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Animal models of cavitation in pulmonary tuberculosis
ARTICLE IN PRESS Tuberculosis (2006) 86, 337–348
Tuberculosis http://intl.elsevierhealth.com/journals/tube
REVIEW
Animal models of cavitation in pulmonary tuberculosis Kris L. Helkea,b, Joseph L. Mankowskia,b, Yukari C. Manabec,d,e, a
Department of Comparative Medicine, Johns Hopkins University School of Medicine, 733 N. Broadway, Room 811, Baltimore, MD 21205, USA b Department of Pathology, Johns Hopkins University School of Medicine c Department of Medicine, Johns Hopkins University School of Medicine, 1503 E. Jefferson Street, Room 108, Baltimore, MD 21231-1004, USA d Department of International Health, Johns Hopkins Bloomberg School of Public Health, USA e Department of Molecular Microbiology & Immunology, Johns Hopkins Bloomberg School of Public Health, USA Received 11 May 2005
KEYWORDS Review; Cavitation; Tuberculosis; Animal models; Rabbit; Guinea pig; Monkey
Summary Transmission of tuberculosis occurs with the highest frequency from patients with extensive, cavitary, pulmonary disease and positive sputum smear microscopy. In animal models of tuberculosis, the development of caseous necrosis is an important prerequisite for the formation of cavities although the immunological triggers for liquefaction are unknown. We review the relative merits and the information gleaned from the available animal models of pulmonary cavitation. Understanding the host–pathogen interaction important to the formation of cavities may lead to new strategies to prevent cavitation and thereby, block transmission. & 2005 Elsevier Ltd. All rights reserved.
Contents Cavitary tuberculosis in humans . . Tuberculosis in mice. . . . . . . . . . Tuberculosis in guinea pigs . . . . . Cavitation in tuberculous primates Cavitation in tuberculous rabbits . Other animal species . . . . . . . . .
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338 339 340 340 341 344
Corresponding author. Department of Medicine, Johns Hopkins University School of Medicine, 1503 E. Jefferson Street, Room 108,
Baltimore, MD 21231-1004, USA. Tel.: +1 410 614 6600; fax: +1 410 614 8173. E-mail address: [email protected] (Y.C. Manabe). 1472-9792/$ - see front matter & 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tube.2005.09.001
ARTICLE IN PRESS 338 Pathogenesis of caseation and cavitation: a comparison Implications for human disease . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
K.L. Helke et al. of the animal models . ............... ............... ...............
Tuberculosis remains a globally important disease in human populations despite effective antimicrobial therapy and extensive programmatic control efforts. Key aspects in the pathogenesis of human tuberculosis are the ability of the bacilli to remain dormant for decades and cause pulmonary disease. Tuberculosis is characterized histopathologically by granulomas with caseous necrosis.1 In some hosts, caseous necrotic sites enlarge and liquefy.2 Once communication with the bronchial tree is established, a cavity is formed and more oxygen enters. Thereafter, bacilli can multiply to high numbers.1,3 Among the different manifestations of pulmonary tuberculosis, the cavity is the most important in the transmission of this disease. Quantification of the liquefied material within a single cavity has shown that 107–109 bacilli can routinely be cultured from human pulmonary cavities.4 Therefore, patients with cavitary tuberculosis can readily transmit the disease to others. In addition, the high titer of bacilli increases the likelihood of developing drug resistant bacterial populations as a result of spontaneous mutations.5,6 Transmission of Mycobacterium tuberculosis from a patient with active pulmonary tuberculosis to case contacts correlates with the lung burden of bacilli as measured by sputum-smear positivity.7–10 Numerous bacilli on the initial smear,11–13 and multiple cavity disease favor persistent smear positivity.14,15 Furthermore, patients with cavitary disease and persistently positive sputum cultures are much more likely to relapse following standard anti-tuberculous chemotherapy.11,13,16,17 A better understanding of the pathogenesis of cavity formation and of the early sterilization of cavities could lead to methods to prevent the transmission of tuberculosis. Despite the clinical implications of cavitary lesions and their irrefutable role in disease transmission, investigators have only recently refocused their attention on this specific aspect of tuberculosis. One notable obstacle to the systematic study of such pulmonary cavities has been the lack of reliable, rapid, and representative animal models for this characteristic human lesion. The relative merits and historical context of these models in comparison to human cavities are discussed in this review.
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344 345 345 346
Cavitary tuberculosis in humans Patients co-infected with human immunodeficiency virus (HIV) are more likely to have impaired DTH responses due to a deficiency of CD4 T cells, and less likely to have cavitary disease.18 These data indirectly point to an important role for the CD4+ T cell in the pathogenesis of cavitation. A study comparing the bronchoalveolar lavage (BAL) fluid of 7 patients with active cavitary disease to 7 others without cavities showed more CD4+ T cells in the BAL of non-cavitary patients.19 However, this study did not examine specifically activated T cells. Other studies have shown increases in macrophages and polymorphonuclear leukocytes in patients with smear positive and/or cavitary disease.20,21 Alphadefensins that are secreted from the neutrophils were assayed in BAL fluid from 6 patients with cavitary TB and 6 patients with non-cavitary pulmonary disease.22 There was a significantly higher concentration of a-defensins in cavitary patient BAL fluid and may correlate with the increased number of neutrophils noted in advanced disease. There are several papers implicating the Th2 cytokine, interleukin-4 (IL-4) in the pathogenesis of human cavitation. In Mazzarella et al.’s study of BAL fluid from patients with cavitary disease, both CD4+ and CD8+ T lymphocytes were polarized to secrete more Th2 cytokine, IL-4, and less likely to secrete interferon-gamma (IFN-g).19,23 Another study of BAL fluid in 43 patients with active disease showed that there were 5 patients that had detectable IL-4 secretion, 3 of whom were in the clinically most severe group (often cavitary).24 In another study examining peripheral blood, there was a significantly higher percentage of CD8+ T cells that stained for intracellular IL-4 in cavitary patients, although no difference was noted in IFN-g. Finally, in a study of 29 patients (15 with large cavity 44 cm, 14 with small o4 cm or no cavity), BAL fluid showed significantly higher amounts of tumor necrosis factor-alpha (TNF-a), IL-1b, and IL-1 receptor agonist in patients with large cavities.25 TNF-a is a well-studied cytokine in tuberculosis and has been implicated both in granuloma formation and maintenance.26,27 In murine studies, mice with a deficiency in TNF-a or its receptors are susceptible to infection by mycobacteria.28 Zganiacz and colleagues showed
ARTICLE IN PRESS Animal models of cavitation in pulmonary tuberculosis that TNF-a is a critical negative regulator of type 1 immune activation and in its absence, uncontrolled CD4+ and CD8+ T cell activation occurred with elevated levels of IFN-g .29 The presence of TNF-a in the BAL of patients with cavitary disease could correlate with lower levels of IFN-g and more type 2 cellular activation locally in the lung segments with cavitation.
Tuberculosis in mice Mice have little measurable delayed-type hypersensitivity (DTH) response and do not form true caseous necrosis in response to aerosol or parenteral infection with M. tuberculosis (Tables 1 and 2). Table 1 Virulence of pathogenic mycobacteria in different animal species.y Species
M. bovisz M. tuberculosisz M. aviumz
Man Monkeys Guinea-pigs Rabbits Cattle Pigs Mice
2 5 5 5 4 4 4
2 5 5 1 1 2 4
1 1 2 4 1 2 3y
Virulence is measured by the likelihood of disease progression and the rate of progression to fatal disease. y Adapted from a table in John Francis’ ‘‘Tuberculosis in Animals in Man’’.44 z Scale: 0 ¼ absent, 5 ¼ high. y Strain dependent virulence—see text under ‘‘Tuberculosis in mice’’.
