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Atrial natriuretic peptide suppresses Th17 development through regulation of cGMP-dependent protein kinase and PI3K–Akt signaling pathways
Regulatory Peptides 181 (2013) 9–16
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Atrial natriuretic peptide suppresses Th17 development through regulation of cGMP-dependent protein kinase and PI3K–Akt signaling pathways Libing Ma a, Jinxiu Li b,⁎, Guyi Wang b, Subo Gong c, Li Zhang d, Keng Li c, Xiaoying Ji c, Yi Liu e, Ping Chen c, Xudong Xiang c,⁎⁎ a
Department of Respiratory Medicine, The Affiliated Hospital of Guilin Medical University, Lequn Road, No. 15, Guilin, Guangxi 541001, PR China Center of Critical Care Medicine, The Second Xiangya Hospital, Central South University, Middle Renmin Road, No. 139, Changsha, Hunan 410011, PR China Department of Respiratory Medicine, The Second Xiangya Hospital, Central South University, Middle Renmin Road, No. 139, Changsha, Hunan 410011, PR China d Department of Respiratory Medicine, The First Hospital Affiliated Luzhou Medical School, Luzhou, Sichuan 646000, PR China e Department of Respiratory Medicine, The First Hospital of Zhuzhou City, Zhuzhou, Hunan 412000, PR China b c
a r t i c l e
i n f o
Article history: Received 30 March 2012 Received in revised form 29 September 2012 Accepted 17 December 2012 Available online 14 January 2013 Keywords: Atrial natriuretic peptide (ANP) Th17 cells Natriuretic peptide receptor A/cGMPdependent protein kinase (NPRA/PKG) PI3K/Akt–FKHR
a b s t r a c t In recent years, accumulating evidence suggests that atrial natriuretic peptide (ANP), a hormone widely known as a result of its significant effects on the cardiovascular system mediated by natriuretic peptide receptor A (NPRA), may play a nonnegligible role in the regulation of immune responses. In this study, we firstly investigated whether ANP signaling could regulate the differentiation and capacity of Th17 cells and discovered ANP-dose (10−8–10−6 M) dependently indeed suppressed the differentiation of Th17 cells along with the reduced IL-17 production by polarizing naïve CD4+ T cells isolated from splenocytes to Th17 phenotype in vitro. Moreover, ANP primarily signals through NPRA and cGMP-dependent protein kinase (PKG) which could be antagonized when pretreated with either ANP/NPRA signaling antagonist or PKG inhibitor. In addition, we also found that ANP signaling could upregulate the levels of phosphorylation of Akt which was hypothesized to be implicated in ANP-induced inhibition of Th17 development in our studies, and the effect of ANP on the development of murine Th17 cells seemed to be partially reversed when an inhibitor of phosphatidylinositol 3′-kinase (PI3K)/Akt had been performed in advance. Briefly, we showed for the first time that ANP signaling could suppress murine Th17 cell development from naïve CD4+ T cells in vitro through NPRA/PKG pathway and the PI3K–Akt signal was implicated in the ANP-mediated suppression of Th17 development. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Atrial natriuretic peptide (ANP), an endocrine hormone with multiple biological effects, is an important member for the natriuretic peptides (NPs) family and comprises residues 99–126 of the pro-ANP hormone with potential to produce several peptides with different functions [1]. Natriuretic peptide receptor A (NPRA) is the major receptor highly expressed in immune tissues such as thymus and T lymphocytes to transmit signals through activating coupled cGMP-dependent protein kinase (PKG), which in turn promotes the expression of genes encoding ion transporters and transcription factors implicated in cell growth and apoptosis [2–4]. Abbreviations: ANP, atrial natriuretic peptide; BATF, B-cell activating transcription factor; FKHR(Foxo1a), forkhead in human rhabdomyosarcoma; NPRA, natriuretic peptide receptor A; PI3K, phosphatidylinositol 3′-kinase; PKG, cGMP-dependent protein kinase; ROR, retinoid-related orphan receptor; Stat, signal transducer and activator of transcription. ⁎ Corresponding author. ⁎⁎ Corresponding author. Tel./fax: +86 731 85533525. E-mail addresses: [email protected] (J. Li), [email protected] (X. Xiang). 0167-0115/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.regpep.2012.12.003
To date, ANP has been mainly studied for its cardiovascular actions such as its implication in volume-pressure homeostasis ever since its discovery [5,6]. However, more and more recent data suggest that ANP signaling is probably involved in the regulation of immune responses. Indeed, ANP pro-hormone has been found in the thymus, spleen, and lymph nodes [7]. In line with this discovery, ANP mRNA can be detected in the thymus and tonsil [8,9]. Furthermore, ANP seems to attenuate the maturation and differentiation of thymocytes, and expression of ANP receptors on rat thymocytes inhibited the proliferation of thymocytes during thymic differentiation [10,11]. Nevertheless, the exact role of ANP in the regulation of immune responses, particularly in Th17 development, has yet to be fully elucidated. Naïve CD4 + T cells can differentiate into at least four distinct effector subsets including Th1, Th2, Th17 and Treg cells manifested by the production of featured cytokines [12]. Among which, the recently identified Th17 cells (IL-17-producing CD4 + T cells) have been suggested to play a significant role in the pathological process of inflammatory diseases [13]. IL-17 (also referred as IL-17A) is the featured cytokine for Th17 cells. Th17 development requires the action of both TGF-β1 and IL-6; however, IL-23 has been suggested to be
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2. Materials and methods 2.1. Animals and reagents
Fig. 1. Expression of NPRA in naïve CD4+ T cells and spleens of mice. Naïve CD4+ T cells were isolated from splenocyte by immunomagnetic bead selection. NPRA protein extracted from CD4+ T cells and spleens of mice were detected by Western-blot analysis. This experiment was independently performed twice.
important for the promotion and maintenance of Th17 cells. While transcription factors retinoid-related orphan receptor γt (ROR-γt) and AP-1 B-cell activating transcription factor (BATF) are essential to polarize naïve CD4 + T cells into Th17 lineage [14–18]. It has been noted that IL-6/Stat3 is an indispensable signaling pathway for the differentiation of Th17 cells [19]. Interestingly, there is evidence suggesting a potential cross-talk between PI3K– Akt signal and IL-6/Stat3 pathway [20,21]. Given that PI3K–Akt signaling is involved in the ANP- and NPRA-mediated cell activities such as cardiomyocyte survival and endothelial proliferation [22–24], we therefore hypothesize that ANP/NPRA/PKG signal affects PI3K–Akt, and by which it regulates Th17 polarization. Our results demonstrate that ANP could suppress the polarization of naïve CD4 + T cells into Th17 lineage along with decreased IL-17 secretion. To our knowledge, it is the first time to suggest a role for ANP in the regulation of homeostasis of Th17 development.
