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Objective:To investigate changes of natural killer (NK) cell subsets and interleukin-18 (IL-18) level in peripheral blood of patients with alopecia areata, and to assess the regulatory effect of IL-18 on NK cell activity.Methods:A total of 67 patients with alopecia areata (alopecia areata group) and 25 healthy volunteers (control group) were collected from Shanxi Provincial People′s Hospital between December 2019 and January 2021. Peripheral blood mononuclear cells (PBMCs) and plasma were isolated. The percentage of NK cell subsets was investigated by flow cytometry, and plasma IL-18 level was measured by enzyme-linked immunosorbent assay. PBMCs were stimulated with recombinant human IL-18, and co-culture systems of PBMCs with 721.221 cells, K562 cells and P815-Ab cells were established separately. NK cell function was assessed by determining the percentage of CD107a-expressing NK cells and fluorescence intensity of CD16 + NK cells. Comparisons between groups were performed using t test or paired t test. Results:Compared with the control group, the alopecia areata group showed significantly decreased percentage of CD56 +CD16 - NK cells (8.12% ± 3.14% vs. 10.78% ± 4.08%, t = 3.33, P = 0.001) , but significantly increased percentage of CD56 +CD16 + NK cells (46.08% ± 15.21% vs. 32.14% ± 10.45%, t = 4.22, P < 0.001) , and there was no significant difference in the percentage of CD56 -CD16 + NK cells between the alopecia areata group and control group (28.81% ± 8.65% vs. 27.09% ± 7.62%, t = 0.88, P = 0.383) . The plasma IL-18 level was significantly higher in the alopecia areata group than in the control group (112.0 ± 23.72 pg/ml vs. 99.34 ± 15.15 pg/ml, t = 2.48, P = 0.015) . After co-culture with 721.221 cells, K562 cells and P815-Ab cells, the percentage of CD107a-expressing NK cells was significantly higher in NK cells from the alopecia areata group (9.53% ± 1.70%, 5.15% ± 1.35%, 6.50% ± 1.64%, respectively) than in those from the control group (5.00% ± 1.17%, 4.40% ± 1.09%, 5.13% ± 1.36%, respectively, all P < 0.05) . After the stimulation with P815-Ab cells, the alopecia areata group showed significantly decreased fluorescence intensity of CD16 + NK cells (151.10% ± 59.30%) compared with the control group (221.90% ± 93.56%, t = 4.31, P < 0.001) . After IL-18 stimulation, the percentage of CD107a-expressing NK cells significantly increased in the co-culture system of NK cells with 721.221 cells compared with the unstimulated co-culture system (14.47% ± 2.67% vs. 9.93% ± 1.94%, t = 6.00, P < 0.001) , while there was no significant difference between the IL-8-stimulated co-culture system of NK cells with K562 cells or P815-Ab cells and the unstimulated co-culture systems (both P > 0.05) . Conclusion:IL-18 could enhance NK cell activity in patients with alopecia areata, likely by promoting natural cytotoxicity receptor-mediated cytotoxicity.
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Objective:To investigate changes in the peripheral interleukin-35 (IL-35) level in patients with alopecia areata, and to assess its modulatory effect on regulatory T (Treg) cell activities.Methods:Totally, 81 patients with alopecia areata (alopecia areata group) and 27 healthy volunteers (control group) were enrolled from Shanxi Provincial People′s Hospital between December 2019 and January 2021. Sera and peripheral blood mononuclear cells (PBMCs) were isolated. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the serum IL-35 level, real-time fluorescence-based quantitative PCR to determine the mRNA expression of IL-35 subunits EBI3 and IL-12p35, and flow cytometry to determine the proportion of CD4 + CD25 + CD127 dim/- Treg cells. Sorted Treg cells were stimulated by recombinant human IL-35, ELISA was performed to detect levels of perforin and granzyme B in the culture supernatant, and real-time fluorescence-based quantitative PCR to determine the mRNA expression of EBI3, IL-12p35, and immune checkpoint molecules, such as programmed death protein 1 (PD-1) , T cell immunoglobulin and mucin protein-3 (Tim-3) , cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and lymphocyte activation gene-3 (LAG-3) in Treg cells. IL-35-stimulated or unstimulated Treg cells were co-cultured with autologous PBMCs, and cell counting kit-8 (CCK8) assay was conducted to assess cellular proliferative activity. Measurement data were compared between 2 groups by using t test, comparisons among multiple groups were carried out by using one-way analysis of variance, correlation analysis was carried out by using Pearson correlation analysis, and enumeration data were compared by using chi-square test. Results:Compared with the control group, the alopecia areata group showed significantly decreased IL-35 levels (90.10 ± 11.98 ng/L vs. 100.74 ± 28.71 ng/L, t= 2.71, P= 0.008) , mRNA expression of EBI3 and IL-12p35 in PBMCs (EBI3: 1.06 ± 0.15 vs. 1.25 ± 0.11, t= 6.09, P < 0.001; IL-12p35: 1.00 ± 0.15 vs. 1.38 ± 0.22, t= 10.16, P < 0.001) , and proportions of Treg cells (5.91% ± 1.17% vs. 6.85% ± 1.23%, t= 3.54, P= 0.001) . In the alopecia areata group, the proportion of Treg cells was positively correlated with the serum IL-35 level ( r= 0.25, P= 0.026) , and the mRNA expression of EBI3 and IL-12p35 in PBMCs ( r= 0.31, 0.24, P= 0.004, 0.032, respectively) . Compared with the control group, the unstimulated Treg cells from the alopecia areata group showed significantly decreased supernatant levels of perforin and granzyme B, mRNA expression of EBI3, IL-12p35 and immune checkpoint molecules ( P < 0.05 or 0.001) , as well as weakened inhibitory effect on the proliferative activity of PBMCs ( P= 0.013) . There was no significant difference in the level of perforin or granzyme B between the recombinant human IL-35-stimulated and unstimulated Treg cells from the patients with alopecia areata (both P > 0.05) . However, the mRNA expression of EBI3, IL-12p35 and immune checkpoint molecules was significantly higher in the IL-35-stimulated Treg cells than in the unstimulated Treg cells in the alopecia areata group ( P < 0.05 or 0.001) , and the inhibitory effect on the proliferative activity of PBMCs was also significantly enhanced in the IL-35-stimulated Treg cells compared with the unstimulated Treg cells ( P= 0.037) . Conclusion:The peripheral IL-35 level was significantly decreased in the patients with alopecia areata, which was closely associated with reduced activities of Treg cells, and IL-35 may be involved in the occurrence of alopecia areata.
