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Autoimmune pancreatitis results from loss of TGFbeta signalling in S100A4-positive dendritic cells.

Boomershine CS, Chamberlain A, Kendall P, Afshar-Sharif AR, Huang H, Washington MK, Lawson WE, Thomas JW, Blackwell TS, Bhowmick NA - Gut (2009)

Bottom Line: DCs were confirmed to express S100A4, a previously reported protein expressed by fibroblasts.Tgfbr2(fspKO) DCs undergo elevated maturation in response to antigen and increased activation of naïve CD4-positive T cells.The loss of immune tolerance in myeloid S100A4(+) DCs can mediate mAIP and may explain some aspects of AIP disease pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Vanderbilt-Ingram Cancer, Vanderbilt University, Nashville 37232, Tennessee, USA.

ABSTRACT

Background and aims: Autoimmune pancreatitis (AIP) is a poorly understood human disease affecting the exocrine pancreas. The goal of the present study was to elucidate the pathogenic mechanisms underlying pancreatic autoimmunity in a murine disease model.

Methods: A transgenic mouse with an S100A4/fibroblast-specific protein 1 (FSP1) Cre-mediated conditional knockout of the transforming growth factor beta (TGFbeta) type II receptor, termed Tgfbr2(fspKO), was used to determine the direct role of TGFbeta in S100A4(+) cells. Immunohistochemical studies suggested that Tgfbr2(fspKO) mice develop mouse AIP (mAIP) characterised by interlobular ductal inflammatory infiltrates and pancreatic autoantibody production. Fluorescence-activated cell sorting (FACS)-isolated dendritic cells (DCs) from diseased pancreata were verified to have S100A4-Cre-mediated DNA recombination.

Results: The Tgfbr2(fspKO) mice spontaneously developed mAIP by 6 weeks of age. DCs were confirmed to express S100A4, a previously reported protein expressed by fibroblasts. Adoptive transfer of bone marrow-derived DCs from Tgfbr2(fspKO) mice into 2-week-old syngenic wild-type C57BL/6 mice resulted in reproduction of pancreatitis within 6 weeks. Similar adoptive transfer of wild-type DCs had no effect on pancreas pathology of the host mice. The inability to induce pancreatitis by adoptive transfer of Tgfbr2(fspKO) DCs in adult mice suggested a developmental event in mAIP pathogenesis. Tgfbr2(fspKO) DCs undergo elevated maturation in response to antigen and increased activation of naïve CD4-positive T cells.

Conclusion: The development of mAIP in the Tgfbr2(fspKO) mouse model illustrates the role of TGFbeta in maintaining myeloid DC immune tolerance. The loss of immune tolerance in myeloid S100A4(+) DCs can mediate mAIP and may explain some aspects of AIP disease pathogenesis.

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Dendritic cells (DCs) lacking the ability to respond to transforming growth factor β (TGFβ) are present in diseased pancreata. β-Galactosidase activity in conjunction with immunohistochemical stains of pancreas from Rosa26/Tgfbr2fspKO mice indicates fibroblast-specific protein (FSP)-Cre-mediated DNA recombination (A–E). (A) β-Galactosidase activity (blue) is associated with pancreatitis with nuclear fast red counterstain (red). Concomitant β-galactosidase with immunostaining (brown) for (B) insulin, (C) F4/80 (for macrophages), (D) CD3 (for T cells) and (E) nestin (for stellate cells) did not suggest DNA recombination in these cell types. (F) PCR analysis for Cre-recombinase activity in flow-sorted CD11c-positive pancreatic cells from wild-type (wt) and Tgfbr2fspKO (KO) suggests specific recombination in DCs (n = 6). Mouse tail DNA from Tgfbr2fspKO and Tgfbr2floxE2/floxE2 mice was used as a positive (+) and negative (−) control, respectively.
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gut-58-09-1267-f05: Dendritic cells (DCs) lacking the ability to respond to transforming growth factor β (TGFβ) are present in diseased pancreata. β-Galactosidase activity in conjunction with immunohistochemical stains of pancreas from Rosa26/Tgfbr2fspKO mice indicates fibroblast-specific protein (FSP)-Cre-mediated DNA recombination (A–E). (A) β-Galactosidase activity (blue) is associated with pancreatitis with nuclear fast red counterstain (red). Concomitant β-galactosidase with immunostaining (brown) for (B) insulin, (C) F4/80 (for macrophages), (D) CD3 (for T cells) and (E) nestin (for stellate cells) did not suggest DNA recombination in these cell types. (F) PCR analysis for Cre-recombinase activity in flow-sorted CD11c-positive pancreatic cells from wild-type (wt) and Tgfbr2fspKO (KO) suggests specific recombination in DCs (n = 6). Mouse tail DNA from Tgfbr2fspKO and Tgfbr2floxE2/floxE2 mice was used as a positive (+) and negative (−) control, respectively.

