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Fragile X syndrome (FXS) is identified by abnormal dendrite morphology and altered synaptic protein expression. Astrocyte secreted factors such as Tenascin C (TNC), may contribute to the synaptic changes, including maturation of the synapse. TNC is a known endogenous ligand of toll-like receptor 4 (TLR4) that has been shown to induce the expression of pro-inflammatory cytokines such as interleukin-6 (IL-6). At the molecular level, elevated IL-6 promotes excitatory synapse formation and increases dendrite spine length. With these molecular changes linked to the phenotype of FXS, we examined the expression and the mechanism of the endogenous TLR4 activator TNC, and its downstream target IL-6 in astrocytes from the FMR1 KO mouse model. Secreted TNC and IL-6 were significantly increased in FMR1 KO astrocytes. Exogenous TNC and lipopolysaccharide (LPS) stimulation of TLR4 induced secreted IL-6, whereas the antagonist of TLR4 (LPS-RS) had an opposing effect. Cortical protein expression of TNC and IL-6 were also significantly elevated in the postnatal FMR1 KO mouse. These results identify TNC as an endogenous ligand of TLR4, capable of effecting IL-6 secretion by astrocytes. In addition, there was an increase in the number of VGLUT1/PSD95 positive synaptic puncta of both WT and FMR1 KO neurons when plated with astrocyte conditioned media from FMR1 KO astrocytes, compared to those plated with media from wild type astrocytes. By assessing the cellular mechanisms involved, a novel therapeutic option could be made available to target abnormalities of synaptic function seen in FXS. / Thesis / Master of Science (MSc) / Autism spectrum disorders (ASDs) are neurodevelopmental disorders which arise from genetic and environmental factors. In the brain, a type of cell called the astrocyte is responsible for proper brain growth and development. Astrocytes release factors that promote inflammation, causing disruption of brain functions that control learning, memory and behaviour. Such factors released by astrocytes are capable of binding to their receptors, in turn impacting downstream targets, which have physiological effects. This research used various biological and genetic techniques to determine if the mechanism of an astrocyte-specific factor called Tenascin C (TNC) is impaired in the Fragile X mouse model. In a normal astrocyte, TNC with its binding partner is able to release molecules responsible for inflammation. Such molecules have been shown to increase the number synapses, where neurons and astrocytes exchange information, to control brain function. This proposed research would be the first to determine a role for TNC in ASDs. By assessing the cellular mechanisms involved between TNC and its binding partner, a novel therapeutic option could be made available in ASDs.

L’inflammation joue un rôle clé dans de nombreuses pathologies, telles que les maladies inflammatoires chroniques, les désordres métaboliques et le cancer. L’un de ses médiateurs le plus puissant est l’interleukine-1β (IL-1β), qui est une cytokine pro-inflammatoire participant à tous les stades de l’inflammation et de l’immunité. Son activation est régulée par un complexe multi-protéique nommé inflammasome, dont la caspase-1 active en découlant est responsable du clivage et de la maturation de l’IL-1β. Huit types d’inflammasomes activant et clivant la pro-caspase-1 ont été identifiés et contiennent tous la protéine ASC (apoptosis-associated speck-like protein containing a CARD). Les inflammasomes partagent un signal intracellulaire commun et le mécanisme menant à leur assemblage et leur activation n’est pas totalement élucidé. L’utilisation de la lignée cellulaire humaine monocytaire, THP-1, différenciée en macrophages grâce à un ester de phorbol, le TPA, a permis la mise en place d’un modèle d’étude de modulateurs de l’inflammasome en conditions stériles. Ce modèle a permis l’étude des mécanismes impliqués suite à des signaux issus de l’inflammation chronique, tels que l’ATP et les espèces réactives de l’oxygène (ROS). Ce travail montre qu’il existe une synergie entre ATP et ROS, qui agissent grâce à une boucle d’activation impliquant probablement plusieurs inflammasomes, dont NLRP3. Des donneurs de NO connus (trinitrine et isosorbide dinitrate) ou nouveau (dérivé de purine) ont montré une activité anti-inflammatoire. D’autres composés ont été identifiés comme de potentiels inhibiteurs d’inflammasome (extraits de dattes et dérivé de purine portant un acide lipoïque) / Inflammation has a pivotal role in several disorders, such as chronic inflammatory diseases, metabolic disorders and cancer. Interleukin-1β (IL-1β) is one of the most powerful mediators in this mechanism. IL-1β is a pro-inflammatory cytokine which is implicated in every step of inflammation and immunity. IL-1β is regulated by a