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Lung carcinogenesis induced by chronic tuberculosis infection: the experimental model and genetic control

Abstract

Coexistence of pulmonary tuberculosis (TB) and lung cancer in clinic poses significant challenges for the diagnostic and treatment of both diseases. Although association of chronic inflammation and cancer is well-documented, causal relationship between TB infection and lung cancer are not understood. We present experimental evidence that chronic TB infection induces cell dysplasia and squamous cell carcinoma (SCC) in a lung-specific manner. First, squamous cell aggregates consistently appeared within the lung tissue associated with chronic TB lesions, and in some cases resembled SCCs. A transplantable tumor was established after the transfer of cells isolated from TB lung lesions into syngeneic recipients. Second, the (Mycobacterium tuberculosis) MTB-infected macrophages play a pivotal role in TB-induced carcinogenesis by inducing DNA damage in their vicinity and by the production of a potent epidermal growth factor epiregulin, which may serve as a paracrine survival and growth factor responsible for squamous metaplasia and tumorigenesis. Third, lung carcinogenesis during the course of chronic TB infection was more pronounced in animals with severe lung tissue damage mediated by TB-susceptibility locus sst1. Together, our experimental findings showed a causal link between pulmonary TB and lung tumorigenesis and established a genetic model for further analysis of carcinogenic mechanisms activated by TB infection.

Introduction

Tuberculosis (TB) remains one of the major causes of death among infectious diseases, posing a global public health threat. The virulent Mycobacterium tuberculosis (MTB) is responsible for over 2 million deaths annually worldwide, with an estimated one-third of the human population harboring the pathogen in its latent form. Outcomes of TB-infection range from the primary progressive disease, which occurs in less than 10% of the infected individuals, to latent infection, which may lead to the re-activation of the disease many years after the primary infection, or remain subclinical for the lifetime of the infected individuals. Pulmonary TB develops in 85% of clinical cases and is the most epidemiologically significant form of the disease. The ability of the pathogen to cause destructive pulmonary disease in immunocompetent hosts enables its efficient transmission within human populations through the respiratory route. Understanding processes initiated by the pathogen in the lungs is a key to deciphering its evolutionary successful virulence strategy and developing effective interventions to control the disease. However, characteristics of TB lesions in the lungs that distinguish them from granulomas in other organs remain unknown.

Pulmonary TB in the majority of immunocompetent adults is a chronic infectious process associated with extensive remodeling of lung tissue. The hallmark of this process is the formation of granulomas containing live mycobacteria as well as myeloid and lymphoid cells. Formation and maintenance of the granulomas are mediated by an active immune response and require the production of interferon-γ and other cytokines by antigen-specific T lymphocytes. Subsequent macrophage activation by combination of cytokines and mycobacterial products results in the massive local production of reactive nitrogen and oxygen species, prostaglandins, proteases and inflammatory cytokines representing an attempt of the host to clear the bacteria (Fujiwara and Kobayashi, 2005; Russell, 2007; Saunders and Britton, 2007), which may also cause an extensive host tissue damage. Unfolding tissue repair processes lead to a varying degree of fibrosis in the vicinity of granulomas. Efficient maintenance of granulomas during chronic phase of persistent or even latent infection also requires continuous action of immunocompetent cells. Decrease in the activity of CD4-positive T cells in HIV-infected individuals, neutralization of tumor necrosis factor (TNF)-α to treat chronic inflammatory diseases in human patients, as well as the elimination of CD4-positive cells, interferon-γ, TNF-α or nitric oxide production in the experimental mouse model result in the reactivation of latent TB infection. Therefore, the task of maintaining the granulomas in vital organs, such as lung, is especially challenging during chronic infection, because production of tissue damaging agents, including DNA-damaging agents, and activation of tissue repair mechanisms must be engaged in parallel to control the pathogen and simultaneously reduce the collateral damage to normal tissues (Ulrichs et al., 2004; Ulrichs and Kaufmann, 2006; Saunders and Britton, 2007). Dysbalance of those processes may generate a microenvironment predisposing to malignant transformation (Dalgleish and O'Byrne, 2002; Naito and Yoshikawa, 2002; Melnikova and Ananthaswamy, 2005; Jin, 2007; Azad et al., 2008). Indeed, association of cancers with chronic inflammation of both infectious and non-infectious origins is well established and a number of mechanisms through which inflammatory mediators contribute to carcinogenesis have been uncovered in recent years (Karin et al., 2006; Lawrence and Gilroy, 2007).

