Distinct Microbiome-Neuroimmune Signatures Correlate With Functional Abdominal Pain in Children With Autism Spectrum Disorder

Abstract

Background & aims: Emerging data on the gut microbiome in autism spectrum disorder (ASD) suggest that altered host-microbe interactions may contribute to disease symptoms. Although gut microbial communities in children with ASD are reported to differ from individuals with neurotypical development, it is not known whether these bacteria induce pathogenic neuroimmune signals.

Methods: Because commensal clostridia interactions with the intestinal mucosa can regulate disease-associated cytokine and serotonergic pathways in animal models, we evaluated whether microbiome-neuroimmune profiles (from rectal biopsy specimens and blood) differed in ASD children with functional gastrointestinal disorders (ASD-FGID, n = 14) compared with neurotypical (NT) children with FGID (NT-FGID, n = 15) and without abdominal pain (NT, n = 6). Microbial 16S ribosomal DNA community signatures, cytokines, and serotonergic metabolites were quantified and correlated with gastrointestinal symptoms.

Results: A significant increase in several mucosa-associated Clostridiales was observed in ASD-FGID, whereas marked decreases in Dorea and Blautia, as well as Sutterella, were evident. Stratification by abdominal pain showed multiple organisms in ASD-FGID that correlated significantly with cytokines (interleukin [IL]6, IL1, IL17A, and interferon-γ). Group comparisons showed that IL6 and tryptophan release by mucosal biopsy specimens was highest in ASD children with abdominal pain, whereas serotonergic metabolites generally were increased in children with FGIDs. Furthermore, proinflammatory cytokines correlated significantly with several Clostridiales previously reported to associate with ASD, as did tryptophan and serotonin.

Conclusions: Our findings identify distinctive mucosal microbial signatures in ASD children with FGID that correlate with cytokine and tryptophan homeostasis. Future studies are needed to establish whether these disease-associated Clostridiales species confer early pathogenic signals in children with ASD and FGID.

Keywords: 5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, serotonin; ASD, autism spectrum disorder; FGID, functional gastrointestinal disorder; GI, gastrointestinal; GM-CSF, granulocyte-macrophage colony-stimulating factor; GROα, growth-related oncogene alpha; Gastrointestinal Disorders; IBS, irritable bowel syndrome; IFN, interferon; IL, interleukin; IP, interferon gamma-induced protein; MCP-1, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; Microbiome; Microbiome–Gut–Brain Axis; Mucosa; NT, neurotypical; OTU, operational taxonomic unit; QPGS-RIII, Questionnaire on Pediatric Gastrointestinal Symptoms-Rome III; Serotonin; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor.

Graphical abstract

Figure 1

Separate clusters for the ASD-FGID (blue squares), NT-FGID (green triangles), and NT (orange circles) groups are evident via principal component analysis.

Figure 2

Microbiome profiles per group (ASD-FGID, NT-FGID, and NT) show subtle differences in overall composition at the order level (left). Greater differentiation between the groups was observed when comparisons were made at the OTU level. Right: Clostridiales OTUs that were further identified to the species level. The separation of the ASD-FGID group from the NT-FGID and NT groups was highly driven by both increases and decreases in specific Clostridiales.

Figure 3

Differences in OTUs related to specific bacteria associated with ASD were identified in the mucosal microbial community. Overall, an increase in Clostridiales (C lituseburense, P = .002; L bolteae, P = .017; L hathewayi, P = .030; C aldenense, P = .038; and F plautii, P = .038) was seen in the ASD-FGID group compared with the NT-FGID and NT groups. A decrease in relative abundance in the ASD-FGID group was seen for D formicigenerans (P =.006), B luti (P = .020), and Sutterella species (P = .025). Plots depict the maximum and minimum (whiskers), upper and lower quartile limits (box), and median (horizontal line).

Figure 4

Bacteria associated with pain in ASD-FGID were identified. When comparing across all groups, a significant increase in OTUs identified as C aldenense (P = .004), C disporicum (P = .049), C lituseburense (P = .003), C tertium (P = .045), F plautii (P = .007), and T sanguinis (P = .033) was noted in the cohort of ASD children in whom abdominal pain had been reported. Comparisons solely between the 2 ASD groups, with and without abdominal pain, also showed 2 additional organisms of interest that were increased in the ASD pain group: P excrementihominis (P = .037) and T nexilis (P = .046). Plots depict the maximum and minimum (whiskers), upper and lower quartile limits (box), and median (horizontal line).

Figure 5

The identification of distinct microbial profiles of the NT-FGID, ASD-FGID, and ASD-FGID with abdominal pain groups was influenced greatly by the differences in relative abundances of the organisms listed (left). Several of these bacteria also were found to correlate with the serotonin pathway (center), as well as with multiple cytokines (right).