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Keywords:

  • acidification;
  • cAMP;
  • interleukin-1β;
  • microglia;
  • protein kinase A;
  • TDAG8

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information
Thumbnail image of graphical abstract

Interleukin-1β (IL-1β) is released from activated microglia and involved in the neurodegeneration of acute and chronic brain disorders, such as stroke and Alzheimer's disease, in which extracellular acidification has been shown to occur. Here, we examined the extracellular acidic pH regulation of IL-1β production, especially focusing on TDAG8, a major proton-sensing G-protein-coupled receptor, in mouse microglia. Extracellular acidification inhibited lipopolysaccharide -induced IL-1β production, which was associated with the inhibition of IL-1β cytoplasmic precursor and mRNA expression. The IL-1β mRNA and protein responses were significantly, though not completely, attenuated in microglia derived from TDAG8-deficient mice compared with those from wild-type mice. The acidic pH also stimulated cellular cAMP accumulation, which was completely inhibited by TDAG8 deficiency. Forskolin and a cAMP derivative, which specifically stimulates protein kinase A (PKA), mimicked the proton actions, and PKA inhibitors reversed the acidic pH-induced IL-1β mRNA expression. The acidic pH-induced inhibitory IL-1β responses were accompanied by the inhibition of extracellular signal-related kinase and c-Jun N-terminal kinase activities. The inhibitory enzyme activities in response to acidic pH were reversed by the PKA inhibitor and TDAG8 deficiency. We conclude that extracellular acidic pH inhibits lipopolysaccharide-induced IL-1β production, at least partly, through the TDAG8/cAMP/PKA pathway, by inhibiting extracellular signal-related kinase and c-Jun N-terminal kinase activities, in mouse microglia.

A number of studies have shown that extracellular acidification in brain is observed in ischemia and neurodegenerative disorders. However, the molecular mechanism by which extracellular acidification regulates the biological activities of microglia remains uncharacterized. Here, we examined the extracellular acidic pH regulation of IL-1β production, especially focusing on TDAG8, a member of OGR1 family receptors. Our results suggest that extracellular acidic pH inhibited lipopolysaccharide -induced IL-1β production at least partly through the TDAG8/cAMP pathway, by inhibiting ERK and JNK activities.

Abbreviations used
8CPT-2Me-cAMP

8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate

ERK

extracellular signal-related kinase

GAPDH

glyceroaldehyde-3-phosphate dehydrogenase

GPCR

G-protein-coupled receptor

H89

N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonmide

IBMX

3-isobutyl-1-methylxanthine

IκB

inhibitor of κB

IL-1β

interleukin-1β

JNK

c-Jun N-terminal kinase

LPS

lipopolysaccharide

myr-PKI

myristoylated PKA inhibitor

N6-Ben-cAMP

N6-benzoyladenosine-3′,5′-cyclic monophosphate

NF-κB

nuclear factor-κB

p38 MAPK

p38 mitogen-activated protein kinase

PKA

cAMP-dependent protein kinase

proIL-1β

cytoplasmic precursor of IL-1β

TLR

toll-like receptor

Microglia fulfill a central role in the innate and adaptive immune system in brain. The activation of microglia and subsequent increase in the synthesis and release of proinflammatory cytokines, such as IL-1, have been shown to occur under acute, e.g., stroke and traumatic brain injury, and chronic conditions, e.g., Alzheimer's disease and Parkinson's disease (Yenari et al. 2010). IL-1 is known to have a causal role in their neurodegeneration, although the cytokine is also thought to be involved in the recovery of the neuronal functions (Spulber and Schultzberg 2010; Smith et al. 2012). A variety of extracellular signals trigger the activation of microglia. Toll-like receptors (TLRs) are important; they monitor changes in the extracellular microenvironment, resulting in the accumulation of inflammatory cytokines and chemokines (Okun et al. 2009; Downes and Crack 2010). Extracellular acidification in brain has also been observed in ischemia and neurodegenerative disorders, in which lactate and by-products of glycolysis are accumulated in association with impairment of mitochondrial function (Wang and Xu 2011). For example, under an ischemic situation, a lack of blood supply causes hypoxia and the inhibition of aerobic respiration and, thereby, increases lactic acid production through glycolysis, causing a decrease in pH to 6.1~6.8 depending on the blood glucose concentration (Siemkowicz and Hansen 1981; Smith et al. 1986). Acidic pH is thought to influence mitochondrial function, free radical formation, synthesis and degradation of cellular components, cell volume control, and endothelial damage; these events lead to energetic dysbalance, dysfunction of membrane integrity, edema, and finally irreversible neuronal cell death (Rehncrona 1985). Extracellular acidic pH has been shown to affect microglial functions as well. Thus, extracellular acidification induces change in membrane depolarization (Chung et al. 1988), inhibition of store-operated Ca2+ influx (Khoo et al. 2001), potentiation of a voltage-gated proton current (Morihata et al. 2000, 2008), and inhibition of the basal microglial motility and C5a-induced chemotaxis via a rearrangement of the cytoskeleton (Faff and Nolte 2000). However, the molecular mechanism by which extracellular acidification regulates the biological activities of microglia remains uncharacterized.

Recent studies revealed that OGR1 family G-protein-coupled receptors (GPCRs), including OGR1, GPR4, G2A, and TDAG8, which have previously been proposed as receptors for lysolipids, sense extracellular protons, resulting in the stimulation of intracellular signaling pathways (Tomura et al. 2005; Seuwen et al. 2006; Mogi et al. 2013; Okajima 2013). For example, extracellular acidification induces Ca2+ mobilization in association with phospholipase C activation through OGR1 (Ludwig et al. 2003; Mogi et al. 2005), activation of the Rho-signaling pathway through G2A (Murakami et al. 2004), and stimulation of cAMP accumulation and the Rho-signaling pathway through TDAG8 (Wang et al. 2004; Ishii et al. 2005; Radu et al. 2005; Hikiji et al. 2013) and GPR4 (Ludwig et al. 2003; Tobo et al. 2007; Liu et al. 2010). TDAG8 is expressed in lymphoid tissues, including peripheral blood leukocytes, spleen, lymph nodes, and thymus (Choi et al. 1996: Kyaw et al. 1998). In this study, we examined whether extracellular acidic pH regulates IL-1β production, with a focus on proton-sensing TDAG8 in mouse microglia. Our results suggest that extracellular acidic pH inhibited lipopolysaccharide (LPS)-induced IL-1β production, at least partly, through the TDAG8/cAMP pathway, by inhibiting extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK).

