Image-Based Measurement of H 2 O 2 Reaction-Diffusion in Wounded Zebrafish Larvae

Abstract

Epithelial injury induces rapid recruitment of antimicrobial leukocytes to the wound site. In zebrafish larvae, activation of the epithelial NADPH oxidase Duox at the wound margin is required early during this response. Before injury, leukocytes are near the vascular region, that is, $100-300 mm away from the injury site. How Duox establishes long-range signaling to leukocytes is unclear. We conceived that extracellular hydrogen peroxide (H 2 O 2 ) generated by Duox diffuses through the tissue to directly regulate chemotactic signaling in these cells. But before it can oxidize cellular proteins, H 2 O 2 must get past the antioxidant barriers that protect the cellular proteome. To test whether, or on which length scales this occurs during physiological wound signaling, we developed a computational method based on reaction-diffusion principles that infers H 2 O 2 degradation rates from intravital H 2 O 2 -biosensor imaging data. Our results indicate that at high tissue H 2 O 2 levels the peroxiredoxin-thioredoxin antioxidant chain becomes overwhelmed, and H 2 O 2 degradation stalls or ceases. Although the wound H 2 O 2 gradient reaches deep into the tissue, it likely overcomes antioxidant barriers only within $30 mm of the wound margin. Thus, Duox-mediated long-range signaling may require other spatial relay mechanisms besides extracellular H 2 O 2 diffusion.

Mapping the phenotypic repertoire of the cytoplasmic 2-Cys peroxiredoxin – Thioredoxin system. 1. Understanding commonalities and differences among cell types

“…The inability of these sinks to maintain a strong transmembrane H 2 O 2 gradient is experimentally demonstrated by the observation that partial Cat inhibition has a substantial effect on the pseudo-first order rate constant ( ) for the consumption of extracellular H 2 O 2 boluses that are sufficient to extensively oxidize PrxI and PrxII [66] , [67] : otherwise H 2 O 2 consumption would be strongly limited by the membrane permeation step and virtually insensitive to Cat inhibition (see SI3.2.1). It is also supported by recent experiments in living zebra fish [59] . In turn, a strong transmembrane gradient when Prx-S - is predominant is predicted by reaction-diffusion models [13] , [68] and was experimentally estimated as ≈650 for HeLa cells [68] .…”

“…First, the threshold must be inversely proportional to the membrane permeability (SF12A,B), as for the same extracellular H 2 O 2 the H 2 O 2 influx rate is proportional to membrane permeability. There is growing evidence that cells’ permeability to H 2 O 2 is largely determined by some aquaporins [58] , [59] , and the previous prediction can thus be tested through the use of aquaporin inhibitors. Second, the threshold should be higher the higher the concentration of Trx available in the cytoplasm (SF12C,D), as Prx-SS reduction in these cells is rate limited by Trx availability.…”

Mapping the phenotypic repertoire of the cytoplasmic 2-Cys peroxiredoxin – thioredoxin system. 1. Understanding commonalities and differences among cell types

“…It is also supported by recent experiments in living zebra fish. [59] In turn, a strong transmembrane gradient when Prx-Sis predominant is predicted by reaction-diffusion models [13,68] and was experimentally estimated as 650 for HeLa cells [68].…”

“…H2O2 chemotaxis by leukocytes far away from wound borders may be an example of H2O2 signaling in this regime. [59] In turn, at sup v above the cytoplasmic capacity to reduce Prx-SS, cytoplasmic H2O2 concentrations are determined by the activities of peroxiredoxin VI, GPx1 and Cat. These activities are, collectively, too low to impose a significant H2O2 gradient over the cytoplasm or even a very large transmembrane gradient.…”

“…First, the threshold must be inversely proportional to the membrane permeability (SF12A,B), as for the same extracellular H2O2 the H2O2 influx rate is proportional to membrane permeability. There is growing evidence that cells' permeability to H2O2 is largely determined by some aquaporins[58,59], and the previous prediction can thus be tested through the use of aquaporin inhibitors.Second, the threshold should be higher the higher the concentration of Trx available in the cytoplasm (SF12C,D), as Prx-SS reduction in these cells is rate limited by Trx availability. Third, increasing the TrxR activity should have qualitatively the same effect as increasing the Trx concentration, but the effect should be much less pronounced in these cells than the latter (SF12C,D) because here the TrxR activity is not rate-limiting for Trx-SS reduction.…”