Reconstruction of IRG1 Gene Regulatory Network in Mammalian Macrophages under Inflammatory Conditions

The immune system is the first line of defence against invading pathogens. Macrophages are the key effector cells of the innate immune system which produce an array of cytokines, antimicrobial peptides, and effector molecules pathogen in response to pathogen invasion. Immunoresponsive gene 1 (Irg1) is highly upregulated when mouse macrophages are stimulated with LPS. Recently, the function of Irg1 has been elucidated as a gene coding for a protein which catalyses the decarboxylation of cis-aconitate, a tricarboxylic acid (TCA) cycle intermediate, to itaconic acid. In turn, itaconic acid selectively inhibits isocitrate lyase, a key enzyme of the glyoxylate shunt, which is a saviour pathway for bacteria to grow on low carbon diets. Thus, Irg1 via the production of itaconic acid plays an important role in combating pathogen invasion. Despite the importance of this recent discovery, the upstream transcriptional machinery of IRG1 has not yet been investigated. Hence, the aim of this thesis was to elucidate the gene regulatory networks of IRG1 in mammalian macrophages under inflammatory conditions. To achieve this aim, the experimental protocols for the isolation of monocytes from peripheral blood and their differentiation into macrophages were implemented. The cells were then characterised based on their morphology and the expression profile of cellular marker genes to confirm their identity. Using this cellular model, I discovered IRG1 expression and itaconic acid production in human macrophages under LPS activation. Both IRG1 and itaconic acid were initially discovered elsewhere in murine macrophages upon LPS stimulation. I further analysed the dynamics of IRG1 expression and itaconic acid production using different bacterial and viral ligands in human and mouse macrophages, showing that IRG1 expression could be upregulated when mammalian cells encounter bacteria and viruses. Owing to the complex upstream transcriptional machinery for IRG1 expression, I implemented a workflow defined as transcription factor identification protocol (TFIP) using both experimental and computational methods to identify potential transcription regulators for IRG1 expression in human and mouse macrophages. siRNA mediated gene silencing experiments in human and mouse macrophages revealed IRF1 as a transcriptional regulator for IRG1 in both the species and CEBPB in mouse macrophages under LPS activation

NS Segment of a 1918 Influenza A Virus-Descendent Enhances Replication of H1N1pdm09 and Virus-Induced Cellular Immune Response in Mammalian and Avian Systems.

The 2009 pandemic influenza A virus (IAV) H1N1 strain (H1N1pdm09) has widely spread and is circulating in humans and swine together with other human and avian IAVs. This fact raises the concern that reassortment between H1N1pdm09 and co-circulating viruses might lead to an increase of H1N1pdm09 pathogenicity in different susceptible host species. Herein, we explored the potential of different NS segments to enhance the replication dynamics, pathogenicity and host range of H1N1pdm09 strain A/Giessen/06/09 (Gi-wt). The NS segments were derived from (i) human H1N1- and H3N2 IAVs, (ii) highly pathogenic- (H5- or H7-subtypes) or (iii) low pathogenic avian influenza viruses (H7- or H9-subtypes). A significant increase of growth kinetics in A549 (human lung epithelia) and NPTr (porcine tracheal epithelia) cells was only notice

Early Lymphocyte Loss and Increased Granulocyte/Lymphocyte Ratio Predict Systemic Spread of in a Mouse Model of Acute Skin Infection.

