Cofilin‐1 participates in the hyperfunction of myeloid dendritic cells in patients with severe aplastic anaemia

Abstract Cofilin‐1 interacts with actin to regulate cell movement. The importance of cofilin‐1 in immunity has been established, and its involvement in a number of autoimmune diseases has been confirmed. However, its role in severe aplastic anaemia (SAA) remains elusive. Thus, the aim of the current study was to investigate the role of cofilin‐1 in patients with SAA. Flow cytometry, Western blotting and real‐time quantitative reverse transcription‐polymerase chain reaction were performed to detect the mRNA and protein expression of cofilin‐1 in myeloid dendritic cells (mDCs) from patients with SAA. The expression of cofilin‐1 was then suppressed via siRNA, and its effects on mDCs and downstream effector T‐cell function were evaluated. Cofilin‐1 expression was higher in mDCs from patients with SAA and correlated with routine blood and immune indexes. Moreover, cofilin‐1 knockdown in mDCs from patients with SAA reduced their phagocytic capacity, migration capacity, and CD86 expression through F‐actin remodelling, downregulating the stimulatory capacity of mDCs on CD4+ and CD8+ T lymphocytes. Collectively, these findings indicate that cofilin‐1 participates in the hyperfunction of mDCs in patients with SAA and that the downregulation of cofilin‐1 in mDCs from patients with SAA could be a novel treatment approach for SAA.

study, we aimed to investigate the origin of SAA immune-related pathogenesis. The results showed that mDCs of patients with SAA are in a state of overactivation with enhanced antigen presentation. 5 Therefore, we proposed that myeloid DCs (mDCs) might play an important role in the primary immune responses related to SAA.
To further investigate the mechanism of mDC overactivation in SAA, Liu et al. 6 carried out a proteomic analysis of mDCs from patients with SAA. The results revealed that cofilin-1, glucose-6phosphate dehydrogenase and the pyruvate kinase enzyme M2 in mDCs from patients with SAA may contribute to mDC overactivation. In this study, we investigated the mechanism of cofilin-1 in the overactivation of mDCs from patients with SAA. Cofilin-1, a member of the actin-depolymerizing factor/cofilin protein family, interacts directly or indirectly with the actin cytoskeletal system to participate in the formation, function, and recombination of the actin skeleton and regulate cell motility. Cofilin-1 is one of the major regulators of intracellular actin remodelling. On one hand, cofilin-1 cleaves actin filaments and causes actin depolymerization. On the other hand, it can generate new free ends and provide more actin monomer sources for the extension of the fast-growing end of F-actin to increase actin polymerization. 7,8 Previous studies have elucidated the role of the cytoskeletal system in autoimmune diseases, including cofilin-1. 9,10 Actin polymerization and depolymerization, mediated by cofilin-1, generate a time-space-coordinated dynamic actin cycle that is critical to various biological processes, including the immune response. It has been confirmed that cofilin-1 plays an important role in the function of DCs. Activation of cofilin can promote phagocytosis 11,12 and inactivation of cofilin-1 reduces the migration ability of DCs and their ability to co-stimulate T cells. 13 Therefore, we speculated that the involvement of cofilin-1 in mDC activation may be of interest for the further exploration of SAA pathogenesis and therapy development. Here, we explored the role of cofilin-1 in patients with SAA and related molecular mechanisms that might be potential targets for SAA treatment. six males and nine females), with a median age of 45 (range  years, were enrolled. There was no significant difference in participant sex or age between groups (p > 0.05).

| Study subjects
Studies involving human participants were reviewed and approved by the Ethics Committee of Tianjin Medical University General Hospital (IRB2019-KY-098). Written informed consent to participate in this study was provided by each participant or his/her legal guardian/next of kin.

| Flow cytometric analysis
To detect cofilin-1 in mDCs, peripheral blood samples were col- Th1-related (IL-2, TNFα and IFNγ) and Th2-related (IL-4, IL-6 and IL-10) cytokine concentrations were measured using the Human Th1/ Th2 Assay Kit (Celgene Biotech, Hangzhou, China). Plasma was obtained by centrifuging the blood samples at 300 × g for 5 min. The FITC Annexin V Apoptosis Kit (BD Pharmingen) was used to detect apoptosis. The cell concentration was adjusted to 1 × 10 6 /mL with binding buffer (1×). Next, 5 μL of FITC Annexin V was added to the cells, which were then incubated in the dark at room temperature for 30 min. After incubation, 5 μL of propidium iodide (PI; 50 μg/mL) was added, and the cells were then incubated in the dark for 5 min.
Annexin V − PI − cells were considered as live cells, Annexin V + PI − cells as early apoptotic cells, Annexin V + PI + cells as late apoptotic cells and Annexin V − PI + cells as dead cells.
FITC-CD8 antibody (BD Pharmingen) was used to label surface CD8 before permeabilization. Intracellular perforin and granzyme B in CD8 + T lymphocytes were detected using PE-perforin and APCgranzyme B (BD Pharmingen), respectively, after permeabilization.
An isotype-matched control antibody (IgG1; BD Pharmingen) was used. Data acquisition was performed on an FACS-Calibur, and the acquired data were analysed using CellQuest 3.1 (Becton Dickinson, Franklin Lakes, NJ, USA).
Protein loading buffer (Solarbio) was added to make proteins fully bind to sodium dodecyl sulphate after denaturation. The protein samples were stored at −80°C until use. The proteins were sepa-

| RNA extraction and qRT-PCR
TRIzol reagent (Invitrogen) was added to the cell suspension to lyse cells, and chloroform was added to collect the total RNA.
Isopropanol and 75% ethanol were used to isolate and purify RNA. The thermal cycling protocol used was 95°C for 30 s, followed by 50 amplification cycles (95°C for 5 s, 54.6°C for 45 s and 70°C for 30 s), and 4°C for 10 s to terminate the reaction. The cycle threshold (Ct) was recorded, and the 2 −ΔΔCt method was used to calculate the relative expression level.

