RESUMEN
There is a reciprocal relationship between extracellular matrix (ECM) remodelling and inflammation that could be operating in the progression of severe COVID-19. To explore the immune-driven ECM remodelling in COVID-19, we in this explorative study analysed these interactions in hospitalised COVID-19 patients. RNA sequencing and flow analysis were performed on peripheral blood mononuclear cells. Inflammatory mediators in plasma were measured by ELISA and MSD, and clinical information from hospitalised COVID-19 patients (N=15) at admission was included in the analysis. Further, we reanalysed two publicly available datasets: (1) lung tissue RNA-sequencing dataset (N=5) and (2) proteomics dataset from PBCM. ECM remodelling pathways were enriched in PBMC from COVID-19 patients compared to healthy controls. Patients treated at the intensive care unit (ICU) expressed distinct ECM remodelling gene profiles compared to patients in the hospital ward. Several markers were strongly correlated to immune cell subsets, and the dysregulation in the ICU patients was positively associated with plasma levels of inflammatory cytokines and negatively associated with B-cell activating factors. Finally, our analysis of publicly accessible datasets revealed (i) an augmented ECM remodelling signature in inflamed lung tissue compared to non-inflamed tissue and (ii) proteomics analysis of PBMC from severe COVID-19 patients demonstrated an up-regulation in an ECM remodelling pathway. Our results may suggest the presence of an interaction between ECM remodelling, inflammation, and immune cells, potentially initiating or perpetuating pulmonary pathology in severe COVID-19.
Asunto(s)
COVID-19 , Matriz Extracelular , Leucocitos Mononucleares , SARS-CoV-2 , Humanos , COVID-19/inmunología , COVID-19/sangre , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Matriz Extracelular/metabolismo , Masculino , Femenino , Persona de Mediana Edad , SARS-CoV-2/fisiología , SARS-CoV-2/inmunología , Anciano , Citocinas/sangre , Proteómica/métodos , Pulmón/inmunología , Pulmón/patología , AdultoRESUMEN
PURPOSE: Triglycerides (TG) and their major transport lipoprotein in the circulation (VLDL) appear to be related to inflammation. Patients with common variable immunodeficiency (CVID) have inflammatory complications associated with gut microbial dysbiosis. We hypothesized that CVID patients have disturbed TG/VLDL profiles associated with these clinical characteristics. METHODS: We measured plasma concentrations of TGs, inflammatory markers, and lipopolysaccharide (LPS) in 95 CVID patients and 28 healthy controls. Additionally, in 40 CVID patients, we explored plasma lipoprotein profiling, fatty acid, gut microbial dysbiosis, and diet. RESULTS: TG levels were increased in CVID patients as compared to healthy controls (1.36 ± 0.53 mmol/l versus 1.08 ± 0.56 [mean, SD], respectively, P = 0.008), particularly in the clinical subgroup "Complications," characterized by autoimmunity and organ-specific inflammation, compared to "Infection only" (1.41 mmol/l, 0.71[median, IQR] versus [1.02 mmol/l, 0.50], P = 0.021). Lipoprotein profile analyses showed increased levels of all sizes of VLDL particles in CVID patients compared to controls. TG levels correlated positively with CRP (rho = 0.256, P = 0.015), IL-6 (rho = 0.237, P = 0.021), IL-12 (rho = 0.265, P = 0.009), LPS (r = 0.654, P = 6.59 × 10-13), CVID-specific gut dysbiosis index (r = 0.315, P = 0.048), and inversely with a favorable fatty acid profile (docosahexaenoic acid [rho = - 0.369, P = 0.021] and linoleic acid [rho = - 0.375, P = 0.019]). TGs and VLDL lipids did not appear to be associated with diet and there were no differences in body mass index (BMI) between CVID patients and controls. CONCLUSION: We found increased plasma levels of TGs and all sizes of VLDL particles, which were associated with systemic inflammation, LPS, and gut dysbiosis in CVID, but not diet or BMI.
Asunto(s)
Inmunodeficiencia Variable Común , Lipopolisacáridos , Humanos , Disbiosis , Lipoproteínas , Triglicéridos , Inflamación , Ácidos GrasosRESUMEN
Background: Results showing that sera from double vaccinated individuals have minimal neutralizing activity against Omicron have been interpreted as indicating the need for a third vaccine dose for protection. However, there is little information about early immune responses to Omicron infection in double vaccinated individuals. Methods: We measured inflammatory mediators, antibodies to the SARS-CoV-2 spike and nucleocapsid proteins, and spike peptide-induced release of interferon gamma in whole blood in 51 double-vaccinated individuals infected with Omicron, in 14 infected with Delta, and in 18 healthy controls. The median time points for the first and second samples were 7 and 14 days after symptom onset, respectively. Findings: Infection with Omicron or Delta led to a rapid and similar increase in antibodies to the receptor-binding domain (RBD) of Omicron protein and spike peptide-induced interferon gamma in whole blood. Both the Omicron- and the Delta-infected patients had a mild and transient increase in inflammatory parameters. Interpretation: The results suggest that two vaccine doses are sufficient to mount a rapid and potent immune response upon infection in healthy individuals of with the Omicron variant. Funding: The study was funded by the Oslo University Hospital, and by grants from The Coalition for Epidemic Preparedness Innovations, Research Council of Norway (no 312780, 324272), South-Eastern Norway Regional Health Authority (no 2019067, 2021071, 10357, 2021047, 33612, 2021087, 2017092), EU Horizon 2020 grant no 848099, a philantropic donation from Vivaldi Invest A/S, and The European Virus Archive Global.
