Effect of IL-34 on T helper 17 cell proliferation and IL-17 secretion by peripheral blood mononuclear cells from rheumatoid arthritis patients

Interleukin (IL)-34 is a new pro-inflammatory cytokine with elevated expression in rheumatoid arthritis (RA) patients. Our previous study showed that the frequency of T helper 17 (Th17) cells was also elevated in RA patients. Our study aimed to determine the effects of IL-34 on the proliferation, transcription factor expression and cytokine secretion of different subgroups of CD4 + T cells [Th1, Th2, Th17 and regulatory T (Treg) cells] in RA patients. Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of 10 RA patients and stimulated with different concentrations of recombinant human (rh) IL-34 (0, 25, 50 and 100 ng/ml). Flow cytometry was used to determine the frequencies of the 4 subgroups of CD4 + T cells. Reverse transcription-PCR, western blotting and enzyme-linked immunosorbent assays were used to determine the mRNA and protein expression levels of transcription factors and cytokines. As a result, the frequency of Th17 cells was obviously increased under IL-34 stimulation. Moreover, the expression of the transcription factor retinoic acid-related orphan receptor (ROR-γt) and secretion of IL-17 by PBMCs were increased by stimulation with IL-34. However, there were no effects of IL-34 on transcription factors or cytokine secretion in Th1, Th2 and Treg cells. In conclusion, IL-34 can improve the proliferation of Th17 cells and expression of IL-17 in RA patients.


Scientific Reports
| (2020) 10:22239 | https://doi.org/10.1038/s41598-020-79312-z www.nature.com/scientificreports/ cells indistinct effector lineages 13 . T-box (T-bet) and GATA-binding protein 3 (GATA-3) are required for the differentiation of Th1 and Th2 cells, respectively. The transcription factor retinoic acid-related orphan receptor (ROR-γt) is essential for Th17 cell differentiation 14 . Moreover, forkhead box protein 3 (Foxp3) is a master regulator of Treg cells. However, whether IL-34 affects the proliferation, transcription factor expression and cytokine expression of different subsets of CD4 + T cells in RA is still not clear. In this study, we evaluated the effects of IL-34 on Th1, Th2, Th17 and Treg cell proliferation and function to further elucidate the pathogenesis of IL-34 in RA.

Materials and methods
Preparation, stimulation and culture of peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from the peripheral blood of 10 newly diagnosed RA patients by centrifugation (20 °C at 1500 r/ min for 10 min) using Lymphoprep (Fresenius KabiNorge AS, Norway). All patients fulfilled the 2010 American College of Rheumatology (ACR) criteria for RA 15 . The mean age of the RA patients (male/female: 4/6) was 54 (54.00 ± 5.03) years, and the mean disease duration was 6.17 (6.17 ± 3.89) years. None of the patients had any other diseases, and no disease-modifying antirheumatic drugs (DMARDs) were administered. All RA patients were in the acute active phase when enrolled. The present study was approved by the ethics committee of The First Affiliated Hospital of Jinzhou Medical University according to the Declaration of Helsinki. Written informed consent was provided by all the patients. All experiments were performed in accordance with relevant guidelines and regulations, including any relevant details.
Reverse transcription-PCR analysis of the gene expression of TBX21, GATA-3, IL-17A, RORC, IL-10 and Foxp3. Total RNA was extracted from cultured PBMCs with TRIzol and examined by evaluating the A260/A280 ratio for values between 1.8 and 2.0. Reverse transcription of the total mRNA was performed using a routine method. A 0.5-μg sample of total RNA was reverse transcribed using the PrimeScript RT Master Kit. The resulting cDNA was used for amplification by RT-PCR with the SYBR Premix Ex TaqTM Kit and ABI Prism 7000 (Applied Biosystems, Norwalk, CT). The cDNA was subjected to 40 cycles of PCR amplification. Each cycle included 30 s of denaturation at 95 °C, 30 s of annealing at 60 °C, and 30 s of extension at 72 °C. β-actin was amplified as an internal control. The primers used in this study are listed in Table 1. The different gene expression levels were calculated using the 2 −ΔΔCt method, as previously described 16  Statistical analysis. Data with a normal distribution are shown as the mean ± standard error of the mean (SEM). Data with a non-normal distribution are shown as the median (IQR). GraphPad Prism 6.0 was used for statistical analysis. Statistical comparisons between four groups with normal distributions were performed by ANOVA. Statistical comparisons between four groups with non-normal distributions were performed with the Kruskal-Wallis test. Differences were considered statistically significant at values of P < 0.05.

Discussion
In previous studies, IL-34 was shown to have a pro-inflammatory effect, and compared with those in healthy people, the levels of IL-34 in the serum and synovial fluid of RA patients were increased 17 . Our previous study showed that the serum levels of IL-34 in RA patients were significantly elevated and associated with disease activity, including the number of tender joints, ESR, CRP and DAS28-CRP 11 . In a pilot study, we showed that IL-34 might promote the secretion of IL-17 by PBMCs in RA patients 11 . These results showed that IL-34 might play a role in RA pathogenesis by regulating Th17 cells.
In our previous studies, we found that IL-34 promoted IL-17 production in PBMCs from RA patients but not in those from healthy donors 18  In this study, we found that IL-34 could stimulate the differentiation of Th17 cells in PBMCs from RA patients. However, IL-34 had no effect on the differentiation of other Th cell subsets, including the Th1, Th2 and Treg   Fig. 1A,B). These data suggested that the effect of IL-34 might be specific to RA. Transcription factors are required for Th cell differentiation 19 . Our data showed that IL-34 led to activation of ROR-γt and thereby promoted the expression of IL-17. In contrast, IL-34 had no effect on the expression of transcription factors for Th1 (T-bet), Th2 (GATA-3) and Treg (Foxp3) cells. We further detected the mRNA expression of TBX21, GATA-3, RORC and FOXP3 by RT-PCR. We showed that IL-34 could increase the mRNA levels of RORC and IL-17. In contrast, IL-34 had no effect on the mRNA expression of TBX21, GATA-3 or FOXP3.
IL-17 is mainly produced by Th17 cells 20 . Our study showed that IL-34 promoted the secretion of IL-17 by PBMCs from RA patients in a dose-dependent manner. However, IL-34 had no effect on the secretion of IFN-γ or IL-10.
CSF-1R is the main receptor of IL-34 21 . A previous study showed that blocking CSF-1R in mice had a protective effect against collagen-induced arthritis and was more effective than a TNF-α antagonist 22 . Anti-CSF-1R PBMCs were stimulated with anti-CD3 and anti-CD28 for 3 days before RNA extraction. The differences between four groups were tested by ANOVA and after post Tukey test.
Our future work may focus on blocking CSF-1R to learn more about the role of IL-34 in Th17 cell differentiation.
In conclusion, IL-34 can promote Th17 cell proliferation, transcription factor expression, and IL-17 secretion. We hypothesized that IL-34 acts upstream of Th17 cell differentiation and plays an important role in RA pathogenesis. This may provide a new target for RA therapy.
Informed consent. We claim that all patients have been given informed consent prior to their inclusion in the study and the research was approved by Ethics Committee of the First Affiliated Hospital of China Medical University.