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There is a gap in the treatment of preterm infants with respiratory distress syndrome. Despite addressing surfactant insufficiency and mechanical instability, currently available exogenous surfactant does not reduce the inflammation that results from aggressive ventilation and hyperoxia. Preterm infants are also deficient in anti-oxidant and anti-inflammatory defenses. All of these factors predispose the infant to bronchopulmonary dysplasia. Recombinant human Club Cell Secretory Protein 10 (rhCC10), known to inhibit sPLA2, has been used to reduce ventilator induced lung inflammation. The long-term goal of this study is to understand the impact of rhCC10 on the pro/anti-inflammatory balance during early development. We will interrogate the link between Toll-Like Receptors (TLR 4) and rhCC10 because TLRs are integral to lung inflammation. By measuring the mRNA expression, protein, and downstream signaling activity in rhCC10 treated preterm lamb lung and then in A549 cells, an alveolar epithelial cell-like system, the anti-inflammatory effect of rhCC10 will be differentiated. This research is significant because it will improve understanding of the effect of rhCC10 on pro/anti-inflammatory regulation and provide insight regarding potential co-therapies when treating with rhCC10. / Physiology

The infections agent of Lyme disease, Borrelia Burgdorferi is a complex microorganism with a highly diverse genome. One of the most remarkable aspects of the B. burgdorferi genome is the large number of sequences encoding predicted or known lipoproteins, including outer-surface proteins. The B. burgdorferi genome encodes no recognizable toxins. Instead, this extracellular pathogen causes pathology by migration through tissues, adhesion to host cells, and evasion of immune clearance. Inflammation elicited by infection with B. burgdorferi depends on the ability of the spirochete to survive in the mammalian host, as well as the immune response that arises upon the interaction of the bacterium with phagocytic, T and other cell types. Innate immune responses are critical in recognition and clearance of pathogens, and also play an important role in the outcome of adaptive immune responses. The regulation of innate immune responses to pathogens occurs through the interaction of Toll-like receptors (TLRs) with pathogen-associated molecular patterns (PAMPs) and the activation of several signaling pathways whose contribution to the overall innate immune response to pathogens is poorly understood. In this study we demonstrate a mechanism of control of murine macrophage responses mediated by TLR1/2 heterodimers through c-Jun N-terminal kinase 1 (JNK1) activity. JNK also controls tumor necrosis factor production and TLR-mediated macrophage responses to B. burgdorferi. We also show that the proximal promoter region of the human tlr1 gene contains an AP-1 binding site that is subjected to regulation by the kinase and binds two complexes that involve the JNK substrates c-Jun, JunD, and ATF-2. These results demonstrate that JNK1 regulates the response to TLR1/2 ligands and suggest a positive feedback loop that may serve to increase the innate immune response to the spirochete. MCJ is a newly identified member of the DnaJ protein family of cochaperones that contains unique features different than the normally described DnaJ proteins. However, there is little known about its function and the role it plays in different cells and systems. It has been previously shown that MCJ is required for the repression of the ABCB1 drug transporter expression in breast cancer cells, and that this repression is mediated through the control of c-Jun protein stability. We were therefore interested in determining the role that MCJ plays in macrophages in response to B. burgdorferi antigens. We now provide evidence that MCJ controls inflammatory responses of macrophages through the regulation of c-Jun protein stability, and the expression and release of the inflammatory cytokine TNF through the regulation of the expression of TNF converting enzyme (TACE) inhibitor tissue inhibitor of metalloproteinase 3 (TIMP-3).

