Figure 1.
Intestinal colonization and systemic spread of S. Typhimurium.
Number of viable bacteria in total (A) small intestine and (B) colon at day 1 and 2 p.i. The results represent the median values from 2–3 independent experiments (n = 3–6 per group). (C) Whole body IVIS imaging of 1-day-old mice orally infected with 5×103 CFU of a lux expressing S. Typhimurium strain. Strength of the detected light emission in p/sec/cm2/sr. 4 mice were used per group in 2 independent experiments. (D and E) Number of viable bacteria in total liver (D) and spleen (E) at day 1, 2, and 4 p.i. with the indicated inoculum. The results represent the median values from 2–3 independent experiments (n = 3–6 per group). (F and G) Colonization of the small intestine and colon (F) as well as organ counts of liver and spleen (G) 4 days after oral infection of 1-day-old (102 CFU), 6-day-old (5×102 CFU) and streptomycin pre-treated adult (6 week-old) mice (5×108 CFU). The values indicate the total organ CFU divided by the inoculum. The results represent the median values from 2–3 independent experiments (n = 3–6 per group).
Figure 2.
Comparative analysis of WT and invasion-deficient Salmonella.
(A–C) Organ counts in liver (A), spleen (B) and MLN (C) 1, 2 and 4 days after oral infection of 1-day-old mice with 102 CFU WT (open circles), ΔinvC SPI1 mutant (filled circles) or complemented ΔinvC pinvC (open squares) S. Typhimurium. The results represent the median values from 3–4 independent experiments (n = 9–15 per group). (D) Immunostaining of S. Typhimurium (red) in small intestinal tissue sections of 1-day-old mice 4 days p.i. with 102 CFU WT or SPI1 mutant (ΔinvC) S. Typhimurium. Counterstaining with E-cadherin (green) and Dapi (blue). Bar, upper panel = 20 µm; lower panel = 5 µm. (E) Viable bacteria cultured from gentamicin treated primary enterocytes isolated at day 4 p.i. with WT or SPI1 mutant (ΔinvC) S. Typhimurium. The results represent the median values from one out of two independent experiments (n = 4–6 per group). (F) Median of the number of intraepithelial gentamicin-protected bacteria as shown in (E) divided by the total number of bacteria cultured from untreated isolated IECs (in %). (G) Flow cytometric analysis of infected enterocytes (Fl-1) isolated at day 4 after infection of 1-day-old mice with 102 CFU WT or SPI1 mutant (ΔinvC) (GFP+) S. Typhimurium. Cells were gated for the epithelial cell marker EpCAM (APC). The number of GFP-positive enterocytes of all EpCAM+ cells is indicated (%). One representative data set of three independent experiments is shown. Note that due to the relatively high autofluorescence of enterocytes, cell infected by low numbers of bacteria may remain undetected; the indicated gating might therefore underestimate the number of Salmonella infected cells. (H) Organ counts in liver, spleen and MLN after oral infection of 6-week-old streptomycin (20 mg) pretreated mice infected with 2×108 CFU WT (open circles, n = 21) or isogenic ΔinvC SPI1 mutant (filled circles, n = 18) S. Typhimurium (n = 6). The results represent the median values from three experiments.
Figure 3.
Absence of mature M cells in the neonatal small intestine.
