Mesenchymal Stromal Cells in the Antimicrobial Host Response of Hematopoietic Stem Cell Recipients with Graft-Versus-Host Disease—Friends Or Foes?

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REVIEW Mesenchymal stromal cells in the antimicrobial host response of hematopoietic stem cell recipients with graft-versus-host disease—friends or foes?

A Balan1,2, G Lucchini1, S Schmidt1, A Schneider1, L Tramsen1, S Kuc¸i1, R Meisel3, P Bader1 and T Lehrnbecher1

Mesenchymal stromal cells (MSCs) are multipotent cells, which exhibit broad immunosuppressive activities. Moreover, they may be administered irrespectively of human leukocyte antigen (HLA) compatibility, without inducing life-threatening immunological reactions, as they express no HLA class II and limited HLA class I antigens under resting conditions. These characteristics have made MSC an appealing candidate for cell therapy after hematopoietic stem cell transplantation (HSCT), for example, for treatment of graft-versus-host disease (GvHD) or for graft rejection prevention/treatment in allogeneic HSCT recipients. Unfortunately, information regarding the effect of MSC infusion on the host response to infectious agents is scarce, and study results on infectious complications in patients receiving MSC are conﬂicting. The present review focuses on the available data from in vitro studies and animal models regarding the interaction of MSC with bacterial, viral and fungal pathogens. In a clinical part, we present the current information on infectious complications in allogeneic HSCT recipients who had received MSCs as prophylaxis or treatment of GvHD disease.

Leukemia (2014) 28, 1941–1948; doi:10.1038/leu.2014.127

INTRODUCTION diabetes mellitus.32,35–38 As MSCs do not elicit immunologic Mesenchymal stromal cells (MSCs) are a heterogeneous cell responses from alloreactive T lymphocytes and/or other effector population endowed with multi-lineage differentiation potential cells, they are also evaluated as an innovative therapeutic tool to and extensive immunomodulatory properties.1 According to the prevent or to treat graft-versus-host disease (GvHD) following 39,40 definition of the International Society for Cellular Therapy, MSCs allogeneic hematopoietic stem cell transplantation (HSCT). In must (I) be plastic adherent in standard culture conditions; (II) be contrast to the promising results of MSCs administration in the capable of in vitro differentiation into adipocytes, chondroblasts different clinical settings, it remains a matter of concern that the and osteoblasts; and (III) express specific immunophenotypic application of immunosuppressive MSCs may inadvertently inhibit markers (CD73, CD90 and CD105), while lacking hematopoietic antimicrobial immune responses and ultimately result in an marker expression (CD14, CD34 and CD45) and class II major increased risk of infection, in particular in immunocompromised 41 histocompatibility complex molecules.2 MSCs have the capacity to patients such as allogeneic HSCT recipients. We therefore interact within the immune system,3–7 an activity that has been reviewed the current evidence of antimicrobial properties of described toward a large number of effector cells of the adaptive MSCs; we report on data from in vitro studies and animal models and innate immunity, including T-cell subsets, B cells, natural killer and focus in a clinical part on infectious complications in cells, monocyte-derived dendritic cells and neutrophils.1 The allogeneic HSCT recipients who had received MSCs as complex cellular interactions are modulated by a broad panel of prophylaxis or treatment of GvHD. soluble molecules, including interferon (IFN)-g,8,9 interleukin-1b (IL-1b),10 transforming growth factor beta,11–13 indoleamine-2, 3-dioxygenase (IDO),8,9,14 IL-6,15,16 IL-10refs. 17,18 and prostaglandin E2.4 IN VITRO STUDIES ON ANTIMICROBIAL PROPERTIES OF MSCS MSCs are also capable of inducing effective tissue repair and Most of the data on antimicrobial properties of MSCs are derived regeneration.19–21 MSCs can be isolated and expanded from many from in vitro studies with bacteria (Table 1). For both unstimulated tissues including bone marrow,22,23 umbilical cord blood,24–26 and stimulated human MSCs (huMSCs), a direct effect against a placenta27,28 and adipose tissue.29,30 Owing to these properties, variety of bacteria has been demonstrated (Figure 1). Unstimu- MSCs are currently investigated as novel cellular therapy in a lated huMSCs are capable to inhibit the growth of Gram-negative multitude of clinical settings.31 For example, in the regenerative bacteria such as Escherichia coli and Pseudomonas aeruginosa, field, MSCs have been widely used for their potential in as well as the growth of Gram-positive pathogens such as orthopedics, dermatology and cardiology.31–34 In addition, owing Staphylococcus aureus.42 For IFN-g-stimulated huMSCs, growth to their immunosuppressive properties, MSCs are currently tested inhibition of Gram-positive bacteria such as S. aureus, S. epidermidis, in autoimmune diseases, such as multiple sclerosis and Crohn’s group B streptococci and Enterococcus faecium has been disease, as well as in systemic lupus erythematosus and type 1 demonstrated.43 In unstimulated huMSCs, the antimicrobial