Introduction: Communication between neuroendocrinne and immune system is arranged by hormones and cytokines in endocrinne, paracrinne and autocrinne manner. One of the factors involved is also prolactin, a pituitary hormone and an immune cytokine. Sepsis is a system reaction to inflammation mediated by Th1 immune response, which is supported by prolactin as well. Primary protection against sepsis is mediated by innate immunity. Toll- like receptors distinguish molecules, which are connected with pathogens. Afterwards this identification of a specific pathogen toll-like receptors trigger immune reaction with the main goal of destroying this pathogen and also with the goal of renewing the balance of the organism. It is supposed that in the organism that is hardly attacked by a pathogen, the PRL, TLR2 and TLR4 gene expression is on the increase. We studied the levels of PRL, TLR2 and TLR4 mRNA production in circulating monocytes derived from septic patients. Simultaneously, the effect of PRL -1149 G/T SNP on physiological levels of PRL mRNA and its expression in the course of sepsis was evaluated. Materials and methods: As a source of monocytes, blood specimens from 43 septic patients and 40 healthy controls were used. The blood of septic patients was taken three times with some time difference and...

Após a resposta imune, a expansão dos linfócitos T CD4 é seguida de uma fase de retração chamada Morte Celular induzida por Ativação (AICD), para que a homeostasia seja reestabelecida. Nosso grupo demonstrou que DCs estimuladas com LPS produzem PGE2 que inibe a expressão de FASL e bloqueia a AICD dos linfócitos T CD4. Nossa hipótese é que a apresentação de antígenos em contexto de infecção tenha um impacto na expressão de FASL e na sobrevivência das células T CD4, de maneira dependente de TLRPGE2. Para comprovar nossa hipótese nós estudamos a apresentação de OVA in vitro e in vivo. Observamos que a adição de LPS durante a apresentação de OVA aumenta a ativação e proliferação das células T CD4 específicas. O pré-tratamento dos camundongos com Indometacina, um inibidor da enzima COX, reduz a frequência das células específicas através do aumento na expressão de FASL e da apoptose, mas sem interferir com a proliferação. Nós sugerimos que a PGE2 produzida em resposta ao LPS regule a sobrevivência dos linfócitos T CD4 durante a persistência do estímulo antigênico. / After immune response, expansion of antigen-specific CD4 T cells is followed by a contraction phase due to Activation-Induced Cell Death (AICD) to reestablish homeostasis. Our group demonstrated that LPS stimulated-DCs produce PGE2 that protects CD4 T cells from AICD by preventing TCR/CD3-mediated FASL upregulation. Our hypothesis is that antigen presentation in the context of infection impacts on FASL expression and survival of CD4 T cells, dependently on TLR-mediated PGE2 release. To approach our hypothesis we studied OVA presentation in vitro and in vivo. We observed that the addition of LPS during OVA presentation increased specific CD4+ T cells activation and proliferation. Pretreatment of mice with indomethacin, an inhibitor of COX enzyme, reduces the frequency of specific T cells by increasing FASL expression and apoptosis, but did not interfere with proliferation. We suggest that PGE2 produced in response to LPS regulates the survival of CD4 T lymphocytes during persistent antigen stimulation.