(A) Comparative gene expression analysis of M cell specific genes (black: Spi-B, Ccl9), antimicrobial peptide or protein genes (red: Defcr3, Defcr4, Defcr6, Defcr20, Defcr22, Defcr23, Defcr-rs1, Defcr-rs7, Defcr-rs12, Reg3g, Saa1), cyclin genes (brown: Ccna2, Ccnb1, Ccnb2, Ccnc, Ccnd1, Ccnd2, Ccnd3, Ccne1, Ccne2, Ccnf), mucin genes (dark red: Muc2, Muc3, Muc4, Muc5b, Muc6, Muc13, Muc20), autophagosome constituents (light green: Atg3, Atg4a, Atg4b, Atg4c, Atg4d, Atg5, Atg7, Atg10, Atg12, Atg16l1, Atg16l2, LC3, p62, NDP52), and SCV constituents (dark green: gp91-phox, p22-phox, iNOS, vATPase, Eea1, Lamp1, Lamp2, Rab5a, Rab5b, Rab5c, Rab7, lysosomal acid phosphatase, mannose-6-phosphate receptor, cathepsin A exopeptidase, cathepsin D endopeptidase, cathepsin L endopeptidase, G6pc, Frap1) obtained from a two-color comparative gene expression array of isolated highly pure primary intestinal epithelial obtained from healthy 3-day-old versus 21-days-old C57BL/6 mice. The expression array data are accessible through GEO Series accession numbers GSE35596 and GSE35597. (B) Fold mRNA expression analysis of Ccl9 and Spi-B in primary enterocytes from 21-day-old mice as compared to 3-day-old neonate animals. The results represent the mean ± SD (n = 4 per group). (C) Quantitative RT-PCR analysis for Spi-B mRNA in total enterocytes isolated from healthy C57BL/6 mice at the indicated age (n = 3–4 per group). (D) Immunostaining for the M cell markers Ccl9, gp2 and UEA-1 (red) in small intestinal tissue sections obtained from healthy 5-, 10-, 15-, 21-, and 56-day-old adult C57BL/6 mice. Peyer's patches were identified using an anti-CD45 antibody (green). Counterstaining with Dapi (blue). Bar = 100 µm.
Figure 4.
Visualization of intraepithelial Salmonella.
(A) Immunostaining in small intestinal tissue 4 days after 102 CFU S. Typhimurium (red) infection of 1-day-old neonate mice. Counterstaining with E-cadherin (green) and Dapi (blue). i and ii, bar = 20 µm; iii–viii, bar = 5 µm. (B) Transmission electron microscopy of enterocytes from 1-day-old mice obtained at day 4 p.i. with S. Typhimurium. Image iii and iv represent enlarged parts of image i and ii, respectively. Image vii and viii represent enlarged images of the bacteria marked with an arrow in image v and vi, respectively. Bar: i and ii = 2 µm; iii–viii = 1 µm.
Figure 5.
Characterization of S. Typhimurium infected enterocytes.
(A) Immunostaining of C57BL/6 mice simultaneously infected with two genetically labelled S. Typhimurium strains orally administrated at a ratio of 1∶1. Staining was performed simultaneously with fluorophore-conjugated anti-GFP (green) and anti-mCherry antibodies (red). Note the presence of equal numbers of both green and red bacteria in the intestinal lumen in (i). Note also that the anti-mCherry antibody cross-reacted to some extend with the epithelial brush border. Counterstaining with Dapi (blue). Bar, i = 50 µm; ii and iii = 10 µm; iv and v = 5 µm. (B) Co-immunostaining of the SCV marker Lamp1 (red) and S. Typhimurium (green). i represents the merged image, ii–iv single color channel images. v shows a three-dimensional reconstruction to illustrate the punctuate Lamp1 staining surrounding the SCV. Counterstaining with Dapi (blue). Bar, i = 5 µm. (C) Quantitative analysis of the percentage of S. Typhimurium microcolonies associated with Lamp1 staining. All detected microcolony positive cells in 3 sections obtained from 3 S. Typhimurium infected animals were analyzed at day 4 p.i. The results represent the mean ± SD. (D) Co-immunostaining for active caspase 3 (white, arrow) and Salmonella (red) in small intestinal tissue sections obtained at day 4 p.i. Image ii represents an enlarged part of image i as indicated. Counterstaining with E-cadherin (green) and Dapi (blue). Bar, i = 50 µm. (E) Quantitative analysis of the percentage of active caspase 3 positive enterocytes among S. Typhimurium-infected and non-infected cells. 20 image areas (Magnification ×20) of intestinal sections from 3 S. Typhimurium infected animals were analyzed at day 4 p.i. The results represent the mean ± SD.
Figure 6.
Invasion-associated epithelial innate immune stimulation.
