RESUMO
Aging causes chronic low-grade inflammation known as inflamm-aging. It is a risk factor for several chronic disorders, including chronic myelomonocytic leukemia (CMML), a hematological malignancy that is most prevalent in older people. Recent studies suggest a critical role for the NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome in inflamm-aging. However, the mechanisms involved in NLRP3 activation in aging and its involvement in CMML progression are not fully understood. In this study, we report that aging increases IL-1ß production upon NLRP3 activation in human CD14+ monocytes. Interestingly, we found that the TLR1/2 agonist Pam3CSK4 directly activates the NLRP3 inflammasome in monocytes from older but not from younger healthy donors. Furthermore, we observed a dichotomous response to NLRP3 inflammasome activation in monocytes from a small cohort of CMML patients, and some patients produced high levels of IL-1ß and some patients produced low levels of IL-1ß compared with older healthy donors. Intriguingly, CMML patients with heightened NLRP3 activation showed increased treatment dependency and disease severity. Collectively, our results suggest that aging causes increased sensitivity to NLRP3 inflammasome activation at a cellular level, which may explain increased inflammation and immune dysregulation in older individuals. Furthermore, NLRP3 inflammasome activation was dysregulated in a small cohort of CMML patients and was positively correlated with disease severity.
Assuntos
Inflamassomos , Leucemia Mielomonocítica Crônica , Humanos , Idoso , Proteína 3 que Contém Domínio de Pirina da Família NLR , Envelhecimento , Inflamação , Gravidade do PacienteRESUMO
BACKGROUND: Despite the introduction of targeted therapies, most patients with myeloid malignancies will not be cured and progress. Genomics is useful to elucidate the mutational landscape but remains limited in the prediction of therapeutic outcome and identification of targets for resistance. Dysregulation of phosphorylation-based signaling pathways is a hallmark of cancer, and therefore, kinase-inhibitors are playing an increasingly important role as targeted treatments. Untargeted phosphoproteomics analysis pipelines have been published but show limitations in inferring kinase-activities and identifying potential biomarkers of response and resistance. METHODS: We developed a phosphoproteomics workflow based on titanium dioxide phosphopeptide enrichment with subsequent analysis by liquid chromatography tandem mass spectrometry (LC-MS). We applied a novel Kinase-Activity Enrichment Analysis (KAEA) pipeline on differential phosphoproteomics profiles, which is based on the recently published SetRank enrichment algorithm with reduced false positive rates. Kinase activities were inferred by this algorithm using an extensive reference database comprising five experimentally validated kinase-substrate meta-databases complemented with the NetworKIN in-silico prediction tool. For the proof of concept, we used human myeloid cell lines (K562, NB4, THP1, OCI-AML3, MOLM13 and MV4-11) with known oncogenic drivers and exposed them to clinically established kinase-inhibitors. RESULTS: Biologically meaningful over- and under-active kinases were identified by KAEA in the unperturbed human myeloid cell lines (K562, NB4, THP1, OCI-AML3 and MOLM13). To increase the inhibition signal of the driving oncogenic kinases, we exposed the K562 (BCR-ABL1) and MOLM13/MV4-11 (FLT3-ITD) cell lines to either Nilotinib or Midostaurin kinase inhibitors, respectively. We observed correct detection of expected direct (ABL, KIT, SRC) and indirect (MAPK) targets of Nilotinib in K562 as well as indirect (PRKC, MAPK, AKT, RPS6K) targets of Midostaurin in MOLM13/MV4-11, respectively. Moreover, our pipeline was able to characterize unexplored kinase-activities within the corresponding signaling networks. CONCLUSIONS: We developed and validated a novel KAEA pipeline for the analysis of differential phosphoproteomics MS profiling data. We provide translational researchers with an improved instrument to characterize the biological behavior of kinases in response or resistance to targeted treatment. Further investigations are warranted to determine the utility of KAEA to characterize mechanisms of disease progression and treatment failure using primary patient samples.
