Pro-inflammatory effects of inhaled Great Salt Lake dust particles.

SUBJECT:cowley-c57bl6-inhalation

Male and female C57Bl/6 mice, whole-body GSLD qPM2.5 inhalation

Male and female C57Bl/6 mice 8-10 weeks of age, housed ~5/cage in an AALAC accredited vivarium and fed and watered ad libitum with standard chow. The age slot records the midpoint of the stated 8-10 week range with the range itself as the variability, because the paper gives a range rather than a single age.

Acute inhalation toxicology study; mice sacrificed and BAL collected 24 h after exposure

SUBJECT:cowley-c57bl6-opa

Male and female C57Bl/6 mice, GSLD qPM2.5 by oropharyngeal aspiration

Male and female C57Bl/6 mice 8-10 weeks of age, housed ~5/cage in an AALAC accredited vivarium and fed and watered ad libitum with standard chow. Given a separate subject id from the inhalation cohort because these are different animals dosed by a different route.

Acute oropharyngeal aspiration dose-response study; BAL collected 24 h after treatment

SUBJECT:cowley-c57bl6-inhalation-3x

Male and female C57Bl/6 mice, 3x GSLD qPM2.5 inhalation, BAL protein

Male and female C57Bl/6 mice 8-10 weeks of age. Given its own subject id because the BAL protein measurements come from the 3x exposure cohort specifically (issue #132).

Acute inhalation toxicology study; BAL collected 24 h after the 3x exposure

SUBJECT:cowley-c57bl6-wildtype

Wild-type C57Bl/6 mice, 3x GSLD qPM2.5 inhalation

Wild-type C57Bl/6 mice carrying the native mouse Trpv1 allele (asparagine at residue 606), the comparator for the humanized Trpv1N606D line.

Genotype comparison arm; BAL collected 24 h after the 3x exposure

SUBJECT:cowley-trpv1n606d

Humanized Trpv1N606D mice, 3x GSLD qPM2.5 inhalation

CRISPR-Cas9 knock-in mice carrying a single N606D substitution in mouse Trpv1, replacing the mouse asparagine with the corresponding human aspartate, plus two silent changes that block re-cutting and introduce an EcoN1 site for genotyping. Generated at the University of Utah Transgenic and Gene Targeting Mouse Core and sequence-confirmed at the N1 generation.

Genotype comparison arm; BAL collected 24 h after the 3x exposure

SUBJECT:cowley-c57bl6-cfa-comparison

Male and female C57Bl/6 mice, GSLD versus CFA qPM2.5 inhalation, 3x paradigm

Male and female C57Bl/6 mice 8-10 weeks of age. Given its own subject id because this cohort includes the CFA-exposed comparator animals (issue #132).

Particle-potency comparison arm; BAL collected 24 h after the 3x exposure

SUBJECT:cowley-c57bl6-lung-rna

Male and female C57Bl/6 mice, left lung lobe RNA after GSLD qPM2.5 inhalation

Male and female C57Bl/6 mice 8-10 weeks of age. Given its own subject id because the RNA specimen is the left lung lobe rather than BAL (issue #132).

Acute inhalation toxicology study; lung taken 24 h after exposure

soma:cowley-hbec3kt-001

HBEC3-KT immortalized human bronchial epithelial cells

soma:cowley-thp1-001

Differentiated (PMA) THP-1 macrophage-like cells

BALF:cowley-gsld-inhalation-dose

BAL total cells and neutrophils after whole-body GSLD qPM2.5 inhalation, 1x to 6x

Fig. 5a and b and Supplemental Fig. 6. Male and female C57Bl/6 mice were exposed whole-body to GSLD qPM2.5 aerosol averaging ~200 mg/m3 post-chamber for 12 min (1x), 24 min (2x) or 36 min (3x) in one day, or 72 min over two days (6x), and BAL was collected 24 h after exposure. Total cell counts and the percentage of neutrophils both rose dose-dependently; the figure legend reports mean and standard deviation with 1-way ANOVA and a Dunnett post-test, significance ** p < 0.01 and **** p < 0.0001. Histology of the post-lavage tissue confirmed pulmonary inflammation, increased neutrophils and alveolar edema, and tissue flow cytometry confirmed Ly6G+ neutrophilia along with changes in eosinophils, alveolar, interstitial and monocyte-derived macrophages, CD3+ and CD8+ T cells and B cells (Supplemental Fig. 8). NO MEASUREMENT VALUES: the paper publishes these counts only as figures, so no output record carries a value and none was read off the figure. The paper states the effects were dose dependent with no difference between male and female mice.

