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  • 1.
    Eklund, Daniel
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Persson, Hans Lennart
    Linköpings universitet, Institutionen för medicin och hälsa, Lungmedicin. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Centrum för kirurgi, ortopedi och cancervård, Lungmedicinska kliniken US.
    Larsson, Marie C.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Welin, Amanda
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Idh, Jonna
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Paues, Jakob
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Infektionsmedicin. Linköpings universitet, Hälsouniversitetet.
    Fransson, Sven-Göran
    Linköpings universitet, Institutionen för medicin och hälsa, Medicinsk radiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Röntgenkliniken i Linköping.
    Stendahl, Olle
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Schön, Thomas
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Lerm, Maria
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Vitamin D enhances IL-1β secretion and restricts growth of Mycobacterium tuberculosis in macrophages from TB patients2013Ingår i: International Journal of Mycobacteriology, ISSN 2212-5531, Vol. 2, nr 1, s. 18-25Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The emergence of multidrug-resistant strains of Mycobacterium tuberculosis (MTB), the bacterium responsible for tuberculosis (TB), has rekindled the interest in the role of nutritional supplementation of micronutrients, such as vitamin D, as adjuvant treatment. Here, the growth of virulent MTB in macrophages obtained from the peripheral blood of patients with and without TB was studied. The H37Rv strain genetically modified to express Vibrio harveyi luciferase was used to determine the growth of MTB by luminometry in the human monocyte-derived macrophages (hMDMs) from study subjects. Determination of cytokine levels in culture supernatants was performed using a flow cytometry-based bead array technique. No differences in intracellular growth of MTB were observed between the different study groups. However, stimulation with 100 nM 1,25-dihydroxyvitamin D significantly enhanced the capacity of hMDMs isolated from TB patients to control the infection. This effect was not observed in hMDMs from the other groups. The interleukin (IL)-1β and IL-10 release by hMDMs was clearly increased upon stimulation with 1,25-dihydroxyvitamin D. Furthermore, the 1,25-dihydroxyvitamin D stimulation also led to elevated levels of TNF-α (tumor necrosis factor-alpha) and IL-12p40. It was concluded that vitamin D triggers an inflammatory response in human macrophages with enhanced secretion of cytokines, as well as enhancing the capacity of hMDMs from patients with active TB to restrict mycobacterial growth.

  • 2.
    Eklund, Daniel
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Hälsouniversitetet.
    Welin, Amanda
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Hälsouniversitetet.
    Andersson, Henrik
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Hälsouniversitetet.
    Verma, Deepti
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för inflammationsmedicin. Linköpings universitet, Hälsouniversitetet.
    Söderkvist, Peter
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk patologi och klinisk genetik.
    Stendahl, Olle
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Hälsouniversitetet.
    Särndahl, Eva
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Lerm, Maria
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Hälsouniversitetet.
    Human gene variants linked to enhanced NLRP3 activity limit intramacrophage growth of Mycobacterium tuberculosis2014Ingår i: The Journal of infectious diseases, ISSN 1537-6613, Vol. 209, nr 5, s. 749-753Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Activation of the NLRP3 inflammasome and subsequent generation of IL-1β is initiated in macrophages upon recognition of several stimuli. In the present work, we show that gain-of-function gene variants of inflammasome components known to predispose individuals to inflammatory disorders have a host-protective role during infection with Mycobacterium tuberculosis. By isolation of macrophages from patients and healthy blood donors with genetic variants in NLRP3 and CARD8 and subsequently infecting the cells by virulent M. tuberculosis, we show that these gene variants, combined, are associated with increased control of bacterial growth in human macrophages.

