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

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

The effect of LAG-3 silencing on cytokine production in co-cultures supplemented with CD4⁺ T-cells isolated the lung of <i>Mtb</i>-infected rhesus macaques.

<p>(<b>A</b>) Concentrations of IFN-γ, (<b>B</b>) IL-6 and (<b>C</b>) CXCL11 in <i>Mtb</i>-infected macrophage culture, untreated co-culture, and LAG-3 silenced co-culture at 48h post-infection. (<b>D</b>) The presence of MIF in <i>Mtb</i>-infected macrophage culture, untreated co-culture, and LAG-3 silenced co-culture at 24h post-infection. All samples were measured in pg/ml. Statistical significance was determined using a one-way ANOVA in Prism 6, using multiple comparisons to compare each mean, with the Tukey multiple comparison test; the mean and SEM are represented by horizontal bars. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.</p

Image_4_Antibody-mediated depletion of select leukocyte subsets in blood and tissue of nonhuman primates.jpg

Understanding the immunological control of pathogens requires a detailed evaluation of the mechanistic contributions of individual cell types within the immune system. While knockout mouse models that lack certain cell types have been used to help define the role of those cells, the biological and physiological characteristics of mice do not necessarily recapitulate that of a human. To overcome some of these differences, studies often look towards nonhuman primates (NHPs) due to their close phylogenetic relationship to humans. To evaluate the immunological role of select cell types, the NHP model provides distinct advantages since NHP more closely mirror the disease manifestations and immunological characteristics of humans. However, many of the experimental manipulations routinely used in mice (e.g., gene knock-out) cannot be used with the NHP model. As an alternative, the in vivo infusion of monoclonal antibodies that target surface proteins on specific cells to either functionally inhibit or deplete cells can be a useful tool. Such depleting antibodies have been used in NHP studies to address immunological mechanisms of action. In these studies, the extent of depletion has generally been reported for blood, but not thoroughly assessed in tissues. Here, we evaluated four depleting regimens that primarily target T cells in NHP: anti-CD4, anti-CD8α, anti-CD8β, and immunotoxin-conjugated anti-CD3. We evaluated these treatments in healthy unvaccinated and IV BCG-vaccinated NHP to measure the extent that vaccine-elicited T cells – which may be activated, increased in number, or resident in specific tissues – are depleted compared to resting populations in unvaccinated NHPs. We report quantitative measurements of in vivo depletion at multiple tissue sites providing insight into the range of cell types depleted by a given mAb. While we found substantial depletion of target cell types in blood and tissue of many animals, residual cells remained, often residing within tissue. Notably, we find that animal-to-animal variation is substantial and consequently studies that use these reagents should be powered accordingly.</p

Log₂ average fold change values of gene expression for select immune function genes amongst biological replicate samples from <i>Mtb</i>-infected macrophages (24h) as well as the two co-culture sets (<i>Mtb</i>-infected macrophages (24h) + infected lung CD4⁺ T-cells; <i>Mtb</i>-infected macrophages (24h) + infected lung CD4⁺ T-cells where LAG-3 expression was silenced by RNAi).

<p>Log₂ average fold change values of gene expression for select immune function genes amongst biological replicate samples from <i>Mtb</i>-infected macrophages (24h) as well as the two co-culture sets (<i>Mtb</i>-infected macrophages (24h) + infected lung CD4⁺ T-cells; <i>Mtb</i>-infected macrophages (24h) + infected lung CD4⁺ T-cells where LAG-3 expression was silenced by RNAi).</p

The effect of LAG-3 silencing on cytokine production in co-cultures supplemented with CD4⁺ T-cells from the blood of <i>Mtb</i>-infected rhesus macaques.

<p>(<b>A</b>) Production of IFN-γ in <i>Mtb</i>-infected macrophage culture, untreated co-cultures, and LAG-3 silenced co-cultures at 0, 24, and 48h post-infection. (<b>B</b>) The presence of IL-6 and (<b>C</b>) CXCL11 in <i>Mtb</i>-infected macrophage culture, untreated co-culture, and LAG-3 silenced co-culture at 48h post-infection. (<b>D</b>) Levels of MIF in <i>Mtb</i>-infected macrophage culture, untreated co-culture, and LAG-3 silenced co-culture supernatant at 24h post-infection. All samples were measured in pg/ml. Statistical significance was determined using a one-way ANOVA in Prism 6, using multiple comparisons to compare each mean, with the Tukey multiple comparison test; the mean and SEM are represented by horizontal bars. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.</p