- Email: [email protected]
HO-1 pathway
Accepted Manuscript Sodium Butyrate Regulates Th17/Treg Cell Balance to Ameliorate Uveitis via the Nrf2/HO-1 Pathway Xiaoqing Chen, Wenru Su, Taoshang Wan, Jianfeng Yu, Wenjie Zhu, Fen Tang, Guangming Liu, Nancy Olsen, Dan Liang, Song Guo Zheng PII: DOI: Reference:
S0006-2952(17)30476-8 http://dx.doi.org/10.1016/j.bcp.2017.06.136 BCP 12862
To appear in:
Biochemical Pharmacology
Received Date: Accepted Date:
11 May 2017 30 June 2017
Please cite this article as: X. Chen, W. Su, T. Wan, J. Yu, W. Zhu, F. Tang, G. Liu, N. Olsen, D. Liang, S.G. Zheng, Sodium Butyrate Regulates Th17/Treg Cell Balance to Ameliorate Uveitis via the Nrf2/HO-1 Pathway, Biochemical Pharmacology (2017), doi: http://dx.doi.org/10.1016/j.bcp.2017.06.136
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Sodium Butyrate Regulates Th17/Treg Cell Balance to Ameliorate Uveitis via the Nrf2/HO1 Pathway Xiaoqing Chen MD*1; Wenru Su MD, PHD*1#; Taoshang Wan, MD1; Jianfeng Yu, MD1; Wenjie Zhu, MD1; Fen Tang, MD1; Guangming Liu, MD1; Nancy Olsen, PHD3; Dan Liang MD, PHD1# and Song Guo Zheng MD, PHD2,3# Running title: NaB attenuates uveitis 1
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen
University, Guangzhou, China; 2 Center for Clinic Immunology, Sun Yat-sen University Third Affiliated Hospital, Guangzhou, China; 3Division of Rheumatology, Department of Medicine, Penn State University Hershey College of Medicine, USA * These authors contributed equally to this work. Author contributions: X-Q. C.: conception and design, data collection and assembly, manuscript writing, and final manuscript approval; W-R. S.: conception and design, data collection and assembly, manuscript writing, final manuscript approval, and financial support; W-J. Z., J-F.Y., F.T., T-S.W., G-M.L.: data collection and assembly; N. O.: conception and design, manuscript writing; D.L., S-G. Z.: conception and design, manuscript writing, final manuscript approval, and financial support. # Correspondence to: Dan
Liang,
MD,
PhD,
Phone:
+86-20-87330341;
Fax:
+86-20-87330341,
Email:
[email protected] or Song Guo Zheng MD, PHD, Phone 717 531 0003, Fax: 717 531 8274, Email: [email protected] or [email protected] or Wenru Su, MD, PhD, Phone: +86-20-87330402; Fax: +86-20-87330402, Email: [email protected] Disclosure of potential conflicts of interest: The authors declare no competing financial conflicts of interest.
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ABSTRACT Autoimmune uveitis, a group of potentially blinding intraocular inflammatory diseases, remains a therapeutic challenge for ophthalmologists. Butyrates, which belong to the short-chain fatty acid family, possess immunomodulatory properties and therapeutic potential in several inflammatory disorders. However, the roles of butyrates in uveitis and their underlying immunomodulatory mechanisms remain elusive. Here, we report that treatment with sodium butyrate (NaB) significantly attenuated the ocular inflammatory response in mice with experimental autoimmune uveitis (EAU) at 14 days after immunization, with significant decreases in inflammatory cell infiltration and inflammatory cytokine production in the retinas. Furthermore, NaB treatment decreased the frequency and number of Th17 cells and increased the frequency and number of T regulatory (Treg) cells in both draining lymph nodes and spleens of EAU mice. In vitro, NaB treatment directly converted the differentiation of naive T cells from Th17 cells toward Treg cells. Mechanistically, the NaB-mediated inhibition of Th17 cell differentiation may occur via inhibition of the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO1)/interleukin-6 receptor pathway. Moreover, the NaB-mediated inhibition on Th17 cell differentiation and uveitis were abrogated when an HO-1 inhibitor, SnPP, was used. These findings suggest that NaB inverts the differentiation of Th17 cells toward Treg cells and attenuates experimental autoimmune uveitis by modulating the Nrf2/HO-1 pathway. Key Words: Uveitis, Butyrate, T regulatory cells, Th17, Heme oxygenase 1, autoimmune diseases Chemical compounds studied in this article: Sodium Butyrate (PubChem CID: 5222465)
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1. INTRODUCTION Autoimmune uveitis is a group of potentially blinding intraocular inflammatory diseases accounting for approximately 10% of cases of severe visual handicaps in the United States, with a higher prevalence in undeveloped countries(1). Due to diverse causes, the care and management of patients with uveitis remain challenges to ophthalmologists(2). Although the pathogenesis of uveitis is not fully understood, the imbalance of CD4+CD25+Forkhead box protein 3(Foxp3)+ T regulatory (Treg) cells and T helper 17 (Th17) cells has been considered a critical part of pathogenesis for autoimmune uveitis
(3, 4)
. The application of corticosteroids and other immune
modulating therapies has improved uveitis treatment. However, long-term exposure to these drugs may result in potentially significant adverse effects(1, 2, 5, 6). Furthermore, many patients cannot tolerate or are resistant to these agents. Therefore, novel safe and effective alternatives are needed for uveitis treatment. Butyrate is a small molecule belonging to the short-chain fatty acid family and is a natural product of the intestinal microbial fermentation of indigestible foods(7). Recent works have found that butyrate is actively involved in a number of pathological processes, including autoimmunity, cancer and neurological disorders(7-9). Moreover, several recent studies have shown that butyrate possesses great potential for Treg induction in vivo and in vitro
(9, 10)
. Sodium butyrate (NaB)
administration to mice can significantly ameliorate inflammation and related symptoms in colitis and diabetes(10,
11)
. Therefore, butyrate may be a potential therapeutic alternative for uveitis
treatment. However, the role of butyrate in uveitis has not yet been explored. Moreover, the mechanisms by which butyrate mediates immunomodulation remain elusive. Thus, in this study, we investigated the therapeutic effect of NaB on experimental autoimmune uveitis (EAU) and the immunomodulatory mechanisms of NaB.
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2. MATERIALS ANDMETHODS 2.1 Animals Inbred female C57BL/6 mice, 6 to 8 weeks old and weighing 20 to 25 g, were obtained from the Guangzhou Animal Testing Center and were maintained in an air-conditioned room with a 12hour light–dark cycle. The animals were provided access to food and water ad libitum until they were used for experiments. All animal experiments were performed according to a protocol approved by the Institutional Animal Care and Use Committee of Zhongshan Ophthalmic Center, Sun Yat-sen University, and all procedures were performed in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 2.2. Treatment of EAU by NaB Mice were subcutaneously immunized in two flank and back regions near the tail with 200 µg of IRBP1–20 (GPTHLFQPSLVLDMAKVLLD, Sangon Biotech Shanghai, China) emulsified in CFA (Difco, Detroit, MI) containing 2.5 mg of Mycobacterium tuberculosis strain H37Ra (Difco) （1：1 v/v）in 0.2ml. 200 ng pertussis toxins (Sigma-Aldrich, St. Louis, MO) were given intraperitoneally on day 0 and day 2. NaB (Sigma-Aldrich) at a dose of 1 g/kg/time point was administered orally every day. Funduscopic examination was performed on day 14 after immunization, and the clinical findings were graded from 0 to 4 as previously described
(12)
.
