Sage Journals: Discover world-class research

Abstract

Mycobacterium tuberculosis is an old enemy of the human race, with evidence of infection observed as early as 5000 years ago. Although more host-restricted than Mycobacterium bovis, which can infect all warm-blooded vertebrates, M. tuberculosis can infect, and cause morbidity and mortality in, several veterinary species as well. As M. tuberculosis is one of the earliest described bacterial pathogens, the literature describing this organism is vast and overwhelming. This review strives to distill what is currently known about this bacterium and the disease it causes for the veterinary pathologist.

Keywords

animal models Bacille Calmette-Guérin granuloma pathogenesis tuberculosis

Naturally, we’re disposed to think about diseases just from our own point of view: what can we do to save ourselves and to kill the microbes? Let’s stamp out the scoundrels, and never mind what their motives are! In life in general, though, one has to understand the enemy in order to beat him, and that’s especially true in medicine.

—Jared Diamond in Guns, Germs, and Steel: The Fates of Human Societies ⁵⁴

Tuberculosis (TB) is an ancient disease with a history interwoven with the evolution and migration of mankind, as well as with the origins of microbiology. The main etiologic agent of TB, Mycobacterium tuberculosis (Mtb), is thought to have evolved from an early progenitor in East Africa as early as 3 million years ago.⁹⁰ By the early 1800s, TB epidemics ravaged much of Europe and North America, resulting in 800 to 1000 deaths per 100 000 per year.¹²⁰ Today, approximately 9 million new cases of TB are identified per year, with almost 2 million deaths related to TB, making Mtb the single greatest cause of mortality due to a bacterial pathogen.⁷⁰

Mtb is a major component of the history of microbiology. In 1882, Robert Koch demonstrated the tubercle bacillus during a presentation of his famous postulates, which have since set the standard for the demonstration of infectious etiology.¹¹⁹ Koch also developed tuberculin, a glycerine extract of pure Mtb cultures, which, although a failure in its proposed use as a therapy, could be used to detect latent infections by the measurement of skin reactions at the injection site. Today, tuberculin has been replaced by purified protein derivative (PPD), a precipitate of sterilized Mtb culture filtrate, for testing in humans.

A major breakthrough in the fight against TB occurred when Calmette and Guérin developed the attenuated vaccine strain, Bacille Calmette-Guérin (BCG), through multiple passages of Mycobacterium bovis on ox bile and glycerol-soaked potato slices between 1906 and 1919.³² Although highly effective in preventing the childhood form of TB, the BCG vaccine has variable efficacy in adults.⁶⁷ As an attenuated strain, BCG has been enormously useful in TB research because, unlike Mtb, BCG does not require biosafety level 3 facilities. The other major breakthrough was the discovery of the antimycobacterial drugs streptomycin, isoniazid, the rifamycins, and pyrazinamide, in 1944, 1952, 1957, and 1980, respectively.²⁰⁷ These discoveries led to a new era in TB prevention, research, and treatment.

Today, the absence of a completely protective TB vaccine, the slow development of new antimycobacterial drugs, the need for prolonged therapy regimens, and the emergence of multidrug-resistant (MDR) and extremely drug-resistant (XDR) strains of Mtb are issues that highlight the reemerging TB crisis. The MDR strains account for almost 5% of TB cases.⁷⁰ Because veterinary species are infected by Mtb, and because we can apply what is known about Mtb to the fight against M. bovis, which has reemerged in the United States and other countries due to persistence within wildlife reservoirs²¹⁸ and for which MDR strains have been reported,¹⁰⁵ it is critical that veterinary pathologists have a good basic understanding of Mtb. This review therefore strives to summarize the key components of the microbiology, cell biology, immunology, and pathology of Mtb that are pertinent for the veterinary pathologist.

The Bacterium

Mycobacteria are nonmotile, nonsporulating, weakly gram-positive, acid-fast bacilli that appear microscopically as straight or slightly curved rods, 1 to 4 μm in length and 0.3 to 0.6 μm wide. Mycobacteria are within the order Actinomycetales, which it shares with bacteria such as Corynebacterium, Nocardia, and Rhodococcus. These bacteria also express unique mycolic acids in the cell envelope that play a critical role in the structure and function of the cell wall.¹⁴ The waxy cell wall confers many of the unique characteristics of this genus: acid-fastness, extreme hydrophobicity, resistance to drying, acidity/alkalinity, and many antibiotics, as well as distinctive immunostimulatory properties.⁴⁵

Mtb is a member of the slow-growing pathogenic mycobacterial species, characterized by a 12- to 24-hour division rate and prolonged culture period on agar of up to 21 days. Why Mtb grows so slowly is not well understood. Proposed mechanisms include limitation of nutrient uptake through the highly impermeable cell wall and slow rates of RNA synthesis.⁹⁶ During experimental infections, its metabolism can shift from an aerobic, carbohydrate-metabolizing mode to one that is microaerophilic and lipid metabolizing.²⁵ Mycobacteria are facultative intracellular bacteria that multiply within phagocytic cells, particularly macrophages and monocytes. Although many mycobacterial species are environmental, Mtb is strictly parasitic.

Mtb is a member of the M. tuberculosis complex, which is defined as the etiologic agents of TB in distinct hosts, and also includes M. bovis, M. africanum, M. canetti, and M. microti, with M. caprae and M. pinnipedii considered variants of M. bovis. It was previously assumed that Mtb evolved from M. bovis during the domestication of cattle,¹⁹⁰ a theory made widespread by the national bestseller, Guns, Germs, and Steel.⁵⁴ The genome sequencing projects for both species, however, revealed that M. bovis has several DNA deletions while maintaining 99.95% identity with Mtb and no new genetic material,³¹ supporting the opposite scenario. Successive DNA deletions of regions of difference (RD) resulted in the branching off of members of the Mtb complex.³¹ These genetic analyses indicate that members of the Mtb complex are the clonal progeny of an ancestral strain of M. canetti, also referred to as Mycobacterium prototuberculosis.⁹⁰

An important genetic difference between Mtb and BCG is the deletion of 16 loci in the BCG genome, designated RD1 through RD16.¹⁹ RD1, specifically, was the primary deletion in the original attenuation of M. bovis to create BCG.¹⁰⁴ RD1 of Mtb is composed of 9 genes, which, together with genes lying outside the RD1 locus, constitute the ESAT-6 secretion system (ESX-1).³⁰ The major function of RD1 is thought to be the encoding and secretion of the major antigenic proteins, early secretory antigenic target–6 (ESAT-6) and culture filtrate protein–10 (CFP-10),¹⁰⁹ which are discussed in Table 1.

Table 1.

Virulence Factors of Mycobacterium tuberculosis

Factor (Gene)	Mechanism
Proteins
	Immunomodulation
ESAT-6 (esxA) and CFP-10 (esxB)	T cell stimulation, elicitation of DTH¹⁸⁸
	Downregulate ROS production in macrophages⁸⁰
	Block TLR2-mediated signaling¹⁶¹
	Pore formation, apoptosis,⁵³ cytolysis¹¹⁸
α-crystallin (acr)	Antigenic; potential role in triggering latency¹⁹³
Antigen 85 complex (fbpA, fbpB, fbpC)	Antigenic; mediates attachment to macrophages²¹⁶
	Intracellular survival/metabolism
Erp (erp)	Required for intracellular growth²³
Cholesterol transporter (Mce4)	Major cholesterol uptake system, important for survival during chronic phase¹⁵⁸
Enzymes and lipid carriers (Igr locus)	Cholesterol metabolism; important for growth³⁷
Isocitrate lyase (Icl1)	Allows shift to use of fatty acids as major carbon source; important for chronicity, persistence¹⁴⁰
	Protection against ROI and RNI
Catalase-peroxidase-peroxynitritase (KatG)	¹³⁵
Alkyl-hydroperoxide reductase (ahpC)	¹⁸⁶
Superoxide dismutases (sodA, sodC)	⁵⁹
Nitric oxide reductase (noxR3)	¹⁸⁰
Cell wall components
Lipoarabinomannan
Mannose-capped (ManLAM)	Inhibition of DC maturation; induction of IL-10⁸⁴
	Inhibition of phagolysosomal fusion¹⁰¹
	Protection against ROI; inhibition of protein kinase C activity; block transcription of IFN-γ-inducible genes³⁵
Mycolic acids	Role in granuloma formation, macrophage activation⁹⁴
	Required for mycobacterial survival¹⁶⁶
	Biofilm formation¹⁵¹
Glycopeptidolipids	Biofilm formation¹⁷⁰
Trehalose dimycolate	Granuloma formation, proinflammatory, cachexia¹⁶⁴
	Decrease in NAD⁸
	Damage to host cell membranes¹²²
	Damage to mitochondria¹¹⁴
	Induction of apoptosis¹⁵⁶
	Inhibition of phagosomal-lysosomal fusion¹⁰
Phenolic glycolipids	Immunosuppression¹⁷¹
Sulfolipids	Increase macrophage infectivity, impair macrophage activation by inhibiting phagosomal maturation, blocking priming by IFN-γ¹⁵⁷

CDP-10, culture filtrate protein–10; DC, dendritic cell; DTH, delayed-type hypersensitivity; ESAT-6, early secretory antigenic target–6; IFN, interferon; IL, interleukin; NAD, nicotinamide adenine dinucleotide; RNI, reactive nitrogen intermediates; ROI, reactive oxygen intermediates; ROS, reactive oxygen species; TLR2, Toll-like receptor 2.

Cell Biology of Mtb

The fate of Mtb in the macrophage is one of the most intriguing aspects of Mtb pathogenesis and deserves explanation before a discussion of virulence factors. Mtb interacts with macrophages through multiple phagocytic receptors (Fig. 1; for an older but excellent review, see Ernst⁶⁰). Although macrophage responses to Mtb ligation of any or all of these receptors in vivo are unclear, some in vitro studies show that the ligation of particular receptors may determine the macrophage response. For instance, ligation of the Fc receptor (FcR) led to production of reactive oxygen intermediates (ROIs) and phagosomal-lysosomal fusion, whereas ligation of complement receptor 3 (CR3) or mannose receptor (MR) inhibited the respiratory burst and prevented phagosomal maturation.^7,111

Figure 1.

