Tuberculosis (TB) is an infectious disease which is caused by strains of genus Mycobacteria. The predominant pathogenic species that infects humans is Mycobacterium tuberculosis (MTb). It is a pulmonary pathogen as its typical life cycle begins in lungs of human but can spread to other organs and tissues and causes extra-pulmonary TB (1). It spreads through the air when diseased person coughs or sneezes (2). In 2014, 9.6 million new cases of TB were diagnosed and TB was a cause of death for 1.5 million patients worldwide (3). One-third of the world’s populations, that is, nearly 2.5 billion individuals are infected with MTb, but remain asymptomatic; among them, only 10% will develop clinically active TB in their lifetime (1). Though TB cases are declining due to effective antibiotic regiments, interest in TB has resurfaced due to the global emergence of multiple drug-resistant (MDR) and extreme drug-resistant (XDR) strains. As new resistant strains are making antibiotics obsolete, it has become pertinent to study the host–pathogen interaction to develop strategies which could augment host to fight against the pathogen. To achieve this, understanding of the host immune dynamics at its fundamental level is essential. The evolutionary arms race has endowed both the pathogen and host with several advantages to counter each other strategies. The fight between pathogen effectors and host immunity determines whether the pathogen is eliminated, made ineffective, or causes disease. When MTb is inside the human lung, phagocytic cells act as antigen-presenting cells (APCs) to neutralize the pathogen. APCs, such as neutrophils, dendritic cells, and macrophages, are known to concomitantly act to initiate T cell-based adaptive immunity to clear out the infection (4). Macrophages are of central importance as they possess a battery of immunological processes to kill the pathogen. Macrophages normally generate toxic nitrogen and oxygen radicals and induce autophagy and apoptosis to eliminate the pathogen. These capabilities are severely compromised by MTb and can, in turn, be manipulated for its survival. MTb has been experimentally shown to evade both apoptosis and autophagy by altering the intracellular machinery of the macrophage (5). In macrophage, a pathogen is normally phagocytosed and gets encased in endosome inside the cytosol. When this endosome matures, it fuses with the lysosome whereby the pathogen is neutralized, but MTb has the capacity to perturb the intracellular trafficking. It incapacitates phagosomal acidification by arresting its fusion with the lysosome (6). This crucial hindrance, which prevents phagolysosome maturation, leads to the survival of MTb inside the macrophage. This feat is achieved by MTb due to its interference with several intracellular pathways. It inhibits Rab5-dependent endosome maturation, modulates Ca²⁺ and PI3P-based signaling along with the modification of the actin cytoskeleton to prevent its fusion with the lysosome (7). By modulating intracellular mediators, MTb establishes an immunological block that leads to a chronic inflammatory condition in which it continues to persist. Host counteracts the onslaught and initiates an adaptive immune response by forming a granuloma, an immunological barricade to contain the further infection. By the time infection progresses, T-helper (T_H1) cell with its cytokine secretions, such as tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), propagates the adaptive limb of the immunity (8). The granuloma is established when a sufficient inflammatory response is generated by T_H1 cells along with the expression of inflammatory chemokine (9). The main effector cells which are affected by cytokines of T_H1 cells are macrophages. T_H1 response primes macrophages by upregulating major histocompatibility complex (MHC)-2 and activating appropriate signaling pathways necessary for the elimination of intracellular pathogens. MTb has the capacity to resist the deadly assault of the macrophage. Other than that, macrophages show a high level of plasticity and can differentiate into two opposite phenotypes (10). M1 macrophages are classically activated macrophages, which are immune-stimulatory and are influenced by cytokines, such as IFN-γ and TNF-α. M2 cells are alternatively activated macrophages, which are characterized by their immuno-suppressive properties, such as enhanced IL4, IL10, and IL-13 response (11). There is a lot of ambiguity in M1/M2 nomenclature, but certain transcription factors and cytokines have been shown to differentiate these extreme phenotypes (Figure 1).

Apart from M1/M2 plasticity, macrophages can also transform into foam cells, which are rich in lipid and cholesterol (12). Foamy macrophages are cells with deregulated lipid metabolism (13, 14). The homeostatic balance between the influx and efflux of low-density lipoprotein (LDL) comprising cholesterol, triacylglycerides, and phospholipids, is compromised in these cells. This modification is achieved in two steps, first, scavenger receptors and CD36, leads to increased uptake of LDLs by the macrophages. Second, transporters like ABCA1 that regulate the efflux of excess cholesterol are affected inside these macrophages. ABCA1 knockdown has been associated with exacerbated conditions of foam cells. Increased uptake of LDLs and later its compromised efflux suits the nutritional requirement of MTb (13). MTb has also been shown to modulate the ketone body synthesis to accumulate lipid bodies inside the macrophages (14). Peroxisome proliferator-activated receptor (PPAR) group of transcription factors have also been implicated in both M2 transition and later in foam cell formation. PPARs upregulate metabolism genes and also represses M1 phenotypic marker (15). All of these factors are susceptible to modulation by MTb for its survival. These modified macrophages in the form of foamy cells are shown to be a predominant niche for MTb in granuloma (16) (Figure 1).

