Treadmill Training Increases SIRT-1 and PGC-1 alpha Protein Levels and AMPK Phosphorylation in Quadriceps of Middle-Aged Rats in an Intensity-Dependent Manner

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Phenotypic and functional evaluations of peripheral blood monocytes from chronic-form paracoccidioidomycosis patients before and after treatment

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Increased il-1 β production in placental villous subjected to hyperglycemic conditions

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Endogenous and uric acid-induced activation of NLRP3 inflammasome in pregnant women with preeclampsia

Preeclampsia (PE) is a specific syndrome of pregnancy, characterized by hypertension and proteinuria. This pathology is associated with hyperuricemia and elevated serum levels of inflammatory cytokines. Uric acid crystals may activate an intracellular complex called inflammasome, which is important for processing and release of inflammatory cytokines. This study investigated the state of monocyte activation, both endogenous and stimulated with monosodium urate (MSU), by gene expression of NLRP1 and NLRP3 receptors as well as their association with inflammatory cytokines expression. Monocytes were obtained from peripheral blood of 23 preeclamptic pregnant women, 23 normotensive pregnant women (NT) and 23 healthy non-pregnant women (NP). Inflammasome activation was evaluated by the gene expression of NLRP1, NLRP3, caspase-1, IL-1 beta, IL-18 and TNF-alpha by RT-qPCR in unstimulated monocytes (endogenous expression), or after cell stimulation with MSU (stimulated expression). The concentration of cytokines was assessed by ELISA. In preeclamptic pregnant women, gene expression of NLRP1, NLRP3, caspase-1, IL-1 beta and TNF-alpha by monocytes stimulated or not with MSU was significantly higher than in NT and NP groups. Stimulation of monocytes from preeclamptic and non-pregnant women with MSU induced increased gene expression of NLRP3, caspase-1 and TNF-alpha in relation to the endogenous expression in these groups, while this was not observed in the NT group. The cytokine determination showed that monocytes from women with PE produced higher endogenous levels of IL-1 beta, IL-18 and TNF-alpha compared to the other groups, while the stimulus with MSU led to higher production of these cytokines in preeclamptic group than in the NT group. In conclusion, the results showed increased basal gene expression of NLRP1 and NLRP3 receptors in monocytes from PE group. These cells stimulation with MSU demonstrates that uric acid plays a role in NLRP3 inflammasome activation, suggesting the participation of this inflammatory complex in the pathogenesis of preeclampsia.

NLRP3 inflammasome-mediated neutrophil recruitment and hypernociception depend on leukotriene B4 in a murine model of gout

Objective Deposition of monosodium urate monohydrate (MSU) crystals in the joints promotes an intense inflammatory response and joint dysfunction. This study evaluated the role of the NLRP3 inflammasome and 5-lipoxygenase (5-LOX)derived leukotriene B4 (LTB4) in driving tissue inflammation and hypernociception in a murine model of gout. Methods. Gout was induced by injecting MSU crystals into the joints of mice. Wild-type mice and mice deficient in NLRP3, ASC, caspase 1, interleukin-1 beta (IL-1 beta), IL-1 receptor type I (IL-1RI), IL-18R, myeloid differentiation factor 88 (MyD88), or 5-LOX were used. Evaluations were performed to assess neutrophil influx, LTB4 activity, cytokine (IL-1 beta, CXCL1) production (by enzyme-linked immunosorbent assay), synovial microvasculature cell adhesion (by intravital microscopy), and hypernociception. Cleaved caspase 1 and production of reactive oxygen species (ROS) were analyzed in macrophages by Western blotting and fluorometric assay, respectively. Results. Injection of MSU crystals into the knee joints of mice induced neutrophil influx and neutrophildependent hypernociception. MSU crystal-induced neutrophil influx was CXCR2-dependent and relied on the induction of CXCL1 in an NLRP3/ASC/caspase 1/IL-1 beta/MyD88-dependent manner. LTB4 was produced rapidly after injection of MSU crystals, and this was necessary for caspase 1-dependent IL-1 beta production and consequent release of CXCR2-acting chemokines in vivo. In vitro, macrophages produced LTB4 after MSU crystal injection, and LTB4 was relevant in the MSU crystalinduced maturation of IL-1 beta. Mechanistically, LTB4 drove MSU crystal-induced production of ROS and ROS-dependent activation of the NLRP3 inflammasome. Conclusion. These results reveal the role of the NLRP3 inflammasome in mediating MSU crystalinduced inflammation and dysfunction of the joints, and highlight a previously unrecognized role of LTB4 in driving NLRP3 inflammasome activation in response to MSU crystals, both in vitro and in vivo.

