研究背景及實驗目的: 非酒精性脂肪性肝炎(NASH)使得肥胖和2 型糖尿病變得複雜，肝臟炎症的持續產生是其主要的發病機理。CXCL10 是一種促進炎症的細胞因數，其在肥胖和2 型糖尿病中的表達顯著升高。CXCL10 以及其受體CXCR3 是否在NASH 的發生發展中起作用尚不清楚。在本研究中，我們探索了CXCL10 以及其受體CXCR3 在脂肪性肝炎中的功能， 並評估了CXCL10 在NASH 中的臨床價值。 / 實驗方法：CXCL10 基因敲除鼠，CXCR3 敲除鼠以及野生型C57BL/6 小鼠給予蛋氨酸膽鹼缺乏食(MCD)4 周或者8 周。CXCL10 的信號通路以及下游靶點通過細胞因數分析，cDNA array, 蛋白DNA 結合實驗，自噬溶酶體系統分析進行檢測。為了闡明CXCL10 抑制對NASH 的預防治療作用，我們給MCD 餵養的小鼠注射抗CXCL10 抗體。用不同濃度的CXCL10 抗體以及CXCR3 抑制劑NIBR2130 幹預MCD 培養的肝細胞株AML-12。臨床研究中，我們收集了147個非酒精性脂肪肝患者以及73 個健康對照的血清，用酶聯免疫吸附試驗檢測血清中CXCL10 的水準。 / 結果：野生型小鼠給予MCD 餵養後，CXCL10 以及CXCR3 的表達升高，並出現脂肪性肝炎的表現。然而，MCD 飼養的CXCL10 以及CXCR3 基因敲除鼠中，脂肪性肝炎明顯減輕。CXCL10 通過促炎細胞因數的產生以及NK-κB 信號通路促進MCD 飼養的小鼠NASH 的發生。CXCL10 通過促進脂質合成的基因SREBP-1c, ChREBP 和 SCD-1 引起脂肪變性，並通過CYP2E1 以及 C/EBPβ 的上調引起氧化應激。值得注意的是，自噬的損傷在CXCL10 以及CXCR3 導致的脂肪性肝炎的進展中起重要作用。 MCD 飼養的野生型小鼠中p62 以及LC3-II 表達明顯高於CXCL10 以及CXCR3 基因敲除鼠。通過抗CXCL10 抗體中和CXCL10 可以減輕MCD 食引起的小鼠脂肪性肝炎以及MCD 培養液引起的AML-12 細胞損傷。高選擇性的CXCR3 抑制劑NIBR2130 也可以抑制MCD 引起的肝細胞損傷。我們進一步研究了CXCL10 的臨床應用價值，發現NASH 患者血清以及肝臟中CXCL10 的水準明顯升高。更重要的是，血液中CXCL10 的水準與肝小葉炎症程度有關，是NASH 的獨立危險因素。 / 結論：我們的研究首次發現CXCL10 以及其受體CXCR3 通過促進炎症，脂質聚集，氧化應激以及自噬缺乏在NASH 的發病中起重要作用。抑制CXCL10 或者CXCR3 為NASH 患者的治療提供了新的方法。CXCL10 可作為NASH 患者非侵入性診斷的標誌物。 / Background and aims: Non-alcoholic steatoheaptitis (NASH) complicates obesity and type 2 diabetes, while recruitment and perpetuation of liver inflammation is central to its pathogenesis. Expression of C-X-C motif chemokine 10 (CXCL10), a proinflammatory cytokine, correlates positively with obesity and type 2 diabetes. Whether CXCL10 and its receptor CXCR3 play a role in NASH is unknown. In this study, we investigated the functional significance of CXCL10 and its receptor CXCR3 in steatoheaptitis. Moreover, the clinical impact of CXCL10 in NASH was examined. / Methods: Gene-deleted CXCL10 (CXCL10-/-), CXCL10 receptor CXCR3 (CXCR3-/-) and C57BL/6 wildtype (WT) mice were fed methionine and choline-deficient (MCD) diet for 4 or 8 weeks. Cytokine profiling assay, cDNA array, protein-DNA binding activity assay and autophagosome-lysosome system analysis of CXCL10 signaling and downstream targets were performed. In other experiments, we injected neutralizing anti-CXCL10 monoclonal antibodies (mAb) into MCD diet-fed WT mice, while AML-12 cells were cultured in MCD medium in the presence of anti-CXCL10 mAb or CXCR3 inhibitor (NIBR2130) for 24 hours. Human serum was obtained from 147 patients with biopsy-proven non-alcoholic fatty liver disease and 73 controls. Circulating CXCL10 levels were determined by enzyme-linked immunosorbent assay. / Results: MCD-fed WT mice developed steatohepatitis with higher hepatic CXCL10 and CXCR3 expression. CXCL10-/- and CXCR3-/- mice were refractory to MCDinduced steatohepatitis. In WT mice with steatohepatitis, but not in CXCL10-/- mice, CXCL10 was associated with the induction of pro-inflammatory chemokines and cytokines, as well as activation of nuclear factor-κB (NF-κB) signaling. CXCL10 expression was linked to steatosis through lipogenic factors, including liver X receptors and its downstream targets (SREBP-1c, ChREBP and SCD-1), and also to oxidative stress (up-regulation of CYP2E1 and C/EBPβ). In particular, autophagy deficiency was involved in CXCL10- and CXCR3-induced steatohepatitis as indicated by p62 and LC3-I/II protein accumulation in MCD-fed WT mice than in CXCL10-/- and CXCR3-/- mice. Moreover, the impaired autophagic function was related to the reduction of lysosomal function in CXCL10- or CXCR3-induced NASH. Blockade of CXCL10 by anti-CXCL10 mAb protected against MCD-induced steatohepatitis in vivo and against MCD-mediated injury to AML-12 cells in vitro. The highly selective CXCR3 antagonist NIBR2130 also inhibited MCD-induced injury in AML-12 hepatocytes. We further investigated the clinical impact of CXCL10 and found circulating and hepatic CXCL10 levels were significantly higher in human NASH. Importantly, circulating CXCL10 level was correlated with the degree of lobular inflammation and was an independent risk factor for NASH patients. / Conclusions: We demonstrate for the first time that CXCL10 and its receptor CXCR3 plays a pivotal role in the pathogenesis of NASH by promoting inflammation, fatty acid accumulation, oxidative stress and autophagy deficiency. Blockade of CXCL10 or CXCR3 is a potential novel approach for NASH intervention. CXCL10 is a noninvasive biomarker for NASH patients. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhang, Xiang. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 145-167). / Abstracts also in Chinese.