  • 3.
    Eklund, Daniel
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Hälsouniversitetet.
    Welin, Amanda
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Lerm, Maria
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    A medium-throughput microplate-based ex vivo model for measuring intramacrophage growth of mycobacterium tuberculosis in CYTOKINE, vol 48, issue 1-2, pp 14-142009Ingår i: CYTOKINE, 2009, Vol. 48, nr 1-2, s. 14-14Konferensbidrag (Refereegranskat)
    Abstract [en]

    n/a

  • 4.
    Eklund, Daniel
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Hälsouniversitetet.
    Welin, Amanda
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Schon, Thomas
    Kalmar County Hospital.
    Stendahl, Olle
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Huygen, Kris
    Science Institute for Public Health, Belgium .
    Lerm, Maria
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Validation of a Medium-Throughput Method for Evaluation of Intracellular Growth of Mycobacterium tuberculosis2010Ingår i: Clinical and Vaccine Immunology, ISSN 1556-6811, E-ISSN 1556-679X, Vol. 17, nr 4, s. 513-517Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Intracellular pathogens such as Mycobacterium tuberculosis have adapted to a life inside host cells, in which they utilize host nutrients to replicate and spread. Ineffective methods for the evaluation of growth of intracellular pathogens in their true environment pose an obstacle for basic research and drug screening. Here we present the validation of a luminometry-based method for the analysis of intramacrophage growth of M. tuberculosis. The method, which is performed in a medium-throughput format, can easily be adapted for studies of other intracellular pathogens and cell types. The use of host cells in drug-screening assays dedicated to find antimicrobials effective against intracellular pathogens permits the discovery of not only novel antibiotics but also compounds with immunomodulatory and virulence-impairing activities, which may be future alternatives or complements to antibiotics.

  • 5.
    Persson, Hans Lennart
    et al.
    Linköpings universitet, Institutionen för medicin och hälsa, Lungmedicin. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Centrum för kirurgi, ortopedi och cancervård, Lungmedicinska kliniken US.
    Eklund, Daniel
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Welin, Amanda
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Paues, Jakob
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Infektionsmedicin. Linköpings universitet, Hälsouniversitetet.
    Idh, Jonna
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Fransson, Sven-Göran
    Linköpings universitet, Institutionen för medicin och hälsa, Medicinsk radiologi. Linköpings universitet, Hälsouniversitetet.
    Stendahl, Olle
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Lerm, Maria
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Schön, Thomas
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Alveolar macrophages from patients with tuberculosis exhibit reduced capacity of restricting growth of Mycobacterium tuberculosis: a pilot study of vitamin D stimulation in vitro2013Ingår i: HOAJ Biology, ISSN 2050-0874Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: The role of vitamin D supplementation as adjuvant treatment of tuberculosis (TB) has lately attracted increasing interest. Our aim was to investigate the capacity of alveolar macrophages (AMs) from patients with or without exposure to TB to control intracellular growth of virulent Mycobacterium tuberculosis (Mtb).

    Methods: AMs were freshly harvested from the bronchoalveolar lavage fluid of 7 patients with a history of TB (4 patients with previous TB and 3 patients with current TB) and 4 non-TB subjects. The H37Rv strain, genetically modified to express Vibrio harveyi luciferase, was used to determine the growth of Mtb by luminometry in the AMs from study subjects. Cytokine levels in culture supernatants were determined using a flow cytometry-based bead array technique.

    Results: AMs from patients with a TB history were less efficient in restricting Mtb growth. Stimulation with 100 nM1, 25-dihydroxyvitamin D (1,25D3) did not significantly influence the capacity of AMs from any study subjects to control the infection. Out of the cytokines evaluated (TNF-α, IL-1β, IL-10 and IL-12p40) only TNF-α demonstrated detectable levels in culture supernatants, but did not respond to stimulation with 1,25D3.

    Conclusions: We conclude that AMs of TB-patients show reduced ability to control mycobacterial growth in vitro, and, that AMs in this pilot study do no respond to 1, 25D3-stimulation. The former observation supports the concept that innate immunity is crucial for the control of TB infection.

