Th17 responses in mice

Accepted Manuscript

Curcumin attenuates the scurfy-induced immune disorder, a model of IPEX syndrome, with inhibiting Th1/Th2/Th17 responses in mice Gihyun Lee , Hwan-Suck Chung , Kyeseok Lee , Hyeonhoon Lee , Minhwan Kim , Hyunsu Bae PII: DOI: Reference:

S0944-7113(17)30017-X 10.1016/j.phymed.2017.01.008 PHYMED 52153

To appear in:

Phytomedicine

Received date: Revised date: Accepted date:

2 March 2016 9 December 2016 18 January 2017

Please cite this article as: Gihyun Lee , Hwan-Suck Chung , Kyeseok Lee , Hyeonhoon Lee , Minhwan Kim , Hyunsu Bae , Curcumin attenuates the scurfy-induced immune disorder, a model of IPEX syndrome, with inhibiting Th1/Th2/Th17 responses in mice, Phytomedicine (2017), doi: 10.1016/j.phymed.2017.01.008

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Curcumin attenuates the scurfy-induced immune disorder, a model of IPEX syndrome, with inhibiting Th1/Th2/Th17 responses in mice

Gihyun Leea,b,1, Hwan-Suck Chungc,1, Kyeseok Leea, Hyeonhoon Leea, Minhwan Kima,

a

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Hyunsu Baea,*

Department of Science in Korean Medicine, College of Korean Medicine, Kyung Hee

University, #1 Hoegi-Dong, Dongdaemoon-gu, Seoul, Republic of Korea

c

National Development Institute of Korean Medicine, Gyeongsan, Republic of Korea

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b

Korean Medicine (KM)-Application Center, Korea Institute of Oriental Medicine (KIOM),

Daegu, Republic of Korea

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Running title: Curcumin intake on IPEX syndrome Both authors contributed equally to this work.

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Corresponding author:

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1

Department of Physiology, College of Korean Medicine, Kyung Hee University, #1 Hoegi-Dong,

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Dongdaemoon-gu, Seoul 130-701, Republic of Korea. Phone: +82-2-961-9316; fax: +82-2-962-9316;

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E-mail address: [email protected]

ABSTRACT Background: Immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX) is a lethal autoimmune disease caused by mutations in the Foxp3 gene scurfin (scurfy). Immunosuppressive therapy for IPEX patients has been generally ineffective and has caused severe

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side effects, however curcumin has shown immune regulation properties for inflammatory diseases, such as rheumatoid arthritis, psoriasis, and inflammatory bowel diseases without side effects. Objective: The aim of this study was to investigate whether curcumin would attenuate symptoms of IPEX in mouse model and would prolong its survival period. Methods: C57BL/6 mice were separated into scurfy or wild-type litter mate groups by genotyping,

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and each group subsequently was separated into 2 subgroups that were fed a 1% curcumin containing or normal diet from the last day of breast-feeding. After weaning, pups were fed either a 1% curcumin containing or normal diet until all scurfy mice die for survival data. To elucidate immune cell proportions in spleen and lymph nodes, cells were analyzed by flowcytometry. Cellular cytokine

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production was accessed to investigate the effects of curcumin in T cell differentiation in vitro.

Results: Scurfy mice fed a 1% curcumin diet survived 4.0-fold longer compared to scurfy (92.5 days) mice fed a normal diet (23 days). A curcumin diet decreased all of the Th1/Th2/Th17 cell populations and attenuated diverse symptoms such as splenomegaly in scurfy mice. In vitro experiments showed

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that curcumin treatment directly decreased the Th1/Th2/Th17 cytokine production of IFN-γ, IL-4, and IL-17A in CD4+ T cells.

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Conclusions: Curcumin diet attenuated the scurfy-induced immune disorder, a model of IPEX

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syndrome, by inhibiting Th1/Th2/Th17 responses in mice. These results have implications for

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improving clinical therapy for patients with IPEX and other T cell related autoimmune diseases.

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Keywords: Curcumin; Tumeric; IPEX; Scurfy; T cell

Abbreviations: HSCT:

Hematopoietic

stem

cell

transplantation;

IPEX

syndrome:

polyendocrinopathy enteropathy X-linked syndrome; Treg: Regulatory T cell

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Immunodysregulation

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Introduction Curcumin, which constitutes 2~5% of dried turmeric root, is the most active component in Curcuma longa. It has also been traditionally recognized as an anti-inflammatory agent in the Orient. It is well known that curcumin down-regulates the expression of various pro-inflammatory cytokines, including tumor necrosis factor-alpha, IL-1β, IL-2, IL-6, IL-8, and IL-12, and chemokines by inactivating the transcription factor NF-

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κB (Jagetia and Aggarwal, 2007). It was recently shown that curcumin modulates the activation of immune cells, such as T cells, B cells, macrophages, neutrophils, natural killer cells, and dendritic cells (Bhaumik et al., 2000; Churchill et al., 2000; Jancinova et al., 2009; Kim et al., 2005).

