A Novel <i>In Vitro</i> Human Granuloma Model of Sarcoidosis and Latent Tuberculosis Infection

Crouser, Elliott D.; White, Peter; Caceres, Evelyn Guirado; Julián, M; Papp, Audrey C.; Locke, Landon W.; Sadée, Wolfgang; Schlesinger, Larry S.

doi:10.1165/rcmb.2016-0321oc

Cited by 57 publications

(85 citation statements)

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“…Newly developed in vitro human granuloma models could help further establish the mechanisms of macrophage polarization during early granuloma formation, maturation, and disintegration of the structures during reactivation of the disease . Confirming similar observations, PBMCs isolated from cohorts of bacillus Calmette‐Guérin (BCG)‐vaccinated individuals, and individuals with LTBI, patients with active TB, and people from contact investigations following recent TB exposure, showed a variable capacity to control mycobacterial growth .…”

Section: Macrophage Heterogeneity and M Tuberculosis Infectionmentioning

confidence: 58%

Macrophage heterogeneity and plasticity in tuberculosis

Khan

Singh

Hunter

et al. 2019

Journal of Leukocyte Biology

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Macrophages are the primary host cells for Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), during its intracellular survival in humans. The pathogen has a remarkable capacity to survive within the hostile environment of macrophages. However, primary infection does not result in active TB disease in most individuals. The majority of individuals remain latently infected, wherein the bacteria are held in check by the host immune response. Nevertheless, such individuals can develop active TB later upon the decline in their immune status. In contrast, in a small fraction of infected individuals, the host immune response fails to control the growth of M. tuberculosis bacilli, and granulomatous TB develops progressively. Elucidating the molecular and phenotypic events that govern the outcome of the infection within macrophages is fundamental to understanding the key features of these cells that could be equally critical in infection control. The molecular details of the M. tuberculosis‐macrophage interaction continue to be discerned, and emerging evidence suggests that macrophage population that participate in infection is heterogeneous. While the local environment and developmental origin could influence the phenotypic heterogeneity and functional plasticity of macrophages, M. tuberculosis has also been demonstrated to modulate the polarization of macrophages. In this review, we draw on work investigating specialized macrophage populations and their interactions with M. tuberculosis with respect to pathogenesis and specific immune responses. Understanding the mechanisms that control the repertoire of macrophage phenotypes and behaviors during infection may provide prospects for novel TB control strategies through modulation of immunobiological functions of macrophages.

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Section: Macrophage Heterogeneity and M Tuberculosis Infectionmentioning

confidence: 58%

Macrophage heterogeneity and plasticity in tuberculosis

Khan

Singh

Hunter

et al. 2019

Journal of Leukocyte Biology

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“…To validate the successful establishment of a microscale in vitro granuloma model within our platform, we used three separate previously reported readouts: 1) aggregate formation, 2) encapsulation of the mycobacterium within host immune cells, and 3) soluble factor analysis. (Birkness et al 2007; Kapoor et al 2013; Crouser et al 2017; Tezera et al 2017) After initiating infection by mixing MDMs and BCG into the ECM (collagen I) and seeding it into the wells, we consistently observed aggregate formation in the granuloma layer containing BCG when wells were fixed and imaged on Day 4 post infection (p.i.) (Figure 2).…”

Section: Resultsmentioning

confidence: 87%

“…(Yu et al 2019) The first layer, herein called the granuloma layer, consists of an infection model of monocyte-derived macrophages (MDMs) and a model mycobacterium strain, Mycobacterium bovis Bacillus Calmette-Guérin (BCG), suspended in a 3D extracellular matrix (ECM) plug to mimic some aspects of in vivo granuloma behavior (e.g., pathogen encapsulation, soluble factor secretion, aggregate formation) previously observed in other in vitro granuloma models. (Birkness et al 2007; Kapoor et al 2013; Crouser et al 2017) The second layer, herein called the endothelial layer, consists of an in vitro angiogenesis model in which endothelial cells are cultured on a hydrogel plug. By placing the primary human endothelial cells on a separate Stacks layer, our model can be used to examine the induction of angiogenic processes around the granuloma layer and how those angiogenic processes are affected by the soluble factor signaling profile of the granuloma layer.…”

