Behavioral Changes in Mice Lacking Interleukin-33

Abstract

Interleukin (IL)-33 is a member of the IL-1 family of cytokines. IL-33 is expressed in nuclei and secreted as alarmin upon cellular damage to deliver a danger signal to the surrounding cells. Previous studies showed that IL-33 is expressed in the brain and that it is involved in neuroinflammatory and neurodegenerative processes in both humans and rodents. Nevertheless, the role of IL-33 in physiological brain function and behavior remains unclear. Here, we have investigated the behaviors of mice lacking IL-33 (Il33^-/- mice). IL-33 is constitutively expressed throughout the adult mouse brain, mainly in oligodendrocyte-lineage cells and astrocytes. Notably, Il33^-/- mice exhibited reduced anxiety-like behaviors in the elevated plus maze (EPM) and the open field test (OFT), as well as deficits in social novelty recognition, despite their intact sociability, in the three-chamber social interaction test. The immunoreactivity of c-Fos proteins, an indicator of neuronal activity, was altered in several brain regions implicated in anxiety-related behaviors, such as the medial prefrontal cortex (mPFC), amygdala, and piriform cortex (PCX), in Il33^-/- mice after the EPM. Altered c-Fos immunoreactivity in Il33^-/- mice was not correlated with IL-33 expression in wild-type (WT) mice nor was IL-33 expression affected by the EPM in WT mice. Thus, our study has revealed that Il33^-/- mice exhibit multiple behavioral deficits, such as reduced anxiety and impaired social recognition. Our findings also indicate that IL-33 may regulate the development and/or maturation of neuronal circuits, rather than control neuronal activities in adult brains.

Keywords: IL-33; anxiety; astrocytes; cytokines; oligodendrocytes; social behavior.

Graphical abstract

Figure 1.

IL-33 expression patterns in the adult mouse brain. A, Validation of specificity of IL-33 immunostaining using brain sections (CC) from WT and Il33 ^−/− mice. B, Western blot analysis of IL-33 expression in the cortex from WT and Il33 ^−/− mice. β-actin was used as an internal control for protein loading. C, Absence of IL-33 proteins in the peripheral blood of WT mice. The levels of serum IL-33 proteins were below the detection threshold of ELISA assay (15.6 pg/ml). N.D., not detected. D, Schematic illustration of the distribution of IL-33-expressing cells. The number of IL-33-expressing cells was quantified at 20× under a fluorescence microscope. Light, medium, and dark green correspond to 0–25, 26–100, and 101+ cells per field, respectively. Small red boxes indicate the area analyzed in E–K and Figs. 3, 4A–D : a, mPFC; b, secondary motor cortex (M2); c, CC; d, PCX; e, primary S1BF; f, Pe; g, CeA; h, BLA; i, CoA; j, ventral hippocampus (vHip); k, vDG. E, Representative pictures of IL-33 expression in several brain regions. Arrows, IL-33⁺ astrocytes (S100β⁺ cells); arrowheads, IL-33⁺ oligodendrocyte-lineage cells (Olig2⁺ cells). F, The average nuclear intensity of IL-33 between Olig2⁺ cells and S100β⁺ cells in the M2. A.U., arbitrary unit. G, Percentages of S100β⁺, Olig2⁺, and other cells among IL-33⁺ cells in each brain region. H, Comparison of IL-33 expression in Olig2⁺ cells across brain regions. I, Comparison of IL-33 expression in S100β⁺ cells across brain regions. We also observed Olig2⁺ and S100β⁺ cells, but these cells are not included in this graph. J, No colocalization of IL-33 signals to neurons (NeuN⁺ cells) or microglia (Iba1⁺ cells) in most brain regions. Representative pictures of the M2 are shown. K, IL-33 colocalization to neurons in the granular layer of vDG. Scale bar, 30 µm. Each bar represents mean ± SEM; *p < 0.05, **p < 0.01 (Student’s t test, one-way ANOVA with post hoc Tukey’s test, and Kruskal–Wallis test with post hoc Dunnett’s test; see Table 2 for the detail of statistical analysis).

