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 Table of Contents  
ARTICLE
Year : 2013  |  Volume : 2  |  Issue : 3  |  Page : 128-134

Alteration of human macrophages microRNA expression profile upon infection with Mycobacterium tuberculosis


Division of Immunity, Transplantation and Infectious Diseases, Emerging Bacterial Pathogens Unit, San Raffaele Scientific Institute, Via Olgettina, 58 20132 Milan, Italy

Date of Web Publication28-Feb-2017

Correspondence Address:
Lucinda Furci
Division of Immunity, Transplantation and Infectious Diseases, Emerging Bacterial Pathogens Unit, San Raffaele Scientific Institute, Via Olgettina, 58 20132 Milan
Italy
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Source of Support: None, Conflict of Interest: None


DOI: 10.1016/j.ijmyco.2013.04.006

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  Abstract 


Background: Mycobacterium tuberculosis (Mtb) has evolved multiple mechanisms to manipulate its cellular niche for its own advantage. Many efforts have been made to understand basal mechanisms of mycobacterial infections. However, the underlying molecular regulation is not fully understood. Recently, a new class of non-coding, small RNAs, called microRNAs (miRNAs), has emerged as important regulators in biological processes, and their involvement in mycobacterial infection has been identified, thus opening a new field of research.
Methods: This study aimed to determine by TaqMan Low Density Array the host genome-wide miRNA expression profile of primary human monocyte-derived macrophages (MDM) infected with two members of the Mtb complex: virulent Mtb H37Rv and the non-virulent vaccine strain Mycobacterium bovis Bacillus Calmette-Guerin (BCG) in comparison with chemically-inactivated Mtb bacilli.
Results: The findings of this study showed that infection of MDM with H37Rv or BCG results in a signature of miRNA expression mostly overlapping between the two mycobacteria. A substantially different signature emerged from infection with killed virulent bacilli, suggesting an active influence of live intracellular bacteria on cellular miRNA metabolism. Specifically, Mtb induced miRNA signature is composed of miRNAs well established in immune regulation, miR-155 and miR-146a, as well as a set of miRNAs newly associated with Mtb infection: miR-145, miR-222*, miR-27a and miR-27b. All of these miRNAs are predicted to target important immune-related genes.
Conclusions: This study signifies the miRNA host response upon intracellular mycobacterial infection in macrophages, providing new aspects of regulation in host-pathogen interactions, at post-transcriptional levels.

Keywords: Mycobacterium tuberculosis, microRNAs, TaqMan Low Density Array, Macrophages


How to cite this article:
Furci L, Schena E, Miotto P, Cirillo DM. Alteration of human macrophages microRNA expression profile upon infection with Mycobacterium tuberculosis. Int J Mycobacteriol 2013;2:128-34

How to cite this URL:
Furci L, Schena E, Miotto P, Cirillo DM. Alteration of human macrophages microRNA expression profile upon infection with Mycobacterium tuberculosis. Int J Mycobacteriol [serial online] 2013 [cited 2019 May 22];2:128-34. Available from: http://www.ijmyco.org/text.asp?2013/2/3/128/201219




  Introduction Top


To establish an infection in a host organism, Mtb must first overcome the barriers of the innate immune system [1]. Macrophages are professional phagocytes that provide a first line of innate immune defense against invading pathogens. As an intracellular bacterium, Mtb depends on the tolerance of the host immune system for its survival and replication [2]. Detection of Mtb by macrophages at the cell surface, within phagosomes, or the cytosol triggers distinct host cell transcriptional responses via pattern recognition receptors (PRRs) [3],[4],[5]. As a consequence, numerous pro-inflammatory mediators and other molecules are expressed that further instruct elicitation of antigen-specific acquired immunity and clearance of infection. To prevent excessive and inappropriate activation of the immune system upon infection with an intracellular pathogen, the host cellular pathways need to be tightly regulated. MicroRNAs (miRNAs) are significant modulators of the immune response that function at post-transcriptional levels [6],[7]. Binding of miRNAs to partially complementary sequences in the 3’ untranslated region (3’ UTR) of their respective protein coding mRNA targets leads to transcript degradation or translational inhibition [8]. MiRNA are ‘fine-tuners’ of the immune response – subtle yet essential regulators in key immune pathways.