Exposure conditions

Outputs

Evidence

BALF:cowley-gsld-opa-dose

BAL total cells and neutrophils after oropharyngeal aspiration of GSLD qPM2.5, 2.5 to 250 ug

Supplemental Fig. 7a-f. GSLD qPM2.5 was delivered by oropharyngeal aspiration in 25 uL at 2.5, 12.5, 62.5 and 250 ug, and BAL was collected 24 h later. Total cells and neutrophils rose dose-dependently, as for the inhalation arm, and the post-lavage histology showed the same pulmonary inflammation, increased neutrophils and alveolar edema. The paper states these doses model potential inhalable doses of ~86.4-375 ug PM2.5 for humans. NO MEASUREMENT VALUES: this arm is published only as a supplemental figure, so each output record carries its dose condition and no value.

Exposure conditions

Outputs

Evidence

BALF:cowley-gsld-3x-cytokines

BAL IL6 and CXCL1 protein after 3x GSLD qPM2.5 inhalation

Fig. 6e and f. CXCL1 and IL6 protein concentrations were measured by ELISA in the first 1 mL of BAL fluid from mice given the 3x inhalation exposure, and both were elevated 2-3-fold relative to control. The figure legend reports mean and standard deviation analysed by 1-way ANOVA with a Dunnett post-test. NO ABSOLUTE VALUES: the per-group concentrations appear only in the figure, so neither output record carries an il6_concentration or il8_concentration value. The stated 2-3-fold rise is recorded in response_comparisons as a fold_change whose variability is the reported range, with derivation: reported_in_paper. Note that CXCL1 (KC) is a mouse chemokine with no named BALFSputumOutput slot of its own; the paper itself describes IL8 as the human analogue of Cxcl1 and Cxcl2, and the CXCL1 comparison below therefore uses il8_concentration as its compared_measurement, following the convention used elsewhere in this corpus for murine KC.

Exposure conditions

Outputs

Evidence

BALF:cowley-gsld-3x-neutrophils-wt

BAL neutrophil percentage in wild-type C57Bl/6 mice after 3x GSLD qPM2.5 inhalation

Fig. 11c. Wild-type C57Bl/6 mice given the 3x GSLD qPM2.5 inhalation paradigm had 58 +/- 9% neutrophils in BAL. The figure legend states the data are the mean and standard deviation, analysed by one-way ANOVA with a Bonferroni post-test. This is the wild-type reference group for the Trpv1N606D comparison recorded in the companion assay.

Exposure conditions

Outputs

Evidence

BALF:cowley-gsld-3x-neutrophils-trpv1n606d

BAL neutrophil percentage in humanized Trpv1N606D mice after 3x GSLD qPM2.5 inhalation

Fig. 11c. CRISPR-Cas9 Trpv1N606D mice, in which the mouse TRPV1 residue N606 is replaced by the corresponding human aspartate, had 76 +/- 6% neutrophils in BAL after the 3x GSLD qPM2.5 inhalation paradigm, higher than the 58 +/- 9% of wild-type C57Bl/6 controls. The mice were made because mouse TRPV1 is intrinsically less sensitive than human TRPV1 to coal fly ash, and the N606D substitution sensitized mouse TRPV1 to both CFA and GSLD qPM2.5 in calcium flux assays. The authors read the greater neutrophilia as support for TRPV1 initiating lung inflammation after GSLD inhalation. The paper also notes that increases in lung cytokine mRNA and BAL cytokine protein in these mice were comparable to, and not significantly different from, wild-type mice, so no such difference is recorded here.

Exposure conditions

Outputs

Evidence

BALF:cowley-gsld-vs-cfa-3x

BAL neutrophilia after 3x inhalation of GSLD qPM2.5 versus coal fly ash qPM2.5

Supplemental Fig. 10. Using the 3x inhalation paradigm, GSLD qPM2.5 produced greater neutrophilia than an equivalent dose of coal fly ash (CFA) qPM2.5, as well as greater Cxcl2 and Muc5ac mRNA expression. CFA is a well-characterized pneumotoxic particulate from the Hunter power plant in Castledale, UT, prepared as qPM2.5 by the same cascade-impactor method, and the authors use it as the reference particle against which GSLD potency is judged. NO MEASUREMENT VALUES: this comparison is published only as a supplemental figure, so both output records carry their condition and no value.

