RESUMEN
Hemolysis drives susceptibility to bacterial infections and predicts poor outcome from sepsis. These detrimental effects are commonly considered to be a consequence of heme-iron serving as a nutrient for bacteria. We employed a Gram-negative sepsis model and found that elevated heme levels impaired the control of bacterial proliferation independently of heme-iron acquisition by pathogens. Heme strongly inhibited phagocytosis and the migration of human and mouse phagocytes by disrupting actin cytoskeletal dynamics via activation of the GTP-binding Rho family protein Cdc42 by the guanine nucleotide exchange factor DOCK8. A chemical screening approach revealed that quinine effectively prevented heme effects on the cytoskeleton, restored phagocytosis and improved survival in sepsis. These mechanistic insights provide potential therapeutic targets for patients with sepsis or hemolytic disorders.
Asunto(s)
Infecciones por Bacterias Gramnegativas/inmunología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Hemo/metabolismo , Hemólisis/inmunología , Macrófagos/inmunología , Fagocitosis , Sepsis/inmunología , Animales , Antibacterianos/uso terapéutico , Citoesqueleto/metabolismo , Femenino , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Factores de Intercambio de Guanina Nucleótido/genética , Hemo-Oxigenasa 1/genética , Hemólisis/efectos de los fármacos , Humanos , Evasión Inmune , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fagocitosis/efectos de los fármacos , Quinina/uso terapéutico , Células RAW 264.7 , Sepsis/tratamiento farmacológico , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus has been causing the COVID-19 pandemic since December 2019, with over 600 million infected persons worldwide and over six million deaths. We investigated the anti-viral effects of polyphenolic green tea ingredients and the synthetic resveratrol analogue 3,3',4,4',5,5'-hexahydroxy-trans-stilbene (HHS), a compound with antioxidant, antitumor and anti-HIV properties. In the TCID50 assay, four out of nine green tea constituents showed minor to modest cell protective effects, whereas HHS demonstrated the highest reduction (1103-fold) of the TCID50, indicating pronounced inhibition of virus replication. HHS was also a highly effective inhibitor of SARS-CoV-2 proliferation in VeroE6 cells with an IC50 value of 31.1 µM. HSS also inhibited the binding of the receptor-binding domain (RBD) of the spike protein to the human angiotensin-converting enzyme 2 (ACE2) receptor (RBD-ACE2) binding with 29% at 100 µM and with 9.2% at 50 µM indicating that the SARS-CoV-2 inhibitory effect might at least in part be attributed to the inhibition of virus binding to ACE2. Based on the chemical similarity to other polyphenols, the oral bioavailability of HHS is likely also very low, resulting in blood levels far below the inhibitory concentration of EGCG against SARS-CoV-2 observed in vitro. However, administration of HHS topically as a nose or throat spray would increase concentrations several-fold above the minimal inhibitory concentration (MIC) in the mucosa and might reduce virus load when administered soon after infection. Due to these promising tissue culture results, further preclinical and clinical studies are warranted to develop HHS as an additional treatment option for SARS-CoV-2 infection to complement vaccines, which is and will be the main pillar to combat the COVID-19 pandemic.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Enzima Convertidora de Angiotensina 2/metabolismo , Resveratrol/farmacología , Pandemias , Unión ProteicaRESUMEN
RATIONALE: Lung transplantation is the ultimate treatment option for patients with end-stage respiratory diseases but bears the highest mortality rate among all solid organ transplantations due to chronic lung allograft dysfunction (CLAD). The mechanisms leading to CLAD remain elusive due to an insufficient understanding of the complex post-transplant adaptation processes. OBJECTIVES: To better understand these lung adaptation processes after transplantation and to investigate their association with future changes in allograft function. METHODS: We performed an exploratory cohort study of bronchoalveolar lavage samples from 78 lung recipients and donors. We analysed the alveolar microbiome using 16S rRNA sequencing, the cellular composition using flow cytometry, as well as metabolome and lipidome profiling. MEASUREMENTS AND MAIN RESULTS: We established distinct temporal dynamics for each of the analysed data sets. Comparing matched donor and recipient samples, we revealed that recipient-specific as well as environmental factors, rather than the donor microbiome, shape the long-term lung microbiome. We further discovered that the abundance of certain bacterial strains correlated with underlying lung diseases even after transplantation. A decline in forced expiratory volume during the first second (FEV1) is a major characteristic of lung allograft dysfunction in transplant recipients. By using a machine learning approach, we could accurately predict future changes in FEV1 from our multi-omics data, whereby microbial profiles showed a particularly high predictive power. CONCLUSION: Bronchoalveolar microbiome, cellular composition, metabolome and lipidome show specific temporal dynamics after lung transplantation. The lung microbiome can predict future changes in lung function with high precision.