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information

Materials

LPS, forskolin, 8-(4-chlorophenylthio)-2′-O-methyladenosine 3′, 5′-cyclic monophosphate (8CPT-2Me-cAMP), N6-benzoyladenosine-3′,5′-cyclic monophosphate (N6-Ben-cAMP), 3-isobutyl-1-methylxanthine (IBMX), N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonmide (H89), and anti-β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA); myristoylated protein kinase A (PKA) inhibitor (myr-PKI) was from Enzo Life Sciences, Inc. (Farmingdale, NY, USA); polyinosinic-polycytidic acid (poly(I:C)) was from IMGENEX (San Diego, CA, USA); fatty acid-free bovine serum albumin (BSA, Fraction V), BAY-11-7082, SB203580, SP600125, and U0126 were from Calbiochem-Novabiochem Co. (San Diego, CA, USA); anti-phosphorylated IκBα (#9246), anti-phosphorylated NF-κB p65 (#3033), and anti-phosphorylated ERK (#9106) antibodies were from Cell Signaling Technology (Beverly, MA, USA); anti-phosphorylated JNK (V7931) and anti-phosphorylated p38 mitogen-activated protein kinase (p38 MAPK) (V1211) antibodies were from Promega (Madison, WI, USA); anti-NF-κB (p65) antibody (#06-418) was from Upstate Biotechnology (Charlottesville, VA, USA); Mouse IL-1β ELISA kit (DuoSet) and anti-rat IL-1β antibody were from R & D Systems (Minneapolis, MN, USA); Cyclic AMP EIA Kit and NF-κB (p65) Transcription Factor Assay Kit were from Cayman Chemical Co. (Ann Arbor, MI, USA); bicinchoninic acid (BCA) Protein Assay was from Thermo (Rockford, IL, USA); HEPES and 3-(4,5-Dimethyl-2-thiazoyl)-2,5-diphenyltetrazolium bromide (MTT) were from Dojindo (Tokyo, Japan); anti-Iba1 antibody was from Wako Chemicals (Osaka, Japan); anti-CD11b antibody (M170.15) was from Acris Antibodies (Herford, Germany); anti-F4/80 antibody (BM8) was from Sanbio BV (Uden, The Netherlands); anti-glial fibrillary acidic protein (GFAP) antibody was from PROGEN (Heidelberg, Germany); and RT-PCR probes specific for G2A (Mm00490809), GPR4 (Mm00558777), OGR1 (Mm01335272), TDAG8 (Mm00433695), TLR4 (Mm00445274), IL-1β (Mm 01336189), and glyceraldehydes 3-phosphate dehydrogenase (4352932E) were from Applied Biosystems (Foster City, CA, USA). TDAG8Tp/Tp was kindly provided by Drs K. Horie and J. Takeda of Osaka University (Osaka, Japan). MG6 cells (RCB 2403) were provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. The sources of all other reagents were the same as described previously (Malchinkhuu et al. 2009; Mogi et al. 2009; Sato et al. 2011).

Animal

TDAG8Tp/Tp mice were obtained by backcrossing to C57BL/6 mice more than five generation from TM88ICR mice, which contains a transposon insertion in the tdag8 (Horie et al. 2003). Wild-type (TDAG8+/+) or gene-deficient (TDAG8Tp/Tp) mice were generated by heterozygous (TDAG8Tp/+) brother–sister mating. Offspring with a single transposon inserted into the tdag8 were identified by a PCR genotyping as described before (Mogi et al. 2009). The mice were maintained in sterile cages on sterile bedding and housed in rooms at a constant temperature and humidity. Sterile food and water were fed to the mice ad libitum. All experiments using animals were performed according to procedures approved by the Gunma University Animal Care Committee.

Cell culture

Mouse type I astroglial cells (evaluated by anti-GFAP antibody) were prepared as described (Sato et al. 2005, 2011). Briefly, the cerebral cortex from 1- to 2-day-old mouse pups was minced and digested with 0.25% trypsin for 20 min at 32°C. Dissociated cells were collected, resuspended and filtered (71 μm) in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), and plated at a density of 1.0 × 106 cells/mL on the poly-d-lysine-coated flask (75 cm2). The cultures were maintained for 20 days until confluent. The growth medium was collected and stored as a glial conditioned medium containing 10% FBS (G-DMEM). Microglial cells were prepared by a mild trypsinization method according to Saura et al. (2003). Briefly, the cell suspension obtained by mincing and digestion with 0.25% trypsin of cerebral cortex as described above was plated on regular culture dishes. The adherent cells are incubated in 0.05% trypsin (Trypsin 25200 from Life Technologies-Gibco (Carlsbad, CA, USA) diluted in DMEM) for 60 min at 37°C to remove astrocytes. The attached microglial cells were recovered by 0.25% trypsin for 10 min at 37°C, resuspended in G-DMEM, and filtered (40 μm). The cell suspension was plated at a density of 3–5 × 105 cells/mL on 6-, 12- or 96-well dishes for following experiments, and cultured for 1 day. The resulting population consisted of > 95% microglia evaluated by anti-Iba1, anti-CD11b, and anti-F4/80 antibodies. MG6 cells were a microglial cell line with human c-myc from C57BL/6 mouse, and were grown in DMEM supplemented with 10% FBS, 100 μmol/L β-mercaptoethanol, and 10 μg/mL insulin as described in Takenouchi et al. (2005). For analysis of IL-1β mRNA, IL-1β protein, and cytoplasmic precursor of IL-1β (proIL-1β) in glial cells, the culture medium was changed to a fresh DMEM containing 0.1% BSA for 16 h. The dishes were then stimulated for the indicated time with HEPES-buffered α-minimum essential medium (MEM) containing 20 mmol/L HEPES, 0.1% BSA and test agents under an appropriate pH.

