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Keywords:

  • Mesenchymal stem cell;
  • Bone marrow stromal cell;
  • Tolerance;
  • Enhanced green fluorescent protein;
  • Autoimmunity;
  • Inflammatory bowel disease;
  • Regulatory T cell

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

Cell-based tolerogenic therapy is a relatively new approach for the treatment of autoimmune diseases. Mesenchymal stem cells (MSCs) have been shown to be potent immunomodulatory agents in a number of experimental and clinical scenarios; however, their use in various autoimmune diseases is undefined. Herein, we report the efficacy of MSC transplantation in a multiorgan autoimmunity model. Mice with defective peripheral tolerance caused by a deficiency in regulatory T cells were used as a testbed for therapy. After screening multiple target tissues of autoimmune attack, we observed an MSC-specific improvement in the histopathology of the distal ileum of treated mice. We then showed that MSCs can reduce mesenteric lymph node (MLN) cellularity in autoimmune mice during active disease and decrease activated T-cell populations in the MLN. Trafficking studies using enhanced green fluorescent protein (eGFP)-reporter MSCs revealed no appreciable engraftment in the intestine, but it did reveal the presence of eGFP+ cells organized in clusters within the MLN, as well as ancillary nodes. Semiquantitative analysis showed no difference in the number of clusters; however, eGFP+ cells in MLNs compared with ancillary nodes had distinct fibroblastoid morphology and formed a network with neighboring eGFP+ cells. Finally, we show evidence that transplantation of MSCs caused global immunosuppression, as measured by increased CD4+ CD8+ thymocyte production and serum interleukin-10 and decreased serum interferon-γ. These data implicate the intestine as a new site of MSC tolerance induction and should motivate additional studies evaluating the use of MSCs as a treatment for autoimmune enteropathies.

Disclosure of potential conflicts of interest is found at the end of this article.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

Author contributions: B.P.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; A.W.T.: conception and design, financial support, data analysis and interpretation, manuscript writing; M.L.Y.: conception and design, financial support, data analysis and interpretation, final approval of manuscript.

Autoimmune disease is the result of a failure of the immune system to regulate self-reactive lymphocytes. There are more than 80 different types of autoimmune disorders, with a variety of genetic/environmental triggers and symptomatology. These disorders affect millions worldwide and represent a staggering burden in quality of life and health care costs. Current clinical approaches to these diseases often involve nonspecific immunosuppressive/modulatory treatments, such as corticosteroids, which do not correct the etiology of the disorder and leave a significant population refractory to therapy.

Cell-based tolerogenic therapy has gained momentum in recent years with the identification of endogenous suppressor cells capable of inhibiting lymphocyte effector functions [1]. Mesenchymal stem cells (MSCs), the precursor to stromal cells in the bone marrow, have been reported to have immunosuppressive effects in vitro and in vivo. The majority of these studies have shown that MSCs can inhibit the activation of CD3+, CD4+ T-cell proliferation, and secretion of interleukin (IL)-2 and interferon (IFN)-γ induced by mixed lymphocyte reactions [2, [3], [4]5], mitogens [3, 6], and receptor engagement [7, 8]. These in vitro studies have revealed that soluble factors secreted by MSCs may be the primary mode of immunomodulation [5, 9]; however, cell contact-dependent mechanisms of suppression may also coexist [10, 11]. Regardless, the efficacy of MSC transplantation in severe acute graft-versus-host disease (GVHD) in clinical trials demonstrates the potency of these cells as immunosuppressive agents [12, [13]14].

