Increased internalization of Staphylococcus aureus and cytokine expression in human Wharton's jelly mesenchymal stem cells.

BACKGROUND: Although a large number of studies have documented the interaction of mesenchymal stem cells (MSCs) with cells of both the innate and adaptive immune systems, not much is known about how bacteria interact with MSCs and how this might influence MSCs behavior. In this study, we investigated the impact of Staphylococcus aureus (S. aureus), on viability and cytokines' production of human Wharton's jelly-MSCs (WJ-MSCs). OBJECTIVE: To investigate if WJ-MSCs: (1) internalize S. aureus; (2) are able to survive and (3) release immunomodulatory mediators after interaction with S. aureus. METHODS: WJ-MSCs were exposed to S. aureus at a multiplicity of infection (MOI) of 10:1 or 30:1 for different designed times. After interaction, intracellular bacteria were quantified as well as MSCs viability. Expression and cytokine-secretion were assessed using quantitative real-time PCR and ELISA. RESULTS: We found that the challenge of WJ-MSCs with S. aureus resulted in increased internalization of S. aureus in a time-dependent manner until six hours post-infection at either MOI of 10:1 and 30:1 and in increased expression of IL-6 mRNA and secretion of TNF-α at six hours and nine hours post-infection (p<0.05). CONCLUSIONS: These results indicate that WJ-MSCs are able to internalize S. aureus and reveal a potential important function of these cells in the immune response.

Safety and efficacy of antigen-specific regulatory T-cell therapy for patients with refractory Crohn's disease.

BACKGROUND & AIMS: New therapeutic strategies are needed for patients with refractory Crohn's disease (CD). We evaluated data from the Crohn's And Treg Cells Study (CATS1) to determine the safety and efficacy of antigen-specific T-regulatory (Treg) cells for treatment of patients with refractory CD. METHODS: We performed a 12-week, open-label, multicenter, single-injection, escalating-dose, phase 1/2a clinical study in 20 patients with refractory CD. Ovalbumin-specific Treg cells (ova-Tregs) were isolated from patients' peripheral blood mononuclear cells (PBMCs), exposed to ovalbumin, and administrated intravenously. Safety and efficacy were assessed using clinical and laboratory parameters. We evaluated proliferation of PBMCs in response to ovalbumin. RESULTS: Injections of ova-Tregs were well tolerated, with 54 adverse events (2 related to the test reagent) and 11 serious adverse events (3 related to the test reagent, all recovered). Overall, a response, based on a reduction in Crohn's Disease Activity Index (CDAI) of 100 points, was observed in 40% of patients at weeks 5 and 8. Six of the 8 patients (75%) who received doses of 10(6) cells had a response at weeks 5 and 8, with a statistically significant reduction in CDAI. In this group, remission (based on CDAI ≤150) was observed in 3 of 8 patients (38%) at week 5 and 2 of 8 patients (25%) at week 8. CONCLUSIONS: Administration of antigen-specific Tregs to patients with refractory CD (CATS1) was well tolerated and had dose-related efficacy. The ovalbumin-specific immune response correlated with clinical response, supporting immune-suppressive mechanisms of ova-Tregs. The consistency of results among different assessment methods supports the efficacy of ova-Tregs; this immune therapy approach warrants further clinical and mechanistic studies in refractory CD. Eudract, Number: 2006-004712-44.

Isolation of functional autologous collagen-II specific IL-10 producing Tr1 cell clones from rheumatoid arthritis blood.

IL-10 producing regulatory type 1 (Tr1) cells represents a subpopulation of CD4+ regulatory cells able to prevent in vitro bystander T-cell proliferation and to inhibit a wide range of inflammatory diseases in mice. Our aim was to evaluate the frequency and function of joint specific Tr1 cells in the peripheral blood of severe Rheumatoid Arthritis (RA) patients. The collagen II protein was chosen to isolate Tr1 cells specific for a joint antigen. We successfully isolated Tr1 clones from 9 out of 11 RA patients. We showed that cells from patients display the same phenotype and surface marker regulation as previously shown for human Tr1 cells, characterized by expression of markers of regulation (FoxP3, CD25) at the activated but not at the resting state. Importantly, cells from patients showed Tr1 cytokine secretion (IL-10 and IFN-γ) and immunosuppressive action on bystander T cell proliferation. Based on these results, we demonstrated that collagen II specific Tr1 cells can be isolated from the blood of severe refractory patients and that these cells are not altered in their phenotype and function.

