RESUMO
Neutrophil extracellular traps (NETs) are a key antimicrobial feature of cellular innate immunity mediated by polymorphonuclear neutrophils (PMNs). NETs counteract microbes but are also linked to inflammation in atherosclerosis, arthritis, or psoriasis by unknown mechanisms. Here, we report that NET-associated RNA (naRNA) stimulates further NET formation in naive PMNs via a unique TLR8-NLRP3 inflammasome-dependent pathway. Keratinocytes respond to naRNA with expression of psoriasis-related genes (e.g., IL17, IL36) via atypical NOD2-RIPK signaling. In vivo, naRNA drives temporary skin inflammation, which is drastically ameliorated by genetic ablation of RNA sensing. Unexpectedly, the naRNA-LL37 'composite damage-associated molecular pattern (DAMP)' is pre-stored in resting neutrophil granules, defining sterile NETs as inflammatory webs that amplify neutrophil activation. However, the activity of the naRNA-LL37 DAMP is transient and hence supposedly self-limiting under physiological conditions. Collectively, upon dysregulated NET release like in psoriasis, naRNA sensing may represent both a potential cause of disease and a new intervention target.
Assuntos
Alarminas , Catelicidinas , Armadilhas Extracelulares , Inflamação , Neutrófilos , Armadilhas Extracelulares/metabolismo , Neutrófilos/metabolismo , Neutrófilos/imunologia , Inflamação/metabolismo , Inflamação/genética , Animais , Humanos , Camundongos , Alarminas/metabolismo , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Queratinócitos/metabolismo , RNA/genética , RNA/metabolismo , Psoríase/genética , Psoríase/metabolismo , Psoríase/patologia , Transdução de Sinais , Ativação de Neutrófilo/genética , Imunidade Inata/genéticaRESUMO
These days not only humans but also freshwater fish battle with infections by RNA viruses (Zou & Nie, 2017). This observation prompted Liao et al to turn their attention to viral recognition in Grass carp (Ctenopharyngodon idella), the most important cultivated freshwater fish with 5.7 million tons and 13 billion USD in fishery exports per year (FAO, 2021). Grass carp and other freshwater fish, such as the model organism Danio rerio (zebrafish), have a sophisticated innate immune system that helps them to detect microbial and viral pathogens by employing a variety of pattern recognition receptors (PRRs; Zou & Nie, 2017). Toll-like receptors (TLRs) are one class of PRRs that detect microbe-associated molecular patterns (MAMPs), such as flagellin or viral double-stranded (ds)RNA. In mammals, TLR3 is specialized in sensing viral dsRNA (Liu et al, 2008), while TLR5 recognizes the MAMP flagellin (Yoon et al, 2012; Fig 1). The well-established notion of TLR5 as a purely "bacterial" flagellin TLR has now been challenged by Liao et al in this issue of EMBO Reports (Liao et al, 2022). The authors' intriguing and unexpected results indicate that fish TLR5 is involved in viral recognition, a function lost in mammals, and shed light on hitherto inexplicable links of mammalian TLR5 to antiviral immune signaling.
