Mutations in the leucine‐rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease, chronic inflammation and mycobacterial infections. Although there is evidence supporting the idea that ...LRRK2 has an immune function, the cellular function of this kinase is still largely unknown. By using genetic, pharmacological and proteomics approaches, we show that LRRK2 kinase activity negatively regulates phagosome maturation via the recruitment of the Class III phosphatidylinositol‐3 kinase complex and Rubicon to the phagosome in macrophages. Moreover, inhibition of LRRK2 kinase activity in mouse and human macrophages enhanced Mycobacterium tuberculosis phagosome maturation and mycobacterial control independently of autophagy. In vivo, LRRK2 deficiency in mice resulted in a significant decrease in M. tuberculosis burdens early during the infection. Collectively, our findings provide a molecular mechanism explaining genetic evidence linking LRRK2 to mycobacterial diseases and establish an LRRK2‐dependent cellular pathway that controls M. tuberculosis replication by regulating phagosome maturation.
Synopsis
Possible immune functions of LRRK2, a kinase frequently mutated in Parkinson's disease, have remained ill‐defined. Genetic, pharmacological and proteomics approaches now reveal it as a negative regulator of phagosome maturation in macrophages, thereby affecting the control of Mycobacterium tuberculosis (Mtb) infection.
LRRK2 loss targets Mtb to phagolysosomes and limits Mtb replication.
LRRK2 inhibition enhances phagosome maturation in macrophages.
LRRK2 activity is required for the recruitment of class III PI3K/Rubicon into phagosomes.
Loss of LRRK2 enhances innate immunity to Mtb in mice.
LRRK2 KO alters inflammatory profiles after Mtb infection in vitro and in vivo.
The Parkinson's disease‐associated kinase LRRK2 affects innate immune control of mycobacterial infections in vitro and in vivo via phagosome recruitment of class III PI3K and Rubicon.
Autophagy in immunity and inflammation LEVINE, Beth; MIZUSHIMA, Noboru; VIRGIN, Herbert W
Nature (London),
01/2011, Letnik:
469, Številka:
7330
Journal Article
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Autophagy is an essential, homeostatic process by which cells break down their own components. Perhaps the most primordial function of this lysosomal degradation pathway is adaptation to nutrient ...deprivation. However, in complex multicellular organisms, the core molecular machinery of autophagy - the 'autophagy proteins' - orchestrates diverse aspects of cellular and organismal responses to other dangerous stimuli such as infection. Recent developments reveal a crucial role for the autophagy pathway and proteins in immunity and inflammation. They balance the beneficial and detrimental effects of immunity and inflammation, and thereby may protect against infectious, autoimmune and inflammatory diseases.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Extracellular vesicles (EVs) have been shown to carry microbial components and function in the host defense against infections. In this study, we demonstrate that Mycobacterium tuberculosis (M.tb) ...RNA is delivered into macrophage‐derived EVs through an M.tb SecA2‐dependent pathway and that EVs released from M.tb‐infected macrophages stimulate a host RIG‐I/MAVS/TBK1/IRF3 RNA sensing pathway, leading to type I interferon production in recipient cells. These EVs also promote, in a RIG‐I/MAVS‐dependent manner, the maturation of M.tb‐containing phagosomes through a noncanonical LC3 pathway, leading to increased bacterial killing. Moreover, treatment of M.tb‐infected macrophages or mice with a combination of moxifloxacin and EVs, isolated from M.tb‐infected macrophages, significantly lowered bacterial burden relative to either treatment alone. We hypothesize that EVs, which are preferentially removed by macrophages in vivo, can be combined with effective antibiotics as a novel approach to treat drug‐resistant TB.
Synopsis
Extracellular vesicles carry microbial components and have roles during host defence. EVs released from M. tuberculosis‐infected macrophages activate the cytosolic RNA sensing pathway, and potentiate antibiotic treatment in an infection model.
EVs from M.tb‐infected macrophages stimulate RIG‐I/MAVS/TBK1/IRF3 RNA sensing pathways.
