Objective Helicobacter pylori infection is the most prevalent bacterial infection worldwide. Besides being the most important risk factor for gastric cancer development, epidemiological data show that infected individuals harbour a nearly twofold increased risk to develop colorectal cancer (CRC). However, a direct causal and functional connection between H. pylori infection and colon cancer is lacking.
Design We infected two Apc-mutant mouse models and C57BL/6 mice with H. pylori and conducted a comprehensive analysis of H. pylori-induced changes in intestinal immune responses and epithelial signatures via flow cytometry, chip cytometry, immunohistochemistry and single cell RNA sequencing. Microbial signatures were characterised and evaluated in germ-free mice and via stool transfer experiments.
Results H. pylori infection accelerated tumour development in Apc-mutant mice. We identified a unique H. pylori-driven immune alteration signature characterised by a reduction in regulatory T cells and pro-inflammatory T cells. Furthermore, in the intestinal and colonic epithelium, H. pylori induced pro-carcinogenic STAT3 signalling and a loss of goblet cells, changes that have been shown to contribute—in combination with pro-inflammatory and mucus degrading microbial signatures—to tumour development. Similar immune and epithelial alterations were found in human colon biopsies from H. pylori-infected patients. Housing of Apc-mutant mice under germ-free conditions ameliorated, and early antibiotic eradication of H. pylori infection normalised the tumour incidence to the level of uninfected controls.
Conclusions Our studies provide evidence that H. pylori infection is a strong causal promoter of colorectal carcinogenesis. Therefore, implementation of H. pylori status into preventive measures of CRC should be considered.
- Helicobacter pylori
- colorectal cancer
- immune response
- colonic microflora
Data availability statement
Data are available in a public, open access repository. Data are available on reasonable request. Raw single cell RNA sequencing and 16S rRNA sequencing data have been deposited with links to BioProject accession number PRJNA808836 in the NCBI BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA808836/).
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WHAT IS ALREADY KNOWN ON THIS TOPIC
Helicobacter pylori infection is the most prevalent bacterial infection worldwide and is the most important risk factor for gastric cancer development.
Infected individuals harbour a nearly twofold increased risk to develop colorectal cancer (CRC).
WHAT THIS STUDY ADDS
H. pylori infection accelerates intestinal tumour development in Apc-mutant mice.
H. pylori infection induces a pro-inflammatory and pro-carcinogenic environment in murine and human colon.
The observed phenotype was normalised upon eradication therapy and is strongly dependent on microbiota.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
We provide evidence that H. pylori infection is a strong causal promoter of colorectal carcinogenesis and should be included into an adapted risk score for CRC.
Eradication of H. pylori infection might be an effective measure to reduce this risk.
Helicobacter pylori infection affects more than half of the world’s population and it is a main risk factor for gastric cancer. H. pylori induces a number of alterations in the gastric mucosa that together result in neoplastic transformation of the epithelium. Thus, H. pylori infection first triggers a complex plethora of immune cascades, directed towards H. pylori and orchestrated by the bacterium itself, which originate from priming at the Peyer’s patches and the mesenteric lymph nodes of the small intestine.1 2 The major pro-inflammatory response towards H. pylori consists of a mixed T helper (Th)1 and Th17 response,1 and is to a large extent related to the presence and activity of a type 4 secretion system,3 which mediates translocation of the oncogenic and highly immunogenic protein CagA into gastric epithelial cells.4 This leads to chronic inflammation and results in the activation of pro-inflammatory signalling pathways such as activating nuclear factor-κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) signalling, which are major drivers of H. pylori-induced gastric carcinogenesis.5 However, H. pylori has evolved counter mechanisms in order to establish and maintain chronic infection, for example, by reprogramming dendritic cells (DCs) to induce regulatory T cells (Treg),6 7 which counterbalance the local pro-inflammatory response in the stomach,8 and are involved in protection from allergic asthma.9 Interestingly, this tolerogenic reprogramming of DCs is partially mediated by CagA, via activation of STAT3.7 Finally, alterations in gastric microbiota are observed on infection, which seem to contribute to the deleterious events leading to gastric cancer following H. pylori infection.10 This idea is supported by studies using animal models as the insulin-gastrin (INS-GAS) mice, which showed more severe gastric pathology and early development of neoplasia when colonised with H. pylori and carrying normal commensal microbiota compared with germ-free INS-GAS mice infected with the bacterium.11
Although H. pylori infection is limited to the stomach, accumulating epidemiological data indicate an association between H. pylori infection and different extragastric diseases.12 Among those, a higher risk of colorectal cancer (CRC) has been reported to be associated with H. pylori infection status.13 However, the mechanisms that could explain this increased risk have not been elucidated.
