La plus grande sensibilité des souris TLR9-/- a révélé le rôle de ce récepteur dans l'infection par Leishmania major. Les cellules dendritiques (DCs) sont activées de manière TLR9-dépendante par l'ADN du L. major et d'autres Trypanosomatidae et non par l'ADN de vertébré. La nature de l'ADN capable d'activer le TLR9 reste controversée quant à la séquence/charpente de l'ADN et l'implication de cofacteurs se liant avec le TLR9 ou l'ADN. Nous avons démontré l'importance de la séquence d'ADN. Contrairement aux génomes de parasites, l'ADN de vertébré présente une contre-sélection des motifs activateurs du TLR9 au profit des motifs inhibiteurs. De plus, l'activation du TLR9 par l'ADN du parasite est augmentée en présence de la protéine HMGB1, qui se fixe mieux sur l'ADN de parasite que de vertébré. La maturation du TLR9 requiert un clivage protéolytique par des protéases endosomales, dont les cathepsines (Cat) B, S, L et l'asparagine endopeptidase (AEP) qui interviennent différemment dans les macrophages et les DCs. Après infection par L. major, nous avons montré que les souris AEP-/-, CatS-/- et CatL-/- ont une pathologie identique aux souris WT, ce qui peut être dû à la redondance de leur fonction. Etonnamment, les souris CatB-/- sont plus résistantes. Leurs lésions et la charge parasitaire dans les ganglions se résolvent plus rapidement, reflétant une réponse immune plus précoce et un contrôle plus rapide de la réaction inflammatoire.En conclusion, ces résultats contribuent à une meilleure compréhension des mécanismes permettant au TLR9 de discriminer entre l'ADN de pathogène et de vertébré et soulèvent le rôle non protecteur de la cathepsine B dans l'infection par L. major. / As TLR9-deficient mice are more sensitive to Leishmania major infection, we have shown previously that TLR9 receptor mediates this parasite infection. Dendritic cells (DCs) are activated by L. major and other Trypanosomatidae DNA and not by vertebrate DNA. There is an ongoing controversy concerning the properties of DNA required for TLR9 activation, regarding the DNA sequence or backbone or the implication of a cofactor interacting with TLR9 or DNA. We have established the importance of DNA sequences. In contrast to parasite genome, vertebrate genome have counter-selected stimulatory sequences and over-represented inhibitory motifs for TLR9. In addition, host proteins contribute to TLR9-dependent DC activation. HMGB1 enhances TLR9 activation only in the presence of L. major DNA and, surprisingly, HMGB1 binds more abundantly L. major than vertebrate DNA. TLR9 activation requires a proteolytic cleavage by endosomal proteases, as cathepsins (Cat) B, S and L and asparagine endopeptidase (AEP) that have a differential activity in macrophages and DCs. After L. major infection, we have showed that AEP-/-, CatS-/- and CatL-/- mice have a similar pathology than WT mice, likely due to their functionnally redundant activites. In contrast, CatB-/- mice are more resistant to the infection. Their lesion sizes and the parasite burdens in lymph nodes are significantly decreased, reflecting an earlier immune response and a more rapid control of the inflammatory response. In conclusion, our results bring further insights into how TLR9 discriminates between Trypanosomatidae and vertebrate DNA and reveal a non protective role of cathepsin B in L. major infection.