Cell biology of Mycobacterium tuberculosis (Mtb) infection. Mtb interacts with macrophages through a number of receptors that have been shown in vitro to differentially regulate the fate of the bacillus. Phagocytic receptors include the Fc receptor (FcR), mannose receptor (MR), complement receptors (CR1, CR3, CR4), surfactant protein receptors (SPR), and scavenger receptors (SR). CD14 is a tethering receptor that aids in Toll-like receptor (TLR) signaling. TLRs are expressed at both the plasma and phagosomal membranes and induce cytokine production via the MyD88 signaling pathway. Mycolylarabinogalactan-peptidoglycan (MAGP) from the Mtb cell wall can be released into the cytoplasm, where it is detected by the pattern recognition receptor, NOD2/CARD15. Both TLR and NOD2 signaling induce cytokine gene transcription via nuclear factor–κB (NF-κB). Once in the phagosome, live Mtb blocks phagosomal maturation (X). The mycobacterial phagosome actively retains the characteristics of an early endosome, such as tryptophan aspartate coat protein (TACO), although this is currently in debate.⁶⁵ Vacuolar proton ATPase, inducible nitric oxide synthase (iNOS), and natural resistance-associated membrane protein 1 (NRAMP1) are inhibited from associating with this phagosome, thereby avoiding pH reduction, reactive nitrogen intermediates (RNIs), and Fe²⁺/Mn²⁺ starvation, respectively.^74,146,195 Phosphatidylinositol 3-phosphate (PI3P), a lipid involved in proper trafficking of lysosomal constituents to phagosomes, is also continuously eliminated from phagosomes containing live Mtb due to the hydrolytic activity of secreted lipid phosphatase, SapM.²¹¹ Early endosomal autoantigen 1 (EEA1) and Hrs, both of which are trafficking molecules that associate with PI3P, are also prevented from associating with the Mtb phagosome.^75,215 The Mtb cell wall component, lipoarabinomannan (LAM), potentially prevents phagosomal maturation through transient inhibition of cytoplasmic Ca²⁺ fluxes, which then impairs recruitment of the kinase (hVPS34) that produces PI3P.²¹⁰ TR, transferrin receptor; LAMP-1, lysosomal-associated membrane protein-1; Rab, small GTPases. Modified from Kaufmann.¹¹⁵

Surface-expressed pattern recognition receptors (PRR), such as Toll-like receptor 2 (TLR2), also bind Mtb and initiate proinflammatory cascades in response to mycobacterial components, such as lipoarabinomannan (LAM), lipomannan, phosphatidylinositol mannosides (PIMs), 19-kDa lipoprotein, and trehalose dimycolate (TDM).^{27,107,143,168} TLR4, TLR9, and the TLR adaptor molecule, MyD88, also appear to play important protective roles during Mtb infection in mice, as mice deficient in MyD88 or doubly deficient in TLR2 and TLR4 or TLR9 are more susceptible to infection and have uncontrolled bacterial growth.^11,77,181

The intracellular PRR, NOD2/CARD15, was shown to recognize the Mtb cell wall component mycolylarabinogalactan-peptidoglycan and to be at least partially required for IL-12p40 responses to this molecule in macrophages and dendritic cells (DCs). NOD2-deficient mice had increased bacterial burdens compared to wild-type mice at 6 months postinfection with Mtb.⁵⁵ NOD2 variants have also been demonstrated to be associated with susceptibility to TB in African American populations.⁹

Both TLRs and NOD2 signal through the NF-κB signaling pathway, and mutations in the leucine zipper domain of NEMO (IκB kinase-γ), which is an activator of this pathway, have been associated with increased susceptibility to mycobacterial disease.¹²¹ These mutations led to profound defects in interleukin (IL)–12 and secondary interferon (IFN)–γ production in monocytes from these patients.⁶⁶

Internalized Mtb resides in a phagosome, where, in resting macrophages, the bacteria block maturation, lysosomal fusion, and acidification. This inhibition is an active process because dead mycobacteria traffic to the lysosome.⁷ Reduced acidification has been attributed to reduced or absent levels of vacuolar proton ATPase.¹⁹⁵ The mycobacterial phagosome retains the characteristics of early endosomes while excluding late endosomal markers.⁴⁰ Mycobacterial phagosomes are also prevented from association with inducible nitric oxide synthase (iNOS), thereby limiting exposure to damaging nitrogen radicals.¹⁴⁶ This protective environment also prevents effective antigen processing of Mtb.²⁰¹

Our understanding of the mechanisms controlling phagosomal maturation is incomplete and controversial. Prevention of mycobacterial phagosome maturation has been attributed to the active retention of tryptophan aspartate coat protein (TACO), as bacilli are readily trafficked to lysosomes and eliminated in TACO-deficient cells, but this is currently in debate.⁶⁵ Phosphatidylinositol 3-phosphate (PI3P), a lipid involved in proper trafficking of lysosomal constituents to phagosomes, is also continuously eliminated from phagosomes containing live Mtb.²¹¹ In this study, it was shown that Mtb secretes a lipid phosphatase, SapM, which hydrolyzes PI3P, thereby inhibiting phagosome-late endosome fusion in vitro.

It is likely that Mtb cell wall lipids, such as LAM and TDM, both of which have been shown to prevent phagosomal maturation in vitro,^10,76 interact directly with the phagosomal membrane to mediate this effect. This likelihood is supported by a recent study that showed that M. avium required a closely apposed phagosomal membrane to prevent phagosomal maturation.⁴⁸ This study also showed that M. avium prevented from closely associating with the phagosomal membrane due to the presence of surface-bound beads, could be rescued from lysosomal conditions when the beads were shed.

Macrophages activated by cytokines such as IFN-γ and tumor necrosis factor (TNF)–α, however, can overcome this mycobacterial defense mechanism, potentially through the stimulation of reactive nitrogen intermediates (RNIs). Nitric oxide (NO), the product of iNOS, is highly toxic to intracellular mycobacteria.³⁶ The iNOS inhibitors aggravate the course of murine TB, and NOS2-deficient mice are highly susceptible to mycobacterial infection.^73,83 The role of iNOS in human TB is less clear, although iNOS expression is documented in human monocytes and alveolar macrophages.¹⁴⁹ The role of ROI is even murkier, although mice deficient in cytosolic p47, which is required for effective superoxide production, were partially impaired in controlling acute aerosol infections.⁴³ This ambiguity may be because certain mycobacterial cell wall components, such as LAM and phenolic glycolipid I, can scavenge oxygen radicals.^35,123 Amazingly, Mtb forced into phagolysosomes through antibody opsonization can still survive.⁷

Natural resistance-associated membrane protein 1 (NRAMP1) is a membrane protein involved in innate immunity that is primarily expressed in macrophages and tissues of the reticuloendothelial system. This protein is expressed in the membrane of lysosomes in macrophages and is rapidly recruited to phagosomes.⁸⁷ The NRAMP1 gene (also called Slc11a1) has been linked with genetic resistance to BCG and other bacterial infections in the mouse by controlling intracellular microbial replication in macrophages.¹²⁸ NRAMP1 does this by acting as an efflux pump for the divalent cations Fe²⁺ and Mn²⁺, thereby preventing the use of the cations by the phagocytosed bacteria.⁷⁴ Mice strains susceptible to BCG infection carry a point mutation that affects NRAMP1 protein folding, leading to rapid degradation of this protein.²¹⁴

IFN-γ can also induce autophagy, the process whereby organelles are degraded and recycled, through the induction of p47 GTPases.⁹¹ Induction of autophagy has been shown to lead to the destruction of intracellular Mtb, likely through forced interaction with antimicrobial autolysosomal contents, such as ubiquitin fragments.⁴ Autophagy of Mtb can be induced by ligation of PRRs, such as TLRs.⁵⁰ Interestingly, Th2 cytokines such as IL-4 and IL-13 both inhibit autophagy and counteract IFN-γ-induced autophagy.⁹⁵

Virulence Factors

The virulence factors of Mtb can generally be divided into 2 groups: proteins and cell wall components. Because the literature on Mtb virulence factors is vast, the factors that have shown a phenotype in animal models when the gene is deleted or mutated, or for which the mechanisms are well described, are summarized in Table 1.

Pathogenesis and Lesion Development

Human Mtb infections usually begin by inhalation of aerosol droplets containing tubercle bacilli directly expectorated from an individual with “open” pulmonary disease. The infectious dose for a person is reported to be between 1 and 200 bacilli; however, as a single aerosol droplet can contain anywhere from 1 to 400 bacilli, it is unclear what is considered a biologically relevant dose.^12,173 The bacilli travel to the alveoli, where they are rapidly phagocytosed by alveolar macrophages. These macrophages are stimulated by ligation of TLRs and other PRRs to produce proinflammatory cytokines and chemokines, driving the recruitment of more leukocytes to the site of infection. Neutrophils and monocytes arrive first, phagocytose additional bacteria, secrete more cytokines and chemokines, and begin to organize the early granuloma. Dendritic cells also phagocytose Mtb and then migrate to regional lymph nodes to present mycobacterial antigens to lymphocytes.

Eventually, a well-organized granuloma develops, consisting of central, infected macrophages, surrounded by epithelioid macrophages, foam cells, and occasional multinucleated giant cells of the Langhans type, with peripheral recruited lymphocytes and a fibrous capsule (Fig. 2a). This structure is a fine balance between host containment of infection and protection of Mtb from IFN-γ-producing lymphocytes. Over time, the center of the granuloma undergoes necrosis, and the high lipid and protein content of the dead macrophages results in the caseous appearance grossly and histologically (Fig. 2a). The granuloma is maintained by a delayed-type hypersensitivity (DTH) response to the persistent presence of antigens and immunostimulatory lipids, as Mtb can survive within macrophages and extracellularly within the granuloma.¹³⁸ The caseum is predominantly composed of host-derived lipids, and a recent analysis of gene regulation in the immediately surrounding cells shows Mtb-driven dysregulation of host lipid metabolism.¹¹⁷ The granuloma environment is considered hypoxic in guinea pigs, rabbits, nonhuman primates, and human beings,^5,213 which is an important consideration for mycobacterial metabolism and antibiotic therapy efficacy. It was previously believed that Mtb were sparse in the caseum, based on Ziehl-Neelsen acid-fast staining, with most of the detectable bacilli persisting at the periphery.¹²⁶ A recently developed acid-fast technique using Auramine O and Rhodamine B revealed that the majority of bacilli are actually in the necrotic core in guinea pig caseous granulomas,¹⁰² but the viability of these bacilli remains unclear. Mtb adapt to this environment by preferentially using fatty acids in their metabolism, slowing down active replication, and increasing cell wall thickness, entering a so-called dormant state.²⁶

Figure 2.

Lung; human. (a) The central caseum is surrounded by epithelioid macrophages, multinucleated giant cells, peripheral lymphocytes and plasma cells, and a fibrous capsule. Hematoxylin and eosin (HE). (b) This higher magnification of a portion of (a) contains foamy macrophages (arrows). HE. Courtesy of Dr. M. Kim.

The lesion at the primary site of implantation is termed the Ghon focus. Bacilli may spread before the formation of these organized granulomas via the lymphatics to regional lymph nodes, resulting in granulomatous lymphangitis and lymphadenitis, which, along with the Ghon focus, is called the primary Ranke’s complex. Hematogenous dissemination within the lung or to other organs can also occur during the early stage of this disease. Interestingly, the upper lung lobes of the human favor bacillary growth due to higher oxygen pressure and delayed immune responses.¹⁶⁰

Despite wide dissemination of Mtb during primary infection, the majority of infected, but otherwise healthy, individuals resolve these lesions without becoming symptomatic. Approximately 10% of infected individuals, however, develop severe, life-threatening disease or primary progressive disease at this stage.⁴¹ This is believed to be caused by high bacterial load, increased bacterial virulence, immunosuppression, or genetic susceptibility.⁴¹ In most cases, however, the initial infection subsides, and the collagen capsule contracts to form a scar, or the granuloma calcifies. Intriguingly, most TB patients have pulmonary lesions with heterogeneous morphology,¹¹² suggesting that each granuloma contains bacilli at different stages of latency and reactivation.^46,64 In chronic lesions, Mtb growth is predominantly in the foamy macrophages at the periphery of lesions or in chronic granulomas that have cavitated and accessed an airway.¹¹²

In the absence of progressive disease, an infected individual can remain asymptomatic for years or decades, with bacilli existing in a poorly understood “latent” state, presumably held in check by the host immune system. This ability to establish a chronic asymptomatic infection, followed by reactivation and transmission years later to new uninfected hosts, lends to the tremendous success of Mtb as a pathogen. Any condition affecting the immune system, such as HIV or other infection, old age, malnutrition, malignant disease, immunosuppressive medication, or new infection, can lead to reactivation or secondary disease.²¹²

Five to 10% of latently infected individuals develop secondary disease at some point in their lives for reasons that are not always clear.²⁰³ These individuals make up to 80% of clinical TB cases (at least in industrialized countries) and almost all cases of transmission.⁸¹ Interestingly, extralesional or extrapulmonary tissues may be persistently infected with Mtb.^98,148 In fact, nearly 15% of reactivating TB cases occur at extrapulmonary sites, such as the central nervous system, the skin, internal organs, and genitourinary tract, without accompanying lung lesions.⁴⁷ How reactivation occurs, particularly whether “latent” bacilli become proliferative or if reinfection or bacilli from other sites are responsible for reactivation, are questions that remain to be answered.

