Primary healthy pediatric HLFs were obtained from Lonza. Cells were expanded until passage #5 and used for all studies described herein. HLFs were seeded at a density of ~2,500 cells/cm² in 12-well plates (Corning^® Life Sciences) and were maintained in 10% FBS DMEM supplemented with Pen/Strep and L-glutamine with media changes every 48 h. LUVA cells were generously provided by Dr. John Steinke, University of Virginia (24) and were maintained in culture flasks using Stempro-34 media (Thermo Fisher Scientific) supplemented with Pen/Strep and L-glutamine with media changes every 48–72 h. RSV (line 19, A strain) was obtained and propagated as described (25). Knockdown of tumor necrosis factor-stimulated gene 6 (TSG-6) in HLFs was achieved using TSG-6 siRNA (4392420, Silencer^® Select, Thermo Fisher) and Lipofectamine™ RNAiMAX Reagent (Thermo Fisher) according to the manufacturer's recommendations. Optimal transfection conditions were assessed based on manufacturer's suggested protocols leading to ~95% reduction of TSG-6 expression detected by RT-PCR. For TSG-6 siRNA knockdown experiments, control conditions included transfection of non-coding scrambled siRNA.

To establish the optimal timeline for experiments, preliminary studies were performed to investigate the time course for mast cell binding at 24, 48, and 96 h (data not shown). Maximal mast cell binding following RSV infection of HLFs was demonstrated at the 48-h timepoint and was unchanged by the 96-h timepoint, thus all additional experiments were conducted at 48 h post-RSV infection.

The timeline for experiments included two different paradigms depending on outcome measures. For all experiments, HLFs were seeded and grown for 3 days to achieve confluence following which HLFs were infected with RSV (MOI = 1) at experimental Day 0. The first set of studies included endpoint LUVA cell adhesion assays after 48 h of HLF RSV infection. In addition to LUVA cell binding assays, samples were isolated at this point for gene expression analysis of HLFs and characterization of HA accumulation and fragment size. The second paradigm included LUVA cells co-cultured with RSV-infected HLFs at the 48-h timepoint and maintained in co-culture for an additional 48 h at which point LUVA cells were collected following a brief hyaluronidase (from Streptomyces hyalurolyticus; Catalog # H1136, MilliporeSigma) treatment to remove adherent LUVA cells from the HA-enriched ECM, leading to ~90% recovery of LUVA cells embedded in the HA-enriched ECM. HLFs and LUVA cell samples were collected and lysed for western blot. A subset of HLFs was treated with 2.5 mM 4-methylumbelliferone (4-MU; Catalog # M1381, MilliporeSigma), a HA synthase (HAS) inhibitor, at the time of RSV infection to inhibit formation of the HA-enriched ECM (26) and was re-dosed with each media change. In parallel, additional LUVA-HLF co-cultures were treated with monoclonal neutralizing antibodies against CD44 (30 μg/mL; Catalog # MA4400, Thermo Fisher) at the time of co-culture to block interactions between LUVAs and HA (27). A separate subset of HLFs was treated with siRNA to knockdown expression of TSG-6 24 h prior to RSV infection. LUVA cells were isolated following 48 h of co-culture for gene expression analysis, binding assays, and immunohistochemistry.

For gene expression analysis experiments, total RNA was isolated from either HLFs or LUVA cells according to manufacturer recommendations (RNAqueous kit, Ambion^®-Applied Biosystems). RNA concentration and quality were determined using the NanoDrop™ One Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific). RNA samples were reverse-transcribed using the SuperScript^® VILO cDNA Synthesis Kit (Life Technologies). Real-time PCR was performed using validated TaqMan^® probes (Life Technologies) for hyaluaronan synthase (HAS) 1, HAS2, HAS3, hyaluronidase (HYAL) 1, HYAL2, CD44, receptor for HA mediated motility (RHAMM), lymphatic vessel endothelial HA receptor 1 (LYVE-1), versican (VCAN), TSG-6, chymase, tryptase, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, see Table 1 for additional details). Assays were performed using the TaqMan^® Fast Advanced Master Mix reagents and the Applied Biosystems StepOnePlus™ Real-Time PCR System (Life Technologies).

