Transcriptome profile and immune infiltrated landscape revealed a novel role of γδT cells in mediating pyroptosis in celiac disease

Celiac disease (CeD) is a primary malabsorption syndrome with no specific therapy, which greatly affects the quality of life. Since the pathogenesis of CeD remains riddled, based on multiple transcriptome profiles, this study aimed to establish an immune interaction network and elucidated new mechanisms involved in the pathogenesis of CeD, providing potentially new evidence for the diagnosis and treatment of CeD.Three microarray and three RNA sequencing datasets of human duodenal tissue with or without CeD were included in Gene Expression Omnibus and respectively merged into derivation and validation cohorts. Differential expression gene and functional enrichment analysis were developed, then pyroptosis enrichment score (PES) model was established to quantify pyroptosis levels. Immune infiltration and co-expression network were constructed based on Xcell database. Protein-protein interaction and weighted gene co-expression network analysis were determined to identify pyroptosis relative hub genes, whose predictive efficiency were tested using a least absolute shrinkage and selection operator (LASSO) regression model. CeD animal and in vitro cell line models were established to verify the occurrence of pyroptosis and molecules expression employing immunofluorescence, western blotting, cell counting kit-8 assay and enzyme-linked immunosorbent assay. Analysis of single-cell RNAseq (scRNAseq) was performed using "Seurat" R package.Differentially expressed genes (DEGs) (137) were identified in derivation cohort whose function was mainly enriched in interferon response and suppression of metabolism. Since an enrichment of pyroptosis pathway in CeD was unexpectedly discovered, a PES model with high efficiency was constructed and verified with two external databases, which confirmed that pyroptosis was significantly upregulated in CeD epithelia. γδT cells exhibited high expression of IFN-γ were the most relevant cells associated with pyroptosis and occupied a greater weight in the LASSO predictive model of CeD. An accumulation of GSDMD expressed in epithelia was identified using scRNAseq, while animal model and in vitro experiments confirmed that epithelium cells were induced to become "pre-pyroptotic" status via IFN-γ/IRF1/GSDMD axis. Furthermore, gluten intake triggered pyroptosis via caspase-1/GSDMD/IL-1β pathway.Our study demonstrated that pyroptosis was involved in the pathogenesis of CeD, and elucidated the novel role of γδT cells in mediating epithelial cell pyroptosis.