Exosomes isolated from mycobacteria-infected mice or cultured macrophages can recruit and activate immune cells in vitro and in vivo

More than 2 billion people are infected with Mycobacterium. tuberculosis; however, only 5-10% of those infected will develop active disease. Recent data suggest that containment is controlled locally at the level of the granuloma and that granuloma architecture may differ even within a single infected individual. Formation of a granuloma likely requires exposure to mycobacterial components released from infected macrophages, but the mechanism of their release is still unclear. We hypothesize that exosomes, which are small membrane vesicles containing mycobacterial components released from infected macrophages, could promote cellular recruitment during granuloma formation. In support of this hypothesis, we found that C57BL/6 mouse-derived bone marrow macrophages treated with exosomes released from M. tuberculosis-infected RAW264.7 cells secrete significant levels of chemokines and can induce migration of CFSE-labeled macrophages and splenocytes. Exosomes isolated from the serum of M. bovis bacillus Calmette-Guérin-infected mice could also stimulate macrophage production of chemokines and cytokines ex vivo, but the level and type differed during the course of a 60-d infection. Of interest, the exosome concentration in serum correlated strongly with mouse bacterial load, suggesting some role in immune regulation. Finally, hollow fiber-based experiments indicated that macrophages treated with exosomes released from M. tuberculosis-infected cells could promote macrophage recruitment in vivo. Exosomes injected intranasally could also recruit CD11b(+) cells into the lung. Overall, our study suggests that exosomes may play an important role in recruiting and regulating host cells during an M. tuberculosis infection.     

Infection of J774A.1 with different Mycobacterium species induces differential immune and miRNA‐related responses

Macrophages act as a reservoir for Mycobacterium tuberculosis, producing latent infection in approximately 90% of infected people. In this study, J774A.1 mouse macrophage cell line response and microRNA (miRNA) expression during infection with the most relevant mycobacterial strains for humans (M. tuberculosis, M. bovis and M. bovis BCG) was explored. No significant differences in bacillary loads were observed between activate and naive macrophages infected with M. tuberculosis and M. bovis. Nitrite production inhibition and infection control were in accordance with the virulence of the strain. Expression of let‐7e, miR‐21, miR‐155, miR‐210 and miR‐223 was opposite in the two species and miR‐146b* and miR‐1224 expression seemed to be part of the general response to infection.     

Exosomal Hsp70 Induces a Pro-Inflammatory Response to Foreign Particles Including Mycobacteria

Background

Exosomes 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 Findings

We 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/Significance

Our results fit into an emerging concept whereby exosomes-containing Hsp70 are effective inducers of inflammation, also in response to mycobacterial infection.     

Mycobacterium tuberculosis Rv3034c regulates mTORC1 and PPAR‐γ dependant pexophagy mechanism to control redox levels in macrophages

Mycobacterium tuberculosis survives inside the macrophages by employing several host immune evasion strategies. Here, we reported a novel mechanism in which M. tuberculosis acetyltransferase, encoded by Rv3034c, induces peroxisome homeostasis to regulate host oxidative stress levels to facilitate intracellular mycobacterial infection. Presence of M. tuberculosis Rv3034c induces the expression of peroxisome biogenesis and proliferation factors such as Pex3, Pex5, Pex19, Pex11b, Fis‐1 and DLP‐1; while depletion of Rv3034c decreased the expression of these molecules, thereby selective degradation of peroxisomes via pexophagy. Further studies revealed that M. tuberculosis Rv3034c inhibit induction of pexophagy mechanism by down‐regulating the expression of pexophagy associated proteins (p‐AMPKα, p‐ULK‐1, Atg5, Atg7, Beclin‐1, LC3‐II, TFEB and Keap‐1) and adaptor molecules (NBR1 and p62). Inhibition was found to be dependent on the phosphorylation of mTORC1 and activation of peroxisome proliferator activated receptor‐γ. In order to maintain intracellular homeostasis during oxidative stress, M. tuberculosis Rv3034c was found to induce degradation of dysfunctional and damaged peroxisomes through activation of Pex14 in infected macrophages. In conclusion, this is the first report which demonstrated that M. tuberculosis acetyltransferase regulate peroxisome homeostasis in response to intracellular redox levels to favour mycobacterial infection in macrophage.     

