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Introduction: People living with HIV (PLHIV) are characterized by functional reprogramming of innate immune cells even after long-term antiretroviral therapy (ART). In order to assess technical feasibility of omics technologies for application to larger cohorts, we compared multiple omics data layers. Methods: Bulk and single-cell transcriptomics, flow cytometry, proteomics, chromatin landscape analysis by ATAC-seq as well as ex vivo drug stimulation were performed in a small number of blood samples derived from PLHIV and healthy controls from the 200-HIV cohort study. Results: Single-cell RNA-seq analysis revealed that most immune cells in peripheral blood of PLHIV are altered in their transcriptomes and that a specific functional monocyte state previously described in acute HIV infection is still existing in PLHIV while other monocyte cell states are only occurring acute infection. Further, a reverse transcriptome approach on a rather small number of PLHIV was sufficient to identify drug candidates for reversing the transcriptional phenotype of monocytes in PLHIV. Discussion: These scientific findings and technological advancements for clinical application of single-cell transcriptomics form the basis for the larger 2000-HIV multicenter cohort study on PLHIV, for which a combination of bulk and single-cell transcriptomics will be included as the leading technology to determine disease endotypes in PLHIV and to predict disease trajectories and outcomes.
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Fármacos Anti-VIH , Infecciones por VIH , Humanos , Fármacos Anti-VIH/uso terapéutico , Estudios de Cohortes , Monocitos , Estudios Multicéntricos como AsuntoRESUMEN
Background: Even during long-term combination antiretroviral therapy (cART), people living with HIV (PLHIV) have a dysregulated immune system, characterized by persistent immune activation, accelerated immune ageing and increased risk of non-AIDS comorbidities. A multi-omics approach is applied to a large cohort of PLHIV to understand pathways underlying these dysregulations in order to identify new biomarkers and novel genetically validated therapeutic drugs targets. Methods: The 2000HIV study is a prospective longitudinal cohort study of PLHIV on cART. In addition, untreated HIV spontaneous controllers were recruited. In-depth multi-omics characterization will be performed, including genomics, epigenomics, transcriptomics, proteomics, metabolomics and metagenomics, functional immunological assays and extensive immunophenotyping. Furthermore, the latent viral reservoir will be assessed through cell associated HIV-1 RNA and DNA, and full-length individual proviral sequencing on a subset. Clinical measurements include an ECG, carotid intima-media thickness and plaque measurement, hepatic steatosis and fibrosis measurement as well as psychological symptoms and recreational drug questionnaires. Additionally, considering the developing pandemic, COVID-19 history and vaccination was recorded. Participants return for a two-year follow-up visit. The 2000HIV study consists of a discovery and validation cohort collected at separate sites to immediately validate any finding in an independent cohort. Results: Overall, 1895 PLHIV from four sites were included for analysis, 1559 in the discovery and 336 in the validation cohort. The study population was representative of a Western European HIV population, including 288 (15.2%) cis-women, 463 (24.4%) non-whites, and 1360 (71.8%) MSM (Men who have Sex with Men). Extreme phenotypes included 114 spontaneous controllers, 81 rapid progressors and 162 immunological non-responders. According to the Framingham score 321 (16.9%) had a cardiovascular risk of >20% in the next 10 years. COVID-19 infection was documented in 234 (12.3%) participants and 474 (25.0%) individuals had received a COVID-19 vaccine. Conclusion: The 2000HIV study established a cohort of 1895 PLHIV that employs multi-omics to discover new biological pathways and biomarkers to unravel non-AIDS comorbidities, extreme phenotypes and the latent viral reservoir that impact the health of PLHIV. The ultimate goal is to contribute to a more personalized approach to the best standard of care and a potential cure for PLHIV.
