Supplemental Information. Commensal Microbes Induce Serum IgA. Responses that Protect against Polymicrobial Sepsis
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1 Cell Host & Microbe, Volume 23 Supplemental Information Commensal Microbes Induce Serum Ig Responses that Protect against Polymicrobial Sepsis Joel R. Wilmore, rian T. Gaudette, Daniela Gomez tria, Tina Hashemi, Derek D. Jones, Christopher. Gardner, Stephen D. Cole, na M. Misic, Daniel P. eiting, and David llman
2 SUPPLEMENTL INFORMTION Number of Plasma cells per 10 5 bone marrow cells PENN-SPF TCONIC-SPF JX-SPF Percentage of PCs within CD138+ gate Ig IgG IgM PENN-SPF TCONIC-SPF JX-SPF 0 Ig IgG IgM Supplemental Figure 1. Frequencies of Ig + bone marrow plasma cells in 6 mice from Taconic Farms, related to Figure week old 6 mice were purchased from Jackson Labs, Taconic farms, or bred in house. () Total number of Ig, IgG, and IgM secreting PCs per 10 5 M cells determined by ELISpot. () Frequencies of the indicated plasma cell subset among all M plasma cells were determined by flow cytometry focusing on Dump - IgD - Thy1.2 - CD138 high cells as illustrated in Figure 1. Horizontal lines indicate means and error bars SEMs for each group.
3 100%! PC2 ( 15 %) PC1 ( 44 %) Penn SPF JX SPF RG Relative abundance 90%! 80%! 70%! 60%! 50%! 40%! 30%! 20%! 10%! Verrucomicrobia! Tenericutes! TM7! Proteobacteria! Firmicutes! Deferribacteres! acteroidetes! ctinobacteria! Other! 0%! WT RG1 -/- Supplemental Figure 2. Fecal microbiota in 6 and 6.RG -/- mice, related to Figure 4. Taxonomy for fecal bacteria in stool samples from individuals in the indicated group was determined by 16S V4 rdn gene sequencing. () Principal coordinate analysis of the resulting data. () Relative abundance of the indicated phyla is shown.
4 ! RG1 -/- feces,! size gated! Serum:! RG1 -/-! PENN 6! SytoC! SSC-! DPI! Ig!! kkermansia! RF39;f_;g_! cinetobacter! Proteobacteria! urkholderia! Sphingomonas! Firmicutes! acteroidetes! ctinobacteria! llobaculum! Ruminococcus! Osciilllospira! Lachnospiraceae;Other! SF! Clostridiaceae;g_! Turcibacter! Lactobacillus! Lactobacillales;Other;Other! acillus! acillales;other;other! S24-7;g_! Prevotella! acteroides! dlercreutzia! Coriobacteriaceae;g_! Corynebacterium! 0.1! 1! 10! 100! 1000! Ig + /Ig -! Supplemental Figure 3. Select taxa among serum Ig-bound bacteria, related to Figure 4. () Fecal bacteria from 6.RG1 -/- mice were stained with sera from the indicated mice, and Ig + bacteria identified with PE-anti-Ig antibodies. () Fecal bacteria from RG1 -/- mice were stained with sera from PENN-6 adults and both Ig + and Ig - fraction within the DPI + SytoC + gate as shown in () sorted twice. DN from these samples was then used for16s V4 sequencing. The ratio of the relative abundance for each taxa in each fraction (Ig +, Ig - ) was used to identify taxa enriched for Ig binding. acterial genera that were highly enriched (Ig + /Ig - 10) are highlighted in red. Genera shown were selected because they possessed ICI scores above or below 3, or because of potential relevance based on previous studies.
5 C57L/6 feces SSC- SytoC SSC- FSC- DPI Ig Proteobacteria Firmicutes Deferribacteres acteroidetes ctinobacteria Enterobacteriaceae Desulfovibrio Ralstonia urkholderia etaproteobacteria Sphingomonas Ruminococcus Osciilllospira SF Streptococcus Lactococcus acillus Mucispiriium S24-7 F12 Prevotella Parabacteroides acteroides dlercreutzia Coriobacteriaceae ifidobacterium 0.1! 1! 10! 100! 1000! 10000! Ig + /Ig - Supplemental Figure 4. Select taxa bound by Ig in 6 mice, related to Figure 4. () Representative flow cytometric data of fecal bacteria stained with DPI, Syto C, and PE-anti- Ig from a Penn-SPF 6 adult. () Twice sorted bacteria from () were sequenced for the 16S V4 region to identify bacterial taxa in each population. s in Figure 5, highly coated bacteria (Ig + /Ig - 10) are in red and the remaining groups were among the most enriched across all taxa.
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