RESUMO
BACKGROUND: Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) cause a wide variety of bacterial infections and coinfections, showing a complex interaction that involves the production of different metabolites and metabolic changes. Temperature is a key factor for bacterial survival and virulence and within the host, bacteria could be exposed to an increment in temperature during fever development. We analyzed the previously unexplored effect of fever-like temperatures (39 °C) on S. aureus USA300 and P. aeruginosa PAO1 microaerobic mono- and co-cultures compared with 37 °C, by using RNAseq and physiological assays including in vivo experiments. RESULTS: In general terms both temperature and co-culturing had a strong impact on both PA and SA with the exception of the temperature response of monocultured PA. We studied metabolic and virulence changes in both species. Altered metabolic features at 39 °C included arginine biosynthesis and the periplasmic glucose oxidation in S. aureus and P. aeruginosa monocultures respectively. When PA co-cultures were exposed at 39 °C, they upregulated ethanol oxidation-related genes along with an increment in organic acid accumulation. Regarding virulence factors, monocultured SA showed an increase in the mRNA expression of the agr operon and hld, pmsα, and pmsß genes at 39 °C. Supported by mRNA data, we performed physiological experiments and detected and increment in hemolysis, staphyloxantin production, and a decrease in biofilm formation at 39 °C. On the side of PA monocultures, we observed an increase in extracellular lipase and protease and biofilm formation at 39 °C along with a decrease in the motility in correlation with changes observed at mRNA abundance. Additionally, we assessed host-pathogen interaction both in vitro and in vivo. S. aureus monocultured at 39οC showed a decrease in cellular invasion and an increase in IL-8-but not in IL-6-production by A549 cell line. PA also decreased its cellular invasion when monocultured at 39 °C and did not induce any change in IL-8 or IL-6 production. PA strongly increased cellular invasion when co-cultured at 37 and 39 °C. Finally, we observed increased lethality in mice intranasally inoculated with S. aureus monocultures pre-incubated at 39 °C and even higher levels when inoculated with co-cultures. The bacterial burden for P. aeruginosa was higher in liver when the mice were infected with co-cultures previously incubated at 39 °C comparing with 37 °C. CONCLUSIONS: Our results highlight a relevant change in the virulence of bacterial opportunistic pathogens exposed to fever-like temperatures in presence of competitors, opening new questions related to bacteria-bacteria and host-pathogen interactions and coevolution.
Assuntos
Infecções por Pseudomonas , Infecções Estafilocócicas , Camundongos , Animais , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Virulência/fisiologia , Pseudomonas aeruginosa , Temperatura , Interleucina-6/metabolismo , Interleucina-6/farmacologia , Interleucina-8/metabolismo , Interleucina-8/farmacologia , Infecções por Pseudomonas/microbiologia , RNA Mensageiro/metabolismo , Biofilmes , Infecções Estafilocócicas/microbiologiaRESUMO
This study investigated the effect of N-acetylcysteine (NAC) and silymarin (SIL) in the liver of mice exposed to ethanol and lipopolysaccharides (LPS). Mice were divided into four groups (n = 6): naive, vehicle, NAC (200 mg/kg), and SIL (200 mg/kg). Treatments were given orally (po) once daily for 10 days. Liver injury was induced by administration of ethanol (30%, po) for 10 days, once daily, followed by a single administration of LPS (2 mg/kg, ip) 24 h before euthanasia. After the treatment period, animals were euthanized, and liver and blood samples were collected. NAC, but not SIL, prevented the increase in oxalacetic glutamic transaminase (OGT) and pyruvic glutamic transaminase (PGT) serum levels. NAC and SIL did not restore levels of reduced glutathione or hepatic malonaldehyde. The treatments with NAC or SIL showed no difference in the activity of glutathione S-transferase, superoxide dismutase, and catalase compared to vehicle group. Myeloperoxidase and N-acetylglucosaminidase activities are increased, as well as the IL-6 and IL-10 levels in the liver. The treatment with NAC, but not SIL, reduced the N-acetylglucosamines activity and the IL-6 and IL-10 amount in the liver. Histological findings revealed microsteatosis in the vehicle group, which was not prevented by SIL but was partially reduced in animals receiving NAC. Unlike other liver injury models, NAC (200 mg/kg) or SIL (200 mg/kg) did not positively affect antioxidant patterns in liver tissue of animals exposed to ethanol plus LPS, but NAC treatment displays anti-inflammatory properties in this model.
