Nontuberculous Mycobacteria, Macrophages, and Host Innate Immune Response

ABSTRACT

Although nontuberculous mycobacteria (NTM) are considered opportunistic infections, incidence and prevalence of NTM infection are increasing worldwide becoming a major public health threat. Innate immunity plays an essential role in mediating the initial host response against these intracellular bacteria. Specifically, macrophages phagocytose and eliminate NTM and act as antigen-presenting cells, which trigger downstream activation of cellular and humoral adaptive immune responses. Identification of macrophage receptors, mycobacterial ligands, phagosome maturation, autophagy/necrosis, and escape mechanisms are important components of this immunity network. The role of the macrophage in mycobacterial disease has mainly been studied in tuberculosis (TB), but limited information exists on its role in NTM. In this review, we focus on NTM immunity, the role of macrophages, and host interaction in NTM infection.

INTRODUCTION

Nontuberculous mycobacteria (NTM) include all bacteria of the Mycobacterium genus with the exception of Mycobacterium tuberculosis complex and Mycobacterium Leprae, which are classified separately (1). NTM are ubiquitous in the environment, including water and soil, tap water, and in-house pipelines (2). The American Thoracic Society (ATS) considers four distinct clinical presentations of NTM. Syndromes include chronic pulmonary disease, disseminated disease, lymphatic disease, and skin/soft tissue/bone disease with chronic pulmonary disease being the most prevalent (1). Despite the fact that NTM are ubiquitous in the environment and, of course, that contact with them is inevitable, NTM infection occurs quite rarely. Immune defense against NTM is often appropriate for preventing disease. Therefore, patients who develop NTM disease have predisposing factors that make them vulnerable to the development of NTM disease after infective exposure (3).

Mycobacterium avium complex (MAC) is a subtype of NTM, which is responsible for the majority of pulmonary isolates and disease worldwide (4). MAC consists of a growing number of species, with the most important human pathogens as follows: Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium chimaera (5). Other important NTM that cause infection in humans are Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium abscessus complex bacteria (Mycobacterium abscessus, Mycobacterium massiliense, and Mycobacterium bolletii), Mycobacterium chelonae, and Mycobacterium fortuitum (6), of which MAC and M. kansasii are slow-growing mycobacteria (SGM) (3).

Mycobacterium celatum is also slow growing, with biochemical and morphologic characteristics resembling those of M. avium, M. intracellulare, and Mycobacterium xenopi; it could be responsible for pulmonary and disseminated infections in patients with AIDS (7). Mycobacterium gordonae (tap water bacillus) is ubiquitous in the environment and is considered an SGM with some reports of it causing disseminated disease with pulmonary and hepatic involvement (8). M. marinum causes a tuberculosis-like illness in fish and can infect humans with exposed skin. M. marinum is the closest genetic relative of the M. tuberculosis complex of the NTM (9).

Mycobacterium abscessus complex is a group of emerging pulmonary pathogens, rapid-growing mycobacteria (RGM), responsible for a wide spectrum of infections (from skin and soft tissue diseases to central nervous system infections), and such clinical presentations are multidrug resistant and difficult to treat (10). M. abscessus, with two variants of rough (R) and smooth (S) (transition capacity from a smooth [S] morphotype with cell surface-associated glycopeptidolipids [GPL] to a rough (R) morphotype lacking GPL, in which R is a hypervirulence phenotype, is frequently associated with severe infections in cystic fibrosis [CF] patients versus the S morphotype) (11, 12), is a major problem in patients with CF and is becoming an alarm for most CF centers worldwide considering person to person transmission (13).

Structural pulmonary disorders like bronchiectasis and chronic obstructive pulmonary disease (COPD) predispose patients to the development of pulmonary NTM disease (14). In the prospective cohort demonstrating heightened susceptibility to NTM in COPD patients, 22% of individuals with a diagnosed COPD exacerbation had positive cultures for NTM (15). Cystic fibrosis (CF), which is often accompanied by bronchiectasis, is strongly associated with pulmonary NTM disease (prevalence rates vary from 6.6% to 13.7%). Prevalence rates trend up to 50% in patients over the age of 40 (16–18). In fact, the cystic fibrosis transmembrane conductance regulator (CFTR) mutation is found in 36 to 50% of NTM disease (19).

