ZC3H12C expression in dendritic cells is necessary to prevent lymphadenopathy of skin-draining lymph nodes.

The role of RNA-binding proteins of the CCCH-containing family in regulating proinflammatory cytokine production and inflammation is increasingly recognized. We have identified ZC3H12C (Regnase-3) as a potential post-transcriptional regulator of tumor necrosis factor expression and have investigated its role in vivo by generating Zc3h12c-deficient mice that express green fluorescent protein instead of ZC3H12C. Zc3h12c-deficient mice develop hypertrophic lymph nodes. In the immune system, ZC3H12C expression is mostly restricted to the dendritic cell (DC) populations, and we show that DC-restricted ZC3H12C depletion is sufficient to cause lymphadenopathy. ZC3H12C can regulate Tnf messenger RNA stability via its RNase activity in vitro, and we confirmed the role of Tnf in the development of lymphadenopathy. Finally, we found that loss of ZC3H12C did not impact the outcome of skin inflammation in the imiquimod-induced murine model of psoriasis, despite Zc3h12c being identified as a risk factor for psoriasis susceptibility in several genome-wide association studies. Our data suggest a role for ZC3H12C in DC-driven skin homeostasis.

A regulatory region on RIPK2 is required for XIAP binding and NOD signaling activity.

Signaling via the intracellular pathogen receptors nucleotide-binding oligomerization domain-containing proteins NOD1 and NOD2 requires receptor interacting kinase 2 (RIPK2), an adaptor kinase that can be targeted for the treatment of various inflammatory diseases. However, the molecular mechanisms of how RIPK2 contributes to NOD signaling are not completely understood. We generated FLAG-tagged RIPK2 knock-in mice using CRISPR/Cas9 technology to study NOD signaling mechanisms at the endogenous level. Using cells from these mice, we were able to generate a detailed map of post-translational modifications on RIPK2. Similar to other reports, we did not detect ubiquitination of RIPK2 lysine 209 during NOD2 signaling. However, using site-directed mutagenesis we identified a new regulatory region on RIPK2, which dictates the crucial interaction with the E3 ligase XIAP and downstream signaling outcomes.

NOD Signaling and Cell Death.

Innate immune signaling and programmed cell death are intimately linked, and many signaling pathways can regulate and induce both, transcription of inflammatory mediators or autonomous cell death. The best-characterized examples for these dual outcomes are members of the TNF superfamily, the inflammasome receptors, and the toll-like receptors. Signaling via the intracellular peptidoglycan receptors NOD1 and NOD2, however, does not appear to follow this trend, despite involving signaling proteins, or proteins with domains that are linked to programmed cell death, such as RIP kinases, inhibitors of apoptosis (IAP) proteins or the CARD domains on NOD1/2. To better understand the connections between NOD signaling and cell death induction, we here review the latest findings on the molecular regulation of signaling downstream of the NOD receptors and explore the links between this immune signaling pathway and the regulation of cell death.

XIAP controls RIPK2 signaling by preventing its deposition in speck-like structures.

The receptor interacting serine/threonine kinase 2 (RIPK2) is essential for linking activation of the pattern recognition receptors NOD1 and NOD2 to cellular signaling events. Recently, it was shown that RIPK2 can form higher order molecular structures in vitro. Here, we demonstrate that RIPK2 forms detergent insoluble complexes in the cytosol of host cells upon infection with invasive enteropathogenic bacteria. Formation of these structures occurred after NF-κB activation and depended on the caspase activation and recruitment domain of NOD1 or NOD2. Complex formation upon activation required RIPK2 autophosphorylation at Y474 and was influenced by phosphorylation at S176. We found that the E3 ligase X-linked inhibitor of apoptosis (XIAP) counteracts complex formation of RIPK2, accordingly mutation of the XIAP ubiquitylation sites in RIPK2 enhanced complex formation. Taken together, our work reveals novel roles of XIAP in the regulation of RIPK2 and expands our knowledge on the function of RIPK2 posttranslational modifications in NOD1/2 signaling.

