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Journal of Histochemistry and Cytochemistry logoLink to Journal of Histochemistry and Cytochemistry
. 2009 Apr;57(4):327–338. doi: 10.1369/jhc.2008.952366

Role of ASC in the Mouse Model of Helicobacter pylori Infection

Bekale N Benoit 1, Motohiro Kobayashi 1, Masatomo Kawakubo 1, Michiko Takeoka 1, Kenji Sano 1, Jian Zou 1, Naoki Itano 1, Hiroko Tsutsui 1, Tetsuo Noda 1, Minoru Fukuda 1, Jun Nakayama 1, Shun'ichiro Taniguchi 1
PMCID: PMC2664985  PMID: 19064716

Abstract

Apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC) is an adaptor molecule activating caspase-1 that stimulates pro-interleukin-1β (pro-IL-1β) and pro-IL-18, two pro-inflammatory cytokines with critical functions in host defense against a variety of pathogens. In this study, we investigated the role of ASC in the host defense against Helicobacter pylori utilizing ASC-deficient mice. Mice were orally inoculated with H. pylori; bacterial load, degree of gastritis, and mucosal levels of inflammatory cytokines were analyzed and compared with those obtained from wild-type mice. We found more prominent H. pylori colonization in ASC-deficient mice, as revealed by colony-forming unit counts. Both groups of mice developed gastritis; however, ASC-deficient mice showed significant attenuation of inflammation despite high H. pylori colonization. ELISA, immunohistochemistry, and quantitative RT-PCR analyses revealed complete suppression of IL-1β and IL-18, and substantial reduction of interferon-γ (IFN-γ) expression, in ASC-deficient mice without apparent upregulation of other cytokines, including IL-10 and tumor necrosis factor-α. These results as a whole indicate that ASC exerts considerable influence on the host defense, acting through IL-1β/IL-18 and subsequent IFN-γ production, which in turn contributes to continuous chronic inflammatory response and consequent reduction of H. pylori colonization. (J Histochem Cytochem 57:327–338, 2009)

Keywords: apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain, IL-1β, IL-18, INF-γ, Helicobacter pylori, gastritis

Apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC) is an adaptor molecule originally identified in an insoluble cytosolic fraction called the speck, found in cells undergoing apoptosis (Taniguchi and Sagara 2007). ASC is composed of an N-terminal pyrin domain and a C-terminal caspase recruitment domain (Masumoto et al. 1999). Recent studies have shown that in the presence of ASC, interleukin 1β (IL-1β)-converting enzyme (ICE) protease-activating factor, cryopirin, and nucleotide-binding oligomerization domain (NOD)-like receptors regulate activation of caspase-1 (Martinon et al. 2002). ASC has been proposed to link intracellular NOD-leucine-rich repeats (LRR) (NOD-LRR) proteins such as cryopirin to caspase-1 through direct physiological association (Martinon et al. 2002). Consistent with this, Nalp1 and cryopirin can form an endogenous multiprotein complex containing ASC and caspase-1 called inflammasome, which promotes caspase-1 activation (Martinon et al. 2002).

Caspase-1 is synthesized as an inactive zymogen that is activated by cleavage after aspartic residues to generate the enzymatically active heterodimer in response to pro-inflammatory stimuli and bacterial infections (Thornberry and Lazebnik 1998). Activated caspase-1 is essential for the processing and release of biologically active IL-1β and IL-18, two pro-inflammatory cytokines with critical roles in host defense against a variety of pathogens (Dinarello 1998). However, ASC is not required for the secretion of tumor necrosis factor-α (TNF-α) and IL-6, which indicates a specific requirement for ASC in caspase-1-dependent secretion of IL-1β and IL-18.

Gene-targeted mice that lack ASC have revealed the in vivo role of ASC in several physiological and pathological settings (Mariathasan et al. 2004; Yamamoto et al. 2004). Among them, ASC-deficient mice showed extreme sensitivity to infection with Francisella tularensis, with markedly increased bacterial burdens and mortality compared with wild-type mice, which highlights the role of ASC in innate immune defense against certain intracellular bacterial pathogens (Mariathasan et al. 2005).

