PM(2.5) Exposure Inhibits Transepithelial Anion Short-circuit Current by Downregulating P2Y2 Receptor/CFTR Pathway.

Calu-3 submerged monolayer on transwell insert

Saline control male Balb/c mouse

6-8 weeks old, 18-22 g, SPF grade, saline control group

OVA plus PM2.5 male Balb/c mouse

6-8 weeks old, 18-22 g, SPF grade, OVA+PM2.5 group

Calu-3 cells in six-well plates

Male Balb/c mouse cohort, five treatment groups

6-8 weeks old, 18-22 g, SPF grade, 50 mice in five groups of 10

ASL volume after 24 h ATP and/or PM2.5, Calu-3 transwell

ASL volume recovered from the apical chamber after 24 h in four groups: PBS control, ATP 10 uM, PM2.5 100 ug/mL and ATP+PM2.5. Reported directions: ASL volume in the ATP group was higher than in the blank control group (p < 0.001), and ASL in the ATP+PM2.5 group was evidently lower than in the ATP group; the authors conclude that PM2.5 reduced ASL production. Transepithelial resistance was measured on the same monolayers: no difference between ATP and control, TER in the PM2.5 group lower than control, and TER in PM2.5+ATP significantly lower than in ATP alone. No numeric volumes or resistances are reported - only figure bars - so no magnitude is recorded here, and the schema has no slot for ASL volume (as opposed to height) or for TER.

Exposure conditions

Outputs

Evidence

ATP-induced transepithelial anion Isc, Calu-3 control vs PM2.5, 24 h

Isc measured in an Ussing chamber. A stable baseline with no Isc peak was seen in both the control and the PM2.5 group before stimulation; adding ATP 10 uM to side A produced a rapidly rising Isc peak in both. The increase in the ATP-induced Isc peak (delta-Isc) in the PM2.5 group was significantly slower than in the control group (p < 0.05), and the average rate (delta-Isc/delta-T) evidently decreased in the PM2.5 group. The paper reports these as figure traces and bars with a significance threshold only, so no current magnitudes are recorded here.

Exposure conditions

Outputs

Evidence

ATP-induced transepithelial anion Isc, control mouse trachea

Isc in freshly excised main tracheal tissue (8 mm x 8 mm) from saline control mice, stimulated with ATP 100 uM on the luminal side. A stable baseline and an ATP-induced Isc peak were recorded. Control and PM2.5 tracheal recordings come from different animals, so they are kept as separate assay records. The paper reports traces and bars only; no numeric current or rate is given.

Exposure conditions

Outputs

Evidence

ATP-induced transepithelial anion Isc, PM2.5-exposed mouse trachea

Isc in main tracheal tissue from OVA mice given nasal instillation of PM2.5 on days 21-27, stimulated with ATP 100 uM on the luminal side. PM2.5 exposure inhibited the ATP-induced transepithelial Isc of tracheal tissue relative to control (Student's t test, p < 0.05, reported in the figure). The paper reports traces and bars only; no numeric current or rate is given.

Exposure conditions

Outputs

Evidence

P2Y2R mRNA in Calu-3 cells at 24 h (qRT-PCR)

P2Y2R mRNA measured by qRT-PCR with GADPH as internal control in six groups (control, ATP 10 uM, PM2.5 100 ug/mL, Suramin 100 uM, ATP+PM2.5, Suramin+PM2.5) after 24 h. At 24 h there was no difference between the ATP group and the control group (p > 0.05). The paper reports relative expression as figure bars only; no numeric fold changes are given.

Exposure conditions

Outputs

Evidence

P2Y2R mRNA in Calu-3 cells at 48 h (qRT-PCR)

P2Y2R mRNA at 48 h. Reported directions: P2Y2R in the ATP group was evidently higher than control; P2Y2R in the ATP+PM2.5 group was significantly lower than in the ATP group; and P2Y2R was lower in the PM2.5+Suramin group than in the Suramin group. The paper reports relative expression as figure bars only; no numeric fold changes are given.

Exposure conditions

Outputs

Evidence

P2Y2R protein in Calu-3 cells at 24 h and 48 h (western blot)

P2Y2R protein by western blot with GADPH loading control, semi-quantified by densitometry. Reported directions: P2Y2R in the Suramin group and the PM2.5 group was significantly lower than control at both 24 h and 48 h (p < 0.05); P2Y2R in the ATP group was elevated relative to control after 48 h (p < 0.05); P2Y2R in the ATP+PM2.5 group was significantly lower than in the ATP group at both timepoints (p < 0.01); and P2Y2R in the PM2.5+Suramin group was obviously lower than in the Suramin group. The paper reports densitometry as figure bars only; no numeric values are given.

