Tight regulation of caspase-9, a key initiator of apoptosis, is required to uphold cellular homeostasis. Although it is controlled on a multifactorial level, misregulation of this process does occur, which is a characteristic of a variety of diseases from ischemic injury to cancer. Therefore it remains important to gain a detailed understanding of the mechanisms behind native caspase-9 regulatory pathways and harness these mechanisms for therapeutic purposes. Based on known mechanisms, such as the unique inhibitory complex of caspase-9 and XIAP-BIR3, development of synthetic regulators can be envisioned, while other mechanisms such as zinc-mediated inhibition and CARD activation of caspse-9 remain undefined. Intrigued by the multiple ways to control caspase-9’s activity, we sought after designing synthetic caspase-9 inhibitors in addition to defining the mechanistic details metal regulation and CARD domain activation. We report the first stabilized α-helical peptides that harness the native regulatory mechanism of caspase-9 and the BIR3 domain which lead to the understanding of the importance of exosites in inhibitory complexes. Our studies also revealed that there are two distinct zinc binding sites, one at the active site and another at a novel zinc binding site of yet unknown function in caspase-9 however this site may have the potential to control caspase-6 based on its regulatory mechanism. Furthermore, an interaction was discovered between CARD and the catalytic core of caspase-9 in the presence of a properly formed substrate binding groove, a potential mechanism utilized by the apoptosome for activation of the enzyme. All in all, the regulation of caspase-9 occurs on a variety of levels that requires almost every surface of the enzyme. Through exploring these underlying molecular details behind the various mechanisms, not only has the field of caspase-9 regulation mechanisms been extended, essential information was gained for further pursuit in an advancement towards the design of caspase-9 activators and inhibitors.

As a front-line treatment for cardiovascular disease, statins are among some of the most widely prescribed drugs worldwide. Statins are effective at lowering cholesterol, but approximately 7-29% of patients report some form of adverse muscle effect during the course of treatment. The severity of these side effects ranges from low-level to life-threatening myopathy. The mechanism of statin myopathy remains ill-defined, but muscle-specific E3 ubiquitin ligases have been implicated. In addition, statins have been shown to activate caspase-1 (and increase IL-1β) in immune cells, which is a key effector of the NLRP3 inflammasome. The relevance of this inflammatory response in statin myopathy remains unknown. Using C2C12 myotubes, an in vitro model of statin-induced myopathy was developed to test the impact of NLRP3 inflammasome activation on markers of statin myopathy. Gene expression of the muscle-specific E3 ubiquitin ligases atrogin-1 and MuRF-1 (atrogenes) were used as markers of statin-induced myopathy. Lipopolysaccharide priming of the NLRP3 inflammasome was found to lower the effective dose of fluvastatin required to augment atrogene expression. This effect correlated with reduced phosphorylation of Akt and FOXO3a, a transcription factor regulating atrogene expression. Statin-induced atrogene expression was also found to be dependent on an isoprenoid that is required for protein prenylation rather than cholesterol biosynthesis pathways. Fluvastatin increased caspase-1 activity in a prenylation-dependent manner and selective inhibitors of NLRP3 and caspase-1 were able to prevent increased atrogene expression with fluvastatin treatment. Therefore, the NLRP3 inflammasome contributes to markers of statin-induced myopathy through a prenylation-dependant pathway in muscle cells. This work presents a novel mechanism involved in statin myopathy, and has shown that the inflammasome may represent a new drug target to mitigate muscle symptoms in patients taking statins. / Thesis / Master of Science (MSc) / Statins are a class of widely prescribed cholesterol-lowering drugs that reduce the risk of heart attack and stroke. However, many patients often complain of statin-induced muscle side effects (myopathy) that impact their quality of life. Symptoms of this statin-induced myopathy can manifest as muscle pain and weakness. The underlying biology causing this condition is still not well understood. Independent of its cholesterol-lowering effect, statins can activate an immune receptor called the NLRP3 inflammasome, indicating that inflammation may contribute to myopathy. Therefore, the primary goal of this study was to determine if this immune response contributes to statin-induced myopathy. It was found that inhibition of the NLRP3 inflammasome lowers markers of statin myopathy. Results from this study will provide further insight into mechanisms regulating this myopathy, and may lead to new treatments that can help alleviate statin side effects in muscle.

No description available.

