The innate immune system are a cooperation of many components – receptors being one of them. Both membrane-bound and cytosolic receptors play a large role in the defence system against pathogens and danger. NLRP3 is a receptor which assembles a protein complex called inflammasome in response to cytosolic stress and is responsible for many autoimmune diseases if it malfunctions. The activation of the NLRP3 inflammasome leads to secretion of inflammatory cytokines and in many cases to programmed cell death. The structure, function and activation of the NLRP3 inflammasome is still not fully understood and the urge to understand the mechanisms behind are important for future medical improvements. The aim was to anchor the NLRP3 inflammasome by the cell membrane - By Overlap PCR, the NLRP3 cDNA was fused extracellular and trans-membrane parts of the TLR4 cDNA to anchor the NLRP3 to the membrane and in turn analyse the inflammasome with LPI™ technology. Multiple primers and a TLR4 nucleotide were designed and the NLRP3 was amplified with specific overhangs by PCR. The fusion protein was successfully linked together by Overlap PCR but not confirmed by sequencing. The gene fusion demands high quality primers for amplification and further evaluation must be made to the details of the laboratory. To anchor the protein complex to the cell membrane, continue to be of full importance and can be an asset in many structural studies and biopharmaceuticals trials.

Autofagia e ativação dos inflamassomas são dois processos celulares autônomos, que podem interagir entre si. Estes processos participam ativamente do controle de infecções ocasionadas por diversos patógenos intracelulares. Anteriormente, descrevemos o inflamassoma NLRP3 no controle do T. cruzi, agente causador da Doença de Chagas. Entretanto, o papel da autofagia nesse controle não era conhecido. Neste trabalho, foi demonstrado que o T. cruzi induz aumento da expressão de LC3-II, formação de autofagossomas e autolisossomos em macrófagos peritoneais (MPs) de camundongos C57BL/6 selvagens. Ainda, a manipulação farmacológica da autofagia interferiu com a capacidade dos MPs em controlar a infecção pelo T. cruzi, apontando esse processo como um mecanismo efetor envolvido no controle do protozoário por macrófagos. Nesse contexto, NLRP3 parece funcionar como um modulador do processo autofágico. Na ausência de NLRP3, a manipulação farmacológica da autofagia não interferiu no controle do T. cruzi por MPs. Isso se correlacionou ao fato do fluxo autofágico se encontrar interrompido em MPs de camundongos deficientes para NLRP3 em resposta à infecção, mas não em resposta à rapamicina e starvation. A razão do bloqueio no fluxo autofágico parece ser a incapacidade de MPs deficientes em NLRP3 em formar autolisossomos, fato visualizado em microscopia confocal e eletrônica. Interessante, NLRP3 parece agir independente de caspase-1/11 na regulação da autofagia. Por outro lado, a análise da expressão de genes autofágicos por PCR-array revelou que MPs de animais deficientes em NLRP3 apresentam alta expressão basal de genes relacionados com a formação e maturação de autofagossomas e autolisossomas. Em contrapartida, a infecção pelo T. cruzi inibe a expressão desses genes na ausência de NLRP3, ao contrário da indução observada em MPs selvagens. Juntos, esses dados mostram que NLRP3 induz autofagia funcional em resposta ao T. cruzi, sendo sua presença fundamental para impedir o escape do parasita pela inibição de genes autofágicos. / Autophagy and inflammasome activation are two cell-autonomous and cross-regulated processes involved in host resistance against infections. Our group previously described that NLRP3 inflammasome is required for the control of T. cruzi, causative agent of Chagas disease. However, the involvement of autophagy in this process was largely unknown. Here, we demonstrated that T. cruzi is able to induce an increase in LC3II expression, formation of autophagosome and autolysosomes in peritoneal macrophages (PMs) from C57BL/6 mice. Moreover, the pharmacological modulation of autophagic machinery influenced the trypanocidal ability of PMs, pointing out autophagy as an effector mechanism to control T. cruzi infection. In this sense, NLRP3 seems to be involved in the modulation of the autophagic process. In the absence of NLRP3, the pharmacological modulation of autophagy did not interfere in the control of T. cruzi by PMs. Furthermore, autophagic flux is blocked in these cells in response to infection, but not in response to rapamycin and starvation. In fact, whereas T. cruzi induces the formation of large autolysosomes (LC3&#43 and Lysotracker&#43)-containing amastigotes in WT macrophages, only small and single positive vesicles are found in the absence of NLRP3. Interesting, NLRP3 appears to act independently of caspase-1/11 on the regulation of autophagy. On the other hand, the PCR-array analysis of autophagic genes demonstrated that NLRP3-/- PMs have higher basal expression of genes related to the formation and maturation of autophagosomes and autolysosomes in comparison to WT cells. In contrast, T. cruzi inhibited the expression of these genes in the absence of NLRP3, unlike the induction observed in WT PMs. Together, these data show that NLRP3 induces functional autophagy in response to T. cruzi being its presence required to overcome the escape of the parasite by preventing its inhibition of autophagic genes.

