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. Author manuscript; available in PMC: 2024 Jul 1.
Published in final edited form as: Environ Pollut. 2023 Apr 14;328:121603. doi: 10.1016/j.envpol.2023.121603

Particulate Air Pollution Exaggerates Diet-Induced Insulin Resistance Through NLRP3 Inflammasome in Mice

Jixin Zhong 1,2, Gang Zhao 3,4, Sabrina Edwards 3, Joanne Tran 3,5, Sanjay Rajagopalan 2, Xiaoquan Rao 1,2,3,*
PMCID: PMC10164710  NIHMSID: NIHMS1894864  PMID: 37062408

Abstract

Air particulate matter 2.5 (PM2.5) has been demonstrated to exaggerate insulin resistance in both human and animal studies. However, the exact molecular mechanisms remain elusive. This study sought to assess the role of NLRP3 inflammasome in PM2.5 exposure-induced insulin resistance and explore the underlying mechanisms. Wild-type (WT), Nlrp3−/−, Tlr4Lps-d, or Nrf2−/− mice, on a normal diet or high-fat diet (HFD), were exposed to PM2.5 or filtered air (FA) in a whole-body exposure facility. Priming (first signal) and assembly (second signal) of NLRP3 inflammasome activation were assessed by measuring the transcription of Nlrp3/Il-1β and detecting the activity of caspase-1 and secretion of IL-1β. We found PM2.5 exposure exaggerated insulin resistance and increased IL-1β production in the HFD-fed WT mice, but not Nlrp3−/− mice. Gene expressions of Nlrp3 and Il-1β in the lungs and peritoneal macrophages were upregulated in WT mice exposed to PM2.5. When stimulated with LPS (first signal) or monosodium urate (second signal), PM2.5 exposure was able to enhance the activity of caspase-1 and IL-1β secretion, suggesting that PM2.5 may serve as a stimulus of either the first or second signal for NLRP3 inflammasome activation. Effects of PM2.5 on caspase-1 activation and IL-1β secretion were partially blocked in Tlr4Lps-d mice. Reactive oxygen species (ROS), co-localization of NLRP3 and mitochondria, and secondary lysosomes in macrophages were increased after PM2.5 exposure, while deficiency of antioxidant gene Nrf2 in mice significantly enhanced PM2.5-induced secretion of IL-1β. Imaging flow cytometry and transmission electron microscopy demonstrated an engulfment of PM2.5 particles by macrophages, while suppression of phagocytosis by cytochalasin D abolished PM2.5-induced transcription of Nlrp3/Il-1β. Our results demonstrated a critical role of NLRP3 inflammasome in PM2.5 exaggerated insulin resistance, and multiple pathways in the first and second signals of NLRP3 inflammasome activation may be involved.

Graphical Abstract

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Introduction

Type 2 diabetes (T2D), characterized by insulin resistance in the peripheral tissue and inadequate insulin secretion from pancreatic β cells, was mainly caused by obesity and was once considered a “western disease”. However, the prevalence of T2D has dramatically increased in developing countries in recent decades. By the year 2030, it is expected that two-thirds of all diabetes cases will occur in low- to middle-income countries, which could not be completely explained by obesity1. Therefore, there is an urgent need to study the emerging risk factors and mechanisms behind the spike in the incidence of T2D. While a number of factors including genetics, diet, physical activity, smoking, and alcohol use have been extensively studied, the role of factors in the physical environment such as air pollution has been underappreciated. Air pollution, especially particulate matter 2.5 (PM2.5) that is rising at an alarming rate particularly in developing countries, has been linked to insulin resistance and T2D in both human and animal studies2–6. It has been postulated that oxidative stress and inflammation may be involved in the pathophysiology of PM2.5-associated insulin resistance5–7. However, the exact molecular mechanism of how PM2.5 causes insulin resistance remains elusive.

Macrophages are the major immune cells involved in the development of insulin resistance8. While the initial inflammation aims to protect cells from stressors, prolonged activation of the immune system can promote insulin resistance9–11. Interleukin 1 beta (IL-1β), a major proinflammatory cytokine produced by macrophages, plays a critical role in insulin resistance and associated chronic inflammation12, 13. The bioactive form of IL-1β is mainly produced by activated inflammasomes, which are a group of cytosolic multiprotein complexes comprising a sensor protein of the NOD-like receptor (NLR) family, an adaptor apoptosis-associated speck-like (ASC) protein, and an effector caspase protein. Among various inflammasome complexes, NLR family pyrin domain containing 3 (NLRP3) inflammasome is the best-characterized inflammasome. Unlike other NLRs, the basal expression of NLRP3 in resting cells is minimal and insufficient for inflammasome activation14. Therefore, a priming signal (first signal), which increases the expressions of Nlrp3 and Il-1β, is a prerequisite for NLRP3 inflammasome activation. In addition, a second signal provided by a plethora of stimuli, such as ATP, toxin, and virus, is required to assemble the NLRP3 inflammasome complex to activate the effector component caspase-115.

Although NLRP3 inflammasome activation has been observed in PM2.5 induced metabolic disorder16, whether NLRP3 inflammasome is a mediator or bystander in PM2.5-induced insulin resistance is not clear. In particular, a cause-and-effect relationship between NLRP3 and PM2.5 in diet-induced insulin resistance using Nlrp3−/− mice has not been established. Furthermore, the underlying mechanisms of how PM2.5 activates NLRP3 inflammasome has not been studied. In this study, we used a state-of-the-art whole-body exposure method and genetic mouse models to test the hypothesis that NLRP3 inflammasome plays a pivotal role in PM2.5-induced insulin resistance. After chronic exposure to PM2.5, insulin sensitivity, NLRP3 inflammasome activation, and IL-1β secretion were examined in both WT and Nlrp3−/− mice. Both the first and second signals of NLRP3 Inflammasome activation were evaluated in primary tissue-resident macrophages, including peritoneal macrophages and bone marrow-derived macrophages, to understand the mechanism of how PM2.5 activates NLRP3 Inflammasome that leads to systemic inflammation.