Exposure conditions

Outputs

Evidence

GENE:cowley-il6-lung-inhalation

Mouse lung Il6 mRNA after whole-body GSLD qPM2.5 inhalation

Fig. 6a. Il6 mRNA in the left lung lobe rose after whole-body inhalation of GSLD qPM2.5, measured by TaqMan qRT-PCR and normalized to Gapdh by the comparative cycle threshold method. The paper states the effects were dose dependent with no differences between male and female mice, and that the same induction occurred after oropharyngeal aspiration (Supplemental Fig. 9a). The figure legend reports mean and standard deviation with 1-way ANOVA and a Dunnett post-test (* p < 0.05, ** p < 0.01, p < 0.001). NO MEASUREMENT VALUES: the relative expression values appear only in the figure, so each output record carries its condition and no mrna_level.

Exposure conditions

Outputs

Evidence

GENE:cowley-cxcl1-lung-inhalation

Mouse lung Cxcl1 mRNA after whole-body GSLD qPM2.5 inhalation

Fig. 6b. Cxcl1 mRNA in the left lung lobe rose after whole-body inhalation of GSLD qPM2.5, measured by TaqMan qRT-PCR and normalized to Gapdh by the comparative cycle threshold method. The paper states the effects were dose dependent with no differences between male and female mice, and that the same induction occurred after oropharyngeal aspiration (Supplemental Fig. 9b). The figure legend reports mean and standard deviation with 1-way ANOVA and a Dunnett post-test (* p < 0.05, ** p < 0.01, p < 0.001). NO MEASUREMENT VALUES: the relative expression values appear only in the figure, so each output record carries its condition and no mrna_level.

Exposure conditions

Outputs

Evidence

GENE:cowley-cxcl2-lung-inhalation

Mouse lung Cxcl2 mRNA after whole-body GSLD qPM2.5 inhalation

Fig. 6c. Cxcl2 mRNA in the left lung lobe rose after whole-body inhalation of GSLD qPM2.5, measured by TaqMan qRT-PCR and normalized to Gapdh by the comparative cycle threshold method. The paper states the effects were dose dependent with no differences between male and female mice, and that the same induction occurred after oropharyngeal aspiration (Supplemental Fig. 9c). The figure legend reports mean and standard deviation with 1-way ANOVA and a Dunnett post-test (* p < 0.05, ** p < 0.01, p < 0.001). NO MEASUREMENT VALUES: the relative expression values appear only in the figure, so each output record carries its condition and no mrna_level.

Exposure conditions

Outputs

Evidence

GENE:cowley-il6-hbec-chelators

IL6 mRNA in HBEC3-KT cells - GSLD qPM2.5 with calcium/metal chelation and TLR4 inhibition

Fig. 8a. HBEC3-KT cells treated for 24 h with GSLD qPM2.5 at 0.25 mg/mL (66 ug/cm2) induced IL6 mRNA, reproducing the Il6 induction seen in mouse lung. The induction was significantly reduced by co-treatment with the metal/calcium chelator EGTA plus the non-selective calcium channel blocker ruthenium red (10 + 50 uM), and was also reduced - though the authors describe the effect as slight - by the TLR4 inhibitor TAK-242 (1 uM) and by the iron chelator deferoxamine (50 uM). The figure legend states the data are mean and standard deviation analysed by 1-way ANOVA with a Dunnett post-test (** p < 0.01, **** p < 0.0001), with all treatment groups corrected for their respective controls and normalized to GSLD qPM2.5 treated cells. Separately (Fig. 8c), pre-extracting the dust with water and ethanol, which sterilized it and removed soluble components, ENHANCED IL6 induction, while heating it to 500 C for 4 h, which sterilized it and removed or degraded semi-volatile organics and oxidized metals, REDUCED IL6 induction. NO MEASUREMENT VALUES: every value in this figure is a figure-only normalized ratio, so no output record carries an mrna_level. Context for potency: in these same cells the LC50 for GSLD qPM2.5 was 0.73 mg/mL (459 ug/cm2) versus 1.13 mg/mL for unfractionated GSLD and 4.36 mg/mL for coal fly ash, so the 66 ug/cm2 used here is well below the cytotoxic range. The schema has no cytotoxicity assay class, so those LC50 values are recorded here and in the quoted evidence rather than being forced into an unrelated assay type.

