RESUMO
Macrophages are important drivers of pathogenesis and progression to AIDS in HIV infection. The virus in the later phases of the infection is often predominantly macrophage-tropic and this tropism contributes to a chronic inflammatory and immune activation state that is observed in HIV patients. Pattern recognition receptors of the innate immune system are the key molecules that recognise HIV and mount the inflammatory responses in macrophages. The innate immune response against HIV-1 is potent and elicits caspase-1-dependent pro-inflammatory cytokine production of IL-1ß and IL-18. Although, NLRP3 has been reported as an inflammasome sensor dictating this response little is known about the pattern recognition receptors that trigger the "priming" signal for inflammasome activation, the NLRs involved or the HIV components that trigger the response. Using a combination of siRNA knockdowns in monocyte derived macrophages (MDMs) of different TLRs and NLRs as well as chemical inhibition, it was demonstrated that HIV Vpu could trigger inflammasome activation via TLR4/NLRP3 leading to IL-1ß/IL-18 secretion. The priming signal is triggered via TLR4, whereas the activation signal is triggered by direct effects on Kv1.3 channels, causing K+ efflux. In contrast, HIV gp41 could trigger IL-18 production via NAIP/NLRC4, independently of priming, as a one-step inflammasome activation. NAIP binds directly to the cytoplasmic tail of HIV envelope protein gp41 and represents the first non-bacterial ligand for the NAIP/NLRC4 inflammasome. These divergent pathways represent novel targets to resolve specific inflammatory pathologies associated with HIV-1 infection in macrophages.
Assuntos
Infecções por HIV/virologia , Inflamassomos/imunologia , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/virologia , Fragmentos de Peptídeos/metabolismo , Comunicação Celular/genética , Comunicação Celular/imunologia , Expressão Gênica/genética , Expressão Gênica/imunologia , Infecções por HIV/metabolismo , Humanos , Imunidade Inata/genética , Imunidade Inata/imunologia , Inflamassomos/metabolismo , Macrófagos/imunologia , Proteína Inibidora de Apoptose Neuronal/genética , Transdução de Sinais/imunologiaRESUMO
Nucleotide-binding domain and leucine-rich repeat receptor (NLR)-mediated inflammasome activation is important in host response to microbes, danger-associated molecular patterns (DAMPs) and metabolic disease. Some NLRs have been shown to interact with distinct cell metabolic pathways and cause negative regulation, tumorigenesis and autoimmune disorders, interacting with multiple innate immune receptors to modulate disease. NLR activation is therefore crucial in host response and in the regulation of metabolic pathways that can trigger a wide range of immunometabolic diseases or syndromes. However, the exact mode by which some of the less well-studied NLR inflammasomes are activated, interact with other metabolites and immune receptors, and the role they play in the progression of metabolic diseases is still not fully elucidated. In this study, we review up-to-date evidence regarding NLR function in metabolic pathways and the interplay with other immune receptors involved in GPCR signalling, gut microbiota and the complement system, in order to gain a better understanding of its link to disease processes.
Assuntos
Doenças Autoimunes/metabolismo , Proteínas do Sistema Complemento/metabolismo , Inflamassomos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Doenças Autoimunes/imunologia , Microbioma Gastrointestinal , Humanos , Imunidade Inata , Receptor Cross-Talk , Transdução de SinaisRESUMO
Different immune activation states require distinct metabolic features and activities in immune cells. For instance, inhibition of fatty acid synthase (FASN), which catalyzes the synthesis of long-chain fatty acids, prevents the proinflammatory response in macrophages; however, the precise role of this enzyme in this response remains poorly defined. Consistent with previous studies, we found here that FASN is essential for lipopolysaccharide-induced, Toll-like receptor (TLR)-mediated macrophage activation. Interestingly, only agents that block FASN upstream of acetoacetyl-CoA synthesis, including the well-characterized FASN inhibitor C75, inhibited TLR4 signaling, while those acting downstream had no effect. We found that acetoacetyl-CoA could overcome C75's inhibitory effect, whereas other FASN metabolites, including palmitate, did not prevent C75-mediated inhibition. This suggested an unexpected role for acetoacetyl-CoA in inflammation that is independent of its role in palmitate synthesis. Our evidence further suggested that acetoacetyl-CoA arising from FASN activity promotes cholesterol production, indicating a surprising link between fatty acid synthesis and cholesterol synthesis. We further demonstrate that this process is required for TLR4 to enter lipid rafts and facilitate TLR4 signaling. In conclusion, we have uncovered an unexpected link between FASN and cholesterol synthesis that appears to be required for TLR signal transduction and proinflammatory macrophage activation.
