Background: Uterine fibriod (UF) or leiomyoma is the most frequent benign tumour upon lower genital tract and represents the most frequent indication for hysterectomy. The aetiology remains still unknown. The genetic factors contributing for the development of UF are being intensively investigated. The aim of our study was to look for possible genetic markers which could be used as prognostic tools for evaluation of an increased risk for development of UF. Methods: The study group enrolled 102 patients diagnosed with UF and 145 healthy controls. Ultrasonographic examination of the pelvis was performed and a single blood sample was taken in all women. Histological verification followed the surgery in the patient group. The principal of the cytokine gene polymorphisms detection is based on PCR reaction with sequence-specific primers. Results: A large spectrum of Th1/Th2 cytokine gene polymorphisms in patients with uterine fibroid was compared with control group. The frequencies of the majority of tested cytokine gene SNP in the patient cohort were not statistically different from the cytokine SNP in the control group. However, an intriguing association between polymorphisms of the IL-4 gene promotor at positions -590 C/T and -33 C/T, and the risk of leiomyoma was observed. The CC genotype of IL-4 at position...

Die Erkrankungen des atopischen Formenkreises sind durch eine hypererge Immunreaktion auf harmlose Umwelt-Antigene gekennzeichnet und umfassen u.a. die klinischen Bilder der allergischen Rhinokonjunktivitis, des allergischen Asthmas und der atopischen Dermatitis. Die Pathogenese atopischer Krankheiten ist zwar noch nicht hinreichend aufgeklärt, aber man schreibt der Regulation des IgE bei der Entstehung und Erhaltung dieser Krankheiten eine zentrale Rolle zu. Auf der Suche nach potentiellen anti-allergischen Therapeutika wurde in dieser Arbeit erstmalig der Einfluß von 1alpha,25-Dihydroxyvitamin-D3 (VD) und dem niedrigkalzämischen Derivat EB1089 in vitro auf die IgE-Produktion humaner B-Zellen gesunder Spender untersucht, die zuvor mit anti-CD40+IL-4 stimuliert wurden, wodurch der Isotypenklassenwechsel nativer B-Zellen nach IgE resultierte. Das Steroidhormon VD ist für die Kalziumhämostase essentiell, jedoch sind die immunregulatorischen Funktionen des VD noch relativ unerforscht. Die Experimente dieser Arbeit zeigen eine starke, dosisabhängige Hemmung der IgE-Produktion in diesem Modell, die spezifisch von VD und dem Derivat EB1089 vermittelt wird. Auch bei PBMC atopischer Spender konnte die basale IgE-Produktion mit VD dosisabhängig signifikant gehemmt werden. Eine mögliche Proliferationshemmung, die diese Wirkung erklären könnte, konnte ausgeschlossen werden. Die Messung der Produktion der anderen Immunglobulinklassen (Ig) IgA, IgG und IgM bestätigte, daß die Zellviabilität und die Ig-Produktion außer der von IgE durch VD und EB1089 in diesem Modell nur wenig beeinflußt wird. Diese Beobachtungen weisen auf eine selektive, spezifische Inhibition der IgE-Synthese durch das Molekül aus der Familie der Steroidhormone hin. Auf der Suche nach möglichen indirekten Mechanismen der IgE-Regulation, die die VD-vermittelte Inhibition der IgE-Synthese erklären könnten, wurde der Einfluß von VD auf die Expression der kostimulatorischen Faktoren der IgE-Produktion CD23 (Fc(-RII), CD54 (ICAM-1) und CD86 (B7.2) sowie auf die Sekretion der Zytokine, IL-6, sCD23 und IFN-gamma untersucht. Die Ergebnisse dieser Arbeit deuteten jedoch nicht auf eine Beteiligung der genannten Faktoren an der Regulation der VD-vermittelten Hemmung der IgE-Produktion hin. Diese in vitro Arbeit stellt einen wichtigen Hinweis auf eine potentielle Nutzung von VD in der anti-allergischen Therapie dar, jedoch deutet die beobachtete Hemmung der IgE-Synthese von PBMC atopischer Spender darauf hin, daß auch in vivo die IgE-Synthese gehemmt werden kann. Es sind daher weitere Studien des Signalweges des VD auf molekularer Ebene, sowie in vivo Versuche notwendig, um zu klären, ob VD selbst oder ein möglichst nicht-kalzitropes VD-Derivat mit ausgeprägten immunologischen Eigenschaften für die anti-allergische Therapie geeignet ist. / Atopic diseases are characterized by a hyperergic immunoreaction towards harmless environmental antigens, expressed by the clinical phenotype of allergic rhinitis, allergic asthma and atopic dermatitis. Even though the pathogenesis of atopic diseases are not completely clear, the regulation of IgE plays a crucial role in the development and maintenance of these disorders. In search of new potentially