BALB/c mice (6 to 8-week old) were purchased from the Shanghai Laboratory Animals Center. All experiments were approved by The Second Xiangya Hospital Animal Research and Care Committee. Mouse ANP 99–126 (ANP) was obtained from California Bioscience Inc. (California, USA), A71915-ANP/NPRA antagonist was purchased from Bachem (California, USA). The PKG inhibitor — KT5823, PI3K– Akt inhibitor — LY294002, monensin, PMA and ionomycin were all derived from Beyotime (Shanghai, China). CD4 +CD25 − T cell isolation kit was purchased from Miltenyi Biotec (Cologne, Germany). Recombinant murine IL-6 and TGF-β1 were obtained from PeproTech Inc. (New Jersey, USA), while IL-23 was derived from eBioscience (California, USA). Antibodies for CD3, CD28, IFN-γ, IL-4, FITC labeled IL-17A, IgG1, κ Isotype control, and FITC labeled CD4 were obtained from BioLegend (California, USA). Antibodies for Western blot analysis such as antibodies against p-Akt (Ser473), p-FKHR (Ser256), p-Stat3 (Tyr705), Akt (Ser473), FKHR (Ser256) and Stat3 (Tyr705) were originated from Signalway (Maryland, USA), while antibodies against anti-NPRA was from Abcam (Cambridge, UK), ROR-γt was from eBioscience (California, USA) and BATF was from Proteintech (Chicago, USA). 2.2. Naïve CD4 + T cell isolation, culture, and differentiation Spleens were collected from mice after anesthesia with 10% chloral hydrate. Mononuclear cells were next separated from splenic cells
Fig. 2. Effect of ANP on the differentiation from murine naïve CD4+ T cells to Th17 cells in vitro. (A) Representative flow cytometry plots for detecting IL-17A- (FITC) producing CD4+ T cells are shown here. (B) Percentage of IL-17-producing CD4+ T cells in anti-CD3/CD28-activated naïve CD4+ T cells determined at 4 days using an intracellular staining. (Meanings of abbreviations used in figures: C, anti-CD3 and anti-CD28 alone; D, differentiation cytokine microenvironment of Th17 cells with anti-CD3/CD28). (C) ROR-γt and BATF proteins extracted from naïve CD4+ T cells after culture for 4 days were analyzed by Western-blot and representative films are showed. (D) Cartogram for ratio of ROR-γt and BATF to β-actin. *, significant to anti-CD3/CD28 alone, p b 0.01; #, significant to differentiation condition, p b 0.01.
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Fig. 3. Effect of ANP on IL-17 secretion of supernatant and changes of the second messenger cGMP. (A) IL-17 production by anti-CD3/CD28-stimulated naïve CD4+ T cells, in response to cytokines-driven Th17 differentiation or in the presence of different concentrations of ANP at 4 days. (B) Measurement of IL-17 secretion in polarized Th17 cells treated with ANP alone or vehicle by ELISA. (Note: #, significant to ANP alone, p b 0.05.) (C) Detection of IL-17 in all experimental groups. (D) Measurement of intracellular cGMP by ELISA. (A7, A71915 and ANP; KT, KT5823 and ANP; LY, LY294002 and ANP; A7 + LY, both A71915 and LY294002 besides ANP.) *, significant to anti-CD3/CD28, p b 0.01; #, significant to differentiation condition, p b 0.01; +, significant to ANP alone, p b 0.05.
using Lymphocyte Separation Medium (Dakewe Biotech, Beijing, China) through density centrifugation as instructed. CD4+ T cells were isolated by negative selection using the CD4 antibody conjugated microbeads, and CD25 − T cells were then depleted from the resulting CD4 + T cells by positive selection with anti-CD25 microbeads. The purity of CD4+CD25− cells was about 95% as confirmed by flow cytometry. The above purified naïve CD4+ (CD4 +CD25 −) T cells were seeded at a density of 1 × 106 cells/ml in 24-well plates and activated with plate-bound anti-CD3 and anti-CD28 Abs (2 μg/ml each). Anti-IFN-γ (10 μg/ml), anti-IL-4 (10 μg/ml), TGF-β1 (5 ng/ml), IL-6 (20 ng/ml), and IL-23 (50 ng/ml) were then added into the cultures for Th17 induction. ANP with concentrations ranging from 10−8 to 10 −6 M was also added into the cultures every 24 h, respectively. The cells were all cultured in RPMI 1640 supplemented with 1% penicillin/streptomycin, 1% glutamine, and 10% heat-inactivated FCS for 4 days. A71915 (1 μM), KT5823 (1 μM), LY294002 (50 μM) or A71915 plus LY294002 were added into the cultures 30 min prior to the addition of ANP and cytokines in each experimental group, respectively. 2.3. Flow cytometry analysis Four days after polarization, the cells were restimulated with 500 ng/ml ionomycin and 50 ng/ml PMA in the presence of 1 μl/ml monensin for 4 h as reported, followed by flow cytometry analysis of Th17 production. For intracellular staining of IL-17A, the cells were fixed, permeabilized, and then labeled with either FITC conjugated anti-IgG1 or FITC labeled anti-IL-17A. The stained cells were then analyzed using a FACSCalibur instrument (BD, Biosciences).
2.4. Western blot analysis The cells were lysed and the resulting protein extracts were resolved by 10% SDS-PAGE. Immunoblot analysis was performed by transferring the proteins onto polyvinylidene difluoride membranes (Millipore) using a Mini Trans-Blot System (Bio-Rad). After blocking with 5% milk in Tris-buffered saline supplemented with Tween 20 (TBST), the membranes were incubated with an indicated primary antibody overnight at 4 °C followed by probing with a horseradish peroxidase-conjugated secondary antibody 1 h at room temperature. The reactive bands detected were visualized according to the manufacturer's instruction. 2.5. Quantitative analysis of cGMP and IL-17 production Cells derived from each experimental group were washed with prewarmed PBS. Lysates from the cells were then subjected to the measurement of second messenger cGMP by using a cGMP ELISA as instructed. IL-17 production in the culture supernatants was analyzed using an IL-17 ELISA kit following the instructions provided by the manufacturer (R&D). 2.6. Statistical analysis All data were analyzed by Student's t test or one-way ANOVA test. The results were expressed as mean±SD. In all cases, pb 0.05 was considered to be statistically significant. All experiments were performed with two independent replications.
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Fig. 4. Effect of NPRA–PKG signal in the process of ANP effecting on differentiation of murine Th17 cells in vitro. (A) Flow cytometry plots when the addition of A71915 or KT5823. (B) Proportion of IL-17-producing CD4+ T cells were detected after conditioned culture. (C and D) Detection of ROR-γt and BATF proteins and the cartogram are showed. *, significant to anti-CD3/CD28 alone, p b 0.01; #, significant to differentiation condition, p b 0.01; +, significant to ANP alone, p b 0.05.