Coexistence of pulmonary TB and carcinoma was described by many pathologists starting with the first report by Bayle in 1810 (Sakuraba et al., 2006; Tan and Coussens, 2007). Since then, it has been reported that pulmonary TB may coexist with a variety of histological types of lung cancer. For example, close association of metastatic squamous cell carcinoma (SCC) of the lung with large-cavitary TB lesions have been documented (Oka and Chan, 2005). A study by Dacosta and Kinare in India reported that TB lesions, either active or healing, were found in the lungs of 30–33% of patients with bronchogenic tumors as compared with 7% in the general population (Dacosta and Kinare, 1991). Kurasawa also reported higher rates of coexistence of pulmonary TB and lung cancer, which in this study was represented mostly by SCC (Kurasawa, 1998). On autopsies, excessive high rates of death from lung cancer were observed both among smoking and non-smoking TB patients (Sakurai et al., 1989). Clinical diagnosis of coexisting TB and lung cancer represents a challenge and often there is a considerable delay in administering adequate treatment for both conditions, which is associated with poor prognosis (Gopalakrishnan et al., 1975; Ting et al., 1976).

Causal link between lung cancer and pulmonary TB is not clear. On the one hand, chronic inflammatory reaction associated with latent TB infection may serve as a cause of malignancy. This possibility is indirectly supported by observations of the so-called ‘scar carcinomas’ (Cagle et al., 1985). Ardies postulated that in many cases lung scarring occurred as a result of spontaneous healing after recurrent TB infection, and by triggering cycles of inflammatory and tissue repair processes generated a favorable environment for tumorigenesis (Ardies, 2003). On the other hand, lung cancer may develop autonomously and then provoke reactivation of latent TB by weakening local immunity. This is also a likely scenario, as almost one-third of the human population is latently infected with MTB. Therefore, inferring causality based exclusively on clinical correlations is impossible. Meanwhile, this distinction is critical for showing specific molecular mechanisms underlying the coexistence of TB with cancer and the development of rational preventive and therapeutic measures.

In this study, we present experimental evidence that chronic TB infection in the lungs is sufficient to cause a multi-step transformation of cells associated with TB lung lesions through squamous cell dysplasia to malignant SCCs. We have showed that in addition to DNA-damaging reactive oxygen and nitrogen species, the MTB-infected macrophages produce a most potent member of the epidermal growth factor family epiregulin, which may serve as a paracrine growth factor at the initial steps of tumorigenesis. We also identified a powerful genetic modifier of MTB-induced lung tumorigenesis in our model: the genetic locus sst1 (Kramnik et al., 2000), which specifically controls tissue damage and TB progression in the lungs.

Results

Squamous cell metaplasia develops in the lungs of mice chronically infected with MTB

Initially clusters of cells resembling SCCs were observed in the lungs of relatively TB-resistant (C3HeB/FeJ × B6.C3H-sst1) F2 progeny after i.v. infection with MTB. In this sst1 susceptible but otherwise genetically heterogeneous population, the survival ranged from 5–6 weeks to more than a year. Seven long-term survivors were killed 15 months post infection. Live MTB was detected in their lungs by plating organ homogenates. On histopathological examination, in four out of seven animals we found clusters of abnormal large cells resembling squamous cells, which were tightly adherent to one another and contained large atypical nuclei. These clusters were adjacent to extensive inflammatory lesions, but clearly distinct from the surrounding inflammatory cells (Figure 1a, arrows). These cells stained positively for cytokeratins 5, 6 and 14 and occupied large contiguous areas of the lung (Figure 1b), whereas staining for cytokeratins 1, 8 and 10 was negative (data not shown).

