Les infections et l’activation du système immunitaire stimulent l’hématopoïèse. L’activation des récepteurs Toll-like (TLRs) des cellules souches hématopoïétiques, par leur reconnaissance de motifs moléculaires portés par des agents infectieux, en oriente la différenciation vers les voies myéloïdes, renforçant la capacité de notre organisme à lutter contre les infections. Ici, nous avons étudié si les agonistes TLRs peuvent, au contraire, induire au sein de la moelle osseuse l’émergence de progéniteurs hématopoïétiques présentant des propriétés immunorégulatrices. Nous montrons que l’incubation de moelle osseuse de souris en présence de l’agoniste TRL-9, CpG-B, entraîne l’émergence d’une population de progéniteurs au stade pro-B (appelée CpG-proBs). Le transfert adoptif de seulement 60,000 CpG-proBs par receveur, à l’apparition des premiers signes cliniques, confère une protection à long terme dans deux modèles expérimentaux de maladies auto-immunes, le Diabète de Type I (T1D) et l’Encéphalomyélite Auto-immune Expérimentale (EAE). La migration, la différenciation, et les mécanismes cellulaires et moléculaires de cette population protectrice sont décrits et comparés entre ces deux modèles. Dans les deux modèles, les CpG-proBs migrent vers le tissu cible de la réponse auto-immune et se différencient en cellules B matures régulatrices. Dans le T1D, l’interféron-γ (IFN-γ) produit par les cellules T s’avère essentiel pour induire la surexpression de FasL à la surface des CpG-proBs, entraînant l’apoptose des cellules T effectrices. De plus, l’IFN-γ produit par les CpG-proBs réduit la production par les cellules T de l’IL-21, une cytokine pathogène majeure dans le T1D. La descendance des CpG-proBs est composée de précurseurs transitionnels B, de cellules B de la zone marginale et de cellules B folliculaires, exprimant de forts niveaux de FasL et toujours capables d’induire l’apoptose des cellules T, prolongeant ainsi le contrôle des cellules effectrices T auto-immunes in vivo. Dans l’EAE, l’IFNγ est indirectement responsable de la rétention des cellules T, par l’internalisation de CCR7, au sein des ganglions lymphatiques, inhibant ainsi leur migration au système nerveux central (SNC). Dans la moelle épinière, tissu cible de l’EAE, les CpG-proBs se différencient en cellules B220+CD5+CD1dhiCD11b+, secrétant la cytokine anti-inflammatoire IL-10. Enfin, la mobilisation des progéniteurs hématopoïétiques par un cocktail de facteurs hématopoïétiques confère à une sous-population multipotente au stade MPP2 la propriété d’augmenter l’expansion des Foxp3+ Tregs et de prévenir la survenue du diabète de type 1. Nous montrons que les MPP2 mobilisés s’avèrent également capables d’exercer un effet protecteur envers l’EAE. Leur capacité à induire l’expansion de Treg Foxp3+ au sein du SNC et à la périphérie joue un rôle essentiel dans la protection des souris envers l’EAE, puisque la déplétion des Treg abolit la protection déjà établie. Pour conclure, nous avons mis en évidence que diverses stimulations de l’hématopoïèse induisent l’émergence de nouvelles populations de progéniteurs hématopoïétiques qui présentent des propriétés immunorégulatrices et constituent de nouveaux outils de thérapie cellulaire des maladies auto-immunes. / It is well known today that various infectious events or other stimuli of the immune system can trigger hematopoiesis. The hematopoeitic stem and/or progenitor cells express on their cell surface Toll-like receptors which can recognize molecular motifs of infectious agents. The stimulation of TLRs on hematopoietic stem cells favors their differentiation into myeloid lineages, reinforcing the capacity of our body to fight against the pathogens. Herein, we have investigated whether the stimulation of TLRs can induce, instead, the emergence within the bone marrow of selective progenitor cells with immunoregulatory properties. We show that incubation of bone marrow cells with the TLR-9 ligand CpG-B can induce a pro-B cell population (named CpG-proBs) whose adoptive transfer at low numbers of 60,000 cells provided long-lasting protection in two models of autoimmune diseases, Type I Diabetes (TID) and Experimental Autoimmune Encephalomyelitis (EAE) at the onset of clinical signs. The migration, differentiation and molecular mechanism of action of this protective population is described and compared between these two models. In both models, the CpG-proBs migrate to the target tissue of autoimmune responses and differentiate into more mature regulatory B cells. In TID, IFN-γ produced by both T and CpG-proB cells is essential for the upregulation of FasL at the surface of CpG-proBs, inducing the apoptosis of the effector T cells. In addition, IFN-γ reduced the T-cell production of IL-21, a major pathogenic cytokine in TID. The progeny of the adoptively transferred CpG-proBs, including transitional precursors B cells, marginal zone and follicular B cells, display high expression of FasL, promote apoptosis of effector T cells and prolong the control of autoimmune effector T cells in vivo. In EAE, IFN-γ was responsible for the restriction of T cells to the lymph nodes, inhibiting their homing to the CNS. IFN-γ indirectly induced the internalization of CCR7, a receptor required for the migration across the blood-brain barrier. In the spinal cord (target tissue in EAE), CpG-proBs differentiated into B220+CD5+CD1dhiCD11b+ cells secreting the anti-inflammatory cytokine IL-10. Finally, hematopoietic progenitor populations mobilized to the periphery by a cocktail of G-CSF and Flt3l, at the stage of MPP2, have already been shown to protect against TID by expanding the Foxp3+ Tregs. We evaluated them in the EAE model, showing that the ability of these mobilized progenitor cells to trigger the expansion of Foxp3+ Treg within the CNS and the periphery was necessary for providing protection to EAE mice since Treg depletion abrogated the protection once established. In conclusion, we provide evidence for the emergence of new populations of hematopoietic progenitor cells which can display immunoregulatory properties and might be used for cell therapy of autoimmune diseases.

