Different modalities of host cell death and their impact on Mycobacterium tuberculosis infection

Abstract

Mycobacterium tuberculosis (Mtb) is the pathogen that causes tuberculosis (TB), a leading infectious disease of humans worldwide. One of the main histopathological hallmarks of TB is the formation of granulomas comprised of elaborately organized aggregates of immune cells containing the pathogen. Dissemination of Mtb from infected cells in the granulomas due to host and mycobacterial factors induces multiple cell death modalities in infected cells. Based on molecular mechanism, morphological characteristics, and signal dependency, there are two main categories of cell death: programmed and nonprogrammed. Programmed cell death (PCD), such as apoptosis and autophagy, is associated with a protective response to Mtb by keeping the bacteria encased within dead macrophages that can be readily phagocytosed by arriving in uninfected or neighboring cells. In contrast, non-PCD necrotic cell death favors the pathogen, resulting in bacterial release into the extracellular environment. Multiple types of cell death in the PCD category, including pyroptosis, necroptosis, ferroptosis, ETosis, parthanatos, and PANoptosis, may be involved in Mtb infection. Since PCD pathways are essential for host immunity to Mtb, therapeutic compounds targeting cell death signaling pathways have been experimentally tested for TB treatment. This review summarizes different modalities of Mtb-mediated host cell deaths, the molecular mechanisms underpinning host cell death during Mtb infection, and its potential implications for host immunity. In addition, targeting host cell death pathways as potential therapeutic and preventive approaches against Mtb infection is also discussed.

INTRODUCTION

Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis (TB), which remains a leading cause of human infectious death worldwide, taking the lives of 1.5 million people in 2021 (1). One-fourth of the world’s population, or approximately two billion people, is estimated to be infected with Mtb, with more than 10 million developing active TB each year (1, 2). Mtb is a highly adapted intracellular pathogen that has evolved multiple mechanisms to manipulate host immune responses. Host cell death pathways are one of the main attributes of host-pathogen interaction in Mtb infection.

Cell death is fundamental to the interaction between Mtb and the phagocytes it infects, including macrophages (MΦ) and other types of myeloid cells, such as dendritic cells (DCs) and neutrophils. Cell death was originally divided into three major groups: type 1 (apoptosis), type II (autophagy), and type III (necrosis) (3). In recent years, several novel types of cell death have been characterized regarding their corresponding stimuli, molecular mechanisms, and morphologies. Some of these types of cell death share overlapping, although not identical, signal pathways and are not categorized into the type I–III groups. The Nomenclature Committee on Cell Death (NCCD) devised guidelines for cell death from morphological, biochemical, and functional perspectives in 2018 (4). Tang et al. (5) provide detailed historical origins and concepts used throughout cell death research development and a comprehensive summary of the molecular machinery involved in regulated cell death (RCD). Furthermore, Yan et al. (6) summarize different key features of different cell death modalities and categorize them into programmed cell death (PCD) and nonprogrammed cell death based on signal dependency.

Several studies have shown multiple and distinct types of cell deaths occurring during Mtb infection. Mtb can manipulate the kinetics and mode of cell death in infected macrophages (7, 8) to evade host immunity, shielding the bacteria from destruction by the host immune systems while allowing the bacteria to disseminate (9–11). Mtb infection of macrophages can result in multiple outcomes, including apoptosis, necrosis, or survival/maintenance of infected cells (8). The role of Mtb virulence factors in the induction of different modes of cell death remains controversial (12–14). Although macrophage apoptosis is required for antigen presentation to T cells and consequent elimination of the pathogen, necrosis can lead to a slow generation of host adaptive responses that result in Mtb dissemination and further infection of alveolar macrophages (14–16). Hence, a detailed understanding of how this successful intracellular pathogen regulates host cell death to promote its survival is crucial for developing alternative therapeutic strategies to target host proteins involved in cell death pathways (17). This article summarizes cell death modalities during in vitro and in vivo Mtb infection, characterizing the implications and consequences for host immunity, and suggests potential therapeutic opportunities and preventive approaches to combat TB.

DIFFERENT MODALITIES OF HOST CELL DEATH IN M. TUBERCULOSIS INFECTION

The interaction between Mtb and macrophages plays a crucial role in TB pathogenesis (18). Mtb grows and replicates within the infected host cells as an intracellular pathogen, mainly within the macrophages (19). Following the establishment of pulmonary infection, the transmission of the bacilli to new hosts requires a transition to the extracellular space and host cell death. Numerous studies have identified many different forms of cell death in the Mtb infection scenario, both in vitro and in vivo. A review on the topic has also listed spectrums of death modes linked to TB, including extrinsic, intrinsic, perforin/granzyme-mediated apoptosis, and several types of necrosis (20). This review discusses recent developments in multiple cell death modalities resulting directly from Mtb infection and the consequences to the host cells. It will also identify mycobacterial and host factors as potential targets for future interventions against TB.

Apoptosis

Apoptosis is an energy-dependent cascade of a highly regulated process for cellular deconstruction that encloses the cytoplasmic contents of dying cells within membrane-bound vesicles called apoptotic bodies (21). These apoptotic bodies express “eat me” signals and therefore are recognized and engulfed by the phagocytic cells via multiple specific surface receptors in a process called efferocytosis, which removes apoptotic/dying cells without causing inflammatory responses (22). Upon activation by physiological and pathological conditions, apoptosis occurs in two major pathways: the extrinsic/ligand and granzyme B-mediated pathway and the intrinsic/mitochondrial pathway (21). The critical players in these apoptotic pathways are caspases (a family of cysteine-dependent aspartate-directed proteases constitutively expressed as zymogens), adaptor proteins, tumor necrosis factor (TNF) receptor (TNF-R) family, and B cell lymphoma-2 (Bcl-2) family proteins (23). Cells that undergo apoptosis exhibit several biochemical modifications, such as protein cleavage and cross linking, DNA breakdown, and phagocytic recognition, resulting in distinctive structural pathology and morphological features (24). Another biochemical characteristic of apoptotic cells is the expression of cell surface markers leading to early phagocytic recognition by neighboring cells, allowing efficient phagocytosis of debris without compromising the surrounding tissue (21).

Host cell apoptosis during M. tuberculosis infection.

Host apoptosis signaling occurs through two major pathways, the intrinsic and the extrinsic pathways, depending on the source (intracellular vs. extracellular) of the activator, as reviewed extensively elsewhere before (22, 25–28). In this review, we discuss the factors underpinning the alterations in apoptotic cascades upon Mtb infection. These include host apoptosis-associated proteins induced following mycobacterial infection (Table 1) and mycobacterial proteins/factors that modulate host cell apoptosis pathways (Table 2).

Table 1.

Host factors modulated by Mtb in various cell death modalities

Cell Death Pathways	Host Factor	Remarks	Cell Type	Reference
Apoptosis	Bcl-2 (B-cell lymphoma-2)	Integral outer mitochondrial membrane protein that blocks apoptosis; upregulated over 2.5-fold following 48 h infection of Mtb	Human alveolar MΦ, alveolar epithelial cells, murine models	(29, 30)
	Bax (Bcl-2 associated X)	Apoptosis regulator protein; downregulated > 2-fold following Mtb infection	Human alveolar MΦ, alveolar epithelial cells, murine models	(29, 30)
	Bad (Bcl-2 agonist of cell death)	Promotes apoptosis by forming heterodimers with Bcl-xl and Bcl-2; downregulated > 2-fold following Mtb infection	Human alveolar MΦ and alveolar epithelial cells	(29)
	Bim (Bcl-2 interacting mediator)	Proapoptotic Bcl-2 family protein; upregulated after 24, 48, and 72 h after Mtb infection	Mouse MΦ J774	(31)
	Bcl-w (Bcl-2l2)	Acts as anti- and proapoptotic regulator; upregulated 1.8–2-fold following Mtb infection	Human alveolar MΦ	(32)
	Calreticulin	Modulates Mtb survival through endoplasmic reticulum stress-mediated apoptosis; upregulated following Mtb infection	THP-1 MΦ	(33)
	Elongation factor 1-alpha (eEF1α)	Modulates TNF-α dependent apoptosis; down-regulated > 2-fold following pathogenic Mtb infection	Human alveolar MΦ	(32)
	Interferon gamma (IFN-γ)	Activates nitric oxide-induced apoptosis; upregulated following mycobacterial infection	Mouse MΦ	(34)
	Tumor necrosis factor-alpha (TNF-α)	Modulates apoptosis and other cell death pathways; expression is inversely proportional to mycobacterial virulence	BALB/c mouse MΦ	(35)
	Myeloid leukemia-1 (Mcl-1)	Provide resistance to apoptosis; upregulated as early as 4 h and peaked (∼6-fold) at 24 h after infection	THP-1 MΦ	(36)
	Mitochondrial mortalin	Modulates macrophage apoptosis signaling by interaction with mitochondrial mortalin; upregulated during Mtb infection.	THP-1 and RAW 264.7 MΦ	(37)
	Nuclear factor kappa-B (NFκB)	Regulates apoptosis following Mtb- antigen stimulation; upregulated during Mtb infection.	HeLa cells	(38)
	RAD23 homolog B	Modulates TNF-α dependent apoptosis; downregulated > 2-fold following pathogenic Mtb infection.	Human alveolar MΦ	(32)
	Retinoblastoma (Rb)	Regulates apoptosis; upregulated over 2.5-fold after 48 h of Mtb infection	Human alveolar MΦ and alveolar epithelial cells	(29)
	Superoxide dismutase-2 (SOD2)	Modulates TNF-α dependent apoptosis; downregulated 1.8 to 2.0-fold after 48 h post-Mtb infection	Human alveolar MΦ	(32)
	Toll-like receptor 2 (TLR-2)	Induces macrophage death; upregulated in Mtb infection	Mouse MΦ	(39)
	Lymphocyte activation gene-3 (LAG-3)	Immunomodulator that affects host cell apoptosis; induced nearly 100-fold following Mtb infection.	Macaque blood derived MΦ/T cell co-culture	(40)
	Kruppel-type zinc finger (ZK1) protein	Modulates TNF-α dependent apoptosis; downregulated > 2-fold following Mtb infection.	Human alveolar MΦ	(32)
	Gasdermin-D (GSDMD)	Mediates apoptosis but not IL-1β secretion in Mtb-infected macrophages	Human MΦ, PBMCs	(41)
Pyroptosis	NOD-, LRR-. and pyrin domain-containing protein 3 (NLRP3)	Activation of the NLRP3 inflammasome to modulate inflammatory disease pathogenicity	Immune cells (T cells)	(42)
	Caspase-1 (CASP-1)	CASP-1 activation leads to chromosomal DNA damage through an endonuclease	H9C2 cardiomyocytes	(43)
Necroptosis	Receptor-interacting serine/threonine-protein kinase 3 (RIPK3)	RIPK3 complex recruits and phosphorylates MLKL to form the necrosome	Mouse embryonic fibroblasts (MEFs)	(44, 45)
	Mixed lineage kinase domain-like (MLKL)	Initiates necroptotic cell death by allowing ion influx, cell swelling, and membrane lysis followed by the uncontrollable release of intracellular material	Mouse embryonic fibroblasts (MEFs)	(45, 46)

Several host factors/proteins regulated following Mtb infection in different cell death modes and their fold expression are listed. MΦ, macrophages; Mtb, Mycobacterium tuberculosis; NOD, nucleotide binding oligomerization domain; PBMCs, peripheral blood mononuclear cells.

Table 2.