Assuntos
Leucemia Mieloide Aguda/tratamento farmacológico , Células Mieloides/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteômica/métodos , Linhagem Celular Tumoral , Humanos , Mutação , FosforilaçãoRESUMO
Interleukin-1ß (IL-1ß) is a potent inflammatory cytokine that is usually cleaved and activated by inflammasome-associated caspase-1. To determine whether IL-1ß activation is regulated by inhibitor of apoptosis (IAP) proteins, we treated macrophages with an IAP-antagonist "Smac mimetic" compound or genetically deleted the genes that encode the three IAP family members cIAP1, cIAP2, and XIAP. After Toll-like receptor priming, IAP inhibition triggered cleavage of IL-1ß that was mediated not only by the NLRP3-caspase-1 inflammasome, but also by caspase-8 in a caspase-1-independent manner. In the absence of IAPs, rapid and full generation of active IL-1ß by the NLRP3-caspase-1 inflammasome, or by caspase-8, required the kinase RIP3 and reactive oxygen species production. These results demonstrate that activation of the cell death-inducing ripoptosome platform and RIP3 can generate bioactive IL-1ß and implicate them as additional targets for the treatment of pathological IL-1-driven inflammatory responses.
Assuntos
Proteínas Inibidoras de Apoptose/metabolismo , Interleucina-1beta/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Apoptose , Proteínas Reguladoras de Apoptose , Proteína 3 com Repetições IAP de Baculovírus , Proteínas de Transporte/agonistas , Proteínas de Transporte/metabolismo , Caspase 1/metabolismo , Inflamassomos/imunologia , Inflamassomos/metabolismo , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose/deficiência , Proteínas Inibidoras de Apoptose/genética , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/agonistas , Mimetismo Molecular , Proteína 3 que Contém Domínio de Pirina da Família NLR , Espécies Reativas de Oxigênio/metabolismo , Ubiquitina-Proteína Ligases , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/deficiência , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
Protein synthesis is a highly complex process executed by well-organized translation machinery. Ribosomes, tRNAs and mRNAs are the principal components of this machinery whereas RNA binding proteins and ribosome interacting partners act as accessory factors. Angiogenin (ANG)-Ribonuclease inhibitor (RNH1) system is one such accessory part of the translation machinery that came into focus afresh due to its unconventional role in the translation. ANG is conventionally known for its ability to induce blood vessel formation and RNH1 as a "sentry" to protect RNAs from extracellular RNases. However, recent studies suggest them to be important in translation regulation. During cell homeostasis, ANG in the nucleus promotes rRNA transcription. While under stress, ANG translocates to the cytosol and cleaves tRNA into fragments which inhibit ribosome biogenesis and protein synthesis. RNH1, which intimately interacts with ANG to inhibit its ribonucleolytic activity, can also bind to the 40S ribosomes and control translation by yet to be known mechanisms. Here, we review recent advancement in the knowledge of translation regulation by the ANG-RNH1 system. We also gather information about this system in cell homeostasis as well as in pathological conditions such as cancer and ribosomopathies. Additionally, we discuss the future research directions and therapeutic potential of this system.
Assuntos
Proteínas de Transporte/metabolismo , Transporte Proteico , RNA de Transferência/genética , Ribonuclease Pancreático/metabolismo , Animais , Núcleo Celular/genética , Humanos , Biossíntese de Proteínas , RNA Ribossômico/genética , Transcrição GênicaRESUMO
A current paradigm proposes that mitochondrial damage is a critical determinant of NLRP3 inflammasome activation. Here, we genetically assess whether mitochondrial signalling represents a unified mechanism to explain how NLRP3 is activated by divergent stimuli. Neither co-deletion of the essential executioners of mitochondrial apoptosis BAK and BAX, nor removal of the mitochondrial permeability transition pore component cyclophilin D, nor loss of the mitophagy regulator Parkin, nor deficiency in MAVS affects NLRP3 inflammasome function. In contrast, caspase-8, a caspase essential for death-receptor-mediated apoptosis, is required for efficient Toll-like-receptor-induced inflammasome priming and cytokine production. Collectively, these results demonstrate that mitochondrial apoptosis is not required for NLRP3 activation, and highlight an important non-apoptotic role for caspase-8 in regulating inflammasome activation and pro-inflammatory cytokine levels.