Like structural pulmonary disease, a compromised immune response can predispose patients to the development of NTM disease. The HIV pandemic highlighted disseminated infection with Mycobacterium avium-intracellulare complex (MAC) as an opportunistic infection in the last few years (20). There are some reports of disseminated NTM in non-HIV patients in Thailand (21) and the United States (22). In a subgroup of NTM patients, no risk factor could be detected. However, noncategorized primary or secondary immunodeficiencies might contribute to the development of NTM in these groups (3).

Mycobacteria are phagocyted by macrophages and other antigen presenting cells (APCs), which secrete interleukin-12 (IL-12) that upregulates gamma interferon (INF-γ) through T-cell stimulation (23). IFN-γ then further activates macrophages and neutrophils to kill intracellular pathogens like mycobacteria. Neutrophils express 1,000 receptors, which bind IFN-γ and induce downstream gene expression of inflammatory cytokines through the Jak-Stat pathway (24). This positive feedback loop between IL-12 and INF-γ plays a crucial role in host immunity against mycobacteria. Disseminated NTM is a definitive result of innate or acquired immunity disorder involving the IL-12/INF-γ pathway, which consequently affects macrophage function (25). In this review, we summarize the current knowledge about macrophage and host response against NTM, focusing on macrophage surface receptors, phagosome maturation, and apoptosis. Although most of our experience is according to M. tuberculosis studies, we have discussed all available data on NTM-based studies. As the incidence and prevalence of lung disease caused by NTM are increasing worldwide (3), this could be an important reason to ongoing research about NTM and host interaction.

HOST INTERACTION AND NTM

NTM present different pathogen-associated molecular patterns (PAMPs), which are recognized by pathogen-recognition receptors (PRRs) at the plasma membrane of immune cells, such as macrophages (26, 27). Mycobacteria are surrounded by a complex cell wall with a thin peptidoglycan layer that protects the cell membrane. An arabinogalactan layer with a dense layer of long-chain beta-hydroxy fatty acids, called mycolic acids, protects mycobacteria from chemical damage, antibiotics, and dehydration (28, 29). The most important PAMPS in mycobacteria are lipomannan (LM), lipoarabinomannan (LAM), mannosylated LAM (ManLAM), trehalose dimycolate (TDM), and hydrophilic PI-mannosides (PIMs) (28–30). Glycopeptidolipids (GPLs), including highly antigenic typeable serovar-specific (ssGPLs) and nonspecific GPLs (nsGPLs are absent in M. tuberculosis), play a critical role in the biology of NTM (31, 32).

SURFACE RECOGNITION RECEPTORS

Extracellular PRRs are the first receptors of phagocytes and APCs to recognize PAMPs. Opsonic or nonopsonic phagocytosis depends on whether or not the microbe is coated with soluble PRRs like complement and antibodies (29). Opsonic receptors include Fc receptors, integrins, and complement receptors (CR) that recognize soluble PRRs as complement and antibodies (33). Although humoral immunity is generally considered not protective in mycobacterial infection, there is some evidence that at least opsonization in M. avium subsp. paratuberculosis induces uptake and killing in the activated macrophage (34).

There are great spectrums of the nonopsonic receptor that recognize PAMPs (Table 1). These receptors are displayed on specific phagocytic cells, including two major districts of C-type lectin receptors (Dectin-1 and −2, macrophage-inducible C-type lectin [Mincle], macrophage C-type lectin [MCL], mannose receptor [MR]) and scavenger receptors (CD36, macrophage receptor with collagenous structure [MARCO], SR-A, apoptosis inhibitor of macrophages [AIM]) (35). The other main receptors that not only facilitate uptake of mycobacteria but also initiate an intracellular signaling cascade are Toll-like receptors (TLRs), which are the link between adaptive and innate immune response (36).

TABLE 1.

