Influence of age and duration of infection on bacterial load and immune responses to Helicobacter pylori infection in a murine model

Abstract

Using a murine model, we previously showed that Helicobacter pylori infects and colonizes offspring via maternal transmission during the nursing period. The aim of this study was to investigate the influence of age and duration of infection on inflammatory and immune responses to H. pylori in infant and adult mice. During the breast-feeding period, the number of bacteria was significantly suppressed in 1-week-old mice infected with H. pylori at an early stage of nursing, compared with adult mice, suggesting that breast-milk induces such low colonization. In addition, these mice had weaker gastric inflammation, especially Th1 cytokine and humoral responses than in mice infected with H. pylori after weaning in spite of elevated levels of Th1 cytokines. Although infant mice showed low inflammatory responses against H. pylori, they produced H. pylori-specific antibodies following vaccination with oral or parenteral adjuvant. Our results suggest the importance of age at the time of primary infection on bacterial load, gastric inflammation and humoral responses in a murine model of H. pylori infection.

Introduction

Helicobacter pylori is a Gram-negative, spiral-shaped, microaerophilic bacterium that infects the human gastric mucosa [1]. Chronic H. pylori infection is thought to be associated with chronic active gastritis, peptic ulceration, and gastric malignancies, such as mucosa-associated B-cell lymphoma and adenocarcinoma [2]. Half of the world's population is estimated to be infected with H. pylori, and the infection is acquired in most patients before the age of 5 years [3]. Rothenbacher et al. [4] reported that acquisition of H. pylori appears to occur mainly between the first and second year of life, that is, after the age of weaning. In contrast, our previous study in a murine maternal-transmission model showed that H. pylori was detectable by polymerase chain reaction (PCR) in almost all infant mice during the breast-feeding period [5]. This discrepancy might be partially due to maternal milk, which might suppress the bacterial burden below the detection level of the enzyme-linked immunosorbent assay (ELISA), which was performed in the study of Rothenbacher et al. using fecal samples (HpSA test). However, there are only a few studies of the bacterial load of H. pylori during the nursing period.

Several studies of the inflammatory responses to H. pylori in children showed that the lack of neutrophil infiltration and appearance of lymphoid follicles in the stomach are more common in children than in adults [6,7]. Other studies showed enhanced expression of proinflammatory cytokines in the gastric mucosa of H. pylori-infected children [8,9]. In these human studies, however, data of children aged from infancy to adolescence were grouped and analysed together. Thus, the characteristics of the inflammatory response in each age group of children remain to be elucidated.

With regard to the humoral response in children with H. pylori infection, Czinn [10] found significantly lower levels of anti-H. pylori antibodies in young children than in older children and adults. In addition, antibody titres in children may not reach adult levels until the age of 7 years [11]. The present study was designed to determine the influence of age and duration of infection on the immune response to H. pylori using a murine model.

Methods

Animals

Experiments were conducted in C57BL/6 mice. Mice were divided into four groups based on their age: group 1, 1-week-old mice (n = 51); group 2, 3-week-old mice (n = 32); group 3, 5-week-old mice (n = 32); and group 4 of negative control (n = 32). Mice of groups 2–4 were purchased from Seac Yoshitomi (Yoshitomi-cho, Fukuoka, Japan). Mice of group 1 consisted of 8 litters and were bred from 8 pairs of C57BL/6 breeders previously obtained from the same supplier. All mice were housed under specific-pathogen free (SPF) conditions. Litters of group 1 were nursed by their mother and received maternal milk. The other groups were allowed free access to food and water. Experiments were performed according to the guidelines of the Ethics Committee for Animal Experiments at Oita Medical University.

All mice from the groups 1–3 were infected with ∼10⁷ colony forming units (CFU) of H. pylori (SS 1) as described previously [12]. Eight mice from each group were sacrificed at 1, 2, 4 and 8 weeks after infection except for group 1. Since in group 1 each litter consisted of different number of neonates, the number of sacrificed mice was 11 at 1 week, 14 at 2 weeks, 12 at 4 weeks, and 14 at 8 weeks after infection. In group 4, eight mice were sacrificed at 2, 3, 5 and 9 weeks of age. A strip from the greater curvature of the stomach was fixed in 10% formalin for histological examination. One portion of the gastric wall from remaining stomach was frozen immediately in liquid nitrogen for cytokine measurement, while another was homogenized to assess bacterial colonization. In addition, serum samples were collected from the heart at sacrifice to titrate humoral responses against H. pylori.

