CCR2⁺ Monocyte-Derived Ly6C^-F4/80⁺ Fibrocytes Inhibit Collagen Degradation and Contribute to the Development of Colon Fibrosis Via Production of TIMP-1

Introduction: Fibrocytes are derived from a subset of monocytes and express mesenchymal markers such as collagen type I (Col I) and hematopoietic markers such as CD45 and CD11b. They also express several chemokine receptors such as CC chemokine receptor (CCR)-1, CCR2, CCR5, CCR7, and CXC chemokine receptor type 4 (CXCR4). They circulate in the peripheral blood (PB) and can be isolated from many fibrotic tissues. Fibrocytes participate in both physiological wound healing and various pathological fibrosis including myelofibrosis, hypertrophic scar, systemic sclerosis, idiopathic pulmonary fibrosis, liver cirrhosis, and progressive kidney disease. In murine and human colitis, fibrocytes are also reported to be associated with the colon fibrosis. It has been described that migration of fibrocytes to the injured sites involves CC chemokine ligand 2 (CCL2)/CCR2 axis in the liver and kidney and CXC chemokine ligand 12 (CXCL12)/CXCR4 axis in the lung. However, there are few reports concerning the role of fibrocytes and their expression of chemokine receptors related to the induction of colon fibrosis.

Methods: We generated bone marrow (BM) chimeric mice by transplantation of BM total-nucleated cells, which were isolated from enhanced green fluorescent protein (EGFP)-transgenic mice or CCR2 knockout (KO) mice, into lethally irradiated C57BL/6J-Ly5.1 mice. Two months after BM transplantation, BM chimeric mice were treated with a single intraperitoneal injection of azoxymethane (10 mg/kg body weight) followed by 3 cycles of 1% dextran sulfate sodium (DSS) in the drinking water. We assessed the level of fibrosis in the colon using Sirius red staining and analyzed the presence of BM-derived CD45⁺CD11b⁺Col I⁺ fibrocytes in the colon lamina propria (LP) using immunofluorescence staining and flow cytometry. Furthermore, we investigated the expressions of Col I, transforming growth factor-ß (TGF- ß), matrix metalloproteinases (MMPs), and tissue inhibitor of MMPs (TIMP)-1 in the colon tissues and fibrocytes sorted from colon LP cells after chronic DSS treatment using quantitative real-time RT-PCR.

Results: During chronic inflammation, infiltration of CCR2⁺ BM-derived monocytes and fibrocytes and production of CCL2 in the colon were particularly increased and colon fibrosis was developed in EGFP BM chimeric mice. Two types of fibrocytes, CCR2⁺CXCR4⁺Ly6C^-F4/80⁺ fibrocytes and CCR2^-CXCR4⁺Ly6C^highF4/80^- fibrocytes, were identified in the colon LP, whereas only the latter fibrocytes were detected in the PB. Adoptive transferred CCR2⁺Ly6C^highCol I^- monocytes migrated to the injured colon and a part of them differentiated into CCR2⁺Col I⁺ fibrocytes. In CCR2KO BM chimeric mice, the numbers of monocytes and fibrocytes in the colon LP were significantly decreased and colon fibrosis was attenuated. However, there was no difference in the mRNA expressions of Col I, TGF-ß, and MMPs (MMP-1a, MMP-8, and MMP-13, known as collagenases) in colon tissues between EGFP BM chimeric mice and CCR2KO BM chimeric mice. Improvement of colon fibrosis in CCR2KO BM chimeric mice was associated with the decreased expression of Timp1 mRNA in colon tissues. We analyzed the expression of Timp1 mRNA in CCR2⁺ cells and CCR2^- cells sorted from colon LP cells and found a high expression of Timp1 in CCR2⁺ monocytes/macrophages and fibrocytes.

Conclusions: Circulating CCR2⁺ monocytes migrate into the inflamed colon via CCL2/CCR2 axis and differentiate into CCR2⁺Ly6C^-F4/80⁺ fibrocytes, which inhibit collagen degradation and contribute to the development of colon fibrosis by the production of TIMP-1.