Assuntos
Carpas , Receptor 5 Toll-Like , Animais , Flagelina , Humanos , Imunidade Inata , Mamíferos/genética , RNA de Cadeia Dupla , RNA Viral/genética , Paladar , Receptor 5 Toll-Like/genética , Peixe-Zebra/genéticaRESUMO
Upon recognition of aberrantly located DNA, the innate immune sensor cyclic GMP-AMP synthase (cGAS) activates stimulator of IFN genes (STING)/IFN regulatory factor (IRF)3-driven antiviral responses. In this study, we characterized the ability of a specific variant of the human cGAS-encoding gene MB21D1, rs610913, to alter cGAS-mediated DNA sensing and viral infection. rs610913 is a frequent G>T polymorphism resulting in a P261H exchange in the cGAS protein. Data from the International Collaboration for the Genomics of HIV suggested that rs610913 nominally associates with HIV-1 acquisition in vivo. Molecular modeling of cGAS(P261H) hinted toward the possibility for an additional binding site for a potential cellular cofactor in cGAS dimers. However, cGAS(wild-type [WT]) or cGAS(P261H)-reconstituted THP-1 cGAS knockout cells shared steady-state expression of IFN-stimulated genes, as opposed to cells expressing the enzymatically inactive cGAS(G212A/S213A). Accordingly, cGAS(WT) and cGAS(P261H) cells were less susceptible to lentiviral transduction and infection with HIV-1, HSV-1, and Chikungunya virus as compared with cGAS knockout or cGAS(G212A/S213A) cells. Upon DNA challenge, innate immune activation appeared to be mildly reduced upon expression of cGAS(P261H) compared with cGAS(WT). Finally, DNA challenge of PBMCs from donors homozygously expressing rs610913 provoked a trend toward a slightly reduced type I IFN response as compared with PBMCs from GG donors. Taken together, the steady-state activity of cGAS maintains a baseline antiviral state rendering cells more refractory to IFN-stimulated gene-sensitive viral infections. rs610913 failed to grossly differ phenotypically from the WT gene, suggesting that cGAS(P261H) and WT cGAS share a similar ability to sense viral infections in vivo.
Assuntos
Imunidade Inata , Viroses , Humanos , DNA Viral/imunologia , Imunidade Inata/genética , Imunidade Inata/imunologia , Nucleotidiltransferases/genética , Nucleotidiltransferases/imunologia , Nucleotidiltransferases/metabolismo , Transdução de Sinais , Viroses/genética , Viroses/imunologia , Viroses/prevenção & controleRESUMO
BACKGROUND: Ischemic stroke immediately evokes a strong neuro-inflammatory response within the vascular compartment, which contributes to primary infarct development under vessel occlusion as well as further infarct growth despite recanalization, referred to as ischemia/reperfusion injury. Later, in the subacute phase of stroke (beyond day 1 after recanalization), further inflammatory processes within the brain parenchyma follow. Whether this second wave of parenchymal inflammation contributes to an additional/secondary increase in infarct volumes and bears the potential to be pharmacologically targeted remains elusive. We addressed the role of the NLR-family pyrin domain-containing protein 3 (NLRP3) inflammasome in the subacute phase of ischemic stroke. METHODS: Focal cerebral ischemia was induced in C57Bl/6 mice by a 30-min transient middle cerebral artery occlusion (tMCAO). Animals were treated with the NLRP3 inhibitor MCC950 therapeutically 24 h after or prophylactically before tMCAO. Stroke outcome, including infarct size and functional deficits as well as the local inflammatory response, was assessed on day 7 after tMCAO. RESULTS: Infarct sizes on day 7 after tMCAO decreased about 35% after delayed and about 60% after prophylactic NLRP3 inhibition compared to vehicle. Functionally, pharmacological inhibition of NLRP3 mitigated the local inflammatory response in the ischemic brain as indicated by reduction of infiltrating immune cells and reactive astrogliosis. CONCLUSIONS: Our results demonstrate that the NLRP3 inflammasome continues to drive neuroinflammation within the subacute stroke phase. NLRP3 inflammasome inhibition leads to a better long-term outcome-even when administered with a delay of 1 day after stroke induction, indicating ongoing inflammation-driven infarct progression. These findings may pave the way for eagerly awaited delayed treatment options in ischemic stroke.
