Dust was collected from hog confinement facilities in the Midwest, United States from surfaces at least 1 meter off the ground to represent the respirable fraction. Dust aliquots were stored at -20°C until use. Dust extracts were prepared as previously described (18, 46). Briefly, whole dust was combined with Hank’s balanced saline solution (HBSS) (HyClone Laboratories) at a ratio of 1 g dust to 10 mL HBSS on a magnetic stir plate for 1 hour at room temperature. The resulting mixture was centrifuged at 2500 rpm for 20 minutes at 4°C. The supernate was collected, while the pellet was discarded. Supernate was transferred to a new tube and centrifuged again at 2500 rpm for 20 minutes at 4°C. Supernate was collected and filtered through 0.22 μm syringe filters to produce 100% dust extract (DE) and stored at -20°C. The resulting extract contains predominantly gram-positive bacterial components, endotoxins, and trace metals (15). Complete analysis of dust extract can be found at Online Repository Methods at www.jacionline.org (15).

All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee (Protocol Number 2887). C57BL/6J (WT) and whole-body IL-22 knock-out [C57BL/6-Il22^{tm1.1(icre)Stck} /J] (KO) (Jackson Labs) mice aged 8-12 weeks were housed in the Colorado State University Painter Facility in a specific pathogen-free environment with free access to standard mouse feed and water. Three pairs of KO animals were purchased to establish a breeding colony of IL-22cre x IL-22cre that produced all experimental animals. Animals from the original breeding pairs and all offspring were genotyped through TransnetYX (Memphis, TN) genotyping PCR service to ensure accurate genotypes. WT mice were purchased directly from Jackson Laboratories, age and sex matched to KO mice. Purchased mice were acclimated for at least 7 days before performing any procedures.

Mice were intranasally (i.n.) instilled with 50 μl 12.5% DE or sterile saline 5 days/week for 3 weeks under light isoflurane sedation. The concentration of DE was previously determined by a dose-response study, which was found to elicit a strong pulmonary inflammatory response without the risk of mortality (47). Sedation for instillations was achieved using a SomnoSuite Small Animal Anesthesia System (Kent Scientific Corporation) fitted with a small animal anesthesia box. Animals were placed into the box with a flow rate of 100 mL/minute at 2.0-3.0% anesthesia. Animals were removed for instillations once breathing appeared slowed and even. Animals were held in a supine position and 50 μl of the appropriate exposure was loaded into a pipette tip and deposited at the tip of the animal’s nose one drop at a time to allow natural inhalation. Afterwards, animals were returned to their enclosures in a supine position to encourage recovery and monitored for several minutes to ensure return of normal behaviors. AT-RvD1 treated mice were administered intraperitoneal (i.p) injections of 50 μl of 250 ng AT-RvD1 or 5% ethanol (EtOH) (AT-RvD1 vehicle) in sterile saline either once per week after the 5^th DE instillation (weekly, for a total of 3 injections) or 5 times per week (daily, for a total of 15 injections) ( Figures 1A, B ). Dosages and controls were determined based on previously published data and a dose-response pilot study (data not shown) (32, 33, 48, 49). Animals were allowed to recover for 72 hours past-last DE instillation and AT-RvD1 injection before sacrifice.

