Enterocolitis causes profound lymphoid depletion in endothelin receptor B- and endothelin 3-null mouse models of Hirschsprung-associated enterocolitis

Abstract

Potentially life-threatening enterocolitis is the most frequent complication in children with colonic aganglionosis (Hirschsprung disease, HSCR), and little is known about the mechanisms leading to enterocolitis. Splenic lymphopenia has been reported in the Endothelin Receptor B (Ednrb)-null mouse model of HSCR that develops enterocolitis. In this study, we sought to identify molecular mechanisms underlying this immune phenotype. We employed the Ednrb(-/-) mouse, and the knockout of its ligand, Edn3 (Edn3(-/-)). The major finding is that enterocolitis in the Ednrb(-/-) and Edn3(-/-) mice lead to thymic involution, splenic lymphopenia, and suppression of B lymphopoiesis as a consequence of colonic aganglionosis, not an intrinsic Edn3-Ednrb signaling defect directly affecting the lymphoid organs. We showed that adoptive transfer of Ednrb(-/-) marrow repopulated the RAG2-null mice marrow, thymus and spleen without development of enterocolitis. We identified the glucocorticoid corticosterone, as a potential mediator of the immune phenotype. This previously unrecognized pattern of immune abnormalities in mouse is nearly identical to lymphoid depletion in neonatal sepsis during severe physiological stress, suggesting that the mouse model used here could be also used for sepsis studies.

Keywords: Corticosterone; Endothelins; Enterocolitis; Glucocorticoids; HAEC; Hirschsprung disease; Lymphoid depletion.

Conflict of interest statement

Conflict of interest

P.F. is a consultant for Karl Storz Endoscopy – America. The remaining authors declare that they have no conflict of interest.

© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Figures

Figure 1

Splenic lymphocyte composition and enterocolitis scores in Ednrb- and Edn3-null mice. (A) Representative FACS profiles and percentages of splenic CD4+, CD8+, and IgM+ cells of WT, Ednrb−/−, and Edn3−/− mice. (B) Total splenic cell numbers, numbers of splenic CD4+, CD8+, and IgM+ cells in Ednrb−/− and Edn3−/− mice. (C) Enterocolitis scores of wild type, Ednrb−/− and Edn3−/−. Data are presented as mean ± SEM of n = 5–6 mice/group. These data are representative of two separate experiments performed with both genotypes. *p < 0.05, **p < 0.01, and ***p < 0.001. Comparison between Ednrb−/− and +/+, Edn3−/− and +/+ was made using Student’s t-test.

Figure 2

Thymocyte composition in Ednrb- and Edn3-null mice. (A) Summary of total numbers of CD4+, CD8+, and CD4+CD8+ cells in thymus. (B) Representative FACS profiles and percentages of CD4+, CD8+, and CD4+CD8+ cells in thymus. Data are shown as mean ± SEM of n = 5–6 mice/group, and are representative of two independent experiments performed with both genotypes. *p < 0.05, **p < 0.01, and ***p < 0.001. Comparisons between Ednrb−/− and +/+, Edn3−/−, and +/+ were performed using Student’s t-test.

Figure 3

Bone marrow B lymphopoiesis in Ednrb- and Edn3-null mice at age P21. (A) Representative FACS profiles and percentages of pro-B and pre-B, and mature B cells of WT, Ednrb−/−, and Edn3−/− mice. Pro-B (CD45R+CD43+IgM−) and pre-B (CD45R+CD43−IgM−) cells were gated from CD45R+IgM− cells. IgM+ cells represent mature B cells. B, Number of pro-B, pre-B, and IgM+ cell numbers in bone marrow. These data are representative of two separate experiments performed with both genotypes. (C) Representative FACS histograms and percentages of HSCs[Lin−CD117 (c-kit)highSca-1highCD150+] and CLPs[Lin−CD117lowSca-1lowCD127+] cells in bone marrow. (D) Number and percentages of HSCs and CLPs in bone marrow of Ednrb−/− and their WT littermates. Data are presented as mean ± SEM of n = 5–6 mice/group. (C and D) Data shown are from a single experiment. *p < 0.05, **p < 0.01, and ***p < 0.001. Comparison between Ednrb−/− and +/+ was performed using Student’s t-test.

