Actinobacteria Relative abundance (%) Co-housed CD300f WT. CD300f KO. Colon length (cm) Day 9. Microscopic inflammation score
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1 y groups y individuals 9 Actinobacteria Relative abundance (%) acteroidetes Cyanobacteria Deferribacteres Firmicutes Proteobacteria TM Tenericutes Unclassified CDf CDf Co-housed CDf Co-housed CDf CDf CDf Co-housed CDf Co-housed CDf C Day 9 D Day 9 DAI score CDf CDf 9 (Days) Colon length (cm) 9 Macroscopic inflammation score..... H O Day 9 E CDf CDf Microscopic inflammation score.... Supplemental Figure : Gut microbiota is not responsible for the enhanced sensitivity to colonic inflammation in Cdf -/- mice. (A) Phylum-level composition of intestinal bacteria in the indicated mice. (-E) Cdf +/+ and Cdf -/- mice were co-housed for weeks, and then given drinking water containing.% for days, followed by unadulterated drinking water for another days. DAI was determined during and after administration (). On day 9, the colon length (C), macroscopic inflammation score (D) and microscopic inflammation score (E) were determined. Images in E show H&E staining of colon tissues; scale bar µm. Data are expressed as means + S.E.M. (n =, each group). Two-tailed paired Student s t test was used to determine statistical significance (p<., p<.).
2 Day 9 % CD + cells/total cells CONT No. of CD + cells (x ) No. of neutrophils (x ) Day % CD + cells/total cells No. of CD + cells (x ) No. of neutrophils (x ) Supplemental Figure : Cell populations in the lamina propria of Cdf +/+ and Cdf -/- mice. (A and ) Cdf +/+ or Cdf -/- mice were given drinking water containing.% for days (A), followed by unadulterated drinking water for another days (). Lamina propria immune cells were isolated from the colon of -treated Cdf +/+ or Cdf -/- mice, and the number and the percentage of total immune cells (CD + ) and neutrophils (CD + LyG + CDc - F/ - ) was determined by flow cytometry. Data are expressed as means + S.E.M. (n =, each group). Two-tailed paired Student s t test was used to determine statistical significance (p<., p<., p<.).
3 CDf CDf CDf CDf MFI (TNF-α) MFI (IFN-γ) (Day ) (Day ) Supplemental Figure : Analysis of TNF-α and INF-γ signal intensities in the colon tissue. Colon sections from Cdf +/+ and Cdf -/- mice, collected on day () or day (), were stained for TNF-α (A) or IFN-γ (). The average pixel intensity in the collected images (n= in each group) was measured and is summarized in the bar graph; error bars show S.E.M. Two-tailed paired Student s t test was used to determine statistical significance (p<., p<.).
4 % of TNF-α+ cells 9 Day CDf CDf No. of TNF-α+ cells Day CDf CDf Mø DC % of IFN-γ+ cells Mast cell T cell cell NK cell NKT cell 9 Day CDf CDf Mø DC Mast cell T cell cell NK cell NKT cell Mø DC Mast cell T cell cell NK cell NKT cell Day CDf CDf Mø DC Mast cell T cell cell NK cell NKT cell No. of IFN-γ + cells Supplemental Figure : Analysis of lamina propria cells producing TNF-α and IFN-γ at the early phase of colonic inflammation. (A and ) Lamina propria cells were isolated from the colons of -treated Cdf +/+ or Cdf -/- mice collected on day of treatment. The intracellular expression of TNF-α (A) and IFN-γ () was determined by flow cytometry in the following cell populations: macrophages (CD + F/ + CDb + CD + LyG - CDc - ), DC (CD + CDc + F/ - LyG - CD - ), neutrophils (CD + CDb + LyG + CDc - F/ - ), mast cells (CD + CDb + FcεRI + ), T cells (CD + CD + ), cells (CD + CD - CD9 + ), NK cells (CD + CD - NK. + ), and NKT cells (CD + CD + NK. + ). The graphs show the percentages (left) and total number (right) of cells expressing TNF-α (A), or IFN-γ (). Data are expressed as means + S.E.M. (n =, each group). Two-tailed paired Student s t test was used to determine statistical significance (p<., p<., p<.).
