Autism spectrum disorder (ASD) is a kind of neurodevelopmental condition and troubles a lot of children in the world, with a prevalence up to 1%–2% (Liu et al., 2022). ASD is characterized by a deficit in social communication and interaction and the restrictive, repetitive pattern of behavior (Lai et al., 2014). What is worse, the coexistence of neuropsychiatric disorders and allergic diseases is not uncommon (Simpson, 2012; Miyazaki et al., 2017). In a large cross-sectional study, ASD children showed a higher odds ratio in association with skin allergy compared with children without ASD (Xu et al., 2018). Billeci et al. retrospectively analyzed 18 studies to assess the relationship between ASD and atopic dermatitis (AD). They found that ASD was positively associated with AD compared with typically developing controls, and vice versa (Billeci et al., 2015). Moreover, the frequencies of AD in ASD varied, ranging from 7% to 64.2%. Atopic dermatitis is a chronically inflammatory skin disease featured with intense itching and relapsing eczema-like skin lesions and is one of the most common skin diseases in children (Weidinger et al., 2018). It is generally accepted that epidermal barrier abnormalities and T-cell abnormal activation are involved in the development of AD, and it shows a relationship with some autoimmune diseases especially those affecting the skin, the gastrointestinal tract, and the connective tissue, such as systemic lupus erythematosus (Narla and Silverberg, 2019; Ivert et al., 2021). AD mostly develops within the first 5 years of life, up to 90% of cases. In accordance with previous studies, in our clinic center, autistic children are prone to suffering from AD, which impairs their daily life and lowers the quality of life further.

The co-occurrence of autism and atopic dermatitis indicates potentially common mechanisms including shared genetic background, common immunologic dysfunction, and autoimmune process. For those with ASD, their siblings were prone to develop atopic diseases (Dai et al., 2019), and children, who suffered from early AD were more likely to develop subsequent ASD (Billeci et al., 2015; Lee et al., 2016). Meanwhile, in prior studies, ASD children also showed an imbalance in inflammatory cytokine and T-cell subsets (Xu et al., 2018). Besides, in recent years, with both incidence increasing, it has come to the realization that environmental factors play an important role in the development of diseases. Digestive symptoms were more easily observed in autistic children including constipation, diarrhea, bloating, and vomiting (Holingue et al., 2018; Vargason et al., 2019; Lasheras et al., 2020). Compared to neurotypical children, autistic children showed lower bacterial diversity, which was inversely associated with the severity of digestive symptoms (Srikantha and Mohajeri, 2019). There are lots of studies revealing intestinal dysbiosis in ASD and, mostly, showing a decreased ratio of Bacteroidetes to Firmicutes (Srikantha and Mohajeri, 2019). Based on prior studies, Xu et al. conducted a meta-analysis and found that autistic children had lower abundance of Enterococcus, Escherichia coli, Bacteroides, and Bifidobacterium and higher abundance of Lactobacillus compared to controls (Xu et al., 2019). Clostridia species were also found richer in ASD and related to the severity of the autistic condition (Iovene et al., 2017). The emergence of the microbiota–gut–brain axis led to more research on the underlying mechanism and the possible strategies to ameliorate the clinical symptoms. Autistic children presented altered metabolic profiles in urinary and blood analyses, which could be the biomarker of gut dysbiosis (Srikantha and Mohajeri, 2019). Meanwhile, there are several studies revealing promising results that the application of antibiotics and probiotics could improve not only the mental conditions of autistic children but also digestive symptoms (Critchfield et al., 2011; Patusco and Ziegler, 2018; Shaaban et al., 2018; Niu et al., 2019), even though a consensus has not been reached. Similarly, the gut–skin axis described the mutual connection between the gut and the skin, fitting the hygiene hypothesis (Sinha et al., 2021). For those with AD, their gut microbial diversity was lower than that in healthy individuals (Fang et al., 2021). Furthermore, many studies have shown intestinal dysbiosis in children with AD, which could be further improved by probiotics (Watanabe et al., 2003; Roessler et al., 2012; Hulshof et al., 2017). The alteration of the gut microbiota may influence the ratio among T-cell subsets and the metabolic pathway, which induces the inflammatory response and the development of AD.