Table 2
339 Mice develop an acquired cell-mediated response that is primarily mediated by T cells. After low-dose aerosol, they develop cellular aggregates in the lung with bacillary proliferation that reaches a plateau around 106 organisms in the lung.30,31 After 4 weeks, an equilibrium is maintained that suppresses further increases in bacillary titer which allows some strains of mice to survive for more than 1 year.32 Mice eventually succumb to progressive, coalescing pulmonary infiltrates. They have been widely used because of their economy of cost, availability of inbred species, reproducibility of infection, and availability of immunologic reagents. Mice are particularly useful for studying the acquired cell-mediated immune response in tuberculosis. Important roles for CD4+ T cells, IFN-g, interleukin-12, and TNF-a in granuloma formation and cell-mediated immune responses have been elucidated in mice and have been well reviewed by others.33 Since mice cannot form appreciable caseous necrosis after infection with M. tuberculosis, their lung lesions do not progress to cavities. Therefore, investigators have used the development of granulomatous necrosis as a surrogate for cavitation in the mouse model. Tissue necrosis and early cavitation have been produced in immunocompetent mice with a highly pathogenic strain of M. avium by 2 different research groups.34–37 The mice formed ill-defined cellular aggregates followed by chronic fibrosis and ultimately death. The M. avium multiplied intracellularly to very large numbers which were apparently the cause of the necrosis. Genetically engineered mice with deletions in CD4, interleukin12 p40, IFN-g or ab T cell receptors (TCR), all had less necrosis when infected with M. avium than
Characteristics of tuberculosis in different animal species. DTHy,z Bacillary burdeny,z Caseationy Calcificationy Cavitationy Transmissiony,
Species
Strainy,y
Man Monkeys Guinea-pigs Rabbits Rabbits Cattle Pigs Mice
M.tb 5 M.tb 2 M.tb/ bovis 4 M.tb 2 bovis 2 bovis 2 bovis 3 M.tb 0
1 2 3 2 4 4 3 5
5 5 5 0–4 5 4 4 0
5 3 2 1 2 5 4 0
4 4 2 2 5 0 4 0
5 5 1 1 1 5 1 0
Adapted from table in John Francis’ ‘‘Tuberculosis in Animals in Man’’.44 y
Scale: 0 ¼ absent, 5 ¼ high or large. DTH ¼ delayed type hypersensitivity response to Old Tuberculin or purified protein derivative. y M.tb ¼ M. tuberculosis, bovis ¼ M. bovis. z Refers to number of culturable bacilli in the lungs. Transmission refers to ability to spread animal-to-animal: man and monkeys primarily spread the disease via the respiratory route; cattle probably spread it through both oral and respiratory routes. Natural spread is not common in the other species. z
ARTICLE IN PRESS 340 wildtype C57BL/6 mice. TNF-a may also be important in the formation of necrosis. However, both the TNF-knockout mouse and the TNF-receptor-1-knockout mice were unable to form granulomas and rapidly succumbed to infection, so it was not possible to discern a specific role for TNF-a in necrosis. Another model used the TNFRp55/ mouse infected with M. avium to show that both CD4+ and CD8+ T cells as well as the IL-12 cytokine are crucially important to the granuloma necrosis and prevented early death as seen in the controls not subjected to monoclonal antibody depletion.34 As noted by the authors, M. avium as a surrogate for M. tuberculosis complex infection is problematic because the pathologic cause of death is different for the 2 organisms in mouse. Furthermore, the mice in the M. avium model never control the infection and bacillary burden increases until death. This is in direct contradistinction to humans where cavitary disease often occurs when the host is effectively controlling the infection except in the cavitary focus.1 Finally, a role for a Th2 polarized response in the formation of fibrosis has been suggested by an interesting murine experiment. Using adoptive transfer of T cells from transgenic mice with an ab TCR specific for peptide 323–339 from OVA presented in the context of I-Ad polarized toward a Th1 or Th2 phenotype in vitro (using either OVA, IL12, and anti-IL-4 (Th1) or OVA and IL-4 (Th2)) adoptive transfer of Th2 polarized CD4+ T cells resulted in increased inflammation and fibrosis secondary to increased levels of hydroxyproline and collagen III expression.38 Taken together, the data from murine models using granuloma necrosis or fibrosis as a surrogate for cavitation point to a role for CD4+, CD8+ T cells, IL-12, and Th2 polarized responses, respectively. These findings compare favorably with BAL fluid from humans with cavitary tuberculosis.
Tuberculosis in guinea pigs Guinea pigs develop good DTH responses to mycobacterial antigens, and have tuberculin sensitivity similar to that of humans. After parenteral and aerosol infection with M. tuberculosis, guinea pigs form impressive caseous necrosis in their lungs (Tables 1 and 2, Fig. 1B). They are also very susceptible hosts and develop chronic progressive disease after very low-dose aerosol infection.39,40 Virulent M. tuberculosis multiplies logarithmically and is followed by a stationary phase where
K.L. Helke et al. bacillary antigens are continually produced due to cycles of bacillary multiplication and bacillary death.39,41–43 The DTH response to the accumulating bacillary antigens causes progressive lung destruction, leading ultimately to the death of the animal. A mature immune system is required for DTH, because guinea pigs infected just after birth do not mount a DTH response and have numerous bacilli causing some necrosis, but very little typical caseation.44 Tuberculosis in guinea pigs is primarily a hematogenously disseminated disease (to lungs, liver and spleen) that can be considerably reduced by BCG vaccination.39,40,45,46 Histologically, tuberculous guinea pigs show coalescing pulmonary lesions with large areas of caseous necrosis, thickened alveolar walls, and fibrous capsules.47 Chronic pulmonary tuberculosis due to low-dose infection (guinea pigs in the same room as other tuberculous animals or aerosol infection with 2–4 cfu) can variably lead to cavitation over several months48–51 (Figs. 2B and 3B).
Cavitation in tuberculous primates Non-human primates have been used as an animal model of tuberculosis primarily for vaccine52–56 and therapeutic drug testing.57–60 Some information on the susceptibility of non-human primates has also been gleaned from outbreaks within colonies and from health reports of monkeys during shipment.61–63 Due to their size, increasingly difficult availability and cost, they lost favor and only recently have become reinvigorated as an animal model mimicking various types of human disease.60,64 After infection with high doses of M. tuberculosis (3000 cfu), both rhesus and cynomolgus macaques develop fulminant, rapidly progressive disease. Infections with lower doses (25 cfu) in cynomolgus macaques were first investigated by Walsh et al.65 and more recently by Capuano et al.64 These macaques developed all the specific stages of human tuberculosis from unapparent infection to chronic progressive disease. DTH responses in the skin of non-human primates are weak.66 After natural infection, monkeys have a low rate of skin test positivity.56,62 Using a more potent Old Tuberculin, most animals will develop a palpebral DTH response, but some animals will remain skin test negative. Although caseation necrosis is routinely seen with primate infection with M. tuberculosis (Fig. 1C), only animals receiving low-dose aerosol go on to form cavities.56,64,67 (Figs. 2C and 3C) The histopathology of the granulomas is similar to that of humans with central
ARTICLE IN PRESS Animal models of cavitation in pulmonary tuberculosis
341
Figure 1 Pulmonary granulomas with central caseous necrosis denoted in (A) human lung infected for an unknown duration with clinical strain of M. tuberculosis, (B) guinea pig lung 29 weeks after aerosol infection with M. tuberculosis H37Rv, (C) cynomolgus macaque intratracheally infected with M. tuberculosis Erdman, (D) rabbit 17 weeks after aerosol infection with M. tuberculosis Erdman.
caseation necrosis surrounded by epithelioid macrophages and Langhans’ giant cells.60 In monkeys with cavitary disease, lesions of widely variable size were observed. Large cavitary lesions with necrosis were also observed to infiltrate the airways with surrounding tuberculous pneumonia.64 Because NHP are not inbred, the pathologic outcome is highly variable and not predictable. Therefore, although cavities have been recognized in NHP, they have not been systematically studied in this model.
Cavitation in tuberculous rabbits Rabbits mount a moderate DTH response and form caseous necrosis after either aerosol or intravenous infection with M. bovis3,68 and with more virulent strains of M. tuberculosis69,70 (Fig. 1D). Sporadic reports of cavitation in rabbits were published as early as the 1900s.51 With virulent M. tuberculosis, rabbits are relatively resistant to infection and require 500–3000 bacilli inhaled to form one grossly visible tubercle at 5 weeks after infection.69 By 6
ARTICLE IN PRESS 342
K.L. Helke et al.
