Crosstalk between Body Microbiota and the Regulation of Immunity.

The microbiome corresponds to the genetic component of microorganisms (archaea, bacteria, phages, viruses, fungi, and protozoa) that coexist with an individual. During the last two decades, research on this topic has become massive demonstrating that in both homeostasis and disease, the microbiome plays an important role, and in some cases, a decisive one. To date, microbiota have been identified at different body locations, such as the eyes, lung, gastrointestinal and genitourinary tracts, and skin, and technological advances have permitted the taxonomic characterization of resident species and their metabolites, in addition to the cellular and molecular components of the host that maintain a crosstalk with local microorganisms. Here, we summarize recent studies regarding microbiota residing in different zones of the body and their relationship with the immune system. We emphasize the immune components underlying pathological conditions and how they interact with local (and distant) microbiota.

Neuropilin-1 is present on Foxp3+ T regulatory cell-derived small extracellular vesicles and mediates immunity against skin transplantation.

Among the mechanisms of suppression that T regulatory (Treg) cells exert to control the immune responses, the secretion of small extracellular vesicles (sEV) has been recently proposed as a novel contact-independent immunomodulatory mechanism. Previous studies have demonstrated that Treg cells produce sEV, including exosomes, able to modulate the effector function of CD4+ T cells, and antigen presenting cells (APCs) such as dendritic cells (DCs) through the transfer of microRNA, cytokines, the production of adenosine, among others. Previously, we have demonstrated that Neuropilin-1 (Nrp1) is required for Tregs-mediated immunosuppression mainly by impacting on the phenotype and function of effector CD4+ T cells. Here, we show that Foxp3+ Treg cells secrete sEV, which bear Nrp1 in their membrane. These sEV modulate effector CD4+ T cell phenotype and proliferation in vitro in a Nrp1-dependent manner. Proteomic analysis indicated that sEV obtained from wild type (wt) and Nrp1KO Treg cells differed in proteins related to immune tolerance, finding less representation of CD73 and Granzyme B in sEV obtained from Nrp1KO Treg cells. Likewise, we show that Nrp1 is required in Treg cell-derived sEV for inducing skin transplantation tolerance, since a reduction in graft survival and an increase on M1/M2 ratio were found in animals treated with Nrp1KO Treg cell-derived sEV. Altogether, this study describes for the first time that Treg cells secrete sEV containing Nrp1 and that this protein, among others, is necessary to promote transplantation tolerance in vivo via sEV local administration.

Time-dependent LPS exposure commands MSC immunoplasticity through TLR4 activation leading to opposite therapeutic outcome in EAE.

BACKGROUND: Mesenchymal stem cells (MSCs) have been recognized for their regenerative and anti-inflammatory capacity which makes them very attractive to cell therapy, especially those ones to treat inflammatory and autoimmune disease. Two different immune-phenotypes have been described for MSCs depending on which Toll-like receptor (TLR) is activated. MSC1 is endowed with a pro-inflammatory phenotype following TLR4 activation with LPS. On the other hand, anti-inflammatory MSC2 is induced by the activation of TLR3 with Poly(I:C). High immunoplasticity of MSCs is a matter of concern in cell-based therapies. In this study, we investigated whether a single stimulus can induce both types of MSCs through a differential activation of TLR4 with LPS. METHODS: MSCs were activated with LPS following a short exposure of 1-h (MSCs-LPS1h) or long-time exposure for 48 h (MSCs-LPS48h), and then, we evaluated the biological response in vitro, the immunosuppressive capacity of MSCs in vitro, and the therapeutic potential of MSCs in an experimental autoimmune encephalomyelitis (EAE) mouse model. RESULTS: Our results showed that 1-h LPS exposure induced a MSC1 phenotype. Indeed, MSCs-LPS1h expressed low levels of NO/iNOS and decreased immunosuppressive capacity in vitro without therapeutic effect in the EAE model. In contrast, MSCs-LPS48h achieved a MSC2-like phenotype with significant increase in the immunosuppressive capacity on T cell proliferation in vitro, together with an improved in the therapeutic effect and higher Treg, compared to unstimulated MSCs. Furthermore, we determine through the MSCs-TLR4KO that the expression of TLR4 receptor is essential for MSCs' suppressive activity since TLR4 deletion was associated with a diminished suppressive effect in vitro and a loss of therapeutic effect in vivo. CONCLUSIONS: We demonstrate that MSCs display a high immunoplasticity commanded by a single stimulus, where LPS exposure time regulated the MSC suppressive effect leading into either an enhanced or an impairment therapeutic activity. Our results underscore the importance of phenotype conversion probably related to the TLR4 expression and activation, in the design of future clinical protocols to treat patients with inflammatory and autoimmune diseases.

