Vitamin D3 regulates PM-driven primary human neutrophil inflammatory responses

Abstract

Recent evidence has demonstrated that both acute and chronic exposure to particulate air pollution are risk factors for respiratory tract infections and increased mortality from sepsis. There is therefore an urgent need to establish the impact of ambient particulate matter (PM) on innate immune cells and to establish potential strategies to mitigate against adverse effects. PM has previously been reported to have potential adverse effects on neutrophil function. In the present study, we investigated the impact of standard urban PM (SRM1648a, NIST) and PM_2.5 collected from Chiang Mai, Thailand, on human peripheral blood neutrophil functions, including LPS-induced migration, IL-8 production, and bacterial killing. Both NIST and the PM_2.5, being collected in Chiang Mai, Thailand, increased IL-8 production, but reduced CXCR2 expression and migration of human primary neutrophils stimulated with Escherichia coli LPS. Moreover, PM-pretreated neutrophils from vitamin D-insufficient participants showed reduced E. coli-killing activity. Furthermore, in vitro vitamin D3 supplementation attenuated IL-8 production and improved bacterial killing by cells from vitamin D-insufficient participants. Our findings suggest that provision of vitamin D to individuals with insufficiency may attenuate adverse acute neutrophilic responses to ambient PM.

Figure 1

PM enhances IL-8 production by human neutrophils in response to LPS. Isolated human neutrophils at 2.5 × 10⁶ cells/ml from vitamin D-sufficient participants (n = 3) were pretreated with (a) SRM 1648a (PM; 0, 5, 10, 20 and 40 μg/ml) or (b) Chiang Mai PM_2.5 (CM PM_2.5; 0, 1.25, 2.5, 5, 10, 20 and 40 μg/ml) with or without 100 ng/ml E. coli LPS stimulation. The supernatant of PM-pretreated neutrophil cultures was harvested at 18 h for IL-8 detection. Each dot indicates the median with the range. Asterisks indicate significant differences between PM with LPS condition (red line) versus LPS alone stimulation (green dashed line) or between PM alone (black line) and medium control at 0 μg/ml PM. Statistical analysis was performed using two-way ANOVA with Šídák’s multiple comparisons test. ****P < 0.001, No asterisk- nonsignificant.

Figure 2

Standard urban PM reduces LPS-induced CXCR2 expression and migration of human neutrophils in response to E. coli LPS. Isolated human neutrophils at 2.5 × 10⁶ cells/ml from vitamin D-sufficient participants were pretreated with SRM 1648a (PM; 0, 5, 10, 20 and 40 μg/ml) with or without 100 ng/ml E. coli LPS stimulation. (a) After 6 h, the cells (n = 5) were stained with anti-human CXCR2-PE and analysed by flow cytometry by gating the percentage of CXCR2-expressing cells compared with unstained cells from the selected neutrophil population. Percentage enhancement was calculated by comparison with the no PM condition. Each bar represents the median with interquartile range. (b) SRM 1648a—pretreated neutrophils (n = 4) were added to upper wells and cocultured with E. coli LPS in lower wells. After coculture for 1 h, transmigrating neutrophils were counted by flow cytometry. The dark circles indicate the migration index or number of transmigrated cells for neutrophils from each donor under each exposure condition. Statistical analysis was performed using Friedman’s test with Dunn’s post hoc analysis to compare among PM concentrations, **P < 0.01.

Figure 3

1α,25-Dihydroxyvitamin D3 reduces the inflammatory response of PM-treated human neutrophils. Isolated human neutrophils at 2.5 × 10⁶ cells/ml from vitamin D-sufficient participants (n = 3) were pretreated with 40 μg/ml SRM 1648a and varying concentrations of 1α,25-dihydroxyvitamin D3 (Vit D; 0, 10, 50, 100, 500 and 1000 nM) for 30 min and then restimulated with (panel (a)) medium control (no LPS) or (panel (b)) 100 ng/ml E. coli LPS. The supernatant of pretreated neutrophil cultures was harvested at 18 h for IL-8 detection. Each dot indicates the median with the range of IL-8 concentrations. Statistical analysis was performed using two-way ANOVA with Šídák's multiple comparisons test between the medium control and 1α,25-dihydroxyvitamin D3 treatments. A cell suspension of pretreated neutrophil cultures at 6 h was stained with anti-human CXCR2-PE and analysed by flow cytometry (panels (c, d)). Each bar indicates the median with the range of % CXCR2-positive cells. The pretreated cells were added to upper wells and cocultured with E. coli LPS in lower wells. After coculture for 1 h, transmigrating neutrophils were counted by flow cytometry, and the migration index was calculated (panel e). Each bar indicates the median with the range of the neutrophil migration index. Grey and white bars indicate untreated and PM-treated conditions, respectively. Statistical analysis was performed using two-way ANOVA with Šídák's multiple comparisons test. **P < 0.01, *P < 0.05.

Figure 4

1α,25-Dihydroxyvitamin D3 reduces IL-8 production and migration of PM-treated neutrophils isolated from vitamin D-sufficient and vitamin D-insufficient participants. Isolated neutrophils at 2.5 × 10⁶ cells/ml from vitamin D-sufficient (n = 5) and vitamin D-insufficient (n = 5) participants were pretreated with 40 μg/ml standard urban PM with or without 1α,25-dihydroxyvitamin D3 (VitD; 10, 100 and 1000 nM) for 30 min and then stimulated with 100 ng/ml E. coli LPS. Panels (a) and (c) show median (interquartile range) IL-8 concentrations in supernatant from pretreated neutrophil cultures at 18 h. Isolated neutrophils were pretreated with 40 μg/ml standard urban PM with or without 100 nM 1α,25-dihydroxyvitamin D3 for 30 min. The pretreated cells were added to upper wells and cocultured with E. coli LPS in lower wells. After coculture for 1 h, transmigrating neutrophils were counted by flow cytometry. Panels (b) and (d) show the median (IQR) of the neutrophil migration index, and each dot represents the value of each participant. Grey and white bars indicate vitamin D-sufficient and vitamin D-insufficient participants, respectively. Statistical analysis was performed using two-way ANOVA with Šídák’s multiple comparisons test among 1α,25-dihydroxyvitamin D3 pretreatments and between sufficient and insufficient participants. ****P < 0.001, **P < 0.01, *P < 0.05, No asterisk- nonsignificant.

Figure 5

1α,25-Dihydroxyvitamin D3 enhances E. coli killing of human neutrophils isolated from vitamin D insufficient participants. Neutrophils isolated from vitamin D-sufficient (n = 10) and vitamin D-insufficient participants (n = 10) were pretreated with 40 μg/ml standard urban PM with or without 100 nM 1alpha,25-dihydroxyvitamin D3 before coculture with live E. coli at an MOI of 0.1:1. The cells were lysed for bacterial counting at 3 h. Total bacteria were quantified by standard colony plating, and the results are expressed as percentages of bacterial survival. Each bar indicates the median with the interquartile range of number of bacteria (panel a) or % bacterial survival (panel b), and each dot represents the value of each participant. Grey and white bars indicate vitamin D-sufficient and vitamin D-insufficient participants, respectively. Statistical analysis was performed using two-way ANOVA with Šídák’s multiple comparisons test. *P < 0.05, No asterisk-nonsignificant.