The Role of Interferon-Gamma and Interferon-Gamma Receptor in Tuberculosis and Nontuberculous Mycobacterial Infections

Abstract

Mycobacterium tuberculosis (Mtb) and nontuberculous mycobacteria (NTM) remain the leading causes of lung disease and mortality worldwide. Interferon-gamma (IFN-γ) and its receptor (IFN-γR) play a key role in mediating immunity against Mtb and NTM. This study was conducted as a systematic review; all information was collected from databases such as: PubMed, Scopus, Medline, SID, and medical databases. Finally, all the collected data were reviewed, and all content was categorized briefly. There is growing evidence that IFN-γ plays an important role in host defense against these two intracellular pathogens by activating macrophages. In addition, IFN-γ has been shown to be an integral part of various antibacterial methods such as granuloma formation and phagosome-lysosome fusion, both of which lead to the death of intracellular Mycobacterium. As a result, its absence is associated with overgrowth of intracellular pathogens and disease caused by Mtb or Mycobacterium nontuberculosis. We also look at the role of IFN-γR in Mtb or NTM because IFN-γ acts through IFN-γR. Finally, we introduce new approaches to the treatment of M. tuberculosis complex (MTC) and NTM disease, such as cell and gene-based therapies that work by modulating IFN-γ and IFN-γR.

INTRODUCTION

Interferon-gamma (IFN-γ) in which belongs to IFN type II is exclusively produced by CD4+ T helper cell type 1 (Th1) lymphocytes, CD8+ cytotoxic lymphocytes, and natural killer cells. Following IFN-γ secretion, it binds to its cell surface receptor which results in dimerization of nonligand-binding receptors subunits IFNR1 and IFNR2. IFNR1 is constitutively expressed in all nucleated cells. However, following bacterial infection IFNR2 is translocated into the cell membrane and forms functional interferon-gamma receptor (IFN-γR). E-selectin was identified as a major contributor in IFNR2 translocation from the Golgi into the cell membrane in an extensive experimental study by Xu et al. in 2018. They identified bruton tyrosine kinase (BTK) as a critical factor in IFNR2 translocation and found decreased IFNR2 expression levels on macrophages in Btk^-/- mice. They further through cell-free kinase assay disclosed that BTK plays a critical role in direct phosphorylation of IFN-γR2 Y289. They also through co-immunoprecipitation experiment illustrated that EFhd2 directly interacts with phosphorylated IFN-γR2 and drives its translocation into the cell membrane. In this context, they observed a decreased expression levels of IFN-γR2 on macrophage cell membrane in Efhd2 knockout (Efhd2^-/-) mice. In this manner, E-selectin through BTK activation induces IFN-γR2 translocation from the Golgi into the cell membrane and resulting in the formation of functional IFNγR.[¹]

The trans-activation of Jaks, which then phosphorylate the receptors' cytoplasmic tails to produce the required docking sites for STATs, occurs as a result of receptor dimerization. This brings Jaks and STATs closer together, allowing the former to mediate the latter's tyrosine-phosphorylation (p-Tyr), leading to nuclear translocation STAT dimerization, DNA binding, and, eventually, gene transcription control [Figure 1].[²] According to recent researches, Stat proteins are thought to play a crucial role in bacterial infections, and these theories result in innate immune reactions. In fact, the JAK/STAT1 pathway is one of the most common methods to activate macrophages, modulating the expression of a variety of genes involved in secondary cell reactions, resulting in macrophage activation or apoptosis. In this context, recent findings demonstrated that STAT1-knockout mice are susceptible to intracellular pathogens. Table 1 represents the major signaling pathways involved in TB and nontuberculous mycobacteria (NTM).

Table 1: Major signaling pathways involved in tuberculosis and nonberculous mycobacteria

Figure 1: Interferon-gamma/IFNR/JAK/STAT signaling pathway

MYCOBACTERIUM TUBERCULOSIS

Genus Mycobacterium with nearly 160 species was firstly introduced by Lehmann and Neuman in 1896. Only a few species are successful as pathogens of higher vertebrates, with the majority existing as free-living saprophytes. Mycobacterium tuberculosis (Mtb) complex is a group of bacteria from the genus Mycobacterium that are responsible for tuberculosis (TB). Mtb complex comprises Mtb, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium microti, Mycobacterium canettii, Mycobacterium caprae, Mycobacterium pinnipedii, Mycobacterium mungi, Mycobacterium orygis, and Mycobacterium suricattae species in which Mtb, M. africanum and M. canettii cause TB in human. Mtb as the intracellular pathogen is transmitted through aerosolized inhalation of bacteria-containing droplets. The WHO statistics in 2017 disclosed that there were almost 1.7 million TB-associated deaths globally.[³] According to the Global Burden of Disease Study, TB is the seventh biggest cause of disability adjusted life years (DALYs) globally and will remain in the top 10 reasons for DALYs in 2020, unlike most infectious disorders.[⁴]

