Extracellular acidosis is a novel danger signal alerting innate immunity via the NLRP3 inflammasome

Abstract

Background: Local acidosis has been demonstrated in ischemic tissues and at inflammatory sites.

Results: Acidic extracellular pH triggers NLRP3 inflammasome activation and interleukin-1β secretion in human macrophages.

Conclusion: Acidic pH represents a novel danger signal alerting the innate immunity.

Significance: Local acidosis may promote inflammation at ischemic and inflammatory sites. Local extracellular acidification has been demonstrated at sites of ischemia and inflammation. IL-1β is one of the key proinflammatory cytokines, and thus, its synthesis and secretion are tightly regulated. The NLRP3 (nucleotide-binding domain leucine-rich repeat containing family, pyrin domain containing 3) inflammasome complex, assembled in response to microbial components or endogenous danger signals, triggers caspase-1-mediated maturation and secretion of IL-1β. In this study, we explored whether acidic environment is sensed by immune cells as an inflammasome-activating danger signal. Human macrophages were exposed to custom cell culture media at pH 7.5-6.0. Acidic medium triggered pH-dependent secretion of IL-1β and activation of caspase-1 via a mechanism involving potassium efflux from the cells. Acidic extracellular pH caused rapid intracellular acidification, and the IL-1β-inducing effect of acidic medium could be mimicked by acidifying the cytosol with bafilomycin A1, a proton pump inhibitor. Knocking down the mRNA expression of NLRP3 receptor abolished IL-1β secretion at acidic pH. Remarkably, alkaline extracellular pH strongly inhibited the IL-1β response to several known NLRP3 activators, demonstrating bipartite regulatory potential of pH on the activity of this inflammasome. The data suggest that acidic environment represents a novel endogenous danger signal alerting the innate immunity. Low pH may thus contribute to inflammation in acidosis-associated pathologies such as atherosclerosis and post-ischemic inflammatory responses.

Keywords: Acidosis; Inflammasome; Inflammation; Innate Immunity; Interleukin; Macrophages; Nod-like Receptors (NLR).

Figures

FIGURE 1.

Acidic pH triggers the secretion of proinflammatory cytokines in LPS-primed primary human macrophages (A) and human THP-1 macrophages (B–F).A–C, E, and F, cytokine secretion into culture medium was analyzed by ELISA, and the data are means ± S.E. from 6 (A), 5 (B), or 3 (C, E, and F) experiments, performed in duplicate in B, C, E, and F. D, Western blots after a 6-h incubation in custom RPMI representative of three experiments. Procaspase-1 (p45), the active p10 subunit of caspase-1, pro-IL-1β (p31), mature p17 IL-1β, and a p20 fragment of IL-1β are indicated. SN, culture medium supernatant. *, p < 0.05; **, p < 0.01; and ***, p < 0.001, compared with control at pH 7.5.

FIGURE 2.

Acidic pH induces the mRNA expression of proinflammatory cytokines in THP-1 macrophages.A–D, the results from quantitative real-time RT-PCR are expressed as the means ± S.E. of relative expression (in arbitrary units, AU) from four experiments. *, p < 0.05, compared with control at pH 7.5.

FIGURE 3.

The mechanism of low pH -induced IL-1β secretion in THP-1 macrophages (A–D and G–I) and LPS-primed primary human macrophages (E and F). Duration of the pH stimulus was 1 h (F), 3 h (G), 4 h (A–D), or 6 h (E, H, and I). In the primary macrophages, the control values of IL-1β secretion at acidic pH were 60 ± 8 pg/ml (E) and 239 ± 29 pg/ml (F). A–G, the data are means ± S.E. from three to seven experiments, performed in duplicate in B, F, and G. The Western blots of IL-1β (H) and caspase-1 (I) are representative of four experiments. Pro-IL-1β (p31), mature p17 IL-1β, a p20 fragment of IL-1β, procaspase-1 (p45), and the active p10 subunit and a p7 fragment of caspase-1 are indicated. SN, culture medium supernatant; TEA, tetraethylammonium chloride; 4-AP, 4-aminopyridine. *, p < 0.05; **, p < 0.01; and ***, p < 0.001, compared with untreated control at pH 6.5/6.0. See “Experimental Procedures” for details on the use of inhibitors.

FIGURE 4.

Acidic pH activates the NLRP3 inflammasome. The relative mRNA expressions of NLRP1 (A) and NLRP3 (B) receptors are means ± S.E. from four experiments in THP-1 macrophages. In THP-1 macrophages transfected with NLRP3 siRNA, the mRNA expression data (C) and cytokine secretion data (D and E) are expressed as fold changes compared with the negative control siRNA transfection. The data are means ± S.E. from five to six experiments (C) or from four to five experiments performed in duplicate (D and E). AU, arbitrary units; NT, nontransfected; Mock, transfection reagent without siRNA; Control, negative control siRNA; NLRP3, NLRP3 siRNA. *, p < 0.05 and **, p < 0.01.

FIGURE 5.

Intracellular acidification is essential for inflammasome activation caused by low pH environment. Bafilomycin A1 (Baf) induces IL-1β secretion in primary human macrophages (A) and THP-1 macrophages (B). In primary macrophages, the control values of IL-1β secretion were 27 ± 8 pg/ml and 66 ± 20 pg/ml at pH 7.5 and 6.5, respectively. The data are means ± S.E. from six to nine (A) and five (B) experiments. C, initial pHi in THP-1 macrophages was recorded at pHe 7.5, followed by perfusion of the indicated test medium (arrow). The data are means from three experiments (50 cells/experiment). D, THP-1 macrophages were pulsed in a medium of pH 7.5 or 6.5, washed, and subsequently incubated for 4 h at indicated pH for measurement of cytokine secretion. The data are means ± S.E. from four experiments performed in duplicate. *, p < 0.05, **, p < 0.01, and ***, p < 0.001, compared with untreated control.

FIGURE 6.

Mildly acidic pH synergizes with other NLRP3 inflammasome activators. THP-1 macrophages were incubated for 4 h at pH 7.5 or pH 7.0 in the absence or presence of 0.5 mg/ml CHC (A) or 0.5 μg/ml SAA (B). The background secretion of IL-1β at pH 7.5 has been subtracted from all values. The data are means ± S.E. from five experiments performed in duplicate. SUM, activator at pH 7.5 + release at pH 7.0 without activator. **, p < 0.01.

FIGURE 7.

Alkaline pH strongly inhibits NLRP3 inflammasome activation in LPS-primed primary human macrophages (A) and in THP-1 macrophages (B–F). Duration of the pH stimulus was 10 h (A), 4 h (B–D), or 16 h (E and F), in the absence or presence of 0.25 mg/ml MSU, 2 μg/ml SAA, or 1 mg/ml CHC. In the primary macrophages, alkaline pH alone did not induce IL-1β secretion, and MSU-induced IL-1β secretion at pH 7.5 was 381 ± 61 pg/ml. The data are means ± S.E. from eight experiments (A) or from five experiments performed in duplicate (B–F). CE, cholesteryl ester. *, p < 0.05; **, p < 0.01; and ***, p < 0.001, compared with control at pH 7.5.