Overall structure of GM of male and female BTBR mice

We analyzed sex-related profiles of GM in BTBR mice, a known mouse model of autism to gain insights into relationship between autistic behavior and dysbiosis. Fecal microbiota of fully symptomatic, 12 months old, female and male BTBR (fBTBR and mBTBR, respectively; n = 6 mice each group) and female and male C57 control mice of same age (fC57 and mC57, respectively; n = 6 mice each group;) was analyzed by next generation sequencing (NGS) technology using the Illumina Miseq system. V3–V4 variable regions of the 16S rRNA gene were amplified and sequenced to characterize total bacterial population; 62,009.83 ± 33,665.39 high-quality sequences/sample were obtained from all 24 fecal samples, representing 3,250 operational taxonomic units (OTUs). The results shown were obtained considering a depth of 32,288 sequences/sample clustered in 2,740 OTUs; Good’s coverage > of 99.3% for all sequences in the four groups indicated good sequencing depth for reliable investigation of differences in fecal microbiota between BTBR and control mice. Among the 2,740 OTUs detected across any of the samples, 245 OTUs discriminated between fBTBR and fC57 mice, while 167 discriminated between mBTBR and mC57 mice. Discriminant OTUs were identified using two complementary analyses, LEfSe algorithm and Metastats comparison (Tables S1 and S2).

We evaluated ecological features of fecal bacterial communities in fBTBR and mBTBR compared to those of control groups. No significant differences in species richness (number of OTUs) and degree of homogeneity abundance of the species (Shannon index) were observed between groups (data not shown), while strong differences in phylogenetic assortment were detected comparing fBTBR and mBTBR with their respective controls (Fig. 1). Phylogenetic distances among samples were assessed by means of Unweighted Unifrac distance metrics, a qualitative phylogenetic measure that considers the presence/absence of a taxon. ANOSIM R statistic revealed a difference in gut bacterial assortment between BTBR of both sexes and their respective controls, with fBTBR vs fC57 displaying a higher R value compared to mBTBR vs mC57. This effect was evident in the PCoA plot, where fBTBR samples clustered to the extreme right of the plot, while mBTBR samples were positioned midway between fBTBR and control samples of both sexes (Fig. 1A, left plot).

Figure 1: Female and male BTBR mice exhibit an altered gut microbial composition. (A) Unweighted UniFrac-based 3D PCoA plot constructed on all OTUs (32,288 reads per sample, left) or all OTUs without Bacteroidetes and Firmicutes reads (850 reads per sample, right) of fecal community of BTBR and C57 mice of both sexes. Analysis of similarity (ANOSIM) with 999 permutations was used to detect the statistical significant differences in microbial community composition between fBTBR and mBTBR compared to their controls (fC57 and mC57); on the top of plots are reported both R statistics and p values. (B) Relative abundance of all identified OTUs classified at phylum level. Mean values ± SEM are plotted (n = 6/group). Significant differences are indicated by *p < 0.05 and **p < 0.01 for comparison of fBTBR vs. fC57 and mBTBR vs. mC57. Abbreviations: fBTBR (BTBR female mice); mBTBR (BTBR male mice); fC57 (C57 female mice); mC57 (C57 male mice). Full size image

Sequencing data revealed that 89.1% of total reads were taxonomically classified in Bacteroidetes and Firmicutes phyla, and the majority of discriminatory OTUs, both in females and males, were classified in these phyla (Tables S1 and S2). The impact of these taxa was evident when the Unweighted Unifrac analysis was repeated after negative filtering of these phyla from total sequences. After subtraction, ANOSIM analysis on remaining OTUs revealed a weaker grouping level among samples (Fig. 1A, right plot), indicating that Bacteroidetes and Firmicutes were the principal contributors to the BTBR and C57 GM differences both in female and male mice.

