Browsing by Author "Guler, Reto"

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    Antisense Therapy for Infectious Diseases
    (Multidisciplinary Digital Publishing Institute, 2023-08-21) Buthelezi, Lwanda Abonga; Pillay, Shandre; Ntuli, Noxolo Nokukhanya; Gcanga, Lorna; Guler, Reto
    Infectious diseases, particularly Tuberculosis (TB) caused by Mycobacterium tuberculosis, pose a significant global health challenge, with 1.6 million reported deaths in 2021, making it the most fatal disease caused by a single infectious agent. The rise of drug-resistant infectious diseases adds to the urgency of finding effective and safe intervention therapies. Antisense therapy uses antisense oligonucleotides (ASOs) that are short, chemically modified, single-stranded deoxyribonucleotide molecules complementary to their mRNA target. Due to their designed target specificity and inhibition of a disease-causing gene at the mRNA level, antisense therapy has gained interest as a potential therapeutic approach. This type of therapy is currently utilized in numerous diseases, such as cancer and genetic disorders. Currently, there are limited but steadily increasing studies available that report on the use of ASOs as treatment for infectious diseases. This review explores the sustainability of FDA-approved and preclinically tested ASOs as a treatment for infectious diseases and the adaptability of ASOs for chemical modifications resulting in reduced side effects with improved drug delivery; thus, highlighting the potential therapeutic uses of ASOs for treating infectious diseases.
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    Genome-wide profiling of transcribed enhancers during macrophage activation
    (BioMed Central, 2017-10-23) Denisenko, Elena; Guler, Reto; Mhlanga, Musa M; Suzuki, Harukazu; Brombacher, Frank; Schmeier, Sebastian
    Background: Macrophages are sentinel cells essential for tissue homeostasis and host defence. Owing to their plasticity, macrophages acquire a range of functional phenotypes in response to microenvironmental stimuli, of which M(IFN-γ) and M(IL-4/IL-13) are well known for their opposing pro- and anti-inflammatory roles. Enhancers have emerged as regulatory DNA elements crucial for transcriptional activation of gene expression. Results: Using cap analysis of gene expression and epigenetic data, we identify on large-scale transcribed enhancers in bone marrow-derived mouse macrophages, their time kinetics, and target protein-coding genes. We observe an increase in target gene expression, concomitant with increasing numbers of associated enhancers, and find that genes associated with many enhancers show a shift towards stronger enrichment for macrophage-specific biological processes. We infer enhancers that drive transcriptional responses of genes upon M(IFN-γ) and M(IL-4/IL-13) macrophage activation and demonstrate stimuli specificity of regulatory associations. Finally, we show that enhancer regions are enriched for binding sites of inflammation-related transcription factors, suggesting a link between stimuli response and enhancer transcriptional control. Conclusions: Our study provides new insights into genome-wide enhancer-mediated transcriptional control of macrophage genes, including those implicated in macrophage activation, and offers a detailed genome-wide catalogue of transcribed enhancers in bone marrow-derived mouse macrophages.
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    IL-4/IL-13-inducible lincRNA-MIR99AHG regulates macrophage polarization and promotes intracellular survival of Mycobacterium tuberculosis
    (2020) Gcanga, Lona; Guler, Reto; Tamgue, Ousman; Brombacher, Frank
    Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) kills 1.6 million people worldwide every year, and there is an urgent need for targeting host-pathogen interactions as a strategy to reduce mycobacterial resistance to current antimicrobials. Non-coding RNAs are emerging as important regulators of numerous biological processes and avenues for exploitation in host-directed therapeutics. Although long non-coding RNAs (lncRNAs) are abundantly expressed in immune cells, their functional role in gene regulation and bacterial infections remains under-studied. Here, we identify an immunoregulatory, lincRNA-MIR99AHG, which is upregulated in macrophages upon IL-4/IL-13 stimulation and downregulated after Mtb infection and in active TB patients. To evaluate the functional role of lincRNA-MIR99AHG, we employed antisense GapmeR-mediated lncRNA knockdown experiments. Knockdown of lincRNA-MIR99AHG with LNA-GapmeRs significantly reduced intracellular Mtb growth in mouse and human macrophages and reduced proinflammatory cytokine production. In addition, in vivo treatment with MIR99AHG LNA-GapmeRs reduced the mycobacterial burden in the lung and spleen. In vivo LNA-GapmeR treatment experiments demonstrated a role of lincRNA-MIR99AHG as a regulator of macrophage polarization and a host-mediated response post Mtb infection. Further, lincRNA-MIR99AHG translocated to the nucleus and interacts with a high affinity to hnRNPA2/B1 following IL-4/IL-13 stimulation and Mtb infection. Together, these findings identify lincRNA-MIR99AHG as a positive regulator of inflammation to promote Mtb growth and a possible for host-directed targeting or for adjunctive therapeutics against TB.