Synthetic CpG containing oligodeoxynucleotides (ODNs) are recognized by Toll like Receptor 9 (TLR9) and induce a strong pro-inflamatory immune response. To date, four different CpG ODN classes have been described. K-Class ODNs (also known as B-ODN) are potent B cell activators and stimulate TNF

Lung dysfunction due to exposure to air in high intensity livestock barn operations is a common problem for workers in these facilities. Exposure to this air has been linked to disorders such as chronic bronchitis, occupational asthma, organic dust toxic syndrome, and chronic cough and phlegm. These symptoms have been linked to higher levels of endotoxins in air in chicken and swine barns. However, there are many other toxic molecules such as bacterial DNA and gases capable of inducing respiratory inflammation. Bacterial molecules are recognized through highly conserved pattern recognition molecules called Toll-like receptors (TLR). While lipopolysaccharides are recognized by TLR4, bacterial unmethylated DNA binds to and signals through TLR9. As a prelude to understanding the biology of TLR9 in lung inflammation, it is important to precisely clarify their in situ expression in the lung. I determined expression of TLR9 in intact lungs from cattle, pigs, dogs, horses, mice, and humans. Two samples from normal lungs of cattle, pigs, dogs, three from horses, and two from inflamed calf lungs were tested. Five normal mouse and three normal human lungs were similarly tested as well as 5 human lungs with diagnosis of asthma. The expression was determined with multiple methods such as Western blots, immunohistology, immunogold electron microscopy and in situ hybridization. Lungs from all the species showed TLR9 expression in the bronchial epithelium, vascular endothelium, alveolar septa, alveolar macrophages, and type-II alveolar epithelial cells. Immuno-electron microscopy detected TLR9 on the plasma membrane, cytoplasm and the nucleus of various cells including macrophages. In situ hybridization demonstrated TLR9 mRNA in the bronchial epithelium, vascular endothelium, alveolar septa, alveolar macrophages, and type-II alveolar epithelial cells of mouse and human. Asthmatic human lungs showed many more inflammatory cells expressing TLR9 compared to healthy lungs. In cattle and horses, pulmonary intravascular macrophages showed robust expression of TLR9. Depletion of pulmonary intravascular macrophages in horses resulted in significant reduction in total TLR9 mRNA in the lungs. Having determined that TLR9 expression is similarly expressed on many lung cell types in mice and humans, I determined the role of TLR9 in barn air induced lung inflammation by exposing TLR9-/- and wild-type mice (6 per group) to single or multiple days (5 and 20) in a chicken barn. Each exposure was of 8 hours/day duration. The TLR9-/- mice exposed 5 and 20 times showed significant reductions in TNF-alpha and IFN-gamma expression in lung lavages as well as cellular changes consistent with reduced lung inflammation such as reductions in the number of lung neutrophils. This suggests that barn dust DNA, acting through TLR9, contributes to lung inflammation seen in response to exposure to chicken barn air. These fundamental data advance our knowledge on the cell-specific expression of TLR9 across a range of species including the humans and demonstrate that TLR9-/- partially regulates lung inflammation induced following exposure to chicken barn air.

Lung dysfunction due to exposure to air in high intensity livestock barn operations is a common problem for workers in these facilities. Exposure to this air has been linked to disorders such as chronic bronchitis, occupational asthma, organic dust toxic syndrome, and chronic cough and phlegm. These symptoms have been linked to higher levels of endotoxins in air in chicken and swine barns. However, there are many other toxic molecules such as bacterial DNA and gases capable of inducing respiratory inflammation. Bacterial molecules are recognized through highly conserved pattern recognition molecules called Toll-like receptors (TLR). While lipopolysaccharides are recognized by TLR4, bacterial unmethylated DNA binds to and signals through TLR9. As a prelude to understanding the biology of TLR9 in lung inflammation, it is important to precisely clarify their in situ expression in the lung. I determined expression of TLR9 in intact lungs from cattle, pigs, dogs, horses, mice, and humans. Two samples from normal lungs of cattle, pigs, dogs, three from horses, and two from inflamed calf lungs were tested. Five normal mouse and three normal human lungs were similarly tested as well as 5 human lungs with diagnosis of asthma. The expression was determined with multiple methods such as Western blots, immunohistology, immunogold electron microscopy and in situ hybridization. Lungs from all the species showed TLR9 expression in the bronchial epithelium, vascular endothelium, alveolar septa, alveolar macrophages, and type-II alveolar epithelial cells. Immuno-electron microscopy detected TLR9 on the plasma membrane, cytoplasm and the nucleus of various cells including macrophages. In situ hybridization demonstrated TLR9 mRNA in the bronchial epithelium, vascular endothelium, alveolar septa, alveolar macrophages, and type-II alveolar epithelial cells of mouse and human. Asthmatic human lungs showed many more inflammatory cells expressing TLR9 compared to healthy lungs. In cattle and horses, pulmonary intravascular macrophages showed robust expression of TLR9. Depletion of pulmonary intravascular macrophages in horses resulted in significant reduction in total TLR9 mRNA in the lungs. Having determined that TLR9 expression is similarly expressed on many lung cell types in mice and humans, I determined the role of TLR9 in barn air induced lung inflammation by exposing TLR9-/- and wild-type mice (6 per group) to single or multiple days (5 and 20) in a chicken barn. Each exposure was of 8 hours/day duration. The TLR9-/- mice exposed 5 and 20 times showed significant reductions in TNF-alpha and IFN-gamma expression in lung lavages as well as cellular changes consistent with reduced lung inflammation such as reductions in the number of lung neutrophils. This suggests that barn dust DNA, acting through TLR9, contributes to lung inflammation seen in response to exposure to chicken barn air. These fundamental data advance our knowledge on the cell-specific expression of TLR9 across a range of species including the humans and demonstrate that TLR9-/- partially regulates lung inflammation induced following exposure to chicken barn air.

