Extracellular vesicles and immune response during pregnancy: A balancing act.

The mechanisms underlying maternal tolerance of the semi- or fully-allogeneic fetus are intensely investigated. Across gestation, feto-placental antigens interact with the maternal immune system locally within the trophoblast-decidual interface and distantly through shed cells and soluble molecules that interact with maternal secondary lymphoid tissues. The discovery of extracellular vesicles (EVs) as local or systemic carriers of antigens and immune-regulatory molecules has added a new dimension to our understanding of immune modulation prior to implantation, during trophoblast invasion, and throughout the course of pregnancy. New data on immune-regulatory molecules, located on EVs or within their cargo, suggest a role for EVs in negotiating immune tolerance during gestation. Lessons from the field of transplant immunology also shed light on possible interactions between feto-placentally derived EVs and maternal lymphoid tissues. These insights illuminate a potential role for EVs in major obstetrical disorders. This review provides updated information on intensely studied, pregnancy-related EVs, their cargo molecules, and patterns of fetal-placental-maternal trafficking, highlighting potential immune pathways that might underlie immune suppression or activation in gestational health and disease. Our summary also underscores the likely need to broaden the definition of the maternal-fetal interface to systemic maternal immune tissues that might interact with circulating EVs.

Skin codelivery of contact sensitizers and neurokinin-1 receptor antagonists integrated in microneedle arrays suppresses allergic contact dermatitis.

BACKGROUND: Allergic contact dermatitis (CD) is a chronic inflammatory skin disease caused by type 1 biased adaptive immunity for which there is an unmet need for antigen (Ag)-specific immunotherapies. Exposure to skin sensitizers stimulates secretion of the proinflammatory neuropeptides substance P and hemokinin 1, which signal via the neurokinin-1 receptor (NK1R) to promote the innate and adaptive immune responses of CD. Accordingly, mice lacking the NK1R develop impaired CD. Nonetheless, the role and therapeutic opportunities of targeting the NK1R in CD remain to be elucidated. OBJECTIVE: We sought to develop an Ag-specific immunosuppressive approach to treat CD by skin codelivery of hapten and NK1R antagonists integrated in dissolvable microneedle arrays (MNA). METHODS: In vivo mouse models of contact hypersensitivity and ex vivo models of human skin were used to delineate the effects and mechanisms of NK1R signaling and the immunosuppressive effects of the contact sensitizer NK1R antagonist MNA in CD. RESULTS: We demonstrated in mice that CD requires NK1R signaling by substance P and hemokinin 1. Specific deletion of the NK1R in keratinocytes and dendritic cells, but not in mast cells, prevented CD. Skin codelivery of hapten or Ag MNA inhibited neuropeptide-mediated skin inflammation in mouse and human skin, promoted deletion of Ag-specific effector T cells, and increased regulatory T cells, which prevented CD onset and relapses locally and systemically in an Ag-specific manner. CONCLUSIONS: Immunoregulation by engineering localized skin neuroimmune networks can be used to treat cutaneous diseases that like CD are caused by type 1 immunity.

Platelet Extracellular Vesicles Drive Inflammasome-IL-1ß-Dependent Lung Injury in Sickle Cell Disease.

Rationale: Intraerythrocytic polymerization of Hb S promotes hemolysis and vasoocclusive events in the microvasculature of patients with sickle cell disease (SCD). Although platelet-neutrophil aggregate-dependent vasoocclusion is known to occur in the lung and contribute to acute chest syndrome, the etiological mechanisms that trigger acute chest syndrome are largely unknown.Objectives: To identify the innate immune mechanism that promotes platelet-neutrophil aggregate-dependent lung vasoocclusion and injury in SCD.Methods:In vivo imaging of the lung in transgenic humanized SCD mice and in vitro imaging of SCD patient blood flowing through a microfluidic system was performed. SCD mice were systemically challenged with nanogram quantities of LPS to trigger lung vasoocclusion.Measurements and Main Results: Platelet-inflammasome activation led to generation of IL-1ß and caspase-1-carrying platelet extracellular vesicles (EVs) that bind to neutrophils and promote platelet-neutrophil aggregation in lung arterioles of SCD mice in vivo and SCD human blood in microfluidics in vitro. The inflammasome activation, platelet EV generation, and platelet-neutrophil aggregation were enhanced by the presence of LPS at a nanogram dose in SCD but not control human blood. Inhibition of the inflammasome effector caspase-1 or IL-1ß pathway attenuated platelet EV generation, prevented platelet-neutrophil aggregation, and restored microvascular blood flow in lung arterioles of SCD mice in vivo and SCD human blood in microfluidics in vitro.Conclusions: These results are the first to identify that platelet-inflammasome-dependent shedding of IL-1ß and caspase-1-carrying platelet EVs promote lung vasoocclusion in SCD. The current findings also highlight the therapeutic potential of targeting the platelet-inflammasome-dependent innate immune pathway to prevent acute chest syndrome.