The most common form of secondary tuberculosis is usually limited to the lung, and lesions begin as an exudative bronchopneumonia and progress to classical caseous granuloma formation, followed by massive necrosis and cavity formation.¹²⁷ Eventually, there is breakdown of the fibrous capsule and communication with airways. These events allow for rapid growth of extracellular bacilli and spread into airways, leading to transmission or intrapulmonary spread.³³ Sputum analysis during this phase of TB reveals increases in macrophages and neutrophils.¹⁵⁵

Although symptoms of primary TB in most cases are generally subtle and easily overlooked, secondary cases are characterized by localized symptoms, such as coughing, hemoptysis, and pleuritic pain, as well as the generalized symptoms of fever, anorexia, night sweats, and cachexia. This propensity of TB to cause wasting away of patients garnered it the name consumption. In immunocompetent individuals, secondary TB can result in dissemination and death in 50% of cases and chronicity in 25% to 30% of cases. If the host is able to reestablish immune control over the disease, recovery is also possible, as is seen in 20% to 25% of cases.¹⁵

Host Response

Cytokines

After phagocytosis of inhaled Mtb, alveolar macrophages are stimulated, primarily by ligation of TLR2 and TLR4, to secrete proinflammatory cytokines, such as TNF-α, IL-1β, IL-6, and IL-12.^28,143 TNF-α is a critical cytokine in the response to Mtb in that it can stimulate neutrophils and macrophages in an autocrine and paracrine fashion to stimulate apoptosis and ROI/RNI production, leading to the destruction of some phagocytosed bacilli.⁷⁹ TNF-α is also an essential cytokine in driving organized granuloma formation, and the recent development of anti-TNF-α drugs for the treatment of chronic inflammatory conditions has led to increased TB reactivation cases.⁷² The role of TNF-α in granuloma formation is in part due to its own leukocyte recruitment activity, as well as TNF-α-dependent chemokine production.³ TNF-α is also at least partially responsible for the fever and wasting that occur in progressive TB.⁷²

IL-1β has also been shown to play an important role in granuloma formation and has been correlated to disease activity and fever in infected patients.^63,202 IL-1 also acts in an autocrine and paracrine fashion to stimulate macrophages to produce TNF-α and IL-6 and upregulates T cell expression of IL-2 and its receptor, thus stimulating T cell proliferation.²⁰⁰ IL-1α has shown some use as a biomarker for patients with pulmonary TB when used in combination with MIP-1β and epidermal growth factor.³⁸

IL-6 is produced in large amounts in the pleural fluid and bronchoalveolar lavage cells from TB patients, but this cytokine tends to antagonize the effects of TNF-α by preventing binding of TNF-α to macrophages and antagonizing its antimycobacterial activity.^21,202,220 IL-6 has also been shown to enhance mycobacterial growth both intra- and extracellularly⁵² while inhibiting TNF-α and IL-1β production at the transcriptional level in vitro.¹⁸³ IL-6 also suppresses T cell proliferation and IL-2 production in response to antigenic stimulation.²⁰⁸

IL-12 is an important cytokine driving T helper type 1 (Th1) differentiation and IFN-γ production, which is required for resistance to mycobacteria and other intracellular pathogens.⁴² IL-12 also enhances cytotoxicity by stimulating the proliferation of antigen-specific cytolytic T cells and natural killer (NK) cells.²² The importance of IL-12 is highlighted by cases of severe nontuberculous mycobacterial disease in patients with autosomal recessive mutations in the genes encoding either IL-12 or its receptor, IL-12Rβ1, most likely via decreased production of IFN-γ.⁵⁸

IFN-γ, a T cell cytokine that activates macrophages to produce reactive oxygen and nitrogen species, is probably the most important cytokine in the immune response to mycobacteria. This cytokine is mainly secreted by Th1 and CD8⁺ cytotoxic lymphocytes, NK cells, natural killer T (NKT) cells (cells with characteristics of both NK and T cells), professional antigen-presenting cells (APCs), and, to a lesser extent, B cells.¹³⁶ Mice defective in IFN-γ or its receptor are extremely susceptible to Mtb and BCG, with earlier death, high bacterial numbers, and progressive destructive granuloma formation.^71,110 Patients with genetic defects in IFN-γ receptor function or its signaling pathway (ie, STAT1) are particularly prone to chronic or uncontrolled nontuberculous mycobacterial infections.⁵⁸ Furthermore, treatment of these patients with exogenous IFN-γ can lead to clinical improvement.¹⁰³ Failure of AIDS patients to produce high levels of IFN-γ in the lung during pulmonary TB most likely contributes to their susceptibility to infection with Mtb.¹²⁴

A separate subset of T cells, termed Th17 cells, which produce IL-17A, IL-17F, IL-21, and IL-22, has been recently described.¹⁵⁹ IL-17, in particular, has been shown to play an important role in the immunopathology of autoimmune disorders and chronic infectious diseases,^56,57 mostly by the induction of chemokines and therefore excessive recruitment of leukocytes to the site of infection, with ensuing tissue damage.¹⁵⁴ This cytokine is produced primarily by γδ T cells in mice, in response to IL-6 and IL-23 during mycobacterial infection.¹²⁹ Studies in human DCs suggest that prior to IFN-γ production during Mtb infection, IL-23 is preferentially produced, suggesting early skewing toward a Th17 response.⁸⁵ In the absence of IL-17, neutrophils are found in lesser numbers, and granuloma formation in response to BCG is delayed,²⁰⁵ suggesting a role for this cytokine in the initial formation of the granuloma. It is likely that a proper Th1-Th17 balance is required for control of Mtb.

Immunosuppressive cytokines such as IL-10 and transforming growth factor (TGF)–β, both of which suppress effective Th1 responses, play a role in active TB and tend to be transiently overexpressed in TB patients.¹⁰⁰ Higher numbers of regulatory T cells, which produce these immunosuppressive cytokines, were reported in the peripheral blood of TB patients and at the site of infection.⁹² TGF-β is also associated with fibrosis during chronic Mtb infection.⁹⁷ IL-27, a member of the IL-12 cytokine family, has been shown to negatively regulate development of Th17-type cells during chronic inflammation.¹⁹⁴ Improved control of mycobacterial growth due to increased inflammatory cytokine responses in the lungs of mice lacking IL-27 receptor signaling has been reported.¹⁶²

IL-4 and other Th2 cytokines are elevated in patients with active TB, and high pulmonary IL-4 expression has correlated with cavitation.²⁰⁶ IL-4 has a pathogenic role during late TB disease by downregulating protective Th1 responses.²⁴ Th2-skewed responses are also more commonly observed in TB patients living closer to the equator, possibly due to helminth coinfection, high exposure to saprophytic mycobacteria, or higher Mtb inoculum.⁹⁹ This effect may be responsible for the uneven efficacy of BCG vaccination in different parts of the world.¹⁷⁹

Cellular Response

TB and DTH lesions are thought to be examples of granulomas composed of classically activated macrophages, also referred to as M1 macrophages, in that they develop in response to concomitant stimulation with IFN-γ and microbial products.² Although Mtb contains several components known to ligate TLRs, which usually results in innately activated macrophages, infection elicits robust IL-12 production, supporting the M1 phenotype. These activated macrophages compose the majority of epithelioid macrophages within TB granulomas. The Langhans-type multinucleated giant cells, typical of Mtb granulomas, are the result of fusion of epithelioid macrophages and have recently been shown to be induced by the cell wall lipid lipomannan.¹⁶⁷ Foamy macrophages (Fig. 2b, arrows) predominate in advanced TB and are only induced by virulent mycobacteria in response to oxygenated mycolic acids.¹⁶⁵ These cells are thought to provide a nutrient-rich, protective reservoir for Mtb persistence, as these cells have reduced microbicidal and phagocytic activity. Foamy macrophages can also induce apoptosis of Th1 cells through the Fas/Fas ligand mechanism while suppressing their own apoptosis through high expression of the antiapoptotic Bcl2 molecule.¹⁷⁶

Neutrophils are one of the first responders to Mtb infection, and although their bactericidal activities, such as phagocytosis, ROI, RNI, antimicrobial peptides, and extracellular traps, are well characterized, their role in the immune response to Mtb is quite controversial. In mouse models, depletion of neutrophils was shown to lead to an increase in mycobacterial growth, whereas stimulation of neutrophilia led to decreases.⁷⁸ There are conflicting reports, however, regarding the ability of neutrophils to kill mycobacteria directly.^51,108 Nevertheless, neutrophils do play a role in cytokine and chemokine production, granuloma formation, and transference of microbicidal molecules to neighboring, infected macrophages.^174,185,197 Neutrophilic inflammation may also be an important part of TB immunopathology, in that there is greater neutrophil accumulation within lesions in TB-susceptible versus resistant animals.⁶¹ This effect has recently been shown to be due to differential chemokine expression between these 2 models and may explain the earlier discrepancies in neutrophil studies.¹¹⁶ Neutrophils have also been shown to be the predominant infected cell type in active TB patients, suggesting a permissive environment for bacillary replication.⁶²

Type 1 and 2 pneumocytes, as well as pulmonary fibroblasts and endothelial cells, have been reported to contain mycobacterial DNA in TB patients,⁹⁸ and both pneumocytes and alveolar endothelial cells can be infected in vitro,¹⁴⁴ but the role of these cells in disease is unclear. Mtb was shown in vitro to enter type 1 pneumocytes by macropinocytosis.⁸² The M cells overlying the luminal aspect of bronchiolar-associated lymphoid tissue is also a proposed site of entry for Mtb.¹⁹⁹ As mentioned in Table 1, ESAT-6 is cytolytic for type 1 pneumocytes,¹¹⁸ and necrosis of alveolar epithelium was shown to be a characteristic of virulent Mtb infection.¹³⁹ Both mouse and human lung and tracheal epithelial cells have been shown to express defensins after infection with Mtb, with data supporting a protective role by these cationic peptides.^177,178

