RELMbeta/FIZZ2 is a goblet cell-specific immune-effector molecule in the gastrointestinal tract

Abstract

Gastrointestinal (GI) nematode infections are an important public health and economic concern. Experimental studies have shown that resistance to infection requires CD4(+) T helper type 2 (Th2) cytokine responses characterized by the production of IL-4 and IL-13. However, despite >30 years of research, it is unclear how the immune system mediates the expulsion of worms from the GI tract. Here, we demonstrate that a recently described intestinal goblet cell-specific protein, RELMbeta/FIZZ2, is induced after exposure to three phylogenetically distinct GI nematode pathogens. Maximal expression of RELMbeta was coincident with the production of Th2 cytokines and host protective immunity, whereas production of the Th1 cytokine, IFN-gamma, inhibited RELMbeta expression and led to chronic infection. Furthermore, whereas induction of RELMbeta was equivalent in nematode-infected wild-type and IL-4-deficient mice, IL-4 receptor-deficient mice showed minimal RELMbeta induction and developed persistent infections, demonstrating a direct role for IL-13 in optimal expression of RELMbeta. Finally, we show that RELMbeta binds to components of the nematode chemosensory apparatus and inhibits chemotaxic function of a parasitic nematode in vitro. Together, these results suggest that intestinal goblet cell-derived RELMbeta may be a novel Th2 cytokine-induced immune-effector molecule in resistance to GI nematode infection.

Fig. 1.

GI nematode infections induce high-level expression of RELMβ. (A) Quantitative RT-PCR analysis of mRNA expression for RELMβ, TFF3, and Muc2 in the GI tract of BALB/c mice infected with T. muris (colon, day 16 after infection), Tri. spiralis (small intestine, day 14 after infection), or N. brasiliensis (small intestine, day 10 after infection). (B) Immunohistochemistry for RELMβ in the small intestine of naïve and Tri. spiralis- or N. brasiliensis-infected BALB/c mice. (C) Immunohistochemistry for RELMβ in the colon of naïve and T. muris-infected BALB/c mice. Results are expressed as mean ± SD and are representative of two independent experiments containing three to four mice in each experimental group.

Fig. 2.

Expulsion of T. muris is associated with production of Th2 cytokines and the induction of RELMβ expression. (A) Immunoblots for RELMβ protein isolated from the stool of naïve or T. muris-infected AKR and BALB/c mice. (B) Analysis of T. muris worm burden in AKR and BALB/c mice on various days after infection. (C) Quantitative RT-PCR analysis of RELMβ mRNA expression in LS174T cells stimulated with either IL-4, IL-13, or IFN-γ for the indicated times (levels normalized to RELMβ expression in unstimulated LS174T cells were arbitrarily assigned a value of 1). (D) Immunoblots for RELMβ protein isolated from the stool of naïve (N) or T. muris-infected wild-type BALB/c (Wt), IL-4^–/–, and IL-4Rα^–/– mice collected on various days after infection.

Fig. 3.

Administration of rIL-13 results in the induction of RELMβ expression and expulsion of T. muris in AKR mice. (A) Immunoblots for RELMβ protein isolated from the stool of naïve (N) or T. muris-infected AKR mice that received either PBS or rIL-13 (10 μg) by i.p. injection. Days p.i., days after injection. (B) Immunolocalization of RELMβ in the cecum and jejunum (small intestine) of naïve and T. muris-infected AKR mice that received either PBS or rIL-13 (day 16 after infection). (C) Quantitative RT-PCR analysis of mRNA expression of RELMβ, TFF3, and Muc2 in the colon of naïve (N) and T. muris-infected AKR mice that received either PBS or rIL-13. (D) Quantification of T. muris worm burden in AKR mice that received either PBS or rIL-13 (16 days after infection). Results are expressed as mean ± SD and are representative of two independent experiments containing three to four mice in each treatment group.

Fig. 4.

RELMβ binds specifically to structures located on the integument of parasitic nematodes and inhibits the chemotaxis of S. stercoralis in vitro. (A) Cy3 (red) immunofluorescent detection of RELMβ binding to the integument of T. muris isolated from BALB/c mice at 14 days after infection. The differential interference contrast is a ×20 magnification. (B) High-power magnification of A (×100). (C) Cy3 (red) immunofluorescence for RELMβ with 4′,6-diamidino-2-phenylindole nuclear counterstain (blue). Image is ×100 magnification. (D) Fluorescent Z-stacked optical images of T. muris stained for RELMβ (red) and 4′,6-diamidino-2-phenylindole (blue) rotated 80°. Image is ×100 magnification. (E) Cy3 (red) immunofluorescent detection of RELMβ binding to paired lateral alae on the cuticle of S. stercoralis. The differential interference contrast is a ×120 magnification. (F) Immunoblot for RELMβ in protein isolated from the stool of a naïve (N) and T. muris-infected (Inf) BALB/c mice (16 days after infection), and various concentrations of rRELMβ. (G) In vitro chemotaxis assay of S. stercoralis L3i preincubated with either rIL-4 (50 ng/ml, control) or rRELMβ (50 ng/ml) for 30 min. Percentage of worms reaching the attractant well was determined at the indicated times. Results are expressed as mean percentage ± SD of three independent experiments.