S100 calcium-binding protein A9 (S100A9) is elevated in plasma and bronchoalveolar lavage fluid (BALF) of patients with chronic obstructive pulmonary disease (COPD), and aging enhances S100A9 expression in several tissues. Currently, the direct impact of S100A9- mediated signaling on lung function and within the aging lung is unknown. Here, we observed that elevated S100A9 levels in human BALF correlated with age. Elevated lung levels of S100A9 were higher in older mice compared with in young animals and coincided with pulmonary function changes. Both acute and chronic exposure to cigarette smoke enhanced S100A9 levels in age-matched mice. To examine the direct role of S100A9 on the development of COPD, S100a9 -/- mice or mice administered paquinimod were exposed to chronic cigarette smoke. S100A9 depletion and inhibition attenuated the loss of lung function, pressure-volume loops, airway inflammation, lung compliance, and forced expiratory volume in 0.05 s/forced vital capacity, compared with age-matched wild-type or vehicleadministered animals. Loss of S100a9 signaling reduced cigarette smoke-induced airspace enlargement, alveolar remodeling, lung destruction, ERK and c-RAF phosphorylation, matrix metalloproteinase- 3 (MMP-3), matrix metalloproteinase-9 (MMP-9), monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), and keratinocyte- derived chemokine (KC) release into the airways. Paquinimod administered to nonsmoked, aged animals reduced age-associated loss of lung function. Since fibroblasts play a major role in the production and maintenance of extracellular matrix in emphysema, primary lung fibroblasts were treated with the ERK inhibitor LY3214996 or the c-RAF inhibitor GW5074, resulting in less S100A9-induced MMP-3, MMP-9, MCP-1, IL-6, and IL-8. Silencing Toll-like receptor 4 (TLR4), receptor for advanced glycation endproducts (RAGE), or extracellular matrix metalloproteinase inducer (EMMPRIN) prevented S100A9-induced phosphorylation of ERK and c-RAF. Our data suggest that S100A9 signaling contributes to the progression of smoke-induced and age-related COPD.

O peptídeo idêntico ao C-terminal da proteína S100A9 murina (pS100A9mH92-G110) inibe a hiperalgesia inflamatória induzida pela carragenina. Em adição, este peptídeo inibe a hiperalgesia inflamatória induzida por tripsina, uma serino protease capaz de ativar receptores ativados por protease do tipo 2 (PAR2). O objetivo inicial deste trabalho foi caracterizar a relação estrutura/ efeito do pS100A9m, a fim de determinar a menor seqüência peptídica dotada de atividade antinociceptiva. Ainda, como parte dos objetivos, neste trabalho foram investigados os mecanismos envolvidos no efeito antinociceptivo do pS100A9m e da menor seqüência ativa sobre a hiperalgesia induzida pela ativação de PAR2. Diferentes seqüências peptídicas homólogas ao pS100A9m foram sintetizadas e avaliadas em ratos submetidos ao modelo de hiperalgesia mecânica induzida por carragenina. Dentre todas as seqüências peptídicas investigadas, o peptídeo denominado AcE97-G102 foi determinado como a menor seqüência ativa com efeito semelhante ao pS100A9m. Com relação aos estudos sobre a ativação de PAR2, os resultados obtidos demonstraram que o pS100A9m bem como o AcE97-G102 inibem a hiperalgesia térmica e mecânica decorrentes da ativação de PAR2 (induzida por um peptídeo agonista deste receptor ? PAR2AP). A análise por imuno-histoquímica demonstrou que a ativação de PAR2 aumenta a expressão da proteína Egr-1 em neurônios nociceptivos, sendo o pS100A9m capaz de inibir este efeito. Em adição, ambos pS100A9m e AcE97-G102 inibiram o influxo de cálcio induzido por PAR2AP ou tripsina, em neurônios sensoriais do gânglio da raiz dorsal da medula espinhal (DRG). Por outro lado, nenhum dos peptídeos apresentou efeito sobre a