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 3

GIV is required for PL fusion and bacterial clearance.

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GIV is required for PL fusion and bacterial clearance. (A and B) Workflo...
(A and B) Workflow for immunofluorescence studies of AIEC-LF82–challenged TGPMs (A) and representative images (B) showing the proximity of AIEC-LF82 (red) to LAMP1-positive lysosomes (green). Insets show magnified, 3D-rendered versions of boxed regions, created using ImageJ. Scale bars = 5 μm. (C) Workflow for TEM studies of infected peritoneal macrophages in D–H. (D) Representative TEM images showing bacterial abundance. Scale bar: 2 μm. (E) Bar graphs quantifying the number of bacteria per cell at 5 and 30 minutes after infection. (F) High-magnification TEM images highlighting PL fusion events (arrowheads). (G and H) Bar graphs display the number of events per cell (G) and number of unfused lysosomes per cell (H); n = 2 repeats. B, bacteria AIEC-LF82N; L, lysosome; nucleus; P, phagosome; PL, phagolysosome. (I and J) Summary of findings in cells with (I) or without (J) GIV. All TEM quantifications were based on ~20–30 fields; n = 2 independent biological repeats. Results are presented as mean ± SEM. Significance was determined using 2-tailed Student’s t test; P ≤ 0.05 is considered significant.