Atherosclerosis, considered a chronic inflammatory disease, is the underlying mechanism for several cardiovascular diseases. Hyperlipidemia is the number one risk factor for atherogenesis. Caspase-1 is an inflammatory caspase, which can be activated by the metabolic stresses through pathogen associated molecular patterns (PAMPs)-recognition receptors, (PRR) recognition and inflammasome assembly. Activated caspase-1 can initiate inflammation in multiple ways. Thus, regulating inflammasome components expression is essential to control caspase-1 activation and its subsequent inflammatory processes. I hypothesized that the readiness of inflammasome component expression for caspase-1 activation in tissues is an index for inflammation privilege. Endothelial cells (EC) which are the innermost layer of the vessel and are the critical gatekeeper for monocyte migration. The first step of atherogenesis is activation of ECs, which allows monocyte adhesion and migration into the sub-endothelial layer. I also hypothesized that caspase-1 can sense hyperlipidemia and regulate EC activation and inflammation during early atherogenesis. I first determined the expression profiles of inflammasome components, pro-inflammatory caspases and PRRs is different among tissues, and cardiovascular tissues express relative less PRRs via a database-mining method. According to the readiness of inflammasome components, tissues could be classified into three tiers. The first tier consists of tissues with constitutively expressed inflammasomes. The second tier of tissues includes potentially inducible expression of one inflammasome component. The third tier of tissues has inducible expression of at least two inflammasome components. This three-tier model can be applied to determine the inflammation privilege of tissues in response to pro-inflammatory stimuli. I also demonstrated that hyperlipidemia induced caspase-1 expression and activation in aorta along with the atherogenesis in apolipoprotein E (ApoE)-/- mice with high fat (HF) diet, experimentally. We then generated the ApoE-/-/Casp-1-/- double knockout mice, and found that the ApoE-/-/Casp-1-/- mice contained significantly less atherosclerotic lesion in aortic sinus and less cytokine and chemokine expression in aortic tissues compared with ApoE-/- mice. ApoE-/-/Casp-1-/- mice also had less CD11b+/F4/80- neutrophil and CD11b+/F4/80+ monocyte recruitments into aorta compared with ApoE-/- mice. However, the percentage of monocyte subsets in peryphery blood remained at the same level in between ApoE-/- mice and ApoE-/-/Casp-1-/- mice. I then proposed that perhaps the caspase-1 activation in vascular cells, in ECs played the essential role of controling monocyte migraion. My in vitro data demonstrated that oxidized low density lipoprotein (ox-LDL) and its componnents could induced caspase-1 activation in human aortic ECs (HAECs) through ROS pathway which then led to EC activation and pyroptotic cell death. Deficiency of caspase-1 in aortic EC attenuated hyperlipidemia induced EC activation and inflammtion. Mechanically, I found that caspase-1 deficiency accumulated an anti-atherogenic protein, Sirt-1 in the aorta. Collectively, our data suggested that caspase-1/inflammasome in ECs can sense hyperlipidemia, become activated, drive EC activation, and promote monocyte recruitment and early atherosclerosis. / Pharmacology

Caspase-3 has demonstrated a non-apoptotic function in several developmental programs including skeletal muscle differentiation, yet the mechanism of action has not been fully elucidated. Under apoptotic conditions Caspase-3 induces DNA fragmentation through activation of CAD. Recent observations have demonstrated CAD activity and the resulting DNA strand breaks are also vital for skeletal muscle differentiation. These breaks are transient in nature, suggesting an active DNA repair program to maintain genomic integrity. The aim of this study was to delineate the DNA repair mechanism coordinated with caspase/CAD mediated DNA damage. It was found that XRCC1 formed punctate nuclear foci early in myoblast differentiation concurrent to the induction of DNA damage. Caspase-3 inhibition caused attenuation of the formation of DNA lesions and XRCC1 foci in differentiating myoblasts. Targeted reduction in XRCC1 expression impaired myoblast differentiation. These results suggest that XRCC1 may play a role in repairing the DNA damage associated with myoblast differentiation.