A number of recent studies describe the regulations of mammalian miRNAs in response to bacterial infection. Helicobacter pylori infection alters the expression of oncogenes, tumor suppressor genes and miRNAs [9]. Listeria monocytogenes, a gram-positive facultative intracellular pathogen, subvert in bone marrow-derived macrophages the expression of miR-155, miR-146a, miR-125a-3p/5p, and miR-149 all of which are implicated in the regulation of immune related genes [10]. Similarly, infections with the protozoan parasite Cryptosporidium parvum in epithelial cells [11] or detection of heat-killed Candida albicans by macrophages [12] leads to the alteration of host miRNA profile, most likely involved in immunoregulation.

Despite several advancements, tuberculosis remains one of the most deadly infectious diseases. Recently, the involvement of miRNAs in mycobacterial infection has been identified, thus opening a new field of research. Several Mycobacterium species, including Mycobacterium avium ssp. Hominissuis, have been shown to actively regulate miRNA expression in human and murine macrophages during infection [13] or through the interaction of their antigens with immune cells. Lipomannan from virulent Mtb has been shown to be a potent inhibitor of TNFα biosynthesis, through the regulation of miR-125b and miR-155 expression in human macrophages [14], while early secreted antigenic target 6kDa protein (ESAT-6) exerts strong suppression of mouse macrophage microbicidal activity through the induction of miR-155 [15]. Thus, miRNAs – by virtue of their ability to fine-tune innate immune gene expression – stand out as candidate regulators of Mtb-induced signaling; however, there is only sketchy knowledge on their role in guiding the outcome of host-pathogen interactions and the mechanisms exploited by virulent Mtb to tilt the balance of the innate immune response in its favor, thereby leaving its role largely unknown. To address this question, this study designed an approach aimed at determining whether the host genome-wide miRNA profile is altered upon infection of primary human macrophages infected with two members of the Mtb complex: virulent Mtb H37Rv and the non-virulent vaccine strain Mycobacterium bovis Bacillus Calmette-Guerin (BCG). To distinguish the modulation on miRNA expression due to passively transferred mycobacterial antigens from intracellular invasion by metabolically active bacteria, parallel infections with chemically inactivated Mtb bacilli were initiated.


  Materials and methods Top


Isolation of human monocytes and differentiation into MDMs

Unpolarized MDMs were prepared as previously described [16]. Briefly, leukocyte fractions from peripheral blood of healthy human donors were obtained from the Ospedale San Raffaele Blood Center, Milan, Italy. Peripheral blood mononuclear cells were isolated by density gradient sedimentation using Lympholyte-H (Cederlane, Hornby, Ontario, Canada) and were maintained in 75 cm2 cell culture flasks (Costar, Cambridge, MA) in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 5% heat-inactivated human AB serum (all from Lonza, Basel, Switzerland). After 24 h at 37 °C non-adherent cells – mostly T lymphocytes – were removed by rinsing with PBS-2%FBS and adherent cells were dislodged with cold PBS by gentle scraping. Detached cells (≥95% monocytes as determined by flow cytometric analysis after staining with anti-CD14 mAbs) were seeded into 12-well plastic plates (Costar) at the concentration of 5 × 105 cells/well and cultivated for an additional 7–8 days in order to obtain macrophages fully differentiated but not polarized [16]. Human study protocols were approved by the institutional review board at the San Raffaele Research Institute (Milan, Italy). Written consent was obtained from all study participants.

Infection assay

MDMs from three different donors were infected with Mtb H37Rv, M. bovis BCG, or chemically inactivated H37Rv in triplicate cultures. M. bovis BCG and Mtb H37Rv (NCTC 7416 and NCTC 5692; American Type Culture Collection, Manassas, VA) were grown to mid log phase in Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI) supplemented with 0.05% Tween 80 (Sigma) and 10% Middlebrook oleic acid albumin dextrose catalase enrichment (Becton Dickinson, Sparks, MD). Chemically killed bacteria were prepared by treatment with 3% cold formaldehyde for 1 hr, followed by extensive washing with PBS. Formaldehyde treatment, as opposed to heat inactivation, kills the bacteria but preserves most of the antigenic structures of the cell [17]. Mycobacteria were washed twice with PBS, suspended by sonication in RPMI without antibiotics and used to infect MDMs at multiplicity of infection (MOI) 5. After 2 h of incubation, MDMs were washed twice to remove unbound bacilli, cultured for another 24 h and total RNA assayed using TaqMan Low Density Array v2.0. For each experiment, a chamber slide with mycobacteria-infected macrophage culture was prepared and stained with Ziehl-Neelsen acid-fast staining in order to check microscopically for intracellular mycobacterial infection.