Assuntos
Colesterol/biossíntese , Ácido Graxo Sintase Tipo I/metabolismo , Ativação de Macrófagos , Macrófagos/enzimologia , Transdução de Sinais , Acil Coenzima A/metabolismo , Animais , Inflamação/enzimologia , Camundongos , Ácido Palmítico/metabolismo , Receptor 4 Toll-Like/metabolismoRESUMO
The innate immune system is an ancient surveillance system able to sense microbial invaders as well as aberrations in normal cell function. No longer viewed as a static and non-specific part of immunity, the innate immune system employs a plethora of specialized pattern recognition sensors to monitor and achieve homeostasis; these include the Toll-like receptors, the retinoic acid-inducible gene-like receptors, the nucleotide-binding oligomerization domain receptors (NLRs), the C-type lectins and the complement system. In order to increase specificity and diversity, innate immunity uses homotypic and heterotypic associations among these different components. Multi-molecular assemblies are formed both on the cell surface and in the cytosol to respond to pathogen and danger signals. Diverse, but tailored, responses to a changing environment are orchestrated depending on the the nature of the challenge and the repertoire of interacting receptors and components available in the sensing cell. It is now emerging that innate immunity operates a system of 'checks and balances' where interaction among the sensors is key in maintaining normal cell function. Complement sits at the heart of this alarm system and it is becoming apparent that it is capable of interacting with all the other pathways to effect a tailored immune response. In this review, we will focus on complement interactions with NLRs, the so-called 'inflammasomes', describing the molecular mechanisms that have been revealed so far and discussing the circumstantial evidence that exists for these interactions in disease states.
Assuntos
Ativação do Complemento , Proteínas do Sistema Complemento/imunologia , Imunidade Inata , Inflamassomos/imunologia , Inflamação/imunologia , Animais , Anti-Inflamatórios/uso terapêutico , Ativação do Complemento/efeitos dos fármacos , Proteínas do Sistema Complemento/metabolismo , Humanos , Imunidade Inata/efeitos dos fármacos , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Inflamação/metabolismo , Inflamação/prevenção & controle , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Ligantes , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Transdução de SinaisRESUMO
Inflammation is mediated mainly by leukocytes that express both Toll-like receptor 4 (TLR4) and Fc γ receptors (FcγR). Dysregulated activation of leukocytes via exogenous and endogenous ligands of TLR4 results in a large number of inflammatory disorders that underlie a variety of human diseases. Thus, differentially blocking inflammatory cells while sparing structural cells, which are FcγR-negative, represents an elegant strategy when targeting the underlying causes of human diseases. Here, we report a novel tethering mechanism of the Fv and Fc portions of anti-TLR4 blocking antibodies that achieves increased potency on inflammatory cells. In the presence of ligand (e.g. lipopolysaccharide (LPS)), TLR4 traffics into glycolipoprotein microdomains, forming concentrated protein platforms that include FcγRs. This clustering produces a microenvironment allowing anti-TLR4 antibodies to co-engage TLR4 and FcγRs, increasing their avidity and thus substantially increasing their inhibitory potency. Tethering of antibodies to both TLR4 and FcγRs proves valuable in ameliorating inflammation in vivo. This novel mechanism of action therefore has the potential to enable selective intervention of relevant cell types in TLR4-driven diseases.