Clinical grade production of IL-10 producing regulatory Tr1 lymphocytes for cell therapy of chronic inflammatory diseases.

IL-10 producing regulatory type 1 (Tr1) cells represents a subpopulation of CD4(+) regulatory cells able to prevent in vitro bystander T-cell proliferation and to cure ongoing chronic colitis in mice. In order to assess the efficacy and tolerance of Tr1 cell therapy in a Phase I/IIa clinical trial in patients displaying severe Crohn's disease, we set up a reproducible manufacturing process for the GMP production of human ovalbumin specific Tr1 cells. Procedures used for Tr1-cell production include the use of Drosophila derived artificial Antigen Presenting Cells transfected with specific stimulatory molecules. Characterization of the human cell therapy product shows an in vitro suppressive activity on T-cell proliferation dependent on the production of both IL-10 and TGF-beta. Manufactured Tr1 cells display a regulatory phenotype including Foxp3, GITR and CTLA-4 surface expression. In vitro toxicity studies of human Tr1 cell product show a safety profile compatible with the use of these regulatory Tr1 lymphocytes for cell therapy.

Saccharomyces boulardii inhibits inflammatory bowel disease by trapping T cells in mesenteric lymph nodes.

BACKGROUND & AIMS: Saccharomyces boulardii is a nonpathogenic yeast used for treatment of diarrhea. We used a mice model of inflammatory bowel disease (IBD) to analyze the effects of S boulardii on inflammation. METHODS: Lymphocyte-transferred SCID mice, displaying IBD, were fed daily with S boulardii. Weight loss and inflammatory status of the colon were monitored. Nuclear factor-kappaB activity was assessed in the colon. The CD4(+) T-cell production of interferon (IFN) gamma was evaluated by enzyme-linked immunosorbent assay, and a comprehensive reverse-transcription polymerase chain reaction (RT-PCR) analysis for both colon and mesenteric lymph nodes was performed. Finally, we analyzed cell migration mechanisms in vitro and in vivo. RESULTS: S boulardii treatment inhibits IBD. S boulardii induces an accumulation of IFN-gamma-producing T-helper 1 cells within the mesenteric lymph nodes correlated with a diminution of CD4(+) T-cell number and IFN-gamma production by CD4+ T cells within the colon. The influence of S boulardii treatment on cell accumulation in mesenteric lymph nodes was also observed in normal BALB/c mice and involves modifications of lymph node endothelial cell adhesiveness by a yeast secretion product. CONCLUSIONS: S boulardii has a unique action on inflammation by a specific alteration of the migratory behavior of T cells, which accumulate in mesenteric lymph nodes. Therefore, S boulardii treatment limits the infiltration of T-helper 1 cells in the inflammed colon and the amplification of inflammation induced by proinflammatory cytokines production. These results suggest that S boulardii administration may have a beneficial effect in the treatment of IBD.

In vivo follow-up of rat tumor models with 2-deoxy-2-[F-18]fluoro-D-glucose/dual-head coincidence gamma camera imaging.

UNLABELLED: Before studying the impact of 2-deoxy-2-[F-18]fluoro-D-glucose (FDG) imaging with a dual-head coincidence gamma camera (DHC) for the follow-up of animal tumor models, we wanted to optimize this technique. METHODS: Three different animal tumor models (osteosarcoma, melanoma, and breast cancer) were studied after FDG injection. Dynamic and dual time point FDG/DHC imaging were studied from one hour to five hours postinjection. In vitro tumor cell FDG uptake was assessed in eight different tumor cell lines. In one model (osteosarcoma), tumor growth, lung metastasis emergence, and survival were assessed by classical clinical follow-up and compared to FDG imaging in a control group (n = 6) and in a group treated by endostatin liposome complexes (n = 6). RESULTS: Images obtained five hours after injection were more reliable for tumor growth follow-up than standard images (one hour). In vitro tumor cell FDG uptake confirmed in vivo imaging studies. In eight different tumor cell lines the FDG uptake was higher after five hours incubation than after one hour (p < 0.002). With FDG follow-up, we found that FDG uptake was strongly correlated with survival and that lung metastasis larger than 5 mm could be detected. CONCLUSION: Using the optimization proposed above, DHC/FDG functional imaging seems to be a powerful tool to study rat tumor models and to help develop novel cancer therapies.

