Regulation of Cutaneous Immunity In Vivo by Calcitonin Gene-Related Peptide Signaling through Endothelial Cells.

Calcitonin gene-related peptide (CGRP) can bias the outcome of Ag presentation to responsive T cells in vitro away from Th1-type immunity and toward the Th2 and Th17 poles through actions on endothelial cells (ECs). To test the in vivo significance of this observation, we engineered a mouse lacking functional CGRP receptors on ECs (EC receptor activity modifying protein 1 [RAMP1] knockout mice). On percutaneous immunization to 1-fluoro-2,4-dinitrobenzene, stimulated CD4+ T cells from draining lymph nodes showed significantly reduced IL-17A expression with significantly increased IFN-γ, IL-4, and IL-22 expression at the protein and mRNA levels compared with control mice. Retinoic acid receptor-related orphan receptor γ t mRNA was significantly reduced, while mRNAs for T-box expressed in T cells and GATA binding protein 3 were significantly increased. In addition, EC RAMP1 knockout mice had significantly reduced contact hypersensitivity responses, and systemic administration of a CGRP receptor antagonist similarly inhibited contact hypersensitivity in wild-type mice. These observations provide compelling evidence that CGRP is a key regulator of cutaneous immunity through effects on ECs and suggest a novel pathway for potential therapeutic manipulation.

Oral administration of whole dihomo-γ-linolenic acid-producing yeast suppresses allergic contact dermatitis in mice.

Dihomo-γ-linolenic acid (DGLA, C20: 3n-6) is known to have an anti-inflammatory activity, but its range of effects was not well studied because of its limited natural sources. We addressed these issues by constructing an yeast Saccharomyces cerevisiae strain having a complete metabolic pathway for DGLA synthesis by introducing two desaturase and one elongase genes to convert endogenous oleic acid to DGLA. Taking advantage of well-known safety of S. cerevisiae, we previously investigated the efficacy of heat-killed whole DGLA-producing yeast cells on irritant contact dermatitis, and showed that oral intake of this yeast significantly suppressed inflammatory reactions, whereas no such suppression was observed by the intake of 25 times the amount of purified DGLA. Since this method is considered to be a simple and efficient way to suppress inflammation, we examined its effectiveness against allergic contact dermatitis (ACD) in this study and showed that this method was also effective against ACD.

Xanthone suppresses allergic contact dermatitis in vitro and in vivo.

Xanthone is a phenolic compound found in a few higher plant families; it has a variety of biological activities, including antioxidant, anti-inflammatory, and anticancer properties. However, the molecular and cellular mechanisms underlying the activity of xanthone in allergic contact dermatitis (ACD) remain to be explored. Therefore, this study aimed to investigate the regulatory effects of xanthone in ACD in human keratinocytes (HaCaT cell), and human mast cell line (HMC-1 cell) in vitro and in an experimental murine model. The results demonstrated that treatment with xanthone reduced the production of pro-inflammatory cytokines and chemokines including interleukin (IL)-1ß, IL-6, IL-8, and expression of chemokines thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC) in tumor necrosis factor (TNF)-α and interferon (IFN)-γ-stimulated HaCaT cells. Xanthone also suppressed the production of pro-inflammatory cytokines, chemokines, and allergic mediators in phorbol myristate acetate/A23187 calcium ionophore (PMACI)-stimulated HMC-1 cells. Xanthone significantly suppressed the phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) and activation of caspase-1 signaling pathway in vitro model. Additionally, xanthone administration alleviated 2,4-dinitrofluorobenzene (DNFB)-induced atopic dermatitis like-skin lesion by reducing the serum levels of immunoglobulin E (IgE), histamine, and pro-inflammatory cytokines and suppressing MAPKs phosphorylation. Xanthone administration also inhibited mortality due to compound 48/80-induced anaphylactic shock and suppressed the passive cutaneous anaphylaxis (PCA) reaction mediated by IgE. Collectively, these results suggest that xanthone has a potential for use in the treatment of allergic inflammatory diseases.

Successful development of methodology for detection of hapten-specific contact hypersensitivity (CHS) memory in swine.

