Int J Stem Cells.  2021 Feb;14(1):58-73. 10.15283/ijsc19121.

TL1A/TNFR2 Axis Enhances Immunoregulatory Effects of Bone Marrow Derived Mesenchymal Stem Cell by Indian Hedgehog Signaling Pathway

• Al-Azab M
• Walana W
• Wei J
• Li W
• Tang Y
• Wei X
• Almoiliqy M
• Shopit A
• Abbas EE
• Adlat S
• Awsh M
• Li X
• Wang B

• KMID: 2513083
• DOI: http://doi.org/10.15283/ijsc19121

Abstract

Background and Objectives
The immunomodulatory potential of mesenchymal stem cells (MSCs) can be regulated by a variety of molecules, especially cytokines. The inflammatory cytokine, TNF-like ligand 1A (TL1A), has been reported as an inflammation stimulator in-multiple autoimmune diseases. Here, we studied the effects of TL1A/TNF-receptor 2 (TNFR2) pathway on the therapeutic potency of bone marrow-derived MSCs (BMSCs).
Methods and Results
BMSCs, fibroblast-like synoviocytes (FLSs), and H9 and jurkat human T lymphocytes were used in this study. BMSCs paracrine activities, differentiation, proliferation, and migration were investigated after stimulation with TL1A, and intervened with anti-TNFR2. Additionally, the effects of TL1A on BMSCs therapeutic potency were evaluated by treating RA-FLSs, and H9 and jurkat T cells with TL1A-stimulated BMSCs conditioned medium (CM). Indian hedgehog (IHH) involvement was determined by gene silencing and treatment by recombinant IHH (rIHH). TL1A induced BMSCs stemness-related genes, COX-2, IL-6, IDO, TGF-β and HGF through TNFR2. Also, TL1A corrected biased differentiation and increased proliferation, and migration through TNFR2. Meanwhile, CM of TL1A-stimulated BMSCs decreased the inflammatory markers of RA-FLSs and T cells. Moreover, TL1A-stimulated BMSCs experienced IHH up-regulation coupled with NF-κB and STAT3 signaling up-regulation, while p53 and oxidative stress were down-regulated. Furthermore, treatment of BMSCs by rIHH increased their anti-inflammatory effects. More importantly, knockdown of IHH decreased the ability of TL1A-stimulated BMSCs to alleviating the inflammation in RA-FLSs and T cells.
Conclusions
This study reports the effects of TL1A/TNFR2 pathway on the biological behaviors and therapeutic potency of BMSCs through IHH. These findings could introduce novel procedures to increase the stemness of MSCs in cellular therapy.

Keyword

Bone marrow-derived mesenchymal stem cells; Tumor necrosis factor-like ligand 1A; TNF-receptor 2; Indian hedgehog

Figure

• 

  Fig. 1 TL1A induced paracrine activity of BMSCs. (a∼f) Cytokines expression in BMSCs. GAPDH used as reference. (g) The expre-ssion of TNFR2 in BMSCs. Results are representative of three inde-pendent experiment, and data presented as mean±SEM. *p< 0.05, **p<0.01. ****p<0.0001 compared to control. ##p<0.01, ###p<0.001 compared to TL1A treatment.

• 

  Fig. 2 TL1A promoted osteogenic differentiation at the cost of adipogenic differentiation. (a, b) Osteoge-nic stain and measure of stain inten-sity. (c, d) Adipogenic stain and measure of stain intensity. (e, f) Flow cytometry of cells apoptosis and apoptotic cells percentages. Data presented as mean±SEM from three independent experiments. ns>0.05, *p<0.05, **p<0.01 compared to control. #p<0.05, ##p<0.01 compared to TL1A treatment.

• 

  Fig. 3 TL1A promoted BMSCs proliferation and migration. (a, b) Colony forming assay after 12 days of treatments and continuous culture. (c, d) Transwell migration assay after 24h of treatment. (e, f) Wound healing assay for 0 h and 24 h after scratch. Results are representative of three independent experiment, and data presented as mean±SEM. *p<0.05, **p<0.01 compared to control. #p<0.05, ##p<0.001 compared to TL1A treatment.

• 

  Fig. 4 TL1A increased anti-inflammatory effect of BMSCs on human T cells. Representative RT-PCR bands and quantification of inflammatory cytokines expressed by H9 T cells (a∼l). GAPDH used as reference. (m∼p) IL-6, IL-18, IL-1β, and IL-8 levels in jurkat T cells measured by ELISA. Results are representative of three independent experiment, and data presented as mean±SEM. ns>0.05, *p<0.05, **p<0.01, ***p<0.001 compared to BMSC-CM.

• 

  Fig. 5 TL1A induced BMSCs anti-inflammatory effect on FLSs from rheumatoid arthritis patients. Representative RT-PCR bands and quantification of inflammatory cytokines expressed by RA-FLSs (a∼j). GAPDH used as reference. (k∼n) IL-6, IL-18, IL-1β, and IL-8 levels in RA-FLSs cells measured by ELISA. Results are representative of three independent experiment, and data presented as mean±SEM. ns>0.05, *p<0.05, **p<0.01, ***p<0.001 compared to BMSC-CM.

• 

  Fig. 6 TL1A modulated IHH expression and stemness-related signaling pathways in BMSCs. (a∼d) Representative western blot bands and relative band intensity of p65-NF-kβ, p-STAT3, and IHH. (e∼h) PCR bands and estimation of mRNA expression of some aging-related genes. β-actin and GAPDH used as references. (i∼k) Representative flow cytometry and microscopic (at 20X magnification) images and quantification of ROS generation. Results are representative of three independent experiment, and data presented as mean±SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to control. #p<0.05, ##p<0.001 compared to TL1A treatment.

• 

  Fig. 7 IHH induced anti-inflammatory power of BMSCs. (a∼d) IL-6, IL-18, IL-1β, and IL-8 levels in jurkat T cells measured by ELISA. (e∼h) IL-6, IL-18, IL-1β, and IL-8 levels in RA-FLSs cells measured by ELISA. Results are representative of three independent experiment, and data presented as mean±SEM. ns>0.05, *p<0.05, **p<0.01, ***p<0.001 compared to BMSC-CM.

• 

  Fig. 8 Effect of TL1A-BMSCs-IHH conditioned medium on H9 T cells and FLSs from rheumatoid arthritis patients. Representative RT-PCR bands and quantification of inflammatory mediators expressed by H9 T cells (a∼h), and RA-FLS (i∼m). GAPDH used as reference. Results are representative of three independent experiment, and data presented as mean±SEM. ns>0.05, *p<0.05, **p<0.01, ***p<0.001 compared to BMSC-CM.

Reference

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