Leukocyte-derived interleukin 10 is required for protection against atherosclerosis in low-density lipoprotein receptor knockout mice.

BACKGROUND: Atherosclerosis is an immunoinflammatory disease. Here we examined the role of leukocyte-derived interleukin 10 (IL-10) on advanced atherosclerosis development in low-density lipoprotein receptor knockout (LDLr-/-) mice. METHODS AND RESULTS: Bone marrow cells harvested from C57BL/6 IL-10-/- and IL-10+/+ mice were transplanted into irradiated male LDLr-/- mice. Four weeks after transplantation, mice were fed a high-fat cholate-free diet for 14 weeks. Despite no differences in weights, serum total, and HDL-cholesterol levels between the 2 groups, IL-10 deficiency in leukocytes induced a >2-fold increase in lesion development in the thoracic aorta compared with controls. We also found a significant 35% increase in aortic root lesion area of IL-10-/- mice compared with IL-10+/+ mice. Furthermore, IL-10 deficiency led to a marked increase in lymphocyte and macrophage accumulation associated with a significant reduction in collagen accumulation. Finally, transfer of IL-10-/- splenocytes to LDLr-/- mice resulted in a 3-fold increase in lesion size in the aortic sinus compared with mice transplanted with IL-10+/+ splenocytes. CONCLUSIONS: IL-10 expressed by leukocytes prevents exaggerated advanced atherosclerosis development and plays a critical role in modulation of cellular and collagen plaque composition, at least in part, through a modulation of the systemic immune response.

Transplantation of bone marrow-derived mononuclear cells in ischemic apolipoprotein E-knockout mice accelerates atherosclerosis without altering plaque composition.

BACKGROUND: Bone marrow-derived mononuclear cells (BM-MNCs) enhance postischemic neovascularization, and their therapeutic use is currently under clinical investigation. We evaluated the safety of BM-MNC-based therapy in the setting of atherosclerosis. METHODS AND RESULTS: Apolipoprotein E (apoE)-knockout (KO) mice were divided into 4 groups: 20 nonischemic mice receiving intravenous injection of either saline (n=10) or 10(6) BM-MNCs from wild-type animals (n=10) and 20 mice with arterial femoral ligature receiving intravenous injection of either saline (n=10) or 10(6) BM-MNCs from wild-type animals (n=10) at the time of ischemia induction. Animals were monitored for 4 additional weeks. Atherosclerosis was evaluated in the aortic sinus. BM-MNC transplantation improved tissue neovascularization in ischemic hind limbs, as revealed by the 210% increase in angiography score (P<0.0001), the 33% increase in capillary density (P=0.01), and the 65% increase in tissue Doppler perfusion score (P=0.0002). Hindlimb ischemia without BM-MNC transplantation or BM-MNC transplantation without ischemia did not affect atherosclerotic plaque size. However, transplantation of 10(6) BM-MNCs into apoE-KO mice with hindlimb ischemia induced a significant 48% to 72% increase in lesion size compared with the other 3 groups (P=0.0025), despite similar total cholesterol levels. Transplantation of 10(5) BM-MNCs produced similar results, whereas transplantation of 10(6) apoE-KO-derived BM-MNCs had neither proangiogenic nor proatherogenic effects. There was no difference in plaque composition between groups. CONCLUSIONS: BM-MNC therapy is unlikely to affect atherosclerotic plaque stability in the short term. However, it may promote further atherosclerotic plaque progression in an ischemic setting.

A comparative study between T regulatory type 1 and CD4+CD25+ T cells in the control of inflammation.