Asunto(s)
Trasplante de Pulmón , Microbiota , Aloinjertos , Estudios de Cohortes , Humanos , Pulmón , ARN Ribosómico 16S/genética , Estudios RetrospectivosRESUMEN
OBJECTIVE: Liver injury impacts hepatic inflammation in part via Toll-like receptor (TLR) signalling. Triggering receptor expressed on myeloid cells 2 (TREM-2) modulates TLR4-mediated inflammation in bone marrow (BM)-derived macrophages but its function in liver injury is unknown. Here we hypothesised that the anti-inflammatory effects of TREM-2 on TLR signalling may limit hepatic injury. DESIGN: TREM-2 expression was analysed in livers of humans with various forms of liver injury compared with control individuals. Acute and chronic liver injury models were performed in wild type and Trem-2-/- mice. Primary liver cells from both genotypes of mice were isolated for in vitro experiments. RESULTS: TREM-2 was expressed on non-parenchymal hepatic cells and induced during liver injury in mice and man. Mice lacking TREM-2 exhibited heightened liver damage and inflammation during acute and repetitive carbon tetrachloride and acetaminophen (APAP) intoxication, the latter of which TREM-2 deficiency was remarkably associated with worsened survival. Liver damage in Trem-2-/- mice following chronic injury and APAP challenge was associated with elevated hepatic lipid peroxidation and macrophage content. BM transplantation experiments and cellular reactive oxygen species assays revealed effects of TREM-2 in the context of chronic injury depended on both immune and resident TREM-2 expression. Consistent with effects of TREM-2 on inflammation-associated injury, primary hepatic macrophages and hepatic stellate cells lacking TREM-2 exhibited augmented TLR4-driven proinflammatory responses. CONCLUSION: Our data indicate that by acting as a natural brake on inflammation during hepatocellular injury, TREM-2 is a critical regulator of diverse types of hepatotoxic injury.
Asunto(s)
Cirrosis Hepática/metabolismo , Hígado/metabolismo , Glicoproteínas de Membrana/fisiología , Receptores Inmunológicos/fisiología , Acetaminofén , Anciano , Animales , Tetracloruro de Carbono , Estudios de Casos y Controles , Femenino , Células Madre Hematopoyéticas/metabolismo , Hepatocitos/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Macrófagos del Hígado/metabolismo , Peroxidación de Lípido/fisiología , Cirrosis Hepática/etiología , Cirrosis Hepática/inmunología , Cirrosis Hepática Experimental/inmunología , Cirrosis Hepática Experimental/metabolismo , Masculino , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones Noqueados , Persona de Mediana Edad , Especies Reactivas de Oxígeno/metabolismo , Receptores Inmunológicos/deficiencia , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Receptor Toll-Like 4/fisiología , Regulación hacia Arriba/fisiologíaRESUMEN
Phagocytosis and inflammation within the lungs is crucial for host defense during bacterial pneumonia. Triggering receptor expressed on myeloid cells (TREM)-2 was proposed to negatively regulate TLR-mediated responses and enhance phagocytosis by macrophages, but the role of TREM-2 in respiratory tract infections is unknown. Here, we established the presence of TREM-2 on alveolar macrophages (AM) and explored the function of TREM-2 in the innate immune response to pneumococcal infection in vivo. Unexpectedly, we found Trem-2(-/-) AM to display augmented bacterial phagocytosis in vitro and in vivo compared to WT AM. Mechanistically, we detected that in the absence of TREM-2, pulmonary macrophages selectively produced elevated complement component 1q (C1q) levels. We found that these increased C1q levels depended on peroxisome proliferator-activated receptor-δ (PPAR-δ) activity and were responsible for the enhanced phagocytosis of bacteria. Upon infection with S. pneumoniae, Trem-2(-/-) mice exhibited an augmented bacterial clearance from lungs, decreased bacteremia and improved survival compared to their WT counterparts. This work is the first to disclose a role for TREM-2 in clinically relevant respiratory tract infections and demonstrates a previously unknown link between TREM-2 and opsonin production within the lungs.