Helicobacter pylori is a Gram-negative microaerophilic bacterium that colonizes the gastric mucosa of about half the world's population (Marshall and Warren 1984; Graham 1997). H. pylori causes persistent gastritis characterized by neutrophilic and lymphoplasmacytic infiltration and induction of pro-inflammatory cytokines, and is directly linked to the development of peptic ulcer disease as well as gastric adenocarcinoma and gastric malignant lymphoma of mucosa-associated lymphoid tissue type (Sipponen and Hyvarinen 1993; Huang et al. 1998; Kobayashi et al. 2004). Fortunately, only a small percentage of the population develops serious disease due to H. pylori infection (Cave 2001). Who develops such disease is determined largely by the inflammatory response to the infection, which is influenced by the virulence of the infecting strain, host genetic predisposition to disease, and environmental cofactors (Fox et al. 2000).

The initial migration into the gastric mucosa and subsequent activation of inflammatory cells are believed to depend on the production of pro-inflammatory cytokines (Yamaoka et al. 1997). Cytokines are important immune mediators in the host defense against microbial pathogens, including H. pylori. The cytokine response in the gastric mucosa of patients chronically infected with H. pylori is thought to be predominantly of the Th1 type (Lindholm et al. 1998). In this regard, pro-inflammatory cytokines such as IL-1β, TNF-α, interferon-γ (IFN-γ), and IL-18 have been implicated in the pathogenesis of and immunity to H. pylori infection.

An important part of the innate immune response is the secretion of IL-1β. IL-1β is a potent inflammatory cytokine that is released as a component of the host response against bacterial infection. It is primarily expressed by activated monocytes, macrophages, and polymorphonuclear (PMN) phagocytes as a precursor molecule, pro-IL-1β, a 31–34-kDa inactive form of cytokine, which is later cleaved by caspase-1 to active 17-kDa IL-1β (Cerretti et al. 1992). Broadly speaking, the biological activities of IL-1β are targeted toward enhancing the host's inflammatory response against a variety of endogenous and exogenous stimuli. This pro-inflammatory cytokine promotes leukocyte infiltration by inducing expression of an array of pro-inflammatory cytokines, chemokines, and adhesion molecules.

Soluble mediators of H. pylori are known to induce IL-1β. Of particular significance is the finding that IL-1β cluster polymorphisms suspected of enhancing production of IL-1β seem to be associated with increased risk of H. pylori-induced hypochlorhydria and gastric cancer (El-Omar et al. 2000; Fiqueiredo et al. 2002).

Formerly called IFN-γ-inducing factor, IL-18 is a potent pro-inflammatory cytokine in the IL-1 superfamily (Okamura et al. 1995; Ushio et al. 1996) that promotes the production of IFN-γ from Th1, B, and NK cells in synergy with IL-12 (Micallef et al. 1996). IL-18 also acts as a costimulant for Th1 cells to augment the production of IFN-γ, IL-2, and granulocyte macrophage colony-stimulating factor (Noguchi et al. 1998). Similar to IL-1β, IL-18 is synthesized as a biologically inactive precursor molecule lacking a signal peptide, which requires cleavage into an active, mature molecule by the intracellular cysteine protease called ICE (also called caspase-1). Previous study has shown that IL-18-deficient mice failed to develop protection after oral immunization with H. pylori lysates and cholera toxin adjuvant, indicating the importance of IL-18 in protection (Akhiani et al. 2004). Well-protected wild-type mice showed moderate gastric inflammation, whereas unprotected IL-18-deficient mice had less gastric inflammation, suggesting that IL-18 has an important role in the pathogenesis of H. pylori-induced gastro-duodenal disease. A recent work by Yamauchi et al. (2008) demonstrated that IL-18 levels in H. pylori-infected gastric mucosa from Colombian patients with non-ulcer dyspepsia were well-correlated with the severity of gastric inflammation, confirming that H. pylori-induced IL-18 plays an important role in gastric injury.

It remains unclear, however, whether ASC is involved in the host defense mechanism against H. pylori infection. In this study, we utilized mice deficient in ASC to examine the role of this adaptor molecule in host defense against H. pylori infection.

Materials and Methods

Animals

The generation of mice deficient in ASC has been described previously (Yamamoto et al. 2004). The mice were backcrossed to C57BL/6 genetic background for over eight generations. Homozygotes for the null allele of ASC were produced by crossing heterozygotes, and genotyping of offspring was confirmed by PCR of genomic DNA. Wild-type littermates were used as controls. A total of 87 male ASC-deficient mice and 87 male wild-type littermate control mice were bred at the animal facility of Shinshu University under specific pathogen-free conditions, housed in plastic cages, and offered commercially available food pellets and water ad libitum. Fifteen mice from each group were used as uninfected controls.

