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

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.

Fig 1. EdnrB ^NCC-/- animals develop intestinal inflammation in the fourth week of life.

Inflammation within proximal colon and mid-colon of P26-29 EdnrB ^NCC+/- (Het) and EdnrB ^NCC-/- (Null) animals was graded using the previously published Murine Enterocolitis Grading System (Cheng, et.al., 2010). (A) There was evidence of inflammation in both the (B) proximal and (C) mid-colon of the EdnrB ^NCC-/- animals at the later time point. Arrows indicate inflammatory cells. Scale bar = 50 μm. (*p<0.05).

Fig 2. Peyer’s Patches of EdnrB ^NCC-/- animals are small in size but have normal architecture.

(A) Gross photographs of representative PP isolated from EdnrB ^NCC+/- and EdnrB ^NCC-/- animals demonstrating the size difference. Scale bar = 1000μm. (B) H&E staining of PP cross-sections from EdnrB ^NCC+/- and EdnrB ^NCC-/- animals. Architectural features are preserved in EdnrB ^NCC-/-. 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 EdnrB ^NCC+/- and EdnrB ^NCC-/-.

Fig 3. Peyer’s Patches of EdnrB ^NCC-/- animals exhibit B-cell lymphopenia.

(A) Total lymphocytes (per PP) are decreased in EdnrB ^NCC-/- vs. EdnrB ^NCC+/- (*p = 0.0196). (B) Representative flow cytometry gating of PP showing CD3 and B220 to identify T- and B-lymphocytes (top) and IgD and IgM to identify B220⁺IgM⁺IgD^high (mature) B-lymphocytes (bottom). (C) The proportion of B-lymphocytes (B220+) per PP is decreased in EdnrB ^NCC-/- vs. EdnrB ^NCC+/- (*p = 0.01). (D) The number of B-lymphocytes per PP is decreased in EdnrB ^NCC-/- vs EdnrB ^NCC+/- (calculated values). (E) The proportion of B220⁺IgM⁺IgD^high mature B-lymphocytes is decreased in EdnrB ^NCC-/- vs.—het PP (*p = 0.015). (F) The number of mature B-lymphocytes per PP is decreased in EdnrB ^NCC-/- vs EdnrB ^NCC+/- (calculated values).

Fig 4. EdnrB ^NCC-/- animals demonstrate a gut-specific deficiency of Secretory IgA (SIgA).

Secretory IgA was measured by ELISA in (A) small intestinal lavage fluid (*p = 0.002), (B) nasal airway lavage fluid, and (C) bronchoalveolar lavage fluid of EdnrB ^NCC+/- and EdnrB ^NCC-/- animals. (D) Additionally, IgM in small intestinal lavage was measured.

Fig 5. The spleen of EdnrB ^NCC-/- animals is small in size and has abnormal architecture.

(A) Gross photographs of representative spleens isolated from EdnrB ^NCC+/- and EdnrB ^NCC-/- animals demonstrating the size difference. (B) H&E staining of spleen cross-sections from EdnrB ^NCC+/- and EdnrB ^NCC-/- animals. EdnrB ^NCC-/- spleens demonstrate decreased red pulp (RP) and white pulp (WP). Scale bar = 600μm. (C) IHC of spleen cross-sections with B220 (green) and CD3 (blue). There are decreased B-cells in the germinal centers and a paucity of these cells within the marginal zone, located outside the ring of MOMA+ (red) staining. Scale bar = 200μm.

Fig 6. The spleen of EdnrB ^NCC-/- animals exhibits B-cell lymphopenia.

(A) Organ weights of the spleen (*p = 0.02) as a proportion of body weight, indicating that the spleens of EdnrB ^NCC-/- animals are small for size. (B) Total lymphocytes (per spleen) are decreased in EdnrB ^NCC-/- vs. EdnrB ^NCC+/- (*p = 0.002). (C) Representative flow cytometry gating of splenocytes showing CD3 and B220 to identify T- and B-lymphocytes (top) and IgD and IgM to identify B220⁺IgM⁺IgD^high (mature) B-lymphocytes (bottom). (D) The proportion of B-lymphocytes (B220+) per spleen is decreased in EdnrB ^NCC-/- vs. EdnrB ^NCC+/- (*p = 0.000053). (E) The number of B-lymphocytes per spleen is decreased in EdnrB ^NCC-/- vs.—het spleens (calculated values). (F) B220⁺IgM⁺IgD^high mature B-lymphocytes are increased in EdnrB ^NCC-/- vs.—het spleen (*p = 0.00023). (G) The number of mature B-lymphocytes per spleen is decreased in EdnrB ^NCC-/- vs.—het spleens (calculated values).

Fig 7. Trafficking of B-lymphocytes from Peripheral Blood to PP is preserved.

(A) The proportion of circulating mature B-lymphocytes expressing L-selectin is unchanged between EdnrB ^NCC+/- and-null animals. (B) Levels of PP Mucosal Addressin Cell Adhesion Molecule-1 (MAdCAM-1) are unchanged between EdnrB ^NCC+/- and EdnrB ^NCC-/- animals.