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In JoVE (1)
Other Publications (26)
- Journal of Immunology (Baltimore, Md. : 1950)
- Cellular Immunology
- Journal of Immunology (Baltimore, Md. : 1950)
- Journal of Immunology (Baltimore, Md. : 1950)
- Infection and Immunity
- Journal of Leukocyte Biology
- Immunology
- Infection and Immunity
- Journal of Immunology (Baltimore, Md. : 1950)
- Journal of Leukocyte Biology
- Journal of Immunology (Baltimore, Md. : 1950)
- Veterinary Immunology and Immunopathology
- Journal of Leukocyte Biology
- Current Protocols in Microbiology
- Journal of Immunology (Baltimore, Md. : 1950)
- Journal of Immunology (Baltimore, Md. : 1950)
- American Journal of Respiratory Cell and Molecular Biology
- Tuberculosis (Edinburgh, Scotland)
- The American Journal of Pathology
- Journal of Biomedical Materials Research. Part A
- American Journal of Respiratory Cell and Molecular Biology
- Journal of Leukocyte Biology
- PloS One
- Tuberculosis (Edinburgh, Scotland)
- Tuberculosis (Edinburgh, Scotland)
- The Journal of Biological Chemistry
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Articles by Mercedes Gonzalez-Juarrero in JoVE
JoVE Immunology and Infection
Não-cirúrgico instilação endotraqueal de camundongos com Análise de Pulmões e Drenagem Linfática Nodes Lung por citometria de fluxo
1Department of Immunology, University of Colorado School of Medicine, 2Division of Cell Biology, Department of Pediatrics, National Jewish Health, 3Department of Microbiology, Immunology, and Pathology, Colorado State University, 4Department of Immunology, National Jewish Health
Nós ilustramos não-cirúrgico de entrega de materiais de teste para os pulmões de ratos anestesiados através da traquéia. Este método permite a exposição de pulmão a patógenos virais e bacterianas, citocinas, anticorpos, miçangas, químicos ou corantes. Iremos descrever colheita e processamento dos pulmões e dos gânglios linfáticos de drenagem de pulmão (LDLNs) para citometria de fluxo.
Other articles by Mercedes Gonzalez-Juarrero on PubMed
In Vivo IL-10 Production Reactivates Chronic Pulmonary Tuberculosis in C57BL/6 Mice
The production of immunosuppressive cytokines, such as IL-10 and TGF-beta, has been documented in individuals diagnosed with active tuberculosis. In addition, IL-10 production is increased within the lungs of mice that have chronic mycobacterial infection. Therefore, we hypothesized that the down-regulatory properties of IL-10 might contribute to the reactivation of chronic Mycobacterium tuberculosis infection in mice. To determine the influence of IL-10 on the course of infection, transgenic mice producing increased amounts of IL-10 under the control of the IL-2 promotor were infected with M. tuberculosis via the respiratory route. Mice that overexpressed IL-10 showed no increase in susceptibility during the early stages of infection, but during the chronic phase of the infection showed evidence of reactivation tuberculosis with a highly significant increase in bacterial numbers within the lungs. Reactivation was associated with the formation of macrophage-dominated lesions, decreased mRNA production for TNF and IL-12p40, and a decrease in Ag-specific IFN-gamma secretion. These data support the hypothesis that IL-10 plays a pivotal role during the chronic/latent stage of pulmonary tuberculosis, with increased production playing a potentially central role in promoting reactivation tuberculosis.
Florid Pulmonary Inflammatory Responses in Mice Vaccinated with Antigen-85 Pulsed Dendritic Cells and Challenged by Aerosol with Mycobacterium Tuberculosis
Mice immunized by the intranasal route with dendritic cells harvested from the lungs and then pulsed with Ag85 (LDC-Ag85) were able to prime naive CD4(+) T cells in vivo. As a result splenic CD4(+) T cells from these immunized mice were able to produce IFNgamma following culture with Mycobacterium tuberculosis-infected antigen presenting cells. Hematoxylin and eosin stained lung sections from LDC-Ag85 immunized mice after they had been exposed to aerosol challenge with M. tuberculosis showed a florid infiltration of macrophages and lymphocytes into granulomas and parenchymal tissues when compared to lung sections from control groups implanted with dendritic cells pulsed with ovalbumin. In addition, using immunohistochemistry, these tissues appeared to have more CD4(+) and CD8(+) cells than the control groups. This was confirmed by flow cytometric analysis which showed that lung cell digests contained increased numbers of CD4 and CD8 interferongamma secreting cells. Despite this increase however, no evidence was seen that indicated that the LDC-Ag85 immunized mice were more resistant to M. tuberculosis infection than mice immunized with LDC pulsed with an irrelevant protein. Instead, the potent inflammatory response in the LDC-Ag85 resulted in serious consolidation of the lung tissue.
