Abstract
IntroductionM2 macrophages promote tissue repair and regeneration through various mechanisms including immunomodulation and scavenging of tissue debris. Delivering increased numbers of these cells to ischemic tissues may limit tissue injury and promote repair. Ixmyelocel-T is an expanded, autologous multicellular therapy cultured from bone-marrow mononuclear cells (BMMNCs). The purpose of this study was to characterize further a unique expanded population of M2-like macrophages, generated in ixmyelocel-T therapy.MethodsApproximately 50 ml of whole bone marrow was obtained from healthy donors and shipped overnight. BMMNCs were produced by using density-gradient separation and cultured for approximately 12 days to generate ixmyelocel-T. CD14+ cells were isolated from ixmyelocel-T with positive selection for analysis. Cell-surface phenotype was examined with flow cytometry and immunofluorescence, and expression of cytokines and chemokines was analyzed with enzyme-linked immunosorbent assay (ELISA). Quantitative real-time PCR was used to analyze expression of genes in BMMNCs, ixmyelocel-T, the CD14+ population from ixmyelocel-T, and M1 and M2 macrophages. Ixmyelocel-T was cultured with apoptotic BMMNCs, and then visualized under fluorescence microscopy to assess efferocytosis.ResultsMacrophages in ixmyelocel-T therapy expressed surface markers of M2 macrophages, CD206, and CD163. These cells were also found to express several M2 markers, and few to no M1 markers. After stimulation with lipopolysaccharide (LPS), they showed minimal secretion of the proinflammatory cytokines interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-α) compared with M1 and M2 macrophages. Ixmyelocel-T macrophages efficiently ingested apoptotic BMMNCs.ConclusionsIxmyelocel-T therapy contains a unique population of M2-like macrophages that are characterized by expression of M2 markers, decreased secretion of proinflammatory cytokines after inflammatory stimuli, and efficient removal of apoptotic cells. This subpopulation of cells may have a potential role in tissue repair and regeneration.
Highlights
M2 macrophages promote tissue repair and regeneration through various mechanisms including immunomodulation and scavenging of tissue debris
Ixmyelocel-T therapy contains a unique population of M2-like macrophages that are characterized by expression of M2 markers, decreased secretion of proinflammatory cytokines after inflammatory stimuli, and efficient removal of apoptotic cells
Macrophages can be classified based on their functional phenotypes; M1 macrophages are classically activated by proinflammatory cytokines such as gamma interferon (IFN-ɣ) and are T-helper 1 (Th1) associated, whereas M2 macrophages are alternatively activated by cytokines such as IL-4 and IL-13, and are T-helper 2 (Th2) associated [2]
Summary
M2 macrophages promote tissue repair and regeneration through various mechanisms including immunomodulation and scavenging of tissue debris. Macrophages can be classified based on their functional phenotypes; M1 macrophages are classically activated by proinflammatory cytokines such as gamma interferon (IFN-ɣ) and are T-helper 1 (Th1) associated, whereas M2 macrophages are alternatively activated by cytokines such as IL-4 and IL-13, and are T-helper 2 (Th2) associated [2] Both in vitro and in vivo studies have demonstrated that M1 macrophages have an inflammatory phenotype that corresponds with the early phases of tissue injury [1], whereas M2 macrophages have an antiinflammatory and tissue-remodeling phenotype corresponding with the late phases of tissue injury [1,3,4,5,6,7,8]. Studies have shown that atherosclerotic lesions are characterized by the presence of proinflammatory M1 macrophages that fail to switch to an antiinflammatory and reparative phenotype, promoting disease progression [5,15,16,17]. Increasing the proportion of M2 macrophages in such disease states could be used to limit tissue injury and promote repair
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