Cellular and molecular approaches to the study of Erdheim-Chester Disease and Chronic Periaortitis

Cellular and molecular approaches to the study of Erdheim-Chester Disease and Chronic Periaortitis Project 1 – Pro-collagen type I expression in foamy histiocytes in Erdheim-Chester Disease ABSTRACT Background. Erdheim-Chester disease (ECD) is a rare systemic disorder characterized by skeletal and extra-skeletal areas where the normal local tissue architecture is replaced by foci of CD68+/CD1a- foamy histiocytes located in a fibrous extracellular matrix. In these foci, the fibroblastic component is often quantitatively minor, notwithstanding the large amounts of fibrous tissue. The aim of this study was to explore whether ECD histiocytes can synthesize collagen type I (Col I) mRNA and subsequently produce this protein. Methods. In this study we clinico-pathologically investigated 10 ECD biopsies and 10 non-ECD xanthomas biopsies (as non-ECD foamy histiocyte lesions). An ultrastructural examination of the ECD biopsies was performed at the time of the original diagnosis. Two anti-pro-collagen I (pro-col I) and anti-PPAR-γ antibodies were immunohistochemically tested in all of the biopsies and in the ECD samples, respectively. Confocal microscopy was performed to evaluate whether ECD histiocytes co-expressed pro-col I and the histiocytic marker CD68-KP1. Two ECD cases and 6 non-ECD xanthomas underwent real time-polymerase chain reaction (RT-PCR) performed after microdissecting histiocytic tissue areas, to explore the ability of these cells to express the collagen genes COL1A1 and COL1A2. Lastly, RNA In Situ Hybridization (RNA-ISH) was carried out in the 10 ECD cases to morphologically detect the expression of COL1A1 and COL1A2 genes. Results. On electron microscopy, sparse thread-like fibrils in keeping with Col I were found in the cytoplasm of the ECD histiocytes. Immunohistochemical analysis showed a strong expression of pro-col I in ECD histiocytes but a negative signal in foamy histiocytes of non-ECD xanthomas. The PPAR-γ antibody was positive in the nucleus of ECD histiocytes. On confocal microscopy, pro-col I and CD68-KP1 co-expressed in the ECD histiocytes. The RT-PCR analysis showed that the COL1A1 and COL1A2 transcripts were present in the ECD histiocytes, while they were undetectable in the histiocytes of the non-ECD xanthomas. In all ECD cases, the RNA-ISH analysis displayed a very high signal for COL1A1 and COL1A2 genes. Conclusions. In this study we demonstrate that the ECD foamy cell, while retaining a histiocytic phenotype, is capable of expressing Col I genes and synthetizing the relative proteins, thereby mimicking a functionally histiocyte/fibroblast hybrid cell. Future developments of this investigation will aim at investigating the pathogenetic mechanisms of foamy phenotype development, Col I synthesis, and Col I gene mRNA post-transcriptional regulation. Project 2 - Fibrocytes in Chronic Periaortitis. A novel mechanism linking inflammation and fibrosis ABSTRACT Objective. Chronic periaortitis (CP) is a rare disease characterised by a peri-aortic and peri-iliac fibro-inflammatory tissue. The pathogenic mechanisms leading to tissue accumulation and activation of fibroblasts are unclear. We explored the role of fibrocytes, circulating precursors of tissue fibroblasts, in patients with CP. Methods. We studied 44 newly diagnosed CP patients and 30 healthy controls. Circulating fibrocytes were identified as collagen type I (Col I)+/CD45+ cells using flow cytometry; CP biopsies were stained with anti-pro-collagen-I (pro-col I), anti-CXCR4 and anti-CD45 antibodies and analysed by confocal microscopy to detect tissue-infiltrating fibrocytes. Circulating levels and tissue expression of CXCL12, a CXCR4 ligand that promotes fibrocyte homing, were investigated respectively using ELISA and immunohistochemistry. We also characterised T-helper (Th) polarisation in CP biopsies and measured serum levels of a panel of cytokines hallmarking Th responses and capable of influencing fibrocyte differentiation. Results. The frequency of circulating Col I+/CD45+ fibrocytes was higher in patients than in controls (P=0.0371). CD45+/pro-col I+ and CXCR4+/pro-col I+ cells were detected in all examined CP biopsies. CXCL12 serum levels were also higher in CP patients (P=0.0056), and tissue-infiltrating inflammatory cells intensely expressed CXCL12. Increased serum levels of Th2-cytokines (e.g., IL-13, IL-10) were found in patients, and immunohistochemistry revealed a dominant infiltration of GATA-3+ cells, also indicating Th2-polarisation; Th2-skewed responses are known to promote fibrocyte differentiation. Conclusions. Fibrocytes are enriched in the peripheral blood of CP patients and infiltrate target lesions; their accumulation in the pathologic tissue might be driven by CXCL12, and Th2-skewed immune responses are likely to facilitate their differentiation.