3. Results 3.1. Expression of NPRA in murine naïve CD4 + T cells and spleens NPRA has been found to be highly expressed in various tissues (e.g., kidney, adrenal gland, intestinal ileum, aortic, lung, pituitary and vascular smooth muscle) and cells such as endothelial cells [1,3,5,6]. It has been confirmed that NPRA is the major effecting receptor for ANP with highly expression in T lymphocytes [2,25]. To define whether naïve CD4 + T cells also express NPRA, we examined NPRA protein levels by Western blot analysis using splenic naïve CD4 + T cell lysates. As expected, we detected NPRA expression in spleen derived naïve CD4 + T cells (Fig. 1). Studies in splenic lysates showed similar results. These results formed the basis for addressing the effect of ANP on Th17 polarization. 3.2. ANP suppresses naïve CD4 + T cells polarizing to Th17 cells To address the effect of ANP on Th17 polarization, naïve CD4+ T cells isolated from spleens were cultured with different doses of ANP in the conditioned medium containing anti-IFN-γ (10 μg/ml), anti-IL-4 (10 μg/ml), TGF-β1 (5 ng/ml), IL-6 (20 ng/ml), and IL-23 (50 ng/ml) as described [26–30]. Interestingly, the addition of ANP significantly inhibited Th17 polarization. Compared with control cells, ANP treatment reduced the percentage of IL-17-producing CD4 + T cells by 50% (20.26± 0.8% vs. 9.94± 0.24%, p b 0.01) when 10−6 M of ANP was added. Moreover, ANP was found dose-dependently (10−8–10 −6 M) attenuated the production of Th17 cells with the highest inhibitory effect when 10 −6 M of ANP was employed (Fig. 2A and B). In line with
these results, ANP treatment decreased the expression for transcription factors ROR-γt and BATF as confirmed by Western blot analysis (Fig. 2C and D). 3.3. ANP attenuates the secretion of IL-17 Given that IL-17 is the principal effector cytokine produced by Th17 cells [18,31], we next sought to address the impact of ANP on IL-17 secretion. To this end, we analyzed IL-17 levels in the culture supernatants of naïve CD4+ T cells after induction of Th17 polarization. Similar as above, ANP dose-dependently concomitantly IL-17 secretion. As shown in Fig. 3A, when 10−6 M ANP was added into the culture, the amount of IL-17 decreased by one-fold as compared with that of control cells (218.32±30.57 pg/ml vs. 381.08±54.23 pg/ml, pb 0.01). Moreover, to further address that the reduced IL-17 production after ANP treatment was caused by the decrease of Th17 cells and/or the reduced capacity of Th17 cells, we treated Th17 cells with either ANP or control vehicle for 24 h, and assayed for IL-17 production by ELISA analysis of culture supernatants (295.03±29.1 pg/ml vs. 413.52±32.02 pg/ml, pb 0.01). The results suggest that the inhibitory capacity of Th17 cells by ANP treatment may be partly responsible for the reduced IL-17 secretion besides the decrease of Th17 cells (Fig. 3B). 3.4. ANP activates NPRA/PKG signaling To dissect the underlying mechanisms by which ANP attenuates Th17 development, we examined the impact of ANP on NPRA/PKG signaling. For this purpose, we employed an ANP/NPRA antagonist, A71915, and a selective PKG inhibitor, KT5823 [32], for the studies. As shown in Fig. 4,
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Fig. 5. Changes of PI3K–Akt/FKHR and Stat3 proteins in the ANP signaling. (A) p-Akt, p-FKHR, p-Stat3 and the respective total proteins extracted from the cultivated CD4+ T cells were analyzed by Western-blot and the representative films are showed. (B) Cartogram for ratio of p-Akt/Akt, p-FKHR/FKHR and p-Stat/Stat3 to β-actin. (C) Changes of total proteins for Akt, FKHR and Stat3 expressions in treated CD4+ T cells which were incubated with ANP, or addition of either A71915 or KT5823. (Note: The expression of total proteins for Akt, FKHR and Stat3 showed no significant differences respectively except compared with control cells.) *, significant to anti-CD3/CD28 alone, p b 0.01; #, significant to differentiation condition, pb 0.05; +, significant to ANP alone, p b 0.05.
the administration of ANP along with A71915 rescued the production of IL-17-producing CD4+ T (Th17) cells. Consistently, the expressions for ROR-γt and BATF were also reversed. To further confirm this result, we examined the levels for the second messenger-cGMP, and a significant decrease of cGMP was noted in cells cultured in the presence of both ANP and A71915 as compared with that of ANP alone (8.58± 1.17 pmol/1×106 cells vs. 65.4±5.13 pmol/1×106 cells; pb 0.01) (Fig. 3D). Similarly, treatment of cells with either A71915 or KT5823 partially enhanced IL-17 production (Fig. 3C). Together, these results show that ANP attenuates Th17 polarization by activation of NPRA/PKG signaling. 3.5. ANP/NPRA/PKG mediates a cross-talk with PI3K–Akt/FKHR signaling As aforementioned, we assume a cross-talk between ANP/NPRA/PKG and PI3K–Akt signaling as well as IL-6/Stat3 pathway that are essential for Th17 development. Given that FKHR is a downstream target of PI3K–Akt signaling and a possible transcriptional coactivator for Stat3 [20,21], ANP may affect FKHR activation as well. To address these questions, we did Western blot analysis to examine the phosphorylated levels for Akt (p-Akt), FKHR (p-FKHR) and Stat3 (p-Stat3) before (0 min) and after A71915 or KT5823 treatment (30 min and 60 min) besides total protein respectively (t-Akt, t-FKHR and t-Stat3). ANP treatment significantly increased levels of p-Akt, p-FKHR along with the decreased p-Stat3 as manifested in Fig. 5A and B. Moreover, the levels for p-Akt, p-FKHR and p-Stat3 were significantly changed upon the addition of A71915, and the most striking effect was observed 30 min after the treatment as shown in Fig. 5, suggesting that Akt/FKHR can be activated upon the conjugation of ANP to NPRA. To our surprise, we failed to detect a discernable change for p-Akt and p-FKHR in cells treated with KT5823, a
PKG inhibitor, despite the increased p-Stat3, suggesting that PI3K–Akt/ FKHR is downstream of ANP signaling independent of PKG signaling. In addition, the Western blot analysis of total proteins for Akt, FKHR and Stat3 in Fig. 5C demonstrated that changes of phosphorylated form for Akt, FKHR and Stat3 were all caused by ANP enhancing the activation of them, because the total proteins showed no significant difference respectively. 3.6. ANP impacts PI3K–Akt signaling to suppress Th17 development Given that ANP can modulate the growth, proliferation and migration of diverse cells such as pancreatic-β cells, endothelial cells and cardiomyocytes [22–25], the above data prompted us to exam whether PI3K–Akt signaling is responsible for ANP-mediated suppression of Th17 development. To test this assumption, we treated naïve CD4+ T cells with ANP along with LY294002, a PI3K–Akt inhibitor. As shown in Fig. 6, indeed, PI3K–Akt signaling is implicated in ANP-mediated suppression of Th17 development. Moreover, blockade of PI3K–Akt signaling partly reversed ANP-mediated effect on p-FKHR and p-Stat3 (Fig. 6D). Although we noticed an increase of IL-17 secretion in cells treated with both ANP and LY294002, but it did not reach a statistical significance compared with the cells treated with ANP alone (Fig. 3C). 4. Discussion ANP is a 28-amino acid endocrine mediator mainly secreted by the heart and, as a result, it has been extensively studied for its impact on the homeostasis of the cardiovascular system. However, ANP and its receptors were also found to be expressed in other tissues and cells such as in immune organs and cells [3,5,9–11]. Indeed, there is
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Fig. 6. Effect of PI3K–Akt signal in the course of ANP's biological actions. (A) Representative flow cytometry plots of addition of LY294002 or A71915 with LY294002. (B) Cartogram of data for flow cytometry analysis. *, significant to anti-CD3/CD28 alone, p b 0.01; #, significant to differentiation condition, p b 0.05; +, significant to ANP, p b 0.05. (C) Expression of transcription factor ROR-γt and BATF proteins. (D) Western-blot analysis for p-Akt/Akt or p-FKHR/FKHR or p-Stat3/Stat3 in all groups.
suggestive evidence indicating a role for ANP in the regulation of immune responses, but its exact impact on immune system largely remains poorly understood. Th17 cells, as a new subset of CD4 + T cells, have been found to play an important role in and the pathogenesis of inflammatory diseases. In the present study, we have demonstrated for the first time that ANP possesses the capability to suppress naïve CD4+ T cells polarizing to Th17 cells, which is associated with decreased IL-17 secretion through the activation of NPRA/PKG signaling. We further demonstrated that ANP-mediated alteration in PI3K/Akt signaling could be partly responsible for the suppression of Th17 development. NPRA is the predominant receptor for ANP to execute its biological function, while the other receptor, NPRC, is responsible for its clearance [25]. Although NPRA has been found to be expressed in T lymphocytes [2,3], but its expression in naïve CD4+ T cells has never been defined. We, therefore, first sought to determine the expression of NPRA in
naïve CD4+ T cells. Indeed, NPRA can be detected in naïve CD4 + T cells, supporting that ANP/NPRA signaling might be implicated in the regulation of T cell polarization. Given that aberrant levels for ANP have been found in some chronic inflammatory diseases such as asthma [1,2,33–35], in which Th17 cells play a predominant role in disease pathogenesis [13], we next intend to address the impact of ANP on Th17 development. We first isolated splenic naïve CD4+ T cells and then induced Th17 polarization in the presence of ANP with different doses. Our studies reached a consistent conclusion that ANP suppresses the capability of naïve T cells polarizing to Th17 lineage in a dose dependent manner. Our studies also suggest a link between the endocrine system and the immune system. To confirm the above results, we then induced Th17 polarization in naïve CD4+ T cells in the presence of ANP along with a ANP/NPRA inhibitor, A71915. We noted that pretreatment of naïve CD4+ T cells with A71915 almost abolished the suppressive effect mediated by ANP.
