Hepatitis C virus (HCV) infection is a leading cause of liver transplantation and hepatocellular carcinoma worldwide. HCV, like all obligate parasites, relies on host pathways to facilitate its pathogenesis. In particular, the virus possesses an intimate link with hepatic lipid metabolism, promoting a lipid-rich cellular environment conducive to HCV propagation. Clinically, these metabolic perturbations manifest as steatosis in over 50% of patients. The majority of research to-date examining how the virus co-opts hepatic lipid pathways has been focused on coding genes and their protein products. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression, which have been implicated in virtually every cellular process. Through interactions with partially complementary mRNAs, each individual miRNA has the capacity to repress the expression of hundreds of genes and induce significant regulatory effects. Herein, we demonstrate that hepatic miRNAs, including miR-7, miR-27a/b, miR-130b, and miR-185, act as crucial regulatory molecules to the maintenance of hepatic lipid homeostasis. These miRNAs cooperate to regulate fatty acid and cholesterol metabolism. HCV modulates the expression of a subset of these miRNAs (miR-27a/b, miR-130b, and miR-185) to promote hepatocellular lipid accumulation and the HCV life cycle. There appears to be a broad viral requirement for lipids, and the mammalian innate immune response strategically targets host metabolic pathways to restrict virus’ access to key lipid species. We demonstrate that 25-hydroxycholesterol, a broadly anti-viral oxysterol produced as part of the innate anti-viral response, activates miR-185 expression in the liver to deplete virus infected cells of lipids. HCV appears to actively counteract this anti-viral response by suppressing miR-185 expression. Collectively, our results highlight the role of microRNAs in hepatic lipid metabolism and the immunometabolic response to viral infection.

Orientadores: Andrei Carvalho Sposito, Eliana Cotta de Faria / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T15:58:25Z (GMT). No. of bitstreams: 1 Virginio_VitorWilsondeMoura_M.pdf: 2355466 bytes, checksum: 77d04b1ff5936c26cd64f6d516447ff7 (MD5) Previous issue date: 2014 / Resumo: Atualmente aproximadamente 17 milhões de pessoas morrem por doenças cardiovasculares (DCV). No Brasil durante 2011 ocorreram cerca de 200 mil óbitos por DCV principalmente em decorrência da aterosclerose, classificada como uma doença imuno-inflamatória crônica que causa estreitamento luminal e gera diversas fatalidades como infarto agudo do miocárdio (IAM), acidente vascular encefálico (AVE), doença arterial periférica (DAP), entre outras. A DAP prediz outras DCV e é a principal causa de amputação devido a obstrução das artérias de membros inferiores. Análises histológicas indicam que a ruptura da placa, capa fibrosa do ateroma, erosão da placa e nódulo calcificado geram as principais fatalidades nas DCV. Até o momento não há biomarcadores plasmáticos para uso clínico afim de avaliar a estabilidade e a gravidade das placas ateroscleróticas, esse fato motiva a exploração de componentes envolvidos em doenças imuno-inflamatórias e no metabolismo lipídico tais como oxisteróis e marcadores da síntese e absorção do colesterol (MSAC). Os oxisteróis, principalmente 27-hidroxicolesterol (27-HDC) e 7-ketocolesterol estão envolvidos em mecanismos fisiopatológicos da aterosclerose e desempenham um papel na inflamação, citotoxicidade e necrose, além de funções no metabolismo lipídico. É relatado que os oxisteróis 24S- e 25-hidroxicolesterol (24S- e 25-HDC) estão envolvidos em doenças neurodegenerativas como Alzheimer. Estudos apontam uma causa em comum entre Alzheimer e aterosclerose, ambas são doenças inflamatórias crônicas, assim são necessárias mais investigações desses oxisteróis na doença aterosclerótica. A proteína de transferência de éster de colesterol (CETP) e proteína de transferência de fosfolípides (PLTP), assim como as lipoproteínas, podem mediar a transferência desses lípides. O objetivo desse trabalho foi quantificar concentrações de oxisteróis (24S-, 25-, 27-HDC e 7-ketocolesterol) e marcadores de síntese (desmosterol e latosterol) e absorção (?