Exposure conditions

Outputs

Evidence

GENE:cowley-il8-hbec-chelators

IL8 mRNA in HBEC3-KT cells - GSLD qPM2.5 with calcium/metal chelation and TLR4 inhibition

Fig. 8b. The companion of the IL6 chelator assay, run on the same cells and conditions but reading IL8, which the paper identifies as the human analogue of mouse Cxcl1 and Cxcl2. GSLD qPM2.5 at 66 ug/cm2 for 24 h induced IL8 mRNA, and the induction was significantly reduced by EGTA plus ruthenium red (10 + 50 uM). THE TWO READOUTS DIVERGE HERE: unlike IL6, IL8 induction was NOT reduced by the TLR4 inhibitor TAK-242 (1 uM) or by the iron chelator deferoxamine (50 uM). Likewise (Fig. 8d), neither water/ethanol pre-extraction nor heating to 500 C affected IL8 induction, although the authors note a trend toward enhanced IL8 induction after heating. The figure legend states the data are mean and standard deviation analysed by 1-way ANOVA with a Dunnett post-test, with all treatment groups corrected for their respective controls and normalized to GSLD qPM2.5 treated cells. NO MEASUREMENT VALUES: every value in this figure is a figure-only normalized ratio, so no output record carries an mrna_level.

Exposure conditions

Outputs

Evidence

GENE:cowley-il6-hbec-trp

IL6 mRNA in HBEC3-KT cells - GSLD qPM2.5 with TRPV1 and TRPV3 antagonists

Fig. 10a, with the initial screen in Supplemental Fig. 12a. HBEC3-KT cells were pre-treated 30 min and co-treated for 24 h with GSLD qPM2.5 (66 ug/cm2) and TRP channel antagonists. Inhibitors of TRPA1 (A967079), TRPM8 and TRPM8 delta-M801 (AMTB) and TRPV4 (HC-067047) did not affect mRNA induction and are therefore not modelled as conditions here. IL6 induction WAS attenuated by the TRPV1 antagonist LJO-328 (20 uM), by a second TRPV1 antagonist AMG-9810 (1 uM), by the TRPV3 antagonist 007 (50 uM), and by the LJO-328 plus 007 combination. Supporting the mechanism, calcium flux assays in HEK-293/GCaMP6s cells showed that GSLD qPM2.5 activates mouse and human TRPA1, TRPM8, the truncated TRPM8 delta-M801 variant and TRPV1, while TRPV3 and TRPV4 activation was not observed - so the TRPV3 antagonist effect on cytokine induction is pharmacological rather than explained by direct TRPV3 activation by the dust. The same pattern of IL6 and IL8 inhibition was seen with Salton Sea dust qPM2.5, though there the antagonist effect was dose dependent. The figure legend states the data are mean and standard deviation analysed by 1-way ANOVA with a Dunnett post-test (* p < 0.05, *** p < 0.001, **** p < 0.0001), normalized to GSLD qPM2.5 treated cells. NO MEASUREMENT VALUES: all values are figure-only normalized ratios.

Exposure conditions

Outputs

Evidence

GENE:cowley-il8-hbec-trp

IL8 mRNA in HBEC3-KT cells - GSLD qPM2.5 with TRPV1 and TRPV3 antagonists

Fig. 10b, with the initial screen in Supplemental Fig. 12b. The IL8 companion of the TRPV1/TRPV3 antagonist assay, run on the same cells and conditions. THE DIRECTION OF EFFECT IS OPPOSITE TO IL6 FOR THE SINGLE ANTAGONISTS: inhibiting either TRPV1 (LJO-328 20 uM) or TRPV3 (007 50 uM) alone appeared to INCREASE IL8 induction, while the LJO-328 plus 007 combination was inhibitory. The screen likewise found IL8 increased by 007. Because the single-antagonist effect is an increase, the output records for LJO-328, AMG-9810 and 007 are annotated as such rather than being read as inhibition. Inhibitors of TRPA1, TRPM8/TRPM8 delta-M801 and TRPV4 did not affect mRNA induction and are not modelled. The figure legend states the data are mean and standard deviation analysed by 1-way ANOVA with a Dunnett post-test, normalized to GSLD qPM2.5 treated cells. NO MEASUREMENT VALUES: all values are figure-only normalized ratios.