Asunto(s)
COVID-19 , Vacunas Virales , Anticuerpos Antivirales , COVID-19/prevención & control , Humanos , Mediadores de Inflamación , Interferón gamma , Proteínas de la Nucleocápside , SARS-CoV-2RESUMEN
BACKGROUND: Although coronavirus disease 2019 (COVID-19) is primarily a respiratory infection, mounting evidence suggests that the gastrointestinal tract is involved in the disease, with gut barrier dysfunction and gut microbiota alterations being related to disease severity. Whether these alterations persist and are related to long-term respiratory dysfunction remains unknown. METHODS: Plasma was collected during hospital admission and after 3 months from the NOR-Solidarity trial (n = 181) and analyzed for markers of gut barrier dysfunction and inflammation. At the 3-month follow-up, pulmonary function was assessed by measuring the diffusing capacity of the lungs for carbon monoxide (DLCO ). Rectal swabs for gut microbiota analyses were collected (n = 97) and analyzed by sequencing the 16S rRNA gene. RESULTS: Gut microbiota diversity was reduced in COVID-19 patients with respiratory dysfunction, defined as DLCO below the lower limit of normal 3 months after hospitalization. These patients also had an altered global gut microbiota composition, with reduced relative abundance of 20 bacterial taxa and increased abundance of five taxa, including Veillonella, potentially linked to fibrosis. During hospitalization, increased plasma levels of lipopolysaccharide-binding protein (LBP) were strongly associated with respiratory failure, defined as pO2 /fiO2 (P/F ratio) <26.6 kPa. LBP levels remained elevated during and after hospitalization and were associated with low-grade inflammation and respiratory dysfunction after 3 months. CONCLUSION: Respiratory dysfunction after COVID-19 is associated with altered gut microbiota and persistently elevated LBP levels. Our results should be regarded as hypothesis generating, pointing to a potential gut-lung axis that should be further investigated in relation to long-term pulmonary dysfunction and long COVID.
Asunto(s)
COVID-19 , Microbioma Gastrointestinal , COVID-19/complicaciones , Ensayos Clínicos como Asunto , Humanos , Inflamación , ARN Ribosómico 16S/genética , SARS-CoV-2 , Síndrome Post Agudo de COVID-19RESUMEN
Thromboembolic events are frequent and associated with poor outcome in severe COVID-19 disease. Anti-PF4/polyanion antibodies are related to heparin-induced thrombocytopenia (HIT) and thrombus formation, but data on these antibodies in unselected COVID-19 populations are scarce. We assessed the presence of anti-PF4/polyanion antibodies in prospectively collected serum from an unselected cohort of hospitalized COVID-19 patients and evaluated if elevated levels could give prognostic information on ICU admission and respiratory failure (RF), were associated with markers of inflammation, endothelial activation, platelet activation, coagulation and fibrosis and were associated with long-term pulmonary CT changes. Five out of 65 patients had anti-PF4/polyanion reactivity with OD ≥0.200. These patients had more severe disease as reflected by ICU admission without any evidence of HIT. They also had signs of enhanced inflammation and fibrinogenesis as reflected by elevated ferritin and osteopontin, respectively, during the first 10 days of hospitalization. Increased ferritin and osteopontin persisted in these patients at 3 months follow-up, concomitant with pulmonary CT pathology. Our finding shows that the presence of anti-PF4/polyanion antibodies in unselected hospitalized COVID-19 patients was not related to HIT, but was associated with disease severity, inflammation, and pulmonary pathology after 3 months.
Asunto(s)
COVID-19 , Trombocitopenia , Anticoagulantes/efectos adversos , Ferritinas/efectos adversos , Heparina/efectos adversos , Humanos , Inflamación , Osteopontina/efectos adversos , Factor Plaquetario 4 , Índice de Severidad de la Enfermedad , Trombocitopenia/diagnósticoRESUMEN
Obesity-related diseases (e.g. type 2 diabetes mellitus and cardiovascular disorders) represent an increasing health problem worldwide. NLRP3 inflammasome activation may underlie obesity-induced inflammation and insulin resistance, and NLRP3 deficient mice exposed to high fat diet (HFD) appear to be protected from left ventricle (LV) concentric remodeling. Herein, we investigated if these beneficial effects were associated with alterations in plasma metabolites, using metabolomic and lipidomic analysis, and gut microbiota composition, using 16S rRNA sequencing of cecum content, comparing NLRP3 deficient and wild type (WT) mice on HFD and control diet. Obese NLRP3 deficient mice had lower systemic ceramide levels, potentially resulting attenuating inflammation, altered hepatic expression of fatty acids (FA) with lower mono-saturated FA and higher polyunsaturated FA levels, potentially counteracting development of liver steatosis, downregulated myocardial energy metabolism as assessed by proteomic analyses of LV heart tissue, and different levels of bile acids as compared with WT mice. These changes were accompanied by an altered composition of gut microbiota associated with decreased systemic levels of tri-methylamine-N-oxide and lipopolysaccharide, potentially inducing attenuating systemic inflammation and beneficial effects on lipid metabolism. Our findings support a role of NLRP3 inflammasome in the interface between metabolic and inflammatory stress, involving an altered gut microbiota composition.