Caspase-9 has two splice variants, pro-apoptotic caspase-9a and anti-apoptotic caspase-9b, and dysregulation of caspase-9 splice variant ratio or expression of caspase-9b isoform has been linked to augmentation of the anchorage-independent growth and tumorigenic capacity of non-small cell lung cancer (NSCLC) cells. This study focuses on cell signaling pathway(s) regulating the alternative splicing of caspase-9 pre-mRNA and mechanistic roles of caspase-9b in a certain oncogenic/survival pathway. In regards to the former, we have identified hnRNP U as a novel splice-enhancer associated with exon 3 of caspase-9 (C9/E3). Moreover, hnRNP U binds specifically to C9/E3 at an RNA cis-element previously reported as the binding site for the splicing repressor, hnRNP L. Phosphorylated hnRNP L interferes with hnRNP U for binding to C9/E3, and our results demonstrate the importance of the phosphoinositide 3-kinase/AKT pathway in modulating the association of hnRNP U to C9/E3. Overall, a mechanistic model has been revealed where hnRNP U competes with hnRNP L for C9/E3 binding to enhance the inclusion of the four-exon cassette, and this splice-enhancing effect is blocked by the AKT pathway via phosphorylation of hnRNP L. As to the latter aim, it is unknown about the mechanistic roles of caspase-9b besides the inhibitory effect on caspase-9a processing. In this study, caspase-9b has been demonstrated to have a dual function in regulating the survival/oncogenic nuclear factor κB (NF-κB) pathway, which is independent from modulating caspase-9a activation. In particular, caspase-9b has been shown to activate the canonical arm and inhibit the non-canonical arm of the NF-κB pathway by destabilizing NF-κB inhibitor alpha (IκB-α) and NF-κB-inducing kinase (NIK). Importantly, this new role for caspase-9b contributes to the enhanced survival and anchorage-independent growth of NSCLC cells conferred by caspase-9b expression. Further mechanistic studies have demonstrated a direct association of caspase-9b with the cellular inhibitor of apoptosis 1 (cIAP1), a regulatory factor in both arms of the NF-κB network, via its IAP-binding motif. Through this interaction, caspase-9b induces the E3 ligase activity of cIAP1, which regulates NF-κB activation, and promotes the survival, anchorage-independent growth and tumorigenicity of NSCLC cells. Overall, a novel tumorigenic mechanism has been identified, by which alternative mRNA processing regulates the NF-κB signaling independent of external agonist.

Plasmodium falciparum est un protozoaire parasite responsable du paludisme causant la mort d’environ un million de personnes par an. La résistance médicamenteuse du parasite augmente la pathogénicité de cette maladie. Il est question ici d’explorer les mécanismes moléculaires impliqués dans la mort cellulaire programmée (apoptose) du parasite en présence de chloroquine (CQ) et de tester l’hypothèse qu’une résistance à la CQ peut s’expliquer en partie par une défaillance de ce mécanisme de mort. Dans un premier temps l’étude des marqueurs de l’apoptose (TUNEL, JC1, formes pyknotiques) montre qu’une souche sensible de parasite (3D7) à la CQ peut subir une apoptose en présence de CQ alors qu’une souche résistante (7G8) présente un défaut d’apoptose. Dans un deuxième temps nous montrons que la protéine PfMCA1 (P. falciparum métacaspase 1) présente une structure et une maturation protéolytique proche de celui des caspases faisant de cette protéine un candidat potentiellement impliqué dans l’apoptose du parasite. Dans un troisième temps nous montrons que l’expression du domaine catalytique de PfMCA1 dans la levure induit une mort cellulaire et un retard de croissance de la levure. Nous montrons également que PfMCA1 présente une activité enzymatique de type arginase alors que les effets induit par sa surexpression peuvent être inhibés par l’ajout d’un inhibiteur de protéases spécifiques des aspartates. Ces résultats suggèrent que PfMCA1 pourrait agir comme une protéine initiatrice induisant l’action d’une protéase effectrice spécifique des aspartates conduisant à la mort cellulaire. Cette hypothèse testée chez la levure reste à confirmée chez P. falciparum / Plasmodium falciparum is a protozoan parasite responsible for malaria causing one million