The Microbiota Protects against Ischemia/Reperfusion-Induced Intestinal Injury through Nucleotide-Binding Oligomerization Domain-Containing Protein 2 (NOD2) Signaling

Nucleotide-binding oligomerization domain-containing protein 2 (NOD2), an intracellular pattern recognition receptor, induces autophagy on detection of muramyl dipeptide (MDP), a component of microbial cell walls. The role of bacteria and NOD2 signaling toward ischemia/reperfusion (I/R)–induced intestinal injury response is unknown. Herein, we report that I/R-induced intestinal injury in germ-free (GF) C57BL/6 wild-type (WT) mice is worse than in conventionally derived mice. More important, microbiota-mediated protection against I/R-induced intestinal injury is abrogated in conventionally derived Nod2^−/− mice and GF Nod2^−/− mice. Also, WT mice raised in specific pathogen-free (SPF) conditions fared better against I/R-induced injury than SPF Nod2^−/− mice. Moreover, SPF WT mice i.p. administered 10 mg/kg MDP were protected against injury compared with mice administered the inactive enantiomer, l-MDP, an effect lost in Nod2^−/− mice. However, MDP administration failed to protect GF mice from I/R-induced intestinal injury compared with control, a phenomenon correlating with undetectable Nod2 mRNA level in the epithelium of GF mice. More important, the autophagy-inducer rapamycin protected Nod2^−/− mice against I/R-induced injury and increased the levels of LC3⁺ puncta in injured tissue of Nod2^−/− mice. These findings demonstrate that NOD2 protects against I/R and promotes wound healing, likely through the induction of the autophagy response.The epithelium lining the intestinal track is composed of a single layer sheet of epithelial cells that provides nutrient absorption, hormone secretion, and innate immune sampling of luminal contents.^1,2 In addition, the epithelium provides a physical barrier between the host and gut microbes, where intestinal epithelial cells (IECs) are stitched together by tight junctions that maintain the architecture of the epithelial sheet and prevent uncontrolled access of luminal content (eg, microbes and dietary toxins) to subepithelial tissues.³ The epithelium is preserved by the homeostatic migration and proliferation of IECs from the base of the intestinal crypts to tips of the villi. Events that disrupt this equilibrium could have deleterious consequences for the host, as seen in patients experiencing intestinal ischemia.⁴Ischemia occurs when blood supply to the small bowel is occluded, which is followed by reperfusion, the return of blood flood flow, and simultaneous re-oxygenation of the tissue. During ischemia, an imbalance of metabolic demand and supply results in hypoxic response with activation of hypoxia-inducible factor-1 as well as cell death programs,⁵ autophagy,^6–8 and necrosis (organelle swelling and plasma membrane rupturing).^4,9 Paradoxically, the restoration of blood flow causes the release of inflammatory mediators, such as IL-6, tumor necrosis factor-α, and IL-1β, which exacerbate the injury.⁴ As a result, extra-intestinal organs, such as liver and the lung, may experience inflammatory activation and fatal multiorgan dysfunction syndrome. In the clinic, causes of intestinal ischemia/reperfusion (I/R)–induced injury include atherosclerosis, hypotension, blood clots, hernias, cardiac and mesenteric surgery, venous thrombosis, and necrotizing enterocolitis.Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is a pattern-recognition receptor whose function is the intracellular reconnaissance of pathogen-associated molecular patterns. NOD2 is important for the recognition of muramyl dipeptide, MDP, a component of peptidoglycan that is present in the cell walls of Gram-positive and Gram-negative bacteria. Loss-of-function mutations of NOD2 have been associated with Crohn''s disease and, recently, NOD2-associated autoinflammatory disease.¹⁰ The mechanism by which NOD2 maintains intestinal homeostasis has yet to be clearly defined, although a current paradigm suggests an involvement of this innate sensor in controlling microbial composition,^11,12 likely through expression of antimicrobial peptides from Paneth cells.¹³ In addition, NOD2 is implicated in other important biological responses, such as inflammasome activation¹⁴ and autophagy.⁶ More important, in a preclinical model of necrotizing enterocolitis, NOD2 signaling was shown to protect against hypoxic stress through down-regulation of the Toll-like receptor (TLR) 4 pathway.¹⁵ However, the role of commensal bacteria and NOD2 signaling in intestinal I/R injury response has not been elucidated.A balance between innate inflammatory responses and cytoprotective mechanisms dictates the extent of end-organ damage during I/R injury. During injury-induced hypoxic stress, cells undergo a prosurvival process called autophagy.¹⁶ This autophagic response occurs on inhibition of mammalian target of rapamycin, thereby inducing the encapsulation of cytoplasmic components in a double membrane (autophagosome), which is delivered to the lysosome for degradation.¹⁶ In hepatic ischemia, autophagy has been shown to be a protective mechanism that favors cell survival and proliferation,⁸ two key processes in epithelial injury response. Interestingly, NOD2 recruits ATG16L1 to the plasma membrane to initiate autophagosome formation in response to MDP and at the site of Shigella flexneri entry.¹⁷ However, the role of commensal bacteria–induced autophagy in the context of hypoxic stress and intestinal damage is currently unknown.Herein, we investigated the role of microbes and NOD2 signaling in I/R-induced intestinal injury using germ-free (GF) and conventionally derived (CONV-D) Nod2^−/− mice. We demonstrate that microbes are important for optimal intestinal response to injury, an effect mediated by NOD2 signaling.