HO‐1 regulates the function of Treg: Association with the immune intolerance in vitiligo

Abstract In vitiligo, cutaneous depigmentation is accompanied by increased T cell cytolytic activity targeting melanocytes, indicating that autoimmune tolerance is disrupted. The inhibited amount and function of Tregs have been indicated to be involved in the autoimmune intolerance in vitiligo, however, with the conclusion still controversial and the involved mechanism unknown. In this study, we explored the molecular and cellular alterations accounting for the impaired Treg response in vitiligo. Our results showed that the amount of Tregs was drastically reduced in peripheral blood of active vitiligo patients. Furthermore, the immunoregulatory function of Tregs was attenuated, with lower expression of CTLA4, IL‐10 and TGF‐β. Moreover, the expression of HO‐1, a functional modulator of Tregs, was decreased in vitiligo Tregs, and the concentrations of HO‐1 metabolites, including bilirubin, CoHb and iron, were correspondingly decreased in serum of vitiligo patients. In addition, we treated the Tregs from vitiligo patients with Hemin, an agonist of HO‐1, and found that enhanced HO‐1 expression restored the function of Tregs by up‐regulating IL‐10 expression. Our study demonstrates the essential role of HO‐1 in the impaired Treg response in vitiligo and indicates the potential of HO‐1 as a therapeutic target in vitiligo management.


| INTRODUCTION
Vitiligo is a chronic skin disease with a worldwide incidence rate of approximately 0.5%-1.0%. 1 The loss of melanocytes in the epidermis is the key process in the development of vitiligo, which is the direct non-lesional, perilesional and lesional skin from vitiligo patients, which was further supported by many other subsequent researches. 5,6 Moreover, increasing the abundance of Tregs by CCL22 overexpression can reduce depigmentation in two mouse models of vitiligo, 7 indicating that replenishing Tregs can repair the disrupted autoimmune tolerance and is a promising treatment for vitiligo.
Recently, it is reported that Tregs are suppressed in vitiligo not only on their amounts but also on their function. The polymorphisms of several immunosuppressive genes expressed in Tregs including transcription factor Forkhead box P3 (Foxp3), 8 interleukin-10 (IL-10) 9 and transforming growth factor b (TGF-b) 10 are associated with susceptibility to vitiligo. Moreover, the function of Tregs has been shown impaired in vitiligo patients, with weaker suppressive effect of Tregs on autologous CD8 + T cells 4 and decreased expression level of Foxp3 and cytotoxic T lymphocyte antigen-4 (CTLA-4) that induces the anergy of effector T cells. 6 Therefore, amending the impaired function of Tregs is another way to reconstruct the autoimmune tolerance and may be applied to the treatment for vitiligo.
Heme oxygenases-1 (HO-1) is a rate-limiting enzyme that degrades heme into biliverdin, yielding carbon monoxide (CO) and free iron. 11 HO-1 exerts antioxidant, anti-apoptotic and immunemodulating functions, leading to overall cytoprotective effect on mammalian cells. HO-1 has been proved to modulate the function of Tregs. 12 For instance, enhanced HO-1 is necessary for the regulation of Tregs on the balance of Th1/Th2 response in Spesis, 13 while the blockage of HO-1 abrogates the protective effect of Treg on immune homoeostasis during murine pregnancy. 14 Given that the level of HO-1 is significantly decreased in vitiligo, 15 we hypothesized the deficiency of HO-1 may be a key reason for the impaired function of Tregs in vitiligo.
To test this hypothesis, we recruited 51 vitiligo patients and 51 age-and sex-matched healthy controls. We compared their Tregs to CD4 + T cells ratio in peripheral blood, and evaluated the association between the Tregs to CD4 + T cells ratio and disease activity or lesional areas. Besides, we analysed the immunoregulatory ability of Tregs towards effector T cells and the proliferative ability of Tregs.
Furthermore, the key immunosuppressive molecules involved in the function of Tregs were also tested, and at last we attempted to restore the Treg function through regulating HO-1 expression.

| Patients
All patients and donors were recruited from Xijing Hospital, Fourth Military Medical University. All vitiligo patients did not receive any systemic treatment including immunosuppressive agents and phototherapy for at least 3 months prior to the procedures. Patients with other autoimmune diseases were excluded from this study. Information on demographics, and other characteristics were obtained by questionnaires. Patients whose lesional skin area progressed in the past 1 month were defined as active patients, and others were defined as stable patients. All the subjects voluntarily agreed to participate in this study and signed informed consent forms. Each of them donated 20 mL of blood, which was collected in heparinized tubes for further separation of PBMCs. This study was approved by the ethics review board of Fourth Military Medical University and was conducted according to the principles of Helsinki Declaration.
The characteristics of the participants were summarized in Table 1.