Samples were lysed with 1× lysis buffer (MPER + 1X Halt protease inhibitor cocktail + 5 μM EDTA; Thermo Scientific, San Jose, CA, USA), and total protein concentration determined using the Pierce BCA protein assay kit (Thermo Scientific), according to the manufacturer's protocol. Equal amounts of protein were electrophoresed on 4–20% polyacrylamide gels and transferred onto PVDF membranes. Tryptase and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were detected with human anti-mast cell tryptase (clone G3, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and human anti-GAPDH (clone GA1R, Cell Sciences) antibodies, respectively.

HA content and hydrodynamic size were assessed using a modification of reported methods (12, 28). Media and cell layer samples were isolated separately and digested with pronase (300 μg/ml, Roche) in 0.5 M Tris buffer (pH 6.5) for 18 h at 37°C. Following digestion, the pronase was heat inactivated by incubation at 100°C for 20 min. Media and cell layer concentrations of HA were measured using an Enzyme-Linked Immunosorbent Assay (ELISA) from R&D Systems^® (kit DY3614-05). To determine the hydrodynamic size of HA fragments contained within the samples, equal amounts of HA were applied to an S-1000 column (GE Healthcare) in a 0.5 M sodium acetate buffer containing 0.025% Chaps, 0.02% sodium azide at pH 7.0. Samples were collected using a microtube fraction collector (Model 2110, BioRad) and were analyzed separately using the HA ELISA described above.

Mast cell binding was assessed using a modification of reported methods (12, 18). Subsets of RSV-infected and control HLFs were used to assess the ability of the secreted ECM to bind LUVA cells. Parallel conditions pre-treated for 30 min at 37°C with hyaluronidase (4 U/mL; from S. hyalurolyticus; Catalog # H1136, MilliporeSigma) were included. LUVA cells were washed twice in phenol-free media and re-suspended (1 × 10⁶ cells/mL) and were then incubated with calcein-AM (0.5 μg/ml; Life Technologies) for 45 min at 37°C. HLF wells were washed with RPMI. Afterward, 1.0 mL of the mast cell suspension was added to the wells and allowed to bind at 4°C for 90 min to inhibit enzymatic HA turnover. Cultures were washed 5 times in cold RPMI to remove non-adherent cells. Adherent cell area was quantified using live-cell fluorescent microscopy (ImageXpress Pico, Molecular Devices). Following live-cell imaging, subsets of cells were fixed using a 10% formalin/70% ethanol/5% acetic acid fixative for 10 min at room temperature, washed with PBS, and stained with biotinylated hyaluronan binding protein (HABP) primary (2.5 μg/ml; Catalog # H0161, MilliporeSigma) and a streptavidin conjugated Alexa Fluor 568 secondary (1:1,000, Thermo Fisher). Plates were then reimaged to using the ImageXpress Pico to highlight interactions between the HA matrix and LUVA cells.

Separate HLF specimens were grown on sterilized, collagen coated 12 mm glass coverslips under the experimental conditions described above. Staining for HA was achieved as described above, HLF specimens were also stained with a primary antibody to inter-alpha-trypsin inhibitor heavy chain 1 (ITIH1, 1:500, Thermo Fisher) and a rabbit secondary antibody conjugated to Alexa Fluor 488 (1:1,000, Thermo Fisher). Imaging was performed by either conventional epi-fluorescence microscopy (DM6000B, Leica, Wetzlar, Germany) or confocal microscopy (TCS SP5, Leica).

Analyses of RT-PCR results were performed using GenEx version 6.0.5 (MultiD Analyses AB,) based on previously described methods (29). For all other data, the unpaired t-test was used for comparisons that were normally distributed within each group. For non-normally distributed data, the Mann-Whitney test was used. Statistical analyses were performed using Prism^® 8.0 software (Graph-Pad Software Inc.). Statistical significance was set at P < 0.05.