Assuntos
Isquemia Encefálica , Inflamassomos , AVC Isquêmico , Traumatismo por Reperfusão , Acidente Vascular Cerebral , Animais , Camundongos , Isquemia Encefálica/metabolismo , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/metabolismo , Inflamassomos/antagonistas & inibidores , Inflamassomos/metabolismo , Inflamação/complicações , AVC Isquêmico/tratamento farmacológico , AVC Isquêmico/complicações , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Traumatismo por Reperfusão/metabolismo , Acidente Vascular Cerebral/metabolismoRESUMO
The NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome is a fascinating cellular machinery endowed with the capacity for rapid proteolytic processing of the pro-inflammatory cytokine IL-1ß and the cell death effector gasdermin D (GSDMD). Although its activity is essential to fight infection and support tissue homeostasis, the inflammasome complex, which consists of the danger sensor NLRP3, the adaptor apoptosis-associated speck-like protein containing a CARD (ASC; also known as PYCARD), caspase-1 and probably other regulatory proteins, also bears considerable potential for detrimental inflammation, as observed in human conditions such as gout, heart attack, stroke and Alzheimer's disease. Thus, multi-layered regulatory networks are required to ensure the fine balance between rapid responsiveness versus erroneous activation (sufficient and temporally restricted versus excessive and chronic activity) of the inflammasome. These involve multiple activation, secretion and cell death pathways, as well as modulation of the subcellular localization of NLRP3, and its structure and activity, owing to post-translational modification by other cellular proteins. Here, we discuss the exciting progress that has recently been made in deciphering the regulation of the NLRP3 inflammasome. Additionally, we highlight open questions and describe areas of research that warrant further exploration to obtain a more comprehensive molecular and cellular understanding of the NLRP3 inflammasome.
Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Apoptose , Caspase 1 , Citocinas , Humanos , Inflamação/genética , Interleucina-1beta , Proteína 3 que Contém Domínio de Pirina da Família NLR/genéticaRESUMO
Lipopolysaccharide (LPS) binding protein (LBP) is an acute-phase protein that initiates an immune response after recognition of bacterial LPS. Here, we report the crystal structure of murine LBP at 2.9 Å resolution. Several structural differences were observed between LBP and the related bactericidal/permeability-increasing protein (BPI), and the LBP C-terminal domain contained a negatively charged groove and a hydrophobic "phenylalanine core." A frequent human LBP SNP (allelic frequency 0.08) affected this region, potentially generating a proteinase cleavage site. The mutant protein had a reduced binding capacity for LPS and lipopeptides. SNP carriers displayed a reduced cytokine response after in vivo LPS exposure and lower cytokine concentrations in pneumonia. In a retrospective trial, the LBP SNP was associated with increased mortality rates during sepsis and pneumonia. Thus, the structural integrity of LBP may be crucial for fighting infections efficiently, and future patient stratification might help to develop better therapeutic strategies.
Assuntos
Proteínas de Fase Aguda/química , Peptídeos Catiônicos Antimicrobianos/química , Proteínas Sanguíneas/química , Proteínas de Transporte/química , Imunidade Inata/genética , Lipopolissacarídeos/química , Glicoproteínas de Membrana/química , Modelos Moleculares , Mutação , Polimorfismo de Nucleotídeo Único , Proteínas de Fase Aguda/genética , Proteínas de Fase Aguda/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Sítios de Ligação , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/imunologia , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Cristalografia por Raios X , Genótipo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lipopolissacarídeos/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Ligação Proteica , Estrutura Terciária de Proteína , Eletricidade Estática , Homologia Estrutural de ProteínaRESUMO
Escherichia coli represents a classical intestinal gram-negative commensal. Despite this commensalism, different E. coli strains can mediate disparate immunogenic properties in a given host. Symbiotic E. coli strains such as E. coli Nissle 1917 (EcN) are attributed beneficial properties, e.g., promotion of intestinal homeostasis. Therefore, we aimed to identify molecular features derived from symbiotic bacteria that might help to develop innovative therapeutic alternatives for the treatment of intestinal immune disorders. This study was performed using the dextran sodium sulphate (DSS)-induced colitis mouse model, which is routinely used to evaluate potential therapeutics for the treatment of Inflammatory Bowel Diseases (IBDs). We focused on the analysis of flagellin structures of different E. coli strains. EcN flagellin was found to harbor a substantially longer hypervariable region (HVR) compared to other commensal E. coli strains, and this longer HVR mediated symbiotic properties through stronger activation of Toll-like receptor (TLR)5, thereby resulting in interleukin (IL)-22-mediated protection of mice against DSS-induced colitis. Furthermore, using bone-marrow-chimeric mice (BMCM), CD11c+ cells of the colonic lamina propria (LP) were identified as the main mediators of these flagellin-induced symbiotic effects. We propose flagellin from symbiotic E. coli strains as a potential therapeutic to restore intestinal immune homeostasis, e.g., for the treatment of IBD patients.
