Atopic dermatitis (AD) is a chronic and recurrent inflammatory skin disorder characterized by symptoms, such as eczema, erythema, persistent itching, and continuous skin damage. Over the past 30 years, the prevalence of AD has shown a notable rise, affecting between 5% and 20% of the child population and approximately 7% of adults in industrialized countries. ^{1 , 2} The pathogenesis of AD is not completely clear; however, allergens can activate an intense Th2 response. ³ Allergenic sources can vary depending on the population; for example, house dust mites are the main source of sensitization in tropical countries, whereas in Europe, pollen is the main trigger for allergies. In addition, depending on the environmental conditions, it is possible to be exposed to different species; therefore, epidemiological studies have shown variations in the frequencies of sensitization. ⁴ The severity of the disease is influenced by several factors, including compromised skin barrier integrity due to reduced levels of filaggrins and ceramides as well as an elevated presence of water channels such as aquaporin, which drives the skin, making the patient more susceptible to atopic dermatitis. ⁵ In general, allergy management includes allergen avoidance, pharmacotherapy, and immunotherapy, where only the latter has a beneficial effect that lasts for years and allows for a reduction in anti-inflammatory treatment. ^{6 – 8}

In addition to other environmental factors, the skin microbiome plays a pivotal role in AD development. Aspergillus fumigatus is an opportunistic pathogen in both poikilothermic and homeothermic animals. In humans, this pathogen has been associated with many pathologies such as invasive cutaneous aspergillosis, saprophytic lung colonization, and aspergillomas. In addition, A. fumigatus can induce IgE-mediated allergic diseases including rhinitis, allergic sinusitis, asthma, hypersensitivity pneumonitis, and AD. ^{9 , 10} It has been characterized as an important allergenic source, and some allergens, including thioredoxins and cyclophilins, share homology with human proteins and are implicated in the autoreactive response in AD.

Despite the identification of sensitization to extracts from allergenic sources, such as fungi, the recognition of specific IgE sensitization to allergens is necessary to carry out a better diagnosis and immunotherapy. ¹¹ However, this represents a challenge, because most fungal allergens cross-react with other taxonomically distant species, making it difficult to prove the clinical relevance of IgE reactivity against these allergens. In addition, it is important to consider that the evolution of AD varies greatly among patients with this disease and can be triggered by different factors. For example, in A. fumigatus and AD, studies suggest that specific IgE-mediated activation is induced not by multiple allergens and not by a few major allergens, as is the case with asthma and rhinitis. ^{12 , 13} Another problem presented by the diagnosis of allergic diseases associated with A. fumigatus is the lack of standardized allergens for preparation of molecular component tests. Therefore, the discovery of new fungal allergens and the identification of the prevalence of these new components may provide an opportunity to learn more about the pathology of atopic dermatitis, improving the diagnosis and treatment of this disease.

In recent years, aquaporin has become a candidate protein for the study of skin and allergic diseases such as AD, given its role in epidermal hydration and the transport of various substances such as glycerol, salts, and exocrine fluids, favoring dry skin and allergen leakage. ¹⁴ They have also been associated with various atopic diseases, such as glaucoma, cancer, epilepsy, obesity, epidermal hyperplasia, and neuromyelitis optical, among others. In AD, an increase in the expression of aquaporin 3 has been observed, which may be related to water loss and the severity of the disease, in contrast to healthy individuals who maintain a balanced expression of this protein in the basal, horny, and spinous layers. ⁵ The consequences of aquaporin 3 (AQP3) overexpression extend beyond cutaneous water retention as it becomes a target for the exacerbated immune response in AD inflammation. This hypothesis suggests that it may be recognized by human autoantibodies, which, due to the persistence of self-protein exposure, contribute to increased inflammation and chronicity of AD.

Additionally, this protein is present in most organisms with a wide variety of isoforms and functions throughout the taxonomic framework. Recently, aquaporin has been identified in highly allergenic species, such as house dust mites. In a study carried out by Mao et al., describing the Dermatophagoides farinae proteome, two-dimensional immunoblotting was performed, which showed binding with IgE antibodies in a band that has the same isoelectric point and molecular weight of the aquaporin family, suggesting its possible role as an allergen. The objective of this study was to perform an in silico analysis to investigate the potential molecular mimicry between human aquaporins and those from A. fumigatus, as well as various allergenic sources.