Materials & Methods

Whole-body inhalational exposure to concentrated ambient fine particulate matter

Eight-week old mice were exposed to either filtered air (FA) or concentrated ambient PM2.5 via inhalation for indicated duration at 6 hours/day, 5 days/week, in a Versatile Aerosol Concentrator and Enrichment System (VACES) at the Case Western Reserve University17. In the concentrated ambient PM2.5 chamber, PM2.5 particles were drawn from ambient air and concentrated 8- to 10-fold, to a PM2.5 level similar to the heavily polluted area, in this whole-body small animal air pollution exposure system. In contrast, particles in the air of the FA chamber were removed by a high-efficiency particulate air filter (Pall Life Sciences, East Hills, NY) positioned in the inlet valve17. The exposure environment and ambient aerosol were monitored as described previously17, 18. The average concentrations of PM2.5 in the exposure chamber and ambient air during the exposure period were 74.97 ± 5.3 and 10.18 ± 0.6 μg/m3 respectively (Supplementary figure 1).

Animals

C57BL/6J (stock #000664), Nlrp3tm1Bhk/J (Nlrp3−/−; stock #021302), C3H/HeJ (Tlr4Lps-d; stock #000659), and Nfe2l2tm1Ywk/J mice (Nrf2−/−; stock #017009) used in this study were purchased from Jackson Laboratory (Bar Harbor, ME). All procedures involving animals were conducted in strict compliance with protocols approved by the IACUC of Case Western Reserve University, Oregon Health & Science University, and Huazhong University of Science and Technology.

Statistical analysis

Data are expressed as mean ± standard error of the mean (SEM) unless otherwise noted. Student’s t-test was used to compare the difference between two groups, such as macrophages from FA or PM2.5 exposed mice treated with LPS. Two-way analysis of variance (ANOVA) followed by Sidak’s or Turkey post-hoc multiple comparisons test was used to compare the means of more than two groups, such as FA and PM2.5 exposure in both WT and knockout mice. All statistical analyses were performed using GraphPad Prism software (version 7). Statistically significant values were considered if the P value < 0.05.

Detailed materials and methods are available in the Supplemental Material.

Results

Effect of 12-week PM2.5 exposure on cytokine profile and IL-1β secretion by peritoneal macrophages

Macrophages are an important mediator of inflammation in PM2.5 exposure. However, how macrophages are activated in air pollution is not clear. To examine the functional activation of macrophages in PM2.5 exposure, we first exposed C57BL/6 (WT) mice to FA or PM2.5 for 12 weeks. At the end of 12-week exposure, peritoneal exudate cells were harvested by lavage with 4 ml cold PBS. Cells were immediately plated in 12-well plates with or without LPS (100 ng/ml) stimulation, and cell culture media were collected 4 hours later for the detection of cytokine expression profile using a mouse cytokine array. Cytokines/chemokines that were secreted into the culture media were analyzed using a Proteome Profiler Mouse Cytokine Array kit, which includes 40 different cytokines and chemokines. A number of cytokines, including interleukin 1 alpha (IL-1α), and interleukin 1 beta (IL-1β), interleukin 6 (IL-6), interleukin 23 (IL-23), granulocyte colony-stimulating factor (G-CSF), and TNFα were elevated after LPS stimulation, in both FA and PM2.5 groups. However, the concentrations of these cytokines/chemokines were similar in the cell media of FA and PM2.5 exposed macrophages without any stimulation (Figure 1A & supplementary figure 3), whereas the levels of G-CSF, IL-1α, and IL-1β were significantly increased in PM2.5 group after LPS stimulation (Figures 1A & 1B). Next, we used ELISA to validate the increase of IL-1β in PM2.5 exposure observed by the semi-quantitative protein array. We harvested peritoneal macrophages from WT mice with FA or PM2.5 exposure and concomitant feeding of a normal diet (ND) or a high hat diet (HFD) for 12 weeks. Cells were treated with PBS (control, Ctrl), LPS, or LPS and ATP to induce NLRP3 inflammasome activation. Cell culture media were collected to measure the secretion of IL-1β 4 hours later. Within the PBS treatment group, PM2.5 exposure increased the secretion of IL-1β in peritoneal macrophages isolated from HFD-fed but not ND-fed mice. However, when stimulated with LPS, or LPS and ATP to induce inflammasome activation, there was a significant increase of IL-1β in PM2.5 exposed peritoneal macrophages under both ND and HFD conditions (Figure 1C). These data indicate that inhalation of PM2.5 may have a weak promotive effect on IL-1β produced by peritoneal resident macrophages, which can be exaggerated with stimulations such as HFD feeding and LPS treatment.

Figure 1. The protein level of IL-1β increased in peritoneal macrophages isolated from PM2.5-exposedosed mice.

Figure 1.

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WT mice were exposed to FA or PM2.5 and simultaneously fed a normal diet (ND) or high-fat diet (HFD) for 12 weeks. A, Peritoneal exudate macrophages (PEM) collected from FA or PM2.5 exposed mice were incubated with PBS (Ctrl) or LPS (100 ng/ml) for 4 hours. Cell medium was collected for the mouse cytokine antibody array (n=3 per group). Representative images of the antibody array were shown and analyzed by ImageJ software. B, The quantitative bar graph of antibody array result in LPS treated FA and PM2.5 group (n=3 per group). C, PEM was collected from FA or PM2.5 exposed mice concomitantly fed with ND) or HFD. Cells were then incubated with PBS (Ctrl) or LPS (100 ng/ml) for 4 hours, with or without ATP (5 mM) treatment. Cell medium was collected for detection of IL-1β by ELISA (n=5 per group). Data was presented as Mean ± SEM; *P<0.05, **P<0.01.

PM2.5 induced insulin resistance and lipid deposition in the liver were ameliorated in Nlrp3−/− mice

As the major producer of cleaved IL-1β, NLRP3 inflammasome is the best characterized inflammasome. To test if NLRP3 inflammasome is involved in PM2.5 induced insulin resistance, 26 WT and 26 Nlrp3−/− mice were randomly exposed to FA or PM2.5, and simultaneously fed normal (ND, n=5 per group) or high fat diet (HFD, n=8 per group) 6 h/d, 5 d/wk, for a total of 12 weeks. The responses to glucose (IPGTT) and insulin challenge (IPITT) were similar between FA and PM in both WT and Nlrp3−/− mice with ND feeding (Figures 2A & 2B). Under HFD condition, PM2.5 exposed WT mice showed small but significantly higher glucose levels in response to both glucose (Figure 2C) and insulin (Figure 2D) challenges, compared with FA group. However, no significant differences in IPGTT and IPITT were observed between PM2.5 and FA groups in Nlrp3−/− mice (Figures 2C & 2D), suggesting that PM2.5 exposure induced insulin resistance was blunted in Nlrp3−/− mice. We also examined HFD induced lipid accumulation in the liver, white adipose tissue, and brown adipose tissue by H&E staining. PM2.5 exposed WT mice, but not Nlrp3−/− mice, have more lipid droplets in the liver, when compared to FA exposed mice (Supplementary Figure 4A). However, the lipid deposition in the white adipose tissue (Supplementary Figure 4B) and the brown adipose tissue (Supplementary Figure 4C) were not affected by either PM2.5 exposure or NLRP3 deficiency. These results suggest that NLRP3 is required in PM2.5 induced insulin resistance and lipid accumulation in the liver.