Figure 1
figure1

Squamous cells in the lungs of mice chronically infected with MTB. (a) Squamous cell carcinoma in the lungs of genetically resistant (C3HeB/FeJ × B6.C3H-sst1) F2 hybrid mouse 15 months after infection with MTB. Hematoxylin and Eosin (H&E) staining of the lung sections. Arrows—clusters of squamous cells. Original magnification— × 40, × 200 and × 600 (left, middle and right panels, respectively). (b) Immunohistochemical analysis of the lung tissue of the F2 mouse using anti-cytokeratin 5 (left panel), anti-cytokeratin 6 (middle panel) and anti-cytokeratin 14 antibodies (right panel). Original magnification— × 200. (c) Lung sections of the sst1 susceptible B6.C3H-sst1 congenic mouse strain 5 months after aerosol infection with MTB, double staining for cytokeratin 6 (left and middle panels, original magnification— × 40 and × 400, respectively) and acid-fast mycobacteria (right panel, × 400). (d) Massive fibrosis associated with the necrotic lung lesion of the B6.C3H-sst1 mouse (arrows). Trichrome staining, original magnification— × 40 and × 200, left and middle panels, respectively. Right panel—large cluster of squamous cells within the tuberculosis lung lesion. H&E, original magnification— × 400.

Next, we investigated whether squamous cell dysplasia emerged within typical necrotic TB lung lesions, which developed in the B6.C3H-sst1 mice after a low-dose aerosol infection. At 5 months post MTB infection large clusters of cytokeratin 6-positive cells were found on the periphery of the TB lesions (Figure 1c, left and middle panels) adjacent to central necrotic area containing mycobacteria (Figure 1c, right panel). Those cells displayed atypical tumor-like morphology (Figure 1d, right panel) and were surrounded by massive fibrotic tissue (Figure 1d, left and middle panels).

In both experiments, our tentative diagnosis was squamous cell metaplasia of alveolar cells with possible malignant transformation.

The rate of squamous cell metaplasia is genetically controlled

We compared the appearance of metaplastic cells within TB lung lesions of the following mouse strains: C3HeB/FeJ (TB-susceptible strain that carry the original susceptible allele of the sst1 locus), the sst1 congenic B6.C3H-sst1. (intermediate TB susceptibility) and C57BL/6J (resistant). As shown in Figure 2, after infection with a low dose of virulent MTB via aerosol these strains differed in their survival (Figure 2a), ability to control multiplication of MTB in the lungs (Figure 2b) and extent of lung inflammation (Figure 2c, shown at 4 months post infection). Squamous cell appearance in the lungs of the MTB-infected mice was followed using immunostaining with cytokeratin 6-specific antibodies, which allowed more sensitive identification of squamous cells within complex TB lesions. Using this technique no squamous cells were found in the lungs of uninfected mice of either strain (not shown).

Figure 2
figure2

Squamous cell metaplasia during tuberculosis infection in MTB-resistant and susceptible mouse strains. (a) Survival curves of the C3HeB/FeJ, B6.C3H-sst1 and C57BL/6J mouse strains after a low-dose aerosol infection with 30–50 CFU of MTB strain Erdman. (b) Mycobacterial burden in the lungs of C3HeB/FeJ, B6.C3H-sst1 and C57BL/6J mouse strains as determined by plating of lung homogenates (CFUs—colony forming units). (c) Inflammatory lung lesions 4 months after the aerosol infection (H&E, original magnification— × 40 for upper panels; × 100 for lower panels). (d) Analysis of cytokeratin 6-positive cells within tuberculosis lung lesions of the C3HeB/FeJ, B6.C3H-sst1 and B6 mice (left, middle and right panels, respectively) at 4 months post infection (original magnification— × 100).

As summarized in Table 1, first cytokeratin 6-positive cells were found at 4 months post MTB infection. At that time we observed extensive inflammatory lesions in the lungs of the susceptible C3HeB/FeJ mice, less prominent lesions in B6.C3H-sst1 and small areas of mild interstitial inflammation in the resistant C57BL/6J mice (Figure 2c). In one out of four mice in the C3HeB/FeJ and B6.C3H-sst1 groups, we found small clusters of cytokeratin 6-positive squamous cells embedded within the inflammatory lung lesions (Figure 2d, left and middle panels), whereas no squamous cells were found within TB lung lesions of the C57BL/6J mice (Figure 2d, right panel). The TB-susceptible C3HeB/FeJ mice succumbed to infection shortly after the 4-month timepoint, meanwhile large clusters of squamous cells were found in the lungs of all B6.C3H-sst1 mice at 5 and 7 months post infection (Figures 1c and d and Table 1). Squamous cell metaplasia was observed in the lungs of C57BL/6J mice at 7 months post infection (Table 1 and Figure 3a, upper panels). At 12 months, squamous cell clusters were found in the lungs of 8 out of 10 C57BL/6J mice, although their lung lesions contained no necrosis or extracellular bacteria. (Figure 3a, lower panels). The acid-fast fluorescent mycobacteria were found intracellularly in cytokeratin 6-negative macrophages adjacent to the clusters of squamous cells at 12 months post infection (Figure 3a, lower right panel).

