Para-hydroxyphenylpyruvate inhibits the pro-inflammatory stimulation of macrophage preventing LPS-mediated nitro-oxidative unbalance and immunometabolic shift

PLOS ONE, Nov 2019

Targeting metabolism is emerging as a promising therapeutic strategy for modulation of the immune response in human diseases. In the presented study we used the lipopolysaccharide (LPS)-mediated activation of RAW 264.7 macrophage-like cell line as a model to investigate changes in the metabolic phenotype and to test the effect of p-hydroxyphenylpyruvate (pHPP) on it. pHPP is an intermediate of the PHE/TYR catabolic pathway, selected as analogue of the ethyl pyruvate (EP), which proved to exhibit antioxidant and anti-inflammatory activities. The results obtained show that LPS-priming of RAW 264.7 cell line to the activated M1 state resulted in up-regulation of the inducible nitric oxide synthase (iNOS) expression and consequently of NO production and in release of the pro-inflammatory cytokine IL-6. All these effects were prevented dose dependently by mM concentrations of pHPP more efficiently than EP. Respirometric and metabolic flux analysis of LPS-treated RAW 264.7 cells unveiled a marked metabolic shift consisting in downregulation of the mitochondrial oxidative phosphorylation and upregulation of aerobic glycolysis respectively. The observed respiratory failure in LPS-treated cells was accompanied with inhibition of the respiratory chain complexes I and IV and enhanced production of reactive oxygen species. Inhibition of the respiratory activity was also observed following incubation of human neonatal fibroblasts (NHDF-neo) with sera from septic patients. pHPP prevented all the observed metabolic alteration caused by LPS on RAW 264.7 or by septic sera on NHDF-neo. Moreover, we provide evidence that pHPP is an efficient reductant of cytochrome c. On the basis of the presented results a working model, linking pathogen-associated molecular patterns (PAMPs)-mediated immune response to mitochondrial oxidative metabolism, is put forward along with suggestions for its therapeutic control.

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Para-hydroxyphenylpyruvate inhibits the pro-inflammatory stimulation of macrophage preventing LPS-mediated nitro-oxidative unbalance and immunometabolic shift

November Para-hydroxyphenylpyruvate inhibits the pro- inflammatory stimulation of macrophage preventing LPS-mediated nitro-oxidative unbalance and immunometabolic shift Rosella Scrima 0 1 Marta Menga 0 1 Consiglia Pacelli 0 1 Francesca Agriesti 1 Olga Cela 0 1 Claudia Piccoli 0 1 Antonella Cotoia 1 Alessandra De Gregorio 1 Julia V. Gefter 1 Gilda Cinnella 1 Nazzareno Capitanio 0 1 0 Department of Clinical and Experimental Medicine, University of Foggia , Foggia , Italy , 2 Laboratory of Pre- Clinical and Translational Research, IRCCS CROB , Rionero in Vulture, Potenza , Italy , 3 Department of Medical and Surgical Sciences, University of Foggia , Foggia , Italy , 4 Department of Critical Care Medicine, University of Pittsburgh School of Medicine , Pittsburgh , United States of America 1 Editor: Alberto G Passi, University of Insubria , ITALY Targeting metabolism is emerging as a promising therapeutic strategy for modulation of the immune response in human diseases. In the presented study we used the lipopolysaccharide (LPS)-mediated activation of RAW 264.7 macrophage-like cell line as a model to investigate changes in the metabolic phenotype and to test the effect of p-hydroxyphenylpyruvate (pHPP) on it. pHPP is an intermediate of the PHE/TYR catabolic pathway, selected as analogue of the ethyl pyruvate (EP), which proved to exhibit antioxidant and anti-inflammatory activities. The results obtained show that LPS-priming of RAW 264.7 cell line to the activated M1 state resulted in up-regulation of the inducible nitric oxide synthase (iNOS) expression and consequently of NO production and in release of the pro-inflammatory cytokine IL6. All these effects were prevented dose dependently by mM concentrations of pHPP more efficiently than EP. Respirometric and metabolic flux analysis of LPS-treated RAW 264.7 cells unveiled a marked metabolic shift consisting in downregulation of the mitochondrial oxidative phosphorylation and upregulation of aerobic glycolysis respectively. The observed respiratory failure in LPS-treated cells was accompanied with inhibition of the respiratory chain complexes I and IV and enhanced production of reactive oxygen species. Inhibition of the respiratory activity was also observed following incubation of human neonatal fibroblasts (NHDF-neo) with sera from septic patients. pHPP prevented all the observed metabolic alteration caused by LPS on RAW 264.7 or by septic sera on NHDF-neo. Moreover, we provide evidence that pHPP is an efficient reductant of cytochrome c. On the basis of the presented results a working model, linking pathogen-associated molecular patterns (PAMPs)-mediated immune response to mitochondrial oxidative metabolism, is put forward along with suggestions for its therapeutic control. Data Availability Statement; All relevant data are within the paper - Funding: This work was supported by local funds from the University of Foggia to RS, MM, FA, OC, CPi, AD, JVG, GC, NC and funds from FIR 20152018 from Apulia Region to AC, CPa. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction Recent studies have highlighted the tight link between metabolic state and phenotype of immune-competent cells both in the innate and acquired immunity [1±4]. The emerging concept is that metabolism not only sustains diverse immune cell phenotypes as a consequence of alterations in cellular signalling, but also feeds back and alters signalling to drive the immune-cell phenotype. A common feature of pro-inflammatory immune cells, such as effector lymphocytes, Tr1 regulatory T cells, M1 macrophages, mature dendritic cells, neutrophils and other granulocytes, is that they adopt a distinct metabolic signature termed ªaerobic glycolysisº to support cellular biosynthetic processes: that is, glucose metabolized to lactate in the presence of abundant oxygen. Aerobic glycolysis is strategic to cells engaged in robust growth and proliferation because it provides fast ATP production (albeit with low efficiency) and biosynthetic precursors that are essential for the synthesis of nucleotides, amino acids, and lipids. Such a metabolic phenotype closely resembles what, since long observed in many cancer cells, known as "Warburg effect". Conversely, other immune cell subsets, such as naïve lymphocytes, memory T cells, FoxP3+ regulatory T cells, M2 macrophages, rely on oxidative metabolism [ 5 ]. In this context mitochondria are being increasingly recognized as signalling organelles for both the maintenance and establishment of immune cell phenotypes [ 6 ]. In addition to the recognized role in cellular bioenergetics mitochondria function as hubs integrating multiple innate immune signalling [ 7 ]. Mitochondria are also a major source and target of reactive oxygen and nitrogen species, establishing oxidative unbalance in various context, including the response to bacterial infection. On this basis, targeting the (...truncated)


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Rosella Scrima, Marta Menga, Consiglia Pacelli, Francesca Agriesti, Olga Cela, Claudia Piccoli, Antonella Cotoia, Alessandra De Gregorio, Julia V. Gefter, Gilda Cinnella, Nazzareno Capitanio. Para-hydroxyphenylpyruvate inhibits the pro-inflammatory stimulation of macrophage preventing LPS-mediated nitro-oxidative unbalance and immunometabolic shift, PLOS ONE, 2017, Volume 12, Issue 11, DOI: 10.1371/journal.pone.0188683