Oral administration of lipoteichoic acid from Lactobacillus rhamnosus GG overcomes UVB-induced immunosuppression and impairs skin tumor growth in mice.

There is increasing evidence of the relevant connection and regulation between the gut and skin immune axis. In fact, oral administration of lipoteichoic acid (LTA) from Lactobacillus rhamnosus GG (LGG) prevents the development of UV-induced skin tumors in chronically exposed mice. Here we aim to evaluate whether this LTA is able to revert UV-induced immunosuppression as a mechanism involved in its anti-tumor effect and whether it has an immunotherapeutic effect against cutaneous squamous cell carcinoma. Using a mouse model of contact hypersensitivity, we demonstrate that LTA overcomes UV-induced skin immunosuppression. This effect was in part achieved by modulating the phenotype of lymph node resident dendritic cells (DC) and the homing of skin migratory DC. Importantly, oral LTA reduced significantly the growth of established skin tumors once UV radiation was discontinued, demonstrating that it has a therapeutic, besides the already demonstrated preventive antitumor effect. The data presented here strongly indicates that oral administration of LTA represents a promising immunotherapeutic approach for different conditions in which the skin immune system is compromised.

Interferon regulatory factor 1-Rab27a regulated extracellular vesicles promote liver ischemia/reperfusion injury.

The role and regulators of extracellular vesicle (EV) secretion in hepatic ischemia/reperfusion (IR) injury have not been defined. Rab27a is a guanosine triphosphatase known to control EV release. Interferon regulatory factor 1 (IRF-1) is a transcription factor that plays an important role in liver IR and regulates certain guanosine triphosphatases. However, the relationships among IRF-1, Rab27a, and EV secretion are largely unknown. Here, we show induction of IRF-1 and Rab27a both in vitro in hypoxic hepatocytes and in vivo in warm IR and orthotopic liver transplantation livers. Interferon γ stimulation, IRF-1 transduction, or IR promoted Rab27a expression and EV secretion. Meanwhile, silencing of IRF-1 decreased Rab27a expression and EV secretion. Rab27a silencing decreased EV secretion and liver IR injury. Ten putative IRF-1 binding motifs in the 1,692-bp Rab27a promoter region were identified. Chromatin immunoprecipitation and electrophoretic mobility shift assay verified five functional IRF-1 binding motifs, which were confirmed by a Rab27a promoter luciferase assay. IR-induced EVs contained higher oxidized phospholipids (OxPL). OxPLs on the EV surface activated neutrophils through the toll-like receptor 4 pathway. OxPL-neutralizing E06 antibody blocked the effect of EVs and decreased liver IR injury. CONCLUSION: These findings provide a novel mechanism by which IRF-1 regulates Rab27a transcription and EV secretion, leading to OxPL activation of neutrophils and subsequent hepatic IR injury. (Hepatology 2018;67:1056-1070).

Graft-infiltrating PD-L1hi cross-dressed dendritic cells regulate antidonor T cell responses in mouse liver transplant tolerance.