Animals were euthanized in accordance with the American Veterinary Medical Association guidelines by isoflurane overdose immediately followed by cervical dislocation, 72 hours following the final DE instillation and AT-RvD1 injection. Bronchoalveolar lavage fluid (BALF) was obtained by inserting a 25G catheter into the trachea, tying it off with suture, and lavaging the lungs three times with 1 mL of ice-cold phosphate-buffered saline (PBS) (HyClone Laboratories) for each wash. The first wash was collected in one 5 mL FACS tube while washes two and three were collected in a second 5 mL FACS tube. Both tubes were centrifuged at 300 x g for 8 minutes to pellet the cells. The supernatant fraction from wash 1 was aliquoted into a separate tube and stored at -80°C for cytokine analysis by enzyme-linked immunosorbent assay (ELISA). The supernates from washes two and three were discarded, and cell pellets from both tubes were combined with 400 µl of red blood cell lysis buffer (Life Technologies Corporation), placed on ice for 5 minutes, then centrifuged at 300 x g for 8 minutes at 4°C. The supernatant fraction was discarded, and the cell pellets were resuspended with 200 μL of PBS. A 10 μl aliquot of the cell suspension was then collected for counting using a Countess 3 FL automatic cell counter to generate total cell infiltrate values. The cell suspensions were diluted with PBS to achieve a concentration of 1x10⁶ cells/mL. 200 µl of the final cell suspension was then added to a Thermo Cytospin 4 cytocentrifuge and centrifuged at 600 rpm for 5 minutes. Slides were dried overnight for further staining and analysis. The left lobes of the lungs were tied off with suture, removed, placed in 800 μl of RNAlater (Invitrogen), and stored at -80°C until use. The right lung lobes were extracted along with the heart and tracheal tissue, then inflated with 10% neutral-buffered formalin (NBF) (Cancer Diagnostics Inc.) before hanging under 20 cm of pressure and submerged in 10% NBF overnight. They were then transferred to cassettes and stored in 10% NBF for at least 48 hours before further processing. Brains were extracted en bloc and immediately fixed whole in tissue cassettes in 10% NBF for at least 48 hours before further processing. All tissues were submitted to the Colorado State University Veterinary Diagnostic Laboratory Experimental Pathology Facility (EPF) for paraffin embedding and sectioning at 5 μm.

Staining was performed on dried slides using a Volu-Sol dip-stain kit, with methanol as the fixative, and eosin and methylene blue (Volu-Sol) as the differential stains. Slides were imaged at 20X magnification using an Olympus BX35 microscope and cellSense software version 4.1. Differential counts were performed by counting 300 cells on each image to evaluate macrophages, neutrophils, eosinophils, and lymphocytes, assisted by QuPath version 0.5.1.

Bronchoalveolar lavage fluid (BALF) was collected, processed, and stored as previously described above. Left lung lobes in RNAlater were homogenized with lysis buffer containing PBS, 1X RIPA lysis buffer (Thermo Scientific, Cat#: J62524.AE), and 1X proteinase inhibitor cocktail (Thermo Scientific, Cat#: 1861279) at a ratio of 50 mg tissue to 1 mL buffer. A Bead Mill 24 bead homogenizer was used to break up the tissues (speed: 5.00, time: 0:05, cycles: 3, duration: 0:03). The samples were then incubated on ice for 30 minutes, with agitation every 10 minutes. Homogenate aliquots were stored at -80°C until use. Cytokine protein concentrations in BALF and lung tissue were quantified using sandwich ELISAs. Kits were purchased from DuoSet (R&D Systems) and used according to the manufacturer’s recommendations with the modification of capture antibody diluted in BupH Carbonate-Bicarbonate Buffer (Voller’s Coating Buffer, Thermo Scientific). High-binding half-well 96-well plates (Greiner Biotech) were coated with manufacturer-specified capture antibody concentrations and incubated overnight at room temperature before progression of assay. All washes were performed using a Tecan HydroFlex™ plate washer. After completion of the assay, plates were read on a FLUOstar Omega spectrophotometer at 450 nm using Omega software version 5.7. Concentration values were calculated from standards using a four-parameter fit model in Omega MARS Software Version 4.00.

