PM(2.5)-induced oxidative stress increases intercellular adhesion molecule-1 expression in lung epithelial cells through the IL-6/AKT/STAT3/NF-κB-dependent pathway.

soma:liu-a549-001

A549 human lung type II alveolar epithelial cell line

SUBJECT:liu-mouse-wt-il6ko

C57BL/6 wild-type and IL-6 knockout mice given intratracheal PMs

Male C57BL/6 wild type (WT) mice and IL-6 knockout (KO) mice from National Taiwan University, 8-12 weeks old, 20-35 g, n = 6 per group. PMs (200-350 ug/mouse in 100 ul sterile PBS) instilled intratracheally under 2% isoflurane; controls received an equal volume of PBS. Sacrificed at Day 7 or Day 14.

Procedures approved under National Taiwan University animal care guidelines (ICCUC: 20160235)

SUBJECT:liu-mouse-wt-nac

C57BL/6 wild-type mice given NAC before intratracheal PMs

WT mice (n = 6) treated with NAC 3-5 mg/mouse (150 mg/kg body weight) in 50 ul PBS intraperitoneally one day before PM intratracheal instillation (200-350 ug/mouse).

SUBJECT:liu-mouse-plasma

C57BL/6 wild-type and IL-6 knockout mice, plasma

Plasma collected from mice (six mice per group) 7 days and 14 days after PMs treatment. Blood collected in heparinized tubes and centrifuged at 3000 rpm for 15 min.

SUBJECT:liu-human-copd

COPD patients and healthy subjects, Taoyuan General Hospital

8 COPD patients (median age 77 years, range 49-88; all with a smoking history) and 8 healthy subjects with normal pulmonary function (median age 68 years, range 51-82; none had ever been smokers). Blood collected in sterile heparinized tubes, centrifuged at 1000 x g for 10 min and stored at -80 C.

Written informed consent obtained from each individual; protocol conformed to the 1975 Declaration of Helsinki and was approved by the Ethics Committee of Taoyuan General Hospital (TYGH99025)

OX:liu-a549-ros-opms-wpms

Intracellular ROS in A549 cells exposed to O-PMs or W-PMs (DCFH-DA)

DCFH-DA fluorescence in confluent A549 cells after 24 h exposure to 50 or 100 ug/ml of the organic-extractable (O-PMs) or water-soluble (W-PMs) fraction of SRM1649b, read by fluorescence microscopy and flow cytometry. Both fractions significantly increased ROS production over control, and O-PMs-induced ROS production was higher than that induced by W-PMs. Fluorescence is shown only as images and flow histograms in Fig. 1b, so no magnitude is recorded.

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Evidence

OX:liu-a549-ros-nac

Effect of N-acetyl cysteine on PMs-induced ROS in A549 cells

A549 cells pretreated with 5 mM N-acetyl cysteine (NAC) for 1 h and then treated with 100 ug/ml O-PMs or W-PMs for 24 h, with ROS measured by DCFH-DA fluorescence microscopy and flow cytometry. NAC pretreatment significantly decreased PMs-induced ROS production, establishing that the ROS signal is the antioxidant-reversible oxidative stress this key event describes. Values appear only in Fig. 1c; none recorded.

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Evidence

GE:liu-a549-icam1-opms-dose

ICAM-1 protein in A549 cells across an O-PMs dose series (Western blot)

A549 cells incubated with 0, 25, 50 or 100 ug/ml O-PMs for 24 h and ICAM-1 protein in cell lysates measured by Western blot with GAPDH as loading control. O-PMs significantly increased ICAM-1 expression in a dose-dependent manner, by 1.3 +/- 0.2 times the untreated control level at 25 ug/ml, 2.2 +/- 0.1 at 50 ug/ml and 2.7 +/- 0.2 at 100 ug/ml. These are the only group-level magnitudes stated numerically in the paper's running text and are recorded here as fold change relative to untreated control, with the spread type taken from the Statistical analysis section's declaration that all data are expressed as the mean +/- SEM. Significance was reported only as a threshold (p < 0.05 vs untreated cells), so no p_value is recorded. The result was consistent with the immunofluorescent images in Fig. 2c.