-sitosterol e campesterol) do colesterol em ateromas e plasma de 10 indivíduos com DAP (grupo DAP) e comparar com artérias sem ateroma de 13 voluntários (grupo CTL). Também relacionar esses esteróis com outros parâmetros como histórico clínico e proteínas de transferência de lípides. Foram realizadas análises clínicas e bioquímicas, classificações histológicas dos ateromas e quantificações de esteróis em artérias e plasma. Para interpretação dos dados obtidos foram usados testes estatísticos de comparação e correlação. Os dois grupos apresentaram uma similaridade nos dados clínicos e bioquímicos, diferenciando apenas em pressão arterial sistólica, atividade da PLTP, glicose e proteína C reativa (PCR), maiores no grupo DAP. A PCR se correlacionou positivamente com 24S-HDC, 25-HDC e 27-HDC plasmáticos. Comparação entre os grupos em relação a concentração de oxisteróis em artérias mostraram o 24S-HDC e o 27-HDC maiores no ateroma do grupo DAP, curiosamente o 25-HDC esteve maior em CTL. Quantificações de oxisteróis em plasma do grupo DAP mostrou um aumento do 25-HDC em relação ao grupo CTL. Não houve diferença significante para os MSAC. Os ateromas classificaram-se em 50% ruptura da placa, 20 % capa fibrosa do ateroma, 10% nódulo calcificado e 10% lesão fibrocálcica. Em conclusão mostramos o acumulo do 24S-HDC em placas de aterosclerose. O 27-HDC assim como outros estudos esteve em maior concentração nos ateromas indicando fortemente sua ação na patogênese dessa doença. Todos os oxisteróis sintetizados enzimaticamente dosados em plasma se correlacionaram positivamente com atividade inflamatória sistêmica, assim como 24S-HDC e 27-HDC em ateromas. Finalmente a atividade da PLTP esteve maior no grupo DAP / Abstract: Currently around 17 million people die from cardiovascular diseases (CVD). In Brazil, durant 2011 nearly 200.000 deaths occur mainly due atherosclerosis, classified as a chronic, immune-inflammatory disease that causes luminal narrowing and generates several outcomes as myocardial infarction, cerebrovascular accidents (stroke), peripheral arterial disease (PAD) and others. DAP predict other CVDs and is the leading cause of amputation due the clogged arteries in inferior members. Histological analysis indicates that plaque rupture, fibrous cap atheroma, plaque erosion and calcified nodules generate major CVD fatalities. So far, no plasma biomarkers for clinical use are available in order to evaluate the stability and extend of atherosclerotic plaques, therefore is necessary the investigation of the components involved in immune-inflammatory diseases and lipids metabolism such as oxysterols and cholesterol synthesis and absorption markers (CSAM). Oxidized sterols or oxysterols, manly 27-hidroxycholesterol and 7-ketocholesterol are involved in pathophysiological mechanisms of atherosclerosis and play a role in inflammation, apoptosis, necrosis and cytotoxicity, in addiction to lipid metabolism. Oxysterols 24S- and 25-hidroxycholesterol are involved with neurodegenerative diseases such Alzheimer¿s and resemble with atherosclerosis development since are chronical inflammatory diseases, for this reason is necessary an investigation of these oxysterols in atherosclerosis is needed. Cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) as well as lipoproteins, could mediate the transfer of the oxysterols. The aim of this work was quantified the levels of oxysterols (24S- 25-, 27- hidroxycholesterol and 7-ketocholesterol) and cholesterol synthesis (desmosterol and lathosterol) and absorption markers (?-sitosterol and campesterol) in atheroma and plaques from 10 individuals with PAD (PAD group) and compare with arteries without atheroma of 13 volunteers (CTL group). Also, relate these sterols with other parameters as clinical history, lipid transfer proteins and histological scores. Clinical and biochemical data were collected and sterols were quantified in arteries and plasma by gas chromatography coupled to a mass spectrophotometer (GCMS) and histological atheroma classification were determined. Comparison anlysis were performed using Mann-Whitney and correlations by Spearman¿s test. Both groups were not different regarding clinical and biochemical data, differencing only for systolic blood pressure, alcohol intake, PLTP activity, glucose and CRP, increased in PAD group. CRP was positively correlated with 24S-HDC, 25-HDC e 27-HDC from plasma. The comparisons of oxysterols levels in atheroma showed that 24-HDC and 27-HDC were increased in PAD group; interestingly, the 25-HDC was higher in CTL group. No significant differences were observed for CSAM between groups. The atheroma were classified as 50% plaque rupture, 20% fibrous cap, 10% calcified nodules and 10% of fibrocalcific injury. From the oxysterols from plasma of PAD group, only 25-HDC was increased compared to the CTL group. In conclusion, we demonstrated the accumulation of 24S-HDC in atherosclerotic plaques. The 27-HDC, as well as other studies, present higher levels in atheroma¿s, strongly indicating is role in the disease pathogenesis. All oxysterols measured in plasma were positively correlated with the systemic inflammatory activity. Finally, all atherosclerotic plaques were classified as lesions of advanced extension, according to the histological analysis / Mestrado / Clinica Medica / Mestre em Clinica Medica