Relevant pattern recognition receptors, cellular localization, and mycobacterial ligand

PRRs	Cellular localization	Mycobacterial ligand	Reference(s)
C-type lectins
Dectin-1	Plasma membrane	Uncharacterized	23, 27
Dectin-2	Plasma membrane	ManLAM	28
Mincle	Plasma membrane	TDM	31, 32
MCL	Plasma membrane	TDM	32, 35, 36
MR	Plasma membrane	ManLAM, higher PIMs, LM	37
Scavenger receptors
MARCO	Plasma membrane	TDM	40, 41
SR-A	Plasma membrane	TDM	40
CD36	Plasma membrane	ManLAM	40
Toll-like receptors
TLR2	Plasma membrane	LAM, LM, PIM, 19- and 24-kDa lipoprotein of M. tuberculosis	52–55
TLR4	Plasma/endosomal membrane	Multiple HSP, M. tuberculosis 50S ribosomal, Cpn 60, 38-kDa glycoprotein of M. tuberculosis	62–64, 66, 67
TLR9	Endosomal membrane	CpG in bacterial DNA	74

C-type lectins receptors.

Dectin-1 (dendritic cell-associated C-type lectin) signaling and Dectin-1/TLR 2 induce proinflammatory cytokine secretion in macrophages with M. abscessus infection (Fig. 1) (37). Dectin-1, MR (mannose receptor), and dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) are the most important transmembrane C-type lectins receptor in the fight against M. tuberculosis (38), although the mycobacterial ligand of Dectin-1 is not accurately clarified (35, 39). Dectin-2 recognizes mycobacterial mannosylated lipoarabinomannan (ManLAM), which triggers intracellular signaling cascades with effects on cellular and immunological processes (40). Dectin-2 recognizes slow-growing mannose-capped strains, such as M. intracellulare and M. gordonae (Man-LAM) but not M. abscessus and Mycobacterium smegmatis, which lack mannose capping. Dectin-2 deficiency results in enhanced pathology in mice infected with nontuberculous M. avium, though the bacterial burden did not significantly change after 3 weeks (41). This is in contrast with Dectin-1-deficient mice, which are resistant to M. tuberculosis infection, similar to wild-type mice, with slightly reduced lung bacterial burdens (42).

FIG 1.

Phagocytosis. Pattern recognition receptors (PRRs) are shown in two main districts, C-type lectin receptors (CLR), such as Destin-1 and Dectin-2, and TLRs. MyD88 is a key responsible factor for the downstream signaling cascade of TLRs, which leads to nuclear factor kappa B (NF-κB) activation and ends in the production of inflammatory cytokines (29, 56). Mycobacterial phagocytosis and phagosome maturation through the accumulation of v-ATPase end in the formation of the phagolysosome and mycobacterial killing (29). Macrophage autophagy (autophagolysosome formation) is also an intracellular mycobacterial killing mechanism.

TDM, a well-known mycobacterial cell wall glycolipid, is the major mycobacterial ligand for macrophage-inducible C-type lectin (Mincle) (43, 44). Mincle and Dectin-2 (Fig. 1) both require the adapter protein FcRγ for Syk activation (45). There is not much agreement about Mincle capacity in mycobacterial infection control. A study by Lee et al. demonstrated that Mincle deficiency induces bacterial lung burden in M. tuberculosis-infected mice (46), while another study implies the same immune response with the wild type (WT), including macrophage effector mechanism, lung bacterial burden, and T-helper cells (47). TDM is also a mycobacterial ligand for MCL (macrophage C-type lectin), which is an endocytic receptor. Downstream signaling of this receptor induces various intracellular responses, including NF-κβ activation and proinflammatory cytokine production (Fig. 1). MCL receptor interaction with TDM ligand also induces phagocytosis, and DC maturation (48, 49). Mannose receptor (MR) recognizes a number of mycobacterial ligands, including ManLAM, higher PIMs, LM, and other mannosylated proteins. It is predominately expressed on alveolar macrophages (AMs), and its interaction with ManLAM induces production of anti-inflammatory cytokines (50). Using quantitative electron and confocal microscopy, it has been shown that M. tuberculosis mannose-capped lipoarabinomannan (ManLAM) blocks phagosome maturation with MR interaction (51). ManLAM presents only in some species of mycobacteria like M. tuberculosis, M. bovis, M. leprae, and M. avium. Its ligand interaction has immunosuppressive effects by stimulating IL-10 production, which ultimately inhibits the production of IL-12. ManLAM also modulates M. tuberculosis-induced macrophage apoptosis, which allows the bacteria to survive and multiply within macrophages (52).

