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Introduction: The role of suppressor of cytokine signaling (SOCS)2 in anti-infective bacterial immunity has been poorly investigated compared to other members of the SOCS family. Methods: We characterized the previously identified loss of function R96C point mutation of SOCS2 using a genome-edited mouse model that resumes the phenotype of Socs2 knockout mice. The response of macrophages to TLR-ligands and Staphylococcus aureus was examined. Results and discussion: Conversely to previously published data using human monocyte-derived macrophages, the stimulation of bone-marrow-derived macrophages with various TLR ligands did not show any difference according to the SOCS2 variant. Upregulation of IL-6 and TNF-α pro-inflammatory cytokines production was only seen when the SOCS2 expression was promoted by the culture of macrophages in the presence of GM-CSF. Furthermore, we showed that the SOCS2 point mutation is associated with heightened STAT5 phosphorylation in a short time frame upon GM-CSF incubation. In mice, recruitment of neutrophil and F4/80int Ly6C+ inflammatory macrophage, as well as IFN-γ and IL-10 concentrations, are significantly increased upon S. aureus peritoneal infection. Altogether, these data support the idea that by lowering the pro-inflammatory environment, SOCS2 favors better control of bacterial burden during a systemic infection caused by S. aureus.
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Macrófagos , Ratones Noqueados , Infecciones Estafilocócicas , Staphylococcus aureus , Proteínas Supresoras de la Señalización de Citocinas , Animales , Staphylococcus aureus/inmunología , Ratones , Proteínas Supresoras de la Señalización de Citocinas/genética , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/genética , Receptores Toll-Like/metabolismo , Receptores Toll-Like/genética , Citocinas/metabolismo , Mutación con Pérdida de Función , Inflamación/inmunología , Inflamación/genética , Ratones Endogámicos C57BL , Ligandos , HumanosRESUMEN
This study evaluated the influence of a temporary nutritional protein restriction (NPR) performed, under commercial conditions, in prepubertal female lambs on first lactation milk production traits and the inflammatory response triggered by an inflammatory challenge of the. From 40 Assaf female lambs, we defined a control group (Cn = 20), which received a standard diet for replacement lambs and the NPR group (n = 20), which received the same diet but without soybean meal between 3 and 5 months of age. About 150 days after lambing, 24 of these ewes (13 NPR, 11C) were subjected to an intramammary infusion of E. coli lipopolysaccharide (LPS). Our dynamic study identified indicator traits of local (SCC) and systemic (rectal Ta, IL-6, CXCL8, IL-10, IL-36RA, VEGF-A) response to the LPS challenge. The NPR did not show significant effects on milk production traits and did not affect the SCC and rectal Ta after the LPS challenge. However, the NPR had a significant influence on 8 of the 14 plasma biomarkers analysed, in all the cases with higher relative values in the C group. The effects observed on VEGF-A (involved in vasculogenesis during mammary gland development and vascular permeability) and IL-10 (a regulatory cytokine classically known by its anti-inflammatory action) are the most remarkable to explain the differences found between groups. Whereas further studies should be undertaken to confirm these results, our findings are of interest considering the current concern about the future world's demand for protein and the need for animal production systems to evolve toward sustainability.
