Lymphocyte-dependent and Th2 cytokine-associated colitis in mice deficient in Wiskott-Aldrich syndrome protein

Abstract

Background & aims: Controversy exists as to whether patients with inflammatory bowel disease have an underlying immunodeficiency. We have focused on a murine model of the Wiskott-Aldrich syndrome, an immunodeficiency in which autoimmunity can manifest in the form of an inflammatory bowel disease-like illness. Wiskott-Aldrich syndrome protein (WASP) deficiency in mice results in similar clinical features. Herein, we characterized the colitis in WASP-deficient mice.

Methods: WASP-deficient mice were followed clinically and histologically. Immunologic studies were performed to determine the pathogenic cell population(s), the predominant cytokine expression pattern, and the role of cytokine(s) in colitis pathogenesis.

Results: All WASP-deficient mice develop colitis by 6 months of age. Lymphocytes are required for disease induction, and CD4(+) T cells from WASP-deficient mice are sufficient to induce disease in lymphocyte-deficient hosts. Lamina propria preparations from WASP-deficient mice demonstrated elevations in interferon-gamma, interleukin (IL)-4, and IL-13 levels but decreased IL-6 and no difference in IL-17 expression in comparison with wild-type controls. Treatment with neutralizing antibody to IL-4, but not to interferon-gamma, abrogated colitis development. However, mice deficient in both WASP and IL-4 showed no difference in histologic colitis scores at 24 weeks of age compared with WASP-deficient mice.

Conclusions: These results demonstrate a critical role for lymphocytes and a relative T helper 2 cytokine predominance in the colitis associated with WASP-deficient mice. This is the only model of colitis with elevated T helper 2 cytokines and aberrant natural regulatory T cell function and is unique in having a human disease counterpart with similar defects.

Figure 1. A majority of WKO mice develop spontaneous colitis and splenomegaly

(A) Upper left panel shows normal WT (top) compared to thickened WKO (bottom) colons. H&E staining reveals crypt elongation and a large inflammatory cell infiltrate into the lamina propria of WKO colon (right, objective: 10x) compared to normal WT colon (lower left, objective: 10x). Splenomegaly is often associated with colitis in WKO mice (upper middle panel). Bar represents 1cm. (B) Prevalence of clinical (weight loss, diarrhea, rectal prolapse, or death) and microscopic (wall thickening, LP infiltration, crypt abscess) signs of colitis by age. Note, none of the 11 WT mice followed concurrently demonstrated any clinical or histologic signs of colitis.

Figure 2. WKO colonic LP is infiltrated with several distinct leukocyte subsets

Note an increase of macrophages (F4/80⁺ cells, black arrowheads), CD8⁺ cells (black arrows), and dendritic cells (CD11c⁺ cells, black curved arrows) in WKO colonic lamina propria. Data are representative of stainings of 2-4 WT and WKO samples each. Staining was performed on WKO colons with severe colitis (age > 6 months). All micrographs were taken with 10x objective.

Figure 3. WKO colonic LP contains activated T cells

Lymphocytes from WT and WKO colonic lamina propria (LP), mesenteric lymph nodes (MLN), subcutaneous lymph nodes (SLN), and spleen (SPL) were analyzed for activation markers. WKO LP is comprised of a higher percentage of T cells (i.e., TCRβ⁺) than WT. There is marked expansion of activated T cells (as evidenced by CD69⁺ and CD62L⁻ staining) in the LP and less striking expansion in other lymphoid compartments in WKO mice compared to WT mice. Data are representative examples of at least three individual experiments. Mice used in the experiment were 3-4 months old.

Figure 4. WKO lymphocytes are required and CD4⁺ T cells are sufficient for colitis induction

(A) Representative cross sections of colons from WKO or WT bone marrow cells (BMC) → RAG-2 KO transfer recipients (objective: 4x). Note the absence of colitis in recipients that received only WT BMCs compared to the markedly thickened colon of recipients of WKO BMCs. (B) WRDKO mice do not develop colitis (objective: 4x). (C) Macroscopic (upper panels) and microscopic analyses (lower panels, objective: 10x) demonstrated that CD4⁺ T cells from WKO (right) but not WT mice (center) can transfer colitis to RAG-2 KO recipients. RAG-2 KO control mouse colons are shown on the left.

Figure 5. Relative Th2 skewing associated with colitis in WKO mice

(A) WKO LP cells secrete markedly elevated levels of IL-4 and IL-13 as well as IFN-γ, TNF-α, and IL-2 with a decrease in IL-6. Results shown are means ± SEM pooling from two to four experiments, *p < 0.05 between WT and WKO stimulated with P+I. (B) Only IL-4 and IL-10 are mildly elevated in WKO MLN compared to WT. Results shown are means ± SEM pooling from two to four experiments, *p<0.05 between WT and WKO stimulated with P+I. (C) Shown are representative sections of two independent experiments of WT (left panel, objective: 10x) and inflamed (5-month-old) WKO (right panel, objective: 10x; inset, objective: 40x) colonic sections stained for IL-4 by immunohistochemistry. US = unstimulated; P+I = stimulated with PMA and ionomycin.

Figure 6. Neutralization of IFN-γ does not alleviate colitis in WKO mice

(A) Mice treated with weekly injections of anti-IFN-γ antibody (n = 7), but not mice treated with control IgG antibodies (n = 11), lost weight. (B) Average histologic score of anti-IFN-γ Ab-treated mice compared to control IgG Ab-treated mice one week after final antibody treatment (p = 0.07).

Figure 7. Neutralization of IL-4 attenuates WKO colitis

(A) Mice treated with weekly injections of anti-IL-4 antibody, but not mice treated with control IgG antibodies, gained weight. (B) Representative sections from mice treated with anti-IL-4 antibody and mice treated with control IgG antibodies that developed colitis (objective: 10x). (C) LP cells from mice treated with anti-IL-4 antibody secreted negligible amounts of cytokines. In contrast, LP cells from mice treated with control rat IgG antibodies that developed severe colitis secreted a moderate amount of IL-10 and a large amount of IL-4, IL-13, and IFN-γ, *p < 0.05 between cells stimulated with P+I from mice treated with anti-IL-4 antibody and mice treated with control IgG antibodies that developed colitis. Shown are means ± SEM pooling from two to four mice in each group. US = unstimulated; P+I = stimulated with PMA and ionomycin. (D) WASP/IL-4 DKO mice demonstrated higher weights and lower clinical colitis scores at 18 weeks of age than WKO counterparts that were followed concurrently.

Figure 8. CD4⁺ T cells from WKO can be skewed to secrete Th1 or Th2 cytokines

(A) Representative flow cytometry plots of intracellular IL-4 and IFN-γ staining in WT (n = 3) and WKO (n = 3) CD4+ T cells polarized to Th0, Th1, or Th2 by appropriate stimulations. (B) Graph representing the mean percentage ± SD of CD4+ T cells expressing IL-4 or IFN-γ in cells isolated from WT (n = 3) and WKO (n = 3) mice polarized in Th0, Th1, or Th2 conditions.