Toll-like receptors (TLRs) recognize microbial components as danger signals and induce immune responses. TLR’s are expressed in many tissues of the host that are involved in immune responses including the intestines where they are abundantly expressed. This situation presents a challenge in the gastrointestinal tract which is constantly exposed to a wide variety of commensal organisms. Therefore, innate immune recognition in the intestine must be tightly regulated to prevent unwanted inflammation against harmless commensal micro-organisms and yet allow for the induction of protective immunity to invading pathogens. A dysregulation of this balance can result in intestinal inflammation. Peyer’s patches (PP) are the primary site for the induction of immune responses in the intestine and abundantly express TLRs. It is not known how PP regulate microbial signals from commensal bacteria and yet mount vigorous immune responses against dangerous pathogens. CpG DNA, an agonist for TLR9, can strongly activate immune cells in blood, lymph nodes and spleen. However, CpG very poorly activates immune cells from Peyer’s patches, although these cells express TLR9 [1, 2]. Understanding how TLR responses are regulated in PP cells will unveil important information on how immune responses are regulated in the intestine. Investigations from our laboratory have revealed a B cell population (CD5-CD11c-CD21+) in PP that spontaneously secrete high levels of IL-10 which in turn down regulates TLR9 induced IFN and IL-12 production. These IL-10-secreting PP B cells represent a novel subset of the recently proposed regulatory B cells (Bregs) in the intestine [1, 3]. Bregs may have a role in maintaining tolerance to commensal bacteria thereby achieving intestinal homeostasis. The overall goal of the work described in this thesis was to improve our understanding of the immunobiology of Bregs. We performed several experiments to achieve this goal. First, we studied the development of regulatory B cells in lambs of different ages. Jejunal PP were collected from 3-4 month old, neonatal and fetal lambs and the production of IL-10 (the immunoregulatory cytokine secreted by Bregs) was assayed. We found that IL-10 was secreted by CD21+ B cells from the PP in all the three age groups, confirming that Bregs develop prior to birth. We then wondered whether our CD21+ B cells might be contaminated with other cells or activated when using MACS to enrich B cells. To address this issue, we prepared very highly purified CD21+ B cell population using high speed cell sorting to negatively enrich for B cells. We also sorted DCs and assayed IL-10 production in both cell populations. Only the PP B cells spontaneously secreted IL-10. In contrast, dendritic cells, T cells, macrophages, neutrophils and NK cells did not secrete detectable IL-10. Since B cells exist as regulatory and effector populations in mice, we wondered whether an effector B cell population also existed in ovine PP that secreted the pro-inflammatory cytokines IFN-, IFN- and IL-12. Therefore, ovine PP B cells were fractionated into CD72+CD21+and CD72+CD21- subpopulations to assess their capacity to secrete pro-inflammatory cytokines. Interestingly, the CD72+CD21- B cell population secreted the cytokines IFN-, IFN- and IL-12 suggesting there was an effector population. We then surveyed for Bregs in different mucosal and peripheral tissues in sheep. We observed the Bregs frequency varied among the different lymphoid tissues. Finally, we investigated whether Bregs were present in PP of other ruminant species. We identified Bregs exist in PP of neonatal calves. In conclusion, our investigations reveal that ovine Bregs develop in utero prior to antigen exposure, and are present in a variety of mucosal and peripheral tissues. We also report the novel observation that two distinct B cell sub-populations are present in ovine jejunal PP’s: Regulatory and effector B cells.

