Association of circulating cytokine levels and tissue-infiltrating myeloid cells with achalasia: results from Mendelian randomization and validation through clinical characteristics and single-cell RNA sequencing

Achalasia is a rare motility disorder of the esophagus often accompanied by immune dysregulation, yet specific underlying mechanisms remain poorly understood.

We utilized Mendelian randomization (MR) to explore the causal effects of cytokine levels on achalasia, with cis-expression/protein quantitative trait loci (cis-eQTLs/pQTLs) for 47 cytokines selected from a genome-wide association study (GWAS) meta-analysis and GWAS data for achalasia obtained from FinnGen. For cytokines significantly linked to achalasia, we analyzed their plasma concentrations and expression differences in the lower esophageal sphincter (LES) using enzyme-linked immunosorbent assay and single-cell RNA sequencing (scRNA-seq) profiling, respectively. We further employed bioinformatics approaches to investigate underlying mechanisms.

We revealed positive associations of circulating Eotaxin, macrophage inflammatory protein-1b (MIP1b), soluble E-selectin (SeSelectin) and TNF-related apoptosis-inducing ligand (TRAIL) with achalasia. When combining MR findings with scRNA-seq data, we observed upregulation of TRAIL (OR = 2.70, 95% CI, 1.20–6.07), encoded by TNFSF10, in monocytes and downregulation of interleukin-1 receptor antagonist (IL-1ra) (OR = 0.70, 95% CI 0.59–0.84), encoded by IL1RN, in FOS_macrophages in achalasia. TNFSF10^high monocytes in achalasia displayed activated type I interferon signaling, and IL1RN^low FOS_macrophages exhibited increased intercellular communications with various lymphocytes, together shaping the proinflammatory microenvironment of achalasia.

We identified circulating Eotaxin, MIP1b, SeSelectin and TRAIL as potential drug targets for achalasia. TNFSF10^high monocytes and IL1RN^low macrophages may play a role in the pathogenesis of achalasia.