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    IL-4Rα-Dependent Alternative Activation of Macrophages Is Not Decisive for Mycobacterium tuberculosis Pathology and Bacterial Burden in Mice
    (Public Library of Science, 2015) Guler, Reto; Parihar, Suraj P; Savvi, Suzana; Logan, Erin; Schwegmann, Anita; Roy, Sugata; Nieuwenhuizen, Natalie E; Ozturk, Mumin; Schmeier, Sebastian; Suzuki, Harukazu; Brombacher, Frank
    Classical activation of macrophages (caMph or M1) is crucial for host protection against Mycobacterium tuberculosis ( Mtb ) infection. Evidence suggests that IL-4/IL-13 alternatively activated macrophages (aaMph or M2) are exploited by Mtb to divert microbicidal functions of caMph. To define the functions of M2 macrophages during tuberculosis (TB), we infected mice deficient for IL-4 receptor α on macrophages (LysM cre IL-4Rα -/lox ) with Mtb . We show that absence of IL-4Rα on macrophages does not play a major role during infection with Mtb H37Rv, or the clinical Beijing strain HN878. This was demonstrated by similar mortality, bacterial burden, histopathology and T cell proliferation between infected wild-type (WT) and LysM cre IL-4Rα -/lox mice. Interestingly, we observed no differences in the lung expression of inducible nitric oxide synthase (iNOS) and Arginase 1 (Arg1), well-established markers for M1/M2 macrophages among the Mtb -infected groups. Kinetic expression studies of IL-4/IL-13 activated bone marrow-derived macrophages (BMDM) infected with HN878, followed by gene set enrichment analysis, revealed that the MyD88 and IL-6, IL-10, G-CSF pathways are significantly enriched, but not the IL-4Rα driven pathway. Together, these results suggest that IL-4Rα-macrophages do not play a central role in TB disease progression.
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    Investigating the role of IL-4/IL-13 signalling through the IL-4 receptor alpha (IL-4Rα) on keratinocytes in murine models of Leishmania major and Schistosoma mansoni
    (2017) Govender, Melissa; Brombacher, Frank; Hurdayal, Ramona; Guler, Reto
    Keratinocytes represent the major cell type in the skin. During cutaneous leishmaniasis (CL) and schistosomiasis, the skin is important during the parasite life cycle. While Th1 immunity is required to control CL, protection during schistosomiasis requires Th2 immunity. Paradoxically, Th2 characteristic IL-4 secreted early during L. major infection in mice, can drive a Th1 response by instructing dendritic cells to produce IL-12. Additionally, keratinocytes at the site of L. major infection in C57BL/6 mice, were postulated to be the source of the IL-4. We investigated if IL-4/IL-13 signalling via the IL-4Rα on keratinocytes contributed to early immunity during CL and schistosomiasis. Keratinocyte-specific IL-4Rα deficient (KRT14creIL-4Rα-/lox) BALB/c and C57BL/6 mice were generated by gene targeting and site-specific recombination (cre/loxP) under control of the KRT14 locus. In the L. major footpad model, KRT14creIL-4Rα-/lox BALB/c mice developed increased swelling, high parasite burdens, and cytokine and antibody secretion similar to littermate controls. L. major-infected KRT14creIL-4Rα-/lox C57BL/6 mice had decreased footpad swelling, low parasite burdens, a dominant Th1 cytokine response, and low type 1 and 2 antibody titres, similar to littermate control and resistant C57BL/6. In the L major ear model, KRT14creIL-4Rα-/lox BALB/c mice developed increased swelling, high parasite burdens, Th1 and Th2 cytokines, and high antibody titres, similar to littermate controls. L. major LV39-infected KRT14creIL-4Rα-/lox BALB/c mice showed significantly decreased parasite burdens in the ear, compared to littermate controls. L. major-infected KRT14creIL-4Rα-/lox C57BL/6 mice in the ear model, had decreased swelling, low parasite burdens, a dominant Th1 immune response, and low type 1 and 2 antibody titres, similar to littermate control and C57BL/6 mice. In the Schistosoma model, survival of S. mansoni-infected KRT14creIL-4Rα-/lox BALB/c mice was similar to littermate controls during mortality studies. During acute infection, S. mansoni-infected KRT14creIL-4Rα-/lox BALB/c mice showed gut pathology, hepatosplenomegaly, cytokine production, low type 1 and high type 2 antibodies, similar to littermate controls. In comparison to littermate controls, S. mansoni-infected KRT14creIL-4Rα-/lox BALB/c mice had smaller granulomas. Collectively, our results indicate that IL-4/IL-13 signalling through the IL-4Rα on keratinocytes is not required for control during CL or acute schistosomiasis, but does contribute to efficient granuloma formation during acute schistosomiasis.