  • 6.
    Raffetseder, Johanna
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Hälsouniversitetet.
    Pienaar, Elsje
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Hälsouniversitetet.
    Blomgran, Robert
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Hälsouniversitetet.
    Eklund, Daniel
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för inflammationsmedicin. Linköpings universitet, Hälsouniversitetet.
    Brodin Patcha, Veronika
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Hälsouniversitetet.
    Andersson, Henrik
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Hälsouniversitetet.
    Welin, Amanda
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Hälsouniversitetet.
    Lerm, Maria
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Hälsouniversitetet.
    Replication Rates of Mycobacterium tuberculosis in Human Macrophages Do Not Correlate with Mycobacterial Antibiotic Susceptibility2014Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, nr 11, s. e112426-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The standard treatment of tuberculosis (TB) takes six to nine months to complete and this lengthy therapy contributes to the emergence of drug-resistant TB. TB is caused by Mycobacterium tuberculosis (Mtb) and the ability of this bacterium to switch to a dormant phenotype has been suggested to be responsible for the slow clearance during treatment. A recent study showed that the replication rate of a non-virulent mycobacterium, Mycobacterium smegmatis, did not correlate with antibiotic susceptibility. However, the question whether this observation also holds true for Mtb remains unanswered. Here, in order to mimic physiological conditions of TB infection, we established a protocol based on long-term infection of primary human macrophages, featuring Mtb replicating at different rates inside the cells. During conditions that restricted Mtb replication, the bacterial phenotype was associated with reduced acid-fastness. However, these phenotypically altered bacteria were as sensitive to isoniazid, pyrazinamide and ethambutol as intracellularly replicating Mtb. In support of the recent findings with M. smegmatis, we conclude that replication rates of Mtb do not correlate with antibiotic tolerance.

  • 7.
    Welin, Amanda
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Survival strategies of Mycobacterium tuberculosis inside the human macrophage2011Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Mycobacterium tuberculosis (Mtb) is the bacterium responsible for tuberculosis (TB). For decades, it was believed that TB was a disease of the past, but the onset of the HIV epidemic resulting in a greatly increased number of TB cases, the emergence of antibiotic resistant Mtb strains, and the relative ineffectiveness of the BCG vaccine have put TB back on the agenda. With almost two million people being killed by TB each year, the World Health Organization has declared it a global emergency. TB is an especially big issue in low-income countries, where crowded living conditions accelerates spread of the disease, and where access to health care and medication is problematic. Mtb spreads by aerosol and infects its host through the airways. The bacterium is phagocytosed by resident macrophages in the lung, and when successful is able to replicate inside these cells, which are actually designed to kill invading microbes. Mtb is able to evade macrophage responses in part by inhibiting the fusion between the phagosome in which it resides and bactericidal lysosomes, as well as by dampening the acidification of the vacuole. The initial macrophage infection results in a pro-inflammatory response and the recruitment of other cells of the innate and adaptive immune systems, giving rise to the hallmark of Mtb infection – the granuloma. It is believed that in up to 50 % of exposed individuals, however, the infection is cleared without the involvement of the adaptive immune system, indicating that the innate immune system may be able to control or clear the infection if activated appropriately. This thesis focuses on the interaction between the host macrophage and Mtb, aiming to understand some of the mechanisms employed by the bacterium to evade macrophage responses to enable replication and spread to new host cells. Furthermore, mechanisms used by the macrophage to keep the infection under control were studied, and a method that could be used to measure the replication of the bacilli inside macrophages in vitro in an efficient way was developed. We found that a mycobacterial glycoprotein, mannose-capped lipoarabinomannan (ManLAM), which is shed from the bacilli during phagocytosis by macrophages, integrates into membrane raft domains of the host cell membrane via its GPI anchor. This integration leads to an inhibition of phagosomal maturation. Subsequently, we developed a luciferase-based method by which intracellular replication of Mtb as well as viability of the host macrophage could be measured in a rapid, inexpensive and quantitative way in a 96-well plate. This method could be used for drug screening as well as for studying the different host and bacterial factors that influence the growth of Mtb inside the host cell. Using this method, we discovered that infection of macrophages with Mtb at a low multiplicity of infection (MOI) led to effective control of bacterial growth by the cell, and that this was dependent on functional lysosomal proteases as well as phagosomal acidification. However, we found no correlation between controlled bacterial growth and the translocation of late endosomal membrane proteins to the phagosome, showing that these markers are poor indicators of phagosomal functionality. Furthermore, we discovered that infection of macrophages with Mtb at a higher MOI led to replication of the bacilli accompanied by host cell death within a few days. We characterized this cell death, and concluded that when replication of Mtb inside macrophages reaches a certain threshold and the bacteria secrete a protein termed ESAT-6, necrotic cell death of the host cell occurs. However, although the bacilli activated inflammasome complexes in the host cell and IL-1β was secreted during infection of macrophages, Mtb infection did not induce either of the recently characterized inflammasome-related cell death types pyroptosis or pyronecrosis. Thus, we have elucidated some of the strategies that Mtb uses to be able to survive and replicate inside the macrophage and spread to new cells, as well as studied the conditions under which the host cell is able to control infection. This knowledge could be used in the future for developing drugs that boost the innate immune system or targets bacterial virulence factors in the macrophage.

