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 PM2.5 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 PM2.5, 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.
Similar content being viewed by others
Introduction
Long-term exposure to PM, especially the fine fraction (PM2.5), is associated with increased risk of cardiovascular disease, diabetes, acute respiratory tract infections (RTIs)1,2,3 and sepsis-related mortality4. All these conditions are linked to local airway and systemic inflammation, which are induced following both short5,6 and long7,8,9 term air pollution exposures. Both PM10 and PM2.5 have been reported to have adverse effects on airway structural and immune cells, including both the recruitment and activation of neutrophils at the epithelial barriers encompassing respiratory tract lining fluid and resident inflammatory cells10,11. After exposure to PM2.5, neutrophils are recruited into the lungs of mice and associated with production of proinflammatory cytokines12. IL-8 or CXCL8 is the most potent neutrophil-recruiting chemokine13. Human exposures to freshly generated diesel exhaust have also reported IL-8 production in the airways, concomitant with increased neutrophil infiltration into the bronchial mucosa and central airways and sustained up to 18 h post exposure14,15,16.
Moreover, cigarette smoke PM challenge has been shown to compromise Escherichia coli LPS-induced proinflammatory cytokine production and the bacterial killing function of murine neutrophils17. During infection, Toll-like receptor 4 (TLR4) responds to the presence of LPS at the site of infection or bloodstream and triggers pro-inflammatory reactions, for instance cytokine/chemokine production, facilitating elimination of the invading gram-negative bacteria18. Nevertheless, relatively few studies have focused on primary human neutrophil responses following particulate pollutant challenge, and the underlying mechanisms by which inhaled PM may affect neutrophil function in response to infection remain unclear.
Vitamin D is a secosteroid hormone that is well known for its role in mineral and skeletal homeostasis. Vitamin D also plays a critical role in modulating the immune response, including the response to respiratory infections19. Vitamin D deficiency has been associated with disease severity in community-acquired pneumonia20 and other RTIs21, while vitamin D supplementation has proven benefits in patients with these conditions22. Moreover, vitamin D is an essential component of innate immune modulation19,23: it promotes pneumococcal killing and modulates inflammatory responses in primary human neutrophils24. Furthermore, vitamin D3 may reduce the predominantly oxidative stress-mediated inflammation induced by PM25,26, and vitamin D3 has been shown to increase human α-1-antitrypsin (AAT), a serine protease inhibitor with immunoregulatory properties that prevents excessive lung damage by neutrophil elastase, in CD4 + T cells27,28. Based on these previous observations we hypothesized that vitamin D3 would modulate the inflammatory responses of PM-exposed neutrophils. We therefore examined whether vitamin D3 was protective against a range of PM-driven human neutrophil responses relevant to infection risk: the production of IL-8, cell migration and bacterial killing.
Results
PM enhances interleukin-8 production by primary human neutrophils in response to lipopolysaccharide (LPS) challenge
Isolated neutrophils from healthy participants were pre-treated with standard reference urban PM (SRM 1648a) prior to stimulation with LPS for 18 h. Initial experiments confirmed no loss of cell viability with PM incubation at concentrations ranging from 1.25 to 40 µg/ml for 30, 60 and 90 min (Supplementary Table S1). We observed that standard urban PM alone significantly induced IL-8 production in a dose-dependent manner (Fig. 1a). Moreover, pretreatment of neutrophils with standard urban PM led to an additive effect of IL-8 production in response to LPS compared with LPS stimulation alone, which was statistically significant at 20 and 40 µg/ml (Fig. 1a). We further determined these effects with Chiang Mai PM2.5. Although treatment with Chiang Mai PM2.5 alone did not induce a significant increase in IL-8 production over the range of concentrations tested, enhancement of IL-8 production in response to LPS compared with LPS stimulation alone was similar to that induced by standard urban PM (Fig. 1b). There was insufficient quantity of the CM PM2.5 sample for further comprehensive analyses and therefore all further experiments were conducted using SRM 1648a.
Pretreatment of primary human neutrophils with PM decreased expression of the IL-8 receptor and reduced the migration index in response to LPS
We further investigated the effect of standard (SRM 1648a) PM on downstream functions affected by IL-8 production, specifically IL-8 receptor (CXCR2) expression and neutrophil migration activity. Isolated neutrophils were pretreated with PM at a range of concentrations, with or without 100 ng/ml LPS stimulation. After 6 h, the cells were stained with anti-human CXCR2-PE and analysed by flow cytometry. We found little evidence that PM alone enhanced the frequency of CXCR2-expressing neutrophils over the concentration range 5–40 ug/ml, though a statistically significant increase was evidence at the highest (40 ug/ml) dose. However, PM pretreatment with LPS stimulation significantly reduced LPS-induced CXCR2 expression in primary human neutrophils in a dose-dependent manner (Fig. 2a).
To determine neutrophil migration activity by Transwell plate, standard (SRM 1648a) PM-pretreated neutrophils were added to the upper wells and cocultured with LPS or medium alone as the control, which was added to the lower wells. The data are represented as both the number of transmigrated neutrophils and the migration index, which was calculated by subtracting the absolute count of transmigrated neutrophils to LPS from the medium control and then dividing this by transmigrated neutrophils in the medium control (Fig. 2b). Firstly, in PM-pretreated neutrophils cocultured with medium alone, the number of transmigrated neutrophils was modestly, but significantly increased at the highest PM concentration tested (Fig. 2b; 1st panel). In contrast, LPS-induced neutrophil migration was much stronger than that induced by PM, and LPS-induced neutrophil migration was inhibited by PM pretreatment in a dose-dependent manner (Fig. 2b). These data indicate that PM treatment alone activates neutrophil functions, but impairs the migration activity of primary human neutrophils in response to LPS.