deaths per year. Drug resistance of the parasite increases the pathogenicity of this disease. In this thesis, it is question to explore the molecular pathway involved in programmed cell death (apoptosis) of the parasite in the presence of chloroquine (CQ) and to test the hypothesis that CQ resistance could be partly explained by a failure of such a mechanism. In a first step, we showed that a sensitive clone (3D7) exhibited the classical hallmarks of apoptosis (DNA fragmentation, mitochondrial depolarization) under a CQ pressure while a resistance clone failed to undergo apoptosis. In a second step we show that the protein PfMCA1 (P. falciparum metacaspase 1) has a structure and a processing similar to the well known caspases which are the key effectors of apoptosis for higher eukaryotic cells. In a third step we show that expression of the catalytic domain of PfMCA1 in yeast induces cell death and growth retardation of yeast. We show that PfMCA1 presented an arginine-specific protease activity while the effects induced by its overexpression were inhibited by an aspartate-specific protease inhibitor (z-VAD-fmk). These results suggest that PfMCA1 might act as an initiator protein inducing an aspartate-specific protease effector leading to cell death. This hypothesis tested in yeast remains to be confirmed in P. falciparum

Introdução: A disfunção erétil (DE) é definida como a incapacidade de alcançar ou manter a ereção do pênis para um desempenho sexual satisfatório, contribuindo significativamente para a baixa qualidade de vida e morbidade psicossocial masculina. A endotelina-1 (ET-1), um potente peptídeo vasoconstritor que promove contração lenta e sustentada em células de músculo liso vascular, possui grande importância na fisiopatologia da DE. Diversos estudos mostram que o aumento da expressão de mediadores inflamatórios está intimamente ligado ao desenvolvimento da DE. O inflamassoma é um complexo multiprotéico do sistema imune inato que atua através da ativação da caspase-1 e resulta na maturação de citocinas pró- inflamatórias, tais como interleucina- IL (IL-l?). O receptor NLRP3 faz parte do inflamassoma e sua ativação leva a clivagem de caspase-1 e consequente secreção de IL-1?. A ET-1, também possui papel importante na inflamação crônica vascular, mediando a liberação de citocinas pró-inflamatórias. No entanto, ainda é desconhecido se a ação pró- inflamatória da ET-1 em células de músculo liso é mediada pela ativação da via do inflamassoma. Hipótese: A ET-1 ativa o NLRP3 em células do músculo liso do corpo cavernoso (CMLCC), promovendo alterações na reatividade do corpo cavernoso (CC). Objetivo: Avaliar o papel da endotelina-1 na ativação do NLRP3 em CMLCC de camundongos. Métodos: CMLCC de camundongos C578BL/6 (WT) e NLRP3-/- foram cultivadas em meio de cultura DMEM acrescido de soro fetal bovino (SFB), 10%, foram pré- incubadas com endotelina-1 nas concentrações de 10-9, 10-8 e 10-7 M, em presença de LPS ou veículo. Avaliamos o efeito da deleção do NLRP3 sobre a reatividade do CC (contratilidade e relaxamento mediante estímulos por campo elétrico e/ou farmacológico). Após, avaliamos o efeito da ET-1 na ativação do NLRP3, nas alterações sobre a reatividade do CC de camundongos WT, e se estas persistiriam nos camundongos NLRP3-/- e caspase1/11-/- . Resultados: As células apresentaram-se fluorescentes para marcação para ?-actina e não para Von Willebrand, caracterizando assim que não houve contaminação com células endoteliais. A incubação com a ET-1 10-7 M por 24 h na presença de LPS ou veículo aumentou a atividade da caspase-1 em CMLCC de camundongos WT e este efeito não ocorreu nas CMLCC de camundongos NLRP3-/-. Não se observou diferença com relação à massa corporal ou massa dos órgãos entre os animais WT e NLRP3-/-. O CC de animais NLRP3-/- apresenta prejuízo para o relaxamento mediado por nitroprussiato de sódio (NPS) quando comparado com as tiras de CC de camundongos WT. A incubação com ET-1 10-7 M por 4 horas promove aumento na contração para fenilefrina (PE) e prejuízo no relaxamento induzido por nitroprussiato de sódio (NPS), e o mesmo efeito não é observado nas tiras de CC de camundongos NLRP3-/- e caspase1/11-/-. Conclusão: O NLRP3 contribui para o aumento na contração e prejuízo no relaxamento