Hapten contact hypersensitivity (CHS) elicits a well-documented inflammation response that can be used to illustrate training of immune cells through hapten-specific CHS memory. The education of hapten-specific memory T cells has been well-established, recent research in mice has expanded the "adaptive" characteristic of a memory response from solely a function of the adaptive immune system, to innate cells as well. To test whether similar responses are seen in a non-rodent model, we used hapten-specific CHS to measure the ear inflammation response of outbred pigs to dinitrofluorobenzene (DNFB), oxazolone (OXA), or vehicle controls. We adapted mouse innate memory literature protocols to the domestic pig model. Animals were challenged up to 32 days post initial sensitization exposure to the hapten, and specific ear swelling responses to this challenge were significant for 7, 21, and 32 days post-sensitization. We established hapten-specific CHS memory exists in a non-rodent model. We also developed a successful protocol for demonstrating these CHS responses in a porcine system.

1-Fluoro-2,4-dinitrobenzene and its derivatives act as secretagogues on rodent mast cells.

Accumulating evidence suggests that activated mast cells are involved in contact hypersensitivity, although the precise mechanisms of their activation are still not completely understood. We investigated the potential of common experimental allergens to induce mast cell activation using murine bone marrow-derived cultured mast cells and rat peritoneal mast cells. Among these allergens, 1-chloro-2,4-dinitrobenzene and 1-fluoro-2,4-dinirobenzene (DNFB) were found to induce degranulation of rat peritoneal mast cells. DNFB-induced degranulation is accompanied by cytosolic Ca2+ mobilization and is significantly inhibited by pertussis toxin, U73122 (a phospholipase C inhibitor), and BAPTA (a Ca2+ chelator), raising the possibility that DNFB acts on the G protein-coupled receptors and activates Gi , which induces activation of phospholipase C, as well as known mast cell secretagogues, such as compound 48/80. DNFB could induce mast cell degranulation in the absence of serum proteins and IgE. Structure-activity relationship analyses revealed an inverse correlation between the degree of degranulation and the electron density of the C1 carbon of the DNFB derivatives. These findings raise a possibility that DNFB functions as a potent contact allergen through induction of cutaneous mast cell degranulation.

Development of interleukin-17-producing Vγ2+ γδ T cells is reduced by ICOS signaling in the thymus.

Co-stimulation is an integral part of T cell signaling involved in almost all facets of T cell biology. While much is known about co-stimulation in differentiation and function of conventional αß T cells, less is known about how co-stimulation affects the development and programming of γδ T cells. In this study, we have investigated the role of inducible T cell co-stimulator (ICOS) on the development of γδ T cells. We show that ICOS is expressed by a population of immature Vγ2+CD45RBlow γδ T cells predisposed to interleukin-17 (IL-17) production. We found that treatment with ICOS specific antibodies drastically reduces fetal development of IL-17-producing γδ T cells by agonistic actions, and that ICOS deficient mice have a significant increase in the population of IL-17-producing Vγ2+ γδ T cells in the thymus, spleen, lymph nodes and skin and exhibit exacerbated sensitization responses to 2,4-dinitrofluorobenzene. In conclusion, this study demonstrates that development of IL-17-producing Vγ2+ γδ T cells is reduced by ICOS signaling in the thymus.

Depletion of regulatory T cells in a hapten-induced inflammation model results in prolonged and increased inflammation driven by T cells.

Regulatory T cells (Tregs ) are known to play an immunosuppressive role in the response of contact hypersensitivity (CHS), but neither the dynamics of Tregs during the CHS response nor the exaggerated inflammatory response after depletion of Tregs has been characterized in detail. In this study we show that the number of Tregs in the challenged tissue peak at the same time as the ear-swelling reaches its maximum on day 1 after challenge, whereas the number of Tregs in the draining lymph nodes peaks at day 2. As expected, depletion of Tregs by injection of a monoclonal antibody to CD25 prior to sensitization led to a prolonged and sustained inflammatory response which was dependent upon CD8 T cells, and co-stimulatory blockade with cytotoxic T lymphocyte antigen-4-immunoglobulin (CTLA-4-Ig) suppressed the exaggerated inflammation. In contrast, blockade of the interleukin (IL)-10-receptor (IL-10R) did not further increase the exaggerated inflammatory response in the Treg -depleted mice. In the absence of Tregs , the response changed from a mainly acute reaction with heavy infiltration of neutrophils to a sustained response with more chronic characteristics (fewer neutrophils and dominated by macrophages). Furthermore, depletion of Tregs enhanced the release of cytokines and chemokines locally in the inflamed ear and augmented serum levels of the systemic inflammatory mediators serum amyloid (SAP) and haptoglobin early in the response.