Mtb is associated with an active form of disease known as TB. Furthermore, in latent TB infection, Mtb is restricted in a quiescent state within granulomas. Moreover, it has been reported that the resister phenotype is associated with polygenic variance in which many individual genetic variants with small individual effects contribute additively to the phenotype.[⁵] However, disrupted immunity due to aging and disease could lead to TB activation.[⁶]

Following inhalation, in the air gaps, the bacilli are mainly phagocytosed by dendritic cells (DCs) and resident alveolar macrophages (AMs). Various classes of pattern recognition receptors including C-type lectin receptors, toll-like receptors (TLRs), and nod-like receptors are linked to inflammasome activation which are involved in the recognition of MTB. Following lung infection, Mtb-infected phagocytes can move from the alveolar space into the lung interstitium, where they can then spread to other organs by lymphatic and hematogenous ways.

When compared to control Balb/C mice, SCID mice are more susceptible to Mtb infection. The host response against Mtb infection in the lungs is improved by genetically modified macrophages that overexpress IFN-.y. SCID mice's mortality was improved and Mtb burden in the lungs was reduced after J774.1 cells overexpressing IFN-y were administered into their lungs. In this way, macrophages overexpressing IFN-y limit Mtb growth in vivo, potentially improving host defense against Mtb infection.[⁷] As a result, IFN-y is vital in the fight against Mtb. IFN-y acts against Mtb through phagosome maturation, programmed death activation of host cells, ROS generation, and other mechanisms. IFN-y acts against Mtb through phagosome maturation, programmed death activation of host cells, ROS generation, and other mechanisms.

Interferon-gamma in apoptosis

Apoptosis is a key natural defensive mechanism against intracellular mycobacterial infection in macrophages. NOS2, an enzyme produced in macrophages activated by proinflammatory cytokines and bacterial cell wall components, produces a large amount of nitric oxide (NO). Recent findings show that NO through cytochrome c structure alteration and releasing from the mitochondria following chemical modification plays a crucial role in macrophages apoptosis induction, regardless of the changes of mitochondrial transmembrane potential (ΔΨm).[⁸] In addition, Herbst et al. discovered that IFN-γ caused apoptosis in mycobacteria-infected macrophages, which was solely dependent on NO, in an experimental research. Following that, NO-mediated apoptosis killed intracellular mycobacteria without the need for autophagy.[⁹] M. bovis as a slow-growing (16- to 20-h generation time) aerobic bacterium was considered the causative agent of TB in cattle. Zhang has explored the apoptotic effects of IFN-γ on monocytic THP-1 cells infected with M. bovis. Their findings revealed that following IFN-γ administration, apoptosis process in THP-1 cells infected with M. bovis was increased in time-dependent manner through translocation of apoptosis-inducing factor (AIF) to the nucleus and caspase activation. They further disclosed that upon addition of anti-TNF-α, the percentage of cells undergoing apoptosis decreased dramatically from 33.6% ± 2.7% to 11.7% ± 1.1%. In addition, significant reductions in caspase-3, -8, and -9 enzymatic activity have been observed following anti-TNF-α Mab addition to the cell medium. In this manner, IFN-γ through TNF-α-mediated death receptor and the AIF apoptotic pathway plays a critical role in apoptosis of THP-1 cells infected with M. bovis.[¹⁰]

