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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 (SP) and hemokinin 1 (HK1), 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: To develop an Ag-specific immunosuppressive approach to treat CD by skin co-delivery 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-antagonists-MNA in CD. RESULTS: We demonstrate in mice that CD requires NK1R signaling by SP and HK1. Specific deletion of the NK1R in keratinocytes and dendritic cells, but not in mast cells prevented CD. Skin co-delivery of hapten- or Ag-NK1R-antagonist-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: Our findings demonstrate that immune-regulation by engineering localized skin neuroimmune networks can be used to treat cutaneous diseases that like CD, are caused by type-1 immunity.

Despite the role of donor-specific antibodies (DSAs) in recognizing major histocompatibility complex (MHC) antigens and mediating transplant rejection, how and where recipient B cells in lymphoid tissues encounter donor MHC antigens remains unclear. Contrary to the dogma, we demonstrated here that migration of donor leukocytes out of skin or heart allografts is not necessary for B or T cell allosensitization in mice. We found that mouse skin and cardiac allografts and human skin grafts release cell-free donor MHC antigens via extracellular vesicles (EVs) that are captured by subcapsular sinus (SCS) macrophages in lymph nodes or analog macrophages in the spleen. Donor EVs were transported across the SCS macrophages, and donor MHC molecules on the EVs were recognized by alloreactive B cells. This triggered B cell activation and DSA production, which were both prevented by SCS macrophage depletion. These results reveal an unexpected role for graft-derived EVs and open venues to interfere with EV biogenesis, trafficking, or function to restrain priming or reactivation of alloreactive B cells.

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. Fil: Friedrich, Adrián David. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Campo, Valeria Evelyn. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Cela, Eliana Maiten. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Morelli, Adrian E.. University of Pittsburgh; Estados Unidos Fil: Shufesky, William J.. University of Pittsburgh; Estados Unidos Fil: Tckacheva, Olga A.. University of Pittsburgh; Estados Unidos Fil: Leoni, Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Paz, Mariela Laura. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Larregina, Adriana T.. University of Pittsburgh; Estados Unidos Fil: Gonzalez Maglio, Daniel Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina

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 HIFl alpha, Notch3 intracellular domain and beta-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 ILKinhibitor 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.

The immune response against transplanted allografts is one of the most potent reactions mounted by the immune system. The acute rejection response has been attributed to donor dendritic cells (DCs), which migrate to recipient lymphoid tissues and directly activate alloreactive T cells against donor MHC molecules. Here, using a murine heart transplant model, we determined that only a small number of donor DCs reach lymphoid tissues and investigated how this limited population of donor DCs efficiently initiates the alloreactive T cell response that causes acute rejection. In our mouse model, efficient passage of donor MHC molecules to recipient conventional DCs (cDCs) was dependent on the transfer of extracellular vesicles (EVs) from donor DCs that migrated from the graft to lymphoid tissues. These EVs shared characteristics with exosomes and were internalized or remained attached to the recipient cDCs. Recipient cDCs that acquired exosomes became activated and triggered full activation of alloreactive T cells. Depletion of recipient cDCs after cardiac transplantation drastically decreased presentation of donor MHC molecules to directly alloreactive T cells and delayed graft rejection in mice. These findings support a key role for transfer of donor EVs in the generation of allograft-targeting immune responses and suggest that interrupting this process has potential to dampen the immune response to allografts.

T-cell receptor (TCR)-signaling triggers intracellular Ca2+ increase required for NFAT1/2-mediated IL-2 secretion. This pathway is necessary for thymocyte maturation and survival of activated T cells in secondary lymphoid organs (SLOs). In T cells, cellular Ca2+ levels are regulated by TCR- and G-protein coupled receptor (GPCR)-signaling via the PLCγ and PLCβ subunits, respectively. Nevertheless, the GPCR(s) involved in this phenomenon has not been identified. The neurokinin-1 receptor (NK1R) is a GPCR that induces Ca2+ flux in neurons, and NK1R-signaling by the neuropetides substance P (SP) and hemokinin 1 (HK1) promotes T-cell immunity. We studied the role of NK1R-signaling in T-cell development in the thymus and after T-cell priming in SLOs. By Imagestream, we found that the NK1R and its ligands localize at the site of dendritic cell (DC)-T cell contact. Following CD3-signaling, the NK1R was required for optimal Ca2+flux and NFAT-mediated IL-2 secretion in T cells, effects that were abrogated in NK1RKO or SP/HK1double KO T cells. In the thymus, absence of NK1R resulted in decreased maturation and survival of TCR+ double positive CD4 CD8, single positive CD4, and single positive CD8thymocytes. In SLOs, the NK1R was required for survival of Ag-activated CD4 Th1 and CD8 T cells. In vivo, in a skin model of Th1-DTH induced in NK1RKO T-cell or HK-1/SPdouble KOT-cell bone marrow chimeras, 73±5% of activated CD4 and CD8 T cells died during priming in skin-draining SLOs, and the remaining T cells died in the skin following elicitation. We conclude that the NK1R cooperates with the TCR to increase intracellular Ca2+ necessary for thymocyte maturation and survival of activated T cells in SLOs. NIH R01 AR068249 and AR071277 to ATL and LDF.

