The onset of immune response against microbial stimuli activates induction of many anti- inflammatory genes and ISGs for effective clearance of the pathogen. This response includes transcriptional activation of several non-coding transcripts such as miRNAs and long non-coding RNAs (lncRNAs). LncRNAs constitutes the largest class of non-coding genome and are arbitrarily described as transcripts greater than 200 base pairs. Similar to protein coding mRNAs, lncRNAs are RNA polymerase II transcripts and undergo mRNA processing such as capping, splicing and polyadenylation. In recent years, high throughput sequencing has enabled an in-depth exploration of the human genome and subsequent discovery of lncRNAs. Several studies have highlighted the crucial role of lncRNAs in many biological processes including as regulators of gene expression as well as molecular effectors of host-pathogen driven immune responses. To date, majority of lncRNAs have been studied in murine models with limited understanding in human cells. In order to elucidate the role of lncRNAs in human immune cell regulation, the goal of this thesis is to identify and characterize novel lncRNAs critical to host-pathogen innate immune responses. RNA sequencing in LPS, IAV and HSV stimulated cells revealed lncRNA LUCAT1 as most differentially regulated lncRNA. CRISPR-cas9 and shRNA mediated depletion of LUCAT1 showed enhanced IFN-I genes signature, which was suppressed upon overexpression of LUCAT1. Additionally, LPS stimulated hDCs showed enrichment of LUCAT1 in the nucleus and its association with the chromatin markers. Further, LUCAT1 depletion contributed to enhanced occupancy of transcriptional coactivators at the promoters of IFN-I genes. Global identification of RNA associated proteins revealed LUCAT1 association with STAT1 in the nucleus thus emphasizing its role in transcriptional regulation of Type I IFN genes in inflammatory responses. This thesis furthers the understanding about the molecular factors affecting immune regulation and describes the novel role of LUCAT1 as an attenuator of immune cell response to pathogens.

No description available.

Doctor of Philosophy / Department of Biology / Stefan Rothenburg / Vaccinia virus is the prototypic poxvirus that has been widely used as a model for investigating poxvirus biology and genetics. Like several members of the Poxviridae family, vaccinia virus can infect several different species including mice, cows and humans. Because the entry of poxviruses into a host cell relies on ubiquitously expressed surface molecules, which are found in many species, the ability of poxviruses to infect and replicate in different host cells primarily depends on their ability to subvert the host’s innate immune response. One critical barrier to infection is overcoming the general shutdown of protein translation initiated by the cellular protein kinase PKR. PKR detects cytoplasmic double-stranded (ds) RNA generated during infection by the replicating virus, which activates it to phosphorylate the alpha-subunit of the eukaryotic translation initiation factor 2 (eIF2) and suppress general translation. Poxviruses are large viruses with dsDNA genomes that encode around 200 genes. Several of these genes are known as host range genes and are important for replication in different host species and many interact with components of the host immune response to promote viral replication. Two genes in vaccinia virus, called E3L and K3L, are known inhibitors of PKR and have previously been shown to be important for virus replication in cells from different species. The molecular explanation behind their host range function, however, is unknown. The main goal of the research presented in this thesis is to determine the molecular mechanisms for the host range function of vaccinia virus E3L and K3L, particularly in different hamster host cells. Along with an analysis of vaccinia virus host range genes, we have used genome-wide comparisons between host-restricted poxviruses in the Leporipoxvirus genus to parse out the potential genomic determinants of host range restriction in this clade of poxviruses. The overarching aim of this thesis work is to better understand the molecular mechanisms for host range in poxviruses.

O papel do complemento na modulação da resposta de células dendríticas não é bem entendido. Neste trabalho observamos que estas células produzirem proteínas do complemento e que o componente C3 regula positivamente a expressão de DC-SIGN, HLA-DR, CD1a, CD80 e CD86 e a produção de IL-6 e IL-12 por células dendríticas humanas. Também observamos, em modelo murino, menor expressão de MHC-II em células dendríticas C5aR-/-, a qual está associada com menor expressão do transativador de MHC-II; menor expressão de moléculas coestimuladoras nas células C3aR-/-, C5aR-/- e C5L2-/- e menor produção de IL-12p40, IL-12p70 e IL-6 em resposta a ligantes de TLR2 pelas células C3aR-/- e C5aR-/-. Conseqüentemente, a ausência de sinal pelos C3aR e C5aR, regula negativamente a habilidade destas células na indução da resposta de linfócitos T CD4+, modificando os níveis de proliferação e citocinas produzidas. De maneira conjunta, observamos participação do complemento na diferenciação e maturação de células dendríticas e no desenvolvimento da resposta imune adaptativa. / The role of complement in the modulation of dendritic cells functions remains elusive. Here we show that these cells are able to produce complement proteins and that complement C3 upregulates the expression of DC-SIGN, HLA-DR, CD1a, CD80 and CD86 and the production of IL-6 e IL-12 by human dendritic cells. We also observed, in a mouse model, lower expression of MHC-II in C5aR-/- dendritic cells, which is correlated to lower expression of MHC-II transactivator; lower expression of costimmulatory molecules in C3aR-/-, C5aR-/- and C5L2-/- cells and lower production of IL-12p40, IL-12p70 and IL-6 by C3aR-/- and C5aR-/- cells in response to TLR2 stimulation. In consequence to the absence of C3aR and/or C5aR signaling we observed that these cells have lower ability to induce CD4+ T cells proliferation and production of Th1 cytokines. Together our data show a participation of complement in dendritic cell differentiation and maturation and in the polarization of adaptative immune responses.

