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.

Sepsis inflammation accelerates myeloid cell generation to compensate for rapid mobilization of the myeloid progenitors from bone marrow. This inflammation-driven myelopoiesis, however, generates myeloid progenitors with immunosuppressive functions that are unable to differentiate into mature, innate immune cells. The myeloid-derived suppressor cells (MDSCs) expand markedly in the later phases of sepsis, suppress both innate and adaptive immunity, and thus, elevate mortality. Using a murine model with myeloid-restricted deletion of the C/EBPβ transcription factor, we show that sepsis-induced generation of MDSCs depends on C/EBPβ. C/EBPβ myeloid cell–deficient mice did not generate MDSCs or develop immunosuppression and survived sepsis. However, septic mice still generated Gr1+ CD11b+ myeloid progenitors at the steady-state levels similar to the control sham mice, suggesting that C/EBPβ is not involved in healthy, steady-state myelopoiesis. C/EBPβ-deficient Gr1+ CD11b+ cells generated fewer monocyte- and granulocyte-like colonies than control mice did, indicating reduced proliferation potential, but differentiated normally in response to growth factors. Adoptive transfer of C/EBPβ-deficient Gr1+ CD11b+ cells from late septic mice exacerbated inflammation in control mice undergoing early sepsis, confirming they were not immunosuppressive. These results show that C/EBPβ directs a switch from proinflammatory to repressor myeloid cells and identifies a novel treatment target.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20253号 / 医博第4212号 / 新制||医||1020(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 戸井 雅和, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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One of the most effective immunological interventions for human disease prevention is the administration of anti-red blood cell (RBC) IgG, more specifically, anti-D IgG, for prevention of hemolytic disease of the fetus and newborn (HDN), a serious and potentially fatal condition caused by the maternal immune response against the Rhesus (Rh) blood group system D antigen on fetal RBC. Despite its widespread clinical use, the mechanism of the suppressive anti-RBC IgG effect is not fully understood. In a murine model of immunity to foreign RBCs, transfusion of mice with IgG-opsonized RBCs strongly attenuated the antibody response compared to transfusion of untreated RBCs. This model was used to study the anti-RBC IgG effect on the host immune response. Contrary to the predominant theories of the anti-D effect, here it is shown that IgG-mediated RBC clearance is not sufficient for the attenuation of antibody responses. IgG-opsonized RBCs internalized by the mononuclear phagocytic cells could stimulate T and B cell responses against RBC antigens. This thesis also shows that the adaptive tolerance at the T or B cell level is not the reason for the attenuation of the antibody response. Instead, IgG selectively prevented the appearance of antigen-primed RBC-specific B cells and, surprisingly, induced the host B cell response against the IgG in complex with RBCs. These results suggest that the inability of RBC-specific B cells to recognize and present RBC-specific epitopes may explain the inhibitory IgG effect.

One of the most effective immunological interventions for human disease prevention is the administration of anti-red blood cell (RBC) IgG, more specifically, anti-D IgG, for prevention of hemolytic disease of the fetus and newborn (HDN), a serious and potentially fatal condition caused by the maternal immune response against the Rhesus (Rh) blood group system D antigen on fetal RBC. Despite its widespread clinical use, the mechanism of the suppressive anti-RBC IgG effect is not fully understood. In a murine model of immunity to foreign RBCs, transfusion of mice with IgG-opsonized RBCs strongly attenuated the antibody response compared to transfusion of untreated RBCs. This model was used to study the anti-RBC IgG effect on the host immune response. Contrary to the predominant theories of the anti-D effect, here it is shown that IgG-mediated RBC clearance is not sufficient for the attenuation of antibody responses. IgG-opsonized RBCs internalized by the mononuclear phagocytic cells could stimulate T and B cell responses against RBC antigens. This thesis also shows that the adaptive tolerance at the T or B cell level is not the reason for the attenuation of the antibody response. Instead, IgG selectively prevented the appearance of antigen-primed RBC-specific B cells and, surprisingly, induced the host B cell response against the IgG in complex with RBCs. These results suggest that the inability of RBC-specific B cells to recognize and present RBC-specific epitopes may explain the inhibitory IgG effect.

Regenerative medicine and cell therapies hold great potential to treat a variety of medical conditions. Product characterisation of these therapies is particularly difficult as they pose regulatory challenges due to donor heterogeneity and the lack of standardised lot release tests that can reliably predict in vivo function. Human mesenchymal stem cells (hMSCs), also called multipotent stem cells or mesenchymal stromal cells, are a viable option in cell therapies due to their immunosuppressive and pro-angiogenic functions. Currently there are no standardised methods or potency assays to quantify these properties. To address this, five individual hMSCs lines from different donors were created and characterised based upon growth rate, differentiation capability and extracellular surface protein expression. A novel multiparameter flow cytometry method to characterise the cells based upon extracellular surface markers was developed that supports high-throughput and high-content analyses. Three candidate lines were taken forward and assessed in multiple in vitro bioassays that examined the hMSC immunosuppressive response to a defined inflammatory environment, effect on T-cell proliferation, and effect on a mixed lymphocyte population. Next, the angiogenic properties were assessed using human umbilical vein endothelial cells (HUVECs) tube formation as a model for cardiac regeneration. This involved utilising automated time lapse microscopy techniques coupled with image analysis software to quantify endothelial to tube formation. Further analysis of the hMSC secretome revealed differences in the levels of pro-angiogenic cytokines such as vascular endothelial growth factor, hepatocyte growth factor and IL-8. Significant differences in angiogenic potency were found between the hMSC lines. This thesis highlights the need to develop specific assays that reflect the intended clinical action. Taken together, these quantitative approaches provide valuable tools to measure hMSC quality and potency, and supports continued efforts to improve characterisation strategies for cellular therapies.