Natural killer (NK) cells are a critical component of the immune response against viral infections. NK cell depletion prior to murine cytomegalovirus (MCMV) infections results in increased susceptibility to infection in several mouse strains. The mechanism of protection in C57Bl/6 mice is dependent on the activation of NK cells by Ly49H recognition of m157. Our previous studies have examined important residues of m157 for Ly49H recognition, as well as the contribution of m157 glycosylation to NK cell activation. However, what role the glycophosphatidyl inositol (GPI) anchor of m157 plays in Ly49H activation was unknown. Here we demonstrate that the GPI anchor of m157 regulates the surface expression of the protein. While the GPI anchor was not required for recognition of m157 by the activating or inhibitory Ly49 receptors, expression of GPI-anchored m157 resulted in greater receptor downregulation on NK cells, as well as increased NK cell cytotoxicity compared to transmembrane m157. In addition to MCMV infections, NK cells have been shown to participate in the immune response to influenza A virus (IAV). However the exact role of NK cells in IAV infection is less clear, as some studies have found NK cells to be protective, while others have shown that NK cells cause lethal immunopathology. It is likely that the severity of IAV infection may dictate the NK cell response to IAV infection (i.e. protective vs. immunopathogenic). Herein we show that NK cell accumulation in IAV-infected lungs and lung-draining lymph nodes (DLN) is regulated by the severity of IAV infection, where there is increased NK cell accumulation in the lungs during high dose IAV infection, and greater NK cell accumulation in the DLN in low dose IAV infections. Despite significant NK cell recruitment to the lung during IAV infection, as well as previously published studies demonstrating the importance of NK cells to IAV immunity, NK cell depletion prior to IAV infection did not result in a significant change in morbidity or mortality. Interestingly, NK cell depletion resulted in a significantly greater number of CD4 T cells in the IAV infected lung. Further, both CD4 and CD8 T cells in NK-depleted mice showed increased IFN-Γ production. Finally, while not statistically significant, NK cell depletion resulted in a trend toward greater protection from heterosubtypic IAV challenge infections. Taken together these results suggest that NK cells may either regulate the adaptive immune response to IAV infection through suppression of CD4 and CD8 T cells, or that the T cell response to IAV infection is able to compensate for the loss of NK cells. Moreover, while NK cell suppression of T cell function during a primary IAV infection does not result in increased susceptibility to primary IAV infections, NK cell regulation of adaptive immune responses may suppress the memory T cell response, and therefore leave the host more susceptible to secondary infections. Overall the studies presented herein demonstrate a complex role for NK cells in the immune response against viral infections. Ly49H+ NK cells directly kill MCMV-infected cells and m157-bearing targets, but NK cell activation is regulated by ligand density, as well as the ligand membrane anchor. Additionally, NK cells suppress adaptive immune responses during a primary IAV infection, resulting in changes to the T cell response during both primary and memory responses.

