RESUMO
Chronic inflammatory diseases are associated with altered hematopoiesis that could result in neutrophilia and anemia. Here we report that genetic or chemical manipulation of different inflammasome components altered the differentiation of hematopoietic stem and progenitor cells (HSPC) in zebrafish. Although the inflammasome was dispensable for the emergence of HSPC, it was intrinsically required for their myeloid differentiation. In addition, Gata1 transcript and protein amounts increased in inflammasome-deficient larvae, enforcing erythropoiesis and inhibiting myelopoiesis. This mechanism is evolutionarily conserved, since pharmacological inhibition of the inflammasome altered erythroid differentiation of human erythroleukemic K562 cells. In addition, caspase-1 inhibition rapidly upregulated GATA1 protein in mouse HSPC promoting their erythroid differentiation. Importantly, pharmacological inhibition of the inflammasome rescued zebrafish disease models of neutrophilic inflammation and anemia. These results indicate that the inflammasome plays a major role in the pathogenesis of neutrophilia and anemia of chronic diseases and reveal druggable targets for therapeutic interventions.
Assuntos
Anemia/imunologia , Doenças dos Peixes/imunologia , Fator de Transcrição GATA1/metabolismo , Inflamassomos/metabolismo , Inflamação/imunologia , Neutrófilos/imunologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Caspase 1/genética , Caspase 1/metabolismo , Diferenciação Celular , Células Eritroides/citologia , Fator de Transcrição GATA1/genética , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese , Humanos , Inflamassomos/genética , Células K562 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteólise , Proteínas de Peixe-Zebra/genéticaRESUMO
Several studies have revealed a correlation between chronic inflammation and nicotinamide adenine dinucleotide (NAD+) metabolism, but the precise mechanism involved is unknown. Here, we report that the genetic and pharmacological inhibition of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the salvage pathway of NAD+ biosynthesis, reduced oxidative stress, inflammation, and keratinocyte DNA damage, hyperproliferation, and cell death in zebrafish models of chronic skin inflammation, while all these effects were reversed by NAD+ supplementation. Similarly, genetic and pharmacological inhibition of poly(ADP-ribose) (PAR) polymerase 1 (Parp1), overexpression of PAR glycohydrolase, inhibition of apoptosis-inducing factor 1, inhibition of NADPH oxidases, and reactive oxygen species (ROS) scavenging all phenocopied the effects of Nampt inhibition. Pharmacological inhibition of NADPH oxidases/NAMPT/PARP/AIFM1 axis decreased the expression of pathology-associated genes in human organotypic 3D skin models of psoriasis. Consistently, an aberrant induction of NAMPT and PARP activity, together with AIFM1 nuclear translocation, was observed in lesional skin from psoriasis patients. In conclusion, hyperactivation of PARP1 in response to ROS-induced DNA damage, fueled by NAMPT-derived NAD+, mediates skin inflammation through parthanatos cell death.
Assuntos
Inflamação/patologia , NAD/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Parthanatos , Poli(ADP-Ribose) Polimerases/metabolismo , Pele/patologia , Animais , Fator de Indução de Apoptose/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Dano ao DNA , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/genética , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/patologia , Larva/metabolismo , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Parthanatos/efeitos dos fármacos , Parthanatos/genética , Poli Adenosina Difosfato Ribose/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas Secretadas Inibidoras de Proteinases/deficiência , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Psoríase/genética , Psoríase/patologia , Espécies Reativas de Oxigênio/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/metabolismoRESUMO
Dyskeratosis congenita (DC) is a rare inherited bone marrow failure and cancer predisposition syndrome caused by mutations in telomerase or telomeric proteins. Here, we report that zebrafish telomerase RNA (terc) binds to specific DNA sequences of master myeloid genes and controls their expression by recruiting RNA Polymerase II (Pol II). Zebrafish terc harboring the CR4-CR5 domain mutation found in DC patients hardly interacted with Pol II and failed to regulate myeloid gene expression in vivo and to increase their transcription rates in vitro. Similarly, TERC regulated myeloid gene expression and Pol II promoter occupancy in human myeloid progenitor cells. Strikingly, induced pluripotent stem cells derived from DC patients with a TERC mutation in the CR4-CR5 domain showed impaired myelopoiesis, while those with mutated telomerase catalytic subunit differentiated normally. Our findings show that TERC acts as a transcription factor, revealing a target for therapeutic intervention in DC patients.
