Human peripheral blood mononuclear cells (PBMCs) from smokers release higher levels of IL-1-like cytokines after exposure to combustion-generated ultrafine particles

Abstract

Ultrafine particles (UFP) generated by combustion processes are often associated with adverse health effects. However, little is known about the inflammatory processes generated by UFP that may underlie their toxicological activity. Murine macrophages (J774.1 cells) and human peripheral blood mononuclear cells (PBMCs) were used to evaluate the molecular mechanism underlying the pro-inflammatory activity of UFP. The addition of soot particles to J774.1 cells induced a concentration-dependent release of IL-1α, IL-1β and IL-33 This effect was not associated with cell death and, in contrast to literature, was pronounced at very low concentrations (5-100 pg/ml). Similarly, UFP induced the release of IL-1α, IL-18 and IL-33 by PBMCs. However, this effect was solely observed in PBMCs obtained from smokers, as the PBMCs from non-smokers instead released higher levels of IL-10. The release of these cytokines after UFP exposure was caspase-1- and NLRP3 inflammasome-dependent in PBMCs from healthy smokers, whereas IL-1α release was calpain-dependent. These results show that UFP at very low concentrations are able to give rise to an inflammatory process that is responsible for IL-1α, IL-18 and IL-33 release, which is pronounced in PBMCs from smokers, confirming that these individuals are especially susceptible to inflammatory-based airway diseases once exposed to air pollution.

Figure 1. The administration of soot particles for 5 hours induced the release of IL-1-like cytokines by murine macrophages.

J774.1 cells (a murine macrophage cell line) were treated with soot particles in a concentration-dependent manner (1 pg/ml–5 ng/ml) for 5 hours. LPS (0.1 μg/ml) and/or ATP (0.5 mM) was used as a positive control. The addition of soot particles to macrophages induced the release of IL-1β (A), IL-1α (B) and IL-33 (C). Data are presented as the means ± SEM (n = 12). Statistically significant differences are denoted by *, ** and ***, indicating p < 0.05, p < 0.01 and p < 0.001, respectively, as determined by one-way ANOVA followed by Bonferroni’s multiple comparison post-test.

Figure 2. PBMCs obtained by healthy smokers were more susceptible to soot particle-induced IL-1-like cytokine release.

PBMCs were isolated from the blood of healthy non-smoking and smoking volunteers. The addition of soot particles (1 pg/ml–5 ng/ml) for 5 hours to PBMCs isolated from non-smokers did not induce the release of IL-1α (A) or IL-33 (C). However, soot particles slightly increased the release of IL-18 (50 pg/ml) (E). In sharp contrast, the addition of soot particles to PBMCs obtained from smokers significantly increased the release of IL-1α (B), IL-33 (D) and IL-18 (F). Data are presented as the means ± SEM (n = 5). Statistically significant differences are denoted by *, ** and ***, indicating p < 0.05, p < 0.01, p < 0.001 and p < 0.001, respectively, as determined by one-way ANOVA followed by Bonferroni’s multiple comparison post-test.

Figure 3. The addition of soot particles induced PBMCs to release IL-10.

PBMCs were isolated from the blood of healthy non-smoking and smoking volunteers. The administration of soot particles (1 pg/ml–5 ng/ml) for 5 hours to PBMCs isolated from non-smokers significantly increased the release of IL-10 (A). In the same way, but to a lower extent, PBMCs isolated from smokers released IL-10 after soot particle addition (B). (C) Comparison of the levels of IL-10 between non-smokers (open white bars) and smokers (dotted bars). Data are presented as the means ± SEM (n = 5). Statistically significant differences are denoted by *, ** and ***, indicating p < 0.05, p < 0.01 and p < 0.001, respectively, as determined by one-way ANOVA followed by Bonferroni’s multiple comparison post-test.

Figure 4. The release of IL-1-like cytokines by soot particle-treated PBMCs obtained from healthy smokers is caspase-1-dependent.

PBMCs obtained from smokers were treated with soot particles (1 pg/ml–5 ng/ml) in the presence or absence of Ac-YVad (Y-Vad: 1 μg/ml), a caspase-1 inhibitor. The inhibition of caspase-1 with Y-Vad significantly reduced the release of IL-1α (A), IL-18 (B) and IL-33 (C) from PBMCs treated for 5 hours. Data are presented as the means ± SEM (n = 5). Statistically significant differences are denoted by *, ** and ***, indicating p < 0.05, p < 0.01, and p < 0.001, respectively, as determined by one-way ANOVA followed by Bonferroni’s multiple comparison post-test.

Figure 5. NLRP3 is involved in the release of IL-1-like cytokines by soot particle-treated PBMCs obtained from healthy smokers.

PBMCs obtained from smokers were treated with soot particles (1 pg/ml–5 ng/ml) in the presence or absence of glybenclamide (Gly: 1 μM), an NLRP3 inhibitor. The inhibition of NLRP3 with Gly significantly reduced the release of IL-1α (A), IL-18 (B) and IL-33 (C) from PBMCs treated for 5 hours. Data are presented as the means ± SEM (n = 5). Statistically significant differences are denoted by * and ***, indicating p < 0.05 and p < 0.001, respectively, as determined by one-way ANOVA followed by Bonferroni’s multiple comparison post-test.

Figure 6. The release of IL-1-α from soot particle-treated PBMCs obtained from healthy smokers is calpain-dependent.

PBMCs obtained from smokers were treated with soot particles (1 pg/ml–5 ng/ml) in the presence or absence of M6690 (M66: 10 μM), a calpain I/II inhibitor. The inhibition of calpain I/II significantly reduced the release of IL-1α from PBMCs treated for 5 hours. Data are presented as the means ± SEM (n = 5). Statistically significant differences are denoted by * and **, indicating p < 0.05 and p < 0.01, respectively, as determined by one-way ANOVA followed by Bonferroni’s multiple comparison post-test.