Group A streptococci may induce lymphopenia, but the value of lymphocyte loss as early biomarkers for systemic spread and severe infection has not been examined systematically. We evaluated peripheral blood cell indices as biomarkers for severity and spread of infection in a mouse model of skin infection, using two isolates of greatly differing virulence. Internal organs were examined histologically. After subcutaneous inoculation, strain AP1 disseminated rapidly to peripheral blood and internal organs, causing frank sepsis. In contrast, seeding of internal organs by 5448 was mild, this strain could not be isolated from blood, and infection remained mostly localized to skin. Histopathologic examination of liver revealed microvesicular fatty change (steatosis) in AP1 infection, and examination of spleen showed elevated apoptosis and blurring of the white pulp/red pulp border late (40 h post infection) in AP1 infection. Both strains caused profound lymphopenia, but lymphocyte loss was more rapid early in AP1 infection, and lymphocyte count at 6 h post infection was the most accurate early marker for AP1 infection (area under the receiver operator curve [AUC] = 0.93), followed by the granulocyte/lymphocyte ratio (AUC = 0.89). The results suggest that virulence of correlates with the degree of early lymphopenia and underscore the value of peripheral blood indices to predict severity of bacterial infections in mice. Early lymphopenia and elevated granulocyte/lymphocyte ratio merit further investigation as biomarkers for systemic spread of skin infections in humans and, possibly, related pyogenic streptococci in humans and animals

Wnt/Tcf1 pathway restricts embryonic stem cell cycle through activation of the Ink4/Arf locus

Understanding the mechanisms regulating cell cycle, proliferation and potency of pluripotent stem cells guarantees their safe use in the clinic. Embryonic stem cells (ESCs) present a fast cell cycle with a short G1 phase. This is due to the lack of expression of cell cycle inhibitors, which ultimately determines naïve pluripotency by holding back differentiation. The canonical Wnt/β-catenin pathway controls mESC pluripotency via the Wnt-effector Tcf3. However, if the activity of the Wnt/β-catenin controls the cell cycle of mESCs remains unknown. Here we show that the Wnt-effector Tcf1 is recruited to and triggers transcription of the Ink4/Arf tumor suppressor locus. Thereby, the activation of the Wnt pathway, a known mitogenic pathway in somatic tissues, restores G1 phase and drastically reduces proliferation of mESCs without perturbing pluripotency. Tcf1, but not Tcf3, is recruited to a palindromic motif enriched in the promoter of cell cycle repressor genes, such as p15Ink4b, p16Ink4a and p19Arf, which mediate the Wnt-dependent anti-proliferative effect in mESCs. Consistently, ablation of β-catenin or Tcf1 expression impairs Wnt-dependent cell cycle regulation. All together, here we showed that Wnt signaling controls mESC pluripotency and proliferation through non-overlapping functions of distinct Tcf factors.We are grateful for the support from ERC grant (242630-RERE) (MPC), the Ministerio de Economia y Competitividad y FEDER (SAF2011-28580, and BFU2014-54717-P, BFU2015-71984-ERC to MPC), an AGAUR grant from Secretaria d´Universitats i Investigació del Departament d´Economia i Coneixement de la Generalitat de Catalunya (2014SGR1137 to MPC), Ministerio de Ciencia e Innovación FPI (to FA), the European Union's Horizon 2020 research and innovation programme under grant agreement CellViewer No 686637 (to MPC), the Spanish Ministry of Economy and Competitiveness, Centro de Excelencia Severo Ochoa 2013-2017, the CERCA Programme/Generalitat de Catalunya (to MPC); KU Leuven starting grant (STG) and KU Leuven C1 funds (C14/16/078) (to FL), AFR Postdoctoral Grant from the Luxembourg National Research Fund (FNR); ANEMO project N. 4001584/PDR 2012-1 and Dutch Province of Limburg (to GE), Short Term Mobility Award, CNR (to AC)

Wnt/Tcf1 pathway restricts embryonic stem cell cycle through activation of the Ink4/Arf locus

