Bovis Bacillus Calmette–Guerin (BCG) infection induces exosomal miRNA release by human macrophages

Background Tuberculosis (TB) remains a significant global health concern and its diagnosis is challenging due to the limitations in the specificity and sensitivity of the current diagnostic tests. Exosomes are bioactive 30–100 nm vesicles produced by most cell types and are found in almost all human body fluids. Exosomal microRNAs (miRNAs) can transfer biological information between cells and tissues and may act as potential biomarkers in many diseases. In this pilot study, we assessed the miRNA profile of exosomes released from human monocyte-derived macrophages upon infection with Mycobacterium bovis Bacillus Calmette–Guerin (BCG). Methods Human monocytes were obtained from the peripheral blood of three healthy subjects and driven to a monocyte-derived macrophage (MDM) phenotype using standard protocols. MDMs were infected with BCG or left uninfected as control. 72 h post-infection, exosomes were collected from the cell culture medium, RNA was isolated and RNA-seq performed. The raw reads were filtered to eliminate adaptor and primer sequences and the sequences were run against the mature human miRNA sequences available in miRBase. MicroRNAs were identified using an E value <0.01. miRNA network analysis was performed using the DIANA miRNA tool, miRDB and functional KEGG pathway analysis. Results Infection of MDMs with BCG leads to the release of several exosomal miRNAs. These included miR-1224, -1293, -425, -4467, -4732, -484, -5094, -6848-6849, -4488 and -96 all of which were predicted to target metabolism and energy production-related pathways. Conclusions This study provides evidence for the release of specific exosomal miRNAs from BCG-infected MDMs. These exosomal miRNAs reflect host-pathogen interaction and subversion of host metabolic processes following infection.

MicroRNAs are important regulatory molecules that play critical roles in pathological conditions [27,28]. The role of miRNAs in modulation of innate and adaptive immunity and cellular responses to bacterial infection has been reported previously [29][30][31][32]. Various bacterial components, such as peptidoglycan (PG), lipoproteins and lipopolysaccharide (LPS) can affect the host's miRNA expression levels [33,34] and trigger inflammatory responses [35]. Functional miRNAs are capsulated in exosomes and delivered to recipient cells and subsequently cause specific modulation of their transcriptome [36]. Several studies have described the role of exosomes in TB [1,8,[37][38][39]. Exosomes released from macrophages infected with M.tb, as well as exosomes isolated from M.tb-infected mice, promote both innate and acquired immune responses in vitro and in vivo [1,8,[37][38][39]. The modulatory effects of exosomes released from M.tb-infected macrophages has been reviewed [40] and indicate that they can stimulate production of inflammatory mediators and induction of apoptosis in recipient cells [28,40].
Exosomal miRNAs have been proposed as potential biomarkers in numerous diseases such as cardiovascular disease, malignancies, and Alzheimer's disease [15,16,[41][42][43] although the role of exosomal miRNAs as potential biomarkers TB are not well described. In this pilot study, we profiled the exosomal miRNA of human macrophages after co-infection with Mycobacterium bovis, Bacillus Calmette-Guerin (BCG). We hypothesized that BCG-infected macrophages would secrete a specific set of exosomal miRNAs that may playing role in the pathogenesis of TB.

Cell culture
Peripheral blood was obtained from three healthy human donors who had no clinical manifestations of disease.
Complete blood count (CBC), ESR, CRP and liver and kidney function tests were evaluated. To investigate prior exposure of TB, QuantiFERON-TB Gold (QFT ® ) and PPD tests were performed and all three healthy subjects were negative for latent TB. An institutional review board (IRB) from Dr. Masih Daneshvari Hospital, Tehran, Iran approved the study.

Infection assay
On day 7 or 8, the GM-CSF medium was removed and replaced with fresh medium without GM-CSF for at least 4 h before infection. Uptake of bacteria was assessed by flow cytometry to determine the infection ratio required to obtain 85% infectivity as described previously [45,46]. Ten flasks each containing 1 × 10 7 cells were infected with opsonized BCG (obtained as a gift from Pasteur Institute of Iran, IPI) at an MOI (Multiplicity of infection) of 10 or were left uninfected as controls. Cells were incubated for 2 h at 37 °C in a 5% CO 2 before washing with 1× PBS containing amikacin (80 μg/ml) to eliminate possible free organisms. Subsequently, the cells were incubated in medium containing exosome-depleted FBS (10% final concentration) (System Bioscience, CA, USA) and 100 ng/ml GM-CSF for 72 h.

