Viral modulation of interferon (IFN) responses to african swine fever virus (ASFV)

Tese de mestrado. Biologia (Biologia Humana e Ambiente). Universidade de Lisboa, Faculdade de Ciências, 2011A imunidade inata constitui a primeira resposta dada por um hospedeiro quando atacado por agentes patogénicos. A resposta imune baseia-se em genes codificados na linha germinativa, chamados receptores de reconhecimento de padrões (PRRs). Estes conseguem distinguir o “Eu” do “não-Eu”, reconhecendo padrões moleculares conservados ao longo da evolução dos vários agentes patogénicos, chamados padrões moleculares associados a agentes patogénicos (PAMPs). No caso dos vírus, um parasita intracelular obrigatório, os PAMPs mais importantes e mais estudados são o seu material genético, tal como o DNA genómico viral, RNA de cadeia dupla (ds) ou simples (ss) ou a estrutura RNA viral, 5’- trisfosfato-RNA. Existem vários PRRs, que podem ser agrupados em classes: os receptores transmembranares do tipo Toll (TLRs), os receptores citoplasmáticos do tipo RIG-I (RLRs), os receptores do tipo Nod (NLRs) e os receptores do tipo AIM2 (ALRs). Os PRRs iniciam uma sinalização em cascata que culmina com a activação de factores de transcrição, que entre outros, vão induzir a produção e excreção duma citoquina, o interferão (IFN). Este grupo de citoquinas é composto por três classes, IFN tipo I (p.e IFN-α/β) , tipo II (p.e IFN-γ) ou do tipo III (p.e. IFN-λ). O IFN pode despoletar variados efeitos anti-virais. A cascata de sinalização estimulada pelo IFN inicia-se com a ligação do IFN ao seu respectivo receptor extra celular que ,através de fosforilações, permite a activação de receptores intracelulares. Já no interior da célula, sinalizadores de transdução e ativadores da transcrição (STATs) são recrutados e fosforilados, o que permite a formação de homo ou heterodímeros que migram para o núcleo. No núcleo, as STATs ligam-se a zonas promotoras de genes estimulados pelo IFN (ISGs), para promover a transcrição de mais de 300 ISGs com propriedades anti-virais. No caso do estímulo causado por IFN do tipo I, os complexos formados pelas STATs vão ligar-se ao elemento de resposta estimulado pelo IFN (ISRE). No caso do IFNs do tipo II, os complexos ligam-se à sequência activada pelo IFN-λ (GAS). Os ISGs facultam ao hospedeiro diversas estratégias para combater a infeção viral. Apesar de os mamíferos possuírem um sistema imune bastante evoluído, os vírus também têm evoluído estratégias para evitar e/ou manipular as defesas do hospedeiro, dedicando uma parte substancial do seu genoma a estas estratégias. Estas podem ir desde uma interferência global na expressão e/ou síntese de proteínas das células do hospedeiro, ou serem mais específicas, diminuindo o impacto dos IFNs. O estudo destas interações, pode não só ser útil para conhecer os mecanismos de infecção do vírus, mas também para perceber melhor os mecanismos de defesa do hospedeiro. Estes conhecimentos podem permitir o desenvolvimento de terapias e tratamentos anti-virais ou mesmo anticancerígenos. A peste suína africana (ASF) é uma doença que nos porcos domésticos (Sus sacrofa) é tipicamente hemorrágica e leva normalmente à morte do hospedeiro. Contudo, as infecções são assintomáticas nos hospedeiros naturais, o javali, o porco selvagem e a carraça, sendo esta última, um dos principais vectores de transmissão do vírus da peste suína africana (ASFV), tornando o seu controlo difícil sem uma vacina. Nos últimos anos, devido ao grande desenvolvimento urbano e consumo de carne de porco, têm havido surtos de ASF em África, causando perdas devastadoras. O ASFV é um virus de DNA de cadeia dupla, o único arbovírus de DNA e o único membro da familia Asfaviridae, infectando principalmente macrófagos e monócitos. Tal como todos os vírus, o ASFV contém genes que manipulam a biologia da célula do hospedeiro, como por exemplo, genes que inibem a apoptose e respostas imunes controladas pelo factor nuclear kappa B (NFκB), entre outros. Contudo, ainda não foi demonstrado que algum gene do ASFV consiga inibir a resposta do IFN. Isto é surpreendente, pois o ASFV infecta macrófagos, um tipo de célula sensível ao IFN e porque a sua infecção persistente, é incompatível com uma resposta efectiva mediada por IFN. O K205R é um gene do ASFV sem função definida, mas ensaios preliminares de luciferase mostraram que este gene consegue inibir a resposta do IFN. Contudo, os mecanismos utilizados pelo K205R nesta inibição são desconhecidos. O objectivo desta dissertação de mestrado é tentar perceber melhor estes mecanismos e determinar a sequência mínima necessária para que o K205R tenha o efeito observado. O K205R foi isolado através de PCR, utilizando como molde o DNA genómico da estirpe do AFSV, BA71. Subsequentemente, foiclonado no plasmídeo pcDNA3, que contém um marcador molecular, a hemaglutinina (HA), a montante da zona de inserção do gene. Para determinar a extensão da ação do K205R, foram feitos ensaios de luciferase utilizando