Cytosolic double stranded DNA (dsDNA) is sensed as a “danger signal” by host cells. Detection of viral and bacterial nucleic acid is emerging as a major route for cells to identify an infection by a pathogen. Recognition of cytoplasmic DNA causes death of some cells and interferon (IFN) and cytokine induction, which are appropriate anti-viral responses. Responses to cytoplasmic DNA may not only be relevant to certain retrovirus, DNA virus and bacterial infections, but could also be generated by reverse transcription of endogenous retro-elements. Introduction of DNA into the cytoplasm of bone marrow derived macrophages (BMM) causes upregulation of MHC Class I, induction of IFNβ and other cytokines and cell death. Both cytokine induction and cell death were independent of recognition of “CpG motifs” through TLR9. In order to determine whether a single receptor was likely to mediate these responses, the types of DNA eliciting these responses was compared. Both cellular activation to produce cytokines and IFNβ, as well as cell death were seen only with dsDNA but not single stranded DNA (ssDNA). Both responses increased with increasing DNA length, with little detectable effect of a double stranded 22bp oligonucleotide (ODN). The sequences of DNA leading to optimal induction of IFNβ and death were different. Although all dsDNA induced death of primary macrophages, poly(dA):(dT) was a particularly potent and rapid pro-death stimulus. In contrast, poly(dA):(dT) was a relatively poor stimulus for IFNβ, even at doses which were minimally toxic, or in cells which are resistant to DNA induced cell death. The alternating co-polymer poly(dA-dT) was the most potent inducer of IFNβ. This data suggests that separate DNA receptors mediate cell death and IFNβ induction in response to dsDNA Transfected dsDNA also rapidly activated caspase 3, a classical pro-apoptotic caspase, in BMM as early as 2½ minutes post-transfection with DNA. Caspase 3 is an effector caspase which is activated by an upstream initiator caspase. Although the apical caspase in the DNA detection system has not been defined, use of Bcl2 overexpressing BMM and caspase 2-/- BMM showed that DNA-dependent caspase 3 activation did not occur via the mitochondrial damage or the caspase 2 activation pathways. The inflammatory caspase, caspase 1 was also activated in response to DNA transfection, although whether caspase 1 is responsible for cleavage of caspase 3 has not been established. Caspase 1 activation suggests the involvement of the inflammasome, which is important for processing pro-inflammatory cytokines such as IL-1β into their biologically active forms. Furthermore, there is recent evidence suggesting that DNA-transfected cells die by a caspase 1-dependent cell death called pyroptosis. Other work in our lab identified the HIN-200 family member and candidate lupus susceptibility factor p202 as a candidate receptor for cytoplasmic dsDNA; p202 bound stably and rapidly to transfected DNA. Here, knockdown studies revealed p202 to be a regulatory protein limiting DNA-induced caspase 1 and 3 activation. Conversely, the related pyrin domain-containing HIN-200 factor AIM2 (p210), a candidate tumour suppressor, was required for caspase 1 and 3 activation by cytoplasmic dsDNA. Recently published work suggests that AIM2 multimerises along the length of the DNA leading to the formation of an inflammasome complex. The pyrin domain of AIM2 recruits the adaptor protein ASC through homotypic pyrin domain interactions. ASC subsequently recruits caspase 1, which results in its auto-activation. The inhibitory effect of p202 on caspase activation is likely to be due to its lack of a pyrin signalling domain. p202 rapidly binds to cytoplasmic DNA, and may reduce the clustering of AIM2 pyrin domains which results in caspase activation. Consistent with this proposal, DNA-dependent caspase activation correlated inversely with p202 expresssion in 3 mouse strains. This work defines HIN-200 proteins as a new class of pattern recognition receptors mediating responses to dsDNA. Work in this thesis aimed to understand the biological role and mechanism of responses to cytoplasmic DNA. Responses to cytoplasmic DNA are likely to be relevant not only to infectious disease but also to autoimmune diseases such as systemic lupus erythmatosus (SLE), where DNA appears to act as an adjuvant, and even tumour progression where there is evidence for a role for active endogenous retro-elements. In addition, responses to DNA may limit transfection efficiency and the efficacy of non-viral gene therapy.

