Immunomodulatory potential of cytokine-licensed human bone marrow-derived mesenchymal stromal cells correlates with potency marker expression profile.

Cytokine(s) pre-activation/licensing is an effective way to enhance the immunomodulatory potency of mesenchymal stromal cells (MSCs). Currently, IFN-γ licensing received the most attention in comparison with other cytokines. After licensing human bone marrow-derived MSCs with pro-/anti-inflammatory cytokines IFN-γ, IL-1ß, TNF-α, TGF-ß1 alone or in combination, the in-vitro immunomodulatory potency of these MSCs was studied by incubating with allogeneic T cells and macrophage-like THP-1 cells. In addition, immunomodulation-related molecules filtered by bioinformatics, complement 1 subcomponent (C1s) and interferon-induced GTP-binding protein Mx2 (MX2), were studied to verify whether to reflect the immunomodulatory potency. Herein, we reported that different cytokines cause different effects on the function of MSC. While TGF-ß1 licensing enhances the capacity of MSCs to induce T cells with an immunosuppressive phenotype, IFN-γ-licensing strengthens the inhibitory effect of MSC on T cell proliferation. Both TGF-ß1 and IFN-γ licensing can enhance the effect of MSC on reducing the expression of pro-inflammatory cytokines by M1 macrophage-like THP-1 cells. Interestingly, IFN-γ upregulates potential potency markers extracellular C1s and kynurenine (KYN) and intracellular MX2. These three molecules have the potential to reflect mesenchymal stromal cell immunomodulatory potency. In addition, we reported that there is a synergistic effect of TGF-ß1 and IFN-γ in immunomodulation.

The pseudo-caspase FLIP(L) regulates cell fate following p53 activation.

p53 is the most frequently mutated, well-studied tumor-suppressor gene, yet the molecular basis of the switch from p53-induced cell-cycle arrest to apoptosis remains poorly understood. Using a combination of transcriptomics and functional genomics, we unexpectedly identified a nodal role for the caspase-8 paralog and only human pseudo-caspase, FLIP(L), in regulating this switch. Moreover, we identify FLIP(L) as a direct p53 transcriptional target gene that is rapidly up-regulated in response to Nutlin-3A, an MDM2 inhibitor that potently activates p53. Genetically or pharmacologically inhibiting expression of FLIP(L) using siRNA or entinostat (a clinically relevant class-I HDAC inhibitor) efficiently promoted apoptosis in colorectal cancer cells in response to Nutlin-3A, which otherwise predominantly induced cell-cycle arrest. Enhanced apoptosis was also observed when entinostat was combined with clinically relevant, p53-activating chemotherapy in vitro, and this translated into enhanced in vivo efficacy. Mechanistically, FLIP(L) inhibited p53-induced apoptosis by blocking activation of caspase-8 by the TRAIL-R2/DR5 death receptor; notably, this activation was not dependent on receptor engagement by its ligand, TRAIL. In the absence of caspase-8, another of its paralogs, caspase-10 (also transcriptionally up-regulated by p53), induced apoptosis in Nutlin-3A-treated, FLIP(L)-depleted cells, albeit to a lesser extent than in caspase-8-proficient cells. FLIP(L) depletion also modulated transcription of canonical p53 target genes, suppressing p53-induced expression of the cell-cycle regulator p21 and enhancing p53-induced up-regulation of proapoptotic PUMA. Thus, even in the absence of caspase-8/10, FLIP(L) silencing promoted p53-induced apoptosis by enhancing PUMA expression. Thus, we report unexpected, therapeutically relevant roles for FLIP(L) in determining cell fate following p53 activation.

Activation of innate-adaptive immune machinery by poly(I:C) exposes a therapeutic vulnerability to prevent relapse in stroma-rich colon cancer.

