Specific bacterial genotypes of Mycobacterium tuberculosis cause extensive

Tuberculosis 90 (2010) 268e277

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Tuberculosis journal homepage: http://intl.elsevierhealth.com/journals/tube

MODEL SYSTEMS

Specific bacterial genotypes of Mycobacterium tuberculosis cause extensive dissemination and brain infection in an experimental model Rogelio Hernandez Pando a, *, Diana Aguilar a, Ingrid Cohen b, Martha Guerrero c, Wellman Ribon c, Patrícia Acosta a, Hector Orozco a, Brenda Marquina a, Citlal Salinas d, Daniel Rembao d, Clara Espitia b a

Experimental Pathology Section, Department of Pathology, National Institute of Medical Sciences, Mexico City, Mexico Department of Immunology, Biomedical Research Institute, National Autonomous University of Mexico, Mexico City, Mexico Mycobacteria Group, Research Division, National Institute of Health, Bogotá, Colombia d Department of Pathology, National Institute of Neurology and Neurosurgery, Mexico City, Mexico b c

a r t i c l e i n f o

s u m m a r y

Article history: Received 15 December 2009 Received in revised form 7 May 2010 Accepted 10 May 2010

Meningeal tuberculosis is a severe type of extrapulmonary disease, which is thought to begin with respiratory infection, followed by hematogenous dissemination and brain infection. Host genetic susceptibility factors and specific mycobacterial substrains could be involved in its development. From an epidemiological study in Colombia, we selected three Mycobacterium tuberculosis clinical strains isolated from the cerebrospinal fluid (CSF) of patients with meningeal tuberculosis, and used them to infect BALB/ c mice through the intratracheal route. These strains showed a distinctive spoligotype pattern. The course of infection in terms of strain virulence (mice survival, bacillary loads in lungs), bacilli dissemination and extrapulmonary infection (bacilli loads in blood, brain, liver, kidney and spleen), and immune responses (cytokine expression determined by real time PCR in brain and lung) was studied and compared with that induced by the laboratory strain H37Rv and other five clinical strains isolated from patients with pulmonary TB. All the clinical isolates from meningeal TB patients disseminated extensively through the hematogenous route infecting the brain, producing inflammation in the cerebral parenchyma and meninges, whereas H37Rv and clinical isolates from pulmonary TB patients showed very limited efficiency to infect the brain. Thus, it seems that mycobacterial strains with a distinctive genotype are able to disseminate extensively after the respiratory infection and infect the brain. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Meningeal tuberculosis Experimental models Mycobacterial hematogenous dissemination Tuberculosis immunopathology

1. Introduction Tuberculosis involvement of the central nervous system (CNS) is a serious type of extrapulmonary disease, it constitutes approximately 5e15% of the extrapulmonary cases and is often fatal.1 There are different clinical/pathological types of cerebral tuberculosis; the most common is tuberculous meningitis.2 It is believed that cerebral tuberculosis, like any other form of tuberculosis, begins with respiratory infection followed by hematogenous dissemination to extrapulmonary sites. On the basis of their clinical and experimental observations, Rich and Mc Cordock suggested that cerebral tuberculosis develops in two stages. Initially small tuberculous

* Corresponding author. Experimental Pathology Section, Department of Pathology, National Institute of Medical Sciences and Nutrition “Salvador Zubirán”, Calle Vasco de Quiroga 15, Tlalpan, CP 14000, Mexico City, Mexico. Tel./fax: þ52 55 54 85 34 91. E-mail addresses: [email protected], [email protected] (R. Hernandez Pando). 1472-9792/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tube.2010.05.002