Assuntos
Escherichia coli/metabolismo , Flagelina/genética , Simbiose/genética , Animais , Colite/induzido quimicamente , Colite/imunologia , Modelos Animais de Doenças , Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Feminino , Flagelina/metabolismo , Mucosa Intestinal , Intestinos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/imunologia , Simbiose/fisiologia , Receptor 5 Toll-Like/metabolismoRESUMO
PURPOSE: Cerebral ischemia induces a profound neuro-inflammatory response, but the underlying molecular mechanisms are poorly understood. Inflammasomes (NLRP1, NLRP3, NLRC4, AIM2) are intracellular multi-protein complexes which can induce sets of pro-inflammatory cyto- and chemokines, and thereby guide inflammation. We, here, assessed the functional role of NLRP3 in ischemia/reperfusion (I/R) injury in a mouse model of transient cerebral ischemia. METHODS: Ischemic stroke was induced in C57Bl/6 mice by 60 min transient middle cerebral artery occlusion (tMCAO) and 3, 7 or 23 h of reperfusion, a paradigm of I/R injury. The expression patterns of inflammasomes in the ischemic hemispheres were evaluated by semiquantitative real-time PCR and Western Blot analysis accompanied by protein localization using immunocytochemistry. Finally, animals were treated with the inflammasome inhibitors Sulforaphane, Genipin, MCC950 or vehicle, directly before or upon recanalization after tMCAO. Stroke outcome was assessed, including infarct size and functional deficits, local inflammatory response, neuronal survival as well as blood-brain barrier function on day 1 after tMCAO. RESULTS: After tMCAO the relative gene expression levels of NLRP3 increased 20-30x within 1 day in the ischemic hemisphere which translated into an increased expression of NLRP3 in neurons. Accordingly, the gene expression levels of the NLRP3-modulator, Bruton's Tyrosine Kinase (BTK), and the NLRP3-inducible cytokine IL-1ß significantly rose. Lesser or non-significant changes were seen for the other inflammasomes. Application of inflammasome inhibitors covering all inflammasomes or specifically NLRP3 significantly reduced infarct volumes when given before or after tMCAO and was accompanied by clear evidence for reduced activation of caspase 1. This stroke attenuating effect coincided with less immune cell infiltration in the ischemic hemisphere and preservation of the blood-brain barrier integrity. CONCLUSIONS: Our data show that induction of the NLRP3 inflammasome in neurons drives neuroinflammation in acute ischemic stroke. Early blockade of NLRP3 protects from I/R injury by mitigating inflammation and stabilizing the blood-brain barrier.
Assuntos
Isquemia Encefálica , Traumatismo por Reperfusão , Acidente Vascular Cerebral , Animais , Infarto da Artéria Cerebral Média , Inflamassomos , Inflamação , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLRRESUMO
OBJECTIVE: To report a chronic recurrent multifocal osteomyelitis (CRMO)-like clinical phenotype with multisystem inflammation associated with a novel gene variant in the spectrum of IL-1-mediated diseases. METHODS: A 3-year-old boy presented with recurrent episodes of fever, serositis, pancreatitis and high inflammatory markers with onset at age 13 months. At age 3 years, he started limping. Imaging revealed multifocal pelvic bone inflammation suggestive of CRMO. Autoinflammation panel testing was non-contributory. Whole exome sequencing (WES) and advanced IL-1 pathway analysis was conducted. RESULTS: WES identified a novel homozygous interleukin receptor 1 (IL1RN) variant (c.62C>G; p. Ser21*) (NM_173842.2). Functional analysis of IL1RN mRNA and IL-1 receptor antagonist (IL-1RA) protein confirmed the diagnosis of a deficiency of the IL-1 receptor antagonist (DIRA). Treatment with the nonselective IL-1 inhibitor anakinra resulting in rapid remission; switch to the selective IL-1ß antagonist canakinumab led to a flare within 6 weeks. Re-start of anakinra recaptured remission, last documented at the recent 19-month follow-up. CONCLUSION: This is the first report of a novel late-onset DIRA confirmed by advanced diagnostic testing. In patients with systemic inflammation and CRMO-like bone lesions, IL1RN testing should be considered; even in the absence of skin manifestations. Non-selective IL-1 inhibition is an effective therapy.
