Modulating Macrophage Polarization through CCR2 Inhibition and Multivalent Engagement

Abstract

Excessive or prolonged recruitment of inflammatory monocytes to damaged tissue can significantly worsen patient outcomes. Monocytes migrate to sites of tissue inflammation in response to high local concentrations of CCL2, a chemokine that binds to and signals through the CCR2 receptor. While the role of CCR2 in cellular migration is well studied, it is unclear how CCR2 inhibition affects macrophage polarization and if multivalency can increase downstream signaling effects. Using affinity selection with a phage library, we identified a novel single-chain variable fragment (scFv) (58C) that binds specifically and with high affinity to the N-terminal domain of CCR2 ( K = 59.8 nM). The newly identified 58C-scFv bound to native CCR2 expressed on macrophages and MDA-MB-231 cells, inhibited migration, and induced a pro-inflammatory M1-phenotype in macrophages. The M1/M2 macrophage phenotype ratio for monomeric 58C-scFv was significantly increased over the negative control by 1.0 × 10-fold (iNOS/Arg-1), 5.1 × 10-fold (iNOS/Mgl2), 3.4 × 10-fold (IL-6/Arg-1), and 1.7 × 10-fold (IL-6/Mgl2). The multivalent display of 58C-scFv on liposomes further reduced migration of both cell types by 25-40% and enhanced M1 polarization by 200% over monomeric 58C-scFv. These studies demonstrate that CCR2 inhibition polarizes macrophages toward an inflammatory M1 phenotype, and that multivalent engagement of CCR2 increases the effects of 58C-scFv on polarization and migration. These data provide important insights into the role of multivalency in modulating binding, downstream signaling, and cellular fate.

miR-511-3p protects against cockroach allergen–induced lung inflammation by antagonizing CCL2

“…A further mechanism elucidated by this study is that Ccl2 induces macrophage polarization to the M1 phenotype through its receptor, Ccr2. The Ccl2/Ccr2 pathway has been reported to skew macrophage polarization at the transcriptomic and functional level in human and mouse macrophages (46)(47)(48)(49). Our studies here provide evidence that Ccl2 can induce Ccr2-dependent expression of M1-associated genes, but not M2-associated genes, and further suggested that the Ccl2/Ccr2 pathway may be associated with M1 macrophage polarization.…”

Boosting the Biogenesis and Secretion of Mesenchymal Stem Cell-Derived Exosomes

“…Cellular viability was analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as previously described [18][19][20]. MSCs were seeded into 96-well plates and cultured overnight.…”

Boosting the biogenesis and secretion of mesenchymal stem cell-derived exosomes

“…Cellular viability was analyzed using the MTT assay as previously described [7,9,10]. MSCs were seeded into 96-well plates and cultured overnight.…”