Les adénocarcinomes lépidiques prédominant (ALP) sont une entité originale sur le plan histologique, clinique et biologique parmi les adénocarcinomes pulmonaires. Il s’agit de tumeurs non-mucineuses mais il existe un variant mucineux, l’adénocarcinome mucineux invasif (AMI), caractérisé par un plus mauvais pronostic et l’absence de traitement efficace dans les formes avancées. L’objectif de ce travail était d’étudier les différences moléculaires distinguant ALP et AMI et d’en dégager les implications biologiques. Après avoir détaillé les caractéristiques cliniques et les altérations oncogéniques d’une cohorte d’ALP et d’AMI, nous avons exploité les banques de prélèvements issus de cette cohorte. Une étude de l’expression en immunohistochimie des mucines MUC1, 2, 5B, 5AC et 6 au sein des pièces opératoires de 27 ALP et 27 AMI montrait un profil d’expression spécifique entre ALP et AMI. L’expression de MUC1 était associée aux ALP, celle de MUC5AC, 5B et 6 aux AMI. L’expression de MUC1 était associée aux mutations EGFR et MUC5B et 5AC aux mutations KRAS. Un réarrangement NRG1 a été détecté par FISH dans 1 AMI sur 25. La chimiokine CXCL10 était surexprimée dans les surnageants de lavages broncho-alvéolaires (LBA) de patients avec AMI (n=38) comparés aux ALP (n=25), et cette surexpression était de mauvais pronostic. La voie cytokine/récepteur CXCL10/CXCR3-A était surexprimée dans les AMI, faisait la promotion de la migration des cellules tumorales mucineuses et gouvernait l’expression tumorale de VEGF. Le VEGF issu du LBA des patients était à l’origine in vitro d’une augmentation significative de la formation de tubes vasculaires inhibée par l’anti-VEGF bevacizumab. Le ciblage de CXCL10/CXCR3-A et du VEGF pourraient être des options thérapeutiques dans les AMI. Ces résultats permettent d’affiner les connaissances biologiques des ALP et des AMI et dégagent des voies de recherche originales qui pourraient amener au développement de nouveaux traitements / Lepidic predominant adenocarcinoma (LPA) represents an original entity in terms of histological, clinical and biological characteristics among adenocarcinomas of the lung. While LPA is typically a non-mucinous adenocarcinoma, a mucinous variant does exist, termed invasive mucinous adenocarcinoma (IMA), associated with a worse prognosis and a lack of effective treatment in advanced diseases. This work sought to study molecular differences between LPA and IMA, and explore their biological meanings. A cohort of LPA and AMI has been studied in regard of clinical characteristics and oncogenic drivers and samples from this cohort were exploited. An immunohistochemical study of expression of mucins MUC1, 2, 5B, 5AC and 6 in surgical samples of 27 LPA and 27 IMA showed different profile of expression between LPA and IMA. MUC1 expression was associated to MUC1 and MUC5AC, 5B and 6 to IMA. MUC1 was associated to EGFR mutations and MUC5B and 5AC to KRAS mutations. One NRG1 rearrangement was detected by FISH in one in 25 IMA. The CXCL10 chemokine was overexpressed in bronchoalveolar lavage fluid (BALF) supernatants of IMA (n=38) compared to LPA (n=25). This overexpression was linked to worse prognosis. The cytokine/receptor axis CXCL10/CXCR3-A was overexpressed in IMA and promoted migration of mucinous tumoral cells and drived tumoral expression of VEGF. VEGF from BALF of patients significantly enhanced human lung endothelial tubes formation in vitro which was inhibited by anti-VEGF bevacizumab. CXCL10/CXCR3 and VEGF could present valuable therapeutic targets in IMA. These results improve knowledge in biology of LPA and AMI and identify new lines of research which could lead to development of new therapies.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23105号 / 医博第4732号 / 新制||医||1050(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 竹内 理, 教授 上野 英樹, 教授 椛島 健治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21636号 / 医博第4442号 / 新制||医||1034(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 生田 宏一, 教授 杉田 昌彦, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

O desenvolvimento gradual e recente de uma epidemia mundial de obesidade alavancou sobremaneira o estudo dessa condição e de suas comorbidades metabólicas. No âmbito fisiopatológico, múltiplos estudos demonstraram a expressão aumentada de mediadores inflamatórios no tecido adiposo de animais e humanos obesos, o acúmulo local de macrófagos, e um papel central da inflamação no desequilíbrio da homeostase metabólica local e sistêmica na obesidade. A definição de um papel ativo dos macrófagos, e portanto da imunidade inata, na rede inflamatória do tecido adiposo, evocou a hipótese de que, similarmente a outras condições inflamatórias crônicas como a aterosclerose, a obesidade também contaria com a importante participação de elementos da imunidade adaptativa, como