1Department of Pediatric Hematology and Oncology, Children’s Hospital, Johann Wolfgang Goethe University, Frankfurt, Germany; 2‘Victor Babes’ University of Medicine and Pharmacy, Timisoara, Romania and 3Department of Pediatric Oncology, Hematology, and Clinical Immunology, Heinrich-Heine University, Du¨sseldorf, Germany. Correspondence: Professor T Lehrnbecher, Department of Pediatric Hematology and Oncology, Children’s Hospital, Johann Wolfgang Goethe University, Theodor-Stern-Kai 7, Frankfurt D-60590, Germany. E-mail: [email protected] Received 2 January 2014; revised 22 February 2014; accepted 21 March 2014; accepted article preview online 3 April 2014; advance online publication, 25 April 2014 MSC and antimicrobial host response A Balan et al 1942 Table 1. In vitro studies on mesenchymal stromal cell activity in the infection setting

MSC type Activity Proposed mechanism Reference of action

Unstimulated huMSC Growth inhibition of Gram-positive and m Cathelicidin LL-37 Krasnodembskaya et al.42 Gram-negative bacteria IFN-g-stimulated huMSC Inhibition of Gram-positive bacteria m Indoleamine Meisel et al.43 Inhibition of intracellular CMV and HSV1 replication 2,3-dioxygenase Unstimulated muMSC Inhibition of bacterial growth m Lipocalin 2 Gupta et al.47 m Phagocytic activity IL-17-producing muMSC Inhibition of growth of C. albicans m IL-17 Yang et al.52 huMSC Enhancement of neutrophil function IL-6, IFN-b and Maqbool et al.55; Cassatella et al.56; Inhibition of neutrophil apoptosis GM-CSF Raffaghello et al.57 Abbreviations: CMV, cytomegalovirus; GM-CSF, granulocyte macrophage-colony stimulating factor; HSV1, herpes simplex virus 1; huMSC, human mesenchymal stromal cells; IFN, interferon; IL, interleukin; muMSC, murine mesenchymal stromal cells; m, increased.

Figure 1. Antimicrobial effects of human (left) and murine (right) MSCs on bacterial, viral and fungal pathogens. CMV, cytomegalovirus; EBV, Epstein–Barr virus; LPS, lipopolysaccharide; PGE2, prostaglandin E2; TH1, T helper cell type 1; TNF, tumor necrosis factor.

