Reactive oxygen species inhibit catalytic activity of peptidylarginine deiminase

<p>Protein citrullination catalysed by peptidylarginine deiminase (PAD) may play an important pathogenic role in several chronic inflammatory diseases and malignancies. PAD2, PAD4, and citrullinated proteins are found in the synovium of rheumatoid arthritis patients. PAD activity is dependent on calcium and reducing conditions. However, reactive oxygen species (ROS) have been shown to induce citrullination of histones in granulocytes. Here we examine the ability of H₂O₂ and leukocyte-derived ROS to regulate PAD activity using citrullination of fibrinogen as read-out. H₂O₂ at concentrations above 40 µM inhibited the catalytic activity of PAD2 and PAD4 in a dose-dependent manner. PMA-stimulated leukocytes citrullinated fibrinogen and this citrullination was markedly enhanced when ROS formation was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium (DPI). In contrast, PAD released from stimulated leukocytes was unaffected by exogenously added H₂O₂ at concentrations up to 1000 µM. The role of ROS in regulating PAD activity may play an important part in preventing hypercitrullination of proteins.</p

Bacterial killing along the depth dimension of the biofilm.

<p>Simulation of a 4 hour treatment scheme with six different doses of ciprofloxacin in a 5 mm biofilm model. The initial concentration ciprofloxacin in the supernatant is indicated in the figure while the equilibrated concentration is 5 times lower. Bacterial density is measured in (CFU/mL). Left: Normoxic treatment. Right: Hyperbaric oxygen treatment.</p

Tobramycin diffusion.

<p>Numerical solution of the reaction-diffusion equation with power-law binding to the biofilm matrix in a spherical bead with radius <i>R</i> and diffusion constant <i>D</i> for the free tobramycin. The free tobramycin is displayed as a function of radius and time. Time is expressed in units of <i>R</i>²/<i>D</i>. a) With binding. The external concentration of tobramycin is kept at <i>a</i> = 4 mg/l from time <i>t</i> = 0. The maximum time shown is <i>t</i> = 5<i>R</i>²/<i>D</i>. b) Without binding. The maximum time shown is <i>t</i> = 0.5<i>R</i>²/<i>D</i>. c) With binding, external concentration is kept at <i>a</i> = 4 mg/l from time <i>t</i> = 0 to <i>t</i> = <i>R</i>²/<i>D</i>. d) Without binding, external concentration is kept at <i>a</i> = 4 mg/l from time <i>t</i> = 0 to <i>t</i> = <i>R</i>²/<i>D</i>.</p

Geometry of ciprofloxacin model coupled with oxygen treatment.

<p>Sketch of the 1-dimensional model implemented in MATLAB. The origin is placed at the interface between the supernatant and the biofilm domains, hence the top of the supernatant has coordinate <i>z</i> = −1.25 mm, the top of the biofilm has <i>z</i> = 0 mm, and the bottom of the biofilm has coordinate <i>z</i> = 5 mm.</p

Oxygen consumption for overnight cultures of PAO1.

<p>Dots represent experimental data and the red lines are the solution from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198909#pone.0198909.e003" target="_blank">Eq (3)</a> with respective parameters.</p

Oxygen profiles sampled after 90 minutes of HBOT.

<p>Oxygen profiles are recorded after 90 minutes of HBOT applied to a 5 mm thick agarose biofilm, hence an unloading process of an already oxygen penetrated biofilm is observed. Oxygen profiling was initiated 4 minutes after the 90 minutes HBOT and every profile takes approximately 3 minutes to record. Horizontal black bars represent supernatant and biofilm surfaces, respectively. The concentration of oxygen in the supernatant immediately after treatment is approximately 1000 <i>μ</i>M as the chamber has to be decompressed to 1 atm. The biofilm is present for depth 0 mm to 5 mm while the supernatant is present in the region from −5 mm to 0 mm. The supernatant is displayed primarily to verify that the mixing is strong in this region. Figure modified from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198909#pone.0198909.ref003" target="_blank">3</a>].</p

Numerical solutions of the reaction-diffusion equation with a michaelis-menten reaction term.

<p>Dots are experimental data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198909#pone.0198909.g001" target="_blank">Fig 1</a> and the coloured lines are matching numerical solutions to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198909#pone.0198909.e008" target="_blank">Eq (4)</a>. The biofilm extends from depth 0 mm to 5 mm, but only the part with measurable oxygen concentration is shown in the figure.</p

Parameter values used for simulation of the oxygen and ciprofloxacin models.

<p>Parameter values used for simulation of the oxygen and ciprofloxacin models.</p

Predicted volume-averaged time kill curves for normoxic oxygen treatment and HBOT.

<p>Different dosing schemes over 4 hours of treatment simulated in a 5 mm biofilm model. The initial ciprofloxacin concentrations in the 1.25 mm supernatant are displayed in the figures while the fully equilibrated concentrations are 5 times lower. Left: Normoxic treatment. Right: Hyperbaric oxygen treatment.</p

Oxygen penetration.

<p>Simulation of oxygen penetration in a 5 mm biofilm following a 4 hour treatment scheme with six different ciprofloxacin dosings. The initial concentration ciprofloxacin in the supernatant is indicated in the figure. The equilibrated concentration is 5 times lower. Left: Normoxic treatment. Right: Hyperbaric oxygen treatment.</p