Assuntos
Disceratose Congênita/genética , Mielopoese/fisiologia , RNA Polimerase II/genética , RNA/metabolismo , Telomerase/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Células Cultivadas , Disceratose Congênita/patologia , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Larva/genética , Mutação , Mielopoese/genética , Regiões Promotoras Genéticas , Domínios Proteicos , RNA/genética , RNA Polimerase II/metabolismo , Telomerase/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Hematopoiesis is a complex process through which immature bone marrow precursor cells mature into all types of blood cells. Although the association of hematopoietic lineage bias (including anemia and neutrophilia) with chronic inflammatory diseases has long been appreciated, the causes involved are obscure. Recently, cytosolic multiprotein inflammasome complexes were shown to activate inflammatory and immune responses, and directly regulate hematopoiesis in zebrafish models; this was deemed to occur via cleavage and inactivation of the master erythroid transcription factor GATA1. Herein summarized are the zebrafish models that are currently available to study this unappreciated role of inflammasome-mediated regulation of hematopoiesis. Novel putative therapeutic strategies, for the treatment of hematopoietic alterations associated with chronic inflammatory diseases in humans, are also proposed.
Assuntos
Hematopoese , Inflamassomos , Modelos Animais , Peixe-Zebra , Animais , Hematopoese/genética , Hematopoese/imunologia , Humanos , Inflamassomos/metabolismo , Pesquisa/tendências , Peixe-Zebra/genética , Peixe-Zebra/imunologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/imunologiaRESUMO
A retrospective study of 200 psoriasis patients and 100 healthy donors in a Spanish cohort was carried out to study the comorbidities associated with psoriasis and their association with the response to phototherapy. The results showed a higher incidence of psychiatric disease, liver disease, kidney disease, hypertension, heart disease, vascular disease, diabetes, gastrointestinal disease, autoimmune and infectious diseases, dyslipidemia, and psoriatic arthritis in patients with psoriasis than in the control group. The incidence of comorbidities was higher in psoriasis patients over 40 years old than in the control individuals of the same age, which could be indicative of premature aging. Phototherapy was seen to be an effective treatment in cases of moderate-severe psoriasis, total whitening being achieved in more than 30% of patients, with women showing a better response than men. Narrow-band ultraviolet B was found to be the most effective type of phototherapy, although achievement of PASI100 was lower in patients with liver disease, hypertension, heart disease, vascular disease, or diabetes. Strikingly, liver disease and anemia comorbidities favored therapeutic failure. Finally, zebrafish and human 3D organotypic models of psoriasis point to the therapeutic benefit of inhibiting the glucose transporter GLUT1 and the major regulator of blood glucose dipeptidyl peptidase 4. Our study reveals that specific comorbidities of psoriasis patients are associated to failure of phototherapy and, therefore, need to be considered when planning treatment for these patients.
