Acid-Sensing Ion Channels Contribute to the Effect of Acidosis on the Function of Dendritic Cells

This information is current as Jing Tong, Wen-Ning Wu, Xiaoling Kong, Peng-Fei Wu, Li of September 27, 2021. Tian, Wenjiao Du, Min Fang, Fang Zheng, Jian-Guo Chen, Zheng Tan and Feili Gong J Immunol published online 14 February 2011 http://www.jimmunol.org/content/early/2011/02/14/jimmun

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 14, 2011, doi:10.4049/jimmunol.1001346 The Journal of Immunology

Acid-Sensing Ion Channels Contribute to the Effect of Acidosis on the Function of Dendritic Cells

Jing Tong,*,1 Wen-Ning Wu,†,1 Xiaoling Kong,* Peng-Fei Wu,† Li Tian,* Wenjiao Du,* Min Fang,* Fang Zheng,* Jian-Guo Chen,† Zheng Tan,* and Feili Gong*

As an H+-gated subgroup of the degenerin/epithelial Na+ channel family, acid-sensing ion channels (ASICs) were reported to be involved in various physiological and pathological processes in neurons. However, little is known about the role of ASICs in the function of dendritic cells (DCs). In this study, we investigated the expression of ASICs in mouse bone marrow-derived DCs and their possible role in the function of DCs. We found that ASIC1, ASIC2, and ASIC3 are expressed in DCs at the mRNA and protein levels, and extracellular acid can evoke ASIC-like currents in DCs. We also demonstrated that acidosis upregulated the expression of CD11c, MHC class II, CD80, and CD86 and enhanced the Ag-presenting ability of DCs via ASICs. Moreover, the effect of

acidosis on DCs can be abolished by the nonsteroidal anti-inflammatory drugs ibuprofen and diclofenac. These results suggest that Downloaded from ASICs are involved in the acidosis-mediated effect on DC function. The Journal of Immunology, 2011, 186: 000–000.