Mitochondrial transfer from MSCs to T cells induces Treg differentiation and restricts inflammatory response.

Mesenchymal stem cells (MSCs) have fueled ample translation for the treatment of immune-mediated diseases. They exert immunoregulatory and tissue-restoring effects. MSC-mediated transfer of mitochondria (MitoT) has been demonstrated to rescue target organs from tissue damage, yet the mechanism remains to be fully resolved. Therefore, we explored the effect of MitoT on lymphoid cells. Here, we describe dose-dependent MitoT from mitochondria-labeled MSCs mainly to CD4+ T cells, rather than CD8+ T cells or CD19+ B cells. Artificial transfer of isolated MSC-derived mitochondria increases the expression of mRNA transcripts involved in T-cell activation and T regulatory cell differentiation including FOXP3, IL2RA, CTLA4, and TGFß1, leading to an increase in a highly suppressive CD25+ FoxP3+ population. In a GVHD mouse model, transplantation of MitoT-induced human T cells leads to significant improvement in survival and reduction in tissue damage and organ T CD4+ , CD8+ , and IFN-γ+ expressing cell infiltration. These findings point to a unique CD4+ T-cell reprogramming mechanism with pre-clinical proof-of-concept data that pave the way for the exploration of organelle-based therapies in immune diseases.

IL17/IL17RA as a Novel Signaling Axis Driving Mesenchymal Stem Cell Therapeutic Function in Experimental Autoimmune Encephalomyelitis.

The therapeutic effect of mesenchymal stem cells (MSCs) in multiple sclerosis (MS) and the experimental autoimmune encephalomyelitis (EAE) model has been well described. This effect is, in part, mediated through the inhibition of IL17-producing cells and the generation of regulatory T cells. While proinflammatory cytokines such as IFNγ, TNFα, and IL1ß have been shown to enhance MSCs immunosuppressive function, the role of IL17 remains poorly elucidated. The aim of this study was, therefore, to investigate the role of the IL17/IL17R pathway on MSCs immunoregulatory effects focusing on Th17 cell generation in vitro and on Th17-mediated EAE pathogenesis in vivo. In vitro, we showed that the immunosuppressive effect of MSCs on Th17 cell proliferation and differentiation is partially dependent on IL17RA expression. This was associated with a reduced expression level of MSCs immunosuppressive mediators such as VCAM1, ICAM1, and PD-L1 in IL17RA-/- MSCs as compared to wild-type (WT) MSCs. In the EAE model, we demonstrated that while WT MSCs significantly reduced the clinical scores of the disease, IL17RA-/- MSCs injected mice exhibited a clinical worsening of the disease. The disability of IL17RA-/- MSCs to reduce the progression of the disease paralleled the inability of these cells to reduce the frequency of Th17 cells in the draining lymph node of the mice as compared to WT MSCs. Moreover, we showed that the therapeutic effect of MSCs was correlated with the generation of classical Treg bearing the CD4+CD25+Foxp3+ signature in an IL17RA-dependent manner. Our findings reveal a novel role of IL17RA on MSCs immunosuppressive and therapeutic potential in EAE and suggest that the modulation of IL17RA in MSCs could represent a novel method to enhance their therapeutic effect in MS.

Differential TLR activation of murine mesenchymal stem cells generates distinct immunomodulatory effects in EAE.