In our study, we identify H. pylori-specific alterations in gut homeostasis that contribute to colorectal carcinogenesis in mouse models of CRC as well as in human samples, and are reversible on H. pylori eradication. These findings provide a basis for assessing H. pylori status for gastric, and for colon cancer prevention programmes.
H. pylori promotes intestinal carcinogenesis in adenomatous polyposis coli mouse models
To determine whether H. pylori infection promotes the development of tumours in the lower gastrointestinal (GI) tract, we infected Apc +/min and Apc +/1638N mice for different time periods (online supplemental figure 1A,B). Unexpectedly, Apc +/min mice were highly susceptible to the infection, with only 60% of the mice surviving after 12 weeks of H. pylori infection (figure 1A). An increased tumour burden in the small intestine and colon was observed in infected Apc +/min mice compared with uninfected controls (figure 1B,C). Similar results were observed in Apc +/1638N mice, which developed twice as many tumours after infection, and showed larger tumours in the small intestine (online supplemental figure 1C). Notably, in Apc +/1638N mice, colonic tumours were exclusively detected in H. pylori-infected mice (online supplemental figure 1C). These observations demonstrate that H. pylori infection promotes the development of intestinal and colonic tumours in tumour-prone mice, while exclusively infecting the stomachs of these mice (online supplemental figure 1B).
H. pylori infection induces a pro-inflammatory response in the intestine
Manipulation of host’s T-cell immune responses characterises H. pylori infection and is one of the main mechanisms contributing to gastric carcinogenesis. To assess whether alterations in intestinal immunity could be related to the increased tumour burden observed in infected Apc mutant mice, we first analysed lymphocyte infiltration in the small intestine of Apc +/min and Apc +/1638N mice upon infection (online supplemental figure 1A). Recruitment of intraepithelial CD3+ T cells to small intestine and colon was increased upon H. pylori infection (figure 2A and online supplemental figure 2A), wich was also confirmed by flow cytometric analysis of T cells (online supplemental figure 2B,C). Furthermore, this revealed a shift towards more CD8+ and less CD4+ T cells upon infection (online supplemental figure 2D). In addition, the abundance and protein level of Foxp3+ Treg cells was reduced in the small intestine from infected mice compared with uninfected controls, as detected by flow cytometry (figure 2B and online supplemental figure 2E).
To further explore the underlying mechanisms promoting intestinal tumourigenesis upon H. pylori infection independently from tumour-prone backgrounds, we infected wild-type C57BL/6 mice (WT) for 24 weeks and analysed immune responses (online supplemental figure 2F). An increased number of intraepithelial CD3+ T cells was also observed in the small intestine as well as in the colon of H. pylori-infected WT mice compared with uninfected controls (figure 2C). Contrasting the balanced immune phenotype observed in the stomach (figure 2C and online supplemental figure 2G), this was accompanied by a reduction in Foxp3+ Treg cells (figure 2D and online supplemental figure 2G). Multiplexed ChipCytometry corroborated an overall reduction of Treg cells, and additionally revealed their compartmentalisation within the lamina propria in infected colonic tissue (figure 2E).
We next confirmed the specificity of these T cells for H. pylori by restimulating lamina propria CD4+ T cells with H. pylori lysate and measuring the release of the pro-inflammatory cytokine IL-17A, which has been previously described to be one of the main players in the immune response to H. pylori.1 A specific IL-17A/CD4+ T cell response was observed in infected C57BL/6 and Apc +/min mice (online supplemental figure 2H).
To characterise in depth the specific intestinal immune response elicited by gastric H. pylori, we investigated the immune cell compartment on a single cell level by performing 10X single-cell RNA sequencing (scRNAseq) (dataset).14 We isolated and sorted single CD45+ immune cells of intestinal and colonic tissue from Apc +/min mice and littermate WT controls that had been infected for 12 weeks with H. pylori, and compared them with non-infected controls (online supplemental figure 2I).
Unsupervised clustering identified 16 clusters according to their transcriptional profiles, which are visualised using Uniform Manifold Approximation and Projection (UMAP)15 (figure 2F and online supplemental figure 2J) and were annotated based on known marker genes (online supplemental figure 2K).
To further characterise the Treg cell compartment, we subclustered and annotated Treg cells, which resulted in three subclusters: activated Treg cells (act. Tregs); peripherally derived Treg cells (pTregs), characterised by high RORγt expression and thymus-derived Treg cells (tTregs), characterised by GATA3 expression16 17 (figure 2G and online supplemental figure 2L). We then computed a Treg effector score18 (figure 2H and online supplemental figure 1), and found significantly increased Th17 differentiation genes in infected act. Tregs (figure 2H and online supplemental table 1), indicating that H. pylori infection reprogrammes Treg cells into potentially pathogenic Foxp3+ IL-17A+ T cells.