s the most potent APCs of the immune system, dendritic upregulates the expression of cell surface proteins involved in cells (DCs) are specialized to capture, process, and Ag presentation of DCs (16), the molecular mechanism through present Ags to T lymphocytes to initiate Ag-specific which DCs are regulated by acidosis remained unknown. A + T cell responses (1, 2). Many reports showed that DCs play an Acid-sensing ion channels (ASICs) represent an H -gated sub- http://www.jimmunol.org/ important role in the development of inflammatory diseases, tu- group of the degenerin/epithelial Na+ channel family of cation mors, and autoimmune diseases (3–6). channels and are activated by extracellular protons (17). Six ASIC pH value is an important physiological indicator of internal subunit proteins, encoded by four genes, have been identified: environment homeostasis, which usually ranges from pH 7.2–7.4 ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, and ASIC4. They are in the physiological state. However, in inflammatory diseases, widely expressed in peripheral sensory neurons and in the neurons pH values in inflamed areas are always lower than those of nor- of the CNS. Activation of these channels by protons plays an mal tissues, ranging from pH 5.5–7 (7–9). Thus, low pH is consid- important role in a variety of physiological and pathological ered a hallmark of inflammatory reactions. In inflammation, as processes, such as nociception, mechanosensation, and acidosis- well as in autoimmune diseases, such as rheumatoid arthritis and mediated neuronal injury. Our previous study also demonstrated by guest on September 27, 2021 asthma, pH values of synovial fluid of affected joints and airway that ASICs are involved in learning and memory (18). Recent vapor condensate are lower than those in normal tissues (10, 11). studies reported that ASICs are also expressed in nonneuronal Moreover, extracellular pH values range from 5.7–7.8 in solid cells and have important effects on their physiological and path- tumors (12–14). However, few studies have focused on the effect ological function (19, 20). Whether ASICs are expressed in DCs of extracellular pH on immune cells and their functions (15), es- and their potential role in DC functions have not been determined. pecially DCs. Although it was reported that extracellular acidosis In the current study, we investigated the expression of ASICs in mouse DCs and the role of ASICs in Ag-presentation–related molecule expression in acidosis. We demonstrated that DCs ex- *Department of Immunology, Tongji Medical College, Huazhong University of Sci- press ASICs, and the cell-surface molecules involved in Ag pre- ence and Technology, Wuhan 430030, People’s Republic of China; and †Depart- sentation are upregulated by acidosis via ASICs. ment of Pharmacology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China 1J.T. and W.-N.W. contributed equally to this work. Materials and Methods Received for publication April 26, 2010. Accepted for publication January 3, 2011. Animals This work was supported by National Development Program (973) for Key Basic Female C57BL/6 and BALB/c mice, 6–8 wk old, were obtained from the Research of China Grant 2007CB512402 (to F.G.), Key Project of National Natural Institute of Organ Transplantation of Tongji Hospital, Huazhong Univer- Science Foundation of China Grant 30930104 (to J.C.), and National Natural Science sity of Science and Technology. Neonatal Sprague-Dawley rats (days 0–3) Foundation of China Grant 30700736 (to Z.T.). were obtained from the Experimental Animal Center of Tongji Medical Address correspondence and reprint requests to Dr. Feili Gong, Dr. Zheng Tan, and College, Huazhong University of Science and Technology. The animals Dr. Jianguo Chen, Department of Immunology, Tongji Medical College, Huazhong were housed under specific pathogen-free conditions. All studies were per- University of Science and Technology, 13 Hangkong Road, Wuhan 430030, People’s formed according to the guidelines of the Animal Care and Use Com- Republic of China (F.G. and Z.T.) and Department of Pharmacology, Tongji Medical mittee of Tongji Medical College, Huazhong University of Science and College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, People’s Republic of China (J.C.). E-mail addresses: flgong@163. Technology. com (F.G.), [email protected] (Z.T.), and [email protected] (J.C.) Cell culture The online version of this article contains supplemental material. DCs were generated as previously described (21, 22), with minor mod- Abbreviations used in this article: ASIC, acid-sensing ion channel; Cap, capsaicin; DC, dendritic cell; iDC, immature dendritic cell; mDC, mature dendritic cell; MFI, ifications. In brief, bone marrow cells were obtained from the femurs and 3 6 mean fluorescence intensity; NSAID, nonsteroidal anti-inflammatory drug; TRPV1, tibias of C57BL/6 mice; suspended at 1 10 cells/ml in RPMI 1640 transient receptor potential vanilloid-1. medium (Invitrogen) supplemented with 10% FCS, penicillin (100 U/ml), streptomycin (100 mg/ml), L-glutamine (2 mM), and 2-ME (50 mM); and Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 then seeded in 100-mm dishes at 10 ml/dish. Cultures were supplemented

