Published ahead of print on November 21, 2007, doi:10.1165/rcmb.2007-0326OC
© 2008 American Thoracic Society DOI: 10.1165/rcmb.2007-0326OC Cytokine Profiles in Primary and Secondary Pulmonary Granulomas of Guinea Pigs with Tuberculosis1 Department of Microbial and Molecular Pathogenesis, Texas A&M University System Health Science Center, College Station, Texas; and 2 Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, Texas Correspondence and requests for reprints should be addressed to Lan H. Ly, Texas A&M Health Science Center, Microbial and Molecular Pathogenesis, 463 Reynolds Medical Building, College Station, TX 77843. E-mail: lhly{at}medicine.tamhsc.edu
The cytokine mRNA profiles of primary (arising from inhaled bacilli) and secondary (arising from hematogenous reseeding of the lung) granulomas from the lung lobes of bacillus Calmette-Guérin (BCG)-vaccinated and unimmunized guinea pigs challenged with virulent Mycobacterium tuberculosis by the pulmonary route were assessed in situ using laser capture microdissection (LCM) at 6 weeks after infection. The challenge dose chosen was so low that some lung lobes did not receive an implant from the airway. In unimmunized guinea pigs, some lobes contained either large, necrotic primary lesions or small, non-necrotic secondary lesions, or both. The lobes of BCG-vaccinated animals contained only non-necrotic primary tubercles, and no secondary lesions were visible. Real-time PCR analysis of the acquired RNA clearly demonstrated that primary tubercles from BCG-vaccinated guinea pigs were overwhelmed with mRNA from the anti-inflammatory cytokine, transforming growth factor (TGF)-β, with some IFN-  and IL-12p40 mRNA. In contrast, primary lesions from unimmunized animals were dominated by proinflammatory TNF- mRNA. The cytokine mRNA profile of secondary lesions from unimmunized animals was strikingly similar to the profile of primary lesions from BCG-vaccinated guinea pigs (i.e., a predominance of TGF-β mRNA with some IL-12p40 and IFN- mRNA), indicating that the lung lobes from which these lesions were retrieved had been naturally "vaccinated" by the time the bloodborne bacilli returned to the lung at 3 to 4 weeks after infection. Furthermore, cytokine mRNA analysis of splenic granulomas from nonvaccinated and vaccinated animals showed close resemblance to primary granulomas recovered from the lungs of the same animal, that is, high levels of TNF- mRNA in unimmunized animals, and mostly TGF-β mRNA in BCG-vaccinated guinea pigs. Taken together, these data indicate that mycobacteria returning to the lungs of unimmunized guinea pigs 3 to 4 weeks after infection induce a local cytokine response that is fundamentally different from the response to inhaled bacilli and is reminiscent of the primary response in a vaccinated animal.
Key Words: guinea pig • vaccine • tuberculosis • granuloma • cytokine
Disease caused by Mycobacterium tuberculosis (M.tb) can result either from the early progression of a primary granuloma that results from the inhalation of an infectious particle, or from the reactivation of a dormant granuloma that the patient may have carried for many years (1, 2). The precise location and nature of the so-called "reactivatable" granulomas remain controversial. However, there is strong evidence to suggest that secondary, bloodborne lesions in the apex or sub-apex of the lung, rather than the primary lesion, are the reactivating lesions responsible for disease manifestation. Single, calcified, primary lesions were found in various locations in the lungs of 105 patients with tuberculosis (TB) (3), whereas cavitary lesions in patients with reactivation TB had a propensity to be found in the apical regions of the lungs (2). In the highly biologically relevant guinea pig model of low-dose pulmonary TB, the virulent bacilli multiply extensively at the site of primary implantation and are transported very early (7–10 d after infection) to the draining (hilar or bronchotracheal) lymph nodes. From this point, the bacilli quickly escape into the bloodstream and are observed within a few days (14–21 d after infection) in the spleen. Several days later, bacilli appear for the first time in all lung lobes, including those that do not contain a primary lesion (4–7). When individual lung lobes were cultured by Ho and coworkers, the bacilli were detected from primary lesion–free lobes (i.e., lobes containing only secondary lesions) only after 22 days after infection (6). Since the arrival of bacteria in previously sterile lobes was detected after the bacilli were detected in the spleen, these data indirectly imply that secondary lesions result from bloodborne reseeding. Intrathoracic spread would have occurred earlier if it was an important route of bacterial dissemination to previously uninfected lobes. Thus, two different types of lesions exist in the lung after hematogenous dissemination: primary (arising from inhaled bacilli) and secondary (bloodborne). Lenaerts and colleagues (8), recently demonstrated that inflammation in secondary lung lesions resolved more completely after antimycobacterial therapy in guinea pigs infected under conditions similar to those used in the present study (8). The timing of arrival in the lung of bloodborne M.tb coincides with the onset of an immune response (3–4 wk after infection) (7). In addition, bacillary replication in the secondary, bloodborne lesion is curtailed at peak levels approximately 10- to 100-fold lower than peak levels in the primary lesions of the same animal (6). These observations suggest that previously uninvolved portions of the lung are actually "vaccinated" by the primary infection (9). Therefore, our hypothesis was that the local immune response in primary and secondary lesions would differ significantly, with the latter reflecting a protective phenotype. Understanding the immunologic differences between the two lesions will help us better understand the process of reactivation and, thus, the management of the estimated 2 billion people at risk for reactivation TB.
