Mycobacterium tuberculosis : Macrophage Takeover and Modulation of Innate Effector Responses Mycobacteriumtuberculosis: Macrophage Takeover and Modulation of Innate Effector Responses

Macrophages mediate the first line of defense in the host against various intracellular path- ogens. They are armed with several immune-effector mechanisms to detect and combat pathogens. However, intracellular pathogens have developed strategies to overcome the macrophage protective immune responses and colonize inside the macrophages. Tuberculosis (TB), both pulmonary and extrapulmonary, is an infectious disease of global concern caused by Mycobacterium tuberculosis . M . tuberculosis is a highly successful pathogen and has acquired various strategies to downregulate critical innate-effector immune responses of macrophages such as phagosome-lysosome fusion, antigen presentation, autophagy, and inhibition of reactive oxygen (ROI) and reactive nitrogen (RNI) species to ensure its longer survival inside the macrophages. In addition to these, the bacilli also modulate Tcell immune response which can help the bacilli to survive inside the host for a long time. In this chapter, we focus to describe important macrophage innate defense mechanisms and the signaling that can influence Tcell adaptive response and the strategies adopted by the bacilli to exploit these signaling cascades to favor its replication and persistence inside the macrophages for establishing a productive infection. by macrophage surveillance their safe which in designing suitable interventions against


Introduction
Macrophages mediate the first line of defense in the host against various intracellular pathogens [1]. They are armed with several immune-effector mechanisms to detect and combat pathogens [2,3]. However, intracellular pathogens have developed strategies to overcome the macrophage protective immune responses and colonize inside the macrophages. Tuberculosis (TB), is an infectious disease caused by a extremely successful pathogen, Mycobacterium tuberculosis, as it has evolved numerous clever strategies over time to modulate important macrophage innateeffector immune responses such as phagosome maturation, antigen presentation, inhibition of reactive oxygen (ROI) and reactive nitrogen (RNI) species, and autophagy to ensure its survival inside the macrophages [4][5][6]. In addition to these, the bacilli also modulate T cell immune response which can help the bacilli to survive inside the host for a long time [7]. In this chapter, we focus to describe important macrophage innate defense mechanisms and the signaling that can influence T cell adaptive response and the strategies adopted by the bacilli to exploit these signaling cascades to favor its replication and persistence inside the macrophages for establishing a productive infection.

Monocytes/macrophages 2.1. History and development
Eli Metchnikoff's obsession, the "phagocyte" [phagos-to eat, cyte-cell], is a constituent of Ludwig Aschoff's reticuloendothelial system (RES) [8]; the macrophage plays a key role at almost all the stages of immune response including innate and adaptive immune responses. Macrophages provide the first line of defense against the invading pathogens. In addition to protecting the body against attacks by foreign organisms, macrophages regulate important physiological functions. Their role in homeostasis has been well established. Macrophages clear almost 2 Â 10 11 erythrocytes per day. This enormous metabolic turnover is crucial for iron homeostasis and to prevent formation of toxic intermediates [9]. Macrophages are equipped with scavenger receptors such as phosphatidylserine receptors, thrombospondin receptor, integrins, and complement receptors to clear the cell debris and rapidly remove the apoptotic cells to help in tissue-remodeling processes. Antigens from the engulfed cells are presented along with the MHC molecules to activate the adaptive immune responses [10]. Thus, macrophage serves as a professional scavenger of the dying cells that not only clears the corpus but also regulates the immune system.
The circulating monocytes that are considered to be the developmental intermediates between bone marrow precursors and tissue macrophages emigrate from the blood vessels and differentiate into tissue macrophages [11]. Macrophages and monocytes originate from hematopoietic stem cell-derived progenitors with myeloid-restricted differentiation potential [1]. The bone marrow progenitors, monocytes, and macrophages collectively were classified into mononuclear phagocytic system, a concept pioneered by van Furth [12]. Monocytes are initially identified by the expression of CD14 molecules and lack of CD16 expression on the surface. These monocytes are termed as "classical monocytes" with CD14 ++ CD16 À phenotype and accounting for about 90% of human blood monocytes. However, later studies have proved the expression of CD16 on the surface of some cell populations that were termed as "nonclassical monocytes" with CD14 + CD16 ++ phenotype [13]. The replenishment of tissue macrophages with the circulating monocytes is well established, but in some instances like in microglial cells of brain, local proliferation of macrophages has been established. Owing to the adaptability and plasticity of macrophages and their responsiveness to different microenvironments in different tissues such as lung, spleen, liver, gut, and brain, a considerable heterogeneity exists among them [14]. For example, lung alveolar macrophages being constantly exposed to a variety of antigens, express a high level of pattern recognition receptors and scavenger receptors on the surface. In contrast, the macrophages of the gut exhibit high levels of phagocytic and antibacterial activities compared to other macrophages [15].