List of Mtb factors associated with host cell death

Actions on host	Mtb Protein/Factor	Remarks	Cell Type	Reference
Apoptosis inducers	Lipomannan (LM); lipoarabinomannan (LAM); mannosylated lipoarabinomannan (manLAM)	Induces apoptosis and IL-12 secretion	Human THP-1	(47)
	Glycolipoprotein (p19; LpqH)	Induces apoptosis through TLR-2 and mitochondrial apoptosis-inducing factor	THP-1/CHO expressing TLR2	(48)
	Mycolactone	Causes apoptosis in guinea pig ulcers and tissue culture cells	macrophage cells J774A.1/guinea pig model	(49)
	Periplasmic phosphate-specific transporter; 38-kDa lipoprotein (Pst-S1)	Causes TNF-α and FasL-mediated caspase-dependent apoptosis and upregulation of cell-death receptors TNFR1, TNFR2, and Fas and TLR2	THP-1 macrophages	(50)
	Early Secreting Antigen Target-6kDa (ESAT-6)-family (esxA, esxT and esxL)	Induces apoptosis through activation of caspase gene expression	THP-1 macrophages	(51)
	PE-PGRS33	Induces apoptosis through TLR2 pathway	RAW 264.7 and HEK293 cells	(52)
	Rv0183	Induces inflammation and apoptosis in RAW macrophages	Macrophage RAW264.7 cells	(53)
	Rv0901	Overexpression induces macrophage apoptosis	THP-1 macrophages	(54)
	Rv3499c (Mce4A)	Induces apoptosis mediated by TNF-α	THP-1 macrophages	(55)
	PPE37	C-terminal domain induces apoptosis	THP-1 macrophages	(56)
	PE9 (Rv1088)/PE10 (Rv1089)	Induces apoptosis by TLR4 pathway	THP-1 macrophages	(57)
	PE_PGRS41	Enhances intracellular survival	THP-1 macrophages	(58)
	PE13/Rv1195	Increases intracellular survival by activating the p38-ERK pathway	THP-1 macrophages	(59)
	PE_PGRS18	Enhances intracellular survival	THP-1 macrophages	(60)
Apoptosis inhibitors	NADH-quinone oxidoreductase subunit G (NuoG)	Inhibits apoptosis of infected host cells	BALB/c or SCID/Ncr (BALB/c mice)	(61)
	Protein Phosphatase 2 Phosphatase Activator (PtpA )	Inhibits phagosome acidification and maturation. Interacts with tripartite motif containing 27 (TRIM27) and suppresses apoptosis	THP-1 macrophages	(62)
	DUF732 domain-containing protein kinase (Rv3354)	Targets the metalloprotease resulting in suppression of apoptosis; destabilizes regulatory cullin-RING ubiquitin E3 enzymatic activity; inhibits constitutive photomorphogenesis 9 (cop9) signaling	THP-1 macrophages	(63)
	MPT64 (Rv1980c)	Inhibits apoptosis of macrophages through the NF-kB-miRNA21-Bcl-2 pathway	RAW264.7 macrophages	(64)
	Protein Kinase E (pknE)	Mutation increases the expression of genes involved in apoptosis	THP-1 macrophages	(65)
	Rv2456c	Inhibits apoptosis through NF-κB and extends the survival of Mtb.	Human epithelial cells (A549) & THP-1 macrophages	(66)
	GroEL2 (Rv0440)	Blocks macrophage apoptosis via interaction with mitochondrial mortalin	THP-1 and RAW 264.7 cell lines	(37)
	SecA2 (Rv1821)	Controls(?) secretion of a specific subset of proteins and blocks macrophage apoptosis	C57BL/6 mice	(67)
	Oligopeptide transporter subunit (OppD)	Transports Mtb and induces apoptosis	THP-1 macrophages	(68)
	Rv3654c/Rv3655c	Interacts with AL017 and blocks the extrinsic pathway	U937 monocyte-derived macrophage	(69)
Necrosis inducers	ESAT-6	Results in necrosis of human macrophages mediated by pyrin domain-containing protein-3 (NLRP3)	THP-1 macrophages	(70)
	Channel protein with necrosis-inducing toxin (CpnT /Rv3903c)	Induce necrosis of human macrophages through its secreted C-terminal domain known as tuberculosis necrotizing toxin (TNT)	RAW 264.7 macrophages	(71)
			THP-1 macrophages
	PE25/PPE41	Recombinant form induces necrosis	RAW 264.7 macrophages	(72)
	Rv2626c	High levels of necrosis were observed in THP-1 macrophages infected with MtbH37Rv over-expressing Rv2626c	THP-1 macrophages	(73)
	PPE68	Inactivation decreases necrosis	THP-1 macrophages	(73)
	PPE31	Shortens the life of macrophages infected by Mtb over-expressing PPE31 and promotes mycobacterial survival	RAW264.7 macrophages	(74)
Pyroptosis inhibitors	Rv3364c	Binds to the cathepsin G, inhibiting its enzymatic activity and the downstream signal caspase-1.	Human monocytic cell line U937-derived macrophages	(75)
		Inhibits host pyroptosis
Pyroptosis inducers	Rv1579c/EST12	Binds to RACK1 to recruit the deubiquitinase UCHL5 leading to NLRP3 inflammasome activation that promotes pyroptosis	Human monocytic THP-1 and mouse BMDMs	(76)
	Zinc metalloprotease 1 (Zmp-1/Rv0198c)	Deletion triggers activation of caspase-1/IL-1β inflammasome leading to pyroptosis signaling and improved mycobacterial clearance	J774A and RAW 264.7 macrophages	(77)
Autophagy inhibitors	Enhanced intracellular survival (Eis)	Infection with Mtb-Δeis elevates ROS generation and renders the cells highly sensitive to autophagy activation	THP-1 macrophages Mouse BMDM	(78)
	ESAT-6	Transient expression of the ESAT-6/CFP-10 fusion inhibits autophagosome formation by decreasing expression levels of ATG genes	RAW264.7 macrophages	(79)
	PtpA	Interacts with active-phosphorylated vacuolar protein sorting 33B (VPS33B) and inhibits phagosome-lysosome fusion, impacting autophagy	THP-1	(80)
	PknG	Inhibits phagosome-lysosome fusion, impacting autophagy	J774 macrophage cells	(81)
ETosis	ESAT-6	Induces a high level of intracellular Ca2+ leading to the formation of NETs characterized by extracellular DNA and myeloperoxidase	Neutrophils from healthy volunteers	(82)

Mycobacterium tuberculosis (Mtb) modulates several types of host cell death upon infection. A list of different Mtb factors and their regulatory role in host cell death are tabulated. TLR2, Toll-like receptor 2; TNFR, tumor necrosis factor-alpha receptor.

Intrinsic apoptosis is engaged by cells that are deprived of growth factors, damaged, or infected. These diverse stimuli can tip the balance between different groups of the Bcl‐2 (B‐cell lymphoma 2) proteins leading to the activation of cell death (83). The Bcl‐2 protein family can be divided into three subfamilies: the antiapoptotic Bcl‐2 proteins, the proapoptotic BH3‐only (BH: Bcl‐2 homology) proteins, and the death effectors Bax (Bcl‐2‐associated X protein), Bak (Bcl‐2 homologous antagonist/killer), and Bok (Bcl‐2‐related ovarian killer) (84). The BH3‐only members can inhibit the anti‐apoptotic Bcl‐2 proteins, or, in some cases, directly engage Bax and Bak (e.g., Bim) (85, 86). Tipping the equilibrium in favor of proapoptotic Bcl‐2 proteins leads to the activation of Bax and Bak and results in mitochondrial outer membrane permeabilization (MOMP) (87). Some BH3‐only proteins are regulated by transcriptional regulation (the p-53 upregulated modulator of apoptosis; PUMA, is regulated by p53) (88) or by post‐translational modifications (Bim, Bid) (89). Bok, which can constitutively induce MOMP, is regulated differently by proteasomal degradation pathways (90, 91). MOMP causes the release of the key mediators of intrinsic apoptosis, cytochrome c (92), an endogenous inhibitor of apoptosis (IAP) antagonist, and SMAC/Diablo (second mitochondria-derived activator of caspases/direct IAP binding protein) (93, 94). Cytochrome c‐bound apoptotic protease activating factor 1 (Apaf1) recruits initiator caspase, caspase‐9, to form apoptosome (a multimeric scaffold protein complex) for the activation of the executioner caspases, caspase‐3 and ‐7 (95). Caspases‐3, ‐7, and ‐9 can be blocked by the major endogenous caspase inhibitor, XIAP (X chromosome‐linked IAP) (96). SMAC can antagonize XIAP and other IAPs, allowing full caspase activation and apoptosis initiation (84, 93). Caspases cleave various cellular proteins to induce characteristic changes of apoptotic death (cellular and nuclear fragmentation, DNA laddering, etc.). For example, an inhibitor of caspase‐activated DNase (ICAD) cleavage leads to the activation of caspase‐activated DNase (CAD) that induces genome fragmentation (84).

Extrinsic apoptosis is triggered by TNF family ligand‐receptor interactions, most prominently by TNF family ligands: TNF, FasL, TRAIL, and TL1A. The receptor complexes either recruit Fas‐associated protein with death domain (FADD) or TNFRSF1A‐associated via death domain (TRADD) to the oligomerized complex (84, 97). FasL‐mediated signaling will be used to describe extrinsic apoptotic signaling, and TNF signaling will be described for necroptotic signaling. FasL binds to its transmembrane receptor Fas, which recruits FADD via death domain (DD) interactions (98). The proximity of multiple caspase‐8 molecules induces the transactivation by proteolytic cleavage, resulting in the p18 and p10 fragments, which activate caspase‐3 and caspase‐7 (type I apoptosis) (84, 99). Insufficient activation of caspase‐3 leads to type II apoptosis in which caspase‐8 cleaves the BH3‐only protein Bid (BH3 interacting domain death agonist) to generate its activated form: truncated Bid (tB499) (100). tB499 stimulates the intrinsic apoptotic pathway by binding directly to Bax/Bak, inducing MOMP (type II apoptosis) (101).

Cytotoxic lymphoid cells (predominantly NK cells and cytotoxic T cells) can induce apoptosis via death receptor ligands (extrinsic apoptosis) or via the granzyme/perforin system (102, 103). After recognizing transformed or infected cells, cytotoxic cells release secretory granules containing perforin and granzyme B. These secreted factors are taken up by endocytosis and released to the cytosol by the perforin‐dependent or ‐independent pathways (104). Once released to the cytosol, granzyme B cleaves caspases and Bid (105), activating the apoptotic pathways described earlier. However, human granzyme B can also directly cleave ICAD, a known caspase‐3 target, to induce DNA fragmentation, thereby evading the need for caspases (106).

Following the encounter with Mtb, the host releases its antimicrobial defenses (Fig. 1). During this host-pathogen interaction, several host proteins/factors are induced due to either host defense mechanisms or bacterial manipulation (7). The apoptosis signaling is mainly regulated by multiple host apoptotic factors including receptor proteins [Toll-like receptor, TLRs (107) and Fas/APO-1 (apoptosis antigen 1) CD95 receptor (108, 109)], effector proteins [caspases (51), TNF-α (110), IFN-γ (111), IL-10 (112, 113), and CCL20 (chemokine C-C motif ligand 20) (114)], Bcl-2 family proteins (29, 115), as well as transcription factors (NFκB) (116).

Figure 1.

Intrinsic and extrinsic host apoptosis pathways in Mycobacterium tuberculosis infection. Apoptosis is induced by the host’s innate immune response against infection by either intrinsic or extrinsic pathways. A range of mycobacterial proteins inhibits host-induced apoptosis during successful infection. Pathogen-associated molecular patterns (PAMPs) are recognized by host signaling proteins that drive the cell toward apoptosis. Specific bacterial factors cause mitochondrial membrane depolarization and lead to the release of mitochondrial factors that trigger apoptosis by the intrinsic pathway. Various stimuli can induce intrinsic apoptosis by shifting the equilibrium of prosurvival B-cell lymphoma-2 (Bcl-2) and Bcl‐2 homology 3 (BH3) proteins. Extrinsic apoptosis can be induced by the binding of a select group of tumor necrosis factor (TNF) family ligands to their receptors leading to death-inducing signaling complex (DISC) formation by recruitment of adapter Fas‐associated protein with death domain (FADD)/TNFRSF1A‐associated via death domain (TRADD) and caspase-8. Caspase-8 auto-processes itself (cCasp8—cleaved/activated caspase-8) and can directly activate caspase-3 or cleave Bid to generate tBid and triggers intrinsic apoptosis. Apaf-1, apoptotic peptidase activating factor 1; Bax, B-cell leukemia/lymphoma-2 associated-X, Bak, B-cell leukemia/lymphoma-2 antagonist/killer; BID, BH3 interacting domain; Bok, B-cell leukemia/lymphoma-2 associated related ovarian killer; CAD, intracellular caspase-activated deoxyribonuclease; CARD, caspase recruitment domain; Casp, caspase; CytC, cytochrome C; FAD, flavin adenine dinucleotide; FASL, FAS ligand; iFAS, FS-7-associated surface antigen; LAM, lipoarabinomannan; SMAC, second mitochondria-derived activator of caspases; tBID, truncated BID; TNFα, tumor necrosis factor-alpha; TNFR, tumor necrosis factor-alpha receptor; XIAP, X-linked inhibitor of apoptosis protein. Figure created with images from smart.servier.com.

