Oral administration of a novel RORγt antagonist attenuates psoriasis-like skin lesion of two independent mouse models through neutralization of IL-17.

BACKGROUND: Targeting the IL-17 pathway represents a highly effective strategy for the treatment of psoriasis, using antibodies against IL-17A and IL-17 receptor, suggesting that Th17 cells essentially contribute to development of psoriasis. Th17 differentiation depends on the key transcription factor, RORγt. OBJECTIVE: To develop a novel RORγt antagonist which is effective on psoriasis via oral administration. METHODS: A chemical library was screened using cell-based high-throughput methods, luciferase reporter assay, competitive binding assay, and T cell differentiation assay. To evaluate in vivo effects of a novel RORγt antagonist, A213, we orally administrated it to two independent mouse models of psoriasis; IL-23-injection model and K5.Stat3C transgenic mouse. RESULTS: Oral administration of A213 resulted in attenuation of skin inflammation in the both mouse models. At the same time, increased levels of IL-17A expression were significantly reduced in the skin lesions and skin-draining lymph nodes. CONCLUSION: These results implicate a new therapeutic application of RORγt antagonist for the treatment of psoriasis.

Targeting cell surface TLR7 for therapeutic intervention in autoimmune diseases.

Toll-like receptor 7 (TLR7) senses microbial-derived RNA but can also potentially respond to self-derived RNA. To prevent autoimmune responses, TLR7 is thought to localize in endolysosomes. Contrary to this view, we show here that TLR7 is present on the cell surface of immune cells and that TLR7 responses can be inhibited by an anti-TLR7 antibody. The anti-TLR7 antibody is internalized with TLR7 and accumulates in endolysosomes as an immune complex. TLR7 responses in dendritic cells, macrophages and B cells are all inhibited by the anti-TLR7 antibody. Furthermore, the anti-TLR7 antibody inhibits in vivo cytokine production induced by a TLR7 ligand. Spontaneous TLR7 activation in Unc93b1(D34A/D34A) mice causes lethal inflammation. Progressive inflammation such as splenomegaly, thrombocytopenia and chronic active hepatitis are ameliorated by anti-TLR7 antibody treatment. These results demonstrate that cell surface TLR7 is a promising target for therapeutic intervention in autoimmune diseases.

Tyk2 is a therapeutic target for psoriasis-like skin inflammation.

Tyrosine kinase 2 (Tyk2), a member of the Jak kinase family, mediates signals triggered by various cytokines, which are related to the pathogenesis of psoriasis. In this study, we investigated the role of Tyk2 in IL-23-induced psoriasis-like skin inflammation. Tyk2(-/-) mice when injected with IL-23 showed significantly reduced ear skin swelling with epidermal hyperplasia and inflammatory cell infiltration compared with wild-type mice. In addition, Tyk2 deficiency reduced production of pro-inflammatory cytokines and psoriasis-relevant anti-microbial peptides. More noteworthy is that Tyk2 directly regulated IL-22-dependent inflammation and epidermal hyperplasia. Taken together with the inhibition of IL-23-induced inflammation by treatment with neutralizing antibodies against IL-17 or IL-22, Tyk2 participates in both IL-23 and IL-22 signal transduction to mediate psoriasis-like skin inflammation. On the basis of these findings, we demonstrated for the first time that a small-molecule Tyk2 inhibitor significantly inhibited IL-23-induced inflammation and cytokine production in the skin. These observations demonstrate the important role of Tyk2 in experimental skin inflammation and indicate the therapeutic potential of Tyk2 inhibition in human psoriasis.

Tyk2 deficiency protects joints against destruction in anti-type II collagen antibody-induced arthritis in mice.

Tyrosine kinase-2 (Tyk2) participates in the signaling pathways of multiple cytokines in innate and acquired immunity. In the present study, we investigated the in vivo involvement of Tyk2 in anti-type II collagen antibody-induced arthritis (CAIA) using Tyk2-deficient mice. Hind paws of wild-type mice showed massive swelling and erythema by arthritogenic antibody injection, whereas Tyk2-deficient mice did not show any signs of arthritis. Indeed, neither the infiltration of inflammatory cells nor the fibrillation of articular cartilages was observed in Tyk2-deficient mice. Tyk2 deficiency also reduced the production of T(h)1/T(h)17-related cytokines, the other proinflammatory cytokines and matrix metalloproteases, which are induced in the CAIA paw. Our results demonstrate a critical contribution of Tyk2 in the development of arthritis, and we propose that Tyk2 might be an important candidate for drug development.

Involvement of tyrosine kinase-2 in both the IL-12/Th1 and IL-23/Th17 axes in vivo.

