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Gut microbiome derived metabolites modulate intestinal epithelial cell damage and mitigate Graft-versus-Host Disease

View Article: PubMed Central - PubMed

ABSTRACT

The impact of alterations in intestinal microbiota on microbial metabolites and on disease processes, such as graft-versus-host disease (GVHD), is not known. Here we performed unbiased analysis to identify novel alterations in gastrointestinal microbiota-derived short chain fatty acids (SCFA) after allogeneic bone marrow transplant (allo-BMT). Alterations in the amounts of only one SCFA, butyrate, were observed only within the intestinal tissue. The reduced butyrate in CD326+ intestinal epithelial cells (IECs) after allo-BMT resulted in decreased histone acetylation, which was restored upon local administration of exogenous butyrate. Butyrate restoration improved IEC junctional integrity, decreased apoptosis, and mitigated GVHD. Furthermore, alteration of the indigenous microbiota with 17 rationally selected strains of high butyrate producing Clostridia also decreased GVHD. These data demonstrate a heretofore unrecognized role of microbial metabolites and suggest that local and specific alteration of microbial metabolites has direct salutary effects on GVHD target tissues and can mitigate its severity.

No MeSH data available.


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Rationally altering the commensal microbiota reduces GVHD(a) 16S rRNA-encoding gene sequencing of stool for percent of 17 Clostridal strains in total GI community on day −1 and day +35, relative to allogeneic (C57BL/6J → BALB/c) BMT on day 0. Vehicle or 17-strain cocktail were administered to recipients every other day via intragastric gavage beginning day −14 and continued through day +21, relative to BMT. ND = not detected. (b)-(c) Recipients of allogeneic BMT gavaged with vehicle or colonized with 17-strain cocktail were sacrificed 21 days following BMT. (b) Intestinal luminal contents (stool) and (c) intestinal tissue were harvested and analyzed via GC/MS. (d) GVHD clinical score and (e) survival following syngeneic (BALB/c → BALB/c) and allogeneic (C57BL/6J → BALB/c) BMT with 17-strain administration, compared to vehicle control; syngeneic n=6, n=12 mice per allogeneic group. (f)-(g) Obligate anaerobes of C57BL/6J (H-2b) mice were targeted with antibiotic mixture (ampicillin 5 mg, metronidazole 4 mg, clindamycin 5 mg, and vancomycin 5 mg) by intragastric gavage for 6 days followed by colonization with cocktails of E. faecium or 17-strains, 4 and 6 days later and subsequently used as recipients of allogeneic BMT (B10.BR → C57BL/6J). 17-strain Clostridial cocktail used at the University of Michigan and Memorial Sloan Kettering was identical. (f) 16S gene sequencing was performed on stool collected from recipients of allogeneic BMT on day −1, relative to BMT. (g) Survival of allo-BMT recipients of intragastric gavage of 17 Clostridial strains cocktail; Recipients of E. faecium n=9 , 17-strain n=10 mice. *P < .05; **P < .01; ***P < .0001 of students t-test a – c, f; Mantel-Cox log-rank test d, e, g. Bars and error bars represent the means and standard errors of the mean, respectively.
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Figure 6: Rationally altering the commensal microbiota reduces GVHD(a) 16S rRNA-encoding gene sequencing of stool for percent of 17 Clostridal strains in total GI community on day −1 and day +35, relative to allogeneic (C57BL/6J → BALB/c) BMT on day 0. Vehicle or 17-strain cocktail were administered to recipients every other day via intragastric gavage beginning day −14 and continued through day +21, relative to BMT. ND = not detected. (b)-(c) Recipients of allogeneic BMT gavaged with vehicle or colonized with 17-strain cocktail were sacrificed 21 days following BMT. (b) Intestinal luminal contents (stool) and (c) intestinal tissue were harvested and analyzed via GC/MS. (d) GVHD clinical score and (e) survival following syngeneic (BALB/c → BALB/c) and allogeneic (C57BL/6J → BALB/c) BMT with 17-strain administration, compared to vehicle control; syngeneic n=6, n=12 mice per allogeneic group. (f)-(g) Obligate anaerobes of C57BL/6J (H-2b) mice were targeted with antibiotic mixture (ampicillin 5 mg, metronidazole 4 mg, clindamycin 5 mg, and vancomycin 5 mg) by intragastric gavage for 6 days followed by colonization with cocktails of E. faecium or 17-strains, 4 and 6 days later and subsequently used as recipients of allogeneic BMT (B10.BR → C57BL/6J). 17-strain Clostridial cocktail used at the University of Michigan and Memorial Sloan Kettering was identical. (f) 16S gene sequencing was performed on stool collected from recipients of allogeneic BMT on day −1, relative to BMT. (g) Survival of allo-BMT recipients of intragastric gavage of 17 Clostridial strains cocktail; Recipients of E. faecium n=9 , 17-strain n=10 mice. *P < .05; **P < .01; ***P < .0001 of students t-test a – c, f; Mantel-Cox log-rank test d, e, g. Bars and error bars represent the means and standard errors of the mean, respectively.

