Defining the Biological Functions of Novel Lipids in Healthy and Wounded Skin

Healthy skin forms a barrier that is essential for preventing infection and loss of water from our bodies. Loss of this barrier occurs during common skin diseases such as psoriasis and eczema, and allows infection during chronic wounds. Lipids (fats) are required to maintain the barrier, but little is known regarding how they do this, with the role of skin lipids in disease a major under-researched area.

In Cardiff and Vanderbilt, we discovered several families of lipids that are generated by skin or by blood cells that migrate to the skin during response to injury, or in skin disease. Our preliminary data suggests that several of these may be important in maintaining the health of skin and preventing disease. They are made by a family of proteins (enzymes) called lipoxygenases (LOXs) and include two types of lipids, called phospholipids and ceramides.

In this study we will use a number of approaches to determine the roles of the lipids in regulating skin biology. The studies may lead to development of new treatments for common skin disorders and chronic wounds, conditions that cause significant morbidity in a large section of our population.

The primary objective of this study is to determine how lipids discovered by the Cardiff and Vanderbilt groups regulate skin biology. To do this we will use reconstituted human skin, mouse models and patient samples.

Aims:
(1) Determine the structure and localization of the lipids in human skin.
In this aim we will determine the structures of all the lipids still to be fully characterized. We will also use imaging and other methods to determine where the lipids are located in skin (which layers of the epidermis). Both healthy skin and samples from patients with inflammatory skin disorders will be studied.

(2) Determine how the lipids regulate skin health and disease at the molecular level.
We propose that the lipids may help regulate skin through two different molecular processes. These include regulation of an important protein called PPAR and through regulating how blood cells that have migrated into the skin behave during health and disease. We will study these events using a 3D cultured human skin system, and in a murine model of wounding in vivo.

(3) Characterise regulation of skin gene expression by LOX-derived lipids.
To identify pathways by which the lipids regulate skin health, the enzymes that generate them will be removed in cultured human skin using an approach called siRNA. A global screening approaches called RNAseq will then determine changes in gene expression in specific skin compartments and how these are mediated.

(4) Discover new lipids formed in skin disease. The total number and types of lipids are present in skin is unknown. As part of a European Research Council grant, we are developing new methods for studying lipids that will be applied to our studies of skin lipids in this project. Lipids will be catalogued in samples of healthy skin, and in biopsies from patients with the common skin disorders, psoriasis and eczema, with a view to identifying new lipids that regulate skin health and disease.

These studies will determine how lipids regulate skin biology, and may lead to design of new therapies for inflammatory skin diseases and wounds.

Research objectives (Aims):
(1) Determine structure and localization of LOX-derived lipids in human skin (healthy, eczema, psoriasis).
Structural characterization (including enantiomeric) of the LOX-derived ceramides will be undertaken using Orbitrap mass spectrometry and chromatography. The localization of all LOX-derived lipids will be determined using MALDI mass spectrometry imaging and LC/MS/MS of human skin separated into distinct epithelial layers, using both healthy skin and samples from patients with inflammatory skin disorders (obtained through dermatology clinics at University Hospital Wales).

(2) Determine regulation of barrier function, innate immune responses and wound healing in skin by LOX-derived lipids.
A 3D cultured human skin system and in vivo murine model of wounding will be used. PPAR/TG activities will be tested using gene expression and immunohistochemistry analysis. Innate immune responses will be tested by flow cytometry analysis of migrating blood cells into skin, and determination of chemokine/cytokine levels using ELISAs.

(3) Characterise regulation of skin gene expression by LOX-derived lipids.
Skin LOXs will be downregulated in cultured human skin by delivery of siRNA using microneedles. RNAseq and network analysis will determine global changes in gene expression in specific skin compartments and transcription factor involvement.

(4) Discover new lipids formed in skin disease. Using a new method (Orbitrap, LipidArray), the total number and diversity of lipids will be catalogued in biopsy samples of healthy skin, and from patients with the common skin disorders, psoriasis and eczema. Samples will be obtained from dermatology clinics at University Hospital Wales.

The main group to benefit from this research would be patients with chronic skin disorders, both common and rare.

Skin inflammation, including psoriasis and atopic eczema affects 15-20% of UK children. Current treatments for these include moisturizers and barrier creams to limit water loss and topical steroids to inhibit keratinocyte proliferation, but these can have significant side effects and are of limited efficacy. Thus, these disorders represent conditions of significant unmet clinical need, causing persistent morbidity, especially in children. In this study, we will characterize the lipids present in the skin of patients with these disorders, an initial first step required to further our understanding of how lipids regulate the basic biology of common skin diseases.

Separate to this, chronic wounds affect 6.5 million patients in the USA, with an annual estimated cost to healthcare services in excess of US$25 billion. Chronic wounds are generally seen in patients with co-morbidities that include diabetes and obesity, while creating wounds is an essential component of surgery. Forty million inpatient surgeries were performed in the USA in 2000, with 31.5 million on outpatients. Thus, there is significant need to ensure that post surgery wound care is effective. The economic and social impact of wounds is significant, but research dedicated to this area is not considered high priority in general, with skin biology being under-researched in comparison with many other high profile diseases, such as heart disease and cancer.

A final important group is patients with the rare disorder, ichthyosis, sometimes caused by mutations in the skin LOX genes, for whom life is dramatically shortened due to absence of an effective barrier function. These patients (non-bullous congenital ichthyosiform erythroderma , NCIE) have thickened, scaly skin, and the disease can be life-threatening. Treatments are limited to topical application of creams and oils, in an attempt to improve hydration. Lactic acid, retinoids and propylene glycol have been used, with some limited efficacy.

Thus, there is a major clinical need to understand skin biology in both health and disease, in order to identify new therapeutic targets for intervention and treatment.

A separate group who may benefit are companies developing treatments for chronic skin inflammatory conditions and wounds. While we do not currently collaborate with industry, we would look to develop any promising candidates for prevention of inflammation/promotion of barrier function, with interested industrial partners, when appropriate.

This research will identify mechanisms by which certain newly identified lipids regulate skin health and disease. If the lipids themselves are found to be anti-inflammatory, or promote wound healing, they could be developed into novel drugs. We already have methodologies established for generating mg amounts, and these could potentially be scaled up to gm level, in collaboration with industrial partners. On the other hand, if the lipids are pro-inflammatory (we believe unlikely), then the LOX isoforms that generate them become potential drug targets themselves.

Development of new treatments for common skin disorders based on understanding and exploiting the biology of these conditions has the potential to be transformational for patients, who suffer extensively from the long term chronic nature of what are often highly-visible conditions. Thus, the research has potential to enhance quality of life significantly, if effective treatments are developed from our results.

Treatments for common conditions would be highly valuable to industry, who would be able to command a significant worldwide market for biological treatments in this area.