Abstract
The gram-negative bacterial pathogen Helicobacter pylori is a major cause of peptic ulcer disease and a risk factor for gastric cancer in humans. Adapted H. pylori strains, such as strain SS1, are able to infect mice and are a useful model for gastric colonization and vaccination studies. In this study we used a streptomycin-resistant derivative of H. pylori SS1 to analyze the colonization behavior and the success of vaccination in wild-type (wt) and various knockout mice of the BALB/c and C57BL/6J genetic backgrounds. We here report that BALB/c interleukin-4 knockout (IL-4−/−) mice are weakly overcolonized compared to the wt strain but that the IL-12−/− knockout results in a strong overcolonization (500%). Unexpectedly, in the C57BL/6J background the same knockouts behaved in diametrically opposed manners. The IL-4−/− mutation caused a 50% reduction and the IL-12−/− knockout caused a 95% reduction compared to the wt colonization rate. For C57BL/6J mice we further analyzed the IL-18−/− and Toll-like receptor 2 knockout mutations, which showed reductions to 66 and 57%, respectively, whereas mice with the IL-10−/− phenotype were hardly infected at all (5%). In contrast, the tumor necrosis factor receptor knockout (p55−/− and p55/75−/−) mice showed an overcolonization compared to the C57BL/6J wt strain. With exception of the low-level infected C57BL/6J IL-10−/− and IL-12−/− knockout mice, all knockout mutants were accessible to a prophylactic vaccination and their vaccination behavior was comparable to that of the wt strains.
The gram-negative spiral-shaped bacterium Helicobacter pylori infects half of the world's population (56). Although most infected individuals develop a chronic active gastritis, which may persist for decades, in the majority of cases the infection remains asymptomatic. In a minority of cases, however, the infection causes various clinical symptoms, ranging from upper abdominal pain to gastric or duodenal ulcer (48). Furthermore, the risk of developing gastric malignancies, such as adenocarcinoma and low-grade B-cell lymphoma, is increased 3- to 12-fold (19, 50).
The present therapy for chronic infection with H. pylori involves treatment with multidrug regimens, which are suspected to induce antibiotic resistance (21) and to cause the risk of a reinfection following eradication (46). Prophylactic or therapeutic vaccination against H. pylori is a desirable alternative to control the H. pylori infection (3), and animal studies have proven the feasibility of this approach (9, 10, 14, 16, 27, 61).
Although significant progress has been made concerning different vaccination protocols in various animal models of infection (11) and human trials have been performed (24, 32), the mechanism of protection against H. pylori infection is still not understood. Nevertheless, some general statements, mostly derived from studies in mice, can be taken into account. (i) The H. pylori infection is characterized by a polarized T-helper cell type 1 (Th1) response that seems to be correlated with pathogenesis (4, 12, 31, 33, 49). (ii) If we assume that Th1-Th2 polarization is a paradigm that has the potential to misroute (2), a shift from a Th1 towards a Th0 or a Th2 response seems to be a prerequisite for protection against the H. pylori infection (34, 42). (iii) major histocompatibility complex class II-restricted CD4+ T cells rather than major histocompatibility complex class I-restricted CD8+ T cells are critical for host protection (15, 38). (iv) B cells (antibodies) are not required for protection (5, 15, 54) but may be beneficial (10, 17, 27). (v) IL-4, a prototypical Th2 cytokine, is not necessary for successful vaccination (1, 28), although Th2 cells show the ability to reduce the bacterial load in the mouse stomach (34). Gamma interferon (IFN-γ), a prototypical Th1 cytokine, is not necessary for successful vaccination, although it has a protective quality (43). Taken together, these data clearly demonstrate the protective role of antigen-specific CD4+ T-helper cells, but less is known about the impact of the innate part of the immune system on infection with and immunization against H. pylori.
