Supplementary Figure 1 NLRP12 is downregulated in biopsy samples from patients with active ulcerative colitis (UC). (a-g) NLRP12 expression in 7 UC mrna profiling studies deposited in NCBI GEO database. Disease stages, GEO accession numbers, subject tissues for mrna purification, therapeutic treatments including corticosteroid or anti-tnf (infliximab) and mrna measuring microarrays and sequencer platforms are listed. For (a), n=5/group; (b), healthy (n=6), UC nonresponse (n=13), UC response (n=8); (c) healthy (n=20), active UC (n=15), inactive UC (n=18); (d) healthy (n=12), active UC (n=13); (e) n=20/group; (f) healthy (n=6), UC before treatment (n=20), UC response to treatment (n=8); (g) healthy (n=42), UC (n=40). Error bars show SEM. *p<0.05, **p<0.01 and n.s. means no significance determined by two-tailed unpaired t test.
Supplementary Figure 2 Conventionally raised Nlrp12 / mice display exacerbated colitis. (a) DAI and (b) colon length (n=17/group) of conventionally-raised mice treated with 3% DSS, compiled from 3 independent experiments. (c) Representative images of H&E-stained colons from conventionally-raised or germ-free mice after DSS-induced colitis. Scale bars represent 1 mm for 40X and 200 μm for 200X. (d) Histopathology scoring of H&E-stained colons (WT, n=7; Nlrp12 /, n=8; GF WT, n=6; GF Nlrp12 /, n=6). (e-g) Representative immunoblots of distal colon proteins and composite densitometry from 2 independent experiments. One dot or one lane represents one mouse. Error bars show SEM. *p<0.05, **p<0.01, ***p<0.001, and n.s. means no significance determined by two-tailed unpaired t test.
Supplementary Figure 3 Nlrp12 defieciency causes significant changes in the intestinal microbiota. (a) Schematic showing fecal DNA collection from mice housed in two distinct vivarium for 16s rrna gene sequencing. (b) Bacterial diversity within WT (n=18) and Nlrp12 / (n=15) mice, (c) PCoA plot showing microbiota compositional differences between WT and Nlrp12 -/- mice from a repeated 16S rrna gene microbiome sequencing experiment conducted in vivarium #2. (d) Schematic showing fecal DNA collection from Nlrp12 +/+ and Nlrp12 / littermates derived from the same parents for 16s rrna gene sequencing. (e) PCoA plots of microbiota compositional differences between Nlrp12 +/+ and Nlrp12 / littermates. Error bars show SEM. One dot represents one mouse. **p<0.01 determined by two-tailed unpaired t test.
Supplementary Figure 4 Nlrp12 / mice cohoused with wild-type mice display attenuated colitis. (a) Representative images of H&E-stained colons from DSS-treated cohoused or single-housed WT and Nlrp12 / mice. (b) Body weight, (c) percent survival ( indicates statistical significance between SiHo WT vs. SiHo Asc /, and * indicates significance between SiHo WT vs. CoHo WT), (d) DAI and (e) colon length of DSS-treated cohoused or single-housed WT and Asc / mice (CoHo WT and CoHo Asc /, n=4; SiHo WT and SiHo Asc /, n=6). (f) Representative images of H&E-stained colons of SiHo and Coho WT and Asc / mice after DSS-induced colitis. (g) PCoA plot showing fecal microbial composition before cohousing (n=9/group). (h) Quantification of UniFrac distance between mice from (g) after cohousing, indicated as dissimilarity values. Error bars show SEM. *p<0.05, ***p<0.001 and n.s. means no significance, determined by two-tailed unpaired t test (b, d, e), Log-rank (Mantel Cox) test (c) and ANOSIM (h). Scale bars represent 1 mm for 40X and 200 μm for 200X (a,f). The Tukey s boxplot indicates first (bottom of the box) and third quartiles (top of the box), and the band inside the box is the median, and the ends of the whiskers indicate 1.5 interquartile range of the upper or lower quartile (h).
Supplementary Figure 5 Inoculation of Lachnospiraceae strains suppresses colitis in Nlrp12 / mice. (a) Representative images of H&E-stained colons from mice administered Lachnospiraceae or BHI vehicle prior to DSS treatment. Scale bars represent 1 mm for 40X and 200 μm for 200X.
Supplementary Figure 6 Treatment with anti-tnf and anti-il6r reverses dysbiosis in Nlrp12 / mice. (a, b) Flow chart showing WT and Nlrp12 / littermates treated with anti-tnf and anti-il6r antibodies (Ab) or PBS, and experimental strategy for Ab dosing (filled triangles) and fecal collection (open triangles) (n=6 mice/group). (c) Microbial diversity and (d) PCoA plots of microbial compositional differences in WT and Nlrp12 / mice before antibody treatment. (e) Quantification of UniFrac distances calculated from (d) represented as dissimilarity values. Error bars show SEM. *p<0.05, **p<0.01, ****p<0.0001 and n.s. means no significance determined by two-tailed unpaired t test (c) or ANOSIM (e).
Supplementary Figure 7 Model for the role of NLRP12 in maintaining intestinal homeostasis between host innate immunity and intestinal commensal symbiosis. Graphic summary of NLRP12 in maintaining intestinal homeostasis between host innate immunity and intestinal commensal symbiosis. The dysbiosis and inflammation caused by dysfunction of NLRP12 are shown in red. And therapeutic intervention points for the treatment of IBD during NLRP12 dysfunction are shown in blue.