Abstract
Autophagy is a cellular stress-induced intracellular process, through which damaged cellular components are decomposed via lysosomal degradation. This process plays important roles in host innate immunity, particularly the elimination of intracellular pathogens inside host macrophages. A more detailed understanding of the roles of autophagic events in the effective manifestation of macrophagic antimycobacterial activity is needed. Furthermore, the effects of medicinal plants on macrophagic autophagy response to mycobacterial infection need to be clarified. We herein examined the significance of autophagic events in the manifestation of host immunity during mycobacterial infection, by performing a literature search using PubMed. Recent studies demonstrated that autophagy up-regulated macrophage functions related to the intracellular killing of mycobacteria, even when pathogens were residing within the cytoplasm of macrophages. The majority of medicinal plants potentiated macrophagic autophagy, thereby enhancing their antimycobacterial functions. In contrast, most medicinal plants down-regulate the development and activation of the Th17 cell population, which reduces macrophage antimycobacterial activity. These opposing effects of medicinal plants on macrophage autophagy (enhancement) and Th17 cell functions (inhibition) may provide a plausible explanation for the clinical observation of their modest efficacy in the treatment of mycobacterial infections.
Introduction
Mycobacterial pathogens, including Mycobacterium tuberculosis (MTB) and Mycobacterium avium-intracellulare complex (MAC), cause intractable diseases in humans [1]. Tuberculosis (TB) is one of the most important public health issues worldwide, with high morbidity and mortality rates, particularly in developing countries. One-third of the world's population is infected with MTB. The World Health Organization estimates that approximately eight to ten million new TB cases occur annually worldwide and the incidence of multidrug-resistant TB is currently increasing [1, 2]. In addition, very refractory infections caused by MAC are increasing globally.
Cellular immunity based on type 1 helper T cells (Th1 cells), macrophages, and natural killer cells (NK cells) plays crucial roles in host defense mechanisms against virulent mycobacterial pathogens [3]. Figure 1 shows the cytokine network in hosts infected with mycobacterial organisms. This network involves very complex events mediated by the following immunocompetent cells [3]: (1) the activation/maturation of Th1 cells and NK cells in response to stimulatory signals due to proinflammatory cytokines, including interleukin (IL)-12, tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ) and, to a lesser extent, IL-1, IL-7, IL-15, IL-18, IL-23, and IL-27, which are mainly produced by macrophages and dendritic cells stimulated by specific components of mycobacterial organisms [4, 5]; (2) the activation of macrophages in response to activating signals by major proinflammatory cytokines, such as IFN-γ, TNF-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by Th1 cells, NK cells, and macrophages [4, 5]; and (3) the suppression of macrophage functions by cytokines, including IL-10, IL-13, and transforming growth factor-beta (TGF-β) [3, 4, 6]. The immunological events mediated by these cytokines are important for the establishment of antimycobacterial immunity and the expression of host resistance in addition to immune unresponsiveness in patients in the advanced stages of TB, thereby enabling us to consider the biological significance of targets of anti-TB drugs in host immune cells and MTB organisms.
Cytokine network in hosts during the course of mycobacterial infection
Citation: European Journal of Microbiology and Immunology 14, 1; 10.1556/1886.2023.00062
Host macrophages play central roles in eliminating invaded mycobacterial pathogens using various types of antimicrobial effector molecules, including reactive oxygen intermediates (ROI), reactive nitrogen intermediates (RNI), long chain unsaturated free fatty acids (FFA), and antimicrobial peptides/proteins, such as cathelicidin and cathepsin G [7–10]. We previously reported that the intracellular growth of MAC in murine peritoneal macrophages was accelerated by scavengers for ROI or RNI and inhibitors of nitric oxide synthase (N(G)-monomethyl-l-arginine) or phospholipase A2 (arachidonyl trifluoromethylketone), indicating the roles of ROI, RNI, and FFA in macrophage anti-MAC functions [8, 9]. FFA generated by phospholipase A2 in macrophage phagosomal vesicles are also important effector molecules against invading mycobacteria [11]. We demonstrated that adenosine 5′-triphosphate (ATP) also acted as an antimycobacterial effector molecule through its iron-chelating effect [12].