Exosomes derived from M. Bovis BCG infected macrophages activate antigen-specific CD4+ and CD8+ T cells in vitro and in vivo

Activation of both CD4(+) and CD8(+) T cells is required for an effective immune response to an M. tuberculosis infection. However, infected macrophages are poor antigen presenting cells and may be spatially separated from recruited T cells, thus limiting antigen presentation within a granuloma. Our previous studies showed that infected macrophages release from cells small membrane-bound vesicles called exosomes which contain mycobacterial lipid components and showed that these exosomes could stimulate a pro-inflammatory response in na?ve macrophages. In the present study we demonstrate that exosomes stimulate both CD4(+) and CD8(+) splenic T cells isolated from mycobacteria-sensitized mice. Although the exosomes contain MHC I and II as well as costimulatory molecules, maximum stimulation of T cells required prior incubation of exosomes with antigen presenting cells. Exosomes isolated from M. bovis and M. tuberculosis infected macrophages also stimulated activation and maturation of mouse bone marrow-derived dendritic cells. Interestingly, intranasal administration of mice with exosomes isolated from M. bovis BCG infected macrophages induce the generation of memory CD4(+) and CD8(+) T cells. The isolated T cells also produced IFN-gamma upon restimulation with BCG antigens. The release of exosomes from infected macrophages may overcome some of the defects in antigen presentation associated with mycobacterial infections and we suggest that exosomes may be a promising M. tuberculosis vaccine candidate.     

Microparticles released from Mycobacterium tuberculosis‐infected human macrophages contain increased levels of the type I interferon inducible proteins including ISG15

Microparticles (MPs) are small membranous particles (100–1000 nm) released under normal steady‐state conditions and are thought to provide a communication network between host cells. Previous studies demonstrated that Mycobacterium tuberculosis (M. tb) infection of macrophages increased the release of MPs, and these MPs induced a proinflammatory response from uninfected macrophages in vitro and in vivo following their transfer into uninfected mice. To determine how M. tb infection modulates the protein composition of the MPs, and if this contributes to their proinflammatory properties, we compared the proteomes of MPs derived from M. tb‐infected (TBinf‐MP) and uninfected human THP‐1 monocytic cells. MP proteins were analyzed by GeLC‐MS/MS with spectral counting revealing 68 proteins with statistically significant differential abundances. The 42 proteins increased in abundance in TBinf‐MPs included proteins associated with immune function (7), lysosomal/endosomal maturation (4), vesicular formation (12), nucleosome proteins (4), and antigen processing (9). Prominent among these were the type I interferon inducible proteins, ISG15, IFIT1, IFIT2, and IFIT3. Exposure of uninfected THP‐1 cells to TBinf‐MPs induced increased gene expression of isg15, ifit1, ifit2, and ifit3 and the release of proinflammatory cytokines. These proteins may regulate the proinflammatory potential of the MPs and provide candidate biomarkers for M. tb infection.     

Proteomic analysis of extracellular vesicles derived from Mycobacterium tuberculosis

The release of extracellular vesicles, also known as outer membrane vesicles, membrane vesicles, exosomes, and microvesicles, is an evolutionarily conserved phenomenon from bacteria to eukaryotes. It has been reported that Mycobacterium tuberculosis releases extracellular vesicles harboring immunologically active molecules, and these extracellular vesicles have been suggested to be applicable in vaccine development and biomarker discovery. However, the comprehensive proteomic analysis has not been performed for M. tuberculosis extracellular vesicles. In this study, we identified a total of 287 vesicular proteins by four LC‐MS/MS analyses with high confidence. In addition, we identified several vesicular proteins associated with the virulence of M. tuberculosis. This comprehensive proteome profile will help elucidate the pathogenic mechanism of M. tuberculosis. The data have been deposited to the ProteomeXchange with identifier PXD001160 ( http://proteomecentral.proteomexchange.org/dataset/PXD001160 ).     