Exposure conditions

Outputs

Evidence

CFTR protein in Calu-3 cells at 24 h and 48 h (western blot)

CFTR protein by western blot with GADPH loading control, semi-quantified by densitometry, in the same blots as P2Y2R. Reported directions: CFTR in the Suramin group and the PM2.5 group was significantly lower than control at 24 h and 48 h (p < 0.05); CFTR in the ATP group was elevated relative to control after 48 h (p < 0.05); and CFTR in the ATP+PM2.5 group was significantly lower than in the ATP group at both timepoints (p < 0.01). The paper reports densitometry as figure bars only; no numeric values are given.

Exposure conditions

Outputs

Evidence

P2Y2R protein in mouse lung tissue (immunohistochemistry)

P2Y2R immunohistochemistry on 5 um paraffin sections of left lung, expressed as the percentage of positive cells / positive area. Reported directions: P2Y2R dramatically decreased in the OVA group relative to control; the OVA+PM2.5 group was significantly lower than the OVA group; and the OVA+ATP group significantly increased relative to the OVA group. The paper reports positive-area percentages as figure bars only; no numeric percentages are given.

Exposure conditions

Outputs

Evidence

CFTR protein in mouse lung tissue (immunohistochemistry)

CFTR immunohistochemistry on the same lung sections, expressed as the percentage of positive cells / positive area, with western blot of right lung tissue in parallel. Reported directions: CFTR dramatically decreased in the OVA group relative to control; the OVA+PM2.5 group was significantly lower than the OVA group; and the OVA+ATP group significantly increased relative to the OVA group. The paper reports positive-area percentages as figure bars only; no numeric percentages are given.

Exposure conditions

Outputs

Evidence

Goblet cell proportion in mouse bronchus (PAS staining)

PAS-positive cells counted as goblet cells on 5 um sections; goblet cells were mainly distributed near the trachea and bronchus. Reported directions: the proportion of goblet cells was significantly higher in OVA mice than in the blank group; the proportion in ATP-treated OVA mice was evidently lower than in the OVA group; and the proportion in the PM2.5 group and the Suramin group was obviously higher than in the OVA group. Semi-quantification is reported as goblet cell positive percentage in a figure only; no numeric percentages are given.

Exposure conditions

Outputs

Evidence

Th2 cytokines (IL-4, IL-5, IL-13) in mouse BALF (ELISA)

IL-4, IL-5 and IL-13 measured by ELISA in BALF from the right lung (left main bronchus ligated, right lung washed twice with 0.5 mL saline). Reported directions: all three were higher in the OVA group than in control; all three increased significantly in the OVA+PM2.5 group relative to OVA; all three were lower in the OVA+ATP group than in OVA; and IL-13 was higher in the OVA+Suramin group than in OVA. The paper reports concentrations as figure bars only and gives no numeric concentrations; the schema's BALF output class also has no IL-4, IL-5 or IL-13 slot, so these outputs carry their group and condition without a magnitude.

Exposure conditions

Outputs

Evidence

Special airway resistance (sRaw) under methacholine challenge

Whole-body plethysmography with methacholine aerosol at 0, 5, 10 and 20 mg/mL; airway responsiveness expressed as sRaw in cmH2O/mL/s. Reported directions: sRaw in OVA mice increased significantly over control, confirming the asthma model; sRaw in the OVA+PM2.5 group was significantly elevated over the OVA group; sRaw at 20 mg/mL methacholine in the OVA+Suramin group was higher than OVA alone (p < 0.05); and sRaw in the OVA+ATP group did not differ from OVA. The paper reports sRaw as figure curves only; no numeric resistances are given, so no magnitude is recorded here.

Exposure conditions

Outputs

Evidence

ATP-induced Isc, PM2.5 vs control (Calu-3, 24 h)

The increase in the ATP-induced Isc peak was significantly slower in the PM2.5 group than in the control group, and the average rate delta-Isc/delta-T evidently decreased. Reported significance: p < 0.05 by one-way ANOVA. The paper gives no magnitude for the change.