Dynamics of Macrophage Cell Populations During Murine Pulmonary Tuberculosis
The influx of macrophages into the lungs is the major component of the granulomatous response to infection with Mycobacterium tuberculosis. In this investigation we used flow cytometric analysis to define macrophage populations entering the airways and lung tissues of infected mice. We demonstrate that by the judicious use of cell surface markers, especially CD11b and CD11c, several cell populations can be distinguished, allowing cell sorting and morphological definition. Primary populations of CD11b(-)/CD11c(+/high) were defined as alveolar macrophages, CD11b(high)/CD11c(+/high) as dendritic cells, and CD11b(+/mid)/CD11c(+/mid) as small macrophages or monocytes, and changes in the activation phenotype of these populations were followed over the early course of the infection. In further studies, these cell populations were compared with cells harvested during the chronic stage of the disease. During the chronic stage of infection, Ag-presenting class II molecules and activation markers were poorly expressed on dendritic, small macrophage, and monocyte cell populations, which may have important implications for the breakdown of the lesions during reactivation disease. This analytical approach may facilitate the further characterization of macrophage populations entering into the lung tissues and their relative contributions to host resistance to tuberculosis infection.
NK Cells Respond to Pulmonary Infection with Mycobacterium Tuberculosis, but Play a Minimal Role in Protection
Both innate and adaptive immune systems contribute to host defense against infection with Mycobacterium tuberculosis. NK cells have been associated with early resistance against intracellular pathogens and are known to be potent producers of the cytokine IFN-gamma. In C57BL/6 mice infected by aerosol exposure with M. tuberculosis, NK cells increased in the lungs over the first 21 days of infection. Expansion of the NK cell subset was associated with increased expression of activation and maturation markers. In addition, NK cells isolated from the infected lungs were capable of producing IFN-gamma and became positive for perforin. In vivo depletion of NK cells using a lytic Ab had no influence on bacterial load within the lungs. These findings indicate that NK cells can become activated during the early response to pulmonary tuberculosis in the mouse model and are a source of IFN-gamma, but their removal does not substantially alter the expression of host resistance.
Stable T-cell Population Expressing an Effector Cell Surface Phenotype in the Lungs of Mice Chronically Infected with Mycobacterium Tuberculosis
Analysis of T-cell subsets accumulating in the lungs of C57BL/6 mice chronically infected with Mycobacterium tuberculosis revealed that both CD4 and CD8 T-cell populations expressed a cell surface phenotype consistent with that of effector T cells and that a significant proportion of these cells were in the process of secreting gamma interferon.
Disruption of Granulocyte Macrophage-colony Stimulating Factor Production in the Lungs Severely Affects the Ability of Mice to Control Mycobacterium Tuberculosis Infection
Mice lacking expression of granulocyte macrophage-colony stimulating factor (GM-CSF KO) are unable to contain Mycobacterium tuberculosis (M. tuberculosis) growth and succumb to infection by 35 days following pulmonary challenge. GM-CSF KO mice do not express normal levels of the inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) nor the chemokines, regulated on activation, normal T expressed and secreted (RANTES), macrophage-inflammatory protein-1beta (MIP-1beta), MIP-1alpha, and lymphotactin, which are required for recruitment of lymphocytes and expression of a T helper cell type 1 (TH1) response within the lungs. In contrast, transgenic mice overexpressing GM-CSF in the lungs but with a lack of GM-CSF in other organs (GM+) are able to recruit lymphocytes and to express a TH1 response with production of TNF-alpha and interferon-gamma in the lungs. However, GM+ mice succumb to infection between 60 and 90 days post-challenge, as they are unable to develop a normal granulomatous response. Although GM+ mice are able to express the chemokine RANTES, they lack the ability to express other inflammatory chemokines such as lymphotactin and MIP-1beta. We conclude that GM-CSF is essential to the recruitment of lymphocytes and expression of a TH1 response in the lung, to the generation of a normal mononuclear granuloma, and most importantly, to the containment of M. tuberculosis bacterial growth.
