Cross-presenting Langerhans cells are required for the early reactivation of resident CD8+ memory T cells in the epidermis.

Tissue-resident memory CD8+ T cells (TRM) reside at sites of previous infection, providing protection against reinfection with the same pathogen. In the skin, TRM patrol the epidermis, where keratinocytes are the entry site for many viral infections. Epidermal TRM react rapidly to cognate antigen encounter with the secretion of cytokines and differentiation into cytotoxic effector cells, constituting a first line of defense against skin reinfection. Despite the important protective role of skin TRM, it has remained unclear, whether their reactivation requires a professional antigen-presenting cell (APC). We show here, using a model system that allows antigen targeting selectively to keratinocytes in a defined area of the skin, that limited antigen expression by keratinocytes results in rapid, antigen-specific reactivation of skin TRM. Our data identify epidermal Langerhans cells that cross-present keratinocyte-derived antigens, as the professional APC indispensable for the early reactivation of TRM in the epidermal layer of the skin.

IL-10-modulated dendritic cells from birch pollen- and hazelnut-allergic patients facilitate Treg-mediated allergen-specific and cross-reactive tolerance.

BACKGROUND: Approximately 70% of individuals allergic to birch pollen (Bet v 1.01 [Bet]) develop a secondary food allergy (e.g., hazelnut: Cor a 1.04 [Cor]), due to allergen cross-reactivity. However, standard immunotherapy for type I allergies often does not improve the food allergy sufficiently. We analyzed the allergen-specific and cross-reactive suppressive capacity of primary human regulatory T cells (Treg) induced by autologous IL-10-modulated dendritic cells (IL-10 DC) in vitro and in vivo. METHODS: CD4+ T cells of patients with birch pollen and associated hazelnut allergies were differentiated into Bet-specific or non-specific induced Treg (iTreg). After Bet- or Cor-specific restimulation the phenotype, proliferation, and suppressive capacity of iTreg subsets were analyzed. iTreg function was further investigated in humanized mouse models of airway and intestinal allergy, generated by engraftment of peripheral blood mononuclear cells from allergic donors into immunodeficient animals. RESULTS: After IL-10 DC priming and allergen-specific restimulation (Bet or Cor), non-specific control iTreg remained anergic, whereas Bet-specific iTreg proliferated extensively and exhibited a regulatory phenotype (enhanced expression of CTLA-4, PD-1, TNFR2, IL-10). Accordingly, activated Bet-specific iTreg displayed a high capacity to suppress Bet- and Cor-induced responder Th2 cell responses in vitro, indicating induction of both allergen-specific (birch) and cross-reactive tolerance (hazelnut). In vivo, the beneficial effect of Bet-specific iTreg was verified in humanized mouse models of allergic airway and intestinal inflammation, resulting in reduced allergen-induced clinical symptoms, and immune responses. CONCLUSION: Human IL-10 DC-induced iTreg facilitate allergen-specific and cross-reactive tolerance. Therefore, they are potential candidates for regulatory cell therapy in allergic and autoimmune diseases.

Specialized regulatory T cells control venous blood clot resolution through SPARC.

The cells and mechanisms involved in blood clot resorption are only partially known. We show that regulatory T cells (Tregs) accumulate in venous blood clots and regulate thrombolysis by controlling the recruitment, differentiation and matrix metalloproteinase (MMP) activity of monocytes. We describe a clot Treg population that forms the matricellular acid- and cysteine-rich protein SPARC (secreted protein acidic and rich in cysteine) and show that SPARC enhances monocyte MMP activity and that SPARC+ Tregs are crucial for blood clot resorption. By comparing different treatment times, we define a therapeutic window of Treg expansion that accelerates clot resorption.

Inflammatory Monocyte Counts Determine Venous Blood Clot Formation and Resolution.