Exosome isolation and characterization
Exosomes were isolated from the culture supernatants of infected and uninfected cells 72 h post infection using total exosome isolation (TEI) reagent according to the manufacturer's instructions (Invitrogen by the Thermo Fisher Scientific corporation, Waltham, MA, USA). Briefly, the cell culture media (CCM) were centrifuged at 300×g for 30 min and filtered twice through a 0.22 μm filter (Merck-Millipore, Billerica, MA, USA) to remove apoptotic bodies, dead cells and cell debris. The CCM was mixed with TEI solution at a 5:1 ratio. The samples were incubated overnight at 4 °C and centrifuged for 1 h at 10,000×g. The pellet was re-suspended in 1 ml of PBS and stored at −20 °C. Purified exosomes were characterized by electron microscopy (Carl Zeiss NTS, Oberkochen, Germany) and nanoparticle tracking analysis (Malvern/Nanosight LM10. CA, USA).

Exosomal RNA isolation and qualification
Prior to RNA extraction, exosomes were again filtered through a 0.22 mm filter and treated with RNase A (5 μg/ μl Fermentase, Thermo-Fisher, Boston, MA, USA) for 90 min at 37 °C to eliminate non-exosomal RNAs. Total RNA was isolated from exosomes using the total exosomal RNA and protein isolation kit (Thermo-Fisher) according to the manufacturer's instructions. RNA concentration and purity was measured using a Nano-drop 2000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA). The quality, yield, and size of extracted RNA was analyzed using capillary electrophoresis (Agilent 2100 Bioanalyzer, Agilent Technologies, Foster City, CA, USA).

Small RNA library construction and miRNA sequencing
Size selection and gel purification of the RNA samples was performed on a 15% Tris-Borate-EDTA (TBE) polyacrylamide/urea gel, RNAs were excised from the gel, purified as above and small RNA libraries constructed as described previously [47]. RNA libraries were run on a Roche 454 Genome Sequencer FLX according to the manufacturer's instructions. The raw reads were filtered to eliminate adaptor sequences and the sequences, in FASTQ format, were run against the mature human miRNA sequence database available at miRBase using BLAST software. MicroRNAs were identified using an E value cutoff <0.01. MicroRNA pathway analysis was performed using the DIANA miRNA tool [48,49], miRDB [50] and functional KEGG pathway database [51].

Exosome characterization and exosomal RNA preparation
Exosomes were isolated from the culture supernatants from paired infected and uninfected monocyte-derived macrophages from healthy subjects. Exosomes were confirmed by scanning (SEM) and transmission electron microscopy (TEM) and characterized for size distribution by nanoparticle analyzer and demonstrated the expected size and morphology (Fig. 1).
Total exosomal RNA was extracted and the concentration measured. The amount of RNA isolated from 10 T75 culture flasks in each group varied from 1.3 to 1.8 µg from the infected and uninfected cell-derived exosomes with no significant differences between groups (p > 0.05). The extracted exosomal RNA was also qualified on an Agilent Bioanalyzer and the RNA population observed in the exosomes were predominantly from small RNAs (Fig. 2).

Differentially expressed miRNAs were associated with pathways related to pathogenesis of mycobacterial infection and intracellular survival
To examine the target pathways affected by the differentially expressed miRNAs in the infected macrophage-like group, miRNA network analysis was performed. Pathway analysis showed differential activation of pathways related to mycobacterium invasion, intracellular survival, energy production machinery and immunity reactions ( Table 3). Most of the target genes regulated by these miRNAs were involved in the cell infection process and energy production pathways (Table 4). A subgroup of these differentially expressed miRNAs (miR-484, -5094, -425, -1293, -6848, -6849) had the most profound effect on the pathways activated by BCG infection (Table 5).

