RESUMO
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.
Assuntos
Mutação com Ganho de Função , Proteína 3 que Contém Domínio de Pirina da Família NLR , Humanos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Mutação com Ganho de Função/genética , Inflamassomos/genética , Desenvolvimento de Medicamentos , SíndromeRESUMO
The metabolic syndrome is a plethora of related disorders that are frequently associated with morbidity and mortality in addition to economic burden. While various treatment options are available, the need to understand the pathology and find new targets still remains. Recent data have suggested GPR75 as one such exciting target that has shown to a highly druggable potential. In this review, we have discussed the recent findings on GPR75 in terms of its expression and signaling and the way it could be a novel target in diseases associated with metabolic syndrome including obesity, dyslipidemia, diabetes, cardiovascular disease, and cerebrovascular disease. In addition, the opportunities and challenges related with the druggable potential of GPR75 have also been highlighted in this review.
Assuntos
Doenças Cardiovasculares , Dislipidemias , Síndrome Metabólica , Humanos , Síndrome Metabólica/tratamento farmacológico , Obesidade , Receptores Acoplados a Proteínas G/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/química , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Caseína Quinase II , Caseína Quinase Ialfa , Caspase 1/metabolismo , Citocinas/metabolismo , Enzimas Desubiquitinantes , Células HEK293 , Humanos , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Quinases Relacionadas a NIMA/metabolismo , Fosforilação , Piroptose , UbiquitinaçãoRESUMO
BACKGROUND AND AIMS: Apolipoprotein (apo) C1 is a 6.6 kDa protein associated with HDL and VLDL. ApoC1 alters triglyceride clearance, and it also favors cholesterol accumulation in HDL, especially by inhibiting CETP in human plasma. Apart from studies in mice, which lack CETP, the impact of apoC1 on atherosclerosis in animal models expressing CETP, like in humans, is not known. This study aimed at determining the net effect of human apoC1 on atherosclerosis in rabbits, a species with naturally high CETP activity but with endogenous apoC1 without CETP inhibitory potential. METHODS: Rabbits expressing a human apoC1 transgene (HuApoC1Tg) were generated and displayed significant amounts of human apoC1 in plasma. RESULTS: After cholesterol feeding, atherosclerosis lesions were significantly less extensive (-22%, p < 0.05) and HDL displayed a reduced ability to serve as CETP substrates (-25%, p < 0.05) in HuApoC1Tg rabbits than in WT littermates. It was associated with rises in plasma HDL cholesterol level and PON-1 activity, and a decrease in the plasma level of the lipid oxidation markers 12(S)-HODE and 8(S)HETE. In chow-fed animals, the level of HDL-cholesterol was also significantly higher in HuApoC1Tg than in WT animals (0.83 ± 0.11 versus 0.73 ± 0.11 mmol/L, respectively, p < 0.05), and it was associated with significantly lower CETP activity (cholesteryl ester transfer rate, -10%, p < 0.05; specific CETP activity, -14%, p < 0.05). CONCLUSIONS: Constitutive expression of fully functional human apoC1 in transgenic rabbit attenuates atherosclerosis. It was found to relate, at least in part, to the inhibition of plasma CETP activity and to alterations in plasma HDL.
