Hypoxia Impairs Mesenchymal Stromal Cell-Induced Macrophage M1 to M2 Transition

Abstract

The transition of macrophages from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype is crucial for the progression of normal wound healing. Persistent M1 macrophages within the injury site may lead to an uncontrolled macrophage-mediated inflammatory response and ultimately a failure of the wound healing cascade, leading to chronic wounds. Mesenchymal stromal cells (MSCs) have been widely reported to promote M1 to M2 macrophage transition; however, it is unclear whether MSCs can drive this transition in the hypoxic environment typically observed in chronic wounds. Here we report on the effect of hypoxia (1% O₂) on MSCs' ability to transition macrophages from the M1 to the M2 phenotype. While hypoxia had no effect on MSC secretion, it inhibited MSC-induced M1 to M2 macrophage transition, and suppressed macrophage expression and production of the anti-inflammatory mediator interleukin-10 (IL-10). These results suggest that hypoxic environments may impede the therapeutic effects of MSCs.

Fig. 1

LPS augments macrophage and MSC secretion. Immobilized MSCs or M1 macrophages were cultured with or without LPS for 48 hours under normoxia or hypoxia. (A) TNF-α and IL-10 proteins secreted by macrophages. N=4; *:p<0.0001 compared to no LPS (B) TNF-α, IL-10, PGE2, and TGF-β1 secreted by MSCs. N=3 – 6; *:p<0.0001 compared to no LPS.

Fig. 2

Hypoxia regulates macrophage function. LPS-stimulated M1 macrophages were cultured for 48 hours under normoxia or hypoxia. (A) HIF-1α protein expression in macrophages was determined by Western blot. GAPDH was used as the loading control. (B) Percent macrophage viability was determined by fluorescent live/dead staining. Calcein AM⁺/EthD-1⁻ cells were deemed viable. N=3. (C) PGE2 secreted by MSCs. N=4. (D) TNF-α, IL-10, and IDO gene expressions by macrophages. N=4; *:p=0.05.

Fig. 3

MSCs upregulate CD206 expression on macrophages. LPS-stimulated M1 macrophages were cultured in MSC-conditioned medium or sham-conditioned medium for 48 hours under normoxia or hypoxia. (A) CD206 (M2 macrophage marker) expression in macrophages was examined by fluorescent microscopy. Scale bar = 20μm. Quantification of CD206⁺ cells. N=6; **:p<0.0001 compared to both MSC groups under normoxia; +:p<0.01 compared to MSC-CM group under hypoxia; *:p<0.001.

Fig. 4

MSCs induce an M2 secretion profile by macrophages. M1 macrophages were co-cultured with MSCs for 48 h under normoxia or hypoxia. (A) TNF-α protein secreted by macrophages. N=6; *:p<0.0001 compared to both MSC groups and the PGE2 group in both normoxia and hypoxia. +:p<0.05 compared to both MSC groups. #:p<0.05. (B) IL-10 protein secreted by macrophages. N=6; *:p<0.0001 compared to both MSC groups and the PGE2 group in normoxia. +:p<0.01 compared to both MSC groups and the PGE2 group in hypoxia. (C) TGF-β1 protein secreted by macrophages. N=6; *:p<0.001 compared to both MSC groups and the PGE2 group in normoxia. +:p<0.01 compared to both MSC groups and the PGE2 group in hypoxia.