BACKGROUND: Recently, it has been observed that mesenchymal stem cells (MSCs) can modulate their immunoregulatory properties depending on the specific in-vitro activation of different Toll-like receptors (TLR), such as TLR3 and TLR4. In the present study, we evaluated the effect of polyinosinic:polycytidylic acid (poly(I:C)) and lipopolysaccharide (LPS) pretreatment on the immunological capacity of MSCs in vitro and in vivo. METHODS: C57BL/6 bone marrow-derived MSCs were pretreated with poly(I:C) and LPS for 1 hour and their immunomodulatory capacity was evaluated. T-cell proliferation and their effect on Th1, Th17, and Treg differentiation/activation were measured. Next, we evaluated the therapeutic effect of MSCs in an experimental autoimmune encephalomyelitis (EAE) model, which was induced for 27 days with MOG35-55 peptide following the standard protocol. Mice were subjected to a single intraperitoneal injection (2 × 106 MSCs/100 µl) on day 4. Clinical score and body weight were monitored daily by blinded analysis. At day 27, mice were euthanized and draining lymph nodes were extracted for Th1, Th17, and Treg detection by flow cytometry. RESULTS: Pretreatment of MSCs with poly(I:C) significantly reduced the proliferation of CD3+ T cells as well as nitric oxide secretion, an important immunosuppressive factor. Furthermore, MSCs treated with poly(I:C) reduced the differentiation/activation of proinflammatory lymphocytes, Th1 and Th17. In contrast, MSCs pretreated with LPS increased CD3+ T-cell proliferation, and induced Th1 and Th17 cells, as well as the levels of proinflammatory cytokine IL-6. Finally, we observed that intraperitoneal administration of MSCs pretreated with poly(I:C) significantly reduced the severity of EAE as well as the percentages of Th1 and Th17 proinflammatory subsets, while the pretreatment of MSCs with LPS completely reversed the therapeutic immunosuppressive effect of MSCs. CONCLUSIONS: Taken together, these data show that pretreatment of MSCs with poly(I:C) improved their immunosuppressive abilities. This may provide an opportunity to better define strategies for cell-based therapies to autoimmune diseases.

Overexpression of Glutamate Decarboxylase in Mesenchymal Stem Cells Enhances Their Immunosuppressive Properties and Increases GABA and Nitric Oxide Levels.

The neurotransmitter GABA has been recently identified as a potent immunosuppressive agent that targets both innate and adaptive immune systems and prevents disease progression of several autoimmunity models. Mesenchymal stem cells (MSCs) are self-renewing progenitor cells that differentiate into various cell types under specific conditions, including neurons. In addition, MSC possess strong immunosuppressive capabilities. Upon cytokine priming, undifferentiated MSC suppress T-cell proliferation via cell-to-cell contact mechanisms and the secretion of soluble factors like nitric oxide, prostaglandin E2 and IDO. Although MSC and MSC-derived neuron-like cells express some GABAergic markers in vitro, the role for GABAergic signaling in MSC-mediated immunosuppression remains completely unexplored. Here, we demonstrate that pro-inflammatory cytokines selectively regulate GAD-67 expression in murine bone marrow-MSC. However, expression of GAD-65 is required for maximal GABA release by MSC. Gain of function experiments using GAD-67 and GAD-65 co-expression demonstrates that GAD increases immunosuppressive function in the absence of pro-inflammatory licensing. Moreover, GAD expression in MSC evokes an increase in both GABA and NO levels in the supernatants of co-cultured MSC with activated splenocytes. Notably, the increase in NO levels by GAD expression was not observed in cultures of isolated MSC expressing GAD, suggesting crosstalk between these two pathways in the setting of immunosuppression. These results indicate that GAD expression increases MSC-mediated immunosuppression via secretion of immunosuppressive agents. Our findings may help reconsider GABAergic activation in MSC for immunological disorders.

Toll-like receptor 3 pre-conditioning increases the therapeutic efficacy of umbilical cord mesenchymal stromal cells in a dextran sulfate sodium-induced colitis model.