Mutations in the Foxp3 gene scurfin (scurfy) can cause Immunodysregulation polyendocrinopathy

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enteropathy X-linked syndrome (IPEX), an autoimmune disease in humans that is associated with eczema, severe enteropathy, type I diabetes, thyroiditis, hemolytic anemia, and thrombocytopenia (Bennett et al., 2001; Ziegler, 2006). Foxp3 is a major transcription factor associated with the development and function of regulatory T cell (Treg) (Hori et al., 2003). Treg plays a pivotal role in immune tolerance. A deficiency in

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Treg can cause autoimmune diseases (Sakaguchi et al., 2006). Scurfy mice have a natural frame-shift

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mutation in the forkhead box region, which causes a premature stop codon and truncation of the Foxp3 protein (Brunkow et al., 2001). These mice are an equivalent mouse model to human IPEX. The scurfy

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mutation leads to the development of severe lymph-adenopathy with a similar spectrum of immune-mediated tissue inflammation as is observed in IPEX patients (Clark et al., 1999; Godfrey et al., 1991). Massive

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autoimmunity in scurfy mice results in death during the first month after birth, and the disease has been shown to be mediated by CD4+ T cells (Blair et al., 1994).

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We hypothesized that a curcumin can rescue scurfy mice by modulating CD4 + T cells based on various reports on the immune-regulatory effects of curcumin. In this research, we demonstrated that a curcumin can attenuate the scurfy-induced immune disorder by inhibiting Th1/Th2/Th17 cells.

Methods 3

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Chemical compound Curcumin isolated from root of curcuma longa was bought from Sigma-Aldrich (1,7-bis(4-hydroxy-3methoxyphenyl)-1,6-heptadiene-3,5-dione; CAS number 458-37-7; St. Louis, MO, USA).

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Mice All experiments were performed in accordance with the approved animal protocol and guidelines established by the Animal Care and Use Committee of Kyung Hee University (KHUASP(SE)-11-025). Scurfy mice (B.Cg-Foxp3sf/J) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). All mice were kept

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under specific pathogen-free conditions under air conditioning and a 12 h light/dark cycle, and food was provided ad libitum. At 21 days after birth, mice were separated from their mother and given a normal diet (Purina Rodent Chow; Purina Co., South Korea) or 1% curcumin containing diet (1% curcumin added Purina Rodent Chow, Supplementary Table 1). 1% curcumin diet was prepared as previous studies in a murine

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Survival test

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every 2 weeks in our laboratory.

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model (Lee et al., 2013a; Limtrakul et al., 1997a). All of the control and experimental diets were prepared

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Scurfy mice and their wild-type litter mates were fed a 1% curcumin-containing or normal diet from weaning day until they died. We checked the mouse colony every day for dead mice until all mice died. The results

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were statistically analyzed using the Prism 5.01 software (GraphPad, San Diego, CA, USA)

Th1/Th2/Th17 cell phenotyping After being fed a 1% curcumin-containing or normal diet for 1 week after weaning, the spleen was isolated from the scurfy mice and their wild-type litter mates. The spleens were ground over a wire mesh. Red blood cells were lysed in 0.85% NH4 in Tris-HCl buffer. Then, splenocytes were washed with PBS 4

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twice and resuspended in RPMI 1640 (WelGENE, Inc., Taegu, Korea) supplemented with 10% fetal bovine serum, 50 IU/ml penicillin, and 50 µg/ml streptomycin (Hyclone, Logan, Utah, USA). Thes e cells were checked for viability using trypan blue and adjusted to a density of 1 × 10 7 cells/ml for further experiments. To evaluate the Th1/Th2/Th17 cell populations, intracellular staining was performed on isolated splenocytes using a cytofix/cytoferm kit (BD Biosiceces) per the manufacturer’s instructions.

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Briefly, cells were restimulated with 50 ng/ml of PMA and 1 μg/ml of ionomycin (both from Sigma-Aldrich) for five hours in the presence of the GolgiStop™ Protein Transport Inhibitor (BD Biosiceces). Then, the cells were stained with antibodies, including anti-CD4 (RM4-5), anti-CD8 (53-6.7), anti-IL-4 (RM4-5), anti-IL17A (eBio17B7), and anti-IFN-r (XMG1.2). After data were acquired on a FACS Calibur flow cytometer

(Tree Star, Ashland, OR, USA).

