Commensal bacteria and expression of two major intestinal chemokines, TECK/CCL25 and MEC/CCL28, and their receptors

Abstract

Background: CCL25/TECK and CCL28/MEC are CC chemokines primarily expressed in thymic dendritic cells and mucosal epithelial cells. Their receptors, CCR9 and CCR10, are mainly expressed on T and B lymphocytes. In human, mouse, pig and sheep CCL25 and CCL28 play an important role in the segregation and the compartmentalization of the mucosal immune system. As evidenced by early comparisons of germ-free and conventional animals, the intestinal bacterial microflora has a marked effect on host intestinal immune functions. However, little is known about the impact of bacterial colonization on constitutive and induced chemokine expressions as well as on the generation of anti-inflammatory mechanisms.

Methodology/principal findings: Therefore, we decided to focus by qPCR on the mRNA expression of two main gut chemokines, CCL25 and CCL28, their receptors CCR9 and CCR10, the Tregs marker Foxp3 and anti-inflammatory cytokines TGF-beta and IL-10 following colonization with different bacterial species within the small intestine. To accomplish this we used an original germ-free neonatal pig model and monoassociated pigs with a representative Gram-negative (Escherichia coli) or Gram-positive (Lactobacillus fermentum) commensal bacteria commonly isolated from the neonatal pig intestine. Our results show a consistent and marked effect of microbial colonization on the mRNA expression of intestinal chemokines, chemokine receptors, Foxp3 and TGF-beta. Moreover, as evidenced by in vitro experiments using two different cell lines, the pattern of regulation of CCL25 and CCL28 expression in the gut appears complex and suggests an additional role for in vivo factors.

Conclusions/significance: Taken together, the results highlight the key role of bacterial microflora in the development of a functional intestinal immune system in an elegant and relevant model for human immune system development.

Figure 1. Expression of CCL25/TECK mRNA in segments collected at 5–95% (pyloric sphincter = 5%; ileo-cecal junction = 95%) of SI length in gnotobiotic and conventional pigs derived by caesarean section and reared in isolators until 13 days of age.

cDNA was synthesized from 50 ng of RNA and subjected to qPCR using primer/probe sets for CCL25/TECK. Levels of expression are expressed in copy number. Copy numbers and median from four animals are shown in each group (GF: germ free; LF: Lactobacillus fermentum; EC: Escherichia coli; CV: conventionalized with fresh adult porcine fecal material). *, P<0.05, **, P<0.01 (Student's t test and Wilcoxon Signed Rank Test (Exact)).

Figure 2. Expression of CCL28/MEC mRNA in segments collected at 5–95% (pyloric sphincter = 5%; ileo-cecal junction = 95%) of SI length in gnotobiotic and conventional pigs derived by caesarean section and reared in isolators until 13 days of age.

cDNA was synthesized from 50 ng of RNA and subjected to qPCR using primer/probe sets for CCL28/MEC. Levels of expression are expressed in copy number. Copy numbers and median from four animals are shown in each group (GF: germ free; LF: Lactobacillus fermentum; EC: Escherichia coli; CV: conventionalized with fresh adult porcine fecal material). *, P<0.05, **, P<0.01 (Student's t test and Wilcoxon Signed Rank Test (Exact)).

Figure 3. Expression of CCR9 mRNA in segments collected at 5–95% (pyloric sphincter = 5%; ileo-cecal junction = 95%) of SI length in gnotobiotic and conventional pigs derived by caesarean section and reared in isolators until 13 days of age.

cDNA was synthesized from 50 ng of RNA and subjected to qPCR using primer/probe sets for CCR9. Levels of expression are expressed in copy number. Copy numbers and median from four animals are shown in each group (GF: germ free; LF: Lactobacillus fermentum; EC: Escherichia coli; CV: conventionalized with fresh adult porcine fecal material). *, P<0.05, **, P<0.01 (Student's t test and Wilcoxon Signed Rank Test (Exact)).

Figure 4. Expression of CCR10 mRNA in segments collected at 5–95% (pyloric sphincter = 5%; ileo-cecal junction = 95%) of SI length in gnotobiotic and conventional pigs derived by caesarean section and reared in isolators until 13 days of age.

cDNA was synthesized from 50 ng of RNA and subjected to qPCR using primer/probe sets for CCR10. Levels of expression are expressed in copy number. Copy numbers and median from four animals are shown in each group (GF: germ free; LF: Lactobacillus fermentum; EC: Escherichia coli; CV: conventionalized with fresh adult porcine fecal material). *, P<0.05, **, P<0.01 (Student's t test and Wilcoxon Signed Rank Test (Exact)).

Figure 5. Comparison of the expression of CCL25/TECK and CCR9 mRNA in Escherichia coli (EC) gnotobiotic and conventional (CV) pigs in segments collected at 5–95% (pyloric sphincter = 5%; ileo-cecal junction = 95%) of SI length.

cDNA was synthesized from 50 ng of RNA and subjected to qPCR using primer/probe sets for CCL25/TECK and CCR9. Levels of expression are expressed in copy number. Copy numbers and median from four animals are shown in each group. *, P<0.05, **, P<0.01 (Student's t test and Wilcoxon Signed Rank Test (Exact)).

Figure 6. Comparison of the expression of CCL28/MEC and CCR10 mRNA in Escherichia coli (EC) gnotobiotic and conventional (CV) pigs in segments collected at 5–95% (pyloric sphincter = 5%; ileo-cecal junction = 95%) of SI length.

cDNA was synthesized from 50 ng of RNA and subjected to qPCR using primer/probe sets for CCL28/MEC and CCR10. Levels of expression are expressed in copy number. Copy numbers and median from four animals are shown in each. *, P<0.05, **, P<0.01 (Student's t test and Wilcoxon Signed Rank Test (Exact)).

Figure 7. Comparison of the expression of Foxp3 mRNA in segments collected at 5–95% (pyloric sphincter = 5%; ileo-cecal junction = 95%) of SI length in gnotobiotic and conventional pigs derived by caesarean section and reared in isolators until 13 days of age.

cDNA was synthesized from 50 ng of RNA and subjected to qPCR using primer/probe sets for Foxp3 and GAPDH. Delta CT was calculated as follows: Foxp3 CT – GAPDH CT. Foxp3 quantities and median are shown for four animals in each group (GF: germ free; LF: Lactobacillus fermentum; EC: Escherichia coli; CV: conventionalized with fresh adult porcine fecal material), with samples expressing the highest levels of Foxp3 mRNA demonstrating the lowest delta CT value. *, P<0.05, **, P<0.01 (Student's t test and Wilcoxon Signed Rank Test (Exact)).

Figure 8. Expression of Cdx-2 and GAPDH mRNA by conventional PCR amplifications.

cDNA was synthesized from 50 ng of RNA extracted from small intestine (SI), IPI-2I, and SJPL cells. After 30 cycles of amplification, the PCR products were run on a 1.5% agarose gel and stained with ethidium bromide. Three DNA molecular weight markers were used (MW1 from Amersham and MW2, MW3 from Fermentas). The PCR product sizes are presented in Table 1.