irC patients exhibit colitogenicity in microbiome prior to irC development in a TCT colitis model. (A and B) Stool collection (black circle) for 25 non-irC (A) and 13 irC patients (B). Day of irC onset (red triangle) and day of irC remission (green circle) are shown as days relative to the first ICI dose (vertical dotted line). (C) MaAsLin2 analysis (species level) examined metagenomic pre-ICI microbiome profiles of irC (red, n = 9) and non-irC patients (blue, n = 15). Features displayed are P < 0.05. The horizontal bar length indicates log10(P value) associated with each species. (D and E) Total colonic histology severity score (D) and representative H&E-stained colon sections (E) 7–8 wk after TCT in Rag1−/− mice colonized with microbiotas from patients who would remain irC-free (blue) or eventually developed irC (red). Each dot in D represents the averaged colonic histology severity score by each microbiota donor (9 irC, 10 non-irC) (Mann-Whitney P = 0.033). (E) Upper: Non-inflamed colon section from the murine recipient of non-irC patient’s microbiota. Lower: Inflamed colon section from murine recipient of pre-colitis microbiota from irC patient. Moderate mucosal and submucosal inflammation with crypt and goblet cell loss, crypt hyperplasia, and muscle thickening. The horizontal bars in E represent the scale bar 200 µm. (F) Weekly body mass percentage change (relative to week 0) following TCT in Rag1−/− mice colonized with microbiota from 9 irC patients (red) or from 10 non-irC patients (blue) with three to seven mice receiving a single fecal microbiota. Bolded blue and red lines represent the mean ± SEM of all irC or non-irC-colonized group of mice, respectively, at each week after TCT. Individual opaque thin lines represent the body mass change of individual mice. The linear mixed-effect model showed no significant difference in weight between the irC- and non-irC-colonized mice (P = 0.63) at week 0 but a significant effect of patient colitis status on raw weight change at weeks five and six after TCT (week 5, P = 1.7 × 10−4; week 6, P = 3.2 × 10−4). Plots D–F are combined from three independent experiments. Analysis included a total of 67 mice (experiment 1: n = 19, 2: n = 24, 3: n = 24) colonized with 19 human microbiotas (n = 10 irC patients, n = 9 non-irC patients). Three to seven mice colonized per microbiota donor. Error bars represent mean ± SEM. *P < 0.05 and ***P < 0.001. Linear mixed model fit by restricted maximum likelihood with P value estimations using Satterthwaite’s method “lmerModLmerTest” was performed to evaluate differences in body mass change between irC and non-irC microbiota-colonized mice groups. Mann-Whitney was performed to assess differences in colonic histological severity scores between irC and non-irC microbiota-colonized mice groups.
Figure 1.

irC patients exhibit colitogenicity in microbiome prior to irC development in a TCT colitis model. (A and B) Stool collection (black circle) for 25 non-irC (A) and 13 irC patients (B). Day of irC onset (red triangle) and day of irC remission (green circle) are shown as days relative to the first ICI dose (vertical dotted line). (C) MaAsLin2 analysis (species level) examined metagenomic pre-ICI microbiome profiles of irC (red, n = 9) and non-irC patients (blue, n = 15). Features displayed are P < 0.05. The horizontal bar length indicates log10(P value) associated with each species. (D and E) Total colonic histology severity score (D) and representative H&E-stained colon sections (E) 7–8 wk after TCT in Rag1−/− mice colonized with microbiotas from patients who would remain irC-free (blue) or eventually developed irC (red). Each dot in D represents the averaged colonic histology severity score by each microbiota donor (9 irC, 10 non-irC) (Mann-Whitney P = 0.033). (E) Upper: Non-inflamed colon section from the murine recipient of non-irC patient’s microbiota. Lower: Inflamed colon section from murine recipient of pre-colitis microbiota from irC patient. Moderate mucosal and submucosal inflammation with crypt and goblet cell loss, crypt hyperplasia, and muscle thickening. The horizontal bars in E represent the scale bar 200 µm. (F) Weekly body mass percentage change (relative to week 0) following TCT in Rag1−/− mice colonized with microbiota from 9 irC patients (red) or from 10 non-irC patients (blue) with three to seven mice receiving a single fecal microbiota. Bolded blue and red lines represent the mean ± SEM of all irC or non-irC-colonized group of mice, respectively, at each week after TCT. Individual opaque thin lines represent the body mass change of individual mice. The linear mixed-effect model showed no significant difference in weight between the irC- and non-irC-colonized mice (P = 0.63) at week 0 but a significant effect of patient colitis status on raw weight change at weeks five and six after TCT (week 5, P = 1.7 × 10−4; week 6, P = 3.2 × 10−4). Plots D–F are combined from three independent experiments. Analysis included a total of 67 mice (experiment 1: n = 19, 2: n = 24, 3: n = 24) colonized with 19 human microbiotas (n = 10 irC patients, n = 9 non-irC patients). Three to seven mice colonized per microbiota donor. Error bars represent mean ± SEM. *P < 0.05 and ***P < 0.001. Linear mixed model fit by restricted maximum likelihood with P value estimations using Satterthwaite’s method “lmerModLmerTest” was performed to evaluate differences in body mass change between irC and non-irC microbiota-colonized mice groups. Mann-Whitney was performed to assess differences in colonic histological severity scores between irC and non-irC microbiota-colonized mice groups.

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