Morphological Differences of Intestinal Tissues and Organs
Compared with CL and SPF grade mice, the intestinal of GF mice have characteristic changes, which are mainly manifested as the enlargement of cecum and the thin intestinal wall (Figure 1A). Moreover, significant differences in the morphology of the small intestine and mesenteric lymph nodes of GF grade mice were observed by H&E staining sections (Figure 1B). In GF mice, the density of small intestinal villi was thinner, the length of small intestinal villi was longer, the depth of crypt was deeper, and the glandular layer and basal layer were thinner. In addition, the original germinal center of the mesenteric lymph nodes became smaller, and the diffuse lymphoid tissue between the superficial cortical lymph nodes under the capsule of the lymph nodes was less. It is suggested that the tissue structure of small intestine and lymph nodes of GF mice are dysplasia.
Sequencing of Intestinal Flora
In order to explore the factors that affect the intestinal mucosal immune system of three different feeding levels mice, we detected the diversity of intestinal microorganisms in GF, SPF and CL mice. The Operational Taxonomic Units (OTU) statistical results of mouse intestinal flora were shown in Additional file 1. The OTU number of intestinal flora in GF, SPF and CL groups were about 50, 280 and 330, respectively. The results of principal component analysis showed that the composition of intestinal microflora was different in three grades of mice, and the composition of intestinal microflora in SPF group and CL group were similar (Additional file 2). OTU Rank analysis showed that the species richness and evenness of intestinal microflora were the lowest in GF mice, and the diversity of intestinal microflora was relatively rich in SPF and CL mice, among which CL mice were the most abundant (Additional file 2).
Species Composition and Clustering of Intestinal Flora
By comparing the sequencing results of intestinal flora with the available database, the features of species classification of intestinal flora were conducted (Figure 2 and Additional file 3). About 99% of the fecal flora of GF grade mice were Brevibacilli in the phylum Firmicutes. The intestinal microflora of SPF and CL mice were mainly composed of Firmicutes and Bacteroidetes, but the proportion of the two groups were different. Bacteroides was the main composition of intestinal microflora in SPF mice, mainly including s24-7, rikenellaceae, prevotellaceae, bacteroideae and paraprevotellaceaes. The intestinal microflora of CL mice was mainly composed of Firmicutes, including clostridia under clostridia. Further analysis showed that about 10% of the unique intestinal microflora in CL group were desulfovibrionaceae and helicobacteraceae under proteobacteria. Moreover，the cluster analysis of six classification levels of intestinal microorganisms also indicated that the species of intestinal microorganisms in GF group were less, and almost all of them were Bacillus brevis.
Species Diversity Analysis of Intestinal Flora
The Chao index, Shannon index, Ace index, Simpson index and Observed species index were used to evaluate the alpha diversity of the samples, and the results showed that there were significant differences in the five diversity indexes of intestinal flora among the three groups (P < 0.05), suggesting that the diversity of intestinal flora were different among the three groups. The diversity of intestinal flora was the most abundant in CL mice, followed by SPF mice, and the lowest in GF mice (Additional file 4).
Detection of BCR Heavy Chain CDR3 Sequence
High-throughput sequencing was performed on memory B cells and total B cells in small intestine of GF, SPF and CL mice to obtain BCR heavy chains CDR3 libraries. Each library was filtered to obtain high-quality reads and compared with databases. The statistical results were shown in Table 1. The ratio of unique nucleotides to total nucleotides and the ratio of productive unique nucleotides to total productive nucleotide in B cells BCR CDR3 sequence were lower than those in memory B cells in all three groups. The ratio of unique CDR3 amino acids to total CDR3 amino acids and the ratio of unique in frame CDR3 amino acids to total in frame CDR3 amino acids were higher than those in memory B cells in GF and SPF groups, and the CL group was the opposite.
Diversity of the BCR Heavy Chain CDR3 Repertoire
The inverse Simpson’s diversity index (1/DS) for small intestine BCR heavy-chain CDR3 repertoire of total and memory B cells were showed in Figure 3. The values of 1/DS were significant in different animal groups among total and memory B cells. The highest index of total B cells was detected in SPF group, followed by GF group, and the lowest was CL group. For memory B cells, the 1/DS index was positively correlated with the feeding level of mice.
Usage Frequency of V, D and J Gene Segments of the BCR Heavy Chain CDR3 Region
The usage frequency of the V, D and J gene segment in the BCR Heavy chain CDR3 were determined among three groups. IGHV03-02、IGHV14-02、IGHV09-03、IGHV05-17、IGHV06-06、IGHV02-09、IGHV01-04、IGHV01-09 and IGHV01-14 were common high frequency (> 2%) IGHV genes of total B cells in all groups. IGHV14-02、IGHV03-02 and IGHV09-03 were common high frequency IGHV genes of memory B cells in all groups. Holm-Sidak Test showed that the distribution of the IGHV gene found in total B cells and memory B cells were significant difference among three groups. In addition, some IGHV genes disappeared in GF and CL groups, for instance, IGHV08-04 and IGHV02-02-1 genes of total B cells, only found in GF and SPF groups (Additional file 5).