Assuntos
Inflamação/imunologia , Macrófagos/imunologia , Receptores de IgG/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Anticorpos Monoclonais/imunologia , Sítios de Ligação , Células CHO , Linhagem Celular , Cricetulus , Dimerização , Feminino , Humanos , Inflamação/metabolismo , Macrófagos/citologia , Microdomínios da Membrana/imunologia , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptores de IgG/metabolismo , Receptor 4 Toll-Like/química , Receptor 4 Toll-Like/metabolismo , Células U937RESUMO
The membrane attack complex of complement (MAC), apart from its classical role of lysing cells, can also trigger a range of non-lethal effects on cells, acting as a drive to inflammation. In the present study, we chose to investigate these non-lethal effects on inflammasome activation. We found that, following sublytic MAC attack, there is increased cytosolic Ca(2+) concentration, at least partly through Ca(2+) release from the endoplasmic reticulum lumen via the inositol 1,4,5-triphosphate receptor (IP3R) and ryanodine receptor (RyR) channels. This increase in intracellular Ca(2+) concentration leads to Ca(2+) accumulation in the mitochondrial matrix via the 'mitochondrial calcium uniporter' (MCU), and loss of mitochondrial transmembrane potential, triggering NLRP3 inflammasome activation and IL-1ß release. NLRP3 co-localises with the mitochondria, probably sensing the increase in calcium and the resultant mitochondrial dysfunction, leading to caspase activation and apoptosis. This is the first study that links non-lethal effects of sublytic MAC attack with inflammasome activation and provides a mechanism by which sublytic MAC can drive inflammation and apoptosis.
Assuntos
Cálcio/metabolismo , Proteínas de Transporte/genética , Membrana Celular/efeitos dos fármacos , Complexo de Ataque à Membrana do Sistema Complemento/farmacologia , Células Epiteliais/efeitos dos fármacos , Inflamassomos/efeitos dos fármacos , Mucosa Respiratória/efeitos dos fármacos , Cálcio/agonistas , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Proteínas de Transporte/imunologia , Membrana Celular/imunologia , Retículo Endoplasmático/metabolismo , Células Epiteliais/citologia , Células Epiteliais/imunologia , Humanos , Inflamassomos/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/imunologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Cultura Primária de Células , Mucosa Respiratória/citologia , Mucosa Respiratória/imunologia , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
The innate immune system is a vital part of the body's defences against viral pathogens. The proteins retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation associated gene 5 (MDA5) function as cytoplasmic pattern recognition receptors that are involved in the elimination of actively replicating RNA viruses. Their location and their differential responses to RNA viruses emphasises the complexity of the innate detection system. Despite the wealth of information on the types of RNA that trigger RIG-I, much less is known about the nature of the RNAs that act as agonists for MDA5. In order to identify which RNA species triggers MDA5 activation during infection, we isolated viral ssRNA and replicative intermediates of RNA from positive sense ssRNA viruses. We reveal that MDA5 recognises not the genomic ssRNA but the dsRNA generated by the replication of these viruses. Furthermore, using fluorescent imaging we present the first report of the visualisation of dsRNA and MDA5, which provides unique evidence of the relationship between viral dsRNA and MDA5 and proves without a doubt that MDA5 is the key sensor for the dsRNA replicative intermediate form of positive sense ssRNA viruses.