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COVID-19 , Infecciones por VIH , Minorías Sexuales y de Género , Masculino , Humanos , Femenino , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/epidemiología , Homosexualidad Masculina , Estudios Prospectivos , Vacunas contra la COVID-19/uso terapéutico , Grosor Intima-Media Carotídeo , Estudios Longitudinales , MultiómicaRESUMEN
BACKGROUND: Gout flares are driven by interleukin (IL)-1ß. Dapansutrile inhibits the NLRP3 inflammasome and subsequent activation of IL-1ß. In this study we aimed to investigate the safety and efficacy of orally administered dapansutrile in patients with a gout flare. METHODS: In this open-label, proof-of-concept, phase 2a trial, adult patients (aged 18-80 years) with a monoarticular monosodium urate crystal-proven gout flare were enrolled at an outpatient clinic in the Netherlands and sequentially assigned using a dose-adaptive design to receive 100 mg/day, 300 mg/day, 1000 mg/day, or 2000 mg/day oral dapansutrile for 8 days. The coprimary outcomes were change in patient-reported target joint pain from baseline to day 3 and from baseline to day 7, assessed in the per-protocol population (all patients who received at least 80% of the study drug and had no major protocol deviations). Safety was assessed in the intention-to-treat population. This trial is registered with the EU Clinical Trials Register, EudraCT 2016-000943-14, and is completed. FINDINGS: Between May 18, 2017, and Jan 21, 2019, 144 patients were assessed for eligibility, of whom 34 were enrolled and 29 were included in the per-protocol population (three patients were excluded due to receiving <80% of study drug and two had major protocol deviations): eight patients received 100 mg/day, seven received 300 mg/day, six received 1000 mg/day, and eight received 2000 mg/day. Between baseline and day 3, there was a mean reduction in patient-reported target joint pain of 52·4% (SD 32·94; p=0â016) for the 100 mg/day group, 68·4% (34·29; p=0â016) for the 300 mg/day group, 55·8% (44·90; p=0â063) for the 1000 mg/day group, and 57·6% (38·72; p=0â016) for the 2000 mg/day group. At day 7, there was a mean reduction of 82·1% (22·68; p=0â031) for the 100 mg/day group, 84·2% (16·33; p=0â016) for the 300 mg/day group, 68·9% (34·89; p=0â031) for the 1000 mg/day group, and 83·9% (15·44; p=0â008) for the 2000 mg/day group, compared to baseline. 25 (73·5%) of 34 patients reported a total of 45 treatment-emergent adverse events, most of which were metabolism and nutrition disorders (17 [37·8%]) and gastrointestinal disorders (ten [22·2%]). Two serious adverse events occurred during the study, admission to hospital because of worsening of gout flare at day 3, and admission to hospital because of coronary stenosis 18 days after the patient received their last dose; these were considered moderate in severity and unrelated to the study drug. INTERPRETATION: Dapansutrile is a specific NLRP3 inflammasome inhibitor with a satisfactory safety profile and efficacy in the reduction of target joint pain in this study. Future studies are needed to confirm the clinical potential of dapansutrile.
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Bacille Calmette-Guérin (BCG) induces long-term boosting of innate immunity, termed trained immunity, and decreases susceptibility to respiratory tract infections. BCG vaccination trials for reducing SARS-CoV-2 infection are underway, but concerns have been raised regarding the potential harm of strong innate immune responses. To investigate the safety of BCG vaccination, we retrospectively assessed coronavirus disease 2019 (COVID-19) and related symptoms in three cohorts of healthy volunteers who either received BCG in the last 5 years or did not. BCG vaccination is not associated with increased incidence of symptoms during the COVID-19 outbreak in the Netherlands. Our data suggest that BCG vaccination might be associated with a decrease in the incidence of sickness during the COVID-19 pandemic (adjusted odds ratio [AOR] 0.58, p < 0.05), and lower incidence of extreme fatigue. In conclusion, recent BCG vaccination is safe, and large randomized trials are needed to reveal if BCG reduces the incidence and/or severity of SARS-CoV-2 infection.