Mtb proteins are involved in both inhibition and induction of host cell apoptosis signaling, depending on the stage of infection. The following sections discuss Mtb proteins/factors involved in host apoptotic pathways. Induction of apoptosis, which occurs following mycobacterial infection, can be considered either as a host defense mechanism to eliminate the bacteria or as a pathogen-induced response to evade the host defense mechanism, which facilitates bacterial survival (117). However, the stage at which host apoptosis is induced influences the progression of the infection (7).

M. tuberculosis factors promote host apoptosis.

Several Mtb factors have been shown to induce host cell apoptosis (Table 2), including secreted proteins [19-kDa protein/LpqH (118) and ESAT-1/RD1 (56)] and surface proteins [PE_PGRS33/Rv1818c (52) and 38-kDa lipoprotein/PstS-1 (68)]. 19-kDa secreted protein is a cell wall-associated and secreted virulence factor. Recombinant p19 protein induces apoptosis in PMA (phorbal 12-myristate 13-acetate)-differentiated THP-1 macrophages in a TLR-dependent pathway (118). Recombinant p19 also appeared to induce both extrinsic and intrinsic pathways of host cell apoptosis (119). In addition, this secreted protein was reported to induce IL-1β in monocyte-derived macrophages infected with knockout (KO) Mtb strain (120).

Early secreted antigenic target of 6 kDa (ESAT-6) (Rv3875) is a well-characterized secreted virulent protein within the RD1 region of Mtb (121). Expression of this antigen is known to be induced in macrophages following Mtb infection, which inhibits T cell immune responses (51, 122). ESAT-6, as an early secretory antigenic target EsxA and phthioceroldimycocerosates (DIM), was shown to cause phagosomal rupture leading to cell death induced by virulent mycobacteria inside host phagocytes (123). ESAT-6 is known to intercept the host defenses at different stages of infection, including autophagosome formation (124), disruption of TLR signaling (125), and inhibition of IFN-γ (126). It has also been shown that ESAT-6 interferes with the NF-κB pathway and autophagy (125, 127). ESAT-6 stimulated A549 cells (human epithelial cells) are known to induce the endoplasmic reticulum (ER) stress response, causing an increase in intracellular Ca²⁺ concentration, which results in ROS accumulation, and therefore inducing the onset of ER stress-induced apoptosis (128). However, it is known that Mycobacterium bovis BCG (Bacillus Calmette-Guerin), with no virulent factors in its RD1 region, can induce greater host cell apoptotic signaling compared with Mtb (129); this leads to an unclear role of ESAT-6 in host cell apoptosis.

PE_PGRS33 is a surface antigen protein of pathogenic mycobacteria, including Mtb, and one of the most studied PE_PGRS proteins (130). This surface protein is known to induce apoptosis in macrophages (52, 131); it interacts with Toll-like receptor 2 (TLR2), leading to inflammatory chemokine and cytokine secretion, which is key in the immunopathogenesis of Mtb infection (130). Another Mtb surface protein involved in apoptosis is PstS-1, also known as 38-kDa lipoprotein. PstS-1 is a mannosylated glycolipoprotein of Mtb that binds to the mannose receptor in macrophages, promoting phagocytosis (132). This cell wall-associated protein acts as an immunodominant antigen that induces macrophage apoptosis in a caspase-dependent manner involving TNF-α and FasL (50). The same study also shows that PstS-1 can induce both intrinsic and extrinsic pathways in apoptotic death of human monocyte-derived macrophages (MDM) by TLR2 activation following Mtb infection (50). Since apoptosis most likely results in bacterial killing, the host cell signaling through the PstS-1 pathway might be detrimental to the intracellular survival of Mtb.

M. tuberculosis interferes with host cell apoptosis signaling.

A striking contradiction exists in the literature regarding the role of apoptosis in TB disease; in some reports, virulent Mtb is described to induce apoptotic host cell death, whereas, in others, the pathogen is thought to inhibit this process (133). These differences can partly be due to the complexity of the experimental system, which relies on the interaction of viable bacteria and host cells. In the early stage of infection, apoptosis inhibition is crucial for the bacteria to survive and multiply within the host cells. Mtb has adopted several mechanisms to evade the host-protective responses (Table 1). Multiple host-induced apoptosis triggers culminate in host cell death resulting in pathogen clearance. These triggers include host-induced-reactive oxygen species (ROS), leading to oxidative stress, cytokines, damage sensors, apoptotic mediators, and cell surface receptors (7). Different strains of mycobacteria have varied abilities to modulate apoptosis resulting in different scenarios of disease outcomes. In addition, host susceptibility factors that regulate host-pathogen interactions determine the clinical outcome of the disease (134–136). Mycobacteria-induced mechanisms that inhibit apoptosis of host cells include those that interfere with pathways of ROS/RNS generation, induction of anti-apoptotic genes (137, 138), TNF-α production (139), and eicosanoid regulation (8).

Several proteins secreted by Mtb, such as superoxide dismutase, heme-dependent catalase-peroxidase KatG, serine/threonine protein kinase PknE, type I NADH dehydrogenase NuoG, Rv3654c, and Rv3655c putative proteins are known to inhibit macrophage apoptosis (69, 140). These proteins regulate the production of NO and proinflammatory cytokines to interfere with TLR mechanisms leading to the inhibition of TNF-α-induced apoptosis (69). Various secreted virulence factors of Mtb interfere with the caspase (86), JAK2/STAT1 (141), TNF-α (142), and Bcl-2 pathways to suppress macrophage apoptosis and increase the survival rate of pathogens (143, 144).

Mtb infection can also inhibit host apoptosis through the blockage of eicosanoids (8). Eicosanoids are locally acting bioactive signaling lipids derived from arachidonic acid and related polyunsaturated fatty acids categorized as prostaglandins (PGE) that regulate a diverse set of homeostatic and inflammatory processes (145). Mtb infection leads to the formation of foamy macrophages with accumulated lipid droplets (146). The lipid droplets support the formation of eicosanoids and contribute toward host defense against Mtb. Thus, Mtb-mediated modulation of eicosanoid production determines the infected macrophage cell death signaling, resulting in a substantial impact on the outcome of infection and skewing the host cell death mode from apoptosis to necrosis (8).

In summary, both intrinsic and extrinsic pathways of host cell apoptosis can be triggered and/or suppressed at various stages of Mtb infection. When apoptosis occurs as an early response to the infection, it contributes primarily to the pro-host response that eliminates the bacterial pathogen. However, apoptosis that occurs at a later stage of infection gravitates toward pro-pathogenic mode, facilitating bacterial survival and dissemination, and averting proinflammatory host immune responses. Apoptosis in Mtb infection has been extensively studied; for more comprehensive reviews on apoptosis signaling, readers may refer to these articles by Briken and Miller (133), Behar et al. (147), Xu et al. (148), Lam et al. (149), Galluzzi et al. (4), Mohareer et al. (7), and Zhai et al. (143).

Necrosis

The definition and concept of necrosis, which was previously viewed as a nonprogrammed and proinflammatory mode of cell death, has expanded over time (150). Traditional and classical necrosis was classified as accidental cell death (ACD) according to NCCD-2018, which defined ACD as a “virtually instantaneous and uncontrollable form of cell death corresponding to the physical disassembly of the plasma membrane caused by extreme physical, chemical, or mechanical cues” (4). Necrosis has also been later categorized into “mitochondrial permeability transition-driven necrosis” (MPT-necrosis) and programmed necrosis, also known as necroptosis (4, 151). Programmed necrosis is tightly interconnected with innate inflammatory responses and is active during infections and tissue injury and repair (152).

Recently, there is a paradigm shift in our understanding of cellular necrosis. Contrary to the earlier conception that necrosis is accidental and unregulated, recent evidence suggests that necrosis may be regulated (4, 6, 153). The ability of specific receptors, such as tumor necrosis factor-α (TNF-α), to trigger necrosis suggests that this is a regulated event with a structured signaling pathway (4, 154). Other signals, including physical and chemical stressors such as ischemia-reperfusion (IR) injury, oxidative stress, calcium (Ca²⁺) overload, extensive DNA damage, and irradiation, can induce necrotic cell death (155). ROS generation, as a consequence of mitochondrial membrane potential loss, plays a central role in the progression of necrosis to osmotic swelling and, ultimately, cell lysis (156). Various host factors, including MPT (MPT-driven necrosis), receptor-interacting protein (RIP) kinases 1 and 3 (RIPK1/RIPK3, mostly involved in necrosis, pyroptosis, necroptosis), mixed lineage kinase domain-like (MLKL, mainly involved in necroptosis), caspase-1 and caspase 11 (primarily involved in pyroptosis), and cathepsin B (mostly involved in pyronecrosis) have been shown to regulate necrosis (151, 157, 158). Furthermore, chemical mediators have been shown to inhibit different necrotic signals, suggesting that necrosis can be regulated by extrinsic factors, such as therapeutic drugs (151). The other forms of regulated necrosis, as per NCCD-2018, include parthanatos, oxytosis (cell death by the accumulation of ROS), ferroptosis, and ETosis, all of which lead to inflammation (4). All these necrotic cell death modes have specific characteristics and overlapping pathways, which are highly relevant to the pathogenesis and treatment of TB.

M. tuberculosis infection promotes host cell necrosis.

Necrosis induction, associated with the granulomatous response during Mtb infection, is central to bacterial survival and dissemination and contributes to the severity and morbidity of the disease (7). Recent evidence shows that necrotic macrophages provide a niche for Mtb growth and survival before entering the extracellular milieu (159). In addition, neutrophils also undergo necroptosis following Mtb infection (160).

The cells that are frequently observed to participate in inflammatory pathology in actively necrotic granulomas are thought to be neutrophils (82). Intra-alveolar neutrophil infiltration and an excessive inflammatory response play critical roles in the progression to active TB, which is characterized by initial caseous and later liquefactive necrosis in the lung (161, 162). Neutrophil necrosis induced by Mtb contributes to bacterial growth in host cells, thus sustaining the infection (160). Mtb infection renders human macrophages necrotic, favoring bacterial replication (159). Mtb has also been shown to induce neutrophil extracellular traps (NETs) that promote the recruitment and activation of effector cells (163).

Several Mtb proteins activate the necrosis of host macrophages in vivo (164). ESAT-6 protein induces intracellular Ca²⁺ influx, triggering neutrophil necrosis and production of NETs, ultimately contributing to necrotic pathology and TB transmission (82). This resulted in ESAT-6 becoming an important therapeutic target (165). In addition, the Mtb PPE11 (Rv0453) protein, found in infected guinea pig lungs, promoted mycobacterial survival under stressful conditions by enhancing inflammation, organ pathology, and host-cell death (166). Recombinant PE17 (Rv1646) inhibits the production of proinflammatory cytokines (IL-6, IL-12, and TNF-α) and enhances macrophage necrosis (167). The degree of tissue necrosis and lung inflammation may be Mtb strain-specific; TLR2-deficient mice infected with Mtb W-Beijing exhibited increased neutrophil infiltration (168). The molecular mechanisms by which Mtb and its effectors aggravate host cell necrosis during TB remain unclear. Further characterization of Mtb protein and lipid functions in inducing host cell necrosis would certainly provide insight into its virulence mechanisms. Understanding the mechanisms of induction of host cell necrosis following Mtb infection would facilitate the development of novel therapeutic approaches.

