Read Anti-inflammatory effects of mesenchymal stem cells: novel concept for future therapies text version

Review

Cell- & Tissue-based Therapy

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Introduction Immunomodulatory and anti-inflammatory effects of mesenchymal stem cells Clinical applications of mesenchymal stem cells Conclusion Expert opinion

Anti-inflammatory effects of mesenchymal stem cells: novel concept for future therapies

Smita S Iyer & Mauricio Rojas

Emory

University, Department of Medicine, Atlanta, GA 30322, USA

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Background: Mesenchymal stem cells (MSC) are multipotent cells that can be isolated from the bone marrow and expanded in culture relatively easily. Culture-expanded MSC have been used in clinical settings to enhance hematopoietic stem cell engraftment in bone marrow transplant patients and in tissue regeneration therapy. More recently, the anti-inflammatory effects of MSC have generated a great deal of interest. Objective/methods: In this review we describe in vitro assays that have demonstrated how MSC regulate immune cell proliferation, differentiation and phenotype. We also highlight effector molecules produced by MSC that drive this function. In addition, we focus on animal models of lung injury, in which administration of MSC attenuates inflammation, and injury revealing a central role for MSC in mitigating pro-inflammatory networks and amplifying anti-inflammatory signals. Conclusions: The discoveries described herein have contributed to the novel concept of MSC as a therapeutic modality in inflammatory diseases, including acute lung injury.

Keywords: acute lung injury, bone marrow-derived mesenchymal stem cells, cytokines, immunomodulation, lung fibrosis, multipotent mesenchymal stromal cells Expert Opin. Biol. Ther. (2008) 8(5):569-581

1.

Introduction

The two fundamental characteristics of stem cells are their capacity for extensive self-renewal and their potential to diversify into cells of multiple lineages [1]. In contrast to the pleuripotent nature of embryonic stem cells, the fate pathways of mesenchymal stems cells (MSC) are somewhat restricted. MSC have the potential to differentiate into various connective tissue lineages which include adipose tissue, marrow stroma, cartilage, tendon and bone [2]. However, several lines of evidence exist that MSC can differentiate across germ layers; neural and myocardial fates have been demonstrated for MSC in vitro [3-5]. While these unorthodox cell fates raise the possibility that the plasticity of MSC may be more extensive then originally ascribed, demonstration of this capacity in vivo is needed. MSC have been isolated from multiple tissues including adipose tissue [6], skeletal muscle [7], synovium [8], spleen, thymus [9], blood, lung, fetal blood [10] and amniotic fluid [11]. The most accessible and by far the best characterized source of MSC is the bone marrow, and much of what we know today about MSC is based on studies of bone marrow-derived mesenchymal stem cells (BMDMSC) [12,13]. Friedenstein and colleagues were the first to describe a population of plastic adherent, spindle shaped, multipotent stromal cells in the bone marrow [14]. These cells were subsequently labeled mesenchymal stem cells by Caplan [15]. However, due to lack

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Anti-inflammatory effects of mesenchymal stem cells: novel concept for future therapies

of compelling evidence supporting the `stemness' of all bone marrow-derived stromal cell populations, the International Society for Cellular Therapy, in their recent position statement, recommends that the term `multipotent mesenchymal stromal cells' be used to designate plastic-adherent cells derived from the bone marrow and that the term `mesenchymal stem cells' be reserved for cells meeting specified stem-cell criteria [16]. Stricter distinctions will emerge in the literature as this definition is universally adopted. Until then, self-renewal capacity may not be assumed for cells labeled mesenchymal stem cells, unless otherwise stated. Nonetheless, the acronym MSC may be used to denote both cell populations. MSC are present in the bone marrow in relatively small numbers with an estimate of about 10 MSC, per million total bone marrow cells [17]. The low numbers of BMDMSC necessitate their expansion in vitro. Enrichment of BMDMSC from crude marrow suspensions is achieved by selection for a plastic-adherent cell population that expresses neither hematopoietic nor endothelial cell surface markers but is positive for the expression of adhesion and stromal markers. Several groups have reported the use of surface markers such as CD49a, CD73, CD90, CD105 and CD166 as markers for human MSC [18] and CD29, Sca-1, CD34, CD106 and CD44 surface antigens to identify mouse MSC [19,20]. More recently, antigens thought to mark embryonic stem cells; stage specific embryonic antigen-1 (SSEA-1) and SSEA-4 have been shown to identify mouse MSC [21,22]. However, despite a wide array of markers used for the detection of MSC by flow cytometry, a defined panel of unambiguous markers distinguishing MSC is lacking. The absence of a standardized MSC marker presents a challenge in the field and requires additional criteria for distinguishing MSC. A gold standard criterion for establishing MSC phenotype is a trilineage differentiation assay where the plasticity of MSC is confirmed by their ability to differentiate into adipocytes, osteocytes and chondrocytes, on stimulation. This plasticity makes BMDMSC attractive tools for tissue regeneration and BMDMSC have been used successfully in clinical settings to treat osteogenesis imperfecta in children [23]. BMDMSC have also been used to facilitate engraftment of hematopoietic cells after myeloablative therapy [24]. More recently, a case study reported a striking response of BMDMSC administration in the attenuation of graft-versus-host disease, demonstrating that BMDMSC can have immunomodulatory effects in vivo [25]. In this review, we discuss in vitro studies that have identified how BMDMSC interact with cells of the innate and adaptive immune systems. Numerous investigators have documented the antiinflammatory effects of exogenously administered BMDMSC in animal models of injury, including lung injury, and these studies will be discussed. Finally the therapeutic use of BMDMSC in humans, the promises and the challenges associated with BMDMSC therapy will be addressed.