Whole right lung coronal sections, collected as previously described above, were stained with hematoxylin and eosin (H&E) following deparaffinization and rehydration with xylene and graded EtOH solutions (xylene, 100% EtOH, 95% EtOH, 80% EtOH, 50% EtOH, and finally 1X PBS). Slides were scanned at 40X on a Vectra Polaris scanning microscope. Images of lungs from animals administered weekly AT-RvD1 injections were blinded and manually scored using a well-established semiquantitative scoring method (19, 32, 45, 46, 50). Images from animals administered daily AT-RvD1 injections were imported into VisioPharm version 2023.09.3.15043 x64 and processed through an artificial intelligence (AI) workflow for quantitative assessment of alveolar space, alveolar septa thickness, peribronchiolar inflammation, perivascular inflammation, total alveolar septa nuclei, and percentage of inducible bronchus-associated lymphoid tissue (iBALT) formations relative to tissue area. AI quantification was performed on the whole lung tissue with output measurements including area, percentages of parameters to total tissue area or total parameter counts. VisioPharm is one of the leading AI histopathology analysis software available. Our workflow and applications were designed, annotated, trained, and verified by a board-certified veterinary anatomic pathologist to identify our desired readouts and ensure accurate results. The workflow is then trained through a large set of sample images by a board-certified veterinary anatomic pathologist where structures are identified by the software and confirmed by the pathologist, allowing the software to “learn” how structures are organized and are identified in the experimental images. Briefly, the workflow consisted of multiple stepwise applications of increasing specificity, starting with identification of the lung tissue, followed by identification of airways and vessels, then inflammation and iBALT, and finally identification of airway epithelial cells and alveolar nuclei. During each step of the workflow, the results are confirmed manually to ensure accurate detection of structures. All parameters are normalized to tissue area to mitigate size and artifact skewing.

Whole brain sagittal paraffin embedded tissue sections, as previously described above, were deparaffinized by baking slides at 60°C for 20 minutes, followed by rehydration through an EtOH gradient (xylene, 50% xylene/50% EtOH, 100% EtOH, 95% EtOH, 70% EtOH), and then incubated in 1.0 M Tris buffered saline (TBS). Antigen retrieval was achieved using 1 nM EDTA buffer with 0.05% Tween20, pH 8.0 at 95°C for 20 minutes in a Biocare Medical Decloaking Chamber™ NxGen (Biocare Medical). Tissues were washed three times for 10 minutes each with 0.05M TBS and blocked using 2% donkey serum in 0.2% Triton-X in 1.0 M TBS for 1 hour at room temperature. Microglia were identified using rabbit anti-ionized calcium binding adaptor molecule 1 (Iba1) (1:1000; Abcam, Cat #: ab178846) diluted in 1.0 M TBS and incubated overnight at 4°C. Tissues were then washed four times for 10 minutes each with 0.05 M TBS. A goat anti-rabbit Alexa Fluor 647 secondary antibody (Invitrogen, Cat#: A21244) diluted 1:500 in 2% donkey serum in 1.0 M TBS, was applied and incubated for 1 hour at room temperature. Sections were washed three times for 10 minutes each with 0.05 M TBS. Nuclear staining was achieved by incubation with Hoechst 33342, diluted 1:2000 in PBS (Invitrogen, Cat #: H3570), for three minutes, then washed three times for 10 minutes each with 0.05 M M TBS. ProLong Diamond Antifade Mountant (Fisher Scientific, Cat #: P36970) was then applied before mounting with glass coverslips. Slides were kept at room temperature, protected from light, for 24 – 48 hours to cure mounting medium, then stored at 4°C in the dark prior to imaging. Stained sections were scanned at 40X using a Vectra Polaris microscope, and the number of microglia per area of each brain region of interest were quantified using QuPath Version 0.5.1. All sections were imaged on the same day with the same exposure settings for each channel.