OBJECTIVE: Stroma-rich tumours represent a poor prognostic subtype in stage II/III colon cancer (CC), with high relapse rates and limited response to standard adjuvant chemotherapy. DESIGN: To address the lack of efficacious therapeutic options for patients with stroma-rich CC, we stratified our human tumour cohorts according to stromal content, enabling identification of the biology underpinning relapse and potential therapeutic vulnerabilities specifically within stroma-rich tumours that could be exploited clinically. Following human tumour-based discovery and independent clinical validation, we use a series of in vitro and stroma-rich in vivo models to test and validate the therapeutic potential of elevating the biology associated with reduced relapse in human tumours. RESULTS: By performing our analyses specifically within the stroma-rich/high-fibroblast (HiFi) subtype of CC, we identify and validate the clinical value of a HiFi-specific prognostic signature (HPS), which stratifies tumours based on STAT1-related signalling (High-HPS v Low-HPS=HR 0.093, CI 0.019 to 0.466). Using in silico, in vitro and in vivo models, we demonstrate that the HPS is associated with antigen processing and presentation within discrete immune lineages in stroma-rich CC, downstream of double-stranded RNA and viral response signalling. Treatment with the TLR3 agonist poly(I:C) elevated the HPS signalling and antigen processing phenotype across in vitro and in vivo models. In an in vivo model of stroma-rich CC, poly(I:C) treatment significantly increased systemic cytotoxic T cell activity (p<0.05) and reduced liver metastases (p<0.0002). CONCLUSION: This study reveals new biological insight that offers a novel therapeutic option to reduce relapse rates in patients with the worst prognosis CC.

TGF-ß1-Licensed Murine MSCs Show Superior Therapeutic Efficacy in Modulating Corneal Allograft Immune Rejection In Vivo.

Mesenchymal stromal cells (MSCs) are a promising therapeutic option for multiple immune diseases/disorders; however, efficacy of MSC treatments can vary significantly. We present a novel licensing strategy to improve the immunosuppressive capacity of MSCs. Licensing murine MSCs with transforming growth factor-ß1 (TGF-ß MSCs) significantly improved their ability to modulate both the phenotype and secretome of inflammatory bone marrow-derived macrophages and significantly increased the numbers of regulatory T lymphocytes following co-culture assays. These TGF-ß MSC-expanded regulatory T lymphocytes also expressed significantly higher levels of PD-L1 and CD73, indicating enhanced suppressive potential. Detailed analysis of T lymphocyte co-cultures revealed modulation of secreted factors, most notably elevated prostaglandin E2 (PGE2). Furthermore, TGF-ß MSCs could significantly prolong rejection-free survival (69.2% acceptance rate compared to 21.4% for unlicensed MSC-treated recipients) in a murine corneal allograft model. Mechanistic studies revealed that (1) therapeutic efficacy of TGF-ß MSCs is Smad2/3-dependent, (2) the enhanced immunosuppressive capacity of TGF-ß MSCs is contact-dependent, and (3) enhanced secretion of PGE2 (via prostaglandin EP4 [E-type prostanoid 4] receptor) by TGF-ß MSCs is the predominant mediator of Treg expansion and T cell activation and is associated with corneal allograft survival. Collectively, we provide compelling evidence for the use of TGF-ß1 licensing as an unconventional strategy for enhancing MSC immunosuppressive capacity.

Nitric Oxide Modulates Metabolic Processes in the Tumor Immune Microenvironment.

The metabolic requirements and functions of cancer and normal tissues are vastly different. Due to the rapid growth of cancer cells in the tumor microenvironment, distorted vasculature is commonly observed, which creates harsh environments that require rigorous and constantly evolving cellular adaption. A common hallmark of aggressive and therapeutically resistant tumors is hypoxia and hypoxia-induced stress markers. However, recent studies have identified alterations in a wide spectrum of metabolic pathways that dictate tumor behavior and response to therapy. Accordingly, it is becoming clear that metabolic processes are not uniform throughout the tumor microenvironment. Metabolic processes differ and are cell type specific where various factors promote metabolic heterogeneity within the tumor microenvironment. Furthermore, within the tumor, these metabolically distinct cell types can organize to form cellular neighborhoods that serve to establish a pro-tumor milieu in which distant and spatially distinct cellular neighborhoods can communicate via signaling metabolites from stroma, immune and tumor cells. In this review, we will discuss how biochemical interactions of various metabolic pathways influence cancer and immune microenvironments, as well as associated mechanisms that lead to good or poor clinical outcomes.