Clinical score was assessed in a masked fashion. Eyes were enucleated on day 14, and inflammation was assessed by histopathologic examination. 2.3. Histopathologic Examination The mice were euthanized 14 days after immunization, and their intact eyes were immediately enucleated, stored in a 10% neutral buffered formalin solution for 24 to 48 hours at room temperature, dehydrated in a graded ethanol series, and embedded in paraffin. Then, 5-µm
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sagittal sections were cut near the optic nerve head and were stained with hematoxylin and eosin (H&E). 2.4. Cell Purification and treatment C57BL/6 splenic naive CD4+T cells were purified by magnetic-activated cell sorting (MACS) bead isolation of CD4+T cells (Miltenyi Biotec, Gladbach, Germany). The purity of CD4+cells was 90%, as assayed by flow cytometry. Naive CD4+T cells (2×105/well) were activated with anti-CD3/CD28 beads (one bead to 5 cells [1:5]) in 96-well plates for 72 hours. Cultures were also supplemented with IL-2 (100 U/mL, PeproTech, Rocky Hill, NJ), IL-6 (20 ng/mL, PeproTech) and TGF-β1 (20 ng/mL, PeproTech) for Th17 cell differentiation. HO-1 siRNA and fluorescein-conjugated control siRNA were obtained from RiboBio (Guangzhou, China). siRNA transfection of T cells was performed according to the manufacturer’s instructions. CD11c+ dendritic cells (DCs) from mouse draining lymph nodes (DLNs) by immunomagnetic positive selection kit (STEMCELL Technologies, Vancouver, BC, Canada). 2.5. Flow Cytometry For intracellular cytokine staining, cells were stimulated in culture medium containing phorbol myristate acetate (PMA, 5 ng/mL; Sigma), ionomycin (500 ng/mL; Sigma), and monensin as a cytokine secretion blocker (Golgi-stop; BD Bioscience, San Jose, CA) in a 10% CO2 incubator at 37°C for 4 hours. Subsequently, the cells were stained using fluorescein isothiocyanate (FITC)conjugated monoclonal antibodies against mouse CD4 (eBioscience, San Diego, CA). The cells were washed, fixed, permeabilized with buffer (Cytofix/Cytoperm; eBioscience), intracellularly stained with phycoerythrin (PE)-conjugated antibodies against IL-17A (eBioscience), and analyzed on a flow cytometer (FACSCalibur; BD Bioscience, San Jose, CA) using acquisition and analysis software (CellQuest; Becton Dickinson, Franklin Lakes, NJ). For Foxp3,
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phosphorylated signal transduction activator 3 (pStat3) and retinoic acid-related orphan receptor (ROR)γt staining, cells were stained with FITC-conjugated antibodies against CD4. The cells were washed, fixed, permeabilized with buffer (eBioscience), intracellularly stained with PEconjugated antibodies against Foxp3, pStat3, and RORγt (BD Bioscience), and analyzed on a flow cytometer. 2.6. Real-time PCR Total RNA was extracted from the tissues and cell lysates using an RNeasy Mini Kit (Qiagen, Valencia, CA), and cDNA was generated using an Omniscript RT Kit (Qiagen). mRNA expression levels were quantified using ABsolute SYBR Green ROX mix (Thermo Fisher Scientific, Waltham, MA). The reactions were performed in triplicate, and relative mRNA expression was determined by normalizing the expression of each target gene to that of β-actin using the 2-∆∆Ct method. 2.7. Western Blot Analysis, ELISA The total proteins of the tissue and cell homogenates were extracted using the KeyGen whole cell lysis assay (KeyGen Biotech, China). Nuclear and cytoplasmic extracts were then prepared using the NE-PERTM nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific), according to manufacturer instructions. Proteins (25–50 µg) were separated on polyacrylamide-SDS gels and electroblotted onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). After blocking with Tris-buffered saline (TBS)/5% nonfat dry milk, the membranes were incubated with an antibody against Nrf2 or HO-1 (Abcam, Cambridge, MA, USA), followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody. The signals were visualized by enhanced chemiluminescence (Pierce, Rockford, IL, USA). The blots were then reprobed with a specific antibody against β-actin or proliferating cell nuclear antigen (PCNA) (Abcam).
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The concentrations of IL-17A, INF-γ, TNF-α, and IL-10 in the serum or supernatants of cultured cells were detected by ELISA (eBioscience). 2.8. Statistical Analysis Statistical analyses were performed with 1-way ANOVA or Student’s t-test, as appropriate, using SPSS software (16.0; SPSS, Chicago, IL). A p-value <0.05 was considered significant.
3. RESULTS 3.1. NaB treatment decreases the severity of experimental uveitis To examine the therapeutic effects of NaB on EAU, NaB was administered orally every day (1 g/kg/day) from day 7 to day 21 after IRBP1–20 immunization, which generated typical signs mimicking human uveitis, including chorioretinal lesions and infiltrations and vasculitis (large sizes and thick walls). Clinical scores were evaluated at day 14 after IRBP1–20 immunization. As shown in Fig. 1A, NaB treatment significantly reduced the clinical scores of the EAU mice compared with the phosphate-buffered saline (PBS)-treated group. Representative images of fundoscopic examinations demonstrated that few infiltrations and trace vasculitis were present in the NaB-treated group (Fig. 1B). Histopathological analyses of uveitis eyes showed heavy inflammatory cell infiltration, extensive retinal folding with detachments, moderate photoreceptor cell damage and medium-sized granulomatous lesions in the PBS-treated control group (Figs. 1C and D). Oral NaB strikingly reduced histopathological severity in EAU mice (Figs. 1C and D). 3.2. NaB modulates local and systemic inflammatory profiles in EAU We investigated the effects of NaB treatment on inflammatory profiles in EAU. The levels of local retinal inflammatory mediators were evaluated by mRNA expression. Significant increases in IL-17A, IFN-γ and TNF-α expression were detected in the retinas of mice with EAU, whereas
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NaB suppressed IL-17A, IFN-γ and TNF-α expression but increased IL-10 expression in the retinas of mice with EAU (Figs. 2A–D). Additionally, CXCL9 (T cells chemokine) and MCP-1 (monocytes chemokines) were also decreased in local retina after NaB treatment (Figs. 2E-F). Serum was prepared from the peripheral blood of IRBP-immunized mice, and inflammatory cytokines were quantified by ELISA. Quantitative analyses revealed significantly reduced IL17A, IFN-γ and TNF-α expression levels but increased IL-10 expression levels in the NaBtreated group (Figs. 2G-J). Overall, EAU inhibition by NaB modulated local and systemic immune responses. 3.3. NaB decreases Th17 cells and increases Treg cells in EAU Because Th17 and Treg cells are crucial for autoimmune uveitis development(3, 4), we assessed the effects of NaB treatment on Th17 and Treg cells in vivo. Analyses of draining lymph nodes (DLNs) and spleens showed that NaB significantly decreased the frequency and number of Th17 cells (Figs. 3A-C) in mice with EAU compared with the PBS treated group. As shown in Figs. 3D-F, treatment with NaB significantly increased the frequencies and numbers of CD4+FoxP3+ cells in the DLNs and spleens in EAU mice. 3.4. NaB suppresses Th17 cell differentiation and reciprocally induces Treg cell differentiation in vitro To determine how NaB affects Th17/Treg cell frequency, we next asked whether NaB regulates Th17 and Treg cell differentiation in vitro. For this purpose, naïve CD4+ cells (CD4+CD62LHighCD44Low) isolated from spleens of C57/B6J mice were cultured under Th17 cell–polarizing conditions with or without NaB (100 µM) for 72 hours. Addition of NaB to the cultures significantly decreased the frequency and numbers of IL-17–expressing CD4+ T cells (Fig. 4A-C). The IL-17 levels in the supernatants measured by Elisa were also significantly
9
reduced when NaB was added to the cultures (Fig. 4D). Similar to the observation made in EAU in vivo, as shown in Fig. 4A, E and F, the frequency and numbers of CD4+ FoxP3+T cells were significantly increased after NaB was added to the culture (Fig.4A, E and F). As RORγt and STAT-3 signaling is critically involved in the Th17 cells differentiation(13), we also demonstrated that in the presence of NaB, both RORγt and pSTAT-3 expression is markedly decreased in CD4+T cells (Fig. 4G and H), highlighting that NaB mainly controls Th17 cell differentiation and eventually diminishes Th17-meidated EAU. 3.5. Nrf2/HO-1 signaling is critically involved in NaB-mediated modulation of Th17 cell differentiation NaB is a potent activator for Nrf2, a governor of antioxidant signaling(14-16). Thus, we determined whether Nrf2 and its downstream signaling factor, HO-1, were involved in NaB-mediated modulation in Th17 cells. Western blot results showed that NaB treatment significantly induced the nuclear translocation of Nrf2 in CD4+T cells under Th17 cell-polarizing conditions (Fig. 5A). NaB treatment also increased HO-1 protein expression compared with vehicle control (Fig. 5B). We found that treatment with NaB inhibited IL-6R expression at the mRNA and protein levels (Figs. 5C and D). Next, SnPP, a HO-1 inhibitor, was used to examine the role of Nrf2/HO-1 signaling in NaB-mediated modulation on Th17 cell differentiation. As shown in Figs. 5E-G, SnPP treatment significantly but incompletely reversed NaB-mediated modulatory effects on Th17 cell differentiation. The inhibitory capacity of NaB on IL-6R expression was significantly decreased after SnPP treatment (Fig. 5H). To further confirm the role of Nrf2/HO-1 in NaB– mediated Th17 cell suppression, we used small interfering RNA (siRNA) to knock down HO-1 expression in CD4+ T cells, and the inhibitory capacity of NaB on Th17 cell differentiation was also significantly decreased after HO-1 siRNA treatment but not control siRNA (Figs. 5I-L) .
