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Objective: To investigate the effect of IL-22 on the expression of Amphiregulin (AREG) in HaCaT and further understand its role in psoriasis vulgaris (PsV). Methods: The mRNA expressions of IL-22, IL-22R1, IL-22BP and AREG were detected by RT-qPCR in PsV lesions (n=26) and healthy control (HC) skin specimens (n=15). RT-qPCR and Western blot were applied to detect the expression of AREG in HaCaT cells stimulated with IL-22 (20 ng/mL) and its soluble receptor IL-22BP (20 ng/mL) for 24 h. Results: The mRNA expressions of IL-22 (P<0.001), IL-22R1 (P<0.001) and AREG (P<0.01) were significantly increased respectively by 36 times, 24 times and 15 times in PsV compared with those in HC. In addition, there were positive correlations between the mRNA levels of AREG and IL-22 (r=0.49, P<0.05). IL-22 could upregulate the mRNA level of AREG by 22 times and protein expression by 6 times in HaCaT cells (P<0.01). IL-22BP could inhibit the effect of IL-22 (P<0.05). Conclusion: IL-22 may regulate positively amphiregulin expression of keratinocytes involved in psoriasis, and IL-22BP may inhibit this role as a blocker in treating psoriasis.
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Objective To explore the relationships of neutrophil gelatinase-associated lipocalin (NGAL) with severity of skin lesions in children with psoriasis and peripheral neutrophil count,and to evaluate in vitro effect of NGAL on expression of tumor necrosis factor-α (TNF-α) and interleukin-22 (IL-22) by a human immortalized keratinocyte cell line HaCaT.Methods From January 1st 2017 to December 31st 2018,98 children who newly developed psoriasis were enrolled from Department of Dermatology of 6 hospitals in China,including 51 males and 47 females.Their age was 7.00 ± 2.99 years (range:3-14 years),and their course of disease was 7.4 ± 5.85 days (range:3-28 days).The serum level of NGAL was detected in all the patients before and two weeks after treatment,and the relationships of NGAL with psoriasis area and severity index (PASI) scores and peripheral neutrophil count were evaluated.Western blot analysis and reverse-transcription (RT)-PCR were performed to determine the protein and mRNA expression of TNF-α and IL-22 in HaCaT cells,respectively,after 12-hour treatment with NGAL at concentrations of 0 (control group),0.125,0.25,0.5,1 mg/L.Statistical analysis was carried out with SPSS 16 software.by using t test and one-way analysis of variance.Results After 2-week treatment,the PASI score,neutrophil count and NGAL level in children with psoriasis significantly decreased (1.80 ± 1.19,[6.16 ± 0.76] × 109/L,90.86 ± 0.75 μ g/L,respectively) compared with those before the treatment (10.38 ± 3.42,[11.01 ± 2.85] × 109/L,113.48 ± 21.26 μ g/L,respectively;t =31.42,18.34,16.37 respectively,all P ＜ 0.001).Before the treatment,the serum level of NGAL in the patients was positively correlated with the PASI score and peripheral neutrophil count (r =0.918,0.799 respectively,both P ＜ 0.05).The mRNA and protein expression of IL-22 in HaCaT cells significantly differed among these groups treated with different concentrations of NGAL (F =176.31,296.96 respectively,both P ＜ 0.001),so did the mRNA and protein expression of TNF-α (F =193.28,318.80 respectively,both P ＜ 0.001).Additionally,the protein and mRNA expression of IL-22 and TNF-α in HaCaT cells was significantly higher in the 0.125-,0.25-,0.5-and 1-mg/L NGAL group than in the control group (all P ＜ 0.05).The NGAL level was positively correlated with the protein and mRNA expression of TNF-α and IL-22 in HaCaT cells (all P ＜ 0.05).Conclusions The serum level of NGAL was high in children with psoriasis,and positively correlated with severity of skin lesions and peripheral neutrophil count.NGAL can upregnlate the expression of TNF-α and IL-22 in HaCaT cells in vitro.