Asunto(s)
Dieta Alta en Grasa/efectos adversos , Microbioma Gastrointestinal , Metaboloma , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Animales , Ceramidas/sangre , Metabolismo Energético , Ácidos Grasos/sangre , Ácidos Grasos/metabolismo , Metabolismo de los Lípidos , Lipopolisacáridos/sangre , Hígado/metabolismo , Masculino , Metilaminas/sangre , Ratones , Ratones Endogámicos C57BL , Miocardio/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/deficiencia , Proteína con Dominio Pirina 3 de la Familia NLR/genéticaRESUMEN
BACKGROUND: Identification of novel biomarkers could provide prognostic information and improve risk stratification in patients with aortic stenosis (AS). YKL-40 (chitinase-3-like protein 1), a protein involved in atherogenesis, is upregulated in human calcific aortic valves. We hypothesized that circulating YKL-40 would be elevated and associated with the degree of AS severity and outcome in patients with symptomatic AS. METHODS: Plasma YKL-40 was analyzed in 2 AS populations, one severe AS (n=572) with outcome measures and one with mixed severity (n=67). YKL-40 expression in calcified valves and in an experimental pressure overload model was assessed. RESULTS: We found (1) patients with AS had upregulated circulating YKL-40 compared with healthy controls (median 109 versus 34 ng/mL, P<0.001), but levels were not related to the degree of AS severity. (2) High YKL-40 levels (quartile 4) were associated with long-term (median follow-up 4.7 years) all-cause mortality (adjusted hazard ratio, 1.93 [95% CI, 1.37-2.73], P<0.001). (3) YKL-40 protein expression in human calcific valves co-localized with its putative receptor IL-13rα2 in close proximity to valve interstitial cells. (4) Myocardial YKL-40 increased in experimental pressure overload (6-fold in decompensated versus sham mice). CONCLUSIONS: YKL-40 levels were elevated in AS and associated with mortality but not with other metrics of disease severity including the degree of AS severity. Despite scientific rationale for its role in AS, the clinical utility of circulating YKL-40 as a biomarker is limited. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT01794832.
Asunto(s)
Estenosis de la Válvula Aórtica/sangre , Válvula Aórtica/metabolismo , Proteína 1 Similar a Quitinasa-3/sangre , Anciano , Anciano de 80 o más Años , Animales , Válvula Aórtica/patología , Estenosis de la Válvula Aórtica/diagnóstico , Estenosis de la Válvula Aórtica/genética , Estenosis de la Válvula Aórtica/mortalidad , Biomarcadores/sangre , Estudios de Casos y Controles , Proteína 1 Similar a Quitinasa-3/genética , Estudios Transversales , Dinamarca , Modelos Animales de Enfermedad , Femenino , Humanos , Subunidad alfa2 del Receptor de Interleucina-13/genética , Subunidad alfa2 del Receptor de Interleucina-13/metabolismo , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Noruega , Pronóstico , Índice de Severidad de la Enfermedad , Regulación hacia ArribaRESUMEN
Objective: The aetiology of thoracic aortic aneurysm (TAA) is largely unknown, but inflammation is likely to play a central role in the pathogenesis. In this present study, we aim to investigate the complement receptors in TAA. Methods: Aortic tissue and blood from 31 patients with non-syndromic TAA undergoing thoracic aortic repair surgery were collected. Aortic tissue and blood from 36 patients with atherosclerosis undergoing coronary artery bypass surgery or aortic valve replacement were collected and served as control material. The expression of the complement anaphylatoxin receptors C3aR1, C5aR1 and C5aR2 in aortic tissue were examined by quantitative RT-PCR and C5aR2 protein by immunohistochemistry. Colocalisation of C5aR2 to different cell types was analysed by immunofluorescence. Complement activation products C3bc and sC5b-9 were measured in plasma. Results: Compared with controls, TAA patients had substantial (73%) downregulated gene expression of C5aR2 as seen both at the mRNA (p=0.005) level and protein (p=0.03) level. In contrast, there were no differences in the expression of C3aR1 and C5aR1 between the two groups. Immunofluorescence examination showed that C5aR2 was colocalised to macrophages and T cells in the aortic media. There were no differences in the degree of systemic complement activation between the two groups. Conclusion: Our findings suggest downregulation of the C5aR2, regarded to act mainly anti-inflammatory, in electively operated TAA as compared with non-aneurysmatic aortas of patients with aortic stenosis and/or coronary artery disease. This may tip the balance towards a relative increase in the inflammatory responses induced by C5aR1 and thus enhance the inflammatory processes in TAA.