Assuntos
Hipertensão , Psoríase , Terapia Ultravioleta , Adulto , Animais , Feminino , Humanos , Masculino , Fototerapia/métodos , Psoríase/tratamento farmacológico , Psoríase/terapia , Estudos Retrospectivos , Terapia Ultravioleta/métodos , Peixe-ZebraRESUMO
Apoptosis is the main mechanism by which multicellular organisms eliminate damaged or unwanted cells. To regulate this process, a balance between pro-survival and pro-apoptotic proteins is necessary in order to avoid impaired apoptosis, which is the cause of several pathologies, including cancer. Among the anti-apoptotic proteins, Bcl-xL exhibits a high conformational flexibility, whose regulation is strictly controlled by alternative splicing and post-transcriptional regulation mediated by transcription factors or microRNAs. It shows relevant functions in different forms of cancer, including melanoma. In melanoma, Bcl-xL contributes to both canonical roles, such as pro-survival, protection from apoptosis and induction of drug resistance, and non-canonical functions, including promotion of cell migration and invasion, and angiogenesis. Growing evidence indicates that Bcl-xL inhibition can be helpful for cancer patients, but at present, effective and safe therapies targeting Bcl-xL are lacking due to toxicity to platelets. In this review, we summarized findings describing the mechanisms of Bcl-xL regulation, and the role that Bcl-xL plays in melanoma pathobiology and response to therapy. From these findings, it emerged that even if Bcl-xL plays a crucial role in melanoma pathobiology, we need further studies aimed at evaluating the involvement of Bcl-xL and other members of the Bcl-2 family in the progression of melanoma and at identifying new non-toxic Bcl-xL inhibitors.
Assuntos
Apoptose , Resistencia a Medicamentos Antineoplásicos , Melanoma/irrigação sanguínea , Melanoma/metabolismo , Neovascularização Patológica/tratamento farmacológico , Proteína bcl-X/metabolismo , Humanos , Melanoma/tratamento farmacológico , Melanoma/patologia , MicroRNAs/metabolismo , Invasividade Neoplásica , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , RNA Neoplásico/metabolismoRESUMO
Reversible protein ubiquitylation plays important roles in various processes including DNA repair. Here, we identify the deubiquitylase USP45 as a critical DNA repair regulator. USP45 associates with ERCC1, a subunit of the DNA repair endonuclease XPF-ERCC1, via a short acidic motif outside of the USP45 catalytic domain. Wild-type USP45, but not a USP45 mutant defective in ERCC1 binding, efficiently deubiquitylates ERCC1 in vitro, and the levels of ubiquitylated ERCC1 are markedly enhanced in USP45 knockout cells. Cells lacking USP45 are hypersensitive specifically to UV irradiation and DNA interstrand cross-links, similar to cells lacking ERCC1. Furthermore, the repair of UV-induced DNA damage is markedly reduced in USP45-deficient cells. ERCC1 translocation to DNA damage-induced subnuclear foci is markedly impaired in USP45 knockout cells, possibly accounting for defective DNA repair. Finally, USP45 localises to sites of DNA damage in a manner dependent on its deubiquitylase activity, but independent of its ability to bind ERCC1-XPF. Together, these results establish USP45 as a new regulator of XPF-ERCC1 crucial for efficient DNA repair.
Assuntos
Núcleo Celular/metabolismo , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Endopeptidases/metabolismo , Ubiquitinação , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/efeitos da radiação , Animais , Linhagem Celular Tumoral , Núcleo Celular/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Endopeptidases/genética , Humanos , Camundongos , Camundongos Knockout , Raios Ultravioleta/efeitos adversosRESUMO
The zebrafish has unique advantages for understanding the evolution of vertebrate immunity and to model human diseases. In this review, we will firstly give an overview of the current knowledge on vertebrate innate immune receptors with special emphasis on the inflammasome and then summarize the main contribution of the zebrafish model to this field, including to the identification of novel inflammasome components and to the mechanisms involved in its activation, assembly and clearance of intracellular bacteria.
