As proteínas S100A8/A9 são expressas em diferentes tipos celulares e quando sozinhas ou complexadas e em baixas concentrações, promoveram proliferação, migração celular e formação de estruturas capilares. Por outro lado, quando em altas concentrações, esse complexo inibe o crescimento de diversos tipos de células tumorais murinas e humanas. Ainda, tanto a proteína S100A9 humana, quanto um peptídeo sintético idêntico a porção C-terminal da proteína S100A9 murina (pS100A9m) possuem efeitos antinociceptivo e imunorregulatório. Apesar dessas evidencias, até o momento não foi investigado o efeito do pS100A9m sobre a angiogênese e a tumorigênese. Portanto, o objetivo do presente estudo foi investigar, in vitro, o efeito do pS100A9m sobre os eventos fundamentais envolvidos com a angiogênese e o desenvolvimento tumoral. Para tanto, a fim de avaliar o efeito do pS100A9m sobre a angiogênese foi utilizada a linhagem de células endoteliais tímicas murinas (tEnd.1) nos ensaios de proliferação, migração da célula endotelial em meio de cultura, avaliada nos modelos de wound healing e transwell ou migração em meio condicionado, obtido de células tumorais LLC WRC256, avaliada no modelo de transwell, ensaio de adesão (aos componentes de matriz, tais como o colágeno tipo I, fibronectina e laminina) e formação de tubos em matrigel tridimensional (3D). Para os estudos sobre o efeito do pS100A9m sobre as células tumorais, foi utilizada a linhagem de células LLC WRC256 para realização dos ensaios funcionais de proliferação, migração (wound healing) e adesão (sobre os componentes da matriz extracelular). Os resultados obtidos demonstraram que o pS100A9m inibe a proliferação, migração, adesão sobre os componentes de matriz e, consequentemente, a formação de estruturas capilares em matriz 3D. Em relação às células tumorais LLC WRC256, foi observada, novamente, a ação inibitória do pS100A9m sobre os eventos de proliferação e migração. Em relação à adesão, o peptídeo aumentou a capacidade de adesão das células tumorais sobre o colágeno tipo I e fibronectina, porém inibiu a adesão dessas células sobre laminina. Em conclusão, os dados aqui obtidos demonstram que o pS100A9m inibe in vitro os eventos fundamentais envolvidos com a angiogênese e com a progressão tumoral. Desta forma, o peptídeo da porção C-terminal da proteína S100A9 pode ser considerado uma nova ferramenta para o estudo da angiogênese e tumorigênese, além apresentar potencial para uma possível aplicação terapêutica nesses processos / The S100A8/A9 proteins are expressed in different cell types and alone or when complexed, and at low concentrations promoted proliferation, cell migration and formation of capillary structures. On the other hand, at higher concentrations, this compound inhibits the growth of many types of murine and human tumor cells. Moreover, both human S100A9 protein and a synthetic peptide identical to the C-terminal portion of murine S100A9 (mS100A9p) present antinociceptive and immunomodulatory effects. Despite these evidences, the effect of mS100A9p on angiogenesis and tumorigenesis has not been investigated. Therefore, the aim of this study was to investigate the in vitro effect of mS100A9p on crucial events involved in angiogenesis and tumor development. For this, in order to evaluate the effect of mS100A9p on angiogenesis was used the murine endothelial cell line derived from thymus hemangioma (tEnd.1) for proliferation assays, endothelial cell migration in the presence of culture medium (scratch wound healing and chemotaxis assays) or in conditioned medium prevenient from LLC WRC256 tumor cells (chemotaxis assays), adhesion assay (on extracellular matrix components, such as type I collagen, fibronectin and laminin) and tube like-structure formation in 3D matrix. For the analyzes of the effect of mS100A9p on tumor cells, the cell line LLC WRC256 was used to perform functional assays such as proliferation, migration (scratch wound healing model) and adhesion (on components of the extracellular matrix). The results showed that the mS100A9p inhibits the proliferation, migration and adhesion of endothelial cells to the matrix components and consequently the formation of capillary structures in 3D matrix. Regarding LLC WRC256 tumor cells, it was observed again the inhibitory action of the mS100A9p on proliferation and migration events. In relation to cellular adhesion, this peptide increased this parameter of tumor cells on type I collagen and fibronectin. However mS100A9p inhibited the adhesion of these cells on laminin. In conclusion, the data obtained show that the mS100A9p inhibits in vitro crucial events involved in angiogenesis and tumor progression. Thus, the C-terminal portion of murine S100A9 protein may be considered as a new tool for the study of tumorigenesis and angiogenesis besides presenting potential to a possible therapeutic application in these processes