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Interestingly, analysis of cell lysates revealed that ANP increased the levels for intracellular second messenger cGMP, we thus pretreated naïve CD4+ T cells with a selective PKG inhibitor, KT5823. Blockade of PKG signaling partially reversed ANP-mediated suppressive effect, indicating the involvement of PKG in ANP/NPRA signaling. These results suggest that cGMP/PKG signaling pathway was implicated in ANP-mediated suppressive effect. Actually, NPRA/PKG pathway has already been confirmed to be the major pathway of ANP signaling in some previous studies, which is similar to our data [2–4]. ROR-γt is the indispensable transcription factor for Th17 development. There is also evidence that BATF acts as a co-factor essential to Th17 lineage as manifested by that mice deficient BATF−/− with normal Th1 and Th2 development but defective Th17 polarization. This notion was further supported by the observation that BATF −/− T cells fail to induce ROR-γt [14–16]. Interestingly, our Western blot analysis provided convincing evidence that ANP suppresses the expression of ROR-γt and BATF, which perfectly explains why ANP treatment impairs Th17 development. Other than the involvement of ROR-γt and BATF, Stat3 has also been noted to be important for Th17 development. Stat3 regulates the transcription of ROR-γt and BATF, and thus, loss of Stat3 activation renders naïve T cells unable to differentiate into Th17 cells. IL-6 and IL-23 are featured cytokines for the development of Th17 cells, and both of them can activate Janus family kinases, which then preferentially activate Stat3 through phosphorylation [19,36,37]. As a result, IL-6-inducible Jak/Stat3 pathway plays a significant role in the development of Th17 cells. Importantly, ANP treatment down-regulated the levels for phosphorylated Stat3 (p-Stat3) along with reduced expression for ROR-γt and BATF. These results suggest that ANP also impacts Stat3 activation to impair Th17 development. Previous studies suggested feasible evidence indicating a potential cross-talk between PI3K–Akt signaling and IL-6/Stat3 pathway, in which FKHR (also named Foxo1a) might act as the connector [20]. It is believed that FKHR, a downstream target of Akt, is held in check by Akt-mediated phosphorylation, and through which it acts as a possible transcriptional coactivator to interact with Stat3 and to modulate its IL-6-induced transcriptional activities [20,21]. Indeed, our studies revealed that ANP treatment increased the levels for p-Akt and p-FKHR but decreased the levels for p-Stat3, while the addition of ANP/NPRA antagonist reversed the suppressive effect of ANP on Stat3, suggesting the existence of a cross-talk between PI3K–Akt signaling and IL-6/Stat3 pathway. Given that PKG inhibitor only partially reversed ANP-mediated suppressive effect in our study and PI3K–Akt/FKHR signaling has been linked with ANP/NPRA-mediated cell proliferation, survival, and so on [22–24], we assumed that PI3K–Akt/FKHR signaling is also involved in ANP-mediated suppressive effect on Th17 development. As expected, when PI3K–Akt inhibitor was administered along with ANP in naïve T cells, the expressions for ROR-γt and BATF were partially reversed along with upregulation of p-Stat3 and increase of IL-17-producing CD4+ T cells. However, we failed to detect a significant change for IL-17 production and the mechanisms underlying this unexpected result remain unknown. Similar to our results, Mandy Pierau et al. reported that Akt signals impair TGF-β1/IL-6-mediated differentiation of naïve CD4+ T cells into the Th17 lineage [38]. Together, these data support that PI3K–Akt signaling is partly responsible for ANP-mediated biological effect on Th17 polarization. However, combining ANP/NPRA antagonist with PI3K–Akt inhibitor failed to observe enhanced reversing effect as compared with either of them alone, suggesting that PI3K–Akt is a downstream signal of NPRA. In summary, we provided feasible evidence that ANP is implicated in the regulation of naïve CD4 + T cells polarizing to Th17 lineage. ANP conjugates to NPRA, and by which it activates PKG and PI3K–Akt pathways to suppress Stat3 activation, which then impairs the expression of ROR-γt and BATF, the two essential factors for Th17
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development. Given the role of Th17 cells played in the pathogenesis of many autoimmune and inflammatory diseases, our data revealed a great potential for ANP to be a potential therapeutic target for treatment of some inflammatory disorders. Acknowledgments This work was supported by a grant from the key project foundation of the Project of Science and Technology of Hunan Province (2010FJ2004) and an Innovation Research Fund for Graduate Students of Hunan Province (CX2011B070). References [1] Mohapatra Shyam S. Role of natriuretic peptide signaling in modulating asthma and inflammation. Can J Physiol Pharmacol 2007;85:754–9. [2] Mohapatra SS, Lockey RF, Vesely DL, Gower Jr WR. Natriuretic peptides and genesis of asthma: an emerging paradigm? J Allergy Clin Immunol 2004;114: 520–6. [3] Misono KS. Natriuretic peptide receptor: structure and signaling. Mol Cell Biochem 2002;230:49–60. [4] Silberbach M, Roberts Jr CT. Natriuretic peptide signalling: molecular and cellular pathways to growth regulation. Cell Signal 2001;13:221–31. [5] Levin ER, Gardner DG, Samson WK. Natriuretic peptides. N Engl J Med 1998;339: 321–8. [6] Beltowski J. N-terminal atrial natriuretic peptides. Postepy Hig Med Dosw 2000;54:895–914. [7] Vollmar AM, Schulz R. Atrial natriuretic peptide in lymphoid organs of various species. Comp Biochem Physiol A 1990;96:459–63. [8] Gerbes AL, Vollmar AM, Thibault G, Arendt RM, Cantin M, Paumgartner G. Different behaviour of the N-terminal and C-terminal fragment of proatrial natriuretic factor in plasma of healthy subjects as well as of patients with cirrhosis. Scand J Clin Lab Invest 1990;50:195–8. [9] Tallerico-Melnyk T, Yip CC, Watt VM. Widespread co-localization of mRNAs encoding the guanylate cyclase-coupled natriuretic peptide receptors in rat tissues. Biochem Biophys Res Commun 1992;189:610–6. [10] Vollmar Angelika M. Influence of atrial natriuretic peptide on thymocyte development in fetal thymic organ culture. J Neuroimmunol 1997;78:90–6. [11] Vollmar AM, Schmidt KN, Schulz R. Natriuretic peptide receptors on rat thymocytes: inhibition of proliferation by atrial natriuretic peptide. Endocrinology 1996;137:1706–13. [12] Chen Zhi, Laurence Arian, O'Shea John J. Signal transduction pathways and transcriptional regulation in the control of Th17 differentiation. Semin Immunol 2007;19: 400–8. [13] Fouser LA, Wright JF, Dunussi-Joannopoulos K, et al. Th17 cytokines and their emerging roles in inflammation and autoimmunity. Immunol Rev 2008;226: 87–102. [14] Harrington LE, Hatton RD, Mangan PR, Turner H, Murphy TL, Murphy KM, Weaver CT. Interleukin 17 producing CD4+ effector T cells develop via a liesge distinct from the T helper type 1 and lineages. Nat Immunol 2005;6:1123–32. [15] Ivanov II, McKenzie BS, Zhou L, Tadokoro CE, Lepelley A, Lafaille JJ, Cua DJ, Littman DR. The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 2006;126:1121–33. [16] Schraml BU, Hildner K, Ise W, Lee WL, Smith WA, Solomon B, Sahota G, Sim J, Mukasa R, Cemerski S, Hatton RD, Stormo GD, Weaver CT, Russell JH, Murphy TL, Murphy KM. The AP-1 transcription factor Batf controls TH17 