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Evidence

GE:liu-a549-icam1-wpms-dose

ICAM-1 protein in A549 cells across a W-PMs dose series (Western blot)

The water-soluble arm of the same experiment: A549 cells exposed to 0, 25, 50 or 100 ug/ml W-PMs for 24 h with ICAM-1 measured by Western blot. In contrast to O-PMs, W-PMs did not have any effect on ICAM-1 expression in the cell lysates, which is the negative result that makes the ICAM-1 induction specific to the organic-extractable fraction. Blots only in Fig. 2b; no value recorded.

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Evidence

GE:liu-a549-icam1-nac

Effect of N-acetyl cysteine on O-PMs-induced ICAM-1 in A549 cells

A549 cells pretreated with 5 mM NAC for 1 h and then treated with 100 ug/ml O-PMs for 24 h; total ICAM-1 measured by Western blot (Fig. 2d) and ICAM-1 surface expression by flow cytometry (Fig. 2e). NAC pretreatment attenuated the O-PMs-induced ICAM-1 expression in both readouts, placing ROS upstream of ICAM-1 induction. ICAM-1 antibody treatment also significantly decreased monocyte adhesion to O-PMs-treated epithelial cells, confirming the adhesion is ICAM-1-mediated. Values appear only in Fig. 2d-f; none recorded.

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Outputs

Evidence

GE:liu-a549-u937-adhesion

U937 monocyte adhesion to PMs-treated A549 cells

BCECF/AM-labelled U937 monocytic cells incubated for 1 h with A549 monolayers pretreated for 24 h with 50 or 100 ug/ml O-PMs or W-PMs, with or without 1 h of 5 mM NAC, and bound cells counted by fluorescence microscopy. Control confluent A549 cells showed minimal binding; adhesion was significantly increased by PMs pretreatment, and the number of monocytes that adhered to O-PMs-treated cells was 2-times higher than to W-PMs-treated cells. NAC pretreatment significantly decreased the O-PMs-increased adhesion, and co-treatment with 1 ug/ml anti-ICAM-1 antibody (against an anti-IgG isotype control) also significantly decreased it, which is what establishes the adhesion as ICAM-1-mediated. The 2-fold O-PMs-over-W-PMs ratio is the only magnitude stated for this assay and is recorded as a response_comparisons entry rather than as an output value, because the paper gives no per-group bound-cell counts. Note that the Fig. 1d legend reports mean +/- SD of three independent experiments, unlike the mean +/- SEM declared in the Statistical analysis section.

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Evidence

GE:liu-a549-akt-stat3-p65-dose

Phosphorylation of AKT, STAT3 and p65 in A549 cells across an O-PMs dose series

Western blot of phospho- and total AKT, STAT3 and p65 in A549 lysates after 24 h exposure to 0, 25, 50 or 100 ug/ml O-PMs. The phosphorylation of AKT, STAT3 and p65 was significantly increased in a concentration-dependent manner, and the increase in p-p65 was more robust than that in p-AKT and p-STAT3. Pretreatment with 5 mM NAC for 1 h significantly attenuated the phosphorylation of all three, placing ROS upstream of the axis. Densitometry appears only in Fig. 3b-c; no value recorded.

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Evidence

GE:liu-a549-mapk-dose

Phosphorylation of ERK, p38 and JNK in A549 cells across an O-PMs dose series

Western blot of phospho- and total ERK1/2, p38 and JNK in A549 lysates after 24 h exposure to 0, 25, 50 or 100 ug/ml O-PMs. O-PMs treatment did not induce phosphorylation of ERK, p38 or JNK, and the p38 inhibitor SB203580 and JNK inhibitor SP600125 did not block O-PMs-induced ICAM-1 expression. This negative MAPK result is what narrows the mechanism to the AKT/STAT3/NF-kB axis. Blots only in Fig. 3a; no value recorded.

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Evidence

GE:liu-a549-icam1-inhibitors

Pathway-inhibitor dependence of O-PMs-induced ICAM-1 and monocyte adhesion in A549 cells

A549 cells pretreated for 1 h with 10 uM BAY11-7082 (NF-kB), LY294002 (AKT), Stattic (STAT3), SB203580 (p38) or SP600125 (JNK), then treated with 100 ug/ml O-PMs for 24 h; ICAM-1 measured in lysates by Western blot and on the surface by flow cytometry, and U937 adhesion by fluorescence microscopy. The AKT, NF-kB and STAT3 inhibitors significantly down-regulated O-PMs-induced ICAM-1 expression and U937 adhesion, while the p38 and JNK inhibitors did not. Values appear only in Fig. 3d-e and Fig. 4d; none recorded.

