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).

Keywords: AmpliSeq; RNA-Seq; Th1; peripheral blood mononuclear cell; purified protein derivative.

Figure 1.

In vitro granuloma-like cell aggregate formation induced by purified protein derivative (PPD)–stimulated peripheral blood mononuclear cells (PBMCs) of patients with sarcoidosis and latent tuberculosis (TB) infection (LTBI). (A) Representative light photomicrographs (×20) obtained 7 days after sarcoidosis PBMCs were incubated with PPD-coated (PPD) or uncoated (UNC) beads. (B) Representative light photomicrographs (×20 and ×40, respectively) obtained 7 days after the indicated treatment of PBMCs in the sarcoidosis, LTBI, and control groups. Uncoated beads elicited no significant cellular aggregation in any of the study groups, and human serum albumin (HSA)–coated beads caused no perceptible aggregation in any group, with the exception of the sarcoidosis group, in which an intermediate response was observed. PPD-coated beads induced prominent cellular aggregation in the sarcoidosis and LTBI groups but not in the control subjects.

Figure 2.

Composite immunofluorescence imaging of granuloma-like structures demonstrates the presence of both macrophages and lymphocytes. Representative photomicrographs of immunostained PBMCs in granuloma-like structures 7 days after bead treatment using confocal microscopy. Composite images of 4′,6-diamidino-2-phenylindole, CD11b, and CD3 are shown in blue, red, and green, respectively. (A) PPD-coated and (B) uncoated bead–stimulated PBMCs obtained from a PPD⁻, healthy control subject showing CD11b⁺ macrophages and CD3⁺ lymphocytes in the absence of granuloma formation. (C and D) CD11b⁺ macrophages and CD3⁺ lymphocytes in a granuloma-like structure of cells obtained from a subject with LTBI after PPD-coated and uncoated bead stimulation, respectively. (E and F) CD11b⁺ macrophages and CD3⁺ lymphocytes in a granuloma-like structure of cells obtained from a patient with sarcoidosis after PPD-coated and uncoated bead stimulation, respectively. Uncoated beads did not induce granuloma-like structures in both the patients with LTBI and those with sarcoidosis. In each panel, a matching differential interference contrast image is shown next to each composite image. All images were acquired at ×60 magnification.

Figure 3.

Extracellular cytokine release from PPD-stimulated PBMCs revealed distinct patterns in sarcoidosis and LTBI. Compared with treatment with uncoated or HSA-coated beads, incubation of PBMCs in the sarcoidosis (SARC), LTBI, and control (CTRL) groups with PPD-coated beads for 7 days was associated with a significant increase in the extracellular release of T helper type 1 cytokines (IFN-γ [B], IL-2 [D]) and other proinflammatory cytokines (TNF-α [A], granulocyte–macrophage colony-stimulating factor [GM-CSF] [C]). Treatment of sarcoidosis PBMCs with HSA-coated beads induced a significant increase in TNF-α and GM-CSF release compared with HSA-coated bead treatment of PBMCs from the LTBI or control groups (*P < 0.05, compared with the corresponding [within group] uncoated and no bead controls; ^†P < 0.05, relative to the matching [between groups] bead treatments).

Figure 4.

Clustering of global transcriptional responses of PBMCs after treatment with PPD-coated beads. (A) Principal component (PC) analysis of differential gene expression (AmpliSeq) performed on PBMCs derived from patients with SARC, LTBI, or normal healthy control subjects (CTRL) after incubation with PPD-coated beads for 7 days compared with paired treatment with uncoated (PBS-washed) beads. (B) Euclidean distance between samples was plotted to assess overall similarity between samples. This analysis revealed a hierarchical clustering of four subjects with SARC, whereas the remaining two SARC samples aligned more closely with CTRL (see text for further discussion).

Figure 5.

Heat map analysis of gene expression data from PPD-treated PBMCs. Differential gene expression was determined in PBMCs derived from patients with SARC, LTBI, or normal healthy control subjects (CTRL) after incubation with PPD-coated beads for 7 days compared with paired treatment with uncoated (PBS-washed) beads. The heat map was generated using the top 10 most up-regulated and top 10 most down-regulated genes within each treatment group. Genes with up-regulated expression are shaded red, whereas those with down-regulated expression are shaded green, based upon the magnitude of the change in expression. Hierarchical clustering demonstrated a grouping of four of six SARC samples, which were readily distinguished from three of four LTBI samples, based upon gene expression patterns (see text for further discussion).

Figure 6.

Gene expression comparison after PPD treatment of PBMCs derived from subjects with sarcoidosis, those with LTBI, and normal, healthy control subjects. The output type “MvA” plots are of normalized gene expression data wherein each point represents a gene, with the y-axis representing a log (base 2) fold change in expression after 7-day treatment with PPD-coated beads compared with uncoated beads for PBMCs derived from normal, healthy control subjects (A), subjects with LTBI (C), and subjects with sarcoidosis (E). The x-axis is the log average of the gene expression level. All genes with an adjusted P value of 0.05 (representing a 5% false discovery rate) and at least a twofold change (highlighted by the two horizontal gray lines) in the magnitude of gene expression between PPD and uncoated beads are shaded red. The blue line represents smoothed local mean expression (fitted using a generalized additive model), with the surrounding 95% confidence level interval shaded light blue. The dotted gray contour lines represent a two-dimensional density estimation. Genes most highly expressed in response to PPD-coated beads are highlighted in the top of the MvA plot, whereas those genes expressed at a lower level are at the bottom (the top five most differentially expressed genes in each case are labeled). Volcano plots of the same data wherein the y-axis corresponds with transcripts with high statistical significance (−log 10 of P value), and the x-axis corresponds with fold-change of gene expression (log base 2) were also generated for normal, healthy control subjects (B), subjects with LTBI (D), and those with sarcoidosis (F). Labeled transcripts on the upper left side of the plot have strong statistical significance with relatively low expression after treatment with PPD-coated versus uncoated beads. Transcripts on the upper right are more highly expressed with strong statistical significance after PPD treatment. The dotted line corresponds with a P value cutoff of 0.05, and genes failing to meet this cutoff are shaded gray.