RESUMEN
Plasma membrane repair is a fundamental homeostatic process of eukaryotic cells. Here, we report a new function for the conserved cytoskeletal proteins known as septins in the repair of cells perforated by pore-forming toxins or mechanical disruption. Using a silencing RNA screen, we identified known repair factors (e.g. annexin A2, ANXA2) and novel factors such as septin 7 (SEPT7) that is essential for septin assembly. Upon plasma membrane injury, the septin cytoskeleton is extensively redistributed to form submembranous domains arranged as knob and loop structures containing F-actin, myosin IIA, S100A11, and ANXA2. Formation of these domains is Ca2+-dependent and correlates with plasma membrane repair efficiency. Super-resolution microscopy revealed that septins and F-actin form intertwined filaments associated with ANXA2. Depletion of SEPT7 prevented ANXA2 recruitment and formation of submembranous actomyosin domains. However, ANXA2 depletion had no effect on domain formation. Collectively, our data support a novel septin-based mechanism for resealing damaged cells, in which the septin cytoskeleton plays a key structural role in remodeling the plasma membrane by promoting the formation of SEPT/F-actin/myosin IIA/ANXA2/S100A11 repair domains.
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Neuroinflammation and accumulation of Amyloid Beta (Aß) accompanied by deterioration of special memory are hallmarks of Alzheimer's disease (AD). Effective preventative and treatment options for AD are still needed. Microglia in AD brains are characterized by elevated levels of microRNA-17 (miR-17), which is accompanied by defective autophagy, Aß accumulation, and increased inflammatory cytokine production. However, the effect of targeting miR-17 on AD pathology and memory loss is not clear. To specifically inhibit miR-17 in microglia, we generated mannose-coated lipid nanoparticles (MLNPs) enclosing miR-17 antagomir (Anti-17 MLNPs), which are targeted to mannose receptors readily expressed on microglia. We used a 5XFAD mouse model (AD) that recapitulates many AD-related phenotypes observed in humans. Our results show that Anti-17 MLNPs, delivered to 5XFAD mice by intra-cisterna magna injection, specifically deliver Anti-17 to microglia. Anti-17 MLNPs downregulated miR-17 expression in microglia but not in neurons, astrocytes, and oligodendrocytes. Anti-17 MLNPs attenuated inflammation, improved autophagy, and reduced Aß burdens in the brains. Additionally, Anti-17 MLNPs reduced the deterioration in spatial memory and decreased anxiety-like behavior in 5XFAD mice. Therefore, targeting miR-17 using MLNPs is a viable strategy to prevent several AD pathologies. This selective targeting strategy delivers specific agents to microglia without the adverse off-target effects on other cell types. Additionally, this approach can be used to deliver other molecules to microglia and other immune cells in other organs.
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Enfermedad de Alzheimer , Encéfalo , Modelos Animales de Enfermedad , Manosa , Ratones Transgénicos , MicroARNs , Microglía , Nanopartículas , Animales , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , MicroARNs/metabolismo , Nanopartículas/administración & dosificación , Ratones , Microglía/metabolismo , Microglía/efectos de los fármacos , Manosa/farmacología , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Péptidos beta-Amiloides/metabolismo , Lípidos , Masculino , Antagomirs/farmacología , Antagomirs/administración & dosificaciónRESUMEN
Influenza virus activates cellular inflammasome pathways, which can be both beneficial and detrimental to infection outcomes. Here, we investigate the function of the inflammasome-activated, pore-forming protein gasdermin D (GSDMD) during infection. Ablation of GSDMD in knockout (KO) mice (Gsdmd-/-) significantly attenuates influenza virus-induced weight loss, lung dysfunction, lung histopathology, and mortality compared with wild type (WT) mice, despite similar viral loads. Infected Gsdmd-/- mice exhibit decreased inflammatory gene signatures shown by lung transcriptomics. Among these, diminished neutrophil gene activation signatures are corroborated by decreased detection of neutrophil elastase and myeloperoxidase in KO mouse lungs. Indeed, directly infected neutrophils are observed in vivo and infection of neutrophils in vitro induces release of DNA and tissue-damaging enzymes that is largely dependent on GSDMD. Neutrophil depletion in infected WT mice recapitulates the reductions in mortality, lung inflammation, and lung dysfunction observed in Gsdmd-/- animals, while depletion does not have additive protective effects in Gsdmd-/- mice. These findings implicate a function for GSDMD in promoting lung neutrophil responses that amplify influenza virus-induced inflammation and pathogenesis. Targeting the GSDMD/neutrophil axis may provide a therapeutic avenue for treating severe influenza.
