RESUMEN
CD8+ T cells provide robust antiviral immunity, but how epitope-specific T cells evolve across the human lifespan is unclear. Here we defined CD8+ T cell immunity directed at the prominent influenza epitope HLA-A*02:01-M158-66 (A2/M158) across four age groups at phenotypic, transcriptomic, clonal and functional levels. We identify a linear differentiation trajectory from newborns to children then adults, followed by divergence and a clonal reset in older adults. Gene profiles in older adults closely resemble those of newborns and children, despite being clonally distinct. Only child-derived and adult-derived A2/M158+CD8+ T cells had the potential to differentiate into highly cytotoxic epitope-specific CD8+ T cells, which was linked to highly functional public T cell receptor (TCR)αß signatures. Suboptimal TCRαß signatures in older adults led to less proliferation, polyfunctionality, avidity and recognition of peptide mutants, although displayed no signs of exhaustion. These data suggest that priming T cells at different stages of life might greatly affect CD8+ T cell responses toward viral infections.
Asunto(s)
Linfocitos T CD8-positivos , Longevidad , Recién Nacido , Humanos , Anciano , Epítopos de Linfocito T/genética , Linfocitos T Citotóxicos , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T/genéticaRESUMEN
As the establishment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell memory in children remains largely unexplored, we recruited convalescent COVID-19 children and adults to define their circulating memory SARS-CoV-2-specific CD4+ and CD8+ T cells prior to vaccination. We analyzed epitope-specific T cells directly ex vivo using seven HLA class I and class II tetramers presenting SARS-CoV-2 epitopes, together with Spike-specific B cells. Unvaccinated children who seroconverted had comparable Spike-specific but lower ORF1a- and N-specific memory T cell responses compared with adults. This agreed with our TCR sequencing data showing reduced clonal expansion in children. A strong stem cell memory phenotype and common T cell receptor motifs were detected within tetramer-specific T cells in seroconverted children. Conversely, children who did not seroconvert had tetramer-specific T cells of predominantly naive phenotypes and diverse TCRαß repertoires. Our study demonstrates the generation of SARS-CoV-2-specific T cell memory with common TCRαß motifs in unvaccinated seroconverted children after their first virus encounter.
Asunto(s)
COVID-19 , SARS-CoV-2 , Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos , Epítopos de Linfocito T , Humanos , Memoria Inmunológica , Receptores de Antígenos de Linfocitos T , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Glicoproteína de la Espiga del CoronavirusRESUMEN
To better understand primary and recall T cell responses during coronavirus disease 2019 (COVID-19), it is important to examine unmanipulated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells. By using peptide-human leukocyte antigen (HLA) tetramers for direct ex vivo analysis, we characterized CD8+ T cells specific for SARS-CoV-2 epitopes in COVID-19 patients and unexposed individuals. Unlike CD8+ T cells directed toward subdominant epitopes (B7/N257, A2/S269, and A24/S1,208) CD8+ T cells specific for the immunodominant B7/N105 epitope were detected at high frequencies in pre-pandemic samples and at increased frequencies during acute COVID-19 and convalescence. SARS-CoV-2-specific CD8+ T cells in pre-pandemic samples from children, adults, and elderly individuals predominantly displayed a naive phenotype, indicating a lack of previous cross-reactive exposures. T cell receptor (TCR) analyses revealed diverse TCRαß repertoires and promiscuous αß-TCR pairing within B7/N105+CD8+ T cells. Our study demonstrates high naive precursor frequency and TCRαß diversity within immunodominant B7/N105-specific CD8+ T cells and provides insight into SARS-CoV-2-specific T cell origins and subsequent responses.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , COVID-19/inmunología , Proteínas de la Nucleocápside de Coronavirus/inmunología , Epítopos Inmunodominantes/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , SARS-CoV-2/inmunología , Adulto , Anciano , Secuencias de Aminoácidos , Linfocitos T CD4-Positivos , Niño , Convalecencia , Proteínas de la Nucleocápside de Coronavirus/química , Epítopos de Linfocito T/química , Epítopos de Linfocito T/inmunología , Femenino , Humanos , Epítopos Inmunodominantes/química , Masculino , Persona de Mediana Edad , Fenotipo , Fosfoproteínas/química , Fosfoproteínas/inmunología , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
Molecular signalling mediated by the phosphatidylinositol-3-kinase (PI3K)-Akt axis is a key regulator of cellular functions. Importantly, alteration of the PI3K-Akt signalling underlies the development of different human diseases, thus prompting the investigation of the pathway as a molecular target for pharmacologic intervention. In this regard, recent studies showed that small molecule inhibitors of PI3K, the upstream regulator of the pathway, reduced the development of inflammation during acute pancreatitis, a highly debilitating and potentially lethal disease. Here we investigated whether a specific reduction of Akt activity, by using either pharmacologic Akt inhibition, or genetic inactivation of the Akt1 isoform selectively in pancreatic acinar cells, is effective in ameliorating the onset and progression of the disease. We discovered that systemic reduction of Akt activity did not protect the pancreas from initial damage and only transiently delayed leukocyte recruitment. However, reduction of Akt activity decreased acinar proliferation and exacerbated acinar-to-ductal metaplasia (ADM) formation, two critical events in the progression of pancreatitis. These phenotypes were recapitulated upon conditional inactivation of Akt1 in acinar cells, which resulted in reduced expression of 4E-BP1, a multifunctional protein of key importance in cell proliferation and metaplasia formation. Collectively, our results highlight the critical role played by Akt1 during the development of acute pancreatitis in the control of acinar cell proliferation and ADM formation. In addition, these results harbour important translational implications as they raise the concern that inhibitors of PI3K-Akt signalling pathways may negatively affect the regeneration of the pancreas. Finally, this work provides the basis for further investigating the potential of Akt1 activators to boost pancreatic regeneration following inflammatory insults. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Asunto(s)
Células Acinares/enzimología , Proliferación Celular , Páncreas Exocrino/enzimología , Conductos Pancreáticos/enzimología , Pancreatitis/enzimología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células Acinares/efectos de los fármacos , Células Acinares/patología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ceruletida , Modelos Animales de Enfermedad , Masculino , Metaplasia , Ratones Endogámicos C57BL , Ratones Noqueados , Páncreas Exocrino/efectos de los fármacos , Páncreas Exocrino/patología , Conductos Pancreáticos/efectos de los fármacos , Conductos Pancreáticos/patología , Pancreatitis/inducido químicamente , Pancreatitis/genética , Pancreatitis/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/análisis , Proteínas Proto-Oncogénicas c-akt/deficiencia , Proteínas Proto-Oncogénicas c-akt/genética , Ratas , Transducción de SeñalRESUMEN
AIMS/HYPOTHESIS: Compared with the general population, individuals with diabetes have a higher risk of developing severe acute pancreatitis, a highly debilitating and potentially lethal inflammation of the exocrine pancreas. In this study, we investigated whether 1-deoxysphingolipids, atypical lipids that increase in the circulation following the development of diabetes, exacerbate the severity of pancreatitis in a diabetic setting. METHODS: We analysed whether administration of an L-serine-enriched diet to mouse models of diabetes, an established method for decreasing the synthesis of 1-deoxysphingolipids in vivo, reduced the severity of acute pancreatitis. Furthermore, we elucidated the molecular mechanisms underlying the lipotoxicity exerted by 1-deoxysphingolipids towards rodent pancreatic acinar cells in vitro. RESULTS: We demonstrated that L-serine supplementation reduced the damage of acinar tissue resulting from the induction of pancreatitis in diabetic mice (average histological damage score: 1.5 in L-serine-treated mice vs 2.7 in the control group). At the cellular level, we showed that L-serine decreased the production of reactive oxygen species, endoplasmic reticulum stress and cellular apoptosis in acinar tissue. Importantly, these parameters, together with DNA damage, were triggered in acinar cells upon treatment with 1-deoxysphingolipids in vitro, suggesting that these lipids are cytotoxic towards pancreatic acinar cells in a cell-autonomous manner. In search of the initiating events of the observed cytotoxicity, we discovered that 1-deoxysphingolipids induced early mitochondrial dysfunction in acinar cells, characterised by ultrastructural alterations, impaired oxygen consumption rate and reduced ATP synthesis. CONCLUSIONS/INTERPRETATION: Our results suggest that 1-deoxysphingolipids directly damage the functionality of pancreatic acinar cells and highlight that an L-serine-enriched diet may be used as a promising prophylactic intervention to reduce the severity of pancreatitis in the context of diabetes.
Asunto(s)
Células Acinares/efectos de los fármacos , Diabetes Mellitus Experimental/metabolismo , Páncreas/efectos de los fármacos , Pancreatitis/metabolismo , Serina/farmacología , Células Acinares/metabolismo , Células Acinares/ultraestructura , Animales , Apoptosis/efectos de los fármacos , Ceruletida/toxicidad , Daño del ADN/efectos de los fármacos , Diabetes Mellitus Experimental/complicaciones , Modelos Animales de Enfermedad , Estrés del Retículo Endoplásmico/efectos de los fármacos , Técnicas In Vitro , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Páncreas/citología , Pancreatitis/etiología , Especies Reactivas de Oxígeno/metabolismo , Índice de Severidad de la Enfermedad , Esfingolípidos/metabolismo , Esfingolípidos/farmacologíaRESUMEN
Proliferation of pancreatic acinar cells is a critical process in the pathophysiology of pancreatic diseases, because limited or defective proliferation is associated with organ dysfunction and patient morbidity. In this context, elucidating the signalling pathways that trigger and sustain acinar proliferation is pivotal to develop therapeutic interventions promoting the regenerative process of the organ. In this study we used genetic and pharmacological approaches to manipulate both local and systemic levels of thyroid hormones to elucidate their role in acinar proliferation following caerulein-mediated acute pancreatitis in mice. In addition, molecular mechanisms mediating the effects of thyroid hormones were identified by genetic and pharmacological inactivation of selected signalling pathways.In this study we demonstrated that levels of the thyroid hormone 3,3',5-triiodo-l-thyronine (T3) transiently increased in the pancreas during acute pancreatitis. Moreover, by using genetic and pharmacological approaches to manipulate both local and systemic levels of thyroid hormones, we showed that T3 was required to promote proliferation of pancreatic acinar cells, without affecting the extent of tissue damage or inflammatory infiltration.Finally, upon genetic and pharmacological inactivation of selected signalling pathways, we demonstrated that T3 exerted its mitogenic effect on acinar cells via a tightly controlled action on different molecular effectors, including histone deacetylase, AKT, and TGFß signalling.In conclusion, our data suggest that local availability of T3 in the pancreas is required to promote acinar cell proliferation and provide the rationale to exploit thyroid hormone signalling to enhance pancreatic regeneration. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Asunto(s)
