Myeloid cell ageing and wound healing: analysing ageing-related changes to myeloid cells and their impact on cutaneous wound healing

Abstract

Wound healing is the process of restoring skin homeostasis following injury. Optimal healing is achieved through coordinated intercellular communication between the skin’s resident cells and infiltrating immune cells that orchestrate the inflammatory, proliferative and remodelling phases of healing. Wound closure is delayed in ageing skin and previous research demonstrates that early healing responses, particularly inflammation, are the most impacted by ageing. Prolonged inflammatory responses are a key feature of non-healing wounds in the elderly. The development of therapies for delayed healing in aged tissues has remained challenging due to the lack of a complete characterisation of ageing-related alterations in skin cellular function and how these changes affect the inflammatory phase of wound healing. To address this, we performed a global characterisation of ageing-related transcriptomic and epigenetic changes in skin resident cells, particularly keratinocytes, fibroblasts and macrophages, from mouse models using a combination of immunostaining, flow cytometry and RNA sequencing. Firstly, we discovered altered keratinocyte differentiation dynamics in aged but not young skin driven by functionally impaired proliferative basal cells. We revealed that impaired keratinocyte differentiation was associated with changes in specific histone modification patterns in the aged epidermis suggesting that epidermal homeostasis is tightly regulated by epigenetic remodelling. Secondly, our data supported that loss of fibroblast functional identity was a hallmark of skin ageing defined by the accumulation of inflammatory or senescent fibroblasts in the skin. Thirdly, we illustrated that dermal macrophages were reduced in the aged skin and aged tissues were mostly colonised by a functionally impaired macrophage subpopulation in comparison to young tissues. We concluded that intrinsic cellular alterations in aged skin keratinocytes, fibroblasts and macrophages drive changes in the skin microenvironment as demonstrated by aberrant intercellular signalling pathways between these cells. To examine if ageing-associated changes in the skin microenvironment impaired the regulation of inflammatory responses, particularly myeloid cell behaviour, during wound healing, we wounded young and aged mice and excised the tissues on Day 3 corresponding to the inflammatory response. Using the same techniques as above, we showed that poor healing aged wounded tissues had an exacerbated inflammatory response determined by an elevated number of neutrophils and reduced distribution of macrophages in comparison to young wounds. Neutrophils in aged wounds had increased chemotaxis but diminished capacity to clear pathogens. In addition, we discovered that macrophages in aged wounds had a hyperinflammatory signature but reduced proliferative potential, consistent with their diminished numbers in the tissue. Moreover, monocyte-to-macrophage transitioning was impaired in aged but not young wounds resulting in differential distribution of macrophage subpopulations. We noted that defective signalling pathways in aged skin were also impaired in aged wounds indicating that ageing-related changes in the skin microenvironment persist and influence cellular behaviour during early healing responses to injury. Altogether, our work provides key knowledge advances into how ageing globally alters skin cellular function and how these changes affect early healing responses to wounding. This project has generated comprehensive datasets to aid further studies into improving early healing responses in aged tissues.

Date of Award	1 Aug 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Kimberly Mace (Supervisor) & Matthew Ronshaugen (Supervisor)