There is now compelling evidence that CD4(+)CD25(+) T cells play a major role in the maintenance of tolerance. Besides CD4(+)CD25(+) T cells, different populations of regulatory CD4(+) T cells secreting high amounts of IL-10 (T regulatory type 1 (Tr1)) or TGF-beta (Th3) have also been described in in vivo models. In the lymphocyte transfer model of inflammatory bowel disease, we show here that the control of inflammation during the first weeks is not due to a complete inhibition of differentiation of aggressive proinflammatory T cells, but is the result of a balance between proinflammatory and Tr cells. We also show that in the first weeks continuous IL-10 secretion was required to actively control inflammation. Indeed, treatment with anti-IL-10R Abs 3 wk after the start of the experiment completely reversed the protective effect of Tr cells. IL-10 secretion and control of inflammation could be provided by late injection of Tr1 cells that efficiently cure ongoing inflammatory responses in two different models of inflammation. In contrast, inflammation was not controlled when high numbers of CD4(+)CD45RB(low) or CD4(+)CD25(+) T cells were injected as early as 1 wk after the start of the experiment. These results confirm in vitro studies showing that CD4(+)CD45RB(low) do not contain high IL-10-producing cells and suggest that CD4(+)CD45RB(low) Tr cells maintain tolerance in vivo, in part indirectly, through the differentiation of IL-10-secreting Tr1 cells.

Rho-associated protein kinase contributes to early atherosclerotic lesion formation in mice.

Members of the Rho family of small GTPases have been recently implicated in inflammatory signaling. We examined the effect of in vivo inhibition of Rho kinase on atherogenesis in mice. Low-density lipoprotein receptor (LDLR) knockout (KO) mice fed a cholate-free high-fat diet received daily intraperitoneal injection of saline (n=8, control group) or Y-27632 (30 mg/kg, n=9), a specific Rho kinase inhibitor. After 9 weeks, Y-27632 treatment resulted in significant in vivo inhibition of Rho kinase activity (P=0.004). Body weights, arterial blood pressures, and plasma cholesterol levels were comparable in both groups. Atherosclerotic lesion size in the aortic sinus and thoracic aorta of mice treated with Y-27632 was reduced by respectively 35% and 29% in comparison with the saline-treated animals (P=0.006 and P=0.03, respectively). This was associated with a significant reduction in T lymphocyte accumulation (P=0.035) and expression of p65 subunit of NF-kappaB within plaques (P<0.05). In vitro, treatment with Y-27632 inhibited p65 phosphorylation and degradation of IkappaBalpha in mouse peritoneal macrophages and significantly inhibited concanavalin A-induced proliferation of spleen-derived T cells (P<0.001). In conclusion, inhibition of Rho kinase significantly limits early atherosclerotic plaque development in the LDLR KO mice. This study identifies Rho kinase inhibitors as potential candidates for the treatment of atherosclerosis.

Induction of a regulatory T cell type 1 response reduces the development of atherosclerosis in apolipoprotein E-knockout mice.

BACKGROUND: T helper type 1 (Th1) response plays a permissive role in atherosclerosis. We hypothesized that adoptive transfer of a novel subtype of T lymphocytes called regulatory T cells type 1 (Tr1) would inhibit Th1 responses by inducing a bystander immune suppression and therefore limit the development of atherosclerosis. METHODS AND RESULTS: Clones of ovalbumin (OVA)-specific Tr1 cells expanded in vitro were administered intraperitoneally (106 cells per mouse) with their cognate antigen (50 microg of OVA subcutaneously in complete Freund's adjuvant [CFA]) to female apolipoprotein E-knockout mice. A group of mice received only (OVA/CFA) immunization without Tr1 cells. Two other control groups received no immunization and were injected with either Tr1 cells or saline. After 9 weeks of treatment, mice injected with (OVA/CFA)+OVA-specific Tr1 cells showed a significant decrease in Th1 responses, as revealed by a decrease in OVA-specific IgG2a serum levels (P<0.0001), a decrease in the production of interferon-gamma (P<0.001), and an increase in interleukin-10 production (P<0.001) by cultured spleen and lymph T cells compared with controls. In addition, cytokine production by concanavalin A-stimulated spleen cells showed a clear switch to a regulatory immune response in mice treated with (OVA/CFA)+Tr1. This was associated with a significant reduction in atherosclerotic lesion size in both the thoracic aorta and aortic sinus of mice treated with (OVA/CFA)+Tr1 compared with controls (P=0.002 to P<0.0001). Plaques of mice injected with (OVA/CFA)+Tr1 showed significantly lower accumulation of macrophages and T cells than plaques of control mice. CONCLUSIONS: Tr1-type regulatory immune response reduces the development of experimental atherosclerosis.

