Phagolysosomal Fusion Research Articles

Interleukin 22 Inhibits Intracellular Growth of Mycobacterium tuberculosis by Enhancing Calgranulin A Expression

Previously, we found that interleukin 22 (IL-22) inhibits intracellular growth of Mycobacterium tuberculosis in human monocyte-derived macrophages (MDMs). In the current study, we determined the mechanisms underlying these effects. We found that W7, a phagolysosomal fusion inhibitor, abrogates IL-22-dependent M. tuberculosis growth inhibition in MDMs, suggesting that IL-22 acts through enhanced phagolysosomal fusion. Our microarray analysis indicated that recombinant IL-22 (rIL-22) enhances the expression of an intracellular signaling molecule, calgranulin A. This was confirmed by real-time polymerase chain reaction, Western blot, and confocal microscopy. Calgranulin A small interfering RNA (siRNA) abrogated rIL-22-dependent growth inhibition of M. tuberculosis in MDMs. IL-22 enhanced Rab7 expression and downregulated Rab14 expression of M. tuberculosis-infected MDMs, and these effects were reversed by calgranulin A siRNA. These results suggest that M. tuberculosis growth inhibition by IL-22 depends on calgranulin A and enhanced phagolysosomal fusion, which is associated with increased Rab7 and reduced Rab14 expression.

Bordetella pertussisentry into respiratory epithelial cells and intracellular survival

Bordetella pertussis is the causative agent of pertussis, aka whooping cough. Although generally considered an extracellular pathogen, this bacterium has been found inside respiratory epithelial cells, which might represent a survival strategy inside the host. Relatively little is known, however, about the mechanism of internalization and the fate of B.pertussis inside the epithelia. We show here that B.pertussis is able to enter those cells by a mechanism dependent on microtubule assembly, lipid raft integrity, and the activation of a tyrosine-kinase-mediated signaling. Once inside the cell, a significant proportion of the intracellular bacteria evade phagolysosomal fusion and remain viable in nonacidic lysosome-associated membrane-protein-1-negative compartments. In addition, intracellular B.pertussis was found able to repopulate the extracellular environment after complete elimination of the extracellular bacteria with polymyxin B. Taken together, these data suggest that B.pertussis is able to survive within respiratory epithelial cells and by this means potentially contribute to host immune system evasion.

The roles of complement receptor 3 and Fcγ receptors during Leishmania phagosome maturation

Leishmania are intracellular parasites adapted to surviving in macrophages, whose primary function is elimination of invading pathogens. Leishmania entry into host cells is receptor-mediated. These parasites are able to engage multiple host cell-surface receptors, including MR, TLRs, CR3, and FcγRs. Here, we investigated the role of CR3 and FcγR engagement on the maturation of Leishmania-containing phagosomes using CD11b-/- and FcγR-/- macrophages, and assessing EEA1 and lysosome-associated proteins is necessary for the phagosome maturation delay, characteristic of Leishmania infection. Leishmania-containing phagosomes do not fuse with lyosomes until 5 h postinfection in WT mice. Phagolysosome fusion occurs by 1 h in CD11b and FcγR common chain KO macrophages, although receptor deficiency does not influence Leishmania entry or viability. We also investigated the influence of serum components and their effects on phagosome maturation progression. Opsonization with normal mouse serum, complement-deficient serum, or serum from Leishmania-infected mice all influenced phagosome maturation progression. Our results indicate that opsonophagocytosis influences phagosomal trafficking of Leishmania without altering the intracellular fate.

Listeria monocytogenes: No Spreading without NO

In infectious disease immunology, inducible nitric oxide (NO) synthase-derived NO is believed to function primarily as an antimicrobial and immunoregulatory molecule. In this issue of Immunity, Cole etal. (2012) show that NO helps the intracellular bacteria Listeria monocytogenes to spread.

