Innate defense regulator peptides (IDR) 1002 (VQRWLIVWRIRK-NH₂), HH2 (VQLRIRVAVIRA-NH₂), and 1 (KSRIVPAIPVSLL-NH₂) were synthesized using solid phase F-moc chemistry by CPC Scientific, Inc. (Sunnyvale, CA, USA) and were >95% pure. RPMI 1640 cell culture medium, Bradford reagent, bovine serum albumin (BSA), Wortmannin (inhibitor of PI3K), Akt IV (inhibitor of Akt), Rapamycin (inhibitor of p70S6K), SB216763 (inhibitor of GSK3), PD98059 (inhibitor of ERK1/2), and Ro-318220 (inhibitor of MSK1) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). SB203580 (inhibitor of p38) and SP600125 (inhibitor of JNK) were purchased from Calbiochem (San Diego, CA, USA). Lymphoprep™ was purchased from Axis-Shield PoC AS (Oslo, Norway). Upon arrival, all inhibitors were solubilized in DMSO and stored in aliquots at −20°C. Newborn calf serum was acquired from Hyclone Laboratories, Inc. (South Logan, UT, USA). Penicillin G and streptomycin were purchased from Gibco-BRL (Gaithesburg, MD, USA). EDTA-free protease inhibitor cocktail (50×) was purchased from Roche Applied Science (Manheim, Germany). Antibodies against β-actin (Sc-47778), COX-2 (Sc-1747), GAPDH (Sc-25778), laminin A/C (6215), IκBα (Sc-203), and phospho-IκBα (Sc-52943) for the detection of phosphorylated Ser32 and Ser36, CREB-1 (Sc-186), NF-κB p65 (Sc-8008), CBP (Sc-7300), anti-rabbit IgG-HRP (Sc-2004), rabbit anti-goat IgG-HRP (Sc-2768), and goat anti-mouse IgG-HRP (Sc-2031) were acquired from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against the phosphorylated forms of CREB at Ser133 (9196), p38 at Thr180/Tyr182 (4511), NF-κB p65 at Ser536 (3033), GSK3α at Ser21 (9316), and GSK3β at Ser9 (9336) were purchased from Cell Signaling Technology (Boston, MA, USA). Antibodies to detect phosphorylated MSK1 at Thr581 (AF2518) were purchased from R&D Systems (Minneapolis, MN, USA). Antibodies to detect the phosphorylated forms of IKKα and β at Thr23 were obtained from Santa Cruz Biotechnology (Cat. Sc-21660, Santa Cruz, CA, USA). Non-fat dry milk was acquired from Bio-Rad (California, CA, USA) and Luminol reagent from Millipore (Billerica, MA, USA).

The mouse leukemic monocyte macrophage cell line RAW 264.7 used was obtained from the American Type Culture Collection (ATCC). This immortalized cell line was grown and maintained in RPMI medium supplemented with 10% FCS, unless otherwise noted.

Venous blood (20 ml) from healthy human donors was collected in tubes containing EDTA in accordance with the approval and ethical guidelines of the Universidad Michoacana de San Nicolás de Hidalgo. Blood was diluted in the same volume of RPMI medium. Then, 20 ml of diluted blood was carefully added to a tube containing 10 ml of Lymphoprep™ and centrifuged at 800 × g for 20 min at room temperature (23°C). The phase containing peripheral blood mononuclear cells (PBMC) was separated and washed 3× with enough PBS to complete 50 ml for each wash and centrifuged at 250 × g for 10 min. After washing, PBMC were resuspended in RPMI medium and seeded in six-well plate at 2 × 10⁶ cells/well. Cells were left resting for at least 3 h before the experiments.

MSK1 and MSK2 gene expression were silenced with siRNA acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The siRNA for MSK1 (sc-35978), MSK2 (sc-75837), and control siRNA (sc-37007) were added to macrophages at the final concentration of 40 nM for 48 h, according to the manufacturer’s instructions. To test for MSK1/MSK2 participation in CREB phosphorylation at Ser133, macrophages were first gene silenced for MSK1 and/or MSK2 and then stimulated with 20 μg/ml of IDR-1002 for 15 min.