Purpose: Neurodevelopmental difficulties are correlated with lower mental well-being and more mental health problems. Issues in social relations such as bullying are a prominent risk factor in a social setting of high school, have also been associated with the status of disability. This thesis investigated the relation between mental health problems, mental well-being, and bullying in adolescents with self-reported neurodevelopmental difficulties and compared these with adolescents with Other disabilities. Materials and methods: Data from a Swedish longitudinal survey study (LoRDIA) was used. Mental health problems were measured through emotional and conduct problems scales of SDQ, mental well-being was measured with MHC-SF, and a bullying questionnaire was used. Results: Adolescents with NDD experience more bullying victimization and perpetration than adolescents with Other disabilities such as physical disability and autoimmune diseases. Significant association to bullying perpetration was found in NDD adolescents. Adolescents with Other disabilities indicated high mental well-being compared to NDD. While gender predicts high mental well-being, disability and emotional problems have a negative relation with it. Conclusion: Adolescents with NDD report more bullying victimization and perpetration experiences in comparison to adolescents with Other disabilities. Emotional problems have an inverse relation for predicting high mental well-being for adolescents with NDD and Other disabilities.

O diabetes mellitus tipo 1 (DM-1) e a esclerose múltipla (EM) são doenças auto-imunes órgão-específicas, inflamatórias, mediadas por células T e B auto-reativas e caracterizadas pela destruição seletiva de células b pancreáticas produtoras de insulina e do sistema nervoso central, respectivamente. Acredita-se que a desregulação da expressão de genes reguladores da maquinaria apoptótica possa contribuir para o desenvolvimento da auto-imunidade, visto que algumas dessas moléculas participam nos processos de tolerância central e periférica de linfócitos auto-reativos. O objetivo deste projeto foi analisar a expressão de moléculas reguladoras das vias intrínseca, extrínseca e da Família de proteínas inibidoras da apoptose (IAP) em 33 indivíduos saudáveis, 15 pacientes com DM-1 e 18 com EM submetidos à terapia de imunossupressão em altas doses seguida do transplante autólogo de células-tronco hematopoéticas (IAD/TACTH). As células mononucleares (CMN) foram isoladas do sangue periférico dos controles e de pacientes nos períodos pré-mobilização (pré-mob), pré-condicionamento (pré-cond), D+180, D+360, D+540 e D+720 pós-transplante. As CMN foram utilizadas para extração de RNA, síntese de cDNA, quantificação da expressão por PCR em tempo real dos genes a1, bcl-2, bcl-w, bcl-xL, mcl-1, bad, bak, bax, bid, bik, bim, bok, noxa, fas, fasL, c-FLIPL, cIAP-1 e cIAP-2 e protéica de Bcl-2, Bcl-xL, Bak, Bim e c-FLIPL por western-blotting. Os resultados de expressão gênica foram representados por unidades relativas de expressão em medianas nas diferentes amostras. Os pacientes com DM-1 apresentaram diminuição da expressão dos genes anti-apoptóticos bcl-2 (mediana: 0,98; p=0,04), bcl-w (0,08; p=0,04), mcl-1 (1254; p=0,03) e cIAP-1 (1,24; p=0,003) nas CMN dos pacientes no período pré-mob em relação aos indivíduos saudáveis (medianas: bcl-2: 7,58; bcl-w: 0,52; mcl-1: 1659; cIAP-1: 14,5), enquanto a expressão de cIAP-2 (60,8; p=0,0005) estava aumentada em relação aos controles (23,3). Foi observada redução significativa na expressão dos genes pró-apoptóticos bad (0,002; p<0,0001), bax (0,01; p=0,002) e fasL (1,66; p=0,001) no período pré-mob comparada aos controles sadios (bad: 0,23; bax: 2,79; fasL: 3,56). Os níveis de RNAm de bid (0,10; p=0,001) e bok (0,72; p=0,006) estavam elevados no pré-mob em relação ao grupo controle (bid: 0,004; bok: 0,31). As moléculas bcl-2, bcl-w, bcl-xL, mcl-1, bad, bax, bok, fasL e cIAP-1 atingiram níveis de RNAm similares aos controles após o TACTH. Foi verificado que a expressão de bcl-w, cIAP-1 e noxa estava maior nos pacientes com DM-1 em remissão quando comparados àqueles em recaída. A diminuição da expressão de a1, bcl-2 e bcl-w e o aumento de fas e noxa correlacionaram-se às porcentagens de hemoglobina glicosilada, concentração de auto-anticorpos GAD65, e aos níveis séricos de peptídeo-C após o transplante. Os pacientes com EM mostraram uma expressão reduzida dos genes anti-apoptóticos bcl-w (0,11; p=0,02) e cIAP-1 (1,87; p=0,04) no pré-mob comparada aos valores dos controles (bcl-w: 0,27; cIAP-1: 7,75) e maior expressão dos genes a1 (90,8; p=0,001) e cIAP-2 (58,8; p=0,009) em relação aos controles (a1: 12,7; cIAP-2: 22,3). As moléculas pró-apoptóticas bad (0,007; p=0,01) e bax (0,0007; p=0,004) mostraram menor expressão nas CMN no período pré-mob do que nos controles (bad: 0,27; bax: 1,24). Os genes bid (20,7; p=0,004), bik (0,84; p=0,02) e bok (1,77; p=0,0001) possuíam maior expressão no período pré-mob em relação aos indivíduos sadios (bid: 2,64; bik: 0,33; bok: 0,26). Não foram observadas diferenças significativas na expressão das moléculas da via extrínseca da apoptose nos pacientes com EM (p>0,05) nos períodos avaliados. Os valores de expressão de bcl-w, bak, bax, bik, bok e cIAP-1 atingiram níveis semelhantes aos controles após o transplante. A expressão dos genes bcl-2, cIAP-1, bad e bax estava maior nos pacientes em remissão da EM quando comparados àqueles em progressão neurológica. O aumento da expressão dos genes pró-apoptóticos bax, bak e bimEL correlacionou-se inversamente aos valores de EDSS dos pacientes com EM após o TACTH. Os resultados de expressão protéica foram equivalentes aos de expressão gênica nas duas doenças, com exceção