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    Investigation of minor groove binders (MGB), non-ionic surfactant vesicles (NIV) delivery systems and IL-4i1 as novel pathogen- and host-directed drug therapy for tuberculosis
    (2019) Hlaka, Lerato; Guler, Reto; Brombacher, Frank; Ozturk, Mumin
    Tuberculosis (TB), caused by Mycobacterium tuberculosis is the leading infectious disease epidemic that claims over 1.6 million lives, while 10 million fell ill in 2017. South Africa is burdened with the third highest global incidences following India and China with high rates of co-infections with HIV and highest numbers of multi-drug resistant (MDR) and extremely resistant (XDR) TB per capita. The current treatment regimen is decades old and requires a prolonged period of 6 months. The lack of efficient TB therapy and the emergence of MDR and XDR TB, there is an urgent need to find new drug targets for TB therapy through understanding the complex host-pathogen interactions. This may then lead to pathogen, host-directed therapies (HDT) or adjunct therapies as well as the development of effective drugs and drug formulations for the treatment of TB. Here we aimed to investigate potential targets for pathogen-and host-directed therapies for TB. We screened the anti-mycobacterial activity of 172 minor groove binder (MGB) compounds that selectively bind to AT-rich regions of the minor groove of bacterial DNA with the helical structure matching that of DNA in Mtb culture. Of the 172 total compounds screened 17 hits were identified, of which 2, MGB 362 and MGB 364 displayed intracellular mycobactericidal activity against Mtb HN878 at an MIC50 of 4.09 and 4.19 μM, respectively, whilst being non-toxic. Encapsulation of MGBs into non- ionic surfactant vesicles (NIVs) demonstrated a 1.6- and 2.1-fold increased intracellular mycobacterial activity, similar to that of rifampicin when compared with MGB alone. Treatment with MGB 364 or MGB 364 formulation did not cause DNA damage in murine infected macrophages as displayed by low expression of γ-H2Ax compared to H2O2 and DMSO. Intranasal administration of MGB 364 and MGB-NIV 364 formulation showed one log reduction in bacterial burden with improved pathology and immune cytokine production when in formulation. However, intranasal administration of 10 mg/kg MGB 362 together with rifampicin had no effect on bacterial loads. In summary, the data demonstrate the potential of MGB as a novel class of drug/chemical entity in anti-TB therapy and NIVs as an effective delivery system in a novel anti-TB formulation. Using deep CAGE and small RNA (CHIP-seq) technologies, International Center for Genetic Engineering and Biotechnology’s Cytokines and Diseases lab in collaboration with the RIKEN Center for Integrative Medical Sciences (Yokohama, Japan) performed a novel transcriptomics study approach by conducting a genome-wide transcriptional analyses of RNA transcripts from classically activated macrophages (caMph) and alternatively activated macrophages (aaMph) during Mtb infection. We identified host target genes that may play a role in host immune subverting mechanism by Mtb to hide away from host effector functions providing a possible target for host-directed therapy for tuberculosis. It is postulated that Mtb modulates the transcriptional landscape of IL-4/IL13 alternatively activated macrophages (aaMph) to escape killing by reactive nitrogen intermediates (NO) and reactive oxygen species (ROS) functions by IFN-γ stimulated classically activated macrophages (caMph). Here we report on the immunoregulatory role of IL-4i1, a candidate gene that was upregulated in aaMph during Mtb infection. IL-4i1 is a secreted L-amino oxidase with antibacterial properties. The enzyme converts Phenylalanine (Phe) into phenylpyruvate releasing toxic products ammonia and hydrogen peroxide (H2O2) which in-turn cause immunosuppression of effector T-cells by directly inhibiting polarization, proliferation and function or by promoting the generation of