O gene Slc11a1 regula a resistência contra S. entérica, L. donovani e M. tuberculosis. Sublinhagens de camundongos AIRmax e AIRmin homozigotas para os alelos R e S do gene Slc11a1 (AIRmax,RR, AIRmaxSS, AIRminRR e AIRminSS) foram utilizadas para avaliar o efeito destes alelos na ativação de macrófagos peritoneais (M<font face=\"Symbol\">F), induzida pelo tioglicolato (TIO) ou infecção por M. bovis BCG.O TIO induz baixa ativação celular, a estimulação com LPS aumentou a ativação nos macrófagos dos animais AIRmaxRR e AIRmaxSS. Assim, na inflamação com TIO, a ativação do M<font face=\"Symbol\">F foi dependente do fundo genético selecionado nos animais AIRmax, independente do alelo do gene Slc11a1. Na infecção com BCG, a sublinhagem AIRmaxRR foi a única capaz de controlar a proliferação da bactéria, secretando altos níveis de IL-1<font face=\"Symbol\">b, IL-12, TNF<font face=\"Symbol\">a e IL-6 que ativam o macrófago. Por outro lado, os AIRminSS susceptíveis à infecção produziram maiores concentrações de NO, H2O2 e IL-10, mostrando o fundo genético para alta ou baixa resposta inflamatória, e os alelos do gene Slc11a1 interferem na resistência à infecção. / The Slc11a1 gene regulates resistance against S. enterica, L. donovani and M. tuberculosis. AIRmax and AIRmin mouse sublines, homozygous for Slc11a1 R and S alleles (AIRmaxRR, AIRmaxSS, AIRminRR and AIRminSS) were used in this work to evaluate the effect of this gene in peritoneal macrophage (M<font face=\"Symbol\">F) activation induced by thioglycollate (TIO) or M. bovis BCG infection. TIO induced weak cellular activation, LPS stimulation increased AIRmaxRR and AIRmaxSS macrophages activation. These results indicate that inflammation and M<font face=\"Symbol\">F activation induced by TIO were dependent on the genetic background for acute inflammatory response, while Slc11a1 alleles have little effect on this phenotype. In BCG infection only AIRmaxRR mice were capable of controlling bacterial proliferation, which was accompanied with high levels of IL-1<font face=\"Symbol\">b, IL-12, TNF and IL-6 produced by activated macrophages. On the other hand, susceptible AIRminSS mice produced higher amounts of NO, H2O2 and IL-10 suggesting that both inflammatory background and Slc11a1 alleles interfere on resistance to BCG infection.

Crotalfina (CRF) é um peptídeo produzido do veneno da serpente Crotalus durissus terrificus com efeito antinociceptivo tipo opióide detectado apenas na presença de inflamação. Estudos sobre mecanismos de analgesia relatam a ação de opióides em células imunes e macrófagos são apontados como alvos para os efeitos destes. Este estudo caracterizou a ação da CRF sobre funções de macrófagos e avaliou a importância de seus receptores opióides nestas ações. Os resultados indicam que CRF inibe fagocitose, liberação de H2O2 e produção de NO&bull; e TNF-a, modula IL-6 e estimula IL-1b por macrófagos residentes e inflamatórios. Ensaios de expressão e ativação mostram que macrófagos residentes expressam receptores opióides k seguido de m e d e macrófagos inflamatórios expressam receptores m seguido de k e d. A CRF interferiu, de maneira distinta, com a expressão destes receptores, dependendo do estado de ativação destas células. Antagonistas destes receptores bloquearam a ação da CRF, mostrando que este peptídeo possui ação imunomodulatória mediada por receptores opióides. / Crotalphine (CRF) is a peptide isolated from the venom of Crotalus durissus terrificus with type opioid antinociceptive effect only detected in the presence of inflammation. Studies on mechanisms of analgesia reported the action of opioids in immune cells and macrophages are considered as targets for these effects. This study characterized the action of CRF on functions of macrophages and assessed the importance of opioid receptors in these actions. The results indicate that CRF inhibits phagocytosis, release of H2O2 and production of NO&bull; and TNF-a, modulates IL-6 and stimulates IL-1b production by resident and inflammatory macrophages. Expression and activation assays show that resident macrophages express k receptors followed by m and d. Inflammatory macrophages express m receptors followed by k and d. CRF interfered differently with the expression of these receptors, depending on the activation state of these cells. Antagonists of these receptors blocked the action of CRF, showing that this peptide has immunomodulatory action mediated by opioid receptors.