    Delarbeten
    1. Incorporation of Mycobacterium tuberculosis lipoarabinomannan into macrophage membrane rafts is a prerequisite for the phagosomal maturation block.
    Öppna denna publikation i ny flik eller fönster >>Incorporation of Mycobacterium tuberculosis lipoarabinomannan into macrophage membrane rafts is a prerequisite for the phagosomal maturation block.
    Visa övriga...
    2008 (Engelska)Ingår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 76, nr 7, s. 2882-2887Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Lipoarabinomannan (LAM) is one of the key virulence factors for Mycobacterium tuberculosis, the etiological agent of tuberculosis. During uptake of mycobacteria, LAM interacts with the cell membrane of the host macrophage and can be detected throughout the cell upon infection. LAM can inhibit phagosomal maturation as well as induce a proinflammatory response in bystander cells. The aim of this study was to investigate how LAM exerts its action on human macrophages. We show that LAM is incorporated into membrane rafts of the macrophage cell membrane via its glycosylphosphatidylinositol anchor and that incorporation of mannose-capped LAM from M. tuberculosis results in reduced phagosomal maturation. This is dependent on successful insertion of the glycosylphosphatidylinositol anchor. LAM does not, however, induce the phagosomal maturation block through activation of p38 mitogen-activated protein kinase, contradicting some previous suggestions.

    Nationell ämneskategori
    Mikrobiologi inom det medicinska området
    Identifikatorer
    urn:nbn:se:liu:diva-20816 (URN)10.1128/IAI.01549-07 (DOI)18426888 (PubMedID)
    Tillgänglig från: 2009-09-22 Skapad: 2009-09-22 Senast uppdaterad: 2018-01-13Bibliografiskt granskad
    2. Validation of a Medium-Throughput Method for Evaluation of Intracellular Growth of Mycobacterium tuberculosis
    Öppna denna publikation i ny flik eller fönster >>Validation of a Medium-Throughput Method for Evaluation of Intracellular Growth of Mycobacterium tuberculosis
    Visa övriga...
    2010 (Engelska)Ingår i: Clinical and Vaccine Immunology, ISSN 1556-6811, E-ISSN 1556-679X, Vol. 17, nr 4, s. 513-517Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Intracellular pathogens such as Mycobacterium tuberculosis have adapted to a life inside host cells, in which they utilize host nutrients to replicate and spread. Ineffective methods for the evaluation of growth of intracellular pathogens in their true environment pose an obstacle for basic research and drug screening. Here we present the validation of a luminometry-based method for the analysis of intramacrophage growth of M. tuberculosis. The method, which is performed in a medium-throughput format, can easily be adapted for studies of other intracellular pathogens and cell types. The use of host cells in drug-screening assays dedicated to find antimicrobials effective against intracellular pathogens permits the discovery of not only novel antibiotics but also compounds with immunomodulatory and virulence-impairing activities, which may be future alternatives or complements to antibiotics.

