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. 2025 Jan 6;73(1):109–119. doi: 10.1165/rcmb.2024-0385OC

Hyaluronan Directs Alveolar Type II Cell Response to Acute Ozone Exposure in Mice

Aaron Vose 1,✉, Anastasiya Birukova 1, Michaela Albright 1, Adam Schlobohm 3, Stavros Garantziotis 2, Purushothama Rao Tata 1, Christina Barkauskas 1, Robert Tighe 1
PMCID: PMC12254692  PMID: 39761597

Abstract

Becoming more frequent because of climate change, ozone (O3) exposures can cause lung injury. Alveolar type 2 (AT2) cells and hyaluronan (HA), a matrix component, are critical to repairing lung injury and restoring homeostasis. Here, we define the impact of HA on AT2 cells after acute O3 exposure. C57BL/6J mice were exposed to filtered air or O3 (2 ppm) for 3 hours. HA was measured in BAL and lung tissue; HAS (HA synthase) 1, 2, and 3 and HYAL (hyaluronidase) 1, 2, and 3 mRNA were measured in lung tissue and BAL cells. At 48 to 72 hours after O3 exposure, HA increased in BAL fluid by ELISA and lung tissue by immunohistochemistry, with new HA deposition localized to the alveolar ducts. This was associated with increased whole-lung HAS2 mRNA expression. Using an AT2 lineage reporter (Sftpc-CreER;Rosa-Tm) mouse strain, we noted that proliferating AT2 cells colocalized with O3-induced HA deposition in the alveolar duct region. In addition, AT2-to-AT1 cell differentiation after O3 was noted. To determine whether O3-induced HA alters AT2 cell function, we inhibited HA–AT2 interaction with a synthetic inhibitor (Pep-1), which diminished AT2 proliferation. Mice treated with Pep-1 after O3 exposure demonstrated increased BAL albumin concentration compared with filtered air exposure, suggesting that inhibition of HA–AT2 cell interactions resulted in persistent alveolar–capillary permeability and diminished resolution of O3-induced lung injury. Overall, the findings suggest that HA increases in the alveolar duct after acute O3 exposure and that HA–AT2 cell interactions are required for resolution of acute O3-induced lung injury.

Keywords: lung injury, climate change, cell–matrix interaction

Clinical Relevance

This article investigates lung repair responses by alveolar type 2 cells after acute ozone exposure. This advances the field by establishing a novel interaction between matrix hyaluronan and alveolar type 2 cells that is required for resolution of acute environmental lung injury. These relationships will allow further investigation into therapeutic targets and risk mitigation strategies for environmental lung injury that will become increasingly important as the climate changes and pollutant exposure increases.

The alveolar epithelium is a mucosal surface that interfaces with the external environment to facilitate efficient gas exchange. A consequence of this ongoing external environment exposure is that the lung is repetitively challenged by potentially injurious agents, including microbes, viruses, and air pollutants. Ozone (O3), a criterion air pollutant, is a ubiquitous environmental toxicant that causes lung injury. In most cases, although not all, this lung injury resolves after exposure. In cases in which the lung injury does not resolve, this can lead to more severe or persistent injury driving incidence or exacerbation of lung disease. Understanding the mechanisms driving this injury is becoming critically important, as climate change is increasing the frequency and severity of air pollution exposures. This is particularly important for O3 exposure, as its generation is favored with increased heat, and therefore O3 concentrations will increase at higher ambient temperatures (1–3). Increased surface temperatures and heat waves resulting from climate change are associated with O3 extremes, which can further exacerbate the effects of heat on human health (4). Therefore, understanding mechanisms that promote resolution of lung injury after O3 exposure provides insight that could lead to therapeutic interventions that address this important public health threat that will continue to evolve because of climate change.

At present, the homeostatic mechanisms by which resolution of O3-induced lung injury occurs are incompletely understood. O3 causes lung injury by generating airspace oxidant stress leading to formation of bioactive mediators and activation of the innate immune system (5). After acute O3 exposure, there is an initial influx of neutrophils into the airspace, followed by macrophages, which function to restore homeostasis by clearing reactive oxidants and apoptotic neutrophils (6). This acute inflammatory response is largely resolved by 48 hours after exposure, with a decrease in airspace neutrophils, cytokines, and markers of oxidant stress (7). However, our prior research supports that bronchoalveolar lavage total protein and/or albumin remains elevated through 72 hours after exposure (7), suggesting a persistence of lung injury/epithelial permeability. This suggests that additional components, beyond immune cells, are important for resolution of O3-induced lung injury. Prior research has established that resolution of lung injury and return to homeostasis requires the function of alveolar type 2 (AT2) cells. After lung injury, AT2 cells serve as stem cells of the adult lung, where they proliferate and differentiate into alveolar type 1 (AT1) cells (8). This is a critical function, as AT1 cell restoration is required for returning the lung to homeostasis, and defects in AT1 cell restoration are associated with lung disease (9–11).