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These results suggest that Nrf2/HO-1 signaling plays an important role in the NaB–mediated inhibition of Th17 cell differentiation. 3.6. NaB Attenuates EAU via Nrf2/HO-1 signaling Our in vitro studies showed that Nrf2/HO-1 signaling plays a critical role in the NaB-mediated modulation of Th17 cell differentiation. Therefore, we determined whether Nrf2/HO-1 signaling was also involved in the NaB-mediated inhibition of EAU. Western blot results showed that NaB treatment significantly induced the nuclear translocation of Nrf2 and increased HO-1 expression in the DLNs of EAU mice (Figs. 6A and B). SnPP, HO-1 inhibitor, was intraperitoneally injected (40 mol/kg) once daily for 7 days after NaB administration. Treatment with the HO-1 inhibitor
significantly blocked the NaB-mediated decrease in the clinical and pathological scores of EAU (Figs. 6C and D). As shown in Figs. 6E-G, the HO-1 inhibitor significantly decreased the inhibitory capacity of NaB on Th17 cell frequency and number in DLNs. Additionally, the HO-1 inhibitor also blocked NaB-mediated inhibition on IL-17A expression in retinas (Fig. 6H) and IL6R expression in DLNs (Fig. 6I). These findings suggest that the upregulation of NRF2/HO-1 signaling may contribute, at least in part, to the NaB-mediated attenuation of EAU. Dendritic cells (DCs) play also important roles in uveitis. Thus, we also evaluate the effects of NaB on DCs in EAU. Our results showed that NaB treatment reduced CD11c+CD80+ DCs in DLNs (Figs. 6J-L). Furthermore, levels of Nrf2/HO-1 were increased in DCs isolated from DLNs in EAU after NaB treatment (Figs. 6M-N). These findings suggest that the multiple targets may contribute to therapeutic effects of NaB in uveitis.
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4. DISCUSSION EAU is an established animal model for human uveitic diseases, including Vogt-KoyanagiHarada disease and Behcet’s disease (12). In this study, EAU was induced via local injection of an IRBP peptide, with inflammation peaking by 14 day after immunization. We investigated the therapeutic effects and mechanisms of NaB in EAU. This study demonstrated that NaB could significantly attenuate the ocular inflammatory response in EAU mice 14 days after immunization, with significant decreases in inflammatory cell infiltration and inflammatory cytokine production in the retina. The main mechanism by which NaB exerts its therapeutic efficacy is the reciprocal regulation of Th17 and Treg cell differentiation. The inhibitory effect of NaB on Th17 cell differentiation depended primarily on IL-6R expression. Interestingly, NaB treatment in EAU mice resulted in the profound activation of the Nrf2/HO-1 signaling pathway, which plays crucial roles in NaB-mediated inhibition on Th17 cell differentiation and EAU. Additionally, NaB treatment also reduced mature DCs in DLNs of EAU mice. These findings suggest that NaB ameliorates EAU mainly through regulating Th17/Treg cells balance. As the main subgroups of T cells, Treg and Th17 cells have always been the focus of immunologists in the area of autoimmune diseases
(4, 17, 18)
. In uveitis, decreased frequency and
diminished function of Treg cells have been associated with active uveitis in patients with Vogt Koyanagi-Harada disease and Behcet’s disease
(19, 20)
. Furthermore, Treg cell depletion studies
have indicated augmented retinal inflammatory responses and support the notion that Treg cells play protective roles in the development of uveitis
(21)
. In contrast, Th17 cells are increased in
active uveitis patients with Vogt Koyanagi-Harada disease and Behcet’s disease and promote uveitis development (22-24). Importantly, accumulating evidence has shown that the Th17 and Treg developmental pathways are reciprocally interconnected and that Treg cells are able to be
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converted to Th17 cells in the context of the inflammatory milieu(13). Thus, the conversion of Th17 cells toward Treg cells to reset the Treg/Th17 cell balance would be a promising treatment strategy for uveitis(13). In this study, we found that NaB directly converted the differentiation of naive T cells from Th17 cells toward Treg cells in vitro. In vivo, NaB treatment decreased the frequencies and numbers of Th17 cells and increased the frequencies and numbers of Treg cells in both the DLNs and spleens of EAU mice. Thus, NaB may be a promising treatment for uveitis. Furthermore, the inversion of the differentiation of Th17 cells toward Treg cells may be the main mechanism underlying NaB action.
Butyrate has been shown to promote Treg cell differentiation in previous studies(9, 10). In 2013, Furusawa and colleagues reported that butyrate treatment enhanced Foxp3 expression in naive T cells under Treg-cell-polarizing conditions and induced the differentiation of colonic regulatory T cells(10). Arpaia et al. reported that butyrate treatment promoted de novo Treg-cell generation and facilitated extrathymic Treg cell generation(9). As reported in these previous studies, in EAU, NaB treatment increased Treg cell generation in DLN and spleen. Importantly, we found that NaB treatment reduced pSTAT-3 and RORγt expression levels, whereas Foxp3 expression increased in naive T cells under Th17-cell-polarizing conditions. This is a novel observation that butyrate directly converted the differentiation of naive T cells from Th17 cells toward Treg cells. IL-6/IL-6R signaling is crucial for Th17 cell differentiation. We found that NaB treatment significantly inhibited IL-6R in naive T cells under Th17-cell-polarizing conditions. Thus, the inhibition of IL-6R expression may be crucial for NaB-mediated modulation of Th17 cell differentiation. Taken together, in addition to directly enhancing Treg cell differentiation, butyrate inverts the differentiation of Th17 cells toward Treg cells by inhibiting IL-6R.
