Mercedes Gonzalez-Juarrero

In Vivo IL-10 Production Reactivates Chronic Pulmonary Tuberculosis in C57BL/6 Mice

The production of immunosuppressive cytokines, such as IL-10 and TGF-β, 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-γ 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.

Temporal and Spatial Arrangement of Lymphocytes within Lung Granulomas Induced by Aerosol Infection with Mycobacterium tuberculosis

ABSTRACT The progression of the immune response in the lungs after aerosol infection with Mycobacterium tuberculosis is a complex cellular event dominated by macrophages and lymphocytes. Although the phenotype of lymphocytes participating in this response is becoming increasingly well characterized, the dynamic influx of these cells during the infection and their spatial arrangements within the lung tissue are still poorly understood. This study shows that in the first month after aerosol infection with M. tuberculosis there was a steady increase in the percentages of total CD3+, CD3+ CD4+ and CD3+ CD8+cells, with consistently larger numbers of CD3+CD4+ cells than of CD3+ CD8+ cells. As granuloma formation continued, the granuloma was found to consist of macrophages, CD4, and CD8 T cells, as well as a smaller number of B cells. Whereas CD4 T cells formed organized aggregates, CD8 T cells were fewer and more scattered and tended to be more prominent toward the periphery of the granulomas. The possible ramifications of the juxtapositions of these two major T-cell subsets are discussed.

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, CD11bhigh/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.

Characterization of murine lung dendritic cells infected with Mycobacterium tuberculosis.

ABSTRACT Lung dendritic cells were identified by immunohistochemistry in lung tissue sections from C57BL/6 mice. Following isolation from the lungs using CD11c magnetic beads, the flow cytometric analysis of I-Ab+ and CD11c+ cells indicated a mixed population of dendritic cells at different stages of maturation, with most expressing an immature phenotype. When cultured for 7 days with recombinant murine granulocyte-macrophage colony-stimulating factor, 99% of cells were CD11c+ and had a morphology typical of immature dendritic cells. These cells were negative for CD34, CD14, and CD8α antigens but expressed low levels of the myeloid marker F4/80 and moderate levels of MAC3. All expressed high levels of CD11a (LFA-1), CD11b (Mac1), and CD54 antigens, with low levels of class II major histocompatibility complex. Most cells expressed CD80 but only a small percentage of cells were positive for CD40 and CD86. Both overnight and 7-day cultures of lung dendritic cells were able to phagocytose Mycobacterium tuberculosis, and this was associated with the production of interleukin-12 and stimulation of both naı̈ve and immune T cells to produce gamma interferon.

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-γ 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.

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 α (TNF‐α) nor the chemokines, regulated on activation, normal T expressed and secreted (RANTES), macrophage‐inflammatory protein‐1β (MIP‐1β), MIP‐1α, 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‐α and interferon‐γ 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‐1β. 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.

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 α. A key difference, however, was the degree of cell cytotoxicity observed with the more virulent strains ater 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.

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.

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.

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+CD11chighMHC class IIhigh, and CD40high, 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.