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TPL-2 kinase induces phagosome acidification to promote macrophage killing of bacteria.

ABSTRACT: Tumour progression locus 2 (TPL-2) kinase mediates Toll-like receptor (TLR) activation of ERK1/2 and p38α MAP kinases in myeloid cells to modulate expression of key cytokines in innate immunity. This study identified a novel MAP kinase-independent regulatory function for TPL-2 in phagosome maturation, an essential process for killing of phagocytosed microbes. TPL-2 catalytic activity was demonstrated to induce phagosome acidification and proteolysis in primary mouse and human macrophages following uptake of latex beads. Quantitative proteomics revealed that blocking TPL-2 catalytic activity significantly altered the protein composition of phagosomes, particularly reducing the abundance of V-ATPase proton pump subunits. Furthermore, TPL-2 stimulated the phosphorylation of DMXL1, a regulator of V-ATPases, to induce V-ATPase assembly and phagosome acidification. Consistent with these results, TPL-2 catalytic activity was required for phagosome acidification and the efficient killing of Staphylococcus aureus and Citrobacter rodentium following phagocytic uptake by macrophages. TPL-2 therefore controls innate immune responses of macrophages to bacteria via V-ATPase induction of phagosome maturation.

SUBMITTER: Breyer F

PROVIDER: S-EPMC8126920 | biostudies-literature |

REPOSITORIES: biostudies-literature

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TPL-2 kinase induces phagosome acidification to promote macrophage killing of bacteria

Project description:Tumor Progression Locus 2 (TPL-2) kinase mediates Toll-like Receptor (TLR) activation of ERK1/2 and p38 MAP kinases in myeloid cells to modulate expression of key cytokines in innate immunity. This study identified a novel MAP kinase-independent regulatory function for TPL-2 in phagosome maturation, an essential process for killing of phagocytosed microbes. TPL-2 catalytic activity was demonstrated to induce phagosome acidification and proteolysis in primary mouse and human macrophages following uptake of latex beads. Quantitative proteomics revealed that blocking TPL-2 catalytic activity significantly altered the protein composition of phagosomes, particularly reducing the abundance of V-ATPase proton pump subunits. Furthermore, TPL-2 stimulated the phosphorylation of DMXL1, a critical regulator of V-ATPases, to induce phagosome acidification. Consistent with these results, TPL-2 catalytic activity was required for phagosome maturation and the efficient killing of Staphylococcus aureus following phagocytic uptake by macrophages. These results indicate that TPL-2 controls the innate immune response of macrophages to bacteria via V-ATPase induction of phagosome maturation.

2021-03-24 | PXD020247 | Pride

TPL-2 kinase induces phagosome acidification to promote macrophage killing of Staphylococcus aureus

Project description:Tumor Progression Locus 2 (TPL-2) kinase mediates Toll-like Receptor (TLR) activation of ERK1/2 and p38-alpha MAP kinases in myeloid cells to modulate expression of key cytokines in innate immunity. This study identified a novel MAP kinase-independent regulatory function for TPL-2 in phagosome maturation, an essential process for killing of phagocytosed bacteria. TPL-2 catalytic activity was demonstrated to induce phagosome acidification and proteolysis in primary mouse and human macrophages following uptake of latex beads. Mass spectrometry analysis revealed that blocking TPL-2 catalytic activity significantly altered the protein composition of phagosomes, particularly reducing the abundance of V-ATPase proton pump subunits. Furthermore, TPL-2 was shown to stimulate the phosphorylation of DMXL1, a critical regulator of V-ATPases, to induce phagosome acidification. Consistent with these results, TPL-2 catalytic activity was required for phagosome acidification, activation of phagosome acid-sensitive cathepsins and the efficient killing of Staphylococcus aureus following phagocytic uptake by macrophages. These results indicate that TPL-2 controls the innate immune response of macrophages to bacteria via MAP kinase regulation of gene expression and V-ATPase induction of phagosome maturation.