Autophagy is a cellular stress-induced intracellular process, through which damaged cellular components and organelles are decomposed by lysosomal degradation (Fig. 2) [13–15]. This process plays important roles in host innate immunity, particularly the elimination of intracellular pathogens inside host macrophages. The induction of autophagy involves three important events assumed by phosphoinositide 3-kinase complex 3 (PI3K), Unc-51-like kinase 1 complex, and autophagy-related (ATG) protein complexes (Fig. 2) [16]. There are three types of autophagy: microautophagy, macroautophagy, and chaperone-mediated autophagy. Macroautophagy is the most common form, where double membrane vesicles called phagophores elongate and form autophagosomes to isolate dysfunctional cell structures and waste [17]. A cytosolic form of the autophagosome biomarker protein light chain 3-I (LC3-I) is lipidated to its membrane-bound form light chain 3-II (LC3-II). The cellular processes of the formation and elongation of LC3-II-bound phagophores and subsequent generation of autophagosomes require the collaboration of autophagy-related proteins, such as the ATG12/ATG5/ATG16L complex. Thereafter, autophagosomes are transported to lysosomes for degradation with the aid of the p62 protein as the cargo receptor [18]. Thus, autophagy, a lysosomal degradation pathway that contributes to the maintenance of intracellular homeostasis, plays important roles in both innate and adaptive immunity [13, 15, 16, 19, 20]. Autophagy was initially shown to act as a stress response that is critical for cell survival during nutrient starvation. It also eliminates damaged organelles and decomposes misfolded and damaged proteins. It therefore plays important roles in the maintenance of intracellular homeostasis [19, 21]. Previous studies demonstrated the innate defense role of autophagy against intracellular bacterial pathogens [15, 19, 20, 22–25]. In this context, autophagy promotes the establishment and manifestation of host-acquired immunity specific to MTB antigens, stimulating crosstalk between innate and adaptive immune responses in MTB-infected hosts by enhancing the presentation of antigens [15, 26].
Molecular mechanisms underlying the autophagic response to mycobacterial infections and pharmacological effects of active chemical components of some medicinal plants
Citation: European Journal of Microbiology and Immunology 14, 1; 10.1556/1886.2023.00062
In this review, we discuss the mechanisms underlying autophagic responses that affect the cellular functions of macrophages against mycobacterial pathogens. Moreover, we describe the effects of various medicinal plants and their active chemical components on macrophage autophagic responses to mycobacterial infection. We also indicate the meaning of these effects on the efficacy of medicinal plants to promote host defense mechanisms when administered to hosts with MAC infection.
Methods
In order to search and collect many reports indicating the most recent findings, on the roles of the autophagic responses of host macrophages to mycobacterial organisms, we performed a literature search mainly using PubMed, which comprises more than 36 million citations for biomedical literature from MEDLINE, life science journals, and online books. We searched the literature, including original papers and review articles, in terms of molecular mechanisms, particularly the signal transduction pathways of autophagic events in the cytoplasm of macrophages, the biological effects of the microRNA system upon autophagy, and the molecular mechanisms of bacterial resistance to cellular autophagic responses in host macrophages. The literature search was performed using the following key words: autophagy, signal transduction, LC3, mechanistic target of rapamycin (mTOR), phosphatidylinositol-3 kinase (PI3k), rat sarcoma viral oncogene (Ras), rapidly accelerated fibrosarcoma 1 (Raf1), adenosine monophosphate-activated protein kinase (AMPK), mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), protein kinase B (Akt), activator protein 1 (AP-1), nuclear factor-kappa B (NF-κB), autophagosome, lysosome, phagolysosome, medicinal plants, and so on.
We carefully read more than 200 articles, most of which have been published in international journals with an impact factor of five or higher, for these several years, and summarized the present status of the features of macrophage autophagy in response to mycobacterial infections. In addition, we performed our own experiments regarding host defense mechanisms against mycobacterial infections, based on macrophage antimicrobial functions against mycobacterial organisms residing with macrophages.
Results
Modulatory effects of autophagy on host defense mechanisms against mycobacterial pathogens, particularly host macrophage activity to eliminate mycobacterial organisms residing in phagocytes
- 1.Outline of roles of autophagy in intracellular antimycobacterial effects of macrophages
Pathogenic mycobacterial pathogens, including MTB and MAC, evade an intramacrophage attack by stopping the normal progression of phagosomes into acidic and hydrolytically active compartment through the inhibition of phagosomal maturation, which leads to phagosome-lysosome fusion [27, 28]. Therefore, the induction of autophagy reduces the intramacrophage survival of mycobacteria by reviving lysosomal antimycobacterial activity (Fig. 2) [29]. Among 74 target genes, 44 were found to be responsible for the autophagy-mediated elimination of MTB organisms in macrophages [30]. Therefore, autophagy is considered to confer protection against TB disease by reducing bacterial growth and inflammation [19, 24, 31, 32].