Quantification of bacterial load

Gastric homogenate (100 µl) was diluted serially from 10⁻¹ to 10⁻³, and 50 µl of each dilution was plated on M-BHM Pylori Agar Medium (Nikken Biomedical Laboratory, Kyoto, Japan) and colonies were counted after 5 or 6 days of culture. Bacterial load was expressed as log CFU per gram of stomach tissue. H. pylori-infected mice that were negative for bacterial culture were assigned a value of 1 CFU/g in order to calculate the log CFU per gram and geometric means. The remainder of the homogenate from these mice negative for bacterial culture was further analysed by the nested PCR method as described previously [5], to detect small amounts of bacteria, if any, that was otherwise below the detection level of culture. Comparisons of bacterial loads among groups were made by analysis of variance with JMP 5·0 (SAS Institute Inc., Cary, NC, USA) with Tukey-Kramer's HSD test.

Inflammatory response to H. pylori infection

Histological examination (haematoxylin and eosin staining) was performed in a blinded fashion independently by two examiners. Because gastric specimens of infant mice were too small to evaluate the extent of inflammation, only the intensity of inflammation was scored on a scale of 0–3 as described previously [12]: grade 0, rare inflammatory cells; grade 1, mild; grade 2, moderate; and grade 3, severe inflammation with marked inflammatory cell infiltration.

The concentrations of proinflammatory cytokines, gamma interferon (IFN-γ ), interleukin (IL)-12, IL-4, and IL-10 in the gastric tissue were measured by ELISA. Frozen gastric specimens were treated as mentioned previously [5,13], and analysed with the OptEIA^TM set for mouse IFN-γ, IL-12, IL-4, and IL-10 (BD Biosciences Pharmingen, San Diego, CA, USA). The results were expressed as pg/mg protein.

Humoral response to H. pylori infection and ability to produce antibody

Serum samples were tested by ELISA, using plates coated with the bacterial whole-cell lysates [12]. Biotinylated antimouse immunoglobulin (Ig) G or IgA (Zymed Laboratories Inc., San Francisco, CA, USA) was used as the secondary antibody. Bound antibody was visualized with the TMB Substrate Reagent Set (BD Biosciences Pharmingen, San Diego, CA, USA). Serum samples obtained from several C57BL/6 mice, hyperimmunized by systemic immunization using bacterial antigen-aluminium hydroxide (AlOH), were mixed, and then used as standards of anti-H. pylori IgG and IgA antibodies. For both IgG and IgA, the A₄₅₀ values of undiluted standard sera were about 1000-times greater than the mean A₄₅₀ value plus two standard deviations (SD) of the negative control sera. Thus, the A₄₅₀ value of undiluted standard sera was defined as 1000 ELISA units (EU).

To confirm the latent ability to produce anti-H. pylori antibodies, infant mice were immunized orally or parenterally and their antibody titres tested. One-week-old mice were immunized and their humoral responses were evaluated 4 weeks after immunization. H. pylori whole-cell lysate was used as an antigen, and cholera toxin (Sigma Chemical Co., St. Louis, MO, USA) or AlOH (Imject Alum, Pierce, IL, USA) was used as an adjuvant [5,12]. One dose consisting of 100 µl of H. pylori whole-cell lysate (4 mg/ml) plus 1 µg of cholera toxin was administered orally as a mucosal vaccine, while one dose consisting of 25 µl of H. pylori whole-cell lysate (2 mg/ml) plus 25 µl of AlOH was intraperitoneally administered as a parenteral vaccine.