Foxp3 T-regulatory cells. Thus suggesting that IL-4i1 is involved in immune-regulatory mechanisms and may be implicated in immune evasion mechanisms by the pathogen. Here we report on the role of IL-4i1 on tissue localized T-cell activation and proliferative status thus maintaining immune local immune homeostasis. Thus showing that the absence of IL-4i1 could cause autoimmunity. To determine the functional role of IL-4i1 during Mtb infection, IL-4i1 deficient mice and wild-type littermate controls were infected with H37Rv and hypervirulent HN878 Mtb strain. IL-4i1 deficient mice were highly resistant to both strains of Mtb at 12- and 21-days post-infection as denoted by significant reduction in bacterial loads, reduced inflammation, reduced tissue iNOS expression reduced recruitment of interstitial macrophages, pro-inflammatory cytokines showed a trend for reduction. Interestingly there was a significant increase in NO production in infected tissues. There was an increase in M1-like macrophages that correlated with increased pro-inflammatory cytokines and chemokines. These data suggested that IL-4i1 regulates macrophage-mediated inflammatory responses during acute Mtb infection thus showing potential as an immunomodulatory target for TB HDT therapy. The study thus provides a framework for new drug targets for the development of new effective drugs and vaccines for TB therapy.
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    Remodelling of Mycobacterial Peptidoglycan During Cell Division and the Epigenetics of Macrophages during M. tuberculosis infection
    (2021) Kieswetter, Nathan Scott; Guler, Reto; Ozturk, Mumin; Brombacher, Frank
    Tuberculosis (TB) has emerged as the world’s most deleterious infectious disease. The etiological agent of TB, Mycobacterium tuberculosis (Mtb), has evolved the ability to evade the host immune system using several mechanisms; emphasising the need for novel treatment strategies. Peptidoglycan (PG) is an important immunomodulatory heteropolysaccharide structure that can be shed during mycobacterial infection with immunological consequences and as such, changes in PG structure are expected to have important implications on disease progression and host responses. Mycobacterial amidases have been shown to have important roles in the remodelling of PG during cell division in M. smegmatis and are implicated in sensitivity to antibiotic treatment. However, their roles in modulating host immunity remain unknown. Herein, we assess the immune responses to Mtb mutants defective for either one of two amidases, Ami1 and Ami4, in bone marrow-derived macrophages (BMDM) and the C57BL/6 murine models of tuberculosis. Both Ami1 and Ami4 deletion resulted in increased pro-inflammatory response in BMDM. Infection with the Mtb Δami1 mutant in mice resulted in differential induction of proinflammatory cytokines and certain chemokines during the acute phase of the infection, an eff ect that was abrogated in chronic phase infection. The Δami1mutant was found to be susceptible to antibiotics in liquid growth culture but this sensitivity was negated in macrophages and reversed to a tolerant phenotype in mice. The Δami4 mutant, by contrast, did not display differential antibiotic susceptibility and did not significantly alter cytokine and chemokine responses relative to the wildtype control in mice. These findings suggest that Ami1 and Ami4 in Mtb play a nonoverlapping role in antibiotic sensitivity and modulating host immunity during tuberculosis. Additionally, the specific epigenetic alterations which occur during host-Mtb infection that contribute to immune evasion remain unknown. Here, we propose a method to elucidate transcriptomic changes in both human primary monocyte-derived macrophages (MDM) and the Mtb bacillus with which they were infected. In this study, we exhibit a dual-RNA-seq proof-of-concept methodology where, from a single donor, we successfully sequence host RNA from infected MDMs as well as Mtb RNA enriched from those same infected MDMs. Utilizing this optimised methodology, we aim to discover and model epigenetic and transcriptional alterations as well as their effector proteins in primary human macrophages following Mtb infection. Further, we aim to identify novel and annotated ncRNAs which are correlated with these epigenetic modifications.