Interleukin-10 Production by Lung Macrophages in CBA Xid Mutant Mice Infected with Mycobacterium Tuberculosis
Mice on the CBA inbred strain background expressing the well characterized mutation designated xid in the cytoplasmic signalling enzyme Bruton's protein kinase have been previously noted to illustrate shifts in T helper type 1 (Th1)/Th2 immunity which is underlined by an apparent failure to produce the regulatory cytokine interleukin-10. In the current study we examined if this extended to infection with Mycobacterium tuberculosis, which also depends on Th1 immunity. Contrary to expectations, xid mice showed evidence of a transient early susceptibility to pulmonary infection, changes in macrophage morphology, and decreased activation of lung natural killer cells, while showing evidence of substantial IL-10 production and accumulation in lung lesions macrophages, but paradoxically this did not influence the course of the chronic disease. In addition, macrophages from the lungs of xid mice also expressed high levels of CD14. These observations suggest that the xid mutation in cellular signalling has much wider effects on the immune system than previously thought.
Factors Associated with Severe Granulomatous Pneumonia in Mycobacterium Tuberculosis-infected Mice Vaccinated Therapeutically with Hsp65 DNA
Resistant C57BL/6 mice infected in the lungs with Mycobacterium tuberculosis and then therapeutically vaccinated with Mycobacterium leprae-derived hsp65 DNA develop severe granulomatous pneumonia and tissue damage. Analysis of cells accumulating in the lungs of these animals revealed substantial increases in T cells secreting tumor necrosis factor alpha and CD8 cells staining positive for granzyme B. Stimulation of lung cells ex vivo revealed very high levels of interleukin-10, some of which was produced by B-1 B cells. This was probably an anti-inflammatory response, since lung pathology was dramatically worsened in B-cell gene-disrupted mice.
Foamy Macrophages Within Lung Granulomas of Mice Infected with Mycobacterium Tuberculosis Express Molecules Characteristic of Dendritic Cells and Antiapoptotic Markers of the TNF Receptor-associated Factor Family
Highly vacuolated or foamy macrophages are a distinct characteristic of granulomas in the lungs of animals infected with Mycobacterium tuberculosis. To date these have usually been considered to represent activated macrophages derived from monocytes entering the lesions from the blood. However, we demonstrate in this study that foamy macrophages express high levels of DEC-205, a marker characteristic of dendritic cells (DCs). In addition to high expression of the DEC-205 marker, these cells were characterized as CD11b(+)CD11c(high)MHC class II(high), and CD40(high), which are additional markers typically expressed by DCs. Up-regulation of CD40 was seen only during the early chronic stage of the lung disease, and both the expression of CD40 and MHC class II markers were down-regulated as the disease progressed into the late chronic phase. Foamy cells positive for the DEC-205 marker also expressed high levels of TNFR-associated factor-1 (TRAF-1), TRAF-2, and TRAF-3, markers associated with resistance to apoptosis. These data indicate that in addition to the central role of DCs in initiating the acquired immune response against M. tuberculosis infection, they also participate in the granulomatous response.
Virulent Clinical Isolates of Mycobacterium Tuberculosis Grow Rapidly and Induce Cellular Necrosis but Minimal Apoptosis in Murine Macrophages
In this study, we investigated the ability of four clinical isolates of Mycobacterium tuberculosis representing a range of virulence for their capacity to grow in bone marrow-derived macrophages. The rate of growth of each of the isolates in macrophages reflected their known virulence, but the most virulent isolates strongly induced production of the cytokine tumor necrosis factor alpha. A key difference, however, was the degree of cell cytotoxicity observed with the more virulent strains after several days in culture. Staining of cell monolayers for DNA fragmentation indicative of apoptosis showed that this was minimal and only evident to any degree in macrophages infected with the most virulent strains. In contrast, electron microscopy revealed damage of macrophages consistent with cell necrosis. These results suggest that rapid intracellular growth rate and induction of necrotic cell death within host macrophages are virulence factors of M. tuberculosis in the early stages of bacterial infection. They further imply that infected cell apoptosis, regarded as a defense mechanism or cross-priming mechanism, plays a minimal role.