Understanding the mechanisms regulating cell cycle, proliferation and potency of pluripotent stem cells guarantees their safe use in the clinic. Embryonic stem cells (ESCs) present a fast cell cycle with a short G1 phase. This is due to the lack of expression of cell cycle inhibitors, which ultimately determines naïve pluripotency by holding back differentiation. The canonical Wnt/β-catenin pathway controls mESC pluripotency via the Wnt-effector Tcf3. However, if the activity of the Wnt/β-catenin controls the cell cycle of mESCs remains unknown. Here we show that the Wnt-effector Tcf1 is recruited to and triggers transcription of the Ink4/Arf tumor suppressor locus. Thereby, the activation of the Wnt pathway, a known mitogenic pathway in somatic tissues, restores G1 phase and drastically reduces proliferation of mESCs without perturbing pluripotency. Tcf1, but not Tcf3, is recruited to a palindromic motif enriched in the promoter of cell cycle repressor genes, such as p15Ink4b, p16Ink4a and p19Arf, which mediate the Wnt-dependent anti-proliferative effect in mESCs. Consistently, ablation of β-catenin or Tcf1 expression impairs Wnt-dependent cell cycle regulation. All together, here we showed that Wnt signaling controls mESC pluripotency and proliferation through non-overlapping functions of distinct Tcf factors.We are grateful for the support from ERC grant (242630-RERE) (MPC), the Ministerio de Economia y Competitividad y FEDER (SAF2011-28580, and BFU2014-54717-P, BFU2015-71984-ERC to MPC), an AGAUR grant from Secretaria d´Universitats i Investigació del Departament d´Economia i Coneixement de la Generalitat de Catalunya (2014SGR1137 to MPC), Ministerio de Ciencia e Innovación FPI (to FA), the European Union's Horizon 2020 research and innovation programme under grant agreement CellViewer No 686637 (to MPC), the Spanish Ministry of Economy and Competitiveness, Centro de Excelencia Severo Ochoa 2013-2017, the CERCA Programme/Generalitat de Catalunya (to MPC); KU Leuven starting grant (STG) and KU Leuven C1 funds (C14/16/078) (to FL), AFR Postdoctoral Grant from the Luxembourg National Research Fund (FNR); ANEMO project N. 4001584/PDR 2012-1 and Dutch Province of Limburg (to GE), Short Term Mobility Award, CNR (to AC)

Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production

Immunoresponsive gene 1 (Irg1) is highly expressed in mammalian macrophages during inflammation, but its biological function has not yet been elucidated. Here, we identify Irg1 as the gene coding for an enzyme producing itaconic acid (also known as methylenesuccinic acid) through the decarboxylation of cis-aconitate, a tricarboxylic acid cycle intermediate. Using a gain-and-loss-of-function approach in both mouse and human immune cells, we found Irg1 expression levels correlating with the amounts of itaconic acid, a metabolite previously proposed to have an antimicrobial effect. We purified IRG1 protein and identified its cis-aconitate decarboxylating activity in an enzymatic assay. Itaconic acid is an organic compound that inhibits isocitrate lyase, the key enzyme of the glyoxylate shunt, a pathway essential for bacterial growth under specific conditions. Here we show that itaconic acid inhibits the growth of bacteria expressing isocitrate lyase, such as Salmonella enterica and Mycobacterium tuberculosis. Furthermore, Irg1 gene silencing in macrophages resulted in significantly decreased intracellular itaconic acid levels as well as significantly reduced antimicrobial activity during bacterial infections. Taken together, our results demonstrate that IRG1 links cellular metabolism with immune defense by catalyzing itaconic acid production

NS Segment of a 1918 Influenza A Virus-Descendent Enhances Replication of H1N1pdm09 and Virus-Induced Cellular Immune Response in Mammalian and Avian Systems