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Vacuna BCG/administración & dosificación , COVID-19/epidemiología , Vacunación/estadística & datos numéricos , Adulto , Anciano , Vacuna BCG/inmunología , COVID-19/inmunología , Femenino , Humanos , Memoria Inmunológica , Incidencia , Masculino , Persona de Mediana Edad , Países Bajos/epidemiología , Estudios Retrospectivos , SARS-CoV-2 , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
OBJECTIVE: Gout is characterised by severe interleukin (IL)-1-mediated joint inflammation induced by monosodium urate crystals. Since IL-37 is a pivotal anti-inflammatory cytokine suppressing the activity of IL-1, we conducted genetic and functional studies aimed at elucidating the role of IL-37 in the pathogenesis and treatment of gout. METHODS: Variant identification was performed by DNA sequencing of all coding bases of IL37 using molecular inversion probe-based resequencing (discovery cohort: gout n=675, controls n=520) and TaqMan genotyping (validation cohort: gout n=2202, controls n=2295). Predictive modelling of the effects of rare variants on protein structure was followed by in vitro experiments evaluating the impact on protein function. Treatment with recombinant IL-37 was evaluated in vitro and in vivo in a mouse model of gout. RESULTS: We identified four rare variants in IL37 in six of the discovery gout patients; p.(A144P), p.(G174Dfs*16), p.(C181*) and p.(N182S), whereas none emerged in healthy controls (Fisher's exact p-value=0.043). All variants clustered in the functional domain of IL-37 in exon 5 (p-value=5.71×10-5). Predictive modelling and functional studies confirmed loss of anti-inflammatory functions and we substantiated the therapeutic potential of recombinant IL-37 in the treatment of gouty inflammation. Furthermore, the carrier status of p.(N182S)(rs752113534) was associated with increased risk (OR=1.81, p-value=0.031) of developing gout in hyperuricaemic individuals of Polynesian ancestry. CONCLUSION: Here, we provide genetic as well as mechanistic evidence for the role of IL-37 in the pathogenesis of gout, and highlight the therapeutic potential of recombinant IL-37 for the treatment of gouty arthritis.
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Gota/genética , Interleucina-1/genética , Adulto , Anciano , Anciano de 80 o más Años , Animales , Estudios de Casos y Controles , Femenino , Predisposición Genética a la Enfermedad , Gota/inmunología , Humanos , Técnicas In Vitro , Interleucina-1/inmunología , Interleucina-1/farmacología , Interleucina-1beta/efectos de los fármacos , Interleucina-1beta/inmunología , Interleucina-6/inmunología , Interleucina-8/efectos de los fármacos , Interleucina-8/inmunología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Masculino , Ratones , Persona de Mediana Edad , Nativos de Hawái y Otras Islas del Pacífico/genética , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Polimorfismo Genético , Proteínas Recombinantes/farmacología , Ácido Úrico/inmunología , Ácido Úrico/farmacología , Población Blanca/genéticaRESUMEN
Sodium butyrate is well-known for its immune-modulatory properties. Studies until now only focused on the in vitro effects of butyrate or assessed local effects in the gut upon butyrate administration. In this trial, we studied the systemic anti-inflammatory effects induced by sodium butyrate supplementation in humans. Nine healthy (Lean) and ten obese (metabolic syndrome group, MetSyn) males were given 4 grams sodium butyrate daily for 4 weeks. PBMCs were isolated before and after supplementation for direct stimulation experiments and induction of trained immunity by oxidized low-density lipoprotein (oxLDL), ß-glucan, or Bacillus Calmette-Guérin vaccine (BCG). Butyrate supplementation moderately affected some of the cytokine responses in the MetSyn group. In the direct stimulation setup, effects of butyrate supplementation were limited. Interestingly, butyrate supplementation decreased oxLDL-induced trained immunity in the MetSyn group for LPS-induced IL-6 responses and Pam3CSK4-induced TNF-α responses. Induction of trained immunity by ß-glucan was decreased by butyrate in the MetSyn group for Pam3CSK4-induced IL-10 production. In this study, while having only limited effects on the direct stimulation of cytokine production, butyrate supplementation significantly affected trained immunity in monocytes of obese individuals with metabolic complications. Therefore, oral butyrate supplementation may be beneficial in reducing the overall inflammatory status of circulating monocytes in patients with metabolic syndrome.