In light of these findings, MSCs may be a viable cellular therapeutic for autoimmune disease because of their tolerogenic properties. Recent reports have stated preclinical efficacy of MSC transplantation in rheumatoid arthritis [15], experimental autoimmune encephalomyelitis [16, 17], and type I diabetes [18], suggesting that MSCs may be useful in a multisystem autoimmune disorder; however, the efficacy of such treatment has not been fully explored. To this end, the goal of the present study was to determine whether MSCs can suppress autoimmune pathology in multiple tissues during active disease in Foxp3sf mice. These animals have a spontaneous mutation in Foxp3, a forkhead-winged helix box transcription factor specifically expressed in CD4+ CD25+ Foxp3+ regulatory T cells (Tregs), which are endogenous suppressor cells of peripheral self-reactive T lymphocytes in vivo [19, [20]21]. Therefore, these animals have a failure in one mode of peripheral tolerance, because of a deficiency in the Treg pool, and do not regulate self-reactive lymphocytes, leading to an associated multiorgan autoimmune disease. Self-reactive T cells can be found in many target organs of Foxp3sf animals, such as skin, endocrine glands, and the gastrointestinal (GI) tract. We screened multiple targets of autoimmune attack after MSC transplantation and compared the specificity of any effects to Treg transplants as a stringent control. On the basis of this comparative analysis, we discovered a new MSC-specific site of therapy, the GI tract. Furthermore, we tracked infused MSCs and observed no engraftment in the end organ, but rather gut-associated and ancillary lymph nodes with notable differences in morphology between the two lymph node sites. Finally, we observed systemic signs of immunosuppression after MSC treatment.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

Mice

C57Bl/6 mice between 4 and 6 weeks of age were purchased from Charles River Laboratories (Wilmington, MA, http://www.criver.com). Foxp3sf mice were purchased from Jackson Laboratory (Bar Harbor, ME, http://www.jax.org).

Animals were maintained in a light-controlled room (12-hour light-dark cycle) at an ambient temperature of 25°C with chow diet and water ad libitum. The animals were cared for in accordance with the guidelines set forth by the NIH Committee on Laboratory Resources. All experimental procedures performed were approved by the Subcommittee on Research Animal Care and Laboratory Animal Resources of Massachusetts General Hospital. Foxp3sf mice were housed and used in a pathogen-free facility at Shriners Hospitals for Children in accordance with all applicable guidelines.

Antibody and Reagents

The following antibodies used for flow cytometry were purchased from BD Pharmingen (San Diego, http://www.bdbiosciences.com/index_us.shtml): CD4-allophycocyanine (APC), CD44-fluorescein isothiocyanate (FITC), CD25-phycoerythrin (PE), CD8-FITC, CD106-FITC, Flk-1-PE, CD90-FITC, and Sca-1-FITC. Biotinylated antibodies to CD4 and CD25 were purchased from eBioscience Inc. (San Diego, http://www.ebioscience.com). Streptavidin microbeads, CD45 and CD11b microbeads, and magnetic columns were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany, http://www.miltenyibiotec.com). For immunocytochemistry, anti-mouse α-smooth muscle actin was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, http://www.scbt.com). MSC expansion medium consisted of α-minimal essential medium without deoxyribonucleosides and ribonucleosides (Gibco, Grand Island, NY, http://www.invitrogen.com), 10% lot selected fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA, http://www.atlantabio.com), 100 U/ml penicillin (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), and 100 μg/ml streptomycin (Sigma-Aldrich).

Isolation and Culture of Bone Marrow-Derived MSCs

Bone marrow was harvested from wild-type mice after euthanization. Tibias and femurs were dissected, and the marrow space was flushed with MSC expansion medium using a 23-gauge needle. Bone marrow plugs were collected on ice, dissociated by repeated passage through an 18-gauge needle, and passed through a 70-μm filter to remove bony spicules and debris. Approximately 50 × 106 bone marrow cells were plated on a 100-mm2 tissue culture dish and cultured for 3 days to allow for differential adhesion of stromal cells. Nonadherent cells were aspirated on day 3, and the adherent population was cultured in MSC expansion medium for a subsequent 4–10 days to achieve the maximal number of colony-forming units-fibroblast prior to initial passage. Cells were passaged using 0.1% trypsin/0.1% EDTA and subcultured at a density of 5 × 103 cells per cm2. Prior to transplantation, stromal cells were then depleted of CD11b and CD45 cells using magnetic-activated cell sorting (MACS) per the vendor's instructions. Enhanced green fluorescent protein (eGFP)-MSCs were kindly donated by the Center for Gene Therapy at Tulane University and grown in MSC expansion medium. All cultures were used between passage 2 and passage 5.