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The transcription factor Nrf2 is regarded as a master regulator in the activation of cellular responses to oxidative stress(25). Under normal conditions, Nrf2 is present as an inactive complex in the cytoplasm with Kelch-like ECH-associated protein 1. When the complex is disrupted by activated signaling, Nrf2 translocates to the nucleus, leading to target gene transcription, including HO-1, a key heme-degrading enzyme(25-27). In addition to well-known antioxidative properties, accumulating evidence shows that HO-1 possesses versatile anti-inflammatory and immune regulatory functions(28). Several previous studies have shown that NaB could activate Nrf2 in intestinal epithelial cells, leading to chronic cerebral hypoperfusion(14-16). Consistent with previous studies, our results showed that NaB treatment promoted Nrf2 nuclear translocation and subsequently increased HO-1 expression in naïve T cells under Th17-cell-polarizing conditions. Furthermore, an HO-1 blockade significantly but incompletely reversed NaB-mediated inhibition on Th17 cell differentiation. Importantly, the HO-1 inhibitor also blocked the NaB-mediated decreases in the clinical and pathological scores of EAU. Collectively, these compelling findings suggest that Nrf2/HO-1 signaling contributes, at least in part, to the NaB-mediated inhibition of Th17 cell differentiation and EAU. In summary, our study was the first to demonstrate the therapeutic effects of NaB on experimental uveitis. Our results showed that NaB directly converted the differentiation of naive T cells from Th17 cells to Treg cells, possibly by inhibiting IL-6R signaling. Importantly, we further determined the immune-regulatory mechanisms of NaB by demonstrating that Nrf2-HO-1 signaling is essential for NaB-mediated immune regulatory effects in vitro and in vivo. These findings provide compelling evidence that NaB can inhibit uveitis and support butyrate as a potential therapeutic option for autoimmune diseases.
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Acknowledgements: This study was supported by the Natural Science Foundation of China (81470670, 81670897, 81671611 and U1601226), Guangdong Natural Science Funds for Distinguished Young Scholar (2016A030306006); Science and technology cooperation project of Hong Kong, Macao and Taiwan (L2015TGA0005); Natural Science Foundation of Guangdong Province (2015A030313189), Science and Technology Planning Project of Guangdong Province (2013B051000027), the Youth Science and Technology Innovation Talents Funds in a Special Support Plan for High Level Talents in Guangdong Province (No. 2015TQ01R261); the Zhu Jiang Star of Science and Technology Foundation in Guangzhou (201610010129). Conflict of interest None.
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Abbreviations: NaB: sodium butyrate; EAU: experimental autoimmune uveitis; Nrf2; Nuclear factor erythroid 2 -related factor 2; HO-1; Heme oxygenase-1; DLN: draining lymphoid nodes; Treg cells: CD4+ T regulatory cells; Foxp3: Forkhead box protein 3; STAT-3: Signal transduction activator 3; pSTAT3: Phosphorylated signal transduction activator 3 RORγt: Retinoic acid-related orphan receptor (ROR)γt IL-6R: Interleukin-6 receptor DC: Dendritic cell
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Figure 1. NaB treatment decreases the severity of experimental uveitis. (A): The clinical scores of EAU were determined at 14 day after immunization (n=7). (B): Representative images of fundoscopic examination in the indicated experimental group at 14 day after immunization (white arrow: vasculitis). (C): The pathological scores of EAU were determined at 14 day after immunization (n=7). (D): Representative images of with histopathologic examination in the indicated experimental group at 14 day after immunization (Scale bars represent 200 µm). The data are presented as the mean ± SD. **P < 0.01. Figure 2. NaB modulates local and systemic inflammatory profiles in EAU. (A-D): The expression of IL-17A, INF-γ, TNF-α and IL-10 mRNA in retinas were measured by realtime PCR at 14 day after immunization (n=6). (E-F): The expression of CXCL9 and MCP-1 in retinas was examined by real-time PCR. (G-J) The levels of IL-17A, INF-γ, TNF-α and IL-10 protein in serum were determined via ELISA at 14 day after immunization (n=6). The data are presented as the mean ± SD. **P < 0.01. Figure 3. NaB decreases Th17 cells and increases Treg cells in EAU. (A-C): CD4+IL-17A+ T cells in the draining lymph node (DLNs) and spleen of EAU mice were determined by flow cytometry at 14 day after immunization (n=3). (D-F): CD4+Foxp3+ T cells in the draining lymph node (DLNs) and spleen of EAU mice were determined by flow cytometry at 14 day after immunization (n=3). The data are presented as the mean ± SD. *P<0.05, **P < 0.01. Figure 4. NaB suppresses Th17 cell differentiation and reciprocally induces Treg cell differentiation in vitro. CD4+CD62L+ cells from C57BL/6J mice polarized to Th17 cells in presence or absence of NaB for 3 days. (A) Expression of IL-17A and Foxp3 were checked by flow cytometry (n=3). (B-C) Quantitative analysis of CD4+IL-17A+T cells. (D) The levels of
21
IL-17A protein in the supernatants were determined via ELISA (n=3). (E-F) Quantitative analysis of CD4+Foxp3+T cells. (G) The same cells were analyzed by flow cytometry for RORγt expression (n=3). (H) CD4+CD62L+ cells were incubated in presence or absence of NaB for 2 h, then IL6 was added for 30min. pSTAT3 expression was determined and compared to unstimulated CD4+CD62L+ cells (Unstim, filled gray histogram, n=3). The data are presented as the mean ± SD. *P<0.05, **P < 0.01.
Figure 5. Nrf2/HO-1 signaling is critically involved in NaB-mediated modulation of Th17 cell differentiation. NaB treatment significantly induced the nuclear translocation of Nrf2 (A) and increased HO-1 expression (B) in CD4+T cells under Th17 cell–polarizing condition (n=3). NaB obviously inhibited IL-6R expression in both mRNA (C) and protein (D) levels in CD4+T cells under Th17 cell–polarizing condition (n=3). (E-G): CD4+IL-17A+ T cells under indicated condition were determined by flow cytometry analysis (n=3). (H): The expression of IL-6R mRNA in CD4+T cells under indicated condition were measured by realtime PCR (n=3). (I) After pretreating CD4+T cells with HO-1 siRNA (kdHO-1) or negative control (NC), expression of IL-17A was checked by flow cytometry (n=3). (J-K) Quantitative analysis of CD4+IL-17A+T cells. (L) After pretreating CD4+T cells with HO-1 siRNA (kdHO-1) or negative control (NC), the expression of IL-6R mRNA was measured by real-time PCR (n=3). The data are presented as the mean ± SD. **P < 0.01. Figure 6. NaB Attenuates EAU via Nrf2/HO-1 signaling. (A-B): NaB treatment significantly induced the nuclear translocation of Nrf2 and increased HO-1 expression in the DLNs of EAU mice. (C-D): SnPP (an HO-1 inhibitor) significantly blocked the NaB-mediated decrease in the
22
clinical and pathological scores of EAU (n=8). (E-G): The SnPP significantly reversed the NaBmediated decreases in the frequencies and numbers of Th17 in the DLN (n=3). (H-I): The IL17A mRNA expression in retinas and IL-6R mRNA expression in DLN were measured via realtime PCR (n=3). (J-L): CD11c+CD80+ DCs under indicated groups were determined by flow cytometry analysis (n=3). (M-N): NaB treatment significantly induced the nuclear translocation of Nrf2 and increased HO-1 expression in DCs isolated from the DLNs of EAU mice (n=3). The data are presented as the mean ± SD. *P<0.05, **P < 0.01.
