| BackgroundPsoriasis is a common kind of chronic inflammatory disease with a long course and recurrent attacks. This incurable disease makes the patients and their families suffer from powerful pressure both on economic as well as on mind.The pathogenesis of psoriasis is inadequately understood, a variety of factors such as environment, spirit, nerve, immunity, endocrine and genetic inheritance are involved in its outbreak, in which the factor of immunity is regarded as the central part. Recent research has shown that psoriasis is a kind of disease mediated by Thl/Th17 cells and various kinds of cytokines. Th17 cells have been found to participate in multiple kinds of autoimmune diseases such as ankylosing spondylitis, rheumatoid arthritis and systemic lupus erythematosus.Th17 cells and the related cytokines such as Interleukin IL-17 and IL-22 play a leading role in the pathogenesis of psoriasis, IL-17 transgenic mice exhibited similar symptoms to human psoriasis patients. The level of protein and mRNA of IL-17 and IL-22 in the serum and lesions of psoriasis patients was higher than that in the normal, and showed an association with severity of the disease. A significant relief in clinical symptoms such as erythema, scales and thickness was observed in mice treated with IL-17 antibody, which indicates the importance of IL-17 in psoriasis.IL-17 participates in the pathogenesis of psoriasis through a variety of ways:to promote multiple kinds of inflammatory factors such as Interferon (IFN)-y, (Tumor necrosis factor)TNF-a, IL-6, IL-9, IL-18, IL-36, GM-CSF and antibacterial peptides to stimulate KC proliferation and local inflammatory response; to stimulate the amount of VEGF to promote capillary proliferation and local vasodilation; to promote a variety of chemokines such as IL-8, (Chemokine-c motif ligand)CCL20, CC chemokine receptor(CCR)6 to attract neutrophils, dendritic cells and lymphocytes to infiltrate into the lesions. Th17 cells also secret IL-22 to stimulate the hyperplasia of keratinocytes, participating in the occurence of psoriasis.The migration of Thl7 to the lesions is the earliest process in the onset of psoriasis. CCR6, which expressed mainly on the surface of Thl7 and dendritic cells, participates in this infiltration. CCL20, which is the specific ligand of CCR6, is mainly expressed on KC. A higher expression of CCL20 and CCR6 was detected in the lesions of psoriasis, indicating the importance of CCL20-CCR6 in this migration. The expression of IL-17 and CCR6 is regulated by numerous of cytokins such as TNF-a, IFN-y. Recent studies have emphasized the importance of IL-9 in the regulation of IL-17 and CCR6.IL-9 is a kind of single-chain glycoprotein which plays a part in maintaining the cloning of T lymphocyte as well as in promoting their growth and function. Studies have shown an increase in both the level of IL-9 in the lesions of psorisis patients and the ratio of Th9 cells in the peripheral blood which indicates a role of IL-9 in the pathogenesis of this disease. IL-9 was originally regarded as a cytokine of Th2 cells, and research of IL-9 mainly focued on asthma, infections of parasites, atopic dermatitis and so on. IL-9 regulates the growth, signal transduction and function of mast cells, eosinophils and airway epithelium. IL-9 participates in the pathogenesis of asthma through the following ways:to accelebrate the differentiation, mature and proliferation of mast cells, eosinophils and lymphocytes through STAT signaling; to strengthen the sensitivity of mast cells to allergens; to augment the quantity of mucin secreted by airway epithelium as well as the proportion of goblet cells; to promote the production of IgE ang IL-13 from inflammatory cells. As for infections of parasites, IL-9 can promote the proliferation of intestinal eosinophils and mast cells, boost the secretion of IgE and IgG and stimulate the muscle contractions to accelerate the process of the excretion of parasitics.Recent research found that except for cells mentioned above, Th9 cells, Th17 cells and Regulatory T cells (Treg) cells can also produce IL-9. IL-9 participates in the differentiation of Th17 cells in vitro. IL-9 acts on the differentiation of native T cells ang the secretion of TNF-α, IFN-y, IL-17 and so on. The level of IL-9 was elevated in the serum, lesions, joint effusion, cerebrospinal fluid of systemic lupus erythematosus, experimental autoimmune encephalomyelitis and experimental autoimmune uveitis and related with the severity of these diseases. In addition, research has found that in melanomas IL-9 attracted CCR6+dendritic cells to the local lesion, playing a role of anti-tumor. In experimental autoimmune encephalomyelitis, IL-9 mediates the migration of CCR6+Th17 cells to the central nervous system and application of IL-9 antibody has been found to relieve symptoms of experimental autoimmune encephalomyelitis, accompanying with a decrease in the level of IL-17 and Th17 cells.Studies above have shown a role of IL-9 in the regulation of Th17 cells. Th17 cells and related cytokines such as IL-17 and CCR6 play a critical role in the pathogenesis of psoriasis. So it is of great importance to analyse the expression of IL-9 in psoriasis-like lesions induced by imiquimod, the effects of IL-9 antibody on the lesions and the related mechanism. Mice model of psoriasis induced by imiquimod were used in our experiment to analyse the expression of IL-9 in the lesions. The amount of IL-17 and CCR6 in the lesions of the mice was detected after intraperitoneal injection of IL-9 antibody to explore the mechanism behind it.Objective1. To compare the expression of IL-9 between psoriasis-like lesions induced by imiquimod in mice and control skins in mice.2. To explore whether the IL-9 antibody can alleviate imiquimod-induced psoriasis lesions in mice.3. To explore the effects of IL-9 antibody on the expression of IL-17 and CCR6 in