Regional differences in colonic mucosa-associated microbiota determine the physiological expression of host heat shock proteins

Abstract

Cytoprotective heat shock proteins (Hsps) are critical for intestinal homeostasis and are known to be decreased in inflammatory bowel diseases. Signals responsible for maintenance of Hsp expression are incompletely understood. In this study, we find that Hsp25/27 and Hsp70 protein expressions are differentially regulated along the longitudinal length of the large intestine, being highest in the proximal colon and decreasing to the distal colon. This longitudinal gradient was similar in both conventionally colonized mouse colon as well as biopsies of human proximal and distal colon but was abolished in the colon of germ-free mice, suggesting a role of intestinal microbiota in the Hsp regional expression. Correspondingly, analysis of 16S ribosomal RNA genes of bacteria from each colonic segment indicated increased bacterial richness and diversity in the proximal colon. The mechanism of regulation is transcriptional, as Hsp70 mRNA followed a similar pattern to Hsp70 protein expression. Lysates of mucosa-associated bacteria from the proximal colon stimulated greater Hsp25 and Hsp70 mRNA transcription and subsequent protein expression in intestinal epithelial cells than did lysates from distal colon. In addition, transrectal administration of cecal contents stimulated Hsp25 and Hsp70 expression in the distal colon. Thus host-microbial interactions resulting in differential Hsp expression may have significant implications for the maintenance of intestinal homeostasis and possibly for development of inflammatory diseases of the bowel.

Fig. 1.

Expression of inducible heat shock protein 27 (Hsp27) and Hsp70 is higher in human ascending (proximal) colon compared with descending (distal) colon. A: immunohistochemical staining for Hsp27 and Hsp70. B: Western blots of Hsp27, Hsp70, Hsc70, and villin. C: real-time PCR analysis of mRNA abundance for Hsp27 and Hsp70 in colonic mucosa. Changes in Hsp mRNA relative to GAPDH were determined as fold change over right colon. Images shown are representative of 4 experiments. Results are means ± SE, n = 4. *P < 0.05 comparing distal and proximal colon by Student's t-test for protein (B) or mRNA (C).

Fig. 2.

Expression of inducible Hsp25 and Hsp70 is higher in mouse proximal compared with distal colonic mucosa. A: immunohistochemical staining for Hsp25 and Hsp70 in proximal and distal colon sections from C57BL/6 mouse. Image shown is representative of 4 individual experiments. B: Western blots of Hsp25, Hsp70, Hsc70, and villin in mucosa of jejunum and proximal and distal colon from C57BL/6 mouse. C: real-time PCR analysis of mRNA abundance of Hsp25 and Hsp70 in intestinal mucosa. Changes in Hsp mRNA relative to GAPDH were determined as fold change over proximal colon. Results are mean ± SE, n = 5. *P < 0.05, +P < 0.01, ++P < 0.001 comparing Hsp levels in different segments of the distal colon by ANOVA (B) or comparing jejunum or distal colon to proximal colon mRNA by ANOVA (C).

Fig. 3.

Hsp25 and Hsp70 expression levels are low in colonic mucosa of germ-free mice, and differential Hsp25 and Hsp70 expression between proximal and distal colon are not present in germ-free mice. A: immunohistochemical staining for Hsp25 and Hsp70 in proximal and distal colon sections from germ-free mice on C57BL/6 background. B: Western blots of Hsp25, Hsp70, Hsc70, and villin in mucosa of jejunum (J) and proximal (P) and distal colon (D) from germ-free mouse. Image shown is representative of 4 individual experiments. C: real-time PCR analysis of mRNA abundance of Hsp25 (top) and Hsp70 (bottom) in intestinal mucosa. Changes in Hsp mRNA relative to GAPDH were determined as fold change over control proximal colon. Results are means ± SE, n = 5. +P < 0.05 comparing Hsp (B) or mRNA (C) from control and germ-free mice by paired Student's t-test.

Fig. 4.

Difference of mucosa-associated bacteria profile in mouse proximal and distal colon. A: terminal restriction fragment-length polymorphism (T-RFLP) profiles of 16S ribosomal RNA (rRNA) genes from mucosa-associated bacteria from murine proximal and distal colon. Images shown are representative of 4 experiments. B: representative phylogenetic tree was built up from 4 mice on the basis of T-RFLP analysis. Similarities between proximal colon and distal colon were compared by Bray-Curtis distance calculations. The scale bar shows the distance of similarity. C: calculated richness and Shannon diversity for different bacteria profiles. Results are means ± SE, n = 4. *P < 0.05 comparing distal and proximal colon by Student's t-test.

Fig. 5.

Bacterial lysates from proximal colonic lumen content induce Hsp expression in young adult mouse colon (YAMC) cells. A: total bacteria 16S rRNA gene content was measured by qPCR using universal primers from mucosa scrapings of the proximal and distal colon. Total mucosa protein was used to normalize the sample sizes. Lysates from mucosa-associated bacteria of the proximal and distal colons of C57BL/6 mice were prepared as described in materials and methods. YAMC cells were incubated with the same amount of bacteria lysates (quantified by Coomassie dye-binding assay) for 16 h before harvest. B: YAMC cell Hsp25 and Hsc70 were analyzed using Western blots. C: real-time PCR analysis of mRNA abundance of Hsp25 and Hsp70 in YAMC cells. D: YAMC cells were treated with butyrate (0.5, 1.5, or 5.0 mM) or LPS (1, 3, or 10 mM) for 24 h before harvest. Cells without treatment were harvested as control. Hsp25, Hsp70, and Hsc70 were analyzed on Western blots. Images shown are representative of 4 experiments. Results are means ± SE, n = 4. *P < 0.05 comparing induction by distal and proximal colon lumen contents by paired Student's t-test (A and B) and 16S rRNA gene content of proximal and distal by paired Student's t-test (C).

Fig. 6.

Treatment of distal colon with cecal lysate by enema induces Hsps. Mice were treated daily by enema for 3 days with saline or cecal contents from conventional or germ-free mice suspended in saline. After treatment, mucosa was washed with saline and scraped, and protein lysates were prepared and analyzed by Western blots for Hsp25 and 70 and Hsc70 as described in materials and methods. Image shown is from 3 individual mice with saline or cecal lysate enema. Densitometric values were obtained using Image J and averaged for mice with saline enema in each group, and this average was used to analyze induction in cecal content in enema-treated mice. +P < 0.01 compared with saline enema-treated mice by paired Student's t-test.