A novel plasma membrane-phillic graphene oxide nanocarrier for neuropeptide delivery to generate tolerogenic dendritic cells in GVHD Immunotherapy

The prevention and the treatment of graft-versus-host disease (GVHD) remains a barrier to allogeneic hematopoietic stem cell transplantation (allo-HSCT). Tolerogenic dendritic cells (TDCs) have become a hotspot in GVHD prevention despite the poor induction efficiency. Herein, we designed a novel size-dependent platform of graphene oxide (GO) nanosheets for neuropeptide delivery for the purpose of TDC generation. GO with a lateral size༞1 µm (L-GO) showed strong affinities to DC membrane, which effectively promoted the recognition between neuropeptide, urocortin (UCN) and its receptor CRHR2 and in turn benefited TDC generation through PKA C/CREB phosphorylation. Simultaneously, L-GO also elevated the expression of CCR7 and enhanced the migration ability of TDCs by mediating cytoskeleton reorganization. In vivo experiments offered direct evidence that TDCs inducted by UCN@L-GO exhibited efficient migration to lymph nodes, abundant generation of Treg, a significant decrease of proinflammatory cytokines and in turn excellent efficiency in GVHD relief. In the current study, we proposed an innovative GO nanosheets based cytomembrane-targeted platform for neuropeptide delivery and subsequent TDC generation. In the meantime, the promoted mobility of TDC pulsed by GOs ensured the high homing efficiency to secondary lymph nodes, attributing to GVHD treatment in vivo. Thus, such work provided a promising strategy that might be applicable more broadly to delivery systems for receptor-mediated drugs, and could meet the changing demand of nanotechnology-based immunotherapy.

Combined Therapy of Vitamin D3-Tolerogenic Dendritic Cells and Interferon-β in a Preclinical Model of Multiple Sclerosis

Autologous antigen-specific therapies based on tolerogenic dendritic cells (tolDC) offer the possibility to treat autoimmune diseases by restoring homeostasis and targeting specifically autoreactive responses. Here, we explore the hypothesis that systemic inflammation occurring in autoimmune diseases, such as multiple sclerosis (MS), can generate a disease-specific environment able to alter the functionality of tolDC. In this context in fact, a combined therapy of tolDC with an immunomodulatory treatment could potentiate the beneficial effect of this antigen-specific cell therapy. For this purpose, we analyzed the efficacy of a combined therapy based on the use of vitamin D3 (VitD3)-tolDC plus interferon beta (IFN-beta) in MS. VitD3-tolDC were generated from healthy donors and MS patients and co-cultured with allogeneic peripheral blood mononuclear cells, in the presence or absence of IFN-beta. In vitro, VitD3-tolDC treatment reduced the percentage of activated T cells and allogeneic proliferation, whereas VitD3-tolDC+IFN-beta treatment enhanced the suppressive ability of VitD3-tolDC and, additionally, induced a shift towards a Th2 profile. To determine the clinical benefit of the combined therapy, C57BL/6-experimental autoimmune encephalomyelitis (EAE)-induced mice were treated with antigen-specific VitD3-tolDC and/or IFN-beta. Treatment of EAE mice with combined therapy ameliorated the disease course compared to each monotherapy. These results suggest that a combined therapy based on antigen-specific VitD3-tolDC and IFN-beta may represent a promising strategy for MS patients.

Retinoic Acid-Containing Liposomes for the Induction of Antigen-Specific Regulatory T Cells as a Treatment for Autoimmune Diseases

The current treatment of autoimmune and chronic inflammatory diseases entails systemic immune suppression, which is associated with increased susceptibility to infections. To restore immune tolerance and reduce systemic side effects, a targeted approach using tolerogenic dendritic cells (tolDCs) is being explored. TolDCs are characterized by the expression of CD11c, the major histocompatibility complex (MHC)II and low levels of co-stimulatory molecules CD40 and CD86. In this study, tolDCs were generated using a human-proteoglycan-derived peptide (hPG) and all-trans retinoic acid (RA). RA-tolDCs not only display a tolerogenic phenotype but also can induce an antigen-specific regulatory T cell (Treg) response in vitro. However, further analysis showed that RA-tolDCs make up a heterogeneous population of DCs, with only a small proportion being antigen-associated tolDCs. To increase the homogeneity of this population, 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG)-containing liposomes were used to encapsulate the relevant antigen together with RA. These liposomes greatly enhanced the proportion of antigen-associated tolDCs in culture. In addition, in mice, we showed that the liposomal co-delivery of antigen and RA can be a more targeted approach to induce antigen-specific tolerance compared to the injection of RA-tolDCs, and that these liposomes can stimulate the generation of antigen-specific Tregs. This work highlights the importance of the co-delivery of an antigen and immunomodulator to minimize off-target effects and systemic side effects and provides new insights in the use of RA for antigen-specific immunotherapy for autoimmune and chronic inflammatory diseases.

