Umbilical cord-derived mesenchymal stromal cells immunomodulate and restore actin dynamics and phagocytosis of LPS-activated microglia via PI3K/Akt/Rho GTPase pathway

Abstract

Microglia are the immune cells in the central nervous system surveying environment and reacting to various injuries. Activated microglia may cause impaired synaptic plasticity, therefore modulating and restoring them to neutral phenotype is crucial to counteract a pro-inflammatory, neurotoxic state. In this study, we focused on elucidating whether human umbilical cord (UC) -derived mesenchymal stromal cells (MSCs) can exert immunomodulatory effect and change the phenotype of activated microglia. Primary culture of microglia was activated by lipopolysaccharide (LPS) and was co-cultured with three lots of MSCs. We investigated immunomodulation, actin dynamics and phagocytic capacity of activated microglia, and examined change of Rho GTPase in microglia as the mechanism. MSCs suppressed the expression of IL-1β and pNFκB in LPS-activated microglia, and conversely elevated the expression of IL-1β in resting-surveying microglia with lot-to-lot variation. Morphological and phagocytotic analyses revealed that LPS stimulation significantly increased active Rho GTPase, Rac1, and Cdc42 levels in the microglia, and their morphology changed to amoeboid in which F-actin spread with ruffle formation. The F-actin spreading persisted after removal of LPS stimulation and reduced phagocytosis. On the other hand, MSC co-culture induced bimodal increase in active Rac1 and Cdc42 levels in LPS-activated microglia. Moreover, extended ruffles of F-actin shrinked and concentrated to form an actin ring, thereby restoring phagocytosis. We confirmed inhibition of the PI3K/Akt pathway attenuated F-actin dynamics and phagocytosis restored by MSCs. Overall, we demonstrated that MSCs immunomodulated microglia with lot-to-lot variation, and changed the phenotype of LPS-activated microglia restoring actin dynamics and phagocytosis by increase of active Rho GTPase.

Fig. 1. Experimental design and characteristics of primary culture of microglia and 3 lots of MSC.

In this experiment, microglia were stimulated with LPS for 6 h and backed to culture in NM. In LPS + MSC group, co-cultured with MSC was started 3 h after LPS stimulation. The same procedure was performed without LPS stimulation for NM and NM + MSC groups (a). Immunocytochemistry of EYFP stained with anti-GFP antibody (green), Iba-1 (red) and Hoechst 33342 (blue) (scale bar = 50 μm) (b). The expression of Iba-1, GFAP, TUBB3 and Olig2 in microglia normalized to whole brain tissue by qPCR (c). Phase contrast images of 3 lots MSC (scale bar = 100 μm) (d). Cell number (line) and PDL (bar) of 3 lots MSC (e). Surface markers of MSC that positive for CD73, CD105, CD90, HLA-ABC, and CD44, and negative for CD45, CD34, CD11b, CD 19 and HLA–DR (f). Abbreviation: NM: normal medium, PDL: population doubling level.

Fig. 2. MSC exert immunomodulatory effect on LPS activated microglia.

qPCR analysis of IL-1β, TNF-α, IL-10 and TGF-β genes in microglia. The expression of each gene was calculated by normalization to GAPDH. Data are presented as mean ± SEM. **p < 0.01; LPS group compared to NM group. ^§p < 0.05; NM + MSC lot1 group compared to the NM group. ^#p < 0.05; LPS + MSC lot1 group compared to the LPS group. ^††p < 0.01; LPS + MSC lot2 group compared to the LPS group. ^‡‡p < 0.01, ^‡p < 0.05; LPS + MSC lot3 group compared to the LPS group (unpaired t-test and One-way ANOVA followed by Sidak’s multiple comparisons test for IL-10, and Mann–Whitney U-test and Dunn’s multiple comparisons test for IL-1β, TNF-α and TGF-β were used) (a). Western blotting results of NFκB, pNFκB, iNOS and Arg1. GAPDH was used as an internal control (b). Quantification of Western blotting. Data are presented as mean ± SEM with *p < 0.05 (One-way ANOVA followed by Sidak’s multiple comparisons test for iNOS and Dunn’s multiple comparisons test for Arg1, pNFκB and NFκB were used) (c). Immunocytochemistry of microglia stained with Iba-1 (white), CD86 (red), CD206 (green), and Hoechst 33342 (blue) (scale bar = 20 μm) (d). FACS analysis of microglia using CD86 and CD206 markers (e). Quantification of CD86 positive cells (f), and CD206 positive cells (g). Data are presented as mean ± SEM (One-way ANOVA followed by Sidak’s multiple comparisons test for CD206, and Dunn’s multiple comparisons test for CD86 were used).