lesions (Rich’s foci) develop in the brain during the bacteremia stage of the primary infection. These early lesions can be located in the subpial or subependymal surface, and may remain dormant for long time. Later, rupture or growth of these lesions into the subarachnoideal space or into the ventricular system produces meningitis.3 Animal models of cerebral tuberculosis have been established in rabbits,4,5 mice6,7 and pigs.8 Although they reproduce the human lesions to some extent, these models are artificial because they use the direct intracerebral or intravenous route of infection, instead of the natural respiratory route. Thus, it is important to establish an experimental model that reproduces more closely the human disease, including the initial respiratory route of infection. However, such a model is difficult to achieve because of the efficient CNS protection conferred by the bloodebrain-barrier (BBB).9 The BBB is composed of tightly associated endothelial cells covered by pericytes and outgrowths of astrocytes, so to produce CNS infection some microorganisms have evolved specific virulence factors that permit endothelial invasion followed by brain

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infection.10e12 Clinicaleepidemiological studies have shown a distinct genotype pattern in strains isolated from tuberculous patients’ cerebrospinal fluid (CSF),13 which suggest strain-dependent neurovirulence. In the present study, we used a model of pulmonary tuberculosis in BALB/c mouse infected intratracheally, with the aim of studying the ability of three clinical isolates obtained from an epidemiologic study in Colombia to infect the brain. These clinical strains were CSF isolated from patients with meningeal tuberculosis, and they showed a distinctive spoligotyping pattern.

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ethylic alcohol for histology, while the other hemisphere was frozen in liquid nitrogen for other determinations. All procedures were performed in a bio-safety level III facility following the Institutional Ethics Committee and the national regulations on Animal Care and Experimentation. 2.3. Preparation of tissues for histology

During the years 1995e2001, more than 500 M. tuberculosis clinical isolates were studied in the Mycobacteria Laboratory at the National Institute of Health in Bogota, Colombia. By spoligotyping, one distinctive genotype was identified in a group of 10 strains, all of them isolated from the CSF of patients with meningeal tuberculosis. The amplified DR locus was further studied by PCR-subtyping, designed to amplify a fragment from non-repetitive spacer 7e12 (nRS7 50 -ctgaggagacgagtactcgg-30 and nRS12 50 -ctcggacagcatctccccggg-30 ).

Fixed lungs were embedded in paraffin, sectioned and stained with hematoxylineeosin. The percentage of the pulmonary area affected by pneumonia was determined by automated morphometry (Q Win Leica, Milton Keynes, UK).15,16 One brain hemisphere from each mouse was fixed and embedded in paraffin. For histological analysis, at least 20 serial sections of 5 mm thickness were stained with hematoxylineeosin and ZiehleNeelsen. For immunohistochemistry, sections were mounted on silanecovered slides, deparaffinized, and the endogenous peroxidase quenched. Sections were incubated overnight at room temperature with an optimal dilution of rabbit polyclonal antibodies against interferon gamma (IFNg), tumor necrosis factor alpha (TNFa), transforming growth factor (TGF-b), acid-fibrillar glycoprotein (AGP), and anti-BCG protein mycobacterial antigens (Invitrogen, Carlsbad, CA, USA).16 Bound antibodies were detected with goat antirabbit IgG labeled with peroxidase (Dako, Carpinteria, CA, USA).