Macrophage activation
Activation is defined as the acquisition of competence to execute a complex function [16]. The factor responsible for macrophage activation was found to be the interferon-gamma (IFN-γ) produced by CD8 + cytotoxic T (Tc1) cells, CD4 + T helper 1 (Th1) T cells, and natural killer (NK) cells. IFN-γ activation leads to conversion of macrophages to potent phagocytotic cells with increased production of reactive oxygen intermediates and reactive nitrogen intermediates, superoxides and proinflammatory cytokines helping the cells to efficiently kill the intracellular pathogens. These macrophages have increased antigen presentation activity, thus they mount an effective immune responses in the host. The IFN-γ-mediated activation is known as "classical activation" and the macrophages are classified as "type 1 or M1 macrophages" [3,18] (Figure 1). IFN-γ stimulation is not enough for the classical activation of macrophages, and may require additional stimulation by TNF-α. As TNF-α is not constitutively present in the environment, specific receptor ligands like lipopolysaccharides (LPS) and various microbial ligands may help in the induction of endogenous expression of TNF-α in macrophages [19,20].
The T helper 2 (Th2) type of cytokines, IL-4 and IL-13, induce a response distinct from the one induced by IFN-γ with distinct set of genes being expressed and is known as "alternative activation" pathway of macrophages, and the cells are named as "alternative activated type 2 or M2 macrophages" [21]. In addition to T cells and B cells, IL-4 and IL-13 are also produced by various other cells such as mast cells, basophils, eosinophils, NK T cells, and macrophages that are involved in regulation of innate immune responses. Hence, alternative activation can be of both innate and acquired origin. Other than these two cytokines, immune complexes, IL-10, glucocorticoid, or secosteroid (vitamin D3) hormone can also contribute to the activation of M2 macrophages [22][23][24][25]. M2 macrophages are characterized by expression of scavenger, mannose [26], and galactose-type receptors, and markers such as dectin-1, arginase 1, Ym1, and FIZZ1 [27]. The M2 macrophages have anti-inflammatory properties and are associated with allergic and anti-parasite responses, and are thought to regulate humoral immunity [27,28]. The alternatively activated macrophages are found to be recruited to wounds and other sites of tissue injury and are programmed to perform a wound healing function by expressing arginase. These macrophages are termed as "repair macrophages" or "wound healing macrophages" [19,29,30]. The M1 and M2 macrophages thus represent two populations of cells with different biological functions [31]. For example, the M1 macrophages, but not the M2 macrophages, produce high levels of reactive oxygen and nitrogen intermediates) and inflammatory cytokines (IL-1β, TNF-α, IL-6), and have low arginase activity, express relatively high levels of CD86, and are efficient APCs. While the M1 cells have an IL-12 high , IL-23 high , and IL-10 low phenotype and play an important role in inducing a dominant Th1 response and provide resistance against intracellular pathogens and tumors [17,23,[32][33][34], the various forms of M2 macrophages share an IL-12 low and IL-23 low phenotype, virtually devoid of the co-stimulatory molecules and fail to mount a strong T cell proliferation [35,36]. The innate and adaptive immune responses can also lead to the production of the "regulatory macrophages" (M reg) ( Figure 1). The M reg cells are shown to be very stable in their phenotype and have regulatory activity. These cells are a novel type of suppressor macrophage which induces tolerance during organ transplantation. They have potent T cell suppressive function [37] and inhibit production of the IL-12 cytokine [38].
The activated macrophages exhibit a profound change in their capacities and functions. In addition to other physiological changes, there is a rapid membrane turnover found in case of macrophages even in the resting stage. This membrane flow is enormously increased in the activated state as a result of enhanced phagocytic activity and lysosomal degradation of the ingested material [39,40]. Phagosomes undergo a series of maturation steps resulting in gradual acidification and increase in the hydrolytic activity. In addition to hydrolases, lethal superoxide generating enzyme activities become prevalent toward the end of phagosome maturation [40]. The NADPH oxidase activity of the enzyme complex leads to formation of H 2 O 2 in presence of superoxide dismutase enzyme [41].

M. tuberculosis: infection and disease
Mycobacteria are rod-shaped bacteria of phylum Actinobacteria mostly found in soil or water. The M. tuberculosis, M. bovis, M. africanum, M. microti, M. canettii, M. caprae, M. pinnipedii, and M. mungi all cause TB disease and are classified as M. tuberculosis complex [42][43][44]. The M. tuberculosis, a facultative intracellular pathogen, was discovered by Robert Koch in 1882 as a causative agent for TB disease in human, those days commonly known as "consumption" or "white plague" [45]. Different strains of M. tuberculosis differ in virulence and in distribution among different human populations. M. tuberculosis W-Beijing strain is one of the most pathogenic strains distributed throughout the world [46,47]. The use of chemotherapeutics against M. tuberculosis has resulted in the appearance of drug-resistant strains [48]. Multiple drug-resistant (MDR), extensive drugresistant (XDR), and total drug-resistant (TDR) strains are becoming increasingly prevalent [49][50][51][52]. The M. tuberculosis bacteria are highly aerobic, non-sporulating, and non-motile bacteria. They have a high guanine plus cytosine (G + C) content (61-71%) in their genomic DNA, and are characterized by the presence of large hydroxylated branched-chain fatty acids called mycolic acids in their cell envelope [53,54]. Although they have been classified with other Gram-positive actinomycetes due to their lack of an outer cell membrane, mycobacteria stain weakly with crystal violet and are resistant to decolorization with acid-alcohol solutions after staining with alkaline arylmethane dyes such as carbol fuchsin, hence called acid-fast. M. tuberculosis primarily infects not only lungs (pulmonary) but can also colonize other body parts (extra-pulmonary). The symptoms of TB disease include chronic cough, blood with sputum, weight loss, fever and night sweats, cavitation, and fibrosis [55,56].
