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. 2015 Aug 17;14(2):180–191. doi: 10.1038/cmi.2015.59

Extracellular calcium elicits feedforward regulation of the Toll-like receptor-triggered innate immune response

Songqing Tang 1,2,4, Taoyong Chen 2,4, Mingjin Yang 2,3, Lei Wang 3, Zhou Yu 1, Bin Xie 1,2, Cheng Qian 2, Sheng Xu 2, Nan Li 2, Xuetao Cao 1,2,3, Jianli Wang 1
PMCID: PMC5301151  PMID: 26277896

Abstract

Despite the expanding knowledge on feedback regulation of Toll-like receptor (TLR) signaling, the feedforward regulation of TLR signaling for the proper innate response to invading microbes is not fully understood. Here, we report that extracellular calcium can coordinate the activation of the small GTPases Ras and Ras-proximate-1 (Rap1) upon TLR stimulation which favors activation of macrophages through a feedforward mechanism. We show that different doses of TLR agonists can trigger different levels of cytokine production, which can be potentiated by extracellular calcium but are impaired by the chelating reagent ethylene glycol tetraacetic acid (EGTA) or by knockdown of stromal interaction molecule 1 (STIM1). Upon TLR engagement, GTP-bound Ras levels are increased and GTP-bound Rap1 is decreased, which can be reversed by EGTA-mediated removal of extracellular calcium. Furthermore, we demonstrate that Rap1 knockdown rescues the inhibitory effects of EGTA on the TLR-triggered innate response. Examination of the TLR signaling pathway reveals that extracellular calcium may regulate the TLR response via feedforward activation of the extracellular signal-regulated kinase signaling pathway. Our data suggest that an influx of extracellular calcium, mediated by STIM1-operated calcium channels, may transmit the information about the intensity of extracellular TLR stimuli to initiate innate responses at an appropriate level. Our study may provide mechanistic insight into the feedforward regulation of the TLR-triggered innate immune response.

Keywords: calcium influx, innate immunity, Rap1, Ras, STIM1, Toll-like receptor

Introduction

Innate immunity guards against invading microbial pathogens at the frontier of host defense. Toll-like receptors (TLRs) are critical pattern recognition receptors expressed by innate immune cells, such as macrophages and granulocytes, that can discriminate the type and different severity of invading pathogens to instruct a host to avoid insufficient or excessive responses.1,2,3 After recognizing the pathogen-associated molecular patterns (PAMP), TLRs initiate signaling pathways by recruiting various combinations of four Toll-interleukin 1 receptor domain-containing adapter molecules, including MyD88, TIRAP (Mal), TRIF, and TRAM, to activate the key transcription factors NF-κB, AP1, and interferon-regulatory factors (IRF) 3/7, leading to the induction of inflammatory cytokines, chemokines, and type I interferons.1,2,3 These linear signaling pathways after TLR activation have been extensively investigated. However, closely related mechanisms may be important for instructing proper TLR responses because TLRs have been implicated in inflammatory and autoimmune diseases. Previously, feedback regulation of the TLR response has been reported through the induction of intracellular ubiquitin-editing protein A20, I kappa B alpha (IkB a), and suppressor of cytokine signaling (SOCSs); that negatively regulate TLR signaling.1,2 However, feedforward regulation of TLR signalling through modules adjacent to the linear machinery and important for TLR activation has not been clearly understood.

Accompanying the activation of TLRs, the extracellular signal-regulated kinases (ERK1/2) pathway is activated sensitively and rapidly in innate immune cells.4 The ERK1/2 pathway may be a convergence point or an indicator for TLR signaling to guide the accurate production of inflammatory cytokines and chemokines, which needs to be investigated. The activation of ERK1/2 in immune cells is affected mainly by small GTPases, especially by Ras, a classical activator of the ERK1/2 pathway.4,5,6 One of our previous studies showed that Ras was activated by CpG ODN in macrophages.7 Whether the Ras/ERK pathway is involved in the feedforward regulation of TLR signaling needs to be further investigated. Rap, containing a homologous functional domain with Ras,8 is another critical small GTPase. The effects of Rap on ERK1/2 activation remain controversial.8 A series of studies have indicated that Rap could inhibit the activation of ERK1/2 and mediate negative regulation of T-cell function.9,10,11,12 However, other reports have shown that Rap could promote the activation of ERK1/2 and mediate the positive regulation of functions of T cells and other cells.13,14,15,16,17 Still other studies have indicated that Rap had no effect on the activation of ERK1/2.18,19 These inconsistent results about the effects of Rap on ERK1/2 signaling may be due to the different experimental methods and different cell types used. However, the roles of Rap and the relationship of Ras and Rap in ERK1/2 activation in macrophages during the TLR innate response remain unclear.