2.2. Experimental mouse model of tuberculosis

2.4. Determination of colony forming units (CFUs)

CSF clinical isolates (strains 28, 136, 209) and H37Rv that was obtained from ATCC and used as a control, were grown in Middlebrook 7H9 broth (Difco, Detroit, MI, USA) for 28 days. Considering that there are phenotypic variations among the H37Rv strains used by different laboratories, five clinical strains isolated from the sputum of patients with pulmonary tuberculosis were also included (strains 290, 1431, 5186, Ven 10, Suda 27). Growth was monitored by densitometry. As soon as the culture reached mid-log phase the bacilli were harvested and suspended in phosphatebuffered saline (PBS) containing 0.05% Tween 80 by shaking for 10 min with glass beads. The suspension was centrifuged for 1 min at 350g to remove large clumps of bacilli. Then a preliminary bacterial count was achieved by smearing the supernatant at a known ratio of volume to area, and counting 10 random fields after staining by the ZiehleNeelsen technique. The suspension was finally diluted to 2.5  105 bacteria in 100 ml of PBS and aliquots stored at 70  C. Before use bacteria were recounted, and viability checked as described.14 Reculture was kept to a minimum (less than 2 passages) to avoid virulence loss. After animal infection, the remnant of the bacterial inoculum was plated to confirm the number of bacilli administrated to the animals. Male BALB/c mice, 8-week-old, were anesthetized and inoculated intratracheally with 2.5  105 bacilli in 100 ml PBS.15,16 Three separate experiments were performed, in each experiment nine groups of 70 mice were infected with the different M. tuberculosis clinical isolates; 20 mice from each group were left undisturbed to record survival. Six animals from each group were euthanized by exsanguination and whole blood samples were collected at 1, 3, 7, 14, 21, 28, 60, and 120 days after infection. Three lung lobes, right or left, from the same number of animals were perfused with 10% formaldehyde dissolved in PBS and prepared for histopathology. The other three lung lobes were snap-frozen in liquid nitrogen, stored at 70  C and used for bacilli loads quantifications, while the other three lung lobes from the same number of different animals were used for immunological analysis. The brain of each infected animal was divided into right and left hemispheres after saggital section of the corpus callosum. One hemisphere was immediately fixed by immersion in absolute

Whole blood, liver, spleen, kidney, right or left lungs and cerebral hemispheres from three mice at each time point were weighed and then homogenized with a Polytron (Kinematica, Luzern, Switzerland). Four dilutions of each homogenate were spread onto duplicate plates containing Bacto Middlebrook 7H10 agar (Difco Lab, Detroit, MI). After 21 days, CFUs were counted and adjusted per gram of tissue.

2. Material and methods 2.1. Selection of Mycobacterium tuberculosis clinical isolates

2.5. Real time PCR analysis of cytokines in lung and brain homogenates Left or right lungs and cerebral hemispheres from three different mice per group in three different experiments were used to isolate mRNA, and determine cytokines expression by real time PCR as described before.17 Sequence of specific primers and cycling conditions have been described elsewhere.17 The mRNA copy number of each cytokine was related to one million copies of mRNA encoding the G3PDH gene. 2.6. Statistical analysis Survival curves were analyzed with KaplaneMeier plots and Long Rank tests. Student’s T-test was used to determine statistical significance of CFU, histopathology and cytokine expression. p < 0.05 was considered significant. 3. Results 3.1. Epidemiology, clinical and molecular information of M. tuberculosis isolates Figure 1A shows the code, year, and geographical origin of the clinical isolates used in this study. Strains 136 and 28 were drug susceptible, whereas isolate 209 was multidrug resistant. Considering that in Colombia the prevalence of tuberculosis is 26.2 cases per 100,000 inhabitants, strain 209 was highly prevalent (42.4 cases/100,000 inhabitants), whereas isolates 136 and 28 showed lower prevalence (12.8 and 6.2/100,000 inhabitants, respectively).

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Figure 1. Clinical, epidemiological, and molecular data on the M. tuberculosis isolates used in this study. A) Code, year of isolation, drug susceptibility, spoligotyping, IS6110 RFLP pattern, geographical origin, prevalence, patient’s gender, age and HIV status. B) Results of the PCR-subtyping, amplifying the DR locus of the clinical isolates from non-repetitive spacer 7e12. C) IS6110 RFLP patterns comparing, BCG, strain H37Rv, and the clinical isolates.

