Critical role of IL-25 in nematode infection-induced alterations in intestinal function

Abstract

IL-25 (IL-17E) is a member of the IL-17 cytokine family. IL-25-deficient mice exhibit impaired Th2 immunity against nematode infection, implicating IL-25 as a key component in mucosal immunity. The sources of IL-25 and mechanisms responsible for the induction of Th2 immunity by IL-25 in the gastrointestinal tract remain poorly understood. There is also little information on the regulation of IL-25 during inflammation or its role in gut function. In the current study, we investigated the regulation of IL-25 during Nippostrongylus brasiliensis infection and the contribution of IL-25 to the infection-induced alterations in intestinal function. We found that epithelial cells, but not immune cells, are the major source of IL-25 in the small intestine. N. brasiliensis infection-induced upregulation of IL-25 depends upon IL-13 activation of STAT6. IL-25(-/-) mice had diminished intestinal smooth muscle and epithelial responses to N. brasiliensis infection that were associated with an impaired Th2 protective immunity. Exogenous IL-25 induced characteristic changes similar to those after nematode infection but was unable to restore the impaired host immunity against N. brasiliensis infection in IL-13(-/-) mice. These data show that IL-25 plays a critical role in nematode infection-induced alterations in intestinal function that are important for host protective immunity, and IL-13 is the major downstream Th2 cytokine responsible for the IL-25 effects.

Figure 1

Nematode-induced IL-25 is IL-13- and STAT6-dependent. Mice were infected with N. brasiliensis (Nb) and studied at day 9 post-infection (A), or injected i.v. with 10μg of mouse recombinant IL-13 daily and studied at day 7 post-injection (B). qPCR was performed to measure mRNA expression in the small intestine. The fold changes were relative to the individual vehicle groups after normalization to 18s rRNA. * p<0.05 versus the respective vehicle group (n≥5 for each group).

Figure 2

Epithelial cells, but not immune cells, are the major cellular source of IL-25 in the small intestine. Mice were infected with N. brasiliensis and studied 9 days later. (A) LCM combined with qPCR technique was performed to determine mRNA expression in specific cell populations from captured intestinal epithelial (EC), lamina propria (LP), and smooth muscle (Muscle). (B) Frozen tissue blocks of mid-jejunum were prepared and the sections were cut for immunofluoresence staining with anti-IL-25mAb. Pictures are representative of each group of at least 5 mice. Original magnification, x600.

Figure 3

Immune-regulated IL-25 receptor expression. Mice were infected with N. brasiliensis and studied 9 days later. qPCR was performed to examine mRNA expression of IL-17RA and IL-17RB on samples from small intestine (A), or LCM-captured intestinal lamina propria (LP), epithelial (EC), or smooth muscle cells (Muscle) (B). The fold changes were relative to the individual vehicle groups (A) or vehicle LP (B) after normalization to 18s rRNA. * p<0.05 versus the respective vehicle group; ^φp<0.05 versus the respective LP (n≥5 for each group).

Figure 4

Impaired host immunity against N. brasiliensis infection in IL-25^−/− mice was associated with a diminished intestinal smooth muscle and epithelial responses to infection. C57BL/6 mice (WT) or mice with IL-25 deficiency (IL-25^−/−) were infected with N. brasiliensis (Nb) and studied at day 10 post-infection. Numbers of adult worms were counted (A). Intestinal strips were suspended longitudinally in organ baths for in vitro contractility studies in response to (B) acetylcholine (ACH, 10nM-0.1mM), (C) EFS (1–20Hz, 80V), or (D) 5-HT (100μM). For epithelial functional studies, muscle-free mucosa was mounted in (E) microsnap-well for the measurement of TEER, or in Ussing chambers for epithelial cell response to (F) acetylcholine (1mM). *p<0.05 versus WT-Nb (A), or the respective vehicle (B–F); ^φp<0.05 versus the respective WT (n≥3 for each group).

Figure 5

Exogenous IL-25 induced intestinal smooth muscle hyper-contractility in mice. BALB/c mice were injected i.v. with 0.7μg of IL-25 daily and studied at day 7 post-injection. Intestinal strips were suspended longitudinally in organ baths for in vitro contractility studies in response to (A) acetylcholine (ACH, 10nM-0.1mM), (B) EFS (1–20Hz, 80V), (C) 5-HT (100μM), or (D) for spontaneous contraction. *p<0.05 versus the respective vehicle (n≥5 for each group).

Figure 6

Exogenous IL-25 increased mucosal permeability and induced stereotypic changes in mucosal epithelial function in mice. BALB/c mice were injected i.v. with 0.7μg of IL-25 daily and studied at day 7 post-injection. Muscle-free mucosa was mounted in (A) microsnap-well for the measurement of TEER, or in (B) Ussing chambers for epithelial secretion in response to acetylcholine (10nM-1mM). *p<0.05 versus the respective vehicle (n≥5 for each group).

Figure 7

Exogenous IL-25 selectively influenced T helper (Th) cell immune responses in the small intestine in mice. BALB/c mice were injected i.v. with 0.7μg of IL-25 daily and studied at day 7 post-injection. qPCR was performed to measure the mRNA expression of major cytokines of Th2 (A), Th1 (C), and IL-25 and IL-17RB (C). ELISA was performed to validate in situ IL-13 production in the small intestine (B). The fold increases for mRNA expression were relative to the individual vehicle groups after normalization to 18s rRNA. * p<0.05 versus the respective vehicle group (n≥5 for each group).

Figure 8

Exogenous IL-25 was unable to restore the impaired host immunity against N. brasiliensis infection in mice with IL-13 deficiency. Mice were infected with N. brasiliensis (Nb) and studied at day 10 post-infection. One separate group of IL-13^−/− mice received daily injection of IL-25 simultaneously with N. brasiliensis infection, starting from day 1 until day 8 post-infection. Intestinal strips were suspended longitudinally in organ baths for in vitro contractility studies in response to (A) acetylcholine (ACH, 10nM-0.1mM), or (B) for spontaneous contraction. *p<0.05 versus the respective vehicle (n≥5 for each group).