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Published in final edited form as: Oncogene. 2014 May 19;34(14):1865–1871. doi: 10.1038/onc.2014.135

Matrix metalloproteinase 7 restrains Helicobacter pylori-induced gastric inflammation and premalignant lesions in the stomach by altering macrophage polarization

MS Krakowiak 1,5, JM Noto 2,5, MB Piazuelo 2, DM Hardbower 2,3, J Romero-Gallo 2, A Delgado 2, R Chaturvedi 2, P Correa 2, KT Wilson 1,2,3,4, RM Peek Jr 1,2
PMCID: PMC4237684  NIHMSID: NIHMS598334  PMID: 24837365

Abstract

Helicobacter pylori is the strongest risk factor for the development of gastric cancer. Although the specific mechanisms by which this pathogen induces carcinogenesis have not been fully elucidated, high-expression interleukin (IL)-1β alleles are associated with increased gastric cancer risk among H. pylori-infected persons. In addition, loss of matrix metalloproteinase 7 (MMP7) increases mucosal inflammation in mouse models of epithelial injury, and we have shown that gastric inflammation is increased in H. pylori-infected MMP7−/− C57BL/6 mice. In this report, we define mechanisms that underpin such responses and extend these results into a genetic model of MMP7 deficiency and gastric cancer. Wild-type (WT) or MMP7−/− C57BL/6 mice were challenged with broth alone as an uninfected control or the H. pylori strain PMSS1. All H. pylori-challenged mice were successfully colonized. As expected, H. pylori-infected MMP7−/− C57BL/6 mice exhibited a significant increase in gastric inflammation compared with uninfected or infected WT C57BL/6 animals. Loss of MMP7 resulted in M1 macrophage polarization within H. pylori-infected stomachs, as assessed by Luminex technology and immunohistochemistry, and macrophages isolated from infected MMP7-deficient mice expressed significantly higher levels of the M1 macrophage marker IL-1β compared with macrophages isolated from WT mice. To extend these findings into a model of gastric cancer, hypergastrinemic WT INS-GAS or MMP7−/− INS-GAS mice were challenged with H. pylori strain PMSS1. Consistent with findings in the C57BL/6 model, H. pylori-infected MMP7-deficient INS-GAS mice exhibited a significant increase in gastric inflammation compared with either uninfected or infected WT INS-GAS mice. In addition, the incidence of gastric hyperplasia and dysplasia was significantly increased in H. pylori-infected MMP7−/− INS-GAS mice compared with infected WT INS-GAS mice, and loss of MMP7 promoted M1 macrophage polarization. These results suggest that MMP7 exerts a restrictive role on H. pylori-induced gastric injury and the development of premalignant lesions by suppressing M1 macrophage polarization.

Introduction

Gastric cancer is the third leading cause of cancer-related death in the world,1,2 and the strongest identified risk factor for this malignancy is chronic gastritis induced by the pathogen, Helicobacter pylori3. The specific mechanisms by which H. pylori initiates gastric carcinogenesis are not completely understood, and the disease outcome results from a complex and orchestrated interplay between environmental factors, bacterial virulence constituents and host responses3. One host response that augments the risk for gastric cancer is increased expression of the proinflammatory cytokine IL-1β, and individuals who harbor high-expression IL-1β alleles are at increased risk for gastric cancer4. Another host factor linked to neoplasia within the intestinal tract is matrix metalloproteinase 7 (MMP7), a minimal-domain MMP secreted preferentially by epithelial cells5–8. Previously, we observed that C57BL/6 mice lacking MMP7 develop increased inflammation following H. pylori infection, although the specific mechanism regulating this phenotype was not identified5. One limitation of this model is that C57BL/6 mice rarely develop premalignant or malignant lesions of the stomach following H. pylori infection. In this report, we now demonstrate that the loss of MMP7 results in M1 macrophage polarization, suggesting MMP7 may suppress inflammation and injury by regulating the immune response through directed macrophage polarization. We have also extended these findings into a mouse model of gastric cancer and MMP7 deficiency and demonstrate that MMP7 deficiency increases H. pylori-induced gastric premalignant lesions and promotes M1 macrophage polarization.

