treatment protected epithelial cells from TNFa-induced cell apoptosis, improving both epithelial cell number and tight junction protein ZO-1 staining.
Conclusion
GB004 modulates key anti-inflammatory and immunomodulatory activities in human co-culture systems. Furthermore, GB004 demonstrates protective effects on human derived monolayer cultures by reducing cell death, promoting tight junction formation, and improving barrier integrity. Targeting both barrier function and local colonic inflammation represents a multi-faceted approach to treatment of inflammatory bowel disease. A phase 2 clinical study of GB004 is ongoing in patients with ulcerative colitis (NCT04556383). Sponsored by GB004, Inc., a wholly owned subsidiary of Gossamer Bio, Inc.
Anionic surfactants and commercial detergents decrease tight junction barrier integrity in human keratinocytes