Specific abrogation of transforming growth factor-beta signaling in T cells alters atherosclerotic lesion size and composition in mice.

A large body of evidence supports a role for proinflammatory mediators in atherosclerotic disease progression and instability. However, only few endogenous mechanisms have been suggested that could alter disease progression. One such mechanism is thought to be mediated by transforming growth factor beta (TGF-beta). Transgenic mice that express a dominant-negative TGF-beta receptor type II under a T-cell-specific promoter were generated. Bone marrow transplantation from transgenic mice into irradiated low density lipoprotein receptor knock-out (LDLr KO) mice, subsequently fed an atherogenic diet, resulted in T-cell-specific blockade of TGF-beta signaling in the recipient mice and increased differentiation of T cells toward both T helper 1 (Th1) and Th2 phenotypes. These mice showed a significant decrease in atherosclerotic lesion size in the aortic sinus compared with mice receiving transplants with the wild-type bone marrow. Atherosclerotic plaques of mice receiving transplants with the transgenic bone marrow showed increased T-cell infiltration and expression of major histocompatability complex (MHC) class II, along with a decrease in smooth muscle cell and collagen content, a plaque phenotype that is potentially vulnerable to rupture. These results identify for the first time an important role for specific and selective T-cell-TGF-beta signaling in atherosclerosis.

Characterization of dendritic cells that induce tolerance and T regulatory 1 cell differentiation in vivo.

Active suppression is mediated by a subpopulation of CD4(+) T cells that prevents autoimmunity. However, the mechanisms involved in their differentiation in vivo are currently under intensive research. Here we show that in vitro culture of bone marrow cells in the presence of IL-10 induces the differentiation of a distinct subset of dendritic cells with a specific expression of CD45RB. These CD11c(low)CD45RB(high) DCs are present in the spleen and lymph nodes of normal mice and are significantly enriched in the spleen of IL-10 Tg mice. These natural or in vitro-derived DCs display plasmacytoid morphology and an immature-like phenotype, and secrete high levels of IL-10 after activation. OVA peptide-pulsed CD11c(low)CD45RB(high) DCs specifically induce tolerance through the differentiation of Tr1 cells in vitro and in vivo. Our findings identify a natural DC subset that induces the differentiation of Tr1 cells and suggest their therapeutic use.

Anti-inflammatory properties of the mu opioid receptor support its use in the treatment of colon inflammation.

The physiologic role of the mu opioid receptor (MOR) in gut nociception, motility, and secretion is well established. To evaluate whether MOR may also be involved in controlling gut inflammation, we first showed that subcutaneous administration of selective peripheral MOR agonists, named DALDA and DAMGO, significantly reduces inflammation in two experimental models of colitis induced by administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) or peripheral expansion of CD4(+) T cells in mice. This therapeutic effect was almost completely abolished by concomitant administration of the opioid antagonist naloxone. Evidence of a genetic role for MOR in the control of gut inflammation was provided by showing that MOR-deficient mice were highly susceptible to colon inflammation, with a 50% mortality rate occurring 3 days after TNBS administration. The mechanistic basis of these observations suggests that the anti-inflammatory effects of MOR in the colon are mediated through the regulation of cytokine production and T cell proliferation, two important immunologic events required for the development of colon inflammation in mice and patients with inflammatory bowel disease (IBD). These data provide evidence that MOR plays a role in the control of gut inflammation and suggest that MOR agonists might be new therapeutic molecules in IBD.