Enhancement of Alternatively Activated Macrophages’ Phenotype and Function

Maria, Osama 2; Kamal, Ahmad 2; Chan, Mable 2; Bhatt, Shashank 2; Gomez-Aristizábal, Alejandro 2; Viswanathan, Sowmya 1, 2

1. IBBME, University of Toronto; 2. Genetics and Development department, Krembil research institute, University Health Network, U of Toronto

BackgroundMacrophages (Mфs) are tissue resident scavenger and antigen-presenting cells that are developed from the peripheral blood mononuclear cells (PBMC), the monocytes (Mcs). Three major categories of Mфs were identified by researchers: classically activated Mфs (M1) with pro-inflammatory properties, alternatively activated macrophages (M2) with anti-inflammatory properties, and naive Mфs (M0). Materials and methodsMurine bone marrow was collected and cultured with macrophage colony stimulating factor (M-CSF) for 5 and 7 days I order to develop Mcs-Mфs population. Cells (5 days mixed Mcs-Mфs or 7 days pure Mфs) were either, polarized to M1 (with IFNγ and LPS), M2 (with IL-10 and TGF-β1), or kept non-polarized in naive status M0. Cells were exposed to a 24 hours conditioned media (CM) of the other polarized phenotypes. Phenotype expression markers and functionality were studied by flow cytometry and PCR.Results7 days pure culture Mфs (only, attached cells collected) showed enhanced expression of phenotype-specific surface markers (CD11b, F4/80, and CD115) compared to 5 days mixed culture Mcs-Mфs (both floating and attached cells collected). All cells were, as expected, negative for CD3є, Ly-6G, and CD45R/B220 surface markers. Polarization verification experiments showed successful differential M2 (CD86low, MHCII IA-IElow and B7H4high) and M1 (CD86high, MHCII IA-IEhigh and B7H4low) in both compared population (Mфs and Mcs-Mфs). Gene expression profiling confirmed the stable polarized phenotypes with Arginasehigh and CD163high in M2 and (IL1-RA, CCR7, IL1-β, Il6, IL12-α and β, TNF-α, and iNOS)high in M1 polarization. Endocytosis functional assay showed superior endocytic capacity of M2 over M1 cells in Mфs pure culture only, but not in Mcs-Mфs mixed culture. Interestingly, M1 Mcs-Mфs challenged with M2 Mcs-Mфs CM for 24 hours showed gene expression shift towards M2 phanorype (TGF-β, CD206, Arginage.1, YAM, and OSM)high and ((IL1-RA, CCR7, IL1-β, Il6, IL12-α and β, TNF-α, and iNOS)low. In the contrary of that, M2 Mфs challenged with M1 Mфs CM for 24 hours showed enhanced phagocytic capacity with higher F4/80 and B7H4 expression with evidence of stable phenotype (unchanged MHCII IA-IE expression).ConclusionPure culture Mфs showed more enhanced alternatively activated M2 phenotype. M2 Mфs showed stable and more dominant phenotype when present with classically activated M1 Mфs.

 

Macrophages (Mфs) are tissue resident scavenger and antigen-presenting cells that are developed from the peripheral blood mononuclear cells (PBMC), the monocytes (Mcs). Three major categories of Mфs were identified by researchers: classically activated Mфs (M1) with pro-inflammatory properties, alternatively activated macrophages (M2) with anti-inflammatory properties, and naive Mфs (M0).

Materials and methods

Murine bone marrow was collected and cultured with macrophage colony stimulating factor (M-CSF) for 5 and 7 days I order to develop Mcs-Mфs population. Cells (5 days mixed Mcs-Mфs or 7 days pure Mфs) were either, polarized to M1 (with IFNγ and LPS), M2 (with IL-10 and TGF-ß1), or kept non-polarized in naive status M0. Cells were exposed to a 24 hours conditioned media (CM) of the other polarized phenotypes. Phenotype expression markers and functionality were studied by flow cytometry and PCR.

Results

7 days pure culture Mфs (only, attached cells collected) showed enhanced expression of phenotype-specific surface markers (CD11b, F4/80, and CD115) compared to 5 days mixed culture Mcs-Mфs (both floating and attached cells collected).

All cells were, as expected, negative for CD3є, Ly-6G, and CD45R/B220 surface markers. Polarization verification experiments showed successful differential M2 (CD86low, MHCII IA-IElow and B7H4high) and M1 (CD86high, MHCII IA-IEhigh and B7H4low) in both compared population (Mфs and Mcs-Mфs).

Gene expression profiling confirmed the stable polarized phenotypes with Arginasehigh and CD163high in M2 and (IL1-RA, CCR7, IL1-ß, Il6, IL12-a and ß, TNF-a, and iNOS)high in M1 polarization.

Endocytosis functional assay showed superior endocytic capacity of M2 over M1 cells in Mфs pure culture only, but not in Mcs-Mфs mixed culture.

Interestingly, M1 Mcs-Mфs challenged with M2 Mcs-Mфs CM for 24 hours showed gene expression shift towards M2 phanorype (TGF-ß, CD206, Arginage.1, YAM, and OSM)high and ((IL1-RA, CCR7, IL1-ß, Il6, IL12-a and ß, TNF-a, and iNOS)low.

In the contrary of that, M2 Mфs challenged with M1 Mфs CM for 24 hours showed enhanced phagocytic capacity with higher F4/80 and B7H4 expression with evidence of stable phenotype (unchanged MHCII IA-IE expression).

Conclusion

Pure culture Mфs showed more enhanced alternatively activated M2 phenotype. M2 Mфs showed stable and more dominant phenotype when present with classically activated M1 Mфs.