Browsing by Author "Ozturk, Mumin"

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    Arteannuin-B and (3-Chlorophenyl)-2-Spiroisoxazoline Derivative Exhibit Anti-Inflammatory Effects in LPS-Activated RAW 264.7 Macrophages and BALB/c Mice-Induced Proinflammatory Responses via Downregulation of NF-κB/P38 MAPK Signaling
    (2022-11-20) Sawhney, Gifty; Rasool, Javeed Ur; Saroch, Diksha; Ozturk, Mumin; Brombacher, Frank; Ahmad, Bilal; Bhagat, Asha; Ali, Asif; Parihar, Suraj P.; Ahmed, Zabeer
    Host inflammatory responses are key to protection against injury; however, persistent inflammation is detrimental and contributes to morbidity and mortality. Herein, we demonstrated the anti-inflammatory role of Arteannuin-B (1) and its new spirocyclic-2-isoxazoline derivative JR-9 and their side effects in acute inflammatory condition in vivo using LPS-induced cytokines assay, carrageenan-induced paw edema, acetic acid-induced writhing and tail immersion. The results show that the spirocyclic-2-isoxazoline derivative is a potent anti-inflammatory agent with minimal cell toxicity as compared to Arteannuin-B. In addition, the efficacies of these compounds were also validated by flow cytometric, computational, and histopathological analysis. Our results show that the anti-inflammatory response of JR-9 significantly reduces the ability of mouse macrophages to produce NO, TNF-α, and IL-6 following LPS stimulation. Therefore, JR-9 is a prospective candidate for the development of anti-inflammatory drugs and its molecular mechanism is likely related to the regulation of NF-κB and MAPK signaling pathway.
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    Biomarkers and cell phenotypes in TB patients with minimal or persisting lung inflammation post-anti-TB treatment and ex-vivo atorvastatin immunomodulatory effects on M. tuberculosis-infected PBMC
    (2024) Motaung, Bongani; Guler, Reto; Thienemann, Friedrich; Ozturk, Mumin
    Unresolved lung inflammation post-anti-TB treatment necessitates the evaluation of additional host-directed therapies (HDT). Statins, recognized for their pleiotropic effects, show potential as immunomodulators to reduce post-TB lung inflammation. The ongoing StatinTB clinical trial utilizes a double-blind, randomized, placebo-controlled approach post-anti-TB treatment to assess the safety and efficacy of atorvastatin in reducing post TB lung inflammation. At the end of TB treatment, Mtb culture-negative participants were stratified into minimal (Arm A, Total Lung Glycolysis (TLG) < 50 SUV) or persisting (Arm B/C, TLG ≥ 50 SUV) lung inflammation using Positron Emission Tomography/Computed Tomography (PET/CT) scan. Arm B/C received atorvastatin (40mg/day) or placebo for 12 weeks, and this data remains currently blinded. Study participants were evaluated for hematological, biochemical, and inflammatory parameters where ALP, proBNP, vitamin D, and CRP showed significant increases in Arm B/C compared to Arm A at
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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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    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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    Omega-3 Fatty Acid and Iron Supplementation Alone, but Not in Combination, Lower Inflammation and Anemia of Infection in Mycobacterium tuberculosis-Infected Mice
    (2020-09-22) Nienaber, Arista; Baumgartner, Jeannine; Dolman, Robin C; Ozturk, Mumin; Zandberg, Lizelle; Hayford, Frank E A; Brombacher, Frank; Blaauw, Renee; Parihar, Suraj P; Smuts, Cornelius M; Malan, Linda
    Progressive inflammation and anemia are common in tuberculosis (TB) and linked to poor clinical outcomes. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have inflammation-resolving properties, whereas iron supplementation in TB may have limited efficacy and enhance bacterial growth. We investigated effects of iron and EPA/DHA supplementation, alone and in combination, on inflammation, anemia, iron status markers and clinical outcomes in Mycobacterium tuberculosis-infected C3HeB/FeJ mice. One week post-infection, mice received the AIN-93 diet without (control) or with supplemental iron (Fe), EPA/DHA, or Fe+EPA/DHA for 3 weeks. Mice supplemented with Fe or EPA/DHA had lower soluble transferrin receptor, ferritin and hepcidin than controls, but these effects were attenuated in Fe+EPA/DHA mice. EPA/DHA increased inflammation-resolving lipid mediators and lowered lung IL-1α, IFN-γ, plasma IL-1β, and TNF-α. Fe lowered lung IL-1α, IL-1β, plasma IL-1β, TNF-α, and IL-6. However, the cytokine-lowering effects in the lungs were attenuated with Fe+EPA/DHA. Mice supplemented with EPA/DHA had lower lung bacterial loads than controls, but this effect was attenuated in Fe+EPA/DHA mice. Thus, individually, post-infection EPA/DHA and iron supplementation lowered systemic and lung inflammation and mitigated anemia of infection in TB, but not when combined. EPA/DHA also enhanced bactericidal effects and could support inflammation resolution and management of anemia.
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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 Cysteinyl leukotriene type 1 receptor (CysLTR1) during Listeria monocytogenes infection in mice
    (2020) Poswayo, Sibongiseni Kwakho Luntukazi; Parihar, Suraj; Ozturk, Mumin; Brombacher, Frank
    South Africa recently experienced a Listeriosis outbreak, which was responsible for over 180 deaths, caused by an intracellular, rod-shaped bacilli called Listeria monocytogenes (LM). LM can infect both phagocytic and non-phagocytic cell types and induces its uptake by expressing internalin A and B, then secretes listeriolysin O (LLO), a virulence factor forming pores on the phagosome membrane to escape into the cytosol. Macrophages can phagocytose invading pathogens and induce innate inflammatory responses. Production of cytokines and eicosanoids by antigen presenting cells activates the adaptive immunity. Eicosanoids (epoxyeicosatreinoic acids, prostanoids and leukotrienes) are generated from metabolites of 20-carbon chained polyunsaturated fatty acids and arachidonic acid. Leukotrienes (LTs) are generated from 5- lipoxygenase-metabolism of arachidonic acid to LTB4 and cysteinyl LTs (cysLTs). CysLTs are pro-inflammatory lipids that have pathobiological functions in asthma. CysLTs function through three G-protein coupled receptors (CysLTR1, CysLTR2 and GPR99). The CysLTR1 and its ligands function has been well elucidated in asthmatic and allergic responses however, its role in bacterial infections is unknown. The aim of our study was to elucidate the role of CysLTR1 on disease progression in mice and macrophages infected with LM. In this study, we showed that CysLTR1 mRNA expression is upregulated by LM infection in WT macrophages and mice. Mice deficient of CysLTR1 had no defects at homeostasis. During time kinetic experiments with LM, CysLTR1 knockout mice displayed increased neutrophil recruitment and decreased lymphocyte cells at 3dpi, however, bacterial burdens were comparable to wild-type mice. In addition, macrophages deficient of CysLTR1 have no effect on the intracellular growth of LM. In conclusion, CysLTR1 signalling plays a role in lymphoid cell activation and neutrophilic recruitment during early LM infection, however, further studies are required to better understand the role of CysLTR1 during inflammatory responses.
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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.