psoriasis-like lesions induced by imiquimod in mice, and to primarily study the role of IL-9 in the mechanism of psoriasis.Methods1. Imiquimod cream was applied on the dorsal skin of BALB/c female mice for consecutive eight days to set up the psoriasis model. HE staining was used to make sure that the modol was successful.Vaseline cream was applied on the control group.2. Western Blot was applied to measure the expression of IL-9 in psoriasis-like lesions induced by imiquimod and in the control skin in mice.3. BALB/c female mice were randomly divided into three groups:vaseline blank control group (short for blank control group), imiquimod model group (short for model group) and IL-9 antibody treatment experimental group (short for experimental group). Blank control group received vaseline on the dorsal skin daily and intraperitoneal injection of 200 μg IL-9 antibody every other day. Model group received imiquimod on the dorsal skin daily and intraperitoneal injection of 200 μg PBS every other day. Experimental group received imiquimod on the dorsal skin daily and intraperitoneal injection of 200 μg IL-9 antibody every other day.4. Psoriasis Area Severity Index (PASI) was scored to measure the severity of the psoriasis-like lesions. HE staining was used to observe the pathologic changes and to measure the epidermis thickness.5. Samples were taken from the dorsal skin of these three groups, one half for paraffin embedding and the other for liquid nitrogen frozen. Immunohistochemistry was performed to determine the expression and distribution of IL-17 and CCR6 in the skin tissue. Western Blot was used to test the level of IL-17 in the lesions of skin.Results1. The expression of IL-9 in imiquimod-induced psoriasis lesions in mice and in the control skin in miceThe immunohistochemistry results showed that compared with control group, the psoriasis group exhibited significantly increased IL-9 expression (P<0.05). Only a small amount of IL-9 can be observed in the control skin in mice, which appeared as light brown in the epidermis. Significantly more stained cells were exhibited in psoriasis-like lesions induced by imiquimod, with IL-9 expressed widely in the epidermis and the dermis in dark brown and brown. The results indicate a role of IL-9 in the model of psoriasis.2. The effect of IL-9 antibody on imiquimod-induced psoriasis lesions in miceAfter daily use of imiquimod for two days, light erythema can be oberserved on the dorsal skin of model group. On the third day, erythema and some scales appeared, then the skin gradually grew thicken and wrinkled. The erythema and scales became more severe continuously until they reached the peak on the seventh to eighth day, appearing as the typical psoriasis-like skins. While in the experimental group which received IL-9 antibody synchronously, only a small proportion of mice revealed spot erythema on the second day. On the third day light erythema and scales appeared in part of the mice. On the fourth to eighth day, the degree of erythema, scales and thickness grew continuously but not to such an extent as to the model group. And no typical psoriasis-like lesion was observed in this group. The blank control group remained normal during the experiment. Results above indicated the therapeutic effect of IL-9 antibodies on psoriasis-like lesions in mice.HE staining revealed a typical psoriasis-like change in the model group, which consisted of severe hyperkeratosis, parakeratosis, Muro micro abscess, acanthosis, capillary prolifreration and subcutaneous infiltration of lymphocytes on the eighth day. While the degree of the hyperkeratosis, acanthosis and infiltration of lymphocytes in the experimental group was moderate. And the HE staining of the blank control group was normal. Epidermis thickness was measured in these three groups, and the result was:blank control group<experimental group<model group. The difference was statistically significant (P<0.05). The results showed that application of IL-9 antibody was able to improve the pathological severity of psoriasis-like lesions in mice induced by imiquidmod.3. The effects of IL-9 antibody on the expression of IL-17 and CCR6 in psoriasis-like lesions induced by imiquimod in miceThe immunohistochemistry results revealed that only a small amount of IL-17 and CCR6 can be observed in the blank group, which appeared as light brown in the dermis. Significantly more stained cells were exhibited in the model group, with IL-17 expressed widely in the epidermis and the dermis in dark brown granulas and CCR6 expressed in epidermis in brown. The number of stained cells in the experimental group was less than the model group, with IL-17 and CCR6 showed in the dermis and the stratum basele in light brown.The immunohistochemistry results showed that compared with blank control group, the model group revealed significantly increased IL-17, CCR6 expression (P <0.05); Compared with the model group, the experimental group showed significantly lower lesion severity score, less inflammatory cells infiltration, and obviously decreased expression of IL-17, CCR6 (P<0.05). The Western Blot results of IL-17 also revealed significant difference between the experimental group and the model group(P< 0.05).Results all above indicate that IL-9 antibody significantly decreased the level of IL-17 and CCR6 in the lesions.Conclusion1. Compared with control skin in mice, the expression of IL-9 was increased in imiquimod-induced psoriasis lesions in mice, which indicates a role of IL-9 in the pathogenesis of psoriasis.2. IL-9 antibody can alleviate the severity of erythema, scaling, and thickness in imiquimod-induced psoriasis lesions in mice, which is probably due to its effects in inhibiting the expression of CCR6 and reducing the number of Th17 cells and DCs, as well as down-regulating the secretion of IL-17 in the lesions. |
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