Isolation and Analysis of Cells from Lymph Nodes, Spleen, and Thymus

Lymphoid organs were dissected from experimental mice and dissociated into cellular components by mechanical disruption of the tissue into a saline solution. The cell suspensions were centrifuged at 1,500 rpm for 10 minutes and were exposed to ammonium chloride (ACK) lysis buffer for 1–2 minutes to remove contaminating erythrocytes. ACK lysis buffer consisted of 8.024 mg of NH4Cl, 1.0 mg of KHCO3, and 3.722 mg of Na2EDTA·2H2O in a 1-l solution of deionized H2O adjusted to a pH of 7.4. The solution was neutralized with serum-containing medium and pelleted. Cells were resuspended in a blocking solution containing 0.5% bovine serum albumin (BSA) and antibodies to the Fc receptor CD16/32. This cellular preparation was incubated with specific primary antibody combinations for 30 minutes at 4°C. After incubations, the cells were pelleted and resuspended in buffer and analyzed using a flow cytometer (FACSCalibur; Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). For the isolation of CD4+ CD25+ splenocytes, we used MACS protocols per the vendor's instructions to enrich splenocytes of regulatory T cells.

Enzyme-Linked Immunosorbent Assays

Peripheral blood was collected from animals by cardiac puncture and centrifuged at 1,500 rpm for 15 minutes to collect serum. Serum was analyzed for IFN-γ and IL-10 by enzyme-linked immunosorbent assay (ELISA). Mouse IFN-γ capture antibody (BD Biosciences, San Diego, http://www.bdbiosciences.com) diluted at 2 μg/ml in carbonate buffer (pH 9.0) was physisorbed on 96-well plates overnight at 4°C. Plates were washed with phosphate-buffered saline (PBS) with 0.1% Triton X-100 (Sigma-Aldrich) and blocked with borate-buffered saline (BBS) (pH 8.0)/2% bovine serum albumin (Sigma-Aldrich) at room temperature for 2 hours. Standards of mouse recombinant IFN-γ (R&D Systems Inc., Minneapolis, http://www.rndsystems.com) and samples were loaded and incubated at 4°C overnight. Plates were washed and incubated with biotin anti-IFN-γ (1 μg/ml; BD Biosciences) at room temperature for 45 minutes. Plates were washed and incubated with horseradish peroxidase conjugated to avidin (1:1,000 in BBS/2% BSA; BD Biosciences) for 45 minutes. Plates were washed and incubated with citrate buffer supplemented with 2,2′-azino-bis(3-ethylbenzthiazoline 6-sulphonic acid) (Sigma-Aldrich) and 30% hydrogen peroxide, colorized, and read at 415 nm on a microplate reader. ELISA for mouse IL-10 (BD Biosciences OptiEIA IL-10 kit) was performed per the vendor's instructions. Each animal's serum was tested in triplicate, and data are representative of four animals per group for each cytokine analyzed.

Histology

Tissue from the distal ileum, pancreas, and liver was harvested from animal groups 1 week after treatments. Tissue was fixed in 10% buffered formalin, embedded in paraffin, sectioned to 6-μm thickness, and stained with hematoxylin and eosin. Images are representative of six animals per group for the distal ileum and four animals for each of the other tissues.

Immunohistochemistry

Tissues of interest were harvested and placed in a solution of 4% paraformaldehyde and 10% sucrose for 3 hours. Samples were then transferred to a 30% sucrose solution and left overnight to allow for full penetration of the cryoprotectant. Tissues were then embedded in Tissue-Tek optimal cutting temperature Compound (Sakura Finetek, Torrance, CA, http://www.sakura.com), frozen, and sectioned. Sections of fixed tissue 8 μm thick were washed three times in PBS for 15 minutes and blocked with a buffer containing 5% donkey serum and 0.1% Triton X-100 for 30 minutes at room temperature. Slides were washed again with blocking buffer and then incubated with a primary anti-eGFP antibody (clone 3E6; Molecular Probes, Eugene, OR, http://probes.invitrogen.com) at a 1:250 dilution overnight at 4°C. After washing with PBS, a secondary donkey anti-rabbit antibody conjugated to Cy3 at a 1:500 dilution in blocking buffer for 30 minutes at room temperature was used for detection. Note that detection of eGFP using this method of indirect immunofluorescence with the stated secondary antibody results in a red signal rather than a green signal, which was better for visualization purposes. The sections were then washed three times with blocking buffer and PBS and developed using 3,3′-diaminobenzidine. All histology images were captured on a Nikon Eclipse E800 upright microscope (Nikon, Tokyo, http://www.nikon.com).

