Pathology of Tuberculosis: How the Pathology of Human Tuberculosis Informs and Directs Animal Models

ABSTRACT

Tuberculosis (TB) is a chronic inflammatory disease caused by the pathogenic bacterium Mycobacterium tuberculosis. A wide variety of host- and pathogen-associated variables influence the clinical manifestation of TB in different individuals within the human population. As a consequence, the characteristic granulomatous lesions that develop within the lung are heterogeneous in size and cellular composition. Due to the lack of appropriate tissues from human TB patients, a variety of animal models are used as surrogates to study the basic pathogenesis and to test experimental vaccines and new drug therapies. Few animal models mimic the clinical course and pathological response of M. tuberculosis seen in the naturally occurring disease in people. In particular, post-primary TB, which accounts for the majority of cases of active TB and is responsible for transmission between individuals via aerosol exposers, cannot be reproduced in animals and therefore cannot be adequately modeled experimentally. This article describes a new paradigm that explains the pathogenesis of post-primary TB in humans. This new evidence was derived from histological examination of tissues from patients with different stages of M. tuberculosis infection and that had not been treated with antimicrobial drugs. Gaining a better understanding of this unique stage of TB disease will lead to more effective treatment, diagnostic, and prevention strategies.

INTRODUCTION

Progress toward developing new strategies to control the spread of Mycobacterium tuberculosis is limited by a poor understanding of the basic pathogenesis of post-primary tuberculosis (TB). Progress is being made in developing more rapid diagnostic assays and implementing new anti-TB drug combinations, but we are failing to answer key scientific questions that will further advance the development of new treatment and prevention strategies. In a recent statement, Dr. Anthony Fauci, head of the National Institute of Allergy and Infectious Diseases, said, “We need to better understand the delicate balance between the host and pathogen in the context of the entire biological system and this requires a radical and transformational approach.” M. tuberculosis has coevolved with its human host for centuries. The more recent emergence of antimicrobial drug-resistant strains represents an additional challenge to controlling the global spread of TB. No new TB vaccines have been shown to be more effective than the original bacillus Calmette-Guérin (BCG) developed over 100 years ago. The development of new anti-TB drugs lags far behind the need, and prospects for host-directed therapies have validated neither targets nor biomarkers.

In nature, M. tuberculosis is a human, obligate, intracellular bacterial pathogen. While it can infect nearly any warm-blooded animal, the majority of experimentally infected laboratory animals cannot easily transmit the infection through aerosol exposure. In addition, while M. tuberculosis infections in small animal models consistently develop the acute stages of primary TB disease, they fail to develop the late stages of post-primary disease. Post-primary TB accounts for 80% of clinical disease and nearly 100% of transmission of infection in humans, yet very little is known about the pathogenesis. Gross and histopathological examination of tissues from human patients was central to the study of TB for 150 years, from 1800 until 1950. However, the use of pathology to study TB fell out of favor as a consequence of scientific advances and changing attitudes. First, the discovery of antibiotics that were effective against M. tuberculosis led many to believe that TB and other bacterial diseases would soon be eradicated and therefore not in need of further study. Second, the advances in the fields of immunology, molecular biology, and genetics in the second half of the 20th century replaced morphologic pathology as a cutting-edge science. Finally, fewer routine autopsies were performed on patients that died of TB or other diseases, which significantly decreased the availability of tissue samples to study early-stage disease. Lung samples from TB patients are still occasionally available following surgical excision as an adjunct to antimicrobial therapy, but these represent the most chronic stages of active TB disease and have limited value as a research resource.

As a consequence, when the value of the pathology of human and animal TB was again realized in the 1980s, appropriate tissue samples were seldom available. As a surrogate, a variety of animal models were used to help advance the basic understanding of TB pathology and pathogenesis as well as to better understand the impact vaccination and antimicrobial drug treatment have on the progression of active TB disease. However, it became increasingly obvious that there were limitations to the use of animal models to study the basic pathogenesis of human pulmonary and extrapulmonary TB.

