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A low-grade and persistent inflammation, which is the hallmark of obesity, requires the participation of NLRP3 and cell death. During Mycobacterium tuberculosis infection, NLRP3 signaling is important for bacterial killing by macrophages in vitro but was shown to be dispensable for host protection in vivo. We hypothesized that during obesity-tuberculosis (TB) comorbidity, NLRP3 signaling might play a detrimental role by inducing excessive inflammation. We employed a model of high-fat-diet-induced obesity, followed by M. tuberculosis infection in C57BL/6 mice. Obese mice presented increased susceptibility to infection and pulmonary immunopathology compared to lean mice. Using treatment with NLRP3 antagonist and Nlrp3-/- mice, we showed that NLRP3 signaling promoted cell death, with no effect in bacterial loads. The levels of palmitate were higher in the lungs of obese infected mice compared to lean counterparts, and we observed that this lipid increased M. tuberculosis-induced macrophage death in vitro, which was dependent on NLRP3 and caspase-1. At the chronic phase, although lungs of obese Nlrp3-/- mice showed an indication of granuloma formation compared to obese wild-type mice, there was no difference in the bacterial load. Our findings indicate that NLRP3 may be a potential target for host-directed therapy to reduce initial and severe inflammation-mediated disease and to treat comorbidity-associated TB. © 2022 The Pathological Society of Great Britain and Ireland.
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Mycobacterium tuberculosis , Tuberculosis , Ratones , Animales , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Palmitatos/metabolismo , Ratones Endogámicos C57BL , Tuberculosis/patología , Pulmón/patología , Inflamación/patología , Obesidad/metabolismo , Muerte Celular , ComorbilidadRESUMEN
Lipid and cholinergic mediators are inflammatory regulators, but their role in the immunopathology of COVID-19 is still unclear. Here, we used human blood and tracheal aspirate (TA) to investigate whether acetylcholine (Ach), fatty acids (FAs), and their derived lipid mediators (LMs) are associated with COVID-19 severity. First, we analyzed the perturbation profile induced by SARS-CoV-2 infection in the transcriptional profile of genes related to the ACh and FA/LM pathways. Blood and TA were used for metabolomic and lipidomic analyses and for quantification of leukocytes, cytokines, and ACh. Differential expression and coexpression gene network data revealed a unique transcriptional profile associated with ACh and FA/LM production, release, and cellular signaling. Transcriptomic data were corroborated by laboratory findings: SARS-CoV-2 infection increased plasma and TA levels of arachidonic acid, 5-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid, 11-hydroxy-5Z,8Z,12E,14Z-eicosatetraenoic acid, and ACh. TA samples also exhibited high levels of PGE2, thromboxane B2, 12-oxo-5Z,8Z,10E,14Z-eicosatetraenoic acid, and 6-trans-leukotriene B4 Bioinformatics and experimental approaches demonstrated robust correlation between transcriptional profile in Ach and FA/LM pathways and parameters of severe COVID-19. As expected, the increased neutrophil-to-lymphocyte ratio, neutrophil counts, and cytokine levels (IL-6, IL-10, IL-1ß, and IL-8) correlated with worse clinical scores. Glucocorticoids protected severe and critical patients and correlated with reduced Ach levels in plasma and TA samples. We demonstrated that pulmonary and systemic hyperinflammation in severe COVID-19 are associated with high levels of Ach and FA/LM. Glucocorticoids favored the survival of patients with severe/critical disease, and this effect was associated with a reduction in ACh levels.
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Acetilcolina , COVID-19 , Ácido Araquidónico , Ácidos Araquidónicos/farmacología , Ácidos Grasos , Glucocorticoides , Humanos , SARS-CoV-2RESUMEN
COVID-19 has a broad spectrum of clinical manifestations associated with the host immune response heterogeneity. Despite the advances in COVID-19 research, it is still crucial to seek a panel of molecular markers that enable accurate stratification of COVID-19 patients. Here, we performed a study that combined analysis of blood transcriptome, demographic data, clinical aspects and laboratory findings from 66 participants classified into different degrees of COVID-19 severity and healthy subjects. We identified a perturbation in blood-leukocyte transcriptional profile associated with COVID-19 aggravation, which was mainly related to processes that disfavoured lymphocyte activation and favoured neutrophil activation. This transcriptional profile stratified patients according to COVID-19 severity. Hence, it enabled identification of a turning point in transcriptional dynamics that distinguished disease outcomes and non-hospitalized from hospitalized moderate patients. Central genes of this unique neutrophil signature were S100A9, ANXA3, CEACAM6, VNN1, OLFM4, IL1R2, TCN1 and CD177. Our study indicates the molecular changes that are linked with the differing clinical aspects presented by humans when suffering from COVID-19, which involve neutrophil activation.