Statistical test: one-way analysis of variance

ATP-induced Isc, PM2.5 vs control (mouse trachea)

Cross-assay comparison: the control and PM2.5 tracheal recordings are separate assay records because they come from different animals. PM2.5 exposure inhibited the ATP-induced transepithelial Isc of tracheal tissue. Reported significance: p < 0.05 by Student's t test. The paper gives no magnitude for the change.

Statistical test: Student's t test

Goblet cell proportion, OVA+PM2.5 vs OVA

The proportion of PAS-positive goblet cells in the PM2.5 group was obviously higher than in the OVA group. Reported by non-parametric test with thresholds p < 0.05, p < 0.001 and p < 0.0001 marked on the figure; the paper gives no magnitude for the change.

Statistical test: non-parametric test

Goblet cell proportion, OVA+ATP vs OVA

The proportion of PAS-positive goblet cells in ATP-treated OVA mice was evidently lower than in the OVA group - the rescue arm of the same experiment. Reported by non-parametric test; the paper gives no magnitude for the change.

Statistical test: non-parametric test

Special airway resistance, OVA+PM2.5 vs OVA

sRaw in the PM2.5 group was significantly elevated over the OVA group across methacholine concentrations. Reported by one-way ANOVA with p < 0.05 marked against the OVA group; the paper gives no magnitude for the change.

Statistical test: one-way analysis of variance

Special airway resistance at 20 mg/mL methacholine, OVA+Suramin vs OVA

sRaw at 20 mg/mL methacholine in the Suramin+OVA group was higher than in the OVA group alone; the paper states the threshold P < 0.05 and no magnitude. This arm is the paper's evidence that blocking P2Y2R alone worsens airway responsiveness.

Statistical test: one-way analysis of variance

Decreased P2Y2R expression leads to decreased intracellular calcium

ATP pre-treatment raised cytoplasmic free calcium in Calu-3 cells, and Suramin (a P2Y2R antagonist) or PM2.5 reduced the ATP-induced rise, which the paper reads as P2Y2R-dependent calcium mobilisation. Measured by Fluo-3/AM flow cytometry; the paper reports mean fluorescence intensity as figure bars only, so it is recorded here as a relationship rather than as an assay with a magnitude (the schema also has no calcium-mobilisation assay type).

Decreased intracellular calcium leads to decreased CFTR function

The paper concludes that PM2.5 inhibited the transepithelial anion Isc by downregulating the P2Y2R/CFTR pathway and the P2Y2/calcium-dependent chloride channel, and suggests that activation of calcium-dependent chloride channels participates in modulating transepithelial Isc.

Decreased P2Y2R expression leads to decreased CFTR function

The central mechanism of the paper: PM2.5 downregulates the P2Y2R/CFTR pathway and so impairs the ATP-induced transepithelial anion short-circuit current, in Calu-3 monolayers and in mouse tracheal tissue alike.

Decreased CFTR function leads to decreased ASL

The ATP-induced anion current and the ASL volume fell together after PM2.5 exposure in the transwell model; the authors attribute the ASL loss to partial inhibition of the ATP-induced anion current, which is mainly controlled by CFTR.

Decreased CFTR function leads to airway inflammation

The paper's in vivo arm: PM2.5 and Suramin, both of which lower P2Y2R/CFTR, raised Th2 cytokines in BALF, lung inflammation, collagen deposition and goblet cell hyperplasia, while ATP administration reduced lung inflammation in the asthma model.

Airway inflammation leads to decreased lung function

sRaw rose in OVA mice over control and rose further with PM2.5, and rose at 20 mg/mL methacholine with Suramin; the paper concludes that impairing the ATP/P2Y2R pathway increases airway responsiveness alongside Th2 inflammatory factors.

Ussing chamber short-circuit current, Calu-3 monolayer

TER measured with an RE1600 epithelium resistor (Kingtech, Beijing). Monolayers loaded into an Ussing chamber (Kingtech, Beijing) with 5 mL K-H solution per side: solution A (NaCl 120 mM, NaHCO3 25 mM, KH2PO4 3.3 mM, K2HPO4 0.128 mM, MgCl2 1.2 mM, CaCl2 1.2 mM, mannitol 10 mM) on side A, solution B (mannitol replaced by glucose 10 mM) on side B. Agar-bridge current and voltage electrodes (1% agar in 3 M KCl). Chamber at 37 C, 95% oxygen / 5% carbon dioxide; an electrode was accepted only if the current exceeded 60 uA, and the A/B resistance difference was compensated to zero. Voltage clamped at zero; baseline Isc recorded once stable (up to 30 min), then ATP added to side A. Analysis of the response from baseline to peak: maximum difference (delta-Isc) and average rate (delta-Isc/delta-T).