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2. Immunomodulatory and anti-inflammatory effects of mesenchymal stem cells

2.1

In vitro observations

Our ability to dissect the immune capabilities of BMDMSC has been greatly advanced by in vitro studies that have investigated the interaction of BMDMSC with cells of the innate and adaptive immune system [26-30]. This reductionist approach has enabled the identification of two principal means by which BMDMSC modulate the immune response, by cell-to-cell contact and by the secretion of regulatory molecules. The mixed lymphocyte reaction (MLR) is the most widely used experimental strategy to investigate the immunomodulatory effects of MSC in vitro. Peripheral blood lymphocytes from unrelated donors (allogeneic lymphocytes) are obtained and irradiated lymphocytes from one donor (stimulating cells) are cultured with lymphocytes from the second donor (responder cells). The immunomodulatory effect of MSC is determined by their ability to inhibit proliferation of the responder cells in the MLR. The MSC are bone marrow-derived, culture-expanded and can be allogeneic, autologous or `third-party' to responder or stimulatory lymphocytes. The ability of BMDMSC to inhibit lymphocyte proliferation in response to mitogens such as phytohemagglutinin (PHA) or concanavalin A (conA) is another widely used technique. Several lines of evidence indicate that BMDMSC inhibit lymphocyte proliferation [31,32]. Bartholomew and colleagues demonstrated that baboon BMDMSC inhibited T lymphocyte proliferation in a MLR [27]. BMDMSC also suppressed proliferation of conA-stimulated lymphocytes in a dose-dependent manner. Suppression of lymphocyte proliferation by BMDMSC was antagonized but not completely inhibited by IL-2, a T-cell mitogen. The observation that T-cell proliferation is inhibited by allogeneic or thirdparty BMDMSC led to the suggestion that BMDMSC do not express the MHC. Recent evidence demonstrates that undifferentiated BMDMSC express intermediate levels of MHC Class I but do not express MHC Class II or costimulatory molecules such as CD40, B7-1 and B7-2 [26,33]. Furthermore, BMDMSC retain their immunosuppressive capabilities after differentiation along adipogenic, chondrogenic, and osteogenic lineages. Clinically, this means that undifferentiated and differentiated BMDMSC are suitable for transplantation, even between MHC incompatible individuals. Transplantation across MHC barriers would also require that allogeneic BMDMSC escape recognition by differentiated effector CD8+ T cells/cytotoxic T lymphocytes (CTL). Evidence that BMDMSC may be able to escape allorecognition by CTL comes from Rasmusson and colleagues who demonstrated that peptide-pulsed BMDMSC are resistant to CTL-mediated cell lysis [34]. BMDMSC can downregulate the activation of CTL and prevent expression of pro-inflammatory cytokines such as IFN- and TNF-.

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Table 1. Immunomodulatory effects of BMDMSC on immune cells determined by in vitro studies.

Immune cell DCs MSC* species Human Immunomodulatory effect Differentiation of monocytes to DC inhibited by MSC at a MSC:monocyte ratio ranging from 1:10 to 1:40. Co-culture of mature DC with MSC resulted in decreased expression of presentation molecules, HLA-DR and CD1a, and costimulatory molecules CD86 and CD80, suggesting that MSC skewed the phenotype of mature DC to an immature status. In a MLR, DC co-cultured with MSC showed a reduced potential to activate CD4 T-cells; MSC/DC reduced T-cell mediated production of IL-12, IFN-, and increased IL-10 NK-mediated lysis of K562 cells not affected by MSC Secretion of IFN- from IL-12 stimulated-NK cells inhibited by MSC by up to 80% MSC inhibited NK-mediated cytotoxicity in a MLR but high numbers of MSC were required, the ratio of MSC: blood lymphocytes was 1:1 MSC inhibited IL-2-induced proliferation of resting NK cells but only partially inhibited proliferation of activated NK cells. Lysis of MSC by IL-2-stimulated NK cells was inhibited by exposing MSC to IFN- B-cells Human Proliferation of stimulated B-cells was inhibited via soluble factors secreted by MSC. MSCs inhibited B-cell mediated IgG, IgM, and IgA production. MSC downregulated the expression of chemokine receptors, CXCR4, CXCR5 and CCR7 and as a consequence B-cell chemotaxis to the respective ligands, CXCL12, CXCL13 and CXCL19 was attenuated. These effects were seen at a 1:1 MSC:B-cell ratio. B-cell expression of co-stimulatory molecules was unaffected by MSC Ref.

[36]

NK cells

Human

[31] [28] [73]

[37]

[38]

*MSC's are bone-marrow-derived, unless specified. BMDMSC: Bone marrow-derived mesenchymal stem cells; DC: Dendritic cells; MSC: Mesenchymal stem cells; NK: Natural killer.

Together, these findings indicate that allogeneic BMDMSC can persist across MHC barriers and that engrafted BMDMSC can elicit immunosuppressive effects. However, the immunosuppressive properties of allogeneic MSC appear to depend on the competence of the host immune system. In immunocompetent mice, MHC-mismatched MSC failed to engraft, and increases in host lymphoid and natural killer cells (NK cells) were detected at the site of infusion [35]. It is also important to note that BMDMSC can elicit immunosuppressive effects only when administered at the initiation of, or early in the MLR. For instance, BMDMSC inhibit the activation of CTL, as measured by lysis of target lymphocytes, only when present in the culture at the initiation of the MLR [31]. When BMDMSC are added on day 3 after initiation of MLR, CTL-mediated lysis of target lymphocytes is not abrogated, indicating that BMDMSC can suppress formation of CTL but cannot inhibit activated CTL. BMDMSC also interact with CTL in a dose-dependent manner. While little inhibition of CTLmediated lymphocyte lysis is observed when BMDMSC constitute 0.1% of responder cells in the MLR, lymphocyte lysis is inhibited by up to 40% when BMDMSC constitute 10% of responder cells. Interestingly, at this concentration BMDMSC are equally effective at inhibiting lymphocyte lysis when the effector CTL to target lymphocyte ratio is as high as 50:1 and when it is as low as 6:1, indicating that secretion of soluble factors by BMDMSC is probably responsible for CTL inhibition. Indeed, CTL lysis is still reduced when BMDMSC and lymphocytes are separated by a transwell system. These observations underscore the

