Background: Ischemic heart disease (IHD) is a leading cause of morbidity and mortality worldwide. Deprivation of oxygen/nutrients to the heart during IHD induces cardiac cell death resulting in an inflammatory response with the migration of neutrophils and macrophages, an essential process for cardiac repair. However, unresolved inflammation can cause adverse cardiac remodeling following myocardial ischemia/reperfusion (I/R) injury. Ubiquitin (UB) is an evolutionarily conserved protein. Previously, our lab has shown that exogenous UB treatment plays a cardioprotective role and significantly reduces infiltration of neutrophils and macrophages 3 days post-I/R injury in mice. Here, we investigated the role of exogenous UB in macrophage phenotype and function. It is hypothesized that exogenous UB modulates the phenotype and function of polarized M1 (pro-inflammatory), and/or M2 (anti-inflammatory) macrophages, thereby playing a cardioprotective role 3 days post-I/R. Methods: Isolated peritoneal macrophages (3 days post-thioglycolate injection) were pretreated with UB (20μg/mL) for 30 minutes followed by treatment with IFNγ (100U/mL), for M1 polarization, or IL-4 (20ng/mL), for M2 polarization for 72 hours. Polarized peritoneal macrophages were used to prepare cell lysates for western blot analyses, immunohistochemistry or migration. Plated and paraformaldehyde-fixed polarized peritoneal macrophages were stained with F4/80 to measure the cell spreading area. A scratch assay was used to measure polarized peritoneal macrophage migration. RAW264.7 macrophage cell line was used to assess polarized macrophage phagocytosis of pHrodo-conjugated E. coli bioparticles (100μg/mL). Results: Western blot analysis of polarized peritoneal macrophage lysates showed that UB treatment increased IFNγ-induced expression of iNOS (a marker of M1 phenotype). Treatment with UB decreased IFNγ induced-phosphorylation of STAT1 (transcription factor activated by IFNγ). IFNγ and IL-4 treatment decreased the expression of CXCR4 (receptor for UB). However, UB treatment had no effect on IFNγ and IL-4-mediated decrease in CXCR4 expression. Treatment with UB significantly reduced the cell spreading area of M1-polarized peritoneal macrophages. The migration of M1-polarized peritoneal macrophages was significantly increased with UB treatment. The migration of M2-polarized peritoneal macrophages was significantly greater compared to non-polarized control macrophages, and UB treatment had no effect on M2-polarized macrophage migration. In RAW264.7 cells, M1-polarized macrophages exhibited significantly increased phagocytosis of E. coli bioparticles vs. non-polarized control, and UB treatment enhanced M1-polarized macrophage phagocytosis. In contrast, UB treatment reduces the phagocytosis of E. coli bioparticles in M2-polarized macrophages. Conclusions: These findings indicate that treatment with exogenous UB can potentially alter the phenotype and function of M1- and M2-polarized macrophages.
| Identifer | oai:union.ndltd.org:ETSU/oai:dc.etsu.edu:asrf-2189 |
| Date | 25 April 2023 |
| Creators | Shook, Paige, Casteel, Jared, Yakubenko, Valentin, Dalal, Suman, Singh, Mahipal, Singh, Krishna |
| Publisher | Digital Commons @ East Tennessee State University |
| Source Sets | East Tennessee State University |
| Detected Language | English |
| Type | text |
| Source | Appalachian Student Research Forum |
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