Scavenger receptor.

Scavenger receptors (SRs) are the other surface receptors in monocytes-macrophages that are involved in recognizing mycobacterial ligands. The SR family includes the following two known members in class A, which are expressed on the lung macrophages and DCs: macrophage receptor with collagenous structure (MARCO) and class A SR (SR-A) (53). MARCO is effective in recognition of TDM (54). Polymorphism in MARCO has been associated with susceptibility to M. tuberculosis in the Gambian (55) and Chinese Han populations (56). SR-A expression is induced in foamy macrophages chronically infected with M. tuberculosis with low levels of expression in AMs (57). Also, SR-A-deficient mice show significantly better survival than the WT to aerosol M. tuberculosis infection (57). Furthermore, mice double deficient for SR-A and CD36, demonstrate early impairment in bacterial burden control, but there is no difference from the WT in survival (58). This issue also explains the decrease in granulomatous formation and initial bacterial expansion after primary infection in this experience, similar to CD36-deficient mice.

CD36, a member of class B SRs, is broadly expressed on monocyte-macrophage and dendritic cells (DCs) (38). CD36 recognizes mycobacterial lipoglycans, such as ManLAM and LM; in vitro treatment of ManLAM increase tumor necrosis factor alpha (TNF-α) expression in lipopolysaccharide (LPS)-induced macrophages (59). Further, CD36-deficient mice are less sensitive to Mycobacterium bovis BCG infection, while in vitro study demonstrated increased CD36^−/− macrophages intracellular killing of M. tuberculosis HR37rv and M. marinum. The mechanism for why CD36-deficient mice are more protected against infection is not clear. Protection cannot be attributed to phagocyte uptake, macrophage apoptosis, or TNF-α or IL-10 production. Indeed, CD36 deficiency causes resistance to mycobacterial infection, but this deficiency was accompanied by a decrease in granulomas in the target tissue (liver and spleen) (40). In contrast, there is another study showing that CD36 gene expression was downregulated with M. marinum infection in zebrafish, while CD36^−/− in zebrafish larva showed more bacterial burden (41).

Apoptosis inhibitor of macrophages (AIM) makes a relevant contribution to the macrophage autophagy mechanisms that lead to intracellular mycobacterial killing (60). In a study by Sanjurjo et al., THP-1 macrophages were infected by the H37Rv strain. These macrophages showed that stable expression of AIM leads to increased macrophage viability and decreased bacterial load upon infection. These data suggest that AIM may play a protective role in M. tuberculosis infection by the induction of autophagy (60). However, a mycobacterial ligand of the AIM scavenger receptor is not specifically demonstrated (61).

Toll-like receptors.

TLRs recognize a wide variety of PAMPs promote uptake and signaling in phagocytosis (33). In humans, 10 TLRs have been evaluated, which activate numerous signaling pathways using different Toll-IL-1 receptor (TIR) domains consisting of adaptor molecules, such as MyD88, TRIF, TIRAP, and TRAM (29). TLR1, TLR2, TLR4, TLR6, and TLR9 have been reported in the recognition of mycobacterial cell wall glycolipids or mycobacterial cell wall-associated components (62–64). TLR2, 4, and 9 and their adaptor molecule MyD88 play the most prominent roles in innate immune response against tuberculosis (Fig. 1) (56).