Orientador: Mário José Abdalla Saad / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T22:12:54Z (GMT). No. of bitstreams: 1 Mittestainer_FrancineCappa_M.pdf: 2379142 bytes, checksum: cb6178fdb335fdabb4a29e4767a5c1b5 (MD5) Previous issue date: 2011 / Resumo: A infusão da insulina durante a inflamação aguda melhora os resultados clínicos, mas o exato mecanismo desse efeito benéfico da insulina ainda não foi bem compreendido. Estudos recentes mostraram que a insulina tem um efeito antiinflamatório de modo que o hormônio também exibe um efeito inibidor sobre a mediação da transcrição de NF-kB em células mononucleares. Assim, o objetivo do presente estudo foi investigar os efeitos agudos da administração de insulina regular sobre a modulação de proteínas da via IKK/IkB/NF-kB e das proteínas chave da via de sinalização da insulina em tecido hepático, muscular e adiposo, a expressão de NF-kB nesses tecidos através de ELISA, o efeito do tratamento com insulina em macrófagos extraídos do peritônio de ratos e a influência dos inibidores da PI3K e MAPK na via inflamatória. Ademais, fizemos um ensaio da proteína fosfatase PP2A usando a imunoprecipitação com anticorpos anti-IKKbeta. Para o experimento, ratos machos adultos Wistar compuseram aleatoriamente 4 grupos diferentes. Três deles foram submetidos à infusão de insulina na veia porta e, então foram estimulados em 1, 3 e 5 minutos, enquanto o outro grupo (0) não foi estimulado com insulina. Em nossos resultados, observamos um aumento da fosforilação de IR, IRS1 e Akt induzida pela insulina nos três tecidos estudados. Em paralelo, houve uma redução na fosforilação de IKK e IkB após o estimulo da insulina. A ativação de NF-kB p65 no núcleo das células mostrou a redução na fosforilação do IKK e IkB no fígado, músculo e tecido adiposo. Na cultura de macrófagos tratada com insulina, após a adição dos inibidores específicos para PI3K e MAPK, observamos um aumento na fosforilação de IKK e IkB. Nossos resultados também mostraram que após estímulo da insulina houve um aumento na atividade da proteina fosfatase PP2A associada ao IKK nos três tecidos estudados. Desta forma, podemos sugerir que insulina pode induzir uma interação entre PP2A e IKK, o que resultará em mais desfosforilação de IKK e, assim, uma inibição da atividade da proteína quinase, revelando um potencial mecanismo de ação efeito anti-inflamatório da insulina / Abstract: Insulin infusion during acute inflammation improves clinical the outcomes but the exact mechanism of this beneficial effect is unclear. Recent studies have suggested that insulin has an anti-inflammatory effect in such a way that this hormone exhibits an inhibitory effect on the mediation of transcription of NF- kB in mononuclear cells. Thus, the aim of this study was to investigate the acute effects of regular insulin administration in modulation of IKK/IkB/NF-kB pathway and insulin signaling pathway (IR, IRS1 and Akt) and the DNA binding of NF-kB p65 in liver, muscle and adipose tissue of rats and also analyze the effect of treatment with insulin on macrophages of rats and the influence of PI3K and MAPK inhibitors on pathway. And finally, we analyze a phosphatase assay by using an immunoprecipitation with anti-IKKbeta antibody and PP2A phosphatase assay. For the experiment, male adult Wistar rats were divided in 4 groups. Three of them were submitted to insulin injection in the portal vein and were stimulated for 1, 3 and 5 minutes and the other group (0) was not stimulated (saline). In our results, we observed an increase in insulin-induced phosphorylation of IR, IRS1 and Akt during insulin injection in the three tissues studied. In parallel, there was a reduction in the phosphorylation of IKK and IkB after insulin stimulation. The DNA binding of NF-kB p65 in the cell nucleus showed a reduction of the IKK and IkB phosphorylation after insulin injection in liver, muscle and adipose tissue. In macrophages culture of treated with insulin and when added the specific inhibitor for PI3K and MAPK we observed an increased on phosphorylation of IKK and IkB. Our results also showed that after insulin stimulus there was an increase in PP2A phosphatase activity associated with IKK in the three tissues studied. Thus, we can suggest that insulin might induce an interaction between PP2A and IKK, which will result in more IKK dephosphorylation and thus an inhibition of this protein kinase activity, showed a possible anti-inflammatory effect of insulin / Mestrado / Mestre em Ciências