anti-allergic drugs, the effect of 1alpha,25-Dihydroxyvitamin-D3 (VD) and the low-calcemic synthetic analogue EB1089 was determined in anti-CD40+Il-4-mediated IgE-production by B-cells of healthy donors in vitro. The essential role of VD in calcium hemostasis is well understood, but the immunoregulatory functions of the hormone are still quite unclear. The present data show for the first time, that VD and the analogue EB1089 mediate a strong, dose-dependent inhibition of IgE-synthesis. In PBMC of atopic donors, the baseline IgE-production was also significantly inhibited by VD. Anti-CD40+IL-4-mediated proliferation of B-cells in the presence of VD was only modestly modulated, as well as the production of the other immunoglobulin-isotypes IgA, IgG and IgM. Taken together these data suggest a specific and selective inhibition of IgE-synthesis by VD and EB1089. In order to explore possible mechanisms of VD induced IgE-inibition, the expression of CD23 (Fc(-RII), CD54 (ICAM-1) and CD86 (B7.2) on anti-CD+IL-4 stimulated B-cells was determined as well as IL-6, sCD23 und IFN-gamma. However, none of these molecules were significantly modulated in the presence of VD. Taken together, these in vitro experiments suggest a potential role of VD as a new anti-allergic drug, which is underlined by the observed inhibition of IgE-production by PBMC in allergic donors.

Plusieurs conditions allergiques, dont certains phénotypes d’asthme et le souffle équin, sont caractérisées par une infiltration neutrophilique. L’interleukine-4 (IL-4), une cytokine clé de la réponse allergique, pourrait contribuer au recrutement de ces cellules inflammatoires lors de ces pathologies. L’objectif de cette étude était d’évaluer si l’administration sous-cutanée d’IL-4 chez des chevaux sains favorise une réponse neutrophilique locale. Trois études ont été réalisées pour 1) évaluer l’effet de concentrations cytokiniques différentes (10 ng, 250 ng et 500 ng) et 2) évaluer l’effet de la durée d'incubation (3 h, 6 h, 12 h, 48 h et 7 jours) sur le recrutement des neutrophiles chez 18 chevaux sains. Le matrigel, une matrice protéique solubilisée, a servi de véhicule pour l’administration des cytokines. Un grade histologique semi-quantitatif a été élaboré pour évaluer la neutrophilie tissulaire pour toutes les études, mais nous avons, en outre, ajouté une analyse par cytométrie de flux dans la troisième étude pour valider les grades histologiques. Nos résultats démontrent que 1) l'IL-4 ne parvient pas à induire une migration neutrophilique significative dans les tissus sous-cutanés de chevaux sains ; 2) la cytométrie de flux s’est révélée être une méthode plus fiable pour estimer la migration des neutrophiles en comparaison avec l'analyse moins sensible des scores histologiques. Nous avons de plus observé que 3) le matrigel entraîne une réaction inflammatoire potentiellement de nature immunogène chez les chevaux. Cette étude est la première incorporant le matrigel et IL-4 dans un protocole in vivo impliquant des chevaux. Ces données suggèrent que l’IL-4 seule ne permet pas le recrutement sous-cutané de neutrophiles chez des chevaux sains. / Many allergic conditions, including asthma and equine heaves, are characterized by a neutrophilic inflammation. Interleukin-4 (IL-4) is a cytokine that plays a key role in allergic responses that may contribute to the recruitment of inflammatory cells in these diseases. The objective of this study was to assess the neutrophilic response following a subcutaneous administration of IL-4 in healthy horses. Three studies were conducted to evaluate the effects 1) of different cytokine concentrations (10 ng, 250 ng and 500 ng) and 2) incubation times (3 h, 6 h, 12 h, 48 h and 7 days) on neutrophil recruitment in 18 healthy horses. A semi-quantitative histological score was developed to assess tissue neutrophilia for all three studies. Flow cytometry analysis was also performed in study 3 in order to validate the histological scoring method. Our results demonstrate that 1) IL-4 fails to induce significant neutrophilic migration and 2) flow cytometry has proved to be a more reliable method in estimating neutrophil migration when compared to histological scoring, which lacked sensitivity. We also observed that 3) matrigel causes an inflammatory reaction in horses, possibly of immunogenic nature. These are the first studies incorporating matrigel and IL-4 in an in vivo protocol involving horses. Our data suggests that IL-4 alone does not induce neutrophil recruitment in the skin of healthy horses.