Presently, little is known about AT2 cell functions after O3 exposure or their functions in alveolar epithelial recovery after exposure. Previous work has identified that O3 exposure affects the alveolar region of the lung in mice and rats. These studies demonstrate that AT2 cells proliferate after O3 exposure and that there is evidence of distal airway remodeling after O3-induced lung injury (12, 13). However, this work does not suggest or demonstrate potential mechanisms by which AT2 cells are directed to proliferate or the role for proliferating AT2 cells after these injuries. Here, we suggest that AT2 cell restorative function is directed by stromal cells and the underlying matrix. Prior research has suggested a role for the matrix component hyaluronan (HA) in AT2 functions (14, 15) and, separately, HA regulation of O3-induced lung inflammation and airway hyperresponsiveness (16–18), but the connections between AT2 cells and HA after O3 exposure are unexplored.

HA is a glycosaminoglycan that is a component of the lung extracellular matrix. In lung homeostasis, HA is localized to the basement membrane of airways and lung vasculature (19), regulates extracellular matrix signaling, and facilitates maintenance of normal epithelial barrier functions (20, 21). In stress responses, HA is synthesized by airway epithelial cells in response to endoplasmic reticular stress (22). This pathologically induced HA functionally differs from homeostatic HA, suggesting the potential for a role in the injury response, although this has previously focused on leukocyte responses. Most published work has focused on pulmonary HA responses during acute inflammation (i.e., in the first 24 h after injury), showing that during this early phase HA is fragmented into low-molecular-weight species, which promote inflammation and airway constriction (23–25). Here, we focus on the role of pulmonary HA responses at later times (i.e., 48–72 h) rather than the previously studied earlier time points. We hypothesized that O3 exposure generates active HA in areas of lung injury that function at delayed time points to direct AT2 proliferation and restoration of alveolar epithelium, thereby resolving O3-induced lung injury.

In the present study, we define interactions between HA and AT2 cells after acute O3 exposure. Mice exposed to O3, when compared with filtered air (FA) control mice, demonstrate elevated BAL fluid and lung tissue HA at later times after exposure. Immunohistochemical staining of O3-exposed lung tissue localized new HA deposition to alveolar spaces just distal to the terminal airway (i.e., the alveolar duct). Using O3-exposed AT2 cell lineage–labeled mice administered 5-ethynyl 2′-deoxyuridine (EdU) before harvest, we associated the regions of new HA deposition with proliferating AT2 cells. In addition, we demonstrate that AT2 cells differentiate into AT1 cells after acute O3 exposure. To define direct AT2 cell–HA interactions, we inhibited HA–AT2 cell interactions with administration of an HA synthetic peptide inhibitor, Pep-1, demonstrating reductions in the number of proliferating AT2 cells and prolonged epithelial permeability 7 days after O3 exposure. Our observations suggest HA regulates AT2 cell proliferation to resolve acute O3-induced lung injury and are critically important to recovery of alveolar epithelial integrity after exposure.

Methods

Experimental Mice

Six- to 8-week-old male C57BL/6J mice (Jackson Laboratory) and mixed-sex Sftpc-CreER;Rosa-Tm mice aged 8–12 weeks from in-house colony, developed by Rock and colleagues (26), were used for experiments. Animal experiments were conducted by National Institute of Health guidelines and approved by the Animal Care and Use Committee at Duke University.

O3 Exposure

C57BL/6J or Sftpc-CreER;Rosa-Tm mice were exposed to FA or 2 ppm O3 for 3 hours. Exposure details are previously published (6) and outlined in the data supplement. For all experiments, the exposure date is Day 0.

Pep-1 and Scrambled Control Peptide, Tamoxifen, and EdU Administration

Mice received 1 mg of Pep-1 or scrambled control (SC) (Genscript) via intraperitoneal injection in sterile PBS. Peptide sequences are provided in Table E1 in the data supplement. For 72-hour experiments, doses were administered 1 day after exposure (D+1) and harvested 3 days after exposure (D+3). For 7-day experiments, doses were administered on D+1, +3, and +5 and harvested on D+7. AT2 lineage labeling was induced by intraperitoneal injection of tamoxifen (20 mg/ml solution in corn oil) daily for 2 weeks. EdU labeling was performed using a Clik-It EdU kit (ThermoFisher, cat# C10340).