Tyrosine kinase-2 (Tyk2), a member of the Jak family of kinases, mediates the signals triggered by various cytokines, including type I IFNs, IL-12, and IL-23. In the current study, we investigated the in vivo involvement of Tyk2 in several IL-12/Th1- and IL-23/Th17-mediated models of experimental diseases, including methylated BSA injection-induced footpad thickness, imiquimod-induced psoriasis-like skin inflammation, and dextran sulfate sodium- or 2,4,6-trinitrobenzene sulfonic acid-induced colitis. In these disease models, Tyk2 deficiency influenced the phenotypes in immunity and/or inflammation. Our findings demonstrate a somewhat broader contribution of Tyk2 to immune systems than previously expected and suggest that Tyk2 may represent an important candidate for drug development by targeting both the IL-12/Th1 and IL-23/Th17 axes.

A novel CC-chemokine receptor 3 antagonist, Ki19003, inhibits airway eosinophilia and subepithelial/peribronchial fibrosis induced by repeated antigen challenge in mice.

CC-chemokine receptor 3 (CCR3) is a chemokine receptor for which major ligands, CC-chemokine ligand (CCL) 11, CCL24, and CCL26, are known to be involved in chemotaxis for eosinophils. In the present study, we evaluated the effect of a low molecular weight CCR3-receptor antagonist, Ki19003 (4-[[5-(2,4-dichlorobenzylureido)pentyl][1-(4-chlorophenyl)ethyl]amino]butanoic acid), on airway remodeling in a mouse model of allergic asthma. BALB/c mice were sensitized twice by intraperitoneal injection of ovalbumin (OA) and exposed daily to 1% OA for 3 weeks. Twenty-four hours after the final antigen challenge, bronchoalveolar lavage and histological examinations were carried out. Ki19003 clearly inhibited antigen-induced increase in the number of eosinophils in bronchoalveolar lavage fluid (BALF), but did not affect the number of other cell types examined in this study. Ki19003 also inhibited the increased production of transforming growth factor-beta1 in BALF and the amount of hydroxyproline in the lungs in a dose-dependent manner. Furthermore, Ki19003 significantly attenuated allergen-induced subepithelial and peribronchial fibrosis. These findings indicate that CCR3 antagonism prevents not only the infiltration of eosinophils into the airways but also the development of allergen-induced subepithelial and peribronchial fibrosis. Therefore, a CCR3 antagonist may be useful in the treatment of airway remodeling, especially subepithelial and peribronchial fibrosis, in allergic asthma.

Nafamostat mesilate, a potent serine protease inhibitor, inhibits airway eosinophilic inflammation and airway epithelial remodeling in a murine model of allergic asthma.

To clarify the involvement of serine proteases in the development of allergic airway inflammation, we investigated the effect of nafamostat mesilate, a serine protease inhibitor, in a murine model of allergic asthma. Mice were sensitized to ovalbumin (OA) with alum and then exposed to 1% OA for 30 min, three times every 4th day. Nafamostat mesilate was administered orally for 10 days during the allergen challenge. In sensitized mice, repeated allergen challenge induced an increase in tryptase proteolytic activity in bronchoalveolar lavage fluid (BALF). In addition, marked increases in the numbers of inflammatory cells, levels of T helper type 2 (Th2) cytokines and eotaxin in BALF, numbers of goblet cells in the epithelium, and level of OA-specific IgE in serum were observed after repetitive allergen inhalation. Treatment with nafamostat mesilate significantly inhibited not only increased proteolytic activities, but also increases in the numbers of eosinophils and lymphocytes in the BALF. Nafamostat mesilate also dose-dependently inhibited increases in the levels of interleukin-13 and eotaxin in BALF and goblet cell hyperplasia. These findings suggest that increased serine protease activity in the airways is involved in the development of antigen-induced allergic eosinophilic inflammation and epithelial remodeling in bronchial asthma.

Production of matrix metalloproteinases in human cultured mast cells: involvement of protein kinase C-mitogen activated protein kinase kinase-extracellular signal-regulated kinase pathway.

BACKGROUND: Matrix metalloproteinases (MMPs) have been reported to play crucial roles in the migration of inflammatory cells through basement membrane components. To confirm the role of mast cells as a source of MMPs, we investigated the production of MMP and its pathway in human cultured mast cells (HCMC). We also investigated the production of tissue inhibitors of metalloproteinase (TIMPs). METHODS: HCMC was stimulated with phorbor 12-miristate 13-acetate (PMA) and/or calcium ionophore A23187 (A23187), and the resulting MMP production was evaluated by gelatin zymography and western blotting. Expression of MMP and TIMP mRNA was also examined. Granulocyte macrophage-colony stimulating factor (GM-CSF) was measured by ELISA and activation of extracellular signal-regulated kinase (ERK) was evaluated by western blotting. RESULTS: We detected the de novo synthesis of MMP-9 in HCMC after stimulation with PMA and found that the synthesis was mediated through protein kinase C-mitogen activated protein kinase kinase (MEK)-ERK pathway. The MMP-9 production induced by PMA was suppressed by simultaneous treatment with A23187, whereas GM-CSF production was potentiated. We also detected the expression of mRNA for membrane-type 1 (MT1)-MMP, TIMP-1 and TIMP-2 after stimulation with PMA. Glucocorticoids and flavonoids inhibited MMP-9 production, and TIMPs and MMP inhibitors inhibited the gelatinolytic activity of mast cell-derived MMP-9. Furthermore, phenylmethylsulfonyl fluoride, a protease inhibitor, inhibited the conversion from proMMP-9 to active MMP-9. CONCLUSIONS: These results suggest that the human mast cell is a leading member of MMP production, and the production, activation and activity are controllable by pharmacological agents.