Equipment: Ussing chamber, electrophysiological detection equipment (Kingtech, Beijing); RE1600 epithelium resistor (Kingtech, Beijing); Agar-bridge current and voltage electrodes (1% agar in 3 M KCl); K-H solution A and solution B

Ussing chamber short-circuit current, mouse tracheal tissue

As for the Calu-3 protocol, with tissue in place of a monolayer: main trachea excised at 8 mm x 8 mm, held in saline to preserve viability, blood vessels and connective tissue removed, mounted on the tissue clip with K-H solution A to the luminal side and solution B to the basal side. The ATP concentration used to stimulate tissue was 100 uM rather than 10 uM.

Equipment: Ussing chamber, electrophysiological detection equipment (Kingtech, Beijing); Tissue clip

Airway surface liquid volume on transwell inserts

At 100% confluency in transwell plates, 100 uL of medium containing ATP or PM2.5 was added per group; the control received 100 uL PBS. ASL volume was recovered from the chamber after 24 h with a pipette accurate to 0.01 uL. The experiment was repeated three times. The paper reports the resulting volumes only as figure bars, not as numbers.

Equipment: Transwell plates (3801, Corning); Pipette with 0.01 uL accuracy

qRT-PCR for P2Y2R in Calu-3 cells

Total RNA extracted from Calu-3 cells by the TRIzol method (Sigma); cDNA synthesised with a reverse transcription kit (Takara, Dalian). GADPH used as internal control. Experiment repeated three times.

Western blot for P2Y2R and CFTR

RIPA lysate (Solarbio, Beijing) used to extract total protein from Calu-3 cells and from right lung tissue of mice; protein quantified with a BCA Protein Assay Kit (Beyotime). 40 ug per lane separated on 10% SDS-PAGE, transferred to PVDF (Millipore), blocked in 5% skim milk / TBS-T for 1.5 h. Bands observed on a ChemiScope series imager (Clinx, Shanghai) and analysed semi-quantitatively by densitometry. Experiment repeated three times.

Fluo-3/AM flow cytometry for cytoplasmic free calcium

Calu-3 cells digested with EDTA-free trypsin and washed twice in calcium-free PBS. Each sample split in half; one half labelled with 0.5 uL Fluo-3/AM probe (Beyotime, Shanghai), the other kept as the unlabelled control. Incubated at 37 C for 30 min, washed twice in calcium-free PBS, resuspended in 400 uL PBS and incubated 20 min. FITC-channel fluorescence read on a BD Accuri C6 Plus flow cytometer; Fluo-3/AM excitation 488 nm, emission 525 nm. Mean fluorescence intensity analysed in FlowJo v10.0. Experiment repeated three times.

Immunohistochemistry for P2Y2R and CFTR in lung tissue

Left lung fixed in 4% paraformaldehyde (Sigma) for 24 h, paraffin-embedded and cut at 5 um. Sections incubated with primary antibodies, washed in PBS, then incubated with mouse anti-rabbit IgG and a streptavidin peroxidase complex for 40 min at 37 C (ZSGB-BIO, Beijing). Expression quantified by staining intensity and expressed as the percentage of positive cells.

Periodic Acid-Schiff staining for goblet cells

Tissue slices (5 um) stained with Periodic Acid-Schiff to assess hyperplasia of goblet cells in the bronchus. PAS-stained cells counted as goblet cells; result expressed as the percentage of positive cells.

ELISA for Th2 cytokines in mouse BALF

After isoflurane anaesthesia (RWD, Guangzhou) the left main bronchus was ligated with surgical thread; the right lung was washed twice with 0.5 mL saline and the lavage liquid collected. IL-4, IL-5 and IL-13 measured with mouse ELISA kits (Absin, Shanghai) per the manufacturer's instructions.

Whole-body plethysmography with methacholine challenge

Mice placed in a whole-body chamber connected to the BUXCO system (DSI, MN). Airway responsiveness expressed as special airway resistance (sRaw, cmH2O/mL/s), obtained with aerosols of methacholine at 0, 5, 10 and 20 mg/mL and analysed with FLexi Vent software.