Comparison of gene expression by HLFs with or without RSV infection demonstrated enhanced expression of mRNA for HA synthetic enzymes (i.e., HAS isoforms) and decreased expression of mRNA for the HA degradation enzyme (HYAL2). HLF expression of HAS1 was essentially undetectable in both control and RSV-infected HLFs, thus precluding statistical comparisons between the groups (not shown). Expression of HAS2, the major HAS isoform expressed by HLFs, was significantly elevated following the 48-h RSV infection (13-fold, P < 0.0006, Figure 1A) as was the expression of HAS3 (5.6-fold, P < 0.0006, Figure 1B). To investigate the contributions of HA degradation mechanisms to the accumulation of HA, expression of HYAL1, HYAL2, and CD44 mRNA transcripts was also assessed with and without RSV infection. No significant differences in HYAL1 and CD44 expression were found between RSV-treated or control HLFs (Figures 1C,E); however, expression of HYAL2 mRNA transcripts by RSV-infected HLFs was significantly decreased (~80% decrease, P < 0.0001, Figure 1D).

In addition to characterization of gene expression of HA synthesis and degradation enzymes by HLFs, accumulation of HA in HLF cultures was assessed by immunofluorescence (Figures 2A,B). Staining for HA revealed an increase in HA accumulation in HLFs infected with RSV compared to control HLFs (Figure 2C; 3.5-fold increase, P < 0.03). Quantitative analysis of HA deposits assessed by ELISA demonstrated an increased total amount of HA contained within samples collected from RSV-infected HLFs compared to control HLFs (8,878 ± 114 ng/mL vs. 5,295 ± 389 ng/mL, P < 0.0009). This difference was driven by increased HA contained in the HLF cell layer following RSV infection (4,477 ± 56 ng/mL vs. 601 ± 128 ng/mL, P < 0.0001) without significant differences in the media compartment HA content (Figure 2D). In both the media and cell layer compartments, HA size was found to be skewed toward HMW-HA in RSV-infected samples compared to control HLFs with a more polydisperse pattern seen in the control samples (Figures 2E,F).

To determine if the HA-enriched ECM produced by HLFs following RSV infection would promote greater adhesion of mast cells compared to that of control HLFs, adhesion of LUVA cells using quantitative fluorescent live-cell imaging was assessed. Compared to control HLFs (Figure 3A) adhesion of LUVA cells to the ECM generated by RSV-infected HLFs (Figure 3B) was significantly greater. The increased binding in RSV-infected conditions compared to controls was reversible by pretreating the wells with Streptomyces hyaluronidase (Figures 3C,D). Quantitative analysis of the staining area in each of these conditions revealed a 2-fold increase in LUVA binding in adhesion studies conducted with RSV-infected HLFs compared to control HLFs (401.6 ± 18.9 units vs. 213.2 ± 9.8 units, P < 0.0001). The enhanced binding was eliminated in samples pre-treated with hyaluronidase (P < 0.0001, Figure 3E).

To assess whether the interactions with the ECM generated by RSV-infected HLFs would lead to alterations in mast cell phenotype beyond increased adhesion, co-cultures of LUVA cells with HLFs following the establishment of the HA-enriched ECM for an additional 48 h were performed. LUVA cells were then isolated and assayed for expression of mast cell proteases as well as expression of HA receptors. Expression of the three major HA receptors (CD44, RHAMM, and LYVE1) was evaluated by PCR. Co-culture with RSV infected HLFs led to a 3-fold increase in CD44 expression by LUVA cells (P = 0.002, Figure 4A) without significant changes in RHAMM or LYVE1 expression (Figures 4B,C). To determine the role of HA in the induction of protease expression, a condition with the HAS inhibitor 4-MU as well as a condition using a monoclonal neutralizing antibody to the HA receptor, CD44, were included (Figure 5). LUVA cells co-cultured with RSV-infected HLFs exhibited an increased expression of chymase mRNA compared to LUVA cells alone (P = 0.01, Figure 5A) or to LUVA cells co-cultured with uninfected controls (P = 0.002). RSV infection of LUVA cells alone lead to a small increase in chymase expression at baseline (1.7-fold, P = 0.03); however, the magnitude of this increase is small in comparison to the HLF/RSV-infected co-culture condition. Pre-treatment with the HAS inhibitor 4-MU decreased the expression of chymase by LUVA cells co-cultured with RSV-infected HLFs to levels similar to LUVA cells alone (P = 0.02). Addition of monoclonal anti-CD44 antibodies also significantly attenuated the expression of chymase by LUVA cells co-cultured with HLFs following RSV infection (P = 0.01). LUVA cells co-cultured with RSV-infected HLFs also demonstrated an increased expression of tryptase mRNA compared to LUVA cells alone (P = 0.01) or to LUVA cells co-cultured with uninfected controls (P = 0.002, Figure 5B). Similar to chymase mRNA expression, RSV infection of LUVA cells alone led to a slight increase in tryptase expression at baseline (P = 0.03). Additionally, pre-treatment with 4-MU also decreased the expression of tryptase by LUVA cells co-cultured with RSV-infected HLFs (P = 0.02). Similarly, the addition of monoclonal CD44 blocking antibodies significantly attenuated the expression of tryptase by LUVA cells co-cultured with HLFs following RSV infection (P = 0.01). Tryptase expression assayed by western blot confirmed a pattern of protein expression that was similar to the gene expression analysis (Figure 5C).

