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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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Butyrate treatment enhances IEC barrier(a) CD326+ purified intestinal epithelial cells (IECs) cultured in the presence or absence of butyrate and withheld (left panel) or subjected to (right panel) irradiation (6 Gy). (b) Allogeneic CD8+ T cell killing assay. CD326+ IECs incubated with or without butyrate overnight followed by co-culture with allo-primed CD8+ T cells. (c) Size (µm2) of cultured primary intestinal organoids following butyrate treatment (0.5 mM and 1 mM), compared to control. Gene expression of (d) anti-apoptotic protein BCL-B (Bcl2l10) and (e) junctional protein JAM (F11r) in primary CD326+ IECs in the presence of butyrate 1mM. (f)-(g) Chromatin immunoprecipitation assay (ChIP) of butyrate treated IECs (CD326+) binding acetylated histone H4 in the promoter region of (f) Bcl2l10 and (g) F11r. (h)-(i) Analysis of IECs (CD326+) isolated from recipients of syngeneic (BALB/c → BALB/c) and allogeneic (C57BL/6J → BALB/c) BMT treated with butyrate or vehicle daily via intragastric gavage for 21 days. (h) Gene expression of pro-apoptotic proteins Bak1 (left) and Bax (right), (i) anti-apoptotic protein BCL-B (Bcl2l10), and (j) junctional proteins in CD326+ purified IECs isolated 21 days following BMT. (k) Immunoblot of CD326+ purified intestinal epithelial cells from allogeneic (C57BL/6J → BALB/c) BMT recipients treated daily with intragastric vehicle or butyrate (10mg/kg) for occludin, JAM, and claudin 5; isolated 21 days following BMT. Densitometric analysis of two experiments combined shown to the right of each blot, compared to α-tubulin loading control; representative immunoblots shown. Syngeneic n=5, n = 10 mice per allogeneic group. *P < .05; **P < .01; ***P < .0001 of students t-test a – g, k; ANOVA h – j. Bars and error bars represent the means and standard errors of the mean, respectively.
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Figure 5: Butyrate treatment enhances IEC barrier(a) CD326+ purified intestinal epithelial cells (IECs) cultured in the presence or absence of butyrate and withheld (left panel) or subjected to (right panel) irradiation (6 Gy). (b) Allogeneic CD8+ T cell killing assay. CD326+ IECs incubated with or without butyrate overnight followed by co-culture with allo-primed CD8+ T cells. (c) Size (µm2) of cultured primary intestinal organoids following butyrate treatment (0.5 mM and 1 mM), compared to control. Gene expression of (d) anti-apoptotic protein BCL-B (Bcl2l10) and (e) junctional protein JAM (F11r) in primary CD326+ IECs in the presence of butyrate 1mM. (f)-(g) Chromatin immunoprecipitation assay (ChIP) of butyrate treated IECs (CD326+) binding acetylated histone H4 in the promoter region of (f) Bcl2l10 and (g) F11r. (h)-(i) Analysis of IECs (CD326+) isolated from recipients of syngeneic (BALB/c → BALB/c) and allogeneic (C57BL/6J → BALB/c) BMT treated with butyrate or vehicle daily via intragastric gavage for 21 days. (h) Gene expression of pro-apoptotic proteins Bak1 (left) and Bax (right), (i) anti-apoptotic protein BCL-B (Bcl2l10), and (j) junctional proteins in CD326+ purified IECs isolated 21 days following BMT. (k) Immunoblot of CD326+ purified intestinal epithelial cells from allogeneic (C57BL/6J → BALB/c) BMT recipients treated daily with intragastric vehicle or butyrate (10mg/kg) for occludin, JAM, and claudin 5; isolated 21 days following BMT. Densitometric analysis of two experiments combined shown to the right of each blot, compared to α-tubulin loading control; representative immunoblots shown. Syngeneic n=5, n = 10 mice per allogeneic group. *P < .05; **P < .01; ***P < .0001 of students t-test a – g, k; ANOVA h – j. Bars and error bars represent the means and standard errors of the mean, respectively.

Mentions: We next sought to explore the potential mechanisms that contribute to butyrate-induced protection from severe GVHD. Because (a) butyrate is decreased in IECs, (b) administration of butyrate mitigated GI GVHD independent of Treg cells, but (c) improved junction integrity, we therefore explored whether butyrate had direct effects on protecting IECs from allo-T cell mediated damage and conditioning. We treated IECs ex vivo with vehicle or butyrate for 24 hours and withheld or subjected the cells to irradiation (6 Gy), followed by 24 hours of additional incubation with butyrate. We observed that butyrate was not toxic to IECs (Fig. 5a, left) and more importantly conferred protection from irradiation-induced apoptosis (Fig. 5a, right).