Table 1 Appearance of squamous cell metaplasia in lungs of mice of different genetic backgrounds during chronic infection with virulent MTB
Figure 3
figure3

Development of squamous cell metaplasia and carcinoma within tuberculosis lung lesions of the C57BL/6J mice. (a) Cytokeratin 6 (K6)-positive cell clusters in the lungs of B6 mice at 7 (upper panels) and 12 (lower panels) months after aerosol infection with MTB. Double staining for cytokeratin 6 (left panels) and acid-fast mycobacteria (right panels). Original magnification— × 400, inset— × 600. (b) Mitotic cells (staining with phosphorylated histone 3 (pH3)-specific antibodies, upper panels) are associated with clusters of squamous cells (cytokeratin 6-specific staining, lower panels). The pH3 and K6 staining were performed on non-serial lung sections of the same C57BL/6J (left panels) or B6-C3H-sst1 (right panels) mouse 7 months after the aerosol infection with MTB. Original magnification— × 400. (c) Transplantable tumor after subcutaneous transplantation of single cell suspension isolated from the tuberculosis lung lesions of the C57BL/6J mice 12 months after aerosol infection with MTB. Tumor at the site of injection 2.5 months after transplantation. Histopathology of the transplantable tumor (second passage)—H&E (original magnification— × 200, lower left panel). Right panels—anti-cytokeratin 6 staining (original magnification— × 100 and × 400, upper and lower panels, respectively). Acid-fast fluorescent staining of mycobacteria was negative in transplantable tumors.

Taken together, our data show that the development of squamous cell metaplasia is a common feature of chronic TB infection in the lungs, whereas no squamous cells were associated with MTB-infected cells in spleens and livers at any stage of the infection (data not shown).

The squamous cell metaplasia results in malignant transformation

Based on the morphological criteria, squamous cell metaplasia may progress towards the formation of SCCs at later stages of the infection (Figure 1). To assess proliferative activity of cells associated with the metaplastic lesions, we used phosphorylated histone 3-specific antibodies, which recognize nuclei of mitotic cells (Hendzel et al., 1997; Inagaki et al., 1997; Preuss et al., 2003). At 7 months post infection, a large proportion of cells closely associated with the typical clusters of cytokeratin 6-postive cells displayed prominent nuclear histone 3 staining (Figure 3b). This was observed within the metaplastic lung lesions of both C57BL/6J and B6.C3H-sst1 mice (Figure 3b, left and right panels, respectively).

As 80% of C57BL/6J mice at 12 months post infection displayed evidence of squamous cell metaplasia, some with morphological signs of malignant transformation (Table 1), we tested the tumorigenic potential of those cells directly by transplanting the cells isolated from the TB lung lesions into syngeneic recipients. A single cell suspension was prepared from the lungs of five C57BL/6J mice 12 months after MTB infection using collagenase digestion, and 10 millions of unseparated lung cells (equivalent to one lung) were injected per mouse either subcutaneously (five animals) or intraperitoneally (five animals). The mice were given an anti-TB drug isoniazid (INH) in drinking water to suppress the growth of MTB. After 2–3 months the cell transfer, we observed formation of tumors in 2 out of 10 mice that received the lung cells, one after subcutaneous and one after intraperitoneal transplantation. As a control, we transplanted single cell suspensions prepared from spleens of the same MTB-infected mice into syngeneic recipients and observed no tumor formation.

The subcutaneous tumor was transplanted into a secondary recipient, which also developed a large tumor at the subcutaneous site of injection 2 months later (Figure 3c, upper left panel). To date, a total of three passages have been performed at 2 months intervals, and no acid-fast mycobacteria were detected within transplantable tumors. As compared with the primary lung tumors, the transplantable tumor exhibited less differentiated morphology (Figure 3c, lower left panel, H&E) and contained fewer cytokeratin 6-positive cells (Figure 3c, right panels). Thus after transfer into syngeneic recipients, cells originating from chronic TB lung lesions gave rise to a transplantable tumor that did not require MTB for its autonomous growth.