Assuntos
Proteínas de Transporte/biossíntese , Caspase 8/biossíntese , Inflamassomos/metabolismo , Mitocôndrias/metabolismo , Apoptose/genética , Autofagia/genética , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Proteínas de Transporte/genética , Caspase 8/genética , Células Cultivadas , Peptidil-Prolil Isomerase F , Ciclofilinas/antagonistas & inibidores , Ciclofilinas/genética , Humanos , Interleucina-1beta/biossíntese , Mitocôndrias/patologia , Mitofagia/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR , Receptores Toll-Like/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Sterile cell death mediated inflammation is linked to several pathological disorders and involves danger recognition of intracellular molecules released by necrotic cells that activate different groups of innate pattern recognition receptors. Toll-like receptors directly interact with their extrinsic or intrinsic agonists and induce multiple proinflammatory mediators. In contrast, the NLRP3 inflammasome is rather thought to represent a downstream element integrating various indirect stimuli into proteolytic cleavage of interleukin (IL)-1ß and IL-18. Here, we report that histones released from necrotic cells induce IL-1ß secretion in an NLRP3-ASC-caspase-1-dependent manner. Genetic deletion of NLRP3 in mice significantly attenuated histone-induced IL-1ß production and neutrophil recruitment. Furthermore, necrotic cells induced neutrophil recruitment, which was significantly reduced by histone-neutralizing antibodies or depleting extracellular histones via enzymatic degradation. These results identify cytosolic uptake of necrotic cell-derived histones as a triggering mechanism of sterile inflammation, which involves NLRP3 inflammasome activation and IL-1ß secretion via oxidative stress.
Assuntos
Proteínas de Transporte/imunologia , Histonas/imunologia , Inflamassomos/imunologia , Neutrófilos/imunologia , Estresse Oxidativo/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Proteínas Reguladoras de Apoptose , Proteínas Adaptadoras de Sinalização CARD , Proteínas de Transporte/genética , Caspase 1/genética , Caspase 1/imunologia , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/imunologia , Deleção de Genes , Histonas/antagonistas & inibidores , Inflamassomos/genética , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interleucina-18/genética , Interleucina-18/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Necrose/genética , Necrose/imunologia , Necrose/patologia , Neutrófilos/patologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Proteólise/efeitos dos fármacosRESUMO
Translation of mRNAs is a fundamental process that occurs in all cell types of multicellular organisms. Conventionally, it has been considered a default step in gene expression, lacking specific regulation. However, recent studies have documented that certain mRNAs exhibit cell type-specific translation. Despite this, it remains unclear whether global translation is controlled in a cell type-specific manner. By using human cell lines and mouse models, we found that deletion of the ribosome-associated protein ribonuclease inhibitor 1 (RNH1) decreases global translation selectively in hematopoietic-origin cells but not in the non-hematopoietic-origin cells. RNH1-mediated cell type-specific translation is mechanistically linked to angiogenin-induced ribosomal biogenesis. Collectively, this study unravels the existence of cell type-specific global translation regulators and highlights the complex translation regulation in vertebrates.
Assuntos
Biossíntese de Proteínas , Ribonuclease Pancreático , Ribossomos , Ribonuclease Pancreático/metabolismo , Ribonuclease Pancreático/genética , Humanos , Animais , Camundongos , Ribossomos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação da Expressão Gênica , Linhagem Celular , Especificidade de Órgãos , Proteínas de TransporteRESUMO
Renal dendritic cells (DCs) form an interstitial network contributing to inflammatory and adaptive immune responses in the kidney. The presence and functional role of DC-like glomerular CD11c(+) mononuclear phagocytes is a matter of debate. Using compartment-specific flow cytometry we found that healthy mouse kidneys contained 1.3 CD11c(+) cells per 100 glomeruli and these increased by 4.6-fold and 13-fold after TNF stimulation and immune complex deposition, respectively. Compartment-specific mRNA expression revealed a predominantly glomerular expression of TNF receptors, chemokines, and adhesion molecules; all upregulated after TNF exposure. Intraperitoneal TNF injection induced influx of neutrophils and mononuclear phagocytes including DC-like CD11c(+) cells into both the glomerular and tubulointerstitial compartments, but reduced in TNF receptor (Tnfr) 1-deficient mice. Additionally, Tnfr2 deficiency impaired glomerular infiltration of CD11c(+) cells, but not neutrophils. Interstitial CD11c(+) cells infiltrated in the presence of Tnfr1 or Tnfr2. TNF exposure also induced similar maturation of glomerular and interstitial CD11c(+) cells as demonstrated by increased surface expression of MHC II, CD54, and costimulatory molecules CD40, CD80, and CD86. Thus, by compartment-specific flow cytometry we could demonstrate the constitutive presence of DC-like CD11c(+) mononuclear phagocytes in normal mouse glomeruli and their TNF-induced accumulation and activation.