Enhanced Macrophage Activity in Granulomatous Lesions of Immune Mice Challenged with Mycobacterium Tuberculosis
In this study, we evaluated the cellular influx and cytokine environment in the lungs of mice made immune by prior vaccination with Mycobacterium bovis bacillus Calmette-Guérin compared with control mice after infection with Mycobacterium tuberculosis to characterize composition of protective lesions in the lungs. Immune mice controlled the growth of the M. tuberculosis challenge more efficiently than control mice. In immune animals, granulomatous lesions were smaller and had a more lymphocytic core, less foamy cells, less parenchymal inflammation, and slower progression of lung pathology than in lungs of control mice. During the chronic stage of the infection, the bacterial load in the lungs of immune mice remained at a level 10 times lower than control mice, and this was associated with reduced numbers of CD4P(+P) and CD8P(+P) T cells, and the lower expression of protective (IL-12, IFN-gamma), inflammatory (TNF-alpha), immunoregulatory (GM-CSF), and immunosuppressive (IL-10) cytokines. The immune mice had higher numbers of CD11b- CD11c(high) DEC-205(low) alveolar macrophages, but lower numbers of CD11b+ CD11c(high) DEC-205(high) dendritic cells, with the latter expressing significantly lower levels of the antiapoptotic marker TNFR-associated factor-1. Moreover, during the early stage of chronic infection, lung dendritic cells from immune mice expressed higher levels of MHC class II and CD40 molecules than similar cells from control mice. These results indicate that while a chronic disease state is the eventual outcome in both control and immune mice infected with M. tuberculosis by aerosol exposure, immune mice develop a protective granulomatous lesion by increasing macrophage numbers and reduced expression of protective and inflammatory cytokines.
Cross-reaction of Anti-human CD Monoclonal Antibodies on Guinea Pig Cells: a Summary of the Guinea Pig Section of the HLDA8 Animal Homologues Data
A panel of 377 commercially available monoclonal antibodies (mAbs) specific for a total of 144 CD antigens was submitted to the animal homologue section of the Eighth International Workshop on Human Leukocyte Differentiation Antigens (HLDA8, Adelaide, Australia) for cross-reactivity studies in a range of vertebrate species. Each of the mAbs in this study was screened for positive reactivity with guinea pig splenocytes by flow cytometry. In the first phase of this study 36 of the total 367 mAbs (9.81%) cross-reacted with splenocyte surface molecules. The majority (26 of 36) of these cross-reactive mAbs were analysed further to confirm appropriate cell subset expression by two-color immunofluorescence. Our results indicate that 15 anti-human CD9, CD10, CD14, CD20 (two clones), CD22, CD25, CD29 (two clones), CD32, CD47 (two clones), CD49d, CD49e, and CD86 mAbs exhibit clear cross-reactivity with guinea pig splenocytes. These mAb can potentially be added to the limited repertoire of reagents available for studies in this model system. This data clearly indicates that mouse anti-human CD mAb guinea pig cross-reactions have been defined and that an aim of this HLDA8 section has been fulfilled, i.e., to identify mAbs which recognize conserved, species-independent CD epitopes. These results will contribute to the availability of mAbs and tools in veterinary medicine and immunology.
XCL1 (lymphotactin) Chemokine Produced by Activated CD8 T Cells During the Chronic Stage of Infection with Mycobacterium Tuberculosis Negatively Affects Production of IFN-gamma by CD4 T Cells and Participates in Granuloma Stability
CD8 T cell immune responses are known not to be essential during the initial stages of infection with Mycobacterium tuberculosis (Mtb), but their presence becomes important as the chronic infection ensues. The basis of this is still not clear. In previous studies, we showed that CD8 T cells have a distinctive positioning in the architecture of the granuloma lesion, with further changes throughout the course of the chronic infection. We have also hypothesized that further movement of lymphocytes once they are within the lung lesions could be associated with the levels of expression of the chemokine XCL1 (lymphotactin). XCL1 is produced mainly by activated CD8 T cells, and its chemotactic activity seems primarily controlling movement of CD4 and CD8 T cells. In this study, using a murine low-dose aerosol infection model coupled with antibody depletion of T cell subsets, we investigated the role of CD8 T cells in the control of the bacterial growth and in the pathogenesis of the disease in mice at early, mid, or late stages of the chronic disease state. Additionally, we also describe for the first time that during Mtb infection, activated CD8 T cells in the lungs produce XCL1 and that this chemokine is capable of controlling IFN-gamma production by CD4 T cells.