A pathogen’s journey in the host cell

Manipulation of the actin cytoskeleton is a commonly used process by which bacterial pathogens and viruses are able to neutralize host defense mechanisms and subvert them in order to replicate in a hostile environment. Diverse bacteria display a wide array of mechanisms of regulation of microfilaments to enter, move within or exit the host cell. A less studied subject is how pathogens may co-opt the actin cytoskeleton to disturb vesicle trafficking pathways, namely phagolysosomal fusion, and avoid degradation. In fact, although actin plays a role in endosomal trafficking and phagosome maturation, the knowledge on the exact mechanisms and additional players is still scarce. Recently, we found that the Legionella pneumophila virulence factor VipA is an actin nucleator, associates with actin filaments and early endosomes during infection, and interferes in yeast organelle trafficking pathways, suggesting it may be linking actin dynamics to endosome biogenesis. Further studies on this protein, together with work on other bacterial effectors, may help shed light in the role of actin in endosomal maturation.

The Identification of Gene Duplication and the Role of Secreted Aspartyl Proteinase 1 in Candida parapsilosis Virulence

In this study, we analyzed the role of Candida parapsilosis-secreted aspartyl proteinase isoenzyme 1 (SAPP1) in virulence. The in silico analysis of SAPP1 sequence revealed a 2871 base pair-duplicated region (SAPP1a and SAPP1b) in the genome of C. parapsilosis. We generated homozygous ΔΔsapp1a, ΔΔsapp1b, and ΔΔsapp1a-ΔΔsapp1b mutants. Notably, Sapp1 production in an inducer medium was reduced by approximately 50% in the ΔΔsapp1a and ΔΔsapp1b mutants, but the other validated SAPP gene (SAPP2) was not affected. In contrast, Sapp2 production was increased in the ΔΔsapp1a-ΔΔsapp1b mutant relative to wild-type (WT) yeast. The ΔΔsapp1a-ΔΔsapp1b strain was hypersusceptible to human serum and was attenuated in its capacity to damage host-effector cells. The phagocytosis and killing of ΔΔsapp1a-ΔΔsapp1b yeasts by human peripheral blood mononuclear cells (PBMCs) and PBMC-derived macrophages (PBMC-DM) was significantly enhanced relative to WT. Phagolysosomal fusion in PBMC-DMs occurred more than twice as frequently with ingested ΔΔsapp1a-ΔΔsapp1b yeast cells compared with WT.

Neutrophil Extracellular Trap Formation Is Associated with IL-1β and Autophagy-Related Signaling in Gout

BackgroundGout is a prevalent inflammatory arthritis affecting 1–2% of adults characterized by activation of innate immune cells by monosodium urate (MSU) crystals resulting in the secretion of interleukin-1β (IL-1β). Since neutrophils play a major role in gout we sought to determine whether their activation may involve the formation of proinflammatory neutrophil extracellular traps (NETs) in relation to autophagy and IL-1β.Methodology/Principal FindingsSynovial fluid neutrophils from six patients with gout crisis and peripheral blood neutrophils from six patients with acute gout and six control subjects were isolated. MSU crystals, as well as synovial fluid or serum obtained from patients with acute gout, were used for the treatment of control neutrophils. NET formation was assessed using immunofluorescence microscopy. MSU crystals or synovial fluid or serum from patients induced NET formation in control neutrophils. Importantly, NET production was observed in neutrophils isolated from synovial fluid or peripheral blood from patients with acute gout. NETs contained the alarmin high mobility group box 1 (HMGB1) supporting their pro-inflammatory potential. Inhibition of phosphatidylinositol 3-kinase signaling or phagolysosomal fusion prevented NET formation, implicating autophagy in this process. NET formation was driven at least in part by IL-1β as demonstrated by experiments involving IL-1β and its inhibitor anakinra.Conclusions/SignificanceThese findings document for the first time that activation of neutrophils in gout is associated with the formation of proinflammatory NETs and links this process to both autophagy and IL-1β. Modulation of the autophagic machinery may represent an additional therapeutic study in crystalline arthritides.

