Characterisation of Myeloid Cells in the Peritoneal Foreign Body Response: Evidence for Phenotypic Plasticity

The implantation of foreign material in the peritoneal cavity initiates a sequence of events including inflammatory cell recruitment and wound healing-like processes, collectively known as the foreign body response (FBR). Previous research has demonstrated that bone marrow-derived cells, many with monocyte/macrophage morphology, are recruited to the implant. This continues until the material is either phagocytosed or encapsulated in tissue comprised predominantly of myofibroblasts. Given that plasticity is a hallmark of cells of the myelomonocytic lineage, and cells intermediate in morphology between macrophage and fibroblast have been detected within the tissue capsule, monocyte/macrophages have been proposed as a possible source of tissue capsule myofibroblasts. However, to date, definitive evidence to support this has not been provided. Thus, this thesis investigates the hypothesis that monocyte/macrophages transdifferentiate to tissue capsule myofibroblasts in the peritoneal FBR. The transgenic MacGreen mouse, in which EGFP expression is controlled by the Cfms (Csf1r) promoter, was used to investigate the time-dependent accumulation of myeloid cells in the peritoneal FBR. An influx of monocytes and neutrophils into the peritoneal cavity within 48 hours of implantation was identified by surface marker expression and FACS analysis of the inflammatory infiltrate. These cells could be distinguished by variable expression levels of EGFP and Ly6C; neutrophils were EGFPlo Ly6Cmed-hi whilst monocytes were EGFPhi and expressed variable levels of Ly6C. Over time (days 7-14), Ly6C expression was down-regulated, concomitant with up-regulation of F4/80, indicative of monocyte-to-macrophage maturation. At the same time, up-regulation of F4/80 on the EGFPlo subset suggested neutrophil-to-macrophage transdifferentiation. The infiltrating EGFP+ Ly6C+ subsets rapidly adhered to the surface of the implanted material, and a similar process of differentiation and maturation was observed for cells in the encapsulating tissue. Over time, EGFP+ cells within the tissue capsule changed morphology from a rounded macrophage-like appearance to an elongated phenotype characteristic of myofibroblasts. Co-expression of EGFP and the myofibroblast marker alpha-smooth muscle (α-SM) actin was also demonstrated with the proportion of EGFP+ α-SM actin+ cells increasing from 11.13 ± 0.67% at day 14 to 50.77 ± 12.85% of total cells at day 28. The critical role of monocyte/macrophages in tissue capsule development was confirmed by clodronate-liposome depletion experiments which resulted in almost complete abrogation of capsule development. Microarray technologies were employed to determine the time-dependent gene expression profile of monocyte/macrophages within the peritoneal FBR. BioLayout Express3D network analysis identified clusters of co-expressed genes associated with EGFPhi cells from the inflammatory infiltrate and tissue capsules over a 28 day time-course. The expression of Acta2 and other genes associated with the actin cytoskeleton (Cnn2, Tnni1 and Tnni2) was demonstrated in early infiltrating cells, suggesting that this transcriptional programme is switched on very early in the inflammatory response, before encapsulation and well before the translated proteins can be detected. Whilst Acta2 was not expressed by tissue capsule cells, a number of other mesenchymal-related genes were expressed including Actc1, Tnni3 and Twist1. Also identified as significant during the process of tissue encapsulation were genes related to angiogenesis and wound repair. Encapsulating cells co-expressed genes characteristic of M1 and M2 macrophages, shifting from an M1-dominated expression profile during the early inflammatory stage of foreign object recognition and encapsulation, to a more reparative M2 phenotype during the later stages of fibrotic tissue capsule formation. Macrophages associated with early encapsulation (day 7) expressed the classical (M1) activation marker Nos2, as well as low levels of the alternative (M2) activation markers Chi3l3 and Chi3l4 (Ym1/2), Ccl17 and Il10. Nos2 expression declined after 7, whilst Chi3l3, Chi3l4, Ccl17 and Il10 expression increased over time, reaching maximal levels after day 21; expression of the classical activation marker Il23a also increased. Taken together, the results show that tissue capsule macrophages possess a mixed M1-M2 activation phenotype that is dynamic and exhibits phenotypic duality. In conclusion, macrophages participating in encapsulation of sterile foreign material implanted in the peritoneal cavity possess significant phenotypic plasticity and display a complex progression of phenotypes, with overlap in M1/M2 gene expression. This M1-M2 switch is coincident with morphological progression from rounded macrophage-like appearance, to a spindle-shaped morphology and acquisition of α-SM actin expression. Whilst the research findings do not support macrophage-to-myofibroblast transdifferentiation, a dual role for macrophages within the peritoneal FBR is clearly established, as evidenced by the progression from a pro-inflammatory to wound healing, or ‘fibroblastoid’, phenotype. Taken together, the results presented in this thesis contribute to the growing body of literature describing distinctive macrophage phenotypes that are dependent on the environmental milieu and coupled to changes in transcriptional output