<p>This study investigated the associations between IL-4 -590 C/T and IL-10 -1087 A/G polymorphisms and malariometric indexes in the Fulani and the Dogon ethnic groups living in sympatry in Mali and differing in susceptibility to malaria. The correlations between antibodies level and parasitological data as well as splenomegaly were assessed. The impact of IL-4 -590 variants on the levels of the studied antibodies was also studied. </p><p>The allele and genotype frequencies of both studied SNPs differed significantly between the two groups. The Fulani IL-4 T allele carriers had a significantly higher infection prevalence compared with those carrying the CC genotype. No correlation between anti-malarial antibody levels and parasite prevalence was seen in any of the communities. In the Fulani, the increase in total IgE levels was related to the presence of infection. Malaria-specific IgG4 levels were negatively correlated to the number of clones within the Fulani. The Fulani IL-4 T allele carriers had higher total and malaria-specific IgE levels, compared to the CC genotype carriers. These results suggest that the amount of antibodies may not be the key element in the protection against malaria. IgG4 might be involved in protection against malaria. The impact of IL-4 -590 variants on the antibody levels may be affected by other genetic/epigenetic/epistatic or environmental factors. </p><p>In the study in Senegal, multiplicity of infection (MOI) increased after the transmission season in all subjects, except in α-thalassaemic and in G6PD-mutated children, suggesting that α-thalassaemia may protect against infection by certain parasite strains. G6PD-mutated individuals may resist against increase in MOI after the transmission season due to rapid clearance of infection at an early stage. HbAs and the ABO system do not affect MOI in asymptomatic individuals. MOI was positively correlated to parasitemia, and did not vary over age (in the range of 2 to 10 years). No relation between MOI and clinical attack was noted. </p>

This study investigated the associations between IL-4 -590 C/T and IL-10 -1087 A/G polymorphisms and malariometric indexes in the Fulani and the Dogon ethnic groups living in sympatry in Mali and differing in susceptibility to malaria. The correlations between antibodies level and parasitological data as well as splenomegaly were assessed. The impact of IL-4 -590 variants on the levels of the studied antibodies was also studied. The allele and genotype frequencies of both studied SNPs differed significantly between the two groups. The Fulani IL-4 T allele carriers had a significantly higher infection prevalence compared with those carrying the CC genotype. No correlation between anti-malarial antibody levels and parasite prevalence was seen in any of the communities. In the Fulani, the increase in total IgE levels was related to the presence of infection. Malaria-specific IgG4 levels were negatively correlated to the number of clones within the Fulani. The Fulani IL-4 T allele carriers had higher total and malaria-specific IgE levels, compared to the CC genotype carriers. These results suggest that the amount of antibodies may not be the key element in the protection against malaria. IgG4 might be involved in protection against malaria. The impact of IL-4 -590 variants on the antibody levels may be affected by other genetic/epigenetic/epistatic or environmental factors. In the study in Senegal, multiplicity of infection (MOI) increased after the transmission season in all subjects, except in α-thalassaemic and in G6PD-mutated children, suggesting that α-thalassaemia may protect against infection by certain parasite strains. G6PD-mutated individuals may resist against increase in MOI after the transmission season due to rapid clearance of infection at an early stage. HbAs and the ABO system do not affect MOI in asymptomatic individuals. MOI was positively correlated to parasitemia, and did not vary over age (in the range of 2 to 10 years). No relation between MOI and clinical attack was noted.