Interferon-gamma in autophagy

Autophagy is a critical homeostatic mechanism that is activated by famine and other cellular stressors and involves the targeting of cytoplasmic cargo for breakdown in specialized structures known as autophagosomes. By bridging adaptive and innate immunological responses, autophagy is now recognized as a critical host-defense system against Mtb.[¹¹] IFN-γ was identified as a master and key regulator of autophagy and following intracellular Mtb clearance. The master modulator of lysosomal biogenesis and autophagy is TFEB (transcription factor EB). The induction of autophagy by TFEB is controlled by its compartmentalization. To trigger lysosome biogenesis and autophagy, the phosphorylated form is found in the cytoplasm, whereas the dephosphorylated form is found in the nucleus. HMOX (heme oxygenase) exists in two isoforms: Hmox 1 and Hmox 2. It catalyzes the oxidative breakdown of heme into carbon monoxide (CO), molecular iron (Fe2+), and biliverdin. N Singh et al. have found that inhibiting HMOX1 activity with a pharmacological inhibitor such as zinc protoporphyrin IX (ZnPP) triggers reduction of autophagosomes formation and maturation in response to INF-γ. Interestingly, they found that exogenous CO administration restored autophagy induction in response to IFN-γ even in the presence of HMOX1 inhibitor. In this manner, HMOX1-generated CO is crucial for autophagy induction. In addition, their findings disclosed that HMOX1-generated CO is essential for autophagosomes (containing Mtb) maturation and autophagosomes-lysosome fusion. In more details, they observed that HMOX1-generated CO through increasing cytoplasmic Ca2⁺ levels, phosphatase PPP3 activation, and finally TFEB dephosphorylation and translocation into the nucleus, involved in autophagy induction and death of Mtb residing in macrophages. Therefore, PPP3-TFEB signaling axis could act as a therapeutic target for suppressing of the intracellular pathogens growth.[¹²] Matsuzawa et al. have discovered that JAK1/2 is required for the early signaling cascade downstream of IFN-y and may enhance autophagy. They also discovered that autophagy activation is essential after IFN-g stimulation through inhibiting p38 MAPK and PI3K.[¹³]

Interferon-gamma in phagosome-lysosome fusion

Phagosome–lysosome (P–L) fusion is a major immune-effector reaction of host in which through pathogen antigen presentation eliminates the intracellular Pathogens. Ca²⁺ signaling has been identified to be associated with phagosome maturation through calcium-dependent kinases regulation such as calmodulin-dependent kinase II (CaMKII) as well as actin assembly on the phagosome surface for homotypic and heterotypic fusion. Furthermore, a recent intensive experimental study have found that exposure of human macrophages and monocytes to IFN-γ triggers significant upregulation of [Ca2+]_i.[¹⁴] Indeed, elevation of [Ca2+] I by IFN-y may result in Phagosome–lysosome (P–L) fusion and the eradication of Mtb.

Signaling lymphocytic activation molecule (SLAM) family with nine distinct receptors including SLAMF1 to SLAMF9 belongs to the immunoglobulin superfamily. SLAMF1 as a type I membrane protein consists of one V domain, one C2 domain, and eight potential N glycosylation sites. SLAMF1 expression has been reported on various immune cells including platelets, DCs, macrophages activated T-cells, B-cells, and central germinal T-follicular helper cells.[¹⁵] Barbero et al. have evaluated SLAMF1 surface expression by flow cytometry and found increased SLAMF1 expression levels following simultaneous treatment of THP-1 cells with Mtb and IFN-γ. These findings indicated that SLAMF1 expression on macrophages is induced mainly by proinflammatory stimuli such as IFN-γ. In addition, SLAMF1 was discovered in both early and late endosomes/lysosomes using confocal microscopy. In conclusion, their observations suggest that IFN-γ through SLAMF1 induction plays a crucial role in Mtb recognition, internalization, and finally phagosome maturation.[¹⁶]

Interferon-gamma in reactive oxygen species generation

Reactive oxygen species (ROS) have been identified as a crucial factor in macrophage-mediated immunity and are toxic to Mtb. Superoxide-generating NADPH oxidase 2 (NOX2) family proteins, such as the catalytic subunits gp22^phox, gp40^phox, gp47^phox, gp67^phox, and gp91^phox involved in ROS generation in phagocytes. Following ROS generation, they are employed in microbial oxidants production such as hydrogen peroxide as well as hypochlorus acid in the macrophage phagosome. Furthermore, NOX2 subunits translocation to the phagosomes are catalyzed by GTPase such as Gbp1, Gbp6, Gbp7, and Gbp10. In addition, Kim et al. in an extensive study have disclosed that IFN-γ by inducing gene expression of GTPase family engages in ROS generation and Mtb clearance.[¹⁷]

Interferon-gamma in metabolic shift

Hypoxia-inducible factor-1a (HIF-1a) plays a crucial role in glycolytic gene expression induction in hypoxic conditions. HIF-1a has been demonstrated for controlling pathogenic organisms such as Pseudomonas aeruginosa, group A Streptococcus, mycobacteria, and uropathogenic Escherichia coli.[¹⁸¹⁹] Furthermore, in vivo and in vitro analysis by J Braverman et al. in 2016 disclosed that HIF-1a is critical for IFN-γ-dependent control of Mtb infection. They via RNA-sequencing analysis found that HIF-1a is required for approximately one-half of the transcriptional responses to IFN-γ during Mtb infection. They also found that during infection, HIF-1a coordinates a metabolic shift to aerobic glycolysis in IFN-γ-activated macrophages in which is crucial for IFN-γ–dependent control of infection in macrophages.[²⁰]