5 % of TNF-α+ Mø CDf CDf % of TNF-α+ DC CDf CDf % of TNF-α+ neutrophils CDf CDf % of TNF-α+ mast cells CDf CDf (Day ) (Day ) (Day ) % of TNF-α+ T cells (Day ) (Day ) (Day ) (Day ) (Day ) CDf CDf CDf CDf CDf CDf CDf CDf (Day ) (Day ) (Day ) (Day ) (Day ) (Day ) (Day ) (Day ) % of TNF-α+ cells % of TNF-α+ NK cells % of TNF-α+ NKT cells Supplemental Figure : Analysis of TNF-α-producing cells in the lamina propria during gut inflammation. Lamina propria cells were isolated from the colons of -treated Cdf +/+ or Cdf -/- mice collected on day,, and. The intracellular expression of TNF-α was determined by flow cytometry in the following cell populations: macrophages (CD + F/ + CDb + CD + LyG - CDc - ), DC (CD + CDc + F/ - LyG - CD - ), neutrophils (CD + CDb + LyG + CDc - F/ - ), mast cells (CD + CDb + FcεRI + ), T cells (CD + CD + ), cells (CD + CD - CD9 + ), NK cells (CD + CD - NK. + ), and NKT cells (CD + CD + NK. + ). The graphs summarize the data from Figure and Supplemental Figure, and illustrate the percentages of cells expressing TNF-α at the indicated times. Data are expressed as means ± S.E.M. (n = -, each group). Two-tailed paired Student s t test was used to determine statistical significance (p<., p<., p<.).
6 Day Day % of TNFα+ cells (in each DC sub-population) CDf CDf CDb + CD - CDb - CD + CDb - CD - pdc % of TNFα+ cells (in each DC sub-population) CDb + CD - CDb - CD + CDb - CD - pdc CDf CDf Supplemental Figure : Analysis of TNF-α production by DC sub-populations in the lamina propria. Lamina propria cells were isolated from the colon of -treated Cdf +/+ or Cdf -/- mice on day (A) or day () after.% treatment. The intracellular levels of TNFα in DC cell sub-populations including myeloid CDb + CD -, CDb - CD +, CDb - CD - DC, and plasmacytoid DC (pdc) were determined by flow cytometry. The graph shows the percentage of cells expressing TNF-α, as means + S.E.M. (n =, each group). Two-tailed paired Student s t test was used to determine statistical significance (p<., p<.).
7 % of IFNγ+ cells (in each T cell sub-population) 9 CD - CD - CD + CD + Foxp - CD + Foxp + CDf CDf Supplemental Figure : CD + T cells constitute the major T cell population producing IFN-γ in the lamina propria of Cdf -/- mice. Lamina propria cells were isolated from the colon of -treated Cdf +/+ or Cdf -/- mice on day after.% treatment. The intracellular levels of IFN-γ in T cell sub-populations including CD - CD -, CD +, CD + FoxP -, and CD + FoxP + T cells were determined by flow cytometry. The graph shows the percentage of cells expressing IFN-γ, as means + S.E.M. (n =, each group). Two-tailed paired Student s t test was used to determine statistical significance (p<.).
8 Undifferentiated M SSC 9.%.% FSC CDf F/ CDb CD Gr- CDc MHCII MMϕ SSC.% 9.% FSC CDf F/ CDb CD Gr- CDc MHCII CDc+ MDC SSC 9.% 9.% FSC CDf F/ CDb CD Gr- CDc MHCII Supplemental Figure : Analysis of MMϕ and MDC purity. one marrow cells (M) were isolated from Cdf +/+ or Cdf -/- mice and differentiated to macrophages (MMϕ) or DC (MDC) as described in Methods section. MMϕ were crudely purified by removal of non-adherent cells, and MDC were purified from non-adherent cells using CDc selection (see Methods for details). Cell identity and population purity were analyzed by flow cytometry. Dot plots show the forward scatter (FSC) and side scatter (SSC) distribution of analyzed cells, and the gating strategy. Histograms illustrate the indicated marker distribution on the surface of cells derived from Cdf +/+ (; black dashed lines) or Cdf -/- mice (; red solid lines). Results are representative of two independent experiments.