As aforementioned, the gut microbiota plays an important role in disease development, which could be affected by diet, living habits, and mental stress. Kong et al. enrolled 20 individuals with ASD, and they found an increased relative abundance of gut Proteobacteria in ASD with allergy compared with those without allergy, which was not observed in neurotypical controls (Kong et al., 2019). Considering the high prevalence of AD in autism, in this study, we aim to explore the different compositions of gut microbiota between autistic children with and without AD. Besides, we further conduct a non-invasive urinary analysis to detect the possible metabolic changes, which may give some clues to the underlying mechanism of gut microbes on AD development in autism and aid in the clinical management of those special individuals.

The coexistence of atopic dermatitis in autism is far from rare, which bothers those children and further lowers their life quality. This is the first study exploring the alterations of gut microbiota and urinary organic acids in autistic children with AD compared with those without AD. We found that there was no significant difference in sex, age, and disease evaluation between both groups, which was reasonable to carry out further analysis.

As expected, in our study, autistic children with AD had more prominent GI symptoms than their control counterparts. GI symptoms showed greater prevalence in patients with ASD compared to those without ASD (McElhanon et al., 2014). As for patients with AD, lots of studies have revealed that AD is related to the increased risk of GI disorders including Crohn’s disease, ulcerative colitis, and celiac disease (Baron et al., 2005; Ress et al., 2014; Augustin et al., 2015). Considering the limitation of verbal communication in children, their underlying GI problems may be presented as sleep or mood disturbance (Maenner et al., 2012). In our study, AD individuals had a higher score in the item about being unhappy or crying than that in controls.

In the analysis of gut microbiota, lower diversity was observed in the AD group, which was consistent with previous studies on autism and atopic dermatitis (Petersen et al., 2019; Bezawada et al., 2020). This may echo the known theory—the hygiene hypothesis—in which the lack of exposure to a high diversity of microbes in early life is related to a higher prevalence of chronic inflammatory disorders as well as psychiatric disorders (Garn et al., 2021). In our study, B. bifidum showed an enrichment in autistic children with AD (0.31% vs. 0.05%). For children with ASD, Xu et al. summarized that the relative abundance of Bifidobacterium was lower compared to controls (Xu et al., 2019). Even though several studies had revealed that Bifidobacterium was less in AD infants compared to healthy controls (Fang et al., 2021), controversy always exists. A cross-sectional study from Brazil revealed that AD children had a greater abundance of Bifidobacterium (OR: 11.09; 95% CI: 2.14; 57.39) (Melli et al., 2020). At species levels, Waligora-Dupriet et al. enrolled 10 allergic infants and 20 controls, and they did not detect any alteration in the diversity of gut Bifidobacterium species (Waligora-Dupriet et al., 2011). However, Suzuki et al. found that B. bifidum had a higher prevalence in allergic infants than healthy controls (70% vs. 12.5%, P < 0.01), which was irrelevant to feeding methods (Suzuki et al., 2007). Furthermore, Huang and coauthors enrolled 13 randomized controlled trials and found it was not robust to conclude that probiotics were beneficial to children with AD (Huang et al., 2017). Similarly, Yang et al. did not observe the therapeutic or immunomodulatory effects of probiotics on the treatment of AD (Yang et al., 2014). Additionally, as mentioned above, as a possible comorbidity of AD, Nistal et al. observed that the diversity of Bifidobacterium species was reduced in treated patients with celiac disease while B. bifidum was enriched in untreated patients (Nistal et al., 2012). Therefore, it is necessary to further investigate the relationship between conventional probiotics and the development of AD in autistic children.

