Fig 2: Lung parenchymal HC-HA formation after IP or IT LPS injury. Abundance of heavy chain (HC)-modified HA in perfused and homogenized lung detected by western blot using IaI antibody (recognizing HC) on lungs before (-) and after (+) hyaluronidase (HAse) liberation of HC linked to HA. Each pair of (-) and (+) lanes represents an individual mouse lung exposed to indicated treatment. Lung HC abundance was determined using densitometry and shown relative to that of total protein. A. Lung HC-HA was assessed 12 h after indicated treatments and quantified. B. TSG-6's role as the exclusive mediator of HC-HA formation was confirmed using WT and KO lungs. n = 4 mice per treatment group. Data analyzed with ANOVA and Tukey's multiple comparisons.

Fig 3: Effect of TSG-6 on circulating neutrophils and TNFa. A. Flow cytometry strategy for distinguishing neutrophils (PMN), mononuclear cells, and eosinophils. Hematopoietic cells (depicted in center) were identified using CD45+TER-119-. PMN were identified as Ly6G+SiglecF-. Mononuclear cells were identified as SSCloLy6G-SiglecF-. Eosinophils were identified as SSChiLy6G-SiglecF+. B-D. Plasma from TSG-6 KO and littermate control (WT & HT) mice was collected 8 h after IP LPS or PBS injection. Plasma was analyzed for PMN (B) and mononuclear cells (C), expressed as percentage of CD45+ cells, and for TNFa cytokine levels assessed by ELISA (D). n = 3–11 mice per treatment group. Data analyzed with ANOVA and Tukey's multiple comparisons.

Fig 4: Plasma HC-HA and TSG-6 induction after IP or IT LPS injury. A. Abundance of HC-modified HA was detected in plasma collected 12 h after indicated treatments. B-C. Endogenous TSG-6 activity in plasma 12 h after indicated treatments was detected by adding 10-oligosacharide HA (HA10) and western blot probing with IaI antibody (recognizing HC) for HC linked to HA10 (HC-HA10). B. Recombinant human TSG-6 (rhTSG-6) was used as a positive control to confirm HC-HA10 formation and that the sodium citrate anticoagulant used for collecting plasma did not abolish TSG-6 activity. As TSG-6 requires divalent metal ions for activity, excess (0.1 M) EDTA was used as a negative control. C. HC-HA10 abundance was detected in plasma and quantified using densitometry and expressed relative to total protein. n = 4 mice per treatment group. Data analyzed with ANOVA and Tukey's multiple comparisons.

Fig 5: Effect of TSG-6 on survival during endotoxic shock. A. Survival was assessed over 3 days after intraperitoneal (IP) LPS (20 mg/kg total body weight or TBW) administration. n = 12–16 mice per treatment group. Analyzed with Log-rank (Mantel-Cox test). B. Expression of msTNFa was assessed by qPCR in whole lung from TSG-6 KO and control (WT & HT) littermate lungs were assessed at 8 h following IP LPS injury. C. Survival and daily TBW change over four days (mean +/- SD, % change from baseline) were assessed following intratracheal (IT) LPS (20 µg/mouse) instillations. D. Expression of msTNFa in whole lung from TSG-6 KO and control lungs at the indicated timepoints following IT LPS injury. n = 4–6 mice per treatment group. Data analyzed with ANOVA and Tukey's multiple comparisons. Wildtype (WT); Heterozygote (HT); Knockout (KO); days post injury (dpi).