A paracoccidioidomicose é uma micose sistêmica de natureza profunda que afeta preferencialmente o tecido pulmonar podendo disseminar via linfo-hematogênica para outros órgãos e tecidos, sendo causada principalmente pelo Paracoccidioides brasiliensis, fungo que apresenta dimorfismo térmico. O sistema imune inato mediado por macrófagos é extremamente importante para o controle de infecções e está envolvido na indução e regulação da resposta imune/inflamatória. Estas células são capazes de reconhecer patógenos por meio de receptores de reconhecimento de padrões (PRRs), tais como receptores Toll-like (TLR). Além desses PRRs, recentemente, demonstrou-se a importância da via de sinalização Notch no sistema imune inato e na regulação da atividade dos macrófagos. Nossos dados demonstram que a cepa Pb18 do P. brasiliensis é capaz de ativar o receptor Notch1 em macrófagos J774. A ativação desse receptor concomitante com a ativação de TLR 4 (via LPS) induz a produção de IL-6, e apresenta elevada carga fúngica e menor fagocitose, o que favorece a patogenia. Ao utilizarmos um inibidor farmacológico da γ-secretase (DAPT) para inibir a ativação do receptor Notch1 em macrófagos, é possível observar diminuição da carga fúngica, diminuição de IL-6, aumento de TNF-α e aumento da fagocitose. Entretanto, a ausência do receptor TLR 4 em macrófagos derivados de medula óssea de camundongos TLR 4-/-, na presença de DAPT, percebe-se diminuição da capacidade fagocítica desses macrófagos e também diminuição da carga fúngica, evidenciando a relação entre TLR 4 e Notch1. Em adição, realizamos um tratamento em camundongos BALB/c com DAPT previamente à infecção com Pb18. Nossos resultados evidenciaram que animais com este tratamento apresentaram diminuição da carga fúngica dos pulmões, diminuição de IL-6, ativação de macrófagos e aumento de IgG, após 45 dias de infecção, indicando um perfil de cura desses animais. O mesmo tratamento foi realizado em camundongos BALB/c NUDE, seguido da infecção com Pb18. Nestes animais, verificamos que há maior produção de citocinas pró-inflamatórias no pulmão, aumento de células CD19+ e a carga fúngica dos animais tratados manteve-se similar ao dos animais não tratados, indicando que o perfil protetor observado em animais com DAPT é dependente da resposta das células T. Juntos, esses resultados evidenciam que o Pb18 é capaz de ativar o receptor Notch1 em macrófagos e utiliza a via de sinalização Notch-TLR 4 como um possível mecanismo de escape, podendo fornecer uma nova abordagem de estudo da imunidade envolvida na paracoccidioidomicose experimental. / Paracoccidioidomycosis is a systemic mycosis of deep nature that primarily affects the lung and can spread via lymphatic and hematogenous to other organs and tissues. It is mainly caused by Paracoccidioides brasiliensis fungus which exhibit thermal dimorphism. The innate immune system mediated by macrophages is extremely important for the control of infection and is involved in the induction and regulation of immune/inflammatory response. These cells are able to recognize pathogens through pattern recognition receptors (PRRs) such as Toll-like receptors (TLR). Beyond these PRRs, the importance of Notch signaling has recently been demonstrated in the innate immune system and the regulation of macrophage activity. Our data demonstrate that the Pb18 strain of P. brasiliensis is able to activate the Notch1 receptor in J774 macrophages. Activation of this receptor with also activation of TLR 4 (via LPS) induces IL-6 production, induces phagocytosis and decreases fungal burden, which favors the pathogenesis. By using a γ-secretase pharmacological inhibitor (DAPT) for inhibiting the activation of Notch1 receptor on macrophages, it is possible to observe decreased fungal burden, less production of IL-6, and increased TNF-α and phagocytosis. However, due to the absence of TLR 4 receptor in bone marrow derived macrophages from TLR 4-/- mice, these macrophages showed decreased phagocytic ability and also reduced fungal burden in the presence of DAPT, showing a relationship between TLR 4 and Notch1. In addition, we made a treatment with DAPT in BALB/c mice prior to infection with Pb18. And our results showed that DAPT-treated animals exhibited a decrease of fungal burden in the lungs, and a decrease of IL-6. Furthermore, we observed an increase of IgG after 45 days of infection, indicating probably a healing of these animals. Same treatment was made in BALB/c NUDE mice, followed by infection with Pb18. In these animals, we observed an increased production of proinflammatory cytokines in the lung and increased CD19+ cells, but fungal burden was similar in both group (treated and untreated), which indicates that treatment with DAPT is dependent on T cell response. Taken together, these results showed that Pb18 is able to activate the Notch 1 receptor on macrophages and uses the Notch-TLR 4 signaling pathway as a possible escape mechanism, and may provide a new immunity study approach in experimental paracoccidioidomycosis.