In the experiments presented here, we examined the H. pylori infection density, the potential of the pathogen to induce histological lesions, and the ability of oral immunization to interfere with H. pylori infection in interleukin-4 knockout (IL-4−/−), IL-10−/−, IL-12−/−, IL-18−/−, and tumor necrosis factor alpha (TNF-α) receptor knockout mice. Furthermore, the influence of the innate immune receptor Toll-like receptor 2 (TLR2) on infection density was investigated. Since it is known that the host genetic background has a profound influence on the immune responses in mice (22, 59), comparative studies with IL-4−/− and IL-12−/− knockout mice with different genetic background (BALB/c versus C57BL/6J) should reveal whether or not the influence of these cytokines is dependent on the genetic background of the animals.
MATERIALS AND METHODS
Bacterial strains and growth conditions.
H. pylori strains were grown on GC agar plates (Difco) supplemented with horse serum (8%), vancomycin (10 mg/liter), trimethoprim (5 mg/liter), and nystatin (1 mg/liter) (serum plates) and incubated for 24 to 48 h in a microaerophilic atmosphere (85% N2, 10% CO2, and 5% O2) at 37°C. The highly mouse-adapted H. pylori strain SS1 (A. Lee, University of Wales, Sydney, Australia) was kindly provided by F. Sommer, University Erlangen, and transformed to streptomycin resistance with plasmid pEG21 to obtain strain SS1S (18). Streptomycin selection was used for optimal quantitative reisloation of SS1S from the infected mouse stomach.
Natural transformation.
The suicide plasmid pEG21 was introduced into H. pylori SS1 by natural transformation as described previously (18). H. pylori transformants were selected on serum plates containing 250 mg of streptomycin per liter.
Animals.
Age-matched (6 to 8 weeks) female BALB/c or C57BL/6J mice were obtained from RCC, Itingen, Switzerland. All protocols involving animal experimentation were approved by the Regierung von Oberbayern (Aktenzeichen 211-2531-60/98). Animals were housed under specific-pathogen-free conditions. All of the following mouse strains were originally obtained from the Jackson Laboratory (Bar Harbor, Maine), except for C57BL/6J IL-18−/− TLR−/− mice, which were kindly provided by Shizuo Akira (Department of Biochemistry and Institute for Advanced Medical Sciences, Hyogo College of Medicine, Hyogo, Japan): BALB/c-IL-4tm2Nnt, BALB/c-Il12atm1Jm, C57BL/6J-IL-4tm1Nnt, B6.129P2-IL-10tm1Cgn, C57BL/6J-Il12atm1Jm, B6.129-Tnfrsf1atm1Mak, B6.129S-Tnfrsf1atm1Imx Tnfrsf1btm1Imx, C57BL/6J IL-18−/−, and TLR2−/− (55).
H. pylori infection and immunization schedules.
Groups (n = 5) of 6- to 8-week-old mice were challenged and immunized orogastrically (0.2 ml) with a stainless steel blunt feeding needle (Eickemeyer, Tuttlingen, Germany). For all experiments except those with IL-18−/− and TLR2−/− mice, the following schedule was applied: mice were immunized on days 0 and 35 (week 5) with 0.2 mg of H. pylori sonicate and 10 μg of cholera toxin (CT) adjuvant (Sigma-Aldrich) in phosphate-buffered saline and challenged on day 63 (week 9) with 107 SS1S organisms; the experiment was terminated from day 210 to 220 (week 30 to 31). For experiments with IL-18−/− and TLR2−/− mice, the following schedule was applied: mice were immunized on days 0 and 21 (week 3) and challenged on day 35 (week 5); the experiment was terminated from day 63 to 65 (week 9).
Growth of H. pylori for oral infection of mice.
For infection of mice, strain SS1S was grown for 2 days on serum plates containing 250 mg of streptomycin per liter at 37°C, harvested, and suspended in brucella broth (Oxoid Ltd., Basingstoke, England), and the final concentration was adjusted to 107 bacteria/200 μl. Mice were inoculated once orogastrically with 0.2 ml of bacterial suspension (107 bacteria).
Reisolation of bacteria and quantitative culture.