Factors predisposing to squamous cell carcinomas are present within TB lung lesions

Next, we wanted to determine which of the factors that might be responsible for the squamous cell dysplasia are present within the microenvironment of the TB lung lesions. One of the candidate growth factors known to be critical in SCC development is epiregulin, a potent member of the epidermal growth factor family (Toyoda et al., 1997). We examined the epiregulin gene expression in the lungs of C57BL/6J mice during the course of infection with MTB using quantitative RT–PCR of total lung tissue. As shown in Figure 4a, epiregulin expression was low for the first 6 months of infection and was significantly upregulated by 12 months, in parallel to the development of squamous cell metaplasia and carcinomas.

Figure 4
figure4

Factors predisposing to squamous cell carcinoma are present within tuberculosis lung lesions. (a) Kinetics of epiregulin mRNA expression in total lung tissue of the C57BL/6J mice after a low-dose aerosol infection with MTB. Upper panel—quantitative RT–PCR, lower panel—semi-quantitative RT–PCR. (b) Expression of epiregulin by macrophage populations isolated from the lungs of MTB-infected C57BL/6J mice, 1—alveolar macrophages, 2—interstitial inflammatory macrophages. Left panel—quantitative RT–PCR, right panel—semi-quantitative RT–PCR. (c) Epiregulin mRNA expression in bone marrow-derived macrophages (BMDM) after infection with MTB in vitro (MOI=1:1). The total RNA was isolated from the C57BL/6J (black bars) and B6.C3H-sst1 (white bars) BMDMs at indicated time points. (d) Tuberculosis lung lesions of the B6.C3H-sst1 congenic mouse 7 months after the aerosol infection. Upper panel—double staining for iNOS expression (left) and AFF mycobacteria (right panel). Lower panel—double staining for nuclear γ-H2AX (DNA damage) and AFF MTB. The lung lesions of the B6 mice also contained clusters of iNOS-positive macrophages and cells with γ-H2AX-positive nuclei in their vicinity, although these lesions contained significantly fewer AFF mycobacteria. Original magnification— × 400.

To determine whether MTB infection can also induce expression of epiregulin in macrophages, bone marrow-derived macrophages isolated from C57BL/6J (black bars) and B6.C3H-sst1 (white bars) strains were infected with MTB in vitro, and the kinetics of epiregulin mRNA expression were analysed by the quantitative RT–PCR. As shown in Figure 4c, an upregulation of epiregulin mRNA was observed 6 h post MTB infection in both the C57BL/6J and B6.C3H-sst1 strains. The kinetics of the epiregulin mRNA expression in MTB-infected macrophages suggests that its expression was triggered by the initial interaction of macrophages with MTB, possibly through toll-like receptors.

Next, we tested whether epiregulin is expressed by macrophages of TB lung lesions in vivo. Two populations of macrophages, alveolar and interstitial inflammatory macrophages, were isolated from the lungs of the B6 mice 12 months post MTB infection. The alveolar macrophages were isolated by bronchoalveolar lavage and interstitial (granuloma) macrophages were isolated using collagenase digestion of interstitial inflammatory lesions. In both populations, macrophages were enriched by plastic adherence for 2 h and total RNA isolation was performed without any additional stimulation. Interstitial macrophages expressed epiregulin mRNA, whereas no detectable epiregulin expression was observed in alveolar macrophages (Figure 4b). Thus in vivo, epiregulin was expressed by inflammatory macrophages within TB lung lesions.

As shown in Figure 4d, macrophages infected with MTB are the major source of inducible nitric oxide synthase within TB lung lesions (upper panels). Reactive oxygen and nitrogen species are potent inducers of oxidative DNA damage. To assess accumulation of cells experiencing nuclear DNA damage in the vicinity of the MTB-infected macrophages, we stained TB lesions with antibodies specific for phosphorylated γ-histone2AX, whose appearance in the nuclei indicates double-stranded DNA breaks (Bewersdorf et al., 2006; Nussenzweig and Paull, 2006). The γ-H2AX staining showed that abundant punctated nuclear staining of non-infected cells surrounded the MTB-infected macrophages. These data shows that the MTB-infected macrophages produce a dual impact on the cells in their immediate surroundings, being both a source of DNA damaging agents and a source of pro-survival growth factor(s).