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001346 2 EXTRACELLULAR ACIDOSIS REGULATES DC FUNCTION VIA ASICs with 10 ng/ml GM-CSF and 5 ng/ml IL-4 (both from PeproTech). The cells 0.5% Triton X-100 and 25 mg/ml proteinase A for 20 min, in sequence. were fed on days 3 and 5, by aspirating 75% of the medium and adding After extensive washing, cells were blocked with normal goat serum for 2 back fresh medium with GM-CSF and IL-4 at appropriate concentrations, h at room temperature. Cells were incubated with rabbit anti-ASIC1, rabbit and cultured for 7 d. By day 7, .85% of the harvested cells expressed anti-ASIC2, or rabbit anti-ASIC3 primary Ab diluted in PBS overnight at CD11c. Immature DCs (iDCs) used in this study were cultured for 7 d. To 4˚C. Then, cells were washed with PBS three times before staining with obtain mature DCs (mDCs), iDCs were activated with 1 mg/ml LPS anti-rabbit IgG (whole molecule)-Gold (Sigma-Aldrich) for 30 min. Cells (Sigma-Aldrich) for 24 h. were postfixed with 1% OsO4 for 1 h and were dehydrated, embedded, Primary rat cortical neurons were isolated as described in our previous made into ultrathin sections, and stained with uranyl acetate, using con- study (23, 24), with some modifications. Briefly, the cortex of newborn ventional methods. Images were taken with an FEI-Tecnai G2 12 trans- Sprague-Dawley rats were dissected and rinsed in ice-cold Dulbecco’s mission electron microscope (FEI). PBS. The dissected tissues were treated with 0.125% trypsin in HBSS for 25 min at 37˚C and mechanically dissociated using fire-polished Pasteur Whole-cell patch-clamp recordings pipettes. Cells were collected by centrifugation and resuspended in The procedure for whole-cell patch-clamp recording was performed as DMEM/F12 (1:1) with 10% FBS. For whole-cell patch-clamp recording, described in our previous studies (28, 29), with minor modification. The cells (20,000–40,000) were seeded on poly-D-lysine–coated coverslips and bath solution for recording ASIC currents contained NaCl 150 mM, KCl 5 kept at 37˚C in a 5%-CO2 incubator. After 24 h, the culture medium was mM, MgCl 1 mM, CaCl 2 mM, HEPES 10 mM, and glucose 10 mM; pH changed with DMEM medium supplemented with 2% B27, and the cor- 2 2 was adjusted to 7.4 with NaOH. Glass pipettes were used with a resistance tical neurons were fed with fresh medium twice a week. Microscopically, of ∼2–4 MV when filled with the following solution: KCl 140 mM, NaCl glial cells were not apparent by using this protocol. The neurons were 10 mM, MgCl2 1 mM, EGTA 5 mM, Mg-ATP 2 mM, and HEPES 10 mM; maintained for 7–10 d in primary culture until used. pH was adjusted to 7.2 with KOH. After establishing a whole-cell con- figuration, the adjustment of capacitance compensation and series re- RT-PCR sistance compensation was done before recording. The current signals Downloaded from Total RNA was isolated from cells using TRIzol reagent (Invitrogen), were acquired at a sampling rate of 10 kHz and were filtered at 3 kHz. according to the manufacturer’s instructions. cDNA synthesis was per- Whole-cell patch-clamp recordings were carried out using an EPC-10 formed using a PrimeScript 1st Strand cDNA Synthesis Kit (Takara Bio- amplifier, driven by Pulse/PulseFit software (both from HEKA Elek- technology). PCR amplifications of cDNA were performed by standard tronik). Drug actions were measured only after steady-state conditions methods. The following specific primers (forward, reverse) were used: were reached, which were judged by the amplitudes and time courses of ASIC1, 59-CAG ATG GCT GAT GAA AAG CA-39,59-AAG TGG CAC currents remaining constant. All recordings were made at room tempera- GAG AGA AGC AT-39; ASIC2, 59-TGA CAT TGG TGG TCA AAT GG- ture (20˚C–22˚C). All experiments were repeated three times using dif- 39,59-ATC ATG GCT CCC TTC CTC TT-39; ASIC3, 59-AGG GAG AAG ferent batches of cells, and at least three or four dishes of cells were used http://www.jimmunol.org/ TCC CAA AGC AT-39,59-GAC ACT CCA TTC CCA GGA GA-39 (25); for recording in different batches of cells. TRPV1, 59-TCG TCT ACC TCG TGT TCT TGT TTG-39,59-CCA GAT GTT CTT GCT CTC TTG TGC-39 (26); and GAPDH 59-TTC ACC ACC Flow cytometry ATG GAG AAG GC-39,59-GGC ATG GAC TGT GGT CAT GA-39 (27). Cells (1 3 106) were stained with PE-conjugated mAbs diluted in PBS GAPDH was used for normalization. PCR products were analyzed by 2% containing 1% BSA (FACS buffer). Before staining, cells were incubated agarose gel electrophoresis. with purified anti-mouse CD16/32 (Biolegend) for 10 min on ice to block FcR. The following PE-conjugated Abs (all from eBioscience) were used: Western blot anti-CD11, anti-IA/IE (MHC class II), anti-CD80, and anti-CD86. After incubating with Abs for 30 min at 4˚C, cells were washed twice with FACS Cells were lysed in lysis buffer (50 mM Tris [pH 8], 150 mM NaCl