The conditional knockdown of the core autophagy component ATG5 in myeloid cells was previously reported to confer extreme susceptibility to MTB in mice, whereas the depletion of other autophagy factors had no effect [33]. Golovkine et al. recently showed that the knockout of ATG16L1 or ATG7 promoted the growth of MTB and increased host susceptibility in mice, with ATG5-depleted mice being more sensitive than ATG16L1- or ATG7-depleted mice [34]. The authors proposed their concept that autophagy protects host macrophages by partially reducing mycobacterial access to the cytosol [34]. Aylan et al. found that the deletion of ATG7 by CRISPR-Cas9 in macrophages increased the replication of wild-type MTB, but not MTB mutant strains, one with the mutation ΔesxBA and the other with the ΔcpsA mutation [35]. MTB ΔesxBA is a mutant that cannot induce canonical autophagy, while MTB ΔcpsA lacks the ability to block non-canonical autophagy. Furthermore, the deletion of ATG14 increased the replication of both the MTB wild-type and MTB ΔesxBA [35]. The authors described the role of ATG14 in regulating the fusion of phagosomes containing MTB with lysosomes, thereby enabling intracellular bacterial restriction.
- 2.Molecular process of autophagic events in macrophages responding to mycobacterial infection
Several types of molecular circuitry are involved in the regulation of autophagy, including: (1) the Ras/Raf1/mitogen-activated protein kinase kinase 1/2 (MEK1/2)/ERK1/2 cascade, (2) the beclin 1/class III phosphatidylinositol 3-kinase (PtdIns3K) cascade, (3) the mTOR-mediated cascade (class I Pxtdlns3K/Akt/mTORC1 pathway) and (4) the AMPK-mediated pathway [36]. Kim et al. also indicated that sirtuin 3 (SIRT3), a mitochondrial protein deacetylase, coordinated mitochondrial functions and the activation of macroautophagy/autophagy, thereby promoting anti-mycobacterial responses through peroxisome proliferator activated receptor α (PPARA) [37]. SIRT3 deficiency enhanced inflammatory responses and mitochondrial dysfunction, leading to defective host defenses and pathological inflammation during mycobacterial infection. In addition, mitochondrial oxidative stress promoted excessive inflammation induced by MTB infection in SIRT3-knockout macrophages. Therefore, SIRT3 is essential for the enhancement of PPARA, a key regulator of mitochondrial homeostasis and autophagy activation in the context of infection. Collectively, these findings show that SIRT3 plays important roles in orchestrating mitochondrial and autophagic functions to promote antimycobacterial responses.
- 3.Dual effects of host microRNA on the mycobacterial induction of macrophage autophagy
Infection with Mycobacterium bovis BCG was previously shown to induce the up-regulation of a miRNA called mmu-miR-25-3p and down-regulation of dual specificity phosphatase 10 (DUSP10) in RAW264.7 macrophages, which further increased the expression of LC3-II and promoted autophagy [38]. The up-regulated expression of mmu-miR-25-3p subsequently decreased DUSP10 levels and enhanced the phosphorylation of ERK1/2, which, in turn up-regulated the expression of LC3-II, Atg5, Atg7, and beclin1 [38]. Therefore, mmu-miR-25-3p appears to promote the phosphorylation of ERK1/2 by inhibiting the expression of DUSP10, thereby enhancing the BCG-induced autophagy of macrophages [38]. On the other hand, other types of microRNA have been shown to play roles in suppressing the autophagy of macrophages. The up-regulation of miR-423-5p, a host microRNA that is a biomarker of TB, was found to inhibit the maturation of autophagosomes by suppressing autophagosome-lysosome fusion in macrophages, which indicated that host macrophages possess a self-regulatory system for the induction of autophagy in response to mycobacterial pathogens [39]. In any case, accumulated experimental evidence supports the concept that autophagy plays an important role in defenses against MTB in mammals.
