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

ABSTRACT: Many aspects of pathogenic granuloma formation are poorly understood, requiring new relevant laboratory models that represent the complexity (genetics and diversity) of human disease. To address this need, we developed an in vitro model of granuloma formation using human peripheral blood mononuclear cells (PBMCs) derived from patients with active sarcoidosis, latent tuberculosis (TB) infection (LTBI), or normal healthy control subjects. PBMCs were incubated for 7 days with uncoated polystyrene beads or beads coated with purified protein derivative (PPD) or human serum albumin. In response to PPD-coated beads, PBMCs from donors with sarcoidosis and LTBI formed robust multicellular aggregates resembling granulomas, displaying a typical T-helper cell type 1 immune response, as assessed by cytokine analyses. In contrast, minimal PBMC aggregation occurred when control PBMCs were incubated with PPD-coated beads, whereas the response to uncoated beads was negligible in all groups. Sarcoidosis PBMCs responded to human serum albumin-coated beads with modest cellular aggregation and inflammatory cytokine release. Whereas the granuloma-like aggregates formed in response to PPD-coated beads were similar for sarcoidosis and LTBI, molecular profiles differed significantly. mRNA expression patterns revealed distinct pathways engaged in early granuloma formation in sarcoidosis and LTBI, and they resemble molecular patterns reported in diseased human tissues. This novel in vitro human granuloma model is proposed as a tool to investigate mechanisms of early granuloma formation and for preclinical drug discovery research of human granulomatous disorders. Clinical trial registered with www.clinicaltrials.gov (NCT01857401).

SUBMITTER: Crouser ED

PROVIDER: S-EPMC5650085 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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

Crouser Elliott D ED White Peter P Caceres Evelyn Guirado EG Julian Mark W MW Papp Audrey C AC Locke Landon W LW Sadee Wolfgang W Schlesinger Larry S LS

American journal of respiratory cell and molecular biology 20171001 4

Many aspects of pathogenic granuloma formation are poorly understood, requiring new relevant laboratory models that represent the complexity (genetics and diversity) of human disease. To address this need, we developed an in vitro model of granuloma formation using human peripheral blood mononuclear cells (PBMCs) derived from patients with active sarcoidosis, latent tuberculosis (TB) infection (LTBI), or normal healthy control subjects. PBMCs were incubated for 7 days with uncoated polystyrene b ...[more]

PMID: 28598206

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Project description:UNLABELLED:Granulomas sit at the center of tuberculosis (TB) immunopathogenesis. Progress in biomarkers and treatment specific to the human granuloma environment is hindered by the lack of a relevant and tractable infection model that better accounts for the complexity of the host immune response as well as pathogen counterresponses that subvert host immunity in granulomas. Here we developed and characterized an in vitro granuloma model derived from human peripheral blood mononuclear cells (PBMCs) and autologous serum. Importantly, we interrogated this model for its ability to discriminate between host and bacterial determinants in individuals with and without latent TB infection (LTBI). By the use of this model, we provide the first evidence that granuloma formation, bacterial survival, lymphocyte proliferation, pro- and anti-inflammatory cytokines, and lipid body accumulation are significantly altered in LTBI individuals. Moreover, we show a specific transcriptional signature of Mycobacterium tuberculosis associated with survival within human granuloma structures depending on the host immune status. Our report provides fundamentally new information on how the human host immune status and bacterial transcriptional signature may dictate early granuloma formation and outcome and provides evidence for the validity of the granuloma model and its potential applications. IMPORTANCE:In 2012, approximately 1.3 million people died from tuberculosis (TB), the highest rate for any single bacterial pathogen. The long-term control of TB requires a better understanding of Mycobacterium tuberculosis pathogenesis in appropriate research models. Granulomas represent the characteristic host tissue response to TB, controlling the bacilli while concentrating the immune response to a limited area. However, complete eradication of bacteria does not occur, since M. tuberculosis has its own strategies to adapt and persist. Thus, the M. tuberculosis-containing granuloma represents a unique environment for dictating both the host immune response and the bacterial response. Here we developed and characterized an in vitro granuloma model derived from blood cells of individuals with latent TB infection that more accurately defines the human immune response and metabolic profiles of M. tuberculosis within this uniquely regulated immune environment. This model may also prove beneficial for understanding other granulomatous diseases.

