Biological functions and diagnostic implications of microRNAs in Mycobacterium tuberculosis infection

Godkowicz Magdalena, Druszczynska Magdalena

1Department of Immunology and Infectious Biology, Institute of Microbiology, Biotechnology and Immunology, Faculty of Biology and Environmental Protection, University of Lodz, Banacha 12/16, 90-237 Lodz, Poland

2The Bio-Med-Chem Doctoral School of the University of Lodz and Lodz Institutes of the Polish Academy of Sciences, Lodz, Poland

ABSTRACT

MicroRNAs (miRNAs), small non-coding RNAs, play important roles in regulating host defense against pathogenic infections.This review provides information on the role of miRNAs in the antimycobacterial immune response and summarizes their possible diagnostic utility. It was compiled using scientific literature retrieved from such databases as PubMed, Scopus, ScienceDirect,Google Scholar, and PubMed Central. Relevant articles published in the English language until December 2020 were taken into consideration. It has been revealed that specific host miRNAs induced by Mycobacterium tuberculosis can target diverse factors and pathways in immune signaling to ensure longer pathogen survival inside the phagocytes. The potential use of miRNAs in tuberculosis diagnosis or therapeutic strategies has been attracting increasing attention in recent years. However, despite considerable efforts devoted to miRNA profiling, further studies are needed to elucidate the full potential of miRNAs as novel tuberculosis biomarkers or therapeutic targets.

KEYWORDS: microRNA; Mycobacterium tuberculosis; Biomarker;Diagnostics; Tuberculosis

1. Introduction

Tuberculosis (TB) is a contagious and infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis). It is estimated that about 2 billion people worldwide are infected with the pathogen,of which 5%-10% develop active TB, and the remaining 90% stay asymptomatically infected with M. tuberculosis within their lifetime[1]. A priority for TB control programs is to strengthen the capacity to diagnose TB and treat its multidrug-resistant (MDR-TB)forms. Standard TB diagnostic methods involving microbial growth in culture media or diverse immunological and genetic techniques are not sensitive enough to confirm M. tuberculosis infection in all TB patients[2]. Therefore, it has become necessary to develop new diagnostic tools, and many different classes of molecules have been studied for this purpose.

The discovery of the mechanism of post-transcriptional silencing of target messenger RNA (mRNA) by small RNA molecules called microRNAs (miRNAs) has altered the understanding of the control of the genetic information expression system. Micro RNAs were first discovered in 1993 in the nematode Caenorhabditis elegans[3],and in the following years, they were identified in plants, viruses,and human cells[4]. These small non-coding RNA molecules regulate many biological processes, mainly by inhibiting translation or inducing mRNA degradation[5,6]. According to the latest analyses,more than 2 600 miRNAs have been identified in the human genome, which together regulates 50% of genes in the genome[7,8].It has been found that a single miRNA molecule can modulate the expression of multiple genes, while one mRNA can be targeted by several different miRNAs[9,10]. The potential use of miRNAs in diagnosis or therapeutic strategies has attracted increasing attention in recent years[11–13]. In this review, we focus on the role of several miRNAs in the antimycobacterial immune response and summarize their utility as potential biomarkers.

2. Research methodology

We searched the following electronic, web-based databases to extract relevant studies from research articles, review articles, and book chapters in English language: PubMed, PubMed Central,Scopus, ScienceDirect, and Google Scholar. Non-English articles and letters to the editor were omitted. The following terms were used to generate the search: “microRNA,” “miRNA” or “miRNA vs.tuberculosis” or “miRNA vs. mycobacteria” or “miRNA function” or“miRNA biomarker,” or “miRNA diagnostics”.

3. Involvement of miRNAs in the regulation of antimycobacterial immune response

M. tuberculosis has developed various strategies to avoid being killed and escape immune surveillance[14,15]. The inhibition of phagosomal maturation, down-regulation of major histocompatibility complex class Ⅱ antigen presentation, attenuation of apoptosis and autophagy processes, and reduction of reactive oxygen and reactive nitrogen intermediates function, are some of the best-characterized M. tuberculosis mechanisms. As essential modifiers of hostpathogen interactions, miRNAs influence both innate and acquired antimycobacterial immunity by regulating maturation, activation,and cell effector functions (Figure 1). They are also involved in cell apoptosis and play a role in M. tuberculosis pathogenesis by the modulation of the lipid metabolic pathway[16,17]. In the next subsections, we review the involvement of several host miRNAs in the regulation of the host immune response to M. tuberculosis infection.