- 4.Molecular mechanisms of the resistance of mycobacteria against the autophagic response of macrophages
A previous study reported that different mycobacterial species varied widely in their autophagy-inducing abilities in macrophages [40]. Low virulent species, including Mycobacterium smegmatis and Mycobacterium fortuitum, induce potent autophagy responses, whereas highly virulent species, including MTB, only caused weak autophagic responses in host macrophages [40]. Eukaryotic-type protein kinase G (PknG) in MTB was recently shown to be involved in mycobacterial survival within macrophages, presumably by suppressing the maturation of phagosomes and autophagosomes [41]. Moreover, PknG inhibited the maturation of autophagosomes, which ultimately blocked autophagy flux and enhanced the intracellular survival of pathogens. PknG prevents the activation of AKT serine/threonine kinase, which induces autophagy [41]. PknG also inhibits the maturation of autophagosomes and, thus, block autophagy flux by targeting the host small GTPase, ras-related protein Rab-24 (RAB14) through the blockade of RAB14-GTP [41]. In macrophages and in vivo, PknG promoted MTB intracellular survival by blocking autophagy, which was dependent on RAB14 [41]. In addition, pathogenic mycobacteria, particularly highly virulent organisms, use various strategies other than the PknG protein to evade attack by the autophagic machinery, including the formation of LC3-II-bound double membrane phagophores and the subsequent generation of autolysosomes in host macrophages [19, 27, 42]. Therefore, various intracellular microorganisms, including mycobacteria, are considered to interfere with the autophagic processes of host cells by modulating these autophagy-related molecular cascades.
Effects of some medicinal plants and their active chemical components on macrophage autophagy
- 1.Summary of the present status of the development of new antimycobacterial drugs
The representative mycobacterial infection TB is currently controlled using standard regimens, consisting of the first-line anti-MTB drugs rifampicin or rifabutin, isoniazid, pyrazinamide, and ethambutol or streptomycin. In some cases of multidrug-resistant TB, second-line anti-TB drugs, including levofloxacin, kanamycin, ethionamide, enviomycin, para-amino salicylic acid, and cycloserine, are used [1]. In some cases of intractable multidrug-resistant TB, the newly developed imidazole drugs, delamanid (a nitro-dihydro-imidazooxazole derivative) and bedaquiline (diarylquinoline), are also used [43–45]. On the other hand, MAC infection is more refractory even with standard regimens, involving clarithromycin (azithromycin), rifampicin (rifabutin), ethambutol, and streptomycin [46]. Some MAC organisms are generally very resistant to most antimycobacterial agents, resulting in the failure of clinical treatment using conventional drug regimens. Amikacin was recently approved for the adjunctive treatment of patients with MAC infection resistant to the above standard regimens.
- 2.Perspectives for the development of host-directed therapeutics (HDT) against mycobacteriosis
Strategies to improve the therapeutic efficacy of existing antimicrobial drugs against intractable bacterial infections by combining them with HDT are promising [4, 47–50]. There is increasing interest in adjunctive immunotherapeutic agents that will aid host immunological defenses against bacterial infections, thereby increasing the efficacy of clinical treatments with antimicrobial agents. Notably, HDT may provide a new clinical approach as an adjunctive therapy against refractory bacterial infections [45]. Some HDT augment the immunological potential of hosts, while other HDT down-regulate an excessive immunological response to bacterial pathogens, which occasionally increases the morbidity and mortality rates of hosts [48, 49]. In cases of TB and MAC infection, granuloma-targeted therapy is advantageous as an adjunctive therapy in combination with antimycobacterial drugs [4, 48, 50]. Notably, immunological events mediated by various cytokines are important for the establishment and expression of host anti-TB resistance, as well as immune unresponsiveness encountered during the advanced stages of TB, thereby enabling us to consider the biological significance of adjunctive immunotherapy against mycobacterial infections [51]. Nevertheless, HDT are usually associated with high costs and occasionally cause strong side effects. In addition, most HDT, including autophagy-inducing agents, display only modest efficacy in potentiating host defense mechanisms, partly due to the induction of immunosuppressive cytokines, including IL-10, IL-13, and TGF-β, during their long-term administration [3, 4, 51].
- 3.Chinese herbal medicines and medicinal plants do not exert sufficient immuno-adjunctive effects in in vitro or in vivo tests
Although some clinical trials to treat patients with intractable MAC infection using Chinese traditional herbal medicines, including medicinal plants, reported modest efficacy, the significance and reproducibility of medicinal effects were insufficient [3, 4, 51]. We examined the immuno-potentiating effects of thirty types of Chinese traditional medicines in in vitro and in vivo experiments using MAC-infected mice, and found that most did not effectively up-regulate the anti-MAC antimicrobial activity of host macrophages. In only one case using the Chinese medicine, Mao-Busi-Saisin-To, we observed a modest increase in the anti-MAC activity of host macrophages and weak therapeutic efficacy against MAC infection in mice [3, 52].