Figure 2. PM2.5-induced insulin resistance was attenuated in Nlrp3−/− mice.

Figure 2.

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WT and Nlrp3−/− mice were exposed to FA or PM2.5 for 12 weeks. Mice were simultaneously fed with ND (A & B) or HFD (C & D). Glucose tolerance test (A & C) and insulin tolerance test (B & D) were examined after exposure. The left panel of each figure is the line chart of blood glucose level or percentage of baseline glucose tested at 0, 30, 60, 90, and 120 min; the right panel of each figure is the bar graph to show the area under the curve (AUC) of the corresponding line chart. Data were presented as Mean ± SEM; n=8 per group; *P<0.05 and **P<0.01 for PM vs. FA; #P<0.05 for Nlrp3−/− vs. WT.

PM2.5 selectively promoted NLRP3 inflammasome gene transcription and increases IL-1β secretion through NLRP3 inflammasome activation

Transcription of nlrp3 and il-1β is a prerequisite for the activation of NLRP3 inflammasome. Therefore, we examined the expression of il-1β and genes encoding components of NLRP3 inflammasome, including nlrp3, asc, and caspase-1, using the middle lobe of the right lung collected from FA or PM2.5 exposed WT and Nlrp3−/− mice (n=6 per group). Expression of nlrp3 was up-regulated in the lungs of PM2.5 exposed WT mice under both HFD (Figure 3A) and ND conditions (Supplementary Figure 5). To determine if PM2.5 can also increase the transcription of other inflammasomes, we next examined the expression of other inflammasome genes in the lung tissue (n=6 per group). The expressions of naip1, naip5, nlrc4, nlrp1, and aim2 were similar between PM2.5 and FA in both WT and Nlrp3−/− mice (Figure 3B), suggesting that PM2.5 selectively enhanced the expression of nlrp3. Subsequently, peritoneal macrophages of FA or PM2.5 exposed WT and Nlrp3−/− mice (n=6 per group) were collected to test the transcription of nlrp3 and il-1β by RT-PCR, and the production of active (cleaved) IL-1β by ELISA. The upregulation of nlrp3 was confirmed in the PM2.5 exposed WT peritoneal macrophages, while the expression of il-1β was up-regulated in both PM2.5 exposed WT and Nlrp3−/− peritoneal macrophages (Figure 3C). Secretion of active IL-1β by peritoneal macrophages was significantly increased in the PM2.5 exposed WT mice with LPS stimulation, and this effect was abolished in the Nlrp3−/− mice (Figure 3D). The increase of il-1β transcription (Figure 3C), but not IL-1β secretion (Figure 3D), in PM2.5 exposed Nlrp3−/− peritoneal macrophages further suggests that PM2.5 induced maturation and secretion of IL-1β depend on NLRP3 inflammasome, rather than other inflammasomes.

Figure 3. PM2.5 exposure selectively activated NLRP3 inflammasome.

Figure 3.

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Lung tissue (A & B) and peritoneal exudate macrophages (PEMs) (C & D) were collected from WT and Nlrp3−/− mice exposed to FA or PM2.5 with HFD for 12 weeks. A, Gene expressions of Nlrp3, Caspase1, Il-1β, and Asc, the major components of Nlrp3 inflammasome, were examined in the lung tissue (n=6 per group). B, Expressions of other inflammasome genes, including Naip1, Naip5, Nlrc4, Nlrp1, and Aim2, were examined in the lung tissue (n=6 per group). C, Expressions of Nlrp3, Caspase1, and Il-1β were also tested in PEMs (n=6 per group). D, Secretion of active (cleaved) IL-1β by PEMs with or without treatment of LPS (100 ng/ml) for 4 hours was evaluated by ELISA (n=6 per group). Data was presented as Mean ± SEM; *P<0.05, **P<0.01.

PM2.5 activated the first signal of NLRP3 inflammasome

Taken together, the above results suggested that PM2.5 enhances the production of inflammatory cytokine IL-1β via activating the NLRP3 inflammasome. We then investigated how PM2.5 activates the NLRP3 inflammasome in macrophages in vivo. The above ex vivo experiments have demonstrated an up-regulation of nlrp3/il-1β expression in PM2.5 exposed animals, suggesting that PM2.5 may activate the first signal of NLRP3 inflammasome. To further test the effect of PM2.5 on the first signal in vivo, WT mice were exposed to FA or PM2.5 by inhalation 6 h/day, 5 d/wk, for a total of 4 weeks and then injected with PBS or monosodium urate (MSU, 1mg/25 mg body weight) into the peritoneal cavity (n=7 per group) (Figure 4A). MSU is a classical endogenous damage-associated molecular pattern (DAMP) that can serve as a second signal stimulus for NLRP3 inflammasome19, 20. After 16 hours, cells in the peritoneal cavity were collected for immediate detection of imaging flow cytometry (Figure 4A). Compared with the solvent control (PBS), MSU evoked significant neutrophil recruitment in both FA and PM2.5 exposed mice (Figures 4B & 4C). Nevertheless, PM2.5 exposure didn’t affect the frequencies of neutrophils (Figure 4C) and macrophages (Figure 4D) in the peritoneal cavity after MSU treatment, when compared with FA exposure. Activation of Caspase-1, the effector of inflammasome assembly, was further examined in neutrophils and macrophages separately by flow cytometry. After MSU stimulation, PM2.5 significantly enhanced the activation of caspase-1 with an about one-fold increase of caspase-1+ cells in both macrophages and neutrophils (Figures 4E & 4F), indicating that PM2.5 activated the first signal of NLRP3 inflammasome in vivo.

Figure 4. PM2.5 activated both 1st and 2nd signals of NLRP3 inflammasome.

Figure 4.