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Antiinfecciosos/administración & dosificación , Ácido Butírico/administración & dosificación , Leucocitos Mononucleares/inmunología , Obesidad/inmunología , Adulto , Antiinfecciosos/farmacología , Vacuna BCG/inmunología , Vacuna BCG/farmacología , Ácido Butírico/farmacología , Estudios de Casos y Controles , Citocinas/metabolismo , Esquema de Medicación , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Lipoproteínas LDL/inmunología , Lipoproteínas LDL/farmacología , Masculino , Persona de Mediana Edad , Adulto Joven , beta-Glucanos/inmunología , beta-Glucanos/farmacologíaRESUMEN
BACKGROUND: Intestinal microbiota have been found to be linked to cardiovascular disease via conversion of the dietary compounds choline and carnitine to the atherogenic metabolite TMAO (trimethylamine-N-oxide). Specifically, a vegan diet was associated with decreased plasma TMAO levels and nearly absent TMAO production on carnitine challenge. METHODS AND RESULTS: We performed a double-blind randomized controlled pilot study in which 20 male metabolic syndrome patients were randomized to single lean vegan-donor or autologous fecal microbiota transplantation. At baseline and 2 weeks thereafter, we determined the ability to produce TMAO from d6-choline and d3-carnitine (eg, labeled and unlabeled TMAO in plasma and 24-hour urine after oral ingestion of 250 mg of both isotope-labeled precursor nutrients), and fecal samples were collected for analysis of microbiota composition. 18F-fluorodeoxyglucose positron emission tomography/computed tomography scans of the abdominal aorta, as well as ex vivo peripheral blood mononuclear cell cytokine production assays, were performed. At baseline, fecal microbiota composition differed significantly between vegans and metabolic syndrome patients. With vegan-donor fecal microbiota transplantation, intestinal microbiota composition in metabolic syndrome patients, as monitored by global fecal microbial community structure, changed toward a vegan profile in some of the patients; however, no functional effects from vegan-donor fecal microbiota transplantation were seen on TMAO production, abdominal aortic 18F-fluorodeoxyglucose uptake, or ex vivo cytokine production from peripheral blood mononuclear cells. CONCLUSIONS: Single lean vegan-donor fecal microbiota transplantation in metabolic syndrome patients resulted in detectable changes in intestinal microbiota composition but failed to elicit changes in TMAO production capacity or parameters related to vascular inflammation. CLINICAL TRIAL REGISTRATION: URL: http://www.trialregister.nl. Unique identifier: NTR 4338.