Exposure conditions

Outputs

Evidence

GENE:cowley-il6-thp1-tlr4

IL6 mRNA in differentiated THP-1 cells - TLR4 inhibition of GSLD, Salton Sea dust and LPS responses

Supplemental Fig. 13a and c. THP-1 monocytes were differentiated for 72 h with phorbol 12-myristate acetate (25 nM) to a macrophage-like phenotype, then treated with GSLD qPM2.5 at 0.045 mg/mL (12 ug/cm2), Salton Sea dust qPM2.5, or LPS, with and without 30 min pre-treatment and co-treatment with the TLR4 inhibitor TAK-242. IL6 mRNA induction was reduced by TAK-242 by more than 95% for LPS (p = 0.000002), by ~22% for GSLD qPM2.5 (p = 0.076, i.e. not significant at the paper's 0.05 threshold) and by ~51% for SSD qPM2.5 (p = 0.044). IL8 mRNA induction was not reduced by TAK-242, and TNFa induction was less than 2-fold over vehicle control in all cases and unaffected by any co-treatment, so neither is modelled as a measurement here. The authors read this as a role for LPS, acting through TLR4, in driving inflammation in THP-1 cells, and contrast it with the epithelial cells: TRPV1 inhibition had no effect on the THP-1 response to GSLD qPM2.5, while deferoxamine enhanced it. NO ABSOLUTE VALUES: the per-group expression levels are figure-only, so the output records carry no mrna_level; only the three reported inhibition percentages are recorded, in response_comparisons. For context on the concentration chosen, the LC50 for GSLD qPM2.5 in differentiated THP-1 cells was ~0.024 mg/mL (15 ug/cm2), so the 12 ug/cm2 used here sits just below the LC50 - these cells are markedly more sensitive than HBEC3-KT, whose LC50 was 0.73 mg/mL.

Exposure conditions

Outputs

Evidence

GOBLET:cowley-muc5ac-lung-inhalation

Mouse lung Muc5ac mRNA after whole-body GSLD qPM2.5 inhalation

Fig. 6d. Muc5ac mRNA in the left lung lobe was induced after whole-body inhalation of GSLD qPM2.5, measured by TaqMan qRT-PCR (Mm01276718_m1) and normalized to Gapdh by the comparative cycle threshold method; the same induction occurred after oropharyngeal aspiration (Supplemental Fig. 9d). The authors note in the discussion that Muc5ac has been shown to contribute to airway obstruction and hypersensitivity in asthma, which is why this readout is tracked separately from the inflammatory cytokines. GSLD qPM2.5 also induced more Muc5ac mRNA than an equivalent dose of coal fly ash under the 3x paradigm. NO MEASUREMENT VALUES: the relative expression values appear only in the figure, so each output record carries its condition and no muc5ac_mrna_expression value. Note the contrast with the in vitro arm, where MUC5AC induction in HBEC3-KT cells was reported as negligible and variable and was not shown.

Exposure conditions

Outputs

Evidence

COMP:cowley-bal-il6-3x

BAL IL6 protein, 3x GSLD qPM2.5 inhalation versus air control

The paper states IL6 and CXCL1 protein in BAL were elevated 2-3-fold following inhalation. change_value is the midpoint of that stated range and the variability records the range itself, because the paper reports a range and extraction must not narrow it to a single number.

COMP:cowley-bal-cxcl1-3x

BAL CXCL1 protein, 3x GSLD qPM2.5 inhalation versus air control

The CXCL1 half of the same 2-3-fold statement. CXCL1 (murine KC) has no named BALFSputumOutput slot, so compared_measurement names il8_concentration, following the paper's own description of IL8 as the human analogue of Cxcl1 and Cxcl2 and the convention used elsewhere in this corpus for murine KC. change_value is the midpoint of the stated range with the range as its variability.

COMP:cowley-thp1-il6-lps-tak242

THP-1 IL6 mRNA induction by LPS, with TAK-242 versus without

TAK-242 reduced LPS-induced IL6 mRNA by more than 95% (p = 0.000002). The paper gives the figure as a lower bound ('> 95%'); change_value records 95 and this description preserves that it is a floor rather than a point estimate. The statistical test is not named for this specific contrast beyond the p-value, so statistical_test is omitted.