Cytokine measurements in vitro

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(BD Biosciences), the results were generated in graphical and tabular formats using the FlowJo software

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Production of IFN-γ, IL-4, and IL-17A from CD4+ T cells was measured using a BD™ Cytometric Bead Array Mouse Th1/Th2/Th17 Cytokine Kit following the manufacturer’s instruction (BD Biosciences, San

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Jose, CA, USA). In brief, CD4+ T cells were isolated from scurfy mice and their wild-type litter mates using magnetic bead separation (CD4 T cell isolation kit; Miltenyi Biotec, Bergisch Gladbach, Germany) 21 days

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after they were born. Isolated CD4+ T cells were resuspended in culture medium and seeded into 96-well

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plates. The cells were treated with various doses (0~5μM) of curcumin in the presence of plate-bound antiCD3 (5 mg/ml, BD Biosciences) and soluble anti-CD28 (2 mg/ml, BD Biosciences) antibodies for 72 h. Cell

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culture supernatants were used for Cytometric Bead Array.

Curcumin blood level monitoring To monitor the level of curcumin in blood, we measured it in plasma from wild type mice at day 0, 1, 2, 7 after being fed a 1% curcumin-containing diet. MeOH added plasma was vortexed for approximately 30 s 5

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and centrifuged at 13,000rpm for 10 min at 4 °C. After centrifuging, the resulting organic layer was transferred into a different tube. All the eluents were evaporated to dryness under reduced pressure in Speed vacuum concentrator (Thermo Fisher, Needham Heights, MA) at 40 °C for 3 hrs. 50 μl of MeOH was added to each tube and vortexed for approximately 30 s. The concentrated solution was transferred to HPLC sample vial and 5 μl of the solution was injected into the LC-qToF-MS system. Data acquisition and processing were

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carried out using Analyst TF 1.7 and PeakVeiw 2.2 software (AB SCIEX), respectively. The data obtained from MRMHR mode were quantitated using MultiQuant 3.0 software (AB SCIEX).

Statistical analysis

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All of the values are expressed as the mean ± S.E.M. The statistical significance (p < 0.05 for all analyses) was assessed by one-way ANOVA followed by the Newman–Keuls multiple comparison test for multiple comparisons or by two-tailed Student’s t test for single comparisons using the Prism 5.01 software

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(GraphPad Software, San Diego, CA).

Results

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Curcumin prolonged the survival of scurfy mice

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Scurfy mice are known to die approximately one month after birth because of their severe autoimmune symptoms. To examine the effects of curcumin on the lifespan of scurfy mice, normal or 1% curcumin diets

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were given to scurfy mice and their wild-type litter mates until they died. As shown in Fig. 1, the survival of scurfy mice fed the curcumin diet was significantly prolonged compared with that of scurfy mice fed the normal diet (n = 12 mice, p < 0.0001). The median survival period of scurfy mice fed the curcumin diet was 4.0-fold longer than that of scurfy mice fed the normal diet (scurfy = 23 days, scurfy + curcumin = 92.5 days). All of the wild-type litter mates survived regardless of the curcumin diet, as expected. The longest surviving scurfy mouse fed the curcumin diet lived for 166 days, even though scurfy mice fed with the normal diet all died within 33 days after birth. 6

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Curcumin attenuated the phenotypes of scurfy mice Scaly and crusty skin on the tail and dermal thickening are distinct phenotypes of scurfy mice. The curcumin diet-fed scurfy mice did not develop scaly and crusty skin on their tails (Fig. 2A upper). Splenomegaly and lymphadenopathy are also known characteristics of scurfy mice. The curcumin diet reduced splenomegaly

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and lymphadenopathy in scurfy mice (Fig. 2A lower). The most distinct characteristic of scurfy mice is an over-proliferation of CD4+ T cells. The relative CD4+ T cell population in the spleen was analyzed after feeding the mice curcumin diets for 7 days. The curcumin diet decreased the relative CD4+ T cell population in scurfy mice. It was recently revealed that CD4+ T cells induced effector CD8+ T cells and enhanced

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autoimmune inflammation in scurfy mice (Iwamoto et al., 2014). Interestingly, the population of CD8+ T cells was increased in scurfy mice, and this increment of CD8+ T cells was reduced in the mice fed with the curcumin diet. Notably, the curcumin diet did not affect the CD4+ and CD8+ T cell populations of wild-type

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littermates (Fig. 2B).