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Bacterias/metabolismo , Colina/metabolismo , Citocinas/metabolismo , Dieta Vegana , Trasplante de Microbiota Fecal , Microbioma Gastrointestinal , Mediadores de Inflamación/metabolismo , Síndrome Metabólico/terapia , Metilaminas/metabolismo , Vasculitis/terapia , Adulto , Anciano , Carnitina/metabolismo , Método Doble Ciego , Trasplante de Microbiota Fecal/efectos adversos , Heces/microbiología , Humanos , Masculino , Síndrome Metabólico/diagnóstico , Síndrome Metabólico/microbiología , Persona de Mediana Edad , Países Bajos , Proyectos Piloto , Factores de Tiempo , Resultado del Tratamiento , Vasculitis/diagnóstico , Vasculitis/microbiología , Adulto JovenRESUMEN
Metabolic triggers are important inducers of the inflammatory processes in gout. Whereas the high serum urate levels observed in patients with gout predispose them to the formation of monosodium urate (MSU) crystals, soluble urate also primes for inflammatory signals in cells responding to gout-related stimuli, but also in other common metabolic diseases. In this study, we investigated the mechanisms through which uric acid selectively lowers human blood monocyte production of the natural inhibitor IL-1 receptor antagonist (IL-1Ra) and shifts production toward the highly inflammatory IL-1ß. Monocytes from healthy volunteers were first primed with uric acid for 24 h and then subjected to stimulation with lipopolysaccharide (LPS) in the presence or absence of MSU. Transcriptomic analysis revealed broad inflammatory pathways associated with uric acid priming, with NF-κB and mammalian target of rapamycin (mTOR) signaling strongly increased. Functional validation did not identify NF-κB or AMP-activated protein kinase phosphorylation, but uric acid priming induced phosphorylation of AKT and proline-rich AKT substrate 40 kDa (PRAS 40), which in turn activated mTOR. Subsequently, Western blot for the autophagic structure LC3-I and LC3-II (microtubule-associated protein 1A/1B-light chain 3) fractions, as well as fluorescence microscopy of LC3-GFP-overexpressing HeLa cells, revealed lower autophagic activity in cells exposed to uric acid compared with control conditions. Interestingly, reactive oxygen species production was diminished by uric acid priming. Thus, the Akt-PRAS40 pathway is activated by uric acid, which inhibits autophagy and recapitulates the uric acid-induced proinflammatory cytokine phenotype.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagia , Monocitos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ácido Úrico/metabolismo , Animales , Modelos Animales de Enfermedad , Gota/metabolismo , Enfermedad Granulomatosa Crónica/metabolismo , Células HeLa , Humanos , Hiperuricemia/metabolismo , Proteína Antagonista del Receptor de Interleucina 1/metabolismo , Interleucina-1beta/metabolismo , Ratones , Especies Reactivas de Oxígeno/metabolismo , Transcriptoma , Urato Oxidasa/antagonistas & inhibidoresRESUMEN
PURPOSE OF REVIEW: Gout is a common debilitating form of arthritis and despite our extensive knowledge on the pathogenesis its prevalence is still rising quickly. In the current review, we provide a concise overview of recent discoveries in factors tuning the inflammatory response to soluble uric acid and monosodium urate crystals. RECENT FINDINGS: It appears that soluble uric acid has a much larger role to play than just being a risk factor for gout. It may have widespread consequences for systemic inflammation and the development of metabolic syndrome. Additionally, a specific gout-related gut microbiome might not only provide us with a new diagnostic tool, but also highlights possible new therapeutic targets. Furthermore, several recent publications further elucidated the roles of mitochondrial dysfunction, production of reactive oxygen species, autophagy, and AMP-dependent protein kinase in monosodium urate-induced NLRP3 inflammasome activation. Finally, neutrophils have been shown to be involved in both the promotion and resolution of gouty inflammation. A new alpha-1-antitrypsin fusion protein may limit the proinflammatory effects of neutrophil-derived serine proteases. SUMMARY: Together, these studies provide us with many new insights in the pathogenesis of gout, important new treatment targets, and a rationale to further study the role of soluble uric acid in inflammatory diseases.
