RESUMO
Cough is an important symptom in children with acute and chronic respiratory disease. Daily cough is common in Cystic Fibrosis (CF) and increased cough is a symptom of pulmonary exacerbation. To date, cough assessment is primarily subjective in clinical practice and research. Attempts to develop objective, automatic cough counting tools have faced reliability issues in noisy environments and practical barriers limiting long-term use. This single-center pilot study evaluated usability, acceptability and performance of a mechanoacoustic sensor (MAS), previously used for cough classification in adults, in 36 children with CF over brief and multi-day periods in four cohorts. Children whose health was at baseline and who had symptoms of pulmonary exacerbation were included. We trained, validated, and deployed custom deep learning algorithms for accurate cough detection and classification from other vocalization or artifacts with an overall area under the receiver-operator characteristic curve (AUROC) of 0.96 and average precision (AP) of 0.93. Child and parent feedback led to a redesign of the MAS towards a smaller, more discreet device acceptable for daily use in children. Additional improvements optimized power efficiency and data management. The MAS's ability to objectively measure cough and other physiologic signals across clinic, hospital, and home settings is demonstrated, particularly aided by an AUROC of 0.97 and AP of 0.96 for motion artifact rejection. Examples of cough frequency and physiologic parameter correlations with participant-reported outcomes and clinical measurements for individual patients are presented. The MAS is a promising tool in objective longitudinal evaluation of cough in children with CF.
RESUMO
Caspase-8 activity controls the switch from cell death to pyroptosis when apoptosis and necroptosis are blocked, yet how caspase-8 inactivation induces inflammasome assembly remains unclear. We show that caspase-8 inhibition via IETD treatment in Toll-like receptor (TLR)-primed Fadd-/-Ripk3-/- myeloid cells promoted interleukin-1ß (IL-1ß) and IL-18 production through inflammasome activation. Caspase-8, caspase-1/11, and functional GSDMD, but not NLRP3 or RIPK1 activity, proved essential for IETD-triggered inflammasome activation. Autophagy became prominent in IETD-treated Fadd-/-Ripk3-/- macrophages, and inhibiting it attenuated IETD-induced cell death and IL-1ß/IL-18 production. In contrast, inhibiting GSDMD or autophagy did not prevent IETD-induced septic shock in Fadd-/-Ripk3-/- mice, implying distinct death processes in other cell types. Cathepsin-B contributes to IETD-mediated inflammasome activation, as its inhibition or down-regulation limited IETD-elicited IL-1ß production. Therefore, the autophagy and cathepsin-B axis represents one of the pathways leading to atypical inflammasome activation when apoptosis and necroptosis are suppressed and capase-8 is inhibited in myeloid cells.
Assuntos
Inflamassomos , Interleucina-18 , Camundongos , Animais , Inflamassomos/metabolismo , Caspase 8/genética , Interleucina-18/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , AutofagiaRESUMO
Promyelocytic leukemia protein (PML) is a tumor suppressor possessing multiple modes of action, including induction of apoptosis. We unexpectedly find that PML promotes necroptosis in addition to apoptosis, with Pml-/- macrophages being more resistant to TNF-mediated necroptosis than wild-type counterparts and PML-deficient mice displaying resistance to TNF-induced systemic inflammatory response syndrome. Reduced necroptosis in PML-deficient cells is associated with attenuated receptor-interacting protein kinase 1 (RIPK1) activation, as revealed by reduced RIPK1[S166] phosphorylation, and attenuated RIPK1-RIPK3-MLKL necrosome complex formation. We show that PML deficiency leads to enhanced TNF-induced MAPK-activated kinase 2 (MK2) activation and elevated RIPK1[S321] phosphorylation, which suppresses necrosome formation. MK2 inhibitor treatment or MK2 knockout abrogates resistance to cell death induction in PML-null cells and mice. PML binds MK2 and p38 MAPK, thereby inhibiting p38-MK2 interaction and MK2 activation. Moreover, PML participates in autocrine production of TNF induced by cellular inhibitors of apoptosis 1 (cIAP1)/cIAP2 degradation, since PML-knockout attenuates autocrine TNF. Thus, by targeting MK2 activation and autocrine TNF, PML promotes necroptosis and apoptosis, representing a novel tumor-suppressive activity for PML.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Serina-Treonina Quinases , Proteína Serina-Treonina Quinases de Interação com Receptores , Transdução de Sinais , Animais , Apoptose , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Necroptose , Fosforilação , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