RNA analysis

Total RNA was prepared from cultured cells according to the manufacturer's instructions for RNAisoPlus (Takara, Japan). Quantitative real-time PCR (RT-qPCR) was performed according to Bustin et al. (2009) using hydrolysis probes of TaqMan technology (Applied Biosystems). The total RNA (5 μg) was treated with DNase I to remove possible trace of genomic DNA and subjected to the RT-qPCR. The thermal cycling conditions were as follows: 2 min at 50°C, 10 min at 95°C, 40 cycles of 15 s at 95°C, and 1 min at 60°C. The expression level of the target mRNA was normalized to the relative ratio of the expression of glyceraldehydes 3-phosphate dehydrogenase mRNA. The RT-qPCR assay was performed with three different RNA concentrations in each sample.

Measurement of IL-1β protein and its precursor protein

The HEPES-buffered culture medium was collected and centrifuged at 14 000 g for 20 min. The pH in the sample was adjusted to around 7.4 by an addition of 0.5 mol/L HCl or NaOH and stored at −80°C until evaluation of IL-1β content. A commercially available ELISA kit was used for determination of IL-1β concentration according to its instruction manual. For detection of proIL-1β, the cells were washed twice with ice-cold PBS and harvested from the dishes with a rubber policeman by adding a lysis buffer composed of PBS, 1% IGEPAL CA-630 (Sigma-Aldrich), 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mmol/L EDTA, and 1% proteinase inhibitor cocktail (Sigma-Aldrich). The lysate was incubated for 30 min on ice and was centrifuged at 14 000 g for 20 min. The protein concentration of the supernatant was determined with the BCA Protein Assay. The recovered lysate was subjected to 12.5% SDS–polyacrylamide gel electrophoresis and analyzed by western blotting with anti-rat IL-1β antibody. The protein bands were detected by alkaline phosphatase method, as described previously (Sato et al. 2005, 2011), and the scanned bands were quantified by Image-J (http://imagej.nih.gov/ij/index.html). The expression level of the target protein was normalized to the relative ratio of actin.

Estimation of IκBα, NF-κB, ERK, JNK, and p38MAPK activation by western blot analysis

Anti-phosphorylated antibodies against IκBα, NF-κB (p65), ERK (p44 and p42), JNK (p54 and p46), and p38MAPK were used for estimation of their activation. The serum-starved cells were incubated at 37°C in the HEPES-buffered α-MEM containing 0.1% BSA together with test substances under an appropriate pH for the indicated times. Reactions were terminated by washing twice with ice-cold PBS containing 1% phosphatase inhibitor cocktail (Sigma-Aldrich) and adding a lysis buffer composed of 50 mmol/L HEPES, pH 7.0, 150 mmol/L NaCl, 0.1% Nonidet P-40, 1% phosphatase inhibitor cocktail, and 1% proteinase inhibitor cocktail. Cytosol fractions were prepared and subjected to the western blot analysis as described above.

Intracellular cAMP accumulation

The serum-starved cells were washed once, pre-incubated for 10 min at 37°C in a HEPES-buffered medium composed of 20 mmol/L HEPES, pH7.6, 134 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L KH2PO4, 1.2 mmol/L MgSO4, 2 mmol/L CaCl2, 2.5 mmol/L NaHCO3, 5 mmol/L glucose, and 0.1% BSA. The medium was then replaced with the same medium containing 0.5 mmol/L IBMX under an appropriate pH. After a 30-min incubation at 37°C, the reaction was terminated by the addition of 0.1 mol/L HCl. Cyclic AMP in the acid fraction (0.2 mL) was diluted with an EIA buffer and measured according to the instruction manual of cAMP EIA Kit. The adherent cells were washed with PBS, followed by treatment with 5% trichloroacetic acid. The trichloroacetic acid-insoluble material was solubilized with a mixture containing 2% Na2CO3, 0.1% SDS, and 0.1 mol/L NaOH. The protein concentration of protein extracts was determined with the BCA Protein Assay.

Cell viability assay

The cell viability was measured by the cellular activity to reduce MTT. The serum-starved microglia in 96-well plates was cultured for 24 h in HEPES-buffered α-MEM containing 0.1% BSA at pH 7.6 to 6.4. The medium was changed to α-MEM containing 0.1% BSA and 0.5 mg/mL MTT at pH 7.4 and incubated for 3 h at 37°C. The reaction was stopped by the addition of 5% trichloroacetic acid (0.015 mL), followed by 2% Na2CO3, 0.1% SDS, and 0.1 mol/L NaOH (0.1 mL), and then completely dissolved by the addition of 0.04 mol/L HCl in isopropanol (0.1 mL). MTT values were measured by absorbance at 570 nm, and data were expressed as a percentage of control cells at pH 7.6.

NF-κB (p65) nuclear translocation with fluorescence microscopy

Mouse microglia were seeded on a sterile cover-slip chamber. The serum-starved cells were stimulated with 1 μg/mL LPS in HEPES-buffered α-MEM (pH 7.6 or 6.8) for appropriate time up to 6 h. Afterward, the cells were fixed in 4% paraformaldehyde, treated with 0.1% TritonX-100, and then incubated in 3% goat serum. The fixed cells were treated with anti-NF-κB (p65) antibody and following Alexa Fluor 488 goat anti-rabbit antibody for estimation of nuclear translocation. The fixed cells were also stained with 4′, 6-diamidino-2-phenylindole, dihydrochloride (DAPI) and the stained specimens were examined by fluorescence microscopy.

DNA-binding assay for NF-κB (p65)

The serum-starved MG6 cells in 6-cm dishes were incubated at 37°C in HEPES-buffered α-MEM (pH 7.6 or 6.8) together with 1 μg/mL LPS for the indicated times. NF-κB (p65) Transcription Factor Assay Kit was used for preparation of nuclear extracts from MG6 cells and NF-κB (p65) DNA-binding assays were performed according to its instruction manual. NF-κB (p65) binding to its DNA response element was evaluated by incubating nuclear extracts with immobilized DNA probes onto the bottom of 96-well plate. In competition experiments, the competitor was included prior to addition of nuclear extracts. Anti-NF-κB (p65) antibody in the kit was used for detection of DNA-binding activity.