Figure 2 Cavitary lesions in the lung of (A) human infected with clinical strain of unknown duration, (B) guinea pig lung 29 weeks after aerosol infection with M. tuberculosis H37Rv, (C) cynomolgus macaque 38 weeks after intratracheal infection with M. tuberculosis Erdman, (D) rabbit 17 weeks after aerosol infection with M. tuberculosis Erdman at low power showing granulomatous lesion with outer layer of lymphocytes, epithelioid macrophages and central necrotic areas undergoing liquefaction.
months, most animals will heal disease with few culturable bacilli.71,72 Occasionally, cavities can form many months after infection with some M. tuberculosis strains (Figs. 2D, 3D).69,70 In contrast, rabbits are much more susceptible to infection with M. bovis and form 1 tubercle for every 1–5 bacilli of M. bovis inhaled.3,73 In 1942, Takeda and Shinpo74 reported on the induction of caseous pneumonia and cavity formation in rabbits infected transbronchially with virulent M. bovis. The most recent reports of the natural pathogenesis of cavities in M. bovis infected rabbits have been published by Converse and Dannenberg.3,73,75 They used M. bovis
in an aerosol model and were able to produce cavities in 9 of 12 rabbits given a moderately low infecting dose (102–103 cfu) and in all animals (6 of 6) given very high dose (103–104 cfu). In all rabbits, the number of caseous lung lesions was impressively high.3,73 A study of cavity formation in rabbits was published by Wells and Lurie76 who immunized 4 rabbits with heat-killed bacteria and then challenged with low-dose M. bovis by aerosol. Over a long course of disease (6–10 months), all 4 animals were found to have pulmonary cavities. Another report by Ratcliffe and Wells77 emphasized this
ARTICLE IN PRESS Animal models of cavitation in pulmonary tuberculosis
343
Figure 3 Cavitary lesions in the lung of (A) human, (B) guinea pig, (C) cynomolgus macaque, (D) rabbit as in Fig. 2 at high power showing many layers of fibrosis (arrows) and epithelioid macrophages surrounding areas of caseation and liquefaction as seen in the lower left corner.
same principle of the importance of pre-sensitization. Using very low-dose aerosol infection with M. bovis, followed by a high-dose reinfection 5 weeks later (a dose that would be lethal in the naı¨ve rabbit), they reported these rabbits showed considerable immunity to the reinfection, and also developed pulmonary cavities. In an attempt to shorten the course of cavity formation and to produce cavities more reliably, Yamamura published an important series of papers51,78 that greatly increased our understanding of cavity formation in rabbits. The materials that elicited the cavities were injected directly into the
lungs through the chest wall. Several important points emerged. First, subcutaneous sensitization with heat-killed bacilli in the skin increased the reliability and rapidity of cavity formation after intrathoracic injection of live or heat-killed bacilli. Without sensitization with heat-killed bacilli, the probability of cavity formation was much lower. Second, M. bovis was better than M. tuberculosis and doses less than 0.5 mg (about 2.5 107 cfu) were ineffective. Third, paraffin oil mixed with anhydrous lanolin improved the reproducibility of cavity formation in both sensitized and non-sensitized rabbits presumably due to local
ARTICLE IN PRESS 344 concentration effects and to a potentiation of the host immune response. The administration of immunosuppressants abolished the ability of intrathoracically injected M. bovis to produce cavities in rabbits that were pre-sensitized subcutaneously with heat-killed M. bovis.79 Yamamura also performed experiments on the particular components of tubercle bacilli that encourage the formation of cavities.78 Because both heat-killed as well as live tubercle bacilli could stimulate the formation of cavities, he postulated that particular soluble proteins were important. In an elegant series of experiments, he showed that a lipid–protein mixture evidently both sensitized and elicited cavities. Mycobacterial proteins alone produced granulomas, but not cavities.80 Interestingly, cavities could be formed by mycobacterial proteins alone (if animals are presensitized with heat-killed M. bovis) or even with egg albumin. However these cavitary lesions lacked the usual amount of necrotic tissue, were relatively thin-walled, and did not liquefy as do true tuberculous cavities.78 Later work suggested that mycobacterial lipids were acting as adjuvants and that the mycobacterial protein combined with synthetic adjuvants were able to elicit cavity formation.81 Desensitization of rabbits to mycobacterial lipoproteins successfully abolished the ability of the animal to form cavities.82
Other animal species Swine are natural hosts for mycobacterial infections83,84 and have been used as an experimental model for tuberculosis.85 After intratracheal infection with M. bovis, swine can form pulmonary tubercles with caseous necrosis, followed by liquefaction and cavity formation. Swine readily develop good tuberculin responses.86,87 Lack of immunologic reagents has precluded further investigation of the immunopathogenesis. Using a low-dose M. bovis respiratory challenge model, cattle form large caseous lesions in lungs and draining hilar lymph nodes.88,89 Cattle do mount a DTH response that can be measured by skin testing, although it is weaker than that produced by guinea pigs.90 Interestingly, cattle, rarely, if ever, form cavitary lesions.44,88 Swine and cattle are cumbersome and expensive to use. Other animals that develop cavitary lesions with M. bovis include elephants, goats, sheep, dogs, and horses.44
K.L. Helke et al.
Pathogenesis of caseation and cavitation: a comparison of the animal models Guinea pigs, monkeys, and rabbits all form granuloma with caseous necrosis with strikingly similar architecture to that of humans (Fig. 1). Caseous necrosis is a pathologic prerequisite for cavity formation. Monkeys and guinea pigs can form cavities, but do not do so invariably.64,91 Rabbits infected by aerosol with low-dose M. bovis, uniformly develop cavities. Fig. 2 shows low-power views of small cavitary lesions in humans (1A), guinea pigs (1B), cynomolgus monkeys (1C), and rabbits (1D). In general, these animal species’ cavitary lesions are similar to those seen in humans, and are composed of liquefied caseum, lymphocytes, epithelioid macrophages, and layers of fibrosis all of which vary with the age of the lesion (Fig. 2, low power, Fig. 3, high power). Although caseous necrosis and DTH are necessary, their presence does not assure that cavities will form. Cattle had measurable DTH responses and developed impressive caseous pulmonary lesions after intratracheal infection with M. bovis, but did not form cavities.88 Guinea pigs vaccinated with BCG and then challenged with low-dose aerosol M. tuberculosis never form liquefied lesions (personal communication Dr. David McMurray). Similarly, guinea pigs infected with a low virulence M. tuberculosis isolate and then challenged with a virulent strain showed protection as well and did not develop cavitation.92 In a model similar to Yamamura’s pre-sensitization and challenge in rabbits, guinea pigs vaccinated with Freund’s incomplete adjuvant with defatted residue of tubercle bacilli protected guinea pigs against subsequent aerosol challenge and did not result in cavities as in Yamamura’s rabbits.40 The specific components and timing of the immune response responsible for caseous necrosis, progressive tissue destruction, and, ultimately, liquefaction and cavity formation in rabbits, humans and NHP in contrast to guinea pigs and mice are not well delineated.93 The cytokines that mediate the evolution to a cavity and the T cell polarization may overlap with those involved in the protective granulomatous response, but evidence from human cavities suggest that Th2 cytokines like IL-4 may be important in addition to TNF-a. Although antigens from M. tuberculosis may play a role in the original formation and maintenance of the granuloma, it is less clear that the pathogen plays a central role in the formation of cavities. Based on the Yamamura model, pre-sensitization with heat-killed bacilli is the key to ensuring the formation of cavities in rabbits. In addition, the heat-killed
ARTICLE IN PRESS Animal models of cavitation in pulmonary tuberculosis bacilli must be suspended in the paraffin lanolin solution that adjuvants the response. Data on heatkilled bacilli from the vaccine literature would suggest that heat-killed bacilli incite only a partially protective immune response, although strong tuberculin skin test responses result.94 It is also known that Freund’s adjuvant potentiates both T and B cell responses. In the bronchoscopic model of rabbit cavity formation (Yoder et al. unpublished),93 rabbits pre-sensitized with heat killed M. bovis suspended in 7H9 medium, then challenged with live M. bovis formed only consolidated bronchopneumonia in the infected lower lung lobe. In contrast, rabbits pre-sensitized with heat-killed M. bovis suspended in paraffin-lanolin, then challenged with the same dose and strain of bacilli uniformly formed cavities (10 of 10 rabbits) in the infected lobe within 3–6 weeks. A comparison of the 2 groups showed that adjuvanted pre-sensitization led to significantly more robust tuberculin skin test responses in the group that ultimately had cavities. Therefore, some immune elements of the DTH response also participate in the formation of cavities in rabbits. The CD4+ T cell plays a critical role in the formation of cavities. AIDS patients with low CD4+ T cells are impaired in their ability to form cavities and are more likely to develop extensive bronchopneumonia or disseminated disease. In rabbits pre-treated with corticosteroids, infected with M. tuberculosis, and then allowed to recover off steroids over 6–9 weeks, all rabbits mounted strong DTH skin test responses and formed extensive, cavitary disease.71 Our data on corticosteroid administration have shown that CD4+ T lymphocytes are preferentially affected by steroids.95 Five weeks after discontinuation of a 5 week course of dexamethasone (given from week 10–15 after infection had been established), CD4+ T cell rebound was accompanied by rapid worsening of tuberculosis (Manabe et al. in preparation). Finally, the tuberculin skin test although insensitive for disseminated, miliary tuberculosis, is positive in 68–83% of human pulmonary disease.96 HIVinfected patients with low CD4 T cell counts are also often tuberculin negative and anergic, and rarely have cavitary disease. We are not aware of any studies that have specifically correlated the tuberculin skin test with cavitary tuberculosis. The data from the rabbit model suggest that the tuberculin skin test may be a reasonable surrogate for the capacity to cavitate, although the exact immunologic triggers and cascade are yet to be fully examined to find a more sensitive and specific marker of this stage of disease. Interestingly, BCG-vaccinated guinea pigs have a faster, larger tuberculin skin test
345 response than unvaccinated controls in the first 4 weeks after virulent M. tuberculosis challenge, but have a waning response by 6 weeks when the unvaccinated controls have larger necrotic tuberculin skin test responses.97,98 Therefore, although the guinea pig has immune components that allow it to mount a strong DTH skin response and caseous necrosis, it does not potentiate this memory response to cause liquefaction as in rabbits. This may illustrate why Koch’s tuberculin was protective and immunomodulatory in infected guinea pigs, but killed some infected humans.