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COVID-19 , Humanos , COVID-19/genética , Neutrófilos , Transcriptoma , BiomarcadoresRESUMEN
BACKGROUND: Sex-determined differences are rarely addressed in the management of diseases, despite well-known contrasting outcomes between female and male patients. In COVID-19 there is a remarkable disparity, with higher rates of mortality and more severe acute disease in men compared to women, who are mostly affected by long COVID-19. Furthermore, whether androgens play a protective or detrimental role in COVID-19 is still a matter of debate. Hence, the adequate management of the disease, especially regarding men presenting acute disease aggravation, still needs important data to elucidate the interplay between sex hormones and host immune responses that drive the worse evolution in male patients. METHODS: A cohort of 92 controls and 198 non-severe and severe COVID-19 patients, from both sexes, was assessed for clinical outcomes, plasma steroids, gonadotropins, sex hormone binding globulin (SHBG) and immune mediators, before vaccination. These data were correlated with the global gene expression of blood leukocytes. The androgen receptor (AR) signaling pathway was investigated by transcriptomics and tracheal aspirate was obtained from severe patients for SARS-COV-2 quantification in the respiratory tract. The interplay among clinical, endocrine and immunological data deciphered the sex differences in COVID-19. Importantly, statistical analyses, using 95% confidence interval, considered confounding factors such as age and comorbidities, to definitely parse the role of androgens in the disease outcome. RESULTS: There were notable contrasting levels of testosterone and dihydrotestosterone (DHT) throughout the disease course in male but not female patients. Inflammatory mediators presented significant negative correlations with testosterone, which was partially dependent on age and diabetes in men. Male subjects with severe COVID-19 had a significant up regulation of the AR signaling pathway, including modulation of TMPRSS2 and SRD5A1 genes, which are related to the viral infection and DHT production. Indeed, men had a higher viral load in the tracheal aspirate and levels of DHT were associated with increased relative risk of death. In contrast, the testosterone hormone, which was notably reduced in severe disease, was significantly related with susceptibility to COVID-19 worsening in male patients. Secondary hypogonadism was ruled out in the male severe COVID-19 subjects, as FSH, LH, and SHBG levels were not significantly altered. Instead, these subjects tended to have increased gonadotropin levels. Most interestingly, in this study we identified, for the first time, combined sets of clinical and immunoendocrine parameters that together predicted progression from non-severe to severe COVID-19 in men. One of the limitations of our study was the low or undetectable levels of DHT in many patients. Then, the evaluation of enzymes related to biosynthesis and signaling by androgens was mandatory and reiterated our findings. CONCLUSIONS: These original results unraveled the disease immunoendocrine regulation, despite vaccination or comorbidities and pointed to the fundamental divergent role of the androgens testosterone and DHT in the determination of COVID-19 outcomes in men. Therefore, sex-specific management of the dysregulated responses, treatments or public health measures should be considered for the control of COVID-19 pandemic.
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Clinical data point to adverse cardiovascular events elicited by testosterone replacement therapy. Testosterone is the main hormone used in gender-affirming hormone therapy (GAHT) by transmasculine people. However, the cardiovascular impact of testosterone in experimental models of GAHT remains unknown. Sex hormones modulate T-cell activation, and immune mechanisms contribute to cardiovascular risk. The present study evaluated whether testosterone negatively impacts female cardiovascular function by enhancing Th17 cell-linked effector mechanisms. Female (8 wk old) C57BL/6J mice received testosterone (48 mg/kg/wk) for 8 wk. Male mice were used for phenotypical comparisons. The hormone treatment in female mice increased circulating testosterone to levels observed in male mice. Testosterone increased lean body mass and body mass index, and decreased perigonadal fat mass, mimicking clinical findings. After 8 wk, testosterone decreased endothelium-dependent vasodilation and increased peripheral Th17 cells. After 24 wk, testosterone increased blood pressure in female mice. Ovariectomy did not intensify phenotypical or cardiovascular effects by testosterone. Female mice lacking T and B cells [Rag1 knockout (-/-)], as well as female mice lacking IL-17 receptor (IL-17Ra-/-), did not exhibit vascular dysfunction induced by testosterone. Testosterone impaired endothelium-dependent vasodilation in female mice lacking γδ T cells, similarly to the observed in wild-type female mice. Adoptive transfer of CD4+ T cells restored testosterone-induced vascular dysfunction in Rag1-/- female mice. Together, these data suggest that CD4+ T cells, most likely Th17 cells, are central to vascular dysfunction induced by testosterone in female mice, indicating that changes in immune-cell balance are important in the GAHT in transmasculine people.NEW & NOTEWORTHY Sex hormone-induced cardiovascular events are important undesirable effects in transgender people under GAHT. Studies addressing the cardiovascular impact of GAHT will certainly contribute to improve healthcare services offered to this population. Our study showing that vascular dysfunction, via Th17 cell-related mechanisms, precedes increased blood pressure induced by testosterone in a GAHT mouse model, reveals potential mechanisms involved in GAHT-related cardiovascular events and may provide new markers/targets for clinical practices in transmasculine people.