Statistical analysis

All experiments were performed in duplicate or triplicate. The results of multiple observations are presented as the mean ± SEM or as representative results from more than three different batches of cells unless otherwise stated. Statistical significance was assessed by the Student's t-test; values were considered significant at *p < 0.05 and **p < 0.01.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information

TDAG8 mRNA is by far the most abundant in proton-sensing GPCR mRNAs

We examined the expression levels of proton-sensing GPCRs in primary mouse microglia, primary mouse astrocytes, and the mouse microglia cell line, MG6. As shown in Fig. 1a, TDAG8 mRNA expression is by far the most abundant among proton-sensing GPCRs in primary microglia and MG cells. TDAG8 mRNA expression was also observed in primary astrocytes but to a significantly lesser degree than that in microglia. Although we cannot completely separate microglia and astrocytes, we used at least more than 95% pure microglia in this study. Therefore, we can exclude the possibility that TDAG8 mRNA expression in the microglia used in this study reflects the mRNA in the contaminated astrocytes, although TDAG8 mRNA expression in astrocytes used here may represent the mRNA in the contaminated microglia. In other proton-sensing GPCRs, a slight expression of GPR4 was detected in astrocytes; however, OGR1 and G2A mRNA expression was at an undetectable or very low level.

image

Figure 1. Expression analysis of OGR1 family G-protein-coupled receptors (GPCRs). (a) RNAs were prepared from mouse glial cells; microglia of wild-type mouse (WT) and TDAG8-deficient mouse (TDAG8Tp/Tp), astrocytes of wild-type mouse, and MG6 cells. Expression of mRNAs for OGR1, TDAG8, GPR4, and G2A were analyzed by RT-qPCR as described in 'Materials and methods'. #The expression of TDAG8 mRNA was undetectable. (b) Microglia from wild-type mouse were stimulated with 1 μg/mL lipopolysaccharide (LPS) in HEPES-buffered α-minimum essential medium (MEM) (pH 7.6) at 37°C for 6 h. The mRNA expression of proton-sensing GPCRs was analyzed by RT-qPCR.

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We further examined whether the mRNA expression of proton-sensing GPCRs is altered by LPS, an agonist for TLR4, which has been shown to regulate microglia activities (Delgado 2002; Nam et al. 2008; Jung et al. 2010; Park et al. 2011; Wang et al. 2011). As shown in Fig. 1b, LPS treatment did not appreciably change the mRNA expression of proton-sensing GPCRs.

Extracellular acidic pH inhibits LPS-induced IL-1β production, which is partly dependent on TDAG8 in microglia

We first examined whether extracellular acidification regulates basal activities of production of IL-1β (Figure S1a) and proIL-1β, a precursor protein of IL-1β, (Figure S1b) in microglia. However, we failed to detect the basal activities of IL-1β and proIL-1β production at pH employed. On the other hand, LPS clearly stimulated these activities and acidic pH 6.8 inhibited the LPS-induced actions.

We therefore examined the acidic pH effects in the presence of LPS (Fig. 2). Extracellular acidic pH from 7.6 to 7.0~6.4 clearly reduced the LPS-induced IL-1β production; 50% inhibition was observed at around pH 7.0 (Fig. 2a). The role of TDAG8 was examined by using microglia derived from TDAG8-deficient mouse. The IL-1β production at pH 7.6 was significantly higher in TDAG8-deficient microglia than in control microglia derived from wild-type mice, and the inhibitory activities by acidic pH of 7.0 and 6.8 were reversed or markedly attenuated by TDAG8 deficiency (Fig. 2a). At more severe acidic pH of 6.4, however, TDAG8 deficiency did not affect the inhibitory actions (Fig. 2a).

image

Figure 2. Effects of extracellular acidification on the lipopolysaccharide (LPS)-induced extracellular production of interleukin-1β (IL-1β) and cell viability in mouse glial cells. (a) Microglia from wild-type mouse (WT, open circle) and TDAG8-deficient mouse (TDAG8Tp/Tp, closed circle) were incubated at 37°C in HEPES-buffered α-minimum essential medium (MEM) with indicated pH containing 1 μg/mL LPS for 24 h. The IL-1β content was measured in the extracellular medium by an ELISA method, as described under 'Materials and methods', and is expressed as pg/mg protein of the adherent cells. The asterisk indicates that the effect of TDAG8 deficiency was significant in cytokine contents (**p < 0.01). (b) Effect of acidification on the viability of microglia. The serum-starved microglia were incubated at 37°C in the indicated pH medium for 24 h. Cell viability was assessed by the MTT reduction assay. The viability was expressed as a percentage of that at pH 7.6 in WT microglia (WT) and TDAG8-deficient microglia (TDAG8Tp/Tp). The viability at pH 7.6 was hardly affected by TDAG8 deficiency. (c) Astrocytes from wild-type mouse (WT, open circle) and TDAG8-deficient mouse (TDAG8Tp/Tp, closed circle), were incubated in the indicated pH medium with 1 μg/mL LPS for 24 h. (d) MG6 cells (open triangle) were incubated with 30 ng/mL LPS for 8 h under the indicated extracellular pH.

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It is unlikely that the inhibitory effect of acidic pH on the cytokine production is because of the change in the viability of the cells. As assessed by an MTT reduction assay (Fig. 2b), there was no significant influence on the cell viability in normal and TDAG8-deficient microglia when the cells were incubated with the test medium at pH 7.6–7.0 for 24 h. Even at pH 6.8–6.4, when present, there was only a small effect (at most, 15% reduction from the viability at pH 7.6), which was again TDAG8 independent.

LPS-induced IL-1β production at pH 7.6 was significantly less in the astrocytes used here (Fig. 2c) than in microglia (Fig. 2a). On the other hand, a comparable IL-1β-producing activity in response to LPS at pH 7.6 and similar pH sensitivity were observed in MG6, a microglia cell line (Fig. 2d). These results suggest that the pH-sensitive IL-1β production observed in Fig. 1a represents the activity of microglia but not the activity of potentially contaminated astrocytes. Henceforth, we characterized the acidic pH effects in primary microglia, unless otherwise specified.

