Immune system dysfunction and inflammation in aging Shank3b mutant mice, a model of autism spectrum disorder

Abstract

Introduction: Autism spectrum disorder (ASD) is a heterogeneous group of neurodevelopmental Q8 conditions characterized by deficits in social interaction/communication and restrictive/repetitive behaviors. Recent studies highlight the role of immune system dysfunction and inflammation in ASD pathophysiology. Indeed, elevated levels of pro-inflammatory cytokines were described in the brain and peripheral blood of ASD individuals. Despite this, how this pro-inflammatory profile evolves with aging and whether it may be associated with behavioral deficits is unknown. In this work, we explored the impact of aging on motor behavior and inflammation using Shank3b mutant mice, a model for syndromic ASD.

Methods: Using RT-qPCR and flow cytometry, we examined the expression of key pro-inflammatory molecules in the cerebellum, bone marrow, spleen, and peripheral blood, comparing adult and old Shank3b ^+/+, Shank3b ^+/-, and Shank3b ^-/- mice.

Results and discussion: Our findings revealed genotype- and age-related differences in inflammation and motor behavior, with Shank3b^-/- mice exhibiting accelerated aging and motor impairments. Correlations between pro-inflammatory molecules and behavioral deficits suggest that a link may be present between systemic inflammation and ASD-related behaviors, underscoring the potential role of age-related inflammation ("inflammaging") in exacerbating ASD symptoms.

Keywords: bone marrow; brain; cerebellum; inflammaging; neurodevelopmental disorder; spleen.

Figure 1

Molecules related to inflammation and damage in the cerebellum of Shank3b ^+/+, Shank3b ^+/-, and Shank3b ^-/- mice. mRNA expression of (A) IFNγ, (B) IL-6, (C) TNF, (D) MMP3, (E) IL-1β, (F) CCL2, (G) CCL3, and (H) NRF2 in the cerebellum (Cb) of adult and old Shank3b^+/+, Shank3b^+/- and Shank3b^-/- mice measured using RT-qPCR. Two-way ANOVA, Tukey post-hoc test. n = 8 per group. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Figure 2

Molecules related to inflammation and damage in the bone marrow of Shank3b ^+/+, Shank3b ^+/-, and Shank3b ^-/- mice. mRNA expression of (A) IFNγ, (B) IL-6, (C) TNF, (D) MMP3, (E) IL-1β, (F) CCL2, (G) CCL3, and (H) NRF2 in the bone marrow (BM) of adult and old Shank3b^+/+, Shank3b^+/- and Shank3b^-/- mice measured using RT-qPCR. Two-way ANOVA, Tukey post-hoc test. n = 8 per group. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Figure 3

Molecules related to inflammation and damage in the spleen of Shank3b ^+/+, Shank3b ^+/-, and Shank3b ^-/- mice. mRNA expression of (A) IFNγ, (B) IL-6, (C) TNF, (D) MMP3, (E) IL-1β, (F) CCL2, (G) CCL3, and (H) NRF2 in the spleen of adult and old Shank3b^+/+, Shank3b^+/- and Shank3b^-/- mice measured using RT-qPCR. Two-way ANOVA, Tukey post-hoc test. n = 8 per group. *p<0.05; *** p<0.001; ****p<0.0001.

Figure 4

The expression pattern of pro-inflammatory cytokines varies with age, according to a genotype- and tissue- dependent logic. a-c, left panels) Heatmaps showing the mean fluorescence cytokine intensity within lymphocytes, CD14⁺ cells, T cells, CD8⁺ and CD4⁺ T cells (defined as shown in Supplementary Figure S1 ) of adult and old Shank3b^+/+, Shank3b^+/- and Shank3b^-/- mice in the bone marrow, spleen and peripheral blood. (A-C), right panels) Scatter plot showing the clustering of the six experimental groups obtained with the discriminant analysis. Two-way ANOVA, Tukey post-hoc test. Adult mice (shown with triangles): n=12 (Shank3b^+/+ ), n=12 (Shank3b^+/ ) ^- , n=12 (Shank3b^-/- ). Old mice (shown with dots): n=14 (Shank3b^+/+ ), n=18 (Shank3b^+/ ) ^- , n=14 (Shank3b^-/- ). Comparisons with p<0.05 are reported in green in the right panels.

Figure 5

Motor behavior in Shank3b ^+/+, Shank3b ^+/-, and Shank3b ^-/- mice. Average speed (cm/s) (A) and distance travelled (B) in the open field test in adult and old Shank3b^+/+ , Shank3b^+/- and Shank3b^-/- mice. (B) Average time on rotarod (latency to fall, s) in the rotarod test. (C) Number of marbles buried in the marble burying test. Two-way ANOVA, Tukey post-hoc test. Adult mice: n=12 (Shank3b^+/+ ), n=12 (Shank3b^+/ ) ^- , n=12 (Shank3b^-/- ). Old mice: n=14 (Shank3b^+/+ ), n=18 (Shank3b^+/ ) ^- , n=14 (Shank3b^-/- ). *p<0.05; *** p<0.001; ****p<0.0001.

Figure 6

Relationship between pro-inflammatory molecules in the BM, spleen, PB and cerebellum and motor behavior. Correlation between IFNγ mean fluorescence intensity (MFI) and time spent moving in the open field (OF) test in adult and old mice in (A) BM, (B) spleen, (C) PB, and (D) cerebellum. Spearman correlation, adult mice n=36, old mice n= 46. Spearman correlation coefficient (r_S) and p values are shown in the graphs.