The aim of this thesis was to study cancer immunotherapy based on combination of TLR7 signalling and activation of phagocytosis of cancer cells. This thesis was focused on the possibility of enhancing its effect. The possibilities of removing immunosuppressive barriers (esp. MDSCs and adenosine) were studied, using a combination with chemotherapy or enzyme - adenosine deaminase and purine nucleoside phosphorylase. The effect of enzymes was studied in both in vivo and in vitro experiments. As another way of optimizing the therapy used so far, the modulation of the therapeutic mixture using lower molecular weight substances was tried.

Após a resposta imune, a expansão dos linfócitos T CD4 é seguida de uma fase de retração chamada Morte Celular induzida por Ativação (AICD), para que a homeostasia seja reestabelecida. Nosso grupo demonstrou que DCs estimuladas com LPS produzem PGE2 que inibe a expressão de FASL e bloqueia a AICD dos linfócitos T CD4. Nossa hipótese é que a apresentação de antígenos em contexto de infecção tenha um impacto na expressão de FASL e na sobrevivência das células T CD4, de maneira dependente de TLRPGE2. Para comprovar nossa hipótese nós estudamos a apresentação de OVA in vitro e in vivo. Observamos que a adição de LPS durante a apresentação de OVA aumenta a ativação e proliferação das células T CD4 específicas. O pré-tratamento dos camundongos com Indometacina, um inibidor da enzima COX, reduz a frequência das células específicas através do aumento na expressão de FASL e da apoptose, mas sem interferir com a proliferação. Nós sugerimos que a PGE2 produzida em resposta ao LPS regule a sobrevivência dos linfócitos T CD4 durante a persistência do estímulo antigênico. / After immune response, expansion of antigen-specific CD4 T cells is followed by a contraction phase due to Activation-Induced Cell Death (AICD) to reestablish homeostasis. Our group demonstrated that LPS stimulated-DCs produce PGE2 that protects CD4 T cells from AICD by preventing TCR/CD3-mediated FASL upregulation. Our hypothesis is that antigen presentation in the context of infection impacts on FASL expression and survival of CD4 T cells, dependently on TLR-mediated PGE2 release. To approach our hypothesis we studied OVA presentation in vitro and in vivo. We observed that the addition of LPS during OVA presentation increased specific CD4+ T cells activation and proliferation. Pretreatment of mice with indomethacin, an inhibitor of COX enzyme, reduces the frequency of specific T cells by increasing FASL expression and apoptosis, but did not interfere with proliferation. We suggest that PGE2 produced in response to LPS regulates the survival of CD4 T lymphocytes during persistent antigen stimulation.

A paracoccidioidomicose é uma micose sistêmica de natureza profunda que afeta preferencialmente o tecido pulmonar podendo disseminar via linfo-hematogênica para outros órgãos e tecidos, sendo causada principalmente pelo Paracoccidioides brasiliensis, fungo que apresenta dimorfismo térmico. O sistema imune inato mediado por macrófagos é extremamente importante para o controle de infecções e está envolvido na indução e regulação da resposta imune/inflamatória. Estas células são capazes de reconhecer patógenos por meio de receptores de reconhecimento de padrões (PRRs), tais como receptores Toll-like (TLR). Além desses PRRs, recentemente, demonstrou-se a importância da via de sinalização Notch no sistema imune inato e na regulação da atividade dos macrófagos. Nossos dados demonstram que a cepa Pb18 do P. brasiliensis é capaz de ativar o receptor Notch1 em macrófagos J774. A ativação desse receptor concomitante com a ativação de TLR 4 (via LPS) induz a produção de IL-6, e apresenta elevada carga fúngica e menor fagocitose, o que favorece a patogenia. Ao utilizarmos um inibidor farmacológico da γ-secretase (DAPT) para inibir a ativação do receptor