importance of both BMDMSC number and timing in curtailing CTL activity and also indicate that because BMDMSC inhibit activation of CTLs by secretion of soluble factors they can be effective over a range of effector: target ratios. The immunoregulatory effects of BMDMSC are not restricted to T cells; BMDMSC interact with multiple immune cells including dendritic cells [28,36], NK cells [28,31,37] and B cells (Table 1) [38]. BMDMSC can also alter the phenotype of immune cells; Aggarwal and Pittenger report that BMDMSC promote a shift from a pro-inflammatory to an anti-inflammatory phenotype in lymphocytes [28]. By decreasing T cell-mediated production of IFN-, BMDMSC induce a T-helper 2 (TH2) shift in immature T cells cultured under TH1-inducing conditions. BMDMSC potentiate a TH2 shift by increasing IL-4 levels in T cells cultured under TH1-inducing conditions. This interaction indicates that BMDMSC can alter the outcome of an ongoing inflammatory response by polarizing the cytokine profile of T cell subsets to an anti-inflammatory phenotype. BMDMSC also skew the phenotype of cells from the innate immune system. BMDMSC decrease TNF-, and increase IL-10 in different dendritic cells subsets. Jiang et al. reported that BMDMSC decrease IL-12 and IFN- production, and increase IL-10 levels in stimulated dendritic cells [36]. BMDMSC deploy a complex array of mechanisms to regulate T cell proliferation and phenotype; these include secretion of soluble regulatory molecules and growth factors, deprivation of tryptophan in the local milieu and production of reactive nitrogen species (Figure 1) [28,36,39-42].

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Anti-inflammatory effects of mesenchymal stem cells: novel concept for future therapies

TNF- IL-12

IL-10

IgG CXCR4 IgM CXCR5 IgA CR7

IFN-

B-cells Dendritic cells IDO

NK cells AA COX-2 PGH2 PGES T-regulatory cells iNOS

Trp Kynurenine HGF TGF-1 IL-10

PGE2 NO

Lymphocytes

Figure 1. Effector molecules produced by BMDMSC that inhibit lymphocyte proliferation. BMDMSC inhibit T lymphocyte proliferation via soluble effector molecules. BMDMSC constitutively express tolerogenic mediators such as hepatocyte growth factor (HGF), TGF-1 and IL-10. BMDMSC also express COX2, the enzyme involved in prostanoid synthesis, and prostaglandin E2 (PGE2) can be detected in unstimulated BMDMSC. PGE2 signals via the prostaglandin E receptor type 2 (EP2) and inhibits T lymphocyte proliferation via accumulation of cAMP. Stimulated BMDMSC produce nitric oxide (NO), which suppresses lymphocyte proliferation by inhibiting signal transducer and activator of transcription 5 (stat5) phosphorylation. The enzyme indoleamine 2,3-dioxygenase (IDO) degrades the essential amino acid, Trp. The increase in IDO mRNA in BMDMSC is detected in mixed lymphocyte reactions (MLRs), and on stimulation with IFN-, TNF- and IL-1. Both depletion of Trp locally and the accumulation of oxidative metabolites of Trp degradation such as kynurenine can inhibit lymphocyte proliferation. BMDMSC also interact with other immune cells. At high concentrations BMDMSC inhibit B-cell proliferation, immunoglobin secretion and chemokine receptor expression. BMDMSC inhibit production of TNF- and IL-12 in stimulated dendritic cells and IFN- production in stimulated NK cells. MSC increase proliferation of T-regulatory cells by elaborating TGF-1 production.

AA: Arachidonic acid; BMDMSC: Bone marrow-derived mesenchymal stem cells; iNOS: Inducible nitric oxide synthase; PGES: PGE synthase.

While such an orchestration of immunosuppressive networks is striking, it is not unique to BMDMSC; activated macrophages and dendritic cells employ similar methods to quell an ongoing immune response and some of the effectors discussed herein promote maternal acceptance of the fetal allograft [43-46]. Human BMDMSC constitutively express tolerogenic factors such as hepatocyte growth factor (HGF), IL-10, and TGF-1, and production of HGF and TGF-1 is increased by as much as twofold by IFN- [39]. Furthermore, administration of HGF, IL-10 and TGF-1 suppresses lymphocyte proliferation in a MLR. However, blocking each of these factors alone or in combination fails to abrogate the antiproliferative effects of BMDMSC, indicating that BMDMSC utilize other distinct mediators

to suppress T-cell proliferation. One such mediator is prostaglandin E2 (PGE2), an enzymatic product of arachidonic acid metabolism. PGE2 is constitutively expressed in cultured human BMDMSC [39]. Interestingly, production of PGE2 is dramatically increased in BMDMSC after stimulation with either TNF- or IFN-, and inhibition of PGE2 synthesis abrogates the antiproliferative effects of BMDMSC on lymphocytes [28]. This makes PGE2 a critical mediator in the immunosuppressive effects of BMDMSC. BMDMSC also adjust extracellular levels of tryptophan (Trp), an essential amino acid. Local depletion of Trp and/or the accumulation of Trp metabolites inhibits T cell proliferation. Stimulation of BMDMSC with IFN- induces the Trp-degrading enzyme, indoleamine 2,3-dioxygenase

