Characterization of Novel Pathogenic Variants Leading to Caspase-8 Cleavage-Resistant RIPK1-Induced Autoinflammatory Syndrome

Pathogenic RIPK1 variants have been described as the cause of two different inborn errors of immunity. Biallelic loss-of-function variants cause the recessively inherited RIPK1 deficiency, while monoallelic variants impairing the caspase-8-mediated RIPK1 cleavage provoke a novel autoinflammatory disease (AID) called cleavage-resistant RIPK1-induced autoinflammatory (CRIA) syndrome. The aim of this study was to characterize the pathogenicity of two novel RIPK1 variants located at the cleavage site of caspase-8 detected in patients with dominantly-inherited, early-onset undefined AID. RIPK1 genotyping was performed by Sanger and next-generation sequencing. Clinical and analytical data were collected from medical charts, and in silico and in vitro assays were performed to evaluate the functional consequences. Genetic analyses identified two novel heterozygous RIPK1 variants at the caspase-8 cleavage site (p.Leu321Arg and p.Asp324Gly), which displayed a perfect intrafamilial phenotype-genotype segregation following a dominant inheritance pattern. Structural analyses suggested that these variants disrupt the normal RIPK1 structure, probably making it less accessible to and/or less cleavable by caspase-8. In vitro experiments confirmed that the p.Leu321Arg and p.Asp324Gly RIPK1 variants were resistant to caspase-8-mediated cleavage and induced a constitutive activation of necroptotic pathway in a similar manner that previously characterized RIPK1 variants causing CRIA syndrome. All these results strongly supported the pathogenicity of the two novel RIPK1 variants and the diagnosis of CRIA syndrome in all enrolled patients. Moreover, the evidences here collected expand the phenotypic and genetic diversity of this recently described AID, and provide interesting data about effectiveness of treatments that may benefit future patients

P2X7 receptor induces mitochondrial failure in monocytes and compromises NLRP3 inflammasome activation during sepsis

International audienceSepsis is characterized by a systemic inflammatory response followed by immunosuppres-sion of the host. Metabolic defects and mitochondrial failure are common in immunocom-promised patients with sepsis. The NLRP3 inflammasome is important for establishing an inflammatory response after activation by the purinergic P2X7 receptor. Here, we study a cohort of individuals with intra-abdominal origin sepsis and show that patient monocytes have impaired NLRP3 activation by the P2X7 receptor. Furthermore, most sepsis-related deaths are among patients whose NLRP3 activation is profoundly altered. In monocytes from sepsis patients, the P2X7 receptor is associated with mitochondrial dysfunction. Furthermore, activation of the P2X7 receptor results in mitochondrial damage, which in turn inhibits NLRP3 activation by HIF-1α. We show that mortality increases in a mouse model of sepsis when the P2X7 receptor is activated in vivo. These data reveal a molecular mechanism initiated by the P2X7 receptor that contributes to NLRP3 impairment during infection

Caracterización filogenética, molecular y funcional de nuevos miembros de la familia de la interluquina-1 de peces teleósteos= Phylogenetic, molecular and functional characterization of new interleukin-1 family members in teleost fish.

1. PALABRAS CLAVE Inmunidad, interleuquina-1, inflamasoma, piroptosis, actividad caspasa-1, Salmonella typhimurium, evolución, filogenia, sintenia, dorada, pez cebra, peces, teleósteos. 2. RESUMEN En la presente tesis doctoral se ha estudiado la evolución de las funciones del inflamasoma en peces, llegando a la conclusión de que tanto el inflamasoma como la caspasa-1 desencadenan muerte celular conocida como piroptosis, pero no son necesarios para el procesamiento de la IL-1. Además se ha desarrollado un modelo de infección en pez cebra para el estudio del papel del inflamasoma en la eliminación de bacterias intracelulares, como Salmonella. Por último, se ha descrito un nuevo miembro de la familia de la IL-1, IL-1Fm2, perteneciente exclusivamente a los peces teleósteos más avanzados evolutivamente. La identificación de este nuevo miembro de la familia de la IL-1, IL-1Fm2, ayuda a entender la evolución de esta familia de citoquinas en vertebrados e indica su gran complejidad. 3. SUMMARY In the present thesis, we have studied the evolution of the inflammasome functions in fish. We observed that inflammasome and caspase-1 trigger pyroptotic cell death but they are not involved in the processing of IL-1. In addition, we have developed a zebrafish-Salmonella infection model to study the role of the inflammasome in the clearance of intracellular bacteria. Finally, we have identified and characterized a new member of the IL-1 family, IL-1Fm2, which is exclusively present in most evolutionarily advanced teleosts. The identification of this new IL-1 family member helps to understand the evolution of this family of cytokines in vertebrates and point out to its complexity