Interferon-gamma in iron exporting

Most life forms, including mycobacterial species, require iron to survive. Therefore, iron-rich diet (a diet high in iron) as well as hemochromatosis will lead to severe case of Mtb and its dysregulation is linked to a worse clinical outcome.[²¹] Mtb via activating the TLR signaling, which results in decreased ferroportin expression and increased hepcidin secretion facilitates induction of intracellular iron sequestration. Furthermore, pathogen-associated intracellular iron sequestration is associated with increased intracellular replication of Mtb.[²²] Low iron conditions also increased the bacteria's ability to obtain restricted host iron by stimulating the expression of genes encoding iron importers, exporters, and siderophore (iron-chelator) biosynthetic enzymes.[²³] In the year 2020, Abreu et al. discovered a new method in which IFN-γ plays an important role in intracellular iron concentration. They analyzed transcriptional expression of the genes for the iron regulator hepcidin (HAMP) and the iron exporter ferroportin (SLC40A1) using quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis and found that it was significantly reduced after IFN therapy. They concluded that IFN-γ through significant reduction in intracellular iron availability to Mtb limits its replication inside the macrophage.[²²]

Lipid droplet formation and interferon-gamma

In an extensive study by Knight et al., it was disclosed that during infection of Mtb, macrophage lipid droplet (LD) formation is part of an adaptive immune response activated by IFN-γ. Furthermore, they also demonstrated Mtb capability in the accumulation of lipid inclusion in the absence of macrophage LDs. Moreover, their findings revealed that IFN-γ addition is accompanied with macrophage LDs production and plays a critical role in limitation of bacterial lipid accumulation. They concluded that macrophage LDs are not the major source from which Mtb acquires host lipids. In addition, they also investigated the mechanisms by which macrophages regulate LD formation during infection. They found that IFN-γ/HIF-1α/Hig2 signaling is the main signaling pathway that leads to LD formation. We found that these IFN-γ induced LDs enhance the macrophage immune responses by serving as an important site for production of a broad range of immunomodulatory eicosanoids.[²⁴]

Interferon-gamma influence in high and low burden of Mycobacterium tuberculosis

Based on intracellular bacilli number, IFN-γ effects on the survival or death of Mtb-infected macrophages varied dramatically. It was demonstrated that IFN-γ leads to apoptosis and necrosis of macrophages with increased intracellular bacillary burden in a Stat-1 dependent manner. In addition, in severely infected macrophages, IFN-γ plays a key role in inducing apoptotic vesicle formation and fragmentation of chromosomal DNA in a caspase-dependent manner and increases the release of high-mobility group box 1 protein (HMGB1) and lactate dehydrogenase in which serves as an early marker of necrosis. On contrary, IFN-γ through inhibition of bacterial replication triggers survival promotion of macrophages with a low intracellular Mtb load.[²⁵]

Interferon-gamma and microbiome

The epidermis, oral, lung, urogenital, and gastrointestinal (GI) tracts all have microbial populations, with the GI tract having the highest number of microbes. The intestinal microbiota is important for shaping and modifying the systemic immune response. Furthermore, the role of the gut microbiota in CD4 T cell maturation as a source of INF-γ has recently been discovered. Moreover, a close association between gut microbiota imbalance and extra-intestinal ailments such as obesity, cardiovascular disease, cancer, and non-alcoholic fatty liver disease has been identified.[²⁶] In this manner, microbial composition could greatly influence our health. Khan et al. have assessed Mtb and its dissemination in mice with disrupted gut microbial community. They observed that disruption of microbiota significantly enhanced the growth of Mtb in the lungs of the infected animals as well as its dissemination to other parts of the body such as spleen and liver. In addition, they also analyzed IFN-γ expression levels at mRNA levels and observed a significant decrease in its expression in a Mtb infected animal with disrupted microbial community.[²⁷] Thereby, gut microbial composition through maintenance of immune homeostasis plays a critical role in restriction of Mtb proliferation and dissemination. Beside, Yang et al. have investigated miRNA involvement in TB and host immune system for the first time. They through miRNA sequencing as well as volcano plot analysis identified increased expression levels of miR-21-3p in both lungs and cecum in the microbiota dysbiosis mice during Mtb infection. They further through Target Scan database as well as dual-luciferase reporter assay revealed that IFN-γ mRNA is targeted by miR-21a-3p. Therefore, intestinal dysbiosis through miRNA dysregulation lead to impaired immunity against Mtb.[²⁸] In addition, S Nadeem has investigated gut dysbiosis on vaccine efficiency against Mtb. They through various experimental assays including flow cytometry, proliferation assay, and reverse transcription quantitative polymerase chain reaction found that gut dysbiosis by hampering CD4⁺ T cell, and CD8⁺ activation, as well as down-regulating memory T cells and lung resident T-cell population, greatly influences the protective efficacy of vaccines.[²⁹]

