Int J Stem Cells.  2022 May;15(2):203-216. 10.15283/ijsc21178.

Diesel Exhaust Particles Impair Therapeutic Effect of Human Wharton’s Jelly-Derived Mesenchymal Stem Cells against Experimental Colitis through ROS/ERK/cFos Signaling Pathway

• Park HS
• Oh MK
• Lee JW
• Chae DH
• Joo H
• Kang JY
• An HB
• Yu A
• Park JH
• Yoo HM
• Jung HJ
• Choi U
• Jung JW
• Kim IS
• Oh IH
• Yu KR

• KMID: 2529835
• DOI: http://doi.org/10.15283/ijsc21178

Abstract

Background and Objectives
Epidemiological investigations have shown positive correlations between increased diesel exhaust particles (DEP) in ambient air and adverse health outcomes. DEP are the major constituent of particulate atmospheric pollution and have been shown to induce proinflammatory responses both in the lung and systemically. Here, we report the effects of DEP exposure on the properties of human Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs), including stemness, regeneration, and immunomodulation.
Methods and Results
Non-apoptotic concentrations of DEP (10 μg/ml) inhibited the migration and osteogenic differentiation capacity of WJ-MSCs. Gene expression profiling showed that DEP increased intracellular reactive oxygen species (ROS) and expression of pro-inflammatory and metabolic-process-related genes including cFos. Furthermore, WJ-MSCs cultured with DEP showed impaired suppression of T cell proliferation that was reversed by inhibition of ROS or knockdown of cFos. ERK inhibition assay revealed that DEP-induced ROS regulated cFos through activation of ERK but not NF-κB signaling. Overall, low concentrations of DEP (10 μg/ml) significantly suppressed the stemness and immunomodulatory properties of WJ-MSCs through ROS/ERK/cFos signaling pathways. Furthermore, WJ-MSCs cultured with DEP impaired the therapeutic effect of WJ-MSCs in experimental colitis mice, but was partly reversed by inhibition of ROS.
Conclusions
Taken together, these results indicate that exposure to DEP enhances the expression of pro-inflammatory cytokines and immune responses through a mechanism involving the ROS/ERK/cFos pathway in WJ-MSCs, and that DEP-induced ROS damage impairs the therapeutic effect of WJ-MSCs in colitis. Our results suggest that modulation of ROS/ERK/cFos signaling pathways in WJ-MSCs might be a novel therapeutic strategy for DEP-induced diseases.

Keyword

Diesel exhaust particle; Mesenchymal stem cell; Oxidative stress; ERK; cFos; Immunomodulation

Figure

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  Fig. 1 Diesel exhaust particles (DEP) affect WJ-MSC function. (a, b) The WJ-MSCs were treated with DEP (0∼40 μg/ml) for 48 hr. (a) Cell viability was assessed using an MTT assay. (b) Early and late apoptosis were measured by labeling with Annexin-V-FITC and 7-AAD for flow cytometry analysis. (c) A scratched wound healing assay of DEP-treated WJ-MSCs (0∼10 μg/ml) was performed to analyze cell migration into the scratched area. After 18 and 36 hr DEP treatment, migration into the scratched area was measured. (Scale bar: 200 μm). (d, e) DEP-treated WJ-MSCs were cultured with osteogenic differentiation medium for 21 days. (d) Representative light microscopic images of osteocytes. Calcium deposits in cells cultured under osteogenesis were stained with Alizarin Red S (scale bar: 100 μm) and quantified by measuring absorbance using a spectrophotometer. Blue arrows indicate DEP. (e) Expression of an osteogenic gene (RUNX2) was quantified by qRT-PCR with β-actin as the reference gene. Data are presented as the mean±S.D. of triplicate experiments (*p<0.05; **p<0.01; ***p<0.001).