The 2009 pandemic influenza A virus (IAV) H1N1 strain (H1N1pdm09) has widely spread and is circulating in humans and swine together with other human and avian IAVs. This fact raises the concern that reassortment between H1N1pdm09 and co-circulating viruses might lead to an increase of H1N1pdm09 pathogenicity in different susceptible host species. Herein, we explored the potential of different NS segments to enhance the replication dynamics, pathogenicity and host range of H1N1pdm09 strain A/Giessen/06/09 (Gi-wt). The NS segments were derived from (i) human H1N1- and H3N2 IAVs, (ii) highly pathogenic- (H5- or H7-subtypes) or (iii) low pathogenic avian influenza viruses (H7- or H9-subtypes). A significant increase of growth kinetics in A549 (human lung epithelia) and NPTr (porcine tracheal epithelia) cells was only noticed in vitro for the reassortant Gi-NS-PR8 carrying the NS segment of the 1918-descendent A/Puerto Rico/8/34 (PR8-wt, H1N1), whereas all other reassortants showed either reduced or comparable replication efficiencies. Analysis using ex vivo tracheal organ cultures of turkeys (TOC-Tu), a species susceptible to IAV H1N1 infection, demonstrated increased replication of Gi-NS-PR8 compared to Gi-wt. Also, Gi-NS-PR8 induced a markedly higher expression of immunoregulatory and pro-inflammatory cytokines, chemokines and interferon-stimulated genes in A549 cells, THP-1-derived macrophages (dHTP) and TOC-Tu. In vivo, Gi-NS-PR8 induced an earlier onset of mortality than Gi-wt in mice, whereas, 6-week-old chickens were found to be resistant to both viruses. These data suggest that the specific characteristics of the PR8 NS segments can impact on replication, virus induced cellular immune responses and pathogenicity of the H1N1pdm09 in different avian and mammalian host species

All possible paths from LPS to <i>IRG1</i> from the contextualised network.

<p>(<b>A</b>) Mouse and (<b>B</b>) human networks hierarchical layouts after calculating all possible paths between LPS and <i>IRG1</i> from the contextualised networks. <i>IRF1</i>, interferon regulatory factor 1; CEBPB, CCAAT/enhancer binding protein (C/EBP) beta; CEBPD, CCAAT/enhancer binding protein (C/EBP) delta; STAT1, signal transducer and activator of transcription 1; JUNB, Jun B proto-oncogene; PRDM1, PR domain containing 1 with ZNF domain; STAT4, signal transducer and activator of transcription 4; FOS, FBJ murine osteosarcoma viral oncogene homolog; ETS2, v-ets avian erythroblastosis virus E26 oncogene homolog 2; VDR, vitamin D (1,25-dihydroxyvitamin D3) receptor; RARA, retinoic acid receptor alpha; RUNX1, runt-related transcription factor 1. Solid line: activation; dashed line: inhibition.</p

Reprogramming of small noncoding RNA populations in peripheral blood reveals host biomarkers for latent and active Mycobacterium tuberculosis infection