effect is mediated by the cathelicidin LL-37 in a dose-dependent of LL-37 by muMSCs, the presence of E. coli significantly increases manner. Co-incubation with E. coli significantly increases the the secretion of the antimicrobial molecule lipocalin 2 by expression of LL-37 in huMSCs, whereas the concentrations of muMSCs.47 Moreover, in contrast to huMSCs, muMSCs are not other antimicrobial peptides such as lipocalin 2, human-beta capable to express IDO even after stimulation with a combination defensins and surfactant protein D remain below the limit of of cytokines, which includes, in addition to IFN-g, both tumor detection.42 LL-37 has been demonstrated to kill many clinical necrosis factor-a and IL-1b.43 It has also been reported that relevant Gram-positive and Gram-negative bacteria such as unstimulated muMSCs exhibit phagocytic activity when co- Streptococcus spp., Staphylococcus spp., E. coli, P. aeruginosa and incubated with bacteria such as E. coli and S. aureus; however, Klebsiella pneumoniae.44 In addition, LL-37 affects fungi such as these data are still conflicting and need further confirmation.47–50 Candida albicans.45,46 In contrast to unstimulated huMSCs, the Little data are published regarding the impact of MSCs on viral antibacterial effect of IFN-g-stimulated huMSCs is mediated and fungal pathogens. One study reported that IFN-g-stimulated through the tryptophan-catabolizing enzyme IDO, which huMSCs reduce in vitro the intracellular replication of both ultimately leads to tryptophan depletion and accumulation of cytomegalovirus (CMV) and herpes simplex virus type 1.43 As toxic kynurenines resulting in growth inhibition. This effect is the antiviral activity of huMSCs could be completely reversed by increased when both tumor necrosis factor-a and IL-1b are added the addition of the IDO-specific inhibitor 1-methyltrypthophan, to IFN-g in order to stimulate huMSCs.43 It is important to note the authors conclude that the antiviral effect of huMSCs can be that the available data suggest a difference between the attributed to IDO, which is upregulated by stimulation with antibacterial activities of human and murine MSCs (muMSCs) IFN-g.43 Importantly, huMSCs do not suppress proliferation (Figure 1). For example, whereas no data exist on the production and IFN-g production of viral-specific T cells against CMV and