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A-F, PM2.5 enhanced inflammasome activation in the presence of extensive 2nd activation signal of NLRP3 inflammasome. WT mice were exposed to FA or PM2.5 for 4 weeks. PBS or monosodium urate (MSU) was then injected into the peritoneal cavity and peritoneal exudate cells were collected 16 hours later, for the detection of enzymatic activity of caspase-1 using flow cytometry. A, Flow chart of experimental design. B, Gating strategy of neutrophils and macrophages isolated from the peritoneal cavity. C & D, Percentage of neutrophils (C) and macrophages (D) in total cells isolated from the peritoneal cavity of mice. N=5 per group for PBS treatments and n=7 per group for MSU treatments. E & F, Representative density plots (E), and statistical analysis (F) of caspase-1 positive macrophages and neutrophils were shown. N=5 per group for PBS treatments and n=7 per group for MSU treatments. G-I, PM2.5 enhanced inflammasome activation in the presence of extensive 1st activation signal of NLRP3 inflammasome. WT mice were exposed to FA or PM2.5 for 4 weeks and then peritoneally injected with LPS or PBS (vehicle control) for 4 hours (G). Immediately after that, the activity of caspase-1 in peritoneal macrophages was detected by flow cytometry (H). The protein level of active IL-1β in the peritoneal lavage fluid was assessed by ELISA (I). N=5 per group. Data was presented as Mean ± SEM; n=5–7; *P<0.05, ****P<0.0001.

PM2.5 induced the second signal of NLRP3 inflammasome

Similarly, to test if PM2.5 affects the second signal of NLRP3 inflammasome activation, WT mice were exposed to FA or PM2.5 by inhalation 6 h/d, 5 d/wk, for a total of 4 weeks, followed by peritoneal injection of PBS or LPS (1.5 mg/kg body weight) (n=5 per group). After 4 hours of LPS stimulation, peritoneal cells and fluid were collected for caspase-1 activation and IL-1β analyses (Figure 4G). Both caspase-1+ peritoneal macrophages (Figure 4H) and the level of IL-1β in the peritoneal lavage fluid (Figure 4I) were increased in PM2.5 exposed mice after LPS injection, suggesting that PM2.5 also induces second signal for NLRP3 inflammasome activation.

Tlr4 loss-of-function mutation partially blocked PM2.5-induced NLRP3 inflammasome activation

As both LPS and PM2.5 can be recognized by toll-like receptor 4 (TLR4) to induce inflammation21, we next evaluated whether the effect of LPS and PM2.5 induced IL-1β release is blunted in TLR4 loss-of-function mutant mice (Tlr4Lps-d). WT and Tlr4Lps-d mice were exposed to FA or PM2.5 6 h/day (n=5 per group), 5 d/wk, for a total of 4 weeks. After 4-week exposure, mice were intraperitoneally injected with LPS or PBS as vehicle control. Four hours later, peritoneal exudate cells, peritoneal fluid, and plasma were collected for analyses of caspase-1 activity and IL-1β. Whereas caspase-1 activity and IL-1β secretion were significantly attenuated in Tlr4Lps-d mice compared with WT mice as expected (Figures 5A – 5C), mutation of Tlr4 in Tlr4Lps-d mice only partially blocked PM2.5 induced caspase-1 activation in peritoneal macrophages (Figure 5A) and IL-1β release in peritoneal fluid (Figure 5B), suggesting that other pattern recognition receptors may also be involved in vivo. The release of IL-1β in the plasma was almost completely blocked in Tlr4Lps-d mice (Figure 5C).

Figure 5. Loss-of-function mutation of TLR4 in Tlr4LPS-d mice abolished PM2.5-induced activation of NLRP3 inflammasome.

Figure 5.

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WT and Tlr4LPS-d mutant mice were exposed to FA or PM2.5 for 4 weeks and then peritoneally injected with LPS or PBS (vehicle control) for 4 hours (n=5 per group). Peritoneal macrophages were then harvested and immediately used for the detection of caspase-1 activity using flow cytometry. Representative density plots were shown (A). Protein levels of IL-1β in the peritoneal lavage fluid (B) and serum (C) were assessed by ELISA (n=5 per group). Data was presented as Mean ± SEM; *P<0.05, **P<0.01.

PM2.5 directly activated NLRP3 inflammasome in vitro

Following our observations in vivo, we sought to evaluate whether NLRP3 inflammasome was directly activated by PM2.5 in an in vitro condition lacking the influence of complex in vivo environment22. Bone marrow derived macrophages (BMDMs) were incubated with PBS or PM2.5 particles (50 μg/mL) for 24 hours, with or without priming with LPS (100 ng/mL) for 4 hours. First, we examined the uptake of PM2.5 by BMDM using imaging flow cytometry. Side scatter parameter is a well-known indicator for cell granularity measured in flow cytometry23, and it has been used to measure the uptake of nanoparticles24. Cell granularity was significantly enhanced in PM2.5 treated macrophages and was further increased in LPS pre-treated cells, suggesting that the particles had been taken up by the macrophages (Figures 6A–6D). Next, we tested the caspase-1 activity in PBS and PM2.5 treated BMDM cells. Without LPS stimulation, no difference was observed between PBS and PM2.5 treated BMDM cells, suggesting that 50 μg/mL PM2.5 alone was not able to activate caspase-1 (Figure 6E–6F). However, a remarkable increase of caspase-1 activity was observed in PM2.5 treated cells if cells were pre-incubated with LPS (n=7 per group), indicating LPS augmented the effect of PM2.5 on the activation of NLRP3 inflammasome (Figures 6E–6F). Suppression of endocytosis with cytochalasin D abolished PM2.5-induced transcription of nlrp3 and il-1β in WT (both nlrp3 and il-1β) and Nlrp3−/− (il-1β) mice (Figures 6G–6H), suggesting that the enhancement of 1st NLRP3 activation signal is dependent on the uptake of PM2.5 particles.

Figure 6. PM2.5 directly activated NLRP3 inflammasome in vitro in an endocytosis-dependent manner.

Figure 6.

