Abstract
Receptor-interacting protein 2 (RIP2) is a kinase that mediates signaling downstream of the bacterial peptidoglycan sensors NOD1 and NOD2. Genetic loss or pharmaceutical inhibition of RIP2 has been shown to be beneficial in multiple inflammatory disease models with the effects largely attributed to reducing proinflammatory signaling downstream of peptidoglycan recognition. However, given the widespread expression of this kinase and its reported interactions with numerous other proteins, it is possible that RIP2 may also function in roles outside of peptidoglycan sensing. In this work, we show that RIP2 undergoes tyrosine phosphorylation and activation in response to engagement of the Fc γ receptor (FcγR). Using bone marrow-derived macrophages from WT and RIP2-KO mice, we show that loss of RIP2 leads to deficient FcγR signaling and reactive oxygen species (ROS) production upon FcγR cross-linking without affecting cytokine secretion, phagocytosis, or nitrate/nitrite production. The FcγR-induced ROS response was still dependent on NOD2, as macrophages deficient in this receptor showed similar defects. Mechanistically, we found that different members of the Src family kinases (SFKs) can promote RIP2 tyrosine phosphorylation and activation. Altogether, our findings suggest that RIP2 is functionally important in pathways outside of bacterial peptidoglycan sensing and that involvement in such pathways may depend on the actions of SFKs. These findings will have important implications for future therapies designed to target this kinase.
Highlights
Receptor-interacting protein 2 (RIP2) is a kinase that mediates signaling downstream of the bacterial peptidoglycan sensors NOD1 and NOD2
Using bone marrow– derived macrophages from WT and RIP2-KO mice, we show that loss of RIP2 leads to deficient Fc ␥ receptor (Fc␥R) signaling and reactive oxygen species (ROS) production upon Fc␥R crosslinking without affecting cytokine secretion, phagocytosis, or nitrate/nitrite production
Other studies have proposed a NOD1/2-independent, T-cell–intrinsic role for RIP2, with some groups suggesting that RIP2 prevents the generation of pathogenic IL-17A–producing T-cells [17] and others that RIP2 can interact with B cell leukemia 10 protein (Bcl10) and directly mediate its phosphorylation, leading to activation of NF-B [18]
Summary
Identification of the tyrosine autophosphorylation site on RIP2 brought to our attention that, when phosphorylated, this site could serve as a Src-family kinase (SFK) SH2-binding motif, pYEX(I/L/P/V) (Fig. 1A) [21]. To assess whether RIP2 is involved in Fc␥R-mediated ROS production, we generated BMDMs from WT or RIP2-KO mice, primed these with IFN-␥, induced Fc␥R cross-linking, and assessed ROS production using a ROS-reactive fluorescent probe In both genotypes, a significant amount of ROS production was observed upon Fc␥R cross-linking compared with unstimulated cells (Fig. 6, A and B, represented as percentage of cells stained positive for the probe and as mean fluorescence intensity (MFI) in the FL-1 channel. BMDMs, Fc␥R cross-linking of NOD2-KO BMDMs led to a significantly lower amount of ROS generated when compared with Fc␥R cross-linking of WT BMDMs (Fig. 8C, expressed as either percentage of positive cells or MFI of the ROS-reactive probe) These data suggest that the involvement of RIP2 in Fc␥R signaling and ROS production depends on NOD2
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