Assuntos
RNA Helicases DEAD-box , Vírus de RNA , RNA de Cadeia Dupla , Adulto , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/imunologia , RNA Helicases DEAD-box/metabolismo , Enterovirus/genética , Enterovirus/metabolismo , Feminino , Células HEK293 , Humanos , Imunidade Inata/genética , Infecções/genética , Infecções/imunologia , Helicase IFIH1 Induzida por Interferon , Melanoma/genética , Melanoma/imunologia , Células Musculares/citologia , Células Musculares/metabolismo , Vírus de RNA/genética , Vírus de RNA/imunologia , Vírus de RNA/metabolismo , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/imunologia , RNA de Cadeia Dupla/metabolismo , Receptores Imunológicos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Replicação Viral/genéticaRESUMO
OBJECTIVE: The pathway by which herpes simplex virus 2 (HSV2) triggers the innate immune system in the urogenital system has not as yet been fully elucidated. In this study, we aimed to determine which pattern recognition receptors (PRRs) recognize HSV2 in primary vaginal epithelial cells. Once we deciphered the receptors involved, we aimed to target them to immunomodulate innate responses as a prophylactic or therapeutic intervention for early HSV2 infection. STUDY DESIGN: To determine which PRRs are involved, receptor silencing as well as confocal microscopy was utilized. For immunomodulation, PRR agonists were utilized to induce a strong, local response to limit the infection, and we used 2 quantitative methods, flow cytometry and plaque assays, to determine their effect on HSV2 replication. RESULTS: Our results show that HSV2 is detected by a plethora of PRRs: Toll-like receptors (TLR) 2 as well as deoxyribonucleic acid (DNA) sensors TLR9, DNA-dependent activator of interferon regulatory factors, and to a lesser extent interferon-inducible 16, which trigger cytokine secretion to protect the host. Using PRR agonists, such as lipoproteins, CpG DNA, and cyclic dinucleotides, we could significantly limit HSV2 replication. CONCLUSION: Different PRRs are strategically placed in different cell locations to detect virus invasion. Use of agonists that target and activate these PRRs appeared to be effective in preventing primary HSV2 infection in vaginal cells and could provide new insights in defense against HSV2 urogenital infections.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Herpes Genital/virologia , Herpesvirus Humano 2/fisiologia , Proteínas Nucleares/fisiologia , Fosfoproteínas/fisiologia , Receptor 2 Toll-Like/fisiologia , Ativação Viral/fisiologia , Feminino , Herpes Genital/imunologia , Humanos , Imunidade Inata , Proteínas de Ligação a RNA , Receptor Toll-Like 9/fisiologia , Vagina/imunologia , Vagina/virologiaRESUMO
Myristoylated alanine-rich C kinase substrate (MARCKS) is an intrinsically unfolded protein with a conserved cationic effector domain, which mediates the cross-talk between several signal transduction pathways. Transcription of MARCKS is increased by stimulation with bacterial LPS. We determined that MARCKS and MARCKS-related protein specifically bind to LPS and that the addition of the MARCKS effector peptide inhibited LPS-induced production of TNF-α in mononuclear cells. The LPS binding site within the effector domain of MARCKS was narrowed down to a heptapeptide that binds to LPS in an extended conformation as determined by nuclear magnetic resonance spectroscopy. After LPS stimulation, MARCKS moved from the plasma membrane to FYVE-positive endosomes, where it colocalized with LPS. MARCKS-deficient mouse embryonic fibroblasts (MEFs) responded to LPS with increased IL-6 production compared with the matched wild-type MEFs. Similarly, small interfering RNA knockdown of MARCKS also increased LPS signaling, whereas overexpression of MARCKS inhibited LPS signaling. TLR4 signaling was enhanced by the ablation of MARCKS, which had no effect on stimulation by TLR2, TLR3, and TLR5 agonists. These findings demonstrate that MARCKS contributes to the negative regulation of the cellular response to LPS.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Leucócitos Mononucleares/imunologia , Lipopolissacarídeos/imunologia , Proteínas de Membrana/imunologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Endossomos/imunologia , Fibroblastos/imunologia , Regulação da Expressão Gênica , Células HEK293 , Humanos , Imunidade Inata , Interleucina-6/biossíntese , Interleucina-6/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Substrato Quinase C Rico em Alanina Miristoilada , Peptídeos/química , Peptídeos/farmacologia , Ligação Proteica , Transporte Proteico/imunologia , RNA Interferente Pequeno/genética , Transdução de Sinais , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Human rhinoviruses have been linked with underlying lung disorders, such as asthma and chronic obstructive pulmonary disease, in children and adults. However, the mechanism of virus-induced airway inflammation is poorly understood. In this study, using virus deletion mutants and silencing for nucleotide-binding oligomerization domain-like receptors (NLRs), we show that the rhinovirus ion channel protein 2B triggers NLRP3 and NLRC5 inflammasome activation and IL-1ß secretion in bronchial cells. 2B protein targets the endoplasmic reticulum and Golgi and induces Ca(2+) reduction in these organelles, thereby disturbing the intracellular calcium homeostasis. NLRP3 and NLRC5 act in a cooperative manner during the inflammasome assembly by sensing intracellular Ca(2+) fluxes and trigger IL-1ß secretion. These results reveal for the first time that human rhinovirus infection in primary bronchial cells triggers inflammasome activation.