IL-1ß-dependent activation of dendritic epidermal T cells in contact hypersensitivity.

Substances that penetrate the skin surface can act as allergens and induce a T cell-mediated inflammatory skin disease called contact hypersensitivity (CHS). IL-17 is a key cytokine in CHS and was originally thought to be produced solely by CD4(+) T cells. However, it is now known that several cell types, including γδ T cells, can produce IL-17. In this study, we determine the role of γδ T cells, especially dendritic epidermal T cells (DETCs), in CHS. Using a well-established model for CHS in which 2,4-dinitrofluorobenzene (DNFB) is used as allergen, we found that γδ T cells are important players in CHS. Thus, more IL-17-producing DETCs appear in the skin following exposure to DNFB in wild-type mice, and DNFB-induced ear swelling is reduced by ∼50% in TCRδ(-/-) mice compared with wild-type mice. In accordance, DNFB-induced ear swelling was reduced by ∼50% in IL-17(-/-) mice. We show that DNFB triggers DETC activation and IL-1ß production in the skin and that keratinocytes produce IL-1ß when stimulated with DNFB. We find that DETCs activated in vitro by incubation with anti-CD3 and IL-1ß produce IL-17. Importantly, we demonstrate that the IL-1R antagonist anakinra significantly reduces CHS responses, as measured by decreased ear swelling, inhibition of local DETC activation, and a reduction in the number of IL-17(+) γδ T cells and DETCs in the draining lymph nodes. Taken together, we show that DETCs become activated and produce IL-17 in an IL-1ß-dependent manner during CHS, suggesting a key role for DETCs in CHS.

Antihelminthic niclosamide modulates dendritic cells activation and function.

Dendritic cells (DCs) link the sensing of the environment by the innate immune system to the initiation of adaptive immune responses. Accordingly, DCs are considered to be a major target in the development of immunomodulating compounds. In this study, the effect of niclosamide, a Food and Drug Administration-approved antihelminthic drug, on the activation of lipopolysaccharide (LPS)-stimulated murine bone marrow-derived DCs was examined. Our experimental results show that niclosamide reduced the pro-inflammatory cytokine and chemokine expression of LPS-activated DCs. In addition, niclosamide also affected the expression of MHC and costimulatory molecules and influenced the ability of the cells to take up antigens. Therefore, in mixed cell cultures composed of syngeneic OVA-specific T cells and DCs, niclosamide-treated DCs showed a decreased ability to stimulate T cell proliferation and IFN-γ production. Furthermore, intravenous injection of niclosamide also attenuated contact hypersensitivity (CHS) in mice during sensitization with 2,4-dinitro-1-fluorobenzene. Blocking the LPS-induced activation of MAPK-ERK, JNK and NF-κB may contribute to the inhibitory effect of niclosamide on DC activation. Collectively, our findings suggest that niclosamide can manipulate the function of DCs. These results provide new insight into the immunopharmacological role of niclosamide and suggest that it may be useful for the treatment of chronic inflammatory disorders or DC-mediated autoimmune diseases.

Development of LLNA:DAE: a new local lymph node assay that includes the elicitation phase, discriminates borderline-positive chemicals, and is useful for cross-sensitization testing.