Results

Quantification of bacterial load

During the first 2 weeks after infection, the amount of colonizing bacteria in group 1 was significantly lower than that of the other two groups (Fig. 1). Particularly in group 1, the majority of animals (8 of 11) were culture negative at 1 week after infection. At 2 weeks after infection, the bacterial load in group 1 was still lower than that of the other two groups (P < 0·01). However, half of these culture-negative mice (4 of 8) were PCR positive. On the other hand, the colonization rates in group 2 at 2 weeks after infection (P < 0·05) and that of group 1 at 4 weeks after infection (P < 0·01) were higher than that of group 3 at the corresponding periods (Fig. 1). Moreover, the bacterial load in group 1 varied among individual mice at 8 weeks after infection; heavy colonization was still noted in 8 mice but the remaining 6 were negative for culture, though PCR positive. To determine whether H. pylori-specific antibodies were present in maternal milk, breast milk samples were tested for bacteriostatic activity by incubation with SS1 culture in vitro. The results showed that the milk samples from H. pylori infected- and naïve-dams had similar bacteriostatic effects against in vitro H. pylori growth (data not shown).

Fig. 1.

The load of colonized bacteria in group 1 (1-week-old) (○), group 2 (3-week-old) (◊) and group 3 (5-week-old) (□); bacterial culture and PCR negative mouse (•). Data are presented as geometric mean ± standard error of the mean. The bacterial load of group 1 was significantly lower than that of the other two groups at 1 week after infection (****P < 0·0001) and at 2 weeks after infection (**P < 0·01). *P < 0·05 and ***P < 0·001, compared with group 3.

Inflammatory response to H. pylori infection

The gastritis score in all mice of the control group 4 were grade 0 throughout the observation period. The extent of gastritis in groups 2 and 3 worsened at 2 weeks after infection and was significantly worse at 4 and 8 weeks after infection than in group 1 (P < 0·05, each) while that of group 1 remained nearly at grade 0.

The concentrations of IFN-γ and IL-12 in group 1 were significantly higher than in group 4 (Fig. 2b). The concentrations of these cytokines in groups 2 and 3 were also greater than in group 4 although no difference was apparent among the three infected groups (data not shown). We also compared Th2 cytokine response by measuring the concentrations of IL-4 and IL-10 by ELISA. The serum levels of IL-10 were almost equal in groups 1 and 4 and our ELISA for IL-4 could not detect any signals (data not shown).

Fig. 2.

(a) Gastritis scores. Scores were graded on a scale of 0–3. Values represent mean ± standard error (SEM) of each group of mice. group 1 (1-week-old) (•), group 2 (3-week-old) (♦) and group 3 (5-week-old) (▪)*P < 0·05, **P < 0·01, ****P < 0·0001, compared with group 3, by analysis of variance with Tukey-Kramer's HSD test. (b) Comparison of production of gastric proinflammatory cytokines between group 1 (•) and group 4 (control mice) (○). *P < 0·05, **P < 0·01 and ****P < 0·0001, compared with the corresponding cytokine level of group 4, by the unpaired t-test.

Humoral response to H. pylori infection and ability to produce antibodies

The IgG and IgA anti-H. pylori antibody titres of group 1 were persistently lower than those of the other two groups (Fig. 3a). These findings indicate a lower humoral response to H. pylori in infant mice compared to adult mice, although the antibody titres were higher in group 1 at 8 weeks after infection than at 1–4 weeks after infection (P < 0·01, each). The humoral response of 1-week-old mice at 4 weeks after  immunization  is  shown  in  Fig. 3b,  compared  to  that at 4 weeks after infection. Immunized mice, especially parenterally immunized mice, exhibited significantly enhanced antibody production of IgG and IgA. In addition, the IgG titres of mucosally immunized mice were significantly higher than those of the infected group (P < 0·01, Fig. 3b).

Fig. 3.

IgG and IgA anti-H. pylori antibody titres in infected and immunized mice. (a) Closed symbols (•, ♦, ▪) are the geometric mean ± standard error (SEM) of each infection group. Analysis of variance with Tukey-Kramer's HSD test was used to compare differences in antibody titre. After 4 weeks post infection, IgG and IgA titres of group 1 were significantly lower than those of the other two groups (**P < 0·01; ***P < 0·001; *P < 0·05). IgG titres of group 2 were lower than those of the other two groups (*P < 0·05) at 8 weeks after infection. (b) Antibody production in 1-week-old mice after infection (▪), mucosal vaccination () and parenteral vaccination (). Data are mean ± SEM. *P < 0·05; **P < 0·01; ****P < 0·0001, by the unpaired t-test.