Assuntos
Brônquios/metabolismo , Brônquios/virologia , Sinalização do Cálcio/fisiologia , Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Rhinovirus/patogenicidade , Asma/etiologia , Asma/metabolismo , Asma/virologia , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Células Cultivadas , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Técnicas de Silenciamento de Genes , Complexo de Golgi/metabolismo , Complexo de Golgi/virologia , Humanos , Inflamassomos/metabolismo , Interleucina-1beta/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR , Infecções por Picornaviridae/complicações , Infecções por Picornaviridae/metabolismo , Infecções por Picornaviridae/virologia , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/virologia , Rhinovirus/genética , Rhinovirus/metabolismo , Proteínas Virais/metabolismoRESUMO
BACKGROUND: Respiratory syncytial virus (RSV) remains the leading cause of serious viral bronchiolitis and pneumonia in infants and young children throughout the world. The burden of disease is significant, with 70% of all infants being infected with RSV within the first year of their life. 40% of those children discharged from hospital have recurrent, repeated respiratory symptoms and wheezing for at least 10 years. The infection is also an important illness in the elderly and immunocompromised individuals. Ongoing symptoms relate to continued lung inflammation. One cytokine that is associated with RSV infection is IL-1ß, but the mechanism of activation remain unclear. OBJECTIVES: In the current study, we set out to decipher the molecular mechanisms of RSV-induced inflammasome activation. METHODS AND RESULTS: Using deletion mutants of the virus and measuring IL-1ß secretion, as well as caspase 1 expression via western blotting, we demonstrate that the NLRP3 inflammasome is activated through the small hydrophobic (SH) RSV viroporin which induces membrane permeability to ions or small molecules. Confocal microscopy revealed that during virus infection, SH seems to accumulate within lipid rafts in the Golgi compartments. CONCLUSIONS: Upon RSV infection, SH gets localised in the cell membranes and intracellular organelle membranes, and then induces permeability by disrupting membrane architecture, thus leading us to believe that formation of viral ion channels in lipid bilayers of cells is a viral recognition pathway used by the host to signal inflammasome activation.
Assuntos
Proteínas de Transporte/metabolismo , Inflamassomos/metabolismo , Vírus Sincicial Respiratório Humano/genética , Adulto , Bronquiolite Viral/genética , Bronquiolite Viral/fisiopatologia , Proteínas de Transporte/genética , Células Cultivadas , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Humanos , Inflamassomos/genética , Pulmão/citologia , Masculino , Microscopia Confocal , Proteína 3 que Contém Domínio de Pirina da Família NLR , Pneumonia Viral/metabolismo , Pneumonia Viral/fisiopatologia , RNA Viral/genética , RNA Viral/metabolismo , Infecções por Vírus Respiratório Sincicial/genética , Infecções por Vírus Respiratório Sincicial/fisiopatologia , Vírus Sincicial Respiratório Humano/fisiologia , Sensibilidade e Especificidade , Transdução de Sinais/genéticaRESUMO
Lipoteichoic acid (LTA), a ubiquitous cell wall component of Gram-positive bacteria, represents a potent immunostimulatory molecule. Because LTA of a mutant Staphylococcus aureus strain lacking lipoproteins (Deltalgt-LTA) has been described to be immunobiologically inactive despite a lack of ascertained structural differences to wild-type LTA (wt-LTA), we investigated the functional requirements for the recognition of Deltalgt-LTA by human peripheral blood cells. In this study, we demonstrate that Deltalgt-LTA-induced immune activation critically depends on the immobilization of LTA and the presence of human serum components, which, to a lesser degree, was also observed for wt-LTA. Under experimental conditions allowing LTA-mediated stimulation, we found no differences between the immunostimulatory capacity of Deltalgt-LTA and wt-LTA in human blood cells, arguing for a limited contribution of possible lipoprotein contaminants to wt-LTA-mediated immune activation. In contrast to human blood cells, TLR2-transfected human embryonic kidney 293 cells could be activated only by wt-LTA, whereas activation of these cells by Deltalgt-LTA required the additional expression of TLR6 and CD14, suggesting that activation of human embryonic kidney 293 cells expressing solely TLR2 is probably mediated by residual lipoproteins in wt-LTA. Notably, in human peripheral blood, LTA-specific IgG Abs are essential for Deltalgt-LTA-mediated immune activation and appear to induce the phagocytic uptake of Deltalgt-LTA via engagement of FcgammaRII. In this study, we have elucidated a novel mechanism of LTA-induced cytokine induction in human peripheral blood cells that involves uptake of LTA and subsequent intracellular recognition driven by TLR2, TLR6, and CD14.
