SUMMARY Sensing of pathogens by Nucleotide oligomerization domain (NOD)-like 2 receptor (NOD2) induces a protective inflammatory response that coordinates bacterial clearance. Polymorphisms in NOD2 impair bacterial clearance, leading to chronic gut inflammation in Crohn’s disease (CD) via mechanisms that remain incompletely understood. We identify GIV/Girdin (CCDC88A) as a NOD2-interactor that shapes bacterial sensing-and-signaling in macrophages. Myeloid-specific GIV depletion exacerbated and protracted infectious colitis and abolished the protective effect of muramyl dipeptide (MDP) in both chemical colitis and severe sepsis. In the presence of GIV, macrophages enhance anti-bacterial pathways downstream of NOD2, clear microbes rapidly and concomitantly suppress inflammation. GIV’s actions are mediated via its C-terminus, which directly binds the terminal leucine-rich repeat (LRR#10) of NOD2; binding is augmented by MDP and ATP, precedes receptor oligomerization, and is abolished by the 1007fs CD-risk variant which lacks LRR#10. Findings illuminate mechanisms that underlie protective NOD2 signaling and loss of function in the major 1007fs variant. GRAPHIC ABSTRACT In brief This work reveals a mechanism by which macrophages use their innate immune sensor, NOD2, to protect the host against overzealous inflammation during bacterial infections, and the consequences of its loss, as occurs in the most important Crohn’s disease-risk variant. HIGHLIGHTS GIV is a functional and direct interactor of the terminal LRR repeat of NOD2 Mice lacking MФ GIV develop dysbiosis, protracted ileocolitis and sepsis MDP/NOD2-dependent protective host responses require GIV CD-risk NOD2 1007fs variant lacking the terminal LRR#10 cannot bind GIV

Coupling of NOD2 to GIV is Required for Bacterial Sensing