To determine whether higher mycobacterial loads or other factors were responsible for more rapid development of squamous cell dysplasia in the lungs of the B6.C3H-sst1 mice, we used anti-mycobacterial chemotherapy with INH to control the bacterial growth. After a 2-month period of the disease progression, INH was administered for 3 months. At the onset of the INH treatment, we observed the development of necrotic lesions and 500–1000 fold higher bacterial loads in the lungs of the sst1-susceptible B6.C3H-sst1 congenic mice. However, no squamous cells were observed in their lungs at this time. The INH therapy reduced the bacterial loads in the organs of the B6.C3H-sst1 mice 500-fold, that is to the levels normally observed in non-treated C57BL/6J mice. Nevertheless, in the lungs of two out of the three B6.C3H-sst1 mice cytokeratin 6-positive metaplastic cells were readily detectable (Figure 5). In one animal, we observed advanced stages of metaplasia adjacent to large clusters of MTB-infected macrophages (Figures 5a–c, left 2 panels, mouse #1). Meanwhile, the lungs of another mouse represented an earlier stage of metaplasia with small clusters of cytokeratin 6-positive cells associated with inflammatory tissue, which contained no acid-fast bacilli (Figures 5a–c, right panels).

Figure 5
figure5

Squamous cell dysplasia associated with tuberculosis lung lesions following anti-mycobacterial chemotherapy with isoniazid (INH). The C57BL/6J and B6.C3H-sst1 congenic mice were infected i.v. with 50 000 CFU of MTB. After 2 months of infection, the animals received INH in drinking water (0.1 mg/ml) for additional 3 months. The animals were killed 7 days after cessation of the INH chemotherapy. The bacterial burden in the lungs of the B6.C3H-sst1 mice after the treatment ranged from 5 × 104 (mouse #2) to 20 × 104 CFU (mouse #1). At that time MTB was below the level of detection (<100 CFU) in the lungs of B6 mice and no cytokeratin 6-positive cells were observed. Panels (a)—H&E staining. Large clusters of squamous cells within the mouse #1 lung lesions are shown by arrow. (b) and (c)—double staining for cytokeratin 6 and AFF mycobacteria, respectively. Mouse #1—original magnification × 100 for left panels, × 200 for upper right panel, and × 400 for middle right and lower right panels, respectively; Mouse #2—original magnification × 400 for all panels.

This experiment showed that a period of initial MTB growth and lung tissue damage in the sst1 susceptible background was sufficient to trigger the metaplastic process, which developed and persisted in the absence of further bacterial growth. It is likely that the metaplastic cells, which form following extensive tissue damage and may remain in the lungs even after complete elimination of the pathogen, later may pose a threat of malignant transformation.

Discussion

In this study, we presented experimental evidence that chronic MTB infection can provoke the development of lung carcinoma. Our data show that the pathogen can trigger a series of events that lead to extensive remodeling of lung tissue, activation of atypical differentiation pathway and, eventually, to malignant transformation. In our model, squamous cell metaplasia was a frequent event specifically associated with TB lesions in the lung, as no signs of squamous cell metaplasia were observed in the vicinity of MTB-infected macrophages in the spleens and livers, as well as in the lungs of non-infected mice.

Tuberculosis lung lesions are composed of heterogeneous cell populations, which include derivatives of myeloid lineage with unusual morphology, such as foamy macrophages, epithelioid cell and giant cells (Ulrichs et al., 2004; Ulrichs and Kaufmann, 2006). Therefore, identification of emergence of additional atypical cells, such as squamous cells, within this heterogeneous population is difficult, unless they undergo malignant transformation and expand to occupy large contiguous areas. Following our initial observation of large areas of SCC-like cells in the lungs of the TB-infected mice, we developed an immunohistochemical double staining technique for simultaneous detection of squamous cells and acid-fast mycobacteria that allowed early detection and monitoring progression of squamous cell dysplasia specifically at the sites of TB infection.