RNAscope^® assays were performed according to the formalin-fixed, paraffin-embedded (FFPE) tissue protocol provided in the RNAscope™ Multiplex Fluorescent Reagent Kit v2 Assay Manual (Document Number: UM 323100). Mouse lung and brain sections previously described above were utilized, with 3 animals per sex, per treatment condition, per genotype selected. Probes for IL-1β (Cat #: 316891-C3), CXCL10 (Cat #: 408921-C3), IL-10 (Cat #: 317261-C2), TGF-β (Cat #: 407751), and AREG (Cat #: 430501) were purchased from Advanced Cell Diagnostics, Inc. TSA Vivid Fluorophore dyes 570 (Cat #: 323272) and 650 (Cat #: 232273) were purchased from Advanced Cell Diagnostics, Inc. and diluted in TSA buffer 1:1500 (Cat #: 322809). Opal Polaris 780 Fluorophore Reagent Pack was purchased from Akoya Biosciences (Cat #: FP1501001KT). TSA-DIG was diluted 1:1500 in TSA buffer for lung sections and 1:750 for brain sections. Opal Polaris 780 Fluorophore was diluted 1:500 in Antibody Diluent/Block (Akoya Biosciences, Cat #: ARD1001EA) for lung sections and 1:200 for brain sections. Positive control slides using RNAscope™ 3-plex positive control probes (CAT #: 320881) and negative control slides using RNAscope™ 3-plex negative control probes (Cat#: 320871) were utilized to validate the assay and results. After completion of the RNAscope^® protocol, sections were incubated with RNAscope^® DAPI (Advanced Cell Diagnostics, Inc.) for 30 seconds, then mounted on glass coverslips with ProLong Diamond Antifade mounting medium (Invitrogen). The mounted sections were kept at room temperature, protected from light, for 24–48 hours to cure mounting medium, before being stored at 4°C until imaging. Slides were scanned at 40X using a Vectra Polaris microscope, with all sections imaged on the same day using the same exposure settings for each channel. QuPath Version 0.5.1 was used according to manufacturer protocols to detect the cell nuclei and quantify subcellular dots per nucleus for the entirety of the lung tissue and for each of the brain regions of interest.

All statistical and graphical analyses were performed using GraphPad Prism Version 10. Outliers were tested for and removed from the datasets using ROUT analysis with Q=1%. Statistical significance was determined by performing 3-way ANOVA analyses with Benjamini, Krieger and Yekutieli post-hoc analysis for pairwise comparisons to reduce false discovery rate. A p-value of less than or equal to 0.05 was used to determine significance and a p-value of less than or equal to 0.1 was considered a trend. Individual animals are represented by a single symbol on figures, with filled shapes representing male animals and unfilled shapes representing female animals. On figures, significance is denoted by, * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001.

We have previously demonstrated that DE exposure leads to increased infiltration of immune cells into the airways (19, 32, 51). One well-established mechanism of AT-RvD1 is its ability to modify immune cell recruitment to sites of infection and injury (49). We aimed to establish an effective dosing strategy to assess the therapeutic applications of AT-RvD1 in a model of repetitive ODE. WT and IL-22 KO mice were instilled i.n. with 50 μl of either 12.5% DE in sterile saline or sterile saline for 5 days per week for 3 weeks (15 total installations) and injected i.p. with 50 μl of either 250 ng AT-RvD1 in sterile saline or 5% EtOH (AT-RvD1 carrier) in sterile saline once per week following the 5^th DE instillation each week (3 total injections) ( Figure 1A ). To determine the impact of AT-RvD1 treatment on immune cell recruitment following DE, we examined cellular infiltration into the airway via BALF cellular analysis ( Figures 2A–E ) and lung histopathology ( Figures 3A–D ).

