Triggering the succinate receptor GPR91 on dendritic cells enhances immunity.

Succinate acts as an extracellular mediator signaling through the G protein-coupled receptor GPR91. Here we show that dendritic cells had high expression of GPR91. In these cells, succinate triggered intracellular calcium mobilization, induced migratory responses and acted in synergy with Toll-like receptor ligands for the production of proinflammatory cytokines. Succinate also enhanced antigen-specific activation of human and mouse helper T cells. GPR91-deficient mice had less migration of Langerhans cells to draining lymph nodes and impaired tetanus toxoid-specific recall T cell responses. Furthermore, GPR91-deficient allografts elicited weaker transplant rejection than did the corresponding grafts from wild-type mice. Our results suggest that the succinate receptor GPR91 is involved in sensing immunological danger, which establishes a link between immunity and a metabolite of cellular respiration.

CCR7 is required for the in vivo function of CD4+ CD25+ regulatory T cells.

CCR7-mediated migration of naive T cells into the secondary lymphoid organs is a prerequisite for their encounter with mature dendritic cells, the productive presentation of cognate antigen, and consequent T cell proliferation and effector differentiation. Therefore, CCR7 was suggested to play an important role in the initiation of adaptive immune responses. In this study, we show that primary immunity can also develop in the absence of CCR7. Moreover, CCR7-deficient knockout (KO) mice display augmented immune responses. Our data cumulatively suggest that enhanced immunity in CCR7 KO mice is caused by the defective lymph node (LN) positioning of FoxP3(+) CD4(+) CD25(+) regulatory T cells (T reg cells) and the consequent impediment of their function. The FoxP3(+) T reg cells express CCR7 and, after their adoptive transfer, migrate into the LNs of wild-type mice. Here, they proliferate in situ upon antigen stimulation and inhibit the generation of antigen-specific T cells. Conversely, transferred CCR7-deficient T reg cells fail to migrate into the LNs and suppress antigen-induced T cell responses. The transfer of combinations of naive and T reg cells from wild-type and CCR7 KO mice into syngeneic severe combined immunodeficient mice directly demonstrates that CCR7-deficient T reg cells are less effective than their wild-type counterparts in preventing the development of inflammatory bowel disease.

The PKC inhibitor AEB071 may be a therapeutic option for psoriasis.

PKC isoforms tau, alpha, and beta play fundamental roles in the activation of T cells and other immune cell functions. Here we show that the PKC inhibitor AEB071 both abolishes the production of several cytokines by activated human T cells, keratinocytes, and macrophages in vitro and inhibits an acute allergic contact dermatitis response in rats. To translate these findings into humans, single and multiple ascending oral doses of AEB071 were administered to healthy volunteers and patients with psoriasis, respectively. AEB071 was well tolerated with no clinically relevant laboratory abnormalities. Ex vivo stimulation of lymphocytes from subjects exposed to single doses of AEB071 resulted in a dose-dependent inhibition of both lymphocyte proliferation and IL2 mRNA expression. Clinical severity of psoriasis was reduced up to 69% compared with baseline after 2 weeks of treatment, as measured by the Psoriasis Area Severity Index (PASI) score. The improvement in psoriasis patients was accompanied by histological improvement of skin lesions and may be partially explained by a substantial reduction of p40+ dermal cells, which are known to mediate psoriasis. These data suggest that AEB071 could be an effective novel treatment regimen for psoriasis and other autoimmune diseases, and that AEB071 warrants long-term studies to establish safety and efficacy.

Inhibition of chronic and acute skin inflammation by treatment with a vascular endothelial growth factor receptor tyrosine kinase inhibitor.