Sterols play many important roles in physiology, including maintaining cell membrane integrity, and producing vitamin D and steroid hormones. Recent studies implicate sterol metabolism in the host innate immune response. Previous work, based on transcriptional profiling studies of mouse cytomegalovirus (MCMV) infection of primary bone-marrow-derived macrophages (BMDM, MΦ), uncovered a previously uncharacterized role of interferon in regulating the cholesterol pathway. Notably, Toll-like receptor (TLR) induced interferon modulates the suppression of SREBP2 (Sterol Regulatory Element-Binding Protein 2) activation, the master transcription factor for sterol biosynthesis. This finding resulted in the downregulation of the sterol biosynthesis pathway. However, how interferon is molecularly linked to sterol metabolism, and what part of the pathway mediates the antiviral effect remains unknown. The central hypothesis of the thesis is that the antiviral effect of interferon is in part mediated by secondary sterol metabolites and the dependency of viral replication on the host mevalonate branch of the sterol biosynthesis pathway. To test this hypothesis, my studies have examined the components of the host sterol pathway and their respective roles in influencing viral replication. Paradigmatically, I used MCMV and BMDM to explore the host- metabolic-virus interactions. Specifically, my findings address the question of how MCMV replication depends on the sterol biosynthesis pathway, and how the pathway is modulated by interferon as an antiviral response. In Chapter 2, the importance of the sterol biosynthesis pathway for viral replication was investigated using a combination of gene silencing and pharmacological inhibitors. These studies demonstrated that resistance to viral infection through suppressing the cholesterol pathway is not due to a requirement of the virus for cholesterol itself, but instead involves the mevalonate-isoprenoid arm of the pathway. This branch of the pathway chemically links lipids to specific host proteins (protein prenylation). These results suggest a new role for the mevalonate arm during viral infection. In Chapter 3, I examined what part of the sterol pathway mediates the antiviral effects. Oxysterols are natural modulators of sterol biosynthesis, and are produced by the oxidation of cholesterol by the enzyme cholesterol hydroxylase. Oxysterol suppression of SREBP2 activation leads to transcriptional repression of the sterol biosynthesis pathway. Additionally, oxysterols also modulate cholesterol homeostasis through cholesterol efflux. My studies led to identifing cholesterol-25-hydroxylase (Ch25h) as an interferon-stimulated gene (ISG). CH25H oxidizes cholesterol to produce a soluble oxysterol metabolite, 25-hydroxycholesterol (25-HC). Treatment of cells with 25-HC resulted in antiviral effects against MCMV and MHV-68. 25-HC was found to have no effects on MCMV entry into the host cell, but rather mediated inhibition of viral gene transcription. In addition, 25-HC-specific antiviral effect partially involved the suppression of the isoprenoid pathway, rather than cholesterol efflux. This work uncovered a physiological role for 25-HC as a sterol-lipid effector of an innate immune pathway. The antiviral activity of 25-HC in a lipid replete condition was found to occur at a concentration higher than the concentration required to inhibit SREBP2 activation. This implies that the antiviral effects of 25-HC is independent of SREBP2 in sterol replete conditions. Conversely, the antiviral action of 25-HC was signifi enhanced in cells under sterol-depleted conditions, suggesting that the antiviral effect of 25- HC is likely mediated through multiple processes involving SREBP2 dependent and independent mechanisms. These sterol dependent and independent mechanisms are examined in Chapter 4, using pathway expression profiling and pharmacological synergy studies. These studies showed that 25-HC suppression of the isoprenoid synthetic pathway is crucial in controlling infection, but also highlighted that other 25-HC dependent antiviral mechanisms are likely to exist. The inhibition of the mevalonate-isoprenoid arm by statins and 25-HC clearly demonstrated that MCMV replication dependents on protein prenylation. Chapter 5 investigation showed that either chemical inhibition of geranylgeranylation of host proteins or limiting mevalonate production led to restriction of MCMV replication. Importantly, through a series of systematic loss of function siRNA screenings demonstrated that specific host RabGTPases mediating vesicular transport pathways play vital roles in the replication and the assembly of the virus. This finding provides new mechanistic insights in to the dependency of cytomegalovirus replication on the host cell trafficking pathways and lays the groundwork for further definition of this important aspect of host-viral interactions. In summary, the overall findings of this research support the original hypothesis, by highlighting the importance of the host mevalonate-isoprenoid pathway, and provide further definition of the mechanisms and components linking sterol metabolism with interferon mediated antiviral effect.

