RESUMEN
The differentiation and specificity of human CD4+ T follicular helper cells (TFH cells) after influenza vaccination have been poorly defined. Here we profiled blood and draining lymph node (LN) samples from human volunteers for over 2 years after two influenza vaccines were administered 1 year apart to define the evolution of the CD4+ TFH cell response. The first vaccination induced an increase in the frequency of circulating TFH (cTFH) and LN TFH cells at week 1 postvaccination. This increase was transient for cTFH cells, whereas the LN TFH cells further expanded during week 2 and remained elevated in frequency for at least 3 months. We observed several distinct subsets of TFH cells in the LN, including pre-TFH cells, memory TFH cells, germinal center (GC) TFH cells and interleukin-10+ TFH cell subsets beginning at baseline and at all time points postvaccination. The shift toward a GC TFH cell phenotype occurred with faster kinetics after the second vaccine compared to the first vaccine. We identified several influenza-specific TFH cell clonal lineages, including multiple responses targeting internal influenza virus proteins, and found that each TFH cell state was attainable within a clonal lineage. Thus, human TFH cells form a durable and dynamic multitissue network.
Asunto(s)
Diferenciación Celular , Centro Germinal , Vacunas contra la Influenza , Gripe Humana , Células T Auxiliares Foliculares , Vacunación , Humanos , Vacunas contra la Influenza/inmunología , Células T Auxiliares Foliculares/inmunología , Gripe Humana/inmunología , Gripe Humana/prevención & control , Centro Germinal/inmunología , Diferenciación Celular/inmunología , Ganglios Linfáticos/inmunología , Adulto , Femenino , Masculino , Persona de Mediana Edad , Interleucina-10/inmunología , Interleucina-10/metabolismo , Memoria Inmunológica/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Adulto JovenRESUMEN
BACKGROUND: We reported that PARP-1 regulates genes whose products are crucial for asthma, in part, by controlling STAT6 integrity speculatively through a calpain-dependent mechanism. We wished to decipher the PARP-1/STAT6 relationship in the context of intracellular trafficking and promoter occupancy of the transcription factor on target genes, its integrity in the presence of calpains, and its connection to autophagy. METHODS: This study was conducted using primary splenocytes or fibroblasts derived from wild-type or PARP-1-/- mice and Jurkat T cells to mimic Th2 inflammation. RESULTS: We show that the role for PARP-1 in expression of IL-4-induced genes (e.g. gata-3) in splenocytes did not involve effects on STAT6 phosphorylation or its subcellular trafficking, rather, it influenced its occupancy of gata-3 proximal and distal promoters in the early stages of IL-4 stimulation. At later stages, PARP-1 was crucial for STAT6 integrity as its inhibition, pharmacologically or by gene knockout, compromised the fate of the transcription factor. Calpain-1 appeared to preferentially degrade JAK-phosphorylated-STAT6, which was blocked by calpastatin-mediated inhibition or by genetic knockout in mouse fibroblasts. The STAT6/PARP-1 relationship entailed physical interaction and modification by poly(ADP-ribosyl)ation independently of double-strand-DNA breaks. Poly(ADP-ribosyl)ation protected phosphorylated-STAT6 against calpain-1-mediated degradation. Additionally, our results show that STAT6 is a bonafide substrate for chaperone-mediated autophagy in a selective and calpain-dependent manner in the human Jurkat cell-line. The effects were partially blocked by IL-4 treatment and PARP-1 inhibition. CONCLUSIONS: The results demonstrate that poly(ADP-ribosyl)ation plays a critical role in protecting activated STAT6 during Th2 inflammation, which may be synthetically targeted for degradation by inhibiting PARP-1.
