New Principles of mycobacterial killing in host macrophages

Tuberculosis is caused by the bacterium Mycobacterium tuberculosis and is a global health problem. At the same time, epidemiological data show that the incidence of infections with non-tuberculous opportunistic mycobacteria like M. avium is increasing, especially in countries where tuberculosis is not endemic and in people with impaired immune function due to immunosuppressive drugs, underlying immune deficiency and old age. The treatment of mycobacterial infections is complicated and long-lasting, and resistance is increasing. There is therefore a great need for new and more effective treatment. To get there, we must get a better understanding of the interaction between pathogenic mycobacteria and the host's immune system. In this project, the main goal has been to understand the molecular mechanisms the macrophage uses in attempting to eliminate mycobacteria, and vice versa, how the mycobacteria circumvent these and take residence in macrophages. We have also used the knowledge we have from research on innate immune responses to mycobacteria to study similar responses in T cells and their importance in HIV infection. Keap1 is a sensor for oxidative stress. In 2015, we published a paper in PNAS showing that Keap1 is a negative regulator of inflammatory signaling during infection with M. avium in human macrophages (Awuh et al, PNAS 2015). We also show that this can probably contribute to an increased risk of infection with opportunists such as M. avium. Pathogenic mycobacteria survive in macrophages, probably by manipulating the space they live in so that they are not sent for degradation. We have previously found that M. avium resides in a compartment that is in contact with recycling endosomes (Halaas et al, JID 2010). Post Doctor Alexandre Gidon has continued the work of tracking how M. avium moves inside the macrophage, and how the localization is related to inflammatory signaling. Our data indicate that M. avium is transported to phagolysosomes where it is recognized by Toll-like receptors (TLRs), but some are able to escape and establish in an intracellular compartment where they are not detected. The work has been published in PLOs Pathogens (Gidon, PLOs Pathogens 2017). Following up on this paper, PhD student Signe Åsberg found that antibiotic treatment causes M. avium to lose control of its compartment and is sent to lysosomes for degradation without a new inflammatory response being initiated. The work is prepared for publication. While studying M. avium trafficking, we observed that uninfected cells were also activated. Gidon has now completed a study showing that this is due to a para-/autocrine signal loop of TNF and IL-6 that inhibits the growth of M. avium in the macrophages. The work is prepared for publication. T cells have also been shown to have innate immune receptors such as TLRs, but it is not clear what their significance is. PhD candidate Hany Meås (formerly Ibrahim) found that T cells respond to TLR ligands with the production of inflammatory cytokines. TLR activation also potentiates HIV replication and can re-activate viral replication in aviremic HIV patients. The work is in revision. We have also made a large CRISPR/Cas9 screen in T cells to identify genes central to HIV infection in collaboration with the research group of Richard Kandasamy at CEMIR. Candidates are now being verified, and the work will be completed in 2019

Resultatene fra prosjektet bidrar til økt forståelse for hvordan immunforsvaret responderer på mykobakterier og HIV, og hvordan mikrobene manipulerer immunsystemet. Karakterisering av hvor og hvordan patogene mykobakterier overlever inni vertscellene vil peke på attraktive mål for behandling. TLR responsen i T-celler kan medvirke til lavgradig inflammasjonen hos HIV pasienter og utgjøre nye behandlingsmål. TLR-aktivering kan være aktuelt å bruke i en «kick-and-kill» strategi for HIV behandling og utnyttes i vaksineutvikling. Kompetanseutvikling: Nye forskningsmetoder er etablert: arbeid med HIV infeksjon i BSL3 laboratorier, etablering av mikroskopi-teknikker, og CRISPR/Cas9 screening for identifisering av gener involvert i infeksjonsforsvar. Flere PhD studenter, post doktorer og forskere har vært involvert i prosjektet og fått veiledererfaring samt knyttet nasjonale og internasjonale kontakter. Prosjektet har lagt grunn for nye prosjekter og tildelinger av forskningsmidler

Tuberculosis is caused by the bacterium Mycobacterium tuberculosis and is a global health problem. Treatment is complicated and lengthy, there is the increasing incidence of resistance, and the only available vaccine, BCG, is not effective against adult pulmonary tuberculosis. There is therefore an urgent need for both new vaccines and more effective drugs against tuberculosis. We believe we can contribute to this by studying the interaction between pathogenic mycobacteria and the host's immune system, and we focus specifically on understanding the molecular mechanisms that enables mycobacteria to survive inside its worst enemy, the macrophage. In July 2015 we published a paper in the prestigious journal PNAS (Awuh et al) which shows that Keap1 is a negative regulator of inflammatory signaling by infection with M. avium in human macrophages. In the current period, we have examined the importance of Keap1 in bacterial sepsis and new interaction partners for Keap1 in regulating inflammation. The work is ongoing and we have just received mice that lack Keap1 in the myeloid compartment. The plan is to set up a urinary tract infection model to study the in vivo impact of Keap1 in sepsis (urosepsis). Post Doctor Alexandre Gidon has charted how M. avium moves within the macrophage and how the location is related to inflammatory signaling. This work was published in PLOS Pathogens in July 2017. Some unresolved questions that arose during this work are which proteins are present in the different cellular compartments under infection. We have established an international cooperation (Germany and India) to do proteomic analyzes of mycobacterial phagosomes in order to clarify this. During this work Gidon also observed that non-infected cells in a M. avium infected culture were as activated and secreted cytokines similar to infected cells. He now explores how cells communicate and how this impacts the infection. Gidon continues with PhD fellow Signe Åsberg to study how macrophages respond to mycobacteria subsequent to treatment with different types of antibiotics. This work will be completed by spring 2018. At the same time, Gidon studies how G-protein coupled receptors modulate the inflammatory response in mycobacterium-infected macrophages and will use this work as a starting point for applying for Young Researcher scholarships in FriMedBio. We have also established 3D electron microscopy (FIBSEM) techniques for studying intracellular environments in mycobacterium-infected macrophages. The first article was published in PLOS ONE in September 2015 (Beckwith et al). In the current period, we have established correlative imaging using fluorescence microscopy and FIBSEM, and we have opened a biosafety level 3 lab where we have a confocal microscope for imaging and filming infection with Mycobacterium tuberculosis (Mtb). We have made exciting discoveries on Mtb mediated killing of macrophages and inflammasomes, which upon activation cause secondary cell death. It is shown that T cells also have innate immune receptors like TLRs, but it is not clear what meaning they have. PhD fellow Hany Ibrahim has data showing that T cells respond to TLR ligands with production of classical inflammatory cytokines. We want to see if this is relevant to HIV. This work is now prepared for submission. We have also done a large CRISPR/Cas9 screen in T cells to identify genes/proteins central in HIV infection. This is done in collaboration with Richard Kandasamy, a new group leader of CEMIR. We have candidates now verified and work is expected to be completed in spring 2018. Ibrahim and others will pursue selected candidate proteins for function in HIV infection.