Bacteria

Mouse-adapted H. pylori Sydney strain 1, first developed by Lee et al. (1997), was used in this study. The H. pylori was plated on 5% sheep blood agar plates (Becton Dickinson; Cockeysville, MD) and incubated at 37C for 4 days under microaerophilic conditions with high humidity. To prepare the bacterial suspension, semi-confluent bacterial colonies on plates were scraped with a cotton swab, transferred into 50 ml of Brucella broth (Becton Dickinson) supplemented with 50 μg/ml cholesterol, and incubated at 37C overnight with agitation at 120 rpm under microaerophilic conditions with high humidity. After the incubation, 10 ml of culture was added to 50 ml of brain-heart infusion broth (Eiken Chemical; Tokyo, Japan) supplemented with 0.5% yeast extract (Becton Dickinson) and 10% fetal bovine serum (HyClone; South Logan, UT), and incubated further for 24 hr. After the morphology and mobility of bacteria were checked using a polarizing microscope, the concentration of the bacterial suspension was adjusted at 1.0 × 108/ml by measuring the optical density at 650 nm with a spectrophotometer.

Experimental Infection and Sample Collection

In three independent experiments, specific pathogen-free 8-week-old male mice deficient in ASC (n=24) and wild-type littermates (n=24) were subjected to H. pylori infection as described (Kobayashi et al. 2007). After overnight fasting, each animal was orogastrically inoculated three times every other day with 0.5 ml of the bacterial suspension by using a gastric intubation needle. Mice were then maintained on fasting a further 4 hr after each inoculation, and housed as described above.

Eight mice from each infected group were sacrificed by cervical dislocation at 2, 10, and 16 weeks after the last inoculation. The stomach was removed, cut along the greater curvature with a separate sterile surgical blade to avoid cross-contamination, and briefly washed with sterile saline. Immediately after macroscopic observation, the stomach was longitudinally dissected into three pieces of tissue fragments so that each fragment contained gastric cardia, body, and antrum. For each stomach, one piece was used for quantitative bacterial cultures, and another piece was fixed in 10% neutral buffered formalin at 4C for 24 hr for histopathological examination. The remaining piece was immediately stored in RNA (Ambion; Austin, TX), later frozen in liquid nitrogen, and stored at −80C for measurement of cytokine mRNA expression levels.

Blood samples were collected by cardiac puncture using 1 ml tuberculin syringes. Sera were separated from blood clots by centrifugation at 3000 rpm for 10 min, and stored at −80C until evaluated for cytokine protein levels. Uninfected control mice from each group (n=5) were sacrificed at 24 weeks of age (to serve as controls for infected animals 16 weeks after H. pylori inoculation) and treated in the same manner.

All animal experiments were conducted according to the protocols approved by the Animal Experiment Committee of Shinshu University School of Medicine.

Assessment of H. pylori Infection

The presence of viable H. pylori in individual mice was determined by quantitative bacterial culture as described previously (Chen et al. 1999). A piece of gastric tissue fragment was weighed and then homogenized in 500 μl of sterile saline with disposable grinders and tubes. Homogenates at a dilution of 1:1, 1:10, and 1:100 with sterile saline were prepared, and 50 μl of each dilution was spread onto H. pylori-selective agar plates (NISSUI Pharmaceutical; Tokyo, Japan). Each dilution was assayed in duplicate. In that way, six selective agar plates were used for each sample.

The plates were incubated at 37C for 7 days under microaerophilic conditions with high humidity. Bacterial colonies were identified by the rapid urease test and Gram staining for morphology. The number of colonies was counted, and the amount of viable H. pylori was expressed as colony-forming unit (CFU)/gram of stomach. For statistical analysis, the data were normalized by log transformation (log10CFU/gram of stomach).

Histopathological Evaluation

Formalin-fixed gastric tissues were embedded in paraffin, cut into 4-μm-thick sections, and stained with hematoxylin and eosin (HE). Additionally, tissue sections were immunostained with rabbit polyclonal anti-H. pylori antibody (DAKO; Kyoto, Japan) using an indirect immunoperoxidase method with peroxidase-labeled goat anti-rabbit IgG antibody (DAKO) as described previously (Kobayashi et al. 2007).

Gastritis-composing factors, e.g., (1) H. pylori density, (2) PMN cell (neutrophil) infiltration, and (3) lymphoplasmacytic infiltration, were categorized into four grades (normal, 0; mild, 1; moderate, 2; marked, 3) using a visual analog scale based upon the updated Sydney system (Dixon et al. 1996). For evaluation of epithelial changes, HE-stained sections were scored based on the degree of epithelial destruction in the corpus, including parietal cell loss and hyperplasia of the surface epithelium as described (Ermak et al. 1997). Briefly, epithelial scores were defined as follows: 0, none; 1, small, focal areas of parietal cell loss in the corpus and/or hyperplasia of the surface epithelium; 2, epithelial changes throughout 75% of the mucosa; 3, epithelial changes throughout the mucosa plus one to three microabscesses or cystic glands; or 4, epithelial changes throughout the mucosa plus four or more microabscesses or cystic glands. All samples were blinded, and grading was carried out.

