Toll-like receptor 3-stimulation and aggregate-formation synergistically enhances anti-inflammatory activity of feline mesenchymal stem cells

Youhei Fujimoto; Shingo Hatoya; Kikuya Sugiura; Yasuhiko Tabata

doi:10.4142/jvs.23330

. 2024 Nov 22;25(6):e86. doi: 10.4142/jvs.23330

Toll-like receptor 3-stimulation and aggregate-formation synergistically enhances anti-inflammatory activity of feline mesenchymal stem cells

Youhei Fujimoto ^1,², Shingo Hatoya ¹, Kikuya Sugiura ¹, Yasuhiko Tabata ^2,^✉

PMCID: PMC11611489 PMID: 39608780

Abstract

Importance

Mesenchymal stem cells (MSCs) used to treat inflammatory diseases in humans show improved clinical outcomes compared to other treatments. On the other hand, feline MSCs have limited therapeutic effects because of their low bioactivity. Successful clinical treatment requires enhancing the anti-inflammatory ability of feline adipose-derived MSCs (fAdMSCs).

Objective

To enhance the anti-inflammatory activity of fAdMSCs.

Methods

fAdMSCs were treated with the toll-like receptor 3 (TLR3) ligand poly (I:C) and aggregated. Indoleamine 2,3-dioxygenase-1 (IDO-1) expression and kynurenine production were measured to evaluate the anti-inflammatory activity. Anti-inflammatory effects were assessed by culturing fAdMSCs with rat macrophages and transplanting them into the kidney capsules of rats.

Results

IDO-1 expression and kynurenine production in fAdMSCs were increased significantly by a poly (I:C) treatment and enhanced using a basic fibroblast growth factor (bFGF) treatment. The level of fAdMSC aggregation increased IDO-1 expression significantly compared to the monolayer. These effects were enhanced by pretreatment with bFGF and poly (I:C). The bFGF and poly (I:C)-pretreated fAdMSC aggregates suppressed tumor necrosis factor-α expression in rat macrophages. During transplantation, the pretreated fAdMSC aggregates avoided leakage, survived in aggregate form, and induced anti-inflammatory macrophages.

Conclusions and Relevance

TLR3-stimulated, bFGF-pretreated fAdMSC aggregates increase the anti-inflammatory activity significantly, providing a potential therapeutic approach for inflammatory diseases in felines.

Keywords: Mesenchymal stem cells, feline, toll-like receptor 3, aggregates, basic fibroblast growth factor

INTRODUCTION

Mesenchymal stem cells (MSC), which differ from other somatic and pluripotent stem cells, have anti-inflammatory, tissue-protective, and anti-fibrotic properties [1]. These abilities are achieved by secreting bioactive substances such as indoleamine 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), hepatocyte growth factor (HGF), and tumor necrosis factor-stimulated gene/protein-6 (TSG-6) [2]. Accordingly, MSCs have been used in human therapies for conditions such as xenograft rejection and graft vs. host disease [3,4]. Furthermore, in the context of chronic kidney disease (CKD), MSCs ameliorate renal blood flow and the glomerular filtration rate [5]. Cats exhibit several inflammatory diseases. In particular, many elderly cats experience significant CKD-related problems [6]. On the other hand, feline MSCs (fMSCs) do not have a considerable effect on this disease [7]. In addition to the pathological conditions that depend on the species, the unsuccessful results in cats may be due to the lower biological activity of fMSC, the earlier loss of proliferative activity and stemness [8], and the poorer ability to produce bioactive mediators without stimulation [9].

A previous study reported that basic fibroblast growth factor (bFGF) significantly promoted the proliferation of fMSC while maintaining stemness and enhancing the production of tissue-protective factors, HGF and TSG-6 [10]. On the other hand, further improvements are required to enhance the anti-inflammatory activity for successful clinical treatment. The stimulation of pro-inflammatory cytokines, such as interferon γ (IFNγ) or toll-like receptors (TLR), was reported to enhance the anti-inflammatory activity of MSCs [9,11,12]. Of these stimulations, TLR3 stimulation elicited the anti-inflammatory activity of human MSCs and ameliorated drug-induced colitis in rats [13]. Aggregate formation by MSCs was reported to improve their anti-inflammatory and angiogenic properties and ameliorate fibrosis and stemness [14]. Moreover, MSCs in aggregate form significantly increased the number of remaining cells at the transplant site compared to those in single-cell form [15].