At the end point of the experiment, mice were anesthetized and sacrificed with CO2. The stomachs were removed, weighed, and opened along the great curvature. For assessment of H. pylori colonization by quantitative culture, weighed stomachs were homogenized in 2 ml of brucella broth with a hand homogenizer (Fisher Scientific, Schwerte, Germany), and 1/200 serial dilutions were spread over the surfaces of serum plates containing 250 mg of streptomycin per liter (performed in triplicate to quadruplicate). The plates were incubated for 5 to 7 days, and colonies were counted to determine the CFU per gram of stomach tissue.
Histopathological evaluation.
One half of the longitudinally dissected murine stomach was formalin fixed and paraffin embedded according to routine protocols. Blocks were cut in 3-μm sections, stained with hematoxylin-eosin, and histologically examined by one experienced histopathologist. Gastric mucosal alterations were classified according to the updated Sydney system (13). Grade and activity of gastritis were scored with respect to the density of the lymphocytic or polymorphonuclear infiltrate from 0 (no infiltrate) to 3 (severe infiltrate). All histological analyses were performed without knowledge of the experimental protocol for the respective tissue.
Statistical analysis.
In the vaccination experiments, statistical significance between the groups was determined by the nonparametric Wilcoxon signed rank (two-tail) test. P values of <0.05 are considered significantly different.
RESULTS
Determination of the colonization capacity of H. pylori SS1S for the infection of mice.
The H. pylori Sydney strain SS1 is widely used as a model strain for the infection of mice in vaccination experiments (26). To facilitate the quantitative reisolation of SS1 from mouse stomach, we generated a streptomycin-resistant derivative of SS1 by transformation with plasmid pEG21. The plasmid introduces a point mutation into the chromosomal rpsL gene, causing resistance to streptomycin (18). The resulting mutant strain was named SS1S. To rule out that SS1S has, due to the rpsL mutation, changed its fitness for the infection of mice, we determined the infectious dose of SS1S. H. pylori SS1S inocula of various doses were determined by quantitative culture (see Materials and Methods). C57BL/6J mice were each orogastrically inoculated twice at 2-day intervals with 0.2 ml of the given suspension. The presence of H. pylori infection was assessed in five mice for each inoculum at 1 month postinfection by urease activity and culture determinations. In each group, five of five mice were infected with an SS1S dose of 103, 104, 105 106, or 107 CFU. Thus, it was demonstrated that 103 CFU of the organism was sufficient to obtain a 100% infection rate.
IL-4 and IL-12 knockout mice in the BALB/c and C57BL/6J background have diametrically opposed colonization behaviors.
The contributions of the cytokines IL-4 and IL-12 to experimental infection and prophylactic vaccination with H. pylori in BALB/c and C57BL/6J mice were examined. The H. pylori infection of knockout and wild-type (wt) mice and the challenge after immunization resulted in 100% infection rates (see Materials and Methods). The experiments were performed as long-term colonizations (immunization on days 0 and 35 [week 5], challenge on day 63 [week 9], and termination from day 210 to 220 [week 30 to 31]). Evaluation of the magnitude of colonization by quantitative bacterial culture revealed opposite effects in the different genetic backgrounds of BALB/c and C57BL/6J IL-4−/− and IL-12−/− mice (Fig. 1). The slightly enhanced colonization density in BALB/c IL-4−/− mice compared to the corresponding wt mice is reminiscent of previously published data on corresponding knockout mice in the C57/BL/6 background (34). Unexpectedly, in C57BL/6J IL-4−/− mice the bacterial burden was decreased to about 50% of the wt colonization rate, compared to the corresponding median. This dichotomy became even more obvious when BALB/c and C57BL/6J IL-12−/− mice were compared to their corresponding wt mice: BALB/c IL-12−/− mice exhibited a strongly enhanced colonization density (500%), but in contrast to that, C57BL/6J IL-12−/− mice could hardly be infected at all with H. pylori (5%). A classical prophylactic immunization with CT as a mucosal adjuvant was successful in both wt and cytokine-deficient mice. In all groups tested except C57BL/6J IL-12−/− mice, the orogastric immunization led to a significant reduction of the bacterial burden. Surprisingly, the immunization of the nearly resistant C57BL/6J IL-12−/− mice did not lead to a further reduction of H. pylori colonization density (Fig. 1). To test reproducibility, the infection experiments were repeated once with the same experimental setting but with a short-term infection schedule (4 instead of 22 weeks). The short-term infections resulted in essentially the same tendency for the colonization behavior as obtained with the long-term infections, but the absolute differences in colonization rates between the corresponding wt and knockout groups were less pronounced (data not shown).