Although a key role of cross-dressing has been established in immunity to viral infection and more recently in the instigation of transplant rejection, its role in tolerance is unclear. We investigated the role of intragraft dendritic cells (DCs) and cross-dressing in mouse major histocompatibility complex (MHC)-mismatched liver transplant tolerance that occurs without therapeutic immunosuppression. Although donor interstitial DCs diminished rapidly after transplantation, they were replaced in the liver by host DCs that peaked on postoperative day (POD) 7 and persisted indefinitely. Approximately 60% of these recipient DCs displayed donor MHC class I, indicating cross-dressing. By contrast, only a very minor fraction (0%-2%) of cross-dressed DCs (CD-DCs) was evident in the spleen. CD-DCs sorted from liver grafts expressed much higher levels of T cell inhibitory programed death ligand 1 (PD-L1) and high levels of interleukin-10 compared with non-CD-DCs (nCD-DCs) isolated from the graft. Concomitantly, high incidences of programed death protein 1 (PD-1)hi T cell immunoglobulin and mucin domain containing 3 (TIM-3)+ exhausted graft-infiltrating CD8+ T cells were observed. Unlike nCD-DCs, the CD-DCs failed to stimulate proliferation of allogeneic T cells but markedly suppressed antidonor host T cell proliferation. CD-DCs were much less evident in allografts from DNAX-activating protein of 12 kDa (DAP12)-/- donors that were rejected acutely. CONCLUSION: These findings suggest that graft-infiltrating PD-L1hi CD-DCs may play a key role in the regulation of alloimmunity and in the induction of liver transplant tolerance. (Hepatology 2018;67:1499-1515).

Antigen Presentation in Transplantation.

Transplantation of solid organs between genetically distinct individuals leads, in the absence of immunosuppression, to T cell-dependent transplant rejection. Activation of graft-reactive T cells relies on the presentation of transplant-derived antigens (intact donor MHC molecules or processed peptides on host MHC molecules) by mature dendritic cells (DCs). This review focuses on novel insights regarding the steps for maturation and differentiation of DCs that are necessary for productive presentation of transplant antigens to host T cells. These steps include the licensing of DCs by the microbiota, their activation and maturation following recognition of allogeneic non-self, and their capture of donor cell exosomes to amplify the presentation of transplant antigens.

Impact of extracellular vesicles on innate immunity.

PURPOSE OF REVIEW: Extracellular vesicles released by prokaryote or eukaryote cells are emerging as mechanisms of cell-to-cell communication, by either physically interacting with the surface of target cells or transferring proteins/peptides, lipids, carbohydrates, and nuclei acids to acceptor cells. Accumulating evidence indicates that extracellular vesicles, among other functions, regulate innate and adaptive immune responses. We revisit here the effects that extracellular vesicles of various origins have on innate immunity. RECENT FINDINGS: Extracellular vesicles comprise a heterogeneous group of vesicles with different biogenesis, composition and biological properties, which include exosomes, microvesicles, apoptotic cell-derived extracellular vesicles, and other extracellular vesicles still not well characterized. Extracellular vesicles released by pathogens, leukocytes, nonhematopoietic cells, tumor cells, and likely allografts, can either stimulate or suppress innate immunity via multiple mechanisms. These include transfer to target leukocytes of pro-inflammatory or anti-inflammatory mediators, membrane receptors, enzymes, mRNAs, and noncoding RNAs; and interaction of extracellular vesicles with the complement and coagulation systems. As a result, extracellular vesicles affect differentiation, polarization, activation, tissue recruitment, cytokine and chemokine production, cytolytic and phagocytic function, and antigen transfer ability, of different types of innate immune cells. SUMMARY: The field of intercellular communication via extracellular vesicles is a rapid evolving area and the effects of pathogen-derived and host-derived extracellular vesicles on innate immunity in particular, have received increasing attention during the past decade. Future studies will be necessary to assess the full potential of the crosstalk between extracellular vesicles and the innate immune system and its use for therapeutic applications to treat chronic inflammation-based diseases and cancer growth and dissemination, among the growing list of disorders in which the innate immune system plays a critical role.

Tolerogenic dendritic cells and the quest for transplant tolerance.

In recent years, there has been a shift from the perception of dendritic cells (DCs) solely as inducers of immune reactivity to the view that these cells are crucial regulators of immunity, which includes their ability to induce and maintain tolerance. Advances in our understanding of the phenotypical and functional plasticity of DCs, and in our ability to manipulate their development and maturation in vitro and in vivo, has provided a basis for the therapeutic harnessing of their inherent tolerogenicity. In this Review, we integrate the available information on the role of DCs in the induction of tolerance, with a focus on transplantation.

Concise Review: Mechanisms Behind Apoptotic Cell-Based Therapies Against Transplant Rejection and Graft versus Host Disease.

