Molecular and functional differentiation of murine macrophage subtypes

Macrophages can be differentiated ex vivo from bone marrow hematopoietic stem cells, so-called bone marrow derived macrophages (BM macrophages), but also from embryonic stem cells resembling the embryonic/fetal development of macrophages, so-called ES macrophages. I hypothesized, that ES macrophages may represent a source of naïve macrophages resembling the embryonic macrophage subtype, which should be non-inflammatory, highly proficient in apoptotic cell clearing and tissue remodeling, and should facilitate scar-free wound healing. In contrast, BM macrophages are thought to represent a macrophage subtype predominantly mediating inflammation related immune responses and antigen presentation. ES macrophages were to be molecularly and functionally characterized in vitro in comparison to BM macrophages. To this end, I employed genome wide expression profiling, activation profiling, surface marker profiling, and functional assays testing endocytosis, adhesion, antigen presentation, and cell migration. Surface marker profiling and gene expression analysis revealed similar expression of the macrophage specific markers CD14, CD115, F4/80, and CD68 in BM and ES macrophages. In contrast, the two macrophage subtypes differed in the expression of the receptor repertoire involved in endocytosis of foreign body particles, calciprotein particles and apoptotic cells. This involved Toll-like receptors 4 and 6 and macrophage scavenger receptor 2, and resulted in higher clearing capacity in BM macrophages in comparison to ES macrophages. Developmental profiling revealed that ES macrophages resembled a liver hematopoietic embryonic or an adult macrophage subtype. Both macrophage subtypes were classified as resident macrophages that could be differentiated into prefusion osteoclasts, and showed differential activation properties concerning the classical and alternative activation pathway. BM macrophages were more sensitive to pro-inflammatory stimuli (classical activation), whereas ES macrophages showed stronger reaction to alternative activation and deactivation stimuli. Gene expression analysis revealed IL-1 and IL-3-dependent differential expression of coagulation factors and MHC I and II molecules in BM and ES macrophages, but an inductor-independent up-regulation of matrix metalloproteinase expression in the latter macrophage subtype. Functional analysis revealed that BM macrophages had a higher and more prolonged cross-presentation capacity in comparison to ES macrophages, whereas ES macrophages showed higher proteolytic activity in cell culture supernatant. The differential expression of integrins was associated with a higher adherence capacity of ES macrophages, e.g. on laminin and collagen types I and III. The transmigration properties of the two macrophage subtypes showed the complex network of the interplay between MMP secretion and integrin adhesion and was influenced by the interaction potential of the two macrophage subtypes with the endothelial monolayer. Macrophages are key players during all phases of wound healing, strongly associated with the severity of inflammation and the level of scar formation. Investigation of the influence of the two macrophage subtypes on wound healing in an in vivo-tail wound model showed diminished wound contraction, delayed eschar shedding and delayed wound closure in both macrophage-treated wound types compared to cell-free treated control wounds. Furthermore, macrophage-treatment of wounds resulted in prolonged neutrophil influx and prolonged inflammatory response. ES macrophage-treatment resulted in a higher cellularity of the wound after complete wound closure, mainly caused by fibroblast accumulation. These findings confirmed the hypothesis that BM macrophages resemble a macrophage subtype predominantly mediating inflammation related immune responses and tissue homeostatis, thus classified as M1 macrophage according to Mantovani and colleagues (Mantovani et al. 2004). My original hypothesis held that ES macrophages should be non-inflammatory and predominantly involved in apoptotic cell clearing and tissue remodeling, resembling an embryonic/fetal macrophage subtypes, thus probably mediating scar-free healing. Contrary to this hypothesis, the present study showed that ES macrophages efficiently mediated inflammation related processes, showed less clearing capacity and were associated with fibroblast accumulation in tail wounds after complete re-epithelialization, eventually resulting in fibrosis. Consistent with the activation profiles, ES macrophages thus do not constitute an embryonic/fetal, but rather an alternatively activated macrophage subtype of the M2 type according to current classification criteria (Mantovani et al. 2004)