Leukemia (2014) 1941 – 1948 & 2014 Macmillan Publishers Limited MSC and antimicrobial host response A Balan et al 1943 Epstein–Barr virus, which is in contrast to the inhibition of combined action of IL-6, IFN-b and granulocyte macrophage functional properties of alloantigen and mitogen-induced T cells colony-stimulating factor.55–57 by huMSCs.51 In conclusion, the available data indicate that both muMSCs and Recently, Yang et al.52 reported on the antifungal activity of a huMSCs exhibit direct and indirect antimicrobial activities. subset of muMSCs. These single-colony derived muMSCs However, several issues are unresolved to date and need to be producing IL-17 inhibit the growth of C. albicans in vitro and the addressed in future studies. For example, it is unclear whether and antifungal effect of these IL-17-positive muMSCs is stronger as to what extent different sub-populations of MSCs are active compared with that of IL-17-negative or bulk muMSCs. The against bacterial, viral and fungal pathogens, respectively. In growth inhibition of C. albicans is mediated by IL-17 in a dose- addition, the different antimicrobial mechanisms (for example, dependent manner and anti-IL-17 antibodies can partially reduce secretion of soluble antimicrobial factors, phagocytosis) by which muMSC-mediated anti-C. albicans activity.52 Notably, in contrast to MSCs inhibit the growth or even kill the pathogens need to be the bulk muMSC population, IL-17-positive muMSCs are unable to better defined. Finally, it is unknown but clinically important how upregulate T regulatory cells or to downregulate T helper cell MSCs are triggered to up- or downregulate the pro- and anti- type 17 (TH17) cells and thus fail to execute MSC-based inflammatory host responses of other effector cells. immunosuppression.52 MSCs not only exhibit a direct antimicrobial effect, but also have an important role in the complex network of the host immune IN VIVO STUDIES ON ANTIMICROBIAL EFFECTS OF MSCS response against pathogens, in particular in the dynamic Animal models coordination of the pro- and anti-inflammatory components of The in vitro data demonstrating an antimicrobial activity of MSCs the immune system. For example, co-culturing of muMSCs and are clearly supported by animal models, although it has to be murine alveolar macrophages in the presence of lipopolysacchar- noted that the effect of MSCs has not been studied in an ide results in a synergistic effect on lipocalin 2 production as immunocompromised setting, in particular not in the setting of compared with lipocalin 2 production by either cell type alone.47 HSCT (Table 2). In immunocompetent mice suffering from At the same time, muMSCs increase in vitro the secretion of polymicrobial sepsis, which was induced by cecal ligation and the anti-inflammatory cytokine IL-10 by murine alveolar puncture and subsequent systemic infection, administration of macrophages.53 This effect is augmented by a direct contact of MSCs results not only in a reduction of the burden of pathogens, muMSC with murine alveolar macrophages and can be attributed but also in a significantly improved survival. These observations to the lipopolysaccharide-induced upregulation of prostaglandin were reported independently whether huMSCs or muMSCs were E2 in muMSCs.53 Taken together, these data suggest that MSCs are administered, independently of the route of administration able to modulate the quality and magnitude of effector responses (intravenously versus intraperitoneally) and independently of the and help to control the delicate balance between activation and strategy (prophylaxis versus therapy).48,49,53,58 In addition, the suppression of the immune response, or, in other words, that antimicrobial effect of MCSs was demonstrated in blood,49,53,58 MSCs are involved in the trade-off between eliminating a peritoneum,48,49,58 liver58 and spleen.48,58 These results are in line pathogen and limiting collateral damage to tissues and restoring with a model of Gram-negative pneumonia in immunocompetent homeostatic environment. mice where the administration of MSCs resulted in a significantly Conflicting results are reported whether or not muMSCs improved survival and in a reduction of bacterial burden both in increase the activity of professional phagocytes. Although Hall the lung and in the blood.42,47,59 et al.49 demonstrated that co-incubation with muMSCs increases There are several reasons that may account for the beneficial the phagocytic activity of peritoneal cavity-derived murine effect of MSCs in these settings. First, MSCs increase the neutrophils, this effect was not observed when neutrophils were antimicrobial host response, which results in a decrease of the derived from the bone marrow.47 In addition, muMSCs do not burden of pathogens. For example, in mice suffering from affect the phagocytic activity of peritoneal macrophages or of the pneumonia, it has been demonstrated that the concentration of peritoneal macrophage cell line J744.a1 when co-cultured with the antimicrobial molecule lipocalin 2 in the bronchoalveolar Gram-positive or Gram-negative bacteria.47,48 On the other hand, lavage is increased 8 h after the intravenous administration of it has been reported that co-incubation of muMSCs with muMSCs; blocking lipocalin 2 by a monoclonal antibody monocytes for several days results in alternative activated significantly decreases the antimicrobial host defence.47 Similar macrophages.54 These cells have both increased phagocytic and results can be observed for LL-37 in huMSCs.42 The decreased anti-inflammatory activity and have been suggested to have a burden of pathogens is further due to the upregulation of the major role in tissue repair.54 In contrast to the murine setting, phagocytic activity of neutrophils and macrophages by MSCs, several studies uniformly demonstrate that huMSCs significantly although these data are conflicting. Although muMSCs, administered prolong the survival and function of human neutrophils by the intraperitoneally to mice, increase in vivo phagocytosis of E. coli by

Table 2. In vivo studies on mesenchymal stromal cell activity in the infection setting

MSC type Activity Proposed mechanism of action Reference

huMSC and muMSC m Survival m Phagocytosis Mei et al.48 k Bacterial burden after cecal ligation m IL-10 Hall et al.49 k Oxidative stress Nemeth et al.53 Gonzalez-Rey et al.58 huMSC and muMSC m Survival m Lipocalin 2 in muMSC Krasnodembskaya et al.42 k Bacterial burden in pneumonia m LL-37 in huMSC Gupta et al.47 Kim et al.59 52 muMSC k Fungal burden in invasive candidiasis m TH17 Yang et al.

Abbreviations: huMSC, human mesenchymal stromal cells; IL interleukin; muMSC, murine mesenchymal stromal cells; TH17, T helper cell type 17; m, increased; k, decreased. All experiments were conducted in immunocompetent mice.