buffer and analyzed by a LSR II flow cytometer (BD Bioscience). The by guest on September 27, 2021 containing 1% Nonidet P-40, 0.02% NaN3, 0.5 mM PMSF, 1 mg/ml aprotinin, and 1 mg/ml leupeptin). Whole-cell lysate was mixed with 53 results are shown as mean fluorescence intensity (MFI). SDS loading buffer and boiled for 5 min. The proteins were separated by MLR electrophoresis on 10% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with Splenic T cells from normal BALB/c mice were stained with CFSE and used 5% (w/v) nonfat milk in TBST for 2 h at 37˚C and then incubated with as responders. A total of 2 3 106/ml T cells was cultured with 2 3 105/ml rabbit anti-ASIC1, rabbit anti-ASIC2, or rabbit anti-ASIC3 Abs (all from DCs treated with mitomycin C (30 mg/ml) and incubated at 37˚C in 5% Alomone Laboratories), as well as mouse anti-actin Ab-5 Ab (BD Bio- CO2 for 5 d. After three washes with PBS, the cells were harvested and sciences), which served as a loading control, diluted in 5% (w/v) nonfat analyzed for T cell proliferation using a LSR II flow cytometer (BD milk in 0.1% TBST overnight at 4˚C. Membranes incubated with Abs that Bioscience). were preincubated with respective antigenic peptides were used to test specificity of Abs. After washing three times (10 min each) in 0.1% TBST, Statistical analysis the membranes were incubated with peroxidase-conjugated goat anti- Data are presented as mean 6 SEM. The Student t test with paired rabbit or goat anti-mouse secondary Ab (both from Pierce) for 2 h at comparisons was used to evaluate differences; p , 0.05 was considered 37˚C. The membrane was washed three times in 0.1% TBST, and the statistically significant. proteins were detected using Peroxide Solution (Millipore) with a Kodak Image Station 4000MM.

Immunocytochemistry Results ASICs are expressed in iDCs We used the immunofluorescence double-staining method to determine the localization of ASICs in DCs. First, DCs were plated on polylysine-coated To determine whether ASIC1, ASIC2, and ASIC3 are expressed in coverslips, cultured for 24 h, washed with PBS once, fixed with 4% iDCs, we performed RT-PCR to detect the expression of mRNA for paraformaldehyde in PBS for 20 min, washed three times in PBS, per- ASICs. Cortex neurons served as positive control. The sizes of the meabilized with 0.3% Triton X-100 in PBS for 20 min, washed three times for 5 min in PBS, and then blocked with 3% BSA for 2 h at 37˚C. Cells were expected PCR products for ASIC1, ASIC2, and ASIC3 were 140, incubated with rabbit anti-ASIC1, rabbit anti-ASIC2, or rabbit anti-ASIC3 139, and 107 bp, respectively. As a result, mRNAs for all three primary Abs diluted in 3% BSA overnight at 4˚C. After washing three ASICs were detected in iDCs (Fig. 1A). Then, we confirmed the times in PBS, cells were incubated with the FITC-conjugated goat anti- expression of ASIC proteins by Western blotting. Abs for ASIC1, rabbit secondary Ab (Pierce) and PE-conjugated anti-CD11c Ab (eBio- science) diluted in 3% BSA for 1 h at room temperature. After extensive ASIC2, and ASIC3 were preincubated with the corresponding Ag washing, coverslips were mounted on glass slides with 50% glycerol. peptide as specific control (Fig. 1B). Furthermore, we used im- Staining was visualized with an Olympus FV500 confocal microscope. munocytochemistry to determine cellular and subcellular dis- tributions of ASICs in iDCs. As shown in Fig. 1C, ASIC1 and Electron microscopy ASIC3 proteins were predominantly expressed in cytoplasm. Most DCs were fixed with a solution of 4% paraformaldehyde and 1.25% glutaral ASIC2 is expressed on the plasma membrane. We further ob- for 30 min; after washing three times in PBS, cells were permeabilized with served, by electron microscopy, that ASIC1 was expressed in The Journal of Immunology 3

FIGURE 1. Expression and localization of ASIC1, ASIC2, and ASIC3 in iDCs. A, ASIC transcripts were detected in iDCs by RT-PCR. Cortex neurons were used as positive control. B, Western blot analysis of ASIC protein expression in iDCs. Cortex protein was used as positive control. For specificity control, Abs for ASICs were preincubated with corresponding peptide Ags. C, Detection of localization of ASIC proteins in iDCs by double-staining immunofluorescence (original magnification 3400). Nuclei were coun- terstained with Hoechst33342 (blue). Green, ASICs; red, CD11c. D, Subcellular localization of ASICs was visualized using a transmission electron mi- croscope. ASIC1 and ASIC3 were found in endo- plasmic reticulum and mitochondrion, respectively, and ASIC2 was located on the cell membrane. Downloaded from Arrows indicate the localization of ASICs.