LPS-induced expression of proIL-1β and IL-1β mRNA is also inhibited by extracellular acidification in a manner partly dependent on TDAG8 in microglia

IL-1β is synthesized by caspase 1-catalyzed cleavage of proIL-1β. To investigate the process(es) affected by extracellular acidification, we measured the acidic pH effect on the expression of proIL-1β. Similar to IL-1β production, acidic pH inhibited proIL-1β expression in a manner partly dependent on TDAG8 (Fig. 3a). Thus, acidic pH inhibited the proIL-1β expression, which was markedly attenuated by TDAG8 deficiency at pH 7.0 and 6.8; however, the inhibitory effect at severe pH of 6.4 was not affected by TDAG8 deficiency (Fig. 3a).

image

Figure 3. Inhibitory effects of acidic pH on the lipopolysaccharide (LPS)-induced cytoplasmic precursor of IL-1β (proIL-1β) and interleukin-1β (IL-1β) mRNA expression in microglia. (a) Microglia from wild-type mouse (WT, open circle) and TDAG8-deficient mouse (TDAG8Tp/Tp, closed circle) were incubated at 37°C in HEPES-buffered α-minimum essential medium (MEM) with the indicated pH containing 1 μg/mL LPS for 24 h. The proIL-1β content was measured in cell lysate by the western blotting as described under 'Materials and methods'. Gel images are representative (upper panel). The results were also expressed as percentages of each basal value at pH 7.6 (lower panel). The asterisk indicates statistically significant difference in each value between TDAG8-deficient and normal cells (**p < 0.01). (b) Microglia from wild-type mouse (WT) and TDAG8-deficient mouse (TDAG8Tp/Tp) were incubated in the medium (pH 7.6 or 6.8) with or without 1 μg/mL LPS for 6 h. The IL-1β mRNA was analyzed by the RT-qPCR. The asterisk indicates statistically significant difference from the value at pH 7.6 (**p < 0.01). #The expression of IL-1β mRNA was undetectable in wild-type microglia.

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The acidic pH effect was also observed for IL-1β mRNA expression. Thus, the LPS-induced IL-1β mRNA expression was significantly inhibited by acidic pH of 6.8 in control microglia, and its effect was not observed in TDAG8-deficient microglia (Fig. 3b). Similar to IL-1β production and proIL-1β expression, basal IL-1β mRNA expression was not detected in control microglia, although a slight but significant basal activity was detected in TDAG8-deficient microglia. It is noteworthy that, similar to IL-1β production, the LPS-induced mRNA expression at pH 7.6 was significantly higher in TDAG8-deficient microglia than in control microglia (Fig. 3b), suggesting that TDAG8 is activated at even pH 7.6, where 25 nmol/L protons exist in the extracellular medium.

Acidic pH inhibited TDAG8 mRNA expression in the presence of LPS but not in its absence (Fig. 4a). On the other hand, LPS inhibited its own receptor TLR4 mRNA expression; however, acidic pH did not appreciably affect its expression regardless of the presence of LPS (Fig. 4b). These results may exclude the possibility that acidic pH-induced inhibition of LPS-induced IL-1β expression is owing to the increased expression TDAG8 or decreased expression of TLR4. Taking together the results shown in Figs 2-4, extracellular protons inhibit IL-1β gene transcription and, thereby, suppress the proIL-1β expression and IL-1β production, at least partly, through TDAG8.

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Figure 4. Effects of lipopolysaccharide (LPS) on the mRNA expression of TDAG8 and toll-like receptor (TLR)4 in wild-type microglia (WT) and TDAG8-deficient microglia (TDAG8Tp/Tp). The cells were incubated at 37°C in HEPES-buffered α-minimum essential medium (MEM) (pH 7.6 or 6.8) with or without 1 μg/mL LPS for 6 h to measure the expression of TDAG8 (a) and TLR4 (b) mRNAs. The asterisk indicates statistically significant difference from the value at pH7.6 (**p < 0.01). #The expression of TDAG8 mRNA was undetectable in microglia of TDAG8-deficient mouse.

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A minor role of TDAG8 in the inhibitory pH effects on poly(I:C), a TLR3 agonist, -induced IL-1β production and proIL-1β expression

We examined whether TDAG8-dependent inhibition of IL-1β production is observed when the cells are stimulated by the TLR agonist other than TLR4. Poly (I:C), a TLR3 agonist, stimulated IL-1β production (Fig. 5a) and proIL-1β expression (Fig. 5b), and acidic pH inhibited these activities. However, the involvement of TDAG8 seems to be minimum, in the pH-induced actions, as evidenced by only a slight recovery, if any, by TDAG8 deficiency.

image

Figure 5. No appreciable recovery by TDAG8 deficiency of the acidic pH-induced inhibitory effect on the interleukin-1β (IL-1β) response to poly(I-C). (a) Microglia from wild-type mouse (WT, open circle) and TDAG8-deficient mouse (TDAG8Tp/Tp, closed circle) were incubated at 37°C in HEPES-buffered α-minimum essential medium (MEM) with the indicated pH containing 100 μg/mL poly(I-C) for 24 h to measure IL-1β production. (b) Microglia from wild-type mouse (WT) and TDAG8-deficient mouse (TDAG8Tp/Tp) were incubated in the medium (pH 7.6 or 6.8) with 1 μg/mL lipopolysaccharide (LPS) or 100 μg/mL poly(I-C) for 24 h to measure cytoplasmic precursor of IL-1β (proIL-1β) content. Gel images are representative (upper panels). The results were also expressed as percentages of each basal value of WT cells at pH 7.6 (lower panels). The asterisks indicate statistically significant difference from the value at pH7.6 (**p < 0.01).

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Involvement of the cAMP/PKA pathway in extracellular acidic pH-induced inhibition of IL-1β production

The results described above suggest that TDAG8 may mediate the proton-induced inhibitory role on LPS-induced cytokine production. However, the signaling mechanism remains unknown. TDAG8 has been shown to be coupled with multisignaling pathways, including the Gs protein/cAMP pathway and the G12/13 protein/Rho pathway (Wang et al. 2004; Ishii et al. 2005; Radu et al. 2005). To examine whether cAMP accumulation occurs by acidification in microglia, we tried to measure the cellular cAMP content under the same experimental condition as that for cytokine production. Unfortunately, however, the cAMP content was too low to be measured by an available cAMP detection kit. We, therefore, measured the cAMP content in the presence of a phosphodiesterase inhibitor, IBMX. As shown in Fig. 6, acidification significantly induced cAMP accumulation in normal microglia, and the cAMP response was almost completely lost in TDAG8-deficient cells. Thus, acid pH stimulates the adenylyl cyclase/cAMP pathway through TDAG8.