Exosomes released from M. tuberculosis infected cells can suppress IFN-γ mediated activation of naïve macrophages

Background

Macrophages infected with Mycobacterium tuberculosis (M.tb) are known to be refractory to IFN-γ stimulation. Previous studies have shown that M.tb express components such as the 19-kDa lipoprotein and peptidoglycan that can bind to macrophage receptors including the Toll-like receptor 2 resulting in the loss in IFN-γresponsiveness. However, it is unclear whether this effect is limited to infected macrophages. We have previously shown that M.tb-infected macrophages release exosomes which are 30–100 nm membrane bound vesicles of endosomal origin that function in intercellular communication. These exosomes contain mycobacterial components including the 19-kDa lipoprotein and therefore we hypothesized that macrophages exposed to exosomes may show limited response to IFN-γ stimulation.

Methodology/Principal Findings

Exosomes were isolated from resting as well as M.tb-infected RAW264.7 macrophages. Mouse bone marrow-derived macrophages (BMMØ) were treated with exosomes +/− IFN-γ. Cells were harvested and analyzed for suppression of IFN-γ responsive genes by flow cytometry and real time PCR. We found that exosomes derived from M.tb H37Rv-infected but not from uninfected macrophages inhibited IFN-γ induced MHC class II and CD64 expression on BMMØ. This inhibition was only partially dependent on the presence of lipoproteins but completely dependent on TLR2 and MyD88. The exosomes isolated from infected cells did not inhibit STAT1 Tyrosine phosphorylation but down-regulated IFN-γ induced expression of the class II major histocompatibity complex transactivator; a key regulator of class II MHC expression. Microarray studies showed that subsets of genes induced by IFN-γ were inhibited by exosomes from H37Rv-infeced cells including genes involved in antigen presentation. Moreover, this set of genes partially overlapped with the IFN-γ-induced genes inhibited by H37Rv infection.

Conclusions

Our study suggests that exosomes, as carriers of M.tb pathogen associated molecular patterns (PAMPs), may provide a mechanism by which M.tb may exert its suppression of a host immune response beyond the infected cell.     

Exosomes released by Brucella-infected macrophages inhibit the intracellular survival of Brucella by promoting the polarization of M1 macrophages

Exosomes, membrane vesicles released extracellularly from cells, contain nucleic acids, proteins, lipids and other components, allowing the transfer of material information between cells. Recent studies reported the role of exosomes in pathogenic microbial infection and host immune mechanisms. Brucella-invasive bodies can survive in host cells for a long time and cause chronic infection, which causes tissue damage. Whether exosomes are involved in host anti-Brucella congenital immune responses has not been reported. Here, we extracted and identified exosomes secreted by Brucella melitensis M5 (Exo-M5)-infected macrophages, and performed in vivo and in vitro studies to examine the effects of exosomes carrying antigen on the polarization of macrophages and immune activation. Exo-M5 promoted the polarization of M1 macrophages, which induced the significant secretion of M1 cytokines (tumour necrosis factor-α and interferon-γ) through NF-κB signalling pathways and inhibited the secretion of M2 cytokines (IL-10), thereby inhibiting the intracellular survival of Brucella. Exo-M5 activated innate immunity and promoted the release of IgG2a antibodies that protected mice from Brucella infection and reduced the parasitaemia of Brucella in the spleen. Furthermore, Exo-M5 contained Brucella antigen components, including Omp31 and OmpA. These results demonstrated that exosomes have an important role in immune responses against Brucella, which might help elucidate the mechanisms of host immunity against Brucella infection and aid the search for Brucella biomarkers and the development of new vaccine candidates.     