Akt plays a role in protecting macrophages from apoptosis induced by some oxysterols. Previously we observed enhanced degradation of Akt in P388D1 moncocyte/macrophages following treatment with 25-hydroxycholesterol (25-OH) or 7-ketocholesterol (7-KC). In the present report we examine the role of the ubiquitin proteasomal pathway in this process. We show that treatment with 25-OH or 7-KC results in the accumulation of poly-ubiquitinated Akt, an effect that is enhanced by co-treatment with the proteasome inhibitor MG-132. Modification of Akt by the addition of a Gly-Ala repeat (GAr), a domain known to block ubiquitin-dependent targeting of proteins to the proteasome, resulted in a chimeric protein that is resistant to turn-over induced by 25-OH or 7-KC and provides protection from apoptosis induced by these oxysterols. These results uncover a new aspect of oxysterol regulation of Akt in macrophages; oxysterol-stimulated poly-ubiquitination of Akt and degradation by the proteasomal pathway.

The oxysterol, 25-hydroxycholesterol (25-HC), is important for sterol metabolism and emerging evidence suggests that 25-HC plays a more critical role in immunity and infection. However, the precise antiviral mechanism and the target of 25- HC remains unclear. Here efforts were made to investigate the link between viral infection and the triggering of the 25-HC associated interferon (IFN) response, and how this dynamically alters the endogenous level of 3-hydroxy- 3-methylglutaryl-CoA reductase (HMGCR), a key enzyme that catalyses the production of the precursor of cholesterol and oxysterols. In this thesis I have sought to specifically explore the temporal changes and role of HMGCR in DNA virus (cytomegalovirus) and RNA (Influenza) virus infections. I hypothesise that HMGCR is a target for 25-HC associated IFN-mediated host defence against viral infection. To characterise HMGCR and test this hypothesis, the following objectives were defined: (1). To establish an experimental system to quantitatively study the endogenous HMGCR protein level; (2). To investigate the mechanism of the down-regulation of HMGCR involved in the IFN-mediated innate immune response; (3). To study the behaviour of HMGCR in the influenza virus induced 25-HC associated IFN-mediated innate immune response; (4). To study the behaviour of HMGCR in the cytomegalovirus induced 25-HC associated IFN-mediated innate immune response. Chapter 3, describes establishing an experimental system for the quantification of endogenous HMGCR levels. Different protein detection methods, including a modified western blot protocol and immunostaining, were tested. The results of RNA interference of HMGCR demonstrate that under lipid-deficient condition with the supplementation of mevastatin (an HMGCR inhibitor) the modified western blot protocol specifically detects endogenous HMGCR. This chapter lays the foundational work for the temporal analysis and testing the role of HMGCR in infection. In Chapter 4, the mechanism of the degradation of HMGCR following 25-HC and IFN treatments, in wild-type and Ch25h−/− mouse bone marrow derived macrophages (BMDMs), was investigated. Similar to 25-HC, IFN-γ treatment results in the drop of both the transcript and protein abundance of HMGCR in wild-type BMDMs. Differential temporal analysis of RNA and protein alterations and the use of proteasome inhibitors reveals that both 25-HC and IFN-γ lead to a marked reduction of HMGCR protein via a proteasomal degradation mechanism within early times of treatments. Further, the immediate reduction of HMGCR levels induced by IFN-γ was completely abrogated in Ch25h−/− BMDMs. Hence, the reduction of HMGCR following IFN-γ treatment is due to the de novo synthesis in macrophages of 25-HC. However, the decrease of Hmgcr gene expression was observed in not only wild-type but also Ch25h−/− BMDMs, suggesting additional mechanisms for regulating Hmgcr RNA levels. These results demonstrate the mechanism of the down-regulation of HMGCR resulted from the induction of IFN response during viral infection, is only partially due the de novo synthesis of 25-HC. In chapter 5, influenza A virus was used to investigate the role of HMGCR in the IFN-mediated innate immune response. The inhibition of HMGCR by RNA interference inhibited viral growth, suggesting the requirement of HMGCR for optimal intracellular viral growth. Viral infection in wild-type murine BMDMs reduced the endogenous HMGCR levels. However, the reduction of HMGCR at early times was