The Alteration of Salivary Immunoglobulin A in Autism Spectrum Disorders

Abstract

Objectives: Autism spectrum disorders (ASD) are neurodevelopmental disorders with changes in the gut and oral microbiota. Based on the intimate relationship between the oral microbiota and oral mucosal immunity, this study aimed to investigate changes in salivary immunoglobulin A (IgA) level in ASD and the underlying mechanism for any such changes. Methods: We recruited 36 children diagnosed with ASD and 35 normally developing children and measured their salivary IgA content using enzyme-linked immunosorbent assay (ELISA). The valproate (VPA) -treated ASD mouse model was established by prenatal exposure to valproate and mouse salivary IgA content was also quantified by ELISA. The submandibular glands of VPA and control mice were isolated and analyzed using qRT-PCR, immunofluorescence staining, and flow cytometry. ASD-related Streptococci were co-incubated with the human salivary gland (HSG) cell line, and western blotting was used to detect the levels of relevant proteins. Results: We found that salivary IgA content was significantly decreased in patients with ASD and had a significant ASD diagnostic value. The salivary IgA content also decreased in VPA mice and was significantly correlated with autistic-like behaviors among them. The mRNA and protein levels of the polymeric immunoglobulin receptor (Pigr) were downregulated in the submandibular glands of VPA mice and the Pigr mRNA level was positively correlated with mouse salivary IgA content. HSG cells treated with ASD-related Streptococci had reduced PIGR protein level. Conclusion: Therefore, protective IgA levels were reduced in the saliva of individuals with ASD, which correlated with the bacteria-induced downregulation of Pigr in salivary glands. This study suggests a new direction for ASD diagnosis and prevention of oral diseases in ASD cohorts and provides evidence for the ASD mucosal immunophenotype in the oral cavity.

Keywords: Streptococcus mutans; autism spectrum disorders; behavior; immunoglobulin A; mucosal immunity; polymeric immunoglobulin receptor; saliva.

Figure 1

Illustration of the formation of salivary IgA. IgA, immunoglobulin A; PIGR, polymeric immunoglobulin receptor.

Figure 2

The outline of animal experiments. VPA, valproate acid; E, embryonic day; PND, postnatal day.

Figure 3

The salivary immunoglobulin A (IgA) content in human saliva samples and the diagnostic value of human salivary IgA in Autism Spectrum Disorders (ASD). (A) The IgA content (presented as optical density at 450 nm) in the saliva of the typically developing group and ASD children was determined by enzyme-linked immunosorbent assay. The above data are presented as the mean ± SEM, n = 35–36 per group, two replicate experiments, each data point represents results from an individual child, Student's t-test, ** denotes p < 0.01. (B) The receiver operator characteristic (ROC) curve and the area under the ROC curve (AUC) of the salivary IgA in ASD diagnosis. The arrow indicates the optimal cutoff point of salivary IgA.

Figure 4

The salivary immunoglobulin A (IgA) content in mouse saliva samples and its correlation with the mouse autistic-like behaviors. (A) In the three-chamber social performance test, the durations of valproate acid (VPA) mice and control mice interacted with the object, Stranger 1 and Stranger 2 in phase 2 and phase 3, and the time they spent in each chamber were recorded. According to which the sociability index and social preference index were calculated (n = 9–11 per group, Mann-Whitney U-test and Student's t-test). (B) The time VPA mice and control mice spent in the central zone and the frequency of them entering the central zone in the open field test (n = 9–11 per group, Student's t-test). (C) The time VPA mice and control mice spent in the lightroom in the light-dark test (n = 9–11 per group, Student's t-test). (D) The time VPA mice and control mice spent in self-grooming (n = 9–11 per group, Student's t-test). (E) A representative picture of the “kink” in the tails of VPA mice. (F) The IgA content (presented as optical density at 450 nm) of mouse saliva samples was determined by enzyme-linked immunosorbent assay (n = 6 per group, two replicate experiments, Student's t-test). The above data are presented as the mean ± SEM, * denotes p < 0.05, ** denotes p < 0.01. (G) The Pearson's correlation analysis of salivary IgA content and mouse autistic-like behaviors, including self-grooming time, time spent in the central zone, central zone entries, time spent in the lightroom, and phase 3 Stranger 2 interaction time.

Figure 5

The polymeric immunoglobulin receptor (Pigr) levels in mouse submandibular glands and the correlation of which with the mouse salivary immunoglobulin A (IgA) content. (A) The qRT-PCR result of the relative mRNA level of Pigr in the submandibular glands of valproate acid (VPA) mice and control mice (n = 5 per group, Mann-Whitney U-test). (B) The Pearson's correlation analysis of the relative Pigr mRNA level and mouse salivary IgA content. (C) The PIGR protein level in the submandibular glands of VPA mice and control mice confirming by immunofluorescence staining and the quantification results (measuring with fluorescence intensity at 400× magnification, n = 5 per group, Student's t-test). Representative images of similar results are shown. (D) The qRT-PCR results of the relative mRNA levels of genes regulating IgA class-switch recombination, IgA synthesis, and the IgA response in the submandibular glands of VPA mice and control mice (n = 5 per group, Mann-Whitney U-test and Student's t-test). (E) The IgA content (presented as optical density at 450 nm) of mouse serum samples was measured by enzyme-linked immunosorbent assay (n = 5–6 per group, two replicate experiments, Mann-Whitney U-test). (F) Flow cytometry results showed the percentage of IgA-producing lymphocytes in the submandibular glands of VPA mice and control mice (n = 5–6 per group, Mann-Whitney U-test). The above data are presented as the mean ± SEM, * denotes p < 0.05, ** denotes p < 0.01.

Figure 6

The effect of Autism spectrum disorders (ASD) -related Streptococci on the polymeric immunoglobulin receptor (PIGR) protein level and the NF-κB signaling pathway in the human salivary gland (HSG) cells. (A) Cytokeratin 14 and PIGR expression in HSG cells was validated by immunofluorescent staining. Representative images of similar results are shown. (B) Control group: normal HSG cells; +BHI group: HSG cells treated with blank brain heart infusion broth; +S. mutans group: HSG cells co-incubated with heat-killed Streptococcus mutans. Western blotting was used to determine the protein levels of PIGR, NF-κB, and phosphorylated NF-κB (p-NF-κB). Representative pictures of western blots with similar results and the quantification of protein levels are shown. The above data are presented as the mean ± SEM, n = 3 per group, One-way ANOVA, * denotes p < 0.05 ** denotes p < 0.01.