IglG and IglI of the Francisella Pathogenicity Island Are Important Virulence Determinants of Francisella tularensis LVS

The Gram-negative bacterium Francisella tularensis is the causative agent of tularemia, a disease intimately associated with the multiplication of the bacterium within host macrophages. This in turn requires the expression of Francisella pathogenicity island (FPI) genes, believed to encode a type VI secretion system. While the exact functions of many of the components have yet to be revealed, some have been found to contribute to the ability of Francisella to cause systemic infection in mice as well as to prevent phagolysosomal fusion and facilitate escape into the host cytosol. Upon reaching this compartment, the bacterium rapidly multiplies, inhibits activation of the inflammasome, and ultimately causes apoptosis of the host cell. In this study, we analyzed the contribution of the FPI-encoded proteins IglG, IglI, and PdpE to the aforementioned processes in F. tularensis LVS. The ΔpdpE mutant behaved similarly to the parental strain in all investigated assays. In contrast, ΔiglG and ΔiglI mutants, although they were efficiently replicating in J774A.1 cells, both exhibited delayed phagosomal escape, conferred a delayed activation of the inflammasome, and exhibited reduced cytopathogenicity as well as marked attenuation in the mouse model. Thus, IglG and IglI play key roles for modulation of the intracellular host response and also for the virulence of F. tularensis.

Contribution of the PhoP/Q regulon to survival and replication of Salmonella enterica serovar Typhimurium in macrophages

The ability of serovars of Salmonella enterica to cause systemic disease is dependent upon their survival and replication within macrophages. To do this, bacteria must withstand or surmount bacteriostatic and bactericidal responses by the host cell, including the delivery of hydrolytic enzymes from lysosomes to the phagosome. The bacterial two-component regulatory system PhoP/Q has been implicated in avoidance of phagolysosomal fusion by S. enterica serovar Typhimurium (S. Typhimurium) in murine macrophages. In this study, the involvement of PhoP/Q-activated genes in avoidance of phagolysosomal fusion was analysed: of all the S. Typhimurium mutant strains tested, only an mgtC mutant strain partially reproduced the phenotype of the phoP mutant strain. As this gene is required for bacterial growth in magnesium-depleted conditions in vitro, the contributions of PhoP/Q to intramacrophage replication and survival were reappraised. Although PhoP/Q was required for both replication and survival of S. Typhimurium within murine macrophages, subsequent analysis of the kinetics of phagolysosomal fusion, taking account of differences in the replication rates of wild-type and phoP mutant strains, provided no evidence for a PhoP/Q-dependent role in this process. PhoP/Q appeared to act subsequent to the process of phagolysosomal avoidance and to promote replication of those bacteria that had already escaped a phagolysosomal fate. Therefore, we conclude that the PhoP/Q regulon enables S. Typhimurium to adapt to intramacrophage stresses other than phagolysosomal fusion.

IL-22 enhances Calgranulin A expression by human macrophages to inhibit virulent Mycobacterium tuberculosis growth (158.21)