& 2014 Macmillan Publishers Limited Leukemia (2014) 1941 – 1948 MSC and antimicrobial host response A Balan et al 1944 peritoneal neutrophils,49 huMSCs given intravenously to mice with huMSCs became an interesting tool in the prophylaxis and P. aeruginosa sepsis do not increase the phagocytic activity of treatment of GvHD in the allogeneic HSCT setting (Table 3). peripheral blood neutrophils.60 Surprisingly, in the same setting, As the first report on a durable complete remission of huMSCs increase the phagocytic activity of blood monocytes/ advanced-stage steroid-resistant acute GvHD in a child macrophages, whereas they do not have an effect on the same treated with two infusions of haploidentical MSCs,63 multiple cell population derived from the spleen.60 The exact reason for studies have been performed in order to explore the potential these discrepancies is currently unknown. benefit of MSCs in GvHD. However, owing to the differences However, not only the enhanced antimicrobial host response not only in donor source, culture methods and expansion, contributes to the beneficial effect of MSCs in immunocompetent but also in the characteristics of transplantation (for example, mice suffering from sepsis, but also the downregulation of conditioning regimen, graft) and in the use of immuno- inflammatory responses that reduces tissue injury and thus suppressants, head-to-head comparisons of the different MSCs improves organ function. These effects have been demonstrated products are difficult to perform, and it is not surprising that the in lung,48 liver,49 spleen,49 bowel49 and kidney.48,49 Mice that have results of these studies are often conflicting.64 In addition, many received prophylactic administration of MSCs before induction of studies did not report in detail on incidence and severity of sepsis exhibited higher levels of the anti-inflammatory cytokine IL- infectious complications, which makes a systematic review of 10 as compared with mice without pre-treatment with MSCs. the data impossible. Blocking the IL-10/IL-10R pathway by antibodies prevented the beneficial effect of MSCs.53 In addition to the increase of the anti- inflammatory cytokine IL-10, MSCs diminish oxidative tissue CO-TRANSPLANTATION OF MSCS WITH THE GRAFT damage by reducing tissue levels of myeloperoxidase in bowel, Co-transplantation of MSCs with the graft has been reported to lung, liver and kidneys of septic mice.53,58,59 shorten the time to neutrophil engraftment65–67 and decrease the Although no in vivo data are published regarding the impact of risk of acute and chronic GvHD.68,69 In this regard, an open MSCs given to animals with viral infections, recent data demonstrate randomized clinical trial demonstrated that the incidence of acute a beneficial effect of the IL-17-producing sub-population of muMSCs grades II–IV and chronic GvHD in 10 patients receiving a median in immunocompetent mice suffering from invasive candidiasis.52 dose of 3.4 Â 105/kg MSCs derived from an human leukocyte Intravenous infusion of this cell population, which comprises o5% antigen-identical sibling donor was lower than in 15 patients not of the bone marrow-derived muMSCs, significantly reduces the receiving MSCs (11% versus 53%, and 14% versus 29%, fungal burden in kidneys as compared with mice, which received respectively).69 Unfortunately, this did not translate into a lower bulk muMSCs or IL17-negative muMSCs. The administration of risk for infectious complications, and the incidences of severely IL17-positive muMSCs also increases the number of TH17 cells in early- and mid-phase infections were even higher in patients who 52 the blood of mice infected with C. albicans. Notably, TH17 cells have received a co-transplantation of hematopoietic stem cells have an important role in the host response against extracellular and MSCs as compared with a control group not receiving MSCs, pathogens including fungi, promoting TH1 type immune although the differences did not reach statistical significance (4/10 61,62 responses and restraining TH2 type responses. (40%) versus 5/15 (33%)). Specifically, two patients each receiving Although these observations impressively demonstrate the MSCs suffered from CMV interstitial pneumonia and bacterial potential benefit of administering MSCs to animals suffering from and/or fungal infection, respectively, whereas this was seen in two severe infection, the data have to be confirmed in immunocom- and three patients, respectively, who did not receive MSCs.69 On promised animals before drawing firm conclusions in the setting the other hand, none of the patients who were treated with MSCs of HSCT. died because of infectious complications, whereas this was the fact in two patients of the control group not receiving MSCs, and one is prompted to speculate whether the severity of infections is lower when MSCs are co-transplanted with the graft. Notably, in CLINICAL OBSERVATIONS WITH MSCS AND INFECTIOUS two other, non-randomized clinical trials including 20 patients70 COMPLICATIONS IN THE SETTING OF ALLOGENEIC HSCT and 14 pediatric patients,71 co-transplantation of MSCs did not Owing to their immunosuppressive properties and their ability result in a higher incidence and severity of infections when to modulate angiogenesis and endogenous tissue repair, compared with historical controls.