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Curcumin inhibited Th1/Th2/Th17 responses Expanded populations of Th1, Th2, and Th17 cells are known characteristics of scurfy mice (Suscovich et al.,

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2012). To elucidate whether the curcumin diet affects Th1/Th2/Th17 responses in vivo, we analyzed the Th1,

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Th2, and Th17 cell populations in spleens from scurfy mice and their wild-type litter mates after 7 days on the curcumin diet. Th1/Th2/Th17 cell populations were all increased in scurfy mice compared to their wild-

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type litter mates. The increased numbers of Th1/Th2/Th17 cells in the scurfy mice were significantly reduced by the curcumin diet (Fig. 3A and 3B). Notably, the curcumin diet did not affect the Th1/Th2/Th17 cell populations in the wild-type littermates (Fig. 3B).

Curcumin inhibited the production of Th1/Th2/Th17 cytokines directly in vitro in CD4+ T cells from scurfy mice 7

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Previous studies have suggested that autoimmunity in scurfy mice may be caused by Th1/Th2/Th17. We measured the most representative cytokines, Th1 (IFN-γ), Th2 (IL-4), and Th17 (IL-17A), in CD4+ T cells in vitro. When we checked CD4+ T cell viability using an MTS assay 24 h after curcumin treatment, the cell viability was affected by curcumin concentrations ≥10 μM (Fig. 4A). Therefore, we treated CD4+ T cells with up to 5 μM of curcumin, which did not cause cytotoxicity. CD4+ T cells from scurfy mice produced

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highly increased levels of IFN-γ, IL-4, and IL-17A compared with CD4+ T cells from wide-type litter mates. Curcumin treatment significantly decreased the secretion of IL-4, IFN-γ and IL-17A (Fig. 4B-D). This result suggested that curcumin inhibits Th1/Th2/Th17 cytokine production directly on CD4+ T cells from scurfy

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mice and that curcumin may have therapeutic effects on scurfy mice.

Discussion

IPEX is a fatal autoimmune disease and is caused by a Foxp3 gene mutation. Currently, there is no drug to treat IPEX. The most common immunosuppressive treatment, a combination of steroids and the calcineurin

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suppressive agents CsA or tacrolimus, is partially effective, and the dose is limited in large part by renal toxicity. There is a report that sirolimus controlled the gastrointestinal and dermatologic symptoms of IPEX

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in three patients without significant side effects (Bindl et al., 2005). Presently, hematopoietic stem cell transplantation (HSCT) is the best hope to cure IPEX even though its success is uncertain. It is critical for

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patients to survive with medication until they receive a successful HSCT, as IPEX is a hereditary disease that

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is inextirpable with drugs. Therefore, a safe and effective new treatment is still required for IPEX patients. There is increasing evidence that curcumin is effective for treating various autoimmune diseases, such as

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multiple sclerosis, inflammatory bowel disease, and type I diabetes, which are caused by defective Treg functions (Jagetia and Aggarwal, 2007). Moreover, humans have taken curcumin safely for a long time as a food ingredient. In this study, we evaluated the effects of curcumin on IPEX using scurfy mice, which resulted in fairly prolonged survival with attenuated phenotypes. Recently, it is known that the immune system undergoes gradual age-related changes in cell populations, which lead to repressed of the immune responses with a decrease in the proportion of CD4 T cells(Heaton et al., 2002; Pinchuk and Filipov, 2008; Salam et al., 2013). However, it is unlikely that these age-related shifts can be relevant with IPEX since 8

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IPEX is a fatal genetic disease showing the symptoms and leading patients to death before childhood’s end. Although previous studies suggested that autoimmunity in scurfy mice may be caused by Th2 responses, Suscovich et al. suggested that Th1 immunity drives early lethality in scurfy mice, with data showing that Th1 cell deficient scurfy mice had an increased median lifespan of 62 days. They also showed that Th2 cell deficient scurfy mice had decreased dermatitis (Suscovich et al., 2012). Similar to the results from previous

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studies demonstrating that curcumin attenuates various autoimmune diseases related to Th1/Th2/Th17 cells, the curcumin diet reduced Th1/Th2/Th17 cell populations and resulted in moderated phenotypes of scurfy mice in our experiment (Natarajan and Bright, 2002; Sanford and McKeage, 2015; Suscovich et al., 2012). In vitro curcumin treatments also inhibited the production of IFN-γ, IL-4, and IL-17 in CD4+ T cells from

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scurfy mice. Surprisingly, the median lifespan of scurfy mice fed the curcumin diet was 92.5 days, which was 4 times longer than that of the scurfy mice fed the normal diet. This increased lifespan with the curcumin diet was longer than that of the Th1 cell deficient scurfy mice. In addition to the increased lifespan, the pathologic phenotypes, such as scales and dermatitis on the tail, splenomegaly and lymphadenopathy,