Gut microbiome derived metabolites modulate intestinal epithelial cell damage and mitigate Graft-versus-Host Disease
Butyrate treatment enhances IEC barrier(a) CD326+ purified intestinal epithelial cells (IECs) cultured in the presence or absence of butyrate and withheld (left panel) or subjected to (right panel) irradiation (6 Gy). (b) Allogeneic CD8+ T cell killing assay. CD326+ IECs incubated with or without butyrate overnight followed by co-culture with allo-primed CD8+ T cells. (c) Size (µm2) of cultured primary intestinal organoids following butyrate treatment (0.5 mM and 1 mM), compared to control. Gene expression of (d) anti-apoptotic protein BCL-B (Bcl2l10) and (e) junctional protein JAM (F11r) in primary CD326+ IECs in the presence of butyrate 1mM. (f)-(g) Chromatin immunoprecipitation assay (ChIP) of butyrate treated IECs (CD326+) binding acetylated histone H4 in the promoter region of (f) Bcl2l10 and (g) F11r. (h)-(i) Analysis of IECs (CD326+) isolated from recipients of syngeneic (BALB/c → BALB/c) and allogeneic (C57BL/6J → BALB/c) BMT treated with butyrate or vehicle daily via intragastric gavage for 21 days. (h) Gene expression of pro-apoptotic proteins Bak1 (left) and Bax (right), (i) anti-apoptotic protein BCL-B (Bcl2l10), and (j) junctional proteins in CD326+ purified IECs isolated 21 days following BMT. (k) Immunoblot of CD326+ purified intestinal epithelial cells from allogeneic (C57BL/6J → BALB/c) BMT recipients treated daily with intragastric vehicle or butyrate (10mg/kg) for occludin, JAM, and claudin 5; isolated 21 days following BMT. Densitometric analysis of two experiments combined shown to the right of each blot, compared to α-tubulin loading control; representative immunoblots shown. Syngeneic n=5, n = 10 mice per allogeneic group. *P < .05; **P < .01; ***P < .0001 of students t-test a – g, k; ANOVA h – j. Bars and error bars represent the means and standard errors of the mean, respectively.
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Figure 5: Butyrate treatment enhances IEC barrier(a) CD326+ purified intestinal epithelial cells (IECs) cultured in the presence or absence of butyrate and withheld (left panel) or subjected to (right panel) irradiation (6 Gy). (b) Allogeneic CD8+ T cell killing assay. CD326+ IECs incubated with or without butyrate overnight followed by co-culture with allo-primed CD8+ T cells. (c) Size (µm2) of cultured primary intestinal organoids following butyrate treatment (0.5 mM and 1 mM), compared to control. Gene expression of (d) anti-apoptotic protein BCL-B (Bcl2l10) and (e) junctional protein JAM (F11r) in primary CD326+ IECs in the presence of butyrate 1mM. (f)-(g) Chromatin immunoprecipitation assay (ChIP) of butyrate treated IECs (CD326+) binding acetylated histone H4 in the promoter region of (f) Bcl2l10 and (g) F11r. (h)-(i) Analysis of IECs (CD326+) isolated from recipients of syngeneic (BALB/c → BALB/c) and allogeneic (C57BL/6J → BALB/c) BMT treated with butyrate or vehicle daily via intragastric gavage for 21 days. (h) Gene expression of pro-apoptotic proteins Bak1 (left) and Bax (right), (i) anti-apoptotic protein BCL-B (Bcl2l10), and (j) junctional proteins in CD326+ purified IECs isolated 21 days following BMT. (k) Immunoblot of CD326+ purified intestinal epithelial cells from allogeneic (C57BL/6J → BALB/c) BMT recipients treated daily with intragastric vehicle or butyrate (10mg/kg) for occludin, JAM, and claudin 5; isolated 21 days following BMT. Densitometric analysis of two experiments combined shown to the right of each blot, compared to α-tubulin loading control; representative immunoblots shown. Syngeneic n=5, n = 10 mice per allogeneic group. *P < .05; **P < .01; ***P < .0001 of students t-test a – g, k; ANOVA h – j. Bars and error bars represent the means and standard errors of the mean, respectively.
Mentions: We next sought to explore the potential mechanisms that contribute to butyrate-induced protection from severe GVHD. Because (a) butyrate is decreased in IECs, (b) administration of butyrate mitigated GI GVHD independent of Treg cells, but (c) improved junction integrity, we therefore explored whether butyrate had direct effects on protecting IECs from allo-T cell mediated damage and conditioning. We treated IECs ex vivo with vehicle or butyrate for 24 hours and withheld or subjected the cells to irradiation (6 Gy), followed by 24 hours of additional incubation with butyrate. We observed that butyrate was not toxic to IECs (Fig. 5a, left) and more importantly conferred protection from irradiation-induced apoptosis (Fig. 5a, right).

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