O papel do complemento na modulação da resposta de células dendríticas não é bem entendido. Neste trabalho observamos que estas células produzirem proteínas do complemento e que o componente C3 regula positivamente a expressão de DC-SIGN, HLA-DR, CD1a, CD80 e CD86 e a produção de IL-6 e IL-12 por células dendríticas humanas. Também observamos, em modelo murino, menor expressão de MHC-II em células dendríticas C5aR-/-, a qual está associada com menor expressão do transativador de MHC-II; menor expressão de moléculas coestimuladoras nas células C3aR-/-, C5aR-/- e C5L2-/- e menor produção de IL-12p40, IL-12p70 e IL-6 em resposta a ligantes de TLR2 pelas células C3aR-/- e C5aR-/-. Conseqüentemente, a ausência de sinal pelos C3aR e C5aR, regula negativamente a habilidade destas células na indução da resposta de linfócitos T CD4+, modificando os níveis de proliferação e citocinas produzidas. De maneira conjunta, observamos participação do complemento na diferenciação e maturação de células dendríticas e no desenvolvimento da resposta imune adaptativa. / The role of complement in the modulation of dendritic cells functions remains elusive. Here we show that these cells are able to produce complement proteins and that complement C3 upregulates the expression of DC-SIGN, HLA-DR, CD1a, CD80 and CD86 and the production of IL-6 e IL-12 by human dendritic cells. We also observed, in a mouse model, lower expression of MHC-II in C5aR-/- dendritic cells, which is correlated to lower expression of MHC-II transactivator; lower expression of costimmulatory molecules in C3aR-/-, C5aR-/- and C5L2-/- cells and lower production of IL-12p40, IL-12p70 and IL-6 by C3aR-/- and C5aR-/- cells in response to TLR2 stimulation. In consequence to the absence of C3aR and/or C5aR signaling we observed that these cells have lower ability to induce CD4+ T cells proliferation and production of Th1 cytokines. Together our data show a participation of complement in dendritic cell differentiation and maturation and in the polarization of adaptative immune responses.

No description available.

Human immunodeficiency virus (HIV) infects 30 million people worldwide. In sub-Saharan Africa, the region most affected by HIV, women comprise 60% of the infected population. Heterosexual transmission is a major mode of viral acquisition, mandating further research of the process and prevention of HIV acquisition via the female reproductive tract (FRT). The FRT is a dynamic environment, protected by host immune mechanisms and commensal microbes. The disruption of either of these elements can increase susceptibility to HIV. Accordingly, one common risk factor for HIV acquisition is the microbial shift condition known as bacterial vaginosis (BV), which is characterized by the displacement of healthy lactobacilli by an overgrowth of pathogenic bacteria. As the bacteria responsible for BV pathogenicity and their interactions with host immunity are not understood, we sought to evaluate the effects of BV-associated bacteria on reproductive epithelia. Here we have characterized the interaction between BV-associated bacteria and the female reproductive tract by measuring cytokine and defensin induction in FRT epithelial cells following bacterial inoculation. Four BV-associated bacteria were evaluated alongside six lactobacilli for a comparative assessment. Our model showed good agreement with clinical BV trends; we observed a distinct cytokine and human β- defensin-2 response to BV-associated bacteria, especially Atopobium vaginae, compared to most lactobacilli. One lactobacillus species, Lactobacillus vaginalis, induced an immune response similar to that elicited by BV-associated bacteria. These iii data provide an important prioritization of BV-associated bacteria and support further characterization of reproductive bacteria and their interactions with host epithelia. We next evaluated the effect of this interaction on HIV infection by investigating the soluble effectors secreted when FRT epithelial cells were cocultured with A. vaginae. We observed increased proviral activity mediated by secreted low molecular weight effectors, and determined that this activity was not likely mediated by cytokine responses. Instead, we identified a complex mixture containing several upregulated host proteins. Selected individual proteins from the mixture exhibited HIV-enhancing activity only when applied with the complex mixture of proviral factors, suggesting that HIV enhancement might be mediated by synergistic effects. In addition to characterizing the immune interactions that mediate the enhanced HIV acquisition associated with BV, we also evaluated the safety and efficacy of RC- 101, a candidate vaginal microbicide being developed for the prevention of HIV transmission. RC-101 has been effective and well tolerated in preliminary cell culture and macaque models. However, the effect of RC-101 on primary vaginal tissues and resident vaginal microflora requires further evaluation. Here, we treated primary vaginal tissues and vaginal bacteria, both pathogenic and commensal, with RC-101 to investigate compatibility of this microbicide with FRT tissue and microflora. RC-101 was well tolerated by host tissues and commensal vaginal bacteria, while BV-associated bacteria were inhibited by RC-101. By establishing vaginal microflora, the specific antibacterial activity of RC-101 may provide a dual mechanism of HIV protection.