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Objective@#To explore the relationships of neutrophil gelatinase-associated lipocalin (NGAL) with severity of skin lesions in children with psoriasis and peripheral neutrophil count, and to evaluate in vitro effect of NGAL on expression of tumor necrosis factor-α (TNF-α) and interleukin-22 (IL-22) by a human immortalized keratinocyte cell line HaCaT.@*Methods@#From January 1st 2017 to December 31st 2018, 98 children who newly developed psoriasis were enrolled from Department of Dermatology of 6 hospitals in China, including 51 males and 47 females. Their age was 7.00 ± 2.99 years (range: 3-14 years) , and their course of disease was 7.4 ± 5.85 days (range: 3-28 days) . The serum level of NGAL was detected in all the patients before and two weeks after treatment, and the relationships of NGAL with psoriasis area and severity index (PASI) scores and peripheral neutrophil count were evaluated. Western blot analysis and reverse-transcription (RT) -PCR were performed to determine the protein and mRNA expression of TNF-α and IL-22 in HaCaT cells, respectively, after 12-hour treatment with NGAL at concentrations of 0 (control group) , 0.125, 0.25, 0.5, 1 mg/L. Statistical analysis was carried out with SPSS 16 software. by using t test and one-way analysis of variance.@*Results@#After 2-week treatment, the PASI score, neutrophil count and NGAL level in children with psoriasis significantly decreased (1.80 ± 1.19, [6.16 ± 0.76] × 109/L, 90.86 ± 0.75 μg/L, respectively) compared with those before the treatment (10.38 ± 3.42, [11.01 ± 2.85] × 109/L, 113.48 ± 21.26 μg/L, respectively; t = 31.42, 18.34, 16.37 respectively, all P < 0.001) . Before the treatment, the serum level of NGAL in the patients was positively correlated with the PASI score and peripheral neutrophil count (r = 0.918, 0.799 respectively, both P < 0.05) . The mRNA and protein expression of IL-22 in HaCaT cells significantly differed among these groups treated with different concentrations of NGAL (F = 176.31, 296.96 respectively, both P < 0.001) , so did the mRNA and protein expression of TNF-α (F = 193.28, 318.80 respectively, both P < 0.001) . Additionally, the protein and mRNA expression of IL-22 and TNF-α in HaCaT cells was significantly higher in the 0.125-, 0.25-, 0.5- and 1-mg/L NGAL group than in the control group (all P < 0.05) . The NGAL level was positively correlated with the protein and mRNA expression of TNF-α and IL-22 in HaCaT cells (all P < 0.05) .@*Conclusions@#The serum level of NGAL was high in children with psoriasis, and positively correlated with severity of skin lesions and peripheral neutrophil count. NGAL can upregulate the expression of TNF-α and IL-22 in HaCaT cells in vitro.
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Objective To evaluate the effects of interleukin-22 (IL-22) on the expression of CC chemokine ligand 27 (CCL27) in human epidermal keratinocytes,and to explore its mechanism.Methods Immunohistochemical study was performed to determine the expression of CCL27 in skin lesions of 10 patients with psoriasis and skin tissues of 5 healthy controls.Cultured HaCaT cells were divided into 8 groups:control group treated with PBS,5 IL-22 groups treated with 12.5,25,50,100 and 200 μg/L IL-22 respectively,2 signaling pathway inhibition groups treated with 50 μrmol/L AG490 (JAK2/STAT3 signaling pathway inhibitor) or PD98059 (MAPK-ERK1/2 signaling pathway inhibitor) for 2 hours followed by the treatment with 50 μg/L IL-22 in the 5％ CO2 incubator at 37 ℃.After 24-hour cultivation,total proteins were extracted,and culture supernatants were collected,and both Western blot analysis and enzyme-linked immunosorbent assay (ELISA) were performed to determine the expression of CCL27.Results Immunohistochemical study showed that the expression of CCL27 was significantly higher in the skin lesions of the patients with psoriasis than in the skin tissues of the healthy controls.Western blot analysis revealed that the protein expression of CCL27 in the 12.5-,25-,50-,100-and 200-μg/L IL-22 groups was 0.491 ± 0.013,0.620 ± 0.019,0.623 ± 0.014,0.802 ± 0.052 and 1.138 ± 0.013 respectively,which were all higher than that in the control group (0.413 ± 0.013,all P ＜ 0.01).The expression of CCL27 was significantly lower in the IL-22 + AG490 group (0.411 ± 0.019) and IL-22 + PD98059 group (0.280 ± 0.012) than in the 50-μg/L IL-22 group (both P ＜ 0.01).ELISA also showed the same trend of changes in the level of CCL27 in the above groups as Western blot showed.Conclusion IL-22 can promote the expression of CCL27 in HaCaT cells,which may be associated with the MAPK-ERK 1/2 and JAK2/STAT3 signaling pathways.