BACKGROUND: Monocytes are thought to be involved in venous thrombosis but the role of individual monocyte subpopulations on thrombus formation, clot inflammation, and degradation is an important unresolved issue. We investigate the role of inflammatory Ly6Chi monocytes in deep vein thrombosis and their potential therapeutic impact. METHODS: Frequencies and compositions of blood monocytes were analyzed by flow cytometry in CCR2-/- (C-C chemokine receptor type 2) and wild-type mice of different ages and after treatment with the NR4A1 (nuclear receptor group 4 family A member 1, Nur77) agonist CnsB (cytosporone B). TF (tissue factor) sufficient and deficient Ly6Chi monocytes were adoptively transferred into aged CCR2-/- mice. Thrombus formation and size were followed by ultrasound over a 3-week period after surgical reduction of blood flow (stenosis) in the inferior vena cava. RESULTS: Reduced numbers of peripheral monocytes in aged (>30 w) CCR2-/- mice are accompanied by reduced thrombus formation after inferior vena cava ligation. Reducing the number of inflammatory Ly6Chi monocytes in wild-type mice by CsnB treatment before ligation, similarly suspends clotting, while later treatment (d1 or d4) reduces thrombus growth and accelerates resolution. We describe how changes in inflammatory monocyte numbers affect the gradual differentiation of monocytes in thrombi and show that only tissue factor-competent Ly6Chi monocytes restore thrombosis in aged CCR2-/- mice. CONCLUSIONS: We conclude that the number of inflammatory Ly6Chi monocytes controls deep vein thrombosis formation, growth, and resolution and can be therapeutically manipulated with a NR4A1 agonist at all disease stages.

Expression of the α7 Nicotinic Acetylcholine Receptor Is Critically Required for the Antifibrotic Effect of PHA-543613 on Skin Fibrosis.

INTRODUCTION: Targeting the α7 nicotinic acetylcholine receptor (α7nAChR) has recently been suggested as a potential new treatment for fibrotic skin diseases. Here, we performed a genetic and pharmacologic approach to clarify the role of this receptor in the bleomycin (BLM) mouse model of skin fibrosis using α7nAChR KO mice. METHODS: We analyzed the expression of extracellular matrix (ECM) components in murine skin using quantitative RT-PCR, pepsin digestion/SDS-PAGE of proteins and performed hydroxyproline assays as well as histological/immunohistochemical staining of skin sections. To identity the target cells of the α7nAChR agonist PHA-543613, we used murine dermal fibroblasts (MDF). We tested their response to the profibrotic cytokine transforming growth factor-ß1 (TGF-ß1) and utilized gene silencing to elucidate the role of the α7nAChR. RESULTS: We confirmed our previous findings on C3H/HeJ mice and detected a suppressive effect of PHA-543613 on BLM-induced skin fibrosis in the mouse strain C57BL/6J. This antifibrotic effect of PHA-543613 was abrogated in α7nAChR-KO mice. Interestingly, α7nAChR-KO animals exhibited a basal profibrotic signature by higher RNA expression of ECM genes and hydroxyproline content than WT mice. In WT MDF, PHA-543613 suppressed ECM gene expression induced by TGF-ß1. Gene silencing of α7nAChR by small interfering RNA neutralized the effects of PHA-543613 on TGF-ß1-mediated ECM gene expression. CONCLUSION: In summary, we have identified the α7nAChR as the essential mediator of the antifibrotic effect of PHA-543613. MDF are directly targeted by PHA-543613 to suppress collagen synthesis. Our findings emphasize therapeutic exploitation of α7nAChR receptor agonists in fibrotic skin diseases.

HSV-1 triggers paracrine fibroblast growth factor response from cortical brain cells via immediate-early protein ICP0.

