RESUMEN
Innate immune responses are linked to key metabolic pathways, yet the proximal signaling events that connect these systems remain poorly understood. Here we show that phosphofructokinase 1, liver type (PFKL), a rate-limiting enzyme of glycolysis, is phosphorylated at Ser775 in macrophages following several innate stimuli. This phosphorylation increases the catalytic activity of PFKL, as shown by biochemical assays and glycolysis monitoring in cells expressing phosphorylation-defective PFKL variants. Using a genetic mouse model in which PFKL Ser775 phosphorylation cannot take place, we observe that upon activation, glycolysis in macrophages is lower than in the same cell population of wild-type animals. Consistent with their higher glycolytic activity, wild-type cells have higher levels of HIF1α and IL-1ß than PfklS775A/S775A after LPS treatment. In an in vivo inflammation model, PfklS775A/S775A mice show reduced levels of MCP-1 and IL-1ß. Our study thus identifies a molecular link between innate immune activation and early induction of glycolysis.
Asunto(s)
Glucólisis , Subunidad alfa del Factor 1 Inducible por Hipoxia , Inmunidad Innata , Interleucina-1beta , Macrófagos , Animales , Macrófagos/metabolismo , Macrófagos/inmunología , Ratones , Fosforilación , Interleucina-1beta/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Receptores de Reconocimiento de Patrones/metabolismo , Receptores de Reconocimiento de Patrones/genética , Fosfofructoquinasa-1/metabolismo , Fosfofructoquinasa-1/genética , Lipopolisacáridos/farmacología , Ratones Endogámicos C57BL , Humanos , Quimiocina CCL2/metabolismo , Quimiocina CCL2/genética , Inflamación/metabolismo , Masculino , Reprogramación MetabólicaRESUMEN
The NLRP3 inflammasome plays a central role in antimicrobial defense as well as in the context of sterile inflammatory conditions. NLRP3 activity is governed by two independent signals: the first signal primes NLRP3, rendering it responsive to the second signal, which then triggers inflammasome formation. Our understanding of how NLRP3 priming contributes to inflammasome activation remains limited. Here, we show that IKKß, a kinase activated during priming, induces recruitment of NLRP3 to phosphatidylinositol-4-phosphate (PI4P), a phospholipid enriched on the trans-Golgi network. NEK7, a mitotic spindle kinase that had previously been thought to be indispensable for NLRP3 activation, was redundant for inflammasome formation when IKKß recruited NLRP3 to PI4P. Studying iPSC-derived human macrophages revealed that the IKKß-mediated NEK7-independent pathway constitutes the predominant NLRP3 priming mechanism in human myeloid cells. Our results suggest that PI4P binding represents a primed state into which NLRP3 is brought by IKKß activity.
Asunto(s)
Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Humanos , Quinasa I-kappa B , Inflamasomas/metabolismo , Ratones Endogámicos C57BL , Quinasas Relacionadas con NIMA/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Red trans-Golgi/metabolismoRESUMEN
CD4+ T cells are central mediators of adaptive and innate immune responses and constitute a major reservoir for human immunodeficiency virus (HIV) in vivo. Detailed investigations of resting human CD4+ T cells have been precluded by the absence of efficient approaches for genetic manipulation limiting our understanding of HIV replication and restricting efforts to find a cure. Here we report a method for rapid, efficient, activation-neutral gene editing of resting, polyclonal human CD4+ T cells using optimized cell cultivation and nucleofection conditions of Cas9-guide RNA ribonucleoprotein complexes. Up to six genes, including HIV dependency and restriction factors, were knocked out individually or simultaneously and functionally characterized. Moreover, we demonstrate the knock in of double-stranded DNA donor templates into different endogenous loci, enabling the study of the physiological interplay of cellular and viral components at single-cell resolution. Together, this technique allows improved molecular and functional characterizations of HIV biology and general immune functions in resting CD4+ T cells.
Asunto(s)
Linfocitos T CD4-Positivos/fisiología , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Infecciones por VIH/genética , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/virología , Proteína 9 Asociada a CRISPR/genética , Movimiento Celular/genética , Células Cultivadas , ADN , Técnicas de Inactivación de Genes , Infecciones por VIH/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Resistencia a Mixovirus/genética , Proteínas de Resistencia a Mixovirus/metabolismo , ARN Guía de Kinetoplastida , Proteína 1 que Contiene Dominios SAM y HD/genética , Transgenes , Factores de Escisión y Poliadenilación de ARNm/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
PURPOSE: To determine risk factors for coronavirus disease 2019 (COVID-19) in healthcare workers (HCWs), characterize symptoms, and evaluate preventive measures against SARS-CoV-2 spread in hospitals. METHODS: In a cross-sectional study conducted between May 27 and August 12, 2020, after the first wave of the COVID-19 pandemic, we obtained serological, epidemiological, occupational as well as COVID-19-related data at a quaternary care, multicenter hospital in Munich, Germany. RESULTS: 7554 HCWs participated, 2.2% of whom tested positive for anti-SARS-CoV-2 antibodies. Multivariate analysis revealed increased COVID-19 risk for nurses (3.1% seropositivity, 95% CI 2.5-3.9%, p = 0.012), staff working on COVID-19 units (4.6% seropositivity, 95% CI 3.2-6.5%, p = 0.032), males (2.4% seropositivity, 95% CI 1.8-3.2%, p = 0.019), and HCWs reporting high-risk exposures to infected patients (5.5% seropositivity, 95% CI 4.0-7.5%, p = 0.0022) or outside of work (12.0% seropositivity, 95% CI 8.0-17.4%, p < 0.0001). Smoking was a protective factor (1.1% seropositivity, 95% CI 0.7-1.8% p = 0.00018) and the symptom taste disorder was strongly associated with COVID-19 (29.8% seropositivity, 95% CI 24.3-35.8%, p < 0.0001). An unbiased decision tree identified subgroups with different risk profiles. Working from home as a preventive measure did not protect against SARS-CoV-2 infection. A PCR-testing strategy focused on symptoms and high-risk exposures detected all larger COVID-19 outbreaks. CONCLUSION: Awareness of the identified COVID-19 risk factors and successful surveillance strategies are key to protecting HCWs against SARS-CoV-2, especially in settings with limited vaccination capacities or reduced vaccine efficacy.
Asunto(s)
COVID-19 , COVID-19/epidemiología , COVID-19/prevención & control , Estudios Transversales , Personal de Salud , Humanos , Masculino , Pandemias/prevención & control , Factores de Riesgo , SARS-CoV-2RESUMEN
The importance of pre-existing immune responses to seasonal endemic coronaviruses (HCoVs) for the susceptibility to SARS-CoV-2 infection and the course of COVID-19 is the subject of an ongoing scientific debate. Recent studies postulate that immune responses to previous HCoV infections can either have a slightly protective or no effect on SARS-CoV-2 pathogenesis and, consequently, be neglected for COVID-19 risk stratification. Challenging this notion, we provide evidence that pre-existing, anti-nucleocapsid antibodies against endemic α-coronaviruses and S2 domain-specific anti-spike antibodies against ß-coronavirus HCoV-OC43 are elevated in patients with COVID-19 compared to pre-pandemic donors. This finding is particularly pronounced in males and in critically ill patients. Longitudinal evaluation reveals that antibody cross-reactivity or polyclonal stimulation by SARS-CoV-2 infection are unlikely to be confounders. Thus, specific pre-existing immunity to seasonal coronaviruses may increase susceptibility to SARS-CoV-2 and predispose individuals to an adverse COVID-19 outcome, guiding risk management and supporting the development of universal coronavirus vaccines.