Assuntos
Apolipoproteína C-I , Aterosclerose , Animais , Apolipoproteína C-I/genética , Aterosclerose/genética , Aterosclerose/prevenção & controle , Proteínas de Transferência de Ésteres de Colesterol/genética , HDL-Colesterol/metabolismo , Técnicas de Transferência de Genes , Humanos , Camundongos , CoelhosRESUMO
Mitochondria have emerged as key actors of innate and adaptive immunity. Mitophagy has a pivotal role in cell homeostasis, but its contribution to macrophage functions and host defense remains to be delineated. Here, we showed that lipopolysaccharide (LPS) in combination with IFN-γ inhibited PINK1-dependent mitophagy in macrophages through a STAT1-dependent activation of the inflammatory caspases 1 and 11. In addition, we demonstrated that the inhibition of mitophagy triggered classical macrophage activation in a mitochondrial ROS-dependent manner. In a murine model of polymicrobial infection (cecal ligature and puncture), adoptive transfer of Pink1-deficient bone marrow or pharmacological inhibition of mitophagy promoted macrophage activation, which favored bactericidal clearance and led to a better survival rate. Reciprocally, mitochondrial uncouplers that promote mitophagy reversed LPS/IFN-γ-mediated activation of macrophages and led to immunoparalysis with impaired bacterial clearance and lowered survival. In critically ill patients, we showed that mitophagy was inhibited in blood monocytes of patients with sepsis as compared with nonseptic patients. Overall, this work demonstrates that the inhibition of mitophagy is a physiological mechanism that contributes to the activation of myeloid cells and improves the outcome of sepsis.
Assuntos
Bactérias/imunologia , Ativação de Macrófagos , Macrófagos Peritoneais/imunologia , Mitofagia/imunologia , Sepse/imunologia , Animais , Feminino , Humanos , Interferon gama/imunologia , Lipopolissacarídeos/imunologia , Macrófagos Peritoneais/microbiologia , Macrófagos Peritoneais/patologia , Masculino , Camundongos , Proteínas Quinases/imunologia , Células RAW 264.7 , Sepse/microbiologia , Sepse/patologiaRESUMO
Lipopolysaccharides (LPS) originate from the outer membrane of Gram-negative bacteria and trigger an inflammatory response via the innate immune system. LPS consist of a lipid A moiety directly responsible for the stimulation of the proinflammatory cascade and a polysaccharide chain of variable length. LPS form aggregates of variable size and structure in aqueous media, and the aggregation/disaggregation propensity of LPS is known as a key determinant of their biological activity. The aim of the present study was to determine to which extent the length of the polysaccharide chain can affect the nature of LPS structures, their pharmacokinetics, and eventually their proinflammatory properties in vivo. LPS variants of Salmonella Minnesota with identical lipid A but with different polysaccharide moieties were used. The physical properties of LPS aggregates were analyzed by zetametry, dynamic light scattering, and microscopy. The stability of LPS aggregates was tested in the presence of plasma, whole blood, and cultured cell lines. LPS pharmacokinetics was performed in wild-type mice. The accumulation in plasma of rough LPS (R-LPS) with a short polysaccharidic chain was lower, and its hepatic uptake was faster as compared to smooth LPS (S-LPS) with a long polysaccharidic chain. The inflammatory response was weaker with R-LPS than with S-LPS. As compared to S-LPS, R-LPS formed larger aggregates, with a higher hydrophobicity index, a more negative zeta potential, and a higher critical aggregation concentration. The lower stability of R-LPS aggregates could be illustrated in vitro by a higher extent of association of LPS to plasma lipoproteins, faster binding to blood cells, and increased uptake by macrophages and hepatocytes, compared to S-LPS. Our data indicate that a long polysaccharide chain is associated with the formation of more stable aggregates with extended residence time in plasma and higher inflammatory potential. These results show that polysaccharide chain length, and overall aggregability of LPS might be helpful to predict the proinflammatory effect that can be expected in experimental settings using LPS preparations. In addition, better knowledge and control of LPS aggregation and disaggregation might lead to new strategies to enhance LPS detoxification in septic patients.