Age-Related Expansion of Tim-3 Expressing T Cells in Vertically HIV-1 Infected Children

As perinatally HIV-1-infected children grow into adolescents and young adults, they are increasingly burdened with the long-term consequences of chronic HIV-1 infection, with long-term morbidity due to inadequate immunity. In progressive HIV-1 infection in horizontally infected adults, inflammation, T cell activation, and perturbed T cell differentiation lead to an "immune exhaustion'', with decline in T cell effector functions. T effector cells develop an increased expression of CD57 and loss of CD28, with an increase in co-inhibitory receptors such as PD-1 and Tim-3. Very little is known about HIV-1 induced T cell dysfunction in vertical infection. In two perinatally antiretroviral drug treated HIV-1-infected groups with median ages of 11.2 yr and 18.5 yr, matched for viral load, we found no difference in the proportion of senescent CD28(-)CD57(+)CD8(+) T cells between the groups. However, the frequency of Tim-3(+)CD8(+) and Tim-3(+)CD4(+) exhausted T cells, but not PD-1(+) T cells, was significantly increased in the adolescents with longer duration of infection compared to the children with shorter duration of HIV-1 infection. PD-1(+)CD8(+) T cells were directly associated with T cell immune activation in children. The frequency of Tim-3(+)CD8(+) T cells positively correlated with HIV-1 plasma viral load in the adolescents but not in the children. These data suggest that Tim-3 upregulation was driven by both HIV-1 viral replication and increased age, whereas PD-1 expression is associated with immune activation. These findings also suggest that the Tim-3 immune exhaustion phenotype rather than PD-1 or senescent cells plays an important role in age-related T cell dysfunction in perinatal HIV-1 infection. Targeting Tim-3 may serve as a novel therapeutic approach to improve immune control of virus replication and mitigate age related T cell exhaustion.

Persistence of Inflammatory Response to Intense Exercise in Diabetic Rats

In this study we evaluated the onset and resolution of inflammation in control and streptozotocin-induced diabetic rats subjected to a single session of intense exercise. The following measurements were carried out prior to, immediately after, and 2 and 24 hours after exercise: plasma levels of proinflammatory cytokines (TNF-alpha, IL-1 beta, IL-6, CINC-2 alpha/beta, MIP-3 alpha, and IL-6), immunoglobulins (IgA and IgM), acute phase proteins (CRP and C3), and creatine kinase (CK) activity. We also examined the occurrence of macrophage death by measurements of macrophages necrosis (loss of membrane integrity) and DNA fragmentation. An increase was observed in the concentration of IL-1 beta (3.3-fold) and TNF-alpha (2.0-fold) and in the proportion of necrotic macrophages (4.5-fold) in diabetic rats 24 hours after exercise, while the control group showed basal measurements. Twenty-four hours after the exercise, serum CK activity was elevated in diabetic rats but not in control animals. We concluded that lesion and inflammations resulting from intense exercise were greater and lasted longer in diabetic animals than in nondiabetic control rats.