Many model species develop early lesions that resemble primary TB in humans (Fig. 1). However, with the possible exception of non-human primates, few animals develop the full spectrum of disease and, in particular, post-primary TB. One of the most important manifestations of post-primary TB is the development of thin-walled cavities that support proliferation of massive numbers of extracellular M. tuberculosis (Fig. 2, Fig. 3), which increases the likelihood of transmission between individuals through aerosols. To survive and spread within the human population, M. tuberculosis induces a nonprotective immune response in some individuals that leads to extensive tissue damage and high bacterial burden, which if left untreated will result in death of the host. Some individuals, however, either clear the infection or remain healthy but harbor small numbers of bacilli that contribute to the development of reactivation disease later in life. In this way, M. tuberculosis persists within the population and serves as a reservoir of infection, thus maintaining the transmission cycle. Therefore, the coevolution of M. tuberculosis with humans has resulted in a complex, multifaceted host response such that individuals that develop active TB transmit soon after exposure, while those that remain asymptomatic have the potential to spread disease following the development of post-primary TB (2). Many attempts to identify the specific immune responses that determine whether the host response will be protective or result in active TB disease have been unsuccessful. What remains the fundamental, unanswered question is a poor understanding of how the tubercle bacillus survives in the face of an aggressive immune response, thus creating a microenvironment that favors M. tuberculosis persistence (2).

FIGURE 1.

Well-delineated foci of granulomatous inflammation (granuloma) within the lung is a common manifestation of primary tuberculosis in humans and many laboratory animals. (A) A low-magnification view of a primary lung granuloma from an M. tuberculosis-infected guinea pig has an area of central necrosis that is partially calcified (C). (B) The wall of the lesion contains active lymphocytic and histiocytic inflammation and is well delineated from the surrounding normal lung parenchyma by a fibrous capsule that contains regenerative airway epithelium (black arrow). A higher-magnification view of the central dystrophic calcification (white arrow) shows residual tissue necrosis that harbors extracellular bacilli. Hematoxylin and eosin (H&E) stain.

FIGURE 2.

An important manifestation of post-primary TB in humans that is not seen in many small animal models is the formation of thin wall cavities. Cavity formation (C) represents one of the most destructive manifestations of active tuberculosis in humans and non-human primates. The lung parenchyma is replaced by an open space that often contains necrotic cellular debris (N) and myriads of extracellular and intracellular bacilli that can be transmitted between individuals though aerosol spread. The wall of the cavity consists of mixed inflammation similar to primary granulomas and similarly is delineated from the more normal parenchyma by a fibrous capsule that impairs the penetration of antimicrobial drugs. H&E stain.

FIGURE 3.

Cavity formation associated with post-primary TB is important in the transmission of bacilli between individuals due to the large numbers of extracellular and intracellular bacilli. (A) High-magnification images of a cavitary lung lesion shown filling the lumen with necrotic cellular debris (white arrow). (B) An acid-fast stain shows that bacilli within the necrotic debris are arranged in small clusters or as individuals (black arrow) and are mostly extracellular. The high numbers of bacilli within these lesions are important in the transmission of M. tuberculosis between individuals, especially when cavities communicate with lung airways. H&E stain.

Due to the relative lack of tissues from M. tuberculosis-infected patients, the current literature pertaining to TB pathology and pathogenesis is based almost entirely on animal studies using models that do not necessarily mimic all the important features of naturally occurring human disease (3–5). Stimulated by inconsistencies in the recent literature, we attempted to understand the pathology of human post-primary TB from studies published during the preantibiotic era as well as through the study of autopsy tissue samples obtained from untreated patients. Many of the books describing human TB pathology are either out of print or use unfamiliar nomenclature. More importantly, gross and histopathological images or illustrations from this literature are either nonexistent or of poor quality and therefore not suitable for primary reexamination. We were fortunate to acquire tissues from untreated TB from medical examiners, historic collections, and international collaborators. Comprehensive study of these samples enabled us to reevaluate and reinterpret findings reported in the older literature and to develop a comprehensive model explaining the pathogenesis of post-primary TB (6).