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Complemento C1q/metabolismo , Modelos Animales de Enfermedad , Pulmón/inmunología , Macrófagos Alveolares/inmunología , Glicoproteínas de Membrana/metabolismo , Neumonía Neumocócica/inmunología , Receptores Inmunológicos/metabolismo , Mucosa Respiratoria/inmunología , Animales , Apoptosis , Línea Celular Transformada , Células Cultivadas , Complemento C1q/genética , Citocinas/metabolismo , Femenino , Pulmón/citología , Pulmón/metabolismo , Pulmón/patología , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patología , Masculino , Glicoproteínas de Membrana/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Infiltración Neutrófila , PPAR gamma/metabolismo , Fagocitosis , Neumonía Neumocócica/metabolismo , Neumonía Neumocócica/patología , Receptores Inmunológicos/genética , Mucosa Respiratoria/citología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Análisis de SupervivenciaRESUMEN
During infections, TLR-mediated responses require tight regulation to allow for pathogen removal, while preventing overwhelming inflammation and immunopathology. The triggering receptor expressed on myeloid cells (TREM)-2 negatively regulates inflammation by macrophages and impacts on phagocytosis, but the function of endogenous TREM-2 during infections is poorly understood. We investigated TREM-2's role in regulating TLR4-mediated inflammation by studying wild-type and TREM-2(-/-) mice challenged with LPS and found TREM-2 to dampen early inflammation. Augmented early inflammation in TREM-2(-/-) animals was followed by an accelerated resolution and ultimately improved survival, associated with the induction of the negative regulator A20. Upon infection with Escherichia coli, the otherwise beneficial effect of an exaggerated early immune response in TREM-2(-/-) animals was counteracted by a 50% reduction in bacterial phagocytosis. In line with this, TREM-2(-/-) peritoneal macrophages (PMs) exhibited augmented inflammation following TLR4 stimulation, demonstrating the presence and negative regulatory functionality of TREM-2 on primary PMs. Significantly, we identified a high turnover rate because TREM-2 RNA is 25-fold down-regulated and the protein proteasomally degraded upon LPS encounter, thus ensuring a tightly regulated and versatile system that modulates inflammation. Our results illustrate TREM-2's effects on infection-triggered inflammation and identify TREM-2 as a potential target to prevent overwhelming inflammation while preserving antibacterial-effector functions.