as células T e suas citocinas, em sua fisiopatologia. Com base nessas considerações, os objetivos principais desse estudo foram: 1) avaliar a presença das células T e o papel do interferon-gama (IFNy), clássica citocina T-helper 1 (ou Th1), na inflamação do tecido adiposo; e 2) estudar mecanismos de acúmulo das células T no tecido adiposo na obesidade, particularmente a participação do receptor CXCR3 nesse processo. Experimentos de citometria de fluxo mostraram que o tecido adiposo visceral de camundongos C57BL/6 obesos após consumo de dieta rica em gorduras apresentou maior número de macrófagos e também de células T, CD4+ e CD8+, em comparação a controles que receberam dieta pobre em gorduras. A expressão de I-Ab, marcador do complexo de histocompatibilidade principal classe II (MHC II) murino, também foi maior no tecido adiposo dos animais obesos, sugerindo a presença local da atividade de apresentação de antígeno com consequente ativação das células T. Quando estimuladas in vitro, células T derivadas do tecido adiposo de camundongos obesos produziram mais IFNy do que aquelas isoladas de controles, novamente sugerindo a ativação dessas células em um contexto de obesidade. Na análise das possíveis funções do IFNy no tecido adiposo, a estimulação da linhagem de células 3T3-L1 diferenciadas em adipócitos com IFNy recombinante resultou na produção aumentada de quimiocinas de macrófagos, como a proteína quimiotática de monócito (MCP-1), e de quimiocinas de células T, como a proteína 10 induzida por IFNy (IP-10) e monocina induzida por IFNy (MIG). A estimulação de adipócitos com o sobrenadante de células Th1 cultivadas in vitro, com abundante concentração de IFNy, também levou à produção aumentada de IP-10. Em análise mais ampla, através de microarray, dos possíveis efeitos do IFNy na expressão gênica de adipócitos, o tratamento dessas células com 100 U/ml de IFNy resultou na expressão aumentada de diversas quimiocinas e seus receptores em comparação ao grupo tratado com placebo. Similarmente à estimulação de células isoladas com IFNy, a incubação de tecido adiposo ex vivo de camundongos com essa citocina também resultou em secreção aumentada de IP-10, MIG e fator de necrose tumoral alfa (TNFy). A investigação do papel do IFNy na inflamação do tecido adiposo in vivo envolveu camundongos com deficiência de IFNy e controles, ambos os grupos submetidos a dieta rica em gorduras (obesos) ou pobre em gorduras (não obesos). Camundongos obesos deficientes em IFNy apresentaram expressão reduzida de mRNA de genes inflamatórios como TNFalfa e MCP-1 no tecido adiposo; acúmulo local reduzido de macrófagos; e melhor tolerância à glicose em comparação aos controles sob mesma dieta. Animais com deficiência de apolipoproteína E (ApoE) e também do receptor de IFNy também apresentaram em seu tecido adiposo a expressão reduzida de mRNA de genes inflamatórios, particularmente relacionados às células T, como IP-10, MIG, e o receptor CXCR3, em comparação aos controles com deficiência única de ApoE. Resultados in vitro e in vivo sugerem conjuntamente um importante papel do IFNy, e portanto, das células T e da imunidade adaptativa, na rede inflamatória do tecido adiposo na obesidade, com consequente impacto metabólico sistêmico. A presença de células T ativadas no tecido adiposo e seu acúmulo diferencial na obesidade motivaram também a pesquisa de potenciais mecanismos quimiotáticos reguladores desse processo. CXCR3, receptor das quimiocinas de células T, IP-10, MIG e quimiocina alfa de células T IFNy-induzida (I-TAC), é expresso preferencialmente em células T ativadas, e detém papel central na migração dessas células em outras condições inflamatórias crônicas, como a aterosclerose. Em camundongos com deficiência de CXCR3 e que receberam dieta rica em gorduras por 8 ou 16 semanas, o tecido adiposo apresentou significativamente menos células T, incluindo as células CD4+ e CD8+, em comparação a controles submetidos a mesma dieta. Os números similares de células T e outras populações de leucócitos no baço e sangue periférico dos animais deficientes em CXCR3 e controles fortalecem o conceito de um efeito do CXCR3 sobre o acúmulo de células T no tecido adiposo, independentemente do número de células circulantes e periféricas. Os camundongos deficientes em CXCR3 apresentaram também maior tolerância à glicose e expressão reduzida de mRNA de mediadores inflamatórios em seu tecido adiposo em comparação aos controles após 8 semanas de dieta rica em gorduras. No entanto, a diferença na tolerância à glicose entre os dois grupos tornou-se não significativa após 16 semanas de dieta gordurosa, coincidindo com redução substancial na expressão de mRNA de mediadores anti-inflamatórios (como interleucina-10 [IL-10] e Arginase 1), e número reduzido de células T regulatórias no tecido adiposo de camundongo s