NLRP3 Inflammasome and Pyroptosis in Liver Pathophysiology: The Emerging Relevance of Nrf2 Inducers

Inflammasomes, particularly the nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain containing 3 (NLRP3) inflammasome, apparently serve as crucial regulators of the inflammatory response through the activation of Caspase-1 and induction of pro-inflammatory cytokines and pyroptotic cell death. Pyroptosis is a type of programmed cell death mediated by Caspase-1 cleavage of Gasdermin D and the insertion of its N-terminal fragment into the plasma membrane, where it forms pores, enabling the release of different pro-inflammatory mediators. Pyroptosis is considered not only a pro-inflammatory pathway involved in liver pathophysiology but also an important pro-fibrotic mediator. Diverse molecular mechanisms linking oxidative stress, inflammasome activation, pyroptosis, and the progression of liver pathologies have been documented. Numerous studies have indicated the protective effects of several antioxidants, with the ability to induce nuclear factor erythroid 2-related factor 2 (Nrf2) activity on liver inflammation and fibrosis. In this review, we have summarised recent studies addressing the role of the NLRP3 inflammasome and pyroptosis in the pathogenesis of various hepatic diseases, highlighting the potential application of Nrf2 inducers in the prevention of pyroptosis as liver protective compounds

Sensing low intracellular potassium by NLRP3 results in a stable open structure that promotes inflammasome activation

The NLRP3 inflammasome is activated by a wide range of stimuli and drives diverse inflammatory diseases. The decrease of intracellular K+ concentration is a minimal upstream signal to most of the NLRP3 activation models. Here, we found that cellular K+ efflux induces a stable structural change in the inactive NLRP3, promoting an open conformation as a step preceding activation. This conformational change is facilitated by the specific NLRP3 FISNA domain and a unique flexible linker sequence between the PYD and FISNA domains. This linker also facilitates the ensemble of NLRP3PYD into a seed structure for ASC oligomerization. The introduction of the NLRP3 PYD-linker-FISNA sequence into NLRP6 resulted in a chimeric receptor able to be activated by K+ efflux–specific NLRP3 activators and promoted an in vivo inflammatory response to uric acid crystals. Our results establish that the amino-terminal sequence between PYD and NACHT domain of NLRP3 is key for inflammasome activation.I.H.-B. would like to acknowledge the funding by the Slovenian Research Agency (project grant J3-1746 and core funding P4-0176), and B.O. would like to acknowledge the funding by the Ministerio de Economía, Industria y Competitividad and ERDF (BIO2017-85329-R). This work was supported by grants to A.T.-A. from the internal support program of the Medical Faculty, University of Tübingen, Fortüne-Antrag Nr. 2615-0-0 and to P.P. from FEDER/Ministerio de Ciencia, Innovación y Universidades—Agencia Estatal de Investigación (grant SAF2017-88276-R), Fundación Séneca (grants 20859/PI/18, 21081/PDC/19, and 0003/COVI/20), and the European Research Council (ERC-2013-CoG grant 614578 and ERC-2019-PoC grant 899636)

Pathogenic NLRP3 mutants form constitutively active inflammasomes resulting in immune-metabolic limitation of IL-1β production