Abstract Previously, we found that IL-22 produced by NK cells inhibits intracellular mycobacterial growth in human macrophages (Mϕ) by enhancing phagolysosomal fusion. In the current study, we determined the mechanism/s by which IL-22 inhibits virulent M. tuberculosis H37Rv (M. tb) growth in Mϕ. First we asked whether IL-22 restricts M. tb growth in phagosomes that leads to enhanced phagolysosomal fusion. W7, a phagolysosomal fusion inhibitor abrogated IL-22 dependent M. tb growth in Mϕ (11.38 ± 3.3 x 106 vs 2.95 ± 1.7 x 106 CFU per well, p=0.05) suggesting IL-22 inhibits M. tb growth after phagolysosomal fusion. In microarray analysis we found that mRNA expression for 41 genes was >1-fold higher in rIL-22 cultured infected Mϕ compared to infected Mϕ. Of these genes, we selected Indolamine 2,3-dioxegenase (IDO1) and Calgranulin A for further study. rIL-22 enhanced Calgranulin A expression in M. tb infected Mϕ by three fold (3.3 ± 0.83 vs 1.0 arbitrary units, p=0.04) compared to infected Mϕ as measured by real time PCR and confirmed by confocal microscopy. In contrast, IDO1 expression of infected Mϕ was not effected by rIL-22. Calgranulin A siRNA abrogated rIL-22 dependent growth inhibition of M. tb in macrophages (18.4 ± 1.8 x 106 vs 7.97 ± 0.8 x 106 CFU per well, p=0.0005). Studies are underway to determine the effect of Calgranulin A siRNA on phagolysosomal fusion using early endosomal, late endosomal and lysosomal markers.

Mycobacterium avium and modulation of the host macrophage immune mechanisms [Review article

Pathogenesis by mycobacteria requires the exploitation of host-cell signaling pathways to enhance intracellular survival and persistence of the pathogen. Among patients with end-stage acquired immune-deficiency syndrome, disseminated infection with Mycobacterium avium, a member of the M. avium complex (MAC), is the most common bacterial infection. The virulence and intrinsic multidrug resistance of this pathogen has been attributed in part to its unique cell wall, which is a complex array of hydrocarbon chains containing the arabinogalactan-peptidoglycan mycolic acid core found in all mycobacteria, surrounded by a second electron-dense layer made up, in part, of serovar-specific glycopeptidolipids (GPLs) found only in MAC. Via cell-surface receptors, M. avium, an intra-macrophage (mφ) pathogen, can modulate various host signaling pathways such as the mitogen-activated protein kinase and nuclear factor κB pathways. The modulation of specific mφ signaling cascades can result in the regulation of pro- and anti-inflammatory cytokine production, and the process of phagolysosome fusion. The outcome of this M. avium-host mφ interaction could result in host disease or death of the invading pathogen. This review will focus on the immunomodulation aspects of M. avium pathogenesis as well as the role of GPLs as virulence factors.

NF-κB activation is critical for bacterial lipoprotein tolerance-enhanced bactericidal activity in macrophages during microbial infection (116.15)

Abstract Tolerance to bacterial cell-wall components including gram-positive bacterial lipoprotein (BLP) represents an essential regulatory mechanism during bacterial infection. Our previous work has demonstrated that BLP-induced tolerance, characterised by hyporesponsiveness in producing proinflammatory cytokines and simultaneously enhanced antimicrobial functions, protects against microbial sepsis-related lethality. However, the underlying mechanisms remain unidentified. In this study we reported that upon S. aureus or E. coli challenge, significantly enhanced IκBα phosphorylation and p65 translocation into the nucleus were observed in BLP-tolerised bone marrow-derived macrophages (BMM), indicating that NF-κB is activated in BLP-tolerised macrophages during bacteria infection. To further clarify whether activation of the NF-κB pathway is required for efficient bacterial killing by BLP-tolerised macrophages, we used two NF-κB inhibitors, SN50 and SC-154. Both inhibitors substantially restrained BLP-tolerised macrophage-induced intracellular bacterial killing, and this was closely associated with delayed phagolysosome fusion. Furthermore, the expression of LAMP-1 and Rab5, two membrane-trafficking regulators participated the process of phagosome maturation was significantly downregulated when the NF-κB pathway was blocked. Collectively, our results highlight a novel role of the NF-κB pathway in bactericidal activity displayed by BLP-tolerised macrophages during microbial infection.