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Enfermedades Cardiovasculares , Testosterona , Animales , Enfermedades Cardiovasculares/tratamiento farmacológico , Modelos Animales de Enfermedad , Femenino , Hormonas Esteroides Gonadales , Proteínas de Homeodominio , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Células Th17RESUMEN
The global emergence of coronavirus disease 2019 (COVID-19) has caused substantial human casualties. Clinical manifestations of this disease vary from asymptomatic to lethal, and the symptomatic form can be associated with cytokine storm and hyperinflammation. In face of the urgent demand for effective drugs to treat COVID-19, we have searched for candidate compounds using in silico approach followed by experimental validation. Here we identified celastrol, a pentacyclic triterpene isolated from Tripterygium wilfordii Hook F, as one of the best compounds out of 39 drug candidates. Celastrol reverted the gene expression signature from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected cells and irreversibly inhibited the recombinant forms of the viral and human cysteine proteases involved in virus invasion, such as Mpro (main protease), PLpro (papain-like protease), and recombinant human cathepsin L. Celastrol suppressed SARS-CoV-2 replication in human and monkey cell lines and decreased interleukin-6 (IL-6) secretion in the SARS-CoV-2-infected human cell line. Celastrol acted in a concentration-dependent manner, with undetectable signs of cytotoxicity, and inhibited in vitro replication of the parental and SARS-CoV-2 variant. Therefore, celastrol is a promising lead compound to develop new drug candidates to face COVID-19 due to its ability to suppress SARS-CoV-2 replication and IL-6 production in infected cells.
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Antivirales , Tratamiento Farmacológico de COVID-19 , Proteasas 3C de Coronavirus , Triterpenos Pentacíclicos , Humanos , Antivirales/farmacología , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Interleucina-6 , Simulación del Acoplamiento Molecular , Triterpenos Pentacíclicos/farmacología , Inhibidores de Proteasas/farmacología , SARS-CoV-2/efectos de los fármacos , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
The non-classical histocompatibility antigen G (HLA-G) is an immune checkpoint molecule that has been implicated in viral disorders. We evaluated the plasma soluble HLA-G (sHLA-G) in 239 individuals, arranged in COVID-19 patients (n = 189) followed up at home or in a hospital, and in healthy controls (n = 50). Increased levels of sHLA-G were observed in COVID-19 patients irrespective of the facility care, gender, age, and the presence of comorbidities. Compared with controls, the sHLA-G levels increased as far as disease severity progressed; however, the levels decreased in critically ill patients, suggesting an immune exhaustion phenomenon. Notably, sHLA-G exhibited a positive correlation with other mediators currently observed in the acute phase of the disease, including IL-6, IL-8 and IL-10. Although sHLA-G levels may be associated with an acute biomarker of COVID-19, the increased levels alone were not associated with disease severity or mortality due to COVID-19. Whether the SARS-CoV-2 per se or the innate/adaptive immune response against the virus is responsible for the increased levels of sHLA-G are questions that need to be further addressed.
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COVID-19 , Antígenos HLA-G , Antígenos de Histocompatibilidad Clase I , Humanos , Proteínas de Punto de Control Inmunitario , Plasma , SARS-CoV-2RESUMEN
OBJECTIVE AND DESIGN: Investigate survival outcomes, and immunological and metabolomic effects of hyaluronidase (Hz) treatment during mouse models of acute inflammation and sepsis. METHODS: Survival of C57Bl/6 mice was monitored after lethal challenge with lipopolysaccharide (LPS) or cecal and ligation puncture (CLP)-induced sepsis and treated with Hz or saline. Mice were also challenged with LPS and treated with Hz for leukocyte counting, cytokine quantification and determination of metabolomic profiles in the peritoneal fluid. RESULTS: Hz treatment improved survival outcomes after lethal challenge with LPS or CLP-induced sepsis. LPS challenge promoted acute neutrophil accumulation and production of interleukin-1ß (IL-1ß) and IL-6 in the peritoneum, whereas Hz treatment suppressed neutrophil infiltration and cytokine production. We further characterized the metabolomic alterations caused by LPS challenge, which predicted activity of metabolic pathways related to fatty acids and eicosanoids. Hz treatment had a profound effect over the metabolic response, reflected by reductions of the relative levels of fatty acids. CONCLUSION: Collectively, these data demonstrate that Hz treatment is associated with metabolic reprogramming of pathways that sustain the inflammatory response.