1α,25-Dihydroxyvitamin D3 reduces the inflammatory responses of primary human neutrophils pre-treated with PM samples
To determine whether 1α,25-dihydroxyvitamin D3 modulates IL-8 production, neutrophils were pretreated with 40 μg/ml PM (SRM 1648a) and various concentrations of 1α,25-dihydroxyvitamin D3. Initial experiments were performed using neutrophils isolated from vitamin D-sufficient participants (Table 1). Production of IL-8 induced by PM was significantly reduced in neutrophils by 1α,25-dihydroxyvitamin D3 in cell culture (Fig. 3a). Next, we investigated the effect of vitamin D3 on PM-pretreated neutrophils in response to LPS, and a similar reduction was observed with LPS-induced IL-8 production, as represented by bar graphs in Fig. 3b. Moreover, in neutrophils pretreated with both PM and the highest concentration of 1α,25-dihydroxyvitamin D3 studied, IL-8 production was modestly, but significantly reduced in response to LPS exposure, as represented by line-dot plots in Fig. 3b.
Next, we observed the effect of 1α,25-dihydroxyvitamin D3 on CXCR2 expression. Without LPS addition, CXCR2 expression was significantly decreased in neutrophils pretreated with PM and 1α,25-dihydroxyvitamin D3 compared to PM alone (Fig. 3c). On the other hand, LPS exposure abrogated the effect of 1α,25-dihydroxyvitamin D3 across all conditions (Fig. 3d), and the migration index of neutrophils pretreated with 1α,25-dihydroxyvitamin D3 in response to LPS exposure was significantly reduced both with (white bars) and without (grey bars) PM pretreatment (Fig. 3e). Taken together, our data indicate that PM alone is not only proinflammatory, but also compromises the inflammatory response to TLR ligation by LPS.
The responses of neutrophils from vitamin D-insufficient participants in response to PM challenge are attenuated by 1α,25-dihydroxyvitamin D3 treatment
We next investigated whether there were any differences in PM-induced responses in neutrophils obtained from vitamin D-sufficient and vitamin D-insufficient (< 5.8 pg/ml)29 participants, and the effect of 1α,25-dihydroxyvitamin D3 on this response. The free 25-OH vitamin D concentrations in plasma, which are strongly correlated to total 25-OH vitamin D in most populations30, were 9.6 ± 1.6 and 4.4 ± 0.9 pg/ml for vitamin D-sufficient and -insufficient subjects, respectively (Table 1).
1α,25-dihydroxyvitamin D3-pretreated neutrophils showed a significantly reduced IL-8 production in response to LPS in both vitamin D-sufficient and vitamin D-insufficient participants (Fig. 4a) and a decreased migration index (Fig. 4b) that achieved significance in the vitamin D-insufficient participants only. However overall, there was no significant difference between the groups. In PM-pretreated neutrophils treated with LPS, neutrophils from vitamin D-insufficient participants secreted lower concentrations of IL-8 in response to LPS than those from vitamin D-sufficient participants. Furthermore, decreased IL-8 production due to vitamin D3 treatment was observed only in vitamin D-sufficient participants (Fig. 4c), and reduction in IL-8 production by 1α,25-dihydroxyvitamin D3 in culture in vitamin D-insufficient participants was less than that in vitamin D-sufficient participants (Supplementary Figure S1). The migration index was also decreased in PM-pretreated neutrophils from both vitamin D-sufficient and -insufficient participants (Fig. 4d).
1α,25-Dihydroxyvitamin D3 enhances the bacterial killing activity of primary human neutrophils isolated from vitamin D-insufficient participants following PM-pretreatment
To investigate the effect of 1α,25-dihydroxyvitamin D3 on the regulation of bacterial killing by PM-pretreated neutrophils, isolated neutrophils from both vitamin D-sufficient and -insufficient participants were pretreated with 1α,25-dihydroxyvitamin D3 alone, PM alone or both 1α,25-dihydroxyvitamin D3 and PM prior to E. coli inoculation. No significant difference in bacterial number was found in untreated neutrophils between both participant groups (Fig. 5a). Interestingly, for neutrophils pretreated with vitamin D3 alone, bacterial number and % bacterial survival were significantly reduced when examining cells from vitamin D-insufficient participants (Fig. 5a,b); PM pretreatment alone significantly enhanced % bacterial survival compared with neutrophils from vitamin D-sufficient participants (Fig. 5b). Moreover, when neutrophils were pretreated with both 1α,25-dihydroxyvitamin D3 and PM, % bacterial survival was similar when using cells from both vitamin D-insufficient and vitamin D-sufficient participants (Fig. 5).
Discussion
Air pollution is the major environmental risk factor for respiratory and cardiovascular disease globally, with the greatest burden experienced in Central and Southeast Asia31. Both PM10 and PM2.5 have been shown to modulate several inflammatory pathways related to the development and exacerbation of chronic cardiopulmonary disease32,33, but recent evidence has suggested that they may also be associated with increased infectious disease risk34,35. Moreover, a meta-analysis and a population-based time series study have shown a positive association between daily levels of ambient air pollution and hospitalization or mortality of children due to pneumonia36,37.