células transfectadas com repórteres de luciferase sobre o controlo dos promotores de IFN- β, ISRE e GAS. O K205R mostrou inibição para todos os reporteres. Para tentar definir a zona do K205R responsável pelo efeito observado, fez-se uma previsão da estrutura secundária da proteína do K205R, recorrendo à bioinformática, que permitiu identificar uma sequência “coiled-coil” putativa, uma estrutura secundária associada a interações entre proteínas. Também é sugerida uma sequência putativa para um sinal de exportação nuclear (NES). Com base nesta análise foram construídos quatro fragmentos do K205R e posteriormente clonados no pcDNA3. Depois de se verificar a sequencia correcta de DNA de cada um dos clones e expressão das suas proteinas em células vero transfectadas , o passo seguinte foi verificar a localização celular destes fragmentos através de imunofluorescência nestas mesmas células. Esta experiência permitiu verificar que de facto, os fragmentos que não tinham a sequência putativa NES, em comparação com células transfectadas com o K205R inteiro, tinham uma maior acumulação nuclear. Para estudar o mecanismo, e a que nivel o K205R actua para inibir a via de sinalização do ISRE, foi feito um “western blot” utilizando extractos proteicos de células VERO transfectadas com os diferentes fragmentos do K205R e posteriormente estimuladas com IFN-β durante 15 minutos e durante 45 minutos. Esta experiência permitiu verificar que a fosforilação da STAT1 diminui na presença do K205R, contudo, apenas um fragmento reproduziu este efeito. Este fragmento de 75 aminoácidos não contém a sequência, nem para a sequência “coiled-coil”, nem para NES. Esta dissertação de mestrado apresenta resultados consistentes com a existência de um NES funcional na sequência do K205R, uma inibição da fosforilação da STAT1 mediada pelo K205R, mas também apresenta uma abordagem para determinar os mecanismos utilizados pelo K205R para inibir a indução e o impacto do IFN-β. Contudo, mais experiências têm de ser feitas para realmente se comprovar a existência de um NES, como por exemplo, ensaios de imunofluorescência de células transfectadas com K205R na presença de Leptomicina B, um inibidor da exportação nuclear. Também será necessário estudar as vias de sinalização inibidas pelo K205R que não foram abordadas neste trabalho, tal como a via de indução de IFN-β e a via do GAS.A key part of the innate response to virus infections is the interferon (IFN) response. This can limit virus replication and dissemination and is a critical factor in controlling virus infections, particularly persistent viruses. Many viruses encode proteins which interfere with induction of IFN and IFN-activated pathways and these can have important roles in virus pathogenesis and persistence. African Swine Fever (ASF) causes major economic losses in many African countries and is a threat to pig farming worldwide. There is no vaccine and therefore options for disease control are limited. In Europe, there is always the danger of accidental introduction of the virus, as indeed occurred in Portugal in 1957, causing severe financial losses. Thus, defining the mechanism of proteins involved in evasion of the host’s defense response and in virus virulence is of extreme interest, so we can understand the virus and try to develop strategies to reduce ASF impact. ASFV is a large cytoplasmic DNA virus which encodes between 160 to 175 open reading frames. Many of its genes are not essential for replication in vitro, but are host evasion strategies facilitating virus replication and transmission in vivo. These include proteins which inhibit host defence systems. Surprisingly, since ASFV can survive as a persistent virus, no ASFV proteins have been described which inhibit the IFN response. However, the early gene K205R, might have an impact on IFN response. Luciferase assays, shown inhibitions of IFN induction (IFN-β) and IFN-signalling (ISRE, GAS) pathways. Using a bioinformatics tool (Jpred), we got a predication of K205R protein secondary structure. Based on this prediction, deletion mutant fragments of K205R were constructed and used in immunofluorescence and western blot assays. The immunofluorescence results suggest the presence of a functional nuclear export signal (NES) motif in the K205R protein sequence. Western blot experiments suggested that K205R is affecting the phosphorylation status of STAT1, in cells stimulated with IFN-β (ISRE pathway). Although it was not possible to clearly determine the minimum sequence needed for all the functions of K205R, the results suggest that K205R inhibition of the impact of IFN type I, depends on a sequence within amino acids 130 and 205, which affects STAT1 phosphorylation. Further experiments should be done to investigate the mechanism of K205R inhibition in the pathways not covered on this thesis (IFN-β induction pathway and GAS pathway). The existence of functional NES also needs confirmation

MicroRNA regulation of CD8+ T cell responses.