2021-02-11 | PXD020401 | Pride

The Bicarbonate Transporter SLC4A7 Plays a Key Role in Macrophage Phagosome Acidification.

Project description:Macrophages represent the first line of immune defense against pathogens, and phagosome acidification is a necessary step in pathogen clearance. Here, we identified the bicarbonate transporter SLC4A7, which is strongly induced upon macrophage differentiation, as critical for phagosome acidification. Loss of SLC4A7 reduced acidification of phagocytosed beads or bacteria and impaired the intracellular microbicidal capacity in human macrophage cell lines. The phenotype was rescued by wild-type SLC4A7, but not by SLC4A7 mutants, affecting transport capacity or cell surface localization. Loss of SLC4A7 resulted in increased cytoplasmic acidification during phagocytosis, suggesting that SLC4A7-mediated, bicarbonate-driven maintenance of cytoplasmic pH is necessary for phagosome acidification. Altogether, we identify SLC4A7 and bicarbonate-driven cytoplasmic pH homeostasis as an important element of phagocytosis and the associated microbicidal functions in macrophages.

| S-EPMC6002608 | biostudies-literature

Bet-hedging antimicrobial strategies in macrophage phagosome acidification drive the dynamics of Cryptococcus neoformans intracellular escape mechanisms.

Project description:The fungus Cryptococcus neoformans is a major human pathogen with a remarkable intracellular survival strategy that includes exiting macrophages through non-lytic exocytosis (Vomocytosis) and transferring between macrophages (Dragotcytosis) by a mechanism that involves sequential events of non-lytic exocytosis and phagocytosis. Vomocytosis and Dragotcytosis are fungal driven processes, but their triggers are not understood. We hypothesized that the dynamics of Dragotcytosis could inherit the stochasticity of phagolysosome acidification and that Dragotcytosis was triggered by fungal cell stress. Consistent with this view, fungal cells involved in Dragotcytosis reside in phagolysosomes characterized by low pH and/or high oxidative stress. Using fluorescent microscopy, qPCR, live cell video microscopy, and fungal growth assays we found that the that mitigating pH or oxidative stress reduced Dragotcytosis frequency, whereas ROS susceptible mutants of C. neoformans underwent Dragotcytosis more frequently. Dragotcytosis initiation was linked to phagolysosomal pH, oxidative stresses, and macrophage polarization state. Dragotcytosis manifested stochastic dynamics thus paralleling the dynamics of phagosomal acidification, which correlated with the inhospitality of phagolysosomes in differently polarized macrophages. Hence, randomness in phagosomal acidification randomly created a population of inhospitable phagosomes where fungal cell stress triggered stochastic C. neoformans non-lytic exocytosis dynamics to escape a non-permissive intracellular macrophage environment.

| S-EPMC9302974 | biostudies-literature

Phosphatidylserine liposomes induce a phagosome acidification-dependent and ROS-mediated intracellular killing of <i>Mycobacterium abscessus</i> in human macrophages.

Project description:Mycobacterium abscessus (Mab) is an opportunistic nontuberculous mycobacterium responsible of difficult-to-treat pulmonary infections in vulnerable patients, such as those suffering from Cystic Fibrosis (CF), where it represents a major cause of morbidity and mortality. Additionally, due to the intrinsic extensive antimicrobial resistance spectrum displayed by this species and the side effects reported for some available antibiotics, the therapeutic management of such infections remains extremely difficult. In the present study, we show that phosphatidylserine liposomes (PS-L) enhance intracellular mycobacterial killing of Mab infected human macrophages with functional or pharmacologically inhibited cystic fibrosis conductance regulator (CFTR), by a mechanism involving phagosome acidification and reactive oxygen species (ROS) production. Additionally, PS-L significantly reduce proinflammatory response of Mab infected macrophages in terms of NF-kB activation and TNF-α production, irrespective of CFTR inhibition. Altogether, these results represent the proof of concept for a possible future development of PS-L as a therapeutic strategy against difficult-to-treat Mab infection.

| S-EPMC11366698 | biostudies-literature

Mycobacterium avium MAV_2941 mimics phosphoinositol-3-kinase to interfere with macrophage phagosome maturation.

