Table 1.
Enterocolitis Score by Anatomic Location.
Fig 1.
EdnrBNCC-/- animals develop intestinal inflammation in the fourth week of life.
Inflammation within proximal colon and mid-colon of P26-29 EdnrBNCC+/- (Het) and EdnrBNCC-/- (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 EdnrBNCC-/- animals at the later time point. Arrows indicate inflammatory cells. Scale bar = 50 μm. (*p<0.05).
Fig 2.
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-/-.
Fig 3.
Peyer’s Patches of EdnrBNCC-/- animals exhibit B-cell lymphopenia.
(A) Total lymphocytes (per PP) are decreased in EdnrBNCC-/- vs. EdnrBNCC+/- (*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+IgDhigh (mature) B-lymphocytes (bottom). (C) The proportion of B-lymphocytes (B220+) per PP is decreased in EdnrBNCC-/- vs. EdnrBNCC+/- (*p = 0.01). (D) The number of B-lymphocytes per PP is decreased in EdnrBNCC-/- vs EdnrBNCC+/- (calculated values). (E) The proportion of B220+IgM+IgDhigh mature B-lymphocytes is decreased in EdnrBNCC-/- vs.—het PP (*p = 0.015). (F) The number of mature B-lymphocytes per PP is decreased in EdnrBNCC-/- vs EdnrBNCC+/- (calculated values).
Table 2.
Summary results of cellularity findings in EdnrBNCC+/- and EdnrBNCC-/- Peyer’s Patches (PP), Spleen (SPL) and Peripheral Blood (PBL).
Fig 4.
EdnrBNCC-/- 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 EdnrBNCC+/- and EdnrBNCC-/- animals. (D) Additionally, IgM in small intestinal lavage was measured.
Fig 5.
The spleen of EdnrBNCC-/- animals is small in size and has abnormal architecture.
(A) Gross photographs of representative spleens isolated from EdnrBNCC+/- and EdnrBNCC-/- animals demonstrating the size difference. (B) H&E staining of spleen cross-sections from EdnrBNCC+/- and EdnrBNCC-/- animals. EdnrBNCC-/- 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 EdnrBNCC-/- 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 EdnrBNCC-/- animals are small for size. (B) Total lymphocytes (per spleen) are decreased in EdnrBNCC-/- vs. EdnrBNCC+/- (*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+IgDhigh (mature) B-lymphocytes (bottom). (D) The proportion of B-lymphocytes (B220+) per spleen is decreased in EdnrBNCC-/- vs. EdnrBNCC+/- (*p = 0.000053). (E) The number of B-lymphocytes per spleen is decreased in EdnrBNCC-/- vs.—het spleens (calculated values). (F) B220+IgM+IgDhigh mature B-lymphocytes are increased in EdnrBNCC-/- vs.—het spleen (*p = 0.00023). (G) The number of mature B-lymphocytes per spleen is decreased in EdnrBNCC-/- 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 EdnrBNCC+/- and-null animals. (B) Levels of PP Mucosal Addressin Cell Adhesion Molecule-1 (MAdCAM-1) are unchanged between EdnrBNCC+/- and EdnrBNCC-/- animals.