RESUMEN
Metabolic and immune systems are among the most fundamental requirements for survival. Many metabolic and immune response pathways or nutrient- and pathogen-sensing systems are evolutionarily conserved throughout species. As a result, the immune response and metabolic regulation are highly integrated and the proper function of each is dependent on the other. This interaction between metabolic disturbances and the immune system has been most extensively studied in disorders related to obesity such as insulin resistance, type 2 diabetes, and nonalcoholic fatty liver disease. Metabolically induced inflammation seems also to play a role in the development and progression of atherosclerosis including its complications such as myocardial infarction (MI) and post-MI remodeling. There are several lines of evidence suggesting that NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is a sensor of metabolic stress linking metabolic disturbances to inflammation. Here, we will discuss the role of the NLRP3 inflammasome in the pathogenesis of obesity and diabetes, 2 important risk factors for atherosclerosis and MI. We will also discuss the role of NLRP3 inflammasome in the interaction between metabolic disturbances and myocardial inflammation during MI and during metabolically induced myocardial remodeling.
Asunto(s)
Aterosclerosis/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Inflamasomas/metabolismo , Mediadores de Inflamación/metabolismo , Inflamación/metabolismo , Infarto del Miocardio/metabolismo , Miocardio/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Obesidad/metabolismo , Animales , Aterosclerosis/epidemiología , Aterosclerosis/inmunología , Aterosclerosis/patología , Diabetes Mellitus Tipo 2/epidemiología , Diabetes Mellitus Tipo 2/inmunología , Diabetes Mellitus Tipo 2/patología , Metabolismo Energético , Microbioma Gastrointestinal , Humanos , Inflamasomas/inmunología , Inflamación/epidemiología , Inflamación/inmunología , Inflamación/patología , Mediadores de Inflamación/inmunología , Resistencia a la Insulina , Infarto del Miocardio/epidemiología , Infarto del Miocardio/inmunología , Infarto del Miocardio/patología , Miocardio/inmunología , Miocardio/patología , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Obesidad/epidemiología , Obesidad/inmunología , Obesidad/patología , Factores de Riesgo , Transducción de SeñalRESUMEN
NAD+ is an essential cofactor in reduction-oxidation metabolism with impact on metabolic and inflammatory diseases. However, data elucidating the effects of NAD+ on the proinflammatory features of human primary monocytes are scarce. In this study, we explored how NAD+ affects TLR4 and NOD-like receptor with a PYD-domain 3 (NLRP3) inflammasome activation, two key innate immune responses. Human primary monocytes were isolated from buffy coats obtained from healthy individuals. Intracellular NAD+ was manipulated by nicotinamide riboside and the NAMPT inhibitor FK866. Cells were primed with LPS with or without subsequent NLRP3 activation with ATP or cholesterol crystals to analyze the effects of NAD+ levels on TLR4-mediated NF-κB activation and NLRP3 activity, respectively. Cytokine release was quantified, and the downstream signal pathway of TLR4 was investigated with Western blot and proteomic analysis. The impact of sirtuin and PARP inhibition was also explored. Our main findings were: 1) elevated NAD+ enhanced IL-1ß release in LPS-primed human monocytes exposed to ATP in vitro, 2) both NLRP3-dependent and -independent inflammatory responses in LPS-exposed monocytes were inhibited by NAD+ depletion with FK866, 3) the inhibition was not caused by suppression of sirtuins or PARP1, and 4) phosphorylation of several proteins TLR4 signal pathway was inhibited by FK866-mediated NAD+ depletion, specifically TAK1, IKKß, IkBα, MEK 1/2, ERK 1/2, and p38. Hence, we suggest a novel mechanism in which NAD+ affects TLR4 signal transduction. Furthermore, our data challenge previous reports of the interaction between NAD+ and inflammation and question the use of nicotinamide riboside in the therapy of inflammatory disorders.
Asunto(s)
Inflamasomas/metabolismo , Inflamación/metabolismo , Monocitos/metabolismo , NAD/metabolismo , Transducción de Señal/fisiología , Receptor Toll-Like 4/metabolismo , Células Cultivadas , Regulación de la Expresión Génica/fisiología , Humanos , Inmunidad Innata/fisiología , Inflamación/inducido químicamente , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Fosforilación/fisiología , Proteómica/métodosRESUMEN
Background: Obesity is an increasingly prevalent metabolic disorder in the modern world and is associated with structural and functional changes in the heart. The NLRP3 inflammasome is an innate immune sensor that can be activated in response to endogenous danger signals and triggers activation of interleukin (IL)-1ß and IL-18. Increasing evidence points to the involvement of the NLRP3 inflammasome in obesity-induced inflammation and insulin resistance, and we hypothesized that it also could play a role in the development of obesity induced cardiac alterations. Methods and Results: WT, Nlrp3-/-, and ASC-/- (Pycard-/-) male mice were exposed to high fat diet (HFD; 60 cal% fat) or control diet for 52 weeks. Cardiac structure and function were evaluated by echocardiography and magnetic resonance imaging, respectively. Whereas, NLRP3 and ASC deficiency did not affect the cardiac hypertrophic response to obesity, it was preventive against left ventricle concentric remodeling and impairment of diastolic function. Furthermore, whereas NLRP3 and ASC deficiency attenuated systemic inflammation in HFD fed mice; long-term HFD did not induce significant cardiac fibrosis or inflammation, suggesting that the beneficial effects of NLRP3 inflammasome deficiency on myocardial remodeling at least partly reflect systemic mechanisms. Nlrp3 and ASC (Pycard) deficient mice were also protected against obesity-induced systemic metabolic dysregulation, as well as lipid accumulation and impaired insulin signaling in hepatic and cardiac tissues. Conclusions: Our data indicate that the NLRP3 inflammasome modulates cardiac concentric remodeling in obesity through effects on systemic inflammation and metabolic disturbances, with effect on insulin signaling as a potential mediator within the myocardium.
