Adult, male, 12-week-old BALB/c mice were obtained from Envigo (Livermore, CA) and randomly divided into 4 exposure groups (n=8/group). After a week of acclimation, mice were anesthetized with isoflurane (Western Medical Supply, Arcadia, CA) and administered a single 50–μl oropharyngeal (OPA) instillation of nanopure water (H₂O; control) or stock PM_UF, PM_2.5, or PM₁₀ solution. Since the stock solutions were all 1 μg/μl, all PM-exposed mice received 50 μg PM in the acute experiments.

Male and female 9-week-old BALB/c mice were randomly divided into 4 exposure groups (n=6/gender/group), anesthetized with isoflurane (Western Medical Supply, Arcadia, CA) as above, and administered a total of six 16.6-μL intranasal instillations of H₂O or stock PM_UF, PM_2.5, or PM₁₀ solution over the course of 14 days as illustrated in the exposure protocol described in Figure 1. PM-exposed mice received a total PM dose of approximately 100 μg.

After pulmonary function testing, each mouse was euthanized with an ip injection of Beuthanasia-D (1 mg/ml; MWI Veterinary Supply, Los Angeles, CA). The left lung was clamped, and the right lung lobes lavaged with two 0.6-mL aliquots of PBS. Bronchoalveolar lavage (BAL) was performed with each PBS aliquot drawn in and out three times prior to collection. BAL fluid (BALF) from each aliquot was combined, the total sample measured and centrifuged at 4° C for 15 min at 2000g. The resulting BALF supernatant was decanted for measurement of protein levels while the cell pellet was resuspended in 0.5 ml PBS. Protein measurements to assess cell permeability in BALF were made using a Lowry assay (BioRad, Hercules, CA) to measure cell permeability within 24 hr after collection. The resuspended cells were utilized for viability and total cell counts using a hemocytometer and 10 μl trypan blue stain. Cytospin slides (n = 3/mouse) were prepared by placing 100-μl cell suspensions in a Shandon Cytospin 4 (ThermoScientific, Kalamazoo, MI) to achieve an approximate count of 1.5 × 10³ cells/slide. Each slide was dried and stained with DiffQuik (American Mastertech, Lodi, CA) for cell differential analysis. A total of 500 cells were counted per mouse to determine the relative % and cells/ml of BAL macrophages, lymphocytes, eosinophils, and neutrophils.

Blood was collected via cardiac-puncture and placed into EDTA-coated tubes during necropsy. Blood samples were centrifuged for 15 min at 2000g, plasma collected and frozen at −80° C for later determinations of the amount of circulating immunoglobulin E (IgE) via enzyme-linked immunosorbent assays (ELISAs; Biolegend, San Diego, CA). IgE, a type of antibody produced in mammals as a result of allergies and other conditions, was measured in the present study to observe whether PM alone, without experimental use of a known allergen (such as ovalbumin or house dust mite), was sufficient to prompt an atopic response.

The left lung was removed along with the cannulated trachea and fixed under hydrostatic pressure at 30 cm/H₂O in 4% paraformaldehyde (PFA) for one hr. The fixed left lungs were then stored in 4% PFA for 24 hr and transferred to 70% ethanol for later sectioning. For each mouse, four serial transverse slices of the left lung were prepared, starting at the left main-stem bronchus. These sections were subsequently processed using an auto-technicon system and embedded 24 hr later in paraffin wax. The embedded tissue blocks were cut into 5-μm sections using a microtome and stained with hematoxylin and eosin (H&E). Stained slides were employed for semi-quantitative histopathological analyses in which the alveolar, pleural, bronchiolar, and perivascular regions of the lungs were scored for inflammation using the ordinal rubric in Table 1.

During necropsy, after the right lung lobes were lavaged, the right mainstem bronchus was closed with a suture and individual lobes were removed, flash frozen in liquid nitrogen, and stored in a −80°C freezer until future use. The caudal lobe was homogenized, and tissues used in plate-based Lowry (BioRad, Hercules, CA) assays and ELISAs (Biolegend, San Diego, CA) to measure total protein and interleukin 1-beta (IL-1ß) protein, respectively. IL-1ß was selected as the cytokine to measure in lung homogenate due to the markedly high levels produced by macrophages when exposed in vitro to PM₁₀ from Imperial Valley (D’Evelyn et al. 2021) IL-1ß, part of the Interleukin 1 family, is involved in the activation of the inflammasome, and has recently been identified as having a role in neutrophilia related to severe asthma (Mahmutovic Persson et al. 2018; Lee and Lawrence 2018). Lowry assays and ELISAs were performed in duplicate. All plates were read using a SpectroMax plate reader at 450 nm. IL-1ß concentrations (ELISA) were standardized to total lung protein as determined by the Lowry assay and expressed as pg/mg lung tissue.

Shapiro-Wilk tests were used to assess the normality of data residuals and transformations were performed to achieve normal distributions as necessary. One-way analyses of variance (ANOVAs) were performed to test the effect of particle size on AHR, BALF cell counts, and IL-1ß and IgE protein levels. Two-way ANOVA was performed to determine whether particle size, MCh dose, or an interaction of both variables affected airway resistance, and whether particle size, lung region, or an interaction of both variables influenced semi-quantitative histopathological scores of inflammation. Post-hoc Tukey’s Honest multiple comparison tests were used with all endpoints to determine statistical differences between specific exposure groups.