produzido pela ET-1 em CC de camundongos, possivelmente através da ativação da caspase-1 / Introduction: Erectile dysfunction (ED) is defined as the inability to achieve or maintain penile erection to perform sexual intercourse, it contributes significantly to the low quality of life and male psychosocial morbidity. Endothelin-1 (ET-1), a potent vasoconstrictor peptide that promotes slow and sustained contraction of vascular smooth muscle cells, has great importance in the pathophysiology of ED. Several studies show that increased expression of inflammatory mediators is closely linked to the development of ED. The inflammasome is a multiproteic complex of the innate immune system that acts through activation of caspase-1, which leads to maturation of pro-inflammatory cytokines such as interleukin-1 beta (IL-l?). The activation of NLRP3 receptor, part of the inflammasome, leads to caspase-1 cleavage and subsequent secretion of IL-1?. ET-1 also plays an important role in chronic vascular inflammation by mediating the release of pro-inflammatory cytokines. However, it is still unknown whether pro-inflammatory actions of ET-1 on smooth muscle cells is mediated by the activation of the inflammasome. Hypothesis: ET-1 activates NLRP3 in smooth muscle cells of the corpora cavernosa (SMCCC), promoting changes in corpus cavernosum (CC) reactivity. Objective: To evaluate the role of endothelin-1 in the activation of the NLRP3 in SMCCC of mice. Methods: SMCCC of C57BL/6 (WT) and NLRP3-/- mice were grown in DMEM culture medium supplemented with bovine fetal serum (FBS) 10%, pre-incubated with endothelin-1 at concentrations of 10-9, 10- 8 and 10-7M, in the presence of LPS or vehicle. We evaluated the effect of the NLRP3 deletion on the reactivity of the CC (contractility and relaxation by electric field and/or pharmacological stimulation). After that, we evaluated the ET-1 effect on activation NLRP3, changes on the reactivity of the CC of WT, and if these alterations would persist NLRP3-/- and caspase1/11-/- mice. Results: The cells presented fluorescent labeling to ?-actin, but not for Von Willebrand factor, characterizing absence of endothelial cells contamination. The incubation with 10-7 M ET-1 for 24 h in the presence of LPS or vehicle increased caspase-1 activity in SMCCC from WT, but not from NLRP3-/- mice. No difference was observed in body mass or weight of the organs between WT and NLRP3-/- animals. The CC from NLRP3-/- animals displayed impaired relaxation mediated by sodium nitroprusside (SNP) when compared to WT CC. The incubation with ET-1 10-7 M for 4 hours promoted an increase in the contraction to phenylephrine (PE) and reduced relaxation induced by sodium nitroprusside (SNP). The same effect was not observed in CC strips from NLRP3-/- and caspase1/11-/- mice. Conclusion: NLRP3 contributes to the increase in contraction and impaired relaxation produced by ET-1 in mice CC, possibly by activation of caspase-1

O estresse causa modificações no epitélio intestinal, tais como o aumento de células caliciformes e da taxa de apoptose. O uso de alimentos nutracêuticos tem sido uma alternativa para amenizar essas modificações sobre o tecido epitelial. Desta forma, este trabalho teve como objetivo avaliar se a inclusão de colostro bovino, o qual é constituído de fatores antioxidantes, imunes e de crescimento, seria capaz de amenizar as consequências do estresse crônico sob o intestino. Para isso, juvenis de pacu (Piaractus mesopotamicus) adensados a 50 kg/m3 foram alimentados duas vezes ao dia até a saciedade com ração peletizada e semi-purificada sem (0%CBL) e com a inclusão de colostro bovino liofilizado em concentrações crescentes (10, 20 e 30%CBL), (n=4). Após 28 dias, foram coletados segmentos do intestino médio, S1 e S2, e reto. Os tecidos foram marcados com corantes histológicos para a quantificação de células caliciformes contendo mucinas neutras, ácidas (incluindo sialo e sulfomucinas) e ácidas-neutras. Também foram mensurados o volume (Vv) e a densidade da superfície (Sv) da mucosa, por análise estereológica, e a espessura da camada muscular. A razão do número de cada tipo e subtipo de célula caliciforme sobre o Vv e Sv foi calculada para estimar a densidade de células caliciformes, Dv e Ds, respectivamente. A