RNA Extraction and Quantitative RT-PCR

Total RNA was extracted from gastric tissue samples using the RNeasy Mini Kit (QIAGEN, Inc.; Valencia, CA) according to the manufacturer's instructions. Single-stranded cDNA was then synthesized using Prime-Script Reverse Transcriptase (Promega; Madison, WI) with random primers (Promega) following the manufacturer's instructions. Negative controls were obtained by omitting the reverse transcriptase.

Quantitative PCR was carried out using the ABI Prism 7900HT sequence detection system (Applied Biosystems; Foster City, CA) with TaqMan Gene Expression Assays (Applied Biosystems) containing the following mouse-specific primers and TaqMan probes: IL-10 (Mm00439616_m1), TNF-α (Mm00443258_ m1), IFN-γ (Mm00801778_m1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Mm99999915_g1) as internal control.

Each reaction comprised 1 × PCR Buffer (Applied Biosystems) with 200 μM deoxy-nucleotide triphosphate (Applied Biosystems), 1.25 μl of TaqMan Gene Expression Assay, 2.5 μl of cDNA template, and 0.1 μl of AmpliTaq Gold (Applied Biosystems) in a final volume of 25 μl. After initial denaturation at 50C for 2 min followed by 95C for 10 min, 50 cycles of amplification with denaturation at 95C for 15 sec and annealing-extension at 60C for 60 sec were performed according to the manufacturer's instructions. The relative amounts of GAPDH were also quantified in the same reactions using TaqMan GAPDH control reagents (Applied Biosystems). Each sample was assayed in duplicate, and all TaqMan PCR data were captured using SDS 2.3 software (Applied Biosystems). Relative cytokine gene expression was determined by normalizing the amount of each cytokine mRNA divided by that of the GAPDH mRNA (Cytokine/GAPDH × 105). The negative controls did not show significant amplifications.

Cytokine ELISA

Serum protein levels of IL-1β and IL-18 in ASC-deficient mice and wild-type littermates were measured using ELISA kits (R and D Systems; Minneapolis, MN), based on the quantitative immunometric sandwich enzyme immunoassay technique, according to the manufacturer's instructions. All sera were assayed in duplicate, and results were reported as pg/ml.

In Situ Immunodetection of Inflammatory Cytokine Expression in the Gastric Mucosa

In situ expression of IL-1β and IL-18 was visualized by immunohistochemistry with specific antibodies and peroxidase detection. The immunohistochemical staining was performed on 10% neutral buffered formalin-fixed, paraffin-embedded sections. Briefly, after deparaffinization in xylene and rehydration through a graded alcohol series, gastric tissue sections were treated for endogenous peroxidase activity by incubation in 1% H2O2 for 10 min, washing with PBS, microwave irradiation for 15 min in 10 mM citrate buffer, then blocking in 1% goat normal serum (DakoCytomation; Glostrup, Denmark) for 20 min. To detect IL-1β and IL-18, sections were incubated with a primary rabbit polyclonal antibody raised against amino acids 117–269 of IL-1β of human origin (diluted 1:500) (Santa Cruz Biotechnology; Santa Cruz, CA) and a primary rabbit anti-mouse IL-18 monoclonal antibody (diluted 1:300) (Rockland Immunochemicals, Inc.; Philadelphia, PA), respectively, for 4 hr at room temperature.

Following three additional washes in PBS, the slides were incubated with a secondary goat anti-mouse antibody (Dako North America, Inc.; Philadelphia, PA) for 30 min at room temperature in the dark according to the manufacturer's instructions. After washing, sections were reacted with 3′-diaminobenzidine terahydrochloride chomogen (DAB) (Dako EnVision System-HRP; Dako) for 3 min and counterstained with hematoxylin. Control slides were incubated in PBS in the first step instead of the first specific antibody and examined for nonspecific staining (negative control).

Statistical Analysis

All values are expressed as means ± standard error of the mean (SEM). Log10CFU/gram of stomach, grading of inflammation, and the cytokine mRNA expression levels were compared between ASC-deficient mice and wild-type littermates using the Mann-Whitney U-test. Statistical significance was assumed for p values less than 0.05.