For the selection of aquaporins, the Aspergillus fumigatus (KEY82230) and human aquaporin 3 (Q92482) sequences of interest were compared with existing allergenic protein sequences using Allermatch ( https://allermatch.org/), an allergen comparative server that allows allergen sequence alignment; therefore, those with high identity values were chosen. The sequence pool was obtained from the UniProt ( https://www.uniprot.org/) and National Center for Biotechnology Information (NCBI) databases. Given the diversity in amino acid origins and lengths across the molecules under examination, we employed coverage and identity values to gauge the extent of their relationships. The identity and coverage values between the molecules used in this study were determined using the EMBOSS Needle ( https://www.ebi.ac.uk/Tools/psa/emboss_needle/), specialized for performing paired alignments, and the web server from Praline ( http://www.ibi.vu.nl/), which allowed for paired and multiple alignments. When carrying out the alignment, it was considered that the sequences were different and came from different sources; therefore, the alignment parameters were set to utilize the BLOSUM62 matrix, which primarily assesses evolutionary divergence, along with adjustments to the conditional scoring matrix. ^{15 , 16}

Numerous diseases, including atopic dermatitis, bullous pemphigoid, chronic spontaneous urticaria, and multiple sclerosis, are associated with the presence of IgE autoantibodies. In the context of allergic responses intertwined with autoimmunity, researchers have categorized autoantigens that bind to IgE into three functional groups: 1) autoantigens that share sequence homology with environmental allergens, 2) autoantigens that lack sequence homology with known environmental allergens, and 3) chemically modified autoantigens. However, the identification of environmental allergens with homology to human proteins and their influence on the allergic response vary among these autoantigens, and their clinical significance often remains unclear. ¹⁹

Aquaporin is a promising protein candidate for studying cutaneous, autoimmune, and allergic diseases, including atopic dermatitis (AD), because of its involvement in epidermal hydration and transportation of various substances such as glycerol, salts, and exocrine fluids. These functions contribute to skin dryness, which, in turn, facilitates the filtration of allergens. ¹⁴ This protein is widely present in numerous organisms, and exhibits a diverse range of isoforms and functions across different taxonomic groups.

Recent research has identified the presence of aquaporins in allergenic species such as dust mites. ²⁰ Mao et al. characterized the proteome of Dermatophagoides farinae, and two-dimensional immunoblotting revealed the binding of IgE antibodies to a band with the same isoelectric point and molecular weight as the aquaporin family, suggesting its potential role as an allergen. ²¹

On the other hand, in a systematic review, a total of 32 articles were examined to assess the role of the microbiota in atopic dermatitis. The microbiome profile revealed substantial variation in bacterial diversity along with an increased presence of fungi. Notably, there was diversification and expansion of species belonging to the genus Aspergillus within the fungal group, whereas a reduction in the number of Malassezia spp. was observed. Research has also demonstrated that A. fumigatus is a prominent source of sensitization in patients with severe atopic dermatitis. Additionally, the presence of new allergens from this species is believed to have a significant impact on the severity of the condition. ²²

The comprehensive analysis of aquaporins provides guidance for considering other potential allergenic sources beyond the main aquaporins studied, namely AQP3 and A. fumigatus aquaporin. We conducted a comparative analysis of 25 aquaporin sequences from various sources, including A. fumigatus aquaporin and human aquaporin 3 (AQP3). A comparison between A. fumigatus aquaporin and AQP3 revealed an identity of 32.6% and similarity of 47.5%, indicating a relevant level of homology between these two proteins despite the evolutionary gap and taxonomic diversification among species. Notably, human AQP3 exhibited the maximum identity and similarity values of 95.5% and 98.6%, respectively. These high values could be attributed to the inclusion of aquaporins from mammalian allergen sources. Given that these species belong to a taxonomic group closer to humans, it is understandable that the APQs are more conserved.

Five clades were constructed for the phylogenetic analysis. A and B showed the highest identity with A. fumigatus aquaporins and AQP3. The alignment results suggest that divergence among aquaporins primarily occurs in smaller species, while it remains conserved in mammals. In addition, the presence of aquaporin isoforms, each with special functions and locations, such as AQP3, indicates functional and phenotypic variability among different cell types within the same organism. However, despite this diversity, the identity of aquaporins within the clades revealed values higher than 30%, particularly in clade B (95%), indicating that if the protein is recognized by antibodies, potential cross-reactivity among these species can occur.

