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Microcalorimetric method to assess phagocytosis: macrophage-nanoparticle interactions.

ABSTRACT: This study evaluated the use of isothermal microcalorimetry (ITMC) to detect macrophage-nanoparticle interactions. Four different nanoparticle (NP) formulations were prepared: uncoated poly(isobutyl cyanoacrylate) (PIBCA), polysorbate-80-coated PIBCA, gelatin, and mannosylated gelatin NPs. Changes in NP formulations were aimed to either enhance or decrease macrophage-NP interactions via phagocytosis. Alveolar macrophages were cultured on glass slabs and inserted in the ITMC instrument. Thermal activities of the macrophages alone and after titration of 100 μL of NP suspensions were compared. The relative interactive coefficients of macrophage-NP interactions were calculated using the heat exchange observed after NP titration. Control experiments were performed using cytochalasin B (Cyto B), a known phagocytosis inhibitor. The results of NP titration showed that the total thermal activity produced by macrophages changed according to the NP formulation. Mannosylated gelatin NPs were associated with the highest heat exchange, 75.4 ± 7.5 J, and thus the highest relative interactive coefficient, 9,269 ± 630 M-1. Polysorbate-80-coated NPs were associated with the lowest heat exchange, 15.2 ± 3.4 J, and the lowest interactive coefficient, 890 ± 120 M-1. Cyto B inhibited macrophage response to NPs, indicating a connection between the thermal activity recorded and NP phagocytosis. These results are in agreement with flow cytometry results. ITMC is a valuable tool to monitor the biological responses to nano-sized dosage forms such as NPs. Since the thermal activity of macrophage-NP interactions differed according to the type of NPs used, ITMC may provide a method to better understand phagocytosis and further the development of colloidal dosage forms.

SUBMITTER: Al-Hallak MH

PROVIDER: S-EPMC3032094 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMobilization of the innate immune response to clear and metabolize necrotic and apoptotic cardiomyocytes is a prerequisite to heart repair after cardiac injury. Suboptimal kinetics of dying myocyte clearance leads to secondary necrosis, and in the case of the heart, increased potential for collateral loss of neighboring non-regenerative myocytes. Despite the importance of myocyte phagocytic clearance during heart repair, surprisingly little is known about its underlying cell and molecular biology.ObjectiveTo determine if phagocytic receptor MERTK is expressed in human hearts and to elucidate key sequential steps and phagocytosis efficiency of dying adult cardiomyocytes, by macrophages.ResultsIn infarcted human hearts, expression profiles of the phagocytic receptor MER-tyrosine kinase (MERTK) mimicked that found in experimental ischemic mouse hearts. Electron micrographs of myocardium identified MERTK signal along macrophage phagocytic cups and Mertk-/- macrophages contained reduced digested myocyte debris after myocardial infarction. Ex vivo co-culture of primary macrophages and adult cardiomyocyte apoptotic bodies revealed reduced engulfment relative to resident cardiac fibroblasts. Inefficient clearance was not due to the larger size of myocyte apoptotic bodies, nor were other key steps preceding the formation of phagocytic synapses significantly affected; this included macrophage chemotaxis and direct binding of phagocytes to myocytes. Instead, suppressed phagocytosis was directly associated with myocyte-induced inactivation of MERTK, which was partially rescued by genetic deletion of a MERTK proteolytic susceptibility site.ConclusionUtilizing an ex vivo co-cultivation approach to model key cellular and molecular events found in vivo during infarction, cardiomyocyte phagocytosis was found to be inefficient, in part due to myocyte-induced shedding of macrophage MERTK. These findings warrant future studies to identify other cofactors of macrophage-cardiomyocyte cross-talk that contribute to cardiac pathophysiology.

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Microcalorimetric method to assess phagocytosis: macrophage-nanoparticle interactions.

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