The main limitations to the success of transplantation are the antigraft response developed by the recipient immune system, and the adverse side effects of chronic immunosuppression. Graft-versus-host disease (GVHD) triggered by donor-derived T lymphocytes against the recipient tissues is another serious obstacle in the field of hematopoietic stem cell transplantation. Several laboratories have tested the possibility of promoting antigen (Ag)-specific tolerance for therapy of graft rejection, GVHD, and autoimmune disorders, by developing methodologies that mimic the mechanisms by which the immune system maintains peripheral tolerance in the steady state. It has been long recognized that the silent clearance of cells undergoing apoptosis exerts potent immune-regulatory effects and provides apoptotic cell-derived Ags to those Ag-presenting cells (APCs) that internalize them, in particular macrophages and dendritic cells. Therefore, in situ-targeting of recipient APCs by systemic administration of leukocytes in early apoptosis and bearing donor Ags represents a relatively simple approach to control the antidonor response against allografts. Here, we review the mechanisms by which apoptotic cells are silently cleared by phagocytes, and how such phenomenon leads to down-regulation of the innate and adaptive immunity. We discuss the evolution of apoptotic cell-based therapies from murine models of organ/tissue transplantation and GVHD, to clinical trials. We make emphasis on potential limitations and areas of concern of apoptotic cell-based therapies, and on how other immune-suppressive therapies used in the clinics or tested experimentally likely also function through the silent clearance of apoptotic cells by the immune system. Stem Cells 2016;34:1142-1150.

HIPPO-Integrin-linked Kinase Cross-Talk Controls Self-Sustaining Proliferation and Survival in Pulmonary Hypertension.

RATIONALE: Enhanced proliferation and impaired apoptosis of pulmonary arterial vascular smooth muscle cells (PAVSMCs) are key pathophysiologic components of pulmonary vascular remodeling in pulmonary arterial hypertension (PAH). OBJECTIVES: To determine the role and therapeutic relevance of HIPPO signaling in PAVSMC proliferation/apoptosis imbalance in PAH. METHODS: Primary distal PAVSMCs, lung tissue sections from unused donor (control) and idiopathic PAH lungs, and rat and mouse models of SU5416/hypoxia-induced pulmonary hypertension (PH) were used. Immunohistochemical, immunocytochemical, and immunoblot analyses and transfection, infection, DNA synthesis, apoptosis, migration, cell count, and protein activity assays were performed in this study. MEASUREMENTS AND MAIN RESULTS: Immunohistochemical and immunoblot analyses demonstrated that the HIPPO central component large tumor suppressor 1 (LATS1) is inactivated in small remodeled pulmonary arteries (PAs) and distal PAVSMCs in idiopathic PAH. Molecular- and pharmacology-based analyses revealed that LATS1 inactivation and consequent up-regulation of its reciprocal effector Yes-associated protein (Yap) were required for activation of mammalian target of rapamycin (mTOR)-Akt, accumulation of HIF1α, Notch3 intracellular domain and ß-catenin, deficiency of proapoptotic Bim, increased proliferation, and survival of human PAH PAVSMCs. LATS1 inactivation and up-regulation of Yap increased production and secretion of fibronectin that up-regulated integrin-linked kinase 1 (ILK1). ILK1 supported LATS1 inactivation, and its inhibition reactivated LATS1, down-regulated Yap, suppressed proliferation, and promoted apoptosis in PAH, but not control PAVSMCs. PAVSM in small remodeled PAs from rats and mice with SU5416/hypoxia-induced PH showed down-regulation of LATS1 and overexpression of ILK1. Treatment of mice with selective ILK inhibitor Cpd22 at Days 22-35 of SU5416/hypoxia exposure restored LATS1 signaling and reduced established pulmonary vascular remodeling and PH. CONCLUSIONS: These data report inactivation of HIPPO/LATS1, self-supported via Yap-fibronectin-ILK1 signaling loop, as a novel mechanism of self-sustaining proliferation and apoptosis resistance of PAVSMCs in PAH and suggest a new potential target for therapeutic intervention.

Donor-derived exosomes: the trick behind the semidirect pathway of allorecognition.