Assuntos
Proteína Antagonista do Receptor de Interleucina 1/genética , Osteomielite/genética , Anticorpos Monoclonais Humanizados/uso terapêutico , Antirreumáticos/uso terapêutico , Pré-Escolar , Substituição de Medicamentos/efeitos adversos , Homozigoto , Humanos , Quimioterapia de Indução/métodos , Proteína Antagonista do Receptor de Interleucina 1/deficiência , Proteína Antagonista do Receptor de Interleucina 1/uso terapêutico , Interleucina-1beta/antagonistas & inibidores , Masculino , Osteomielite/diagnóstico por imagem , Fenótipo , Exacerbação dos Sintomas , Sequenciamento do ExomaRESUMO
Activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) is an aggressive lymphoproliferative disorder involving chronic NF-κB activation. Several mutations in the BCR and MyD88 signaling pathway components, such as MyD88 L265P, are implicated in this aberrant activation. Among heat shock proteins, HSP110 has recently been identified as a prosurvival and/or proliferation factor in many cancers, but its role in ABC-DLBCL survival mechanisms remained to be established. We observed that short hairpin RNA-mediated HSP110 silencing decreased the survival of several ABC-DLBCL cell lines and decreased immunoglobulin M-MyD88 co-localization and subsequent NF-κB signaling. Conversely, overexpression of HSP110 in ABC-DLBCL or non-DLBCL cell lines increased NF-κB signaling, indicating a tight interplay between HSP110 and the NF-κB pathway. By using immunoprecipitation and proximity ligation assays, we identified an interaction between HSP110 and both wild-type MyD88 and MyD88 L265P. HSP110 stabilized both MyD88 forms with a stronger effect on MyD88 L265P, thus facilitating chronic NF-κB activation. Finally, HSP110 expression was higher in lymph node biopsies from patients with ABC-DLBCL than in normal reactive lymph nodes, and a strong correlation was found between the level of HSP110 and MyD88. In conclusion, we identified HSP110 as a regulator of NF-κB signaling through MyD88 stabilization in ABC-DLBCL. This finding reveals HSP110 as a new potential therapeutic target in ABC-DLBCL.
Assuntos
Proteínas de Choque Térmico HSP110/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Fator 88 de Diferenciação Mieloide/química , NF-kappa B/metabolismo , Estudos de Coortes , Proteínas de Choque Térmico HSP110/genética , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/genética , Estabilidade Proteica , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Mammalian Nod-like receptor (NLR) proteins contribute to the regulation and induction of innate and adaptive immunity in mammals, although the function of about half of the currently identified NLR proteins remains poorly characterized. Here we analyzed the function of the primate-specific NLRP11 gene product. We show that NLRP11 is highly expressed in immune cells, including myeloid cells, B cells, and some B cell lymphoma lines. Overexpression of NLRP11 in human cells did not trigger key innate immune signaling pathways, including NF-κB and type I interferon responses. NLRP11 harbors a pyrin domain, which is responsible for inflammasome formation in related NLR proteins. However, NLRP11 did not interact with the inflammasome adaptor protein ASC, and it did not trigger caspase-1 activation. By contrast, expression of NLRP11 specifically repressed NF-κB and type I interferon responses, two key innate immune pathways involved in inflammation. This effect was independent of the pyrin domain and ATPase activity of NLRP11. siRNA-mediated knockdown of NLRP11 in human myeloid THP1 cells validated these findings and revealed enhanced lipopolysaccharide and Sendai virus-induced cytokine and interferon responses, respectively, in cells with reduced NLRP11 expression. In summary, our work identifies a novel role of NLRP11 in the regulation of inflammatory responses in human cells.