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Interleucina-10 , Leche , Animales , Ovinos , Femenino , Leche/metabolismo , Interleucina-10/metabolismo , Lipopolisacáridos/farmacología , Lipopolisacáridos/metabolismo , Escherichia coli , Factor A de Crecimiento Endotelial Vascular/metabolismo , Lactancia/fisiología , Oveja Doméstica , Proteínas en la Dieta/metabolismoRESUMEN
Concurrent infections with multiple pathogens are often described in cattle with respiratory illness. However, how the host-pathogen interactions influence the clinical outcome has been only partially explored in this species. Influenza D virus (IDV) was discovered in 2011. Since then, IDV has been detected worldwide in different hosts. A significant association between IDV and bacterial pathogens in sick cattle was shown in epidemiological studies, especially with Mycoplasma bovis. In an experimental challenge, IDV aggravated M. bovis-induced pneumonia. However, the mechanisms through which IDV drives an increased susceptibility to bacterial superinfections remain unknown. Here, we used the organotypic lung model precision-cut lung slices to study the interplay between IDV and M. bovis coinfection. Our results show that a primary IDV infection promotes M. bovis superinfection by increasing the bacterial replication and the ultrastructural damages in lung pneumocytes. In our model, IDV impaired the innate immune response triggered by M. bovis by decreasing the expression of several proinflammatory cytokines and chemokines that are important for immune cell recruitment and the bacterial clearance. Stimulations with agonists of cytosolic helicases and Toll-like receptors (TLRs) revealed that a primary activation of RIG-I/MDA5 desensitizes the TLR2 activation, similar to what was observed with IDV infection. The cross talk between these two pattern recognition receptors leads to a nonadditive response, which alters the TLR2-mediated cascade that controls the bacterial infection. These results highlight innate immune mechanisms that were not described for cattle so far and improve our understanding of the bovine host-microbe interactions and IDV pathogenesis. IMPORTANCE Since the spread of the respiratory influenza D virus (IDV) infection to the cattle population, the question about the impact of this virus on bovine respiratory disease (BRD) remains still unanswered. Animals affected by BRD are often coinfected with multiple pathogens, especially viruses and bacteria. In particular, viruses are suspected to enhance secondary bacterial superinfections. Here, we use an ex vivo model of lung tissue to study the effects of IDV infection on bacterial superinfections. Our results show that IDV increases the susceptibility to the respiratory pathogen Mycoplasma bovis. In particular, IDV seems to activate immune pathways that inhibit the innate immune response against the bacteria. This may allow M. bovis to increase its proliferation and to delay its clearance from lung tissue. These results suggest that IDV could have a negative impact on the respiratory pathology of cattle.
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Enfermedades de los Bovinos , Interacciones Microbiota-Huesped , Infecciones por Mycoplasma , Infecciones por Orthomyxoviridae , Transducción de Señal , Thogotovirus , Animales , Bovinos , Enfermedades de los Bovinos/inmunología , Enfermedades de los Bovinos/virología , Pulmón/inmunología , Pulmón/microbiología , Pulmón/virología , Mycoplasma bovis/inmunología , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/veterinaria , Infecciones por Orthomyxoviridae/virología , Transducción de Señal/inmunología , Sobreinfección/inmunología , Sobreinfección/veterinaria , Receptor Toll-Like 2 , Interacciones Microbiota-Huesped/inmunología , Infecciones por Mycoplasma/inmunología , Infecciones por Mycoplasma/virologíaRESUMEN
Type 2 diabetes is associated with an inflammatory phenotype in the pancreatic islets. We previously demonstrated that proinflammatory cytokines potently activate the tryptophan/kynurenine pathway (TKP) in INS-1 cells and in normal rat islets. Here we examined: (1) the TKP enzymes expression in the diabetic GK islets; (2) the TKP enzymes expression profiles in the GK islets before and after the onset of diabetes; (3) The glucose-stimulated insulin secretion (GSIS) in vitro in GK islets after KMO knockdown using specific morpholino-oligonucleotides against KMO or KMO blockade using the specific inhibitor Ro618048; (4) The glucose tolerance and GSIS after acute in vivo exposure to Ro618048 in GK rats. We report a remarkable induction of the kmo gene in GK islets and in human islets exposed to proinflammatory conditions. It occurred prominently in beta cells. The increased expression and activity of KMO reflected an acquired adaptation. Both KMO knockdown and specific inhibitor Ro618048 enhanced GSIS in vitro in GK islets. Moreover, acute administration of Ro618048 in vivo improved glucose tolerance, GSIS and basal blood glucose levels in GK rats. These results demonstrate that targeting islet TKP is able to correct defective GSIS. KMO inhibition could represent a potential therapeutic strategy for type 2 diabetes.