BACKGROUND: Herpes simplex virus-1 (HSV-1) infections of the central nervous system (CNS) can result in HSV-1 encephalitis (HSE) which is characterized by severe brain damage and long-term disabilities. Different cell types including neurons and astrocytes become infected in the course of an HSE which leads to an activation of glial cells. Activated glial cells change their neurotrophic factor profile and modulate inflammation and repair. The superfamily of fibroblast growth factors (FGFs) is one of the largest family of neurotrophic factors comprising 22 ligands. FGFs induce pro-survival signaling in neurons and an anti-inflammatory answer in glial cells thereby providing a coordinated tissue response which favors repair over inflammation. Here, we hypothesize that FGF expression is altered in HSV-1-infected CNS cells. METHOD: We employed primary murine cortical cultures comprising a mixed cell population of astrocytes, neurons, microglia, and oligodendrocytes. Astrocyte reactivity was morphometrically monitored by an automated image analysis algorithm as well as by analyses of A1/A2 marker expression. Altered FGF expression was detected by quantitative real-time PCR and its paracrine FGF activity. In addition, HSV-1 mutants were employed to characterize viral factors important for FGF responses of infected host cells. RESULTS: Astrocytes in HSV-1-infected cortical cultures were transiently activated and became hypertrophic and expressed both A1- and A2-markers. Consistently, a number of FGFs were transiently upregulated inducing paracrine neurotrophic signaling in neighboring cells. Most prominently, FGF-4, FGF-8, FGF-9, and FGF-15 became upregulated in a switch-on like mechanism. This effect was specific for CNS cells and for a fully functional HSV-1. Moreover, the viral protein ICP0 critically mediated the FGF switch-on mechanism. CONCLUSIONS: HSV-1 uses the viral protein ICP0 for the induction of FGF-expression in CNS cells. Thus, we propose that HSV-1 triggers FGF activity in the CNS for a modulation of tissue response upon infection.

Dietary wheat amylase trypsin inhibitors exacerbate murine allergic airway inflammation.

BACKGROUND: Wheat amylase trypsin inhibitors (ATI) are dietary non-gluten proteins that activate the toll-like receptor 4 on myeloid cells, promoting intestinal inflammation. AIM OF THE STUDY: We investigated the effects of dietary ATI on experimental allergic airway inflammation. METHODS: Mice on a gluten and ATI-free diet (GAFD), sensitized with PBS or ovalbumin (OVA) and challenged with OVA, were compared to mice on a commercial standard chow, a gluten diet naturally containing ~ 0.75% of protein as ATI (G+AD), a gluten diet containing ~ 0.19% of protein as ATI (G-AD) and a GAFD with 1% of protein as ATI (AD). Airway hyperreactivity (AHR), inflammation in bronchoalveolar lavage (BAL) and pulmonary tissue sections were analyzed. Allergic sensitization was assessed ex vivo via proliferation of OVA-stimulated splenocytes. RESULTS: Mice on a GAFD sensitized with PBS did not develop AHR after local provocation with methacholine. Mice on a GAFD or on a G-AD and sensitized with OVA developed milder AHR compared to mice fed a G+AD or an AD. The increased AHR was paralleled by increased BAL eosinophils, IL-5 and IL-13 production, and an enhanced ex vivo splenocyte activation in the ATI-fed groups. CONCLUSIONS: Dietary ATI enhance allergic airway inflammation in OVA-challenged mice, while an ATI-free or ATI-reduced diet has a protective effect on AHR. Nutritional wheat ATI, activators of intestinal myeloid cells, may be clinically relevant adjuvants to allergic airway inflammation.

Protein corona-mediated targeting of nanocarriers to B cells allows redirection of allergic immune responses.

BACKGROUND: Nanoparticle (NP)-based vaccines are attractive immunotherapy tools because of their capability to codeliver antigen and adjuvant to antigen-presenting cells. Their cellular distribution and serum protein interaction ("protein corona") after systemic administration and their effect on the functional properties of NPs is poorly understood. OBJECTIVES: We analyzed the relevance of the protein corona on cell type-selective uptake of dextran-coated NPs and determined the outcome of vaccination with NPs that codeliver antigen and adjuvant in disease models of allergy. METHODS: The role of protein corona constituents for cellular binding/uptake of dextran-coated ferrous nanoparticles (DEX-NPs) was analyzed both in vitro and in vivo. DEX-NPs conjugated with the model antigen ovalbumin (OVA) and immunostimulatory CpG-rich oligodeoxynucleotides were administered to monitor the induction of cellular and humoral immune responses. Therapeutic effects of this DEX-NP vaccine in mouse models of OVA-induced anaphylaxis and allergic asthma were assessed. RESULTS: DEX-NPs triggered lectin-induced complement activation, yielding deposition of activated complement factor 3 on the DEX-NP surface. In the spleen DEX-NPs targeted predominantly B cells through complement receptors 1 and 2. The DEX-NP vaccine elicited much stronger OVA-specific IgG2a production than coadministered soluble OVA plus CpG oligodeoxynucleotides. B-cell binding of the DEX-NP vaccine was critical for IgG2a production. Treatment of OVA-sensitized mice with the DEX-NP vaccine prevented induction of anaphylactic shock and allergic asthma accompanied by IgE inhibition. CONCLUSIONS: Opsonization of lectin-coated NPs by activated complement components results in selective B-cell targeting. The intrinsic B-cell targeting property of lectin-coated NPs can be exploited for treatment of allergic immune responses.

