Luminescent probes for detection and imaging of hydrogen peroxide

Reactive oxygen species (ROS) have been shown to be toxic but also function as signalling molecules. This biological paradox underlies mechanisms that are important for the integrity and fitness of living organisms and their ageing. The pathways that regulate ROS homeostasis are crucial for mitigating the toxicity of ROS and provide strong evidence about specificity in ROS signalling. By taking advantage of the chemistry of ROS, highly specific mechanisms have evolved that form the basis of oxidant scavenging and ROS signalling systems.

Inflammation is a complex set of interactions among soluble factors and cells that can arise in any tissue in response to traumatic, infectious, post-ischaemic, toxic or autoimmune injury. The process normally leads to recovery from infection and to healing, However, if targeted destruction and assisted repair are not properly phased, inflammation can lead to persistent tissue damage by leukocytes, lymphocytes or collagen. Inflammation may be considered in terms of its checkpoints, where binary or higher-order signals drive each commitment to escalate, go signals trigger stop signals, and molecules responsible for mediating the inflammatory response also suppress it, depending on timing and context. The non-inflammatory state does not arise passively from an absence of inflammatory stimuli; rather, maintenance of health requires the positive actions of specific gene products to suppress reactions to potentially inflammatory stimuli that do not warrant a full response.

Barrier structures (e.g. epithelia around tissues, plasma membranes around cells) are required for internal homeostasis and protection from pathogens. Wound detection and healing represent a dormant morphogenetic program that can be rapidly executed to restore barrier integrity and tissue homeostasis. In animals, initial steps include recruitment of leukocytes to the site of injury across distances of hundreds of micrometers within minutes of wounding. The spatial signals that direct this immediate tissue response are unknown. Due to their fast diffusion and versatile biological activities, reactive oxygen species (ROS), including hydrogen peroxide (H2O2), are interesting candidates for wound-to-leukocyte signalling. We probed the role of H2O2 during the early events of wound responses in zebrafish larvae expressing a genetically encoded H2O2 sensor1. This reporter revealed a sustained rise in H2O2 concentration at the wound margin, starting ∼3 min after wounding and peaking at ∼20 min, which extended ∼100−200 μm into the tail fin epithelium as a decreasing concentration gradient. Using pharmacological and genetic inhibition, we show that this gradient is created by Dual oxidase (Duox), and that it is required for rapid recruitment of leukocytes to the wound. This is the first observation of a tissue-scale H2O2 pattern, and the first evidence that H2O2 signals to leukocytes in tissues, in addition to its known antiseptic role.