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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 ( KD = 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 × 104-fold (iNOS/Arg-1), 5.1 × 104-fold (iNOS/Mgl2), 3.4 × 105-fold (IL-6/Arg-1), and 1.7 × 106-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.

SUBMITTER: Deci MB

PROVIDER: S-EPMC6499372 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Modulating Macrophage Polarization through CCR2 Inhibition and Multivalent Engagement.

Deci Michael B MB Ferguson Scott W SW Scatigno Sydney L SL Nguyen Juliane J

Molecular pharmaceutics 20180604 7

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 selecti ...[more]

PMID: 29791797

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Project description:BackgroundAccumulating evidence suggests that M2-polarized tumor-associated macrophages (TAMs) play an important role in cancer progression and metastasis, making M2 polarization of TAMs an ever more appealing target for therapeutic intervention. Astragaloside IV (AS-IV), a saponin component isolated from Astragali radix, has been reported to inhibit the invasion and metastasis of lung cancer, but its effects on TAMs during lung cancer progression have not been investigated.MethodsHuman THP-1 monocytes were induced to differentiate into M2 macrophages through treatments with IL-4, IL-13, and phorbol myristate acetate (PMA). We used the lung cancer cell lines A549 and H1299 cultured in conditioned medium from M2 macrophages (M2-CM) to investigate the effects of AS-IV on tumor growth, invasion, migration, and angiogenesis of lung cancer cells. Macrophage subset distribution, M1 and M2 macrophage-associated markers, and mRNA expression were analyzed by flow cytometry and quantitative PCR. The activation of adenosine monophosphate-activated protein kinase (AMPK) signaling pathways that mediate M2-CM-promoted tumor migration was detected using western blotting.ResultsHere we found that AS-IV significantly inhibited IL-13 and IL-4-induced M2 polarization of macrophages, as illustrated by reduced expression of CD206 and M2-associated genes, and that AS-IV suppressed the M2-CM-induced invasion, migration, and angiogenesis of A549 and H1299 cells. In vivo experiments demonstrated that AS-IV greatly inhibited tumor growth and reduced the number of metastases of Lewis lung cancer. The percentage of M2 macrophages was decreased in tumor tissue after AS-IV treatment. Furthermore, AS-IV inhibited AMPKα activation in M2 macrophages, and silencing of AMPKα partially abrogated the inhibitory effect of AS-IV.ConclusionsAS-IV reduced the growth, invasion, migration, and angiogenesis of lung cancer by blocking the M2 polarization of macrophages partially through the AMPK signaling pathway, which appears to play an important role in AS-IV's ability to inhibit the metastasis of lung cancer.

Dataset Information

Modulating Macrophage Polarization through CCR2 Inhibition and Multivalent Engagement.

Publications

Modulating Macrophage Polarization through CCR2 Inhibition and Multivalent Engagement.

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