Autophagy and ATP-induced anti-apoptosis in antigen presenting cells (APC) follows the cytokine storm in patients after major trauma

Severe trauma and the systemic inflammatory response syndrome (SIRS) occur as a result of a cytokine storm which is in part due to ATP released from damaged tissue. This pathology also leads to increased numbers of immature antigen presenting cells (APC) sharing properties of dendritic cells (DC) or macrophages (MΦ). The occurrence of immature APC appears to coincide with the reactivation of herpes virus infections such as Epstein Barr virus (EBV). The aim of this study was the comparative analysis of the ultrastructural and functional characteristics of such immature APC. In addition, we investigated EBV infection/ reactivation and whether immature APC might be targets for natural killers (NK). Significant macroautophagy, mitochondrial degradation and multivesicular body formation together with the identification of herpes virus particles were morphological findings associated with immature APC. Exogenous stressors such as ATP further increased morphological signs of autophagy, including LC3 expression. Functional tests using fluorescent bacteria proved impaired phagolysosome fusion. However, immature APC were susceptible to NK-92-mediated cytolysis. We found evidence for EBV latency state II infection by detecting EBV-specific LMP1 and EBNA2 in immature APC and in whole blood of these patients. In summary, trauma-induced cytokine storms may induce maturation arrest of APC, promote ATP-induced autophagy, support EBV persistence and impair the degradation of phagocytozed bacteria through inefficient phagolysosome fusion. The susceptibility to NK-mediated cytolysis supports the hypothesis that NK function is likely to contribute to immune reconstitution after major trauma by regulating immature APC, and ATP-induced autophagy and survival.Electronic supplementary materialThe online version of this article (doi:10.1007/s12079-010-0113-z) contains supplementary material, which is available to authorized users.

Phagocytosis and digestion of pH-sensitive fluorescent dye (Eos-FP) transfected E. coli in whole blood assays from patients with severe sepsis and septic shock

The function of phagocytic and antigen presenting cells is of crucial importance to sustain immune competence against infectious agents as well as malignancies. We here describe a reproducible procedure for the quantification of phagocytosis by leukocytes in whole blood. For this, a pH-sensitive green-fluorescent protein- (GFP) like dye (Eos-FP) is transfected into infectious microroganisms. After UV-irradiation, the transfected bacteria emit green (≈5160 nm) and red (≈581 nm) fluorescent light at 490 nm excitation. Since the red fluorescent light is sensitive to acidic pH, the phagocytosed bacteria stop emitting red fluorescent light as soon as the phagosomes fuse with lysosomes. The green fluorescence is maintained in the phagolysosome until pathogen degradation is completed. Fluorescence emission can be followed by flow cytometry with filter settings documenting fluorescence 1 (FL 1, FITC) and fluorescence 2 (FL 2, phycoerythrin, PE). Eos-FP transfected bacteria can also be traced within phagocytes using microscopical techniques. A standardized assay has been developed which is suitable for clinical studies by providing clinicians with syringes pre-filled with fixed and appropriately UV-irradiated Eos-FP E. coli (TruCulture™). After adding blood or body fluids to these containers and starting the incubation at 37°C, phagocytosis by granulocytes proceeds over time. Cultures can be terminated at a given time by lysing red blood cells followed by flow cytometry. A pilot study demonstrated that Eos-FP E. coli phagocytosis and digestion was up-regulated in the majority of patients with either severe sepsis or septic shock as compared to healthy donors (p < 0.0001 after o/n incubation). Following treatment with recombinant human granulocyte colony-stimulating factor (rhG-CSF) in selected patients with sepsis, phagolysosome fusion appeared to be accelerated.Electronic supplementary materialThe online version of this article (doi:10.1007/s12079-010-0112-0) contains supplementary material, which is available to authorized users.