Asunto(s)
Infecciones por Bacterias Gramnegativas/inmunología , Glicoproteínas de Membrana/metabolismo , Receptores Inmunológicos/metabolismo , Sepsis/inmunología , Animales , Carga Bacteriana , Regulación hacia Abajo , Endotoxemia/etiología , Endotoxemia/inmunología , Infecciones por Escherichia coli/etiología , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Femenino , Infecciones por Bacterias Gramnegativas/etiología , Infecciones por Bacterias Gramnegativas/microbiología , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/toxicidad , Macrófagos Peritoneales/inmunología , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Peritonitis/etiología , Peritonitis/inmunología , Peritonitis/microbiología , Fagocitosis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Inmunológicos/deficiencia , Receptores Inmunológicos/genética , Sepsis/etiología , Receptor Toll-Like 4/metabolismoRESUMEN
Sepsis is a life-threatening condition characterized by uncontrolled systemic inflammation and coagulation, leading to multiorgan failure. Therapeutic options to prevent sepsis-associated immunopathology remain scarce. Here, we established a mouse model of long-lasting disease tolerance during severe sepsis, manifested by diminished immunothrombosis and organ damage in spite of a high pathogen burden. We found that both neutrophils and B cells emerged as key regulators of tissue integrity. Enduring changes in the transcriptional profile of neutrophils include upregulated Cxcr4 expression in protected, tolerant hosts. Neutrophil Cxcr4 upregulation required the presence of B cells, suggesting that B cells promoted disease tolerance by improving tissue damage control via the suppression of neutrophils' tissue-damaging properties. Finally, therapeutic administration of a Cxcr4 agonist successfully promoted tissue damage control and prevented liver damage during sepsis. Our findings highlight the importance of a critical B-cell/neutrophil interaction during sepsis and establish neutrophil Cxcr4 activation as a potential means to promote disease tolerance during sepsis.
Asunto(s)
Infecciones Bacterianas , Sepsis , Animales , Infecciones Bacterianas/metabolismo , Modelos Animales de Enfermedad , Ratones , Insuficiencia Multiorgánica/metabolismo , Insuficiencia Multiorgánica/patología , Neutrófilos/metabolismo , Sepsis/metabolismoRESUMEN
As ACE2 is the critical SARS-CoV-2 receptor, we hypothesized that aerosol administration of clinical grade soluble human recombinant ACE2 (APN01) will neutralize SARS-CoV-2 in the airways, limit spread of infection in the lung, and mitigate lung damage caused by deregulated signaling in the renin-angiotensin (RAS) and Kinin pathways. Here, after demonstrating in vitro neutralization of SARS-CoV-2 by APN01, and after obtaining preliminary evidence of its tolerability and preventive efficacy in a mouse model, we pursued development of an aerosol formulation. As a prerequisite to a clinical trial, we evaluated both virus binding activity and enzymatic activity for cleavage of Ang II following aerosolization. We report successful aerosolization for APN01, retaining viral binding as well as catalytic RAS activity. Dose range-finding and IND-enabling repeat-dose aerosol toxicology testing were conducted in dogs. Twice daily aerosol administration for two weeks at the maximum feasible concentration revealed no notable toxicities. Based on these results, a Phase I clinical trial in healthy volunteers has now been initiated (NCT05065645), with subsequent Phase II testing planned for individuals with SARS-CoV-2 infection.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , Aerosoles , Enzima Convertidora de Angiotensina 2 , Angiotensinas , Animales , Ensayos Clínicos Fase I como Asunto , Perros , Humanos , Ratones , Nebulizadores y Vaporizadores , Peptidil-Dipeptidasa A/metabolismo , Renina/metabolismo , Sistema Renina-Angiotensina , SARS-CoV-2RESUMEN
Despite tremendous progress in the understanding of COVID-19, mechanistic insight into immunological, disease-driving factors remains limited. We generated maVie16, a mouse-adapted SARS-CoV-2, by serial passaging of a human isolate. In silico modeling revealed how only three Spike mutations of maVie16 enhanced interaction with murine ACE2. maVie16 induced profound pathology in BALB/c and C57BL/6 mice, and the resulting mouse COVID-19 (mCOVID-19) replicated critical aspects of human disease, including early lymphopenia, pulmonary immune cell infiltration, pneumonia, and specific adaptive immunity. Inhibition of the proinflammatory cytokines IFNγ and TNF substantially reduced immunopathology. Importantly, genetic ACE2-deficiency completely prevented mCOVID-19 development. Finally, inhalation therapy with recombinant ACE2 fully protected mice from mCOVID-19, revealing a novel and efficient treatment. Thus, we here present maVie16 as a new tool to model COVID-19 for the discovery of new therapies and show that disease severity is determined by cytokine-driven immunopathology and critically dependent on ACE2 in vivo.