All the strains were CSF isolated from adult male patients with meningeal tuberculosis, all were HIV negative. All the clinical isolates share the same spoligotype, which consists of 35 copies of DRs; when compared with H37Rv pattern, these clinical isolates did not hybridize with DR 8e11 or 33e36. Interestingly, when the region between non-repeat spacer 7e12 was amplified, three different groups were established according to the size of the fragments: group 1 showed DNA fragments from 189 to 194 bp; group 2, fragments from 262 to 266 bp; and group 3, from 409 to 411 bp (Figure 1B). From each group, strains 28, 136, and 209 were chosen and used for animal infections. The RFLP with IS6110 showed three different patterns (Figure 1C). For comparison purposes, five clinical isolates from patients with pulmonary TB were also used for animal infection, three were from the Latin American Mediterranean (LAM) genotype family (strains 290, 1431 and 5186), their clinical and epidemiological characteristics, as well as their virulence level and type of immune response induced in the same murine model have been described elsewere.17 The other two strains belong to the Beijing genotype family, they were prevalent strains from big clusters in Venezuela (strain Ven 10), and Cape Town South Africa (strain Suda 27). 3.2. Survival, lung histopathology, and pulmonary and extrapulmonary bacillary loads Strains isolated from CSF of meningeal TB patients showed diverse virulence. Strain 209 produced significantly higher mortality than H37Rv and the other two clinical strains (p < 0.001) (Figure 2A). In comparison with mice infected with strain H37Rv, the survival rate after 16 weeks was significantly higher in mice infected with strains 136 and 28 (p < 0.001). In agreement with the survival rate, animals infected with strain 209 showed the highest lung bacillary

loads (Figure 2B) and tissue damage (Figure 2C), whereas mice infected with the two other clinical isolates showed similar bacillary loads and pneumonia (Figure 2B, C). Animals infected with any of the three CSF clinical isolates showed rapid and progressive increase of CFU in the blood, significantly higher than in H37Rv-infected mice and any other of the TB sputum isolated strains; the highest CFU loads were in animals infected with strain 209. Similar kinetics were determined in spleen, kidney and liver (Figures 3 and 4). Strains isolated from pulmonary TB patients also showed variability in their virulence level. Both prevalent Beijing strains were highly virulent, they started to kill infected mice after 3 weeks of infection, and after 7 weeks all the animals died (Figure 4). In contrast, animals infected with LAM strains started to died after one month post-infection and all the animals died 10e16 weeks postinfection (Figure 4). Bacilli loads in blood, kidney and spleen were very low in comparison with the strains isolated from the CSF of meningeal TB patients (Figures 3 and 4). 3.3. Brain histopathology and bacillary loads Brains collected from mice infected with any of the three clinical strains isolated from meningeal TB patients showed the presence of bacteria from day one post-infection. Bacterial growth, which progressively increased, was the highest in animals infected with strain 209, and the lowest in mice infected with strain 136; an intermediate bacterial load was seen in animals infected with strain 28, and these animals exhibited intermediate survival rates. In striking contrast, very low bacilli loads were detected in the brain homogenates from control mice infected with the H37Rv strain or with any of the clinical strains isolated from the sputum of pulmonary TB patients (Figures 3 and 4).

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Figure 2. Survival (A), lung bacillary loads (B), and (C) morphometry (% of lung surface area affected by pneumonia) in BALB/c mice infected by an intratracheal injection (2.5  105 bacilli) of M. tuberculosis isolates from patients with meningeal tuberculosis. Reference strain H37Rv was used as control. Survival curves were constructed with 20 infected mice and analyzed with KaplaneMeier plots and Long Rank tests. Data are the mean  SD of measurements from three mice per time point in three different experiments. Asterisks represent statistical significance (p < 0.05) comparing with H37Rv strain infection.

