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Toll-like Receptor–Mediated Signaling in Human Adipose-Derived Stem Cells: Implications for Immunogenicity and Immunosuppressive Potential

Human adipose-derived stem cells (hASCs) are mesenchymal stem cells with reduced immunogenicity and the capability to modulate immune responses. These properties make hASCs of special interest as therapeutic agents in the settings of chronic inflammatory and autoimmune diseases. Exogenous and endogenous toll-like receptor (TLR) ligands have been linked with the perpetuation of inflammation in a number of chronic inflammatory diseases such as inflammatory bowel disease and rheumatoid arthritis because of the permanent exposure of the immune system to TLR-specific stimuli. Therefore, hASCs employed in therapy are potentially exposed to TLR ligands, which may result in the modulation of hASC activity and therapeutic potency. In this study, we demonstrate that hASCs possess active TLR2, TLR3, and TLR4, because activation with specific ligands resulted in induction of nuclear factor kappa B-dependent genes, such as manganese superoxide dismutase and the release of interleukin (IL)-6 and IL-8. TLR3 and TLR4 ligands increased osteogenic differentiation, but no effect on adipogenic differentiation or proliferation was observed. Moreover, we show that TLR activation does not impair the immunogenic and immunosuppressive properties of hASCs. These results may have important implications with respect to the safety and efficacy of hASC-based cell therapies.

Human Adipose‐Derived Mesenchymal Stem Cells Modulate Experimental Autoimmune Arthritis by Modifying Early Adaptive T Cell Responses

Mesenchymal stem cells (MSCs) are multipotent stromal cells with immunosuppressive properties. They have emerged as a very promising treatment for autoimmunity and inflammatory diseases such as rheumatoid arthritis. Recent data have identified that GM‐CSF‐expressing CD4 T cells and Th17 cells have critical roles in the pathogenesis of arthritis and other inflammatory diseases. Although many studies have demonstrated that MSCs can either prevent or suppress inflammation, no studies have addressed their modulation on GM‐CSF‐expressing CD4 T cells and on the plasticity of Th17 cells. To address this, a single dose of human expanded adipose‐derived mesenchymal stem cells (eASCs) was administered to mice with established collagen‐induced arthritis. A beneficial effect was observed soon after the infusion of the eASCs as shown by a significant decrease in the severity of arthritis. This was accompanied by reduced number of pathogenic GM‐CSF+CD4+ T cells in the spleen and peripheral blood and by an increase in the number of different subsets of regulatory T cells like FOXP3+CD4+ T cells and IL10+IL17−CD4+ T cells in the draining lymph nodes (LNs). Interestingly, increased numbers of Th17 cells coexpressing IL10 were also found in draining LNs. These results demonstrate that eASCs ameliorated arthritis after the onset of the disease by reducing the total number of pathogenic GM‐CSF+CD4+ T and by increasing the number of different subsets of regulatory T cells in draining LNs, including Th17 cells expressing IL10. All these cellular responses, ultimately, lead to the reestablishment of the regulatory/inflammatory balance in the draining LNs. Stem Cells 2015;33:3493–3503

Tryptophan concentration is the main mediator of the capacity of adipose mesenchymal stromal cells to inhibit T-lymphocyte proliferation in vitro

BACKGROUND AIMS Mesenchymal stromal cells (MSCs) have immunomodulatory properties that are mediated by cell-to-cell interactions and paracrine effects through soluble factors, among which tryptophan (Trp) conversion into kynurenine (Kyn) through the enzymatic activity of indoleamine 2,3-dioxygenase has been proven to be of special relevance. However, the respective role of Trp depletion and/or Kyn accumulation on the inhibition of T-cell proliferation by MSCs remains unclear. METHODS The effect of supplementation with increasing concentrations of Trp on the capacity of MSCs to inhibit T-lymphocyte proliferation in vitro was investigated. RESULTS We report that Trp supplementation impairs the capacity of adipose mesenchymal stromal cells (ASCs) to inhibit T-cell proliferation, despite the accumulation of very high concentrations of Kyn in the medium (>200 μmol/L). Moreover, Trp supplementation after 72 h of peripheral blood mononuclear cell:ASC co-culture, once the inhibitory effect of ASCs was established, reverted ASC inhibition and restored T-cell proliferation. Addition to stimulated lymphocytes of Kyn inhibited T proliferation in 3 of 10 peripheral blood mononuclear cell donors, but at different concentrations, suggesting that sensitivity of lymphocytes to Kyn might be donor-dependent. CONCLUSIONS Our results confirm the relevance of Trp metabolism as a key mediator of the immunomodulatory properties of ASCs and clarify the respective roles of the Trp/Kyn balance.

