Indirubin modulates CD4+ T‐cell homeostasis via PD1/PTEN/AKT signalling pathway in immune thrombocytopenia

Abstract Immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by an immune mediated decrease in platelet number. Disturbance of CD4+ T‐cell homeostasis with simultaneous decrease of CD4+CD25+Foxp3+ regulatory T cells (Tregs) as well as unrestricted proliferation and activation of peripheral CD4+ effector T cells underpin the pathophysiology of ITP. Indirubin is an active ingredient of a traditional Chinese herb called Indigofera tinctoria L. which is clinically used for the treatment of ITP patients. Whether indirubin targets the Tregs/effector T cell‐axis to restore platelet number is unknown. In our in vitro studies, Indirubin could significantly enhance the number and function of Tregs and meanwhile dampen the activation of effector T cells in a dose‐dependent manner. Indirubin was observed to restore the expression of programmed cell‐death 1 (PD1) and phosphatase and tensin homolog (PTEN) on the CD4+ T cells of ITP patients, leading to the subsequent attenuation of the AKT/mTOR pathway. Furthermore, these observations were recapitulated in an active murine model of ITP with a prominent platelet response. Thus, our results identified a potentially novel mechanism of the therapeutic action of indirubin in the treatment of ITP through regulating the homeostasis of CD4+ T cells in a PD1/PTEN/AKT signalling pathway.


| INTRODUCTION
Immune thrombocytopenia (ITP) is an autoimmune disease predominantly characterized by autoantibody-mediated platelet destruction and/or impaired platelet production. 1 Abnormalities in T-cell populations are one of the major mechanisms for the pathogenesis of ITP. [2][3][4] In ITP patients, platelet auto-reactive CD4 + effector T cells are excessively activated with decreased apoptosis, while the CD4 + regulatory T cells (Tregs) are numerically and functionally impaired. [3][4][5][6][7][8] Skewed subsets of CD4 + T helper cells manifested as the imbalance of Th1/Th2, as well as elevated Th17, Th22 and Th9 levels were reported in ITP patients, contributing to a cytokine imbalance that can lead to lower levels and abnormal function of Tregs. [9][10][11][12][13] Tregs play an important role in maintaining self-tolerance and are phenotypically defined as CD4 + CD25 + Foxp3 + . One facet of the pathophysiology of ITP and other autoimmune diseases involves deficiencies in number and function of peripheral Tregs. [14][15][16][17][18][19][20][21] It has been suggested that ITP murine models have retention of Tregs in the thymus, which may suppress the proliferation and function of Tregs in peripheral lymphoid organs, such as spleen and lymph nodes. 22 Peripheral Tregs deficiencies could result in unrestricted proliferation and function of auto-reactive effector T cells, contributing to the breakdown of immune homeostasis and autoimmmune diseases. 8 The importance of T-cell homeostasis in attenuating autoimmunity is further evidenced in the current clinical treatment of ITP. Both clinical and experimental studies show treatments such as intravenous immunoglobulin, dexamethasone, rituximab, and thrombopoietic mimetics, exert their therapeutic effects at least partially through modulating Tregs activation and expansion, and/or dampening the survival and proliferation of effector T cells. 14,17,[22][23][24] Indirubin is an active ingredient of a traditional Chinese herb called Indigofera tinctoria L. and is most well-known for its clinical use in the treatment of chronic myelogenous leukemia. 25 There are also reports of indirubin possessing anti-viral, anti-bacterial and antiinflammatory properties. [26][27][28][29][30] In murine models of ITP and ulcerative colitis, indirubin was effective therapeutically through down-regulating the immune response and increasing the percentage of Tregs. 27,31 Clinically, I tinctoria L. is used as a traditional Chinese medical therapy for thrombocytopenia patients. However, the efficacy of I tinctoria L. in the treatment of ITP still needs to be confirmed by appropriate clinical trials and the endogenous mechanisms of action remains to be explored.
Programmed cell-death 1 (PD1) is a co-receptor that is inducibly expressed on T cells. Tregs activation and expansion in parallel with PD1 signalling are critical for the maintenance of CD4 + T-cell homeostasis and elimination of either can result in its breakdown. 32 Dysregulation of the PD1 pathway can be an important contributor to autoimmune pathogenesis, as has been shown in rheumatoid arthritis (RA), systemic lupus erythematosus, and multiple sclerosis (MS). [33][34][35][36][37][38] Furthermore, PD1 and its ligand PDL1 were reported to be decreased in the PBMCs of ITP patients, which suggested the important role of the PD1 pathway in the pathogenesis of ITP. 39 Here we demonstrate that the therapeutic effects of indirubin may be achieved through the regulation of CD4 + T-cell homeostasis in ITP by promoting the development and function of Tregs as well as reducing the activation of effector T cells via a PD1/phosphatase and tensin homolog (PTEN)/AKT signalling pathway.  40 The patient's platelet counts ranged between 1 × 10 9 /L and 95 × 10 9 /L, with a median count of 15 × 10 9 /L. The demographic and key clinical information of ITP patients are summarized in Table 1 and Table 2. Previous therapy were completed at least 3 weeks prior to enrollment. In parallel, 28 consenting healthy volunteers (13 females and 15 males; age range, 23-71 years; median age, 40 years) were recruited to establish healthy controls. And their platelet counts ranged between 143 × 10 9 /L and 358 × 10 9 /L, with a median count of 256 × 10 9 /L ( Table 2). All study subjects donated 4 or 5 mL of heparinized venous peripheral blood. Our study was approved by the Institutional Review Boards of Qilu Hospital, Shandong University, China.