Figure 1. A schematic presentation of miRNA regulation of host immune responses against Mycobacterium tuberculosis (M. tuberculosis) infection. The depicted miRNAs promote or inhibit important pathways in the cellular response in macropages and T cells. BCG: Mycobacterium bovis bacillus Calmette-Guérin.

3.1. miRNA-20

The specific effect of miRNA-20b on M. tuberculosis infection has been documented by Lou et al.[18]. They observed a reduced expression of miRNA-20b-5p accompanied by the activation of nod-like receptor(NLR) family pyrin domain containing 3 (NLRP3)-mediated immune response, in TB patient macrophages[18]. Downregulation of miR-20b-5p expression was also found in M. tuberculosis-infected mice.Targeting the NLRP3/caspase-1/IL-1β pathway via injection of mice with the miR-20b mimics inhibited the inflammatory response of murine alveolar epithelial cells co-cultured with macrophages[18].

3.2. miRNA-21

miRNA-21 is one of the most commonly deregulated miRNAs in almost all cancer cell types and is therefore called an “oncomiRNA”[19]. In recent years, additional roles of miRNA-21 in pulmonary and cardiovascular diseases have been described. It has been suggested that increased miRNA-21 activity may contribute to the attenuation of the antimycobacterial immune response and longer pathogen survival. Wu et al. reported that miR-21 was upregulated in alveolar macrophages obtained from Mycobacterium bovis (M.bovis) bacillus Calmette-Guérin (BCG)-vaccinated mice, as well as in macrophages and dendritic cells experimentally infected with M.bovis BCG[20]. miRNA-21 impaired IL-12 production and promoted dendritic cell apoptosis by targeting IL-12p35 and B-cell lymphoma 2 (Bcl-2) proteins. In another study, it was found that in a RAW264.7 cell line, miRNA-21 expression, enhanced after the stimulation with M. tuberculosis protein, MPT64, might have participated in the inhibition of apoptosis due to an increased NF-κB expression and upregulation of Bcl-2[21]. Moreover, miRNA-21 was proved to boost M. tuberculosis survival and apoptosis and impair the production of inflammatory cytokines (IL-1β, IL-6, and TNF-α) by targeting the Bcl-2 and Toll-like receptor 4 in RAW264.7 and THP-1 cells[22].Sheedy et al. observed that miRNA-21 impaired the development of a pro-inflammatory immune response by upregulating IL-10 production[23].

3.3. miRNA-26

The miRNA-26 family consists of miRNA-26a-1, miRNA-26a-2,and miRNA-26b members, which are involved in various biological processes including cell growth, development, and tumorigenesis[24].Ni et al. found that M. tuberculosis-induced overexpression of miRNA-26a and miRNA-132 decreased the levels of the p300 transcriptional coactivator, thereby diminishing the transcription of IFN-γ–induced genes and macrophage response to this cytokine[25].On the contrary, Sahu et al. showed downregulation of miRNA-26a-5p in macrophages infected with M. tuberculosis and in the lungs,spleen, and lymph nodes of mice infected with M. tuberculosis[26].The decrease in miRNA-26a-5p expression was correlated with the upregulation of the Kruppel-like factor 4, which is involved in the polarization of macrophages towards the M2 phenotype[26].Kleinsteuber et al. confirmed a significant reduction in the expression of miRNA-26a, along with miRNA-29a and miRNA-142-3p, in the peripheral blood of TB patients compared to individuals latently infected with M. tuberculosis[27]. An increase in miR-26a expression during treatment pointed to miR-26a as a potential biomarker not only indicating M. tuberculosis infection but also confirming the effectiveness of the applied therapy.

3.4. miRNA-27

miRNA-27a and miRNA-27b play a vital role in modulating tumorigenesis, proliferation, apoptosis, and angiogenesis[28,29].Liu et al. demonstrated that miRNA-27a was upregulated in M. tuberculosis-infected mice and macrophages as well as in patients with active TB[30]. miRNA-27a-knockout mice had fewer histological lesions and inflammatory infiltrates after M. tuberculosis infection in the lungs than wild-type mice. The target of miRNA-27a was CACNA2D3, a component of a voltage-dependent calcium transporter, present in the endoplasmic reticulum. Targeting CACNA2D3 inhibited autophagosome formation and promoted intracellular survival of M. tuberculosis. The role of miRNA-27b in the macrophage response to M. tuberculosis infection was investigated by Liang et al.[31]. They showed that miRNA-27b expression targeted Bcl-2–associated athanogene 2 in macrophages and inhibited the production of pro-inflammatory factors via TLR-2/MyD88/NF-κB signaling cascade. Besides, miRNA-27b decreased the mycobacterial load and, at the same time, it increased cell apoptosis and the production of reactive oxygen intermediates[31].