Upon analysis of BALF cellular infiltrates, we observed an increase in the total number of cellular infiltrates in both WT (p=0.0011) and KO (p<0.0001) mice exposed to DE compared to saline controls ( Figure 2A ). Additionally, we observed a trend of increased total cells in KO mice exposed to DE compared to WT mice exposed to DE (p=0.0702) ( Figure 2A ). KO mice exposed to DE and treated with AT-RvD1 exhibited reduced total cell counts compared to KO mice exposed to DE and treated with saline (p=0.0265) ( Figure 2A ). Macrophage differential counts revealed increased influx in WT (p=0.0162) and KO (p=0.0015) mice exposed to DE compared to WT and KO mice exposed to saline, respectively ( Figure 2B ). We also observed a trend between KO mice exposed to DE and WT mice exposed to DE (p=0.0966) ( Figure 2B ). KO mice exposed to DE and treated with AT-RvD1 demonstrated significantly reduced macrophage differential counts compared to KO mice exposed to DE and treated with AT-RvD1 (p=0.0457) ( Figure 2B ). We did not observe any significant differences or trends in the number of neutrophils, regardless of exposure or treatment condition ( Figure 2C ). Eosinophil differential counts revealed a decrease in the number of eosinophil influx in KO mice exposed to DE and treated with AT-RvD1 compared to KO mice exposed to DE and treated with saline (p=0.0180) ( Figure 2D ). We also observed elevated trafficking of lymphocytes in WT (p=0.0013) and KO (p<0.0001) mice exposed to DE compared to saline exposed controls as well as significantly increased lymphocyte counts in KO mice exposed to DE compared to WT mice exposed to DE (p=0.0454) ( Figure 2E ).

We also examined lung pathology of mice injected with 250 ng AT-RvD1 once per week to assess the effectiveness of AT-RvD1 at reducing DE-induced lung pathology. We evaluated inducible bronchus associated lymphoid tissue (iBALT), peribronchiolar inflammation, and alveolar inflammation using a semiquantitative scoring method previously utilized in our laboratory (19, 32, 50, 52, 53). Statistical analysis revealed a significant main effect of DE exposure in all three parameters evaluated: iBALT (p=0.0002), peribronchiolar inflammation (p<0.0001) and alveolar inflammation (p<0.0001) ( Figures 3B–D ). However, on post-hoc analysis, we observed no statistically significant differences or trends between AT-RvD1 i.p. and saline i.p. treatment groups ( Figures 3B–D ). Interestingly, we observed a similar pattern in all parameters evaluated. We found a significant increase in the mean inflammatory score of iBALT in both WT (p=0.0446) and KO (p=0.0025) animals exposed to DE compared to saline-exposure controls ( Figure 3B ). Additionally, we observed a significantly increased peribronchiolar inflammation in WT (p=0.0064) and KO (p=0.0005) animals exposed to DE compared to saline-exposed controls ( Figure 3C ), as well as a significant increase in the mean score for alveolar inflammation in KO animals exposed to DE compared to saline-exposure controls (p=0.0004) and a trend in WT animals exposed to DE compared to saline-exposure controls (p=0.0596) ( Figure 3D ). We also did not observe any significant changes in mean pathological scores of WT compared to KO animals regardless of exposure or treatment group.

Following our observations of no statistically significant changes in animals exposed to DE and treated with AT-RvD1 once weekly ( Figures 3A–D ), we developed a 5 day/week AT-RvD1 injection regimen, which we found yielded more significant therapeutic efficacy, through histopathology resolution, and was thus employed as our model for further analysis of the pulmonary and neurological inflammatory response to DE. Briefly, WT and IL-22 KO mice were instilled i.n. with 50 μl of either 12.5% DE in sterile saline or sterile saline for 5 days per week for 3 weeks, and then injected i.p. with 50 μl of either 250 ng AT-RvD1 in sterile saline or 5% EtOH (AT-RvD1 carrier) in sterile saline 5 days per week for 3 weeks ( Figure 1B ).