We previously examined the effects of various Chinese medicines, including medicinal plants, on the differentiation of type 17 helper T cells (Th17 cells) from type 0 helper T cells (Th0 cells) and the modulatory effects of these natural medicines on cellular immunity based on Th17 cell functions. By performing our own experiments and searching for related literature using PubMed, we noted the following: mycobacterial infections induced in mice caused the expansion of suppressor-type macrophages, which strongly induced the differentiation of Th17 cells from Th0 cells in mice [53]. Most medicinal plants and their active chemical components significantly reduced Th17 cell polarization from Th0 cells and the cellular activity of Th17 cells to eliminate mycobacterial pathogens from host macrophages [53]. These findings indicate that medicinal plants generally suppress the antimycobacterial activity of host macrophages, suggesting their antagonistic effects against the clinical treatment of mycobacterial infections. However, some types of medicinal plants and decoctions consisting of medicinal plants are still known to exhibit modest therapeutic efficacy against mycobacterial infections [3]. This predicament warrants some explanation. The reasons for the following contradictory conditions need to be clarified: most medicinal plants generally do not exhibit immuno-potentiating effects in hosts with mycobacteria infection; however, a few medicinal plants up-regulated the host macrophage function of eliminating infected mycobacteria.
- 4.Effects of some medicinal plants and their active components on autophagic events in macrophages infected with mycobacteria
We herein newly attempted to assess the effects of various medicinal plants and their active chemical components on macrophage autophagy by performing a literature-search using PubMed. The following findings were obtained:
- (1)Curcumin
Curcumin (diferuloylmethane) is the major active component of Curcuma longa, which has traditionally been used as a dietary spice [54]. This agent has numerous implications for medical purposes, particularly in relation to inflammation, neoplasms, anorexia, and dyslipidemia [54, 55]. Many studies indicated the effects of curcumin on cellular autophagy and the curcumin-mediated induction of cellular autophagic responses was based on various molecular mechanisms. For example, curcumin activated autophagy by down-regulating the PI3K/Akt/mTOR signaling pathway [56]. Curcumin is a potent inducer of cellular apoptosis, which is an effector mechanism used by macrophages to kill intracellular MTB organisms. However, low doses of curcumin exerted protective effects against macrophage-mediated apoptosis induced by the 19-kDa lipoprotein of MTB through the regulation of the c-Jun N-terminal kinase (JNK) pathway [57]. Bai et al. also reported that curcumin enhanced the clearance of MTB in the THP-1 human macrophage cell line and in primary human alveolar macrophages [58]. The authors also found that curcumin was an inducer of caspase-3-dependent apoptosis and autophagy. Gupta et al. recently demonstrated that curcumin induced autophagy in uninfected and MTB-infected macrophages based on the conversion of LC3-I to LC3-II and the degradation of p62 [59]. The inhibition of autophagy by the pharmacological inhibitor 3-methyl adenine resulted in the significant inhibition of the intracellular killing activity of curcumin, suggesting the involvement of autophagy in the intracellular clearance of MTB with curcumin [59]. This effect of curcumin was accompanied by increase in the expression of the pro-apoptotic protein Bax. Curcumin mediates these anti-MTB cellular functions, in part, by inhibiting the activation of NF-κB. Ahmad et al. indicated that curcumin up-regulated autophagy in MTB-infected macrophages, resulting in the enhancement of their antigen-presenting functions, costimulatory activity, and production of inflammatory cytokines (TNF-α and IL-12) and other mediators, such as nitric oxide [60]. Due to these effects on macrophage functions, curcumin may potentiate the capacity of BCG vaccine efficacy in hosts.