Only a limited number of studies have investigated the relationship between aquaporins derived from allergenic sources and their potential relevance in human diseases. Zhou et al. conducted an assessment using transcriptomics to identify potential AQPs in Blomia tropicalis using molecular cloning techniques, followed by a comparative analysis of identity among mite aquaporin sequences and human aquaporins. The author identified five putative AQP-coding sequences, known as BlotAQP1-5, which were indexed into all three subgroups: AQPs, aquaglyceroporins, and superAQPs. BlotAQP1, BlotAQP2, and BlotAQP5 were clustered with the aquaglyceroporins hAQP3, hAQP7, hAQP9, and hAQP10, with a common sequence consensus ‘N-G-NPSRD-PRL.’ The molecular weight and isoelectric point of these molecules were consistent with the findings reported by Mao et al. in their two-dimensional electrophoresis study. ²³ This observation suggests the potential recognition of aquaporins, specifically aquaglycerins such as AQP3, by IgE in patients with allergies. These results are consistent with those presented in our findings, where despite the evolutionary distance between species, there is an identity exceeding 30% and consensus sequences that may serve as potential antigenic patches.

In contrast, in regions where higher conservation was observed among the sequences, neither linear nor conformational epitopes were identified. This is attributed to the fact that these sequences are in the transmembrane region of the protein, which limits their accessibility to antibodies. However, despite the absence of epitopes, these conserved regions are interesting. During cell lysis caused by mechanical injury or microbial effects, these proteins can be linearly exposed, leading to the emergence of neoepitopes that can be recognized by IgE or phagocytic cells. ²⁴

Although little is known about the role of AQP3 as an allergen or autoantigen, other aquaporins have been studied for their relevance as antigens in autoimmune diseases. Sagan et al. investigated the relevance of AQP4 as an autoantigen in neuromyelitis optica, recognized by T lymphocytes, in an animal model. ²⁵ This aquaporin is a water channel expressed in astrocytes in areas in contact with the blood-brain barrier, and 75% of patients with neuromyelitis have antibodies capable of recognizing AQP4. Therefore, the relevance of human aquaporins found in other tissues, such as AQP3 in the skin, can be considered as potential autoantigens and, through molecular mimicry, triggers relevant skin diseases such as atopic dermatitis. ²⁶ Evidence indicates cross-reactivity with aquaporins expressed by bacteria and mycobacteria, including Escherichia coli and Mycobacterium species, in relation to this specific autoantigen. ²⁷ This indicated that antibodies targeting aquaporins from A. fumigatus could potentially recognize and target this autoantigen. In a previous study, we found that AQP4 exhibits only one predicted cross-reactive epitope. It has been postulated that the recognition of a single epitope by autoantibodies could trigger an epitope spreading mechanism, leading to the involvement of additional autoantigens in the immune response. ²⁸ For both A. fumigatus aquaporins and AQP3, a total of four linear epitopes and three discontinuous epitopes were described. Structural superimposition highlighted a notable degree of similarity between the proteins, indicating that the epitopes could reside within the corresponding antigenic regions. This observation supports the idea of a potential molecular mimicry between these proteins.

It is important to acknowledge that our study has several limitations. In silico modeling and epitope prediction analyses are not definitive, and there may be variations in the actual structure compared with our proposed models. Nonetheless, bioinformatic analyses offer several advantages in directing research resources efficiently. They play a crucial role in the preliminary assessment of hypotheses and help to determine whether it is justified to proceed with in vitro or ex vivo experiments.

During alignment of aquaporin sequences from A. fumigatus and AQP3, specific conserved epitopes were observed. Analysis revealed that the local sequence identity exceeded 70%, indicating molecular mimicry in these regions of both aquaporins. Further investigation of epitopes and domain identification revealed the most conserved region in transmembrane domains 12 and 14 of the respective aquaporins. This study represents a groundbreaking contribution to the exploration and analysis of aquaporin as a potential allergen and autoallergen in atopic dermatitis, as it is the first known study to focus on this subject. To confirm cross-reactivity, future in vitro studies are needed to demonstrate the IgE-binding capacity in these regions.

By acquiring knowledge regarding the sequences and structures of potential epitopes from different allergenic sources, we can modify and synthesize these molecules for future in vitro and in vivo studies. This not only expands the literature in the field of immunotherapy but also facilitates the diagnosis and treatment of autoimmune diseases such as atopic dermatitis. It is important to note that in silico tests optimize these studies by providing rapid results and circumventing the high costs associated with laboratory testing.