Implications for human disease The pathogenesis of smear-positive cavitary tuberculosis remains an important area of study because of the high likelihood for these patients to transmit disease to close contacts.7,8 Animal models have identified host immune components that may be important in human cavity formation. In rabbits, host recognition of both cell wall and protein components of the bacilli were needed for cavity formation.80 Corroborative evidence for pre-sensitization exist in humans: alcoholic homeless patients who were tuberculin-positive from a prior infection and who had documented exogenous reinfection developed cavitary disease more readily than those who were not previously tuberculinpositive.99 A cell-mediated response to bacillary components followed by caseous necrosis is necessary for the formation of cavities. However, cavities only form in a percentage of tuberculin-positive humans with caseous tuberculous lesions. The clear difference in outcome between guinea pigs and rabbits with pre-sensitization and challenge with virulent mycobacteria (protection and cavitation, respectively) is intriguing. Immune differences between species that form granuloma that caseate but do not cavitate (cattle, guinea pigs) and those that do (rabbit and NHP) as well as the study of murine granulomatous necrosis and fibrosis may hold clues to the pathogenesis of liquefaction. Understanding the pathogenesis of cavities and the host factors important to their formation may lead to new strategies to block transmission and assess the effectiveness of new chemotherapeutic agents against cavitary tuberculosis.6
Acknowledgments The authors would like to thank Dr. Arthur M. Dannenberg, Jr. for his invaluable advice and
ARTICLE IN PRESS 346 editorial assistance. The authors would also like to thank Dr. David N. McMurray and Dr. JoAnne L. Flynn for graciously providing pulmonary tissue sections of guinea pig and cynomolgus macaques respectively and for invaluable scientific input. This work was supported by funding from the National Institutes of Health, 1R01 HL71554 (YCM) and RR07002 (KLH).
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K.L. Helke et al. 18. Brindle RJ, et al. Quantitative bacillary response to treatment in HIV-associated pulmonary tuberculosis. Am Rev Respir Dis 1993;147:958–61. 19. Mazzarella G, et al. T lymphocyte phenotypic profile in lung segments affected by cavitary and non-cavitary tuberculosis. Clin Exp Immunol 2003;132:283–8. 20. Condos R, Rom WN, Liu YM, Schluger NW. Local immune responses correlate with presentation and outcome in tuberculosis. Am J Respir Crit Care Med 1998;157: 729–35. 21. Ozaki T, et al. Differential cell analysis in bronchoalveolar lavage fluid from pulmonary lesions of patients with tuberculosis. Chest 1992;102:54–9. 22. Ashitani J, et al. Elevated levels of alpha-defensins in plasma and BAL fluid of patients with active pulmonary tuberculosis. Chest 2002;121:519–26. 23. van Crevel R, et al. 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;181:1194–7. 24. Somoskovi A, et al. Different cytokine patterns correlate with the extension of disease in pulmonary tuberculosis. Eur Cytokine Network 1999;10:135–42. 25. Tsao TC, et al. Imbalances between tumor necrosis factoralpha and its soluble receptor forms, and interleukin-1beta and interleukin-1 receptor antagonist in BAL fluid of cavitary pulmonary tuberculosis. Chest 2000;117:103–9. 26. Roach DR, et al. TNF regulates chemokine induction essential for cell recruitment, granuloma formation, and clearance of mycobacterial infection. J Immunol 2002;168:4620–7. 27. Mohan VP, et al. Effects of tumor necrosis factor alpha on host immune response in chronic persistent tuberculosis: possible role for limiting pathology. Infect Immun 2001;69:1847–55. 28. Bean AG, et al. Structural deficiencies in granuloma formation in TNF gene-targeted mice underlie the heightened susceptibility to aerosol Mycobacterium tuberculosis infection, which is not compensated for by lymphotoxin. J Immunol 1999;162:3504–11. 29. Zganiacz A, et al. TNF-alpha is a critical negative regulator of type 1 immune activation during intracellular bacterial infection. J Clin Invest 2004;113:401–13. 30. Orme I, Collins FM. In: Bloom BR, editor. Tuberculosis: pathogenesis, protection, and control. Washington, DC: American Society for Microbiology; 1994. p. 113–34. 31. Flynn JL, Cooper AM, Bishai WR. In: Cole ST, Eisenach KD, McMurray DN, Jacobs Jr. WR, editors. Tuberculosis and the tubercle bacillus. Washington, DC: American Society for Microbiology; 2005. p. 547–60. 32. Dunn PL, North RJ. Virulence ranking of some Mycobacterium tuberculosis and Mycobacterium bovis strains according to their ability to multiply in the lungs, induce lung pathology, and cause mortality in mice. Infect Immun 1995;63:3428–37. 33. Flynn JL, Chan J. Immunology of tuberculosis. Annu Rev Immunol 2001;19:93–129. 34. Ehlers S, Kutsch S, Ehlers EM, Benini J, Pfeffer K. Lethal granuloma disintegration in mycobacteria-infected TNFRp55/ mice is dependent on T cells and IL-12. J Immunol 2000;165:483–92. 35. Ehlers S, et al. Alphabeta T cell receptor-positive cells and interferon-gamma, but not inducible nitric oxide synthase, are critical for granuloma necrosis in a mouse model of mycobacteria-induced pulmonary immunopathology. J Exp Med 2001;194:1847–59.
ARTICLE IN PRESS Animal models of cavitation in pulmonary tuberculosis 36. Benini J, Ehlers EM, Ehlers S. Different types of pulmonary granuloma necrosis in immunocompetent vs. TNFRp55-genedeficient mice aerogenically infected with highly virulent Mycobacterium avium. J Pathol 1999;189:127–37. 37. Florido M, Cooper AM, Appelberg R. Immunological basis of the development of necrotic lesions following Mycobacterium avium infection. Immunology 2002;106:590–601. 38. Wangoo A, et al. Contribution of Th1 and Th2 cells to protection and pathology in experimental models of granulomatous lung disease. J Immunol 2001;166:3432–9. 39. Smith DW, McMurray DN, Wiegeshaus EH, Grover AA, Harding GE. 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;102:937–49. 40. Wiegeshaus EH, McMurray DN, Grover AA, Harding GE, Smith DW. Host-parasite relationships in experimental airborne tuberculosis. 3. Relevance of microbial enumeration to acquired resistance in guinea pigs. Am Rev Respir Dis 1970;102:422–9. 41. Alsaadi AI, Smith DW. The fate of virulent and attenuated Mycobacteria in guinea pigs infected by the respiratory route. Am Rev Respir Dis 1973;107:1041–6. 42. Balasubramanian V, Wiegeshaus EH, Smith DW. Growth characteristics of recent sputum isolates of Mycobacterium tuberculosis in guinea pigs infected by the respiratory route. Infect Immun 1992;60:4762–7. 43. Balasubramanian V, Wiegeshaus EH, Smith DW. Mycobacterial infection in guinea pigs. Immunobiology 1994;191: 395–401. 44. Francis J. Tuberculosis in animals and man. A study in comparative pathology. London: Cassell and Company, Ltd.; 1958. 45. Fok JS, Ho RS, Arora PK, Harding GE, Smith DW. Hostparasite relationships in experimental airborne tuberculosis. V. Lack of hematogenous dissemination of Mycobacterium tuberculosis to the lungs in animals vaccinated with Bacille Calmette–Guerin. J Infect Dis 1976;133:137–44. 46. Ho RS, Fok JS, Harding GE, Smith DW. Host-parasite relationships in experimental airborne tuberculosis. VII. Fate of Mycobacterium tuberculosis in primary lung lesions and in primary lesion-free lung tissue infected as a result of bacillemia. J Infect Dis 1978;138:237–41. 47. Turner OC, Basaraba RJ, Orme IM. Immunopathogenesis of pulmonary granulomas in the guinea pig after infection with Mycobacterium tuberculosis. Infect Immun 2003;71:864–71. 48. Lurie M. Experimental epidemiology of tuberculosis: the effect of eliminating exposure of enteric infection on the incidence and course of tuberculosis acquired by normal guinea pigs confined with tuberculous cage mates. J Exp Med 1930;51:753–68. 49. Lurie M. Experimental epidemiology of tuberculosis: airborne contagion of tuberculosis in an animal room. J Exp Med 1930;51:743–51. 50. Smith DW, Harding GE. Animal model of human disease. Pulmonary tuberculosis. Animal model: Experimental airborne tuberculosis in the guinea pig. Am J Pathol 1977;89: 273–6. 51. Yamamura Y. The Pathogenesis of Tuberculous Cavities. Adv Tuberc Res 1958;9:13–37. 52. Good RC. Biology of the mycobacterioses. Simian tuberculosis: immunologic aspects. Ann N Y Acad Sci 1968;154: 200–13. 53. Barclay WR, et al. Protection of monkeys against airborne tuberculosis by aerosol vaccination with bacillus Calmette– Guerin. Am Rev Respir Dis 1973;107:351–8.