deficientes em CXCR3 em relação a controles. Esses resultados sugerem que o CXCR3 é capaz de regular o acúmulo de células T de diferentes subtipos, com perfil proinflamatório ou anti-inflamatório. Em conclusão, nossos resultados revelam um importante papel da citocina Th1 IFNy na rede inflamatória do tecido adiposo na obesidade em camundongos, sugerindo a participação fundamental das células T e portanto, da imunidade adaptativa nesse cenário. Além disso, o receptor CXCR3 contribui significativamente para o acúmulo das células T, incluindo as células T regulatórias, no tecido adiposo desses animais / The gradual and recent development of a worldwide epidemic of obesity greatly leveraged the study of this condition and its metabolic comorbidities. In the pathophysiologic context, multiple studies have demonstrated increased expression of inflammatory mediators in adipose tissue of obese animals and humans, the local macrophage accumulation, and a central role of inflammation in the imbalance of local or systemic metabolic homeostasis in obesity. The concept of an active role of macrophages and thus of innate immunity in the inflammatory network of adipose tissue, suggested the hypothesis that, similar to other chronic inflammatory conditions such as atherosclerosis, obesity also count on the participation of important elements of adaptive immunity such as T cells and their cytokines in its pathophysiology. Based on these considerations, the main objectives of this study were: 1) to evaluate the presence of T cells and the role of interferon-gamma (IFNy), classic T-helper 1 (Th1) cytokine, in adipose tissue inflammation, and 2) to study mechanisms of T cell accumulation in adipose tissue in the context of obesity, particularly the involvement of CXCR3 receptor in this process. Flow cytometry experiments showed that the visceral fat tissue of C57BL/6 obese mice fed a high fat diet showed a greater number of macrophages and also T cells, including CD4+ and CD8+ cells, compared to controls fed a low-fat diet. The expression of I-Ab, murine marker of class II major histocompatibility complex (MHC II), was also higher in adipose tissue of obese animals, suggesting the presence of local antigen presentation and consequent T cell activation. When stimulated in vitro, T cells derived from adipose tissue of obese mice produced more IFNy than those isolated from controls, again suggesting the activation of these cells in the context of obesity. In the analysis of possible functions of IFNy in adipose tissue, stimulation of 3T3 -L1 cells differentiated into adipocytes with recombinant IFNy resulted in enhanced production of macrophage chemokines, such as monocyte chemotactic protein-1 (MCP-1) and T-cell chemokines, such as interferon gamma-induced protein 10 (IP-10) and monokine induced by gamma interferon (MIG). The stimulation of adipocytes with the supernatant of in vitro cultured Th1 cells, with abundant levels of IFNy, has also led to increased IP-10 production. In a broader analysis, by microarray, of the possible effects of IFNy on adipocyte gene expression, treatment of these cells with 100 U/ml of IFNy resulted in increased expression of chemokines and their receptors in comparison to the placebo group. Similarly to the stimulation of isolated cells with IFNy, incubation of ex vivo adipose tissue with this cytokine also resulted in increased IP-10, MIG and tumor necrosis factor alpha (TNFalpha) secretion

O desenvolvimento gradual e recente de uma epidemia mundial de obesidade alavancou sobremaneira o estudo dessa condição e de suas comorbidades metabólicas. No âmbito fisiopatológico, múltiplos estudos demonstraram a expressão aumentada de mediadores inflamatórios no tecido adiposo de animais e humanos obesos, o acúmulo local de macrófagos, e um papel central da inflamação no desequilíbrio da homeostase metabólica local e sistêmica na obesidade. A definição de um papel ativo dos macrófagos, e portanto da imunidade inata, na rede inflamatória do tecido adiposo, evocou a hipótese de que, similarmente a outras condições inflamatórias crônicas como a aterosclerose, a obesidade também contaria com a importante participação de elementos da imunidade adaptativa, como as células T e suas citocinas, em sua fisiopatologia. Com base nessas considerações, os objetivos principais desse estudo foram: 1) avaliar a presença das células T e o papel do interferon-gama (IFNy), clássica citocina T-helper 1 (ou Th1), na inflamação do tecido adiposo; e 2) estudar mecanismos de acúmulo das células T no tecido adiposo na obesidade, particularmente a participação do receptor CXCR3 nesse processo. Experimentos de citometria de fluxo mostraram que o tecido adiposo visceral de camundongos C57BL/6 obesos após consumo de dieta rica em gorduras apresentou maior número de macrófagos e também de