Asunto(s)
Inflamasomas/metabolismo , Miocardio/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Obesidad/metabolismo , Animales , Dieta Alta en Grasa/efectos adversos , Inflamación/metabolismo , Insulina/metabolismo , Resistencia a la Insulina/fisiología , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal/fisiologíaRESUMEN
Background: IL-1ß is a highly potent pro-inflammatory cytokine and its secretion is tightly regulated. Inactive pro-IL-1ß is transcribed in response to innate immune receptors activating NFκB. If tissue damage occurs, danger signals released from necrotic cells, such as ATP, can activate NLRP3-inflammasomes (multiprotein complexes consisting of NLRP3, ASC, and active caspase-1) which cleaves and activates pro-IL-1ß. NLRP3 activation also depends on NEK7 and mitochondrial ROS-production. Thus, IL-1ß secretion may be regulated at the level of each involved component. We have previously shown that NLRP3-dependent IL-1ß release can be induced in cardiac fibroblasts by pro-inflammatory stimuli. However, anti-inflammatory mechanisms targeting IL-1ß release in cardiac cells have not been investigated. mTOR is a key regulator of protein metabolism, including autophagy and proteasome activity. In this study we explored whether autophagy or proteasomal degradation are regulators of NLRP3 inflammasome activation and IL-1ß release from cardiac fibroblasts. Methods and Results: Serum starvation selectively reduced LPS/ATP-induced IL-1ß secretion from cardiac fibroblasts. However, no other inflammasome components, nor mitochondrial mass, were affected. The mTOR inhibitor rapamycin restored pro-IL-1ß protein levels as well as LPS/ATP-induced IL-1ß release from serum starved cells. However, neither serum starvation nor rapamycin induced autophagy in cardiac fibroblasts. Conversely, chloroquine and bafilomycin A (inhibitors of autophagy) and betulinic acid (a proteasome activator) effectively reduced LPS-induced pro-IL-1ß protein levels. Key findings were reinvestigated in human monocyte-derived macrophages. Conclusion: In cardiac fibroblasts, mTOR inhibition selectively favors pro-IL-1ß synthesis while proteasomal degradation and not autophagy is the major catabolic anti-inflammatory mechanism for degradation of this cytokine.
Asunto(s)
Fibroblastos/metabolismo , Expresión Génica , Inflamasomas/metabolismo , Interleucina-1beta/genética , Serina-Treonina Quinasas TOR/metabolismo , Animales , Biomarcadores , Línea Celular , Células Cultivadas , Cloroquina , Citocinas , Interleucina-1beta/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Mitocondrias/metabolismo , Proteolisis , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: Following birth asphyxia there is a robust inflammatory response. NLRP3 is a receptor of the innate immune system. Upon activation, NLRP3 forms an inflammasome together with ASC and procaspase-1 to mediate release of IL-1ß and IL-18. NLRP3 has previously been shown to be upregulated following neonatal hypoxic-ischemic (HI) brain injury in mice, but with no early effect on brain injury. OBJECTIVE: We aimed to evaluate if deficiency of NLRP3 or ASC protects against neonatal HI brain damage 7 days after hypoxia-ischemia. METHODS: C57BL/6J, NLRP3-/-, and ASC-/- mice were subjected to unilateral common carotid artery ligation followed by hypoxia at P9. Brain infarction, apoptosis, and microglial response were evaluated, as well as total RNA sequencing and examination of plasma levels of systemic proinflammatory cytokines. RESULTS: NLRP3-/- mice showed significantly increased brain infarction volumes compared to wild-type (Wt) mice, while ASC-/- mice showed reduced brain infarction volumes after neonatal hypoxia-ischemia. The amount of activated microglia was increased in NLRP3-/- mice, while decreased in ASC-/- mice compared to Wt mice. Total RNA sequencing showed an impaired inflammatory transcriptional response in the hippocampus of NLRP3-/- mice. Plasma levels of IL-1ß and IL-18 were not affected, but TNF was lower in NLRP3-/- and ASC-/- mice compared to Wt mice. CONCLUSION: ASC deficiency is neuroprotective in neonatal HI brain damage in mice, while NLRP3 deficiency increases brain damage.
