HGF prevents the nuclear translocation of phosphorylated p65.

<p>BMM were (A) untreated (B) stimulated with LPS (1 µg/ml) (C) treated with 10 pg HGF and stimulated with LPS (1 µg/ml) or (D) treated with the MET kinase inhibitor, SU11274, 10 pg HGF and stimulated with LPS (1 µg/ml). Cytospin preparations were then stained by immunoflourescence for phosphorylated p65 (Ser 276) and for nuclei with DAPI. The blue staining indicates nuclei, red staining indicates phosphorylated p65, and purple staining indicates colocalization of phospho-p65 within the nucleus.</p

HGF modulates IL-6 production in LPS stimulated macrophages.

<p>BMM derived from C57BL6 mice were pretreated with or without 10 pg and 10 ng HGF for 24 hours and stimulated with 1 µg/ml LPS. Cell culture media was collected (24 h) and IL-6 levels were measured by ELISA. Results are representative of the mean (± SEM) of three independent experiment done in triplicate, * indicates <0.001.</p

Proposed mechanism of HGF-mediated suppression.

<p>The canonical signaling pathway of LPS-TLR engagement leads to NFκB dependent pro-inflammatory cytokine production through the interaction of CBP with NFκB. However, TLR signaling has also been shown to weakly activate alternative signaling through PI3K, resulting in phosphorylation and inactivation of GSK3β, subsequent sequestration of CBP from NFκB to phospho-CREB, and resultant anti-inflammatory (IL-10) production<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015384#pone.0015384-Martin1" target="_blank">[7]</a>. Our results show that the presence of HGF enhances the IL-10 pathway. We postulate that in order to resolve inflammation, the generation of IL-6 by LPS-TLR signaling leads to the production of HGF, ultimately leading to the inhibition of inflammation. Hence, we propose HGF acts as an internal rheostat for resolving acute phase inflammatory responses.</p

Deletion of the HGF receptor MET demonstrates a reversal in the effects of HGF on LPS stimulated BMM.

<p>BMM derived from either macrophage specific MET conditional knockout mice (MET^fl/fl:cre^lysZ+/−) or their wild type littermate controls (MET^fl/fl:cre^lysZ−/−) were pretreated with or without 1, 10 and 100 pg HGF for 24 hours and stimulated with 1 µg/ml LPS. Cell culture media was collected (24 h) and IL-6 levels were measured by ELISA. Results are representative of two independent experiments done in triplicate.</p

Treatment with HGF leads to increased GSK3β phosphorylation.

<p>(A) Cytoplasmic lysates prepared from BMM were incubated with 10 ng HGF for 24 hours prior to stimulation with LPS (1 µg/ml) for 15 minutes. The lysates were separated by SDS-PAGE and probed with a phospho-specific GSK3β antibody before re-probing for β-actin. (B) Densitometric analysis for phospho-GSK3β fold induction normalized to β-actin is shown for 3 separate experiments. Note that all experiments show induction greater than 1 when HGF is present.</p

HGF promotes the interaction of phosphorylated CREB with CBP along with an increased production of IL-10.

<p>(A) Whole cell lysates prepared from BMM were incubated overnight with HGF (10 ng) prior to stimulation LPS (1 µg/ml) for 15 minutes, then subjected to immunoprecipitation with a CBP antibody. The lysates were separated by SDS-PAGE and probed with a phospho-specific CREB antibody before re-probing for β-actin. (B) Densitometric analysis for phospho-CREB fold induction normalized to β-actin. Is shown for 3 separate experiments. Note that all experiments show induction greater than 1 when HGF is present. (C) BMM were pretreated with or without 1 µM of the MET kinase inhibitor (SU11274) for 2 h prior to incubation with HGF (1, 10 and 100 pg) for 24 hours followed by stimulation with 1 µg/ml LPS. Cell culture media was collected (24 h) and IL-10 levels were measured by ELISA. Results are representative of two independent experiments done in triplicate.</p

A MET kinase inhibitor abrogates HGF suppression of IL-6.

<p>Using an optimal dose of the MET inhibitor SU11274 (1 µg), BMM were pretreated for 2 hours before an overnight incubation with 10 and 100 pg HGF and 24 hour stimulation with LPS. Results are representative of the mean (± SEM) of three independent experiments done in triplicate. *, p = 0.02 vs. the respective control group.</p

RAGE protein levels are decreased in the lungs after injury.

<p>Aliquots of 10 µg of protein from membrane or soluble protein fractions from lung homogenates were assayed for RAGE expression by western blot analysis of RAGE and β-actin. Net intensity of RAGE expression was normalized to β-actin. Membrane RAGE expression was significantly decreased after both LPS and <i>E. coli</i> challenge (A). Soluble RAGE expression was significantly decreased after <i>E. coli</i> injury, and markedly, but not significantly decreased after LPS injury (p = 0.057 for LPS treatment). Normalized net intensities were analyzed by a Mann-Whitney test. (* p<0.05, <i>n of 4 mice per group</i>).</p

HMGB1 levels are increased in the BALF of wild type and RAGE KO mice after <i>E. coli</i> injury.

<p>In addition, soluble RAGE is increased in the BALF of wild type mice after <i>E. coli</i> injury. Aliquots of 35 µL BALF were loaded per lane. By western blot there is a significant increase in the levels of soluble RAGE and HMGB1 in the BALF from wild type mice (A) and a significant increase of HMGB1 protein in the BALF of RAGE KO mice (B) after <i>E. coli</i> injury. Direct comparison of HMGB1 levels between wild type and RAGE KO mice after <i>E. coli</i> injury (C) shows a similar level of HMGB1 in BALF from both strains. Comparisons of protein levels were done by band densitometry analysis and analyzed by a Mann-Whitney test (* p<0.05, <i>n of 5 mice per group</i>).</p

H&E stained lung sections from wild type and RAGE KO control, LPS and <i>E. coli</i> treated mice.

<p>The first column shows wild type and the second column shows RAGE KO lungs. Macrophages (arrows) and neutrophils (arrowheads) in alveoli are indicated. First row is control treated from both strains. A few macrophages are present in the alveoli of both control treated strains. LPS treatment, second row, led to a significant infiltration of neutrophils into the alveolar space; however no immediate difference in degree of infiltration was noticeable between the strains. The third row shows lung histology from <i>E. coli</i> treated mice. <i>E. coli</i> treated RAGE KO mice have fewer neutrophils compared to <i>E. coli</i> treated wild type mice. Black bars represent 30 µm.</p