Assuntos
Adjuvantes Imunológicos/sangue , Lipopolissacarídeos/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/imunologia , Ácidos Teicoicos/metabolismo , Receptor 2 Toll-Like/metabolismo , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/fisiologia , Reações Antígeno-Anticorpo , Linhagem Celular , Membrana Celular/imunologia , Membrana Celular/metabolismo , Membrana Celular/microbiologia , Citocinas/biossíntese , Citocinas/sangue , Citocinas/metabolismo , Humanos , Imunoglobulina G/metabolismo , Líquido Intracelular/imunologia , Líquido Intracelular/metabolismo , Líquido Intracelular/microbiologia , Receptores de Lipopolissacarídeos/biossíntese , Receptores de Lipopolissacarídeos/fisiologia , Lipopolissacarídeos/sangue , Lipopolissacarídeos/imunologia , Lipoproteínas/deficiência , Lipoproteínas/genética , Proteínas Opsonizantes/metabolismo , Receptores de IgG/fisiologia , Staphylococcus aureus/metabolismo , Ácidos Teicoicos/sangue , Ácidos Teicoicos/imunologia , Receptor 2 Toll-Like/sangue , Receptor 2 Toll-Like/fisiologia , Receptor 6 Toll-Like/biossíntese , Receptor 6 Toll-Like/fisiologiaRESUMO
The complement system is an ancient and critical part of innate immunity. Recent studies have highlighted novel roles of complement beyond lysis of invading pathogens with implications in regulating the innate immune response, as well as contributing to metabolic reprogramming of T-cells, synoviocytes as well as cells in the CNS. These findings hint that complement can be an immunometabolic regulator, but whether this is also the case for the terminal step of the complement pathway, the membrane attack complex (MAC) is not clear. In this study we focused on determining whether MAC is an immunometabolic regulator of the innate immune response in human monocyte-derived macrophages. Here, we uncover previously uncharacterized metabolic changes and mitochondrial dysfunction occurring downstream of MAC deposition. These alterations in glycolytic flux and mitochondrial morphology and function mediate NLRP3 inflammasome activation, pro-inflammatory cytokine release and gasdermin D formation. Together, these data elucidate a novel signalling cascade, with metabolic alterations at its center, in MAC-stimulated human macrophages that drives an inflammatory consequence in an immunologically relevant cell type.
Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Humanos , Inflamassomos/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Macrófagos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismoRESUMO
Human rhinovirus (HRV), like coronavirus (HCoV), are positive-strand RNA viruses that cause both upper and lower respiratory tract illness, with their replication facilitated by concentrating RNA-synthesizing machinery in intracellular compartments made of modified host membranes, referred to as replication organelles (ROs). Here we report a non-canonical, essential function for stimulator of interferon genes (STING) during HRV infections. While the canonical function of STING is to detect cytosolic DNA and activate inflammatory responses, HRV infection triggers the release of STIM1-bound STING in the ER by lowering Ca2+, thereby allowing STING to interact with phosphatidylinositol 4-phosphate (PI4P) and traffic to ROs to facilitates viral replication and transmission via autophagy. Our results thus hint a critical function of STING in HRV viral replication and transmission, with possible implications for other RO-mediated RNA viruses.