Digital Cell Cluster Quantification

Quantification of cell clusters in stained sections was performed on five random ×40 images from each section in which at least one cluster was found. Ten sections were made for each tissue from each animal, and three animals were used in cell trafficking studies. Clusters were visually distinct and defined as a local aggregate of at least 10 eGFP+ cells.

Statistical Analysis

For flow cytometry data, median values ± SDs are reported. For cytokine analysis, results were analyzed using an unpaired Student's t test given an unskewed data set and assuming a normal distribution. Significance values of p < .05 were considered statistically significant. Results are given as mean ± SEM.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

Purification and Characterization of Marrow Stromal Subpopulation

The anchorage-dependent population of bone marrow cells, also known as stromal cells, consists of a relatively heterogeneous mixture of cells (supplemental online Fig. 1A). Evidence exists that a subpopulation of this heterogeneous mixture, which displays phenotypic similarities to MSCs, is responsible for the immunosuppressive effect of the marrow stroma. For the current study, we purified this subpopulation by immunodepleting adherent bone marrow cells of CD11b+ and CD45+ cells using MACS. The CD11b+ CD45+ cell fraction resembled macrophage-like cells (supplemental online Fig. 1B), whereas the negative fraction had a more fibroblastoid morphology (supplemental online Fig. 1C). These fibroblastoid cells were found to be positive for α-smooth muscle actin expression by immunocytochemistry (supplemental online Fig. 1D). The immunophenotype of the CD11b− CD45− fraction was CD106+, Flk-1+, Sca-1+, and CD90−, as determined by cytofluorimetry (supplemental online Fig. 1E–1H). The phenotype of these cells is identical to that of C57Bl/6 MSCs [22, 23], and they will be referred to as MSCs herein for the sake of convenience.

MSCs Suppress Histopathological Changes of Target Organs in Foxp3Sf Mice

We evaluated the immunosuppressive efficacy of different cell-based transplantation strategies in Foxp3sf mice, which have widespread autoimmunity due to inefficient peripheral tolerance. Formulations using MSCs were compared with Tregs − the suppressor cells that are deficient in Foxp3sf mice because of the genetic mutation. At 4 weeks of age, Foxp3sf mice were infused with 3 × 105 MSCs intraperitoneally (i.p.) and were sacrificed 1 week postinfusion. For comparison, we used age- and sex-matched mice of the following groups: (a) wild-type C57Bl/6, (b) Foxp3sf treated with vehicle, and (c) Foxp3sf treated with MACS-selected CD4+ CD25+ T lymphocytes (3 × 105 cells) (a Treg phenotype, and hence the most stringent control). Self-reactive T cells can be found in many target organs of Foxp3sf animals, such as skin, endocrine glands, and the GI tract. We harvested the distal ileum, pancreas, and liver, as representative tissue targets of autoimmunity, and examined histopathology. The most dramatic histological change was found in the distal ileum. Foxp3sf animals treated with MSCs regenerated crypt structures similar to wild-type, whereas untreated and Treg-treated animals failed to do so (Fig. 1A–1D). We observed this GI finding in four of six animals tested, whereas no vehicle-treated mice and only one Treg-treated mouse showed any signs of regrowth. Histopathology of the pancreas and liver were not qualitatively improved by either cell treatment (data not shown).

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Figure Figure 1.. Histological changes in ileal tissue of Foxp3sf mice after cell treatments during active disease. Cross-sections of distal ileum comparing WT (A) with Foxp3sf mice treated with i.p. infusions of saline (B), Tregs(C), or MSCs (D). Inset images present higher magnification of boxed regions in original micrographs. Scale bar = 250 μm (magnification, ×4). Data are representative of three independent trials, with a total of n = 6 per group. Abbreviations: MSC, mesenchymal stem cell; Treg, regulatory T cell; WT, wild-type.

