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Extracellular matrix-associated cytokines regulate CD4+ effector T-cell responses in the human intestinal mucosa.

Huff KR, Akhtar LN, Fox AL, Cannon JA, Smith PD, Smythies LE - Mucosal Immunol (2011)

Bottom Line: In this study, we uncovered a role for intestinal stromal products in the innate regulation of effector T cells.Stroma-conditioned media (S-CM) derived from the normal human intestinal stroma (transforming growth factor-β (TGF-β)(hi)/interleukin (IL)-6(lo)/IL-1β(lo)) significantly downregulated T-cell proliferation and interferon-γ (IFN-γ) production compared with S-CM derived from the inflamed Crohn's mucosa (TGF-β(hi)/IL-6(hi)/IL-1β(hi)).These findings implicate stromal TGF-β in the downregulation of T-cell 2 responses in the normal intestinal mucosa, and stromal IL-6 and IL-1β in the promotion of Th1 and Th17 responses in the inflamed Crohn's mucosa, suggesting an innate regulatory function for the intestinal extracellular matrix.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine (Gastroenterology), University of Alabama at Birmingham, Birmingham, Alabama, USA.

ABSTRACT
Extracellular matrix (stroma) regulation of mucosal T-cell function is incompletely understood. In this study, we uncovered a role for intestinal stromal products in the innate regulation of effector T cells. Stroma-conditioned media (S-CM) derived from the normal human intestinal stroma (transforming growth factor-β (TGF-β)(hi)/interleukin (IL)-6(lo)/IL-1β(lo)) significantly downregulated T-cell proliferation and interferon-γ (IFN-γ) production compared with S-CM derived from the inflamed Crohn's mucosa (TGF-β(hi)/IL-6(hi)/IL-1β(hi)). Antibody neutralization studies showed that TGF-β in normal S-CM inhibited T-cell proliferation and IFN-γ production, whereas IL-6 plus IL-1β in Crohn's S-CM promoted T-cell proliferation, and IL-1β alone promoted IFN-γ and IL-17 release. Importantly, normal S-CM inhibited T-bet expression, whereas Crohn's S-CM activated signal transducer and activator of transcription 3, suggesting that discordant T-cell responses are regulated at the transcription factor and signaling levels. These findings implicate stromal TGF-β in the downregulation of T-cell 2 responses in the normal intestinal mucosa, and stromal IL-6 and IL-1β in the promotion of Th1 and Th17 responses in the inflamed Crohn's mucosa, suggesting an innate regulatory function for the intestinal extracellular matrix.

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S-CM inhibition of CD4+ T-cell responses(A) CD3/CD28-stimulated T-cells cultured in media alone or the presence of a representative normal or Crohn’s S-CM were analyzed on day 4 for proliferation by flow cytometry for CFSE dilution. The percent of cells that proliferated is indicated in the top left corner of the plots. (B) Percent inhibition of proliferation for CD3/CD28-stimulated T-cells cultured in normal S-CM (n=7) or Crohn’s S-CM (n=6). (C) IFN-γ release by CD3/CD28-stimulated T-cells cultured for 4 days in the presence of media, normal S-CM (n=5) or Crohn’s S-CM (n=6). (D) Percent inhibition of IFN-γ release by CD3/CD28-stimulated T-cells cultured for 4 days in the presence of normal S-CM (n=5) or Crohn’s S-CM (n=6) compared to stimulated T-cells cultured in media alone. (E) Western blot analysis of T-bet protein expression in CD3/CD28-stimulated T-cells cultured 24 hr in the presence of a representative normal S-CM or Crohn’s S-CM (n=3 each). (F) Densitometric analysis of T-bet relative to β-actin (n=3). For B–D and F, error bars indicate the standard error of the mean. * p ≤ 0.05, ** p ≤ 0.01.
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Figure 1: S-CM inhibition of CD4+ T-cell responses(A) CD3/CD28-stimulated T-cells cultured in media alone or the presence of a representative normal or Crohn’s S-CM were analyzed on day 4 for proliferation by flow cytometry for CFSE dilution. The percent of cells that proliferated is indicated in the top left corner of the plots. (B) Percent inhibition of proliferation for CD3/CD28-stimulated T-cells cultured in normal S-CM (n=7) or Crohn’s S-CM (n=6). (C) IFN-γ release by CD3/CD28-stimulated T-cells cultured for 4 days in the presence of media, normal S-CM (n=5) or Crohn’s S-CM (n=6). (D) Percent inhibition of IFN-γ release by CD3/CD28-stimulated T-cells cultured for 4 days in the presence of normal S-CM (n=5) or Crohn’s S-CM (n=6) compared to stimulated T-cells cultured in media alone. (E) Western blot analysis of T-bet protein expression in CD3/CD28-stimulated T-cells cultured 24 hr in the presence of a representative normal S-CM or Crohn’s S-CM (n=3 each). (F) Densitometric analysis of T-bet relative to β-actin (n=3). For B–D and F, error bars indicate the standard error of the mean. * p ≤ 0.05, ** p ≤ 0.01.