Results

Defect in Bacterial Clearance in ASC-deficient Mice

To examine the effect of ASC deficiency on H. pylori clearance, we compared CFU/gram of stomach between ASC-deficient mice and wild-type littermates at 2, 10, and 16 weeks postinoculation. The bacterial load of both groups of mice showed a gradual decrease with time, and there was a tendency for ASC-deficient mice to show higher CFU values (Figure 1A). We found that the difference in log10CFU/gram of stomach between ASC-deficient mice and wild-type littermates was not statistically significant by 10 weeks postinoculation; however, the difference became apparent at a later time point. At 16 weeks postinoculation, although wild-type mice showed a substantial drop in bacterial load, the degree of colonization in ASC-deficient mice was significantly higher, with a mean value of log10CFU/gram of stomach 4.52 ± 0.08 vs 3.87 ± 0.09 in wild-type mice (p=0.0043) (Figure 1A). Additionally, immunostaining for H. pylori showed numbers of bacteria on the surface mucosa and in the gastric proper glands in both ASC-deficient and wild-type mice (Figure 1B). However, bacteriological and immunohistochemical examination showed no detectable H. pylori in uninfected control mice (data not shown). Taken together, these findings indicate that the deficiency of ASC is associated with a defect in bacterial clearance, suggesting a protective role of ASC against H. pylori infection.

Figure 1.

Figure 1

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Defect in H. pylori clearance in the stomach of apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC)-deficient mice. (A) Bacterial loads, assessed as log10 colony-forming unit (CFU)/gram of stomach, of both wild-type (open bars) and ASC-deficient mice (closed bars) show gradual decrease with time, and there is a tendency in ASC-deficient mice to show higher CFU values. Colonization in ASC-deficient mice is significantly higher than that in wild-type mice at 16 weeks postinfection. **p<0.01; NS, not significant. (B) Immunostaining for H. pylori. Tissue sections were obtained from the stomachs of wild-type (left) and ASC-deficient mice (right) 16 weeks postinoculation. H. pylori colonization was detected on the surface mucosa and in the gastric proper glands in both groups of mice.

H. pylori-induced Gastritis Is Attenuated in ASC-deficient Mice

To evaluate the impact of ASC deficiency on the pathogenesis of H. pylori-induced gastritis, tissue sections obtained from the stomach of ASC-deficient and wild-type mice infected with H. pylori were examined histologically. We found that both ASC-deficient and wild-type mice developed some gastric inflammation at all time points examined. At 2 and 10 weeks after infection, both groups of mice showed very mild neutrophilic and lymphoplasmacytic infiltrations, with a small number of eosinophils, in the lamina propria, particularly in the vicinity of muscularis mucosae (Figures 2C–2F). It was noted that neutrophilic infiltrations were almost always conspicuous in the fundic gland area and minimal in the pyloric gland area in both ASC-deficient and wild-type mice, consistent with a previous report that neutrophilic infiltrations are site dependent in mice (Lee et al. 1990). There were no statistical differences in the scores of inflammatory cell infiltrate between the two groups at 10 weeks after infection (Figures 3A and 3B). However, at 16 weeks after infection, wild-type mice showed moderate corpus gastritis. The inflammation was characterized by a chronic inflammatory cell infiltrate composed mostly of lymphocytes, which formed discrete foci in the deeper portions of mucosa and submucosa. Mild neutrophilic infiltration was also present in the gastric corpus (Figure 2G). In contrast to wild-type mice, the degree of inflammation in ASC-deficient mice was significantly attenuated (Figure 2H). The score for activity in ASC-deficient mice was significantly lower than that in wild-type mice (p=0.0427) (Figure 3B). Scores for mononuclear cell infiltration were also lower in ASC-deficient mice compared with wild-type mice, although the difference was not statistically significant (Figure 3A). No significant difference between ASC-deficient and wild-type mice was detected when epithelial changes were examined 16 weeks after inoculation (Figure 3C). The epithelial change observed in the corpus region of both groups of mice was a mild hyperplasia of the gastric pit epithelium. Parietal cell density was well-maintained despite local inflammation. There was only a moderate loss of parietal cell mass in the corpus mucosa, resulting in extensive replacement by mucous cells in one mouse from the ASC-deficient group (Figure 2H). No glandular cystic lesions were observed in any mice of both groups.

Figure 2.