Results and Discussion

Our previous studies using a mouse model of H. pylori-induced gastritis demonstrated that MMP7−/− C57BL/6 mice develop more severe inflammation in response to H. pylori infection than wild-type (WT) C57BL/6 mice5. To extend these findings and identify mechanisms that regulate this phenotype, WT C57BL/6 and MMP7−/− C57BL/6 mice were challenged with broth alone as an uninfected control or the H. pylori strain PMSS1, and stomachs were harvested and analyzed 12 weeks post challenge. Twelve weeks were selected for analysis on the basis of previous studies indicating that infection with H. pylori strain PMSS1 reproducibly induces inflammation and injury in C57BL/6 mice at this time point9. To assess efficacy of bacterial colonization, colonization efficiency (% of challenged animals successfully colonized) and colonization density (colony-forming units/gram of gastric tissue) were determined. Colonization efficiency was 100% for all H. pylori-challenged mice (data not shown). Colonization density was significantly (P < 0.05) lower in infected MMP7-deficient mice compared with infected WT C57BL/6 mice (Figure 1a). Consistent with our previous findings, H. pylori-infected MMP7−/− C57BL/6 exhibited significantly higher levels of gastric inflammation and increased mucosal thickening compared with either uninfected mice or infected WT C57BL/6 mice (Figures 1b–f), indicating that the loss of MMP7 results in more severe inflammation within the context of H. pylori infection. Consistent with previous reports,10 an inverse association was observed between H. pylori colonization density and the severity of gastric inflammation (P < 0.005, r = −0.5732, data not shown).

Figure 1.

Figure 1

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Loss of MMP7 results in a significant increase in gastric inflammation in H. pylori-infected C57BL/6 mice. Eight-week-old specific pathogen free WT C57BL/6 (N = 16) and MMP7−/− C57BL/6 (N = 18) mice were challenged with broth alone as an uninfected control or with H. pylori strain PMSS1. Twelve weeks post challenge, mice were euthanized and stomachs harvested. (a) Gastric tissue from H. pylori-challenged mice was homogenized and plated on selective trypticase soy agar plates with 5% sheep blood for isolation of H. pylori. Plates were incubated for 5–7 days, and colonization density was determined and expressed as log colony-forming units (CFU) per gram of tissue. Each data point represents colonization density from an individual animal. (b–f) Linear strips of gastric tissue, extending from the squamocolumnar junction through the proximal duodenum, were fixed in 10% neutral-buffered formalin, embedded in paraffin and stained with hematoxylin and eosin. A pathologist (MBP), masked to the treatment groups, assessed indices of inflammation. (b) Severity of acute and chronic inflammation was graded 0–3 (absence of, mild, moderate or marked inflammation) in both the gastric antrum and corpus, for a cumulative score ranging from 0–12. Each data point represents inflammation scores from an individual animal. (c–f) Representative histologic images from uninfected WT C57BL/6 (c), uninfected MMP7−/− C57BL/6 (d), PMSS1-infected WT (e) and PMSS1-infected MMP7−/− C57BL/6 (f) mice at × 200 magnification. Student's t-test (a) and one-way analysis of variance with a Bonferroni post test (b) were used to determine statistical significance among groups.

We next sought to define mechanisms through which the loss of MMP7 leads to increased inflammation among H. pylori-infected mice. Gastritis is an initiating event for the development of most gastric adenocarcinomas3, and macrophages are required for the development of gastritis in response to H. pylori11. Several functional macrophage phenotypes have been described, including M1, M2 and Mreg. M1 macrophages are classically activated macrophages, which function to clear pathogens via intracellular microbicidal activity and by secreting inflammatory mediators that promote a Th1-type response, while simultaneously dampening Th2-type responses12,13. M2, or alternatively activated macrophages, promote wound healing by secreting components of the extracellular matrix12,14 as well as anti-inflammatory effectors that promote Th2-directed responses, while dampening Th1 responses13. Mregs or regulatory macrophages are also anti-inflammatory, but fail to deposit extracellular matrix12. Alterations in the composition of functional macrophage phenotypes within a specific inflamed niche can significantly alter the risk for carcinogenesis12.