Assuntos
Evolução Biológica , Proteínas de Peixes/genética , Imunidade Inata , Receptores Imunológicos/genética , Peixe-Zebra/imunologia , Animais , Modelos Animais de Doenças , Evolução Molecular , Proteínas de Peixes/metabolismo , Imunidade Inata/genética , Modelos Animais , Receptores Imunológicos/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
The protein bcl-xL is able to enhance the secretion of the proinflammatory chemokine interleukin 8 (CXCL8) in human melanoma lines. In this study, we investigate whether the bcl-xL/CXCL8 axis is important for promoting melanoma angiogenesis and aggressiveness in vivo, using angiogenesis and xenotransplantation assays in zebrafish embryos. When injected into wild-type embryos, bcl-xL-overexpressing melanoma cells showed enhanced dissemination and angiogenic activity compared with control cells. Human CXCL8 protein elicited a strong proangiogenic activity in zebrafish embryos and zebrafish Cxcr2 receptor was identified as the mediator of CXCL8 proangiogenic activity using a morpholino-mediated gene knockdown. However, human CXCL8 failed to induce neutrophil recruitment in contrast to its zebrafish homolog. Interestingly, the greater aggressiveness of bcl-xL-overexpressing melanoma cells was mediated by an autocrine effect of CXCL8 on its CXCR2 receptor, as confirmed by an shRNA approach. Finally, correlation studies of gene expression and survival analyses using microarray and RNA-seq public databases of human melanoma biopsies revealed that bcl-xL expression significantly correlated with the expression of CXCL8 and other markers of melanoma progression. More importantly, a high level of co-expression of bcl-xL and CXCL8 was associated with poor prognosis in melanoma patients. In conclusion, these data demonstrate the existence of an autocrine CXCL8/CXCR2 signaling pathway in the bcl-xL-induced melanoma aggressiveness, encouraging the development of novel therapeutic approaches for high bcl-xL-expressing melanoma.
Assuntos
Interleucina-8/metabolismo , Melanoma/irrigação sanguínea , Proteína bcl-X/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular Tumoral , Xenoenxertos , Humanos , Interleucina-8/biossíntese , Interleucina-8/genética , Interleucina-8/farmacologia , Melanoma/genética , Melanoma/metabolismo , Neovascularização Patológica/metabolismo , Proteínas Recombinantes/farmacologia , Microambiente Tumoral , Peixe-Zebra , Proteína bcl-X/biossíntese , Proteína bcl-X/genéticaRESUMO
The melanocortin 1 receptor (MC1R) is a G-protein-coupled receptor (GPCR) crucial for the regulation of melanocyte proliferation and differentiation. MC1R activation by melanocortin hormones triggers the cAMP pathway and stimulates the extracellular-signal-regulated protein kinases ERK1 and ERK2 to promote synthesis of photoprotective eumelanin pigments, among other effects. Signaling from most GPCRs is regulated by the ß-arrestin (ARRB) family of cytosolic multifunctional adaptor proteins, which mediate signal termination and endocytosis of GPCR-agonist complexes. The ubiquitously expressed non-visual ß-arrestin1 (ARRB1) and ß-arrestin2 (ARRB2) are highly similar but not functionally equivalent. Their role in the regulation of MC1R is unknown. Using a combination of co-immunoprecipitation, gel filtration chromatography, confocal microscopy, siRNA-mediated knockdown and functional assays, we demonstrated agonist-independent competitive interactions of ARRB1 and ARRB2 with MC1R, which might also be independent of phosphorylation of Ser/Thr residues in the C-terminus of the MC1R. The effects of ARRBs were isoform specific; ARRB2 inhibited MC1R agonist-dependent cAMP production but not ERK activation, stimulated internalization and showed prolonged co-localization with the receptor in endocytic vesicles. By contrast, ARRB1 had no effect on internalization or functional coupling, but competed with ARRB2 for binding MC1R, which might increase signaling by displacement of inhibitory ARRB2. These data suggest a new mechanism of MC1R functional regulation based on the relative expression of ARRB isoforms, with possible activatory ARRB1-dependent effects arising from partial relief of inhibitory ARRB2-MC1R interactions. Thus, competitive displacement of inhibitory ARRBs by functionally neutral ARRB isoforms might exert a paradigm-shifting signal-promoting effect to fine-tune signaling downstream of certain GPCRs.