multi-protein complex named inflammasome, in which active caspase-1 is responsible for IL-1β processing and maturation. Eight inflammasomes processing and activating pro-caspase-1 has been identified and all contain the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD). Inflammasomes share a common intracellular signal and the mechanism for their structural assembly and activation is not totally clear. The human monocytic cell line, THP-1, has been used and cells have been differentiated in macrophages by the phorbol ester, TPA. Based on this, a cellular pattern has been established for studying inflammasome modulators under sterile conditions. This pattern allowed the study of implicated mechanisms in chronic inflammatory signals, such as ATP and reactive oxygen species (ROS). This study shows that ATP and ROS synergize for activating inflammasomes and NLRP3 and act in an autocrine loop. Stimulation with others natural or synthetic compounds has been realized for characterization of their inflammatory activity. Therapeutic NO donors (trinitrin and isosorbide dinitrate) and a new NO donor (purine nitrate ester) have been characterized for their anti-inflammatory activity. Others compounds from a new category of inflammasome inhibitors have been identified (date fruit extracts and lipoic acid purine derivative)

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Os tecidos periodontais estão em confronto continuo com microorganismos capazes de disparar mecanismos da resposta imune inata, dando origem ao infiltrado inflamatório. Estudos recentes mostraram a importancia dos receptores do tipo Toll (TLRs) na fase inicial de reconhecimento de diferentes patogenos. A participação de receptores tipo Toll (TLRs) na resposta de neutrófilos e macrófagos frente a periodontopatógenos precisa ser determinada. Nesse estudo procuramos caracterizar o infiltrado inflamatório presente no peritônio de animais deficientes de TLR4-/-, avaliar a atividade fagocítica, bem como a produção de óxido nítrico (NO) e a atividade de mieloperoxidase (MPO) no curso da infecção por Aggregatibacter actinomycetemcomitans. A ausência de TLR4 não influenciou a quimiotaxia de neutrófilos e macrófagos para o local da infecção, a produção de óxido nítrico, a atividade de MPO e a viabilidade celular. No entanto, neutrófilos e macrófagos de animais TLR4-/- apresentaram menor atividade fagocítica quando comparado ao grupo controle (camundongos WT). Em relação a doença periodontal induzida experimentalmente com Aggregatibacter actinomycetemcomitans em camundongos deficientes de TLR4, os resultados mostraram que 100% dos animais deficientes de TLR4 sobreviveram a infecção durante o período de observação. Em relação a análise de perda óssea, os dados revelaram uma menor perda progressiva de osso alveolar na região dos molares de animais deficientes de TLR4. A ausência do receptor interferiu na disseminação da bactéria, uma vez que se observou um grande número de bacilos no linfonodo e baco dos animais que não expressaram TLR4, diferente do observado para os animais selvagens (WT). Os resultados indicam a importância da sinalização via TLR4 durante a resposta imune contra Aggregatibacter actinomycetemcomitans. / Aggregatibacter actinomycetemcomitans is an oral gram negative bacteria that contributes to periodontitis progression. Isolated antigens from A. actinomycetemcomitans could be activating innate immune cells through Toll-like receptors (TLRs), molecules that recognize structural components conserved among microorganisms. In this study, we evaluated the role of TLR4 in the recognition of Aggregatibacter actinomycetemcomitans. Neutrophils and macrophage from TLR4 deficient mice and WT mice were collected and used for the subsequent assays. The phagocytosis of leukocytes against A. actinomycetemcomitans and the presence of apoptotic cells were determined by flow cytometry. The in vivo and in vitro production of NO and MPO was evaluated 24h after A. actinomycetemcomitans challenge. In addition, we examined the mechanisms that modulate the outcome of A. actinomycetemcomitans-induced periodontal disease in TLR4-/- mice. The results show that inflammatory cells influx in peritoneal cavity of TLR4-/- mice was similar to that observed into their littermate controls. The phagocytic activity was diminished by cells from TLR4-/- mice. In addition, we did not observe difference in NO and MPO production and the frequency of apoptotic cells between cells from TLR4-/- and WT mice. The results showed that TLR4-deficient mice developed less severe periodontitis after A. actinomycetemcomitans infection, characterized by significantly lower bone loss and inflammatory cell migration to periodontal tissues. Together, these data demonstrate the role TLR4 signals for neutrophils activation after A. actinomycetemcomitans infection and development of periodontal disease.

