Prednisolone and salbutamol synergistically suppress HMGB1-induced TNFα secretion.

<p>RAW 264.7 cells were pretreated with prednisolone and salbutamol at the indicated concentrations and exposed to HMGB1 (5 µg/ml) for 18 hours. TNFα secretion (<b>A, B</b>) and LDH release (<b>E, F</b>) were measured in the supernatant. Cell viability (<b>C, D</b>) was measured by the MTT assay. (^§p<0.05 HMGB1-treated group compared to vehicle treated control, *p<0.05 compared to HMGB1 group, ^#p<0.05 compared to the respective first compound treatment).</p

List of hit compounds identified in the primary screen.

<p>Non-toxic compounds that reduced the HMGB1-induced TNFα production by 2 standard deviation values are listed in order of potency, according to their inhibitory potency for TNFα secretion. The source library of the compounds, their known biological activity and the respective viability values are shown. Viability was measured by the MTT assay. (Abbreviations: MAP kinase: Mitogen-activated protein kinase, U0126∶1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene, MEK: mitogen-activated protein kinase kinase, STAT3: Signal transducer and activator of transcription 3, ST057244∶1-[(2E)-3-(3,4,5-trimethoxyphenyl)prop-2-enoyl]piperidin-2-one, Bay 11-7085: (2<i>E</i>)-3-[[4-(1,1-dimethylethyl)pheny?l]sulfonyl]-2-propenenitrile, Bay 11-7082∶3- [(4- methylphenyl)sulfonyl]- (2E)- propenenitrile, ST009819: (2R,3R,13R,14R)-3-(phenylcarbonyl)-17,19-dioxa-4-azapentacyclo[14.2.1.0<2,14>. 0<4,13>.0<7,12>]nonadeca-5,7(12),8,10-tetraen-15-one, MNS: 3,4-methylenedioxy-β-nitrostyrene, IkB: inhibitor of nuclear factor κB kinase, NF-κB: nuclear factor κB, HIV: human immunodeficiency virus, Src: sarcoma tyrosine kinase, Syk: Spleen tyrosine kinase).</p

HMGB1 induces time-dependent caspase activation in RAW 264.7 macrophages.

<p>RAW 264.7 cells were exposed to HMGB1 (5 µg/ml) for 24, 48 or 72 hours. Activated Caspase-3 was detected in cell extracts by Western blotting. Tubulin was used for loading control. The graph shows relative Caspase-3 activation values, normalized to tubulin. (**p<0.01 shows significant caspase activation compared to vehicle-treated cells).</p

Inhibition of the HMGB-induced TNFα production by catecholamines and glucocorticoids <i>in vivo</i>.

<p>Balb/c male mice (Charles River Laboratories) were injected with 0.5 mg/kg HMGB1 in the presence of 60 min pretreatment of either vehicle, or 20 mg/kg prednisolone, 10 mg/kg salbutamol, the combination of prednisolone and salbutamol (doses as above), or the glucocorticoid receptor blocker mifepristone (30 mg/kg) or the β-receptor antagonist propranolol (10 mg/kg). At 8 hours after HMGB1 injection, animals were sacrificed and serum levels of TNFα were measured. ^#p<0.05 represents a significant increase in TNFα serum levels in response to HMGB1; *p<0.05 represents significant inhibition of HMGB1-induced TNFα production by the various pharmacological agents indicated. n = 7 animals per group.</p

List of confirmed hit compounds.

<p>Hit compounds of the primary screen were retested in replicates at 3 and 10 µM against HMGB1 and LPS and compounds are shown that decreased the HMGB<b>-</b>induced TNFα production by at least 40% in the hit confirmation experiments. (The majority of the glucocorticoids were not retested during the hit confirmation studies, since all glucocorticoids showed similar activity, confirming their class action.) TNFα production and viability values are shown for the primary screen and the TNFα production is shown for the hit confirmation experiments (Mean±SD). Compounds that reduced cell viability by at least 25% are labeled with an asterisk. (Abbreviations: Bay 11-7085: (2<i>E</i>)-3-[[4-(1,1-dimethylethyl)phenyl]sulfonyl]-2-propenenitrile, MNS: 3,4-methylenedioxy-β-nitrostyrene, PABA potassium salt: para-aminobenzoic acid potassium salt, NF-κB: nuclear factor κB, HIV: human immunodeficiency virus, Src: sarcoma tyrosine kinase, Syk: Spleen tyrosine kinase).</p

MAPK activation and IκB phosphorylation in response to HMGB1 are ameliorated in synergy by prednisolone and salbutamol.

