Antimicrobial effects of murine mesenchymal stromal cells directed against Toxoplasma gondii and Neospora caninum: role of immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs).

Mesenchymal stromal cells (MSCs) have a multilineage differentiation potential and provide immunosuppressive and antimicrobial functions. Murine as well as human MSCs restrict the proliferation of T cells. However, species-specific differences in the underlying molecular mechanisms have been described. Here, we analyzed the antiparasitic effector mechanisms active in murine MSCs. Murine MSCs, in contrast to human MSCs, could not restrict the growth of a highly virulent strain of Toxoplasma gondii (BK) after stimulation with IFN-γ. However, the growth of a type II strain of T. gondii (ME49) was strongly inhibited by IFN-γ-activated murine MSCs. Immunity-related GTPases (IRGs) as well as guanylate-binding proteins (GBPs) contributed to this antiparasitic effect. Further analysis showed that IFN-γ-activated mMSCs also inhibit the growth of Neospora caninum, a parasite belonging to the apicomplexan group as well. Detailed studies with murine IFN-γ-activated MSC indicated an involvement in IRGs like Irga6, Irgb6 and Irgd in the inhibition of N. caninum. Additional data showed that, furthermore, GBPs like mGBP1 and mGBP2 could have played a role in the anti-N. caninum effect of murine MSCs. These data underline that MSCs, in addition to their regenerative and immunosuppressive activity, function as antiparasitic effector cells as well. However, IRGs are not present in the human genome, indicating a species-specific difference in anti-T. gondii and anti-N. caninum effect between human and murine MSCs.

Monitoring bacterial contamination of blood components in Germany: effect of contamination reduction measures.

BACKGROUND AND OBJECTIVES: National guidelines for monitoring bacterial contamination of blood components were introduced in Germany in 1997. Between 1998 and 2002, numerous measures were implemented to prevent bacterial contamination. This study investigates their impact on contamination rates. MATERIALS AND METHODS: Culture-based testing for bacterial detection on a random sample of blood components is part of routine quality control in German blood establishments. Using standardized questionnaires, data from the production periods 1998, 2001 and 2005/2006 were collected and analysed. RESULTS: The bacterial contamination rate of RBCs was reduced from 0·157% in 1998 to 0·029% in 2005/2006 (P<0·001). While the contamination rate of apheresis PCs remained nearly unchanged over the years, it dramatically decreased for pooled PCs by 70% to a contamination rate of 0·158% (P=0·001) within the last observation period, similar to that of apheresis PCs. The contamination rate of plasma decreased from 0·100% in 1998 to 0·019% in 2005/2006 (P=0·002). CONCLUSIONS: Precautionary measures significantly reduced bacterial contamination rates of blood components. Long-term monitoring with standardized methods is appropriate to evaluate the cumulative effect of contamination-preventing measures.

Necrotic cell-derived high mobility group box 1 attracts antigen-presenting cells but inhibits hepatocyte growth factor-mediated tropism of mesenchymal stem cells for apoptotic cell death.

Tissue damage due to apoptotic or necrotic cell death typically initiates distinct cellular responses, leading either directly to tissue repair and regeneration or to immunological processes first, to clear the site, for example, of potentially damage-inducing agents. Mesenchymal stem cells (MSC) as well as immature dendritic cells (iDC) and monocytes migrate to injured tissues. MSC have regenerative capacity, whereas monocytes and iDC have a critical role in inflammation and induction of immune responses, including autoimmunity after tissue damage. Here, we investigated the influence of apoptotic and necrotic cell death on recruitment of MSC, monocytes and iDC, and identified hepatocyte growth factor (HGF) and the alarmin high mobility group box 1 (HMGB1) as key factors differentially regulating these migratory responses. MSC, but not monocytes or iDC, were attracted by apoptotic cardiomyocytic and neuronal cells, whereas necrosis induced migration of monocytes and iDC, but not of MSC. Only apoptotic cell death resulted in HGF production and HGF-mediated migration of MSC towards the apoptotic targets. In contrast, HMGB1 was predominantly released by the necrotic cells and mediated recruitment of monocytes and iDC via the receptor of advanced glycation end products. Moreover, necrotic cardiomyocytic and neuronal cells caused an HMGB1/toll-like receptor-4-dependent inhibition of MSC migration towards apoptosis or HGF, while recruitment of monocytes and iDC by necrosis or HMGB1 was not affected by apoptotic cells or HGF. Thus, the type of cell death differentially regulates recruitment of either MSC or monocytes and iDC through HGF and HMGB1, respectively, with a dominant, HMGB1-mediated role of necrosis in determining tropism after tissue injury.

