Respiratory Syncytial Virus Infection of Human Lung Fibroblasts Induces a Hyaluronan-Enriched Extracellular Matrix That Binds Mast Cells and Enhances Expression of Mast Cell Proteases

Abstract

Human lung fibroblasts (HLFs) treated with the viral mimetic polyinosine-polycytidylic acid (poly I:C) form an extracellular matrix (ECM) enriched in hyaluronan (HA) that avidly binds monocytes and lymphocytes. Mast cells are important innate immune cells in both asthma and acute respiratory infections including respiratory syncytial virus (RSV); however, the effect of RSV on HA dependent mast cell adhesion and/or function is unknown. To determine if RSV infection of HLFs leads to the formation of a HA-enriched ECM that binds and enhances mast cell activity primary HLFs were infected with RSV for 48 h prior to leukocyte binding studies using a fluorescently labeled human mast cell line (LUVA). Parallel HLFs were harvested for characterization of HA production by ELISA and size exclusion chromatography. In separate experiments, HLFs were infected as above for 48 h prior to adding LUVA cells to HLF wells. Co-cultures were incubated for 48 h at which point media and cell pellets were collected for analysis. The role of the hyaladherin tumor necrosis factor-stimulated gene 6 (TSG-6) was also assessed using siRNA knockdown. RSV infection of primary HLFs for 48 h enhanced HA-dependent LUVA binding assessed by quantitative fluorescent microscopy. This coincided with increased HLF HA synthase (HAS) 2 and HAS3 expression and decreased hyaluronidase (HYAL) 2 expression leading to increased HA accumulation in the HLF cell layer and the presence of larger HA fragments. Separately, LUVAs co-cultured with RSV-infected HLFs for 48 h displayed enhanced production of the mast cell proteases, chymase, and tryptase. Pre-treatment with the HA inhibitor 4-methylumbelliferone (4-MU) and neutralizing antibodies to CD44 (HA receptor) decreased mast cell protease expression in co-cultured LUVAs implicating a direct role for HA. TSG-6 expression was increased over the 48-h infection. Inhibition of HLF TSG-6 expression by siRNA knockdown led to decreased LUVA binding suggesting an important role for this hyaladherin for LUVA adhesion in the setting of RSV infection. In summary, RSV infection of HLFs contributes to inflammation via HA-dependent mechanisms that enhance mast cell binding as well as mast cell protease expression via direct interactions with the ECM.

Keywords: airway inflammation; airway remodeling; extracellular matrix; human lung fibroblasts; hyaluronan; mast cells; respiratory syncytial virus; tumor necrosis factor-stimulated gene 6.

Figure 1

Expression of hyaluronan synthase (HAS) and hyaluronidase (HYAL) isoforms by human lung fibroblasts (HLFs) infected with respiratory syncytial virus (RSV). Expression of (A) HAS2 and (B) HAS3 were both significantly increased following RSV infection (13-fold, ^*P < 0.0006 and 5.6-fold, ^*P < 0.0006, respectively). HLF expression of (C) HYAL1 was not significantly altered by RSV infection. Expression of (D) HYAL2 was significantly decreased (~80% reduction, ^**P < 0.0001) following RSV infection. Expression of CD44 was not significantly altered (E). Gene expression was normalized to GAPDH and is shown as normalized mean ± SD relative to control HLFs for N = 7 replicates/group.

Figure 2

Imaging studies were performed to label HA deposition (red) by HLFs in (A) control HLFs and (B) RSV-infected HLFs. (C) Quantitative analysis of staining area demonstrated a 3.5-fold increase in the staining of HA (^#P < 0.03). (D) Quantitative analysis of HA accumulation revealed that HLFs infected with RSV displayed increased total HA accumulation (8,878 ± 114.4 ng/mL vs. 5,295 ± 389.6 ng/mL, ^*P < 0.0009). Differences were driven by increased HA contained in the HLF cell layer following RSV infection (4,477 ± 56.13 ng/mL vs. 600.8 ± 128.3 ng/mL, ^**P < 0.0001) without significant differences in the media compartment HA content. Data shown as mean ± SD for N = 3 replicates/group. HLFs infected with RSV demonstrated a greater abundance of HMW-HA species compared to HLF controls in both (E) media and (F) cell layer compartments. A representative plot of HA profiles measured by S-1000 size exclusion chromatography is shown. Data is plotted as ng HA vs. the partition coefficient (K_av) for N = 3 replicates/group.

Figure 3

Adhesion of LUVA cells (green) to the ECM generated by (A) control HLFs compared to (B) RSV-infected HLFs. Note that LUVA cells aggregated in clusters in a pattern similar to that shown in Figure 2. Panels (C,D) depict LUVA binding following pre-treatment of the wells with Streptomyces hyaluronidase for control HLFs and RSV-infected HLFs, respectively. (E) Quantitative analysis of the staining area revealed a 2-fold increase in LUVA binding in adhesion studies conducted with RSV-infected HLFs compared to control HLFs (401.6 ± 18.94 units vs. 213.2 ± 9.818 units, ^*P < 0.0001). The enhanced binding was eliminated in samples pre-treated with hyaluronidase (^*P < 0.0001). Data shown as mean ± SD for N = 6 replicates/group.

Figure 4

Gene expression of HA receptors by LUVA cells co-cultured with HLFs with or without RSV infection for 48 h. (A) LUVA cells co-cultured with RSV-infected HLFs demonstrated an increased expression of CD44 mRNA compared to LUVA cells alone (^*P = 0.002). No differences in LUVA cell expression of RHAMM or LYVE1 were observed (B,C). Gene expression was normalized to GAPDH and is shown as normalized mean ± SD relative to control LUVA cells for N = 6 replicates/group.

