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1.
J Immunol ; 210(9): 1209-1221, 2023 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-36961448

RESUMEN

Autosomal recessive PRKCD deficiency has previously been associated with the development of systemic lupus erythematosus in human patients, but the mechanisms underlying autoimmunity remain poorly understood. We introduced the Prkcd G510S mutation that we previously associated to a Mendelian cause of systemic lupus erythematosus in the mouse genome, using CRISPR-Cas9 gene editing. PrkcdG510S/G510S mice recapitulated the human phenotype and had reduced lifespan. We demonstrate that this phenotype is linked to a B cell-autonomous role of Prkcd. A detailed analysis of B cell activation in PrkcdG510S/G510S mice shows an upregulation of the PI3K/mTOR pathway after the engagement of the BCR in these cells, leading to lymphoproliferation. Treatment of mice with rapamycin, an mTORC1 inhibitor, significantly improves autoimmune symptoms, demonstrating in vivo the deleterious effect of mTOR pathway activation in PrkcdG510S/G510S mice. Additional defects in PrkcdG510S/G510S mice include a decrease in peripheral mature NK cells that might contribute to the known susceptibility to viral infections of patients with PRKCD mutations.


Asunto(s)
Autoinmunidad , Lupus Eritematoso Sistémico , Humanos , Animales , Ratones , Serina-Treonina Quinasas TOR/metabolismo , Linfocitos B , Proliferación Celular
2.
J Exp Med ; 221(5)2024 May 06.
Artículo en Inglés | MEDLINE | ID: mdl-38530241

RESUMEN

NLRP3-associated autoinflammatory disease is a heterogenous group of monogenic conditions caused by NLRP3 gain-of-function mutations. The poor functional characterization of most NLRP3 variants hinders diagnosis despite efficient anti-IL-1 treatments. Additionally, while NLRP3 is controlled by priming and activation signals, gain-of-functions have only been investigated in response to priming. Here, we characterize 34 NLRP3 variants in vitro, evaluating their activity upon induction, priming, and/or activation signals, and their sensitivity to four inhibitors. We highlight the functional diversity of the gain-of-function mutants and describe four groups based on the signals governing their activation, correlating partly with the symptom severity. We identify a new group of NLRP3 mutants responding to the activation signal without priming, associated with frequent misdiagnoses. Our results identify key NLRP3 residues controlling inflammasome activity and sensitivity to inhibitors, and antagonistic mechanisms with broader efficacy for therapeutic strategies. They provide new insights into NLRP3 activation, an explanatory mechanism for NLRP3-AID heterogeneity, and original tools for NLRP3-AID diagnosis and drug development.


Asunto(s)
Mutación con Ganancia de Función , Proteína con Dominio Pirina 3 de la Familia NLR , Humanos , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Mutación con Ganancia de Función/genética , Inflamasomas/genética , Desarrollo de Medicamentos , Síndrome
3.
Nat Commun ; 12(1): 5862, 2021 10 06.
Artículo en Inglés | MEDLINE | ID: mdl-34615873

RESUMEN

NLRP3 controls the secretion of inflammatory cytokines IL-1ß/18 and pyroptosis by assembling the inflammasome. Upon coordinated priming and activation stimuli, NLRP3 recruits NEK7 within hetero-oligomers that nucleate ASC and caspase-1 filaments, but the apical molecular mechanisms underlying inflammasome assembly remain elusive. Here we show that NEK7 recruitment to NLRP3 is controlled by the phosphorylation status of NLRP3 S803 located within the interaction surface, in which NLRP3 S803 is phosphorylated upon priming and later dephosphorylated upon activation. Phosphomimetic substitutions of S803 abolish NEK7 recruitment and inflammasome activity in macrophages in vitro and in vivo. In addition, NLRP3-NEK7 binding is also essential for NLRP3 deubiquitination by BRCC3 and subsequently inflammasome assembly, with NLRP3 phosphomimetic mutants showing enhanced ubiquitination and degradation than wildtype NLRP3. Finally, we identify CSNK1A1 as the kinase targeting NLRP3 S803. Our findings thus reveal NLRP3 S803 phosphorylation status as a druggable apical molecular mechanism controlling inflammasome assembly.