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S0006-2952(17)30476-8 http://dx.doi.org/10.1016/j.bcp.2017.06.136 BCP 12862
To appear in:
Biochemical Pharmacology
Received Date: Accepted Date:
11 May 2017 30 June 2017
Please cite this article as: X. Chen, W. Su, T. Wan, J. Yu, W. Zhu, F. Tang, G. Liu, N. Olsen, D. Liang, S.G. Zheng, Sodium Butyrate Regulates Th17/Treg Cell Balance to Ameliorate Uveitis via the Nrf2/HO-1 Pathway, Biochemical Pharmacology (2017), doi: http://dx.doi.org/10.1016/j.bcp.2017.06.136
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Sodium Butyrate Regulates Th17/Treg Cell Balance to Ameliorate Uveitis via the Nrf2/HO1 Pathway Xiaoqing Chen MD*1; Wenru Su MD, PHD*1#; Taoshang Wan, MD1; Jianfeng Yu, MD1; Wenjie Zhu, MD1; Fen Tang, MD1; Guangming Liu, MD1; Nancy Olsen, PHD3; Dan Liang MD, PHD1# and Song Guo Zheng MD, PHD2,3# Running title: NaB attenuates uveitis 1
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen
University, Guangzhou, China; 2 Center for Clinic Immunology, Sun Yat-sen University Third Affiliated Hospital, Guangzhou, China; 3Division of Rheumatology, Department of Medicine, Penn State University Hershey College of Medicine, USA * These authors contributed equally to this work. Author contributions: X-Q. C.: conception and design, data collection and assembly, manuscript writing, and final manuscript approval; W-R. S.: conception and design, data collection and assembly, manuscript writing, final manuscript approval, and financial support; W-J. Z., J-F.Y., F.T., T-S.W., G-M.L.: data collection and assembly; N. O.: conception and design, manuscript writing; D.L., S-G. Z.: conception and design, manuscript writing, final manuscript approval, and financial support. # Correspondence to: Dan
Liang,
MD,
PhD,
Phone:
+86-20-87330341;
Fax:
+86-20-87330341,
Email:
[email protected] or Song Guo Zheng MD, PHD, Phone 717 531 0003, Fax: 717 531 8274, Email: [email protected] or [email protected] or Wenru Su, MD, PhD, Phone: +86-20-87330402; Fax: +86-20-87330402, Email: [email protected] Disclosure of potential conflicts of interest: The authors declare no competing financial conflicts of interest.
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ABSTRACT Autoimmune uveitis, a group of potentially blinding intraocular inflammatory diseases, remains a therapeutic challenge for ophthalmologists. Butyrates, which belong to the short-chain fatty acid family, possess immunomodulatory properties and therapeutic potential in several inflammatory disorders. However, the roles of butyrates in uveitis and their underlying immunomodulatory mechanisms remain elusive. Here, we report that treatment with sodium butyrate (NaB) significantly attenuated the ocular inflammatory response in mice with experimental autoimmune uveitis (EAU) at 14 days after immunization, with significant decreases in inflammatory cell infiltration and inflammatory cytokine production in the retinas. Furthermore, NaB treatment decreased the frequency and number of Th17 cells and increased the frequency and number of T regulatory (Treg) cells in both draining lymph nodes and spleens of EAU mice. In vitro, NaB treatment directly converted the differentiation of naive T cells from Th17 cells toward Treg cells. Mechanistically, the NaB-mediated inhibition of Th17 cell differentiation may occur via inhibition of the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO1)/interleukin-6 receptor pathway. Moreover, the NaB-mediated inhibition on Th17 cell differentiation and uveitis were abrogated when an HO-1 inhibitor, SnPP, was used. These findings suggest that NaB inverts the differentiation of Th17 cells toward Treg cells and attenuates experimental autoimmune uveitis by modulating the Nrf2/HO-1 pathway. Key Words: Uveitis, Butyrate, T regulatory cells, Th17, Heme oxygenase 1, autoimmune diseases Chemical compounds studied in this article: Sodium Butyrate (PubChem CID: 5222465)
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1. INTRODUCTION Autoimmune uveitis is a group of potentially blinding intraocular inflammatory diseases accounting for approximately 10% of cases of severe visual handicaps in the United States, with a higher prevalence in undeveloped countries(1). Due to diverse causes, the care and management of patients with uveitis remain challenges to ophthalmologists(2). Although the pathogenesis of uveitis is not fully understood, the imbalance of CD4+CD25+Forkhead box protein 3(Foxp3)+ T regulatory (Treg) cells and T helper 17 (Th17) cells has been considered a critical part of pathogenesis for autoimmune uveitis
(3, 4)
. The application of corticosteroids and other immune
modulating therapies has improved uveitis treatment. However, long-term exposure to these drugs may result in potentially significant adverse effects(1, 2, 5, 6). Furthermore, many patients cannot tolerate or are resistant to these agents. Therefore, novel safe and effective alternatives are needed for uveitis treatment. Butyrate is a small molecule belonging to the short-chain fatty acid family and is a natural product of the intestinal microbial fermentation of indigestible foods(7). Recent works have found that butyrate is actively involved in a number of pathological processes, including autoimmunity, cancer and neurological disorders(7-9). Moreover, several recent studies have shown that butyrate possesses great potential for Treg induction in vivo and in vitro
(9, 10)
. Sodium butyrate (NaB)
administration to mice can significantly ameliorate inflammation and related symptoms in colitis and diabetes(10,
11)
. Therefore, butyrate may be a potential therapeutic alternative for uveitis
treatment. However, the role of butyrate in uveitis has not yet been explored. Moreover, the mechanisms by which butyrate mediates immunomodulation remain elusive. Thus, in this study, we investigated the therapeutic effect of NaB on experimental autoimmune uveitis (EAU) and the immunomodulatory mechanisms of NaB.
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2. MATERIALS ANDMETHODS 2.1 Animals Inbred female C57BL/6 mice, 6 to 8 weeks old and weighing 20 to 25 g, were obtained from the Guangzhou Animal Testing Center and were maintained in an air-conditioned room with a 12hour light–dark cycle. The animals were provided access to food and water ad libitum until they were used for experiments. All animal experiments were performed according to a protocol approved by the Institutional Animal Care and Use Committee of Zhongshan Ophthalmic Center, Sun Yat-sen University, and all procedures were performed in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 2.2. Treatment of EAU by NaB Mice were subcutaneously immunized in two flank and back regions near the tail with 200 µg of IRBP1–20 (GPTHLFQPSLVLDMAKVLLD, Sangon Biotech Shanghai, China) emulsified in CFA (Difco, Detroit, MI) containing 2.5 mg of Mycobacterium tuberculosis strain H37Ra (Difco) （1：1 v/v）in 0.2ml. 200 ng pertussis toxins (Sigma-Aldrich, St. Louis, MO) were given intraperitoneally on day 0 and day 2. NaB (Sigma-Aldrich) at a dose of 1 g/kg/time point was administered orally every day. Funduscopic examination was performed on day 14 after immunization, and the clinical findings were graded from 0 to 4 as previously described
(12)
.
