IL-13–regulated Macrophage Polarization during Granuloma Formation in anIn VitroHuman Sarcoidosis Model

Abstract

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 controls were incubated for 7 days with purified protein derivative (PPD)-coated polystyrene beads. Molecular responses were analyzed, as reflected by differential expression (DE) of genes, extracellular cytokine patterns, and cell surface receptor expression. Unbiased systems biology approaches were used to identify signaling pathways engaged during granuloma formation. Model findings were compared to human lung and mediastinal lymph node gene expression profiles. Compared to identically treated PBMCs of controls (n = 5), PPD-treated sarcoidosis PBMCs (n = 6) were distinguished by the formation of cellular aggregates resembling granulomas. Pathway Analysis of DE 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.

Current Sarcoidosis Models and the Importance of Focusing on the Granuloma

“…Macrophage features observed in our in vitro human granuloma model strongly align with this theory, including a significant increase in >50 different M2-like macrophage gene transcripts and elevated expression of CD163 in sarcoidosis granulomas at the protein level ( Figure 5 ). Pathway analysis predicted the cytokine IL-13 as being an important upstream regulator of the observed gene expression changes in sarcoidosis compared to healthy controls after antigen stimulation, and we verified this gene network to be highly overrepresented in human sarcoidosis lung and lymph node tissues ( 45 ) ( Figure 6 ). IL-13 signaling is associated with a Th2-biased immune response and is a known promoter of alternative or M2-like macrophage activation ( 46 , 47 ).…”

“…Other data from the model that point to a functional imbalance in favor of Th2-immune signaling in sarcoidosis was the relatively blunted INF-γ production associated with sarcoidosis following M.tb antigen stimulation and the significant inhibition of granuloma formation in sarcoidosis through pharmacologic signal transducer and activator of transcription 6 (STAT6) inhibition (45). IL-13-mediated activation of STAT6 is a well-established pathway for M2-like macrophage polarization (52).…”

GM-CSF and IL-33 Orchestrate Polynucleation and Polyploidy of Resident Murine Alveolar Macrophages in a Murine Model of Allergic Asthma

“…However, while tAMs from naive mice express the receptor for GM-CSF [ 19 ], IL-13, and IL-33, we did not observe the expression of TSLPR or IL-17RB (the specific receptor chain involved in the formation of the IL-25 receptor) in steady state alveolar macrophages, therefore limiting our study to IL-13 and IL-33. Intriguingly, IL-13 has been reported to act as an inducer of fusion-driven MNGC formation [ 5 ] and contributes to macrophage polarization in sarcoidosis-associated granuloma formation in vitro [ 39 ]. However, in tAMs, our data suggest that IL-13 drives polyploidy.…”

Models Contribution to the Understanding of Sarcoidosis Pathogenesis: “Are There Good Models of Sarcoidosis?”

“…Crouser et al showed that macrophages from sarcoidosis patients maintained a M2 phenotype in the presence of PPD beads compared to non-coated beads, compared to control patients for whom macrophages acquired M1 characteristics [ 60 ]. Likewise, Locke et al showed an increase in M2 polarization with the expression of CD163 and an IL13 gene expression profile in sarcoidosis patients compared to control patients in response to PPD beads [ 61 ]. In the MAB model, granuloma formation was observed 72 h after exposure with a marked Th1 and Th17 signature and NFκB activation [ 62 ].…”