The development of cancer is a multi-step process in which normal cells evolve through a series of pre-malignant steps into autonomous invasive cancers. A role for the microenvironment in supporting or preventing this evolution is well documented (reviewed in (Nelson and Bissell, 2006)). Chronic inflammation is known to generate microenvironment that favors tumor initiation and progression (reviewed in (Coussens and Werb, 2002; van Kempen et al., 2006; Lawrence, 2007)). Hanahan and Weinberg summarized general rules that govern gradual transformation of normal cells into malignant cancers (Hanahan and Weinberg, 2000). Two of them, accumulation of genome alterations and accessibility of growth signals, were shown in our studies. First, using the immunohistochemical technique, we showed that MTB-infected macrophages express the highest levels of inducible nitric oxide synthase within TB lung lesions. Therefore, production of reactive nitrogen and oxygen species must be the highest in their vicinity leading to DNA damage. Indeed, cells with double-strand DNA breaks (shown by H2AX phosphorylation) accumulate in the vicinity of the infected macrophages starting between 2 and 4 months post infection. Besides, constitutive activation of transcription factor Nrf2 by oxidative stress may directly induce squamous cell metaplasia (Wakabayashi et al., 2003). Second, normally proliferation of cells with DNA damage would have been blocked through G2/M checkpoint activation and those cells would either persist indefinitely in the absence of the ‘promotion’ signal or would be eliminated through p53-mediated pro-apoptotic pathway. However, this pathway is antagonized by activation of the NF-κB-mediated pro-inflammatory pathway (Komarova et al., 2005; Karin et al., 2006), which allows cells with unrepaired DNA damage to avoid apoptosis and enter the cell cycle. In TB lesions, activation of the NF-κB pathway in macrophages as well as in epithelial cells may be triggered by at least three activation pathways, namely oxidative damage, toll-like receptor ligands of bacterial origin and TNF-α. All of those pathways are known to be activated during the course of chronic TB infection. Thus, the factors that are essential for protective immunity and adaptation to oxidative stress within TB lung lesions, may also participate in the initiation and promotion of lung tumorigenesis.

Finally, we found that MTB-infected macrophages produce a potent epithelial growth factor epiregulin. Epiregulin is a member of the epidermal growth factor superfamily and one of the ligands for the epidermal growth factor receptor, which was shown to stimulate proliferation of fibroblasts, hepatocytes, smooth muscle cells and keratinocytes (Harris et al., 2003). In its secreted form, epiregulin stimulated proliferation of keratinocytes in an autocrine and paracrine manner in vitro (Shirakata et al., 2000; Takahashi et al., 2003). Mice deficient in epiregulin-developed dermatitis, further implicating epiregulin in regulating keratinocyte growth in vivo (Shirasawa et al., 2004). High expression levels of the epiregulin gene have been shown in a number of cancer cell lines and activation of the Ki-Ras signaling pathway in colon cancer cells has been linked to the deregulation of epiregulin expression (Baba et al., 2000). Among the epidermal growth factor receptor ligands, epiregulin is distinguished by its more potent mitotic activity, the ability to bind various heterodimeric ErbB receptors (Shelly et al., 1998), and its unusual expression pattern, mainly in peripheral blood monocytes, macrophages and the placenta in normal human tissues, as well as in various types of epithelial tumor cell lines (Toyoda et al., 1997). In the mouse, epiregulin is expressed by tissue resident and peritoneal macrophages in vivo and its expression in macrophages is induced in response to the toll-like receptor agonists LPS and peptidoglycan in vitro (Shirasawa et al., 2004). Production of epiregulin by inflammatory macrophages may be a beneficial reaction that helps repair tissue damage at the sites of inflammation. Indeed, the epiregulin knockout mice are highly susceptible to intestinal damage caused by oral administration of aseptic inflammatory stimulus dextran sulfate (Lee et al., 2004). However, in the presence of live persisting pathogen this adaptive response is eventually turned against the host, as MTB-infected macrophages producing epiregulin provide potent growth factor to pre-malignant epithelial and, possibly, stromal cells surrounding them.