TNF-α/IL-1ß-licensed mesenchymal stromal cells promote corneal allograft survival via myeloid cell-mediated induction of Foxp3+ regulatory T cells in the lung.

Mesenchymal stromal cells (MSCs) have shown promise as a therapy for immune-mediated disorders, including transplant rejection. Our group previously demonstrated the efficacy of pretransplant, systemic administration of allogeneic but not syngeneic MSCs in a rat cornea transplant model. The aim of this study was to enhance the immunomodulatory capacity of syngeneic MSCs. In vitro, MSCs licensed with TNF-α/IL-1ß (MSCsTNF-α/IL-1ß) suppress syngeneic lymphocyte proliferation via NO production. In vivo, when administered post-transplantation, nonlicensed syngeneic MSCs improved graft survival from 0 to 50% and MSCsTNF-α/IL-1ß, in an NO-dependent manner, improved survival to 70%. Improved survival was associated with increased CD4+CD25+forkhead box P3+ regulatory T (Treg) cells and decreased proinflammatory cytokine expression in the draining lymph node. MSCsTNF-α/IL-1ß demonstrated a more potent immunomodulatory capacity compared with nonlicensed MSCs, promoting an immune-regulatory CD11b+B220+ monocyte/macrophage population and significantly expanding Treg cells in the lungs and spleen. Ex vivo, we observed that lung-derived myeloid cells act as intermediaries of MSC immunomodulatory function. MSC-conditioned myeloid cells suppressed stimulated lymphocyte proliferation and promoted expansion of Treg cells from naive lymphocytes. This work illustrates how syngeneic MSC therapy can be enhanced by licensing and optimization of timing strategies and further highlights the important role of myeloid cells in mediating MSC immunomodulatory capacity.-Murphy, N., Treacy, O., Lynch, K., Morcos, M., Lohan, P., Howard, L., Fahy, G., Griffin, M. D., Ryan, A. E., Ritter, T. TNF-α/IL-1ß-licensed mesenchymal stromal cells promote corneal allograft survival via myeloid cell-mediated induction of Foxp3+ regulatory T cells in the lung.

Coexpression of NOS2 and COX2 accelerates tumor growth and reduces survival in estrogen receptor-negative breast cancer.

Proinflammatory signaling pathways are commonly up-regulated in breast cancer. In estrogen receptor-negative (ER-) and triple-negative breast cancer (TNBC), nitric oxide synthase-2 (NOS2) and cyclooxygenase-2 (COX2) have been described as independent predictors of disease outcome. We further explore these findings by investigating the impact of their coexpression on breast cancer survival. Elevated coexpression of NOS2/COX2 proteins is a strong predictor of poor survival among ER- patients (hazard ratio: 21). Furthermore, we found that the key products of NOS2 and COX2, NO and prostaglandin E2 (PGE2), respectively, promote feed-forward NOS2/COX2 crosstalk in both MDA-MB-468 (basal-like) and MDA-MB-231 (mesenchymal-like) TNBC cell lines in which NO induced COX2 and PGE2 induced NOS2 proteins. COX2 induction by NO involved TRAF2 activation that occurred in a TNFα-dependent manner in MDA-MB-468 cells. In contrast, NO-mediated TRAF2 activation in the more aggressive MDA-MB-231 cells was TNFα independent but involved the endoplasmic reticulum stress response. Inhibition of NOS2 and COX2 using amino-guanidine and aspirin/indomethacin yielded an additive reduction in the growth of MDA-MB-231 tumor xenografts. These findings support a role of NOS2/COX2 crosstalk during disease progression of aggressive cancer phenotypes and offer insight into therapeutic applications for better survival of patients with ER- and TNBC disease.