The histopathology study of the brain infected with any CSF strain from meningeal TB did not show any evident abnormality during the first and second week of infection. After 3 and 4 weeks of infection, ZN staining showed occasional capillaries with acid fast bacilli on the surface or crossing the cytoplasm of endothelial cells (Figure 5A), and occasional astrocytes or microglial cells with intracellular bacilli (Figure 5B) or dispersed extracellular bacteria in areas without inflammatory cells (Figure 5C). In the base (mesencephalus, thalamus) or in the cortex of the brain, as well as below the choroid plexus epithelium, occasional small inflammatory nodules containing lymphocytes and a few macrophages were found (Figure 5D, E). These lesions were surrounded by interstitial edema and activated microglia. In these areas, immunohistochemistry to detect mycobacterial antigens showed strong immunostaining in astrocytes, activated microglia, and ependymal cells (Figure 5F). These histopathological features were more evident in animals infected with strain 209. At day 60 and 120 post-infection, bigger inflammatory nodules were seen (Figure 6A), and some of these lesions were connected to the sub-arachnoideal space producing mild or extensive inflammatory infiltrates there (Figure 6B, C), in coexistence with dilatation of cerebral ventricles and mild inflammation in the central areas of the choroid plexus. At this time point, the hippocampal area showed numerous necrotic neurons and cellular depopulation (Figure 6D), with extensive gliosis demonstrated by numerous GFP immunostained cells (Figure 6E). Our immunohistochemical study to detect cytokines showed strong immunostaining for IFNg in lymphocytes and TNFa in macrophages located in nodules and meningeal inflammation. Ependymal cells also showed strong immunoreactivity for both cytokines (Figure 6F). Macrophages and meningothelial cells showed strong TNFa immunoreactivity (Figure 6G). Some macrophages located in the inflammatory infiltrates and activated microglia exhibited TGF-b immunoreactivity. Interestingly, the

endothelium from numerous capillaries near the inflammatory areas or in apparently normal brain tissue also showed strong immunostaining to TGF-b (Figure 6E). Brain sections from animals infected with the laboratory strain H37Rv or with any of the clinical strains from pulmonary TB patients did not show any apparent histological abnormality. 3.4. Cytokine gene expression determined by real time RT-PCR in the lung and brain during infection with clinical strains isolated from CSF and H37Rv strain In comparison with control mice infected with the H37Rv strain, in animals infected with strain 209, the induction of IFNg expression in the lungs was significantly lower, but there was rapid and high IL-4 expression during the first and second weeks postinfection. Interestingly, strain 209 also induced high but transient TNFa expression and the lowest levels of iNOS expression (Figure 7). Overall, these results indicate that this strain is more virulent and does not induce a protective immune response in our animal model. In contrast, the lungs of mice infected with the less virulent strain 136 showed the opposite kinetics, with the highest IFNg expression during the first and second week of infection, and the lowest IL-4 expression throughout the experiment (Figure 7). Animals infected with strain 28 showed higher expression of IFNg and TNFa than control mice, whereas similar expression of IL-4 was seen during the early phase of infection, followed by progressive increase during late disease (Figure 7). The brains of mice infected with strain 209 showed a progressive increase and the highest expression of IFNg, whereas TNFa expression was very rapid and highest during the first week post-infection followed by a slow progressive decrease; similar kinetics was observed in iNOS expression (Figure 8). The brain from animals infected with the less virulent strain 136 showed low but stable expression of IFNg and TNFa, but with progressive increase of iNOS

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DAYS POST-INFECTION Figure 3. Bacillary loads per 1 ml of total blood and 1 g of tissue from different organs of mice infected with M. tuberculosis strains isolated from the CSF of patients with meningeal tuberculosis. The indicated organs were collected after euthanizing mice at different time points after the infection with the indicated M. tuberculosis strain. Organs were weighed and plated for bacillary colony forming units determination. Data are the mean  SD of measurements from three mice per time point in three different experiments. Asterisks represent statistical significance (p < 0.05) comparing with H37Rv strain infection.

Figure 4. Survival and bacillary loads per 1 ml of total blood and 1 g of tissue from different organs in mice infected with different strains isolated from the sputum of patients with pulmonary tuberculosis. The indicated organs were collected after euthanizing mice at different time points after the infection with the indicated M. tuberculosis strain. Organs were weighed and plated for bacillary colony forming units determination. Data are the mean  SD of measurements from three mice per time point in three different experiments.