Necroptosis as a prototype of regulated necrosis with proinflammatory functions.

Necroptosis, considered a link between host cell death and inflammation, is initiated by perturbations of the intracellular or extracellular microenvironment detected by specific death receptors such as TNF receptor, Fas receptor, pattern recognition receptors (PRRs) (TLR3, TLR4, Z-DNA binding domain protein-1), adaptor proteins, including MLKL, and RIPK1/RIPK3 (169). Necroptosis is activated downstream of death domain receptors, such as TNFR and Fas (170). Activation of these receptors recruits adapter proteins, including FADD, TRADD, and Toll/interleukin-1 receptor (TIR)-domain-containing adapter inducing interferon-β (TRIF), which interact with RIPK1 and caspase-8 or -10 (171). Necroptosis starts with the ubiquitylation of RIPK1 by IAPs, keeping it nonfunctional and enabling proinflammatory downstream activity via NFκB (170). Following the detection of cell death signaling, RIPK1 is deubiquitylated by CYLD and can thus recruit and phosphorylate RIPK3, forming a complex called ripoptosome (172, 173). The RIPK1/RIPK3 complex further recruits and phosphorylates MLKL. The oligomerization of phosphorylated MLKL occurs in the presence of highly phosphorylated inositol phosphate (IP₆), which leads to the formation of the necrosome (44, 45). The MLKL oligomers translocate to regions in the plasma membrane enriched with phosphatidylinositol phosphate (PIP), forming pores. These processes ultimately lead to necroptotic cell death due to ion influx, cell swelling, and membrane lysis, followed by the dysregulated release of intracellular material (170, 174). The cytosolic nucleic acid sensors, such as RIG-I and cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING), also contribute to necroptotic cell death, as they induce the production of type 1 interferons (IFN-I) and TNF-α and thus promote necroptosis via an autocrine feedback loop (170, 175, 176). In macrophages, downstream of TNFR or TLR engagement, active caspase-8 cleaves the cytokine blocker N4BP1, thus promoting an increase in cytokine release (177). Following activation, RIPK3 phosphorylates the pyruvate dehydrogenase complex (PDC) in mitochondria and promotes aerobic respiration and mitochondrial ROS production (170). The DNA-dependent activator of IFN regulatory factor (DAI) recruits RIPK3 in the presence of cytosolic microbial DNA released during infection, which bypasses RIPK1-dependent activation of MLKL and leads to the formation of necrosome complex (45, 170). Necroptosis can also be triggered by the activation of pattern recognition receptors (PRRs), such as TLR3 and TLR4, intracellular sensing proteins including DAI, RIG-I, and MDA-5, and interferon signaling (178).

Several types of mycobacterial proteins have been reported to induce necroptosis in human cells, including THP-1 cells and peripheral blood mononuclear cells (PBMCs) based on propidium iodide (PI) staining and lactate dehydrogenase (LDH) assays (72–74). Mtb infection promotes necroptosis as it is a favorable outcome as necrosis for mycobacterial survival (7). Necroptosis is linked to apoptosis with caspase-8 activity, which functions as a switch between apoptosis and necroptosis. Active caspase-8 cleaves RIP1K/RIP3K, inhibiting necroptosis, whereas inactive caspase-8 promotes necroptosis (179). Mtb induces the secretion of TNF-α and at high bacterial load can signal toward necroptosis of infected host cells. Other Mtb factors that have been reported to induce necroptosis include early secretion system-1 (ESX-1) and CpnT (180). The activation of RIP1K/RIP3K/MLKL has been established in Mtb infection. Following Mtb infection, the depletion of bacterial nicotinamide adenine dinucleotide (NAD⁺) activates RIPK3 and MLKL, the key mediators of necroptosis, leading to the death of host macrophages in a RIPK3- and MLKL-dependent manner (180).

Pyroptosis as microbicidal and caspase-dependent necrosis and regulator of inflammation.

Pyroptosis is a distinct type of cell death culminating in the loss of plasma membrane integrity and induced by activation of inflammasome sensors that includes the nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) family, the DNA receptor absent in melanoma 2 (AIM2), and the pyrin receptor (170). This cell death mechanism depends on caspase-1 and can cause plasma membrane rupture (181, 182). The formation of a supramolecular assembly pyroptosome, also known as an inflammasome, is the hallmark of pyroptosis (183). The inflammatory caspase-1 acts on the substrate gasdermin and releases a 31 kDa N-terminal domain of the protein that has a pore-forming activity, which is the final and direct executor of pyroptotic cell death (43). The plasma membrane rupture causes cellular swelling and osmotic lysis releasing cytoplasmic contents resulting in inflammation. Caspase-1 activation leads to chromosomal DNA damage through an endonuclease but does not cause the characteristic DNA fragmentation. Apart from caspase-1, other caspases involved in pyroptosis include caspase-4, -5, and -11, whose mechanism of action is poorly characterized (43).

Upon internalization of Mtb in the phagosome, phagosomal pores are formed through which bacterial factors, including mycobacterial extracellular DNA, trigger the cGAS/STING pathway leading to IFN-γ response (184) inducing inflammasome formation. Extensive studies have shown the importance of the inflammasome in TB pathogenesis using pyrin domain-containing protein-3 (NLRP3) deficient mice, which effectively controls the disease progress (26, 185). The role of inflammasomes as antipathogen or antihost responses depends on the delicate balance of its activity between pyroptosis induction and the production of proinflammatory cytokines, such as IL-1β and IL-18, which contribute to inflammation and cell death (181, 186, 187). The induction of host inflammasomes depends on the nature of infecting Mtb strain and the activation status of the macrophages (188). In general, macrophages activated with IFN-γ induce greater inflammasomes, and IL-1β, IL-18, and IL-1R deficient mice with defective inflammasome activation develop more significant pathological symptoms upon Mtb infection (185–187). Mtb mutants lacking the early secreted antigenic target of 6 kDa (ESAT-6) and early secretion system-1 (ESX-1) fail to induce inflammasomes suggesting the association of mycobacterial factors such as ESAT-6 and extracellular DNA with inflammasome formation and activity (189). In addition, recent studies have found that the molecular mechanisms of NLRP3 inflammasome sensing of Mtb involve type VII secretion system ESX-1, cell surface lipids (trehalose-6,6-dimycolate/trehalose-6,6-dibehenate;TDM/TDB), secreted effector proteins (LpqH, PPE13, EST12, EsxA), and double-stranded RNA acting on the priming and/or activation steps of inflammasome activation (188, 190). Thus, it appears that ESAT-6 and ESX-1 systems-mediated activation of inflammasome causes more disease that favors Mtb survival and transmission since ESAT-6 mutants are attenuated for intracellular survival. In contrast, Mtb also mediates inhibition of the NLRP3 inflammasome by limiting exposure of cell surface ligands via Hip1 hydrolase by inhibiting the host cell cathepsin G protease through the secreted Mtb effector Rv3364c and by limiting intracellular triggers (K⁺ and Cl⁻ efflux and cytosolic reactive oxygen species production) using serine/threonine kinase PknF (188).

Pyroptosis serves as an efficient antimicrobial defense pathway. As in other microbial infections, mycobacteria induce inflammasome by recognizing pathogen-associated molecular patterns (PAMPs) (191). Pyroptosis plays a crucial role in reducing the bacterial burden. The role of inflammasome formation and IL-β processing in bacterial clearance and its inhibition by Mtb was demonstrated in a murine infection model (77, 192). Secretion of IL-1β and IL-18 by macrophages infected with Mtb was reported to be dependent on NLRP3 and ASC but not NLRC4 (193). IL-1r1^−/− and IL-18^−/− mice were very susceptible to Mtb infection (194–196). Casp1^−/− and Asc^−/− mice were also more susceptible than WT mice due to defective granuloma formation (197). However, the resistance of Nlrp3^−/− mice to Mtb infection was not significantly different from that of WT mice (197). This observation suggests the involvement of other inflammasome activation pathways in TB pathogenesis. Surprisingly, the production of IL-1β during Mtb infection was reported to occur also in a caspase-1-independent manner (198). Pathogenic Mtb counteracts macrophage pyroptosis through its secreted protein Rv3364c, inhibiting the activity of cathepsin G, a serine protease downstream of caspase-1 (158). Another secreted factor of Mtb, zinc metalloprotease-1 (Zmp-1), inhibits inflammasome and IL-β activation, thus contributing to bacterial defense against host antimicrobial responses during infection (8, 77).

Pyronecrosis is another form of pyroptosis involving inflammasome activity, which is caspase-1 independent but cathepsin B-dependent cell death pathway (199, 200). The pyronecrosis signaling pathway overlaps with pyroptosis up to the stage of inflammasome formation utilizing the NOD, LRR (leucine-rich repeat protein), NLRP3, and apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain-D(ASC) proteins (201). The caspase independence of pyronecrosis was demonstrated in a caspase-1-deficient mouse model (200, 202, 203). However, the role of this host cell death pathway in the host-pathogen interactions during TB is poorly characterized.

Other types of regulated necrosis involved in M. tuberculosis infection.

Ferroptosis.

Ferroptosis is another example of RCD pathways, driven by perturbed intracellular iron homeostasis resulting in excessive accumulation of iron-dependent ROS and lipid peroxides, surpassing cellular antioxidation capacity and damaging the membrane structure (204–206). The imbalance of oxidation stress and antioxidant status leads to lipid peroxidation causing lethal damage to nucleic acids, proteins, and lipids, which leads to ferroptosis with shrunken cell volume, condensed mitochondrial membrane densities, and reduction or absence of mitochondria crista that differ from apoptosis and necrosis morphologically (204, 207, 208).

Recent reports provide the first evidence that ferroptosis, a newly described form of regulated cell death, is detrimental for the host during Mtb infection, that the finding has important implications for the development of host-directed therapies for TB (209). Upon macrophage infection by Mtb, the increased labile iron levels promote ROS-dependent lipid peroxidation. Accumulation of lipid peroxides induces plasma membrane destabilization, leading to ferroptosis-mediated cell death. Ferroptosis drives tissue necrosis and allows Mtb to thrive and spread, probably due to iron availability and the lack of an efficient macrophage antimicrobial immune response (209, 210).

ETosis.

ETosis refers to a cell death mode involving the formation of extracellular traps (ETs) in response to bacterial and viral infections (211). Mtb induces ETs through ETosis in different host cells, including neutrophils (NETosis), macrophages (METosis), and eosinophils (EETosis) (212–214).

ETosis was first described in neutrophils, which differs from apoptosis or necrosis in releasing decondensed chromatin filled with antimicrobial peptides, microbicidal enzymes, and proteinases (215). These neutrophil extracellular traps (NETs) contain eliminated microorganisms, from viruses and bacteria to yeasts and parasites, during infection of neutrophils by respective microbe (212). Upon infection, Mtb can induce NETs in vitro, as demonstrated in isolated human neutrophils and in vivo in guinea pig lungs (163, 216). Induction of NETs by Mtb is ROS- and phagocytosis-dependent (217). However, NETs were able to trap Mtb, though it is unclear if these NETs can effectively clear the bacteria (163). The NETs had Hsp72 molecule interspersed, which were suggested to transfer danger signals from neutrophils to macrophages, thus stimulating these cells for a more aggressive innate response (217). In addition to neutrophils, macrophages generate extracellular traps called METs, which constitute a phagocytosis-independent mode of antimicrobial mechanism (213). There is limited information on mycobacterium-induced METosis. However, recent studies show that only the cord-forming phenotype of Mtb induces METosis; notably, METosis is dependent on bacterial ESAT-6 expression (218).

The eosinophil-mediated extracellular traps (EETs) are structurally different from neutrophil-induced traps and can also induce cell death in a process referred to as EETosis (214). EETosis forms thicker fibers with globular structures containing intact eosinophil granules and presents lesser susceptibility to proteolytic degradation (214). EETosis presents a more complex pathophysiology than NETosis in patients with chronic obstructive pulmonary disease, as the debris is less likely to be degraded by proteolytic activity (219). Notably, the role of EETosis in the pathogenesis of mycobacterial infection remains unknown.