Specifically, we reexamined the paradigm that the host responses resulting in the formation of caseous granulomas represent the early stages of both primary and post-primary TB as suggested by animal studies of the late 20th century. We concluded that this interpretation is not supported by the data or generated by pathologists or radiologists who extensively studied the pathology of human pulmonary TB in the preantibiotic era (7–23). The current paradigm is that caseous granulomas are the origin of both primary and post-primary TB and that cavities arise from erosion of necrotic granulomas into bronchi, which is not supported by the literature or our studies. Moreover, the use of mice and other small animal models to study the basic in vivo pathogenesis and immunological response of M. tuberculosis infection has further complicated our understanding of TB disease progression. Through extensive review of the literature and re-examination of tissues from untreated human TB patients, we have concluded that post-primary TB develops as an obstructive lobular pneumonia that spreads asymptomatically via bronchi within individuals with a high degree of M. tuberculosis-specific immunity (Fig. 4) (3, 6). This revised description of the pathology is supported by hundreds of publications from Rene Laënnec using gross pathology as early as 1804 through more recent histological studies and advanced imaging techniques such as high-resolution magnetic resonance imaging and computed tomography (24).

FIGURE 4.

Intrapulmonary spread of mixed inflammatory cells within the lung parenchyma results in an obstructive lobar pneumonia and is involved in the early pathogenesis of post-primary TB. In contrast to primary lesions, the filling of alveoli with mixed inflammatory cells, including foamy macrophages, in the absence of necrosis contributes to airway obstruction and the development of post-primary TB. H&E stain.

The revised interpretation based on more recent examination of tissue sections from untreated human patients is also supported by two other observations, which explains the pathogenesis of post-primary TB (25). Post-primary TB is consistently associated with bronchial obstruction from any number of causes, resulting in a postobstructive lipid pneumonia that progresses to cavitary lesions (26). Moreover, in the early stages of post-primary TB, lipid pneumonia is in part due to intracellular and extracellular accumulation of secreted mycobacterial antigens as well as excess host lipids in asymptomatic patients. These observations led to the formulation of a new model of TB pathogenesis using the metaphor of a three-act play. The critical new component is act 2, or what we refer to as the sneak attack. This is the process in which accumulation of mycobacterial antigens and host lipids ultimately trigger a massive necrotizing reaction that leads to cavitary lesions that communicate with conducting airways. As a consequence, the rapid proliferation and accumulation of infectious bacilli overwhelm host defenses and contribute to efficient transmission of the organism to new hosts.

The dictionary defines paradigm as “a framework containing the basic assumptions, ways of thinking, and methodology that are commonly accepted by members of the scientific community.” Accordingly, a new paradigm calls for new experimental approaches and validation. This article attempts to briefly summarize our current understanding of human post-primary TB. It will then describe how different animal models have or can be used to specifically address the knowledge gaps that continue to slow progress toward preventing, diagnosing, and effectively treating human TB.

