PPAR-alpha in cutaneous inflammation

Abstract

Peroxisome proliferator-activated receptor (PPAR)-alpha is a fatty acid activated transcription factors that belongs to the nuclear hormone receptor family. Primarily PPAR-alpha serves as a lipid sensor. While PPAR-alpha controls enzymes from the lipid and glucose metabolism in the liver, heart and muscles, PPAR-alpha is also involved in skin homeostasis. PPAR-alpha controls keratinocyte proliferation/differentiation, contributes to wound healing and regulates skin inflammation. PPAR-alpha activation exerts anti-inflammatory effects in various skin conditions such as irritant and allergic contact dermatitis, atopic dermatitis and UV-induced erythema, rendering investigations into the functions of PPAR-alpha necessary to provide better understandings to treat many inflammatory skin disorders.

PPAR-alpha Expression in Skin Cells

PPAR-alpha is expressed in rodent^9–11 and human keratinocytes.^12,13 PPAR-alpha ligands promote epidermal differentiation, restore epidermal homeostasis in hyperproliferative mouse epidermis and regulate apoptosis.¹⁴ In human keratinocytes, however, low doses of the PPAR-alpha selective ligand Wy-14643 seem to have negligible effects on the expression of differentiation markers.¹³ In contrast, higher doses of clofibrate, another PPAR-alpha ligand, increase the expression of involucrin and transglutaminase-1, which are essential for the formation of the stratum corneum.¹⁰ Furthermore, PPAR-alpha activators profoundly influence epidermal lipid metabolism and they enhance the formation of lamellar bodies (syn. membrane coating granules or Odland bodies), which are lipid-containing organelles secreting their content into the extracellular spaces of the stratum corneum, thus forming the epidermal permeability barrier.^12,15 PPAR-alpha is expressed in cultured sebocytes and in human sebaceous glands.^16–18 In addition, PPAR-alpha is expressed in mouse¹¹ and human¹³ dermis. It is expressed in immature epidermal Langerhans cells (LC) and expression is downregulated in mature LC in mouse skin.¹⁹ PPAR-alpha is also expressed in other cutaneous immune cell types, such as macrophages²⁰ and T-lymphocytes,^21–23 where it regulates inflammatory responses. The clinical relevance of these findings for inflammatory skin disease is under active investigation.

Expression of PPAR-alpha in Inflamed Skin

The expression pattern of PPAR-alpha is altered in inflamed skin. For example, PPAR-alpha decreases in skin after topical treatment with 2,4-dinitrofluorobenzene (DNFB) in a mouse model of allergic contact dermatitis.²⁴ PPAR-alpha expression is reduced by approximately 50% in lesional atopic dermatitis (AD), when compared to normal skin.²⁵ These results are confirmed by previous work showing that PPAR-alpha expression is moderately decreased in non-lesional AD.²⁶ Several of the PPAR-alpha target genes are profoundly decreased in inflamed skin.²⁶ It was suggested that downregulation of PPAR-alpha could be involved in AD development or maintenance by affecting the lipid pathway.²⁶

Rivier et al. showed a moderate decrease in PPAR-alpha expression in non-lesional and lesional psoriatic epidermis.¹² In contrast, Westergaard et al. did not observe any consistent changes of PPAR-alpha expression in involved psoriatic skin when compared to non-involved psoriatic skin.²⁷ The subcellular localization of PPAR-alpha in non-lesional skin from psoriasis patients resembles normal skin.²⁷ In the thickened epidermis of lesional psoriatic skin, a cytoplasmic localization of PPAR-alpha extends through most of the suprabasal layers with only the outermost layer of suprabasal cells exhibiting a nuclear localization.²⁷ Although the function of PPAR-alpha may be altered in psoriasis, no associations were found between genetic PPAR-alpha variants and the disease.²⁸

Taken together these studies show that PPAR-alpha expression is reduced in inflamed skin, independent of the nature of the inflammatory response, i.e., regardless of Th₁ vs. Th₂ dominance. Whether this downregulation of PPAR-alpha causes the disease or whether it is a consequence of the disease remains to be clarified. However, there are a few observations indicating that PPAR-alpha is downregulated very early, e.g., when the skin inflammation is experimentally induced by challenge with an allergen. For example, topical application of sodium lauryl sulphate, which is known to be an irritant and to damage the skin barrier, reduces PPAR-alpha mRNA levels in the skin as early as 6 hours after the insult.²⁹ In this latter work, the authors demonstrate that expression of ALOXE3, but not of ALOX12B, is similarly regulated,²⁹ pointing to an early dysregulation of PPAR-alpha related-pathways in inflammatory skin disorders.

