ERK/MEK Pathway Regulates Th17 Cell Differentiation in Patients with Pemphigus Vulgaris

Abstract

Background: T helper (Th) cells are involved in the pathogenesis of pemphigus vulgaris (PV). However, the mechanism still needs more exploration.

Aims: This study aimed to evaluate the molecular mechanism of the dysregulation of Th17 cells in the peripheral blood of patients with PV.

Materials and methods: Serum levels of IL-17 and anti-Dsg3 titres in patients with PV were analysed using ELISA. The mRNA expression of retinoic acid orphan receptor γt (RORγt) in CD4⁺ T cells was detected using reverse transcription-quantitative PCR (qPCR). The number of Th17 cells was examined using flow cytometry. Western blot analysis and immunofluorescent staining were also performed to investigate the expression levels of ERK/MAPK signalling proteins and Th17 lineage-associated proteins.

Results: The proportion of Th17 cells and Th17 spectrum-associated proteins (p-STAT3, RORγt and IL-17) were upregulated in CD4+ cells in PV patients. The increased transcriptional levels of RORγt and IL-17 correlated positively with the severity of PV. Elevated phosphorylation of the ERK signalling factors was found in the collected CD4+ T cells in PV patients. The inhibition of the ERK signalling pathway significantly reduced the differentiation of Th17 cells in PV patients in vitro.

Conclusion: Th17 cells are essential in the dysregulation of PV, and ERK signalling is involved in Th17-type immunity and promotes the development of PV. The study here provides us with a potential therapeutic target for PV.

Keywords: ERK signalling; Th17 cell; pemphigus vulgaris.

Figure 1

Abnormality of Th17 cells in the peripheral blood of PV patients. (a) The proportion of Th17 cells (CD3+, CD8-, IL-17A+) to CD4+ T cells (CD3+, CD8-) between healthy controls and the PV patients, which was detected by flow cytometry and illustrated in a histogram. (b) The serum levels of IL-17 were detected using ELISA. (c) Relative mRNA levels of RORγt in CD4+ T cells were measured by qPCR. (d) Immunoblot analysis of Th17 lineage-associated proteins (STAT3, p-STAT3, RORγt and IL-17) in CD4+ T cells. The relative intensity was normalised to the levels of GAPDH and showed in a histogram. (e) Immunofluorescence staining of accumulated p-STAT3 in Th17 cells. p-STAT3 was stained in red, and nuclei were stained by DAPI in blue. Magnification: 400×. Scale bar: 50 μm. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Figure 2

Correlation between the characteristics of Th17 cells (RORγt mRNA levels and IL-17 serum levels) and the clinical severity of PV. (a) The correlation between relative expression of RORγt and the titres of autoantibody against Dsg3. (b) The relationship between relative expression of RORγt and the ABSIS score of PV patients. (c and d) The correlation between serum IL-17 levels and disease activity-related factors

Figure 3

Expression of the ERK signalling pathway molecules in CD4+ T cells from PV patients compared with healthy controls. (a) Detection of MEK, p-MEK, ERK and p-ERK by Western blot. The relative intensity was normalised to GAPDH. (b and c) Immunofluorescence staining of accumulated p-MEK and p-ERK in CD4+ T cells. Both p-MEK and p-ERK were stained red; nuclei were stained by DAPI in blue. Magnification: 400×. Scale bar: 50 μm. ***, P < 0.001

Figure 4

Inhibition of the ERK signalling prevented Th17 cell differentiation in PV. The inhibition was performed in vitro by the addition of PD98059. DMSO was used as a reagent control. (a) The proportion of Th17 cells (CD3+, CD8-, IL-17A+) to CD4+ T cells (CD3+, CD8-), which was detected by flow cytometry. (b) The mRNA levels of RORγt were determined using qPCR. (c) The IL-17 levels in cell culture supernatant were detected by ELISA. *, P < 0.05; **, P < 0.01