Thy-1 is a glycosylphosphatidylinositol-anchored protein that is expressed on murine T lymphocytes and is involved in T cell-mediated immune responses. In the presence of costimulatory signals, monoclonal antibody (mAb)-induced signaling through Thy-1 is associated with hallmarks of T cell activation, including IL-2 production and T cell proliferation. Thy-1-induced signaling promotes cytotoxic effector molecule expression, but is unable to trigger delivery of the lethal hit to target cells, suggesting that Thy-1 provides an incomplete T cell receptor (TcR)-like signal. However, the effect of Thy-1 signaling on cytokine production and the development of T helper (Th) cell phenotypes (Th1, Th2, Th17) remains unclear. The purpose of this work was to further our understanding of Thy-1-mediated signal transduction and the role that Thy-1 plays in the development of effector T cell responses. I found that, in the context of costimulatory signals, anti-Thy-1 mAb induced significantly less IL-2 production, CD25 expression and T cell proliferation than anti-TcR? mAb. Several key signaling molecules, including protein tyrosine kinases, zeta chain-associated protein-70 and extracellular signal-regulated kinase were activated with delayed kinetics during Thy-1-mediated T cell activation. The delayed signaling kinetics resulted in the delayed acquisition of cytotoxic effector function and also delayed delivery of the lethal hit to target cells. Interestingly, Thy-1-mediated signaling induced significantly more IL-17 and IL-4 synthesis and less IFN-? synthesis in comparison to TcR-mediated signaling. Moreover, Thy-1-activated CD4+ T cells produced high levels of IL-17 and IL-4 but minimal IFN? when restimulated with anti-Thy-1 mAb or anti-TcR? mAb with or without costimulatory signals. The unique ability of Thy-1 signaling to induce IL-17 production correlated with the expression of the Th17 lineage-specific transcription factor, retinoic orphan receptor gamma t. These observations show that Thy-1 signaling differs from TcR signaling in its ability to induce Th cell cytokines. Taken together, my findings show that Thy-1 signaling can provide the full TcR-like signal required for both the differentiation and triggering of Th cells and cytotoxic T lymphocytes, albeit with delayed kinetics in comparison to TcR signaling. They also suggest that Thy-1 signaling may be important in the development of Th2 and Th17 responses.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2018-02-15T12:44:59Z No. of bitstreams: 1 DanielCostaDeSouza_DISSERT.pdf: 1625998 bytes, checksum: 824c5486dd368ffb01c3d3a1163f37f1 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2018-02-19T21:26:44Z (GMT) No. of bitstreams: 1 DanielCostaDeSouza_DISSERT.pdf: 1625998 bytes, checksum: 824c5486dd368ffb01c3d3a1163f37f1 (MD5) / Made available in DSpace on 2018-02-19T21:26:44Z (GMT). No. of bitstreams: 1 DanielCostaDeSouza_DISSERT.pdf: 1625998 bytes, checksum: 824c5486dd368ffb01c3d3a1163f37f1 (MD5) Previous issue date: 2017-07-10 / Introdu??o: O excesso de gordura corporal ? associado ? inflama??o cr?nica de baixo grau e uma maior incid?ncia de doen?as infecciosas. Estudos recentes, indicam que o exerc?cio intervalado de alta intensidade (EIAI) ? uma estrat?gia eficiente em fun??o do tempo para melhorar par?metros de sa?de de indiv?duos com obesidade. No entanto, pouco ? conhecido sobre o efeito de uma sess?o de EIAI no equil?brio de citocinas pro e anti-inflamat?rias em obesos. Objetivo: O objetivo do presente estudo foi comparar uma ?nica sess?o de EIAI e exerc?cio cont?nuo de moderada intensidade (ECMI) sobre os n?veis s?ricos de interferon-y (INF-y), interleucina 4 e 6 (IL-4) (IL-6) e INF-y/IL-4 em homens com obesidade. M?todos: Dez homens com obesidade (IMC>30 