Interspecies Incompatibilities Limit the Immunomodulatory Effect of Human Mesenchymal Stromal Cells in the Rat.

Mesenchymal stem/stromal cells (MSC) are an immunomodulatory cell population which are under preclinical and clinical investigation for a number of inflammatory conditions including transplantation. In this study, a well-established rat corneal transplantation model was used to test the ability of human MSC to prolong corneal allograft rejection-free survival using a pre-transplant intravenous infusion protocol previously shown to be efficacious with allogeneic rat MSC. Surprisingly, pre-transplant administration of human MSC had no effect on corneal allograft survival. In vitro, human MSC failed to produce nitric oxide and upregulate IDO and, as a consequence, could not suppress rat T-cell proliferation. Furthermore, human MSC were not activated by rat pro-inflammatory cytokines. Thus, interspecies incompatibility in cytokine signaling leading to failure of MSC licensing may explain the lack of in vivo efficacy of human MSC in a rat tissue allotransplant model. Interspecies incompatibilities should be taken into consideration when interpreting preclinical data efficacy data in the context of translation to clinical trial. Stem Cells 2018;36:1210-1215.

Secreted factors from metastatic prostate cancer cells stimulate mesenchymal stem cell transition to a pro-tumourigenic 'activated' state that enhances prostate cancer cell migration.

Mesenchymal stem cells (MSCs) are a heterogeneous population of multipotent cells that are capable of differentiating into osteocytes, chondrocytes and adipocytes. Recently, MSCs have been found to home to the tumour site and engraft in the tumour stroma. However, it is not yet known whether they have a tumour promoting or suppressive function. We investigated the interaction between prostate cancer cell lines 22Rv1, DU145 and PC3, and bone marrow-derived MSCs. MSCs were 'educated' for extended periods in prostate cancer cell conditioned media and PC3-educated MSCs were found to be the most responsive with a secretory profile rich in pro-inflammatory cytokines. PC3-educated MSCs secreted increased osteopontin (OPN), interleukin-8 (IL-8) and fibroblast growth factor-2 (FGF-2) and decreased soluble fms-like tyrosine kinase-1 (sFlt-1) compared to untreated MSCs. PC3-educated MSCs showed a reduced migration and proliferation capacity that was dependent on exposure to PC3-conditioned medium. Vimentin and α-smooth muscle actin (αSMA) expression was decreased in PC3-educated MSCs compared to untreated MSCs. PC3 and DU145 education of healthy donor and prostate cancer patient-derived MSCs led to a reduced proportion of FAP+ αSMA+ cells contrary to characteristics commonly associated with cancer associated fibroblasts (CAFs). The migration of PC3 cells was increased toward both PC3-educated and DU145-educated MSCs compared to untreated MSCs, while DU145 migration was only enhanced toward patient-derived MSCs. In summary, MSCs developed an altered phenotype in response to prostate cancer conditioned medium which resulted in increased secretion of pro-inflammatory cytokines, modified functional activity and the chemoattraction of prostate cancer cells.

Mesenchymal Stem Cell-derived Extracellular Vesicles: Toward Cell-free Therapeutic Applications.

Mesenchymal stem (stromal) cells (MSCs) are multipotent cells with the ability to differentiate into several cell types, thus serving as a cell reservoir for regenerative medicine. Much of the current interest in therapeutic application of MSCs to various disease settings can be linked to their immunosuppressive and anti-inflammatory properties. One of the key mechanisms of MSC anti-inflammatory effects is the secretion of soluble factors with paracrine actions. Recently it has emerged that the paracrine functions of MSCs could, at least in part, be mediated by extracellular vesicles (EVs). EVs are predominantly released from the endosomal compartment and contain a cargo that includes miRNA, mRNA, and proteins from their cells of origin. Recent animal model-based studies suggest that EVs have significant potential as a novel alternative to whole cell therapies. Compared to their parent cells, EVs may have a superior safety profile and can be safely stored without losing function. In this article, we review current knowledge related to the potential use of MSC-derived EVs in various diseases and discuss the promising future for EVs as an alternative, cell-free therapy.