Animal Killing and BAL Protocol

Animal killing and BAL protocols are provided in the data supplement. BAL fluid and BAL cells were used for ELISA and real-time PCR as detailed below.

Histopathology and Immunohistochemistry/Immunofluorescence

Antibodies are described in Table E2. All primary antibodies were incubated at 4°C overnight. HA staining followed a protocol generously provided by Ronald J. Midura and provided in the data supplement. For quantitative immunofluorescence, a 250-μm region of interest (ROI) centered around the terminal airway was created. Cells were manually counted for presence of SFTPC+, EdU+, SFTPC/EdU+, and DAPI+. Slides were imaged on a confocal fluorescent microscope (Olympus Fluoview FV3000 or Zeiss 780 Upright Confocal).

ELISA Protocol for Lung Tissue and BAL Fluid HA

Harvested left lung underwent bead tube homogenization. Lung tissue and BAL fluid HA concentrations were determined with HA ELISA (Echelon, cat# K-1200).

Real-Time PCR for HA Synthase and Hyaluronidase

RNA was extracted from lung tissue and BAL cells using RNeasy kit (Qiagen). A total of 500 ng (lung) and 250 ng (cell) of RNA per sample was reverse transcribed using a cDNA synthesis kit (Bio-Rad) and amplified for HAS (HA synthase) and HYAL (hyaluronidase) expression with iTaq SYBR green master mix (Bio-Rad) in an ABI real-time PCR machine (Applied Biosciences). Housekeeping gene 18s mRNA was used as endogenous control. See the data supplement for analytical details. Primers are listed in Table E3.

Statistics

Data are expressed as mean ± SE. The statistical difference between groups was assessed by one-way ANOVA followed by Tukey test for multiple comparisons. A P value of <0.05 was considered statistically significant. All statistics were performed using GraphPad Prism.

Results

Acute O3 Exposure Increases BAL Fluid HA Levels at 48–72 Hours after Exposure

To define a time course of HA after acute O3 exposure, 6- to 8-week-old C57BL/6J male mice were exposed to either filtered air or O3 (2 ppm) for 3 hours. After exposure, mice underwent BAL, and lung tissue was collected and preserved at 6, 12, 24, 48, and 72 hours and 7 days after exposure. BAL fluid and lung tissue homogenates were assessed for HA concentration by ELISA. In the BAL fluid, O3 exposure increased BAL fluid HA concentration at 12 and 24 hours (Figure 1A). This is consistent with previous findings that demonstrated an early increase in HA directly after O3 exposure (16). Interestingly, after the 12- and 24-hour time points, we identified a further ∼10-fold increase in HA levels at 48 and 72 hours (Figure 1A). We then assessed the differences between BAL HA levels and lung tissue HA by measuring HA in homogenized lung tissue. After O3 exposure, lung tissue HA was unchanged at 12 and 24 hours after exposure, but at 48 and 72 hours there was a measurable, although not statistically significant, increase in lung tissue HA (Figure 1B). This was consistent with our observed increase in BAL fluid HA, suggesting that the increased BAL HA maybe driven by the increase in lung tissue HA. Overall, these findings suggest further increases in O3-induced BAL HA at a time point after exposure when prior data has suggested O3-induced inflammation has largely resolved.

Figure 1.

Figure 1.

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Acute ozone (O3) exposure results in increased BAL fluid hyaluronan (HA) and nonsignificant increases in whole lung HA. C57BL/6J mice were exposed to filtered air (FA) or O3 (2 ppm) for 3 hours. O3-exposed mice were harvested at 6, 12, 24, 48, and 72 hours and 7 days after exposure. HA levels were measured in (A) BAL fluid and (B) digested lung tissue. BAL HA levels were maximally elevated at delayed time points, 48–72 hours (*P < 0.05). Whole-lung tissue HA levels were not statistically increased at 48–72 hours. n = 5/group/time point.