The present study examined the effects of TLR3 stimulation and aggregate formation on the anti-inflammatory activity of fMSCs. The synergy between bFGF and these treatments was also investigated.

METHODS

Preparation of fMSC

Subcutaneous adipose tissue was isolated during an ovariohysterectomy in a one-year-old client-owned cat in a clinically healthy condition. Standard clinical assessments were used to evaluate the health of the cat. Feline immunodeficiency virus and feline leukemia virus infections were ruled out using rapid diagnostic kits (Snap FIV/FeLV combo test, IDEXX Japan, Japan). Consent was obtained from the owner. All the experiments and procedures were conducted in accordance with the guidelines of the Japanese Society for Veterinary Regenerative Medicine. As described previously [10], feline adipose-derived MSCs (fAdMSCs) were obtained from adipose tissue cultures. The purity of fAdMSCs was examined by analyzing the resulting cells using flow cytometry for the markers of MSC using the following monoclonal antibodies (mAbs) conjugated with FITC: anti-CD29 mAb (clone TS2/16, Thermo Fisher Scientific K.K., Japan), anti-CD44 mAb (clone IM7, BD Biosciences, USA), anti-CD90 mAb (clone 5E10, BD Biosciences), and anti-CD105 mAb (clone SN6, Bio-Rad Laboratories, USA). As a negative control, the cells were examined for macrophage markers using FITC-anti CD14 mAb (clone TUK4, GeneTex, USA). The ability of the resulting cells to differentiate into osteoblasts or adipocytes was examined, as previously reported [10].

Animals

Ten- to eleven-week-old female Sprague–Dawley (SD) rats were purchased from Shimizu Laboratory Supplies (Japan). The Kyoto University Animal Experimentation Committee approved all animal experiments, which were performed in accordance with the guidelines for animal experiments of the Institute for Life and Medical Sciences, Kyoto University (approval number: F-21-252).

Treatment of fAdMSC

TLR3 was stimulated by suspending fAdMSCs were suspended in α-MEM (Nacalai Tesque, Japan) containing 15% fetal bovine serum (HyClone, GE Healthcare UK Ltd., England), penicillin and streptomycin (100 units/mL and 100 µg/mL, respectively, FUJIFILM Wako Pure Chemical Corporation, Japan) (hereafter, culture medium), seeding in wells of a six-well plate (AGC Techno Glass, Japan), and incubating them with a TLR3 ligand (poly [I:C], 1 μg/mL, FUJIFILM Wako Pure Chemical Corporation) for 1 h at 37°C in 5% CO₂ and 95% air. After incubation, the cells were washed twice with phosphate-buffered saline without Ca²⁺ or Mg²⁺ (phosphate-buffered saline [PBS] [−]).

The effects of bFGF on TLR3 stimulation were examined by culturing fAdMSCs with various concentrations of bFGF (ReproCELL, Japan) for three days, as described elsewhere [10].

Assay of anti-inflammatory activity of fAdMSCs

IDO, especially IDO-1, exhibits immunosuppressive effects [16] and produces kynurenines from tryptophan, which also exhibit immunosuppressive activity [17]. IDO-1 is a key factor responsible for the anti-immune effects of human MSCs [18,19]. Therefore, this study evaluated the anti-inflammatory activity of feline fAdMSCs based on IDO-1 expression and kynurenine production. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed or IDO-1 expression using the feline IDO-1-specific primers described in Supplementary Table 1 using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) and QuantStudio 3 (Thermo Fisher Scientific). As a reference, feline-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was similarly quantified (primers are listed in Supplementary Table 1). The results are expressed as the relative IDO-1 expression, which was calculated using the Ct values of IDO-1 and GAPDH. Kynurenine in the fAdMSC culture was evaluated using an ELISA pack (Immusmol, France) and expressed as the kynurenine/tryptophan ratio because kynurenine production is dependent on the tryptophan concentration. The expression of cyclooxygenase-2 (COX-2), which synthesizes PGE2 in MSCs and induces immunosuppression [20], was evaluated by qRT-PCR using the specific primers described in Supplementary Table 1. The results are expressed as the relative expression of COX-2, which was calculated as described above using the rat-actin gene as a reference.

Construction of adipose-derived (AdMSC) aggregates

fAdMSCs were detached from the culture dish using trypsin-EDTA and resuspended in a culture medium. Five thousand fAdMSCs were added to each well of a non-treated 96-well plate (Violamo, Japan), in which the cells did not adhere easily to the wall and remained in suspension. The plates were centrifuged for 1 min at 200 ×g to induce cell aggregation and incubated for 24 h at 37°C in 5% CO₂ to construct stable aggregates.