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Objective To determine the serum levels of tumor necrosis factor (TNF)-α,interleukin (IL)-17,IL-22 and IL-17F in patients with palmoplantar pustulosis (PP),and to estimate their relationship with disease activity in PP.Methods Venous blood samples were collected from 30 patients with PP at both active stage and stationary stage and from 20 healthy human controls.Enzyme-linked immunosorbent assay (ELISA) was conducted to determine the serum levels of TNF-α,IL-17,IL-22 and IL-17F.The paired Wilcoxon signed rank test was carried out to compare the serum levels of cytokines between patients at active stage and at stationary stage,and the Mann-Whitney U test to compare those among different groups.Results The median serum levels of TNF-α,IL-17 and IL-22 in patients with PP at active stage were 186.35 (range,113.48-412.69) ng/L,420.45 (range,278.55-748.73) ng/L and 106.48 (range,69.13-251.86) ng/L respectively,significantly higher than those at stationary stage (42.52(18.83-95.37) ng/L,48.11 (36.43-80.04) ng/L,20.32 (10.55-48.75) ng/L,respectively,all P ＜ 0.05) and those in the controls (24.30 (12.0-61.56) ng/L,10.49 (6.24-24.44) ng/L,2.58 (1.41-5.78) ng/L,respectively,all P ＜ 0.05).Moreover,the patients at stationary stage showed a significant elevation in serum levels of TNF-α,IL-17 and IL-22 compared with the controls (u =2.71,3.53,2.18,respectively,all P ＜ 0.05).No statistical difference was noted in the serum level of IL-17F among the patients at different stages and controls (P ＞ 0.05).Conclusion The circulating levels of TNF-α,IL-17 and IL-22 were associated with disease activity in PP,hinting that they may be involved in the development of PP.
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Objective To investigate the mechanisms underlying intedeukin-22 (IL-22)-induced expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in HaCaT cells.Methods Some HaCaT cells were divided into several inverention groups treated with IL-22 at concentrations of 12.5,25,50,100 μg/L,respectively and a control group treated with phosphate buffer saline (PBS).After 24-hour culture,total proteins were extracted from the HaCaT cells,and Western blot was performed to measure the expression of phosphorylated extracellular signalregulated kinase 1/2 (P-ERK1/2) in the mitogen-activated protein kinase (MAPK)-ERK1/2 pathway,as well as phosphorylated-JAK2 (P-JAK2) and phosphorylated-signal transducer and activator of transcription 3 (P-STAT3) in the JAK2/STAT3 pathway.In a blocking experiment,some HaCaT cells were divided into 4 groups to be treated with PBS,IL-22,PD98059 (an inhibitor of MAPK-ERK1/2) combined with IL-22 (PD98059 group),AG490 (an inhibitor of JAK2/STAT3) combined with IL-22 (AG490 group),respectively.After 24-hour treatment,total proteins and mRNAs were extracted from the HaCaT cells followed by Western blot and real-time quantitative reverse transcription-PCR for the measurement of protein and mRNA expressions of HB-EGF respectively.Statistical analysis was carried out with the software SPSS 16.0 by one-way analysis of variance (ANOVA) for intergroup comparisons and by Bonferroni's test for multiple comparisons.Results After treatment with IL-22 at the above 4 concentrations,the expressions of P-ERK1/2,P-JAK2 and P-STAT3 in HaCaT cells were all increased compared with the control group (all P ＜ 0.05).The protein and mRNA expression levels (expressed as the HB-EGF/β-actin ratio and 2-△△Cr respectively) of HB-EGF were both significantly decreased in the PD98059 group and AG490 group than in the IL-22 group (protein:0.183 ± 0.020 and 0.199 ± 0.011 vs.0.924 ± 0.032,F =37.700,36.400,respectively,both P ＜ 0.05; mRNA:1.034 ± 0.072 and 0.989 ± 0.038 vs.1.844 ± 0.135,F =11.271,13.429,respectively,both P ＜ 0.05).Conclusions IL-22 can activate the MAPK-ERK1/2 and JAK2/STAT3 signaling pathways in HaCaT cells,which may contribute to IL-22-induced expression of HB-EGF in HaCaT cells.