FIG. 1.
Colonization and prophylactic immunization of wt and knockout mice of BALB/c and C57BL/6J backgrounds with H. pylori SS1S. The rates of colonization of BALB/c (A and C) and C57BL/6J (B and D) wt, IL-4−/−, and IL-12−/− mice with or without prophylactic vaccination are shown. (A and B) The medians for groups are given as percentages of H. pylori reisolation compared to the corresponding wt mice (set to 100%). (C and D) For single mice data are given as CFU per gram of stomach. The experiment lasted for 31 weeks (see Materials and Methods). Bars: 1, wt, infected; 2, wt, immunized and infected; 3, IL-4−/−, infected; 4, IL-4−/−, immunized and infected; 5, IL-12−/−, infected; 6, IL-12−/−, immunized and infected. When infected mice were compared to the corresponding immunized mice, all groups differed significantly (P < 0.05), with exception of the C57BL/6J IL-12−/− mice (P = 0.138) (panel D, bars 5 and 6). The rate of colonization of infected knockout mice differed significantly from that of the corresponding infected wt group, with exception of the BALB/c IL-4−/− (panel C, bars 1 and 3) (P = 0.892) and C57BL/6J IL-4−/− (panel D, bars 1 and 3) (P = 0.0796) groups. *, P < 0.05
C57BL/6J IL-10 knockout mice are hardly able to be infected by H. pylori, and TNF-α receptor knockout mice are overcolonized.
Next we were interested in analyzing the contributions of IL-10 and the TNF-α receptor to experimental infection and prophylactic oral vaccination with H. pylori in C57BL/6J mice (see Materials and Methods for details). As seen before, the challenge of groups of five mice resulted in 100% infection rates. C57BL/6J IL-10−/− mice infected with H. pylori displayed a significantly reduced bacterial burden (Fig. 2), and they tended to be resistant against this extracellular pathogen. This is consistent with previously published data (8), and again, as already seen with C57BL/6J IL-12−/− mice, vaccination had no effect in diminishing the colonization density further (Fig. 2).
FIG. 2.
Colonization and prophylactic immunization of wt and knockout mice of the C57BL/6J background with H. pylori SS1S. (A) The rates of colonization of C57BL/6J wt, IL-10−/−, p55−/−, and p55/75−/− mice are shown. The medians for groups are given as percentages of H. pylori recovery. (B) Data for single mice are given as CFU per gram of stomach. The experiment lasted for 31 weeks (see Materials and Methods). Bars: 1, wt, infected; 2, wt, immunized and infected; 3, IL-10−/−, infected; 4, IL-10−/−, immunized and infected; 5, p55−/−, infected; 6, p55−/−, immunized and infected; 7, p55/p75−/−, infected; 8, p55/p75−/−, immunized and infected. When the colonization rates of infected mice were compared to those of the corresponding immunized and infected mice, all groups differed significantly (P < 0.05), with the exception of the IL-10−/− mice (panel B, bars 3 and 4) (P = 0.715). Rates of colonization of infected wt mice differed significantly from those of the knockout groups (P < 0.05), with the following exceptions: p55−/− infected only (panel B, bars 1 and 5) (P = 0.5002), p55/75−/− infected only (panel B, bars 1 and 7) (P = 0.0796), and p55/75−/− immunized and infected (panel B, bars 2 and 8) (P = 0.5002). The groups presented in Fig. 1 and 2 were evaluated in the same experiment, and the wt control experiments were therefore with the same animals. *, P < 0.05
In contrast, C57BL/6J p55−/− and p55/75−/− mice (Fig. 2) were susceptible to H. pylori infection. Infection with SS1S was increased (700 to 800%), but again, like for IL-4−/− mice, no statistical significance could be shown for these groups (Fig. 2). However, the comparable results for p55−/− and p55/75−/− mice may indicate a suppressive effect of TNF on the infection with H. pylori. Immunization of these mice led to a significant reduction of the bacterial burden, although the colonization density still remained higher than in vaccinated wt C57BL/6J animals.