Asunto(s)
Poli ADP Ribosilación , Poli(ADP-Ribosa) Polimerasas , Humanos , Ratones , Animales , Poli(ADP-Ribosa) Polimerasas/metabolismo , Calpaína/genética , Calpaína/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Interleucina-4/farmacología , Interleucina-4/metabolismo , Autofagia , Inflamación , Factor de Transcripción STAT6/metabolismoRESUMEN
BACKGROUND AND OBJECTIVE: Endothelial microparticles (EMP) are submicron vesicles released from endothelial cells. We aimed to determine the utility of EMP as biomarkers of pulmonary arterial hypertension (PAH) in systemic sclerosis (SSc) patients and the pathogenic role of microparticles (MP) in vascular inflammation. METHODS: Levels of EMP (CD144+, CD31+, CD62E+ and CD143+) were compared between three groups (10 SSc patients with PAH, 10 SSc patients without pulmonary hypertension (no-PH) and 10 healthy age- and sex-matched controls). Human pulmonary artery endothelial cells (HPAEC) were exposed in vitro to MP obtained from SSc patients or healthy controls, and levels of cytokines and inflammatory adhesion molecules were compared. RESULTS: CD144+ EMP were significantly higher in the SSc-PAH group compared to either the SSc-no PH or healthy controls (diagnostic accuracy 80%, P = 0.02). Compared to controls, SSc patients had higher CD31+/CD62E+ ratios, indicating larger contributions of apoptosis to EMP release (P = 0.04). Patients with limited SSc had significantly higher levels of CD143+ EMP compared to those with diffuse subtype (P = 0.008). When HPAEC were exposed to MP from SSc patients, there was a significant increase in inflammatory cytokines and adhesion molecules. Interestingly, exposure to healthy control MP caused a reduction in inflammatory markers. CONCLUSION: EMP (particularly CD144+) are promising biomarkers of PAH in SSc but require further study. MP isolated from SSc patients induced an increase in endothelial cell inflammation and may be an important pathogenic factor in SSc.
Asunto(s)
Micropartículas Derivadas de Células , Células Endoteliales/metabolismo , Hipertensión Pulmonar/metabolismo , Esclerodermia Sistémica/metabolismo , Biomarcadores/metabolismo , Citocinas/metabolismo , Femenino , Humanos , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/patología , Masculino , Persona de Mediana Edad , Proyectos Piloto , Arteria Pulmonar/patología , Esclerodermia Sistémica/complicaciones , Esclerodermia Sistémica/patologíaRESUMEN
Dendritic cells (DCs) are critical in asthma and many other immune diseases. We previously demonstrated a role for PARP-1 in asthma. Evidence on PARP-1 playing a role in Th2-associated DC function is not clear. In this study, we examined whether PARP-1 is critical for DC differentiation and function using bone marrow progenitors and their migration to the lung in an ovalbumin-based mouse model of asthma. Results show that changes in PARP-1 levels during GM-CSF-induced DC differentiation from bone marrow progenitors were cyclic and appear to be part of an array of changes that included STAT3/STAT5/STAT6/GRAIL/RAD51. Interestingly, PARP-1 gene deletion affected primarily STAT6 and γH2AX. PARP-1 inhibition significantly reduced the migration of DCs to the lungs of ovalbumin-challenged mice, which was associated with a concomitant reduction in lung levels of the adhesion molecule VCAM-1. The requirement of PARP-1 for VCAM-1 expression was confirmed using endothelial and lung smooth muscle cells. PARP-1 expression and activity were also required for VCAM-1 in differentiated DCs. An assessment of CD11b+/CD11c+/MHCIIhigh DCs in spleens and lymph nodes of OVA-sensitized mice revealed that PARP-1 inhibition genetically or by olaparib exerted little to no effect on DC differentiation, percentage of CD80+/CD86+/CD40+-expressing cells, or their capacity to promote proliferation of ovalbumin-primed (OTII) CD4+ T cells. These findings were corroborated using GM-CSF-induced differentiation of DCs from the bone marrow. Surprisingly, the PARP-1-/- DCs exhibited a higher intrinsic capacity to induce OTII CD4+ T cell proliferation in the absence of ovalbumin. Overall, our results show that PARP-1 plays little to no role in DC differentiation and function and that the protective effect of PARP-1 inhibition against asthma is associated with a prevention of DC migration to the lung through a reduction in VCAM-1 expression. Given the current use of PARP inhibitors (e.g., olaparib) in the clinic, the present results may be of interest for the relevant therapies.