Assuntos
Separação Celular/métodos , Quimiotaxia , Células Dendríticas/imunologia , Citometria de Fluxo , Mediadores da Inflamação/metabolismo , Glomérulos Renais/imunologia , Nefrite Intersticial/imunologia , Fagócitos/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Biomarcadores/metabolismo , Antígeno CD11c/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Quimiocinas/genética , Quimiocinas/metabolismo , Modelos Animais de Doenças , Feminino , Glomérulos Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nefrite Intersticial/genética , Nefrite Intersticial/patologia , Fenótipo , RNA Mensageiro/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/deficiência , Receptores Tipo II do Fator de Necrose Tumoral/genética , Transdução de Sinais , Obstrução Ureteral/complicaçõesRESUMO
Clinical use of antibiotics is based on their capacity to inhibit bacterial growth via bacteriostatic or bacteriocidal effects. In this article, we show that the aminoglycoside antibiotic neomycin, the cyclic lipopeptide antibiotic polymyxin B, and the cyclic peptide antibiotics gramicidin and tyrothricin can induce IL-1ß secretion in bone marrow dendritic cells and macrophages. LPS priming was required to trigger the transcription and translation of pro-IL-1ß but was independent of TNFR or IL-1R signaling. All four antibiotics required the NLRP3 inflammasome, the adaptor ASC, and caspase-1 activation to secrete IL-1ß, a process that depended on potassium efflux but was independent of P2X7 receptor. All four antibiotics induced neutrophil influx into the peritoneal cavity of mice, which required NLRP3 only in the case of polymyxin B. Together, certain antibiotics have the potential to directly activate innate immunity of the host.
Assuntos
Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Proteínas de Transporte/metabolismo , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Peptídeos Cíclicos/farmacologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Proteínas de Transporte/genética , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Humanos , Inflamassomos/genética , Inflamassomos/fisiologia , Interleucina-1beta/biossíntese , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/imunologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Peritonite/imunologia , Peritonite/metabolismo , Peritonite/patologia , Precursores de Proteínas/biossíntese , Precursores de Proteínas/metabolismo , Receptores de Interleucina-1/deficiência , Receptores de Interleucina-1/genéticaRESUMO
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease leading to inflammatory tissue damage in multiple organs (e.g., lupus nephritis). Current treatments including steroids, antimalarials, and immunosuppressive drugs have significant side effects. Activated protein C is a natural protein with anticoagulant and immunomodulatory effects, and its recombinant version has been approved by the U.S. Food and Drug Administration to treat severe sepsis. Given the similarities between overshooting immune activation in sepsis and autoimmunity, we hypothesized that recombinant activated protein C would also suppress SLE and lupus nephritis. To test this concept, autoimmune female MRL-Fas(lpr) mice were injected with either vehicle or recombinant human activated protein C from week 14-18 of age. Activated protein C treatment significantly suppressed lupus nephritis as evidenced by decrease in activity index, glomerular IgG and complement C3 deposits, macrophage counts, as well as intrarenal IL-12 expression. Further, activated protein C attenuated cutaneous lupus and lung disease as compared with vehicle-treated MRL-Fas(lpr) mice. In addition, parameters of systemic autoimmunity, such as plasma cytokine levels of IL-12p40, IL-6, and CCL2/MCP-1, and numbers of B cells and plasma cells in spleen were suppressed by activated protein C. The latter was associated with lower total plasma IgM and IgG levels as well as lower titers of anti-dsDNA IgG and rheumatoid factor. Together, recombinant activated protein C suppresses the abnormal systemic immune activation in SLE of MRL-Fas(lpr) mice, which prevents subsequent kidney, lung, and skin disease. These results implicate that recombinant activated protein C might be useful for the treatment of human SLE.