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Objective To assess the role of T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) in regulation of interleukin(IL)-12 secretion by CD14+ peripheral blood monocytes from patients with psoriasis vulgaris.Methods From November 2017 to March 2018,a total of 47 patients with psoriasis vulgaris (psoriasis group) and 19 healthy volunteers (control group) were enrolled from Shanxi Provincial People's Hospital.Peripheral blood mononuclear cells (PBMC) were isolated,and stimulated with Toll-like receptor (TLR) ligands.TIM-3 expression and IL-12 secretion by CD14+ monocytes were measured by flow cytometry.After blockade of TIM-3 pathway by anti-TIM-3 neutralizing antibody,changes in the downstream signaling pathway molecules in and IL-12 secretion by CD14+ monocytes were investigated.Two independent samples t-test was used for comparison between two groups,and Pearson correlation test for correlation analysis.Results Under the unstimulated condition,the level of IL-12 secreted by CD14+ monocytes was very low,and the proportion of CD14+TIM-3+ cells was significantly higher in the psoriasis group (12.20％ ± 2.83％) than in the control group (9.91％ ± 1.77％,t =3.270,P =0.001 7).After the stimulation with TLR ligands,the proportion of CD14+IL-12+ cells was significantly lower in the psoriasis group (13.49％ ± 2.80％) than in the control group (28.97％ ± 8.97％,t =10.71,P ＜0.000 1),but the proportion of CD14+TIM-3+ cells was still higher in the psoriasis group (6.80％ ± 1.11％)than in the control group (4.85％ ± 1.37％,t =6.064,P ＜ 0.000 1).The proportion of CD14+TIM-3+ cells was negatively correlated with that of CD14+IL-12+ cells in both the control group and psoriasis group (r =-0.473,-0.371 respectively,both P ＜ 0.05).The TIM-3 pathway blockade could induce the decrease of suppressor of cytokine signaling 1 in CD14+ monocytes,increase the phosphorylation of signaling transducers and activators of transcription 1,and promote IL-12 secretion by CD 14+ monocytes induced by keratinocytes isolated from psoriatic skin lesions.Conclusion TIM-3 plays a crucial role in the negative regulation of CD 14+ monocyte-induced innate immune response in psoriasis.
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Objective@#To assess the role of T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) in regulation of interleukin (IL) -12 secretion by CD14+ peripheral blood monocytes from patients with psoriasis vulgaris.@*Methods@#From November 2017 to March 2018, a total of 47 patients with psoriasis vulgaris (psoriasis group) and 19 healthy volunteers (control group) were enrolled from Shanxi Provincial People′s Hospital. Peripheral blood mononuclear cells (PBMC) were isolated, and stimulated with Toll-like receptor (TLR) ligands. TIM-3 expression and IL-12 secretion by CD14+ monocytes were measured by flow cytometry. After blockade of TIM-3 pathway by anti-TIM-3 neutralizing antibody, changes in the downstream signaling pathway molecules in and IL-12 secretion by CD14+ monocytes were investigated. Two independent samples t-test was used for comparison between two groups, and Pearson correlation test for correlation analysis.@*Results@#Under the unstimulated condition, the level of IL-12 secreted by CD14+ monocytes was very low, and the proportion of CD14+ TIM-3+ cells was significantly higher in the psoriasis group (12.20% ± 2.83%) than in the control group (9.91% ± 1.77%, t = 3.270, P = 0.001 7) . After the stimulation with TLR ligands, the proportion of CD14+ IL-12+ cells was significantly lower in the psoriasis group (13.49% ± 2.80%) than in the control group (28.97% ± 8.97%, t = 10.71, P < 0.000 1) , but the proportion of CD14+TIM-3+ cells was still higher in the psoriasis group (6.80% ± 1.11%) than in the control group (4.85% ± 1.37%, t = 6.064, P < 0.000 1) . The proportion of CD14+TIM-3+ cells was negatively correlated with that of CD14+ IL-12+ cells in both the control group and psoriasis group (r = -0.473, -0.371 respectively, both P < 0.05) . The TIM-3 pathway blockade could induce the decrease of suppressor of cytokine signaling 1 in CD14+ monocytes, increase the phosphorylation of signaling transducers and activators of transcription 1, and promote IL-12 secretion by CD14+ monocytes induced by keratinocytes isolated from psoriatic skin lesions.@*Conclusion@#TIM-3 plays a crucial role in the negative regulation of CD14+ monocyte-induced innate immune response in psoriasis.