Infection and vaccination of C57BL/6J IL-18−/− and TLR2−/− mutant mice.
In extension of the above-described experiments, the contributions of IL-18 and TLR2 to the experimental infection and prophylactic oral vaccination with H. pylori in C57BL/6J mice were studied in an experiment with a shorter duration (immunization on days 0 and 21, challenge on day 35, and termination from day 63 to 65 [week 9]) (see Materials and Methods). IL-18−/− and TLR2−/− mice, both on a C57BL/6J genetic background, displayed a slightly decreased colonization density compared to the C57BL/6J wt control strain (Fig. 3). Whereas the IL-18 knockout mice did not have a significant effect on the colonization density, the TLR2 knockout mice displayed a significantly lower bacterial burden. Both types of mutations did not have a significant effect on the efficiency of oral immunization to reduce the colonization density of H. pylori (Fig. 3).
FIG. 3.
Colonization and prophylactic immunization of wt and knockout mice of the C57BL/6J background with H. pylori SS1S. (A) The rates of colonization of C57BL/6J wt, IL-18−/−, and TLR2−/− mice are shown. The medians for groups are given as percentages of H. pylori recovery. (B) Data for single mice are given as CFU per gram of stomach. The experiment lasted for 9 weeks (see Materials and Methods). Bars 1, wt, infected; 2, wt, immunized and infected; 3, IL-18−/−, infected; 4, IL-18−/−, immunized and infected; 5, TLR2−/−, infected; 6, TLR2−/−, immunized and infected. When infected-only mice were compared to the corresponding immunized and infected mice, all groups differed significantly (P < 0.05). When compared to infected-only wt mice, the IL-18−/− group (P = 0.2249) did not differ significantly (P < 0,05). *, P < 0.05.
Histology of the stomachs of colonized and immunized mice of different genetic backgrounds and genotypes.
The stomachs of the mice were examined by histology to determine the grade and activity of gastritis according to the updated Sydney system (13). We were mainly interested in the question of whether the infected groups of wt or knockout mice differed in their grade and activity of gastritis compared to the corresponding prophylactically vaccinated and infected groups. The esophageal-gastral transition zone was chosen to determine gastritis, and for each group the average for five animals was determined (Table 1). For BALB/c mice we did not find appreciable gastritis scores, independent of the knockout or vaccination status of the animals (Table 1). In C57BL/6J mice the situation was different. In the group of H. pylori-infected wt mice, we generally found a lower grade and activity of gastritis than in the immunized group. In the IL-4−/− and IL-12−/− groups, the grade of gastritis was high but there was no significant difference between colonized and immunized groups (Table 1). A clear difference in the grade and activity of gastritis was seen in the TNF receptor p55−/− and p55/75−/− groups between mice that were colonized only and those that were vaccinated. The vaccination caused a strong increase (Table 1). The strongest gastritis score was seen in IL-10−/− mice, which did not differ much between the colonized and the vaccinated status.
TABLE 1.