Adipose-derived mesenchymal stromal cells modulate experimental autoimmune arthritis by inducing an early regulatory innate cell signature.

Modulation of innate immune responses in rheumatoid arthritis and other immune‐mediated disorders is of critical importance in the clinic since a growing body of information has shown the key contribution of dysregulated innate responses in the progression of the disease. Mesenchymal stromal cells (MSCs) are the focus of intensive efforts worldwide due to their key role in tissue regeneration and modulation of inflammation. In this study, we define innate immune responses occurring during the early course of treatment with a single dose of expanded adipose‐derived MSCs (eASCs) in established collagen‐induced arthritis. eASCs delay the progression of the disease during the early phase of the disease. This is accompanied by a transient induction of Ly6C+ monocytes that differentiate into IL10+F4/80+ cells in arthritic mice. Strikingly, the induced IL10+F4/80+ myeloid cells preferentially accumulated in the draining lymph nodes. This effect was accompanied with a concomitant declining of their frequencies in the spleens. Our results show that eASCs attenuate the arthritic process by inducing an early innate cell signature that involves a transient induction of Ly6C+ monocytes in periphery that differentiate into IL10+F4/80+ macrophages. Our findings demonstrate that early regulatory innate cell responses, involving the monocyte compartment, are targeted by the eASCs during the onset of collagen‐induced inflammation.

Intralymphatic Administration of Adipose Mesenchymal Stem Cells Reduces the Severity of Collagen-Induced Experimental Arthritis

Mesenchymal stem cells (MSCs) are multipotent stromal cells with immunomodulatory properties. They have emerged as a very promising treatment for autoimmunity and inflammatory diseases such as rheumatoid arthritis. Previous studies have demonstrated that MSCs, administered systemically, migrate to lymphoid tissues associated with the inflammatory site where functional MSC-induced immune cells with a regulatory phenotype were increased mediating the immunomodulatory effects of MSCs. These results suggest that homing of MSCs to the lymphatic system plays an important role in the mechanism of action of MSCs in vivo. Thus, we hypothesized that direct intralymphatic (IL) (also referred as intranodal) administration of MSCs could be an alternative and effective route of administration for MSC-based therapy. Here, we report the feasibility and efficacy of the IL administration of human expanded adipose mesenchymal stem cells (eASCs) in a mouse model of collagen-induced arthritis (CIA). IL administration of eASCs attenuated the severity and progression of arthritis, reduced bone destruction and increased the levels of regulatory T cells (CD25+Foxp3+CD4+ cells) and Tr1 cells (IL10+CD4+), in spleen and draining lymph nodes. Taken together, these results indicate that IL administration of eASCs is very effective in modulating established CIA and may represent an alternative treatment modality for cell therapy with eASCs.

Biodistribution and Efficacy of Human Adipose-Derived Mesenchymal Stem Cells Following Intranodal Administration in Experimental Colitis

Mesenchymal stem cells (MSCs) have a large potential in cell therapy for treatment of inflammatory and autoimmune diseases, thanks to their immunomodulatory properties. The encouraging results in animal models have initiated the translation of MSC therapy to clinical trials. In cell therapy protocols with MSCs, administered intravenously, several studies have shown that a small proportion of infused MSCs can traffic to the draining lymph nodes (LNs). This is accompanied with an increase of different types of regulatory immune cells in the LNs, suggesting the importance of migration of MSCs to the LNs in order to contribute to immunomodulatory response. Intranodal (IN), also referred as intralymphatic, injection of cells, like dendritic cells, is being proposed in the clinic for the treatment of cancer and allergy, showing that this route of administration is clinically safe and efficient. In this study, we investigated, for the first time, the biodistribution and the efficacy of Luciferase+ adipose-derived MSCs (Luci-eASCs), infused through the inguinal LNs (iLNs), in normal mice and in inflamed mice with colitis. Most of the Luci-eASCs remain in the iLNs and in the adipose tissue surrounding the inguinal LNs. A small proportion of Luci-eASCs can migrate to other locations within the lymphatic system and to other tissues and organs, having a preferential migration toward the intestine in colitic mice. Our results show that the infused Luci-eASCs protected 58% of the mice against induced colitis. Importantly, a correlation between the response to eASC treatment and a higher accumulation of eASCs in popliteal, parathymic, parathyroid, and mesenteric LNs were found. Altogether, these results suggest that IN administration of eASCs is feasible and may represent an effective strategy for cell therapy protocols with human adipose-derived MSCs in the clinic for the treatment of immune-mediated disorders.