Assuntos
Linfócitos B/metabolismo , Regulação para Baixo , Regulação da Expressão Gênica , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células Mieloides/metabolismo , Proteínas NLR/metabolismo , Substituição de Aminoácidos , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter/efeitos dos fármacos , Humanos , Imunidade Inata/efeitos dos fármacos , Interferon Tipo I/agonistas , Interferon Tipo I/antagonistas & inibidores , Interferon Tipo I/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lipopolissacarídeos/toxicidade , Masculino , Mutação , Células Mieloides/citologia , Células Mieloides/efeitos dos fármacos , Células Mieloides/imunologia , NF-kappa B/agonistas , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Proteínas NLR/genética , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Especificidade de Órgãos , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Oncogenic MYD88 mutations, most notably the Leu 265 Pro (L265P) mutation, were recently identified as potential driver mutations in various B-cell non-Hodgkin Lymphomas (NHLs). The L265P mutation is now thought to be common to virtually all NHLs and occurs in between 4 and 90% of cases, depending on the entity. Since it is tumor-specific, the mutation, and the pathways it regulates, might serve as advantageous therapeutic targets for both conventional chemotherapeutic intervention, as well as immunotherapeutic strategies. Here, we review recent progress on elucidating the molecular and cellular processes affected by the L265P mutation of MYD88, describe a new in vivo model for MyD88 L265P-mediated oncogenesis, and summarize how these findings could be exploited therapeutically by specific targeting of signaling pathways. In addition, we summarize current and explore future possibilities for conceivable immunotherapeutic approaches, such as L265P-derived peptide vaccination, adoptive transfer of L265P-restricted T cells, and use of T-cell receptor-engineered T cells. With clinical trials regarding their efficacy rapidly expanding to NHLs, we also discuss potential combinations of immune checkpoint inhibitors with the described targeted chemotherapies of L265P signaling networks, and/or with the above immunological approaches as potential ways of targeting MYD88-mutated lymphomas in the future.
Assuntos
Antineoplásicos/uso terapêutico , Linfoma/tratamento farmacológico , Linfoma/genética , Terapia de Alvo Molecular , Mutação , Fator 88 de Diferenciação Mieloide/genética , HumanosRESUMO
The adaptor protein MYD88 is critical for relaying activation of Toll-like receptor signaling to NF-κB activation. MYD88 mutations, particularly the p.L265P mutation, have been described in numerous distinct B-cell malignancies, including diffuse large B-cell lymphoma (DLBCL). Twenty-nine percent of activated B-cell-type DLBCL (ABC-DLBCL), which is characterized by constitutive activation of the NF-κB pathway, carry the p.L265P mutation. In addition, ABC-DLBCL frequently displays focal copy number gains affecting BCL2 Here, we generated a novel mouse model in which Cre-mediated recombination, specifically in B cells, leads to the conditional expression of Myd88(p.L252P) (the orthologous position of the human MYD88(p.L265P) mutation) from the endogenous locus. These mice develop a lymphoproliferative disease and occasional transformation into clonal lymphomas. The clonal disease displays the morphologic and immunophenotypical characteristics of ABC-DLBCL. Lymphomagenesis can be accelerated by crossing in a further novel allele, which mediates conditional overexpression of BCL2 Cross-validation experiments in human DLBCL samples revealed that both MYD88 and CD79B mutations are substantially enriched in ABC-DLBCL compared with germinal center B-cell DLBCL. Furthermore, analyses of human DLBCL genome sequencing data confirmed that BCL2 amplifications frequently co-occurred with MYD88 mutations, further validating our approach. Finally, in silico experiments revealed that MYD88-mutant ABC-DLBCL cells in particular display an actionable addiction to BCL2. Altogether, we generated a novel autochthonous mouse model of ABC-DLBCL that could be used as a preclinical platform for the development and validation of novel therapeutic approaches for the treatment of ABC-DLBCL.
