Mechanical Growth Factor Promotes FOXP3 Expression in Regulatory T Cells and Enhances its Function in Treating Ankylosing Spondylitis


 BackgroundTo verify that mechanical growth factor (MGF) may be an effective target for treating ankylosing spondylitis. MethodsFOXP3 expression was measured in Treg cells from healthy male subjects treated with MGF. A rat model of ankylosing spondylitis was established, and the levels of ankylosing spondylitis-related factors (tumor necrosis factor [TNF]-α, interleukin [IL]-2, and IL-10) were measured. ResultsWe found that the proliferation and total number of Treg cells, as well as FOXP3 expression levels, increased significantly in the MGF-treated groups versus in the control. The levels of inflammation, bone destruction, and new bone formation were significantly decreased in animals treated with MGF compared to in the control group. TNF-α expression decreased significantly, while IL-2 and IL-10 levels increased significantly in the MGF group compared to in the control. Conclusions﻿MGF may delay disease progression in ankylosing rats by inducing FOXP3 expression, promote FOXP3+ Treg cell proliferation and differentiation and reducing the expression of TNF-α and increasing the expression of IL-10 and IL-2.


Introduction
Ankylosing spondylitis (AS) is an autoimmune disease that causes in ammation of the sacroiliac joints and muscle attachment points and is closely related to the HLA-B27 gene. It typically begins to develop around the age of 30 years, affecting the central axis; in ammation of the sacroiliac joint is an important feature of this disease [1]. As the disease progresses, calci cation and rigidity of the annulus brosus and nearby ligaments often occur. The clinical manifestations include lower back pain and reduced spine mobility, which frequently lead to long-term patient disability and cause serious psychological and physiological damage. Non-steroidal antiin ammatory drugs remain the rst-line recommended treatment for AS [2]. Conventional synthetic disease-modifying antirheumatic drugs, such as methotrexate and sulfadiazine, may slightly improve peripheral symptoms but are ineffective for treating axial manifestations [3]. For patients who fail to respond to non-steroidal anti-in ammatory drugs, biological disease anti-rheumatic drugs, mainly tumor necrosis factor (TNF) inhibitors, have become among the most powerful treatment options [2]. However, TNF-α antagonists are only effective in 40% of patients, and their associated high risks of infection and tumor development [4] have prompted researchers to identify safer and more effective treatments. Among them, regulatory T (Treg) cells have attracted increasing attention [5].
Regulatory T lymphocytes comprise a small subset of CD4+ T cells and play crucial roles in maintaining immune tolerance and preventing autoimmunity [6]. In 1995, Sakaguchi rst identi ed Treg cells as a subset of CD4+ T cells with constitutive CD25 expression [7]. In 2003, the forkhead box P3 (FOXP3) transcription factor was identi ed as a key transcription factor of Treg cells, and was later identi ed as an essential component for the development, maturation, and immunosuppressive functions of Treg cells [8]. CD4, CD25, and CD127 antibodies can be labeled to effectively isolate CD4+CD25+FOXP3+ Treg cells [9,10]. A previous study showed that Treg cells from patients with AS have functional defects and play important roles in AS [5]. Therefore, it is important to determine how the proliferation and immunetolerance functions of Treg cells can be enhanced in AS. Enhancement of Treg cell functions has emerged as a promising strategy and new direction for treating AS.
AS mostly affects the central axis of the spine, sacroiliac joint, and Achilles tendon, which are subjected to the highest mechanical loads in the body. In addition, ossi cation starts at the attachment points of muscles, which also experience concentrated mechanical loads. Therefore, a relationship exists between mechanical load and AS. In terms of disease treatment, functional exercise can effectively alleviate the symptoms of AS in affected patients [11]. The painful symptoms in patients with AS are aggravated at night, and patients cannot remain in bed for a long time. However, if patients get up and move their waist, the symptoms are rapidly relieved. In addition, if a patient engages in su cient physical activity during the day, the night pain is reduced or even disappears, and the patient's sleep quality is greatly improved.