dos dados das proteínas Bcl-2 e Bim. Em conjunto, os resultados demonstraram a desregulação da expressão de várias moléculas anti- e pró-apoptóticas nas CMN dos pacientes com DM-1 e EM. Esses achados sugerem a associação de alterações nos processos de apoptose celular com o surgimento e persistência de células auto-reativas no DM-1 e EM. Os dados indicam que essas alterações, principalmente a diminuição da expressão de moléculas pró-apoptóticas, como bak e bax, possam contribuir para a patogênese do DM-1 e EM. Além disso, a terapia de IAD/TACTH foi capaz de modular a expressão da maioria dos genes anormalmente expressos nas CMN dos pacientes com DM-1 e EM, já que esses atingiram níveis de expressão similares ao grupo controle após o transplante. Esta normalização da expressão de vários genes analisados correlacionou-se com a remissão clínica da doença na maioria dos pacientes / Type 1 diabetes mellitus (T1DM) and multiple sclerosis (MS) are inflammatory, organ-specific autoimmune diseases characterized by selective destruction of insulin-producing pancreatic -cells and central nervous system, respectively, by autoreactive B and T cells. Deregulation of apoptotic machinery is supposed to contribute to self-tolerance breakdown and autoimmune diseases pathogenesis, since apoptotic molecules have an important role in B and T lymphocytes central and peripheral tolerance mechanisms. The aim of this study was to evaluate the expression of pro and anti-apoptotic molecules from intrinsic and extrinsic apoptotic pathways and IAP Family members in 33 healthy individuals, 15 T1DM and 18 MS patients submitted to high-dose immunossupression therapy followed by autologous hematopoietic stem cell transplantation (HDI/AHSCT). Peripheral blood mononuclear cells (PBMC) were isolated from controls and patients at pre-mobilization (pre-mob), pre-conditioning (pre-cond), D+180, D+360, D+540 and D+720 post-transplantation. PBMC were used for RNA extraction, cDNA synthesis, gene quantification of a1, bcl-2, bcl-w, bcl-xL, bad, bak, bax, bid, bik, bimEL, bok, noxa, fas, fasL, c-FLIPL, cIAP-1 and cIAP-2 by Real Time PCR and Bcl-2, Bcl-xL, Bak, BimEL and c-FLIPL proteins detection by western-blotting. Results are expressed as median of relative expression units. Results from T1DM patients indicated that antiapoptotic molecules bcl-2 (median: 0,98; p=0,04), bcl-w (0,08; p=0,04), mcl-1 (1254; p=0,03) and cIAP-1 (1,24; p=0,003) were downregulated at pre-mob compared with healthy controls (medians bcl-2: 7,58; bcl-w: 0,52; mcl-1: 1659; cIAP-1: 14,5), while cIAP-2 (60,8; p=0,0005) gene expression was upregulated compared to healthy controls (23,3). We observed a significant decrease in proapoptotic bad (0,002; p<0,0001), bax (0,01; p=0,002) and fasL (1,66; p=0,001) genes expression in patients PBMC at pre-mob period compared to healthy subjects (bad: 0,23; bax: 2,79; fasL: 3,56). mRNA levels of bid (0.10; p=0.001) and bok (0.72; p=0.006) were elevated at pre-mob period when compared to control group (bid: 0.004; bok: 0.31). The bcl-2, bcl-w, bcl-xL, mcl-1, bad, bak, bax, bok, fasL and cIAP-1 mRNA levels reached controls levels after HDI/AHSCT. We observed that bcl-w, cIAP-1 and noxa gene expression were increased in T1DM patients in remission when compared to relapsed patients. The decreased antiapoptotic gene expression and increased in proapoptotic molecules correlated with decreased glicosilated hemoglobin percentages (Hb A1C) and anti-GAD65 antibodies and increased peptide-C levels. Results from MS patients showed decreased bcl-w (0,11; p=0,02) and cIAP-1 gene expression (1,87; p=0,04) in patients PBMC at pre-mob period compared to healthy controls (bcl-w: 0,27; cIAP-1: 7,75) and increased expression of a1 (90,8; p=0,001) and cIAP-2 (58,8; p=0,009) compared to controls (a1: 12,7; cIAP-2: 22,3). Proapoptotic molecules bad (0.007; p=0.01) and bax (0.0007; p=0.004) showed decreased gene expression at pre-mob compared to control group (bad: 0.27; bax: 1.24). bid (20.7; p=0.004), bik (0.84; p=0.01) and bok genes (1.77; p=0.0001) showed increased expression at pre-mob compared to healthy controls (bid: 2.64; bik: 0.33; bok: 0.26). Significant differences were not observed in the expression of the extrinsic pathway genes in pre-mob and healthy controls samples (p>0.05). bcl-w, bak, bax, bik, bok and cIAP-1 expression values reached healthy control values after transplantation. We observed that bcl-2, cIAP-1, bad and bax gene expression was increased in MS patients in disease remission when compared to patients with neurologic progression. Significant correlation of increased proapoptotic genes expression with decreased EDSS values in MS patients after HDI/AHSCT was observed. Results of protein quantification of apoptotic molecules in PBMC of T1DM and MS patients were similar to the gene expression results of these molecules, except for Bcl-2 and Bim proteins. Taken together, these data indicate a deregulated expression of anti- and proapoptotic genes in T1DM and MS patients PBMC. These data suggest an association of deregulated apoptosis with emergence and maintenance of autoreactive lymphocytes in analyzed patients. Based on these results, we suggest that this altered gene expression profile, mainly the decreased proapoptotic genes expression, as bak and bax, may contribute to T1DM and MS pathogenesis. Furthermore, we showed that the HDI/AHSCT therapy was able to modulate and normalize the expression of most genes abnormally expressed in T1DM and MS patients at pre-transplant period. Many analyzed genes achieved expression levels similar to healthy controls. The normalization of the expression of many evaluated genes correlated to disease remission in the majority of the patients.