Submitted by Simone Souza (simonecgsouza@hotmail.com) on 2017-08-21T13:59:05Z No. of bitstreams: 1 DISS_2014_Celma Maria Ferreira.pdf: 2069185 bytes, checksum: 3327660ede858ab91f20c4f9da62e4de (MD5) / Approved for entry into archive by Jordan (jordanbiblio@gmail.com) on 2017-08-24T14:44:49Z (GMT) No. of bitstreams: 1 DISS_2014_Celma Maria Ferreira.pdf: 2069185 bytes, checksum: 3327660ede858ab91f20c4f9da62e4de (MD5) / Made available in DSpace on 2017-08-24T14:44:49Z (GMT). No. of bitstreams: 1 DISS_2014_Celma Maria Ferreira.pdf: 2069185 bytes, checksum: 3327660ede858ab91f20c4f9da62e4de (MD5) Previous issue date: 2014-03-10 / PROPEQ-UFMT / Objetivou-se estudar se o uso de probiótico a base de Bacillus spp, veiculado na ração e dissolvido em água, é capaz de alterar as características histomorfométricas da mucosa intestinal de tambaqui e minimizar as respostas de estresse desta espécie durante o procedimento de transporte. Um total de 510 juvenis de tambaqui (peso e comprimento total médio inicial de 83,26 ± 28,14 g e 17,39 ± 1,90 cm, respectivamente) foi distribuído em três tanques de alvenaria. Durante 60 dias, os peixes de dois tanques foram alimentados com ração comercial e os peixes do terceiro tanque com ração comercial suplementada com probiótico. Após este período, os peixes de cada tanque foram divididos em sacos plásticos e transportados por 4 horas de acordo com os seguintes tratamentos: T1: alimentação com ração comercial (controle); T2: alimentação com ração comercial e probiótico adicionado na água durante o transporte (20 mg/L); T3: alimentação com ração comercial suplementada com probiótico (1,0 g/kg de ração). Antes do transporte (basal), 24 e 96 horas após o transporte, oito peixes de cada tratamento foram submetidos à eutanásia e o intestino retirado para pesagem e mensuração do comprimento para avaliação da relação comprimento corporal/intestino. Em seguida, foi realizada coleta da porção anterior e posterior do intestino, para avaliação das características morfo-histológicas da mucosa intestinal (altura e comprimento das vilosidades). Nestes mesmos tempos de amostragem e imediatamente após transporte (chegada)ocorreram as coletas de sangue para avaliação de parâmetros metabólicos, hematológicos, iônicos e imunológicos. Os resultados foram analisados por ANOVA usando o software SAS e os dados expressos em médias comparadas pelo teste de Tukey, com 5% de significância. A suplementação com probiótico (Bacillus spp.) não exerceu efeito no peso, peso relativo, altura e comprimento das vilosidades do intestino do tambaqui submetido ao transporte. Além disso, as respostas de estresse durante este procedimento foram similares entre os tratamentos testados, indicando que o probiótico não foi eficiente em suprimir as respostas de estresse e melhorar o sistema imunológico inato no tambaqui. / The aim of this study was to evaluate the intestinal mucosal integrity and the effectiveness of addition of a probiotic as a potential stress reducer during the transport of tambaqui juveniles. In a completely randomized design with three treatments and seven replications, a control treatment (fish fed a commercial diet without probiotic) with was compared with two other treatments involving the use of a commercial probiotic incorporated into the diet or in the transport water. A total of 510 juvenile tambaqui (83.26 ± 28.14 g and 17.39 ± 1.90 cm) were distributed into three cement tanks. For 60 days, fish from the two tanks were fed a commercial diet, and the fish from the third tank received a commercial diet supplemented with a probiotic. After this period, the fish from each tank were divided into plastic bags and transported for four hours, receiving the following treatments: T1: commercial diet (control); T2: commercial diet and probiotic dissolved in the transport water (20 mg/L); T3: commercial diet supplemented with probiotic (1.0 g/kg diet). Before transport (baseline), and 24 and 96 hours after transport, fish from each treatment were euthanized and their intestine was removed for morphological and histological evaluation of the intestinal mucosa. In these same sampling times and immediately after transport, blood was collected to evaluate metabolic, hematologic, immunologic, and ionic parameters. The results were analyzed by ANOVA using the SAS software and data were expressed as means compared by Tukey’s test at 5% significance. In all treatments, the intestine length increased with increase in thefish size. Supplementation with probiotic (Bacillus spp.) had no effect on weight (g), relative weight (%), villus height and villus length of tambaqui challenged with transport. The stress response of the tambaqui fish was similar among the tested treatments, indicating that the probiotic used was not efficient in suppressing stress during transportation.