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(IDO), which metabolizes Trp to kynurenine. Accumulation of kynurenine occurs in a MLR with BMDMSC and treatment of T cells with kynurenine alone can suppress proliferation. Thus, in addition to the production of cytokines, growth factors and lipid mediators, BMDMSC can employ the restriction of local amino acid concentrations to regulate T cell proliferation. Recently Sato et al., reported that activated BMDMSC produce nitric oxide (NO), which suppresses T cell proliferation by preventing signal transducer and activator of transcription 5 (stat5) phosphorylation [47]. Pharmacological and genetic inhibition of inducible nitric oxide synthase (iNOS2) led to impaired suppression of T cell proliferation by BMDMSC. Furthermore, the effects were greater when BMDMSC were in contact with T cells compared with when they were separated by a transwell system. The criterion for cell-to-cell contact for NO release is intuitive, as target specificity would be enhanced and bystander effects such as non-selective oxidation of extracellular and membrane proteins would be mitigated [47]. Thus, BMDMSC can inhibit lymphocyte proliferation, phenotype and differentiation in vitro. While the precise molecular mechanisms that drive the immunomodulatory and immunosuppressive effects of BMDMSC are yet to be unraveled, data generated in animal models of injury show that MSC elicit anti-inflammatory effects in vivo. In the next section we describe how administration of BMDMSC attenuates the systemic inflammatory response in animal models of injury, and examine in some detail how BMDMSC protect against lung injury.

2.2 Anti-inflammatory effects of mesenchymal stem cells in animal models of injury

The ability of BMDMSC to create a tolerogenic niche by direct interaction with immune cells and by secretion of regulatory molecules makes them attractive therapeutic candidates in the regulation of the inflammatory response to infection and injury. Several animal studies have demonstrated that exogenously administered BMDMSC augment tissue repair and facilitate regeneration of injured tissue. These effects appear to be mediated largely by soluble factors produced by BMDMSC because the numbers of donor BMDMSC engrafting to the injured tissue are too low to account for the magnitude of protection conferred by BMDMSC. The immunomodulatory effects of BMDMSC has been demonstrated in numerous animal models of injury including myocardial injury [48], renal ischemia and reperfusion injury [49], hepatic failure [50], autoimmune encephalomyelitis [51] and burn wounds [52]. Ohnishi et al. reported that administration of BMDMSC intravenously to rats during acute myocarditis decreased levels of monocyte chemotactic protein-1 in the myocardium, increased angiogenesis, and improved cardiac function [48]. BMDMSC have also been shown to reverse hepatic failure in rats [50]. In a

novel experimental setup, plasma from rats treated with D-galactosamine, a hepatotoxin, was perfused through an extracorporeal bioreactor, seeded with either BMDMSC or fibroblasts. Serum biomarkers of liver injury decreased, and survival dramatically improved with the BMDMSC bioreactor. Therapy with BMDMSC-conditioned medium resulted in a significant reduction in leukocyte infiltration in the liver compared with fibroblast-conditioned medium. Furthermore, when leukocyte trafficking was monitored in real-time, by single-photon emission computed tomography, a distinct decrease in signal intensity was observed in the liver in BMDMSC-treated animals indicating that BMDMSC actively prevented leukocyte homing and infiltration at the site of injury. The efficacy of MSC in attenuating renal damage triggered by ischemia and reperfusion injury in rats has also been documented [49]. Serum creatinine and plasma urea levels were normalized within 24 h of MSC infusion; levels of IL-1, a pro-inflammatory cytokine, decreased and levels of IL-4, an anti-inflammatory cytokine, increased in renal tissue. While infusion of MSC confers substantial benefits in many animal models of injury at least two studies failed to find benefits with MSC infusion [53,54]. MSC failed to protect against rheumatoid arthritis (RA) in a mouse model of collagen-induced arthritis. In fact, when given at a high dose, MSC worsened the clinical symptoms. Although MSC increased IL-4 levels, IFN- also increased so that the TH1 response was exaggerated. MSC could not be detected in the articular environment of the knee suggesting that local concentrations of MSC may have been too low to exert immunosuppressive effects. Treatment of MSC in vitro with TNF-, a predominant cytokine in RA, inhibited the antiproliferative effect of MSC on T cells and increased levels of IL-6, another pro-inflammatory cytokine. Although these results indicate that MSC may not be suitable as a therapeutic modality in RA, it should be noted that the effects observed in this study may relate to the use of an immortalized MSC cell line. Indeed more recently, using a similar model of RA, Augello et al. reported that intraperitoneal infusion of allogeneic BMDMSC decreased serum TNF- levels and prevented damage to the bone and cartilage [55]. Future studies dissecting the mechanistic basis for the heterogeneity in outcomes between MSC-cell lines versus primary BMDMSC will undoubtedly shed more light on the properties of MSC that are critical in modulating the inflammatory response. In the setting of graft-versus-host disease (GVHD) Sudres et al. reported that allogeneic BMDMSC failed to protect against GVHD after allogeneic bone marrow transplant in mice. In contrast, preliminary data from bone marrow transplant patients administered allogenic BMDMSC suggest that BMDMSC protect against GVHD [56]. These differences probably relate to species-specific differences in immunosuppressive properties of MSC. Because human

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Anti-inflammatory effects of mesenchymal stem cells: novel concept for future therapies

BMDMSC are more immunosuppressive than murine BMDMSC, animal models may not always predict clinical effects.