Figure 2.

Reduced anxiety-like behavior in Il33 ^−/− mice. A, Increased open arm entries into the EPM. B, Increased time spent in the open arms in the EPM. C, No difference in total entries between WT and Il33 ^−/− mice in the EPM. WT mice, n = 12; Il33 ^−/− mice, n = 9. D, Increased time spent in the center during the OFT. WT mice, n = 9; Il33 ^−/− mice, n = 7. Each bar represents mean ± SEM; n.s, not significant; **p < 0.01 (Student’s t test; see Table 2 for the detail of statistical analysis).

Figure 3.

Altered c-Fos immunoreactivity in brain regions related to anxiety in Il33 ^−/− mice. A, Representative images of c-Fos expression in NeuN⁺ neurons. Location of each brain region is indicated in Fig. 1D [a, mPFC; b, M2; d, PCX; e, S1BF; g, CeA; h, BLA; i, CoA; k, ventral hippocampus (vHip)]. B, Quantification of c-Fos expression in various brain regions. Percentages of c-Fos-expressing neurons among all neurons (NeuN⁺ cells) were calculated and compared between WT and Il33 ^−/− mice (n = 3-4). Scale bar, 100 µm. Each bar represents mean ± SEM; n.s., not significant; *p < 0.05, **p < 0.01 (Student’s t test; see Table 2 for the detail of statistical analysis).

Figure 4.

Relationship between IL-33 expression and c-Fos immunoreactivity. A, Correlation between the number of IL-33⁺ cells per field in WT mice and fold change of % c-Fos⁺ neurons (NeuN⁺ cells) per field in WT mice normalized with average % c-Fos⁺ neurons per field in Il33 ^−/− mice. Each dot refers to the brain region of an individual mouse (WT mice, n = 5; Il33^−/− mice, n = 5). B, Correlation between the % c-Fos⁺ neurons per field in WT mice and the number of IL-33⁺ cells per field in WT mice based on c-Fos elevated brain regions in Il33^−/− mice after EPM. Each dot refers to the brain region of an individual mouse. C, D, No difference in IL-33 expression in the mPFC and ventral hippocampus (vHip) between WT mice with and without the EPM (EPM⁺ and EPM^–). E, No difference in the Il33 mRNA expression in the fontal cortex between EPM⁺ and EPM^-. EPM⁺ n = 4, EPM^– n = 4. A.U., arbitrary unit. Each bar represents mean ± SEM; n.s., not significant (Pearson correlation, Student’s t test; see Table 1 for the detail of statistical analysis).

Figure 5.

Altered social behaviors in Il33 ^−/− mice. A, No significant difference in sociability between WT and Il33 ^−/− mice. (F_{genotype x chamber(1,22)} = 0.9178, p = 0.3485; F_{genotype(1,22)} = 0.09669, p = 0.7588; F_{Chamber(1,22)} = 54.44, p < 0.001). Both WT and Il33 ^−/− mice preferred mice to objects (**p < 0.01, post hoc Sidak test). B, Preference index data for sociability behaviors (p = 0.3587, Student’s t test). C, Reduced preference to novel mice in Il33 ^−/− mice. WT and Il33 ^−/− mice differed significantly in preference trial (F_{genotype x chamber(1,22)} = 4.681, p < 0.05; F_{genotype(1,22)} = 0.00224, p = 0.8824; F_{Chamber(1,22)} = 26.34; p < 0.0001). Only WT mice showed a significant preference to novel mice (**p < 0.01, post hoc Sidak test). D, Preference index data for social novelty preference. Il33 ^−/− group showed a significantly lower preference index than the WT group (*p < 0.05, Student’s t test). Each bar represents mean ± SEM; n.s., not significant. See Table 2 for the detail of statistical analysis.