347 54. Chaparas SD, Good RC, Janicki BW. Tuberculin-induced lymphocyte transformation and skin reactivity in monkeys vaccinated or not vaccinated with Bacille Calmette–Guerin, then challenged with virulent Mycobacterium tuberculosis. Am Rev Respir Dis 1975;112:43–7. 55. Janicki BW, Good RC, Minden P, Affronti LF, Hymes WF. Immune responses in rhesus monkeys after bacillus Calmette–Guerin vaccination and aerosol challenge with Mycobacterium tuberculosis. Am Rev Respir Dis 1973;107:359–66. 56. Ribi E, et al. Efficacy of mycobacterial cell walls as a vaccine against airborne tuberculosis in the Rhesus monkey. J Infect Dis 1971;123:527–38. 57. Schmidt LH, Hoffman R, Hughes HB. The toxicity of isoniazid for the rhesus monkey. Am Rev Tuberc 1953;67:798–807. 58. Schmidt LH. Studies on the antituberculous activity of ethambutol in monkeys. Ann N Y Acad Sci 1966;135:747–58. 59. Wolf RH, Gibson SV, Watson EA, Baskin GB. Multidrug chemotherapy of tuberculosis in rhesus monkeys. Lab Anim Sci 1988;38:25–33. 60. Langermans JA, et al. Divergent effect of bacillus Calmette–Guerin (BCG) vaccination on Mycobacterium tuberculosis infection in highly related macaque species: implications for primate models in tuberculosis vaccine research. Proc Natl Acad Sci USA 2001;98:11497–502. 61. Corcoran KD, Jaax GP. An attempt to predict anergy in tuberculosis suspect cynomolgus monkeys. Lab Anim Sci 1991;41:57–62. 62. Schroeder CR. Acquired tuberculosis in the primate in laboratories and zoological collections. Am J Public Health 1938;28:469–75. 63. Zumpe D, Silberman MS, Michael RP. Unusual outbreak of tuberculosis due to Mycobacterium bovis in a closed colony of rhesus monkeys (Macaca mulatta). Lab Anim Sci 1980;30:237–40. 64. Capuano III SV, et al. Experimental Mycobacterium tuberculosis infection of cynomolgus macaques closely resembles the various manifestations of human M. tuberculosis infection. Infect Immun 2003;71:5831–44. 65. Walsh GP, et al. The Philippine cynomolgus monkey (Macaca fasicularis) provides a new nonhuman primate model of tuberculosis that resembles human disease. Nat Med 1996;2:430–6. 66. Schmidt LH. Some observations on the utility of simian tuberculosis in defining the therapeutic potentialities of isoniazid. Annu. Rev. Tuberc. Pulm. Dis. 1955;74: 138–53. 67. Francis J. Natural and experimental tuberculosis in monkeys. J. Comp. Pathol. 1956;66:123–35. 68. Lurie MB. The fate of human and bovine tubercle bacilli in various organs of the rabbit. J Exp Med 1928;48:155–82. 69. Manabe YC, et al. Different strains of Mycobacterium tuberculosis cause various spectrums of disease in the rabbit model of tuberculosis. Infect Immun 2003;71: 6004–11. 70. Heppleston AG. Quantitative air-borne tuberculosis in the rabbit. J Exp Med 1949;89:597–610. 71. Lurie MB. Resistance to tuberculosis. Cambridge: Harvard University Press; 1964. 72. Lurie MB, Abramson S, Heppleston AG. 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;95:119–34. 73. Converse PJ, et al. Cavitary tuberculosis produced in rabbits by aerosolized virulent tubercle bacilli. Infect Immun 1996;64:4776–87.
ARTICLE IN PRESS 348 74. Takeda K, Shinpo K. On the meaning of the allergic tissue reaction as regards the development of pulmonary tuberculosis. Kekkaku 1942;20:208–21, 275–90, 472–89. 75. Dannenberg Jr. AM, Collins FM. Progressive pulmonary tuberculosis is not due to increasing numbers of viable bacilli in rabbits, mice and guinea pigs, but is due to a continuous host response to mycobacterial products. Tuberculosis 2001;81:229–42. 76. Wells W, Lurie MB. Experimental airborne disease: quantitative natureal respiratory contagion of tuberculosis. Am J Hyg 1941;34:21. 77. Ratcliffe HL, Wells WF. Tuberculosis of rabbits induced by droplet nuclei infection. J Exp Med 1948;87:575–84. 78. Yamamura Y, et al. Studies on the experimental tuberculous cavity. Med. J. Osaka Univ. 1954;5:187–97. 79. Yamamura Y, Ogawa Y, Yamagata H. Prevention of tuberculous cavity formation by immunosuppressive drugs. Am Rev Respir Dis 1968;98:720–3. 80. Maeda H, Yamamura Y, Ogawa Y, Maeda J. Mycobacterial antigens relating to experimental pulmonary cavity formation. Am Rev Respir Dis 1977;115:617–23. 81. Yamamura Y, Maeda H, Ogawa Y, Hashimoto T. Experimental pulmonary cavity formation by mycobacterial components and synthetic adjuvants. Microbiol Immunol 1986;30: 1175–87. 82. Yamamura Y, Ogawa Y, Maeda H, Yamamura Y. Prevention of tuberculous cavity formation by desensitization with tuberculin-active peptide. Am Rev Respir Dis 1974;109:594–601. 83. Alfredsen S, Saxegaard F. An outbreak of tuberculosis in pigs and cattle caused by Mycobacterium africanum. Vet Rec 1992;131:51–3. 84. Corner LA, Barrett RH, Lepper AW, Lewis V, Pearson CW. A survey of mycobacteriosis of feral pigs in the Northern Territory. Austral Vet J 1981;57:537–42. 85. Bolin CA, et al. Infection of swine with Mycobacterium bovis as a model of human tuberculosis. J Infect Dis 1997;176: 1559–66. 86. Muscoplat CC, Thoen CO, Chen AW, Rakich PM, Johnson DW. Development of specific lymphocyte immunostimulation and tuberculin skin reactivity in swine infected with Mycobacterium bovis and Mycobacterium avium. Am J Vet Res 1975;36:1167–71. 87. Ray JA, Mallmann VH, Mallmann WL, Morrill CC. Pathologic and bacteriologic features and hypersensitivity of pigs given
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Tuberculosis http://intl.elsevierhealth.com/journals/tube
REVIEW
Animal models of cavitation in pulmonary tuberculosis Kris L. Helkea,b, Joseph L. Mankowskia,b, Yukari C. Manabec,d,e, a
Department of Comparative Medicine, Johns Hopkins University School of Medicine, 733 N. Broadway, Room 811, Baltimore, MD 21205, USA b Department of Pathology, Johns Hopkins University School of Medicine c Department of Medicine, Johns Hopkins University School of Medicine, 1503 E. Jefferson Street, Room 108, Baltimore, MD 21231-1004, USA d Department of International Health, Johns Hopkins Bloomberg School of Public Health, USA e Department of Molecular Microbiology & Immunology, Johns Hopkins Bloomberg School of Public Health, USA Received 11 May 2005
KEYWORDS Review; Cavitation; Tuberculosis; Animal models; Rabbit; Guinea pig; Monkey
Summary Transmission of tuberculosis occurs with the highest frequency from patients with extensive, cavitary, pulmonary disease and positive sputum smear microscopy. In animal models of tuberculosis, the development of caseous necrosis is an important prerequisite for the formation of cavities although the immunological triggers for liquefaction are unknown. We review the relative merits and the information gleaned from the available animal models of pulmonary cavitation. Understanding the host–pathogen interaction important to the formation of cavities may lead to new strategies to prevent cavitation and thereby, block transmission. & 2005 Elsevier Ltd. All rights reserved.
Contents Cavitary tuberculosis in humans . . Tuberculosis in mice. . . . . . . . . . Tuberculosis in guinea pigs . . . . . Cavitation in tuberculous primates Cavitation in tuberculous rabbits . Other animal species . . . . . . . . .