Project description:Mycobacterium avium subsp hominissuis (M. avium) is a pathogen that infects and survives in macrophages. Previously, we have identified the M. avium MAV_2941 gene encoding a 73 amino acid protein exported by the oligopeptide transporter OppA to the macrophage cytoplasm. Mutations in MAV_2941 were associated with significant impairment of M. avium growth in THP-1 macrophages. In this study, we investigated the molecular mechanism of MAV_2941 action and demonstrated that MAV_2941 interacts with the vesicle trafficking proteins syntaxin-8 (STX8), adaptor-related protein complex 3 (AP-3) complex subunit beta-1 (AP3B1) and Archain 1 (ARCN1) in mononuclear phagocytic cells. Sequencing analysis revealed that the binding site of MAV_2941 is structurally homologous to the human phosphatidylinositol 3-kinase (PI3K) chiefly in the region recognized by vesicle trafficking proteins. The β3A subunit of AP-3, encoded by AP3B1, is essential for trafficking cargo proteins, including lysosomal-associated membrane protein 1 (LAMP-1), to the phagosome and lysosome-related organelles. Here, we show that while the heat-killed M. avium when ingested by macrophages co-localizes with LAMP-1 protein, transfection of MAV_2941 in macrophages results in significant decrease of LAMP-1 co-localization with the heat-killed M. avium phagosomes. Mutated MAV_2941, where the amino acids homologous to the binding region of PI3K were changed, failed to interact with trafficking proteins. Inactivation of the AP3B1 gene led to alteration in the trafficking of LAMP-1. These results suggest that M. avium MAV_2941 interferes with the protein trafficking within macrophages altering the maturation of phagosome.

| S-EPMC4554883 | biostudies-literature

A small molecule deubiquitinase inhibitor increases localization of inducible nitric oxide synthase to the macrophage phagosome and enhances bacterial killing.

Project description:Macrophages are key mediators of antimicrobial defense and innate immunity. Innate intracellular defense mechanisms can be rapidly regulated at the posttranslational level by the coordinated addition and removal of ubiquitin by ubiquitin ligases and deubiquitinases (DUBs). While ubiquitin ligases have been extensively studied, the contribution of DUBs to macrophage innate immune function is incompletely defined. We therefore employed a small molecule DUB inhibitor, WP1130, to probe the role of DUBs in the macrophage response to bacterial infection. Treatment of activated bone marrow-derived macrophages (BMM) with WP1130 significantly augmented killing of the intracellular bacterial pathogen Listeria monocytogenes. WP1130 also induced killing of phagosome-restricted bacteria, implicating a bactericidal mechanism associated with the phagosome, such as the inducible nitric oxide synthase (iNOS). WP1130 had a minimal antimicrobial effect in macrophages lacking iNOS, indicating that iNOS is an effector mechanism for WP1130-mediated bacterial killing. Although overall iNOS levels were not notably different, we found that WP1130 significantly increased colocalization of iNOS with the Listeria-containing phagosome during infection. Taken together, our data indicate that the deubiquitinase inhibitor WP1130 increases bacterial killing in macrophages by enhancing iNOS localization to the phagosome and suggest a potential role for ubiquitin regulation in iNOS trafficking.

| S-EPMC3232648 | biostudies-literature

Staphylococcus aureus lipoproteins promote abscess formation in mice, shielding bacteria from immune killing.