TB is a major public health burden. Despite the availability of effective short-course chemotherapy (DOTS) and M. bovis bacillus Calmette-Guérin (BCG) vaccine, more than 9 million new cases of M. tuberculosis infections are reported every year that accounts to more than 2 billion (one third of world population) being positive for the infection, resulting in 2 million deaths every year and one fifth of all adult deaths in developing countries. Developing countries are the most affected by this pandemic with 30% of the cases being reported from Africa and 55% from Asia. India and China alone are harboring 35% of the cases. With no new drug in use for a while, TB has become increasingly resistant to drugs and multi-, extensive-, and total-drug resistant TB have emerged. Interaction with other infectious diseases like HIV is making it challenging to handle the disease [57]. Other pathological conditions and risk factors associated with TB such as diabetes mellitus, renal diseases, hematological disorders and use of anti-TNF-a drugs has complicated the problem [58]. Socioeconomic factors and variable efficacy of BCG vaccination are also responsible for further aggravating the already complex problem [55,59].
After infection with M. tuberculosis, an individual may not necessarily develop active disease. In case the immune system is competent enough, an individual will either clear the infection or remain latently infected with no clinical signs of disease throughout the life or can have reactivation of TB during weakening of immune system or co-infection with other pathogens like HIV [60,61]. The molecular factors or environmental conditions that influence the progression of latent phase to active disease are not well understood. During latency, the M. tuberculosis bacterium remains inside the infected macrophages in granulomas. These tiny granulomas show no clinical symptoms although they may be visible in chest X-rays and give positive tuberculin skin test [62,63]. In 5% of the cases where immune system is weakened, the microscopic primary lesion progresses to a larger primary caseous lesion. The caseation of the primary lesion may lead to hematogenous spread of bacteria causing miliary TB or extrapulmonary TB, where the infection spreads to liver, spleen, and kidneys [63]. In case the bacteria find their way into brain, they may cause tuberculous meningitis [63]. Patients with active pulmonary TB are diagnosed most commonly by sputum smear microscopy where bacteria are directly observed under microscope in the sputum samples of patients or culturing the samples to check for colony forming units and by chest X-ray. Diagnosis of extrapulmonary TB patients is carried out by tissue biopsy, urine culture, cerebrospinal fluid test, CT scan, or MRI. Latent TB has long been diagnosed by tuberculin skin test; however, its specificity has been questioned due to false positive results as a result of infection with other non-tuberculous bacteria or prior vaccination with BCG [64,65]. Therefore, in recent years, interferon gamma release assays (IGRAs) have been used as an alternative for the diagnosis of both latent TB infection and active TB cases [66].

Infection of macrophages with M. tuberculosis and host immune responses 4.1. The host-bacilli interplay
M. tuberculosis infection is transmitted via aerosol route. Prolonged and close contacts with infected patients result in the transmission of the pathogen in healthy persons as it is known that survival of the bacterium ranges from one to few hours in the aerosol droplets, which are about 1-2 μm or less size [67,68]. Once the pathogen enters the respiratory track, it is finally engulfed by the alveolar macrophages of lung through surface receptors. A number of studies reveal that complement receptors and complement-mediated opsonization are majorly involved in the entry of M. tuberculosis inside the macrophages. One of the most important receptors for mycobacteria is complement receptor 3 (CR3), while other receptors such as CR1 and CR4, mannose receptor, surfactant protein A receptor, CD14, Fcγ receptor, scavenger receptors, etc., have also been implicated in phagocytosis and internalization of the bacteria inside the macrophages [69][70][71]. For alveolar dendritic cells (DC), DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is the main receptor for M. tuberculosis [72]. Though complement receptors are found to be important for phagocytosis of both the avirulent and virulent strains of M. tuberculosis, the decline of mannose receptors was found to be associated with reduced binding of only the virulent strains [73]. The mycobacterial surface glycoprotein, mannose-capped lipoarabinomannan (Man-LAM) is recognized by the C-type lectins and the macrophage mannose receptor (MMR) [74,75]. An important role of toll-receptors, mainly the TLR2, has been demonstrated for the attachment of mycobacteria to macrophages [76]. Interestingly, a large number of surface proteins of M. tuberculosis interact with the TLR2 receptors [76]. After binding, the bacteria are internalized and engulfed into phagosomes, where they can be killed by several defense mechanisms. Thoma-Uszynski et al., (2001) have shown a role of the TLR2-triggered signaling to induce cytotoxicity against M. tuberculosis in alveolar macrophages [77]. Soon after the first contact of M. tuberculosis with alveolar macrophages, generally a robust proinflammatory immune response is induced that confers protection against the bacilli. It is observed that dampening of the proinflammatory signaling can increase M. tuberculosis infection burden in mice [78]. Following intracellular infection, adaptive immunity is generated against the invading pathogen via activation of CD4 + T cells and CD8 + T cells. Many times this adaptive immunity fails to