taxa apoptótica foi analisada qualitativamente através da intensidade (alta, média e baixa) da imunomarcação da caspase-3 nas células epiteliais. As dietas não influenciaram os parâmetros zootécnicos analisados (P>0,05). No reto, os grupos que receberam 20 e 30%CBL apresentaram menor número de células caliciformes contendo sulfomucinas e menor Ds em relação a 0 e 10% (P=0,0148 e 0,0198, respectivamente). No RT, Dv total e Dv de células caliciformes contendo mucinas ácidas foi maior em 0 e 30%CBL em relação a 20%CBL (P=0,0155 e 0,225, respectivamente). No S1, 10 e 30%CBL apresentaram maior Dv em relação a 20%CBL (P=0,0540). A espessura da camada muscular, o Vv e a Sv não diferiram entre os tratamentos (P>0,05). No S2 e RT, a taxa de apoptose teve relação inversa à concentração de colostro bovino liofilizado adicionado na ração. Nos três segmentos, houve maior proporção de células caliciformes contendo mucinas ácidas do que neutras, sendo a maioria representada por sulfomucinas. Assim, a inclusão de colostro bovino liofilizado nas rações de juvenis de pacu adensados diminuiu a apoptose nos segmentos intestinais S2 e RT e também diminuiu o número de células caliciformes contendo sulfomucinas no RT, indicando que o colostro bovino liofilizado pode ser utilizado como alimento nutracêutico para pacus (Piaractus mesopotamicus) adensados, a fim de diminuir a taxa apoptótica e proteger o intestino contra enzimas bacterianas, uma das principais funções das sulfomucinas. / The stress causes changes in the intestinal epithelium, such as the increase in the number of goblet cells and on the rate of apoptosis. The use of nutraceutical foods has been an alternative to soften these modifications on the epithelial tissue. Thus, this study aimed to evaluate if the inclusion of bovine colostrum, which is composed of antioxidant, immune and growth factors, would be able to attenuate the consequences of chronic stress on the intestine. For this, pacu juveniles (Piaractus mesopotamicus), stocked at density of 50 kg/m3, were fed twice daily until satiety with pelleted and semi-purified diet without (0% LBC) and with the inclusion of lyophilized bovine colostrum in increasing concentrations (10, 20 and 30% LBC), (n = 4). After 28 days, segments of the middle gut, S1 and S2, and rectum (RT) were collected. The tissues were stained with histological dyes for the quantification of goblet cells containing neutral, acidic (including sialo and sulphomucins) and acid-neutral mucins. The volume (Vv) and surface density (Sv) of the mucosa were also measured by stereological analysis and the thickness of the muscular layer. The ratio between the number of each goblet cell type and subtype and the Vv or Sv was calculated to estimate the density of goblet cells, Dv and Ds, respectively. The apoptotic rate was analyzed qualitatively according to the intensity (high, medium and low) of caspase-3 immunostaining in epithelial cells. The diets did not influence the zootechnical parameters analyzed (P> 0.05). In the rectum, the groups that received 20 and 30% LBC presented lower number of goblet cells containing sulphomucins and lower Ds in relation to 0 and 10% (P = 0.0148 and 0.0198, respectively). In RT, total Dv and Dv of goblet cells containing acid mucins were higher in 0 and 30% LBC in relation to 20% LBC (P = 0.0155 and 0.225, respectively). In S1, 10 and 30% LBC presented higher Dv in relation to 20% LBC (P = 0.0540). Muscle layer thickness, Vv and Sv did not differ between treatments (P> 0.05). In S2 and RT, the rate of apoptosis was inversely related to the concentration of lyophilized bovine colostrum added in the diet. In the three segments, there was higher proportion of goblet cells containing acidic than neutral mucins, most of them being sulphomucins. Thus, the inclusion of lyophilized bovine colostrum in diets of pacu juveniles reduced apoptosis in the intestinal segments S2 and RT and also decreased the number of goblet-containing sulphomucins in the RT, indicating that lyophilized bovine colostrum can be used as a nutraceutical feed for pacus (Piaractus mesopotamicus) under high stocking density to decrease the apoptotic rate and protect the intestine against bacterial enzymes, one of the main functions of sulphomucins.