Os tecidos periodontais estão em confronto continuo com microorganismos capazes de disparar mecanismos da resposta imune inata, dando origem ao infiltrado inflamatório. Estudos recentes mostraram a importancia dos receptores do tipo Toll (TLRs) na fase inicial de reconhecimento de diferentes patogenos. A participação de receptores tipo Toll (TLRs) na resposta de neutrófilos e macrófagos frente a periodontopatógenos precisa ser determinada. Nesse estudo procuramos caracterizar o infiltrado inflamatório presente no peritônio de animais deficientes de TLR4-/-, avaliar a atividade fagocítica, bem como a produção de óxido nítrico (NO) e a atividade de mieloperoxidase (MPO) no curso da infecção por Aggregatibacter actinomycetemcomitans. A ausência de TLR4 não influenciou a quimiotaxia de neutrófilos e macrófagos para o local da infecção, a produção de óxido nítrico, a atividade de MPO e a viabilidade celular. No entanto, neutrófilos e macrófagos de animais TLR4-/- apresentaram menor atividade fagocítica quando comparado ao grupo controle (camundongos WT). Em relação a doença periodontal induzida experimentalmente com Aggregatibacter actinomycetemcomitans em camundongos deficientes de TLR4, os resultados mostraram que 100% dos animais deficientes de TLR4 sobreviveram a infecção durante o período de observação. Em relação a análise de perda óssea, os dados revelaram uma menor perda progressiva de osso alveolar na região dos molares de animais deficientes de TLR4. A ausência do receptor interferiu na disseminação da bactéria, uma vez que se observou um grande número de bacilos no linfonodo e baco dos animais que não expressaram TLR4, diferente do observado para os animais selvagens (WT). Os resultados indicam a importância da sinalização via TLR4 durante a resposta imune contra Aggregatibacter actinomycetemcomitans. / Aggregatibacter actinomycetemcomitans is an oral gram negative bacteria that contributes to periodontitis progression. Isolated antigens from A. actinomycetemcomitans could be activating innate immune cells through Toll-like receptors (TLRs), molecules that recognize structural components conserved among microorganisms. In this study, we evaluated the role of TLR4 in the recognition of Aggregatibacter actinomycetemcomitans. Neutrophils and macrophage from TLR4 deficient mice and WT mice were collected and used for the subsequent assays. The phagocytosis of leukocytes against A. actinomycetemcomitans and the presence of apoptotic cells were determined by flow cytometry. The in vivo and in vitro production of NO and MPO was evaluated 24h after A. actinomycetemcomitans challenge. In addition, we examined the mechanisms that modulate the outcome of A. actinomycetemcomitans-induced periodontal disease in TLR4-/- mice. The results show that inflammatory cells influx in peritoneal cavity of TLR4-/- mice was similar to that observed into their littermate controls. The phagocytic activity was diminished by cells from TLR4-/- mice. In addition, we did not observe difference in NO and MPO production and the frequency of apoptotic cells between cells from TLR4-/- and WT mice. The results showed that TLR4-deficient mice developed less severe periodontitis after A. actinomycetemcomitans infection, characterized by significantly lower bone loss and inflammatory cell migration to periodontal tissues. Together, these data demonstrate the role TLR4 signals for neutrophils activation after A. actinomycetemcomitans infection and development of periodontal disease.