ABSTRACT: We tested whether aerobic exercise training altered morphine analgesic responses or reduced morphine dosages necessary for adequate analgesia. Patients with chronic back pain were randomized to an 18-session aerobic exercise intervention (n = 38) or usual activity control (n = 45). Before and after the intervention, participants underwent 3 laboratory sessions (double-blinded, crossover) to assess effects of saline placebo, i.v. morphine (0.09 mg/kg), and i.v. naloxone (12 mg) on low back pain and evoked heat pain responses. Differences in evoked and back pain measures between the placebo and morphine conditions indexed morphine analgesia, with pre-post intervention changes the primary outcome. Endogenous opioid analgesia was indexed by differences in evoked and low back pain measures between the naloxone and placebo conditions. A Sex X Intervention interaction on the analgesic effects of morphine on visual analogue scale back pain intensity was observed (P = 0.046), with a similar trend for evoked pain threshold (P = 0.093). Male exercisers showed reduced morphine analgesia pre-post intervention, whereas male controls showed increased analgesia (with no differences in females). Of clinical significance were findings that relative to the control group, aerobic exercise produced analgesia more similar to that observed after receiving ≈7 mg morphine preintervention (P < 0.045). Greater pre-post intervention increases in endogenous opioid function (from any source) were significantly associated with larger pre-post intervention decreases in morphine analgesia (P < 0.046). The overall pattern of findings suggests that regular aerobic exercise has limited direct effects on morphine responsiveness, reducing morphine analgesia in males only.
Assuntos
Analgésicos Opioides , Dor Lombar , Analgésicos Opioides/uso terapêutico , Método Duplo-Cego , Exercício Físico , Feminino , Humanos , Dor Lombar/tratamento farmacológico , Masculino , Morfina/uso terapêutico , Dor Pós-OperatóriaRESUMO
Aerobic exercise is believed to be an effective chronic low back pain (CLBP) intervention, although its mechanisms remain largely untested. This study evaluated whether endogenous opioid (EO) mechanisms contributed to the analgesic effects of an aerobic exercise intervention for CLBP. Individuals with CLBP were randomized to a 6-week, 18-session aerobic exercise intervention (n = 38) or usual activity control (n = 44). Before and after the intervention, participants underwent separate laboratory sessions to assess responses to evoked heat pain after receiving saline placebo or intravenous naloxone (opioid antagonist) in a double-blinded, crossover fashion. Chronic pain intensity and interference were assessed before and after the intervention. Endogenous opioid analgesia was indexed by naloxone-placebo condition differences in evoked pain responses (blockade effects). Relative to controls, exercise participants reported significantly greater pre-post intervention decreases in chronic pain intensity and interference (Ps < 0.04) and larger reductions in placebo condition evoked pain responsiveness (McGill Pain Questionnaire-Short Form [MPQ]-Total). At the group level, EO analgesia (MPQ-Total blockade effects) increased significantly pre-post intervention only among female exercisers (P = 0.03). Dose-response effects were suggested by a significant positive association in the exercise group between exercise intensity (based on meeting heart rate targets) and EO increases (MPQ-Present Pain Intensity; P = 0.04). Enhanced EO analgesia (MPQ-Total) was associated with a significantly greater improvement in average chronic pain intensity (P = 0.009). Aerobic exercise training in the absence of other interventions appears effective for CLBP management. Aerobic exercise-related enhancements in endogenous pain inhibition, in part EO-related, likely contribute to these benefits.
Assuntos
Dor Crônica , Dor Lombar , Analgésicos Opioides/uso terapêutico , Dor Crônica/terapia , Método Duplo-Cego , Exercício Físico , Feminino , Humanos , Dor Lombar/terapia , Peptídeos OpioidesRESUMO
Death-associated protein kinase 1 (DAPK1, DAPk, DAPK) is known for its involvement in apoptosis and autophagy-associated cell death. Here, we identified an unexpected function of DAPK1 in suppressing necroptosis. DAPK1-deficiency renders macrophages and dendritic cells susceptible to necroptotic death. We also observed an inhibitory role for DAPK1 in necroptosis in HT-29 cells, since knockdown or knockout of DAPK1 in such cells increased their sensitivity to necroptosis. Increased necroptosis was associated with enhanced formation of the RIPK1-RIPK3-MLKL complex in these DAPK1-deficient cells. We further found that DAPK1-deficiency led to decreased MAPK activated kinase 2 (MK2) activation and reduced RIPK1 S321 phosphorylation, with this latter representing a critical step controlling necrosome formation. Most TNF signaling pathways, including ERK, JNK, and AKT, were not regulated by DAPK. In contrast, DAPK bound p38 MAPK and selectively promoted p38 MAPK activation, resulting in enhanced MK2 phosphorylation. Our results reveal a novel role for DAPK1 in inhibiting necroptosis and illustrate an unexpected selectivity for DAPK1 in promoting p38 MAPK-MK2 activation. Importantly, our study suggests that modulation of necroptosis and p38/MK2-mediated inflammation may be achieved by targeting DAPK1.