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Corresponding author. Department of Medicine, Johns Hopkins University School of Medicine, 1503 E. Jefferson Street, Room 108,
Baltimore, MD 21231-1004, USA. Tel.: +1 410 614 6600; fax: +1 410 614 8173. E-mail address: [email protected] (Y.C. Manabe). 1472-9792/$ - see front matter & 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tube.2005.09.001
ARTICLE IN PRESS 338 Pathogenesis of caseation and cavitation: a comparison Implications for human disease . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
K.L. Helke et al. of the animal models . ............... ............... ...............
Tuberculosis remains a globally important disease in human populations despite effective antimicrobial therapy and extensive programmatic control efforts. Key aspects in the pathogenesis of human tuberculosis are the ability of the bacilli to remain dormant for decades and cause pulmonary disease. Tuberculosis is characterized histopathologically by granulomas with caseous necrosis.1 In some hosts, caseous necrotic sites enlarge and liquefy.2 Once communication with the bronchial tree is established, a cavity is formed and more oxygen enters. Thereafter, bacilli can multiply to high numbers.1,3 Among the different manifestations of pulmonary tuberculosis, the cavity is the most important in the transmission of this disease. Quantification of the liquefied material within a single cavity has shown that 107–109 bacilli can routinely be cultured from human pulmonary cavities.4 Therefore, patients with cavitary tuberculosis can readily transmit the disease to others. In addition, the high titer of bacilli increases the likelihood of developing drug resistant bacterial populations as a result of spontaneous mutations.5,6 Transmission of Mycobacterium tuberculosis from a patient with active pulmonary tuberculosis to case contacts correlates with the lung burden of bacilli as measured by sputum-smear positivity.7–10 Numerous bacilli on the initial smear,11–13 and multiple cavity disease favor persistent smear positivity.14,15 Furthermore, patients with cavitary disease and persistently positive sputum cultures are much more likely to relapse following standard anti-tuberculous chemotherapy.11,13,16,17 A better understanding of the pathogenesis of cavity formation and of the early sterilization of cavities could lead to methods to prevent the transmission of tuberculosis. Despite the clinical implications of cavitary lesions and their irrefutable role in disease transmission, investigators have only recently refocused their attention on this specific aspect of tuberculosis. One notable obstacle to the systematic study of such pulmonary cavities has been the lack of reliable, rapid, and representative animal models for this characteristic human lesion. The relative merits and historical context of these models in comparison to human cavities are discussed in this review.
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344 345 345 346
Cavitary tuberculosis in humans Patients co-infected with human immunodeficiency virus (HIV) are more likely to have impaired DTH responses due to a deficiency of CD4 T cells, and less likely to have cavitary disease.18 These data indirectly point to an important role for the CD4+ T cell in the pathogenesis of cavitation. A study comparing the bronchoalveolar lavage (BAL) fluid of 7 patients with active cavitary disease to 7 others without cavities showed more CD4+ T cells in the BAL of non-cavitary patients.19 However, this study did not examine specifically activated T cells. Other studies have shown increases in macrophages and polymorphonuclear leukocytes in patients with smear positive and/or cavitary disease.20,21 Alphadefensins that are secreted from the neutrophils were assayed in BAL fluid from 6 patients with cavitary TB and 6 patients with non-cavitary pulmonary disease.22 There was a significantly higher concentration of a-defensins in cavitary patient BAL fluid and may correlate with the increased number of neutrophils noted in advanced disease. There are several papers implicating the Th2 cytokine, interleukin-4 (IL-4) in the pathogenesis of human cavitation. In Mazzarella et al.’s study of BAL fluid from patients with cavitary disease, both CD4+ and CD8+ T lymphocytes were polarized to secrete more Th2 cytokine, IL-4, and less likely to secrete interferon-gamma (IFN-g).19,23 Another study of BAL fluid in 43 patients with active disease showed that there were 5 patients that had detectable IL-4 secretion, 3 of whom were in the clinically most severe group (often cavitary).24 In another study examining peripheral blood, there was a significantly higher percentage of CD8+ T cells that stained for intracellular IL-4 in cavitary patients, although no difference was noted in IFN-g. Finally, in a study of 29 patients (15 with large cavity 44 cm, 14 with small o4 cm or no cavity), BAL fluid showed significantly higher amounts of tumor necrosis factor-alpha (TNF-a), IL-1b, and IL-1 receptor agonist in patients with large cavities.25 TNF-a is a well-studied cytokine in tuberculosis and has been implicated both in granuloma formation and maintenance.26,27 In murine studies, mice with a deficiency in TNF-a or its receptors are susceptible to infection by mycobacteria.28 Zganiacz and colleagues showed
ARTICLE IN PRESS Animal models of cavitation in pulmonary tuberculosis that TNF-a is a critical negative regulator of type 1 immune activation and in its absence, uncontrolled CD4+ and CD8+ T cell activation occurred with elevated levels of IFN-g .29 The presence of TNF-a in the BAL of patients with cavitary disease could correlate with lower levels of IFN-g and more type 2 cellular activation locally in the lung segments with cavitation.
Tuberculosis in mice Mice have little measurable delayed-type hypersensitivity (DTH) response and do not form true caseous necrosis in response to aerosol or parenteral infection with M. tuberculosis (Tables 1 and 2). Table 1 Virulence of pathogenic mycobacteria in different animal species.y Species
M. bovisz M. tuberculosisz M. aviumz
Man Monkeys Guinea-pigs Rabbits Cattle Pigs Mice
2 5 5 5 4 4 4
2 5 5 1 1 2 4
1 1 2 4 1 2 3y
Virulence is measured by the likelihood of disease progression and the rate of progression to fatal disease. y Adapted from a table in John Francis’ ‘‘Tuberculosis in Animals in Man’’.44 z Scale: 0 ¼ absent, 5 ¼ high. y Strain dependent virulence—see text under ‘‘Tuberculosis in mice’’.
Table 2
339 Mice develop an acquired cell-mediated response that is primarily mediated by T cells. After low-dose aerosol, they develop cellular aggregates in the lung with bacillary proliferation that reaches a plateau around 106 organisms in the lung.30,31 After 4 weeks, an equilibrium is maintained that suppresses further increases in bacillary titer which allows some strains of mice to survive for more than 1 year.32 Mice eventually succumb to progressive, coalescing pulmonary infiltrates. They have been widely used because of their economy of cost, availability of inbred species, reproducibility of infection, and availability of immunologic reagents. Mice are particularly useful for studying the acquired cell-mediated immune response in tuberculosis. Important roles for CD4+ T cells, IFN-g, interleukin-12, and TNF-a in granuloma formation and cell-mediated immune responses have been elucidated in mice and have been well reviewed by others.33 Since mice cannot form appreciable caseous necrosis after infection with M. tuberculosis, their lung lesions do not progress to cavities. Therefore, investigators have used the development of granulomatous necrosis as a surrogate for cavitation in the mouse model. Tissue necrosis and early cavitation have been produced in immunocompetent mice with a highly pathogenic strain of M. avium by 2 different research groups.34–37 The mice formed ill-defined cellular aggregates followed by chronic fibrosis and ultimately death. The M. avium multiplied intracellularly to very large numbers which were apparently the cause of the necrosis. Genetically engineered mice with deletions in CD4, interleukin12 p40, IFN-g or ab T cell receptors (TCR), all had less necrosis when infected with M. avium than
Characteristics of tuberculosis in different animal species. DTHy,z Bacillary burdeny,z Caseationy Calcificationy Cavitationy Transmissiony,
Species
Strainy,y
Man Monkeys Guinea-pigs Rabbits Rabbits Cattle Pigs Mice
M.tb 5 M.tb 2 M.tb/ bovis 4 M.tb 2 bovis 2 bovis 2 bovis 3 M.tb 0
1 2 3 2 4 4 3 5
5 5 5 0–4 5 4 4 0
5 3 2 1 2 5 4 0
4 4 2 2 5 0 4 0
5 5 1 1 1 5 1 0
Adapted from table in John Francis’ ‘‘Tuberculosis in Animals in Man’’.44 y
Scale: 0 ¼ absent, 5 ¼ high or large. DTH ¼ delayed type hypersensitivity response to Old Tuberculin or purified protein derivative. y M.tb ¼ M. tuberculosis, bovis ¼ M. bovis. z Refers to number of culturable bacilli in the lungs. Transmission refers to ability to spread animal-to-animal: man and monkeys primarily spread the disease via the respiratory route; cattle probably spread it through both oral and respiratory routes. Natural spread is not common in the other species. z
ARTICLE IN PRESS 340 wildtype C57BL/6 mice. TNF-a may also be important in the formation of necrosis. However, both the TNF-knockout mouse and the TNF-receptor-1-knockout mice were unable to form granulomas and rapidly succumbed to infection, so it was not possible to discern a specific role for TNF-a in necrosis. Another model used the TNFRp55/ mouse infected with M. avium to show that both CD4+ and CD8+ T cells as well as the IL-12 cytokine are crucially important to the granuloma necrosis and prevented early death as seen in the controls not subjected to monoclonal antibody depletion.34 As noted by the authors, M. avium as a surrogate for M. tuberculosis complex infection is problematic because the pathologic cause of death is different for the 2 organisms in mouse. Furthermore, the mice in the M. avium model never control the infection and bacillary burden increases until death. This is in direct contradistinction to humans where cavitary disease often occurs when the host is effectively controlling the infection except in the cavitary focus.1 Finally, a role for a Th2 polarized response in the formation of fibrosis has been suggested by an interesting murine experiment. Using adoptive transfer of T cells from transgenic mice with an ab TCR specific for peptide 323–339 from OVA presented in the context of I-Ad polarized toward a Th1 or Th2 phenotype in vitro (using either OVA, IL12, and anti-IL-4 (Th1) or OVA and IL-4 (Th2)) adoptive transfer of Th2 polarized CD4+ T cells resulted in increased inflammation and fibrosis secondary to increased levels of hydroxyproline and collagen III expression.38 Taken together, the data from murine models using granuloma necrosis or fibrosis as a surrogate for cavitation point to a role for CD4+, CD8+ T cells, IL-12, and Th2 polarized responses, respectively. These findings compare favorably with BAL fluid from humans with cavitary tuberculosis.