Using laser capture microdissection (LCM), we have previously demonstrated that the cytokine profile of pulmonary granulomas taken from individual lung lobes of M.tb-infected guinea pigs were affected significantly by bacillus Calmette-Guérin (BCG) vaccination (10). Primary lesions microdissected from unimmunized guinea pigs were overwhelmed by the proinflammatory TNF- In the current study, we examined the cytokine milieu of primary and secondary lesions of the lungs of nonvaccinated and BCG-vaccinated guinea pigs infected with a dose of virulent M.tb too low to result in the initial infection of all lung lobes. For comparison, the cytokine mRNA profiles of granulomas from the spleen were also studied. These results, for the first time, allow us to compare the immunological differences between primary and secondary lesions, view a map of the lobes in which these lesions exist, compare the cytokine profile of granulomas from the lung and spleen of the same animal, and evaluate the effect of BCG vaccination status on these variables in the guinea pig.
Animals and Vaccination Specific pathogen–free outbred Hartley strain guinea pigs (250–300 g) from Charles River Breeding Laboratories, Inc. (Willmington, MA) were housed individually in polycarbonate cages under conventional conditions and rested for at least 7 days. Half of the animals were vaccinated via intradermal injection in the left inguinal region with 0.1 ml (103 colony-forming units [CFU]) of viable Mycobacterium bovis BCG (Danish 1331 strain; Statens Seruminstitut) and allowed to rest for 6 weeks before aerosol infection. After virulent infection, the guinea pigs were housed in a BSL-3 containment facility. All protocols were approved by the Texas A&M University Laboratory Animal Care Committee.
Aerosol Infection
Necropsy and Tissue Processing
Laser Capture Microdissection
Total RNA Isolation and Real-Time PCR
All data were normalized to HPRT mRNA expression and then normalized to the values derived from nongranulomatous microdissected cells. For relative percentages of cytokines expressed in Figures 3–6
Statistical Analysis
BCG-vaccinated and nonvaccinated guinea pigs were infected with virulent M.tb at a dose so low that not all lung lobes in each animal were infected by the airway. This infectious dose created an in vivo phenotype in which the individual lung lobes were populated with either large, primary (1°) or small, secondary (2°) lesions, or both (6). The 1° and 2° lesions were characterized according to their relative size and physical appearance as described previously (12). To determine whether the cytokine mRNA expression levels differed between 1° and 2° pulmonary tubercles, real-time PCR analysis of cells captured by laser microdissection from these granulomas was performed. Lung lobes were kept separate to determine whether the cytokine milieu was different among the two types of granulomas. The 6-week interval after infection was chosen to allow for complete hematogenous dissemination of bacilli and reseeding of the lung to occur. Primary granulomas from non-vaccinated guinea pigs stained with H&E were large, with an intensely necrotic center surrounded by loosely grouped scatterings of epithelioid macrophages (Figure 1A). Large, necrotic primary granulomas were not found in the UL lobes of the lung in the nonvaccinated guinea pigs. Conversely, 2° granulomas from nonvaccinated guinea pigs were smaller and remained highly cellular with clusters of fibronecrotic debris (Figure 1B). Secondary granulomas were found in all lung lobes of nonvaccinated animals. Primary lesions were found in the lung lobes of BCG-vaccinated guinea pigs, but no secondary lesions were found. These primary lesions were quite different histologically, as they were smaller than the primary lesions from nonvaccinated animals, and were non-necrotic (Figure 1C).