Figure 5

Gene expression of mast cell proteases by LUVA cells co-cultured (CC) with HLFs with or without RSV infection for 48 h (RSV CC and CTL CC, respectively). (A) LUVA cells co-cultured with RSV-infected HLFs demonstrated an increased expression of chymase mRNA compared to LUVA cells alone (^**P = 0.01) and LUVA cells co-cultured with uninfected HLFs (^***P = 0.002). RSV infection of LUVA cells alone did lead to a small increase in chymase expression at baseline (^*1.7-fold, P = 0.03). Treatment with either 4-MU or neutralizing antibody to CD44 significantly decreased chymase expression by LUVA cells co-cultured with RSV-infected HLFs (^#P = 0.02; ^##P = 0.01, respectively). (B) Expression of tryptase by LUVA cells co-cultured with RSV-infected HLFs was increased compared to LUVA cells alone (^**P = 0.01) and LUVA cells co-cultured with uninfected HLFs (^***P = 0.002). RSV infection of LUVA cells alone led to a slight increase in tryptase expression at baseline (^*1.7-fold, P = 0.03). Treatment with either 4-MU or neutralizing antibody to CD44 decreased the expression of tryptase by LUVA cells co-cultured with RSV-infected HLFs (^#P = 0.02; ^##P = 0.01, respectively). (C) Tryptase protein assayed by western blot confirmed a similar pattern as the gene expression analysis. Gene expression was normalized to GAPDH and is shown as normalized mean ± SD relative to control LUVA cells for N = 3–6 replicates/group.

Figure 6

Time course of versican and versicanase expression by HLFs following infection with RSV. (A) Beginning at the 12-h timepoint, versican gene expression was decreased in RSV-infected HLFs compared to control HLFs (P > 0.0001, each comparison). (B) No significant differences were observed for ADAMSTS1 gene expression between the groups at 6 h. Expression of ADAMSTS1 was decreased at 12 h (^#P < 0.005), 24 h (^†P < 0.05), and 48 h (^‡P < 0.002) following RSV infection. (C) Gene expression of ADAMSTS4 was significantly increased at 6 h, 24 h, and 48 h in RSV-infected HLFs compared to control HLFs (^*P < 0.0001, each comparison). (D) Gene expression of ADAMSTS5 was significantly decreased in RSV-infected HLFs at 6 h (^§P < 0.01), 12 h (^*P < 0.0001), and 24 h (^§P < 0.01), but not at the 48-h timepoint.

Figure 7

The effect of TSG-6 expression by HLFs following RSV infection on mast cell binding. (A) TSG-6 expression by HLFs was significantly increased following 6, 24, and 48 h of RSV infection (^*P < 0.0001, each comparison). Gene expression was normalized to GAPDH and is shown as normalized mean ± SD relative to control HLFs at 6 h. (B) Quantitative analysis of LUVA cells following knockdown of TSG-6 in a live-cell binding assay revealed a significant reduction in the binding of LUVA cells observed in HLF following RSV infection (54% reduction, ^**P < 0.002). Immunohistochemistry of adherent LUVA cells (green) to the HA ECM (red) generated by (C) control HLFs compared to (D) RSV-infected HLFs. Binding of LUVA cell aggregates is demonstrated in the RSV-infected HLF cultures along HA cable structures (E). Aggregates of bound LUVA cells decreased following pre-treatment with siRNA to inhibit TSG-6 expression (F). Data shown as mean ± SD for N = 6 replicates/group.

Figure 8

Immunohistochemistry for HA (red) and ITIH1, i.e., HC1 (green), accumulated in the ECM generated by HLFs following a 48-h RSV infection. (A) Uninfected control HLFs displayed a modest amount of HA production and relatively little incorporation of ITIH1 into the ECM. (B) RSV-infected HLFs demonstrated increased HA staining and organization of the ECM in higher order structures that also stained positively for ITIH1. Inhibition of TSG-6 expression did not alter the pattern observed in control conditions (C); however, inhibition of TSG-6 expression decreased the amount of staining for ITIH1 and decreased the number of higher order HA structures observed in the RSV-infected HLFs (D).

Figure 9

Gene expression by LUVA cells co-cultured with RSV-infected HLFs with or without knockdown of TSG-6 expression by siRNA. (A) LUVA chymase expression was increased following co-culture with RSV-infected HLFs (^*P = 0.03) but was found to be significantly reduced by treatment of HLFs with TSG-6 siRNA (^*P = 0.03), HLFs (^*P = 0.03). (B) Expression of tryptase was also increased following co-culture with RSV-infected HLFs (^*P = 0.03); however, it was not significantly different following treatment of HLFs with TSG-6 siRNA. Gene expression was normalized to GAPDH and is shown as normalized mean ± SD relative to control HLFs for N = 4 replicates/group.

Figure 10

Summary of findings. In this study, we have shown that infection of HLFs with RSV induces upregulation of HAS2 and HAS3 with concomitant downregulation of HYAL2 leading to greater accumulation of HA in the HLF cell layer that displays greater adherence of mast cells via direct interactions with the HA-enriched ECM via a CD44 dependent mechanism. Furthermore, RSV infected HLFs display increased expression of TSG-6, which in turn catalyzes the transfer of ITIH1 to form HC-HA and enhances the binding of mast cells to the ECM. Mast cells bound to the RSV-induced ECM upregulate expression mast cell proteases contributing to the proinflammatory milieu. Blocking the formation of the HA-enriched ECM or blocking the CD44 receptor with neutralizing antibodies attenuates this process. Figure created with BioRender.com.