Assuntos
Anti-Inflamatórios/uso terapêutico , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Proteína C/uso terapêutico , Animais , Autoanticorpos/sangue , Separação Celular , Citocinas/análise , Citocinas/sangue , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Imunoglobulina G/sangue , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Nefrite Lúpica/tratamento farmacológico , Nefrite Lúpica/imunologia , Nefrite Lúpica/patologia , Camundongos , Camundongos Endogâmicos MRL lpr , Proteínas Recombinantes/uso terapêutico , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In AKI, dying renal cells release intracellular molecules that stimulate immune cells to secrete proinflammatory cytokines, which trigger leukocyte recruitment and renal inflammation. Whether the release of histones, specifically, from dying cells contributes to the inflammation of AKI is unknown. In this study, we found that dying tubular epithelial cells released histones into the extracellular space, which directly interacted with Toll-like receptor (TLR)-2 (TLR2) and TLR4 to induce MyD88, NF-κB, and mitogen activated protein kinase signaling. Extracellular histones also had directly toxic effects on renal endothelial cells and tubular epithelial cells in vitro. In addition, direct injection of histones into the renal arteries of mice demonstrated that histones induce leukocyte recruitment, microvascular vascular leakage, renal inflammation, and structural features of AKI in a TLR2/TLR4-dependent manner. Antihistone IgG, which neutralizes the immunostimulatory effects of histones, suppressed intrarenal inflammation, neutrophil infiltration, and tubular cell necrosis and improved excretory renal function. In summary, the release of histones from dying cells aggravates AKI via both its direct toxicity to renal cells and its proinflammatory effects. Because the induction of proinflammatory cytokines in dendritic cells requires TLR2 and TLR4, these results support the concept that renal damage triggers an innate immune response, which contributes to the pathogenesis of AKI.
Assuntos
Injúria Renal Aguda/metabolismo , Histonas/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Injúria Renal Aguda/imunologia , Animais , Permeabilidade Capilar , Citocinas/metabolismo , Células Endoteliais/fisiologia , Células Epiteliais/metabolismo , Injeções Intra-Arteriais , Rim/patologia , Túbulos Renais/metabolismo , Leucócitos/fisiologia , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Necrose , Artéria Renal , Traumatismo por Reperfusão/prevenção & controleRESUMO
Systemic lupus erythematosus (SLE) is a polyclonal autoimmune syndrome directed against multiple nuclear autoantigens. Although RNA and DNA seem to have identical immunostimulatory effects on systemic and intrarenal inflammation, each seems to differ with regard to the propensity to induce mitogenic effects such as lymphoproliferation. To identify potential mechanisms by which DNA specifically contributes to the pathogenesis of lupus nephritis, we stimulated cells with immunostimulatory DNA or RNA in vitro and used microarray to compare the transcriptomes of RNA- and DNA-induced genes. Immunostimulatory DNA, but not RNA, induced Mdm2, which is a negative regulator of p53. In vivo, we observed greater expression and activation of Mdm2 in the spleen and kidneys in a mouse model of lupus (MRL-Fas(lpr) mice) than healthy controls. Treatment of MRL-Fas(lpr) mice with the Mdm2 inhibitor nutlin-3a prevented nephritis and lung disease and significantly prolonged survival. Inhibition of Mdm2 reduced systemic inflammation and abrogated immune complex disease by suppressing plasma cells and the production of lupus autoantibodies. In addition, nutlin-3a suppressed the abnormal expansion of all T cell subsets, including CD3(+)CD4(-)CD8(-) T cells, which associated with attenuated systemic inflammation. However, inhibiting Mdm2 did not cause myelosuppression or affect splenic regulatory T cells, neutrophils, dendritic cells, or monocytes. Taken together, these data suggest that the induction of Mdm2 promotes the expansion of plasma cells and CD3(+)CD4(-)CD8(-) T cells, which cause autoantibody production and immune complex disease in MRL-Fas(lpr) mice. Antagonizing Mdm2 may have therapeutic potential in lupus nephritis.