mobilização de cálcio em células HEK-293, que naturalmente expressam PAR2, ou em células KNRK transfectadas com este tipo de receptor, sugerindo que o efeito tanto do pS100A9m quanto do AcE97-G102, sobre a ativação de PAR2, seja específico para neurônios sensoriais. O pS100A9m e o AcE97-G102 inibiram o influxo de cálcio nos neurônios DRG estimulados com bradicinina, capsaicina ou KCl. Ainda, o pS100A9m inibiu a liberação de substância P induzida por PAR2. Os resultados obtidos com o tratamento de neurônios DRG com tapsigaragina ou com ionóforo de cálcio sugerem um efeito direto do pS100A9m sobre os canais de cálcio. Desta forma, foi avaliada atividade do pS100A9m e do AcE97-G102 sobre culturas de células HEK-tsA transfectadas com canais de cálcio dependente de voltagem do tipo N ou do tipo L. Os resultados obtidos demonstraram que ambos peptídeos inibirem o influxo de cálcio em células transfectadas com receptores do tipo N. Em conjunto, os dados aqui obtidos demonstram que o efeito do C-terminal da proteína S100A9 murina sobre a nocicepção experimental é devido a uma inibição de canais de cálcio do tipo N, por uma ação direta em neurônios sensoriais. Ainda, a seqüência responsável por este efeito está localizada na porção E97-G102 do domínio C-terminal da proteína S100A9 murina. / Peptide identical to the C-terminus of S100A9 protein (mS100A9pH92-G110) inhibits inflammatory hyperalgesia induced by carrageenan and trypsin, a serine protease that activates protease-activated receptors 2 (PAR2). The aim of this work was to characterize the relationship between structure and function of mS100A9p in order to identify the shortest peptide sequence endowed with antinociceptive effect. Furthermore, the mechanisms involved on the antinociceptive effect of both mS100A9p and the shortest homologous sequence on PAR2-induced hyperalgesia were also evaluated. Different peptide sequences homologous to mS100A9p were synthesized and evaluated in rats submitted to the carrageenan-induced mechanical hyperalgesia model. Among all evaluated sequences, the peptide AcE97-G102 was found to be the shortest sequence that showed an antinociceptive effect similar to that induced by mS100A9p. In regard to PAR2 activation, data obtained herein demonstrated that both mS100A9p and AcE97-G102 inhibit PAR2-induced mechanical and thermal hyperalgesia, induced by the selective agonist peptide ? PAR2AP. Imunohistochemical evaluation demonstrated that PAR2 activation increased Egr-1 protein expression on sensory neurons and mS100A9p inhibited this effect. In addition, both mS100A9p and AcE97-G102 inhibited PAR2- and trypsin-induced calcium influx in dorsal root ganglia neurons (DRG). On the other hand, no effect on the calcium influx of the peptides were observed on HEK-293 cells or KNRK-PAR2 transfected cells, suggesting that the effects of mS100A9p and AcE97-G102 on PAR2 activation are specific for sensory neurons. Both mS100A9p and AcE97-G102 inhibited DRG calcium flux when cells were stimulated with bradykinin, capsaicin or KCl. Also, mS100A9p inhibited PAR2-induced substance P release in DRG. Treatment of DRG with either thapsigargin or calcium ionophore suggest a direct effect of mS100A9p on calcium channels. To evaluate this hypothesis the effects of mS100A9p and AcE97-G102 were evaluated on N-type or L-type voltage-dependent calcium channel transfected HEK-tsA cells. Both peptides inhibited calcium influx of N-type transfected cells. In conclusion, data presented herein demonstrate that the C-terminus of murine S100A9 protein inhibits experimental nociception through a block of N-type voltage-dependent calcium channels, directly on sensory neurons. Also, the domain involved in this effect is localized on the sequence E97-G102 of the C-terminus of murine S100A9 protein.