In TB, granuloma formation is an immunologically dynamic process which involves both innate and adaptive mechanisms. Few studies have focused on M1 and M2 macrophages bias in granuloma (17). The confirmed presence of M1, M2, and foamy macrophages in granuloma warrants further investigations to understand its transition and its role in the pathology of the disease. We have suspected the role of microRNAs against some transcription factors which transcribe critical genes that affect the M1/M2/Foamy macrophage continuum. miRNAs are evolutionarily conserved, 20–24 nucleotide small non-coding RNA, which inhibits the expression of genes by binding to the 3′-UTR of the transcribed mRNAs. The recognition of mRNA nucleotides by 6–8 nucleotides long seed region of miRNA is sufficient to inhibit the gene expression (18). The miRNA–mRNA complex leads to mRNA destabilization, translational inhibition, or mRNA degradation. miRNA has been shown to play diverse roles from affecting developmental pathways to immunological mechanisms (19). The specific variations in miRNA expression during immune cell development are increasingly being appreciated. Its aberrant expression has been linked to infections, aging, and other pathological outcomes (20). As monocyte transition to macrophage is a continuous process, the perturbed expression of miRNA can influence its phenotype under certain temporal and spatial conditions. In this study, we have identified transcription factors which can regulate macrophage polarization during MTb infection. In fact, several studies have corroborated that levels of transcription factors present in cytosol influence the gene expression profile of a particular cell (21, 22). MTb-associated miRNA modulation can lead to a quantitative variation of transcription factors inside the macrophage either by directly altering the transcription factor or by influencing other proteins interacting with the transcription factors. There has been evidence that PtpA secreted by MTb directly dephosphorylates p-Jnk and p-p38, thus modulating cytokine expression (23).

In this computational study, we have identified key miRNAs that can be upregulated or repressed to skew the macrophage polarity to pro-survival M2 phenotype. Transcription factors like IRF5, IRF1, SOCS1, STAT1, and STAT2 promotes inflammatory M1 macrophage, and its MTb-based suppression enhances its survival. Transcription factors expressed by M2 macrophages, such as IRF4, SP1, SP3, STAT6, SOCS 2, SOCS 3, PPAR, and KLF6, were also analyzed in this study. Their upregulation boosts the survival of MTb (Figure 1). This study revealed novel MTb-associated miRNAs which bind to the 3′-UTR of the aforesaid transcription factors. The variation in the miRNA profile decides the fate of the macrophage to which survival of MTb is intertwined. Later, we analyzed few microRNAs which fulfills the dual role of transition from M2 phenotype to nutritional rich foam cells. Similar computational approaches have been used in deciphering the role of miRNAs in other human disorders (24, 25).

Tuberculosis involves combined effort of innate and adaptive immunity to clear out the infection. Macrophages are the central immune cells, as they possess an arsenal of toxic mechanisms sufficient to kill the pathogen. MTb-dependent modulation of macrophage, whereby it not only survives but also thrives, is one of the great evolutionary adaptations. In the host, macrophages’ plasticity yields predominantly pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages. For MTb, the advantage of M2 cells could be gauged by the fact that it downregulates the antigen presentation molecules and co-stimulatory receptors, in turn impairing the activation of T_H1 cells (41). The absence of T_H1 response coupled with immune manipulation to generate M2 creates a comfortable niche for this pathogen. Add to it, the capacity to transform macrophage into a foam cell, MTb successfully manipulates the host to generate a safe harbor for its long-term survival (16). The crucial molecular factors which are essential for deciding the phenotypic outcome of a macrophage is of great interest as the survival of pathogens is dictated by the lethality of macrophage response. As infection by MTb progresses, the defensive action of host creates an immunological barricade called a granuloma. There has been evidence suggesting the presence of both types of macrophages in the granuloma (17). During the initial stages of infection, M1 type of macrophages predominates, but loses out to M2 as the infection progresses (17). M1/M2 polarization is based on the differential expression of genes in a macrophage. For example, IL-23- and IL-12-secreting M1 macrophages promote mycobacterial killing, but IL-10-secreting M2 expression subverts its killing (41). Expression of these genes is controlled by specific transcription factors, which express a single gene or a set of genes (42). The fine-tuning of gene expression with respect to transcription factors could be achieved in two ways. First, the downregulation or upregulation of the transcription factors in the cell leads to the corresponding effect on the quantity of the gene product. Second, modification of transcription factor also influences the gene expression. Certain transcription factor such as NFAT must be dephosphorylated before they can translocate to the nucleus (43). Some studies have shown that the quantity of transcription factor as a key variable in deciding the inflammatory status of the macrophage (21, 22). We have pointed out that MTb-dependent miRNAs modulation of transcription factors might be one of the molecular factors that have the capacity to tip the balance toward pro-survival M2 phenotype.