MiRNA profiling by TaqMan Low Density Array (TLDA)

Total RNA containing the small RNA fraction was isolated by mirVana miRNA Isolation Kit (Ambion, Austin, Texas) and RNA quality and concentration measured on the 2100 Bioanalyzer (Agilent, Santa Clara, CA). Average RNA integrity number (RIN) was 9.3. MiRNA reverse transcription cDNA synthesis for TLDA was performed according to manufacturer's recommendation. Briefly, 105ng RNA was reverse transcribed using the miRNA reverse transcription kit in combination with stem-loop Megaplex primer pools. MiRNA profiling assays were performed using TaqMan Low-Density Array v2.0 (Applied Biosystem, CA, USA). Each sample was analyzed with an A and B card for the detection of 667 miRNAs together with endogenous controls. In order to increase the sensitivity of the TLDA, a pre-amplification was performed after the RT procedure using the TaqMan PreAmp Mastermix and the Megaplex Pre Amp Primer Pools A+B. qRT-PCR was carried out on a 7900HT Fast Real-Time PCR System (Applied Biosystems).

Statistical analysis

Data analyses were performed using the SDS RQ Manager 1.2 software and DataAssist v2.0 software (Applied Biosystems). Real-time PCR data on miRNA expression levels were normalized on the most stable candidate endogenous controls selected using the NormFinder algorithm [18]. Fold changes of miRNA expression relative to the non-infected control were calculated by the 2−ΔΔCT method. Statistical significance of miRNA expression between infected and non-infected was determined according to unpaired Student t-test of three biological replicates.


  Results and discussion Top


M. tuberculosis infection in human macrophages induces significant host miRNA expression

In order to identify miRNAs differentially expressed in Mtb-infected human macrophages, virulent strain H37Rv was compared with the non-virulent vaccine strain M. bovis BCG, both extensively characterized in vivo and in vitro. Furthermore, parallel infections with chemically inactivated Mtb H37Rv bacilli allowed insight as to whether the effects of Mtb on miRNAs expression required the viable bacteria.

Among the 667 miRNAs characterized in the TLDA assays, 358 (53.6%) distinct miRNAs were expressed in MDMs, either control or treated, as determined by cycle threshold (Ct) values of <35. When infected MDMs were compared with untreated cells, 52 (14.5%) out of 358 miRNAs were found significantly different (p < 0.05 unpaired t test of three biological replicates) with two-or-more-fold changes in expression ([Figure 1]). Interestingly, as depicted in the Venn diagram in [Figure 2]C, the 52 differentially expressed miRNAs segregated in discrete groups related to infection conditions. Several general observations can be drawn from this data: (i) The largest group, 26 (51.9%), represents miRNAs modulated in response to killed H37Rv, of these only 7 were shared with either BCG or H37Rv, while 19 were specific to killed H37Rv ([Figure 2]B and [Figure 3]A); (ii) a pool of 33 miRNAs were modulated in response to live mycobacteria infection (i.e., H37Rv and BCG), of these 12 were common to both mycobacteria. Three miRNAs for H37Rv and 4 for BCG were shared with killed H37Rv ([Figure 2]B); and (iii) The last pool included miRNAs specifically modulated by only one infection condition ([Figure 3]A,[Figure 3]B,[Figure 3]C).
Figure 1: Heat map representation of differentially expressed miRNAs in MDM infected with M. tuberculosis H37Rv, M. bovis BCG and M. tuberculosis H37Rv killed. Each column represents the mean of three different donors and each row shows miRNAs. The relative up and down-regulation of miRNAs, expressed as log2 (fold change) is indicated by red and blue, respectively. OnlymiRNAs significantlymodulated (p < 0.05) with more than two-fold change expression compared with non-infected MDM are included in the map.