Assuntos
Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Nefrite Lúpica/imunologia , Nefrite Lúpica/patologia , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/imunologia , Animais , Autoanticorpos/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citosol/fisiologia , DNA/imunologia , Feminino , Expressão Gênica/imunologia , Mesângio Glomerular/imunologia , Mesângio Glomerular/patologia , Imidazóis/farmacologia , Lúpus Eritematoso Sistêmico/genética , Nefrite Lúpica/genética , Camundongos , Camundongos Endogâmicos MRL lpr , Células NIH 3T3 , Necrose , Piperazinas/farmacologia , Plasmócitos/citologia , Plasmócitos/imunologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Baço/imunologiaRESUMO
Inflammasomes are cytosolic innate immune sensors of pathogen infection and cellular damage that induce caspase-1-mediated inflammation upon activation. Although inflammation is protective, uncontrolled excessive inflammation can cause inflammatory diseases and can be detrimental, such as in coronavirus disease (COVID-19). However, the underlying mechanisms that control inflammasome activation are incompletely understood. Here we report that the leucine-rich repeat (LRR) protein ribonuclease inhibitor (RNH1), which shares homology with LRRs of NLRP (nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain containing) proteins, attenuates inflammasome activation. Deletion of RNH1 in macrophages increases interleukin (IL)-1ß production and caspase-1 activation in response to inflammasome stimulation. Mechanistically, RNH1 decreases pro-IL-1ß expression and induces proteasome-mediated caspase-1 degradation. Corroborating this, mouse models of monosodium urate (MSU)-induced peritonitis and lipopolysaccharide (LPS)-induced endotoxemia, which are dependent on caspase-1, respectively, show increased neutrophil infiltration and lethality in Rnh1 -/- mice compared with wild-type mice. Furthermore, RNH1 protein levels were negatively related with disease severity and inflammation in hospitalized COVID-19 patients. We propose that RNH1 is a new inflammasome regulator with relevance to COVID-19 severity.
Assuntos
COVID-19/patologia , Proteínas de Transporte/metabolismo , Inflamassomos/metabolismo , Proteínas de Repetições Ricas em Leucina/metabolismo , Animais , COVID-19/imunologia , Caspase 1/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Gravidade do Paciente , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
Ischemia-reperfusion (IR) triggers tissue injury by activating innate immunity, for example, via TLR2 and TLR4. Surprisingly, TLR signaling in intrinsic renal cells predominates in comparison to intrarenal myeloid cells in the postischemic kidney. We hypothesized that immune cell activation is specifically suppressed in the postischemic kidney, for example, by single Ig IL-1-related receptor (SIGIRR). SIGIRR deficiency aggravated postischemic acute renal failure in association with increased renal CXCL2/MIP2, CCL2/MCP-1, and IL-6 mRNA expression 24 h after IR. Consistent with this finding interstitial neutrophil and macrophage counts were increased and tubular cell necrosis was aggravated in Sigirr-deficient vs wild-type IR kidneys. In vivo microscopy revealed increased leukocyte transmigration in the postischemic microvasculature of Sigirr-deficient mice. IL-6 and CXCL2/MIP2 release was much higher in Sigirr-deficient renal myeloid cells but not in Sigirr-deficient tubular epithelial cells after transient hypoxic culture conditions. Renal IR studies with chimeric mice confirmed this finding, as lack of SIGIRR in myeloid cells largely reproduced the phenotype of renal IR injury seen in Sigirr(-/-) mice. Additionally, clodronate depletion of dendritic cells prevented the aggravated renal failure in Sigirr(-/-) mice. Thus, loss of function mutations in the SIGIRR gene predispose to acute renal failure because SIGIRR prevents overshooting tissue injury by suppressing the postischemic activation of intrarenal myeloid cells.
Assuntos
Injúria Renal Aguda/etiologia , Células Dendríticas/fisiologia , Receptores de Interleucina-1/fisiologia , Traumatismo por Reperfusão/complicações , Animais , Contagem de Células , Quimiotaxia de Leucócito , Células Epiteliais , Túbulos Renais/patologia , Macrófagos/fisiologia , Camundongos , Camundongos Knockout , Microcirculação , Células Mieloides , Neutrófilos/fisiologia , Receptores de Interleucina-1/deficiência , Receptores de Interleucina-1/genética , Traumatismo por Reperfusão/imunologiaRESUMO
Myeloid malignancies including myelodysplastic syndromes, myeloproliferative neoplasms and acute myeloid leukemia are heterogeneous disorders originating from mutated hematopoietic stem and progenitor cells (HSPCs). Genetically, they are very heterogeneous and characterized by uncontrolled proliferation and/or blockage of differentiation of abnormal HSPCs. Recent studies suggest the involvement of inflammasome activation in disease initiation and clonal progression. Inflammasomes are cytosolic innate immune sensors that, upon activation, induce caspase-1 mediated processing of interleukin (IL) -1-cytokine members IL-1ß and IL-18, as well as initiation of gasdermin D-dependent pyroptosis. Inflammasome activation leads to a pro-inflammatory microenvironment in the bone marrow, which drives proliferation and may induce clonal selection of mutated HSPCs. However, there are also contradictory data showing that inflammasome activation actually counteracts leukemogenesis. Overall, the beneficial or detrimental effect of inflammasome activation seems to be highly dependent on mutational, environmental, and immunological contexts and an improved understanding is fundamental to advance specific therapeutic targeting strategies. This review summarizes current knowledge about this dichotomous effect of inflammasome activation in myeloid malignancies and provides further perspectives on therapeutic targeting.