Histological grade of esophageal-gastric transition area gastritis in BALB/c and C57BL/6J mouse mutants
| Mouse strain | Mouse mutant | Prophylactic Immunization | Gastritisa
|
|
|---|---|---|---|---|
| Grade (mean ± SD) | Activity (mean ± SD) | |||
| BALB/c | wt | − | 0 ± 0 | 0 ± 0 |
| + | 0.5 ± 0.87 | 0 ± 0 | ||
| IL-4−/− | − | 0.6 ± 0.42 | 0 ± 0 | |
| + | 0 ± 0.29 | 0 ± 0 | ||
| IL-12−/− | − | 0.6 ± 0.25 | 0 ± 0 | |
| + | 0.3 ± 0.25 | 0 ± 0 | ||
| C57BL/6J | wt | − | 1 ± 0.63 | 0.5 ± 0.41 |
| + | 1.8 ± 0.45 | 1.5 ± 0.98 | ||
| IL-4−/− | − | 1.9 ± 0.42 | 1.6 ± 0.42 | |
| + | 1.6 ± 0.55 | 1.2 ± 0.76 | ||
| IL-10−/− | − | 2 ± 0 | 1.4 ± 0.42 | |
| + | 2.2 ± 0.45 | 1.4 ± 0.45 | ||
| IL-12−/− | − | 1.7 ± 0.27 | 1.2 ± 0.45 | |
| + | 1.6 ± 0.42 | 1 ± 0.35 | ||
| p55−/− | − | 1.1 ± 0.22 | 0.9 ± 0.27 | |
| + | 2 ± 0 | 1.8 ± 0.29 | ||
| p55/75−/− | − | 0.7 ± 0.29 | 0.5 ± 0.29 | |
| + | 1.8 ± 0.57 | 1.7 ± 0.45 | ||
Data are for three to five mice for each group.
DISCUSSION
It has been well established that vaccination against H. pylori in mice is feasible. Different routes of immunization, different antigens, and different adjuvants have been used (51). One major problem for transferring the H. pylori vaccine technology to humans is the highly toxic adjuvants used, such as the heat-labile enterotoxin of Escherichia coli or the CT of Vibrio cholerae, which are well tolerated in mice but not in humans. CT is able to induce Th2 cells and IL-4 in mice (30) and to suppress IL-12 production and IL-12 receptor β1 and β2 chain expression (6). Taken together, these data fit with the observation that a shift from a polarized Th1 towards a Th0 or Th2 immune response is necessary for protection against infection with H. pylori (12, 20, 42).
In this context it seemed interesting to test the ability of H. pylori to establish and maintain an infection in IL-4 and IL-12 knockout mice on different genetic backgrounds. The experiments diagrammed in Fig. 1 show a clear dichotomy of response in mice of the H2d haplotype (BALB/c) versus mice of the H2b haplotype (C57BL/6J). BALB/c IL-4−/− and C57BL/6J IL-4−/− mice showed a weak but opposed effect when challenged with H. pylori. These results might be explained by the known different immune responses of these mice upon an infection (25, 52). Specific immunity induced by vaccination was effective in reducing bacterial counts in both mouse strains but was not dependent on the IL-4−/− mutation. Although our experiments were performed with IL-4−/− mice and not IL-4 receptor knockout strains, they are consistent with and extend the findings of Lucas et al. (28). They could show that adoptively transferred urease-specific CD4+ T cells reduce H. pylori stomach colonization in the absence of IL-4/IL-13 receptor signaling in BALB/c mice. In contrast, the effect of the IL-12 gene knockout was very pronounced. BALB/c IL-12−/− mice were found to be highly susceptible to infection with H. pylori, whereas C57BL/6J IL-12−/− mice were hardly able to be infected with H. pylori. It is interesting that infected C57BL/6J IL-12−/− mice were not accessible to a further reduction of bacterial load by vaccination. A possible explanation may be that H. pylori resides in two different niches in the mouse stomach, one accessible to the immune system and another which may not be reached by the immune response. The immunization-induced specific immunity in IL-4−/− and IL-12−/− mice does not seem to be hampered in its ability to control infection with H. pylori, but the immune system is not able to reduce the colonization density below a minimal bacterial load. The mechanism by which the IL-12−/− gene knockout renders BALB/c mice more susceptible and C57BL/6J mice partly resistant to H. pylori has to be evaluated in future experiments. Because one of the most important properties of IL-12 is its ability to induce the production of large amounts of IFN-γ from resting and activated T and NK cells (7, 23, 57), it was interesting to answer the question of whether C57BL/6J IL-18−/− mice, which are negative in inducing IFN-γ, would behave similarly