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Histologic scoring of ALI lungs. A. Formalin-fixed, paraffin-embedded mice lungs (4 day post LPS instillation) were stained with hematoxylin and eosin and scored at high power fields (400X magnification). Neutrophils marked with blue arrowhead. B. Representative images of lung sections from LPS injured TSG-6 KO and WT mice compared to PBS control taken at lower power fields (100X and 200X). Scale bars: 25 μm (400X), 50 μm (200X), and 100 μm (100X). (DOCX 984 kb)

HC-modification of HA after LPS and PA injury. A. HC-modified HA following LPS injury at 1 and 4 dpi measured as in Fig. 1a. B. HC-modified HA following PA injury (2*106Â CFU) at 2 and 4 dpi measured as in Fig. 1b. HT denotes heterozygous TSG-6 control littermate. (DOCX 110 kb)

Effect of TSG-6 deficiency on lung HA molecular weight distribution and lavage HA levels following LPS injury. A. HA was extracted from lung tissue (LPS 1 dpi), separated by agarose gel, and visualized as described in Fig. 3. HT and KO denote TSG-6 heterozygous and knockout mice. Select-HA consisting of 2500, 1000, 500, and 250 kDa HA was used to determine the molecular weight. B. HA levels in bronchoalveolar lavage were measured by ELISA. n = 5–10 mice per group. (DOCX 87 kb)

TSG-6 is conserved for catalyzing HC-modification of HA. A. Modifying HA with heavy chains (HC) of the serum protein inter alpha inhibitor (IaI), also known as serum-derived HA-associated protein (SHAP), is the only covalent modification HA can undergo. B. TNFα-stimulated gene-6 (TSG-6) is an inflammation-induced secreted protein that exclusively mediates formation of HC-modified HA. Through two transesterification reactions, TSG-6 transfers HC from IαI onto itself and then onto HA. To form the TSG-6-HC intermediate, HC is covalently linked to a conserved serine residue adjacent to the TSG-6 Link domain that binds HA and facilitates HC transfer [1–5]. The serum protein IaI consists of a chondroitin sulfate that is covalently linked to the light chain bikunin and two heavy chains (HC1 and HC2) that can be removed by TSG-6. C. Alignment of TSG-6 pro-protein (residues 1–47 depicted, hTSG-6 numbering) containing the signal peptide (highlighted in gray) and start of the HA-binding Link domain (highlighted in green). The serine residue (highlighted in yellow) responsible for removing HC from serum IaI and transferring HC onto HA is evolutionarily conserved across all vertebrates including fish, reptile, and bird: Homo sapiens (human), Mus musculus (mouse), Equus caballus (horse), Bos taurus (cattle), Pelodiscus sinensis (chinese softshell turtle), and Danio rerio (zebrafish). CLUSTAL multiple sequence alignment by MUSCLE 3.8 (MUltiple Sequence Comparison by Log-Expectation); “*” (asterisk) indicates fully conserved residue. “:” (colon) indicates residues with strongly similar properties (>0.5 in Gonnet PAM 250); “.” (period) indicates residues with weakly similar properties (≤0.5 in Gonnet PAM 250). (DOCX 95 kb)

Correlation between HC-HA and markers of alveolar permeability. Correlation plot of HC-HA abundance, measured in arbitrary units (AU) versus BAL fluid albumin (A) or RAGE (B) levels in individual mice exposed to LPS for 1 or 4Â days compared to PBS control with simple linear regression line and coefficient of determination (R-squared). (DOCX 36 kb)

Effect of LPS on HA staining. A. Representative images of paraformaldehyde-fixed, frozen lung sections immunostained as described for Fig. 4 shown here in detail. Changes in HA staining can be seen in the peri-broncho-vascular interstitium (white arrow). B. Representative sections from independent animals (n = 3 mice per group). Br depicts a bronchial airway and V pulmonary vessels. Scale bar 50 μm. (DOCX 821 kb)

TSG-6 expression in resident and recruited mouse alveolar macrophages. Resident and recruited mouse alveolar macrophages (msAM) were isolated from bronchoalveolar lavage of LPS treated mice (20 μg intratracheal; 0, 3, 6, 9, and 12 dpi). Expression level of msTSG-6 was assessed by RNA-seq and shown as transcripts per million (TPM). Mean (n = 3 independently pooled samples per time point, 4–7 mice for each pool) and error bar (SD) plotted. ND, not detected; NA, not applicable; SD, standard deviation. (DOCX 42 kb)

Effect of LPS on HA staining. A. Representative images of paraformaldehyde-fixed, frozen lung sections immunostained as described for Fig. 4 shown here in detail. Changes in HA staining can be seen in the peri-broncho-vascular interstitium (white arrow). B. Representative sections from independent animals (n = 3 mice per group). Br depicts a bronchial airway and V pulmonary vessels. Scale bar 50 μm. (DOCX 821 kb)

TSG-6 expression in resident and recruited mouse alveolar macrophages. Resident and recruited mouse alveolar macrophages (msAM) were isolated from bronchoalveolar lavage of LPS treated mice (20 μg intratracheal; 0, 3, 6, 9, and 12 dpi). Expression level of msTSG-6 was assessed by RNA-seq and shown as transcripts per million (TPM). Mean (n = 3 independently pooled samples per time point, 4–7 mice for each pool) and error bar (SD) plotted. ND, not detected; NA, not applicable; SD, standard deviation. (DOCX 42 kb)