Chondrogenic differentiation increases antidonor immune response to allogeneic mesenchymal stem cell transplantation.

Allogeneic mesenchymal stem cells (allo-MSCs) have potent regenerative and immunosuppressive potential and are being investigated as a therapy for osteoarthritis; however, little is known about the immunological changes that occur in allo-MSCs after ex vivo induced or in vivo differentiation. Three-dimensional chondrogenic differentiation was induced in an alginate matrix, which served to immobilize and potentially protect MSCs at the site of implantation. We show that allogeneic differentiated MSCs lost the ability to inhibit T-cell proliferation in vitro, in association with reduced nitric oxide and prostaglandin E2 secretion. Differentiation altered immunogenicity as evidenced by induced proliferation of allogeneic T cells and increased susceptibility to cytotoxic lysis by allo-specific T cells. Undifferentiated or differentiated allo-MSCs were implanted subcutaneously, with and without alginate encapsulation. Increased CD3(+) and CD68(+) infiltration was evident in differentiated and splenocyte encapsulated implants only. Without encapsulation, increased local memory T-cell responses were detectable in recipients of undifferentiated and differentiated MSCs; however, only differentiated MSCs induced systemic memory T-cell responses. In recipients of encapsulated allogeneic cells, only differentiated allo-MSCs induced memory T-cell responses locally and systemically. Systemic alloimmune responses to differentiated MSCs indicate immunogenicity regardless of alginate encapsulation and may require immunosuppressive therapy for therapeutic use.

Donor bone marrow-derived dendritic cells prolong corneal allograft survival and promote an intragraft immunoregulatory milieu.

Investigations into cell therapies for application in organ transplantation have grown. Here, we describe the ex vivo generation of donor bone marrow-derived dendritic cells (BMDCs) and glucocorticoid-treated BMDCs with potent immunomodulatory properties for application in allogeneic transplantation. BMDCs were treated with dexamethasone (Dexa) to induce an immature, maturation-resistant phenotype. BMDC and Dexa BMDC phenotype, antigen presenting cell function, and immunomodulatory properties were fully characterized. Both populations display significant immunomodulatory properties, including, but not limited to, a significant increase in mRNA expression of programmed death-ligand 1 and indoleamine 2,3-dioxygenase. BMDCs and Dexa BMDCs display a profound impaired capacity to stimulate allogeneic lymphocytes. Moreover, in a fully MHC I/II mismatched rat corneal transplantation model, injection of donor-derived, untreated BMDC or Dexa BMDCs (1 × 10(6) cells, day -7) significantly prolonged corneal allograft survival without the need for additional immunosuppression. Although neovascularization was not reduced and evidence of donor-specific alloantibody response was detected, a significant reduction in allograft cellular infiltration combined with a significant increase in the ratio of intragraft FoxP3-expressing regulatory cells was observed. Our comprehensive analysis demonstrates the novel cellular therapeutic approach and significant effect of donor-derived, untreated BMDCs and Dexa BMDCs in preventing corneal allograft rejection.

Tumor-associated mesenchymal stromal cells modulate macrophage phagocytosis in stromal-rich colorectal cancer via PD-1 signaling.

