The paracaspase MALT1 plays an important role in immune receptor-driven signaling pathways leading to NF-κB activation. MALT1 promotes signaling by acting as a scaffold, recruiting downstream signaling proteins, as well as by proteolytic cleavage of multiple substrates. However, the relative contributions of these two different activities to T and B cell function are not well understood. To investigate how MALT1 proteolytic activity contributes to overall immune cell regulation, we generated MALT1 protease-deficient mice (Malt1PD/PD) and compared their phenotype with that of MALT1 knockout animals (Malt1−/−). Malt1PD/PD mice displayed defects in multiple cell types including marginal zone B cells, B1 B cells, IL-10–producing B cells, regulatory T cells, and mature T and B cells. In general, immune defects were more pronounced in Malt1−/− animals. Both mouse lines showed abrogated B cell responses upon immunization with T-dependent and T-independent Ags. In vitro, inactivation of MALT1 protease activity caused reduced stimulation-induced T cell proliferation, impaired IL-2 and TNF-α production, as well as defective Th17 differentiation. Consequently, Malt1PD/PD mice were protected in a Th17-dependent experimental autoimmune encephalomyelitis model. Surprisingly, Malt1PD/PD animals developed a multiorgan inflammatory pathology, characterized by Th1 and Th2/0 responses and enhanced IgG1 and IgE levels, which was delayed by wild-type regulatory T cell reconstitution. We therefore propose that the pathology characterizing Malt1PD/PD animals arises from an immune imbalance featuring pathogenic Th1- and Th2/0-skewed effector responses and reduced immunosuppressive compartments. These data uncover a previously unappreciated key function of MALT1 protease activity in immune homeostasis and underline its relevance in human health and disease.

In mammals, ceramide kinase (CerK)-mediated phosphorylation of ceramide is the only known pathway to ceramide-1-phosphate (C1P), a recently identified signaling sphingolipid metabolite. To help delineate the roles of CerK and C1P, we knocked out the gene of CerK in BALB/c mice by homologous recombination. All in vitro as well as cell-based assays indicated that CerK activity is completely abolished in Cerk−/− mice. Labeling with radioactive orthophosphate showed a profound reduction in the levels of de novo C1P formed in Cerk−/− macrophages. Consistently, mass spectrometry analysis revealed a major contribution of CerK to the formation of C16-C1P. However, the significant residual C1P levels in Cerk−/− animals indicate that alternative routes to C1P exist. Furthermore, serum levels of proapoptotic ceramide in these animals were significantly increased while levels of dihydroceramide as the biosynthetic precursor were reduced. Previous literature pointed to a role of CerK or C1P in innate immune cell function. Using a variety of mechanistic and disease models, as well as primary cells, we found that macrophage- and mast cell-dependent readouts are barely affected in the absence of CerK. However, the number of neutrophils was strikingly reduced in blood and spleen of Cerk−/− animals. When tested in a model of fulminant pneumonia, Cerk−/− animals developed a more severe disease, lending support to a defect in neutrophil homeostasis following CerK ablation. These results identify ceramide kinase as a key regulator of C1P, dihydroceramide and ceramide levels, with important implications for neutrophil homeostasis and innate immunity regulation.

Ceramide kinase (CERK) produces the bioactive lipid ceramide-1-phosphate (C1P) and is a key regulator of ceramide and dihydroceramide levels. It is likely that CERK and C1P play a role in inflammatory processes but the cells involved and the mechanisms used remain to be clarified. In particular, the impact of CERK on T-cell biology has not been studied so far. Here, we used Cerk-/- mice backcrossed with DO11.10/RAG1-/- mice to probe the effect of CERK ablation on T-cell activation. Levels of interleukin (IL)-2, IL-4, IL-5, IL-13, of tumor necrosis factor (TNF)-α, and of interferon (INF)-γ were recorded following ovalbumin challenge in vivo and using ovalbumin-treated splenocytes ex- vivo. Absence of CERK led to a significant decrease in the production of IL-4, thus suggesting that CERK may polarize T cells towards the TH2 cell subtype. However, the importance of CERK to TH2 cell biology will have to be investigated further because in a model of asthma, which is TH2-cell driven, Cerk-/- mice responded like wild-type animals.

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