Backgrounds: Creeping fat, a hallmark of Crohn's disease (CD), is intimately associated with intestinal fibrosis, and the formation of strictures. Histology shows that extracellular matrix (ECM) remodeling exists in the area of creeping fat. However, the pathophysiological mechanisms underlying its development and subsequent impact on intestinal fibrosis remain largely elusive. Our study aims to unravel the role of ECM remodeling in creeping fat dysfunction, thereby enhancing our understanding of the pathogenesis of Crohn's disease.

Methods: We analyzed the average area of collagen in the mesenteric adipose tissue (MAT) from 27 CD patients using Image J. RNA-seq was conducted on 12 pairs of MAT samples from CD patients, encompassing creeping fat surrounding the strictured (CF) and relatively normal (MAT) parts of the intestine for each patient. Differentially expressed gene (DEG) analysis and functional pathway analyses were carried out on these samples. Single-cell sequencing analysis of MAT from both CD patients and healthy controls has revealed the pivotal cells involved in the fibrosis of creeping fat.

Results: The collagen area fraction in the CF was significantly larger than that in the MAT (p＜0.001) of relatively normal areas and it exhibited a positive correlation with the severity of intestinal inflammation and fibrosis (Figure 1). Differentially expressed gene (DEG) analysis and functional pathway analyses of RNA Seq highlighted a substantial upregulation of the B-cell chemokine CXCL13 in creeping fat, along with a marked activation of B-cell signaling. This was further validated by flow cytometry (p=0.0463) and immunohistochemistry (Figure 2). The spatial co-localization of B-cells with a-SMA was observed. Single-cell analysis of the ECM score revealed that adipose mesenchymal stem cells and progenitor cells were the primary contributors to ECM production in mesenteric fat.

Conclusions: Our study illuminates the substantial pathophysiological implications of CF fibrosis in the context of CD. Besides, B-cells might play a crucial role in the fibrosis process occurring within creeping fat. Future in vitro/vivo experiments will be performed to validate the function of B cells in the pathogenesis of creeping fat fibrosis.

$Figure 1: Increased collagen area fraction in creeping fat A. Schematic representation of collagen area scoring. B. Increased collagen area fraction in creeping fat tissue compared to relatively normal parts. C. Positive correlation between collagen area fraction in mesenteric adipose tissue and intestinal inflammation score. D. Positive correlation between collagen area fraction in mesenteric adipose tissue and intestinal fibrosis score.$

Figure 1: Increased collagen area fraction in creeping fat
A. Schematic representation of collagen area scoring.
B. Increased collagen area fraction in creeping fat tissue compared to relatively normal parts.
C. Positive correlation between collagen area fraction in mesenteric adipose tissue and intestinal inflammation score.
D. Positive correlation between collagen area fraction in mesenteric adipose tissue and intestinal fibrosis score.

<b>Figure 2: Increased B cell signaling in creeping fat</b><br /> A, B. Flow cytometry data shows a significant increase in the proportion of B cells in creeping fat compared to relatively normal MAT.<br /> C. Immunohistochemistry shows a significant increase in the number of B cells in creeping fat compared to normal MAT.

Figure 2: Increased B cell signaling in creeping fat
A, B. Flow cytometry data shows a significant increase in the proportion of B cells in creeping fat compared to relatively normal MAT.
C. Immunohistochemistry shows a significant increase in the number of B cells in creeping fat compared to normal MAT.

Jie Li

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