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Neutrófilos , Orthomyxoviridae , Animales , Ratones , Neutrófilos/metabolismo , Gasderminas , Inflamasomas/genética , Inflamasomas/metabolismo , Inflamación/genética , Inflamación/metabolismo , Orthomyxoviridae/metabolismo , Proteínas de Unión a Fosfato/genética , Proteínas de Unión a Fosfato/metabolismoRESUMEN
Alzheimer's disease (AD) is the sixth leading cause of death in the USA. It is established that neuroinflammation contributes to the synaptic loss, neuronal death, and symptomatic decline of AD patients. Accumulating evidence suggests a critical role for microglia, innate immune phagocytes of the brain. For instance, microglia release pro-inflammatory products such as IL-1ß which is highly implicated in AD pathobiology. The mechanisms underlying the transition of microglia to proinflammatory promoters of AD remain largely unknown. To address this gap, we performed reduced representation bisulfite sequencing (RRBS) to profile global DNA methylation changes in human AD brains compared to no disease controls. We identified differential DNA methylation of CASPASE-4 (CASP4), which when expressed promotes the generation of IL-1ß and is predominantly expressed in immune cells. DNA upstream of the CASP4 transcription start site was hypomethylated in human AD brains, which was correlated with increased expression of CASP4. Furthermore, microglia from a mouse model of AD (5xFAD) express increased levels of CASP4 compared to wild-type (WT) mice. To study the role of CASP4 in AD, we developed a novel mouse model of AD lacking the mouse ortholog of CASP4 and CASP11, which is encoded by mouse Caspase-4 (5xFAD/Casp4-/-). The expression of CASP11 was associated with increased accumulation of pathologic protein aggregate amyloid-ß (Aß) and increased microglial production of IL-1ß in 5xFAD mice. Utilizing RNA-sequencing, we determined that CASP11 promotes unique transcriptomic phenotypes in 5xFAD mouse brains, including alterations of neuroinflammatory and chemokine signaling pathways. Notably, in vitro, CASP11 promoted generation of IL-1ß from macrophages in response to cytosolic Aß through cleavage of downstream effector Gasdermin D (GSDMD). Therefore, here we unravel the role for CASP11 and GSDMD in the generation of IL-1ß in response to Aß and the progression of pathologic inflammation in AD. Overall, our results demonstrate that overexpression of CASP4 due to differential DNA methylation in AD microglia contributes to the progression of AD pathobiology. Thus, we identify CASP4 as a potential target for immunotherapies for the treatment and prevention of AD.