RESUMO
BACKGROUND: The inflammasome plays an essential role in lower risk MDS and immune subversion, with the up-regulation of immune checkpoint molecules in the progression to higher-risk disease. In this study, we explored the utility of immune-related biomarkers for the diagnosis and prognosis of MDS. METHODS: We performed an exploratory, case-control study with 20 randomly selected MDS patients and nine controls with non-inflammatory (n = 3) and inflammatory conditions (n = 6). Patients were stratified in groups of lower (n = 10) and higher risk (n = 10) using IPSS-R. For the exploration of inflammasome and immune checkpoint activities, the expression of caspase-1 (Casp1), programmed cell death protein 1 (PD-1) and its ligand (PD-L1) were assessed in bone marrow samples using immunohistochemistry. RESULTS: In multivariate analysis, we observed significant differences for Casp1 but not PD1/PD-L1 expression in our four conditions (p = 0.003). We found a discordant co-expression of Casp1/PD-L1 in MDS (rho = -0.41, p = 0.07) compared with a concordant co-expression in controls (rho = 0.64, p = 0.06). Neutrophil counts correlated directly with Casp1 (rho = 0.57, p = 0.009) but inversely with PD-L1 expression (rho = -0.58, p = 0.007). CONCLUSION: We identified characteristic discordant co-expression patterns in lower- (Casp1high/PD-L1low) and higher-risk MDS (Casp1low/PD-L1high), contrasting with concordant patterns in the non-inflammatory (Casp1low/PD-L1low) and inflammatory conditions (Casp1high/PD-L1high). Further validation is warranted in larger, prospective studies.
RESUMO
Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal disorders caused by sequential accumulation of somatic driver mutations in hematopoietic stem and progenitor cells (HSPCs). MDS is characterized by ineffective hematopoiesis with cytopenia, dysplasia, inflammation, and a variable risk of transformation into secondary acute myeloid leukemia. The advent of next-generation sequencing has revolutionized our understanding of the genetic basis of the disease. Nevertheless, the biology of clonal evolution remains poorly understood, and the stochastic genetic drift with sequential accumulation of genetic hits in HSPCs is individual, highly dynamic and hardly predictable. These continuously moving genetic targets pose substantial challenges for the implementation of precision medicine, which aims to maximize efficacy with minimal toxicity of treatments. In the current postgenomic era, allogeneic hematopoietic stem cell transplantation remains the only curative option for younger and fit MDS patients. For all unfit patients, regeneration of HSPCs stays out of reach and all available therapies remain palliative, which will eventually lead to refractoriness and progression. In this review, we summarize the recent advances in our understanding of MDS pathophysiology and its impact on diagnosis, risk-assessment and disease monitoring. Moreover, we present ongoing clinical trials with targeting compounds and highlight future perspectives for precision medicine.
RESUMO
Type I interferons, interferon-alpha and interferon-beta, are central regulators of antiviral immunity and autoimmunity, but little is known about their role in renal inflammation. Recent work documents that viral nucleic acids are potent inducers of interferon-alpha and interferon-beta in mesangial cells and glomerular endothelial cells. This review discusses the available evidence on the role of interferon-alpha and interferon-beta in viral nephropathies, in kidney diseases triggered by extrarenal infections, in lupus nephritis, and in other kidney disease entities. Finally, we propose areas of research that may help unravel the roles of type I interferons and interferon-related genes in the renal field.