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Figure 2: Shared miRNAs expression. (A and B) Represent miRNA commonly expressed in different infection conditions, BCG white bars, H37Rv gray bars and killed H37Rv black bars. Data represents the mean ± SEM from three biological replicates. (C) A Venn diagram showing the number of strain specific and shared miRNAs. Only miRNAs significantly modulated (p < 0.05) with more than two-fold change expression compared with non-infected MDM are included in the diagram. The online tool Data Overlapping and Area-Proportional Venn Diagram (http://bioinforx.com/free/bxarrays/overlap.php) was used to generate the Venn diagram. Statistical significance was calculated according to unpaired t test of three biological replicates.

Click here to view
Figure 3: Specific miRNA expression after infection with BCG, H37Rv and killed H37Rv. (A, B and C) Represent miRNAs specifically modulated upon infection with, BCG white bars, H37Rv gray bars and killed H37Rv black bars. Only miRNAs significantly modulated (p < 0.05) with more than two-fold change expression compared with non-infected MDM are included in the diagram. Statistical significance was calculated according to unpaired t test of three biological replicates.

Click here to view


MDMs infected with killed Mtb H37Rv respond with a vigorous modulation of miRNA expression

Infection of human primary macrophages with killed H37Rv resulted in the strongest miRNA response among the three infection conditions considered, both in terms of number of miRNAs and fold change ([Figure 3]A). A large number of statistically significant changes in miRNA abundance induced by killed H37Rv occurred in miRNA passenger strands (marked with an asterisk after the miRNA name) rather than in the active guide strand (no asterisk). Passenger strands are usually excluded from miRNA-induced silencing complex and thus are thought to lack biological functions [19] although exceptions have been noted [20]. Alterations in passenger strand abundance were mirrored by smaller, statistically insignificant changes in abundance of the corresponding miRNA guide strands in MDMs (data not shown). These data are consistent with a recent report by Graff et al. [21] that demonstrated with kinetic analyses that miRNA passenger strand expression provides a transient marker for delayed increases in the functional guide strand miRNA expression following acute stimulation of macrophages.

The set of miRNAs responding to ingestion of killed H37Rv overlapped only to a small extent (7 miRNAs) with the larger set responding to viable mycobacteria, [Figure 2]B and C. A similar pattern of gene expression has been previously reported in mouse macrophage responses to live and inactivated Mtb [22]. Heat-killed mycobacteria are likely to present carbohydrates, glycolipids, and DNA sequences that can be powerful stimuli for innate immunity. However, the findings of this study direct attention to those molecules whose production or release by mycobacteria requires their viability. These include secreted proteins and lipids, heat-labile proteins and lipopeptides on the surface, and substances synthesized after the adaptation of Mtb to the phagosomal environment [23].

A common pool of miRNAs responds in MDM to both virulent and avirulent mycobacteria infection