Assuntos
Doença Hepática Crônica Induzida por Substâncias e Drogas , Silimarina , Camundongos , Animais , Acetilcisteína/farmacologia , Silimarina/farmacologia , Lipopolissacarídeos/toxicidade , Interleucina-10 , Etanol/toxicidade , Doença Hepática Crônica Induzida por Substâncias e Drogas/patologia , Interleucina-6/farmacologia , Fígado/patologia , Antioxidantes/farmacologia , Glutationa , Transaminases/farmacologiaRESUMO
Exosomes, organelles measuring 30-200nm, are secreted by various cell types. Leishmania exosomes consist of many proteins, including heat shock proteins, annexins, Glycoprotein 63, proteins exerting signaling activity and those containing mRNA and miRNA. Studies have demonstrated that Leishmania donovani exosomes downregulate IFN-γ and inhibit the expression of microbicidal molecules, such as TNF and nitric oxide, thus creating a microenvironment favoring parasite proliferation. Despite lacking immunological memory, data in the literature suggest that, following initial stimulation, mononuclear phagocytes may become "trained" to respond more effectively to subsequent stimuli. Here we characterized the effects of macrophage sensitization using L. braziliensis exosomes prior to infection by the same pathogen. Human macrophages were stimulated with L. braziliensis exosomes and then infected with L. braziliensis. Higher levels of IL-1ß and IL-6 were detected in cultures sensitized prior to infection compared to unstimulated infected cells. Moreover, stimulation with L. braziliensis exosomes induced macrophage production of IL-1ß, IL-6, IL-10 and TNF. Inhibition of exosome secretion by L. braziliensis prior to macrophage infection reduced cytokine production and produced lower infection rates than untreated infected cells. Exosome stimulation also induced the consumption/regulation of NLRP3 inflammasome components in macrophages, while the blockade of NLRP3 resulted in lower levels of IL-6 and IL-1ß. Our results suggest that L. braziliensis exosomes stimulate macrophages, leading to an exacerbated inflammatory state that may be NLRP3-dependent.
Assuntos
Exossomos , Leishmania braziliensis , Leishmania donovani , Humanos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Interleucina-6/farmacologia , MacrófagosRESUMO
Sulfite predominantly accumulates in the brain of patients with isolated sulfite oxidase (ISOD) and molybdenum cofactor (MoCD) deficiencies. Patients present with severe neurological symptoms and basal ganglia alterations, the pathophysiology of which is not fully established. Therapies are ineffective. To elucidate the pathomechanisms of ISOD and MoCD, we investigated the effects of intrastriatal administration of sulfite on myelin structure, neuroinflammation, and oxidative stress in rat striatum. Sulfite administration decreased FluoromyelinTM and myelin basic protein staining, suggesting myelin abnormalities. Sulfite also increased the staining of NG2, a protein marker of oligodendrocyte progenitor cells. In line with this, sulfite also reduced the viability of MO3.13 cells, which express oligodendroglial markers. Furthermore, sulfite altered the expression of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-10 (IL-10), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1), indicating neuroinflammation and redox homeostasis disturbances. Iba1 staining, another marker of neuroinflammation, was also increased by sulfite. These data suggest that myelin changes and neuroinflammation induced by sulfite contribute to the pathophysiology of ISOD and MoCD. Notably, post-treatment with bezafibrate (BEZ), a pan-PPAR agonist, mitigated alterations in myelin markers and Iba1 staining, and IL-1ß, IL-6, iNOS and HO-1 expression in the striatum. MO3.13 cell viability decrease was further prevented. Moreover, pre-treatment with BEZ also attenuated some effects. These findings show the modulation of PPAR as a potential opportunity for therapeutic intervention in these disorders.