Assuntos
Enterovirus , Vírus de RNA , Humanos , Organelas , Rhinovirus , Replicação Viral/fisiologiaRESUMO
In the last twenty years, the general view of the plasma membrane has changed from a homogeneous arrangement of lipids to a mosaic of microdomains. It is currently thought that islands of highly ordered saturated lipids and cholesterol, which are laterally mobile, exist in the plane of the plasma membrane. Lipid rafts are thought to provide a means to explain the spatial segregation of certain signalling pathways emanating from the cell surface. They seem to provide the necessary microenvironment in order for certain specialised signalling events to take place, such as the innate immune recognition. The innate immune system seems to employ germ-lined encoded receptors, called pattern recognition receptors (PRRs), in order to detect pathogens. One family of such receptors are the Toll-like receptors (TLRs), which are the central "sensing" apparatus of the innate immune system. In recent years, it has become apparent that TLRs are recruited into membrane microdomains in response to ligands. These nanoscale assemblies of sphingolipid, cholesterol, and TLRs stabilize and coalesce, forming signalling platforms, which transduce signals that lead to innate immune activation. In the current paper, we will investigate all past and current literature concerning recruitment of extracellular and intracellular TLRs into lipid rafts and how this membrane organization modulates innate immune responses.
Assuntos
Compartimento Celular/imunologia , Membrana Celular/imunologia , Sistema Imunitário/imunologia , Microdomínios da Membrana/imunologia , Receptores Toll-Like/imunologia , Animais , HumanosRESUMO
Lipid rafts are envisaged as islands of highly ordered saturated lipids and cholesterol that are laterally mobile in the plane of the plasma membrane. Lipid rafts are thought to provide a means to explain the spatial segregation of certain signalling pathways emanating from the cell surface. They seem to provide the necessary microenvironment in order for certain specialised signalling events to take place- such as the innate immune recognition. The innate immune system seems to employ germ-lined encoded receptors, called pattern recognition receptors (PRRs) in order to "sense" pathogens. One family of such receptors are the Toll like receptors (TLRs), which are the central "sensing" apparatus of the innate immune system. In recent years, it has become apparent that TLRs are recruited into membrane microdomains in response to ligands and these constitute signalling platforms, which transducer singals that lead to innate immune activation. In this chapter will review all past and current literature concerning recruitment of TLRs into lipid rafts and how this membrane compartmentalization is crucial for innate immune responses.
Assuntos
Endotoxinas/imunologia , Microdomínios da Membrana/química , Receptores Toll-Like/metabolismo , Transporte Biológico , Humanos , Imunidade Inata/imunologia , Microdomínios da Membrana/metabolismoRESUMO
We report a mechanism of microbial evasion of Toll-like receptor (TLR)-mediated immunity that depends on CXCR4 exploitation. Specifically, the oral/systemic pathogen Porphyromonas gingivalis induces cross-talk between CXCR4 and TLR2 in human monocytes or mouse macrophages and undermines host defense. This is accomplished through its surface fimbriae, which induce CXCR4/TLR2 co-association in lipid rafts and interact with both receptors: Binding to CXCR4 induces cAMP-dependent protein kinase A (PKA) signaling, which in turn inhibits TLR2-mediated proinflammatory and antimicrobial responses to the pathogen. This outcome enables P. gingivalis to resist clearance in vitro and in vivo and thus to promote its adaptive fitness. However, a specific CXCR4 antagonist abrogates this immune evasion mechanism and offers a promising counterstrategy for the control of P. gingivalis periodontal or systemic infections.
Assuntos
Porphyromonas gingivalis/imunologia , Porphyromonas gingivalis/patogenicidade , Receptor Cross-Talk/imunologia , Receptores CXCR4/imunologia , Receptor 2 Toll-Like/imunologia , Animais , Infecções por Bacteroidaceae/imunologia , Sobrevivência Celular , Células Cultivadas , Cricetinae , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Fímbrias/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Monócitos/imunologia , NF-kappa B/metabolismo , Óxidos de Nitrogênio/metabolismo , Ligação Proteica , Receptor 2 Toll-Like/deficiência , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismoRESUMO
TLRs detect conserved molecular patterns that are unique to microbes, enabling tailored responses to invading pathogens and modulating a multitude of immunopathological conditions. We investigated the ability of a naturally occurring stearoyl-arachidonoyl form of phosphatidylserine (SAPS) to inhibit the proinflammatory effects of TLR agonists in models of inflammation investigating the interaction of leukocytes with epithelial and endothelial cells. The responses to LPS of both epithelial and endothelial cells were highly amplified in the presence of PBMCs. Coincubation with SAPS markedly inhibited activation of cocultures by LPS, principally through inhibition of the TLR4 signaling pathway in PBMCs; however, this was not through downmodulation of TLR4 or coreceptor expression, nor was IL-1beta-induced cytokine release affected. SAPS also impaired Pam(3)CSK(4) (TLR2/1), Gardiquimod (TLR7/8), and Streptococcus pneumoniae-induced cytokine release, but had only modest effects on poly(I:C) (TLR3)-induced responses. Fluorescence resonance energy transfer analysis of molecular associations revealed that SAPS disrupted the association of both TLR4 and TLR2 with their respective membrane partners that are required for signaling. Thus, our data reinforce the existence and importance of cooperative networks of TLRs, tissue cells, and leukocytes in mediating innate immunity, and identify a novel disrupter of membrane microdomains, revealing the dependence of TLR signaling on localization within these domains.
