Fig 2: 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.

Fig 3: 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.

Fig 4: Qualitative results after training a CNN to separate cellular structures in superposed images of a nuclear stain and an antibody against a plasma membrane protein. (A) Training input and benchmark images of a MeT5A cell that was dual labelled with the nuclear stain SYTOX Green and an anti-CD44 antibody, and corresponding reconstructions after training a CNN with an L3S-SSIM. Scale bar: 10 µm. The image pairs were obtained via sequential imaging by changing the excitation wavelength. (B) Qualitative result for an ROI (5 µm×5 µm). Prediction success is dependent on the level of superposition of both labels. Structures appear slightly blurry in the restorations compared to the benchmark, but image noise and jitter are reduced. The images shown were excluded from the training.

Fig 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.