Regulation of Macrophage Phenotype in Crohn's Disease Hypertrophied Adipose Tissue

Abstract

Background: Mutations within the gene loci, encoding protein tyrosine phosphatase nonreceptor type 2 (PTPN2) and nucleotide oligomerization domain 2 (NOD2), have been associated with Crohn's disease (CD). We have recently shown that PTPN2 limits proinflammatory effects in human intestinal epithelial cells (IEC) and monocytes. A dysregulated immune response to bacterial antigens, such as the NOD2-ligand, muramyl-dipeptide (MDP), has been strongly implicated in the pathogenesis of CD. The transcription factor T-bet is upregulated in NOD2-deficient mice, activated during CD and controls the secretion of proinflammatory cytokines, such as IFNγ. Here, we studied whether PTPN2 controls NOD2mediated effects in human THP-1 monocytes. Methods: Protein analysis was performed by Western blotting, mRNA analysis by real-time PCR, cytokine secretion by ELISA and visual imaging by immunofluorescence (IF) studies. PTPN2 or NOD2 knock-down was induced by siRNA. Primary intestinal colonic lamina propria fibroblasts (CLPF) were isolated from surgical specimens derived from patients with active CD (n=5) and were genotyped for the CD-associated PTPN2 variant. Results: MDP (100 ng/ml) increased PTPN2 mRNA (p<0.05; n=3) and protein expression (p<0.05; n=3) by 24 h treatment. This effect was absent in NOD2 knock-down cells (n=3). Levels of the transcription factor T-bet (p<0.01) were also enhanced by MDP stimulation and this effect was potentiated by PTPN2 knock-down (p<0.05; n=3). While MDP treatment induced secretion of TNF, IL-8 and IFNγ (p<0.01; n=3 each) in THP-1 cell supernatants, knock-down of PTPN2 by siRNA resulted in diminished secretion of all three cytokines in response to MDP (p<0.01; n=3 each). We next studied whether PTPN2 regulates NOD2-mediated autophagy. PTPN2 knock-down prevented the MDP-induced expression of microtubule-associated protein 1 light-chain 3 B-II (LC3B-II), a marker of autophagy activity, in THP-1 cells (n=3; p<0.05). Though MDP was able to increase PTPN2 and LC3B-II protein levels in PTPN2 wild-type (WT) CLPF (n=3), in CLPF carrying the CD-associated PTPN2 variant (n=2), MDP was unable to increase PTPN2 or LC3B-II expression. By IF studies we found elevated numbers of LC3B+ vesicles, indicating autophagosome assembly, in MDP-treated PTPN2-WT cells. However, in PTPN2-variant cells LC3B+ vesicles were less numerous, but larger in size, when compared to PTPN2WT cells, suggesting that defective autophagosome formation occurred. Conclusions: We demonstrate that PTPN2 is regulated by MDP via a NOD2-dependent mechanism and controls NOD2-mediated cytokine secretion and autophagy. These findings demonstrate for the first time a functional interaction between the two CD candidate genes, PTPN2 and NOD2, and suggest that the CD-associated PTPN2 variant could favour a dysregulated immune response to bacterial antigens.