Noncoding RNA and interferon-gamma

MicroRNAs (miRNAs) are noncoding RNAs that influence the immunological response in macrophages infected with Mtb.[³⁰] In this context, B Ni via NanoString and quantitative real-time RT-PCR analysis reported 31 differentially expressed miRNAs in primary human macrophages during Mtb infection. Among them, miR-132 and miR-26a were increased upon Mtb infection. It was demonstrated that Knockdown of miR-132 and miR-26a is associated with an increased in the p300 protein levels as well as improvement of transcriptional, translational, and functional responding to IFN-γ in human macrophages. Altogether, these findings identify that p300 is a direct target for miR-132 and miR-26a, and that Mtb through alteration of host miRNA expression can restrict macrophage responses to IFN-γ.[³¹] Moreover, Shi et al. have found that Mtb infection elevated miR-1178 expression in human macrophages, including THP-1 and U937 cells, and that this impact was dosage and time dependent. They discovered a link between enhanced mycobacteria survival in human macrophages and the overexpression of miR-1178. Furthermore, they through transfection study and luciferase report assay revealed that miR-1178 overexpression via direct targeting of TLR4 lead to decreased mRNA and protein levels of IFN-γ.[³²]

Long noncoding RNA (lncRNA) is a type of noncoding RNA that has more than 200 nucleotides. It accounts for 80% of noncoding RNAs. There is substantial evidence that lncRNAs are involved in host immunological responses to invasive pathogens, and dysregulated lncRNAs have been linked to a number of infectious diseases. Wang et al. have discovered that active TB infection stimulated CD244 signaling cascades in CD8+ T cells, which then triggered lymphocytic activation molecule (SLAM)-associated protein (SAP) and EWS-Fli1-activated transcript 2. They discovered that CD244 signaling cause's higher production of lncRNA-CD244 by maintaining a more permissive chromatin condition in the lncRNA-CD244 locus throughout active TB infection, using ncRNA microarray and hierarchical clustering analysis. They revealed that lncRNA-CD244 physically binds the polycomb protein regulator of zeste homolog 2 (EZH2) to control H3K27Me3 at the IFN-γ and TNF-a loci, allowing chromatin to establish repressive states and block IFN-y and TNF-a gene transcription in CD244+ CD8+ T cells.[³³] Furthermore, Pawar et al. hypothesize that mycobacterial infection and IFN-γ stimulated human macrophages activate autophagy through lncRNA-mediated induction to battle intracellular microorganisms. They observed that MEG3 Knockdown leads to autophagy induction in human macrophage infected with M. bovis BCG. In this manner, IFN-γ in infected macrophages via significant MEG3 down regulation played a crucial role in autophagy induction and enhanced eradication of intracellular M. bovis BCG.[³⁴]

NONTUBERCULOUS MYCOBACTERIA

NTM refers to all Mycobacterium species that do not cause TB, but are significant sources of lung morbidity and mortality around the world, particularly in developed countries. NTM lung disease, unlike TB, is not a notifiable sickness, making it impossible to assess incidence correctly. Furthermore, unlike TB, the disease induced by NTM has a wide range of clinical symptoms and is less virulent than TB. Non-TB mycobacteria that cause lung disease in humans are Mycobacterium avium, Mycobacterium intracellulare complex, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium fortuitum, Mycobacterium chelonae complex, Mycobacterium abscessus, and Mycobacterium scrofulaceum. Furthermore, the most frequent NTM in humans is M. avium subsp. avium (MAC) and M. abscessus subsp. abscessus (MAB). In addition, there is no record of NTM transmission from animal to human (zoonosis) or human to human (human-to-human), and human infections are typically derived from environmental exposure. Matsuyama et al. have investigated transcriptomic changes in MAC and MAB infected cell in vitro. They have discovered that ciliary genes were downregulated, but that genes involved with cytokines/chemokines, like IL-32, and cholesterol production were upregulated. Moreover, MAB infection tended to upregulate inflammatory response genes more than MAC infection.[³⁵]