Animal Models of M. Tuberculosis Infection
Animal models of tuberculosis infection continue to provide useful information about the nature of the disease process, including specific information about the immune response to the infection and the disease pathology. In addition, standardized animal models are now used extensively to test the capacity of new vaccines to inhibit the course of the infection, as well as test the capacity of new drugs to sterilize the infection. This unit describes some basic protocols illustrating established protocols for infecting animals with tuberculosis, subsequent processes for analysis, and various aspects of biosafety that must be observed. Using these protocols the course of infection, the basic immune response, and the extent of lung pathology can be determined in mouse and guinea pig models of experimental tuberculosis.
Relative Levels of M-CSF and GM-CSF Influence the Specific Generation of Macrophage Populations During Infection with Mycobacterium Tuberculosis
Members of the CSF cytokine family play important roles in macrophage recruitment and activation. However, the role of M-CSF in pulmonary infection with Mycobacterium tuberculosis is not clear. In this study, we show the lungs of mice infected with M. tuberculosis displayed a progressive decrease in M-CSF in contrast to increasing levels of GM-CSF. Restoring pulmonary M-CSF levels during infection resulted in a significant decrease in the presence of foamy macrophages and increased expression of CCR7 and MHC class II, specifically on alveolar macrophages. In response to M-CSF, alveolar macrophages also increased their T cell-stimulating capacity and expression of DEC-205. These studies show that the levels of expression of M-CSF and GM-CSF participate in the progression of macrophages into foamy cells and that these cytokines are important factors in the differentiation and regulation of expression of dendritic cell-associated markers on alveolar macrophages. In addition, these studies demonstrate that M-CSF may have a role in the adaptive immune response to infection with M. tuberculosis.
Interleukin-10 Promotes Mycobacterium Tuberculosis Disease Progression in CBA/J Mice
IL-10 is a potent immunomodulatory cytokine that affects innate and acquired immune responses. The immunological consequences of IL-10 production during pulmonary tuberculosis (TB) are currently unknown, although IL-10 has been implicated in reactivation TB in humans and with TB disease in mice. Using Mycobacterium tuberculosis-susceptible CBA/J mice, we show that blocking the action of IL-10 in vivo during chronic infection stabilized the pulmonary bacterial load and improved survival. Furthermore, this beneficial outcome was highly associated with the recruitment of T cells to the lungs and enhanced T cell IFN-gamma production. Our results indicate that IL-10 promotes TB disease progression. These findings have important diagnostic and/or therapeutic implications for the prevention of reactivation TB in humans.
Intrapulmonary Delivery of XCL1-targeting Small Interfering RNA in Mice Chronically Infected with Mycobacterium Tuberculosis
Mice infected for 60 days with Mycobacterium tuberculosis were treated with aerosolized XCL1-targeting small interfering RNA (siRNA) to induce local and transient suppression of XCL1/lymphotactin (an important chemokine in tuberculoid granuloma formation). The local pulmonary siRNA therapy resulted in a 50% decrease in the total amount of xcl1 gene transcripts at 3 days, and 40 to 50% protein suppression 3 and 5 days after treatment. Reduced XCL1 expression in the lungs was associated with decreased numbers of T lymphocytes, reduction in the IFN-gamma response, disorganized granulomatous lesions, and higher fibrosis when compared with control mice treated with either PBS or nontargeting siRNA. This indicates that a transient but strong modulation of the production of XCL1 in the lungs has a significant effect on the influx of IFN-gamma-secreting T cells, as well as local pathology, but without significantly altering containment of the infection.