Qualitative differences in T‐cell activation by dendritic cell‐derived extracellular vesicle subtypes

Exosomes, nano‐sized secreted extracellular vesicles (EVs), are actively studied for their diagnostic and therapeutic potential. In particular, exosomes secreted by dendritic cells (DCs) have been shown to carry MHC‐peptide complexes allowing efficient activation of T lymphocytes, thus displaying potential as promoters of adaptive immune responses. DCs also secrete other types of EVs of different size, subcellular origin and protein composition, whose immune capacities have not been yet compared to those of exosomes. Here, we show that large EVs (lEVs) released by human DCs are as efficient as small EVs (sEVs), including exosomes, to induce CD4⁺ T‐cell activation in vitro. When released by immature DCs, however, lEVs and sEVs differ in their capacity to orient T helper (Th) cell responses, the former favouring secretion of Th2 cytokines, whereas the latter promote Th1 cytokine secretion (IFN‐γ). Upon DC maturation, however, these functional differences are abolished, and all EVs become able to induce IFN‐γ. Our results highlight the need to comprehensively compare the functionalities of EV subtypes in all patho/physiological systems where exosomes are claimed to perform critical roles.     

Highly fluorescent GFPm2+‐based genome integration‐proficient promoter probe vector to study Mycobacterium tuberculosis promoters in infected macrophages

Study of activity of cloned promoters in slow‐growing Mycobacterium tuberculosis during long‐term growth conditions in vitro or inside macrophages, requires a genome‐integration proficient promoter probe vector, which can be stably maintained even without antibiotics, carrying a substrate‐independent, easily scorable and highly sensitive reporter gene. In order to meet this requirement, we constructed pAKMN2, which contains mycobacterial codon‐optimized gfp_m²⁺ gene, coding for GFP_m²⁺ of highest fluorescence reported till date, mycobacteriophage L5 attP‐int sequence for genome integration, and a multiple cloning site. pAKMN2 showed stable integration and expression of GFP_m²⁺ from M. tuberculosis and M. smegmatis genome. Expression of GFP_m²⁺, driven by the cloned minimal promoters of M. tuberculosis cell division gene, ftsZ (MtftsZ), could be detected in the M. tuberculosis/pAKMN2‐promoter integrants, growing at exponential phase in defined medium in vitro and inside macrophages. Stable expression from genome‐integrated format even without antibiotic, and high sensitivity of detection by flow cytometry and fluorescence imaging, in spite of single copy integration, make pAKMN2 useful for the study of cloned promoters of any mycobacterial species under long‐term in vitro growth or stress conditions, or inside macrophages.     

Granzyme B‐expressing neutrophils correlate with bacterial load in granulomas from Mycobacterium tuberculosis‐infected cynomolgus macaques

The role of neutrophils in tuberculosis (TB), and whether neutrophils express granzyme B (grzB), a pro‐apoptotic enzyme associated with cytotoxic T cells, is controversial. We examined neutrophils in peripheral blood (PB) and lung granulomas of Mycobacterium tuberculosis‐infected cynomolgus macaques and humans to determine whether mycobacterial products or pro‐inflammatory factors induce neutrophil grzB expression. We found large numbers of grzB‐expressing neutrophils in macaque and human granulomas and these cells contained more grzB+ granules than T cells. Higher neutrophil, but not T cell, grzB expression correlated with increased bacterial load. Although unstimulated PB neutrophils lacked grzB expression, grzB expression increased upon exposure to M. tuberculosis bacilli, M. tuberculosis culture filtrate protein or lipopolysaccharide from Escherichia coli. Perforin is required for granzyme‐mediated cytotoxicity by T cells, but was not observed in PB or granuloma neutrophils. Nonetheless, stimulated PB neutrophils secreted grzB as determined by enzyme‐linked immunospot assays. Purified grzB was not bactericidal or bacteriostatic, suggesting secreted neutrophil grzB acts on extracellular targets, potentially enhancing neutrophil migration through extracellular matrix and regulating apoptosis or activation in other cell types. These data indicate mycobacterial products and the pro‐inflammatory environment of granulomas up‐regulates neutrophil grzB expression and suggests a previously unappreciated aspect of neutrophil biology in TB.     