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    The role of scavenger receptor B1 in infection with Mycobacterium tuberculosis in a murine model
    (Public Library of Science, 2009) Schäfer, Georgia; Guler, Reto; Murray, Graeme; Brombacher, Frank; Brown, Gordon D
    BACKGROUND: The interaction between Mycobacterium tuberculosis (Mtb) and host cells is complex and far from being understood. The role of the different receptor(s) implicated in the recognition of Mtb in particular remains poorly defined, and those that have been found to have activity in vitro were subsequently shown to be redundant in vivo . Methods and FINDINGS: To identify novel receptors involved in the recognition of Mtb, we screened a macrophage cDNA library and identified scavenger receptor B class 1 (SR-B1) as a receptor for mycobacteria. SR-B1 has been well-described as a lipoprotein receptor which mediates both the selective uptake of cholesteryl esters and the efflux of cholesterol, and has also recently been implicated in the recognition of other pathogens. We show here that mycobacteria can bind directly to SR-B1 on transfected cells, and that this interaction could be inhibited in the presence of a specific antibody to SR-B1, serum or LDL. We define a variety of macrophage populations, including alveolar macrophages, that express this receptor, however, no differences in the recognition and response to mycobacteria were observed in macrophages isolated from SR-B1 −/− or wild type mice in vitro . Moreover, when wild type and SR-B1 −/− animals were infected with a low dose of Mtb (100 CFU/mouse) there were no alterations in survival, bacterial burdens, granuloma formation or cytokine production in the lung. However, significant reduction in the production of TNF, IFNγ, and IL10 were observed in SR-B1 −/− mice following infection with a high dose of Mtb (1000 CFU/mouse), which marginally affected the size of inflammatory foci but did not influence bacterial burdens. Deficiency of SR-B1 also had no effect on resistance to disease under conditions of varying dietary cholesterol. We did observe, however, that the presence of high levels of cholesterol in the diet significantly enhanced the bacterial burdens in the lung, but this was independent of SR-B1. CONCLUSION: SR-B1 is involved in mycobacterial recognition, but this receptor plays only a minor role in anti-mycobacterial immunity in vivo . Like many other receptors for these pathogens, the loss of SR-B1 can be functionally compensated for under normal conditions.
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    A role of statins against listeria monocytogenes and Mycobacterium tuberculosis infection
    (2011) Parihar, Suraj P; Brombacher, Frank; Guler, Reto
    Cholesterol has been shown to play important role in the pathogenesis and persistence of intracellular pathogens. Here, we modulate host cholesterol biosynthesis pathway using pharmacological agent statins, which are reversible inhibitors of HMG†CoA reductase enzyme. The aim of the study was to investigate the role of statins in inducing host protective responses against intracellular pathogens. We report reduced growth of Listeria monocytogenes (LM) and Mycobacterium tuberculosis (Mtb) in murine macrophages. We show prominent immunomodulatory activity induced by statins, mainly increased phagosomal maturation and autophagy resulting in decreased bacterial growth in macrophages. Subsequently, statin†treated mice showed decrease in bacterial loads, accompanied by reduced histopathology in the acute phase of infection during listeriosis and tuberculosis. Furthermore, we found decreased growth of Mtb in peripheral blood mononuclear cells (PBMC) and monocyte†derived macrophages (MDM) isolated from patients with familial hypercholesterolemia (FH) on statin therapy when compared to healthy subjects. Together, our results show that statins induces protection against Mtb in murine macrophages, mice and human mononuclear cells and monocyte†derived macrophages.