células T, CD4+ e CD8+, em comparação a controles que receberam dieta pobre em gorduras. A expressão de I-Ab, marcador do complexo de histocompatibilidade principal classe II (MHC II) murino, também foi maior no tecido adiposo dos animais obesos, sugerindo a presença local da atividade de apresentação de antígeno com consequente ativação das células T. Quando estimuladas in vitro, células T derivadas do tecido adiposo de camundongos obesos produziram mais IFNy do que aquelas isoladas de controles, novamente sugerindo a ativação dessas células em um contexto de obesidade. Na análise das possíveis funções do IFNy no tecido adiposo, a estimulação da linhagem de células 3T3-L1 diferenciadas em adipócitos com IFNy recombinante resultou na produção aumentada de quimiocinas de macrófagos, como a proteína quimiotática de monócito (MCP-1), e de quimiocinas de células T, como a proteína 10 induzida por IFNy (IP-10) e monocina induzida por IFNy (MIG). A estimulação de adipócitos com o sobrenadante de células Th1 cultivadas in vitro, com abundante concentração de IFNy, também levou à produção aumentada de IP-10. Em análise mais ampla, através de microarray, dos possíveis efeitos do IFNy na expressão gênica de adipócitos, o tratamento dessas células com 100 U/ml de IFNy resultou na expressão aumentada de diversas quimiocinas e seus receptores em comparação ao grupo tratado com placebo. Similarmente à estimulação de células isoladas com IFNy, a incubação de tecido adiposo ex vivo de camundongos com essa citocina também resultou em secreção aumentada de IP-10, MIG e fator de necrose tumoral alfa (TNFy). A investigação do papel do IFNy na inflamação do tecido adiposo in vivo envolveu camundongos com deficiência de IFNy e controles, ambos os grupos submetidos a dieta rica em gorduras (obesos) ou pobre em gorduras (não obesos). Camundongos obesos deficientes em IFNy apresentaram expressão reduzida de mRNA de genes inflamatórios como TNFalfa e MCP-1 no tecido adiposo; acúmulo local reduzido de macrófagos; e melhor tolerância à glicose em comparação aos controles sob mesma dieta. Animais com deficiência de apolipoproteína E (ApoE) e também do receptor de IFNy também apresentaram em seu tecido adiposo a expressão reduzida de mRNA de genes inflamatórios, particularmente relacionados às células T, como IP-10, MIG, e o receptor CXCR3, em comparação aos controles com deficiência única de ApoE. Resultados in vitro e in vivo sugerem conjuntamente um importante papel do IFNy, e portanto, das células T e da imunidade adaptativa, na rede inflamatória do tecido adiposo na obesidade, com consequente impacto metabólico sistêmico. A presença de células T ativadas no tecido adiposo e seu acúmulo diferencial na obesidade motivaram também a pesquisa de potenciais mecanismos quimiotáticos reguladores desse processo. CXCR3, receptor das quimiocinas de células T, IP-10, MIG e quimiocina alfa de células T IFNy-induzida (I-TAC), é expresso preferencialmente em células T ativadas, e detém papel central na migração dessas células em outras condições inflamatórias crônicas, como a aterosclerose. Em camundongos com deficiência de CXCR3 e que receberam dieta rica em gorduras por 8 ou 16 semanas, o tecido adiposo apresentou significativamente menos células T, incluindo as células CD4+ e CD8+, em comparação a controles submetidos a mesma dieta. Os números similares de células T e outras populações de leucócitos no baço e sangue periférico dos animais deficientes em CXCR3 e controles fortalecem o conceito de um efeito do CXCR3 sobre o acúmulo de células T no tecido adiposo, independentemente do número de células circulantes e periféricas. Os camundongos deficientes em CXCR3 apresentaram também maior tolerância à glicose e expressão reduzida de mRNA de mediadores inflamatórios em seu tecido adiposo em comparação aos controles após 8 semanas de dieta rica em gorduras. No entanto, a diferença na tolerância à glicose entre os dois grupos tornou-se não significativa após 16 semanas de dieta gordurosa, coincidindo com redução substancial na expressão de mRNA de mediadores anti-inflamatórios (como interleucina-10 [IL-10] e Arginase 1), e número reduzido de células T regulatórias no tecido adiposo de camundongo s deficientes em CXCR3 em relação a controles. Esses resultados sugerem que o CXCR3 é capaz de regular o acúmulo de células T de diferentes subtipos, com perfil proinflamatório ou anti-inflamatório. Em conclusão, nossos resultados revelam um importante papel da citocina Th1 IFNy na rede inflamatória do tecido adiposo na obesidade em camundongos, sugerindo a participação fundamental das células T e portanto, da imunidade adaptativa nesse cenário. Além disso, o receptor CXCR3 contribui significativamente para o acúmulo das células T, incluindo as células T regulatórias, no tecido adiposo desses animais / The gradual