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 2

GIV dampens inflammation and promotes bacterial clearance in MDP-stimulated macrophages.

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GIV dampens inflammation and promotes bacterial clearance in MDP-stimula...
(A) Immunoblot of control (shC) or shGIV RAW 264.7 cells. (B and C) Workflow of the NF-κB reporter assay in RAW 264.7 cells (B). Bar graphs display the fold change in NF-κB activity (C). (D) Immunoblot of WT (control) and GIV-KO THP1-NF-kB SEAP reporter cell line–derived macrophages. (E–G) Workflow of the NF-κB reporter assay in CRISPR-depleted human THP-1 cells expressing an NF-κB activity–tracking reporter (E). Line graphs (F) and AUC bar graphs (G) display the fold change in NF-κB activity relative to WT control. (H) Workflow of the bacterial clearance assay (H). (I) Line graphs show the viable bacterial counts in the peritoneal macrophages. (J) Immunoblot of WT or GIV-KO THP1 monocyte–derived macrophage. (K) Workflow of the gentamicin protection assay in THP1 cells. (L) Bar graphs show the viable bacterial counts in the THP1 monocyte–derived macrophage. (M) Immunofluorescence images display representative fields of TGPMs challenged with live AIEC-LF82 (MOI 1:30) for 1 hour. Scale bar: 20 μM. Bar graphs display quantification of intracellular AIEC-LF82; n = 4–6. (N) Workflow for multiplexed proteomics analyses. (O and P) Bar graphs showing biological process as determined by Gene Ontology biological process (GOBP) analysis (red indicates the pathways cited in the text). (Q and R) Schematics summarizing findings in cells with GIV (Q) and without GIV (R). All results are displayed as mean ± SEM (n = 3 biological replicates). Significance was tested using 1-way ANOVA with Tukey’s test (C and G), 2-way ANOVA with Tukey’s test (F and I), and 2-tailed Student’s t test (L and N). P ≤ 0.05 is considered significant. Luc, luciferase; Reg, regulation.