CD4+ T cells drive goblet cell depletion during Citrobacter rodentium infection

Abstract

Both idiopathic and infectious forms of colitis disrupt normal intestinal epithelial cell (IEC) proliferation and differentiation, although the mechanisms involved remain unclear. Recently, we demonstrated that infection by the attaching and effacing murine pathogen Citrobacter rodentium leads to a significant reduction in colonic goblet cell numbers (goblet cell depletion). This pathology depends on T and/or B cells, as Rag1(-/-) mice do not suffer this depletion during infection, instead suffering high mortality rates. To address the immune mechanisms involved, we reconstituted Rag(-/-) mice with either CD4(+) or CD8(+) T cells. Both T cell subsets increased Rag1(-/-) mouse survival during infection, with mice that received CD8(+) T cells developing colonic ulcers but not goblet cell depletion. In contrast, mice that received CD4(+) T cells showed goblet cell depletion in concert with exaggerated IEC proliferation. To define the possible involvement of T cell-derived cytokines, we infected gamma interferon receptor gene knockout (IFN-γR(-/-)) mice and wild-type mice given interleukin 17A (IL-17A) neutralizing antibodies and found that IFN-γ signaling was required for both goblet cell depletion and increased IEC proliferation. Immunostaining revealed that C. rodentium cells preferentially localized to nonhyperplastic crypts containing numerous goblet cells, whereas hyperplastic, goblet cell-depleted crypts appeared protected from infection. To address whether goblet cell depletion benefits the C. rodentium-infected host, we increased goblet cell numbers using the γ-secretase inhibitor dibenzazepine (DBZ), which resulted in greatly increased pathogen burdens and mortality rates. These results demonstrate that goblet cell depletion reflects host immunomodulation of IEC homeostasis and reflects a novel host defense mechanism against mucosal-adherent pathogens.

Fig 1

CD4⁺ and CD8⁺ T cell reconstitution reduces mortality in Rag1^−/− mice. (A) Percent survival of Rag1^−/− mice during infection is greatly reduced compared to that of CD4⁺ and CD8⁺ T cell-reconstituted and C57BL/6 control mice. Each symbol represents the mean of three independent infections. Error bars indicate ±1 SEM. Asterisks show significance at a P value of <0.05. (B) No differences in C. rodentium CFU/gram of tissue were identified among the cecae and distal colons from Rag1^−/− and CD4⁺ and CD8⁺ T cell-reconstituted mice at day 12 p.i. Cecae and distal colons from control C57BL/6 mice yielded significantly lower bacterial burdens than those of Rag1^−/− counterparts. Error bars indicate SEM from at least six mice. Asterisks show significance at a P value of <0.05.

Fig 2

Increased pathology in Rag1^−/− mice reconstituted with CD4⁺ or CD8⁺ T cells. (A) Comparative pathological scores of control Rag1^−/− and CD4⁺ and CD8⁺ T cell-reconstituted mice. CD4⁺ reconstitution greatly increased hyperplasia, while CD8⁺ reconstitution resulted in worsened epithelial integrity. Bars represent the average pathology scores of at least 3 experiments, each with 5 to 8 mice. Error bars indicate SEM. Asterisks show significance at a P value of <0.05. (B to D) Representative H&E staining of distal colonic tissues removed on day 12 p.i. from nonreconstituted Rag1^−/− (B), CD4⁺ T cell-reconstituted (C), and CD8⁺ T cell-reconstituted (D) mice exhibiting different associated pathologies. Yellow-bordered areas are enlarged to the right. Double-headed arrows highlight increased hyperplasia in CD4⁺ reconstitution, while CD8⁺ T cell reconstitution exhibits severe focal ulceration. (C and D) Anti-CD3 staining (red) in serial sections showing aggregates of CD3-positive cells (arrows) in the lamina propria and regions of mucosal ulceration. Original magnification was ×200. Scale bars = 50 μm.