Receptor‐mediated recognition of mycobacterial pathogens

Mycobacteria are a genus of bacteria that range from the non‐pathogenic Mycobacterium smegmatis to Mycobacterium tuberculosis, the causative agent of tuberculosis in humans. Mycobacteria primarily infect host tissues through inhalation or ingestion. They are phagocytosed by host macrophages and dendritic cells. Here, conserved pathogen‐associated molecular patterns (PAMPs) on the surface of mycobacteria are recognized by phagocytic pattern recognition receptors (PRRs). Several families of PRRs have been shown to non‐opsonically recognize mycobacterial PAMPs, including membrane‐bound C‐type lectin receptors, membrane‐bound and cytosolic Toll‐like receptors and cytosolic NOD‐like receptors. Recently, a possible role for intracellular cytosolic PRRs in the recognition of mycobacterial pathogens has been proposed. Here, we discuss currentideas on receptor‐mediated recognition of mycobacterial pathogens by macrophages and dendritic cells.     

Differential expression of NF-κB in mycobacteria infected THP-1 affects apoptosis

The present study was conducted to see the role of NF-κB in virulent (Mycobacterium tuberculosis H37Rv) and avirulent (M. tuberculosis H37Ra) mycobacterial infection in THP-1 cells. To inactivate NF-κB, pCMV-IκBαM dn containing THP-1 cell line was generated which showed marked increase in apoptosis with M. tuberculosis H37Rv and M. tuberculosis H37Ra. Infected THP-1-IκBαM dn cells showed decrease in mitochondrial membrane potential, cytochrome c release, activation of caspase-3 and enhanced TNF-α production. Increase in apoptosis of infected THP-1-IκBαM dn cells resulted in inhibition of intracellular mycobacterial growth. Differential NF-κB activation potential was observed with M. tuberculosis H37Rv and M. tuberculosis H37Ra. Both the strains activated NF-κB after 4 h in THP-1 cells however after 48 h only M. tuberculosis H37Rv activated NF-κB which lead to up-regulation of bcl-2 family anti-apoptotic member, bfl-1/A1. Our results indicated that NF-κB activation may be a determinant factor for the success of virulent mycobacteria within macrophages.     

Arrestin‐Domain Containing Protein 1 (Arrdc1) Regulates the Protein Cargo and Release of Extracellular Vesicles

Extracellular vesicles (EVs) are lipid‐bilayered vesicles that are released by multiple cell types and contain nucleic acids and proteins. Very little is known about how the cargo is packaged into EVs. Ubiquitination of proteins is a key posttranslational modification that regulates protein stability and trafficking to subcellular compartments including EVs. Recently, arrestin‐domain containing protein 1 (Arrdc1), an adaptor for the Nedd4 family of ubiquitin ligases, has been implicated in the release of ectosomes, a subtype of EV that buds from the plasma membrane. However, it is currently unknown whether Arrdc1 can regulate the release of exosomes, a class of EVs that are derived endocytically. Furthermore, it is unclear whether Arrdc1 can regulate the sorting of protein cargo into the EVs. Exosomes and ectosomes are isolated from mouse embryonic fibroblasts isolated from wild type and Arrdc1‐deficient (Arrdc1^?/?) mice. Nanoparticle tracking analysis–based EV quantitation shows that Arrdc1 regulates the release of both exosomes and ectosomes. Proteomic analysis highlights the change in protein cargo in EVs upon deletion of Arrdc1. Functional enrichment analysis reveals the enrichment of mitochondrial proteins in ectosomes, while proteins implicated in apoptotic cleavage of cell adhesion proteins and formation of cornified envelope are significantly depleted in exosomes upon knockout of Arrdc1.     

NK cells inhibit anti‐Mycobacterium bovis BCG T cell responses and aggravate pulmonary inflammation in a direct lung infection mouse model

Tuberculosis remains a threat to public health. The major problem for curing this disease is latent infection, of which the underlying mechanisms are still not fully understood. Previous studies indicate that natural killer (NK) cells do not play a role in inhibiting the growth of Mycobacterium tuberculosis in the lung, and recent studies have revealed that NK cells regulate the adaptive immunity during mycobacterial infection. By using a mouse model of direct lung infection with Mycobacterium bovis bacillus Calmette‐Guerin (BCG), we found that the presence of NK cells postponed the priming and activation of T cells after BCG infection. In addition, depletion of NK cells before infection alleviated pulmonary pathology. Further studies showed that NK cells lysed BCG‐infected macrophages in an NKG2D dependent manner. Thus, NK cells did not play a direct role in control BCG, but aggravated the pulmonary inflammation and impaired anti‐BCG T cell immunity, likely through killing BCG‐infected macrophages. Our results may have important implications for the design of immune therapy to treat tuberculosis.     