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Figure 6. Accumulation of cAMP in response to extracellular acidification and its inhibition by TDAG8 deficiency. Microglia from wild-type mouse (WT) and TDAG8-deficient mouse (TDAG8Tp/Tp) were incubated at 37°C in HEPES-buffered medium with the indicated pH containing 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) for 30 min. Cyclic AMP accumulation is expressed as ng/mg protein of the adherent cells. The asterisk indicates statistically significant difference in each value between TDAG8-deficient and normal cells (**p < 0.01).

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We next examined the role of the cAMP-signaling pathway in the acidic pH-induced action. As shown in Fig. 7a, N6-Ben-cAMP, a PKA-specific cAMP derivative, but not 8CPT-2Me-cAMP, an Epac-specific cAMP derivative, effectively attenuated the LPS-induced IL-1β production at pH7.6. The inhibitory role of cAMP on IL-1β production was further confirmed by forskolin inhibition of IL-1β production (Fig. 7b). As expected, the inhibitory effects of N6-Ben-cAMP and forskolin were not suppressive to TDAG8 deficiency, whereas the effect of acidic pH of 6.8 was almost completely reversed by knockout of TDAG8 (Fig. 7b). To confirm the role of PKA, we performed experiments using H89 (Fig. 7c) and myr-PKI (Fig. 7d), specific PKA inhibitors. Consistent with the results of Fig. 3b, acidification to pH 6.8 resulted in roughly 50% inhibition of IL-1β mRNA expression at pH 7.6 (Fig. 7c and d). In the presence of these PKA inhibitors, however, the inhibitory pH effects were completely reversed (Fig. 7c and d).

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Figure 7. Effects of cAMP derivatives and cAMP-increasing agents on lipopolysaccharide (LPS)-induced interleukin-1β (IL-1β) production. (a) Wild-type microglia were incubated at 37°C in HEPES-buffered α-minimum essential medium (MEM) (pH 7.6) with 1 μg/mL LPS in the presence or absence of the indicated concentrations of N6-Ben-cAMP and 8CPT-2Me-cAMP to measure IL-1β production. (b) Microglia from wild-type mouse (WT) and TDAG8-deficient mouse (TDAG8Tp/Tp) were similarly incubated with 1 μg/mL LPS in the presence of 100 μmol/L cAMP derivatives and 10 μmol/L Forskolin to measure IL-1β production. The asterisk indicates statistically significant difference from the basal value (**p < 0.01). (c, d) PKA inhibitors reversed acidic pH-induced inhibition of IL-1β mRNA expression. Wild-type microglial cells were incubated in the medium (pH 7.6 or 6.8) with 1 μg/mL LPS in the presence of 1 μmol/L H89 (or 0.1% DMSO as a vehicle) in (c) or 10 μmol/L myr-PKI (or PBS as a vehicle) in (d) for 8 h to measure IL-1β mRNA. The RT-qPCR was performed with three different RNA concentrations in each sample, and result is a representative from two different batches. The asterisks indicate statistically significant difference from the value at pH7.6 (**p < 0.01).

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TDAG8/PKA signaling inhibits LPS-induced ERK and JNK and, thereby, inhibits IL-1β production

To elucidate the signaling pathways involved in TDAG8-dependent inhibition of TLR4-mediated actions, we examined the effects of inhibitors for NF-κB and MAPK signaling molecules on LPS-induced proIL-1β expression (Fig. 8). U0126, an inhibitor of p44/42 ERK kinase, SP600125, an inhibitor of JNK, and BAY-11-7082, an inhibitor of IκB kinase (IKK), all significantly inhibited the LPS-induced action (Fig. 8a), suggesting the involvement of ERK, JNK, and NF-κB signaling pathways in the LPS-induced action. Unexpectedly, however, SB203580, an inhibitor of p38 MAPK, failed to inhibit the LPS action at the concentration (up to 3 μM) employed in the experiments. Increase in its concentration inhibited the β-actin expression, suggesting a non-specific action of the inhibitor.

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Figure 8. MAPK signaling pathway including extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK) in addition to nuclear factor-κB (NF-κB) signaling pathway are important for lipopolysaccharide (LPS)-induced cytoplasmic precursor of IL-1β (proIL-1β) expression. (a) Microglial cells were incubated at 37°C in HEPES-buffered α-minimum essential medium (MEM) (pH 7.6) with 1 μg/mL LPS in the presence of 10–30 μmol/L U0126, 3–30 μmol/L SP600125, or 3–10 μmol/L BAY-11-7082 for 24 h to measure proIL-1β content. Gel images are representative (upper panels). The results with 30 μmol/L U0126, 30 μmol/L SP600125, and 10 μmol/L BAY-11-7082 were also expressed as percentages of control values (lower panel). The asterisk indicates statistically significant difference from control (*p < 0.05, **p < 0.01). (b) SB203580 failed to inhibit the LPS action. The microglial cells were treated with 1–3 μmol/L SB203580. Other experimental procedures are the same as those described in (a). Gel images are representative results (left panel). Effect of SB203580 at 3 μmol/L on the LPS action was quantified by densitometry, and the results were expressed as percentages of control value (right panel).

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We next measured which signaling molecules are practically regulated by LPS and/or acidification (Fig. 9). Phosphorylation of IκBα and NF-κB (p65), which may reflect NF-κB signaling activity, was appreciably affected by LPS but the LPS-induced action was hardly affected by extracellular acidification (Fig. 9a). The lack of acidic pH effect was also observed in LPS-induced translocation of NF-κB (p65) from cytosol to nuclear; LPS remarkably stimulated the nuclear translocation of NF-κB but the acidic pH 6.8 failed to modulate the LPS-induced translocation (Figure S2a). We also tried to evaluate the effect on the NF-κB binding to the transcription factor-specific binding sites of DNA using nuclear extract of microglia; however, it was technically difficult to obtain the amount of nuclear extract required for measurement of the activity. We therefore used mouse microglia cell line MG6 for this purpose. As shown in Figure S2b, the LPS-stimulated NF-κB binding activity was not appreciably affected by acidic pH 6.8, either. These results suggest that NF-κB signaling may be important for LPS-induced IL-1β expression but may not be the target of acidic pH-induced actions. Similarly, p38 MAPK was phosphorylated by LPS but the LPS action was hardly affected by extracellular acidification (Fig. 9a). Thus, p38 MAPK does not seem to be involved in the acidic pH-induced inhibitory actions on the IL-1β response, either.

