Altered stool cytokine profiles and pro-onflammatory/anti-inflammatory imbalance in children with autism spectrum disorder : a developmental analysis

Finderle, Petra (author), Jekovec-Vrhovšek, Maja (author), Prosenc Zmrzljak, Uršula (author), Osredkar, Damjan (author), Avguštin, Gorazd (author), Osredkar, Joško (author)

Background: Immune dysregulation and gut dysbiosis are increasingly implicated in autism spectrum disorder (ASD), but compartment-specific intestinal cytokine profiles remain poorly defined. Aim: To characterize stool cytokine profiles and pro-/anti-inflammatory balance in children with ASD across development. Methods: We analyzed stool samples from 283 children (109 controls, 104 mild ASD, 70 severe ASD; age 0.9–21.5 years) recruited at a tertiary centre. Nine cytokines (IFN-γ, IL-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-17, TNF-α) were measured using a Luminex multi-plex assay; IL-15 was excluded due to >70% missing values. Group comparisons used Mann–Whitney U tests, with age stratification at the cohort median (≤9.5 vs. >9.5 years). A composite pro-/anti-inflammatory ratio (IL-1α + IL-1β + IL-6 + IL-8 + IL-17 + TNF-α + IFN-γ divided by IL-4 + IL-10) was calculated. Results: In the overall cohort, stool IL-8 and IL-4 were significantly decreased in ASD versus controls (IL-8: median 0.36 vs. 0.49 ng/L, p = 0.0041; IL-4: 0.28 vs. 0.30 ng/L, p = 0.0316), with a graded reduction from controls to mild and severe ASD. Age-stratified analysis revealed that IL-8 reduction was confined to younger children (≤9.5 years; p = 0.0025) and absent in older children, while IL-1β was significantly reduced in younger ASD children and tended to reverse in older ASD children. The pro-/anti-inflammatory ratio was markedly elevated in severe ASD (median 491 vs. 209 in controls; p = 0.059), particularly in older children. Stool IL-8 and IL-1β correlated inversely with CARS scores within the ASD group. Conclusions: Children with ASD show decreased stool IL-8 and IL-4 and a shift toward a pro-inflammatory cytokine balance, with the strongest alterations during early childhood. These findings are consistent with the hypothesis of a developmental window of intestinal immune dysregulation in ASD, with stool IL-8 as the primary FDR-corrected finding. The present cross-sectional data do not establish causality and independent replication is required before clinical conclusions are drawn.

• Language: English
• Keywords: autism spectrum disorder, cytokines, stool biomarkers, IL-8, IL-4, interleukin, gut–immune axis, CARS, developmental immunology
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: FFA - Faculty of Pharmacy; MF - Faculty of Medicine; BF - Biotechnical Faculty
• Publication status: Published
• Publication version: Version of Record
• Year: 2026
• Number of pages: 24 str.
• Numbering: Vol. 14, iss. 7, art. 1559
• PID: 20.500.12556/RUL-185225
• UDC: 616.896+616-097
• ISSN on article: 2227-9059
• DOI: 10.3390/biomedicines14071559
• COBISS.SI-ID: 286211331
• Publication date in RUL: 29.07.2026

Record is a part of a journal

• Title: Biomedicines
• Shortened title: Biomedicines
• Publisher: MDPI
• ISSN: 2227-9059
• COBISS.SI-ID: 523006745

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: biomarkerji v blatu, IL-8, IL-4, interlevkin, črevesno-imunska os, CARS, razvojna imunologija, motnje avtističnega spektra, citokini, imunologija

Projects

• Funder: ARRS - Slovenian Research Agency
• Project number: P3-0124
• Name: Metabolni in prirojeni dejavniki reproduktivnega zdravja, porod III

• Funder: ARRS - Slovenian Research Agency
• Project number: J3-1756
• Name: Okoljski in genetski dejavniki pri motnjah avtističnega spektra