Background. Toll-like receptors (TLRs) play a role in the pathophysiology of sepsis and multiple organ failure. This study examined the effect of CpG oligodeoxynucleotide (CpG-ODN), the TLR9 ligand, on polymicrobial sepsis-induced cardiac dysfunction.Methods. Male C57BL/6 mice were treated with CpG-ODN, control CpG-ODN (control-ODN), or inhibitory CpG-ODN (iCpG-ODN) 1 hour prior to cecal ligation and puncture (CLP)-induced sepsis. Mice that underwent sham surgery served as sham controls. Cardiac function was examined by echocardiography before and 6 hours after CLP.Results. Cardiac function was significantly decreased 6 hours after CLP. CpG-ODN prevented CLP-induced cardiac dysfunction, as evidenced by maintenance of the ejection fraction and fractional shortening. Control-ODN or iCpG-ODN did not alter CLP-induced cardiac dysfunction. CpG-ODN significantly attenuated CLP-induced myocardial apoptosis and increased myocardial Akt and extracellular-signal-related kinase (ERK) phosphorylation levels following CLP. In vitro experiments demonstrated that CpG-ODN promotes an association between TLR9 and Ras, resulting in Akt and ERK phosphorylation. Inhibition of phosphoinositide 3-kinase (PI3K) by Ly294002 or inhibition of ERK by U0126 in vivo abolished CpG-ODN attenuation of CLP-induced cardiac dysfunction.Conclusions. CpG-ODN prevents CLP-induced cardiac dysfunction, in part through activation of PI3K/Akt and ERK signaling. Modulation of TLR9 could be an effective approach for treatment of cardiovascular dysfunction in patients with sepsis or septic shock.

Background. Toll-like receptors (TLRs) play a role in the pathophysiology of sepsis and multiple organ failure. This study examined the effect of CpG oligodeoxynucleotide (CpG-ODN), the TLR9 ligand, on polymicrobial sepsis-induced cardiac dysfunction.Methods. Male C57BL/6 mice were treated with CpG-ODN, control CpG-ODN (control-ODN), or inhibitory CpG-ODN (iCpG-ODN) 1 hour prior to cecal ligation and puncture (CLP)-induced sepsis. Mice that underwent sham surgery served as sham controls. Cardiac function was examined by echocardiography before and 6 hours after CLP.Results. Cardiac function was significantly decreased 6 hours after CLP. CpG-ODN prevented CLP-induced cardiac dysfunction, as evidenced by maintenance of the ejection fraction and fractional shortening. Control-ODN or iCpG-ODN did not alter CLP-induced cardiac dysfunction. CpG-ODN significantly attenuated CLP-induced myocardial apoptosis and increased myocardial Akt and extracellular-signal-related kinase (ERK) phosphorylation levels following CLP. In vitro experiments demonstrated that CpG-ODN promotes an association between TLR9 and Ras, resulting in Akt and ERK phosphorylation. Inhibition of phosphoinositide 3-kinase (PI3K) by Ly294002 or inhibition of ERK by U0126 in vivo abolished CpG-ODN attenuation of CLP-induced cardiac dysfunction.Conclusions. CpG-ODN prevents CLP-induced cardiac dysfunction, in part through activation of PI3K/Akt and ERK signaling. Modulation of TLR9 could be an effective approach for treatment of cardiovascular dysfunction in patients with sepsis or septic shock.

Depending on the duration and severity, psychological tension and physical stress can enhance or suppress the immune system in both humans and animals. Although it has been established that chronic stress exerts a significant suppressive effect on immune function, the mechanisms by which affects immune responses remain elusive. By employing an in vivo murine system, we revealed that TGF-β1/Smad2/3/Foxp3 axis was remarkably activated following chronic stress. Furthermore, TLR9 and p38 MAPK played a critical role in the activation of TGF-β1/Smad2/3/Foxp3 signaling cascade. Moreover, inhibition of TGF-β1/Smad2/3/Foxp3 or p38 significantly attenuated chronic stress-induced lymphocyte apoptosis and apoptosis-related proteins, as well as the differentiation of T regulatory cells in spleen. Interestingly, disequilibrium of pro-inflammatory and anti-inflammatory cytokines balance caused by chronic stress was also rescued by blocking TGF-β1/Smad2/3/Foxp3 axis. These findings yield insight into a novel mechanism by which chronic stress modulates immune functions and identifies new targets for the development of novel anti-immune suppressant medications.