In this present study, we observed different distributions of species from family Lachnospiraceae in both groups, among which A. caccae and E. hallii predominated in AD individuals, with R. intestinalis in controls (8.22 × 10^-4 vs. 1.18 × 10^-5; 0.28% vs. 0.08%; 0.30% vs. 0.95%). All these microbes were involved in the production of short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate (Louis et al., 2014). SCFAs, the major products from the fermentation activity of gut microbes, are proved to play a crucial role in host health (Wong et al., 2006; Koh et al., 2016; Sivaprakasam et al., 2016; Gill et al., 2018; Hu et al., 2018). They could promote the differentiation of regulatory T cells and the production of the anti-inflammatory cytokines, avoiding colonic inflammation and cancer development, as well as modulate host energy metabolism (Sleeth et al., 2010; Singh et al., 2014; Koh et al., 2016; Hu et al., 2018). Additionally, it became increasingly accepted that SCFAs could mediate epigenetic modification, which was negatively associated with allergic sensitization and events (Acevedo et al., 2021). Reddel et al. showed that AD children were characterized as a reduction of SCFA-producing bacteria (Reddel et al., 2019). Moreover, the severity of atopic eczema was inversely correlated with the abundance of butyrate-producing bacteria (r = -0.52, P = 0.005) (Nylund et al., 2015). Meanwhile, in an ovalbumin-induced mouse model of AD, the use of antibiotics before primary ovalbumin sensitization would reduce gut SCFA levels and aggravate AD symptoms mediated by gut dysbiosis (Kim et al., 2020). Nevertheless, in autism, SCFA is a double-edged sword. Compared to healthy controls, autistic children had a lower concentration of SCFAs, among which the level of butyrate was dramatically reduced while propionate and acetate were in higher levels (De Angelis et al., 2013). The injection of propionate into rats could cause ASD-like symptoms, and it was associated with Clostridia species, which was generally acknowledged involved in the pathophysiology of ASD (Srikantha and Mohajeri, 2019). Butyrate is considered as the most important SCFA in hosts. Lots of studies had shown its role in brain protection and its positive effects on neurodegenerative diseases (Mohajeri et al., 2018).

R. intestinalis, a butyrate-producing bacterium in the colon, has come to the fore due to its role in the prevention of intestinal inflammation and the maintenance of host homeostasis in many digestive, autoimmune, and neurological diseases (Nie et al., 2021). Of note, one of the fermentative products of B. bifidum is lactate (de Vries et al., 1967), which could be the substrate for producing butyrate by A. caccae and E. hallii besides acetate, while R. intestinalis just uses acetate for producing butyrate (Koh et al., 2016). Additionally, B. bifidum is the only species to degrade mucin into monosaccharides in genus Bifidobacterium and enable the growth of E. hallii to produce SCFAs by cross-feeding (Bunesova et al., 2017). To some extent, these may explain why two SCFA-producing bacteria were richer in AD individuals, unexpectedly in our study.

As a promising probiotic, A. muciniphila was found richer in autistic children without AD compared to those with AD (2.31% vs. 0.61%). According to previous studies, compared to healthy children, atopic children showed a significant decrease in or even deletion of A. muciniphila ( Candela et al., 2012; Drell et al., 2015; Sung et al., 2022). Lee et al. found that the colonization of A. muciniphila reduced in infants with AD compared to the control group (Lee et al., 2018). Besides atopy, the lower relative abundance of A. muciniphila in the gut was also reported to be related to autism (Wang et al., 2011), even though the relative abundance of genus Akkermansia was not significantly different between ASD infants and healthy controls (Inoue et al., 2016). Characterized by mucolytic ability, A. muciniphila could degrade host mucin to promote the production of SCFAs and regulate the immune system, maintaining the integrity of the gut barrier (Belzer et al., 2017; Ottman et al., 2017; Zhang et al., 2019; Zhou and Zhang, 2019). Meanwhile, SCFAs should bind to G protein-coupled receptors to achieve the normal resolution of inflammatory response (Maslowski et al., 2009), the expression of which was regulated by A. muciniphila ( Lukovac et al., 2014). Compared to children with persistent AD, the proportion of Akkermansia was higher in transient AD cases, indicating its potential role in the remission of AD (Park et al., 2020).

