TRPA1 Is Involved in Inflammatory Skin Diseases Through Inflammation Phenotype Skewing

Inflammation is a physiological process aiming to eliminate harmful factors entering the body, clean up damaged tissues and initiate resolution to heal. It can be correctly initiated by recognition of the invading organism or trauma to host tissues, which will lead to a straightforward process of acute inflammation followed by resolution. However, in many diseases like allergies and autoimmune diseases the immune system functions abnormally, and the inflammation is not resolved but becomes chronic. In most cases chronic inflammation is an ineffective response and detrimental to the host. Chronic inflammatory diseases are alternately driven by different subtypes of immune cells especially lymphocytes (Th1/Th2/Th17) and macrophages (M1/M2) and sometimes the imbalance between the subtypes is believed to maintain the chronic inflammatory state.

Transient receptor potential ankyrin 1 (TRPA1) belongs to the TRP ion channel family which consists of homo- and heterotetrameric non-selective cation channels consisting of proteins with six transmembrane domains. It is abundantly expressed in non-myelinated nerve endings where its activation by pungent exogenous and endogenous substances including inflammatory mediators causes pain, itch and neurogenic inflammation. TRPA1 is also known to be expressed in non-neuronal cells such as skin cells and some immune cells, and TRPA1-mediated interventions have been shown to influence many models of inflammatory diseases. TRPA1 expression is also associated with human inflammatory diseases, especially skin diseases where TRPA1 expression is commonly upregulated in patient skin samples. Preclinical studies have yielded variable results regarding the role of TRPA1 as either a pro- or anti-inflammatory factor. Our preliminary experiments and results from others have shown that TRPA1 activation or ablation might affect inflammatory processes driven by different inflammation subtypes toward opposing directions. Therefore, we hypothesized that TRPA1 is an inflammation phenotype skewing factor that drives inflammation towards the subtype driven by Th2 lymphocytes and M2 macrophages. In the present study, we set out to investigate how TRPA1 deficiency affects models of Th2-driven contact allergy and M2-driven scleroderma-type skin fibrosis.

In part I, a known contact allergen methylisothiazolinone (MIT) was studied for TRPA1 activation, and the role of TRPA1 was investigated in MIT-induced models of allergic contact dermatitis (ACD) and acute inflammatory paw edema. The effects in wild-type and TRPA1-deficient mice were compared. The results showed for the first time that MIT is a potent TRPA1 activator, and that MIT-induced acute inflammatory paw edema is reduced with genetic or pharmacological ablation of TRPA1. Mutation of cysteine in position 621 was shown to downregulate MIT-induced activation of TRPA1 proposing a putative molecular mechanism acting on cysteine residues. In MIT-induced model of allergic contact dermatitis, TRPA1-deficient mice had reduced elevation of the expression of the Th2-cytokine IL-4.

In part II, wild-type and TRPA1-deficient mice were compared in 2,4-dinitrochlorobenzene (DNCB)-induced model of allergic contact dermatitis. Like MIT, DNCB was also shown to activate TRPA1 in Fluo 3 calcium measurements. In DNCB-induced allergic contact dermatitis model, TRPA1-deficient mice had lower expression of Th2-cytokines IL-4 and IL-13 as well as IL-19 and IFN-γ than wild-type mice. In mechanistic studies, the TRPA1 blocker HC-030031 was found to inhibit THP-1 monocyte transformation into antigen-presenting cells in response to DNCB measured as apical expression of CD54 and CD86. In HaCaT keratinocytes, HC-030031 was shown to inhibit the DNCB-induced expression of oxidative stress related genes SRXN1 and HMOX1.

In part III, wild-type and TRPA1-deficient mice were compared in bleomycin-induced model of M2 macrophage mediated rheumatoid disease scleroderma. Intradermal bleomycin injection caused dermal thickening and collagen accumulation which were attenuated in TRPA1-deficient mice. Upregulation of pro-fibrotic and M2 markers in skin samples were also reduced. In cultured macrophages, bleomycin was discovered to polarize macrophages towards M2 phenotype especially when administered on top of IL-4, and this was possibly due to prolonged upregulation of PPARγ expression. Bleomycin did not effectively amplify M2 marker expression in macrophages collected from TRPA1-deficient mice proposing a TRPA1-mediated mechanism.

In part IV, a group of triterpenoids modified from the naturally occurring triterpene betulin were investigated for their activity on TRPA1. Six out of fourteen studied triterpenoids blocked TRPA1 at 10 µM concentration in a statistically significant manner. The two triterpenoids modified to contain a pyrazine ring were most effective and were studied in further detail to demonstrate an IC50 value of ~0.3 µM in whole-cell patch clamp recordings. TRPA1-blocking activity was confirmed in vivo using the selective TRPA1 activator AITC-induced inflammatory paw edema model, in which the two pyrazine-fused triterpenoids attenuated edema formation in a statistically significant manner.

The results introduce TRPA1 as a potential drug target for Th2- and M2-driven type 2 inflammatory diseases, especially those present in the skin. Blocking TRPA1 might reduce the risk of sensitization and alleviate inflammation and symptoms related to allergic contact dermatitis and mitigate disease severity in scleroderma. Known TRPA1 activators should be considered as suspected contact allergens in clinical practice. We also introduce two novel compounds with potential as TRPA1-blocking agents.