Inflammatory mediators drive neuroinflammation in autism spectrum disorder and cerebral palsy

Abstract

Inflammation conditions are associated with autism spectrum disorder (ASD) and cerebral palsy (CP), primarily observed in the peripheral immune system. However, the extent of neuro-inflammation and neuro-immune dysregulation remains poorly studied. In this study, we analyzed the composition of cerebrospinal fluid (CSF) to uncover the inflammatory mediators driving the neuro-immune system in ASD and CP patients. Our findings revealed that ASD patients had elevated levels of four inflammatory cytokines (TNF-α, IL-4, IL-21, and BAFF) compared to controls, while CP patients exhibited increased levels of eight inflammatory cytokines (IFN-γ, GM-CSF, TNF-α, IL-2, IL-4, IL-6, IL-17A and IL-12), one anti-inflammatory cytokine (IL-10), and five growth factors (GFs) (NGF-β, EGF, GDF-15, G-CSF and BMP-9) compared to both controls and ASD patients. Additionally, intrathecal infusion of autologous bone marrow mononuclear cells (BMMNCs) led to a slight decrease in TGF-β and GDF-15 levels in the CSF of ASD and CP patients, respectively. Our study provides new insights into the molecular composition of CSF in ASD and CP patients, with the potential to develop more effective diagnosis methods and improved treatment for these diseases.Clinical trial registration CSF samples used in this study are from clinical trials NCT03225651, NCT05307536, NCT02569775, NCT03123562, NCT02574923, NCT05472428 and previous reports [7, 9, 17-19].

Figure 1

Elevated levels of inflammatory cytokines in CSF from ASD patients. (A) Experiment design. CSF samples were collected at baseline (before the first autologous BMMNC infusion) for CSF composition analysis by Luminex assay. (B) Graphs show the concentration of indicated inflammatory cytokines quantified by Luminex assay from CSF samples of ASD compared to SB and CP patients. Only cytokines significantly increased in the CSF samples from ASD patients compared with SB or CP were shown. Only samples in which cytokine concentration was detected by Luminex assay as indicated in Table 2, were included in the analysis. Groups were compared using ANOVA and Turkey HSD tests. Data were presented as Mean ± SEM for the concentration of indicated cytokines. Considered p-values were: *p < 0.05; **p < 0.01; *** p < 0.001; **** p < 0.0001. p values > 0.05 are not shown.

Figure 2

Elevated levels of inflammatory cytokines in CSF from CP patients. Graphs show the concentration of indicated inflammatory cytokines, including IFN-γ, GM-CSF, IL-2, IL-6, IL-17A, and IL-12, in the CSF samples from CP patients compared to those from ASD and SB patients. Only cytokines significantly increased in the CSF samples from CP patients compared with those from ASD and SB were shown. Only samples in which cytokine concentration was detected by Luminex assay as indicated in Table 2, were included in the analysis. Groups were compared using ANOVA and Turkey HSD tests. Data were presented as Mean ± SEM for the concentration of indicated cytokines. Considered p-values were: *p < 0.05; **p < 0.01; **** p < 0.0001. p values > 0.05 are not shown.

Figure 3

Increased levels of growth factors in CSF from CP patients. Graphs show the concentration of indicated growth factors, including NGF-β, EGF, GDF-15, G-CSF, and BMP-9, in the CSF samples from CP compared to ASD and SB patients. Only growth factors significantly increased in the CSF samples from CP patients compared with those from ASD and SB were shown. Only samples in which cytokine concentration was detected by Luminex assay as indicated in Table 2, were included in the analysis. Groups were compared using ANOVA and Turkey HSD tests. Data were presented as Mean ± SEM for the concentration of indicated cytokines. Considered p-values were: *p < 0.05; **p < 0.01; *** p < 0.001; **** p < 0.0001. p values > 0.05 are not shown.

Figure 4

Changes in cytokines and growth factors in CSF from ASD and CP patients after autologous BMMNC infusion. (A) Experiment design. CSF samples were collected from ASD and CP patients at day 0 as the baseline (before the first BMMNC infusion) and at day 150–360 (after the first BMMNC infusion and before the second BMMNC infusion) for CSF composition analysis by Luminex assay. (B) Graphs show the concentration of 27 analyzed molecules in the CSF from ASD and CP patients at baseline compared with those at 150–360 days after BMMNC infusion. Only samples in which cytokine concentration was detected by Luminex assay were included in the analysis. Groups were compared using paired T-test or Wilcoxon Signed Rank Test. Data were presented as Mean ± SEM for the concentration of indicated cytokines. Considered p-values were: *p < 0.05. p values > 0.05 are not shown.