Dendritic cells in the lung can phagocytose Mtb that has crossed the epithelial barrier. Mtb enters by binding the receptor DC-SIGN via the cell wall component ManLAM, with a lesser role played by CR3 and MR.¹⁹⁶ After phagocytosis, the DCs mature and migrate to regional lymph nodes, where they present mycobacterial antigens in the context of major histocompatibility complex (MHC) molecules, as well as CD1, to prime CD4⁺ and CD8⁺ T cells.^13,89 Dendritic cells infected with Mtb have an impaired ability to present lipid antigens, possibly due to delay of maturation mediated by cell wall components, and are retained at the lung for prolonged periods compared to DCs that have taken up model antigens.¹⁶³ Interestingly, it has been shown in vitro that more virulent strains of Mtb, the Beijing strains in particular, inhibit the maturation of DCs and the ability of DCs to present lipid antigens through decreased expression of CD1.¹⁹²

The activated T cells specific for mycobacterial antigens migrate to the lungs to participate in granuloma formation. The localized immune response to Mtb or the tuberculin reaction is the classical model for DTH. Mycobacterial antigens drive the differentiation of CD4⁺ T cells to Th1 cells. Th1 cells preferentially secrete IFN-γ, which is responsible for the development of DTH via macrophage activation. IL-12 is also critical for the induction of Th1 and IFN-γ responses and therefore DTH. Other important cytokines in this response are IL-2, which causes proliferation of the Th1 cells, and TNF-α and lymphotoxin, which mediate many of the vasodilatory effects and induce the secretion of more chemokines to recruit leukocytes to the reaction site. CD4⁺ T cells are important in the prevention of reactivation and both primary and secondary disease, as decreased CD4⁺ T cells accelerate TB in HIV patients.²¹⁹ CD4⁺ T cells are also relatively absent in cavitated TB lesions, HIV/TB coinfected patients are less likely to have cavitary disease,²⁹ and CD4-deficient mice have less necrosis in M. avium–induced lesions,⁶⁹ suggesting a role for CD4⁺ T cells in cavitation. CD4⁺ T cells in the bronchoalveolar lavage (BAL) fluid of patients with cavitary disease are also more Th2-skewed, secreting more IL-4 than IFN-γ.²⁰⁶

αβ T cell receptor (TCR)–expressing CD4⁺ T cells are required for granuloma formation; transfer of these cells, but not CD8⁺ T cells, can protect immunodeficient mice from Mtb.⁴⁴ CD4⁺ T cells may also reduce bacterial numbers by the induction of apoptosis through the Fas (CD95) ligand system.¹⁵⁰ CD8⁺ T cells do play an important role, however, by destroying infected cells or killing intracellular bacteria via the secretion of granulysin and perforin, which lyse host cells and attack Mtb directly.¹⁹¹ Recent studies in the nonhuman primate model, show an important role for CD8⁺ T cells in that both BCG-vaccinated or previously infected and antibiotic-cured rhesus macaques had markedly reduced protection against challenge with Mtb after CD8⁺ T cell depletion.³⁹

The CD1 family is composed of MHC class I–like molecules that bind distinct lipid antigens, particularly glycolipids, which are recognized by T cells.¹⁷ CD1 molecules are therefore of great interest to TB researchers because Mtb abundantly releases lipid antigens during infection.¹⁶ CD1 molecules have been implicated in the presentation of mycobacterial lipids and the immune response to Mtb. CD1d binds and presents the phosphatidylinositol mannoside, PIM₄, to invariant NKT cells, which have recently been shown to be able to suppress intracellular Mtb replication both in vitro and in vivo through the innate production of IFN-γ in response to infection.^68,182 Mycobacterial diacylated sulfoglycolipids were shown to be presented by CD1b molecules and to stimulate specific T cells to recognize Mtb-infected cells and kill intracellular bacteria.⁸⁶ CD1b molecules also present free mycolic acids and LAM.^17,187 Treatment of mice with anti-CD1 antibody resulted in exacerbated early pathology and decreased reduction of type 1 cytokines after Mtb infection, although CD1d-deficient mice showed no greater susceptibility to infection.¹⁸ In the context of human infection, CD1-restricted T cell responses to mycobacterial lipid preparations were observed in Mtb-infected individuals.²⁰⁴

Natural killer cells are also mycobactericidal and can be activated in the absence of APCs. Stimulated NK cells secrete IFN-γ, IL-15, and IL-18, which play a crucial role in the regulation of CD8⁺ T cells during Mtb infection.²⁰⁹ Natural killer cells also improve the cytolytic activity of γδ T cells and induce them to secrete IFN-γ.²²²

Although cell-mediated immunity is far more critical for effective responses against Mtb, B cells and antibodies have been shown to play a role during TB. B cell–deficient mice were shown to have higher bacillary burden and exacerbated pulmonary lesions due to higher neutrophil recruitment after aerosol infection with Mtb.¹³² Passive immunization of Mtb-infected SCID mice with sera from mice treated with Mtb extracts also reduced the number of bacilli in the lungs and the extent of granulomatous infiltration.⁸⁸ Treatment with antibodies specific for particular Mtb components, such as 19-kDa lipoprotein,¹³⁰ α-crystallin,²¹⁷ and LAM,⁹³ resulted in reduction of bacterial load and lesions in the lungs of infected mice. Antibody-opsonized mycobacteria are more efficiently internalized and killed by neutrophils and macrophages⁴⁹ and more effectively processed for antigen presentation by DCs.¹⁹⁸ Interestingly, although antibody production is typically associated with a shift to a Th2 response, BCG-induced antibodies were shown to enhance proliferation of IFN-γ production in mycobacterium-specific CD4+ and CD8+ T cells.⁴⁹ Unfortunately, diagnostic testing for antibody responses to Mtb components has poor sensitivity and specificity, rendering these tests relatively ineffective.¹

Animal Models of TB

The following is a description of animal models of TB that specifically use Mtb. There are several important TB models using Mycobacterium marinum (zebrafish) and M. bovis (rabbits, swine, calves), for which the reader is directed to other reviews.

Mice

The study of TB in laboratory animals has been challenging in that mice, for which the most reagents are available, are relatively resistant to Mtb and do not typically develop TB or a DTH response to Mtb in a manner similar to humans. Mice can be infected by aerosol, but their more complicated nasal scrolls, relative to humans, affect the final dose that is administered to the lungs.¹⁷² Furthermore, mice can harbor high numbers of Mtb within lung tissue without showing clinical signs.¹⁴¹ Mice also do not cough or form cavitary lesions, making them a poor model for transmission studies.¹¹³ It is important to recognize, however, that the mouse model has been critical in the development of the standard anti-TB therapy that is used today (for an excellent review of this history, see Mitchison¹⁴⁷) and is still a required step in the development of new vaccines.

The histologic appearance of lesions in mice infected with Mtb by aerosol typically consists of multifocal aggregates of epithelioid and foamy, infected macrophages, with scattered multinucleated giant cells, lymphocytes, and plasma cells flooding alveoli, with significant lymphoplasmacytic perivascular cuffing. Often, there is the appearance of “inverted” granulomas, where the lymphocytes and plasma cells are centrally located, surrounding a venule or arteriole, with the infected macrophages located peripherally. This has potential significance for the pathogenesis of Mtb in mice, in that the centrally located, IFN-γ-producing T cells have greater access to the infected macrophages and may thus allow for better control of infection and reduced necrosis.¹⁵³ Fibrous capsules are also not observed histologically, which can affect the validity of antibiotic studies, as Mtb would be more easily accessed by drugs in the mouse lung. Nonetheless, chronic infection can be experimentally produced in mice, and caseous granulomas have been shown in sensitized mice in response to Mtb when infections were in proximity to lipids, such as adipose tissue or if preceded by lipid pneumonia,¹⁰⁶ or in certain mouse strains, such as I/St.¹⁶⁹ Necrotizing and cavitating granulomas with fibrosis can also be produced in mice by infection with M. avium, but in this model, infection is never controlled and bacillary burden increases until death.²⁰ In addition, because of their short life span, mice are poor models for the study of latent infection. Latency can be studied to a degree using the Cornell mouse model, in which mice are treated with antibiotics until a paucibacillary state of infection is reached, followed by immunosuppression by steroid administration.¹³⁷

Guinea Pigs

Guinea pigs develop robust DTH responses to mycobacterial antigens and, after infection with Mtb, reproduce many of the aspects of human infection, such as caseous and mineralized granulomas, primary and hematogenous pulmonary lesions, fibrous capsule formation, and dissemination.¹⁴² Cavitation can occur with low-dose, chronic infections of guinea pigs but not as reliably as in the rabbit model.²²¹ They are also exceptionally vulnerable to infection by as little as a few inhaled mycobacteria, making them well suited for airborne Mtb transmission studies and as sentinel animals.¹⁷⁵

Guinea pigs, however, are not suitable for studies of latency because they are very susceptible hosts and often develop chronic progressive disease with extensive tissue destruction after very low-dose aerosol infection.¹⁸⁹ TB is also a primarily hematogenously disseminated disease in the guinea pig.¹⁸⁹ Pulmonary lesions in the guinea pig also contain a high proportion of granulocytes, particularly eosinophils,¹⁵² which are not common features of the human disease. Furthermore, although inbred strains of guinea pigs are available, they are not well characterized with respect to Mtb infection, and immune responsiveness and reagents, although improving, are limited. It is also important to remember that guinea pigs have heterophils, rather than neutrophils, and that heterophils lack myeloperoxidase but instead contain antimicrobial cationic peptides that are not present in human neutrophils.¹²⁵ This difference is commonly overlooked by researchers and may be important when interpreting results in these species.

Rabbits

Rabbits infected with Mtb mount a moderate DTH response and form caseous granulomas and cavitary lesions.¹³³ Rabbits, including currently available inbred strains, are relatively resistant to Mtb, however, requiring the inhalation of 500 to 3000 bacilli to form one grossly visible tubercle at 5 weeks postinfection.¹³³ Most rabbits will also overcome disease completely, with few culturable bacilli.¹³¹ This model is useful in the study of latent or paucibacillary TB states, however, without the use of antibiotics as in the Cornell model. Rabbits do need to be experimentally immunosuppressed, however, as they will not spontaneously reactivate disease.¹³⁴ There are minimal immune reagents, however, for this model, and the larger size of rabbits makes them more costly to use. There are inbred strains of rabbits, such as the Lurie and Thorbecke rabbits, which are more susceptible to Mtb infection. This susceptibility has been linked to suppressed macrophage antimycobacterial activity, decreased MHC class II expression, and impaired development of type IV hypersensitivity.^131,145 Rabbits, like guinea pigs, also have heterophils as opposed to neutrophils.

Nonhuman Primates

Nonhuman primates, such as the cynomolgus macaque, although expensive and challenging to work with, do provide an excellent model to study TB. Fifty percent of cynomolgus macaques infected via bronchoscope develop primary TB, whereas the other half become latently infected and can reactivate, and the full spectrum of granuloma types can be observed.³⁴ This model is also important for the study of coinfection with simian immunodeficiency virus and Mtb, as coinfection with HIV is particularly common in sub-Saharan Africa, and TB is the most common cause of death in HIV patients.⁶ DTH responses in nonhuman primates are typically weak, with low rates of skin test positivity unless the more potent old tuberculin is used.¹⁸⁴ Although human reagents can often be used for analyses, because of the expense and difficulties of handling nonhuman primates, as well as the lack of inbred strains and potential for horizontal transmission within a colony, this animal model has not been widely used except in industry, as part of vaccine and treatment studies.