A peritonite neutrofílica induzida por glicogênio acarreta antinocicepção em camundongos submetidos ao teste de contorção abdominal, a qual é mediada por uma proteína ligante de cálcio, com peso molecular de 14 kDa, denominada S100A9. O objetivo do presente trabalho foi aprofundar o estudo sobre o envolvimento dos neutrófilos na antinocicepção induzida pelo glicogênio em ratos submetidos ao teste de pressão de pata e avaliar a expressão da proteína S100A9 nos tempos onde foi detectado esse efeito. O glicogênio induz antinocicepção em ratos entre 2 e 12 horas após sua injeção intraplantar. O pré-tratamento dos animais com fucoidina, um inibidor de selectinas, não só reverte o efeito antinociceptivo observado como também induz hiperalgesia entre 2 e 6 horas após a injeção do glicogênio. Após 8 horas do tratamento com glicogênio, a fucoidina apenas inibiu a antinocicepção induzida pelo agente inflamatório. A análise histológica demonstrou um aumento na migração de células polimorfonucleares entre 2 e 8 horas após a administração de glicogênio, a qual foi inibida pelo pré-tratamento com fucoidina. Tanto a injeção subcutânea como intraplantar de naloxona, um inibidor inespecífico de receptores opioides, não interferiram no efeito antinociceptivo induzido pelo glicogênio, em nenhum dos tempos avaliados. Quanto à expressão da S100A9, analisada por "Western Blotting", foi observado que as amostras obtidas do coxim plantar dos animais injetados com o glicogênio, entre 2 e 12 horas, apresentaram uma banda com peso molecular aproximado de 14 kDa, o qual equivale ao peso da proteína S100A9. A quantificação das bandas marcadas com o anticorpo anti-S100A9, nos tempos entre 2 e 12 horas, demonstrou um aumento significativo da expressão dessa proteína nas amostras obtidas dos animais tratados com glicogênio, em comparação com os tratados com salina. A injeção intraperitoneal de glicogênio induziu um aumento significativo no número total de células presentes na cavidade abdominal dos animais entre a 2º e a 12º hora após o tratamento, representado pelo aumento do número de células polimorfonucleares migradas. Os sobrenadantes obtidos do exsudato peritoneal entre 2 e 12 horas após a injeção de glicogênio, administrados via intraplantar, não só reverteram a hiperalgesia induzida pela carragenina (Cg) como induziram efeito antinociceptivo. Já, o sobrenadante obtido após 24 horas da injeção de glicogênio reverteu apenas parcialmente o efeito hiperalgésico induzido pela Cg. O tratamento do sobrenadante obtido 4 horas após a injeção do glicogênio com o anticorpo anti-S100A9 aboliu totalmente o efeito antinociceptivo observado com esse sobrenadante sobre a hiperalgesia induzida pela Cg. Esses dados sugerem que a antinocicepção acarretada pelo glicogênio em ratos submetidos ao modelo de pressão de pata é dependente da migração neutrofílica, não está relacionado à liberação de peptídeos opioides, mas possivelmente à secreção da proteína S100A9 por essas células. Ainda, os resultados obtidos com os sobrenadantes do exsudato peritoneal após a injeção do glicogênio, demonstram que durante a peritonite neutrofílica é secretada uma molécula capaz tanto de inibir a hiperalgesia acarretada pela carragenina quanto induzir antinocicepção, a qual possivelmente é a proteína S100A9. / Neutrophilic peritonitis induced by glycogen causes antinociception in mice subjected to the writhing test, which is médiated by a calcium-binding protein with a molecular mass of 14 kDa, named S100A9. The purpose of this study was to deepen the study on the involvement of neutrophils in glycogen-induced antinociception in rats subjected to the paw pressure test and evaluate the expression of S100A9 protein in time periods when this effect was detected. Glycogen induces antinociception in rats between 2 and 12 hours after intraplantar injection. Pretreatment of animals with fucoidan, a selectin inhibitor, not only reversed the antinociceptive effect, but also induces hyperalgesia between 2 and 6 hours after glycogen injection. Eight hours after treatment with glycogen, fucoidan only inhibited the antinociception induced by the inflammatory agent. Histological analysis showed an increased migration of polymorphonuclear cells between 2 and 8 hours after glycogen administration, which was inhibited by pretreatment with fucoidan. Both intraplantar and subcutaneous injection of naloxone, a nonspecific inhibitor of opioid receptors, did not affect the antinociceptive effect induced by glycogen at all evaluated times. In relation to the expression of S100A9 analyzed by Western blotting, it was observed that the samples obtained from the footpad injected with glycogen, between 2 and 12 hours, had a band with a molecular weight of 14 kDa, which is similar to molecular weight of S100A9. Relative quantification of the bands marked with anti-S100A9 in the time periods between 2 and 12 hours showed a significant increase in protein expression in samples obtained from animals treated with glycogen, compared with those treated with saline. Intraperitoneal injection of glycogen induced a significant increase in the total number of cells in the abdominal cavity of animals between 2 and 12 hours after treatment, represented by increased numbers of migrated polymorphonuclear cells. The supernatants obtained from peritoneal exudate between 2 and 12 hours after injection of glycogen, administered intraplantarly, not only reversed the hyperalgesia induced by carrageenan (Cg) but also induced antinociceptive effect. Already, the supernatant obtained 24 hours after injection of glycogen only partially reversed the hyperalgesic effect induced by Cg. The treatment of the supernatant obtained 4 hours after injection of glycogen with anti-S100A9 abolished the antinociceptive effect observed with the supernatant on hyperalgesia induced by Cg. These data suggest that antinociception entailed by glycogen in rats submitted to the paw pressure is dependent on neutrophil migration. Moreover, this effect is not related to the release of opioid peptides but possibly to the S100A9 protein secretion by these cells. In addition, the results obtained with the supernatants of peritoneal exudate after glycogen injection show that during neutrophilic peritonitis a molecule able to inhibit carrageenan-induced hyperalgesia is secreted and induce antinociception entailed by glycogen, which is possibly the S100A9 protein.

