When looking at the canonical pathways in which the genes were upregulated via available literature, we noticed an alteration in the balance of Th1 vs Th2 immune expression in particular genes. The genes of interest, ALDH3A1, ACVR1C, and IL-13RA2, were examined closer in literature for identification of pathways involved in AD. These genes and findings are listed below.
Aldehyde Dehydrogenase 3 Family Member A1(ALDH3A1)
ALDH3A1 is a gene that encodes the enzyme aldehyde dehydrogenase 3, member A1. Aldehyde dehydrogenases play a major role in the detoxification of medium and long chain aldehydes into non-toxic carboxylic acids.[8] These aldehydes are often very reactive and byproducts of oxidative stress capable of causing cellular damage.[9] Aldehyde dehydrogenases are also involved in the metabolism of corticosteroids, biogenic amines, neurotransmitters, and products of lipid peroxidation. ALDH3A1 was originally known to protect the cornea from UV light induced damage and accounts for about 50% of the soluble proteins in the cornea.[8] Now evidence is arising that ALDH3A1 may also protect other tissues from oxidative stress.[10] Chronic skin inflammation is associated with oxidative stress and an overproduction of ROS such as superoxide and hydrogen peroxide which cause cellular damage.[4] Byproducts of ROS can be metabolized by enzymes such as ALDH3A1 before they have the chance to cause damage, providing an opportunity to treat oxidative stress induced skin inflammation.[9] ALDH3A1 is upregulated by partial Rlip loss and has therapeutic potential in the treatment of ROS in AD.
4-HNE is a byproduct of lipid peroxidation and is toxic to cells. It can inhibit cell growth and lead to apoptosis if left unchecked. ALDH3A1 metabolizes 4-HNE and prevents it from causing damage.[10] Thus, ALDH3A1 can reduce the amount of ROS in cells protecting them from damage induced by oxidative stress. One study utilized stably transfected V79 cell lines that express either human class 1 or class 3 ALDH to examine whether ALDH expression was sufficient to confer resistance to specific toxic aldehydes such as 4-HNE and others.[9] It was found that 6–9 carbon aldehydes were excellent substrates for ALDH3A1 expression and protected against exhaustion of the critical antioxidant buffer system. ALDH3A1 was able to prevent depletion of glutathione by aldehydes in cells.[9] Glutathione is an antioxidant and prevents ROS damage in cells. Thus, the ability of ALDH3A1 to prevent glutathione depletion further confirms ALDH3’s protective role against oxidative stress. Furthermore, studies comparing non-expressing ALDH3A1 control V79 cells with the transfectant line expressing ALDH3A1 showed a 21-fold resistance to 4-HNE toxicity in the ALDH3 cell line.[10] In addition, the 4-HNE-induced apoptosis was completely prevented in the ALDH3A1-transfected up to a concentration of 70mM 4-HNE.[10] Expression of ALDH3A1 has been proposed as a defensive mechanism against toxic lipid aldehydes generated during lipid peroxidation. Consistent with this view, the results of these studies demonstrate that ALDH3A1 protects well against the damaging and growth-inhibitory effects of medium chain-length aliphatic aldehydes produced during lipid peroxidation.[9] Upregulation of ALDH3A1 via Rlip loss could prevent oxidative stress from causing extensive skin inflammation mitigating the symptoms and pathogenesis of AD.
Activin Receptor Type-1C (ACVR1C)
ACVR1C, originally known as ALK7, is a gene which encodes a serine/threonine protein kinase which forms a receptor complex upon ligand binding. ACVR1C is the receptor for ligands activin AB, activin B and NODAL. The receptor complex is composed of two type II and two type I transmembrane serine/threonine kinases. Once the ligand binds, the type II receptors phosphorylate and activate the type I receptors which auto-phosphorylate, then bind and activate SMAD transcriptional regulators, SMAD2 and SMAD3. It’s been established that ACVR1C plays a role in processes such as cell differentiation, growth arrest and apoptosis via activation of TGF-Beta. Recently, ACVR1C was found to play a role in the symptoms of AD by regulating ion balance and intracellular calcium concentrations in cells of the skin.[11] Atopic skin is a hyperactive skin condition where skin has an exaggerated reaction in response to internal stimulants and external irritants. This exaggerated reaction may be due to sensory neural changes and disruption of the skin barrier by a decreased pH.[12] The ACVR1C gene is upregulated by conversion of WT mice to heterozygous Rlip loss mice and increased expression of ACVR1C seems to be associated with a decrease in dermatitis symptoms. Further, a deficiency of ACVR1C was correlated with an increase in atopic skin.