Table 3. Clinical trials on the administration of MSCs in hematopoietic stem cell recipients, which also evaluated infectious complications

Study purpose Patients number Infectious complications Reference in pts receiving MSCs

GvHD prevention 25 m Infections (bacterial and fungal; CMV reactivation) Ning et al.69 GvHD prevention 20 No impact on incidence of infections Baron et al.70 GvHD prevention 14 No impact on incidence of infections Ball et al.71 Treatment of steroid-resistant GvHD 53 m Risk for pneumonia-related death for patients Forslow et al.77 receiving MSC, no impact on incidence of mold infections Treatment of steroid-resistant GvHD 31 30% of patients receiving MSCs died of late infectious Von Bahr et al.78 or hemorrhagic cystitis complications; no control group Treatment of steroid-resistant GvHD 15 m Incidence of invasive fungal disease, no increased Remberger and Ringden79 risk for viral reactivation Treatment of steroid-resistant GvHD 64 No significant risk for increased fungal infection Kurtzberg et al.80 Treatment of steroid-resistant GvHD 6 No significant risk for increased fungal infection Fang et al.81 Treatment of steroid-resistant GvHD 24 No significant risk for increased viral infection Lucchini et al.83 Abbreviations: CMV, cytomegalovirus; GvHD, graft-versus-host disease; MSC, mesenchymal stromal cell; m, increased.