Figure 2

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Representative histopathology of the gastric mucosa of wild-type (left panels) and ASC-deficient (right panels) mice. Histological findings of non-infected controls (A,B), and 2 weeks (C,D), 10 weeks (E,F), and 16 weeks (G,H) after H. pylori infection. There was no inflammation in the gastric mucosa of non-infected wild-type (A) or non-infected knockout (KO) (B) mice throughout the experiment. At 2 weeks after infection, both wild-type (C) and ASC-deficient mice (D) developed gastritis, with mild to moderate inflammatory cell infiltrate present within the lamina propria. At 10 weeks postinfection, the gastric mucosa from both groups of mice showed an inflammatory cell infiltrate similar in composition but slightly larger than that seen at 2 weeks postinfection (E,F). No significant histopathological difference was found between the two groups. At 16 weeks after infection, wild-type mice developed moderate gastritis (G), whereas ASC-deficient mice had only mild gastritis (H). Inset in (G) shows inflammatory cells composed of lymphocytes and neutrophils.

Figure 3.

Figure 3

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Assessment of chronic inflammation (A), activity (B), and epithelial changes (C) induced by H. pylori infection in the stomachs of wild-type (open bars) and ASC-deficient mice (closed bars). Gastric mucosal changes were assessed histologically based on the updated Sydney system. Scores for respective mononuclear and polymorphonuclear cell infiltrates at 10 weeks postinfection do not differ between the two groups. At 16 weeks postinfection, however, the score of activity in ASC-deficient mice is significantly lower than that in wild-type mice (B). The score for mononuclear cell infiltration is also lower in ASC-deficient mice compared with wild-type mice, although the difference is not statistically significant (A). The degree of epithelial changes was evaluated 16 weeks after infection as described in Materials and Methods. No significant difference was found between the two groups (C). Data are expressed as means ± SEM. Each group consisted of eight mice. *p<0.05; NS, not significant.

In brief, there were no visible changes in the gastric mucosa of any uninfected mice (Figures 2A and 2B). Lesions consistent with dysplasia and/or carcinoma were not seen in any mice examined during the 16-week time period. Taken together, these data demonstrate that deficiency of ASC in mice leads to a less severe inflammatory response against H. pylori infection, suggesting that ASC contributes to host resistance against H. pylori infection by increasing the gastric inflammatory response.

ASC Deficiency Resulted in Complete Suppression of IL-18 and Substantial Reduction of INF-γ Expression

We further investigated the host response elicited by H. pylori in ASC-deficient mice by measuring cytokine responses, including IL-10, TNF-α, IFN-γ, IL-1β, and IL-18 in the gastric mucosa and in the serum at 16 weeks after infection, by quantitative PCR and ELISA, and compared with data obtained from wild-type littermates. ASC-deficient mice had significantly (p=0.0286) lower levels of mRNA for the Th1 cytokine IFN-γ than did wild-type mice (Figure 4A), whereas IL-10 and TNF-α mRNA expression levels did not differ significantly between the two groups (Figures 4B and 4C).

Figure 4.

Figure 4

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Relative gene expression levels of mRNA for interferon-γ (IFN-γ) (A), tumor necrosis factor-α (TNF-α) (B), and interleukin-10 (IL-10) (C) in gastric tissues from uninfected (HP−) and infected (HP+) wild-type and ASC-deficient mice were measured by RT-PCR. The expression of all IFN-γ, TNF-α, and IL-10 genes was elevated in both the infected wild-type and the infected ASC-deficient mice compared with that in the non-infected wild-type and non-infected ASC-deficient mice (A–C). RT-PCR shows lower levels of IFN-γ mRNA expression in infected ASC-deficient mice vs infected wild-type littermates with statistical significance (p=0.029) (A). On the other hand, the expression of TNF-α and IL-10 does not differ significantly between the infected groups (B,C). Data are expressed as the means ± SEM. *p<0.05; NS, not significant. (D) Serum protein level of IL-18 as assessed by ELISA. Sera were taken from ASC-deficient (closed bar) and wild-type mice (open bar) at 16 weeks after infection. ASC deficiency completely abolishes IL-18 secretion. Data are represented as means ± SEM. ND, not detected.

We next measured the serum concentration of IL-1β and IL-18 by ELISA in both ASC-deficient and wild-type mice. As expected, we found that ASC deficiency completely abolished IL-18 secretion (Figure 4D). However, we did not detect measurable serum IL-1β following infection with H. pylori in both groups of mice (data not shown).

ASC Deficiency Completely Abolished IL-1β and IL-18 Expression in the Gastric Tissue Sections

The immunohistochemical expression of inflammatory cytokines IL-1β and IL-18 was compared between the absence and the presence of H. pylori using tissues from ASC-deficient and wild-type mice obtained 16 weeks after the last inoculation.