In this computational study, we have uncovered few critical transcription factors which influence the M1/M2 plasticity and are potential targets modulated by MTb. Indeed, many other studies are also pointing at the role of microRNAs in various immune cells, including macrophages (20). Experimentally several studies have documented MTb-dependent miRNA modulation (44, 45). The pathogen downregulates inflammatory molecules IFN-γ, TNF-α, and IRAK1 by modulating several miRNAs (46–48). In this study, by analyzing multiple databases, we have filtered critical micro RNAs that can affect the availability of TFs. Initial hits were selected from TargetScan as it uses P_CT and context ++ percentile score. Higher score translates to a better probability of binding with the given mRNA. These filtered miRNAs were further analyzed by other databases.

The interferon regulatory elements (IRF) are transcription factors which upregulate the transcription of a specific set of genes and play a significant role in deciding the outcome of macrophage phenotype. For M1 macrophage, IRF5 is the critical transcription factor which influences the inflammatory response of the macrophage (26). IRF5 depended production of IL-12 by M1 macrophage is required to IFN-γ-dependent pathway in T_H1 cells. IFN-γ then potentiates the macrophages to unleash its antibacterial arsenal (49). IRF5 also inhibits the transcription of IL10, thus negatively regulates M2 phenotype. Its upregulation is synonymous with M1 and down regulation leads to M2 cells. IRF1 also plays its part by inducing Myd88-dependent pathway, but recent evidence has also suggested its association with BATF1 which leads to the expression of several host protective genes against MTb (50). Its knockdown lead to severe downregulation of IFN-γ-dependent pro-inflammatory limb (50). IRF5 and IRF1 down-modulation by miRNAs prolong the survival of MTb in macrophages.

IRF4, another transcription factor is essential for generating M2 phenotype. IRF 4 is known to associate with Jmjd3, an H3K27 demethylase, and has been implicated in the promotion of M2 phenotype (51). We found out that miR-302c putatively controls IRF5 expression, and miR-27a and miR-27b regulate IRF4 expression. For the promotion of M2 macrophage, it is essential to downregulate IRF5 by upregulating miR-302c. Expression of IRF4 can be affected by downregulating miR-27a and miR-27b. When perturbed by MTb, these miRNA can significantly alter the macrophage polarization status, thus providing a means to MTb for its survival.

STATs are an intracellular transducer that regulates the expression of several immune genes in macrophages. These participate by expressing higher MHC-2, IL-12, and Nitric oxide synthase (NOS), necessary for killing of the pathogen (52). STAT1 and STAT2, transducers play their part in M1 polarization, they form a heterodimer to induce transcription of several microbicidal genes. miR-1252 and miR-3202 against STAT1 and STAT2 will downregulate M1-specific response.

Suppressor of cytokine signaling proteins has been shown to play a key role in M1 and M2 macrophage polarization. Expression of SOCS is highly context dependent and is actively triggered once the macrophage is activated. SOCS plays a dampening effect on JAK/STAT signaling pathways through its association with phosphorylated tyrosine. SOCS also utilizes proteasome machinery to degrade signaling molecules. High SOCS1 and SOCS2 have been associated with M2 polarization, and high SOCS3 promotes M1 phenotype (53, 54). Downregulation of miR-155-5p against SOCS1 will result in corresponding increase in SOCS1. The down-modulation of miR-132 will increase SOCS2. High SOCS1 and SOCS2 will promote pro-survival M2 phenotype. At the same time, MTb has the capacity to increase miR-455 expression which possesses conserved complementarity against SOCS3 and thus, prevents the cell from generating M1-specific response.

As IFN-γ and TNF-α are associated with M1 macrophages, IL-4 and IL-10 are known to be associated with M2 macrophages (10). SP1 and STAT6 both play an important role in the outcome of the M2 phenotype. STAT6 plays its part in IL-4-dependent cell signaling (55). SP1 has been shown to bind to IL10 motif, thus regulating its expression which is critical for M2 cell (30). IL-10 promotes anti-inflammatory genes and is associated with a dampening of the immune response. Our computation study predicted the affinity of miR-135a and miR-135b to these mRNA. MTb-dependent downregulation of this microRNA will be advantageous to MTb as it greatly enhances the formation of M2 phenotype.

KLF4 is also essential for M2 as it is highly expressed in M2 macrophages but is significantly reduced in M1 macrophages (33). It binds to STAT6 and sequesters co-activators required for NF-κB. As it is an important transcription factor which expresses an inflammatory gene, its unavailability shifts the macrophage toward M2 path. Our study found that miR-34c and miR-449 have conserved targets against KLF4. MTb-dependent upregulation of KLF4 through negative modulation by these microRNAs might lead to M2 phenotype.

PA, RP, PS, and VP conceived and designed the experiments, contributed reagents/materials/analysis tools, and wrote the paper. PA and RP performed the experiments. PA, RP, and VP analyzed the data.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.