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Evidence

GE:liu-a549-p65-translocation

NF-kB p65 nuclear translocation in O-PMs-treated A549 cells

Western blot of NF-kB p65 in cytosolic and nuclear fractions of A549 cells treated with or without 100 ug/ml O-PMs for 24 h, with Lamin A and alpha-Tubulin as fraction markers. p65 expression in the nuclear portion was increased by O-PMs compared with control cells, but not in the cytosolic portion, demonstrating O-PMs-driven nuclear translocation. Blots only in Fig. 4a; no value recorded.

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Evidence

GE:liu-a549-p65-sirna

p65 siRNA knockdown and O-PMs-induced ICAM-1, p-AKT and p-STAT3 in A549 cells

A549 cells transfected with 1 uM p65-specific siRNA for 24 h (knockdown confirmed by Western blot after 48 h of transfection across a siRNA concentration series) and then stimulated with 100 ug/ml O-PMs for 24 h. O-PMs-induced ICAM-1 expression and AKT and STAT3 phosphorylation were attenuated in the p65 siRNA-treated cells, and U937 adhesion was markedly inhibited. This places NF-kB p65 upstream of both ICAM-1 and the AKT/STAT3 phosphorylation. Blots only in Fig. 4b-d; no value recorded.

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Evidence

GE:liu-a549-il6-dose

IL-6 expression and secretion in A549 cells across an O-PMs dose series

A human cytokine antibody array on conditioned media from A549 cells with or without 100 ug/ml O-PMs for 24 h identified IL-6 as the most significantly changed cytokine between treated and untreated cells. IL-6 was then measured in cell lysates by Western blot and in conditioned media by ELISA across 0-100 ug/ml O-PMs: 25-100 ug/ml O-PMs increased IL-6 expression and secretion. Values appear only in Fig. 5a-c and Additional file 1 Table S1; none recorded.

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Evidence

GE:liu-a549-il6-blockade

IL-6 receptor blockade and IL-6 knockdown versus O-PMs-induced ICAM-1 in A549 cells

A549 cells pretreated with the IL-6 receptor inhibitor tocilizumab (TCZ, 20 ug/ml), the IL-6 trans-signalling blocker sgp130Fc (5 ug/ml), or IL-6 siRNA (1 uM), then treated with 100 ug/ml O-PMs for 24 h. TCZ significantly reduced ICAM-1 secretion and expression and also reduced the phosphorylation of AKT, p65 and STAT3. IL-6 siRNA likewise attenuated O-PMs-induced ICAM-1 expression and AKT/p65/STAT3 phosphorylation. sgp130Fc, which blocks IL-6 trans-signalling without affecting classic signalling, did not affect ICAM-1 expression, indicating the effect runs through classic IL-6 signalling. Values appear only in Fig. 5c-e and Additional file 1 Fig. S1; none recorded.

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Evidence

GE:liu-mouse-lung-icam1-il6

ICAM-1 and IL-6 in lung tissue of PMs-instilled WT and IL-6 KO mice

WT and IL-6 KO C57BL/6 mice untreated or injected intratracheally with 200-350 ug PMs per mouse and examined at Day 7 and Day 14 (n = 6 per group). Lung tissues assessed by immunohistochemical staining and Western blot. PMs significantly induced ICAM-1 and IL-6 expression in lung tissues at Day 7 and Day 14 in WT mice, along with the phosphorylation of AKT, p65 and STAT3, whereas in IL-6 KO mice the expression of ICAM-1 and IL-6 was not obvious by immunohistochemistry. This is the in vivo genetic test that IL-6 is required for the PMs-induced ICAM-1 response. Values appear only in Fig. 6a-c; none recorded.

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Evidence

GE:liu-mouse-lung-nac

Effect of N-acetyl cysteine on PMs-induced ICAM-1 and IL-6 in mouse lung

WT mice (n = 6) given NAC 3-5 mg/mouse (150 mg/kg body weight) intraperitoneally one day before intratracheal instillation of 200-350 ug PMs per mouse, assessed at Day 7. Pre-administration of NAC significantly attenuated PMs-induced ICAM-1 and IL-6 expression in lung tissues by immunohistochemical staining, and attenuated the expression of ICAM-1 and IL-6 as well as the phosphorylation of AKT, p65 and STAT3 by Western blot. This reproduces in vivo the ROS dependence established in A549 cells. Values appear only in Fig. 6f-g; none recorded.