In this study, we found that PM enhanced IL-8 production alone. PM also enhanced IL-8 production in response to LPS stimulation. PM modestly enhanced both CXCR2 expression and the neutrophil migration response at the highest concentration tested. In contrast PM clearly inhibited CXCR2 expression and the migration activity of neutrophils in response to LPS. In particular, preexposure to Chiang Mai PM2.5 strongly enhanced IL-8 production in LPS-induced neutrophils compared to LPS alone. However, Chiang Mai PM2.5 alone did not induce IL-8 production. Urban PM is total suspended material and contains particles up to 30 µm in diameter. Therefore, the different results might be due to differences in size distribution, composition and extraction methods between the two samples used. Moreover, these data would suggest that the contemporary PM2.5 sample from Chiang Mai is less immunostimulatory than the historic SRM 1648a. Our study suggests that preexposure to PM alters the response of human neutrophils stimulated by LPS, consistent with findings in a recent study using a mouse model38.
LPS is a potent microbial inducer of inflammation, and recognition of LPS by the innate immune system is a crucial step in identifying invading gram-negative bacteria and in initiating a protective immune response; any impairment of this process may enhance susceptibility to certain infectious agents. The capacity of PM to increase susceptibility to infections has previously been shown for various gram-positive and gram-negative bacteria such as Mycobacterium tuberculosis39, Streptococcus pneumoniae40, and Pseudomonas aeruginosa41. Furthermore, case-crossover analyses conducted in the United States found that short-term exposure to PM2.5 is associated with increased risk of hospital admissions due to septicaemia42. However, the current evidence of the effect of PM2.5 air pollution on sepsis is insufficient and warrants further investigation. Our results revealed that PM increased IL-8, but reduced IL-8 receptor expression and migration in response to LPS stimulation. This is relevant to a previous study in a mouse model in which PM reduced neutrophil migration to the lungs in response to LPS by decreasing gene expression of TLR-4 and MyD8838. Moreover, TLR4-induced loss of IL-8 receptor (CXCR2) was modified by enhanced LPS presentation efficiency43. However, there are no direct evidence demonstrated in those studies that LPS is able to bind IL-8 receptor. Together these data suggest that PM interferes with neutrophil migration in response to LPS, an area that warrants more detailed investigation.
Vitamin D3, a lipophilic micronutrient, has been well reported to both attenuate oxidative stress26,44 and modulate inflammation45, including in neutrophils46,47. In CD4 + T cells, it has been shown to increase human α-1-antitrypsin, a serine protease inhibitor that acts to limit excessive lung damage by neutrophil elastase and also has immunoregulatory activities27,28. Vitamin D3 also plays a role in the control of airway remodelling, which is a hallmark of severe asthma and COPD48,49,50. Moreover, vitamin D3 has been reported to potentially modify neutrophil function by modulating expression of neutrophil genes via functional vitamin D receptors51.
Although the biological roles of vitamin D3 and the adverse effects of PM are well known, the association between vitamin D3 and PM on human neutrophil function has not been addressed. A previous study found that 95 nM of total serum vitamin D was the cut-off for beneficial effects against viral respiratory tract infections52, and another in vitro study using human primary neutrophils showed that the effective concentration of vitamin D supplementation for killing activity is 100 nM24. The concentrations of 1α,25-dihydroxyvitamin D3 studied here were varied to cover the concentrations from both previous studies and we investigated the potential effects of 1α,25-dihydroxyvitamin D3 on the PM-induced inflammatory response in primary human neutrophils from vitamin D-sufficient individuals as a baseline response. Our data showed that the pretreatment with 100 to 1000 nM of 1α,25-dihydroxyvitamin D3 reduced PM-induced IL-8 production. A high dose at 1000 nM of 1α,25-dihydroxyvitamin D3 likely only occurs during hypercalcemia53; however, 100 to 1000 nM of vitamin D is commonly used in in vitro studies. Furthermore, we were able to observe the effect of 1α,25-dihydroxyvitamin D3 from 100 nM.
Overall, our data that PM reduces LPS-induced migration are robust, and this is presumed to be detrimental for the host. Vitamin D is seen as a homeostatic regulator23 and the capacity of vitamin D to reduce, but not completely eliminate LPS-induced migration is arguably a good thing to ensure that an inflammatory response is mounted to a pathogen, but is not over-exuberant leading to tissue pathology. In contrast, where PM reduced the LPS-induced migratory response, and then vitamin D reduced this even further we postulate this may compromise the host, leaving it without an appropriate and protective inflammatory response. So, we suggest that a detrimental effect on LPS-induced migration is seen when both PM and vitamin D are present, but not when only vitamin D was present. Future work could test this in an in vivo (animal) challenge model.
Due to sufficient sunlight year-round, the Thai population should not be at risk for vitamin D deficiency, but high levels of insufficiency due to lifestyle and dietary factors have been reported54. Our current study is the first to examine the effect of vitamin D on PM-driven human neutrophil function in vitamin D-insufficient participants living in high PM-exposure areas. Interestingly, we found that PM-pretreated neutrophils from vitamin D-insufficient participants had impaired IL-8 production and bacterial killing activity compared with those from vitamin D-sufficient participants. A previous study in mice showed that vitamin D deficiency reduces the immune response, phagocytosis rate, and intracellular killing rate of microglial cells against E. coli infection55, and severe vitamin D deficiency failed to significantly minimize cellular inflammation in bronchoalveolar lavage samples by instillation of LPS56. Thus, the mechanisms underlying this phenomenon need further investigation.