Discussion

In the present study, we examined the influence of age and duration of infection on H. pylori infection status in a murine model. Results of group 1 (1-week-old) with a low bacterial load during the early stage of life suggest that maternal milk reduces the number of colonizing bacteria in the stomach since the breast-feeding period in mice is the first 3 weeks of neonatal life. Anti-bacterial components in the maternal milk, such as lactoferrin [13], glycoconjugate [14], and secretory IgA [15] were hypothesized as bacteriostatic substances. In our preliminary experiment, the addition of purified lactoferrin resulted in complete growth inhibition of live H. pylori in vitro (data not shown). In this regard, Corthesy-Theulaz and coworkers [15] showed persistence of H. pylori infection in neonatal mice at undetectable levels of bacteria by passive immunity from milk from the H. pylori urease-immunized dams. We speculate that while milk obtained from H. pylori urease-immunized dams may reduce bacterial colonization, that from H. pylori-infected dams does not. However, there might be no apparent difference in the bacteriostatic effect of milk obtained from H. pylori-infected dams and naïve dams. Again, there was a wide variation in H. pylori colonization among mice of group 1 (1-week-old) at 8 weeks after infection. Interestingly, the 8 culture-positive mice originated from one family while the 6 culture-negative mice were from another family. The reason for this difference is not clear but may be related to familial genetic factors, since these two families were treated in the same fashion. Another possible explanation might be differences in the breastfeeding period or the amount of maternal milk among families. In the present study, mice infected with H. pylori were followed experimentally only until 8 weeks after infection. The selection of this period was based on previous kinetic studies in 4- to 8-week-old mice, which showed that the number of bacteria in the stomachs decreased and the variation between individual mice increased beyond 8 weeks after infection [16,17]. Thus, it is possible that the bacterial load among the individual stomach could have varied markedly in 1-week-old mice when examined at more than 8 weeks after infection. Possible mechanisms for such variability among families might be due to subtle differences of physiological functions or maturation of receptors for bacterial adhesion in the gastric epithelium.

While the reason for the high levels of gastric proinflammatory cytokines and the simultaneous mild infiltration of inflammatory cells in group 1 is not clear, it is clear that a Th1 cell-mediated immune response occurs even in infant mice with H. pylori infection. We speculate that such discrepancy is due to induction of tolerance against H. pylori within very early age, especially nursing period. Finally, the bacterial load in the stomach increased despite the low grade of gastritis scores.

The humoral response in group 1 was weak and delayed. This finding in mice is in agreement with that in humans [10,11]. We also evaluated the ability of infant mice for the latent antibody-production by vaccination. The findings of this experiment suggest that lymphocytes, even in young mice, can potentially produce anti-H. pylori antibodies. However, mice of group 2 exhibited a weaker response than those of group 3 at 8 weeks after infection (Fig. 3a). In addition, the humoral response of mice parenterally immunized at 3 weeks of age was greater than that of mice immunized at the age of 1 week (P < 0·0001, data not shown). These results suggest that humoral immunity against H. pylori infection at a younger age is immature or tolerant to such mucosal pathogens.

In the present study, the inoculum of H. pylori was administered only once. This is because several attempts to challenge 1-week-old mice twice resulted in the death of all mice, probably due to toxic shock (data not shown). No adverse symptoms were observed in groups 2 and 3. Eisenberg et al. [18] reported that repeated systemic administration of AlOH with whole-cell lysates resulted in the death of all neonatal mice. H. pylori is thought to contain a component that induces toxic shock in infants. There is a great deal of controversy about the association between sudden infant death syndrome (SIDS) and H. pylori infection [19], although at present the two conditions are generally thought to be unrelated [20,21]. However, our findings might support such a relationship.

In summary, our study provided new information on childhood H. pylori infection. In our murine model, the nursing period was apparently critical for maternal protection against H. pylori infection of the child. The present study showed that the pathological manifestations of mice infected during the breastfeeding period were notably different from those of mice infected after weaning. We believe that infant C57BL/6 mice with H. pylori infection represent a useful tool for investigating the pathology of human childhood H. pylori infection.