Assuntos
Arrestinas/metabolismo , Receptor Tipo 1 de Melanocortina/metabolismo , Arrestinas/genética , Diferenciação Celular/efeitos dos fármacos , Células HEK293 , Humanos , Isoformas de Proteínas , Receptor Tipo 1 de Melanocortina/genética , Transdução de Sinais , Transfecção , beta-Arrestina 1 , beta-Arrestina 2 , beta-ArrestinasRESUMO
BACKGROUND: Macrophages take center stage in the tumor microenvironment, a niche composed of extracellular matrix and a heterogeneous group of cells, including immune ones. They can evolve during tumor progression and acquire Tumor-Associated Macrophage (TAMs) phenotype. The release of cytokines by tumor and stromal cells, influence the secretion of cytokines by TAMs, which can guarantee tumor progression and influence the response to therapy. Among all factors able to recruit and polarize macrophages, we focused our attention on Bcl-xL, a multifaceted member of the Bcl-2 family, whose expression is deregulated in melanoma. It acts not only as a canonical pro-survival and anti-apoptotic protein, but also as a promoter of tumor progression. METHODS: Human melanoma cells silencing or overexpressing Bcl-xL protein, THP-1 monocytic cells and monocyte-derived macrophages were used in this study. Protein array and specific neutralizing antibodies were used to analyze cytokines and chemokines secreted by melanoma cells. qRT-PCR, ELISA and Western Blot analyses were used to evaluate macrophage polarization markers and protein expression levels. Transwell chambers were used to evaluate migration of THP-1 and monocyte-derived macrophages. Mouse and zebrafish models were used to evaluate the ability of melanoma cells to recruit and polarize macrophages in vivo. RESULTS: We demonstrated that melanoma cells overexpressing Bcl-xL recruit macrophages at the tumor site and induce a M2 phenotype. In addition, we identified that interleukin-8 and interleukin-1ß cytokines are involved in macrophage polarization, and the chemokine CCL5/RANTES in the macrophages recruitment at the tumor site. We also found that all these Bcl-xL-induced factors are regulated in a NF-kB dependent manner in human and zebrafish melanoma models. CONCLUSIONS: Our findings confirmed the pro-tumoral function of Bcl-xL in melanoma through its effects on macrophage phenotype.
Assuntos
Melanoma , Peixe-Zebra , Proteína bcl-X , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Citocinas/metabolismo , Macrófagos/metabolismo , Melanoma/patologia , Microambiente TumoralRESUMO
Sickle cell disease (SCD) is an inherited autosomal recessive hemoglobin disorder caused by the presence of hemoglobin S, a mutant abnormal hemoglobin caused by a nucleotide change in codon 6 of the ß-globin chain gene. SCD involves a chronic inflammatory state, exacerbated during vaso-occlusive crises, which leads to end-organ damage that occurs throughout the lifespan. SCD is associated with premature mortality in the first years of life. The process of sickling provokes asplenia in the first years of life with an increased risk of infection by encapsulated germs. These complications can be life-threatening and require early diagnosis and management. The most important interventions recommend an early diagnosis of SCD to ensure that affected newborns receive immediate care to reduce mortality and morbidity. The newborn screening program in the region of Murcia for SCD began in March 2016. We aimed to determine the incidence of sickle cell anemia and other structural hemoglobinopathies in the neonatal population of the region of Murcia, an area of high migratory stress, and to systematically assess the benefit of newborn screening for SCD, leading to earlier treatment, as well as to offer genetic counseling to all carriers. The prevalence of SCD in our region is similar to others in Spain, except for Catalonia and Madrid. The newborns with confirmed diagnoses of SCD received early attention, and all the carriers received genetic counseling.