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Reduction of Cellularity and Activated T Cells in Mesenteric Lymph Nodes of Foxp3sf Mice After MSC Infusion

We then analyzed lymphoid tissue associated with the intestine for changes in disease. Mesenteric lymph nodes (MLNs) are typically enlarged when there is adjacent inflammation of intestinal tissue. We harvested MLNs and measured total cellularity at 7 days postinfusion of cell transplants. Representative gross histology for MLNs within each group is shown in Figure 2A. Draining MLNs remained hypercellular in Foxp3sf mice when treated with vehicle (73.5 ± 8.1 × 106 cells) compared with wild-type (17.8 ± 2.8 × 106 cells), whereas cellularity was reduced by MSC (48.1 ± 7.3 × 106 cells) or Treg treatment (63.2 ± 5.3 × 106 cells) compared with mutant mice (Fig. 2B). Lymph node cells were isolated, gated for CD4 expression, and analyzed for the cell surface activation marker CD44 using flow cytometry. Compared with wild-type mice (18.8% ± 3.8%), the majority of CD4+ lymph node cells were activated in mutant mice (83.4% ± 4.0%; Fig. 2C). The CD44hi population was reduced in both cellular treatments (57.9% ± 8.1% vs. 69.2% ± 7.0%, respectively). Overall, treatment with MSCs was qualitatively more remarkable in effect than with Tregs with respect to suppressing local inflammation in the MLN.

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Figure Figure 2.. MSC treatment reduces mesenteric lymph node cellularity and CD4+ CD44hi lymph node cell number. Lymph nodes were harvested 7 days post-cell infusion of MSCs or Tregs and compared with untreated and WT nodes. (A): Gross histology of lymph nodes from mice. (B): After tissue harvesting, cellularity was determined using a Coulter counter (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com). (C): Lymph node cells were analyzed for CD4 and CD44 expression using flow cytometry. Data represent mean ± SEM of n = 5 per group. *, p < .01. Abbreviations: MSC, mesenchymal stem cell; Treg, regulatory T cell; WT, wild-type.

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MSCs Do Not Engraft in the Intestine but Rather in Ancillary and Gut-Associated Lymph Nodes

After a tissue- and cell-specific effect of MSC treatment was observed, we attempted to delineate whether this therapy was due to MSC-mediated regeneration of gut tissue versus an alteration of the immunological attack at the intestine. We infused 3 × 105 eGFP-labeled MSCs i.p. and harvested the distal ileum at 7 days post-treatment. We found no appreciable eGFP+ cells in the intestinal tissue at the 7-day time point (Fig. 3A). On the contrary, eGFP+ cells were detected in the MLN at a significant proportion of the graft relative to intestinal tissue (Fig. 3B). Clusters of eGFP+ cells were found in a network, with each cell having a distinct fibroblastoid morphology (Fig. 3B, inset). To determine whether this engraftment was specific to MLNs in this model of autoimmune disease, we harvested inguinal lymph nodes as an ancillary site. Transplanted cells were also found in inguinal nodes (Fig. 3C). Semiquantitative image analysis of lymph node engraftment, as assessed by the number of clusters counted, showed no differences between the two sites (Fig. 3D). However, the majority of the cells found within inguinal lymph nodes were not fibroblastoid in morphology and did not form a network.

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Figure Figure 3.. Evaluation of eGFP+ mesenchymal stem cell engraftment in intestinal and lymphoid tissue in Foxp3sf mice. Tissues were harvested at 7 days post-treatment. Representative immunofluorescent images of the distal ileum (A), mesenteric lymph node (MLN) (B), and inguinal lymph node (C). eGFP is depicted in red, and 4,6-diamidino-2-phenylindole was used as a nuclear counterstain. (D): Semiquantitative analysis of the number of eGFP+ cell clusters in MLNs and inguinal nodes. Abbreviation: eGFP, enhanced green fluorescent protein.

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Systemic Evidence of Immunosuppression in Foxp3Sf Mice After MSC Treatment

Since we had observed engraftment of MSCs in lymph nodes in two anatomically remote sites, we hypothesized that the cell transplant may have had systemic immunological effects. Two circumstantial measures of systemic immunosuppression are as follows: (a) an increase in the percentage of newly formed CD4+ CD8+ thymocytes, and (b) a change in the serum cytokine profile. After cell treatment we observed no difference in total thymocyte number (data not shown) but found that the number of CD4+ CD8+ thymocytes of animals treated with MSCs (74.0% ± 5.6%) were increased relative to wild-type (69.2% ± 5.5%), untreated (63.0% ± 7.6%), and Treg-treated (69.2% ± 3.1%) animals (Fig. 4A). In addition, serum IFN-γ (Fig. 4B), an indicator of cell-mediated immune responses, and IL-10 (Fig. 4C), a potent immunosuppressive cytokine, were shifted in favor of a global downregulation of the immune system after MSC treatment.