kg/m?) foram submetidos a tr?s sess?es experimentais com uma semana de intervalo em ordem aleat?ria: 1) EIAI: 10 x 60 s a 90% da FCm?x alternados por 60 s de recupera??o ativa; 2) ECMI: 20 min a 70% da FCmax; 3) Controle. As coletas de sangue para analisar os n?veis s?ricos de citocinas foram realizadas nos momentos pr?, p?s e 60 min ap?s as sess?es de exerc?cio ou controle. Os dados foram apresentados em m?dia ? DP. ANOVA fatorial com medidas repetidas e post hoc de Bonferoni foi utilizado para avaliar as diferen?as entre os momentos e condi??es de exerc?cio. O n?vel de signific?ncia aceito foi de P< 0.05. Resultados: O EIAI reduziu os n?veis de INF-y imediatamente ap?s o exerc?cio (41,09 ? 14,99; P=0,032) e 60 min ap?s o exerc?cio (43,45 ? 11,76; P=0,003) em rela??o ao pr?-exerc?cio (46,64 ? 13,14), ao mesmo tempo que promoveu aumento nos n?veis de IL-4 imediatamente ap?s o exerc?cio (36,47 ? 11,09; P=0,007) em rela??o ao pr?-exerc?cio (32,04 ? 8,5). O ECMI promoveu aumento nos n?veis de INF-y imediatamente ap?s o exerc?cio (47,48 ? 8,42; P=0,025) e 60 min ap?s o exerc?cio (50,13 ? 7,99; P=0,004) em rela??o ao valor pr?-exerc?cio (44,21 ? 8,11). Ambas as condi??es de exerc?cio aumentaram os n?veis de IL-6 at? 60 min ap?s o exerc?cio (P<0.05). A raz?o INF-Y/IL-4 reduziu imediatamente ap?s (1,24 ? 0,60; P=0,002) e 60 min ap?s o exerc?cio (1,32 ? 0,53; P=0,005) em rela??o ao pr?-exerc?cio (1,58 ? 0,64) apenas para o EIAI. Conclus?o: Uma sess?o de EIAI induziu um padr?o de resposta anti-inflamat?ria e est? associado a preju?zos na resposta imune intracelular contra pat?genos. Por outro lado, uma breve sess?o de ECMI induziu altera??es no padr?o de citocinas seguindo um padr?o pr?-inflamat?rio o que pode favorecer ? melhora da resposta imune intracelular em homens com obesidade. / Introduction: The excess of body fat is associated with chronic low-grade inflammation and high rates of infectious disease. Emerging evidences indicate that high intensity interval exercise (HIIE) is a time-efficient approach to promote health in obese population. However, little is known about the influence of HIIE on pro and anti-inflammatory cytokine balance in obesity. Purpose: Our purpose was to compare the acute effects of HIIE and moderate-intensity continuous exercise (MICE) on cytokine levels, include interferon-y (INF-y), interleukin 4 and 6 (IL-4) (IL-6) levels and INF-y/IL-4 ratio in obese males. Methods: Ten obese males (BMI>30 kg/m?) were submitted into two experimental sessions with a week interval in a randomized order: 1) HIIE: 10 x 60 s at 90% of HRm?x interspersed by active recovery; 2) MICE: 20 min at 70% of HRmax;. Cytokines was analyzed before, immediately after and 60 min after-exercise. Data was presented in mean ? SD. ANOVA 2-way with repeated measures and post hoc Bonferoni was used to assess differences between moments and exercise conditions. Statistical significance was accepted at a p value of ? 0.05. Results: The HIIE results in decreased INF-Y levels immediately after-exercise (41,09 ? 4,74; P=0,032) and 60 min after-exercise (43,45 ? 3,72) (P=0,003) compared with before-exercise (46,64 ? 4,15), while an significant elevation in IL-4 levels was observed immediately after-exercise (36,47 ? 3,5) (P=0,007). The MICE results in increase of INF-Y levels after exercise (P=0,025) and 60 min post-exercise (P=0,004). Both exercise conditions results in increase of IL-6 levels after exercise (P<0.05). The INF-Y/IL-4 ratio decreases immediately after (P=0,002) and 60 min after-exercise (P=0,005) only for HIIE. Conclusion: HIIE induces imbalance in INF-Y/IL-4 ratio 60 min after exercise following an anti-inflammatory pattern. For other hand, MICE did not promote imbalance in INF-Y/IL-4 to 60 min after exercise in obese males.

