ABSTRACT
Intestinal colonization with the protozoan Giardiacauses diffuse brush border microvillous alterations and disaccharidase deficiencies, which in turn are responsible for intestinal malabsorption and maldigestion. The role of T cells and/or cytokines in the pathogenesis of Giardia-induced microvillous injury remains unclear. The aim of this study was to assess the role of T cells and interleukin-6 (IL-6) in the brush border pathophysiology of acute murine giardiasis in vivo. Athymic nude (nu−/nu−) CD-1 mice and isogenic immunocompetent (nu+/nu+) CD-1 mice (4 weeks old) received an axenic Giardia muris trophozoite inoculum or vehicle (control) via orogastric gavage. Weight gain and food intake were assessed daily. On day 6, segments of jejunum were assessed for parasite load, brush border ultrastructure, IL-6 content, maltase and sucrase activities, villus-crypt architecture, and intraepithelial lymphocyte (IEL) infiltration. Despite similar parasitic loads on day 6, infected immunocompetent animals, but not infected nude mice, showed a diffuse loss of brush border microvillous surface area, which was correlated with a significant reduction in maltase and sucrase activities and a decrease in jejunal IL-6 concentration. In both athymic control and infected mice, jejunal brush border surface area and disaccharidases were high, but levels of tissue IL-6 were low and comparable to the concentration measured in immunocompetent infected animals. In both immunocompetent and nude mice, infection caused a small but significant increase in the numbers of IELs. These findings suggest that the enterocyte brush border injury and malfunction seen in giardiasis is, at least in part, mediated by thymus-derived T lymphocytes and that suppressed jejunal IL-6 does not necessarily accompany microvillous shortening.
Intestinal infection withGiardia spp. decreases electrolyte, glucose, and fluid absorption and causes disaccharidase deficiencies at least in part via diffuse brush border microvillus shortening (7, 8, 19). Together, these abnormalities lead to malabsorption and maldigestion. Similar pathophysiology, including loss of brush border surface area, is observed in bacterial enteritis (9, 10), chronic food anaphylaxis (14), celiac disease (47), and Crohn's disease (18). Moreover, giardiasis has been reported to mimic inflammatory bowel disease in humans (25). These similarities make experimental giardiasis a useful model for the study of the pathogenesis of epithelial brush border injury. The fact that some of the above-mentioned disorders do not involve colonization by a microbial pathogen suggests that host immune factors may be involved in pathogenesis.
Gut-associated lymphoid tissue-derived immunity is necessary to clearGiardia infections from the gut (12), and unlike immunocompetent mice, nude athymic (nu−/nu−) mice which are infected with Giardia muris fail to clear the infection and develop chronic giardiasis (45). Immunocompetent mice also become immune to reinfection, while nude mice gain no immunity to Giardia infection and are susceptible to secondary infections. In addition, reconstitution of athymic mice with T cells leads to decreased parasite load as well as further villus atrophy (45). Villus atrophy can also be caused by activated T cells in the absence of Giardia infection (19), and T lymphocytes have been implicated in the manifestation of villous atrophy during other disorders (15, 22, 34). Together, these observations imply a protective function for T cells, as well as a role for T cells in the pathogenesis of intestinal villous injury. The role of T lymphocytes and their cytokines in the pathogenesis of the injury to brush border microvilli rather than villi remains unknown, and yet it is the ultrastructural changes of the latter that represent the limiting factor for absorption and digestion in a number of intestinal disorders (8, 14, 19).
Interleukin-6 (IL-6) is a prominent cytokine of the Th2 subset which is commonly associated with antibody-dependent immunity, as opposed to the leukocyte infiltration observed during Th1-dependent immunity. IL-6 is produced by a large number of cell types, including epithelial enterocytes, Th2 lymphocytes, monocytes, and macrophages (26, 31, 35, 42). Among its pleiotropic effects, IL-6 induces B-cell differentiation, immunoglobulin synthesis, T-cell proliferation, and enhancement of immunoglobulin A (IgA) responses (35, 42). IL-6 has been implicated as a possible pathogenic factor in diseases, such as Crohn's disease (39, 43). Previous studies have shown that excess IL-6 is associated with decreased activity of sucrase-isomaltase (57), a brush border enzyme which is also markedly decreased in patients with active Crohn's disease or giardiasis. Hosts infected with Giardia commonly mount a humoral immune response, and there is no noticeable granulocytic cell influx into the intestine during the acute phase of giardiasis although the immune response to Giardia unquestionably involves activation of T lymphocytes (20, 24, 27, 29, 49, 53, 54). The antibody response to Giardia is predominantly of the IgA and IgG isotypes (4, 27). The effects of giardiasis on intestinal IL-6 and the implications for pathogenesis have not been investigated.