Although vascular remodeling is a hallmark of many chronic inflammatory disorders, antivascular strategies to treat these conditions have received little attention to date. We investigated the effects of a newly identified vascular endothelial growth factor (VEGF) receptor tyrosine-kinase inhibitor, NVP-BAW2881, on endothelial cell function in vitro and its anti-inflammatory activity in different animal models. NVP-BAW2881 inhibited proliferation, migration, and tube formation by human umbilical vein endothelial cells and lymphatic endothelial cells in vitro. In a transgenic mouse model of psoriasis, NVP-BAW2881 reduced the number of blood and lymphatic vessels and infiltrating leukocytes in the skin, and normalized the epidermal architecture. NVP-BAW2881 also displayed strong anti-inflammatory effects in models of acute inflammation; pretreatment with topical NVP-BAW2881 significantly inhibited VEGF-A-induced vascular permeability in the skin of pigs and mice. Furthermore, topical application of NVP-BAW2881 reduced the inflammatory response elicited in pig skin by UV-B irradiation or by contact hypersensitivity reactions. These results demonstrate for the first time that VEGF receptor tyrosine-kinase inhibitors might be used to treat patients with inflammatory skin disorders such as psoriasis.

Signal transducer and activator of transcription 1 decoy oligodeoxynucleotide suppression of contact hypersensitivity.

BACKGROUND: Cytokines play a pivotal role in allergy development through activating signaling mechanisms, such as the Janus kinase/signal transducer and activator of transcription (STAT) pathway, which controls the expression of numerous proinflammatory genes. OBJECTIVE: In comparison with 2 different corticosteroids and a calcineurin inhibitor, the efficacy of a STAT1 decoy oligodeoxynucleotide (dODN)-containing ointment on hapten-induced contact hypersensitivity was examined in 3 different animal models. METHODS: After sensitization, the test compounds were administered before hapten challenge, after hapten challenge, or both to different sites of the animal skin. Subsequent erythema and edema formation was scored macroscopically, microscopically, or by a shift in ear weight. Biopsy specimens were taken and processed for histopathology, immunohistochemistry, and real-time PCR analyses. RESULTS: Treatment with the STAT1 dODN but not the corresponding control ODN markedly improved the clinical signs of inflammation in all 3 animal models in a dose-related manner. In guinea pig skin this was accompanied by a distinct decrease in leukocyte infiltration into the dermis after 24 hours. In addition, expression of CD40, IFN-gamma, IL-1beta, IL-8, IL-12, and TNF-alpha was strongly attenuated. The dODN was equally effective in the domestic pig model when administered therapeutically, and its preventive effect in the mouse model lasted for more than 48 hours. CONCLUSIONS: Altogether, treatment with the dODN proved to be at least as effective as treatment with the reference compounds.

Pimecrolimus inhibits the elicitation phase but does not suppress the sensitization phase in murine contact hypersensitivity, in contrast to tacrolimus and cyclosporine A.

Pimecrolimus (SDZ ASM 981, Elidel) is a nonsteroid inflammatory cytokine inhibitor specifically developed for the treatment of inflammatory skin diseases. Its effect on the elicitation and sensitization phases of oxazolone-induced contact hypersensitivity was compared with tacrolimus and cyclosporine A (CyA) in BALB/c mice using the ear swelling assay. The compounds were administered orally. Elicitation was dose-dependently inhibited by all three compounds. The minimal effective doses were 30 mg per kg (pimecrolimus, tacrolimus) and 90 mg per kg (CyA), respectively. There was no impairment of sensitization by pimecrolimus up to the highest dose tested (120 mg per kg), in contrast to CyA (60% inhibition at 60 mg per kg) and tacrolimus (71% inhibition at 30 mg per kg). Weight and cellularity of the draining lymph nodes in mice treated with tacrolimus or CyA during sensitization were reduced. In addition, proliferation of T cells after secondary stimulation was inhibited in cell cultures from lymph nodes of mice treated with tacrolimus or CyA. Thus, in contrast to tacrolimus and CyA, pimecrolimus exerts a more selective immunomodulatory effect. It does not impair the primary immune response (sensitization phase) but effectively inhibits the secondary phase, the elicitation phase that is the clinical manifestation of contact hypersensitivity.

Corticosteroids but not pimecrolimus affect viability, maturation and immune function of murine epidermal Langerhans cells.