Conclusion

The tuberculous granuloma can be viewed as either a dance or a battleground between the pathogen and the host that has evolved over millennia. Changes in Mtb and in the granuloma environment can easily tip the scale in favor of one side versus the other. Although a tremendous amount of research has been performed in the study of TB, this disease continues to be a scourge upon humanity and veterinary species. Until the factors affecting each side of this battle are better understood, we are likely to continue to suffer this plague. The continued development of animal models and reagents, as well as the wide use of genetic and other screens to study virulence factors in Mtb, will help us in this endeavor. The veterinary pathologist can play an important role on the front lines, diagnostically, as well as in the interpretation of lesions produced during experimental infection.

Footnotes

Acknowledgements

I thank Dr. Elizabeth Rhoades for her help editing the parts of this review that composed the literature review of my dissertation and Drs. Kyle Rohde, Melinda Camus, and Andrew Moorhead for their help in editing this version. Thanks also to Dr. Mi-Jeong Kim for providing the human TB photomicrographs and to Dr. Helen Wainwright and Ms. Annalie Visser at the University of Cape Town, South Africa, for providing Dr. Kim with access to human pulmonary TB lesions. I also thank Dr. David Russell for his guidance and support during my graduate studies. I am grateful to the editors and reviewers of this manuscript, who provided excellent suggestions and constructive criticism. Finally, I would like to recognize and apologize to all of the researchers whose work I could not include in this review due to restrictions in the number of references I could include (my original reference list contained almost 500 citations). This review and my own research could not have happened without the tremendous effort of TB researchers past and present.

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The author(s) received no financial support for the research, authorship, and/or publication of this article.

References

Abebe

Holm-Hansen

Wiker

Progress in serodiagnosis of Mycobacterium tuberculosis infection. Scand J Immunol. 2007; 66: 176–191.

Adams

. Molecular interactions in macrophage activation. Immunol Today. 1989; 10: 33–35.

Algood

Lin

Yankura

TNF influences chemokine expression of macrophages in vitro and that of CD11b+ cells in vivo during Mycobacterium tuberculosis infection. J Immunol. 2004; 172: 6846–6857.

Alonso

Pethe

Russell

Lysosomal killing of Mycobacterium mediated by ubiquitin-derived peptides is enhanced by autophagy. Proc Natl Acad Sci U S A. 2007; 104: 6031–6036.

Aly

Wagner

Keller

Oxygen status of lung granulomas in Mycobacterium tuberculosis–infected mice. J Pathol. 2006; 210: 298–305.

Anandaiah

Dheda

Keane

Novel developments in the epidemic of HIV and TB co-infection. Am J Respir Crit Care Med. 2011; 183: 987–997.

Armstrong

Hart

. Phagosome-lysosome interactions in cultured macrophages infected with virulent tubercle bacilli: reversal of the usual nonfusion pattern and observations on bacterial survival. J Exp Med. 1975; 142: 1–16.

Artman

Bekierkunst

Goldenberg

. Tissue metabolism in infection: biochemical changes in mice treated with cord factor. Arch Biochem Biophys. 1964; 105: 80–85.

Austin

Graviss

. Common nonsynonymous polymorphisms in the NOD2 gene are associated with resistance or susceptibility to tuberculosis disease in African Americans. J Infect Dis. 2008; 197: 1713–1716.

10.

Axelrod

Oschkinat

Enders

Delay of phagosome maturation by a mycobacterial lipid is reversed by nitric oxide. Cell Microbiol. 2008; 10: 1530–1545.

11.

Bafica

Scanga

Feng

TLR9 regulates Th1 responses and cooperates with TLR2 in mediating optimal resistance to Mycobacterium tuberculosis . J Exp Med. 2005; 202: 1715–1724.

12.

Balasubramanian

Wiegeshaus

Taylor

Pathogenesis of tuberculosis: pathway to apical localization. Tuber Lung Dis. 1994; 75: 168–178.

13.

Banchereau

Steinman

. Dendritic cells and the control of immunity. Nature. 1998; 392: 245–252.

14.

Barry

CE III

Lee

Mdluli

Mycolic acids: structure, biosynthesis and physiological functions. Prog Lipid Res. 1998; 37: 143–179.

15.

Bates

. Transmission and pathogenesis of tuberculosis. Clin Chest Med. 1980; 1: 167–174.

16.

Beatty

Rhoades

Ullrich

Trafficking and release of mycobacterial lipids from infected macrophages. Traffic. 2000; 1: 235–247.

17.

Beckman

Porcelli

Morita

Recognition of a lipid antigen by CD1-restricted alpha beta+ T cells. Nature. 1994; 372: 691–694.

18.

Behar

Dascher

Grusby

Susceptibility of mice deficient in CD1D or TAP1 to infection with Mycobacterium tuberculosis . J Exp Med. 1999; 189: 1973–1980.

19.

Behr

Wilson

Gill

Comparative genomics of BCG vaccines by whole-genome DNA microarray. Science. 1999; 284: 1520–1523.

20.

Benini

Ehlers

. Different types of pulmonary granuloma necrosis in immunocompetent vs. TNFRp55-gene-deficient mice aerogenically infected with highly virulent Mycobacterium avium . J Pathol. 1999; 189: 127–137.

21.

Bermudez

Petrofsky

Interleukin-6 antagonizes tumor necrosis factor–mediated mycobacteriostatic and mycobactericidal activities in macrophages. Infect Immun. 1992; 60: 4245–4252.

22.

Bertagnolli

Lin

Young

IL-12 augments antigen-dependent proliferation of activated T lymphocytes. J Immunol. 1992; 149: 3778–3783.

23.

Berthet

Lagranderie

Gounon

Attenuation of virulence by disruption of the Mycobacterium tuberculosis erp gene. Science. 1998; 282: 759–762.

24.

Biedermann

Mailhammer

Mai

Reversal of established delayed type hypersensitivity reactions following therapy with IL-4 or antigen-specific Th2 cells. Eur J Immunol. 2001; 31: 1582–1591.

25.

Bloch

Segal

. Biochemical differentiation of Mycobacterium tuberculosis grown in vivo and in vitro. J Bacteriol. 1956; 72: 132–141.

26.

Boshoff

Barry

CE III

. Tuberculosis: metabolism and respiration in the absence of growth. Nat Rev Microbiol. 2005; 3: 70–80.

27.

Bowdish

Sakamoto

Kim

MARCO, TLR2, and CD14 are required for macrophage cytokine responses to mycobacterial trehalose dimycolate and Mycobacterium tuberculosis . PLoS Pathog. 2009; 5: e1000474.

28.

Brightbill

Libraty

Krutzik

Host defense mechanisms triggered by microbial lipoproteins through Toll-like receptors. Science. 1999; 285: 732–736.

29.

Brindle

Nunn

Githui

Quantitative bacillary response to treatment in HIV-associated pulmonary tuberculosis. Am Rev Respir Dis. 1993; 147: 958–961.

30.

Brodin

Rosenkrands

Andersen

ESAT-6 proteins: protective antigens and virulence factors?

Trends Microbiol. 2004; 12: 500–508.

31.

Brosch

Gordon

Marmiesse

A new evolutionary scenario for the Mycobacterium tuberculosis complex. Proc Natl Acad Sci U S A. 2002; 99: 3684–3689.

32.

Calmette

Boquet

Negre

. Contribution à l’étude du bacille tuberculeux bilié. Ann Inst Pasteur. 1921; 35: 561–570.

33.

Canetti

. Exogenous reinfection and pulmonary tuberculosis a study of the pathology. Tubercle. 1950; 31: 224–233.

34.

Capuano

SV III

Croix

Pawar

Experimental Mycobacterium tuberculosis infection of cynomolgus macaques closely resembles the various manifestations of human M. tuberculosis infection. Infect Immun. 2003; 71: 5831–5844.

35.

Chan

Fan

Hunter

Lipoarabinomannan, a possible virulence factor involved in persistence of Mycobacterium tuberculosis within macrophages. Infect Immun. 1991; 59: 1755–1761.

36.

Chan

Xing

Magliozzo

Killing of virulent Mycobacterium tuberculosis by reactive nitrogen intermediates produced by activated murine macrophages. J Exp Med. 1992; 175: 1111–1122.

37.

Chang

Miner

Pandey

igr Genes and Mycobacterium tuberculosis cholesterol metabolism. J Bacteriol. 2009; 191: 5232–5239.

38.

Chegou

Black

Kidd

Host markers in QuantiFERON supernatants differentiate active TB from latent TB infection: preliminary report. BMC Pulm Med. 2009; 9: 21.

39.

Chen

Huang

Wang

A critical role for CD8 T cells in a nonhuman primate model of tuberculosis. PLoS Pathog. 2009; 5: e1000392.

40.

Clemens

Horwitz

. The Mycobacterium tuberculosis phagosome interacts with early endosomes and is accessible to exogenously administered transferrin. J Exp Med. 1996; 184: 1349–1355.

41.

Comstock

. Epidemiology of tuberculosis. Am Rev Respir Dis. 1982; 125: 8–15.

42.

Cooper

Roberts

Rhoades

The role of interleukin-12 in acquired immunity to Mycobacterium tuberculosis infection. Immunology. 1995; 84: 423–432.

43.

Cooper

Segal

Frank

Transient loss of resistance to pulmonary tuberculosis in p47(phox–/–) mice. Infect Immun. 2000; 68: 1231–1234.

44.

D’Souza

Denis

Scorza

CD4+ T cells contain Mycobacterium tuberculosis infection in the absence of CD8+ T cells in mice vaccinated with DNA encoding Ag85A. Eur J Immunol. 2000; 30: 2455–2459.

45.

Daffe

Draper

. The envelope layers of mycobacteria with reference to their pathogenicity. Adv Microb Physiol. 1998; 39: 131–203.

46.

Dannenberg

. Pathophysiology: basic aspects. In: Schlossberg

, ed. Tuberculosis and Nontuberculous Mycobacterial Infections. 4th ed. Philadelphia, PA: WB Saunders; 1999: 17–47.

47.

De Backer

Mortele

De Keulenaer

Tuberculosis: epidemiology, manifestations, and the value of medical imaging in diagnosis. JBR-BTR. 2006; 89: 243–250.

48.

de Chastellier

Forquet

Gordon

Mycobacterium requires an all-around closely apposing phagosome membrane to maintain the maturation block and this apposition is re-established when it rescues itself from phagolysosomes. Cell Microbiol. 2009; 11: 1190–1207.

49.

de Valliere

Abate

Blazevic

Enhancement of innate and cell-mediated immunity by antimycobacterial antibodies. Infect Immun. 2005; 73: 6711–6720.

50.

Delgado

Elmaoued

Davis

Toll-like receptors control autophagy. EMBO J. 2008; 27: 1110–1121.

51.

Denis

. Human neutrophils, activated with cytokines or not, do not kill virulent Mycobacterium tuberculosis . J Infect Dis. 1991; 163: 919–920.

52.

Denis

Gregg

. Recombinant interleukin-6 increases the intracellular and extracellular growth of Mycobacterium avium . Can J Microbiol. 1991; 37: 479–483.

53.