Allergic asthma is an immunological disease, mediated by CD4+ Th2 cells, and its prevalence has increased over recent decades. Features of allergic asthma include airway hyperresponsiveness (AHR), airway eosinophilia, excessive airway mucus production, and increased IgE and Th2 cytokine levels. Airway remodeling with pulmonary fibrosis is noted in the progress of asthma. In this thesis, a murine model of allergic asthma was used to investigate the effect of cyclophosphamide (CY) on asthma and the involvement of regulatory T cells (Treg), and the role of Granulocyte-macrophage colony stimulating-factor (GM-CSF) in allergic asthma by using GM-CSF knockout mice. CY is a cytotoxic agent, which paradoxically augments several immune responses. The first part of this thesis was aimed to study the effects of CY in a murine model of allergic airway inflammation. BALB/c mice were immunized with ovalbumin (OVA) on days 0 and 14, and challenged with aerosolized OVA from days 21 to 27. Some mice additionally received CY on days -2 and 12. In the CY-treated animals, pronounced worsening of inflammatory features was noted, including increases in eosinophil infiltration, epithelial thickness, mucus occlusion and eosinophil numbers in bronchoalveolar lavage fluid (BALF). Increased total and OVA-specific serum IgE were also noted in the CY-treated animals. In cell cultures from peritracheal lymph nodes, the Th2 cytokines IL-4 and IL-5 were elevated in animals treated with CY. It was hypothesized that the effects of CY could be caused by reduced immunosuppression mediated by Treg. mRNA expression of the immunosuppressive cytokines IL-10 and TGF-beta was reduced in the lungs of CY-treated mice. The expression of FoxP3, a marker of naturally occurring Treg, was significantly reduced in spleens, thymuses and peritracheal lymph nodes after the second injection of CY, and in the lung tissue after allergen challenge in CY-treated mice. Furthermore, lung IL-10-producing CD4+ T cells and CTLA-4+-bearing CD4+ T cells were reduced after allergen aerosol challenge in CY-treated mice. Thus CY worsened the features of allergic pulmonary inflammation in this model, in association with increased production of IgE and Th2 cytokines. The reduction in expression of FoxP3 and immunosuppressive cytokines by CY suggests that toxicity to Treg may contribute to the increased inflammation. GM-CSF plays a role in the growth, development, and maturation of bone marrow hemopoietic cells into mature blood cells, and has been proposed to be involved in potentiating the function of inflammatory cells in allergic inflammation. In the second part of this thesis, GM-CSF knockout (KO) mice were used to investigate the role of GM-CSF. In allergic KO mice, airway eosinophils were only shown in the perivascular, but not peribronchial areas in the lung, compared to the allergic wild-type (WT) mice in which eosinophil infiltration appeared in both areas. Eosinophil numbers were drastically reduced in the bronchoalveolar lavage fluid (BALF) of KO mice. IL-5 production in the lung tissue and BALF in allergic KO mice was reduced; similar results were also found in peritracheal draining lymph nodes after in vitro stimulation assays. However, IL-4 and IL-13 production, airway hyperresponsiveness (AHR), and serum IgE production were not affected in allergic KO mice. Surprisingly, lung IFN-gamma mRNA and BALF levels were increased in allergic KO mice. Lung mRNA levels of CCR3, a key chemokine receptor on eosinophils, were significantly reduced in allergic KO mice, whereas expression of the chemokines eotaxin and RANTES were at similar levels in allergic KO and WT mice. Lung mRNA levels of the IFN-gamma-inducible chemokines Mig (CXCL9) and IP-10 (CXCL10), which are antagonists of CCR3, and their receptor CXCR3 were increased in allergic KO mice, compared with allergic WT mice. Data obtained from flow cytometry showed more eosinophils survived in the lung of WT mice than KO mice. Another allergy model, a peritoneal allergy model was performed to investigate inflammation in a different model. Leukocyte subpopulations such as neutrophils, eosinophils, macrophages, and lymphocytes were reduced in the peritoneal lavage fluid of allergic KO mice. The findings revealed that GM-CSF is