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Figure 9. The acidic pH inhibited the lipopolysaccharide (LPS)-induced interleukin-1β (IL-1β) production by suppressing extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK) activities via the PKA-dependent pathway in microglia. (a) Time course of nuclear factor-kappa B (NF-κB) and MAPK signaling molecules activation by LPS under the indicated pH. Normal microglia were stimulated by 1 μg/mL LPS in HEPES-buffered α-minimum essential medium (MEM) at 37°C for the indicated time, and then analyzed by the western blotting for detection of phosphorylated IκBα, NF-κB (p65), ERK (p44 and p42), JNK (p54 and p46), and p38MAPK as described under 'Materials and methods'. Gel images are representative from separate experiments. (b) Microglia were pre-incubated in the medium at pH 7.6 with or without 10 μmol/L myr-PKI for 30 min, and then treated with or without 1 μg/mL LPS at pH 7.6 or 6.8 for 30 min to measure phosphorylation of ERK (p44 and p42) and JNK (p54 and p46). Gel images are representative results (upper panels). Phosphorylation activities were quantified by densitometry and were expressed as percentages of control value at pH 7.6 in the presence of LPS (lower panels). The effect of acidic pH was significant (**p < 0.01) in control cells.

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On the other hand, ERK (either p44 or p42) and JNK (either p54 or p46) were phosphorylated by LPS and their phosphorylation was significantly inhibited by extracellular acidic pH 6.8 (Fig. 9b). The PKA inhibitor reversed the inhibitory effects of acidic pH on LPS-induced phosphorylation of these enzymes (Fig. 9b). Moreover, the inhibitory effects on ERK and JNK phosphorylation were reversed by TDAG8 deficiency (Fig. 10). These results, together with the results of the specific enzyme inhibitors (Fig. 8a), suggest that both ERK and JNK signaling pathways are involved in acidic pH-induced inhibition of IL-1β responses to LPS.

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Figure 10. Acidic pH inhibits the lipopolysaccharide (LPS)-induced phosphorylation of extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK) in a TDAG8-dependent manner. Microglia from wild-type mouse (WT) and TDAG8-deficient mouse (TDAG8Tp/Tp) were stimulated with or without 1 μg/mL LPS in HEPES-buffered α-minimum essential medium (MEM) (pH 7.6 or 6.8) at 37°C for 30 min to measure phosphorylation of ERK (p44 and p42) and JNK (p54 and p46). Gel images are representative results in duplicate (upper panels). Phosphorylation activities were quantified by densitometry and were expressed as percentages of control value at pH 7.6 in the presence of LPS (lower panels). The effect of acidic pH was significant (*p < 0.05) in wild-type cells.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information

Hypoxia and acidosis are hallmarks of acute brain damage, such as ischemia and traumatic injury. As for hypoxia, the role of HIF-1 as a mediator of hypoxia-induced actions has been well characterized. Thus, hypoxia induces increase in HIF-1α protein expression, which was accompanied by the expression of VEGF and other HIF-1 target genes in primary microglia (Weinstein et al. 2010). Acidosis occurs under not only acute brain damage but also chronic neurodegenerative diseases, such as Alzheimer's and Parkinson's (Wang and Xu 2011). As for the molecular targets for acidosis or extracellular acidification, proton-sensing channels, such as acid-sensing ion channels and transient receptor potential vanilloid type 1 (TRPV1), have been suggested in acidosis-induced neuron cell death (Wang and Xu 2011); however, the mechanisms involved in the extracellular acidic effects on microglial functions have been poorly understood. In this study, we examined the effects of extracellular acidification on the LPS-induced IL-1β production in mouse microglia and showed that acidic pH inhibited the TLR-mediated IL-1β production through TDAG8, a proton-sensing GPCR. This is the first indication, to our knowledge, of the extracellular or cell surface proton-sensing molecule to regulate microglial functions. We also found that proton/TDAG8 inhibits IL-1β production, through the cAMP/PKA pathway, by inhibiting ERK and JNK signaling pathways (Fig. 11).

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Figure 11. A postulated mechanism for inhibition of toll-like receptor (TLR)4-mediated interleukin-1β (IL-1β) production by extracellular acidification in mouse microglia. The acidic pH stimulates the TDAG8/cAMP/PKA pathway. The activation of PKA may inhibit signaling pathways involving extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK) and, thereby, inhibit the lipopolysaccharide (LPS)/TLR4-mediated transcriptional regulation of IL-1β gene and subsequent cytoplasmic precursor of IL-1β (proIL-1β) and IL-1β production. Nuclear factor-kappa B (NF-κB) signal pathways may not be appreciably influenced by the acidic pH environment of the cells. See text for more detail.

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The following observations support our conclusion. First, in microglia, TDAG8 is the most significant proton-sensing GPCRs. The TDAG8-deficient microglia showed the attenuation of acidic pH-induced protein production of proIL-1β and IL-1β, which was associated with the attenuation of IL-1β mRNA expression, suggesting the transcriptional regulation of IL-1β by proton/TDAG8. Second, extracellular acidification induced cAMP accumulation in a manner dependent on TDAG8 expression. Third, the PKA-specific cAMP derivative, but not the Epac-specific cAMP derivative, inhibited the IL-1β production. The role of PKA was confirmed by the finding that PKA-specific inhibitors attenuated the acidic pH-induced inhibition of IL-1β mRNA expression. Thus, we clearly showed the role of TDAG8/cAMP/PKA in the acidic pH-induced inhibition of IL-1β mRNA and protein production. The inhibitory role of TDAG8-mediated cAMP/PKA in inflammatory cytokine production is quite consistent with that reported in recent studies. Thus, acidic pH-induced inhibition of tumor necrosis factor-α (TNF-α) and IL-6 production by LPS was attenuated by TDAG8 deficiency in mouse macrophages (Mogi et al. 2009), and a TDAG8 agonist, BTB09089, suppressed anti-CD3-stimulated IL-2 production in mouse splenocytes and LPS-induced inflammatory cytokine production in mouse macrophages in a TDAG8-dependent manner (Onozawa et al. 2012). The role of the cAMP-signaling pathway in acidic pH was also observed in eosinophils and neutrophils; eosinophil viability is increased by acidic pH through a TDAG8/cAMP-signaling pathway (Kottyan et al. 2009), and the production of superoxide anion in neutrophil is inhibited by the acidic pH/cAMP/PKA pathway, possibly through TDAG8 (Murata et al. 2009).

