Pediatric Encephalopathy: Clinical, Biochemical and Cellular Insights into the Role of Gln52 of GNAO1 and GNAI1 for the Dominant Disease.

Heterotrimeric G proteins are immediate transducers of G protein-coupled receptors-the biggest receptor family in metazoans-and play innumerate functions in health and disease. A set of de novo point mutations in GNAO1 and GNAI1, the genes encoding the α-subunits (Gαo and Gαi1, respectively) of the heterotrimeric G proteins, have been described to cause pediatric encephalopathies represented by epileptic seizures, movement disorders, developmental delay, intellectual disability, and signs of neurodegeneration. Among such mutations, the Gln52Pro substitutions have been previously identified in GNAO1 and GNAI1. Here, we describe the case of an infant with another mutation in the same site, Gln52Arg. The patient manifested epileptic and movement disorders and a developmental delay, at the onset of 1.5 weeks after birth. We have analyzed biochemical and cellular properties of the three types of dominant pathogenic mutants in the Gln52 position described so far: Gαo[Gln52Pro], Gαi1[Gln52Pro], and the novel Gαo[Gln52Arg]. At the biochemical level, the three mutant proteins are deficient in binding and hydrolyzing GTP, which is the fundamental function of the healthy G proteins. At the cellular level, the mutants are defective in the interaction with partner proteins recognizing either the GDP-loaded or the GTP-loaded forms of Gαo. Further, of the two intracellular sites of Gαo localization, plasma membrane and Golgi, the former is strongly reduced for the mutant proteins. We conclude that the point mutations at Gln52 inactivate the Gαo and Gαi1 proteins leading to aberrant intracellular localization and partner protein interactions. These features likely lie at the core of the molecular etiology of pediatric encephalopathies associated with the codon 52 mutations in GNAO1/GNAI1.

The severity of imiquimod-induced mouse skin inflammation is independent of endogenous IL-38 expression.

The IL-1 cytokine family includes eleven members, among which Il-36α, ß and γ, IL-36Ra and IL-38. The IL-36 cytokines are involved in the pathogenesis of psoriasis. IL-38 is also expressed in the skin and was previously proposed to act as an IL-36 antagonist. In this study, we thus examined expression and function of Il-38 in a mouse model of imiquimod (IMQ)-induced skin inflammation. Il-38 mRNA was detected in the epidermis and in primary mouse keratinocytes, but not in dermal fibroblasts. At the peak of IMQ-induced inflammation, skin Il-38 mRNA levels were reduced, whereas Il-36ra mRNA expression increased. The severity of IMQ-induced skin inflammation, as assessed by recording ear thickness and histological changes, was similar in Il-38 KO and WT littermate control mice, while, in contrast, Il-36ra-deficient mice displayed more severe skin pathology than their WT littermates. Il-38-deficiency had no impact on IMQ-induced expression of proinflammatory mediators in the skin in vivo, on the basal expression of various cytokines or chemokines by cultured primary keratinocytes and dermal fibroblasts in vitro, or on the response of these cells to Il-36ß. Finally, after cessation of topical IMQ application, the resolution of skin inflammation was also not altered in Il-38 KO mice. In conclusion, Il-38-deficiency did not impact the development or resolution of IMQ-induced skin inflammation. Our observations further suggest that endogenous Il-38 does not exert Il-36 inhibitory activity in this model, or in cultured skin cells. A potential anti-inflammatory function of Il-38 in mouse skin thus still remains to be demonstrated.

Unopposed IL-18 signaling leads to severe TLR9-induced macrophage activation syndrome in mice.

The term macrophage activation syndrome (MAS) defines a severe, potentially fatal disorder characterized by overwhelming inflammation and multiorgan involvement. Interleukin-18 (IL-18) is a proinflammatory cytokine belonging to the IL-1 family, the activity of which is regulated by its endogenous inhibitor IL-18 binding protein (IL-18BP). Elevated IL-18 levels have been reported in patients with MAS. Herein, we show that on repeated toll-like receptor 9 (TLR9) stimulation with unmethylated cytosine guanine dinucleotide containing single-stranded DNA (CpG), IL-18BP-/- mice display severe MAS manifestations, including increased weight loss, splenomegaly, anemia, thrombocytopenia, hyperferritinemia, and bone marrow hemophagocytosis as compared with wild-type mice. Serum-free IL-18 was detected in CpG-treated IL-18BP-/- mice only. Levels of interferon-γ (IFN-γ) and of IFN-γ signature genes, such as the chemokine Cxcl9 or the transcription factor CIIta, were significantly increased in IL-18BP-/- mice. Blocking IL-18 receptor signaling attenuated the severity of MAS and IFN-γ responses in IL-18BP-/- mice. Blocking IFN-γ had comparable effects to IL-18 inhibition on most MAS manifestations. Our data indicate that endogenous IL-18BP exerts a protective role in CpG-induced MAS and that IL-18, which acts upstream of IFN-γ, is involved in the severity of MAS.

IL-36-Induced Toxicity in Neonatal Mice Involves TNF-α Production by Liver Myeloid Cells.

Human and mouse neonates exhibit limited vaccine responses characterized by predominant Th2 and limited Th1 responses. Because IL-36 exerts a synergic adjuvant effect with IL-12, enhancing Th1 polarization in adult (AD) mice, we administered IL-36ß to neonatal (1-wk old) and AD control mice at the time of immunization with tetanus toxoid adsorbed to aluminum hydroxide (TT/Alum). Unexpectedly, the combination of IL-36ß with TT/Alum, which was well tolerated in AD mice, proved toxic and even lethal in neonates. This neonatal toxicity was associated with high Il36r mRNA expression in neonatal liver, resulting in increased cytokine production. Liver Il36r mRNA expression decreased with the termination of fetal liver hematopoiesis, and this decrease correlated with a complete protection from TT/Alum/IL-36ß-induced mortality. The combination of IL-36ß and TT/Alum induced the rapid production of TNF-α and IFN-γ by liver myeloid and lymphoid cells, respectively. These responses were less marked when IL-36ß was used alone, with no adverse effect. The toxicity of IL-36ß + TT/Alum was abrogated by the administration of a neutralizing anti-TNF-α Ab, confirming causality. In conclusion, liver myeloid cells in neonatal mice are an important source of proinflammatory cytokines that may lead to TNF-α-mediated toxicity and even lethality.