to C57BL/6J-IL-12−/− mice. As shown in Fig. 3, in a short-term colonization experiment C57BL/6J-IL-18−/− animals exhibited a diminished colonization density, which is in contrast to the data of Sawai et al., who showed that IFN-γ has a weak protective influence in C57BL/6J mice (43). No influence of the gene knockout when mice were immunized against H. pylori could be shown. One explanation again could be that the observed IL-18 effect is due to a modulated innate immune response. In analogy to the C57BL/6J IL-12−/− mice, C57BL/6J IL-10−/− mice also were partly resistant to infection with H. pylori, a confirmation of the data of Chen et al. (8) and Sutton et al. (53), who classified the immunoregulatory and potent anti-inflammatory cytokine IL-10 as an inhibitor of the protective immune response to H. pylori infection. These data mimic the situation with a variety of intracellular pathogens (35) and are in contrast to gastrointestinal helminth infection (45). Again, the low bacterial burden could not be further reduced by vaccination of these mice, supporting the idea that H. pylori colonizes two different gastric niches. Another explanation would be that H. pylori exists in two phase variants, one of which may be able to evade the immune response.
It is interesting that the extracellular bacterium Yersinia enterocolitica is able to evade the host innate immune system by V antigen-induced IL-10 production of macrophages and that evasion is eliminated in IL-10-deficient mice (47). The V antigen of pathogenic Yersinia species is part of the type III translocation apparatus, which is required to deliver antihost effector proteins into host cells (39). It is tempting to speculate that H. pylori may use a similar, unknown mechanism to tune the host immune response, utilizing its type IV secretion apparatus encoded on the cag pathogenicity island (36, 37). It is known that the anti-inflammatory cytokine IL-10 suppresses the secretion of TNF-α, a potent and pleiotropic proinflammatory cytokine that plays a crucial role in limiting bacterial infections (35, 60). To evade the host immune defense, the inhibition of TNF-α would be a powerful strategy that a pathogen might develop. The observation that C57BL/6J p55 and p55/75 receptor mutant mice are more susceptible to infection with H. pylori could be in agreement with the in vivo effects observed for IL-10 and fits into this assumption.
It is obvious that TLRs (58) might have an influence on the immune response against H. pylori. C3H/HeJ mice, which carry a missense mutation in the TLR4 gene (tlr4) (40) that makes them unable to sense bacterial lipopolysaccharide, appear to be normally colonized by H. pylori and Helicobacter felis (29, 41). Despite a heavy colonization, the gastritis and gastric atrophy were strongly reduced in the H. felis-infected C3H/HeJ mice. This indicates that TLR4 might play a role in sensing of H. pylori lipopolysaccharide in the gastric mucosa. We were interested in analyzing the role of another innate immune receptor, TLR2, and we compared the C57BL/6J TLR2−/− mutants with the corresponding C57BL/6J wt mice. In a short-term experiment we were able to show that C57BL/6J TLR2−/− mice have a moderately but significantly reduced colonization density (Fig. 3), which makes this mutation an interesting candidate for future experiments to clarify its role as a potential key player involved in the control of the H. pylori infection. Microbial components that activate TLRs usually induce the production of IL-12 by dendritic cells, leading to the development of Th1 cells but not Th2 responses (44). Further research defining the modulated innate and adaptive immune responses in these mice may reveal valuable data that may help to define requirements for future vaccines to be developed against infection with H. pylori.
Acknowledgments
We thank S. Akira for providing our institute with the C57BL/6J IL-18−/− mice.
This work was supported by the Deutsche Forschungsgemeinschaft (grant HA 2697/4-1 to R.H.).
Editor: D. L. Burns
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