The Kinase Activity of Rip2 Determines Its Stability and Consequently Nod1- and Nod2-mediated Immune Responses

Rip2 (RICK, CARD3) has been identified as a key effector molecule downstream of the pattern recognition receptors, Nod1 and Nod2; however, its mechanism of action remains to be elucidated. In particular, it is unclear whether its kinase activity is required for signaling or for maintaining protein stability. We have investigated the expression level of different retrovirally expressed kinase-dead Rip2 mutants and the role of Rip2 kinase activity in the signaling events that follow Nod1 and Nod2 stimulation. We show that in primary cells expressing kinase-inactive Rip2, protein levels were severely compromised, and stability could not be reconstituted by the addition of a phospho-mimetic mutation in its autophosphorylation site. Consequently, inflammatory cytokine production in response to Nod1 and Nod2 ligands was abrogated both in vitro and in vivo in the absence of Rip2 kinase activity. Our results highlight the central role that Rip2 kinase activity plays in conferring stability to the protein and thus in the preservation of Nod1- and Nod2-mediated innate immune responses.A key step in the initiation of effector immune responses is the recognition of highly conserved molecules expressed by microbial pathogens. The immune system has developed specific receptors that sense these so-called pathogen-associated molecular patterns and initiate appropriate immune responses. One key family of pattern recognition receptors is the Nod-like receptor (NLR)² family (1–3), of which two members, Nod1 and Nod2, have been implicated in the recognition of bacterial peptidoglycan derivatives released into the cytosol upon bacterial infection (4–6). Several studies have shown that Nod1 plays a role in host defense against invasive pathogens such as Helicobacter pylori and Escherichia coli (7, 8), and Nod2 mutations have been associated with a higher incidence of Crohn disease (9, 10), thus highlighting these NLRs as important regulators of inflammatory immune responses.Rip2, also called CARD3, RICK, or CARDIAK, is a serine/threonine kinase, which was implicated in the induction of NF-κB activation and apoptosis (11–13). Rip2 has been described to be critical for responses against Toll-like receptor ligands such as LPS (14, 15), although findings from recent studies did not support this conclusion (16). Rip2 contains a caspase-recruitment domain (CARD), which mediates interaction with other CARD-containing proteins such as Nod1 and Nod2, in addition to an N-terminal kinase domain and an intermediate domain. Nod1 and Nod2 associate with Rip2 upon peptidoglycan ligation (17) leading to downstream signaling events that culminate in NF-κB and mitogen-activated protein kinase activation (15, 18–20). Recent reports have suggested that the mitogen-activated protein kinase kinase kinase family member TAK1 provides the link between Rip2 and NF-κB activation upon Nod1 and Nod2 stimulation (21–23). However, the exact role of Rip2 and in particular its kinase activity in mediating downstream effector activation in NLR signaling still remains unclear. Notably, in vitro investigations have suggested that Rip2 kinase activity may be dispensable for the induction of immune responses initiated by NLR-ligands (21, 24, 25) and that disruption of Rip2 kinase activity is associated with a loss in protein stability (23); however, such studies utilized protein overexpression in cell lines and are yet to be tested in primary cells or in vivo.In the current investigation we sought to elucidate the role of Rip2 kinase activity in transducing inflammatory signals upon NLR stimulation in vitro and in vivo. To this end, we utilized both Rip2 knock-out (15) and Rip2 kinase-dead knock-in mice (24) in addition to Rip2 deficient primary cells that were retrovirally reconstituted with different kinase-inactive mutants. We show here that in the absence of intact kinase activity, Rip2 protein is not stable and that insertion of a phospho-mimetic mutation is not sufficient to restore stability. Moreover, pharmacological abrogation of Rip2 kinase activity in primary cells similarly leads to destabilization of the molecule. As a consequence, signaling downstream of Nod1 and Nod2 and inflammatory cytokine production is impaired both in vivo and in vitro. Our results highlight Rip2 kinase activity as a central regulator of protein stability and consequently innate immune responses triggered by Nod1 and Nod2 ligands.     

