Distinct colitis-associated macrophages drive NOD2-dependent bacterial sensing and gut homeostasis
Gajanan D. Katkar, Mahitha Shree Anandachar, Stella-Rita C. Ibeawuchi, Ella G. McLaren, Megan L. Estanol, Kennith Carpio-Perkins, Shu-Ting Hsu, Celia R. Espinoza, Jane E. Coates, Yashaswat S. Malhotra, Madhubanti Mullick, Vanessa Castillo, Daniella Vo, Saptarshi Sinha, Pradipta Ghosh
Gajanan D. Katkar, Mahitha Shree Anandachar, Stella-Rita C. Ibeawuchi, Ella G. McLaren, Megan L. Estanol, Kennith Carpio-Perkins, Shu-Ting Hsu, Celia R. Espinoza, Jane E. Coates, Yashaswat S. Malhotra, Madhubanti Mullick, Vanessa Castillo, Daniella Vo, Saptarshi Sinha, Pradipta Ghosh
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Research Article Gastroenterology Immunology Microbiology

Distinct colitis-associated macrophages drive NOD2-dependent bacterial sensing and gut homeostasis

Abstract

Single-cell studies have revealed that intestinal macrophages maintain gut homeostasis through the balanced actions of reactive (inflammatory) and tolerant (noninflammatory) subpopulations. How such balance is impaired in inflammatory bowel diseases (IBDs), including Crohn’s disease (CD) and ulcerative colitis (UC), remains unresolved. Here, we define colon-specific macrophage states and reveal the critical role of noninflammatory colon-associated macrophages (niColAMs) in IBD recovery. Through trans-scale analyses—integrating computational transcriptomics, proteomics, and in vivo interventional studies—we identified GIV (CCDC88A) as a key regulator of niColAMs. GIV emerged as the top-ranked gene in niColAMs that physically and functionally interacts with NOD2, an innate immune sensor implicated in CD and UC. Myeloid-specific GIV depletion exacerbates infectious colitis, prolongs disease, and abolishes the protective effects of the NOD2 ligand muramyl dipeptide in colitis and sepsis models. Mechanistically, GIV’s C-terminus binds the terminal leucine-rich repeat 10 (LRR 10) of NOD2 and is required for NOD2 to dampen inflammation and clear microbes. The CD-associated 1007fs NOD2 variant, which lacks LRR 10, cannot bind GIV, which provides critical insights into how this clinically relevant variant impairs microbial sensing and clearance. These findings illuminate a critical GIV•NOD2 axis essential for gut homeostasis and highlight its disruption as a driver of dysbiosis and inflammation in IBD.

Authors

Gajanan D. Katkar, Mahitha Shree Anandachar, Stella-Rita C. Ibeawuchi, Ella G. McLaren, Megan L. Estanol, Kennith Carpio-Perkins, Shu-Ting Hsu, Celia R. Espinoza, Jane E. Coates, Yashaswat S. Malhotra, Madhubanti Mullick, Vanessa Castillo, Daniella Vo, Saptarshi Sinha, Pradipta Ghosh

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Figure 1

Identification of CCDC88A as a putative NOD2 modulator in IBD-associated macrophages.

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Identification of CCDC88A as a putative NOD2 modulator in IBD-associated...
(A) Key steps of a previously published workflow (3) used to develop the computational model of macrophage continuum states—SMaRT, which identifies invariant gene clusters representing reactive (R) and tolerant (T1, T2) states across >12,500 diverse transcriptomic datasets. The schematic illustrates their opposing roles: reactive macrophages act as accelerators, while tolerant states serve as brakes, working antagonistically to fine-tune inflammatory responses to perceived threats. (B) Key steps used to refine SMaRT in the context of the gut mucosa and derive ColAM signatures using a dataset (24) comprising both healthy and IBD samples. SMaRT genes were trained to derive a subset of ColAMs that could classify healthy versus IBD samples, achieving an AUC ≥ 0.7 and P ≤ 0.05 (Fisher’s exact test). (C) iColAM- and niColAM-defining genes identified in healthy and IBD samples. (D and E) Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis for SMaRT (D) and the ColAM-IBD (E) gene sets. (F and G) The receiver operating characteristic–AUC (circle size) and regulation (red, up; blue, down) for classifying healthy versus CD and healthy versus UC in human (H) colonic lamina propria (GEO GSE123141) (F) and DSS-induced acute, chronic, and healing phases of murine colitis models (G). Classification was based on macrophage gene signatures of reactivity (R) and tolerance (T), identified in the SMaRT model and the iColAMs and niColAMs, used independently. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001, Welch’s 2-sample unpaired t test. (H) Venn diagram of ColAM genes identified in B and C with NOD1 and NOD2 interactors identified by independent studies. CCDC88A (GIV; white circle) emerges as a NOD2-specific interactor linked to tolerant ColAMs. (I) Correlation coefficient of normalized gene expression of CCDC88A with NOD2 and NOD1 across independent transcriptomic datasets of healthy and IBD tissues. (J) MDP/NOD2-induced NF-κB activity observed during a functional genomic (siRNA-based) screen. The impact of depletion of iColAM and niColAM genes is presented. The dashed line marks 75% enhancement relative to MDP-stimulated controls. Luc, luciferase; Mϕ, macrophage.