Our major findings on 1α,25-dihydroxyvitamin D3 supplementation in vitro suggest that 1α,25-dihydroxyvitamin D3 improved the bacterial killing activity of PM-pretreated neutrophils from vitamin D-insufficient participants. These data are consistent with another in vitro study of primary human neutrophils demonstrating that vitamin D supplementation enhances S. pneumoniae killing24. Notably, local nebulization of vitamin D in severely vitamin D-deficient mice failed to attenuate the cytokine storm against LPS challenge and was only effective in mice with normal levels of vitamin D56. Moreover, we found that PM-pretreated neutrophils from vitamin D-insufficient participants could not respond to 1α,25-dihydroxy vitamin D3 treatment in terms of decreasing IL-8 production. These observations suggest that the route of vitamin D3 supplementation and establishing the effective dose should be considered in further studies addressing the potential of vitamin D to protect against the harmful effects of PM exposure.
Possible mechanisms that may explain how 1α,25-dihydroxyvitamin D3 regulates PM-driven neutrophil inflammation include the following: (1) inhibition of TLR2, 4, and 9 expression, as previously reported for monocytes57; (2) reduction in oxidative stress-mediated inflammation induced via the p38/NF-κB/NLRP3 signalling pathway, as previously reported for human bronchial epithelial cells25; and (3) regulation of the cathelicidin antimicrobial peptide gene, as reported for macrophages58. Moreover, vitamin D is known to promote expression of the antimicrobial peptides cathelicidin, the alpha-defensins (HNP1–3) and beta-defensins, all mediators of the antibacterial activity from primary human neutrophils for pneumococcal killing24. In this study, 100 nM 1α,25-dihydroxyvitamin D3 demonstrated a trend to enhance the killing activity of neutrophils from vitamin D-insufficient individuals (p = 0.1823).
Taken together, in vitro short-term exposure to PM, including PM2.5 collected in Chiang Mai, Thailand, enhanced IL-8 production stimulated by LPS. In addition, standard urban PM was able to modify the neutrophil inflammatory response to LPS and reduce bacterial killing activity. Importantly, vitamin D3 attenuated IL-8 production and restored bacterial killing in neutrophils from vitamin D-insufficient participants. These results suggest that 1α,25-dihydroxyvitamin D3 is a promising therapeutic target to regulate the PM-driven human neutrophil inflammatory response, and may represent a low cost strategy to protect the population from infectious disease risks in high PM exposure areas.
Methods
Participants
We enrolled 20 healthy participants from the Khon Kaen and Chiang Mai provinces. All participants were age matched, and none had signs of acute infectious disease within a 3-months period prior to blood donation. Each participant provided 20 ml of venous blood, which was heparinized, for neutrophil isolation and measurement of free 25-hydroxy vitamin D by ELISA (DIAsource) according to the manufacturer’s instructions.
Ethics statement
The protocol for blood collection and sample analysis was approved by the Ethics Committee for Human Research of the Faculty of Associated Medical Sciences, Chiang Mai University (AMSEC63EX036) and the Khon Kaen University Ethics Committee for Human Research (HE631315). The study was performed under the approved guidelines and regulations. All subjects provided written informed consent.
Chiang Mai PM2.5 collection and standard reference PM (SRM1648a) sample studied
Two sets of the Tisch model TE-WILBUR-2.5 Low Volume Federal Reference Method (FRM) Ambient Air Particulate Samplers (Tisch Environmental) with the adjusted flow rate at 16.7 l/min were operated to collect PM2.5 samples during March 20–27, 2020, at the open rooftop on the 12th floor of Building 2, Faculty of Associated Medical Sciences, Chiang Mai University located in Su Thep Sub district, Mueang Chiang Mai District, Chiang Mai, Thailand (UTM (WGS84) 47Q 497444 E, 2077573 N). This building is approximately 90 m away from Suthep main road, and the height from the rooftop floor to the centre of the inlets is 1.96 m. The activities considered as significant sources of PM during the sampling period were not only from regular traffic and transboundary PM from biomass burning but also PM from the forest fire within the Doi Suthep Mountain region (starting on March 24th), which was 9.31 km from the sampling point. Masses of PM2.5 samples on the 47 mm diameter PTFE filters were determined gravimetrically using a 7-digit place ultramicrobalance (Mettler Toledo: METLER UMX 2), and the concentrations were calculated prior to extraction. Extraction was performed by the addition of HPLC grade methanol (3 mL) to the filter in 50 mL falcon tubes, followed by vortexing for 1 min and sonication for 10 min before decanting into fresh falcon tubes. This step was repeated three times with fresh methanol and regular changing of ultrasonic bath water to ensure constant temperature. Filters and extracted PM-methanol solutions were then dried under nitrogen. Extracted filters were then reweighed as described above. The extracted PM2.5 was then resuspended as a stock at 1 mg/ml in Milli-Q system (Merck Millipore)-purified water with a resistivity ≥ 18.2 MΩ cm and pretreated with Chelex-100 resin to reduce background metal contamination, as previously described59. Stock PM suspensions were stored at − 80 °C prior to use.
Urban particulate matter samples SRM 1648a were purchased from National Institute of Standards and Technology in the USA. The samples were composed of particulate matter collected over a period of 1 year (1976–1977) in the St. Louis (MO, USA) area into a specially designed dust collector. SRM 1648a is a conglomeration of fine and ultrafine particles with the mean particle diameter 5.85 µm49.