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Bone marrow-derived macrophages (BMDMs) were harvested from WT mice, and incubated with PBS (n=10) or PM2.5 (50 μg/ml, n=10) for 16 hours with or without pre-administration of LPS (100 ng/mL) for 4 hours. N=3 per group without LPS treatment and n=7 per group with LPS treatment. A, Bright-field images captured at the end of the treatment showing the PM2.5 particles in the cells. B-D, Histogram (B), representative images (C), and bar graphs (D) showing the granularity as indicated by the parameter of side scatter (SSC) detected by imaging flow cytometry. Purple color in the representative images indicates SSC, red color indicates F4/80, and blue color indicates nucleus. N=3 per group without LPS treatment and n=7 per group with LPS treatment. E, Representative density plots, histogram, and cell images showing the enzymatic activity of caspase-1 in the 4 groups. F, Quantitative bar graphs showing the percentage of caspase-1 positive cells (left panel) and mean fluorescence intensity (MFI, right panel) of caspase-1. N=3 per group without LPS treatment and n=7 per group with LPS treatment. G & H, WT or Nlrp3−/− BMDMs were treated with PBS or PM2.5 (50 μg/ml) for 16 hours with or without the presence of cytochalasin D, an endocytosis inhibitor. Cells were then subjected to RT-PCR analysis of nlrp3 (G) and il-1β (H). N=3 per group. The ΔΔCT method was used to calculate the relative mRNA expression, using beta-actin as the reference gene. Fold difference was calculated by comparing the relative expression of target genes in each group with WT Ctrl group. Data was presented as Mean ± SEM; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Reactive oxygen species (ROS) production, NLRP3 translocation, and lysosomes may be involved in PM2.5-induced NLRP3 activation

The precise mechanism of NLRP3 inflammasome remains to be elucidated. However, it’s been proposed that ROS and lysosome rupture may play a major role in inflammasome activation25. First, we investigated whether ROS is involved in the activation of the NLRP3 inflammasome by PM2.5 exposure. LPS-primed BMDMs were exposed to PBS or PM2.5 particles for 16 hours and subjected to ROS analysis by flow cytometry (n=3 per group). ROS was significantly enhanced by PM particles in the macrophages (Figure 7A). Further, we tested PM2.5-induced activation of NLRP3 inflammasome in WT and nuclear factor erythroid 2-related factor 2 deficient mice (Nrf2−/−), which is a key regulator in protecting against oxidative stress induced by various toxicants26. 20 WT and 20 Nrf2−/− mice were randomly exposed to FA or PM2.5 for 12 wks and then intraperitoneally injected with or without LPS to induce inflammasome activation. Compared with WT mice, PM2.5 exposure dramatically enhanced IL-1β secretion in the peritoneal of Nrf2−/− mice after LPS injection (Figure 7B), suggesting that ROS was critical for PM2.5-induced activation of NLRP3 inflammasome. Next, BMDMs treated with PBS (Control), LPS, PM2.5, and a combination of PM2.5 and LPS were stained with NLRP3 (green), mitochondria staining dye MitoTracker (red), and nuclear staining dye DAPI (blue). Compared with the control group, the expression of NLRP3 located in mitochondria (yellow fluorescence) and cytoplasm (green fluorescence) was enhanced by LPS, PM2.5, or LPS and PM2.5 (Figure 7C). In addition, unlike the homogenous distribution of NLRP3 in the cytosol of PBS- or LPS-treated macrophages, treatments with PM2.5 or LPS+PM2.5 induced a clustering of NLRP3, which may indicate the assembly of NLRP3 inflammasome. Finally, the ultrastructure of peritoneal macrophages collected from 4-week FA or PM2.5 exposed mice was observed under transmission electron microscopy (TEM). Particles, indicated by red arrows, were revealed in the peritoneal macrophages of PM2.5-exposed mice, suggesting that inhaled PM2.5 particles may be engulfed by the tissue resident macrophages (Figure 7D). Moreover, secondary lysosomes indicated by yellow arrows, which are formed by the fusion of primary lysosomes with phagocytotic vesicles, were also significantly increased after exposure (Figure 7D). These results suggest that PM2.5 may activate NLRP3 inflammasome through multiple pathways.

Figure 7. PM2.5 activated NLRP3 inflammasome by inducing ROS and phagocytosis.

Figure 7.

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A, WT BMDMs were stimulated with LPS (100 ng/ml) for 4 hours, followed by incubations with or without PM2.5 (50 μg/ml) for 16 hours (n=3 per group). Reactive oxygen species (ROS) were measured by flow cytometry. B, WT and Nrf2−/− mice were exposed to FA or PM2.5 for 12 weeks (n=5 per group). At the end of exposure, mice were intraperitoneally injected with PBS (vehicle control) or LPS (1.5 mg/kg body weight) for 4 hours. Peritoneal lavage fluid was then collected for ELISA detection of active IL-1β. C, WT BMDMs were primed with or without LPS (100 ng/ml) for 4 hours and treated with PBS (vehicle control) or PM2.5 (50 μg/ml) for 16 hours (n=5 per group). Cells were stained with NLRP3 (green), MitoTracker (red), and DAPI (blue) to show the expression and location of NLRP3, mitochondria, and nucleus. Area of green (NLRP3+) and yellow (NLRP3+ in mitochondria) fluorescence were quantified in the bar graphs (n=5 per group). D, Transmission Electron Microscopy (TEM) was performed using peritoneal macrophages isolated from FA or PM2.5 exposed WT mice to show the particles (upper panel) and the lysosomes (bottom panel) within the cells (n=3 per group). Number of particles/cell and lysosome/cell were quantified in the bar graphs (n=6–9 per group). Data were presented as Mean ± SEM; *P<0.05, ** P< 0.01, ***P< 0.001.

Discussion

Exposure to ambient PM2.5, even at levels deemed acceptable, has been associated with diabetes and insulin resistance in both humans and animals in the past few decades5, 27–29. As a leading environmental risk factor, the contribution of PM2.5 to the global health burden including metabolic diseases is rapidly rising30. Despite increasing awareness, the hazardous health effect of PM2.5 remains underestimated, and there were inadequate measures to stop this momentum, especially in low-income countries31. As one of the most challenging environmental problems, exposure to PM2.5 is inevitable and usually lifelong for most people. Therefore, developing strategies to decrease the harmful health effects of PM2.5 is urgently needed. Although building’s air handling equipment such as air purifiers and filters could be used to control indoor air quality, limited approaches are available to block outdoor pollution. Furthermore, there is currently no specific therapeutic approach to reduce the adverse health effect of PM2.5, partially due to our limited understanding of the molecular mechanisms underlying the health effect of air pollutants. Although recent evidence has revealed a role of inflammation and oxidative stress in PM2.5-associated cardiometabolic disease, the detailed molecular mechanisms remain elusive. In our study, we found that NLRP3 inflammasome activation appears to be critical for PM2.5-accelerated inflammation and insulin resistance using a small animal PM2.5 whole body in vivo exposure system. By activating priming and assembling signals, PM2.5 may induce the NLRP3 inflammasome formation in macrophages to enhance systemic inflammation. Deletion of NLRP3 blunted PM2.5-induced acceleration of HFD-induced insulin resistance in mice.