[ES] El consumo de altas cantidades de etanol durante en un corto período de tiempo, conocido también como consumo en atracón, causa importantes alteraciones en el sistema nervioso central del adolescente, activando la respuesta innata inflamatoria, que puede causar muerte neuronal y alteraciones a nivel estructural y de la conducta. Esta respuesta se produce mediante la activación de los receptores de membrana Toll-like, y específicamente por los receptores TLR4, localizados en células gliales. Cuando dicho receptor se une a su ligando y dimeriza, desencadena una cascada de señalización que finaliza con la translocación al núcleo del factor de transcripción NF-κB, donde se promueve la síntesis y liberación de citoquinas y quimioquinas pro-inflamatorias al medio extracelular. Entre los mecanismos que podrían participar en la amplificación de la respuesta neuroinflamatoria se encontrarían un tipo de vesículas extracelulares (VEs), denominadas exosomas. Los exosomas son micropartículas de 30-150 nm de tamaño con un contenido de carácter bioactivo, formado por proteínas, lípidos y ácidos nucleicos, y que cumplen un papel importante en la comunicación intercelular. Por tanto, la hipótesis que planteamos en esta tesis doctoral es que las VEs ejercen un papel en el mantenimiento y propagación de la neuroinflamación causada por el consumo de alcohol en forma de atracón. Utilizando VEs de cultivos primarios de astrocitos demostramos que el etanol induce una mayor secreción de VEs y altera los niveles de determinadas proteínas y microARNs (miARNs) asociados con la neuroinflamacion. Además, demostramos que cuando las neuronas corticales en cultivo se incuban con VEs procedentes de los astrocitos WT tratados con etanol, se inducen marcadores inflamatorios en las neuronas y presentan mayores niveles de apoptosis. Durante el proceso de biogénesis exosomal, se ha demostrado la participación de una familia de enzimas, llamadas esfingomielinasas, que estarían relacionadas con la biogénesis y secreción de las VEs. Estudios previos de este laboratorio han demostrado que el etanol es capaz de activar las esfingomielinasas, aunque el mecanismo por el cual esto ocurre se desconoce. En este proyecto de tesis proponemos a las membranas asociadas a mitocondrias (MAM) como mecanismo regulador de la secreción de VEs mediada por esfingomielinasas. Mediante el análisis de la actividad de transferencia de fosfolípidos, marcador de actividad de MAM, observamos que el etanol, tanto a nivel tisular como en cultivo, es capaz de activar MAM. Además, demostramos que, inhibiendo la actividad tanto de MAM como de las esfingomielinasas, se revierte el aumento en secreción de VEs causado por el tratamiento con etanol. Estos resultados sugieren que el etanol promueve una mayor liberación de VEs mediante la activación de las enzimas esfingomielinasas a través de MAM. Además, puesto que las VEs tienen la capacidad de cruzar la barrera hematoencefálica (BBB) y tener estabilidad en la circulación, se han considerado como posibles candidatos a biomarcadores de situaciones patológicas. Uno de los elementos presentes en las VEs, que se ha utilizado recientemente como biomarcador en diversos estudios, son los miARNs, moléculas de ARN no codificante de cadena corta implicados en la regulación génica. Se ha descrito que, en pacientes con enfermedades neurodegenerativas como el Alzheimer, Parkinson u otras, se detectan patrones de expresión diferencial de miARNs en las VEs circulantes, en comparación con pacientes control. En este estudio demostramos que el etanol es capaz de alterar los perfiles de miARNs relacionados con la inflamación presentes en VEs circulantes de jóvenes con intoxicación etílica aguda (IEA). Estos efectos presentan diferencias de género, siendo las mujeres/hembras más vulnerables a los efectos del alcohol, ya que la expresión de marcadores inflamatorios en cerebro y en VEs circulantes son más elevadas en mujeres/hembras que en hombres/machos. Estos resultados ponen de manifiesto que las VEs circulantes y sus perfiles de miARNs son posibles candidatos a biomarcadores de la neuroinflamación asociadas con el abuso de alcohol. / [CA] El consum d'elevades quantitats d'alcohol durant un curt període de temps, conegut també com a consum en afartament, causa importants alteracions en el sistema nerviós central de l'adolescent, activant la resposta innata inflamatòria, que pot causar mort neuronal i alteracions a nivell estructural i de conducta. Aquesta resposta es produeix mitjançant l'activació dels