Stromal interaction molecule 1 (STIM1), located on the endoplasmic reticulum (ER) membrane, is a sensor of ER-resident Ca2+.20,21 After ligation with TLR ligands, inositol-1,4,5-trisphosphate and diacylglycerol are produced via breakdown of phosphatidylinositol-4,5-bisphosphate by phospholipase C coupled with TLRs.1,2,3 Inositol-1,4,5-trisphosphate then diffuses to the ER and binds to receptors situated on the ER membrane to release Ca2+ from the ER lumen, leading to a reduction of ER-resident Ca2+.20,21 This reduction of ER-resident Ca2+ promotes STIM1 oligomerization and relocalization on the ER membrane near the plasma membrane of the macrophage polarization area. There, STIM1 binds directly to the calcium release-activated calcium channel protein 1 (ORAI1) located on the plasma membrane, opening Ca2+-permeable ORAI1 ion channels and initiating Ca2+ entry.20,21 This entrance of Ca2+ could affect the activation of ERK1/2,22 indicating that Ca2+ may play an important role in regulating TLR-triggered activation of ERK1/2.

TLRs can quickly trigger an appropriate innate response according to the strength of TLR stimuli, which indicates that TLR signaling is likely modulated by feedforward regulation. However, the mechanism for feedforward regulation of the TLR-triggered innate response remains unclear. Here, we report that extracellular Ca2+ coordinates the activation of the small GTPases Ras and Ras-proximate-1 (Rap1) and consequently regulates the activation of ERK1/2 to initiate appropriate innate response, which may provide mechanistic insight into the feedforward regulation of the TLR-triggered innate response.

Materials and methods

Mice, cells, and reagents

C57BL/6J mice were purchased from Joint Ventures Sipper BK Experimental Animals (Shanghai, China). The mice were kept and bred in pathogen-free conditions. All animal experiments were undertaken in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and with the approval of the Scientific Investigation Board of Second Military Medical University, Shanghai. Thioglycolate-elicited mouse peritoneal macrophages were prepared as described12,23 and cultured in RPMI-1640 medium with 10% (vol/vol) FCS at a density of 2 × 105 cells per well for cytokine assays and 1 × 106 cells per well for immunoblot analysis. Human peripheral blood monocyte-derived macrophages (MDM) were prepared and cultured as described previously.12 Lipopolysaccharide (LPS) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Poly(inosine-cytidine) (Poly (I:C)) and CpG oligodeoxynucleotides (CpG ODN) were purchased from Invivogen (San Diego, CA, USA). Antibodies (Abs) specific to AKT, phosphorylated AKT (Ser473), ERK1/2, and phosphorylated ERK1/2 (Thr202/Tyr204) were from Cell Signaling Technology (Beverly, MA, USA). Ab specific to β-actin were from Sigma-Aldrich. The HRP-coupled secondary Ab was from Santa Cruz Biotechnology (Dallas, TX, USA).

RNA interference

Four small interfering RNA (siRNA) duplexes were synthesized (GenePharma Co., Shanghai, China) and selected for the knockdown of Stim1. For transient knockdown, 21-nucleotide sequences of siRNA were synthesized as follows: 5′-GCUGCUGGUUUGCCUAUAUTT-3′ (sense) and 5′-AUAUAGGCAAACCAGCAGCTT-3′(antisense). Another set of siRNA sequences for knocking down STIM1 were reported previously.23 The sequences 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) and 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense) were used as a scrambled RNA interference control (Ctrl). The sequences used for Rap1a knockdown were reported previously.12 siRNA duplexes were transfected into macrophages using INTERFERin according to the standard protocol (Polyplus-Transfection, Illkirch, France).

Quantitative PCR

Total cellular RNA was extracted using an RNA extraction kit (Fastagen Biotech Co., Shanghai, China). The specific primers used for quantitative PCR assays were 5′-CCTCAGTATGAGGAGACCTT-3′ (sense) and 5′-TCCTGAAGGTCATGCAGACT-3′ (antisense) for STIM1, 5′-CCCTCACACTCAGATCATCTTCT-3′ (sense) and 5′-GCTACGACGTGGGCTACAG-3′ (antisense) for TNFα, 5′-GAGTTGTGCAATGGCAATTCTG-3′ (sense) and 5′-GCAAGTGCATCATCGTTGTTCAT-3′ (antisense) for IL-6, 5′-GAAATGCCACCTTTTGACAGTG-3′ (sense) and 5′-CTGGATGCTCTCATCAGGACA-3′ (antisense) for IL-1β, 5′-TTTGCCTACCTCTCCCTCG-3′ (sense) and 5′-CGACTGCAAGATTGGAGCACT-3′ (antisense) for CCL-5, 5′-CCAAGTGCTGCCGTCATTTTC-3′ (sense) and 5′-GGCTCGCAGGGATGATTTCAA-3′ (antisense) for CXCL10, 5′-ACATCGACCCGTCCACAGTAT-3′ (sense) and 5′-CAGAGGGGTAGGCTTGTCTC-3′ for iNOS, and 5′-AGTGTGACGTT-GACATCCGT-3′ (sense) and 5′-GCAGCTCAGTAACAGTCCGC-3′ (antisense) for mouse β-actin. The primer sequences used for human cytokine mRNA quantification were as previously described.12 Quantitative PCR was performed using a LightCycler (Roche Diagnostic, Indianapolis, IN) as previously described.12,24 The data were normalized by the level of β-actin expression in each sample.