Notch1 em macrófagos, é possível observar diminuição da carga fúngica, diminuição de IL-6, aumento de TNF-α e aumento da fagocitose. Entretanto, a ausência do receptor TLR 4 em macrófagos derivados de medula óssea de camundongos TLR 4-/-, na presença de DAPT, percebe-se diminuição da capacidade fagocítica desses macrófagos e também diminuição da carga fúngica, evidenciando a relação entre TLR 4 e Notch1. Em adição, realizamos um tratamento em camundongos BALB/c com DAPT previamente à infecção com Pb18. Nossos resultados evidenciaram que animais com este tratamento apresentaram diminuição da carga fúngica dos pulmões, diminuição de IL-6, ativação de macrófagos e aumento de IgG, após 45 dias de infecção, indicando um perfil de cura desses animais. O mesmo tratamento foi realizado em camundongos BALB/c NUDE, seguido da infecção com Pb18. Nestes animais, verificamos que há maior produção de citocinas pró-inflamatórias no pulmão, aumento de células CD19+ e a carga fúngica dos animais tratados manteve-se similar ao dos animais não tratados, indicando que o perfil protetor observado em animais com DAPT é dependente da resposta das células T. Juntos, esses resultados evidenciam que o Pb18 é capaz de ativar o receptor Notch1 em macrófagos e utiliza a via de sinalização Notch-TLR 4 como um possível mecanismo de escape, podendo fornecer uma nova abordagem de estudo da imunidade envolvida na paracoccidioidomicose experimental. / Paracoccidioidomycosis is a systemic mycosis of deep nature that primarily affects the lung and can spread via lymphatic and hematogenous to other organs and tissues. It is mainly caused by Paracoccidioides brasiliensis fungus which exhibit thermal dimorphism. The innate immune system mediated by macrophages is extremely important for the control of infection and is involved in the induction and regulation of immune/inflammatory response. These cells are able to recognize pathogens through pattern recognition receptors (PRRs) such as Toll-like receptors (TLR). Beyond these PRRs, the importance of Notch signaling has recently been demonstrated in the innate immune system and the regulation of macrophage activity. Our data demonstrate that the Pb18 strain of P. brasiliensis is able to activate the Notch1 receptor in J774 macrophages. Activation of this receptor with also activation of TLR 4 (via LPS) induces IL-6 production, induces phagocytosis and decreases fungal burden, which favors the pathogenesis. By using a γ-secretase pharmacological inhibitor (DAPT) for inhibiting the activation of Notch1 receptor on macrophages, it is possible to observe decreased fungal burden, less production of IL-6, and increased TNF-α and phagocytosis. However, due to the absence of TLR 4 receptor in bone marrow derived macrophages from TLR 4-/- mice, these macrophages showed decreased phagocytic ability and also reduced fungal burden in the presence of DAPT, showing a relationship between TLR 4 and Notch1. In addition, we made a treatment with DAPT in BALB/c mice prior to infection with Pb18. And our results showed that DAPT-treated animals exhibited a decrease of fungal burden in the lungs, and a decrease of IL-6. Furthermore, we observed an increase of IgG after 45 days of infection, indicating probably a healing of these animals. Same treatment was made in BALB/c NUDE mice, followed by infection with Pb18. In these animals, we observed an increased production of proinflammatory cytokines in the lung and increased CD19+ cells, but fungal burden was similar in both group (treated and untreated), which indicates that treatment with DAPT is dependent on T cell response. Taken together, these results showed that Pb18 is able to activate the Notch 1 receptor on macrophages and uses the Notch-TLR 4 signaling pathway as a possible escape mechanism, and may provide a new immunity study approach in experimental paracoccidioidomycosis.