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Hialuronoglucosaminidasa/farmacología , Sepsis/inmunología , Sepsis/metabolismo , Enfermedad Aguda , Animales , Líquido Ascítico/citología , Líquido Ascítico/inmunología , Líquido Ascítico/metabolismo , Modelos Animales de Enfermedad , Eicosanoides/metabolismo , Ácidos Grasos/metabolismo , Inmunomodulación , Interleucina-1beta/inmunología , Interleucina-6/inmunología , Recuento de Leucocitos , Lipopolisacáridos , Masculino , Redes y Vías Metabólicas/efectos de los fármacos , Metabolómica , Ratones Endogámicos C57BLRESUMEN
Prostaglandin E2 (PGE2) suppresses macrophage effector mechanisms; however, little is known about the function of PGD2 in infected alveolar macrophages (AMs). Using serum-opsonized Histoplasma capsulatum (Ops-H. capsulatum) in vitro, we demonstrated that AMs produced PGE2 and PGD2 in a time-dependent manner, with PGE2 levels exceeding those of PGD2 by 48 h postinfection. Comparison of the effects of both exogenous PGs on AMs revealed that PGD2 increased phagocytosis and killing through the chemoattractant receptor-homologous molecule expressed on Th2 lymphocytes receptor, whereas PGE2 had opposite effects, through E prostanoid (EP) receptor 2 (EP2)/EP4-dependent mechanisms. Moreover, PGD2 inhibited phospholipase C-γ (PLC-γ) phosphorylation, reduced IL-10 production, and increased leukotriene B4 receptor expression. In contrast, exogenous PGE2 treatment reduced PLC-γ phosphorylation, p38 and nuclear factor κB activation, TNF-α, H2O2, and leukotriene B4, but increased IL-1ß production. Using specific compounds to inhibit the synthesis of each PG in vitro and in vivo, we found that endogenous PGD2 contributed to fungicidal mechanisms and controlled inflammation, whereas endogenous PGE2 decreased phagocytosis and killing of the fungus and induced inflammation. These findings demonstrate that, although PGD2 acts as an immunostimulatory mediator to control H. capsulatum infection, PGE2 has immunosuppressive effects, and the balance between these two PGs may limit collateral immune damage at the expense of microbial containment.
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Dinoprostona/farmacología , Histoplasma/efectos de los fármacos , Histoplasmosis/tratamiento farmacológico , Macrófagos Alveolares/efectos de los fármacos , Prostaglandina D2/farmacología , Animales , Células Cultivadas , Macrófagos Alveolares/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Ratas , Ratas WistarRESUMEN
Coagulopathies following snakebite are triggered by pro-coagulant venom toxins, in which metalloproteases play a major role in envenomation-induced coagulation disorders by acting on coagulation cascade, platelet function and fibrinolysis. Considering this relevance, here we describe the isolation and biochemical characterization of moojenactivase (MooA), a metalloprotease from Bothrops moojeni snake venom, and investigate its involvement in hemostasis in vitro. MooA is a glycoprotein of 85,746.22 Da, member of the PIIId group of snake venom metalloproteases, composed of three linked disulfide-bonded chains: an N-glycosylated heavy chain, and two light chains. The venom protease induced human plasma clotting in vitro by activating on both blood coagulation factors II (prothrombin) and X, which in turn generated α-thrombin and factor Xa, respectively. Additionally, MooA induced expression of tissue factor (TF) on the membrane surface of peripheral blood mononuclear cells (PBMC), which led these cells to adopt pro-coagulant characteristics. MooA was also shown to be involved with production of the inflammatory mediators TNF-α, IL-8 and MCP-1, suggesting an association between MooA pro-inflammatory stimulation of PBMC and TF up-regulation. We also observed aggregation of washed platelets when in presence of MooA; however, the protease had no effect on fibrinolysis. Our findings show that MooA is a novel hemostatically active metalloprotease, which may lead to the development of coagulopathies during B. moojeni envenomation. Moreover, the metalloprotease may contribute to the development of new diagnostic tools and pharmacological approaches applied to hemostatic disorders.