Surprisingly, we found that the amounts of H. parainfluenzae and R. mucilaginosa were significantly higher in controls compared with AD individuals (1.17% vs. 0.68%; 4.42 × 10^-5 vs. 3.28 × 10^-5). Both two species are usually regarded as opportunistic pathogens, which could lead to a variety of diseases, such as pneumonia, endocarditis, and bacteremia (Spernoga, 1980; Bruminhent et al., 2013; Maraki and Papadakis, 2015; Cobo et al., 2017; Faure et al., 2017; Poyer et al., 2019). However, Zheng and coauthors identified that genus Haemophilus was richer in healthy infants compared to infants with eczema (Zheng et al., 2016). In a study by Arrieta et al., the reduced amount of genus Rothia in the gut during the first 100 days of life was associated with a higher risk of childhood asthma by means of reducing the level of fecal acetate (Arrieta et al., 2015). In ASD, Kang et al. revealed that a decrease in the abundance of H. parainfluenzae in the gut was related to more severe GI symptoms in children with ASD (Kang et al., 2018), similar to the study conducted by Zou et al. (2020). By far, no study has yet been reported on the relationship between these two species and AD.

The gut dysbiosis may come along with the disruption of the host barrier and the alteration of metabolism, which could be further reflected by the metabolic analysis in feces, blood, and urine. In our study, for urinary organic acids, only adipic acid was statistically significant in the multivariate analysis (OR = 1.513, P = 0.030). Adipic acid was produced from the omega-oxidation pathway of fatty acids, a process which was normally an alternative to the beta-oxidation pathway in mitochondria (Puig-Alcaraz et al., 2016). This pathway could be augmented when mitochondrial function was impaired. Compared to healthy controls, the increase of adipic acid in urine was significantly correlated with ASD and its severity (Puig-Alcaraz et al., 2016; Khan et al., 2022). Furthermore, in the correlation analysis, aconitic acid and adipic acid were positively related. Aconitic acid was an intermediate product in the Krebs cycle and produced by the dehydration citric acid (Mussap et al., 2016). The increase of aconitic acid in urine was observed in individuals with autism, depression, or autoimmune diseases compared to healthy controls (Jones et al., 2005; Noto et al., 2014). Both indicated mitochondrial dysfunction. Mitochondria critically modulated host immune response, and its dysfunction could induce oxidative stress favoring systemic inflammation, which was observed to be involved in allergic diseases including atopic dermatitis, allergic rhinitis, and asthma (Iyer et al., 2017; Trinchese et al., 2018). Zhang et al. found that the deficiency of the detoxification pathway of the gut microbiota was linked to the biomarkers of mitochondrial dysfunction (Zhang et al., 2020). This was in line with the enrichment of the pathway of xenobiotics biodegradations in the control group, and this pathway of the gut microbiota seems to have a negative correlation with adipic acid (r = -0.42, P = 0.18). Besides, we also found that R. intestinalis showed a negative correlation with aconitic acid, which was the same with their distributions in different groups. The exposure of toxic substances may contribute to mitochondrial dysfunction (Lee et al., 2010), finally leading to disease development. Meanwhile, with the dramatical increase in the incidence of AD, epigenetic regulation was brought to the fore and considered as a link between the changing environment and genetic changes, which had been explored extensively (Sroka-Tomaszewska and Trzeciak, 2021). The disorders of xenobiotic biodegradation may increase the risk of environmental exposure and detrimental epigenetic modifications, shifting the host toward a higher risk of allergic diseases. The specific mechanisms of gut dysbiosis on AD development in autism require more evidence in the future.

