Nchiolar cellular inflammation was present in the lungs of recovering Sftpc2/2 mice (Figure E1B). A semiquantitative scoring of all mice in each control PBS or repetitive LPS exposure groups was performed to indicate the general observable histopathology, and is reported in Table E1. These findings indicate that, upon repetitive LPS challenge, the Sftpc2/2 mice didn’t resolve LPS-induced inflammation as quickly as Sftpc1/1mice.SP-C Null Mice Express Transcription Factors Related with Goblet Cell Transformation immediately after LPS InjuryPreparation of SP-C hospholipid ComplexesNative SP-C was purified by C8 liquid chromatography of bovine lung lavage, as previously described within the supplemental Materials AND Techniques (15).Determination of SP-C and E. coli LPS InteractionsThe synthetic phospholipid liposomes with or without the need of the incorporated 5 wt/wt SP-C (prepared as described in supplemental Supplies AND Techniques) have been incubated with commercially available E. coli 0111:B4 LPS onjugated with FITC (Sigma F-3665; Sigma-Aldrich, St. Louis, MO) and the fluorescence monitored to detect LPS binding.Isolation of Alveolar Type II Cells for CLEC2B Proteins Recombinant Proteins microarray Analysis and In Vitro LPS ResponseCell isolation procedures for culture and RNA extraction and microarray evaluation are offered inside the on the internet supplement.Immunostaining for the transcription element, SPDEF, was detected inside the airway epithelia of Sftpc2/2 mice at Day 3 after LPS exposures, whereas no expression was detected in Sftpc1/1 mice (compare Figures 2C and 2D). Faint immunostaining for the transcription issue, Foxa3, was detected inside a handful of cells lining the airways of saline-treated Sftpc2/2 mice. These information are consistent with preceding studies showing that the airways of Sftpc2/2 mice are predisposed to inflammatory alterations. The intensity of staining and number of Foxa3-positive cells was elevated in the airways of your LPS-Toll-like Receptor 1 Proteins Accession exposed Sftpc2/2 mice in comparison to the exposed Sftpc1/1 mice at Day three (Figure 2E versus Figure 2F, black nuclei). Cytoplasmic alcian blue staining that denotes acidic mucin glycoprotein production was similarly improved in intensity and colocalized with the Foxa3-positive and adjacent airway epithelia of LPS-exposed Sftpc2/2 mice (Figures 2E and 2F).Effect of SP-C Deficiency on Long-Term Recovery soon after LPS ExposureCell Transfection and SP-C Effect on NF-kB SignalingHuman embryonic kidney (HEK) 293T cells had been transiently transfected with plasmids to reconstruct the TLR4-mediated signaling. LPS-stimulated TLR4 signaling was detected by monitoring luciferaseThe lungs of LPS-exposed mice were examined 30 days soon after the sequential LPS exposures to identify if long-term recovery isGlasser, Maxfield, Ruetschilling, et al.: LPS-Induced Lung Injury in SP-C eficient MiceFigure 1. Lung histopathology of surfactant protein-C Sftpc1/1 and Sftpc2/2 mice in the course of recovery from repeated LPS exposure. Photos are of hematoxylin and eosin (H E) staining of lung sections from Sftpc1/1 (A, C, and E, left) or Sftpc2/2 (B, D, and F, right) after the last of three doses of PBS (A and B, best) and either 3 days (C and D, middle) or 5 days right after final LPS dose (E and F, bottom). Day 3–arrowheads indicate morphology of airway epithelium. Arrows determine alveolar accumulation of inflammatory cells and area of alveolar septal fragmentation indicative of airspace injury. Day 5–diffuse alveolar infiltrates have been present in LPS-exposed Sftpc2/2 mice. Partial airway obstruction by inflammatory cells was pre.