Role of interleukin-5 and eosinophils in allergen-induced airway remodeling in mice.

Asthma is a chronic inflammatory disease characterized by variable bronchial obstruction, hyperresponsiveness, and by tissue damage known as airway remodeling. In the present study we demonstrate that interleukin (IL)-5 plays an obligatory role in the airway remodeling observed in experimental asthma. BALB/c mice sensitized by intraperitoneal injections of ovalbumin and exposed daily to aerosol of ovalbumin for up to 3 wk, develop eosinophilic infiltration of the bronchi and subepithelial and peribronchial fibrosis. The lesions are associated with increased amounts of hydroxyproline in the lungs and elevated levels of eosinophils and transforming growth factor (TGF)-beta1 in the bronchoalveolar lavage fluid. After 1 wk of allergen challenge, TGF-beta is mainly produced by eosinophils accumulated in the peribronchial and perivascular lesions. At a later stage of the disease, the main source of TGF-beta is myofibroblasts, identified by alpha-smooth muscle actin mAb. We show that all these lesions, including fibrosis, are abolished in sensitized and allergen-exposed IL-5 receptor-null mice, whereas they are markedly accentuated in IL-5 transgenic animals. More importantly, treatment of wild-type mice with neutralizing anti-IL-5 antibody, administered before each allergen challenge, almost completely prevented subepithelial and peribronchial fibrosis. These findings demonstrated that eosinophils are involved in allergen-induced subepithelial and peribronchial fibrosis probably by producing a fibrogenic factor, TGF-beta1.

Effects of RS-601, a novel leukotriene D(4)/thromboxane A(2) dual receptor antagonist, on asthmatic responses in guinea pigs.

The effects of 4-[4-[5,5,6,6,6-pentafluoro-1-(4-fluorobenzene-sulfonamido)hexyl]phenyl]butyric acid (RS-601), a novel leukotriene D(4) (LTD(4))/thromboxane A(2) (TxA(2)) dual receptor antagonist, on bronchial asthmatic responses in guinea pigs were examined. The effects were compared with those of pranlukast (LTD(4) receptor antagonist) and S-1452 (TxA(2) receptor antagonist). RS-601 inhibited the increase in airway resistance caused by LTD(4) and TxA(2) mimetic compound, U-46619, but not by histamine. RS-601 and pranlukast but not S-1452 inhibited an antigen-induced late asthmatic response. In addition, RS-601 inhibited an antigen-induced airway hyperresponsiveness (AHR), whereas pranlukast and S-1452 had no effect on the AHR. The antigen-induced increase in inflammatory cells in airway was not affected by all examined agents. Furthermore, bacterial lipopolysaccharide-induced AHR in guinea pigs was clearly suppressed by RS-601 but not by pranlukast and S-1452. The increase in airway inflammatory cells caused by lipopolysaccharide was not affected by all three drugs. These findings indicate that RS-601 has a potent antiasthmatic efficacy, especially on AHR, but does not affect accumulation of eosinophils in the airways.

Role of Th2 responses in the development of allergen-induced airway remodelling in a murine model of allergic asthma.

(1) To clarify the involvement of Th2 responses in the development of allergen-induced airway remodelling, we investigated the effect of anti-CD4 monoclonal antibody (mAb) and anti-CD8 mAb, and the responses of IL-4 gene-knockout (KO) mice in a murine model of allergic asthma. (2) Mice were immunized twice by intraperitoneal injections of ovalbumin (OA), and exposed to aeroallergen (OA, 1% w v(-1)) for 3 weeks. Twenty-four hours after the final challenge, airway responsiveness to acetylcholine was measured, and bronchoalveolar lavage (BAL) and histological examinations were carried out. (3) Anti-CD4 mAb (1 mg kg(-1)) clearly inhibited allergen-induced increases in airway responsiveness to acetylcholine, the number of eosinophils in BAL fluid, serum OA-specific IgE levels, IL-13 and transforming growth factor-beta1 levels in BAL fluid, and amount of hydroxyproline in the lung by 100, 99, 100, 100, 84, and 60%, respectively. Furthermore, the antibody (1 mg kg(-1)) also attenuated allergen-induced goblet cell hyperplasia in the epithelium and subepithelial fibrosis by 72 and 83%, respectively. In contrast, anti-CD8 mAb (1 mg kg(-1)) showed no effect on each parameter. Furthermore, all these parameters were attenuated in IL-4KO mice by 57, 93, 100, 45, 84 and 60%, and also 72 and 83%, respectively. (4) These findings suggest that Th2 responses play a critical role for the development of allergen-induced airway remodelling, and that the inhibition of Th2 responses, e.g. using anti-CD4 mAb, is a therapeutic approach for the treatment of airway remodelling in asthma.