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Impaired Cellular Immunity in the Murine Neural Crest Conditional Deletion of Endothelin Receptor-B Model of Hirschsprung's Disease.

Gosain A, Barlow-Anacker AJ, Erickson CS, Pierre JF, Heneghan AF, Epstein ML, Kudsk KA - PLoS ONE (2015)

Bottom Line: We found that EdnrBNCC-/- display lymphopenia of their Peyer's Patches, the major inductive site of GI mucosal immunity.In the spleen, which is the primary source of IgA-producing Mature B-lymphocytes, EdnrBNCC-/- animals display decreased B-lymphocytes, but an increase in Mature B-lymphocytes.EdnrBNCC-/- spleens are also small and show altered architecture, with decreased red pulp and a paucity of B-lymphocytes in the germinal centers and marginal zone.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin, United States of America; Department of Neuroscience, University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin, United States of America.

ABSTRACT
Hirschsprung's disease (HSCR) is characterized by aganglionosis from failure of neural crest cell (NCC) migration to the distal hindgut. Up to 40% of HSCR patients suffer Hirschsprung's-associated enterocolitis (HAEC), with an incidence that is unchanged from the pre-operative to the post-operative state. Recent reports indicate that signaling pathways involved in NCC migration may also be involved in the development of secondary lymphoid organs. We hypothesize that gastrointestinal (GI) mucosal immune defects occur in HSCR that may contribute to enterocolitis. EdnrB was deleted from the neural crest (EdnrBNCC-/-) resulting in mutants with defective NCC migration, distal colonic aganglionosis and the development of enterocolitis. The mucosal immune apparatus of these mice was interrogated at post-natal day (P) 21-24, prior to histological signs of enterocolitis. We found that EdnrBNCC-/- display lymphopenia of their Peyer's Patches, the major inductive site of GI mucosal immunity. EdnrBNCC-/- Peyer's Patches demonstrate decreased B-lymphocytes, specifically IgM+IgDhi (Mature) B-lymphocytes, which are normally activated and produce IgA following antigen presentation. EdnrBNCC-/- animals demonstrate decreased small intestinal secretory IgA, but unchanged nasal and bronchial airway secretory IgA, indicating a gut-specific defect in IgA production or secretion. In the spleen, which is the primary source of IgA-producing Mature B-lymphocytes, EdnrBNCC-/- animals display decreased B-lymphocytes, but an increase in Mature B-lymphocytes. EdnrBNCC-/- spleens are also small and show altered architecture, with decreased red pulp and a paucity of B-lymphocytes in the germinal centers and marginal zone. Taken together, these findings suggest impaired GI mucosal immunity in EdnrBNCC-/- animals, with the spleen as a potential site of the defect. These findings build upon the growing body of literature that suggests that intestinal defects in HSCR are not restricted to the aganglionic colon but extend proximally, even into the ganglionated small intestine and immune cells.

No MeSH data available.


Related in: MedlinePlus

Peyer’s Patches of EdnrBNCC-/- animals are small in size but have normal architecture.(A) Gross photographs of representative PP isolated from EdnrBNCC+/- and EdnrBNCC-/- animals demonstrating the size difference. Scale bar = 1000μm. (B) H&E staining of PP cross-sections from EdnrBNCC+/- and EdnrBNCC-/- animals. Architectural features are preserved in EdnrBNCC-/-. Scale bar = 400μm (applies to B,C,D). (C) IHC of PP cross-sections with CD3 labeling T cells. (D) IHC of PP cross-sections with B220 labeling B cells. The spatial distribution of B-cells and T-cells is similar between EdnrBNCC+/- and EdnrBNCC-/-.
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pone.0128822.g002: Peyer’s Patches of EdnrBNCC-/- animals are small in size but have normal architecture.(A) Gross photographs of representative PP isolated from EdnrBNCC+/- and EdnrBNCC-/- animals demonstrating the size difference. Scale bar = 1000μm. (B) H&E staining of PP cross-sections from EdnrBNCC+/- and EdnrBNCC-/- animals. Architectural features are preserved in EdnrBNCC-/-. Scale bar = 400μm (applies to B,C,D). (C) IHC of PP cross-sections with CD3 labeling T cells. (D) IHC of PP cross-sections with B220 labeling B cells. The spatial distribution of B-cells and T-cells is similar between EdnrBNCC+/- and EdnrBNCC-/-.