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Evidence

GE:liu-mouse-plasma-sicam1-il6

Plasma sICAM-1 and IL-6 in PMs-instilled WT and IL-6 KO mice (ELISA)

Plasma sICAM-1 and IL-6 measured by ELISA in WT and IL-6 KO mice at Day 7 and Day 14 after intratracheal PMs (n = 6 per group). Plasma concentrations of sICAM-1 and IL-6 were higher in the WT mice, with or without PMs treatment, than in the IL-6 KO mice at both timepoints, and NAC attenuated the release of PMs-induced sICAM-1 and IL-6 in plasma. This is the systemic compartment of the same response measured in lung tissue. Values appear only in Fig. 6d-e and 6h-i; none recorded.

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Evidence

GE:liu-human-copd-plasma

Plasma sICAM-1, IL-6, WBC and CRP in COPD patients versus healthy subjects

Plasma from 8 COPD patients with a smoking history and 8 never-smoking healthy subjects with normal pulmonary function, assayed for sICAM-1 and IL-6 by ELISA alongside white blood cell count and C-reactive protein. COPD patients had higher plasma levels of sICAM-1 and IL-6 than healthy subjects, consistent with the animal model, and plasma WBC and CRP also showed a highly statistically significant increase. Table 1 lists sICAM-1 and IL-6 for each of the 16 individuals, but the paper reports no group mean or spread for either marker and the group-level results appear only as Fig. 7a-d, so no numeric value is recorded here rather than deriving one from the individual rows. IMPORTANT - this cohort was NOT exposed to ambient PM2.5: the paper enrolled long-term smokers with COPD explicitly in place of PMs-inhaled humans, so the exposure recorded for it is cigarette smoke. The arm therefore corroborates that the sICAM-1/IL-6 signature seen in PMs-treated mice is also present in human COPD, but it is not itself a PM2.5 exposure measurement.

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Outputs

Evidence

COMP:liu-icam1-opms25

ICAM-1 fold change, O-PMs 25 ug/ml vs untreated control

Reported directly in the Results text as a fold change relative to untreated control levels. Significance is given only as a threshold (p < 0.05 vs untreated cells), so no p_value is recorded.

COMP:liu-icam1-opms50

ICAM-1 fold change, O-PMs 50 ug/ml vs untreated control

Reported directly in the Results text as a fold change relative to untreated control levels. Significance is given only as a threshold (p < 0.05 vs untreated cells and vs O-PMs 25 ug/ml), so no p_value is recorded.

COMP:liu-icam1-opms100

ICAM-1 fold change, O-PMs 100 ug/ml vs untreated control

Reported directly in the Results text as a fold change relative to untreated control levels, the top of a dose-dependent series. Significance is given only as a threshold (p < 0.05 vs untreated cells and vs O-PMs 50 ug/ml), so no p_value is recorded.

COMP:liu-u937-adhesion-opms-vs-wpms

U937 monocyte adhesion, O-PMs-treated vs W-PMs-treated A549 cells

The paper states that the number of monocytes adhering to O-PMs-treated cells was 2-times higher than to W-PMs-treated cells. Two things about this entry are unusual and deliberate. First, the comparison is fraction-vs-fraction rather than treated-vs-untreated: control_output here is the W-PMs 100 ug/ml record, which is the comparator the paper used, not an unexposed control. Second, compared_measurement is omitted: the measured quantity is a count of bound U937 cells, which has no named measurement slot on GeneExpressionOutput, and neither referenced output record carries a measurement slot at all, so naming one would point at a field that does not exist. No per-group bound-cell counts are reported, so the ratio is recorded only at this analysis layer.

KER:liu-oxidative-stress-to-pi3k-akt

Increased oxidative stress leads to PI3K/AKT pathway activation

Tested directly rather than assumed: 24 h of O-PMs raised DCFH-DA ROS and, in the same cells, raised phospho-AKT, phospho-STAT3 and phospho-p65 in a concentration-dependent manner, and 1 h of 5 mM NAC before the exposure attenuated both the ROS and all three phosphorylation events. An antioxidant arm that removes the upstream event and abolishes the downstream one is the strongest design available in a single in vitro study, but the exposure is a complex particulate extract and no ROS-donor arm was run in the other direction, so support is recorded as moderate rather than strong.