Suitability of Sludge Biotic Index (SBI), Sludge Index (SI) and filamentous bacteria analysis for assessing activated sludge process performance: the case of piggery slaughterhouse wastewater

Piggery slaughterhouse wastewater poses serious issues in terms of disposal feasibility and environmental impact, due to its huge organic load and variability. It is commonly treated by means of activated sludge processes, whose performance, in case of municipal wastewater, can be monitored by means of specific analyses, such as Sludge Biotic Index (SBI), Sludge Index (SI) and floc and filamentous bacteria observation. Therefore, this paper was aimed at assessing the applicability of these techniques to piggery slaughterhouse sewage. A plant located in Northern Italy was monitored for 1 year. Physical, chemical and operation parameters were measured; the activated sludge community (ciliates, flagellates, amoebae and small metazoa) was analysed for calculating SBI and SI. Floc and filamentous bacteria were examined and described accordingly with internationally adopted criteria. The results showed the full applicability of the studied techniques for optimizing the operation of a piggery slaughterhouse wastewater treatment plant.     

TLR ligands act directly upon T cells to restore proliferation in the absence of protein kinase C-theta signaling and promote autoimmune myocarditis

The serine/threonine kinase, protein kinase C-theta (PKC-theta), plays a central role in the activation and differentiation of Th2 cells while being redundant in CD4+ and CD8+ antiviral responses. Recent evidence indicates that PKC-theta may however be required for some T cell-driven autoimmune responses. We have investigated the role of PKC-theta in the induction of autoimmune myocarditis induced by either Coxsackie B3 virus infection or immunization with alpha-myosin/CFA (experimental autoimmune myocarditis (EAM)). PKC-theta-deficient mice did not develop EAM as shown by impaired inflammatory cell infiltration into the heart, reduced CD4+ T cell IL-17 production, and the absence of a myosin-specific Ab response. Comparatively, PKC-theta was not essential for both early and late-phase Coxsackie virus-induced myocarditis. We sought to find alternate pathways of immune stimulation that might reconcile the differential requirements for PKC-theta in these two disease models. We found systemic administration of the TLR ligand CpG restored EAM in PKC-theta-deficient mice. CpG could act directly upon TLR9-expressing T cells to restore proliferation and up-regulation of Bcl-x(L), but exogenous IL-6 and TGF-beta was required for Th17 cell differentiation. Taken together, these results indicate that TLR-mediated activation of T cells can directly overcome the requirement for PKC-theta signaling and, combined with the dendritic cell-derived cytokine milieu, can promote the development of autoimmunity.     

VEGF-C promotes immune tolerance in B16 melanomas and cross-presentation of tumor antigen by lymph node lymphatics

Tumor expression of the lymphangiogenic factor VEGF-C is correlated with metastasis and poor prognosis, and although VEGF-C enhances transport to the draining lymph node (dLN) and antigen exposure to the adaptive immune system, its role in tumor immunity remains unexplored. Here, we demonstrate that VEGF-C promotes immune tolerance in murine melanoma. In B16 F10 melanomas expressing a foreign antigen (OVA), VEGF-C protected tumors against preexisting antitumor immunity and promoted local deletion of OVA-specific CD8(+) T cells. Naive OVA-specific CD8(+) T cells, transferred into tumor-bearing mice, were dysfunctionally activated and apoptotic. Lymphatic endothelial cells (LECs) in dLNs cross-presented OVA, and naive LECs scavenge and cross-present OVA in vitro. Cross-presenting LECs drove the proliferation and apoptosis of OVA-specific CD8(+) T cells ex vivo. Our findings introduce a tumor-promoting role for lymphatics in the tumor and dLN and suggest that lymphatic endothelium in the local microenvironment may be a target for immunomodulation.     

Characterization of microsatellite loci in the wall lizard Podarcis muralis (Sauria: Lacertidae)

In natural populations the mating success of males depends on different factors. By enhancing the intensity of secondary sexual characters, testosterone can play a role in mate choice. However, paradoxically, testosterone can also decrease immunocompetence and thus potentially diminish attractiveness. To estimate the influence of testosterone on male mating success in the wall lizard, Podarcis muralis, we characterized nine polymorphic microsatellite loci. There were three to 12 alleles per locus in the five to 16 individuals screened. These microsatellites will also be useful in determining population structure in this species.     

Mechanisms of neonatal mucosal antibody protection

Following an abrupt transition at birth from the sterile uterus to an environment with abundant commensal and pathogenic microbes, neonatal mammals are protected by maternal Abs at mucosal surfaces. We show in mice that different Ab isotypes work in distinct ways to protect the neonatal mucosal surface. Secretory IgA acts to limit penetration of commensal intestinal bacteria through the neonatal intestinal epithelium: an apparently primitive process that does not require diversification of the primary natural Ab repertoire. In contrast, neonatal protection against the exclusively luminal parasite Heligmosomoides polygyrus required IgG from primed females. This immune IgG could either be delivered directly in milk or retrotransported via neonatal Fc receptor from the neonatal serum into the intestinal lumen to exert its protective effect.     