Mentions: The endogenous HDACi butyrate is a by-product of microbial fermentation33. Therefore, we next tested the hypothesis that altering the composition of indigenous GI microbiota in hosts to those that can produce high levels of butyrate will mitigate GVHD. We utilized 17 rationally selected strains of Clostridia that have been shown to increase butyrate both in vitro and in vivo17,34. We administered these strains via intragastric gavage every other day to naive mice starting 14 days prior to allo-BMT and continued administration of the 17-strain cocktail for 21 days post-BMT. We characterized the microbiota in feces collected from animals that received vehicle and 17-strain administration by 16S rRNA-encoding gene sequencing. In animals that received the 17 Clostridial strains, 16S analysis35-37 revealed an important biologically significant shift in the microbiota indicating that these organisms could be detected (Fig. 6a and Supplementary Fig. 6a). Furthermore, GC/MS analysis 21 days following allo-BMT revealed a significant increase of butyrate in the luminal contents (Fig. 6b) and significantly increased butyrate in the intestinal tissues (Fig. 6c) of animals that received intragastric gavage of the 17 Clostridial strains. The recipients of intragastric gavage of the 17 strains and allo-BMT exhibited significantly decreased GVHD (Fig. 6d-e). Detectable levels of the 17 Clostridial strains were diminished within 2 weeks (day +35) of ceasing intragastric administration (Fig. 6a).