Acute O3 Exposure Increased HAS2 mRNA 48–72 Hours after Exposure in Lung Tissue, and HYAL1 mRNA Levels in BAL Fluid Cells

Based on the evidence of increased HA at 48 and 72 hours after O3 exposure, we were interested to determine if this was a result of increased expression of individual HA synthetic (i.e., HAS) or a reduction in degradative (i.e., HYAL) enzymes. Using the same BAL and lung tissue samples from the HA experiments above, RNA was extracted to generate cDNA. Gene expression was assessed by real-time PCR for HA synthases (HAS1, 2, and 3). In BAL cells, high cycle threshold values were observed for HAS1, HAS2, and HAS3 mRNA, consistent with low to nonexistent BAL cell transcription of these genes (Figure E1). Alternatively, in lung tissue, HAS1–3 genes were well expressed. Consistent with prior publications (16), HAS2 was increased at 6 hours after exposure. However, at 48 hours after exposure, HAS2 further increased from 6 hours and then returned to baseline by 7 days after exposure (Figure 2A). This increase was specific to HAS2, as minimal changes were observed in HAS1 and HAS3 mRNA expression after O3 exposure. HAS1 mRNA demonstrated a non–statistically significant increase at the 6-hour time point, whereas HAS3 mRNA was similarly expressed throughout the time points (Figure 2A). HYAL1, 2, and 3 were measured by real-time PCR from BAL cells and lung tissue. Lung tissue did not demonstrate increases in HYAL1 at 6-hour, 24-hour, and 7-day time points or HYAL2 at 6-hour and 72-hour time points (Figure E1). BAL cell HYAL expression demonstrated increased HYAL1 mRNA increases starting at 12 hours, peaking at 24 hours, and resolving by 7 days after exposure. HYAL3 mRNA peaked at 12 hours and resolved. HYAL2 mRNA expression was unchanged throughout the time points (Figure 2B). These data suggest cell-type specificity in HAS2 (i.e., digested whole-lung tissue) and HYAL1 (i.e., BAL cells) that mirror evidence of increased HA in the BAL fluid and highlight dynamic changes in HA synthesis and degradative enzymes after acute O3 exposure.

Figure 2.

Figure 2.

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Acute O3 exposure results in increased HAS2 (HA synthase 2) mRNA in whole lung tissue and increased HYAL1 (hyaluronidase 1) mRNA in BAL fluid cells. (A) Lung tissue HAS1, 2, and 3 were assessed by real-time PCR at 6, 12, 24, 48, and 72 hours and 7 days after O3 exposure (2 ppm for 3 h). HAS1 mRNA was elevated at the 6- and 12-hour time points, whereas HAS2 mRNA was elevated at 12, 24, 48, and 72 hours (*P < 0.05); HAS3 mRNA did not significantly change. HYAL1 mRNA levels were not statistically significant based on time point, whereas HYAL2 mRNA was elevated at the 6-hour time point. (B) BAL cell HYAL1, 2, and 3 were assessed by real-time PCR. HYAL1 was increased at 12, 24, 48, and 72 hours (*P < 0.05) after exposure, HYAL2 was not different between the post-exposure time points, and HYAL3 was increased at 6 hours (*P < 0.05). n = 5/group.

HA Immunohistochemical Staining Identifies Increased HA Deposition at the Alveolar Duct after O3 Exposure

Given the suggestion of increased HA in O3-exposed lung tissue, we performed immunohistochemistry on lung tissue to localize areas of new HA deposition. The left lung harvested from mice exposed to FA or O3 (2 ppm) for 3 hours was formalin fixed and paraffin embedded before sectioning. After paraffinization, hematoxylin and eosin and HA immunohistochemical staining were performed at 24, 48, and 72 hours after exposure based on the time points of elevated BAL and lung tissue HA (Figure 3). In FA-exposed lung tissues, HA was visualized in peribronchovascular regions underlying the airways and the lung vasculature (Figure 3A, Filtered air). At 24 hours after O3 exposure, no new HA deposition was noted, and staining remained peribronchovascular (Figure 3A, Ozone). However, at 48 hours and 72 hours after exposure, in addition to peribronchovascular HA staining, new areas of HA deposition were identified in the alveolar duct (Figure 3A, Ozone, bold arrows). This coincided with the post-exposure time point when BAL HA and alveolar duct HA immunostaining were increased (Figure 1). To quantify our observed O3-induced alveolar duct HA deposition, we imaged 10 bronchoalveolar duct junction (BADJ) regions per exposure time point and measuring the area of HA deposition as a fraction of the total imaged field. This quantification confirmed the increased HA deposition in the alveolar duct regions after acute O3 exposure at 24, 48, and 72 hours after exposure (Figure 3B). These findings suggest that the alveolar duct regions are the primary site of newly deposited HA after acute O3-induced lung injury.

Figure 3.

Figure 3.