COMP:cowley-thp1-il6-gsld-tak242

THP-1 IL6 mRNA induction by GSLD qPM2.5, with TAK-242 versus without

TAK-242 reduced GSLD-induced IL6 mRNA by ~22%, with p = 0.076 - above the paper's own 0.05 significance threshold, so this is a non-significant trend and should not be read as an established effect. It is recorded because the paper states it, and the p-value is carried so downstream users can see it is not significant.

COMP:cowley-thp1-il6-ssd-tak242

THP-1 IL6 mRNA induction by Salton Sea dust qPM2.5, with TAK-242 versus without

TAK-242 reduced SSD-induced IL6 mRNA by ~51% (p = 0.044), which is significant at the paper's 0.05 threshold. The larger and significant effect for Salton Sea dust than for Great Salt Lake dust is the basis for the authors' conclusion that the THP-1 response is only partially TLR4-mediated and varies between the two dusts.

KER:cowley-trp-to-airway-inflammation

TRP channel activation leads to airway inflammation after lakebed dust inhalation

The paper's central proposed mechanism for the epithelial response. GSLD qPM2.5 activates human and mouse TRPV1 (and TRPA1 and TRPM8) in calcium flux assays; blocking TRPV1 with LJO-328 or AMG-9810, or TRPV3 with 007, attenuates IL6 mRNA induction in HBEC3-KT cells; EGTA plus the non-selective calcium channel blocker ruthenium red attenuates both IL6 and IL8 induction, consistent with a calcium-entry-dependent step. The strongest support is genetic and in vivo: humanized Trpv1N606D mice, whose TRPV1 is sensitized to the dust, developed more BAL neutrophilia than wild-type C57Bl/6 mice after the same 3x inhalation exposure. Evidence support is moderate rather than strong because the IL8 readout moved in the opposite direction under single-antagonist treatment, TRPV3 activation by the dust was not itself observed in the calcium assays, and cytokine mRNA and BAL protein did not differ significantly between the two mouse genotypes. Quantitative understanding is low: no dose-response relating channel activation to inflammation magnitude is reported.

KER:cowley-tlr4-to-airway-inflammation

TLR4 activation leads to airway inflammation after lakebed dust exposure

The parallel mechanism the paper proposes for the myeloid compartment. GSL sediment and its qPM2.5 contain LPS and bacteria, and the TLR4 inhibitor TAK-242 reduced IL6 mRNA induction in differentiated THP-1 cells by ~22% for GSLD qPM2.5 (p = 0.076) and ~51% for SSD qPM2.5 (p = 0.044), against more than 95% for LPS itself. TAK-242 also slightly reduced IL6 induction in HBEC3-KT cells, though not IL8. Evidence support is moderate: the inhibition is partial, the GSLD effect did not reach the paper's own significance threshold, and the authors describe the THP-1 responses as only partially TLR4-mediated. Quantitative understanding is low.

PROTOCOL:cowley-gsld-inhalation-001

Whole-body inhalation exposure of mice to GSLD qPM2.5

GSL sediment collected July 2022 near the Great Saltair, Great Salt Lake State Park and the Kennecott Copper mine, dried 10 days at 40 C, resuspended with compressed filtered air and size-fractionated on a 10-stage Andersen cascade impactor at 1 L/min; the pooled PM < 3.1 um fraction is quasi-PM2.5 (qPM2.5). For exposure, 1.25 g GSLD qPM2.5 was resuspended at ~100 mg/min with a PALAS RBG 1000ID at 1,200 RPM, 7 mm piston, 200 mm/h feed rate, bias airflow 2 L/min, into an inExpose whole-body system (Scireq). Aerosol monitored in real time with a Casella CEL-712 Microdust Pro pre-chamber and gravimetrically post-chamber with an impactor and DustTrack II with a PM2.5 inlet. PM concentrations averaged ~1,800 mg/m3 pre-chamber and ~200 mg/m3 post-chamber with ~75% being <= 2.5 um. Mice were exposed for ~12 min intervals: 1x, 2x or 3x in one day, or 6x over two days. The paper's own estimate of the inhaled mass, quoted verbatim, is: 'Inhalable doses of 76-86, 150-170, 23-26, and 460-520 ug were estimated for the 1x, 2x, 3x, and 6x exposures' using a mouse tidal volume of 0.18 mL, 243 breaths/min, a 12 min exposure time and a PM2.5 value of 195-220.8 mg/m3. The 3x figure of 23-26 ug is lower than the 1x figure and appears to be a typographical error, so it is recorded here only as the paper's own words and is not used as a structured exposure value. Applying deposition estimates of ~4%, ~12.5% and ~50% for the deep lung/alveolar, tracheobronchial and nasal/oropharyngeal regions, the paper estimates deposited doses of ~3.2, ~10.1 and ~40.5 ug of PM for a 1x exposure. In all cases mice were sacrificed 24 h after exposure by Euthasol injection. Protocol typed as MolecularAssayProtocol because the schema offers no exposure-protocol subclass.