GM profiling of BTBR female and male mice

Over the total of 9 bacterial phyla identified, comparison of mean abundances (by nonparametric Kruskal-Wallis test) of primary (Bacteroidetes, Firmicutes) and most of the less abundant phyla showed no significant differences between fBTBR and mBTBR compared to their respective controls (Fig. 1B). Exception was made for Proteobacteria which were found significantly more abundant in fBTBR with respect to fC57 (relative abundance 13.2% ± 2.6% and 4.7% ± 1.2%, respectively), and for TM7 phylum which was found significantly less abundant in fBTBR with respect to fC57 (relative abundance of 0.1% ± 0.04% in fBTBR and 0.3% ± 0.05% in fC57). No significant differences were found for all phyla identified in mBTBR compared to mC57 (Fig. 1B). Gram-negative Proteobacteria include lots of gut commensal species with potential pathogenic features, as the lipopolysaccharide expression. Although comparison between animal model and human disease needs to be under great scrutiny, given the known heterogeneity of ASD in both clinical and basic aspects, it is interesting to note that several families among Proteobacteria were found in the intestine of children with autism and gastrointestinal disturbances9. Even though the role of Proteobacteria in the pathogenesis of autism remains unclear, it is possible that alteration of commensal microbes could activate Proteobacteria as infectious agents for example through their ability to trigger immunological response.

Key phylotypes driving GM profiles of male and female BTBR mice

In order to identify GM key phylotypes responsible for differences between fBTBR and mBTBR compared to controls we applied LEfSe algorithm. 17/30 key genera were found within Bacteroidetes and Firmicutes phyla, mainly within Bacteroidales and Clostridiales orders, even though the comparison of the relative abundances of these phyla did not show significant differences (Fig. 1B). This result confirmed that Bacteroidetes and Firmicutes taxa reassortment mainly marks the differences in the GM between BTBR and C57 mice in both sexes accordingly to ANOSIM results on Unweighted Unifrac analysis (Fig. 1A). Alteration of Clostridial and Bacteroidial OTUs was found to drive the major changes in GM composition also in maternal immune activation (MIA) model of autism16. Among key genera with relative abundance >0.1%, Bacteroides and Parabacteroides (order Bacteroidales) were significantly more abundant in BTBR female and male compared to control mice (Table 1). Conversely, the genus Dehalobacterium (order Clostridiales) was significantly less abundant in BTBR mice of both sexes compared to controls (Table 1). Notably, the identified differences in relative abundance of Bacteroides and Parabacteroides were more pronounced in fBTBR as indicated by fold change (Table 1). Bacteroides and Parabacteroides genera are producers of propionic acid as endproduct of their metabolism and previous studies reported that intraventricular injection of propionate in rats induced pathologic changes characteristic of ASD33. Moreover these microorganisms are lipopolysaccharide producers, and high levels of serum enodotoxin were detected in autistic patients34. Finegold et al.8 also detected high levels of Bacteroides (Bacteroides vulgatus) in fecal samples of severely autistic children8.

Table 1 Relative abundance of key genera discriminating female and male BTBR from their sex-matched control mice. Full size table

Among the other key genera, Prevotella, Coprobacillus, Sutterella, Akkermansia (muciniphila) and unclassified members of Desulfovibrionaceae and Enterobacteriaceae significantly increased, while Oscillospira and members of Rikenellaceae and TM7 (AF12 and U. F16, respectively) significantly decreased in fBTBR, possibly driving a female-specific microbial signature in BTBR mice. Key genera specifically altered in mBTBR were Lactobacillus, Ruminococcus, Desulfovibrio and unclassified member of Helicobacteriaceae (Table 1). The alteration in the abundance of key genera that we mainly found in fBTBR was consistent, for a subset of specific taxa, with previously reported data both in human ASD patients and in a different murine model of ASD9,10,13,15. Genus Sutterella, within Proteobacteria, was found to be elevated both in feces of ASD children and in intestinal biopsies from children with autism and intestinal disturbances35,36. Furthermore, high levels of Akkermansia muciniphila were reported in BTBR fed a chow diet, while the ketogenic diet resulted in a normalization of its levels in association with improvement of behavioral symptoms37. Genus Oscillospira accounted for 13.7% in fC57 and was found significantly reduced to 5.4% in fBTBR. Oscillospira, recenlty identified by 16S RNA sequencing, is considered an enigmatic component of the human gut microbiota and, possibly based on butyrate production it was positively associated with human health38. Interestingly butyrate has recently been shown to improve repetitive behavior in BTBR mice39, thus it would be of interest to functionally characterize Oscillospira, and individual species in this genus, to examine their impact on autistic phenotype.