Moreover, PPAR-alpha expression is upregulated in keratinocytes at the edge of the wounded skin as early as 24 h after injury.¹¹ It remains expressed in the epidermis of the wound edges during the entire wound healing process. After closure of the wound, PPAR-alpha expression remains detectable for approximately 3 days after the injury, but it is not observed thereafter.¹¹ Thus, the pattern of PPAR-alpha expression, mainly in the very first days after injury, overlaps with the inflammatory stage of wound healing.

Anti-inflammatory Effects of PPAR-alpha in Skin

Since PPAR-alpha is expressed not only in keratinocytes but also in key cells of the adaptive immune system, specific PPAR-alpha ligands generated by the pharmaceutical industry (for treatment of disorders, such as lipid disorders and diabetes) were explored for their anti-inflammatory potential in the skin. Anti-inflammatory effects of PPAR-alpha activation were reported in mouse models of irritant and allergic contact dermatitis.³⁰ Topical treatment with three different PPAR-alpha agonists, clofibrate, Wy-14643 and linoleic acid, ameliorates ear swelling in these models, which correlates with a reduction of the lymphocytic infiltrates and the expression of the pro-inflammatory cytokines TNFalpha and IL-1alpha in allergic contact dermatitis.³⁰ The lack of these effects in PPAR-alpha deficient mice demonstrated receptor specificity.³⁰ In this experimental setting, PPAR-alpha ligands were applied 45 min and 4 h after skin challenge with the inflammatory stimulus, showing that PPAR-alpha activation is able to prevent ear swelling, when applied within a short time period after challenge.³⁰ Similarly, another selective PPAR-alpha agonist, Palmitoylethanolamide (PEA), with a potency comparable to that of the synthetic Wy-14643, is able to reduce TPA-induced irritant contact dermatitis.³² However, PEA was not only shown to activate PPAR-alpha but also to increase its expression in TPA-challenged skin.³¹ Interestingly, relatively high concentrations of PEA (high nanomolar to low micromolar levels) are present in many animal tissues, in which this substance undergoes active biosynthesis and breakdown.^32–34 This suggests that PEA might serve as an endogenous activator of PPAR-alpha in many organs including skin. It has recently been shown that 2% sunflower oleodistillate (SOD) prevents TPA-induced ear inflammation by decreasing the production of TNFalpha and IL-1beta in mouse skin.³⁵ In a luciferase reporter assay, SOD was shown to activate PPAR-alpha, but not PPAR-delta or -gamma.³⁵ The effects of Wy-14643 were also investigated in a mouse model of AD/chronic allergic dermatitis consisting of repeated topical application of ovalbumin (OVA). Topical Wy-14643 when applied shortly before OVA challenge prevents skin inflammation.²⁵ Wy-14643 reduces epidermal hyperplasia and inflammatory dermal infiltrates, with the most pronounced effects on CD4⁺ T cell infiltration.²⁵ Wy-14643 dramatically reduces the expression of IL-1beta and IL-6 and to a lesser extent IL-4, IL-31 and eotaxin/CCL11.²⁵ However, Wy-14643 does not reduce the amount of IL-5 in mouse skin.²⁵ In addition, in this model Wy-14643 does not affect the humoral response (OVA-specific IgE or IgG1 production) induced by epicutaneous sensitization.²⁵ Of note, in this study, Wy-14643 from a DMSO stock was diluted in a mix acetone/olive oil (4:1) while control mice were treated with DMSO only.²⁵ The effects of PPAR-alpha activators were also investigated in another mouse model of AD; i.e., chronic allergic dermatitis induced by repeated topical application of oxazolone (total of 12 challenges). In this model, the PPAR-alpha activator Wy-14643 was topically applied while the phenotype of AD-like, chronic allergic dermatitis was established by repeated allergen exposure (after the 10^th challenge). Wy-14643 decreases epidermal thickness and hyperplasia in this model.³⁶ Moreover, PPAR-alpha normalizes epidermal function by decreasing transepidermal water loss (TEWL) and pH of the inflamed skin.³⁶ In addition, these authors show that Wy-16463 reduces serine protease activity and normalizes lamellar body secretion.³⁶ In addition, PPAR-alpha activators largely reverse the immunologic abnormalities in chronic allergic dermatitis by decreasing the amount of eosinophils and mast cells in the skin of mice as well as the serum levels of TARC/CCL17, yet it does not alter serum IgE levels,³⁶ indicating that the effects of topical PPAR-alpha activators appear to be limited to the skin.