Measurement of cytokines

Enzyme-linked immunosorbent assay (ELISA) kits for mouse interleukin-6 (IL-6) and TNFα and human IL-6 were obtained from R&D Systems, Minneapolis, MN. Cytokine concentrations in culture supernatants were measured by ELISA as previously described.12,24

Western blotting assays

Total cell lysates were prepared as previously described,12 and their protein concentrations were determined by the bicinchoninic acid protein assay (Pierce, Rockford, IL, USA). Cell extracts were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes, and blotted as previously described.12

Ca2+ influx

Macrophages were labeled for 30 min at 37 °C with 10 μM Cal-520 and 0.04% Pluronic F-127 (AAT Bioquest, Sunnyvale, CA) and then washed with normal saline and resuspended in normal saline. Labeled cells were stimulated with LPS, Poly (I:C), or CpG ODN before flow cytometry. Mean fluorescence ratios were plotted after analysis with FlowJo software (TreeStar, Ashland, CA).

Ras and Rap1 activation assays

The levels of GTP-bound Ras and Rap1 were determined as described previously,12 using kits from Pierce or Millipore (Billerica, MA, USA) according to the standard protocol.

In vivo administration of TLR agonists

The indicated amounts of LPS, Poly (I:C), or CpG ODN, mixed with CaCl2 alone or with both CaCl2 and ethylene glycol tetraacetic acid (EGTA) in 200 μL endotoxin-free H2O, were injected intraperitoneally into C57BL/6J mice. Serum concentrations of IL-6 were determined by ELISA.

Statistical analysis

All the experiments were independently repeated at least three times. The results are given as the means ± S.D. or the means ± S.E.M. Comparisons between two groups were performed using Student's t-test. Statistical significance was determined as p < 0.05.

Results

Macrophages respond according to different intensities of TLR stimuli

To investigate the ability of macrophages to distinguish the intensity of TLR stimuli, we first used gradient doses of LPS, Poly (I:C), or CpG ODN to stimulate macrophages. We found that macrophages could produce graded levels of cytokines and chemokines (Figure 1 and Supplementary Figure S1), indicating that the strength of innate responses to graded intensities of TLR stimuli was properly communicated to macrophages.

Figure 1.

Figure 1

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Macrophages respond according to the intensity of TLR stimuli. Peritoneal macrophages were treated with different doses of LPS (0, 0.5, 2, 8, 32, or 128 ng mL−1) for 4.5 h, Poly (I:C) (0, 5, 10, 20, 40, or 80 μg mL−1) for 5 h, or CpG ODN (0, 1, 2, 4, 8, or 16 μg mL−1) for 6 h, and the IL-6 (a, c) and TNFα (b, d) levels were measured by quantitative PCR (a, b) and ELISA (supernatant; c, d). The data are shown as the means ± S.E.M. of three independent experiments (a, b) or the means ± S.D. of triplicate samples (c, d).

This observation raised an interesting question of how innate immune cells, including macrophages, distinguish the intensity of a stimulus, tune the activation of TLR signaling pathways, and trigger the production of inflammatory cytokines at appropriate levels. TLRs may transmit information about the intensity of a stimulus to the signaling pathway via unclear mechanisms. With high-intensity TLR stimuli, immune cells may adequately sense the stimuli. At low levels of TLR agonism, however, the immune cells may require feedforward machinery to ensure a proper innate response. The underlying mechanisms may thus require further investigations.

Extracellular calcium instructs the activation of macrophages upon TLR stimulation