Recent studies have proposed a few mechanisms by which PKA exerts an inhibitory effect on the expression of inflammatory cytokines at transcription levels in macrophages and dendritic cells. Koga et al. have reported that prostaglandin E2 and cAMP-increasing agents increase c-Fos protein levels in a collaboration with LPS-induced activation of IκB kinase (IKK) and thereby inhibit the p65, a component of NF-κB, -mediated transcriptional activity of TNF-α by a physical interaction of p65 with the c-Fos protein in dendritic cells and macrophage cell lines (Koga et al. 2009). Wall et al. (2009) have shown a specific role for PKA/A kinase-anchoring protein 95 in the suppression of TNF-α gene expression, which involved the phosphorylation of p105, a potential protein with an IκB function, by PKA at a site adjacent to the region targeted by IKK. The phosphorylation of p105 by PKA seems to inhibit the IKK-mediated phosphorylation and subsequent degradation of p105 in macrophages. In this study, we showed that NF-κB signaling pathways do not seem to be the sites for the proton/TDAG8/PKA-mediated inhibitory actions on IL-1β expression in microglia. Our results suggest that ERK and JNK signaling pathways may be involved in the TDAG8/PKA-induced inhibition of IL-1β mRNA expression and subsequent responses, although the upstream and downstream signaling pathways of ERK and JNK remain unknown. The critical role of JNK and ERK in the LPS/TLR-mediated proinflammatory cytokine production has been reported in microglia and its cell lines (Delgado 2002; Nam et al. 2008; Jung et al. 2010; Park et al. 2011; Wang et al. 2011).

TDAG8-dependent acidic pH effects are observed at around 6.8. This pH level is as low as that under neurodegenerative conditions. For example, in ischemic brain, pH falls to 6.1~6.8 (Siemkowicz and Hansen 1981; Smith et al. 1986). Similarly, brain pH is reduced from ~ 7.4 to 6.8 during seizure, and cerebral acidosis (~ pH 6.6) is supposed to occur in Alzheimer's disease (Wang and Xu 2011). At pH less than 6.8, however, acidic pH further inhibited IL-1β production in this study; this severe acidic pH effect was insensitive to TDAG8. It is unlikely that the TDAG8-insensitive inhibition of IL-1β production is owing to the non-specific loss of cell viability by severe acidification because cell viability measured by MTT was lost by, at most, 15% at pH 6.4 compared with that at pH 7.6.

In microglia, the expression of proton-sensing GPCRs other than TDAG8 is very low or undetectable even treatment with LPS. The severe acidic pH-induced inhibition of cytokine production may not be mediated by cAMP because cAMP accumulation induced by even a severe acidic pH was completely inhibited by TDAG8 deficiency. TRPV1, but not acid-sensing ion channel, seems to be expressed in microglia. However, Ca2+ mobilization, which is usually induced directly or indirectly by the activation of the pH-sensing channel, was not observed in response to extracellular acidic pH in microglia (Khoo et al. 2001). Extracellular acidification has been shown to be associated with acute intracellular acidification to a lower level than extracellular pH level in microglia (Morihata et al. 2000, 2008). Some of the previous results, i.e., extracellular acidification-induced inhibition of the motility (Faff and Nolte 2000) and increase in the voltage-gate H+ channel activity in association with swelling of the microglia (Morihata et al. 2000), have been suggested to be explained by intracellular pH decreases. Thus, we tentatively speculate that intracellular acidification may cause the inhibition of TLR4 signaling pathways under such a severe extracellular pH of less than 6.8. Further experiments are required to clarify the mechanism underlying the TDAG8-independent inhibitory actions of acidic pH.

Brief periods of severe hypoxia resulted in phosphorylation of p38MAPK, and pharmacologic inhibition of p38MAPK significantly attenuated the hypoxia-induced increases in nitric oxide, inducible nitric oxide synthase (iNOS), and TNF-α in microglia (Park et al. 2002). Hypoxia has also been shown to induce expression of CXCR4, a chemokine receptor for microglia, (Wang et al. 2008) and potentiate LPS-induced increases in both iNOS and TNF-α (Guo and Bhat 2006). Thus, hypoxia seems to potentiate microglial activation and exert detrimental actions on the nervous system. On the other hand, acidic pH inhibited migration of microglia (Faff and Nolte 2000), store-operated Ca2+ influx (Khoo et al. 2001), and pro-inflammatory cytokine production (this study) in microglia; these inhibitory actions in microglia seem to be neuroprotective and anti-inflammatory. Thus, the elucidation of the molecular targets for acidic pH actions in microglia may provide therapeutic targets of neurodegenerative disorders, including ischemia and Alzheimer's disease. TDAG8 may be a potential target of these disorders.

In conclusion, extracellular acidification inhibits IL-1β production, in association with the inhibition of expression of proIL-1β and IL-1β mRNA, in microglia. The inhibitory actions induced by acidic pH 7.6–6.8 are mediated by TDAG8/cAMP/PKA-induced inhibition of ERK and JNK signaling pathways, whereas those at severe pH of 6.4 are independent of TDAG8, the mechanism of which is currently unknown.

Acknowledgments and conflict of interest disclosure

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information

We are grateful to Drs Kyoji Horie and Junji Takeda of Osaka University, and Dr Takao Shimizu of University of Tokyo for providing us TDAG8-deficient mice. We also appreciate Ms. Mutsumi Takano for maintenance of mice and technical assistance. This work was funded by grants from a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (21390016 to F.O. and 24500435 to K.S.). This work was also supported by Global COE Program from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to Y.J.) and the Japan–Korea basic scientific cooperation program for FY 2010 (to F.O. and DS.I.).

All experiments were conducted in compliance with the ARRIVE guidelines. The authors have no conflict of interest to declare.

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  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information
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jnc12661-sup-0001-FigS1-S2.pdfapplication/PDF1387K

Figure S1 Effects of extracellular acidification on the LPS-induced production of IL-1β in microglia.

Figure S2 LPS-induced NF-κB (p65) nuclear translocation and binding to DNA do not seem to be affected by acidic pH.

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