Mentions: To recapitulate in vitro the exposure of newly recruited blood T-cells to the intestinal lamina propria and determine whether stroma-associated molecules in the lamina propria regulate T-cells, we cultured blood T-cells in intestinal stroma-conditioned media (S-CM) and measured T-cell proliferation and IFN-γ production. Blood CD4+ T-cells from normal subjects pre-incubated with increasing concentrations of normal or Crohn’s S-CM showed differential dose-dependent reductions in CD3/CD28- (Figure 1A) and PHA- (Supplemental Figure 1A) stimulated proliferation. Inhibition of T-cell proliferation was significantly greater for T-cells cultured in normal S-CM than T-cells cultured in Crohn’s S-CM at both 250 µg/mL (p < 0.05) and 500 µg/mL (p < 0.05) (Figure 1B and Supplemental Figure 1B). Moreover, stromal inhibition of T-cell proliferation by normal S-CM was evident not only in a reduced percentage of proliferating T-cells, but also in a reduced number of generations of daughter cells.


Extracellular matrix-associated cytokines regulate CD4+ effector T-cell responses in the human intestinal mucosa.

Huff KR, Akhtar LN, Fox AL, Cannon JA, Smith PD, Smythies LE - Mucosal Immunol (2011)

S-CM inhibition of CD4+ T-cell responses(A) CD3/CD28-stimulated T-cells cultured in media alone or the presence of a representative normal or Crohn’s S-CM were analyzed on day 4 for proliferation by flow cytometry for CFSE dilution. The percent of cells that proliferated is indicated in the top left corner of the plots. (B) Percent inhibition of proliferation for CD3/CD28-stimulated T-cells cultured in normal S-CM (n=7) or Crohn’s S-CM (n=6). (C) IFN-γ release by CD3/CD28-stimulated T-cells cultured for 4 days in the presence of media, normal S-CM (n=5) or Crohn’s S-CM (n=6). (D) Percent inhibition of IFN-γ release by CD3/CD28-stimulated T-cells cultured for 4 days in the presence of normal S-CM (n=5) or Crohn’s S-CM (n=6) compared to stimulated T-cells cultured in media alone. (E) Western blot analysis of T-bet protein expression in CD3/CD28-stimulated T-cells cultured 24 hr in the presence of a representative normal S-CM or Crohn’s S-CM (n=3 each). (F) Densitometric analysis of T-bet relative to β-actin (n=3). For B–D and F, error bars indicate the standard error of the mean. * p ≤ 0.05, ** p ≤ 0.01.
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Related In: Results  -  Collection