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Figure 5. Representative BALB/c mice brain histopathology after one month of infection with M. tuberculosis strain 209, isolated from the cerebrospinal fluid of a patient with meningeal tuberculosis. (A) Acid fast bacilli attached to capillary endothelial cells (1000 magnification). (B) Numerous tuberculous bacilli in the cytoplasm of glial cells from the base of the brain. (C) Extracellular acid fast bacilli in the white substance of the base of the brain. Note the lack of inflammatory cells (D) Small inflammatory nodule in the brain cortex near the meningeal pia layer (arrows). (E) Chronic inflammatory infiltrates (arrows) located below the choroid plexus epithelium. (F) Microglial cells and astrocytes show strong immunoreactivity to mycobacterial antigens in the base of the brain. There are no inflammatory cells. (micrographs A, B, and C at 1000 magnification, micrographs D, E, and F 200 magnification).

expression, peaking at day 120 post-infection. Similar cytokine kinetics, but with higher number of transcripts, was observed in animals infected with strain 28 (Figure 8). Although control animals infected with strain H37Rv showed minimal brain infection, they also expressed pro-inflammatory and anti-inflammatory cytokines. Brains infected with strain 209 showed the highest IL-4 expression during the first week post-infection, followed by similar expression as compared to mice infected with either strain 136 or 28 (Figure 8). Strain 209 also induced the highest expression of TGF-b (Figure 8). Strain 136 and 28 induced stable high IL-4 expression, and progressive expression of TGF-b and IL-10 which was much higher than detected in H37Rv-infected mice (Figure 8). 4. Discussion Our results demonstrate that these selected M. tuberculosis isolates from the CSF of meningeal tuberculous patients with a distinctive spoligotype pattern, produced disseminated disease and brain tuberculosis after intratracheal infection in BALB/c mice. In meningeal tuberculosis, mycobacteria reach the CNS by the hematogenous route secondary to pulmonary infection.3 This experimental model reproduces this situation. Comprehensive clinicaleepidemiological studies have identified several risk factors for meningeal tuberculosis, these include age less than 40 years, HIV infection, and certain ethnic populations.13,18,19 This latter factor suggests participation of the host

genetic background.13 An association between the development of tuberculous meningitis and single nucleotide polymorphism in the Toll-interleukin-1 receptor domain containing adaptor protein and Toll-like receptor-2 genes has been reported.20,21 Epidemiologic studies have suggested that certain bacterial genotypes are associated with meningeal tuberculosis,13 our study demonstrated certain bacterial genotypes were associated with increased virulence and dissemination in an experimental animal model, but the study was not designed to show a clear epidemiological association between a given strain and high frequency of meningeal disease in our human population. Interestingly, the IS6110 pattern of our strains is different from that exhibited by type-1 Canadian strains, which have been epidemiologically related with meningeal tuberculosis.13 Moreover, our strains are from the EuroAmerican lineage, which was recently reported to be more pulmonary than meningeal, due to the lack of pks 15/1.22 The Pks gene participates in the production of phenolic glycolipid (PGL), which inhibits the innate immune response and may be responsible for dissemination and CNS infection.23 Our Euro-American isolates are unable to express PGL, however they produced brain infection, suggesting that other mycobacterial molecules must participate in this process. We consider mycobacterial heparinbinding hemaglutinin/adhesin as a good candidate, because it triggers receptor-mediated bacilli adherence and invasion to epithelial cells, and extrapulmonary dissemination.24 Another potential participating molecule is the histone-like protein, which

