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 6

GIV is required for the emergence of healing niColAMs in MDP-treated WT mice.

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GIV is required for the emergence of healing niColAMs in MDP-treated WT ...
(A) Study design of DSS-induced acute, chronic, and recovery phases in mouse models of colitis (C57/BL6; all WT). (B) Line graphs display the temporal patterns of the emergence of iColAMs and niColAMs in the colon samples in A. The gray dotted line indicates the composite scores of iColAM and niColAM genes in the control mice. (C) Study design for DSS-induced colitis in WT versus GIV mice (n = 3 each). See also Figure 5, A–E, for the detailed study design and disease pathology. (D) Bar plots show the classification accuracy of composite scores derived from iColAM and niColAM gene signatures in DSS-challenged mouse samples, comparing with or without L18-MDP treatment groups. Classification strength within each cohort is measured using receiver operating characteristic AUC analyses. (E) Violin plots show composite scores for niColAMs (for blue border in D) in WT and iColAMs (for brown border in D) in GIV-KO mice, treated with or without L18-MDP. (F and G) Gene Ontology Biological Process (GOBP) pathway enrichment analyses of genes downregulated in WT (F) or GIV-KO (G) mice treated with L18-MDP compared to their respective untreated controls. (H and I) GOBP (H) and Go Molecular Function (GO MF; I) analyses of genes downregulated in L18-MDP-treated WT vs GIV-KO samples. (J and K) Schematic (J) of bulk RNA sequencing in silico deconvolution analysis of distal colons from DSS-treated mice in C. Bar plots (K) show normalized percentage abundances of M1 and M2 macrophages in WT and GIV-KO mice, with and without MDP treatment. Statistics: P-values were calculated using an unpaired multiple t-test (K). P-value ≤ 0.05 is considered as significant. GOBP, Gene Ontology biological process; proc, processes; Reg, regulation; veh, vehicle.