Asunto(s)
Asma/metabolismo , Células Dendríticas/metabolismo , Pulmón/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Animales , Citometría de Flujo , Ratones , Ratones Mutantes , Poli(ADP-Ribosa) Polimerasa-1/genética , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT5/metabolismo , Factor de Transcripción STAT6/metabolismoRESUMEN
BACKGROUND: The efficacy of heparins and low-MW-heparins (LMWH) against human asthma has been known for decades. However, the clinical utility of these compounds has been hampered by their anticoagulant properties. Much effort has been put into harnessing the anti-inflammatory properties of LMWH but none have been used as therapy for asthma. Sulfated-non-anticoagulant heparin (S-NACH) is an ultra-LMWH with no systemic anticoagulant effects. OBJECTIVE: The present study explored the potential of S-NACH in blocking allergic asthma and examined the potential mechanism by which it exerts its effects. METHODS: Acute and chronic ovalbumin-based mouse models of asthma, splenocytes, and a lung epithelial cell line were used. Mice were challenged with aerosolized ovalbumin and administered S-NACH or saline 30 min after each ovalbumin challenge. RESULTS: Sulfated-non-anticoagulant heparin administration in mice promoted a robust reduction in airway eosinophilia, mucus production, and airway hyperresponsiveness even after chronic repeated challenges with ovalbumin. Such effects were linked to suppression of Th2 cytokines IL-4/IL-5/IL-13/GM-CSF and ovalbumin-specific IgE without any effect on IFN-γ. S-NACH also reduced lung fibrosis in mice that were chronically-exposed to ovalbumin. These protective effects of S-NACH may be attributed to modulation of the IL-4/JAK1 signal transduction pathway through an inhibition of STAT6 phosphorylation and a subsequent inhibition of GATA-3 and inducible NO synthase expression. The effect of the drug on STAT6 phosphorylation coincided with a reduction in JAK1 phosphorylation upon IL-4 treatment. The protective effects of S-NACH treatment was associated with reduction of the basal expression of the two isoforms of arginase ARG1 and ARG2 in lung epithelial cells. CONCLUSIONS: Our study demonstrates that S-NACH constitutes an opportunity to benefit from the well-known anti-asthma properties of heparins/LMWH while bypassing the risk of bleeding. Our results show, for the first time, that such anti-asthma effects may be associated with reduction of the IL-4/JAK1/STAT6 pathway.
Asunto(s)
Asma/tratamiento farmacológico , Heparina/efectos adversos , Interleucina-4/metabolismo , Janus Quinasa 1/metabolismo , Factor de Transcripción STAT6/metabolismo , Células Th2/metabolismo , Células A549 , Animales , Antiinflamatorios/química , Anticoagulantes/química , Asma/complicaciones , Línea Celular , Separación Celular , Modelos Animales de Enfermedad , Citometría de Flujo , Hemorragia/tratamiento farmacológico , Humanos , Hipersensibilidad/complicaciones , Hipersensibilidad/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/química , Transducción de Señal , Bazo/citologíaRESUMEN
Pulmonary endothelial prostacyclin appears to be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). The effect of treatment with a prostacyclin analog in animal models of previously established COPD is unknown. We evaluated the short- and long-term effect of iloprost on inflammation and airway hyperresponsiveness (AHR) in a murine model of COPD. Nineteen mice were exposed to LPS/elastase, followed by either three doses of intranasal iloprost or saline. In the long-term treatment experiment, 18 mice were exposed to LPS/elastase and then received 6 wk of iloprost or were left untreated as controls. In the short-term experiment, iloprost did not change AHR but significantly reduced serum IL-5 and IFN-γ. Long-term treatment with iloprost for both 2 and 6 wk significantly improved AHR. After 6 wk of iloprost, there was a reduction in bronchoalveolar lavage (BALF) neutrophils, serum IL-1ß (30.0 ± 9.2 vs. 64.8 ± 7.4 pg/ml, P = 0.045), IL-2 (36.5 ± 10.6 vs. 83.8 ± 0.4 pg/ml, P = 0.01), IL-10 (75.7 ± 9.3 vs. 96.5 ± 3.5 pg/ml, P = 0.02), and nitrite (15.1 ± 5.4 vs. 30.5 ± 10.7 µmol, P = 0.01). Smooth muscle actin (SMA) in the lung homogenate was also significantly reduced after iloprost treatment (P = 0.02), and SMA thickness was reduced in the small and medium blood vessels after iloprost (P < 0.001). In summary, short- and long-term treatment with intranasal iloprost significantly reduced systemic inflammation in an LPS/elastase COPD model. Long-term iloprost treatment also reduced AHR, serum nitrite, SMA, and BALF neutrophilia. These data encourage future investigations of prostanoid therapy as a novel treatment for COPD patients.
Asunto(s)
Antiinflamatorios/administración & dosificación , Iloprost/administración & dosificación , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Administración Intranasal , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Lipopolisacáridos/farmacología , Masculino , Ratones Endogámicos C57BL , Infiltración Neutrófila , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Hipersensibilidad Respiratoria/tratamiento farmacológico , Hipersensibilidad Respiratoria/inmunologíaRESUMEN
Although expression of inducible NO synthase (iNOS) in the lungs of asthmatics and associated nitrosative damage are established, iNOS failed as a therapeutic target for blocking airway hyperresponsiveness (AHR) and inflammation in asthmatics. This dichotomy calls for better strategies with which the enzyme is adequately targeted. Here, we confirm iNOS expression in the asthmatic lung with concomitant protein nitration and poly(ADP-ribose) polymerase (PARP) activation. We show, for the first time, that iNOS is highly expressed in peripheral blood mononuclear cells (PBMCs) of asthmatics with uncontrolled disease, which did not correspond to protein nitration. Selective iNOS inhibition with L-NIL protected against AHR upon acute, but not chronic, exposure to ovalbumin or house dust mite (HDM) in mice. Supplementation of NO by nitrite administration significantly blocked AHR in chronically HDM-exposed mice that were treated with L-NIL. Protection against chronic HDM exposure-induced AHR by olaparib-mediated PARP inhibition may be associated with the partial but not the complete blockade of iNOS expression. Indeed, L-NIL administration prevented olaparib-mediated protection against AHR in chronically HDM-exposed mice. Our study suggests that the amount of iNOS and NO are critical determinants in the modulation of AHR by selective iNOS inhibitors and renews the potential of iNOS as a therapeutic target for asthma.
