Figure 3
ZBTB32-deficiency minimally impacts IgA responses to intestinal bacteria. (A) Serum IgA levels in 8 week-old Zbtb32−/− or Zbtb32+/+ mice, measured by ELISA. Mean values ± SEM are shown. No significant differences were observed by 2-way ANOVA followed by post-hoc Sidak’s multiple comparisons test. (B) Bacterial content in fecal pellets of Zbtb32−/− and Zbtb32+/− littermates as revealed by 16S rDNA sequencing. Data shown are family level taxa for individual mice. No statistically-significant differences between genotypes were observed at the family or OTU level by permutation ANOVA. (C) Representative flow cytometric plot of IgA-bound bacteria in fecal pellets. Bacteria were gated as DAPI+ and isotype control negative events and assessed for IgA and IgG staining. Data are representative of two independent experiments comparing littermates (Zbtb32−/−, n = 5; Zbtb32+/−, n = 4). (D) Volcano plot showing the IgA-enrichment (log2 (% of OTU in IgA+/IgA−) vs t-test p-value. As ratios are very susceptible to small denominators, data are calculated using a filtered dataset (139 OTUs present in ≥2 samples for each genotype at >0.1% frequency). In addition, IgA enrichment per individual was arbitrarily capped at log2(50 or 1/50) to limit effects of small denominators. No OTU comparison passes FDR < 0.25. (E) IgA enrichment values for top 8 OTUs based on greatest differences between genotypes by p-value, and for those with largest relative abundance. Taxonomic assignments at the genus level for each OTU are shown if available. IgA-enrichment is calculated as per (D). (F) Rarefaction plot shows the average species diversity and 95% confidence limits at different sampling intervals. Unc, unclassified at the taxa level presented, with the best higher level assignment noted.