Figure 2 shows the in situ mRNA expression levels of IFN- , TNF-, TGF-β, IL-12p40, and iNOS in primary lesions retrieved from the individual lung lobes of nonvaccinated and BCG-vaccinated guinea pigs infected with M.tb for 6 weeks. In general, levels of mRNA for IFN- and TGF-β in the primary granulomas from BCG-vaccinated guinea pigs were much higher in all lung lobes compared with the unimmunized guinea pigs (Figures 2A and 2B). For TNF- and iNOS, mRNA expression levels were dramatically higher (20- to 1,000-fold) in the primary lesions of nonvaccinated animals than their BCG-vaccinated counterparts (Figures 2C and 2E). Primary lesions from BCG-vaccinated guinea pigs had much higher levels of IL-12p40 mRNA in the LR, but reduced levels were seen in both the UR and LL lobes of the lung (Figure 2D). Comprehensive charts (Figures 3 and 4) were constructed to illustrate the relative proportions of mRNA transcripts for each of the five cytokines measured. It is clear that 1° lesions from all lobes of the lungs from BCG-vaccinated guinea pigs are dominated by TGF-β, followed by IFN- and IL-12p40 cytokine mRNA (Figure 3). In primary lesions from nonvaccinated guinea pigs, TNF- mRNA was the prevalent cytokine present (> 50%) in the mRNA pool, followed by IL-12p40 (7–25%) and TGFβ (17%) (Figure 4). No primary lesions were found in the UL lobes of nonvaccinated guinea pigs.
Figure 5 illustrates similar measurements taken from small, non-necrotic secondary lesions found only in the UL lung lobes of unimmunized animals. Relative mRNA expression levels of TGF-β were highest (  20-fold) among the five cytokines measured, indicating a more anti-inflammatory phenotype than the primary lesions found within the same animal. The corresponding comprehensive chart reveals that the 2° lesions from nonvaccinated guinea pigs were overwhelmed with TGF-β mRNA (80%), and to a lesser extent, by IL-12p40 (11%) and IFN- (4%) mRNA. This cytokine profile is very different from that observed in primary lesions from the same (nonvaccinated) guinea pigs (Figure 4), but strikingly similar to that seen in the primary lesions found in BCG-vaccinated animals (Figure 3).
To determine the cytokine milieu of granulomas that developed in extrapulmonary organs after dissemination from the lung during the first 2 to 3 weeks of infection, granulomas were also microdissected from the spleen using LCM (Figure 6). The arrows in Figure 6A indicate the presence of splenic granulomas surrounding the lymphoid follicles of nonvaccinated guinea pigs. In contrast, fewer lesions were seen in the spleens of BCG-vaccinated animals (lesions per section: BCG = 2.3 ± 1.67, non-vacc = 10.54 ± 3.57; n = 20 sections ± SEM) and the white pulp zone, located around a central arteriole, was well-organized in the spleens of BCG-vaccinated guinea pigs (arrowhead, Figure 6B). Real-time PCR analysis of splenic granulomas from nonvaccinated guinea pigs revealed a phenotype much like that seen in the large, pulmonary granulomas from the same animals in which the cytokine mRNA pool was dominated by TNF- and to a much lesser extent, IL-12p40 (Figure 7). Splenic lesions from previously BCG-immunized guinea pigs showed a dramatic increase (200- to 1,400-fold) in IFN- and TGF-β mRNA expression compared with the nonvaccinated group (Figure 7), which was similar to the profile seen in the pulmonary primary lesions found in the same animal (Figure 4). These results strongly suggest that the cytokine milieu of both pulmonary and extrapulmonary granulomas are almost identical and are driven by the pre-challenge immune status of the guinea pigs.