Interferon-gamma and high-mobility group N2

High-mobility group (HMG) as a resident nuclear protein is composed of five subtypes including HMGN1, HMGN2, HMGN3, HMGN4. and HMGN5. They perform numerous biological functions such as regulation of gene transcription, replication, recombination, and DNA repair. HMGN2 as an antimicrobial peptide exists in the mononuclear leukocytes cytoplasm and is released to the extracellular environment following IL-2 stimulation.[³⁶] Furthermore, Wang et al. in 2019 investigated HMGN2 role in regulation of anti-NTM innate immunity of macrophages. They found that knock-down of HMGN2 via siRNA leads to increased expression levels of IFN-γ and enhanced M1 polarization in infected macrophages. In this manner, HMGN2 via INF-γ regulates M1 macrophage polarization and promotes NTM survival in macrophages.[³⁷]

Interferon-gamma and CXCR3

Three Glu-Leu-Arg (ELR)-negative CXC chemokines, CXCL9, CXCL10 and CXCL11, can together be referred to as the IFN-γ-inducible CXCR3 ligands. They share a common inflammatory function; they do collectively gain fine control over leukocyte trafficking. CXCL10 also known as IFN-γ-induced protein (IP)-10 is expressed by antigen-presenting cells in and promotes the attraction of monocytes and activated T-cells to foci of inflammation, further enhancing Th1 response. Dipeptidyl peptidase IV (DPP-IV) through cleavage of two amino acids at the IP-10 N-terminus triggers IP-10 inactivation.[³⁸] I Palucci et al. in 2019 investigated the contribution of IP-10 in restricting non-NTM replication. They found that endogenous DPP-IV inhibition leads to a significant reduction in M. abscessus and increased expression levels of IP-10. They further analyzed IP-10 role against M. abscessus activity and found that IP-10 indirectly exerted antibacterial activity against M. abscessus. In this manner, we could consider IFN-γ/IP-10/CXCR3 pathway as a therapeutic target in NTM infections.[³⁹]

Interferon-gamma role in granuloma formation and phagosome maturation

Nuclear erythroid 2 p45-related factor (Nrf2) with seven functional domains, Neh (Nrf2-ECH homology) 1 to Neh7 belong to the Cap “n” Collar (CNC) subfamily. Nrf2 through regulation of inflammatory cells migration and infiltration to inflamed tissue plays a key role in the inflammatory process.[⁴⁰] Activation of Nrf2 programmatically activates hundreds of genes by binding to a consensus sequence known as the anti-oxidant response element (ARE) in the promoter regions of Nrf2-responsive genes. Staitieh et al. through transfection-mediated silencing of Nrf2 RNA have found that si-Nrf2 treatment has significantly decreased the hydrogen peroxide scavenging capacity of the IFN-γ polarized M1 macrophages. They conclude that AMs activation with IFN-γ enhance Antioxidant Defenses via the Nrf2-ARE Pathway.[⁴¹] Furthermore, Nakajima et al. have investigated Nrf2 protection role against MAC by performing comprehensive transcriptome analysis in wild-type and Nrf2-deficient mice. They disclosed that Nrf2-deficient mice are highly susceptible to MAC infection. They through homology analysis identified Nrf2 binding sites in the promoter regions of both Nramp1 and HO-1 genes. In the case of granuloma formation, they observed small and disorganized granuloma in the lungs of Nrf2^-/- mice that is associated with reduced expression of HO-1. Moreover, they through confocal microscopy and quantitative analysis disclosed that the proportion of phagolysosomes is significantly lower in AMs of Nrf2^-/- mice than in those of wild-type mice. They also observed that Nramp1 gene transfection into Nrf2^-/- AMs results in enhanced formation of phagolysosomes in Nrf2^-/- macrophages. Therefore, following MAC infection, Nrf2 by upregulating lung expression of Nramp1 and HO-1 mediates granuloma formation and phagosome maturation, respectively, which results in increased resistance to MAC infection. Altogether, IFN-γ by augmenting Nrf2 signaling pathway might lead to protection against pulmonary MAC disease.[⁴²]

Noncoding RNA and interferon-gamma

Recent microRNAome and transcriptome analysis have disclosed that miRNAs are involved in regulation of host responses to MAC infection. In this context, Liang et al. via RNA-seq analysis have revealed that bta-miR-196 b, bta-miR-146 b, and bta-miR-146 b play a critical role in proliferation of endothelial cells, bacteria recognition, and regulation of the inflammatory response, respectively.[⁴³] Moreover, Liu et al. through computations approaches and transfection analysis have disclosed that miR-144 directly targets IFN-γ and influences T cell proliferation.[⁴⁴] Furthermore, Kim et al. via miRNA expression assay as well as gene expression analysis on macrophage from MAB-infected patients has found that increased expression levels of miR-144-3p lead to up-regulation of proinflammatory cytokines/chemokines and enhanced intracellular growth of MAB.[⁴⁵] In this manner, increased expression levels of miR-144 through IFN-γ inhibition are accompany with impaired immunity against MAB and can be used as a potential molecular biomarker in NTM disease.