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Objective To explore the therapeutic mechanism of narrow-band ultraviolet B (NB-UVB)in psoriasis. Methods Forty-two patients with psoriasis vulgaris and 20 healthy controls were enrolled into this study. All the patients received 20 sessions of NB-UVB radiation. Psoriasis area severity index (PASI)was used to evaluate the severity of psoriasis. Blood samples were collected from all the patients before and 15 cured patients after the treatment as well as from 20 healthy controls, and skin samples from 10 patients before and after the treatment as well as from 10 healthy controls. Enzyme-linked immunosorbent assay (ELISA)was performed to determine the serum levels of IL-17 and IL-22, and real-time fluorescence-based quantitative PCR to measure the mRNA expressions of IL-17 and IL-22 in skin specimens. Statistical analysis was carried out by using the two-sample t-test, paired t-test and Pearson correlation analysis. Results After 20 sessions of NB-UVB radiation, 15 out of the 42 patients were cured with a significant decrease in PASI. Compared with the healthy controls, the 15 cured patients showed a significant elevation in the levels of IL-17and IL-22 proteins(IL-17: 34.26 ± 10.05 ng/L vs. 16.34 ± 4.73 ng/L, t = 7.016, P < 0.01; IL-22: 13.72 ± 4.45 ng/L vs. 5.03 ± 1.84 ng/L, t = 8.282, P < 0.01)and mRNAs (IL-17: 13.43 ± 2.12 vs. 5.26 ± 0.87, t = 6.312, P < 0.01; IL-22:16.53 ± 2.65 vs. 7.72 ± 2.13, t = 6.823, P < 0.01)before the treatment. The PASI score was positively correlated with the levels of IL-17 and IL-22 proteins in sera (r = 0.76, 0.70, respectively, both P < 0.05)and their mRNAs in skin lesions (r = 0.65, 0.68, respectively, both P < 0.05)in these patients. The serum levels and mRNA expressions of IL-17 and IL-22 all significantly reduced in the cured patients after the treatment compared with those before the treatment(all P < 0.05). Conclusion NB-UVB may treat psoriasis by downregulating the levels of IL-17 and IL-22 in peripheral blood and skin lesions in patients with psoriasis.
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Objective To determine the frequency of T helper type 22 (Th22) cells and expression level of interleukin-22 (IL-22) in peripheral blood of patients with psoriasis vulgaris,and to investigate their relationship with disease severity and clinical course.Methods Peripheral blood samples were obtained from 40 patients with psoriasis vulgaris and 30 healthy human controls.Five-color flow cytometry was performed to determine the percentage of Th22 cells in peripheral blood,and enzyme-linked immunosorbent assay (ELISA) to measure the expression of serum IL-22.Statistical analysis was carried out by t test and Pearson correlation analysis.Results Both the percentage of Th22 cells and serum level of IL-22 in peripheral blood were significantly higher in patients with psoriasis vulgaris than in healthy human controls (Th22 cells:0.65％ ± 0.48％ vs.0.33％ ± 0.15％,t =3.89,P ＜ 0.01; IL-22:(67.96 ± 14.32) vs.(40.59 ± 9.91) ng/L,t =9.45,P ＜ 0.01).Further more,Th22 cell percentage and IL-22 serum level in peripheral blood were both positively correlated with psoriasis area and severity index (PASI) in these patients (r =0.38,0.94,P ＜ 0.05 and 0.01,respectively),but neither of them correlated with clinical course of psoriasis vulgaris (r =0.20,0.10,respectively,both P ＞ 0.05).Conclusions The percentage of Th22 cells and level of IL-22 are increased in peripheral blood of patients with psoriasis vulgaris,and both of them are correlated with disease severity.
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Th22,a new subset of helper T cells,which is char-acterized by the secretion of interleukin-22(IL-22),could infil-trate to the epidermis in individuals with inflammatory skin disor-ders.This article introduces the action of Th22 and IL-22 in in-flammatory skin diseases,including psoriasis,atopic dermatitis, systemic lupus erythematosus,systemic sclerosis,aiming at re-vealing the role of Th22 and IL-22 in these diseases,which would not only provide some novel targets of drugs for inflamma-tory skin diseases,but also promote the researches on the pre-vention and treatment of these diseases.