The relative abundance of phosphorylated and non-phosphorylated proteins was evaluated in protein extracts from macrophages grown in six-well culture plates to ~90% confluence before serum starvation for at least 4 h. For each assay, total protein (cytosolic plus nuclear from control and treated cells) was obtained by washing cells with cold PBS (2×) and lysing them with 80 μl of a cold lysis buffer containing 20 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% Igepal CA-930, 10 mM Na-pyrophosphate, and 50 mM NaF, supplemented with 1 mM Na-orthovanadate and 1× protease inhibitor cocktail that was added immediately before use. The lysates were centrifuged at 13,000 × g for 20 min at 4°C, and the supernatant was transferred to ice-cold Eppendorf tubes. Nuclear and cytoplasmic fractions were prepared from cells using the NE-PER Reagent kit from Pierce (Rockford, IL, USA), and stored in aliquots at −80°C. Protein concentration was measured by the Bradford method (33) using BSA as standard. Then, 40 μg of protein was separated by electrophoresis in 10% sodium dodecyl sulfate polyacrylamide gels and electroblotted in a wet chamber to 0.45 μm nitrocellulose membrane (Bio-Rad) at 250–300 mA for 1 h. Membranes were then probed with the indicated antibody and the abundance of the phosphorylated and unphosphorylated forms of each protein was detected using the Immobilon Western Chemiluminescent HRP substrate kit from Millipore (Billerica, MA, USA). Membranes were exposed to an X-ray film (Kodak) with two intensifying screens (DuPont) at room temperature (23°C).

Macrophages Raw 264.7 were seeded in 10-cm cell plate culture until confluence, left in serum-free media for 4 h, and then stimulated as indicated. For immunoprecipitation technique, we use the kit Pierce™ Protein A/G Magnetic Beads (Thermo Fischer Scientific). In brief, ~1 × 10⁷ cells were lysed in washing/lysis buffer supplemented with 1× protease inhibitor cocktail, and cells debris were collected by centrifugation at 18,000 × g for 10 min at 4°C. The supernatant was collected and quantified for protein concentration. Then, 4 μg of each immunoprecipitation antibody (CBP or IκBα) was added to 500 μg of protein extract and the volume adjusted to 500 μl. The mixture was incubated with continuous agitation all night at 4°C. Next day, 25 μg of magnetic beads were added and incubated 1 h in agitation at room temperature (23°C); beads were washed 2× with washing buffer and 1× with water. The target antigen was eluted with lane marker buffer 1× plus 50 mM DTT in the final volume of 100 μl. For immunoblots, 20 μl of target antigen in elution buffer was used and detected as described in the western blot analysis section.

Mouse cytokines, IL-4, IL-6, IL-10, IL-13, TNF-α, and PGE2 levels, were measured by ELISA, according to the manufacturer’s instructions (R&D, Minneapolis, MN, USA).

Macrophages were incubated for 1 h with 20 μg/ml of IDR-1, IDR-HH2, or IDR-1002 and then stimulated for 2–24 h with 10 ng/ml of LPS. Supernatants were collected and cytokines were detected using a mouse cytokine antibody pair-based array spotted on a membrane (Abcam, ab133993) according to the manufacturer’s instructions (Cambridge, UK). Briefly, analysis of cytokines present in the macrophages supernatant is carried out by chemiluminescent western blot assay, using biotinylated detector antibodies and streptavidin HRP. Targets of this ELISA-like array are granulocyte-colony stimulating factor (GCSF), granulocyte-macrophage colony stimulating factor (GM-CSF), IL-2, 3, 4, 5, 6, 9, 10, 12 p40/p70, 12p70, 13, 17, interferon gamma (IFN-γ), monocyte chemoattractant protein 1 and 5 (MCP-1 and MCP-5), regulated on activation normal T cell expressed and secreted (RANTES), stem cell factor (SCF), soluble tumor necrosis receptor factor 1 (sTNFR1), tumor necrosis factor alpha (TNF-α), thrombopoietin, and vascular endothelial growth factor (VEGF).

