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Regional mucosa-associated microbiota determine physiological expression of TLR2 and TLR4 in murine colon - PubMed

  • ️Fri Jan 01 2010

Regional mucosa-associated microbiota determine physiological expression of TLR2 and TLR4 in murine colon

Yunwei Wang et al. PLoS One. 2010.

Abstract

Many colonic mucosal genes that are highly regulated by microbial signals are differentially expressed along the rostral-caudal axis. This would suggest that differences in regional microbiota exist, particularly mucosa-associated microbes that are less likely to be transient. We therefore explored this possibility by examining the bacterial populations associated with the normal proximal and distal colonic mucosa in context of host Toll-like receptors (TLR) expression in C57BL/6J mice housed in specific pathogen-free (SPF) and germ-free (GF) environments. 16S rRNA gene-based terminal restriction fragment length polymorphism (T-RFLP) and clone library analysis revealed significant differences in the community structure and diversity of the mucosa-associated microbiota located in the distal colon compared to proximal colon and stool, the latter two clustering closely. Differential expression of colonic TLR2 and TLR4 along the proximal-distal axis was also found in SPF mice, but not in GF mice, suggesting that enteric microbes are essential in maintaining the regional expression of these TLRs. TLR2 is more highly expressed in proximal colon and decreases in a gradient to distal while TLR4 expression is highest in distal colon and a gradient of decreased expression to proximal colon is observed. After transfaunation in GF mice, both regional colonization of mucosa-associated microbes and expression of TLRs in the mouse colon were reestablished. In addition, exposure of the distal colon to cecal (proximal) microbiota induced TLR2 expression. These results demonstrate that regional colonic mucosa-associated microbiota determine the region-specific expression of TLR2 and TLR4. Conversely, region-specific host assembly rules are essential in determining the structure and function of mucosa-associated microbial populations. We believe this type of host-microbial mutualism is pivotal to the maintenance of intestinal and immune homeostasis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. T-RFLP analysis of microbial communities in mouse colonic tissue and stool samples.

(A) Representative T-RFLP patterns of bacterial populations in mouse stool sample (S), proximal (PC) and distal colon (DC). 16S rRNA genes were obtained from amplification of DNA template (50 ng DNA), digested by restriction enzyme Msp I and analyzed by capillary electrophoresis. Fragment size in base pairs is shown at the top and peak height is shown as relative fluorescence. (B) A representative phylogenetic tree was built up from 4 littermate mice based on T-RFLP analysis. Similarities of bacterial populations between stool sample (S), proximal colon (PC) and distal colon (DC) were compared by Bray-Curtis distance calculations. The scale bar shows the distance of similarity.

Figure 2
Figure 2. 16S rRNA gene clone library sequence analysis of microbial communities in mouse colon and stool samples.

(A) Relative bacterial composition in colonic and stool samples from 3 mice (M1–M3) bred at the University of Chicago (C) and 3 obtained from the Jackson laboratories (J) were shown at the phylum level. 16S rRNA gene sequences were grouped into different phyla using the RDP classifier tool at a default confidence threshold (80%). (B) Principal coordinate analysis (PCA) of clone libraries was performed on samples collected from mice of the University of Chicago (triangles (green)) and mice obtained from the Jackson Laboratories (squares (blue)). Mice from the two different facilities clustered separately. Clustering of distal colon (DC) samples was also distinct from those of the proximal colon (PC) and feces (S) in each individual mouse. (C) OTUs were assigned and calculated by DOTUR analysis at 97% cutoff level. Fewer OTUs were found in mouse distal colon compared to those found in the proximal colon and feces.

Figure 3
Figure 3. Expression of TLR2 and TLR4 in mouse colon.

(A) and (B) Colonic mucosal scrapings from SPF and GF mice were used to extract RNA. Expressions of TLR2 and TLR4 between mouse proximal colon and distal colon were compared by real-time PCR as described in Methods (n = 12). (C) Expression of TLR2 and TLR4 in mucosal scrapings by Western blot. Cell lysates from mucosal scrapings of mouse proximal colon (P) and distal colon (D) were separated on a 10% SDS-PAGE gel and blot by anti-TLR2 and anti-TLR4 specific antibodies respectively. Relative protein expression was quantified by measuring the densitometry using NIH Image J 1.54 software which was denoted below each sample. Expression of actin was used as an internal control for each sample.

Figure 4
Figure 4. Analyses of expression of TLR2 and TLR4 in mucosal epithelial cells by LCM.

Laser capture microdissection (LCM) was performed on frozen sections of mouse proximal colon and distal colon. RNA was extracted from microdissected mucosal epithelial cells and transcribed into cDNA which were used to PCR-amplify TLR2 and TLR4, respectively. The expression of GAPDH was used as an internal control. (A) and (B) Quantitative real-time PCR was used to compare the mRNA expression of TLR2 and TLR4 in microdissected mucosal epithelial cells as described in Methods (n = 6).

Figure 5
Figure 5. Microbiota transplantation (transfaunation) in germ-free (GF) mice.

GF mice on C57Bl/6J background were transferred with cecal content of SPF mice by gavage. After 3 weeks colonization, mice were sacrificed and colonic tissues and stool samples were collected for bacterial and gene expression analysis. (A) Microbiota in stool (S), proximal colon (PC) and distal colon (DC) of GF recipient mice (GF-R) was established after 3 weeks transplantation. The T-RFLP profiles are similar between donor (SPF-D, n = 1) and recipient mice (n = 3) for corresponding stool and tissue samples. Proximal colon and distal colon clustered differently in transfaunated GF recipient mice. Similar to donor mice, the richness of the distal colonic microbiota was less than that of proximal colon and stool (B). The regional expression of TLR2 and TLR4 (C) was restored in GF mice following transfaunation. Relative protein expression was denoted below each sample. Actin was used as the internal loading control.

Figure 6
Figure 6. Transrectal administration of cecal microbiota into the distal colon stimulates TLR2 expression.

Mice on C57Bl/6J background housed in SPF conditions were treated by rectal enemas with sterile saline or cecal content prepared as a slurry. Expression of TLR2 and TLR4 in distal colon was compared by real-time PCR (A) and Western blot (B) analysis. Relative protein expression assessed by densitometry was denoted below each sample. Actin was used as the internal loading control (n = 3).

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