Investigating the immunomodulatory and wound healing effects of reactive oxygen species

Abstract

The estimated annual cost of wounds has reached over £8 billion pounds annually and is increasing. There is a need to develop treatments to improve wound healing, particularly in the case of chronic non-healing wounds. At the same time cases of antimicrobial resistance that are a major risk for non-healing wounds are increasing and it is estimated that anti-microbial resistance (AMR) related deaths could exceed cancer related deaths by 2050. There is therefore a need for new treatments that improve the wound healing and treat bacterial infection. Reactive oxygen species (ROS) form part of the innate immune system and are known to induce immunomodulation. Hydrogen peroxide (H2O2), a type of ROS, is a disinfectant often used at high concentrations that are detrimental to cells when used topically. Matoke Holdings Ltd. have designed a bioengineered honey - SurgihoneyRO™ -which improved wound healing outcomes in trials and successfully produces H2O2 over time. RO1O1®, a synthetic equivalent to the honey-based product has also been created with the goal of being used for immunomodulatory and bactericidal activity.
SurgihoneyRO™ and RO1O1® were able to produce steady amounts of H2O2 over 24hrs but SurgihoneyRO™ caused cell death after treatment times longer than 2h compared to 6h with RO1O1®. Only H2O2 treatment of keratinocytes showed increased expression of pro-inflammatory Interleukin 1 beta (IL1b) whereas both H2O2 and RO1O1® treatments resulted in increased expression of pro-angiogenic vascular endothelial growth factor (VEGF). Effects on wound healing, however, were non-conclusive according to wound scratch assays. Dermal fibroblasts, crucial cells for wound healing showed no significant responses in genes related to the remodelling of the extra cellular matrix (ECM) ROS treatment in matrix metalloproteinase 1 (MMP1), or it’s inhibitors TIMP1 and TIMP3 following treatments with H2O2 or RO1O1®. Furthermore, no changes were seen in genes related to the deposition of ECM collagen 1 and 3 (COL1A1 or COL3A1 respectively), or in the angiogenic gene VEGF following fibroblast treatment with H2O2 or RO1O1®. Wound scratch assays showed an increased time to wound resolution. However, when treated with pro-inflammatory cytokine IL1b, which this study showed to be upregulated in keratinocytes following H2O2 treatment, the expression of COL1A1 and TGFb decreased, both of which are involved in wound healing pathways. Following the changes in expressions seen in keratinocytes, RNA bulk sequencing was conducted to determine which pathways responded to treatments. Results did not show upregulation of pro-inflammatory pathways in keratinocytes, but instead
downregulation of multiple pathways related to cell proliferation (senescence, p53 and FoxO signalling). Bulk RNA sequencing from treated fibroblasts showed consistent downregulation of senescence, apopstosis, TGFb, and leukocyte transendothelial migration. These pathways intersect with wound resolution pathways, implying ROS treatments may impact wound healing, as downregulation of some of these pathways may result in reduced complications in wound healing namely: apopstosis, senescence and the TGFb pathway. RO1O1® did not show any antibacterial properties in Pseudomonas aeruginosa or Staphylococcus aureus, whereas H2O2 treatment delayed growth. In conclusion, ROS based treatments targeting immunomodulation and wound healing should be investigated further. These further investigations could use a combination of different concentrations of H2O2 and RO1O1® prototypes, different cell types, ex-vivo or 3D wound models, and bacterial biofilms.

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Identifiers

ORCID for Olivia Kate Lee: orcid.org/0009-0000-7214-9355

ORCID for Sylvia Pender: orcid.org/0000-0001-6332-0333

ORCID for Jeremy Webb: orcid.org/0000-0003-2068-8589

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