Vaccinia virus (VACV) is the most intensively studied orthopoxvirus and acts as an excellent model to investigate host-pathogen interactions. VACV encodes about 200 proteins, many of which modulate the immune response. This study focusses on two of these: C16 and C4, that share 43.7 % amino acid identity. Given the sequence similarity, we explored whether C16 and C4 have any shared functions, whilst also searching for novel functions. To gain mechanistic insight, we sought to identify binding partners and determine the residues responsible. C16 has two reported functions. Firstly, it inhibits DNA-PK-mediated DNA sensing, and this study found that C4 can perform this function as well. Like C16, C4 associates with the Ku heterodimer to block its binding to DNA leading to reduced production of cytokines and chemokines. For both proteins, the function localised to the C termini and was abrogated by mutating three residues. Secondly, C16 induces a hypoxic response by binding to PHD2. This function was mapped to the N-terminal 156 residues and a full length C16 mutant (D70K,D82K) lost the ability to induce a hypoxic response. In contrast, C4 did not bind PHD2. C4 inhibits NF-κB signalling by an unknown mechanism. Reporter gene assays showed that C16 also suppresses NF-κB activity and, intriguingly, this was carried out by both the N and C termini. C16 acts at or downstream of p65 and the N terminus of C16 associated with p65 independently of PHD2-binding. Conversely, C4 acted upstream of p65, did not display an interaction with p65, and the function was restricted to its C-terminal region. Novel binding partners were identified by a screen utilising tandem mass tagging and mass spectrometry, and selected hits were validated. The C terminus of C16 associated with VACV protein K1, a known NF-κB inhibitor. Additionally, C16 bound to the transcriptional regulator ARID4B. C4 did not interact with these proteins, but the N-terminal region of C4 associated with filamins A and B. The functional consequences of these interactions remain to be determined. In vivo, C4 and C16 share some redundancy in that a double deletion virus exhibits an attenuated virulence phenotype that is not observed by single deletion viruses in the intradermal model of infection. However, non-redundant functions also contribute to virulence in that both single deletion viruses display attenuated virulence compared to a wild-type Western Reserve virus in the intranasal model of infection. Data presented also reveal that C4 inhibits the recruitment of immune cells to the site of infection, as was previously described for C16. Overall, this investigation highlights the complexity of host-pathogen interactions showing that VACV encodes two multifunctional proteins with both shared and unique functions. Moreover, their inhibition of DNA-PK emphasises the importance of this PRR as a DNA sensor in vivo.

Effectively treating infectious diseases often requires a multi-step approach to target different components involved in disease pathogenesis. Similarly, the COVID-19 pandemic has become a global health crisis that requires a comprehensive understanding of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) infection to develop effective therapeutics. One potential strategy to instill greater immune protection against COVID-19 is boosting the innate immune system. This boosting, termed trained immunity, employs immune system modulators to train innate immune cells to produce an enhanced, non-specific immune response upon reactivation following exposure to pathogens, a process that has been studied in the context of and clinical studies prior to the COVID-19 pandemic. Evaluation of the underlying pathways that are essential to inducing protective trained immunity will provide insight into identifying potential therapeutic targets that may alleviate the COVID-19 crisis. Here we review multiple immune training agents, including Bacillus Calmette-Guérin (BCG), β-glucan, and lipopolysaccharide (LPS), and the two most popular cell types involved in trained immunity, monocytes and natural killer (NK) cells, and compare the signaling pathways involved in innate immunity. Additionally, we discuss COVID-19 trained immunity clinical trials, emphasizing the potential of trained immunity to fight SARS-CoV-2 infection. Understanding the mechanisms by which training agents activate innate immune cells to reprogram immune responses may prove beneficial in developing preventive and therapeutic targets against COVID-19.