Interfering with Rac1-activation during neonatal monocyte-macrophage differentiation influences the inflammatory responses of M1 macrophages

Necrotizing enterocolitis (NEC) is a life-threatening, inflammatory disease affecting premature infants with intestinal necrosis, but the mechanism remains unclear. Neonatal macrophages are thought to play an important role in the pathogenesis of NEC through the production of proinflammatory cytokines. Restriction of cytokine expression in macrophages of NEC tissues may be beneficial. In adult macrophages, interfering with Rac1 has been shown to influence the expression of cytokines. Here, we investigated whether interfering with Rac1 in neonatal macrophages affects their inflammatory responses. First, we found that Rac1-activation was upregulated in the macrophages of rats with NEC model induction compared to controls. The M1 macrophages derived from human neonatal monocytes showed greater Rac1-activation than the M2 macrophages derived from the same monocytes. Inhibition of Rac1-activation by NSC23766 potently reduced the production of proinflammatory cytokines in these M1 macrophages. While neonatal monocytes differentiated into M1 macrophages in vitro, NSC23766 significantly altered cell function during the first six days of incubation with GM-CSF rather than during the subsequent stimulation phase. However, the same effect of NSC23766 was not observed in adult macrophages. Using mass spectrometry, Y-box binding protein 1 (YB1) was identified as being downregulated upon inhibition of Rac1-activation in the neonatal macrophages. Moreover, we found that inhibition of Rac1-activation shortens the poly A tail of PABPC1 mRNA, thereby reducing the translation of PABPC1 mRNA. Consequently, the downregulation of PABPC1 resulted in a reduced translation of YB1 mRNA. Furthermore, we found that TLR4 expression was downregulated in neonatal macrophages, while YB1 expression was reduced. Adding resatorvid (TLR4 signaling inhibitor) to the macrophages treated with NSC23766 did not further reduce the cytokine expression. These findings reveal a novel Rac1-mediated pathway to inhibit cytokine expression in neonatal M1 macrophages and suggest potential targets for the prevention or treatment of NEC.