Lack of IL-10 Alters Inflammatory and Immune Responses During Pulmonary Mycobacterium Tuberculosis Infection
Failure of mice to produce IL-10 has no effect on the bacterial burden of Mycobacterium tuberculosis infection in the lungs over the first 4-5 months of the disease. We show here that after 185 days of the infection, IL-10 gene disrupted (IL-10 KO) mice showed evidence of bacterial regrowth, began to show signs of wasting, and were moribund. We assessed the status of the acquired immune response and compared the lung lymphocyte cell populations, as well as the expression of Th1 (IL-12 and IFNgamma) and immunosuppressive (TGFbeta) cytokines, between IL-10 KO and wild type mice. The results demonstrated that at 60 days of the infection in the absence of IL-10 there was an increased expression of Th1 type immunity and an overall lack of control of the inflammatory responses. After 185 days of the infection, in the absence of IL-10 there was excessive pulmonary inflammation and increased expression of inflammatory cytokine TNFalpha. These results imply therefore that IL-10 plays a central role in dampening of the Th1 response and protection against chronic lung inflammation in the M. tuberculosis infected lung, and the complete removal of this regulatory component eventually leads to disease progression.
Localized Immunosuppressive Environment in the Foreign Body Response to Implanted Biomaterials
The implantation of synthetic biomaterials initiates the foreign body response (FBR), which is characterized by macrophage infiltration, foreign body giant cell formation, and fibrotic encapsulation of the implant. The FBR is orchestrated by a complex network of immune modulators, including diverse cell types, soluble mediators, and unique cell surface interactions. The specific tissue locations, expression patterns, and spatial distribution of these immune modulators around the site of implantation are not clear. This study describes a model for studying the FBR in vivo and specifically evaluates the spatial relationship of immune modulators. We modified a biomaterials implantation in vivo model that allowed for cross-sectional in situ analysis of the FBR. Immunohistochemical techniques were used to determine the localization of soluble mediators, ie, interleukin (IL)-4, IL-13, IL-10, IL-6, transforming growth factor-beta, tumor necrosis factor-alpha, interferon-gamma, and MCP-1; specific cell types, ie, macrophages, neutrophils, fibroblasts, and lymphocytes; and cell surface markers, ie, F4/80, CD11b, CD11c, and Ly-6C, at early, middle, and late stages of the FBR in subcutaneous implant sites. The cytokines IL-4, IL-13, IL-10, and transforming growth factor-beta were localized to implant-adherent cells that included macrophages and foreign body giant cells. A better understanding of the FBR in vivo will allow the development of novel strategies to enhance biomaterial implant design to achieve better performance and safety of biomedical devices at the site of implant.
Phenotypic Non-equivalence of Murine (monocyte-) Macrophage Cells in Biomaterial and Inflammatory Models
Cells of the mononuclear phagocytic system including monocytes and macrophages (e.g., pooled human monocytes, bone marrow-derived macrophages, etc.) are often employed for in vitro assessment of novel biomaterials and to assay anti-inflammatory drug activity. In this context, numerous macrophage cells are treated interchangeably in the literature despite a lack of demonstrated equivalence among immortalized cell lines and further, between cell lines and primary-derived macrophages of different species. Three murine (monocyte-) macrophage cell lines (IC-21, J774A.1, and RAW 264.7), commonly utilizedin biomaterial and pharmaceutical screening research, have been compared with primary-derived murine bone marrow macrophages. Significant differences were discovered in the expression of cell surface proteins requisite for cell adhesion and activation among cell lines and primary-derived cells as well as between the different cell lines. Results demonstrate activation but with reduced cytokine expression to chemical stimulus (lipopolysaccharide) by cell lines compared with that of primary-derived macrophages. Limited correlation between cultured primary and immortalized cells in cytokine production, phenotype and intrinsic activation states has relevance to fidelity for in vitro testing. These differences warrant justification for selection of various cell lines for specific assay purposes, and merit caution if comparisons to primary cell types (i.e., for biocompatibility) are required.