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Objective To investigate the expressions and significance of IL-22 and related cytokines (IL-23pl9 and IL-6) in sera and PBMCs of patients with psoriasis. Methods Sera and PBMCs were obtained from the venous blood samples from 58 patients with psoriasis vulgaris and 20 normal human controls. The PBMCs were subjected to culture for 5 hours followed by the collection of cells and culture supernatant. Then,quantitative real-time RT-PCR was used to examine the mRNA expressions of IL-22, IL-23pl9 and IL-6 in PBMCs, enzyme-linked immunosorbent assay (ELJSA) to detect the level of IL-22 protein in the sera and culture supernatant of PBMCs. Results In the patients with psoriasis and controls, the relative expression level in PBMCs was 4.48 ± 2.64 and 2.35 ± 0.91 respectively for IL-22 mRNA, 6.07 ± 4.09 and 2.61 ± 1.46 respectively for IL-23pl9 mRNA, 3.87 ± 1.49 and 1.48 ± 0.62 respectively for IL-6 mRNA; significant differences were observed between the two groups in all the above parameters (all P < 0.01). ELISA revealed that the level of IL-22 protein in the patients and controls was (86.23 ± 25.58) ng/L and (43.67 ± 14.82) ng/L respectively in the sera (P< 0.01), (119.11 ± 21.51) ng/L and (57.70 ± 13.17) ng/L respectively in the culture supernatant of PBMCs (P< 0.01). Conclusion There is an overexpression of IL-22 in the PBMCs and sera of patients with psoriasis, implying that IL-22 is involved in the pathogenesis of psoriasis.
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Objective To detect the quantity of Th17 cells and expressions of related cytokines including interleukin (IL)-17 and IL-22, in peripheral blood and skin lesions of patients with psoriasis vulgaris, and to analyze their correlation with disease severity and clinical course. Methods Peripheral blood was obtained from 44 patients with progressive psoriasis vulgaris and 28 normal human controls. Three-color flow cytometry was carried out to detect the quantity of Th17 cells, and ELISA to examine the levels of serum IL-17 and -22.Skin samples were obtained from 20 patients with psoriasis vulgaris and 8 normal human controls, and a quantum dot-based double labled immumofluorescence method was used to determine the quantity of Th17 cells.Results The percentage of peripheral blood Th17 cells was higher in patients with psoriasis vulgaris than in normal human controls (4.71% ± 2.55% vs. 0.55% ± 0.39%, P ＜ 0.01 ). Elevated expressions of IL-17 and IL-22 were noted in the patients compared with the normal human controls (24.02 ± 12.31 ng/L vs. 7.16 ±4.04 ng/L, P ＜ 0.05; 18.32 ± 8.14 ng/L vs. 6.52 ± 4.15 ng/L, P ＜ 0.01 ). The percentage of peripheral blood Th17 cells and serum levels of IL-17 and IL-22 were positively correlated with psoriasis area and severity index (r= 0.53, 0.47, 0.53, respectively, all P ＜ 0.01 ), but unrelated to the clinical course of psoriasis (r = 0.09,0.03, 0.19, respectively, all P ＞ 0.05). There was an infiltrate of Th17 cells in psoriatic lesions, which was mainly distributed around the blood vessels in superficial dermis, whereas there were only a small number of CD4+ T cells in the normal control skin with the absence of Th17 cells. Conclusions Th17 cells are involved in the development of psoriasis, and Th17 cell-secreted cytokines, such as IL-17 and IL-22, may serve as a new therapeutic target for psoriasis.
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In order to study the expression of intedeukin-22 (IL-22) and S100A7, A8, A9 mRNA in the skin lesions of patients with psoriasis vulgaris and their relationship, the biopsies were taken from skin lesions in 35 patients with psoriasis vulgaris and the skin of 16 normal controls, and the expres- sion levels of IL-22 and S 100A7, A8 and A9 mRNA were detected by semi-quantitative RT-PCR. The results showed that (1) IL-22 and SI00A8, A9 mRNA were positively expressed in the psoriatic skin lesions but negatively expressed in the normal controls; The expression level of S100A7 was (1.133±0.040) in the psoriatic skin lesions, significantly higher than that in the normal controls (0.744±0.037, P<0.01). (2) There were significantly positive correlations between the expression of IL-22/S100A7 mRNA, IL-22/S100A8 mRNA, IL-22/S100A9 mRNA in the psoriasis vulgaris (r1=0.543, r2=0.774, r3=0.621, P<0.01). It was concluded that IL-22 and S100A7, A8, A9 might play important roles in the occurrence and progression of psoriasis.