2.2.1 Anti-inflammatory effects of mesenchymal stem cells in animal models of lung injury

The immunoregulatory effects of BMDMSC confer substantial protection in the setting of lung injury. Several studies, including some from our group, have demonstrated compelling benefits from the administration of BMDMSC in animal models of lung injury. In a murine model of acute lung injury (ALI), initiated by the administration of bacterial lipopolysaccharide (endotoxin/LPS), exogenous infusion of BMDMSC prevents the systemic inflammatory response to endotoxin and attenuates lung injury. In humans, ALI is initiated by an acute inflammatory response to a physical trauma or infection [57], most commonly sepsis [58], and often leads to severe respiratory failure termed the acute respiratory distress syndrome [59,60]. ALI is characterized by sequestration of neutrophils in the lung, lung edema, and upregulation of pro-inflammatory mediators, systemically and locally, in the lung. Administration of LPS to mice produces pathophysiologic changes similar to those seen in ALI in humans. We have shown recently that administration of BMDMSC attenuates the massive inflammatory response to LPS and protects the lung from injury. We obtained BMDMSC from syngeneic donors expressing green fluorescent protein (GFP) to facilitate in vivo tracking of donorderived BMDMSC. The cells were expanded in vitro and were depleted of hematopoietic and macrophage markers (CD45 and CD11b, respectively), prior to administration. BMDMSC were infused intravenously (i.v) immediately after intraperitoneal challenge with 1 mg LPS/kg body weight. This dose of LPS causes structural alterations in the lung between 6 and 48 h post-endotoxin and injury begins to resolve by 48 h [61]. BMDMSC infusion protected against pulmonary edema, a hallmark of ALI. Histological examination of lung sections demonstrated that infusion of BMDMSC completely attenuated neutrophil infiltration in the lung between 6 and 48 h. Plasma levels of proinflammatory cytokines, IL-1, IFN-, IL-6 and macrophage inflammatory protein-1 (MIP-1) were significantly decreased with BMDMSC infusion. Levels of IL-10 were maintained and G-CSF increased acutely. Thus, BMDMSC altered the pattern of cytokine responses to LPS; TH1 responses were decreased and levels of TH2 cytokines were maintained. The protection conferred by BMDMSC was not related to clearance of endotoxin, and appeared to be at least partly independent of BMDMSC engraftment into the lung. To further characterize the interaction between injured lung cells and BMDMSC, we co-cultured lung cells obtained from LPS-treated animals and from untreated animals with GFP+ BMDMSC. BMDMSC, plated on the bottom of a chamber, were separated from lung cells by a transwell insert with a

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pore size large enough to allow BMDMSC migration. Significantly higher numbers of BMDMSC migrated in response to LPS-treated lungs, suggesting that injured lung cells produce soluble factors driving BMDMSC chemotaxis. Next, we determined if injured lung cells could respond to factors produced by BMDMSC. For these experiments lung cells obtained from LPS-treated mice were separated from BMDMSC by a transwell insert that either prevented or permitted cell contact. Under both conditions, lung cells produced significantly lower amounts of MIP-1, IL-1, IL-12, IL-6 and regulated upon activation normal T cell expressed and secreted (RANTES). A greater suppression in levels of MIP-1 and RANTES was observed when contact with BMDMSC was permitted. What emerged from these findings was that injured lung cells induced BMDMSC migration, and that BMDMSC regulated inflammatory signaling processes in injured lung cells. To determine if BMDMSC alter regulatory networks mediating cytokine production in lung cells in vivo we used a porcine model of endotoxin-induced lung injury. Because BMDMSC protect against the early inflammatory response to LPS in this model (our unpublished observations) we examined changes in gene expression 30 min after administration of endotoxin plus BMDMSC. Animals receiving endotoxin plus buffy coat served as controls. RNA was isolated from the lung and transcriptional analysis was performed using the GeneChip porcine genome array, which represents 20,201 genes. The data were processed using the RMA algorithm, which performs a background correction and normalization. Normalized data were filtered based on fold changes and genes whose expression changed twofold or more with BMDMSC infusion, relative to treatment with buffy coat, were examined. Of the 303 genes that showed at least a twofold increase or decrease, 114 were annotated. To identify candidate signaling pathways and gene networks regulated by BMDMSC infusion during endotoxemia we used ingenuity pathway analysis (IPA) to evaluate relationships between genes. With the 114 genes entered, the IPA database formed 5 networks. We examined these networks to specifically identify ones containing signaling factors with known roles in immunomodulation. Figure 2 shows expression profiles for genes in networks belonging to TNF- and IL-1 signaling. Genes encoding TNF-, IL-1 and IL-1 underwent a > 2-fold decrease with BMDMSC infusion and expression of the IL-1 receptor antagonist (IL1RN) increased over the same period. Interestingly, of the genes downregulated, 20 were identified as belonging to the canonical acute phase response pathway. Expression of positive acute-phase proteins, such as C-reactive protein (CRP), Amyloid protein C, serum (APCS), Vitronectin (VTN), Fibrinogen gamma chain (FGG), and Orosomucoid 1 (ORM1) were downregulated with BMDMSC infusion. Tables 2 and 3 provide the annotated lists of genes that underwent a > twofold change in expression with BMDMSC infusion. These microarray

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A. PTHLH 2.086 CRP -5.222 APCS -7.722 SULT1E1 2.141 ORM1 ITIH1 -3.345 IL1B -3.160 GSTA2 -12.831 CD163 VIPR1 ALOX15 -4.341 -11.656 TF -4.106 ENTPD1 -2.082 2.430 VTN -8.902 CXCL2 2.469 -11.177 F2 -1.999 APOA1 -2.849 PROC -3.100 ITIH2 -3.488 A2M -3.718 RBP4 -3.294 TTR -7.365 Calpain FGG -23.742 CAPNS1 -2.317

B.

ASS1

-2.078 HAMP -2.116 AMBP -9.894 IL1A* -3.590

IGFBP1 -10.455

GLS

-2.353

SERPINA1 -8.502 KNG1 (includes EG:3827) -3.735 SPP1 4.912

RGSS -2.227

TNF -8.604

DMBT1 IL1RN* 2.246

-3.342

IL10 -4.923

OAS1

IGFBR3 -2.542

KLKB1 -2.523

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22.040

Figure 2. Infusion of BMDMSC during endotoxemia induces expression of anti-inflammatory mediators in the lung. (A) IPA gene network displaying potential interactions between genes in TNF- and (B) IL-1 signaling pathways. Unbroken lines represent direct interactions and dashed lines indicate indirect interactions. Fold change expression values for each gene represent change in expression with BMDMSC infusion relative to change in expression with infusion of buffy coat. Genes colored green displayed twofold or larger decreases and genes in red underwent twofold or larger increases, 30 min after BMDMSC infusion.