Statistical significance was evaluated using Student’s paired t-test using the SIGMASTAT program version 3.0 (SPSS Inc., Chicago, IL, USA). Band densitometric analysis was performed using the Image Processing and Analysis module in Java Program ImageJ (http://rsbweb.nih.gov/ij).

To explore the effect of IDR-1, IDR-HH2, and IDR-1002 on cytokine expression, we used an antibody array of 22 cytokines. After 12 h of incubation with each of the IDR peptides, we evaluated the level of cytokine expression in the supernatants. Compared to control (Figure 1A), IDR-1 had a modest effect on TNF-α expression (Figure 1B). However, when macrophages were treated with IDR-HH2 or IDR-1002, the basal levels of TNF-α were significantly reduced. We observed that IDR-1002 was a somewhat stronger inhibitor than IDR-HH2 (Figures 1C,D). A map of the cytokine array is shown in Figure 1E. To determine the kinetics of the effect of IDR-1002 on TNF-α production, macrophages were pre-incubated with IDR-1002 for 1 h and then stimulated, for periods of 2–24 h, with LPS from E. coli, an agonist that induced a strong release of TNF-α. We observed that production of TNF-α activated by LPS was strongly reduced in the presence of IDR-1002 (Figure 1F). These results indicate that IDR-1002 had an inhibitory effect on the basal and LPS-induced expression of TNF-α.

Another important gene expressed in the inflammatory response and induced by LPS is COX-2. This enzyme catalyzes the conversion of arachidonic acid into prostaglandins and thromboxane, playing an important role in the inflammatory response (34). To determine the effect of IDR-1002 on COX-2 production, macrophages were pre-incubated with IDR-1002 for 1 h and then stimulated with LPS for a period of 2–24 h. Under this condition, we observed that COX-2 expression was strongly and significantly reduced in the presence of IDR-1002 (Figure 2A). Incubation of macrophages with IDR-1002 alone for 2, 4, and 8 h did not have any effect on COX-2 (Figure 2B). These results indicate that IDR-1002 had an inhibitory effect on the LPS-induced expression of COX-2.

Activation of NF-κB stimulates its nuclear translocation leading to the expression of many pro-inflammatory genes, among which TNF-α and COX-2 play important roles in the inflammatory response. To explore if IDR-1002 inhibited TNF-α and COX-2 expression by inhibiting NF-κB activity, we evaluated phosphorylation of the p65 subunit at Ser536 (a residue located in the transactivation domain), and its nuclear relative abundance. An ~20–40% reduction of p65 phosphorylation at Ser536 was observed when macrophages were incubated with IDR-1002 for 5, 30, or 60 min (Figure 3A). Next, we investigated the relative abundance of p65 in the cytoplasm and nucleus of macrophages incubated with LPS and IDR-1002. From data shown in Figure 3B, it was clear that LPS stimulated p65 nuclear translocation, which was inhibited by IDR-1002. The co-immunoprecipitation of NF-κB and coactivator CBP was detected exclusively in macrophages stimulated with LPS, but not in those stimulated with IDR-1002 followed by LPS or IDR-1002 alone (Figure 3C). These results indicate that IDR-1002 reduced the relative abundance of p65 phosphorylated at Ser536 and its LPS-induced nuclear translocation and interaction with CBP.