Assuntos
Proteínas Quinases Associadas com Morte Celular/metabolismo , Necroptose , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Caspase 8/metabolismo , Sobrevivência Celular , Proteínas Quinases Associadas com Morte Celular/deficiência , Regulação para Baixo , Ativação Enzimática , Proteína de Domínio de Morte Associada a Fas/metabolismo , Técnicas de Silenciamento de Genes , Células HT29 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/enzimologia , Células Mieloides/patologia , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Choque Séptico/metabolismo , Choque Séptico/patologia , Transdução de Sinais , Fator de Necrose Tumoral alfaRESUMO
The NLRP3 inflammasome is assembled in macrophages and monocytes in response to inflammatory and danger stimuli. The atypical nature of the NLRP3 complex impedes detection of NLRP3 inflammasome formation by conventional biochemical and cell biology methods. In situ proximity ligation assay (PLA) provides an alternative method of detection, localization, and quantification of protein-protein interactions in tissue and cell samples. Two primary antibodies raised in different species detect the two proteins of interest. When the proteins are in close proximity, secondary antibodies conjugated with specific DNA probes hybridize with linking oligonucleotides to form a DNA bridge between the two proteins. Amplification of the DNA bridge then facilitates detection by microscopy using fluorescence probes. Here, we describe application of in situ PLA to detect NLRP3 inflammasome assembly in mouse bone marrow-derived macrophages and human monocyte cell line THP1.
Assuntos
Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/química , Animais , Bioensaio , Células Cultivadas , DNA/metabolismo , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Mapeamento de Interação de Proteínas , Multimerização Proteica , Células THP-1RESUMO
Cellular FLIP (c-FLIP) specifically inhibits caspase-8 and suppresses death receptor-induced apoptosis. c-FLIP has also been reported to transmit activation signals. In this study, we report a novel function of c-FLIP involving inhibition of myeloid cell activation through antagonizing the selective innate signaling pathway. We found that conditional knockout of c-FLIP in dendritic cells (DCs) led to neutrophilia and splenomegaly. Peripheral DC populations, including CD11b(+) conventional DCs (cDCs), CD8(+) cDCs, and plasmacytoid DCs, were not affected by c-FLIP deficiency. We also found that c-FLIP knockout cDCs, plasmacytoid DCs, and bone marrow-derived DCs (BMDCs) displayed enhanced production of TNF-α, IL-2, or G-CSF in response to stimulation of TLR4, TLR2, and dectin-1. Consistent with the ability of c-FLIP to inhibit the activation of p38 MAPK, the enhanced activation of c-FLIP-deficient BMDCs could be partly linked to an elevated activation of p38 MAPK after engagement of innate receptors. Increased activation was also found in c-FLIP(+/-) macrophages. Additionally, the increased activation in c-FLIP-deficient DCs was independent of caspase-8. Our results reveal a novel inhibitory role of c-FLIP in myeloid cell activation and demonstrate the unexpected anti-inflammatory activity of c-FLIP. Additionally, our observations suggest that cancer therapy targeting c-FLIP downregulation may facilitate DC activation and increase T cell immunity.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/imunologia , Caspase 8/imunologia , Células Dendríticas/imunologia , Células Mieloides/imunologia , Animais , Anti-Inflamatórios , Apresentação de Antígeno/imunologia , Apoptose/imunologia , Antígeno B7-1/biossíntese , Antígeno B7-2/biossíntese , Células da Medula Óssea/imunologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Caspase 8/genética , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Fator Estimulador de Colônias de Granulócitos/biossíntese , Antígenos de Histocompatibilidade Classe II/biossíntese , Inflamação/imunologia , Interleucina-2/biossíntese , Lectinas Tipo C/imunologia , Contagem de Leucócitos , Camundongos , Camundongos Knockout , Células Mieloides/citologia , Neutrófilos/citologia , Neutrófilos/imunologia , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais/imunologia , Esplenomegalia/imunologia , Linfócitos T/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The functional activities of the tumor suppressor promyelocytic leukemia protein (PML) are mostly associated with its nuclear location. In the present study, we discovered an unexpected role of PML in NLRP3 inflammasome activation. In PML-deficient macrophages, the production of IL-1ß was strongly impaired. The expression of pro-IL-1ß, NLRP3, ASC, and procaspase-1 was not affected in Pml(-/-) macrophages. PML deficiency selectively reduced the processing of procaspase-1. We further showed that PML is required for the assembly of the NLRP3 inflammasome in reconstitution experiment. All PML isoforms were capable of stimulating NLRP3 inflammasome activation. In Pml(-/-) macrophages, the generation of reactive oxygen species and release of mitochondrial DNA were decreased. The involvement of PML in inflammasome activation constitutes an important activity of PML and reveals a new mechanism underlying the inflammasome activation. In addition, downregulation of PML by arsenic trioxide suppressed monosodium urate (MSU)-induced IL-1ß production, suggesting that targeting to PML could be used to treat NLRP3 inflammasome-associated diseases.