Células Acinares/metabolismo , Proliferación Celular , Hipertiroidismo/metabolismo , Páncreas Exocrino/metabolismo , Pancreatitis/metabolismo , Triyodotironina/metabolismo , Células Acinares/patología , Animales , Ceruletida , Modelos Animales de Enfermedad , Histona Desacetilasas/metabolismo , Hipertiroidismo/genética , Hipertiroidismo/patología , Yoduro Peroxidasa/deficiencia , Yoduro Peroxidasa/genética , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Páncreas Exocrino/patología , Pancreatitis/inducido químicamente , Pancreatitis/genética , Pancreatitis/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta/deficiencia , Receptor Tipo II de Factor de Crecimiento Transformador beta/genética , Transducción de Señal , Tiroxina/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia ArribaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC), which is the primary cause of pancreatic cancer mortality, is poorly responsive to currently available interventions. Identifying new targets that drive PDAC formation and progression is critical for developing alternative therapeutic strategies to treat this lethal malignancy. Using genetic and pharmacological approaches, we investigated in vivo and in vitro whether uptake of the monoamine serotonin [5-hydroxytryptamine (5-HT)] is required for PDAC development. We demonstrated that pancreatic acinar cells have the ability to readily take up 5-HT in a transport-mediated manner. 5-HT uptake promoted activation of the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1), which is required for transdifferentiation of acinar cells into acinar-to-ductal metaplasia (ADM), a key determinant in PDAC development. Consistent with the central role played by Rac1 in ADM formation, inhibition of the 5-HT transporter Sert (Slc6a4) with fluoxetine reduced ADM formation both in vitro and in vivo in a cell-autonomous manner. In addition, fluoxetine treatment profoundly compromised the stromal reaction and affected the proliferation and lipid metabolism of malignant PDAC cells. We propose that Sert is a promising therapeutic target to counteract the early event of ADM, with the potential to stall the initiation and progression of pancreatic carcinogenesis. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Asunto(s)
Carcinoma Ductal Pancreático/enzimología , Proliferación Celular , Genes ras , Neuropéptidos/metabolismo , Páncreas/enzimología , Neoplasias Pancreáticas/enzimología , Serotonina/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/prevención & control , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Transdiferenciación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Embrión de Pollo , Modelos Animales de Enfermedad , Activación Enzimática , Fluoxetina/farmacología , Predisposición Genética a la Enfermedad , Humanos , Metaplasia , Ratones Endogámicos C57BL , Ratones Transgénicos , Neovascularización Patológica , Páncreas/efectos de los fármacos , Páncreas/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/prevención & control , Fenotipo , Ratas , Proteínas de Transporte de Serotonina en la Membrana Plasmática/efectos de los fármacos , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Transducción de Señal , Triptófano Hidroxilasa/genética , Triptófano Hidroxilasa/metabolismoRESUMEN
OBJECTIVE: Chronic pancreatitis (CP) and autoimmune pancreatitis (AIP) are characterised by different inflammatory processes. If pancreatic inflammation is a prerequisite for autoimmunity is still unclear. AIP is considered mostly a T cell-mediated disease; however, in induction of CP, macrophages play a pivotal role. p21-a member of cyclin-dependent kinase inhibitors-can influence inflammatory processes, in particular can regulate T cell activation and promote macrophage development. We therefore examined the role of p21-mediated inflammation in AIP. DESIGN: We intercrossed lymphotoxin (LT) overexpressing mice (Tg(Ela1-LTa,b))-a model to study AIP development-with p21-deficient mice. Furthermore, we characterised p21 expression in human AIP and non-AIP specimens. RESULTS: p21 deficiency in LT mice (LTp21-/-) prevented early pancreatic injury and reduced inflammation. In acinar cells, diminished proliferation and abrogated activation of non-canonical nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) pathway was observed. In contrast, 12-month-old LT mice with and without p21 had similar inflammatory signatures and T-B cell infiltration. Interestingly, LT and LTp21-/- mice had comparable tertiary lymphoid organs (TLOs), autoantibodies and elevated IgG levels. However, acinar cell proliferation, acinar-to-ductal metaplasia and acinar non-canonical NF-κB pathway activation remained impaired in LTp21-/- pancreata. CONCLUSIONS: Our findings indicate that p21 is crucial for pancreatic inflammation in LT-driven pancreatic injury. p21 is involved in early acinar secretion of inflammatory mediators that attract innate immune cells. However, p21 is not essential for humoral immune response, accountable for autoimmunity. Remarkably, p21 renders acinar cells less susceptible to proliferation and transdifferentiation. We therefore suggest that AIP can also develop independent of chronic inflammatory processes.