Inhibition of human herpes simplex virus type 2 by interferon gamma and tumor necrosis factor alpha is mediated by indoleamine 2,3-dioxygenase.

Genital herpes simplex virus type 2 (HSV-2) is a significant clinical problem. Infection in pregnancy may result in disseminated infection of the newborn with encephalitis. We analyzed the antiviral effects induced by interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in cervix carcinoma cells (HeLa) and astrocytoma cells (86HG39). We found that replication of HSV-2 in HeLa cells and in 86HG39 cells is inhibited after stimulation of the cells by IFN-gamma and TNF-alpha. The antiviral effect of IFN-gamma is enhanced in the presence of TNF-alpha, while stimulation by TNF-alpha alone did not induce antiviral activity. We found that IFN-gamma induces a strong activation of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) and in addition, that the IFN-gamma-induced IDO activity was enhanced in the presence of TNF-alpha. Furthermore, we found that the induction of IDO activity is responsible for the inhibition of herpes simplex virus replication, since the presence of excess amounts of l-tryptophan abrogates the antiviral effect induced by IFN-gamma and the combination of IFN-gamma and TNF-alpha. We therefore conclude that the antiviral effect against HSV-2 mediated by type II interferon and TNF-alpha are dependent on IDO activation.

Protamine enhances the activity of human recombinant interferon-gamma.

Recombinant interferon-gamma (IFN-gamma) is a potent immune regulatory cytokine and is involved in the defense against several intracellular organisms, such as Chlamydia and Toxoplasma. Furthermore IFN-gamma is able to inhibit the growth of human tumor cell lines. The ability to inhibit the growth of intracellular organisms makes the therapeutic use of recombinant human IFN-gamma in certain patient groups, such as those with chronic granulomatous disease, leprosy, and HIV infection, very attractive. We have shown recently that IFN-gamma-mediated effects can be blocked by heparin and that this inhibitory effect can be abrogated by the addition of protamine. In this report, we show that the antagonistic effect of protamine on heparin-mediated inhibition of IFN-gamma activity is mainly due to the capacity of protamine to enhance IFN-gamma activity. We found that protamine enhances the capacity of IFN-gamma to inhibit the growth of different brain tumor cell lines, to induce indolamine 2, 3-dioxygenase activity, to induce toxoplasmostasis, and to induce MHC class II antigen expression in human glioblastoma cells and in human native fibroblasts. We were able to demonstrate that IFN-gamma binds to protamine, and, therefore, we assume that the effect of protamine on IFN-gamma is due to a direct interaction between the two molecules.

A safe and efficient method for elimination of cell culture mycoplasmas using ciprofloxacin.

The antibacterial activity of ciprofloxacin, a 4-fluoroquinolone antibiotic, in the control of mycoplasma contamination in experimentally infected cell lines has been investigated. Seven mycoplasma species, including M. hyorhinis, M. gallisepticum, M. orale, M. salivarium, M. hominis, M. fermentans, and M. arginini, which had chronically infected the murine plasmocytoma line X63-Ag8 653, were eradicated with 10 micrograms/ml ciprofloxacin. Wild type laboratory infections of two human cell lines, HL-60 and U-937, were eliminated by 12 days of such treatment. Mycoplasma decontamination of cell cultures was monitored by the cultivation method 4 weeks after treatment. No side effects were seen in cell cultures and complex proliferation assays with cells of human and murine origin, using ciprofloxacin in doses up to 2.5 times the usual bactericidal concentration.

A new, simple, bioassay for human IFN-gamma.

IFN-gamma induces the production of N-formyl-kynurenine from L-tryptophan in various cell types by the induction of the enzyme indoleamine 2,3-dioxygenase (IDO). The IFN-gamma induced IDO activity in the glioblastoma cell line 86HG39 and cells of clone 2D9 derived from this cell line was found to be greater than that in Hela cells and U373MG cells. Consequently 2D9 cells were used in all subsequent experiments. The determination of kynurenine in the supernatant of IFN-gamma activated cells was performed photometrically using a microplate reader. It was found that the amount of kynurenine produced was directly proportional to the amount of IFN-gamma used to activate cells. The detection limit for IFN-gamma of this assay was 20 U/ml. The induction of L-tryptophan degradation was specific for IFN-gamma since neither IFN-alpha, IFN-beta, IL-1, IL-2, IL-6, GM-CSF nor TNF alpha induced the production of detectable amounts of kynurenine by 86HG39 and 2D9 cells. Furthermore, a mab directed against IFN-gamma was able to completely block the IFN-gamma induced IDO activation. This bioassay was used to determine the IFN-gamma content of supernatants harvested from toxoplasma antigen specific human T cell lines and clones. This assay gave reproducible results which correlated well with the IFN-gamma content detected in the same samples using a commercially available ELISA kit. Furthermore in the case of T cell supernatant stimulated 2D9 cells a mab directed against IFN-gamma was able to completely block IDO induction. We conclude that the measurement of kynurenine production induced by IFN-gamma can be used to determinate IFN-gamma content. This is a simple bioassay which can be performed with standard laboratory equipment.