MicroRNAs (miRNAs) are a class of short noncoding RNAs that play critical roles in the regulation of a broad range of biological processes. Like transcription factors, miRNAs exert their effects by modulating the expression of networks of genes that operate in common or convergent pathways. CD8+ T cells are critical agents of the adaptive immune system that provide protection from infection and cancer. Here, we review the important roles of miRNAs in the regulation of CD8+ T cell biology and provide perspectives on the broader emerging principles of miRNA function

The bacillary and macrophage response to hypoxia in tuberculosis and the consequences for T cell antigen recognition

M. tuberculosis is a facultative anaerobe and its characteristic pathological hallmark, the granuloma, exhibits hypoxia in humans and in most experimental models. Thus the host and bacillary adaptation to hypoxia is of central importance in understanding pathogenesis and thereby to derive new drug treatments and vaccines

Vaccinia protein C16 blocks innate immune sensing of DNA by binding the Ku complex

VACV gene C16L encodes a 37-kDa protein that is highly conserved in orthopoxviruses and functions as an immunomodulator. Intranasal infection of mice with a virus lacking C16L (vΔC16) induced less weight loss, fewer signs of illness and increased infiltration of leukocytes to the lungs compared with wild-type virus. To understand C16’s mechanism of action, tandem affinity purification and mass spectrometry were used to identify C16 binding partners. This revealed that Ku70, Ku80 and PHD2 interact with C16 in cells. Ku70 and Ku80 constitute the Ku heterodimer, a well characterised DNA repair complex. MEFs lacking Ku, or the other component of the DNA-dependent protein kinase (DNA-PK) complex, the catalytic subunit of DNA-PK (DNA-PKcs), were shown to be deficient in the upregulation of IRF-3-dependent genes such as Cxcl10, Il6 and Ifnb in response to transfection of DNA, but not poly (I:C). Furthermore, following infection of MEFs with VACV strain MVA the activation of Cxcl10 or Il6 transcription was dependent on DNA-PK. Therefore, DNA-PK is a DNA sensor capable of detecting poxvirus DNA and activating IRF-3-dependent innate immunity. C16 inhibited the binding of Ku to DNA, and therefore inhibited DNA-mediated induction of Cxcl10 and Il-6 in MEFs. The role of C16 in vivo was also examined: infection with vΔC16 led to increased production of Cxcl10 and Il-6 following intranasal infection of mice compared with wild-type virus. C16 is therefore an inhibitor of DNA-PK-mediated DNA sensing and innate immune activation. C16 was also shown to bind to PHD2, an enzyme involved in regulation of hypoxic signalling. VACV was found to activate the transcription of hypoxia-related genes, and C16 expression in cells was also capable of doing this. The role of hypoxic signalling in VACV infection remains poorly understood

Interferon-γ induces immunoproteasomes and the presentation of MHC I-associated peptides on human salivary gland cells.

A prominent histopathological feature of Sjögren's syndrome, an autoimmune disease, is the presence of lymphocytic infiltrates in the salivary and lachrymal glands. Such infiltrates are comprised of activated lymphocytes and macrophages, and known to produce multiple cytokines including interferon-gamma (IFN-γ). In this study, we have demonstrated that IFN-γ strongly induces the expression of immunoproteasome beta subunits (β1i, β2i and β5i) and immunoproteasome activity but conversely inhibits the expression of proteasome beta subunits (β1, β2 and β5) in human salivary gland (HSG) cells. Mass spectrometric analysis has revealed potential MHC I-associated peptides on the HSG cells, including a tryptic peptide derived from salivary amylase, due to IFN-γ stimulation. These results suggest that IFN-γ induces immunoproteasomes in HSG cells, leading to enhanced presentation of MHC I-associated peptides on cell surface. These peptide-presenting salivary gland cells may be recognized and targeted by auto-reactive T lymphocytes. We have also found that lactacystin, a proteasome inhibitor, inhibits the expression of β1 subunit in HSG cells and blocks the IFN-γ-induced expression of β1i and immunoproteasome activity. However, the expression of β2i and β5i in HSG cells is not affected by lactacystin. These results may add new insight into the mechanism regarding how lactacystin blocks the action of proteasomes or immunoproteasomes