provide a sterilizing immunity resulting in longer persistence of the infection and reactivation of M. tuberculosis bacteria. The bacilli are found to inhibit the class I, class II, and cross presentation of mycobacterial antigen to T cells, thus avoiding immune recognition by T cells. It has been observed that integrity of bacterial cell wall is important for M. tuberculosis in evading adaptive responses. At the site of infection, proinflammatory IFNs and cytokines are secreted, which help in the recruitment of CD4 + T cells, CD8 + T cells, natural killer T cells, and neutrophils [79]. Many a time, the induction of acquired immune response against M. tuberculosis is slow and the establishment of infection wins against the induction of full-fledged response [4,80]. The cell-mediated immune response initiated at the sites of infection is found to be modulated by the bacilli. After establishing the infection, the M. tuberculosis antigens move with the help of alveolar dendritic cells to the draining lymph node, which leads to the stimulation of naive CD4 + T cells. The active role of CD4 + T cells in fighting against M. tuberculosis infection was proposed in 1974 in mice. In addition, the HIV mediated depletion of CD4 + T cells and a defective macrophage activation in some genetic disorders has been associated with worst TB prognosis [4,81]. Furthermore, CD4 À/À knockout and MHC-II À/À knockout mice have been found to be prone to infection by M. tuberculosis [82]. The inhibitory effect of stimulated Th1type CD4 + T cells is by the production proinflammatory cytokines such as IFN-γ and TNF-α, which inhibit bacillary growth [81]. In comparison, when stimulated in the context of MHC class II, Th2-type CD4 + T cells proliferate and produce anti-inflammatory cytokines such as IL-4, IL-5, and IL-10, which are favorable for the bacilli to establish a productive infection [79,81]. Many studies indicate that M. tuberculosis bacilli suppress the pro-inflammatory cytokines such as IL-12 and IFN-γ and activate production of anti-inflammatory cytokines like IL-10 to skew the antimycobacterial immune response from a protective Th1 to a non-protective Th2-type. At the early stages of infection, activation of CD8 + T cells by APC leads to bacterial killing. However, the role of CD8 + T cells at later stages of infection has not been established [82]. The CD8 + T cells can also be activated by the cross presentation of M. tuberculosis antigen along with MHC class I molecules. Post infection, CD8 + T cells migrate to the infected tissue and produce IFN-γ. This migration of CD8 + T cells is a characteristic of the granuloma establishment [83]. Higher bacterial load was found in mice deficient in some of the components of class I presentation and CD8 + T cell activation pathways like β 2 microglobulin, transporter associated with antigen processing protein (TAP), and T cell co-receptor CD8α [83].

Formation of granuloma
M. tuberculosis infection of alveolar macrophages leads to the activation of alveolar dendritic cells which migrate to lymph nodes. In the lymph nodes, CD4 + T cells, CD8 + T cells, and γδ T cells proliferate in response to activation by the alveolar dendritic cells. At the site of infection, the resulting immune activation leads to a microenvironment of cytokines and chemokines which induces the expression of integrins, selectins, and addressins on the surfaces of lymphocytes and endothelial cells. This facilitates a mass migration of immune cells resulting in the formation of a focus of immune cells called "tubercle" or granuloma around the primary site of infection. Macrophages and giant nucleated epithelioid cells form layers around the granuloma. While the inner core of granuloma becomes necrotic, the outer surface is covered by fibrous tissue and vasculature is developed. The granuloma is formed by the immune system to contain M. tuberculosis to the site of infection and not allowing its spread to other normal tissues. The latent infection may persist and remain dormant for life time without proceeding to diseased condition [79,84]. At the nascent stage of granuloma, the recruitment of uninfected and susceptible macrophages to the site of infection may lead to their infection due to apoptosis of the existing infected macrophages. This may lead to initial proliferation of the bacteria till equilibrium is attained by containment of bacteria in the granuloma and control of infection due to adaptive immune response. Some of the newly infected macrophages egress from granuloma and nucleate the formation of new granulomas at other uninfected parts of the lungs (Figure 2) [85]. It has been noticed that bacterial dissemination and rapid disease progression are related with larger necrotic granulomas [57], however, containment of disease is found to be associated with smaller solid granulomas [86]. Furthermore, M. tuberculosis contributes to the formation of granuloma by secreting pathogenic factors as RD1 deficient bacteria show attenuated granuloma formation and macrophage migration to the primary site of infection in zebra fish model [85]. Studies in non-human primate, rabbit, and guinea pig models indicate hypoxic inside the environment of granuloma. In combination with nutrient deficiency, hypoxia induces a dormancy program in the bacilli which is characterized by changes in gene expression and alterations in the bacterial metabolism. Hence probably a latent infection is established which may be activated latter during immunocompromization. Thus, after entry in the human lungs, M. tuberculosis faces a series of host defense attacks. However, the overall outcome of infection with M. tuberculosis depends on the balance between (i) outgrowth and killing of M. tuberculosis and (ii) the extent of tissue necrosis, fibrosis, and regeneration.