Mentions: PP are distinct collections of immune cells located along the anti-mesenteric surface of the bowel; they display follicular architecture similar to lymph nodes and are the primary inductive site for gut mucosal immunity [42]. Previous observations that PP are absent in RET-/- animals and are diminished in RET51/51 hypomorphic animals [29] prompted us to investigate whether the EdnrBNCC-/- animals exhibit alterations in their GI immune apparatus. We harvested small bowel from EdnrBNCC-/- and EdnrBNCC+/- at P21-24 [19]. We found that the PP of EdnrBNCC-/- animals are smaller than those of EdnrBNCC+/- animals on gross examination (Fig 2). To quantify this, cross-sectional measurements of PP height and width were taken and area calculated. We found that EdnrBNCC-/- PP have significantly smaller cross-sectional area than EdnrBNCC+/- (EdnrBNCC+/- 1.15±0.05 mm2 vs. EdnrBNCC-/- 0.49±0.04 mm2, p = 4.1x10-17). Although smaller in size, H&E staining demonstrates that the architecture of EdnrBNCC-/- PP is preserved, with distinct germinal centers, subepithelial domes and follicle-associated epithelium (Fig 2). Additionally, while the extent of B220+ and CD3+ staining is decreased in the EdnrBNCC-/- animals, suggesting fewer cells, the distribution of B220+ B-lymphocytes and CD3+ T-lymphocytes in distinct B- and T-cell zones within the PP appears unchanged between EdnrBNCC+/- and EdnrBNCC-/- animals (Fig 2). Finally, unlike the observation in RET mutant models, we did not observe a difference in the number of PP between groups (EdnrBNCC+/- 9.75±0.85 vs. EdnrBNCC-/- 7.25±1.3, p = 0.16).


Impaired Cellular Immunity in the Murine Neural Crest Conditional Deletion of Endothelin Receptor-B Model of Hirschsprung's Disease.

Gosain A, Barlow-Anacker AJ, Erickson CS, Pierre JF, Heneghan AF, Epstein ML, Kudsk KA - PLoS ONE (2015)

Peyer’s Patches of EdnrBNCC-/- animals are small in size but have normal architecture.(A) Gross photographs of representative PP isolated from EdnrBNCC+/- and EdnrBNCC-/- animals demonstrating the size difference. Scale bar = 1000μm. (B) H&E staining of PP cross-sections from EdnrBNCC+/- and EdnrBNCC-/- animals. Architectural features are preserved in EdnrBNCC-/-. Scale bar = 400μm (applies to B,C,D). (C) IHC of PP cross-sections with CD3 labeling T cells. (D) IHC of PP cross-sections with B220 labeling B cells. The spatial distribution of B-cells and T-cells is similar between EdnrBNCC+/- and EdnrBNCC-/-.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4465674&req=5