Assuntos
Interferons/imunologia , Animais , Antivirais/imunologia , Autoimunidade/imunologia , Infecções/imunologia , Inflamação/imunologia , Interferon Tipo I/imunologia , Interferon-alfa/genética , Interferon-alfa/imunologia , Interferon beta/imunologia , Rim/imunologia , Rim/metabolismo , Nefrite Lúpica/imunologia , Células Mesangiais/imunologia , Células Mesangiais/metabolismo , Camundongos , Proteínas RecombinantesRESUMO
Viral DNA induces potent antiviral immunity by activating dendritic cells; however, the mechanism governing viral DNA-mediated triggering or aggravation of glomerulonephritis is unknown. Glomerular endothelial cells (GEnCs) do not express toll-like receptor (TLR)9, the only DNA-specific TLR. We therefore hypothesized that DNA could activate GEnCs via the recently discovered TLR-independent viral DNA recognition pathway. Indeed, double-stranded non-CpG (B-) DNA activated GEnCs to produce interleukin-6, CCL5/RANTES, CCL2/MCP-1, CXCL10/IP10, interferon (IFN)-alpha, and IFN-beta when cationic lipids facilitated intracellular DNA uptake. This cytokine production was inhibited by chlorpromazine, suggesting that clathrin-dependent endocytosis is required for B-DNA entry. However, chloroquine and MyD88 inhibition did not affect GEnC activation, suggesting TLR-independent DNA recognition. In addition, IFN-beta activated cytokine and chemokine mRNA expression, although only CXCL10/IP10 was induced at the protein level, and type I IFN did not activate GEnC in an autocrine-paracrine auto-activation loop. B-DNA complexes induced intercellular adhesion molecule-1 expression at the GEnC surface and increased intercellular adhesion molecule-1-dependent leukocyte adhesion and microvascular extravasation in vivo. Furthermore, B-DNA complexes increased albumin permeability of GEnC monolayers in culture or microvascular dextran leakage in vivo. In addition, B-DNA complexes impaired GEnC proliferation. Thus, complexed B-DNA activates GEnC to produce cytokines, chemokines, and type I IFNs, increases leukocyte adhesion and microvascular permeability, and reduces GEnC proliferation via a MyD88-independent cytosolic DNA recognition pathway. This innate antiviral response program suggests a novel pathomechanism regulating DNA virus-mediated induction or aggravation of glomerulonephritis.
Assuntos
Permeabilidade Capilar/fisiologia , DNA/imunologia , Células Endoteliais/fisiologia , Glomérulos Renais , Albumina Sérica/metabolismo , Transdução de Sinais/fisiologia , Receptores Toll-Like/imunologia , Animais , Adesão Celular , Comunicação Celular/fisiologia , Quimiocinas/imunologia , Citocinas/imunologia , DNA Viral/imunologia , Endocitose/fisiologia , Endossomos/metabolismo , Células Endoteliais/citologia , Células Endoteliais/imunologia , Molécula 1 de Adesão Intercelular/metabolismo , Interferon Tipo I/imunologia , Glomérulos Renais/citologia , Glomérulos Renais/metabolismo , Leucócitos/citologia , Leucócitos/metabolismo , Camundongos , Fator 88 de Diferenciação Mieloide/metabolismo , Interferência de RNA , Receptores Toll-Like/genéticaRESUMO
Viral RNA can trigger interferon signaling in dendritic cells via the innate recognition receptors melanoma-differentiation-associated gene (MDA)-5 and retinod-inducible gene (RIG)-I in the cytosol or via Toll-like receptors (TLRs) in intracellular endosomes. We hypothesized that viral RNA would also activate glomerular mesangial cells to produce type I interferon (IFN) via TLR-dependent and TLR-independent pathways. To test this hypothesis, we examined Toll/Interleukin-1 receptor domain-containing adaptor-inducing interferon-beta (TRIF)-deficient mice, which lack a key adaptor for TLR3 signaling. In primary mesangial cells, poly I:C RNA-mediated IFN-beta induction was partially TRIF dependent; however, when poly I:C RNA was complexed with cationic lipids to enhance cytosolic uptake, mesangial cells produced large amounts of IFN-alpha and IFN-beta independent of TRIF. Mesangial cells expressed RIG-I and MDA-5 and their mitochondrial adaptor IFN-beta promoter stimulator-1 as well, and small interfering RNA studies revealed that MDA5 but not RIG-I was required for cytosolic poly I:C RNA signaling. In addition, mesangial cells produced Il-6 on stimulation with IFN-alpha and IFN-beta, suggesting an autocrine proinflammatory effect. Indeed, blockade of IFN-alphabeta or lack of the IFNA receptor reduced viral RNA-induced Il-6 production and apoptotic cell death in mesangial cells. Furthermore, viral RNA/cationic lipid complexes increased focal necrosis in murine nephrotoxic serum nephritis in association with increased renal mRNA expression of IFN-related genes. Thus, TLR-independent recognition of viral RNA is a potent inducer of type I interferon in mesangial cells, which can be an important mediator of virally induced glomerulonephritis.