Transcriptional and Molecular Pathways Activated in Mesenteric Adipose Tissue and Intestinal Mucosa of Crohn's Disease Patients

Abstract

Crohn's disease (CD) is a chronic inflammatory disorder, characterized by cytokine imbalance and transcription signaling pathways activation. In addition, the increase of mesenteric adipose tissue (MAT) near the affected intestinal area is a hallmark of CD. Therefore, we evaluated the transcription signaling pathways and cytokines expression in intestinal mucosa and MAT of active CD patients. Ten patients with ileocecal CD and eight with noninflammatory diseases were studied. The biopsies of intestinal mucosa and MAT were snap-frozen and protein expression was determined by immunoblotting. RNA levels were measured by qPCR. The pIkB/IkB ratio and TNFα level were significantly higher in intestinal mucosa of CD when compared to controls. However, STAT1 expression was similar between intestinal mucosa of CD and controls. Considering the MAT, the pIkB/IkB ratio was significantly lower and the anti-inflammatory cytokine IL10 was significantly higher in CD when compared to controls. Finally, the protein content of pSTAT1 was higher in MAT of CD compared to controls. These findings reinforce the predominance of the proinflammatory NF-kB pathway in CD intestinal mucosa. For the first time, we showed the activation of STAT1 pathway in MAT of CD patients, which may help to understand the physiopathology of this immune mediated disease.

Figure 1

Haematoxylin and Eosin (H&E) staining of intestinal mucosa and mesenteric adipose tissues segments of a representative Crohn's disease patient. (a) Intestinal mucosa of a normal control subject (IC Group). (b) Intestinal mucosa of CD patient (ICD Group), showing crypt distortion, ulcers, and inflammatory infiltrate. (c) Mesenteric adipose tissue of a normal control subject (AC Group). (d) Mesenteric adipose tissue of CD patient (ACD Group) revealing characteristic morphometric features, with reduced adipocyte size. Nuclear counterstaining: Mayer's haematoxylin. Original magnification ×20.

Figure 2

Evaluation of cytokines gene expressions in the intestinal mucosa of Crohn's disease patients. Inflammatory signaling proteins reveal a proinflammatory pattern in the intestinal mucosa of Crohn's disease patients. mRNA levels (qRT-PCR) of IL6 (a), IL8 (b), IL23A (c), IL10 (d), and TNFα (e), in intestinal mucosa of CD patients (ICD Group) compared to controls (IC Group). Western blot analysis of TNFα (f), IL1β (g), and IL23 (h) in intestinal mucosa of CD patients (ICD Group) compared to controls (IC Group). Each band represents one patient. For ICD, n = 10; for IC, n = 8,  ^∗p < 0.05,  ^∗∗p < 0.01, and ^∗∗∗p < 0.001 versus control. TA: transcription amount. ASU: arbitrary scanning unit.

Figure 3

Evaluation of cytokines gene expressions in the mesenteric adipose tissue of Crohn's disease patients. Inflammatory signaling pattern in the mesenteric adipose tissue of Crohn's disease patients. mRNA levels (qRT-PCR) of TNFα (a), IL6 (b), IL8 (c), IL23A (d), and IL10 (e) in the mesenteric adipose tissue (MAT) of CD patients (ACD Group) compared to controls (AC Group). Western blot analysis of TNFα (f), IL17 (g), IL23 (h), and IL10 (i) in the mesenteric adipose tissue (MAT) of CD patients (ACD Group) compared to controls (AC Group). Each band represents one patient. For ACD, n = 10; for AC, n = 8,  ^∗p < 0.05,  ^∗∗p < 0.01, and ^∗∗∗p < 0.001 versus control. TA: transcription amount. ASU: arbitrary scanning unit.

Figure 4

Intestinal mucosa of Crohn's disease patients shows inflammation driven by NF-KB. Higher SOCS3 protein expression counterbalances the STAT1 activation. Western blot analysis of pIκB/IκB ratio (a), pSTAT1 (b), and SOCS3 (d) in intestinal mucosa of CD patients (ICD Group) compared to controls (IC Group). mRNA levels (qRT-PCR) of STAT1 (c) in intestinal mucosa of CD patients (ICD Group) compared to controls (IC Group). Each band represents one patient. For ICD, n = 10; for IC, n = 8;  ^∗p < 0.05,  ^∗∗p < 0.01, and ^∗∗∗p < 0.001 versus control. ASU: arbitrary scanning unit; TA: transcription amount.

Figure 5

Mesenteric adipose tissue of Crohn's disease patients shows activation of STAT1 transcriptional pathway. Western blot analysis of pIκB/IκB ratio (a) and pSTAT1 (b) in the mesenteric adipose tissue (MAT) of CD patients (ACD Group) compared to controls (AC Group). mRNA levels (qRT-PCR) of STAT1 (c) in MAT of CD patients (ACD Group) when compared to controls (AC Group). Each band represents one patient. For ACD, n = 10; and, for AC, n = 8,  ^∗p < 0.05,  ^∗∗p < 0.01, and ^∗∗∗p < 0.001 versus control. ASU: arbitrary scanning unit; TA: transcription amount.