Asunto(s)
Alérgenos/inmunología , Asma/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa/inmunología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Hipersensibilidad Respiratoria/metabolismo , Animales , Asma/inmunología , Células Cultivadas , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II/genética , Nitritos/farmacología , Óxidos de Nitrógeno/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Hipersensibilidad Respiratoria/inducido químicamente , Hipersensibilidad Respiratoria/inmunología , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: We reported that DNA-dependent protein kinase (DNA-PK) is critical for the expression of nuclear factor κB-dependent genes in TNF-α-treated glioblastoma cells, suggesting an involvement in inflammatory diseases. OBJECTIVE: We sought to investigate the role of DNA-PK in asthma. METHODS: Cell culture and ovalbumin (OVA)- or house dust mite-based murine asthma models were used in this study. RESULTS: DNA-PK was essential for monocyte adhesion to TNF-α-treated endothelial cells. Administration of the DNA-PK inhibitor NU7441 reduced airway eosinophilia, mucus hypersecretion, airway hyperresponsiveness, and OVA-specific IgE production in mice prechallenged with OVA. Such effects correlated with a marked reduction in lung vascular cell adhesion molecule 1 expression and production of several cytokines, including IL-4, IL-5, IL-13, eotaxin, IL-2, and IL-12 and the chemokines monocyte chemoattractant protein 1 and keratinocyte-derived chemokine, with a negligible effect on IL-10/IFN-γ production. DNA-PK inhibition by gene heterozygosity of the 450-kDa catalytic subunit of the kinase (DNA-PKcs(+/-)) also prevented manifestation of asthma-like traits. These results were confirmed in a chronic model of asthma by using house dust mite, a human allergen. Remarkably, such protection occurred without causing severe combined immunodeficiency. Adoptive transfer of TH2-skewed OT-II wild-type CD4(+) T cells reversed IgE and TH2 cytokine production but not airway hyperresponsiveness in OVA-challenged DNA-PKcs(+/-) mice. DNA-PK inhibition reduced IL-4, IL-5, IL-13, eotaxin, IL-8, and monocyte chemoattractant protein 1 production without affecting IL-2, IL-12, IFN-γ, and interferon-inducible protein 10 production in CD3/CD28-stimulated human CD4(+) T cells, potentially by blocking expression of Gata3. These effects occurred without significant reductions in T-cell proliferation. In mouse CD4(+) T cells in vitro DNA-PK inhibition severely blocked CD3/CD28-induced Gata3 and T-bet expression in CD4(+) T cells and prevented differentiation of TH1 and TH2 cells under respective TH1- and TH2-skewing conditions. CONCLUSION: Our results suggest DNA-PK as a novel determinant of asthma and a potential target for the treatment of the disease.