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Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/virología , Interferón gamma/farmacología , SARS-CoV-2/patogenicidad , Inmunidad Adaptativa/inmunología , Animales , Modelos Animales de Enfermedad , Interferón gamma/metabolismo , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Peptidil-Dipeptidasa A/genética , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
The non-canonical inflammasome is an emerging crucial player in the development of inflammatory and neurodegenerative diseases. It is activated by direct sensing of cytosolic lipopolysaccharide (LPS) by caspase-11 (CASP11), which then induces pyroptosis, an inflammatory form of regulated cell death. Here, we report that tyrosine kinase 2 (TYK2), a cytokine receptor-associated kinase, is a critical upstream regulator of CASP11. Absence of TYK2 or its kinase activity impairs the transcriptional induction of CASP11 in vitro and in vivo and protects mice from LPS-induced lethality. Lack of TYK2 or its enzymatic activity inhibits macrophage pyroptosis and impairs release of mature IL-1ß and IL-18 specifically in response to intracellular LPS. Deletion of TYK2 in myeloid cells reduces LPS-induced IL-1ß and IL-18 production in vivo, highlighting the importance of these cells in the inflammatory response to LPS. In support of our data generated with genetically engineered mice, pharmacological inhibition of TYK2 reduced LPS-induced upregulation of CASP11 in bone marrow-derived macrophages (BMDMs) and of its homolog CASP5 in human macrophages. Our study provides insights into the regulation of CASP11 in vivo and uncovered a novel link between TYK2 activity and CASP11-dependent inflammation.
Asunto(s)
Caspasas Iniciadoras/metabolismo , Inflamasomas/efectos de los fármacos , Macrófagos/efectos de los fármacos , Piroptosis/efectos de los fármacos , TYK2 Quinasa/farmacología , Animales , Endotoxemia/tratamiento farmacológico , Femenino , Humanos , Inflamasomas/inmunología , Inflamasomas/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Lipopolisacáridos/toxicidad , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Células Mieloides/efectos de los fármacos , Células Mieloides/inmunología , Células Mieloides/metabolismo , Células U937RESUMEN
To develop a universal strategy to block SARS-CoV-2 cellular entry and infection represents a central aim for effective COVID-19 therapy. The growing impact of emerging variants of concern increases the urgency for development of effective interventions. Since ACE2 is the critical SARS-CoV-2 receptor and all tested variants bind to ACE2, some even at much increased affinity (see accompanying paper), we hypothesized that aerosol administration of clinical grade soluble human recombinant ACE2 (APN01) will neutralize SARS-CoV-2 in the airways, limit spread of infection in the lung and mitigate lung damage caused by deregulated signaling in the renin-angiotensin (RAS) and Kinin pathways. Here we show that intranasal administration of APN01 in a mouse model of SARS-CoV-2 infection dramatically reduced weight loss and prevented animal death. As a prerequisite to a clinical trial, we evaluated both virus binding activity and enzymatic activity for cleavage of Ang II following aerosolization. We report successful aerosolization for APN01, retaining viral binding as well as catalytic RAS activity. Dose range-finding and IND-enabling repeat-dose aerosol toxicology testing were conducted in dogs. Twice daily aerosol administration for two weeks at the maximum feasible concentration revealed no notable toxicities. Based on these results, a Phase I clinical trial in healthy volunteers can now be initiated, with subsequent Phase II testing in individuals with SARS-CoV-2 infection. This strategy could be used to develop a viable and rapidly actionable therapy to prevent and treat COVID-19, against all current and future SARS-CoV-2 variants.