Project description:The mechanisms underlying abnormal granuloma formation in patients with sarcoidosis are complex and remain poorly understood. A novel in vitro human granuloma model was used to determine the molecular mechanisms of granuloma genesis in patients with sarcoidosis in response to putative disease-causing mycobacterial antigens. Peripheral blood mononuclear cells (PBMCs) from patients with active sarcoidosis and from normal, disease-free control subjects were incubated for 7 days with purified protein derivative-coated polystyrene beads. Molecular responses, as reflected by differential expression of genes, extracellular cytokine patterns, and cell surface receptor expression, were analyzed. Unbiased systems biology approaches were used to identify signaling pathways engaged during granuloma formation. Model findings were compared with human lung and mediastinal lymph node gene expression profiles. Compared with identically treated PBMCs of control subjects (n = 5), purified protein derivative-treated sarcoidosis PBMCs (n = 6) were distinguished by the formation of cellular aggregates resembling granulomas. Ingenuity Pathway Analysis of differential expression gene patterns identified molecular pathways that are primarily regulated by IL-13, which promotes alternatively activated (M2) macrophage polarization. M2 polarization was further demonstrated by immunohistochemistry performed on the in vitro sarcoidosis granuloma-like structures. IL-13-regulated gene pathways were confirmed in human sarcoidosis lung and mediastinal lymph node tissues. The in vitro human sarcoidosis granuloma model provides novel insights into early granuloma formation, particularly IL-13 regulation of molecular networks that regulate M2 macrophage polarization. M2 macrophages are predisposed to aggregation and multinucleated giant cell formation, which are characteristic features of sarcoidosis granulomas. Clinical trial registered with www.clinicaltrials.gov (NCT01857401).

Project description:BackgroundIn Australia, demand for specialist infectious diseases services exceeds capacity to provide timely management of latent tuberculosis infection (LTBI) in areas of high refugee and asylum seeker settlement. A model for treating LTBI patients in primary care has been developed and piloted in a refugee-focused primary health service (Monash Health Refugee Health and Wellbeing [MHRHW]) and a universal primary care clinic. This study reports on the development and evaluation of the model, focusing on the model feasibility, and barriers and enablers to its success.MethodsA convergent mix-methods design was used to evaluate the model for treating LTBI patients in primary care, where a prospective cohort study of patients commencing treatment either at MHRHW or the universal primary care clinic determined the model feasibility, while focus groups with clinicians directly involved in treating these patients explored barriers and enablers to sustainability and success of the model.ResultsFrom January 2017 to April 2018, 65 patients with confirmed LTBI presented at participating clinics. Treatment was accepted by 31 (48%) patients, of whom 15(48%) were treated at MHRHW and 16 (52%) at the universal primary care clinic. The 6-months' treatment completion rate was higher at MHRHW compared to the universal primary care clinic (14 (93%) compared to 9 (56%) respectively, p = 0.0373). Reasons for non-completion included adverse reaction, opting out and relocation. At the completion of the pilot, 15 clinicians participated in two focus groups. Clinicians identified barriers and enablers for successful LTBI management at patient, provider, organisational and clinical levels. While barriers for treatment completion and adherence were consistent across the two pilot sites, enablers, such as resources to facilitate patient education and follow-up, were available only at MHRHW.ConclusionScreening and management of LTBI patients can be achieved within the primary care setting, considerate of barriers and enablers at patient, provider, organisational and clinical levels. Upscaling of a primary care response to the management of LTBI will require supporting primary care clinics with resources to employ dedicated clinical staff for patient education, follow-up communication and monitoring medication adherence.

Dataset Information

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

Publications

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

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