In human skin, Kippenberger et al. showed an inhibition of UV-induced skin erythema by pre-treatment with topical application of Wy-14643.³⁷ Moreover, in 20 adult volunteers, 2% SOD improved atopic dermatitis (AD) when applied twice daily for 4 weeks.³⁸ In a study of 227 children with mild-to-moderate AD, not treated with topical corticosteroids, treatment with 2% SOD cream twice-daily for 30 days induces significant improvements in several relevant clinical parameters, including reduction of skin dryness by 88%, skin flaking by 88% and itching and erythema by 80%.³⁹ Finally, an emollient containing PEA, a PPAR-alpha selective activator,³¹ successfully improves AD symptoms in a multinational, multicentre, observational, non-controlled, prospective cohort study, in patients between 2 and 70 years of age.⁴⁰

Mechanistically, PPAR-alpha ligands block the nuclear factor kappaB pathway and thereby modulate the immune response in both keratinocytes and LC.^19,25,41 Because LC are positioned at the crossroad between the skin barrier, the environment and the cutaneous immune system, they are of particular importance for skin inflammation in AD.^42,43 Interestingly, pharmacologic PPAR-alpha activation inhibits the maturation of LC, their migratory capacity, cytokine expression and their ability to drive T cell proliferation.¹⁹ In LC, PPAR-alpha activation inhibits NFkappaB, but not stress-activated protein kinase/JNK, p38MAPK and ERK1/2.¹⁹ Notably, PPAR-alpha activators could be beneficial for the treatment of inflammatory skin diseases by acting on both keratinocytes and LC.

Inflammation in PPAR-alpha Deficient Skin

PPAR-alpha deficient mice do not exhibit a spontaneous proinflammatory skin phenotype, presumably due to compensatory mechanisms via other PPAR isoforms and/or redundant pathways. The consequences of PPAR-alpha deficiency on skin inflammation only become evident after challenge to the skin. For example, PPAR-alpha deficient mice show a delay in wound healing during the first 4 days after injury, as the wound size in the PPAR-alpha deficient animals decreases more slowly than in wild-type mice.¹¹ Interestingly, in the later stages of wound healing there is no difference between control and PPAR-alpha deficient mice.¹¹ This transient delay of wound healing in PPAR-alpha deficient mice correlates with the window of increased PPAR-alpha expression that normally occurs in control mice as described above and it corresponds to the inflammatory phase of wound healing.¹¹ Accordingly, the recruitment of both neutrophils and monocytes is impaired in PPAR-alpha deficient mice at day 1 post-wounding. The difference in the recruitment of these immune cells is overcome during later stages of wound healing, allowing normal wound repair to take place in PPAR-alpha deficient mice.¹¹