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24139号 / 医博第4879号 / 新制||医||1060(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 森信 暁雄, 教授 上野 英樹, 教授 金子 新 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Abstract Background and aims: Stage and grade are the most widely used prognostic parameters for renal cell carcinoma (RCC). The clinical course of this disease is not, however, always predictable by traditional prognostic factors. In the era of new molecular targeted therapies a more accurate prognostication of RCC patient survival is important for the individualization of treatment and follow-up of patients. Despite exhaustive research there are still no prognostic biomarkers for RCC in clinical practice. In order to find novel prognostic tissue markers for RCC, we examined the expression of 14 biomarkers involved in carcinogenesis and clarified their prognostic significance in RCC. Material and methods: Out of 189 consecutive patients who underwent surgery for kidney cancer at Oulu University Hospital in the 1990s, 152 patients with histologically verified RCC were included in this study. The stage distribution was 70 (46%), 12 (8%), 51 (34%) and 19 (12%) patients with stages I-IV, respectively. The majority of the tumours (83 tumours, 55%) were nuclear grade II and 5 (3%), 40 (27%) and 22 (15%) of the tumours were grades I, III and IV, respectively. Clinical and follow-up data were obtained from patient records, the Finnish Cancer Registry and on demand from the Population Register Centre of Finland. The biomarkers studied included markers of the oxidative and neuroendocrine systems as well as proteins related to cell adhesion and migration, invasion, metastasis, inflammation and immune responses. The expression of various biomarkers was characterized via immunohistochemical tests of archival tumour material. The staining intensity was compared to clinicopathological parameters and patient RCC-specific survival. Results: The 5-year RCC-specific survival was 77%. The expression of Toll-like receptor 9 (TLR9) was an independent marker of favourable RCC-specific survival whereas cytoplasmic myosin VI expression was found to be an independent prognostic factor of poor RCC-specific survival. Cell culture experiments showed how cyclooxygenase-2 (COX-2) expression is regulated by HuR in RCC. HuR and COX-2 immunoexpression were also related to decreased RCC-specific survival. Immunostaining of Keap1 was associated with advanced RCC and a marker of a poorer RCC-specific prognosis. The expression of different neuroendocrine markers was evaluated but we could not establish any prognostic value for them. Conclusions: In particular, TLR9, HuR and myosin VI can be regarded as promising novel prognostic biomarkers in RCC. Stage, however, is the most important single prognostic factor for RCC. / Tiivistelmä Munuaissyöpä on vuosikymmenten ajan jatkuvasti yleistynyt. Vaikka se diagnosoidaan nykyisin useimmiten sattumalöydöksenä vatsan alueen kuvantamistutkimuksissa ja hoitomenetelmät ovat viime vuosikymmenten aikana kehittyneet, munuaissyöpäkuolleisuus ei ole laskenut. Munuaissyövän ennusteen määrittäminen voi olla haasteellista. Perinteiset ennustetekijät, levinneisyys ja erilaistumisaste, eivät riitä selittämään kaikkien potilaiden taudinkulkua, eikä munuaissyövälle vielä ole kliinisessä käytössä ennusteellista merkkiainetta. Munuaissyöpähoitojen kehittyessä taudinkulun ennustaminen on yhä tärkeämpää, jotta potilaiden hoito ja seuranta voidaan yksilöidä. Tämän väitöskirjatyön tarkoituksena oli etsiä uusia ennusteellisia kudosmerkkiaineita munuaissyöpäkasvaimille. Väitöskirjatutkimus perustuu 1990-luvulla Oulun yliopistollisessa sairaalassa leikatun 152 munuaissyöpäpotilaan aineistoon. Lähes puolet aineiston kasvaimista edusti levinneisyysluokkaa I, ja yli puolet munuaissyöpäkasvaimista oli hyvin erilaistuneita (tumagradus I ja II). Tutkimuspotilaista kerättiin kattavat seurantatiedot. Leikkauksessa poistettujen munuaissyöpäkasvainten arkistomateriaalista tutkittiin eri merkkiaineiden ilmenemistä. Tutkitut merkkiaineet käsittivät oksidatiivisen ja neuroendokriinisen järjestelmän merkkiaineita sekä valkuaisaineita, jotka liittyvät keskeisiin syövän ominaisuuksiin, kuten solujen välisiin liitoksiin ja solujen liikkumiseen sekä etäpesäkkeiden syntymiseen. Lisäksi tutkittiin merkkiaineita, jotka liittyvät tulehdusreaktioihin ja immuunipuolustukseen. Väitöskirjatutkimus paljasti useita uusia kudosmerkkiaineita, joiden ilmeneminen munuaissyöpäkasvaimessa on yhteydessä potilaan ennusteeseen. Näistä merkittävimpiä ovat myosiini VI, joka liittyy syöpäkasvainten metastasointiin, sekä immuunipuolustuksessa vaikuttava Tollin kaltainen reseptori 9 (Toll-like receptor 9, TLR9). Molemmat merkkiaineet osoittautuivat itsenäisiksi ennustetekijöiksi munuaissyövässä. Muita ennusteeseen vaikuttavia merkkiaineita ovat tutkimuksen mukaan oksidatiivista stressiä aistiva Keap1 sekä immunologisiin reaktioihin liittyvä syklo-oksigenaasi 2 (COX-2) ja sen ilmenemistä säätelevä HuR.