Primary human neutrophil isolation and stimulation
Primary human neutrophils were isolated from heparinized venous blood by dextran sedimentation and Ficoll-Paque centrifugation, as previously described60. Cell viability was > 98%, as determined by trypan blue exclusion. Unless stated otherwise, isolated neutrophils at 2.5 × 106 cells/ml in RPMI 1640 culture medium (Gibco) were pretreated at 37 °C and 5% CO2 for 30 min with Chiang Mai PM2.5 or standard (STD) urban PM (SRM Number: 1648a, National Institute of Standards & Technology) at concentrations ranging from 5 to 40 µg/ml. PM-pretreated neutrophils were treated with 100 ng/ml LPS (from E. coli, Sigma) for varying periods depending on the indicated measurement. In certain experiments, varying concentrations of 1α,25-dihydroxy vitamin D3 (Sigma‒Aldrich) were added to isolated neutrophils for 30 min during PM pretreatment.
Measurement of interleukin-8 production
The supernatant from PM-pretreated neutrophil cultures (with or without 10–1000 nM 1α,25-dihydroxy vitamin D3) was harvested 18 h after the addition of LPS and stored at − 80 °C prior to measurement. The IL-8 concentration was assessed in duplicate by ELISA (BD Biosciences) according to the manufacturer’s instructions. The lower limit of detection achieved for this assay is approximately 5 pg/ml.
Measurement of CXCR2 expression
Following incubations, PM-treated neutrophils, with or without 1α, 25-dihydroxy vitamin D3, were stimulated with 100 ng/ml LPS at 37 °C for 6 h. The suspended cells were then centrifuged and washed with 1 ml 10% FBS in PBS. The pelleted cells were surface stained with anti-human CXCR2-PE (BioLegend) for 30 min at 4 °C. After washing with 10% FBS in PBS, the cells were fixed with 1% paraformaldehyde (Sigma) for 10 min at 4 °C and kept at 4 °C until analysis. Data were acquired by flow cytometry (FACSCalibur; BD Biosciences).
Measurement of neutrophil migration
PM-pretreated neutrophils at a concentration of 2.5 × 106 cells/ml were incubated in the upper chamber of 3-μm-pore size Transwell plates (Corning Life Sciences), with 100 ng/ml LPS placed in the lower 0.5 ml chamber; plates were incubated at 37 °C for 1 h. Neutrophils transmigrating into the lower chamber with reference beads were counted by flow cytometry (FACSCalibur; BD Biosciences) for 10,000 events gated on the neutrophil population. The absolute count of transmigrated neutrophils was calculated by relating the number of counted cells to the total number of bead events. In certain experiments, isolated neutrophils were pretreated with 40 μg/ml of STD urban PM with or without 100 and 1000 nM of 1α,25-dihydroxyvitamin D3 at 37 °C for 30 min prior to the test for migration. The migration index was calculated by subtracting the absolute count of transmigrated neutrophils to LPS from the medium control and dividing by transmigrated neutrophils to the medium control with the following formula61:
Microorganisms
Pathogenic E. coli isolated from clinical samples was grown to mid-logarithmic phase at 37 °C in Luria–Bertani (LB) broth. Bacterial growth was assessed by measuring the optical density at 600 nm. In general, an absorbance index of 1 is equivalent to 109 CFU/ml of bacteria, and the number of viable bacteria (colony-forming units) in inocula was determined by retrospective plating of serial tenfold dilutions on LB agar.
Total killing assay
Isolated neutrophils were pretreated with 40 μg/ml STD urban PM with or without 100 nM 1α,25-dihydroxyvitamin D3 at 37 °C for 30 min before coculturing with live E. coli at an MOI of 0.1:1. The cells were lysed for bacterial counting (initial inoculum time) after 3 h. Total bacteria were quantified by colony plating at the indicated time points, and the results are expressed as percentages of bacterial survival, as calculated by dividing the decreased number of bacteria from PM-treated neutrophils with or without 1α,25-dihydroxyvitamin D3 by the number of bacteria following treatment with non-PM-treated neutrophils.
Statistics
Comparisons between treatment groups were performed using Friedman’s test with Dunn’s post hoc analysis or two-way ANOVA with Šídák's multiple comparisons test. All analyses were performed using GraphPad PRISM statistical software version 9 (GraphPad 9). P values ≤ 0.05 were considered significant.
Data availability
The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.
References
Miller, K. A. et al. Long-term exposure to air pollution and incidence of cardiovascular events in women. N. Engl. J. Med. 356, 447–458. https://doi.org/10.1056/NEJMoa054409 (2007).
Sun, Q. et al. Ambient air pollution exaggerates adipose inflammation and insulin resistance in a mouse model of diet-induced obesity. Circulation 119, 538–546. https://doi.org/10.1161/CIRCULATIONAHA.108.799015 (2009).
Xing, Y. F., Xu, Y. H., Shi, M. H. & Lian, Y. X. The impact of PM2.5 on the human respiratory system. J. Thorac. Dis. 8, E69-74. https://doi.org/10.3978/j.issn.2072-1439.2016.01.19 (2016).
Honda, T. J., Kazemiparkouhi, F., Henry, T. D. & Suh, H. H. Long-term PM2.5 exposure and sepsis mortality in a US medicare cohort. BMC Public Health 22, 1214. https://doi.org/10.1186/s12889-022-13628-5 (2022).