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Enfermedad de Alzheimer , Péptidos beta-Amiloides , Caspasas Iniciadoras , Animales , Humanos , Ratones , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Modelos Animales de Enfermedad , Metilación de ADN , Inflamación/patología , Ratones Transgénicos , Microglía/metabolismo , Caspasas Iniciadoras/metabolismoRESUMEN
Mammalian cells are frequently exposed to mechanical and biochemical stressors resulting in plasma membrane injuries. Repair mechanisms reseal the plasma membrane to restore homeostasis and prevent cell death. In the present work, a silencing RNA screen was performed to uncover plasma membrane repair mechanisms of cells exposed to a pore-forming toxin (listeriolysin O). This screen identified molecules previously known to repair the injured plasma membrane such as annexin A2 (ANXA2) as well as novel plasma membrane repair candidate proteins. Of the novel candidates, we focused on septin 7 (SEPT7) because the septins are an important family of conserved eukaryotic cytoskeletal proteins. Using diverse experimental approaches, we established for the first time that SEPT7 plays a general role in plasma membrane repair of cells perforated by pore-forming toxins and mechanical wounding. Remarkably, upon cell injury, the septin cytoskeleton is extensively redistributed in a Ca 2+ -dependent fashion, a hallmark of plasma membrane repair machineries. The septins reorganize into subplasmalemmal domains arranged as knob and loop (or ring) structures containing F-actin, myosin II, and annexin A2 (ANXA2) and protrude from the cell surface. Importantly, the formation of these domains correlates with the plasma membrane repair efficiency. Super-resolution microscopy shows that septins and actin are arranged in intertwined filaments associated with ANXA2. Silencing SEPT7 expression prevented the formation of the F-actin/myosin II/ANXA2 domains, however, silencing expression of ANXA2 had no observable effect on their formation. These results highlight the key structural role of the septins in remodeling the plasma membrane and in the recruitment of the repair molecule ANXA2. Collectively, our data support a novel model in which the septin cytoskeleton acts as a scaffold to promote the formation of plasma membrane repair domains containing contractile F-actin and annexin A2.
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As SARS-CoV-2 variants of concern (VoCs) that evade immunity continue to emerge, next-generation adaptable COVID-19 vaccines which protect the respiratory tract and provide broader, more effective, and durable protection are urgently needed. Here, we have developed one such approach, a highly efficacious, intranasally delivered, trivalent measles-mumps-SARS-CoV-2 spike (S) protein (MMS) vaccine candidate that induces robust systemic and mucosal immunity with broad protection. This vaccine candidate is based on three components of the MMR vaccine, a measles virus Edmonston and the two mumps virus strains [Jeryl Lynn 1 (JL1) and JL2] that are known to provide safe, effective, and long-lasting protective immunity. The six proline-stabilized prefusion S protein (preS-6P) genes for ancestral SARS-CoV-2 WA1 and two important SARS-CoV-2 VoCs (Delta and Omicron BA.1) were each inserted into one of these three viruses which were then combined into a trivalent "MMS" candidate vaccine. Intranasal immunization of MMS in IFNAR1-/- mice induced a strong SARS-CoV-2-specific serum IgG response, cross-variant neutralizing antibodies, mucosal IgA, and systemic and tissue-resident T cells. Immunization of golden Syrian hamsters with MMS vaccine induced similarly high levels of antibodies that efficiently neutralized SARS-CoV-2 VoCs and provided broad and complete protection against challenge with any of these VoCs. This MMS vaccine is an efficacious, broadly protective next-generation COVID-19 vaccine candidate, which is readily adaptable to new variants, built on a platform with a 50-y safety record that also protects against measles and mumps.
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COVID-19 , Sarampión , Paperas , Cricetinae , Animales , Humanos , Ratones , SARS-CoV-2/genética , Vacunas contra la COVID-19 , COVID-19/prevención & control , Vacuna contra el Sarampión-Parotiditis-Rubéola , Anticuerpos Antivirales , Anticuerpos ampliamente neutralizantes , Inmunoglobulina G , Mesocricetus , Anticuerpos Neutralizantes , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
Alzheimer's Disease (AD) is the 6th leading cause of death in the US. It is established that neuroinflammation contributes to the synaptic loss, neuronal death, and symptomatic decline of AD patients. Accumulating evidence suggests a critical role for microglia, innate immune phagocytes of the brain. For instance, microglia release proinflammatory products such as IL-1ß which is highly implicated in AD pathobiology. The mechanisms underlying the transition of microglia to proinflammatory promoters of AD remain largely unknown. To address this gap, we performed Reduced Representation Bisulfite Sequencing (RRBS) to profile global DNA methylation changes in human AD brains compared to no disease controls. We identified differential DNA methylation of CASPASE-4 (CASP4), which when expressed, can be involved in generation of IL-1ß and is predominantly expressed in immune cells. DNA upstream of the CASP4 transcription start site was hypomethylated in human AD brains, which was correlated with increased expression of CASP4. Furthermore, microglia from a mouse model of AD (5xFAD) express increased levels of CASP4 compared to wild-type (WT) mice. To study the role of CASP4 in AD, we developed a novel mouse model of AD lacking the mouse ortholog of CASP4, CASP11, which is encoded by mouse Caspase-4 (5xFAD/Casp4-/-). The expression of CASP11 was associated with increased accumulation of pathologic protein aggregate amyloid-ß (Aß) and increased microglial production of IL-1ß in 5xFAD mice. Utilizing RNA sequencing, we determined that CASP11 promotes unique transcriptomic phenotypes in 5xFAD mouse brains, including alterations of neuroinflammatory and chemokine signaling pathways. Notably, in vitro, CASP11 promoted generation of IL-1ß from macrophages in response to cytosolic Aß through cleavage of downstream effector Gasdermin D (G SDMD). We describe a role for CASP11 and GSDMD in the generation of IL-1ß in response to Aß and the progression of pathologic inflammation in AD. Overall, our results demonstrate that overexpression of CASP4 due to differential methylation in AD microglia contributes to the progression of AD pathobiology, thus identifying CASP4 as a potential target for immunotherapies for the treatment of AD.