Anesthesia-induced spatial learning impairments in aged rats model postoperative cognitive dysfunction (POCD) in the elderly surgical population. Mechanisms underlying both normal age-related cognitive decline and anesthesia-induced spatial learning deficits in aged rats were investigated. With respect to the involvement of inflammasome activation and age-related cognitive decline, I hypothesized that the aged hippocampus exhibits an elevated activation of inflammasome components contributing to elevated levels of IL-1β in the aged brain. Age-related cognitive decline was identified in a subpopulation of male Fischer 344 rats. Activation of the NLRP1 inflammasome was elevated in the aged brain, contributing to spatial learning deficits in aged rats. With respect to anesthesia-induced spatial learning impairment in aged rats, I hypothesized that an increase in NR2B subunit in the hippocampus and cortex during and following isoflurane anesthesia exposure resulting in spatial learning impairment in aged rats via disruption of downstream signaling molecule, extracellular-signal regulated protein kinase (ERK). Anesthesia exposure resulted in chronic spatial learning impairment in aged rats that were previously unimpaired in spatial learning tasks. Additionally, anesthesia induced elevated levels of N-methyl-D-aspartate (NMDA) receptor NR2B subunit protein expression in aged. It was concluded that various factors contribute to age-related spatial impairment including: NLRP1 inflammasome activation and NMDA receptor NR2B protein expression elevation. It was also concluded that anesthesia exposure exacerbates the elevation in NR2B protein expression in the aged brain, with subsequent disruption of ERK activation leading to chronic spatial learning deficits in aged rats. In the final chapter, a relationship for the interplay between inflammation and NMDA receptor function in the aged brain is discussed. In addition, a novel mechanism for anesthesia-induced cognitive deficits is presented. Therapeutic treatments for cognitive decline and anesthesia-induced cognitive deficits are explored. Finally, future lines of research are proposed.

Lethal toxin (LeTx) of Bacillus anthracis kills murine macrophages in a caspase-1 and Nod-like-receptor-protein 1b (Nlrp1b)-dependent manner. Nlrp1b detects intoxication, and self-associates to form a macromolecular complex called the inflammasome, which activates the pro-caspase-1 zymogen. I heterologously reconstituted the Nlrp1b inflammasome in human fibroblasts to characterize the role of the FIIND domain of Nlrp1b in pro-caspase-1 activation. Amino-terminal truncation analysis of Nlrp1b revealed that Nlrp1b1100-1233, containing the CARD domain and amino-terminal 42 amino acids within the FIIND domain was the minimal region that self-associated and activated pro-caspase-1. Residues 1100EIKLQIK1106 within the FIIND domain were critical for self-association and pro-caspase-1 activation potential of Nlrp1b1100-1233, but not for binding to pro-caspase-1. Furthermore, residues 1100EIKLQIK1106 were critical for cell death and pro-caspase-1 activation potential of full-length Nlrp1b upon intoxication. These data suggest that after Nlrp1b senses intoxication, the FIIND domain promotes self-association of Nlrp1b, which activates pro-caspase-1 zymogen due to induced pro-caspase-1 proximity.