Assuntos
Linfócitos B/metabolismo , Transformação Celular Neoplásica/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , Mutação de Sentido Incorreto , Fator 88 de Diferenciação Mieloide/biossíntese , Neoplasias Experimentais/metabolismo , Animais , Linfócitos B/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/genética , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genéticaRESUMO
BACKGROUND: The Nod-like receptor NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) and Bruton tyrosine kinase (BTK) are protagonists in innate and adaptive immunity, respectively. NLRP3 senses exogenous and endogenous insults, leading to inflammasome activation, which occurs spontaneously in patients with Muckle-Wells syndrome; BTK mutations cause the genetic immunodeficiency X-linked agammaglobulinemia (XLA). However, to date, few proteins that regulate NLRP3 inflammasome activity in human primary immune cells have been identified, and clinically promising pharmacologic targeting strategies remain elusive. OBJECTIVE: We sought to identify novel regulators of the NLRP3 inflammasome in human cells with a view to exploring interference with inflammasome activity at the level of such regulators. METHODS: After proteome-wide phosphoproteomics, the identified novel regulator BTK was studied in human and murine cells by using pharmacologic and genetic BTK ablation. RESULTS: Here we show that BTK is a critical regulator of NLRP3 inflammasome activation: pharmacologic (using the US Food and Drug Administration-approved inhibitor ibrutinib) and genetic (in patients with XLA and Btk knockout mice) BTK ablation in primary immune cells led to reduced IL-1ß processing and secretion in response to nigericin and the Staphylococcus aureus toxin leukocidin AB (LukAB). BTK affected apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and caspase-1 cleavage and interacted with NLRP3 and ASC. S aureus infection control in vivo and IL-1ß release from cells of patients with Muckle-Wells syndrome were impaired by ibrutinib. Notably, IL-1ß processing and release from immune cells isolated from patients with cancer receiving ibrutinib therapy were reduced. CONCLUSION: Our data suggest that XLA might result in part from genetic inflammasome deficiency and that NLRP3 inflammasome-linked inflammation could potentially be targeted pharmacologically through BTK.
Assuntos
Agamaglobulinemia/genética , Síndromes Periódicas Associadas à Criopirina/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas Tirosina Quinases/metabolismo , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Imunidade Adaptativa , Proteínas Adaptadoras de Transdução de Sinal , Tirosina Quinase da Agamaglobulinemia , Animais , Proteínas Reguladoras de Apoptose , Proteínas de Bactérias/imunologia , Células Cultivadas , Humanos , Imunidade Inata , Leucocidinas/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Terapia de Alvo Molecular , Proteínas NLR , Nigericina/imunologia , Proteínas Tirosina Quinases/genética , Proteômica , Domínio Pirina/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptor de Lamina BRESUMO
OBJECTIVE: As pathogen sensors, Toll-like receptors (TLR) play a role in the first defence line during HCV infection. However, the impact of the DNA sensor TLR9 on the natural course of HCV infection is unknown. To address this, TLR9 promoter polymorphisms (single nucleotide polymorphisms (SNPs)) rs187084 and rs5743836 were investigated for their effect on disease progression. DESIGN: Therefore, the TLR9 SNPs and the interferon lambda 4 (IFNL4) rs12979860 were genotyped in chronically HCV type 1 infected (n=333), in patients who spontaneously cleared the infection (n=161), in the Swiss HCV cohort (n=1057) and the well-characterised German (n=305) and Irish (n=198) 'anti-D' cohorts. Functional analyses were done with promoter reporter constructs of human TLR9 in B cells and assessing TLR9 mRNA levels in whole blood of healthy volunteers. RESULTS: The TLR9 rs187084 C allele was associated with spontaneous virus clearance in women of the study cohort (OR=2.15 (95% CI 1.18 to 3.90) p=0.012), of the Swiss HCV cohort (OR=2.06 (95% CI 1.02 to 4.18) p=0.044) and in both 'anti-D' cohorts (German: OR=2.01 (95% CI 1.14 to 3.55) p=0.016; Irish: OR=1.93 (95% CI 1.10 to 3.68) p=0.047). Multivariate analysis in the combined study and Swiss HCV cohorts supported the results (OR=1.99 (95% CI 1.30 to 3.05) p=0.002). Functional analyses revealed higher transcriptional activities for both TLR9 variants and an association of the C allele of rs5743836 with allele-specific TLR9 mRNA regulation by oestrogens in women. CONCLUSIONS: TLR9 promoter SNPs are associated with the natural course of HCV infection and show higher transcriptional activities. Our results imply the DNA sensor TLR9 in natural immunity against the RNA virus, HCV.