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Previous urinary tract infections (UTIs) can predispose one to future infections; however, the underlying mechanisms affecting recurrence are poorly understood. We previously found that UTIs in mice cause differential bladder epithelial (urothelial) remodelling, depending on disease outcome, that impacts susceptibility to recurrent UTI. Here we compared urothelial stem cell (USC) lines isolated from mice with a history of either resolved or chronic uropathogenic Escherichia coli (UPEC) infection, elucidating evidence of molecular imprinting that involved epigenetic changes, including differences in chromatin accessibility, DNA methylation and histone modification. Epigenetic marks in USCs from chronically infected mice enhanced caspase-1-mediated cell death upon UPEC infection, promoting bacterial clearance. Increased Ptgs2os2 expression also occurred, potentially contributing to sustained cyclooxygenase-2 expression, bladder inflammation and mucosal wounding-responses associated with severe recurrent cystitis. Thus, UPEC infection acts as an epi-mutagen reprogramming the urothelial epigenome, leading to urothelial-intrinsic remodelling and training of the innate response to subsequent infection.
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Infecciones por Escherichia coli , Infecciones Urinarias , Escherichia coli Uropatógena , Ratones , Animales , Escherichia coli Uropatógena/genética , Inmunidad Entrenada , Infecciones Urinarias/microbiología , Vejiga Urinaria/microbiología , Infecciones por Escherichia coli/microbiologíaRESUMEN
Influenza virus activates cellular inflammasome pathways, which can be either beneficial or detrimental to infection outcomes. Here, we investigated the role of the inflammasome-activated pore-forming protein gasdermin D (GSDMD) during infection. Ablation of GSDMD in knockout (KO) mice significantly attenuated virus-induced weight loss, lung dysfunction, lung histopathology, and mortality compared with wild type (WT) mice, despite similar viral loads. Infected GSDMD KO mice exhibited decreased inflammatory gene signatures revealed by lung transcriptomics, which also implicated a diminished neutrophil response. Importantly, neutrophil depletion in infected WT mice recapitulated the reduced mortality and lung inflammation observed in GSDMD KO animals, while having no additional protective effects in GSDMD KOs. These findings reveal a new function for GSDMD in promoting lung neutrophil responses that amplify influenza virus-induced inflammation and pathogenesis. Targeting the GSDMD/neutrophil axis may provide a new therapeutic avenue for treating severe influenza.