INFLAMMATORY RESPONSE AND NEUTROPHIL FUNCTIONS IN PLAYERS AFTER A FUTSAL MATCH

de Moura, NR, Cury-Boaventura, MF, Santos, VC, Levada-Pires, AC, Bortolon, JR, Fiamoncini, J, Pithon-Curi, TC, Curi, R, and Hatanaka, E. Inflammatory response and neutrophil functions in players after a futsal match. J Strength Cond Res 26(9): 2507-2514, 2012-Futsal players suffer injuries resulting from muscle fatigue and contact or collision among players. Muscle lesions can be detected by measuring muscle lesion markers such as creatine kinase (CK) and lactate dehydrogenase (LDH) in plasma. After an initial lesion, there is an increase in the plasma levels of C-reactive protein (CRP) and proinflammatory cytokines. These mediators may activate neutrophils and contribute to tissue damage and increase susceptibility to invasive microorganisms. In this study, we investigated the effect of a futsal match on muscle lesion markers, cytokines, and CRP in elite players. The basal and stimulated neutrophil responsiveness after a match was also evaluated based on measurements of neutrophil necrosis, apoptosis, phagocytic capacity, reactive oxygen species (ROS) production, and cytokines (tumor necrosis factor-alpha [TNF-alpha], interleukin [IL]-8, IL-1 beta, IL-10, and IL-1ra) production. Blood samples were taken from 16 players (26.4 +/- 3.2 years, 70.2 +/- 6.9 kg, 59.7 +/- 5.1 ml.kg(-1).min(-1), sports experience of 4.4 +/- 0.9 years) before and immediately after a match. Exercise increased the serum activities of CK (2.5-fold) and LDH (1.3-fold). Playing futsal also increased the serum concentrations of IL-6 (1.6-fold) and CRP (1.6-fold). The TNF-alpha, IL-1 beta, IL-8, IL-1ra, and IL-10 serum levels were not modified in the conditions studied. The futsal match induced neutrophil apoptosis, as indicated by phosphatidylserine externalization (6.0-fold). The exercise induced priming of neutrophils by increasing ROS (1.3-fold), TNF-alpha (5.8-fold), and IL-1 beta (4.8-fold) released in nonstimulated cells. However, in the stimulated condition, the exercise decreased neutrophil function, diminishing the release of ROS by phorbol myristate acetate-stimulated neutrophils (1.5-fold), and the phagocytic capacity (1.6-fold). We concluded that playing futsal induces inflammation, primes and activates neutrophils, and reduces the efficiency of neutrophil phagocytosis immediately after a match.

Anti-inflammatory Effects of Aerobic Exercise in Mice Exposed to Air Pollution

VIEIRA, R. D. P., A. C. TOLEDO, L. B. SILVA, F. M. ALMEIDA, N. R. DAMACENO-RODRIGUES, E. G. CALDINI, A. B. G. SANTOS, D. H. RIVERO, D. C. HIZUME, F. D. T. Q. S. LOPES, C. R. OLIVO, H. C. CASTRO-FARIA-NETO, M. A. MARTINS, P. H. N. SALDIVA, and M. DOLHNIKOFF. Anti-inflammatory Effects of Aerobic Exercise in Mice Exposed to Air Pollution. Med. Sci. Sports Exerc., Vol. 44, No. 7, pp. 1227-1234, 2012. Purpose: Exposure to diesel exhaust particles (DEP) results in lung inflammation. Regular aerobic exercise improves the inflammatory status in different pulmonary diseases. However, the effects of long-term aerobic exercise on the pulmonary response to DEP have not been investigated. The present study evaluated the effect of aerobic conditioning on the pulmonary inflammatory and oxidative responses of mice exposed to DEP. Methods: BALB/c mice were subjected to aerobic exercise five times per week for 5 wk, concomitantly with exposure to DEP (3 mg.mL (1); 10 mu L per mouse). The levels of exhaled nitric oxide, reactive oxygen species, cellularity, interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) were analyzed in bronchoalveolar lavage fluid, and the density of neutrophils and the volume proportion of collagen fibers were measured in the lung parenchyma. The cellular density of leukocytes expressing IL-1 beta, keratinocyte chemoattractant (KC), and TNF-alpha in lung parenchyma was evaluated with immunohistochemistry. The levels of IL-1 beta, KC, and TNF-alpha were also evaluated in the serum. Results: Aerobic exercise inhibited the DEP-induced increase in the levels of reactive oxygen species (P < 0.05); exhaled nitric oxide (P < 0.01); total (P < 0.01) and differential cells (P < 0.01); IL-6 and TNF-alpha levels in bronchoalveolar lavage fluid (P < 0.05); the level of neutrophils (P < 0.001); collagen density in the lung parenchyma (P < 0.05); the levels of IL-6, KC, and TNF-alpha in plasma (P < 0.05); and the expression of IL-1 beta, KC, and TNF-alpha by leukocytes in the lung parenchyma (P < 0.01). Conclusions: We conclude that long-term aerobic exercise presents protective effects in a mouse model of DEP-induced lung inflammation. Our results indicate a need for human studies that evaluate the pulmonary responses to aerobic exercise chronically performed in polluted areas.