Assuntos
Bezafibrato , Receptores Ativados por Proliferador de Peroxissomo , Ratos , Animais , Bezafibrato/farmacologia , Receptores Ativados por Proliferador de Peroxissomo/farmacologia , Bainha de Mielina , Doenças Neuroinflamatórias , Interleucina-6/farmacologia , Estresse Oxidativo , Sulfitos/farmacologiaRESUMO
The oxidized low-density lipoprotein receptor 1 (LOX-1) is one of the most important receptors for modified LDLs, such as oxidated (oxLDL) and acetylated (acLDL) low-density lipoprotein. LOX-1 and oxLDL are fundamental in atherosclerosis, where oxLDL/LOX1 promotes ROS generation and NF-κB activation inducing the expression of IL-6, a STAT3 activator. Furthermore, LOX-1/oxLDL function has been associated with other diseases, such as obesity, hypertension, and cancer. In prostate cancer (CaP), LOX-1 overexpression is associated with advanced stages, and its activation by oxLDL induces an epithelial-mesenchymal transition, increasing angiogenesis and proliferation. Interestingly, enzalutamide-resistant CaP cells increase the uptake of acLDL. Enzalutamide is an androgen receptor (AR) antagonist for castration-resistant prostate cancer (CRPC) treatment, and a high percentage of patients develop a resistance to this drug. The decreased cytotoxicity is promoted in part by STAT3 and NF-κB activation that induces the secretion of the pro-inflammatory program and the expression of AR and its splicing variant AR-V7. Here, we demonstrate for the first time that oxLDL/LOX-1 increases ROS levels and activates NF-κB, inducing IL-6 secretion and the activation of STAT3 in CRPC cells. Furthermore, oxLDL/LOX1 increases AR and AR-V7 expression and decreases enzalutamide cytotoxicity in CRPC. Thus, our investigation suggests that new factors associated with cardiovascular pathologies, such as LOX-1/oxLDL, may also promote important signaling axes for the progression of CRPC and its resistance to drugs used for its treatment.
Assuntos
Antineoplásicos , Neoplasias de Próstata Resistentes à Castração , Masculino , Humanos , NF-kappa B/metabolismo , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Receptores Androgênicos/metabolismo , Espécies Reativas de Oxigênio/farmacologia , Interleucina-6/genética , Interleucina-6/farmacologia , Antineoplásicos/farmacologia , Nitrilas/farmacologia , Lipoproteínas LDL/farmacologia , Transdução de Sinais , Antagonistas de Receptores de Andrógenos/farmacologia , Receptores Depuradores Classe E/genética , Receptores Depuradores Classe E/metabolismo , Linhagem Celular TumoralRESUMO
BACKGROUND: Type I Diabetes mellitus (T1D) is characterized by a specific destruction of ß-cells by the immune system. During this process pro-inflammatory cytokines are released in the pancreatic islets and contribute for ß-cells demise. Cytokine-induced iNOS activation, via NF-κB, is implicated in induction of ß-cells death, which includes ER stress activation. Physical exercise has been used as an adjunct for better glycemic control in patients with T1D, since it is able to increase glucose uptake independent of insulin. Recently, it was observed that the release of IL-6 by skeletal muscle, during physical exercise, could prevent ß-cells death induced by pro-inflammatory cytokines. However, the molecular mechanisms involved in this beneficial effect on ß-cells are not yet completely elucidated. Our aim was to evaluate the effect of IL-6 on ß-cells exposed to pro-inflammatory cytokines. RESULTS: Pre-treatment with IL-6 sensitized INS-1E cells to cytokine-induced cell death, increasing cytokine-induced iNOS and Caspase-3 expression. Under these conditions, however, there was a decrease in cytokines-induced p-eIF2-α but not p-IRE1expression, proteins related to ER stress. To address if this prevention of adequate UPR response is involved in the increase in ß-cells death markers induced by IL-6 pre-treatment, we used a chemical chaperone (TUDCA), which improves ER folding capacity. Use of TUDCA increased cytokines-induced Caspase-3 expression and Bax/Bcl-2 ratio in the presence of IL-6 pre-treatment. However, there is no modulation of p-eIF2-α expression by TUDCA in this condition, with increase of CHOP expression. CONCLUSION: Treatment with IL-6 alone is not beneficial for ß-cells, leading to increased cell death markers and impaired UPR activation. In addition, TUDCA has not been able to restore ER homeostasis or improve ß-cells viability under this condition, suggesting that other mechanisms may be involved.