EVs promote LC3‐associated phagosome maturation in M.tb‐infected macrophages.
EVs inhibit M.tb survival in macrophages via a MAVS‐dependent pathway.
EVs significantly increase the efficacy of the antibiotic moxifloxacin in M.tb infected mice.
Extracellular vesicles carry microbial components and have roles during host defence. EVs released from M. tuberculosis‐infected macrophages activate the cytosolic RNA sensing pathway, and potentiate antibiotic treatment in an infection model.
Systemic inflammatory reactions have been postulated to exacerbate neurodegenerative diseases via microglial activation. We now demonstrate in vivo that repeated systemic challenge of mice over four ...consecutive days with bacterial LPS maintained an elevated microglial inflammatory phenotype and induced loss of dopaminergic neurons in the substantia nigra. The same total cumulative LPS dose given within a single application did not induce neurodegeneration. Whole-genome transcriptome analysis of the brain demonstrated that repeated systemic LPS application induced an activation pattern involving the classical complement system and its associated phagosome pathway. Loss of dopaminergic neurons induced by repeated systemic LPS application was rescued in complement C3-deficient mice, confirming the involvement of the complement system in neurodegeneration. Our data demonstrate that a phagosomal inflammatory response of microglia is leading to complement-mediated loss of dopaminergic neurons.
Phagocytosis is a fundamental cellular process that is pivotal for immunity as it coordinates microbial killing, innate immune activation and antigen presentation. An essential step in this process ...is phagosome acidification, which regulates many functions of these organelles that allow phagosomes to participate in processes that are essential to both innate and adaptive immunity. Here we report that acidification of phagosomes containing Gram-positive bacteria is regulated by the NLRP3 inflammasome and caspase-1. Active caspase-1 accumulates on phagosomes and acts locally to control the pH by modulating buffering by the NADPH oxidase NOX2. These data provide insight into a mechanism by which innate immune signals can modify cellular defenses and establish a new function for the NLRP3 inflammasome and caspase-1 in host defense.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Autophagy is a fundamental biological process of the eukaryotic cell contributing to diverse cellular and physiological functions including cell-autonomous defense against intracellular pathogens. ...Here, we screened the Rab family of membrane trafficking regulators for effects on autophagic elimination of Mycobacterium tuberculosis var. bovis BCG and found that Rab8b and its downstream interacting partner, innate immunity regulator TBK-1, are required for autophagic elimination of mycobacteria in macrophages. TBK-1 was necessary for autophagic maturation. TBK-1 coordinated assembly and function of the autophagic machinery and phosphorylated the autophagic adaptor p62 (sequestosome 1) on Ser-403, a residue essential for its role in autophagic clearance. A key proinflammatory cytokine, IL-1β, induced autophagy leading to autophagic killing of mycobacteria in macrophages, and this IL-1β activity was dependent on TBK-1. Thus, TBK-1 is a key regulator of immunological autophagy and is responsible for the maturation of autophagosomes into lytic bactericidal organelles.