PURPOSE OF REVIEW: The passenger leukocyte hypothesis predicts that after transplantation, donor antigen-presenting cells (APCs) from the graft present donor MHC molecules to directly alloreactive T cells in lymphoid organs. However, in certain transplantation models, recent evidence contradicts this long-standing concept. New findings demonstrate that host, instead of donor, APCs play a prominent role in allosensitization against donor MHC molecules via the semidirect pathway. A similar mechanism operates in development of T-cell split tolerance to noninherited maternal antigens. RECENT FINDINGS: Following fully mismatch skin or heart transplantation in mice, no or extremely few donor migrating APCs (i.e. conventional dendritic cells) are detected in the draining lymphoid organs. Instead, recipient dendritic cells that have captured donor extracellular vesicles (i.e. exosomes) carrying donor MHC molecules and APC costimulatory signals present donor MHC molecules to directly alloreactive T cells. This semidirect pathway can also give rise to a form of 'split' tolerance during chronic alloantigen exposure, as indirectly alloreactive T helper cells and directly alloreactive T-cell effectors are differentially impacted by host dendritic cells 'cross-dressed' with extracellular vesicles/exosomes derived from maternal microchimerism. SUMMARY: Acquisition by recipient APCs of donor exosomes (and likely other extracellular vesicles) released by passenger leukocytes or the graft explains the potent T-cell allosensitization against donor MHC molecules, in the absence or presence of few passenger leukocytes in lymphoid organs. It also provides the basic mechanism and in-vivo relevance of the elusive semidirect pathway. Its degree of coordination with the allopeptide - specific, indirect pathway of T-cell help may determine whether semidirect allopresentation results in a sustained, effective, acute rejection response, or rather, in abortive acute rejection and 'split' tolerance.

Toll-like Receptor 4 Signaling on Dendritic Cells Suppresses Polymorphonuclear Leukocyte CXCR2 Expression and Trafficking via Interleukin 10 During Intra-abdominal Sepsis.

BACKGROUND: Toll-like receptor 4 (TLR4) is a critical receptor involved in the sensing of gram-negative bacterial infection. However, the roles of TLR4 in sepsis are cell-type specific. Dendritic cells (DCs) are known to play a central role in microbial detection, alerting the immune system to the presence of infection and coordinating adaptive immune response. The goal of this study was to investigate the impact of DC-specific TLR4 signaling on host defense against intra-abdominal polymicrobial sepsis. METHODS: C57BL/6, global Tlr4 knockout, cell-specific knockout control, and CD11c-specific Tlr4(-/-) mice underwent cecal ligation and puncture (CLP). RESULTS: Specific deletion of TLR4 on DCs in mice improved survival and enhanced bacterial clearance. Deletion of TLR4 on DCs was associated with lower levels of circulating interleukin 10 (IL-10), higher polymorphonuclear leukocyte (PMN) accumulation in the peritoneal cavity, and higher expression of chemokine (C-X-C motif) receptor 2 (CXCR2) on PMNs after CLP. In vitro studies of DC and neutrophil cocultures confirmed that TLR4-dependent secretion of IL-10 from DCs regulated neutrophil CXCR2 expression. CONCLUSIONS: Our data shed light on a previously unrecognized role for TLR4 signaling on DCs in driving IL-10 secretion during sepsis and, through this pathway, regulates PMN recruitment via suppression of CXCR2 expression.

Human placental trophoblasts confer viral resistance to recipient cells.

Placental trophoblasts form the interface between the fetal and maternal environments and serve to limit the maternal-fetal spread of viruses. Here we show that cultured primary human placental trophoblasts are highly resistant to infection by a number of viruses and, importantly, confer this resistance to nonplacental recipient cells by exosome-mediated delivery of specific microRNAs (miRNAs). We show that miRNA members of the chromosome 19 miRNA cluster, which are almost exclusively expressed in the human placenta, are packaged within trophoblast-derived exosomes and attenuate viral replication in recipient cells by the induction of autophagy. Together, our findings identify an unprecedented paracrine and/or systemic function of placental trophoblasts that uses exosome-mediated transfer of a unique set of placental-specific effector miRNAs to directly communicate with placental or maternal target cells and regulate their immunity to viral infections.

Autocrine hemokinin-1 functions as an endogenous adjuvant for IgE-mediated mast cell inflammatory responses.