O peptídeo idêntico ao C-terminal da proteína S100A9 murina (pS100A9mH92-G110) inibe a hiperalgesia inflamatória induzida pela carragenina. Em adição, este peptídeo inibe a hiperalgesia inflamatória induzida por tripsina, uma serino protease capaz de ativar receptores ativados por protease do tipo 2 (PAR2). O objetivo inicial deste trabalho foi caracterizar a relação estrutura/ efeito do pS100A9m, a fim de determinar a menor seqüência peptídica dotada de atividade antinociceptiva. Ainda, como parte dos objetivos, neste trabalho foram investigados os mecanismos envolvidos no efeito antinociceptivo do pS100A9m e da menor seqüência ativa sobre a hiperalgesia induzida pela ativação de PAR2. Diferentes seqüências peptídicas homólogas ao pS100A9m foram sintetizadas e avaliadas em ratos submetidos ao modelo de hiperalgesia mecânica induzida por carragenina. Dentre todas as seqüências peptídicas investigadas, o peptídeo denominado AcE97-G102 foi determinado como a menor seqüência ativa com efeito semelhante ao pS100A9m. Com relação aos estudos sobre a ativação de PAR2, os resultados obtidos demonstraram que o pS100A9m bem como o AcE97-G102 inibem a hiperalgesia térmica e mecânica decorrentes da ativação de PAR2 (induzida por um peptídeo agonista deste receptor ? PAR2AP). A análise por imuno-histoquímica demonstrou que a ativação de PAR2 aumenta a expressão da proteína Egr-1 em neurônios nociceptivos, sendo o pS100A9m capaz de inibir este efeito. Em adição, ambos pS100A9m e AcE97-G102 inibiram o influxo de cálcio induzido por PAR2AP ou tripsina, em neurônios sensoriais do gânglio da raiz dorsal da medula espinhal (DRG). Por outro lado, nenhum dos peptídeos apresentou efeito sobre a mobilização de cálcio em células HEK-293, que naturalmente expressam PAR2, ou em células KNRK transfectadas com este tipo de receptor, sugerindo que o efeito tanto do pS100A9m quanto do AcE97-G102, sobre a ativação de PAR2, seja específico para neurônios sensoriais. O pS100A9m e o AcE97-G102 inibiram o influxo de cálcio nos neurônios DRG estimulados com bradicinina, capsaicina ou KCl. Ainda, o pS100A9m inibiu a liberação de substância P induzida por PAR2. Os resultados obtidos com o tratamento de neurônios DRG com tapsigaragina ou com ionóforo de cálcio sugerem um efeito direto do pS100A9m sobre os canais de cálcio. Desta forma, foi avaliada atividade do pS100A9m e do AcE97-G102 sobre culturas de células HEK-tsA transfectadas com canais de cálcio dependente de voltagem do tipo N ou do tipo L. Os resultados obtidos demonstraram que ambos peptídeos inibirem o influxo de cálcio em células transfectadas com receptores do tipo N. Em conjunto, os dados aqui obtidos demonstram que o efeito do C-terminal da proteína S100A9 murina sobre a nocicepção experimental é devido a uma inibição de canais de cálcio do tipo N, por uma ação direta em neurônios sensoriais. Ainda, a seqüência responsável por este efeito está localizada na porção E97-G102 do domínio C-terminal da proteína S100A9 murina. / Peptide identical to the C-terminus of S100A9 protein (mS100A9pH92-G110) inhibits inflammatory hyperalgesia induced by carrageenan and trypsin, a serine protease that activates protease-activated receptors 2 (PAR2). The aim of this work was to characterize the relationship between structure and function of mS100A9p in order to identify the shortest peptide sequence endowed with antinociceptive effect. Furthermore, the mechanisms involved on the antinociceptive effect of both mS100A9p and the shortest homologous sequence on PAR2-induced hyperalgesia were also evaluated. Different peptide sequences homologous to mS100A9p were synthesized and evaluated in rats submitted to the carrageenan-induced mechanical hyperalgesia model. Among all evaluated sequences, the peptide AcE97-G102 was found to be the shortest sequence that showed an antinociceptive effect similar to that induced by mS100A9p. In regard to PAR2 activation, data obtained herein demonstrated that both mS100A9p and AcE97-G102 inhibit PAR2-induced mechanical and thermal hyperalgesia, induced by the selective agonist peptide ? PAR2AP. Imunohistochemical evaluation demonstrated that PAR2 activation increased Egr-1 protein expression on sensory neurons and mS100A9p inhibited this effect. In addition, both mS100A9p and AcE97-G102 inhibited PAR2- and trypsin-induced calcium influx in dorsal root ganglia neurons (DRG). On the other hand, no effect on the calcium influx of the peptides were observed on HEK-293 cells or KNRK-PAR2 transfected cells, suggesting that the effects of mS100A9p and AcE97-G102 on PAR2 activation are specific for sensory neurons. Both mS100A9p and AcE97-G102 inhibited DRG calcium flux when cells were stimulated with bradykinin, capsaicin or KCl. Also, mS100A9p inhibited PAR2-induced substance P release in DRG. Treatment of DRG with either thapsigargin or calcium ionophore suggest a direct effect of mS100A9p on calcium channels. To evaluate this hypothesis the effects of mS100A9p and AcE97-G102 were evaluated on N-type or L-type voltage-dependent calcium channel transfected HEK-tsA cells. Both peptides inhibited calcium influx of N-type transfected cells. In conclusion, data presented herein demonstrate that the C-terminus of murine S100A9 protein inhibits experimental nociception through a block of N-type voltage-dependent calcium channels, directly on sensory neurons. Also, the domain involved in this effect is localized on the sequence E97-G102 of the C-terminus of murine S100A9 protein.