Transplantation of fAdMSC and assay of cell-remaining

The Slc:SD rats were anesthetized with medetomidine (0.3 mg/kg), midazolam (4.0 mg/kg), and butorphanol (5.0 mg/kg) (all from Meiji Seika Pharma, Japan). fAdMSCs (5 × 10⁵ cells) in aggregate or single-cell form were transplanted by injection into the space under the capsule of the left kidney using a 1 mL syringe with a 29G needle (Terumo, Japan). The injection hole was plugged with a finger for 30 sec. The remaining cells that avoided leakage from the injection site were evaluated by extracting the injected kidney 1 h after the injection and washing the fAdMSC-transplanted site under the renal capsule thoroughly with PBS (−) to collect the remaining cells. The number of collected cells was evaluated by qRT-PCR using the primers for feline-specific GAPDH (Supplementary Table 1), in which the Ct value of the sample was applied to a standard curve plotted with those obtained with known numbers of fAdMSCs (5.0 × 10⁵, 1.0 × 10⁵, 2.0 × 10⁴, and 5 × 10³). The results are expressed as the percentage of cells that remained at the injection site of the total number of injected cells (5 × 10³).

The surviving fAdMSCs at the transplantation site were traced by incubating the fAdMSCs with 1.2 mM carboxyfluorescein succinimidyl ester (CFSE; Dojin Kagaku, Japan) for 15 min before constructing the aggregate. CSFE-labeled fAdMSCs in aggregate or single-cell forms were injected as described above. Twenty-four hours after the injection, the injected kidneys were extracted and fixed with 3.7% formaldehyde in 0.1 M phosphate buffer (pH 7.4) (hereafter buffered formalin) for 24 h. The CSFE-labeled fAdMSCs in the frozen sections were detected using a fluorescence microscope (Keyence, Japan). In each experiment, six rats per group were used.

Evaluation of anti-inflammatory effects of fAdMSCs

The anti-inflammatory effects of fAdMSCs were evaluated in vitro by their ability to suppress the expression of tumor necrosis factor-α (TNF-α), an inflammatory factor in rat macrophages, as described by Nii and Tabata [21]. Briefly, macrophages (1 × 10⁵) collected from the peritoneal cavity of SD rats (six rats/group) were suspended in the culture medium and added to the wells of a 24-well culture plate (Corning, USA) with or without 10 ng/mL lipopolysaccharide (LPS) for 1 h. A transwell insert was placed in each well after washing. fAdMSCs (4 × 10⁴ cells) were added to the inserts and incubated for 48 h at 37°C in air containing 5% CO₂. Rat macrophages were incubated without fAdMSCs as a control. After incubation, TNF-α mRNA expression in rat macrophages was evaluated by qRT-PCR as described above using the specific primers shown in Supplementary Table 1. Rat β-actin mRNA expression was quantified as a reference.

For an in vivo evaluation of the anti-inflammatory effects of fAdMSCs, rat kidneys transplanted with fAdMSCs were collected five days after transplantation and fixed with buffered formalin. The types of macrophages that infiltrated the fAdMSC-injected site under the kidney capsule were examined in serial sections using immunohistochemistry. The macrophages or anti-inflammatory (M2) macrophages were detected by staining each adjacent section with rabbit anti-human CD68 antibody (Abcam, USA) [22] or a mAb against CD163 (clone EPR19518, Abcam) [23]. The expressing cells were visualized using peroxidase-labeled goat immunoglobulin G F (ab’) Ab (Vector Laboratories, USA) against primary antibodies. The CD68⁺ cells or CD163⁺ cells were evaluated using the Fiji/ImageJ plugin (https://fiji.sc/).

Statistical analysis

The quantitative data are expressed as the mean ± standard deviation. Statistical analysis among different groups was performed using one-way analysis of variance. A Bonferroni’s multiple comparison test and A student’s t-test were performed to compare pairs of groups. The differences between groups were considered significant at p < 0.05.

RESULTS

Characterization of adipose tissue-derived cells

First, this study examined the cells derived from adipose tissue cultures. Almost all cells expressed the MSC markers, such as CD29, CD44, CD90, and CD105, whereas few expressed the macrophage marker CD14, as shown in Supplementary Fig. 1A. The cells differentiated into osteoblasts, which were stained with alizarin red, and adipocytes, which were stained with Oil Red O (Supplementary Fig. 1B). These results are consistent with previous research (8), suggesting that almost all cells from the adipose tissue culture were MSCs. Therefore, the cultured cells were used as fAdMSCs for subsequent experiments.