Given that MMP7 has the potential to alter the microenvironment by cleaving components of the extracellular matrix and cytokines,15,16 we sought to determine whether gastric tissue from H. pylori-challenged MMP7−/− C57BL/6 mice harbored different cytokine and chemokine profiles than gastric tissue harvested from H. pylori-infected WT C67BL/6 mice, as such differences may affect macrophage polarization. Protein lysates of homogenized gastric tissue from H. pylori-challenged WT C57BL/6 and MMP7−/− C57BL/6 mice were analyzed by a multiplex cytokine assay using Luminex technology17. Levels of IL-1β, MIP-1β, tumor necrosis factor (TNF)-α, IP-10, RANTES and IL-17 were significantly increased in gastric tissue isolated from H. pylori-challenged MMP7−/− C57BL/6 mice compared with either uninfected MMP7−/− C57BL/6 mice, or with uninfected or H. pylori-challenged WT C57BL/6 mice (Figure 2). However, loss of MMP7 did not globally augment cytokine production, as levels of IL-1α and IL-7 were no different between H. pylori-infected WT and MMP7−/− C57BL/6 mice (data not shown). IL-1β and MIP-1β are inflammatory effectors secreted from M1 macrophages, whereas TNF-α is a potent inducer of M1 macrophage polarization18. Consistent with this pattern, several other proinflammatory cytokines implicated in macrophage-induced inflammation were also increased in H. pylori-infected MMP7−/− C57BL/6 versus WT C57BL/6 mice, including the macrophage product IP-10, and the T-cell cytokines RANTES and IL-17 (Figures 2d–f), supporting the concept that the loss of MMP7 leads to increased M1 macrophage activation within the context of H. pylori infection.

Figure 2.

Figure 2

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Loss of MMP7 alters the gastric cytokine and chemokine profiles of H. pylori-infected C57BL/6 mice. Gastric tissue was harvested 12 weeks post challenge and used for a multiplex cytokine assay using Luminex technology. Briefly, gastric tissue was homogenized in CelLytic MT Mammalian Tissue Lysis/Extraction Reagent supplemented with protease inhibitors using a Qiagen TissueLyser (Qiagen, Venlo, Limburg). Cytokine and chemokine concentrations were determined using a Millipore Luminex kit (Billerica, MA, USA) according to the manufacturer's instructions. Concentrations of IL-1β (a), MIP-1β (b), TNF-α (c), IP-10 (d), RANTES (e) and IL-17 (f) were determined as pg/mg of gastric tissue (log transformed). Each data point represents cytokine/chemokine levels in gastric tissue from an individual animal. A one-way analysis of variance with a Bonferroni post test was used to determine statistical significance among groups.

Because H. pylori-infected MMP7−/− C57BL/6 mice harbored an M1 macrophage polarization cytokine profile, we next quantified macrophages and expression of IL-1β, an effector produced by M1 macrophages, using gastric tissue harvested from WT C57BL/6 and MMP7−/− C57BL/6 mice. Consistent with increased levels of gastric inflammation, H. pylori-infected MMP7-deficient mice had significantly higher numbers of macrophages within the gastric mucosa compared with either uninfected or H. pylori-infected WT C57BL/6 mice, as assessed by F4/80 immunostaining (Figures 3a–c). Consistent with increased inflammation and increased IL-1β expression in gastric tissue homogenates (Figures 1 and 2), H. pylori-infected MMP7−/− C57BL/6 mice exhibited significantly higher numbers of immune cells expressing IL-1β, compared with uninfected mice or H. pylori-infected WT mice (Figures 3d–f).

Figure 3.