RESUMEN
Cytomegalovirus (CMV) has a high prevalence worldwide, is often fatal for immunocompromised patients, and causes bone marrow suppression. Deficiency of signal transducer and activator of transcription 1 (STAT1) results in severely impaired antiviral immunity. We have used cell-type restricted deletion of Stat1 to determine the importance of myeloid cell activity for the defense against murine CMV (MCMV). We show that myeloid STAT1 limits MCMV burden and infection-associated pathology in the spleen but does not affect ultimate clearance of infection. Unexpectedly, we found an essential role of myeloid STAT1 in the induction of extramedullary hematopoiesis (EMH). The EMH-promoting function of STAT1 was not restricted to MCMV infection but was also observed during CpG oligodeoxynucleotide-induced sterile inflammation. Collectively, we provide genetic evidence that signaling through STAT1 in myeloid cells is required to restrict MCMV at early time points post-infection and to induce compensatory hematopoiesis in the spleen.
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Hematopoyesis Extramedular , Infecciones por Herpesviridae/fisiopatología , Muromegalovirus , Células Mieloides/fisiología , Factor de Transcripción STAT1/fisiología , Animales , Células Cultivadas , Femenino , Eliminación de Gen , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/metabolismo , Células Asesinas Naturales/inmunología , Masculino , Ratones Endogámicos C57BL , Muromegalovirus/fisiología , Receptor de Interferón alfa y beta/genética , Receptores de Interferón/genética , Receptores de Interleucina/genética , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Bazo/patología , Bazo/virología , Estrés Fisiológico , Replicación ViralRESUMEN
Lipid metabolism is increasingly being appreciated to affect immunoregulation, inflammation and pathology. In this study we found that mice infected with lymphocytic choriomeningitis virus (LCMV) exhibit global perturbations of circulating serum lipids. Mice lacking the lipid-sensing surface receptor triggering receptor expressed on myeloid cells 2 (Trem2 -/-) were protected from LCMV-induced hepatitis and showed improved virus control despite comparable virus-specific T cell responses. Non-hematopoietic expression of TREM2 was found to be responsible for aggravated hepatitis, indicating a novel role for TREM2 in the non-myeloid compartment. These results suggest a link between virus-perturbed lipids and TREM2 that modulates liver pathogenesis upon viral infection. Targeted interventions of this immunoregulatory axis may ameliorate tissue pathology in hepatitis.
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Hepatitis/metabolismo , Hepatitis/virología , Metabolismo de los Lípidos , Virus de la Coriomeningitis Linfocítica/fisiología , Glicoproteínas de Membrana/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Citocinas , Modelos Animales de Enfermedad , Hepatitis/patología , Glicoproteínas de Membrana/genética , Metaboloma , Metabolómica/métodos , Ratones , Ratones Noqueados , Agregado de Proteínas , Receptores Inmunológicos/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Carga ViralAsunto(s)
Betacoronavirus , Infecciones por Coronavirus/cirugía , Trasplante de Pulmón/métodos , Neumonía Viral/cirugía , Síndrome de Dificultad Respiratoria/cirugía , Síndrome de Dificultad Respiratoria/virología , Adulto , COVID-19 , Prueba de COVID-19 , Técnicas de Laboratorio Clínico , Infecciones por Coronavirus/complicaciones , Infecciones por Coronavirus/diagnóstico , Femenino , Humanos , Pandemias , Selección de Paciente , Neumonía Viral/complicaciones , Neumonía Viral/diagnóstico , Reacción en Cadena de la Polimerasa , Síndrome de Dificultad Respiratoria/diagnóstico , SARS-CoV-2RESUMEN
Lipid metabolism and receptor-mediated signaling are highly intertwined processes that cooperate to fulfill cellular functions and safeguard cellular homeostasis. Activation of Toll-like receptors (TLRs) leads to a complex cellular response, orchestrating a diverse range of inflammatory events that need to be tightly controlled. Here, we identified the GPI-anchored Sphingomyelin Phosphodiesterase, Acid-Like 3B (SMPDL3B) in a mass spectrometry screening campaign for membrane proteins co-purifying with TLRs. Deficiency of Smpdl3b in macrophages enhanced responsiveness to TLR stimulation and profoundly changed the cellular lipid composition and membrane fluidity. Increased cellular responses could be reverted by re-introducing affected ceramides, functionally linking membrane lipid composition and innate immune signaling. Finally, Smpdl3b-deficient mice displayed an intensified inflammatory response in TLR-dependent peritonitis models, establishing its negative regulatory role in vivo. Taken together, our results identify the membrane-modulating enzyme SMPDL3B as a negative regulator of TLR signaling that functions at the interface of membrane biology and innate immunity.