The idea that donor dendritic cells (DCs) from the graft present donor MHC Ag to naïve T cells has been challenged. Evidence suggests that donor DCs are undetectable or found are low numbers in graft-draining lymphoid organs. Thus, the aim of our study is to elucidate how donor MHC molecules are recognized so efficiently by recipient naïve T cells. Results After transplantation of CD45.2 BALB/c hearts in CD45.1 B6 mice, very few donor DCs were detected in the spleen, and no donor DCs were found in lymph nodes draining BALB/c skin grafted in B6 mice. In both cases, donor intact MHC molecules were detected by electron microscopy on exosomes attached to recipient conventional DCs in graft-draining lymphoid organs. The transferred MHC Ag induced proliferation and differentiation of CD8 T cells against the BALB/c H2Ld Ag. Cultures of BALB/c DCs with B6 DCs with inhibitors of exosome release (Rab27a siRNA) confirmed that passage of BALB/c MHC occurred via exosomes. Importantly, transfer of exosomes released by donor DCs (unlike other types of vesicles) promoted maturation of recipient DCs. By high resolution confocal microscopy, BALB/c DCs, engineered to release RFP+ exosomes and injected in CD11c-YFP B6 mice, transferred RFP+ exosomes to recipient YFP+ DCs in lymph nodes and spleen. Accordingly, depletion of recipient DCs in CD11c-DTR B6 recipients prevented presentation of donor intact MHC Ag to T cells and delayed heart allograft rejection. Conclusion Our results elucidate the ultrastructural basis of the still elusive semi-direct pathway of allorecognition, by demonstrating that donor exosomes (released by the graft or donor migrating DCs) cross-dress recipient APCs and promote the potent T-cell allosensitization seen in transplantation.

Initiation of cellular immunity relies on T cell activation by Ag-presenting dendritic cells (DCs). By signaling via the neurokinin-1 receptor (NK1R), the neuropeptides hemokinin-1 (HK-1) and substance P (SP) potentiate cellular immunity by mechanisms not fully elucidated. Although SP is mainly secreted by sensory nerves, it is also synthesized and released by T cells. Here, we investigated the role of NK1R and autocrine SP and HK-1 on T cell activation. Using Imagestream flow cytometry (Amnis) to visualize couplets of OVA loaded WT or SP/HK1double KO DCs and responder OT-II CD4 T cells, we observed that the NK1R, SP and HK-1 colocalize with phalloidin at the site of DC-T cell contact. Following CD3/CD28 activation in vitro, we demonstrate that T cells express the full-length NK1R, and that NK1R signaling induces Ca2 flux, activation of calcineurin, NFAT1/2 and NFκB, which results in IL-2 secretion and survival of CD4 and CD8 T cells. These effects were abrogated in NK1RKO orSP/HK1double KO T cells, or by soluble NK1R antagonists. Addition of exogenous SP or HK-1 to CD3/CD28 activated WT T cells did not increase IL-2 secretion indicating that autocrine SP and HK1 released at the DC-T cell synapse suffice to promote T cell survival. In a model of skin DTH in NK1RKO or HK-1/SPdouble KOT cell chimeras, 73 ± 5% of activated (CD44high) CD4 and CD8 T cells die in the draining lymph nodes after priming, and 79%± 7% of the remaining T cells recruited to the skin die in situ following elicitation. In summary, NK1R signaling by autocrine SP and HK1 at the DC-T cell synapse is necessary for the survival of activated T cells and the development of potent cellular immunity.

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 FceRI cross-linking by antigen-IgE complexes (FceRI-activated mast cells [FceRI-MCs]). Additionally, MCs express the NK1R, suggesting an adjuvant role for NK1R agonists in FceRI-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 FceRI-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 FceRI-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 FceRI-MC–mediated passive local and systemic anaphylaxis and on airway inflammation. Results FceRI-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 FceRI stimulation of MCs promotes autocrine secretion of HK-1, which signals through NK1R to provide adjuvancy for efficient development of FceRI-MC–mediated disorders.