CMS4 colorectal cancer (CRC), based on the consensus molecular subtype (CMS), stratifies patients with the poorest disease-free survival rates. It is characterized by a strong mesenchymal stromal cell (MSC) signature, wound healing-like inflammation and therapy resistance. We utilized 2D and 3D in vitro, in vivo, and ex vivo models to assess the impact of inflammation and stromal cells on immunosuppression in CMS4 CRC. RNA sequencing data from untreated stage II/III CRC patients showed enriched TNF-α signatures in CMS1 and CMS4 tumors. Secretome from TNF-α treated cancer cells induced an immunomodulatory and chemotactic phenotype in MSC and cancer-associated fibroblasts (CAFs). Macrophages in CRC tumours migrate and preferentially localise in stromal compartment. Inflammatory CRC secretome enhances expression of PD-L1 and CD47 on both human and murine stromal cells. We demonstrate that TNF-α-induced inflammation in CRC suppresses macrophage phagocytosis via stromal cells. We show that stromal cell-mediated suppression of macrophage phagocytosis is mediated in part through PD-1 signaling. These data suggest that re-stratification of CRC by CMS may reveal patient subsets with microsatellite stable tumors, particularly CMS4-like tumors, that may respond to immunotherapies.

Targeting the EP1 receptor reduces Fas ligand expression and increases the antitumor immune response in an in vivo model of colon cancer.

Despite studies demonstrating that inhibition of cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2 ) has significant chemotherapeutic benefits in vitro and in vivo, inhibition of COX enzymes is associated with serious gastrointestinal and cardiovascular side effects, limiting the clinical utility of these drugs. PGE2 signals through four different receptors (EP1-EP4) and targeting individual receptor(s) may avoid these side effects, while retaining significant anticancer benefits. Here, we show that targeted inhibition of the EP1 receptor in the tumor cells and the tumor microenvironment resulted in the significant inhibition of tumor growth in vivo. Both dietary administration and direct injection of the EP1 receptor-specific antagonist, ONO-8713, effectively reduced the growth of established CT26 tumors in BALB/c mice, with suppression of the EP1 receptor in the tumor cells alone less effective in reducing tumor growth. This antitumor effect was associated with reduced Fas ligand expression and attenuated tumor-induced immune suppression. In particular, tumor infiltration by CD4(+) CD25(+) Foxp3(+) regulatory T cells was decreased, whereas the cytotoxic activity of isolated splenocytes against CT26 cells was increased. F4/80(+) macrophage infiltration was also decreased; however, there was no change in macrophage phenotype. These findings suggest that the EP1 receptor represents a potential target for the treatment of colon cancer.

Anti-donor immune responses elicited by allogeneic mesenchymal stem cells: what have we learned so far?

Mesenchymal stem (stromal) cells (MSCs) have potent anti-inflammatory/immunosuppressive properties which underlie much of their therapeutic potential. This fact has led to the widely accepted belief that MSCs from genetically unrelated individuals (allogeneic (allo)-MSCs) can be used therapeutically with equal efficacy to autologous MSCs and without triggering the donor-specific immune responses that are typically associated with allo-transplants. In this article, we critically review available experimental data to determine whether good in vivo evidence exists in support of the 'immune privileged' status of allo-MSCs. We also examine published studies regarding the immunogenicity of allo-MSCs following activation ('licensing') by inflammatory stimuli or following differentiation. Among the identified studies which have addressed in vivo immunogenicity of allo-MSCs, there was substantial variability as regards experimental species, disease model, route of MSC administration, cell dose and stringency of the immunological assays employed. Nonetheless, the majority of these studies has documented specific cellular (T-cell) and humoral (B-cell/antibody) immune responses against donor antigens following administration of non-manipulated, interferon-γ-activated and differentiated allo-MSCs. The consequences of such anti-donor immune responses were also variable and ranged from reduced in vivo survival of allo-MSCs with accelerated rejection of subsequent allogeneic transplants to apparent promotion of donor-specific tolerance. On the basis of these findings and on existing knowledge of allo-antigen recognition from the field of transplant immunology, we propose that the concept of the immune privileged nature of allo-MSCs should be reconsidered and that the range and clinical implications of anti-donor immune responses elicited by allo-MSCs be more precisely studied in human and animal recipients.

Effects of intratracheal mesenchymal stromal cell therapy during recovery and resolution after ventilator-induced lung injury.

