title: Anti-TNF certolizumab pegol induces antioxidant response in human monocytes via reverse signaling.
abstract: BACKGROUND
  Anti TNF drugs have been widely used in rheumatoid arthritis (RA) but only 70 to 80 % of patients respond to this therapy. Exploring the mode of action of anti-TNF drugs remains important in order to improve the efficiency of the treatment and enhance our knowledge of inflammation. TNF-α exists as classical soluble cytokine as well as transmembrane protein (tmTNF-α). Evidence suggests that tmTNF-α can induce reverse signaling. In the present study, we have explored consequences of reverse signaling in human monocytes using certolizumab pegol (CZP).
  
  
  METHODS
  Monocytes were purified from healthy blood donors and were incubated with CZP. Nuclear translocation of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) was evaluated by wide-field microscopy and cell fractionation. Heme oxygenase 1 (HO-1) was assessed by RT-qPCR and western blot. Monocytes were stimulated with lipopolysaccharide (LPS). IL-1β was quantitated by RT-qPCR. Reactive oxygen species (ROS) were evaluated by flow cytometry using the H2DCFDA fluorescent marker.
  
  
  RESULTS
  CZP induced rapid minimal ROS production and Nrf2 nuclear translocation. This was followed by HO-1 mRNA and protein production. IL-1β induction by LPS was inhibited at the mRNA and protein level. At a later time-point, CZP was able to counteract the strong production of ROS induced by LPS. Reverse signaling was suggested by short kinetics of Nrf2 translocation, extensive washing of CZP and the use of anti-TNF-Rs antibodies.
  
  
  CONCLUSIONS
  Our data suggest a novel mechanism of ROS modulation by CZP. This observation sheds new light on the function of reverse signaling and on potential mechanisms of action of anti-TNF drugs.
authors.affiliation: Université Paul Sabatier Toulouse III, Toulouse, France. jeanfred.boyer@gmail.com.|INSERM - CNRS U1043, CPTP, CHU Purpan, 1, Place Baylac, 31300, Toulouse, France. jeanfred.boyer@gmail.com.|Centre de Rhumatologie, Hopital Pierre Paul Riquet, Toulouse, France. jeanfred.boyer@gmail.com.
authors.forename: Jean Frédéric
authors.initials: JF
authors.lastname: Boyer
authors.affiliation: Université Paul Sabatier Toulouse III, Toulouse, France. michel.baron@inserm.fr.|INSERM - CNRS U1043, CPTP, CHU Purpan, 1, Place Baylac, 31300, Toulouse, France. michel.baron@inserm.fr.
authors.forename: Michel
authors.initials: M
authors.lastname: Baron
authors.affiliation: Université Paul Sabatier Toulouse III, Toulouse, France. arnaud.constantin@univ-tlse3.fr.|INSERM - CNRS U1043, CPTP, CHU Purpan, 1, Place Baylac, 31300, Toulouse, France. arnaud.constantin@univ-tlse3.fr.|Centre de Rhumatologie, Hopital Pierre Paul Riquet, Toulouse, France. arnaud.constantin@univ-tlse3.fr.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Constantin
authors.affiliation: Université Paul Sabatier Toulouse III, Toulouse, France. degboe@gmail.com.|INSERM - CNRS U1043, CPTP, CHU Purpan, 1, Place Baylac, 31300, Toulouse, France. degboe@gmail.com.|Centre de Rhumatologie, Hopital Pierre Paul Riquet, Toulouse, France. degboe@gmail.com.
authors.forename: Yannick
authors.initials: Y
authors.lastname: Degboé
authors.affiliation: Université Paul Sabatier Toulouse III, Toulouse, France. alain.cantagrel@wanadoo.fr.|INSERM - CNRS U1043, CPTP, CHU Purpan, 1, Place Baylac, 31300, Toulouse, France. alain.cantagrel@wanadoo.fr.|Centre de Rhumatologie, Hopital Pierre Paul Riquet, Toulouse, France. alain.cantagrel@wanadoo.fr.
authors.forename: Alain
authors.initials: A
authors.lastname: Cantagrel
authors.affiliation: Université Paul Sabatier Toulouse III, Toulouse, France. jean-luc.davignon@inserm.fr.|INSERM - CNRS U1043, CPTP, CHU Purpan, 1, Place Baylac, 31300, Toulouse, France. jean-luc.davignon@inserm.fr.|Centre de Rhumatologie, Hopital Pierre Paul Riquet, Toulouse, France. jean-luc.davignon@inserm.fr.
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Davignon
mesh_terms: D000975:Antioxidants / Q000494:pharmacology; D018501:Antirheumatic Agents / Q000494:pharmacology*; D015153:Blotting, Western; D002478:Cells, Cultured; D000068582:Certolizumab Pegol / Q000494:pharmacology*; D005434:Flow Cytometry; D006801:Humans; D008856:Microscopy, Fluorescence; D009000:Monocytes / Q000187:drug effects* / Q000378:metabolism; D051267:NF-E2-Related Factor 2 / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors
chemical_list: D000975:Antioxidants; D018501:Antirheumatic Agents; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D000068582:Certolizumab Pegol