in Innate Immunity Production and Responses g Regulates IFN-The RON Receptor Tyrosine Kinase

Receptor tyrosine kinases are emerging as a class of key regulators of innate immune responses. We have shown previously that the RON receptor tyrosine kinases (murine Stk), expressed on tissue-resident macrophages, inhibit classical macrophage activation while promoting hallmarks of alternative activation, thus regulating the critical balance between the inﬂammatory and wound-healing properties of activated macrophages. We have also shown previously that RON (cid:2) / (cid:2) mice are more susceptible to in vivo endotoxin challenge than wild-type mice, suggesting that the expression of this receptor confers a degree of endotoxin resistance to these animals. Here we demonstrate that, in response to in vivo LPS challenge, RON (cid:2) / (cid:2) mice harbor signiﬁcantly increased systemic levels of IFN- (cid:1) and IL-12p70 and increased levels of IL-12p40 transcript in their spleen. This elevation of IFN- (cid:1) can be attributed to splenic NK cells responding to the elevated levels of IL-12. Analysis of RON and IFN- (cid:1) receptor double-knockout mice indicates that the enhanced susceptibility of RON (cid:2) / (cid:2) mice to endotoxin challenge is dependent on IFN- (cid:1) -mediated signals. In vitro studies demonstrate that stimulation of primary peritoneal macrophages with macrophage-stimulating protein, the ligand for RON, inhibits IFN- (cid:1) -induced STAT1 phosphorylation and CIITA expression, resulting in reduced surface levels of MHC class II. Further studies demonstrating the induction of suppressor of cytokine signaling 1 via macrophage-stimulating protein/RON signaling provide a potential mechanistic insight into this regulatory pathway. These results indicate that the RON receptor regulates both the production of and response to IFN- (cid:1) , resulting in enhanced susceptibility to endotoxin assayed using a murine multiplex cytokine assay kit, LINCOplex (Millipore). It is a well-studied fact that SOCS1 is strongly induced by activated STAT1 and SOCS3 by STAT3 (11). The analysis of the SOCS3 promoter has shown that besides the two STAT3-binding IFN- (cid:3) activation site elements, a proximal AP-1 binding site is critical for LPS-induced SOCS3 expression in macrophages and microglial cells (40). These studies also show that the inhibition of the ERK1/2 MAPK, compared with JNK and p38, results in the greatest inhibition of LPS-induced SOCS3 production. Further-more, macrophages from c-Fos (cid:1) / (cid:1) mice are defective in SOCS1 and SOCS3 production in response to LPS stimulation or Salmonella infection (41). Taken together, these studies have shown the importance of STAT and AP-1 transcription factors in SOCS production. To determine whether signaling through RON can induce STAT1/3 and or MAPK activation in primary peritoneal macrophages, we stimulated peritoneal macrophages with MSP and assessed STAT1 and 3, ERK1/2, JNK, and p38 phosphorylation by Western blot over time. Our data show that within 30 min the activation of the RON receptor by its ligand, MSP, results in the robust induction of ERK1/2 phosphorylation. In comparison, we see only a minor induction of p38 and JNK activation even after 4.5 h of MSP stimulation. Finally, we show that MSP induces signiﬁcant STAT3 but not STAT1 phosphorylation in peritoneal macrophages within 1 h ofreceptor activation (Fig. 7). This induction was not observed in primary peritoneal macrophages from RON (cid:1) / (cid:1) mice (data not shown). These results provide an insight into the mechanism by which