A2M: -2-Macroglobulin; ALOX15: Arachidonate 15-lipoxygenase; AMBP: -1-Microglobulin; APCS; Amyloid P component, serum; APOA1: Apolipoprotein A-I; ASS1: Argininosuccinate synthetase 1; BMDMSC: Bone marrow-derived mesenchymal stem cells; CAPNS1: Calpain, small subunit 1; CRP: C-reactive protein; CXCL2: Chemokine (C-X-C motif) ligand 2; DMBT1: Deleted in malignant brain tumors 1; ENTPD1: Ectonucleoside triphosphate diphosphohydrolase 1; F2: Coagulation factor II (thrombin); FGG: Fibrinogen gamma chain; GLS: Glutaminase; GSTA2: Glutathione S-transferase A2; HAMP: Hepcidin antimicrobial peptide; IGFBP: Insulin-like growth factor binding protein; IL1RN: IL-1 receptor antagonist; ITIH: Inter- (globulin) inhibitor; KLKB1: Kallikrein B, plasma; KNG1: Kininogen 1; ORM1: Orosomucoid 1; PTHLH: Parathyroid hormone-like hormone; PROC: Protein C; RBP4: Retinol binding protein 4; RGS5: Regulator of G-protein signaling 5; SERPINA1: Serpin peptidase inhibitor; SPP1: Secreted phosphoprotein 1; SULT1E1: Sulfotransferase family 1E, estrogen-preferring, member 1; TF: Transferrin; TTR: Transthyretin; VIPR1: Vasoactive intestinal peptide receptor 1; VTN: Vtronectin.

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Table 2. Genes shown in Figure 2A displaying 2-fold change with BMDMSC infusion during endotoxemia.

Gene ID ENTPD1 IGFBP1 AMBP TNF APCS TTR CRP IL-10 IL-1a ITIH2 DMBT1 RBP4 ITIH1 GLS RGS5 ASS1 VIPR1 HAMP OAS1 CXCL2 IL1RN PTHLH Name Ectonucleoside triphosphate diphosphohydrolase 1 Insulin-like growth factor binding protein 1 Alpha-1-microglobulin Tumor Necrosis Factor, alpha Amyloid P component, serum Transthyretin C-reactive protein IL-10 IL-1 Inter- (globulin) inhibitor H2 Deleted in malignant brain tumors 1 Retinol binding protein 4 Inter-alpha (globulin) inhibitor H1 Glutaminase Regulator of G-protein signaling 5 Argininosuccinate synthetase 1 Vasoactive intestinal peptide receptor 1 Hepcidin antimicrobial peptide 2,5-Oligoadenylate synthetase 1 Chemokine (C-X-C motif) ligand 2 IL-1 receptor antagonist Parathyroid hormone-like hormone Fold change -11.6 -10.4 -9.9 -8.6 -7.7 -7.4 -5.2 -5.0 -3.6 -3.5 -3.3 -3.3 -3.3 -2.3 -2.2 -2.1 -2.1 -2.1 22.0 2.5 2.2 2.1

BMDMSC: Bone marrow-derived mesenchymal stem cells.

analyses confirm the anti-inflammatory effects of BMDMSC reported in vitro, and extend these findings by providing molecular insights into the regulation of inflammatory process by BMDMSC. Gupta and colleagues recently reported attenuation of lung injury, and improved survival via intrapulmonary delivery of BMDMSC in an endotoxin model [62]. Infusion of BMDMSC 4 h after intratracheal LPS decreased lung edema at 24 and 48 h. Protein infiltration into the lung, a measure of leakiness of the alveolar capillary barrier, was decreased at 48 but not at 24 h. The authors also demonstrated a decrease in MIP-2 levels in the lung lining fluid by 24 h followed by a decrease in TNF- at 48 h. An acute increase (8 h) in IL-10 was noted in the plasma and lining fluid after BMDMSC infusion. Significant histological improvement in lung injury was observed despite low levels of donor BMDMSC engraftment in the lung. The differences in mediator production observed by Gupta et al.

versus those observed by us probably relate to differences both in the dose and mode of administration of endotoxin and BMDMSC. Nevertheless, it is evident that both intravenous and intratracheal infusion of BMDMSC curbs the severe acute inflammatory response systemically and in the lung, and significantly attenuates lung injury. Using an innovative cell-based and gene-based approach, Mei et al. report that the protective effect of BMDMSC in the LPS-injured mouse lung is greatly potentiated by infusion of BMDMSC overexpressing angiopoietin 1 (ANGPT1), a vasculoprotective gene [63]. BMDMSC infusion, 30 min after i.v LPS, significantly decreased airspace neutrophil count 3 days after LPS administration. In mice given BMDMSC expressing ANGPT1 (BMDMSC-pANGPT1), the level of inflammation was further reduced and could not be distinguished from that in control animals. Infusion of BMDMSC-pANGPT1 resulted in a significantly greater suppression of IFN- and IL-1 compared with BMDMSC alone. Indeed TNF- in the lining fluid was significantly reduced only with BMDMSC-pANGPT1 infusion. In concert with our observations and those by Gupta et al., the protective effect seen with BMDMSC therapy did not require high level of engraftment in the lung. These results demonstrate that protection conferred by BMDMSC can be augmented by gene therapy approaches, where a synergy of the anti-inflammatory effect of BMDMSC with improved preservation of endothelial function by overexpressing ANGPT1, can improve outcomes. Taken together, these findings demonstrate a role for BMDMSC in mitigating the inflammatory response to LPS and as a consequence, attenuating lung injury. These results indicate the potential for BMDMSC as a therapy in ALI, a disease with a staggering mortality rate and limited treatment options. While the previous studies demonstrate an acute protective effect of BMDMSC in ameliorating the systemic cytokine storm induced by LPS, we and others have shown that BMDMSC infusion also protects the lung from localized inflammation and aberrant repair induced by bleomycin [61,64,65]. Endotracheal administration of bleomycin leads to lung fibrosis and occurs in three stages. Bleomycininduced cytotoxicity leads to apoptosis and necrosis of the alveolar epithelial cells followed by an inflammatory phase characterized by infiltration of neutrophils and macrophages in the lung microenvironment which peaks at day 7. An aberrant repair and remodeling process ensues resulting in enhanced deposition of matrix molecules such as collagen at day 14. The fibrosis, together with impaired re-epithelialization of the alveolar wall, is a hallmark of the fibrotic process [66]. A protective effect of BMDMSC in reducing inflammation and moderating the lung remodeling in response to bleomycin was first reported by Ortiz and colleagues [64]. Male BMDMSC were injected into female mice immediately following bleomycin challenge. Co-purification of donor BMDMSC with type II epithelial cells indicated

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Table 3. Genes shown in Figure 2B displaying 2-fold change with BMDMSC infusion during endotoxemia.