Clinical score was assessed in a masked fashion. Eyes were enucleated on day 14, and inflammation was assessed by histopathologic examination. 2.3. Histopathologic Examination The mice were euthanized 14 days after immunization, and their intact eyes were immediately enucleated, stored in a 10% neutral buffered formalin solution for 24 to 48 hours at room temperature, dehydrated in a graded ethanol series, and embedded in paraffin. Then, 5-µm
5
sagittal sections were cut near the optic nerve head and were stained with hematoxylin and eosin (H&E). 2.4. Cell Purification and treatment C57BL/6 splenic naive CD4+T cells were purified by magnetic-activated cell sorting (MACS) bead isolation of CD4+T cells (Miltenyi Biotec, Gladbach, Germany). The purity of CD4+cells was 90%, as assayed by flow cytometry. Naive CD4+T cells (2×105/well) were activated with anti-CD3/CD28 beads (one bead to 5 cells [1:5]) in 96-well plates for 72 hours. Cultures were also supplemented with IL-2 (100 U/mL, PeproTech, Rocky Hill, NJ), IL-6 (20 ng/mL, PeproTech) and TGF-β1 (20 ng/mL, PeproTech) for Th17 cell differentiation. HO-1 siRNA and fluorescein-conjugated control siRNA were obtained from RiboBio (Guangzhou, China). siRNA transfection of T cells was performed according to the manufacturer’s instructions. CD11c+ dendritic cells (DCs) from mouse draining lymph nodes (DLNs) by immunomagnetic positive selection kit (STEMCELL Technologies, Vancouver, BC, Canada). 2.5. Flow Cytometry For intracellular cytokine staining, cells were stimulated in culture medium containing phorbol myristate acetate (PMA, 5 ng/mL; Sigma), ionomycin (500 ng/mL; Sigma), and monensin as a cytokine secretion blocker (Golgi-stop; BD Bioscience, San Jose, CA) in a 10% CO2 incubator at 37°C for 4 hours. Subsequently, the cells were stained using fluorescein isothiocyanate (FITC)conjugated monoclonal antibodies against mouse CD4 (eBioscience, San Diego, CA). The cells were washed, fixed, permeabilized with buffer (Cytofix/Cytoperm; eBioscience), intracellularly stained with phycoerythrin (PE)-conjugated antibodies against IL-17A (eBioscience), and analyzed on a flow cytometer (FACSCalibur; BD Bioscience, San Jose, CA) using acquisition and analysis software (CellQuest; Becton Dickinson, Franklin Lakes, NJ). For Foxp3,
6
phosphorylated signal transduction activator 3 (pStat3) and retinoic acid-related orphan receptor (ROR)γt staining, cells were stained with FITC-conjugated antibodies against CD4. The cells were washed, fixed, permeabilized with buffer (eBioscience), intracellularly stained with PEconjugated antibodies against Foxp3, pStat3, and RORγt (BD Bioscience), and analyzed on a flow cytometer. 2.6. Real-time PCR Total RNA was extracted from the tissues and cell lysates using an RNeasy Mini Kit (Qiagen, Valencia, CA), and cDNA was generated using an Omniscript RT Kit (Qiagen). mRNA expression levels were quantified using ABsolute SYBR Green ROX mix (Thermo Fisher Scientific, Waltham, MA). The reactions were performed in triplicate, and relative mRNA expression was determined by normalizing the expression of each target gene to that of β-actin using the 2-∆∆Ct method. 2.7. Western Blot Analysis, ELISA The total proteins of the tissue and cell homogenates were extracted using the KeyGen whole cell lysis assay (KeyGen Biotech, China). Nuclear and cytoplasmic extracts were then prepared using the NE-PERTM nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific), according to manufacturer instructions. Proteins (25–50 µg) were separated on polyacrylamide-SDS gels and electroblotted onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). After blocking with Tris-buffered saline (TBS)/5% nonfat dry milk, the membranes were incubated with an antibody against Nrf2 or HO-1 (Abcam, Cambridge, MA, USA), followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody. The signals were visualized by enhanced chemiluminescence (Pierce, Rockford, IL, USA). The blots were then reprobed with a specific antibody against β-actin or proliferating cell nuclear antigen (PCNA) (Abcam).
7
The concentrations of IL-17A, INF-γ, TNF-α, and IL-10 in the serum or supernatants of cultured cells were detected by ELISA (eBioscience). 2.8. Statistical Analysis Statistical analyses were performed with 1-way ANOVA or Student’s t-test, as appropriate, using SPSS software (16.0; SPSS, Chicago, IL). A p-value <0.05 was considered significant.
3. RESULTS 3.1. NaB treatment decreases the severity of experimental uveitis To examine the therapeutic effects of NaB on EAU, NaB was administered orally every day (1 g/kg/day) from day 7 to day 21 after IRBP1–20 immunization, which generated typical signs mimicking human uveitis, including chorioretinal lesions and infiltrations and vasculitis (large sizes and thick walls). Clinical scores were evaluated at day 14 after IRBP1–20 immunization. As shown in Fig. 1A, NaB treatment significantly reduced the clinical scores of the EAU mice compared with the phosphate-buffered saline (PBS)-treated group. Representative images of fundoscopic examinations demonstrated that few infiltrations and trace vasculitis were present in the NaB-treated group (Fig. 1B). Histopathological analyses of uveitis eyes showed heavy inflammatory cell infiltration, extensive retinal folding with detachments, moderate photoreceptor cell damage and medium-sized granulomatous lesions in the PBS-treated control group (Figs. 1C and D). Oral NaB strikingly reduced histopathological severity in EAU mice (Figs. 1C and D). 3.2. NaB modulates local and systemic inflammatory profiles in EAU We investigated the effects of NaB treatment on inflammatory profiles in EAU. The levels of local retinal inflammatory mediators were evaluated by mRNA expression. Significant increases in IL-17A, IFN-γ and TNF-α expression were detected in the retinas of mice with EAU, whereas
8
NaB suppressed IL-17A, IFN-γ and TNF-α expression but increased IL-10 expression in the retinas of mice with EAU (Figs. 2A–D). Additionally, CXCL9 (T cells chemokine) and MCP-1 (monocytes chemokines) were also decreased in local retina after NaB treatment (Figs. 2E-F). Serum was prepared from the peripheral blood of IRBP-immunized mice, and inflammatory cytokines were quantified by ELISA. Quantitative analyses revealed significantly reduced IL17A, IFN-γ and TNF-α expression levels but increased IL-10 expression levels in the NaBtreated group (Figs. 2G-J). Overall, EAU inhibition by NaB modulated local and systemic immune responses. 3.3. NaB decreases Th17 cells and increases Treg cells in EAU Because Th17 and Treg cells are crucial for autoimmune uveitis development(3, 4), we assessed the effects of NaB treatment on Th17 and Treg cells in vivo. Analyses of draining lymph nodes (DLNs) and spleens showed that NaB significantly decreased the frequency and number of Th17 cells (Figs. 3A-C) in mice with EAU compared with the PBS treated group. As shown in Figs. 3D-F, treatment with NaB significantly increased the frequencies and numbers of CD4+FoxP3+ cells in the DLNs and spleens in EAU mice. 3.4. NaB suppresses Th17 cell differentiation and reciprocally induces Treg cell differentiation in vitro To determine how NaB affects Th17/Treg cell frequency, we next asked whether NaB regulates Th17 and Treg cell differentiation in vitro. For this purpose, naïve CD4+ cells (CD4+CD62LHighCD44Low) isolated from spleens of C57/B6J mice were cultured under Th17 cell–polarizing conditions with or without NaB (100 µM) for 72 hours. Addition of NaB to the cultures significantly decreased the frequency and numbers of IL-17–expressing CD4+ T cells (Fig. 4A-C). The IL-17 levels in the supernatants measured by Elisa were also significantly
9
reduced when NaB was added to the cultures (Fig. 4D). Similar to the observation made in EAU in vivo, as shown in Fig. 4A, E and F, the frequency and numbers of CD4+ FoxP3+T cells were significantly increased after NaB was added to the culture (Fig.4A, E and F). As RORγt and STAT-3 signaling is critically involved in the Th17 cells differentiation(13), we also demonstrated that in the presence of NaB, both RORγt and pSTAT-3 expression is markedly decreased in CD4+T cells (Fig. 4G and H), highlighting that NaB mainly controls Th17 cell differentiation and eventually diminishes Th17-meidated EAU. 3.5. Nrf2/HO-1 signaling is critically involved in NaB-mediated modulation of Th17 cell differentiation NaB is a potent activator for Nrf2, a governor of antioxidant signaling(14-16). Thus, we determined whether Nrf2 and its downstream signaling factor, HO-1, were involved in NaB-mediated modulation in Th17 cells. Western blot results showed that NaB treatment significantly induced the nuclear translocation of Nrf2 in CD4+T cells under Th17 cell-polarizing conditions (Fig. 5A). NaB treatment also increased HO-1 protein expression compared with vehicle control (Fig. 5B). We found that treatment with NaB inhibited IL-6R expression at the mRNA and protein levels (Figs. 5C and D). Next, SnPP, a HO-1 inhibitor, was used to examine the role of Nrf2/HO-1 signaling in NaB-mediated modulation on Th17 cell differentiation. As shown in Figs. 5E-G, SnPP treatment significantly but incompletely reversed NaB-mediated modulatory effects on Th17 cell differentiation. The inhibitory capacity of NaB on IL-6R expression was significantly decreased after SnPP treatment (Fig. 5H). To further confirm the role of Nrf2/HO-1 in NaB– mediated Th17 cell suppression, we used small interfering RNA (siRNA) to knock down HO-1 expression in CD4+ T cells, and the inhibitory capacity of NaB on Th17 cell differentiation was also significantly decreased after HO-1 siRNA treatment but not control siRNA (Figs. 5I-L) .