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HA immunohistochemical staining demonstrates HA enrichment at the terminal airways after acute O3 exposure. (A) Filtered air samples demonstrated HA staining in the perivascular and peribronchiolar interstitium (panel 1). After acute O3 exposure (2 ppm for 3 h), increased HA deposition occurred at terminal airways at 24, 48, and 72 hours (black arrows) after exposure (panel 2). (B) Alveolar duct HA deposition was quantified by measuring fractional area of HA deposition in 10 peribronchiolar regions per time point. These demonstrated statistically significant increases in HA fractional area at 24, 48, and 72 hours after exposure (**P < 0.005).

Acute O3 Exposure Results in AT2 Proliferation at the Alveolar Duct, Colocalized with Regions of O3-induced HA Deposition

As prior research suggests that HA signaling is important for AT2 cell functions (27), and we localized new HA deposition to the alveolar duct region, we then defined the impact of O3 exposure on AT2 cell proliferation. To do this, we used an AT2 lineage–reporting mouse strain (Sftpc-CreER;Rosa-Tm). Lineage labeling of AT2 cells was induced by tamoxifen (20 mg/ml), and, after labeling induction, mice were exposed to FA or O3 (2 ppm) for 3 hours and harvested at 72 hours after exposure. Three hours before harvest, EdU was administered via intraperitoneal injection to label proliferating cells. Immunofluorescence imaging was performing by staining club cells (anti-SCGB1A1 antibodies), AT2 cells (SFTPC-tdTomato fluorescence), and proliferative cells (EdU staining). Fluorescence microscopy performed on thick sections of lung tissue identified enrichment of actively proliferating airway epithelial cells (defined by SCGB1A1 staining) at airway branch points (Figure 4A). This was consistent with proliferation of airway epithelial cells. In addition, in O3-exposed mice, we identified lineage-positive, proliferating cells (SFTPC-tdTomato+/Edu+) in the alveolar duct region, suggesting active proliferation of AT2 cells (Figure 4A). These cells were not identified in FA-exposed mice (Figure 4B, panel 1). To confirm that proliferating AT2 cells colocalized with areas of HA deposition, thin-section staining was performed to define HA staining in alveolar duct regions. We identified colocalization of AT2 cells (SFTPC-tdTomato+) in areas of HA staining (Figure 4B, panel 2) that were not present in FA-exposed mice. These data suggest that areas of new HA deposition in the alveolar duct are enriched with AT2 cells after acute O3 exposure at 72 hours after the exposure time point. These results demonstrate a spatial relationship between newly deposited HA and proliferating AT2 cells after acute O3 exposure.

Figure 4.

Figure 4.

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Alveolar type 2 (AT2) cells proliferate at the terminal airway after acute O3 exposure, colocalizing with areas of HA deposition. Sftpc-CreER;Rosa-Tm mice after tamoxifen induction were exposed to O3 (2 ppm) for 3 hours and harvested at 72 hours after exposure. Before harvest, mice were injected with 5-ethynyl 2′-deoxyuridine (EdU) to label proliferating cells. (A) Areas of EdU+ proliferative labeling were identified at airway branch points and the terminal airway, consistent with enrichment of Tm-positive lineage cells (SFTPC-tdTomato+) and absence of SCGB1a1 staining (yellow arrows). (B) Image at 72 hours after FA exposure demonstrates absence of new HA deposition and lack of proliferating AT2 cells (panel FA). Image at 72 hours after O3 exposure demonstrates colocalization of HABP (HA-Binding Protein) with SFTPC-tdTomato+ lineage cells at the terminal airway/alveolar duct (panel O3, yellow arrows). Representative image shown; all experiments had n = 3 mice/group.

AT2 Cells Differentiate into AT1 Cells after Acute O3 Exposure

Next, we clarified that proliferating AT2 cells differentiated into AT1 cells after acute O3 exposure. Sftpc-CreER;Rosa-Tm mice were induced with tamoxifen and exposed to FA or O3 (2 ppm) for 3 hours. To ensure adequate time for AT2-to-AT1 differentiation, mice were harvested at Day 7, and lungs were preserved for histology as described above. Immunofluorescence staining was performed to define if cells of AT2 lineage (SFTPC-tdTomato+) express markers consistent with AT1 cells stained with PDPN (podoplanin) antibody; 10× tiled confocal images were obtained of 5-μm mouse lung sections (Figure 5). When compared with FA-exposed mice (Figure 5A), O3-exposed mice demonstrated dual-positive (SFTPC-tdTomato+/PDPN+) cells suggestive of AT2-to-AT1 cell transition (Figure 5B, yellow arrow). These cells demonstrated AT1 cell morphology and were enriched in alveolar duct regions, suggesting they derive from previously identified proliferating AT2 cells after O3 exposure.

