Serum cytokine levels in septic (2CLP) rats normalized to sham controls (value of 1) as determined by microarray.

<p>Increased concentrations of Cinc-2, IL-6, IL-10, LIX, MCP-1, MIP-3, and TIMP-1 show activation of the immune response to the bacterial infection. (N = 6, μ±SE).</p

Staining for phenotypic markers of alveolar type II (Toluidin Blue stain of lamellar bodies) and type I (PAI-1, RT1-40) in freshly isolated healthy cells, healthy day 5-6 cells, and 2CLP day 5–6 cells.

<p>These images demonstrate a loss of type II markers in healthy cells and expression of type I markers by day 5. Staining in 2CLP cells on day 5–6 is similar to that in healthy cells, indicating that they have also take on a alveolar type I-like epithelial phenotype. Scale bar 50 µm.</p

Inhibition of transcellular ion pathways modulates TER in sham not 2CLP monolayers.

<p>Following initial TER measurements, the apical fluid was replaced with 1mM amiloride (N = 9) or the basal fluid replaced with 1mM ouabain (N = 10) to block Na⁺K⁺-ATPase and ENaC activity. DMEM was used as a control media (N = 8). Ouabain, not amiloride, treatment of sham wells resulted in significant increases above DMEM controls (-). Neither treatment had a significant affect on TER in 2CLP wells. Significance defined as p<0.05.</p

Area of sham and 2CP cells plated on fibronectin coated glass slides was analyzed on day 2.

<p>Images (60× objective) were taken of phalloidin stained actin to visualize the cell boundary. No differences in epithelial size was observed, indicated that the growth rates of sham and 2CLP cells was not significantly different. (Scale bar = 10 µm, N≥18 cells, μ ± SE).</p

Western analysis of MAPk signaling in 2CLP and sham monolayers.

<p>We found that activation (ratio of phospho-MAPk to total MAPk) of the JNK and ERK kinases were significantly elevated in the 2CLP monolayers compared to sham (*, p<0.05, N≥12). We include representative western blots showing the phosphorylated bands as well as their respective totals. (mean ± SE).</p

Expression levels of the TJ proteins claudin 3, 4, 5, 7, 8, 18, ZO-1, Occludin, and JAM-A were determined via western blot in sham (S) and 2CLP (C) monolayers.

<p>Significant reductions in claudin 4, 18, and occludin were observed (p<0.05, N≥3). Representative Western blots of all proteins analyzed are show along with their respective actin bands to which they were normalized. (mean ± SE).</p

Analysis of Complete Blood Count and Bronchoalveolar lavage fluid.

<p>(Left) Complete Blood Count (CBC) data (normalized per vol or %). Elevated hematocrit (HCT) is likely related to dehydration, while decreased levels of platelets (PLT) and lymphocytes are observed with sepsis. (Right) Bronchoalveolar lavage (BAL) fluid data (expressed as % total cells). The total number of cells in the BAL was not different between sham and 2CLP, however more neutrophils and less macrophages were observed in 2CLP lungs than sham lungs. Significance (-) is defined as p<0.05 as determined by a Mann-Whitney nonparametric test.</p

Transepithelial resistance of sham and 2CLP monolayers following treatment with either U0126 or SP600125 to inhibit ERK or JNK activation respectively.

<p>Monolayers were serum deprived for 2 hours prior to the application of the inhibitor (time = 0). Measurements were taken at 1, 1.5, and 2 hours following treatment. 2CLP monolayers have significantly lower TER values than sham, and sham monolayer TER was unaffected by any of the treatments. Only U0126 significantly improved TER in 2CLP monolayers above initial values by 1.5 hours, and this persisted to 2 hours (*, p<0.05). (μ ± SE) Western blots of lysate obtained following 2 hours of treatment demonstrating inhibition of JNK and ERK are also shown.</p

Permeability analysis of monolayers from sham and 2CLP animals.

<p>(Top) Monolayer permeability to the molecular tracer BODIPY-ouabain (∼20 Å) is shown normalized to the percent area stained in the sham monolayers. No significant differences were observed. (Middle) Monolayer permeability to the small molecular tracer Carboxyfluorescein (∼5 Å) was not significantly different between the two groups. (Bottom) Transepithelial resistance measurements show significant (p<0.05) differences between groups, and indicate that the 2CLP monolayers are more permeable to ions. (mean ± SE).</p

Immunofluorescent staining of the tight junction proteins occluding, ZO-1, claudin 4, and claudin 18 in 2CLP and sham monolayers treated for 1 hour with the ERK inhibitor U0126 or DMSO control.

<p>Scale bar 50 µm.</p