| Patients and controls
All blood samples were collected after obtaining the informed consent from each participant before being included in the study. Medicine. For the establishment of ITP murine models, blood was drawn by retro-orbital bleeding, and centrifuged at 60 g for 10 minutes to collect the platelet rich plasma (PRP). Platelets were gathered from PRP via centrifugation at 800 g for 5 minutes, and adjusted to a concentration of 10 9 /mL for immunization. C57BL/6 CD61 KO mice were transfused weekly with 100 µL of 10 8 platelets from WT mice for three consecutive weeks. The immunized CD61 KO mice were euthanized, and their spleens were harvested and single-cell suspension prepared. 1 On the day of splenocytes transfer, SCID mice were subjected to 180 cGy total body irradiation to inhibit recipient

| Flow cytometry
Tregs were detected using a regulatory T-cell staining kit (eBioscience). Briefly, a total of 1 × 10 6 cultured human PBMCs were harvested from 24-well plates following respective treatments. For the preparation of mouse single-cell suspensions, the spleens, thymuses and bone marrow from ITP mice were smashed and lysed using ACK lysing buffer to remove red blood cells. Cells were then washed two times by centrifugation at 300 g for 10 minutes and adjusted to a concentration of 1 ×
The data was analyzed using Flow Jo software. Semi-quantitative evaluation of a particular protein's total level was analyzed by calculating the band of interest with respect to the housekeeping GAPDH signal. The level of phosphorylated proteins was measured using the ratio of phosphorylated protein to total protein. Results were expressed as fold increase relative to control (DMSO treated CD4 + T cells).

| Quantitative real-time PCR
Trizol reagent (Takara, Japan) was used to isolate total RNA from cultured CD4 + T cells. RNA was reverse transcribed into cDNA using Science, Mannheim, Germany). In order to calculate relative changes in gene expression, target genes were compared to the housekeeping gene GAPDH using the comparative delta Ct (ΔΔC t ) method.

| Statistical analysis
Data were reported as mean values ±SD or Median with interquartile range, and were assessed for statistical significance using SPSS.
Differences between pre-and post-treatment groups were determined by paired Student's t test and differences between two independent groups were compared using the Student's t test, and the data were expressed as mean values ±SD. For the data not normally distributed, the Wilcoxon matched-pairs test and Mann-Whitney test were used, and the data were expressed as Median with interquartile range. The P values <0.05 were considered to be of significance.