differentiation. Nature 2009;460:405–9. [17] Bettelli E, Korn T, Kuchroo VK. Th17: the third member of the effector T cell trilogy. Curr Opin Immunol 2007;19:652–7. [18] Kolls JK, Linden A. Interleukin-17 family members and inflammation. Immunity 2004;21:467–76. [19] Yang XO, Panopoulos AD, Nurieva R, Chang SH, Wang D, Watowich SS, Dong C. STAT3 regulates cytokine-mediated generation of inflammatory helper T cells. J Biol Chem 2007;282:9358–63. [20] Kortylewski M, Feld F, Krüger KD, Bahrenberg G, Roth RA, Joost HG, Heinrich PC, Behrmann I, Barthel A. Akt modulates Stat3-mediated gene expression through a FKHR(Foxo1a)-dependent mechanism. J Biol Chem 2003;278:5242–9. [21] Kops GJ, Burgering BM. Forkhead transcription factors: new insights into protein kinase B (c-akt) signaling. J Mol Med 1999;77:656–65. [22] Kato T, Muraski J, Chen Y, Tsujita Y, Wall J, Glembotski CC, Schaefer E, Beckerle M, Sussman MA. Atrial natriuretic peptide promotes cardiomyocyte survival by cGMP-dependent nuclear accumulation of zyxin and Akt. J Clin Invest 2005;115: 2716–30. [23] Kook H, Itoh H, Choi BS, Sawada N, Doi K, Hwang TJ, Kim KK, Arai H, Baik YH, Nakao K. Physiological concentration of atrial natriuretic peptide induces endothelial regeneration in vitro. Am J Physiol Heart Circ Physiol 2003;284: H1388–97. [24] You Hui, Laychock Suzanne G. Atrial natriuretic peptide promotes pancreatic islet β-cell growth and Akt/Foxo1a/cyclin D2 signaling. Endocrinology 2009;150: 5455–65. [25] Garbers DL. Guanylyl cyclase receptors and their endocrine, paracrine, and autocrine ligands. Cell 1992;71:1–4.
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Regulatory Peptides journal homepage: www.elsevier.com/locate/regpep
Atrial natriuretic peptide suppresses Th17 development through regulation of cGMP-dependent protein kinase and PI3K–Akt signaling pathways Libing Ma a, Jinxiu Li b,⁎, Guyi Wang b, Subo Gong c, Li Zhang d, Keng Li c, Xiaoying Ji c, Yi Liu e, Ping Chen c, Xudong Xiang c,⁎⁎ a
Department of Respiratory Medicine, The Affiliated Hospital of Guilin Medical University, Lequn Road, No. 15, Guilin, Guangxi 541001, PR China Center of Critical Care Medicine, The Second Xiangya Hospital, Central South University, Middle Renmin Road, No. 139, Changsha, Hunan 410011, PR China Department of Respiratory Medicine, The Second Xiangya Hospital, Central South University, Middle Renmin Road, No. 139, Changsha, Hunan 410011, PR China d Department of Respiratory Medicine, The First Hospital Affiliated Luzhou Medical School, Luzhou, Sichuan 646000, PR China e Department of Respiratory Medicine, The First Hospital of Zhuzhou City, Zhuzhou, Hunan 412000, PR China b c
a r t i c l e
i n f o
Article history: Received 30 March 2012 Received in revised form 29 September 2012 Accepted 17 December 2012 Available online 14 January 2013 Keywords: Atrial natriuretic peptide (ANP) Th17 cells Natriuretic peptide receptor A/cGMPdependent protein kinase (NPRA/PKG) PI3K/Akt–FKHR
a b s t r a c t In recent years, accumulating evidence suggests that atrial natriuretic peptide (ANP), a hormone widely known as a result of its significant effects on the cardiovascular system mediated by natriuretic peptide receptor A (NPRA), may play a nonnegligible role in the regulation of immune responses. In this study, we firstly investigated whether ANP signaling could regulate the differentiation and capacity of Th17 cells and discovered ANP-dose (10−8–10−6 M) dependently indeed suppressed the differentiation of Th17 cells along with the reduced IL-17 production by polarizing naïve CD4+ T cells isolated from splenocytes to Th17 phenotype in vitro. Moreover, ANP primarily signals through NPRA and cGMP-dependent protein kinase (PKG) which could be antagonized when pretreated with either ANP/NPRA signaling antagonist or PKG inhibitor. In addition, we also found that ANP signaling could upregulate the levels of phosphorylation of Akt which was hypothesized to be implicated in ANP-induced inhibition of Th17 development in our studies, and the effect of ANP on the development of murine Th17 cells seemed to be partially reversed when an inhibitor of phosphatidylinositol 3′-kinase (PI3K)/Akt had been performed in advance. Briefly, we showed for the first time that ANP signaling could suppress murine Th17 cell development from naïve CD4+ T cells in vitro through NPRA/PKG pathway and the PI3K–Akt signal was implicated in the ANP-mediated suppression of Th17 development. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Atrial natriuretic peptide (ANP), an endocrine hormone with multiple biological effects, is an important member for the natriuretic peptides (NPs) family and comprises residues 99–126 of the pro-ANP hormone with potential to produce several peptides with different functions [1]. Natriuretic peptide receptor A (NPRA) is the major receptor highly expressed in immune tissues such as thymus and T lymphocytes to transmit signals through activating coupled cGMP-dependent protein kinase (PKG), which in turn promotes the expression of genes encoding ion transporters and transcription factors implicated in cell growth and apoptosis [2–4]. Abbreviations: ANP, atrial natriuretic peptide; BATF, B-cell activating transcription factor; FKHR(Foxo1a), forkhead in human rhabdomyosarcoma; NPRA, natriuretic peptide receptor A; PI3K, phosphatidylinositol 3′-kinase; PKG, cGMP-dependent protein kinase; ROR, retinoid-related orphan receptor; Stat, signal transducer and activator of transcription. ⁎ Corresponding author. ⁎⁎ Corresponding author. Tel./fax: +86 731 85533525. E-mail addresses: [email protected] (J. Li), [email protected] (X. Xiang). 0167-0115/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.regpep.2012.12.003
To date, ANP has been mainly studied for its cardiovascular actions such as its implication in volume-pressure homeostasis ever since its discovery [5,6]. However, more and more recent data suggest that ANP signaling is probably involved in the regulation of immune responses. Indeed, ANP pro-hormone has been found in the thymus, spleen, and lymph nodes [7]. In line with this discovery, ANP mRNA can be detected in the thymus and tonsil [8,9]. Furthermore, ANP seems to attenuate the maturation and differentiation of thymocytes, and expression of ANP receptors on rat thymocytes inhibited the proliferation of thymocytes during thymic differentiation [10,11]. Nevertheless, the exact role of ANP in the regulation of immune responses, particularly in Th17 development, has yet to be fully elucidated. Naïve CD4 + T cells can differentiate into at least four distinct effector subsets including Th1, Th2, Th17 and Treg cells manifested by the production of featured cytokines [12]. Among which, the recently identified Th17 cells (IL-17-producing CD4 + T cells) have been suggested to play a significant role in the pathological process of inflammatory diseases [13]. IL-17 (also referred as IL-17A) is the featured cytokine for Th17 cells. Th17 development requires the action of both TGF-β1 and IL-6; however, IL-23 has been suggested to be
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2. Materials and methods 2.1. Animals and reagents
Fig. 1. Expression of NPRA in naïve CD4+ T cells and spleens of mice. Naïve CD4+ T cells were isolated from splenocyte by immunomagnetic bead selection. NPRA protein extracted from CD4+ T cells and spleens of mice were detected by Western-blot analysis. This experiment was independently performed twice.