Introdução: A síndrome antifosfolípide é uma doença autoimune caracterizada por eventos trombóticos e/ou obstétricos adversos associados à presença de anticorpos antifosfolípides. Considera-se síndrome antifosfolípide primária (SAFP) quando não há outra doença autoimune ou inflamatória associada. Objetivos: Avaliar marcadores de reserva ovariana em mulheres com SAFP e a associação entre estes marcadores e dados clínicos, laboratoriais e anticorpo anti-corpo lúteo (anti-CoL). Métodos: Realizou-se estudo transversal em 18 pacientes com SAFP e 24 controles. A reserva ovariana foi avaliada na fase folicular precoce através das dosagens de hormônio folículo estimulante (FSH), hormônio luteinizante (LH), estradiol e hormônio anti-Mülleriano (HAM), pela técnica de ELISA, e contagem ultrassonográfica de folículos antrais (CFA). O anti-CoL foi avaliado através de immunoblot. Todas as análises foram realizadas após suspensão de contraceptivo hormonal por, no mínimo, 6 meses e retorno das menstruações. O teste t foi utilizado para comparar médias ± desvio padrão e o teste de Mann-Whitney, para comparar medianas (variação). O teste exato de Fisher foi utilizado para comparar diferenças entre variáveis categóricas. Adotou-se nível de significância de 5% (p<0,05). Resultados: A média da idade foi similar em pacientes com SAFP e controles (33,0 ± 5,0 vs. 30,4 ± 7,0 anos, p=0,19). Houve maior frequência de CFA baixa (<=10) (56% vs. 22%, p=0,04) e muito baixa (<=5) (37% vs. 9%, p=0,04) em pacientes com SAFP quando comparadas aos controles. Em relação ao HAM, observou-se tendência de maior frequência de concentração sérica reduzida (<1,0 ng/mL), baixa (<0,5 ng/mL) e muito baixa (<0,2 ng/mL) em pacientes com SAFP (p=0,08; p=0,07 and p=0,07; respectivamente). As concetrações séricas de FSH, LH e estradiol foram semelhantes em pacientes e controles (p>0,05). Não houve associação entre baixa reserva ovariana e tipos específicos de anticorpos antifosfolípides. A presença do anti-CoL foi observada apenas em pacientes com SAFP (11% vs. 0%, p=0,177) e não foi relacionada a parâmetros de reserva ovariana. Conclusões: Pacientes com SAFP apresentaram reserva ovariana diminuída, com prevalência maior do que 50%, o que reforça o aconselhamento reprodutivo e planejamento familiar / Introduction: Antiphospholipid syndrome is an autoimmune disease characterized by thrombosis and/or pregnancy morbidity associated with antiphospholipid antibodies. Primary APS (PAPS) is diagnosed when no other autoimmune or inflammatory diseases are present. Objective: to determine ovarian reserve in PAPS women and to evaluate the association between ovarian reserve tests and clinical and laboratorial parameters, and anti-corpus luteum antibody (anti-CoL). Methods: In this cross sectional study, 18 PAPS patients and 24 healthy women were evaluated at early follicular phase with measurement of follicle stimulating hormone (FSH), estradiol, and anti-Müllerian hormone (AMH), carried out by ELISA test, and sonographic antral follicle count (AFC). Serum measurement of anti-CoL was determined by immunoblot analysis. All analyses were performed after at least 6 months from the last intake of hormonal contraceptive and resumption of menstruation. Data were compared by t-test and Mann-Whitney test in continuous variables and by Fisher\'s exact test in categorical variables. The level of significance was set at 5% (p<0.05). Results: The mean age was comparable in PAPS and controls (33.0 ± 5.0 vs. 30.4 ± 7.0 years; p=0.19). Regarding ovarian reserve tests, the frequencies of low AFC (<= 10) (56% vs. 22%, p=0.04) and very low AFC (<= 5) (37% vs. 9%, p=0.04) were significantly higher in PAPS patients than controls. Trends of higher frequencies of reduced (<1.0 ng/mL), low (<0.5 ng/mL) and negligible (<0.2 ng/mL) AMH levels were found in PAPS patients (p=0.08; p=0.07 and p=0.07; respectively). FSH, LH and estradiol were similar in patients and controls. There was no association between low ovarian reserve and specific types of antiphospholipid antibodies. Anti-CoL was solely observed in PAPS patients (11% vs. 0%; p=0.177) and was not related to ovarian reserve tests. Conclusion: Women suffering from PAPS possessed reduced ovarian reserve, with prevalence greater than 50%, emphasizing fertility counseling and family planning

Introdução: A síndrome antifosfolípide é uma doença autoimune caracterizada por eventos trombóticos e/ou obstétricos adversos associados à presença de anticorpos antifosfolípides. Considera-se síndrome antifosfolípide primária (SAFP) quando não há outra doença autoimune ou inflamatória associada. Objetivos: Avaliar marcadores de reserva ovariana em mulheres com SAFP e a associação entre estes marcadores e dados clínicos, laboratoriais e anticorpo anti-corpo lúteo (anti-CoL). Métodos: Realizou-se estudo transversal em 18 pacientes com SAFP e 24 controles. A reserva ovariana foi avaliada na fase folicular precoce através das dosagens de hormônio folículo estimulante (FSH), hormônio luteinizante (LH), estradiol e hormônio anti-Mülleriano (HAM), pela técnica de ELISA, e contagem ultrassonográfica de folículos antrais (CFA). O anti-CoL foi avaliado através de immunoblot. Todas as análises foram realizadas após suspensão de contraceptivo hormonal por, no mínimo, 6 meses e retorno das menstruações. O teste t foi utilizado para comparar médias ± desvio padrão e o teste de Mann-Whitney, para comparar medianas (variação). O teste exato de Fisher foi utilizado para comparar diferenças entre variáveis categóricas. Adotou-se nível de significância de 5% (p<0,05). Resultados: A média da idade foi similar em pacientes com SAFP e controles (33,0 ± 5,0 vs. 30,4 ± 7,0 anos, p=0,19). Houve maior frequência de CFA baixa (<=10) (56% vs. 22%, p=0,04) e muito baixa (<=5) (37% vs. 9%, p=0,04) em pacientes com SAFP quando comparadas aos controles. Em relação ao HAM, observou-se tendência de maior frequência de concentração sérica reduzida (<1,0 ng/mL), baixa (<0,5 ng/mL) e muito baixa (<0,2 ng/mL) em pacientes com SAFP (p=0,08; p=0,07 and p=0,07; respectivamente). As concetrações séricas de FSH, LH e estradiol foram semelhantes em pacientes e controles (p>0,05). Não houve associação entre baixa reserva ovariana e tipos específicos de anticorpos antifosfolípides. A presença do anti-CoL foi observada apenas em pacientes com SAFP (11% vs. 0%, p=0,177) e não foi relacionada a parâmetros de reserva ovariana. Conclusões: Pacientes com SAFP apresentaram reserva ovariana diminuída, com prevalência maior do que 50%, o que