| Indirubin increased the number and suppressive function of Tregs among ITP patients
Tregs were reported to be quantitively and functionally impaired in ITP patients, which was thought to contribute to disregulation of

| Indirubin decreased fidelity of effector T cells in a dose-dependent manner
Whether in addition to the induction of Tregs expansion, indirubin could influence the function of effector T cells was unknown. In the culturing of PBMCs from ITP patients, we found the expression of CD25 in CD4 + T cells (Figure 2A

| Indirubin normalized decreased PD1 and PTEN expression on CD4 + T cells of ITP patients
The PD1-PDL1 pathway, a pivotal modulator of T-cell homeostasis and Tregs development, is critical for limiting the immune response and thwarting the development of autoimmunity. 32,[41][42][43] Not surprisingly, it has been reported that PD1 is found to be decreased on the surface of PBMCs of ITP patients. 39 However, the expression of PD1 on CD4 + T cells in ITP patients has not been reported. We found reduced PD1 expression on CD4 + T cells and increased PD1 expression on CD14 + monocytes of ITP patients compared with healthy controls (Figure 3A,B). The mRNA expression level of PD1 on PBMCs from ITP patients was higher than that from healthy controls ( Figure 3C). Furthermore, we also tested the PD1 ligand, PDL1 expression on CD14 + monocytes and CD4 + T cells, but no significant differences were observed ( Figure 3D,E). Phosphatase and ten-

| Indirubin ameliorated thrombocytopenia in an active murine model
It has been reported previously that indirubin was effective therapeutically in an ITP murine model. 31 However, the animal model

| Indirubin regulated the homeostasis of CD4 + T cells via a PTEN/AKT/mTOR signalling pathway
The downstream signalling events of PD1 include augmenting PTEN expression which consequently leads to attenuation of the phos-

| DISCUSSION
I tinctoria L. is an herb used in ancient China for the treatment of thrombocytopenia. Its effect is mild and persistent, and therefore is usually used as an assistant therapy for the treatment of ITP nowadays. Indirubin is an active ingredient of I tinctoria L., and is well known for its anti-cancer and anti-inflammatory effects. 25 In addition, indirubin has also been reported to be an effective treatment in autoimmune diseases. 52 ITP is a multifaceted autoimmune disease, and its complex pathogenic mechanisms still remain to be fully elucidated. One of the major contributing factors to the immuno-pathogenesis of ITP is the disturbance of the T-cell homeostasis including the peripheral deficiency of Tregs and over-activation of effector T cells. 5  We established the active murine ITP model, and significant amelioration of thrombocytopenia was found 7 days after indirubin administration, implicating a therapeutic role of indirubin in ITP. It has been reported in murine models of ITP that thrombocytopenia was correlated with a significant splenic Tregs deficiency and a concomitant thymic retention of Tregs, which was reversed upon amelioration of ITP. 22 Similarly, in our murine ITP model, we found successful indirubin treatment correlated with an increase of splenic Tregs percentage and a concurrent reduction in the thymic Tregs.
Furthermore, in accordance with our in vitro study, our in vivo study revealed the upregulated expression of PD1 on CD4 + T cells in the spleens from ITP mice treated with indirubin. Moreover, the expression of PDL1 was also increased on the CD4 + T cells and APCs after indirubin treatment.
To the best of our knowledge, this is the first time to demonstrate that indirubin may exert its effects through the PD1-PDL1 axis which has a significant role in reinstating T-cell homeostasis and re-establishing peripheral tolerance. In our future study, we plan to use the PD1 -/mice to establish the ITP murine model and further elucidate the correlation between ITP and PD1 signalling pathway, and the mechanisms of indirubin in a deeper facet. We also plan to register a randomized controlled clinical trial to investigate the therapeutic effect of indirubin on ITP patients clinically.
In summary, indirubin modulated the T-cell homeostasis by enhancing the development and function of Tregs and reducing the activation of effector T cells via a PD1/PTEN/AKT signalling pathway. This novel mechanism of indirubin underlies the potential therapeutic strategy for ITP.