One study indicated that ACVR1C mRNA and protein expression were significantly decreased in sensitive skin when compared with non-sensitive skin.[12] ACVR1C was also found to be down regulated in atopic skin via microarrays. Further, it was shown that atopic skin showed elevated intracellular calcium concentrations when compared to non-atopic skin.[12] To confirm the link between decreased expression of ACVR1C and increased intracellular calcium, researchers transfected cells with ACVR1C siRNA to knock down ACVR1C expression and measured calcium levels after knockdown. ACVR1C cells had an elevated calcium influx and a higher intracellular Ca2+ concentration following calcium ionophore when compared to normal wild type cells.[12] The researchers hypothesized that this elevated intracellular calcium level may increase symptoms of atopic skin via a decreased pain threshold. They found this decrease in pain threshold was related to an increase in the expression proteins such as transient potential cation channel subfamily V member 1 (TRPV1), acid-sensing ion channel 3 (ASIC3), and the pain-related neurotransmitter calcitonin gene-related peptide (CGRP).[13] Thus, an increased intracellular calcium level in sensitive skin could be related to increased expression of TRPV1. This increased intracellular calcium could then be related to a decrease in pH via ASICS3 and lowered threshold for pain tolerance via increased expression of CGRP. CGRP expression was found to be increased by knockdown of ACVR1C. [14] TPRV1 not only affects intracellular calcium levels but may also contribute to a disrupted skin barrier- a major component of AD. [13] In a study, a TRPV1 inhibitor cream was used in the treatment of perioral dermatitis with success. Topical application of this cream to the peri-oral area showed evidence of an improved skin barrier function with decreased transepidermal water loss in previously dermatitis afflicted skin.[13] Additionally, skin redness was improved after use of the cream.[13]
Next it was examined whether upregulation of the ACVR1C pathway posed a therapeutic option for minimizing atopic skin.[13] This was tested by treatment of rhabdomyosarcoma cells with Nodal, an ACVR1C ligand. Nodal treatment was found to induce a substantial decrease in intracellular calcium influx and TRPV1 expression.[13] Thus, upregulation of ACVR1C was found to relieve symptoms of dermatitis and hypersensitivity in skin. Partial Rlip loss would induce upregulation of the ACVR1C gene. Since deficiencies of this gene are associated with an increase in AD symptoms such as hyperactive skin, it’s proposed that upregulating the amount and activity of ACVR1C receptors via Rlip loss would pose a therapeutic target for AD and atopic skin. Increasing signaling via increased ACVR1C ligands would also pose a treatment option.
Interleukin-13 Receptor Subunit Alpha-2 (IL-13RA2)
IL-13RA2 encodes a membrane bound receptor for IL-13 and this receptor is closely related to IL-13RA1, a subunit of the interleukin 13 receptor complex. IL-13RA2 binds IL13 with a high affinity but lacks the cytoplasmic domain and doesn’t partake in IL-13 mediated signaling. Rather it’s known to play a role in the internalization of IL13 and is thought of as a “decoy receptor” due to its ability to regulate IL-13 signaling.[15] IL-13 additionally binds the IL-13RA1 / IL-4Ra receptor complex which is also a component of the IL-4 receptor. Once this receptor complex is activated it can mediate signaling processes that lead to the activation of JAK1, STAT3 and STAT6.[16] IL-13 is an immunoregulatory cytokine that is produced primarily by activated Th2 cells. Overexpression of STAT6 and the IL-13 pathway has been implicated in the pathogenesis of inflammation and AD. Further, IL-13 was found to enhance IgE-mediated responses by upregulating IgE receptors on B lymphocytes, mast cells, and basophils.[15]
IL-13 acts directly on keratinocytes of the skin to enhance production of CCL26, a chemokine which contributes to the recruitment of eosinophils to eczematous skin.[17] Thus, it’s proposed that upregulation of IL-13RA2 via Rlip loss would decrease the eosinophilic inflammation associated elevated IL-13 in AD. IL-13 and IL-4 were also found to inhibit eosinophil apoptosis and promote eosinophilic inflammation by inducing chemotaxis and activation.[12, 15] Elevated IgE and eosinophils are markers of allergic inflammation and are elevated in conditions such as asthma and AD.