Equipment: inExpose whole-body inhalation system (Scireq); PALAS RBG 1000ID dust disperser; Casella CEL-712 Microdust Pro; TSI DustTrack II with PM2.5 inlet filter; 10-stage Andersen cascade impactor

PROTOCOL:cowley-cfa-inhalation-001

Whole-body inhalation exposure of mice to CFA qPM2.5, 3x paradigm

Coal fly ash collected from the Hunter power plant in Castledale, UT and prepared as qPM2.5 exactly as for GSLD. Inhalation exposure to CFA qPM2.5 was performed using the 3x exposure paradigm only, as an equivalent-dose comparator for GSLD qPM2.5, with sacrifice 24 h after exposure. Protocol typed as MolecularAssayProtocol because the schema offers no exposure-protocol subclass.

Equipment: inExpose whole-body inhalation system (Scireq); PALAS RBG 1000ID dust disperser

PROTOCOL:cowley-gsld-opa-001

Oropharyngeal aspiration exposure of mice to GSLD qPM2.5

Mice were dosed with GSLD qPM2.5 by oropharyngeal (OPA) aspiration in a total volume of 25 uL. Doses of 2.5, 12.5, 62.5 and 250 ug qPM2.5 were used to establish a dose-response and to allow comparison with the authors' prior OPA studies of other particulate matter. The paper states these doses model potential inhalable doses of ~86.4-375 ug PM2.5 for humans, estimated from PM2.5 = 50 ug/m3 with either resting (tidal volume 450 mL, 12 breaths/min, 240 min) or exercising (2500 mL, 50 breaths/min, 60 min) ventilation. Mice were sacrificed 24 h after treatment. Protocol typed as MolecularAssayProtocol because the schema offers no exposure-protocol subclass.

PROTOCOL:cowley-bal-diffcount-001

Bronchoalveolar lavage collection and Giemsa differential cell counting

A cannula was inserted through a small incision in the trachea. Cold saline (1 mL) was infused slowly, first 0.5 mL, retracting, then the entire 1 mL. This first 1 mL was placed on ice, clarified by centrifugation and the supernatant frozen at -80 C for ELISA. A further 4 mL was collected 1 mL at a time and placed on ice. Cells from the initial 1 mL and the 4 mL sample were pooled, concentrated by centrifugation, resuspended in 1 mL cold saline, counted, and 5,000 cells fixed to slides by cytospin. Differential counts were also made by tissue flow cytometry on a Cytek Aurora using the antibody panel and singlet/viable/CD45+ gating of Nguyen et al., analysed in FlowJo.

Equipment: Cytospin; Cytek Aurora flow cytometer

PROTOCOL:cowley-bal-elisa-il6-001

Mouse IL6 ELISA on bronchoalveolar lavage supernatant

The first 1 mL of BAL fluid was clarified by centrifugation and the supernatant frozen at -80 C for protein analysis. Mouse IL6 was measured with an Invitrogen kit performed as specified by the supplier. Given its own protocol id separate from the CXCL1 ELISA because the two use different kits and antibody pairs (issue #131).

PROTOCOL:cowley-bal-elisa-cxcl1-001

Mouse CXCL1 (KC) ELISA on bronchoalveolar lavage supernatant

The first 1 mL of BAL fluid was clarified by centrifugation and the supernatant frozen at -80 C for protein analysis. Mouse CXCL1 (KC) was measured with an Invitrogen kit performed as specified by the supplier. Given its own protocol id separate from the IL6 ELISA because the two use different kits and antibody pairs (issue #131).

