Radulovic, K. | Search bioRxiv

bioRxiv2026

A crypt-operating DNA repair checkpoint for uncoupling regeneration and tumorigenesis

The rapid turnover of the intestinal epithelium increases its vulnerability to genomic instability and environmental insults such as irradiation. Defects in DNA damage resolution can compromise epithelial regeneration, promote chronic tissue injury, and predispose to colorectal tumorigenesis. However, the intrinsic mechanisms that coordinate DNA repair with epithelial regeneration at the crypt level remain poorly defined. Here, we identify the Nod-like receptor protein 6 (Nlrp6) as a key epithelial regulator of genome surveillance and regenerative control in intestinal crypts. Nlrp6 is strategically expressed in crypt base columnar cells, where it preserves crypt homeostasis by restraining proliferation under genotoxic stress conditions. Loss of Nlrp6 in crypt base columnar cells results in uncontrolled oncogenic stress, defective epithelial regeneration, and accumulation of unrepaired DNA damage, features associated with poor prognosis in colorectal cancer. Conversely, aberrant Nlrp6 overexpression induces cytoplasmic retention of Csnk2 catalytic subunits, limiting their nuclear availability when DNA repair is required. These findings position Nlrp6 as a non-canonical, cell-intrinsic surveillance mechanism that links DNA damage responses to epithelial regeneration through Csnk2-dependent signaling. Collectively, our study reveals a crypt-intrinsic DNA repair pathway that governs epithelial regeneration and disease outcomes, providing new insight into how genome instability and regenerative failure contribute to colorectal cancer progression.

cell biology↗

bioRxiv2022

Monocyte intrinsic NOD2 signalling inhibits pathogenic macrophage differentiation and its loss in inflammatory macrophages improves intestinal inflammation.

ObjectiveIt is believed that intestinal recruitment of monocytes from Crohns Disease (CD) patients who carry NOD2 risk alleles may repeatedly give rise to recruitment of pathogenic macrophages. We investigated an alternative possibility that NOD2 may rather inhibit their differentiation from intravasating monocytes. DesignThe monocyte fate decision was examined by using germ-free mice, mixed bone marrow chimeras and a culture system yielding macrophages and monocyte-derived dendritic cells (mo-DCs). We next asked whether Nod2 in either monocytes or tissue macrophages have distinct resolving properties in colitis. ResultsDespite a similar abundance of monocytes, the intestinal frequency of mo-DCs from Nod2-deficient mice was lowered independently of the changes in the gut microbiota that are caused by Nod2 deficiency. Similarly, the pool of mo-DCs was poorly reconstituted with mobilized bone marrow Nod2-deficient cells. The use of pharmacological inhibitors revealed that activated NOD2 at an early stage of development dominantly inhibits mTOR-mediated macrophage differentiation in a TNFalpha-dependent manner. These observations were supported by the identification of a TNFalpha-dependent response to MDP that is specifically lost in CD14-expressing blood cells bearing the frameshift mutation in NOD2. Accordingly, loss of NOD2 in monocytes lowers glycolytic reserve, CD115 expression and pro-resolving features. Dietary intake of aryl hydrocarbon receptor (AHR) agonists that promotes mo-DCs generation improves colitis in Nod2-deficient mice to the same extent as what is observed upon macrophage ablation of Nod2. ConclusionNOD2 negatively regulates a macrophage developmental program through a feed-forward loop that could be exploited for overcoming resistance to anti-TNF therapy in CD. Significance of this studyO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LILoss of NOD2 function is predisposing to Crohns disease. C_LIO_LIRapamycin, a serine/THR kinase inhibitor of mammalian target (mTOR) has been reported as potentially effective treatment in discrete subset of CD patients with refractory colitis. C_LIO_LIThe NOD2 protein promotes the chemokine CCL2-dependent recruitment of inflammatory monocytes in response to tissue injury. C_LIO_LIAn accumulation of CCR2-expressing monocytes and inflammatory macrophages is observed within the intestinal mucosa of CD patients including those resistant to anti-TNF therapy. C_LIO_LIActivated NOD2 enhances proinflammatory activity of CX3CR1intLy6Chi effector monocytes. C_LIO_LIThe monocyte fate toward mo-DCs is orchestrated by the aryl hydrocarbon receptor. C_LI What are the new findings?O_LINOD2 has a hierarchically dominant negative role on the mTORC-driven monocyte conversion to inflammatory macrophages independently of the changes in the gut microbiota that are caused by Nod2 deficiency. C_LIO_LIA defect in monocytes fate at the early stage allow the expansion of pathogenic macrophages in Nod2-deficient mice at the expense of mo-DC. C_LIO_LIAdoptive transfer of Nod2-deficient monocytes into wild-type mice was sufficient to exacerbate DSS-induced intestinal damage. C_LIO_LIThe glycolytic reserve of monocytes and their ability to respond to M-CSF is lowered upon loss of NOD2 signalling. C_LIO_LIDeletion of NOD2 in macrophage improves colitis to the same extent as dietary supplementation of AHR agonist in mice. C_LIO_LIThe recognition of the gut microbiota by NOD2 is required for de novo reconstitution of mo-DCs in the lamina propria of the murine intestine, while having minimal effect on the mobilization of their precursors to the intestinal mucosa. C_LI How might it impact on clinical practice in the foreseeable future?This study might contribute to the development of novel mTORC-based therapeutic strategies for improving the response to biologics by restoring the ability of circulating monocytes to reconstitute the pool of mo-DCs during homeostatic turnover and upon tissue injury. It may thereby prevent the accumulation of pathogenic macrophages in patients with loss-of-function NOD2 alleles, which fail to respond to anti-TNF and are at greater risk of developing stricturing disease.

immunology↗