(A and B) Quantitative RT PCR for (A) Cxcl2 and (B) Cxcl5 mRNA in enterocytes isolated at 1, 2 and 4 days p.i. with 102 CFU WT (open circles, n = 13), ΔinvC SPI1 mutant (filled circles, n = 6) or complemented ΔinvC pinvC (semi-filled circles, n = 12) S. Typhimurium. The results represent the median values from 2–5 independent experiments (n = 6–13 per group). (C) Heat map of the relative gene expression in enterocytes of each 4 individual WT or SPI1 mutant (ΔinvC) S. Typhimurium infected animals (#1–#4) as compared to 4 non-infected control animals (fold increase). The 50 most highly expressed genes following WT S. Typhimurium infection are shown. (D) COG analysis of >2.5-fold induced genes in primary isolated intestinal epithelial cells following low dose (102 CFU) wildtype S. Typhimurium infection. (E and F) Quantitative RT PCR for (A) Cxcl2 and (B) Cxcl5 mRNA in enterocytes isolated at 4 days p.i. after infection of 1-day-old neonate (1–2×102 CFU), 6-day-old neonate (5×102 CFU), 6 week-old adult (2×108 CFU) mice with WT S. Typhimurium (open circles) or uninfected age-matched control animals (open squares). The results represent the median values from three independent experiments (n = 3–6 per group).
Figure 7.
Epithelial innate immune stimulation during neonatal Salmonella infection.
(A and B) Quantitative RT PCR for (A) Cxcl2 and (B) Reg3γ mRNA in enterocytes isolated from the indicated gene-deficient mice at 4 days p.i. with 102 CFU WT S. Typhimurium. The results represent the median values from 2–3 independent experiments (n = 3–14 per group: wt, n = 14; Tlr4−/−, n = 9; Tlr5−/−, n = 6; Myd88−/−, n = 3; Trif−/−, n = 12; TLR2−/−, n = 12; TLR9−/−, n = 12; 3d (Unc93B1 H412R), n = 3; NOD2−/−, n = 12). (C and D) Organ counts in (C) colon, spleen, liver and (D) mesenteric lymph nodes (MLN) of 1-day-old Tlr4−/− neonate mice after oral infection with 102 CFU WT S. Typhimurium. The results represent the median values from 3 independent experiments (n = 3–6 per group). (E) Immunostaining of small intestinal tissue 4 days after infection of 1-day-old Tlr4−/− neonate mice with 102 CFU S. Typhimurium (red). Counterstaining with E-cadherin (green) and Dapi (blue). i, bar = 20 µm; ii, bar = 10 µm. (F) Viable bacteria cultured from primary enterocytes isolated from small intestinal tissue at day 4 p.i. with WT S. Typhimurium and left untreated or incubated in gentamicin. The results represent the median values from one out of two independent experiments (n = 7–9 per group). (G) Flow cytometric analysis of enterocytes isolated at day 4 p.i. from 1-day-old Tlr4−/− mice infected with 102 CFU WT (GFP+) S. Typhimurium or from age-matched control animals. Cells were gated for the epithelial cell marker EpCAM (APC). The number of GFP-positive enterocytes of all EpCAM+ cells is indicated (%). One representative data set of three independent experiments is shown.
Figure 8.
Mucosal tissue response following S. Typhimurium infection in neonate mice.
(A) Flow cytometric analysis of lamina propria myeloid immune cells prepared from 1-day-old neonates infected for 4 days with 102 CFU S. Typhimurium or non-infected age-matched control animals. Representative images are shown. (B) Quantitative analysis of CD11b+ F4/80+ MHCII− CD11c− tissue invading macrophages and CD11b+ F4/80− MHCII− CD11c− polymorphonuclear cells relative to total CD45+ cells in the lamina propria of 1-day-old neonates infected for 4 days with 102 CFU S. Typhimurium or non-infected age-matched control animals. The results represent the mean ± SD from 2 independent experiments (n = 4). (C) Hematoxilin and eosin staining of intestinal tissue sections of neonatal mice (small intestine) left untreated or 4 day p.i. with S. Typhimurium (102 CFU). Bar = 50 µm.