Abstract Cryopyrin-associated periodic syndrome (CAPS) is an autoinflammatory condition resulting from monoallelic NLRP3 variants that facilitate IL-1β production. Although these are gain-of-function variants characterized by hypersensitivity to cell priming, patients with CAPS and animal models of the disease may present inflammatory flares without identifiable external triggers. Here we find that CAPS-associated NLRP3 variants are forming constitutively active inflammasome, which induce increased basal cleavage of gasdermin D, IL-18 release and pyroptosis, with a concurrent basal pro-inflammatory gene expression signature, including the induction of nuclear receptors 4 A. The constitutively active NLRP3-inflammasome of CAPS is responsive to the selective NLRP3 inhibitor MCC950 and its activation is regulated by deubiquitination. Despite their preactivated state, the CAPS inflammasomes are responsive to activation of the NF-κB pathway. NLRP3-inflammasomes with CAPS-associated variants affect the immunometabolism of the myeloid compartment, leading to disruptions in lipids and amino acid pathways and impaired glycolysis, limiting IL-1β production. In summary, NLRP3 variants causing CAPS form a constitutively active inflammasome inducing pyroptosis and IL-18 release without cell priming, which enables the host’s innate defence against pathogens while also limiting IL-1β–dependent inflammatory episodes through immunometabolism modulation

Characterization of novel pathogenic variants leading to caspase-8 cleavage-resistant RIPK1-induced autoinflammatory syndrome

Pathogenic RIPK1 variants have been described as the cause of two different inborn errors of immunity. Biallelic loss-of-function variants cause the recessively inherited RIPK1 deficiency, while monoallelic variants impairing the caspase-8-mediated RIPK1 cleavage provoke a novel autoinflammatory disease (AID) called cleavage-resistant RIPK1-induced autoinflammatory (CRIA) syndrome. The aim of this study was to characterize the pathogenicity of two novel RIPK1 variants located at the cleavage site of caspase-8 detected in patients with dominantly-inherited, early-onset undefined AID. RIPK1 genotyping was performed by Sanger and next-generation sequencing. Clinical and analytical data were collected from medical charts, and in silico and in vitro assays were performed to evaluate the functional consequences. Genetic analyses identified two novel heterozygous RIPK1 variants at the caspase-8 cleavage site (p.Leu321Arg and p.Asp324Gly), which displayed a perfect intrafamilial phenotype-genotype segregation following a dominant inheritance pattern. Structural analyses suggested that these variants disrupt the normal RIPK1 structure, probably making it less accessible to and/or less cleavable by caspase-8. In vitro experiments confirmed that the p.Leu321Arg and p.Asp324Gly RIPK1 variants were resistant to caspase-8-mediated cleavage and induced a constitutive activation of necroptotic pathway in a similar manner that previously characterized RIPK1 variants causing CRIA syndrome. All these results strongly supported the pathogenicity of the two novel RIPK1 variants and the diagnosis of CRIA syndrome in all enrolled patients. Moreover, the evidences here collected expand the phenotypic and genetic diversity of this recently described AID, and provide interesting data about effectiveness of treatments that may benefit future patients.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work has been partially funded by the following: Grants from the Spanish Ministry of Science, Innovation and Universities and co-funded by the European Regional Development Fund / Agencia Estatal de Investigación: RTI2018-096824-B-C21 (JIA); RTI2018-096824-B-C22 (FC); SAF2017-88276-R (PP). Grant from the Spanish Ministry of Science and Innovation / Agencia Estatal de Investigación (10.13039/501100011033): PID2020-116709RB-I00 (PP). Grant from Instituto de Salud Carlos III and co-funded by the European Union: PI19/01567 (AM-V). Grants from Fundación Séneca: 20859/PI/18 (PP); 21081/PDC/19 (PP); 0003/COVI/20 (PP). Grants from European Research Council: ERC-2013-CoG 614578 (PP); ERC-2019-PoC 899636 (PP