NLRX1/NOD5 deficiency does not affect MAVS signalling

Dear Editor,

NLRX1/NOD5 belongs to the NOD clade of the NOD-like receptor (NLR) family. NLRs have emerged as key intracellular sensors for pathogen-derived molecules and endogenous danger signals to activate innate immune responses and inflammation. However, the function of most of these receptors still remains unclear.¹ Beside the NACHT and LRR domains present in most NLRs, NLRX1/NOD5 possesses a N-terminal mitochondrial targeting sequence.^{2, 3} The function of NLRX1/NOD5 is still controversial; one study proposed that NLRX1/NOD5 inhibits the RIG-like receptor (RLR) antiviral pathways by binding the adaptor protein MAVS/IPS-1/Cardif/VISA,² whereas another report implicated NLRX1/NOD5 in the generation of reactive oxygen species (ROS) and the amplification of NF-κB and JNK activation triggered by different stimuli such TNF-α, poly (I:C) and Shigella infection.³ Interestingly, a recent study proposed that NLRX1/NOD5 is imported to the mitochondrial matrix, making NLRX1/NOD5 interaction with the outer mitochondrial membrane protein MAVS difficult to explain.⁴

To clarify the role of NOD5 in vivo, we generated NOD5-deficient mice by replacing the first four coding exons with a neomycin selection cassette. Mutant mice are viable and born at the expected Mendelian ratios (data not shown). Considering the proposed role for NOD5 in modulating RLR–MAVS signalling,² we tested whether NOD5 deficiency potentiates responses triggered by intracellular poly (I:C). However, when primary NOD5-deficient mouse embryonic fibroblasts (MEFs) were transfected with poly (I:C), we did not observe any major difference in MAVS-dependent IRF3 phosphorylation and IFN-β or IP-10 mRNA induction (Supplementary Figure 1 and data not shown). Moreover, IFN-β and IP-10 mRNA induction upon Sendai virus infection was normal in NOD5-deficient bone marrow-derived macrophages (BMDMs) (Supplementary Figure 1 and data not shown). NOD5-deficient MEFs also produced normal levels of IFN-β and IL-6 upon poly (I:C) stimulation (Supplementary Figure 1 and data not shown). To corroborate these observations in vivo, we assessed the effect of NOD5 deficiency on serum levels of IFN-β and IL-6 induced by intravenous injection of poly (I:C), responses largely dependent on MAVS (data not shown and⁵). Importantly, production of these cytokines was not affected by NOD5 deficiency (Supplementary Figure 1 and data not shown). Thus, NOD5 deficiency does not affect MAVS-dependent responses. A second report links NOD5 to ROS production and amplification of NF-κB and JNK signalling upon TNF-α stimulation or Shigella infection.³ Interestingly, no major differences in TNF-α-induced NF-κB, JNK and p38 activation were observed in NOD5 knockout cells (data not shown). In a complementary approach to define NOD5 function, we carried out a proteomic screen to identify new NOD5 interaction partners. As a significant hit, we found UQCRC2 (ubiquinol-cytochrome-c reductase complex core protein 2), a subunit of the complex III of the mitochondrial respiratory chain. UQCRC2 was previously identified as an NOD5-binding partner.⁴ In contrast, we failed to detect MAVS in the NOD5 immunoprecipitates. In keeping with this, endogenous UQCRC2, but not MAVS, could be co-immunoprecipitated with NOD5 (Supplementary Figure 1 and data not shown).

Collectively, our data indicate that NOD5 deficiency does not affect RLR signalling in vitro and in vivo, at least under the conditions tested. The reasons for the discrepancy between our findings and the in vitro characterisation of NOD5 as a RLR inhibitor are unclear, but are reminiscent of reports investigating NLRC5/NOD4 function; NLRC5/NOD4 was also proposed as RLR inhibitor based on in vitro studies, but no alteration in RLR responses were observed in NLRC5/NOD4-deficient mice.^{6, 7} Although our findings indicate that NOD5 deficiency does not affect TNF-induced signalling, the confirmation of UQCRC2 as a ‘bonafide' interaction partner of NOD5 is consistent with the proposed link between NOD5 and ROS generation.^{3, 4, 8} Mitochondrial ROS has been recently shown to trigger NLRP3 inflammasome activation, and thus modulation of ROS would be an attractive function of NOD5. With the availability of NOD5-deficient mice, this hypothesis is now testable.

The authors declare no conflict of interest.