Asunto(s)
Asma/inmunología , Linfocitos T CD4-Positivos/inmunología , Proteína Quinasa Activada por ADN/antagonistas & inhibidores , Mucosa Respiratoria/inmunología , Traslado Adoptivo , Alérgenos/inmunología , Animales , Asma/metabolismo , Asma/patología , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/metabolismo , Hiperreactividad Bronquial/patología , Adhesión Celular , Citocinas/metabolismo , Proteína Quinasa Activada por ADN/genética , Proteína Quinasa Activada por ADN/metabolismo , Modelos Animales de Enfermedad , Eosinófilos/inmunología , Eosinófilos/metabolismo , Células Epiteliales/metabolismo , Factor de Transcripción GATA3/genética , Factor de Transcripción GATA3/metabolismo , Expresión Génica , Heterogeneidad Genética , Humanos , Inmunoglobulina E/inmunología , Activación de Linfocitos , Masculino , Ratones , Ratones Noqueados , Tamaño de los Órganos , Ovalbúmina/efectos adversos , Ovalbúmina/inmunología , Fenotipo , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Pyroglyphidae/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Inmunodeficiencia Combinada Grave , Bazo/anatomía & histología , Bazo/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Células Th2/inmunología , Células Th2/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
BACKGROUND: An important portion of asthmatics do not respond to current therapies. Thus, the need for new therapeutic drugs is urgent. We have demonstrated a critical role for PARP in experimental asthma. Olaparib, a PARP inhibitor, was recently introduced in clinical trials against cancer. The objective of the present study was to examine the efficacy of olaparib in blocking established allergic airway inflammation and hyperresponsiveness similar to those observed in human asthma in animal models of the disease. METHODS: We used ovalbumin (OVA)-based mouse models of asthma and primary CD4(+) T cells. C57BL/6J WT or PARP-1(-/-) mice were subjected to OVA sensitization followed by a single or multiple challenges to aerosolized OVA or left unchallenged. WT mice were administered, i.p., 1 mg/kg, 5 or 10 mg/kg of olaparib or saline 30 min after each OVA challenge. RESULTS: Administration of olaparib in mice 30 min post-challenge promoted a robust reduction in airway eosinophilia, mucus production and hyperresponsiveness even after repeated challenges with ovalbumin. The protective effects of olaparib were linked to a suppression of Th2 cytokines eotaxin, IL-4, IL-5, IL-6, IL-13, and M-CSF, and ovalbumin-specific IgE with an increase in the Th1 cytokine IFN-γ. These traits were associated with a decrease in splenic CD4(+) T cells and concomitant increase in T-regulatory cells. The aforementioned traits conferred by olaparib administration were consistent with those observed in OVA-challenged PARP-1(-/-) mice. Adoptive transfer of Th2-skewed OT-II-WT CD4(+) T cells reversed the Th2 cytokines IL-4, IL-5, and IL-10, the chemokine GM-CSF, the Th1 cytokines IL-2 and IFN-γ, and ovalbumin-specific IgE production in ovalbumin-challenged PARP-1(-/-)mice suggesting a role for PARP-1 in CD4(+) T but not B cells. In ex vivo studies, PARP inhibition by olaparib or PARP-1 gene knockout markedly reduced CD3/CD28-stimulated gata-3 and il4 expression in Th2-skewed CD4(+) T cells while causing a moderate elevation in t-bet and ifn-γ expression in Th1-skewed CD4(+) T cells. CONCLUSIONS: Our findings show the potential of PARP inhibition as a viable therapeutic strategy and olaparib as a likely candidate to be tested in human asthma clinical trials.
Asunto(s)
Asma/tratamiento farmacológico , Asma/inmunología , Linfocitos T CD4-Positivos/inmunología , Técnicas de Inactivación de Genes , Ftalazinas/uso terapéutico , Piperazinas/uso terapéutico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Traslado Adoptivo , Animales , Antígenos CD/metabolismo , Asma/complicaciones , Hiperreactividad Bronquial/complicaciones , Hiperreactividad Bronquial/tratamiento farmacológico , Hiperreactividad Bronquial/inmunología , Linfocitos T CD4-Positivos/efectos de los fármacos , Citocinas/metabolismo , Modelos Animales de Enfermedad , Eosinofilia/complicaciones , Eosinofilia/tratamiento farmacológico , Eosinofilia/inmunología , Factor de Transcripción GATA3/genética , Factor de Transcripción GATA3/metabolismo , Humanos , Inmunoglobulina E/biosíntesis , Ratones Endogámicos C57BL , Moco/metabolismo , Ovalbúmina/inmunología , Ftalazinas/farmacología , Piperazinas/farmacología , Bazo/inmunología , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Células TH1/efectos de los fármacos , Células TH1/metabolismo , Células Th2/efectos de los fármacos , Células Th2/metabolismoRESUMEN
Our laboratory established a role for poly(ADP-ribose)polymerase (PARP) in asthma. To increase the clinical significance of our studies, it is imperative to demonstrate that PARP is actually activated in human asthma, to examine whether a PARP inhibitor approved for human testing such as olaparib blocks already-established chronic asthma traits in response to house dust mite (HDM), a true human allergen, in mice and to examine whether the drug modulates human cluster of differentiation type 4 (CD4(+)) T-cell function. To conduct the study, human lung specimens and peripheral blood mononuclear cells (PBMCs) and a HDM-based mouse asthma model were used. Our results show that PARP is activated in PBMCs and lung tissues of asthmatics. PARP inhibition by olaparib or gene knockout blocked established asthma-like traits in mice chronically exposed to HDM including airway eosinophilia and hyper-responsiveness. These effects were linked to a marked reduction in T helper 2 (Th2) cytokine production without a prominent effect on interferon (IFN)-γ or interleukin (IL)-10. PARP inhibition prevented HDM-induced increase in overall cellularity, weight and CD4(+) T-cell population in spleens of treated mice whereas it increased the T-regulatory cell population. In CD3/CD28-stimulated human CD4 (+)T-cells, olaparib treatment reduced Th2 cytokine production potentially by modulating GATA binding protein-3 (gata-3)/IL-4 expression while moderately affecting T-cell proliferation. PARP inhibition inconsistently increased IL-17 in HDM-exposed mice and CD3/CD28-stimulated CD4(+) T cells without a concomitant increase in factors that can be influenced by IL-17. In the present study, we provide evidence for the first time that PARP-1 is activated in human asthma and that its inhibition is effective in blocking established asthma in mice.