Discussion
In the current study, we assessed the exosomal miRNAs released from human macrophages following infection with BCG. We detected a group of 11 exosomal miRNAs miRs-1224, -1293, -425, -4467, -4732, -484, -5094, -6848, -6849, -96 and -4488) that were differentially expressed in infected cells. These miRNAs are involved in several key pathways including central carbon metabolism, fatty acids and sugar metabolism, amino acid metabolism, bacterial invasion related pathways and cell signaling pathways. This suggests that host pathways implicated in immune surveillance are modulated to enable bacterial survival within infected macrophages.
Recent studies have highlighted the role of exosomes as a vehicle for the transfer of proteins, lipids as well as biologically active miRNAs to distant cells. Exosomes may also act as novel biomarkers in several diseases such as acute myeloid leukemia (AML), ovarian cancer, asthma and sarcoidosis [20,24,36]. The miRNA profile and some aspects of exosomal content has recently been examined in TB patients [18,37,52,53]. In these studies, the levels of serum free miRNAs [54], macrophage cell miRNAs [55] and exosomal protein content [39] were evaluated. The expression of 14 miRNAs in M.tb-infected macrophages were significantly altered and depended upon the infective strain (Beijing/W or non-Beijing/W strains) [55] and did not overlap with those reported here from M.tb-infected macrophage-derived exosomes. There were no overlapping miRNAs found in the serum of infected patients [54] and the target pathways were distinct from those seen here. This highlights the potential for detecting strain-specific infection using exosomal miRNAs.
Exosomal transport of miRNAs enables their stability and delivery throughout the body [56]. Exposure of cells to various bacterial components affects the host miRNA profile [34] and the this is also evident in the miRNA profile of released exosomes reported in this study. This will potentially cause significant functional modulation of

Table 1 Exosomal miRNA content in infected and uninfected macrophage-derived exosomes
Differentially expressed exosomal miRNAs (p < 0.05) obtained from three independent experiments. 44 and 47 miRNAs were identified in the M.tb-infected and -uninfected macrophage-derived exosomes with a copy number >20
Dysregulated miRNAs released into exosomes from BCG-infected macrophages also affected amino acid synthesis and metabolism pathways (Table 3). Metabolic profiling demonstrated increased levels of amino acids and activation of pyrimidine and purine nucleotide biosynthesis within M.tb-infected lung tissue [71].
Another group of differentially expressed miRNAs in exosomes from infected macrophages were associated with cell membrane and communication pathways such as adherens junction, gap junction, glycosaminoglycan biosynthesis and heparan sulfate/keratin sulfate metabolism. MicroR-1293 for example targets the tissue inhibitors of metalloproteinases (TIMPs) [72]. TIMP-1 is an inhibitor of matrix metalloproteinases and is involved in the invasion and spreading of bacteria through the epithelial cell [73]. M.tb infection upregulates the expression of matrix metalloproteinases (MMP) and perturbs the MMP/TIMP balance in human monocytes [74]. The up-regulation of miR-1293 in exosomes from BCG-infected macrophages may reflect the ability of mycobacterium antigens to alter the host cell membrane structure and subsequently affect macrophage survival.
miR-484 and miR-425 were preferentially found in exosomes of BCG-infected macrophages. miR-484 regulates intermediate metabolic pathways by targeting the mitochondrial fission protein 1 (Fis1) [75] and altered miR-425 expression is linked to insulin resistance. The role of these miRNAs in infected macrophages remains unclear at this point although it is evident that miR-425 regulates several metabolic pathways and has been associated with metabolic disorders [76]. miRs-1224, -1293, -4467, -4732, -5094, -6848 and -6849 are human mirtrons which are produced via splicing of introns from mRNA coding genes rather than by the formation of hairpin loops by Drosha [77]. The expression of these mirtrons were significantly higher in the exosomes released from infected macrophages. This suggests that mycobacteria may recognize, at least in part, the pattern of miRNA production within the host cell and program over-expression of these mirtrons in order to recruit host metabolic pathways that favour M.tb infection.

Conclusion
This pilot study demonstrated the differential expression of many miRNAs within exosomes released from BCGinfected macrophages. These miRNAs indicate that metabolic reprogramming may occur to favour M.tb survival. Further studies are needed in large cohorts of patients to test for the presence of these 11 miRNAs in blood exosomes to determine their true value as a possible diagnostic biomarker for TB infection. The profiling of miR-NAs upon BCG infection may shed additional light on the host-pathogen interaction and changes in cellular function. Future studies on miRNA expression and function in TB may provide greater understanding of M.tb pathogenesis.