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Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Animales , Glucemia/metabolismo , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glucosa/metabolismo , Glucosa/farmacología , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Quinurenina/metabolismo , Quinurenina 3-Monooxigenasa/metabolismo , Morfolinos , Ratas , Ratas Wistar , Triptófano/metabolismoRESUMEN
The mammary gland is unique in female mammals. Mammary tissue undergoes development and remodeling during lactation, a stage associated with high susceptibility to bacterial infections, inducing an inflammatory condition called mastitis. Although the immune response of the mammary gland has been the subject of intense research to improve prevention and treatment efficacy, the precise definition of its immune composition at this particular physiological stage is still missing. We combined single-cell RNA-Seq, flow cytometry, and three-dimensional confocal microscopy techniques to characterize the immune landscape of lactating murine mammary tissue. Macrophages dominated the immune cell repertoire and could be subdivided into at least two subsets: ductal and stromal macrophages. Ductal macrophages represented approximately 80% of the total CD45pos immune cells and co-expressed F4/80 and CD11c, with high levels of MHC class II molecules. They were strategically poised below the alveolar basal cells in contact with the myoepithelial cell network. Adaptive T and B lymphocytes were remarkably less numerous at this stage, which could explain the limited efficacy of vaccination against mastitis. These results support the view that new strategies to increase mammary immunity and prevent mastitis should be devised.
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Lactancia/inmunología , Macrófagos/inmunología , Glándulas Mamarias Animales/inmunología , Animales , Femenino , Ratones , Ratones Endogámicos C57BLRESUMEN
Impaired type I interferons (IFNs) production or signaling have been associated with severe COVID-19, further promoting the evaluation of recombinant type I IFNs as therapeutics against SARS-CoV-2 infection. In the Syrian hamster model, we show that intranasal administration of IFN-α starting one day pre-infection or one day post-infection limited weight loss and decreased viral lung titers. By contrast, intranasal administration of IFN-α starting at the onset of symptoms three days post-infection had no impact on the clinical course of SARS-CoV-2 infection. Our results provide evidence that early type I IFN treatment is beneficial, while late interventions are ineffective, although not associated with signs of enhanced disease.
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Antivirales/administración & dosificación , Tratamiento Farmacológico de COVID-19 , Interferón Tipo I/administración & dosificación , Administración Intranasal , Animales , Chlorocebus aethiops , Cricetinae , Modelos Animales de Enfermedad , Mesocricetus , SARS-CoV-2RESUMEN
Vaccination against bovine mastitis lags behind despite high demand from the dairy industry and margin for efficacy improvement. We previously compared two immunization protocols against E. coli using either only the intramuscular route or a combination of intramuscular and mammary ductal routes, also known as 'prime and pull' strategy. A homologous mammary challenge during the memory phase showed that immunization favorably modified the mastitis course, notably in locally immunized cows in comparison to intramuscular and control adjuvant-only groups. Here, we performed whole-blood profiling through RNA-seq transcriptome and plasma cytokine 15-plex analyses at time points of the E. coli mastitis that showed significant clinical and laboratory differences among the groups. Diminished production of inflammatory cytokines and increased IFNγ were detected in the blood of immunized cows, where a T lymphocyte activation profile was evidenced at 12-h post infection. Acute phase neutropenia was less severe in these cows, and pathways related to neutrophil diapedesis and monocyte activation were also present. Furthermore, three intramammary-immunized cows showing faster healing and shorter mastitis duration had gene profiles that differed from their counterparts, but without any clue for the mastitis susceptibility difference. Inasmuch, when gene expression of CD4 T cells was assessed in mammary tissue, enrichment of IL-17-associated pathways was identified in the quarters of intramammary-immunized cows not only after challenge but also in the control quarters that were not infected. These findings indicate that local immunization mobilizes protective mechanisms that rely on the settlement of type 3 immunity-related CD4 T cells prior to infection.