Figure 5.

Figure 5.

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AT2 cells transition to AT1 cells by 7-day time point after acute O3 exposure. Sftpc-CreER;Rosa-Tm mice were induced with tamoxifen, then exposed to FA or O3 (2 ppm) for 3 hours and harvested at the 7-day time point. Left lung was collected for histology. Sections measuring 5 μm were labeled with DAPI, PDPN (Podoplanin), SFTPC-tdTomato, and EdU. The 20× confocal images of whole lung demonstrated evidence of AT2-to-AT1 transition in O3-exposed groups, as cells colabeled with PDPN and SFTPC-tdTomato (yellow arrows). Representative images shown; n = 6 mice/group.

Inhibition of HA–AT2 Interaction by Synthetic Peptide Inhibitor Pep-1 Decreases O3-induced AT2 Cell Proliferation

As HA deposition colocalized with AT2 cell proliferation in the alveolar duct after O3 exposure, we hypothesized that HA was driving AT2 cell proliferation. To test this hypothesis, we inhibited HA signaling with a synthetic peptide (Pep-1). Pep-1 is a synthesized protein that binds to HA and prevents cell interactions, with prior data demonstrating inhibition of HA-induced inflammatory responses, cell adhesion, and Langerhans cell migration (28). Sftpc-CreER;Rosa-Tm mice were induced with tamoxifen and then treated with Pep-1 or SC peptide. Mice were then exposed to either FA or O3 (2 ppm) for 3 hours, and Pep-1 or SC peptide was readministered on D+1. EdU was administered 3 hours before harvest at 72 hours after exposure (Figure 6A). Left lung was collected, formalin fixed, and paraffin embedded. Labeling was performed with SFTCPC-tdTomato (AT2 cells), EdU (proliferation), and DAPI (cell nucleus). In blinded samples, terminal airways/alveolar ducts were defined histologically, and a 250-μm ROI was centered around the terminal airway (Figure 6B). AT2 (SFTPC-tdTomato+) cells and proliferating AT2 (SFTPC-tdTomato+/EdU+) cells were manually counted in a blinded fashion within the ROI to generate a proliferating AT2/total AT2 ratio. The total number of cells in this region was identified by DAPI stain positivity. The AT2 cell number was similar in FA- and O3-exposed groups independent of PEP-1 or SC administration. However, mice treated with Pep-1 inhibitor demonstrated a statistically significant reduction in the proliferating AT2 cell/total AT2 cell ratio compared with SC after O3 exposure (Figure 5C). These data suggest that inhibition of the HA–AT2 cell interaction reduces AT2 cell proliferation at the terminal airway after acute O3 exposure.

Figure 6.

Figure 6.

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AT2 cell proliferation after acute O3 exposure is reduced by administration of HA–binding site inhibitor Pep-1. (A) Sftpc-CreER;Rosa-Tm mice were induced with tamoxifen and treated with synthetic peptide (Pep-1) to block HA interaction versus scrambled control (SC) at two time points (green arrow) followed by intraperitoneal EdU injection (pink arrow) before harvest. (B) Immunofluorescent staining was performed to define cells (DAPI), AT2 cells (SFTPC-tdTomato+), and airway epithelial cells (SCGB1A1+). A 250-μm perimeter was drawn around terminal airway, and cells were manually counted to create the ratio of (SFTPC-tdTomato/EdU+) cells. (C) Pep-1 administration did not affect total number of AT2 cells in the region of the terminal airway. However, Pep-1 administration before O3 exposure resulted in a significant decrease in the ratio of proliferating AT2 cells to total AT2 cells in the region of the terminal airway (P < 0.05). n = 5 BADJ/mouse from 3 mice/exposure group. BADJ = bronchoalveolar duct junction; QOD = every other day; TMX = tamoxifen. **P <0.005.