We developed a new local lymph node assay (LLNA) that includes the elicitation phase termed LLNA:DAE for discrimination of borderline-positive chemicals as classified by the LLNA modified by Daicel based on ATP content (LLNA:DA) and for cross-sensitization testing. Although the LLNA:DA method could help identify skin sensitizers, some skin irritants classified as non-sensitizers by the LLNA were classified as borderline positive. In addition, the evaluation for the cross-sensitization potential between chemicals was impossible. In the LLNA:DAE procedure, test group of mice received four applications of chemicals on the dorsum of the right ear for induction and one application on the dorsum of the left ear for elicitation. Control group of mice received one chemical application on the dorsum of the left ear. We evaluated the sensitizing potential by comparing the weights of the lymph nodes from the left ears between the two groups. The results of using the LLNA:DAE method to examine 24 chemicals, which contained borderline-positive chemicals, were consistent with those from the LLNA method, except for nickel chloride (NiCl2). Two chemical pairs, 2,4-dinitrochlorobenzene (DNCB) with 2,4-dinitrofluorobenzene (DNFB) and hydroquinone (HQ) with p-benzoquinone (p-BQ), showed clear cross-sensitization with each other, while another chemical pair, DNFB with hexylcinnamic aldehyde (HCA) did not. Taken together, our results suggest that the LLNA:DAE method is useful for discriminating borderline-positive chemicals and for determining chemical cross-sensitization.

microRNA miR-17-92 cluster is highly expressed in epidermal Langerhans cells but not required for its development.

Langerhans cells (LCs) are bone marrow-derived immature skin-residential dendritic cells (DCs) with a life cycle distinct from that of other types of DCs. The mechanisms involved in LC homeostasis and immunological functions are still not clear. MicroRNAs (miRNAs) are a class of short noncoding RNAs that regulate gene expression through either translational repression or mRNA degradation. A recent study showed that specific deletion of total miRNAs in DCs affects the homeostasis and function of only LCs, but not of other types of DCs. The roles of specific individual miRNA in LC development are still lacking. The miRNA miR-17-92 class, encoding miR-17, miR-18, miR-19a, miR-19b, miR-20 and miR-92, plays a very important role in B- and T-cell development and function. Here, we first report that epidermal LCs highly express the miR-17-92 class compared with spleen naive T cells. To further characterize the role of miR-17-92 in LC development, we generated LC-specific miR-17-92 knockout and knock-in mice. Interestingly, LC-specific gain- and loss-of-function of miR-17-92 cluster did not significantly change LC homeostasis, maturation ability, antigen capture and migration to draining lymph nodes. Thus, the miR-17-92 cluster may be functionally redundant and not critically required for LC development and function.

Antagonistic effect of the inflammasome on thymic stromal lymphopoietin expression in the skin.

BACKGROUND: Innate immune sensors control key cytokines that regulate T-cell priming and T-cell fate. This is particularly evident in allergic reactions, which represent ideal systems to study the interplay of innate and adaptive immunity. In patients with contact dermatitis, inflammasome-mediated IL-1 activation is responsible for a TH1 immune response. Surprisingly, the IL-1 signaling pathway was also proposed to control the activation of thymic stromal lymphopoietin (TSLP), a cytokine implicated in development of the T(H)2 response in patients with atopic dermatitis (AD) and asthma. OBJECTIVES: We sought to assess the effect of the inflammasome on TSLP expression levels and the development of AD. METHODS: We studied the effect of the inflammasome activator 2,4-dinitrofluorobenzene, and IL-1ß on TSLP mRNA expression levels in mouse and human cell lines (in vitro assays), as well as in live mice and on human skin transplants. We also assessed the effect of 2,4-dinitrofluorobenzene on TSLP and the TH2 response in mice in which the inflammasome and IL-1 signaling pathways were blocked, either genetically or pharmacologically, in 2 models of AD. RESULTS: We provide in vitro and in vivo evidence that inflammasome activation has an inhibitory role on TSLP mRNA expression and T(H)2 cell fate in the skin. We also show that solvents influence the activation of TSLP and IL-1ß and direct the T-cell fate to a given hapten. CONCLUSION: Our observations strongly suggest that the TH1 versus TH2 cell fate decision is regulated at multiple levels and starts with innate immune events occurring within peripheral epithelial tissues.

CXCR3 deficiency prolongs Th1-type contact hypersensitivity.

