Kuss-Duerkop, S. K. | bioRxiv Science

bioRxiv2025

NOD2 modulates MDA5 signaling to promote coxsackievirus B3 replication

Coxsackievirus is an enteric virus that encounters intestinal epithelial cells (IECs) after ingestion and can cause a range of illnesses from mild to severe, including gastroenteritis, pancreatitis, central nervous system diseases and myocarditis. Sensing of CVB3 RNA by melanoma differentiation-associated gene (MDA5) induces a Type 1 Interferon (T1IFN) response that is critical for reducing viral replication and dissemination within cells and the host. The cytosolic nucleotide oligomerization domain-containing protein 2 (NOD2), however, has been implicated in a proviral role in CVB3-induced myocarditis, but the underlying mechanism is not well understood. Because NOD2 is especially important in maintaining intestinal immune homeostasis and CVB3 is an enteric virus that initially infects the intestinal tract leading to virus dissemination to other tissues, we sought to determine whether NOD2 expressed in IECs would impact CVB3 infection. Here, we demonstrate that IEC NOD2 promotes CVB3 replication. Replication of a related Enterovirus, poliovirus, was also enhanced by NOD2. We discovered that NOD2-/- IECs have increased T1IFNs and interferon stimulated gene (ISG) expression, including IFIH1, the gene encoding for MDA5. Replication of CVB3 was rescued when MDA5 expression was reduced in NOD2-/- IECs. CVB3 replication was further increased, and the T1IFN response reduced, in NOD2+/+IECs treated with the mitophagy inducer carbonyl cyanide m-chlorphenyl hydrazone (CCCP). Mitophagy induction in NOD2-/- IECs, however, had no effect on CVB3 replication, or the T1IFN response, suggesting a role for NOD2 in mitophagy. Overall, our data demonstrate that NOD2 promotes CVB3 replication in IECs by suppressing T1IFN expression and MDA5 activation via NOD2-mediated mitophagy. Since T1IFN restricts enteroviruses in vivo, CVB3 and other enteroviruses might exploit NOD2 activation in the intestinal tract to evade antiviral T1IFN responses. This evasion could enhance their replication in the intestine and promote their spread to other organs, potentially leading to diseases in the pancreas, heart, and central nervous system. ImportanceCoxsackievirus B3 (CVB3) is a significant human intestinal virus capable of spreading to other organs. It is closely linked to the development of type I diabetes and can cause severe heart infections, particularly in children. Our research demonstrates that CVB3 exploits the immunity protein NOD2, which usually protects against infections, to infect and replicate in intestinal cells. NOD2 also enhances the replication of poliovirus, another related human enteric virus. When expressed in the intestinal epithelial cells, NOD2 reduces the immune response to certain viral infections by inhibiting expression of the viral sensor MDA5. Overall, CVB3 benefits from NOD2-mediated dampening of antiviral immunity to promote its replication in intestinal cells during fecal-oral transmission.

immunology↗

bioRxiv2023

Nitrate-mediated luminal expansion of Salmonella Typhimurium is dependent on the ER stress protein CHOP

Salmonella Typhimurium is an enteric pathogen that employs a variety of mechanisms to exploit inflammation resulting in expansion in the intestinal tract, but host factors that contribute to or counteract the luminal expansion are not well-defined. Endoplasmic reticulum (ER) stress induces inflammation and plays an important role in the pathogenesis of infectious diseases. However, little is known about the contribution of ER stress-induced inflammation during Salmonella pathogenesis. Here, we demonstrate that the ER stress markers Hspa5 and Xbp1 are induced in the colon of S. Typhimurium infected mice, but the pro-apoptotic transcription factor Ddit3, that encodes for the protein CHOP, is significantly downregulated. S. Typhimurium-infected mice deficient for CHOP displayed a significant decrease in inflammation, colonization, dissemination, and pathology compared to littermate control mice. Preceding the differences in S. Typhimurium colonization, a significant decrease in Nos2 gene and iNOS protein expression was observed. Deletion of Chop decreased the bioavailability of nitrate in the colon leading to reduced fitness advantage of wild type S. Typhimurium over a napA narZ narG mutant strain (deficient in nitrate respiration). CD11b+ myeloid cells, but not intestinal epithelial cells, produced iNOS resulting in nitrate bioavailability for S. Typhimurium to expand in the intestinal tract in a CHOP-dependent manner. Altogether our work demonstrates that the host protein CHOP facilitates iNOS expression in CD11b+ cells thereby contributing to luminal expansion of S. Typhimurium via nitrate respiration. Author SummarySalmonella Typhimurium is a gastroenteric bacterium that replicates to large numbers within the gastrointestinal (GI) tract allowing for efficient host-to-host transmission. One strategy that allows Salmonella to expand in the GI tract is via nitrate respiration that is generated during Salmonella infections. Our results presented here provide more insight into the role of the host protein CHOP in the production of nitrate and the subsequent growth of Salmonella in the GI tract. CHOP expression is regulated within the unfolded protein response (UPR), an adaptive response pathway that is activated when cells are undergoing endoplasmic reticulum (ER) stress. ER stress has been implicated in several infectious and inflammatory diseases; however, little is known about the contribution of ER stress and the UPR during Salmonella infections. Here, we demonstrate that Chop is downregulated in mice infected with S. Typhimurium, and that mice deficient for CHOP have reduced bacterial numbers in the colon, suggesting that downregulation of Chop is a host response to resist intestinal colonization by Salmonella. Our results further show that CHOP contributes to increased expression of iNOS, responsible for nitrate production, thereby increasing the bioavailability of nitrate that allows for Salmonella growth. Altogether, our research provides a better understanding of the contribution of the ER stress protein CHOP in intestinal health and disease.

microbiology↗