- (2)Sophora flavescens
The root of the medicinal plant, S. flavescens, contains flavonoids, a major group of bioactive polyphenols in this plant. These compounds exhibit anti-inflammatory and anti-angiogenic activities that may be used to treat allergic asthma and chronic arthritis [61]. It was reported that the flavonoids of S. flavescens suppressed the formation of granuloma in the lungs of mice injected with trehalose dimycolate, one of the major pathogenic components of the cell wall of MTB [62]. Flavonoids of S. flavescens suppressed the production of proinflammatory cytokines (TNF-α and IL-6) and chemokines (CCL5) by M. bovis BCG-stimulated alveolar macrophages and peritoneal macrophages, and this effect was associated with the activation of cellular autophagy by these flavonoids through a signaling pathway consisting of the autophagic proteins LC3 and P62 [63]. S. flavescens flavonoid-induced autophagy may be dependent on lysosomal degradation. In addition, flavonoids were shown to serve as a mediator for autophagy-related gene transcription and autophagy-related protein synthesis in M. bovis BCG-stimulated macrophages [63].
- (3)Larrea tridentata
The medicinal plant L. tridentata (creosote bush) is widely used to treat a number of illnesses, including infertility, rheumatism, arthritis, diabetes, gallbladder and kidney stones, pain, and inflammation [64]. The major active component of this medicinal plant is a lignan called nordihydroguaiaretic acid (NDGA), which selectively inhibits arachidonic acid 5-lipoxygenase activity. This suppresses the synthesis of leukotrienes and prostaglandins, leading to a reduction in inflammatory reactions. A previous study demonstrated that NDGA induced the autophagy of human macrophages through the PI3K/AKT/mTOR signaling pathway, which suppressed the growth of intramacrophagial MTB organisms [65]. In this context, a cochlioquinone B derivative from the medicinal plant, Salvia miltiorrhiza, also induced autophagy by blocking the p21-activated kinase 1 (PAK1)/Akt1/mTOR pathway in mice infected with Pseudomonas aeruginosa, which decreased the expression of PAK1, causing a reduction in the phosphorylation level of the Akt protein [66].
- (4)Garcinia mangostana Linn
The phytochemicals present in the plant G. mangostana Linn (mangosteen) are isoprenylated xanthones, which exhibit the following biological activities: antioxidant, pro-apoptotic, anti-proliferative, antinociceptive, anti-inflammatory, neuroprotective, hypoglycemic, and anti-obesity effects [67]. α-Mangostin, one of the xanthone derivatives produced by this plant, exerts antioxidant, antitumor, anti-inflammatory, antiallergy, antibacterial, and antiviral effects. Moreover, it was shown to enhance autophagic events in target cells, including the expression of LC3-II in autophagosomes [68]. It was reported that α-mangostin induced autophagy in uninfected or MTB-infected THP-1 macrophages 24 h after treatment, and up-regulated the colocalization of autophagic vesicles with MTB, thereby promoting the elimination of organisms residing within host macrophages [65].
- (5)Eurycoma longifolia Jack
Pasakbumin A is isolated from the medicinal plant E. longifolia Jack, which is commonly used to treat diarrhea, fever, malaria, ulcers, and cancer [69]. A recent study reported that pasakbumin A significantly inhibited the intracellular growth of MTB by inducing autophagy via the ERK1/2-mediated signaling pathway in MTB-infected macrophages [70]. Treatment with pasakbumin A and the anti-TB drug rifampicin induced the synergistic suppression of intracellular MTB killing by promoting autophagy as well as TNF-α production through the ERK1/2- and NF-κB-mediated signaling pathways in MTB-infected macrophages.
- (6)Andrographis paniculata
Andrographolide is the major bioactive component of the medicinal plant, A. paniculata, and is considered to be responsible for the majority of the biological effects of this plant. Andrographolide is a diterpene that exhibits many biological activities, including anti-inflammatory, antioxidant, and antineoplastic effects [71]. Clinical studies indicated the potential of andrographolide in treatment for a wide range of diseases, such as osteoarthritis, upper respiratory diseases, and multiple sclerosis [71]. Andrographolide targets distinct molecules within the transcription factors, NF-κB and AP-1, the PI3K/Akt pathway, and the MAPK cascade [71]. The following findings on the anti-inflammatory effects of andrographolide in MTB-infected hosts were recently reported: andrographolide inhibited the production of IL-1β and other inflammatory cytokines [72]. The down-regulation of IL-1β expression reduced the expression of IL-8 and monocyte chemotactic protein 1 in lung epithelial cells co-cultured with MTB-infected macrophages [72]. The inhibition of the NF-κB pathway, but not that of the MAPK signaling pathway, accounts for the anti-inflammatory role of andrographolide. Further studies by the authors revealed that andrographolide activated autophagy to degrade Nod-like receptor (NLR) family pyrin domain containing 3 (NLRP3), which ultimately suppressed the activation of inflammasomes and subsequent production of IL-1β. Andrographolide was also shown to inhibit the Notch1 pathway, thereby down-regulating the phosphorylation of Akt/mTOR and the NF-κB p65 subunit [72].