Figure 3

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Loss of MMP7 results in increased macrophage recruitment and IL-1β expression following H. pylori infection. (a–f) Five micrometers thick paraffin-embedded gastric tissue sections were mounted and deparaffinized. Endogenous peroxidases were quenched with 3% hydrogen peroxide, and slides were blocked with protein block serum-free solution before incubation with an F4/80 antibody (a–c) or a polyclonal rabbit anti-mouse-IL-1β antibody (d–f). (a) A pathologist, (MBP), masked to the treatment groups, enumerated F4/80-postive cells in 10 high-powered fields in the antrum and corpus. (b, c) Representative images of F4/80-positive cells in WT (b) or MMP7−/− (c) C57BL/6 mice infected with H. pylori strain PMSS1 at × 400 magnification. (d, e) Representative images of IL-1β-positive immune cells in WT (d) or MMP7−/− (e) C57BL/6 mice infected with PMSS1 at × 400 magnification. (f) A pathologist, (MBP), masked to the treatment groups, enumerated IL-1β-positive immune cells in 10 high-powered fields in the antrum and corpus. (g) F4/80-postive cells were isolated from the stomachs of WT and MMP7−/− C57BL/6 mice challenged with PMSS1, by magnet-assisted cell sorting. Briefly, gastric tissue was mechanically disrupted and enzymatically digested. Digested cells were resuspended in flow cytometry staining buffer solution with a biotin-conjugated F4/80 antibody, washed, resuspended with streptavidin-conjugated micrometal beads and washed again. F4/80-positive cells were collected using a magnetic apparatus and lysed with PerfectPure Lysis Solution (5PRIME, Hamburg, Germany). RNA was isolated from macrophages using the 5PRIME PerfectPure RNA 96 cell CS kit and quantitative real-time RT–PCR was performed to analyze expression of the M1 macrophage marker, IL-1β, normalized to expression levels of GAPDH. Each point represents data from an individual animal. A one-way analysis of variance with a Bonferroni post test (a, f) or a Student's t-test (g) were used to determine statistical significance among groups.

MMP7 has been shown to induce distinct transcriptional programs in host cells19. Therefore, we next sought to determine whether gastric macrophages harvested from mice that lacked MMP7 would preferentially induce M1-polarized transcriptional programs in response to H. pylori. To test this ex vivo, F4/80-positive cells were isolated from the stomachs of H. pylori-infected WT and MMP7−/− C57BL/6 mice by magnet-assisted cell sorting, and expression of IL-1β was determined by quantitative real-time RT–PCR (PCR with reverse transcription). Levels of IL-1β mRNA were significantly higher in gastric macrophages isolated from infected MMP7−/− C57BL/6 mice compared with macrophages isolated from H. pylori-infected WT C57BL/6 mice (Figure 3g), consistent with increased expression levels of IL-1β in gastric tissue from MMP7−/− C57BL/6 mice (Figure 2a). Furthermore, naïve bone marrow-derived macrophages isolated from MMP7−/− C57BL/6 mice expressed significantly higher levels of IL-1β and iNOS mRNA, classical proinflammatory M1 markers, following infection with H. pylori compared with macrophages isolated from WT C57BL/6 mice (Supplementary Figures 1A and B). In contrast, mRNA expression levels of the M2 markers YM1, Arg1, IL-12 or the Mreg markers LIGHT, TGF-β, IL-10 were no different between these groups (Supplementary Figures 1C–H).