Gene ID FGG GSTA2 ORM VTN SERPINA1 ALOX15 TF A2M IL-1b PROC APOA1 IGFBP3 KLKB1 KNG1 CAPNS1 F2 SPP1 CD163 SULT1E1 Name Fibrinogen gamma chain Glutathione S-transferase A2 Orosomucoid 1 Vitronectin Serpin peptidase inhibitor Arachidonate 15-lipoxygenase Transferrin -2-Macroglobulin IL-1 Protein C Apolipoprotein A-I Insulin-like growth factor binding protein 3 Kallikrein B, plasma Kininogen 1 Calpain, small subunit 1 Coagulation factor II (thrombin) Secreted phosphoprotein 1 Scavenger receptor activity Sulfotransferase family 1E, estrogen-preferring, member 1 Fold change -23.7 -12.8 -11.2 -8.9 -8.5 -4.3 -4.1 -3.7 -3.2 -3.1 -2.8 -2.5 -2.5 -3.7 -2.3 -1.99 4.9 2.4 2.1

BMDMSC: Bone marrow-derived mesenchymal stem cells.

that donor BMDMSC engrafted to the injured lung. BMDMSC infusion resulted in a decrease in lung matrix metalloprotease mRNA and in lung collagen content. Little protection was seen with BMDMSC infusion 7 days after bleomycin challenge. In this study, systemic changes in mediator production after BMDMSC infusion were not detected. We felt that a thorough characterization of the local and systemic response to BMDMSC infusion would provide additional insights into mechanisms by which BMDMSC confer protection in the bleomycin model. We administered bleomycin to myelosuppressed mice, and to mice with an intact bone marrow. Some mice in each group received infusion of GFP+ BMDMSC 6 h after bleomycin-treatment. BMDMSC infusion conferred a substantial survival benefit in myelosuppressed bleomycintreated mice. Morphometric analysis of the lung at day 14 revealed that BMDMSC infusion protected against bleomycin-induced lung injury. Engraftment of BMDMSC in the lung was observed at day 14, and the intensity of GFP staining in the lung was greatest in myelosuppressed animals that received BMDMSC compared with mice that had an intact bone marrow. mRNA levels of TH1 cytokines

(IL-2, IL-1 and IFN-) were significantly decreased in the lung 14 days after bleomycin treatment, and IL-4 expression was upregulated. BMDMSC infusion also increased circulating levels of G-CSF and GM-CSF at day 14. These results indicate that BMDMSC alter the cytokine milieu in favor of repair and together with evidence form Ortiz et al. support a role of BMDMSC in ameliorating a local inflammatory response to bleomycin-induced injury. While the time window is a critical factor in optimizing the protective effect of BMDMSC transplantation, recent data by Yan et al. indicate that infusion of BMDMSC at a later stage of lung injury can in fact be deleterious [67]. GFP+ BMDMSC were infused at 4 h, 60 days or 120 days after lung irradiation. Cells infused early (4 h) engrafted to the lung at low levels and were distributed around alveolar and bronchial epithelium. In contrast, cells injected at a later stage (60 and 120 days) were detected in the interstitium as myofibroblasts, indicating that differentiation of BMDMSC occurred in response to mediators produced in the injured tissue. These data point to the conclusion that infusion of BMDMSC during an ongoing fibrotic response may augment fibrosis. Thus, time window is a critical factor in BMDMSC infusion and must be given due consideration in order to optimize the protective effects of BMDMSC in lung injury. In end stage pulmonary diseases, lung transplantation is a viable treatment option. However, development of obliterative bronchiolitis (OB), in transplant patients reduces survival, accounting for 30% of deaths after the third year. In an animal model of heterotopic tracheal transplantation (our unpublished observations), we demonstrate that systemic administration of BMDMSC prevents the development of OB. We observed a complete inhibition of inflammation and fibrosis when mice received a single dose of BMDMSC immediately after tracheal transplant. This effect was independent of the strain of mice from where the BMDMSC were obtained. Because proliferation of T-regulatory cells (T-regs) represents a mechanism by which BMDMSC can attenuate graft rejection, we studied the interaction of BMDMSC with T-regs in vitro. We found that BMDMSC indeed induced proliferation of T-regs via the secretion of soluble factors such as TGF-1 and IL1-RN. In summary, in vivo studies in animal models of injury show that exogenously administered BMDMSC can protect from injury by attenuating inflammation.

