Interleukin-18 modulation in autism spectrum disorders

Abstract

Background: Autism spectrum disorder (ASD) is a neurodevelopmental disease which affects 1 in 88 children. Its etiology remains basically unknown, but it is apparent that neuroinflammation is involved in disease development. Great attention has been focused on pro-inflammatory cytokines, and several studies have reported their dysfunction unbalance in serum as well as in the brain. The present work aimed at evaluating putative dysregulation of interleukin-18 (IL-18), a pro-inflammatory cytokine of the IL-1 family in the sera of patients with ASD of different grades, compared to healthy controls, as well as in postmortem brain samples obtained from patients with tuberous sclerosis as well as acute inflammatory diseases. Moreover, quantitative analysis of IL-18 was performed in the sera and brain obtained from Reeler mice, an experimental model of autism.

Methods: Serum IL-18 levels were measured by ELISA. IL-18 was localized by immunohistochemical analysis in brain sections obtained from tuberous sclerosis and encephalitis patients, as well as from gender- and age-matched controls, and in the brain sections of both Reeler and wild-type mice. IL-18 was also quantified by Western blots in homogenates of Reeler and wild-type mice brains. IL-18 binding protein (IL-18BP) was evaluated in Reeler and wild-type mice plasma as well as in their brains (sections and homogenates).

Results: IL-18 content decreased in the sera of patients with autism compared to healthy subjects and in Reeler sera compared to wild-type controls. IL-18 was detected within glial cells and neurons in the brain of subjects affected by tuberous sclerosis and encephalitis whereas in healthy subjects, only a weak IL-18 positivity was detected at the level of glial cells. Western blot identified higher amounts of IL-18 in Reeler brain homogenates compared to wild-type littermates. IL-18BP was expressed in higher amounts in Reeler brain compared to the brain of wild-type mice, whereas no significant difference was detected comparing IL-18BP plasma levels.

Conclusions: IL-18 is dysregulated in ASD patients. Further studies seemed necessary to clarify the molecular details behind IL-18 increase in the brain and IL-18 decrease in the sera of patients. An increase in the size of the patient cohort seems necessary to ascertain whether decreased IL-18 content in the sera can become a predictive biomarker of ASD and whether its measure, in combination with other markers (e.g., increased levels of brain-derived neurotrophic factor (BDNF)), may be included in a diagnostic panel.

Fig. 1

IL-18 serum levels in autism patients and healthy controls. a Values observed in autism patients aged below 10 years were significantly lower compared to those of age- and sex-matched healthy controls (p < 0.0013); b values observed in autism patients above the age of 10 years were significantly lower compared to those of age- and sex-matched healthy controls (p < 0.002)

Fig. 2

Human brain IL-18 immunoreactivity (IR). a–b Control white matter (Wm; a) and gray matter (Gm; b) showing the absence of detectable labeling. c Cortical specimen of a patient with viral encephalitis (herpes simplex encephalitis) with strong IR in astrocytes (arrows in c and inset). d, e Dentate gyrus (DG) of control (d) and hippocampal sclerosis (HS, e) showing increased expression in HS; inset in e shows positive astrocytes; arrows); gcl granular cell layer, ml molecular layer. f–h TSC specimens (cortical tuber) showing IL-18-positive reactive astrocytes (arrows in f; Wm) and giant cells (arrows in g and h). Microglia in (e, f). Sections are counterstained with hematoxylin. Scale bar in h, a–b: 80 μm; d–e: 160 μm; f–h: 40 μm

Fig. 3

Immunolocalization of IL-18 and IL-18BP in the mice brain. Wild-type mice: IL-18-associated positivity. Neurons as well as astrocytes and microglia are stained. a Magnification 4×; b magnification 10×; c magnification 20×; IL-18BP-associated positivity IL-18. d Magnification 10×; e magnification 20×. Reeler mice: much neurons and glial cells are stained, and the reaction is stronger. IL-18-associated positivity. f Magnification 4×; g magnification 10×; h magnification 20×; IL-18BP-associated positivity. i Magnification 10×; l magnification 20×

Fig. 4

Immunolocalization of IL-18 in the mice brain. Negative control a (20×), e (40×). The reaction associated to astrocytes and microglia within wild-type brain (b, c, d) and astrocytes and microglia as well as neural cells within Reeler brain (f, g, h). The h inset depicts positive astrocytes. The positivity was stronger and was detected in higher number of cells within the Reeler brain (b 10×, c 20×, d 40×, f 10×, g 20×, h 40×). Calibration bar 50 μm

Fig. 5

IL-18 and IL-18BP detected by ELISA in the plasma of wild-type and Reeler mice (values expressed as mean ± SD). a IL-18 concentrations (pg/mL) and b IL-18BP concentrations (ng/mL) in Reeler mice versus wild-type group. Plasma IL-18 was reduced in Reeler mice (p = 00.0556). No significant difference was detected in IL-18BP levels in Reeler compared to wild-type mice

Fig. 6

IL-18 and IL-18BP Western blots of mice brain homogenates. Representative Western blot analysis (summarized view corresponding to four animals per group) of IL-18, IL-18BP, and β-actin proteins in cortices of WT and Reeler mice. Bottom: semi-quantitative densitometric analysis, obtained by optical density (OD) of IL-18 (left) and IL-18BP (right) normalized with OD of β-actin bands; N = 11, p < 0.01

Fig. 7

IL-18 and BDNF concentrations (pg/ml) in the sera of autism patients. Autistic patients were classified as severe, based on a Childhood Autism Rating Scale (CARS) score of 37 or more; mild-to-moderate disease as determined by CARS score between 32 and 37; and mild, according to CARS score below 32. An inverse relationship between IL-18 and BDNF was observed in the group of patients with severe autism