Orientador: Paulo Cesar Ciarlini. / Banca: Raimundo de Souza Lopes. / Banca: Gisele Fabrino Machado. / Resumo: À semelhança do que ocorre em homens e gatos, o estresse oxidativo associado à insuficiência renal crônica (IRC) canina pode afetar a função dos neutrófilos. Em cães urêmicos com IRC ocorre aumento do estresse oxidativo e os neutrófilos tornam-se menos viáveis e funcionais. Dezoito cães adultos urêmicos com insuficiência renal crônica foram comparados com 10 cães adultos controle. Foram determinados o hemograma, a urinálise, o perfil bioquímico plasmático, a peroxidação lipídica plasmática, a capacidade antioxidante total (CAT) do plasma, a produção de superóxido, a taxa de viabilidade e a apoptose dos neutrófilos. As taxas plasmáticas de uréia (P<0,01), creatinina (p<0,01) e colesterol (p<0,001) de cães urêmicos foram significativamente superiores aos do controle. Em cães com uremia o volume globular (p<0,0001), a densidade urinária (p<0,015) e a peroxidação lipídica plasmática (p<0,001) foram significativamente menores quando comparado aos do grupo controle. Os neutrófilos de cães urêmicos apresentaram uma maior taxa de apoptose espontânea (0,018±0,016 vs 0,30± 0,23, p< 0,001) e induzida com Camptotecina (4,34±3,93 vs 26,29±18,52, p<0,01). Comparado aos do grupo controle, os valores médios da fluorescência gerada pela produção de superóxido dos neutrófilos isolados de cães urêmicos foi menor na presença (162,93±173,09 vs 49,10±41,22, p< 0,001) e na ausência de estimulo com PMA (1182,5± 1253,5 vs 212,9±294,25 p< 0,01). Durante o estresse oxidativo em cães urêmicos portadores de IRC ocorre uma aceleração da apoptose que diminui o número de neutrófilos viáveis e a produção neutrofílica de superóxido. Os mecanismos de como o estresse oxidativo da uremia acelera a apoptose e diminui o metabolismo oxidativo dos neutrófilos em cães permanecem a ser determinados / Abstract: Similar to what occurs in men and cats oxidative stress associated with chronic renal failure (CRF) can affect the function of canine neutrophils. In uremic dogs with CRF is increased oxidative stress and neutrophils become less viable and functional. Eighteen dogs adult uremic patients with chronic renal failure were compared with 10 control adult dogs. We measured the blood count, urinalysis, serum biochemical profile, lipid peroxidation, plasma total antioxidant capacity (TAC) of plasma, the production of superoxide, the rate of viability and apoptosis of neutrophils. Plasma levels of urea (P <0.01), creatinine (p <0.01) and cholesterol (p <0.001) in uremic dogs were significantly above baseline. In dogs with uremia the packed cell volume (p <0.0001), urine specific gravity (p <0.015) and plasma lipid peroxidation (p <0.001) were significantly lower when compared to the control group. Neutrophils in uremic dogs showed a higher rate of spontaneous apoptosis (0.018 ± 0.016 vs. 0.30 ± 0.23, p <0.001) and induced with Camptothecin (4.34 ± 3.93 vs 26.29 ± 18.52, p <0.01). Compared to the control group the mean fluorescence generated by the superoxide production of neutrophils isolated from uremic dogs was lower in the presence (162.93 ± 49.10 vs 173.09 ± 41.22, p <0.001) and in the absence of stimulation with PMA (1182.5 ± 212.9 vs. 1253.5 ± 294.25 p <0.01). During the oxidative stress in uremic dogs with CRF is an acceleration of apoptosis which reduces the number of viable neutrophils and neutrophil production of superoxide. As the mechanisms of oxidative stress in uremia accelerates apoptosis and oxidative metabolism of neutrophils in dogs it remains to be determined / Mestre