Phagosome maturation response
After phagocytosis, M. tuberculosis bacteria reside inside the endosomes of the macrophages. Normally, endosome fuses with lysosome to degrade the pathogens, but M. tuberculosis bacteria are capable of inhibiting the process of phagosome maturation, as a result of which acidification of phagosome is compromised. The intracellular survival and persistence of the tubercle bacilli rests upon its ability to prevent phagosome-lysosome fusion, thus avoiding degradation, antigen processing, and cidal properties of the phagolysosome. Lipoarabinomannan capped with mannose (Man-LAM), a cell wall component of M. tuberculosis and SapM, a phosphatidylinositol 3phosphate (PI3P) phosphatase secreted by the bacilli, are found to interfere with phosphoinositide metabolism of macrophages by depleting PI3P in phagosome [87,88]. The latter is used as a docking molecule by peripheral proteins of lysosomes [7]. M. tuberculosis also possess protein phosphatases (Ptp A and B) that may interfere with host trafficking process possibly by modulating vacuolar sorting proteins [89].
The transport of M. tuberculosis containing vacuoles to lysosomes is mediated by a class of GTPases called Rab GTPases. A phagosome, when it normally matures into the phagolysosome, undergoes a transition between the stages marked by early endocytic Rabs (e.g., Rab5) and late endocytic GTPases (e.g., Rab7). Under normal circumstances, Rab proteins are actively recruited to the vesicles and assembled resulting in fusion of different vesicular compartments. In case of M. tuberculosis infection, it was observed that recruitment of Rab7 to the vacuole containing M. tuberculosis was inhibited while Rab5 was recruited normally indicating a maturation block between a Rab5 and Rab7 stage in infected macrophages that causes inhibition of phagosomelysosome fusion. The exchange of Rab5 protein with Rab7 was later named as Rab conversion [90,91]. Rab5 interacts with early endosomal autoantigen 1 (EEA1), which in turn interacts with phosphatidylinositol 3-phosphate (PI3P). Thus, inhibition of PI3P production by M. tuberculosis appears to be also critical for the inhibition of maturation of endolysosomes [91]. A series of Rab proteins are bind to phagosomes to ensure its acidification and recruitment of cathepsin D, while the process is severely inhibited in case of M. tuberculosis infection. A soluble eukaryotic-like protein kinase PknG of pathogenic M. tuberculosis is shown to be crucial for the prevention of phagosome-lysosome fusion [92]. A recent study has also shown that the M. tuberculosis secretory proteins, ESAT-6 and CFP-10 encoded by RD1 region play crucial roles in preventing phagolysosomal fusion [93]. Genetic screens using comprehensive mutant libraries of M. tuberculosis and BCG suggest that additional mycobacterial products directly or indirectly can influence trafficking processes which probably are important for intracellular survival [94,95]. Coronin1, exclusively recruited to M. tuberculosis containing phagosome, is an important host factor that specifically prevents the lysosomal delivery and death of mycobacteria inside macrophage [96]. Coronin1 prevents phagosome-lysosome fusion by regulating Ca 2+ -dependent signaling processes when macrophages are infected with M. tuberculosis [97]. The Nramp1 (naturalresistance-associated macrophage protein 1) gene involved in macrophage activation and mycobacterial killing [98] becomes part of the phagosome following phagocytosis and displays reduced phagosomal maturation and acidification [99].
A family of IFN-γ-inducible GTPases, also called immunity-related GTPases (IRGs), was found to play a critical role in host innate immunity against intracellular pathogens [100][101][102]. A member of IRG family, 47 kDa Irgm1 (also called LRG-47) protein (which is strongly inducible by IFN-γ and M. tuberculosis infection in mice) is an important anti-M. tuberculosis protein [100]. The anti-mycobacterial role of Irgm1 is due to its interaction with phosphatidylinositol-3,4-bisphosphate (PtdIns (3,4)P2) and PtdIns(3,4,5)P3 present on the phagosomal surface harboring M. tuberculosis. Irgm1 also increases phosphorylation of lipids by augmenting the PI3K activity. Normally, Irgm1 interacts with a membrane trafficking protein Snapin, which interacts with SNARE to ensure the fusion of phagosomes with lysosomes resulting in the elimination of bacilli. M. tuberculosis has developed a way to counter Irgm1 effect by exploiting a natural pathway of Irgm1 inhibition by Rab14 protein that is critical for phagosome-lysosome fusion. It has been shown that the mycobacterial phagosomes recruit and retain Rab14 [103]. Rab 14 is inhibited by unphosphorylated form of AS160 protein.
Manipulation of Rab14-pathway by M. tuberculosis is mediated by the activation of Akt1 that phosphorylates AS160. Phosphorylated AS160 is unable to inhibit Rab14 hence leaving it free to inhibit Irgm1 recruitment to phagolysosomal compartment resulting in the failure of phagolysosome maturation (Figure 3) [103][104][105]. The fusion of phagosomes with lysosomes has been shown to be dependent on Ca 2+ ions. Ca 2+ ions and Ca-binding protein calmodulin are critical for the delivery of lysosomal components to phagosomes using PI3P-dependent pathways. Blockade of Ca 2+ /calmodulin pathway by M. tuberculosis is also found to be one of the ways to block phagosomal maturation [70].

