The neurorepellent SLIT2 inhibits LPS-induced proinflammatory signaling in macrophages

Macrophages are important mediators of immune responses with critical roles in the recognition and clearance of pathogens, as well as in the resolution of inflammation and wound healing. The neuronal guidance cue SLIT2 has been widely studied for its effects on immune cell functions, most notably directional cell migration. Recently, SLIT2 has been shown to directly enhance bacterial killing by macrophages, but the effects of SLIT2 on inflammatory activation of macrophages are less known. Using RNA sequencing analysis, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay, we determined that in murine bone marrow-derived macrophages challenged with the potent proinflammatory mediator lipopolysaccharide (LPS), exposure to the bioactive N-terminal fragment of SLIT2 (NSLIT2) suppressed production of proinflammatory cytokines interleukin (IL)-6 and IL-12 and concurrently increased the anti-inflammatory cytokine IL-10. We found that NSLIT2 inhibited LPS-induced MyD88- and TRIF-mediated signaling cascades and did not inhibit LPS-induced internalization of Toll-like receptor 4 (TLR4), but instead inhibited LPS-induced upregulation of macropinocytosis. Inhibition of macropinocytosis in macrophages attenuated LPS-induced production of proinflammatory IL-6 and IL-12 and concurrently enhanced anti-inflammatory IL-10. Taken together, our results indicate that SLIT2 can selectively modulate macrophage response to potent proinflammatory stimuli, such as LPS, by attenuating proinflammatory activation and simultaneously enhancing anti-inflammatory activity. Our results highlight the role of macropinocytosis in proinflammatory activation of macrophages exposed to LPS. Given that LPS-producing bacteria cause host illness through synergistic direct bacterial infection and excessive LPS-induced systemic inflammation, our work suggests a novel therapeutic role for SLIT2 in combatting the significant morbidity and mortality of patients with Gram-negative bacterial sepsis.