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Figure 6. Representative BALB/c mice brain histopathology and immunohistochemistry during late infection with M. tuberculosis strain 209, isolated from the cerebrospinal fluid of a patient with meningeal tuberculosis. (A) Large inflammatory cortical nodule. (B) Extension of the cortical inflammation to the sub-arachnoideal space. (C) Massive inflammation in the sub-arachnoideal space. (D) Hippocampal area with neuronal depopulation and numerous necrotic neurons (arrows). (E) Immunohistochemistry detection of acid glial protein in the same hippocampal area shows numerous positive cells denoting extensive gliosis. (F) Epithelial cells from the choroid plexus exhibit strong IFNg immunostaining. (H) Meningothelial cells and macrophages in the sub-arachnoideal space show strong TNFa immunoreactivity. (I) In the base of the brain, in areas without inflammation, there are numerous capillaries with TGF-b immunostaining.

permits Mycobacterium leprae to invade Schwann cells.25 This study is now in process in our laboratory. The clinical isolate 209 was epidemiologically a prevalent strain and it showed higher virulence in our experimental model, inducing the highest bacillary loads in lung, blood and brain, suggesting that CNS infection was the consequence of high bacilli number in circulating blood. It seems that higher virulence is not related to extensive extrapulmonary dissemination, since strains 136 and 28 efficiently infected the brain but infected mice showed higher survival than control animals infected with H37Rv strain. Considering that there is significant phenotypic variation among the laboratory strain of H37Rv, we also infected mice with five different clinical isolates from pulmonary TB patients, some of them were previously tested in the same murine model.17,26 The blood and brains from these mice showed very low bacilli loads, in a similar fashion to animals infected with H37Rv strain. When microglia and astrocytes are infected in-vitro with mycobacteria, they produce high amounts of pro-inflammatory cytokines.27 Our results were in agreement with these observations, showing mycobacteria in the cytoplasm of these cells, as well as high expression of TNFa and IFNg, these being highest in animals infected with the more virulent strain and the lowest in animals infected by the less virulent strain. Interestingly, iNOS expression in the brain from animals infected with strain 209 was very high during the first week of infection, followed by progressive decrease, whereas animals infected with the low or intermediate virulence strains showed progressive iNOS expression, peaking at 4 months post-

infection. This is in agreement with a better control of bacilli growth and the smaller inflammatory response, because in the CNS, NO can down-regulate inflammation by inhibiting T cell proliferation and migration.28 The most distinctive early histological feature in the infected brain was small inflammatory nodules located near to the pia mater or below the ependymal cell layer. These lesions were described by Rich many years ago (Rich’s nodules) and, in agreement with his original description,3 we found rupture and connection of these nodules to the sub-arachnoideal space, producing mild or extensive inflammatory infiltrate in this compartment. Our immunohistochemistry study showed strong immunostaining to IFNg and TNFa not only in inflammatory cells and activated microglia, but also in meningothelial and ependymal cells. Thus, pro-inflammatory cytokines have different sources during experimental mycobacterial CNS infection. This high TNFa expression is in agreement with the previous demonstration that it is related with extensive mycobacterial brain damage in animal models5,6 and human disease.29 Pro-inflammatory cytokines are also involved in the induction of astrogliosis and we demonstrated it by numerous GFP positive cells near the inflammatory infiltrate and in the hippocampus. Interestingly, even during late infection when high bacillary loads in the brain were determined, none of the infected mice developed evident clinical signs of neurological damage. The same situation has been reported in mice infected with high bacilli doses through the intravenous route,7 or directly in the brain.6 However,

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Figure 7. Gene expression of cytokines determined by real time RT-PCR in the infected lungs. BALB/c mice were infected with the indicated M. tuberculosis strain and euthanized at different time points. The lungs from three different animals in each time point were used to determine the gene expression of the indicated factor. The more virulent clinical isolate 209 induced the lowest expression of IFNg and iNOS, rapid and high IL-4 expression, and strong but ephemeral TNFa expression. In contrast, less virulent strains 28 and 136 induced higher expression of IFNg and TNFa in coexistence with lower IL-4 expression. Data are the means and standard deviation. Asterisks represent statistical significance (p < 0.05) when compared with mice infected with control H37Rv strain.