Au cours d’une infection, les cellules de l’immunité innée sont capables de reconnaître via les Toll-like receptors (TLR) des motifs appelés pathogen-associated molecular patterns. Les cellules natural killer (NK) contribuent au processus inflammatoire en produisant de nombreuses cytokines. Chez la souris, nous avons montré que l’expression du TLR2 et du TLR4 dans les cellules NK spléniques est intracellulaire, comme pour le TLR9. La réponse des NK aux agonistes des TLR2, 4 et 9 nécessite la présence de cytokines accessoires (IL-15 et IL-18), afin d’obtenir une production significative des cytokines pro-inflammatoires IFN- et GM-CSF. En revanche, dans un modèle de sepsis polymicrobial, les NK spléniques de souris présentent une diminution dramatique de leur production d’IFN- et de GM-CSF en réponse aux agonistes des TLR. Cette diminution est sous le contrôle des cellules T régulatrices (Treg) et due au TGF-1. L’analyse des voies de signalisation nous a permis de montrer que la production de GM-CSF est abolie chez les cellules NK de souris déficientes pour STING en réponse au CpG-DNA. Ces résultats mettent en lumière une voie alternative et cytoplasmique pour la détection de l’ADN bactérien dans les cellules NK, différente de la voie classique TLR9-MyD88 dépendante. De plus, nous avons montré un trafic du récepteur TLR2 depuis l’intérieur vers la surface des cellules NK. La migration du TLR2 à la surface des NK nécessite la molécule UNC93B1, précédemment décrite comme transporteur endosomal de TLR.Chez les cellules NK humaines circulantes (sous-populations CD3-CD56bright et CD3-CD56dim), nous avons montré que l’expression des TLR2 et 4 est majoritairement intracellulaire, comme pour le TLR9 et comme chez la souris. La production d’IFN- par les NK de sujets sains en réponse aux agonistes des TLR nécessite également la présence de cytokines accessoires. Nous montrons que cette production est fortement altérée pour les NK des patients admis en soins intensifs et ayant un sepsis ou un syndrome de réponse inflammatoire systémique (SIRS). De même nous avons trouvé des différences entre les patients et les sujets sains dans l’expression du CD69 (marqueur d’activation précoce) et des TLR eux-mêmes. Cette étude indique que les NK des patients sepsis et SIRS deviennent tolérants aux agonistes des TLR en terme de production d’IFN-, de manière similaire à ce qui a été décrit pour d’autres cellules comme les monocytes / As sensors of infection, innate immune cells are able to recognize pathogen-associated molecular patterns by receptors such as Toll-like receptors (TLR). NK cells contribute to inflammatory processes by the production of numerous cytokines. In mice, we have shown that the protein expression of TLR2 and TLR4 in naive NK cells from spleen is predominantly intracellular, similarly to TLR9. The responsiveness of purified NK cells to TLR2, 4 or 9 agonists in vitro requires the presence of accessory cytokines (IL-15 and 18) to trigger a significant production of IFN- and GM-CSF. In contrast, NK cells purified from a model of in vivo polymicrobial sepsis, showed a dramatic reduction in their capacity to respond to TLR agonists in terms of IFN- and GM-CSF release due an inhibitory cross talk with Treg cells mediated by TGF-1. Analyzing the signaling pathways involved in cytokine production in response to CpG-DNA, we found that GM-CSF production was abolished in NK cells from STING-deficient mice, revealing that this intracytoplasmic receptor acts as a TLR9/MyD88-independent alternative sensor to bacterial DNA in NK cells. Additionally we show that intracellularly expressed TLR2 traffics to the cell surface of NK cells, by a mechanism involving UNC93B1, a protein previous described as an endosomal TLR carrier.In human peripheral blood NK cells (CD3-CD56bright and CD3-CD56dim subsets), we show that TLR2 and 4 protein expression is primarily intracellular, similar to TLR9, and similar to our findings in murine NK cells. The ex vivo responsiveness of human blood NK cells to TLR2, 4 or 9 agonists also requires accessory cytokines, to promote secretion of IFN-. In intensive care patients diagnosed with systemic inflammatory response syndrome (SIRS) and sepsis, IFN- production was significantly decreased. We also discovered modulations in the expression of CD69 (early activation marker) and in that of TLR themselves. This study indicates that NK cells undergo tolerance in response to TLR agonists during SIRS or sepsis, similarly to other cells, such as monocytes.