and recent development of a worldwide epidemic of obesity greatly leveraged the study of this condition and its metabolic comorbidities. In the pathophysiologic context, multiple studies have demonstrated increased expression of inflammatory mediators in adipose tissue of obese animals and humans, the local macrophage accumulation, and a central role of inflammation in the imbalance of local or systemic metabolic homeostasis in obesity. The concept of an active role of macrophages and thus of innate immunity in the inflammatory network of adipose tissue, suggested the hypothesis that, similar to other chronic inflammatory conditions such as atherosclerosis, obesity also count on the participation of important elements of adaptive immunity such as T cells and their cytokines in its pathophysiology. Based on these considerations, the main objectives of this study were: 1) to evaluate the presence of T cells and the role of interferon-gamma (IFNy), classic T-helper 1 (Th1) cytokine, in adipose tissue inflammation, and 2) to study mechanisms of T cell accumulation in adipose tissue in the context of obesity, particularly the involvement of CXCR3 receptor in this process. Flow cytometry experiments showed that the visceral fat tissue of C57BL/6 obese mice fed a high fat diet showed a greater number of macrophages and also T cells, including CD4+ and CD8+ cells, compared to controls fed a low-fat diet. The expression of I-Ab, murine marker of class II major histocompatibility complex (MHC II), was also higher in adipose tissue of obese animals, suggesting the presence of local antigen presentation and consequent T cell activation. When stimulated in vitro, T cells derived from adipose tissue of obese mice produced more IFNy than those isolated from controls, again suggesting the activation of these cells in the context of obesity. In the analysis of possible functions of IFNy in adipose tissue, stimulation of 3T3 -L1 cells differentiated into adipocytes with recombinant IFNy resulted in enhanced production of macrophage chemokines, such as monocyte chemotactic protein-1 (MCP-1) and T-cell chemokines, such as interferon gamma-induced protein 10 (IP-10) and monokine induced by gamma interferon (MIG). The stimulation of adipocytes with the supernatant of in vitro cultured Th1 cells, with abundant levels of IFNy, has also led to increased IP-10 production. In a broader analysis, by microarray, of the possible effects of IFNy on adipocyte gene expression, treatment of these cells with 100 U/ml of IFNy resulted in increased expression of chemokines and their receptors in comparison to the placebo group. Similarly to the stimulation of isolated cells with IFNy, incubation of ex vivo adipose tissue with this cytokine also resulted in increased IP-10, MIG and tumor necrosis factor alpha (TNFalpha) secretion

Le récepteur de chimiokine CXCR3 est un récepteur couplé à la protéine G (RCPG) exprimé, entre autre, sur les cellules T activées lors d’une réponse immune. CXCR3 est activé par trois ligands inductibles par l’interféron-γ (CXCL9, 10, 11) et, plus récemment, il a été découvert que CXCL4 liait CXCR3. Nous savons que CXCR3 joue un rôle dans la chimiotaxie des leucocytes, mais peu d’attention a été portée sur la signalisation biaisée induite par ces quatre ligands. Alors que l’homodimérisation entre récepteurs de chimiokine est un concept grandement observé, l’hétéromérisation entre deux récepteurs reste un domaine de recherche active. La signalisation biaisée et l’hétéromérisation ont été testées grâce à la technique de bioluminescene resonance energy transfer (BRET) dans des cellules HEK293E. Nous présentons une caractérisation pharmacologique des quatre ligands de CXCR3 et démontrons l’hétéromérisation de CXCR3 avec CXCR4 et avec CXCR7. Nos résultats suggèrent que les ligands de CXCR3 n’agissent pas de manière redondante. / The chemokine receptor CXCR3 is a G-protein-coupled receptor (GPCR) rapidly induced on naïve T cells upon activation. CXCR3 is activated by three interferon-γ inducible ligands (CXCL9, 10, 11) and, more recently, CXCL4 has been discovered as a functional ligand for CXCR3. It is known that CXCR3 acts as a chemotactic receptor, but limited attention has been directed to the biased signaling induced by all four ligands. Chemokine receptor homodimerization is now a widely accepted concept, but the extent to which heterodimerization is prevalent remain matter of active research. In this work, biased signalling and heterodimerization were assessed with bioluminescence resonance energy transfer (BRET) in HEK293E cells. We present pharmacological characterization of all four ligands of CXCR3 and heterodimerization of CXCR3 with CXCR4 or CXCR7. Our results suggest that CXCR3 ligands are not redundant and that CXCR3 heterodimerizes with CXCR4 and with CXCR7.