TB DISEASE PROGRESSION IN ANIMAL MODELS

The host response to M. tuberculosis infection in all mammalian species is almost universally characterized as granulomatous inflammation. The timeline of the basic host response to experimental M. tuberculosis infection in the various animal models has been reviewed (27, 28). It is widely accepted that progressive M. tuberculosis infection in mice, rabbits, guinea pigs, and non-human primates as well as humans is somewhat limited in the early stages of infection by an adaptive immune response that has a characteristic histological appearance. However, there is increasing evidence to suggest that M. tuberculosis simultaneously adapts to a changing microenvironment as a consequence of the changing host immune responses. The adaptations of M. tuberculosis to immune pressure results in the selection of small populations of nonreplicating, drug-tolerant bacilli that are able to persist for months or years in asymptomatic hosts. These bacteria maintain a low metabolic state, and the persistence of secreted and nonsecreted antigens is able to continually manipulate the host’s response. In both untreated adult humans and animals, death in the late stages of disease is due to progressive pulmonary and extrapulmonary inflammation and, ultimately, multiorgan failure. Several lines of evidence, especially the observation that bacterial numbers may not increase significantly even in the late stages of disease, suggest that the tissue damage is due largely to ongoing responses to accumulated mycobacterial antigens (Fig. 5). What accounts for the disconnect between bacterial numbers and lesion severity in animal models compared to humans is likely the result of an unnaturally high challenge dose combined with the ongoing bacterial replication in species with minimal resistance combined with the persistence of M. tuberculosis antigens. According to North, “a central problem in tuberculosis research is the inability to explain why immunity to infection does not enable mice, guinea pigs, rabbits or susceptible humans to resolve lung infection and thereby stop development of disease” (28).

FIGURE 5.

Similarities in the time course of humans and animal models of TB demonstrating that pathology does not correlate with increased numbers of bacteria. A central problem in TB research is to explain why immunity to infection does not enable mice, guinea pigs, rabbits, or susceptible humans to resolve lung infection and thereby stop the development of disease.

THE PRIMARY HOST RESPONSE TO M. TUBERCULOSIS INFECTION

After decades of animal experimentation, it has become clear that no one animal model adequately mimics all the complexities of human TB. Animal models are not only limited by the differences in the inherent host response to experimental M. tuberculosis infection but are also subject to experimental differences in challenge strain, dose, and route of infection. In addition, the choice of animal model is heavily influenced by practical considerations given the cost of maintaining certain species in specialized, biocontainment facilities that are not readily available to most investigators. Accepting the experimental variables that influence the host responses to M. tuberculosis infection, investigators involved in anti-TB drug research and development have attempted to standardize animal model protocols to increase the reproducibility of data between laboratories and institutions (29). While this has helped to ensure that drug treatment responses in mice are reproducible, the use of models that better mimic the naturally occurring disease in humans continues to be overlooked. With improved knowledge of the pathology of human TB, it is increasingly recognized that all model species have advantages and disadvantages and can be experimentally manipulated to answer specific questions. However, what remains imperative is that data from animal models be interpreted in the context of naturally occurring TB in humans. “One should never forget the limitations of experimental studies in animals. They provide useful hypotheses and certain facts not observable in man, but in no case can they replace observations in man for ultimate understanding of the disease in human beings” (21).

We argue here that the foundation by which to compare data derived from TB animal models be based on our fundamental understanding of human TB, particularly in patients in which the clinical course has not been altered by antimicrobial drug treatment. Due to obvious ethical considerations, the availability of samples that meet this criteria are limited to postmortem cases from patients that have died from either progressive TB or unrelated accidents or diseases. In a series of recently published studies, Hunter et al. outlined how the primary literature describing the pathology of human TB patients combined with more contemporary approaches including advanced imaging to redefine the relationship, or lack thereof, between primary and post-primary TB disease (3, 4, 6). The challenge is to determine how to apply the technological advances we now have available in combination with what we have learned from studying TB animal models to fill the critical knowledge gaps.

Each of the animal strains or species used for TB research has unique characteristics, and none fully reproduce the human disease, especially the late disease that produces most adult disease and nearly all transmission of infection. However, as pointed out by North, many share common characteristics with the human disease. In particular, mice, rabbits, guinea pigs, and humans all develop immunity sufficient to limit proliferation of organisms in a few weeks but are unable to eliminate all organisms (Fig. 1). The infection then persists asymptomatically for months before the animals rapidly develop inflammation and disease and die, frequently with little or no increase in numbers of viable M. tuberculosis organisms (2, 30). The tissue damage is thought to be an immunopathology somehow triggered by mycobacterial antigens, not by live M. tuberculosis itself. A better understanding of the commonalities of how M. tuberculosis produces disease without increasing in number in quite different histologic lesions would be a major advance.

