Environmental Particulate (PM2.5) Augments Stiffness-Induced Alveolar Epithelial Cell Mechanoactivation of Transforming Growth Factor Beta

<div><p>Dysfunctional pulmonary homeostasis and repair, including diseases such as pulmonary fibrosis (PF), chronic obstructive pulmonary disease (COPD), and tumorigenesis have been increasing over the past decade, a fact that heavily implicates environmental influences. Several investigations have suggested that in response to increased transforming growth factor - beta (TGFβ) signaling, the alveolar type II (ATII) epithelial cell undergoes phenotypic changes that may contribute to the complex pathobiology of PF. We have previously demonstrated that increased tissue stiffness associated with PF is a potent extracellular matrix (ECM) signal for epithelial cell activation of TGFβ. The work reported here explores the relationship between tissue stiffness and exposure to environmental stimuli in the activation of TGFβ. We hypothesized that exposure of ATII cells to fine particulate matter (PM2.5) will result in enhanced cell contractility, TGFβ activation, and subsequent changes to ATII cell phenotype. ATII cells were cultured on increasingly stiff substrates with or without addition of PM2.5. Exposure to PM2.5 resulted in increased activation of TGFβ, increased cell contractility, and elongation of ATII cells. Most notably, on 8 kPa substrates, a stiffness greater than normal but less than established fibrotic lung, addition of PM2.5 resulted in increased cortical cell stiffness, enhanced actin staining and cell elongation; a result not seen in the absence of PM2.5. Our work suggests that PM2.5 exposure additionally enhances the existing interaction between ECM stiffness and TGFβ that has been previously reported. Furthermore, we show that this additional enhancement is likely a consequence of intracellular reactive oxygen species (ROS) leading to increased TGFβ signaling events. These results highlight the importance of both the micromechanical and biochemical environment in lung disease initiation and suggest that individuals in early stages of lung remodeling during fibrosis may be more susceptible than healthy individuals when exposed to environmental injury adjuvants.</p></div

Average Source Apportionment for South Dekalb PM2.5 Collection.

<p>Average Source Apportionment for South Dekalb PM2.5 Collection.</p

Exposure of RLE-6TN cells to increased stiffness and PM2.5 increases intracellular ROS.

<p>RLE-6TN cells were cultured on PA gels of increasing substrate stiffness with either no PM2.5 (A) or 10µg/cm² PM2.5 (B) and intracellular ROS levels were measured by the DCFH₂-DA oxidation assay. Three independent triplicate experiments were performed and statistical significance is shown between substrates (A) and in comparison to the matched no PM2.5 controls (B). ** (p<0.01) and *** (p<0.001).</p

Stiffness mediated ROS production is TGFβ dependent.

<p>RLE-6TN cells were cultured on PA gels of increasing stiffness with or without the addition of 10 µg/cm² and exposure to a TGFβ blocking antibody for 5 days. Levels of intracellular ROS were measured by the DCFH₂-DA oxidation and assay. Three independent triplicate experiments were performed and statistical significance is shown between the PM2.5 groups with or without the TGFβ antibody. *** (p<0.001)</p

RLE-6TN cells are viable up to a concentration of 10µg/cm².

<p>(A) Schematic illustrating process of PM2.5 collection for cell culture experiments. (B) RLE-6TN cells were cultured with increasing concentrations of isolated PM2.5 for 24 hours and analyzed using the trypan blue assay. Approximately 100% of cells were viable through a concentration of 10µg/cm². Treatment with concentrations above 10µg/cm² resulted in significant cell death. Significance shown for * (p<0.01)</p

Stiffness-mediated activation of TGFβ is increased by addition of PM2.5.

<p>RLE-6TN cells were cultured for 5 days on substrates of increasing stiffness with or without the addition of each concentration of PM2.5 and levels of TGFβ activation were determined using the MLEC bioluminescence co-culture assay. Statistical significance is shown for each concentration of PM2.5 within its same substrate stiffness (* p<0.001).</p

Average Comparison of AQS and ASACA Particle Composition for Species of Interest.

<p>Average Comparison of AQS and ASACA Particle Composition for Species of Interest.</p

Exposure to PM2.5 results in elongated cell phenotype and increased cell cortical stiffness.

<p>RLE-6TN cells were cultured on Fn-PA gels or Fn- or Ln-coated glass for 5 days with the addition of 10µg/cm², 1µg/cm², or 0.1µg/cm² concentrations of PM2.5, and changes in the actin cytoskeleton were analyzed by phalloidin staining of actin filaments and quantified as mean fluorescent staining of actin per cell (A-R). Single cell cortical stiffness of RLE-6TN cells cultured on 8 kPa gels were measured by AFM and significance shown for each group compared to the No PM2.5 control (S). Cell circularity was calculated from acquired images. Values closer to 1 indicate a more rounded, epithelial like cell (T). Experiments were performed in triplicate, representative images are presented and significance shown for * (p<0.05), ** (p<0.01) and *** (p<0.001).</p

TGFβ activation is only partially mediated by cell contractility with PM2.5 exposure.

<p>RLE-6TN cells were cultured in each condition with the addition of the ROCK inhibitor, Y-27632, for 5 days and analyzed for TGFβ activation using the MLEC assay. Statistical significance is reported for differences between same substrate stiffness conditions compared to no PM2.5 control. (* p<0.001)</p

Combinatorial treatment with the ROCK inhibitor, Y-27632, and NAC restore low levels of TGFβ activation seen on epithelial maintaining substrates.

<p>RLE-6TN cells were cultured on increasing substrate stiffnesses (A) with 10 µg/cm² PM2.5 (B) with or without treatment with the antioxidant NAC and levels of TGFβ activation measured . RLE-6TN cells were treated with a combination of the Y-27632 ROCK inhibitor and NAC and TGFβ activation measured (C). Three independent triplicate experiments were performed and statistical significance is shown between substrate stiffnesses (A) and to same substrate stiffness PM controls (B,C) *** (p<0.001).</p