important for the promotion and maintenance of Th17 cells. While transcription factors retinoid-related orphan receptor γt (ROR-γt) and AP-1 B-cell activating transcription factor (BATF) are essential to polarize naïve CD4 + T cells into Th17 lineage [14–18]. It has been noted that IL-6/Stat3 is an indispensable signaling pathway for the differentiation of Th17 cells [19]. Interestingly, there is evidence suggesting a potential cross-talk between PI3K– Akt signal and IL-6/Stat3 pathway [20,21]. Given that PI3K–Akt signaling is involved in the ANP- and NPRA-mediated cell activities such as cardiomyocyte survival and endothelial proliferation [22–24], we therefore hypothesize that ANP/NPRA/PKG signal affects PI3K–Akt, and by which it regulates Th17 polarization. Our results demonstrate that ANP could suppress the polarization of naïve CD4 + T cells into Th17 lineage along with decreased IL-17 secretion. To our knowledge, it is the first time to suggest a role for ANP in the regulation of homeostasis of Th17 development.
BALB/c mice (6 to 8-week old) were purchased from the Shanghai Laboratory Animals Center. All experiments were approved by The Second Xiangya Hospital Animal Research and Care Committee. Mouse ANP 99–126 (ANP) was obtained from California Bioscience Inc. (California, USA), A71915-ANP/NPRA antagonist was purchased from Bachem (California, USA). The PKG inhibitor — KT5823, PI3K– Akt inhibitor — LY294002, monensin, PMA and ionomycin were all derived from Beyotime (Shanghai, China). CD4 +CD25 − T cell isolation kit was purchased from Miltenyi Biotec (Cologne, Germany). Recombinant murine IL-6 and TGF-β1 were obtained from PeproTech Inc. (New Jersey, USA), while IL-23 was derived from eBioscience (California, USA). Antibodies for CD3, CD28, IFN-γ, IL-4, FITC labeled IL-17A, IgG1, κ Isotype control, and FITC labeled CD4 were obtained from BioLegend (California, USA). Antibodies for Western blot analysis such as antibodies against p-Akt (Ser473), p-FKHR (Ser256), p-Stat3 (Tyr705), Akt (Ser473), FKHR (Ser256) and Stat3 (Tyr705) were originated from Signalway (Maryland, USA), while antibodies against anti-NPRA was from Abcam (Cambridge, UK), ROR-γt was from eBioscience (California, USA) and BATF was from Proteintech (Chicago, USA). 2.2. Naïve CD4 + T cell isolation, culture, and differentiation Spleens were collected from mice after anesthesia with 10% chloral hydrate. Mononuclear cells were next separated from splenic cells
Fig. 2. Effect of ANP on the differentiation from murine naïve CD4+ T cells to Th17 cells in vitro. (A) Representative flow cytometry plots for detecting IL-17A- (FITC) producing CD4+ T cells are shown here. (B) Percentage of IL-17-producing CD4+ T cells in anti-CD3/CD28-activated naïve CD4+ T cells determined at 4 days using an intracellular staining. (Meanings of abbreviations used in figures: C, anti-CD3 and anti-CD28 alone; D, differentiation cytokine microenvironment of Th17 cells with anti-CD3/CD28). (C) ROR-γt and BATF proteins extracted from naïve CD4+ T cells after culture for 4 days were analyzed by Western-blot and representative films are showed. (D) Cartogram for ratio of ROR-γt and BATF to β-actin. *, significant to anti-CD3/CD28 alone, p b 0.01; #, significant to differentiation condition, p b 0.01.
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Fig. 3. Effect of ANP on IL-17 secretion of supernatant and changes of the second messenger cGMP. (A) IL-17 production by anti-CD3/CD28-stimulated naïve CD4+ T cells, in response to cytokines-driven Th17 differentiation or in the presence of different concentrations of ANP at 4 days. (B) Measurement of IL-17 secretion in polarized Th17 cells treated with ANP alone or vehicle by ELISA. (Note: #, significant to ANP alone, p b 0.05.) (C) Detection of IL-17 in all experimental groups. (D) Measurement of intracellular cGMP by ELISA. (A7, A71915 and ANP; KT, KT5823 and ANP; LY, LY294002 and ANP; A7 + LY, both A71915 and LY294002 besides ANP.) *, significant to anti-CD3/CD28, p b 0.01; #, significant to differentiation condition, p b 0.01; +, significant to ANP alone, p b 0.05.
using Lymphocyte Separation Medium (Dakewe Biotech, Beijing, China) through density centrifugation as instructed. CD4+ T cells were isolated by negative selection using the CD4 antibody conjugated microbeads, and CD25 − T cells were then depleted from the resulting CD4 + T cells by positive selection with anti-CD25 microbeads. The purity of CD4+CD25− cells was about 95% as confirmed by flow cytometry. The above purified naïve CD4+ (CD4 +CD25 −) T cells were seeded at a density of 1 × 106 cells/ml in 24-well plates and activated with plate-bound anti-CD3 and anti-CD28 Abs (2 μg/ml each). Anti-IFN-γ (10 μg/ml), anti-IL-4 (10 μg/ml), TGF-β1 (5 ng/ml), IL-6 (20 ng/ml), and IL-23 (50 ng/ml) were then added into the cultures for Th17 induction. ANP with concentrations ranging from 10−8 to 10 −6 M was also added into the cultures every 24 h, respectively. The cells were all cultured in RPMI 1640 supplemented with 1% penicillin/streptomycin, 1% glutamine, and 10% heat-inactivated FCS for 4 days. A71915 (1 μM), KT5823 (1 μM), LY294002 (50 μM) or A71915 plus LY294002 were added into the cultures 30 min prior to the addition of ANP and cytokines in each experimental group, respectively. 2.3. Flow cytometry analysis Four days after polarization, the cells were restimulated with 500 ng/ml ionomycin and 50 ng/ml PMA in the presence of 1 μl/ml monensin for 4 h as reported, followed by flow cytometry analysis of Th17 production. For intracellular staining of IL-17A, the cells were fixed, permeabilized, and then labeled with either FITC conjugated anti-IgG1 or FITC labeled anti-IL-17A. The stained cells were then analyzed using a FACSCalibur instrument (BD, Biosciences).
