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 9

Characterization of the NOD2•GIV interface exploiting CD-associated NOD2 mutants.

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Characterization of the NOD2•GIV interface exploiting CD-associated NOD2...
(A and B) Schematic shows CD-associated mutations (A) and their positions in NOD2, depicted as a domain map, and alignment (B) of the aa sequence of the 10th LRR repeat of human NOD2 and the CD-risk associated NOD2 variant (NOD2-1007fs). Residues mutated to evaluate potential participating residues in the NOD2•GIV interaction are highlighted. (C) GST–GIV-CT was pulled down using glutathione beads from equal aliquots of lysates of HEK293T coexpressing GST–GIV-CT (aa 1660–1870; mammalian p-CEFL vector) and WT or 3 indicated CD-risk associated variants of HA-NOD2. Bound NOD2 proteins and similar expression of GIV-CT was assessed by IB using anti-HA (NOD2) and anti-GST (GIV-CT) Abs. (D) FLAG-tagged GIV was IP with anti-FLAG mAb from equal aliquots of lysates of HEK293T cells expressing GIV-FLAG and either WT or 1007fs variant of HA-NOD2, stimulated (+) or not (–) with MDP for the indicated time points. IP complexes and input lysates were analyzed for NOD2 and GIV by IB, using anti-HA (NOD2) and anti-FLAG (GIV-CT) Abs. (E) Workflow for assessing NF-κB activity. (F) NF-κB reporter assay in HeLa cells. Cells were preincubated with MDP (10 μg/mL) and then stimulated with LPS (100 ng/mL) and the percentage change of NF-κB activity was detected using a dual-cell reporter assay. (G) Workflow for assessing bacterial clearance via flow cytometry. (H) The flow cytometry panel detects CM-Dil–labeled AIEC-LF82 bacteria (MOI 1:30) in THP1-derived macrophages transfected with HA-NOD2-WT or 1007fs mutant. (I) AIEC-LF82 bacterial load normalized to NOD2-WT. P ≤ 0.05 was considered significant, 1-way ANOVA followed by Tukey’s multiple comparison test. (J and K) Schematic summarizing key findings in this work. Magenta solid and interrupted lines indicate the GIV-dependent impact on NOD2. (J) In physiology, bacterial sensing and signaling by NOD2 requires GIV to limit inflammation. (K) In pathology, dysregulated inflammation results when either WT NOD2 cannot bind GIV (e.g., GIV is low or absent) or when the CD-risk associated 1007fs variant cannot bind GIV. P values were calculated using 1-way ANOVA with Tukey’s test (I) and 2-way ANOVA with Tukey’s test (F) and indicated with P values are shown. P ≤ 0.05 was considered significant.