A bactéria Legionella pneumophila é um bacilo Gram-negativo, flagelado causador da doença dos legionários e febre de Pontiac. O inflamassoma mais importante no controle da replicação desta bactéria é o composto por Naip5/NLRC4, que é responsável pelo reconhecimento de flagelina. A ativação do inflamassoma de Naip5/NLRC4 pela flagelina induz a ativação de caspase-1, induzindo a formação de poros na membrana, piroptose e controle da replicação desta bactéria. A participação da proteína adaptadora ASC é essencial para a nucleação deste complexo e secreção de citocinas inflamatórias como IL-1? e IL-18 por esta via. Além do controle da replicação de L. pneumophila pelo inflamassoma NLRC4 dependente de caspase-1, foi demonstrado que existe uma via induzida por NLRC4 independente de caspase- 1/11. Dessa forma, camundongos e células Nlrc4-/- são mais susceptíveis à infecção por esta bactéria do que as células Casp1/11-/-. Neste trabalho, nós identificamos que a via independente de caspase-1/11 é composta por Naip5/NLRC4/ASC/Caspase-8 e é essencial para o controle da replicação de Legionella spp. flageladas em macrófagos e in vivo. Através da utilização de BMDMs Casp1/11-/- e Asc/Casp1/11-/- transduzidos com NLRC4-GFP ou ASC-GFP, identificamos que a formação de punctas de NLRC4 e ASC dependem do reconhecimento de flagelina e que ASC é essencial para a formação desses punctas. Também foi identificado que a infecção com L. pneumophila que expressa flagelina leva à ativação de caspase-8 de maneira dependente de ASC e Naip5, mas independente de caspase-1/11. De acordo com esses dados, o silenciamento de caspase-8 em macrófagos Casp1/11-/- aumentou a susceptibilidade dessas células à infecção com L. pneumophila flagelada. Além disso, macrófagos e camundongos Asc/Casp1/11-/- foram tão susceptíveis quanto os Nlrc4- /- e mais susceptíveis que os Casp1/11-/-. Nós observamos que o inflamassoma de NLRC4/ASC/Caspase-8 induz formação de poros e morte celular independente de gasdermina-D (GSDMD). Por meio da utilização de células de camundongos C57BL/6, foi observado que caspase-8 é recrutada para o inflamassoma de Naip5/NLRC4/ASC/Caspase-1. Entretanto, a ativação de caspase-8 só ocorre na 10 ausência de caspase-1 ou GSDMD. Nossos dados sugerem que a ativação de caspase-8 no inflamassoma composto por NLRC4/ASC/Caspase-8 representa uma via alternativa que opera para garantir o controle da replicação de bactérias flageladas em situações nas quais ou caspase-1 ou GSDMD estão inibidas. / Legionella pneumophila is a flagellated Gram-negative bacillus that is the causative agent of the legionnaire\'s disease and Pontiac fever. The most important inflammasome for the control of L. pneumophila replication is the Naip5/NLRC4, responsible for the flagellin recognition. The activation of the Naip5/NLRC4 inflammasome leads to caspase-1 activation, consequently pore formation, pyroptosis and control of bacterial replication. The participation of the adaptor molecule ASC is essential for this complex nucleation and the secretion of inflammatory cytokines like IL-1? and IL-18 by this pathway. Besides the control of L. pneumophila replication by Naip5/NLRC4/Caspase-1 inflammasome, it was demonstrated there are NLRC4 responses independent of caspase-1/11. These explain why mice and macrophages Nlrc4-/- are more susceptible than Casp1/11-/-. In this work, we identified that the caspase-1/11-independent pathway is composed of Naip5/NLRC4/ASC/Caspase-8 and it is essential for the control of flagellated Legionella spp. replication in macrophages and in vivo. Infection of Casp1/11-/- and Asc/Casp1/11-/- macrophages, transduced with NLRC4-GFP or ASC-GFP, showed that flagellin-positive bacteria triggered puncta formation that is ASC-dependent. Accordingly, Naip5 and ASC, but not caspase-1/11, were required for caspase-8 activation in response to flagellated bacteria. Silencing caspase-8 in Casp1/11-/- BMDMs increased the susceptibility to L. pneumophila infection. Furthermore, the macrophages and mice Asc/Casp1/11-/- are as susceptible as Nlrc4-/-, but more susceptible than Casp1/11-/-. We also found that the NLRC4/ASC/Caspase-8 inflammasome induces GSDMD-independent pore formation and cell death. Using C57BL/6 cells, we observed that caspase-8 is recruited to Naip5/NLRC4/ASC/Caspase-1 inflammasome. However, caspase-8 is just activated in the absence of caspase-1 or GSDMD. Our data suggest that caspase-8 activation in the NLRC4/ASC/Caspase-8 inflammasome represents an alternative pathway that operates to ensure the control of flagellated bacteria replication in situations which either caspase-1 or GSDMD are inhibited.