Nutritional Wheat Amylase-Trypsin Inhibitors Promote Intestinal Inflammation via Activation of Myeloid Cells.

BACKGROUND & AIMS: Wheat amylase-trypsin inhibitors (ATIs) are nutritional activators of innate immunity, via activation of the toll-like receptor 4 (TLR4) on myeloid cells. We aimed to characterize the biologic activity of ATIs in various foods and their effect on intestinal inflammation. METHODS: We selected 38 different gluten-containing and gluten-free products, either unprocessed (such as wheat, rye, barley, quinoa, amaranth, soya, lentils, and rice) or processed (such as pizza, pasta, bread, and biscuits). ATIs were extracted and their biological activities determined in TLR4-responsive mouse and human cell lines. Effects of oral ATIs on intestinal inflammation were determined in healthy C57BL/6 mice on a gluten-free or ATI-free diet and in mice given low-level polyinosinic:polycytidylic acid or dextran sodium sulfate to induce colitis. Parameters of innate and adaptive immune activation were determined in duodenum, ileum, colon, and mesenteric lymph nodes. RESULTS: Modern gluten-containing staples had levels of TLR4-activating ATIs that were as much as 100-fold higher than in most gluten-free foods. Processed or baked foods retained ATI bioactivity. Most older wheat variants (such as Emmer or Einkorn) had lower bioactivity than modern (hexaploid) wheat. ATI species CM3 and 0.19 were the most prevalent activators of TLR4 in modern wheat and were highly resistant to intestinal proteolysis. Their ingestion induced modest intestinal myeloid cell infiltration and activation, and release of inflammatory mediators-mostly in the colon, then in the ileum, and then in the duodenum. Dendritic cells became prominently activated in mesenteric lymph nodes. Concentrations of ATIs found in a normal daily gluten-containing diet increased low-level intestinal inflammation. CONCLUSIONS: Gluten-containing cereals have by far the highest concentrations of ATIs that activate TLR4. Orally ingested ATIs are largely resistant to proteases and heat, and increase intestinal inflammation by activating gut and mesenteric lymph node myeloid cells.

IL-10-Modulated Human Dendritic Cells for Clinical Use: Identification of a Stable and Migratory Subset with Improved Tolerogenic Activity.

Dendritic cells (DCs) are key regulators of protective immune responses and tolerance to (self-)Ags. Therefore, the scientific rationale for the use of tolerogenic DC therapy in the fields of allergies, autoimmunity, and transplantation medicine is strong. In this study, we analyzed the tolerogenic capacity of IL-10-modulated DC (IL-10DC) subpopulations to identify a DC subset that combines potent immunosuppressive activities with valuable immune properties for clinical implementation. IL-10DCs consist of two phenotypically distinct subpopulations: CD83highCCR7+ IL-10DCs and CD83lowCCR7- IL-10DCs. Suppressor assays with activated effector T cells revealed that CD4+ regulatory T cells generated by CD83high IL-10DCs (iTreg+) exhibited a significantly higher suppressive capacity compared with CD4+ regulatory T cells generated by CD83low IL-10DCs (iTreg-). In this context, iTreg+ displayed a more activated phenotype (proliferation, cytokine production) compared with iTreg- In contrast to CD83low IL-10DCs, CD83high IL-10DCs exerted a strong migratory capacity toward the secondary lymphoid organ chemokine CCL21 and retained a functionally stable phenotype under inflammatory conditions. In addition, CD83high IL-10DCs expressed significantly higher levels of surface and soluble CD25. Functional analysis demonstrated that IL-10DC-related soluble CD25 efficiently inhibited the proliferation of activated T cells and that blockade of CD25 function abolished the induction of regulatory T cells by IL-10DCs, indicating a critical role for IL-10DC-related CD25 in shifting the immune response toward an iTreg- controlled tolerance reaction. In conclusion, the selective use of the CD83high IL-10DC subset may result in a higher efficacy of tolerance induction in vivo and may support the development of novel DC vaccination strategies for transplantations, as well as for allergic and autoimmune diseases.