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Artritis Gotosa/inmunología , Microbioma Gastrointestinal/inmunología , Hiperuricemia/inmunología , Ácido Úrico/inmunología , Autofagia/inmunología , Gota/inmunología , Humanos , Inflamasomas/inmunología , Inflamación , Mitocondrias/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Neutrófilos/inmunología , Estrés Oxidativo , Fosfotransferasas (Aceptor del Grupo Fosfato)/inmunología , Especies Reactivas de Oxígeno/inmunologíaRESUMEN
Type 2 diabetes mellitus confers a threefold increased risk for tuberculosis, but the underlying immunological mechanisms are still largely unknown. Possible mediators of this increased susceptibility are short-chain fatty acids, levels of which have been shown to be altered in individuals with diabetes. We examined the influence of physiological concentrations of butyrate on cytokine responses to Mycobacterium tuberculosis (Mtb) in human peripheral blood mononuclear cells (PBMCs). Butyrate decreased Mtb-induced proinflammatory cytokine responses, while it increased production of IL-10. This anti-inflammatory effect was independent of butyrate's well-characterised inhibition of HDAC activity and was not accompanied by changes in Toll-like receptor signalling pathways, the eicosanoid pathway, or cellular metabolism. In contrast blocking IL-10 activity reversed the effects of butyrate on Mtb-induced inflammation. Alteration of the gut microbiota, thereby increasing butyrate concentrations, can reduce insulin resistance and obesity, but further studies are needed to determine how this affects susceptibility to tuberculosis.
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Butiratos/farmacología , Complicaciones de la Diabetes/inmunología , Leucocitos Mononucleares/efectos de los fármacos , Mycobacterium tuberculosis/inmunología , Tuberculosis/inmunología , Células Cultivadas , Citocinas/inmunología , Complicaciones de la Diabetes/microbiología , Interacciones Huésped-Patógeno , Humanos , Mediadores de Inflamación/inmunología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/microbiología , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Tuberculosis/microbiologíaRESUMEN
OBJECTIVES: Acute gouty arthritis is caused by endogenously formed monosodium urate (MSU) crystals, which are potent activators of the NLRP3 inflammasome. However, to induce the release of active interleukin (IL)-1ß, an additional stimulus is needed. Saturated long-chain free fatty acids (FFAs) can provide such a signal and stimulate transcription of pro-IL-1ß. In contrast, the short-chain fatty acid butyrate possesses anti-inflammatory effects. One of the mechanisms involved is inhibition of histone deacetylases (HDACs). Here, we explored the effects of butyrate on MSU+FFA-induced cytokine production and its inhibition of specific HDACs. METHODS: Freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated with MSU and palmitic acid (C16.0) in the presence or absence of butyrate or a synthetic HDAC inhibitor. Cytokine responses were measured with ELISA and quantitative PCR. HDAC activity was measured with fluorimetric assays. RESULTS: Butyrate decreased C16.0+MSU-induced production of IL-1ß, IL-6, IL-8 and IL-1ß mRNA in PBMCs from healthy donors. Similar results were obtained in PBMCs isolated from patients with gout. Butyrate specifically inhibited class I HDACs. The HDAC inhibitor, panobinostat and the potent HDAC inhibitor, ITF-B, also decreased ex vivo C16.0+MSU-induced IL-1ß production. CONCLUSIONS: In agreement with the reported low inhibitory potency of butyrate, a high concentration was needed for cytokine suppression, whereas synthetic HDAC inhibitors showed potent anti-inflammatory effects at nanomolar concentrations. These novel HDAC inhibitors could be effective in the treatment of acute gout. Moreover, the use of specific HDAC inhibitors could even improve the efficacy and reduce any potential adverse effects.
