<i>P. aeruginosa</i> induction of IFNλ signaling inhibits bacterial clearance.

<p>(A) ELISA analysis of IFNλ in BAL of WT mice following 4 and 18 hours of infection with PAK. (B) Numbers (CFU) of PAK recovered from BAL of WT and IL-28R^−/− mice following 4 and 18 hours of infection. (C) Numbers (CFU) of PAK recovered from lung tissue of WT and IL-28R^−/− mice following 4 and 18 hours of infection. (D) Numbers of PAK recovered from lung tissue of WT and IFNAR^−/− mice following 18 hours of infection. (E) Trichrome stained lungs from WT and IL-28R^−/− mice following and 18 hour PAK infection (original magnification 100x, insert 400x). (F) Numbers of dendritic cells (DC), macrophages (MAC), and neutrophils in BAL of unstimulated (NS) WT and IL-28R^−/− mice or following 4 or 18 hours of infection. (G) Numbers of dendritic cells (DC), macrophages (MAC), and neutrophils in lung tissue of unstimulated (NS) WT and IL-28R^−/− mice or following 4 or 18 hours of infection. Data are representative of at least 2 independent experiments.</p

IFNλ regulation of miR-21 and PDCD4.

<p>(A) qRT-PCR analysis of IL-8 in 16HBE cells treated with control or PDCD4 siRNA and infected with USA300, µ ± SD. (B) qRT-PCR analysis of miR-21 in 16HBE cells treated with recombinant IFNλ for 1 hour, µ ± SD. (C) Western blot analysis of PDCD4 in 16HBE cells treated with recombinant IFNλ. (D) qRT-PCR analysis of miR-21 in 16HBE cells pretreated with STAT3 inhibitor or DMSO treated with recombinant IFNλ for 1 hour, µ ± SD. (E) Western blot analysis of PDCD4 in 16HBE cells pretreated with STAT3 inhibitor or DMSO treated with recombinant IFNλ for 1 hour. Lysate from 16HBE cells treated with DMSO and IFNλ was used as a positive control (pos). (F) Western blot analysis of PDCD4 in human epithelial cells (16HBE) following stimulation with <i>S. aureus</i> protein A (SpA), µ ± SD. (G) Western blot analysis of PDCD4 in 16HBE cells treated with anti-TNFR1 or control IgG and SPA for 2 hours, µ ± SD. Data are representative of at least 2 independent experiments.</p

IFNλ regulates PDCD4 in vivo. (A) qRT-PCR analysis of miR-21 in the lungs of WT and IL-28R^−/− mice following infection with USA300, µ ± SD.

<p>(B) qRT-PCR analysis of PDCD4 mRNA in the lungs of WT and IL-28R^−/− mice following infection with USA300, µ ± SD. (C) Western blot analysis of PDCD4 in the lungs of WT and IL-28R^−/− mice following infection with USA300, µ ± SD. (D) Numbers (CFU) of USA300 recovered from BAL of WT and PDCD4^−/− mice following 18 hours of infection. (E) Numbers (CFU) of USA300 recovered from lung tissue of WT and PDCD4^−/− mice following 18 hours of infection. (F) Trichrome stained lungs from WT and PDCD4^−/− mice following and 18 hour USA300 infection (original magnification 100x, insert 400x). (G) ELISA analysis of individual cytokines in BAL of WT and PDCD4^−/− mice. Data are representative of at least 2 independent experiments.</p

Cytokine production during USA300 infection contributes to pathology.

<p>(A) ELISA analysis of individual cytokines in BAL of WT and IL-28R^−/− mice. (B) Western blot analysis of MX1 expression in lungs of WT and IL-28R^−/− mice following 4 and 18 hours of infection with USA300. (C) Numbers (CFU) of USA300 recovered from the BAL of from PBS control and Anakinra pre-treated mice following an 18 hour infection. (D) Numbers of USA300 (CFU) recovered from the lung of from PBS control and Anakinra pre-treated mice following an 18 hour infection. (E) Trichrome stained lungs from PBS control and Anakinra pre-treated mice following and 18 hour USA300 infection (original magnification 100x, insert 400x). Data are representative of at least 2 independent experiments.</p

Induction of IFNλ by <i>S. aureus</i> USA300 inhibits bacterial clearance.

<p>(A) qRT-PCR analysis of IFNλ mRNA in BMDCs stimulated with heat killed USA300, µ ± SD. (B) ELISA analysis of IFNλ in BAL of WT mice following 4 and 18 hours of infection with USA300. (C) Numbers (CFU) of USA300 recovered from BAL of WT and IL-28R^−/− mice following 4 and 18 hours of infection. (D) Numbers (CFU) of USA300 recovered from lung tissue of WT and IL-28R^−/− mice following 4 and 18 hours of infection. (E) Trichrome stained lungs from WT and IL-28R^−/− mice following and 18 hour USA300 infection (original magnification 100x, insert 400x). (F) Numbers of dendritic cells (DC), macrophages (MAC), and neutrophils in BAL of WT and IL-28R^−/− in unstimulated (NS) mice or following 4 or 18 hours of infection. (G) Numbers of dendritic cells (DC), macrophages (MAC), and neutrophils in lung tissue of WT and IL-28R^−/− in unstimulated (NS) mice or following 4 or 18 hours of infection. Data are representative of at least 2 independent experiments.</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

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

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

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

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