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Figure 1: S-CM inhibition of CD4+ T-cell responses(A) CD3/CD28-stimulated T-cells cultured in media alone or the presence of a representative normal or Crohn’s S-CM were analyzed on day 4 for proliferation by flow cytometry for CFSE dilution. The percent of cells that proliferated is indicated in the top left corner of the plots. (B) Percent inhibition of proliferation for CD3/CD28-stimulated T-cells cultured in normal S-CM (n=7) or Crohn’s S-CM (n=6). (C) IFN-γ release by CD3/CD28-stimulated T-cells cultured for 4 days in the presence of media, normal S-CM (n=5) or Crohn’s S-CM (n=6). (D) Percent inhibition of IFN-γ release by CD3/CD28-stimulated T-cells cultured for 4 days in the presence of normal S-CM (n=5) or Crohn’s S-CM (n=6) compared to stimulated T-cells cultured in media alone. (E) Western blot analysis of T-bet protein expression in CD3/CD28-stimulated T-cells cultured 24 hr in the presence of a representative normal S-CM or Crohn’s S-CM (n=3 each). (F) Densitometric analysis of T-bet relative to β-actin (n=3). For B–D and F, error bars indicate the standard error of the mean. * p ≤ 0.05, ** p ≤ 0.01.
Mentions: To recapitulate in vitro the exposure of newly recruited blood T-cells to the intestinal lamina propria and determine whether stroma-associated molecules in the lamina propria regulate T-cells, we cultured blood T-cells in intestinal stroma-conditioned media (S-CM) and measured T-cell proliferation and IFN-γ production. Blood CD4+ T-cells from normal subjects pre-incubated with increasing concentrations of normal or Crohn’s S-CM showed differential dose-dependent reductions in CD3/CD28- (Figure 1A) and PHA- (Supplemental Figure 1A) stimulated proliferation. Inhibition of T-cell proliferation was significantly greater for T-cells cultured in normal S-CM than T-cells cultured in Crohn’s S-CM at both 250 µg/mL (p < 0.05) and 500 µg/mL (p < 0.05) (Figure 1B and Supplemental Figure 1B). Moreover, stromal inhibition of T-cell proliferation by normal S-CM was evident not only in a reduced percentage of proliferating T-cells, but also in a reduced number of generations of daughter cells.

Bottom Line: In this study, we uncovered a role for intestinal stromal products in the innate regulation of effector T cells.Stroma-conditioned media (S-CM) derived from the normal human intestinal stroma (transforming growth factor-β (TGF-β)(hi)/interleukin (IL)-6(lo)/IL-1β(lo)) significantly downregulated T-cell proliferation and interferon-γ (IFN-γ) production compared with S-CM derived from the inflamed Crohn's mucosa (TGF-β(hi)/IL-6(hi)/IL-1β(hi)).These findings implicate stromal TGF-β in the downregulation of T-cell 2 responses in the normal intestinal mucosa, and stromal IL-6 and IL-1β in the promotion of Th1 and Th17 responses in the inflamed Crohn's mucosa, suggesting an innate regulatory function for the intestinal extracellular matrix.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine (Gastroenterology), University of Alabama at Birmingham, Birmingham, Alabama, USA.

ABSTRACT
Extracellular matrix (stroma) regulation of mucosal T-cell function is incompletely understood. In this study, we uncovered a role for intestinal stromal products in the innate regulation of effector T cells. Stroma-conditioned media (S-CM) derived from the normal human intestinal stroma (transforming growth factor-β (TGF-β)(hi)/interleukin (IL)-6(lo)/IL-1β(lo)) significantly downregulated T-cell proliferation and interferon-γ (IFN-γ) production compared with S-CM derived from the inflamed Crohn's mucosa (TGF-β(hi)/IL-6(hi)/IL-1β(hi)). Antibody neutralization studies showed that TGF-β in normal S-CM inhibited T-cell proliferation and IFN-γ production, whereas IL-6 plus IL-1β in Crohn's S-CM promoted T-cell proliferation, and IL-1β alone promoted IFN-γ and IL-17 release. Importantly, normal S-CM inhibited T-bet expression, whereas Crohn's S-CM activated signal transducer and activator of transcription 3, suggesting that discordant T-cell responses are regulated at the transcription factor and signaling levels. These findings implicate stromal TGF-β in the downregulation of T-cell 2 responses in the normal intestinal mucosa, and stromal IL-6 and IL-1β in the promotion of Th1 and Th17 responses in the inflamed Crohn's mucosa, suggesting an innate regulatory function for the intestinal extracellular matrix.

Show MeSH
Related in: MedlinePlus