Similarly, common high-frequency IGHD genes were found in the total B cells (Figure 4 A) and memory B cells (Figure 4 B) of the three groups of mice. However, both in total B cells and memory B cells, the difference of IGHD genes distribution among three groups were insignificant (P >0.05).
IGHJ04-01 and IGHJ03-01 were common high-frequency IGHJ genes both in total B cells (Figure 4 C) and memory B cells (Figure 4 D) in three groups. Statistical analysis showed that the usage frequency of IGHJ04-01 gene in BCR heavy chain CDR3 repertoire of total B cells of small intestine in CL group was significantly lower than that in GF and SPF group (P <0.05), while the usage frequency of IGHJ04-01 gene of memory B cells in GF groups was the lowest and did differ significantly between GF and SPF groups in terms of IGHJ04-01 gene distribution.
The Combinations of the BCR Heavy Chain V and J Genes
Hierarchical clustering heat map was created to comparatively analyze the combinations of total B cells (Figure 4 E) and memory B cells (Figure 4 F) BCR heavy chain V and J genes in the three groups, and T-test was performed to examine the distribution ratio of combinations. Five (IGHV06-06-IGHJ03-01、IGHV03-02-IGHJ01-01、IGHV03-02-IGHJ03-01、IGHV03-02-IGHJ02-01、IGHV02-09-IGHJ03-01) and three (IGHV14-02-IGHJ03-01、IGHV09-03-IGHJ04-01、IGHV09-03-IGHJ02-01)common combinations were found in total B cells and memory B cells of all groups. The combinations in total B cells between GF group and SPF group was relatively close, while SPF and CL group was relatively close in memory B cells. Compared with GF groups, one combination (IGHV03-02- IGHJ03-01) of total B cells and two combinations (IGHV03-02-IGHJ03-01and IGHV14-02-IGHJ04-01) of memory B cells were significantly up-regulated both in CL and SPF groups. In addition, the combinations (IGHV01-80- IGHJ04-01) of total B cells was significantly down-regulated both in GF and SPF groups by comparing with CL groups.
Amino Acid Sequences in the BCR Heavy Chain CDR3 Repertoire
In analysis of amino acid sequences, Tyrosine was found as most popular amino acid in the BCR heavy chain CDR3 repertoire of total (Figure 5 A) and memory B cells (Figure 5 B) in all three groups, the usage frequency was about 20%. Statistical analysis showed that the usage frequency of methionine was significantly decreased in total B cells of both CL and SPF groups by comparing to GF group, while was significantly increased in memory B cells of SPF groups. The average CDR3 length of nine BALB/c mice were 12 amino acid-based Gaussian distribution, and statistical analysis showed that the difference in the CDR3 length distribution of total (Figure 5 C) and memory B cells (Figure 5 D) among the three groups was insignificant (P > 0.05).
The overlap amino acid sequence was also analyzed both in total B cells (Figure 5 E) and memory B cells (Figure 5 F). 37 amino acid sequences were shared in the total B cells of the nine BALB/c mice, and only one amino acid sequence was shared in the memory B cells of the nine BALB/c mice. However, the frequency of all the common amino acid sequences was low (<0.5%). Top 10 most frequent amino acid sequences in each mouse total B cells and memory B cells were also detected (data not shown), the highly conserved amino acid motif "WYFDV " was found in all three group’s total B cells, and the most was found in GF group. Two highly conserved amino acid motifs (“RYFDV” and “WYFDV”) were detected in all three group’s memory B cells. Interestingly, the most was also found in GF groups.
Mutations Frequency of BCR Heavy Chain CDR3 Repertoire
The statistical results of nucleotide polymorphism of CDR3 repertoire of total B cells and memory B cells in three experimental groups were shown in Figure 6. The insertion and deletion of nucleotides in total B cells were significantly different in three experimental groups (P <0.05), including the number of total nucleotide insertion, D5 'deletion, J gene deletion, V-D gene insertion and D-J gene insertion. In contrast, amino acid insertion and deletion in memory B cells were significantly different only at the D3 ' deletion (P <0.05).
Further analysis of high frequency mutation of V gene showed that the proportion of V gene mutation in total B cells was different in three groups of experimental animals, and the proportion of mutation sequence in CL was significantly higher than that in SPF and GF groups with 400-800 mutations per 10,000 bases. The proportion of mutation sequences in memory B cells was about 70%, and there was no significant difference among the three experimental groups.