    Ort, förlag, år, upplaga, sidor
    American Society for Microbiology, 2010
    Nationell ämneskategori
    Medicin och hälsovetenskap
    Identifikatorer
    urn:nbn:se:liu:diva-54868 (URN)10.1128/CVI.00446-09 (DOI)000276170900004 ()
    Tillgänglig från: 2010-04-16 Skapad: 2010-04-16 Senast uppdaterad: 2017-10-31
    3. Importance of phagosomal functionality for growth restriction of Mycobacterium tuberculosis in primary human macrophages
    Öppna denna publikation i ny flik eller fönster >>Importance of phagosomal functionality for growth restriction of Mycobacterium tuberculosis in primary human macrophages
    Visa övriga...
    2011 (Engelska)Ingår i: Journal of Innate Immunity, ISSN 1662-811X, E-ISSN 1662-8128, Vol. 3, nr 5, s. 508-518Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The best characterized survival mechanism of Mycobacterium tuberculosis inside the macrophage is the inhibition of phagosomal maturation. Phagosomal maturation involves several steps including fusion with lysosomes and acidification. However, it has not been elucidated which components of phagosomal maturation correlate with growth restriction of virulent mycobacteria in human macrophages, and we aimed to study this. We infected human monocyte-derived macrophages with M. tuberculosis and assessed bacterial replication, translocation of CD63 to the phagosome, and phagosomal acidification. We found that unstimulated macrophages were able to control infection with M. tuberculosis upon inoculation at a low, but not high, multiplicity of infection (MOI). H37Rv and H37Ra infection, at both high and low MOI, led to equally ineffective translocation of CD63 to the phagosome. This was true despite the impaired growth ability of H37Rv at the low MOI and of H37Ra even at the high MOI, indicating that inhibition of CD63 translocation was not sufficient for growth to occur. On the other hand, acidification of mycobacterial phagosomes was more efficient at a low MOI with both mycobacterial strains, consistent with a role for phagosomal acidification in restricting M. tuberculosis growth. Inhibition of the vacuolar H+-ATPase as well as of cathepsin D led to enhanced mycobacterial replication inside the macrophage. We conclude that acidification and related functional aspects of the mature phagosome are important factors for restriction of M. tuberculosis replication in human macrophages.

    Ort, förlag, år, upplaga, sidor
    S. Karger, 2011
    Nationell ämneskategori
    Medicinska och farmaceutiska grundvetenskaper
    Identifikatorer
    urn:nbn:se:liu:diva-65447 (URN)10.1159/000325297 (DOI)000294572500008 ()21576918 (PubMedID)
    Anmärkning

    Funding Agencies|Swedish Research Council|529-2003-5994,2005-7046,2006-5968,2007-2673,2009-3821|Bill and Melinda Gates Foundation||SIDA/SAREC||Ekhaga Foundation||Carl Trygger Foundation||King Gustaf V 80-Year Memorial Foundation||County Council of Ostergotland||Swedish Heart Lung Foundation||Oskar II Jubilee Foundation||Clas Groschinsky Foundation||Soderbergs Foundation||Colorado State University, Fort Collins (NIH, NIAID)|HHSN26620040 0091C|

    Tillgänglig från: 2011-02-08 Skapad: 2011-02-08 Senast uppdaterad: 2018-01-12Bibliografiskt granskad
    4. Human macrophages infected with virulent Mycobacterium tuberculosis undergo ESAT-6-dependent necrosis, but not pyroptosis or pyronecrosis
    Öppna denna publikation i ny flik eller fönster >>Human macrophages infected with virulent Mycobacterium tuberculosis undergo ESAT-6-dependent necrosis, but not pyroptosis or pyronecrosis
    (Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Mycobacterium tuberculosis (Mtb) infects lung macrophages, which instead of killing the pathogen can be manipulated by the bacilli, creating an environment suitable for intracellular replication and spread to adjacent cells. The role of host cell death during Mtb infection is debated because the bacilli have been shown to be both anti-apoptotic, keeping the host cell alive to avoid the antimicrobial effects of apoptosis, and pro-necrotic, killing the host macrophage to allow infection of neighboring cells. Since mycobacteria are able to activate the NLRP3 inflammasome, we investigated whether Mtb could induce one of the recently described inflammasome-linked cell death modes pyroptosis and pyronecrosis, in human monocyte-derived macrophages. Cells were infected with virulent (H37Rv) Mtb at a multiplicity of infection (MOI) of 1 or 10. The higher MOI resulted in strongly enhanced release of IL-1β, while a low MOI gave no IL-1β response. The infected macrophages were collected and cell viability in terms of the integrity of DNA, mitochondria and the plasma membrane was determined. We found that infection with H37Rv at MOI 10, but not MOI 1, over two days led to extensive DNA fragmentation, loss of mitochondrial membrane potential and loss of plasma membrane integrity. Although we observed plasma membrane permeabilization and IL-1 β release from infected cells, the cell death induced by Mtb was not pyroptosis or pyronecrosis, as it was independent of caspase-1 and cathepsin B. Instead, we conclude that as virulent Mtb reaches a threshold number of bacilli inside the macrophage, ESAT-6-dependent necrosis occurs, activating caspase-1 in the process.