Effect of TLR3-stimulation on anti-inflammatory activity of fAdMSCs

This study examined the effects of TLR3 stimulation on the anti-inflammatory activity of fAdMSCs. The expression of IDO-1 mRNA was evaluated because IDO-1 is a key factor contributing to the anti-inflammatory activity of MSCs [18]. Treatment with poly (I:C), a TLR3 ligand, significantly increased IDO-1 expression, as shown in Fig. 1A. bFGF enhanced the anti-inflammatory activity of fAdMSCs and promoted their proliferation [10]. Therefore, fAdMSCs were treated with bFGF before the poly (I:C) treatment. Pretreatment with 100 ng/mL bFGF significantly enhanced the increase in ferine-specific IDO-1 expression by the poly (I:C) treatment, as shown in Fig. 1B. Moreover, kynurenines secreted by fAdMSCs, which were produced from tryptophan by the catalysis of IDO and exhibited immunosuppressive activity, were increased significantly by the bFGF and poly (I:C) treatment compared to the poly (I:C) treatment alone (Fig. 1C). These results suggest that TLR3 stimulation significantly increases the anti-inflammatory activity of fAdMSCs and that bFGF synergistically augments the TLR3-stimulated activity.

Fig. 1 — IDO-1, indoleamine 2,3-dioxygenase-1; bFGF, basic fibroblast growth factor; TLR3, toll-like receptor 3; fAdMSC, feline adipose-derived mesenchymal stem cell.

^*p < 0.05.

Effect of TLR3-stimulation on anti-inflammatory activity of fAdMSC-aggregate

The effects of TLR3 stimulation on the inflammatory activity of the fAdMSC aggregates were examined. Poly (I:C)-treated fAdMSCs formed aggregates similar to those of non-treated fAdMSCs (Fig. 2A). Aggregate formation increased IDO-1 expression significantly (p = 0.021), as shown in Fig. 2B. Moreover, in addition to the fAdMSC monolayer, aggregates constricted with poly (I:C)-treated fAdMSCs (poly [I:C]-pretreated fAdMSC aggregates) exhibited relatively higher IDO-1 expression (p = 0.078) compared to the untreated fAdMSC aggregates. The increase in IDO-1 expression in poly (I:C)-pretreated fAdMSC aggregates was enhanced significantly by the bFGF-treatment treatment before the poly (I:C) treatment, as shown in Fig. 3A. In addition, kynurenine production from the poly (I:C)-pretreated fAdMSC aggregates was enhanced significantly by the bFGF treatment (Fig. 3B). The effects of the increased bFGF treatment time were examined. fAdMSCs were treated with bFGF before and after the poly (I:C) treatment and aggregated. The 2×bFGF and poly (I:C)-pretreated fAdMSC aggregates did not increase IDO-1 expression or kynurenine production further compared to the 1×bFGF and poly (I:C)-pretreated fAdMSC aggregates (Figs. 4A and B). On the other hand, the expression of COX-2, which produces PGE2 in human MSC to induce immunosuppression [20], was enhanced significantly by the 2×bFGF treatment (Fig. 4C). These results suggest that TLR3 stimulation increases the anti-inflammatory activity of fAdMSC aggregates and is synergistically augmented by bFGF. Therefore, 2×bFGF and poly (I:C)-pretreated fAdMSC aggregates were used in subsequent experiments.

Fig. 2 — IDO-1, indoleamine 2,3-dioxygenase-1; TLR3, toll-like receptor 3; fAdMSC, feline adipose-derived mesenchymal stem cell.

Fig. 3 — IDO-1, indoleamine 2,3-dioxygenase-1; bFGF, basic fibroblast growth factor; TLR3, toll-like receptor 3; fAdMSC, feline adipose-derived mesenchymal stem cell.

^*p < 0.05.

Fig. 4 — IDO-1, indoleamine 2,3-dioxygenase-1; bFGF, basic fibroblast growth factor; COX-2, cyclooxygenase-2; TNF-α, tumor necrosis factor-α; LPS, lipopolysaccharide; TLR3, toll-like receptor 3; fAdMSC, feline adipose-derived mesenchymal stem cell.

