SRA

Allergic conjunctivitis is a chronic inflammatory disease that is characterized by severe itch in the conjunctiva; but how neuro-immune interactions shape the pathogenesis of severe itch remains unclear. We identified a subset of memory-type pathogenic Th2 cells that preferentially expressed Il1rl1-encoding ST2 and Calca-encoding calcitonin gene-related peptide (CGRP) in the inflammatory conjunctiva using a single-cell analysis. The IL-33-ST2 axis in memory Th2 cells controlled the axonal elongation of the peripheral sensory C-fiber and the induction of severe itch. Pharmacological blockade and genetic deletion of CGRP signaling in vivo attenuated scratching behavior. The analysis of giant papillae from patients with severe allergic conjunctivitis revealed ectopic lymphoid structure formation with the accumulation of IL-33-producing epithelial cells and CGRP-producing pathogenic CD4+ T cells accompanied by peripheral nerve elongation. Thus, the IL-33-ST2-CGRP axis directs severe itch with neuro-reconstruction in the inflammatory conjunctiva and is a potential therapeutic target for severe itch in allergic conjunctivitis. Overall design: White blood cells of CD45+CD31- fractions that were freshly prepared from mice conjunctival tissue and sorted using a BD FACS Aria III (10,000 cells each, cell viability >98%) were encapsulated into droplets, and libraries were prepared using Chromium Single Cell 3' Reagent Kits v3 according to manufacturer's protocol (10X Genomics). The generated scRNA-seq libraries were sequenced using a 128 cycle (paired-end reads) with a NovaSeq 6000 (Illumina). Neuron Cells of CD45-CD31- and live cell+ fractions that were freshly prepared from trigeminal nerve tissue of mice and sorted using a BD FACS Aria III (10,000 cells each, cell viability >98%) were encapsulated into droplets, and libraries were prepared using Chromium Single Cell 3' Reagent Kits v3 according to manufacturer's protocol (10X Genomics). The generated scRNA-seq libraries were sequenced using a 128 cycle (paired-end reads) with a NovaSeq 6000 (Illumina). White blood cells of CD45+ and CD4+ fractions that were freshly prepared from Giant papillae from patients and sorted using a BD FACS Aria III (10,000 cells each, cell viability >98%) were encapsulated into droplets, and libraries were prepared using Chromium Single Cell 3' Reagent Kits v3 according to manufacturer's protocol (10X Genomics). The generated scRNA-seq libraries were sequenced using a 128 cycle (paired-end reads) with a NovaSeq 6000 (Illumina).