BACKGROUND: Mesenchymal stromal cells (MSCs) have been demonstrated to attenuate acute lung injury when delivered by intravenous or intratracheal routes. The authors aimed to determine the efficacy of and mechanism of action of intratracheal MSC therapy and to compare their efficacy in enhancing lung repair after ventilation-induced lung injury with intravenous MSC therapy. METHODS: : After induction of anesthesia, rats were orotracheally intubated and subjected to ventilation-induced lung injury (respiratory rate 18(-1) min, P insp 35 cm H2O,) to produce severe lung injury. After recovery, animals were randomized to receive: (1) no therapy, n = 4; (2) intratracheal vehicle (phosphate-buffered saline, 300 µl, n = 8); (3) intratracheal fibroblasts (4 × 10 cells, n = 8); (4) intratracheal MSCs (4 × 10(6) cells, n = 8); (5) intratracheal conditioned medium (300 µl, n = 8); or (6) intravenous MSCs (4 × 10(6) cells, n = 4). The extent of recovery after acute lung injury and the inflammatory response was assessed after 48 h. RESULTS: Intratracheal MSC therapy enhanced repair after ventilation-induced lung injury, improving arterial oxygenation (mean ± SD, 146 ± 3.9 vs. 110.8 ± 21.5 mmHg), restoring lung compliance (1.04 ± 0.11 vs. 0.83 ± 0.06 ml · cm H2O(-1)), reducing total lung water, and decreasing lung inflammation and histologic injury compared with control. Intratracheal MSC therapy attenuated alveolar tumor necrosis factor-α (130 ± 43 vs. 488 ± 211 pg · ml(-1)) and interleukin-6 concentrations (138 ± 18 vs. 260 ± 82 pg · ml(-1)). The efficacy of intratracheal MSCs was comparable with intravenous MSC therapy. Intratracheal MSCs seemed to act via a paracine mechanism, with conditioned MSC medium also enhancing lung repair after injury. CONCLUSIONS: Intratracheal MSC therapy enhanced recovery after ventilation-induced lung injury via a paracrine mechanism, and was as effective as intravenous MSC therapy.

Immunomodulatory function of licensed human bone marrow mesenchymal stromal cell-derived apoptotic bodies.

BACKGROUND: Mesenchymal stromal cells (MSCs) show great potential for immunomodulatory and anti-inflammatory treatments. Clinical trials have been performed for the treatment of Type 1 diabetes, graft-versus-host disease and organ transplantation, which offer a promise of MSCs as an immunomodulatory therapy. Nevertheless, their unstable efficacy and immunogenicity concerns present challenges to clinical translation. It has emerged that the MSC-derived secretome, which includes secreted proteins, exosomes, apoptotic bodies (ABs) and other macromolecules, may have similar therapeutic effects to parent MSCs. Among all of the components of the MSC-derived secretome, most interest thus far has been garnered by exosomes for their therapeutic potential. However, since MSCs were reported to undergo apoptosis after in vivo transplantation and release ABs, we speculated as to whether ABs have immunomodulatory effects. In this study, cytokine licensing was used to enhance the immunomodulatory potency of MSCs and ABs derived from licensed MSCs in vitro were isolated to explore their immunomodulatory effects as an effective non-viable cell therapy. RESULTS: IFN-γ and IFN-γ/TGF-ß1 licensing enhanced the immunomodulatory effect of MSCs on T cell proliferation. Further, TGF-ß1 and IFN-γ licensing strengthened the immunomodulatory effect of MSC on reducing the TNF-α and IL-1ß expression by M1 macrophage-like THP-1 cells. Additionally, we discovered the immunomodulatory effect mediated by MSC-derived apoptotic bodies. Licensing impacted the uptake of ABs by recipient immune cells and importantly altered their phenotypes. CONCLUSION: ABs derived from IFN-γ/TGF-ß1-licensed apoptotic MSCs significantly inhibited T cell proliferation, induced more regulatory T cells, and maintained immunomodulatory T cells but reduced pro-inflammatory T cells.

Targeting stromal cell sialylation reverses T cell-mediated immunosuppression in the tumor microenvironment.