Fig. 3. Actin dynamics of LPS activated microglia change after co-culture with MSC.

Phase contrast images of four groups (scale bar = 100 μm) (a). Immunocytochemistry of F-Actin (black), vinculin (purple) and Hoechst 33342 (blue) (scale bar = 20 μm) (b). For skeleton analysis, the images underwent background subtraction, binarized and skeletonized for quantification (c). Quantification on the number of branches (d), junctions (e), processes (f), and F-Actin (g). (Dunn’s multiple comparisons test was used). Data are presented as mean ± SEM with **p < 0.01, *p < 0.05. Abbreviation: A.U., arbitrary units.

Fig. 4. Co-culture with MSC enhance phagocytosis in LPS activated microglia.

Immunocytochemistry of E. coli bioparticles (red) and Hoechst 33342 (blue) (a), and merge with EYFP (green) (b) (scale bar = 100 μm). High power field of Fig. 4b (c) (scale bar = 20 μm). F-Actin (black), E. coli bioparticles (red) and Hoechst 33342 (blue) (d) (scale bar = 20 μm). Quantification of phagocytosis (Dunn’s multiple comparisons test was used). Data are presented as mean ± SEM with **p < 0.01, *p < 0.05 (e). Abbreviation: A.U., arbitrary units.

Fig. 5. Co-culture with MSC enhance the activation and expression of Rho GTPase in LPS activated microglia.

Western blotting images of Rho GTPase, Rac1 and Cdc42 in 2 min, 5 min, and 3 h after LPS stimulation and 5 min, 30 min, 3 h and 24 h after MSC co-culture. The data represent at least three individual experiments (a). Quantification of Western blotting showed by time course. *p < 0.05; LPS group compared to NM group. ^§p < 0.05; NM + MSC lot1 group compared to the NM group. ^##p < 0.01; LPS + MSC lot1 group compared to the LPS group. ^†p < 0.05; LPS + MSC lot2 group compared to the LPS group. ^‡‡p < 0.01, ^‡p < 0.05; LPS + MSC lot3 group compared to the LPS group. The expression level of Rac1-GTP and Cdc42-GTP were calculated by normalization to total Rac1 and total Cdc42, and the expression level of total Rac1 and total Cdc42 were calculated by normalization to GAPDH. (Mann–Whitney U-test and Dunn’s multiple comparisons test were used) (b–e).

Fig. 6. Inhibition of PI3K-Akt pathway blocked the activation of Rho GTPase and change of phenotype in LPS activated microglia.

Western blotting images of pAkt, Akt, and Rho GTPase in 30 min after MSC co-culture. The data represent at least three individual experiments (a). Phase contrast images of four groups (scale bar = 100 μm) and immunocytochemistry of F-Actin (black), bioparticles (purple) and Hoechst 33342 (blue) (scale bar = 20 μm) (b). Quantification of the number of branches (c), junctions (d), processes (e), and phagocytosis (f). (Dunn’s multiple comparisons test were used). Data are presented as mean ± SEM with **p < 0.01, *p < 0.05. Abbreviation: A.U., arbitrary units.