endoplasmic reticulum and perinuclear regions, whereas ASIC3 fect was 6.08 6 0.05, and the Hill coefficient was 1.03 6 0.07 http://www.jimmunol.org/ was found in mitochondria (Fig. 1D). (n = 6) (Fig. 2C). Electrophysiological characteristics of ASIC-like currents in iDCs Transient receptor potential vanilloid-1 is not functionally A whole-cell patch-clamp recording was performed to determine expressed in iDCs whether the expression of ASIC proteins in iDCs has a function. As a nonselective cation channel, transient receptor potential Cells were held at 260 mV and then a rapid decrease in extra- vanilloid-1 (TRPV1) also can evoke membrane currents at low cellular pH, from 7.4, was applied to the bath. In 85% (n = 36/42) extracellular pH (,6). Thereby, we examined the effect of cap- by guest on September 27, 2021 of the recorded iDCs, the rapid decrease in extracellular pH eli- saicin (Cap), a selective agonist of TRPV1, on iDCs with the cited inward ASIC-like currents, indicating the presence of whole-cell patch-clamp technique. As shown in Fig. 3A,10mM functional ASIC channels. Usually, this transient inward current Cap significantly evoked an inward current in cortex neurons. contained two phases: early fast and late slow inactivation (Fig. However, 10 or 100 mM Cap failed to induce any inward currents 2A). The mean amplitude of ASIC-like currents induced by pH 6 in iDCs, indicating that the inward currents induced by a decrease was 329.63 6 52.36 pA. Similar to the proton-gated currents in in extracellular pH may not be mediated by TRPV1 channels in sensory and central neurons, the ASIC-like currents in iDCs were iDCs. To further confirm this ratiocination, RT-PCR was per- reversibly inhibited by amiloride (100 mM), a blocker of ASICs formed to investigate whether TRPV1 is expressed in iDCs, with (n =6,p , 0.05; Fig. 2A). As shown in Fig. 2B, the response of cortex neurons as positive control. We did not detect TRPV1 iDCs to acidosis was pH dependent. The threshold extracellular mRNA expression in iDCs (Fig. 3B). These results suggested that pH to elicit the inward current was ∼7, and the maximum re- TRPV1 is not involved in the elicitation of inward currents in- sponse appeared at pH 5. The extracellular pH producing 50% ef- duced by low extracellular pH in iDCs.

FIGURE 2. Electrophysiological characteristics of ASIC-like currents recorded in iDCs by whole-cell patch clamp. A, pH 6 elicited ASIC-like currents in iDCs, and the currents were reversibly inhibited by amiloride (100 mM) (n = 36/42). B and C, ASIC-like currents elicited by acid in iDCs were pH dependent (n = 6 for each pH). 4 EXTRACELLULAR ACIDOSIS REGULATES DC FUNCTION VIA ASICs

FIGURE 3. TRPV1 is not functionally expressed in iDCs. A, Cap (10 or 100 mM) did not elicit current in iDCs. Currents evoked by Cap (10 mM) served as positive control. B, No TRPV1 expression at the mRNA level in iDCs was detected by RT-PCR. Cortex neurons were used as positive control.