Mentions: To identify specific cell type(s) knocked out for Tgfbr2 by S100A4-Cre, Tgfbr2fspKO mice were further crossed with Rosa26 mice. β-Galactosidase reporter activity of the pancreata from Rosa26/Tgfbr2fspKO mice illustrated the presence of cells positive for Cre-mediated DNA recombination (fig 5A). Positive staining was exclusively found in the exocrine pancreas (fig 5B), with the suggested Tgfbr2-knockout cells co-localised to infiltrating inflammatory cells in the pancreatic parenchyma. The lack of co-immunostaining for F4/80, CD3 and nestin with β-galactosidase activity suggested that Tgfbr2-knockout cells were not macrophages, T cells or stellate cells, respectively (fig 5C–E). Due to their morphology and the autoimmune nature of mAIP, we hypothesised that Tgfbr2-knockout cells in the pancreas could be myeloid DCs. Efforts to immunostain for the DC marker CD11c on paraffin sections were unsuccessful, so the presence of Tgfbr2-knockout DCs in disease pancreata was assessed by FACS of pancreatic single-cell suspensions for cells expressing CD11c on their surfaces. Cre-mediated Tgfbr2 recombination was confirmed by PCR on the genomic DNA from the sorted pancreatic DCs (fig 5E). Thus, S100A4-positive DCs lacking TGFβ regulation were present in the pancreata of Tgfbr2fspKO mice.


Autoimmune pancreatitis results from loss of TGFbeta signalling in S100A4-positive dendritic cells.

Boomershine CS, Chamberlain A, Kendall P, Afshar-Sharif AR, Huang H, Washington MK, Lawson WE, Thomas JW, Blackwell TS, Bhowmick NA - Gut (2009)

Dendritic cells (DCs) lacking the ability to respond to transforming growth factor β (TGFβ) are present in diseased pancreata. β-Galactosidase activity in conjunction with immunohistochemical stains of pancreas from Rosa26/Tgfbr2fspKO mice indicates fibroblast-specific protein (FSP)-Cre-mediated DNA recombination (A–E). (A) β-Galactosidase activity (blue) is associated with pancreatitis with nuclear fast red counterstain (red). Concomitant β-galactosidase with immunostaining (brown) for (B) insulin, (C) F4/80 (for macrophages), (D) CD3 (for T cells) and (E) nestin (for stellate cells) did not suggest DNA recombination in these cell types. (F) PCR analysis for Cre-recombinase activity in flow-sorted CD11c-positive pancreatic cells from wild-type (wt) and Tgfbr2fspKO (KO) suggests specific recombination in DCs (n = 6). Mouse tail DNA from Tgfbr2fspKO and Tgfbr2floxE2/floxE2 mice was used as a positive (+) and negative (−) control, respectively.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2719085&req=5

