ApoE-deficient mice on cholate-containing high-fat diet reveal a pathology similar to lung sarcoidosis

Abstract

Sarcoidosis is a chronic disease of unknown etiology characterized by the formation of non-necrotizing epithelioid granulomas in various organs, especially in the lungs. The lack of an adequate animal model reflecting the pathogenesis of the human disease is one of the major impediments in studying sarcoidosis. In this report, we describe ApoE-/- mice on a cholate-containing high-fat diet that exhibit granulomatous lung inflammation similar to human sarcoidosis. Histological analysis revealed well-defined and non-necrotizing granulomas in about 40% of mice with the highest number of granulomas after 16 weeks on a cholate-containing high-fat diet. Granulomas contained CD4+ and CD8+ T cells, and the majority of the cells in granulomas showed immunoreactivity for the macrophage marker Mac-3. Cells with morphological features of epithelioid cells expressed angiotensin-converting enzyme, osteopontin, and cathepsin K, all characteristics of epithelioid and giant cells in granulomas of human sarcoidosis. Giant cells and nonspecific inclusions such as Schaumann's bodies and crystalline deposits were also detected in some lungs. Granulomatous inflammation resulted in progressive pulmonary fibrosis. Removal of cholate from the diet prevented the formation of lung granulomas. The observed similarities between the analyzed mouse lung granulomas and granulomas of human sarcoidosis, as well as the chronic disease character leading to fibrosis, suggest that this mouse model might be a useful tool to study sarcoidosis.

Figure 1

Trichrome staining revealed non-necrotizing granulomas with sharp borders. Mice lungs after 16 weeks of CHFD (A), average number of granulomas per lung tissue section (B), tight fibrotic lung granuloma with abundant collagen fibers (C), and less fibrotic granuloma with more prominent epithelioid cells (D) are shown. Scale bars: 260 μm (A); 40 μm (C and D). *P < 0.05.

Figure 2

Cellular characterization of granulomas in mouse lungs. (A) CD4 expression, (B) CD8 expression, (C) myofibroblast marker (smooth muscle cell α-actin) expression, (D) macrophage marker (Mac-3) expression, (E) angiotensin-converting enzyme expression, (F) osteopontin expression (red) with Mac-3 co-staining (green), (G) cathepsin K expression (red) with Mac-3 co-staining (green) are shown. ACE, osteopontin, and cathepsin K appear to be most strongly expressed in epitheliod cells. H: Percentage of granuloma area stained positive for macrophage marker (Mac-3), osteopontin, highly expressed in epithelioid cells, and T-cell markers is shown. Lower magnification of lung granulomas shows the predominant expression of ACE (I), osteopontin (J), cathepsin K (K), and the macrophages marker, Mac-3, within granulomas (L). Scale bars: 40 μm (A–G); 130 μm (I–L).

Figure 3

In some mouse lungs, Schaumann's bodies (A, arrows) and crystalline structures (B) were recognizable by a dark brown appearance in trichrome stained sections. Crystals were often observed inside of multinucleated giant cells with strong cathepsin K staining (C; red: cathepsin K; blue: nuclei and autofluorescence of crystals). Multinucleated giant cells were also present inside (D and E, arrows), and they were also cathepsin K-positive (F; red: cathepsin K; blue: nuclei). The presence of iBALTs in bronchovascular bundles (G and H, arrows) is shown. Occasionally granulomas were observed inside of blood vessels (I, arrow). The percentage of lung area occupied by iBALT (J) is shown. iBALTs consisted predominantly of CD8-cells and were attached to blood vessels (K; red: CD8; green: smooth muscle cell α-actin). Dense accumulation of CD8⁺ cells in iBALT and fewer Mac-3-positive cells in granulomas (L; red: CD8; green: Mac-3) are shown. Decreased amount of CD4⁺ (M; red: CD4) and the prevalence of CD8⁺ cells in iBALT (N; red: CD8) are shown. CD4/CD8 ratio in BAL fluid was decreased after 16 weeks and increased after 19 weeks of CHFD when compared with control mice (O). Scale bars: 20 μm (A–F); 130 μm (G); 65 μm (H and I); 30 μm (K–N). *P < 0.05.

Figure 4

Organ pathology induced by CHFD. Cholate-containing diet induced hypertrophy in thymus and fibrosis and induced formation of MGCs in thymus, liver, stomach, and skin. After 16 weeks of CHFD, thymus size was significantly increased (A; left: 22-week-old mice on normal diet; right: 16 weeks of CHFD). Trichrome-stained sections of thymus in control mice (B) and highly fibrotic acellular thymus after 19 weeks on CHFD (C) are shown. Foreign body-type MGCs were observed in thymus (D) and stomach (F). Lipid-filled cells with two to three nuclei were seen in liver (E). Mice on prolonged CHFD developed skin lesions with xanthogranulomas (G). Touton-type MGCs with a central and circular distribution of nuclei (H, arrow) and foreign body-like MGCs (I, arrow) in skin sections were frequently observed. Scale bars: 130 μm (B, C, and G); 30 μm (E); 20 μm (D, F, H, and I).

Figure 5

Granulomatous inflammation resulted in progressive pulmonary fibrosis. Lung tissue section of a 22-week-old mice on normal diet (A), granulomas surrounded by single fibrotic masses and hyaline membranes after 16 weeks of CHFD (B, arrows show hyaline membranes), and confluent fibrotic masses after 9 weeks of CHFD (C) are shown. Fibrosis with honeycombing appearance (D) is shown. Immunostaining for smooth muscle cell α-actin revealed the presence of myofibroblasts foci (E) and the deposition of intraalveolar proteinaceous material as a result of acute lung injuries (F). In some cases granulomas were characterized by an unusual high cell density (G, asterisk shows area magnified in H) with the prevalence of CD8-positive cells (I). Quantification of fibrotic changes (J) is shown. Hydroxyproline content in lungs (K) is shown. Scale bars: 130 μm (A–D, F, and G); 20 μm (E); 30 μm (H and I). *P < 0.05.

Figure 6

Human tissue sections from patients with sarcoidosis show similar characteristics regarding cathepsin K expression in granulomas as described in mice in this report. Epithelioid cells in skin granulomas (A, H&E staining) are cathepsin K immunoreactive (B), lymph node granuloma with epithelioid and giant cells (C, H&E staining) containing strong cathepsin K staining (D). Cathepsin K-positive Langhans-type MGC in lymph node (E) and foreign body-like MGC in lung (F) are shown. Crystals and inclusions with dark brown appearance (G, H&E staining) were often seen adjacent or within cathepsin K-positive MGCs (H) in sections with lung sarcoidosis. Scale bars: 130 μm (A, B, G, and H); 30 μm (C and D); 20 μm (E and F).