Human adipose tissue–derived mesenchymal stromal cells promote B-cell motility and chemoattraction

BACKGROUND AIMS Mesenchymal stromal cells hold special interest for cell-based therapy because of their tissue-regenerative and immunosuppressive abilities. B-cell involvement in chronic inflammatory and autoimmune pathologies makes them a desirable target for cell-based therapy. Mesenchymal stromal cells are able to regulate B-cell function; although the mechanisms are little known, they imply cell-to-cell contact. METHODS We studied the ability of human adipose tissue-derived mesenchymal stromal cells (ASCs) to attract B cells. RESULTS We show that ASCs promote B-cell migration through the secretion of chemotactic factors. Inflammatory/innate signals do not modify ASC capacity to mediate B-cell motility and chemotaxis. Analysis of a panel of B cell-related chemokines showed that none of them appeared to be responsible for B-cell motility. Other ASC-secreted factors able to promote cell motility and chemotaxis, such as the cytokine interleukin-8 and prostaglandin E2, did not appear to be implicated. CONCLUSIONS We propose that ASC promotion of B-cell migration by undefined secreted factors is crucial for ASC regulation of B-cell responses.

Endoscopic submucosal injection of adipose-derived mesenchymal stem cells ameliorates TNBS-induced colitis in rats and prevents stenosis

BackgroundMesenchymal stem cells have potential applications in inflammatory bowel disease due to their immunomodulatory properties. Our aim was to evaluate the feasibility, safety and efficacy of endoscopic administration of adipose-derived mesenchymal stem cells (ASCs) in a colitis model in rats.MethodsColitis was induced in rats by rectal trinitrobenzenesulfonic acid (TNBS). After 24 h ASCs (107 cells) or saline vehicle were endoscopically injected into the distal colon. Rats were followed for 11 days. Daily weight, endoscopic score at days 1 and 11, macroscopic appearance at necropsy, colon length and mRNA expression of Foxp3 and IL-10 in mesenteric lymph nodes (MLN) were analyzed.ResultsEndoscopic injection was successful in all the animals. No significant adverse events or mortality due to the procedure occurred. Weight evolution was significantly better in the ASC group, recovering initial weight by day 11 (− 0.8% ± 10.1%, mean ± SD), whereas the vehicle group remained in weight loss (− 6.7% ± 9.2%, p = 0.024). The endoscopic score improved in the ASC group by 47.1% ± 5.3% vs. 21.8% ± 6.6% in the vehicle group (p < 0.01). Stenosis was less frequent in the ASC group (4.8% vs. 41.2%, p < 0.01). Colon length significantly recovered in the ASC group versus the vehicle group (222.6 ± 17.3 mm vs. 193.6 ± 17.9 mm, p < 0.001). The endoscopic score significantly correlated with weight change, macroscopic necropsy score and colon length. Foxp3 and IL-10 mRNA levels in MLN recovered with ASC treatment.ConclusionsASC submucosal endoscopic injection is feasible, safe and ameliorates TNBS-induced colitis in rats, especially stenosis.

Comparative Analysis between the In Vivo Biodistribution and Therapeutic Efficacy of Adipose-Derived Mesenchymal Stromal Cells Administered Intraperitoneally in Experimental Colitis

Mesenchymal stem cells (MSCs) have emerged as a promising treatment for inflammatory diseases. The immunomodulatory effect of MSCs takes place both by direct cell-to-cell contact and by means of soluble factors that leads to an increased accumulation of regulatory immune cells at the sites of inflammation. Similar efficacy of MSCs has been described regardless of the route of administration used, the inflammation conditions and the major histocompatibility complex context. These observations raise the question of whether the migration of the MSCs to the inflamed tissues is a pre-requisite to achieve their beneficial effect. To address this, we examined the biodistribution and the efficacy of intraperitoneal luciferase-expressing human expanded adipose-derived stem cells (Luci-eASCs) in a mouse model of colitis. Luci-eASC-infused mice were stratified according to their response to the Luci-eASC treatment. According to the stratification criteria, there was a tendency to increase the bioluminescence signal in the intestine at the expense of a decrease in the bioluminescence signal in the liver in the “responder” mice. These data thus suggest that the accumulation of the eASCs to the inflamed tissues is beneficial for achieving an optimal modulation of inflammation.