Correlations between GM alterations and behavioral phenotype, gut permeability and cytokine expression in female and male BTBR mice

Along with description of BTBR GM profile we investigated the possible correlation between the levels of specific bacterial taxa and peculiar pathological traits dysregulated in ASD patients such as behavioral abnormalities, gut permeability and immune abnormalities5,40.

Three chamber paradigm, marble burying and spontaneous self-grooming tests were used to evaluate social, stereotyped and repetitive behavior in control and BTBR mice. For all behavioral tests, both female and male BTBR mice showed deficits compared to controls, however a significant sex-related alteration was observed with fBTBR displaying higher self-grooming scores (Fig. 2A–C).

Figure 2: Analysis of social interaction, stereotyped and repetitive behavior in female and male BTBR mice. (A) Three-chamber social interaction test showing time spent in each chamber by BTBR and C57 mice of both sexes (n = 12/group; p = 0.7424 for chamber, p = 0.0077 for strain, p = 0.7761 for sex, p < 0.0001 for chamber x strain and p = 0.1671 for chamber x strain x sex, by three-way ANOVA). (B) Number of buried marble by BTBR and C57 mice of both sexes after a 30 min testing session (n = 12/group; p < 0.0001 for strain, p = 0.1322 for sex and p = 0.2689 for strain x sex, by two-way ANOVA). (C) Seconds spent in repetitive grooming measured for BTBR and C57 mice of both sexes during 10 min test session (n = 12/group; p < 0.0001 for strain, p = 0.0372 for sex, and p = 0.0269 for strain x sex, by two-way ANOVA). Significant differences are indicated by *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 using Bonferroni post-hoc tests following three-way ANOVA with chamber, strain and sex as factors (A) or two-way ANOVA with strain and sex as factors (B and C). Abbreviations: fBTBR (BTBR female mice); mBTBR (BTBR male mice); fC57 (C57 female mice); mC57 (C57 male mice). Data are shown as mean values ± SEM. Full size image

Alteration of gut permeability, as evidenced by increased FITC-dextran translocation across the intestinal epithelium into blood, was observed both in female and male BTBR mice (Fig. 3A). Consistent with deficit in intestinal barrier integrity, a significant reduction of occludin and zonuline mRNA levels was detected in colon of male BTBR mice and a similar trend was observed in females (Fig. 3B). In addition, expression of a subset of cytokines (TNF alpha, IL-6 and IL-10) and CD11c integrin was determined in colon tissue of male and female BTBR and C57 mice. Increased expression of TNF alpha was observed in BTBR mice of both sexes (Fig. 3C). Significant increase of IL-6 and CD11c was observed in mBTBR compared to both sex-mached controls and fBTBR (Fig. 3C). These results indicate that, although with different marks, BTBR mice of both sexes present an increased gut permeability and altered cytokines pattern in colon tissue. In addition, histological evaluation of colon tissues showed tissue damage and evident inflammatory cells infiltration in both mBTBR and fBTBR (Fig. 4).