essential for IL-5 production, pulmonary airway eosinophilia and eosinophil survival. In the absence of GM-CSF, over-production of IFN-???? may induce chemokines, including Mig and IP-10, which are antagonists for CCR3 and may reduce airway eosinophil infiltration. In this thesis, a murine model of allergic asthma has been used to obtain novel findings on the regulation of allergic inflammation. The results with CY are relevant to the treatment of asthma patients with CY and other cytotoxic agents. The findings in the GM-CSF KO mice suggest that GM-CSF is a potential therapeutic target in asthma, and that in assessment of new therapeutic agents for asthma, effects on GM-CSF should be considered.

Inflammatory processes in, or in close vicinity of, the skeleton often lead to loss of bone tissue. Different cytokines have been shown to be involved as stimulators of inflammatory induced osteoclastic bone resorption. During inflammatory processes also the kallikrein-kinin system is activated, leading to production of kinins that can cause pain, vasodilation and increased permeability of vessels. Kinins can also induce bone resorption in vitro. All cytokines and kinins that stimulate bone resorption stimulate in parallell prostaglandin synthesis, and prostaglandins, per se, have also been shown to induce bone resorption. The aim of this project was to increase the knowledge about the mechanisms involved in the interactions between different inflammatory mediators (i.e. kinins, cytokines and prostaglandins) suggested to be involved in the pathogenesis of inflammatory bone resorbing diseases. Human osteoblasts (MG-63) are equipped with both kinin B1 and B2 receptors linked to prostaglandin release and the stimulation of prostaglandin release are likely mediated via separate molecular mechanisms (Paper I). Activation of B1 or B2 receptors causes synergistic stimulation of PGE2 synthesis induced by either interleukin-1b (IL-1b) or tumour necrosis factor-a (TNF-a) (Paper II). The molecular mechanism involves increased expression of cyclooxygenase-2 (COX-2) and results in synergistic potentiation of receptor activator of NF-kB ligand (RANKL) protein expression. The synergistic interaction is dependent on the activation of NF-kB and the mitogen-activated protein kinases (MAPK) p38 and JNK (Paper II). The synergistic increase in RANKL expression might be an explanation why kinins potentiate IL-1b induced bone resorption, a mechanism likely to be important in inflammation induced bone resorption in diseases such as periodontal disease and rheumatoid arthritis. The synergism between kinins and IL-1b or TNF-a might also be dependent on regulation of kinin receptors, since both IL-1b and TNF-a markedly upregulated B1 and B2 receptors, both at the mRNA level and protein level (Paper III). This upregulation is not further potentiated by the kinins, and different kinin receptor agonists do not regulate the receptors for IL-1b or TNF-a, in MG-63 cells. No other cytokines known to stimulate bone resorption regulates the expressions of B1 and B2 receptors. The IL-1b- or TNF-a-induced enhancements of B1 and B2 receptor expressions involve activation of NF-kB and MAPK. The enhancement of kinin receptors may also be an important mechanism in the synergistic interactions between the two pro-inflammatory cytokines and kinins (paper III). IL-4 and IL-13 are two cytokines that have been shown to inhibit bone resorption. We have shown that COX-2 and both B1 and B2 receptors are down-regulated by IL-4 and IL-13, via a ‘signal transducer and activator of transcription6’ (STAT6) dependent pathway, which might be an important regulatory mechanism in inflammation induced bone resorption (paper IV). In conclusion, the mechanisms behind the synergistic potentiation of prostaglandin formation and increased bone resorption caused by co-stimulation with kinins and IL-1b or TNF-a seem to involve both potentiation of COX-2 and subsequently increased levels of RANKL, as well as upregulation of B1 and B2 kinin receptors. Interestingly, IL-4 and IL-13 decreased the expressions of COX-2 and both B1 and B2 receptors. These events might be important in the regulation of inflammation induced bone resorption in diseases such as periodontitis and rheumatoid arthritis.

