Promotion of Myofibroblast Differentiation and Tissue Fibrosis by the Leukotriene B4-Leukotriene B4 Receptor Axis in Systemic Sclerosis

Objective To investigate the role of the inflammatory lipid mediator leukotriene B-4 (LTB4) and its receptor, BLT1, in the development and progression of systemic sclerosis (SSc). Methods Serum levels of LTB4 were compared in 64 patients with SSc and 80 healthy controls. Skin and lung tissue sections from patients with SSc and healthy donors were immunostained for leukotriene A(4) hydrolase (LTA(4)H), the critical enzyme for LTB4 synthesis, and BLT1, in combination with different cell markers. In mouse models of SSc using bleomycin or angiotensin II challenge or immunization with the DNA topoisomerase I, genetic or pharmacologic interruption of the LTB4-BLT1 axis in mice was carried out to assess its effects on systemic disease features and myofibroblast markers. Immunoblotting was performed to examine the signaling pathway in fibroblasts and endothelial cells following stimulation with LTB4 or with serum from SSc patients. Results Serum LTB4 levels were 44.93% higher in patients with SSc than in matched healthy controls (mean +/- SD 220.3 +/- 74.75 pg/ml versus 152.0 +/- 68.05 pg/ml; P < 0.0001), and this was associated with the patient subsets of SSc-associated interstitial lung disease and diffuse cutaneous SSc. Levels of LTA(4)H and BLT1 were increased in lesional areas of the skin and lungs of SSc patients, and both were abundant in myofibroblasts and endothelial cells. Interruption of the LTB4-BLT1 axis in mouse models of SSc significantly mitigated dermal and pulmonary fibrosis, with 54.00% and 52.65% fewer alpha-smooth muscle actin-positive myofibroblasts accumulating in the skin and lungs of mice, respectively, after bleomycin challenge. Immunoblotting of cultures with recombinant LTB4-stimulated fibroblasts and endothelial cells or with serum from SSc patients showed that fibroblast-myofibroblast and endothelial-mesenchymal transitions were promoted via BLT1, and that this was dependent on activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) pathway but independent of the release of transforming growth factor beta (TGF beta) by fibroblasts or endothelial cells. Conclusion The LTB4-BLT1 axis may contribute to fibrosis in SSc by directly promoting myofibroblast differentiation via the PI3K/Akt/mTOR pathway, and this appears to operate independently of autocrine secretion of TGF beta.