Immune–Fibrotic Remodeling States in Achalasia Defined by Integrative Physiologic, Histologic, and Transcriptomic Profiling Restricted; Files Only

Abstract

Achalasia is a primary esophageal motility disorder characterized by impaired lower esophageal sphincter (LES) relaxation, absent peristalsis, and progressive esophageal dilation. Although degeneration of the myenteric plexus is well established, the relationship between immune activation, tissue fibrosis, and physiologic remodeling remains incompletely defined. This study sought to define biologically coherent immune–fibrotic states in achalasia through integrative physiologic, histologic, and transcriptomic profiling of LES tissue. Consecutive patients undergoing surgical therapy underwent high-resolution physiologic assessment and quantitative fibrosis scoring. The cohort included 46 patients (mean age 57.8 ± 17.3 years, 52% female, mean Eckardt symptom score 7.31 ± 2.46). Transcriptomic analyses were performed on specimens meeting RNA quality control criteria. Principal component analysis and unsupervised hierarchical clustering of integrated physiologic and fibrosis variables identified four distinct remodeling phenotypes corresponding to structural stages of disease: Stage I (n = 17), Stage II (n = 15), Stage III (n = 8), and Stage IV (n = 6). Fibrosis burden increased across stages (Stage I 1.50 ± 0.76; Stage II 3.87 ± 0.99; Stage III 4.40 ± 0.84; Stage IV 3.00 ± 1.87; Kruskal–Wallis p < 0.001).  Early, low-fibrosis states demonstrated predominant innate immune activation, whereas intermediate states exhibited enrichment of Th1/Th17-associated signaling. High-fibrosis states demonstrated increased Th2-associated pathway activation, while the most structurally advanced phenotype showed relative attenuation of immune signaling despite persistent fibrosis, suggesting transition to fixed remodeling. To establish a mechanistic platform for modeling cytokine-driven phenotype transitions, primary esophageal smooth muscle cells (ESMCs) were isolated from early-stage achalasia tissue and successfully expanded in culture, with smooth muscle identity confirmed by expression of contractile markers. This platform enables ongoing experimental evaluation of inflammatory and profibrotic cytokine effects on phenotype switching and extracellular matrix remodeling. Together, these findings support a stage-linked immune–fibrotic remodeling model of achalasia and provide a translational framework for understanding disease progression and identifying potential stage-specific therapeutic targets.

Table of Contents

Introduction 1

Objectives 5

Methods 6

Results 9

Discussion 14

Conclusions 17

References 18

About this Master's Thesis

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School	Laney Graduate School
Department	Clinical Research
Degree	M.S.
Submission	Master's Thesis
Language	English
Research Field	Biology, Physiology Health Sciences, Immunology Health Sciences, Medicine and Surgery
Keyword	Esophageal motility disorders Immune remodeling Fibrosis Achalasia Smooth muscle biology
Committee Chair / Thesis Advisor	Amita Manatunga, Emory University
Committee Members	Shanthi Srinivasan, Emory University Jordan Kempker, Emory University Raymond Kim, Emory University

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