BACKGROUND: Efficient development of atopic diseases requires interactions between allergen and adjuvant to initiate and amplify the underlying inflammatory responses. Substance P (SP) and hemokinin-1 (HK-1) are neuropeptides that signal through the neurokinin-1 receptor (NK1R) to promote inflammation. Mast cells initiate the symptoms and tissue effects of atopic disorders, secreting TNF and IL-6 after FcεRI cross-linking by antigen-IgE complexes (FcεRI-activated mast cells [FcεRI-MCs]). Additionally, MCs express the NK1R, suggesting an adjuvant role for NK1R agonists in FcεRI-MC-mediated pathologies; however, in-depth research addressing this relevant aspect of MC biology is lacking. OBJECTIVE: We sought to investigate the effect of NK1R signaling and the individual roles of SP and HK-1 as potential adjuvants for FcεRI-MC-mediated allergic disorders. METHODS: Bone marrow-derived mast cells (BMMCs) from C57BL/6 wild-type (WT) or NK1R(-/-) mice were used to investigate the effects of NK1R signaling on FcεRI-MCs. BMMCs generated from Tac1(-/-) mice or after culture with Tac4 small interfering RNA were used to address the adjuvancy of SP and HK-1. WT, NK1R(-/-), and c-Kit(W-sh/W-sh) mice reconstituted with WT or NK1R(-/-) BMMCs were used to evaluate NK1R signaling on FcεRI-MC-mediated passive local and systemic anaphylaxis and on airway inflammation. RESULTS: FcεRI-activated MCs upregulated NK1R and HK-1 transcripts and protein synthesis, without modifying SP expression. In a positive signaling loop HK-1 promoted TNF and IL-6 secretion by MC degranulation and protein synthesis, the latter through the phosphoinositide 3-kinase/Akt/nuclear factor κB pathways. In vivo NK1R signaling was necessary for the development of passive local and systemic anaphylaxis and airway inflammation. CONCLUSIONS: FcεRI stimulation of MCs promotes autocrine secretion of HK-1, which signals through NK1R to provide adjuvancy for efficient development of FcεRI-MC-mediated disorders.

Neurokinin-1 receptor agonists bias therapeutic dendritic cells to induce type 1 immunity by licensing host dendritic cells to produce IL-12.

Substance-P and hemokinin-1 are proinflammatory neuropeptides with potential to promote type 1 immunity through agonistic binding to neurokinin-1 receptor (NK1R). Dendritic cells (DCs) are professional antigen-presenting cells that initiate and regulate the outcome of innate and adaptive immune responses. Immunostimulatory DCs are highly desired for the development of positive immunization techniques. DCs express functional NK1R; however, regardless of their potential DC-stimulatory function, the ability of NK1R agonists to promote immunostimulatory DCs remains unexplored. Here, we demonstrate that NK1R signaling activates therapeutic DCs capable of biasing type 1 immunity by inhibition of interleukin-10 (IL-10) synthesis and secretion, without affecting their low levels of IL-12 production. The potent type 1 effector immune response observed following cutaneous administration of NK1R-signaled DCs required their homing in skin-draining lymph nodes (sDLNs) where they induced inflammation and licensed endogenous-conventional sDLN-resident and -recruited inflammatory DCs to secrete IL-12. Our data demonstrate that NK1R signaling promotes immunostimulatory DCs, and provide relevant insight into the mechanisms used by neuromediators to regulate innate and adaptive immune responses.

Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes.

Dendritic cells (DCs) are the most potent APCs. Whereas immature DCs down-regulate T-cell responses to induce/maintain immunologic tolerance, mature DCs promote immunity. To amplify their functions, DCs communicate with neighboring DCs through soluble mediators, cell-to-cell contact, and vesicle exchange. Transfer of nanovesicles (< 100 nm) derived from the endocytic pathway (termed exosomes) represents a novel mechanism of DC-to-DC communication. The facts that exosomes contain exosome-shuttle miRNAs and DC functions can be regulated by exogenous miRNAs, suggest that DC-to-DC interactions could be mediated through exosome-shuttle miRNAs, a hypothesis that remains to be tested. Importantly, the mechanism of transfer of exosome-shuttle miRNAs from the exosome lumen to the cytosol of target cells is unknown. Here, we demonstrate that DCs release exosomes with different miRNAs depending on the maturation of the DCs. By visualizing spontaneous transfer of exosomes between DCs, we demonstrate that exosomes fused with the target DCs, the latter followed by release of the exosome content into the DC cytosol. Importantly, exosome-shuttle miRNAs are functional, because they repress target mRNAs of acceptor DCs. Our findings unveil a mechanism of transfer of exosome-shuttle miRNAs between DCs and its role as a means of communication and posttranscriptional regulation between DCs.