To directly implicate MMP7 in regulation of production of the M1 marker IL-1β in response to H. pylori, murine macrophage RAW 264.7 cells treated with nontargeting or MMP7-targeting siRNA were cocultured with H. pylori strain PMSS1. The effectiveness of siRNA treatment on MMP7 mRNA expression was assessed by quantitative real-time RT–PCR. Treatment with specific MMP7-targeting siRNA resulted in a significant decrease in MMP7 expression as compared with treatment with the nontargeting control, and treatment with MMP7-targeting siRNA significantly inhibited H. pylori-induced expression of MMP7 in macrophages (Supplementary Figure 2A). To determine whether reductions in MMP7 resulted in altered iNOS or IL-1β expression in response to H. pylori infection, quantitative real-time RT–PCR for iNOS or IL-1β mRNA levels and ELISA (enzyme-linked immunosorbent assay) for IL-1β protein levels were used. Consistent with our in vivo and ex vivo findings, iNOS mRNA levels were significantly increased following H. pylori infection and were further increased with siRNA-mediated reductions in MMP7 (Supplementary Figure 2B). Furthermore, siRNA-mediated reductions in MMP7 expression resulted in a significant increase in IL-1β mRNA and protein expression (Supplementary Figures 2C and D) following H. pylori infection. Furthermore, overall these data indicate that MMP7 has a critical role in H. pylori-induced expression of iNOS and the proinflammatory cytokine, IL-1β.

Host genetic differences can influence disease outcome in mouse models of H. pylori infection;20 therefore, we next sought to determine whether the increased inflammatory phenotype induced by the loss of MMP7 in C57BL/6 mice could be recapitulated in H. pylori-infected mice on a different genetic background that are predisposed to gastric cancer. Unlike C57BL/6 mice, which do not develop cancer before 15 months post H. pylori infection,5 hypergastrinemic INS-GAS mice on an FVB/N background rapidly develop pre-neoplastic lesions as early as 6 weeks and gastric cancer as early as 24 weeks post H. pylori challenge21. WT INS-GAS and MMP7−/− INS-GAS mice on a FVB/N background were generated, genotyped and challenged with broth as an uninfected control or H. pylori strain PMSS1, and stomachs were harvested and analyzed 12 weeks post challenge. Colonization efficiency was 100% for all H. pylori-challenged mice and, in contrast to infected C57BL/6 mice, bacterial burden was similar among infected WT INS-GAS and MMP7−/− INS-GAS mice (data not shown). Consistent with our findings in the C57BL/6 model, H. pylori-infected MMP7−/− INS-GAS exhibited significantly higher levels of gastric inflammation compared with uninfected mice or infected WT INS-GAS mice (Figure 4a). As expected, both H. pylori-infected WT INS-GAS and MMP7−/− INS-GAS mice developed gastric hyperplasia; however, loss of MMP7 resulted in significantly higher levels of gastric hyperplasia among infected mice (Figure 4b). In contrast to C57BL/6 mice, H. pylori-infected WT INS-GAS and MMP7−/− INS-GAS mice developed gastric dysplasia (Figures 4c and d). Consistent with the pattern observed in C57BL/6 mice and with increased levels of gastric inflammation and hyperplasia observed in H. pylori-infected MMP7−/− INS-GAS mice, loss of MMP7 resulted in an increased incidence of gastric dysplasia (78% versus 20%, Figure 4d).

Figure 4.

Figure 4

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Loss of MMP7 augments the development of pre-neoplastic lesions in H. pylori-infected INS-GAS mice. Eight-week old WT INS-GAS (N = 15) and MMP7−/− INS-GAS (N = 17) mice were challenged with broth alone as an uninfected control or with H. pylori strain PMSS1. Twelve weeks post challenge, mice were euthanized and stomachs harvested. Linear strips of gastric tissue, extending from the squamocolumnar junction through the proximal duodenum, were fixed in 10% neutral-buffered formalin, embedded in paraffin and stained with hematoxylin and eosin. (a) A pathologist, (MBP), masked to the treatment groups, assessed indices of inflammation. Severity of acute and chronic inflammation was graded 0–3, in both the gastric antrum and corpus, for a cumulative score ranging from 0–12. (b) Gastric tissues were scored for hyperplasia on a scale of 0–3 (absent, mild, moderate or marked) in both the antrum and corpus, for a cumulative score ranging from 0–6. (c) Representative histologic image showing the presence of gastric dysplasia in an H. pylori-infected MMP7−/− INS-GAS mouse at × 200 magnification. (d) The presence of gastric dysplasia (% of mice with dysplastic lesions) was determined in H. pylori-infected WT (N = 10) and MMP7−/− (N = 9) INS-GAS mice. (e, f) Five micrometers thick paraffin-embedded gastric tissue sections were mounted and deparaffinized. Endogenous peroxidases were quenched with 3% hydrogen peroxide, and slides were blocked with protein block serum-free solution before incubation with an F4/80 antibody (e) or a polyclonal rabbit anti-mouse-IL-1β antibody (f). A pathologist, (MBP), masked to the treatment groups, enumerated F4/80-postive cells (e) and IL-1β-positive immune cells (f) in 10 high-powered fields in the antrum and corpus. (g) Naïve bone marrow-derived macrophages were isolated from WT and MMP7−/− INS-GAS mice22,23. Macrophages were then cocultured ex vivo with H. pylori strain PMSS1 and IL-1β mRNA levels were determined by quantitative real-time RT–PCR and normalized to expression levels of GAPDH. Each point represents data from an individual animal. A one-way analysis of variance with a Bonferroni post test (a, b, e, f), Χ2 test (d), or a Student's t-test (g) were used to determine statistical significance among groups.

