TLR5 expression in the small intestine depends on the adaptors MyD88 and TRIF, but is independent of the enteric microbiota

Abstract

In our recent article Hörmann and coworkers have reported a role for epithelial cell-intrinsic TLR2 signaling for proliferation and renewal of the small intestinal epithelium. In this study, MyD88 and TRIF expression in the small intestine were affected by gut microbiota. Here, we report that in contrast to TLR2 and its co-receptor TLR1, TLR5 transcripts are not changed by presence of gut microbiota nor regulated through TLR2 or TLR4. Similar to TLR2 also TLR5 depends on MyD88 and TRIF adaptors. Our results indicate that TLR adaptor molecules could be determinants of TLR expression in the small intestine.

Keywords: MyD88; TLR2; TLR5; TOLLIP; TRIF; germ-free; gut microbiota; small intestine.

Figure 1.

The gut microbiota does not change TLR5 transcript levels and TLR5 expression does not depend on intact TLR2 or TLR4 signaling in the small intestine. (A) Small intestinal TLR5 mRNA levels of 10–14 week old male GF and CONV-R C57BL/6 mice (N = 6–8 mice per group) were quantified by qRT-PCR relative to L32 (primers: TLR5 forward GCA GGA TCA TGG CAT GTC AAC; TLR5 reverse ATC TGG GTG AGG TTA CAG CCT). (B) and (C) Small intestinal TLR5 mRNA levels were quantified by qRT-PCR relative to L32 in female Tlr2^-/- (N = 7 mice per group) and Tlr4^-/- C57BL/6J mice (N = 7 mice per group). Results are shown as means +− s.e.m. ns = not significant; independent samples Student's t-test.

Figure 2.

TLR5 deficiency does not impact on mRNA expression of bacteria sensing TLRs in the small intestine. Small intestinal mRNA levels of TLR2 (A), TLR1 (B), TLR6 (C), TLR4 (D) of 10–14 week old male Tlr5^-/- C57BL/6 mice (N = 5–7 per group) were quantified by qRT-PCR relative to L32 (primers see ref. One). Results are shown as means +− s.e.m. ns = not significant; independent samples Student's t-test.

Figure 3.

Transcripts of TLR adaptors are changed by the microbiota and MyD88 interferes with TRIF and TOLLIP. (A) Small intestinal mRNA expression levels of the TLR adaptor TOLLIP in 10–14 week old male GF and CONV-R C57BL/6 mice (N = 4–6 mice per group) was quantified by qRT-PCR relative to L32 (primers: Tollip forward CCC AGG ACT TCC TCC GTA TAA; Tollip reverse AGT CT GCC ATA ATT CTT TGC CA). (B) and (C) Small intestinal TOLLIP mRNA levels were quantified by qRT-PCR relative to L32 in female Myd88^-/- (N = 7 per group) and Trif^-/- C57BL/6J mice (N = 6–7 per group). (D) TRIF mRNA levels quantified by qRT-PCR relative to L32 in female Myd88^-/- C57BL/6 mice (N = 7 per group). (E) MyD88 mRNA levels quantified by qRT-PCR relative to L32 in female Trif^-/- C57BL/6 mice (N = 7 per group). mRNA levels of the cell cycle marker Cyclin D1 in (F) female Myd88^-/- C57BL/6 mice (N = 6–7 per group) and (G) female Trif^-/- C57BL/6 mice (N = 7 per group). Results are shown as means +− s.e.m. Two asterisks, P<0.01; 4 asterisks, P<0.0001; independent samples Student's t-test.

Figure 4.

TLR5 transcript levels are dependent on TLR adaptor proteins. (A) MyD88-deficiency reduces TLR5 transcript levels in the small intestine (N = 3–7 mice per group). (B) TRIF-deficiency reduces TLR5 transcript levels in the small intestine (N = 7 mice per group). Results are shown as means +− s.e.m. Three asterisks, P<0.001; 4 asterisks, P<0.0001; independent samples Student's t-test.

Figure 5.

Cell-intrinsic TLR2 signaling in the small intestinal epithelium is induced by the gut microbiota. TLR adaptors impact on TLR expression.