Our analysis of lung histopathology via VisioPharm AI applications revealed an increase in the percentage of iBALT relative to lung tissue area in both WT (p=0.0014) and KO mice (p<0.0001) exposed to DE ( Figure 4B ). Interestingly, we also observed a significant increase in iBALT percentage in KO mice compared to WT mice exposed to DE (p=0.0002) ( Figure 4B ). Additionally, KO mice exposed to DE and treated with AT-RvD1 displayed a significant decrease in iBALT percent of lung tissue compared to KO mice exposed to DE and treated with saline (p<0.0001) ( Figure 4B ). Furthermore, we discovered significant sex differences in the iBALT percentages displayed by both WT and KO animals. We found that female animals exposed to DE and treated with saline exhibited a significantly higher iBALT percentage than male animals exposed to DE and treated with saline in both WT (p<0.0001) and KO (p<0.0001) animals ( Figures 4C, D ). Additionally, both WT (p=0.0021) and KO (p<0.0001) female animals exposed to dust and treated with AT-RvD1 displayed significantly reduced iBALT compared to saline treated female animals ( Figures 4C, D ). Evaluation of peribronchiolar and perivascular inflammation percentage revealed a significant increase in both WT (p=0.0013) and KO (p<0.0001) mice exposed to DE ( Figure 4E ). We observed a trend of reduced peribronchiolar and perivascular inflammation in KO animals exposed to DE and treated with saline and KO mice exposed to DE and treated with AT-RvD1 (p=0.0791) ( Figure 4E ). Bronchiolar epithelium area in KO mice exposed to DE was significantly increased compared to KO mice exposed to saline (p=0.0474) and was further increased in KO mice exposed to DE and treated with AT-RvD1 compared to KO mice exposed to DE and treated with saline (p=0.0233) ( Figure 4F ). Bronchiolar epithelium area was also decreased in WT mice exposed to DE and treated with AT-RvD1 compared to KO mice exposed to DE and treated with AT-RvD1 (p=0.0353) ( Figure 4F ). We observed a significant decrease in alveolar air space in KO mice exposed to saline compared to WT mice exposed to saline (p=0.0301) ( Figure 4G ). Total alveolar nuclear counts were observed to be elevated in WT mice exposed to DE compared to WT mice exposed to saline (p=0.0377), with an observed trend of increased nuclei in KO mice exposed to DE compared to KO mice exposed to saline (p=0.0598) ( Figure 4H ).

We have previously demonstrated that ODE in mice increases both pro- and anti-inflammatory cytokine production (32, 45, 47). In our recovery model, we found that after 15 DE instillations, mice displayed altered cytokine and inflammatory mediator production in the airway and pulmonary tissue compartments at the protein level ( Figures 5A–F ).

Evaluation of amphiregulin (AREG) concentrations in BALF showed that KO mice exposed to DE exhibited decreased concentrations compared to saline controls (p=0.0130) and that KO mice exposed to DE displayed a trend of decreased AREG concentrations compared to WT mice exposed to DE (p=0.0887) ( Figure 5A ). Concentrations of interleukin-10 (IL-10), a classic anti-inflammatory, pro-resolution cytokine was decreased in the BALF of WT mice exposed to DE compared to saline controls (p=0.0009) and a trend of decreased concentrations in KO mice exposed to DE compared to saline controls (p=0.0535) was also observed ( Figure 5B ). Transforming growth factor-β (TGF-β) concentrations in BALF did not yield any significant differences regardless of genotype or treatment groups, however we did observe a significant main effect of DE exposure (p=0.0021) ( Figure 5C ). In the tissue compartment, IL-10 was observed to be elevated in KO mice exposed to saline compared to WT mice exposed to saline (p=0.0003) and in KO mice exposed to DE compared to WT mice exposed to DE (p=0.0002). WT mice exposed to DE and treated with AT-RvD1 exhibited increased tissue IL-10 concentrations compared to WT mice exposed to DE and treated with saline (p=0.0070) ( Figure 5D ). AREG quantification in lung tissues of WT mice exposed to DE displayed increased concentrations compared to WT saline controls (p=0.0005) and were observed to be decreased in KO mice exposed to DE compared to WT mice exposed to DE (p=0.0051) ( Figure 5E ). We also observed a significant decrease in AREG concentrations in KO mice exposed to DE and treated with AT-RvD1 compared to WT mice exposed to DE and treated with AT-RvD1 (p<0.0001). Lung tissue TGF-β concentrations demonstrated a trend of increased concentrations in KO mice exposed to saline compared to WT mice exposed to saline (p=0.0533) and a significant increase in KO mice exposed to DE and treated with AT-RvD1 compared to WT mice exposed to DE and treated with AT-RvD1 (p<0.0003) ( Figure 5F ).