Briefly, the characteristic response to primary infection with M. tuberculosis in humans and most animal models involves the localized accumulation of mixed inflammatory and immune cells into a discrete nodular mass referred to as a granuloma. “Granulomatous inflammation” is a broadly used term to describe an infiltrate of primarily mononuclear cells composed mostly of macrophages and lymphocytes. Granulomatous inflammation is not a unique response to mycobacterial infections but can be seen in fungal infections as well as in response to foreign bodies (31). In addition, multinucleated giant cells are a frequent feature of TB granulomas, but they too are not unique to M. tuberculosis infection. The primary TB granuloma in humans does have important morphological characteristics that are shared with some commonly used animal models. Specifically, the distribution of different mononuclear cell populations combined with the development of central caseous necrosis, fibrous encapsulation, and dystrophic calcification are features seen in immunologically naive non-human primates and guinea pigs and less often in certain strains of mice and rabbits. Developing post-primary TB in humans has a different pathology that is an asymptomatic obstructive lobular pneumonia that progresses to cavity formation in the most aggressive response or serves as a focus of limited granuloma formation (3). As reported in detail by Canetti and others, granulomas in post-primary TB arise only in response to caseation necrosis and are never the cause of it (19, 21, 32). Nevertheless, the disease in humans and many animals shares the common feature of a prolonged asymptomatic period before development of clinical disease that is frequently not accompanied by a large increase in bacterial load.

HYPERSENSITIVITY IN THE PATHOGENESIS OF POST-PRIMARY TB

An important contribution to the pathogenesis of human post-primary TB involves hypersensitivity to mycobacterial antigens (33, 34). Guinea pigs are among the model species that develop the most robust hypersensitivity responses to M. tuberculosis infection. Studies by McMurray et al. showed that the morphological features of experimental M. tuberculosis infection in guinea pigs differed depending on whether animals were immunologically naive or vaccinated with BCG prior to aerosol exposure (35). In nonvaccinated guinea pigs, the primary response was that of the prototypical TB granuloma that frequently developed central necrosis (36). The similarity between TB granulomas in humans and guinea pigs compared to other model species has been recognized for many decades, which explains why it remains a mainstay in TB research. McMurray showed, however, that the initial response to aerosol infection of BCG-vaccinated guinea pigs differed in that the granulomatous response at the site of infection in the lung was composed primarily of mature lymphocytes which failed to organize into discrete granulomatous masses and rarely developed central necrosis. Moreover, the investigators equated the primary response in BCG-vaccinated animals to secondary lesions resulting from endogenous reinfection of the lung following dissemination of bacilli in naive guinea pigs (35). They further concluded that the differences in lesion morphology were a consequence of the combined effects of hematogenous dissemination of bacilli to extrapulmonary sites including peripheral lymphoid tissues. In the case of guinea pigs, sensitization with BCG prior to M. tuberculosis infection provided partial protection resulting in delayed progression of active disease and a less destructive inflammatory response.

In contrast, immunologically naive rabbits are relatively resistant to infection with less virulent laboratory strains of M. tuberculosis but are more susceptible to Mycobacterium bovis (37). Unlike guinea pigs, a more aggressive proinflammatoy response develops in rabbits that are first sensitized by repeated BCG vaccination (38). The resulting lesions more closely resemble TB abscesses and occasionally form cavitary lesions when lesions communicate with conducting airways. Due to the potential for TB lesions in rabbits to cavitate, this animal model has been used in an attempt to link initial granuloma formation to the development of post-primary TB pathogenesis in humans (39). This overlooks the fact that the pathology produced by M. bovis is different from that of M. tuberculosis (3). Moreover, this model has been used recently to study the impact lesion morphology, specifically abscesses or cavitary lesions, has on TB drug penetration (40–45). These studies have been facilitated by the discovery that rabbits are susceptible to more virulent M. tuberculosis challenge strains including human clinical strains (39, 46). Studies from rabbits support the hypothesis that in humans, cavity formation is linked to an aggressive proinflammatory response, which can be triggered in individuals first sensitized with M. tuberculosis or nontuberculous mycobacterium. However, these data fail to support the direct relationship between the primary granulomas and post-primary TB disease.