9 C DAI score CDf G I MMϕ transfer CDf CDf (Days) Gr- MMϕ transfer.% mouse mouse MFI (Gr-) Sacrifice (Days) CFSE Gr- Merge IgG control D Colon length (cm) No MMϕ transfer 9 Day. mouse E Macroscopic inflammation score mouse White spots: CFSE-labeled MMϕ H CFSE mouse Day TUNEL TUNEL/DAPI F mouse.%.% mouse mouse % of transferred macrophages engulfing apoptotic neutrophils Gr- mouse mouse Microscopic inflammation score TUNEL+ cells/field.... Day Day No. of engulfed Gr-+ cells in transferred macrophages... MMΦ MMΦ transfer transfer Supplemental Figure 9: Impaired macrophage efferocytosis drives the excessive colonic inflammation in Cdf -/- mice. (A) CFSEstained MMϕ ( x cells) derived from Cdf +/+ or Cdf -/- mice were intravenously injected into Cdf -/- mice on days and during administration. The presence of the transferred MMϕ (white spots) in the colon tissues was verified by confocal microscopy (scale bars: µm). () Representative immunofluorescence staining for TUNEL (red) in frozen colon sections from -treated Cdf -/- mice injected with MMϕ derived from Cdf +/+ or Cdf -/- mice. Nuclei were stained with DAPI (blue). Scale bars: µm. The graph shows the quantification of TUNEL + cells per field of view ( fields per colon section). (C) DAI scored during administration; the arrows indicate the time of MMϕ transfer. (D-F) On day, the colon length (D), macroscopic inflammation score (E) and microscopic inflammation score (F) were determined. The pictures in F illustrate representative images of the colon in the indicated mice (H&E-staining, scale bars: µm). (G) Cell surface expression of Gr- on MMϕ derived from Cdf +/+ and Cdf -/- mice, or neutrophils. The histograms show a representative flow cytometry result. The bar graph shows the quantification of Gr- mean fluorescence intensity (MFI) values. (H and I) Spleens were collected from treated Cdf -/- mice i.v. injected with CFSE-labeled MMϕ derived from Cdf +/+ or Cdf -/- mice. (H) The percentage of transferred MMϕ engulfing AC (CFSE + F/ + Gr- + ) in the spleen was determined using flow cytometry. (I) Images show representative examples of transferred MMϕ (CFSE + ; green) engulfing apoptotic neutrophils (Gr- + ; red); scale bars: µm. Data are expressed as means + S.E.M (n =, each group in, n =, each group in D-F, n = in H, n = in I). Two-tailed paired Student s t test was used to determine statistical significance (p<., p<.).
10 TNF-α (pg/ml) IFN-γ (pg/ml) CDf MDC CDf MDC.. (%).. (%) IL- (pg/ml).. (%) IL- (pg/ml).. (%) Supplemental Figure : alone does not alter cytokine production by Cdf +/+ or Cdf -/- MDC. Purified CDc + MDC from Cdf +/+ or Cdf -/- mice were treated with. or.% for h. The cell culture media were collected, and the levels of the indicated cytokines were determined. Data are expressed as means + S.E.M. from separate experiments.
11 (A) Macrophage DC Mast cell T cell NK cell NKT cell cell CONT CDf Relative CDf expression (compared to CDf cells) CONT Macrophage DC Mast cell () CDb+CD- CDb-CD+ CDb-CD- pdc CONT Relative CDf expression (compared to CDf cells) CONT CDf CDb+CD- CDb-CD+ CDb-CD- pdc Supplementary figure. CDf expression on lamina propria cells. Lamina propria cells were isolated from the colon of Cdf +/+ or Cdf -/- mice treated without or with on day. (A) Surface expression of CDf on colonic macrophages (CD + F/ + CDb + CD + LyG - CDc - ), DC (CD + CDc + F/ - LyG - CD - ), neutrophils (CD + CDb + LyG + CDc - F/ - ), mast cells (CD + CDb + FcεRI + ), T cells (CD + CD + ), NK cells (CD + CD - NK. + ), and NKT cells (CD + CD + NK. + ) was determined by flow cytometry. () Surface expression of CDf on DC sub-populations was determined by flow cytometry. Histograms in (A) and () illustrate the expression level of CDf on the surface of cells derived from Cdf +/+ (black solid line) or Cdf -/- mice (gray histograms). The graph shows the fold changes in CDf expression on cells derived from Cdf +/+ mice compared to cells derived from Cdf -/- mice. Results are representative of three independent experiments.
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