3.5. miRNA-29

The miRNA-29 family members (miRNA-29a, miRNA-29b-1,miRNA-29b-2, and miRNA-29c) demonstrate anti-fibrotic activity and are involved in the regulation of many extracellular matrix proteins such as collagen, fibronectin, and laminin[32]. Recent studies showed alterations in miRNA-29 expression during mycobacterial infection[33,34]. Ma et al. noted that infection with the attenuated M.bovis (BCG) strain increased miRNA-29a-3p expression in natural killer cells, CD4(+), and CD8(+) T lymphocytes[33]. A direct target of miRNA-29a-3p was found to be IFN-γ mRNA, and miRNA-29a-3p expression inversely correlated with IFN-γ production. On the contrary, Afum-Adjei et al. did not observe any correlation between miRNA-29a and IFN-γ expression in active TB patients, suggesting that decreased IFN-γ expression had not been caused by differences in miRNA-29a expression[35]. A recent study by Ndzi et al. showed that miRNA-29a was significantly elevated in the blood of active TB patients compared to individuals latently infected with M. tuberculosis and healthy controls, and this miRNA exhibited the potential to be a diagnostic biomarker for TB. It was also upregulated in active TB patients who had not yet started the treatment compared to those who had completed anti-TB therapy, which demonstrated its potential as a biomarker for TB treatment follow-up[36].

3.6. miRNA-144

Many studies have documented the overexpression of miRNA-144-5p in the serum, peripheral blood mononuclear cells (PBMCs),and sputum from active TB patients, suggesting its involvement in the regulation of anti-TB immunity[37–39]. M. tuberculosis-induced upregulation of miRNA-144-5p was found to decrease phagosomal maturation in human monocytes[39], while miRNA-144-5p overexpressed in T cells impaired cell proliferation and production of cytokines[37]. Significantly lower levels of miRNA-144 observed in the sputum and serum after a successful TB treatment suggest a potential of miRNA-144-5p as a new biomarker in the evaluation of the effects of antituberculous therapy[38].

3.7. miRNA-145

miRNA-145, commonly downregulated in many types of cancer cells, regulates many cellular processes including proliferation,apoptosis, and cell cycle[40]. Some studies reported downregulation of miRNA-145 expression during M. tuberculosis infection[41,42].Significantly lower serum miRNA-145 levels were negatively correlated with serum levels of IL-1β and TNF-α, which were observed in patients with active TB compared with healthy subjects,including those latently infected with M. tuberculosis[42]. The decreased expression of miRNA-145 showed its relatively high potential in TB diagnosis, providing evidence that miRNA-145 could serve as a candidate diagnostic biomarker.

3.8. miRNA-146a

Some studies found elevated expression of miRNA-146a in M.tuberculosis-infected human dendritic cells and human monocytederived macrophages, whereas in PBMCs and mononuclear pleural fluid cells as well as plasma samples from TB patients, a decrease in miRNA-146a levels was noted[41,43–45]. In macrophages, BCG-induced miRNA-146a inhibited the expression of inducible nitric oxide (NO) synthase (iNOS) and the production of NO, which was due to the targeting of interleukin-1 receptor-associated kinase-1 and tumor necrosis factor receptor-associated factor 6 via miR-146a-5p[44,46]. Changes in mycobacteria-induced miRNA-146a expression depending on the infection dose and duration could modulate the inflammatory response and facilitate mycobacterial replication in macrophages[46].