Project description:Despite being a major bacterial factor in alerting the human immune system, the role of Staphylococcus aureus (S. aureus) lipoproteins (Lpp) in skin infections remains largely unknown. Here, we demonstrated that subcutaneous injection of S. aureus Lpp led to infiltration of neutrophils and monocytes/macrophages and induced skin lesions in mice. Lipid-moiety of S. aureus Lpp and host TLR2 was responsible for such effect. Lpp-deficient S. aureus strains exhibited smaller lesion size and reduced bacterial loads than their parental strains; the altered phenotype in bacterial loads was TLR2-independent. Lpp expression in skin infections contributed to imbalanced local hemostasis toward hypercoagulable state. Depletion of leukocytes or fibrinogen abrogated the effects induced by Lpp in terms of skin lesions and bacterial burden. Our data suggest that S. aureus Lpp induce skin inflammation and promote abscess formation that protects bacteria from innate immune killing. This suggests an intriguing bacterial immune evasion mechanism.

| S-EPMC8010101 | biostudies-literature

Chronic Iron Overload Results in Impaired Bacterial Killing of THP-1 Derived Macrophage through the Inhibition of Lysosomal Acidification.

Project description:Iron is essential for living organisms and the disturbance of iron homeostasis is associated with altered immune function. Additionally, bacterial infections can cause major complications in instances of chronic iron overload, such as patients with transfusion-dependent thalassemia. Monocytes and macrophages play important roles in maintaining systemic iron homoeostasis and in defense against invading pathogens. However, the effect of iron overload on the function of monocytes and macrophages is unclear. We elucidated the effects of chronic iron overload on human monocytic cell line (THP-1) and THP-1 derived macrophages (TDM) by continuously exposing them to high levels of iron (100 μM) to create I-THP-1 and I-TDM, respectively. Our results show that iron overload did not affect morphology or granularity of I-THP-1, but increased the granularity of I-TDM. Bactericidal assays for non-pathogenic E. coli DH5α, JM109 and pathogenic P. aeruginosa all revealed decreased efficiency with increasing iron concentration in I-TDM. The impaired P. aeruginosa killing ability of human primary monocyte derived macrophages (hMDM) was also found when cells are cultured in iron contained medium. Further studies on the bactericidal activity of I-TDM revealed lysosomal dysfunction associated with the inhibition of lysosomal acidification resulting in increasing lysosomal pH, the impairment of post-translational processing of cathepsins (especially cathepsin D), and decreased autophagic flux. These findings may explain the impaired innate immunity of thalassemic patients with chronic iron overload, suggesting the manipulation of lysosomal function as a novel therapeutic approach.

| S-EPMC4886970 | biostudies-literature

Regulation of experimental autoimmune encephalomyelitis by TPL-2 kinase.

Project description:Tumor progression locus 2 (TPL-2) expression is required for efficient polarization of naive T cells to Th1 effector cells in vitro, as well as for Th1-mediated immune responses. In the present study, we investigated the potential role of TPL-2 in Th17 cells. TPL-2 was found to be dispensable for Th17 cell differentiation in vitro, and for the initial priming of Th17 cells in experimental autoimmune encephalomyelitis (EAE), a Th17 cell-mediated disease model for multiple sclerosis. Nevertheless, TPL-2-deficient mice were protected from EAE, which correlated with reduced immune cell infiltration, demyelination, and axonal damage in the CNS. Adoptive transfer experiments demonstrated that there was no T cell-intrinsic function for TPL-2 in EAE, and that TPL-2 signaling was not required in radiation-sensitive hematopoietic cells. Rather, TPL-2 signaling in radiation-resistant stromal cells promoted the effector phase of the disease. Importantly, using a newly generated mouse strain expressing a kinase-inactive form of TPL-2, we demonstrated that stimulation of EAE was dependent on the catalytic activity of TPL-2 and not its adaptor function to stabilize the associated ubiquitin-binding protein ABIN-2. Our data therefore raise the possibility that small molecule inhibitors of TPL-2 may be beneficial in multiple sclerosis therapy.

| S-EPMC3979668 | biostudies-literature

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