Leukemia (2014) 1941 – 1948 & 2014 Macmillan Publishers Limited MSC and antimicrobial host response A Balan et al 1945 MSCS AS TREATMENT OF ACUTE GVHD Although the type of conditioning did not impact on the risk of Acute GvHD is the most frequent complication after allogeneic pneumonia-associated death, the multivariate analysis revealed HSCT and is associated with considerable morbidity and mortality, that grades II–IV acute GvHD, CMV infection and having received in particular in patients who did not respond to primary therapy.72 MSCs were factors associated with death from pneumonia. Approaches to therapy of patients refractory to standard Overall, 27.5% of the 53 patients with MSCs treatment died from treatment, which usually consists of glucocorticosteroids, include pneumonia, whereas this was only seen in 7.7% of the patients polyclonal or monoclonal antibodies, immunotoxins, additional who had not received MSCs (Po0.001). One-third of the patients immunosuppressive or chemotherapeutic interventions and with pneumonia-related death had a mold infection (19/60), but phototherapy.73 Unfortunately, in most of the studies, long-term here was no significant preponderance of mold infections in survival was not improved in comparison with the use of steroids patients with MSCs treatment. Overall, 85% of pneumonia-related alone, and infectious complications remain a major problem. deaths in patients having received MSCs occurred 4100 days Owing to their immunomodulatory properties in vitro and in vivo, after HSCT. These data corroborate with another study, which MSCs have therefore become an attractive option for cellular included 31 patients receiving MSCs for steroid-refractory acute therapy in acute GvHD.74 GvHD (n ¼ 23) or for hemorrhagic cystitis (n ¼ 8); 7 patients (30%) from this study died because of late infectious complications.78 Two patients died from bacterial infection, one and three patients MSCS AS TREATMENT OF DE NOVO ACUTE GVHD from viral and invasive fungal infections, respectively, and in one patient, no pathogen could be isolated. Although the study is One study, enrolling 32 adult patients with de novo acute GvHD clearly limited by the small number of patients, it is surprising that (grade II n ¼ 21, grade III n ¼ 8 and grade IV n ¼ 3) evaluated the death occurred between 4 months and 2 years after MSCs administration of commercially available third-party MSCs 75 treatment, when acute GvHD had resolved. However, it has also to (Prochymal, Osiris Therapeutics, Columbia, MD, USA). Patients be noted (I) that four out of these seven patients had been treated had received GvHD prophylaxis with tacrolimus, cyclosporine or with other second- or third-line immunosuppressive agents in mycophenolate mofetil, and treatment of acute GvHD consisted of addition to MSCs, including infliximab, daclizumab, photopheresis, conventional systemic corticosteroid therapy in combination with sirolimus and mycophenolate mofetil, and (II) that MSCs do not MSCs given at 24 and 48 h after acute GvHD diagnosis. Although engraft and some authors even suggest repetitive administration randomized in regards to MSCs dose levels (high-dose 8 Â 106 6 of MSCs to reach a long-lasting effect. All these observations make MSCs/kg versus low-dose 2 Â 10 MSCs/kg), the trial did not a conclusion on the impact of MSCs on late infectious include a control group, which is a major limitation. Viral complications extremely difficult. reactivation occurred in eight patients (CMV n ¼ 5, adenovirus An increased risk for invasive fungal infections in patients n ¼ 1 and BK-virus n ¼ 2) and bacteremia in four patients. Three receiving MSCs was suggested in a recent analysis including 15 additional patients died from infections (pseudomonas adult patients who were treated with MSCs between 2002 and pneumonia, enterococcal meningitis and Aspergillus enteritis). 2006 for steroid-refractory grades III–IV acute GvHD and were The authors conclude that the administration of MSCs as therapy compared with patients treated in the same time period but did for de novo acute GvHD is safe and may improve clinical not receive MSCs.79 According to the study results, the incidence symptoms. Unfortunately, in addition to the lack of a control of proven and probable invasive fungal infections was significantly group, it remains unclear whether non-responders to MSCs higher in patients treated with MSCs as compared with patients therapy have a higher risk for infections than patients who without immunotherapy (10 versus 3; P ¼ 0.02). Unfortunately, the benefit from MSCs.75 Notably, a pivotal phase III study evaluating 6 authors did not give important details such as whether antifungal Prochymal given at 2 Â 10 /kg twice weekly failed to meet its prophylaxis was given, the time infection occurred and fungal primary end point of achieving a durable complete response in 76 isolates. The results of this report are in contrast with other acute GvHD response as compared with placebo control. studies, which did not document an increased risk for invasive Although the results of this study were reported in 2010 in fungal infection in MSC-treated stem cell recipients. For example, abstract form, the full study has unfortunately not been published a recent open label, single-arm, prospective multi-center study to date. evaluated Prochymal in 64 pediatric patients (age between 2 months and 17 years) with severe steroid-refractory acute GvHD.80 Although no data were reported on the incidence of infectious MSCS AS TREATMENT OF STEROID-REFRACTORY ACUTE GVHD complications, death due to aspergillosis and mucormycosis Based on the observation that ex vivo-expanded haploidentical occurred in only two patients each (2.7%).80 These data MSCs resulted in an impressive clinical response in a patient with corroborate a small case series, which reported that only one grade IV treatment-resistant acute GvHD of the gut and liver,63 invasive fungal infection occurred in six transplant recipients who there was growing interest in donor-derived and/or unrelated had received adipose-derived MSCs for steroid-refractory acute third-party MSCs as therapeutic strategy in steroid-refractory acute GvHD.81 Although this population of MSCs seems to possess more GvHD, with currently 420 clinical trials being registered. Although potent immunosuppressive properties in vitro in comparison with the majority of the published results report on satisfactory MSCs isolated from other sites,82 the number of patients is by far response rates, the MSC-induced immunosuppression can too small to draw a firm conclusion, and future studies have to potentially increase the incidence and severity of infectious demonstrate whether and to what extent MSCs derived from complications in patients who are already severely immuno- different sources may influence the risk of infectious compromised. Unfortunately, only few of published clinical trials complications. results report in detail on infectious complications in patients The impact of MSCs on the incidence and severity of viral treated with MSCs for steroid-refractory acute GvHD. reactivations was addressed in a cohort of 24 patients treated with A retrospective cohort study was performed to assess the third-party MSCs for steroid-refractory acute GvHD.83 A total of 11 influence of a reduced-intensity conditioning regimen on the risk patients (45%) presented with at least one viral reactivation within of death from pneumonia.77 Importantly, among the 336 patients 100 days following MSCs administration as assessed by who had received reduced-intensity conditioning and the 355 polymerase chain reaction in whole blood. In comparison with patients who had undergone myeloablative conditioning, 53 a historical control of patients treated with second-line patients had been treated with MSCs, the majority for steroid- immunosuppressive drugs, but not receiving MSCs, the refractory acute GvHD (n ¼ 22) or for hemorrhagic cystitis (n ¼ 12). percentage of viral reactivation did not significantly differ