    Nationell ämneskategori
    Medicin och hälsovetenskap
    Identifikatorer
    urn:nbn:se:liu:diva-65451 (URN)
    Anmärkning
    Submitted manuscriptTillgänglig från: 2011-02-08 Skapad: 2011-02-08 Senast uppdaterad: 2011-02-22Bibliografiskt granskad
  • 8.
    Welin, Amanda
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Eklund, Daniel
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi.
    Stendahl, Olle
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Lerm, Maria
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Human Macrophages Infected with a High Burden of ESAT-6-Expressing M. tuberculosis Undergo Caspase-1-and Cathepsin B-Independent Necrosis2011Ingår i: PLOS ONE, ISSN 1932-6203, Vol. 6, nr 5Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Mycobacterium tuberculosis (Mtb) infects lung macrophages, which instead of killing the pathogen can be manipulated by the bacilli, creating an environment suitable for intracellular replication and spread to adjacent cells. The role of host cell death during Mtb infection is debated because the bacilli have been shown to be both anti-apoptotic, keeping the host cell alive to avoid the antimicrobial effects of apoptosis, and pro-necrotic, killing the host macrophage to allow infection of neighboring cells. Since mycobacteria activate the NLRP3 inflammasome in macrophages, we investigated whether Mtb could induce one of the recently described inflammasome-linked cell death modes pyroptosis and pyronecrosis. These are mediated through caspase-1 and cathepsin-B, respectively. Human monocyte-derived macrophages were infected with virulent (H37Rv) Mtb at a multiplicity of infection (MOI) of 1 or 10. The higher MOI resulted in strongly enhanced release of IL-1 beta, while a low MOI gave no IL-1 beta response. The infected macrophages were collected and cell viability in terms of the integrity of DNA, mitochondria and the plasma membrane was determined. We found that infection with H37Rv at MOI 10, but not MOI 1, over two days led to extensive DNA fragmentation, loss of mitochondrial membrane potential, loss of plasma membrane integrity, and HMGB1 release. Although we observed plasma membrane permeabilization and IL-1 beta release from infected cells, the cell death induced by Mtb was not dependent on caspase-1 or cathepsin B. It was, however, dependent on mycobacterial expression of ESAT-6. We conclude that as virulent Mtb reaches a threshold number of bacilli inside the human macrophage, ESAT-6-dependent necrosis occurs, activating caspase-1 in the process.

  • 9.
    Welin, Amanda
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Eklund, Daniel
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Stendahl, Olle
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Lerm, Maria
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Human macrophages infected with virulent Mycobacterium tuberculosis undergo ESAT-6-dependent necrosis, but not pyroptosis or pyronecrosisManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Mycobacterium tuberculosis (Mtb) infects lung macrophages, which instead of killing the pathogen can be manipulated by the bacilli, creating an environment suitable for intracellular replication and spread to adjacent cells. The role of host cell death during Mtb infection is debated because the bacilli have been shown to be both anti-apoptotic, keeping the host cell alive to avoid the antimicrobial effects of apoptosis, and pro-necrotic, killing the host macrophage to allow infection of neighboring cells. Since mycobacteria are able to activate the NLRP3 inflammasome, we investigated whether Mtb could induce one of the recently described inflammasome-linked cell death modes pyroptosis and pyronecrosis, in human monocyte-derived macrophages. Cells were infected with virulent (H37Rv) Mtb at a multiplicity of infection (MOI) of 1 or 10. The higher MOI resulted in strongly enhanced release of IL-1β, while a low MOI gave no IL-1β response. The infected macrophages were collected and cell viability in terms of the integrity of DNA, mitochondria and the plasma membrane was determined. We found that infection with H37Rv at MOI 10, but not MOI 1, over two days led to extensive DNA fragmentation, loss of mitochondrial membrane potential and loss of plasma membrane integrity. Although we observed plasma membrane permeabilization and IL-1 β release from infected cells, the cell death induced by Mtb was not pyroptosis or pyronecrosis, as it was independent of caspase-1 and cathepsin B. Instead, we conclude that as virulent Mtb reaches a threshold number of bacilli inside the macrophage, ESAT-6-dependent necrosis occurs, activating caspase-1 in the process.