Previous work with HLFs demonstrated an important role for versican (VCAN), a large chondroitin sulfate proteoglycan that interacts with HA, in the enhanced binding of leukocytes following the application of the viral mimetic poly I:C (17). Thus, we examined the role of VCAN in our model system as well. We found that VCAN mRNA expression by HLFs was significantly suppressed following a 48-h infection with RSV (10-fold decrease, P < 0.0001, Figure 6A). HLF gene expression of versicanases (ADAMSTS1, ADAMSTS4, and ADAMSTS5) was elevated following the 48-h RSV infection (Figures 6B–D). Given the decreased VCAN synthesis and enhanced expression of VCAN degradation enzymes it seems unlikely that VCAN is driving the HA related effects in this model system. In contrast, we observed a 6-fold increase in the expression of mRNA for the hyaladherin TSG-6 in RSV-infected HLFs by 48 h (P < 0.0001, Figure 7A). Given that TSG-6 has been shown to be an important mediator of HA cable formation and HA-dependent inflammatory changes (30), and that it plays a critical role in the establishment of airway inflammation in murine models (31), we hypothesized that TSG-6 may also be contributing to the enhanced HA-dependent mast cell binding that we have demonstrated.

To determine the role of TSG-6 in our model, we repeated the mast cell adhesion assays following siRNA knockdown of TSG-6 expression in control and RSV-infected HLFs. This led to a significant reduction in the HA-dependent binding of LUVA cells observed in the RSV-infected HLF condition (54% reduction, P < 0.002, Figure 7B). Following live-cell adhesion assays, samples were fixed, stained, and re-examined for HA staining (representative images shown in Figures 7C,D). Wells from the RSV-infected HLFs demonstrated increased HA accumulation with LUVA cells embedded in the HA-enriched ECM (Figure 7D). Closer inspection revealed that LUVA cells were associated with dense cable-like structures of HA (Figure 7E) and that siRNA knockdown of TSG-6 expression prevented this association, contributing to the decreased binding of LUVA cells in that condition (Figure 7F).

Immunofluorescent staining for HA and ITIH1, a heavy chain (HC) covalently bound to HA via TSG-6 activity, in the ECM by HLFs during control conditions demonstrated a modest amount of HA production and relatively little incorporation of ITIH1 into the ECM (Figure 8A). Conversely, RSV infected HLFs demonstrated increased HA staining and organization of the ECM in higher order structures that stained positively for ITIH1, i.e., HC-HA (Figure 8B). Inhibition of TSG-6 expression did not alter the pattern observed in control HLFs (Figure 8C); however, inhibition of TSG-6 expression decreased staining for ITIH1 and decreased the presence of higher order HA structures observed in the RSV infected HLFs (Figure 8D).

Finally, we examined the role of TSG-6 knockdown on mast cell protease expression by the LUVA cells after a 48-h co-culture period. In these studies, we observed a modest 1.3-fold reduction in chymase expression (P = 0.03) and no significant changes in tryptase expression (Figure 9).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.