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Coagulación Sanguínea/efectos de los fármacos , Bothrops/metabolismo , Coagulantes/farmacología , Venenos de Crotálidos/enzimología , Factor Xa/metabolismo , Leucocitos/efectos de los fármacos , Metaloendopeptidasas/farmacología , Metaloproteasas/farmacología , Protrombina/metabolismo , Tromboplastina/metabolismo , Adulto , Secuencia de Aminoácidos , Animales , Coagulantes/aislamiento & purificación , Venenos de Crotálidos/aislamiento & purificación , Venenos de Crotálidos/farmacología , Estabilidad de Enzimas , Femenino , Humanos , Concentración de Iones de Hidrógeno , Mediadores de Inflamación/metabolismo , Cinética , Leucocitos/metabolismo , Masculino , Metaloendopeptidasas/aislamiento & purificación , Metaloproteasas/aislamiento & purificación , Persona de Mediana Edad , Temperatura , Adulto JovenRESUMEN
Puerperal sepsis is a leading cause of maternal mortality worldwide. Streptococcus pyogenes [group A Streptococcus; (GAS)] is a major etiologic agent of severe postpartum sepsis, yet little is known regarding the pathogenesis of these infections. Tissue macrophages provide innate defense against GAS, and their actions are highly regulated. The intracellular second messenger cAMP can negatively regulate macrophage actions against GAS. Because leukotriene (LT) B(4) has been shown to suppress intracellular cAMP in macrophages, we hypothesized that it could enhance innate defenses against GAS. We assessed the capacity of LTB(4) to modulate antistreptococcal actions of human macrophages, including placental and decidual macrophages and used a novel intrauterine infection model of GAS in mice lacking the 5-lipoxygenase enzyme to determine the role of endogenous LTs in host defense against this pathogen. Animals lacking 5-lipoxygenase were significantly more vulnerable to intrauterine GAS infection than were wild-type mice and showed enhanced dissemination of bacteria out of the uterus and a more robust inflammatory response than did wild-type mice. In addition, LTB(4) reduced intracellular cAMP levels via the BLT1 receptor and was a potent stimulant of macrophage phagocytosis and NADPH oxidase-dependent intracellular killing of GAS. Importantly, interference was observed between the macrophage immunomodulatory actions of LTB(4) and the cAMP-inducing lipid PGE(2), suggesting that interplay between pro- and anti-inflammatory compounds may be important in vivo. This work underscores the potential for pharmacological targeting of lipid mediator signaling cascades in the treatment of invasive GAS infections.
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Leucotrieno B4/fisiología , Infección Puerperal/inmunología , Infección Puerperal/microbiología , Sepsis/inmunología , Regulación hacia Arriba/inmunología , Adolescente , Adulto , Animales , Araquidonato 5-Lipooxigenasa/deficiencia , Araquidonato 5-Lipooxigenasa/genética , Células Cultivadas , Femenino , Predisposición Genética a la Enfermedad , Humanos , Inmunidad Innata/genética , Leucotrieno B4/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infección Puerperal/genética , Sepsis/genética , Sepsis/microbiología , Infecciones Estreptocócicas/genética , Infecciones Estreptocócicas/inmunología , Regulación hacia Arriba/genética , Adulto JovenRESUMEN
Previous studies indicate that snake venom contains glycan-binding proteins (GBPs), although the binding specificity and biological activities of many of these GBPs is unclear. Here we report our studies on the glycan binding specificity and activities of galatrox, a Bothrops atrox snake venom-derived GBP. Glycan microarray analysis indicates that galatrox binds most strongly to glycans expressing N-acetyllactosamine (LacNAc), with a significant preference for Galß1-4GlcNAcß over Galß1-3GlcNAcß compounds. Galatrox also bound immobilized laminin, a LacNAc-dense extracellular matrix component, suggesting that this GBP can bind LacNAc-bearing glycoproteins. As several endogenous mammalian GBPs utilize a similar binding LacNAc binding preference to regulate neutrophil and monocyte activity, we hypothesized that galatrox may mediate B. atrox toxicity through regulation of leukocyte activity. Indeed, galatrox bound neutrophils and promoted leukocyte chemotaxis in a carbohydrate-dependent manner. Similarly, galatrox administration into the mouse peritoneal cavity induced significant neutrophil migration and the release of pro-inflammatory cytokines IL-1α and IL-6. Exposure of bone marrow-derived macrophages to galatrox induced generation of pro-inflammatory mediators IL-6, TNF-α, and keratinocyte-derived chemokine. This signaling by galatrox was mediated via its carbohydrate recognition domain by activation of the TLR4-mediated MyD88-dependent signaling pathway. These results indicate that galatrox has pro-inflammatory activity through its interaction with LacNAc-bearing glycans on neutrophils, macrophages and extracellular matrix proteins and induce the release of pro-inflammatory mediators.
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Venenos de Crotálidos/química , Inflamación/inducido químicamente , Lectinas/metabolismo , Polisacáridos/metabolismo , Venenos de Víboras/metabolismo , Animales , Bothrops , Secuencia de Carbohidratos , Lectinas/efectos adversos , Lectinas/química , Datos de Secuencia Molecular , Venenos de Víboras/efectos adversos , Venenos de Víboras/químicaRESUMEN
BACKGROUND: Phospholipases C (PLCs) are virulence factors found in several bacteria. In Mycobacterium tuberculosis (Mtb) they exhibit cytotoxic effects on macrophages, but the mechanisms involved in PLC-induced cell death are not fully understood. It has been reported that induction of cell necrosis by virulent Mtb is coordinated by subversion of PGE2, an essential factor in cell membrane protection. RESULTS: Using two Mtb clinical isolates carrying genetic variations in PLC genes, we show that the isolate 97-1505, which bears plcA and plcB genes, is more resistant to alveolar macrophage microbicidal activity than the isolate 97-1200, which has all PLC genes deleted. The isolate 97-1505 also induced higher rates of alveolar macrophage necrosis, and likewise inhibited COX-2 expression and PGE2 production. To address the direct effect of mycobacterial PLC on cell necrosis and PGE2 inhibition, both isolates were treated with PLC inhibitors prior to macrophage infection. Interestingly, inhibition of PLCs affected the ability of the isolate 97-1505 to induce necrosis, leading to cell death rates similar to those induced by the isolate 97-1200. Finally, PGE2 production by Mtb 97-1505-infected macrophages was restored to levels similar to those produced by 97-1200-infected cells. CONCLUSIONS: Mycobacterium tuberculosis bearing PLCs genes induces alveolar macrophage necrosis, which is associated to subversion of PGE2 production.