Expression, characterization and serological reactivity of a 41 kDa excreted-secreted antigen (ESA) from Toxoplasma gondii.

A Toxoplasma gondii tachyzoite expression library was screened with immune sera from T. gondii infected patients. Among others, one gene product reacted strongly with human sera and was further investigated. The gene called B10 was shown to encode a 41 kDa antigen. The complete genomic nucleotide sequence of the B10 protein has been analysed and was shown to contain one intron with conserved splice junctions. Southern blot analysis indicated that B10 is a single-copy gene. The corresponding 1.5 kb cDNA encodes a 318 amino acid sequence of mainly hydrophilic character with a putative signal sequence of 19 amino acids and no further trans-membrane domain. Immunofluorescence assays and immunoblots with a preparation of excreted-secreted antigens (ESA) suggested that the native protein is secreted into the parasitophorous vacuole and its delimiting membrane, indicating that B10 is a member of the ESA family of T. gondii. Recombinant B10 protein exhibited a strong reactivity with human serum samples both in ELISA and in immunoblots.

Molecular characterization of TgMIC5, a proteolytically processed antigen secreted from the micronemes of Toxoplasma gondii.

During invasion of host cells, Toxoplasma gondii discharges the contents of small, apically located secretory organelles called micronemes. Micronemal proteins are known to be necessary for both parasite motility and invasion of host cells. To further define the contents of Toxoplasma micronemes, we used cell fractionation and secretion-modulating drugs to identify six novel, putative micronemal proteins. In this paper we describe preliminary characterization of one of these novel proteins, TgMIC5. Molecular cloning and DNA sequence analysis of the TgMIC5 cDNA and gene revealed that it encodes a previously identified immunodominant antigen called H4. TgMIC5 also possesses a consensus sequence unique to members of the parvulin family of peptidyl-prolyl cis-trans isomerases (PPIases). TgMIC5 is expressed as a preproprotein, which is proteolytically processed to a proprotein by signal peptidase before being further processed to a mature protein of 22 kDa. Using a combination of protein secretion experiments, immunofluorescence and immunoelectron microscopy, we demonstrated that TgMIC2 is stored in the micronemes of T. gondii tachyzoites before it is secreted into the surrounding medium. Based on its homology with parvulin-like PPIases, TgMIC5 may assist in the folding of other micronemal proteins that function in invasion of host cells by T. gondii tachyzoites.

Lipopolysaccharides and leukotriene B4 induce independently regulated electrophysiological and immunological responses in cultured astrocytes.

Astrocytes play an important role in immunological processes within the central nervous system. They are able to produce cytokines like interleukin 6 (IL-6) and depolarize substantially after stimulation by lipopolysaccharides (LPS) or leukotriene B4 (LTB4). Therefore, we investigated the coupling between these immunological and electrophysiological processes. Amiloride (250 microM), a blocker of various Na+ transport systems, inhibited LPS (5 micrograms/ml)-induced depolarization, whereas the LPS-induced release of IL-6 was unaffected, indicating different intracellular regulatory mechanisms. LTB4 (1.0 microM) induced a depolarization of a similar degree but mediated by a different ionic mechanism and failed to induce a detectable IL-6 release. Dexamethasone (1.0 microM) and cycloheximide (2.0 microM) specifically reduced LTB4-induced depolarization, while LPS-induced depolarization was unaffected, providing further evidence for different regulatory pathways. Neither the depolarization nor the immunological stimuli served as a proliferation signal. These data demonstrate that independent immunological and electrophysiological responses with specific intracellular regulation are evoked after stimulation with LPS or LTB4. With respect to functional disturbance of depolarized glial cells, e.g. in maintaining local ionic homeostasis, neuronal excitability may be affected indirectly and by this way account for the appearance of neurological symptoms during inflammatory CNS diseases.