We hypothesized that second messengers, such as Ca2+, diacylglycerol, cyclic adenylate monophosphate, and cyclic guanylate monophosphate, in immune cells may be involved in the fine-tuning of proper innate responses upon gradient TLR stimulation. Calcium is a rapid, sensitive, and dynamic second messenger that plays crucial roles in various signaling pathways. The difference between extracellular (1–10 mM) and intracellular (0.1–1 μM) Ca2+ concentrations is huge and Ca2+ can transmit from extracellular space into intracellular compartment through Ca2+-permeable ORAI1 ion channels mediated by STIM1.20,21 Calcium thus possesses the ability to quickly change its intracellular concentration, making it a potential messenger for instructing proper TLR responses. To test this hypothesis, we pre-treated macrophages with a calcium chelator, BAPTA (1,2-bis(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid), to remove intracellular free Ca2+, labeled them with Cal-520, and then stimulated these cells with LPS in a 2 mM Ca2+ extracellular environment and detected the Ca2+ influx by confocal microscopy. We found that LPS can trigger different intensities of Ca2+ influx very quickly in single cells (Figure 2a–2f), indicating that these single cells may capture a different number of LPS to produce different intensities of Ca2+ influx. To confirm that this Ca2+ influx derives from extracellular Ca2+, we primed macrophages with LPS, Poly (I:C), or CpG ODN, in a Ca2+-free environment and found that there was no obvious Ca2+ flux until 2 mM Ca2+ was added (Figure 2g–2l). Furthermore, we observed that LPS, Poly (I:C), or CpG ODN can induce different intensities of Ca2+ influx (Figure 2g–2i) in a dose-dependent manner and that the concentration of extracellular Ca2+ can affect this role of LPS, Poly (I:C), or CpG ODN (Figure 2j–2l). These data further suggested that the different concentrations of LPS, Poly (I:C), or CpG ODN can trigger different intensities of Ca2+ influx from extracellular.

Figure 2.

Figure 2

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The strength of Ca2+ influx was decided by the number of stimuli. Peritoneal macrophages were pre-treated with the chelator BAPTA and Cal-520, The macrophages were then stimulated with LPS in a 2 mM Ca2+ extracellular environment, and Ca2+ influx was detected by confocal microscopy (a–e), six cells were selected to compare the strength of their Ca2+ influx (f). Peritoneal macrophages pre-labeled by Cal-520 were primed with different doses of LPS (1, 10, or 100 ng mL−1), Poly (I:C) (5, 20, or 80 μg mL−1), or CpG ODN (2.5, 10, or 40 μg mL−1) for 3 min; then, 2 mM Ca2+ were added and Ca2+ influx was detected by flow cytometry (g–h). Peritoneal macrophages pre-labeled by Cal-520 were primed with 100 ng mL−1 LPS, 80 μg mL−1 Poly (I:C), or 40 μg mL−1 CpG ODN for 3 min; then, 2 mM or 0.5 mM Ca2+ were added and Ca2+ influx was detected by flow cytometry (j–l). The data are representative of three independent experiments.

To test whether extracellular Ca2+ affected the strength of macrophage response, we added different concentrations of Ca2+ to the medium and then stimulated macrophages with LPS (1 ng mL−1), Poly (I:C) (10 μg mL−1), or CpG ODN (5 μg mL−1). We found that adding Ca2+ to the medium significantly increased, in a dose-dependent manner, the cytokine and chemokine production by macrophages in response to LPS, Poly (I:C), or CpG ODN stimuli (Figure 3 and Supplementary Figure S2). We changed the concentrations of the stimuli and found that adding Ca2+ to the medium also significantly increased cytokine and chemokine production by macrophages in a dose-dependent manner (data not shown). These data suggest that the different concentrations of Ca2+, similar to the different intensities of TLR ligands, can affect the response strength of macrophages.

Figure 3.

Figure 3

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Extracellular Ca2+ potentiates TLR-triggered responses in a dose-dependent manner. Peritoneal macrophages were treated with LPS (1 ng mL−1), Poly (I:C) (10 μg mL−1), or CpG ODN (5 μg mL−1) for 6 h, and the IL-6 (a, c) and TNFα (b, d) levels were measured by quantitative PCR (a, b) and ELISA (supernatant; c, d). EGTA (2 mM) was incubated with medium for 2 h to remove the initial Ca2+ before adding Ca2+ (0, 1, 2, or 4 mM) and TLR ligands. The data are shown as the means ± S.E.M. of three independent experiments (a, b) or the means ± S.D. of triplicate samples (c, d). *p < 0.05; **p < 0.01; ***p < 0.001.

To further confirm the role of Ca2+, we used the chelator EGTA to remove Ca2+ from the medium and then analyzed the TLR response of macrophages. We found that removing Ca2+ in the medium by EGTA inhibited the TLR-triggered response in macrophages (Figure 4 and Supplementary Figure S2).

Figure 4.

Figure 4

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Removal of extracellular Ca2+ inhibits TLR-triggered responses. Peritoneal macrophages were treated with LPS (1 ng mL−1), Poly (I:C) (10 μg mL−1), or CpG ODN (5 μg mL−1) for 6 h, and the IL-6 (a, c) and TNFα (b, d) levels were measured by quantitative PCR (a, b) and ELISA (supernatant; c, d). The RPMI-1640 medium was incubated with EGTA (0, 0.1, 0.5, or 1 mM) for 2 h to remove Ca2+ before adding the TLR ligands. The data are shown as the means ± S.E.M. of three independent experiments (a, b) or the means ± S.D. of triplicate samples (c, d). **p < 0.01; ***p < 0.001.