Assuntos
Interleucina-1beta/imunologia , Leucócitos Mononucleares/imunologia , Microdomínios da Membrana/imunologia , Modelos Imunológicos , Fosfatidilserinas/imunologia , Transdução de Sinais/imunologia , Receptores Toll-Like/imunologia , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Células Endoteliais/imunologia , Células Epiteliais/imunologia , Humanos , Imunidade Inata/efeitos dos fármacos , Inflamação/imunologia , Interleucina-1beta/farmacologia , Lipopeptídeos , Peptídeos/farmacologia , Fosfatidilserinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Streptococcus pneumoniae/imunologia , Receptores Toll-Like/agonistasRESUMO
The fimbriae of Porphyromonas gingivalis mediate critical roles in host colonization and evasion of innate defenses and comprise polymerized fimbrilin (FimA) associated with quantitatively minor accessory proteins (FimCDE) of unknown function. We now show that P. gingivalis fimbriae lacking FimCDE fail to interact with the CXC-chemokine receptor 4 (CXCR4), and bacteria expressing FimCDE-deficient fimbriae cannot exploit CXCR4 in vivo for promoting their persistence, as the wild-type organism does. Consistent with these loss-of-function experiments, purified FimC and FimD (but not FimE) were shown to interact with CXCR4. However, significantly stronger binding was observed when a combination of all three proteins was allowed to interact with CXCR4. In addition, FimC and FimD bound to fibronectin and type 1 collagen, whereas FimE failed to interact with these matrix proteins. These data and the fact that FimE is required for the association of FimCDE with P. gingivalis fimbriae suggest that FimE may recruit FimC and FimD into a functional complex, rather than directly binding host proteins. Consistent with this notion, FimE was shown to bind both FimC and FimD. In summary, the FimCDE components cooperate and impart critical adhesive and virulence properties to P. gingivalis fimbriae.
Assuntos
Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Fímbrias Bacterianas/fisiologia , Porphyromonas gingivalis/patogenicidade , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/genética , Colágeno Tipo I/metabolismo , Fibronectinas/metabolismo , Deleção de Genes , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Receptores CXCR4/metabolismo , Virulência , Fatores de Virulência/genéticaRESUMO
Mammalian responses to bacterial products can lead to an uncontrolled inflammatory response that can be deadly for the host. It has been shown that the innate immune system employs at least three cell surface receptors, TLR4, CD14 and MD2, in order to recognize bacterial products. We have previously shown that heat shock proteins (HSPs) are also involved in the innate immune recognition. HSPs are a family of highly conserved proteins that act as molecular chaperones and assist in proper folding, assembly and intracellular trafficking of proteins. How HSPs reach the cell surface and how they are involved in the innate immune response still remain unclear. In the present study we investigated their association with the TLR4/CD14/MD2 complex in response to bacterial products and provide evidence that the Hsp70 and Hsp90 associate with TLR4 on the cell surface in response to stimulation by bacterial products. These associations seem to take place within lipid rafts. The addition of exogenous recombinant Hsp70 to cells in vitro results in a dose-responsive inhibition of the inflammatory signal cascade and cytokine production. Our studies reveal that HSPs may play an important role as endogenous regulators of the innate immune response.