Association between mannose-binding lectin and interleukin-1 receptor antagonist gene polymorphisms and recurrent vulvovaginal candidiasis

Objective The influence of functional polymorphisms in the genes coding for mannose-binding lectin (MBL) and interleukin-1 receptor antagonist (IL-1ra) on recurrent vulvovaginal candidiasis (RVVC) were examined in an urban Brazilian population. Methods DNA was isolated from buccal swabs of 100 women with RVVC and 100 control women and tested by gene amplification for a single nucleotide polymorphism in codon 54 of the MBL2 gene and for a length polymorphism in intron 2 of the IL1RN gene. Genotype and allele frequencies were compared between groups. Results The frequency of the variant MBL2 B allele, associated with reduced circulating and vaginal MBL concentrations, was 27.0% in RVVC and 8.5% in control women (p < .0001). The MBL2 B, B genotype was present in 12% of RVVC patients and 1% of controls (p = .0025). The IL1RN 2 allele frequency, associated with the highest level of unopposed IL-1 beta activity, was 24.0% in RVVC and 23.4% in controls. The IL1RN genotype distribution was also similar in both groups. Conclusion Carriage of the MBL2 codon 54 polymorphism, but not the IL1RN length polymorphism, predisposes to RVVC in Brazilian women.

Increased TLR2 expression in patients with type 1 diabetes: evidenced risk of microalbuminuria

Objective: To study the activation of an inflammatory cascade through leukocyte mRNA expression of TLR2, TLR4, MyD88, and pro-inflammatory cytokines in individuals with childhood onset type 1 diabetes. Design and methods: Seventy-six type 1 diabetic patients and 100 normoglycemic subjects (NG) 6 to 20 years old were recruited. Type 1 diabetic patients (DM1) were considered to have good (DM1G) or poor (DM1P) glycemic control according to the values of glycated hemoglobin. TLR2, TLR4, MyD88, interleukin-1 beta (IL-1 beta), IL-6, and tumor necrosis factor alpha (TNF-alpha) mRNA expressions were measured in peripheral blood leukocytes (PBL) by real-time polymerase chain reaction (PCR). Urea, creatinine, albumin, and total protein serum levels were determined. Urinary albumin-to-creatinine ratio (ACR) was calculated. Results: DM1 and DM1P patients showed higher glycated hemoglobin (10 and 11%, respectively) and serum glucose concentrations (208 and 226 mg/ dL, respectively) compared to NG (Glycated hemoglobin: 7% and glucose: 76 mg/ dL) (p < 0.05). PBL mRNA expressions of TLR2, MyD88, IL-1 beta, IL-6, and TNF-alpha were higher in DM1 and TLR2, IL-1 beta, and IL-6 expressions were higher in DMP1 compared to NG (p < 0.05). In DM1, serum albumin and total protein were lower, while serum urea and ACR were higher in comparison to NG (p < 0.05). However, these differences compared to NG were more pronounced in DM1P, which included nine individuals with microalbuminuria. Conclusions: Increased mRNA expression of TLR2, MyD88, and pro-inflammatory cytokines in leukocytes of patients with childhood onset type 1 diabetes indicates the development of a TLR2-mediated pro-inflammatory process, which may also be associated with an early inflammatory process in the kidney and the occurrence of microalbuminuria.

Nitric oxide modulates metalloproteinase-2, collagen deposition and adhesion rate after polypropylene mesh implantation in the intra-abdominal wall