RESUMO
Chronic inflammatory diseases are associated with hematopoietic lineage bias, including neutrophilia and anemia. We have recently identified that the canonical inflammasome mediates the cleavage of the master erythroid transcription factor GATA1 in hematopoietic stem and progenitor cells (HSPCs). We report here that genetic inhibition of Nlrp1 resulted in reduced number of neutrophils and increased erythrocyte counts in zebrafish larvae. We also found that the NLRP1 inflammasome in human cells was inhibited by LRRFIP1 and FLII, independently of DPP9, and both inhibitors regulated hematopoiesis. Mechanistically, erythroid differentiation resulted in ribosomal stress-induced activation of the ZAKα/P38 kinase axis which, in turn, phosphorylated and promoted the assembly of NLRP1 in both zebrafish and human. Finally, inhibition of Zaka with the FDA/EMA-approved drug Nilotinib alleviated neutrophilia in a zebrafish model of neutrophilic inflammation and promoted erythroid differentiation and GATA1 accumulation in K562 cells. In conclusion, our results reveal that the NLRP1 inflammasome regulates hematopoiesis and pave the way to develop novel therapeutic strategies for the treatment of hematopoietic alterations associated with chronic inflammatory and rare diseases.
RESUMO
In this review, we have summarized classical post-translational modifications (PTMs) such as phosphorylation, ubiquitylation, and SUMOylation of the different components of one of the most studied NLRP3, and other emerging inflammasomes. We will highlight how the discovery of these modifications have provided mechanistic insight into the biology, function, and regulation of these multiprotein complexes not only in the context of the innate immune system but also in adaptive immunity, hematopoiesis, bone marrow transplantation, as well and their role in human diseases. We have also collected available information concerning less-studied modifications such as acetylation, ADP-ribosylation, nitrosylation, prenylation, citrullination, and emphasized their relevance in the regulation of inflammasome complex formation. We have described disease-associated mutations affecting PTMs of inflammasome components. Finally, we have discussed how a deeper understanding of different PTMs can help the development of biomarkers and identification of novel drug targets to treat diseases caused by the malfunctioning of inflammasomes.
RESUMO
ß-thalassemia is a disease caused by genetic mutations including a nucleotide change, small insertions or deletions in the ß-globin gene, or in rare cases, gross deletions into the ß-globin gene. These mutations affect globin-chain subunits within the hemoglobin tetramer what induces an imbalance in the α/ß-globin chain ratio, with an excess of free α-globin chains that triggers the most important pathogenic events of the disease: ineffective erythropoiesis, chronic anemia/chronic hypoxia, compensatory hemopoietic expansion and iron overload. Based on advances in our knowledge of the pathophysiology of ß-thalassemia, in recent years, emerging therapies and clinical trials are being conducted and are classified into three major categories based on the different approach features of the underlying pathophysiology: correction of the α/ß-globin disregulation; improving iron overload and reverse ineffective erythropoiesis. However, pathways such as the dysregulation of transcriptional factors, activation of the inflammasome, or approach to mechanisms of bone mineral loss, remain unexplored for future therapeutic targets. In this review, we update the main pathophysiological pathways involved in ß-thalassemia, focusing on the development of new therapies directed at new therapeutic targets.
