Table 1.
Primer Table.
Figure 1.
Microbial colonization leads to induction of TLR2 receptor expression in the small intestine.
a, Relative TLR2 mRNA levels in small intestinal tissues from GF, CONV-D and CONV-R mice (n = 7 Swiss Webster mice per group). b, TLR2 immunoblot of isolated small-intestinal enterocyte lysates from GF and CONV-R mice (n = 6 mice per group; shown is one representative blot). c–e, Relative TLR1, 6 and 4 mRNA levels in small intestinal tissues from GF, CONV-D and CONV-R mice (n = 6–7 mice per group). f, qPCR analyses of feces samples of control mice and mice treated with antibiotics for 7 days. qPCR was performed using universal primers for 16S bacterial sequences and normed to E. coli bacterial counts (cfu/µl). g–j, Relative mRNA levels of TLR2, 1, 6 and 4 in small intestinal tissues from CONV-R mice treated with a cocktail (ABX) of ampicillin (1 g/L) and neomycin (0.5 g/L) for 7 days (n = 6–7 mice per group). Female Swiss Webster mice or cells isolated from these mice were analyzed. Results are shown as means ± s.e.m. One asterisk, P<0.05; two asterisks, P<0.01; three asterisks, P<0.005.
Figure 2.
Adapter molecules MyD88 and TRIF alter expression of TLRs in the ileum – indications for TLR receptor cross-talk in the small intestine.
a+b, Relative MyD88 and TRIF mRNA levels in small intestine from GF, CONV-D and CONV-R mice (n = 6–7 Swiss Webster mice per group). c+d, Relative MyD88 and TRIF mRNA levels in small intestine from CONV-R mice treated for 7 days with an antibiotic cocktail (ABX) (n = 6–8 Swiss Webster mice per group). e–h, Relative TLR2, 1, 6 and 4 mRNA levels in small intestine from MyD88−/− mice compared to wildtype (WT) controls (n = 6–7 C57BL/6J mice per group). i–l, Relative TLR2, 1, 6 and 4 mRNA levels in small intestine from TRIF−/− mice compared to WT controls (n = 6–7 mice per group). m–o, Relative TLR1, 6 and 4 mRNA levels in small intestine from TLR2−/− mice compared to WT controls (n = 6–7 mice per group). p, Relative TLR2 mRNA levels in small intestine from TLR4−/− mice compared to WT controls (n = 6–7 mice per group). Female mice were analyzed. Results are shown as means ± s.e.m. One asterisk, P<0.05; two asterisks, P<0.01; three asterisks, P<0.005; four asterisks, P<0.001.
Figure 3.
Agonist-specific orchestration of the TLR expression profile in a mouse small intestinal epithelial cell line.
a+b, Relative mRNA expression of TLR2 in MODE-K cells stimulated with PG (50 µg/ml) or Pam3CSK4 (0.5 µg/ml) for 2, 4 or, 8 hours (n = 4). c, TLR2 immunoblot of PG or Pam3CSK4 stimulated MODE-K cells. Cells were treated with or without (CTR) PG or Pam3CSK4 for 2, 4, or 8 hours (n = 3, representative blot). d–f, Relative TLR1, 6 and 4 mRNA expression in MODE-K cells stimulated with Pam3CSK4 or LPS for 2, 4, or 8 hours (n = 4). Results are shown as means ± s.e.m. One asterisk, P<0.05; two asterisks, P<0.01; three asterisks, P<0.005; four asterisks, P<0.001.
Figure 4.
Monocolonization with E. coli JP313 decreases TLR6 transcript levels.
a–d, Relative TLR2, 1, 6, and 4 mRNA levels in small intestine from mice colonized for 14 days with E. coli JP313 (n = 7 male C57BL/6 mice per group). Results are shown as means ± s.e.m. Two asterisks, P<0.01.
Figure 5.
MODE-K cells are reactive to TLR2 activating PAMPs and LPS.
a, Detection of ERK1/2-phosphorylation of TLR-agonist (LTA, 10 µg/ml; Pam3CSK4, 0.5 µg/ml; HKLM, 2×105 cells per well; PG, 50 µg/ml; MALP-2, 2 µg/ml; LPS, 100 ng/ml) treated MODE-K cells (n = 4–10). b, Detection of IkB-phosphorylation of TLR-agonist treated MODE-K cells (n = 3). c, Detection of AKT-phosphorylation of Pam3CSK4 treated MODE-K cells (n = 3).One representative immunoblot is shown for each agonist and kinase.
Figure 6.
TLR2 signals in the small intestinal epithelium evoke a proliferation response and increase signs of apoptosis in terminally differentiated epithelium.
a–c, Relative Ki67, Cyclin D1 or Caspase-3 mRNA levels in small intestine from GF and CONV-R mice (n = 7 Swiss Webster mice per group). d–f, Relative Ki67, Cyclin D1 or Caspase-3 mRNA levels in small intestine from Tlr2−/− mice compared with WT controls (n = 7 C57BL/6J mice per group). g, Number of Caspase-3 positive cells per 10 villi in small intestinal samples from WT or Tlr2−/− mice. h–j, Relative Ki67, Cyclin D1 and Caspase-3 mRNA levels in small intestine from mice colonized for 14 days with E. coli JP313 (n = 7 C57BL/6 mice per group). k, Relative Ki67 mRNA levels in MODE-K cells stimulated with PG (50 µg/ml) for 2, 4, or 8 hours (n = 4). l, Relative proliferation of PG treated MODE-K cells measured by incorporation of BrdU compared to untreated control cells (n = 3). m, Relative proliferation of MODE-K cells transfected with siRNA against TLR2 or scrambled control RNA measured by incorporation of BrdU (n = 4). n, TLR2 immunoblot of siRNA transfected MODE-K cells. Results are shown as means ±s.e.m. One asterisk, P<0.05; two asterisks, P<0.01; three asterisks, P<0.005; four asterisks, P<0.001.