<p><b>A:</b> RAW 264.7 cells were exposed to HMGB1 (5 µg/ml) for the indicated length and the phosphorylation of ERK1/2, p38 and IκB was detected. <b>B:</b> RAW 264.7 cells pretreated with prednisolone (1 µM) and salbutamol (1 µM) were exposed to HMGB1 (5 µg/ml) for 30 min (ERK1/2, p38) or 1 hour (IκB) and the activation was detected as phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), phospho-p38 (Thr 180) or phospho-IκB-α (Ser 32/36). <b>C:</b> Bar graph shows the phosphorylation signal normalized to the total amount of the respective protein. (CTL: vehicle treated control, HMGB: cells exposed to HMGB1, Pred: cells pretreated with prednisolone and exposed to HMGB1, Salb: cells pretreated with salbutamol and exposed to HMGB1, Pred+Salb: cells pretreated with both prednisolone and salbutamol and exposed to HMGB1. ^§p<0.05 HMGB1-treated group compared to vehicle treated control, *p<0.05 compared to HMGB1 group).</p

Time-dependence of the HMGB1-induced suppression of cellular bioenergetics in RAW 264.7 macrophages.

<p>RAW 264.7 cells were exposed to HMGB1 (5 µg/ml) for 24, 48 or 72 hours. Cellular bioenergetic parameters were measured with Seahorse extracellular fluid analysis. <b>A:</b> Time-dependent decrease in basal cellular respiration (Oxygen Consumption Rate, OCR). (**p<0.01 compared to vehicle treated cells) <b>B:</b> Time-dependent decrease in maximal cellular respiration. (*p<0.05 and **p<0.01 compared to vehicle treated cells). <b>C:</b> Representative tracing comparing cellular respiration (Oxygen Consumption Rate) in response to sequential administration of pharmacological modulators of cell metabolism in vehicle-treated cells or cells treated with HMGB1 for 72 hours. Basal Respiration, Calculated ATP Turnover, Proton Leak and Maximal Respiration areas are indicated and demonstrate a marked suppression of cellular bioenergetic parameters.</p

Compounds that enhance the HMGB1-induced TNFα production of RAW264.7 cells.

<p>Compounds augmenting the HMGB1-induced TNFα production by 2 standard deviation values are listed are listed in order of potency, according to their enhancing effect on TNFαα secretion. The source library of the compounds, their known biological activity and the respective viability values are shown. Viability was measured by the MTT assay. (Abbreviations: PKC: protein kinase C, HIV: human immunodeficiency virus, PDGF: platelet-derived growth factor, HMG-CoA: 3-hydroxy-3-methylglutaryl-coenzyme A, TrkA: TRK1-transforming tyrosine kinase protein).</p

Inhibition of the HMGB-induced inflammatory response by endogenous catecholamines and glucocorticoids at physiological concentrations.

<p>RAW 264.7 cells were pretreated with cortisol (0.7 µM), noradrenaline (0.5 ng/ml), adrenaline (0.5 ng/ml), dexamethasone (1 µM) and salbutamol (1 µM) and exposed to HMGB1 (5 µg/ml) for 18 hours. TNFα secretion was measured in the supernatant. (^§p<0.05 HMGB1-treated group compared to vehicle treated control, *p<0.05 compared to HMGB1 group, ^#p<0.05 cells treated with all compounds in combination versus treated with a combination of two.).</p

HMGB1 induces an inflammatory response in RAW 264.7 macrophages.

<p><b>A–B:</b> RAW 264.7 cells were treated with the indicated amount of HMGB1 and IFN-γ for 18 hours and the TNFα secretion was measured in the supernatant. The viability of the cells was measured by the MTT assay. (*p<0.05 compared to vehicle treated cells, ^#p<0.05 IFN-γ treated group compared to the respective HMGB1-treated group) <b>C:</b> RAW 264.7 cells were treated with HMGB1 (5 µg/ml) for 1.5 hours and the expression of TLR-associated genes was analyzed with TLR signaling pathways array. The gene symbols and the average fold-expression values are shown compared to vehicle-treated cells in the color-scale, according to the their relative expression. (*p<0.05 compared to vehicle-treated cells.).</p