^*p < 0.05.

Anti-inflammatory effects of the fAdMSC-aggregate

Finally, this study examined the anti-inflammatory effects of bFGF and poly (I:C)-pretreated fAdMSC aggregates both in vitro and in vivo. An in vitro examination using transwell inserts was performed using the pretreated fAdMSC aggregates in noncontact incubation with rat peritoneal macrophages. The mRNA expression of TNF-α in rat macrophages was decreased significantly by the noncontact incubation with the pretreated fAdMSC-aggregates, as shown in Fig. 4D. This decrease was also observed in LPS-stimulated rat macrophages. These results suggest that the pretreated fAdMSC aggregates have anti-inflammatory effects by secreting anti-inflammatory factors as a mode of action.

For the in vivo examination, the pretreated fAdMSC aggregates were injected into the rat kidney capsule. Significantly more AdMSCs in aggregate form avoided leakage and remained at the injection site than single cells (Fig. 5A). The remaining fAdMSC aggregates survived for at least 24 h while maintaining their aggregate form (Fig. 5B). The type of cells that infiltrated in response to the transplantation stimuli five days after transplantation was then investigated, but no injected AdMSC aggregates were observed. The cells infiltrated were mainly CD68⁺ macrophages at the fAdMSC aggregate injection site and the PBS injection site (Fig. 6A), while more macrophages were observed at the aggregate injection site. On the other hand, in the adjacent serial section, CD163⁺ anti-inflammatory (M2) macrophages appeared to be a large population of those infiltrating the aggregate injection site. In contrast, a few CD163⁺ macrophages were observed in the PBS injection site. The ratio of CD163⁺ M2 macrophages to CD68⁺ macrophages in the section of the aggregate injection site was approximately 20% (Fig. 6B), which was significantly higher than that of the PBS injection site (< 0.05%). These results suggest that the transplanted fAdMSC aggregates induce anti-inflammatory effects in anti-inflammatory macrophages.

Fig. 5 — fAdMSC, feline adipose-derived mesenchymal stem cell; bFGF, basic fibroblast growth factor; CFSE, carboxyfluorescein succinimidyl ester.

^*p < 0.05.

Fig. 6 — fAdMSC, feline adipose-derived mesenchymal stem cell; PBS, phosphate-buffered saline; bFGF, basic fibroblast growth factor.

^*p < 0.05.

DISCUSSION

This study attempted to obtain fAdMSCs with potent therapeutic ability to treat chronic inflammatory diseases, especially CKD, for which no other method has been found.

Previous research has been conducted to enhance the therapeutic potential of MSCs for treating CKD by stimulating or aggregating TLR3, and it has been reported to improve the pathological condition of CKD [15,24]. The present study explored the combination of these two approaches for the first time.

First, this study assessed effective methods to enhance the anti-inflammatory activity of fAdMSCs. Stimulation of TLRs and pro-inflammatory cytokines, such as TNF-α, enhances the anti-inflammatory activity of MSCs [13]. TLR3-stimulation was reported to enhance the anti-inflammatory activity of human MSCs more effectively than TNF-α and IFNγ and ameliorate drug-induced colitis [13]. Hence, fAdMSCs were stimulated with a TLR3-ligand, poly (I:C) at a concentration of 1 μg/mL (cytotoxicity was observed at 10 and 100 μg/mL poly [I:C]), and significant enhancement of anti-inflammatory activity was obtained according to IDO-1 expression and kynurenine production. As mentioned earlier, IDO plays a central role in immune suppression through TLR signaling [19]. IDO generates kynurenine, which exerts inhibitory effects on numerous immune cells [25]. In addition, kynurenine, such as PGE2, is a small molecule that can act universally across different animal species. This study conducted analyses on vascular endothelial growth factor, HGF, TSG-6, interleukin (IL)-10, IL-6, TNF-α, and IL-1β, in addition to IDO. On the other hand, no significant changes were observed for any of these factors. TSG-6 and IL-10 showed an increasing trend, but reproducibility could not be achieved. LIF could not be measured as suitable primers. Therefore, this study focused the analysis on IDO. As mentioned in the main text, the anti-inflammatory capability of IDO holds particular significance among other anti-inflammatory factors. Evaluating IDO holds substantial value. A previous study found that bFGF significantly enhances the proliferation and tissue-protective activity of fAdMSCs. Therefore, this study examined the effects of bFGF on poly (I:C) treatment and found that a bFGF pretreatment significantly enhanced the anti-inflammatory activity of poly (I:C)-treated fAdMSCs. TLR3 stimulation was reported to increase PGE2 production by activating the Jagged 1-Notch 1 signaling pathway [13], and bFGF was reported to activate the Jagged 1-Notch 1 pathway [26]. Although it is unclear if the Jagged 1-Notch 1 pathway is involved in the increase in IDO-1 expression, the overlap of the signaling pathways might be related to the synergistic effects of the poly (I:C) and bFGF treatment. Therefore, the combination of poly (I:C) and bFGF is an efficient and effective method to augment the anti-inflammatory activity of fAdMSCs. This study assessed IDO expression at the mRNA level owing to the unavailability of commercially available antibodies specific to feline IDO protein. Furthermore, kynurenine, a metabolic product of IDO, was measured using a commercially available ELISA kit, allowing an indirect confirmation of the potential expression of the IDO protein. This study did not confirm whether the characteristics of stem cells change with incubation with the TLR3 ligand. Retaining the same characteristics as untreated MSCs is not necessary for MSCs sensitized with the TLR3 ligand because enhancing the anti-inflammatory ability of MSCs alters their original properties. This is considered sufficient, provided they function effectively as cells with anti-inflammatory activity.