The characteristics of ASICs in mDCs 6B). The inhibitory percentage was 51.49 6 3.75%, and the in- After demonstrating the characteristics of ASIC expression in hibition could be reversed by extensive washing with bath solu- m iDCs, we further investigated that in mDCs. As shown in Fig. 4A tion. Similarly, diclofenac (200 M) significantly reduced ASIC 6 6 and 4B, the distribution and expression of ASICs were similar in currents from 353.97 51.58 pA to 166.7 18.57 pA (Fig. 6C, iDCs and mDCs. However, the amplitude of ASIC currents in- 6D). The inhibition was also reversible, and the inhibitory per- 6 duced by extracellular pH 6 was increased significantly in mDCs centage was 51.67 8.3%. These data suggested that ASIC (Fig. 4C), suggesting an enhanced function of ASICs in mDCs. currents in iDCs are also sensitive to NSAIDs. Therefore, ASICs in DCs are possible targets for NSAIDs in inflammation. ASICs are involved in acidosis-induced DC maturation characterized by upregulation of CD11c, MHC, CD80, and NSAIDs inhibit DC maturation induced by acidosis Downloaded from CD86 After determining the inhibitory effect of NSAIDs on acid-sensing It was reported that exposure to pH 6.5 for 4 h resulted in a sig- channels in DCs, we examined the effect of NSAIDs on DC nificant maturation of DCs, characterized by a significant increase maturation induced by acidosis. DCs were preincubated with in CD11c, MHC class II, and CD86 (16). In addition to those ibuprofen or diclofenac at pH 7.3 for 30 min at 37˚C and were molecules, CD80 on DCs was upregulated after exposure to pH exposed to pH 6.5 for 4 h. Ibuprofen and diclofenac had no effect 6.5 for 4 h (Fig. 5); however, it is still unknown how acidosis on the expression of CD11c, MHC II, CD80, or CD86 at pH 7.3 http://www.jimmunol.org/ induces DC maturation. Because ASICs are proton-gated cation (Supplemental Fig. 2), but they significantly abrogated the up- channels and can be activated by extracellular H+, we hypothe- regulation of CD11c, MHC class II, CD80, and CD86 molecules sized that the effect of acidosis on DCs is mediated via ASICs. To induced by acidosis (Fig. 7), indicating that NSAIDs can inhibit examine this hypothesis, DCs were incubated at pH 7.3 for 30 min the maturation of DCs stimulated by acidosis. at 37˚C in the presence of amiloride (100 mM), a blocker of Acidosis increases the Ag-presenting ability of DCs via ASICs ASICs, before being exposed to pH 6.5 for 4 h at 37˚C. Amiloride had no effect on CD11c, MHC class II, CD80, or CD86 at pH 7.3 The data above demonstrated that ASICs are involved in the up- (Supplemental Fig. 1); however, upregulation of CD11c, MHC regulation of cell-surface molecules related to Ag presentation class II, CD80, and CD86 stimulated by acidosis was significantly induced by acidosis in DCs. MLR was used to examine the effect of by guest on September 27, 2021 inhibited by amiloride (Fig. 5), suggesting that ASICs are involved acidosis on the Ag-presenting ability of DCs and the role of ASICs. in DC maturation induced by acidosis. DCs were incubated in pH 7.3 medium for 30 min in the presence or absence of amiloride (100 mM), ibuprofen (200 mM), or diclofenac Nonsteroidal anti-inflammatory drugs suppress inward ASIC (200 mM) before being exposed to pH 6.5 for 4 h; DCs incubated currents in DCs at pH 7.3 for 4.5 h served as controls. After washing, DCs were Previous studies demonstrated that nonsteroidal anti-inflammatory pretreated with mitomycin C and cultured with T cells stained drugs (NSAIDs) significantly inhibited ASIC currents in hippo- with CFSE at pH 7.3 for 5 d, and T cell proliferation was detected campus and sensory neurons (30, 31). To investigate the effect of by flow cytometry. As expected, pH 6.5 increased the Ag- NSAIDs on ASICs in iDCs, the responses of ASIC currents to presenting ability of DCs, whereas amiloride and NSAIDs (ibu- NSAIDs (ibuprofen or diclofenac) were tested. Extracellular ap- profen or diclofenac) abrogated this effect (Fig. 8), suggesting that plication of 200 mM ibuprofen significantly reduced ASIC cur- ASICs play a critical role in the regulation of the Ag-presenting rents from 364.17 6 57.15 pA to 176.07 6 28.77 pA (Fig. 6A, ability of DCs by acidosis.

FIGURE 4. ASIC1, ASIC2, and ASIC3 are functionally expressed in mDCs. A, Localization of ASICs in mDCs was detected by double-staining immunofluorescence (original magnification 3400). Nuclei were counterstained with Hoechst33342 (blue). Green, ASICs; red, CD11c. B, Expression of ASICs in mDCs. iDCs served as control and b-actin was used for normalization. C, ASIC-like currents elicited by pH 6 in iDCs and mDCs (n = 6 for each group). *p , 0.05, significant difference between the two groups. The Journal of Immunology 5

FIGURE 5. pH 6.5 increases the expression of cell-surface molecules CD11c, MHC class II, CD80, and CD86 in DCs, and this effect was abrogated by Downloaded from amiloride. DCs were incubated with or without amiloride (100 mM) at pH 7.3 for 30 min at 37˚C, 5% CO2 before exposure to pH 6.5 for 4 h at 37˚C, 7% CO2. A–D, Graphs from a representative experiment. E–H, MFI values of CD11c, MHC class II, CD80, and CD86 were analyzed by flow cytometry. The data represent mean 6 SEM of six independent experiments. #p , 0.05, significant difference for pH 6.5 compared with pH 7.3; *p , 0.05, significant difference for amiloride treatment compared with pH 6.5.