KER:liu-pi3k-akt-to-airway-inflammation

PI3K/AKT pathway activation leads to airway inflammation

The pathway dependence was established pharmacologically and genetically: LY294002 (AKT), BAY11-7082 (NF-kB) and Stattic (STAT3) each significantly down-regulated O-PMs-induced ICAM-1 expression and U937 monocyte adhesion, p65 siRNA attenuated ICAM-1 together with AKT and STAT3 phosphorylation, and p65 translocated to the nucleus on exposure. The p38 and JNK inhibitors did not, which rules out the parallel MAPK route. Three independent inhibitors plus a knockdown converging on the same endpoint is strong in vitro evidence; it is recorded as moderate because the inflammation endpoint here is epithelial ICAM-1 and monocyte adherence in culture rather than inflammation in an intact airway.

KER:liu-oxidative-stress-to-airway-inflammation

Increased oxidative stress leads to airway inflammation

The paper's overall conclusion, and the edge its title names: PMs-induced ROS contribute to ICAM-1 expression through IL-6/AKT/STAT3/NF-kB. Supported in both directions of intervention and in both compartments - NAC lowered ROS, ICAM-1 and monocyte adhesion in A549 cells, and pre-administration of NAC attenuated PMs-induced ICAM-1 and IL-6 in mouse lung. Recorded as indirect because the authors place IL-6 and the kinase cascade between the two events rather than treating the link as immediate.

KER:liu-airway-inflammation-to-systemic-inflammation

Airway inflammation is accompanied by increased systemic inflammation

The same intratracheal exposure that raised ICAM-1 and IL-6 in mouse lung tissue also raised plasma sICAM-1 and IL-6, and NAC lowered both compartments together; in the human arm, COPD patients had higher plasma sICAM-1, IL-6, WBC and CRP than healthy subjects. The airway and systemic compartments were measured in parallel and nothing tests which drives which, and the human arm is cross-sectional with eight subjects per group, so this is recorded as an association with weak support.

PROTOCOL:liu-pm-extraction-001

Preparation of water-soluble (W-PMs) and organic-extractable (O-PMs) fractions of SRM1649b

The particulate matter was NIST standard reference material 1649b (SRM1649b), composed of urban dust. 50 mg of PMs was dispersed in 1 ml of milli-Q water or in 1 ml of dimethyl sulfoxide (DMSO), shaken and ultra-sonicated for 2 h in an ultrasonic bath, then centrifuged at 13000 x g for 30 min; the supernatant was collected as the water-soluble (W-PMs) or organic-extractable (O-PMs) fraction. All extracts were stored at -20 C until biological analysis.

Equipment: Ultrasonic bath; Refrigerated centrifuge (13000 x g); NIST SRM1649b urban dust

PROTOCOL:liu-mtt-001

MTT cytotoxicity assay in A549 cells

A549 cells seeded in 96-well plates at 1 x 10^4 cells per well in 100 ul medium, cultured 24 h, then treated with 0, 25, 50, 100, 200 or 400 ug/ml of PMs for 24, 48 or 72 h. 20 ul of MTT (1 mg/ml in PBS) added per well and incubated 2 h at 37 C, then 100 ul DMSO added and absorbance read at 570 nm on a microplate spectrophotometer (Thermo MK3). Data expressed as a percentage of the solvent control (0.1% DMSO), set to 100%. Experiments performed in triplicate and repeated 3 times.

Equipment: Microplate spectrophotometer (Thermo MK3); MTT reagent

PROTOCOL:liu-dcfhda-001

DCFH-DA detection of intracellular ROS in A549 cells

Confluent A549 cells on 12-well plates treated with 50 or 100 ug/ml of W-PMs or O-PMs for 24 h, then 10 uM 2',7'-dichlorofluorescein-diacetate (DCFH-DA) added for 20 min at 37 C in the dark. Cells washed twice with ice-cold PBS. Fluorescence observed with a fluorescence microscope (excitation 488 nm, emission 519 nm) and analysed on a FACScan flow cytometer (Becton Dickinson).

Equipment: Fluorescence microscope (excitation 488 nm, emission 519 nm); FACScan flow cytometer (Becton Dickinson); DCFH-DA probe

PROTOCOL:liu-flow-icam1-001

Flow cytometry for ICAM-1 surface expression on A549 cells

A549 cells treated or untreated for 1 h with 10 uM Bay11-7082 (NF-kB inhibitor), LY294002 (AKT inhibitor), Stattic (STAT3 inhibitor), SB203580 (p38 inhibitor) or SP600125 (JNK inhibitor), then treated with 100 ug/ml O-PMs for 24 h and washed twice with PBS. After trypsinization, 10^5 suspended A549 cells incubated in 100 ul PBS containing 5 ul FITC-conjugated anti-human ICAM-1 antibody (Dako) or FITC-conjugated control IgG for 15 min, resuspended in 500 ul PBS and analysed on a FACScan flow cytometer. Experiments performed in triplicate and repeated 3 times.