Mycobacterium tuberculosis alters the differentiation of monocytes into macrophages in vitro

This paper shows that in vitro infection of human monocytes by Mycobacterium tuberculosis affected monocyte to macrophage differentiation. Despite the low bacterial load used, M. tuberculosis-infected monocytes had fewer granules, displayed a reduced number of cytoplasmic projections and decreased HLA class II, CD68, CD86 and CD36 expression compared to cells differentiated in the absence of mycobacteria. Infected cells produced less IL-12p70, TNF-α, IL-10, IL-6 and high IL-1β in response to lipopolysaccharide and purified protein M. tuberculosis-derived. Reduced T-cell proliferative response and IFN-γ secretion in response to phytohemagglutinin and culture filtrate proteins from M. tuberculosis was also observed in infected cells when compared to non-infected ones. The ability of monocytes differentiated in the presence of M. tuberculosis to control mycobacterial growth in response to IFN-γ stimulation was attenuated, as determined by bacterial plate count; however, they had a similar ability to uptake fluorescent M. tuberculosis and latex beads compared to non-infected cells. Recombinant IL-1β partially altered monocyte differentiation into macrophages; however, treating M. tuberculosis-infected monocytes with IL-1RA did not reverse the effects of infection during differentiation. The results indicated that M. tuberculosis infection altered monocyte differentiation into macrophages and affected their ability to respond to innate stimuli and activate T-cells.     

Mycobacterium tuberculosis Mce3C promotes mycobacteria entry into macrophages through activation of β2 integrin‐mediated signalling pathway

Establishment of infection by facultative intracellular pathogen Mycobacterium tuberculosis (Mtb) requires adherence to and internalisation by macrophages. However, the effector molecules exploited by Mtb for entry into macrophages remain to be fully understood. The mammalian cell entry (Mce) proteins play an essential role in facilitating the internalisation of mycobacteria into mammalian cells. Here, we characterized Mtb Mce3C as a new mycobacterial surface protein that could promote mycobacterial adhesion to and invasion of macrophages in an RGD motif‐dependent manner. We then further demonstrated that β2 integrin was required for Mce3C‐mediated cell entry. In addition, we found that binding of Mce3C recruited β2 integrin‐dependent signalling adaptors and induced local actin rearrangement at the site of mycobacterial invasion. By using specific antibodies and pharmacological inhibitors, we further demonstrated the involvement of Src‐family tyrosine kinases, spleen tyrosine kinase, Vav, Rho, and Rho‐associated kinase in Mce3C‐mediated mycobacterial invasion. Our results reveal a novel mechanism by which Mtb Mce3C exploits integrin‐mediated signalling cascade for Mce, providing potential targets for the development of therapies against Mtb infection.     

Visualizing of the cellular uptake and intracellular trafficking of exosomes by live‐cell microscopy

Cells release exosomes to transfer various molecules to other cells. Exosomes are involved in a number of physiological and pathological processes. They are emerging great potential utility for diseases diagnosis and treatment recently. However, the internalization and intracellular trafficking of exosomes have not been described clearly. In this work, exosomes were isolated from the culture medium of PC12 cells, labeled by lipophilic dye and amino‐reactive fluorophore, incubated with resting PC12 cells. The results of live‐cell microscopy indicated that exosomes were internalized through endocytosis pathway, trapped in vesicles, and transported to perinuclear region. Particle tracking fluorescent vesicles suggested that the active transport of exosomes may be mediated by cytoskeleton. The proteins on exosome membrane were found to be released from exosomes and trapped in lysosome. The inverted transport of lipophilic dye from perinuclear region to cell peripheries was revealed, possibly caused by recycling of the exosome lipids. This study provides new sight into the mechanisms of exosome uptake and intracellular fate. J. Cell. Biochem. 111: 488–496, 2010. © 2010 Wiley‐Liss, Inc.