| Small interfering RNA (siRNA)
mDCs were divided into control, cofilin-1 siRNA and scrambled siRNA groups. Sorted mDCs were counted, and 2.5 × 10 5 cells were suspended in 400 μL of culture medium and seeded in a 24well plate. Thereafter, 100 μL of transfection reagent mixture con-

| Immunofluorescence assay
The cell concentration was adjusted to 4 × 10 5 /mL with medium.

| Co-culture of mDCs and lymphocytes
Suspensions of mDCs and T lymphocytes, which were from the same individual, were adjusted to 1 × 10 6 /mL and mixed at a ratio of 1:1 in each well of 24-well plates. CD3 and CD28 monoclonal antibodies were added to the medium at a final concentration of 400 ng/mL. The 24-well culture plate was placed in an incubator at 37°C with 5% CO 2 for 72 h.

| Detection of T lymphocyte proliferation using carboxyfluorescein diacetate succinimidyl ester (CFSE)
Carboxyfluorescein diacetate succinimidyl ester is a new dye that can fluorescently label live cells. The basic mechanism is as follows: CFSE can easily penetrate cell membranes, covalently bind to intracellular proteins in live cells, and release green fluorescence upon hydrolysis. In the process of cell division and proliferation, its fluorescence intensity decreases step by step with cell division. The labelled fluorescence can be equally divided between two progenitor cells, so its fluorescence intensity is half of that of parental cells.
According to this characteristic, we used CFSE to detect cell proliferation. CD8 + T lymphocytes were counted, and the cell concentration was adjusted to 1 × 10 7 /mL. CFSE (BD Pharmingen) storage solution (10 mM) was diluted 1:1000 with PBS to obtain CFSE reaction solution (10 µM). An equal amount of CSFE reaction solution was mixed with the CD8 + T lymphocyte suspension and incubated at 37°C in dark for 15 min to stain the cells. The mean fluorescence intensity (MFI) of CFSE in the CD8 + T lymphocytes was detected using flow cytometry.

| Statistical analysis
SPSS version 21.0 was used for statistical analyses. Results are expressed as mean ± standard deviation. All data were normally distributed. The one-way ANOVA was used for comparison between groups. Turkey test was used for statistical analysis between two groups. Spearman's test was used to assess the linear correlation of the results. Results with P value less than 0.05 were considered statistically significant.

| RE SULTS
3.1 | Cofilin-1 is upregulated in the mDCs of patients with SAA mDCs harvested after 7 days of culture exhibited typical irregular morphology, with varying cell sizes and many dendritic protrusions of different lengths, thicknesses and densities ( Figure 1A). As revealed by flow cytometry, the percentage of CD11c + HLA-DR + mDCs among all cultured cells was 50%-70%. After sorting, the purity of mDCs was >90% ( Figure 1B).

| Cofilin-1 affects the function of mDCs in patients with SAA
The expression of cofilin-1 in mDCs was successfully reduced by siRNA, as confirmed by FACS, qRT-PCR and Western blotting ( Figure 2D-F).
To assess phagocytic activity, FITC-dextran, which is phagocytosed by mDCs and can be detected by FACS or under a fluorescence microscope, was employed. The difference in the FITC-positive rate between 37°C and 4°C was 22.64% ± 12.53%, 40.07% ± 11.90% and 44.83% ± 17.33% in the cofilin-1 siRNA, scrambled siRNA, and control groups, respectively. The downregulation of cofilin-1 led to significantly lower phagocytic capacity (p < 0.05). CD86 expression on mDCs from the cofilin-1 siRNA group was significantly lower than that on mDCs from the scrambled siRNA group (73.80% ± 17.18% vs. 77.26% ± 14.39%, p = 0.034). However, there was no difference in CD80 expression among the three groups ( Figure 4A). As cofilin-1 is involved in cytoskeleton regulation through Factin, we compared the morphology and distribution of F-actin in the groups using immunofluorescence. F-actin was mostly distributed under the membrane in the control and scrambled siRNA groups. In contrast, both F-actin content and cell protrusion density were increased in the cofilin-1 siRNA group, resulting in significant remodelling ( Figure 4B).

| Cofilin-1 in mDCs from patients with SAA participates in the activation of CD4 + and CD8 + T lymphocytes
The purity of CD4 + and CD8 + T lymphocytes obtained by MACS was over 90%. After co-culture of mDCs and CD4 + or CD8 + T lymphocytes, which were from the same individual for 72 h, the cells proliferated and aggregated into clusters floating in the medium. mDCs from the control, cofilin-1 siRNA and scrambled siRNA groups were co-cultured with CD4 + T lymphocytes. The concentrations of Th1-and Th2-related cytokines in the co-culture supernatant were measured, and they are shown in Tables 1 and 2. The knockdown of cofilin-1 in mDCs led to a decrease in their ability to stimulate the production of Th1-related (IL-2, TNFα and IFNγ) and Th2-related (IL-6) cytokines by CD4 + T cells.

| DISCUSS ION
In patients with SAA, undetermined antigens stimulate an increase in the number and function of mDCs, and this contributes to a Th1/Th2 imbalance and CTL overactivation, leading to pancytopenia. 16 The dysregulated immune response observed in patients with SAA involves a rapid and potent response by numerous acti-

CO N FLI C T O F I NTE R E S T
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.