Assuntos
Proteínas de Transporte/imunologia , Inflamassomos/imunologia , Proteínas Nucleares/imunologia , Fatores de Transcrição/imunologia , Proteínas Supressoras de Tumor/imunologia , Animais , Trióxido de Arsênio , Arsenicais/farmacologia , Proteínas de Transporte/genética , Caspase 1/imunologia , Linhagem Celular , Células Cultivadas , DNA Mitocondrial/imunologia , Regulação para Baixo/efeitos dos fármacos , Deleção de Genes , Inibidores do Crescimento/farmacologia , Humanos , Interleucina-1beta/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas Nucleares/genética , Óxidos/farmacologia , Proteína da Leucemia Promielocítica , Espécies Reativas de Oxigênio/imunologia , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Syndecan is the major transmembrane proteoglycan in cells. Of the four syndecans, syndecan-1 is the dominant form expressed in multiple myeloma and is an indicator of poor prognosis. In the current study, we observed that early TRAIL-induced apoptotic processes were accompanied by cleavage of syndecan-1 intracellular region, and explored the possibility whether removal of syndecan-1 promotes apoptotic processes. We found that syndecan-1 knockdown by specific small interfering RNA in multiple myeloma enhanced TRAIL-induced apoptosis, even though the expression of TRAIL receptors and several apoptosis-associated molecules was unaffected. The enhanced TRAIL-mediated apoptosis in syndecan-1-deficient cells was not due to a decrease in surface heparan sulfate or a reduction in TRAIL receptor endocytosis. The increase in TRAIL-induced cell death was accompanied by an elevated caspase-8 activation and an enhanced formation of death-inducing signaling complexes, which could be attributed to an increased expression of TRAIL receptor O-glycosylation enzyme in syndecan-1-deficient cells. We also found that in H9 lymphoma and Jurkat cells, knockdown of the predominant syndecan member also led to an increase in Fas ligand-induced apoptosis. Our results demonstrate that syndecan plays a negative role in death receptor-mediated cell death, suggesting potential application of syndecan downregulation in the treatment of myeloma in combination with TRAIL.
Assuntos
Apoptose/fisiologia , Mieloma Múltiplo/metabolismo , Sindecana-1/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/imunologia , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Imunoprecipitação , Mieloma Múltiplo/imunologia , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Sindecana-1/imunologia , Ligante Indutor de Apoptose Relacionado a TNF/imunologiaRESUMO
The molecular process underlying T cell anergy is incompletely understood. Deltex1 (DTX1) is a Notch target with unknown physiological function. Here we show that Dtx1 was a transcription target of nuclear factor of activated T cells (NFAT) and participated in T cell anergy. DTX1 protein was upregulated during T cell anergy, and transgenic expression of Dtx1 attenuated T cell activation. DTX1 inhibited T cell activation by both E3-dependent and E3-independent mechanisms. In addition, DTX1 suppressed T cell activation in the absence of its Notch-binding domain. Importantly, DTX1 regulated the expression of two anergy-associated molecules, growth arrest and DNA-damage-inducible 45 beta (Gadd45 beta) and Cbl-b. DTX1 interacted with early growth response 2 (Egr-2) for optimum expression of Cbl-b. Furthermore, deficiency of DTX1 augmented T cell activation, conferred resistance to anergy induction, enhanced autoantibody generation, and increased inflammation. DTX1 therefore represents a component downstream of calcium-NFAT signaling that regulates T cell anergy.