PROTOCOL:cowley-qpcr-mouse-il6

TaqMan qRT-PCR for mouse Il6 in lung tissue

Before fixation the left bronchus was tied closed and the left lung lobe removed into RNALater at 4 C. Within one week the tissue was homogenized in Trizol (1 mL per 50-100 mg), phase separated with chloroform, the RNA precipitated with ethanol and purified on a PureLink RNA Mini Kit. RNA (2 ug) was reverse transcribed with the ABI High-Capacity cDNA Kit plus RNase inhibitor. Quantification used a Life Technologies QuantStudio 6 Flex and the TaqMan probe for mouse Il6 (Mm00446190_m1). Each TaqMan target has its own protocol id because each uses a distinct probe (issue #131).

PROTOCOL:cowley-qpcr-mouse-cxcl1

TaqMan qRT-PCR for mouse Cxcl1 in lung tissue

Lung RNA prepared as for the mouse Il6 protocol. Quantification used a Life Technologies QuantStudio 6 Flex and the TaqMan probe for mouse Cxcl1 (Mm04207460_m1). Each TaqMan target has its own protocol id because each uses a distinct probe (issue #131).

PROTOCOL:cowley-qpcr-mouse-cxcl2

TaqMan qRT-PCR for mouse Cxcl2 in lung tissue

Lung RNA prepared as for the mouse Il6 protocol. Quantification used a Life Technologies QuantStudio 6 Flex and the TaqMan probe for mouse Cxcl2 (Mm00436450_m1). Each TaqMan target has its own protocol id because each uses a distinct probe (issue #131).

PROTOCOL:cowley-qpcr-mouse-muc5ac

TaqMan qRT-PCR for mouse Muc5ac in lung tissue

Lung RNA prepared as for the mouse Il6 protocol. Quantification used a Life Technologies QuantStudio 6 Flex and the TaqMan probe for mouse Muc5ac (Mm01276718_m1). Each TaqMan target has its own protocol id because each uses a distinct probe (issue #131).

PROTOCOL:cowley-hbec-treatment-001

HBEC3-KT culture, GSLD qPM2.5 treatment and pathway-inhibitor co-treatment

Immortalized human bronchial epithelial HBEC3-KT cells (ATCC) were grown in Airway Epithelial Basal Medium with the Bronchial Epithelial Cell Growth Kit, plated in 12-well plates at 25,000 cells/cm2 and treated 72 h later. GSLD qPM2.5 treatments were suspended in media, sonicated 20 min, vortex mixed 1 min and vortex mixed again immediately before application to keep the suspension homogeneous. A concentration of 0.25 mg/mL (66 ug/cm2) was used for HBEC3-KT cells, for 24 h. For pathway inhibitor studies cells were pre-treated for 30 min and then co-treated. Inhibitor concentrations were EGTA + ruthenium red (10 + 50 uM), TAK-242 (1 uM), deferoxamine (50 uM), LJO-328 (20 uM), AMG-9810 (1 uM), 007 (50 uM), and LJO-328 + 007 (20 + 50 uM). All treatment groups were corrected for their respective controls and normalized to GSLD qPM2.5 treated cells.

PROTOCOL:cowley-thp1-treatment-001

THP-1 differentiation, dust/LPS treatment and TAK-242 co-treatment

THP-1 cells (ATCC) were maintained in RPMI 1640 with 10% fetal bovine serum and 0.05 mM 2-mercaptoethanol at 0.3-1 million cells/mL, plated in 12-well plates and differentiated for 72 h with phorbol 12-myristate acetate (25 nM) to a macrophage-like phenotype before treatment. GSLD qPM2.5 was applied at 0.045 mg/mL (12 ug/cm2); SSD qPM2.5 and LPS (E. coli O111:B4) were used in parallel. For inhibitor studies cells were pre-treated 30 min with TAK-242 and then co-treated.

PROTOCOL:cowley-qpcr-human-il6

TaqMan qRT-PCR for human IL6

At the end of treatment the media was aspirated and stored, cells were washed with PBS and frozen at -80 C. RNA was isolated with the PureLink RNA Mini Kit and quantified with the TaqMan probe for human IL6 (Hs00174131_m1) on a Life Technologies QuantStudio 6 Flex. Each TaqMan target has its own protocol id because each uses a distinct probe (issue #131).

PROTOCOL:cowley-qpcr-human-il8

TaqMan qRT-PCR for human IL8

RNA prepared as for the human IL6 protocol and quantified with the TaqMan probe for human IL8 (Hs00174103_m1) on a Life Technologies QuantStudio 6 Flex. Each TaqMan target has its own protocol id because each uses a distinct probe (issue #131).