The Formation of Glycan-Specific Natural Antibodies Repertoire in GalT-KO Mice Is Determined by Gut Microbiota

Abstract

Gut commensal bacteria are known to have a significant role in regulating the innate and adaptive immune homeostasis. Alterations in the intestinal microbial composition have been associated with several disease states, including autoimmune and inflammatory conditions. However, it is not entirely clear how commensal gut microbiota modulate and contribute to the systemic immunity, and whether circulating elements of the host immune system could regulate the microbiome. Thus, we have studied the diversity and abundance of specific taxons in the gut microbiota of inbred GalT-KO mice during 7 months of animal life by metagenetic high-throughput sequencing (16S rRNA gene, variable regions V3-V5). The repertoire of glycan-specific natural antibodies, obtained by printed glycan array technology, was then associated with the microbial diversity for each animal by metagenome-wide association studies (MWAS). Our data show that the orders clostridiales (most abundant), bacteriodales, lactobacillales, and deferribacterales may be associated with the development of the final repertoire of natural anti-glycan antibodies in GalT-KO mice. The main changes in microbiota diversity (month-2 and month-3) were related to important changes in levels and repertoire of natural anti-glycan antibodies in these mice. Additionally, significant positive and negative associations were found between the gut microbiota and the pattern of specific anti-glycan antibodies. Regarding individual features, the gut microbiota and the corresponding repertoire of natural anti-glycan antibodies showed differences among the examined animals. We also found redundancy in different taxa associated with the development of specific anti-glycan antibodies. Differences in microbial diversity did not, therefore, necessarily influence the overall functional output of the gut microbiome of GalT-KO mice. In summary, the repertoire of natural anti-carbohydrate antibodies may be partially determined by the continuous antigenic stimulation produced by the gut bacterial population of each GalT-KO mouse. Small differences in gut microbiota diversity could determine different repertoire and levels of natural anti-glycan antibodies and consequently might induce different immune responses to pathogens or other potential threats.

Keywords: 16S rRNA gene; GalT-KO mice; gut microbiota; metagenetic high-throughput sequencing; metagenome-wide association studies; natural anti-glycan antibodies; printed glycan array.

Figure 1

The levels of anti-αGal antibodies are variable between genetically identical GalT-KO mice. IgM and IgG were determined by ELISA and expressed in absorbance units (OD 492 nm). Each mark corresponds to a different mouse and represents the arithmetic mean of three replicates. The short horizontal line represents the arithmetic mean resulting from these determinations (n = 11). ^*3 weeks of age.

Figure 2

The repertoire of natural circulating anti-carbohydrate antibodies in GalT-KO is a function of age. GalT-KO mouse (n = 5) serum (1:15) was incubated with chips printed with 682 different glycans (6 replicates). Chips were scanned using a ScanArray GX Plus reader and data were analyzed with the ScanArray Express Microarray Analysis System (PerkinElmer). The binding results for IgM+IgG+IgA were expressed in RFU as the median ± IQR (25–75th). In the heat map, blue and white colors represent binding signals in RFU lower than 4,000 (background); red color signals ≥ 4,000 RFU (positive binding). ^*3 weeks of age.

Figure 3

Individual differences in the conserved top-rank repertoire of circulating anti-glycan antibodies of GalT-KO mice during lifetime (n = 5). The first ten anti-glycan antibodies showing the strongest binding signals are represented. GalT-KO mice serum (1:15) was incubated with chips printed with 682 different glycans (6 replicates). Chips were scanned using a ScanArray GX Plus reader and data were analyzed with the ScanArray Express Microarray Analysis System (PerkinElmer). The binding results for IgM+IgG+IgA are expressed in RFU as the median.

Figure 4

GalT-KO mice bacterial gut population is dynamic. The diversity of gut bacterial population was estimated by months with Chao1 diversity estimator (n = 77).

Figure 5

Analysis of gut microbiota diversity in GalT-KO mice during the first 7 months of life. The different microbial proportions corresponding to each phylum, class, order, and family are shown in colors (n = 77).

Figure 6

For P-value < 0.01, bar circle graph showing the number of natural anti-glycan antibodies structures associated with the bacterial taxa at a taxonomic level of order and family. Between brackets, in the central circle are the numbers of mice related to these observations.