3.9. miRNA-155

miRNA-155 is a major regulator of inflammation, and increased expression of miRNA-155 has been found in a variety of inflammatory diseases and various cancers. In vivo and in vitro studies have demonstrated that miRNA-155 also plays an important role in the regulation of host immune responses to mycobacteria[47].Iwai et al. found that miRNA-155-/- knockout mice were more susceptible to M. tuberculosis infection and were characterized by higher bacterial loads in the lungs and decreased numbers of antigenspecific CD4+T cells as well as an impaired IFN-γ production compared to wild-type animals[47]. An increased expression of miRNA-155 has been observed in many types of M. tuberculosisinfected cells[47–50], but different effects induced by miRNA-155 in the early and chronic phases of M. tuberculosis infection suggest that miRNA-155 should be considered a pleiotropic immune regulator[49]. In macrophages, M. tuberculosis-induced miRNA-155-5p targeted a negative regulator of autophagy, Rheb (Ras homologue enriched in brain), leading to the inhibition of intracellular survival of the bacteria[48]. Upregulation of miRNA-155-5p in T cells and macrophages during M. tuberculosis infection was found to suppress the expression of Src homology-2 domain-containing inositol 5-phosphatase 1, a negative regulator of the PI3K/Akt pathway involved in TNF biosynthesis, which induces cell apoptosis[49,51]. In turn, in M. tuberculosis-infected human dendritic cells, miRNA-155 impaired the autophagy process via targeting ATG3, an E2-ubiquitinlike conjugating enzyme involved in autophagosome formation[50],while targeting the forkhead box O3 by miRNA-155 was reported to inhibit apoptosis of monocytes[52]. Moreover, overexpression of miRNA-155 was negatively correlated with cytotoxic activity and the production of TNF-α by natural killer cells[53].

3.10. miRNA-223

miRNA-223 plays an important role in promoting granulocytic differentiation[54]. The upregulation of miRNA-223 in blood and lung parenchyma has been confirmed in both human and murine TB[55]. miRNA-223 has been found to control lung recruitment of myeloid cells and neutrophil-driven inflammation by acting on the chemokines CXCL2 (C-X-C motif ligand 2), CCL3 (C-C motif ligand 3), and IL-6 in myeloid cells. Significantly increased expression of miR-223 in patients with TB was accompanied by the inhibition of NF-κB activation in monocyte-derived macrophages,suggesting a role for miRNA-223 in the control of excessive inflammation during M. tuberculosis infection[56].

3.11. miRNA-889

miRNA profiling in PBMCs from subjects with latent M.tuberculosis infection recognized miRNA-889 as one of the most overexpressed miRNAs[57]. The TNF-related weak inducer of apoptosis (TWEAK) was identified as the target of miRNA-889.M. tuberculosis infection upregulated TWEAK expression, which induced autophagy and promoted autophagosome maturation through the activation of AMP-activated protein kinase (AMPK).It has been suggested that the overexpression of miRNA-889 that inhibits autophagy via posttranscriptional suppression of TWEAK expression is responsible for maintaining the survival of mycobacteria in granulomas.

4. miRNAs as potential TB biomarkers

TB detection, along with prevention and treatment, remains an essential element of most of the world’s disease control programs.Despite the availability of many different diagnostic techniques,the diagnosis of TB is still based on the presence of acid-fast bacilli in microscopic smears, observation of bacterial growth in 6-8-week-old cultures, and the result of a skin tuberculin test.However, this standard TB diagnostic procedure does not make it possible to diagnose all patients infected with M. tuberculosis. The microscopic examination is characterized by low sensitivity and the inability to distinguish M. tuberculosis from non-tuberculous mycobacteria. The sensitivity and specificity of methods based on the amplification of mycobacterial nucleic acids are variable, and their use requires specialized equipment, which is often unavailable in routine mycobacterial laboratories. The lack of diagnosis has serious consequences, forcing clinicians to undertake empirical therapy, which, in the case of patients infected with drug-resistant mycobacteria undergoing standard treatment, is sometimes ineffective and may additionally exacerbate the phenomenon of M.tuberculosis drug resistance.

Several advantages that characterize miRNAs make them suitable biomarker candidates. These molecules are present in various body fluids with high stability despite repeated freezing and defrosting,and are not difficult to extract[58]. Calin et al. were the first to investigate the potential use of miRNAs as disease biomarkers and demonstrate that miRNA expression levels were altered in patients with chronic lymphocytic leukemia[59]. Shortly after this discovery,many studies revealed differentially expressed miRNA profiles in a variety of diseases, including TB, suggesting the use of miRNAs as potential diagnostic or prognostic biomarkers[59–66]. There is also a growing interest in using miRNAs as appropriate indicators of therapeutic efficacy[14,45]. Potential miRNA biomarkers in the diagnosis of M. tuberculosis infection are presented in Table 1.