Lethal toxin (LeTx) of Bacillus anthracis kills murine macrophages in a caspase-1 and Nod-like-receptor-protein 1b (Nlrp1b)-dependent manner. Nlrp1b detects intoxication, and self-associates to form a macromolecular complex called the inflammasome, which activates the pro-caspase-1 zymogen. I heterologously reconstituted the Nlrp1b inflammasome in human fibroblasts to characterize the role of the FIIND domain of Nlrp1b in pro-caspase-1 activation. Amino-terminal truncation analysis of Nlrp1b revealed that Nlrp1b1100-1233, containing the CARD domain and amino-terminal 42 amino acids within the FIIND domain was the minimal region that self-associated and activated pro-caspase-1. Residues 1100EIKLQIK1106 within the FIIND domain were critical for self-association and pro-caspase-1 activation potential of Nlrp1b1100-1233, but not for binding to pro-caspase-1. Furthermore, residues 1100EIKLQIK1106 were critical for cell death and pro-caspase-1 activation potential of full-length Nlrp1b upon intoxication. These data suggest that after Nlrp1b senses intoxication, the FIIND domain promotes self-association of Nlrp1b, which activates pro-caspase-1 zymogen due to induced pro-caspase-1 proximity.

Cholesterol crystals have been linked to activation of the NLRP3 inflammasome and the formation of foreign body giant cells (FBGCs). It has been hypothesized that FBGCs have a role in advanced atherosclerotic plaque formation. This thesis examined the feasibility of producing stable cultures of FBGCs starting with human monocytes with the goal to examine pterin production by these cells in comparison to human monocyte derived macrophages (HMDMs). The study also investigated the effect of cholesterol crystals on 7,8-dihydroneopterin (7,8-NP) production and modulation of IL-1β levels in macrophages. 7,8-Dihydroneopterin is a potent antioxidant generated by macrophages which also down regulates the expression of macrophage scavenger receptor CD36. The use of alpha-tocopherol and IL-4 as FBGC fusion mediators was explored. Using these mediators, large numbers of FBGC were successfully cultured. The rates of fusion achieved in the cultures were low, and the cells had poor adhesion, which prevented pterin measurement. FBGC, which are thought to remove crystallized cholesterol from the plaque, cleared 21% of cholesterol crystal compared to 50% cleared by HMDM cells. Due to this result, the effect of cholesterol crystals on pterin production in monocytes and macrophages was explored. Cholesterol crystals cause inflammation through the activation of the NLRP3 inflammasome, however, it was unknown whether they could modulate 7,8-NP production. Cholesterol crystals caused an intracellular dose-dependent loss of 7,8-NP to its oxidized form, neopterin, in HMDM cells. Cholesterol crystals induced intracellular synthesis of 7,8-NP in HMDMs. 7,8-NP was released into the supernatant and oxidized to neopterin in media. Monocytes treated with cholesterol crystals released up to 100 nM of neopterin and 120 nM of 7,8-NP in the media after 48 hours. The combination of IFN- and cholesterol crystals appeared to inhibit the release of 7,8-NP into the media for the first 48 hours, after this time 7,8-NP release rapidly increased. The addition of exogenous 200 μM 7,8-NP showed that in the presence of monocytes, cholesterol crystals did not cause the oxidation of 7,8-NP to neopterin, as seen in HMDMs but possibly to 7,8-dihydroxanthopterin or xanthopterin. The presence of 7,8-NP increased IL-1β expression in the presence of cholesterol crystals after 24 hours incubation. FBGCs and the removal of cholesterol crystals may be a key process in the resolution of atherosclerotic plaques. It appears that cholesterol crystals are able to modulate inflammatory processes including activation of the inflammasome and balance of 7,8-dihydroneopterin to the oxidized neopterin. The infiltrating monocytes may provide antioxidant protection against the inflammation induced by cholesterol crystals and the activity of the infammasome.