Karatay et al. [12] found that functional exercise can effectively increase the expression levels of insulinlike growth factor-1 (IGF-1) in the peripheral blood, suggesting that IGF1 can relieve disease symptoms in patients with AS.
Mechanical growth factor (MGF, also known as mechano growth factor or force growth factor) is an alternative splicing variant of IGF-1. All mRNA splice variants of IGF-1 contain exons 3 and 4. Direct splicing of mRNA exons 4 and 6 results in the formation of IGF-1 Ea, whereas continuous splicing of exons 4-6 generates MGF, which is referred to as IGF-1 Ec in humans and IGF-1 Eb in rodents [13]. Previous ndings con rmed that mechanical loading and muscle damage can induce MGF expression, and that MGF is sensitive to mechanical stimulation [14]. Interestingly, IGF-1 can speci cally promote Treg cell proliferation and differentiation and elicits a therapeutic effect by enhancing immune tolerance in various animal models of immune diseases [15]. Therefore, we hypothesized that MGF can enhance or restore the body's immune tolerance and immune suppression functions by increasing the number of Treg cells and their functionality, thereby improving their therapeutic effects against AS. In the present study, we used a rat model of AS to explore the effects of MGF in AS to establish whether a treatment strategy targeting MGF and Treg cells is be effective for AS. Medical University, and all subjects signed an informed consent form. Fresh peripheral venous blood was drawn from volunteers and stored in a heparin-anticoagulation tube at 4°C. Next, 4 mL of lymphocyte separation solution (Stemcell Technologies, Vancouver, Canada) was added to a 15-mL centrifuge tube, slowly added 8 mL of blood at 1 cm above the liquid surface of the centrifuge tube to avoid the liquids from mixing with each other, and then centrifuged at 2000 rpm for 30 min in a low-speed centrifuge at 25℃. After centrifugation, cells in the centrifuge tube were divided into four layers from top to bottom. The rst layer was blood plasma, second layer was ring-shaped milky white lymphocytes, third layer was transparent separation liquid, and fourth layer was red blood cells. CD4+CD25+CD127-Treg cells (CD4+CD25+) were isolated from the second layer. Brie y, a CD127 MicroBead Kit (Miltenyi Biotec, Gladbach Bergisch, Germany) was used to isolate CD4+CD127-cells, and a CytoFLEX ow cytometer (Beckman Coulter, Brea, CA, USA) was used to sort CD4+CD25+ Treg cells. To determine the purity of the isolated Treg cells, we used an uorescein isothiocyanate (FITC)-conjugated CD4 antibody, allophycocyanin-conjugated CD25 antibody, phycoerythrin-conjugated CD127 antibody, phycoerythrinconjugated rat IgG2K isotype control, allophycocyanin-conjugated rat IgG1K isotype control, and FITC-conjugated rat IgG2K isotype control (eBioscience, San Diego, CA, USA). The experimental procedures were performed in accordance with the manufacturer's instructions (Supplementary Data Fig  1).

Expansion of Treg cells in culture
The Treg cell expansion medium, containing 500 IU/mL Human IL2 Improved Sequence, premium grade (Miltenyi Biotec), 5% human AB serum (GEMINI, West Sacramento, CA, USA), 0.01 mM βmercaptoethanol (Solebold, Beijing, China), 1% penicillin (Solebold), and 100 nmol/L rapamycin (Solebold), was prepared, and the mixture was added to 500 mL of TexMACS GMP Medium (Miltenyi Biotec). The extracted Treg cells were resuspended in the expansion medium at a concentration of 1 × 106 cells/mL. Aliquots of the cell suspension were added to a U-shaped 96-well plate (100 µL per well), followed by addition of anti-CD3/CD28 MACSiBeadTM particles (Miltenyi Biotec). The 96-well plates were incubated at 37°C in 5% carbon dioxide for 7 days. During the incubation period, spent medium was removed, and fresh medium was added based on the color of the culture medium and morphology of the cells under a microscope. After 7 days, the expansion was complete, and magnetic beads were removed using a MACSiMAG Separator (Miltenyi Biotec).