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BACKGROUND: Abnormal macrophage function caused by dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) is a critical contributor to chronic airway infections and inflammation in people with cystic fibrosis (PWCF). Elexacaftor/tezacaftor/ivacaftor (ETI) is a new CFTR modulator therapy for PWCF. Host-pathogen and clinical responses to CFTR modulators are poorly described. We sought to determine how ETI impacts macrophage CFTR function, resulting effector functions and relationships to clinical outcome changes. METHODS: Clinical information and/or biospecimens were obtained at ETI initiation and 3, 6, 9 and 12â months post-ETI in 56 PWCF and compared with non-CF controls. Peripheral blood monocyte-derived macrophages (MDMs) were isolated and functional assays performed. RESULTS: ETI treatment was associated with increased CF MDM CFTR expression, function and localisation to the plasma membrane. CF MDM phagocytosis, intracellular killing of CF pathogens and efferocytosis of apoptotic neutrophils were partially restored by ETI, but inflammatory cytokine production remained unchanged. Clinical outcomes including increased forced expiratory volume in 1â s (+10%) and body mass index (+1.0â kg·m-2) showed fluctuations over time and were highly individualised. Significant correlations between post-ETI MDM CFTR function and sweat chloride levels were observed. However, MDM CFTR function correlated with clinical outcomes better than sweat chloride. CONCLUSION: ETI is associated with unique changes in innate immune function and clinical outcomes.
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Fibrosis Quística , Humanos , Fibrosis Quística/tratamiento farmacológico , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Cloruros/metabolismo , Agonistas de los Canales de Cloruro/uso terapéutico , Mutación , Macrófagos/metabolismoRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) is a worldwide health concern, and new treatment strategies are needed. Targeting inflammatory innate immunity pathways holds therapeutic promise, but effective molecular targets remain elusive. Here, we show that human caspase-4 (CASP4) and its mouse homolog, caspase-11 (CASP11), are up-regulated in SARSCoV-2 infections and that CASP4 expression correlates with severity of SARSCoV-2 infection in humans. SARSCoV-2infected Casp11−/− mice were protected from severe weight loss and lung pathology, including blood vessel damage, compared to wild-type (WT) mice and mice lacking the caspase downstream effector gasdermin-D (Gsdmd−/−). Notably, viral titers were similar regardless of CASP11 knockout. Global transcriptomics of SARSCoV-2infected WT, Casp11−/−, and Gsdmd−/− lungs identified restrained expression of inflammatory molecules and altered neutrophil gene signatures in Casp11−/− mice. We confirmed that protein levels of inflammatory mediators interleukin (IL)-1ß, IL-6, and CXCL1, as well as neutrophil functions, were reduced in Casp11−/− lungs. Additionally, Casp11−/− lungs accumulated less von Willebrand factor, a marker for endothelial damage, but expressed more Kruppel-Like Factor 2, a transcription factor that maintains vascular integrity. Overall, our results demonstrate that CASP4/11 promotes detrimental SARSCoV-2induced inflammation and coagulopathy, largely independently of GSDMD, identifying CASP4/11 as a promising drug target for treatment and prevention of severe COVID-19.
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COVID-19 , Caspasas Iniciadoras/metabolismo , SARS-CoV-2 , Tromboinflamación , Animales , COVID-19/enzimología , COVID-19/patología , Caspasas Iniciadoras/genética , Progresión de la Enfermedad , Humanos , Pulmón/patología , Ratones , Ratones Noqueados , Índice de Severidad de la Enfermedad , Tromboinflamación/enzimología , Tromboinflamación/genéticaRESUMEN
Cystic fibrosis (CF) human and mouse macrophages are defective in their ability to clear bacteria such as Burkholderia cenocepacia. The autophagy process in CF (F508del) macrophages is halted, and the underlying mechanism remains unclear. Furthermore, the role of CFTR in maintaining the acidification of endosomal and lysosomal compartments in CF cells has been a subject of debate. Using 3D reconstruction of z-stack confocal images, we show that CFTR is recruited to LC3-labeled autophagosomes harboring B. cenocepacia. Using several complementary approaches, we report that CF macrophages display defective lysosomal acidification and degradative function for cargos destined to autophagosomes, whereas non-autophagosomal cargos are effectively degraded within acidic compartments. Notably, treatment of CF macrophages with CFTR modulators (tezacaftor/ivacaftor) improved the autophagy flux, lysosomal acidification and function, and bacterial clearance. In addition, CFTR modulators improved CFTR function as demonstrated by patch-clamp. In conclusion, CFTR regulates the acidification of a specific subset of lysosomes that specifically fuse with autophagosomes. Therefore, our study describes a new biological location and function for CFTR in autophago-lysosomes and clarifies the long-standing discrepancies in the field.
