Limits...
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.


Loss of butyrate decreases histone acetylation in IECs(a) Protein expression of acetyl-histone H4 (top blot) and densitometric analysis normalized to presence of α-tubulin (shown right of blot) 21 days following syngeneic (BALB/c → BALB/c) or allogeneic (C57BL/6J → BALB/c) BMT. Densitometric analysis of 3 experiments combined; each experiment had n=5-6 mice per group. (b) Gene expression of representatives of class I, II, and IV HDAC enzymes, (c) histone acetyltransferase (HAT) enzyme levels, (d) HDAC activity, and (e) HAT activity in syngeneic and allogeneic CD326+ IECs. A = absorbance. (f) Gene expression and (g) protein levels of SLC5A8 (monocarboxylate transporter of butyrate) in IECs (CD326+) of syngeneic and allogeneic transplant recipients 21 days following BMT. Representative immunoblots; densitometric analysis of three similar experiments combined is shown right of blots. *P < .05; **P < .01 of students t-test. Bars and error bars represent the means and standard errors of the mean, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4836986&req=5

Figure 2: Loss of butyrate decreases histone acetylation in IECs(a) Protein expression of acetyl-histone H4 (top blot) and densitometric analysis normalized to presence of α-tubulin (shown right of blot) 21 days following syngeneic (BALB/c → BALB/c) or allogeneic (C57BL/6J → BALB/c) BMT. Densitometric analysis of 3 experiments combined; each experiment had n=5-6 mice per group. (b) Gene expression of representatives of class I, II, and IV HDAC enzymes, (c) histone acetyltransferase (HAT) enzyme levels, (d) HDAC activity, and (e) HAT activity in syngeneic and allogeneic CD326+ IECs. A = absorbance. (f) Gene expression and (g) protein levels of SLC5A8 (monocarboxylate transporter of butyrate) in IECs (CD326+) of syngeneic and allogeneic transplant recipients 21 days following BMT. Representative immunoblots; densitometric analysis of three similar experiments combined is shown right of blots. *P < .05; **P < .01 of students t-test. Bars and error bars represent the means and standard errors of the mean, respectively.

Mentions: In light of the reduction of butyrate in allogeneic animals only in the intestinal tissue, we next analyzed the potential functional impact of reduced butyrate in IECs. Because butyrate is an HDACi10,12,19, we examined the degree of histone acetylation by immunoblotting purified CD326+ IECs after BMT. The degree of acetylation of histone H4 was significantly decreased on day 7 (Supplementary Fig. 3a) and day 21 (Fig. 2a) following allo-BMT demonstrating that reduced butyrate resulted in decreased histone acetylation. Therefore to confirm if the decreased acetylation is secondary to decreased HDAC inhibition from reduction in butyrate and not due to potential alterations in HDAC and HAT enzyme levels20 following transplant, we analyzed the expression of HDACs and HATs in IECs after BMT. We observed similar levels of several HDACs (Hdac 1,4,7,9, and 10) (Fig. 2b) and HATs (p300 and TIP60) (Fig. 2c) by qPCR in the IECs (CD326+) of both syngeneic and allogeneic BMT recipients. Furthermore both HDAC (Fig. 2d) and HAT (Fig. 2e) enzyme activity were not different in these animals. These data suggest that reduction in histone acetylation in the IECs after allo-BMT is from reduced levels of butyrate.


Gut microbiome derived metabolites modulate intestinal epithelial cell damage and mitigate Graft-versus-Host Disease
Loss of butyrate decreases histone acetylation in IECs(a) Protein expression of acetyl-histone H4 (top blot) and densitometric analysis normalized to presence of α-tubulin (shown right of blot) 21 days following syngeneic (BALB/c → BALB/c) or allogeneic (C57BL/6J → BALB/c) BMT. Densitometric analysis of 3 experiments combined; each experiment had n=5-6 mice per group. (b) Gene expression of representatives of class I, II, and IV HDAC enzymes, (c) histone acetyltransferase (HAT) enzyme levels, (d) HDAC activity, and (e) HAT activity in syngeneic and allogeneic CD326+ IECs. A = absorbance. (f) Gene expression and (g) protein levels of SLC5A8 (monocarboxylate transporter of butyrate) in IECs (CD326+) of syngeneic and allogeneic transplant recipients 21 days following BMT. Representative immunoblots; densitometric analysis of three similar experiments combined is shown right of blots. *P < .05; **P < .01 of students t-test. Bars and error bars represent the means and standard errors of the mean, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836986&req=5

Figure 2: Loss of butyrate decreases histone acetylation in IECs(a) Protein expression of acetyl-histone H4 (top blot) and densitometric analysis normalized to presence of α-tubulin (shown right of blot) 21 days following syngeneic (BALB/c → BALB/c) or allogeneic (C57BL/6J → BALB/c) BMT. Densitometric analysis of 3 experiments combined; each experiment had n=5-6 mice per group. (b) Gene expression of representatives of class I, II, and IV HDAC enzymes, (c) histone acetyltransferase (HAT) enzyme levels, (d) HDAC activity, and (e) HAT activity in syngeneic and allogeneic CD326+ IECs. A = absorbance. (f) Gene expression and (g) protein levels of SLC5A8 (monocarboxylate transporter of butyrate) in IECs (CD326+) of syngeneic and allogeneic transplant recipients 21 days following BMT. Representative immunoblots; densitometric analysis of three similar experiments combined is shown right of blots. *P < .05; **P < .01 of students t-test. Bars and error bars represent the means and standard errors of the mean, respectively.
Mentions: In light of the reduction of butyrate in allogeneic animals only in the intestinal tissue, we next analyzed the potential functional impact of reduced butyrate in IECs. Because butyrate is an HDACi10,12,19, we examined the degree of histone acetylation by immunoblotting purified CD326+ IECs after BMT. The degree of acetylation of histone H4 was significantly decreased on day 7 (Supplementary Fig. 3a) and day 21 (Fig. 2a) following allo-BMT demonstrating that reduced butyrate resulted in decreased histone acetylation. Therefore to confirm if the decreased acetylation is secondary to decreased HDAC inhibition from reduction in butyrate and not due to potential alterations in HDAC and HAT enzyme levels20 following transplant, we analyzed the expression of HDACs and HATs in IECs after BMT. We observed similar levels of several HDACs (Hdac 1,4,7,9, and 10) (Fig. 2b) and HATs (p300 and TIP60) (Fig. 2c) by qPCR in the IECs (CD326+) of both syngeneic and allogeneic BMT recipients. Furthermore both HDAC (Fig. 2d) and HAT (Fig. 2e) enzyme activity were not different in these animals. These data suggest that reduction in histone acetylation in the IECs after allo-BMT is from reduced levels of butyrate.

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.