Asunto(s)
Enfermedades Autoinmunes/genética , Mutagénesis , Pancreatitis Crónica/genética , Linfocitos T/metabolismo , Quinasas p21 Activadas/genética , Animales , Enfermedades Autoinmunes/complicaciones , Biomarcadores/sangre , Modelos Animales de Enfermedad , Ratones , Ratones Noqueados , Pancreatitis Crónica/complicacionesRESUMEN
Pancreatic fibrosis is the hallmark of chronic pancreatitis, a highly debilitating disease for which there is currently no cure. The key event at the basis of pancreatic fibrosis is the deposition of extracellular matrix proteins by activated pancreatic stellate cells (PSCs). Transforming growth factor ß (TGFß) is a potent profibrotic factor in the pancreas as it promotes the activation of PSC; thus, pharmacologic interventions that effectively reduce TGFß expression harbor considerable therapeutic potential in the treatment of chronic pancreatitis. In this study, we investigated whether TGFß expression is reduced by pharmacologic inhibition of the epigenetic modifiers histone deacetylases (HDACs). To address this aim, chronic pancreatitis was induced in C57BL/6 mice with serial injections of cerulein, and the selective class 1 HDAC inhibitor MS-275 was administered in vivo in a preventive and therapeutic manner. Both MS-275 regimens potently reduced deposition of extracellular matrix and development of fibrosis in the pancreas after 4 weeks of chronic pancreatitis. Reduced pancreatic fibrosis was concomitant with lower expression of pancreatic TGFß and consequent reduced PSC activation. In search of the cell types targeted by the inhibitor, we found that MS-275 treatment abrogated the expression of TGFß in acinar cells stimulated by cerulein treatment. Our study demonstrates that MS-275 is an effective antifibrotic agent in the context of experimental chronic pancreatitis and thus may constitute a valid therapeutic intervention for this severe disease.
Asunto(s)
Benzamidas/administración & dosificación , Inhibidores de Histona Desacetilasas/administración & dosificación , Páncreas/efectos de los fármacos , Pancreatitis Crónica/inducido químicamente , Pancreatitis Crónica/tratamiento farmacológico , Piridinas/administración & dosificación , Factor de Crecimiento Transformador beta/metabolismo , Animales , Benzamidas/farmacología , Línea Celular , Ceruletida/efectos adversos , Modelos Animales de Enfermedad , Fibrosis/prevención & control , Regulación de la Expresión Génica , Inhibidores de Histona Desacetilasas/farmacología , Ratones , Ratones Endogámicos C57BL , Páncreas/patología , Células Estrelladas Pancreáticas/citología , Células Estrelladas Pancreáticas/efectos de los fármacos , Células Estrelladas Pancreáticas/metabolismo , Pancreatitis Crónica/metabolismo , Piridinas/farmacología , RatasRESUMEN
Determining signalling pathways that regulate pancreatic regeneration following pancreatitis is critical for implementing therapeutic interventions. In this study we elucidated the molecular mechanisms underlying the effects of transforming growth factor-ß (TGFß) in pancreatic epithelial cells during tissue regeneration. To this end, we conditionally inactivated TGFß receptor II (TGFß-RII) using a Cre-LoxP system under the control of pancreas transcription factor 1a (PTF1a) promoter, specific for the pancreatic epithelium, and evaluated the molecular and cellular changes in a mouse model of cerulein-induced pancreatitis. We show that TGFß-RII signalling does not mediate the initial acinar cell damage observed at the onset of pancreatitis. However, TGFß-RII signalling not only restricts acinar cell replication during the regenerative phase of the disease but also limits ADM formation in vivo and in vitro in a cell-autonomous manner. Analyses of molecular mechanisms underlying the observed phenotype revealed that TGFß-RII signalling stimulates the expression of cyclin-dependent kinase inhibitors and intersects with the EGFR signalling axis. Finally, TGFß-RII ablation in epithelial cells resulted in increased infiltration of inflammatory cells in the early phases of pancreatitis and increased activation of pancreatic stellate cells in the later stages of pancreatitis, thus highlighting a TGFß-based crosstalk between epithelial and stromal cells regulating the development of pancreatic inflammation and fibrosis. Collectively, our data not only contribute to clarifying the cellular processes governing pancreatic tissue regeneration, but also emphasize the conserved role of TGFß as a tumour suppressor, both in the regenerative process following pancreatitis and in the initial phases of pancreatic cancer.