Abstract Chlamydia pneumoniae is an obligate intracellular gram-negative bacterium, which causes respiratory infections in humans and may participate in the development of chronic diseases like atherosclerosis, chronic obstructive lung disease, adult-onset asthma and late-onset Alzheimer’s disease. It can infect various cell types, e.g. vascular endothelial cells, smooth muscle cells and monocyte-derived macrophages in vitro. It has been speculated that circulating macrophages disseminate the infection in the body, and that genetically susceptible individuals become chronically infected. Quantification of C. pneumoniae growth inside cultured cells is needed when studying e.g. the effect of drugs or host cell factors on infectivity and replication. Conventionally this has been done by immunofluorescence staining and microscopic counting of chlamydial inclusions. However, this method is usable only if the cell numbers do not fluctuate in cell culture vials and the inclusions are uniform. In macrophages, inclusions are often aberrant, their sizes vary and multiple inclusions are also seen. Therefore we developed a new method based on the real-time PCR quantification of chlamydial genomes adjusted to the number of human genomes and used it to quantify the exact amounts of C. pneumoniae in infected cells. The susceptibility of monocyte-macrophages from healthy individuals to C. pneumoniae infection in vitro was studied first. Intracellular growth of C. pneumoniae was used as an indicator of susceptibility to infection, and it was compared to serum levels of CRP, soluble CD14, human HSP-IgG, human HSP-IgA, C. pneumoniae IgG and IgA antibodies. The growth of C. pneumoniae in infected macrophages was highly variable, ranging from 0 to 638 chlamydial genomes per human genome. C. pneumoniae growth associated positively with serum C. pneumoniae IgA (titer ≥10) and hHSP-IgG and negatively with soluble CD14 concentration. The association between chlamydial IgA antibodies, hHSP-IgG and C. pneumoniae growth was statistically significant only among men. Age did not correlate with the growth. Therefore we hypothesize that persons whose macrophages cannot restrict the growth of C. pneumoniae are more prone to chronic infection by this agent. In the next study, we evaluated the effects of innate immunity genes CD14 -260 C>T, TLR2 Arg753Gln, TLR4 Asp299Gly, LBP Phe436Leu and IL-6 -174 G>C polymorphisms on C. pneumoniae growth in human macrophages in vitro. The growth of C. pneumoniae was highest in CD14 -260 C>T TT genotype cells and the difference to CC or CT genotype was statistically significant. The G-allele of the IL6 -174 G>C polymorphism had a positive influence on chlamydial growth; the difference was statistically significant only between CC and GC genotypes. TLR2 Arg753Gln, TLR4 Asp299Gly, LBP Phe436Leu polymorphisms showed no effect on chlamydial growth.