Myeloid cell populations and fibrogenic parameters in bleomycin- and HOCl-induced fibrosis.

Mouse models resembling systemic sclerosis can be chemically induced by application of bleomycin or hypochloric acid (HOCl). To date, little is known about inflammatory cells and their potential role in scleroderma (Scl)-related fibrosis. Therefore, we compared both Scl models to define the early immune cell subsets in relation to fibrosis-related parameters. Both agents induced a significant increase in dermal thickness and collagen deposition after 4 weeks, as hallmarks of Scl. However, clinical skin thickness, densely packed, sirius red-stained collagen bundles and collagen cross-links were more pronounced in HOCl-induced Scl. In parallel, there was a significant upregulation of procollagen α1(I), α-SMA and TGF-ß transcripts in HOCl animals, whereas IL-1ß and MMP-13 mRNA levels were significantly increased in bleomycin-treated mice. Flow cytometric analysis of the Scl skin demonstrated an early cellular infiltrate containing mainly CD19+ B cells, CD4+ T cells, CD11c+ DC and CD11b+ myeloid cells, the latter ones being significantly more prominent after HOCl injection. Subanalysis revealed that Scl mice exhibited a significant increase of inflammatory myeloid CD11b+  Ly6Clow-high  CD64low-high cells (HOCl>bleomycin). In particular, in the HOCl model, activated dermal macrophages (CCR2low  MHCIIhigh ) and monocyte-derived DC (CCR2high MHCIIhigh ) predominated over less activated CD11b+ myeloid cells. In conclusion, the two models differ in certain aspects of the murine and human scleroderma but in the HOCl model, myeloid CD11b+  MHCIIhigh cells correlate with some fibrosis-related parameters. Therefore, analysis of both models is suggested to cover a comprehensive profile of Scl symptoms but with focus on the HOCl model when the role of early myeloid immune cells will be evaluated.

Research in practice: the impact of interferon-α therapy on immune tolerance.

Interferon-α (IFN-α) is the only drug approved for adjuvant therapy of malignant melanoma and is also used in the treatment of hematological and solid tumors. Along with its proven clinical efficacy, IFN-α produces several side effects, particularly with regard to autoimmune disorders. Curious about symptoms of autoimmunity during IFN-α therapy, we asked whether IFN-α directly impacts on immune tolerance. We found that IFN-α does alter the function of tolerogenic dendritic cells (DC) as well as of induced and naturally occurring T-regulatory cells (nTregs). IFN-α blocks the tolerogenic phenotype of DC by inducing maturation and thus preventing the induction of inducible Tregs by DC. It also has direct effects on nTregs. IFN-α reduces cAMP in Tregs via ERK/phosphodiesterase-mediated pathways. Since cAMP is essentially involved in suppression by nTregs, the IFN-α-dependent reduction of cAMP levels abolishes the suppressive capacity of nTregs. Therefore, Tregs are incapable of suppressing the activity of effector T cells and natural killer cells, resulting in tumor rejection. Thus, IFN-α overcomes immunological tolerance processes, leading to an improved immunostimulation and efficient tumor rejection, but also increases the risk of autoimmunity.

Targeting ACC1 in T cells ameliorates psoriatic skin inflammation.

Psoriasis is a chronic inflammatory skin disease driven by the IL-23/IL-17 axis. It results from excessive activation of effector T cells, including T helper (Th) and cytotoxic T (Tc) cells, and is associated with dysfunctional regulatory T cells (Tregs). Acetyl-CoA carboxylase 1 (ACC1), a rate-limiting enzyme of fatty acid synthesis (FAS), directs cell fate decisions between Th17 and Tregs and thus could be a promising therapeutic target for psoriasis treatment. Here, we demonstrate that targeting ACC1 in T cells by genetic ablation ameliorates skin inflammation in an experimental model of psoriasis by limiting Th17, Tc17, Th1, and Tc1 cells in skin lesions and increasing the frequency of effector Tregs in skin-draining lymph nodes (LNs). KEY MESSAGES : ACC1 deficiency in T cells ameliorates psoriatic skin inflammation in mice. ACC1 deficiency in T cells reduces IL-17A-producing Th17/Tc17/dysfunctional Treg populations in psoriatic lesions. ACC1 deficiency in T cells restrains IFN-γ-producing Th1/Tc1 populations in psoriatic skin lesions and skin-draining LNs. ACC1 deficiency promotes activated CD44+CD25+ Tregs and effector CD62L-CD44+ Tregs under homeostasis and psoriatic conditions.