RESUMO
BACKGROUND AND AIMS: LPCAT3 plays a major role in phospholipid metabolism in the liver and intestine. However, the impact of LPCAT3 on hematopoietic cell and macrophage functions has yet to be described. Our aim was to understand the functions of LPCAT3 in macrophages and to investigate whether LPCAT3 deficiency in hematopoietic cells may affect atherosclerosis development. METHODS: Mice with constitutive Lpcat3 deficiency (Lpcat3-/-) were generated. We used fetal hematopoietic liver cells to generate WT and Lpcat3-/- macrophages in vitro and to perform hematopoietic cell transplantation in recipient Ldlr-/- mice. RESULTS: Lpcat3-deficient macrophages displayed major reductions in the arachidonate content of phosphatidylcholines, phosphatidylethanolamines and, unexpectedly, plasmalogens. These changes were associated with altered cholesterol homeostasis, including an increase in the ratio of free to esterified cholesterol and a reduction in cholesterol efflux in Lpcat3-/- macrophages. This correlated with the inhibition of some LXR-regulated pathways, related to altered cellular availability of the arachidonic acid. Indeed, LPCAT3 deficiency was associated with decreased Abca1, Abcg1 and ApoE mRNA levels in fetal liver cells derived macrophages. In vivo, these changes translated into a significant increase in atherosclerotic lesions in Ldlr-/- mice with hematopoietic LPCAT3 deficiency. CONCLUSIONS: This study identifies LPCAT3 as a key factor in the control of phospholipid homeostasis and arachidonate availability in myeloid cells and underlines a new role for LPCAT3 in plasmalogen metabolism. Moreover, our work strengthens the link between phospholipid and sterol metabolism in hematopoietic cells, with significant consequences on nuclear receptor-regulated pathways and atherosclerosis development.
Assuntos
1-Acilglicerofosfocolina O-Aciltransferase/deficiência , Aterosclerose/enzimologia , Colesterol/metabolismo , Células-Tronco Hematopoéticas/enzimologia , Macrófagos/enzimologia , Fosfolipídeos/metabolismo , Placa Aterosclerótica , 1-Acilglicerofosfocolina O-Aciltransferase/genética , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Ácido Araquidônico/metabolismo , Aterosclerose/genética , Aterosclerose/patologia , Células Cultivadas , Modelos Animais de Doenças , Predisposição Genética para Doença , Transplante de Células-Tronco Hematopoéticas , Receptores X do Fígado/metabolismo , Macrófagos/transplante , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Receptores de LDL/deficiência , Receptores de LDL/genéticaRESUMO
Dietary polyphenols, derived from natural products, have received a great interest for their chemopreventive properties against cancer. In this study, we investigated the effects of phenolic extract of the oleaster leaves (PEOL) on tumor growth in mouse model and on cell death in colon cancer cell lines. We assessed the effect of oleaster leaf infusion on HCT116 (human colon cancer cell line) xenograft growth in athymic nude mice. We observed that oleaster leaf polyphenol-rich infusion limited HCT116 tumor growth in vivo. Investigations of PEOL on two human CRC cell lines showed that PEOL induced apoptosis in HCT116 and HCT8 cells. We demonstrated an activation of caspase-3, -7 and -9 by PEOL and that pre-treatment with the pan-caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), prevented PEOL-induced cell death. We observed an involvement of the mitochondrial pathway in PEOL-induced apoptosis evidenced by reactive oxygen species (ROS) production, a decrease of mitochondrial membrane potential, and cytochrome c release. Increase in intracellular Ca2+ concentration induced by PEOL represents the early event involved in mitochondrial dysfunction, ROS-induced endoplasmic reticulum (ER) stress and apoptosis induced by PEOL, as ruthenium red, an inhibitor of mitochondrial calcium uptake inhibited apoptotic effect of PEOL, BAPTA/AM inhibited PEOL-induced ROS generation and finally, N-acetyl-L-cysteine reversed ER stress and apoptotic effect of PEOL. These results demonstrate that polyphenols from oleaster leaves might have a strong potential as chemopreventive agent in colorectal cancer.
Assuntos
Apoptose/efeitos dos fármacos , Caspases/metabolismo , Neoplasias do Colo/patologia , Mitocôndrias/efeitos dos fármacos , Olea/química , Extratos Vegetais/farmacologia , Folhas de Planta/química , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Nus , Mitocôndrias/patologia , Fenol/química , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição CHOP/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0170823.].