A peritonite neutrofílica induzida por glicogênio acarreta antinocicepção em camundongos submetidos ao teste de contorção abdominal, a qual é mediada por uma proteína ligante de cálcio, com peso molecular de 14 kDa, denominada S100A9. O objetivo do presente trabalho foi aprofundar o estudo sobre o envolvimento dos neutrófilos na antinocicepção induzida pelo glicogênio em ratos submetidos ao teste de pressão de pata e avaliar a expressão da proteína S100A9 nos tempos onde foi detectado esse efeito. O glicogênio induz antinocicepção em ratos entre 2 e 12 horas após sua injeção intraplantar. O pré-tratamento dos animais com fucoidina, um inibidor de selectinas, não só reverte o efeito antinociceptivo observado como também induz hiperalgesia entre 2 e 6 horas após a injeção do glicogênio. Após 8 horas do tratamento com glicogênio, a fucoidina apenas inibiu a antinocicepção induzida pelo agente inflamatório. A análise histológica demonstrou um aumento na migração de células polimorfonucleares entre 2 e 8 horas após a administração de glicogênio, a qual foi inibida pelo pré-tratamento com fucoidina. Tanto a injeção subcutânea como intraplantar de naloxona, um inibidor inespecífico de receptores opioides, não interferiram no efeito antinociceptivo induzido pelo glicogênio, em nenhum dos tempos avaliados. Quanto à expressão da S100A9, analisada por "Western Blotting", foi observado que as amostras obtidas do coxim plantar dos animais injetados com o glicogênio, entre 2 e 12 horas, apresentaram uma banda com peso molecular aproximado de 14 kDa, o qual equivale ao peso da proteína S100A9. A quantificação das bandas marcadas com o anticorpo anti-S100A9, nos tempos entre 2 e 12 horas, demonstrou um aumento significativo da expressão dessa proteína nas amostras obtidas dos animais tratados com glicogênio, em comparação com os tratados com salina. A injeção intraperitoneal de glicogênio induziu um aumento significativo no número total de células presentes na cavidade abdominal dos animais entre a 2º e a 12º hora após o tratamento, representado pelo aumento do número de células polimorfonucleares migradas. Os sobrenadantes obtidos do exsudato peritoneal entre 2 e 12 horas após a injeção de glicogênio, administrados via intraplantar, não só reverteram a hiperalgesia induzida pela carragenina (Cg) como induziram efeito antinociceptivo. Já, o sobrenadante obtido após 24 horas da injeção de glicogênio reverteu apenas parcialmente o efeito hiperalgésico induzido pela Cg. O tratamento do sobrenadante obtido 4 horas após a injeção do glicogênio com o anticorpo anti-S100A9 aboliu totalmente o efeito antinociceptivo observado com esse sobrenadante sobre a hiperalgesia induzida pela Cg. Esses dados sugerem que a antinocicepção acarretada pelo glicogênio em ratos submetidos ao modelo de pressão de pata é dependente da migração neutrofílica, não está relacionado à liberação de peptídeos opioides, mas possivelmente à secreção da proteína S100A9 por essas células. Ainda, os resultados obtidos com os sobrenadantes do exsudato peritoneal após a injeção do glicogênio, demonstram que durante a peritonite neutrofílica é secretada uma molécula capaz tanto de inibir a hiperalgesia acarretada pela carragenina quanto induzir antinocicepção, a qual possivelmente é a proteína S100A9. / Neutrophilic peritonitis induced by glycogen causes antinociception in mice subjected to the writhing test, which is médiated by a calcium-binding protein with a molecular mass of 14 kDa, named S100A9. The purpose of this study was to deepen the study on the involvement of neutrophils in glycogen-induced antinociception in rats subjected to the paw pressure test and evaluate the expression of S100A9 protein in time periods when this effect was detected. Glycogen induces antinociception in rats between 2 and 12 hours after intraplantar injection. Pretreatment of animals with fucoidan, a selectin inhibitor, not only reversed the antinociceptive effect, but also induces hyperalgesia between 2 and 6 hours after glycogen injection. Eight hours after treatment with glycogen, fucoidan only inhibited the antinociception induced by the inflammatory agent. Histological analysis showed an increased migration of polymorphonuclear cells between 2 and 8 hours after glycogen administration, which was inhibited by pretreatment with fucoidan. Both intraplantar and subcutaneous injection of naloxone, a nonspecific inhibitor of opioid receptors, did not affect the antinociceptive effect induced by glycogen at all evaluated times. In relation to the expression of S100A9 analyzed by Western blotting, it was observed that the samples obtained from the footpad injected with glycogen, between 2 and 12 hours, had a band with a molecular weight of 14 kDa, which is similar to molecular weight of S100A9. Relative quantification of the bands marked with anti-S100A9 in the time periods between 2 and 12 hours showed a significant increase in protein expression in samples obtained from animals treated with glycogen, compared with those treated with saline. Intraperitoneal injection of glycogen induced a significant increase in the total number of cells in the abdominal cavity of animals between 2 and 12 hours after treatment, represented by increased numbers of migrated polymorphonuclear cells. The supernatants obtained from peritoneal exudate between 2 and 12 hours after injection of glycogen, administered intraplantarly, not only reversed the hyperalgesia induced by carrageenan (Cg) but also induced antinociceptive effect. Already, the supernatant obtained 24 hours after injection of glycogen only partially reversed the hyperalgesic effect induced by Cg. The treatment of the supernatant obtained 4 hours after injection of glycogen with anti-S100A9 abolished the antinociceptive effect observed with the supernatant on hyperalgesia induced by Cg. These data suggest that antinociception entailed by glycogen in rats submitted to the paw pressure is dependent on neutrophil migration. Moreover, this effect is not related to the release of opioid peptides but possibly to the S100A9 protein secretion by these cells. In addition, the results obtained with the supernatants of peritoneal exudate after glycogen injection show that during neutrophilic peritonitis a molecule able to inhibit carrageenan-induced hyperalgesia is secreted and induce antinociception entailed by glycogen, which is possibly the S100A9 protein.