reforça o aconselhamento reprodutivo e planejamento familiar / Introduction: Antiphospholipid syndrome is an autoimmune disease characterized by thrombosis and/or pregnancy morbidity associated with antiphospholipid antibodies. Primary APS (PAPS) is diagnosed when no other autoimmune or inflammatory diseases are present. Objective: to determine ovarian reserve in PAPS women and to evaluate the association between ovarian reserve tests and clinical and laboratorial parameters, and anti-corpus luteum antibody (anti-CoL). Methods: In this cross sectional study, 18 PAPS patients and 24 healthy women were evaluated at early follicular phase with measurement of follicle stimulating hormone (FSH), estradiol, and anti-Müllerian hormone (AMH), carried out by ELISA test, and sonographic antral follicle count (AFC). Serum measurement of anti-CoL was determined by immunoblot analysis. All analyses were performed after at least 6 months from the last intake of hormonal contraceptive and resumption of menstruation. Data were compared by t-test and Mann-Whitney test in continuous variables and by Fisher\'s exact test in categorical variables. The level of significance was set at 5% (p<0.05). Results: The mean age was comparable in PAPS and controls (33.0 ± 5.0 vs. 30.4 ± 7.0 years; p=0.19). Regarding ovarian reserve tests, the frequencies of low AFC (<= 10) (56% vs. 22%, p=0.04) and very low AFC (<= 5) (37% vs. 9%, p=0.04) were significantly higher in PAPS patients than controls. Trends of higher frequencies of reduced (<1.0 ng/mL), low (<0.5 ng/mL) and negligible (<0.2 ng/mL) AMH levels were found in PAPS patients (p=0.08; p=0.07 and p=0.07; respectively). FSH, LH and estradiol were similar in patients and controls. There was no association between low ovarian reserve and specific types of antiphospholipid antibodies. Anti-CoL was solely observed in PAPS patients (11% vs. 0%; p=0.177) and was not related to ovarian reserve tests. Conclusion: Women suffering from PAPS possessed reduced ovarian reserve, with prevalence greater than 50%, emphasizing fertility counseling and family planning

Les interférons de type I (IFN I) sont des cytokines antivirales aux propriétés puissantes. L’induction, la transmission et la résolution de la réponse immunitaire engendrée par les IFN I est minutieusement régulée. Le concept d’interféronopathie de type I, récemment individualisé par notre équipe, repose sur l’hypothèse que certaines pathologies seraient secondaires au déséquilibre de ces voies de signalisation complexes et à la sécrétion excessive et inappropriée d’IFN I. L’inhibition de celle-ci par des thérapeutiques ciblées permettrait de valider cette hypothèse, si les symptômes allégués s’amélioraient, voire disparaissaient. Ce travail de thèse s’est initialement concentré sur la caractérisation clinique et biologique des interféronopathies monogéniques et polygéniques, et secondairement sur l’identification moléculaire de nouvelles mutations du gène TMEM173 à l’origine de l’interféronopathie liée à STING, également appelée SAVI (STING associated vasculopathy with onset in infancy), syndrome auto-inflammatoire associant une atteinte sévère cutanée et pulmonaire. De nouvelles techniques ont permis la sélection de patients présentant une augmentation de l’IFN I en comparaison à des contrôles sains : la signature IFN I, qPCR de 6 gènes stimulés par l’IFN (IFN stimulated genes – ISGs) et le dosage d’IFN alpha sérique ou plasmatique par méthode du SIMOA (single molecule array) permettant la détection de molécules d’IFN de l’ordre du femtogramme (10-18g). Ces méthodes nous ont ainsi permis d’élargir le spectre clinique phénotypique des interféronopathies de type I, initialement considéré comme essentiellement neurologique. Les patients atteints du syndrome d’Aicardi-Goutières, première interféronopathie monogénique décrite, présentaient les signes suivants : dystonie, spasticité, décalage des acquisitions, calcifications intra-cérébrales et anomalies de la substance blanche. Cependant, l’utilisation systématique de nos méthodes de criblage associée à l’avènement des technologies de séquençage à haut débit (next generation sequencing – NGS) a permis de révéler un phénotype plus large, caractéristique des interféronopathies de type I : sur le plan cutané (engelures, vascularite nécrosante des extrémités, sclérodermie), pulmonaire (pneumopathie interstitielle isolée ou non), musculo-squelettique (arthralgies, arthrites, arthropathie de Jaccoud, myalgies et myosites), ophtalmologique (glaucome), néphrologique (néphropathies lupiques), gastro-entérologique (maladies inflammatoires chroniques intestinales précoces), associées à de l’auto-immunité ou un déficit immunitaire inconstants. Notre méthode de sélection nous a notamment permis d’identifier des patients présentant de manière variable des signes cardinaux de SAVI et une de trois nouvelles mutations activatrices dans une région spécifique du gène TMEM173 (codant pour STING). Ces mutations circonscrivent une région de la protéine à ce jour encore jamais impliquée dans le contrôle de la voie de l’IFN I. STING est une protéine du réticulum endoplasmique qui agit comme adaptateur cytosolique de senseurs intracellulaires d’ADN viral dans une voie de signalisation de l’IFN I. STING active TBK1 (TANK-binding kinase) et permet la transcription des IFN I par la phosphorylation d’IRF3. La Janus Kinase 1 (JAK1) et la tyrosine kinase 2 (TYK2) sont activées suite à la stimulation des récepteurs de l’IFN I et phosphorylent les facteurs de transcription STAT1 et STAT2, conduisant à l’expression de nombreux ISGs. Les analyses génétiques, de conformation tridimensionnelle, sur un modèle cellulaire in vitro (HEK293T) et ex vivo sur cellules mononuclées périphériques des patients nous ont ainsi permis de mettre en évidence pour ces mutations un caractère constitutionnellement activé, indépendant de la liaison au ligand cGAMP, mais transmettant ce signal à travers la voie d’aval par TBK1. (...) / Type I interferons (IFN I) are antiviral cytokines with potent properties. Hence, the induction, transmission and resolution of the immune response generated by IFN I is tightly regulated. The concept of the type I interferonopathies, recently formulated by our team, rests on the assumption that some diseases arise from a disturbance of this complex signalling pathway, leading to excessive and inappropriate IFN I secretion. On this basis, targeted therapeutics should improve or cure features of such type I interferonopathies, thereby providing a validation