The aims of this study were (i) to assess the role played by T cells in the pathogenesis of Giardia-induced changes in jejunal brush border microvillous architecture and disaccharidase activities and (ii) to characterize IL-6 production in association with these alterations.
MATERIALS AND METHODS
Animal model.Specific pathogen-free CD-1 mice (nu+/nu+) were obtained from a colony maintained by the Life and Environmental Sciences Animal Resource Centre at the University of Calgary. Isogenic athymic (nude) nu−/nu− mice were obtained from Charles River Laboratories (Wilmington, Mass.). Female mice aged 4 weeks and weighing 17 to 20 g were used in all experiments. Animals were housed in pairs and were given water and fed commercial mouse diet ad libitum. Cages were covered with microisolator lids to ensure disease containment. Room environment was maintained at 22°C and 40% rH with 12-h photoperiods. Maintenance of animals and experimental procedures were carried out in accordance with guidelines of the Canadian Council on Animal Care.
Experimental protocol.Animals of each strain were either infected with axenic Giardia muris trophozoites (see below) or given vehicle alone (control). Mice of the infected groups received 2 × 105 trophozoites suspended in 0.2 ml of sterile phosphate-buffered saline (PBS) by orogastric gavage. Mice of sham-treated control groups received 0.2 ml of sterile PBS. Food intake by each pair of mice was measured daily, and mean daily food intake per animal was calculated. Mean individual weight gain was also calculated for each group, and the percent increase in weight relative to that on day zero was calculated. Animals were killed by cervical dislocation 6 days after inoculation. This time point occurs during the peak of intestinal colonization and reproduces mucosal abnormalities reported in previous studies (6). The jejunum, 1 to 10 cm distal to the ligament of Treitz, was excised and prepared as described below. Blood was collected via cardiac puncture.
Axenic isolation.As bacterial and/or viral contaminants of the inoculum may affect the outcome of these studies, G. muris trophozoites were axenized as described previously (51), with a few modifications. Briefly, 3 days after mice received G. muris trophozoites by orogastric gavage, the duodenum and proximal jejunum of the mouse were removed and opened longitudinally. The 15-cm gut segment was placed in a tube containing chilled, sterile TYI-S33 Giardia medium containing 3.0 mg of piperacillin (Piperacil; Cyanamid Canada, Montreal, Quebec, Canada) and vortexed for 30 s. Tube contents were filtered through sterile gauze and centrifuged, and the pellet was suspended in new sterile TYI-S33 Giardia medium containing piperacillin in polystyrene centrifuge tubes and incubated on an orbital shaker at 150 rpm and 37°C for 2 h to allow for trophozoite adherence to the walls of the tube. The medium was decanted, and trophozoites were detached by cold shock with chilled, sterile PBS and centrifuged at 800 × g for 10 min at 4°C. Pure Giardiatrophozoites were diluted to 2 × 105 trophozoites/ml for use in further experiments.
The axenic status of the trophozoite suspension was confirmed by plating serial dilutions of the inoculum on Columbia blood agar and MacConkey agar (Bacto; Difco Laboratories, Detroit, Mich.) and incubating for 48 h both aerobically and anaerobically at 37°C. In addition, serum of control and infected mice was collected 6 and 35 days postinoculation and sent to Charles River Laboratories for a complete assessment of viral titers (Mouse Assessment Plus service package).
Trophozoite quantification.A 1-cm segment of jejunum was removed, placed in 1 ml of chilled sterile PBS, and vortexed for 30 s to release trophozoites from the intestinal wall. Trophozoites were counted in a hemocytometer, and colonization was expressed as the number (log10) of trophozoites per centimeter of jejunum.
Transmission electron microscopy.Jejunal enterocyte ultrastructure was assessed with transmission electron microscopy. Intestinal segments (1 cm) taken 2 cm distal to the ligament of Treitz were removed and fixed in 5% glutaraldehyde overnight at 4°C. Samples were cut into 1-mm squares and postfixed in 1% OsO4 for 2 h, dehydrated in ethanol, cleared with propylene oxide, and embedded in Spurr low-viscosity medium (Sigma, St. Louis, Mo.). Semithin sections were stained with basic toluidine blue for light microscopy to select midvillus areas for thin sectioning for electron microscopy. Sections (80 nm) were double stained with saturated uranyl acetate in 50% ethanol and 0.4% lead citrate (52). Sections were examined at 75 kV on a Hitachi H7000 transmission electron microscope. Micrographs of midvillus enterocyte apical membranes were obtained at the same magnification (×12,000), and the height, width, and density of microvilli were calculated. Surface area of the epithelial brush border was determined as described previously (7). To avoid observer bias, micrographs were coded and observations were recorded in a blind fashion. For each group, 14 to 21 micrographs were obtained from a total of three animals in each group and microvillus brush border surface area was calculated. Previous studies have demonstrated that Giardia-induced microvillus injury in the midvillus area is representative of the changes along the entire villus axis (7, 8).