Asunto(s)
Infarto Encefálico/patología , Encéfalo/patología , Proteínas Adaptadoras de Señalización CARD/genética , Hipoxia-Isquemia Encefálica/patología , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Factores de Necrosis Tumoral/sangre , Animales , Animales Recién Nacidos , Apoptosis , Infarto Encefálico/genética , Regulación hacia Abajo , Hipoxia-Isquemia Encefálica/genética , Interleucina-18/sangre , Interleucina-1beta/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/patología , Análisis de Secuencia de ARN , Regulación hacia ArribaRESUMEN
Inflammasomes are multiprotein inflammatory platforms that induce caspase-1 activation and subsequently interleukin (IL)-1ß and IL-18 processing. The NLRP3 inflammasome is activated by different forms of oxidative stress, and, based on the central role of IL-1ß in the destruction of pancreatic islets, it could be related to the development of diabetes. We therefore investigated responses in wild-type C57Bl/6 (WT) mice, NLRP3-/- mice, and mice deficient in apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) after exposing islets to short-term hypoxia or alloxan-induced islet damage. NLRP3-deficient islets compared with WT islets had preserved function ex vivo and were protected against hypoxia-induced cell death. Furthermore, NLRP3 and ASC-deficient mice were protected against oxidative stress-induced diabetes caused by repetitive low-dose alloxan administration, and this was associated with reduced ß-cell death and reduced macrophage infiltration. This suggests that the beneficial effect of NLRP3 inflammasome deficiency on oxidative stress-mediated ß-cell damage could involve reduced macrophage infiltration and activation. To support the role of macrophage activation in alloxan-induced diabetes, we injected WT mice with liposomal clodronate, which causes macrophage depletion before induction of a diabetic phenotype by alloxan treatment, resulting in improved glucose homeostasis in WT mice. We show here that the NLRP3 inflammasome acts as a mediator of hypoxia and oxidative stress in insulin-producing cells, suggesting that inhibition of the NLRP3 inflammasome could have beneficial effects on ß-cell preservation.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Inflamasomas/metabolismo , Islotes Pancreáticos/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Estrés Oxidativo/fisiología , Animales , Apoptosis/fisiología , Células Secretoras de Insulina/metabolismo , Interleucina-1beta/metabolismo , Activación de Macrófagos/fisiología , Ratones , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/genéticaRESUMEN
Aim. Inflammation is important in heart failure (HF). The role of the immune receptor toll-like receptor 9 (TLR9) in HF is not understood and not investigated in diastolic HF. We investigated the role of TLR9 in a murine diastolic HF model caused by cardiomyocyte SERCA2a excision. Methods and Results. We crossed SERCA2a KO and TLR9 KO mice to generate four mouse lines. Tamoxifen-induced cardiomyocyte SERCA2a gene excision was carried out in mice, causing diastolic HF. After 7.6 weeks, cardiac functions and dimensions were analyzed by echocardiography and heart tissues were processed. HF mice depleted of TLR9 demonstrated reduced survival compared to SERC2a KO mice, with a median life expectancy of 58 days compared to 63 days. Both HF groups displayed increased left atrium size, lung weight, fetal gene expressions, monocyte/macrophage infiltration, and fibrosis. However, there were no significant differences between the groups. Conclusion. In mice with SERCA2a KO-induced diastolic HF, the absence of TLR9 reduced median life expectancy. The cause remains elusive, as all investigated HF parameters were unaltered. Still, these findings support a salutary role of TLR9 in some subsets of HF conditions and underline the importance for future studies on the mechanisms of TLR9 in diastolic HF.
Asunto(s)
Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/mortalidad , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Receptor Toll-Like 9/metabolismo , Animales , Modelos Animales de Enfermedad , Ecocardiografía , Femenino , Insuficiencia Cardíaca/genética , Masculino , Ratones , Ratones Noqueados , Miocitos Cardíacos/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/deficiencia , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Receptor Toll-Like 9/genéticaRESUMEN
OBJECTIVE: Extracellular nicotinamide phosphoribosyltransferase (eNAMPT) mediates inflammatory and potentially proatherogenic effects, whereas the role of intracellular NAMPT (iNAMPT), the rate limiting enzyme in the salvage pathway of nicotinamide adenine dinucleotide (NAD)+ generation, in atherogenesis is largely unknown. Here we investigated the effects of iNAMPT overexpression in leukocytes on inflammation and atherosclerosis. APPROACH AND RESULTS: Low-density lipoprotein receptor-deficient mice with hematopoietic overexpression of human iNAMPT (iNAMPThi), on a western type diet, showed attenuated plaque burden with features of lesion stabilization. This anti-atherogenic effect was caused by improved resistance of macrophages to apoptosis by attenuated chemokine (C-C motif) receptor 2-dependent monocyte chemotaxis and by skewing macrophage polarization toward an anti-inflammatory M2 phenotype. The iNAMPThi phenotype was almost fully reversed by treatment with the NAMPT inhibitor FK866, indicating that iNAMPT catalytic activity is instrumental in the atheroprotection. Importantly, iNAMPT overexpression did not induce any increase in eNAMPT, and eNAMPT had no effect on chemokine (C-C motif) receptor 2 expression and promoted an inflammatory M1 phenotype in macrophages. The iNAMPT-mediated effects at least partly involved sirtuin 1-dependent molecular crosstalk of NAMPT and peroxisome proliferator-activated receptor γ. Finally, iNAMPT and peroxisome proliferator-activated receptor γ showed a strong correlation in human atherosclerotic, but not healthy arteries, hinting to a relevance of iNAMPT/peroxisome proliferator-activated receptor γ pathway also in human carotid atherosclerosis. CONCLUSIONS: This study highlights the functional dichotomy of intracellular versus extracellular NAMPT, and unveils a critical role for the iNAMPT-peroxisome proliferator-activated receptor γ axis in atherosclerosis.