IL-13RA2 is upregulated by Rlip loss and provides a potential target for AD treatment due to its ability to regulate expression of IL-13. IL-13RA2 binds with a higher affinity to IL-13 than IL-13RA1, another IL-13 receptor.[16] This allows IL-13RA2 to bind circulating IL-13 and prevent it from binding IL-13RA1. IL-13 when bound to IL-13RA1 can exert pathogenic signaling effects.
In one study, mice were treated epicutaneously with 200 mg of Aspergillus fumigatus extract in saline or saline alone (control) applied to their backs.[18] It was found that mice lacking IL-13RA2 had significantly higher levels of AD characteristics. These IL-13RA2 deficient mice had elevated transepidermal water loss, cutaneous inflammation, peripheral eosinophilia, and IgG1 and IgE levels after allergen challenge compared to wild-type mice.[18] IL13-RA2 deficiency was thus found to contribute to the inflammatory environment of AD. Further, depletion of IL-13RA2 in a keratinocyte cell line resulted in increased STAT6 signaling in response to IL-13.[18] Here it’s shown that IL-13RA2 plays a protective role in the pathogenesis of allergic inflammation and loss of skin barrier function in a mouse model of AD. This implies that IL-13RA2 may be a critical regulator of IL-13–induced cutaneous inflammation in humans.
In addition, IL-13RA2 has been found to be upregulated by the act of scratching.[19] An itchy sensation and scratching are central to the symptoms of AD. Further, IL-13 also was increased by scratching and was found to induce IL-13RA2 expression in a negative feedback loop. Here upregulated IL-13RA2 expression is induced in response against IL-13-mediated atopic inflammation to minimize IL-13 levels before too much damage has been done.[17] But, the response of upregulated IL-13RA2 is not enough to compensate for the pathogenic effects of IL-13 and scratching can eventually form a lichenified eruption.[19] Blocking IL-13 signaling through the IL-13/ IL-4 receptor complex has already shown success in the treatment of AD. Dupilumab is a monoclonal antibody against the IL-4 receptor component of the shared IL-13 / IL-4 receptor complex.[20] This antibody is able to inhibit the signaling of both IL-4 and IL-13 which are key drivers of Th2 inflammation. Dupilumab has been shown to be effective in treating patients with atopic/allergic disease such as AD. It progressively improved disease activity, suppressed cellular and molecular cutaneous markers of inflammation, systemic measures of type 2 inflammation, and reversed AD-associated epidermal abnormalities in patients with AD. [20]
Dupilumab treatment also increased expression of a protein called filaggrin, which holds in water and helps keep the skin’s outmost layer intact.[20] AD is marked by a dysfunctional epithelial barrier that allows the skin release moisture and remain dry. Low levels of filaggrin leave skin dry and prone to damage and sensitization. After 16 weeks of dupilumab treatment, it was found that FLG expression (the gene that encodes filaggrin protein) was stronger and more continuous in the granular layer of skin in samples that had AD lesions initially.[20] Upregulation of IL-13RA2 via Rlip loss would be able to regulate IL-13 signaling and mitigate the Th2 inflammation IL-13 activates. This response is involved in many allergic conditions such as AD and asthma.[15] Furthermore, IL-13RA2 shows evidence of maintaining the epithelial skin barrier and reduce eosinophil migration. These studies provide evidence that IL-13RA2 can treat the vital symptoms and characteristics of the AD disease process.