Derrick

Morris

. The ESAT6 protein of Mycobacterium tuberculosis induces apoptosis of macrophages by activating caspase expression. Cell Microbiol. 2007; 9: 1547–1555.

54.

Diamond

. Guns, Germs, and Steel: The Fates of Human Societies. New York: W. W. Norton; 1997.

55.

Divangahi

Mostowy

Coulombe

NOD2-deficient mice have impaired resistance to Mycobacterium tuberculosis infection through defective innate and adaptive immunity. J Immunol. 2008; 181: 7157–7165.

56.

Diveu

McGeachy

Cua

. Cytokines that regulate autoimmunity. Curr Opin Immunol. 2008; 20: 663–668.

57.

Dong

. Regulation and pro-inflammatory function of interleukin-17 family cytokines. Immunol Rev. 2008; 226: 80–86.

58.

Dorman

Holland

. Interferon-gamma and interleukin-12 pathway defects and human disease. Cytokine Growth Factor Rev. 2000; 11: 321–333.

59.

Dussurget

Stewart

Neyrolles

Role of Mycobacterium tuberculosis copper-zinc superoxide dismutase. Infect Immun. 2001; 69: 529–533.

60.

Ernst

. Macrophage receptors for Mycobacterium tuberculosis . Infect Immun. 1998; 66: 1277–1281.

61.

Eruslanov

Lyadova

Kondratieva

Neutrophil responses to Mycobacterium tuberculosis infection in genetically susceptible and resistant mice. Infect Immun. 2005; 73: 1744–1753.

62.

Eum

Kong

Hong

Neutrophils are the predominant infected phagocytic cells in the airways of patients with active pulmonary TB. Chest. 2010; 137: 122–128.

63.

Fantuzzi

Dinarello

. The inflammatory response in interleukin-1 beta-deficient mice: comparison with other cytokine-related knock-out mice. J Leukoc Biol. 1996; 59: 489–493.

64.

Fenhalls

Wong

Bezuidenhout

In situ production of gamma interferon, interleukin-4, and tumor necrosis factor alpha mRNA in human lung tuberculous granulomas. Infect Immun. 2000; 68: 2827–2836.

65.

Ferrari

Langen

Naito

A coat protein on phagosomes involved in the intracellular survival of mycobacteria. Cell. 1999; 97: 435–447.

66.

Filipe-Santos

Bustamante

Haverkamp

X-linked susceptibility to mycobacteria is caused by mutations in NEMO impairing CD40-dependent IL-12 production. J Exp Med. 2006; 203: 1745–1759.

67.

Fine

. Variation in protection by BCG: implications of and for heterologous immunity. Lancet. 1995; 346: 1339–1345.

68.

Fischer

Scotet

Niemeyer

Mycobacterial phosphatidylinositol mannoside is a natural antigen for CD1d-restricted T cells. Proc Natl Acad Sci U S A. 2004; 101: 10685–10690.

69.

Florido

Cooper

Appelberg

. Immunological basis of the development of necrotic lesions following Mycobacterium avium infection. Immunology. 2002; 106: 590–601.

70.

Floyd

. Global Tuberculosis Control. Geneva, Switzerland: World Health Organization; 2009.

71.

Flynn

Chan

Triebold

An essential role for interferon gamma in resistance to Mycobacterium tuberculosis infection. J Exp Med. 1993; 178: 2249–2254.

72.

Flynn

Goldstein

Chan

Tumor necrosis factor–alpha is required in the protective immune response against Mycobacterium tuberculosis in mice. Immunity. 1995; 2: 561–572.

73.

Flynn

Scanga

Tanaka

Effects of aminoguanidine on latent murine tuberculosis. J Immunol. 1998; 160: 1796–1803.

74.

Forbes

Gros

. Iron, manganese, and cobalt transport by Nramp1 (Slc11a1) and Nramp2 (Slc11a2) expressed at the plasma membrane. Blood. 2003; 102: 1884–1892.

75.

Fratti

Backer

Gruenberg

Role of phosphatidylinositol 3-kinase and Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest. J Cell Biol. 2001; 154: 631–644.

76.

Fratti

Chua

Vergne

Mycobacterium tuberculosis glycosylated phosphatidylinositol causes phagosome maturation arrest. Proc Natl Acad Sci U S A. 2003; 100: 5437–5442.

77.

Fremond

Yeremeev

Nicolle

Fatal Mycobacterium tuberculosis infection despite adaptive immune response in the absence of MyD88. J Clin Invest. 2004; 114: 1790–1799.

78.

Fulton

Reba

Martin

Neutrophil-mediated mycobacteriocidal immunity in the lung during Mycobacterium bovis BCG infection in C57BL/6 mice. Infect Immun. 2002; 70: 5322–5327.

79.

Gan

Duan

Enhancement of antimycobacterial activity of macrophages by stabilization of inner mitochondrial membrane potential. J Infect Dis. 2005; 191: 1292–1300.

80.

Ganguly

Giang

Gupta

Mycobacterium tuberculosis secretory proteins CFP-10, ESAT-6 and the CFP10: ESAT6 complex inhibit lipopolysaccharide-induced NF-kappaB transactivation by downregulation of reactive oxidative species (ROS) production. Immunol Cell Biol. 2008; 86: 98–106.

81.

Garay

. Pulmonary Tuberculosis. London: Little, Brown; 1996.

82.

Garcia-Perez

Mondragon-Flores

Luna-Herrera

. Internalization of Mycobacterium tuberculosis by macropinocytosis in non-phagocytic cells. Microb Pathog. 2003; 35: 49–55.

83.

Garcia

Guler

Vesin

Lethal Mycobacterium bovis Bacillus Calmette Guerin infection in nitric oxide synthase 2–deficient mice: cell-mediated immunity requires nitric oxide synthase 2. Lab Invest. 2000; 80: 1385–1397.

84.

Geijtenbeek

Van Vliet

Koppel

Mycobacteria target DC-SIGN to suppress dendritic cell function. J Exp Med. 2003; 197: 7–17.

85.

Gerosa

Baldani-Guerra

Lyakh

Differential regulation of interleukin 12 and interleukin 23 production in human dendritic cells. J Exp Med. 2008; 205: 1447–1461.

86.

Gilleron

Stenger

Mazorra

Diacylated sulfoglycolipids are novel mycobacterial antigens stimulating CD1-restricted T cells during infection with Mycobacterium tuberculosis . J Exp Med. 2004; 199: 649–659.

87.

Gruenheid

Pinner

Desjardins

Natural resistance to infection with intracellular pathogens: the Nramp1 protein is recruited to the membrane of the phagosome. J Exp Med. 1997; 185: 717–730.

88.

Guirado

Amat

Gil

Passive serum therapy with polyclonal antibodies against Mycobacterium tuberculosis protects against post-chemotherapy relapse of tuberculosis infection in SCID mice. Microbes Infect. 2006; 8: 1252–1259.

89.

Gumperz

Brenner

. CD1-specific T cells in microbial immunity. Curr Opin Immunol. 2001; 13: 471–478.

90.

Gutierrez

Brisse

Brosch

Ancient origin and gene mosaicism of the progenitor of Mycobacterium tuberculosis . PLoS Pathog. 2005; 1: e5.

91.

Gutierrez

Master

Singh

Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages. Cell. 2004; 119: 753–766.

92.

Guyot-Revol

Innes

Hackforth

Regulatory T cells are expanded in blood and disease sites in patients with tuberculosis. Am J Respir Crit Care Med. 2006; 173: 803–810.

93.

Hamasur

Haile

Pawlowski

A mycobacterial lipoarabinomannan specific monoclonal antibody and its F(ab’) fragment prolong survival of mice infected with Mycobacterium tuberculosis . Clin Exp Immunol. 2004; 138: 30–38.

94.

Han

Oka

Relationships between structure and biological activity of the mycolic acid–containing glycolipids from Nocardia asteroides “sensu stricto” and related species. Acta Leprol. 1989; 7(suppl 1): 130–132.

95.

Harris

De Haro

Master

T helper 2 cytokines inhibit autophagic control of intracellular Mycobacterium tuberculosis . Immunity. 2007; 27: 505–517.

96.

Harshey

Ramakrishnan

. Rate of ribonucleic acid chain growth in Mycobacterium tuberculosis H37Rv. J Bacteriol. 1977; 129: 616–622.

97.

Hernandez-Pando

Aguilar

Hernandez

Pulmonary tuberculosis in BALB/c mice with non-functional IL-4 genes: changes in the inflammatory effects of TNF-alpha and in the regulation of fibrosis. Eur J Immunol. 2004; 34: 174–183.

98.

Hernandez-Pando

Jeyanathan

Mengistu

Persistence of DNA from Mycobacterium tuberculosis in superficially normal lung tissue during latent infection. Lancet. 2000; 356: 2133–2138.

99.

Hernandez-Pando

Pavon

Arriaga

Pathogenesis of tuberculosis in mice exposed to low and high doses of an environmental mycobacterial saprophyte before infection. Infect Immun. 1997; 65: 3317–3327.

100.

Hirsch

Hussain

Toossi

Cross-modulation by transforming growth factor beta in human tuberculosis: suppression of antigen-driven blastogenesis and interferon gamma production. Proc Natl Acad Sci U S A. 1996; 93: 3193–3198.

101.

Hmama

Sendide

Talal

Quantitative analysis of phagolysosome fusion in intact cells: inhibition by mycobacterial lipoarabinomannan and rescue by an 1alpha,25-dihydroxyvitamin D3-phosphoinositide 3-kinase pathway. J Cell Sci. 2004; 117: 2131–2140.

102.

Hoff

Ryan

Driver

Location of intra- and extracellular M. tuberculosis populations in lungs of mice and guinea pigs during disease progression and after drug treatment. PLoS One. 20111; 6: e17550.

103.

Holland

Eisenstein

Kuhns

Treatment of refractory disseminated nontuberculous mycobacterial infection with interferon gamma: a preliminary report. N Engl J Med. 1994; 330: 1348–1355.

104.

Hsu

Hingley-Wilson

Chen

The primary mechanism of attenuation of bacillus Calmette-Guerin is a loss of secreted lytic function required for invasion of lung interstitial tissue. Proc Natl Acad Sci U S A. 2003; 100: 12420–12425.

105.

Hughes

Skuce

Doig

Analysis of multidrug-resistant Mycobacterium bovis from three clinical samples from Scotland. Int J Tuberc Lung Dis. 2003; 7: 1191–1198.

106.

Hunter

Olsen

Jagannath

Trehalose 6,6′-dimycolate and lipid in the pathogenesis of caseating granulomas of tuberculosis in mice. Am J Pathol. 2006; 168: 1249–1261.

107.

Jones

Means

Heldwein

Different Toll-like receptor agonists induce distinct macrophage responses. J Leukoc Biol. 2001; 69: 1036–1044.

108.

Jones

Amirault

Andersen

. Killing of Mycobacterium tuberculosis by neutrophils: a nonoxidative process. J Infect Dis. 1990; 162: 700–704.

109.

Kaku

Kawamura

Uchiyama

RD1 region in mycobacterial genome is involved in the induction of necrosis in infected RAW264 cells via mitochondrial membrane damage and ATP depletion. FEMS Microbiol Lett. 2007; 274: 189–195.

110.