Equipment: FACScan flow cytometer

PROTOCOL:liu-adhesion-001

Epithelial cell-leukocyte (A549-U937) adhesion assay

A549 cells in 24-well plates untreated or pretreated for 1 h with 10 uM Bay11-7082, LY294002 or Stattic, 5 mM N-acetyl cysteine, 1 ug/ml anti-ICAM-1 antibody, or p65 siRNA, then treated with or without 100 ug/ml O-PMs for 24 h. U937 cells labelled for 1 h at 37 C with 10 mM BCECF/AM (Boehringer Mannheim) in DMSO and suspended in A549 culture medium. 10^6 labelled U937 cells added to 10^6 adherent PM-treated A549 cells and incubated 1 h; nonadherent cells removed by two gentle PBS washes and bound U937 cells counted by fluorescence microscopy.

Equipment: Fluorescence microscope; BCECF/AM (Boehringer Mannheim)

PROTOCOL:liu-cytokinearray-001

Human cytokine antibody array on A549 conditioned media

A human cytokine antibody array (RayBiotech) used to identify cytokines secreted from A549 cells with or without O-PMs treatment, following the manufacturer's instructions. The signal intensity of each spot was analysed with ImageJ and normalized to the average of the positive control spots to determine the variation of the cytokines.

Equipment: Human cytokine antibody array (RayBiotech); ImageJ densitometry

PROTOCOL:liu-wb-001

Western blot for ICAM-1, IL-6, MAPKs, AKT, STAT3 and p65

Cells harvested in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride) with protease and phosphatase inhibitor; lysates centrifuged 14000 x g for 30 min at 4 C. Cytoplasmic and nuclear fractions extracted with cytoplasmic extraction and NE-PER Nuclear reagents (Thermo Fisher Scientific). 20 ug of protein per lane resolved by SDS-PAGE and transferred to PVDF membranes (Pall), blocked 30 min at room temperature in 5% nonfat milk in Tris-buffered saline with 0.2% Tween 20. Membranes incubated overnight at 4 C with primary antibodies, then 1 h at room temperature with HRP-conjugated goat anti-rabbit IgG (Sigma, 1:2000) and detected by chemiluminescence (NEN). Images captured on a UVP BioSpectrum 600 system and band intensity quantified by densitometry. The same lysis and blotting procedure was applied to mouse lung tissue.

Equipment: SDS-PAGE apparatus; UVP BioSpectrum 600 imaging system; NE-PER nuclear and cytoplasmic extraction reagents

PROTOCOL:liu-sirna-001

siRNA knockdown of p65 and IL-6 in A549 cells

Accell SMART pool siRNAs (Dharmacon) targeting p65 or IL-6. A 100 uM siRNA stock prepared in RNase-free water and stored at -20 C. A549 cells cultured in 6-well plates at 70-80% confluence for 24 h, then 1 uM of p65 or IL-6 siRNA added in Turbofect (Thermo Fisher Scientific). After 24 h of transfection cells were stimulated with 100 ug/ml O-PMs for 24 h. Knockdown of p65 confirmed in cell lysates by Western blot; knockdown of IL-6 confirmed in conditioned medium by ELISA.

Equipment: Accell SMART pool siRNA against p65 and IL-6 (Dharmacon); Turbofect transfection reagent (Thermo Fisher Scientific)

PROTOCOL:liu-elisa-sicam1-001

ELISA for soluble ICAM-1 in conditioned media and plasma

Conditioned media collected from A549 cells (2 x 10^5) with and without 100 ug/ml O-PMs for 24 h; plasma collected from mice and from patients. sICAM-1 determined with ELISA kits (R&D Systems) following the manufacturer's protocols. Absorbance read at 450 nm on an EL808 microplate absorbance reader (BioTek). Cell samples run in triplicate and repeated three times. Plasma was collected from 8 COPD patients and 8 healthy subjects, and from mice (six mice per group) at 7 and 14 days after PMs treatment.