Figure 3: Analysis of intestinal integrity and inflammation levels in female and male BTBR mice. (A) Intestinal epithelial permeability to fluorescein isothiocyanate (FITC)-dextran 4 kDa of BTBR and C57 mice of both sexes. Data are represented as plasma concentration of FITC dextran (nM) (n = 5/group; p = 0.0002 for strain, p = 0.0469 for sex and p = 0.8187 for strain x sex, by two-way ANOVA). (B) Colon occludin (OCLN) and zonuline-1 (Tjp1), gene expression normalized to GAPDH gene in BTBR and C57 mice of both sexes. Data were normalized to mC57 control (n = 6/group; OCLN: p = 0.0067 for strain, p = 0.9105 for sex and p = 0.1623 for strain x sex; Tjp1: p = 0.0004 for strain, p = 0.6202 for sex and p = 0.9105 for strain x sex, by two-way ANOVA). (C) Colon mRNA levels of inflammatory markers (CD11c, IL-10, IL-6 and TNF-alpha) normalized to GAPDH in BTBR and C57 mice of both sexes. Data for each gene were normalized to mC57 controls (n = 6/group; CD11c: p = 0.0529 for strain, p = 0.0018 for sex and p = 0.019 for strain x sex; IL-10: p = 0.0041 for strain, p = 0.7827 for sex and p = 0.9739 for strain x sex; IL-6: p = 0.0106 for strain, p = 0.0107 for sex and p = 0.002 for strain x sex; TNF-alpha: p < 0.0001 for strain, p = 0.2196 for sex and p = 0.0354 for strain x sex, by two-way ANOVA). In bar charts all data are expressed as mean values ± SEM; significant differences are indicated by *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001; near-significant differences are also reported (Bonferroni post-hoc tests following two-way ANOVA with strain and sex as factors). Abbreviations: fBTBR (BTBR female mice); mBTBR (BTBR male mice); fC57 (C57 female mice); mC57 (C57 male mice). Full size image

Figure 4: Histological evaluation of colon inflammatory cell infiltration in female and male BTBR mice. Representative hematoxylin and eosin–stained sections from colon tissues of mice. (a) Colon tissue from mC57 mice showing absence of inflammatory cells. (b) Colon tissue of mBTBR group showing leukocyte infiltration in the mucosa and submucosa. (c) Colon tissue of fC57 group, showing absence of inflammatory cells. (d) Colon tissue from fBTBR group showing moderate leukocyte infiltration in the mucosa. Original magnification 10x. Histological evaluation of inflammatory cells infiltration was scored along the entire colon length, inspecting the colon mucosa, submucosa and transmural areas considering the following parametres: leukocyte density (mC57 = 0.33 ± 0.58, mBTBR = 2 ± 1, fC57 = 0, fBTBR = 2.67 ± 0.58) and expansion of leukocyte infiltration (mC57 = 0.33 ± 0.58, mBTBR = 1.33 ± 0.58, fC57 = 0, fBTBR = 1.67 ± 0.58). The histologic scoring system is reported in Material and Methods section. Data reported as mean ± SD, n = 3/group. Full size image

Pearson correlation was applied to correlate the abundance of key genera, that discriminated fBTBR and mBTBR from sex-matched controls, with behavioral tests, colon mRNA expression of occludin, zonuline and immune-markers (Fig. 5). In particular, increase of Parabacteroides and Sutterella, together with decrease of Dehalobacterium, Oscillospira and unclassified member of TM7 were strongly associated to altered behavior and TNF-alpha expression in fBTBR. Onore and collegues (2013) already reported a relationship between the increase in inflammation levels and repetitive grooming behavior in BTBR mice41. Here we add the observation that remodeling of gut microbiota composition by reassortment of specific bacterial genera, may contribute to the immune dysregulation and possibly to the altered behavior of BTBR mice. Unclassified members of Helicobacteriaceae associated with abnormal behavior and low IL-10 expression in mBTBR. Finally, in mBTBR mice lower levels of Dehalobacterium, Ruminococcus and Desulfovibrio were associated with increased gut permeability (Fig. 5).