Les chimiokines sont des petites protéines secrétées dont la fonction principale est la stimulation de la migration de cellules immunitaires vers différents organes et tissus. Elles sont souvent impliquées lors des maladies inflammatoires, auto-immunes et des cancers. Ainsi, les chimiokines et leurs récepteurs couplés aux protéines G (RCPG) sont la cible pharmacologique de plusieurs molécules, actuellement testées en essais cliniques. Nous avons pris comme modèle, lors de notre étude, le récepteur atypique CXCR7. Ce récepteur est dit atypique, car il ne signalise pas via la voie classique des protéines G, mais plutôt via la voie de la β-arrestine. CXCR7 est impliqué dans de nombreux cancers, favorise la progression métastatique et est un co-récepteur pour le virus de l’immunodéficience humaine (VIH). Cependant, aucune donnée sur son mode de liaison avec ses ligands CXCL11/ITAC et CXCL12/SDF-1 n’existe à date. Nous pensons que cette information est essentielle pour le développement efficace d’agonistes et d’antagonistes, et nous nous sommes intéressés à identifier les résidus essentiels à la liaison des deux ligands de CXCR7 et à son activation par ces derniers. Pour cela, nous avons créé une série de mutants par substitution ou délétion d’acides aminés de la partie N-terminale, des boucles extracellulaires et des domaines transmembranaires du récepteur. Nous avons testé leur marquage en surface cellulaire par cytométrie en flux, leur liaison des deux ligands par expériences de radio-liaison, et leur capacité à recruter la β-arrestine en réponse aux ligands par essais BRET. Les résultats obtenus ont permis d’identifier des résidus importants à l’interaction des systèmes CXCR7/SDF-1 et CXCR7-ITAC et suggèrent des modes de liaison à CXCR7 différents entre ITAC et SDF-1. Tout comme la liaison d’ITAC à son autre récepteur CXCR3, sa liaison à CXCR7 suivrait le mode conventionnel de liaison en deux étapes des récepteurs de chimiokines. Cependant, la liaison de SDF-1 à CXCR7 suivrait un autre mode de liaison, contrairement à sa liaison à son autre récepteur, CXCR4. / Chemokines are small secreted proteins whose major function is to stimulate the migration of immune cells to different organs and tissues. They are often involved in inflammatory and auto-immune diseases as well as cancers. Thus, chemokines and their G proteins Coupled Receptors (GPCR) are the pharmacological target of multiples molecules, currently tested in clinical trials. The model of our study is the atypical receptor CXCR7. This receptor is called atypical because it doesn’t signalize through the classical G protein pathway but rather signalizes through the β-arrestin pathway. CXCR7 is involved in many cancers, promotes metastatic progression and is a co-receptor for human immunodeficiency virus (HIV). However, there are, to date, no data concerning its binding mode to its ligands CXCL11/ITAC and CXCL12/SDF-1. We think that this information is crucial for the efficient development of agonists and antagonists and thus decided to identify the residus that are important for the binding of the two ligands of CXCR7 to the receptor and its subsequent activation. For that, we created a set of mutants by substitution or deletion of amino acids of the N-terminus, extracellular loops and transmembrane domains of the receptor. We then tested their surface expression by antibody staining and flow cytometry, their binding of the two ligands by binding assays and their capability to recruit β-arrestin in response to the ligands by BRET assays. The results obtained allowed us to identify important residues for the interaction of the systems CXCR7/SDF-1 and CXCR7/ITAC. They also suggest different binding modes of the chemokines ITAC and SDF-1 to CXCR7. Just like the binding of ITAC to its other receptor CXCR3, the binding mode of ITAC to CXCR7 follows the conventional two steps binding model of chemokine receptors. However, the binding of SDF-1 to CXCR7 follows another binding mode than the classical two step model, unlike its binding to its other receptor CXCR4.

Suite à leur activation par des cellules présentatrices d’antigène, les lymphocytes T expriment le récepteur de chimiokines CXCR3 et peuvent alors infiltrer les tissus enflammés. Pour ce faire, CXCR3 permet aux cellules de détecter et de chimiotaxer vers des gradients de concentration croissants des chimiokines CXCL11 et CXCL10 retrouvées dans le milieu extracellulaire. Les gradients de chimiokines doivent être régulés afin d’assurer une réponse immunitaire adéquate. Toutefois, comment ces gradients sont formés, maintenus et éliminés n’est pas très bien compris. Il a été montré que certaines cellules participent à la régulation des gradients de chimiokines dans d’autres contextes. Pour ce faire, elles expriment des récepteurs de chimiokines spécialisés qui séquestrent les chimiokines, réduisant leur niveau dans le milieu extracellulaire. Ces récepteurs sont classés comme étant atypiques et n’induisent pas la chimiotaxie. Dans ce travail, nous proposons un mécanisme de régulation pour les chimiokines CXCL11 et CXCL10 qui est en ligne avec un mécanisme d’auto-génération de gradients de chimiokines par les lymphocytes T activés. / During the immune response, T lymphocytes are activated in lymph nodes by antigen-presenting cells. Following activation, T cells up-regulate the chemokine receptor CXCR3, which allows them to infiltrate inflamed tissues by a migratory process named chemotaxis. To do so, CXCR3 allows the cells to detect concentration gradients of the chemokines CXCL11 and CXCL10 which are secreted locally, at the inflammation site. The chemokine gradient represents a directional cue, indicating to cells in which direction to migrate. Chemokine gradients must be regulated to assure an appropriate immune response. However, how these gradients are formed, maintained and eliminated is not well understood. It has been shown in other contexts that cells participate in gradient shaping. To do so, they express specialised chemokine receptors who scavenge chemokines, reducing chemokine levels in the environment. These receptors are considered atypical because they do not induce chemotaxis. In this work, we provide evidence for the regulation of the chemokines CXCL11 and CXCL10 that is in line with self-generation of chemokine gradients by chemotaxing activated T cells.