Preconditioning of Rat Bone Marrow-Derived Mesenchymal Stromal Cells with Toll-Like Receptor Agonists

Abstract

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are dynamic cells that can sense the environment, adapting their regulatory functions to different conditions. Accordingly, the therapeutic potential of BM-MSCs can be modulated by preconditioning strategies aimed at modifying their paracrine action. Although rat BM-MSCs (rBM-MSCs) have been widely tested in preclinical research, most preconditioning studies have employed human and mouse BM-MSCs. Herein, we investigated whether rBM-MSCs modify their phenotype and paracrine functions in response to Toll-like receptor (TLR) agonists. The data showed that rBM-MSCs expressed TLR3, TLR4, and MDA5 mRNA and were able to internalize polyinosinic-polycytidylic acid (Poly(I:C)), a TLR3/MDA5 agonist. rBM-MSCs were then stimulated with Poly(I:C) or with lipopolysaccharide (LPS, a TLR4 agonist) for 1 h and were grown under normal culture conditions. LPS or Poly(I:C) stimulation did not affect the viability or the morphology of rBM-MSCs and did not modify the expression pattern of key cell surface markers. Poly(I:C) did not induce statistically significant changes in the release of several inflammatory mediators and VEGF by rBM-MSCs, although it tended to increase IL-6 and MCP-1 secretion, whereas LPS increased the release of IL-6, MCP-1, and VEGF, three factors that were constitutively secreted by unstimulated cells. The neurotrophic activity of the conditioned medium from unstimulated and LPS-preconditioned rBM-MSCs was investigated using dorsal root ganglion explants, showing that soluble factors produced by unstimulated and LPS-preconditioned rBM-MSCs can stimulate neurite outgrowth similarly, in a VEGF-dependent manner. LPS-preconditioned cells, however, were slightly more efficient in increasing the number of regrowing axons in a model of sciatic nerve transection in rats. In conclusion, LPS preconditioning boosted the production of constitutively secreted factors by rBM-MSCs, without changing their mesenchymal identity, an effect that requires further investigation in exploratory preclinical studies.

TLR3 Agonist Amplifies the Anti‐Inflammatory Potency of ADSCs via IL‐10‐Mediated Macrophage Polarization in Acute Pancreatitis

“…Further research revealed that MSC2 adopted an immune-suppressive phenotype by secreting anti-inflammatory soluble factors, including IDO, IL-10, PGE2, and TGF- β [ 24 ]. Conversely, MSC1 exhibited a phenotype characterized by high levels of CCL3, CCL4, CCL9, CCL10, IL-1 β , and TNF- α and low levels of IDO [ 25 ]. These findings suggest that the concept of MSCs polarization into an anti-inflammatory phenotype through distinct TLR agonists is an attractive strategy to enhance the anti-inflammatory capabilities of MSCs, and some studies have demonstrated poly(I:C)-pretreated MSCs exhibit strengthened effects in decreasing kidney ischemia/reperfusion injury [ 26 ].…”

Preconditioning mesenchymal stromal cells with flagellin enhances the anti‑inflammatory ability of their secretome against lipopolysaccharide‑induced acute lung injury

“…Although serum-free incubation decreased the viability of ADSCs, flagellin-activated ADSCs retained 73% viability after 48 h. Previous studies have reported that LPS, a TLR5 ligand, could protect MSCs from serum deprivation by stabilizing cell membrane and activating ERK and PI3K/Akt signaling pathways ( 35 , 36 ). Given the similarities between TLR4 and TLR5, flagellin may exert its beneficial effects in similar ways ( 23 ). Therefore, ultrafiltration units with a 3-kDa molecular weight cutoff were used to concentrate CM, to retain the highest number of inflammatory regulators as possible.…”

“…TLR ligations can activate the immune defensive function of MSCs and alter their secretome profiles. For example, TLR4 ligands can activate the NF-κB pathway in MSCs and induce MSCs to release a variety of molecules, including IL-6 and vascular endothelial growth factor ( 23 ). Therefore, priming MSCs with TLR ligations may be a promising method for enhancing their therapeutic effects.…”

“…Flagellin is a subunit protein of the flagellum, a whip-like appendage that enables bacterial motility, and it is a commonly used TLR5 ligand ( 24 ). The majority of previous studies have focused on preconditioning MSCs with TLR3 and TLR4 ligations ( 22 , 23 , 25 ). While Linard et al ( 21 ) reported the superior anti-inflammatory effect of flagellin-activated MSCs in an irradiation-induced proctitis model compared with normal MSCs, whether MSCs preconditioned with flagellin are effective in other inflammation-related diseases and the mechanism underlying the beneficial effects of flagellin preconditioning on the MSCs remain elusive.…”

Erratum to “Preconditioning of Rat Bone Marrow-Derived Mesenchymal Stromal Cells with Toll-Like Receptor Agonists”

“…In the article titled “Preconditioning of Rat Bone Marrow-Derived Mesenchymal Stromal Cells with Toll-Like Receptor Agonists” [ 1 ], there was an error in the production of Figure 2(b) which resulted in some colouration being lost. The publisher apologises for introducing this error, and the corrected figure is shown below and is listed as Figure 1 :…”