Sphingolipid-mediated Inhibition of Apoptotic Cell Clearance by Alveolar Macrophages

A decreased clearance of apoptotic cells (efferocytosis) by alveolar macrophages (AM) may contribute to inflammation in emphysema. The up-regulation of ceramides in response to cigarette smoking (CS) has been linked to AM accumulation and increased detection of apoptotic alveolar epithelial and endothelial cells in lung parenchyma. We hypothesized that ceramides inhibit the AM phagocytosis of apoptotic cells. Release of endogenous ceramides via sphingomyelinase or exogenous ceramide treatments dose-dependently impaired apoptotic Jurkat cell phagocytosis by primary rat or human AM, irrespective of the molecular species of ceramide. Similarly, in vivo augmentation of lung ceramides via intratracheal instillation in rats significantly decreased the engulfment of instilled target apoptotic thymocytes by resident AM. The mechanism of ceramide-induced efferocytosis impairment was dependent on generation of sphingosine via ceramidase. Sphingosine treatment recapitulated the effects of ceramide, dose-dependently inhibiting apoptotic cell clearance. The effect of ceramide on efferocytosis was associated with decreased membrane ruffle formation and attenuated Rac1 plasma membrane recruitment. Constitutively active Rac1 overexpression rescued AM efferocytosis against the effects of ceramide. CS exposure significantly increased AM ceramides and recapitulated the effect of ceramides on Rac1 membrane recruitment in a sphingosine-dependent manner. Importantly, CS profoundly inhibited AM efferocytosis via ceramide-dependent sphingosine production. These results suggest that excessive lung ceramides may amplify lung injury in emphysema by causing both apoptosis of structural cells and inhibition of their clearance by AM.

Cell-mediated immunity in human brucellosis

Brucella can parasitize within human antigen-presenting cells modifying phagocytosis, phagolysosome fusion, antigen presentation, cytokine secretion, and apoptosis. Subversion of innate immune mechanisms by Brucella leads to defective Th1 immune responses and T-cell anergy in chronic brucellosis patients. This review summarizes the cellular immune responses in brucellosis, based on data derived exclusively from human cells or cell lines.

ArgR-Regulated Genes Are Derepressed in the Legionella -Containing Vacuole

Legionella pneumophila is an intracellular pathogen that infects protozoa in aquatic environments and when inhaled by susceptible human hosts replicates in alveolar macrophages and can result in the often fatal pneumonia called Legionnaires' disease. The ability of L. pneumophila to replicate within host cells requires the establishment of a specialized compartment that evades normal phagolysosome fusion called the Legionella-containing vacuole (LCV). Elucidation of the biochemical composition of the LCV and the identification of the regulatory signals sensed during intracellular replication are inherently challenging. L-Arginine is a critical nutrient in the metabolism of both prokaryotic and eukaryotic organisms. We showed that the L. pneumophila arginine repressor homolog, ArgR, is required for maximal intracellular growth in the unicellular host Acanthamoeba castellanii. In this study, we present evidence that the concentration of L-arginine in the LCV is sensed by ArgR to produce an intracellular transcriptional response. We characterized the L. pneumophila ArgR regulon by global gene expression analysis, identified genes highly affected by ArgR, showed that ArgR repression is dependent upon the presence of L-arginine, and demonstrated that ArgR-regulated genes are derepressed during intracellular growth. Additional targets of ArgR that may account for the argR mutant's intracellular multiplication defect are discussed. These results suggest that L-arginine availability functions as a regulatory signal during Legionella intracellular growth.

Macrophage activation differentially modulates particle binding, phagocytosis and downstream antimicrobial mechanisms