Another mode of antimicrobial defense mediated by inflammatory cell death includes the generation of pore-induced intracellular traps (PITs) analogous to extracellular traps. PITs trap viable intracellular bacteria within macrophages that are killed by efferocytosis and subsequently phagocytosed by neutrophils (220).

Parthanatos.

Parthanatos is a modality of RCD initiated by hyperactivation of a specific component of the DNA damage response (DDR) machinery called poly(ADP-ribose) polymerase 1 (PARP1) and precipitated by the consequent bioenergetic catastrophe coupled to apoptosis-inducing factor mitochondria associated 1 (AIFM1, also known as AIF)-dependent and macrophage migration inhibitory factor (MIF)-dependent DNA degradation (4). Notably, parthanatos appears to occur not only as a consequence of severe/prolonged alkylating DNA damage but also in response to oxidative stress, hypoxia, hypoglycemia, and inflammatory cues (221–223).

One of the key processes of parthanatos is the binding of PARP1 to AIF, which promotes the release of AIF into the cytosol and its translocation into the nucleus, where it mediates large-scale DNA fragmentation and chromatin condensation (221, 224–226). Recent studies show that MIF functions as the main nuclease precipitating parthanatos in a recent screening for AIF-binding proteins (227). Thus, cytosolic AIF reportedly promotes the translocation of MIF into the nucleus, where MIF precipitates parthanatos by catalyzing DNA cleavage. In addition, MIF depletion or specific mutations in its nuclease domain confer protection against parthanatos in vitro and in vivo (227).

Inhibitors of PARP1, such as BMN 673 (talazoparib), ABT-888 (veliparib), and AG-014699 (rucaparib), are often used to inhibit parthanatos (228). Following Mtb infection, treatment with these three compounds failed to inhibit Mtb-induced macrophage cell death, suggesting that parthanatos is unlikely to account for the death of the Mtb-infected macrophages and is not the dominant cell death mode or that the Mtb-activated parthanatos pathway is not amenable for inhibition by these drugs (229). In Mtb infection, intracellular stress can activate PARP1, which signals mitochondrial dysfunction potentially involving receptor-interacting protein kinases 1 (RIPK1), highlighting a connection between death receptors and TLR signaling of host cells (11, 155, 230).

Autophagy

Autophagy is a fundamental, innate cellular homeostasis process that recycles defective or malfunctioning cellular organelles and proteins into an energy conservation mechanism (231). Currently, 32 different autophagy-related genes (Atg) have been identified by genetic screening, with many of these genes conserved in multiple organisms, emphasizing the importance of the autophagic process in responses to starvation across phylogeny (232). The three primary types of autophagy occurring in mammalian cells are microautophagy, macroautophagy, and chaperone-mediated autophagy (CMA) (233). Among these three modes, macroautophagy is the most studied and synonymous with autophagy. Macroautophagy involves the formation of a cargo lined by phagophore, a double-layered membrane derived from the endoplasmic reticulum (ER) that isolates the autophagic contents from the cytoplasm, which then fuses with lysosome (233, 234).

Macroautophagy occurs constitutively at a low level but can further be induced under stress conditions, such as nutrient or energy starvation, to degrade cytoplasmic materials into metabolites that can be used in biosynthetic processes or energy production, allowing for cell survival (235). Under normal conditions, macroautophagy assists in cellular maintenance by specifically degrading damaged or superfluous organelles (236). Macroautophagy eliminates intracellular pathogens as part of host defense and is called “xenophagy,” whereby bacteria are engulfed by autophagosomes and degraded after fusion with lysosomes to form autolysosomes (237–239). Recent studies have demonstrated that xenophagy is essential in multiple diseases such as cancer, diabetes, cardiomyopathy, neurodegeneration, liver disease, autoimmune diseases, and pathogen infections, including Mtb (240). In this review, we focus on xenophagy, which will hereafter be referred to as “autophagy,” and its roles in host defense against Mtb.

Autophagy as an innate immune defense mechanism against M. tuberculosis infection.

As an immune mechanism, autophagy controls inflammation and acts as a cell-autonomous defense against intracellular microbes, including Mtb. Another significant role of autophagy is its anti-inflammatory and tissue-sparing function. This combination of antimicrobial and anti-inflammatory actions prevents active disease in animal models (241). In humans, genetic links between autophagy and susceptibility to TB provide further support for these combined roles of autophagy (241, 242). The formation of a double membrane phagosome, which fuses with a lysosome, is a hallmark of autophagy (233). It is a constitutive cellular process induced under stress conditions such as nutrient starvation, oxidative stress, pathogen infection, and hypoxia, which degrades cytoplasmic material into metabolites and degrades cytoplasmic foreign bodies (243, 244).

Autophagy is activated by microbial pattern receptors, including the Toll-like receptors (TLRs), a retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), and nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs), which activate the downstream signaling pathways like p38 mitogen-activated protein kinase (p38MAPK), RIP1/extracellular signal-regulated kinase (ERK), and subsequent activation of the vacuolar protein sorting 34 (VPS-34) complex (245, 246), as illustrated in Fig. 2. TLR4 was the first PRR linked to autophagy through Toll/interleukin-1 receptor (TIR)-domain-containing adapter inducing interferon-β (TRIF)-p38 rather than myeloid differentiation primary response 88 (MyD88) (247–249). The innate and adaptive immune responses converge at autophagy to successfully eliminate pathogens. Cytokines such as IL-1β and IFN-γ induce autophagy and are host-protective (250, 251). Autophagy is also induced by immune cell-cell contact between specific T-cells and Mtb-infected macrophages (252). The autophagy signaling ultimately kills mycobacteria through lytic and antimicrobial properties, which are more robust than antimicrobial compartments of the conventional phagosomes (253). A fraction of bacteria escape the phagosome into the cytosol through permeabilization of the phagosomal membrane and are targeted for selective autophagic process, wherein the bacteria are ubiquitinated (248).

Figure. 2.

Mycobacterium tuberculosis (Mtb) modulation of host autophagy signaling. Multiple surface receptors and intracellular stress signals, including Mtb factors, modulate autophagy signaling. Microbial surface pattern receptors such as Toll-like receptors (TLRs), RIG-I like receptors (RLRs), and NOD-like receptors (NLRs) recognize mycobacterial secreted factors, which further activate p38 mitogen-activated protein kinases (MAPK) and receptor-interacting serine/threonine-protein kinase 1 (RIP1)/extracellular signal-regulated kinases (ERK) that phosphorylate phosphoinositide 3-kinases (PI3K) and signal Beclin-1 phosphorylation to trigger autophagy. Autophagy culminates in the fusion of lysosomes with autophagosomes. Early secreted antigenic target of 6 kDa (ESAT-6), lipoarabinomannan (LAM), enhanced intracellular survival protein 2 (Eis), serine/threonine-protein phosphatase 2 A activator (PtpA), protein kinase G (PknG), early secretion system-1 (Esx-1), LAM/mannose-capped lipoarabinomannan (ManLAM) inhibit autophagy. P in the blue circle represents forward signaling by phosphorylation, while P in the red circle represents inhibition of phosphorylation and, thereby, inhibition of downstream signaling. Following phagocytosis, Mtb resides in the phagosome and blocks phagosome maturation. Mtb secretes Esx-1, promoting phagosome damages that trigger ubiquitination, recruitment of autophagic adaptors and mycobacterial capture via stimulator of interferon genes (STING). NOD, nucleotide binding and oligomerization domain; RIG-I, retinoic acid-inducible gene-I. Figure created with images from smart.servier.com.

Mtb replicates in macrophages (MΦ) in part by inhibiting phagosome-lysosome fusion until IFN-γ activates the MΦ to traffic Mtb to the lysosome (254, 255). The role of autophagy in host cell death is mainly inferred from studies performed in cultured MΦ or dendritic cells (DC) where Mtb colocalizes with autophagy-related (ATG) proteins, such as ATG5, ATG12, ATG16L1, p62 (SQSTM1), NDP52, calcium binding and coiled-coil domain 2 (CALCOCO2/NDP52), Beclin-1, and microtubule-associated protein 1A/1B-light chain 3 (LC3) (248, 256–259), stimulation of autophagy increases bacterial killing (259–261), and inhibition of autophagy allows for increased bacterial survival (241, 248, 256, 259, 260). To understand the role of autophagy in Mtb elimination in vivo, individual Atg genes were genetically deleted in mice using the Cre-loxP system, which knocked out the concerned gene in a specific cell type and was used for infection studies (262). Following the Mtb challenge, of all the atg gene knockouts, only atg5 significantly altered mycobacterial persistence, suggesting the dispensable nature of ATG-associated proteins for the progression of TB (262). Furthermore, ATG5 has a crucial role in autophagosome formation, and atg5 knockout (KO) mice (atg5^fl/fl-LysM-Cre) showed increased Mtb burden and succumbed to infection at 30 days postinfection (248, 262, 263). Thus, autophagy confers host cell protection by activating several antimycobacterial responses in the infected cells. In addition, atg5^fl/fl-LysM-Cre mice lacking ATG5 in monocyte-derived cells and neutrophils (polymorphic mononuclear cells, PMN) have an extremely severe phenotype similar to mice lacking IFN-γ signaling (264).

Among the various ATG factors involved in autophagy, ATG5 is the most well studied during Mtb infection; further, autophagy-independent functions of ATG5 have also been reported (265–270). A genetic approach was utilized to determine the role of Atg and the requirement of autophagy in the host resistance to Mtb infection in vivo. Recent studies have discovered that, contrary to expectation, autophagic capacity does not correlate with the outcome of Mtb infection (262). Instead, ATG5 is involved in preventing PMN-mediated immunopathology and protects against Mtb infection (262). By analyzing different Cre-mediated deletion strains, it is revealed that loss of Atg5 in PMN, but not alveolar MΦ or DC, can result in loss of control of Mtb infection, but the severe susceptibility of the Atg5^fl/fl-LysM-Cre mice relies on the deletion of atg5 in multiple LysM⁺ cell types. These findings also show a PMN-intrinsic role for ATG5 during Mtb infection. Notably, the reversal of all phenotypes in the Atg5^fl/fl-LysM-Cre mice upon PMN depletion positions PMN as a significant driver in the dysfunctional response in these mice. These results point to a model where infection with Mtb induces a proinflammatory response that leads to the recruitment of PMN to the lung. The absence of Atg5 expression within the responding myeloid cells leads to uncontrolled accumulation of PMN in the lung, which causes increased pathology and likely provides an expanded niche for bacterial infection. The animal then succumbs to infection before the adaptive immune response can control the inflammation and bacterial replication. Together, the in vivo genetic analyses presented here shift the understanding of the role of ATG5 during Mtb infection, revealing a new outcome of ATG5 activity in early events of the innate immunity required to regulate TB disease pathology and Mtb replication (262).

M. tuberculosis inhibits autophagy to promote survival in host cells.

The formation of the autophagosome following Mtb infection and the fusion to a lysosome is broken down into five main steps: 1) initiation, 2) elongation, 3) maturation, 4) fusion, and 5) degradation (271). Autophagy initiation is regulated by the master regulator, the mammalian/mechanistic target of Rapamycin (mTOR). It is a negative regulator of autophagy, meaning its dephosphorylation is responsible for autophagy induction. Dephosphorylation of mTOR results in the translocation of the Unc-51-like autophagy activating kinase (Ulk1/2)-ATG13-FAK family-interacting protein (FIP200)-ATG101 complex to the endoplasmic reticulum (272, 273). Class III phosphatidylinositol-3-kinase (PI3K) activates the VPS34-Beclin1-VPS15-ATG14 complex. The PI3K complex induces phosphatidylinositol-3-phosphate, which then recruits double Fab1, YOTB/ZK632.12, Vac1, and EEA1 (FYVE)-containing protein 1 (DFCP1) and WD-repeat domain phosphoinositide-interacting (WIPI) family proteins to initiate the omegasome/phagophore formation (272, 274–276). Elongation of the phagophore into the autophagosome is conducted by the ATG7–ATG10 complex and then conjugated to ATG12-ATG5-ATG16L1 on the phagophore membrane (277). ATG4 cleaves LC3 into LC3-I, while the ATG7–ATG3 complex lipidates LC3-I into LC3-II by conjugating phosphatidyl-ethanolamine (PE). The completed autophagosome fuses with the lysosome to degrade the autophagosome cargo for subsequent metabolite recycling or antigen presentation (Fig. 2) (278–282).