This study assessed the appropriate conditions for transplanting AdMSCs. Aggregate formation facilitates cell–cell and cell–extracellular matrix interactions that are essential for maintaining cellular functions and survival, preventing apoptosis at the transplanted site [27]. Moreover, aggregates of human MSCs showed more potent anti-inflammatory activity than monolayers [14]. In this study, fAdMSC aggregates showed significantly higher anti-inflammatory activity than monolayer aggregates. Furthermore, the anti-inflammatory activity was enhanced when the aggregate was constructed with poly (I:C)-treated fAdMSCs. Moreover, the enhancement was promoted by a bFGF treatment performed before poly (I:C) treatment. The two-time treatment of bFGF, which was carried out before and after the poly (I:C) treatment, significantly enhanced COX-2 expression, which suppresses the production of pro-inflammatory factors, including TNF-α, from macrophages [13]. In addition, cell aggregation has been reported to produce mild hypoxic conditions, which increase PGE2 production [28]. Indeed, the 2×bFGF and poly (I:C)-treated fAdMSCs aggregate suppressed the TNF-α production from macrophages. These results suggest that bFGF- and poly (I:C)-pretreated fAdMSCs have potent anti-inflammatory activities. The highlighted kynurenine and PGE2 in this study are small-molecule bioactive substances not constrained by differences in animal species, enabling an assessment of the anti-inflammatory capabilities even in rat macrophages. Similar anti-inflammatory effects can be expected in feline macrophages.

During transplantation, fAdMSCs were injected under the kidney capsule but not into the kidney tissue, as performed by Burst et al. [29] and Xu et al. [15]. This method does not injure the kidney tissue, but the chance for transplanted cells to leak out from the injection site is increased. Nevertheless, more than 20% of fAdMSC aggregates avoided leakage, while less than 3% of fAdMSCs in single-cell form remained. The remaining aggregates survived for at least 24 h while maintaining their aggregate form. Although this study did not use a CKD mouse model directly, the aim was to investigate the underlying mechanisms responsible for the enhanced anti-inflammatory effects of fAdMSCs aggregates. The use of a CKD model could provide more clinically relevant insights. Therefore, this study focused on elucidating cellular and molecular pathways rather than replicating the complex pathophysiology of CKD. Future studies using CKD models will be needed to validate these findings in a more disease-specific context. The transplantation of bFGF and poly (I:C)-pretreated fAdMSC aggregates changed the population of infiltrating macrophages from the pro-inflammatory M1 type to the anti-inflammatory M2 type. PGE2 has been reported to induce M2 macrophage polarization [30]. A pretreatment with bFGF and poly (I:C) increased COX-2 expression significantly. These results suggest that bFGF- and poly (I:C)-treated fAdMSC aggregates exhibit anti-inflammatory effects in vivo by inducing M2 macrophages. bFGF enhances HGF and TSG-6 expression, which have anti-apoptotic and anti-inflammatory functions [10]. These cytokines protect kidney tissue from ischemia/reperfusion (I/R)-induced injury [15]. The bFGF- and poly (I:C)-pretreated fAdMSC aggregates were expected to enhance the expression of these cytokines. Injecting PBS or cell suspensions can act as inflammatory stimuli. Moreover, the introduction of heterologous cells can induce inflammation to some extent. Therefore, the emergence of CD168-positive macrophages holds significant implications. Overall, bFGF- and poly (I:C)-pretreated fAdMSC aggregates can exhibit significantly enhanced anti-inflammatory activity. Therefore, studies to investigate the specific functions of the kidneys and assess the therapeutic effects of pretreated fAdMSCs in a rat I/R model are currently underway. Single-donor experiments have limitations. Nevertheless, the primary objective of this study was to prove the concept that TLR3 stimulation and aggregated MSCs can have immunosuppressive effects. Therefore, using a single donor is sufficient for this purpose. In this study, protein expression was examined because of the unavailability of antibodies. On the other hand, confirmation of the increase in secondary products indirectly serves as validation for the expression. The characteristics of stem cell changes were not addressed in this study because they did not affect the function of the cells as a therapeutic agent.