receptors de membrana Toll-like, i específicament pels receptors TLR4, localitzats en cèl·lules glials. Quan aquest receptor s'uneix al seu lligant i dimeritza, desencadena una cascada de senyalització que finalitza amb la translocació al nucli del factor de transcripció NF-κB, on es promou la síntesi i alliberament de citoquines i quimioquines pro-inflamatòries al medi extracel·lular. Dins dels mecanismes que podrien participar en l'amplificació de la resposta neuroinflamatòria es trobarien un tipus de vesícules extracel·lulars (VEs), denominades exosomes. Els exosomes són micropartícules de 30-150 nm de grandària amb un contingut de caràcter bioactiu, format per proteïnes, lípids i àcids nucleics, i que compleixen un paper important en la comunicació intercel·lular. Per tant, la hipòtesi que plantegem en aquesta tesi doctoral és que les VEs exerceixen un paper en el manteniment i propagació de la neuroinflamació causada pel consum d'alcohol en forma d'afartament. Utilitzant VEs de cultius primaris d'astròcits demostràrem que l'etanol indueix una major secreció de VEs i una alteració dels nivells de determinades proteïnes i microARNs (miARNs) associats amb la neuroinflamació. A més a més, també observàrem que quan les neurones corticals en cultiu s'incubaben amb VEs procedents dels astròcits WT tractats amb etanol, s'induïen marcadors inflamatoris en les neurones i aquestes presentaven majors nivells d'apoptosi. Durant el procés de biogènesi exosomal, s'ha demostrat la participació d'una família d'enzims, anomenats esfingomielinases, que estarien relacionats amb la biogènesi i secreció de les VEs. Estudis previs d'aquest laboratori han demostrat que l'etanol és capaç d'activar les esfingomielinases, encara que el mecanisme pel qual això passa es desconeix. En aquest projecte de tesi proposem a les membranes associades a mitocòndries (MAM), juntament amb les esfingomielinases com el mecanisme regulador de la secreció de VEs induïda pel consum d'alcohol. Mitjançant l'anàlisi de l'activitat de transferència de fosfolípids, marcador d'activitat de MAM, observàrem que l'etanol, tant a nivell tissular com en cultiu, era capaç d'activar MAM. A més, demostràrem que inhibint l'activitat tant de MAM com de les esfingomielinases, revertiem l'augment en la secreció de VEs causat pel tractament amb etanol. Aquests resultats suggereixen, que l'etanol promou una major alliberament de VEs mitjançant l'activació dels enzims esfingomielinases a través de MAM. A més, ja que les VEs tenen la capacitat de creuar la barrera hematoencefàlica (BBB) i ser estables en circulació, s'han considerat bons candidats a biomarcadors de situacions patològiques. Un dels elements presents en les VEs que s'ha utilitzat recentment com a biomarcador en diversos estudis, són els miARNs, molècules d'ARN no codificant de cadena curta implicats en la regulació gènica. S'ha descrit que, en pacients amb malalties neurodegeneratives, com l'Alzheimer o Parkinson, es detecten patrons d'expressió diferencial de miARNs a les VEs circulants, en comparació amb pacients control. A aquest estudi demostrem que l'etanol és capaç d'alterar els perfils de miARNs relacionats amb la inflamació presents en VEs circulants de joves amb intoxicació etílica aguda (IEA). Aquests efectes presenten diferències de gènere, sent les dones / noies més vulnerables als efectes de l'alcohol, ja que l'expressió de marcadors inflamatoris en cervell i en VEs circulants són més elevades en noies que en nois. / [EN] Heavy alcohol intake during a short period of time, also known as binge drinking, has been proved to produce negative effects on the individual's central nervous system by activating an inflammatory response that can lead to neuronal death and structural and behavioral alterations. This response is produced by glial cells, the main component of the neuroimmune system, through the activation of TLR4, a transmembrane receptor of the TLR family. When TLR4 binds to its ligand and dimerizes, it triggers a signaling cascade that ends up with the translocation of NF-κB to the nucleus, acting as a transcription factor, where it promotes the synthesis and release of pro-inflammatory cytokines and chemokines to the extracellular milieu. Among the mechanisms responsible for the transmission and amplification of this neuroinflammatory response, one candidate could be a kind of extracellular vesicles (VEs) called exosomes. Exosomes are microparticles of 30-150 nm in size, with a bioactive content, composed mostly by proteins, lipids and nucleic