2.4. Western blot analysis The cells were lysed and the resulting protein extracts were resolved by 10% SDS-PAGE. Immunoblot analysis was performed by transferring the proteins onto polyvinylidene difluoride membranes (Millipore) using a Mini Trans-Blot System (Bio-Rad). After blocking with 5% milk in Tris-buffered saline supplemented with Tween 20 (TBST), the membranes were incubated with an indicated primary antibody overnight at 4 °C followed by probing with a horseradish peroxidase-conjugated secondary antibody 1 h at room temperature. The reactive bands detected were visualized according to the manufacturer's instruction. 2.5. Quantitative analysis of cGMP and IL-17 production Cells derived from each experimental group were washed with prewarmed PBS. Lysates from the cells were then subjected to the measurement of second messenger cGMP by using a cGMP ELISA as instructed. IL-17 production in the culture supernatants was analyzed using an IL-17 ELISA kit following the instructions provided by the manufacturer (R&D). 2.6. Statistical analysis All data were analyzed by Student's t test or one-way ANOVA test. The results were expressed as mean±SD. In all cases, pb 0.05 was considered to be statistically significant. All experiments were performed with two independent replications.
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Fig. 4. Effect of NPRA–PKG signal in the process of ANP effecting on differentiation of murine Th17 cells in vitro. (A) Flow cytometry plots when the addition of A71915 or KT5823. (B) Proportion of IL-17-producing CD4+ T cells were detected after conditioned culture. (C and D) Detection of ROR-γt and BATF proteins and the cartogram are showed. *, significant to anti-CD3/CD28 alone, p b 0.01; #, significant to differentiation condition, p b 0.01; +, significant to ANP alone, p b 0.05.
3. Results 3.1. Expression of NPRA in murine naïve CD4 + T cells and spleens NPRA has been found to be highly expressed in various tissues (e.g., kidney, adrenal gland, intestinal ileum, aortic, lung, pituitary and vascular smooth muscle) and cells such as endothelial cells [1,3,5,6]. It has been confirmed that NPRA is the major effecting receptor for ANP with highly expression in T lymphocytes [2,25]. To define whether naïve CD4 + T cells also express NPRA, we examined NPRA protein levels by Western blot analysis using splenic naïve CD4 + T cell lysates. As expected, we detected NPRA expression in spleen derived naïve CD4 + T cells (Fig. 1). Studies in splenic lysates showed similar results. These results formed the basis for addressing the effect of ANP on Th17 polarization. 3.2. ANP suppresses naïve CD4 + T cells polarizing to Th17 cells To address the effect of ANP on Th17 polarization, naïve CD4+ T cells isolated from spleens were cultured with different doses of ANP in the conditioned medium containing anti-IFN-γ (10 μg/ml), anti-IL-4 (10 μg/ml), TGF-β1 (5 ng/ml), IL-6 (20 ng/ml), and IL-23 (50 ng/ml) as described [26–30]. Interestingly, the addition of ANP significantly inhibited Th17 polarization. Compared with control cells, ANP treatment reduced the percentage of IL-17-producing CD4 + T cells by 50% (20.26± 0.8% vs. 9.94± 0.24%, p b 0.01) when 10−6 M of ANP was added. Moreover, ANP was found dose-dependently (10−8–10 −6 M) attenuated the production of Th17 cells with the highest inhibitory effect when 10 −6 M of ANP was employed (Fig. 2A and B). In line with
these results, ANP treatment decreased the expression for transcription factors ROR-γt and BATF as confirmed by Western blot analysis (Fig. 2C and D). 3.3. ANP attenuates the secretion of IL-17 Given that IL-17 is the principal effector cytokine produced by Th17 cells [18,31], we next sought to address the impact of ANP on IL-17 secretion. To this end, we analyzed IL-17 levels in the culture supernatants of naïve CD4+ T cells after induction of Th17 polarization. Similar as above, ANP dose-dependently concomitantly IL-17 secretion. As shown in Fig. 3A, when 10−6 M ANP was added into the culture, the amount of IL-17 decreased by one-fold as compared with that of control cells (218.32±30.57 pg/ml vs. 381.08±54.23 pg/ml, pb 0.01). Moreover, to further address that the reduced IL-17 production after ANP treatment was caused by the decrease of Th17 cells and/or the reduced capacity of Th17 cells, we treated Th17 cells with either ANP or control vehicle for 24 h, and assayed for IL-17 production by ELISA analysis of culture supernatants (295.03±29.1 pg/ml vs. 413.52±32.02 pg/ml, pb 0.01). The results suggest that the inhibitory capacity of Th17 cells by ANP treatment may be partly responsible for the reduced IL-17 secretion besides the decrease of Th17 cells (Fig. 3B). 3.4. ANP activates NPRA/PKG signaling To dissect the underlying mechanisms by which ANP attenuates Th17 development, we examined the impact of ANP on NPRA/PKG signaling. For this purpose, we employed an ANP/NPRA antagonist, A71915, and a selective PKG inhibitor, KT5823 [32], for the studies. As shown in Fig. 4,
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Fig. 5. Changes of PI3K–Akt/FKHR and Stat3 proteins in the ANP signaling. (A) p-Akt, p-FKHR, p-Stat3 and the respective total proteins extracted from the cultivated CD4+ T cells were analyzed by Western-blot and the representative films are showed. (B) Cartogram for ratio of p-Akt/Akt, p-FKHR/FKHR and p-Stat/Stat3 to β-actin. (C) Changes of total proteins for Akt, FKHR and Stat3 expressions in treated CD4+ T cells which were incubated with ANP, or addition of either A71915 or KT5823. (Note: The expression of total proteins for Akt, FKHR and Stat3 showed no significant differences respectively except compared with control cells.) *, significant to anti-CD3/CD28 alone, p b 0.01; #, significant to differentiation condition, pb 0.05; +, significant to ANP alone, p b 0.05.
the administration of ANP along with A71915 rescued the production of IL-17-producing CD4+ T (Th17) cells. Consistently, the expressions for ROR-γt and BATF were also reversed. To further confirm this result, we examined the levels for the second messenger-cGMP, and a significant decrease of cGMP was noted in cells cultured in the presence of both ANP and A71915 as compared with that of ANP alone (8.58± 1.17 pmol/1×106 cells vs. 65.4±5.13 pmol/1×106 cells; pb 0.01) (Fig. 3D). Similarly, treatment of cells with either A71915 or KT5823 partially enhanced IL-17 production (Fig. 3C). Together, these results show that ANP attenuates Th17 polarization by activation of NPRA/PKG signaling. 3.5. ANP/NPRA/PKG mediates a cross-talk with PI3K–Akt/FKHR signaling As aforementioned, we assume a cross-talk between ANP/NPRA/PKG and PI3K–Akt signaling as well as IL-6/Stat3 pathway that are essential for Th17 development. Given that FKHR is a downstream target of PI3K–Akt signaling and a possible transcriptional coactivator for Stat3 [20,21], ANP may affect FKHR activation as well. To address these questions, we did Western blot analysis to examine the phosphorylated levels for Akt (p-Akt), FKHR (p-FKHR) and Stat3 (p-Stat3) before (0 min) and after A71915 or KT5823 treatment (30 min and 60 min) besides total protein respectively (t-Akt, t-FKHR and t-Stat3). ANP treatment significantly increased levels of p-Akt, p-FKHR along with the decreased p-Stat3 as manifested in Fig. 5A and B. Moreover, the levels for p-Akt, p-FKHR and p-Stat3 were significantly changed upon the addition of A71915, and the most striking effect was observed 30 min after the treatment as shown in Fig. 5, suggesting that Akt/FKHR can be activated upon the conjugation of ANP to NPRA. To our surprise, we failed to detect a discernable change for p-Akt and p-FKHR in cells treated with KT5823, a
PKG inhibitor, despite the increased p-Stat3, suggesting that PI3K–Akt/ FKHR is downstream of ANP signaling independent of PKG signaling. In addition, the Western blot analysis of total proteins for Akt, FKHR and Stat3 in Fig. 5C demonstrated that changes of phosphorylated form for Akt, FKHR and Stat3 were all caused by ANP enhancing the activation of them, because the total proteins showed no significant difference respectively. 3.6. ANP impacts PI3K–Akt signaling to suppress Th17 development Given that ANP can modulate the growth, proliferation and migration of diverse cells such as pancreatic-β cells, endothelial cells and cardiomyocytes [22–25], the above data prompted us to exam whether PI3K–Akt signaling is responsible for ANP-mediated suppression of Th17 development. To test this assumption, we treated naïve CD4+ T cells with ANP along with LY294002, a PI3K–Akt inhibitor. As shown in Fig. 6, indeed, PI3K–Akt signaling is implicated in ANP-mediated suppression of Th17 development. Moreover, blockade of PI3K–Akt signaling partly reversed ANP-mediated effect on p-FKHR and p-Stat3 (Fig. 6D). Although we noticed an increase of IL-17 secretion in cells treated with both ANP and LY294002, but it did not reach a statistical significance compared with the cells treated with ANP alone (Fig. 3C). 4. Discussion ANP is a 28-amino acid endocrine mediator mainly secreted by the heart and, as a result, it has been extensively studied for its impact on the homeostasis of the cardiovascular system. However, ANP and its receptors were also found to be expressed in other tissues and cells such as in immune organs and cells [3,5,9–11]. Indeed, there is
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Fig. 6. Effect of PI3K–Akt signal in the course of ANP's biological actions. (A) Representative flow cytometry plots of addition of LY294002 or A71915 with LY294002. (B) Cartogram of data for flow cytometry analysis. *, significant to anti-CD3/CD28 alone, p b 0.01; #, significant to differentiation condition, p b 0.05; +, significant to ANP, p b 0.05. (C) Expression of transcription factor ROR-γt and BATF proteins. (D) Western-blot analysis for p-Akt/Akt or p-FKHR/FKHR or p-Stat3/Stat3 in all groups.