As proteínas S100A8/A9 são expressas em diferentes tipos celulares e quando sozinhas ou complexadas e em baixas concentrações, promoveram proliferação, migração celular e formação de estruturas capilares. Por outro lado, quando em altas concentrações, esse complexo inibe o crescimento de diversos tipos de células tumorais murinas e humanas. Ainda, tanto a proteína S100A9 humana, quanto um peptídeo sintético idêntico a porção C-terminal da proteína S100A9 murina (pS100A9m) possuem efeitos antinociceptivo e imunorregulatório. Apesar dessas evidencias, até o momento não foi investigado o efeito do pS100A9m sobre a angiogênese e a tumorigênese. Portanto, o objetivo do presente estudo foi investigar, in vitro, o efeito do pS100A9m sobre os eventos fundamentais envolvidos com a angiogênese e o desenvolvimento tumoral. Para tanto, a fim de avaliar o efeito do pS100A9m sobre a angiogênese foi utilizada a linhagem de células endoteliais tímicas murinas (tEnd.1) nos ensaios de proliferação, migração da célula endotelial em meio de cultura, avaliada nos modelos de wound healing e transwell ou migração em meio condicionado, obtido de células tumorais LLC WRC256, avaliada no modelo de transwell, ensaio de adesão (aos componentes de matriz, tais como o colágeno tipo I, fibronectina e laminina) e formação de tubos em matrigel tridimensional (3D). Para os estudos sobre o efeito do pS100A9m sobre as células tumorais, foi utilizada a linhagem de células LLC WRC256 para realização dos ensaios funcionais de proliferação, migração (wound healing) e adesão (sobre os componentes da matriz extracelular). Os resultados obtidos demonstraram que o pS100A9m inibe a proliferação, migração, adesão sobre os componentes de matriz e, consequentemente, a formação de estruturas capilares em matriz 3D. Em relação às células tumorais LLC WRC256, foi observada, novamente, a ação inibitória do pS100A9m sobre os eventos de proliferação e migração. Em relação à adesão, o peptídeo aumentou a capacidade de adesão das células tumorais sobre o colágeno tipo I e fibronectina, porém inibiu a adesão dessas células sobre laminina. Em conclusão, os dados aqui obtidos demonstram que o pS100A9m inibe in vitro os eventos fundamentais envolvidos com a angiogênese e com a progressão tumoral. Desta forma, o peptídeo da porção C-terminal da proteína S100A9 pode ser considerado uma nova ferramenta para o estudo da angiogênese e tumorigênese, além apresentar potencial para uma possível aplicação terapêutica nesses processos / The S100A8/A9 proteins are expressed in different cell types and alone or when complexed, and at low concentrations promoted proliferation, cell migration and formation of capillary structures. On the other hand, at higher concentrations, this compound inhibits the growth of many types of murine and human tumor cells. Moreover, both human S100A9 protein and a synthetic peptide identical to the C-terminal portion of murine S100A9 (mS100A9p) present antinociceptive and immunomodulatory effects. Despite these evidences, the effect of mS100A9p on angiogenesis and tumorigenesis has not been investigated. Therefore, the aim of this study was to investigate the in vitro effect of mS100A9p on crucial events involved in angiogenesis and tumor development. For this, in order to evaluate the effect of mS100A9p on angiogenesis was used the murine endothelial cell line derived from thymus hemangioma (tEnd.1) for proliferation assays, endothelial cell migration in the presence of culture medium (scratch wound healing and chemotaxis assays) or in conditioned medium prevenient from LLC WRC256 tumor cells (chemotaxis assays), adhesion assay (on extracellular matrix components, such as type I collagen, fibronectin and laminin) and tube like-structure formation in 3D matrix. For the analyzes of the effect of mS100A9p on tumor cells, the cell line LLC WRC256 was used to perform functional assays such as proliferation, migration (scratch wound healing model) and adhesion (on components of the extracellular matrix). The results showed that the mS100A9p inhibits the proliferation, migration and adhesion of endothelial cells to the matrix components and consequently the formation of capillary structures in 3D matrix. Regarding LLC WRC256 tumor cells, it was observed again the inhibitory action of the mS100A9p on proliferation and migration events. In relation to cellular adhesion, this peptide increased this parameter of tumor cells on type I collagen and fibronectin. However mS100A9p inhibited the adhesion of these cells on laminin. In conclusion, the data obtained show that the mS100A9p inhibits in vitro crucial events involved in angiogenesis and tumor progression. Thus, the C-terminal portion of murine S100A9 protein may be considered as a new tool for the study of tumorigenesis and angiogenesis besides presenting potential to a possible therapeutic application in these processes