INFR1/2 IN MYCOBACTERIUM TUBERCULOSIS AND NONTUBERCULOUS MYCOBACTERIA

IFN-γ primarily communicates with the IFN-γ receptor (IFNR). IFN-γR is a tetramer composed of two IFN-γR1 strands in combination with two IFN-γR2 strands, where IFN-γR1 is the IFN-γR's ligand-binding tail and IFN-γR2 is the IFN-γ receptor's signal-transducing tail. MSMD stands for Mendelian susceptibility to mycobacterial disease, a category of primary immunodeficiency marked by higher vulnerability to mycobacteria. Mutation in approximately 19 genes involve in IFN-γ signaling pathway such as IFN-γR1, and IFN-γR2 lead to MSMD.[⁴⁶] Rapkiewicz et al. by examining three children with autosomal recessive IFN-γR2 deficiency have found that IFN-γR2 deficiency is associated with diffuse MAC infection, which in turn through compressing venous lumina leads to hepatoportal venopathy.[⁴⁷] Bossi et al. through genetic analysis have detected a INFGR1 homozygous 9-bp deletion which involves the splice acceptor site at the 5′ end of exon 2 on the paternal allele as well as a 77.6-Kb heterozygous deletion entirely spanning INFGR1 and part of IL22RA2 on the maternal allele in an 18-month-old child presenting with a clinical picture of MSMD. This compound heterozygosity completely abolished induction of INFGR1 and resulted in disseminated M. avium.[⁴⁸⁴⁹] In addition, in patients diagnosed with partly recessive IFN-γR1 (RP-IFN-γR1), Sologuren have discovered that IFN-γ binding and clearance were impaired, but not completely eliminated. RP-IFN-γR1 had greater levels of STAT1 translocation than Autosomal recessive complete IFN-γR1 (RC-IFN-γR1), as well as a less serious symptoms phenotype.[⁵⁰] Furthermore, Mohamed et al. through molecular tools such as RFLP have found that polymorphism in IFN-γ R1 gene is associated with development of pulmonary TB among Sudanese patients.[⁵¹] Interestingly, Farnia et al. via genotyping IFNγR1 gene in Iranians patients with pulmonary disease have disclosed that there is a close relationship between SNP of IFNGR1 at position-56 and susceptibility to NTM infection. They also by investigating 95 patients with non-TB pulmonary constructed a close association between SNP of IFNGR1 at–56 and susceptibility to rapid grower infection. They finally suggested that SNP of IFNGR1 at position-56 could function as a powerful tool for detecting populations at higher risk risks of NTM infection.[⁵²⁵³] In this manner, beside IFN-γ deficiency, IFN-γR deficiency should therefore also be considered in people who suffer from mycobacterial diseases.

CELL AND GENE BASED THERAPY IN MYCOBACTERIUM TUBERCULOSIS COMPLEX AND NONTUBERCULOUS MYCOBACTERIA

The recent invention of the (CRISPR)/CRISPR-associated protein 9 (Cas9) system as a genome-editing technology has greatly enabled gene alterations in both pathogen and host cells, allowing for a more in-depth investigation of the molecular pathways engaged in infection pathogenesis. In E. coli, a customized CRISPR method was recently established for targeted gene regulation through using endonuclease inadequate Cas9, dCas9, which has two mutations in the nuclease domains: D10A and H840A. The CRISPRi system was named after the dCas9's ability to interfere with DNA transcription by blocking RNA polymerase from the specific promoter or causing a steric barrier to transcription elongation, which can inhibit gene expression by thousands of times. The activity of CRISPRi on mycobacteria gene expression has been investigated by Choudhary et al. They have shown that combining Streptococcus pyogenes' codon-optimized dCas9 with sequence-specific guide RNA causes full repression of single or multiple sites in mycobacteria. However, the Cas9-based CRISPRi method based on S. pyogenes is of limited use due of off-target effects and proteotoxicity.[⁵⁴] Furthermore, owing of off-target impacts and proteotoxicity, the Cas9-based CRISPRi method based on S. pyogenes is of limited use. To solve these constraints, Rock et al. evaluated 11 different Cas9 orthologues in 2017 and found four that are generally effective in mycobacteria for targeted gene silencing. The most effective of these proteins, CRISPR1 Cas9 from Streptococcus thermophilus (dCas9Sth1), demolishes endogenous gene expression by 20-100-fold with negligible proteotoxicity. As a result, CRISPRi may provide a straightforward, quick, and cost-effective technique for controlling gene expression in Mycobacterium.[⁵⁵⁵⁶]