A connection between air pollution and NLRP3 inflammasome activation has been recently found. Duan et al. showed that intratracheal instillation of PM2.5 for 5 days increased immune cell infiltration and fibrosis in the heart tissue of BALB/c mice, accompanied by upregulations of NLRP3, IL-1β, IL-18, cleaved caspase-1, and cleaved IL-1β32. In another study, Jia et al. demonstrated activation of NLRP3/caspase-1 in the lung tissue of mice intratracheally instilled with PM2.5 suspension for 14 days33. In our study, by using a 12-week concentrated ambient PM2.5 whole-body inhalation exposure system, a more physiologically relevant air pollution exposure model, we demonstrated a systemic activation of the macrophage NLRP3 inflammasome by PM2.5. PM2.5 exposure not only enhanced the expression of NLRP3, the rate-limiting step of inflammasome activation, but also the transcription and secretion of downstream IL-1β. There are several other proteins that have the ability to form inflammasome besides NLRP3, including NLRP1, NLRC4, AIM2, and some less well-characterized inflammasome proteins34. We found here that inhalational PM2.5 exposure resulted in specific activation of the NLRP3 inflammasome, without any effect on other inflammasome genes such as Naip1, Naip5, Nlrc4, Nlrp1, and Aim2.

In addition to the lung tissue, macrophages isolated from the peritoneal cavity, distant from the lung tissue that have direct contact with inhaled pollutants, also showed increased activation of the NLRP3 pathway in PM2.5-exposed mice. Interestingly, peritoneal macrophages isolated from PM2.5-exposed mice exhibited significant amounts of dark particles within the cytosol under the transmission electron microscope. It is believed that small particulate matter particles, especially those with a diameter smaller than 100 nm (ultrafine particles), can pass through the lungs and travel into the bloodstream and remote organs35, 36. Therefore, macrophages in these remote organs may have direct contact with these particles, which further induces NLRP3 inflammasome activation. This may explain how PM2.5 signal transmits across the lung barrier to induce systemic inflammation and how PM2.5 activates the second signal of NLRP3 inflammasome.

Priming signal (also called the first signal) and assembling signal (also known as the second signal) are both required to activate NLRP3 inflammasome. Priming signal is conferred by PAMPs or cytokine activation, initiating the transcription of NLRP3 inflammasome components. Previous studies have reported an increased expression of NLRP3 and its downstream molecules in PM2.5 exposure16, 32, 33, suggesting an activation of the priming signal of NLRP3 inflammasome. However, the underlying mechanisms of how PM2.5 primes NLRP3 inflammasome remain elusive. We found that in vivo whole-body inhalation exposure to PM2.5 increased the transcription of Nlrp3 and Il1β in both peritoneal macrophages and lung tissue. Additionally, although the maturation/secretion of IL-1β was abolished in PM2.5-exposed Nlrp3−/− peritoneal macrophages due to the lack of essential component for NLRP3 inflammasome, PM2.5 exposure did enhance the transcription of il-1β in NLRP3 deficient macrophages, further indicating that PM2.5 activates the first signal for inflammasome activation. To confirm the effect of PM2.5 in vivo, PM2.5-exposed mice were injected with MSU, a classical inflammasome second signal activator, to provide an overwhelming second signal for inflammasome activation. As a result, PM2.5 significantly enhanced the activation of caspase-1 in the presence of MSU, by providing the first signal. Toll-like receptors (TLRs) have been identified as important receptors mediating the priming signal for NLRP3 inflammasome37. Kampfrath et al. reported that TLR4 mediates PM2.5-induced monocyte ROS and systemic inflammation response21. In this study, we also demonstrated that disruption of TLR4 signaling partially abolished PM2.5-induced NLRP3 inflammasome activation, supporting the role of TLR4 in mediating the priming signal of NLRP3 inflammasome.

By initiating the transcriptionof critical genes, the priming signal prepares the inflammasome for the subsequent activation and assembly of the inflammasome complex. The assembling signal is provided by various cellular stimuli, such as particulate matter (uric acid crystals, silica, asbestos, alum, etc.), extracellular ATP, and pore-forming toxins15. These stimuli induce inflammasome assembly by triggering potassium (K+) or chloride ions (Cl−) efflux, calcium ions flux (Ca2+), lysosomal disruption, and/or mitochondrial dysfunction38. Although PM2.5 has been associated with inflammasome activation, which indicates an activation of the first signal, such as enhancing the transcription of Nlrp3 and Il1β16, 32, whether PM2.5 affects the assembling signal, the second signal indispensable for NLRP3 inflammasome activation, has not been reported. In the presence of an LPS-induced overwhelming priming signal, PM2.5-exposed mice showed a significantly increased level of caspase-1 activation and IL-1β production, suggesting that PM2.5 may activate the second signal for NLRP3 inflammasome. It has been shown that crystalline/particulate structures can activate the assembling signal for NLRP3 inflammasome39. We confirmed in this study that macrophages were able to directly uptake PM2.5 and form particulate structures within the cytosol of macrophages as evidenced by light microscopy, flow cytometry, and transmission electron microscopy. Immunofluorescent staining also showed that PM2.5 stimulation resulted in the clustering of NLRP3 in the cytosol of macrophages, suggesting the assembling of NLRP3 inflammasome. Intracellular reactive oxygen species (ROS) and lysosome rupture are critical common pathways of the inflammasome assembling signal25. We found that in vitro PM2.5 particle treatment increased the production of ROS in BMDM. In vivo experiments also indicate that lack of Nrf2-antioxidant response significantly enhanced PM2.5-induced NLRP3 inflammasome activation. These results suggest that PM2.5 is able to activate the assembling signal of NLRP3 inflammasome. Therefore, both priming and assembling signals were activated by exposure to PM2.5. Although PM2.5 alone induces weak activation in vivo, which is probably due to the low levels of PM2.5 in the circulation, it was able to markedly increase the activation of inflammasome with the presence of either signal 1 or 2.

In summary, our study reported evidence for underlying mechanisms by which PM2.5 activates the NLRP3 inflammasome. There were some limitations within this study. First, the PM2.5 components responsible for the induction of NLRP3 inflammasome activation were not investigated in this study. PM2.5 is a complex mixture of solids and aerosols. We did not analyze the composition of PM2.5 particles and examine the major components responsible for the activation of NLRP3. In addition, we also did not differentiate the effects of PM2.5 and PM0.1 in this study. Second, the effects of PM2.5 on other pathways in inflammasome activation need further investigation. In this study, we observed the critical role of ROS production and lysosome destabilization in the activation of PM2.5-induced inflammasome activation. Our data are consistent with previous studies that observed activation of NLRP3 inflammasome upon PM2.5 exposure32, 33. However, the activation of the inflammasome is a very complicated process involving a variety of pathways such as ion fluxes and post-translational modification of NLRP3. Whether there are other pathways involved and how important those pathways are not clear. Also, obesity has also been reported to activate NLRP3 inflammasome. Whether NLRP3 activation plays a role in the enhanced susceptibility of obese individuals to PM2.5 remains elusive. Therefore, future studies are required to address these unanswered questions.