RESUMO
Prostaglandins (PGs) are highly reactive small lipophilic molecules derived from polyunsaturated fatty acids of the cell membrane and play a key role in the resolution of inflammation processes. 15-deoxy-Δ12,14-PGJ2 (15dPGJ2) is a cyclopentenone PG (CyPG) of the J series with anti-inflammatory, anti-proliferative and pro-apoptotic effects. This CyPG can signal through: (i) the PGD2 receptor (DP2) and peroxisome proliferator-activated receptor γ (PPARγ) or (ii) by covalent binding to protein nucleophiles, such as, thiols groups of cysteine, lysine or histidine via a Michael addition reaction, modifying its structure and function. In this work we show that acidophilic granulocytes (AGs) of gilthead seabream (Sparus aurata L.), the functional equivalent to mammalian neutrophils, constitutively expressed ppara, pparb and pparg genes, the latter showing the highest expression and up-regulation when stimulated by bacterial DNA. In addition, we tested the ability of 15dPGJ2, and its biotinylated analog, as well as several PPARγ ligands, to modulate reactive oxygen species (ROS) and/or cytokines production during a Toll like receptor (TLR)-mediated granulocyte response. Thus, 15dPGJ2 was able to significantly decrease bacterial DNA-induced ROS production and transcript levels of pparg, interleukin-1ß (il1b) and prostaglandin-endoperoxide synthase 2 (ptgs2). In contrast, its biotinylated analog was less potent and a higher dose was required to elicit the same effects on ROS production and cytokine expression. In addition, different PPARγ agonists were able to mimic the effects of 15dPGJ2. Conversely, the PPARγ antagonist T007097 abolished the effect of 15dPGJ2 on DNA bacterial-induced ROS production. Surprisingly, transactivation assays revealed that both 15dPGJ2 and its biotinylated analog signaled via Pparα and Pparß, but not by Pparγ. These results were further confirmed by HPLC/MS analysis, where Pparß was identified as an interactor of biotin-15dPGJ2 in naïve and DNA-stimulated leukocytes. Taken together, our data show that 15dPGJ2 acts both through Ppar activation and covalent binding to proteins in fish granulocytes and identify for the first time in vertebrates a role for Pparα and Pparß in the resolution of inflammation mediated by 15dPGJ2.
Assuntos
PPAR beta , Dourada , Animais , Ciclo-Oxigenase 2/metabolismo , Ciclopentanos , DNA Bacteriano , Granulócitos/metabolismo , Inflamação , Mamíferos , PPAR alfa , PPAR gama/genética , PPAR gama/metabolismo , Prostaglandina D2/química , Prostaglandina D2/farmacologia , Prostaglandinas , Espécies Reativas de Oxigênio , Dourada/metabolismoRESUMO
Chronic diseases and hematopoietic disorders are associated with dysregulation of the inflammasome. Our group has recently reported the relevance of the inflammasome in the differentiation of hematopoietic stem and progenitor cells. However, the impact of the inflammasome of myeloid cells in the regulation of hematopoiesis is largely unknown. In this study, we used the unique advantages of the zebrafish model to demonstrate that genetic inhibition of macrophage inflammasome resulted in increased number of macrophages in larvae with skin inflammation without affecting erythrocyte and neutrophil counts. Similarly, the inhibition of the neutrophil inflammasome by the same strategy resulted in increased number of neutrophils in larvae with skin inflammation but did not affect erythrocytes and macrophages. Consistently, hyperactivation of the inflammasome in neutrophils in this model promoted neutrophil death, which was recovered by pharmacological inhibition of Gasdermin E. We conclude that the myeloid inflammasome autonomously regulates pyroptotic cell death in chronic inflammation through a Gasdermin E-dependent pathway in zebrafish.
Assuntos
Piroptose , Peixe-Zebra , Animais , Doença Crônica , Inflamassomos/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Neutrófilos/metabolismo , Peixe-Zebra/metabolismoRESUMO
We have tested the usefulness of several commercial anti-CD33 monoclonal antibodies (mAb) to determine the expression and localization of the two CD33 isoforms on several hematopoietic cell lines. The expression of the isoform CD33m, a CD33 transmembrane splice variant lacking the ligand-binding V immunoglobulin (Ig)-like domain, was detected by RT-polymerase chain reaction, western blot, confocal microscopy and flow cytometry on the membrane of several human cell types. CD33m was only detected by the anti-CD33 mAb HIM3-4 on the cell surface, whereas WM53, P67.6, 4D3, HIM3-4, WM54, D3HL60.251 or MY9 detected the CD33M isoform, indicating that HIM3-4 is the only mAb recognizing CD33 C(2) Ig domain. Accordingly, HIM3-4 binding to CD33 did not interfere with the binding of other antibodies against the CD33 V-domain. P67.6 mAb interfered with recognition by the rest of antibodies specific for the V domain. HIM3-4 staining could be increased after the sialidase treatment of all CD33(+) cells. However, this increase was stronger in activated T cells, suggesting a CD33 masking state in this cell population. Confocal microscopy analysis of CD33m HEK 293T-transfected cells revealed that this protein is expressed on the cell membrane and also detected in the Golgi compartment. CD33 is constitutively located outside the lipid raft domains, whereas cross-linked CD33 is highly recruited to this signaling platform. The unique ability of HIM3-4 mAb to detect the masking state of CD33 on different cell lineages makes it a good tool to improve the knowledge of the biological role of this sialic acid-binding Ig-like lectin.
