The current state of animal models and genomic approaches towards identifying and validating molecular determinants of Mycobacterium tuberculosis infection and tuberculosis disease

Animal models are important in understanding both the pathogenesis of and immunity to tuberculosis (TB). Unfortunately, we are beginning to understand that no animal model perfectly recapitulates the human TB syndrome, which encompasses numerous different stages. Furthermore, Mycobacterium tuberculosis infection is a very heterogeneous event at both the levels of pathogenesis and immunity. This review seeks to establish the current understanding of TB pathogenesis and immunity, as validated in the animal models of TB in active use today. We especially focus on the use of modern genomic approaches in these models to determine the mechanism and the role of specific molecular pathways. Animal models have significantly enhanced our understanding of TB. Incorporation of contemporary technologies such as single cell transcriptomics, high-parameter flow cytometric immune profiling, proteomics, proteomic flow cytometry and immunocytometry into the animal models in use will further enhance our understanding of TB and facilitate the development of treatment and vaccination strategies

Antiretroviral therapy timing impacts latent tuberculosis infection reactivation in a Mycobacterium tuberculosis/SIV coinfection model

Studies using the nonhuman primate model of Mycobacterium tuberculosis/simian immunodeficiency virus coinfection have revealed protective CD4+ T cell-independent immune responses that suppress latent tuberculosis infection (LTBI) reactivation. In particular, chronic immune activation rather than the mere depletion of CD4+ T cells correlates with reactivation due to SIV coinfection. Here, we administered combinatorial antiretroviral therapy (cART) 2 weeks after SIV coinfection to study whether restoration of CD4+ T cell immunity occurred more broadly, and whether this prevented reactivation of LTBI compared to cART initiated 4 weeks after SIV. Earlier initiation of cART enhanced survival, led to better control of viral replication, and reduced immune activation in the periphery and lung vasculature, thereby reducing the rate of SIV-induced reactivation. We observed robust CD8+ T effector memory responses and significantly reduced macrophage turnover in the lung tissue. However, skewed CD4+ T effector memory responses persisted and new TB lesions formed after SIV coinfection. Thus, reactivation of LTBI is governed by very early events of SIV infection. Timing of cART is critical in mitigating chronic immune activation. The potential novelty of these findings mainly relates to the development of a robust animal model of human M. tuberculosis/HIV coinfection that allows the testing of underlying mechanisms

Education for the Use of Emergency Egress Lifts

The scope of this project was to develop an educational seminar series that effectively conveys information on issues surrounding the use of evacuation lifts. Evacuation lifts will not only improve evacuation times for high-rise buildings, but also provide equitable egress for persons with disabilities. Key informants and stakeholders were interviewed and surveyed to determine the concerns and misconceptions that they had regarding the use of evacuation lifts. The seminar series focused on considerations that address evacuation planning, building design and management, and maintenance issues. The goal of our project is to educate members of the design team on issues surrounding the use of lifts to ensure the safe and effective use of evacuation lifts in the future

LAG-3 potentiates the survival of Mycobacterium tuberculosis in host phagocytes by modulating mitochondrial signaling in an in-vitro granuloma model.

CD4+ T-cell mediated Th1 immune responses are critical for immunity to TB. The immunomodulatory protein, lymphocyte activation gene-3 (LAG-3) decreases Th1-type immune responses in T-cells. LAG-3 expression is significantly induced in the lungs of macaques with active TB and correlates with increased bacterial burden. Overproduction of LAG-3 can greatly diminish responses and could lead to uncontrolled Mtb replication. To assess the effect of LAG-3 on the progression of Mtb infection, we developed a co-culture system wherein blood-derived macrophages are infected with Mtb and supplemented with macaque blood or lung derived CD4+ T-cells. Silencing LAG-3 signaling in macaque lung CD4+ T-cells enhanced killing of Mtb in co-cultures, accompanied by reduced mitochondrial electron transport and increased IFN-γ expression. Thus, LAG-3 may modulate adaptive immunity to Mtb infection by interfering with the mitochondrial apoptosis pathway. Better understanding this pathway could allow us to circumvent immune features that promote disease

Control of Mycobacterium tuberculosis Infection in Lungs is Associated with Recruitment of Antigen-Specific Th1 and Th17 cells Co-expressing CXCR3 and CCR6