BACKGROUND AIMS: Immunomodulatory properties of human umbilical cord-derived mesenchymal stromal cells (UCMSCs) can be differentially modulated by toll-like receptors (TLR) agonists. Here, the therapeutic efficacy of short TLR3 and TLR4 pre-conditioning of UCMSCs was evaluated in a dextran sulfate sodium (DSS)-induced colitis in mice. The novelty of this study is that although modulation of human MSCs activity by TLRs is not a new concept, this is the first time that short TLR pre-conditioning has been carried out in a murine inflammatory model of acute colitis. METHODS: C57BL/6 mice were exposed to 2.5% dextran sulfate sodium (DSS) in drinking water ad libitum for 7 days. At days 1 and 3, mice were injected intraperitoneally with 1 × 10(6) UCMSCs untreated or TLR3 and TLR4 pre-conditioned UCMSCs. UCMSCs were pre-conditioned with poly(I:C) for TLR3 and LPS for TLR4 for 1 h at 37°C and 5% CO2. We evaluated clinical signs of disease and body weights daily. At the end of the experiment, colon length and histological changes were assessed. RESULTS: poly(I:C) pre-conditioned UCMSCs significantly ameliorated the clinical and histopathological severity of DSS-induced colitis compared with UCMSCs or LPS pre-conditioned UCMSCs. In contrast, infusion of LPS pre-conditioned UCMSCs significantly increased clinical signs of disease, colon shortening and histological disease index in DSS-induced colitis. CONCLUSIONS: These results show that short in vitro TLR3 pre-conditioning with poly(I:C) enhances the therapeutic efficacy of UCMSCs, which is a major breakthrough for developing improved treatments to patients with inflammatory bowel disease.

Intravenous administration of bone marrow-derived mesenchymal stem cells induces a switch from classical to atypical symptoms in experimental autoimmune encephalomyelitis.

Potent immunosuppressive and regenerative properties of mesenchymal stem cells (MSCs) position them as a novel therapy for autoimmune diseases. This research examines the therapeutic effect of MSCs administration at different disease stages in experimental autoimmune encephalomyelitis (EAE). Classical and atypical scores of EAE, associated with Th1 and Th17 response, respectively, and also Treg lymphocytes, were evaluated. MSCs administration at the onset (EAE+MSConset) induced an important amelioration of the clinical signs and less lasting effect at the peak of EAE (EAE+MSCpeak). No effect was observed when MSCs were applied after EAE stabilization (EAE+MSClate). Surprisingly, EAE atypical signs were detected in EAE+MSCpeak and EAE+MSClate mice. However, no correlation was found in Th17/Th1 ratio. Interestingly, regardless of time administration, MSCs significantly reduced IL-6 and also T-bet, RORγT, and Foxp3 mRNA levels in brain samples of EAE mice. The downregulation of IL-6 could restore the well-functioning of the blood-brain barrier of EAE mice, correlated with a decreased number of brain infiltrating leukocytes. These results suggest that the inflammatory status is important to be considered for administering MSCs in autoimmune pathologies, leading to a further research to clarify the effect of MSCs for multiple sclerosis.

Mesenchymal stem cells generate a CD4+CD25+Foxp3+ regulatory T cell population during the differentiation process of Th1 and Th17 cells.

