Macrophages with ITIH4 overexpression attenuate inflammatory responses and regulate intestinal epithelial cells

Inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) is an acute phage protein and secretory protein that is highly related to inflammation. However, the expression profile and function of ITIH4 in macrophage-mediated inflammation are still unclear.

Quantitative proteomics based on mass spectrometry was performed to explore differential protein expression in bone marrow-derived macrophages (BMDMs) subjected to the indicated treatments. Western blot and qRT-PCR assays were performed to confirm the expression of ITIH4 in macrophages. ITIH4 was overexpressed and knocked down in Raw264.7 cells, and ITIH4 was knocked down in THP-1 cells, after which the function and molecular mechanism of ITIH4 in macrophages were investigated. Immunohistochemical staining and online bioinformatics tools were used to explore the expression of ITIH4 in the inflamed colon. In addition, CCK8, scratch wound healing, RNA-seq, qRT-PCR and Western blot assays were performed to explore the function and mechanism of macrophages with ITIH4 overexpression in colonic epithelial cells.

Our findings demonstrated that the expression of full-length ITIH4 increased in pro-inflammatory macrophages resulting from a decrease in cleavage, whereas treatment with 4-octyl itaconate (OI) promoted the synthesis and secretion of full-length ITIH4. We overexpressed or knocked down ITIH4 in macrophages, subsequently revealed that ITIH4 modulated anti-inflammatory responses via the NF-κB signaling pathway in macrophages. Moreover, macrophages with ITIH4 overexpression activated cAMP signaling pathway to enhance the viability and tight junction integrity of colonic epithelial cells.

This study suggests that ITIH4 plays crucial roles in the inflammatory response of macrophages and may be an important molecule that regulates macrophage-epithelium interactions. Our findings shed new light into the molecular mechanism underlying macrophage drives intestinal inflammation and highlight ITIH4 as a therapeutic target for mitigating macrophages-induced intestinal damage.