Given the importance of dendritic cells in the immune response, we investigated the effect of corticosteroids (CS) on the integrity, survival, and function of murine Langerhans cells (LC) in comparison with pimecrolimus, a novel anti-inflammatory drug for the topical treatment of atopic dermatitis. BALB/c mice were treated twice on one day with ethanolic solutions of the compounds. At 24-72 h after the last application, we observed fragmented DNA, caspase-3 activity, and an upregulation of CD95 expression in LC from mice treated with CS but not in LC of pimecrolimus- or vehicle-treated animals. CS-epidermal cell (EC) supernatants but not pimecrolimus-EC supernatants contained significantly lower amounts of soluble factors (GM-CSF, TNF-alpha, IL-1alpha) required for LC survival and maturation than EC supernatants from vehicle-treated mice. With regard to LC maturation, CS but not pimecrolimus inhibited the expression of CD25, CD205, and costimulatory molecules. In line with this, LC from pimecrolimus-treated mice were similar to LC from vehicle-treated mice in their capacity to stimulate antigen-presenting function and migration, whereas LC from CS-treated mice were greatly impaired in these abilities. In summary, our data show for the first time that CS but not pimecrolimus induce apoptosis in LC in situ, implying that the prolonged use of CS could have adverse effects on the skin immune system.

6-[2-(adamantylidene)-hydroxybenzoxazole]-O-sulfamate, a steroid sulfatase inhibitor for the treatment of androgen- and estrogen-dependent diseases.

Steroid sulfatase (STS) offers a new target for the treatment of steroid hormone-dependent diseases, such as breast and prostate cancer and androgen-dependent skin diseases. We here characterize a novel non-estrogenic inhibitor of the enzyme, namely 6-[2-(adamantylidene)-hydroxybenzoxazole]-O-sulfamate (AHBS), with special attention to its potential use in the treatment of acne. The compound blocks STS activity in homogenates of human skin with IC(50)=16 nM. Following a single oral dose (5 mg/kg) in rats, the compound blocks STS in the skin by 95% at 8 h, followed by recovery of activity over 5 days. Following topical application to the skin, both in vitro and in vivo, AHBS passes through the stratum corneum leading to inhibition of STS activity in the dermal compartment with rapid onset and long duration. Topical application of AHBS to Göttingen minipigs for a period of 2 weeks does not induce symptoms of ichthyosis as seen in STS-deficient human subjects, but leads to a reduction of sebum secretion to the skin surface. Based on these data, clinical studies with AHBS in acne patients are warranted, in order to verify the hypothesis on the importance of the sulfatase pathway in androgen-dependent skin diseases.

Topical treatment of Basal cell carcinomas in nevoid Basal cell carcinoma syndrome with a smoothened inhibitor.

Basal cell carcinoma (BCC) is a distinctive manifestation in nevoid basal cell carcinoma syndrome (NBCCS) patients. Both inherited and acquired mutations of patched 1 (PTCH1), a tumor-suppressor gene controlling the activity of Smoothened (SMO), are the primary cause of the constitutive activation of the Hedgehog (HH) pathway, leading to the emergence of BCCs in NBCCS. LDE225, a distinct, selective antagonist of SMO, showed potent inhibition of basaloid tumor nest formation and mediated regression of preformed basaloid tumors in organ cultures of skin derived from Ptch1 heterozygous knockout mice. In a double-blind, randomized, vehicle-controlled, intraindividual study, a total of 8 NBCCS patients presenting 27 BCCs were treated twice daily with 0.75% LDE225 cream or vehicle for 4 weeks. Application of 0.75% LDE225 cream was well tolerated and showed no skin irritation. Of 13 LDE225-treated BCCs, 3 showed a complete, 9 a partial, and only 1 no clinical response. Except for one partial response, the vehicle produced no clinical response in any of the 14 treated BCCs. Treatment with 0.75% LDE225 cream in NBCCS patients was very well tolerated and caused BCC regression, thus potentially offering an attractive therapeutic alternative to currently available therapies for this indication.JID JOURNAL CLUB ARTICLE: For questions, answers, and open discussion about this article, please go to http://www.nature.com/jid/journalclub.

Differential effects of corticosteroids and pimecrolimus on the developing skin immune system in humans and mice.