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Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Inmunidad Innata/genética , Inflamación/genética , Peritonitis/genética , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/inmunología , Modelos Animales de Enfermedad , Humanos , Inflamación/inmunología , Inflamación/patología , Lípidos/inmunología , Macrófagos/inmunología , Ratones , Peritonitis/inmunología , Peritonitis/patología , Receptores Toll-Like/genética , Receptores Toll-Like/inmunologíaRESUMEN
Clearance of invading pathogens is essential to preventing overwhelming inflammation and sepsis that are symptomatic of bacterial peritonitis. Macrophages participate in this innate immune response by engulfing and digesting pathogens, a process called phagocytosis. Oxidized phospholipids (OxPL) are danger-associated molecular patterns (DAMPs) generated in response to infection that can prevent the phagocytic clearance of bacteria. We investigated the mechanism underlying OxPL action in macrophages. Exposure to OxPL induced alterations in actin polymerization, resulting in spreading of peritoneal macrophages and diminished uptake of E. coli. Pharmacological and cell-based studies showed that an anchored pool of PKA mediates the effects of OxPL. Gene silencing approaches identified the A-kinase anchoring protein (AKAP) WAVE1 as an effector of OxPL action in vitro. Chimeric Wave1(-/-) mice survived significantly longer after infection with E. coli and OxPL treatment in vivo. Moreover, we found that endogenously generated OxPL in human peritoneal dialysis fluid from end-stage renal failure patients inhibited phagocytosis via WAVE1. Collectively, these data uncover an unanticipated role for WAVE1 as a critical modulator of the innate immune response to severe bacterial infections.
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Infecciones por Escherichia coli/inmunología , Macrófagos Peritoneales/inmunología , Peritonitis/inmunología , Fagocitosis , Fosfolípidos/fisiología , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismo , Animales , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dimiristoilfosfatidilcolina/farmacología , Activación Enzimática , Escherichia coli/inmunología , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Humanos , Inmunidad Innata , Fallo Renal Crónico/inmunología , Fallo Renal Crónico/metabolismo , Fallo Renal Crónico/terapia , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidación-Reducción , Diálisis Peritoneal , Peritonitis/metabolismo , Peritonitis/microbiología , Fosfatidilcolinas/farmacología , Fosfatidilcolinas/fisiología , Familia de Proteínas del Síndrome de Wiskott-Aldrich/genéticaRESUMEN
Macrophages play a key role in responding to pathogens and initiate an inflammatory response to combat microbe multiplication. Deactivation of macrophages facilitates resolution of the inflammatory response. Deactivated macrophages are characterized by an immunosuppressive phenotype, but the lack of unique markers that can reliably identify these cells explains the poorly defined biological role of this macrophage subset. We identified lipocalin 2 (LCN2) as both a marker of deactivated macrophages and a macrophage deactivator. We show that LCN2 attenuated the early inflammatory response and impaired bacterial clearance, leading to impaired survival of mice suffering from pneumococcal pneumonia. LCN2 induced IL-10 formation by macrophages, skewing macrophage polarization in a STAT3-dependent manner. Pulmonary LCN2 levels were tremendously elevated during bacterial pneumonia in humans, and high LCN2 levels were indicative of a detrimental outcome from pneumonia with Gram-positive bacteria. Our data emphasize the importance of macrophage deactivation for the outcome of pneumococcal infections and highlight the role of LCN2 and IL-10 as determinants of macrophage performance in the respiratory tract.