Post-Primary Lung Reinfection

The term “post-primary TB” denotes an infection that begins after a primary infection. This host response in previously infected and immunized individuals accounts for 80% of all clinical human pulmonary TB. Consequently, studies of infection of previously sensitized hosts are important to understanding human TB but are rarely conducted. The pathology of developing human post-primary TB has been unequivocally established by scores of papers by dozens of authors, each of whom had personally studied hundreds of cases from a period of nearly 200 years (47). In our proposed pathogenesis of human post-primary TB, intrabronchial spread of infection and airway obstruction factors prominently in the pathogenesis of post-primary cavity formation. Reinfection of the lung following hematogenous dissemination of bacilli has been studied in both guinea pig and mouse TB models, where lung infection can spread through intrabronchiolar and lymphatic routes. The role of intrapulmonary lymphatic spread of M. tuberculosis has been best characterized in guinea pigs. Guinea pigs and non-human primates, like humans, have prominent peribronchial and perivascular connective tissue that supports well-developed and inducible bronchus-associated lymphoid tissue as well as an extensive network of intrapulmonary lymphatics (48–50). The intralymphatic spread of M. tuberculosis from a primary lung infection was first described in guinea pigs in the mid to late 19th century (51, 52). Studies by Klein showed that following experimental infection of guinea pigs with exudate obtained from human TB patients, spread along lymphatic vasculature was among the earliest manifestations of M. tuberculosis infection (48, 51). More recent studies have confirmed that granulomatous lymphangitis is also an early manifestation following low-dose aerosol exposure of guinea pigs and non-human primates to M. tuberculosis (49, 50). With the progression of lymphangiocentric lesions, vascular obstruction is followed by granuloma expansion, which accounts for the consistent perivascular and peribronchial distribution of primary granulomas in animals and humans.

Little is known about the significance of lymphangiocentric lesions in the pathogenesis of post-primary TB in humans. The close association with pulmonary arteries, veins, and airways raises the possibility that the expansion of these lesions may be among the first to communicate with conducting airways and predispose to cavity formation. The reason why cavitary disease is not a consistent finding in M. tuberculosis-infected guinea pigs in contrast to primates may be related to the well-developed connective tissue stroma that supports airways as well as blood and lymphatic vasculature (53). Another pathway to direct airway involvement that may predispose to cavity formation is endobronchial TB, which is a rare manifestation of experimental M. tuberculosis infections in animals but is universally found in developing human post-primary TB (54). The potential role of M. tuberculosis-induced pulmonary lymphangitis may be an early manifestation of post-primary TB in humans but requires further investigation.

In the proposed pathogenesis of human post-primary TB, bronchial obstruction and endobronchial TB factor significantly in the development of endogenous lipid pneumonia and, subsequently, lesion cavitation. Based on extensive review of the human pathology literature and more recent histopathological examination of human lung lesions, airway obstruction contributes to the localized accumulation of foamy macrophages, which are a consistent feature of M. tuberculosis infection in animals and humans (55, 56). Foam cell formation is not unique to TB but is a morphological feature of cellular degeneration. Foam cells are a prominent feature of atherosclerosis and are an important component of intravascular plaque formation in patients with peripheral and coronary vascular disease. The foamy appearance of macrophages can be due to swollen cytoplasmic organelles as a result of dysregulated fluid homeostasis (hydropic degeneration). In addition, an imbalance between lipid accumulation and efflux can also account for cytoplasmic vacuolization and foam cell formation (57). Whereas foam cell formation has been directly linked to obstructed airways in human TB, foamy macrophage accumulation within primary granulomas is a consistent finding in animals even in the absence of airway obstruction (58, 59). Moreover, in model species that fail to develop granuloma necrosis, foam cells that contain intracellular bacilli are a prominent histological feature (58, 60–63). These data suggest that while endogenous lipid pneumonia may contribute to the pathogenesis of post-primary TB in humans, there remains a knowledge gap linking foam cell formation with the proinflammatory response that progresses to cavitary disease (55, 64).

