Duodenal helminth infection alters barrier function of the colonic epithelium via adaptive immune activation

Abstract

Chronic infection with intestinal helminth parasites is a major public health problem, particularly in the developing world, and can have significant effects on host physiology and the immune response to other enteric infections and antigens. The mechanisms underlying these effects are not well understood. In the current study, we investigated the impact of infection with the murine nematode parasite Heligmosomoides polygyrus, which resides in the duodenum, on epithelial barrier function in the colon. We found that H. polygyrus infection produced a significant increase in colonic epithelial permeability, as evidenced by detection of elevated serum levels of the tracer horseradish peroxidase following rectal administration. This loss of normal barrier function was associated with clear ultrastructural changes in the tight junctions of colonic epithelial cells and an alteration in the expression and distribution of the junctional protein E-cadherin. These parasite-induced abnormalities were not observed in SCID mice but did occur in SCID mice that were adoptively transferred with wild-type T cells, indicating a requirement for adaptive immunity. Furthermore, the helminth-induced increase in gut permeability was not seen in STAT6 knockout (KO) mice. Taken together, the results demonstrate that one of the mechanisms by which helminths exert their effects involves the lymphocyte- and STAT6-dependent breakdown of the intestinal epithelial barrier. This increase in epithelial permeability may facilitate the movement of lumenal contents across the mucosa, thus helping to explain how helminth infection can alter the immune response to enteric antigens.

Fig. 1.

Helminth infection results in an increase in colonic epithelial permeability in BALB/c mice. Control mice or animals infected 7 days earlier with H. polygyrus (H.p.) were subjected to intrarectal administration of HRP. The appearance of HRP in serum was measured 0, 45, and 120 min later. Data shown represent the time course of change in serum HRP concentration. *, P < 0.05; n = 3 to 6/group. The data shown here are from one of two experiments showing similar results.

Fig. 2.

Infection with H. polygyrus, a small intestinal parasite, induces alterations in colonic epithelial cell-cell junctions. Colon tissues were collected from individual uninfected control (a and b) and H. polygyrus-infected (7 days after infection, c and d) BALB/c mice, and their morphology was examined by EM. Arrows point to cell-cell junction. (a) Tight junction; (b) adherens junction; (c) desmosome; (d) gap junction. An asterisk indicates a disrupted cell junction.

Fig. 3.

Helminth infection induces disorganized distribution and reduced expression of E-cadherin in colonic tissues. BALB/c mice were infected with H. polygyrus for 7 days. Colon tissues were collected in OCT. Frozen sections were prepared and stained by immunofluorescence with anti-ZO-1 or anti-E-cadherin antibodies with Cy3 (A and B)- or FITC (C and D)-conjugated secondary antibodies. The distribution of ZO-1 and E-cadherin from three to five individual mice in each group was examined. Representative figures from each group are shown in panels A to D. (A and C) Tissues from uninfected mice. (B and D) Tissue sections from H. polygyrus-infected mice. (E) Colonic epithelial cells were isolated, and cellular lysates were prepared. Colonic cellular proteins were separated by SDS-PAGE and transferred to nitrocellulose. Immunoblots were performed using anti-E-cadherin and anti-GAPDH antibodies. Each lane represents the colonic epithelial cell sample from an individual mouse. (F) Densitometry on E-cadherin showed a decrease of E-cadherin expression after normalization with GAPDH. *, P < 0.05. The data represent the means ± SEM for three mice in each group.

Fig. 4.

Helminth-induced changes in colonic epithelial permeability in SCID mice. (A) Reconstitution of helminth-infected SCID mice with total lymphocytes or purified CD4⁺ T cells results in increased colonic epithelial permeability. Unfractionated lymphocytes or CD4⁺ T cells were prepared from spleen and lymph nodes of normal BALB/c mice and adoptively transferred into SCID mice, which were infected with H. polygyrus or left uninfected. Seven days later, HRP was administered rectally to the lumen of the colon, and blood HRP levels were measured at 0 and 45 min after HRP inoculation. *, P < 0.05. n = 3 to 5/group. The data shown here are from one of two experiments showing similar results. (B and C) Helminth infection fails to induce alteration in distribution of E-cadherin in colon tissues of SCID mice. SCID mice were infected with H. polygyrus for 7 days. Colon sections were stained by anti-E-cadherin or anti ZO-1 with FITC- or Cy3-conjugated secondary antibodies. (D) Adoptive transfer of total lymphocytes into helminth-infected SCID mice results in alteration in distribution of E-cadherin in colon tissues. SCID mice that were infected with helminth and adoptively transferred with lymphocytes (on the same day; 5 × 10⁶ cells/mouse) were sacrificed at 7 days postinfection. Colon sections were stained by anti-E-cadherin or anti ZO-1 with FITC- or Cy3-conjugated secondary antibodies. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) (in blue). Representative images display the distribution of E-cadherin and ZO-1 in colonic sections for one mouse from each of the treatment groups. There were 3 to 5 mice examined in each group, showing similar results. (B) Normal SCID mice. (C) H. polygyrus-infected SCID mice. (D) H. polygyrus-infected, cell transferred SCID mice (Hp + cells). Green, E-cadherin; red, ZO-1. Magnification, ×200.

Fig. 5.

T-cell cytokine profile in SCID mice with lymphocyte reconstitution and H. polygyrus infection. SCID mice were adoptively transferred with lymphocytes isolated from normal BALB/c mice and infected with or without H. polygyrus. SCID recipient mice were sacrificed 7 days after helminth infection. Lymphocyte suspensions were prepared from MLN and restimulated with anti-CD3 MAb (10 μg/ml). Th2 cytokine production (left panel, IL-4; right panel, IL-10) was measured using ELISA. The data shown are from one of two experiments performed showing similar results (n = 3 to 5 mice per group).

Fig. 6.

H. polygyrus infection results in unchanged colonic epithelial permeability in STAT6 KO mice. (A) Uninfected STAT6 KO or STAT6 KO mice infected 7 days earlier with H. polygyrus were subjected to intrarectal administration of HRP. The appearance of HRP in serum was measured 0, 45, and 120 min later. Data shown represent the time course of change in serum HRP concentration. n = 3 to 6/group. Significant differences were detected in serum HRP levels between uninfected and infected BALB/c mice. *, P < 0.05. Such differences were not seen in STAT6 KO mice. The data shown here are from one of two experiments showing similar results. (B to E) Distribution of E-cadherin was examined using methods described in the legend to Fig. 3. (B) Tissue from uninfected STAT6 KO mice. (C) Tissue section from H. polygyrus-infected STAT6 KO mice. (D) Colonic tissue section from normal BALB/c mice. (E) Tissue section from H. polygyrus-infected BALB/c mice. (F and G) Helminth infection induces disorganized distribution and reduced expression of E-cadherin in colonic tissues of BALB/c mice but not in STAT6 KO mice. *, P < 0.05; n = 3/group. Data for BALB/c mice shown in panels F and G represent the same results as data presented in Fig. 3E and F.