Conclusions

It remains unknown how air pollution exposure induces inflammation and accelerates obesity-induced insulin resistance. In this study, by using NLRP3 knockout animals and whole body PM2.5 inhalation exposure system, the mechanisms by which PM2.5 activates the NLRP3 inflammasome and promotes metabolic inflammation were investigated. We found that PM2.5 results in NLRP3 inflammasome activation, an important risk factor of obesity-induced insulin resistance. We demonstrated that PM2.5 particles are able to activate both the priming and assembling signals for NLRP3 inflammasome. The PM2.5-induced priming signal is partially dependent on TLR4 and the assembling signal involves the uptake of particulate substance and ROS production. In conclusion, this study confirmed the cause-and-effect relationship between PM2.5 exposure and NLRP3 inflammasome activation, and suggested that PM2.5 prompted both priming and assembly signal of NLRP3 inflammasome via activating TLR4 and ROS.

Supplementary Material

1

Highlights.

  • PM2.5-exaggerated insulin resistance was attenuated in NLRP3−/− mice.

  • PM2.5 prompted priming and assembly signal of NLRP3 inflammasome.

  • PM2.5-induced IL-1β was decreased in Tlr4Lps-d mice and increased in Nrf2−/− mice.

  • PM2.5 increased NLRP3 translocation and lysosome destabilization in macrophages.

Acknowledgements:

This work was supported by the National Institutes of Health (R00 ES026241) and the National Natural Science Foundation of China (82170470, 81974254, and 82270903).

Footnotes

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Competing Financial Interests

The authors declare they have no actual or potential competing financial interests.

