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Microbial genomic analysis reveals the essential role of inflammation in bacteria-induced colorectal cancer.

Arthur JC, Gharaibeh RZ, Mühlbauer M, Perez-Chanona E, Uronis JM, McCafferty J, Fodor AA, Jobin C - Nat Commun (2014)

Bottom Line: RNA-sequence analysis indicates significant changes in E. coli gene catalogue in Il10(-/-) mice, with changes mostly driven by adaptation to the intestinal environment.Expression of specific genes present in the tumour-promoting E. coli pks island are modulated by inflammation/CRC development.Thus, progression of inflammation in Il10(-/-) mice supports Enterobacteriaceae and alters a small subset of microbial genes important for tumour development.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27713, USA [2].

ABSTRACT
Enterobacteria, especially Escherichia coli, are abundant in patients with inflammatory bowel disease or colorectal cancer (CRC). However, it is unclear whether cancer is promoted by inflammation-induced expansion of E. coli and/or changes in expression of specific microbial genes. Here we use longitudinal (2, 12 and 20 weeks) 16S rRNA sequencing of luminal microbiota from ex-germ-free mice to show that inflamed Il10(-/-) mice maintain a higher abundance of Enterobacteriaceae than healthy wild-type mice. Experiments with mono-colonized Il10(-/-) mice reveal that host inflammation is necessary for E. coli cancer-promoting activity. RNA-sequence analysis indicates significant changes in E. coli gene catalogue in Il10(-/-) mice, with changes mostly driven by adaptation to the intestinal environment. Expression of specific genes present in the tumour-promoting E. coli pks island are modulated by inflammation/CRC development. Thus, progression of inflammation in Il10(-/-) mice supports Enterobacteriaceae and alters a small subset of microbial genes important for tumour development.

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Inflammation is required for E. coli-enhanced tumorigenesis in Il10−/− miceHistologic scoring of (a) inflammation and (b) tumorigenesis. c) Representative H&E histology at 40X magnification, scale bars indicate 1.0mm, and neoplastic lesion indicated with arrowhead. d) Luminal E. coli load by qPCR of fecal genomic DNA. (a, b, d) Each symbol represents an individual mouse, line at mean, P-values by Kruskal-Wallis with Dunn’s test for multiple comparisons.
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Figure 4: Inflammation is required for E. coli-enhanced tumorigenesis in Il10−/− miceHistologic scoring of (a) inflammation and (b) tumorigenesis. c) Representative H&E histology at 40X magnification, scale bars indicate 1.0mm, and neoplastic lesion indicated with arrowhead. d) Luminal E. coli load by qPCR of fecal genomic DNA. (a, b, d) Each symbol represents an individual mouse, line at mean, P-values by Kruskal-Wallis with Dunn’s test for multiple comparisons.

Mentions: RNA-seq analysis revealed substantial changes to the E. coli transcriptome over the time of intestinal colonization in Il10−/− mice (Fig. 3), but it was unclear if these changes were due to inflammation/cancer or adaptation to the mammalian intestine (i.e. colonization time). Moreover, the functional implication for these E. coli transcriptional responses in mediating this carcinogenic effect was unclear. To determine what changes in the E. coli transcriptome are induced by inflammation and may impact the development of CRC, we used Il10−/−;Rag2−/− mice that lack functional T and B cells, essential cellular components for development of chronic colitis25. One cohort of Il10−/− mice was not injected with AOM in order to evaluate the pro-carcinogenic effect of E. coli NC101 in the absence of initiation by a carcinogen. Histological analysis revealed high levels of inflammation that did not differ between AOM/Il10−/− and Il10−/− mice, but a complete absence of inflammation in AOM/Il10−/−;Rag2−/− mice at 20 weeks post-colonization (Fig. 4a). The number of macroscopic tumors was higher in AOM/Il10−/− vs. Il10−/− mice, and invasive tumors (neoplasia score of 4 or 5) were only detected in AOM-treated animals (Fig. 4b and c), supporting our earlier observations6 that E. coli NC101 rarely induces invasive tumors in Il10−/− mice in the time frame tested. Both AOM/Il10−/− and Il10−/− mice exhibited a significantly higher tumor burden than AOM/Il10−/−;Rag2−/− mice (Fig. 4b). In these 9 non-inflamed AOM/Il10−/−;Rag2−/− mice, 4 exhibited no macroscopic tumors, and 5 exhibited 1 non-invasive macroscopic tumor each. In agreement with histological inflammation, expression of inflammatory cytokines was lower in AOM/Il10−/−;Rag2−/− vs. AOM/Il10−/− or Il10−/− mice (Supplementary Fig. 4). Fecal E. coli load did not differ between AOM/Il10−/−;Rag2−/−, AOM/Il10−/− or Il10−/− mice, as measured by 16S PCR analysis, suggesting that low inflammation and tumor loads in AOM/Il10−/−;Rag2−/− mice are not due to fluctuation in E. coli NC101 abundance (Fig. 4d). These data demonstrate that the sole presence of E. coli is not sufficient to induce CRC in Il10−/− mice and that inflammation is essential to the tumorigenic process.