We found no virtual expression of IL-1β and IL-18 cytokines in the gastric mucosa of ASC-deficient mice in either the presence or the absence of H. pylori infection (Figures 5B, 5D, 5F, and 5H). Cytokine expression was also absent in the uninfected wild-type littermate group (Figures 5A and 5E). However, an immunoreactivity for both cytokines was observed in the gastric mucosa of H. pylori-infected wild-type mice. As shown in Figures 5C and 5G, the specific immunohistochemical staining for the pro-inflammatory cytokines IL-1β and IL-18 were found to be distributed in the lamina propria as well as in the superficial epithelium. All of the tissue sections stained positively for IL-1β or Il-18 had mild to moderate inflammatory cell infiltrate recognized as monocytes.

Figure 5.

Figure 5

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Effects of ASC deficiency on IL-1β and IL-18 expression in gastric mucosa of wild-type (left panels) and ASC-deficient mice (right panels) in the absence or presence of H. pylori. Specific immunohistochemical staining for IL-1β (A–D) and IL-18 (E–H) in gastric tissue sections from non-infected (A,B,E,F) and infected (C,D,G,H) samples at 16 weeks, is shown. Both pro-inflammatory cytokines IL-1β and IL-18 are not expressed in the gastric mucosa of ASC-deficient mice regardless of H. pylori status (B,D,F,H). The expression of both cytokines is also absent in uninfected wild-type mice (A,E). Strong immunoreactivity for IL-1β and IL-18 can be observed in the lamina propria as well as in the epithelium in infected wild-type samples (C,G).

Discussion

To our knowledge, this study is the first to analyze the role of ASC in the host defense mechanism against H. pylori infection. We found that ASC-deficient mice had defects in bacterial clearance and inflammatory lesions, corresponding to a mild-to-moderate gastritis, that were significantly attenuated in ASC-deficient mice at 16 weeks postinoculation, compared with wild-type mice. This was supported by the finding that ASC-deficient mice had lower IFN-γ expression levels in the stomach than did wild-type mice.

However, the expression levels of TNF-α and IL-10 in both groups did not differ significantly. The significant decreased local IFN-γ mRNA in ASC-deficient mice was probably related to deficiency of IL-18, a potent inducer of IFN-γ (Okamura et al. 1995). This is consistent with the requirement of ASC for caspase-1 activation leading to IL-18 secretion in vivo.

Although colonization showed wide inter-individual variation at 16 weeks postinfection, the level of colonization was substantially higher in ASC-deficient mice compared with wild-type mice. The relatively low H. pylori colonization in wild-type littermates was histologically associated with a strong inflammatory cell infiltrate, as compared with ASC-deficient mice. This is consistent with the previous report that IFN-γ-deficient mice showed higher gastric colonization by H. pylori than did wild-type mice in a C57BL/6 background (Sawai et al. 1999). Thus, the lower levels of gastric IFN-γ expression observed in our study may provide an explanation for higher colonization of H. pylori in ASC-deficient mice. Also supportive of our findings is the observation that despite a chronic 15-month infection with H. pylori, the IFN-γ-deficient mice had minimal gastritis, compared with severe inflammation in the stomachs of C57BL/6 mice (Censini et al. 1996). Furthermore, IFN-γ levels measured by RT-PCR have been recognized as a useful predictor of the degree of gastritis in C57BL/6 mice infected with H. pylori (Goto et al. 1999).

In addition, immunohistochemical analysis clearly showed no obvious staining for IL-18 and IL-1β cytokines in the gastric mucosa of H. pylori-infected and uninfected ASC-deficient mice compared with infected wild-type littermates, suggesting the in vivo requirement of ASC for IL-1β and IL-18 expression.

Histological analysis demonstrated that PMN cell infiltration 16 weeks after H. pylori infection was significantly lower in ASC-deficient mice compared with wild-type mice. These data are relevant not only to the lower levels of inflammation observed but also to the level of colonization, because neutrophils are involved primarily in the host response against H. pylori infection. Our discovery is supported by the finding that mice deficient in ASC and IL-1β developed markedly larger skin lesions with higher bacterial counts and decreased neutrophil recruitment, compared with wild-type mice after skin inoculation with Staphylococcus aureus (Miller et al. 2007). Taken together, these data indicate that ASC-mediated production of IL-1β is required for adequate neutrophil recruitment following H. pylori infection, and subsequent bacterial clearance.