Atopic dermatitis arises primarily in early infancy. In these patients, corticosteroids are used especially with great caution because of their side effects. Calcineurin inhibitors such as pimecrolimus (PIM) could be useful, but safety concerns have been raised in particular because of the lack of knowledge about their effects on the developing skin immune system. This study was designed to investigate the impact of PIM and corticosteroids on epidermal cells (EC) in infants and newborn mice. We found that the percentage of unfractionated viable infant ECs was significantly decreased in the presence of beta-methasone-17-valerate (BMV) but not PIM. Exposure of unfractionated infant ECs to BMV but not to PIM and vehicle control caused a significant inhibition of the upregulation of CD86 molecules on Langerhans cells (LC). The release of cytokines by LCs and ECs, cultured in the presence of BMV and PIM, was not significantly reduced compared with controls. Topical corticosteroid but not PIM application onto newborn mice induced apoptosis in some LC precursors. Our data suggest that similar to the situation in adult skin, corticosteroids may impair LC maturation as well as viability of ECs in infants, effects not seen with PIM.

Neutropenia with impaired immune response to Streptococcus pneumoniae in ceramide kinase-deficient mice.

In mammals, ceramide kinase (CerK)-mediated phosphorylation of ceramide is the only known pathway to ceramide-1-phosphate (C1P), a recently identified signaling sphingolipid metabolite. To help delineate the roles of CerK and C1P, we knocked out the gene of CerK in BALB/c mice by homologous recombination. All in vitro as well as cell-based assays indicated that CerK activity is completely abolished in Cerk-/- mice. Labeling with radioactive orthophosphate showed a profound reduction in the levels of de novo C1P formed in Cerk-/- macrophages. Consistently, mass spectrometry analysis revealed a major contribution of CerK to the formation of C16-C1P. However, the significant residual C1P levels in Cerk-/- animals indicate that alternative routes to C1P exist. Furthermore, serum levels of proapoptotic ceramide in these animals were significantly increased while levels of dihydroceramide as the biosynthetic precursor were reduced. Previous literature pointed to a role of CerK or C1P in innate immune cell function. Using a variety of mechanistic and disease models, as well as primary cells, we found that macrophage- and mast cell-dependent readouts are barely affected in the absence of CerK. However, the number of neutrophils was strikingly reduced in blood and spleen of Cerk-/- animals. When tested in a model of fulminant pneumonia, Cerk-/- animals developed a more severe disease, lending support to a defect in neutrophil homeostasis following CerK ablation. These results identify ceramide kinase as a key regulator of C1P, dihydroceramide and ceramide levels, with important implications for neutrophil homeostasis and innate immunity regulation.

Discovery of topical calcineurin inhibitors and pharmacological profile of pimecrolimus.

Using a newly developed model of allergic contact dermatitis in pigs, calcineurin inhibitors of the tacrolimus and ascomycin type were shown to have a highly anti-inflammatory action after topical application. These findings provided the first pharmacological evidence of the efficacy of this novel class of topical agents in the treatment of inflammatory skin diseases, and, thus, their potential to become the first alternative to corticosteroids in more than 40 years. As a result of a large research program into ascomycins, pimecrolimus (Elidel(R), SDZ ASM 981) was selected for development due to its favorable pharmacology and safety profile, alongside tacrolimus (Protopic(R), FK 506). In vitro, pimecrolimus inhibits the transcription and release of pro-inflammatory cytokines in T cells. Similar to the corticosteroids, betamethasone-17-valerate and dexamethasone, pimecrolimus is effective at nanomolar concentrations. Targeting mainly T cells, pimecrolimus has, however, a more specific mode of action. Moreover, in contrast to corticosteroids, pimecrolimus has no effect on Langerhans' cells, the professional antigen- presenting dendritic cells of the skin that are crucial for local immunosurveillance. When applied topically, pimecrolimus exerts a high and selective anti-inflammatory activity in the skin, shows minimal percutaneous absorption, and has a low potential to affect systemic immunoreactions. Pimecrolimus cream 1% has proven to be well tolerated, safe, and highly effective in clinical studies in patients with atopic dermatitis.

Pimecrolimus permeates less than tacrolimus through normal, inflamed, or corticosteroid-pretreated skin.