  • 10.
    Welin, Amanda
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Lerm, Maria
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Inside or outside the phagosome? The controversy of the intracellular localization of Mycobacterium tuberculosis2012Ingår i: Tuberculosis, ISSN 1472-9792, E-ISSN 1873-281X, Vol. 92, nr 2, s. 113-120Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    The localization of Mycobacterium tuberculosis (Mtb) inside the macrophage has been a matter of debate in recent years. Upon inhalation, the bacterium is taken up into macrophage phagosomes, which are manipulated by the bacterium. Subsequent translocation of the bacilli into the cytosol has been observed by several groups, while others fail to observe this phenomenon. Here, we review the available literature in favour of and against this idea, and scrutinize the existing data on how human macrophages control Mtb infection, relating this to the robustness of the host cell. We conclude that both phagosomal maturation inhibition and escape from the phagosome are part of the greater infection strategy of Mtb. The balance between the host cell and the infecting bacterium is an important factor in determining the outcome of infection as well as whether phagosomal escape occurs and can be captured.

  • 11.
    Welin, Amanda
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Raffetseder, Johanna
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Eklund, Daniel
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Stendahl, Olle
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Lerm, Maria
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Importance of phagosomal functionality for growth restriction of Mycobacterium tuberculosis in primary human macrophages2011Ingår i: Journal of Innate Immunity, ISSN 1662-811X, E-ISSN 1662-8128, Vol. 3, nr 5, s. 508-518Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The best characterized survival mechanism of Mycobacterium tuberculosis inside the macrophage is the inhibition of phagosomal maturation. Phagosomal maturation involves several steps including fusion with lysosomes and acidification. However, it has not been elucidated which components of phagosomal maturation correlate with growth restriction of virulent mycobacteria in human macrophages, and we aimed to study this. We infected human monocyte-derived macrophages with M. tuberculosis and assessed bacterial replication, translocation of CD63 to the phagosome, and phagosomal acidification. We found that unstimulated macrophages were able to control infection with M. tuberculosis upon inoculation at a low, but not high, multiplicity of infection (MOI). H37Rv and H37Ra infection, at both high and low MOI, led to equally ineffective translocation of CD63 to the phagosome. This was true despite the impaired growth ability of H37Rv at the low MOI and of H37Ra even at the high MOI, indicating that inhibition of CD63 translocation was not sufficient for growth to occur. On the other hand, acidification of mycobacterial phagosomes was more efficient at a low MOI with both mycobacterial strains, consistent with a role for phagosomal acidification in restricting M. tuberculosis growth. Inhibition of the vacuolar H+-ATPase as well as of cathepsin D led to enhanced mycobacterial replication inside the macrophage. We conclude that acidification and related functional aspects of the mature phagosome are important factors for restriction of M. tuberculosis replication in human macrophages.

  • 12.
    Welin, Amanda
    et al.
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Winberg Tinnerfelt, Martin
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Abdalla, Hana
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Särndahl Lindblom, Eva
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Rasmusson, Birgitta
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Stendahl, Olle
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Lerm, Maria
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Incorporation of Mycobacterium tuberculosis lipoarabinomannan into macrophage membrane rafts is a prerequisite for the phagosomal maturation block.2008Ingår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 76, nr 7, s. 2882-2887Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Lipoarabinomannan (LAM) is one of the key virulence factors for Mycobacterium tuberculosis, the etiological agent of tuberculosis. During uptake of mycobacteria, LAM interacts with the cell membrane of the host macrophage and can be detected throughout the cell upon infection. LAM can inhibit phagosomal maturation as well as induce a proinflammatory response in bystander cells. The aim of this study was to investigate how LAM exerts its action on human macrophages. We show that LAM is incorporated into membrane rafts of the macrophage cell membrane via its glycosylphosphatidylinositol anchor and that incorporation of mannose-capped LAM from M. tuberculosis results in reduced phagosomal maturation. This is dependent on successful insertion of the glycosylphosphatidylinositol anchor. LAM does not, however, induce the phagosomal maturation block through activation of p38 mitogen-activated protein kinase, contradicting some previous suggestions.

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