Asunto(s)
Aterosclerosis/prevención & control , Diferenciación Celular , Citocinas/metabolismo , Leucocitos/enzimología , Macrófagos/metabolismo , Monocitos/metabolismo , Nicotinamida Fosforribosiltransferasa/metabolismo , PPAR gamma/metabolismo , Anciano , Animales , Apoptosis , Aterosclerosis/enzimología , Aterosclerosis/genética , Aterosclerosis/patología , Células Cultivadas , Citocinas/antagonistas & inhibidores , Citocinas/genética , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Femenino , Predisposición Genética a la Enfermedad , Humanos , Leucocitos/efectos de los fármacos , Leucocitos/patología , Activación de Macrófagos , Macrófagos/efectos de los fármacos , Macrófagos/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Monocitos/efectos de los fármacos , Monocitos/patología , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Nicotinamida Fosforribosiltransferasa/genética , Fenotipo , Receptores de LDL/deficiencia , Receptores de LDL/genética , Transducción de Señal , Sirtuina 1/metabolismo , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Wnt signaling is dysregulated in heart failure (HF) and may promote cardiac hypertrophy, fibrosis, and inflammation. Blocking the Wnt ligand Wnt5a prevents HF in animal models. However, the role of Wnt5a in human HF and its functions in cardiac cells remain unclear. Here, we investigated Wnt5a regulation in HF patients and its effects on primary mouse and human cardiac fibroblasts. Serum Wnt5a was elevated in HF patients and associated with hemodynamic, neurohormonal, and clinical measures of disease severity. In failing human hearts, Wnt5a protein correlated with interleukin (IL)-6 and tissue inhibitor of metalloproteinase (TIMP)-1. Wnt5a messenger RNA (mRNA) levels were markedly upregulated in failing myocardium and both mRNA and protein levels declined following left ventricular assist device therapy. In primary mouse and human cardiac fibroblasts, recombinant Wnt5a dose-dependently upregulated mRNA and protein release of IL-6 and TIMP-1. Wnt5a did not affect ß-catenin levels, but activated extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. Importantly, inhibition of ERK1/2 activation attenuated Wnt5a-induced release of IL-6 and TIMP-1. In conclusion, our results show that Wnt5a is elevated in the serum and myocardium of HF patients and is associated with measures of progressive HF. Wnt5a induces IL-6 and TIMP-1 in cardiac fibroblasts, which might promote myocardial inflammation and fibrosis, and thereby contribute to HF progression. KEY MESSAGES: ⢠Wnt5a is elevated in serum and myocardium of HF patients and is associated with measures of progressive HF. ⢠In cardiac fibroblasts, Wnt5a upregulates interleukin (IL)-6 and tissue inhibitor of metalloproteinase (TIMP)-1 through the ERK pathway. ⢠Wnt5a-mediated effects might promote myocardial inflammation and fibrosis, and thereby contribute to HF progression.
Asunto(s)
Fibroblastos/metabolismo , Insuficiencia Cardíaca/metabolismo , Proteína Wnt-5a/metabolismo , Adulto , Anciano , Animales , Femenino , Fibroblastos/patología , Insuficiencia Cardíaca/sangre , Insuficiencia Cardíaca/patología , Humanos , Interleucina-6/metabolismo , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Persona de Mediana Edad , Miocardio/metabolismo , Miocardio/patología , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Vía de Señalización Wnt , Proteína Wnt-5a/sangreRESUMEN
Palmitate triggers inflammatory responses in several cell types, but its effects on cardiac fibroblasts are at present unknown. The aims of the study were to (1) assess the potential of palmitate to promote inflammatory signaling in cardiac fibroblasts through TLR4 and the NLRP3 inflammasome and (2) characterize the cellular phenotype of cardiac fibroblasts exposed to palmitate. We examined whether palmitate induces inflammatory responses in cardiac fibroblasts from WT, NLRP3-/- and ASC-/-mice (C57BL/6 background). Exposure to palmitate caused production of TNF, IL-6 and CXCL2 via TLR4 activation. NLRP3 inflammasomes are activated in a two-step manner. Whereas palmitate did not prime the NLRP3 inflammasome, it induced activation in LPS-primed cardiac fibroblasts as indicated by IL-1ß, IL-18 production and NLRP3-ASC co-localization. Palmitate-induced NLRP3 inflammasome activation in LPS-primed cardiac fibroblasts was associated with reduced AMPK activity, mitochondrial reactive oxygen species production and mitochondrial dysfunction. The cardiac fibroblast phenotype caused by palmitate, in an LPS and NLRP3 independent manner, was characterized by decreased cellular proliferation, contractility, collagen and MMP-2 expression, as well as increased senescence-associated ß-galactosidase activity, and consistent with a state of cellular senescence. This study establishes that in vitro palmitate exposure of cardiac fibroblasts provides inflammatory responses via TLR4 and NLRP3 inflammasome activation. Palmitate also modulates cardiac fibroblast functionality, in a NLRP3 independent manner, resulting in a phenotype related to cellular senescence. These effects of palmitate could be of importance for myocardial dysfunction in obese and diabetic patients.