Chen, X. et al. Respiratory inflammation and short-term ambient air pollution exposures in adult Beijing residents with and without prediabetes: A panel study. Environ. Health Perspect. 128, 67004. https://doi.org/10.1289/EHP4906 (2020).
Neophytou, A. M. et al. Traffic-related exposures and biomarkers of systemic inflammation, endothelial activation and oxidative stress: A panel study in the US trucking industry. Environ. Health 12, 105. https://doi.org/10.1186/1476-069X-12-105 (2013).
Hajat, A. et al. Long-term exposure to air pollution and markers of inflammation, coagulation, and endothelial activation: A repeat-measures analysis in the multi-ethnic study of atherosclerosis (MESA). Epidemiology 26, 310–320. https://doi.org/10.1097/EDE.0000000000000267 (2015).
Tripathy, S. et al. Long-term ambient air pollution exposures and circulating and stimulated inflammatory mediators in a cohort of midlife adults. Environ. Health Perspect 129, 57007. https://doi.org/10.1289/EHP7089 (2021).
Green, R. et al. Long- and short-term exposure to air pollution and inflammatory/hemostatic markers in midlife women. Epidemiology 27, 211–220. https://doi.org/10.1097/EDE.0000000000000421 (2016).
Glencross, D. A., Ho, T. R., Camina, N., Hawrylowicz, C. M. & Pfeffer, P. E. Air pollution and its effects on the immune system. Free Radic. Biol. Med. 151, 56–68. https://doi.org/10.1016/j.freeradbiomed.2020.01.179 (2020).
Valderrama, A., Zapata, M. I., Hernandez, J. C. & Cardona-Arias, J. A. Systematic review of preclinical studies on the neutrophil-mediated immune response to air pollutants, 1980–2020. Heliyon 8, e08778. https://doi.org/10.1016/j.heliyon.2022.e08778 (2022).
Lin, C. M. et al. N-acetylcysteine alleviates fine particulate matter (PM2.5)-induced lung injury by attenuation of ROS-mediated recruitment of neutrophils and Ly6C(high) monocytes and lung inflammation. Ecotoxicol. Environ. Saf. 239, 113632. https://doi.org/10.1016/j.ecoenv.2022.113632 (2022).
Fox, S. E., Lu, W., Maheshwari, A., Christensen, R. D. & Calhoun, D. A. The effects and comparative differences of neutrophil specific chemokines on neutrophil chemotaxis of the neonate. Cytokine 29, 135–140. https://doi.org/10.1016/j.cyto.2004.10.007 (2005).
Salvi, S. et al. Acute inflammatory responses in the airways and peripheral blood after short-term exposure to diesel exhaust in healthy human volunteers. Am. J. Respir. Crit. Care Med. 159, 702–709. https://doi.org/10.1164/ajrccm.159.3.9709083 (1999).
Salvi, S. S. et al. Acute exposure to diesel exhaust increases IL-8 and GRO-alpha production in healthy human airways. Am. J. Respir. Crit. Care Med. 161, 550–557. https://doi.org/10.1164/ajrccm.161.2.9905052 (2000).
Behndig, A. F. et al. Proinflammatory doses of diesel exhaust in healthy subjects fail to elicit equivalent or augmented airway inflammation in subjects with asthma. Thorax 66, 12–19. https://doi.org/10.1136/thx.2010.140053 (2011).
Zhang, Y., Geng, S., Prasad, G. L. & Li, L. Suppression of neutrophil antimicrobial functions by total particulate matter from cigarette smoke. Front. Immunol. 9, 2274. https://doi.org/10.3389/fimmu.2018.02274 (2018).
Ciesielska, A., Matyjek, M. & Kwiatkowska, K. TLR4 and CD14 trafficking and its influence on LPS-induced pro-inflammatory signaling. Cell Mol. Life Sci. 78, 1233–1261. https://doi.org/10.1007/s00018-020-03656-y (2021).
Bleakley, A. S., Licciardi, P. V. & Binks, M. J. Vitamin D modulation of the innate immune response to paediatric respiratory pathogens associated with acute lower respiratory infections. Nutrients https://doi.org/10.3390/nu13010276 (2021).
Pletz, M. W. et al. Vitamin D deficiency in community-acquired pneumonia: Low levels of 1,25(OH)2 D are associated with disease severity. Respir. Res. 15, 53. https://doi.org/10.1186/1465-9921-15-53 (2014).
de Sa Del Fiol, F., Barberato-Filho, S., Lopes, L. C. & de Cassia Bergamaschi, C. Vitamin D and respiratory infections. J. Infect. Dev. Ctries 9, 355–361. https://doi.org/10.3855/jidc.5711 (2015).
Ganmaa, D. et al. Vitamin D, respiratory infections, and chronic disease: Review of meta-analyses and randomized clinical trials. J. Intern. Med. 291, 141–164. https://doi.org/10.1111/joim.13399 (2022).
Pfeffer, P. E. & Hawrylowicz, C. M. Vitamin D in asthma: Mechanisms of action and considerations for clinical trials. Chest 153, 1229–1239. https://doi.org/10.1016/j.chest.2017.09.005 (2018).
Subramanian, K., Bergman, P. & Henriques-Normark, B. Vitamin D promotes pneumococcal killing and modulates inflammatory responses in primary human neutrophils. J. Innate Immun. 9, 375–386. https://doi.org/10.1159/000455969 (2017).
Xin, L. et al. 1,25-Dihydroxy vitamin D3 attenuates the oxidative stress-mediated inflammation induced by PM2.5via the p38/NF-kappaB/NLRP3 pathway. Inflammation 42, 702–713. https://doi.org/10.1007/s10753-018-0928-y (2019).