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

REFERENCES

  • 1.Hu FB. Globalization of diabetes: the role of diet, lifestyle, and genes. Diabetes Care. 2011;34:1249–57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Chen Z, Salam MT, Toledo-Corral C, Watanabe RM, Xiang AH, Buchanan TA, Habre R, Bastain TM, Lurmann F, Wilson JP, Trigo E and Gilliland FD. Ambient Air Pollutants Have Adverse Effects on Insulin and Glucose Homeostasis in Mexican Americans. Diabetes Care. 2016;39:547–54. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Brook RD, Jerrett M, Brook JR, Bard RL and Finkelstein MM. The relationship between diabetes mellitus and traffic-related air pollution. J Occup Environ Med. 2008;50:32–8. [DOI] [PubMed] [Google Scholar]
  • 4.Kramer U, Herder C, Sugiri D, Strassburger K, Schikowski T, Ranft U and Rathmann W. Traffic-related air pollution and incident type 2 diabetes: results from the SALIA cohort study. Environ Health Perspect. 2010;118:1273–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Sun Q, Yue P, Deiuliis JA, Lumeng CN, Kampfrath T, Mikolaj MB, Cai Y, Ostrowski MC, Lu B, Parthasarathy S, Brook RD, Moffatt-Bruce SD, Chen LC and Rajagopalan S. Ambient air pollution exaggerates adipose inflammation and insulin resistance in a mouse model of diet-induced obesity. Circulation. 2009;119:538–46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Haberzettl P, O’Toole TE, Bhatnagar A and Conklin DJ. Exposure to Fine Particulate Air Pollution Causes Vascular Insulin Resistance by Inducing Pulmonary Oxidative Stress. Environ Health Perspect. 2016;124:1830–1839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Rao X, Zhong J, Brook RD and Rajagopalan S. Effect of Particulate Matter Air Pollution on Cardiovascular Oxidative Stress Pathways. Antioxid Redox Signal. 2018;28:797–818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Chawla A, Nguyen KD and Goh YP. Macrophage-mediated inflammation in metabolic disease. Nat Rev Immunol. 2011;11:738–49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Wellen KE and Hotamisligil GS. Inflammation, stress, and diabetes. J Clin Invest. 2005;115:1111–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Hotamisligil GS, Shargill NS and Spiegelman BM. Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science. 1993;259:87–91. [DOI] [PubMed] [Google Scholar]
  • 11.Donath MY and Shoelson SE. Type 2 diabetes as an inflammatory disease. Nat Rev Immunol. 2011;11:98–107. [DOI] [PubMed] [Google Scholar]
  • 12.Gao D, Madi M, Ding C, Fok M, Steele T, Ford C, Hunter L and Bing C. Interleukin-1beta mediates macrophage-induced impairment of insulin signaling in human primary adipocytes. Am J Physiol Endocrinol Metab. 2014;307:E289–304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Boni-Schnetzler M and Donath MY. Increased IL-1beta activation, the culprit not only for defective insulin secretion but also for insulin resistance? Cell Res. 2011;21:995–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Latz E, Xiao TS and Stutz A. Activation and regulation of the inflammasomes. Nat Rev Immunol. 2013;13:397–411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Franchi L, Munoz-Planillo R and Nunez G. Sensing and reacting to microbes through the inflammasomes. Nat Immunol. 2012;13:325–32. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Song L, Lei L, Jiang S, Pan K, Zeng X, Zhang J, Zhou J, Xie Y, Zhou L, Dong C and Zhao J. NLRP3 inflammasome is involved in ambient PM2.5-related metabolic disorders in diabetic model mice but not in wild-type mice. Inhal Toxicol. 2021;33:260–267. [DOI] [PubMed] [Google Scholar]
  • 17.Rao X, Asico LD, Zanos P, Mahabeleshwar GH, Singh Gangwar R, Xia C, Duan L, Cisse YM, Rengasamy P, Jose PA, Gould TD, Nelson R, Biswal S, Chen LC, Zhong J and Rajagopalan S. Alpha2B-Adrenergic Receptor Overexpression in the Brain Potentiate Air Pollution-induced Behavior and Blood Pressure Changes. Toxicol Sci. 2019;169:95–107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Edwards S, Zhao G, Tran J, Patten KT, Valenzuela A, Wallis C, Bein KJ, Wexler AS, Lein PJ and Rao X. Pathological Cardiopulmonary Evaluation of Rats Chronically Exposed to Traffic-Related Air Pollution. Environ Health Perspect. 2020;128:127003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Giamarellos-Bourboulis EJ, Mouktaroudi M, Bodar E, van der Ven J, Kullberg BJ, Netea MG and van der Meer JW. Crystals of monosodium urate monohydrate enhance lipopolysaccharide-induced release of interleukin 1 beta by mononuclear cells through a caspase 1-mediated process. Ann Rheum Dis. 2009;68:273–8. [DOI] [PubMed] [Google Scholar]
  • 20.Martinon F, Petrilli V, Mayor A, Tardivel A and Tschopp J. Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature. 2006;440:237–41. [DOI] [PubMed] [Google Scholar]
  • 21.Kampfrath T, Maiseyeu A, Ying Z, Shah Z, Deiuliis JA, Xu X, Kherada N, Brook RD, Reddy KM, Padture NP, Parthasarathy S, Chen LC, Moffatt-Bruce S, Sun Q, Morawietz H and Rajagopalan S. Chronic fine particulate matter exposure induces systemic vascular dysfunction via NADPH oxidase and TLR4 pathways. Circ Res. 2011;108:716–26. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Miller MR, Raftis JB, Langrish JP, McLean SG, Samutrtai P, Connell SP, Wilson S, Vesey AT, Fokkens PHB, Boere AJF, Krystek P, Campbell CJ, Hadoke PWF, Donaldson K, Cassee FR, Newby DE, Duffin R and Mills NL. Inhaled Nanoparticles Accumulate at Sites of Vascular Disease. ACS Nano. 2017;11:4542–4552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Haynes MK, Strouse JJ, Waller A, Leitao A, Curpan RF, Bologa C, Oprea TI, Prossnitz ER, Edwards BS, Sklar LA and Thompson TA. Detection of intracellular granularity induction in prostate cancer cell lines by small molecules using the HyperCyt high-throughput flow cytometry system. J Biomol Screen. 2009;14:596–609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Ibuki Y and Toyooka T. Nanoparticle uptake measured by flow cytometry. Methods Mol Biol. 2012;926:157–66. [DOI] [PubMed] [Google Scholar]
  • 25.Yang Y, Wang H, Kouadir M, Song H and Shi F. Recent advances in the mechanisms of NLRP3 inflammasome activation and its inhibitors. Cell Death Dis. 2019;10:128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Ma Q Role of nrf2 in oxidative stress and toxicity. Annu Rev Pharmacol Toxicol. 2013;53:401–26. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Zhang S, Mwiberi S, Pickford R, Breitner S, Huth C, Koenig W, Rathmann W, Herder C, Roden M, Cyrys J, Peters A, Wolf K and Schneider A. Longitudinal associations between ambient air pollution and insulin sensitivity: results from the KORA cohort study. Lancet Planet Health. 2021;5:e39–e49. [DOI] [PubMed] [Google Scholar]
  • 28.Eze IC, Hemkens LG, Bucher HC, Hoffmann B, Schindler C, Kunzli N, Schikowski T and Probst-Hensch NM. Association between ambient air pollution and diabetes mellitus in Europe and North America: systematic review and meta-analysis. Environ Health Perspect. 2015;123:381–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Bowe B, Xie Y, Li T, Yan Y, Xian H and Al-Aly Z. The 2016 global and national burden of diabetes mellitus attributable to PM2.5 air pollution. Lancet Planet Health. 2018;2:e301–e312. [DOI] [PubMed] [Google Scholar]
  • 30.Collaborators GBDRF. Global burden of 87 risk factors in 204 countries and territories, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019. Lancet. 2020;396:1223–1249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Landrigan PJ, Fuller R, Acosta NJR, Adeyi O, Arnold R, Basu NN, Balde AB, Bertollini R, Bose-O’Reilly S, Boufford JI, Breysse PN, Chiles T, Mahidol C, Coll-Seck AM, Cropper ML, Fobil J, Fuster V, Greenstone M, Haines A, Hanrahan D, Hunter D, Khare M, Krupnick A, Lanphear B, Lohani B, Martin K, Mathiasen KV, McTeer MA, Murray CJL, Ndahimananjara JD, Perera F, Potocnik J, Preker AS, Ramesh J, Rockstrom J, Salinas C, Samson LD, Sandilya K, Sly PD, Smith KR, Steiner A, Stewart RB, Suk WA, van Schayck OCP, Yadama GN, Yumkella K and Zhong M. The Lancet Commission on pollution and health. Lancet. 2018;391:462–512. [DOI] [PubMed] [Google Scholar]
  • 32.Duan S, Wang N, Huang L, Zhao Y, Shao H, Jin Y, Zhang R, Li C, Wu W, Wang J and Feng F. NLRP3 inflammasome activation is associated with PM2.5 -induced cardiac functional and pathological injury in mice. Environ Toxicol. 2019;34:1246–1254. [DOI] [PubMed] [Google Scholar]
  • 33.Jia H, Liu Y, Guo D, He W, Zhao L and Xia S. PM2.5-induced pulmonary inflammation via activating of the NLRP3/caspase-1 signaling pathway. Environ Toxicol. 2021;36:298–307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Broz P and Dixit VM. Inflammasomes: mechanism of assembly, regulation and signalling. Nat Rev Immunol. 2016;16:407–20. [DOI] [PubMed] [Google Scholar]
  • 35.Fongsodsri K, Chamnanchanunt S, Desakorn V, Thanachartwet V, Sahassananda D, Rojnuckarin P and Umemura T. Particulate Matter 2.5 and Hematological Disorders From Dust to Diseases: A Systematic Review of Available Evidence. Front Med (Lausanne). 2021;8:692008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Du Y, Xu X, Chu M, Guo Y and Wang J. Air particulate matter and cardiovascular disease: the epidemiological, biomedical and clinical evidence. J Thorac Dis. 2016;8:E8–E19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.He Y, Hara H and Nunez G. Mechanism and Regulation of NLRP3 Inflammasome Activation. Trends Biochem Sci. 2016;41:1012–1021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Swanson KV, Deng M and Ting JP. The NLRP3 inflammasome: molecular activation and regulation to therapeutics. Nat Rev Immunol. 2019;19:477–489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Hari A, Zhang Y, Tu Z, Detampel P, Stenner M, Ganguly A and Shi Y. Activation of NLRP3 inflammasome by crystalline structures via cell surface contact. Sci Rep. 2014;4:7281. [DOI] [PMC free article] [PubMed] [Google Scholar]

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