DUOX2-derived reactive oxygen species are effectors of NOD2-mediated antibacterial responses

Abstract

Generation of microbicidal reactive oxygen species (ROS) is a pivotal protective component of the innate immune system in many eukaryotes. NOD (nucleotide oligomerisation domain containing protein)-like receptors (NLRs) have been implicated as phylogenetically ancient sensors of intracellular pathogens or endogenous danger signals. NOD2 recognizes the bacterial cell wall component muramyldipeptide leading to NFκB and MAPK activation via induced proximity signalling through the serine-threonine kinase RIP2. In addition to the subsequent induction of cytokines and antimicrobial peptides, NOD2 has been shown also to exert a direct antibacterial effect. Using a fluorescence-based ROS detection assay we demonstrate controlled ROS generation as an integral component of NOD2-induced signalling in epithelial cells. We demonstrate that the NAD(P)H oxidase family member DUOX2 is involved in NOD2-dependent ROS production. Coimmunoprecipitation and fluorescence microscopy were used to show that DUOX2 interacts and colocalizes with NOD2 at the plasma membrane. Moreover, simultaneous overexpression of NOD2 and DUOX2 was found to result in cooperative protection against bacterial cytoinvasion using the Listeria monocytogenes infection model. RNAi-based studies revealed that DUOX2 is required for the direct bactericidal properties of NOD2. Our results demonstrate a new role of ROS as effector molecules of protective cellular signalling in response to a defined danger signal carried out by a mammalian intracellular NLR system.

The protein Nod2: An innate receptor more complex than previously assumed

“…This hypothesis is consistent with two observations: first, the reduced expression of a-defensins at ileal mucosa of CD patients with mutant Nod2 [89] and secondly, the correlation between the increased susceptibility of Nod2-deficient mice to L. monocytogenes oral infection and diminished expression of Paneth cell-derived antimicrobial peptides [68]. On the other hand, the Nod2-dependent bacteria clearance mechanisms such as autophagy and bactericidal ROS production can be compromised in intestinal epithelial and dendritic cells expressing a CD-associated Nod2 mutant [57,58,63]. Interestingly, a ''loss-of-function'' mutation (T300A) in ATG16L1, a key regulator of autophagy, was also associated with a higher susceptibility for CD [90].…”

“…Given the link between plant R proteins and pathogen-induced reactive oxygen species (ROS) production [61] as well as the interaction between NOX family members and components of the TLR signalling cascade [62], Lipinski et al explored whether Nod2 signalling mediated ROS production and if this mechanism contributed to host defense [63]. They showed a rapid and significant Nod2-dependent ROS production in MDP-stimulated intestinal epithelial cells (IECs) which was mainly mediated by DUOX2, a dual oxidase generating hydrogen peroxide.…”

RNAi screening identifies mediators of NOD2 signaling: Implications for spatial specificity of MDP recognition

“…S2B). Because NOD2 signaling is linked to antimicrobial mechanisms (10,22,24), we also analyzed bacterial cytoinvasion using fluorescence-labeled Listeria monocytogenes. Knockdown of all six genes significantly altered bacterial entry and (except for KPNB1) led to an increased intracellular bacterial load (Fig.…”

Immune Recognition in Lung Diseases: Basic Research and Clinical Application

“…NOD2 senses S. pneumoniae, S. aureus, Escherichia coli, C. pneumoniae and M. tuberculosis [41][42][43]. NOD1 and NOD2 both activated downstream signaling through Rip2 kinase, leading to the expression, NFkB dependent, of pro-inflammatory mediators and the production of reactive oxygen species (ROS) [44].…”