Children with elevated wheat IgG4 antibody titer in autism spectrum disorder: Clinical presentation and findings associated with gut microbiota

Autism spectrum disorder (ASD) is a common neurodevelopmental disorder associated with a high prevalence of food allergies.¹ Amidst ongoing debate and conflicting data on the role of food-specific IgG4 antibodies,² our study delved into the clinical importance of IgG4 in ASD and related food hypersensitivities, providing new research perspectives on non-IgE-mediated conditions.

A cohort of 43 children with ASD was enrolled, and biochemical and immunologic indices (n = 43), and fecal macrogenomic and 16S sequencing data (n = 38) were collected (Table S1). None of the participants presented with eosinophilic esophagitis, inflammatory bowel disease, or celiac disease, and no child manifested overt clinical symptoms indicative of wheat-specific IgE-mediated allergy. Their allergic reactions to common allergens are listed in Table S2. Considering that a high titer of antibodies may be more likely to lead to the deposition of circulating immune complexes in the intestine, patients were stratified into three groups based on their wheat IgG4 titer (Figure 1A). Changes across the three gradient groups were observed in intestinal susceptibility to the pathogens and carbohydrate-metabolizing capacity. We found that higher titers of wheat IgG4 antibodies were associated with reduced hepatic metabolism, such as decreased albumin synthesis (Figure S1A).

Furthermore, elevated wheat IgG4 titers were associated with increased intestinal mucosal susceptibility to pathogens, notably Shigella and Escherichia coli (Figure 1B), and were accompanied by a significant increase in the activity of both the bacterial invasion of epithelial cells pathway and the NOD-like receptor signaling pathway (Figure 1C). Using the Kruskal–Wallis test, we distinguished 153 pathogens or opportunistic pathogens with unique distribution profiles across diverse sample sets, visualized in the heatmap in Figure 1D. Subsequently, we used random forest's (RF) feature importance (mean decrease accuracy) to select seven key microorganisms as model inputs (Figure 1E) and reconstructed the RF model. The confusion matrix (Figure 1F) validated the model's strong predictive performance and the microorganisms' discriminatory power.

Notably, linear regression analyses supported the observations of Shigella and E. coli employing specific invasion mechanisms in the intestinal epithelium (Figure 1G, Figure S2B), underscoring the relevance of these specific mechanisms to pathogenesis. We identified E. coli DSM 30083 as the predominant intestinal microbe in children, potentially explaining the intergroup variance in E. coli abundance (Appendix S2). This strain's genome encodes a complete protein secretion system, including a type IV secretion system, and capabilities for exotoxin production (Figure S3A,B, Table S3), contributing to barrier dysfunction. Its adaptation to d-cysteine and resistance to antibiotics and heavy metals (Figure S3B, Table S3) reflects its survival strategy. According to previous studies,^{3, 4} E. coli DSM 30083 was classified as type B2, serotype O1:K1:H7, a classification that further confirms its potential pathogenicity.

Additionally, our analysis indicated that high wheat IgG4 titers corresponded to altered gut microbial carbohydrate-activated enzymes (CAZymes) profiles, particularly affecting dietary fiber metabolism. Specifically, there was decreased degradation of pectin, β-glucan, and cellulose, but increased hydrolysis of fructans and inulin (Appendix S3, Figure 2A). Notably, CAZymes with Kruskal–Wallis p-values <.2 constituted a consistently substantial proportion (19.7%–20.5%) of the total CAZymes genes across the LT, MT, and HT groups, implying a pivotal role in intestinal carbohydrate metabolism (Figure 2B). Spearman correlation analysis indicated that 14 out of 62 CAZymes had negative correlations with wheat IgG4 (p < .05), whereas 3 exhibited positive correlation (Figure 2C). The RF model effectively distinguished the three sample groups, reinforcing the observed distinctions (Figure S4A–D).

Further analyses revealed a robust correlation between diminished pectin degradation and a decrease in key enzymes for pectin main chain cleavage and side chain glycosidic bond hydrolysis. KEGG pathway analysis corroborated this, showing a significant decline in the ko00040 pathway (pentose and glucuronate interconversions), essential for pectin breakdown, in children with elevated wheat IgG4 titers (Figure 1C). Given the body's inability to digest complex carbohydrates and the gut microbiota's critical role in dietary fiber degradation, this discovery may significantly affect patients' health.⁵

Subsequently, covariate analyses were conducted to examine the impact of various food-specific IgG4, IgG, and IgE allergens on indicators pertaining to intestinal infections and to determine their role as confounders or independent covariates (analytical framework, see Figure S5). No covariates influencing the results were identified (Appendix S4). Importantly, additional to wheat, elevated IgG4 titers of other foods such as milk, yogurt, sheep milk, and eggs were also correlated with a high abundance of E. coli in the fecal samples (Figure S7A,C). This discovery accentuates the broad relevance and implications of our research findings. Nevertheless, while titers of food-specific IgG4 antibodies were associated with intestinal infections, akin associations were absent with food IgGs (Figure S7B,D).

Our study emphasizes the clinical relevance of high titers of food-specific IgG4 and high concentrations of food antigens. It stresses the importance of targeting individuals with notably high titers of food-specific IgG4, rather than solely focusing on positivity for food-specific IgG4, when designing dietary intervention studies. In our study, we set a low threshold of 85 U/L for the HT group, suggesting that wheat IgG4 titers exceeding this threshold might indicate health implications. Additionally, we observed an association between the increased intestinal susceptibility and foods consumed in large quantities such as wheat and dairy products, as opposed to foods consumed in small quantities such as hazelnuts (Figure S7A,C).

These observations led us to hypothesize that excessive accumulation of immune complexes in the mucosa may be a mechanism for intestinal dysfunction, especially when clearance thresholds are exceeded. To delve deeper into this issue, we plan to conduct additional studies to investigate the deposition of immune complexes formed by IgG4 and food antigens in the intestinal mucosa of children with ASD. A prior study conducted by Torrente et al. has identified IgG deposition on the duodenal basolateral epithelial surface in children with regressive ASD.⁶ However, these studies did not distinguish food-specific antibodies, and the proportion of IgG4 remained undisclosed.

In conclusion, wheat IgG4 in ASD is associated with significant physiological and pathological processes, suggesting that children with high titers of wheat IgG4 may improve intestinal function by limiting wheat intake. These findings indicate that markedly elevated food-specific IgG4 titers may affect intestinal function. In clinical management of chronic disease, it is advisable to consider the effect of markedly elevated levels of food-specific IgG4 on symptoms, rather than simply categorizing food-specific IgG4 titers as negative or positive.

XY, the project leader, conceived the study. XY and JL and supervised the study. XL and HW collected the samples and conducted the laboratory experiments. YT, XL, JC, and BL analyzed and interpreted the data. YT worked on visualization and wrote the first draft with all the other authors editing and approving the final manuscript.

This research was supported by Autism Special Fund from Peking Union Medical Foundation, CAMS Innovation Fund for Medical Sciences (CIFMS) (2017-I2M-3-017 and 2023-I2M-C&T-B-042), Non-profit Central Research Institute Fund from Chinese Academy of Medical Sciences (2019XK320030), Peking Natural Science Foundation (L222085), and National High Level Hospital Clinical Research Funding (2022-PUMCH-A-006 and 2022-PUMCH-C-068).

The authors declare no conflict of interests.