Section: Resultsmentioning

confidence: 99%

“…Previous in vitro granuloma models successfully recapitulated important components of granulomatous infections including leukocyte recruitment and signaling, establishment of dormancy and resuscitation, and genetic diversity at scales ranging from 12 well plates to 96 well plates. (Birkness et al 2007; Crouser et al 2017; Kapoor et al 2013) Our model adds to these existing techniques through miniaturization and introduction of modularity to enable examination of the signaling phenomena between a mycobacterial infection and its surrounding microenvironment. We reduce the volume of our cultures (4 μL/well) more than 10-fold from previous examples (50-500 μL/well) to decrease cell and reagent usage in our model; further, we mix BCG and MDMs together in a 3D extracellular matrix (ECM), without pre-infecting the MDMs, to establish the infection in our granuloma layer after a minimum of 24 hours (Figure 1).…”

Section: Resultsmentioning

confidence: 99%

“…(Peyron et al 2008; Deb et al 2009; Birkness et al 2007; Kapoor et al 2013; Elkington et al 2019) These models, which often rely on infection of patient-derived peripheral blood mononuclear cells (PBMCs) with mycobacterial strains, have successfully mimicked granuloma formation and behavior through soluble factor signaling between immune cells (Birkness et al 2007; Puissegur et al 2004; Crouser et al 2017), Mtb reactivation(Kapoor et al 2013), and PBMC differentiation. (Peyron et al 2008) However, many of these in vitro models consist of granulomas grown inside of well plates(Peyron et al 2008; Birkness et al 2007; Kapoor et al 2013; Puissegur et al 2004; Crouser et al 2017), limiting the ability of the researchers to easily manipulate the microenvironment of the granulomas and increase the complexity of their granuloma models through multiculture and introduction of key components of the microenvironment on demand. Recently, more complex, biomimetic models have been developed that have successfully recapitulated important biological phenomena(Venkata Ramanarao Parasa et al 2014; Venkata R. Parasa et al 2017) and examined novel therapeutic approaches to combat TB infection (Tezera et al 2017; Bielecka et al 2017), while simultaneously demonstrating innovative and tractable platforms.…”

Section: Introductionmentioning

confidence: 99%

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A modular microscale granuloma model for immune-microenvironment signaling studiesin vitro

Berry

Gower

et al. 2020

Preprint

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Tuberculosis (TB) is one of the most potent infectious diseases in the world, causing more deaths than any other single infectious agent. TB infection is caused by inhalation of Mycobacterium tuberculosis (Mtb) and subsequent phagocytosis and migration into the lung tissue by innate immune cells (e.g., alveolar macrophages, neutrophils, dendritic cells), resulting in the formation of a fused mass of immune cells known as the granuloma. Considered the pathological hallmark of TB, the granuloma is a complex microenvironment that is crucial for pathogen containment as well as pathogen survival. Disruption of the delicate granuloma microenvironment via numerous stimuli, such as variations in cytokine secretions, nutrient availability, and the makeup of immune cell population, can lead to an active infection. Herein, we present a novel in vitro model to examine the soluble factor signaling between a mycobacterial infection and its surrounding environment. Adapting a newly developed suspended microfluidic platform, known as Stacks, we established a modular microscale infection model containing human immune cells and a model mycobacterial strain that can easily integrate with different microenvironmental cues through simple spatial and temporal “stacking” of each module of the platform. We validate the establishment of suspended microscale (4 μL) infection cultures that secrete increased levels of proinflammatory factors IL-6, VEGF, and TNFα upon infection and form 3D aggregates (granuloma model) encapsulating the mycobacteria. As a proof of concept to demonstrate the capability of our platform to examine soluble factor signaling, we cocultured an in vitro angiogenesis model with the granuloma model and quantified morphology changes in endothelial structures as a result of culture conditions (P < 0.05 when comparing infected vs. uninfected coculture systems). We envision our modular in vitro granuloma model can be further expanded and adapted for studies focusing on the complex interplay between granulomatous structures and their surrounding microenvironment, as well as a complementary tool to augment in vivo signaling and mechanistic studies.

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Cell Lines as In Vitro Model for Studying Microbial Pathogenesis

Chattopadhyay

2020

Model Organisms for Microbial Pathogenesis, Biofilm Formation and Antimicrobial Drug Discovery

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A Novel In Vitro Human Granuloma Model of Sarcoidosis and Latent Tuberculosis Infection

Cited by 57 publications

References 54 publications

Macrophage heterogeneity and plasticity in tuberculosis

Macrophage heterogeneity and plasticity in tuberculosis

A modular microscale granuloma model for immune-microenvironment signaling studiesin vitro

Cell Lines as In Vitro Model for Studying Microbial Pathogenesis

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