Ethics statement

All animal experiments were performed in compliance with UCLA Institutional Animal Care and Use Committee (IACUC) protocols, under a project license also approved by the UCLA IACUC. Humane care and use of animals were observed to minimize distress and discomfort.

Collection and chemical analysis of UFP

Ultrafine particles (UFP, defined as particles with an aerodynamic diameter less than 0.8 μm) were collected on 8″ × 10″ Teflon filters using a high-volume ultrafine particle (HVUP) sampler57 at flow rate of 400 L/min, about 150 meters downwind of the I-110 Freeway in central Los Angeles, during January and February of 2015. The UFP represent a mixture of pollution sources, including fresh ambient PM from areas impacted by heavy-duty diesel trucks, light duty gasoline vehicles and ship emissions, as well as PM generated by photochemical oxidation of primary organic vapors58. The collection and characterization of chemical composition was previously described5. Mass fraction of the measured chemical species (in units of ng/μg UFP), including organic matter (estimated as total organic carbon content based on the method by Turpin et al.)59 and individual elements and metals, are reported in Supplemental Table S1. The most abundant elements in our samples included Ca (29.4 ng/μg PM), Na (23.2 ng/μg PM), S (22.6 ng/μg PM), Al (11.3 ng/μg PM) and Fe (10.0 ng/μg PM). As shown in Table S1, organic matter and metals/trace elements had cumulative mass fractions of 197 ng/μg PM and 116 ng/μg PM (i.e. about 20% and 12% of the UFP mass, respectively). The remaining UFP mass is mostly comprised of inorganic ions (most importantly nitrate, sulfate and ammonium)60,61. While these ions constitute a large fraction of the UFP mass, toxic properties of UFPs in the Los Angeles basin as well as most other urban areas of the world is primarily driven by the organic compounds and redox active metal species62 and therefore the chemical analyses in this study were focused on these UFP fractions.

Mouse exposure to gavaged UFP

Age and weight-matched male low density lipoprotein receptor-null mice (Ldlr–/–) (at the age of 90 days and an average weight of 24.76 ± 0.37 g) on a C57BL/6 background (stock #002207, Jackson Laboratory, FA) were grouped randomly, and were orally administered with either vehicle control (11 mice) or 40 μg ambient UFP (12 mice) in 100 μL saline 3 days per week for 10 weeks via 22 gauge gavage needles. The orally administrated dosage of UFP at about 1.6 ug/g per session was determined based on our previous inhalation exposure at 400 μg/m3 assuming humans inhale 16 m3 air per day63. This dosage is about 5–10 folder lower than reported studies by Mutlu et al. and Kish et al.17,56. The mice were fed a Western-type high-fat diet (TD88137, 21% milk fat, 0.2% cholesterol, Harlan Laboratory) throughout the exposure period.

Measurement of plasma cytokines

Mice were euthanized by inhalation of isofluorane and cervical dislocation following completion of the 10 weeks exposure. Plasma was collected using plasma separators (BD Biosciences) as previously described64. Plasma levels of TNF-α, MCP-1 and IL-6 were analyzed by a Luminex assay (Millipore: MCYTOMAG-70K-04. Mouse Cytokine MAGNETIC Kit).

Immunohistochemistry

Ileum segments were harvested, fixed in PBS/4% paraformaldehyde, and embedded in paraffin blocks. Macrophages and neutrophils were stained using F4/80 antibody (Invitrogen, diluted at 1:100) and antibody against Ly6G (Biolegend, diluted at 1:100), respectively65. Intensity of F4/80 and Ly6G staining was quantified by the NIH ImageJ software (http://imagej.nih.gov/ij/) and presented as staining to background ratio.

Quantification of lipid metabolites

Approximately 0.2–0.6 ml of blood was drawn for plasma preparation using plasma separators (BD Biosciences). The small intestines were dissected and a portion of the ileum was cut out and rinsed with cold saline. The extraction of lipid contents and measurement of lipid metabolites in plasma and intestine extracts were performed as previously described4,64. Lysophsophotidylcholine (LPC), lysophosphatidic acid (LPA), and oxidized phospholipids including 1-palmitoyl-2-epoxyisoprostane E2-sn-glycero-3-phosphorylcholine (PEIPC), 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC), and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC) were analyzed and quantified by liquid chromatography, electron spray ionization, and tandem mass spectrometry as described previously (LC-ESI-MS/MS)4,33,64.

Analysis of cecal bile acids and sterols

Cecum content was suspended in methanol, vortexed for 10 min, centrifuged at 14,000 g for 10 min at room temperature, and the supernatant was collected. Silylation of sterols and bile acids was carried out simultaneously using supernatant. After silylation, samples were centrifuged to collect supernatants for gas chromatography (GC). Samples were injected into RTX-5 column (Restek corp. 30 m × 0.25 mm × 0.25 i.d.) and simultaneous quantification of sterols and bile acids was performed using GC (Agilent 7890A) coupled to a FID. Peak identification was based on comparison of retention times with commercial standards of 5α-cholestane (internal standard), 5β-cholanic acid (internal standard), cholesterol, coprostanol, cholestanol, cholic acid, deoxycholic acid, chenodeoxycholic acid and lithocholic acid.

Analysis of gut microbes via MiSeq sequencing

Cecum contents were used for DNA extraction using a commercial extraction system (PowerSoil DNA isolation kit, MO Bio laboratories, Inc). The quality of the DNA samples was confirmed using a Bio-Rad Experion system (Bio-Rad Laboratories, CA, USA). The 16S rRNA gene V4 variable region PCR primers 530/926 with barcode on the forward primer were used in a 30 cycle PCR using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 94 °C for 3 min, followed by 28 cycles of 94 °C for 30 s, 53 °C for 40 s and 72 °C for 1 min, after which a final elongation step at 72 °C for 5 min was performed. Following amplification, PCR products were checked in 2% agarose gel to determine the success of amplification and the relative intensity of bands. Sequencing was performed at MR DNA (www.mrdnalab.com, Shallowater, TX, USA) on a MiSeq following the manufacturer’s guidelines. Sequence data were processed using a proprietary analysis pipeline (MR DNA, Shallowater, TX, USA). Operational taxonomic units (OTUs) were defined by clustering at 3% divergence (97% similarity). Final OTUs were taxonomically classified using BLASTn against a curated Green Genes database66.

Principle component analysis (PCA) of microbiota

The dimensionality of the interrelated variables exhibiting the bacteria abundance was reduced by PCA67. Due to huge abundance differences among the bacteria, natural logarithm value of the abundance was input to the PCA to form a matrix of the number of bacteria in the taxonomic classification and the number of test samples. After obtaining the Eigen values and the Eigen vectors, the test samples were re-plotted in the coordinates of constructed by the first two Eigen vectors. The control and UFP groups were separated in the first two principal components. The PCA was applied to different taxonomic classifications; namely, phylum, class, order, family, genus, and species, respectively.

Statistical analysis

Data were expressed as mean ± SEM unless otherwise stated. Statistical analysis was done with Matlab or Graphpad Prism. Multiple comparisons were performed by one-way analysis of variance (ANOVA), and statistical significance for comparison between two groups was determined by student t-test or Wilcoxon rank-sum test when data was not normally distributed. The association of lipids and metabolites with gut microbes was assessed by Spearman’s rank correlation analysis among all animals in both control and UFP exposed groups. A p-value < 0.05 was considered statistically significant. Sample size for different assays may differ from original mouse numbers due to issues of sample preparation, capacity of device, or sample limitations, which were specified per individual assay.