Visceral leishmaniasis (VL) causes fatal life-threatening disease, if left untreated. The current drugs have various limitations; hence, natural products from medicinal plants are being focused in search of new drugs to treat leishmaniasis. The aim of the present study was to evaluate the antileishmanial and immunomodulatory activities of F5 and F6 alcoholic fractions from Withania somnifera leaves and purified withaferin-A in Leishmania donovani-infected peritoneal macrophages and BALB/c mice. We observed that F5 (15 mu g/mL), F6 (10 mu g/mL), and withaferin-A (1.5 mu M) reduce amastigote count in peritoneal macrophages and induce reactive oxygen species and significant decrease in IL-10 mRNA expression compared to control upon treatment. Subsequently, in vivo study mice were treated with F5 (25 and 50 mg/kg b.wt.), F6 (25 and 50 mg/kg b.wt.) orally, and withaferin-A (2 mg/kg b.wt.) intraperitoneally for 10 consecutive days and a drastic reduction in parasite burden in both spleen and liver were observed. The treatment resulted in the reduction in IL-10, IL-4, and TGF-beta mRNA expression and a significant increase in IFN-gamma /IL-10 expression ratio in the treated group compared to control. The humoral response of these alcoholic fractions and withaferin-A shows increased IgG2a levels when compared with IgG1 in treated mice. Taken together, our result concludes that withanolides in alcoholic fractions demonstrate a potent antileishmanial and immunomodulatory activities in experimental VL.

Mycobacter avium complex serovars 4 and 20 were cultured in the presence of [3H] fucose, [3H]-methionine, and [3H]-mannose to specifically radiolabel the oligosaccharide of the glycopeptidolipid (GPL) antigens. Distribution of radioactivity in lipid was determined by thin-layer chromatographic methods. Examination of acid hydrolysates from radiolabeled antigens revealed that [3H]-methionine incorporated into methylated sugars in polar and apolar GPL components, whereas [3H]-mannose incorporated exclusively into the oligosaccharide of polar GPL antigens. Least incorporation of radiolabel into antigens was observed with [3H]-fucose. Use of radiolabeled serovar 4 antigens in macrophage uptake studies revealed maximum uptake to be slightly above 250 gg/ 3.2 x 105 cells. Timed experiments demonstrated that GPL antigens were relatively inert to degradation by resident peritoneal macrophages.

Wear particle-induced inflammation leading to periprosthetic osteolysis remains a major cause of hip implant failure. As polyethylene particles from conventional metal-on-polyethylene implants have been associated with these failures, an interest in lower wear metal-on-metal (MM) bearings has emerged. However, the biological effects of nanometer-size chromium oxide particles, predominant type of wear particles produced by MM implants, remain mostly unknown. Therefore, this study aimed to determine the cytotoxicity of nanometer-size Cr2O3 particles on macrophages in vitro, by analyzing their effects on cell mortality and cytokine release and comparing them with those of similarly-sized alumina (Al2O3) particles (known to be relatively bioinert). Results showed that at high concentrations, nanometer-size Cr2O3 particles can be cytotoxic to macrophages, inducing significant decreases in total cell numbers and increases in necrosis. Results also showed that, at high concentrations, the cytotoxicity of Cr2O3 particles was overall higher than that of Al2O3 particles, even though Cr2O3 and Al2O3 are both stable forms of ceramic materials. However, it appeared to be lower than that of previously reported conventional polyethylene and CoCrMo particles. Therefore, chromium oxide particles may not be the main culprit in initiating the inflammatory reaction in MM periprosthetic tissues.

ABCA1 is a key plasma membrane protein required for the efflux of cellular cholesterol to extracellular acceptors, particularly to apoA-I. This process is essential to maintain cholesterol homeostasis in the body. The detailed molecular mechanisms, however, are still insufficiently understood. Also, the molecular identity of ABCA1, i.e. channel, pump or flippase, remains unknown. In this study we analyzed the extracellular ATP levels in the medium of ABCA1-expressing BHK cells and RAW macrophages and compared them to the medium of relevant non-expressing cells. We found that the extracellular ATP concentrations are significantly elevated when cells express ABCA1. Importantly, a dysfunctional ABCA1 mutant (A937V), when expressed similarly as WT-ABCA1, is unable to raise extracellular ATP concentration. This suggests a causal relationship between functional ABCA1 and elevated extracellular ATP. To explore the physiological role of elevated extracellular ATP, we analyzed ABCA1-mediated cholesterol efflux under the conditions where extracellular ATP levels were modulated. We found that increasing extracellular ATP within the physiological range, i.e. < μM, promotes cholesterol efflux to apoA-I. On the other hand, removing extracellular ATP, either by adding apyrase to the medium or by expressing a plasma membrane bound ecto-nucleotidase CD39, abolishes cholesterol efflux to apoA-I. Based on these results we conclude that, through direct or indirect mechanisms, ABCA1 functions to raise ATP levels in the medium. This elevated extracellular ATP is required for ABCA1-mediated cholesterol efflux to apoA-I.