Background Traumatic brain injury (TBI) is a complex disease with a spectrum of symptoms and disabilities. Over the past decade TBI has become the focus of research due to growing epidemiological and clinical evidences that TBI incidences are strong risk factors for Alzheimer’s disease (AD). Major pathological hallmarks of AD are massive accumulations of amyloid-β peptide (Aβ) toxic oligomers and plaques. Neuroinflammation is also considered as a common denominator in AD and aging. The epidemiological and experimental studies have supported that non-steroidal anti-inflammatory drugs markedly reduce the age-related prevalence of AD and can slow amyloid deposition by mechanisms that still remain elusive. S100A9 is a multifunctional cytokine with diverse roles in the cell signaling pathways associated with inflammation and cancers. A widespread expression of S100A9 was also reported in many other ailments involving inflammatory processes, such as AD, malaria, cerebral ischemia and TBI, implying that S100A9 may be a universal biomarker of inflammation. The distinctive feature of S100A9 compared to other pro-inflammatory cytokines is its ability to self-assemble into amyloids, which may lead to the loss of its signaling functions and acquired amyloid cytotoxicity, exceeding that of Aβ. Methods S100A9 properties was studied under various ex vivo and in vitro conditions. First, human and mouse tissues with TBI and AD were subjected to microscopic, immunohistochemical and immunofluorescent techniques. Then, aged mouse treated with native, oligomeric and fibrillary S100A9 was also studied by using behavioral and neurochemical analysis. Moreover, S100A9 was established as a biomarker of dementia progression and compared with others such as Aβ42 and tau proteins, by studying cerebrospinal fluid (CSF) samples from different stages of dementia. Finally, in vitro experiments on S100A9 amyloidogenesis, co-aggregation with Aβ40 and Aβ42, digestion and cytotoxicity were also performed by using spectroscopic, atomic force microscopy and cell biology methods. Results S100A9-driven amyloid-neuroinflammatory cascade serves as a link between TBI and AD. We have found that S100A9 contributes to the plaque formation and intraneuronal responses in AD, being a part of the amyloid-neuroinflammatory cascade. In TBI we have found that extensive S100A9 neuronal production and amyloid self-assembly is triggered immediately after injury, leading to apoptotic pathways and neuronal loss. S100A9 is an integral component of both TBI precursor-plaques, formed prior to Aβ deposition, and AD plaques, characterized by different degree of amyloid maturation, indicating that all plaques are associated with inflammation. Both intra- and extracellular amyloid-neuroinflammatory cascades are intertwined and showed similar tendencies in human and mouse tissues in TBI and AD. Ex vivo findings are further supported by in vitro experiments on S100A9 amyloidogenesis, digestion and cytotoxicity. Importantly, being highly amyloidogenic itself, S100A9 can trigger and aggravate Aβ amyloid self-assembly and significantly contribute to amyloid cytotoxicity. Moreover, the CSF dynamics of S100A9 levels matches very closely the content of Aβ42 in AD, vascular dementia and mild cognitive impairment due to AD, emphasizing the involvement of S100A9 together with Aβ in the amyloid-neuroinflammatory cascade in these ailments. Conclusions The conclusions of this thesis is that the inflammatory pathways and S100A9 specifically represent a potential target for the therapeutic interventions during various post-TBI stages and far prior AD development to halt and reverse these damaging processes. / Role of pro-inflammatory S100A9 protein in amyloid-neuroinflammatory cascade in Alzheimer’s disease and traumatic brain injury

O peptídeo sintético idêntico ao C-terminal da proteína S100A9 murina (pS100A9m) possui efeito antinociceptivo em diferentes modelos de dor inflamatória aguda. No presente estudo, o efeito do pS100A9m foi avaliado sobre a dor neuropática induzida pela constrição crônica (CCI) do nervo ciático em ratos. Ainda, foram investigados os possíveis mecanismos envolvidos neste efeito. A nocicepção foi avaliada pelos testes de hiperalgesia, alodinia e dor espontânea. Os animais foram tratados com diferentes doses do pS100A9m pelas vias intraplantar, oral ou intratecal 14 dias após a CCI e a nocicepção avaliada após 1 hora. As três vias de administração bloquearam a hiperalgesia, a alodinia e a dor espontânea decorrentes da dor neuropática. A duração do efeito do pS100A9m varia de acordo com a via utilizada e com o fenômeno nociceptivo testado. Ainda, a injeção intraplantar do peptídeo, na pata contralateral à CCI, inibiu a hiperalgesia e a alodinia observadas após a constrição do nervo. Quando o pS100A9m foi administrado pela via intraplantar no 7° dia após a CCI, ele também induziu inibição da hiperalgesia inflamatória que é observada nesse período. Os prováveis mecanismos envolvidos no efeito antinociceptivo do pS100A9m foram investigados pela administração de antagonistas de receptores de serotonina, noradrenalina, GABA (A e B) e opióides. Os resultados obtidos demonstraram que apenas o antagonista de receptor GABAB reverteu completamente o efeito antinociceptivo do pS100A9m sobre a dor neuropática, detectada no 14º dia pós-cirúrgico. Além disso, foram avaliadas as expressões das proteínas Egr-1, Fos e TNFα na medula dos ratos submetidos à CCI e tratados com o peptídeo 7 ou 14 dias do procedimento cirúrgico. O aumento na expressão das proteínas Egr-1 e Fos foi evidenciado tanto no 7º como no 14º dia após a CCI, em animais que não receberam nenhum tratamento ou aqueles que foram tratados com o veículo do peptídeo. Por outro lado, o pS100A9m inibiu a expressão destas duas proteínas no lado ipsolateral à CCI no corno dorsal da medula espinhal dos animais. Com relação ao TNFα, apenas no 7º dia após a CCI foi detectado o aumento na expressão desta proteína. Ainda, foi neste período que o pS100A9m acarretou inibição da expressão do TNFα no corno ventral de animais submetidos ao procedimento cirúrgico. Estes resultados demonstram que o C-terminal da S100A9 murina inibe a dor neuropática experimental por uma ação dependente de receptores GABAB, sugerindo que este peptídeo possivelmente promova uma ativação dos mecanismos inibitórios espinhais, acarretando em redução da ativação de neurônios na medula. Desta forma, o pS100A9m demonstra um potencial terapêutico para o tratamento de dores persistentes. / The synthetic peptide identical to the