From the analysis of this study emerged that during MDM infection with live mycobacteria, 12 miRNAs were modulated by both M. bovis BCG and Mtb H37Rv. These changes in miRNA expression were similar both in terms of strength (fold change) than direction (up- or down-modulation). Only miR-145 was different between the two bacteria and just in terms of strength (p < 0.05). The modulation of a set of miRNAs, which have proven to be key in many immune and inflammatory pathways was observed in this study [24]. Among the most characterized miRNAs, miR-155 and miR-146a were significantly up-regulated both in H37Rv and BCG infected macrophages. This is consistent with recent findings on the induction of miR-155 and miR-146a by ESAT-6 stimulation of macrophages [15]. On the other hand, BCG that has lost the RD1 region and as a consequence the capacity to secrete ESAT-6, could induce miR-155 through TLR2-triggering [25]. MiR-145 has been recently described as a potent inducer of apoptosis [26]. It binds to Toll – interleukin-1 receptor domain – containing adaptor protein (TIRAP) that is known to lie upstream of tumor necrosis factor receptor – associated factor-6 (TRAF6), the target of miR-146a, in the innate immune signaling of myeloid cells [27]. TIRAP is the key upstream activator of the innate immune cascade in response to TLR2 and TLR4, while TRAF6 lies at the hub of the pathway activated by multiple receptors to activate downstream signaling to NF-κB [28]. As shown in [Figure 1], miR-145 was up-regulated (FC 1.631; p = 0.317), although non-significantly, upon infection with killed H37Rv, and this is consistent with a regular inflammatory response of the macrophage. However, upon infection with BCG, miR-145 was significantly down-regulated (FC 0.3061; p = 0.0469). Loss of miR-145 has been reported to result in an elevated expression of its targets, TIRAP and inhibition of apoptosis [27]. Virulent H37Rv infection was associated with an even lower level of miR-145 (FC 0.1176; p = 0.006) consistent with a lower capacity of virulent Mtb strain to induce apoptosis [29],[30]. Furthermore, miR-27a and miR-27b, which control inflammation and lipid metabolism through peroxisome proliferator-activated receptors (PPARgamma) [31] were both significantly down-modulated. MiR-222* was strongly down-modulated; interestingly, miR-222* has been shown to be strongly reduced in macrophages under M1-polarizing conditions [21]. This is consistent with previous data reporting that during the early phase of Mtb infection, macrophages are polarized toward an M1 profile [22],[32], which is also in agreement with clinical data collected from patients with active tuberculosis [33].

MiRNAs specifically modulated by BCG, H37Rv and killed H37Rv

Over 52 miRNAs significantly changed in MDM after infection; three sets of miRNAs modulated specifically by each of the infection condition were identified.

Infection of MDM with killed H37Rv resulted in the strong up-regulation of 8 miRNAs (FC range from 31.01 to 37647; p < 0.05) and down-modulation of 11 miRNAs, most of which were passenger strands ([Figure 3]A). This type of modulation suggests a miRNA response representing a conventional onset of inflammatory response of infected macrophages. Interestingly, live virulent Mtb H37Rv appeared to elicit a milder inflammatory activation of the host target cell. Only 4 miRNAs were up-regulated and the range of increase was significantly smaller (FC range from 2.06 to 4.11; p < 0.05); similarly, down-modulated miRNAs were 6, of which 4 ranging from 0.2448 to 0.4265; p < 0.05. MiR-503 (FC 0.0637; p = 0.0316) and miR-551b* (FC 0.0711; p = 0.2649), that represent less characterized miRNAs, showed a more pronounced down-modulation ([Figure 3]B). It can be speculated that this set of miRNA, specifically linked to H37Rv infection, could represent virulence markers. MiRNAs specifically associated with BCG infection were only 4, with a small FC range (2.08 to 2.72; p < 0.05). MiR-645, a less characterized miRNA, was highly modulated (FC 31.5676; p = 0.0068) ([Figure 3]C).

Notably, induction of miR-29a, miR-125b and miR-21 [14],[34],[13], previously reported to be linked to mycobacterial infection, was not confirmed in our experimental system.


  Conclusions Top


This study reports the first genome-wide Mtb infection induced miRNA expression profile in primary human macrophages. The findings show that infection of human macrophages with virulent Mtb H37Rv and avirulent M. bovis BCG results in a pattern of miRNA expression mostly overlapping between the two live mycobacteria considered, while a substantially different pattern emerged from infection with killed Mtb bacilli suggesting an active influence of live intracellular bacteria on cell target miRNA-metabolism.

The Mtb-induced miRNA signature was composed of the well established in immune regulation miR-155 and miR-146a, as well as a set of miRNAs newly associated with Mtb infection: miR-145, miR-222* and miR-27a, miR-27b, all of which are predicted to target important immune-related genes. Moreover, a set of less characterized miRNAs were identified, which are elicited only by virulent H37Rv infection. This study signifies the miRNA host response upon intracellular Mtb complex spp. infection in macrophages, providing new aspects of regulation in host-pathogen interactions, at post-transcriptional levels.


  Conflict of interest Top


The authors have no conflict of interest to declare.


  Acknowledgments Top


The authors thank Dr. Di Serio C. and Dr. P.M. Rancoita for the scientific support and for the kind help with digital graphical elaboration. This work was partially supported by the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement FP7-223681 to DMC. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]


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