ACKNOWLEDGMENTS

The authors wish to express their gratitude to Dr. Atsushi Yokoyama, Yokozeki Takeaki, and other staff members at the Sakura Animal Clinic for their dedicated support. The authors also acknowledge the Tabata Laboratory members for their invaluable comments and encouragement.

Footnotes

Conflict of Interest: The authors declare no conflicts of interest.

Author Contributions:

Data curation: Fujimoto Y.
Investigation: Fujimoto Y.
Project administration: Tabata Y.
Writing - original draft: Fujimoto Y.
Writing - review & editing: Hatoya S, Sugiura K, Tabata Y.

SUPPLEMENTARY MATERIALS

Supplementary Table 1

The sequences of primers used for real time polymerase chain reaction

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Supplementary Fig. 1

Characterization of fAdMSCs used in this study. (A) Flow cytometry analysis of MSC markers (CD29, CD44, CD90, and CD105) and a macrophage marker (CD14). Black peaks indicate expression of markers after staining with antibodies to the indicated markers, while gray peaks are unstained controls. Numbers in data indicate percentage of expressing cells. Reproducible results were obtained (n = 3). (B) Alizarin red and oil red O staining images of feline MSC after osteogenic and adipogenic induction cultures. The scale bar indicates 100 μm.

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27.Jauković A, Abadjieva D, Trivanović D, Stoyanova E, Kostadinova M, Pashova S, et al. Specificity of 3D MSC spheroids microenvironment: impact on MSC behavior and properties. Stem Cell Rev Rep. 2020;16(5):853–875. doi: 10.1007/s12015-020-10006-9. [DOI] [PubMed] [Google Scholar]
28.Ylöstalo JH, Bartosh TJ, Coble K, Prockop DJ. Human mesenchymal stem/stromal cells cultured as spheroids are self-activated to produce prostaglandin E2 that directs stimulated macrophages into an anti-inflammatory phenotype. Stem Cells. 2012;30(10):2283–2296. doi: 10.1002/stem.1191. [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Burst VR, Gillis M, Pütsch F, Herzog R, Fischer JH, Heid P, et al. Poor cell survival limits the beneficial impact of mesenchymal stem cell transplantation on acute kidney injury. Nephron, Exp Nephrol. 2010;114(3):e107–e116. doi: 10.1159/000262318. [DOI] [PubMed] [Google Scholar]
30.Yamamichi K, Fukuda T, Sanui T, Toyoda K, Tanaka U, Nakao Y, et al. Amelogenin induces M2 macrophage polarisation via PGE2/cAMP signalling pathway. Arch Oral Biol. 2017;83:241–251. doi: 10.1016/j.archoralbio.2017.08.005. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Table 1

The sequences of primers used for real time polymerase chain reaction

jvs-25-e86-s001.xls^{(38.5KB, xls)}

Supplementary Fig. 1

jvs-25-e86-s002.ppt^{(850.5KB, ppt)}

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[B17] 17.Fallarino F, Grohmann U, Vacca C, Orabona C, Spreca A, Fioretti MC, et al. T cell apoptosis by kynurenines. Adv Exp Med Biol. 2003;527:183–190. doi: 10.1007/978-1-4615-0135-0_21. [DOI] [PubMed] [Google Scholar]

[B18] 18.Jones BJ, Brooke G, Atkinson K, McTaggart SJ. Immunosuppression by placental indoleamine 2,3-dioxygenase: a role for mesenchymal stem cells. Placenta. 2007;28(11-12):1174–1181. doi: 10.1016/j.placenta.2007.07.001. [DOI] [PubMed] [Google Scholar]