Prosthetic meshes are commonly used to correct abdominal wall defects. However, the inflammatory reaction induced by these devices in the peritoneum is not completely understood. We hypothesized that nitric oxide (NO), produced by nitric oxide synthase 2 (NOS2) may modulate the response induced by mesh implants in the abdominal wall and, consequently, affect the outcome of the surgical procedure. Polypropylene meshes were implanted in the peritoneal side of the abdominal wall in wild-type and NOS2-deficient (NOS2(-/-)) mice. After 15 days tissues around the mesh implant were collected, and inflammatory markers (the cytokine interleukin 1 beta (IL-1 beta) and NO) and tissue remodeling (collagen and metalloproteinases (MMP) 2 and 9) were analyzed. The lack of NOS2-derived NO induced a higher incidence of visceral adhesions at the mesh implantation site compared with wild-type mice that underwent the same procedure (P < 0.05). Additionally, higher levels of IL-1 beta were present in the mesh-implanted NOS2(-/-) animals compared with control and wild-type mice. Mesh implantation induced collagen I and III deposition, but in smaller amounts in NOS2(-/-) mice. MMP-9 activity after the surgical procedure was similarly increased in both groups. Conversely, MMP-2 activity was unchanged in mesh-implanted wild-type mice, but was significantly increased in NOS2(-/-) mice (P < 0.01), due to decreased S-nitrosylation of the enzyme in these animals. We conclude that NOS2-derived NO is crucial for an adequate response to and integration of polypropylene mesh implants in the peritoneum. NO deficiency results in a prolonged inflammatory reaction to the mesh implant, and reduced collagen deposition may contribute to an increased incidence of visceral adhesions. (C) 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Oral Administration of Oleic or Linoleic Acids Modulates the Production of Inflammatory Mediators by Rat Macrophages

Oleic (OLA) and linoleic (LNA) acids are commonly consumed fatty acids and they can modulate the inflammatory response, in which macrophages play an important role. The aim of this study was to investigate the effects of these two fatty acids on the production of inflammatory mediators by macrophages. Rats received oral administration of water (control), OLA or LNA (0.22 g/kg body weight) daily for 10 days and peritoneal resident macrophages were then isolated. Subsequently, they were seeded in culture plates and the production of various inflammatory mediators was assessed. Oral administration with OLA decreased the production of IL-1 beta, IL-6 and CINC-2 alpha beta by resident macrophages and LNA decreased the production of IL-1 beta, IL-6 and VEGF in the absence of lipopolysaccharide (LPS), although it accelerated IL-1 beta release and decreased IL-10 synthesis when cells were stimulated with LPS. Neither fatty acid affected the production of superoxide anion, hydrogen peroxide, nitrite, TNF-alpha, PGE(2), LTB4 or 15(S)-HETE. Thus, OLA and LNA influence the production of several inflammatory mediators by macrophages.

Tributyrin attenuates obesity-associated inflammation and insulin resistance in high-fat-fed mice

Vinolo MA, Rodrigues HG, Festuccia WT, Crisma AR, Alves VS, Martins AR, Amaral CL, Fiamoncini J, Hirabara SM, Sato FT, Fock RA, Malheiros G, dos Santos MF, Curi R. Tributyrin attenuates obesity-associated inflammation and insulin resistance in high-fat-fed mice. Am J Physiol Endocrinol Metab 303: E272-E282, 2012. First published May 22, 2012; doi:10.1152/ajpendo.00053.2012.-The aim of this study was to investigate whether treatment with tributyrin (Tb; a butyrate prodrug) results in protection against diet-induced obesity and associated insulin resistance. C57BL/6 male mice fed a standard chow or high-fat diet were treated with Tb (2 g/kg body wt, 10 wk) and evaluated for glucose homeostasis, plasma lipid profile, and inflammatory status. Tb protected mice against obesity and obesity-associated insulin resistance and dyslipidemia without food consumption being affected. Tb attenuated the production of TNF alpha and IL-1 beta by peritoneal macrophages and their expression in adipose tissue. Furthermore, in the adipose tissue, Tb reduced the expression of MCP-1 and infiltration by leukocytes and restored the production of adiponectin. These effects were associated with a partial reversion of hepatic steatosis, reduction in liver and skeletal muscle content of phosphorylated JNK, and an improvement in muscle insulin-stimulated glucose uptake and Akt signaling. Although part of the beneficial effects of Tb are likely to be secondary to the reduction in body weight, we also found direct protective actions of butyrate reducing TNF alpha production after LPS injection and in vitro by LPS- or palmitic acid-stimulated macrophages and attenuating lipolysis in vitro and in vivo. The results, reported herein, suggest that Tb may be useful for the treatment and prevention of obesity-related metabolic disorders.