Probiotic-Induced Tolerogenic Dendritic Cells: A Novel Therapy for Inflammatory Bowel Disease?

Inflammatory bowel diseases (IBDs) are immune-mediated, chronic relapsing diseases with a rising prevalence worldwide in both adult and pediatric populations. Treatment options for immune-mediated diseases, including IBDs, are traditional steroids, immunomodulators, and biologics, none of which are capable of inducing long-lasting remission in all patients. Dendritic cells (DCs) play a fundamental role in inducing tolerance and regulating T cells and their tolerogenic functions. Hence, modulation of intestinal mucosal immunity by DCs could provide a novel, additional tool for the treatment of IBD. Recent evidence indicates that probiotic bacteria might impact immunomodulation both in vitro and in vivo by regulating DCs’ maturation and producing tolerogenic DCs (tolDCs) which, in turn, might dampen inflammation. In this review, we will discuss this evidence and the mechanisms of action of probiotics and their metabolites in inducing tolDCs in IBDs and some conditions associated with them.

Emerging Cellular Therapies for Anti-myeloperoxidase Vasculitis and Other Autoimmune Diseases

Anti-myeloperoxidase vasculitis (MPO-AAV) is a life-threatening autoimmune disease which causes severe inflammation of small blood vessels, mainly in the kidney. As for many other autoimmune diseases, current treatments, which consist of general immunosuppressants, are partially effective, toxic and broadly immunosuppressive, causing significant and serious adverse effects in many patients. Therefore, there is an urgent need for more targeted and less harmful therapies. Tolerogenic dendritic cells, regulatory T cells and stem cells have emerged as attractive, new and safer options for the treatment for various autoimmune diseases due to their unique and selective immunosuppressive capacity. In this review, we will discuss how these cellular therapies offer potential to become novel and safer treatments for MPO-AAV.

Butyrate and the Intestinal Epithelium: Modulation of Proliferation and Inflammation in Homeostasis and Disease

The microbial metabolite butyrate serves as a link between the intestinal microbiome and epithelium. The monocarboxylate transporters MCT1 and SMCT1 are the predominant means of butyrate transport from the intestinal lumen to epithelial cytoplasm, where the molecule undergoes rapid β-oxidation to generate cellular fuel. However, not all epithelial cells metabolize butyrate equally. Undifferentiated colonocytes, including neoplastic cells and intestinal stem cells at the epithelial crypt base preferentially utilize glucose over butyrate for cellular fuel. This divergent metabolic conditioning is central to the phenomenon known as “butyrate paradox”, in which butyrate induces contradictory effects on epithelial proliferation in undifferentiated and differentiated colonocytes. There is evidence that accumulation of butyrate in epithelial cells results in histone modification and altered transcriptional activation that halts cell cycle progression. This manifests in the apparent protective effect of butyrate against colonic neoplasia. A corollary to this process is butyrate-induced inhibition of intestinal stem cells. Yet, emerging research has illustrated that the evolution of the crypt, along with butyrate-producing bacteria in the intestine, serve to protect crypt base stem cells from butyrate’s anti-proliferative effects. Butyrate also regulates epithelial inflammation and tolerance to antigens, through production of anti-inflammatory cytokines and induction of tolerogenic dendritic cells. The role of butyrate in the pathogenesis and treatment of intestinal neoplasia, inflammatory bowel disease and malabsorptive states is evolving, and holds promise for the potential translation of butyrate’s cellular function into clinical therapies.

Transfection of Vitamin D3-Induced Tolerogenic Dendritic Cells for the Silencing of Potential Tolerogenic Genes. Identification of CSF1R-CSF1 Signaling as a Glycolytic Regulator

The use of autologous tolerogenic dendritic cells (tolDC) has become a promising strategy to re-establish immune tolerance in autoimmune diseases. Among the different strategies available, the use of vitamin D3 for the generation of tolDC (VitD3-tolDC) has been widely tested because of their immune regulatory properties. To identify molecules and pathways involved in the generation of VitD3-tolDC, we established an easy and fast gene silencing method based on the use of Viromer blue to introduce siRNA into monocytes on day 1 of culture differentiation. The analysis of the effect of CD209 (DC-SIGN) and CD115 (CSF1R) down-modulation on the phenotype and functionality of transfected VitD3-tolDC revealed a partial role of CD115 in their tolerogenicity. Further investigations showed that CSF1R-CSF1 signaling is involved in the induction of cell metabolic reprogramming, triggering glycolysis to produce high amounts of lactate, a novel suppressive mechanism of T cell proliferation, recently found in autologous tolerogenic dendritic cells (ATDCs).