In unstimulated cells, NF-κB is anchored to IκBα in the cytoplasm. Release of NF-κB requires phosphorylation of IκBα, which marks it to become ubiquitinated and then degraded in the proteosome. This mechanism implies that if phosphorylation of IκBα is inhibited, then NF-κB would not be released and its nuclear translocation would not occur. Our data indicate that IDR-1002 strongly inhibited the LPS-induced phosphorylation of IκBα (Figure 4A) and its degradation (Figure 4B). This effect of IDR-1002 on IκBα seemed to be independent of the stimulus because inhibition of IκBα phosphorylation and degradation by IDR-1002 was equally effective in macrophages stimulated with TNF-α or IL-1β (Figures 4A,B). Interaction of NF-κB p65 and IκBα was stabilized as a consequence of IDR-1002 inhibition of IκBα phosphorylation and degradation. Figure 4C shows that there was an increase in co-immunoprecipitation NF-κB p65 bound to IκBα in macrophages stimulated with IDR-1002. These data may explain the inhibitory effect exerted by IDR-1002 on TNF-α and COX-2 expression.

A major mechanism for controlling the inflammatory response is the suppression of pro-inflammatory cytokine expression, which can be promoted when NF-κB forms a molecular complex with CBP (16, 17). This process takes place during the inflammatory response when CREB is also activated by phosphorylation at Ser133. Phosphorylation of CREB enables it to compete with NF-κB for the same interaction domain on CBP (18). The consequence of NF-κB separation from CBP is a reduction in inflammatory cytokine expression and simultaneously an increment, in many but not all cases, of anti-inflammatory cytokine expression due to CREB activity. To explore if IDR-1002 activated CREB phosphorylation at Ser133, macrophages were incubated with IDR-1002 for 5– 60 min. Under these conditions, a significant increase in CREB phosphorylation was observed for up to 45 min of incubation with IDR-1002 (Figure 5A). Because CREB phosphorylation can be mediated by Akt (PKB), ERK1/2, p38, and MSK1/2, we performed experiments utilizing specific inhibitors for each one of these enzymes. Macrophages stimulated with IDR-1002 showed an increased relative abundance of CREB phosphorylated at Ser133 and this was reduced by specific inhibitors of p38 and ERK1/2 (Figures 5B,C). Correspondingly, IDR-1002 induced a strong phosphorylation of ERK1/2 and p38 (Figures S1A,B in Supplementary Material), as described previously (27). However, specific inhibitors of PI3K, GSK3, and Akt did not have any effect on CREB phosphorylation (Figures S2A,B in Supplementary Material).

Other authors have reported that ERK1/2 and p38 do not directly phosphorylate CREB but instead they first phosphorylate MSK1/2, which in turn phosphorylates CREB at Ser133 (35). To test if MSK1/2 linked the activation of ERK1/2 and p38 with CREB phosphorylation, we silenced the MSK1 and MSK2 genes using siRNA. When these genes were independently or simultaneously silenced, a significant reduction in CREB phosphorylation was observed (Figure 6A), which indicated that both MSK1 and MSK2 could participate. However, only MSK1 (Figure 6B), but not MSK2 (data not shown), phosphorylation was detected in macrophages stimulated with IDR-1002. Evidence of MSK1 gene knockout by siRNA is presented in Figure 6A. Furthermore, incubation of macrophages with either ERK1/2- or p38-specific inhibitors almost completely prevented phosphorylation of MSK1 (Figure 6B).

However, although CREB was strongly phosphorylated, we observed no increase in the anti-inflammatory cytokines IL-4, IL-10, and IL-13 as measured by ELISA (data not shown). The lack of expression of these cytokines associated with M2 macrophage response might be due to the absence of interaction between phosphorylated CREB and CBP. The co-immunoprecipitation of CREB with CBP from macrophages stimulated with IDR-1002 showed that no complex was formed between CBP and CREB (Figure 6C). Together, these genetic and biochemical data indicated that IDR-1002-induced phosphorylation of CREB at Ser133 depended on the ERK1/2/p38–MSK1 signaling pathway, and that, at least in our system, phosphorylation of CREB by MSK1 did not promote interaction between CREB and CBP.