Assuntos
Antígenos CD/biossíntese , Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/biossíntese , Antígenos de Diferenciação Mielomonocítica/imunologia , Mapeamento de Epitopos , Linfócitos/metabolismo , Células Mieloides/metabolismo , Processamento de Proteína Pós-Traducional , Anticorpos Monoclonais/imunologia , Reações Antígeno-Anticorpo , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/metabolismo , Linhagem Celular , Células HL-60 , Humanos , Linfócitos/imunologia , Células Mieloides/imunologia , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Lectina 3 Semelhante a Ig de Ligação ao Ácido SiálicoRESUMO
Nicotinamide adenine dinucleotide (NAD+) is the most important hydrogen carrier in cell redox reactions. It is involved in mitochondrial function and metabolism, circadian rhythm, the immune response and inflammation, DNA repair, cell division, protein-protein signaling, chromatin remodeling and epigenetics. Recently, NAD+ has been recognized as the molecule of life, since, by increasing NAD+ levels in old or sick animals, it is possible to improve their health and lengthen their lifespan. In this review, we summarize the contribution of NAD+ metabolism to inflammation, with special emphasis in the major NAD+ biosynthetic enzyme, nicotinamide phosphoribosyl transferase (NAMPT), and the NAD+-consuming enzyme, poly(ADP-ribose) polymerase (PARP). The extracurricular roles of these enzymes, i.e. the proinflammatory role of NAMPT after its release, and the ability of PARP to promote a novel form of cell death, known as parthanatos, upon hyperactivation are revised and discussed in the context of several chronic inflammatory diseases.
Assuntos
Inflamação/imunologia , Nicotinamida Fosforribosiltransferase/metabolismo , Parthanatos/imunologia , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Doença Crônica , Modelos Animais de Doenças , Humanos , Inflamação/tratamento farmacológico , NAD/metabolismo , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Transdução de Sinais/imunologiaRESUMO
Prostaglandin E2 (PGE2) plays an important role in immune activities in teleost fish, including seabream. However, receptors involved in PGE2 signaling, as well as the pathways activated downstream, are largely unknown. In this study, one ortholog of mammalian PTGER1, PTGER3 and PTGER4, and two of PTGER2 (Ptger2a and Ptger2b) were identified and characterized in gilthead seabream. In silico analysis showed that all these receptors possessed the organization domain of G protein-coupled receptors, with the exception of Ptger2b. The corresponding in vivo studies revealed that they were expressed in all the tissues examined, the highest mRNA levels of ptger1 and ptger3 being observed in the spleen and of ptger2a and ptger4 in the blood. Bacterial infection induced higher mRNA levels of ptger2a, ptger3 and ptger4 in peritoneal exudate (the site of bacterial injection). In addition, head kidney acidophilic granulocytes and macrophages displayed different ptger1, ptger2a, ptger3 and ptger4 expression profiles. Furthermore, in macrophages the expression of the receptors was weakly affected by stimulation with bacterial DNA or with PGE2, while in acidophilic granulocytes stimulation resulted in the upregulation of ptger2a and ptger4. Taken together, these results suggest different roles for seabream PGE2 receptors in the regulation of the immune responses.