Asunto(s)
Células Acinares/patología , Páncreas/patología , Pancreatitis/patología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Amilasas/metabolismo , Animales , Carcinoma Ductal Pancreático/patología , Puntos de Control del Ciclo Celular/fisiología , Proliferación Celular/fisiología , Transformación Celular Neoplásica/patología , Células Cultivadas , Ceruletida/toxicidad , Células Epiteliales/patología , Fibrosis/patología , Irritantes/toxicidad , Lipasa/metabolismo , Masculino , Metaplasia/patología , Ratones Noqueados , Ratones Transgénicos , Páncreas/enzimología , Neoplasias Pancreáticas/patología , Pancreatitis/enzimología , Receptor Tipo II de Factor de Crecimiento Transformador betaRESUMEN
Trans-differentiation of pancreatic acinar cells into ductal-like lesions, a process defined as acinar-to-ductal metaplasia (ADM), is observed in the course of organ regeneration following pancreatitis. In addition, ADM is found in association with pre-malignant PanIN lesions and correlates with an increased risk of pancreatic adenocarcinoma (PDAC). Human PDAC samples show down-regulation of p21(WAF1) (/Cip1) , a key regulator of cell cycle and cell differentiation. Here we investigated whether p21 down-regulation is implicated in controlling the early events of acinar cell trans-differentiation and ADM formation. p21-mediated regulation of ADM formation and regression was analysed in vivo during the course of cerulein-induced pancreatitis, using wild-type (WT) and p21-deficient (p21(-/-) ) mice. Biochemical and immunohistochemical methods were used to evaluate disease progression over 2 weeks of the disease and during a recovery phase. We found that p21 was strongly up-regulated in WT acinar cells during pancreatitis, while it was absent in ADM areas, suggesting that p21 down-regulation is associated with ADM formation. In support of this hypothesis, p21(-/-) mice showed a significant increase in number and size of metaplasia. In addition, p21 over-expression in acinar cells reduced ADM formation in vitro, suggesting that the protein regulates the metaplastic transition in a cell-autonomous manner. p21(-/-) mice displayed increased expression and relocalization of ß-catenin both during pancreatitis and in the subsequent recovery phase. Finally, loss of p21 was accompanied by increased DNA damage and development of senescence. Our findings are consistent with a gate-keeper role of p21 in acinar cells to limit senescence activation and ADM formation during pancreatic regeneration.
Asunto(s)
Diferenciación Celular/fisiología , Senescencia Celular/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , Páncreas/patología , Páncreas/fisiopatología , Pancreatitis/patología , Pancreatitis/fisiopatología , Animales , Ciclo Celular/fisiología , Proliferación Celular/fisiología , Ceruletida/efectos adversos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/deficiencia , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Modelos Animales de Enfermedad , Regulación hacia Abajo/fisiología , Técnicas In Vitro , Metaplasia , Ratones , Ratones Noqueados , Pancreatitis/inducido químicamente , Regeneración/fisiología , beta Catenina/fisiologíaRESUMEN
The exocrine pancreas exhibits a distinctive capacity for tissue regeneration and renewal following injury. This regenerative ability has important implications for a variety of disorders, including pancreatitis and pancreatic cancer, diseases associated with high morbidity and mortality. Thus, understanding its underlying mechanisms may help in developing therapeutic interventions. Serotonin has been recognized as a potent mitogen for a variety of cells and tissues. Here we investigated whether serotonin exerts a mitogenic effect in pancreatic acinar cells in three regenerative models, inflammatory tissue injury following pancreatitis, tissue loss following partial pancreatectomy, and thyroid hormone-stimulated acinar proliferation. Genetic and pharmacological techniques were used to modulate serotonin levels in vivo. Acinar dedifferentiation and cell cycle progression during the regenerative phase were investigated over the course of 2 weeks. By comparing acinar proliferation in the different murine models of regeneration, we found that serotonin did not affect the clonal regeneration of mature acinar cells. Serotonin was, however, required for acinar dedifferentiation following inflammation-mediated tissue injury. Specifically, lack of serotonin resulted in delayed up-regulation of progenitor genes and delayed the formation of acinar-to-ductal metaplasia and defective acinar cell proliferation. We identified serotonin-dependent acinar secretion as a key step in progenitor-based regeneration, as it promoted acinar cell dedifferentiation and the recruitment of type 2 macrophages. Finally, we identified a regulatory Hes1-Ptfa axis in the uninjured adult pancreas, activated by zymogen secretion. Our findings indicated that serotonin plays a critical role in the regeneration of the adult pancreas following pancreatitis by promoting the dedifferentiation of acinar cells.