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Muerte Celular , Dinoprostona/antagonistas & inhibidores , Macrófagos Alveolares/microbiología , Macrófagos Alveolares/fisiología , Mycobacterium tuberculosis/enzimología , Fosfolipasas de Tipo C/metabolismo , Factores de Virulencia/metabolismo , Proteínas Bacterianas/genética , Humanos , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis/microbiología , Fosfolipasas de Tipo C/genéticaRESUMEN
In the present work we examine the contribution of 5-lipoxygenase- (5-LO-) derived lipid mediators to immune responses during the acute phase of Trypanosoma cruzi infection in 5-LO gene knockout (5-LO(-/-)) mice and wild-type (WT) mice. Compared with WT mice, the 5-LO(-/-) mice developed less parasitemia/tissue parasitism, less inflammatory cell infiltrates, and a lower mortality. This resistance of 5-LO(-/-) mice correlated with several differences in the immune response to infection, including reduced PGE2 synthesis; sustained capacity of splenocytes to produce high levels of interleukin (IL)-12 early in the infection; enhanced splenocyte production of IL-1ß, IL-6, and IFN-γ; rapid T-cell polarization to secrete high quantities of IFN-γ and low quantities of IL-10; and greater numbers of CD8(+)CD44(high)CD62L(low) memory effector T cells at the end of the acute phase of infection. The high mortality in WT mice was associated with increased production of LTB4/LTC4, T cell bias to produce IFN-γ, high levels of serum nitrite, and marked protein extravasation into the peritoneal cavity, although survival was improved by treatment with a cys-LT receptor 1 antagonist. These data also provide evidence that 5-LO-derived mediators negatively affect host survival during the acute phase of T. cruzi infection.
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Enfermedad de Chagas/enzimología , Enfermedad de Chagas/patología , Trypanosoma cruzi/patogenicidad , Animales , Araquidonato 5-Lipooxigenasa , Enfermedad de Chagas/genética , Enfermedad de Chagas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Interleucina-6/metabolismo , Masculino , Ratones , Ratones Noqueados , Nitritos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Insufficient oxygen delivery to organs leads to tissue dysfunction and cell death. Reperfusion, although vital to organ survival, initiates an inflammatory response that may both aggravate local tissue injury and elicit remote organ damage. Polymorphonuclear neutrophil (PMN) trafficking to remote organs following ischaemia/reperfusion (I/R) is associated with the release of lipid mediators, including leucotriene (LT) B4 , cysteinyl-LTs (CysLTs) and platelet-activating factor (PAF). Yet, their potentially cooperative role in regulating I/R-mediated inflammation has not been thoroughly assessed. The present study aimed to determine the cooperative role of lipid mediators in regulating PMN migration, tissue oedema and injury using selective receptor antagonists in selected models of I/R and dermal inflammation. Our results show that rabbits, pre-treated orally with BIIL 284 and/or WEB 2086 and MK-0571, were protected from remote tissue injury following I/R or dermal inflammation in an additive or synergistic manner when the animals were pre-treated with two drugs concomitantly. The functional selectivity of the antagonists towards their respective agonists was assessed in vitro, showing that neither BIIL 284 nor WEB 2086 prevented the inflammatory response to IL-8, C5a and zymosan-activated plasma stimulation. However, these agonists elicited LTB4 biosynthesis in isolated rabbit PMNs. Similarly, a cardioprotective effect of PAF and LTB4 receptor antagonists was shown following myocardial I/R in mice. Taken together, these results underscore the intricate involvement of LTB4 and PAF in each other's responses and provide further evidence that targeting both LTs and PAF receptors provides a much stronger anti-inflammatory effect, regulating PMN migration and oedema formation.