Made available in DSpace on 2014-06-11T19:27:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-08-23Bitstream added on 2014-06-13T20:08:22Z : No. of bitstreams: 1 silva_acra_me_araca.pdf: 170186 bytes, checksum: 3af21aa14d120cf2727d86a2dd2a16b9 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / À semelhança do que ocorre em homens e gatos, o estresse oxidativo associado à insuficiência renal crônica (IRC) canina pode afetar a função dos neutrófilos. Em cães urêmicos com IRC ocorre aumento do estresse oxidativo e os neutrófilos tornam-se menos viáveis e funcionais. Dezoito cães adultos urêmicos com insuficiência renal crônica foram comparados com 10 cães adultos controle. Foram determinados o hemograma, a urinálise, o perfil bioquímico plasmático, a peroxidação lipídica plasmática, a capacidade antioxidante total (CAT) do plasma, a produção de superóxido, a taxa de viabilidade e a apoptose dos neutrófilos. As taxas plasmáticas de uréia (P<0,01), creatinina (p<0,01) e colesterol (p<0,001) de cães urêmicos foram significativamente superiores aos do controle. Em cães com uremia o volume globular (p<0,0001), a densidade urinária (p<0,015) e a peroxidação lipídica plasmática (p<0,001) foram significativamente menores quando comparado aos do grupo controle. Os neutrófilos de cães urêmicos apresentaram uma maior taxa de apoptose espontânea (0,018±0,016 vs 0,30± 0,23, p< 0,001) e induzida com Camptotecina (4,34±3,93 vs 26,29±18,52, p<0,01). Comparado aos do grupo controle, os valores médios da fluorescência gerada pela produção de superóxido dos neutrófilos isolados de cães urêmicos foi menor na presença (162,93±173,09 vs 49,10±41,22, p< 0,001) e na ausência de estimulo com PMA (1182,5± 1253,5 vs 212,9±294,25 p< 0,01). Durante o estresse oxidativo em cães urêmicos portadores de IRC ocorre uma aceleração da apoptose que diminui o número de neutrófilos viáveis e a produção neutrofílica de superóxido. Os mecanismos de como o estresse oxidativo da uremia acelera a apoptose e diminui o metabolismo oxidativo dos neutrófilos em cães permanecem a ser determinados / Similar to what occurs in men and cats oxidative stress associated with chronic renal failure (CRF) can affect the function of canine neutrophils. In uremic dogs with CRF is increased oxidative stress and neutrophils become less viable and functional. Eighteen dogs adult uremic patients with chronic renal failure were compared with 10 control adult dogs. We measured the blood count, urinalysis, serum biochemical profile, lipid peroxidation, plasma total antioxidant capacity (TAC) of plasma, the production of superoxide, the rate of viability and apoptosis of neutrophils. Plasma levels of urea (P <0.01), creatinine (p <0.01) and cholesterol (p <0.001) in uremic dogs were significantly above baseline. In dogs with uremia the packed cell volume (p <0.0001), urine specific gravity (p <0.015) and plasma lipid peroxidation (p <0.001) were significantly lower when compared to the control group. Neutrophils in uremic dogs showed a higher rate of spontaneous apoptosis (0.018 ± 0.016 vs. 0.30 ± 0.23, p <0.001) and induced with Camptothecin (4.34 ± 3.93 vs 26.29 ± 18.52, p <0.01). Compared to the control group the mean fluorescence generated by the superoxide production of neutrophils isolated from uremic dogs was lower in the presence (162.93 ± 49.10 vs 173.09 ± 41.22, p <0.001) and in the absence of stimulation with PMA (1182.5 ± 212.9 vs. 1253.5 ± 294.25 p <0.01). During the oxidative stress in uremic dogs with CRF is an acceleration of apoptosis which reduces the number of viable neutrophils and neutrophil production of superoxide. As the mechanisms of oxidative stress in uremia accelerates apoptosis and oxidative metabolism of neutrophils in dogs it remains to be determined

RUNX1 overlapping RNA (RUNXOR) is a long non-coding RNA and plays a pivotal role in the differentiation of myeloid cells via targeting runt-related transcription factor 1 (RUNX1). We and others have previously reported that myeloid-derived suppressor cells (MDSCs) expand and inhibit host immune responses during chronic viral infections; however, the mechanisms responsible for MDSC differentiation and suppressive functions, in particular the role of RUNXOR-RUNX1, remain unclear. Here, we demonstrated that RUNXOR and RUNX1 expressions are significantly upregulated and associated with elevated levels of immunosuppressive molecules, such as arginase 1 (Arg1), inducible nitric oxide synthase (iNOS), signal transducer and activator of transcription 3 (STAT3), and reactive oxygen species (ROS) in MDSCs during chronic hepatitis C virus (HCV) infection. Mechanistically, we discovered that HCV-associated exosomes (HCV-Exo) can induce the expressions of RUNXOR and RUNX1, which in turn regulates miR-124 expression via STAT3 signaling, thereby promoting MDSC differentiation and suppressive functions. Importantly, overexpression of RUNXOR in healthy CD33+ myeloid cells promoted differentiation and suppressive functions of MDSCs. Conversely, silencing RUNXOR or RUNX1 expression in HCV-derived CD33+ myeloid cells significantly inhibited their differentiation and expressions of suppressive molecules and improved the function of co-cultured autologous CD4 T cells. Taken together, these results indicate that the RUNXOR-RUNX1-STAT3-miR124 axis enhances the differentiation and suppressive functions of MDSCs and could be a potential target for immunomodulation in conjunction with antiviral therapy during chronic HCV infection.