gut-58-09-1267-f05: Dendritic cells (DCs) lacking the ability to respond to transforming growth factor β (TGFβ) are present in diseased pancreata. β-Galactosidase activity in conjunction with immunohistochemical stains of pancreas from Rosa26/Tgfbr2fspKO mice indicates fibroblast-specific protein (FSP)-Cre-mediated DNA recombination (A–E). (A) β-Galactosidase activity (blue) is associated with pancreatitis with nuclear fast red counterstain (red). Concomitant β-galactosidase with immunostaining (brown) for (B) insulin, (C) F4/80 (for macrophages), (D) CD3 (for T cells) and (E) nestin (for stellate cells) did not suggest DNA recombination in these cell types. (F) PCR analysis for Cre-recombinase activity in flow-sorted CD11c-positive pancreatic cells from wild-type (wt) and Tgfbr2fspKO (KO) suggests specific recombination in DCs (n = 6). Mouse tail DNA from Tgfbr2fspKO and Tgfbr2floxE2/floxE2 mice was used as a positive (+) and negative (−) control, respectively.
Mentions: To identify specific cell type(s) knocked out for Tgfbr2 by S100A4-Cre, Tgfbr2fspKO mice were further crossed with Rosa26 mice. β-Galactosidase reporter activity of the pancreata from Rosa26/Tgfbr2fspKO mice illustrated the presence of cells positive for Cre-mediated DNA recombination (fig 5A). Positive staining was exclusively found in the exocrine pancreas (fig 5B), with the suggested Tgfbr2-knockout cells co-localised to infiltrating inflammatory cells in the pancreatic parenchyma. The lack of co-immunostaining for F4/80, CD3 and nestin with β-galactosidase activity suggested that Tgfbr2-knockout cells were not macrophages, T cells or stellate cells, respectively (fig 5C–E). Due to their morphology and the autoimmune nature of mAIP, we hypothesised that Tgfbr2-knockout cells in the pancreas could be myeloid DCs. Efforts to immunostain for the DC marker CD11c on paraffin sections were unsuccessful, so the presence of Tgfbr2-knockout DCs in disease pancreata was assessed by FACS of pancreatic single-cell suspensions for cells expressing CD11c on their surfaces. Cre-mediated Tgfbr2 recombination was confirmed by PCR on the genomic DNA from the sorted pancreatic DCs (fig 5E). Thus, S100A4-positive DCs lacking TGFβ regulation were present in the pancreata of Tgfbr2fspKO mice.

Bottom Line: DCs were confirmed to express S100A4, a previously reported protein expressed by fibroblasts.Tgfbr2(fspKO) DCs undergo elevated maturation in response to antigen and increased activation of naïve CD4-positive T cells.The loss of immune tolerance in myeloid S100A4(+) DCs can mediate mAIP and may explain some aspects of AIP disease pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Vanderbilt-Ingram Cancer, Vanderbilt University, Nashville 37232, Tennessee, USA.

ABSTRACT

Background and aims: Autoimmune pancreatitis (AIP) is a poorly understood human disease affecting the exocrine pancreas. The goal of the present study was to elucidate the pathogenic mechanisms underlying pancreatic autoimmunity in a murine disease model.

Methods: A transgenic mouse with an S100A4/fibroblast-specific protein 1 (FSP1) Cre-mediated conditional knockout of the transforming growth factor beta (TGFbeta) type II receptor, termed Tgfbr2(fspKO), was used to determine the direct role of TGFbeta in S100A4(+) cells. Immunohistochemical studies suggested that Tgfbr2(fspKO) mice develop mouse AIP (mAIP) characterised by interlobular ductal inflammatory infiltrates and pancreatic autoantibody production. Fluorescence-activated cell sorting (FACS)-isolated dendritic cells (DCs) from diseased pancreata were verified to have S100A4-Cre-mediated DNA recombination.

Results: The Tgfbr2(fspKO) mice spontaneously developed mAIP by 6 weeks of age. DCs were confirmed to express S100A4, a previously reported protein expressed by fibroblasts. Adoptive transfer of bone marrow-derived DCs from Tgfbr2(fspKO) mice into 2-week-old syngenic wild-type C57BL/6 mice resulted in reproduction of pancreatitis within 6 weeks. Similar adoptive transfer of wild-type DCs had no effect on pancreas pathology of the host mice. The inability to induce pancreatitis by adoptive transfer of Tgfbr2(fspKO) DCs in adult mice suggested a developmental event in mAIP pathogenesis. Tgfbr2(fspKO) DCs undergo elevated maturation in response to antigen and increased activation of naïve CD4-positive T cells.

Conclusion: The development of mAIP in the Tgfbr2(fspKO) mouse model illustrates the role of TGFbeta in maintaining myeloid DC immune tolerance. The loss of immune tolerance in myeloid S100A4(+) DCs can mediate mAIP and may explain some aspects of AIP disease pathogenesis.

Show MeSH
Related in: MedlinePlus