Various bacterial effector proteins are known to modulate autophagy. Many of these effectors are secreted through the type I to type VII and type IX secretion systems (283). Mycobacteria have numerous Type VII secretion systems (Esx1–Esx5). Mtb Esx1 is responsible for the puncture of the phagosome, allowing for mycobacterial escape (284). Mtb cytosolic DNA is recognized by the cytosolic DNA sensor, cyclic GMP-AMP synthase (cGAS), resulting in the release of cyclic guanosine monophosphate (cGAMP). cGAMP is recognized by the stimulator of interferon genes (STING), leading to type I IFN release and the recruitment of autophagy receptors p62, NDP52, and optineurin (248, 285). These receptors are recruited to the ubiquitinated pathogen, thereby allowing for specific targeting by the autophagosome (Fig. 2). The receptors contain an LC3 interaction region (LIR) to bind the LC3 autophagy protein (286–288).

EspB is a part of the Esx1 secretory apparatus responsible for the secretion of early secretory antigenic target-6 (ESAT-6). Treatment of macrophages with EspB protein demonstrates downregulation of the IFN-γ receptor IFN-γR1, resulting in the inhibition of STAT-1 activation even in the presence of IFN-γ (289). EspB and ESAT-6 are not the only Mtb proteins linked to the inhibition of autophagy. The “enhanced intracellular survival” (eis) gene of Mtb confers enhanced survival of nonpathogenic Mycobacterium smegmatis in macrophages, although it is not required for the persistence of pathogenic Mtb in these cells (78, 290). During Mtb infection, eis significantly inhibits the activation of c-jun N-terminal kinase (JNK), which prevents the induction of non-canonical autophagy through ATG7. JNK activation also induced reactive oxygen species (ROS) generation and significantly increased type 2 macrophage cell death by Mtb eis deletion mutant (78). Studies have revealed that Mtb eis is an efficient N(ε)-acetyltransferase that acetylates Lys55 of dual-specificity protein phosphatase 16 (DUSP16)/MAPK phosphatase-7 (MKP-7), a JNK-specific phosphatase, thus inhibiting JNK phosphorylation and inflammatory cytokine generation (291). Mtb eis was also found to substantially inhibit the production of TNF-α, IL-4, and IL-6, while simultaneously stimulating IFN-γ and IL-10 secretion (78, 292, 293).

Mtb inhibits autophagy to protect against bacterial clearance and host cell death, which impedes antigen presentation (271). The Mtb PE_PGRS47 protein inhibits autophagy and limits MHC (major histocompatibility complex) class II antigen presentation (281). Other Mtb PE/PPE proteins are also known to inhibit autophagy, including PE_PGRS41 (58) and PE_PGRS29 (294), by interacting with autophagy machinery. Mtb also secretes a probable ligase (CpsA) to inhibit the noncanonical autophagy pathway designated as LC3-associated phagocytosis (LAP) and NADPH oxidase (295). In contrast to canonical autophagosomes, LAP does not result in double-membrane structures and instead promotes rapid phagosome maturation (296). This cellular process limits the phagocytosed ability of the pathogen to replicate by accelerating phagosome maturation while regulating the IFN pathway and antigen presentation (271). In addition, the maturation of Mtb-containing autophagosomes into autolysosomes was inhibited by blocking the recruitment of the late endosome marker Rab7 (124). Inhibition of Rab5 conversion to Rab7 in endosomes is a well-established method in which mycobacteria inhibit lysosomal fusion (297).

An interplay between autophagy and innate immunity following M. tuberculosis infection.

TLR signaling and autophagy are linked; while TLR signaling recognizes pathogen-associated molecular patterns through pattern-recognition receptors (PRRs), the autophagic pathways eliminate harmful pathogens (298). Previous studies show that many Mtb molecules act as ligands for TLRs and non-TLRs, and the recognition of mycobacterial molecules may initiate or regulate autophagy (299–301). Multiple TLR stimuli, in addition to NOD-like receptor-induced signals or CD40, can activate autophagic responses to induce host innate immunity and overcome a mycobacterial phagosome arrest (247, 302–305). It is of interest that, regardless of the TLR ligand stimulus, host autophagy activation can lead to the clearance of mycobacteria-containing phagosomes through the recruitment of autophagosomes and induction of phagosomal maturation (247, 304). Furthermore, several crucial components of TLR signaling, such as MyD88, TIR-domain-containing adapter-inducing interferon-β (TRIF), and mitogen-activated protein kinase (MAPK) are involved in the regulation of autophagy activation (247, 304, 306).

Several mycobacterial factors can activate or modulate host autophagy. The well-known mycobacterial TLR2 ligand, 19-kDa lipoprotein, activates autophagic responses in monocytes/macrophages. This only occurs with enough active vitamin D metabolites or with Cyp27b1 gene activation, followed by functional vitamin D signaling (307). In non-TLR-mediated autophagy, muramyl dipeptide (MDP) leads to the formation of autophagosomes through receptor-interacting serine-threonine kinase-2 (RIPK-2), ATG5, ATG7, and ATG16L1 (308, 309). Both NOD1 and NOD2 are involved in autophagy activation and bactericidal responses by recruitment of ATG16L1 to the plasma membrane and bacterial wrapping by autophagosomes (309). Data show that NOD2 activation leads to the expression of autophagy proteins such as LRGM, LC3, and ATG16L1 in human alveolar macrophages, which contribute to improved intracellular control of virulent Mtb (257). In addition, mutant mice harboring variants of NOD2 (3020insC) showed elevated NF-κB activation in response to MDP and upregulated IL-1β secretion (310). Mutant NOD2 (Crohn’s disease-associated NOD2 frameshift mutation) mice also failed to recruit ATG16L1 to the plasma membrane and induce autophagy, suggesting that NOD2 and autophagic pathways are functionally linked to increased bacterial killing and maintaining host immune homeostasis (298).

The mycobacterial NOD2 ligand MDP, which is N-glycosylated by N-acetyl muramic acid hydroxylase, shows more potent NOD2-stimulating immune activation in mouse models (311). Thus, mycobacterial N-glycosylated MDP may induce autophagy to a greater extent than N-acetylated MDP, the most common form in bacteria. It remains unclear whether mycobacterial signaling induced by the C-type lectin receptor Dectin-1 or Mincle and by scavenger receptors is involved in activating or regulating the autophagic pathway during infection (298). Understanding the receptor-specific regulation and induction of autophagy by individual mycobacterial ligands or antigens may facilitate the development of effective therapeutic and vaccine strategies against human TB.

Autophagy modulates the M. tuberculosis-induced cytokine response.

Multiple studies have provided insight into the roles of various protective cytokines in activating autophagy, thus promoting antimycobacterial immune defenses. IFN-γ enhances the autophagic control of Mtb, whereas the Th2 cytokines IL-4 and IL-13 inhibit those effects (312). IFN-γ and its effector mechanisms are mediated through multiple pathways: the induction of antimicrobial effectors, including inducible nitric oxide synthase 2, which may produce nitric oxide; the IFN-inducible GTPase family LRG47 (Irgm) in mice (313); and upregulated expression of MHC class II molecules, which play a central role in antigen processing and presentation (314). Autophagy activation promotes antigen processing and presentation by MHC class II molecules and results in the bridging of innate and adaptive immune responses. In this aspect, IFN-γ may facilitate host protective immune responses through the connection of autophagy activation and innate immune activation for controlling Mtb infection (298).

Proinflammatory TNF-α plays an important role in the activation of phagosome maturation because TNF-α neutralization suppresses IFN-γ-induced phagosome maturation in primary human peripheral blood monocyte-derived macrophages (BMDMs) (315, 316). These results indicate that TNF-α may synergize IFN-γ-induced antimicrobial and autophagic responses against intracellular mycobacteria (317). Other cytokines, including IL-2, CCL2, IL-6, and TGF-β, play key roles in positively regulating autophagy in various cell types, such as IL-2 in CD4⁺ T cells, CCL2 and IL-6 in CD11b⁺ peripheral blood mononuclear cells (PBMCs), and TGF-β in hepatocarcinoma cell lines (317). Various innate cytokines, including CCL2, IL-6, and TNF-α, are increased during the initiation of mycobacterial infection. These cytokines play a role in granuloma formation, pathophysiology, and local immunity in the lungs during Mtb infection (318). Thus, it is crucial to determine the roles of each and/or combined cytokines in controlling the progression of Mtb infection.

Mtb-induced IL-12p40 and IL-23p19 expression are negatively regulated in human macrophages by the mammalian targets of rapamycin (mTOR)/70-kDa ribosomal S6 kinase 1 (S6K1) pathway (319). These findings suggest that IL-12/IL-23 is likely induced during autophagic pathway activation in mycobacterial infection. In addition, IL-23 production by Mtb in dendritic cells is mediated by the combined sensing of NOD2 and TLR2, whereas IL-12p70 production requires IFN-γ or the TLR7/8 ligand R848 (320), suggesting a mechanistic link between protective cytokines and the induction of autophagy.

Autophagy negatively regulates the activation of transcription, processing, and secretion of several proinflammatory cytokines, including IL-1α, IL-1β, and IL-18 (321–325). Negative regulation of the autophagy pathway in controlling IL-1β production involves two mechanisms: targeting pro-IL-1β for degradation in lysosomal compartments and inhibiting NLRP3 inflammasome activation (323, 326). In contrast, it was revealed that autophagy played an important role in the induction of TNF-α transcription and secretion (322). Another finding showed an under-appreciated contribution of autophagy to the synthesis and secretion of the proinflammatory cytokine IL-1β (327). The cross talk between autophagic pathways and various activating/inhibiting cytokine stimuli may be influenced by the microenvironment, which affects the behavior of immune cells, or by functional properties of the innate receptors or signaling molecules that contribute to cytokine generation and the autophagic process (298).

PANoptosis

PANoptosis is an inflammatory programmed cell death (PCD) pathway activated by specific triggers and regulated by the PANoptosome complex that has key features of pyroptosis, apoptosis, and/or necroptosis; hence the term PANoptosis. However, it cannot be classified in any of the PCD pathways mentioned earlier (328). The PANoptosome provides a molecular scaffold for the contemporaneous engagement of key molecules from pyroptosis, apoptosis, and/or necroptosis (329–331). PANoptosis has been observed in various viral, bacterial, and fungal infections, autoinflammatory diseases, and cancer. This mode of PCD has very significant pathophysiological relevance to TB since this pathway can module the host cell response and plays a crucial role in determining the outcome of host-pathogen interactions (332). The characterization of PANoptosis was also central to the molecular understanding of cytokine storm, a life-threatening condition caused by excessive cytokine production due to inflammatory cell death (333). In addition to pathogenic and inflammatory disease triggers, cytokines can induce PANoptosis (334).

Mtb infection results in Esx-1-mediated plasma membrane damage responses that cause potassium ion (K⁺) efflux, leading to the activation of caspase-1/NLRP3/Gasdermin-D (GSDMD)-mediated pyroptosis in human monocytes and macrophages (335). Phospholipids, platelet-activating factor (PAF), and PAF-like lipids can activate the canonical NLRP3 inflammasome through mechanisms involving K⁺ efflux and Ca²⁺ influx independent of the PAF receptor (336). Although K⁺ efflux is required for the NEK7-NLRP3 association, the mechanisms underlying K⁺ efflux-mediated NLRP3 inflammasome activation are not fully understood (337). Thus, more studies are needed to elucidate how K⁺ efflux triggered by multiple signals activates the assembly of the NLRP3 inflammasome complex.