Squamous cell carcinoma is one of the four major types of lung cancers in humans. However, it is a very rare type of lung tumor in mice. In the laboratory mice, SCCs do not occur spontaneously. To induce SCC, Wang et al. (Wang et al., 2004) treated eight inbred mouse strains with a chemical carcinogen by skin painting and found SCC in five mouse strains. Interestingly, in this model of chemical carcinogenesis the C57BL/6J mice failed to develop any SCC. To dissect the genetic control of carcinogen-induced SCC, these authors performed whole-genome linkage disequilibrium analysis and mapped three genetic loci significantly associated with susceptibility to carcinogen-induced SCC on chromosomes 1 (at 15 cM, that is, in position distinct from the sst1 locus), 3 and 18. We also observed genetic control of SCC development induced by TB infection. In our TB model, however, the major TB-susceptibility locus sst1 played a role of a genetic modifier of the rate of the SCC progression. The B6.C3H-sst1 mouse strain that carries the C3H-derived susceptible allele at the sst1 locus on the genetically resistant genetic background C57BL/6J represents a unique mouse model of human chronic pulmonary TB. In their lungs, TB lesions contain large areas of necrosis surrounded by extensive fibrotic tissue and a cell wall composed of myeloid and lymphoid cells. The B6.C3H-sst1 mice developed squamous cell metaplasia much faster than the resistant parental strain B6 (Table 1). Thus, the sst1 locus controlled tumorigenesis in a quantitative manner, as a modifier locus. A possibility that the sst1 locus controlled the levels of epiregulin expression by the MTB-infected macrophages has been excluded, at least in vitro (Figure 4c). Using anti-mycobacterial drug treatment, we excluded another possibility that the effect of the sst1 locus could be explained by higher MTB loads in the lungs of the sst1 susceptible hosts (Figure 5). Most likely, lung tissue damage inflicted by the MTB infection in the sst1 susceptible genetic background is responsible for the accelerating effect of this locus on SCC progression.

Earlier squamous metaplasia has been reported in experimental model of TB reactivation caused by neutralization of TNF-α. Injection of TNF-α-specific neutralizing monoclonal antibodies into genetically resistant B6 mice infected with low dose of MTB resulted in reactivation of persistent TB infection, severe tissue deterioration and appearance of squamous cell metaplasia (Mohan et al., 2001). No tumor formation was observed in this study as the mice rapidly succumbed to the infection after the TNF-α neutralization. The sst1 locus does not affect TNF-α production by macrophages, but causes extensive necrosis within the TB granulomas in the lungs. Thus, MTB-inflicted lung tissue damage seems to be a common feature in these two models, which accelerates the formation or accumulation of squamous cells in the vicinity of TB lesions in the lungs.

Taken together, our studies experimentally proved that chronic TB lung lesions represent an environment that is highly conducive to tumor development. They show that coexistence of lung cancer with pulmonary TB documented in clinic, at least in some cases, is causal, not coincidental. As approximately one-third of the world population is estimated to be latently infected with MTB, powerful surveillance mechanisms counteract cancer development in the majority of latently infected individuals (Klein and Klein, 2005). Our findings provide a model for mechanistic dissection of alterations of the tumor surveillance mechanisms during the course of TB infection. In addition, they may suggest an earlier unrecognized mechanism of reactivation of latent TB infection- as keratinocytes and tumor cells are known to produce a battery of pro-inflammatory and immunosuppressive cytokines (Pastore et al., 2006; Moutsopoulos et al., 2008), those cells may substantially contribute to the breakdown of local immunity. This, yet mostly hypothetical, mechanism may, however, represent a novel facet of a general virulence strategy of MTB—the ability to persist in a face of active immunity and hijack host responses to modify the local microenvironment to the pathogen's benefit.

Materials and methods

Mice and breeding

C57BL/6J (B6) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The congenic B6.C3H-sst1 strain was obtained by transferring the C3HeB/FeJ-derived susceptible sst1 allele on the C57BL/6J genetic background. All mice were housed in the Harvard Medical School Animal Care Facilities under specific pathogen-free conditions. All procedures were performed with the full knowledge and approval of the Standing Committee on Animals at Harvard Medical School (Protocols # 03000).

Infection of mice with MTB and anti-TB drug treatment, histopathology and immunohistochemistry techniques, isolation and infection of bone marrow-derived macrophages with MTB in vitro, RNA isolation and quantitative real-time PCR, transplantation of cells isolated from TB lung lesions are presented in Supplementary information 1.

Conflict of interest

The authors declare no conflict of interest.

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Acknowledgements

We thank Drs Raju Kucherlapati, Steve Elledge, David Christiani and Barry Bloom for helpful discussions. The authors are grateful to Dr Mari Kuraguchi for technical advice.

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Correspondence to I Kramnik.

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Supplementary information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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Nalbandian, A., Yan, BS., Pichugin, A. et al. Lung carcinogenesis induced by chronic tuberculosis infection: the experimental model and genetic control. Oncogene 28, 1928–1938 (2009). https://doi.org/10.1038/onc.2009.32

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Keywords

  • Mycobacterium tuberculosis
  • sst1
  • chronic inflammation
  • squamous cell carcinoma
  • macrophages
  • epiregulin

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