IAPs Regulate Distinct Innate Immune Pathways to Co-ordinate the Response to Bacterial Peptidoglycans.

Inhibitors of apoptosis (IAPs) proteins are critical regulators of innate immune signaling pathways and therefore have potential as drug targets. X-linked IAP (XIAP) and cellular IAP1 and IAP2 (cIAP1 and cIAP2) are E3 ligases that have been shown to be required for signaling downstream of NOD2, an intracellular receptor for bacterial peptidoglycan. We used genetic and biochemical approaches to compare the responses of IAP-deficient mice and cells to NOD2 stimulation. In all cell types tested, XIAP is the only IAP required for signaling immediately downstream of NOD2, while cIAP1 and cIAP2 are dispensable for NOD2-induced nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) activation. However, mice lacking cIAP1 or TNFR1 have a blunted cytokine response to NOD2 stimulation. We conclude that cIAPs regulate NOD2-dependent autocrine TNF signaling in vivo and highlight the importance of physiological context in the interplay of innate immune signaling pathways.

Genetic evidence for prevalence of alloparental care in a socially monogamous biparental cichlid fish, Perissodus microlepis, from Lake Tanganyika supports the "selfish shepherd effect" hypothesis.

Alloparental care - care for unrelated young - is rare in animals, and its ecological or evolutionary advantages or, alternative maladaptive nature, remain unclear. We investigate alloparental care in the socially monogamous cichlid fish Perissodus microlepis from Lake Tanganyika that exhibits bi-parental care. In a genetic parentage analysis, we discovered a surprisingly high percentage of alloparental care represented by brood mixing, extra-pair paternity and extra-pair maternity in all broods that we investigated. The percentage of nondescendant juveniles of other parents, i.e., brood mixing, ranged from 5% to 57% (mean = 28%). The distribution of genetic parentage also suggests that this socially monogamous species has, in fact, polygamous mating system. The prevalence of genetically mixed broods can be best explained by two, not mutually exclusive hypotheses on farming-out and fostering behaviors. In the majority of broods, the sizes of the parents' own (descendant) offspring were significantly larger than those of the adopted (nondescendant) juveniles, supporting the 'selfish shepherd effect' hypothesis, i.e., that foster parents preferentially accept unrelated "smaller or not larger" young since this would tend to lower the predation risks for their own larger offspring. There was also a tendency for larger parents particularly mothers, more so than smaller parents, to care predominantly for their own offspring. Larger parents might be better at defending against cuckoldry and having foreign young dumped into their broods through farming-out behavior. This result might argue for maladaptive effects of allopatric care for the foster parents that only larger and possibly more experienced pairs can guard against. It needs to be determined why, apparently, the ability to recognize one's own young has not evolved in this species.

Genetic and environmental effects on the morphological asymmetry in the scale-eating cichlid fish, Perissodus microlepis.

The scale-eating cichlid fish, Perissodus microlepis, from Lake Tanganyika are a well-known example of an asymmetry dimorphism because the mouth/head is either left-bending or right-bending. However, how strongly its pronounced morphological laterality is affected by genetic and environmental factors remains unclear. Using quantitative assessments of mouth asymmetry, we investigated its origin by estimating narrow-sense heritability (h (2) ) using midparent-offspring regression. The heritability estimates [field estimate: h (2)  = 0.22 ± 0.06, P = 0.013; laboratory estimate: h (2)  = 0.18 ± 0.05, P = 0.004] suggest that although variation in laterality has some additive genetic component, it is strongly environmentally influenced. Family-level association analyses of a putative microsatellite marker that was claimed to be linked to gene(s) for laterality revealed no association of this locus with laterality. Moreover, the observed phenotype frequencies in offspring from parents of different phenotype combinations were not consistent with a previously suggested single-locus two-allele model, but they neither were able to reject with confidence a random asymmetry model. These results reconcile the disputed mechanisms for this textbook case of mouth asymmetry where both genetic and environmental factors contribute to this remarkable case of morphological asymmetry.