Immune Response to Mycobacterium Tuberculosis and Identification of Molecular Markers of Disease
The complex molecular events that occur within the host during the establishment of a Mycobacterium tuberculosis infection are poorly defined, thus preventing identification of predictive markers of disease progression and state. To identify such molecular markers during M. tuberculosis infection, global changes in transcriptional response in the host were assessed using mouse whole genome arrays. Bacterial load in the lungs, the lesions associated with infection, and gene expression profiling was performed by comparing normal lung tissue to lungs from mice collected at 20, 40, and 100 days after aerosol infection with the H37Rv strain of M. tuberculosis. Quantitative, whole lung gene expression identified signature profiles defining different signaling pathways and immunological responses characteristic of disease progression. This includes genes representing members of the interferon-associated gene families, chemokines and cytokines, MHC, and NOS2, as well as an array of cell surface markers associated with the activation of T cells, macrophages, and dendritic cells that participate in immunity to M. tuberculosis infection. More importantly, several gene transcripts encoding proteins that were not previously associated with the host response to M. tuberculosis infection, and unique molecular markers associated with disease progression and state, were identified.
Differential Polarization of Alveolar Macrophages and Bone Marrow-derived Monocytes Following Chemically and Pathogen-induced Chronic Lung Inflammation
Alveolar macrophages and BDMCs undergo sequential biochemical changes during the chronic inflammatory response to chemically induced lung carcinogenesis in mice. Herein, we examine two chronic lung inflammation models-repeated exposure to BHT and infection with Mycobacterium tuberculosis-to establish whether similar macrophage phenotype changes occur in non-neoplastic pulmonary disease. Exposure to BHT or M. tuberculosis results in pulmonary inflammation characterized by an influx of macrophages, followed by systemic effects on the BM and other organs. In both models, pulmonary IFN-gamma and IL-4 production coincided with altered polarization of alveolar macrophages. Soon after BHT administration or M. tuberculosis infection, IFN-gamma content in BALF increased, and BAL macrophages became classically (M1) polarized, as characterized by increased expression of iNOS. As inflammation progressed in both models, the amount of BALF IFN-gamma content and BAL macrophage iNOS expression decreased, and BALF IL-4 content and macrophage arginase I expression rose, indicating alternative/M2 polarization. Macrophages present in M. tuberculosis-induced granulomas remained M1-polarized, implying that these two pulmonary macrophage populations, alveolar and granuloma-associated, are exposed to different activating cytokines. BDMCs from BHT-treated mice displayed polarization profiles similar to alveolar macrophages, but BDMCs in M. tuberculosis-infected mice did not become polarized. Thus, only alveolar macrophages in these two models of chronic lung disease exhibit a similar progression of polarization changes; polarization of BDMCs was specific to BHT-induced pulmonary inflammation, and polarization of granuloma macrophages was specific to the M. tuberculosis infection.
Multiple M. Tuberculosis Phenotypes in Mouse and Guinea Pig Lung Tissue Revealed by a Dual-staining Approach
A unique hallmark of tuberculosis is the granulomatous lesions formed in the lung. Granulomas can be heterogeneous in nature and can develop a necrotic, hypoxic core which is surrounded by an acellular, fibrotic rim. Studying bacilli in this in vivo microenvironment is problematic as Mycobacterium tuberculosis can change its phenotype and also become acid-fast negative. Under in vitro models of differing environments, M. tuberculosis alters its metabolism, transcriptional profile and rate of replication. In this study, we investigated whether these phenotypic adaptations of M. tuberculosis are unique for certain environmental conditions and if they could therefore be used as differential markers. Bacilli were studied using fluorescent acid-fast auramine-rhodamine targeting the mycolic acid containing cell wall, and immunofluorescence targeting bacterial proteins using an anti-M. tuberculosis whole cell lysate polyclonal antibody. These techniques were combined and simultaneously applied to M. tuberculosis in vitro culture samples and to lung sections of M. tuberculosis infected mice and guinea pigs. Two phenotypically different subpopulations of M. tuberculosis were found in stationary culture whilst three subpopulations were found in hypoxic culture and in lung sections. Bacilli were either exclusively acid-fast positive, exclusively immunofluorescent positive or acid-fast and immunofluorescent positive. These results suggest that M. tuberculosis exists as multiple populations in most conditions, even within seemingly a single microenvironment. This is relevant information for approaches that study bacillary characteristics in pooled samples (using lipidomics and proteomics) as well as in M. tuberculosis drug development.