Taken together, our findings demonstrate that extracellular Ca2+ affects the response strength of macrophages and that Ca2+ may be a suitable second messenger for TLRs to transmit information about the intensity of extracellular stimuli to instruct the innate response.

Knockdown of STIM1 impairs the innate response of macrophages upon TLR stimulation

STIM1 can control the strength of calcium influx by directly binding to ORAI1 and opening Ca2+-permeable ORAI1 ion channels.20,21,25 To further demonstrate that Ca2+ is the most suitable second messenger for the transmission of intensity information about extracellular stimuli, we silenced STIM1 expression with siRNA in macrophages (Figure 5a) and found that knockdown of STIM1 significantly decreased the strength of the innate responses induced by LPS, Poly (I:C), or CpG ODN (Figure 5b–5e and Supplementary Figure S3). To avoid the off-target effects of STIM1 siRNA, we also confirmed our results using another previously reported siRNA sequence to knockdown STIM1 (Supplementary Figure S4). These results indicate that knocking down STIM1 decreased the strength of the TLR response, possibly by reducing calcium influx.

Figure 5.

Figure 5

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Knocking down STIM1 impairs the innate response of macrophages upon TLR stimuli. (a) Peritoneal macrophages were transiently transfected with control (Ctrl) or STIM1-specific siRNA. After 48 h, the efficiency of STIM1 knockdown was examined by Western blot. (b–e) Peritoneal macrophages were treated with LPS (1 ng mL−1), Poly (I:C) (10 μg mL−1), or CpG ODN (5 μg mL−1) for 6 h after STIM1 knockdown, and the IL-6 (b, d) and TNFα (c, e) levels were measured by quantitative PCR (b, c) and ELISA (supernatant; d, e). EGTA (2 mM) was incubated with medium for 2 h to remove the initial Ca2+ before adding Ca2+ (2 mM) and TLR ligands. The data are shown as the means ± S.E.M. of three independent experiments (b, c) or the means ± S.D. of triplicate samples (d, e). *p < 0.05; **p < 0.01; ***p < 0.001.

As further evidence, we added Ca2+ in the medium of STIM1-silenced macrophages and examined the response strength of the macrophages after TLR stimulation. We found that adding Ca2+ can reverse the effects of STIM1 knockdown (Figure 5b–5e and Supplementary Figures S3 and S4). Furthermore, we also observed that knocking down STIM1 significantly decreased the strength of Ca2+ influx induced by LPS, Poly (I:C), or CpG ODN (Supplementary Figure S5). These data suggest that STIM1 affect the response strength of macrophages by modulating calcium influx.

Extracellular Ca2+ reciprocally coordinates TLR-triggered activation of the small GTPases, Ras, and Rap1

The monomer of Ca2+-promoted Ras inactivator (CAPRI), a well-known GTPase-activating protein, mainly inactivates Ras, and the homodimer of CAPRI mainly inactivates Rap. Ca2+ can switch the monomer to the dimer of CAPRI.26 To investigate whether Ca2+ can affect the activation of the small GTPases Ras and Rap1, we examined the activation of Ras and Rap1 in macrophages stimulated by 1 ng mL−1 or 10 ng mL−1 LPS with or without Ca2+ in the medium. We found that both doses of LPS increased the levels of GTP-bound Rap1 and that only an LPS concentration greater than 10 ng mL−1 could activate Ras (Figure 6a–6d), indicating that Rap1 but not Ras may be the candidate small GTPase responsive to low-intensity TLR stimuli. Adding Ca2+, however, inhibited Rap1 activation induced by 1 ng mL−1 LPS but potentiated Ras activation induced by 10 ng mL−1 LPS (Figure 6e–6h). To confirm the function of extracellular Ca2+, we used EGTA to deplete Ca2+ in the medium. We found that EGTA could reverse the effects of Ca2+ on Ras and Rap1 activation (Figure 6e–6h). These data suggest that calcium influx triggered by low-intensity TLR stimuli, may inhibit Rap1 but enhance Ras activation to coordinate the TLR innate response.

Figure 6.

Figure 6

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Extracellular Ca2+ coordinates TLR-triggered activation of Ras and Rap1. (a, b) Peritoneal macrophages were stimulated with 1 ng mL−1 LPS for 30 sec or 10 ng mL−1 LPS for 2 min, and the GTP-bound levels of Rap1 (a) and Ras (b) were examined by Western blot. (e, f) Peritoneal macrophages were stimulated with 1 ng mL−1 LPS for 30 sec or 10 ng mL−1 LPS for 2 min with or without adding Ca2+ (2 mM) or EGTA (2 mM) as indicated, and the GTP-bound levels of Rap1 (e) and Ras (f) were examined by Western blot. The band intensities of the indicated molecules were determined and calculated as GTP-bound Rap (c, g) or Ras (d, h) to total Rap or Ras. The data (c, d, g, h) are presented as the means ± S.E.M. of three repetitions. (i–l) Peritoneal macrophages were transfected with control siRNA (Ctrl) or Rap1a siRNA for 48 h and then treated with LPS (1 ng mL−1), Poly (I:C) (10 μg mL−1), or CpG ODN (5 μg mL−1) for 6 h. The IL-6 (i, j) and TNFα (k, l) levels were measured by quantitative PCR (i, k) and ELISA (supernatant; j, l). The data are shown as the means ± S.E.M. of three independent experiments (i, k) or the means ± S.D. of triplicate samples (j, h). **p < 0.01; ***p < 0.001.