Regulatory T-cell deficiency leads to features of autoimmune liver disease overlap syndrome in scurfy mice.

Introduction: Scurfy mice have a complete deficiency of functional regulatory T cells (Treg) due to a frameshift mutation in the Foxp3 gene. The impaired immune homeostasis results in a lethal lymphoproliferative disorder affecting multiple organs, including the liver. The autoimmune pathology in scurfy mice is in part accompanied by autoantibodies such as antinuclear antibodies (ANA). ANA are serological hallmarks of several autoimmune disorders including autoimmune liver diseases (AILD). However, the underlying pathogenesis and the role of Treg in AILD remain to be elucidated. The present study therefore aimed to characterize the liver disease in scurfy mice. Methods: Sera from scurfy mice were screened for ANA by indirect immunofluorescence assay (IFA) and tested for a wide range of AILD-associated autoantibodies by enzyme-linked immunosorbent assay, line immunoassay, and addressable laser bead immunoassay. CD4+ T cells of scurfy mice were transferred into T cell-deficient B6/nude mice. Monoclonal autoantibodies from scurfy mice and recipient B6/nude mice were tested for ANA by IFA. Liver tissue of scurfy mice was analyzed by conventional histology. Collagen deposition in scurfy liver was quantified via hepatic hydroxyproline content. Real-time quantitative PCR was used to determine fibrosis-related hepatic gene expression. Hepatic immune cells were differentiated by flow cytometry. Results: All scurfy mice produced ANA. AILD-associated autoantibodies, predominantly antimitochondrial antibodies, were detected at significantly higher levels in scurfy sera. CD4+ T cells from scurfy mice were sufficient to induce anti-dsDNA autoantibodies and ANA with an AILD-related nuclear envelope staining pattern. Liver histology revealed portal inflammation with bile duct damage and proliferation, as in primary biliary cholangitis (PBC), and interface hepatitis with portal-parenchymal necroinflammation, as found in autoimmune hepatitis (AIH). In scurfy liver, TNFα and fibrosis-related transcripts including Col1a1, Timp1, Acta2, Mmp2, and Mmp9 were upregulated. The level of proinflammatory monocytic macrophages (Ly-6Chi) was increased, while M2-type macrophages (CD206+) were downregulated compared to wildtype controls. Despite severe hepatic inflammation, fibrosis did not develop within 25 days, which is close to the lifespan of scurfy mice. Discussion: Our findings suggest that Treg-deficient scurfy mice spontaneously develop clinical, serological, and immunopathological characteristics of AILD with overlapping features of PBC and AIH.

Commensal bacteria weaken the intestinal barrier by suppressing epithelial neuropilin-1 and Hedgehog signaling.

The gut microbiota influences intestinal barrier integrity through mechanisms that are incompletely understood. Here we show that the commensal microbiota weakens the intestinal barrier by suppressing epithelial neuropilin-1 (NRP1) and Hedgehog (Hh) signaling. Microbial colonization of germ-free mice dampens signaling of the intestinal Hh pathway through epithelial Toll-like receptor (TLR)-2, resulting in decreased epithelial NRP1 protein levels. Following activation via TLR2/TLR6, epithelial NRP1, a positive-feedback regulator of Hh signaling, is lysosomally degraded. Conversely, elevated epithelial NRP1 levels in germ-free mice are associated with a strengthened gut barrier. Functionally, intestinal epithelial cell-specific Nrp1 deficiency (Nrp1ΔIEC) results in decreased Hh pathway activity and a weakened gut barrier. In addition, Nrp1ΔIEC mice have a reduced density of capillary networks in their small intestinal villus structures. Collectively, our results reveal a role for the commensal microbiota and epithelial NRP1 signaling in the regulation of intestinal barrier function through postnatal control of Hh signaling.