A potential cause of airway obstruction that is shared by humans and animals with TB is the migration and accumulation of mixed inflammatory cells within airway lumens. Processes associated with lesion resolution or healing accompany the progression of inflammation. As mentioned previously, the proliferation of fibroblasts and the deposition of extracellular matrix proteins as well as the deposition of dystrophic calcification is obvious evidence of lesion resolution. An important step in the resolution of inflammation is the elimination of senescent inflammatory cells as they approach the end of their functional life span. Neutrophils are among the first cells to respond to M. tuberculosis and other bacterial infections but also have one of the shortest life spans of all white blood cells (65–67). Senescent neutrophils and other inflammatory cells migrate and are eliminated from the body through mucosal surfaces including airways. Leukocytes near the end of their life-span are eliminated by transepithelial migration into the lumens of mucosal surfaces including the lung (65, 68–70). In TB patients and animal models, infected and noninfected senescent leukocytes accumulate within airway lumens and reach the oropharynx through mucociliary clearance and contribute to the formation of sputum to be swallowed or expectorated. The accumulation of mixed inflammatory cells within the lumens of small airways is a consistent histological finding in M. tuberculosis-infected animals but may not necessarily represent functional obstruction (58, 61). These findings suggest that the accumulation and potential obstruction of small airways in animal models of M. tuberculosis infection is a common feature across species and therefore does not fully explain the development of human post-primary TB.

DISCUSSION AND CONCLUSIONS

Much has been learned from studying the basic pathogenesis and immune response to M. tuberculosis infection in animals. However, one of the major differences is that none of the commonly used animal models mimic the natural transition from primary to post-primary disease seen in humans. Post-primary TB is arguably the most important stage of active TB disease, yet very little is known about the factors that determine whether M. tuberculosis exposure results in an established infection and disease. Extensive study of human TB pathology combined with review of the literature from the preantibiotic era has suggested hypotheses about the factors that contribute to post-primary disease that can be tested in animal models. Collectively, studies in animals demonstrate the patterns of disease that contribute to post-primary TB, yet no one animal model recapitulates the naturally occurring disease in humans. The possibility exists that experimental manipulation of individual or a combination of different animal models can be used to systematically fill the knowledge gaps pertaining to post-primary TB. Gaining a better understanding of the factors that promote or limit TB progression through the combined study of human and animal TB will contribute significantly to development of new diagnostics and means of prevention and treatment of TB.

It has long been recognized that the immune response in tuberculosis can both provide protection and contribute to tissue damage. Many attempts to identify the specific immune responses responsible for these opposing responses have been unsuccessful. The new paradigm provides an explanation of how the same immune responses can be responsible for both. Protection is provided when isolated mycobacteria are engulfed and killed by macrophages in an immune individual. The tissue damaging response occurs upon release of secreted antigens that have been stored within alveolar macrophages in asymptomatic patients. The sudden release of these antigens in sensitized people produces massive reactions (otherwise known as the Koch phenomena) and may represent the early manifestations of post-primary cavity formation.