Previous studies have found that Ras can promote the innate response via the MAP kinase pathway.4,5 However, the function of Rap1 in innate immune cells has not been established.12,26,27 To make the role of Rap1 clear, we used siRNA to silence Rap1a (the major member of Rap in macrophages) and found that Rap1a knockdown promoted the production of IL-6 and TNFα by macrophages stimulated by LPS, Poly (I:C), or CpG ODN (Figure 6i–6l). These data suggest that Rap1 can inhibit the response of macrophages to TLR stimuli.

To test whether Ca2+ can potentiate the response of macrophages by inhibiting Rap1, we examined the response of macrophages after silencing Rap1 by adding Ca2+ or EGTA in the medium. We found that Rap1 knockdown enhanced the effects of Ca2+ (Figure 6i–6l) but abolished the effects of removing Ca2+ (Supplementary Figure S6). These results indicate that Rap1 inhibition by extracellular calcium may be a feedforward mechanism to promote the innate response of macrophages.

Taken together, our data show that Ca2+ can reciprocally coordinate the activation of the small GTPases Ras and Rap1, which may regulate the response strength of macrophages in a feedforward mechanism.

Extracellular Ca2+ modulates the TLR response of macrophages via the ERK1/2 pathway

MAP kinase is the main downstream signaling pathway of the small GTPases Ras and Rap1.4,5,8 Therefore, we further investigated the effects of extracellular Ca2+ on TLR-triggered MAP kinase signaling pathways. We found that adding Ca2+ alone to the medium accelerated the dephosphorylation and rephosphorylation of AKT and promoted the activation of ERK1/2 in macrophages by LPS (Figure 7a–7c). These data indicate that Ca2+ may relieve the inhibition of AKT on ERK1/2 signaling by promoting the dephosphorylation of AKT and that Ca2+ may regulate the response strength of macrophages by affecting the extent of activation of ERK1/2. To verify this function of Ca2+, we used EGTA to block the effect of Ca2+ and found that EGTA can reverse the effects of Ca2+ in affecting the phosphorylation levels of AKT and ERK1/2 (Figure 7d–7f).

Figure 7.

Figure 7

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Extracellular Ca2+ potentiates TLR-triggered responses via ERK1/2 pathway. (a, d, g) Peritoneal macrophages were stimulated with 1 ng mL−1 LPS (a, d, g) or 10 ng mL−1 LPS (g) in the presence of added Ca2+ (2 mM, a) or EGTA (1 mM, d) as indicated. Phosphorylated AKT or ERK1/2 was examined by Western blot. (b, c, e, f, h, i) The results in a, d, and g were quantified by determining the band intensity and calculated as the ratio of phosphorylated signaling molecules to total corresponding molecules. The data are presented as the means ± S.E.M. of three repetitions. (j–m) Peritoneal macrophages were pre-treated with vehicle (dimethyl sulfoxide) or wortmannin (1 μM) or PD98059 (20 μM) for 30 min and then treated with LPS (1 ng mL−1), Poly (I:C) (10 μg mL−1), or CpG ODN (5 μg mL−1) for 6 h. The IL-6 (j, l) and TNFα (k, m) levels were measured by quantitative PCR (j, k) and ELISA (supernatant; l, m). The data are shown as the means ± S.E.M. of three independent experiments (j, k) or the means ± S.D. of triplicate samples (l, m). *p < 0.05; **p < 0.01; ***p < 0.001.

When we used different doses of LPS to treat the macrophages, we observed that high-dose LPS could also promote the dephosphorylation and rephosphorylation of AKT and the early phosphorylation of ERK1/2 compared to low-dose LPS (Figure 7g–7i), which resembled the effects of added Ca2+. These results suggest that high doses of LPS may initiate more calcium influx to affect the phosphorylation levels of AKT and ERK1/2.

Next, we pre-treated macrophages with the PI(3)K/AKT inhibitor wortmannin or the MEK/ERK inhibitor PD98059 and added Ca2+ to the medium when we stimulated macrophages with LPS, Poly (I:C), or CpG ODN. We found that wortmannin promoted TLR-triggered responses and enhanced the effects of Ca2+ but that PD98059 repressed TLR-triggered responses and reversed the effects of Ca2+ in TLR-triggered IL-6 and TNFα production in macrophages (Figure 7j–7m), indicating that Ca2+ signaling may affect the response of macrophages through the AKT-ERK1/2 signaling pathway.