Inhibition of group B streptococcal growth by IFN gamma-activated human glioblastoma cells.

Group B streptococci are the most important bacteria inducing neonatal septicemia and meningitis. The aim of this study was to assess the role of IFNgamma in the induction of anti-microbial effector mechanisms in human brain tumor cells. Different human glioblastoma/astrocytoma cell lines, stimulated with IFNgamma, restricted the growth of group B streptococci. In addition, we found that TNF alpha is able to enhance the IFNgamma-mediated anti-microbial effect. In contrast to group B streptococci, other bacteria which are also capable of inducing meningitis, like E. coli and all but one of the tested Streptococcus pneumoniae strains, were not influenced by the IFNgamma treated cells. We found that the IFNgamma or the IFNgamma/TNF alpha induced activation of indoleamine 2,3-dioxygenase is responsible for the inhibition of streptococcal growth, since the addition of supplemental L-tryptophan completely blocks the IFNgamma induced bacteriostasis.

Human glioblastoma cell line 86HG39 activates T cells in an antigen specific major histocompatibility complex class II-dependent manner.

The capacity of three different human glioblastoma cell lines to activate human T cells was analysed by measuring major histocompatibility complex (MHC) antigen expression, monokine secretion and lectin, mAb OKT3 and antigen-driven T cell proliferation. All glioblastoma cells tested were able to induce PHA and concanavalin A (ConA)-driven T cell proliferation in a dose-dependent fashion, while all failed to induce T cell activation with mAb OKT3. In addition, the glioblastoma cell line 86HG39 was able to induce tetanus toxoid and toxoplasma lysate antigen-specific T cell proliferation. The responding T cell lines originated from only one out of five different donors. This foreign antigen-specific T cell proliferation induced by 86HG39 cells could be inhibited with mAb L243 directed against HLA-DR molecules. The study of monokine secretion by 86HG39 cells showed a strong interleukin (IL)-6 secretion after lipopolysaccharide (LPS) treatment, whilst no IL-1 secretion was observed. Furthermore, only 86HG39 cells were positive for HLA-DR molecules, whereas interferon (IFN) gamma treatment of 87HG28 and 87HG31 cells was necessary for the induction of class II antigen expression. Thus, cell line 86HG39 shows many features of an antigen presenting cell and the interaction of these cells with MHC compatible human T cells might be a useful model to study cellular immune reactions within the central nervous system.

Functional dichotomy of mouse microglia developed in vitro: differential effects of macrophage and granulocyte/macrophage colony-stimulating factor on cytokine secretion and antitoxoplasmic activity.

After differentiation either with exogenous macrophage (M) or with granulocyte/macrophage (GM) colony-stimulating factor (CSF) microglial cells were isolated from neonatal mouse brain cell cultures and were comparatively tested for secretory immune effector cell functions. Both factors obviously do not promote the development of cells with biased growth requirement; however, the two microglia populations displayed distinct potentials to produce inflammatory cytokines. Upon gradual stimulation by lipopolysaccharide, the cells harvested from M-CSF-driven culture released more interleukin-1 and tumor necrosis factor activity, GM-CSF-grown cells on the contrary proved superior in interleukin-6 secretion. This pattern was paralleled by corresponding different kinetics of cytokine release in both types of microglial cells. When infected with Toxoplasma gondii only GM-CSF-differentiated cells were able to restrict the intracellular multiplication of tachyzoites in the absence of external stimuli. As described for interferon-gamma-treated macrophages, the antiparasitic activity of this microglia population is due to the synthesis of reactive nitrogen intermediates, since it was antagonized by NG-monomethyl-L-arginine, a competitive inhibitor of the arginine-dependent metabolic pathway. Complementary to previous data which attest an intrinsic capability for antigen presentation to GM-CSF-grown microglia, the functional state of the cells elicited by M-CSF and GM-CSF, respectively, may correspond to the resting and an activated form of microglia as distinguished in vivo.

Induction of toxoplasmostasis in a human glioblastoma by interferon gamma.

