Anti-Inflammatory Diet and Probiotic Supplementation as Strategies to Modulate Immune Dysregulation in Autism Spectrum Disorder

Abstract

Background/Objectives: Autism spectrum disorder (ASD) is a neurodevelopmental condition associated with behavioral and cognitive impairments. Increasing evidence also links ASD with systemic immune dysregulation, including abnormal cytokine profiles and chronic low-grade inflammation. Emerging evidence suggests that targeted dietary strategies and probiotic supplementation may modulate immune responses and gut-brain interactions in patients with ASD. This study aimed to evaluate the immunomodulatory effects of a structured anti-inflammatory diet (NeuroGutPlus) compared to multi-strain probiotics in children with ASD. NeuroGutPlus is a nutritionally complete anti-inflammatory dietary protocol that targets gut integrity, inflammation, and mitochondrial function. It includes a diet low in gluten, FODMAPs, casein, and artificial additives, and a high intake of omega-3 fatty acids, polyphenols, and fermentable fibers. Methods: A total of 30 children with ASD and 12 neurotypical controls were enrolled in a 12-week randomized controlled nutritional trial. Participants received either a NeuroGutPlus anti-inflammatory diet, probiotic supplementation (16 strains of Lactobacillus and Bifidobacterium), or no intervention. Plasma levels of 20 cytokines and chemokines were measured pre- and post-intervention using multiplex Luminex immunoassays. Principal component analysis (PCA) was used to explore shifts in the immune profile. Results: Patients treated with the NeuroGutPlus diet significantly reduced IFN-γ levels (p = 0.0090) and showed a stabilizing effect on immune profiles, as evidenced by PCA clustering. Probiotic supplementation led to a significant increase in IL-8 (+66.6 pg/mL; p = 0.0350) and MIP-1β (+74.5 pg/mL; p = 0.0100), along with a decrease in IFN-γ (p = 0.0070), suggesting reconfiguration of innate immune responses. Eight out of eleven biomarkers showed significant post-intervention differences between groups, indicating distinct immunological effects. Conclusions: This study provides evidence that anti-inflammatory diets exert broader and more consistent immunoregulatory effects than probiotics alone in children with ASD. These findings support the inclusion of precision dietary strategies as non-pharmacological interventions to mitigate immune-related dysfunction in patients with ASD.

Keywords: anti-inflammatory diet; autism spectrum disorder; cytokines; immune modulation; nutritional intervention; probiotics.

Figure 1

Study design illustrating dietary and probiotic interventions across ASD and control groups. GE1—children diagnosed with ASD who received a diet designed under the principles of inflammatory restriction (NeuroGutPlus). GE2—neurotypical controls who also received an anti-inflammatory diet NeuroGutPlus. GC1—control group with ASD consuming their usual diet without modifications. GC2—neurotypical control group fed a conventional diet. GE3—participants with ASD who received their usual diet along with probiotics such as Lactobacillus and Bifidobacterium.

Figure 2

Changes in cytokine levels before and after probiotic intervention in children with ASD. Statistical markers were interpreted as * p < 0.05, ** p < 0.01. Each bar graph represents the mean concentration (±standard error) of a specific biomarker before (PRE) and after (POST) intervention.

Figure 3

Reduction in IFN-γ and RANTES levels after standard care in the ASD Control group. Statistical markers were interpreted as * p < 0.05, *** p < 0.001.

Figure 4

Cytokine and chemokine modulation following anti-inflammatory diet in ASD and natural variation in TD controls. Statistical markers were interpreted as * p < 0.05, ** p < 0.01.

Figure 5

Changes in cytokine levels (Δ POST–PRE) were statistically significant (* p < 0.05).

Figure 6

PCA clustering of immune profiles: PRE- and POST-intervention. PC1 captures the main axis of immunological variability, separating groups with high systemic cytokine activation (e.g., ASD Probiotic POST) from those with stable or regulated immune profiles (e.g., TD Control and ASD Diet). PC2 reflects secondary patterns, likely related to subtle differences in cytokine balance and specific immune signaling pathways. Each point represents the global immune profile of a group at a specific time (PRE or POST), with the color indicating the group and shape distinguishing the time point. The relative positions of the points illustrate immunological similarities or divergences across conditions.