Kamijo

Shapiro

Mice that lack the interferon-gamma receptor have profoundly altered responses to infection with Bacillus Calmette-Guerin and subsequent challenge with lipopolysaccharide. J Exp Med. 1993; 178: 1435–1440.

111.

Kang

Azad

Torrelles

The human macrophage mannose receptor directs Mycobacterium tuberculosis lipoarabinomannan-mediated phagosome biogenesis. J Exp Med. 2005; 202: 987–999.

112.

Kaplan

Post

Moreira

Mycobacterium tuberculosis growth at the cavity surface: a microenvironment with failed immunity. Infect Immun. 2003; 71: 7099–7108.

113.

Karlsson

Sant’Ambrogio

Widdicombe

. Afferent neural pathways in cough and reflex bronchoconstriction. J Appl Physiol. 1988; 65: 1007–1023.

114.

Kato

. Site II–specific inhibition of mitochondria oxidative phosphorylation by trehalose-6,6′-dimycolate (cord factor) of Mycobacterium tuberculosis . Arch Biochem Biophys. 1970; 140: 379–390.

115.

Kaufmann

. How can immunology contribute to the control of tuberculosis? Nat Rev Immunol. 2001; 1: 20–30.

116.

Keller

Hoffmann

Lang

Genetically determined susceptibility to tuberculosis in mice causally involves accelerated and enhanced recruitment of granulocytes. Infect Immun. 2006; 74: 4295–4309.

117.

Kim

Wainwright

Locketz

Caseation of human tuberculosis granulomas correlates with elevated host lipid metabolism. EMBO Mol Med. 2010; 2: 258–274.

118.

Kinhikar

Verma

Chandra

Potential role for ESAT6 in dissemination of M. tuberculosis via human lung epithelial cells. Mol Microbiol. 2010; 75: 92–106.

119.

Koch

. die aetiologie der tuberculose, a translation by Berna Pinner and Max Pinner with an introduction by Allen K. Krause. Am Rev Tuberc. 1932; 25: 285–323.

120.

Krause

. Tuberculosis and public health. Am Rev Tuberc. 1928; 18: 271–322.

121.

Dupuis-Girod

Dittrich

NEMO mutations in 2 unrelated boys with severe infections and conical teeth. Pediatrics. 2005; 115: e615–619.

122.

Laneelle

Tocanne

. Evidence for penetration in liposomes and in mitochondrial membranes of a fluorescent analogue of cord factor. Eur J Biochem. 1980; 109: 177–182.

123.

Launois

Blum

Dieye

Phenolic glycolipid-1 from M. leprae inhibits oxygen free radical production by human mononuclear cells. Res Immunol. 1989; 140: 847–855.

124.

Law

Jagirdar

Weiden

Tuberculosis in HIV-positive patients: cellular response and immune activation in the lung. Am J Respir Crit Care Med. 1996; 153: 1377–1384.

125.

Lehrer

Ladra

Hake

. Nonoxidative fungicidal mechanisms of mammalian granulocytes: demonstration of components with candidacidal activity in human, rabbit, and guinea pig leukocytes. Infect Immun. 1975; 11: 1226–1234.

126.

Lenaerts

Hoff

Aly

Location of persisting mycobacteria in a guinea pig model of tuberculosis revealed by r207910. Antimicrob Agents Chemother. 2007; 51: 3338–3345.

127.

Levine

. Classification of Reinfection Pulmonary Tuberculosis. Springfield, IL: Charles C Thomas; 1949.

128.

Lissner

Weinstein

O’Brien

. Mouse chromosome 1 Ity locus regulates microbicidal activity of isolated peritoneal macrophages against a diverse group of intracellular and extracellular bacteria. J Immunol. 1985; 135: 544–547.

129.

Lockhart

Green

Flynn

. IL-17 production is dominated by gammadelta T cells rather than CD4 T cells during Mycobacterium tuberculosis infection. J Immunol. 2006; 177: 4662–4669.

130.

Lopez

Yero

Falero-Diaz

Induction of a protective response with an IgA monoclonal antibody against Mycobacterium tuberculosis 16kDa protein in a model of progressive pulmonary infection. Int J Med Microbiol. 2009; 299: 447–452.

131.

Lurie

Abramson

Heppleston

. On the response of genetically resistant and susceptible rabbits to the quantitative inhalation of human type tubercle bacilli and the nature of resistance to tuberculosis. J Exp Med. 1952; 95: 119–134.

132.

Maglione

Chan

. B cells moderate inflammatory progression and enhance bacterial containment upon pulmonary challenge with Mycobacterium tuberculosis . J Immunol. 2007; 178: 7222–7234.

133.

Manabe

Dannenberg

Jr Tyagi

Different strains of Mycobacterium tuberculosis cause various spectrums of disease in the rabbit model of tuberculosis. Infect Immun. 2003; 71: 6004–6011.

134.

Manabe

Kesavan

Lopez-Molina

The aerosol rabbit model of TB latency, reactivation and immune reconstitution inflammatory syndrome. Tuberculosis (Edinb). 2008; 88: 187–196.

135.

Manca

Paul

Barry

CE III

Mycobacterium tuberculosis catalase and peroxidase activities and resistance to oxidative killing in human monocytes in vitro. Infect Immun. 1999; 67: 74–79.

136.

Martinez

Helming

Gordon

. Alternative activation of macrophages: an immunologic functional perspective. Annu Rev Immunol. 2009; 27: 451–483.

137.

McCune

Feldmann

Lambert

Microbial persistence, I: the capacity of tubercle bacilli to survive sterilization in mouse tissues. J Exp Med. 1966; 123: 445–468.

138.

McCune

Feldmann

McDermott

. Microbial persistence, II: characteristics of the sterile state of tubercle bacilli. J Exp Med. 1966; 123: 469–486.

139.

McDonough

Kress

. Cytotoxicity for lung epithelial cells is a virulence-associated phenotype of Mycobacterium tuberculosis . Infect Immun. 1995; 63: 4802–4811.

140.

McKinney

Honer zu Bentrup

Munoz-Elias

Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature. 2000; 406: 735–738.

141.

McMurray

. Disease model: pulmonary tuberculosis. Trends Mol Med. 2001; 7: 135–137.

142.

McMurray

. Guinea Pig Model of Tuberculosis. Washington, DC: American Society for Microbiology; 1994.

143.

Means

Wang

Lien

Human Toll-like receptors mediate cellular activation by Mycobacterium tuberculosis . J Immunol. 1999; 163: 3920–3927.

144.

Mehta

Karls

White

Entry and intracellular replication of Mycobacterium tuberculosis in cultured human microvascular endothelial cells. Microb Pathog. 2006; 41: 119–124.

145.

Mendez

Hatem

Kesavan

Susceptibility to tuberculosis: composition of tuberculous granulomas in Thorbecke and outbred New Zealand White rabbits. Vet Immunol Immunopathol. 2008; 122: 167–174.

146.

Miller

Fratti

Poschet

Mycobacteria inhibit nitric oxide synthase recruitment to phagosomes during macrophage infection. Infect Immun. 2004; 72: 2872–2878.

147.

Mitchison

. The diagnosis and therapy of tuberculosis during the past 100 years. Am J Respir Crit Care Med. 2005; 171: 699–706.

148.

Neyrolles

Hernandez-Pando

Pietri-Rouxel

Is adipose tissue a place for Mycobacterium tuberculosis persistence?

PLoS One. 2006; 1: e43.

149.

Nicholson

Bonecini-Almeida Mda

Lapa e Silva

Inducible nitric oxide synthase in pulmonary alveolar macrophages from patients with tuberculosis. J Exp Med. 1996; 183: 2293–2302.

150.

Oddo

Renno

Attinger

Fas ligand-induced apoptosis of infected human macrophages reduces the viability of intracellular Mycobacterium tuberculosis . J Immunol. 1998; 160: 5448–5454.

151.

Ojha

Anand

Bhatt

GroEL1: a dedicated chaperone involved in mycolic acid biosynthesis during biofilm formation in mycobacteria. Cell. 2005; 123: 861–873.

152.

Ordway

Palanisamy

Henao-Tamayo

The cellular immune response to Mycobacterium tuberculosis infection in the guinea pig. J Immunol. 2007; 179: 2532–2541.

153.

Orme

. The immunopathogenesis of tuberculosis: a new working hypothesis. Trends Microbiol. 1998; 6: 94–97.

154.

Ouyang

Kolls

Zheng

. The biological functions of T helper 17 cell effector cytokines in inflammation. Immunity. 2008; 28: 454–467.

155.

Ozaki

Nakahira

Tani

Differential cell analysis in bronchoalveolar lavage fluid from pulmonary lesions of patients with tuberculosis. Chest. 1992; 102: 54–59.

156.

Ozeki

Kaneda

Fujiwara

In vivo induction of apoptosis in the thymus by administration of mycobacterial cord factor (trehalose 6,6′-dimycolate). Infect Immun. 1997; 65: 1793–1799.

157.

Pabst

Gross

Brozna

Inhibition of macrophage priming by sulfatide from Mycobacterium tuberculosis . J Immunol. 1988; 140: 634–640.

158.

Pandey

Sassetti

. Mycobacterial persistence requires the utilization of host cholesterol. Proc Natl Acad Sci U S A. 2008; 105: 4376–4380.

159.

Park

Yang

A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat Immunol. 2005; 6: 1133–1141.

160.

Park

Myers

Marzella

. Oxygen tensions and infections: modulation of microbial growth, activity of antimicrobial agents, and immunologic responses. Clin Infect Dis. 1992; 14: 720–740.

161.

Pathak

Basu

Direct extracellular interaction between the early secreted antigen ESAT-6 of Mycobacterium tuberculosis and TLR2 inhibits TLR signaling in macrophages. Nat Immunol. 2007; 8: 610–618.

162.

Pearl

Khader

Solache

IL-27 signaling compromises control of bacterial growth in mycobacteria-infected mice. J Immunol. 2004; 173: 7490–7496.

163.

Pedroza-Gonzalez

Garcia-Romo

Aguilar-Leon

In situ analysis of lung antigen-presenting cells during murine pulmonary infection with virulent Mycobacterium tuberculosis . Int J Exp Pathol. 2004; 85: 135–145.

164.

Perez

Roman

Roser

Cytokine message and protein expression during lung granuloma formation and resolution induced by the mycobacterial cord factor trehalose-6,6′-dimycolate. J Interferon Cytokine Res. 2000; 20: 795–804.

165.

Peyron

Vaubourgeix

Poquet

Foamy macrophages from tuberculous patients’ granulomas constitute a nutrient-rich reservoir for M. tuberculosis persistence. PLoS Pathog. 2008; 4: e1000204.

166.

Portevin

Sousa-D'

Auria

Houssin

A polyketide synthase catalyzes the last condensation step of mycolic acid biosynthesis in mycobacteria and related organisms. Proc Natl Acad Sci U S A. 2004; 101: 314–319.

167.

Puissegur

Lay

Gilleron

Mycobacterial lipomannan induces granuloma macrophage fusion via a TLR2-dependent, ADAM9- and beta1 integrin-mediated pathway. J Immunol. 2007; 178: 3161–3169.

168.

Quesniaux

Nicolle

Torres

Toll-like receptor 2 (TLR2)–dependent-positive and TLR2-independent-negative regulation of proinflammatory cytokines by mycobacterial lipomannans. J Immunol. 2004; 172: 4425–4434.