Fig 3

Restored hyperplasia, proliferation, and goblet cell depletion in CD4⁺ T cell-reconstituted Rag1^−/− mice. (A) Representative H&E and anti-Ki67 (red) staining of day-12-p.i. distal colons from nonreconstituted Rag1^−/− and CD4⁺ and CD8⁺ T cell-reconstituted mice. Carets indicate representative mature goblet cells. Original magnification was ×200. Scale bars = 50 μm. (B) Quantification of goblet cells per 100 intestinal epithelial cells (IEC) in distal colons of mice at day 12 p.i. Bars represent the means of 3 experiments, each with 5 to 8 mice, accounting for 20 sections. (C) Fold increase in crypt lengths in distal colons of mice at day 12 p.i. Bars represent mean fold increase of crypt length relative to crypt length in Rag1^−/− mice. (D) Enumeration of Ki67-positive cells per crypt in distal colons of mice at day 12 p.i. Bars represent the mean number of Ki67-positive cells per crypt. Error bars indicate SEM. Asterisks show significance at a P value of <0.05.

Fig 4

CD4⁺ T cell reconstitution affects mRNA transcript levels during C. rodentium infection. Bars represent mean mRNA transcript levels of Muc2, Tff3, Relm-β, IFN-γ, and IL-17A relative to transcript levels of control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) determined by quantitative RT-PCR in 3 independent experiments. Significant differences in gene expression of Muc2, IFN-γ, and IL-17A were found with CD4⁺ reconstitution. Error bars indicate SEM. Asterisks show significance at a P value of <0.05.

Fig 5

IFN-γ signaling affects goblet cell depletion and IEC proliferation during C. rodentium infection. (A) Quantification of goblet cells per 100 IEC in C57BL/6 and IFN-γR^−/− mice with or without C. rodentium infection. Error bars indicate SEM. Asterisks show significance at a P value of <0.05. (B) Distal colons stained with H&E and anti-Ki67 (red), showing high numbers of goblet cells (H&E) and reduced proliferation (Ki67). Carets indicate representative mature goblet cells. Original magnification was ×200. Scale bars = 50 μm. (C) C57BL/6 mice treated with IL-17A neutralizing antibody (anti-IL-17A) carried heavier C. rodentium burdens than control mice at day 12 p.i. Horizontal bars and error bars indicate means and SEM from at least six mice. (D) H&E staining of distal colons of control and IL-17A-neutralized mice at day 12 p.i. No changes in goblet cell number or hyperplasia were observed. Original magnification was ×200. Scale bars = 50 μm.

Fig 6

mRNA transcript levels of HES1 (A) and Math1 (B) during C. rodentium infection. Bars represent mean expression of HES1 and Math1 relative to expression of control GAPDH by quantitative RT-PCR from 3 independent experiments. No significant differences were found between tissues compared (colon versus colon, cecum versus cecum). Error bars indicate SEM.

Fig 7

Impaired goblet cell depletion is associated with deep penetration of crypts by C. rodentium cells. (A) Serial sections stained with H&E and Ki67 (red) exhibited patchy goblet cell depletion in the distal colons of CD4⁺ T cell-reconstituted mice at day 12 p.i. Crypts displaying goblet cell depletion also display high numbers of Ki67-positive cells (yellow-bordered area), while crypts with mature goblet cells (carets) exhibit low numbers of Ki67-positive cells (green-bordered area). Original magnification, ×200. Scale bars = 50 μm. (B and C) Immunofluorescence staining for C. rodentium translocated effector Tir (green), Ki67 (red in panel B), Muc2 (red in panel C), and DNA (blue) in distal colonic tissues at day 12 p.i. Nonreconstituted Rag1^−/− mice have deeply filled crypts containing C. rodentium cells (arrows) and few Ki67-positive cells (B, left) but increased Muc2 expression (C, left). CD4⁺ T cell reconstitution prevents C. rodentium cells from associating deep within crypts while dramatically increasing the numbers of Ki67-positive IEC (B, right) but decreasing Muc2 expression (C, right). Original magnification was ×200. Scale bars = 50 μm.

Fig 8

Treatment with γ-secretase inhibitor DBZ increases mortality and C. rodentium burden. (A) Representative H&E-stained images from infected distal colons showing increased goblet cell numbers in DBZ-treated C57BL/6 mice. Carets indicate representative mature goblet cells. Original magnification was ×200. Scale bars = 50 μm. (B) Percent survival of DBZ-treated mice is reduced beginning at day 9 p.i. Each symbol indicates the mean of three independent infections. Error bars indicate SEM. Asterisks show significance at a P value of <0.05. (C) DBZ-treated mice carry increased C. rodentium CFU/gram of tissue in distal colons at day 12 p.i. Error bars indicate SEM from at least six mice. Asterisks show significance at a P value of <0.05.