Investigating The Roles of Gasdermin B in Epithelial Cell Inflammation and Repair

Abstract

The Gasdermins (GSDMs) are a family of proteins consisting of GSDMA, GSDMB, GSDMC, GSDMD, GSDME, and GSDMF. They are mainly expressed in epithelial and immune cells and are involved in an inflammatory form of cell death known as pyroptosis. GSDMB is the least studied of the GSDMs, partially due to its lack of mouse ortholog. Its roles in pyroptosis and inflammation have been controversial. GSDMB polymorphism markers are found to be associated with asthma severity and exacerbations. GSDMB is overexpressed in various breast and gastric cancers, promoting poor prognosis and increasing therapy resistance. However, GSDMB has also shown to be beneficial as it was found to stimulate epithelial cell restitution and repair. In this study, A GSDMB overexpression (OE) model was established using plasmid expressing human GSDMB. A GSDMB knockout (KO) model created using the CRISPR/Cas9 system in the epithelial cell line HBEC3-KT was also investigated. TNFα, lipopolysaccharide (LPS), poly I:C, calcitriol, and CD14 were used to stimulate inflammation in the cell lines. Inflammation markers Interleukin (IL)-6 and IL-8 release were measured with enzyme-linked immunosorbent assay (ELISA) at 0, 4, 10, and 24 hours after treatment. It was found that TNFα, poly I:C, and a combination of LPS, calcitriol, and CD14 are the most effective inflammatory stimulators. IL-8 levels did not show a significant change between wildtype (WT), OE, and KO within the same time point group, but did show a significance 24 hours after treatment. GSDMB KO released more IL-6 following TNFα and poly I:C stimulation (p<0.05 respectively). Expression of inflammasome associated genes NLRP3, AIM2, NOD2, Caspase 5, and GZMB were measured using RT-qPCR 24 hours following treatment with TNFα. AIM2 showed to express significantly higher in the OE group compared to WT group (p<0.01) before treatment. Following TNFα treatment, AIM2 levels significantly 5 decreased in the OE group (p<0.05). NOD2 expression was significantly higher in the KO group comparing to the WT and OE group (p<0.001, p<0.01 respectively). After treatment, NOD2 levels decreased in KO group (p<0.01). Caspase 5 showed a significant decrease across all samples compared with the WT (p<0.001). NLRP3, on the other hand, significantly increased in just KO compared to all other samples (p<0.001). As results are not consistent, further investigation is required to investigate the discrepancies across the different genes. Scratch assay was used to assess for GSDMB’s wound healing properties by imaging the cells over 48 hours following scratching in untreated and TNFα treated cells in WT and KO cells. GSDMB was found to induce wound healing in epithelial cells, with WT cells showing more efficient wound healing when treated with TNFα compared to KO, indicating both GSDMB and TNFα to be involved in the wound healing process. The results of this study warrant further investigations of GSDMB roles in pyroptosis and inflammation in the epithelium. Further investigation will provide new insights into inflammatory diseases and cancers, potentially allowing for the development of novel therapeutic GSDMB targets for the diseases.