• 

  Fig. 2 DEP-treated WJ-MSCs express ROS and pro-inflammatory genes. (a) Heatmap of hierarchical clustering. Differentially expressed gene (DEG) profile for the control and DEP-treated WJ-MSCs were analyzed (p-value<0.05). (b) Volcano plot highlighting DEGs for the control and DEP-treated WJ-MSCs and notable genes of interest. The blue dots represent significantly upregulated genes, and the red dots represent significantly downregulated genes (|log2 FC|≥1 and FDR<0.01). (c) Transcription of cFos was quantified by qRT-PCR. (d) The WJ-MSCs were incubated with DEP at the indicated concentrations for 6 hr. Intracellular ROS levels were measured using the ROS-sensitive fluorophore 2’,7’-dichlorofluorescin diacetate (DCFDA) and analyzed by flow cytometry. (e) Transcription of ROS-related genes was determined by qRT-PCR and normalized to β-actin expression. (f) Transcription of inflammation-related genes was determined by qRT-PCR and normalized to β-actin expression. The data are presented as the mean±S.D. of three independent experiments (*p<0.05; **p<0.01; ***p<0.001).

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  Fig. 3 DEP-induced ROS inhibits the immunomodulatory effects of WJ-MSCs. (a∼c) WJ-MSCs were pretreated for 30 min with NAC (5 mM) and then were mitotically inactivated after treating with various concentrations of DEP for 48 hr. Cells were co-cultured with CFSE-labeled human mononuclear cells (MNCs) that were activated by CD3/CD28 Dynabeads and IL-2. After six days of co-culture, the percentage of T cell proliferation was measured using flow cytometry. (a) Representative histogram of total lymphocyte proliferation. (b) Proliferation of total, CD4＋ T, and CD8＋ T lymphocytes was quantified. (c) The number of cell divisions of total, CD4＋ T, and CD8＋ T lymphocytes was quantified based on dilution of CFSE. (d, e) Cells were pretreated for 30 min with NAC and then were stimulated with 10 μg/ml DEP for 24 hr. (d) cFos mRNA was determined by qRT-PCR and normalized to β-actin mRNA. (e) cFos expression was quantified by western blot analysis. The data represent results from three independent experiments and are presented as mean±SD (*p<0.05; **p<0.01; ***p<0.001).

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  Fig. 4 DEP-induced cFos expression inhibits the immunomodulatory function of WJ-MSCs. WJ-MSCs were transfected with control siRNA (si-cont) or cFos siRNA (si-cFos) for 4 hr and then were stimulated with 10 μg/ml DEP. After 12 hr, knockdown of cFos was confirmed by qRT-PCR (a) and western blot (b). (c∼e) After 48 hr of incubation, cells were mitotically inactivated and co-cultured with MNCs as previously described. (c) Representative histogram of total lymphocyte proliferation. (d) Proliferation of total, CD4＋ T, and CD8＋ T lymphocytes was quantified. (e) Number of cell divisions of total, CD4＋ T, and CD8＋ T lymphocytes was quantified based on dilution of CFSE. (f) si-cFos-transfected WJ-MSCs were stimulated with DEP for 24 hr and then cultured with osteogenic differentiation medium for 14 days. Calcium deposits were stained with Alizarin Red S. Light microscopy images of osteocytes show representative images from three independent experiments (Scale bar: 100 μm). Absorbance of calcium deposits was measured using a spectrophotometer. (g) Transcription of IL-1β, IL-6, IL-8, IL-18, and NLRP3 was determined by qRT-PCR and normalized to β-actin transcription. Data are presented as mean±S.D. of triplicate experiments (*p<0.05; **p<0.01; ***p<0.001).

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  Fig. 5 DEP regulates cFos through ERK signaling. (a) WJ-MSCs were treated with DEP (0∼10 μg/ml) for 1 hr. Activation of p-ERK and p-IκBα was determined by western blot analysis. (b, c) WJ-MSCs were pretreated with U0126 (10 μM) or PTL (5 μM) and then were treated with 10 μg/ml of DEP for 1 hr. Expression of p-ERK, p-IκBα, and cFos was analyzed and quantified by western blotting (ns>0.05; *p<0.05; **p<0.01).

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  Fig. 6 DEP-induced ROS impairs the therapeutic effect of WJ-MSCs in colitis model. (a) The percentage of body weight change during entire experiment. (b) DAI scores were measured on day 7. (c) The survival rate of mice. (d) Mice were sacrificed on day 12, the length of colons was measured. (e) MPO activity was measured on day 12. (f) Colon tissue were examined using H&E staining and (g) histological scores were shown. The scale bars represent 500 μm and 200 μm. Expression levels of inflammatory cytokines (h) and Th1, Th17 and Treg associated cytokines (i) in colonic tissues were analyzed by qRT‐PCR (n=3 mice per group; *p<0.05; **p<0.01; ***p<0.001).

Reference

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