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Burkholderia cenocepacia , Fibrosis Quística , Animales , Burkholderia cenocepacia/metabolismo , Fibrosis Quística/microbiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Concentración de Iones de Hidrógeno , Lisosomas/metabolismo , Macrófagos/microbiología , RatonesRESUMEN
Asthma is an inflammatory lung disorder characterized by mucus hypersecretion, cellular infiltration, and bronchial hyper-responsiveness. House dust mites (HDM) are the most prevalent cause of allergic sensitization. Canonical and noncanonical inflammasomes are multiprotein complexes that assemble in response to pathogen or danger-associated molecular patterns (PAMPs or DAMPs). Murine caspase-11 engages the noncanonical inflammasome. We addressed the role of caspase-11 in mediating host responses to HDM and subsequent allergic inflammation using caspase-11-/- mice, which lack caspase-11 while express caspase-1. We found that HDM induce caspase-11 expression in vitro. The presence of IL-4 and IL-13 promote caspase-11 expression. Additionally, caspase-11-/- macrophages show reduced release of IL-6, IL-12, and KC, and express lower levels of costimulatory molecules (e.g., CD40, CD86 and MHCII) in response to HDM stimulation. Notably, HDM sensitization of caspase-11-/- mice resulted in similar levels of IgE responses and hypothermia in response to nasal HDM challenge compared to WT. However, analysis of cell numbers and cytokines in bronchiolar alveolar lavage fluid (BALF) and histopathology of representative lung segments demonstrate altered inflammatory responses and reduced neutrophilia in the airways of the caspase-11-/- mice. These findings indicate that caspase-11 regulates airway inflammation in response to HDM exposure.
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Caspasas Iniciadoras/inmunología , Hipersensibilidad/inmunología , Neumonía/inmunología , Pyroglyphidae/inmunología , Animales , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Rapid synovial fluid-induced aggregation of Staphylococcus aureus is currently being investigated as an important factor in the establishment of periprosthetic joint infections (PJIs). Pathogenic advantages of aggregate formation have been well documented in vitro, including recalcitrance to antibiotics and protection from host immune defenses. The objective of the present work was to determine the strain dependency of synovial fluid-induced aggregation by measuring the degree of aggregation of 21 clinical S. aureus isolates cultured from either PJI or bloodstream infections using imaging and flow cytometry. Furthermore, by measuring attached bacterial biomass using a conventional crystal violet assay, we assessed whether there is a correlation between the aggregative phenotype and surface-associated biofilm formation. While all of the isolates were stimulated to aggregate upon exposure to bovine synovial fluid (BSF) and human serum (HS), the extent of aggregation was highly variable between individual strains. Interestingly, the PJI isolates aggregated significantly more upon BSF exposure than those isolated from bloodstream infections. While we were able to stimulate biofilm formation with all of the isolates in growth medium, supplementation with either synovial fluid or human serum inhibited bacterial surface attachment over a 24 h incubation. Surprisingly, there was no correlation between the degree of synovial fluid-induced aggregation and quantity of surface-associated biofilm as measured by a conventional biofilm assay without host fluid supplementation. Taken together, our findings suggest that synovial fluid-induced aggregation appears to be widespread among S. aureus strains and mechanistically independent of biofilm formation. IMPORTANCE Bacterial infections of hip and knee implants are rare but devastating complications of orthopedic surgery. Despite a widespread appreciation of the considerable financial, physical, and emotional burden associated with the development of a prosthetic joint infection, the establishment of bacteria in the synovial joint remains poorly understood. It has been shown that immediately upon exposure to synovial fluid, the viscous fluid in the joint, Staphylococcus aureus rapidly forms aggregates which are resistant to antibiotics and host immune cell clearance. The bacterial virulence associated with aggregate formation is likely a step in the establishment of prosthetic joint infection, and as such, it has the potential to be a potent target of prevention. We hope that this work contributes to the future development of therapeutics targeting synovial fluid-induced aggregation to better prevent and treat these infections.