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    Simvastatin enhances protection against Listeria monocytogenes infection in mice by counteracting Listeria-induced phagosomal escape
    (Public Library of Science, 2013) Parihar, Suraj P; Guler, Reto; Lang, Dirk M; Suzuki, Harukazu; Marais, A David; Brombacher, Frank
    Statins are well-known cholesterol lowering drugs targeting HMG-CoA-reductase, reducing the risk of coronary disorders and hypercholesterolemia. Statins are also involved in immunomodulation, which might influence the outcome of bacterial infection. Hence, a possible effect of statin treatment on Listeriosis was explored in mice. Statin treatment prior to subsequent L. monocytogenes infection strikingly reduced bacterial burden in liver and spleen (up to 100-fold) and reduced histopathological lesions. Statin-treatment in infected macrophages resulted in increased IL-12p40 and TNF-α and up to 4-fold reduced bacterial burden within 6 hours post infection, demonstrating a direct effect of statins on limiting bacterial growth in macrophages. Bacterial uptake was normal investigated in microbeads and GFP-expressing Listeria experiments by confocal microscopy. However, intracellular membrane-bound cholesterol level was decreased, as analyzed by cholesterol-dependent filipin staining and cellular lipid extraction. Mevalonate supplementation restored statin-inhibited cholesterol biosynthesis and reverted bacterial growth in Listeria monocytogenes but not in listeriolysin O (LLO)-deficient Listeria . Together, these results suggest that statin pretreatment increases protection against L. monocytogenes infection by reducing membrane cholesterol in macrophages and thereby preventing effectivity of the cholesterol-dependent LLO-mediated phagosomal escape of bacteria.
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    The role and host-directed targeting of long non-coding RNAs in macrophage polarization during Mycobacterium tuberculosis infection
    (2022) Pillay, Shandré; Brombacher, Frank; Guler, Reto; Tamgue, Ousman
    In 2020, the World Health Organization (WHO) reported 1.5 million tuberculosis (TB)- associated deaths and an incidence of 10 million new cases. The causative, Mycobacterium tuberculosis (Mtb), evades host immune responses by skewing macrophage polarization towards a less microbicidal alternative state to avoid classical effector killing functions. However, the molecular details underlying these evasion mechanisms remain incomplete and current therapy is challenged with drug resistance. Host-directed therapy (HDT) has recently gained attention, with long non-coding RNAs (lncRNAs) as potential targets due to their emerging roles in pathogenic immune responses. We previously performed cap analysis gene expression (CAGE) transcriptomics on IFN-γ stimulated (classically activated) and IL-4/IL-13 stimulated (alternatively activated) mouse macrophages, identifying 151 differentially expressed lncRNAs following Mtb infection. We validated the top 11 differentially expressed lncRNAs and two were chosen for this study, lncRNA-125, whose expression was regulated at different levels unstimulated and in response to IFN-γ and IL-4/IL-13, and lncRNA-612 whose expression was only induced by IFN-γ stimulation. Interestingly, the expression of lncRNA125 and lncRNA-612 was downregulated following Mtb infection. Therefore, this study aimed at functionally validating these lncRNAs in unstimulated, IFN-γ and IL-4/IL-13 stimulated and/or Mtb-infected mouse and human macrophages by a loss-of-function approach using chemically engineered antisense oligonucleotides (gapmeRs). Knockdown of lncRNA-125 by gapmeRs reduced Mtb growth and anti-inflammatory cytokine production mediated by increased apoptosis, nitrite and pro-inflammatory cytokine production in IL-4/IL-13 prestimulated mouse macrophages. Whereas knockdown of lncRNA-125 in IFN-γ pre-stimulated mouse macrophages favoured Mtb growth and anti-inflammatory cytokine production, with reduction of apoptosis, nitrite and pro-inflammatory cytokine production. Therefore, indicating that lncRNA-125 regulates macrophage polarization during Mtb infection. Knockdown of lncRNA-125 in human macrophages resulted in reduced Mtb growth and increased proinflammatory cytokine production in unstimulated, IFN-γ and IL-4/IL-13 pre-stimulated BMDMs infected with Mtb. Comparatively, gapmeR knockdown of lncRNA-612 reduced Mtb growth and increased pro-inflammatory cytokine production in IFN-γ pre-stimulated mouse and human macrophages. In mouse macrophages, these responses were mediated by increased apoptosis and nitrite production, with reduced anti-inflammatory cytokine production. Overall, these findings highlight lncRNAs as novel host factors to be further investigated as targets for TB diagnostics and adjunctive HDTs.