Figure 4

B lineage lymphopoiesis in neonatal (P10) Ednrb-null mice. (A) Representative FACS profiles and percentages of pro-B, pre-B, and mature B cells; Pro-B (CD45R+CD43−IgM−) and pre-B (CD45R+CD43+IgM−) cells were gated from CD45R+IgM− cells. (B) Number of IgM+ cells. (C) Number of pro-B and pre-B cells in bone marrow. These data are from a single experiment and presented as mean ± SEM of n = 5–6 mice/group). Comparison between Ednrb−/− and +/+ was made using Student’s t-test. None reached statistical significance.

Figure 5

Reconstitution of splenic B and T lymphocytes in RAG2−/− recipients after adoptive transfer of Ednrb-null and wild type donor derived bone marrow. (A) Representative FACS profiles and percentages of CD4+, CD8+, and IgM+ cells in spleens of WT, RAG2−/− control, and RAG2−/− recipients with bone marrow transplantation of Ednrb+/+ and Ednrb−/− mice. (B) Summary of total lymphocyte numbers in spleens of RAG2−/− recipient mice. (C) Summary of total numbers of CD4+, CD8+, and IgM+ cells in spleens of RAG2−/− recipient mice. These data are from a single experiment and are presented as mean ± SEM of n = 6–8 mice/group. Comparison between groups was made using Student’s t-test.

Figure 6

Bone marrow reconstitution of HSCs and CLPs in RAG2−/− recipients after adoptive transfer of Ednrb-null and wild-type donor derived bone marrow. (A) Representative FACS profiles and percentages of HSCs and CLPs in bone marrow of WT, RAG2−/− control, and RAG2−/− recipients with bone marrow transplantation of Ednrb+/+ and Ednrb−/− mice. (B and C) Graphs show (B) the cell number and (C) the percentage of HSCs and CLPs. These data are from a single experiment and are presented as mean ± SEM of n = 6–8 mice/group. Comparison between groups was made using Student’s t-test. No significant differences were found.

Figure 7

Serum corticosterone concentrations in neonatal Ednrb-null mice, weanling Ednrb- and Edn3-null mice, and wild-type mice with induced megacolon. Serum corticosterone concentrations (ng/mL) of: (A) age P10WTand Ednrb−/− mice. (B) Age P21 WT, Ednrb−/−, and Edn3−/− mice. (C) WT and wild type with induced megacolon (WT-IM). (A–C) Data are from a single experiment and are presented as mean ± SEM of n = 5–6 mice/group. *p < 0.05, **p < 0.01, and ***p < 0.001. Comparison between groups was made using Student’s t-test.

Figure 8

Splenic and thymic lymphocyte populations in wild-type mice with surgically induced megacolon (WT-IM). (A) Representative FACS profiles and percentages of CD4+, CD8+, and IgM+ cells in spleens of WT and WT-IM mice. (B) Representative FACS profiles and percentages of CD4+, CD8+, and CD4+CD8+ cells in thymus of WT and WT-IM mice. (C) Summary of mean number of CD4+, CD8+, and IgM+ cells in spleens of WT and WT-IM mice. (D) Summary of mean number of CD4+, CD8+, and CD4+CD8+ cells in thymus of WT and WT-IM mice. (A and B) Data are from a single experiment and are presented as mean ± SEM of n = 5–6 mice/group. *p < 0.05, **p < 0.01, and ***p < 0.001. Comparison between groups was made using Student’s t-test.

Figure 9

Pro-B, pre-B, and mature B-cell populations of wild-type mice with surgically induced megacolon. (A) Representative FACS profiles and percentages of pro-B, pre-B, and mature B cells; pro-B (CD45R+CD43−IgM−), and pre-B (CD45R+CD43+IgM−) cells were gated from CD45R+IgM− cells in bone marrow of WT and WT-IM mice; (B) Total numbers of pro-B, pre-B, and mature B cells in bone marrow of WT-IM with appropriate controls. Data are from a single experiment and are presented as mean ± SEM of n = 5–6 mice/group. *p < 0.05, **p < 0.01, and ***p < 0.001. Comparison between groups was made using Student’s t-test.