To evaluate mRNA transcript expression in animals exposed to DE and treated with AT-RvD1, we utilized RNAscope technology to visualize and quantify mRNA transcripts in whole lung tissue sections. We evaluated areg, il10, and C-X-C motif chemokine ligand 10 (cxcl10) transcripts in the alveolar and airway compartments to assess the recovery of IL-22 KO mice and the therapeutic impacts of AT-RvD1 and to assess the differences between alveolar and airway inflammatory markers ( Figures 6A–E ). In the alveolar compartment, we observed a significant increase in areg expression in KO mice exposed to saline and treated with AT-RvD1 compared to KO mice exposed to saline and treated with saline (p=0.0051) ( Figure 6B ). Assessment of il10 expression in the alveolar compartment yielded no significant differences or trends of, regardless of DE exposure or AT-RvD1 treatment or genotype ( Figure 6C ). Expression of cxcl10 in the alveolar compartments of KO mice exposed to DE displayed a trend of increased expression compared to saline-exposed KO controls (p=0.0559) ( Figure 6D ). In the airway compartment, we again observed a significant increase in areg expression in KO mice exposed to saline and treated with AT-RvD1 compared to KO mice exposed to saline and treated with saline (p=0.0187) ( Figure 6F ). We again observed no significant differences or trends of il10 expression in the airway compartment ( Figure 6G ). Interestingly, we did observe significantly elevated cxcl10 expression in WT mice exposed to DE compared to saline-exposed WT controls (p=0.0156), which was then revealed to be decreased in WT animals exposed to DE and treated with AT-RvD1 (p=0.0107) ( Figure 6H ).

Brains of WT and IL-22 KO animals exposed to DE and treated with AT-RvD1 were assessed for microglia proliferation via immunofluorescence for Iba1⁺ cells. Cells were counted and represented as a function of the area of each brain region of interest: olfactory bulb, frontal cortex, isocortex, hippocampus, cerebellum, and hindbrain. The olfactory bulb was chosen due to its close proximity to the nasal passages and based upon previous investigations that have shown that intranasal instillations of other particulate matter sources result in gliosis in this region (54). The frontal cortex, isocortex, and hippocampus regions of interest were evaluated due to their functions involving memory and cognition, the decline of which is a hallmark of neurodegenerative disease. The cerebellum was chosen as it controls motor function, and changes in motor function can also be a pathological marker seen in neurodegenerative disease. The hindbrain region of the brain stem is the innervation site for the vagus nerve, which has been implicated in neuroinflammatory models of chronic lung inflammation (2). Increased numbers of Iba1⁺ microglia were detected in areas involved in olfactory sensing and cognition including the olfactory bulb, frontal cortex, and isocortex in WT and KO mice ( Figures 7A–F ). KO mice displayed increased Iba1⁺ microglia in the cerebellum, which is associated with motor function ( Figures 7I, J ). These data indicate that KO mice have altered responses to DE compared to WT controls, but that all mice regardless of genotype, experienced microgliosis in their brains following DE exposure. The increased number of microglia cells detected in brain tissue, is a hallmark sign of neuroinflammation and is a reliable marker for detecting neuroinflammatory processes (55–57).