Moreover, leukotriene (LT) B₄- and arachidonic acid-induced ear swelling is prolonged in PPAR-alpha deficient mice when compared to wild-types.⁶ LTB₄ is presumably cleared more slowly from the site of inflammation in PPAR-alpha deficient mice. This suggests that PPAR-alpha would be able to sense LTB₄ levels and to control its oxidative degradation.⁶ Indeed, LTB₄ is a potent and selective PPAR-alpha activator that promotes its own catabolism as a negative feedback loop via omega and beta-oxidation pathways.⁶ Interestingly, PPAR-alpha deficient mice respond in the same way as wild-type controls to topical application of TPA which involves prostaglandin pathways.⁶ In another study, it was shown that PPAR-alpha deficient mice exhibit increased carrageenan-induced paw oedema in association with enhanced immunostaining for FAS ligand in the inflamed paws.⁴⁴ Because it was reported that PPAR-alpha deficiency preferentially results in the induction of Th₁ cytokines in T-lymphocytes,^21,22 it could be expected that PPAR-alpha deficient mice are more likely to develop Th₁-mediated allergic contact dermatitis. However, Staumont-Salle et al. showed that PPAR-alpha deficient mice exhibit exacerbated skin inflammation in a mouse model of AD consisting of repeated allergen exposure, leading to chronic allergic contact dermatitis, and that both Th₁ and Th₂ cytokines were expressed.²⁵ Likewise, PPAR-alpha deficient mice show increased numbers of mast cells and CD4⁺ T cells in the dermis, but no difference in eosinophil counts when compared to controls.²⁵ Interestingly, the inflammatory phenotype of PPAR-alpha deficient mice is not due to a defect in epidermal barrier function since TEWL remains unchanged. Rather the authors observed activation of the nuclear factor kappaB pathway.²⁵ Recently, we showed that PPAR-alpha deficient mice exhibit higher and prolonged allergic contact dermatitis due to a defect in regulatory T-cells with diminished IL-2 production.⁴⁵ These results confirm previous reports showing that PPAR-alpha deficient mice develop more severe clinical symptoms than control mice in a gender-dependent manner (predilection of females/males) after induction of experimental autoimmune encephalomyelitis.²¹ It is likely that the pro-inflammatory skin phenotype of PPAR-alpha mice is due to an alteration of lipid pathways i.e., the degradation of pro-inflammatory signal molecules such as LTB₄ and to abnormal T-lymphocytes and regulatory T-cells. Notably, the main pro-inflammatory down-stream genes altered by PPAR-alpha deficiency belong to the beta and omega-lipid oxidation pathways and to the nuclear factor kappaB and p38^MAPK pathways.^{6,19,21,22,25}

Finally, the effects of glucocorticoid receptor activation were recently explored in PPAR-deficient animals. Dexamethasone-mediated anti-inflammatory activity is weakened in PPAR-alpha deficient mice.⁴⁶ Indeed, dexamethasone is less effective in PPAR-alpha deficient mice, as evaluated by measuring inhibition of NFkappaB, TNFalpha, cell migration, cycloxygenase-2 (COX-2) and inducible nitric-oxide synthase activation.⁴⁶ Furthermore, macrophages from PPAR-alpha deficient mice are less susceptible to dexamethasone-induced COX-2 inhibition when compared to wild type controls, which is reversible by transfection of PPAR-alpha into PPAR-alpha deficient macrophages.⁴⁶ These results indicate that PPAR-alpha may contribute to the anti-inflammatory activity of glucocorticoids.

Conclusion

PPAR-alpha is not only involved in keratinocyte differentiation, but also in regulating skin inflammation. The anti-inflammatory effects of PPAR-alpha are now well established. Investigations into the function of PPAR-alpha provide an opportunity to better understand and treat many inflammatory skin disorders. Indeed co-administration of PPAR-alpha activators with well-known anti-inflammatory drugs such as glucocorticoids may have beneficial effect/adverse effect profiles. Therefore PPAR-alpha activators have great potential as pharmaceutical drugs in the field of dermatology.

Abbreviations

AD

atopic dermatitis

ALOXE3

epidermis-type lipoxygenase 3

ALOX12B

arachidonate 12-lipoxygenase, 12R type

DMSO

dimethyl sulfoxide

IL

interleukin

LTB₄

leukotriene B4

OVA

ovalbumin

PEA

palmitoylethanolamide

PPAR

peroxisome proliferator-activated receptor

TPA

tissue plasminogen activator

SOD

sunflower oleodistillate