169.

Radaeva

Kondratieva

Sosunov

A human-like TB in genetically susceptible mice followed by the true dormancy in a Cornell-like model. Tuberculosis (Edinb). 2008; 88: 576–585.

170.

Recht

Kolter

. Glycopeptidolipid acetylation affects sliding motility and biofilm formation in Mycobacterium smegmatis . J Bacteriol. 2001; 183: 5718–5724.

171.

Reed

Domenech

Manca

A glycolipid of hypervirulent tuberculosis strains that inhibits the innate immune response. Nature. 2004; 431: 84–87.

172.

Reznik

. Comparative anatomy, physiology, and function of the upper respiratory tract. Environ Health Perspect. 1990; 85: 171–176.

173.

Rich

. The Pathogenesis of Tuberculosis. 2nd ed. Springfield, IL: Charles C Thomas; 1951.

174.

Riedel

Kaufmann

. Chemokine secretion by human polymorphonuclear granulocytes after stimulation with Mycobacterium tuberculosis and lipoarabinomannan. Infect Immun. 1997; 65: 4620–4623.

175.

Riley

. Aerial dissemination of pulmonary tuberculosis. Am Rev Tuberc. 1957; 76: 931–941.

176.

Rios-Barrera

Campos-Pena

Aguilar-Leon

Macrophage and T lymphocyte apoptosis during experimental pulmonary tuberculosis: their relationship to mycobacterial virulence. Eur J Immunol. 2006; 36: 345–353.

177.

Rivas-Santiago

Contreras

Sada

The potential role of lung epithelial cells and beta-defensins in experimental latent tuberculosis. Scand J Immunol. 2008; 67: 448–452.

178.

Rivas-Santiago

Schwander

Sarabia

Human {beta}-defensin 2 is expressed and associated with Mycobacterium tuberculosis during infection of human alveolar epithelial cells. Infect Immun. 2005; 73: 4505–4511.

179.

Rook

Dheda

Zumla

. Immune responses to tuberculosis in developing countries: implications for new vaccines. Nat Rev Immunol. 2005; 5: 661–667.

180.

Ruan

St John

, Ehrt S, et al. noxR3, a novel gene from Mycobacterium tuberculosis, protects Salmonella typhimurium from nitrosative and oxidative stress. Infect Immun. 1999; 67: 3276–3283.

181.

Ryffel

Fremond

Jacobs

Innate immunity to mycobacterial infection in mice: critical role for Toll-like receptors. Tuberculosis (Edinb). 2005; 85: 395–405.

182.

Sada-Ovalle

Chiba

Gonzales

Innate invariant NKT cells recognize Mycobacterium tuberculosis–infected macrophages, produce interferon-gamma, and kill intracellular bacteria. PLoS Pathog. 2008; 4: e1000239.

183.

Schindler

Mancilla

Endres

Correlations and interactions in the production of interleukin-6 (IL-6), IL-1, and tumor necrosis factor (TNF) in human blood mononuclear cells: IL-6 suppresses IL-1 and TNF. Blood. 1990; 75: 40–47.

184.

Schroeder

. Acquired tuberculosis in the primate in laboratories and zoological collections. Am J Public Health Nations Health. 1938; 28: 469–475.

185.

Seiler

Aichele

Bandermann

Early granuloma formation after aerosol Mycobacterium tuberculosis infection is regulated by neutrophils via CXCR3-signaling chemokines. Eur J Immunol. 2003; 33: 2676–2686.

186.

Sherman

Mdluli

Hickey

Compensatory ahpC gene expression in isoniazid-resistant Mycobacterium tuberculosis . Science. 1996; 272: 1641–1643.

187.

Sieling

Chatterjee

Porcelli

CD1-restricted T cell recognition of microbial lipoglycan antigens. Science. 1995; 269: 227–230.

188.

Skjot

Oettinger

Rosenkrands

Comparative evaluation of low-molecular-mass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as immunodominant T-cell antigens. Infect Immun. 2000; 68: 214–220.

189.

Smith

McMurray

Wiegeshaus

Host-parasite relationships in experimental airborne tuberculosis, IV: early events in the course of infection in vaccinated and nonvaccinated guinea pigs. Am Rev Respir Dis. 1970; 102: 937–949.

190.

Stead

Eisenach

Cave

When did Mycobacterium tuberculosis infection first occur in the New World? An important question with public health implications. Am J Respir Crit Care Med. 1995; 151: 1267–1268.

191.

Stegelmann

Bastian

Swoboda

Coordinate expression of CC chemokine ligand 5, granulysin, and perforin in CD8+ T cells provides a host defense mechanism against Mycobacterium tuberculosis . J Immunol. 2005; 175: 7474–7483.

192.

Stenger

Niazi

Modlin

. Down-regulation of CD1 on antigen-presenting cells by infection with Mycobacterium tuberculosis . J Immunol. 1998; 161: 3582–3588.

193.

Stewart

Rivera

Karls

Increased pathology in lungs of mice after infection with an alpha-crystallin mutant of Mycobacterium tuberculosis: changes in cathepsin proteases and certain cytokines. Microbiology. 2006; 152: 233–244.

194.

Stumhofer

Laurence

Wilson

Interleukin 27 negatively regulates the development of interleukin 17–producing T helper cells during chronic inflammation of the central nervous system. Nat Immunol. 2006; 7: 937–945.

195.

Sturgill-Koszycki

Schlesinger

Chakraborty

Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase. Science. 1994; 263: 678–681.

196.

Tailleux

Schwartz

Herrmann

DC-SIGN is the major Mycobacterium tuberculosis receptor on human dendritic cells. J Exp Med. 2003; 197: 121–127.

197.

Tan

Meinken

Bastian

Macrophages acquire neutrophil granules for antimicrobial activity against intracellular pathogens. J Immunol. 2006; 177: 1864–1871.

198.

Teitelbaum

Glatman-Freedman

Chen

A mAb recognizing a surface antigen of Mycobacterium tuberculosis enhances host survival. Proc Natl Acad Sci U S A. 1998; 95: 15688–15693.

199.

Teitelbaum

Schubert

Gunther

The M cell as a portal of entry to the lung for the bacterial pathogen Mycobacterium tuberculosis . Immunity. 1999; 10: 641–650.

200.

Toossi

Sedor

Lapurga

Expression of functional interleukin 2 receptors by peripheral blood monocytes from patients with active pulmonary tuberculosis. J Clin Invest. 1990; 85: 1777–1784.

201.

Torres

Ramachandra

Rojas

Role of phagosomes and major histocompatibility complex class II (MHC-II) compartment in MHC-II antigen processing of Mycobacterium tuberculosis in human macrophages. Infect Immun. 2006; 74: 1621–1630.

202.

Tsao

Hong

Huang

Increased TNF-alpha, IL-1 beta and IL-6 levels in the bronchoalveolar lavage fluid with the upregulation of their mRNA in macrophages lavaged from patients with active pulmonary tuberculosis. Tuber Lung Dis. 1999; 79: 279–285.

203.

Tufariello

Chan

Flynn

. Latent tuberculosis: mechanisms of host and bacillus that contribute to persistent infection. Lancet Infect Dis. 2003; 3: 578–590.

204.

Ulrichs

Moody

Grant

T-cell responses to CD1-presented lipid antigens in humans with Mycobacterium tuberculosis infection. Infect Immun. 2003; 71: 3076–3087.

205.

Umemura

Yahagi

Hamada

IL-17-mediated regulation of innate and acquired immune response against pulmonary Mycobacterium bovis bacille Calmette-Guerin infection. J Immunol. 2007; 178: 3786–3796.

206.

van Crevel

Karyadi

Preyers

Increased production of interleukin 4 by CD4+ and CD8+ T cells from patients with tuberculosis is related to the presence of pulmonary cavities. J Infect Dis. 2000; 181: 1194–1197.

207.

Van Scoy

Wilkowske

. Antimycobacterial therapy. Mayo Clin Proc. 1999; 74: 1038–1048.

208.

VanHeyningen

Collins

Russell

. IL-6 produced by macrophages infected with Mycobacterium species suppresses T cell responses. J Immunol. 1997; 158: 330–337.

209.

Vankayalapati

Klucar

Wizel

NK cells regulate CD8+ T cell effector function in response to an intracellular pathogen. J Immunol. 2004; 172: 130–137.

210.

Vergne

Chua

Deretic

. Tuberculosis toxin blocking phagosome maturation inhibits a novel Ca2+/calmodulin-PI3K hVPS34 cascade. J Exp Med. 2003; 198: 653–659.

211.

Vergne

Chua

Lee

Mechanism of phagolysosome biogenesis block by viable Mycobacterium tuberculosis . Proc Natl Acad Sci U S A. 2005; 102: 4033–4038.

212.

Verver

Warren

Beyers

Rate of reinfection tuberculosis after successful treatment is higher than rate of new tuberculosis. Am J Respir Crit Care Med. 2005; 171: 1430–1435.

213.

Via

Lin

Ray

Tuberculous granulomas are hypoxic in guinea pigs, rabbits, and nonhuman primates. Infect Immun. 2008; 76: 2333–2340.

214.

Vidal

Pinner

Lepage

Natural resistance to intracellular infections: Nramp1 encodes a membrane phosphoglycoprotein absent in macrophages from susceptible (Nramp1 D169) mouse strains. J Immunol. 1996; 157: 3559–3568.

215.

Vieira

Harrison

Scott

Acquisition of Hrs, an essential component of phagosomal maturation, is impaired by mycobacteria. Mol Cell Biol. 2004; 24: 4593–4604.

216.

Wilkinson

DesJardin

Islam

An increase in expression of a Mycobacterium tuberculosis mycolyl transferase gene (fbpB) occurs early after infection of human monocytes. Mol Microbiol. 2001; 39: 813–821.

217.

Williams

Reljic

Naylor

Passive protection with immunoglobulin A antibodies against tuberculous early infection of the lungs. Immunology. 2004; 111: 328–333.

218.

Witmer

Fine

Gionfriddo

Epizootiologic survey of Mycobacterium bovis in wildlife and farm environments in northern Michigan. J Wildl Dis. 2010; 46: 368–378.

219.

Wolday

Hailu

Girma

Low CD4+ T-cell count and high HIV viral load precede the development of tuberculosis disease in a cohort of HIV-positive Ethiopians. Int J Tuberc Lung Dis. 2003; 7: 110–116.

220.

Xirouchaki

Tzanakis

Bouros

Diagnostic value of interleukin-1alpha, interleukin-6, and tumor necrosis factor in pleural effusions. Chest. 2002; 121: 815–820.

221.

Yamamura

. The pathogenesis of tuberculous cavities. Bibl Tuberc. 1958; 13: 13–37.

222.

Zhang

Zheng

Human NK cells positively regulate gammadelta T cells in response to Mycobacterium tuberculosis . J Immunol. 2006; 176: 2610–2616.

The Pathology of Mycobacterium tuberculosis Infection

Abstract

Keywords

The Bacterium

Cell Biology of Mtb

Virulence Factors

Pathogenesis and Lesion Development

Host Response

Cytokines

Cellular Response

Animal Models of TB

Mice

Guinea Pigs

Rabbits

Nonhuman Primates

Conclusion

Footnotes

Acknowledgements

References