Asunto(s)
Células Acinares/citología , Desdiferenciación Celular/fisiología , Páncreas Exocrino/fisiología , Serotonina/metabolismo , Envejecimiento , Animales , Modelos Animales de Enfermedad , Immunoblotting , Inmunohistoquímica , Metaplasia , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Pancreatitis/patología , RegeneraciónRESUMEN
OBJECTIVE: Serotonin (5-hydroxytryptamine, 5-HT) is a potent bioactive molecule involved in a variety of physiological processes. In this study, the authors analysed whether 5-HT regulates zymogen secretion in pancreatic acinar cells and the development of pancreatic inflammation, a potentially lethal disease whose pathophysiology is not completely understood. METHODS: 5-HT regulation of zymogen secretion was analysed in pancreatic acini isolated from wild-type or tryptophan hydoxylase-1 knock-out (TPH1(-/-)) mice, which lack peripheral 5-HT, and in amylase-secreting pancreatic cell lines. Pancreatitis was induced by cerulein stimulation and biochemical and immunohistochemical methods were used to evaluate disease progression over 2 weeks. RESULTS: Absence and reduced intracellular levels of 5-HT inhibited the secretion of zymogen granules both ex vivo and in vitro and altered cytoskeleton dynamics. In addition, absence of 5-HT resulted in attenuated pro-inflammatory response after induction of pancreatitis. TPH1(-/-) mice showed limited zymogen release, reduced expression of the pro-inflammatory chemokine MCP-1 and minimal leucocyte infiltration compared with wild-type animals. Restoration of 5-HT levels in TPH1(-/-) mice recovered the blunted inflammatory processes observed during acute pancreatitis. However, cellular damage, inflammatory and fibrotic processes accelerated in TPH1(-/-) mice during disease progression. CONCLUSIONS: These results identify a 5-HT-mediated regulation of zymogen secretion in pancreatic acinar cells. In addition, they demonstrate that 5-HT is required for the onset but not for the progression of pancreatic inflammation. These findings provide novel insights into the normal physiology of pancreatic acinar cells and into the pathophysiology of pancreatitis, with potential therapeutic implications.
Asunto(s)
Células Acinares/patología , Amilasas/metabolismo , Precursores Enzimáticos/metabolismo , Pancreatitis/enzimología , Serotonina/fisiología , Células Acinares/enzimología , Citoesqueleto de Actina/patología , Animales , Línea Celular , Ceruletida/efectos adversos , Quimiotaxis de Leucocito , Progresión de la Enfermedad , Fibrosis , Inmunohistoquímica , Inflamación/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pancreatitis/inducido químicamente , Pancreatitis/patologíaRESUMEN
Autoantibodies to multiple targets are found during acute COVID-19. Whether all, or some, persist after 6 months, and their correlation with sustained anti-SARS-CoV-2 immunity, is still controversial. Herein, we measured antibodies to multiple SARS-CoV-2 antigens (Wuhan-Hu-1 nucleoprotein (NP), whole spike (S), spike subunits (S1, S2 and receptor binding domain (RBD)) and Omicron spike) and 102 human proteins with known autoimmune associations, in plasma from healthcare workers 8 months post-exposure to SARS-CoV-2 (n=31 with confirmed COVID-19 disease and n=21 uninfected controls (PCR and anti-SARS-CoV-2 negative) at baseline). IgG antibody responses to SARS-CoV-2 antigens were significantly higher in the convalescent cohort than the healthy cohort, highlighting lasting antibody responses up to 8 months post-infection. These were also shown to be cross-reactive to the Omicron variant spike protein at a similar level to lasting anti-RBD antibodies (correlation r=0.89). Individuals post COVID-19 infection recognised a common set of autoantigens, specific to this group in comparison to the healthy controls. Moreover, the long-term level of anti-Spike IgG was associated with the breadth of autoreactivity post-COVID-19. There were further moderate positive correlations between anti-SARS-CoV-2 responses and 11 specific autoantigens. The most commonly recognised autoantigens were found in the COVID-19 convalescent cohort. Although there was no overall correlation in self-reported symptom severity and anti-SARS-CoV-2 antibody levels, anti-calprotectin antibodies were associated with return to healthy normal life 8 months post infection. Calprotectin was also the most common target for autoantibodies, recognized by 22.6% of the overall convalescent cohort. Future studies may address whether, counter-intuitively, such autoantibodies may play a protective role in the pathology of long-COVID-19.
Asunto(s)
Anticuerpos Antivirales , COVID-19 , Glicoproteína de la Espiga del Coronavirus , Anticuerpos Antivirales/inmunología , Autoanticuerpos/inmunología , Autoantígenos , COVID-19/complicaciones , COVID-19/inmunología , Humanos , Inmunoglobulina G , Complejo de Antígeno L1 de Leucocito/inmunología , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/inmunología , Síndrome Post Agudo de COVID-19RESUMEN
Histone modification is an important mechanism regulating both gene expression and the establishment and maintenance of cellular phenotypes during development. Regulation of histone acetylation via histone acetylases and deacetylases (HDACs) appears to be particularly crucial in determining gene expression patterns. In this study we explored the effect of HDAC inhibition on the life cycle of the human pathogen Giardia lamblia, a highly reduced parasitic protozoan characterized by minimized cellular processes. We found that the HDAC inhibitor FR235222 increased the level of histone acetylation and induced transcriptional regulation of approximately 2% of genes in proliferating and encysting parasites. In addition, our analyses showed that the levels of histone acetylation decreased during differentiation into cysts, the infective stage of the parasite. Importantly, FR235222 treatment during encystation reversed this histone hypo-acetylation and potently blocked the formation of cysts. These results provide the first direct evidence for epigenetic regulation of gene expression in this simple eukaryote. This suggests that regulation of histone acetylation is involved in the control of Giardia stage differentiation, and identifies epigenetic mechanisms as a promising target to prevent Giardia transmission.