A very low-dose aerosol infection allowed us to compare primary and secondary, bloodborne tubercles that develop after early infection in the lungs of guinea pigs (6). Primary lesions (from vaccinated and nonvaccinated guinea pigs) were clearly distinguished from secondary lesions (from nonvaccinated guinea pigs only) based on their size and cellular complexity (12). This low infectious dose allowed some lung lobes to escape without receiving an infectious particle by the airway. In these lobes, only secondary lesions were present. We chose the 6-week post-challenge interval to ensure that the secondary lesions had developed to sufficient size to allow analysis by LCM.
In situ microdissection of primary lesions by LCM clearly show that the cytokine mRNA profile in nonvaccinated guinea pigs was overwhelmed by the proinflammatory cytokine, TNF- Previous analysis of the individual lung lobes at 6 weeks after infection suggested that some heterogeneity existed in the cytokine profiles between different lobes of the lung in nonvaccinated guinea pigs, specifically the UL lobes compared with the others (10). In the current study, we were able to distinguish between the cytokine profiles of 1° and 2° lesions based on their size and location in the lungs in nonvaccinated animals. In those animals, primary and secondary lesions were found in all lobes of the lung except for the UL lobe, in which only small, 2° lesions were found. These data imply that the UL lobe of the guinea pig lung did not become implanted with the inhaled bacilli, but rather only with the disseminated bacilli, that returned to the lung via the bloodstream during the "silent bacillemia" (1, 6, 12). In contrast, only typical 1° tubercles were observed in the lung lobes of BCG-vaccinated guinea pigs and no small, 2° lesions were seen. This supports previous studies suggesting that BCG vaccination prevents or delays hematogenous reseeding of the lung after low-dose pulmonary infection of guinea pigs (16). The precise mechanism by which this occurs remains to be elucidated.
Analysis of secondary, bloodborne lesions from the lungs of nonvaccinated guinea pigs revealed a cytokine mRNA profile (predominantly TGF-β mRNA; Figure 5) that was very different from the primary tubercles (predominantly TNF-
It has been suggested that 2° lung lesions might behave like primary lesions from the spleen, since the granulomas in the spleen develop at about the same time after infection as the secondary granulomas in the lungs in guinea pigs (7). The ability of antimycobacterial drugs to significantly reduce bacillary loads in guinea pig tubercles was remarkably similar in 1° lesion-free (i.e., 2° lesion-containing) lung lobes and the spleen (17). The similarity in response to chemotherapy implied that these two types of granulomas might share other similarities (e.g., cytokine profile). However, the present study shows that the cytokine mRNA profile in the lesions found in the spleens of nonvaccinated and vaccinated guinea pigs (Figure 7) closely resembled the profiles of primary lesions retrieved from the lungs of the same animals. We expected the spleen granulomas in the nonvaccinated guinea pigs would be like 2° lung granulomas because they develop at about the same time (i.e., during the onset of acquired resistance and a strong T cell response). This discrepancy may be explained, in part, by the duration of infection studied. Smith and coworkers reported that the tubercle bacilli recovered from primary lung lesions and spleens respond similarly to chemotherapy up to 40 to 50 days after infection, which coincides with our study interval (17). Furthermore, maximum bacterial loads in the spleens of nonvaccinated guinea pigs are as high as those seen in the lung lobes in the same animals in spite of the strong T cell responses (7). Thus, the cytokine profiles in the splenic granulomas may be driven to the over expression of TNF-
iNOS plays an essential role in the killing of M.tb by mononuclear phagocytes in mice (18). However, its role remains to be proven in the immunopathogenesis of TB in guinea pigs. Previously, we and others have been unable to detect iNOS using the nitrate assay in supernatants of IFN- These data, for the first time, confirm that two fundamentally different types of lesions exist in the nonvaccinated guinea pig lung during the pathogenesis of early pulmonary TB and that cytokine mRNA profiles of the secondary, bloodborne granulomas from those animals resemble primary lesions in BCG-vaccinated guinea pigs. Further studies will be needed to determine the relationship between the cytokine profiles and bacillary loads within these two types of lesions.
This work was supported in part, by the National Institutes of Health Grant RO1 AI-15495 to D.N.M. and P30ES0910607 to the Center of Environmental and Rural Health at Texas A&M University. Originally Published in Press as DOI: 10.1165/rcmb.2007-0326OC on November 21, 2007 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form September 5, 2007 Accepted in final form October 26, 2007
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Abstract
CLINICAL RELEVANCE