Sensitization and challenge using dinitrofluorobenzene (DNFB) induce contact hypersensitivity (CHS) with Th1 cell infiltration, whereas those using FITC generate CHS with Th2 cell infiltration. In this study, we attempted to determine the role of CXCR3, a chemokine receptor, in Th1- and Th2-type CHS induced by DNFB or FITC using CXCR3-deficient (CXCR3(-/-)) mice. Ear swelling was prolonged after DNFB challenge in CXCR3(-/-) mice, which was accompanied by increased Th1 cytokines and decreased TGF-ß and IL-10 expression at a late time point of CHS, whereas there was no significant difference between wild-type and CXCR3(-/-) mice in FITC-induced CHS. In Th1-type CHS, the number of regulatory T cells (Tregs) was decreased in the challenged ear of CXCR3(-/-) mice compared with that of wild-type mice, suggesting that CXCR3 would be important in migration of Tregs into the site of inflammation. Moreover, we examined the characteristics of CXCR3(+) Tregs both in vitro and in vivo, revealing that CXCR3(+) Tregs expressed high levels of TGF-ß and IL-10 as well as IFN-γ compared with CXCR3(-) Tregs. When CXCR3(-/-) mice were injected with CXCR3(+) Tregs, the prolonged ear swelling induced by DNFB was normalized. Taken together, our results suggest that CXCR3(+) Tregs play a key role for quenching Th1-type CHS.

Local and systemic effects of co-stimulatory blockade using cytotoxic T lymphocyte antigen-4-immunoglobulin in dinitrofluorobenzene- and oxazolone-induced contact hypersensitivity in mice.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4)-immunoglobulin (Ig) has immunosuppressive properties both in vivo and in vitro, but much is still unknown about the mechanisms by which CTLA-4-Ig exerts its immunosuppressive activities in vivo. The aim of this study was to investigate the effect of CTLA-4-Ig in a mouse model of contact hypersensitivity (CHS). The inflammatory response in the presence or absence of CTLA-4-Ig was evaluated by measuring the increase in ear thickness in sensitized animals after challenge. We observed a dose-dependent suppression of the ear swelling in both dinitrofluorobenzene (DNFB)- and oxazolone-induced CHS. The suppressive effect was still present 3 weeks after administration, even in the absence of circulating levels of CTLA-4-Ig. It was further shown that CTLA-4-Ig inhibits activation of T cells in the draining lymph node after sensitization and affects the maturation level of both dendritic cells and B cells. Furthermore, CTLA-4-Ig reduces infiltration of activated CD8(+) T cells into the inflamed ear tissue and suppresses both local and systemic inflammation, as illustrated by reduced expression of cytokines and chemokines in the inflamed ear and a reduced level of acute-phase proteins in circulation. Finally, our results suggest that CTLA-4-Ig has a mainly immunosuppressive effect during the sensitization phase. We conclude that CTLA-4-Ig induces long-term immunosuppression of both DNFB- and oxazolone-induced inflammation and our data are the first to compare the effect of this compound in both DNFB- and oxazolone-induced CHS and to show that CTLA-4-Ig exerts an immunosuppressive effect on both local and systemic inflammatory mediators which is mediated principally during the sensitization phase.

Complementary methods for contact hypersensitivity (CHS) evaluation in mice.

Contact hypersensitivity (CHS) is an experimental model of allergic contact dermatitis (ACD) that can be studied in mice. For CHS responses, mice are immunized by painting with a reactive hapten, such as 1-fluoro-4,6-dinitrobenzene (DNFB), on the shaved abdominal and chest skin. Subsequently, the ears are challenged with diluted hapten, eliciting 'hypersensitive' ear-swelling reactions, which can be measured with a micrometer. In this manuscript we present complementary methods that can be used to evaluate CHS in mice that include: ear weight, vascular permeability, myeloperoxidase (MPO) activity, IFN-γ concentration in ear extracts and also IFN-γ production by auricular lymph node cells (ELNC). The biochemical evaluation of CHS can be also supported by proliferation assay, measurement of IFN-γ production by skin-draining lymph node cells employing ELISA test and by evaluation of IFN-γ(+) TCRαß CD8 cells with the use of flow cytometry.

Neutrophil expression of Fas ligand and perforin directs effector CD8 T cell infiltration into antigen-challenged skin.