Assuntos
Citocinas , Diabetes Mellitus Tipo 1 , Humanos , Citocinas/metabolismo , Caspase 3 , Interleucina-6/farmacologia , Diabetes Mellitus Tipo 1/metabolismo , Fator de Iniciação 2 em Eucariotos/farmacologia , Morte Celular/fisiologiaRESUMO
Perivascular adipose tissue (PVAT) exerts anticontractile effect, but under non-physiological conditions it may contribute to vascular dysfunction by releasing pro-inflammatory cytokines. Since PVAT is an important source of interleukin (IL)-6, we evaluated whether this cytokine would contribute to ethanol-induced vascular dysfunction. With this purpose, male C57BL/6 wild-type (WT) or IL-6-deficient mice (IL-6-/-) were treated with ethanol for 12 weeks. Increased blood pressure was evidenced after 4 and 6 weeks of treatment with ethanol in WT and IL-6-/- mice, respectively. In WT mice, ethanol increased plasma and PVAT levels of IL-6. Ethanol favoured pro-contractile phenotype of PVAT in mesenteric arteries from WT, but not IL-6-deficient mice. Functional studies showed that tiron [(a scavenger of superoxide (O2-)] reversed the pro-contractile effect of PVAT in mesenteric arteries from ethanol-treated mice. Ethanol increased the levels of O2- in PVAT from WT mice. Ethanol-induced increase in O2- generation was higher in arteries with PVAT from WT mice when compared to IL-6-deficient mice. Treatment with ethanol augmented myeloperoxidase activity in the mesenteric arterial bed (MAB; with or without PVAT) from WT, but not IL-6-deficient mice. In conclusion, IL-6 contributes to the pro-contractile effect of PVAT by a mechanism that involves increase in ROS generation. Additionally, IL-6 mediates intravascular recruitment of neutrophils in response to ethanol and plays a role in the early stages of ethanol-induced hypertension. Collectively, our findings provide novel evidence for a role of IL-6 in the vascular dysfunction induced by ethanol.
Assuntos
Interleucina-6 , Obesidade , Masculino , Camundongos , Animais , Interleucina-6/farmacologia , Camundongos Endogâmicos C57BL , Artérias Mesentéricas , Fenótipo , Etanol/toxicidade , Tecido AdiposoRESUMO
Bone marrow-derived mesenchymal stromal cells (BMSCs) have been used for treating inflammatory disorders. Due to the large size of BMSCs compared to nanoparticles, BMSCs cannot be loaded into the nanoparticles. It is hypothesized that BMSCs lysate loading into the nanocarriers will effectively deliver cellular contents and regulatory elements of BMSCs at the injury site. This study aimed to investigate nanostructured lipid carriers (NLC) loading with BMSCs lysate through basic characterization and morphological analysis. Moreover, this study was mainly designed to investigate the role of NLC loaded BMSCs lysate in reducing inflammation via in-vitro and in-vivoassays. The in-vitro study involves cell viability assays, p53, annexin V and VEGF expression through ELISA and immunocytochemistry, real-time BAX, caspase-3, IL-6, IL-8, TOP2A, PCNA, and Ki-67 gene expression analysis. Additionally, to evaluate in-vivo anti-inflammatory activity, the carrageenan-induced rat paw oedema model was used. In-vitro results showed that NLC loaded BMSCs lysate increased cell viability, decreased apoptosis and pro-inflammatory genes expression and up-regulated angiogenesis and proliferation in H2O2 pre-stimulated cells. Findings of the in-vivo assay also indicated a reduction in rat's paw oedema volume in NLC-loaded BMSCs lysate, and downregulation of BAX, Caspase-3, IL-6, and IL-8 was observed. Enhanced expressions of TOP2A, PCNA, and Ki-67 were obtained. Concluding the results of this study, NLC-loaded BMSCs lysate could reduce inflammation and possibly regenerate damaged tissue mainly via increasing cell viability, angiogenesis and proliferation, and reducing apoptosis and pro-inflammatory cytokines.