Asunto(s)
Senescencia Celular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Corazón/efectos de los fármacos , Inflamación/inducido químicamente , Palmitatos/farmacología , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Quimiocina CXCL2/metabolismo , Fibroblastos/metabolismo , Inflamasomas/metabolismo , Inflamación/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
BACKGROUND: The NLR family, pyrin domain containing 3 (NLRP3) inflammasome is an interleukin (IL)-1ß and IL-18 cytokine processing complex that is activated in inflammatory conditions. The role of the NLRP3 inflammasome in the pathogenesis of atherosclerosis and myocardial infarction is not fully understood. METHODS AND RESULTS: Atherosclerotic plaques were analyzed for transcripts of the NLRP3 inflammasome, and for IL-1ß release. The Swedish First-ever myocardial Infarction study in Ac-county (FIA) cohort consisting of DNA from 555 myocardial infarction patients and 1016 healthy individuals was used to determine the frequency of 4 single nucleotide polymorphisms (SNPs) from the downstream regulatory region of NLRP3. Expression of NLRP3, Apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1 (CASP1), IL1B, and IL18 mRNA was significantly increased in atherosclerotic plaques compared to normal arteries. The expression of NLRP3 mRNA was significantly higher in plaques of symptomatic patients when compared to asymptomatic ones. CD68-positive macrophages were observed in the same areas of atherosclerotic lesions as NLRP3 and ASC expression. Occasionally, expression of NLRP3 and ASC was also present in smooth muscle cells. Cholesterol crystals and ATP induced IL-1ß release from lipopolysaccharide-primed human atherosclerotic lesion plaques. The minor alleles of the variants rs4266924, rs6672995, and rs10733113 were associated with NLRP3 mRNA levels in peripheral blood mononuclear cells but not with the risk of myocardial infarction. CONCLUSIONS: Our results indicate a possible role of the NLRP3 inflammasome and its genetic variants in the pathogenesis of atherosclerosis.
Asunto(s)
Aterosclerosis/genética , Infarto del Miocardio/genética , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Placa Aterosclerótica/genética , ARN Mensajero/metabolismo , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Proteínas Adaptadoras de Señalización CARD/genética , Caspasa 1/genética , Quimiocina CCL2/inmunología , Genotipo , Humanos , Inmunohistoquímica , Inflamasomas/genética , Inflamasomas/inmunología , Interleucina-18/genética , Interleucina-18/inmunología , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Leucocitos Mononucleares/metabolismo , Infarto del Miocardio/inmunología , Infarto del Miocardio/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Placa Aterosclerótica/inmunología , Placa Aterosclerótica/metabolismo , Polimorfismo de Nucleótido Simple , Suecia , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
AIM: Cardiac inflammation is important in the pathogenesis of heart failure. However, the consequence of systemic inflammation on concomitant established heart failure, and in particular diastolic heart failure, is less explored. Here we investigated the impact of systemic inflammation, caused by sustained Toll-like receptor 9 activation, on established diastolic heart failure. METHODS AND RESULTS: Diastolic heart failure was established in 8-10 week old cardiomyocyte specific, inducible SERCA2a knock out (i.e., SERCA2a KO) C57Bl/6J mice. Four weeks after conditional KO, mice were randomized to receive Toll-like receptor 9 agonist (CpG B; 2µg/g body weight) or PBS every third day. After additional four weeks, echocardiography, phase contrast magnetic resonance imaging, histology, flow cytometry, and cardiac RNA analyses were performed. A subgroup was followed, registering morbidity and death. Non-heart failure control groups treated with CpG B or PBS served as controls. Our main findings were: (i) Toll-like receptor 9 activation (CpG B) reduced life expectancy in SERCA2a KO mice compared to PBS treated SERCA2a KO mice. (ii) Diastolic function was lower in SERCA2a KO mice with Toll-like receptor 9 activation. (iii) Toll-like receptor 9 stimulated SERCA2a KO mice also had increased cardiac and systemic inflammation. CONCLUSION: Sustained activation of Toll-like receptor 9 causes cardiac and systemic inflammation, and deterioration of SERCA2a depletion-mediated diastolic heart failure.