Phagocytosis provides a critical first line of defense against invading pathogens. Engagement of particles through receptor-mediated binding precedes internalization and induction of cellular antimicrobial responses. Phagocytes have the capacity to differentially regulate binding and internalization processes through changes in their receptor profile and modulation of downstream events. This is necessary for the intricate control of phagocytic antimicrobial responses. Several methods are available for evaluation of phagocytosis. Unfortunately, none allow for accurate quantitation of both binding and internalization events. To overcome these limitations, we have developed a novel phagocytosis assay based on a multi-spectral imaging flow cytometry platform. This assay discriminates between internalized and surface-bound particles in a statistically robust manner and allows multi-parametric analysis of phagocytosis and downstream anti-microbial responses. We also devised a novel approach for examination of phagolysosome fusion, which provides an improved capacity for quantitative assessment of phagolysosome fusion in mixed populations of intact cells. Importantly, our approaches are likely amenable to a broad range of comparative model systems based on our examination of murine RAW 264.7 cells and a goldfish primary kidney macrophage (PKM) model system. The latter allowed us to examine the evolutionary conservation of phagocytic antimicrobial responses in a lower vertebrate model. While it has been previously reported that mixed populations of these macrophage cultures are phagocytic, it remained unclear if sub-populations within them contributed differentially to this activity. In accordance with higher vertebrate models, we found that differentiation along the macrophage pathway leads to an increased capacity for phagocytosis in goldfish PKM. Interestingly, cellular activation differentially regulated particle internalization in PKM monocyte and mature macrophage subsets. We also found differential regulation of phagolysosome fusion and downstream production of reactive oxygen intermediates (ROI). The temporal activation of specific phagocytic antimicrobial responses at distinct stages of PKM differentiation suggests specialization within the macrophage compartment early in evolution, geared to meet specific host immunity requirements within specialized niches.

Initial receptor–ligand interactions modulate gene expression and phagosomal properties during both early and late stages of phagocytosis

The receptors engaged during recognition and phagocytic uptake of microorganisms and particles influence signaling events and diverse subcellular responses that occur during phagosome formation and maturation. However, pathogens generally have multiple ligands on their surface, making it difficult to dissect the roles of individual receptors during phagocytosis. Moreover, it remains elusive to which extent receptor–ligand interactions and early binding events define the subsequent intracellular fate of phagosomes. Here, we used latex beads coupled to single ligands, focusing on immunoglobulin G, mannan, bacterial lipopolysaccharides and avidin, and monitored: (1) phagocytic uptake rates, (2) fusion of phagosomes with lysosomal compartments, (3) the gene expression profile during phagocytosis, (4) the protein composition of mature phagosomes and (5) time-dependent dynamics of protein association with phagosomes in J774.A1 mouse macrophages. The differently coated latex beads were internalized at different rates and exhibited different kinetics of phagolysosomal fusion events dependent on their specific ligand. Furthermore, less than 60% of identified phagosomal proteins and only 10–15% of changes in gene expression were common to all investigated ligands. These findings demonstrate that each single ligand induced a distinct pattern of genes and a different protein composition of phagosomes. Taken together, our data argue that phagocytic receptor-specific programs of signaling events direct phagosomes to different physiological states and support the existence of a specific receptor–ligand ‘signature’ during the whole process of phagocytosis.

Exosomal Hsp70 induces a pro-inflammatory response to foreign particles including mycobacteria.

BackgroundExosomes are endosome-derived vesicles that are released when multi-vesicular bodies (MVBs) fuse with the plasma membrane. Exosomes released from mycobacteria-infected cells have recently been shown to be pro-inflammatory. A prominent host molecule that is found within these exosomes is Hsp70, a member of the heat-shock family of proteins.Methodology/Principal FindingsWe first characterized the exosomes purified from control and mycobacteria-infected cells. We found that relative to uninfected cells, macrophages infected with M. smegmatis and M. avium release more exosomes and the exosomes they released had more Hsp70 on their surface. Both exosomes and exogenous Hsp70 treatment of macrophages led to NF-κB activation and TNFα release in uninfected macrophages; Hsp70 levels were elevated in mycobacteria-infected cells. Macrophage treatment with Hsp70 also led to increase in the phagocytosis and maturation of latex-bead phagosomes. Finally, Hsp70 pre-incubation of M. smegmatis- and M. avium-infected cells led to increased phago-lysosome fusion, as well as more killing of mycobacteria within macrophages.Conclusions/SignificanceOur results fit into an emerging concept whereby exosomes-containing Hsp70 are effective inducers of inflammation, also in response to mycobacterial infection.