Assuntos
Peróxido de Hidrogênio , Interleucina-6 , Ratos , Animais , Caspase 3/metabolismo , Caspase 3/farmacologia , Peróxido de Hidrogênio/farmacologia , Proteína X Associada a bcl-2/metabolismo , Proteína X Associada a bcl-2/farmacologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Antígeno Nuclear de Célula em Proliferação/farmacologia , Interleucina-6/metabolismo , Interleucina-6/farmacologia , Interleucina-8/metabolismo , Interleucina-8/farmacologia , Antígeno Ki-67/metabolismo , Inflamação/induzido quimicamente , Inflamação/metabolismo , Lipídeos , Células da Medula Óssea , Edema/metabolismoRESUMO
AIM: Natural substances such as omega-3 have been used in the medical field due to their numerous properties and, in particular, modulating effect on the systemic and local inflammatory processes. Thus, this study evaluated the influence of omega-3 supplementation on the subcutaneous tissue response of endodontic sealers in Wistar Rats. METHODOLOGY: Polyethylene tubes were implanted in the subcutaneous tissue of 48 animals (one empty for control and three filled with Sealapex, AH Plus or Endofill). The animals were treated with omega-3 (TO) or water (TW). Treatments started 15 days before implantation until euthanasia. After 5, 15 and 30 days (n = 8), animals were euthanized and polyethylene tubes and surrounding tissue were removed and processed for histological analysis. The inflammatory reaction was analysed by Haematoxylin and Eosin stain and immunolabelling for IL-6 and TNF-α. The collagen maturity was analysed by picrosirius red stain and calcium deposition by von Kossa stain and polarized light. Results were statistically analysed (p < .05). RESULTS: Amongst TW sealer groups, Endofill evoked a more intense inflammatory infiltrate compared with AH Plus and control in the 30-day period (p = .009). However, in TO sealer groups, there was no difference amongst the sealers and control in all periods (p > .05). Comparing each sealer as a function of the supplementation with water or omega-3, there are differences for Endofill (p = .001) and Sealapex (p = .005) in the 30-day period, presenting lower inflammatory infiltrate in the animals treated with omega-3. A higher percentage of immature fibres was observed at 15 and 30 days in the TO group, compared with the TW group (p < .05). The deposition of calcium particles was observed only by Sealapex in all periods, despite the supplementation procedure. CONCLUSIONS: Omega-3 supplementation influence the tissue reactions of endodontic sealers, modulating inflammation, the immunolabelling of IL-6 and TNF-α, the repair process and it does not interfere with calcium deposition.
Assuntos
Materiais Restauradores do Canal Radicular , Tela Subcutânea , Animais , Cálcio , Suplementos Nutricionais , Resinas Epóxi , Inflamação , Interleucina-6/farmacologia , Teste de Materiais , Polietilenos/farmacologia , Ratos , Ratos Wistar , Materiais Restauradores do Canal Radicular/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , ÁguaRESUMO
Acute systemic inflammation can lead to life-threatening organ dysfunction. In patients with sepsis, systemic inflammation is triggered in response to infection, but in other patients, a systemic inflammatory response syndrome (SIRS) is triggered by non-infectious events. IL-6 is a major mediator of inflammation, including systemic inflammatory responses. In homeostatic conditions, when IL-6 engages its membrane-bound receptor on myeloid cells, it promotes pro-inflammatory cytokine production, phagocytosis, and cell migration. However, under non-physiologic conditions, such as SIRS and sepsis, leucocyte dysfunction could modify the response of these cells to IL-6. So, our aim was to evaluate the response to IL-6 of monocytes from patients diagnosed with SIRS or sepsis. We observed that monocytes from patients with SIRS, but not from patients with sepsis, produced significantly more TNF-α than monocytes from healthy volunteers, after stimulation with IL-6. Monocytes from SIRS patients had a significantly increased baseline phosphorylation of the p65 subunit of NF-κB, with no differences in STAT3 phosphorylation or SOCS3 levels, compared with monocytes from septic patients, and this increased phosphorylation was maintained during the IL-6 activation. We found no significant differences in the expression levels of the membrane-bound IL-6 receptor, or the serum levels of IL-6, soluble IL-6 receptor, or soluble gp130, between patients with SIRS and patients with sepsis. Our results suggest that, during systemic inflammation in the absence of infection, IL-6 promotes TNF-α production by activating NF-κB, and not the canonical STAT3 pathway.