suggestive evidence indicating a role for ANP in the regulation of immune responses, but its exact impact on immune system largely remains poorly understood. Th17 cells, as a new subset of CD4 + T cells, have been found to play an important role in and the pathogenesis of inflammatory diseases. In the present study, we have demonstrated for the first time that ANP possesses the capability to suppress naïve CD4+ T cells polarizing to Th17 cells, which is associated with decreased IL-17 secretion through the activation of NPRA/PKG signaling. We further demonstrated that ANP-mediated alteration in PI3K/Akt signaling could be partly responsible for the suppression of Th17 development. NPRA is the predominant receptor for ANP to execute its biological function, while the other receptor, NPRC, is responsible for its clearance [25]. Although NPRA has been found to be expressed in T lymphocytes [2,3], but its expression in naïve CD4+ T cells has never been defined. We, therefore, first sought to determine the expression of NPRA in
naïve CD4+ T cells. Indeed, NPRA can be detected in naïve CD4 + T cells, supporting that ANP/NPRA signaling might be implicated in the regulation of T cell polarization. Given that aberrant levels for ANP have been found in some chronic inflammatory diseases such as asthma [1,2,33–35], in which Th17 cells play a predominant role in disease pathogenesis [13], we next intend to address the impact of ANP on Th17 development. We first isolated splenic naïve CD4+ T cells and then induced Th17 polarization in the presence of ANP with different doses. Our studies reached a consistent conclusion that ANP suppresses the capability of naïve T cells polarizing to Th17 lineage in a dose dependent manner. Our studies also suggest a link between the endocrine system and the immune system. To confirm the above results, we then induced Th17 polarization in naïve CD4+ T cells in the presence of ANP along with a ANP/NPRA inhibitor, A71915. We noted that pretreatment of naïve CD4+ T cells with A71915 almost abolished the suppressive effect mediated by ANP.
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Interestingly, analysis of cell lysates revealed that ANP increased the levels for intracellular second messenger cGMP, we thus pretreated naïve CD4+ T cells with a selective PKG inhibitor, KT5823. Blockade of PKG signaling partially reversed ANP-mediated suppressive effect, indicating the involvement of PKG in ANP/NPRA signaling. These results suggest that cGMP/PKG signaling pathway was implicated in ANP-mediated suppressive effect. Actually, NPRA/PKG pathway has already been confirmed to be the major pathway of ANP signaling in some previous studies, which is similar to our data [2–4]. ROR-γt is the indispensable transcription factor for Th17 development. There is also evidence that BATF acts as a co-factor essential to Th17 lineage as manifested by that mice deficient BATF−/− with normal Th1 and Th2 development but defective Th17 polarization. This notion was further supported by the observation that BATF −/− T cells fail to induce ROR-γt [14–16]. Interestingly, our Western blot analysis provided convincing evidence that ANP suppresses the expression of ROR-γt and BATF, which perfectly explains why ANP treatment impairs Th17 development. Other than the involvement of ROR-γt and BATF, Stat3 has also been noted to be important for Th17 development. Stat3 regulates the transcription of ROR-γt and BATF, and thus, loss of Stat3 activation renders naïve T cells unable to differentiate into Th17 cells. IL-6 and IL-23 are featured cytokines for the development of Th17 cells, and both of them can activate Janus family kinases, which then preferentially activate Stat3 through phosphorylation [19,36,37]. As a result, IL-6-inducible Jak/Stat3 pathway plays a significant role in the development of Th17 cells. Importantly, ANP treatment down-regulated the levels for phosphorylated Stat3 (p-Stat3) along with reduced expression for ROR-γt and BATF. These results suggest that ANP also impacts Stat3 activation to impair Th17 development. Previous studies suggested feasible evidence indicating a potential cross-talk between PI3K–Akt signaling and IL-6/Stat3 pathway, in which FKHR (also named Foxo1a) might act as the connector [20]. It is believed that FKHR, a downstream target of Akt, is held in check by Akt-mediated phosphorylation, and through which it acts as a possible transcriptional coactivator to interact with Stat3 and to modulate its IL-6-induced transcriptional activities [20,21]. Indeed, our studies revealed that ANP treatment increased the levels for p-Akt and p-FKHR but decreased the levels for p-Stat3, while the addition of ANP/NPRA antagonist reversed the suppressive effect of ANP on Stat3, suggesting the existence of a cross-talk between PI3K–Akt signaling and IL-6/Stat3 pathway. Given that PKG inhibitor only partially reversed ANP-mediated suppressive effect in our study and PI3K–Akt/FKHR signaling has been linked with ANP/NPRA-mediated cell proliferation, survival, and so on [22–24], we assumed that PI3K–Akt/FKHR signaling is also involved in ANP-mediated suppressive effect on Th17 development. As expected, when PI3K–Akt inhibitor was administered along with ANP in naïve T cells, the expressions for ROR-γt and BATF were partially reversed along with upregulation of p-Stat3 and increase of IL-17-producing CD4+ T cells. However, we failed to detect a significant change for IL-17 production and the mechanisms underlying this unexpected result remain unknown. Similar to our results, Mandy Pierau et al. reported that Akt signals impair TGF-β1/IL-6-mediated differentiation of naïve CD4+ T cells into the Th17 lineage [38]. Together, these data support that PI3K–Akt signaling is partly responsible for ANP-mediated biological effect on Th17 polarization. However, combining ANP/NPRA antagonist with PI3K–Akt inhibitor failed to observe enhanced reversing effect as compared with either of them alone, suggesting that PI3K–Akt is a downstream signal of NPRA. In summary, we provided feasible evidence that ANP is implicated in the regulation of naïve CD4 + T cells polarizing to Th17 lineage. ANP conjugates to NPRA, and by which it activates PKG and PI3K–Akt pathways to suppress Stat3 activation, which then impairs the expression of ROR-γt and BATF, the two essential factors for Th17
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