Tuberculosis in guinea pigs Guinea pigs develop good DTH responses to mycobacterial antigens, and have tuberculin sensitivity similar to that of humans. After parenteral and aerosol infection with M. tuberculosis, guinea pigs form impressive caseous necrosis in their lungs (Tables 1 and 2, Fig. 1B). They are also very susceptible hosts and develop chronic progressive disease after very low-dose aerosol infection.39,40 Virulent M. tuberculosis multiplies logarithmically and is followed by a stationary phase where
K.L. Helke et al. bacillary antigens are continually produced due to cycles of bacillary multiplication and bacillary death.39,41–43 The DTH response to the accumulating bacillary antigens causes progressive lung destruction, leading ultimately to the death of the animal. A mature immune system is required for DTH, because guinea pigs infected just after birth do not mount a DTH response and have numerous bacilli causing some necrosis, but very little typical caseation.44 Tuberculosis in guinea pigs is primarily a hematogenously disseminated disease (to lungs, liver and spleen) that can be considerably reduced by BCG vaccination.39,40,45,46 Histologically, tuberculous guinea pigs show coalescing pulmonary lesions with large areas of caseous necrosis, thickened alveolar walls, and fibrous capsules.47 Chronic pulmonary tuberculosis due to low-dose infection (guinea pigs in the same room as other tuberculous animals or aerosol infection with 2–4 cfu) can variably lead to cavitation over several months48–51 (Figs. 2B and 3B).
Cavitation in tuberculous primates Non-human primates have been used as an animal model of tuberculosis primarily for vaccine52–56 and therapeutic drug testing.57–60 Some information on the susceptibility of non-human primates has also been gleaned from outbreaks within colonies and from health reports of monkeys during shipment.61–63 Due to their size, increasingly difficult availability and cost, they lost favor and only recently have become reinvigorated as an animal model mimicking various types of human disease.60,64 After infection with high doses of M. tuberculosis (3000 cfu), both rhesus and cynomolgus macaques develop fulminant, rapidly progressive disease. Infections with lower doses (25 cfu) in cynomolgus macaques were first investigated by Walsh et al.65 and more recently by Capuano et al.64 These macaques developed all the specific stages of human tuberculosis from unapparent infection to chronic progressive disease. DTH responses in the skin of non-human primates are weak.66 After natural infection, monkeys have a low rate of skin test positivity.56,62 Using a more potent Old Tuberculin, most animals will develop a palpebral DTH response, but some animals will remain skin test negative. Although caseation necrosis is routinely seen with primate infection with M. tuberculosis (Fig. 1C), only animals receiving low-dose aerosol go on to form cavities.56,64,67 (Figs. 2C and 3C) The histopathology of the granulomas is similar to that of humans with central
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Figure 1 Pulmonary granulomas with central caseous necrosis denoted in (A) human lung infected for an unknown duration with clinical strain of M. tuberculosis, (B) guinea pig lung 29 weeks after aerosol infection with M. tuberculosis H37Rv, (C) cynomolgus macaque intratracheally infected with M. tuberculosis Erdman, (D) rabbit 17 weeks after aerosol infection with M. tuberculosis Erdman.
caseation necrosis surrounded by epithelioid macrophages and Langhans’ giant cells.60 In monkeys with cavitary disease, lesions of widely variable size were observed. Large cavitary lesions with necrosis were also observed to infiltrate the airways with surrounding tuberculous pneumonia.64 Because NHP are not inbred, the pathologic outcome is highly variable and not predictable. Therefore, although cavities have been recognized in NHP, they have not been systematically studied in this model.
Cavitation in tuberculous rabbits Rabbits mount a moderate DTH response and form caseous necrosis after either aerosol or intravenous infection with M. bovis3,68 and with more virulent strains of M. tuberculosis69,70 (Fig. 1D). Sporadic reports of cavitation in rabbits were published as early as the 1900s.51 With virulent M. tuberculosis, rabbits are relatively resistant to infection and require 500–3000 bacilli inhaled to form one grossly visible tubercle at 5 weeks after infection.69 By 6
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Figure 2 Cavitary lesions in the lung of (A) human infected with clinical strain of unknown duration, (B) guinea pig lung 29 weeks after aerosol infection with M. tuberculosis H37Rv, (C) cynomolgus macaque 38 weeks after intratracheal infection with M. tuberculosis Erdman, (D) rabbit 17 weeks after aerosol infection with M. tuberculosis Erdman at low power showing granulomatous lesion with outer layer of lymphocytes, epithelioid macrophages and central necrotic areas undergoing liquefaction.
months, most animals will heal disease with few culturable bacilli.71,72 Occasionally, cavities can form many months after infection with some M. tuberculosis strains (Figs. 2D, 3D).69,70 In contrast, rabbits are much more susceptible to infection with M. bovis and form 1 tubercle for every 1–5 bacilli of M. bovis inhaled.3,73 In 1942, Takeda and Shinpo74 reported on the induction of caseous pneumonia and cavity formation in rabbits infected transbronchially with virulent M. bovis. The most recent reports of the natural pathogenesis of cavities in M. bovis infected rabbits have been published by Converse and Dannenberg.3,73,75 They used M. bovis
in an aerosol model and were able to produce cavities in 9 of 12 rabbits given a moderately low infecting dose (102–103 cfu) and in all animals (6 of 6) given very high dose (103–104 cfu). In all rabbits, the number of caseous lung lesions was impressively high.3,73 A study of cavity formation in rabbits was published by Wells and Lurie76 who immunized 4 rabbits with heat-killed bacteria and then challenged with low-dose M. bovis by aerosol. Over a long course of disease (6–10 months), all 4 animals were found to have pulmonary cavities. Another report by Ratcliffe and Wells77 emphasized this
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Figure 3 Cavitary lesions in the lung of (A) human, (B) guinea pig, (C) cynomolgus macaque, (D) rabbit as in Fig. 2 at high power showing many layers of fibrosis (arrows) and epithelioid macrophages surrounding areas of caseation and liquefaction as seen in the lower left corner.