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OBJECTIVE: Type 2 diabetes (T2D) occurs by deterioration in pancreatic ß-cell function and/or progressive loss of pancreatic ß-cell mass under the context of insulin resistance. α7 nicotinic acetylcholine receptor (nAChR) may contribute to insulin sensitivity but its role in the pathogenesis of T2D remains undefined. We investigated whether the systemic lack of α7 nAChR was sufficient to impair glucose homeostasis. METHODS: We used an α7 nAChR knock-out (α7-/-) mouse model fed a standard chow diet. The effects of the lack of α7 nAChR on islet mass, insulin secretion, glucose and insulin tolerance, body composition, and food behaviour were assessed in vivo and ex vivo experiments. RESULTS: Young α7-/- mice display a chronic mild high glycemia combined with an impaired glucose tolerance and a marked deficit in ß-cell mass. In addition to these metabolic disorders, old mice developed adipose tissue inflammation, elevated plasma free fatty acid concentrations and presented glycolytic muscle insulin resistance in old mice. Finally, α7-/- mice, fed a chow diet, exhibited a late-onset excessive gain in body weight through increased fat mass associated with higher food intake. CONCLUSION: Our work highlights the important role of α7 nAChR in glucose homeostasis. The constitutive lack of α7 nAChR suggests a novel pathway influencing the pathogenesis of T2D.
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Intolerancia a la Glucosa/genética , Hiperglucemia/genética , Resistencia a la Insulina , Receptor Nicotínico de Acetilcolina alfa 7/genética , Animales , Línea Celular , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Femenino , Eliminación de Gen , Glucosa/metabolismo , Intolerancia a la Glucosa/metabolismo , Hiperglucemia/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratas , Receptor Nicotínico de Acetilcolina alfa 7/metabolismoRESUMEN
Islet inflammation is associated with defective ß cell function and mass in type 2 diabetes (T2D). Glycogen synthase kinase 3 (GSK3) has been identified as an important regulator of inflammation in different diseased conditions. However, the role of GSK3 in islet inflammation in the context of diabetes remains unexplored. In this study, we investigated the direct implication of GSK3 in islet inflammation in vitro and tested the impact of GSK3 inhibition in vivo, on the reduction of islet inflammation, and the improvement of glucose metabolism in the Goto-Kakizaki (GK) rat, a spontaneous model of T2D. GK rats were chronically treated with infra-therapeutic doses of lithium, a widely used inhibitor of GSK3. We analyzed parameters of glucose homeostasis as well as islet inflammation and fibrosis in the endocrine pancreas. Ex vivo, we tested the impact of GSK3 inhibition on the autonomous inflammatory response of non-diabetic rat and human islets, exposed to a mix of pro-inflammatory cytokines to mimic an inflammatory environment. Treatment of young GK rats with lithium prevented the development of overt diabetes. Lithium treatment resulted in reduced expression of pro-inflammatory cytokines in the islets. It decreased islet fibrosis and partially restored the glucose-induced insulin secretion in GK rats. Studies in non-diabetic human and rat islets exposed to inflammatory environment revealed the direct implication of GSK3 in the islet autonomous inflammatory response. We show for the first time, the implication of GSK3 in islet inflammation and suggest this enzyme as a viable target to treat diabetes-associated inflammation.
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Diabetes Mellitus Tipo 2/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Islotes Pancreáticos/metabolismo , Animales , Modelos Animales de Enfermedad , Fibrosis , Glucosa/metabolismo , Humanos , Inflamación , Secreción de Insulina , Masculino , Ratas , Ratas WistarRESUMEN
Down syndrome is characterized by premature aging and dementia with neurological features that mimic those found in Alzheimer's disease. This pathology in Down syndrome could be related to inflammation, which plays a role in other neurodegenerative diseases. We previously found a link between the NFkB pathway, long considered a prototypical proinflammatory signaling pathway, and the dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A). DYRK1A is associated with early onset of Alzheimer's disease in Down syndrome patients. Here, we sought to determine the role of DYRK1A on regulation of the NFkB pathway in the mouse brain. We found that over-expression of Dyrk1A (on a C57BL/6J background) stabilizes IκBα protein levels by inhibition of calpain activity and increases cytoplasmic p65 sequestration in the mouse brain. In contrast, Dyrk1A-deficient mice (on a CD1 background) have decreased IκBα protein levels with an increased calpain activity and decreased cytoplasmic p65 sequestration in the brain. Taken together, our results demonstrate a role of DYRK1A in regulation of the NFkB pathway. However, decreased IκBα and DYRK1A protein levels associated with an increased calpain activity were found in the brains of mice over-expressing Dyrk1A after lipopolysaccharide treatment. Although inflammation induced by lipopolysaccharide treatment has a positive effect on calpastatin and a negative effect on DYRK1A protein level, a positive effect on microglial activation is maintained in the brains of mice over-expressing Dyrk1A.