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    Transcriptionally induced enhancers in the macrophage immune response to Mycobacterium tuberculosis infection
    (BioMed Central, 2019-01-22) Denisenko, Elena; Guler, Reto; Mhlanga, Musa; Suzuki, Harukazu; Brombacher, Frank; Schmeier, Sebastian
    Background Tuberculosis is a life-threatening infectious disease caused by Mycobacterium tuberculosis (M.tb). M.tb subverts host immune responses to build a favourable niche and survive inside of host macrophages. Macrophages can control or eliminate the infection, if acquire appropriate functional phenotypes. Transcriptional regulation is a key process that governs the activation and maintenance of these phenotypes. Among the factors orchestrating transcriptional regulation during M.tb infection, transcriptional enhancers still remain unexplored. Results We analysed transcribed enhancers in M.tb-infected mouse bone marrow-derived macrophages. We established a link between known M.tb-responsive transcription factors and transcriptional activation of enhancers and their target genes. Our data suggest that enhancers might drive macrophage response via transcriptional activation of key immune genes, such as Tnf, Tnfrsf1b, Irg1, Hilpda, Ccl3, and Ccl4. We report enhancers acquiring transcription de novo upon infection. Finally, we link highly transcriptionally induced enhancers to activation of genes with previously unappreciated roles in M.tb infection, such as Fbxl3, Tapt1, Edn1, and Hivep1. Conclusions Our findings suggest the importance of macrophage host transcriptional enhancers during M.tb infection. Our study extends current knowledge of the regulation of macrophage responses to M.tb infection and provides a basis for future functional studies on enhancer-gene interactions in this process.
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    Tuberculosis transcriptomics: host protection and immune evasion mechanisms
    (2017) Ozturk, Mumin; Brombacher, Frank; Guler, Reto
    Mycobacterium tuberculosis (Mtb) is the leading cause of death from an infectious disease. The success of the pathogen lies in its ability to subvert hostile intracellular macrophage environment. We performed genome-wide transcriptional deep sequencing on total RNA in murine bone marrow-derived macrophages (BMDM) infected with hypervirulent Beijing strain (HN878) in an extensive time kinetic manner using single molecule sequencer and cap analysis gene expression (CAGE) technique. CAGE analysis revealed nearly 36000 unique RNA transcripts with approximately 16000 are not unannotated to a specific gene. This thesis addressed global changes in RNA expression levels in macrophages infected with Mtb in a time kinetic manner to pinpoint novel host protection and immune evasion genes and elucidate the role of these genes in vitro macrophage assays and in vivo knockout mouse studies. The data in this thesis showed that basic leucine zipper transcription factor 2 (Batf2) was an important factor that regulates inflammatory responses in Mtb infection. Deletion of Batf2 led to the survival of mice with reduced lung inflammation and histopathology due to reduced recruitment of inflammatory macrophages. We also showed that Batf2 was highly expressed in peripheral blood from adolescents who progressed from infection to tuberculosis disease and a predictive human biomarker for tuberculosis disease. In contrast to Batf2, we showed that Protein Kinase C-delta (PKC-δ) deficient mice are highly susceptible to tuberculosis and human lung proteomics dataset revealed that PKC-δ was highly upregulated in the necrotic and cavitory regions of human granulomas in multi-drug resistant subjects. PKC-δ deficient mice had a significant reduction in alveolar macrophages and dendritic cells, reduced accumulation of lipid bodies and serum fatty acids. In vitro experiments showed that PKCδ was required for optimal killing effector functions which were independent of phagosome maturation and autophagy in primary murine macrophages. Our studies suggested that these novel genes play a role in the immune response to Mtb and should be studied more thoroughly to evaluate their potential in possible TB interventions.