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which are distinctive symptoms of scurfy mice, were improved by the curcumin diet. However, we cannot rule out the possibility that the decline of Th1/2/17 cells in the scurfy mice fed the curcumin diet might be

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related to apoptotic cell death caused by curcumin as previously reported (Park et al., 2013). Although many researchers used curcumin at high doses (≥50 μM) for in vitro studies (Cong et al., 2009;

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Prakobwong et al., 2011; Xie et al., 2014), possible false positive results from the natural fluorescence of curcumin when measured by fluorescence or optical density should be considered. In our experiment, a high

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dose of curcumin (≥10 μM concentration) showed cytotoxicity in T cells, and this result suggests that a

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high dose of curcumin treatment may not be translatable to therapeutic effects. Curcumin is safe even at high doses (12g/day) of intake, but is hardly absorbed into body (Anand et al., 2007). In animals, 1% curcumin dietary supplement has been tested for cancer and non-cancer disease models (Farid et al., 2005; Lee et al., 2013b; Lee et al., 2010; Limtrakul et al., 1997b). We monitored the curcumin levels in the plasma during curcumin feeding. The levels were 2.51±0.46 ng/ml, 6.93±1.54 ng/ml, and 11.16±3.79 ng/ml at day 1, 2, and 7, respectively (Supplementary Fig. 1). In summary, curcumin prolonged the lifespan of scurfy mice by inhibiting T cell populations, including 9

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Th1/Th2/Th17 cells and CD8+ cells. Based on these results, it may be feasible to use curcumin as a functional supplement for IPEX patients until they receive a successful HSCT.

Acknowledgments

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We thank Korean Medicine Clinical Trial Center (K-CTC), Kyung Hee University Korean Medicine Hospital for monitoring the curcumin levels in the blood. This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korean government (MEST) (No. 2009-0063466) and the National Research Foundation of Korea (NRF) grant funded by the

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Korean government (MSIP) (No. NRF-2013R1A2A2A01068954).

Author contributions

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G.L. and H.B. designed the study. G.L. and H.C. performed the laboratory work. G.L and K.L.

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analyzed the data. G.L., H.C., and H.B. wrote the manuscript. All authors revised and approved the

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manuscript.

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Conflict of interest

Authors wish to confirm that there are no known conflicts of interest associated with this

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publication and there has been no significant financial support for this work that could have influenced its outcome.

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Figure legends

Fig. 1. Curcumin prolonged the survival of scurfy mice. Scurfy mice and their wild-type litter mates were fed a 1% curcumin-containing diet or a normal diet from weaning day until all of the

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mice died. ***P < 0.0001 vs. indicated control, n = 12

Fig. 2. Curcumin attenuated the phenotypes of scurfy mice. Scurfy mice and their wild-type litter mates were fed a 1% curcumin-containing diet or a normal diet for one week after weaning. (A)

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Representative figures of mouse, spleen, and lymph nodes. (B) Splenocytes were stained with antiCD4 and anti-CD8 antibodies and then analyzed by flow cytometry. **P < 0.01, ***P < 0.001 vs. indicated control, n = 5.

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Fig. 3. Curcumin inhibited Th1/Th2/Th17 responses in scurfy mice. Scurfy mice and their wild-

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type litter mates were fed a 1% curcumin-containing diet or a normal diet for one week after weaning. (A) Representative figures of flow cytometry. (B) The absolute numbers of Th1/Th2/Th17

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control, n = 5.

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cells counted from the CD4+ T cells using flow cytometry data. *P < 0.05, **P < 0.01 vs. indicated

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Fig. 4. Curcumin inhibited the production of Th1/Th2/Th17 cytokines directly in CD4+ T cells from scurfy mice. CD4+ T cells were isolated from the spleens of scurfy mice or their litter mates with magnetic bead linked-CD4 antibodies. (A) To determine the cytotoxicity of curcumin in CD4+ T cells, a MTS assay was performed 24 hours after the curcumin treatment. (B-D) Cells were incubated with curcumin and anti-CD3/28 antibodies for 72 hours, and the cytokines in medium were analyzed by the Cytometric Bead Array TH1/Th2/Th17 assay kit. *P < 0.05, **P < 0.01 vs. indicated control. 13

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Supplementary Fig. 1. Curcumin plasma concentrations The levels of curcumin in blood were measured from wild type mice at day 0, 1, 2, 7 after being fed a 1% curcumin-containing diet using LC-

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Graphical Abstract

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