Pfeffer, P. E. et al. Effects of vitamin D on inflammatory and oxidative stress responses of human bronchial epithelial cells exposed to particulate matter. PLoS ONE 13, e0200040. https://doi.org/10.1371/journal.pone.0200040 (2018).
Chen, Y. H. et al. The induction of Alpha-1 antitrypsin by vitamin D in human T cells is TGF-beta dependent: A proposed anti-inflammatory role in airway disease. Front. Nutr. 8, 667203. https://doi.org/10.3389/fnut.2021.667203 (2021).
Dimeloe, S. et al. Vitamin D (1,25(OH)2D3) induces alpha-1-antitrypsin synthesis by CD4(+) T cells, which is required for 1,25(OH)2D3-driven IL-10. J Steroid Biochem. Mol. Biol. 189, 1–9. https://doi.org/10.1016/j.jsbmb.2019.01.014 (2019).
van Hoof, H. J., Swinkels, L. M., Ross, H. A., Sweep, C. G. & Benraad, T. J. Determination of non-protein-bound plasma 1,25-dihydroxyvitamin D by symmetric (rate) dialysis. Anal. Biochem. 258, 176–183. https://doi.org/10.1006/abio.1998.2586 (1998).
Bikle, D., Bouillon, R., Thadhani, R. & Schoenmakers, I. Vitamin D metabolites in captivity? Should we measure free or total 25(OH)D to assess vitamin D status? J. Steroid Biochem. Mol. Biol. 173, 105–116. https://doi.org/10.1016/j.jsbmb.2017.01.007 (2017).
Diseases, G. B. D. & Injuries, C. Global burden of 369 diseases and injuries in 204 countries and territories, 1990–2019: A systematic analysis for the Global Burden of Disease Study 2019. Lancet 396, 1204–1222. https://doi.org/10.1016/S0140-6736(20)30925-9 (2020).
Schraufnagel, D. E. et al. Air pollution and noncommunicable diseases: A review by the forum of international respiratory societies’ environmental committee, part 2: Air pollution and organ systems. Chest 155, 417–426. https://doi.org/10.1016/j.chest.2018.10.041 (2019).
Arias-Perez, R. D. et al. Inflammatory effects of particulate matter air pollution. Environ. Sci. Pollut. Res. Int. 27, 42390–42404. https://doi.org/10.1007/s11356-020-10574-w (2020).
Zhang, S. et al. Particulate air pollution and respiratory Haemophilus influenzae infection in Mianyang, Southwest China. Environ. Sci. Pollut. Res. Int. https://doi.org/10.1007/s11356-021-13103-5 (2021).
Zhang, S., Chen, X., Dai, C., Wang, J. & Wang, H. Associations between air pollutants and risk of respiratory infection: Patient-based bacterial culture in sputum. Environ. Geochem. Health 44, 4007–4016. https://doi.org/10.1007/s10653-021-01154-z (2022).
Wang, J. et al. Associations between ambient air pollution and mortality from all causes, pneumonia, and congenital heart diseases among children aged under 5 years in Beijing, China: A population-based time series study. Environ. Res. 176, 108531. https://doi.org/10.1016/j.envres.2019.108531 (2019).
Nhung, N. T. T. et al. Short-term association between ambient air pollution and pneumonia in children: A systematic review and meta-analysis of time-series and case-crossover studies. Environ. Pollut. 230, 1000–1008. https://doi.org/10.1016/j.envpol.2017.07.063 (2017).
de Souza Xavier Costa, N. et al. LPS response is impaired by urban fine particulate matter. Int. J. Mol. Sci. https://doi.org/10.3390/ijms23073913 (2022).
Lin, Y. J. et al. Association between ambient air pollution and elevated risk of tuberculosis development. Infect. Drug Resist 12, 3835–3847. https://doi.org/10.2147/IDR.S227823 (2019).
Hakansson, A. P. A particulate matter: How environmental irritants and particulate matter increase sensitivity to bacterial respiratory tract infections: Commentary for Underground railway particulate matter and susceptibility to pneumococcal infection. EBioMedicine 81, 104116. https://doi.org/10.1016/j.ebiom.2022.104116 (2022).
Liu, J. et al. Particulate matter disrupts airway epithelial barrier via oxidative stress to promote Pseudomonas aeruginosa infection. J. Thorac. Dis. 11, 2617–2627. https://doi.org/10.21037/jtd.2019.05.77 (2019).
Wei, Y. et al. Short term exposure to fine particulate matter and hospital admission risks and costs in the Medicare population: Time stratified, case crossover study. BMJ 367, l6258. https://doi.org/10.1136/bmj.l6258 (2019).
Sabroe, I., Jones, E. C., Whyte, M. K. & Dower, S. K. Regulation of human neutrophil chemokine receptor expression and function by activation of Toll-like receptors 2 and 4. Immunology 115, 90–98. https://doi.org/10.1111/j.1365-2567.2005.02133.x (2005).
Uberti, F. et al. Vitamin D protects human endothelial cells from oxidative stress through the autophagic and survival pathways. J. Clin. Endocrinol. Metab. 99, 1367–1374. https://doi.org/10.1210/jc.2013-2103 (2014).
Adams, J. S. & Hewison, M. Update in vitamin D. J. Clin. Endocrinol. Metab. 95, 471–478. https://doi.org/10.1210/jc.2009-1773 (2010).
