Iqbal, Nadia and Jackson-Jones, Lucy (2024) Pleural cavity inflammation : fibres & FALCs, macrophages & malignancy. PhD thesis, Lancaster University.
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Abstract
Malignant pleural mesothelioma (MPM) is a devastating cancer of the mesothelial linings caused most commonly by inhalational exposure to asbestos fibres. In humans there is a delay of 25-40 years between fibre exposure & mesothelioma development. The role of the local immune response in the development of mesothelioma in the months and years proceeding fibre exposure is unclear. In this work, we aimed to understand the sequence of inflammation precipitating events which occur following arrival of asbestos-like fibres within the pleural space, to do this we utilised NM-401 multi-walled carbon nanotubes (MWCNTs/CNTs). CNTs share a similar fibrous shape, chemical characteristics, and bio-persistent properties to asbestos fibres. CNTs are manufactured for use in industries such as automotive, textiles, industrial and engineering. Environmental & occupational exposure to CNTs is increasing annually despite these structures having been shown to mimic asbestos exposure leading to mesothelioma-like tumours in murine models. In our study, CNTs were injected intra-pleurally (i.pl) into wild type mice, pleural cavity lavage and adipose tissues were isolated at various time points after injection. Immune cell composition, cytokine and chemokine expression and release were determined by flow-cytometry, confocal microscopy & multi-plex array. Intrapleural CNT delivery resulted in increases in small cavity macrophages (SCMs), specifically the Ly6C+ inflammatory macrophage (Inf-Mθ) population within the pleural lavage. The population defined as Inf-Mθ were recruited to Fat-Associated Lymphoid Clusters (FALCs) of the pericardium, FALCs revealed a significant loss of antigen presentation capacity observed by a reduction in MHC-II. The secretome of pericardial FALCs in mice exposed to CNTs was compared with matched serum & pleural fluid from patients with mesothelioma. A noticeably similar secretome to that of human pleural fluid but not serum was found, including elevations in CXCL10 and IL-6. We reasoned that FALCs in the pleural adipose tissues may contribute to the inflammatory response after fibre exposure and in so doing, FALCs of the pleural cavity may play a significant role in the early formation of the mesothelioma tumour microenvironment. Fluorescence activated cell sorting (FACS) and quantitative polymerase chain reaction (qPCR) revealed higher expression of Cxcl10, Il-6 and Ccl2 expression within CD45- Podaplanin+ (P+) stromal cells of pleural adipose tissue compared to the CD45+PDPN- leukocytes following CNT exposure in vivo. Further flow cytometric investigations revealed the early release of CXCL10 into the pleural cavity from the platelet derived growth factor receptor α+ (PDGFRα+) stromal compartment in FALCs. Local delivery of anti-CXCL10 blocking antibody into the pleural cavity simultaneously during CNT exposure reduced inflammatory myeloid cell recruitment to pericardial FALCs. This revealed a mechanistic relationship between FALC stromal cell release of CXCL10 and myeloid cell recruitment. In vitro mechanistic studies addressed the role of CXCL10 in co-ordination of the myeloid cell response; the human monocyte cell line THP-1 was differentiated into macrophages and exposed to CNTs. Confocal imaging revealed the activation of the DNA damage sensing Stimulator of Interferon Genes (STING) pathway. Complementary in vitro studies unravelled combinatorial effects of mesothelioma-relevant chemokines (CXCL10, IL-6, IL-4) on myeloid cell activation and functionality to improve understanding of immunoregulation during fibre-driven inflammation. THP-1 cells were polarised to an anti-inflammatory pro-tumorigenic phenotype when incubated with CXCL10, IL-4 and IL-6 overnight. Analysis of human MPM and other cancers that caused effusion within the pleural space, revealed a considerably higher level of CXCL10 in MPM pleural fluid. Flow cytometric analysis of cells isolated from patient pleural fluid uncovered that the cellular infiltrate in MPM and other cancers of the pleural fluid is composed of a high level of CD68+ CD14+ macrophages which we believe are phenotypically similar to SCMs found in mice.