Marcelo Ribeiro Alves. Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Documento produzido em parceria ou por autor vinculado à Fiocruz, mas não consta a informação no documento.Submitted by Janaína Nascimento ([email protected]) on 2019-12-16T13:51:37Z No. of bitstreams: 1 ve_Araujo_Leonardo_etal_INI_2019.pdf: 4561523 bytes, checksum: 31fe5ecf90ccb4d45f353481d14207d7 (MD5)Approved for entry into archive by Janaína Nascimento ([email protected]) on 2019-12-16T14:15:20Z (GMT) No. of bitstreams: 1 ve_Araujo_Leonardo_etal_INI_2019.pdf: 4561523 bytes, checksum: 31fe5ecf90ccb4d45f353481d14207d7 (MD5)Made available in DSpace on 2019-12-16T14:15:20Z (GMT). No. of bitstreams: 1 ve_Araujo_Leonardo_etal_INI_2019.pdf: 4561523 bytes, checksum: 31fe5ecf90ccb4d45f353481d14207d7 (MD5) Previous issue date: 2019TWINCORE Centre for Experimental and Clinical Infection Research. Research Group Biomarkers for Infectious Diseases. Hannover, Germany / Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Rio de Janeiro, RJ, Brasil / Helmholtz Centre for Infection Research. Braunschweig, Germany.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.Federal University of Rio de Janeiro. Thoracic Diseases Institute. Rio de Janeiro, RJ, Brazil.TWINCORE Centre for Experimental and Clinical Infection Research. Institute for Experimental Infection Research. Hannover, Germany.TWINCORE Centre for Experimental and Clinical Infection Research. Research Group Biomarkers for Infectious Diseases. Hannover, Germany / Zagazig University. Faculty of Veterinary Medicine. Department of Zoonoses. Zagazig, Egypt.TWINCORE Centre for Experimental and Clinical Infection Research. Research Group Biomarkers for Infectious Diseases. Hannover, Germany / Hannover Medical School. Institute for Laboratory Animal Science. Hannover, Germany.TWINCORE Centre for Experimental and Clinical Infection Research. Research Group Biomarkers for Infectious Diseases. Hannover, Germany.Federal University of Rio de Janeiro. Thoracic Diseases Institute. Rio de Janeiro, RJ, Brazil.Helmholtz Centre for Infection Research. Braunschweig, Germany.Fundação Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.TWINCORE Centre for Experimental and Clinical Infection Research. Research Group Biomarkers for Infectious Diseases. Hannover, Germany / Helmholtz Centre for Infection Research. Braunschweig, Germany / Centre for Individualised Infection Medicine, Hannover, Germany.In tuberculosis (TB), as in other infectious diseases, studies of small noncoding RNAs (sncRNA) in peripheral blood have focused on microRNAs (miRNAs) but have neglected the other major sncRNA classes in spite of their potential functions in host gene regulation. Using RNA sequencing of whole blood, we have therefore determined expression of miRNA, PIWI-interacting RNA (piRNA), small nucleolar RNA (snoRNA), and small nuclear RNA (snRNA) in patients with TB (n = 8), latent TB infection (LTBI; n = 21), and treated LTBI (LTBItt; n = 6) and in uninfected exposed controls (ExC; n = 14). As expected, sncRNA reprogramming was greater in TB than in LTBI, with the greatest changes seen in miRNA populations. However, substantial dynamics were also evident in piRNA and snoRNA populations. One miRNA and 2 piRNAs were identified as moderately accurate (area under the curve [AUC] = 0.70 to 0.74) biomarkers for LTBI, as were 1 miRNA, 1 piRNA, and 2 snoRNAs (AUC = 0.79 to 0.91) for accomplished LTBI treatment. Logistic regression identified the combination of 4 sncRNA (let-7a-5p, miR-589-5p, miR-196b-5p, and SNORD104) as a highly sensitive (100%) classifier to discriminate TB from all non-TB groups. Notably, it reclassified 8 presumed LTBI cases as TB cases, 5 of which turned out to have features of Mycobacterium tuberculosis infection on chest radiographs. SNORD104 expression decreased during M. tuberculosis infection of primary human peripheral blood mononuclear cells (PBMC) and M2-like (P = 0.03) but not M1-like (P = 0.31) macrophages, suggesting that its downregulation in peripheral blood in TB is biologically relevant. Taken together, the results demonstrate that snoRNA and piRNA should be considered in addition to miRNA as biomarkers and pathogenesis factors in the various stages of TB.IMPORTANCE Tuberculosis is the infectious disease with the worldwide largest disease burden and there remains a great need for better diagnostic biomarkers to detect latent and active M. tuberculosis infection. RNA molecules hold great promise in this regard, as their levels of expression may differ considerably between infected and uninfected subjects. We have measured expression changes in the four major classes of small noncoding RNAs in blood samples from patients with different stages of TB infection. We found that, in addition to miRNAs (which are known to be highly regulated in blood cells from TB patients), expression of piRNA and snoRNA is greatly altered in both latent and active TB, yielding promising biomarkers. Even though the functions of many sncRNA other than miRNA are still poorly understood, our results strongly suggest that at least piRNA and snoRNA populations may represent hitherto underappreciated players in the different stages of TB infection