ProBDNF promotes LPS-induced pro-inflammatory polarization of alveolar macrophages via the Notch1 signaling pathway.

Macrophage polarization is involved in the pathogenesis of acute respiratory distress syndrome (ARDS). However, the upstream regulators that shape macrophage inflammatory phenotypes under pathological conditions remain poorly understood. Pro-brain-derived neurotrophic factor (proBDNF) has recently been implicated in immune regulation, but its role in macrophage polarization and the underlying mechanisms are still unclear. Lipopolysaccharide (LPS) stimulation of NR8383 alveolar macrophages was used to model inflammation in vitro. ProBDNF expression was manipulated via adenoviral overexpression or knockdown. Polarization-related gene expression was measured by RT-qPCR, cytokine secretion was assessed by ELISA, and protein levels were examined by Western blotting and immunofluorescence. Neurotrophin receptor expression, including Ngfr/p75NTR and Ntrk2/TrkB, was also evaluated by RT-qPCR. Notch1 pathway activation was assessed by examining NICD levels and the expression of its downstream targets, Hes1 and Hes5. To further assess pathway involvement, NICD knockdown experiments were performed. LPS stimulation significantly increased proBDNF expression in NR8383 cells. Overexpression of proBDNF promoted a shift toward a pro-inflammatory (M1) phenotype, as indicated by increased expression of Cxcl9 and Nos2 and reduced expression of M2 markers (Arg1 and Mrc1). In contrast, proBDNF knockdown produced the opposite effect. Consistently, proBDNF overexpression increased TNF-α secretion and reduced IL-10 production, whereas proBDNF knockdown produced the opposite trend. NR8383 cells expressed Ngfr/p75NTR and Ntrk2/TrkB, with Ngfr showing a more inflammation-responsive pattern. These changes were accompanied by corresponding alterations in Notch1 signaling activity, reflected by changes in NICD, Hes1, and Hes5 expression. Importantly, silencing NICD markedly attenuated the pro-inflammatory polarization induced by proBDNF overexpression. In contrast, inhibition of Notch1 signaling did not affect proBDNF expression, supporting the interpretation that Notch1 functions downstream of proBDNF in this model. ProBDNF promotes macrophage polarization toward pro-inflammatory phenotypes under LPS stimulation, at least in part via Notch1 pathway activation. These findings identify a proBDNF-Notch1 regulatory axis in macrophage-driven inflammation and offer new insight into neuroimmune mechanisms underlying inflammatory lung injury.