ABSTRACT Although there is ample evidence linking chronic inflammation with cancer, the cellular mechanisms involved in early events leading to tumor development remain unclear. Myeloid cells are an intricate part of inflammation. They consist of mature cells represented by macrophages, dendritic cells and granulocytes and a population of Immature Myeloid Cells (IMC), which in healthy individuals are cells in transition to mature cells. There is a substantial expansion of IMC in cancer and many other pathological conditions which is associated with pathologic activation of these cells. As a result, these cells acquire the ability to suppress immune responses and are termed Myeloid-derived Suppressor Cells (MDSCs). Although the role of MDSC in immune suppression in cancer and tumor progression is well established, their contribution to tumor development is still uncertain. The fact that cells with MDSC phenotype and function are observed in chronic inflammation raised the possibility that these cells can contribute to initial stages of tumor development. To address this question, we used an experimental system where the number of IMC was regulated by the expression of S100A9 protein. In this project, we used two different models of chronic inflammation in S100A9 transgenic (S100A9tg) and S100A9 knock-out (S100A9KO) mice. In the first model, we created the conditions for topical accumulation of these cells in the skin in the absence of infection or tissue damage using S100A9tg mice. Accumulation of IMC in the skin resulted in a dramatic increase in the formation of skin tumors during epidermal carcinogenesis. Conversely, lack of myeloid cell accumulation in S100A9KO mice substantially reduced the formation of skin papillomas. The effect of IMC was not associated with immune suppression but with the recruitment of CD4+ T cells mediated by CCL4 chemokine released by activated IMC. Elimination of CD4+ T cells or blockade of CCL4 abrogated the increase in tumor formation caused by myeloid cells. Thus, this study implicates the accumulation of IMC as an initial step in facilitating of tumor formation, which can mediate the recruitment of CD4+ T cells via the release of CCL4 chemokine. In the second model, we used inflammation-associated lung cancer caused by the chemical lung carcinogen urethane in combination with exposure to cigarette smoke referred to throughout as CS. Exposure of mice to CS alone resulted in a significant accumulation of cells with typical MDSC phenotype in different organs; however, these cells lacked immune suppressive activity and could not be defined as bona fide MDSC. When CS was combined with the single dose of urethane, it led to the accumulation of immune suppressive cells. The expansion of MDSC followed the onset of lung tumors development. This suggests that MDSC in this model is not the preceding factor but rather a consequence of tumor formation. Further studies are necessary to determine the relevance of targeting these cells for cancer treatment and prevention.

Rheumatoid arthritis is a chronic inflammatory autoimmune disease. Measurement of the level of serum markers (sRAGE, S100A9, S100A8 and S100A12) and genetic testing for the presence of the PTPN22 genetic polymorphism could help elucidate the underlying cause of inflammation and complications in RA, such as atherosclerosis. Therefore, serum levels of sRAGE, S100A9, S100A8 and S100A12 were measured by ELISA in patients with established RA (n=138). The associations between the serum levels of these molecules; and inflammatory markers and RA complications were analysed by multiple linear regression modelling. Established RA patients (n=192) were investigated for the PTPN22 C1858T genetic polymorphism by PCR-RFLP. Multiple logistic regression modelling was used to examine the association between PTPN22 C1858T genetic polymorphism and inflammatory markers and RA complications. In RA patients, we found that serum levels of S100A9 were associated with the body mass index (BMI); and the presence of S100A8 and S100A12. The serum levels of S100A8 in RA patients were associated with the presence of anti-citrullinated peptide antibodies, rheumatoid factor and S100A9. The serum levels of S100A12 in RA patients were associated with the presence of anti-citrullinated peptide antibodies and S100A9; and a history of diabetes. Inflammatory markers and RA complications were not associated with the PTPN22 genetic polymorphism in established RA patients; serum level of triglyceride was the only variable associated with PTPN22 C1858T in multiple logistic regression analysis. Taken together, these data suggest that serum levels of sRAGE, S100A9 and S100A12 protein may be useful correlates of inflammation and autoantibody production in RA patients. Further studies are recommended to determine whether these markers predict clinical outcomes when measured at the onset of RA.