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Leucotrienos/metabolismo , Factor de Activación Plaquetaria/metabolismo , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Amidinas/farmacología , Animales , Azepinas/farmacología , Bioensayo , Carbamatos/farmacología , Dermis/patología , Modelos Animales de Enfermedad , Extravasación de Materiales Terapéuticos y Diagnósticos/metabolismo , Extravasación de Materiales Terapéuticos y Diagnósticos/patología , Extremidades/irrigación sanguínea , Extremidades/patología , Inflamación/patología , Leucotrieno B4/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patología , Infiltración Neutrófila/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/agonistas , Glicoproteínas de Membrana Plaquetaria/metabolismo , Propionatos/farmacología , Quinolinas/farmacología , Conejos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Leucotrienos/agonistas , Receptores de Leucotrienos/metabolismo , Triazoles/farmacologíaRESUMEN
The transient receptor potential vanilloid type-1 (TRPV1) channels have been implicated in the modulation of aversive responses. The endocannabinoid anandamide acts as an endogenous TRPV1 agonist, exerting opposite functions at TRPV1 and type-1 cannabinoid receptors (CB1R). Here we tested the hypothesis that hippocampal TRPV1 modulates contextual fear memory retrieval and investigated the influence of the aversive stimulus intensity as well as the role of endocannabinoid signaling. Male C57BL/6J mice were tested for contextual fear memory after low-, moderate-, or high-intensity shock protocols. The selective TRPV1 blockers SB366791 (1-10 nmol) and 6-I-NC (2 nmol) were infused via intra-dorsal hippocampus before the retrieval test session. The local levels of endocannabinoids and Arc and Zif268 mRNAs, involved in synaptic plasticity and memory, were quantified. First, both TRPV1 blockers reduced memory retrieval in animals exposed to moderate or high (but not low) intensity training protocols. In the second series of results, the magnitude of the freezing responses positively correlated with the hippocampal anandamide levels; TRPV1 and CB1R were found co-localized in this brain region; and the CB1R antagonist, AM251, prevented the effects of SB366791. Thus, endocannabinoid signaling possibly mediates the effects of TRPV1 blockers. Finally, inhibition of memory retrieval by TRPV1 blockers increased Arc and Zif268 mRNAs and impaired fear memory reinstatement. In conclusion, the modulation of fear memories by dorsal hippocampal TRPV1 channels may depend on the aversive stimulus intensity and occur via anandamide/CB1 signaling. Moreover, TRPV1 blockers promote Arc and Zif268 transcription, with subsequent attenuation of aversive memory reinstatement.
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Endocannabinoides , Miedo , Ratones , Animales , Masculino , Endocannabinoides/farmacología , Ratones Endogámicos C57BL , Hipocampo , Receptor Cannabinoide CB1 , Canales Catiónicos TRPV/metabolismoRESUMEN
Leukotrienes (LTs) are derived from arachidonic acid metabolism by the 5-lipoxygenase (5-LO) enzyme. The production of LTs is stimulated in the pathogenesis of rheumatoid arthritis (RA), osteoarthritis, and periodontitis, with a relevant contribution to bone resorption. However, its role in bone turnover, particularly the suppression of bone formation by modulating the function of osteoclasts and osteoblasts, remains unclear. We investigated the effects of LTs on bone metabolism and their impact on osteogenic differentiation and osteoclastogenesis using a 5-LO knockout (KO) mouse model. Results from micro-computed tomography (µCT) analysis of femur from 8-week-old 5-LO-deficient mice showed increased cortical bone and medullary region in females and males and decreased trabecular bone in females. In the vertebra, we observed increased marrow area in both females and males 5-LO KO and decreased trabecular bone only in females 5-LO KO. Immunohistochemistry (IHC) analysis showed higher levels of osteogenic markers tissue-nonspecific alkaline phosphatase (TNAP) and osteopontin (OPN) and lower expression of osteoclastogenic marker tartrate-resistant acid phosphatase (TRAP) in the femurs of 5-LO KO mice versus wild-type (WT). Alkaline phosphatase activity and mineralization assay results showed that the 5-LO absence enhances osteoblasts differentiation and mineralization but decreases the proliferation. Alkaline phosphatase (ALP), Bglap, and Sp7 gene expression were higher in 5-LO KO osteoblasts compared to WT cells. Eicosanoids production was higher in 5-LO KO osteoblasts except for thromboxane 2, which was lower in 5-LO-deficient mice. Proteomic analysis identified the downregulation of proteins related to adenosine triphosphate (ATP) metabolism in 5-LO KO osteoblasts, and the upregulation of transcription factors such as the adaptor-related protein complex 1 (AP-1 complex) in long bones from 5-LO KO mice leading to an increased bone formation pattern in 5-LO-deficient mice. We observed enormous differences in the morphology and function of osteoclasts with reduced bone resorption markers and impaired osteoclasts in 5-LO KO compared to WT osteoclasts. Altogether, these results demonstrate that the absence of 5-LO is related to the greater osteogenic profile. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).