10
These results suggest that Nrf2/HO-1 signaling plays an important role in the NaB–mediated inhibition of Th17 cell differentiation. 3.6. NaB Attenuates EAU via Nrf2/HO-1 signaling Our in vitro studies showed that Nrf2/HO-1 signaling plays a critical role in the NaB-mediated modulation of Th17 cell differentiation. Therefore, we determined whether Nrf2/HO-1 signaling was also involved in the NaB-mediated inhibition of EAU. Western blot results showed that NaB treatment significantly induced the nuclear translocation of Nrf2 and increased HO-1 expression in the DLNs of EAU mice (Figs. 6A and B). SnPP, HO-1 inhibitor, was intraperitoneally injected (40 mol/kg) once daily for 7 days after NaB administration. Treatment with the HO-1 inhibitor
significantly blocked the NaB-mediated decrease in the clinical and pathological scores of EAU (Figs. 6C and D). As shown in Figs. 6E-G, the HO-1 inhibitor significantly decreased the inhibitory capacity of NaB on Th17 cell frequency and number in DLNs. Additionally, the HO-1 inhibitor also blocked NaB-mediated inhibition on IL-17A expression in retinas (Fig. 6H) and IL6R expression in DLNs (Fig. 6I). These findings suggest that the upregulation of NRF2/HO-1 signaling may contribute, at least in part, to the NaB-mediated attenuation of EAU. Dendritic cells (DCs) play also important roles in uveitis. Thus, we also evaluate the effects of NaB on DCs in EAU. Our results showed that NaB treatment reduced CD11c+CD80+ DCs in DLNs (Figs. 6J-L). Furthermore, levels of Nrf2/HO-1 were increased in DCs isolated from DLNs in EAU after NaB treatment (Figs. 6M-N). These findings suggest that the multiple targets may contribute to therapeutic effects of NaB in uveitis.
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4. DISCUSSION EAU is an established animal model for human uveitic diseases, including Vogt-KoyanagiHarada disease and Behcet’s disease (12). In this study, EAU was induced via local injection of an IRBP peptide, with inflammation peaking by 14 day after immunization. We investigated the therapeutic effects and mechanisms of NaB in EAU. This study demonstrated that NaB could significantly attenuate the ocular inflammatory response in EAU mice 14 days after immunization, with significant decreases in inflammatory cell infiltration and inflammatory cytokine production in the retina. The main mechanism by which NaB exerts its therapeutic efficacy is the reciprocal regulation of Th17 and Treg cell differentiation. The inhibitory effect of NaB on Th17 cell differentiation depended primarily on IL-6R expression. Interestingly, NaB treatment in EAU mice resulted in the profound activation of the Nrf2/HO-1 signaling pathway, which plays crucial roles in NaB-mediated inhibition on Th17 cell differentiation and EAU. Additionally, NaB treatment also reduced mature DCs in DLNs of EAU mice. These findings suggest that NaB ameliorates EAU mainly through regulating Th17/Treg cells balance. As the main subgroups of T cells, Treg and Th17 cells have always been the focus of immunologists in the area of autoimmune diseases
(4, 17, 18)
. In uveitis, decreased frequency and
diminished function of Treg cells have been associated with active uveitis in patients with Vogt Koyanagi-Harada disease and Behcet’s disease
(19, 20)
. Furthermore, Treg cell depletion studies
have indicated augmented retinal inflammatory responses and support the notion that Treg cells play protective roles in the development of uveitis
(21)
. In contrast, Th17 cells are increased in
active uveitis patients with Vogt Koyanagi-Harada disease and Behcet’s disease and promote uveitis development (22-24). Importantly, accumulating evidence has shown that the Th17 and Treg developmental pathways are reciprocally interconnected and that Treg cells are able to be
12
converted to Th17 cells in the context of the inflammatory milieu(13). Thus, the conversion of Th17 cells toward Treg cells to reset the Treg/Th17 cell balance would be a promising treatment strategy for uveitis(13). In this study, we found that NaB directly converted the differentiation of naive T cells from Th17 cells toward Treg cells in vitro. In vivo, NaB treatment decreased the frequencies and numbers of Th17 cells and increased the frequencies and numbers of Treg cells in both the DLNs and spleens of EAU mice. Thus, NaB may be a promising treatment for uveitis. Furthermore, the inversion of the differentiation of Th17 cells toward Treg cells may be the main mechanism underlying NaB action.
Butyrate has been shown to promote Treg cell differentiation in previous studies(9, 10). In 2013, Furusawa and colleagues reported that butyrate treatment enhanced Foxp3 expression in naive T cells under Treg-cell-polarizing conditions and induced the differentiation of colonic regulatory T cells(10). Arpaia et al. reported that butyrate treatment promoted de novo Treg-cell generation and facilitated extrathymic Treg cell generation(9). As reported in these previous studies, in EAU, NaB treatment increased Treg cell generation in DLN and spleen. Importantly, we found that NaB treatment reduced pSTAT-3 and RORγt expression levels, whereas Foxp3 expression increased in naive T cells under Th17-cell-polarizing conditions. This is a novel observation that butyrate directly converted the differentiation of naive T cells from Th17 cells toward Treg cells. IL-6/IL-6R signaling is crucial for Th17 cell differentiation. We found that NaB treatment significantly inhibited IL-6R in naive T cells under Th17-cell-polarizing conditions. Thus, the inhibition of IL-6R expression may be crucial for NaB-mediated modulation of Th17 cell differentiation. Taken together, in addition to directly enhancing Treg cell differentiation, butyrate inverts the differentiation of Th17 cells toward Treg cells by inhibiting IL-6R.