Collectively, our data suggest that different intensities of TLR stimuli can trigger different strengths of calcium influx to instruct macrophages to elicit different strength responses by coordinating the activation of Ras and Rap1 and subsequently affecting the phosphorylation levels of ERK1/2.

Ca2+ potentiates TLR response in vivo

Our data have suggested that calcium influx, mediated by STIM1, can coordinate the activation of Ras and Rap1 to elicit the feedforward regulation of TLR-triggered innate responses in macrophages. However, the physiological significance of these observations has not been elucidated. To determine whether these mechanisms also exist in vivo, we diluted LPS in endotoxin-free H2O containing Ca2+ or EGTA, intraperitoneally challenged wild-type mice, and detected the levels of IL-6 in serum by ELISA. We found that Ca2+ supplementation potentiated the in vivo effects of low doses of LPS and that EGTA significantly impaired IL-6 production (Figure 8). When Poly (I:C) and CpG ODN were similarly administered, similar effects of Ca2+ and EGTA on IL-6 production were observed (Figure 8). These results indicate that Ca2+ may be capable of mediating the feedforward regulation of TLR responses in vivo.

Figure 8.

Figure 8

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Ca2+ potentiates TLR-triggered IL-6 production in vivo. Wild-type mice (n = 10 per group) were injected intraperitoneally with the indicated amounts of LPS, Poly (I:C), or CpG ODN mixed with Ca2+ or not. After 2.5 h, IL-6 levels in sera were determined by ELISA. EGTA was used to remove the effect of calcium. *p < 0.05; **p < 0.01; ***p < 0.001.

Extracellular Ca2+ potentiates TLR response in human monocyte-derived macrophages

To test whether Ca2+ regulates TLR responses in human immune cells through a feedforward mechanism, we added different concentrations of Ca2+ to the medium and then stimulated human MDM with LPS, Poly (I:C), or CpG ODN, respectively. We found that adding Ca2+ directly to the medium could significantly increase the response strength of human MDM in a dose-dependent manner (Figure 9a and 9b; Supplementary Figure S7). Moreover, we found that removing Ca2+ from the medium by EGTA inhibited the innate response in MDM stimulated by LPS, Poly (I:C) or CpG ODN (Figure 9c and 9d; Supplementary Figure S8). These data indicate that extracellular Ca2+ can modulate different strengths of TLR innate response in MDM.

Figure 9.

Figure 9

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The roles of extracellular Ca2+ in TLR-triggered responses in human monocyte-derived macrophages. Human MDM were treated with LPS (1 ng mL−1), Poly (I:C) (10 μg mL−1), or CpG ODN (5 μg mL−1) for 6 h, and the IL-6 levels were measured by quantitative PCR (a, c) and ELISA (supernatant; b, d). EGTA (2 mM) was incubated with the medium for 2 h to remove the initial Ca2+ before adding Ca2+ (0, 1, 2, or 4 mM) and TLR ligands (a, b). The RPMI-1640 medium was incubated with EGTA (0, 1, 2, and 4 mM) for 2 h to remove Ca2+ before adding TLR ligands (c, d). The data are shown as the means ± S.E.M. of three independent experiments (a, c) or the means ± S.D. of triplicate samples (b, d). ***p < 0.001.

Discussion

Host immune cells are subjected to a broad range of PAMP and must discriminate differential intensities and stimuli and elicit proper innate responses.1,2,3 Tuning TLR responses is rather complicated. Accumulating studies have outlined the process of TLR-triggered signaling pathways.1,2,3 However, whether feedforward regulatory mechanisms, in addition to feedback mechanisms,1,2,3 exist to coordinate TLR responses has been unclear. Our study outlined a potential feedforward machinery for assisting proper activation of the TLR signaling pathway: TLR stimuli, especially at low intensities, may trigger STIM1-mediated calcium influx, which subsequently inhibits Rap1 but activates Ras to modulate the ERK1/2 signaling pathway, thus facilitating proper induction of cytokines and chemokines. Our study thus provides insights into a feedforward regulation mechanism for the TLR-triggered innate immune response.