Evaluation of the olfactory bulbs of animals exposed to DE revealed significant increases in the number of microglia in both WT (p<0.0001) and KO (p=0.0109) animals. We also observed an increase in the number of microglia in mice exposed to saline and treated with AT-RvD1 compared to with saline-exposed, saline-treated controls in both WT (p=0.0002) and KO (p=0.0223) genotypes ( Figure 7B ). Additionally, in the olfactory bulbs we observed a trend of increased microglia numbers in KO mice exposed to saline and treated with saline compared to their WT saline-exposed, saline-treated controls (0.0645) ( Figure 7B ). Animals exposed to DE and treated with AT-RvD1 displayed decreased numbers of Iba1+ cells compared to DE-exposed, saline-treated controls in both WT (p=0.0090) and KO (p=0.0027) genotypes ( Figure 7B ). In the frontal cortex, both WT (p=0.0094) and KO (p=0.0009) animals exposed to DE displayed increased microglia numbers ( Figure 7D ). Saline-exposed, AT-RvD1-treated KO animals also displayed increased numbers compared to saline-exposed, saline-treated KO animals (p=0.0030) ( Figure 7D ). DE-exposed, AT-RvD1-treated WT animals displayed a trend of decreased numbers compared to WT DE-exposed, saline-treated animals (p=0.0633) ( Figure 7D ). Microglia numbers in the isocortex revealed a similar pattern as the olfactory bulb, with both WT (p=0.0051) and KO (p=0.0052) animals exposed to DE and treated with saline demonstrating increased microglia numbers compared to their saline controls ( Figure 7F ). We did not observe any significant differences in the number of microglia in the hippocampus, regardless of exposure or treatment condition ( Figures 7G, H ). Additionally, animals treated with AT-RvD1 exhibited decreased microglia numbers compared to their saline-treated controls for both WT (p=0.0247) and KO (p=0.0117) animals ( Figure 7F ). Saline-exposed KO animals displayed increased microglia counts in their cerebellums compared to WT saline-exposed animals (p=0.0093) and increased counts in DE-exposed, saline treated KO animals compared to saline-exposed controls (p=0.0313) ( Figures 7I, J ). WT animals exposed to saline and treated with AT-RvD1 displayed increased microglia numbers compared to saline-exposed, saline-treated WT animals in the hindbrain (p=0.0253) ( Figures 7K, L ).

We again utilized RNAscope technology to visualize and quantify mRNA transcript expression in WT and KO animals exposed to DE and treated with AT-RvD1 using whole brain tissue sections to evaluate individual regions of interest. We evaluated tgfb, il10, and il1β transcripts in the olfactory bulb, frontal cortex, isocortex, hippocampus, cerebellum, and hindbrain ( Figures 8A–X ).

Transcript quantification in the olfactory bulb revealed a trend of increased tgfb expression between WT saline-exposed mice and KO saline-exposed mice (p=0.0929) ( Figure 8B ). Additionally, a trend was observed for increased il10 transcription in KO mice exposed to DE and treated with AT-RvD1 compared to WT mice exposed to DE and treated with AT-RvD1 (p=0.0669) ( Figure 8C ). Finally, il1b was significantly increased in WT DE-exposed, AT-RvD1-treated mice compared to KO DE-exposed, AT-RvD1-treated mice (p=0.0228) ( Figure 8D ). In the frontal cortex, tgfb displayed a trend of increased transcription between WT and KO mice exposed to saline (p=0.0600), and a trend of reduced transcription between KO mice exposed to DE and KO mice exposed to saline (p=0.0560) ( Figure 8F ). Evaluation of the isocortex revealed significantly increased tgfb transcription between WT and KO saline-exposed mice (p=0. 0355), as well as significantly increased tgfb expression in KO mice exposed to DE compared to KO saline-exposed controls (p=0.0355) ( Figure 8J ). We also found that il1b expression was significantly reduced in WT DE-exposed, AT-RvD1 treated mice compared to KO DE-exposed, AT-RvD1 treated mice (p=0.0245) ( Figure 8L ). Transcript evaluation of the hippocampus revealed significantly increased tgfb expression in KO saline-exposed, saline-treated mice compared to WT saline-exposed, saline-treated mice (p=0.0049) ( Figure 8N ) and a significant decrease in tgfb expression in KO DE-exposed, saline-treated mice compared to KO saline-exposed, saline-treated controls (p=0.0198) ( Figure 8N ). We did not observe any significant differences or trends in the cerebellum. We did observe a change in the hindbrain, with il1b transcripts significantly decreased in WT saline-exposed and AT-RvD1 treated mice compared to WT saline-exposed, saline-treated (p=0.0252) ( Figure 8X ).