AbstractMycobacterium tuberculosis (Mtb)-specific T cell responses associated with immune control during asymptomatic latent tuberculosis infection (LTBI) remain poorly understood. Using a non-human primate (NHP) aerosol model, we studied the kinetics, phenotypes and functions of Mtb antigen-specific T cells in peripheral and lung compartments of Mtb-infected asymptomatic rhesus macaques by longitudinally sampling blood and bronchoalveolar lavage (BAL), for up to 24 weeks post-infection. We found significantly higher frequencies of Mtb-specific effector and memory CD4 and CD8 T cells producing IFN-γ in the airways compared to peripheral blood, which were maintained throughout the study period. Moreover, Mtb-specific IL-17+ and IL-17/IFN-γ double-positive T cells were present in the airways but were largely absent in the periphery, suggesting that balanced mucosal Th1/Th17 responses are associated with LTBI. The majority of Mtb-specific CD4 T cells that homed to the airways expressed the chemokine receptor CXCR3 and co-expressed CCR6. Notably, CXCR3+CD4+ cells were found in granulomatous and non-granulomatous regions of the lung and inversely correlated with Mtb burden. Our findings provide novel insights into antigen-specific T cell responses associated with asymptomatic Mtb infection that are relevant for developing better strategies to control TB.</jats:p

The role of lymphocyte subsets in preventing tuberculosis following intravenous vaccination with BCG

Abstract Background We previously demonstrated in non-human primates (NHP) that intravenous (IV) administration of BCG induces substantial T cell responses and often sterilizing immunity against Mycobacterium tuberculosis (Mtb) infection. Here, we investigated the role of T cell populations in this unmatched response in the context of necessary immune mechanisms for protection against tuberculosis (TB). Methods Beginning 20 weeks after vaccination and through the end of the study, rhesus macaques were infused with antibodies to deplete CD4, CD8α, or CD8β expressing T cells, or with control IgG/saline. NHPs inoculated with ~15 CFU Mtb Erdman via bronchoscope were monitored for 8 weeks. Granulomas, areas of disease, lung tissue and thoracic lymph nodes (LNs) were harvested at necropsy using FDG PET CT as a map to identify inflamed lesions. Immune responses were analyzed by spectral flow cytometry and homogenates were plated for colony forming unit (CFU) quantification. Results Successful depletion of expected cell types was seen in blood, airways, LNs and lung tissue. Depletion of CD4 T cells ablated IV BCG mediated protection in all NHPs. CD8α and CD8β depleted NHPs retained varying levels of protection, leading to mixed outcomes by bacterial burden. Preliminary results indicate the loss of protection following CD8α (both innate and adaptive cells) and CD8β (mainly adaptive cells) depletion is similar, suggesting a key role for conventional CD8 T cells in vaccination. Conclusions Our results provide critical insight regarding roles played by CD4 and CD8 T cell populations in terms of defining desired responses for vaccination against progressive TB. Functionality of these cells in immunized NHPs will clarify the purposes of T cell subsets. Supported by grants from Bill and Melinda Gates Foundation and NIH NIAID (75N93019C00071) </jats:sec

LAG-3 silencing and its effect on CD4⁺ T cells within the <i>Mtb</i>-infected co-culture.

<p>This data illustrates the mean frequency of CD4⁺ T cells positive for LAG-3 (<b>A</b>, <b>B</b>), IFN-γ (<b>C</b>, <b>D</b>), IL-10 (<b>E</b>, <b>F</b>), and Treg frequency (<b>G</b>,<b>H</b>) over the course of 96h in the <i>Mtb</i>-infected macrophage co-culture. In all images, gray squares indicate the <i>Mtb</i>-infected co-culture, where CD4⁺ T cell were untreated, and the black circles represent the <i>Mtb</i>-infected co-culture, where CD4⁺ T cell were silenced for LAG-3 before being added to the culture. In <b>A</b>, <b>C</b>, <b>E</b>, and <b>G</b> the CD4⁺ T cells used for co-culture were derived from blood of <i>Mtb</i>-infected rhesus macaques, whereas in <b>B</b>, <b>D</b>, <b>F</b>, and <b>H</b> the CD4⁺ T cells were isolated from lung of <i>Mtb</i>-infected rhesus macaques. Multiple t-tests corrected for multiple comparisons using the Holm-Sidak method were utilized to determine significance between time points. Horizontal bars represent the SEM. *<i>P</i> < 0.05.</p

Interaction between macaque macrophages and CD4⁺ T-cells during co-culture is shown using multilabel confocal microscopy.

<p>Immunostaining of cells positive for <i>Mtb</i> (red), macrophages (green), nuclei (blue) and a merge images (far right) (A). A representative image of macrophage (green):CD4⁺ T-cell (white) co-culture (lower left panel) and an infected macrophage with <i>Mtb</i> and associated with T-cell (lower right panel) (B); scale bars- 100 μm (A), 20 μm and 40 μm (B).</p