Asunto(s)
Antialérgicos/farmacología , Antiasmáticos/farmacología , Antígenos Dermatofagoides , Asma/prevención & control , Pulmón/efectos de los fármacos , Ftalazinas/farmacología , Piperazinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Animales , Asma/enzimología , Asma/inmunología , Asma/fisiopatología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Activación Enzimática , Humanos , Mediadores de Inflamación/metabolismo , Pulmón/enzimología , Pulmón/inmunología , Pulmón/fisiopatología , Activación de Linfocitos/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones Noqueados , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/deficiencia , Poli(ADP-Ribosa) Polimerasas/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/efectos de los fármacos , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/enzimología , Linfocitos T Reguladores/inmunología , Células Th2/efectos de los fármacos , Células Th2/enzimología , Células Th2/inmunologíaRESUMEN
Caspase-activated DNase (CAD) is the most favorable candidate for chromatin degradation during apoptosis. Ca(2+)-dependent endonucleases are equally important in internucleosomal DNA fragmentation (INDF), including the PARP-1-regulated DNAS1L3. Despite the elaborate work on these endonucleases, the question of whether these enzymes cooperate during INDF was not addressed. Here, we show a lack of correlation between INDF and CAD expression levels and inactivation by cleavage of its inhibitor (ICAD) during apoptosis. The cells that failed to induce INDF accumulated large amounts of 50-kb breaks, which is suggestive of incomplete chromatin processing. Similarly, INDF was blocked by Ca(2+) chelation without a block in ICAD cleavage or caspase-3 activation, which is consistent with the involvement of CAD in 50-kb DNA fragmentation and its Ca(2+) independence. However, DNAS1L3 expression in INDF-deficient cells promoted INDF during apoptosis and was blocked by Ca(2+) chelation. Interestingly, expression of DNAS1L3 in ICAD-deficient cells failed to promote tumor necrosis factor α-induced INDF but required the coexpression of ICAD. These results suggest a cooperative activity between CAD and DNAS1L3 to accomplish INDF. In HT-29 cells, endogenous DNAS1L3 localized to the endoplasmic reticulum (ER) and translocated to the nucleus upon apoptosis induction but prior to INDF manifestation, making it the first reported Ca(2+)-dependent endonuclease to migrate from the ER to the nucleus. The nuclear accumulation of DNAS1L3, but not its exit out of the ER, required the activity of cysteine and serine proteases. Interestingly, the endonuclease accumulated in the cytosol upon inhibition of serine, but not cysteine, proteases. These results exemplify the complexity of chromatin degradation during apoptosis.
