Fig. 2: Deregulated ileum epithelial SAA activates peripheral innate immune response and Th1-skewed inflammation.

a Volcano plot shows differentially expressed genes (DEGs) of the ileum tissue of 7-month-old Tg recipient mice that received 41-week-age WT (+WT feces) and 5XFAD (Tg) feces (+Tg feces) in FMT assays. Up, down: significantly expressed genes up-regulated and down-regulated respectively in ileum tissue of 7-month-old Tg recipient mice that received 41-week-age Tg feces (+ Tg feces) vs WT feces (+ WT feces). NoDiff, not differentially expressed genes. The significance level is defined as P_adj < 0.05 and the absolute value of log₂FC of +Tg feces vs +WT feces > 1. b Heatmap shows the differential gene expression of the ileum tissue. The cut-off P value is 0.05, while the cut-off P_adj value is 0.3. Red or blue colors indicate up- or down-regulation in the ileum tissue of Tg mice that received Tg fecal samples compared to those receving WT feces, respectively. c Representative IHC images stained with SAA antibody of the ileum tissue slices of 7-month-old Tg recipient mice that received 41-week-age WT (+WT feces) and Tg feces (+Tg feces) in FMT assays. Data are represented as mean ± standard error of the mean (SEM). The yellow staining area is the positive SAA staining signal. The positive rate is calculated as the ratio of positive area × positive staining intensity. *P < 0.05, by Student’s t-test, n = 5–6. d Heatmap shows the changes of cytokines secreted by THP1-Dual cells stimulated by hSAA. THP1-Dual cells were treated with 0.8 µg/mL hSAA for 24 h, and the secretion of cytokines in the culture supernatant was determined by cytokine array. n = 3. e Vehicle-induced or hSAA-induced (10 µg/mL, 24 h) THP1-Dual cells were co-cultured with naïve CD4⁺ T cells for 3 days, and the frequency of Th1 cells was detected by the flow cytometer. Data are mean ± SEM. *P < 0.05, by Student’s t-test, n = 3. f BMDCs were incubated with 10 µg/mL mSAA for 24 h, and the secretion of cytokines in the culture supernatant was determined by cytokine array, and the significantly changed cytokines in mSAA-treated group vs control group were shown as heatmap. n = 3. g Level of the blood SAA of WT and Tg mice at w41 measured by enzyme-linked immune absorbent (ELISA) assays. Data are represented as mean ± SEM. *P < 0.05 by Student’s t-test, n = 7. WT: n = 7, 3 M + 4 F; Tg: n = 7, 3 M + 4 F. M, male mice; F, female mice. h Changes in the frequency of blood Th1 cells of WT and Tg mice at w 41 measured by flow cytometry. Data are represented as mean ± SEM, **P < 0.01 by Student’s t-test, n = 5–6. WT: n = 5, 5 M; Tg: n = 6, 4 M + 2 F. M, male mice; F, female mice. i Mouse blood SAA levels of the 12-month-old C57 WT recipient mice that received 41-week-age WT or Tg fecal samples in FMT assays. Data are represented as mean ± SEM. n = 6–7. j Cell frequency of the blood monocyte of the recipient 7-month-old Tg mice that received 41-week-age WT or Tg feces in FMT assay. Data are represented as mean ± SEM. *P < 0.05 by Student’s t-test, n = 4–6. k Frequency of blood Th1 cells of the 12-month-old C57 WT recipient mice that received 41-week-age WT (+ WT feces) or Tg fecal samples (+ Tg feces) in FMT assays. Data are represented as mean ± SEM. *P < 0.05, by Student’s t-test, n = 7. All recipient mice in FMT assays are male mice.