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Adhesión Bacteriana/fisiología , Biopelículas/crecimiento & desarrollo , Infecciones Relacionadas con Prótesis/microbiología , Staphylococcus aureus/crecimiento & desarrollo , Líquido Sinovial/microbiología , Animales , Bovinos , Prótesis de Cadera/microbiología , Humanos , Prótesis de la Rodilla/microbiología , Suero/microbiología , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/aislamiento & purificación , Membrana Sinovial/microbiologíaRESUMEN
Autophagy is a proposed route of amyloid-ß (Aß) clearance by microglia that is halted in Alzheimer's Disease (AD), though mechanisms underlying this dysfunction remain elusive. Here, primary microglia from adult AD (5xFAD) mice were utilized to demonstrate that 5xFAD microglia fail to degrade Aß and express low levels of autophagy cargo receptor NBR1. In 5xFAD mouse brains, we show for the first time that AD microglia express elevated levels of microRNA cluster Mirc1/Mir17-92a, which is known to downregulate autophagy proteins. By in situ hybridization in post-mortem AD human tissue sections, we observed that the Mirc1/Mir17-92a cluster member miR-17 is also elevated in human AD microglia, specifically in the vicinity of Aß deposits, compared to non-disease controls. We show that NBR1 expression is negatively correlated with expression of miR-17 in human AD microglia via immunohistopathologic staining in human AD brain tissue sections. We demonstrate in healthy microglia that autophagy cargo receptor NBR1 is required for Aß degradation. Inhibiting elevated miR-17 in 5xFAD mouse microglia improves Aß degradation, autophagy, and NBR1 puncta formation in vitro and improves NBR1 expression in vivo. These findings offer a mechanism behind dysfunctional autophagy in AD microglia which may be useful for therapeutic interventions aiming to improve autophagy function in AD.
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Enfermedad de Alzheimer/inmunología , Péptidos beta-Amiloides/inmunología , Autofagia/inmunología , Regulación de la Expresión Génica/inmunología , MicroARNs/inmunología , Microglía/inmunología , Proteolisis , Animales , Femenino , Humanos , Masculino , RatonesRESUMEN
Alum, used as an adjuvant in injected vaccines, promotes T helper 2 (Th2) and serum antibody (Ab) responses. However, it fails to induce secretory immunoglobulin (Ig) A (SIgA) in mucosal tissues and is poor in inducing Th1 and cell-mediated immunity. Alum stimulates interleukin 1 (IL-1) and the recruitment of myeloid cells, including neutrophils. We investigated whether neutrophil elastase regulates the adjuvanticity of alum, and whether a strategy targeting neutrophil elastase could improve responses to injected vaccines. Mice coadministered a pharmacological inhibitor of elastase, or lacking elastase, developed high-affinity serum IgG and IgA antibodies after immunization with alum-adsorbed protein vaccines, including the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). These mice also developed broader antigen-specific CD4+ T cell responses, including high Th1 and T follicular helper (Tfh) responses. Interestingly, in the absence of elastase activity, mucosal SIgA responses were induced after systemic immunization with alum as adjuvant. Importantly, lack or suppression of elastase activity enhanced the magnitude of anti-SARS-CoV-2 spike subunit 1 (S1) antibodies, and these antibodies reacted with the same epitopes of spike 1 protein as sera from COVID-19 patients. Therefore, suppression of neutrophil elastase could represent an attractive strategy for improving the efficacy of alum-based injected vaccines for the induction of broad immunity, including mucosal immunity.