3. Clinical applications of mesenchymal stem cells

A number of ongoing randomized Phase I/II clinical trials are evaluating the effects of BMDMSC transplantation in patients with multiple sclerosis, GVHD, Crohn's disease, and severe chronic myocardial ischemia. BMDMSC were first bought into clinical use for the purpose of enhancing

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hematopoietic recovery in bone marrow transplant patients [24,68]. This application was based on evidence that BMDMSC produced a variety of cytokines and growth factors that supported the growth of hematopoietic progenitors. Koc et al. evaluated the efficacy of autologous BMDMSC in enhancing hematopoietic engraftment, after myeloablation, in 28 breast cancer patients. BMDMSC were obtained from bone marrow aspirates, were expanded in culture over 20 ­ 50 days and administered via i.v infusion (1 × 106 ­ 2.2 × 106 BMDMSC/kg) at the time of hematopoietic cell transplantation. No toxicities were associated with BMDMSC infusion and rapid reconstitution of hematopoietic cells was reported. While the lack of a control group complicates the interpretation of the data, the results do demonstrate that administration of autologous, ex vivo-expanded BMDMSC is safe. The ability of BMDMSC to differentiate into osteoblasts led to a trial examining the effect of BMDMSC transplantation in children with osteogenesis imperfecta (OI), a disease resulting from defective type I collagen [23]. OI is characterized by retarded bone growth, skeletal deformities and occurrence of numerous fractures. Six children with OI received 2 i.v infusions of culture-expanded, allogeneic BMDMSC, at a dose of 1 × 106 ­ 5 × 106 BMDMSC/kg. In five of the six patients, infused BMDMSC engrafted in bone, marrow stroma and skin. These patients demonstrated increases in growth velocity, formation of new dense bone and a decrease in fractures, within the first 6 months after BMDMSC therapy. These studies show that therapeutic application of BMDMSC in enhancing hematopoietic engraftment, and in regenerating bone tissue is clinically safe and feasible. The application of BMDMSC as a treatment modality in GVHD emerged when transplantation of BMDMSC to a patient with grade IV acute GVHD resulted in striking clinical improvement [25,69]. Preliminary data show an overall response rate of 69% with BMDMSC in the treatment of steroid-resistant acute GVHD [56]. Currently numerous studies are evaluating the immunomodulatory effects of BMDMSC in attenuating GVHD. Thus, early clinical trials demonstrate that the antiinflammatory properties of BMDMSC can be harnessed therapeutically in the field of bone marrow transplantation, transplantation immunology and tissue regeneration.

4.

As our understanding of the cellular and biochemical events driving the immunomodulatory properties of BMDMSC evolves; so will the applications of BMDMSC in attenuating disease and improving health.

5.

Expert opinion

Conclusion

In summary, in vitro studies have shown that BMDMSC employ a dynamic network of effectors to regulate immune cell function and phenotype. The immunomodulatory properties of BMDMSC translate into anti-inflammatory effects in vivo and numerous animal studies have demonstrated that exogenously administered BMDMSC attenuate inflammation and facilitate regeneration of injured tissue. Therefore, ex vivo-expanded BMDMSC offer tremendous therapeutic potential in various areas of human disease.

578

From their original description in the bone marrow, 40 years ago, to the current applications in regenerative medicine; BMDMSC truly represent a bench-to-bedside paradigm. As the clinical applications for BMDMSC unfold, the challenge is to understand the biochemical, cellular and molecular basis of the anti-inflammatory effects of these cells. While BMDMSC have been shown to consistently attenuate inflammation in numerous experimental models of injury, the deleterious effects of MSC, at high doses, in rheumatoid arthritis [53] and recent evidence documenting B cell activation by MSC [70] point to areas that need further research. The key to understanding the effects of BMDMSC in vivo may lie in answers to two simple questions. What are the recipient factors that dictate BMDMSC response, and what inherent characteristics of BMDMSC potentiate this response? Evidence that recipient factors determine BMDMSC response exists; Yan et al. demonstrate that infusion of BMDMSC during an ongoing fibrotic response augments fibrosis [67]. Age, nutritional status and cytokine profile of the recipient represent important parameters because these factors determine polarization of the immune response relevant to resolution of injury or fibrosis. The second question relates to properties of BMDMSCs themselves. The application of BMDMSC in clinical settings requires culture expansion. However, the effects of long-term culture on MSC properties are unknown. Tolar et al. recently detected cytogenetic aberrations in BMDMSC after several passages [71] revealing that extensive culture can lead to mutations and clonal selection of a transformed population. Indeed, infusion of transformed MSC led to lung sarcomas and decreased survival in recipient animals. These findings raise concerns about the long-term stability of MSC in culture. It has also been shown that BMDMSC cultures are responsive to culture media. In one report, MSCs cultured in the presence of a demethylating agent showed increased expression of fibrocyte cell surface marker [72]. Therefore, culture conditions must be optimized to put into place a standard protocol for BMDMSC expansion. Whether culturing BMDMSC with specific growth factors and nutritional supplements can augment BMDMSC viability and immunosuppressive properties in vivo and/or prevent spontaneous transformation is an important question that needs to be addressed. In conclusion, a greater understanding of both recipient factors and the properties of BMDMSC that drive the anti-inflammatory effects of these cells will provide better

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ways to control and optimize the immune response for the benefit of the recipient.

endotoxin experiments and Jianguo Xu for his work with the heterotopic tracheal transplant model.

Acknowledgements

The authors acknowledge the contributions of Richard Parker, Natalie Thorn, and Claudia Corredor to the porcine Bibliography

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Declaration of interest

The authors have no conflict of interest to declare and no fee has been received for the payment of this manuscript.

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Affiliation

Smita S Iyer1,3,5 & Mauricio Rojas2,3,4,6 MD Author for correspondence 1Nutrition and Health Sciences Program, Atlanta, GA 30322, USA 2Division of Pulmonary, Allergy, and Critical Care Medicine, Atlanta, GA 30322, USA 3Center for Translational Research in the Lung, Atlanta, GA 30322, USA 4McKelvey Center for Lung Transplantation, Atlanta, GA 30322, USA 5Emory University, Clinical Biomarkers Laboratory, Department of Medicine, Atlanta, GA 30322, USA 6Emory University School of Medicine, Department of Medicine/Pulmonary, Whitehead Research Building, Suite 205j, Atlanta, GA 30322, USA Tel: +1 404 712 2169; Fax: +1 404 712 2974; E-mail: [email protected]

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Anti-inflammatory effects of mesenchymal stem cells: novel concept for future therapies