Dendritic cells of myeloid lineage: the masterminds behind acute allograft rejection.

PURPOSE OF REVIEW: Advances in surgery, patient management, and pharmacologic immunosuppression have reduced the incidence of acute allograft rejection. However, generation of therapies to promote donor-specific immunosuppression with minimal side-effects has proved to be a difficult task. To some extent, this is because of our limited knowledge on how Ag-presenting cells (APCs) like dendritic cells initiate and maintain the antidonor response in vivo. Herein, we link the classic concepts on the role of donor's dendritic cells as passenger leukocytes with the state-of-the-art findings in the field. RECENT FINDINGS: Numerous studies are starting to unveil the plethora of mediators and interactions with leukocytes that trigger maturation of donor's dendritic cells in the grafts. The concept that donor's dendritic cells migrate from the grafts to secondary lymphoid organs to prime T cells has been challenged in murine models of lung or intestine transplantation, in which T cells can also be primed in the allograft. Increasing evidence suggests that recipient's dendritic cells present donor's intact major histocompatibility complex (MHC) molecules in lymphoid organs and that they infiltrate the grafts. SUMMARY: A more complete understanding of the role of dendritic cells in allosensitization will help to develop better dendritic cell-based therapies to achieve the final goal of promoting donor-specific immunosuppression.

Orchestration of transplantation tolerance by regulatory dendritic cell therapy or in-situ targeting of dendritic cells.

PURPOSE OF REVIEW: Extensive research in murine transplant models over the past two decades has convincingly demonstrated the ability of regulatory dendritic cells (DCregs) to promote long-term allograft survival. We review important considerations regarding the source of therapeutic DCregs (donor or recipient) and their mode of action, in-situ targeting of DCregs, and optimal therapeutic regimens to promote DCreg function. RECENT FINDINGS: Recent studies have defined protocols and mechanisms whereby ex-vivo-generated DCregs of donor or recipient origin subvert allogeneic T-cell responses and promote long-term organ transplant survival. Particular interest has focused on how donor antigen is acquired, processed and presented by autologous dendritic cells, on the stability of DCregs, and on in-situ targeting of dendritic cells to promote their tolerogenic function. New evidence of the therapeutic efficacy of DCregs in a clinically relevant nonhuman primate organ transplant model and production of clinical grade DCregs support early evaluation of DCreg therapy in human graft recipients. SUMMARY: We discuss strategies currently used to promote dendritic cell tolerogenicity, including DCreg therapy and in-situ targeting of dendritic cells, with a view to improved understanding of underlying mechanisms and identification of the most promising strategies for therapeutic application.

Endogenous dendritic cells mediate the effects of intravenously injected therapeutic immunosuppressive dendritic cells in transplantation.

The prevailing idea regarding the mechanism(s) by which therapeutic immunosuppressive dendritic cells (DCs) restrain alloimmunity is based on the concept that they interact directly with antidonor T cells, inducing anergy, deletion, and/or regulation. However, this idea has not been tested in vivo. Using prototypic in vitro-generated maturation-resistant (MR) DCs, we demonstrate that once MR-DCs carrying donor antigen (Ag) are administered intravenously, they decrease the direct and indirect pathway T-cell responses and prolong heart allograft survival but fail to directly regulate T cells in vivo. Rather, injected MR-DCs are short-lived and reprocessed by recipient DCs for presentation to indirect pathway CD4(+) T cells, resulting in abortive activation and deletion without detrimental effect on the number of indirect CD4(+) FoxP3(+) T cells, thus increasing the regulatory to effector T cell relative percentage. The effect on the antidonor response was independent of the method used to generate therapeutic DCs or their viability; and in accordance with the idea that recipient Ag-presenting cells mediate the effects of therapeutic DCs in transplantation, prolongation of allograft survival was achieved using donor apoptotic MR-DCs or those lacking surface major histocompatibility complex molecules. We therefore conclude that therapeutic DCs function as Ag-transporting cells rather than Ag-presenting cells to prolong allograft survival.