same principle of the importance of pre-sensitization. Using very low-dose aerosol infection with M. bovis, followed by a high-dose reinfection 5 weeks later (a dose that would be lethal in the naı¨ve rabbit), they reported these rabbits showed considerable immunity to the reinfection, and also developed pulmonary cavities. In an attempt to shorten the course of cavity formation and to produce cavities more reliably, Yamamura published an important series of papers51,78 that greatly increased our understanding of cavity formation in rabbits. The materials that elicited the cavities were injected directly into the
lungs through the chest wall. Several important points emerged. First, subcutaneous sensitization with heat-killed bacilli in the skin increased the reliability and rapidity of cavity formation after intrathoracic injection of live or heat-killed bacilli. Without sensitization with heat-killed bacilli, the probability of cavity formation was much lower. Second, M. bovis was better than M. tuberculosis and doses less than 0.5 mg (about 2.5 107 cfu) were ineffective. Third, paraffin oil mixed with anhydrous lanolin improved the reproducibility of cavity formation in both sensitized and non-sensitized rabbits presumably due to local
ARTICLE IN PRESS 344 concentration effects and to a potentiation of the host immune response. The administration of immunosuppressants abolished the ability of intrathoracically injected M. bovis to produce cavities in rabbits that were pre-sensitized subcutaneously with heat-killed M. bovis.79 Yamamura also performed experiments on the particular components of tubercle bacilli that encourage the formation of cavities.78 Because both heat-killed as well as live tubercle bacilli could stimulate the formation of cavities, he postulated that particular soluble proteins were important. In an elegant series of experiments, he showed that a lipid–protein mixture evidently both sensitized and elicited cavities. Mycobacterial proteins alone produced granulomas, but not cavities.80 Interestingly, cavities could be formed by mycobacterial proteins alone (if animals are presensitized with heat-killed M. bovis) or even with egg albumin. However these cavitary lesions lacked the usual amount of necrotic tissue, were relatively thin-walled, and did not liquefy as do true tuberculous cavities.78 Later work suggested that mycobacterial lipids were acting as adjuvants and that the mycobacterial protein combined with synthetic adjuvants were able to elicit cavity formation.81 Desensitization of rabbits to mycobacterial lipoproteins successfully abolished the ability of the animal to form cavities.82
Other animal species Swine are natural hosts for mycobacterial infections83,84 and have been used as an experimental model for tuberculosis.85 After intratracheal infection with M. bovis, swine can form pulmonary tubercles with caseous necrosis, followed by liquefaction and cavity formation. Swine readily develop good tuberculin responses.86,87 Lack of immunologic reagents has precluded further investigation of the immunopathogenesis. Using a low-dose M. bovis respiratory challenge model, cattle form large caseous lesions in lungs and draining hilar lymph nodes.88,89 Cattle do mount a DTH response that can be measured by skin testing, although it is weaker than that produced by guinea pigs.90 Interestingly, cattle, rarely, if ever, form cavitary lesions.44,88 Swine and cattle are cumbersome and expensive to use. Other animals that develop cavitary lesions with M. bovis include elephants, goats, sheep, dogs, and horses.44
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Pathogenesis of caseation and cavitation: a comparison of the animal models Guinea pigs, monkeys, and rabbits all form granuloma with caseous necrosis with strikingly similar architecture to that of humans (Fig. 1). Caseous necrosis is a pathologic prerequisite for cavity formation. Monkeys and guinea pigs can form cavities, but do not do so invariably.64,91 Rabbits infected by aerosol with low-dose M. bovis, uniformly develop cavities. Fig. 2 shows low-power views of small cavitary lesions in humans (1A), guinea pigs (1B), cynomolgus monkeys (1C), and rabbits (1D). In general, these animal species’ cavitary lesions are similar to those seen in humans, and are composed of liquefied caseum, lymphocytes, epithelioid macrophages, and layers of fibrosis all of which vary with the age of the lesion (Fig. 2, low power, Fig. 3, high power). Although caseous necrosis and DTH are necessary, their presence does not assure that cavities will form. Cattle had measurable DTH responses and developed impressive caseous pulmonary lesions after intratracheal infection with M. bovis, but did not form cavities.88 Guinea pigs vaccinated with BCG and then challenged with low-dose aerosol M. tuberculosis never form liquefied lesions (personal communication Dr. David McMurray). Similarly, guinea pigs infected with a low virulence M. tuberculosis isolate and then challenged with a virulent strain showed protection as well and did not develop cavitation.92 In a model similar to Yamamura’s pre-sensitization and challenge in rabbits, guinea pigs vaccinated with Freund’s incomplete adjuvant with defatted residue of tubercle bacilli protected guinea pigs against subsequent aerosol challenge and did not result in cavities as in Yamamura’s rabbits.40 The specific components and timing of the immune response responsible for caseous necrosis, progressive tissue destruction, and, ultimately, liquefaction and cavity formation in rabbits, humans and NHP in contrast to guinea pigs and mice are not well delineated.93 The cytokines that mediate the evolution to a cavity and the T cell polarization may overlap with those involved in the protective granulomatous response, but evidence from human cavities suggest that Th2 cytokines like IL-4 may be important in addition to TNF-a. Although antigens from M. tuberculosis may play a role in the original formation and maintenance of the granuloma, it is less clear that the pathogen plays a central role in the formation of cavities. Based on the Yamamura model, pre-sensitization with heat-killed bacilli is the key to ensuring the formation of cavities in rabbits. In addition, the heat-killed
ARTICLE IN PRESS Animal models of cavitation in pulmonary tuberculosis bacilli must be suspended in the paraffin lanolin solution that adjuvants the response. Data on heatkilled bacilli from the vaccine literature would suggest that heat-killed bacilli incite only a partially protective immune response, although strong tuberculin skin test responses result.94 It is also known that Freund’s adjuvant potentiates both T and B cell responses. In the bronchoscopic model of rabbit cavity formation (Yoder et al. unpublished),93 rabbits pre-sensitized with heat killed M. bovis suspended in 7H9 medium, then challenged with live M. bovis formed only consolidated bronchopneumonia in the infected lower lung lobe. In contrast, rabbits pre-sensitized with heat-killed M. bovis suspended in paraffin-lanolin, then challenged with the same dose and strain of bacilli uniformly formed cavities (10 of 10 rabbits) in the infected lobe within 3–6 weeks. A comparison of the 2 groups showed that adjuvanted pre-sensitization led to significantly more robust tuberculin skin test responses in the group that ultimately had cavities. Therefore, some immune elements of the DTH response also participate in the formation of cavities in rabbits. The CD4+ T cell plays a critical role in the formation of cavities. AIDS patients with low CD4+ T cells are impaired in their ability to form cavities and are more likely to develop extensive bronchopneumonia or disseminated disease. In rabbits pre-treated with corticosteroids, infected with M. tuberculosis, and then allowed to recover off steroids over 6–9 weeks, all rabbits mounted strong DTH skin test responses and formed extensive, cavitary disease.71 Our data on corticosteroid administration have shown that CD4+ T lymphocytes are preferentially affected by steroids.95 Five weeks after discontinuation of a 5 week course of dexamethasone (given from week 10–15 after infection had been established), CD4+ T cell rebound was accompanied by rapid worsening of tuberculosis (Manabe et al. in preparation). Finally, the tuberculin skin test although insensitive for disseminated, miliary tuberculosis, is positive in 68–83% of human pulmonary disease.96 HIVinfected patients with low CD4 T cell counts are also often tuberculin negative and anergic, and rarely have cavitary disease. We are not aware of any studies that have specifically correlated the tuberculin skin test with cavitary tuberculosis. The data from the rabbit model suggest that the tuberculin skin test may be a reasonable surrogate for the capacity to cavitate, although the exact immunologic triggers and cascade are yet to be fully examined to find a more sensitive and specific marker of this stage of disease. Interestingly, BCG-vaccinated guinea pigs have a faster, larger tuberculin skin test
345 response than unvaccinated controls in the first 4 weeks after virulent M. tuberculosis challenge, but have a waning response by 6 weeks when the unvaccinated controls have larger necrotic tuberculin skin test responses.97,98 Therefore, although the guinea pig has immune components that allow it to mount a strong DTH skin response and caseous necrosis, it does not potentiate this memory response to cause liquefaction as in rabbits. This may illustrate why Koch’s tuberculin was protective and immunomodulatory in infected guinea pigs, but killed some infected humans.
Implications for human disease The pathogenesis of smear-positive cavitary tuberculosis remains an important area of study because of the high likelihood for these patients to transmit disease to close contacts.7,8 Animal models have identified host immune components that may be important in human cavity formation. In rabbits, host recognition of both cell wall and protein components of the bacilli were needed for cavity formation.80 Corroborative evidence for pre-sensitization exist in humans: alcoholic homeless patients who were tuberculin-positive from a prior infection and who had documented exogenous reinfection developed cavitary disease more readily than those who were not previously tuberculinpositive.99 A cell-mediated response to bacillary components followed by caseous necrosis is necessary for the formation of cavities. However, cavities only form in a percentage of tuberculin-positive humans with caseous tuberculous lesions. The clear difference in outcome between guinea pigs and rabbits with pre-sensitization and challenge with virulent mycobacteria (protection and cavitation, respectively) is intriguing. Immune differences between species that form granuloma that caseate but do not cavitate (cattle, guinea pigs) and those that do (rabbit and NHP) as well as the study of murine granulomatous necrosis and fibrosis may hold clues to the pathogenesis of liquefaction. Understanding the pathogenesis of cavities and the host factors important to their formation may lead to new strategies to block transmission and assess the effectiveness of new chemotherapeutic agents against cavitary tuberculosis.6
Acknowledgments The authors would like to thank Dr. Arthur M. Dannenberg, Jr. for his invaluable advice and
ARTICLE IN PRESS 346 editorial assistance. The authors would also like to thank Dr. David N. McMurray and Dr. JoAnne L. Flynn for graciously providing pulmonary tissue sections of guinea pig and cynomolgus macaques respectively and for invaluable scientific input. This work was supported by funding from the National Institutes of Health, 1R01 HL71554 (YCM) and RR07002 (KLH).
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