AZD6738 reverses the LPS-induced corneal inflammatory transcriptional program through a conserved transcription factor network converging on NF-κB
TL;DR
AZD6738 reverses a substantial part of the LPS-induced corneal inflammatory transcriptome and suppresses an inflammatory TF program whose regulons are conserved in human bacterial keratitis, supporting ATR inhibition as an adjunct to antimicrobial therapy in bacterial keratitis.
Abstract
Vision loss in bacterial keratitis arises primarily from the host inflammatory response rather than the infection itself, and antimicrobial resistance narrows the benefit achievable with antibiotics alone. AZD6738, an inhibitor of ataxia telangiectasia and Rad3-related kinase (ATR), suppresses lipopolysaccharide (LPS)- induced corneal inflammation in mice, but the transcriptional basis of this effect is uncharacterized. Mice received intrastromal LPS injection and were assigned to control (CTL), LPS and AZD6738-treated (ATRi) groups ( n = 3 each). Corneas harvested at 48 h were analyzed by RNA sequencing with DESeq2-based differential expression, KEGG enrichment, regression-based reversal quantification and TRRUST v2 regulon gene set enrichment analysis (GSEA). Cross-species conservation was assessed in a human bacterial keratitis transcriptome (GSE241715; 4 cases, 7 controls). In RAW264.7 macrophages, p65-3 × Flag overexpression with LPS stimulation tested the effect of AZD6738 on NF-κB-driven transcription and Ser536 phosphorylation; tolerability was assessed in HCE-T corneal epithelial cells. LPS upregulated 1,622 and downregulated 140 corneal genes (padj <0.05, |log 2 FC| > 1), enriched in 80 immune-related pathways. Regression of the treatment effect on the LPS effect indicated reversal of approximately 57% of LPS-induced changes, and 51.2% of LPS-upregulated genes were significantly reversed. Regulon analysis defined an inflammatory transcription factor (TF) program spanning NF-κB, IRF, STAT, AP-1 and myeloid families; cross-species normalized enrichment score (NES) values were rank-correlated (Spearman ρ = 0.62, n = 214, P = 3.4 × 10 −24 ). NFKB1, RELA, SPI1 and STAT1 were significant in both species, and AZD6738 shifted their regulon activity to negative values (NES −2.81 to −2.56). In p65-overexpressing RAW264.7 cells, AZD6738 reduced NF-κB-driven transcription and Ser536 phosphorylation. AZD6738 reverses a substantial part of the LPS-induced corneal inflammatory transcriptome and suppresses an inflammatory TF program whose regulons are conserved in human bacterial keratitis, supporting ATR inhibition as an adjunct to antimicrobial therapy in bacterial keratitis.