In studying TB further, we believe that it would be profitable to focus on the commonalities of different models rather than their differences. Our hypothesis to explain the common course of disease in these various species in spite of markedly different pathologies is as follows. In humans mycobacterial antigens are sequestered within alveolar microphages until they are released to produce caseous pneumonia. We believe a key to understanding TB is an understanding of how M. tuberculosis stores and then releases the antigens to produce a massive necrotizing reaction. In mice a similar type of obstructive alveolar pneumonia is observed with accumulation of antigen, but it leads to a progressive fibrosis rather than caseous necrosis. Since certain immunocompetent mice are able to produce caseating granulomas when challenged appropriately, we believe their failure to do so following low-dose infection may be due to a lack of an antigen load sufficient to promote a proinflammatory response (71). Rabbits and guinea pigs, however, do develop progressive caseating granulomas in their lungs rather than the alveolar pneumonia (2). These slowly progress until the animals die, even though the numbers of viable M. tuberculosis organisms do not markedly increase. We hypothesize that this may be explained by the inability of the macrophages in these animals to limit the production of mycobacterial antigens and to keep that production retained within macrophages. Consequently, there is a continual release of the antigens, which drives the progressive granulomas even though the number of organisms is not changing. The key question again, as stated by North, is “to explain why immunity to infection does not enable mice, guinea pigs, rabbits or susceptible humans to resolve lung infection and thereby stop development of disease” (28). How do small numbers of M. tuberculosis organisms persist in all of these models in spite of different pathologies and produce the conditions for massive clinical disease?

There are many impediments to progress in understanding the pathogenesis of post-primary tuberculosis. Many investigators have studied granulomas following the first infection, trying to extrapolate that through the entire process. However, it has been clear for over a century that the most important parameter determining the course of infection is the existence of previous infection. Post-primary tuberculosis is thus named because it occurs after the primary disease. It is characterized by much greater necrosis development of cavities and transmission. The belief that granulomas are the characteristic lesion of both primary and post-primary tuberculosis has encouraged investigators to concentrate only on early lesions that are inappropriate for post-primary TB.

A second element that has impeded progress is the insistence on infecting animals with a low-dose aerosol of M. tuberculosis to simulate the natural infection. While this provides some standardization and facilitates comparison among studies, there has been no explanation of why the route of infection should be the major determining factor in a disease that occurs 10 to 30 years after infection. Even reinfection tuberculosis develops 1 to 2 years after infection. A better approach is to attempt to reproduce in animals the conditions that occur in humans at particular times of infection. For example, we read that caseous granulomas occur when large numbers of organisms are found in localized areas of immunized individuals. Simulating this in mice by injecting large numbers of organisms into a sensitized animal produced classic caseating granulomas (71). This demonstrated that the inability of mice to produce caseous granulomas following low-dose aerosol infection is due not to their inability to develop appropriate immune responses, but rather to accumulating sufficient proinflammatory antigens at a particular site. In another example, injection of viable M. tuberculosis into sensitized animals stimulates accumulation of foamy macrophages in alveoli within 24 hours (1). Study of the pathology of human TB suggests numerous other areas where animal models can be developed to simulate and study individual phases of the human disease.

We have recently reported that the pathology of post-primary TB in the human lung is quite different from that in animal models and that tissues demonstrating such lesions are exceedingly scarce. While the difficulties in obtaining tissue are immense, the benefits and potential are substantial. Most importantly, TB is the human disease and autopsies are the only place where it can be seen in its developing untreated form. Surgical resections, with very few exceptions, are for chronic TB that has been treated and no longer shows the characteristic lesions of developing post-primary TB. Second, modern technology has produced means of identifying and quantitatively measuring parameters on tissue sections that were not possible a few years ago. We can measure DNA sequences, RNA expression, proteins, cytokines, and cells to better understand the changing microenvironment at various stages of M. tuberculosis infection. In the case of TB, more focused studies of human tissues is essential for directing, focusing, and correlating studies on animal models. Consequently, it will be important to develop tissue banks of such tissues. It is also necessary to develop new animal models that can be used in conjunction with a more accurate version of human pathology to finally answer major questions about this disease.