Discussion To test this hypothesis, the expression of ASICs in mouse iDCs The results demonstrated that ASICs are expressed in DCs and are and mDCs was investigated. As expected, iDCs expressed ASIC1, http://www.jimmunol.org/ involved in the maturation of DCs induced by acidosis. ASIC2, and ASIC3 at the mRNA and protein levels (Fig. 1A,1B). As the most potent APC, DCs are specialized to capture and Then, immunofluorescence confocal microscopy and electron mi- process Ags and present them to T cells to induce immune croscopy were used to examine their localization in iDCs. Un- responses. Therefore, DCs are critical for the induction of immune like the distribution in neurons, ASIC1 and ASIC3 are found responses triggered by microorganisms, tumors, and autoantigens. mainly in cytoplasm rather than on the cell membrane, whereas Recently, there is increasing evidence to suggest that inflammation ASIC2 is located exclusively on the cell membrane (Fig. 1C,1D). plays an important role in various diseases, such as tumors and Consistently, the distribution and expression of ASICs in mDCs were similar to those in iDCs (Fig. 4A,4B).

autoimmune diseases (32–35). Inflammation is always accompa- by guest on September 27, 2021 nied by tissue acidification, which means that the microenviron- We further examined the electrophysiological characteristics ment of pathological sites is acidic compared with normal sites; of ASICs in DCs. We found that the inward ASIC-like currents in however, the effect of an acidic microenvironment on the physi- DCs were reversibly inhibited by a blocker of ASICs, amiloride ological functions of DCs is less well known. Although a few (Fig. 2), suggesting that ASICs are functionally expressed in DCs. studies have focused on the role of acidosis in DC function, the Interestingly, the amplitude of ASIC currents was larger in mDCs mechanism underlying the effect remains unclear (16, 36). than in iDCs (Fig. 4C), indicating that the function of ASICs is Because ASICs are H+-gated cation channels and are activated enhanced in mDCs. by extracellular protons, and the expression of ASICs in T cells, TRPV1 is a nonselective cation channel with high permeability B cells, and macrophages of mice was reported recently (37), we for Ca2+. It was reported to be responsive to a variety of stimuli, proposed that ASICs serve as sensors for extracellular acidosis such as noxious heat, Cap, endovanilloids, and acid in neurons and mediate the responses of DCs to acidosis. (38). A recent study suggested that TRPV1 is expressed in human