Figure 8. Gene expression of cytokines determined by real time RT-PCR in the infected brains. BALB/c mice were infected with the indicated strain and euthanized at different time points. The right or left cerebral hemisphere from three different animals in each time point was used to determine the gene expression of the indicated cytokine. Data are the means and standard deviation. Asterisks represent statistical significance (p < 0.05) when compared with mice infected with control H37Rv strain.

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we observed in the hippocampus many neurons with acidophilic necrosis and extensive gliosis in complete absence of inflammation. These histological abnormalities can be related with memory and cognitive disturbances, and they have been reported in other types of infectious meningitis.30 Another interesting histological observation was the presence of acid fast bacilli both extracellular and intracellularly in astrocytes or microglia cells without inflammatory response. Immunohistochemical detection of mycobacterial antigens showed strong positivity in these cells and in ependymal and meningothelial cells, indicating that all these different cells can be infected or are able to phagocytose bacterial debris. Indeed, under physiological conditions, basal expression of innate immunity receptors involved in mycobacterial phagocytosis, like TLR2 and TLR4, was detected in meninges, choroid plexus, and circumventricular brain area, which lack BBB and are more exposed to pathogens.31,32 Microglia and astrocytes can also express these receptors,33,34 as well as manosereceptors.35 Thus, specific bacterial antigens and their host cell receptors must participate in CNS mycobacterial infection. However, this activity apparently does not induce a strong inflammatory response. Thus, an efficient modulation of inflammation is produced in the brain that could avoid or delay tissue damage and signs of neurological lesion. Type 2 CD4þ cells could participate in this process, due to their efficient suppression of Th-1 excessive activity that induces immunopathology.36,37 The Th-2 response also has beneficial activity for CNS healing after injury, supporting neuronal survival.38,39 We found high IL-4 expression in the infected brain, being higher during late disease in animals infected with the lower and intermediate virulent strains; even in H37Rv-infected mice, which showed minimal brain infection, we detected mild cytokines expression, probably as a consequence of nonspecific mechanisms such as cardiovascular and respiratory dysfunction caused by the lung inflammation, which induce cytokine production by nervous cells without BBB disruption.40 Indeed, both IFNg and IL-4 have neuroprotective activities,38 and Th-2 cells are more efficient conferring neuroprotection and regeneration than Th-1 cells.37,38 Interestingly, CNS injury is associated with systemic Th-2 shift,41 which may be important in down-regulating the Th-1 response and facilitating the progression of pulmonary tuberculosis. Another significant neuroprotective and antinflammatory cytokine localized in activated microglia is TGF-b.42 We found high gene expression of TGF-b in the brains of mice infected with any of the clinical isolates and our histological studies showed positive immunostaining in macrophages and capillary endothelial cells from distant areas of the inflammatory response. Another immunomodulatory cytokine that was highly expressed in infected brains was IL-10, which is mainly produced by T regulatory cells, suggesting that this cell type could be involved in the modulation of neuroinflammation. Besides the immune system, bacterial factors could also participate decreasing inflammation. It has been reported that during in-vitro brain endothelial invasion by M. tuberculosis, there is an extensive shut-down of bacterial genes related to diverse metabolic pathways, which suggest dormant infection,43 a condition related with minimal inflammation.44 In conclusion, we demonstrated that some specific M. tuberculosis isolates from patients with meningeal tuberculosis are able to disseminate and infect the mouse brain. This model could be a useful tool to study host and bacillary factors involved in the pathogenesis of the most severe form of tuberculosis.

Acknowledgments This study was supported by the European Community (STREP contract no 37919, TB-adapt).

Funding: The funding was obtained from the European Community. Competing interests: Ethical approval:

None declared. Not required.

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