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Encéfalo/efectos de los fármacos , Inflamación/inducido químicamente , Lipopolisacáridos/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Enfermedad de Alzheimer/patología , Animales , Encéfalo/metabolismo , Calpaína/metabolismo , Síndrome de Down/metabolismo , Inflamación/metabolismo , Ratones , Fosforilación/efectos de los fármacos , Proteínas tau/metabolismo , Quinasas DyrKRESUMEN
Actin dynamics in pancreatic ß-cells is involved in insulin exocytosis but the molecular mechanisms of this dynamics and its role in biphasic insulin secretion in pancreatic ß-cells is largely unknown. Moreover, the impact of a glucotoxic environment on the sub-cortical actin network dynamics is poorly studied. In this study, we investigate the behavior of insulin granules and the subcortical actin network dynamics in INS-1 832/13 ß-cells submitted to a normal or glucotoxic environment. Our results show that glucose stimulation leads to a reorganization of the subcortical actin network with a rupture of its interactions with t-SNARE proteins (Syntaxin 1A and SNAP-25), promoting insulin secretion in INS-1 832/13 ß-cells. Prolonged exposure of INS-1 832/13 ß-cells to high-glucose levels (glucotoxicity) leads to the densification of the cortical actin network, which prevents its reorganization under acute glucose, and diminishes the glucose-stimulated insulin secretion, as shown by the decreased number of fusion events. The most interesting in our results is the partial restoration by GLP-1 of the insulin secretion ability from high-glucose treated INS-1 832/13 cells. This improved insulin exocytosis is associated with partial restored actin dynamics and fusion events during the two phases of the secretion, with a preferential involvement of Epac2 signaling in the first phase and a rather involvement of PKA signaling in the second phase of insulin exocytosis. All these data provide some new insights into the mechanism by which current therapeutics may be improving insulin secretion.
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Actinas/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Glucosa/farmacología , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Transducción de Señal/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/patología , Animales , Línea Celular Tumoral , Exocitosis/efectos de los fármacos , Glucosa/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células Secretoras de Insulina/patología , Masculino , Ratas , Ratas WistarRESUMEN
Desminopathies, a subgroup of myofibrillar myopathies (MFMs), the progressive muscular diseases characterized by the accumulation of granulofilamentous desmin-positive aggregates, result from mutations in the desmin gene (DES), encoding a muscle-specific intermediate filament. Desminopathies often lead to severe disability and premature death from cardiac and/or respiratory failure; no specific treatment is currently available. To identify drug-targetable pathophysiological pathways, we performed pharmacological studies in C2C12 myoblastic cells expressing mutant DES. We found that inhibition of the Rac1 pathway (a G protein signaling pathway involved in diverse cellular processes), antioxidant treatment, and stimulation of macroautophagy reduced protein aggregation by up to 75% in this model. Further, a combination of two or three of these treatments was more effective than any of them alone. These results pave the way towards the development of the first treatments for desminopathies and are potentially applicable to other muscle or brain diseases associated with abnormal protein aggregation.