prevented in Ch25h−/− BMDMs. Intriguingly, the decrease of HMGCR at late time points was still observed in Ch25h−/− BMDMs. These results indicate that the down-regulation of HMGCR with influenza virus infection in BMDMs at early times is completely due to the de novo synthesis of 25-HC; whereas at late times alternative pathways or mechanisms exist. Additionally, human epithelial A549 cells and A549/PIV5-V cells that are deficient in STAT1 were used to study the role of IFN pathway in the down-regulation of HMGCR at late times during viral infection. Results from these studies show that at late times the reduction of HMGCR is due to IFN-independent mechanisms. Chapter 6, extends these investigations to the herpes virus murine cytomegalovirus and infection of BMDMs. HMGCR is known to be essential for cytomegaloviral infections and 25-HC, statin and RNAi inhibition of HMGCR restrict viral growth. 25-HC is shown to reduce HMGCR at immediate early times of infection. However, most notably, the down-regulation of HMGCR was also observed in Ch25h−/− BMDMs at late times with murine cytomegalovirus infected BMDMs. These results confirm that alternative pathways or mechanisms exist, playing roles in the crosstalk between cholesterol metabolism and innate immune response. Collectively, this study characterises the role of HMGCR in the 25-HC associated IFN-mediated host defence against viral infection. Results indicate that, in addition to the IFN-mediated host response, alternative pathways or other mechanisms also result in the down-regulation of HMGCR during viral infection. HMGCR is at the crossroad of different pathways or mechanisms, and is therefore not only targeted by 25-HC. Hence, further questions can be addressed from these results: (1). What are the alternative pathways or mechanisms for the down-regulation of HMGCR? (2). How do these pathways or mechanisms work in hosts’ immune system? Answering these questions can contribute to refining the pathway map of innate immunity and understanding the precise role of HMGCR, or even the sterol biosynthesis pathway, in hosts’ immune response against pathogens.

The ATP-binding cassette (ABC) transporter, ABCA1, plays a pivotal role in reverse cholesterol transport, which is the elimination of excess sterols from peripheral cells and their transport to the liver for elimination. Early studies failed to detect significant ATPase activity, prompting the suggestion that ABCA1 was an ATP-regulated receptor, rather than an active transporter. We have provided evidence that ABCA1 can bind ATP and trap its hydrolysis product, ADP, in the presence of either ortho-vanadate or beryllium fluoride and Mg2+ or Mn2+. We have also shown that both nucleotide-binding domains (NBDs) trap nucleotide comparably, suggesting that ABCA1 is a functional ATPase. In addition, we have shown that ABCA1 can directly transport 25-hydroxycholesterol (25-OHC) in an ATP-dependent manner using a membrane vesicle uptake assay, and can do so when the physiological substrate acceptor apoA-I is replaced with BSA as a non-specific binding protein. Although more than 50 naturally occurring missense mutations and polymorphisms in ABCA1 have been identified in individuals with HDL-C levels within the lowest 5th percentile of the general population, the extent to which many of these mutations affect ABCA1 function is not known and cannot be predicted. Naturally occurring extracellular loop (ECL) mutations W590S and C1477R have both been shown to effectively eliminate the ability to mediate lipid efflux, despite the fact that the W590S mutant protein retains the ability to bind apoA-I. We show that neither mutant can transport nor efflux 25-OHC, whether in the presence of apoA-I or BSA, despite apparently full retention of the ability to bind and trap nucleotide. This suggests that these two ECL mutations inhibit transport by a mechanism that is independent of their effect on apoA-I binding. By introduction of naturally occurring mutations in the NBDs, we show that although some mutations associated with Tangier Disease, such as N935S, essentially eliminate nucleotide trapping and substrate translocation, other polymorphisms such as L1026P and T2073A associated with low HDL-C, appear to be fully functional. Lastly, we observed differences in the behaviour of both wild-type and mutant forms of ABCA1-GFP depending on whether they were expressed in insect or mammalian cell lines. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2010-02-12 11:14:11.381