Immunosuppressive tumor microenvironments (TMEs) reduce the effectiveness of immune responses in cancer. Mesenchymal stromal cells (MSCs), precursors to cancer-associated fibroblasts (CAFs), promote tumor progression by enhancing immune cell suppression in colorectal cancer (CRC). Hyper-sialylation of glycans promotes immune evasion in cancer through binding of sialic acids to their receptors, Siglecs, expressed on immune cells, which results in inhibition of effector functions. The role of sialylation in shaping MSC/CAF immunosuppression in the TME is not well characterized. In this study, we show that tumor-conditioned stromal cells have increased sialyltransferase expression, α2,3/6-linked sialic acid, and Siglec ligands. Tumor-conditioned stromal cells and CAFs induce exhausted immunomodulatory CD8+ PD1+ and CD8+ Siglec-7+/Siglec-9+ T cell phenotypes. In vivo, targeting stromal cell sialylation reverses stromal cell-mediated immunosuppression, as shown by infiltration of CD25 and granzyme B-expressing CD8+ T cells in the tumor and draining lymph node. Targeting stromal cell sialylation may overcome immunosuppression in the CRC TME.

Autophagosomal IkappaB alpha degradation plays a role in the long term control of tumor necrosis factor-alpha-induced nuclear factor-kappaB (NF-kappaB) activity.

Transcription factor NF-κB is persistently activated in many chronic inflammatory diseases and cancers. The short term regulation of NF-κB is well understood, but little is known about the mechanisms of its long term activation. We studied the effect of a single application of TNF-α on NF-κB activity for up to 48 h in intestinal epithelial cells. Results show that NF-κB remained persistently activated up to 48 h after TNF-α and that the long term activation of NF-κB was accompanied by a biphasic degradation of IκBα. The first phase of IκBα degradation was proteasome-dependent, but the second was not. Further investigation showed that TNF-α stimulated formation of autophagosomes in intestinal epithelial cells and that IκBα co-localized with autophagosomal vesicles. Pharmacological or genetic blockade of autophagosome formation or the inhibition of lysosomal proteases decreased TNF-α-induced degradation of IκBα and lowered NF-κB target gene expression. Together, these findings indicate a role of autophagy in the control of long term NF-κB activity. Because abnormalities in autophagy have been linked to ineffective innate immunity, we propose that alterations in NF-κB may mediate this effect.

Mesenchymal stem cells enhance recovery and repair following ventilator-induced lung injury in the rat.

BACKGROUND: Bone-marrow derived mesenchymal stem cells (MSCs) reduce the severity of evolving acute lung injury (ALI), but their ability to repair the injured lung is not clear. A study was undertaken to determine the potential for MSCs to enhance repair after ventilator-induced lung injury (VILI) and elucidate the mechanisms underlying these effects. METHODS: Anaesthetised rats underwent injurious ventilation which produced severe ALI. Following recovery, they were given an intravenous injection of MSCs (2×10(6) cells) or vehicle immediately and a second dose 24 h later. The extent of recovery following VILI was assessed after 48 h. Subsequent experiments examined the potential for non-stem cells and for the MSC secretome to enhance VILI repair. The contribution of specific MSC-secreted mediators was then examined in a wound healing model. RESULTS: MSC therapy enhanced repair following VILI. MSCs enhanced restoration of systemic oxygenation and lung compliance, reduced total lung water, decreased lung inflammation and histological lung injury and restored lung structure. They attenuated alveolar tumour necrosis factor α concentrations while increasing concentrations of interleukin 10. These effects were not seen with non-stem cells (ie, rat fibroblasts). MSC-secreted products also enhanced lung repair and attenuated the inflammatory response following VILI. The beneficial effect of the MSC secretome on repair of pulmonary epithelial wounds was attenuated by prior depletion of keratinocyte growth factor. CONCLUSION: MSC therapy enhances lung repair following VILI via a paracrine mechanism that may be keratinocyte growth factor-dependent.