Autophagy
Autophagy (also called xenophagy) is an evolutionary conserved basic homeostatic mechanism of a cell to digest intracellular organelles and large protein aggregates that are difficult to digest by normal proteasomal pathway. The engagement of TLRs with mycobacterial ligands induces autophagy using both MyD88-dependent and TRIF-dependent pathways [106,107]. This suggests that autophagy is an effector of innate immune response. The IFN-γ induces IRG proteins that are mainly involved in induction of autophagy and elimination of M. tuberculosis bacilli [108] and polymorphism at IRG locus is shown to be associated with resistance to M. tuberculosis [109]. Many autophagy-associated proteins are shown to be involved in phagosome-lysosome fusion process [110]. Expression of a number of host genes involved in autophagy is shown to be modulated at the onset of M. tuberculosis infection. The pathogen has therefore acquired mechanisms to subvert the autophagy to induce a favorable condition for its persistence inside the host [111]. M. tuberculosis can also inhibit autophagy by skewing the immune response toward the Th2-type. While proinflammatory cytokines such as TNF-α and IFN-γ promote autophagy, Th2 cytokines such as IL-4 and IL-13 inhibit autophagy in human and murine macrophages, and this is dependent on the Akt-STAT6-signaling pathway [112]. Autophagy is known to help in antigen presentation via both MHC class I and class II pathways and, by inhibiting autophagy, M. tuberculosis also achieves the goal of suppression of MHC class I-and class II-mediated antigen presentation [112]. Vaccines that are designed to elicit a strong autophagic response can prove to be effective against latent TB infection and also drugs designed to modulate autophagy can be effective against the drug-resistant strain of M. tuberculosis [113].

Apoptosis
Macrophages use apoptosis as an effector mechanism to eliminate M. tuberculosis and to constrain the spreading of infection [114]. The apoptotic vesicles are readily engulfed by the neighboring dendritic cells. The dendritic cells in turn activate CD8 + T cells by cross-presenting the processed antigens in the context of MHC class I. This results in an effective immune response against the bacilli. Necrosis, however, is favorable for the dissemination of bacteria and spread of infection. Hence, M. tuberculosis has developed mechanisms to suppress apoptosis and favor necrosis during active infection state [6,63,115,116]. Inhibition of host cell apoptosis by M. tuberculosis has been implicated as a potential virulence mechanism. In fact, an inverse correlation between virulence of mycobacterial strains and their capacity to induce apoptosis has been reported [114]. Infection with virulent M. tuberculosis strain H37Rv is found to be associated with reduced expression of several pro-apoptotic genes and increased expression of the anti-apoptotic gene as compared to uninfected macrophages [117]. Studies using H37Rv and H37Ra strains of M. tuberculosis to infect alveolar macrophages have indicated that although both could induce apoptosis, the virulent H37Rv induce less apoptosis than the avirulent H37Ra by upregulating expression of the anti-apoptotic gene Bcl-2 in macrophages [118]. TNF-α is shown to play important role in host cell apoptosis infected with avirulent H37Ra strain [114,119]. Also, the role of Bfl-1/A1 is realized in inhibition of apoptosis by M. tuberculosis, as decreased intracellular H37Rv growth was observed in Bfl-1/A1 siRNA-treated macrophages [120]. These cells show enhancement of phagosome-lysosome fusion and Caspase-3 activity indicating that expression of Bfl-1/A1 in H37Rv-infected macrophages provides the bacteria a survival strategy to overcome host defense. Nuclear factor-kappaB (NF-κB) activation is shown to play an essential role in the inhibition of host cell apoptosis by M. tuberculosis H37Rv [119]. Infection of macrophages with virulent M. tuberculosis confers resistance to apoptotic stimuli like Fas ligand (FasL) or TNFα by reducing the cell surface expression of Fas receptors or secreting soluble TNF-α-receptor, respectively [121,122]. Interestingly, Divangahi et al. have shown that virulent M. tuberculosis strains inhibit the production of prostaglandin E 2 by interfering with lipoxigenase pathway leading to inhibition of apoptosis and promotion of necrosis [115]. The other mechanism by which M. tuberculosis could possibly inhibit apoptotic process is via the nuoG gene, which can neutralize the NOX-2-mediated increase in ROS and TNF-α production in phagosomes containing M. tuberculosis thereby inhibiting apoptosis [123,124]. Also, secA2 and pknE play roles in resistance against apoptosis as mutants of these genes induce more apoptosis in macrophages as compared to the wild-type M. tuberculosis strains [124][125][126].