C-terminus of murine S100A9 protein (mS100A9p) has antinociceptive effect on different acute inflammatory pain models. In this study, the effect of mS100A9p was evaluated on neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve in rats, and the possible mechanisms involved in this effect were investigated. Hyperalgesia, allodynia, and spontaneous pain were assessed to evaluate nociception. Rats were treated with different doses of mS100A9p by intraplantar, oral, or intrathecal routes on day 14 after CCI, and nociception was evaluated 1 hour later. These three routes of administration blocked hyperalgesia, allodynia and spontaneous pain. The duration of mS100A9p effect depends on the route used and the phenomenon analyzed. Moreover, intraplantar injection of mS100A9p in the contralateral paw inhibited the hyperalgesia and allodynia induced by CCI. When mS100A9p was administered by intraplantar route on day 7 after CCI, it reversed the inflammatory hyperalgesia observed in this period. The mechanisms likely involved in the antinociceptive effect of mS100A9p were investigated by administration of antagonists of serotonin, norepinephrine, GABA (A and B) and opioid receptors. Only the GABAB receptor antagonist completely reversed the antinociceptive effect of mS100A9p on neuropathic pain on day 14 after CCI. Besides, the expression of Egr-1, Fos and TNFα proteins was evaluated in the spinal cord of rats submitted to CCI and treated with mS100A9p on days 7 or 14 after CCI. The expression of Egr-1 and Fos was increased in animals not treated or treated with vehicle on days 7 and 14 after CCI. On the other hand, mS100A9p inhibited the expression of these two proteins in the dorsal horn of spinal cord ipsilateral to CCI. The increase in TNFα expression was observed exclusively on day 7 after CCI. In the same time period, mS100A9p nhibited the expression of TNFα in the ventral horn of spinal cord of animals submitted to CCI. The results obtained herein demonstrate that the C-terminus of murine S100A9 protein inhibits the experimental neuropathic pain by a GABAB-dependent action, suggesting that this peptide promotes the activation of spinal inhibitory mechanisms leading to the reduction of activation of spinal neurons. Therefore, mS100A9p demonstrates a potential therapeutic use in persistent pain syndromes.

O peptídeo sintético idêntico ao C-terminal da proteína S100A9 murina (pS100A9m) possui efeito antinociceptivo em diferentes modelos de dor inflamatória aguda. No presente estudo, o efeito do pS100A9m foi avaliado sobre a dor neuropática induzida pela constrição crônica (CCI) do nervo ciático em ratos. Ainda, foram investigados os possíveis mecanismos envolvidos neste efeito. A nocicepção foi avaliada pelos testes de hiperalgesia, alodinia e dor espontânea. Os animais foram tratados com diferentes doses do pS100A9m pelas vias intraplantar, oral ou intratecal 14 dias após a CCI e a nocicepção avaliada após 1 hora. As três vias de administração bloquearam a hiperalgesia, a alodinia e a dor espontânea decorrentes da dor neuropática. A duração do efeito do pS100A9m varia de acordo com a via utilizada e com o fenômeno nociceptivo testado. Ainda, a injeção intraplantar do peptídeo, na pata contralateral à CCI, inibiu a hiperalgesia e a alodinia observadas após a constrição do nervo. Quando o pS100A9m foi administrado pela via intraplantar no 7° dia após a CCI, ele também induziu inibição da hiperalgesia inflamatória que é observada nesse período. Os prováveis mecanismos envolvidos no efeito antinociceptivo do pS100A9m foram investigados pela administração de antagonistas de receptores de serotonina, noradrenalina, GABA (A e B) e opióides. Os resultados obtidos demonstraram que apenas o antagonista de receptor GABAB reverteu completamente o efeito antinociceptivo do pS100A9m sobre a dor neuropática, detectada no 14º dia pós-cirúrgico. Além disso, foram avaliadas as expressões das proteínas Egr-1, Fos e TNFα na medula dos ratos submetidos à CCI e tratados com o peptídeo 7 ou 14 dias do procedimento cirúrgico. O aumento na expressão das proteínas Egr-1 e Fos foi evidenciado tanto no 7º como no 14º dia após a CCI, em animais que não receberam nenhum tratamento ou aqueles que foram tratados com o veículo do peptídeo. Por outro lado, o pS100A9m inibiu a expressão destas duas proteínas no lado ipsolateral à CCI no corno dorsal da medula espinhal dos animais. Com relação ao TNFα, apenas no 7º dia após a CCI foi detectado o aumento na expressão desta proteína. Ainda, foi neste período que o pS100A9m acarretou inibição da expressão do TNFα no corno ventral de animais submetidos ao procedimento cirúrgico. Estes resultados demonstram que o C-terminal da S100A9 murina inibe a dor neuropática experimental por uma ação dependente de receptores GABAB, sugerindo que este peptídeo possivelmente promova uma ativação dos mecanismos inibitórios espinhais, acarretando em redução da ativação de neurônios na medula. Desta forma, o pS100A9m demonstra um potencial terapêutico para o tratamento de dores persistentes. / The synthetic peptide identical to the C-terminus of murine S100A9 protein (mS100A9p) has antinociceptive effect on different acute inflammatory pain models. In this study, the effect of mS100A9p was evaluated on neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve in rats, and the possible mechanisms involved in this effect were investigated. Hyperalgesia, allodynia, and spontaneous pain were assessed to evaluate nociception. Rats were treated with different doses of mS100A9p by intraplantar, oral, or intrathecal routes on day 14 after CCI, and nociception was evaluated 1 hour later. These three routes of administration blocked hyperalgesia, allodynia and spontaneous