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/EBPb 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/EBPb protein complex that activates the miRNA promoters. Reconstituting Gr1+CD11b+ cells from the S100A9 knockout mice with late sepsis with S100A9 protein restores the Stat3-C/EBPb 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.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第21929号 / 生博第414号 / 新制||生||54(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 杉田 昌彦, 教授 松田 道行, 教授 原田 浩 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Myeloid precursor cell reprogramming into a myeloid-derived suppressor cell (MDSC) contributes to high mortality rates in mouse and human sepsis. S100A9 mRNA and intracellular protein levels increase during early sepsis and remain elevated in Gr1+CD11b+ MDSCs after pro-inflammatory sepsis transitions to the later chronic anti-inflammatory and immunosuppressive phenotype. The purpose of this study was to determine whether intracellular S100A9 protein might sustain Gr1+CD11b+ MDSC repressor cell reprogramming during sepsis. We used a chronic model of sepsis in mice to show that S100A9 release from MDSCs and circulating phagocytes decreases after early sepsis and that targeting the S100a9 gene improves survival. Surprisingly, we find that intracellular S100A9 protein translocates from the cytosol to nucleus in Gr1+CD11b+ MDSCs during late sepsis and promotes expression of miR-21 and miR-181b immune repressor mediators. We further provide support of this immunosuppression pathway in human sepsis. This study may inform a new therapeutic target for improving sepsis outcome.

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/EBPb 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/EBPb protein complex that activates the miRNA promoters. Reconstituting Gr1+CD11b+ cells from the S100A9 knockout mice with late sepsis with S100A9 protein restores the Stat3-C/EBPb 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.

Le choc septique reste une pathologie grave, associée à des taux de mortalité et d'infections nosocomiales (IN) secondaires élevés. La prédiction du pronostic est de la plus haute importance pour sélectionner les patients qui pourraient bénéficier de traitements visant à moduler la réponse immunitaire. Le système immunitaire, classiquement active par des agents externes, peut également être activé par des médiateurs endogènes exprimés à la suite d'une agression d'origine septique ou non. Les protéines S100 font partie de ces signaux de danger endogènes (ou alarmines). Le but de ce travail est d'évaluer la capacité de l'ARNm de S100A9 mesuré dans le sang total de patients en choc septique à prédire la survie et la survenue d'IN. Nous avons également étudié la régulation de l'expression des ARN messagers de S100A8 et S100A9 dans un modèle ex vivo de tolérance à l'endotoxine qui reproduit partiellement les dysfonctions de l'immunité innée induites par le sepsis. L'ARNm de S100A9 est surexprimé dans le sang des patients en choc septique. Un taux élevé entre le 7eme et le 10eme jour du début du choc septique est associé à la survenue d'IN secondaires. Ex vivo, l'expression des ARNm de S100A8 et S100A9 est augmentée durant le phénomène de tolérance à l'endotoxine. Le blocage de l IL-10 et l'administration d'IFN-γ réduisent l'augmentation de ces ARNm dans ce modèle. Apres confirmation dans des études cliniques, ces résultats préliminaires suggèrent que l'expression des ARNm de S100A8 et S100A9 puisse être utilisée comme marqueur du phénomène de tolérance à l'endotoxine et comme outils pour évaluer la dysfonction immunitaire des patients en choc septique. Ces patients pourraient alors bénéficier de thérapies visant à restaurer leurs fonctions immunitaires / Septic shock remains a serious disease with high mortality and increased risk of hospital-acquired infection. The prediction of outcome is of the utmost importance for selecting patients for therapeutic strategies aiming to modify the immune response. Immune system, typically activated by external agents, can also be activated by endogenous mediators induced by various types of stress (trauma, infection, burns). S100 proteins are part of the alarmins family. The aim of this study was to assess the capability of S100A9 messenger RNA in whole blood from patients with septic shock to predict survival and the occurrence of hospital-acquired infection. We also investigate the regulation of S100A8 and S100A9 mRNA expressions in an ex vivo model of endotoxin tolerance which partially reproduces sepsis-induced innate immune alterations. S100A9 messenger RNA is increased in septic shock and its delayed overexpression is associated with the occurrence of secondary hospital-acquired infection. Ex vivo, S100A8 and S100A9 mRNA expressions are increased during endotoxin tolerance. IL-10 blockade and rIFN-γ treatment partially abrogated S100A8/A9 mRNA increases in this model. Pending confirmation in larger, independent clinical studies, these preliminary results suggest that S100A8 and S100A9 mRNA levels might be used as surrogate markers of endotoxin tolerance and as evaluation tools for immune dysfunctions in septic shock patients. These patients could be selected for therapeutic aiming to restore immune functions