| Regulatory activity assay
To test the regulatory activity of Tregs, 3.5 9 10 4 CD8 + T cells isolated from healthy subjects were firstly labelled with carboxyfluorescein diacetate, succinimidyl ester (CFSE 2 lmol/L, Invitrogen, Life Technologies) and the labelled CD8 + T were seeded in 96-well plates in 200 lL RPMI medium containing rhIL-2 (50 ng/mL, Peprotech, Rocky Hill, NJ); then, the Treg cells isolated from patients or another healthy controls were added into each well at a mixing ratio of 1:1.
After co-culturing for 7 days, the proliferation rate of CD8 + T cells was analysed by FACS through signalling of CFSE. The results were calculated by FlowJo software version 7.6.1 (Tree Star, Ashland, OR).
The reduction in the proliferation rate of CD8 + T cell was used to measure the inhibitory capacity of Tregs. The calculation method is as follows: the proliferation ratio of CD8 + T cell minus the proliferation ratio of CD8 + T cell with VP-Treg or HC-Treg under the same conditions (with or without Hemin).

| Proliferation assays in vitro
To test the proliferating ability of Tregs, Tregs were isolated from

| Elisa assay for serum cytokines
The serum levels of anti-inflammatory cytokines including TGF-b, IL-

| Hemin treatment
To assess the effect of Hemin, a HO-1 inducer, on Treg cell function, Tregs were isolated from patients or controls and cultured in RPMI 1640 in the presence of Hemin at a concentration of 10 lmol/L for 24 hours. Tregs were then washed with PBS, and the proliferation of Tregs was detected by flow cytometry as described previously. The co-culture system of CD8 + T cells and Hemin-treated Tregs was established, and the reduction in CD8 + T cell proliferation rate was detected to evaluate the inhibitory capacity of Hemintreated Tregs.

| Statistics
Data were presented as mean AE SD throughout the manuscript and analysed for statistical significance of differences between groups using Student's t test accounting to unequal variance.  Figure 1A,B). However, upon dividing the patients into two groups based on their disease activity, the percentage of Tregs showed significant decrease in active patients compared with healthy controls or stable patients, whereas there was no significant difference between healthy controls and stable patients (Figure 1C). We also compared the percentage of Tregs in patients with different lesional areas, however, with no significant difference observed ( Figure 1D). Therefore, the amount of Tregs can be decreased in active vitiligo patients, rather than in stable vitiligo patients.

| The immunoregulatory function and proliferative ability of Tregs were suppressed in vitiligo patients
The homoeostasis of the immune system depends on proper function of Treg cells. We compared the suppressive ability of Tregs towards CD8 + effector T cells. It turned out that the proliferation rate of effector T cells decreased more than 10% with the co-culture of Tregs isolated from healthy controls, but only reduced less than 5% with the co-culture of Tregs from vitiligo patients (Figure 2A,B).

| The expression of HO-1 and the levels of its reactive products were decreased in vitiligo
HO-1 and its reactive products have been described as critical modulators for the function of Tregs. To evaluate the expression of HO-1 in vitiligo patients, we tested the level of HO-1 in both Tregs and serum samples from both vitiligo patients and healthy controls. Flow cytometric analysis revealed a significant decrease in the percentage of CD4 + Foxp3 + HO-1 + T cells in vitiligo patients ( Figure 4A,B). Taken together, these results demonstrated that the expression and the function of HO-1 in Tregs are suppressed in vitiligo.

| Effects of HO-1 modification on Tregs suppression and their proliferation capacity
Given the deficiency of HO-1 in Tregs from vitiligo patients, we assumed that up-regulating HO-1 could restore the function of Tregs in vitiligo. We isolated CD4 + CD25 high T cells and treated these cells with Hemin, a HO-1 inducer and performed flow cytometry analysis.
We found that the expressions of foxp3 is significantly up-regulated  The function of Tregs in vitiligo has been proved impaired by multiple studies. 16,17 However, the mechanism involved in Treg dysfunction in vitiligo is still not clarified before. In our study, we found that the weakened immunoregulatory function and proliferative abil- In summary, our study demonstrated that HO-1 is responsible for the impaired function of regulatory T cells in vitiligo patients.
Although further studies are warranted to determine the involved mechanisms in more details, HO-1 is of great potential to be used as a novel therapeutic target, offering a promising alternative to our current approaches to managing vitiligo.