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Antioxidantes/farmacología , Artritis Gotosa , Butiratos/farmacología , Citocinas/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Ácido Palmítico/farmacología , ARN Mensajero/efectos de los fármacos , Ácido Úrico/farmacología , Adulto , Carbamatos/farmacología , Cristalización , Citocinas/genética , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Histona Desacetilasas , Humanos , Ácidos Hidroxámicos/farmacología , Indoles/farmacología , Interleucina-1beta/efectos de los fármacos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Interleucina-8/efectos de los fármacos , Interleucina-8/metabolismo , Leucocitos Mononucleares , Masculino , Persona de Mediana Edad , Panobinostat , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVES: The study of the proinflammatory role of uric acid has focused on the effects of its crystals of monosodium urate (MSU). However, little is known whether uric acid itself can directly have proinflammatory effects. In this study, we investigate the priming effects of uric acid exposure on the cytokine production of primary human cells upon stimulation with gout-related stimuli. METHODS: Peripheral blood mononuclear cells (PBMCs) were harvested from patients with gout and healthy volunteers. Cells were pretreated with or without uric acid in soluble form for 24â h and then stimulated for 24â h with toll-like receptor (TLR)2 or TLR4 ligands in the presence or absence of MSU crystals. Cytokine production was measured by ELISA; mRNA levels were assessed using qPCR. RESULTS: The production of interleukin (IL)-1ß and IL-6 was higher in patients compared with controls and this correlated with serum urate levels. Proinflammatory cytokine production was significantly potentiated when cells from healthy subjects were pretreated with uric acid. Surprisingly, this was associated with a significant downregulation of the anti-inflammatory cytokine IL-1 receptor antagonist (IL-1Ra). This effect was specific to stimulation by uric acid and was exerted at the level of gene transcription. Epigenetic reprogramming at the level of histone methylation by uric acid was involved in this effect. CONCLUSIONS: In this study we demonstrate a mechanism through which high concentrations of uric acid (up to 50â mg/dL) influence inflammatory responses by facilitating IL-1ß production in PBMCs. We show that a mechanism for the amplification of IL-1ß consists in the downregulation of IL-1Ra and that this effect could be exerted via epigenetic mechanisms such as histone methylation. Hyperuricaemia causes a shift in the IL-1ß/IL-1Ra balance produced by PBMCs after exposure to MSU crystals and TLR-mediated stimuli, and this phenomenon is likely to reinforce the enhanced state of chronic inflammation.
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Citocinas/inmunología , Epigénesis Genética/inmunología , Gota/inmunología , Leucocitos Mononucleares/inmunología , ARN Mensajero/metabolismo , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 4/inmunología , Ácido Úrico/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Antioxidantes/farmacología , Estudios de Casos y Controles , Citocinas/efectos de los fármacos , Citocinas/genética , Ensayo de Inmunoadsorción Enzimática , Epigénesis Genética/genética , Gota/genética , Código de Histonas , Histonas/metabolismo , Humanos , Inflamación , Proteína Antagonista del Receptor de Interleucina 1/efectos de los fármacos , Proteína Antagonista del Receptor de Interleucina 1/genética , Proteína Antagonista del Receptor de Interleucina 1/inmunología , Interleucina-10/genética , Interleucina-10/inmunología , Interleucina-1beta/efectos de los fármacos , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Leucocitos Mononucleares/efectos de los fármacos , Metilación , Persona de Mediana Edad , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Ácido Úrico/farmacologíaRESUMEN
OBJECTIVES: In the present study, we generated a new protein, recombinant human alpha-1-anti-trypsin (AAT)-IgG1 Fc fusion protein (AAT-Fc), and evaluated its properties to suppress inflammation and interleukin (IL)-1ß in a mouse model of gouty arthritis. METHODS: A combination of monosodium urate (MSU) crystals and the fatty acid C16.0 (MSU/C16.0) was injected intra-articularly into the knee to induce gouty arthritis. Joint swelling, synovial cytokine production and histopathology were determined after 4â h. AAT-Fc was evaluated for inhibition of MSU/C16.0-induced IL-1ß release from human blood monocytes and for inhibition of extracellular IL-1ß precursor processing. RESULTS: AAT-Fc markedly suppressed MSU/C16.0-induced