Besides, as Table 2 shows, there were some urinary organic acids, known for the markers of metabolic disorders in amino acids, enriching in the AD group, including 3-hydroxyglutaric acid (Al-Dirbashi et al., 2011) and 2-hydroxyphenylacetic acid (Armstrong et al., 1955), even though both have become statistically insignificant in the multivariate analysis. This was consistent with the result that the pathway of amino acid metabolism was found predominant in the control group. Autistic children showed a perturbation of phenylalanine metabolism, with elevated concentrations of 3-(3-hydroxyphenyl)-3-hydroxypropionic acid, 3-hydroxyphenylacetic acid, and 3-hydroxyhippuric acid in urinary analysis (Xiong et al., 2016). Similarly, 2-hydroxyhippuric acid was richer in the AD group, which was produced in the liver during the process of detoxification of salicylic acid (Suh et al., 1986). Its level generally increased with the intake of exogenous salicylic acid such as aspirin. Nevertheless, Finnie et al. enrolled children with digestive symptoms, to whom no salicylate-containing drugs were administrated. They found that 2-hydroxyhippuric acid was detected in the urine of sick children and its level was associated with GI disorders (Finnie et al., 1976). Considering that salicylic acid showed an anti-inflammatory effect in colitis (Miquel et al., 2015), it was reasonable to deduce that the production of salicylic acid may rise with the severity of GI disorders owning to self-defense mechanisms, and we really observed more GI dysfunctions in autistic children with AD.

We acknowledge that our study has several limitations. Firstly, it was a single-center study and the sample size was limited. Secondly, in this study, we only enrolled autistic children with AD and without AD, lacking those healthy or only with AD as controls. It is impossible for us to exclude the potential effect of autism as a confounder. Thirdly, due to the limitation of technology, we just took urinary organic acids into consideration, not including fecal or blood samples, which may affect the final result to some extent. Apart from the complex network among gut microbiota and their interactions with the host, this may explain why we did not observe evident correlations between urinary organic acids and gut microbiota. Lastly but very importantly, we just limited the usage of antibiotics and probiotics before sample collection, while the diets of enrolled children were not obtained and analyzed, which would introduce deviation in the final results. The question of causality of the alteration of the gut microbiota and the development of AD remains an egg or chicken problem. All of these will make our study preliminary and exploratory. Large-scale and well-designed studies in the future are needed.

Altogether, lots of studies have shed light on the role of the gut microbiota in the pathogenesis of disease. Herein, we identified significant differences of gut microbiota and urinary organic acids between autistic children with and without AD, and the underlying mechanism on AD occurrence in autism needs further investigation. A more comprehensive understanding of the relationship between gut microbiota and AD in autistic children will be beneficial for clinicians to manage those children in the prevention and treatment of AD better.

The data presented in the study are deposited in the National Center for Biotechnology Information (NCBI) Bioproject repository, accession number PRJNA845014.

The studies involving human participants were reviewed and approved by the Peking Union Medical College Hospital Ethics Committee. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

All authors contributed to the study conception and design. Material preparation and data collection were performed by XY, X-jX, X-fZ, and XZ. Data analysis and interpretation were performed by R-pH, Y-yH, GT, Y-fJ, and J-dL. The first draft of the manuscript was written by R-pH, Y-yH, and X-jX, and all authors commented on previous versions of the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by the Autism Special Fund from the Peking Union Medical Foundation, the CAMS Innovation Fund for Medical Science (CIFMS) (2017-I2M-3-017), and the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (2019XK320030). The study sponsors had no involvement in the study design, the collection, analysis, and interpretation of data, the writing of the report, and the decision to submit the paper for publication.

Authors J-dL and GT were employed by the company Geneis (Beijing) Co., Ltd.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.