To summarize these findings, the activation of cellular autophagy by some medicinal plants is generally based on the up-regulation of various cellular processes of autophagy. This induces autophagy via PI3K-, Unc-51-like kinase 1 complex-, and ATG complex-mediated pathways [16], and also the following two types of autophagic processes: xenophagy, which is triggered by mycobacterial components and involves the formation of double membrane autophagosomes [22, 23], and the LC3-associated phagocytosis (LAP) pathway [24]. In the case of MTB, CpsA, an exported virulence factor of the pathogen, blocks LAP by interfering with the recruitment of CYBB/NOX2 to mycobacterial phagosomes [73].
Apart from topics related to macrophages and mycobacterial infections, the following findings are of interest. Law et al. [36] described the potential pharmacological functions of various traditional Chinese herbal medicines via the regulation of autophagy. They listed 39 herbal medicines and their active components, such as Radix sp. (including baicalin, fangchinoline, and licochalcone), Cortex sp. (berberine and magnolol), and Rhizoma sp. (gingerolis and alisol). Most of the herbal medicines examined and their active components (37 of the 39 listed herbal medicines) up-regulated autophagy in various target cells, including cancer cells, podocytes, and glomerular mesangial cells, mainly through the PI3K/Akt/mTOR signaling pathway, AMPK pathways, ERK1/2 cascades, and PTEN-induced putative kinase 1 (PINK1)/Parkin-mediated signaling pathways [36]. Only Radix Ophiopogon japonicus (ophiopogonin) and L. tridentate (lignin) inhibited cellular autophagic activity [36].
Discussion
We herein reviewed the roles of autophagic events in manifesting the elimination of mycobacterial pathogens residing within the cytoplasm of host macrophages. MTB promotes its survival in infected host cells by blocking the maturation of phagosomes in which it resides; this, in turn, results in the failure of bacilli to reach lysosomes [28]. The enhanced intracellular survival (Eis) protein released from MTB organisms inhibits phagosome maturation, ROI production, and autophagy through the direct acetylation of a JNK-specific phosphatase [74]. In addition, virulent mycobacterial organisms, such as MTB, frequently evaded macrophage defenses in mature phago-lysosomes by escaping from phagosomes to the cytoplasm through the phagosomal membrane [27, 28]. Macrophage autophagy appears to play important roles in the elimination of bacteria from the cytoplasm by re-engulfing intracytoplasmic mycobacterial bacilli and then killing these organisms after autophagosome-lysosome fusion [19, 24, 29, 31, 32]. In the case of autophagy in macrophages infected with mycobacteria, several types of molecular circuitry are involved in the regulation of autophagy, including the Ras-Raf1-MEK1/2-ERK1/2 cascade, beclin 1/class III PtdIns3K cascade, class I Pxtdlns3K/Akt/mTORC1 pathway, and AMPK-mediated pathway [36]. Highly virulent mycobacteria, such as MTB, M. bovis BCG, and Mycobacterium kansasii, were reported to induce markedly weaker autophagic responses in host macrophages than avirulent mycobacteria, including M. smegmatis [40].
As described above, autophagy up-regulates host immune effector responses against MTB infection. Many factors are involved; however, the process of autophagy is widely recognized to affect the course of infection. Autophagy plays important roles in both innate and adaptive immunity to various types of microbial pathogens [75]. In eukaryotic cells, autophagy is considered to be one of the most powerful and efficient ways of eliminating intracellular pathogens, including mycobacteria [15, 16, 19, 23, 25, 29]. Many studies have suggested that autophagy acts as an important immune effector mechanism, resulting in autophagosomal maturation that mediates mycobacterial clearance [29]. However, highly virulent mycobacteria are pathogens that have successfully evolved strategies for evading host defense mechanisms by arresting phagosome maturation and inhibiting later stages of the autophagic pathway in infected host cells [15, 19, 27, 40, 42, 76].