H. pylori-infected MMP7−/− INS-GAS mice also exhibited more robust macrophage infiltration into the gastric musoca compared with H. pylori-infected WT INS-GAS mice (Figure 4e), which was coupled with a significant increase in the number of infiltrating IL-1β-positive immune cells (Figure 4f). We next sought to determine whether macrophages harvested from INS-GAS mice lacking MMP7 preferentially induced an M1 macrophage polarized transcription profile. Naïve bone marrow-derived macrophages isolated from WT INS-GAS and MMP7−/− INS-GAS mice were cocultured with H. pylori strain PMSS1 ex vivo and cell lysates were collected for analysis of IL-1β. Consistent with our findings in the C57BL/6 model, levels of IL-1β mRNA were significantly higher in ex vivo H. pylori-stimulated bone marrow-derived macrophages isolated from MMP7−/− INS-GAS mice compared with H. pylori-stimulated macrophages isolated from WT INS-GAS mice (Figure 4g). Furthermore, naïve bone marrow-derived macrophages isolated from MMP7−/− INS-GAS mice and then infected with H. pylori exhibited significantly higher levels of the M1 macrophage markers iNOS and TNF-α mRNA compared with ex vivo-infected macrophages isolated from WT INS-GAS mice (Supplementary Figures 3A and B). Although mRNA expression level of the M2 marker YM1 was no different between WT and MMP7−/− macrophages (Supplementary Figure 3C), expression levels of the M2 markers Arg1 and LIGHT were significantly decreased in MMP7−/− macrophages compared with WT macrophages (Supplementary Figures 3D and E).

In conclusion, our studies demonstrate that MMP7−/− mice on two different genetic backgrounds develop more severe gastric inflammation and injury in response to H. pylori infection and that this is likely mediated by M1 type macrophages, suggesting that MMP7 may function to suppress M1 macrophage polarization. Our findings also identify a putative mechanism through which high-expression alleles of IL-1β may increase gastric cancer risk in human populations. These results raise the testable hypothesis that MMP7 exerts an important role in limiting damage initiated by H. pylori, which may promote persistent infection over years to decades.

Supplementary Material

Supplementary Figure 1
Supplementary Figure 2
Supplementary Figure 3
Supplementary Figure Legends

Acknowledgments

This study was supported by T32CA009592 (MSK), F32CA153539 (JMN), T32GM008554 (DMH), K01AT007324 (RC), P01CA028842 (KTW and PC), R01DK053620 and 1I01BX001453 from the Department of Veterans Affairs (KTW), R01DK058587, R01CA077955, P30DK058404 and P01CA116087 (RMP).

Footnotes

Conflict of Interest: The authors declare no conflict of interest.

Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)

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Supplementary Materials

Supplementary Figure 1
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Supplementary Figure 2
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Supplementary Figure 3
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Supplementary Figure Legends
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