pain. The duration of mS100A9p effect depends on the route used and the phenomenon analyzed. Moreover, intraplantar injection of mS100A9p in the contralateral paw inhibited the hyperalgesia and allodynia induced by CCI. When mS100A9p was administered by intraplantar route on day 7 after CCI, it reversed the inflammatory hyperalgesia observed in this period. The mechanisms likely involved in the antinociceptive effect of mS100A9p were investigated by administration of antagonists of serotonin, norepinephrine, GABA (A and B) and opioid receptors. Only the GABAB receptor antagonist completely reversed the antinociceptive effect of mS100A9p on neuropathic pain on day 14 after CCI. Besides, the expression of Egr-1, Fos and TNFα proteins was evaluated in the spinal cord of rats submitted to CCI and treated with mS100A9p on days 7 or 14 after CCI. The expression of Egr-1 and Fos was increased in animals not treated or treated with vehicle on days 7 and 14 after CCI. On the other hand, mS100A9p inhibited the expression of these two proteins in the dorsal horn of spinal cord ipsilateral to CCI. The increase in TNFα expression was observed exclusively on day 7 after CCI. In the same time period, mS100A9p nhibited the expression of TNFα in the ventral horn of spinal cord of animals submitted to CCI. The results obtained herein demonstrate that the C-terminus of murine S100A9 protein inhibits the experimental neuropathic pain by a GABAB-dependent action, suggesting that this peptide promotes the activation of spinal inhibitory mechanisms leading to the reduction of activation of spinal neurons. Therefore, mS100A9p demonstrates a potential therapeutic use in persistent pain syndromes.

Note: Portions of this abstract have been previously published in the journal Blood, Basiorka et al. Blood. 2016 Oct 13, and has been reproduced in this manuscript with permission from the publisher. Myelodysplastic syndromes (MDS) are genetically diverse hematopoietic stem cell malignancies that share a common phenotype of cytological dysplasia, ineffective hematopoiesis and aberrant myeloid lineage maturation. Apoptotic cell death potentiated by inflammatory cytokines has been considered a fundamental feature of MDS for over two decades. However, this non-inflammatory form of cell death cannot account for the inflammatory nature of these disorders. We report that a hallmark of lower-risk (LR) MDS is activation of the NLRP3 inflammasome, which drives clonal expansion and pyroptosis, a caspase-1-dependent programmed cell death induced by danger-associated molecular pattern (DAMP) signals. Independent of genotype, MDS hematopoietic stem and progenitor cells (HSPC) overexpress pyroptosis-related transcripts, inflammasome proteins and manifest activated NLRP3 inflammasome complexes that direct caspase-1 activation, IL-1β and IL-18 maturation and pyroptotic cell death. Using the S100A9 transgenic (S100A9Tg) mouse model that phenocopies human MDS, we demonstrated that forced expression of S100A9 was sufficient to drive pyroptosis in vivo, implicating pyroptosis as the principal mechanism of HSPC cell death in S100A9Tg mice. The lytic cell death releases intraceullar contents that include alarmins and catalytically active ASC specks, which can propagate bystander inflammation. Notably, MDS mesenchymal stromal cells (MSC) and stromal-derived linages were found to predominantly undergo pyroptosis, with marked activation of caspase-1 and NLRP3 inflammasome complexes. These findings may account for the clusters of both HSPC and stromal cell death previously described in the bone marrows of patients with MDS. Mechanistically, pyroptosis is triggered by the alarmin S100A9 that is found in excess in MDS HSPC and bone marrow (BM) plasma. Further, both somatic gene mutations and S100A9-induced signaling activate NADPH oxidase (NOX), generating reactive oxygen species (ROS) that initiate cation influx, cell swelling and β-catenin activation. Accordingly, ROS and active β-catenin were significantly increased in MDS BM mononuclear cells (BM-MNC) and S100A9Tg mice compared to normal controls, as well as in human cell lines harboring gene mutations and in murine models of gene mutation knock-in or gene loss. ROS and β-catenin nuclear translocation were significantly reduced by NLRP3 or NOX inhibition, indicating that S100A9 and somatic gene mutations prime cells to undergo NOX1/4-dependent NLRP3 inflammasome assembly, pyroptosis and β-catenin activation. Together, these data explain the concurrent proliferation and inflammatory cell death characteristic of LR-MDS. Given that loss of a gene-rich area in del(5q) disease results in derepression of innate immune signaling, we hypothesized that this genetic deficit would trigger assembly of the NLRP3 inflammasome complex, akin to the pathobiological mechanism characteristic of non-del(5q) MDS. To this end, we utilized two distinct murine models of del(5q) disease, namely in the context of Rps14 haploinsufficiency and concurrent loss of mDia1 and microRNA (miR)-146a. In both models, pyroptosis was not evident in the HSPC compartment; however, early erythroid progenitors displayed high fractions of pyroptotic cells. This was associated with significant increases in caspase-1 and NLRP3 inflammasome activation, ROS and nuclear localization of β-catenin, which was extinguished by inflammasome or NOX complex inhibition. These data suggest that early activation of the inflammasome drives cell death and prevents terminal maturation of erythroid precursors, accounting for the progressive anemia characteristic of del(5q) disease, whereby hematopoietic defects are primarily restricted to the erythroid compartment. Importantly, these data implicate a similar pathobiological