Carboxy uorescein succinimidyl ester (CFSE) labeling of Treg cells
Sorted CD4+CD25+CD127-Treg cells were divided into two groups. One group was xed with 2% paraformaldehyde and regarded as negative cells. The other group was stained with CFSE (eBioscience) and xed in 2% paraformaldehyde, after which the uorescence intensity of the two groups was measured and compared.

Co-culture of Treg cells and MGF
CFSE-stained Treg cells were divided into ve groups, each of which was sub-divided into three fractions.
The ve groups were the control group, 100 nM MGF group, 200 nM MGF group, 400 nM MGF group, and 800 nM MGF group. Each group was mixed with the same amount of Treg cell suspension and anti-CD3/CD28 MACSiBeadTM particles (Miltenyi Biotec) and saline or 100, 200, 400, or 800 nM MGF (Kangtide Biotechnology, Beijing, China). The Treg cells were cultured for 7 days, the magnetic beads were removed, and cell proliferation was analyzed by measuring uorescence in the FITC channel by ow cytometry.

Effect of MGF on FOXP3 expression in Treg cells
Based on the results obtained after co-culturing the Treg cells with MGF, we co-cultured Treg cells in normal saline or 800 nM MGF. RNA extraction, reverse transcription, and ampli cation of FOXP3 cDNA from both groups of Treg cells were performed using a micro RNA extraction kit, high-e ciency reverse transcription kit, and uorescent quantitative PCR kit (eBioscience). FOXP3 cDNA was ampli ed using the forward primer 5′-CTCTTCTTCCTTGAACCCCAT3′ and reverse primer 5′-CTGGAGGAGTGCCTGTAAG-3′, which were obtained from Shanghai Shenggong Biological Company (Shanghai, China).

Animals
We obtained 6-12-week-old male and female HLA-B27/Huβ2m transgenic rats weighing 250-300 g from the Rheumatology Team of the University of Texas Southwestern Medical Center (Dallas, TX, USA) [16].
This rat strain possesses 20 copies of the HLA-B27 gene and 50 copies of the Hu-β2m microglobulin gene [16][17][18], and is the most representative available animal model of AS. All animal breeding and experiments were carried out in the speci c pathogenfree laboratory of the Animal Experiment Center of Guangxi Medical University and were approved by the Institutional Ethics Committee.

Orchiectomy
Under normal circumstances, male HLA-B27/Huβ2m transgenic rats have an 80% incidence rate of ankle arthritis between days 100 and 200 and 40% incidence rate of spondylitis between days 125 and 225; in contrast, female B27/Huβ2m transgenic rats do not develop the disease. The reason for this difference is that epididymo-orchitis is crucial for the pathogenesis of subsequent SA in male rats [17]. Therefore, to avoid interference, we performed bilateral epididymal testis castration on 4-week-old male HLA-B27/Huβ2m transgenic rats based on a standard method [19].

Immunization
To increase the incidence of AS and shorten the test period, 90 µg of inactivated Mycobacterium tuberculosis (Solarbio, Beijing, China), dissolved in incomplete Freund's adjuvant, was injected subcutaneously at the base of the tail of 6-week-old female and castrated male rats; this was performed to increase the incidence of rat SA to 100% and greatly shorten the time of onset [18].

Preventive treatment with MGF
Eighteen HLA-B27/Huβ2m transgenic rats were immunized with M. tuberculosis and randomly divided into a control group, low-dose MGF group, or high-dose MGF group (n = 6 rats/group). Preventive treatment with MGF began 1 week after M. tuberculosis immunization. The control group was intraperitoneally injected with 3 mL/kg normal saline, low-dose MGF group was intraperitoneally injected with 3 µg MGF/kg, and the high-dose MGF group was intraperitoneally injected with 10 µg MGF/kg every 3 days. MGF preventive treatment lasted for 7 weeks, during which time the rats' ankles and tail vertebrae were photographed and recorded to measure the severity of symptoms.