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► TBK-1 controls autophagy in cell-autonomous defense against M. tuberculosis ► TBK-1 controls autophagic maturation ► TBK-1 phosphorylates the autophagic adaptor p62 ► IL-1β induces TBK-1 dependent autophagic killing of M. tuberculosis
Coxsackievirus B3 (CVB3), a member of the picornavirus family and enterovirus genus, causes viral myocarditis, aseptic meningitis, and pancreatitis in humans. We genetically engineered a unique ...molecular marker, "fluorescent timer" protein, within our infectious CVB3 clone and isolated a high-titer recombinant viral stock (Timer-CVB3) following transfection in HeLa cells. "Fluorescent timer" protein undergoes slow conversion of fluorescence from green to red over time, and Timer-CVB3 can be utilized to track virus infection and dissemination in real time. Upon infection with Timer-CVB3, HeLa cells, neural progenitor and stem cells (NPSCs), and C2C12 myoblast cells slowly changed fluorescence from green to red over 72 hours as determined by fluorescence microscopy or flow cytometric analysis. The conversion of "fluorescent timer" protein in HeLa cells infected with Timer-CVB3 could be interrupted by fixation, suggesting that the fluorophore was stabilized by formaldehyde cross-linking reactions. Induction of a type I interferon response or ribavirin treatment reduced the progression of cell-to-cell virus spread in HeLa cells or NPSCs infected with Timer-CVB3. Time lapse photography of partially differentiated NPSCs infected with Timer-CVB3 revealed substantial intracellular membrane remodeling and the assembly of discrete virus replication organelles which changed fluorescence color in an asynchronous fashion within the cell. "Fluorescent timer" protein colocalized closely with viral 3A protein within virus replication organelles. Intriguingly, infection of partially differentiated NPSCs or C2C12 myoblast cells induced the release of abundant extracellular microvesicles (EMVs) containing matured "fluorescent timer" protein and infectious virus representing a novel route of virus dissemination. CVB3 virions were readily observed within purified EMVs by transmission electron microscopy, and infectious virus was identified within low-density isopycnic iodixanol gradient fractions consistent with membrane association. The preferential detection of the lipidated form of LC3 protein (LC3 II) in released EMVs harboring infectious virus suggests that the autophagy pathway plays a crucial role in microvesicle shedding and virus release, similar to a process previously described as autophagosome-mediated exit without lysis (AWOL) observed during poliovirus replication. Through the use of this novel recombinant virus which provides more dynamic information from static fluorescent images, we hope to gain a better understanding of CVB3 tropism, intracellular membrane reorganization, and virus-associated microvesicle dissemination within the host.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Once across the barrier of the epithelium, macrophages constitute the primary defense against microbial invasion. For most microbes, the acidic, hydrolytically competent environment of the ...phagolysosome is sufficient to kill them. Despite our understanding of the trafficking events that regulate phagosome maturation, our appreciation of the lumenal environment within the phagosome is only now becoming elucidated through real-time functional assays. The assays quantify pH change, phagosome/lysosome fusion, proteolysis, lipolysis, and β-galactosidase activity. This information is particularly important for understanding pathogens that successfully parasitize the endosomal/lysosomal continuum. Mycobacterium tuberculosis infects macrophages through arresting the normal maturation process of the phagosome, retaining its vacuole at pH 6.4 with many of the characteristics of an early endosome. Current studies are focusing on the transcriptional response of the bacterium to the changing environment in the macrophage phagosome. Manipulation of these environmental cues, such as preventing the pH drop to pH 6.4 with concanamycin A, abrogates the majority of the transcriptional response in the bacterium, showing that pH is the dominant signal that the bacterium senses and responds to. These approaches represent our ongoing attempts to unravel the discourse that takes place between the pathogen and its host cell.
Mycobacterium tuberculosis (M. tb) is an intracellular pathogen that can replicate within infected macrophages. The ability of M. tb to arrest phagosome maturation is believed to facilitate its ...intracellular multiplication. Rab GTPases regulate membrane trafficking, but details of how Rab GTPases regulate phagosome maturation and how M. tb modulates their localization during inhibiting phagolysosome biogenesis remain elusive. We compared the localization of 42 distinct Rab GTPases to phagosomes containing either Staphylococcus aureus or M. tb. The phagosomes containing S. aureus were associated with 22 Rab GTPases, but only 5 of these showed similar localization kinetics as the phagosomes containing M. tb. The Rab GTPases responsible for phagosome maturation, phagosomal acidification and recruitment of cathepsin D were examined in macrophages expressing the dominant‐negative form of each Rab GTPase. LysoTracker staining and immunofluorescence microscopy revealed that Rab7, Rab20 and Rab39 regulated phagosomal acidification and Rab7, Rab20, Rab22b, Rab32, Rab34, Rab38 and Rab43 controlled the recruitment of cathepsin D to the phagosome. These results suggest that phagosome maturation is achieved by a series of interactions between Rab GTPases and phagosomes and that differential recruitment of these Rab GTPases, except for Rab22b and Rab43, to M. tb‐containing phagosomes is involved in arresting phagosome maturation and inhibiting phagolysosome biogenesis.
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