CROSS TALK BETWEEN DIFFERENT TYPES OF HOST CELL DEATH FOLLOWING M. TUBERCULOSIS INFECTION

Various reports have demonstrated that virulent Mtb can evade the immune response by modulating cell death mechanisms. Caspase-8 was initially identified as a component of extrinsic apoptotic signaling platform death-inducing signaling complex (DISC) (338, 339) and later discovered as part of the cytosolic TNF‐induced complex II (340). Soon, it became apparent that caspase‐8 has a more complex role in regulating multiple cell death pathways (84). The enzymatic activity of caspase‐8 determines if cells survive or die via apoptosis or necrosis. Pharmacological inhibition of caspase‐8 (e.g., by zVAD‐FMK or Emricasan) or inhibition by FLIP(S/R) leads to necroptosis and RIP1 activation (341). Interestingly, RIP1 also restricts caspase‐8‐mediated cell death as the loss of RIP1 is sensitized to TNF‐induced apoptosis (46, 342). The interplay of apoptosis and necrosis is apparent in many inflammatory in vivo models where they are frequently simultaneously activated (343), given that the majority of signaling proteins are common to both pathways, and the balance of expression or activation of critical factors can tip the balance in favor of apoptosis or necrosis (84).

The overlapping mechanisms of cell death between apoptosis and necrosis are evidence for the cross talk between the two different pathways of immune responses. Other types of cell death, such as pyroptosis and necroptosis, have overlapping characteristics of apoptosis and necrosis and are the pinnacle of cross talk between these pathways (4, 7, 344). Several host factors interconnect the cell death pathways by interacting with molecules and regulators involved in all these different pathways (345). TNF-α is known for its pleiotropic effects, leading to various host cell death modalities, including apoptosis, necrosis, and necroptosis (7, 346). cFLIP, an inhibitor of caspase-8 (hence inhibitor of extrinsic apoptosis), can interact with ATG3 and inhibit the promotion of autophagy. Interaction of cFLIP_L (cellular Fas-associated death domain-like interleukin-1-beta-converting enzyme), one of the isoforms of cFLIP, with caspase-8 leads to caspase-8 inactivation, but its ability to cleave RIP1 is retained and can therefore inhibit necroptosis (347). In addition, Bcl-2 interacts with Beclin-1, a crucial player in the autophagy pathway. However, Bcl-2 is also known to be upregulated in mycobacterial infection, which not only inhibits apoptosis but also inhibits autophagy. Other factors such as caspases-3, -7, and -8 cleave Beclin-1 and class III phosphoinositol-3-kinase (PI3K) no longer signal the autophagy pathway. The cleaved Beclin-1 localizes to mitochondria and reinforces apoptosis, and inhibits autophagy. The autophagic protein ATG5, a calpain substrate, functions similarly by localizing to mitochondria, associates with Bcl-xL, and induces apoptosis (269). Furthermore, Mtb secretes factors such as ESAT-6, which either causes apoptosis (at lower bacterial load) or necrosis (at higher bacterial load). ESAT-6 can also induce NETosis that can be attributed to the pore-forming nature of ESAT-6 along with PDIM (Phthiocerol dimycocerosates), which causes the destabilization of membranes. At lower bacterial load, ESAT-6 induces mild inflammation, which is sufficient to induce apoptosis, whereas, at higher bacterial load, it causes loss of membrane integrity, triggering necrosis or its forms, including necroptosis and pyroptosis (7, 147, 149). Esx-1 and LAM induce most of the cell death modalities, including apoptosis, necrosis, and autophagy (19, 20). The schematic representation of the cross talk involving multiple cell death pathways in Mtb infection is presented in Fig. 3.

Figure 3.

Cross talk of mycobacterial factors during different forms of host cell death. Various cell death modalities may result from either mycobacteria-induced events or host-induced innate defense mechanisms. The dynamics of these modalities determine the outcome of Mycobacterium tuberculosis (Mtb) infection. Several host factors are involved in multiple cell death pathways, and Mtb factors can inhibit host defense mechanisms at various levels. Arrows indicate activation, deactivation, and forward signaling, the color of the signaling components defines the particular pathway as indicated. The pathways are simplified to accommodate the cross connections showing the mycobacterial factors that increase the complexity of multiple cell death networks in eliminating mycobacteria. ASC-1, alanine/serine/cysteine transporter-1; ATG3, autophagy related 3; Bcl-2, B-cell leukemia/lymphoma 2; Bid, BH3 interacting domain; Casp, caspase; cGAMP, cyclic guanosine monophosphate/adenosine monophosphate; CytC, cytochrome C; DAMPs, death associated molecular patterns; DAPK, death associated protein kinase; eDNA, extracellular DNA; ER, endoplasmic reticulum; GSDMD, Gasdermin D; LC3, light chain 3; MLKL, mixed lineage kinase domain-like pseudokinase; MMP, matrix metalloproteinase; mTORC, mammalian target of rapamycin-C; NLR, NOD-like receptor; NOD, nucleotide binding oligomerization domain; p53, protein 53; PAMPs, pathogen associated molecular patterns; PI3K, phosphatidylinositol 3-kinase; RIPK, receptor interacting serine/threonine kinase; ROS, reactive oxygen species; tBid, truncated BH3 interacting domain; TBK, Tank-binding kinase; TLR, Toll-like receptor; TNF, tumor necrosis factor-alpha; TNFR, tumor necrosis factor-alpha receptor; TRADD, TNF receptor superfamily member 1 A associated via death domain; ULK, Unc-51 like autophagy activating kinase; VPS34, vacuolar protein sorting 34; ZMP-1, zinc metalloproteinase-1. Figure created with images from smart.servier.com.

A recent study presents evidence supporting the simultaneous activation of several cell death pathways by virulent Mtb strains in infected macrophages (348). Mtb H37Ra (avirulent) and H37Rv (virulent) strains were used to infect human monocyte-derived macrophages (MDM) in vitro, and the molecules involved in apoptosis, necroptosis, and pyroptosis were measured. Results revealed that Mtb H37Rv infection increased the Bcl-2 transcript and protein, decreased the BAX transcript, and increased phosphorylated Bcl-2 at the protein level. H37Rv infection was also shown to increase the expression of necroptotic pathway molecules, including ASK1, p-38, RIPK1, RIPK3, and caspase-8. In contrast, avirulent Mtb H37Ra infection increased the expression of caspase-8 and decreased RIPK3 expression. In that study, while IL-1β expression was independent of Mtb virulence, NLRP3 and CASP1 expression was increased at a low multiplicity of infection (MOI) in both strains. These observations suggest a role for bacterial MOI in activating host cell pyroptosis. In addition, virulent Mtb inhibits apoptosis mediated by Bcl-2 family molecules; but, at the same time, increases the expression of molecules involved in apoptosis, necroptosis, and pyroptosis at the transcriptional and protein levels, which might function as a mechanism to avoid the immune response and promote bacterial survival inside the host cells (348).

It is important to note that other pathways get activated or modulated when Mtb subverts one cell death pathway. This clearly defines the host signaling toward a particular outcome. Apoptosis appears as the first mode of the host cell’s innate defense mechanism that induces cell death, whereas the autophagy-dependent cell death pathway is the final option when other death modalities are subverted. Mtb blocks several host cell death mechanisms that lead to bacterial clearance (apoptosis, autophagy, and pyroptosis) while promoting those that support mycobacterial persistence (necrosis, necroptosis, apoptosis in contextual mode), leading to successful infection. Thus, the cross talk between the various cell death modalities reflects the complex dynamics during host-pathogen interaction in TB.

THE ROLE OF MICRORNAS AND LONG NONCODING RNAs IN HOST CELL DEATH PATHWAYS FOLLOWING M. TUBERCULOSIS INFECTION

MicroRNAs (miRNAs) are small noncoding RNAs that regulate host defense mechanisms against viruses, bacteria, and fungi. It is known that bacteria modulate host miRNA expression and, thereby, the host response to innate and adaptive immunity (349). miRNAs are involved in the delicate interplay between Mtb and its host, which contributes to dynamic changes in gene expression patterns and influences host-pathogen interactions (Table 3). Differential expression of miRNAs upon infection with Mtb regulates host signaling pathways linked to inflammation, autophagy, apoptosis, and polarization of macrophages (389).

Table 3.

miRNAs influencing multiple host cell death pathways in M. tuberculosis infection

miRNA	Remarks	Cell Death Pathway	Reference
miR-155	Highly overexpressed in Mtb infected mice 30-day postinfection	Apoptosis	(350)
	ESAT-6 promotes apoptosis of macrophages through miR-155-SOCS1 interaction		(351)
	Inhibits apoptosis of infected cells by targeting FOXO3		(352)
	Induces apoptosis following BCG infection		(353)
miR-155-5p	Promotes the intracellular Mtb replication during early innate immune responses and helps the survival of infected macrophages	Apoptosis	(354, 355)
miR-196b	Increased expression in patients with active TB and functions as a marker for active TB	Apoptosis	(356)
	Induces apoptosis by targeting c-Myc and Bcl2		(357)
miR-325-3p	Promotes Mtb persistence and latency in the host cell	Apoptosis	(358)
	Inhibits apoptosis through activation of STAT3 signaling		(359)
miR-20a-5p	Downregulated by Mtb infection, triggers cell apoptosis to facilitate mycobacterial clearance through targeting JNK2 in human macrophages	Apoptosis	(360)
miR-708	Upregulated in sera of patients with TB	Apoptosis	(361)
	Induces apoptosis by targeting E-cadherin regulators		(362)
miR-let-7e	Inhibits apoptosis by targeting the executioner of programmed cell death Caspase-3	Apoptosis	(363)
miR-29a	Inhibits apoptosis upon mycobacterial infection via Caspase-3 and -7	Apoptosis	(363)
miR-582-5p	Upregulated in patients with active TB, inhibits apoptosis of monocytes by targeting FOXO1	Apoptosis	(364)
miR-21	Inhibits apoptosis, targets several genes of both intrinsic and extrinsic apoptosis pathways	Apoptosis	(365)
	Decreased expression in CD4 T cells and peripheral blood of patients with TB		(366)
	Functions as a key switch in the inflammatory response		(367)
	Inhibits apoptosome formation by targeting Bcl-2 pathway		(64)
miR-145	Downregulation of miR-145 leads to overexpression of its targets and inhibition of apoptosis	Apoptosis	(368)
miR-125b	Mtb lipomannan destabilizes TNF biosynthesis by miR-125b regulation	Apoptosis	(369)
miR-223	Upregulated in patients with active TB and inhibits apoptosis of macrophages by targeting FOXO3	Apoptosis	(370)
miR-27b	Modulates inflammatory response and apoptosis upon Mtb infection	Apoptosis	(371)
miR-155	Promotes autophagy to eliminate intracellular Mtb by targeting Rheb	Autophagy	(261)
miR-99b	Downregulates the production of TNF-α and TNF-α receptor	Autophagy	(372)
miR-26a	Impairs Mtb survival in macrophages by targeting the transcription factor Klotho-like factor-4 (KLF4)	Autophagy	(373)
miR-23a-5p	Overexpression of miR-23a-5p prevented Mtb-induced autophagy in macrophages by modulating TLR2/MyD88/NF-κB signaling	Autophagy	(374)
miR-33	Enables Mtb intracellular survival and persistence inside the host	Autophagy	(375)
	Targets autophagy effectors (ATG5, ATG12, LC3B and LAMP1), inhibits autophagy, and reprogram host lipid metabolism		(375)
miR-27a-5p	Downregulates Ca2+ signaling leading to inhibition of autophagosome formation and xenophagy; promotes intracellular survival of Mtb	Autophagy	(376)
miR-144-3p	Inhibits autophagy activation by targeting ATG4 in RAW264.7 macrophages	Autophagy	(377)
miR-144-5p	Inhibits phagosome maturation and T cell function	Autophagy	(378)
	Upregulated in human MDMs upon Mtb infection		(379)
	Targets the 3′-UTR of DNA damage-regulated autophagy modulator 2 (DRAM2) in PBMC-derived macrophages and THP-1 cells		(379)
miR-889-5p	Overexpression inhibits autophagy and maintains bacterial survival in granulomas	Autophagy	(380)
miR-125a	Increases robustly in TB and inhibits autophagy by targeting UVRAG and anti-microbial responses	Autophagy	(381)
miR-30a	Inhibits autophagy and promotes Mtb survival mechanism in human macrophages by targeting Beclin-1	Autophagy	(382)
miR-3619-5p	Targets cathepsin S and affects autophagy in THP-1 macrophages	Autophagy	(383)
miR-17-5p	Involved in autophagy by targeting ULK-1, controls the survival of mycobacteria in macrophages	Autophagy	(384)
	Regulates autophagy in Mtb-infected macrophages by targeting Mcl-1 and STAT3		(385)
miR-20a	Inhibits autophagy by targeting ATG7 and ATG16L1 and promotes mycobacterial survival in macrophages	Autophagy	(386)
miR-142-3p	Inhibits autophagy upon Mtb infection, targets N-Wasp leading to reduced phagocytosis	Autophagy	(387)
miR-20b	Inhibits Mtb-induced inflammation in mouse lung by targeting NLRP3	Pyroptosis	(388)

Several miRNAs are regulated following mycobacterial infection from several in vitro and in vivo studies. ATG, autophagy related; Bcl2, B-cell leukemia/lymphoma-2; ESAT-6, early secreted antigenic target of 6 kDa; LC3, light chain 3; MDM, monocyte-derived macrophages; Mtb, Mycobacterium tuberculosis; NLRP3, NOD-, LRR- and pyrin domain-containing protein 3; PBMC, peripheral blood mononuclear cell; TB, tuberculosis.