Modelling a wireless connected swarm of mobile robots

It is a characteristic of swarm robotics that modelling the overall swarm behaviour in terms of the low-level behaviours of individual robots is very difficult. Yet if swarm robotics is to make the transition from the laboratory to real-world engineering realisation such models would be critical for both overall validation of algorithm correctness and detailed parameter optimisation. We seek models with predictive power: models that allow us to determine the effect of modifying parameters in individual robots on the overall swarm behaviour. This paper presents results from a study to apply the probabilistic modelling approach to a class of wireless connected swarms operating in unbounded environments. The paper proposes a probabilistic finite state machine (PFSM) that describes the network connectivity and overall macroscopic behaviour of the swarm, then develops a novel robot-centric approach to the estimation of the state transition probabilities within the PFSM. Using measured data from simulation the paper then carefully validates the PFSM model step by step, allowing us to assess the accuracy and hence the utility of the model.     

Strong TCR signaling, TLR ligands, and cytokine redundancies ensure robust development of type 1 effector T cells

T cell effector function is a central mechanism of adaptive immunity, and accordingly, protection of the host against pathogens. One of the primary effector molecules produced by T cells in response to such pathogens is the cytokine, IFN-gamma. Although the signaling pathways associated with the production of IFN-gamma are well established, disparate in vivo and in vitro results indicate that distinct pathways may become more prominent dependent upon the nature of the infection, inflammatory milieu and tissue localization. We have examined the roles and requirements of the major IFN-gamma-inducing pathways in vivo and in vitro, specifically: strength of TCR signal; paracrine release of IL-12, IL-23, and IL-18; and autocrine production of IFN-gamma. Our data show a dynamic interaction between these activation pathways, which allows the host a degree of flexibility and redundancy in the induction of IFN-gamma. Upon strong signaling through the TCR, IL-12, IL-18, and IL-23 play negligible roles in the induction of IFN-gamma, whereas autocrine IFN-gamma is an important component in sustaining its own secretion. However, the absence of any one of these factors during a weaker TCR signal, results in strikingly impaired T cell IFN-gamma production. Of note, TLR-activated dendritic cells (DCs) were capable of overcoming the absence of a strong TCR signal, IL-12, IL-23, or IL-18 revealing an important additional mechanism for ensuring a robust IFN-gamma response. Our findings clarify the hierarchical requirements of the major IFN-gamma inducing pathways and highlight the important role TLR ligand-activated DCs have to preserve them.     

The manganese cycle in Lac Léman,Switzerland: the role ofMetallogenium

The manganese pathways in Lac Léman have been investigated on the basis of chemical analyses undertaken on water, suspended solids, bottom sediments and interstitial water samples. The various modes of occurrence of Mn have been determined by means of visual examination using SEM and TEM (scanning and transmission electron microscopy), by microanalysis (EDAX) of various sediment fractions, by chemical analysis of the dissolved phase, and by chemical speciation and XRD of bottom sediments. Fluxes to and from the sediment have also been computed. The time-depth variations of Mn in the water column are characterized by (a) a very steep gradient of Mn sol. from the sediment interstitial water (15 mg l⁻¹) to the overlying water, 2 m above the bottom(500 μg l⁻¹), (b) an accumulation of Mn part. between 280 m and the interface at 310 m, consisting of mineralized colonies ofMetallogenium. The abundance ofMetallogenium colonies is inversely related to O₂ concentration; the optimal value for the bacterium Mn fixation is around 1 mg l⁻¹. Because of the quasi-anoxic state of the bottom sediments and overlying water, Mn diffuses from a ‘source layer’, 2–5 cm below the interface (a) upwards across the interface, before being taken up byMetallogenium, and (b) downwards to a ‘sink layer’, in which large amounts of Mn-carbonate precipitate. Particulate Mn sedimentation rates measured in traps show that downwards Mn flux due toMetallogenium settling approximately balances the upwards soluble flux due to diffusion. Quantitatively, the process of Mn cycling in Lac Léman is, therefore, limited to the lowermost part of the hypolimnion. Although Zn and Cd seem to follow the same cycle as Mn, Fe behaves in a different manner; it was not taken up byMetallogenium at the time of our study.