Serological analysis
Rats were sacri ced by cervical dislocation. Rat ankles and spine joints were cut into bone fragments (≤1 cm) and placed in ethylenediaminetetraacetic acid decalci ed solution (Sangon, Shanghai, China). The ratio of the solution to the volume of bone tissue was greater than 20. The bone fragments were incubated in ethylenediaminetetraacetic acid decalci cation solution at room temperature, and the solution was replaced every 3 days. Decalci cation was considered as complete when a syringe needle could easily penetrate the bone compact without resistance. Bone sections were stained with hematoxylin and eosin (H&E) using standard methods to observe in ammation and bone destruction, and the sections were stained with safranin O to observe new bone formation.

Histological analysis
Rats were sacri ced by cervical dislocation.Rat ankles and spine joints were cut into bone fragments (≤1 cm) and placed in ethylenediaminetetraacetic acid (EDTA) decalci ed solution (Sangon, Shanghai). The ratio of the solution to the volume of bone tissue was greater than 20. The bone fragments were incubated in the EDTA decalci cation solution at room temperature, and the solution was replaced every 3 days. Decalci cation was considered to be complete when a syringe needle could easily penetrate the bone compact without resistance. Bone sections were stained with H&E according to standard methods to observe in ammation and bone destruction, and sections were stained with safranin O to observe new bone formation.

Histological score
Each joint sample was treated as an independent sample for histological analysis. Two independent observers (QHY and YSS) semi-quantitatively scored the stained samples. Based on similar research [20], the score was based on three factors: in ammation, bone destruction, and new bone formation. Each parameter was assigned a score ranging from 0 to 3 (0 = normal, 1 = mild in uence, 2 = moderate in uence, 3 = severe in uence), and the average sum of 10 elds under a microscope was determined to be the nal joint score of each sample.

Statistical analysis
FlowJo software (version 10; TreeStar, Ashland, OR, USA) was used to graph the cell proliferation data obtained by ow cytometry. GraphPad software (version 9.0, GraphPad, Inc., La Jolla, CA, USA) and SPSS Statistics software (version 24, SPSS, Inc., Chicago, IL, USA) were used for statistical analysis.
Differences between groups were compared by independent sample t-test, and differences among multiple groups were compared by one-way analysis of variance. P < 0.05 was used as the threshold for statistically signi cant differences (*P < 0.05; **P < 0.01;***P < 0.001).
Compared with the control group, the percentage of proliferating cells (relative to the total number of cells) increased signi cantly (P < 0.01) in the 400 and 800 nM MGF groups (Fig. 1F). The effect of MGF treatment on Treg cell proliferation was measured by comparing FOXP3 expression levels in the control and experimental groups (800 nM MGF). FOXP3 expression in the MGF experimental group increased signi cantly to 1.47 ± 0.12-fold that of the control group (Fig. 1G), indicating that MGF increased both the number and activity of Treg cells.
3.2 Effect of MGF on clinical symptoms of HLA-B27/Huβ2m transgenic rats and differences in serum IL-10, IL-2, and TNF-α levels in rats before and after MGF treatment After immunizing rats in different groups with inactivated M. tuberculosis, we scored the degree of in ammation in the wrists and arthritis in the ankles and spinal joints. After 7 weeks of prophylactic MGF treatment, the symptom severity of the wrists, ankles, and spinal joints signi cantly differed in each group. Swelling of the front paw, back grasping joints, and spine joints was most severe in the control group ( Fig. 2A-C) and was lowest in the high-dose MGF group (Fig. 2G-I), followed by the low-dose MGF group (Fig. 2D-F).
In addition, the skin around the wrists and ankle joints in the highdose MGF group had a normal color and the tail structure was normal, whereas the skin around the spine in the low-dose MGF and control groups showed persistent erythema and bamboo-like morphology. The arthritis score in the high-dose MGF group was signi cantly lower (P < 0.05) than that in the control group (Fig. 3A). Our results show that high-dose MGF intraperitoneal injection reduced the severity of SA in rats.
Before MGF intervention, the serum levels of IL-2, IL-10, and TNFα in the rats in each group did not signi cantly differ. After 7 weeks of MGF intervention, the serum IL-2 levels of rats in the high-dose MGF group (1117.95 ± 9.86 pg/mL) were higher than those in the control group (952.50 ± 30.29 pg/mL; P < 0.01; Fig. 3B). The serum IL-10 levels in the high-dose MGF (9.02 ± 0.85 pg/mL) and low-dose MGF (8.53 ± 0.83 pg/mL) groups were higher than those in the control group (5.27 ± 1.03 pg/mL; P < 0.05; Fig. 3C).
H&E and safranin O staining of the rat spinal joint sections suggested that in ammation was decreased, the number of new bone-forming cells was decreased, and bone destruction was decreased signi cantly in a dose-dependent manner in the MGFtreated groups compared to in the control group (Fig. 4).

