Anti-TNF Therapies Suppress Adipose Tissue Inflammation in Crohn's Disease

Abstract

Anti-TNF biologics have been shown to markedly improve the quality of life for patients with Crohn's disease (CD), yet one-third of patients fail to benefit from this treatment. Patients with CD develop a characteristic wrapping of visceral adipose tissue (VAT) in the inflamed intestinal area, termed creeping fat, and it is known that adipose tissue expansion influences the efficacy of anti-TNF drugs. We questioned whether anti-TNF therapies impact the creeping fat in CD, which might affect the outcome of the disease. Adipose tissue biopsies were obtained from a cohort of 14 patients with CD that received anti-TNF drugs and from 29 non-anti-TNF-treated patients (control group) matched by sex, age, and body mass index undergoing surgical interventions for symptomatic complications. We found that anti-TNF therapies restored adipose tissue morphology and suppressed immune cell infiltration in the creeping fat. Additionally, anti-TNF treatments appeared to markedly improve the pro-inflammatory phenotype of adipose-tissue macrophages and adipose-tissue-derived stem cells. Our study provides evidence that anti-TNF medications influence immune cells and progenitor cells in the creeping of patients with CD, suppressing inflammation. We propose that perilesional VAT should be considered when administering anti-TNF therapy in patients with CD.

Keywords: TNF; adalimumab; adipose tissue; creeping fat; infliximab.

Figure 1

Anti-TNF therapy remodels adipose tissue, restoring normal adipocyte size and suppressing immune cell infiltration. (A) Representative histological images obtained from randomly selected 304,558-µm² circular sections. Left panel: hematoxylin & eosin (H&E) staining. Central panel: CD68 immunohistochemistry (brown staining). Right panel: double CD68 (magenta stain) and CD3 immunohistochemistry (brown stain). (B) Adipocyte area in the creeping fat of control group and anti-TNF-treated group. (C) Percentage of large-size (>3000 µm²) and small-size (<3000 µm²) adipocytes detected in creeping fat. (D) Number of CD68-positive macrophages per section detected in creeping fat. (E) CD14 protein expression analyzed by Western blotting in creeping fat of control and anti-TNF-treated group. (F) Number of CD3-positive T lymphocytes in the creeping fat of control and anti-TNF-treated group. (G) Ratio of CD68- to CD3-positive cells in creeping fat. Results are shown as mean ± SEM from independent donors’ experiments. * p < 0.05, *** p < 0.001, and **** p < 0.0001 vs. control group.

Figure 2

Anti-TNF treatment suppresses inflammation in different cell subtypes of creeping fat. (A) Gene expression of pro-inflammatory genes (TNFA, IL6, and IL1B) in VAT-derived adipose tissue macrophages (ATMs). (B) Gene expression of antigen-presentation markers in VAT-derived ATMs. (C) Gene expression of the inflammatory cytokines TNFA, IL6, IL1B, CCL2, and IL12B; anti-inflammatory cytokines TGFB1, IL10, and ADIPOQ; the invasion markers MMP2 and MMP9 in the creeping fat of patients treated with anti-TNF or control group. (D) NFKB-p52 protein expression analyzed by Western blotting in the creeping fat of patients treated with anti-TNF or control group (E) Gene expression of the pro-inflammatory cytokines TNFA, IL6, and IL1B in VAT explants isolated from patients treated with anti-TNF or control group. (F) TNFA and IL6 abundance in 24 h conditioned medium of VAT explants from patients treated with anti-TNF or control group. (G) Inflammatory cytokine gene expression in mature adipocytes isolated from patients treated with anti-TNF or control group. Results are shown as mean ± SEM from independent donors’ experiments. * p < 0.05 and ** p < 0.01 vs. control group.

Figure 3

Anti-TNF therapies modify the inflammatory status of creeping fat-derived ASCs. (A) Gene expression of pro-inflammatory genes (TNFA, IL6, IL1B, and CCL2) in adipose-stem cells (ASCs) and (B) TNFA and IL6 abundance in 24 h conditioned medium of ASCs from patients treated with anti-TNF or control group. (C) Gene expression of anti-inflammatory genes (IL10, CGSF, and TGFB1) in ASCs from patients treated with anti-TNF or control group. (D) TGFB1 protein abundance in 24 h conditioned medium of ASCs from patients treated with anti-TNF or control group. (E) Analysis of the ASC protein secretome and protein abundance differences between ASCs isolated from control group (n = 6) and anti-TNF-treated group (n = 3). (F) Gene expression of antigen-presentation markers (HLA-DM and HLA-DPB) and co-stimulatory molecules (CD40, CD74, and CD80) in ASCs from patients treated with anti-TNF or control group. (G) ASCs isolated from patients treated with anti-TNF or control group co-cultured or not with ovalbumin (OVA) were stained with a panel of antibodies and analyzed by flow cytometry to compare the percentage of surface expression of antigen-presenting markers. Results are shown as mean ± SEM from independent donors’ experiments. * p < 0.05 vs. control group. $ p < 0.05 control group without OVA vs. control group with OVA.