Small GTPases usually exist in two forms: the active form (GTP-bound) and the inactive form (GDP-bound). These forms are switched mainly by two classes of molecules: the guanine nucleotide exchange factors (GEFs), which can activate small GTPases by promoting the exchange of GDP for GTP; and the GTPase-activating proteins (GAPs), which can inactivate small GTPases by enhancing their intrinsic GTPase activity to promote the hydrolysis of GTP back to GDP.29,30 Some GEFs and GAPs can regulate both Ras and Rap,5,8,29,30 which indicates that coordinators may exist to instruct the predominant activation of small GTPases Ras and/or Rap, tuning the activation of ERK1/2 pathway. The roles of Ras in TLR signaling have been elucidated.7,31,32,33,34 However, how Rap1 is activated and the effects of Rap1 in TLR signaling are not completely understood.12,26,27 Our study previously identified a Ras/Rap GEF molecule, RasGRP3, as a negative regulator of the TLR response in macrophages by activating Rap1 and inhibiting ERK1/2 activation.12 Our current study has expanded the view of Rap1 in the TLR response: Rap1 is a sensitive small GTPase upon low-intensity TLR stimuli and is a negative regulator of the TLR-triggered innate response. The findings that extracellular calcium potentiates Rap1 inhibition and ERK1/2 activation during TLR signaling may indicate a potential linkage between extracellular calcium and RasGRP3. TLR-triggered calcium influx, in contrast to TLR-triggered RasGRP3 activation, may relieve RasGRP3-mediated ERK1/2 inhibition and facilitate the TLR innate response, highlighting the influx of extracellular calcium as a feedforward mechanism for a proper TLR response. Whether calcium influx-mediated Rap1 inhibition as a feedforward mechanism also works in other cells or under other stimuli may require further investigation.

Ca2+ signals are crucial for the control of a broad range of cellular functions.20,21 Upon TLR stimulation, intracellular calcium levels are increased,34,35,36,37,38,39 either from internal stores in the ER lumen or from influx from the extracellular space. Previous studies have suggested that increased intracellular calcium plays important roles in mediating the TLR-triggered immune response.35,36,37,38,39,40,41,42,43,44,45,46,47 In most studies, attention has been paid to intracellular calcium mobilization. However, TLRs may trigger calcium release from the ER and then STIM-ORAI-mediated calcium entry. Little is known about the effects of extracellular calcium on TLR signaling, especially under low-intensity TLR stimulation. Our data show that adding calcium potentiates the TLR innate response while knocking down STIM1 or removing calcium from the medium impairs cytokine/chemokine production triggered by low-intensity TLR stimulation. Our findings that the MEK/ERK inhibitor PD98059 inhibits calcium-potentiated TLR responses indicate that calcium may mediate the feedforward regulation of the TLR response by affecting the activation of ERK1/2. How calcium influx finally potentiates TLR-induced ERK1/2 activation is still not fully understood. However, our study suggests that calcium influx, mediated by STIM1, may be the central step in feedforward regulation of TLR signaling. Previous studies have illustrated the essential roles of the STIM-ORAI system in the regulation of T cells, B cells, and mast cells.22,48,49 STIM1 has also been implicated in regulating phagocytosis of bone marrow cells.50 Our study is significant because it reveals an important role for STIM1 in coordinating the TLR innate response in macrophages.

Moreover, our study indicates that extracellular calcium may affect the TLR response by coordinating Ras and Rap1 activation. Ca2+-dependent monomer and dimer formation in CAPRI, a well-known GAP, can switch CAPRI's function between Ras GAP and Rap GAP.26 The monomer of CAPRI mainly inactivates Ras and the dimer of CAPRI mainly inactivates Rap. Increasing the concentration of Ca2+ can promote the dimerization of CAPRI. Our study demonstrates that adding Ca2+ can increase the activation of Ras but decrease the activation of Rap1, indicating that CAPRI may be involved in this process, which needs further study in the future. However, TLR-triggered calcium influx may be low upon low-intensity TLR stimulation, which may favor Rap1 inhibition and ERK1/2 activation; whereas more intense calcium influx, expected to be mobilized upon high-intensity TLR stimulation, may favor Ras activation. The activation levels of Ras or Rap1 may thereafter coordinate TLR responses. Therefore, calcium influx coordinates Ras and Rap1 activation to facilitate feedforward TLR signaling, ensuring adequate ERK1/2 activation and proper innate responses under conditions of different intensities of pathogen infection.

In sum, our results make clear that extracellular calcium, after entrance mediated by STIM1, can guide feedforward regulation of TLR signaling, which is mainly executed by coordinating the activation of small GTPases Ras and Rap1. Moreover, feedforward regulation may widely exist, such as in TCR and BCR signaling, which may be of interest in the future. The influx of extracellular calcium as a feedforward mechanism of the TLR response may be useful for the design of vaccines against infection or tumor.

Acknowledgments

This work was supported by grants from the National Key Basic Research Program of China (2010CB911903 and 2013CB530502), the National Natural Science Foundation of China (81172851, 81222039, 31270944, and 31370902), and the National High Technology Research and Development Program (2012AA020900). We thank Dr. Xingguang Liu for helpful discussion and assistance with manuscript writing, and Ms. Mei Jin and Ms. Hao Shen for their excellent technical assistance.

Footnotes

Supplementary information of this article can be found on Cellular & Molecular Immunology website: http://www.nature.com/cmi.

The authors declare they have no financial conflicts of interests.

Supplementary Information

Supplementary Information
Click here for additional data file. (2MB, pdf)

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