Table 1. Candidate miRNA biomarkers in Mycobacterium tuberculosis infection.

Many studies have shown the alteration of miRNAs expression profiles in clinical samples (sputum, serum, pleural effusion, PBMC)in patients with active TB[14,41,67,68]. In PBMCs from active TB patients, Cao et al. identified a set of 26 differentially expressed miRNAs, among which miRNA-23a-5p, miR-NA-183-5p, miRNA-193a-5p, and miRNA-941 were overexpressed, and miRNA-16-1-3p was reduced compared to healthy subjects, suggesting their potential utility in the diagnosis of active TB[69]. Expression patterns of miRNA in pleural effusions from pulmonary TB patients showed upregulation of miRNA-378i compared to individuals with lung cancer or pneumonia[70]. An increase miRNA-625-3p level in the urine was demonstrated in M. tuberculosis smear-positive compared to M. tuberculosis smear-negative patients[71]. Profiling of miRNAs in the sputum of active TB patients showed overexpression of miRNA-3179 and miRNA-147 and underexpression of miRNA-19b-2[68]. A recent study has shown that miRNA-155 in the sputum may serve as another potential biomarker of active pulmonary TB[72].

The diagnosis of latent M. tuberculosis infection can also be challenging, due to the limitations of available tests – the tuberculin skin test and interferon-gamma release assays. In this context,several miRNA signatures distinguishing active TB from latent TB have been identified[73–75]. Lyu et al. observed unique sets of serum miRNAs for both latent TB (hsa-let-7e-5p, hsa-let-7d-5p, hsa-miR-450a-5p and hsa-miR-140-5p) and active TB (hsa-miR-1246, hsamiR-2110, hsa-miR-370-3P, hsa-miR-28-3p and hsa-miR-193b-5p)[74]. Interestingly, using the expression profile of eight different miRNAs (hsa-miR-122-5p, miR-151a-3p, miR-451a, miR-486-5p,hsa-let-7i-5p, miR-148a-3p, miR-21-5p, and miR-423-5p), latent TB could be distinguished from active disease with an accuracy of 71.8%[75]. Moreover, Wang et al. identified a specific miRNA profile composed of hsa-miR223, hsa-miR-424, hsa-miR-451, and hsa-miR-144 that is capable of distinguishing between latent M.tuberculosis infection and active TB[73]. The lack of consistency in the miRNA signatures reported may be due to the differences in the characteristics of the study population (age, sex, comorbidities), type of samples taken for miRNA isolation (sputum, serum, urine) as well as the applied study protocols.

There is also growing evidence on using miRNA expression profiles as indicators of antituberculous treatment efficacy[2,38,76].The upregulation of the serum miR-125a-5p level accompanied by downregulation of the miR-NA-21-5p, miRNA-92a-3p, and miRNA-148b-3p levels was found in TB patients, who responded properly to the applied therapy[76]. Moreover, it was noticed that miRNA-144,miRNA-16, and miRNA-155 expression that was altered during TB returned to the levels observed in healthy volunteers after the end of treatment, suggesting the possibility of using these proteins as markers of therapy success[2,38].

In recent years, much effort has been devoted to the use of miRNAs in novel therapeutic approaches including the delivery of exogenous miRNAs to host cells[77–79]. However, since miRNAs target the expression of many genes, miRNA therapy can have various unpredictable systemic consequences. Another problem is the efficient delivery of miRNAs, as miRNA molecules can be rapidly degraded. Accurate characterization of the individual roles of miRNAs is needed to assess their therapeutic potential in the future.

5. Conclusions

The recognition of the role of miRNAs as gene silencers and their active participation in the regulation of the immune response against M. tuberculosis has opened a new chapter in TB diagnosis, treatment,and prevention. However, despite the growing number of studies,the high variability of miRNA expression profiles that depend on the experimental conditions and protocol variations, makes it difficult to apply them in practice. Therefore, although some miRNAs are proposed as potential diagnostic biomarkers for TB, further extensive studies in this field are urgently needed.

Conflict of interest statement

The authors declare no conflict of interest.

Authors’ contributions

MD developed the theoretical formalism, performed the analytic calculations and performed the numerical simulations. Both MD and MG contributed to the final version of the manuscript. MD supervised the project.