In this study, our findings suggest that the defect in bacterial clearance and the lower levels of inflammation observed in ASC-deficient mice could be due to a reduced inflammatory environment, e.g., lack of IL-18 and IL-1β, less IFN-γ expression, and PMN cell infiltration.

The hallmark of gastric inflammatory response to H. pylori is its capacity to persist for decades (Blaser et al. 1995; Everhart 2000). This is in contrast to inflammatory reactions induced by other mucosal pathogens, such as Salmonella spp., that either resolve within days or progress to eliminate the host (Gordon et al. 2001; Zhang et al. 2003). Previous study in ASC-deficient mice has focused on the role of ASC in the innate immune response against F. tularensis infection, which causes a plague-like disease in humans after exposure to as few as 10 cells (Mariathasan et al. 2004). Important differences have arisen, which may be due to the site of infection and/or to the persistent nature of H. pylori infection. Mariathasan et al. reported that F. tularensis-infected ASC-deficient mice showed markedly increased bacterial burdens and mortality as compared with wild-type mice, demonstrating a key role for ASC in innate defense against infection by this pathogen (Mariathasan et al. 2004). In contrast, in our hands, the severity of gastritis induced by H. pylori infection was clearly attenuated in ASC-deficient mice compared with wild-type littermates in spite of heavy colonization at 16 weeks postinoculation.

Our experiment specifies a concrete immune defect that impairs the ability of ASC-deficient mice to orchestrate a normal mucosal inflammatory response to H. pylori infection. This may occur via more than one mechanism. However, our available data so far indicate that ASC is critical for processing of the pro-inflammatory cytokines IL-1β and IL-18 and subsequent IFN-γ expression in response to H. pylori infection, contributing to a continuous chronic inflammatory response and consequent reduction of H. pylori colonization.

The association between chronic inflammation and cancer is now well established (Coussens and Werb 2002). This association has recently received renewed interest with the recognition that microbial pathogens can be responsible for the chronic inflammation observed in many cancers, particularly those originating in the gastrointestinal system (Macarthur et al. 2004). A prime example is H. pylori, which infects more than 50% of the world's population and is known to be responsible for inducing chronic gastritis that progresses to atrophy, intestinal metaplasia, dysplasia, and gastric cancer (Correa 1995).

H. pylori infection in both humans and C57BL/6 mice is associated with a Th1-like immune response (Mohammadi et al. 1996). Several studies have provided direct experimental evidence that Th1-type immune response is responsible for the progression of gastric pre-neoplastic lesions (Fox et al. 2000). In C57BL/6 mice, chronic infection with H. pylori is associated with considerable gastric inflammation and corpus atrophy, which is considered a pre-malignant lesion (Fox et al. 2000). However, deletion of the gene encoding the Th1 cytokine IFN-γ protects mice from gastric atrophy induced by infection with H. pylori. Here, we show that deficiency of ASC in mice completely abolished the expression of IL-18 and IL-1β, and drastically reduced IFN-γ expression in the gastric mucosa; this was histologically associated with a weak inflammatory response against H. pylori infection. These findings suggest that ASC-deficient mice may avoid H. pylori-induced gastric cancer.

There is mounting evidence that ASC mediates the clustering and activation of caspase-1 and subsequent maturation of pro-inflammatory cytokines IL-1β and IL-18 (Martinon et al. 2002; Stehlik et al. 2003). These cytokines promote cell-mediated immune responses and are capable of generating anti-tumor responses. For example, IL-18 has been shown to stimulate macrophages to elicit a potent cytotoxic response against glioma cells (Kikuchi et al. 2000).

On the other hand, loss of ASC expression by methylation in breast and other cancers has been indicated to correlate with carcinogenesis, suggesting a tumor suppressor role in those cell types (McConnell and Vertino 2004). Based on these reports, ASC-deficient mice chronically infected with H. pylori may have an increased risk for developing gastric cancer. It is of great interest whether ASC-deficient mice with H. pylori infection represent a useful tool for investigating how the loss of ASC contributes to carcinogenesis.

In conclusion, this study identified a new mechanism that may contribute to H. pylori pathogenesis. Loss of the ASC protein abolished the expression of IL-1β and IL-18 and drastically reduced IFN-γ expression in response to H. pylori infection, contributing to the attenuation of the inflammatory response and the defect in H. pylori clearance. Further studies are necessary to clarify the role of ASC in gastric carcinogenesis during persistent H. pylori infection.

Acknowledgments

This work was supported by Grants-in-Aid for Scientific Research C-19590385 and C-20012020 (to ST) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

We thank Tomoko Nishizawa and Matsuko Watanabe for technical assistance.

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