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Resorción Ósea , Osteogénesis , Masculino , Femenino , Ratones , Animales , Fosfatasa Alcalina/metabolismo , Microtomografía por Rayos X , Proteómica , Osteoclastos/metabolismo , Osteoblastos/metabolismo , Resorción Ósea/patología , Diferenciación Celular , Ratones Noqueados , Leucotrienos/metabolismo , Leucotrienos/farmacologíaRESUMEN
COVID-19 is associated with a dysregulated immune response. Currently, several medicines are licensed for the treatment of this disease. Due to their significant role in inhibiting pro-inflammatory cytokines and lipid mediators, glucocorticoids (GCs) have attracted a great deal of attention. Similarly, the endocannabinoid (eCB) system regulates various physiological processes including the immunological response. Additionally, during inflammatory and thrombotic processes, phospholipids from cell membranes are cleaved to produce platelet-activating factor (PAF), another lipid mediator. Nonetheless, the effect of GCs on this lipid pathway during COVID-19 therapy is still unknown. This is a cross-sectional study involving COVID-19 patients (n = 200) and healthy controls (n = 35). Target tandem mass spectrometry of plasma lipid mediators demonstrated that COVID-19 severity affected eCBs and PAF synthesis. This increased synthesis of eCB was adversely linked with systemic inflammatory markers IL-6 and sTREM-1 levels and neutrophil counts. The use of GCs altered these lipid pathways by reducing PAF and increasing 2-AG production. Corroborating this, transcriptome analysis of GC-treated patients blood leukocytes showed differential modulation of monoacylglycerol lipase and phospholipase A2 gene expression. Altogether, these findings offer a breakthrough in our understanding of COVID-19 pathophysiology, indicating that GCs may promote additional protective pharmacological effects by influencing the eCB and PAF pathways involved in the disease course.
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COVID-19 , Factor de Activación Plaquetaria , Humanos , Estudios Transversales , Endocannabinoides , Glucocorticoides/uso terapéuticoRESUMEN
SARS-CoV-2 infection triggers distinct patterns of disease development characterized by significant alterations in host regulatory responses. Severe cases exhibit profound lung inflammation and systemic repercussions. Remarkably, critically ill patients display a "lipid storm", influencing the inflammatory process and tissue damage. Sphingolipids (SLs) play pivotal roles in various cellular and tissue processes, including inflammation, metabolic disorders, and cancer. In this study, we employed high-resolution mass spectrometry to investigate SL metabolism in plasma samples obtained from control subjects (n = 55), COVID-19 patients (n = 204), and convalescent individuals (n = 77). These data were correlated with inflammatory parameters associated with the clinical severity of COVID-19. Additionally, we utilized RNAseq analysis to examine the gene expression of enzymes involved in the SL pathway. Our analysis revealed the presence of thirty-eight SL species from seven families in the plasma of study participants. The most profound alterations in the SL species profile were observed in patients with severe disease. Notably, a predominant sphingomyelin (SM d18:1) species emerged as a potential biomarker for COVID-19 severity, showing decreased levels in the plasma of convalescent individuals. Elevated SM levels were positively correlated with age, hospitalization duration, clinical score, and neutrophil count, as well as the production of IL-6 and IL-8. Intriguingly, we identified a putative protective effect against disease severity mediated by SM (d18:1/24:0), while ceramide (Cer) species (d18:1/24:1) and (d18:1/24:0)were associated with increased risk. Moreover, we observed the enhanced expression of key enzymes involved in the SL pathway in blood cells from severe COVID-19 patients, suggesting a primary flow towards Cer generation in tandem with SM synthesis. These findings underscore the potential of SM as a prognostic biomarker for COVID-19 and highlight promising pharmacological targets. By targeting sphingolipid pathways, novel therapeutic strategies may emerge to mitigate the severity of COVID-19 and improve patient outcomes.
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COVID-19 , Esfingomielinas , Humanos , Pronóstico , SARS-CoV-2/metabolismo , Ceramidas/metabolismo , Esfingolípidos/metabolismo , BiomarcadoresRESUMEN
Helminths are known to elicit a wide range of immunomodulation characterized by dominant Th2-type immune responses. Our group previously showed that a DNA vaccine encoding the mycobacterial 65-kDa heat shock protein (DNA-hsp65) showed immunomodulatory properties. We also showed, using a helminth-tuberculosis (TB) co-infection model, that the DNA-hsp65 vaccine protected mice against TB. We next investigated the mechanistic role of the vaccine during helminth-TB co-infection. Clinically, helminth infection causes type 2 granulomas in the lung. Mice were immunized with DNA-hsp65 while they were submitted to the type 2 granuloma induction protocol by Schistosoma mansoni eggs infusion. In this work we investigated the effects of DNA-hsp65 on the pathology and immune response during the development of type 2 granuloma induced by S. mansoni eggs. Histologic analyses of lung parenchyma showed that the DNA-hsp65 vaccine protected mice against exacerbated fibrosis induced by Schistosoma eggs, and decreased the size of the granulomas. These changes were correlated with a reduction in the number of T cells specific for the egg antigens in the lung and also with modulation of Th2 cytokine expression. Taken together, our results showed that the adjuvant properties of the DNA-hsp65 vaccine regulated the immune response in this Th2 model, and resulted in a preserved lung parenchyma.