Pathogen-dependent role of turbot (Scophthalmus maximus) interferon-gamma

11 páginas, 7 figurasInterferon-gamma has been typically described as a pro-inflammatory cytokine playing an important role in the resolution of both viral and bacterial diseases. Nevertheless, some anti-inflammatory functions are also attributed to this molecule. In this work we have characterized for the first time the turbot (Scophthalmus maximus) interferon-gamma gene (ifng) and its expression pattern under basal conditions, after type I IFNs administration, and viral and bacterial infection. The intramuscular injection of an expression plasmid encoding turbot Ifng (pMCV1.4-ifng) was not able to affect the transcription of numerous immune genes directly related to the activity of IFN-gamma, with the exception of macrophage-colony stimulating factor (csf1). It was also unable to reduce the mortality caused by a Viral Hemorrhagic Septicemia Virus (VHSV) or Aeromonas salmonicida challenge. Interestingly, at 24 h post-infection, turbot previously inoculated with pMCV1.4-ifng and infected with VHSV showed an increase in the expression of pro-inflammatory cytokines and type I IFNs compared to those fish not receiving expression plasmid, indicating a synergic effect of Ifng and VHSV. On the other hand, some macrophage markers, such as the macrophage receptor with collagenous structure (marco), were down-regulated by Ifng during the viral infection. Ifng had the opposite effect in those turbot infected with the bacteria, showing a reduction in the transcription of pro-inflammatory and type I IFNs genes, and an increase in the expression of genes related to the activity of macrophagesThis work has been funded by the projects CSD2007-00002 “Aquagenomics”, 201230E057 (CSIC) and AGL2014-51773-C3 from the Spanish Ministerio de Economía y Competitividad. P. Pereiro received a predoctoral grant from the gs3:Ministerio de Educación [F.P.U. fellowship AP2010-2408]Peer reviewe

Analysis of the role of the p47 GTPase IIGP1 in Resistance against Intracellular Pathogens

IIGP1 is a member of the p47 GTPase family of IFNγ-induced proteins, which are among the most potent presently known mediators of cell-autonomous resistance against intracellular bacterial and protozoan pathogens in the mouse. From all studied members of this family IIGP1 is the best characterized with respect to biochemical characteristics and enzymatic activity in vitro, as well as membrane binding properties and dynamic behavior in cells. The role of the protein in intracellular defense was however, unknown and this study was set as an initial attempt to reveal it. This thesis describes the generation of an IIGP1 deficient mouse and analysis of the susceptibility of this animal to pathogens from protozoan and bacterial origin, which employ diverse strategies for host cell invasion and intracellular survival and replication. Despite having intact adaptive immune system, the IIGP1 deficient mice showed higher incidence of development of cerebral malaria after infection with Plasmodium berghei sporozoites. In addition, IIGP1 deficient astrocytes exhibited a partial loss of IFNγ-induced inhibition of Toxoplasma gondii growth. IIGP1 deficient animals were not susceptible to infection with Leishmania major, Listeria monocytogenes, Chlamydia trachomatis and Anaplasma phagocytophilum. From the analysis of the obtained data in the context of the intracellular lifestyle of the pathogens involved in this study, we concluded that IIGP1 seems to be specifically involved in defense against protozoan parasites, which like Pl. berghei and T. gondii reside in non-fusigenic parasitophorous vacuoles after entering cells. The mechanisms of IIGP1-dependent protection of cells against these pathogens remain to be studied

The peripheral blood transcriptome in septic cardiomyopathy: an observational, pilot study.

BACKGROUND:Septic cardiomyopathy (SCM) is common in sepsis and associated with increased morbidity and mortality. Left ventricular global longitudinal strain (LV GLS), measured by speckle tracking echocardiography, allows improved identification of impaired cardiac contractility. The peripheral blood transcriptome may be an important window into SCM pathophysiology. We therefore studied the peripheral blood transcriptome and LV GLS in a prospective cohort of patients with sepsis. RESULTS:In this single-center observational pilot study, we enrolled adult patients (age > 18) with sepsis within 48 h of admission to the ICU. SCM was defined as LV GLS > - 17% based on echocardiograms performed within 72 h of admission. We enrolled 27 patients, 24 of whom had high-quality RNA results; 18 (75%) of 24 had SCM. The group was 50% female and had a median (IQR) age of 59.5 (48.5-67.0) years and admission APACHE II score of 21.0 (16.0-32.3). Forty-six percent had septic shock. After filtering for low-expression and non-coding genes, 15,418 protein coding genes were expressed and 73 had significantly different expression between patients with vs. without SCM. In patients with SCM, 43 genes were upregulated and 30 were downregulated. Pathway analysis identified enrichment in type 1 interferon signaling (adjusted p < 10-5). CONCLUSIONS:In this hypothesis-generating study, SCM was associated with upregulation of genes in the type 1 interferon signaling pathway. Interferons are cytokines that stimulate the innate and adaptive immune response and are implicated in the early proinflammatory and delayed immunosuppression phases of sepsis. While type 1 interferons have not been implicated previously in SCM, interferon therapy (for viral hepatitis and Kaposi sarcoma) has been associated with reversible cardiomyopathy, perhaps suggesting a role for interferon signaling in SCM

Identification of co-regulated candidate genes by promoter analysis.

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