Asunto(s)
Epigénesis Genética , Regulación de la Expresión Génica , Giardia lamblia/crecimiento & desarrollo , Giardia lamblia/genética , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Proteínas Protozoarias/metabolismo , Antiprotozoarios/farmacología , Inhibidores Enzimáticos/farmacología , Giardia lamblia/efectos de los fármacos , Péptidos Cíclicos/farmacología , Proteínas Protozoarias/antagonistas & inhibidoresRESUMEN
Chronic pancreatitis is a severe inflammation of the pancreas associated with destruction of the parenchyma, fibrosis, and persistent abdominal pain. Cyclooxygenase-2 (COX-2) and COX-2-derived prostaglandins, key mediators of the inflammatory response, are elevated in patients with chronic pancreatitis. Previous studies investigated COX-2 as a therapeutic target. These reports showed a reduced pathology in COX-2-deficient mice with a better outcome. Here we compared the role of COX-2 in acute and chronic pancreatic inflammation using the same COX-2(-/-) mouse model of cerulein-induced pancreatitis. In a setting of acute pancreatitis, juvenile COX-2(-/-) mice exhibited a reduced histopathological score compared with wild-type littermates; on the contrary, adult mice did not show any difference in the development of the disease. Similarly, in a setting of chronic pancreatitis induced over a period of 4 wk, adult mice of the two strains showed comparable histological score and collagen deposition. However, the abundance of mRNAs coding for profibrotic genes, such as collagen, α-smooth muscle actin, and transforming growth factor-ß was consistently lower in COX-2(-/-) mice. In addition, comparable histological scores and collagen deposition were observed in wild-type mice treated with a COX-2 inhibitor. We conclude that, in contrast to what was observed in the rat pancreatitis models, COX-2 has a limited and age-dependent effect on inflammatory processes in the mouse pancreas. These results suggest that COX-2 modulates the inflammatory process during the development of pancreatitis in a species-specific manner. Thus the pathophysiological roles of COX-2 and its therapeutic implications in patients with pancreatitis should be reexamined.
Asunto(s)
Ciclooxigenasa 2/metabolismo , Páncreas/enzimología , Pancreatitis Crónica/enzimología , Actinas/genética , Actinas/metabolismo , Factores de Edad , Animales , Ceruletida , Colágeno/genética , Colágeno/metabolismo , Ciclooxigenasa 2/deficiencia , Ciclooxigenasa 2/genética , Inhibidores de la Ciclooxigenasa 2/farmacología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Lactonas/farmacología , Ratones , Ratones Noqueados , Páncreas/efectos de los fármacos , Páncreas/patología , Pancreatitis Crónica/inducido químicamente , Pancreatitis Crónica/genética , Pancreatitis Crónica/patología , Pancreatitis Crónica/prevención & control , Especificidad de la Especie , Sulfonas/farmacología , Factores de Tiempo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Identifying the fundamental molecular factors that drive weight gain even in the absence of hypercaloric food intake, is crucial to enable development of novel treatments for the global pandemic of obesity. Here we investigated both adipose tissue-specific and systemic events that underlie the physiological weight gain occurring during early adulthood in mice fed a normocaloric diet. In addition, we used three different genetic models to identify molecular factors that promote physiological weight gain during normocaloric and hypercaloric diets. We demonstrated that normal physiological weight gain was accompanied by an increase in adipose tissue mass and the presence of cellular and metabolic signatures typically found during obesity, including adipocyte hypertrophy, macrophage recruitment into visceral fat and perturbed glucose metabolism. At the molecular level, this was associated with an increase in adipose tissue tryptophan hydroxylase 1 (Tph1) transcripts, the key enzyme responsible for the synthesis of peripheral serotonin. Genetic inactivation of Tph1 was sufficient to limit adipose tissue expansion and associated metabolic alterations. Mechanistically, we discovered that Tph1 inactivation resulted in down-regulation of cyclin-dependent kinase inhibitor p21Waf1/Cip1 expression. Single or double ablation of Tph1 and p21 were equally effective in preventing adipocyte expansion and systemic perturbation of glucose metabolism, upon both normocaloric and hypercaloric diets. Our results suggest that serotonin and p21 act as a central molecular determinant of weight gain and associated metabolic alterations, and highlights the potential of targeting these molecules as a pharmacologic approach to prevent the development of obesity.