joint inflammation by 85-91% (p<0.001). Ex vivo production of IL-1ß and IL-6 from cultured synovia were similarly reduced (63% and 65%, respectively). The efficacy of 2.0â mg/kg AAT-Fc in reducing inflammation was comparable to 80â mg/kg of plasma-derived AAT. Injection of AAT-Fc into mice increased circulating levels of endogenous IL-1 receptor antagonist by fourfold. We also observed that joint swelling was reduced by 80%, cellular infiltration by 95% and synovial production of IL-1ß by 60% in transgenic mice expressing low levels of human AAT. In vitro, AAT-Fc reduced MSU/C16.0-induced release of IL-1ß from human blood monocytes and inhibited proteinase-3-mediated extracellular processing of the IL-1ß precursor into active IL-1ß. CONCLUSIONS: A single low dose of AAT-Fc is highly effective in reducing joint inflammation in this model of acute gouty arthritis. Considering the long-term safety of plasma-derived AAT use in humans, subcutaneous AAT-Fc emerges as a promising therapy for gout attacks.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Artritis Gotosa/tratamiento farmacológico , Supresores de la Gota/uso terapéutico , Fragmentos Fc de Inmunoglobulinas/uso terapéutico , Proteína Antagonista del Receptor de Interleucina 1/biosíntesis , Interleucina-1beta/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/uso terapéutico , alfa 1-Antitripsina/uso terapéutico , Animales , Artritis Experimental/inmunología , Artritis Experimental/patología , Artritis Gotosa/inmunología , Artritis Gotosa/patología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Supresores de la Gota/administración & dosificación , Supresores de la Gota/farmacología , Humanos , Fragmentos Fc de Inmunoglobulinas/administración & dosificación , Fragmentos Fc de Inmunoglobulinas/farmacología , Inyecciones Intraarticulares , Inyecciones Intraperitoneales , Interleucina-1beta/metabolismo , Receptores de Lipopolisacáridos/análisis , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Monocitos/efectos de los fármacos , Monocitos/inmunología , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/farmacología , alfa 1-Antitripsina/administración & dosificación , alfa 1-Antitripsina/farmacologíaRESUMEN
Monosodium urate (MSU) monohydrate crystals synergize with various toll-like receptor (TLR) ligands to induce interleukin-(IL)-1ß production. Data are shown from a young male with mitochondriopathy in Kearns-Sayre syndrome (KSS) who developed gout and underwent urate-lowering therapy (ULT) versus a group of common gout patients. Peripheral blood mononuclear cells (PBMCs) are exposed in vitro to MSU crystals in the presence/absence of TLR2 ligands palmitic acid (C16:0) or palmitoyl-3-cysteine (Pam3Cys); proinflammatory cytokine production (IL-1ß, IL-6, IL-8) is assessed by specific ELISA's. MSU crystals alone failed to induce IL-1beta, IL-6, or IL-8 in both the KSS patient and gout controls. A strong synergy between MSU crystals and C16:0 or Pam3Cys for induction of IL-1 beta/IL-6 is found in gout patients, but in gout with KSS, we found even more response than in control gout patients. Pam3Cys exposure reveals an enhanced response in cells originating from the KSS patient, indicating a high producer phenotype in response to TLR2 stimulation. During ULT, serum urate levels dropped in the KSS case. The hyperresponse of TLR2 may be secondary to the high serum urate concentration of 0.92 mmol/l that was initially found in circulation in vivo. Within a 6-month period, the serum urate concentration dropped, and the in vitro stimulation tests improved but did not fully normalize yet. The ex vivo cytokine production in gout patients is promising a novel gout test; PBMCs' responses in the mitochondriopathic gout patient is enhanced when compared with common gout patients, indicating a supersensitive gout patient profile. The non-inflammatory presentation in the KSS case with bulky gout is due to less inflammatory MSU crystals, i.e., specific crystal stereochemical/conformational properties. For developing gout attacks, the serum urate level and specific crystal properties both are of importance. Key Messages 1. Ex vivo cell tests are promising to serve as a novel gout lab test for screening purposes. 2. Ex vivo cellular responses are reduced following intraarticular glucocorticoid injection and/or urate-lowering therapy. 3. Crystal conformation properties play a role in the inflammatory in vivo and ex vivo response in gout. 4. A young male with Kearns-Sayre syndrome is described with less pronounced inflammatory PMBC responses to his own MSU crystals which explains the advanced stage of urate accumulation in this individual.