Disaccharidase activity.As a number of studies have established that disaccharidase impairment represents a reliable marker of Giardia-induced mucosal injury, sucrase and maltase activities were measured. Jejunal segments (12 cm) were blotted dry, weighed, homogenized in 2.5 mM EDTA (4 ml/1 g of tissue), frozen in liquid nitrogen, and stored at −70°C until needed. Sucrase and maltase activities were determined as previously described by Dahlqvist (16) and Belosevic et al. (5) and expressed as units per gram of protein. Total protein content was determined by the Bradford protein assay (Bio-Rad, Mississauga, Ontario, Canada).
IL-6.IL-6 concentrations were measured in the jejunal homogenates (dilution of 1:4 in 2.5 mM EDTA), and in neat serum samples by using a commercial sandwich enzyme-linked immunosorbent assay sensitive to 5 pg/ml and specific to IL-6 (no detectable cross-reactivity with IL-1α, -1β, -2, -4, -5, -7, or -10, leukemia inhibitory factor (LIF), tumor necrosis factor alpha, granulocyte macrophage-colony-stimulating factor, and gamma interferon) (Intertest-6x; Genzyme, Cambridge, Mass.). Jejunal protein concentration was determined by the Bio-Rad Bradford protein assay, and tissue IL-6 was expressed as picograms per milligram of protein. Serum IL-6 concentration was expressed as picograms per milliliter.
Intestinal histology.Jejunal samples were processed for light microscopy. Samples were fixed in 10% neutral buffered formalin (pH 7.3) and embedded in paraffin wax, and sections were stained with hematoxylin and eosin. Crypts and villi were measured by using a calibrated micrometer, and intraepithelial lymphocytes (IELs) were counted along villus units. IELs were expressed as number per 100 epithelial enterocytes.
Statistical analysis.All values were expressed as mean ± standard error (SE). Means were compared by one-way analysis of variance and Tukey's compromise test for multiple comparison when applicable. Significance levels were established at P values of <0.05 and <0.01.
RESULTS
Axenic status of inoculum and parasitic colonization.No bacterial growth was observed after 24 or 48 h when the inoculum was streaked onto Columbia blood or MacConkey agar and grown aerobically or anaerobically at 37°C (not shown). The presence of viral pathogens of control mice and mice infected with the inoculum assessed 6 and 35 days postinoculation is illustrated in Table1.
Results from assessments of anti-viral titers in serum samples of control or G. muris-infected micea
All animals gavaged with a G. muris trophozoite inoculum became infected. Parasitic load in immunocompetent (5.99 log10 ± 0.11 log10 [mean ± SE] trophozoites/cm, n = 31) and athymic (6.24 log10 ± 0.05 log10 trophozoites/cm, n = 14) mice infected with 2 × 105axenic G. muris trophozoites 6 days postinoculation were not significantly different. No trophozoites were found in any sham-treated control animals.
Clinical observations.Infection with Giardia spp. is known to affect weight gain and food intake in some cases, but not in others. As previous studies have demonstrated that gut epithelial ultrastructure and function may be affected by malnutrition (6), experiments assessed whether G. murisinfection altered food intake in the present model. In the present study, daily food intake of immunocompetent infected (3.61 to 4.35 g/day) or athymic infected (2.40 to 3.23 g/day) animals was not significantly lower than that of their respective controls (immunocompetent control [3.23 to 3.85 g/day] or athymic control [2.60 to 3.48 g/day]) throughout the study. Similarly, weight gain was not significantly different between either infected group and its respective control group at any time during the study (data not shown).