Asunto(s)
Tejido Adiposo/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Dieta Saludable/métodos , Eliminación de Gen , Obesidad/metabolismo , Serotonina/biosíntesis , Transducción de Señal/genética , Adipocitos/patología , Animales , Tamaño de la Célula , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Dieta Alta en Grasa , Glucosa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/genética , Triptófano Hidroxilasa/genética , Triptófano Hidroxilasa/metabolismo , Aumento de Peso/genéticaRESUMEN
Metabolic dysfunction, dysregulated differentiation, and atrophy of skeletal muscle occur as part of a cluster of abnormalities associated with the development of Type 2 diabetes mellitus (T2DM). Recent interest has turned to the attention of the role of 1-deoxysphingolipids (1-DSL), atypical class of sphingolipids which are found significantly elevated in patients diagnosed with T2DM but also in the asymptomatic population who later develop T2DM. In vitro studies demonstrated that 1-DSL have cytotoxic properties and compromise the secretion of insulin from pancreatic beta cells. However, the role of 1-DSL on the functionality of skeletal muscle cells in the pathophysiology of T2DM still remains unclear. This study aimed to investigate whether 1-DSL are cytotoxic and disrupt the cellular processes of skeletal muscle precursors (myoblasts) and differentiated cells (myotubes) by performing a battery of in vitro assays including cell viability adenosine triphosphate assay, migration assay, myoblast fusion assay, glucose uptake assay, and immunocytochemistry. Our results demonstrated that 1-DSL significantly reduced the viability of myoblasts in a concentration and time-dependent manner, and induced apoptosis as well as cellular necrosis. Importantly, myoblasts were more sensitive to the cytotoxic effects induced by 1-DSL rather than by saturated fatty acids, such as palmitate, which are critical mediators of skeletal muscle dysfunction in T2DM. Additionally, 1-DSL significantly reduced the migration ability of myoblasts and the differentiation process of myoblasts into myotubes. 1-DSL also triggered autophagy in myoblasts and significantly reduced insulin-stimulated glucose uptake in myotubes. These findings demonstrate that 1-DSL directly compromise the functionality of skeletal muscle cells and suggest that increased levels of 1-DSL observed during the development of T2DM are likely to contribute to the pathophysiology of muscle dysfunction detected in this disease.
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Movimiento Celular/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Mioblastos Esqueléticos/efectos de los fármacos , Esfingolípidos/farmacología , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Línea Celular , Glucosa/metabolismo , Resistencia a la Insulina/fisiología , Ratones , Músculo Esquelético/metabolismo , Mioblastos Esqueléticos/metabolismoRESUMEN
Synthesis of glucosylceramide via glucosylceramide synthase (GCS) is a crucial event in higher eukaryotes, both for the production of complex glycosphingolipids and for regulating cellular levels of ceramide, a potent antiproliferative second messenger. In this study, we explored the dependence of the early branching eukaryote Giardia lamblia on GCS activity. Biochemical analyses revealed that the parasite has a GCS located in endoplasmic reticulum (ER) membranes that is active in proliferating and encysting trophozoites. Pharmacological inhibition of GCS induced aberrant cell division, characterized by arrest of cytokinesis, incomplete cleavage furrow formation, and consequent block of replication. Importantly, we showed that increased ceramide levels were responsible for the cytokinesis arrest. In addition, GCS inhibition resulted in prominent ultrastructural abnormalities, including accumulation of cytosolic vesicles, enlarged lysosomes, and clathrin disorganization. Moreover, anterograde trafficking of the encystations-specific protein CWP1 was severely compromised and resulted in inhibition of stage differentiation. Our results reveal novel aspects of lipid metabolism in G. lamblia and specifically highlight the vital role of GCS in regulating cell cycle progression, membrane trafficking events, and stage differentiation in this parasite. In addition, we identified ceramide as a potent bioactive molecule, underscoring the universal conservation of ceramide signaling in eukaryotes.
Asunto(s)
Ciclo Celular/fisiología , Diferenciación Celular/fisiología , Membrana Celular/metabolismo , Giardia lamblia/fisiología , Glucosilceramidas/biosíntesis , Secuencia de Aminoácidos , Animales , Transporte Biológico/fisiología , Ceramidas/metabolismo , Ceramidas/farmacología , Clatrina/metabolismo , Retículo Endoplásmico/enzimología , Giardia lamblia/efectos de los fármacos , Giardia lamblia/ultraestructura , Glucosiltransferasas/metabolismo , Humanos , Metabolismo de los Lípidos , Meperidina/análogos & derivados , Meperidina/farmacología , Datos de Secuencia Molecular , Alineación de Secuencia , Esfingolípidos/metabolismo , Trofozoítos/metabolismoRESUMEN
Type 2 diabetes mellitus (T2DM) is a disease associated with dysfunctional metabolic processes that lead to abnormally high levels of blood glucose. Preceding the development of T2DM is insulin resistance (IR), a disorder associated with suppressed or delayed responses to insulin. The effects of this response are predominately mediated through aberrant cell signalling processes and compromised glucose uptake into peripheral tissue including adipose, liver and skeletal muscle. Moreover, a major factor considered to be the cause of IR is endoplasmic reticulum (ER) stress. This subcellular organelle plays a pivotal role in protein folding and processes that increase ER stress, leads to maladaptive responses that result in cell death. Recently, zinc and the proteins that transport this metal ion have been implicated in the ER stress response. Specifically, the ER-specific zinc transporter ZIP7, coined the "gate-keeper" of zinc release from the ER into the cytosol, was shown to be essential for maintaining ER homeostasis in intestinal epithelium and myeloid leukaemia cells. Moreover, ZIP7 controls essential cell signalling pathways similar to insulin and activates glucose uptake in skeletal muscle. Accordingly, ZIP7 may be essential for the control of ER localized zinc and mechanisms that disrupt this process may lead to ER-stress and contribute to IR. Accordingly, understanding the mechanisms of ZIP7 action in the context of IR may provide opportunities to develop novel therapeutic options to target this transporter in the treatment of IR and subsequent T2DM.