TLR2, in synergy with other surface receptors, enhances ligand delivery or recognition (“coreceptors” and “accessory receptors”). The tlr2-mediated response to M. tuberculosis infection involves multiple actions, such as promoting DC maturation; promoting Th1, Th2, and Th17 type immune response; and regulating macrophage activation (63). TLR2 has also been described as the major receptor for mycobacterial ligand, including LAM, LM, and PIM, as well as the 19- and 24-kDa lipoprotein of M. tuberculosis (65–68). TLR2 engagement and activation by M. abscessus was confirmed by experiments with both human and mouse cells, similar to what has been shown for M. avium (69). However, nsGPL surface molecules of M. abscessus S and M. avium S have been shown to prevent TLR2 signaling in respiratory epithelial cells (70, 71). Indeed, there is some evidence that GPL surface molecules in the M. abscessus cell wall mask underlying phosphatidyl-myo-inositol mannosides, which are responsible for stimulating the innate immune response (TLR2), thereby facilitating colonization of this bacteria (72). This colonization in the lung airways is an important step before the development of invasive lung disease by most of the NTM (1). Nevertheless, GPLs demonstrate a wide range of immune modulation from downregulating (Th1-type responses [73]) to induce the release of various proinflammatory mediators (prostaglandins, leukotrienes, IL-1, IL-6, and TNF-α) by ssGPLs (73–75) through MyD88 and TLR2 dependence (75).

MyD88- and TLR2-deficient macrophages showed profound defects in IL-6, TNF, and IL-12p40 responses to M. avium stimulation in vitro (76). It has been shown that mice lacking TLR2 were more susceptible to M. avium infection (77). There is a study that demonstrates an association between TLR gene mutations and increased susceptibility to M. avium subsp. paratuberculosis infection followed by measurement of cytokine expression (IL-4, IL-8, IL-10, IL-12, and IFN-γ) in mutant and wild-type monocyte-derived dendritic cells that was confirmed after inducing M. avium subsp. paratuberculosis or LPS cells (78). It was shown in a Korean population that the GT repeat microsatellite polymorphisms in intron II of the human TLR2 gene were associated with the development of NTM lung disease, especially MAC lung disease (79).

TLR4 is well studied for its recognition of lipopolysaccharide (LPS) as a homodimeric complex with myeloid differentiation 2 (MD2) proteins from Gram-negative bacteria (80). TLR4 is known to recognize cell wall lipids, glycoproteins, and secreted proteins. Other mycobacterial proteins signal through TLR4, including multiple heat shock proteins (HSP) (81–83), an M. tuberculosis 50S ribosomal protein Rv0652 (84), chaperonin (Cpn) 60 proteins (85), and the 38-kDa glycoprotein of M. tuberculosis H37Rv (86). Although several molecules can activate TLR4 in vitro, the actual ligands during mycobacterial infection still remain unknown (36). The M. avium subsp. paratuberculosis MAP1305 protein induces DC maturation and the production of proinflammatory cytokines (IL-6, TNF-α, and IL-1β) through direct binding with TLR4 (87). Mycobacterium scrofulaceum recognition is mediated through TLR4 and Raf-1 signal pathway-dependent DC-SIGN. M. scrofulaceum causes cervical lymphadenitis and lung infections in children as well as disseminated disease in immunosuppressed individuals (88). TLR4 signaling is involved in the maturation and activation of DCs stimulated by the M. abscessus MAB2560 protein (87). TLR4 agonist adjuvant, glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE), promotes potent polyfunctional TH1 responses in vivo characterized by IFN-γ-, TNF-α-, and IL-2-producing cells. GLA-SE is currently being considered in numerous vaccines trials, such as tuberculosis (TB), leishmaniasis, hookworm, malaria, and HIV (89), to explain how stimulation of TLR4 induces immunity.

Myeloid differentiation factor 88 (MyD88) is the adapter molecule for signal transduction by all of the known TLRs (90). In a study by Feng et al., MyD88^−/− mice failed to control acute and chronic M. avium growth. Infected TLR2^−/− but not TLR4 mice also showed increased susceptibility. Massive destruction is demonstrated in the histopathological examination of MyD88^−/− mice but not in WT, TLR2^−/−, or TLR4^−/− mice. These findings indicate that resistance to mycobacterial infection is regulated by multiple MyD88-dependent signals (76).