Brush border ultrastructure.Brush border ultrastructure has been shown to correlate with enterocyte function in health and disease. Figure 1 presents representative transmission electron micrographs of enterocyte brush border membranes in each experimental group. In immunocompetent mice, but not in athymic animals, infection with G. muris caused a significant shortening of brush border microvilli along the entire epithelium, at sites of trophozoite attachment as well as in other areas (Fig. 1). Table 2 describes the different physical attributes of the microvilli and total brush border surface area for all groups. In immunocompetent mice, but not in athymic mice, infection caused a twofold decrease in microvillus height compared to that of controls, as well as a significant reduction in microvillus density per square micrometer of epithelium (Table 2). Localized effacement of epithelial microvilli in immunocompetent infected mice is illustrated in Fig. 2. The widths of individual microvilli were not different between any groups. Reduction of microvillus height and density in immunocompetent animals lead to a significant (P < 0.01) reduction of microvillous surface area per square micrometer of epithelial surface, which was not observed in athymic animals. Ultrastructural characteristics of jejunal microvilli in athymic infected mice were not different from those in their respective controls, nor were they different between control groups (Table 2).
Representative transmission electron micrographs, obtained at the same magnification, of the apical membrane of midvillus enterocytes from the jejunum of immunocompetent control (A), immunocompetent infected (B), athymic control (C), and athymic infected (D) mice 6 days postinoculation. Trophozoites (T) can be seen in two of the micrographs. Microvillous characteristics were consistent along the entire epithelium, at sites of trophozoite colonization as well as in other areas. Bar, 1 μm.
Brush border microvillus characteristics of midvillus enterocytes in the jejunum of micea
Transmission electron micrograph of the jejunal epithelium from an infected immunocompetent mouse demonstrating localized microvillus effacement (arrowheads). Two trophozoites (T) can be seen in the micrograph. Bar, 2 μm.
Disaccharidase activity.As the results indicated that theGiardia-induced microvillous shortening is not observed in athymic animals, a functional marker needed to be assessed to confer physiological relevance to this observation. Disaccharidase activities were measured as a marker of epithelial digestive function. In immunocompetent mice, but not in athymic animals, infection caused a significant (P < 0.01) decrease in sucrase and maltase activities (Fig. 3). There was no significant difference in sucrase or maltase activities between athymic control mice and immunocompetent control mice (Fig. 3). Overall, the differences in disaccharidase activities between groups paralleled those measured in the ultrastructural characteristics of the brush border.
Sucrase and maltase activities in the jejunum of immunocompetent (I) or athymic nude (N) mice. Animals were sham-treated controls (░) or infected with G. muris (■) (n = 16 to 24 per group). Values are expressed as means (± SE) of units/gram of protein. ∗, P value of <0.05 versus paired control.
IL-6.To determine whether jejunal abnormalities associated with giardiasis involved changes in IL-6 production, IL-6 was measured in tissue and serum samples. Infection with G. muris caused a significant (P < 0.05) decrease in the level of jejunal tissue IL-6 (Fig. 4A). This decrease was not seen in the jejunum of nude mice, and IL-6 concentrations found in both infected and control athymic mice were comparable to those in immunocompetent infected mice (Fig. 4A.). Serum IL-6 levels were assessed in order to determine mucosal versus systemic cytokine concentrations. There was no significant difference in the levels of serum IL-6 between any experimental groups (Fig. 4B).
IL-6 concentrations in jejunal homogenates (A) and serum (B) of immunocompetent control (IC, n = 14), immunocompetent infected (II, n = 14), athymic control (NC, n = 4), and athymic infected (NI, n = 9) mice 6 days postinoculation. Values are means (± SE) of picograms/milligram of protein (A) or picograms/milliliter (B). ∗, P value of <0.05 versus paired control; †, P value of <0.05 for nude control versus immunocompetent control.
Intestinal histology.While several host species have been shown to exhibit intestinal villous atrophy and crypt hyperplasia in response to giardiasis, clinical signs have also been reported in infected humans and rodents in the absence of villous atrophy (8, 19). In the present study, villus height was not significantly different between any group (Fig. 5). Crypt depth was increased in athymic infected mice compared with that in athymic controls (P < 0.05) but did not differ between the other groups (Fig. 5). Giardiasis is known to increase the numbers of intraepithelial lymphocytes, particularly during the clearance phase of the infection. In an attempt to verify whether the changes of IL-6 concentration correlated with IEL infiltration, IELs were counted in all tissue samples. Upon infection, there was a small but significant increase in the number of IELs in both immunocompetent (P < 0.05) and nude (P < 0.01) animals (8.71 ± 0.49 [mean ± SE] and 4.08 ± 0.39 IELs/100 epithelial cells, respectively) when compared to their respective controls (10.93 ± 0.67 and 8.58 ± 0.34 IELs/100 epithelial cells for immunocompetent and athymic mice, respectively). IEL numbers were significantly (P < 0.01) lower in athymic control mice than in immunocompetent control mice.
