Polarization of macrophages into pro-inflammatory or anti-inflammatory states has distinct metabolic requirements, with mechanistic target of rapamycin (mTOR) kinase signaling playing a critical role. However, it remains unclear how mTOR regulates metabolic status to promote polarization of these cells. Here we show that an mTOR-Semaphorin 6D (Sema6D)-Peroxisome proliferator receptor γ (PPARγ) axis plays critical roles in macrophage polarization. Inhibition of mTOR or loss of Sema6D blocked anti-inflammatory macrophage polarization, concomitant with severe impairments in PPARγ expression, uptake of fatty acids, and lipid metabolic reprogramming. Macrophage expression of the receptor Plexin-A4 is responsible for Sema6D-mediated anti-inflammatory polarization. We found that a tyrosine kinase, c-Abl, which associates with the cytoplasmic region of Sema6D, is required for PPARγ expression. Furthermore, Sema6D is important for generation of intestinal resident CX3CR1 macrophages and prevents development of colitis. Collectively, these findings highlight crucial roles for Sema6D reverse signaling in macrophage polarization, coupling immunity, and metabolism via PPARγ.

ObjectivesInappropriate activation of neutrophils plays a pathological role in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). The aim of this study was to investigate the functions of semaphorin 4D (SEMA4D) in regulation of neutrophil activation, and its involvement in AAV pathogenesis.MethodsSerum levels of soluble SEMA4D were evaluated by ELISA. Blood cell-surface expression of membrane SEMA4D was evaluated by flow cytometry. To determine the functional interactions between neutrophil membrane SEMA4D and endothelial plexin B2, wild-type and SEMA4D −/− mice neutrophils were cultured with an endothelial cell line (MS1) stained with SYTOX green, and subjected to neutrophil extracellular trap (NET) formation assays. The efficacy of treating human neutrophils with recombinant plexin B2 was assessed by measuring the kinetic oxidative burst and NET formation assays.ResultsSerum levels of soluble SEMA4D were elevated in patients with AAV and correlated with disease activity scores. Cell-surface expression of SEMA4D was downregulated in neutrophils from patients with AAV, a consequence of proteolytic cleavage of membrane SEMA4D. Soluble SEMA4D exerted pro-inflammatory effects on endothelial cells. Membranous SEMA4D on neutrophils bound to plexin B2 on endothelial cells, and this interaction decreased NET formation. Recombinant plexin B2 suppressed neutrophil Rac1 activation through SEMA4D’s intracellular domain, and inhibited pathogen-induced or ANCA-induced oxidative burst and NET formation.ConclusionsNeutrophil surface SEMA4D functions as a negative regulator of neutrophil activation. Proteolytic cleavage of SEMA4D as observed in patients with AAV may amplify neutrophil-mediated inflammatory responses. SEMA4D is a promising biomarker and potential therapeutic target for AAV.

Leukotriene B (LTB) receptor type 1 (BLT1) is abundant in phagocytic and immune cells and plays crucial roles in various inflammatory diseases. BLT1 is phosphorylated at several serine and threonine residues upon stimulation with the inflammatory lipid LTB Using Phos-tag gel electrophoresis to separate differentially phosphorylated forms of BLT1, we identified two distinct types of phosphorylation, basal and ligand-induced, in the carboxyl terminus of human BLT1. In the absence of LTB, the basal phosphorylation sites were modified to various degrees, giving rise to many different phosphorylated forms of BLT1. Different concentrations of LTB induced distinct phosphorylation events, and these ligand-induced modifications facilitated additional phosphorylation events at the basal phosphorylation sites. Because neutrophils migrate toward inflammatory sites along a gradient of LTB, the degree of BLT1 phosphorylation likely increases in parallel with the increase in LTB concentration as the cells migrate. At high concentrations of LTB, deficiencies in these two types of phosphorylation events impaired chemotaxis and β-hexosaminidase release, a proxy for degranulation, in Chinese hamster ovary (CHO-K1) and rat basophilic leukemia (RBL-2H3) cells, respectively. These results suggest that an LTB gradient around inflammatory sites enhances BLT1 phosphorylation in a stepwise manner to facilitate the precise migration of phagocytic and immune cells and the initiation of local responses, including degranulation.