Microbial genomic analysis reveals the essential role of inflammation in bacteria-induced colorectal cancer.

Arthur JC, Gharaibeh RZ, Mühlbauer M, Perez-Chanona E, Uronis JM, McCafferty J, Fodor AA, Jobin C - Nat Commun (2014)

Inflammation is required for E. coli-enhanced tumorigenesis in Il10−/− miceHistologic scoring of (a) inflammation and (b) tumorigenesis. c) Representative H&E histology at 40X magnification, scale bars indicate 1.0mm, and neoplastic lesion indicated with arrowhead. d) Luminal E. coli load by qPCR of fecal genomic DNA. (a, b, d) Each symbol represents an individual mouse, line at mean, P-values by Kruskal-Wallis with Dunn’s test for multiple comparisons.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4155410&req=5

Figure 4: Inflammation is required for E. coli-enhanced tumorigenesis in Il10−/− miceHistologic scoring of (a) inflammation and (b) tumorigenesis. c) Representative H&E histology at 40X magnification, scale bars indicate 1.0mm, and neoplastic lesion indicated with arrowhead. d) Luminal E. coli load by qPCR of fecal genomic DNA. (a, b, d) Each symbol represents an individual mouse, line at mean, P-values by Kruskal-Wallis with Dunn’s test for multiple comparisons.
Mentions: RNA-seq analysis revealed substantial changes to the E. coli transcriptome over the time of intestinal colonization in Il10−/− mice (Fig. 3), but it was unclear if these changes were due to inflammation/cancer or adaptation to the mammalian intestine (i.e. colonization time). Moreover, the functional implication for these E. coli transcriptional responses in mediating this carcinogenic effect was unclear. To determine what changes in the E. coli transcriptome are induced by inflammation and may impact the development of CRC, we used Il10−/−;Rag2−/− mice that lack functional T and B cells, essential cellular components for development of chronic colitis25. One cohort of Il10−/− mice was not injected with AOM in order to evaluate the pro-carcinogenic effect of E. coli NC101 in the absence of initiation by a carcinogen. Histological analysis revealed high levels of inflammation that did not differ between AOM/Il10−/− and Il10−/− mice, but a complete absence of inflammation in AOM/Il10−/−;Rag2−/− mice at 20 weeks post-colonization (Fig. 4a). The number of macroscopic tumors was higher in AOM/Il10−/− vs. Il10−/− mice, and invasive tumors (neoplasia score of 4 or 5) were only detected in AOM-treated animals (Fig. 4b and c), supporting our earlier observations6 that E. coli NC101 rarely induces invasive tumors in Il10−/− mice in the time frame tested. Both AOM/Il10−/− and Il10−/− mice exhibited a significantly higher tumor burden than AOM/Il10−/−;Rag2−/− mice (Fig. 4b). In these 9 non-inflamed AOM/Il10−/−;Rag2−/− mice, 4 exhibited no macroscopic tumors, and 5 exhibited 1 non-invasive macroscopic tumor each. In agreement with histological inflammation, expression of inflammatory cytokines was lower in AOM/Il10−/−;Rag2−/− vs. AOM/Il10−/− or Il10−/− mice (Supplementary Fig. 4). Fecal E. coli load did not differ between AOM/Il10−/−;Rag2−/−, AOM/Il10−/− or Il10−/− mice, as measured by 16S PCR analysis, suggesting that low inflammation and tumor loads in AOM/Il10−/−;Rag2−/− mice are not due to fluctuation in E. coli NC101 abundance (Fig. 4d). These data demonstrate that the sole presence of E. coli is not sufficient to induce CRC in Il10−/− mice and that inflammation is essential to the tumorigenic process.

Bottom Line: RNA-sequence analysis indicates significant changes in E. coli gene catalogue in Il10(-/-) mice, with changes mostly driven by adaptation to the intestinal environment.Expression of specific genes present in the tumour-promoting E. coli pks island are modulated by inflammation/CRC development.Thus, progression of inflammation in Il10(-/-) mice supports Enterobacteriaceae and alters a small subset of microbial genes important for tumour development.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27713, USA [2].

ABSTRACT
Enterobacteria, especially Escherichia coli, are abundant in patients with inflammatory bowel disease or colorectal cancer (CRC). However, it is unclear whether cancer is promoted by inflammation-induced expansion of E. coli and/or changes in expression of specific microbial genes. Here we use longitudinal (2, 12 and 20 weeks) 16S rRNA sequencing of luminal microbiota from ex-germ-free mice to show that inflamed Il10(-/-) mice maintain a higher abundance of Enterobacteriaceae than healthy wild-type mice. Experiments with mono-colonized Il10(-/-) mice reveal that host inflammation is necessary for E. coli cancer-promoting activity. RNA-sequence analysis indicates significant changes in E. coli gene catalogue in Il10(-/-) mice, with changes mostly driven by adaptation to the intestinal environment. Expression of specific genes present in the tumour-promoting E. coli pks island are modulated by inflammation/CRC development. Thus, progression of inflammation in Il10(-/-) mice supports Enterobacteriaceae and alters a small subset of microbial genes important for tumour development.

Show MeSH
Related in: MedlinePlus