Hepatocyte Growth Factor Modulates Interleukin-6 Production in Bone Marrow Derived Macrophages: Implications for Inflammatory Mediated Diseases

The generation of the pro-inflammatory cytokines IL-6, TNF-α, and IL-1β fuel the acute phase response (APR). To maintain body homeostasis, the increase of inflammatory proteins is resolved by acute phase proteins via presently unknown mechanisms. Hepatocyte growth factor (HGF) is transcribed in response to IL-6. Since IL-6 production promotes the generation of HGF and induces the APR, we posited that accumulating HGF might be a likely candidate for quelling excess inflammation under non-pathological conditions. We sought to assess the role of HGF and how it influences the regulation of inflammation utilizing a well-defined model of inflammatory activation, lipopolysaccharide (LPS)-stimulation of bone marrow derived macrophages (BMM). BMM were isolated from C57BL6 mice and were stimulated with LPS in the presence or absence of HGF. When HGF was present, there was a decrease in production of the pro-inflammatory cytokine IL-6, along with an increase in the anti-inflammatory cytokine IL-10. Altered cytokine production correlated with an increase in phosphorylated GSK3β, increased retention of the phosphorylated NFκB p65 subunit in the cytoplasm, and an enhanced interaction between CBP and phospho-CREB. These changes were a direct result of signaling through the HGF receptor, MET, as effects were reversed in the presence of a selective inhibitor of MET (SU11274) or when using BMM from macrophage-specific conditional MET knockout mice. Combined, these data provide compelling evidence that under normal circumstances, HGF acts to suppress the inflammatory response

Lack of the Receptor for Advanced Glycation End-Products Attenuates E. coli Pneumonia in Mice

Background: The receptor for advanced glycation end-products (RAGE) has been suggested to modulate lung injury in models of acute pulmonary inflammation. To study this further, model systems utilizing wild type and RAGE knockout (KO) mice were used to determine the role of RAGE signaling in lipopolysaccharide (LPS) and E. coli induced acute pulmonary inflammation. The effect of intraperitoneal (i.p.) and intratracheal (i.t.) administration of mouse soluble RAGE on E. coli injury was also investigated. Methodology/Principal Findings: C57BL/6 wild type and RAGE KO mice received an i.t. instillation of LPS, E. coli, or vehicle control. Some groups also received i.p. or i.t. administration of mouse soluble RAGE. After 24 hours, the role of RAGE expression on inflammation was assessed by comparing responses in wild type and RAGE KO. RAGE protein levels decreased in wild type lung homogenates after treatment with either LPS or bacteria. In addition, soluble RAGE and HMGB1 increased in the BALF after E. coli instillation. RAGE KO mice challenged with LPS had the same degree of inflammation as wild type mice. However, when challenged with E. coli, RAGE KO mice had significantly less inflammation when compared to wild type mice. Most cytokine levels were lower in the BALF of RAGE KO mice compared to wild type mice after E. coli injury, while only monocyte chemotactic protein-1, MCP-1, was lower after LPS challenge. Neither i.p. nor i.t. administration of mouse soluble RAGE attenuated the severity of E. coli injury in wild type mice. Conclusions/Significance: Lack of RAGE in the lung does not protect against LPS induced acute pulmonary inflammation, but attenuates injury following live E. coli challenge. These findings suggest that RAGE mediates responses to E. coli-associated pathogen-associated molecular pattern molecules other than LPS or other bacterial specific signaling responses. Soluble RAGE treatment had no effect on inflammation. © 2011 Ramsgaard et al

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

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

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

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

Lymphocyte Activation Gene-3 Maintains Mitochondrial and Metabolic Quiescence in Naive CD4+ T Cells

Summary: Lymphocyte activation gene-3 (LAG-3) is an inhibitory receptor expressed by CD4+ T cells and tempers their homeostatic expansion. Because CD4+ T cell proliferation is tightly coupled to bioenergetics, we investigate the role of LAG-3 in modulating naive CD4+ T cell metabolism. LAG-3 deficiency enhances the metabolic profile of naive CD4+ T cells by elevating levels of mitochondrial biogenesis. In vivo, LAG-3 blockade partially restores expansion and the metabolic phenotype of wild-type CD4+ T cells to levels of Lag3−/− CD4+ T cells, solidifying that LAG-3 controls these processes. Lag3−/− CD4+ T cells also demonstrate greater signal transducer and activator of transcription 5 (STAT5) activation, enabling resistance to interleukin-7 (IL-7) deprivation. These results implicate this pathway as a target of LAG-3-mediated inhibition. Additionally, enhancement of STAT5 activation, as a result of LAG-3 deficiency, contributes to greater activation potential in these cells. These results identify an additional mode of regulation elicited by LAG-3 in controlling CD4+ T cell responses. : Previte et al. show that LAG-3 expression regulates the metabolic profile of naive CD4+ T cells during homeostatic expansion. They observed that Lag3-deficient CD4+ T cells are resistant to Interleukin-7 deprivation due to enhanced STAT5 activation. Increased STAT5 signaling also mediated greater activation potential in these T cells following stimulation. Keywords: LAG-3, CD4+ T cell, metabolism, mitochondria, STAT