TLR9 is located on the endosomal membrane and recognizes undermethylated CG motifs (CpG) in bacterial DNA, like that of M. tuberculosis; TLR9-deficient mice are more susceptible to a low-dose aerosol infection than WT (91). There are a few reports in humans that TLR9 polymorphisms have been associated with increased susceptibility to M. tuberculosis (92, 93). These studies demonstrated that TLR9 is required for efficient control of Mycobacterium infection. However, the role of TLR9 is controversial. Chen et al. (94) shows that inhibition of TLR9 prevents M. tuberculosis-induced apoptosis in mice, but Hölscher et al. (95) demonstrates that MyD88, but not TLR2 or TLR9, is necessary for the induction of protective immune responses in macrophage effector mechanism manner. Overall, the role of the MyD88 adaptor and the collaborative effect of TLRs should be considered in obtaining an appropriate conclusion.

PHAGOSOME MATURATION AND MYCOBACTERIUM

Mycobacteria are highly specialized facultative intracellular pathogens that infect macrophages. Briefly, formed phagosomes interact with different types of endosomes to gradually mature from an early phagosome into a late phagosome and then into phagolysosomes, which leads to degradation (Fig. 1) (29). The phagolysosome is an acidic environment with a high enzymatic content (lipases, hydrolases, proteases) and some antimicrobial molecules (e.g., defensins) (96). Essential mechanisms for macrophage maturation include phagosome acidification, reactive oxygen and nitrogen species formation, antimicrobial peptides/proteins (AMPs) formation, and degradative enzymes, such as cathepsins formation (29). Mycobacteria could arrest phagosome maturation to enhance their survival and replication or lead to phagosomal escape. This is not specific for mycobacteria, as even all viruses, some other bacteria (e.g., Listeria monocytogenes and Salmonella typhi), and protozoa can survive inside phagolysosomes (97). Mycobacterial cell wall lipids such as LAM in M. avium (98) and M. tuberculosis (99), ManLAM (100), the phenolic glycolipid phenolphthiocerol diester (PGL-1) (101), the isoprenoid edaxadiene (102), and TDM (103) have all been demonstrated to modulate phagosome maturation.

Acidification is the hallmark of phagosome maturation, which leads to the mature phagolysosome pH of ∼5.0 or less. This intensive decrease in pH is catalyzed by the vacuolar ATPase, wherein an H⁺ pump extrudes H⁺ into the phagosome lumen from the cytosol (104–106). The proton pump v-ATPase is involved in luminal acidification and activates lysosomal hydrolases and cathepsins, which degrade phagolysosomal content (26). M. tuberculosis maintains an almost neutral pH by excluding v-ATPases (107) and preventing with lysosomes (108) while still accessing essential nutrients like iron and fats (109).

Nitrogen oxide (NO) and reactive oxygen species (ROS) produce a highly reactive substance that could destroy microbial structures, such as cell wall, DNA, and tyrosine residues, by oxidation. NO can directly target the iron-sulfur clusters of bacterial enzymes (29). Two pathways activated by IFN-γ that are capable of killing M. tuberculosis are nitric oxide production and phagosome-lysosome fusion, concluding acidification of bacterial phagosome (110), but the role of NO is controversial. Additionally, it does not seem to play an important role in the M. avium infection (111). Further, the GPL surface molecules of M. avium complex (112) and M. abscessus (12) can delay phagosome-lysosome fusion following its ligation to the mannose receptor. However, M. abscessus, M. smegmatis, and M. chelonae all express GPL surface molecules, but only M. abscessus and M. chelonae can survive inside the macrophages (113). There is some evidence that NO brings up the transformation of macrophages into multinucleated giant cells, which are relatively permissive for mycobacterial persistence (114). Also, knockout NOS2 gene mice that cannot produce NO were more resistant than WT to M. avium following stimulation by IFN-γ and TNF-α and showed increased survival of CD4⁺ T cells (115). M. tuberculosis shows inherent resistance to ROS by the thick cell wall containing LAM and mycolic acids or phenolic glycolipid I (PGL-1), which acts as potent scavengers of oxygen radicals (116). Lsr2, a DNA binding protein, could directly protect M. tuberculosis DNA from ROS (117). A study by Helguera-Repetto et al. revealed that viable slow-growing M. celatum persisted inside macrophages without inducing reactive oxygen species (ROS), which indicates a possible immune evasion strategy. In contrast, rapid-growing mycobacteria induced high levels of ROS, which destroyed cell structure (118). This also happened for other SGM like MAC (119) and M. tuberculosis, which could actively block ROS production and persist inside macrophages (107, 120, 121). M. abscessus is a rapid-growing mycobacterium that shows both SGM and RGM characteristics. Roux et al.’s study demonstrates how M. abscessus S variant is able to restrict the intraphagosomal acidification and prevent processing into phagolysosomes. It also induces less apoptosis and autophagy than R variants. By contrast, the R variant has a strong tendency to duplicate in phagocytic cups instead, which will process into phagolysosomes, but cannot degrade the bacilli. Furthermore, the macrophage tolerates apoptosis, and it causes the R variant to reach the extracellular environment with high replication capacity. This replication in the macrophage was not affected by functional cystic fibrosis conductance transmembrane regulator (CFTR) defects (12).