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Apoptosis , Núcleo Celular/enzimología , Fragmentación del ADN , Desoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/metabolismo , Retículo Endoplásmico/enzimología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Emparejamiento Base , Calcio/metabolismo , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Proteasas de Cisteína/metabolismo , Fragmentación del ADN/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Etopósido , Proteína Vmw65 de Virus del Herpes Simple/metabolismo , Humanos , Ratones , Nucleosomas/efectos de los fármacos , Nucleosomas/metabolismo , Inhibidores de Proteasas/farmacología , Transporte de Proteínas/efectos de los fármacos , Serina Proteasas/metabolismoRESUMEN
BACKGROUND: The DNA-dependent protein kinase (DNA-PK) complex comprises a catalytic (PRKDC) and two requisite DNA-binding (Ku70/Ku80) subunits. The role of the complex in repairing double-stranded DNA breaks (DSBs) is established, but its role in inflammation, as a complex or individual subunits, remains elusive. While only ~ 1% of PRKDC is necessary for DNA repair, we reported that partial inhibition blocks asthma in mice without causing SCID. METHODS: We investigated the central role of PRKDC in inflammation and its potential association with DNA repair. We also elucidated the relationship between inflammatory cytokines (e.g., TNF-α) and PRKDC by analyzing its connections to inflammatory kinases. Human cell lines, primary human endothelial cells, and mouse fibroblasts were used to conduct the in vitro studies. For animal studies, LPS- and oxazolone-induced mouse models of acute lung injury (ALI) and delayed-type hypersensitivity (DHT) were used. Wild-type, PRKDC+/-, or Ku70+/- mice used in this study. RESULTS: A ~ 50% reduction in PRKDC markedly blocked TNF-α-induced expression of inflammatory factors (e.g., ICAM-1/VCAM-1). PRKDC regulates Th1-mediated inflammation, such as DHT and ALI, and its role is highly sensitive to inhibition achieved by gene heterozygosity or pharmacologically. In endothelial or epithelial cells, TNF-α promoted rapid PRKDC phosphorylation in a fashion resembling that induced by, but independent of, DSBs. Ku70 heterozygosity exerted little to no effect on ALI in mice, and whatever effect it had was associated with a specific increase in MCP-1 in the lungs and systemically. While Ku70 knockout blocked VP-16-induced PRKDC phosphorylation, it did not prevent TNF-α - induced phosphorylation of the kinase, suggesting Ku70 dispensability. Immunoprecipitation studies revealed that PRKDC transiently interacts with p38MAPK. Inhibition of p38MAPK blocked TNF-α-induced PRKDC phosphorylation. Direct phosphorylation of PRKDC by p38MAPK was demonstrated using a cell-free system. CONCLUSIONS: This study presents compelling evidence that PRKDC functions independently of the DNA-PK complex, emphasizing its central role in Th1-mediated inflammation. The distinct functionality of PRKDC as an individual enzyme, its remarkable sensitivity to inhibition, and its phosphorylation by p38MAPK offer promising therapeutic opportunities to mitigate inflammation while sparing DNA repair processes. These findings expand our understanding of PRKDC biology and open new avenues for targeted anti-inflammatory interventions.
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In recent years, there has been an explosion of interest in how fibroblasts initiate, sustain, and resolve inflammation across disease states. Fibroblasts contain heterogeneous subsets with diverse functionality. The phenotypes of these populations vary depending on their spatial distribution within the tissue and the immunopathologic cues contributing to disease progression. In addition to their roles in structurally supporting organs and remodeling tissue, fibroblasts mediate critical interactions with diverse immune cells. These interactions have important implications for defining mechanisms of disease and identifying potential therapeutic targets. Fibroblasts in the respiratory tract, in particular, determine the severity and outcome of numerous acute and chronic lung diseases, including asthma, chronic obstructive pulmonary disease, acute respiratory distress syndrome, and idiopathic pulmonary fibrosis. Here, we review recent studies defining the spatiotemporal identity of the lung-derived fibroblasts and the mechanisms by which these subsets regulate immune responses to insult exposures and highlight past, current, and future therapeutic targets with relevance to fibroblast biology in the context of acute and chronic human respiratory diseases. This perspective highlights the importance of tissue context in defining fibroblast-immune crosstalk and paves the way for identifying therapeutic approaches to benefit patients with acute and chronic pulmonary disorders.
Asunto(s)
Asma , Neumonía , Enfermedad Pulmonar Obstructiva Crónica , Humanos , Inflamación , FibroblastosRESUMEN
We profiled blood and draining lymph node (LN) samples from human volunteers after influenza vaccination over two years to define evolution in the T follicular helper cell (TFH) response. We show LN TFH cells expanded in a clonal-manner during the first two weeks after vaccination and persisted within the LN for up to six months. LN and circulating TFH (cTFH) clonotypes overlapped but had distinct kinetics. LN TFH cell phenotypes were heterogeneous and mutable, first differentiating into pre-TFH during the month after vaccination before maturing into GC and IL-10+ TFH cells. TFH expansion, upregulation of glucose metabolism, and redifferentiation into GC TFH cells occurred with faster kinetics after re-vaccination in the second year. We identified several influenza-specific TFH clonal lineages, including multiple responses targeting internal influenza proteins, and show each TFH state is attainable within a lineage. This study demonstrates that human TFH cells form a durable and dynamic multi-tissue network.