Contact hypersensitivity (CHS) is a T cell response to hapten skin challenge of sensitized individuals proposed to be mediated by hapten-primed CD8 cytolytic T cells. Effector CD8 T cell recruitment into hapten challenge sites to elicit CHS requires prior CXCL1- and CXCL2-mediated neutrophil infiltration into the site. We investigated whether neutrophil activities directing hapten-primed CD8 T cell skin infiltration in response to 2,4-dinitro-1-fluorobenzene (DNFB) required Fas ligand (FasL) and perforin expression. Although DNFB sensitization of gld/perforin-/- mice induced hapten-specific CD8 T cells producing IFN-γ and IL-17, these T cells did not infiltrate the DNFB challenge site to elicit CHS but did infiltrate the challenge site and elicit CHS when transferred to hapten-challenged naive wild-type recipients. Hapten-primed wild-type CD8 T cells, however, did not elicit CHS when transferred to naive gld/perforin-/- recipients. Wild-type bone marrow neutrophils expressed FasL and perforin, and when transferred to sensitized gld/perforin-/- mice, they restored hapten-primed CD8 T cell infiltration into the challenge site and CHS. The FasL/perforin-mediated activity of wild-type neutrophils induced the expression of T cell chemoattractants, CCL1, CCL2, and CCL5, within the hapten-challenged skin. These results indicate FasL/perforin-independent functions of hapten-primed CD8 T cells in CHS and identify new functions for neutrophils in regulating effector CD8 T cell recruitment and immune responses in the skin.

Histone deacetylase 3 mediates allergic skin inflammation by regulating expression of MCP1 protein.

We have shown the induction of histone deacetylase 3 (HDAC3) in antigen-stimulated rat basophilic leukemia cells via NF-κB. We investigated the role of HDAC3 in allergic skin inflammation. We used a BALB/c mouse model of triphasic cutaneous anaphylaxis (triphasic cutaneous reaction; TpCR) and passive cutaneous anaphylaxis (PCA) to examine the role of HDAC3 in allergic skin inflammation. Triphasic cutaneous reaction involved induction of HDAC3 and was mediated by HDAC3. HDAC3 showed an interaction with FcεRIß. Trichostatin A (TSA), an inhibitor of HDAC(s), disrupted this interaction. Cytokine array analysis showed that the down-regulation of HDAC3 led to the decreased secretion of monocyte chemoattractant protein 1 (MCP1). FcεRI was necessary for induction of HDAC3 and MCP1. ChIP assays showed that HDAC3, in association with Sp1 and c-Jun, was responsible for induction of MCP1 expression. TSA exerted a negative effect on induction of MCP1. HDAC3 exerted a negative regulation on expression of HDAC2 via interaction with Rac1. The down-regulation of HDAC3 or inactivation of Rac1 induced binding of HDAC2 to MCP1 promoter sequences. TSA exerted a negative effect on HDAC3-mediated TpCR. The BALB/c mouse model of PCA involved induction of HDAC3 and MCP1. HDAC3 and MCP1 were necessary for PCA that involved ear swelling, enhanced vascular permeability, and angiogenesis. Recombinant MCP1 enhanced ß-hexosaminidase activity and histamine release and also showed angiogenic potential. TSA exerted a negative effect on PCA. Our data show HDAC3 as a valuable target for the development of allergic skin inflammation therapeutics.

Topical application of Pseudolaric acid B improve DNFB-induced contact hypersensitivity via regulating the balance of Th1/Th17/Treg cell subsets.

Pseudolaric acid B (PAB) is a novel diterpenoid, isolated from Pseudolarix kaempferi Gorden, which roots are widely used to treat inflammatory and microbial skin diseases for centuries, but the underlying mechanism remains elusive. To address the immunoregulatory mechanisms of PAB, we first investigated the effect of PAB in 2,4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity (CHS) mice. The expression of specific transcription factors for T-helper (Th)1, Th2, Th17 and regulatory T cells (Tregs) was determined by RT-PCR, and the capacities for PAB to modulate Th1/Th17/Treg cell subsets was furtherly analyzed by flow cytometric analysis, ELISA and Western blotting assay. The results showed that topical application of PAB could suppress ear swelling, block inflammatory infiltration, and interfere in Th1 response. Furthermore, PAB-treated CHS mice exhibited the impaired Th17 development and enhanced Tregs generation, associated with the up-regulation of peroxisome proliferator activated-receptor γ (PPAR γ) expression. These findings suggest that PAB exerts its immunoregulatory activity through regulating the balance of Th1/Th17/Treg cell subsets, which would provide a novel therapeutic application for PAB on CHS and other inflammatory-associated diseases.