SHIP Blocks (MDP+LPS)-Induced Synergy in Macrophages Independent of Catalytic Activity

Macrophages coordinate cytokine responses by integrating signals from pattern recognition receptors including NOD2 and TLR4. NOD2 detects muramyl dipeptide derived from bacterial peptidoglycan, and TLR4 recognizes lipopolysaccharide in the outer membrane of Gram-negative bacteria. NOD2 and TLR4 signals synergize to enhance cytokine production in myeloid cells. While synergy can support host defense, it must be regulated to minimize the risk of excessive inflammation. The lipid phosphatase SHIP, a negative regulator of Class I PI3Ks, reduces inflammatory signaling, but its role during NOD2-TLR4 co-stimulation remains undefined. We found that SHIP limits IL-1β production by blocking synergy in bone marrow-derived macrophages co-stimulated with muramyl dipeptide and lipopolysaccharide. SHIP-/- macrophages showed a synergistic increase in IL-1β that was not evident in SHIP+/+ macrophages. Moreover, reducing SHIP protein concentrations by differentiation in different growth factors, IL-4 treatment, or siRNAs, enabled synergy for IL-1β production in SHIP+/+ macrophages. Pharmacologic inhibition of SHIP's catalytic activity did not promote synergy, and similarly, blocking PI3K had no effect, suggesting that the response is independent of SHIP's phosphatase activity. Synergy for IL-1β production was dependent on NOD2 signaling despite NOD2 stimulation alone resulting in little to no IL-1β. Moreover, IL-1β was selectively enhanced in SHIP-/- macrophages during co-stimulation with MDP and LPS, or when MDP stimulation preceded LPS. These findings identify SHIP's adaptor function as a negative regulator of IL-1β induced by NOD2-TLR4 co-stimulation in macrophages, and suggest that SHIP acts as a gatekeeper for macrophage IL-1β during early innate immune activation.