Asunto(s)
Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/química , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Animales , Quinasa de la Caseína II , Caseína Quinasa Ialfa , Caspasa 1/metabolismo , Citocinas/metabolismo , Enzimas Desubicuitinizantes , Células HEK293 , Humanos , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Quinasas Relacionadas con NIMA/metabolismo , Fosforilación , Piroptosis , Ubiquitinación
4.
Cell Mol Immunol ; 18(9): 2140-2152, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-32398809

RESUMEN

T cell development proceeds under the influence of a network of transcription factors (TFs). The precise role of Zeb1, a member of this network, remains unclear. Here, we report that Zeb1 expression is induced early during T cell development in CD4-CD8- double-negative (DN) stage 2 (DN2). Zeb1 expression was further increased in the CD4+CD8+ double-positive (DP) stage before decreasing in more mature T cell subsets. We performed an exhaustive characterization of T cells in Cellophane mice that bear Zeb1 hypomorphic mutations. The Zeb1 mutation profoundly affected all thymic subsets, especially DN2 and DP cells. Zeb1 promoted the survival and proliferation of both cell populations in a cell-intrinsic manner. In the periphery of Cellophane mice, the number of conventional T cells was near normal, but invariant NKT cells, NK1.1+ γδ T cells and Ly49+ CD8 T cells were virtually absent. This suggested that Zeb1 regulates the development of unconventional T cell types from DP progenitors. A transcriptomic analysis of WT and Cellophane DP cells revealed that Zeb1 regulated the expression of multiple genes involved in the cell cycle and TCR signaling, which possibly occurred in cooperation with Tcf1 and Heb. Indeed, Cellophane DP cells displayed stronger signaling than WT DP cells upon TCR engagement in terms of the calcium response, phosphorylation events, and expression of early genes. Thus, Zeb1 is a key regulator of the cell cycle and TCR signaling during thymic T cell development. We propose that thymocyte selection is perturbed in Zeb1-mutated mice in a way that does not allow the survival of unconventional T cell subsets.


Asunto(s)
Receptores de Antígenos de Linfocitos T alfa-beta , Subgrupos de Linfocitos T , Animales , Diferenciación Celular , Proliferación Celular , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Transducción de Señal/genética , Timo
5.
Nat Microbiol ; 6(3): 401-412, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33432150

RESUMEN

Inflammasomes are signalling platforms that are assembled in response to infection or sterile inflammation by cytosolic pattern recognition receptors. The consequent inflammasome-triggered caspase-1 activation is critical for the host defence against pathogens. During infection, NLRP3, which is a pattern recognition receptor that is also known as cryopyrin, triggers the assembly of the inflammasome-activating caspase-1 through the recruitment of ASC and Nek7. The activation of the NLRP3 inflammasome is tightly controlled both transcriptionally and post-translationally. Despite the importance of the NLRP3 inflammasome regulation in autoinflammatory and infectious diseases, little is known about the mechanism controlling the activation of NLRP3 and the upstream signalling that regulates the NLRP3 inflammasome assembly. We have previously shown that the Rho-GTPase-activating toxin from Escherichia coli cytotoxic necrotizing factor-1 (CNF1) activates caspase-1, but the upstream mechanism is unclear. Here, we provide evidence of the role of the NLRP3 inflammasome in sensing the activity of bacterial toxins and virulence factors that activate host Rho GTPases. We demonstrate that this activation relies on the monitoring of the toxin's activity on the Rho GTPase Rac2. We also show that the NLRP3 inflammasome is activated by a signalling cascade that involves the p21-activated kinases 1 and 2 (Pak1/2) and the Pak1-mediated phosphorylation of Thr 659 of NLRP3, which is necessary for the NLRP3-Nek7 interaction, inflammasome activation and IL-1ß cytokine maturation. Furthermore, inhibition of the Pak-NLRP3 axis decreases the bacterial clearance of CNF1-expressing UTI89 E. coli during bacteraemia in mice. Taken together, our results establish that Pak1 and Pak2 are critical regulators of the NLRP3 inflammasome and reveal the role of the Pak-NLRP3 signalling axis in vivo during bacteraemia in mice.


Asunto(s)
Bacteriemia/metabolismo , Toxinas Bacterianas/metabolismo , Infecciones por Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Animales , Bacteriemia/inmunología , Bacteriemia/microbiología , Carga Bacteriana , Toxinas Bacterianas/genética , Escherichia coli/genética , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Inmunidad Innata , Ratones , Fosforilación , Transducción de Señal , Quinasas p21 Activadas/metabolismo , Proteínas de Unión al GTP rac/genética , Proteína RCA2 de Unión a GTP
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