acids, which play an important role in intercellular communication. Therefore, the hypothesis of this thesis is that VEs play an important role in the transmission of the neuroinflammatory response caused by ethanol binge drinking. Using VEs from primary cultures of astrocytes, we show that ethanol is able to induce a higher secretion of VEs and alters their composition of inflammatory related protein and microRNAs (miARNs). Furthermore, incubation of these VEs in primary cultures of neurons lead to the development of inflammatory protein and gene markers, and higher apoptosis levels. Exosomal release has been shown to be partly regulated by a family of enzymes called sphingomyelinases, since inhibition of these enzymes resulted in a reduction of secreted VEs. Previous studies from this laboratory have shown that ethanol is able to activate sphingomyelinases, but the mechanism involved in the process is currently unknown. We propose membrane-associated mitochondria (MAM), along with sphingomyelinases, as the responsible for the increased VEs release after ethanol intake. We show that ethanol is capable of increasing phospholipid transfer activity, a marker of MAM activity. Moreover, MAM and sphingomyelinase inhibition resulted in depleted VEs secretion. These results suggest that ethanol promotes increased release of VEs by activating sphingomyelinase enzymes through MAM. VEs display certain biological characteristics, like the ability to cross the blood-brain barrier (BBB) or their high stability in serum, which make them good candidates for biomarkers of pathological situations. One of the elements present in VEs that has recently been used as a biomarker in various studies are miARNs, which are short-chain non-coding RNA molecules involved in gene regulation. It has been described that differential expression patterns of miARNs in circulating VEs can be detected in patients with neurodegenerative diseases, such as Alzheimer's, Parkinson's or others, when compared to healthy patients. In this study we demonstrate that ethanol is able to alter the inflammatory-related miARNs expression patterns in circulating VEs of young people with acute alcohol intoxication (IEA). Notably, the alterations in miARNs are dependent on the patient's gender, being women/females more affected by alcohol than men/males, since women/females showed lower presence of anti-inflammatory miARNs and a higher expression of inflammatory markers in brain tissue than men/males. / Ibáñez Cabanes, F. (2021). Papel de las vesículas extracelulares en la propagación y mantenimiento de la neuroinflamación inducida por el consumo de alcohol en la adolescencia [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/174214 / TESIS

Adipose tissue inflammation has an impact on liver health and it has been demonstrated that chronic alcohol consumption leads to the expression of pro-inflammatory markers in the adipose tissue. A thorough characterization of alcohol-induced adipose inflammation is lacking, and is important to understand in order to identify immune-related mechanisms that drive this phenomenon. Current therapeutic regimens for alcoholic liver disease are ineffective. It is critical to understand how other organs influence liver injury in this disease when developing novel and effective therapies in the future. Alcoholic liver disease exhibits a sexual dimorphism; women are more susceptible to liver injury than men and the same paradigm exists in rodent models. Here, I demonstrate that female mice have greater alcohol-induced adipose tissue inflammation than male mice, evidenced by greater expression of pro-inflammatory cytokines and cell markers. Further, female mice also exhibit higher expression of toll-like receptor genes in the adipose tissue, suggesting a potential role for the innate immune system in alcohol-induced adipose inflammation. Toll-like receptor 4 (TLR4) has been demonstrated to drive inflammation in both the liver and adipose tissue. I used both germline and conditional knockouts of Tlr4 to characterize alcohol-induced changes in the immune cell composition of adipose tissue. Alcohol increased the number of pro-inflammatory adipose tissue macrophages. This macrophage phenotype switching is partially dependent on TLR4; germline, but not myeloid-specific, Tlr4-deletion prevents macrophage phenotype switching. Overall, my work demonstrates that alcohol-induced adipose tissue inflammation is related to liver injury and that TLR4 contributes to adipose macrophage phenotype switching.