MSCs are a heterogeneous subgroup of stromal stem cells that may be easily extracted from a variety of adult tissues such as dental pulp, adipose tissue, amniotic fluid, and bone marrow. They are multipotent cells with the ability to distinguish into adipocytes, osteoblasts, chondrocytes, and neuronal cells, all of which have an effect on tissue deterioration. As a result, they are capable cellular therapy suitors.[⁵⁷] Moreover, MSCs were shown to reduce the amount of bacteria and result in bacterial removal by multiple methods including antimicrobial peptide synthesis, boosting antimicrobial response, and modifying regulatory T cells.[⁵⁸] MSCs have therapeutic promise in the treatment of M. abscessus, according to Kim. They discovered that MSCs boosted the recruitment of inflammatory cells like CD11bhigh macrophages, monocytes, and CD4+ and CD8+ T cells to the lungs in M. abs-R-infected mice compared to M. abs-R-infected animals who were not given with MSCs. MSC infusion boosted the synthesis of proinflammatory cytokines such as IFN-y, IL-6, TNF-a, and MCP-1 in the lungs of M. abs-R-infected mice. They have also found that IFN-γ through PGE2 induction leads to increased production of NO, which in turn resulted in higher intracellular pathogen clearance. Thereby, MSCs injection via recruitment of inflammatory cells, cytokine production, and finally NO production enhances M. abscessus clearance.[⁵⁹⁶⁰] As a result, treating NTM patients with activated MSCs is a new therapeutic alternative.

Apart from their clinical significance, stem cell and especially induced pluripotent stem cell cells are extremely valuable tools for disease modeling.[⁵⁶⁶¹] Patient-specific iPSCs are also of significant relevance for diseases affecting cells which cannot be obtained from individuals, such as those that affect cells other than erythrocytes, leukocytes, and platelets. In 2020, Haake et al. built an illness modeling platform with patient-derived iPSCs to explore IFN-γ reaction in macrophages. They employed iPSCs from individuals with total and partial IFN-γR2 insufficiency, partial IFN-γR1 deficiency, and full STAT1 insufficiency, all of which are autosomal recessive. Macrophages from all patient iPSCs had regular shape and IFN-γ independent functions such as bio particle phagocytic absorption and cytokine internalization. We have found that defects in IFN-γ-dependent activities manifested themselves at various levels of the IFN pathway, with total IFN-γR2 and total STAT1 deficient cells exhibiting the most extreme phenotypes.[⁶²⁶³] Scientists can explore the role of macrophages in individuals with inborn defects of IFN-γ immunity using macrophage from client's iPSC with IFNγ-/IFNR insufficiency in this way.

Viral vectors have proven to be effective and safe delivery vehicles for therapeutic gene therapy, especially in the case of monogenic recessive diseases. Lentiviral vectors have important characteristics such as nondividing cell transduction and a safer implantation character, making them an effective method for introducing genes into mature T cells in CAR T-cell treatment.[⁴⁹⁶⁴] The very first lentiviral-based gene therapy method to maintain expression and activity of the human IFN-γR-downstream signaling pathway was presented by Hahn et al. They transduced human myeloid cell line K562 with third-generation self-inactivating (SIN) lentiviral vector in which composed of human complementary DNA of IFNGR1, enhanced green fluorescent protein, and SFFV promoter (Lv.SFFV.IFNgR1.iGFP) and identified enhanced IFN-gR1 expression without any potential side effects. Furthermore, they through phosphor-STAT1 detection found that constitutive overexpression of IFN-γR1 is linked with restored IFN-γ signaling pathway. They also examined lv.SFFV.IFN-γR1.iGFP efficacy on fibroblast from an IFN.γR1^-/- MSMD patient and disclosed its functionality on restoration of IFN-γ signaling pathway. In addition, Hetzel et al. have established a hematopoietic stem cell gene therapy (HSCGT) to restore the anti-mycobacterial function in hematopoietic cells in vivo. They have found that macrophage stimulation with IFN-γ is associated with increased expression levels of downstream components of IFN-γ such as nitric oxide synthase 2 (Nos2), interferon regulatory factor 1 (Irf1), indoleamine 2,3-dioxygenase, and finally increased intracellular degredation of M. avium in macrophages transduced with Lv.SFFV.IFNγR1 construct. They further demonstrated that Lv.SFFV. Ifnγr1-transduced cells transplantation into Ifnγr1^-/- mice triggers normalized lung and spleen morphology. In this manner, lentiviral gene therapy via IFN-γ signaling pathway stimulation is able to correct MSMD phenotype.[⁶⁵⁶⁶]

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