FIGURE 6. Inhibitory effect of NSAIDs on ASICs in iDCs. A and B, ASIC currents in iDCs were reversibly inhibited by ibuprofen (200 mM). C and D, ASIC currents in iDCs were reversibly inhibited by diclofenac (200 mM). Data shown are mean 6 SEM (n = 6 for each group). *p , 0.05 versus control. 6 EXTRACELLULAR ACIDOSIS REGULATES DC FUNCTION VIA ASICs Downloaded from FIGURE 7. Upregulation of cell-surface molecules CD11c, MHC class II, CD80, and CD86 induced by pH 6.5 in DCs is inhibited by ibuprofen and diclofenac. DCs were incubated with or without ibuprofen (200 mM) or diclofenac (200 mM) at pH 7.3 for 30 min at 37˚C, 5% CO2 before exposure to pH 6.5 for 4 h at 37˚C, 7% CO2. A–D, Graphs from a representative experiment. E–H, The MFI values for CD11c, MHC class II, CD80, and CD86 were analyzed by flow cytometry. Data shown are mean 6 SEM of six independent experiments. #p , 0.05, significant difference for pH 6.5 compared with pH 7.3; *p , 0.05, **p , 0.01, significant difference for treatment with ibuprofen or diclofenac compared with pH 6.5. http://www.jimmunol.org/ iDCs and plays a role in the differentiation and activation of iDCs periphery tissues into the draining lymph nodes to induce primary (39). However, previous studies reported conflicting data on the immune responses. One important feature of mDCs is the increased existence and function of TRPV1 in mouse iDCs (26, 40). Using expression of MHC molecules and costimulatory molecules on the Cap, a specific agonist for TRPV1, we tried to elicit currents in cell membrane. Vermeulen et al. (16) reported that extracellular iDCs but were unsuccessful (Fig. 3A). Furthermore, we could not acidosis upregulates the expression of cell-surface proteins that detect TRPV1 expression in iDCs using RT-PCR (Fig. 3B). These are involved in Ag presentation of DCs, indicating that acidosis results suggested that currents evoked by acid in iDCs are not via has an important role in the maturation of DCs. Consistent with TRPV1. the data of Vermeulen et al., we found that the surface expression In periphery tissues, DCs are in an immature stage and have of CD11c, MHC class II, and CD86 on DCs was upregulated in an by guest on September 27, 2021 phagocytic abilities. After encountering pathogens, they undergo acidic environment (Fig. 5). In addition to those molecules, the a process of maturation to become efficient APCs and migrate from expression of costimulatory molecule CD80 was increased at pH 6.5 (Fig. 5). The upregulations were inhibited by amiloride, a blocker for ASICs (Fig. 6), suggesting that ASICs mediate the maturation of DCs induced by acidosis. Enhanced migration is another characteristic of DC maturation. However, the results suggested that extracellular acidosis does not influence the ex- pression of CCR7 on DCs (Supplemental Fig. 2). It was suggested that NSAIDs inhibit ASIC currents in various neurons and may protect neurons from damage associated with acidosis. Although Dorofeeva et al. (30) demonstrated that NSAIDs reduced the maximal response to a decrease in pH but did not induce a significant decrease in ASICs’ sensitivity to protons in hippocampal interneurons, the mechanism of the NSAID action is still not clear. Similar to previous reports, the results showed that ibuprofen and diclofenac significantly inhibited ASIC cur- rents in DCs (Fig. 6). Moreover, we found that the maturation of DCs induced by acidosis was also inhibited by ibuprofen and diclofenac (Fig. 7), indicating that the inhibitory effect of NSAIDs on acidosis-induced maturation is mediated by ASICs. However, the mechanism by which NSAIDs inhibit ASICs of DCs needs to FIGURE 8. Effect of pH 6.5 on the Ag-presenting ability of DCs is be investigated further. Additionally, acidosis increased the ability inhibited by amiloride, ibuprofen, and diclofenac. DCs (from C57BL/6 of DCs to stimulate the proliferation of allogeneic T cells; this m mice) were incubated or not with amiloride (100 M), ibuprofen (200 effect was significantly inhibited by amiloride, ibuprofen, and mM), or diclofenac (200 mM) at pH 7.3 for 30 min before exposure to pH diclofenac (Fig. 8), indicating that ASICs mediate the maturation 6.5 for 4 h; subsequently, the DCs were treated with mitomycin C and cultured with allogeneic T cells (from BALB/c mice) stained with CFSE at of DCs induced by acidosis and that ASICs in DCs may be a novel pH 7.3 for 5 d. Results are expressed as proliferating index and are shown target for NSAIDs in the anti-inflammation reaction. as mean 6 SEM of six independent experiments. ##p , 0.01, significant In conclusion, the results showed that ASICs play an important difference for pH 6.5 versus pH 7.3; **p , 0.01, significant difference for role in the physiologic function of mouse bone marrow-derived drug treatment compared with pH 6.5. DCs as receptors for extracellular acidosis. Functional ASICs The Journal of Immunology 7 are expressed in DCs and mediate the upregulation of CD11c, MHC 18. Wemmie, J. A., J. Chen, C. C. Askwith, A. M. Hruska-Hageman, M. P. Price, class II, CD80, and CD86 induced by acidosis. ASICs also mediate B. C. Nolan, P. G. Yoder, E. Lamani, T. Hoshi, J. H. Freeman, Jr., and M. J. Welsh. 2002. The acid-activated ion channel ASIC contributes to synaptic the Ag-presenting ability of DCs induced by acidosis. ASICs may plasticity, learning, and memory. 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