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Figure Figure 4.. Alterations in thymocyte production and serum levels of IFN-γ and IL-10 in Foxp3sf mice after cell therapy. (A): Thymii from Foxp3sf mice treated with vehicle or cells were analyzed for CD4 and CD8 expression using flow cytometry and compared with those of WT animals. Data are representative of n = 5 per group. Serum was analyzed for the cytokines (B) IFN-γ and (C) IL-10 one week post-treatment by enzyme-linked immunosorbent assay. Data represent mean ± SEM of two independent trials of a total of n = 2 per group. *, p < .01. Abbreviations: IFN, interferon; MSC, mesenchymal stem cell; Treg, regulatory T cell; WT, wild-type.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

The bone marrow stroma has been identified as a unique site of regenerative and immunosuppressive cells. Many studies have reported inhibition of T lymphocyte functions when cocultured with MSCs by cell contact-dependent and -independent mechanisms. However, the use of MSCs as a cellular therapeutic for autoimmune diseases has not been fully explored. We chose to stringently test the efficacy of MSCs as a treatment for autoimmune disease by transplanting these cells in Foxp3sf mice, which lack one mode of peripheral tolerance because of a genetic mutation in the transcription factor Foxp3 that leads to a deficiency in regulatory T cells. Since it has been reported that MSCs can induce the proliferation of CD4+ CD25+ T lymphocytes in vitro [7, 24, 25], albeit without rigorous analysis of Foxp3 protein expression, the use of Foxp3sf mice as a testbed should not be confounded by such an indirect pathway of therapeutic benefit. However, this study does not rule out an MSC-Treg interaction in vivo that can amplify the immunosuppressive efficacy of MSC transplantation by secondary effects of boosting endogenous Treg-mediated peripheral tolerance.

We infused MSC transplants in a multiorgan autoimmunity model and screened various tissue targets of autoimmune attack. Since Foxp3sf mice are essentially moribund 4–6 weeks after birth because of the magnitude and nature of their autoimmunity, this study looked at short-term benefits of cell transplantation during active disease. We provide the first evidence that MSCs can specifically ameliorate autoimmune enteropathy. We have shown that MSCs infused into these autoimmune mice led to striking improvements in the distal ileum. The regrowth of villous structures was observed 7 days after MSC treatment, whereas Treg-treated animals still had visible disease. There are different potential explanations for ileal regrowth, based on experimental evidence. Previous studies have shown that bone marrow-derived mesenchymal cells can give rise to newly formed myofibroblasts and vasculature in a physiological response to chemically induced colitis in mice and humans [26, 27]. Furthermore, we and other have shown that MSCs can promote regeneration by paracrine stimulation of endogenous self-replicating tissue cells [28] and stem cell populations [29]. Taken together, our results could have been due to cell homing to the distal ileum and (a) a direct regenerative response of MSCs, (b) paracrine signals to promote intestinal stem cell expansion and differentiation, or (c) inhibition of immunological attack on the tissue and allowance for natural regeneration of villi. We did not observe any infused eGFP-MSCs in the distal ileum, ruling out direct or local, paracrine interactions between MSCs and intestinal cells. The natural replenishment of cells in the GI tract during normal tissue turnover is primarily due to an endogenous stem cell population located in the crypt, which in the mouse can occur in 2–3 days [30, [31]32]. Thus, it is likely that the regeneration of villi was not due to MSC differentiation or paracrine actions but rather to an inhibition of the immune response to the tissue and repopulation of parenchymal cells by crypt stem cells.