Le thymus est un organe permettant le développement des lymphocytes T, partie intégrante du système immunitaire. Ces cellules sont communément associées au système immunitaire adaptatif, bien que certaines populations, dont les lymphocytes iNKT et Tγδ, soient associées au système immunitaire inné. De manière générale, ces lymphocytes " innés " sont capables de répondre très rapidement à différents signaux d'activation, par la production rapide et massive d'IL-4, d'IFN-γ et d'IL-17. Notre laboratoire a mis en évidence l'existence de deux sous-populations de lymphocytes iNKT, iNKT conventionnels et iNKT17, ayant deux voies de différenciation thymique bien distinctes, mais dont les mécanismes de détermination sont encore inconnus. Il a été montré que " SLAM-associated protein " (SAP) est indispensable au développement des lymphocytes iNKT, puisqu'ils sont absents chez les souris déficientes en SAP. D'autre part, ces mêmes souris montrent également une déficience de la réponse Th2. Nous avons alors émis l'hypothèse que SAP pourrait être impliqué dans la production d'IL-4 par les lymphocytes iNKT, et dans la détermination des deux sous-populations de lymphocytes iNKT conventionnel ou producteur d'IL-17. Dans une première partie, nous avons utilisé des souris triple mutantes Sap-/- Vα14Tg-ROR(γt)-Egfp, permettant l'étude des sous-populations de lymphocytes iNKT malgré la déficience en SAP. Nous avons ainsi montré que SAP est indispensable à l'acquisition thymique de la capacité de production d'IL-4 par les lymphocytes iNKT conventionnels. Chez ces souris déficientes en SAP, nous avons observé une augmentation de la fréquence des lymphocytes iNKT17 RORγtpos producteurs d'IL-17, ce qui montre clairement que SAP n'est pas nécessaire pour l'acquisition des propriétés fonctionnelles des lymphocytes iNKT17. Nous avons ainsi mis en évidence une nouvelle fonction de SAP dans le développement thymique des cellules iNKT productrices d'IL-4. De plus, nos résultats montrent que SAP est un point de contrôle obligatoire déterminant l'orientation de la différenciation thymique des cellules iNKT vers les cellules iNKT17 ou vers les cellules iNKT conventionnelles. En parallèle des lymphocytes iNKT17 présents dans le thymus, nous avons également analysé une autre population T particulière : des thymocytes TCRαβposCD4pos matures et producteurs d'IL-17, les lymphocytes Th17 naturels. Nous avons mis en évidence que ces lymphocytes expriment le facteur de transcription RORγt. Ces lymphocytes T CD4posCD8negCD44hiRORγtpos sont capables de produire rapidement et massivement de l'IL-17, mais requièrent l'IL-23 pour co-produire l'IL-22. De plus, ils se distinguent des lymphocytes Th17 induits, ou conventionnels, par l'expression du facteur de transcription PLZF et par leur capacité à répondre très rapidement à des stimuli pro-inflammatoires, nommément l'IL-23 associé à l'IL-1β et un agoniste TLR4. Les résultats obtenus durant cette thèse ouvrent donc de nombreuses perspectives de recherches fondamentale et thérapeutique, domaine dans lequel l'IL-17 est devenu une cible privilégiée pour le traitement des maladies auto-immunes.