mechanism in del(5q) MDS as is observed in non-del(5q) patients. The identification of the NLRP3 inflammasome as a pathobiological driver of the LR non-del(5q) and del(5q) MDS phenotype allows for novel therapeutic agent development. Notably, knockdown of NLRP3 or caspase-1, neutralization of S100A9, and pharmacologic inhibition of NLRP3 or NOX suppresses pyroptosis, ROS generation and nuclear β-catenin in MDS, and are sufficient to restore effective hematopoiesis. In del(5q) murine models, inhibition of the NLRP3 inflammasome significantly improved erythroid colony forming capacity by a mechanism distinct from that of lenalidomide, highlighting the translational potential for targeting this innate immune complex in this subset of MDS. Thus, alarmins and founder gene mutations in MDS license a common redox-sensitive inflammasome circuit, which suggests new avenues for therapeutic intervention. Furthermore, aggregated clusters of the NLRP3 adaptor protein ASC [apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CARD)] are referred to as ASC specks. During pyroptosis, ASC specks are released from dying cells and function as DAMP signals that propagate inflammation. In this way, specks are a surrogate marker for NLRP3 inflammasome activation and pyroptotic cell death. Given that pyroptosis is the predominant mechanism of cell death in MDS and ASC specks are readily quantified by flow cytometry, we hypothesized that BM or peripheral blood (PB) plasma-derived ASC specks may be a biologically rational biomarker for the diagnosis of MDS. The percentage of ASC specks were significantly increased in MDS BM plasma compared to normal, healthy donors, which was validated by confocal microscopy. PB plasma-derived ASC specks were significantly greater in LR- versus HR-MDS, consistent with the greater extent of cell death and myeloid-derived suppressor cell (MDSC) expansion in LR disease. As hyperglycemia induces NLRP3 inflammasome activation, plasma glucose levels were measured to adjust for this confounding variable. Subsequently, the percentage of glucose-adjusted, PB plasma-derived ASC specks was measured in a panel of specimens of varied hematologic malignancies. The corrected percentage of ASC specks was significantly increased in MDS compared to normal donors and to each other malignancy investigated, including other myeloid and lymphoid leukemias, myeloproliferative neoplasms and overlap syndromes, like chronic myelomonocytic leukemia (CMML). These data indicate that the glucose-adjusted ASC speck percentage is MDS-specific and may be a valuable diagnostic biomarker. At a cutoff of 0.039, the biomarker minimizes misclassification error and achieves 95% sensitivity and 82% specificity in classifying MDS from normal donors, other hematologic malignancies and T2D. Lastly, the biomarker declined with treatment response to lenalidomide in LR-MDS patients, but not to erythropoietin stimulating agent (ESA) or hypomethylating agent (HMA) therapy. As such, the percentage of ASC specks represents the first biologically rational, diagnostic biomarker for MDS that can be implemented with current diagnostic practices to reduce diagnostic error.

Myeloid-derived suppressor cells (MDSC)expand during sepsis, suppress both innate and adaptive immunity, and promote chronic immunosuppression, which characterizes the late/chronic phase of sepsis. We previously reported that the transcription factors Stat3 and C/EBPβ synergize to induces the expression of microRNA (miR)-21 and miR-181b to promote MDSC expansion in a mouse model of polymicrobial sepsis that progresses from an early/acute proinflammatory phase to a late/chronic immunosuppressive stage. We also showed that Gr1+CD11b+ cells, the precursors of MDSCs, from mice genetically deficient in the inflammatory protein S100A9 lack miR-21 or miR-181b in late sepsis, and are not immunosuppressive. In the present study, we show that S100A9 induces miR-21 and miR-181b during the late sepsis phase. We find that S100A9 associates with and stabilizes the Stat3-C/EBPβ protein complex that activates the miRNA promoters. Reconstituting Gr1+CD11b+ cells from S100A9 knockout mice with late sepsis with S100A9 protein restores the Stat3-C/EBPβ protein complex and miRNA expressions, and switches the Gr1+CD11b+ cells into the immunosuppressive, MDSC phenotype. Importantly, we find that this process requires IL-10 mediated signaling, which induces S100A9 translocation from the cytosol to the nucleus. These results demonstrate that S100A9 promotes MDSC expansion and immunosuppression in late/chronic sepsis by inducing the expression of miR-21 and miR-181b.

The myeloid-related protein S100A9 reprograms Gr1+CD11b+ myeloid precursors into myeloid-derived suppressor cells (MDSCs) during murine sepsis. Here, we show that the immunosuppressive cytokine IL-10 supports S100A9 expression and its nuclear localization in MDSCs to function as immune repressors. To support this new concept, we showed that antibody mediated IL-10 blockade in wild-type mice after sepsis induction inhibited MDSC expansion during late sepsis, and that ectopic expression of S100A9 in Gr1+CD11b+ cells from S100A9 knockout mice switched them into the MDSC phenotype only in the presence of IL-10. Knockdown of S100A9 in MDSCs from wild-type mice with late sepsis confirmed our findings in the S100A9 knockout mice. We also found that while both IL-6 and IL-10 can activate S100A9 expression in naive Gr1+CD11b+ cells, only IL-10 can induce S100A9 nuclear localization. These results support that IL-10 drives the molecular path that generates MDSCs and enhances immunosuppression during late sepsis, and inform that targeting this immune repressor path may improve sepsis survival in mice.