Assuntos
Hepatite C Crônica/genética , Interleucinas/genética , RNA Mensageiro/sangue , Receptor Toll-Like 9/genética , Adulto , Idoso , Alelos , Progressão da Doença , Feminino , Regulação da Expressão Gênica/genética , Alemanha , Haplótipos , Humanos , Irlanda , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Remissão Espontânea , Estudos Retrospectivos , Fatores Sexuais , Suíça , Transcrição GênicaRESUMO
MicroRNAs are important posttranscriptional regulators of gene expression, which have been shown to fine-tune innate immune responses downstream of pattern recognition receptor (PRR) signaling. This study identifies miR-650 as a novel PRR-responsive microRNA that is downregulated upon stimulation of primary human monocyte-derived dendritic cells (MDDCs) with a variety of different microbe-associated molecular patterns. A comprehensive target search combining in silico analysis, transcriptional profiling, and reporter assays reveals that miR-650 regulates several well-known interferon-stimulated genes, including IFIT2 and MXA. In particular, downregulation of miR-650 in influenza A infected MDDCs enhances the expression of MxA and may therefore contribute to the establishment of an antiviral state. Together these findings reveal a novel link between miR-650 and the innate immune response in human MDDCs.
Assuntos
Células Dendríticas/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Vírus da Influenza A/imunologia , MicroRNAs/imunologia , Proteínas de Resistência a Myxovirus/biossíntese , Células Cultivadas , Células Dendríticas/metabolismo , Regulação para Baixo , Citometria de Fluxo , Regulação da Expressão Gênica/genética , Humanos , Imunidade Inata/imunologia , Immunoblotting , MicroRNAs/genética , Microscopia Confocal , Proteínas de Resistência a Myxovirus/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , Receptores de Reconhecimento de Padrão/imunologia , Transdução de Sinais , TransfecçãoRESUMO
Cleavage of interleukin-1ß (IL-1ß) is a key inflammatory event in immune cells and platelets, which is mediated by nucleotide-binding domain leucine rich repeat containing protein (NLRP3)-dependent activation of caspase-1. In immune cells, NLRP3 and caspase-1 form inflammasome complexes with the adaptor proteins apoptosis-associated speck-like protein containing a CARD (ASC) and bruton's tyrosine kinase (BTK). In platelets, however, the regulatory triggers and the functional effects of the NLRP3 inflammasome are unknown. Here, we show in vitro that the platelet NLRP3 inflammasome contributes to platelet activation, aggregation, and thrombus formation. NLRP3 activity, as monitored by caspase-1 activation and cleavage and secretion of IL-1ß, was upregulated in activated platelets, which was dependent on platelet BTK. Pharmacological inhibition or genetic ablation of BTK in platelets led to decreased platelet activation, aggregation, and in vitro thrombus formation. We identify a functionally relevant link between BTK and NLRP3 in platelets, with potential implications in disease states associated with abnormal coagulation and inflammation.
Assuntos
Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Ativação Plaquetária/fisiologia , Proteínas Tirosina Quinases/metabolismo , Trombose/metabolismo , Tirosina Quinase da Agamaglobulinemia , Animais , Benzamidas/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Células Cultivadas , Humanos , Interleucina-1beta/metabolismo , Camundongos Endogâmicos CBA , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Nigericina/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/fisiologia , Proteínas Tirosina Quinases/genéticaRESUMO
Neutrophils, the most abundant human immune cells, are rapidly recruited to sites of infection, where they fulfill their life-saving antimicrobial functions. While traditionally regarded as short-lived phagocytes, recent findings on long-term survival, neutrophil extracellular trap (NET) formation, heterogeneity and plasticity, suppressive functions, and tissue injury have expanded our understanding of their diverse role in infection and inflammation. This review summarises our current understanding of neutrophils in host-pathogen interactions and disease involvement, illustrating the versatility and plasticity of the neutrophil, moving between host defence, immune modulation, and tissue damage.