Inhibition of HA–AT2 Interaction Results in Persistent Bronchoalveolar Lavage Albumin at 7 Days after O3 Exposure

As our previous experiments suggested that Pep-1 inhibits AT2 cell proliferation, we hypothesized that reduced AT2 proliferation would impair AT1 differentiation leading to prolonged lung injury, as assessed by BAL fluid albumin. To test this, we performed two experiments. First, we treated male C57BL/6J mice with Pep-1 or SC before exposure on Days −5, −3, and −1 and then exposed them to FA or O3 (2 ppm) for 3 hours (Figure E2A). In a second experiment, we treated male C57BL/6J mice after exposure with Pep-1 or SC on D+1, D+3, and D+5 (Figure 7A). In each arm, BAL was performed on D+7. Mice pretreated with Pep-1 demonstrated a nonsignificant increase in BAL albumin level compared with mice treated with SC at D+7 (Figure E2B). Mice treated with Pep-1 after O3 exposure demonstrated increased BAL albumin levels compared with mice treated with Pep-1 after FA exposure (Figure 7B). These findings suggest that inhibition of HA–AT2 cell interaction appears to result in persistent alveolar–capillary permeability evidenced by increased BAL fluid albumin.

Figure 7.

Figure 7.

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Inhibition of HA–AT2 cell interaction results in increased BAL fluid albumin at 7 days after O3 exposure. (A) C57BL/6J mice were exposed to FA or O3 (2 ppm) for 3 hours and administered Pep-1 or SC peptide every other day for 7 days after the exposure. BAL fluid was obtained 7 days after exposure to assess for albumin by ELISA (image created with BioRender). (B) Raw BAL fluid albumin data or (C) BAL fluid albumin normalized to FA demonstrate significantly increased BAL fluid albumin in the Pep-1–treated group at the 7-day time point. n = 6 mice/group, analysis by t test (P < 0.05). Fold change is calculated by Fold Change = (Pep-1 albumin − SC albumin)/SC albumin. *P <0.005.

Discussion

The burning of fossil fuels has resulted in increasing levels of greenhouse gases and increasing global temperatures and is significantly impacting human health (29). O3, a criterion air pollutant, is a byproduct of fossil fuel combustion, and its formation is chemically favored in warmer temperatures, suggesting that O3 exposure will increase with climate change. This is concerning, as O3 exposure is associated with an increased risk of exacerbation of cardiopulmonary diseases (30–33). Therefore, defining mechanisms by which the lung recovers from O3-induced injury will be critical as the global population risk of elevated O3 exposure increases because of climate change. In the present study, we demonstrate that new deposition of HA is a critical feature of alveolar epithelial recovery after acute O3-induced lung injury. We demonstrate that HA is deposited at the bronchoalveolar duct region, and this region is enriched with proliferating AT2 cells, which differentiate to AT1 cells. We also show that inhibition of HA binding sites with Pep-1 results in decreased AT2 cell proliferation and persistent lung injury after O3 exposure. Together, these findings suggest that newly synthesized HA may promote AT2 cell proliferation and alveolar regeneration after acute O3 exposure in mice, and this serves to resolve O3-induced lung injury.

In the present study, we focused on acute O3 exposures as our environmental lung injury model. Acute exposure was chosen because it has been previously demonstrated that short-term, acute exposures have critical effects on lung health in both animal and human exposure studies and in epidemiologic exposure data (34–37). The O3 concentrations used in this study are reflective of our prior research and previous work that demonstrate 2-ppm exposure in rodents is similar to 0.4 ppm in humans (38). These levels have been studied in humans, resulting in 8.2-fold increases in BAL neutrophil levels and enhanced inflammatory mediators (39). When able, we used mixed-sex littermate control mice, but male mice were predominantly used in the present study. This was based on our prior research demonstrating sex differences in O3 responses, wherein male mice had more vigorous and reproducible O3 exposure responses (7). However, future studies will need to consider sex differences in O3-induced AT2 proliferative responses.

We measured HA levels 6–72 hours after O3 exposure and made the novel observation that HA levels were substantially increased at later times after exposure (Figure 1). Previous work has identified increased HA in the first 24 hours after acute O3 exposure (16). Given this delayed increase in HA, we hypothesized that delayed HA generation might be regulating epithelial cell recovery. Limited information exists on HA-mediated epithelial effects after O3 exposure. Prior research by Hollingsworth and coworkers (40–43) has demonstrated that airway epithelial cells are injured after O3 exposure, but the potential role of HA in this context, and the impact of HA on alveolar epithelial cells, had not been clarified. Here, we identified a potential role for newly deposited HA in directing alveolar epithelial repair after acute environmental lung injury. Using immunohistochemistry, we observed at 48 and 72 hours after O3 exposure that HA deposition localized to the alveolar duct, a region just distal to the terminal airway representing the transition point from airway to alveolar epithelium. This transitional region in the airspace is especially vulnerable to environmental toxicants and other irritants (44) but is also known to be integral in lung repair (45, 46). These data suggest that localization of HA to the alveolar duct region is relevant to the O3-induced injury response.