To support the concept that MSCs may have modulated the immune reaction at the GI tissue target, we analyzed gut-associated lymph nodes. We observed a decrease in MLN cellularity and activated CD4+ lymph node cells, which supports this hypothesis. Inhibition of T-cell activation by MSCs may be the result of selective apoptosis of activated T cells, prevention of further T-cell activation by direct or indirect (e.g., licensing tolerance-inducing antigen-presenting cells [33]) mechanisms, or dedifferentiation of activated T cells, although more mechanistic studies are warranted. These results are consistent with the benefit seen with MSCs in severe GVHD, a condition that is thought to matriculate from the Peyer's patches of intestinal tissue [34].

We identified MSCs organized in clusters in engrafted tissues, specifically lymph nodes. Others have also described clusters of engrafted human fetal MSCs that had homed to the bone marrow after in utero treatment of children with osteogenesis imperfecta [35], although it was not determined whether this was a clonal population derived from a single engrafted cell or a local distribution of transplanted cells based on circulatory patterns intrinsic to the tissue of study. Interestingly, there were no relative differences in the number of clusters in MLNs compared with an anatomically distinct lymph node bed in the inguinal space. Instead, MSCs were morphologically different within the MLN tree, displaying spiculated projections, and formed a fibroblastoid network. The relevance of this morphological difference is unclear, although we speculate that the engrafted cells seemed more differentiated and integrated into the stroma of the MLN and that this may be relevant to MSC functions necessary for therapeutic gains. In the treatment of experimental encephalomyelitis, MSCs were also localized in secondary lymphoid organs, including the spleen and lymph nodes [17], the latter finding of which was reproduced and extended to other lymph nodes in this study. Prior work has shown trafficking of peritoneal infused lymphocytes to MLNs and pancreatic lymph nodes [36], suggesting that these may be likely sites of tolerance induction after MSC transplantation, assuming that similar homing mechanisms exist in MSCs. Ultimately, these data suggest certain “tropic” aspects of MSC transplantation efficacy that may hinge upon endogenous properties of the host, namely: (a) a tissue-associated lymphoid bed for engraftment and immunomodulation, and (b) a host-autonomous system of regeneration to restore tissue function and homeostasis.

Moreover, we observed an increase in the number of CD4+ CD8+ thymocytes and changes in the serum cytokine profile after MSC infusion. T cells are born in the thymus, and studies have shown that the mesenchyme is integral in the proper education of T cells [37]. In addition, other investigators have shown that infused MSCs can migrate and functionally engraft at lower numbers in the thymus [38]. Our study examined only engraftment in lymph node tissue, so a more comprehensive view of MSC engraftment in this and other autoimmunity models may elucidate the role of transplanted cells on thymic cell output and function. In a separate report, we have shown that human MSCs secrete immunomodulatory molecules that can provide a significant survival benefit to rats undergoing acute liver failure [39] and also shift the serum cytokine profile [28] independently of cell contact mechanisms. We infer that the change in the cytokine profile in the present study may be a paracrine effect of engrafted or lysed infused cells that resulted in global serological differences, although there are obvious pathological and pharmacological differences between the former study and the present one to consider. More importantly, these studies may indicate a systemic immunosuppressive effect after MSC transplantation. It may be argued that successful treatment of autoimmunity may require a systemic-scale approach, and this study validates MSC therapy within such a context; however, general concerns of immunocompromise should be considered. Other reports have observed a loss of immunosurveillance of cancer growth [40, 41] and pathogens after MSC infusion [42], which is a major concern with MSC immunomodulation. Comparative studies between MSCs and other immunosuppressive treatments for autoimmune diseases are needed to determine the relative differences in risk of cell-based versus molecular therapeutic strategies.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

We report the first use of MSCs in a multiorgan model of autoimmunity, including an MSC-specific amelioration of autoimmune enteropathy. These results will hopefully empower future investigations into the use of MSCs for the treatment of inflammatory bowel disease, as well as other autoimmune disorders.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

We thank Robert Crowther for histological preparation and Donald Poulsen for figure preparation. Imaging studies were made possible by the special Morphology Core Facility at the Shriners Hospitals for Children. B.P. was supported by a National Science Foundation predoctoral fellowship. This work was partially supported by grants from the NIH (K08 DK66040, K18 DK076819 and R01 DK43371) and the Shriners Hospitals for Children.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information
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SC-07-0790_Supplemental_Figure.tif1256KSupplemental Figure

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