The permeabilities of normal human and normal, inflamed, or corticosteroid (CS) pretreated skin of young domestic pigs for pimecrolimus and tacrolimus were compared in vitro, using Franz-type diffusion cells. The test articles were either used as 1.0% solutions or as the marketed formulations (Elidel 1% cream, Protopic 0.1%, and 0.03% ointment). In normal human skin, the permeation rate of pimecrolimus from the 1% cream was about sixfold lower than that of tacrolimus from 0.1% ointment and by a factor of 4.3 lower compared with tacrolimus from Protopic 0.03%. In pigs, sodium laurylsulfate-induced irritant contact dermatitis resulted in significantly faster skin permeation of both drugs from applied solutions. The permeation rate for pimecrolimus was lower than that for tacrolimus. Thus, at 24 h, pimecrolimus concentrations in the receptor fluid were 2.8-fold lower than the tacrolimus levels. Compared with normal porcine skin, permeation of drugs through hydrocortisone (1.0%)-, mometasone (0.1%)-, or clobetasol-17-butyrate (0.05%)-pretreated skin was increased by factors of 3.6 (pimecrolimus, applied as 1% cream) and 1.7 (tacrolimus, applied as 0.1% ointment). In normal pig skin, the permeation rate of tacrolimus was found to be 11.2 times higher than that of pimecrolimus and 3.5- to 7.1-fold higher in CS-pretreated skin, independent of the potency of the CSs. The present in vitro data suggest that in patients with acute skin inflammation or after therapy with topical CSs, percutaneous absorption and, as a consequence, systemic drug exposure will be lower with Elidel 1% cream as compared with Protopic 0.1% and 0.03% ointment.

The effect of topical pimecrolimus on keratoconjunctivitis sicca and chronic superficial keratitis in dogs: results from an exploratory study.

OBJECTIVE: Pimecrolimus is an ascomycin derivative that interferes selectively with the activation of T cells and mast cells and inhibits the production of inflammatory cytokines. This study evaluated the efficacy of an experimental ophthalmic formulation of pimecrolimus in treating keratoconjunctivitis sicca (KCS) and chronic superficial keratitis (CSK) in dogs. ANIMALS AND PROCEDURES: Eight dogs with KCS and six with CSK were included. The dogs were of various breeds, suffered from chronic conditions, and had been pretreated unsuccessfully. The affected eyes were treated with 1 drop of an experimental, corn oil-based pimecrolimus 1% formulation three times a day. Parameters evaluated included Schirmer tear test (STT), ocular discharge, conjunctival inflammation, corneal inflammatory cell infiltrate and scarring, and comfort level. RESULTS: The effect of pimecrolimus 1% was pronounced (increase in STT values to higher than 4 mm/min, no signs of inflammation) or moderate (increase in STT values of 3-4 mm/min, mild signs of corneal/conjunctival inflammation) in a total of 6/8 animals with KCS. In 4/6 animals with CSK, the effect was either pronounced (total regression of fibrovascular infiltration into the cornea, no corneal scarring) or moderate (distinct regression of pannus, mild corneal scarring). The response to treatment was unsatisfactory in four of 14 animals. CONCLUSION: Results of this exploratory study suggest that topical 1% pimecrolimus may be a new effective treatment for keratoconjunctivitis sicca and chronic superficial keratitis in dogs.

Statin-derived 1,3-oxazinan-2-ones as submicromolar inhibitors of LFA-1/ICAM-1 interaction: stabilization of the metabolically labile vanillyl side chain.

Modification of the vanillyl substituent on a potent, semisynthetic lymphocyte function-associated antigen (LFA)-1/intercellular adhesion molecule (ICAM)-1 binding inhibitor of the statin family resulted in metabolically more stable analogues that displayed submicromolar inhibitory activity in vitro and considerable anti-inflammatory activity in vivo. The benzodioxole derivative 2b emerged with the best overall profile.

Synthesis of dammarane-type triterpenoids with anti-inflammatory activity in vivo.

The 17-alpha-substituted triterpene 1 [(17alpha)-23-(E)-dammara-20,23-diene-3beta,25-diol] showed promising activity in animal models of immunosuppression and inflammation. Using a mouse model for inflammatory skin diseases (oxazolone-induced allergic contact dermatitis, ACD) as the directing in vivo test system, Structure-activity-relationship studies with the aim to understand the necessary structural requirements for the biological activity of 1 were conducted. Furthermore, we anticipated to identify biologically active compounds with the 17beta configuration, which are thermodynamically more stable and much easier to synthesize. This was achieved by identifying the 17-beta substituted dammarane 5B and its analogues.