Cell death is an imperative mechanism for the development, homeostasis and survival of an organism. Various forms of cell death have been documented and recent reports indicate that the mode of cell death elicited can have a profound influence on the development and perpetuation of inflammation. Apoptosis is the predominant, programmed pathway of cell death, which ensures physiological elimination of unwanted cells. On the other hand, another cell death pathway described as programmed necrosis (necroptosis), has recently been revealed. The induction of necroptosis and its impact in host biology is not clear. Herein I have evaluated the mechanisms of necroptosis in macrophages, an important cell type of the immune system. My experiments indicate that type I interferon (IFN-I) signaling through transcription factors STAT1, STAT2 and IRF9, collectively described as the ISGF3 complex, is indispensable for necroptosis of macrophages. Furthermore, my results indicate that IFN-I signaling promotes the sustained phosphorylation of receptor interacting protein kinase 3 (Rip3), a key protein required for the execution of necroptosis. My findings also reveal that dynamin-dependent endocytosis following IFNβ stimulation and caspase inhibition is necessary for the induction of necroptosis. The results presented in this thesis provide new insights into the molecular mechanisms of necroptosis and therefore contribute to a deeper understanding of multiple inflammatory pathologies.

Monocyte-derived macrophages (MDMs) from HIV-infected patients and MDMs infected in vitro with HIV manifest inhibition of various cytokines including IL 12. Recently, IL-27 was shown to inhibit HIV replication in macrophages. Whether HIV infection or HIV regulatory proteins such as tat, impact IL-23 or IL-27 production in macrophages remains unknown. I have demonstrated that intracellular HIV-tat expression as well as HIV-tat basic domain peptides inhibited LPS-induced IL-23 and IL-27 proteins and their subunits in MDMs. First I investigated the signalling pathways involved in the regulation of LPS-induced IL-23 and IL-27 production in MDMs infected with control pLXIN retrovirus-infected MDMs. The p38 MAPK, SHP-1 and PI3K signalling molecules positively regulated LPS-induced IL-23 expression. In contrast, Src kinases and JNK MAPK negatively regulated LPS-induced IL-23 production. On the other hand, LPS-induced IL-27 production was positively regulated by the PI3K, p38 MAPKs and SHP-1 and Src kinases. Src kinases positively regulated LPS-induced IL-27 production whereas Src kinases and JNK negatively regulated LPS-induced IL-23 production. HIV-Tat significantly inhibited p38 MAPK and PI3K which were implicated in HIV-Tat-mediated inhibition of LPS-induced IL-23 and IL-27 production. Even though HIV-Tat inhibited ERK and JNK MAPK activation, these kinases were not involved in HIV-Tat-mediated inhibition of LPS-induced IL-23 and IL-27 production. While SHP-1 regulated LPS-induced IL-23 and IL-27 production, HIV-Tat did not inhibit SHP-1 and therefore were not involved in HIV-Tat-mediated inhibition of LPS-induced IL-23 and IL-27 production. HIV-Tat did not inhibit Src kinases and hence were not involved in HIV-Tat-mediated inhibition of LPS-induced IL-27 production. Furthermore, HIV-Tat did not inhibit the expression of upstream TLR4-activated signaling molecules including TRAF3, TRIF, MyD88, IRAK1, IRAK3, IRAK4, TRAF-1, TRAF-2, cIAP-1, cIAP-2 and, xIAP. These results suggest association of IL-23 and IL-27 inhibition by HIV with decreased HIV-specific immune responses, and increased viral replication. These results further suggest novel strategies to improve cellular immune responses and inhibition of HIV replication.