AT2 cells are the stem cell of the adult lung and proliferate and differentiate into AT1 cells after lung injury. This process is required for resolution of lung injury and maintenance of epithelial barrier functions. Given the evidence of HA deposition and AT2 cell proliferation in the alveolar duct, we hypothesized that new HA promoted O3-induced AT2 cell proliferation. We showed that inhibition of HA binding reduced AT2 cell proliferation, suggesting that alveolar duct HA deposition drives O3-induced AT2 cell proliferation. AT2 cell proliferation and differentiation have been repeatedly demonstrated to be integral to alveolar repair after injury (47–49). Our data suggest that potential defects in AT2 cell proliferation and subsequent differentiation into AT1 cells could drive defects in lung injury resolution after O3 exposure and that new HA deposition after O3 exposure facilitates proliferation of AT2 cells and differentiation into AT1 cells, a key step in resolution of lung injury. In aggregate, our results highlight that HA has important spatial- and temporal-specific roles in inflammation and homeostasis after O3 exposure and provide important insights into mechanisms of alveolar epithelial injury resolution after O3 exposure.

Given the centrality of HA in O3-induced proliferation of AT2 cells, we defined potential sources of the HA and/or its degradation. We identified that HAS2 was increased in lung tissue and HYAL1 was increased in BAL cells. To further determine cell specificity of HA synthesis and degradation, we performed real-time PCR of whole-lung tissue and BAL cells, assessing for enzyme expression. Whole-lung real-time PCR, but not BAL cells, demonstrated increased HAS2 mRNA in a bimodal distribution at 12 and 48 hours. This suggests that HAS2 may have two distinct sources or kinetics after O3 exposure, although the specific HAS2 source in lung tissue was not identified in the present study. In addition, we measured HA-degrading enzymes (i.e., HYALs). We demonstrate, in BAL cells but not lung tissue, increased HYAL1 levels that peak at 24 hours after O3 exposure and return to baseline by 7 days (Figure 2B). Alternatively, we did not observe differences in BAL HYAL2 or HYAL3 expression after exposure. The significance of the Hyal1 in BAL cells and Has2 in lung tissue was not defined in the present study but does highlight that there is an active process of HA synthesis and degradation after exposure that appears cell-type specific. Future studies will need to consider the specific functional role of these genes in O3 responses and HA signaling.

In summary, we demonstrate regional deposition of HA at the alveolar duct at later times after O3 exposure. Within areas of HA deposition at the alveolar duct, we were successfully able to identify an enrichment of AT2 cells that are actively proliferating, suggesting functional consequences of this anatomically restricted HA deposition. Inhibition of HA–AT2 cell interaction with a synthetic peptide resulted in impairment of AT2 cell proliferation at the terminal airway, as defined by reduced proliferating AT2/total AT2 ratio. These data define a novel relationship between HA and AT2 cells and suggest a potential mechanism for lung injury repair after acute O3 exposure. This has important implications for understanding how exposure to air pollutants leads to lung injury and disease and potential methods to mitigate their adverse health effects in a rapidly warming climate.

Supplemental Materials

Supplementary
rcmb.2024-0385OCS1.docx (32KB, docx)
DOI: 10.1165/rcmb.2024-0385OC

Acknowledgments

Acknowledgment

The authors thank the Cleveland Clinic National Heart, Lung, and Blood Institute Award P01HL1071457 for the HA staining protocol.

Footnotes

Supported by National Heart, Lung, and Blood Institute grant T32HL007538 (A.V.); Duke Office of Physician-Scientist Development Technician Support Award (A.V.); School of Medicine, Duke University Office of Physician-Scientist Development Strong-Start Award (A.V.); and National Institutes of Health grants R01ES027574 (R.T.) and R01ES034350 (R.T.).

Author Contributions: A.V., C.B., and R.T. assisted with the experimental design, performed experiments, and assisted with drafting the manuscript. A.B. and A.S. assisted with assay completion and statistical analysis. M.A. and A.S. led animal experiments and sample collection. S.G. and P.R.T. guided the studies and gave critical feedback during the drafting of the manuscript. C.B., P.R.T., S.G., and R.T. conceived of the area of investigation, analyzed data, and drafted the manuscript. A.V. led studies, performed experiments, collected and analyzed data, and drafted the manuscript.

This article has a data supplement, which is accessible at the Supplements tab.

Originally Published in Press as DOI: 10.1165/rcmb.2024-0385OC on January 6, 2025

Author disclosures are available with the text of this article at www.atsjournals.org.

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DOI: 10.1165/rcmb.2024-0385OC

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