Optimization of Inhaled Therapies for Tuberculosis: the Role of Macrophages and Dendritic Cells
Inhaled therapies in the form of drugs or vaccines for tuberculosis treatment were reported about a decade ago. Experts around the world met to discuss the scientific progress in inhaled therapies at the international symposium "Optimization of inhaled Tuberculosis therapies and implications for host-pathogen interactions" held in New Delhi, India on November 3-5, 2009. The meeting was organized by the Central Drug Research Institute (CDRI) Lucknow, India. The lung is the main route for infection with Mycobacterium tuberculosis bacilli and the primary site of reactivation of latent disease. The only available vaccine BCG is relatively ineffective at preventing tuberculosis disease and current therapy requires prolonged treatment with drugs which results in low patient compliance. Consequently, there is a need to design new vaccines and therapies for this disease. Recently there has been increased interest in the development of inhaled formulations to deliver anti-mycobacterial drugs and vaccines directly to the lung and many of these therapies are designed to target lung macrophages and dendritic cells. However, the development of effective inhaled therapies requires an understanding of the unique function and immunosuppressive environment of the lung which is driven, in part, by alveolar macrophages and dendritic cells. In this review, we will discuss the role of alveolar macrophages and dendritic cells in the host immune response to M. tuberculosis infection and the ways in which inhaled therapies might enhance the anti-microbial response of phagocytes and boost pulmonary immunity.
Local Pulmonary Immunotherapy with SiRNA Targeting TGFβ1 Enhances Antimicrobial Capacity in Mycobacterium Tuberculosis Infected Mice
In this study we demonstrate that it is possible to shift the immune system during a chronic infection with Mycobacterium tuberculosis. TGFβ and IL10 cytokines inhibit the Th1 response during chronic pulmonary infection with M. tuberculosis. We show that intrapulmonary delivery of siRNA targeting TGFβ1 is able to reduce the pulmonary bacillary load in mice chronically infected with M. tuberculosis: an effect that appears to be partly dependent on IL10 expression. To demonstrate this, we induced gene silencing of tgfβ1 in the lungs of wild type and IL10 knockout mice using a non-invasive aerosolized intrapulmonary delivery of siRNA targeting TGFβ1. Five days after the last treatment with siRNA, the levels of tgfb1 transcripts and TGFβ1 protein were reduced when compared with control groups treated with RNase-free water or non-targeting siRNA. Mice treated with siRNA also had increased expression of the antimicrobial mediators (NO and iNOS) which effectively reduced the bacterial load by 0.17 and 0.47 log(10) in C57BL/6 and IL-10 KO mice respectively when compared with their respective control mice. More importantly, the bacterial load in siRNA treated IL-10 KO mice four weeks after the last treatment remained 0.32 log(10) lower than in control mice.
Glycolytic and Non-glycolytic Functions of Mycobacterium Tuberculosis Fructose-1,6-bisphosphate Aldolase, an Essential Enzyme Produced by Replicating and Non-replicating Bacilli
The search for antituberculosis drugs active against persistent bacilli has led to our interest in metallodependent class II fructose-1,6-bisphosphate aldolase (FBA-tb), a key enzyme of gluconeogenesis absent from mammalian cells. Knock-out experiments at the fba-tb locus indicated that this gene is required for the growth of Mycobacterium tuberculosis on gluconeogenetic substrates and in glucose-containing medium. Surface labeling and enzymatic activity measurements revealed that this enzyme was exported to the cell surface of M. tuberculosis and produced under various axenic growth conditions including oxygen depletion and hence by non-replicating bacilli. Importantly, FBA-tb was also produced in vivo in the lungs of infected guinea pigs and mice. FBA-tb bound human plasmin(ogen) and protected FBA-tb-bound plasmin from regulation by α(2)-antiplasmin, suggestive of an involvement of this enzyme in host/pathogen interactions. The crystal structures of FBA-tb in the native form and in complex with a hydroxamate substrate analog were determined to 2.35- and 1.9-Å resolution, respectively. Whereas inhibitor attachment had no effect on the plasminogen binding activity of FBA-tb, it competed with the natural substrate of the enzyme, fructose 1,6-bisphosphate, and substantiated a previously unknown reaction mechanism associated with metallodependent aldolases involving recruitment of the catalytic zinc ion by the substrate upon active site binding. Altogether, our results highlight the potential of FBA-tb as a novel therapeutic target against both replicating and non-replicating bacilli.
