Anti-Inflammatory Effect of Specialized Proresolving Lipid Mediators on Mesenchymal Stem Cells: An In Vitro Study

Abstract

An interconnection between tissue inflammation and regeneration has been established through the regulation of defense and repair mechanisms within diseased dental tissue triggered by the release of immune-resolvent mediators. To better our understanding of the role of specific pro-resolving mediators (SPMs) in inflamed human bone marrow-derived mesenchymal stem cells (hBMMSCs), we studied the effects of Resolvin E1 (RvE1) and Maresin 1 (MaR1) in lipopoly-saccharide (LPS) stimulated hBMMSCs. The hBMMSCs were divided into five different groups, each of which was treated with or without SPMs. Group-1: negative control (no LPS stimulation), Group-2: positive control (LPS-stimulated), Group-3: RvE1 100 nM + 1 μg/mL LPS, Group-4: MaR1 100 nM + 1 µg/mL LPS, and Group-5: RvE1 100 nM + MaR1100 nM + 1 μg/mL LPS. Cell proliferation, apoptosis, migration, colony formation, Western blotting, cytokine array, and LC/MS analysis were all performed on each group to determine the impact of SPMs on inflammatory stem cells. According to our data, RvE1 plus MaR1 effectively reduced inflammation in hBMMSCs. In particular, IL-4, 1L-10, and TGF-β1 activation and downregulation of RANKL, TNF-α, and IFN-γ compared to groups receiving single SPM were shown to be significantly different (Group 3 and 4). In addition, the LC/MS analysis revealed the differentially regulated peptide's role in immunological pathways that define the cellular state against inflammation. Inflamed hBMMSCs treated with a combination of Resolvin E1 (RvE1) and Maresin 1 (MaR1) promoted the highest inflammatory resolution compared to the other groups; this finding suggests a potential new approach of treating bacterially induced dental infections.

Keywords: LPS; Maresin1; Resolvin E1; SPMs; hBMMSCs; inflammation; stem cells.

Figure 1

Comparison of mean proliferation values among the five study groups across different time points. The proliferation and viability assays experiments were repeated three times (n = 3) and the average values used to confirm the findings. The proliferation rate of * Group-5 (RvE1 + MaR1 + LPS) was significantly higher than the other four groups (p ≤ 0.0001).

Figure 2

Rates of apoptosis in hBMMSCs in the control and experimental groups after RvE1 and MaR1 treatment alone or together in response to LPS-induced inflammation using Annexin V/propidium iodide double staining by flow cytometry. The experiments were repeated three times (n = 3) and there were no significant differences in apoptosis between the five study groups (p = 0.71). Representative graph shows the fractions of viable and dead cells across the different study groups.

Figure 3

Comparison of mean migration values among the five study groups across different time points. The migration assays experiments were repeated three times (n = 3) and the mean value of migration of * Group-5 (RvE1 100 nM + MaR1 100 nM + 1 μg/mL LPS), was significantly higher than the other four study groups across 1 h to 3.30 h (p ≤ 0.0001).

Figure 4

Clonogenic assay and plating efficiency at day 14 shows the mean values of CFUs among the five study groups, (a) Negative controls, (b) Positive controls, (c) RvE1 00 nM + 1 μg/mL LPS, (d) MaR1 100 nM + 1 μg/mL LPS and (e) RvE1 100 nM + MaR1 100 nM + 1 μg/mL LPS. Colony-forming unit (CFU) experiment was conducted three times (n = 3) for all experimental groups on three different plates simultaneously and average values calculated. Shown above is a representation of one of the stained plates (a–e) and quantitative histograms of averaged intensity representations. (f) CFU mean values of groups (3, 4, and 5) were significantly higher than controls as indicated in the figure. (**** p ≤ 0.0001).

Figure 5

The right panel of the graph shows the expression of IL-4, 1L-10 and TGF-β1 between the five study groups and the left panel shown the normalized average values of three experimental repeats (n = 3). The expression of TGF-β1, IL4 and IL-10 was significantly higher in Group-5 (RvE1 100 nM + MaR1 100 nM + 1 μg/mL LPS) compared to the other four groups (**** p ≤ 0.0001).

Figure 6

The right panel of the graph depicts the levels of protein expressions of RANKL, TNF-α, and IFN- γ by Western blot across the five study groups. Positive control has significantly higher RANKL and TNF- expression than the other four groups (Negative control, RvE1 + LPS, MaR1 + LPS, and RvE1 + MaR1 + LPS) (p ≤ 0.0001). Negative control group and Group 5 (RvE1+ MaR1 + LPS) have comparable levels of expression (p > 0.05). The left panel represents the normalized average values of three experimental repeats (n = 3) with their significant expressions. (**** p ≤ 0.0001).

Figure 7

The left panel of the graphs show the expression of anti-inflammatory cytokines (IL-4 and TGF-β1), and the right panel pro-inflammatory cytokines (IFN-γ and TNF-α) by human cytokine array assessment in the three analyzed groups. The experiment was repeated three times (n = 3). (*** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05).

Figure 8

Human cytokines Antibody array membranes showing the expression of IL-4, TGF-β1, TNF-α, and IFN-γ among the three SPM treated study groups. Owing to limited supply of membranes for multiple repeats for all samples and groups, the triplicate runs (n = 3) were performed only for the experimental groups.

Figure 9

(A) Principal components analysis using the expression dataset of 464 differentially expressed proteins between Negative control, Positive control, RvE1 + LPS, MaR1 + LPS, and RvE1+ MaR1 + LPS. (B) Hierarchical cluster analysis using the expression dataset of the 464 differentially expressed proteins as described above. (C) Hierarchical cluster analysis using the expression dataset of 33 anti-inflammatory and 5 pro-inflammatory proteins with their expression changes. The heat maps show the expression changes of these proteins among the five sample groups. The figures were partly generated using Qlucore Omics Explorer version 3.7 (Lund, Sweden).

Figure 10

Ingenuity pathway analysis (IPA) of differential proteins linked with inflammatory response, immune complex and cytokines/inflammasome. The figure was generated using a licensed Ingenuity pathway analysis program (www.qiagen.com, accessed on 15 May 2022).