Understanding the molecular mechanism behind this survival in macrophages, the M. abscessus eis2 gene is essential. Comparing the transcriptomic activities of M. abscessus in the amoeba and the mouse macrophage model, Laencina et al. demonstrated how the upregulated genes in the amoeba end in proteins that enable M. abscessus to resist environmental stresses and induce defense mechanisms, such as intracellular survival, phagosome acidification and phagosomal escape, H₂O₂ degradation, cell death, autophagy, metabolic adaptations, and controlling ROS production (122).

The antimicrobial peptide (AMP) cathelicidin (LL-37) binds and disrupts the negatively charged bacterial membrane by forming pores in a variety of Gram-negative and Gram-positive bacteria (123–125). LL-37 has also been shown to kill M. tuberculosis (124, 126). Despite its broad-spectrum of activity, several species of NTM, such as M. avium subsp. hominissuis and M. abscessus, show wide resistance to the antimicrobial peptide cathelicidin (LL-37), which is likely due to LL-37 inactivation by lipid component(s) of the NTM cell envelope (127). Cathepsins are proteolytic enzymes involved in several cell functions, such as antigen processing and presentation (128), the lysosomal killing of Mycobacterium (129), and macrophage apoptosis (130, 131). The Rv3364c protein secreted by M. tuberculosis could bind to the serine protease cathepsin G and inhibit its enzymatic activity and the downstream activation of caspase-1-dependent apoptosis (132). Also, M. avium-containing vacuoles retain cathepsin D in an immature form, concluding the residing of bacteria (133).

Macrophages can induce cell death programs to prevent further microbial intracellular replication and increase free exposure. These apoptotic cells and the contained pathogen reside within their immature phagosomes to be cleared by the other uninfected macrophages through efferocytosis (134). Virulent M. tuberculosis inhibits apoptosis and triggers necrosis of host macrophages to evade innate immunity (135). This can be a double-edged sword; some mycobacteria can evade macrophage response and continue to reside in the other cells. There is some evidence that M. avium uses apoptotic macrophages as a tool for cell-to-cell spreading and survival in the host (136). Whang et al. demonstrates that highly virulent clinical M. abscessus strains significantly induce apoptosis, more so than the smooth (S) variant, as a result of GPL-mediated inhibition of macrophage apoptosis (137). Moreover, another study demonstrates that the release of M. abscessus rough (R) variant from apoptotic macrophages initiated the formation of cords that seem too large to be phagocytized by macrophages or neutrophils. These cords cause abscess formation leading to rapid zebrafish larval death (13).

CONCLUSION

The host immune response to mycobacteria involves a variety of different host cell types to successfully protect against these intracellular pathogens. Surface recognition receptors play an important role in the recognition and imitation of the immune response through specific or nonspecific mycobacterial cell components. Mycobacterial species demonstrate numerous mechanisms that enable them to survive inside of immune cells. Phagosomal maturation and apoptosis are two important mechanisms that could be manipulated by these pathogens through intracellular survival. A better understanding of various mycobacterial cell wall components, innate immune processes, and evasion mechanisms of NTM species could empower us against these bacterial infections.