Discussion
AS is an autoimmune disease involving in ammation of the joints and muscle attachment points. As the disease progresses, the annulus brosus and surrounding ligaments gradually become in amed and calci ed, leading to progressive stiffness, bone destruction, and new bone formation. Studies of autoimmune diseases have mainly focused on Treg cells and Th17 cells [20]. Importantly, Treg cells are the key to immune tolerance and are important in maintaining the immune system. Defects in Treg cell function play a vital role in the pathogenesis of autoimmune diseases [21]. Previous data con rmed that the percentage of Treg cells in the blood of patients with certain autoimmune diseases may decrease, increase, or remain unchanged. However, it is generally accepted that Treg cells function abnormally in patients with autoimmune diseases. The number of Treg cells in the peripheral blood circulation or target organs of patients with autoimmune diseases is normal but their immune suppression and immune tolerance functions are reduced [22]. CD4+CD25+ Treg cells expressing FOXP3 maintain immune regulation and immune tolerance by removing abnormal substances harmful to the host or preventing excessive immune system responses to maintain immunity [23]. FOXP3 plays a key role in the development and function of Treg cells [24]. Mutations in human FOXP3 can lead to incomplete development of Treg cells and affect their normal immune tolerance and immune regulation functions. In turn, this can promote the emergence of various immune diseases, including AS, in ammatory bowel disease, psoriatic arthritis, systemic lupus erythematosus, and dry synthesis by allowing Treg cells to perform their normal functions. In this study, we evaluated the effect of MGF on the proliferation of human CD4+CD25+CD127-Treg cells in vitro. Additionally, the expression level of FOXP3 in Treg cells through MGF intervention signi cantly exceeded the above. This indicates that MGF can promote the proliferation and function of Treg cells.
Previous studies reported that 100-1,000 µg of heat-inactivated M. tuberculosis can only induce arti cial organs in wild-type rats [25,26,27,28]. A paper published in 1961 reported that immunization with a large dose of heat-inactivated M. tuberculosis ( ve times with 500 μg) could cause spondylitis in wild-type rats [29], which requires consideration in terms of safety and ethics. We used the HLA-B27 transgene and established methods for suture, bone destruction, and new bone formation of rat spinal joints after MGF intervention. The severity of histopathological manifestations was reduced by MGF treatment. The highdose MGF group did not produce new bone tissue, and local resection and in ltration was signi cantly improved. This suggests that MGF can relieve the symptoms of AS.
To determine the molecular mechanism by which MGF exerts its obvious protective effect during AS, we measured the levels of the cell death markers TNF-α, IL-2, and IL-10 in the serum of AS model rats. TNF-α is a pro-in ammatory cytokine mainly produced by in ammatory cells such as monocytes, macrophages, and activated T cells. TNF-α enhances the in ammatory response of AS, intervertebral disc degeneration [30], and thrombosis [31]. Our experiments showed that before MGF intervention, there was no signi cant difference in the serum TNF-α levels of rats in each group; After MGF intervention, MGF showed reduced serum TNF-α levels in AS rats, leading to reduced symptoms of AS.
IL-2 is one of the most widely studied cytokines and has a wide range of functions, particularly in the immune system, which plays an important role in maintaining immune resistance [32]. IL-2 also promotes the proliferation of CD4+CD25+ Treg cells. The combination of IL-2 and IL-2 receptors and downstream IL-2 pathway promote the proliferation and differentiation of Tregs, maintain stability, and continue to express FoxP3 and exert immunosuppressive functions [15]. Our results show that after MGF intervention, IL-2 levels in the MGF high-dose group were signi cantly higher than those in the control group. MGF may promote the proliferation and differentiation of Treg cells by promoting IL-2 expression. Promotion of FOXP3 expression may enhance the immunosuppressive function of Treg cells, thereby alleviating the symptoms of AS in rats. The speci c pathway involved requires further analysis.
IL-10 is a pleiotropic anti-in ammatory and immunosuppressive cytokine. An imbalance in IL-10 levels plays an important role in the development of autoimmune diseases such as rheumatoid arthritis, Crohn's disease, and multiple sclerosis [33]. In the presence of proin ammatory signals, almost all immune cells (including cytotoxic T lymphocytes, CD4+ T cells, NK cells, B cells, and macrophages) produce IL-10 to exert anti-in ammatory effects. Recent studies showed that IL-10 can enhance the function of Treg and may be useful for treating autoimmune diseases [34]. In our study, before MGF intervention, serum IL-10 levels in rats in each group were not signi cantly different; after MGF intervention, IL-10 levels were signi cantly increased in the MGF group and lowest in the control group. This indicates that MGF can promote the immune cells in AS rats to produce more IL-10 to inhibit excessive in ammation and relieve the symptoms of AS.