of the underlying hypothesis. This PhD project initially focused on the clinical and biological characterisation of monogenic and polygenic interferonopathies, and secondarily on the molecular identification of novel mutations in the gene TMEM173 causing the interferonopathy called STING associated vasculopathy with onset in infancy (SAVI), an auto-inflammatory syndrome with severe cutaneous and pulmonary features. Our selection of patients in comparison to healthy controls was made possible through the use of novel screening tools: IFN signature (qPCR of 6 IFN stimulated genes – ISGs), and measurement of IFN alpha protein levels in serum or plasma (SIMOA-single molecule array - enabling the detection of molecules of IFN in the femtogram [10-18g]) range. In this way, we have been able to expand the phenotypic spectrum of the interferonopathies, which was initially considered as primarily neurological. Patients with Aicardi-Goutières syndrome (AGS), the first described of the monogenic interferonopathies, exhibit dystonia, spasticity, developmental delay, intra-cranial calcifications and white matter abnormalities. However, the systematic use of our interferon screening assays, plus the advent of next-generation sequencing technology, has revealed a much broader set of features relevant to this novel disease grouping – involving the skin (chilblains, necrotising vasculitis, scleroderma), lungs (isolated lung interstitial disease or associated with other signs), musculoskeletal system (joint pain, arthritis, Jaccoud’s arthropathy, muscle pain and myositis), eyes (glaucoma), kidneys (lupus nephritis) and gastro-intestinal tract (early inflammatory bowel disease), as well features of autoimmunity and immunodeficiency. Using our screening assays enabled us to identify three patients variably exhibiting the core features of SAVI, all of whom were found to harbour distinct novel activating mutations in STING. These mutations highlight a protein domain not previously implicated in the control of IFN I signalling. STING is an endoplasmic reticulum protein, acting as a cytosolic adaptor of intracellular sensors of viral DNA in the type I IFN signalling pathway. STING activates TANK-binding kinase (TBK1), allowing transcription of IFN I through phosphorylation of IRF3. Janus kinase 1 (JAK1) and tyrosine kinase 2 (TYK2) are activated following stimulation of the IFN I receptor, leading to phosphorylation of the transcription factors STAT1 and STAT2 and the subsequent induction of a large number of ISGs. Genetic analysis, conformational studies, an in vitro cellular model (HEK293T) and ex vivo experimental data (using patient peripheral blood mononuclear cells - PBMCs) enabled us to confirm the constitutive activating nature of these variants, and show that this activation did not require binding with cGAMP, but was dependent on signalling through TBK1. Ruxolitinib, a JAK1/2 inhibitor, could antagonise this constitutive activation ex vivo. These results indicate a promising therapeutic approach in such patients, and more widely in the monogenic, and perhaps even, polygenic, interferonopathy context.

O diabetes mellitus tipo 1 (DM-1) e a esclerose múltipla (EM) são doenças auto-imunes órgão-específicas, inflamatórias, mediadas por células T e B auto-reativas e caracterizadas pela destruição seletiva de células b pancreáticas produtoras de insulina e do sistema nervoso central, respectivamente. Acredita-se que a desregulação da expressão de genes reguladores da maquinaria apoptótica possa contribuir para o desenvolvimento da auto-imunidade, visto que algumas dessas moléculas participam nos processos de tolerância central e periférica de linfócitos auto-reativos. O objetivo deste projeto foi analisar a expressão de moléculas reguladoras das vias intrínseca, extrínseca e da Família de proteínas inibidoras da apoptose (IAP) em 33 indivíduos saudáveis, 15 pacientes com DM-1 e 18 com EM submetidos à terapia de imunossupressão em altas doses seguida do transplante autólogo de células-tronco hematopoéticas (IAD/TACTH). As células mononucleares (CMN) foram isoladas do sangue periférico dos controles e de pacientes nos períodos pré-mobilização (pré-mob), pré-condicionamento (pré-cond), D+180, D+360, D+540 e D+720 pós-transplante. As CMN foram utilizadas para extração de RNA, síntese de cDNA, quantificação da expressão por PCR em tempo real dos genes a1, bcl-2, bcl-w, bcl-xL, mcl-1, bad, bak, bax, bid, bik, bim, bok, noxa, fas, fasL, c-FLIPL, cIAP-1 e cIAP-2 e protéica de Bcl-2, Bcl-xL, Bak, Bim e c-FLIPL por western-blotting. Os resultados de expressão gênica foram representados por unidades relativas de expressão em medianas nas diferentes amostras. Os pacientes com DM-1 apresentaram diminuição da expressão dos genes anti-apoptóticos bcl-2 (mediana: 0,98; p=0,04), bcl-w (0,08; p=0,04), mcl-1 (1254; p=0,03) e cIAP-1 (1,24; p=0,003) nas CMN dos pacientes no período pré-mob em relação aos indivíduos saudáveis (medianas: bcl-2: 7,58; bcl-w: 0,52; mcl-1: 1659; cIAP-1: 14,5), enquanto a expressão de cIAP-2 (60,8; p=0,0005) estava aumentada em relação aos controles (23,3). Foi observada redução significativa na expressão dos genes pró-apoptóticos bad (0,002; p<0,0001), bax (0,01; p=0,002) e fasL (1,66; p=0,001) no período pré-mob comparada aos controles sadios (bad: 0,23; bax: 2,79; fasL: 3,56). Os níveis de RNAm de bid (0,10; p=0,001) e bok (0,72; p=0,006) estavam elevados no pré-mob em relação ao grupo controle (bid: 0,004; bok: 0,31). As moléculas bcl-2, bcl-w, bcl-xL, mcl-1, bad, bax, bok, fasL e cIAP-1 atingiram níveis de RNAm similares aos controles após o TACTH. Foi verificado que a expressão de bcl-w, cIAP-1 e noxa estava maior nos pacientes com DM-1 em remissão quando comparados àqueles em recaída. A diminuição da expressão de a1, bcl-2 e bcl-w e o aumento de fas e noxa correlacionaram-se às porcentagens de hemoglobina glicosilada, concentração de auto-anticorpos GAD65, e aos níveis séricos de peptídeo-C após o transplante. Os pacientes com EM mostraram uma expressão reduzida dos genes anti-apoptóticos bcl-w (0,11; p=0,02) e cIAP-1 (1,87; p=0,04) no pré-mob comparada aos valores dos controles (bcl-w: 0,27; cIAP-1: 7,75) e maior expressão dos genes a1 (90,8; p=0,001) e cIAP-2 (58,8; p=0,009) em relação aos controles (a1: 12,7; cIAP-2: 22,3). As moléculas pró-apoptóticas bad (0,007; p=0,01) e bax (0,0007; p=0,004) mostraram menor expressão nas CMN no período pré-mob do que nos controles (bad: 0,27; bax: 1,24). Os genes bid (20,7; p=0,004), bik (0,84; p=0,02) e bok (1,77; p=0,0001) possuíam maior expressão no período pré-mob em relação aos indivíduos sadios (bid: 2,64; bik: 0,33; bok: 0,26). Não foram observadas diferenças significativas na expressão das moléculas da via extrínseca da apoptose nos pacientes com EM (p>0,05) nos períodos avaliados. Os valores de expressão de bcl-w, bak, bax, bik, bok e cIAP-1 atingiram níveis semelhantes aos controles após o transplante. A expressão dos genes bcl-2, cIAP-1, bad e bax estava maior nos pacientes em remissão da EM quando comparados àqueles em progressão neurológica. O aumento da expressão dos genes pró-apoptóticos bax, bak e bimEL correlacionou-se inversamente aos valores de EDSS dos pacientes com EM após o TACTH. Os resultados de expressão protéica foram equivalentes aos de expressão gênica nas duas doenças, com exceção dos dados das proteínas Bcl-2 e Bim. Em conjunto, os resultados demonstraram a desregulação da expressão de várias moléculas anti- e pró-apoptóticas nas CMN dos pacientes com DM-1 e EM. Esses achados sugerem a associação de alterações nos processos de apoptose celular com o surgimento e persistência de células auto-reativas no DM-1 e EM. Os dados indicam que essas alterações, principalmente a diminuição da expressão de moléculas pró-apoptóticas, como bak e bax, possam contribuir para a patogênese do DM-1 e EM. Além disso, a terapia de IAD/TACTH foi capaz de modular a expressão da maioria dos genes anormalmente expressos nas CMN dos pacientes com DM-1 e EM, já que esses atingiram níveis de expressão similares ao grupo controle após o transplante. Esta normalização da expressão de vários genes analisados correlacionou-se com a remissão clínica da doença na maioria dos pacientes / Type 1 diabetes mellitus (T1DM) and multiple sclerosis (MS) are inflammatory, organ-specific autoimmune diseases characterized by selective destruction of insulin-producing pancreatic -cells and central nervous system, respectively, by autoreactive B and T cells. Deregulation of apoptotic machinery is supposed to contribute to self-tolerance breakdown and autoimmune diseases pathogenesis, since apoptotic molecules have an important role in B and T lymphocytes central and peripheral tolerance mechanisms. The aim of this study was to evaluate the expression of pro and anti-apoptotic molecules from intrinsic and extrinsic apoptotic pathways and IAP Family members in 33 healthy individuals, 15 T1DM and 18 MS patients submitted to high-dose immunossupression therapy followed by autologous hematopoietic stem cell transplantation (HDI/AHSCT). Peripheral blood mononuclear cells (PBMC) were isolated from controls and patients at pre-mobilization (pre-mob), pre-conditioning (pre-cond), D+180, D+360, D+540 and D+720 post-transplantation. PBMC were used for RNA extraction, cDNA synthesis, gene quantification of a1, bcl-2, bcl-w, bcl-xL, bad, bak, bax, bid, bik, bimEL, bok, noxa, fas, fasL, c-FLIPL, cIAP-1 and cIAP-2 by Real Time PCR and Bcl-2, Bcl-xL, Bak, BimEL and c-FLIPL proteins detection by western-blotting. Results are expressed as median of relative expression units. Results from T1DM patients indicated that antiapoptotic molecules bcl-2 (median: 0,98; p=0,04), bcl-w (0,08; p=0,04), mcl-1 (1254; p=0,03) and cIAP-1 (1,24; p=0,003) were downregulated at pre-mob compared with healthy controls (medians bcl-2: 7,58; bcl-w: 0,52; mcl-1: 1659; cIAP-1: 14,5), while cIAP-2 (60,8; p=0,0005) gene expression was upregulated compared to healthy controls (23,3). We observed a significant decrease in proapoptotic bad (0,002; p<0,0001), bax (0,01; p=0,002) and fasL (1,66; p=0,001) genes expression in patients PBMC at pre-mob period compared to healthy subjects (bad: 0,23; bax: 2,79; fasL: 3,56). mRNA levels of bid (0.10; p=0.001) and bok (0.72; p=0.006) were elevated at pre-mob period when compared to control group (bid: 0.004; bok: 0.31). The bcl-2, bcl-w, bcl-xL, mcl-1, bad, bak, bax, bok, fasL and cIAP-1 mRNA levels reached controls levels after HDI/AHSCT. We observed that bcl-w, cIAP-1 and noxa gene expression were increased in T1DM patients in remission when compared to relapsed patients. The decreased antiapoptotic gene expression and increased in proapoptotic molecules correlated with decreased glicosilated hemoglobin percentages (Hb A1C) and anti-GAD65 antibodies and increased peptide-C levels. Results from MS patients showed decreased bcl-w (0,11; p=0,02) and cIAP-1 gene expression (1,87; p=0,04) in patients PBMC at pre-mob period compared to healthy controls (bcl-w: 0,27; cIAP-1: 7,75) and increased expression of a1 (90,8; p=0,001) and cIAP-2 (58,8; p=0,009) compared to controls (a1: 12,7; cIAP-2: 22,3). Proapoptotic molecules bad (0.007; p=0.01) and bax (0.0007; p=0.004) showed decreased gene expression at pre-mob compared to control group (bad: 0.27; bax: 1.24). bid (20.7; p=0.004), bik (0.84; p=0.01) and bok genes (1.77; p=0.0001) showed increased expression at pre-mob compared to healthy controls (bid: 2.64; bik: 0.33; bok: 0.26). Significant differences were not observed in the expression of the extrinsic pathway genes in pre-mob and healthy controls samples (p>0.05). bcl-w, bak, bax, bik, bok and cIAP-1 expression values reached healthy control values after transplantation. We observed that bcl-2, cIAP-1, bad and bax gene expression was increased in MS patients in disease remission when compared to patients with neurologic progression. Significant correlation of increased proapoptotic genes expression with decreased EDSS values in MS patients after HDI/AHSCT was observed. Results of protein quantification of apoptotic molecules in PBMC of T1DM and MS patients were similar to the gene expression results of these molecules, except for Bcl-2 and Bim proteins. Taken together, these data indicate a deregulated expression of anti- and proapoptotic genes in T1DM and MS patients PBMC. These data suggest an association of deregulated apoptosis with emergence and maintenance of autoreactive lymphocytes in analyzed patients. Based on these results, we suggest that this altered gene expression profile, mainly the decreased proapoptotic genes expression, as bak and bax, may contribute to T1DM and MS pathogenesis. Furthermore, we showed that the HDI/AHSCT therapy was able to modulate and normalize the expression of most genes abnormally expressed in T1DM and MS patients at pre-transplant period. Many analyzed genes achieved expression levels similar to healthy controls. The normalization of the expression of many evaluated genes correlated to disease remission in the majority of the patients.