Age-related macular degeneration (AMD) is a principal cause of blindness in the United States and other industrialized nations. An estimated 10 million Americans are afflicted with AMD, which is comparable in scope to the 12 million living with cancer, or the 5 million with Alzheimer’s disease. The prevalence of AMD steadily increases with age, affecting 2% of the population at age 40, and one in four people by age 80. For reasons that are not fully understood, AMD is more common in lightly-pigmented and female populations. Treatment of AMD is largely an unmet need: There are no FDA approved therapies except for a small percentage of individuals with end-stage disease. This dissertation investigates the mechanisms of AMD pathogenesis and offers insight into novel therapeutic strategies for this disease.

La théorie du danger développée par Polly Matzinger stipule que l'attrait principal du système immunitaire ne réside pas dans la distinction entre le soi " à protéger " et le non-soi " à combattre ". Toute situation potentiellement délétère pour l'hôte, avec émission de signaux de dangers endogènes, est à ce titre capable de générer une réponse immunitaire afin de mobiliser les acteurs capables de permettre un retour à une situation basale. L'exposition des poumons de façon répétée à des agents toxiques environnementaux se traduit par une inflammation et une fibrose pulmonaire en condition stérile, c'est-à-dire sans intervention de micro-organisme. L'administration de Bléomycine dans les poumons de souris est un bon modèle pour étudier les molécules endogènes ou signaux de dangers engagés et les voies de signalisations associées. Nous avons identifié l'ATP extracellulaire et l'acide urique, provenant des cellules stressées ou endommagées, comme capables d'induire l'activation d'un complexe protéique cytoplasmique appelé inflammasome Nlrp3. Celui-ci conduit à la maturation de l'interleukine-1β, cytokine pro-inflammatoire. Dans une seconde partie, nous nous sommes intéressés aux mécanismes moléculaires d'activation de l'inflammasome Nlrp3 en réponse à des agents particulaires, responsables notamment de pathologies pulmonaires après inhalation. Nos résultats montrent que de l'ATP endogène est libéré activement par les cellules mises en contact avec des particules. Par un mécanisme autocrine/paracrine l'ATP va ensuite signaler sur plusieurs récepteurs purinergiques membranaires. Cette signalisation purinergique est importante dans la capacité des cellules à produire de l'interleukine-1β mature. Au final, les travaux présentés dans ce manuscrit attestent du rôle critique de molécules endogènes dans la mise en place d'une réponse immunitaire innée basée sur l'activité de l'inflammasome Nlrp3.

Chlamydia trachomatis (C. trachomatis) is the most prevalent sexually transmitted bacteria with devastating reproductive consequences that lead to tubal factor infertility (TFI). Recent studies have implicated apoptosis – associated speck – like protein containing a caspase recruitment domain (ASC) as an adaptor of inflammasomes that stimulate IL – 1β and IL – 18 secretion, pro – inflammatory cytokines with critical functions in host defense against a variety of pathogens. Therefore, for the first time, we are reporting the use of ASC-/- mice in a mouse model of Chlamydia infection that might provide some information on the role of inflammasomes in the pathogenesis of Chlamydia infection. In this study, wild type (WT) and ASC-/- mice were infected with Chlamydia. Infectivity, pathology of the upper genital tract (UGT), and, fertility were evaluated. In addition, expression of ASC – dependent inflammasomes and the activation of immune cells within the genital tract (GT) were studied. Results showed that Chlamydia infectivity in ASC-/- mice was significantly higher (p-/- mice which, when compared to infected WT mice, was exhibited by decrease in average number of pups and percent pregnancy. There was also severe UGT damage in ASC-/- mice compared to WT mice, correlating with the higher number of hydrosalpinx observed on the UGT of Chlamydia infected ASC-/- mice. Furthermore, IL – 1β and IL – 18 production as well as immune cell activation were down regulated in the GT of Chlamydia infected ASC-/- mice. This finding indicates that in absence of ASC, host innate and adaptive immunity is impaired. Results imply that ASC plays a protective role in the mucosal immunity against GT Chlamydia infection.