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The transcription factor Nrf2 is regarded as a master regulator in the activation of cellular responses to oxidative stress(25). Under normal conditions, Nrf2 is present as an inactive complex in the cytoplasm with Kelch-like ECH-associated protein 1. When the complex is disrupted by activated signaling, Nrf2 translocates to the nucleus, leading to target gene transcription, including HO-1, a key heme-degrading enzyme(25-27). In addition to well-known antioxidative properties, accumulating evidence shows that HO-1 possesses versatile anti-inflammatory and immune regulatory functions(28). Several previous studies have shown that NaB could activate Nrf2 in intestinal epithelial cells, leading to chronic cerebral hypoperfusion(14-16). Consistent with previous studies, our results showed that NaB treatment promoted Nrf2 nuclear translocation and subsequently increased HO-1 expression in naïve T cells under Th17-cell-polarizing conditions. Furthermore, an HO-1 blockade significantly but incompletely reversed NaB-mediated inhibition on Th17 cell differentiation. Importantly, the HO-1 inhibitor also blocked the NaB-mediated decreases in the clinical and pathological scores of EAU. Collectively, these compelling findings suggest that Nrf2/HO-1 signaling contributes, at least in part, to the NaB-mediated inhibition of Th17 cell differentiation and EAU. In summary, our study was the first to demonstrate the therapeutic effects of NaB on experimental uveitis. Our results showed that NaB directly converted the differentiation of naive T cells from Th17 cells to Treg cells, possibly by inhibiting IL-6R signaling. Importantly, we further determined the immune-regulatory mechanisms of NaB by demonstrating that Nrf2-HO-1 signaling is essential for NaB-mediated immune regulatory effects in vitro and in vivo. These findings provide compelling evidence that NaB can inhibit uveitis and support butyrate as a potential therapeutic option for autoimmune diseases.
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Acknowledgements: This study was supported by the Natural Science Foundation of China (81470670, 81670897, 81671611 and U1601226), Guangdong Natural Science Funds for Distinguished Young Scholar (2016A030306006); Science and technology cooperation project of Hong Kong, Macao and Taiwan (L2015TGA0005); Natural Science Foundation of Guangdong Province (2015A030313189), Science and Technology Planning Project of Guangdong Province (2013B051000027), the Youth Science and Technology Innovation Talents Funds in a Special Support Plan for High Level Talents in Guangdong Province (No. 2015TQ01R261); the Zhu Jiang Star of Science and Technology Foundation in Guangzhou (201610010129). Conflict of interest None.
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Abbreviations: NaB: sodium butyrate; EAU: experimental autoimmune uveitis; Nrf2; Nuclear factor erythroid 2 -related factor 2; HO-1; Heme oxygenase-1; DLN: draining lymphoid nodes; Treg cells: CD4+ T regulatory cells; Foxp3: Forkhead box protein 3; STAT-3: Signal transduction activator 3; pSTAT3: Phosphorylated signal transduction activator 3 RORγt: Retinoic acid-related orphan receptor (ROR)γt IL-6R: Interleukin-6 receptor DC: Dendritic cell
16
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Figure 1. NaB treatment decreases the severity of experimental uveitis. (A): The clinical scores of EAU were determined at 14 day after immunization (n=7). (B): Representative images of fundoscopic examination in the indicated experimental group at 14 day after immunization (white arrow: vasculitis). (C): The pathological scores of EAU were determined at 14 day after immunization (n=7). (D): Representative images of with histopathologic examination in the indicated experimental group at 14 day after immunization (Scale bars represent 200 µm). The data are presented as the mean ± SD. **P < 0.01. Figure 2. NaB modulates local and systemic inflammatory profiles in EAU. (A-D): The expression of IL-17A, INF-γ, TNF-α and IL-10 mRNA in retinas were measured by realtime PCR at 14 day after immunization (n=6). (E-F): The expression of CXCL9 and MCP-1 in retinas was examined by real-time PCR. (G-J) The levels of IL-17A, INF-γ, TNF-α and IL-10 protein in serum were determined via ELISA at 14 day after immunization (n=6). The data are presented as the mean ± SD. **P < 0.01. Figure 3. NaB decreases Th17 cells and increases Treg cells in EAU. (A-C): CD4+IL-17A+ T cells in the draining lymph node (DLNs) and spleen of EAU mice were determined by flow cytometry at 14 day after immunization (n=3). (D-F): CD4+Foxp3+ T cells in the draining lymph node (DLNs) and spleen of EAU mice were determined by flow cytometry at 14 day after immunization (n=3). The data are presented as the mean ± SD. *P<0.05, **P < 0.01. Figure 4. NaB suppresses Th17 cell differentiation and reciprocally induces Treg cell differentiation in vitro. CD4+CD62L+ cells from C57BL/6J mice polarized to Th17 cells in presence or absence of NaB for 3 days. (A) Expression of IL-17A and Foxp3 were checked by flow cytometry (n=3). (B-C) Quantitative analysis of CD4+IL-17A+T cells. (D) The levels of
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IL-17A protein in the supernatants were determined via ELISA (n=3). (E-F) Quantitative analysis of CD4+Foxp3+T cells. (G) The same cells were analyzed by flow cytometry for RORγt expression (n=3). (H) CD4+CD62L+ cells were incubated in presence or absence of NaB for 2 h, then IL6 was added for 30min. pSTAT3 expression was determined and compared to unstimulated CD4+CD62L+ cells (Unstim, filled gray histogram, n=3). The data are presented as the mean ± SD. *P<0.05, **P < 0.01.
Figure 5. Nrf2/HO-1 signaling is critically involved in NaB-mediated modulation of Th17 cell differentiation. NaB treatment significantly induced the nuclear translocation of Nrf2 (A) and increased HO-1 expression (B) in CD4+T cells under Th17 cell–polarizing condition (n=3). NaB obviously inhibited IL-6R expression in both mRNA (C) and protein (D) levels in CD4+T cells under Th17 cell–polarizing condition (n=3). (E-G): CD4+IL-17A+ T cells under indicated condition were determined by flow cytometry analysis (n=3). (H): The expression of IL-6R mRNA in CD4+T cells under indicated condition were measured by realtime PCR (n=3). (I) After pretreating CD4+T cells with HO-1 siRNA (kdHO-1) or negative control (NC), expression of IL-17A was checked by flow cytometry (n=3). (J-K) Quantitative analysis of CD4+IL-17A+T cells. (L) After pretreating CD4+T cells with HO-1 siRNA (kdHO-1) or negative control (NC), the expression of IL-6R mRNA was measured by real-time PCR (n=3). The data are presented as the mean ± SD. **P < 0.01. Figure 6. NaB Attenuates EAU via Nrf2/HO-1 signaling. (A-B): NaB treatment significantly induced the nuclear translocation of Nrf2 and increased HO-1 expression in the DLNs of EAU mice. (C-D): SnPP (an HO-1 inhibitor) significantly blocked the NaB-mediated decrease in the
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clinical and pathological scores of EAU (n=8). (E-G): The SnPP significantly reversed the NaBmediated decreases in the frequencies and numbers of Th17 in the DLN (n=3). (H-I): The IL17A mRNA expression in retinas and IL-6R mRNA expression in DLN were measured via realtime PCR (n=3). (J-L): CD11c+CD80+ DCs under indicated groups were determined by flow cytometry analysis (n=3). (M-N): NaB treatment significantly induced the nuclear translocation of Nrf2 and increased HO-1 expression in DCs isolated from the DLNs of EAU mice (n=3). The data are presented as the mean ± SD. *P<0.05, **P < 0.01.
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