Gut microbiome derived metabolites modulate intestinal epithelial cell damage and mitigate Graft-versus-Host Disease
Rationally altering the commensal microbiota reduces GVHD(a) 16S rRNA-encoding gene sequencing of stool for percent of 17 Clostridal strains in total GI community on day −1 and day +35, relative to allogeneic (C57BL/6J → BALB/c) BMT on day 0. Vehicle or 17-strain cocktail were administered to recipients every other day via intragastric gavage beginning day −14 and continued through day +21, relative to BMT. ND = not detected. (b)-(c) Recipients of allogeneic BMT gavaged with vehicle or colonized with 17-strain cocktail were sacrificed 21 days following BMT. (b) Intestinal luminal contents (stool) and (c) intestinal tissue were harvested and analyzed via GC/MS. (d) GVHD clinical score and (e) survival following syngeneic (BALB/c → BALB/c) and allogeneic (C57BL/6J → BALB/c) BMT with 17-strain administration, compared to vehicle control; syngeneic n=6, n=12 mice per allogeneic group. (f)-(g) Obligate anaerobes of C57BL/6J (H-2b) mice were targeted with antibiotic mixture (ampicillin 5 mg, metronidazole 4 mg, clindamycin 5 mg, and vancomycin 5 mg) by intragastric gavage for 6 days followed by colonization with cocktails of E. faecium or 17-strains, 4 and 6 days later and subsequently used as recipients of allogeneic BMT (B10.BR → C57BL/6J). 17-strain Clostridial cocktail used at the University of Michigan and Memorial Sloan Kettering was identical. (f) 16S gene sequencing was performed on stool collected from recipients of allogeneic BMT on day −1, relative to BMT. (g) Survival of allo-BMT recipients of intragastric gavage of 17 Clostridial strains cocktail; Recipients of E. faecium n=9 , 17-strain n=10 mice. *P < .05; **P < .01; ***P < .0001 of students t-test a – c, f; Mantel-Cox log-rank test d, e, g. Bars and error bars represent the means and standard errors of the mean, respectively.
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Figure 6: Rationally altering the commensal microbiota reduces GVHD(a) 16S rRNA-encoding gene sequencing of stool for percent of 17 Clostridal strains in total GI community on day −1 and day +35, relative to allogeneic (C57BL/6J → BALB/c) BMT on day 0. Vehicle or 17-strain cocktail were administered to recipients every other day via intragastric gavage beginning day −14 and continued through day +21, relative to BMT. ND = not detected. (b)-(c) Recipients of allogeneic BMT gavaged with vehicle or colonized with 17-strain cocktail were sacrificed 21 days following BMT. (b) Intestinal luminal contents (stool) and (c) intestinal tissue were harvested and analyzed via GC/MS. (d) GVHD clinical score and (e) survival following syngeneic (BALB/c → BALB/c) and allogeneic (C57BL/6J → BALB/c) BMT with 17-strain administration, compared to vehicle control; syngeneic n=6, n=12 mice per allogeneic group. (f)-(g) Obligate anaerobes of C57BL/6J (H-2b) mice were targeted with antibiotic mixture (ampicillin 5 mg, metronidazole 4 mg, clindamycin 5 mg, and vancomycin 5 mg) by intragastric gavage for 6 days followed by colonization with cocktails of E. faecium or 17-strains, 4 and 6 days later and subsequently used as recipients of allogeneic BMT (B10.BR → C57BL/6J). 17-strain Clostridial cocktail used at the University of Michigan and Memorial Sloan Kettering was identical. (f) 16S gene sequencing was performed on stool collected from recipients of allogeneic BMT on day −1, relative to BMT. (g) Survival of allo-BMT recipients of intragastric gavage of 17 Clostridial strains cocktail; Recipients of E. faecium n=9 , 17-strain n=10 mice. *P < .05; **P < .01; ***P < .0001 of students t-test a – c, f; Mantel-Cox log-rank test d, e, g. Bars and error bars represent the means and standard errors of the mean, respectively.
Mentions: The endogenous HDACi butyrate is a by-product of microbial fermentation33. Therefore, we next tested the hypothesis that altering the composition of indigenous GI microbiota in hosts to those that can produce high levels of butyrate will mitigate GVHD. We utilized 17 rationally selected strains of Clostridia that have been shown to increase butyrate both in vitro and in vivo17,34. We administered these strains via intragastric gavage every other day to naive mice starting 14 days prior to allo-BMT and continued administration of the 17-strain cocktail for 21 days post-BMT. We characterized the microbiota in feces collected from animals that received vehicle and 17-strain administration by 16S rRNA-encoding gene sequencing. In animals that received the 17 Clostridial strains, 16S analysis35-37 revealed an important biologically significant shift in the microbiota indicating that these organisms could be detected (Fig. 6a and Supplementary Fig. 6a). Furthermore, GC/MS analysis 21 days following allo-BMT revealed a significant increase of butyrate in the luminal contents (Fig. 6b) and significantly increased butyrate in the intestinal tissues (Fig. 6c) of animals that received intragastric gavage of the 17 Clostridial strains. The recipients of intragastric gavage of the 17 strains and allo-BMT exhibited significantly decreased GVHD (Fig. 6d-e). Detectable levels of the 17 Clostridial strains were diminished within 2 weeks (day +35) of ceasing intragastric administration (Fig. 6a).

View Article: PubMed Central - PubMed

ABSTRACT

The impact of alterations in intestinal microbiota on microbial metabolites and on disease processes, such as graft-versus-host disease (GVHD), is not known. Here we performed unbiased analysis to identify novel alterations in gastrointestinal microbiota-derived short chain fatty acids (SCFA) after allogeneic bone marrow transplant (allo-BMT). Alterations in the amounts of only one SCFA, butyrate, were observed only within the intestinal tissue. The reduced butyrate in CD326+ intestinal epithelial cells (IECs) after allo-BMT resulted in decreased histone acetylation, which was restored upon local administration of exogenous butyrate. Butyrate restoration improved IEC junctional integrity, decreased apoptosis, and mitigated GVHD. Furthermore, alteration of the indigenous microbiota with 17 rationally selected strains of high butyrate producing Clostridia also decreased GVHD. These data demonstrate a heretofore unrecognized role of microbial metabolites and suggest that local and specific alteration of microbial metabolites has direct salutary effects on GVHD target tissues and can mitigate its severity.

No MeSH data available.


Related in: MedlinePlus