Equipment: sICAM-1 ELISA kit (R&D Systems); EL808 microplate absorbance reader (BioTek)

PROTOCOL:liu-elisa-il6-001

ELISA for IL-6 in conditioned media and plasma

IL-6 determined by ELISA in A549 conditioned media and in mouse and human plasma. Human IL-6 kits from BioLegend and mouse IL-6 kits from R&D Systems, used according to the manufacturers' protocols, with absorbance read at 450 nm on an EL808 microplate absorbance reader (BioTek). Cell samples run in triplicate and repeated three times.

Equipment: Human IL-6 ELISA kit (BioLegend); Mouse IL-6 ELISA kit (R&D Systems); EL808 microplate absorbance reader (BioTek)

PROTOCOL:liu-if-icam1-001

Immunofluorescent staining of ICAM-1 in A549 cells

A sterilized coverslip with 0.1% gelatin coating placed in a 24-well plate and A549 cells seeded at 1 x 10^5 cells/ml. Confluent cells treated with 100 ug/ml W-PMs or O-PMs for 24 h, washed with PBS, fixed with 4% formaldehyde for 15 min and permeabilized with 0.3% Triton X-100 for 1 min at room temperature. Blocked in PBS with 1% bovine serum albumin for 1 h, incubated with ICAM-1 antibody (1:500, Abcam) overnight at 4 C, washed, then DyLight 488-conjugated secondary antibody (1:1000, Abcam) for 1 h at room temperature. Counterstained with 1 ug/ml DAPI for 5 min and photographed by fluorescence microscopy.

Equipment: Fluorescence microscope

PROTOCOL:liu-instillation-001

Intratracheal instillation of PMs in WT and IL-6 KO mice

Procedures followed National Taiwan University animal care guidelines (ICCUC: 20160235) and the Guide for the Care and Use of Laboratory Animals, NIH publication No. 86-23 (revised 1985). Male C57BL/6 wild-type and IL-6 knockout mice, 8-12 weeks old, 20-35 g. Mice anesthetized with inhaled 2% isoflurane, necks shaved and sterilized with 75% alcohol, a vertical 5-mm incision made and the trachea exposed; the anterior tracheal wall between the second and third cartilage rings punctured with an insulin syringe at 45 degrees to avoid damaging the posterior wall. PMs thawed at room temperature and diluted in sterile PBS to 200-350 ug per 100 ul; a 100 ul suspension slowly instilled intratracheally followed by 100 ul of clean air (n = 6 per group). In a separate WT arm, 50 ul of NAC (3-5 mg/mouse, 150 mg/kg body weight) in PBS was injected intraperitoneally one day before PMs administration. Control mice received an equal volume of PBS. Forelegs held to keep the animal upright for 10 s, then the mice were rotated to help the solution enter the lungs and the skin incision was sutured. Mice were anesthetized with Zoletil/Xylazine (2 mg/kg + 5 mg/kg) and sacrificed at Day 7 or Day 14. Blood was collected for plasma sICAM-1 and IL-6; part of the lung was immersion-fixed in 4% buffered paraformaldehyde and paraffin-embedded for immunohistochemistry, and the remainder frozen in liquid nitrogen for protein isolation.

Equipment: Isoflurane anesthesia system; Insulin syringe

PROTOCOL:liu-ihc-001

Immunohistochemistry for ICAM-1 and IL-6 in mouse lung

5-um-thick lung sections incubated overnight at 4 C with ICAM-1 or IL-6 antibodies (1:100, Abcam), then with biotin-conjugated goat anti-rabbit IgG (1:200, Vector lab) for 1 h at room temperature. Sections stained with 3,3-diaminobenzidine tetrahydrochloride (DAB), counterstained with hematoxylin and examined by light microscopy.

Equipment: Microtome; Light microscope

PROTOCOL:liu-tem-001

Transmission electron microscopy of O-PMs-treated A549 cells

A549 cells treated with 100 ug/ml O-PMs for 24 h collected by centrifugation, washed with PBS, fixed with 2% glutaraldehyde and 2% paraformaldehyde in PBS for 1 min and post-fixed with 1% osmic acid for 30 min. Samples dehydrated in graded ethanol, washed with propylene oxide and embedded in epoxy resin. Ultrathin sections cut on a Reichert ultramicrotome, stained with lead citrate and uranyl acetate and examined in a HITACHI H-7100 at 100 kV.

Equipment: Reichert ultramicrotome; HITACHI H-7100 transmission electron microscope