Reactive oxygen and nitrogen intermediates (ROIs and RNIs)
ROIs and RNIs are produced in cells like macrophages in response to proinflammatory cytokines [127,128]. ROIs and RNIs being small molecules defuse easily through the membranes and have a detrimental effect on the pathogens been engulfed in phagocytic vacuoles [129]. Studies have indicated that these molecules are important in providing innate host defense against M. tuberculosis [4,129]. ROIs are produced by phagocytes, particularly the polymorphonuclear leukocytes and the activated macrophages, while RNIs are produced mainly by the activated macrophages. ROIs (O 2 •ˉ, H 2 O 2 , OH • ,) are generated through the action of phagocyte oxidase (also called NADPH-oxidase). They further react with halides and amines to generate more reactive species (Figure 4). The important role of NADPH-oxidase in defense against M. tuberculosis and other pathogens is proven by the susceptibility of mice deficient in NADPH oxidase [129][130][131]. Children carrying mutations in gp91phox subunit of phagocyte oxidase enzyme are found to be more susceptible to TB infection than normal population [132]. In macrophages, IFN-γ in synergy with the TNF-α induces production of nitric oxide (•NO) with the help of inducible nitric oxide synthase (iNOS) (Figure 4). Not only does NO exert its effects on its own but also reacts with ROIs and the reaction products like peroxynitrite (OONO¯) and other reaction intermediates can be even more toxic [127,133]. After production, NO is not restricted to the area but readily defuses to other areas by using S-nitrosothiols, S-nitrosylated proteins, and nitrosyl-metal complexes as transport vehicles. NO produces heterogenous and diverse effects owing to its non-specific reactivity with a number of regulatory proteins [129,134]. In mouse macrophages, activation of TLR2 by various bacterial ligands induces iNOS promoter activity, production of NO, and killing of intracellular M. tuberculosis. However, protective role of NO in human TB is controversial. An essential role of NO/iNOS in antimycobacterial immunity was established by infection studies using iNOS knock-out mice [135,136]. Mouse homozygous for knock-out allele for iNOS gene (iNOSÀ/À) when challenged with M. tuberculosis showed a very high susceptibility to infection [135]. Although most of the studies were conducted in mouse model, recent studies reveal that NO/iNOS is also important in killing the bacteria in human TB [134,137,138]. The iNOS activation through TLR2 pathway was found to enhance the killing of intracellular M. tuberculosis [77,139]. All these studies indicate that probably iNOS also plays crucial role in anti-mycobacterial immunity in human. A comparative study of mice deficient either in phagocyte oxidase or in iNOS showed higher anti-mycobacterial activities by RNIs as compared to ROIs [131]. ROIs and RNIs damage the DNA and react with variety of other chemical moieties such as Fe-S clusters, tyrosyl radicals, hemes, sulfhydryls, thioethers, and alkenes to inactivate important components of the invading pathogens to compromise its survival inside the host. Like other effector mechanisms, M. tuberculosis has evolved effective strategies to counter RNIsand ROIs-mediated toxicity. The inhibition of the recruitment of iNOS to the infected phagosome would be one of the ideal strategies used by the bacilli [140]. EBP50, a scaffold protein in activated macrophages, targets iNOS to phagosomes [141]. M. tuberculosis downregulate EBP50 in the infected macrophages thereby reducing the transport of iNOS to the infected phagosome [141]. The bacteria also induce various genes to protect them from intracellular oxidative stress [109]. For example, mycobacterial catalase peroxidase (katG) and alkyl hydroperoxide reductase (ahpC) have role in antioxidant defenses, defend bacteria from intracellular oxidative stress [142,143], and play roles in the virulence of M. tuberculosis [144,145]. It has been shown that virulence of different clinical as well as recombinant strains of M. tuberculosis is correlated with varying expression levels of the peroxidase enzymes [143,144]. NO induces a low oxygen state (hypoxia) in mycobacteria resulting in the overexpression of katG and ahpC [129,146]. These genes were also reported to be overexpressed in vivo during infection with mycobacteria and studies indicate that these responses are abrogated in the phagosomal environment of NOS2 À/À mice [147][148][149]. Oxidative stress has been established to induce a two-component system called DosR/DosS, consisting of a sensor histidine kinase DosS/DosT and a response regulator DosR [150][151][152]. The system helps the bacilli to cope oxidative and other stress (S-nitrosoglutathione, ethanol, etc.) conditions by initiating a complex response.
The DosR is characterized as a transcription factor responsible for transcription of the genes in response to oxidative stress. Genes expressed during the stress response, like α-crystallin was shown to carry sequences in the regulatory regions for DosR binding. In response to upstream activation signals, DosR is phosphorylated at Asp54 that results in its binding to DNA via its C-terminal domain and subsequent activation of DosR responsive genes. The sensor kinases, DosS and DosT, respond to redox environment and hypoxia, respectively. Both the proteins contain two GAF domains at N-terminal harboring a heme prosthetic group which interacts with O 2 , NO or CO to induce autophosphorylation of the kinases and induce transcription of genes by activating DosR (Figure 5). CO is produced by heme oxygenase (HO) enzyme of macrophages. The enzyme is significantly upregulated during M. tuberculosis infection, oxidative stress, hypoxia, and stimulation with various cytokines. The enzyme catalyzes degradation of heme to biliverdin, free iron, and produces sufficient physiological amount of CO to induce dormancy program via binding primarily to DosS while DosT plays a little role in CO sensing. Although CO is sufficient to induce dormancy regulon, it was established that iNOS is required for optimal induction of response. Under aerobic condition, both the sensors are blocked and maintain a basal level of DosR responsive genes, while in anaerobic condition, they respond to the ligands and upregulate the DosR responsive genes to help the pathogen to survive in the stress caused by ROIs and RNIs ( Figure 5) [153][154][155].