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Antioxidantes/farmacología , Autofagia , Cardiomiopatías/metabolismo , Desmina/metabolismo , Distrofias Musculares/metabolismo , Animales , Cardiomiopatías/patología , Línea Celular , Desmina/genética , Cinética , Ratones , Modelos Biológicos , Distrofias Musculares/patología , Transducción de Señal , Tocoferoles/farmacologíaRESUMEN
Mutations within the human desmin gene are responsible for a subcategory of myofibrillar myopathies called desminopathies. However, a single inherited mutation can produce different phenotypes within a family, suggesting that environmental factors influence disease states. Although several mouse models have been used to investigate organ-specific desminopathies, a more general mechanistic perspective is required to advance our knowledge toward patient treatment. To improve our understanding of disease pathology, we have developed cellular models to observe desmin behaviour in early stages of disease pathology, e.g., upon formation of cytoplasmic desmin aggregates, within an isogenic background. We cloned the wildtype and three mutant desmin cDNAs using a Tet-On Advanced® expression system in C2C12 cells. Mutations were selected based on positioning within desmin and capacity to form aggregates in transient experiments, as follows: DesS46Y (head domain; low aggregation), DesD399Y (central rod domain; high aggregation), and DesS460I (tail domain; moderate aggregation). Introduction of these proteins into a C2C12 background permitted us to compare between desmin variants as well as to determine the role of external stress on aggregation. Three different types of stress, likely encountered during muscle activity, were introduced to the cell models-thermal (heat shock), redox-associated (H2O2 and cadmium chloride), and mechanical (stretching) stresses-after which aggregation was measured. Cells containing variant DesD399Y were more sensitive to stress, leading to marked cytoplasmic perinuclear aggregations. We then evaluated the capacity of biochemical compounds to prevent this aggregation, applying dexamethasone (an inducer of heat shock proteins), fisetin or N-acetyl-L-cysteine (antioxidants) before stress induction. Interestingly, N-acetyl-L-cysteine pre-treatment prevented DesD399Y aggregation during most stress. N-acetyl-L-cysteine has recently been described as a promising antioxidant in myopathies linked to selenoprotein N or ryanodin receptor defects. Our findings indicate that this drug warrants further study in animal models to speed its potential development as a therapy for DesD399Y-linked desminopathies.
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Acetilcisteína/metabolismo , Cardiomiopatías/metabolismo , Desmina/metabolismo , Distrofias Musculares/metabolismo , Estrés Fisiológico , Acetilcisteína/farmacología , Animales , Cardiomiopatías/genética , Línea Celular , Codón , Citoesqueleto/genética , Citoesqueleto/metabolismo , Desmina/genética , Modelos Animales de Enfermedad , Estrés del Retículo Endoplásmico , Expresión Génica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Ratones , Distrofias Musculares/genética , Mutación , Unión Proteica , Estrés Fisiológico/efectos de los fármacosRESUMEN
Patients with sickle cell disease suffer from painful crises associated with disseminated vaso-occlusions, increased circulating erythrocyte microparticles (MPs), and thrombospondin-1 (TSP1). MPs are submicron membrane vesicles shed by compromised or activated cells. We hypothesized that TSP1 mediates MP shedding and participates in vaso-occlusions. We injected TSP1 to transgenic SAD mice with sickle cell disease and characterized circulating phosphatidylserine+ MPs by FACS. TSP1 stimulated MPs in plasma and initiated vaso-occlusions within minutes. In vitro, TSP1 triggered rapid erythrocyte conversion into spicule-covered echinocytes, followed by MP shedding. MP shedding was recapitulated by peptides derived from the TSP1 carboxyterminus. We purified MPs shed by erythrocytes in vitro and administered them back to SAD mice. MPs triggered immediate renal vaso-occlusions. In vitro, MPs triggered the production of radical oxygen species by endothelial monolayers, favored erythrocyte adhesion, and induced endothelial apoptosis. MPs also compromised vasodilation in perfused microvessels. These effects were inhibited by saturating MP phosphatidylserine with annexin-V, or with inhibitors of endothelial ROS production. We conclude that TSP1 triggers erythrocyte MP shedding. These MPs induce endothelial injury and facilitate acute vaso-occlusive events in transgenic SAD mice. This work supports a novel concept that toxic erythrocyte MPs may connect sickle cell anemia to vascular disease.