Conceived and designed the experiments: GMC WMM JDP. Performed the experiments: GMC JH. Analyzed the data: GMC JH WMM JDP. Contributed reagents/materials/analysis tools: MT JDP. Wrote the paper: GMC WMM JDP.
The authors have declared that no competing interests exist.
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.
As a first line of defense in response to infection, tissue injury and stress, macrophages generate the NFκB-dependent pro-inflammatory cytokines, TNF-α, IL-1β and IL-6
IL-6 induction serves a dual role in the transition between propagation of the inflammatory response and initiation of the APR
To investigate this hypothesis we sought to assess the role of HGF and its cognate receptor, MET, with regard to innate immune activation of LPS-stimulated BMM. Our results demonstrate that in the presence of HGF there is a significant decrease in the secreted levels of IL-6, suggesting that HGF suppresses inflammation after injury. The suppression of IL-6 is achieved through HGF-dependent inactivation of GSK3β, a powerful governor of inflammatory signaling. This inactivation of GSK3β enhances the anti-inflammatory pathway by promoting the interaction of phospho-CREB with CBP and, occurs in concert with an overall decrease of phospho-p65 (Ser 276) and elevated levels of the anti-inflammatory cytokine IL-10
In order to determine that HGF plays a role in regulating the APR to suppress inflammation, we utilized a well-defined
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.
To further confirm that the inhibition of IL-6 production was a result of HGF signaling, we repeated the
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.
To rule out any offsite pharmacological effects by the MET inhibitor as the cause for the IL-6 inhibition and to further study the important role HGF plays in tempering the acute inflammatory response, conditional MET flox mice specific for the macrophage lineage were generated.
BMM derived from either macrophage specific MET conditional knockout mice (METfl/fl:crelysZ+/−) or their wild type littermate controls (METfl/fl:crelysZ−/−) 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.
To further understand the mechanism in which HGF suppresses the inflammatory response, we looked downstream of HGF-MET signaling at potential regulatory targets. One target, GSK3β, is known to regulate inflammation through activation of NFκB, resulting in pro-inflammatory cytokine production
(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.
To further determine the downstream signaling that results from HGF's regulation of GSK3β, we investigated the interaction of NFκB with the co-activator protein (CBP). Interactions between NFκB and CBP are facilitated by activated GSK3β when there is promotion of pro-inflammatory cytokine production
(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.
The enhanced interaction between CBP and phospho-CREB during the HGF-induced anti-inflammatory response suggests CBP is being sequestered away from NFκB. Normally, the transcriptional activity of NFκB occurs through the phosphorylation of p65 at Serine 276 whereby a conformational change takes place that allows its nuclear association with CBP/p300
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.
IL-6, a key pro-inflammatory cytokine, is upregulated “as a defense mechanism” in order to promote the APR and to initiate homeostasis as quickly as possible after an acute injury. In cases of chronic stress; however, IL-6 changes its role by modulating the leukocytic repertoire resulting in a chronic inflammatory state
Using LPS stimulated primary BMM cell cultures as a source of IL-6, we demonstrate that addition of HGF is in fact anti-inflammatory (
Traditionally, GSK3β is known for regulating glycogen synthase and the storage of glycogen into peripheral sites
Pharmacological inhibitors of PI3K, Akt, and GSK3β induce inactivation of GSK3β. Inactive GSK3β can then promote the association of phospho-CREB (Ser 133) with CBP and sequester the CBP away from NFκB p65 (Ser 276). These signaling changes are associated with a switch from a pro- to anti-inflammatory pathway with a resultant increase in IL-10 production
In addition to the canonical pathway, TLR signaling has been shown to weakly activate PI3K. This then leads to anti-inflammatory events by altering the cytokine repertoire
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
C57BL/6J and B6.129P2-Lyz2tm1(cre)Ifo/J mice were purchased from The Jackson Laboratory and the MET floxed mice were a gift from Dr. Snorri Thorgeirsson
Bone marrow derived macrophages (BMM) were cultured as previously described
BMM plated in 24-well plates were pretreated with recombinant mouse HGF (R&D Systems) for 24 h at 37°C prior to stimulation with 1 µg/ml lipopolysacharride (LPS) from
BMM were prepared as above and cultured in 100 mm dishes. At 15 minutes post-LPS stimulation, the BMM were washed with ice cold PBS and cells were harvested with gentle scraping and centrifugation. Cellular extracts were harvested using RIPA buffer supplemented with phosphatase and protease inhibitor cocktails (Roche) and 1 mM PMSF (Sigma-Aldrich). After the addition of 0.3 ml of lysis buffer, the macrophages were incubated on ice for 20 min, vortexed 3 times and centrifuged for 5 min at maximum speed at 4°C. The supernatant (whole cell lysate) was collected and the protein concentration of the lysates was determined by the bicinchonninic acid (BCA) protein assay according to the manufacturer's instruction (Pierce).
Supernatants from BMM pretreated with HGF and/or SU11274 and stimulation with LPS were collected at 24 h. IL-6 and IL-10 cytokines secreted by BMM were measured by ELISA using purified capture and biotinylated detection antibody pairs (BD Pharmingen). The ELISA plates were read using a SpectraMax M2 plate reader, and the data was analyzed using SoftMax Pro software (Molecular Devices).
Protein lysates were assayed by Western blot as previously described, with minor exceptions
1 µg of CBP antibody (Santa Cruz) was added to 100 mg protein from whole cell lysates and incubated at 4°C overnight. The following day, Protein G-conjugated sepharose beads were added to the protein/antibody complex tube and incubated for 2 h. Immunoprecipitates were collected by centrifugation, washed twice with PBS, and then boiled in Laemmli sample buffer. Western blot was performed as described above.
BMMs were washed with PBS and gently scraped from multi-well tissue culture dishes. Cells were collected into centrifuge tubes and spun at high speed for 10 min. Cell pellets were resuspended to 1×103 cells per ml and cytospins were prepared with 100 µl of cells per slide. Slides were dried and stored at −80°C until staining.
Cytospun slides were fixed in 2% paraformaldehyde for 5 minutes and blocked with 20% non-immune normal goat serum for 1 hour at room temperature. After immune stained with primary antibody (Rabbit anti-Phospho-NFκBp65 (Ser276) 1∶100, (Cell Signaling Technology), slides were washed 5 times in PBS and incubated with secondary antibodies Cy3-conjugated goat anti-rabbit (Jackson ImmunoResearch). Nuclear staining (Hoechest staining, Molecular Probes) was performed. Images were viewed at 40x magnification and captured by using a Nikon confocal microscope (Nikon D-ECLIPSE C1, Japan).
G.M.C. is a PhD candidate at the University of Pittsburgh and this work is submitted in partial fulfillment of the requirement for the PhD. We thank Snorri Thorgeirsson for the gift of the MET floxed mice, and Dr. Hubert Tse and Meghan Delmastro for critical reading of this manuscript.