INTRODUCTION: Mesenchymal stem cells (MSCs) are adult, multipotent, stem cells with immunomodulatory properties. The mechanisms involved in the capacity of MSCs to inhibit the proliferation of proinflammatory T lymphocytes, which appear responsible for causing autoimmune disease, have yet to be fully elucidated. One of the underlying mechanisms studied recently is the ability of MSCs to generate T regulatory (Treg) cells in vitro and in vivo from activated peripheral blood mononuclear cells (PBMC), T-CD4+ and also T-CD8(+) cells. In the present work we investigated the capacity of MSCs to generate Treg cells using T-CD4(+) cells induced to differentiate toward the proinflammatory Th1 and Th17 lineages. METHODS: MSCs were obtained from mouse bone marrow and characterized according to their surface antigen expression and their multilineage differentiation potential. CD4(+) T cells isolated from mouse spleens were induced to differentiate into Th1 or Th17 cells and co-cultured with MSCs added at day 0, 2 or 4 of the differentiation processes. After six days, CD25, Foxp3, IL-17 and IFN-γ expression was assessed by flow cytometry and helios and neuropilin 1 mRNA levels were assessed by RT-qPCR. For the functional assays, the 'conditioned' subpopulation generated in the presence of MSCs was cultured with concanavalin A-activated CD4(+) T cells labeled with carboxyfluorescein succinimidyl ester. Finally, we used the encephalomyelitis autoimmune diseases (EAE) mouse model, in which mice were injected with MSCs at day 18 and 30 after immunization. At day 50, the mice were euthanized and draining lymph nodes were extracted for Th1, Th17 and Treg detection by flow cytometry. RESULTS: MSCs were able to suppress the proliferation, activation and differentiation of CD4(+) T cells induced to differentiate into Th1 and Th17 cells. This substantial suppressive effect was associated with an increase of the percentage of functional induced CD4(+)CD25(+)Foxp3(+) regulatory T cells and IL-10 secretion. However, using mature Th1 or Th17 cells our results demonstrated that while MSCs suppress the proliferation and phenotype of mature Th1 and Th17 cells they did not generate Treg cells. Finally, we showed that the beneficial effect observed following MSC injection in an EAE mouse model was associated with the suppression of Th17 cells and an increase in the percentage of CD4(+)CD25(+)Foxp3(+) T lymphocytes when administrated at early stages of the disease. CONCLUSIONS: This study demonstrated that MSCs contribute to the generation of an immunosuppressive environment via the inhibition of proinflammatory T cells and the induction of T cells with a regulatory phenotype. Together, these results might have important clinical implications for inflammatory and autoimmune diseases.

A synthetic peptide homologous to IL-10 functional domain induces monocyte differentiation to TGF-ß+ tolerogenic dendritic cells.

We have previously demonstrated that IT9302, a nonameric peptide homologous to the C-terminal domain of human IL-10, mimics several effects of the cytokine including down-regulation of the antigen presentation machinery and increased sensitivity of tumor cells to NK-mediated lysis. In the present report, we have explored a potential therapeutic utility for IT9302 related to the ex vivo production of tolerogenic dendritic cells (DCs). Our results indicate that IT9302 impedes human monocyte response to differentiation factors and reduces antigen presentation and co-stimulatory capacity by DCs. Additionally, peptide-treated DCs show impaired capacity to stimulate T-cell proliferation and IFN-γ production. IT9302 exerts its effect through mechanisms, in part, distinct from IL-10, involving STAT3 inactivation and NF-κB intracellular pathway. IT9302-treated DCs display increased expression of membrane-associated TGF-ß, linked to a more effective induction of foxp3+ regulatory T cells. These results illustrate for the first time that a short synthetic peptide can promote monocytes differentiation to tolerogenic DCs with therapeutic potential for the treatment of autoimmune and transplantation-related immunopathologic disease.

A synthetic peptide homologous to functional domain of human IL-10 down-regulates expression of MHC class I and Transporter associated with Antigen Processing 1/2 in human melanoma cells.

Tumor cells treated with IL-10 were shown to have decreased, but peptide-inducible expression of MHC class I, decreased sensitivity to MHC class I-restricted CTL, and increased NK sensitivity. These findings could be explained, at least partially, by a down-regulation of TAP1/TAP2 expression. In this study, IT9302, a nanomeric peptide (AYMTMKIRN), homologous to the C-terminal of the human IL-10 sequence, was demonstrated to mimic these previously described IL-10 effects on MHC class I-related molecules and functions. We observed a dose-dependent down-regulation of MHC class I at the cell surface of melanoma cells after 24-h treatment with IT9302. The IL-10 homologue peptide also caused a dose-dependent inhibition of the IFN-gamma-mediated surface induction of MHC class I in a melanoma cell line. We demonstrated, using Western blot and flow cytometry, that IT9302 inhibits the expression of TAP1 and TAP2 proteins, but not MHC class I H chain or low molecular protein molecules. Finally, peptide-treated melanoma cells were shown to be more sensitive to lysis by NK cells in a dose-dependent way. Taken together, these results demonstrate that a small synthetic peptide derived from IL-10 can mimic the Ag presentation-related effects mediated by this cytokine in human melanomas and increase tumor sensitivity to NK cells, which can be relevant in the designing of future strategies for cancer immune therapy.