Statistical comparisons between subacute and acute exposure scenarios were not statistically analyzed due to inherent differences in the exposure scenarios. Although the same PM samples were used, the acute and sub-acute exposures involved unique exposure methodologies (OPA versus IN, respectively) and different cumulative doses. For these reasons, comparisons between the two exposure paradigms were limited to qualitative analyses of the biological responses in male mice since females were not included in the acute exposures.

Overall, no data were excluded from the acute exposure experiments, however in the sub-acute exposure scenarios, MCh values above 2 mg/ml were excluded due to an insufficient number of data points to run the ANOVA. Lack of data above 2 mg/mL of MCh was due to a decision to stop testing after an animal resistance doubled, which, for the majority of animals, occurred at or prior to reaching 2 mg/ml. All cell differential and histopathological data underwent log transformation. All significant differences were assessed at a p-value < 0.05 for ANOVAs and Tukey’s tests. All statistical analyses were performed using GraphPad Prism 8 (San Diego, CA). All data were expressed in terms of the exposure group mean ± standard error of mean (SEM).

At baseline, after acute exposure, there were no significant differences in lung mechanics measurements of lung resistance, compliance and elastance, central airway resistance, tissue hysteresis, or tissue elastance due to PM size. A significantly different response was observed between the H₂O and PM_2.5 groups exposed to 4 mg/mL of MCh (Figure 2A), the difference was primarily due to a single animal in the latter group. AHR, measured with MCh challenge illustrated in Figure 2B presented as EC₂₀₀RL, was also not significantly different between groups exposed acutely to the various PM size fractions.

Results of the acute exposure experiments revealed no marked significant differences between H₂O- and PM-instilled groups in terms of total BAL cells or BAL cell differentials (Figure 3). A Lowry assay on the BALF supernatant detected a significant rise in protein levels in mice exposed to PM_2.5 compared to H₂O (not shown). This elevation suggested increased cell permeability in lungs of the former versus the latter as more proteins moved from within the cells into the mucosal lining and thus collected by BAL. No marked alteration was noted in neutrophil counts with acute exposure to PM₁₀ (Figure 4B). Lymphocyte levels were highest when males were acutely exposed to PM_UF (Figure 4).

Circulating IgE levels were not detectable in the blood serum of any acute exposure group (data were not shown). A difference in the response to size-fractioned PM was found in the amount of IL-1β protein present in lung homogenate after acute exposure (Figure 5). PM_UF-exposed males demonstrated increased levels of IL-1β protein versus control and other exposure groups (Figure 6).

Semi-quantitative histopathological scoring of the fixed left lung tissue sections from acutely exposed animals demonstrated significant rise in bronchiolitis and pleuritis in mice treated with PM₁₀ compared to H₂O, but no other marked exposure-related differences were detected (Figure 7).

Similar to the acute exposure experiment, no significant differences were observed in baseline lung mechanics measurements conducted following sub-acute exposures to PM_UF, PM_2.5 or PM₁₀ (Figures 8A and 8C). Both male and female mice showed the highest change from baseline resistance, with increasing doses of MCh, when exposed to PM_2.5 as opposed to H₂O (Figures 8A and 8C). This was only significant in female mice, and only at the highest shown MCh dose of 2 mg/ml (Figure 8C). Qualitatively, AHR were non-significantly different (Figure 8D) associated with no obvious PM size-related effects in males (Figure 8B). No significant differences were observed in male mice sub-acutely exposed.

After sub-acute exposures, no significant differences were noted between male and female mice with respect to BALF cell endpoints. However, more endpoints were changed in male versus female mice (Figure 9 versus Figure 10) exposed to PM as opposed to H₂O. PM₁₀-exposed male mice exhibited significantly more eosinophils, neutrophils, and lymphocytes than H₂O-exposed controls (Figures 9C–9E). The eosinophil and lymphocyte numbers (Figures 9C and 9E) observed in PM₁₀-exposed male mice were also significantly higher than those in PM_UF and PM_2.5-exposed males. BALF endpoint comparisons in PM₁₀- and H₂O-exposed female mice yielded only increased lymphocyte numbers in the former (Figure 10E). Differences between PM-exposed females were limited to an elevation in BALF neutrophils of PM_2.5-exposed mice versus PM_UF-exposed counterparts (Figure 10D). Sub-acute exposure to PM₁₀ produced a significant increase in eosinophils, neutrophils, and lymphocytes when compared to all other exposure groups (Figures 11D–F, respectively).

With sub-acute exposure, male versus female mice produced different trends in the levels of IgE in blood serum. Levels of IgE in serum were significantly higher in males exposed to PM_UF or PM_2.5 versus H₂O (Figure 12B). In contrast, no marked inter-group differences were found in serum IgE levels of female mice (Figures 12C–12D). PM₁₀-exposed males exhibited elevated levels of IL-1β compared to control and other exposure groups. No marked differences in IL-1β protein were detected in sub-acutely exposed female mice.

Similar analysis of tissues from sub-acute exposure groups showed that PM₁₀-exposed male mice displayed greater magnitude of bronchiolitis and pleuritis relative to all other exposure groups (Figure 13A). Perivasculitis was significantly greater in males exposed to PM₁₀ or PM_UF versus H₂O (Figure 13A). The only significant differences noted among female mice were greater levels of bronchiolitis relative to controls when exposed to PM₁₀ or PM_2.5, and perivasculitis relative to controls when exposed to PM₁₀ or PM_2.5 (Figure 13B). In both lung regions in females, PM_2.5-related inflammation was significantly greater than that due to PM_UF, but not significantly different from the PM₁₀ exposure group (Figure 13B).