In the course of human toxoplasmosis central nervous system involvement often occurs. As a model for toxoplasma growth within human brain cells the proliferation of Toxoplasma gondii strain BK within the human glioblastoma cell line 86HG39 was analysed. We found that 86HG39 cells support the growth of toxoplasma similar to human monocyte derived macrophages and in contrast to human monocytes. The growth of Toxoplasma gondii within interferon gamma (IFN gamma) treated 86HG39 cells is reduced due to toxoplasmostasis and not due to toxoplasmocide effects. The mechanism of IFN gamma induced toxoplasmostasis was also investigated. It was found that IFN gamma did not induce O2- production and/or nitrite oxide production, and inhibitors of O2- and NO2- did not influence IFN gamma induced toxoplasmostasis. In contrast, the supplementation of L-tryptophan to the culture medium completely abolished the IFN gamma effect. We therefore conclude that the induction of L-tryptophan degradation in 86HG39 cells by IFN gamma, possibly by activation of the indoleamine-2,3-dioxygenase, is responsible for the IFN gamma induced toxoplasmostasis within the glioblastoma cell line.

Differentiation driven by granulocyte-macrophage colony-stimulating factor endows microglia with interferon-gamma-independent antigen presentation function.

The antigen presentation function of microglial cells was analyzed after differentiation in neonatal mouse brain cell cultures supplemented either with macrophage (M) or granulocyte/macrophage (GM) colony-stimulating factor (CSF). The cells separated from concomitant astrocytes in both culture systems turned out to exhibit cytological characteristics of macrophages and bore MAC-1 and F4/80 markers in a similar way. When comparatively tested for accessory cell function, only microglia developed with GM-CSF were able to efficiently induce antigen-directed proliferation of a series of helper T cell lines representing both the TH1 and TH2 subtype. Antigenic T cell activation by this microglia population was performed without prior stimulation and exceeded that of M-CSF-dependently grown microglial cells, even if those had been pretreated with interferon-gamma (IFN-gamma). In contrast to such difference in function, low cell surface expression of MHC class II or intercellular adhesion molecule-1 determinants proved to coincide in both populations. Correlating with the capacity for antigen presentation, expression of membrane-bound interleukin-1 (IL1)--a costimulatory signal for TH2 cells--was augmented significantly in GM-CSF-grown microglia. In parallel, the interaction only of this microglia population with a selected TH1 cell line was accompanied by maximal release of T cell-stimulating factor, a cytokine recently identified as an IL1-analogous second signal for TH1 cells. Thus, a developmental process is suggested which produces a form of microglia specialized in antigen presentation and thereby acting uncoupled from IFN-gamma.

Postnatal development of functional T cell subsets in the mouse: a frequency analysis of mitogen reactive precursors of proliferating, of cytotoxic and of IL 2 producing T cells.

In order to study the postnatal development of functional T cell subsets in the mouse, a mitogen-driven limiting dilution culture system was used for a precursor frequency analysis of proliferating, of cytolytic and of IL 2-producing T cells, respectively, present in spleen and thymus of mice from neonatal to adult age. In adult mice, the majority (up to 100%) of splenic T cells was capable to respond to Concanavalin A. In contrast, an up to tenfold lower frequency of mitogen-reactive precursors was found within positively selected Thy-1+ spleen cells of neonatal mice. Within this fraction of Con A reactive neonatal T cells, there was an apparent imbalance in the CTLp/PTLp ratio within the first to second week after birth. Accordingly, significant numbers of immunocompetent precursors of HTLp were detected in the spleen shortly after birth, while the vast majority of CTLp developed later on. This differential development of CTLp and PTLp was not seen with thymocytes of the same mice, where from the age of two to three days on up to the adult age the frequency of both CTLp and PTLp remained largely unchanged. Analysis of the Lyt-antigen expression, in addition, revealed phenotypical differences between neonatal and adult Thy-1+ spleen cells, such that Lyt-2 antigen density but not the proportion of Lyt-2 positive T cells, was considerably lower in newborn mice. An age related increase in both Lyt-2 antigen density and CTLp frequency was parallelled by the rapid increase in the total number of splenic T cells during the second and third postnatal week, reaching 60-70% of adult values. At this time, a normalisation of the CTLp/PTLp ratio at approximately 0.4 had occurred.

Studies of persistent infection by Chlamydia trachomatis serovar K in TPA-differentiated U937 cells and the role of IFN-gamma.