Chen, L., Eapen, M. S. & Zosky, G. R. Vitamin D both facilitates and attenuates the cellular response to lipopolysaccharide. Sci. Rep. 7, 45172. https://doi.org/10.1038/srep45172 (2017).
Liao, X. et al. Vitamin D enhances neutrophil generation and function in Zebrafish (Danio rerio). J. Innate Immun. 14, 229–242. https://doi.org/10.1159/000519183 (2022).
Bolcas, P. E. et al. Vitamin D supplementation attenuates asthma development following traffic-related particulate matter exposure. J. Allergy Clin. Immunol. 143, 386-394 e383. https://doi.org/10.1016/j.jaci.2018.04.042 (2019).
Mann, E. H. et al. Vitamin D counteracts an IL-23-dependent IL-17A(+)IFN-gamma(+) response driven by urban particulate matter. Am. J. Respir. Cell Mol. Biol. 57, 355–366. https://doi.org/10.1165/rcmb.2016-0409OC (2017).
Gupta, A. et al. Relationship between serum vitamin D, disease severity, and airway remodeling in children with asthma. Am. J. Respir. Crit. Care Med. 184, 1342–1349. https://doi.org/10.1164/rccm.201107-1239OC (2011).
Takahashi, K. et al. Human neutrophils express messenger RNA of vitamin D receptor and respond to 1alpha,25-dihydroxyvitamin D3. Immunopharmacol. Immunotoxicol. 24, 335–347. https://doi.org/10.1081/iph-120014721 (2002).
Sabetta, J. R. et al. Serum 25-hydroxyvitamin d and the incidence of acute viral respiratory tract infections in healthy adults. PLoS ONE 5, e11088. https://doi.org/10.1371/journal.pone.0011088 (2010).
Hausler, D., Torke, S. & Weber, M. S. High-dose vitamin d-mediated hypercalcemia as a potential risk factor in central nervous system demyelinating disease. Front. Immunol. 11, 301. https://doi.org/10.3389/fimmu.2020.00301 (2020).
Siwamogsatham, O., Ongphiphadhanakul, B. & Tangpricha, V. Vitamin D deficiency in Thailand. J. Clin. Transl. Endocrinol. 2, 48–49. https://doi.org/10.1016/j.jcte.2014.10.004 (2015).
Djukic, M. et al. Vitamin d deficiency reduces the immune response, phagocytosis rate, and intracellular killing rate of microglial cells. Infect. Immun. 82, 2585–2594. https://doi.org/10.1128/IAI.01814-14 (2014).
Serre, J. et al. Local nebulization of 1alpha,25(OH)2D3 attenuates LPS-induced acute lung inflammation. Respir. Res. 23, 76. https://doi.org/10.1186/s12931-022-01997-9 (2022).
Sadeghi, K. et al. Vitamin D3 down-regulates monocyte TLR expression and triggers hyporesponsiveness to pathogen-associated molecular patterns. Eur. J. Immunol. 36, 361–370. https://doi.org/10.1002/eji.200425995 (2006).
Gois, P. H. F., Ferreira, D., Olenski, S. & Seguro, A. C. Vitamin D and infectious diseases: Simple bystander or contributing factor? Nutrients https://doi.org/10.3390/nu9070651 (2017).
Kelly, F. et al. The impact of the congestion charging scheme on air quality in London. Part 2. Analysis of the oxidative potential of particulate matter. Res Rep Health Eff Inst, 73–144 (2011).
Kewcharoenwong, C. et al. Glibenclamide reduces pro-inflammatory cytokine production by neutrophils of diabetes patients in response to bacterial infection. Sci. Rep. 3, 3363. https://doi.org/10.1038/srep03363 (2013).
Kewcharoenwong, C. et al. Glibenclamide impairs responses of neutrophils against Burkholderia pseudomallei by reduction of intracellular glutathione. Sci. Rep. 6, 34794. https://doi.org/10.1038/srep34794 (2016).
Acknowledgements
The authors would like to acknowledge Prof. Dr. Sakorn Pornprasert, Dean of the Faculty of Associated Medical Sciences, Chiang Mai University for permission to collect Chiang Mai PM2.5. We thank Sarasanant Bungadaeng, Department of Environmental Science, Faculty of Science, Chulalongkorn University, Bangkok, Thailand for PM2.5 collection. This research project was supported by the Newton Fund and Thailand Ministry of Higher Education, Science, Research and Innovation Institutional Links 2020–2021 (CH, IM, TPa, TPr and GL) and the Fundamental Fund, Chiang Mai University 2565. IM and CH received funding from the National Institute for Health Research (NIHR) Health Protection Research Unit in Environmental Exposures and Health, a partnership between the UK Health Security Agency and Imperial College, with IM receiving additional funding from the MRC Centre for Environment and Health, which is funded by the Medical Research Council (MR/S0196669/1, 2019–2024).
Author information
Authors and Affiliations
Contributions
G.L., C.H., I.M., T.P. and T.P. Led the design and setup of the project. C.K., A.K. and A.N. Performed the experiments and analysed the data. CK wrote the original draft. GL, C.H., I.M., T.P. and T.P. Reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Kewcharoenwong, C., Khongmee, A., Nithichanon, A. et al. Vitamin D3 regulates PM-driven primary human neutrophil inflammatory responses. Sci Rep 13, 15850 (2023). https://doi.org/10.1038/s41598-023-43252-1
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-023-43252-1
- Springer Nature Limited