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BACKGROUND: Poly(ADP-ribose) polymerase (PARP) inhibitors (eg, olaparib) are effective against BRCA-mutated cancers at/near maximum tolerated doses by trapping PARP-1 on damaged chromatin, benefitting only small patient proportions. The benefits of targeting non-DNA repair aspects of PARP with metronomic doses remain unexplored. METHODS: Colon epithelial cells or mouse or human bone marrow (BM)-derived-myeloid-derived suppressor cells (MDSCs) were stimulated to assess the effect of partial PARP-1 inhibition on inflammatory gene expression or immune suppression. Mice treated with azoxymethane/four dextran-sulfate-sodium cycles or APCMin/+ mice bred into PARP-1+/- or treated with olaparib were used to examine the role of PARP-1 in colitis-induced or spontaneous colon cancer, respectively. Syngeneic MC-38 cell-based (microsatellite instability, MSIhigh) or CT-26 cell-based (microsatellite stable, MSS) tumor models were used to assess the effects of PARP inhibition on host responses and synergy with anti-Programmed cell Death protein (PD)-1 immunotherapy. RESULTS: Partial PARP-1 inhibition, via gene heterozygosity or a moderate dose of olaparib, protected against colitis-mediated/APCMin -mediated intestinal tumorigenesis and APCMin -associated cachexia, while extensive inhibition, via gene knockout or a high dose of olaparib, was ineffective or aggravating. A sub-IC50-olaparib dose or PARP-1 heterozygosity was sufficient to block tumorigenesis in a syngeneic colon cancer model by modulating the suppressive function, but not intratumoral migration or differentiation, of MDSCs, with concomitant increases in intratumoral T cell function and cytotoxicity, as assessed by granzyme-B/interferon-γ levels. Adoptive transfer of WT-BM-MDSCs abolished the protective effects of PARP-1 heterozygosity. The mechanism of MDSC modulation involved a reduction in arginase-1/inducible nitric oxide synthase/cyclo-oxygenase-2, but independent of PARP-1 trapping on chromatin. Although a high-concentration olaparib or the high-trapping PARP inhibitor, talazoparib, activated stimulator of interferon gene (STING) in BRCA-proficient cells and induced DNA damage, sub-IC50 concentrations of either drug failed to induce activation of the dsDNA break sensor. STING expression appeared dispensable for MDSC suppressive function and was not strictly required for olaparib-mediated effects. Ironically, STING activation blocked human and mouse MDSC function with no additive effects with olaparib. A metronomic dose of olaparib was highly synergistic with anti-PD-1-based immunotherapy, leading to eradication of MSIhigh or reduction of MSS tumors in mice. CONCLUSIONS: These results support a paradigm-shifting concept that expands the utility of PARP inhibitor and encourage testing metronomic dosing of PARP inhibitor to enhance the efficacy of checkpoint inhibitor-based immunotherapies in cancer.
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Colitis/complicaciones , Neoplasias del Colon/tratamiento farmacológico , Inhibidores de Puntos de Control Inmunológico/administración & dosificación , Ftalazinas/administración & dosificación , Piperazinas/administración & dosificación , Inhibidores de Poli(ADP-Ribosa) Polimerasas/administración & dosificación , Administración Metronómica , Animales , Azoximetano/efectos adversos , Línea Celular Tumoral , Colitis/inducido químicamente , Neoplasias del Colon/etiología , Sulfato de Dextran/efectos adversos , Sinergismo Farmacológico , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Ratones , Células Supresoras de Origen Mieloide/metabolismo , Ftalazinas/farmacología , Piperazinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND AND OBJECTIVE: We tested whether the prostacyclin analog inhaled iloprost modulates dead space, dynamic hyperinflation (DH), and systemic inflammation/oxidative stress during maximal exercise in subjects with chronic obstructive pulmonary disease (COPD) who were not selected based on pulmonary hypertension (PH). METHODS: Twenty-four COPD patients with moderate-severe obstruction (age 59 ± 7 years, FEV1 53 ± 13% predicted) participated in a randomized, double-blind, placebo-controlled crossover trial. Each subject received a single nebulized dose of 5.0 µg iloprost or placebo on non-consecutive days followed by maximal cardiopulmonary exercise tests. The primary outcome was DH quantified by end-expiratory lung volume/total lung capacity ratio (EELV/TLC) at metabolic isotime. RESULTS: Inhaled iloprost was well-tolerated and reduced submaximal alveolar dead-space fraction but did not significantly reduce DH (0.70 ± 0.09 vs 0.69 ± 0.07 following placebo and iloprost, respectively, p = 0.38). Maximal exercise time (9.1 ± 2.3 vs 9.3 ± 2.2 min, p = 0.31) and peak oxygen uptake (17.4 ± 6.3 vs 17.9 ± 6.9 mL/kg/min, p = 0.30) were not significantly different following placebo versus iloprost. CONCLUSIONS: A single dose of inhaled iloprost was safe and reduced alveolar dead space fraction; however, it was not efficacious in modulating DH or improving exercise capacity in COPD patients who were not selected for the presence of PH.