Inoculation of phorbol ester-differentiated U937 cells as a model for human macrophages with Chlamydia trachomatis of the urogenital serovar K resulted in a persistent infection, with maximal growth at day 7, until day 10 post-infection. At these times inclusion bodies were present in 0.5-2% of the cells. Typical inclusion bodies containing elementary bodies and reticulate bodies were observed by electron microscopy. Furthermore, single chlamydial particles resembling atypical elementary or intermediate bodies were identified in the cytoplasm in > 80% of the host cells. IFN-gamma exerts antichlamydial activity in epithelial and fibroblastoid cells, but the infection of U937 cells by C. trachomatis was not affected by IFN-gamma. The activity of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) was not detected in untreated or in IFN-gamma-treated or chlamydiae-infected or mock-infected U937 cells. The presence of atypical persisting chlamydiae and the lack of IDO expression in U937 cells indicates that the development of these atypical bacteria is independent from IFN-gamma-mediated tryptophan deprivation and other IFN-gamma-mediated effects. Evaluation of persistently infected cells revealed that the expression of the chlamydial major outer-membrane protein, heat-shock protein (hsp60) and lipopolysaccharide (LPS) antigens was not significantly altered in the course of the culture. An intense staining of the LPS on the surface of the host cells was demonstrated by immunofluorescence. The data show that phorbol ester-differentiated U937 cells restrict chlamydial growth strongly but not completely through a mechanism distinct from IDO-mediated tryptophan deprivation. The mechanisms of persistence of chlamydiae in monocytes, which differ considerably from those described for other cells, require further investigation.

IFN-gamma activated indoleamine 2,3-dioxygenase activity in human cells is an antiparasitic and an antibacterial effector mechanism.

In nearly all human cells IFN-gamma stimulation leads to an activation of indoleamine 2,3-dioxygenase (IDO) activity, which is responsible for anti-toxoplasma and anti-chlamydia effects. We have recently shown that IDO activation is also a defense mechanism against extracellular beta-hemolytic streptococci groups A, B, C and G in human glioblastoma cells, fibroblasts and macrophages. Similar effects were also seen with enterococci and in approximately 65% of staphylococci tested, including multiresistant strains of both species. In addition, we have found that IDO activity is differentially regulated in different cells. For example we have found that TNF-alpha enhances IFN-gamma induced IDO activity and antimicrobial effect in human glioblastoma cells whereas both IFN-gamma mediated effects were blocked by TNF-alpha as well as by IL-1 in a human uroepithelial cell line. We were able to show that the IL-1 and TNF-alpha mediated inhibition of IFN-gamma-induced IDO activity in uroepithelial cells is due to stimulation of inducible nitric oxide synthase. In human astrocytoma cells, IL-1 and TNF-alpha did not inhibit IDO activity and in concordance with this finding these cells did not show a detectable nitric oxide production.

Cytokine mediated regulation of interferon-gamma-induced IDO activation.

Stimulation of human monocyte-derived-macrophages (MDM) with interferon gamma induces the L-tryptophan degrading enzyme indoleamine 2,3-dioxygenase (IDO). It has been well documented that the growth of some intra-cellular parasites such as Chlamydia and Toxoplasma in human fibroblasts and glioblastoma cells is inhibited by IDO mediated L-tryptophan depletion. We have recently shown that IDO induction in cord blood MDM is also responsible for the growth inhibition of extra-cellular group B streptococci and thus for the first time shown an anti-bacterial effect of IDO activation. In view of this immunological function we sought to investigate the regulation, and in particular the downregulation of IDO by the immune system. We describe here the effect of cytokines on IDO activation and in particular the inhibitory function of IL-10, TGF beta and IL-4.

Human but not murine multipotent mesenchymal stromal cells exhibit broad-spectrum antimicrobial effector function mediated by indoleamine 2,3-dioxygenase.

Human multipotent mesenchymal stromal cells (MSCs) exhibit multilineage differentiation potential, support hematopoiesis, and inhibit proliferation and effector function of various immune cells. On the basis of these properties, MSC are currently under clinical investigation in a range of therapeutic applications including tissue repair and immune-mediated disorders such as graft-versus-host-disease refractory to pharmacological immunosuppression. Although initial clinical results appear promising, there are significant concerns that application of MSC might inadvertently suppress antimicrobial immunity with an increased risk of infection. We demonstrate here that on stimulation with inflammatory cytokines human MSC exhibit broad-spectrum antimicrobial effector function directed against a range of clinically relevant bacteria, protozoal parasites and viruses. Moreover, we identify the tryptophan catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) as the underlying molecular mechanism. We furthermore delineate significant differences between human and murine MSC in that murine MSC fail to express IDO and inhibit bacterial growth. Conversely, only murine but not human MSC express inducible nitric oxide synthase on cytokine stimulation thus challenging the validity of murine in vivo models for the preclinical evaluation of human MSC. Collectively, our data identify human MSC as a cellular immunosuppressant that concurrently exhibits potent antimicrobial effector function thus encouraging their further evaluation in clinical trials.