The roles of various miRNAs have been demonstrated in mycobacterial infection (390, 391). In vitro studies to identify differentially expressed miRNAs during Mtb infection using H37Rv and H37Ra revealed nine genes that were differentially expressed (392). One of these genes includes miR-155, which is consistently induced across studies and is known to be upregulated by mycobacterial components lipomannan (LM) and ESAT-6, which are very well associated with apoptosis (351, 353). miR-155 is known to target PKA signaling, activation of caspase-3, Bak-1, and translocation of cytochrome C through MAPK signaling. Loss of miR-155 through KO in BCG caused compromised apoptosis of host cells (353). miR-155 is also known to enhance autophagy of mycobacteria-infected macrophages by targeting Ras homolog enriched in brain (Rheb) (261). A study involving differential expression of miRNAs using PBMCs from patients with TB compared with healthy individuals revealed the role of miR-582, which caused inhibition of apoptosis by downregulating FoxO1 that regulates host response to Mtb infection (364).

miR-29 targets Bcl-2, MCL-1, p85a complex (kinase), CDC42 and thereby regulates apoptosis pathway in immune cells; inhibits IFN-γ pathway and increases apoptosis of cells involved in anti-tubercular responses. miR-29 contributes to the inhibition of autophagy by downregulating the secretion of IFN-γ (393). Another miRNA molecule, miR-21, is activated upon Mtb infection mediated by ESAT-6 and inhibits the secretion of proinflammatory cytokines like IL-12, IL-6, and TNF-α. It also upregulates the production of anti-inflammatory cytokine IL-10 and promotes dendritic cell apoptosis by targeting Bcl-2 (366). Several miRNAs induced during Mtb infection and the host cell modalities affected are listed in Table 3. Recent studies suggest that miRNAs regulate innate immune response against Mtb infection. Moreover, differential miRNA expression in Mtb infection can reflect the disease progression and help distinguish between active and latent Mtb infection (LTBI) (391). These findings helped in the identification and application of miRNAs as potential biomarkers of TB (391). In addition, the active participation of miRNAs in modulating autophagy and apoptosis responses against Mtb provides a better understanding of miRNAs as host-directed therapy (HDT) against TB.

POTENTIAL THERAPEUTICS AND TRANSLATIONAL OPPORTUNITIES TARGETING HOST CELL DEATH IN M. TUBERCULOSIS INFECTION

Since cell death pathways significantly impact the outcome of host-pathogen interactions during Mtb infection, several of the molecules involved in these pathways have been tested as targets for potential intervention for TB. IFN-alfacon-1 (Infergen/IFG) is a synthetic form of IFN-α2b that has immunomodulatory activity and has been shown to enhance the maturation and activation of macrophages (394). IFG exerts bactericidal activity through autophagy and, thus, attenuates Mtb survival in human macrophages (395). Therefore, targeting autophagy through adjunctive immunotherapy with IFG may provide a novel treatment strategy to control Mtb infection. Similarly, peroxisome proliferator-activated receptor alpha (PPARα) agonists (e.g., fibrates), which regulate energy homeostasis and inflammation, promote autophagy, lysosomal biogenesis, phagosome maturation, and antimicrobial defense against Mtb and, therefore, can be used in TB treatment (396). Loperamide, an antidiarrheal drug that increases the degradation of long-lived cellular proteins, induces autophagy in macrophages, and reduces Mtb growth and burden (397, 398). Furthermore, it was shown that vitamin D supplementation in patients with pulmonary TB without cavitation could enhance autophagy in macrophages and improve innate immune function and, therefore, may help to control the intracellular growth of mycobacteria (399). Interestingly, adjunct therapy using CD40 and TLR4 agonists (C40.T4) also significantly enhanced the bactericidal potency of anti-TB drugs by increasing autophagy, thus providing a novel therapeutic avenue to target drug resistance in anti-TB therapy (394). Various TB treatments targeting host cell death mechanisms are listed in Table 4.

Table 4.

List of potential therapeutic compounds that target host cell death pathways in M. tuberculosis infection

Drug Name	Drug Class	Mechanism of Action	References
2-Phospho-l-ascorbic acid	Vitamin	Inhibitor of SapM via metal oxidation	(400)
Aspirin	Nonsteroidal anti-inflammatory drug	Adjunctive host-directed therapy for pulmonary TB	(401, 402)
AX20017	Research compound	PknG inhibitor	(81)
AZD4462	Research compound	PknG inhibitor	(403, 404)
AZD7762	Research compound	PknG inhibitor	(405)
Carbamazepine	Anti-convulsant	Autophagy inducer	(406)
Concanavalin A	Research compound	Necrosis activator	(407)
Cyclosporin A	Immunosuppressant	Inhibitor of calcineurin	(408)
FK506	Immunosuppressive agent	Inhibitor of calcineurin	(408)
Imatinib	Cancer growth blocker	Inhibitor of Abelson tyrosine kinase	(400)
Isoniazid	Antitubercular antibiotic	Prodrug, cell-wall growth inhibitor, an autophagy inducer	(409)
l-ascorbic acid	Vitamin	Competitive inhibitor of SapM, redox destabilizer	(400)
L335-M34	Research compound	PtpA inhibitor	(410)
Levofloxacin	Fluoroquinolone antibiotic	Apoptosis inducer	(411)
Loperamide	Anti-motility (gut)/anti-diarrheal	HDT, autophagy inducer	(398)
Metformin	Type 2-diabetic	HDT, oxidative stress inducer	(412)
Nitazoxanide/tizodanide	Antiparasitic	Autophagy modulator	(413, 414)
NU-6027	Research compound	PknG inhibitor	(415)
OA-NO2	Research compound	PknG inhibitor	(416)
Pretomanid/PA824	Nitroimidazole	Nitric oxide stress inducer	(417, 418)
Pyrazinamide	Antitubercular antibiotic	Autophagy inducer	(419)
R406	Research compound	PknG inhibitor	(403)
Rapamycin	Macrolide antibiotic	Apoptosis inducer	(420)
Rifampicin	Antitubercular Agent	DNA replication inhibitor	(421)
Saxifragifolin D	Research compound	Accumulator of PI3K	(422)
Sclerotiorin	Natural product	Aldose reductase, lipoxygenase and PknG inhibitor	(423)
Streptomycin	Aminoglycoside antibiotic	Necrosis inhibitor	(424)
Vitamin D3	Vitamin	Adjunctive host-directed therapy for pulmonary TB	(401)

HDT, host-directed therapy; PI3K, phosphatidylinositol-3-kinase; PknG, protein kinase G.

Apoptosis as a Direct Target of Adjunctive TB Therapy

Manipulation of apoptosis in the context of vaccination holds a promising future of therapeutic goals for the early translation of knowledge about cell death in Mtb infection. The proapoptotic recombinant strain BCG ΔureC::hly⁺ (rBCG;BCG ΔureC::hly⁺) expresses the pore-forming listeriolysin of Listeria monocytogenes and lacks urease C to ensure an optimal intraphagosomal pH for listeriolysin activity (19). This strain has higher efficacy and is more immunogenic than the parental BCG in inducing Th1 and Th17 responses in preclinical models, with more profound CD4 and CD8 T cell stimulation (425, 426). Although the responsible immune mechanisms are yet to be characterized, cross priming of CD8 T cells has been suggested as a critical mechanism underpinning the protection conferred by rBCGBCG ΔureC::hly⁺ over the parental BCG strain (425).

Opportunities for adjunctive TB therapies that directly enhance apoptosis are less noticeable and could risk damaging necessary cells (e.g., T cells) and/or generating an excess of apoptotic bodies at risk for secondary necrosis. Nonetheless, a more comprehensive understanding of how different fates for mononuclear phagocytes (MPs) and neutrophils influence the effectiveness of host defense and the quantity and quality of immune pathology in TB is vital to understanding TB pathogenesis and new targets for treatment and diagnosis.

Targeting Autophagy for Host-Directed Therapy

Successful infection of Mtb occurs when it subverts the host-induced death programs. The mammalian target of rapamycin (mTOR), a negative regulator of autophagy, can be activated by virulent mycobacteria (427). Macrophage reprogramming causes multiple lipid biogenesis and interruption of the autophagy pathway mediated by Esx-1 (127, 260, 428, 429). IFN-γ can induce autophagy, whereas Th2 cytokines can dominantly inhibit autophagy 162, which in turn activates AMP-activated protein kinase (AMPK) (251, 419, 430). Anti-TB drugs, such as isoniazid and pyrazinamide, also induce autophagy by exposing mycobacterial products within the cell, supporting the potential of the autophagy pathway for host-directed therapy (431, 432). In addition, mTOR inhibitors, such as rapamycin and everolimus that induce autophagy, also possess antimicrobial effects in in vitro Mtb infection models. Notably, these two drugs showed promising results as an adjunctive to standard antibiotics therapy in a clinical trial of human pulmonary TB (433–436).

SUMMARY AND CONCLUSIVE REMARKS

Despite advances made in our understanding of disease pathogenesis, TB remains a global health challenge and a significant cause of morbidity and mortality. Increasing rates of multi- and extreme-drug resistance Mtb strains, coupled with extended multidrug treatment regimens and co-occurrence of other diseases such as diabetes and AIDS, have impeded efforts to control TB worldwide effectively. In recent years, extensive research in host cell death mechanisms have contributed important information on various cell death pathways during Mtb infection and their impact on TB treatment (Fig. 3 and Table 4).

Further understanding of Mtb pathology in the context of host cell death pathways and developing novel therapies are urgently needed. In this regard, targeting molecular pathways that regulate various host cell death modalities would benefit the treatment of infectious diseases such as TB. Recently, universal guidelines for using and interpreting assays to monitor host cell death by autophagy have been released (437). This would facilitate integrating and synchronizing results generated by laboratories worldwide using various autophagy-related assay platforms. Such measures may accelerate translational research from bench to bedside and contribute significantly to developing novel treatment measures to control TB.

GRANTS

This study was supported by funding from the Bill and Melinda Gates Foundation under Grant OPP1157210 (to S.S.) and the National Institute for Allergy and Infectious Diseases (NIAID) of the US National Institute of Health (NIH) under Grant R01AI161822 (to S.S. and A.K.). S.T. acknowledges support from NIGMS (SC1GM140968) and the Border Biomedical Research Center (BBRC).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

A.N. and S.S. conceived and designed research; A.N. prepared figures; A.N. drafted manuscript; A.N., F.C.K., D.P., S.T., A.K., and S.S. edited and revised manuscript; A.N., F.C.K., D.P., S.T., A.K., and S.S. approved final version of manuscript.