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Adyuvantes Inmunológicos/farmacología , Compuestos de Alumbre/farmacología , COVID-19/inmunología , COVID-19/terapia , Inhibidores Enzimáticos/farmacología , Elastasa de Leucocito/antagonistas & inhibidores , SARS-CoV-2/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Formación de Anticuerpos/efectos de los fármacos , COVID-19/metabolismo , Células HEK293 , Humanos , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/inmunología , Inmunidad Mucosa/efectos de los fármacos , Inmunidad Mucosa/inmunología , Inmunoglobulina A/inmunología , Elastasa de Leucocito/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , SARS-CoV-2/efectos de los fármacos , Glicoproteína de la Espiga del Coronavirus/inmunología , Porcinos , Células TH1/inmunología , Tratamiento Farmacológico de COVID-19RESUMEN
Burkholderia cenocepacia (B. cenocepacia) is an opportunistic bacterium; causing severe life threatening systemic infections in immunocompromised individuals including cystic fibrosis patients. The lack of gasdermin D (GSDMD) protects mice against endotoxin lipopolysaccharide (LPS) shock. On the other hand, GSDMD promotes mice survival in response to certain bacterial infections. However, the role of GSDMD during B. cenocepacia infection is not yet determined. Our in vitro study shows that GSDMD restricts B. cenocepacia replication within macrophages independent of its role in cell death through promoting mitochondrial reactive oxygen species (mROS) production. mROS is known to stimulate autophagy, hence, the inhibition of mROS or the absence of GSDMD during B. cenocepacia infections reduces autophagy which plays a critical role in the restriction of the pathogen. GSDMD promotes inflammation in response to B. cenocepacia through mediating the release of inflammasome dependent cytokine (IL-1ß) and an independent one (CXCL1) (KC). Additionally, different B. cenocepacia secretory systems (T3SS, T4SS, and T6SS) contribute to inflammasome activation together with bacterial survival within macrophages. In vivo study confirmed the in vitro findings and showed that GSDMD restricts B. cenocepacia infection and dissemination and stimulates autophagy in response to B. cenocepacia. Nevertheless, GSDMD promotes lung inflammation and necrosis in response to B. cenocepacia without altering mice survival. This study describes the double-edged functions of GSDMD in response to B. cenocepacia infection and shows the importance of GSDMD-mediated mROS in restriction of B. cenocepacia.
Asunto(s)
Infecciones por Burkholderia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Unión a Fosfato/metabolismo , Animales , Autofagia/fisiología , Infecciones por Burkholderia/prevención & control , Burkholderia cenocepacia/patogenicidad , Caspasas Iniciadoras/genética , Caspasas Iniciadoras/metabolismo , Muerte Celular , Femenino , Inflamasomas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/fisiología , Lipopolisacáridos/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Proteínas de Unión a Fosfato/genética , Proteínas de Unión a Fosfato/fisiología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: Mitochondria play a key role in immune defense pathways, particularly for macrophages. We and others have previously demonstrated that cystic fibrosis (CF) macrophages exhibit weak autophagy activity and exacerbated inflammatory responses. Previous studies have revealed that mitochondria are defective in CF epithelial cells, but to date, the connection between defective mitochondrial function and CF macrophage immune dysregulation has not been fully elucidated. Here, we present a characterization of mitochondrial dysfunction in CF macrophages. METHODS: Mitochondrial function in wild-type (WT) and CF F508del/F508del murine macrophages was measured using the Seahorse Extracellular Flux analyzer. Mitochondrial morphology was investigated using transmission electron and confocal microscopy. Mitochondrial membrane potential (MMP) as well as mitochondrial reactive oxygen species (mROS) were measured using TMRM and MitoSOX Red fluorescent dyes, respectively. All assays were performed at baseline and following infection by Burkholderia cenocepacia, a multi-drug resistant bacterium that causes detrimental infections in CF patients. RESULTS: We have identified impaired oxygen consumption in CF macrophages without and with B. cenocepacia infection. We also observed increased mitochondrial fragmentation in CF macrophages following infection. Lastly, we observed increased MMP and impaired mROS production in CF macrophages following infection with B. cenocepacia. CONCLUSIONS: The mitochondrial defects identified are key components of the macrophage response to infection. Their presence suggests that mitochondrial dysfunction contributes to impaired bacterial killing in CF macrophages. Our current study will enhance our understanding of the pathobiology of CF and lead to the identification of novel mitochondrial therapeutic targets for CF.