Vitamin D3
Vitamin D has long been known to be one of the nutritional therapeutic agents with a capacity to modulate the immune system in the case of an M. tuberculosis infection [156]. There has been a greater incidence of TB during the spring/summer months in temperate countries like UK [157], which can be correlated to a decrease in Vitamin D production on account of lower sun exposure in the winter months. Dietary composition also seemed to have a bearing with a predisposition toward TB, since a study conducted on an immigrant Gujarati Indian population in UK showed a marked increase in TB cases. It must be mentioned here that this population is a vegetarian one and hence dietary intake of Vitamin D is markedly less [158]. The genetic reason for this preponderance has been worked out to be an association with the VDR polymorphisms associated with the Gujarati Asian population concerned and Vitamin D deficiency [159]. Various studies indicate that Vitamin D can help the body fight against M. tuberculosis infections [160]. In a recent study, it has been found that patients administered with Vitamin D in combination with antibiotics recovered from TB more quickly than the patients administered with only antibiotics [161].
The mechanisms for a correlation of Vitamin D and TB are unknown, but it could be the antimicrobial peptides in association of Vitamin D generated by the pattern receptor stimulation in lieu of an infection with M. tuberculosis [162]. This stimulation then induces expression of cathelicidin [162] and β-defensin 2 (DEFB4) [163]. Cathelicidin induces phagolysosomal fusion which is necessary for killing of M. tuberculosis. In addition, 1,25(OH)2D, which is another downstream biochemical produced in Vitamin D biosynthetic pathway, induces autophagy [160,164] and downregulates metalloproteinases (MMPs) [165]. All these processes help in the formation of phagolysosomes and the subsequent killing of M. tuberculosis. Vitamin D also seems to affect the adaptive immune system albeit in a regulatory manner. This is ensued as the downstream product 1,25(OH)2D upregulates regulatory responses with a skew toward the Th2phenotype pattern. This can be ascertained by the anti-proliferative effects of 1,25(OH)2D on CD4 + T cells [166]. It also seems to inhibit Th1 cytokine production [167,168], while promoting T regulatory function at the same time [169]. It also seemingly upregulates Th2 cytokine production [170]. Studies using 1α,25-dihydroxyvitamin D3 indicate that vitamin D3 increases generation of oxygen intermediates via NADPH-dependent phagocyte oxidase involving the phosphatidylinositol 3-kinase [171]. In addition to this, Vitamin D3 can downregulate transcription of tryptophan-aspartate containing coat protein [172], which is important for the entry and survival of M. tuberculosis in human macrophages [173]. Thus, the role of proper nutrition in the control of M. tuberculosis is evident. Vitamin D which has otherwise been associated as the "anti-cold" Vitamin seems to have critical roles in the control of M. tuberculosis infection at the innate and the adaptive levels of the immune system of the host.

Conclusion
TB remains a global pandemic and despite thorough and constructive measures to eradicate TB, it has flourished and continues killing people. It has evolved into various MDR, XDR, and TDR strains, notwithstanding the best healthcare available, which are resistant to the obsolete group of drugs. This necessitates the need to find new drug targets as well as drugs to counter the menace of TB. Therefore, it becomes imperative to understand the biology of M. tuberculosis and the host response modulation mechanisms it has evolved. The same can be achieved by dissecting the biochemical processes throughout the life cycle of the pathogen and by understanding the host-pathogen interaction mechanisms in TB, both of which are prerequisites for the development of effective anti-TB vaccines/drugs. More importantly, the processes associated with the so-called "dormant stage" needs to be identified since this stays the biggest challenge in identifying asymptomatic TB patients, and understanding this Trojan can therefore escalate our steps to eradicate this menace by eons. In this chapter, we have attempted to address various host processes that are subverted by M. tuberculosis to survive inside its host as well as launch an assault when the host immune defenses are weakened. Right from when M. tuberculosis enters inside the body, it counters the host innate defenses by downregulating the oxidative burst inside the macrophages. It also subverts other macrophage effector functions like inhibition of phagolysosomal fusion which is critical for the action of lytic enzymes and therefore forms an important block where it escapes the host defenses. Most importantly, it modulates the host signaling targeting the PAMP receptors, more importantly the TLR2 or to downregulate the proinflammatory signaling cascade known to be detrimental for its intracellular survival. The adaptive responses are similarly affected as one of the major mechanisms, viz. antigen presentation seems to be downregulated. Both MHC class I and class II and even cross presentation are affected. This results in a very less or delayed outcome of protective adaptive immune response which again helps the pathogen to survive very efficiently inside the macrophages. It also affects critical host processes like apoptosis which could clear the pathogen in a controlled manner without allowing it to spread. Downregulating the proinflammatory signaling cascade also involves skewing the macrophage responses from a protective Th1-to a non-protective Th2type. This involves most importantly a shift in the cytokine secretion pattern which subverts the host favorable macrophage signaling and effector functions in favor of the host. We have described important pathophysiological events during M. tuberculosis infection and the virulence processes by which the bacilli can escape the macrophage surveillance mechanisms, and use it as their safe refuge which may help in designing suitable interventions against M. tuberculosis infection.