Fig. 4.

Heavy chain-hyaluronan complexes are generated after viral infection.

BAL supernatants from influenza infected mice were incubated at 60 °C for 30 min in the presence (+) or absence (−) of Streptomyces hyaluronidase to release free HCs from HC·HA complexes, and intact IαI and free HCs detected by western blot (A). Additional BAL samples from day 8 after infection were either incubated on ice (N) or incubated for 30 min at 60 °C in the absence (−) or presence (+) of hyaluronidase, then free HCs detected by western blot and relative band densities quantified using ImageJ (B). HC·HA synthesised in vitro [94] was also hyaluronidase treated in parallel as a control (A,B). The expression of tsg6 mRNA was determined in whole-lung (C) and whole-BAL (D) RNA extracts from influenza-infected mice, and in isolated alveolar macrophages stimulated ex vivo with LPS, LTA or Poly(IC) (E), by qPCR. Representative blots shown from n = 2 (A) or n = 5 (B) mice, with boundaries between individual blots highlighted. Error bars represent mean ± SEM. n = 5 (B), n = 9–12 (C), n = 6–7 (D) and n = 3–5 (E). * = p < 0.05, ** = p < 0.01, *** = p < 0.001 by Mann-Whitney U test (B) or ANOVA with Holm-Sidak post-test (C-E).