& 2014 Macmillan Publishers Limited Leukemia (2014) 1941 – 1948 MSC and antimicrobial host response A Balan et al 1946 (45% versus 50%). In addition, all patients responded to complications in large and homogenous patient populations who conventional antiviral treatment and no viral disease-related receive MSCs for prophylaxis or treatment of GvHD. These data death was registered. Similarly, Remberger and Ringden79 will ultimately help to further define safety and efficacy of MSCs reported on no increase in the incidence of CMV reactivation transferred to patients after allogeneic HSCT. and disease and of Epstein–Barr virus-induced posttransplant lymphoproliferative disease in 15 adult patients treated with MSCs for grades III–IV acute GvHD. These observations are in line with CONFLICT OF INTEREST analyses suggesting that MSCs do not have a negative impact on The authors declare no conflict of interest. both the number and function of virus-specific T cells.51,83 Although the available data suggest that treatment of steroid- refractory acute GvHD with MSCs potentially increases the risk of ACKNOWLEDGEMENTS mold infections, but does not affect the incidence of viral AB was supported by the European Social Fund POSDRU/107/1.5/S/78702. infections, it remains unclear to what extent the improvement of acute GvHD by the administration of MSCs may outweigh the immunosuppression by these cells. For example, in a small cohort REFERENCES of 12 pediatric patients receiving MSCs for treatment-resistant 84 1 Krampera M. Mesenchymal stromal cell ’licensing’: a multistep process. Leukemia acute GvHD, 5 patients died because of infections. 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Biochem Biophys Res Commun 2007; 361: response to MSCs may influence the risk of infectious complica- 745–750. tions (for example, by the cessation of immunosuppressants), but 16 Djouad F, Charbonnier LM, Bouffi C, Louis-Plence P, Bony C, Apparailly F et al. many questions on the impact of MSCs on the host response Mesenchymal stem cells inhibit the differentiation of dendritic cells against infectious pathogens are unsolved. Although both in vitro through an interleukin-6-dependent mechanism. Stem Cells 2007; 25: data and studies in immunocompetent animals clearly demon- 2025–2032. strate that MSCs exhibit antibacterial activity, there is a paucity of 17 Zhang W, Ge W, Li C, You S, Liao L, Han Q et al. Effects of mesenchymal stem cells both in vitro and in vivo data regarding the antiviral effect of MSCs. on differentiation, maturation, and function of human monocyte-derived In addition, although clinical studies suggest that the administra- dendritic cells. Stem Cells Dev 2004; 13: 263–271. tion of MSCs is associated with a higher risk of invasive fungal 18 Jiang XX, Zhang Y, Liu B, Zhang SX, Wu Y, Yu XD et al. Human mesenchymal stem infections, the available data are difficult to interpret, and there is cells inhibit differentiation and function of monocyte-derived dendritic cells. Blood 2005; 105: 4120–4126. a lack of in vitro data evaluating the antifungal effect of MSCs. 19 Deans RJ, Moseley AB. Mesenchymal stem cells: biology and potential clinical Therefore, future studies have to address (I) the in vitro effect of uses. Exp Hematol 2000; 28: 875–884. MSCs, alone and in combination with other immune cells, against 20 Horwitz EM, Gordon PL, Koo WK, Marx JC, Neel MD, McNall RY et al. Isolated different viral and fungal pathogens; (II) the in vivo effect of MSCs allogeneic bone marrow-derived mesenchymal cells engraft and stimulate in immunocompromised animals, in particular in animals under- growth in children with osteogenesis imperfecta: implications for cell therapy of going allogeneic HSCT; and (III) the detailed analysis of infectious bone. Proc Natl Acad Sci USA 2002; 99: 8932–8937.

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