Indiana University-Purdue University Indianapolis (IUPUI) / The non-hematopoietic, stationary stromal cells located inside and surrounding skin-draining lymph nodes play a key role in regulating immune responses. We studied distinct populations of lymph node stromal cells from both human subjects and animal models in order to describe their phenotype and function. In the mouse model, we studied two distinct populations: an endothelial cell population expressing Ly51 and MHC-II, and an epithelial cell population expressing the epithelial adhesion molecule EpCAM. Analysis of intra-nodal and extra-nodal lymph node (CD45-) stromal cells through flow cytometry and qPCR provides a general phenotypic profile of the distinct populations. My research focused on the EpCAM+ epithelial cell population located in the fat pad surrounding the skin draining lymph nodes. The EpCAM+ population has been characterized by surface marker phenotype, anatomic location, and gene expression profile. This population demonstrates the ability to inhibit the activation and proliferation of both CD4 and CD8 T cells. This population may play a role in suppressing overactive inflammation and auto-reactive T cells that escaped thymic deletion. The other major arm of my project consisted of identifying a novel endothelial cell population in human lymph nodes. Freshly resected lymph nodes were processed into single cell suspensions and selected for non-hematopoietic CD45- stromal cells. The unique endothelial population expressing CD34 HLA-DR was then characterized and analyzed for anatomic position, surface marker expression, and gene profiles. Overall, these studies emphasize the importance of stationary lymph node stromal cells to our functioning immune systems, and may have clinical relevance to autoimmune diseases, inflammation, and bone marrow transplantation.

Indiana University-Purdue University Indianapolis (IUPUI) / IL-17-producing T cells are critical to the development of pathogen and tumor immunity, but also contribute to the pathology of autoimmune diseases and allergic inflammation. CD8+ (Tc17) and CD4+ (Th17) IL-17-secreting T cells develop in response to a cytokine environment that activates Signal Transducer and Activator of Transcription (STAT) proteins, though the mechanisms underlying Tc17/Th17 development and stability are still unclear. In vivo, Tc17 cells clear vaccinia virus infection and acquire cytotoxic potential, that is independent of IL-17 production and the acquisition of IFN-γ-secreting potential, but partially dependent on Fas ligand, suggesting that Tc17-mediated vaccinia virus clearance is through cell killing independent of an acquired Tc1 phenotype. In contrast, memory Th cells and NKT cells display STAT4-dependent IL-23-induced IL-17 production that correlates with Il23r expression. IL-23 does not activate STAT4 nor do other STAT4-activating cytokines induce Il23r expression in these populations, suggesting a T cell-extrinsic role for STAT4 in mediating IL-23 responsiveness. Although IL-23 is important for the maintenance of IL-17-secreting T cells, it also promotes their instability, often resulting in a pathogenic Th1-like phenotype in vitro and in vivo. In vitro-derived Th17 cells are also flexible when cultured under polarizing conditions that promote Th2 or Th9 differentiation, adopting the respective effector programs, and decreasing IL-17 production. However, in models of allergic airway disease, Th17 cells do not secrete alternative cytokines nor adopt other effector programs, and remain stable IL-17-secretors. In contrast to Th1-biased pro-inflammatory environments that induce Th17 instability in vivo, during allergic inflammatory disease, Th17 cells are comparatively stable, and retain the potential to produce IL-17. Together these data document that the inflammatory environment has distinct effects on the stability of IL-17-secreting T cells in vivo.

Indiana University-Purdue University Indianapolis (IUPUI) / The transcriptional repressor BCL6 has been shown to be essential for the differentiation of germinal center (GC) B cells and follicular T helper (TFH) cells. The interaction of TFH and GC B cells is necessary for the development of high affinity antibodies specific for an invading pathogen. Germline BCL6-deficient mouse models limit our ability to study BCL6 function in T cells due to the strong inflammatory responses seen in these mice. To overcome this, our lab has developed a new BCL6 conditional knockout (cKO) mouse using the cre/lox system, wherein the zinc finger region of the BCL6 gene is flanked by loxP sites. Mating to a CD4-Cre mouse allowed us to study the effects of BCL6 loss specifically in T cells, without the confounding effects seen in germline knockout models. Using this cKO model, we have reaffirmed the necessity of BCL6 for TFH differentiation, including its role in sustained CXCR5 surface expression, a signature marker for TFH cells. This model also allowed us to recognize the role of BCL6 in promoting the expression of PD-1, another key surface marker for TFH cells. Without BCL6, CD4+ T cells cannot express PD-1 at the high levels seen on TFH cells. Our discovery of DNMT3b as a target for BCL6 suggests BCL6-deficient T cells have increased DNA methyltransferase activity at the PD-1 promoter. This data establishes a novel pathway for explaining how BCL6, a transcriptional repressor, can activate genes. Experiments with the BCL6 cKO model have also established a role for BCL6 in naïve CD4+ T cell activation. Furthermore, we did not observe increased differentiation of other helper T cell subsets, in contrast to what has been reported elsewhere with germline BCL6-deficient models. Unexpectedly, we found decreased T helper type 2 (Th2) cells, whereas mouse models with a germline mutation of BCL6 have increased Th2 cells. These results indicate that BCL6 activity in non-T cells is critical for controlling T cell differentiation. Finally, using an HIV-1 gp120 immunization model, we have, for the first time, shown BCL6-dependent GCs to be limiting for antibody development and affinity maturation in a prime-boost vaccine scheme.