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

Abstract

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.

Macrophage Polarization Status Impacts Nanoceria Cellular Distribution but Not Its Biotransformation or Ferritin Effects

“…Cell polarization was also assessed using the standard Seahorse XF Cell Mito Stress Test on a Seahorse XFe96 Analyzer (Agilent, Santa Clara, CA, USA). LPS exposure produced almost complete mitochondrial shutdown [ 46 ], consistent with other studies [ 47 , 48 ]. Cell polarization was further assessed by IL-1β release assay by Western blotting.…”

Dichloroacetate Affects Mitochondrial Function and Stemness-Associated Properties in Pancreatic Cancer Cell Lines

“…Aberrant metabolic processes generally occur in cancer cells, and therefore targeting metabolism represents an emerging strategy to treat tumors, including pancreatic cancer [31,32,33]. Tumor heterogeneity may result in malignant cells with distinct metabolic phenotype, and consequently, different sensitivity to metabolic drugs as in the case of gemcitabine toward the majority of patients with pancreatic cancer develop resistance [17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34].…”

“…The oxygen consumption rate (OCR) and extra-cellular acidification rate (ECAR) were measured in adherent PANC-1 and BXPC-3 cells with an XF96 extracellular flux analyzer (Seahorse Bioscience, Billerica, MA, USA) as previously described [19]. Briefly, for the OCR analysis, after measuring basal respiration, oligomycin (1 μM), FCCP (1 μM), and rotenone + antimycin A (1 μM + 1 μM) were injected into each well sequentially to assess, respectively, the coupling of the respiratory chain, and the maximal and non-mitochondrial oxygen consumption.…”

Anabolic Steroids-Driven Regulation of Porcine Ovarian Putative Stem Cells Favors the Onset of Their Neoplastic Transformation

“…Detailed protocols and their justification can be found at (accessed from June 2020 to May 2021). Additionally, detailed protocols were previously published [ 70 , 71 , 72 ].…”