Based on these findings, we expect autophagy-activating agents to be beneficial for the effective elimination of highly virulent mycobacterial pathogens from hosts by enhancing the antimycobacterial functions of macrophages [3, 4, 51]. Therefore, these agents may be useful in the treatment of mycobacterial infections. Based on our original findings and a literature search using PubMed, most medicinal plants and their active chemical components were shown to up-regulate autophagic responses in macrophages during infection with pathogenic mycobacteria. In contrast, our subsequent studies and literature search indicated that most medicinal plants down-regulated the differentiation and activation of Th17 cell lineages induced by infection with mycobacterial organisms [53]. Th17 cells are known to play important roles in the elimination of mycobacterial pathogens in hosts, particularly in macrophages [53].
We previously reported that immunosuppressive macrophages were induced in the spleen of mice infected with MAC or MTB and suppressor macrophages reduced the activation of Th1 cells. The same macrophage population strongly induced the differentiation of Th17 cells from Th0 cells [77]. This immunosuppressive, but Th17 cell-inducing macrophage population, which possesses unique phenotypes (IL-12 (+), IL-1β (high), IL-6 (+), TNF-α (+), nitric oxide synthase (+), CCR7 (high), IL-10 (high), arginase-1 (−), mannose receptor (low), Ym1 (high), Fizz (low), and CD163 (high)), is considered to belong to neither M1 type macrophages nor M2 type macrophages, and, thus, we called it the M17 type macrophage population [77].
In any case, it is suggested that most medicinal plants exert dual effects in host defenses against mycobacterial infections as follows: the stimulation and suppression of the manifestation of the host antimycobacterial immune system mainly based on macrophage antimicrobial functions. This concept may partly explain the modest therapeutic efficacy of most medicinal plants and also Chinese traditional medicines when administered to patients with intractable mycobacterial diseases.
Conclusions
Autophagic events induced in host macrophages due to infections with mycobacterial pathogens play important roles in the up-regulation of macrophage activity to effectively kill and eliminate intracellular mycobacterial organisms. To the best of our knowledge, most medicinal plants and their active chemical components up-regulated macrophage autophagy, which indicates their modest potentiation of host defense mechanisms against mycobacterial infections.
Funding sources
Nothing declared.
Conflicts of interest
The authors declare no conflicts of interest, financial or otherwise.
Authors' contributions
Supervision: H.T. Writing manuscript: H.T. and Y.T. Review and editing: T.S. and C.S.
Abbreviations
| Akt | protein kinase B |
| AMPK | adenosine monophosphate-activated protein kinase |
| AP-1 | activator protein 1 |
| ATG | autophagy-related |
| ATP | adenosine 5′-triphosphate |
| DUSP10 | dual specificity phosphatase 10 |
| Eis | enhanced intracellular survival |
| ERK1/2 | extracellular signal-regulated kinase 1/2 |
| FFA | free fatty acids |
| GM-CSF | granulocyte-macrophage colony-stimulating factor |
| HDT | host-directed therapeutics |
| IFN-γ | interferon-gamma |
| IL | interleukin |
| JNK | c-Jun N-terminal kinase |
| LAP | LC3-associated phagocytosis |
| LC3-I | light chain 3-I |
| LC3-II | light chain 3-II |
| MAC | Mycobacterium avium-intracellulare complex |
| MAPK | mitogen-activated protein kinase |
| MEK1/2 | mitogen-activated protein kinase kinase 1/2 |
| MTB | Mycobacterium tuberculosis |
| mTOR | mechanistic target of rapamycin |
| NDGA | nordihydroguaiaretic acid |
| NF-κB | nuclear factor-kappa B |
| NK cells | natural killer cells |
| NLR | Nod-like receptor |
| NLRP3 | NLR family pyrin domain containing 3 |
| PAK1 | p21-activated kinase 1 |
| PI3K | phosphatidylinositol-3 kinase |
| PINK1 | PTEN-induced putative kinase 1 |
| PknG | protein kinase G |
| PPARA | peroxisome proliferator activated receptor α |
| PtdIns3K | class III phosphatidylinositol 3-kinase |
| RAB14 | ras-related protein Rab-24 |
| Raf1 | rapidly accelerated fibrosarcoma 1 |
| Ras | rat sarcoma viral oncogene |
| RNI | reactive nitrogen intermediates |
| ROI | reactive oxygen intermediates |
| SIRT3 | sirtuin 3 |
| TB | tuberculosis |
| TGF-β | transforming growth factor-beta |
| Th0 cells | type 0 helper T cells |
| Th1 cells | type 1 helper T cells |
| Th17 cells | type 17 helper T cells |
| TNF-α | tumor necrosis factor-alpha |
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