Conclusion
Our results indicate that MGF can promote the proliferation and differentiation of human peripheral blood Treg cells, increasing the expression of FOXP3, and ultimately enhance immunosuppressive function. In animal experiments, we showed that MGF effectively alleviated the symptoms of AS in rats. This suggests that MGF can be used to treat AS. However, MGF increases the expression of Foxp3 and inhibits TNF-α and IL. The impact of IL10 and IL-2 levels require further analysis.
Abbreviations AS, Ankylosing spondylitis; FOXP3, forkhead box P3; H&E, hematoxylin and eosin; IGF-1, insulin-like growth factor-1; IL, interleukin; MGF, mechanical growth factor; PBMC, peripheral blood mononuclear cell; TNF, tumor necrosis factor; Treg, regulatory T cell Declarations Ethics approval and consent to participate Ethics approval has been obtained for the study (KY-E-141). Informed consent was obtained from all patients to participate in the study.

Consent for publication
Informed written consent was obtained from the patient for publication of this case report and accompanying images.

Availability of data and materials
All the data needed to achieve the conclusion are presented in the paper.

Con icts of interest
The authors have no con icts of interest to disclose in relation to this article.

Funding
This work was supported by the Guangxi Natural Science Foundation (No. 2017GXNSFAA198127). The funding agency fund the collection and analysis of this study and had no role in the collection, analysis and interpretation of data, in the writing of the report, or in the decision to submit the article for publication.
Authors' contributions YJS designed and supervised the overall research. QHY and YSS conducted rat feeding and rat experiment. LH conducted human peripheral blood collection and analysis. ZH and QHY provided pathology assistance and image editing. QHY and YJS wrote and revised the manuscript. All authors read and approved the manuscript.