[B19] 19.Opitz CA, Litzenburger UM, Lutz C, Lanz TV, Tritschler I, Köppel A, et al. Toll-like receptor engagement enhances the immunosuppressive properties of human bone marrow-derived mesenchymal stem cells by inducing indoleamine-2,3-dioxygenase-1 via interferon-beta and protein kinase R. Stem Cells. 2009;27(4):909–919. doi: 10.1002/stem.7. [DOI] [PubMed] [Google Scholar]

[B20] 20.Wang J, Liu Y, Ding H, Shi X, Ren H. Mesenchymal stem cell-secreted prostaglandin E2 ameliorates acute liver failure via attenuation of cell death and regulation of macrophage polarization. Stem Cell Res Ther. 2021;12(1):15. doi: 10.1186/s13287-020-02070-2. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B21] 21.Nii T, Tabata Y. Immunosuppressive mesenchymal stem cells aggregates incorporating hydrogel microspheres promote an in vitro invasion of cancer cells. Regen Ther. 2021;18:516–522. doi: 10.1016/j.reth.2021.11.006. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B22] 22.Holness CL, Simmons DL. Molecular cloning of CD68, a human macrophage marker related to lysosomal glycoproteins. Blood. 1993;81(6):1607–1613. [PubMed] [Google Scholar]

[B23] 23.Buechler C, Ritter M, Orsó E, Langmann T, Klucken J, Schmitz G. Regulation of scavenger receptor CD163 expression in human monocytes and macrophages by pro- and antiinflammatory stimuli. J Leukoc Biol. 2000;67(1):97–103. [PubMed] [Google Scholar]

[B24] 24.Chen T, Jiang Y, Xu S, Cheuk YC, Wang J, Yang C, et al. Poly(I:C)-induced mesenchymal stem cells protect the kidney against ischemia/reperfusion injury via the TLR3/PI3K pathway. Front Med (Lausanne) 2021;8:755849. doi: 10.3389/fmed.2021.755849. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B25] 25.Krupa A, Kowalska I. The kynurenine pathway-new linkage between innate and adaptive immunity in autoimmune endocrinopathies. Int J Mol Sci. 2021;22(18):9879. doi: 10.3390/ijms22189879. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B26] 26.Kiec-Wilk B, Grzybowska-Galuszka J, Polus A, Pryjma J, Knapp A, Kristiansen K. The MAPK-dependent regulation of the Jagged/Notch gene expression by VEGF, bFGF or PPAR gamma mediated angiogenesis in HUVEC. J Physiol Pharmacol. 2010;61(2):217–225. [PubMed] [Google Scholar]

[B27] 27.Jauković A, Abadjieva D, Trivanović D, Stoyanova E, Kostadinova M, Pashova S, et al. Specificity of 3D MSC spheroids microenvironment: impact on MSC behavior and properties. Stem Cell Rev Rep. 2020;16(5):853–875. doi: 10.1007/s12015-020-10006-9. [DOI] [PubMed] [Google Scholar]

[B28] 28.Ylöstalo JH, Bartosh TJ, Coble K, Prockop DJ. Human mesenchymal stem/stromal cells cultured as spheroids are self-activated to produce prostaglandin E2 that directs stimulated macrophages into an anti-inflammatory phenotype. Stem Cells. 2012;30(10):2283–2296. doi: 10.1002/stem.1191. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B29] 29.Burst VR, Gillis M, Pütsch F, Herzog R, Fischer JH, Heid P, et al. Poor cell survival limits the beneficial impact of mesenchymal stem cell transplantation on acute kidney injury. Nephron, Exp Nephrol. 2010;114(3):e107–e116. doi: 10.1159/000262318. [DOI] [PubMed] [Google Scholar]

[B30] 30.Yamamichi K, Fukuda T, Sanui T, Toyoda K, Tanaka U, Nakao Y, et al. Amelogenin induces M2 macrophage polarisation via PGE2/cAMP signalling pathway. Arch Oral Biol. 2017;83:241–251. doi: 10.1016/j.archoralbio.2017.08.005. [DOI] [PubMed] [Google Scholar]

PERMALINK

Toll-like receptor 3-stimulation and aggregate-formation synergistically enhances anti-inflammatory activity of feline mesenchymal stem cells

Youhei Fujimoto

Shingo Hatoya

Kikuya Sugiura

Yasuhiko Tabata