pone.0128822.g002: Peyer’s Patches of EdnrBNCC-/- animals are small in size but have normal architecture.(A) Gross photographs of representative PP isolated from EdnrBNCC+/- and EdnrBNCC-/- animals demonstrating the size difference. Scale bar = 1000μm. (B) H&E staining of PP cross-sections from EdnrBNCC+/- and EdnrBNCC-/- animals. Architectural features are preserved in EdnrBNCC-/-. Scale bar = 400μm (applies to B,C,D). (C) IHC of PP cross-sections with CD3 labeling T cells. (D) IHC of PP cross-sections with B220 labeling B cells. The spatial distribution of B-cells and T-cells is similar between EdnrBNCC+/- and EdnrBNCC-/-.
Mentions: PP are distinct collections of immune cells located along the anti-mesenteric surface of the bowel; they display follicular architecture similar to lymph nodes and are the primary inductive site for gut mucosal immunity [42]. Previous observations that PP are absent in RET-/- animals and are diminished in RET51/51 hypomorphic animals [29] prompted us to investigate whether the EdnrBNCC-/- animals exhibit alterations in their GI immune apparatus. We harvested small bowel from EdnrBNCC-/- and EdnrBNCC+/- at P21-24 [19]. We found that the PP of EdnrBNCC-/- animals are smaller than those of EdnrBNCC+/- animals on gross examination (Fig 2). To quantify this, cross-sectional measurements of PP height and width were taken and area calculated. We found that EdnrBNCC-/- PP have significantly smaller cross-sectional area than EdnrBNCC+/- (EdnrBNCC+/- 1.15±0.05 mm2 vs. EdnrBNCC-/- 0.49±0.04 mm2, p = 4.1x10-17). Although smaller in size, H&E staining demonstrates that the architecture of EdnrBNCC-/- PP is preserved, with distinct germinal centers, subepithelial domes and follicle-associated epithelium (Fig 2). Additionally, while the extent of B220+ and CD3+ staining is decreased in the EdnrBNCC-/- animals, suggesting fewer cells, the distribution of B220+ B-lymphocytes and CD3+ T-lymphocytes in distinct B- and T-cell zones within the PP appears unchanged between EdnrBNCC+/- and EdnrBNCC-/- animals (Fig 2). Finally, unlike the observation in RET mutant models, we did not observe a difference in the number of PP between groups (EdnrBNCC+/- 9.75±0.85 vs. EdnrBNCC-/- 7.25±1.3, p = 0.16).

Bottom Line: We found that EdnrBNCC-/- display lymphopenia of their Peyer's Patches, the major inductive site of GI mucosal immunity.In the spleen, which is the primary source of IgA-producing Mature B-lymphocytes, EdnrBNCC-/- animals display decreased B-lymphocytes, but an increase in Mature B-lymphocytes.EdnrBNCC-/- spleens are also small and show altered architecture, with decreased red pulp and a paucity of B-lymphocytes in the germinal centers and marginal zone.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin, United States of America; Department of Neuroscience, University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin, United States of America.

ABSTRACT
Hirschsprung's disease (HSCR) is characterized by aganglionosis from failure of neural crest cell (NCC) migration to the distal hindgut. Up to 40% of HSCR patients suffer Hirschsprung's-associated enterocolitis (HAEC), with an incidence that is unchanged from the pre-operative to the post-operative state. Recent reports indicate that signaling pathways involved in NCC migration may also be involved in the development of secondary lymphoid organs. We hypothesize that gastrointestinal (GI) mucosal immune defects occur in HSCR that may contribute to enterocolitis. EdnrB was deleted from the neural crest (EdnrBNCC-/-) resulting in mutants with defective NCC migration, distal colonic aganglionosis and the development of enterocolitis. The mucosal immune apparatus of these mice was interrogated at post-natal day (P) 21-24, prior to histological signs of enterocolitis. We found that EdnrBNCC-/- display lymphopenia of their Peyer's Patches, the major inductive site of GI mucosal immunity. EdnrBNCC-/- Peyer's Patches demonstrate decreased B-lymphocytes, specifically IgM+IgDhi (Mature) B-lymphocytes, which are normally activated and produce IgA following antigen presentation. EdnrBNCC-/- animals demonstrate decreased small intestinal secretory IgA, but unchanged nasal and bronchial airway secretory IgA, indicating a gut-specific defect in IgA production or secretion. In the spleen, which is the primary source of IgA-producing Mature B-lymphocytes, EdnrBNCC-/- animals display decreased B-lymphocytes, but an increase in Mature B-lymphocytes. EdnrBNCC-/- spleens are also small and show altered architecture, with decreased red pulp and a paucity of B-lymphocytes in the germinal centers and marginal zone. Taken together, these findings suggest impaired GI mucosal immunity in EdnrBNCC-/- animals, with the spleen as a potential site of the defect. These findings build upon the growing body of literature that suggests that intestinal defects in HSCR are not restricted to the aganglionic colon but extend proximally, even into the ganglionated small intestine and immune cells.

No MeSH data available.


Related in: MedlinePlus