The Dendritic Cell Dilemma in the Skin: Between Tolerance and Immunity.

Dendritic cells (DC) are uniquely capable of initiating and directing immune responses. The range of their activities grounds in the heterogeneity of DC subsets and their functional plasticity. Numerical and functional DC changes influence the development and progression of disease, and correction of such dysregulations has the potential to treat disease causally. In this review, we discuss the major advances in our understanding of the regulation of DC lineage formation, differentiation, and function in the skin. We describe the alteration of DC in disease as well as possibilities for therapeutic reprogramming with a focus on tolerogenic DC. Because regulatory T cells (Treg) are indispensable partners of DC in the induction and control of tolerance, we pay special attention to the interactions with these cells. Above all, we would like to arouse fascination for this cell type and its therapeutic potential in skin diseases.

Protease- and cell type-specific activation of protease-activated receptor 2 in cutaneous inflammation.

BACKGROUND: Protease-activated receptor 2 (PAR2) signaling controls skin barrier function and inflammation, but the roles of immune cells and PAR2-activating proteases in cutaneous diseases are poorly understood. OBJECTIVE: To dissect PAR2 signaling contributions to skin inflammation with new genetic and pharmacological tools. METHODS/RESULTS: We found markedly increased numbers of PAR2+ infiltrating myeloid cells in skin lesions of allergic contact dermatitis (ACD) patients and in the skin of contact hypersensitivity (CHS) in mice, a murine ACD model for T cell-mediated allergic skin inflammation. Cell type-specific deletion of PAR2 in myeloid immune cells as well as mutation-induced complete PAR2 cleavage insensitivity significantly reduced skin inflammation and hapten-specific Tc1/Th1 cell response. Pharmacological approaches identified individual proteases involved in PAR2 cleavage and demonstrated a pivotal role of tissue factor (TF) and coagulation factor Xa (FXa) as upstream activators of PAR2 in both the induction and effector phase of CHS. PAR2 mutant mouse strains with differential cleavage sensitivity for FXa versus skin epithelial cell-expressed proteases furthermore uncovered a time-dependent regulation of CHS development with an important function of FXa-induced PAR2 activation during the late phase of skin inflammation. CONCLUSIONS: Myeloid cells and the TF-FXa-PAR2 axis are key mediators and potential therapeutic targets in inflammatory skin diseases.

Therapeutic melanoma inhibition by local micelle-mediated cyclic nucleotide repression.

The acidic tumor microenvironment in melanoma drives immune evasion by up-regulating cyclic adenosine monophosphate (cAMP) in tumor-infiltrating monocytes. Here we show that the release of non-toxic concentrations of an adenylate cyclase (AC) inhibitor from poly(sarcosine)-block-poly(L-glutamic acid γ-benzyl ester) (polypept(o)id) copolymer micelles restores antitumor immunity. In combination with selective, non-therapeutic regulatory T cell depletion, AC inhibitor micelles achieve a complete remission of established B16-F10-OVA tumors. Single-cell sequencing of melanoma-infiltrating immune cells shows that AC inhibitor micelles reduce the number of anti-inflammatory myeloid cells and checkpoint receptor expression on T cells. AC inhibitor micelles thus represent an immunotherapeutic measure to counteract melanoma immune escape.

Targeted Activation of T Cells with IL-2-Coupled Nanoparticles.

Interleukin-2 (IL-2) is a T cell growth factor particularly required in regulatory T cell maintenance and memory T cell responses. High-dose IL-2 treatment was the first FDA-approved immunotherapy for cancer, while low-dose IL-2 administration has shown promise in allograft rejection and autoimmune and inflammatory diseases. However, its pleiotropic nature and the existence of IL-2 receptors with different binding affinity limit its therapeutic application. For an improved clinical applicability of the cytokine, a targeted receptor assignment must, therefore, be achieved. Nanoparticles allow controlling the location and dose of immunomodulating compounds and to specifically address specific receptors through targeted drug binding. In this review article we discuss the IL-2 biology and current clinical application with regard to nanoparticle-based IL-2-mediated manipulation of T cell responses in autoimmunity, chronic inflammation, and cancer.