Finally, to understand cell dynamics of T cells in infected mice, we calculated RNA velocity vectors, which predict future states of individual cells based on ratios of spliced and unspliced messenger RNAs.19 In line with our previous findings, when looking at the CD4 clusters, it was apparent that less CD4 cells were projected towards CD4 Treg cells in infected Apc mice (online supplemental figure 2M).
In summary, our results show that H. pylori infection induces a H. pylori-specific pro-inflammatory immune response in the small intestine and colon of infected mice, that is characterised by loss of Treg cells and their differentiation into Foxp3+ IL-17A+ T cells.
Activation of carcinogenic signalling pathways and loss of goblet cells characterise the intestinal epithelial response to H. pylori infection
Considering the alterations induced by H. pylori in intestinal immune cells independently of adenomatous polyposis coli (APC) mutations in WT mice, we analysed the effect on signalling pathways putatively mediating the pro-carcinogenic effects of H. pylori infection in the epithelium. Therefore, we assessed transcriptomic profiles of EPCAM+ epithelial cells from Apc +/min mice in our scRNAseq data (online supplemental figure 2I) (dataset).14 Unsupervised clustering revealed 15 clusters according to their transcriptional profiles, which were visualised as UMAP (figure 3A and online supplemental figure 3A) and annotated based on known marker genes (online supplemental figure 3B).
Here, we specifically explored signalling pathways associated with CRC initiation and development, namely STAT3 and NF-κB. Notably, these pathways also orchestrate key inflammatory mechanisms in inflammation-driven colon cancer and have been extensively related to H. pylori infection.22–24 We computed a score of genes involved in the Jak-STAT signalling pathway, which revealed significantly higher scores in enterocytes of WT and Apc +/min mice upon H. pylori infection (figure 3B, online supplemental figure 3D and online supplemental table 1). Increased STAT3 signalling was only detected in stem cells of WT mice and not in Apc +/min mice upon H. pylori infection (online supplemental figure 3D), which can be linked to the binary role of STAT3 in tumourigenesis and a reduced availability of STAT3-inducing receptors during tumour progression, as we found a decreased expression of the IL-22 receptor Il22ra1 on stem cells of Apc mutant mice upon H. pylori infection, but not in infected wt mice or in enterocytes (online supplemental figure 3E). When assessing NF-κB signalling in enterocytes, higher scores were observed upon H. pylori infection (online supplemental figure 3F and online supplemental figure 1).
As it has been shown that activation of epithelial STAT3 favours recruitment of lymphocytes, while inhibiting infiltration of Treg cells in the colon,25 we confirmed hyperactivation of STAT3 in tissue samples from 24 weeks infected H. pylori WT (figure 3C) and Apc mutant mice (online supplemental figure 1A, figure 3D), which was accompanied by enhanced proliferation as detected by Ki67 staining (figure 3C).
Given that a functional intestinal barrier is depending on mucus replenishment by goblet cells, we next assessed their status by periodic acid-Schiff (PAS) staining. We observed reduced number of mucus producing cells in the small intestine and in the colon of H. pylori-infected WT (figure 3C) and Apc mutant mice (figure 3E) compared with uninfected controls.
To explore in depth the effects of H. pylori infection on the goblet cells, we clustered and annotated goblet cells in our scRNAseq dataset based on goblet cell differentiation markers.26 This revealed immature, characterised by high expression of Tff3; intermediate, highly expressing Oasis and terminal goblet cells, with high expression of Muc2 and Klf4 (online supplemental figure 3G,H). Maturational states were distinctly affected by H. pylori infection, with a switch to less differentiated goblet cells (online supplemental figure 3I). To assess goblet cell functionality, we assessed the expression of genes encoding for antimicrobial peptides Reg3b and Reg3g, which are known to play a role in response to pathogens and inflammation27 (online supplemental table 1). We found them to be reduced upon H. pylori infection (online supplemental figure 3J). As those genes are known to be downstream targets of STAT3, we checked for STAT3 signalling specifically in the goblet cell cluster, which was—in contrast to the increased STAT3 signalling in enterocytes and stem cells—not induced upon H. pylori infection (online supplemental figure 3K). These findings are consistent with a compromised intestinal barrier integrity induced by H. pylori infection, independent of APC status.
To explain the absolute loss of goblet cells we observed in infected mice, we studied cellular dynamics of goblet cells by means of RNA velocities. In the colon, we observed less directionality from the stem cell cluster towards the goblet cell cluster, and at the same time a higher projection towards the colonocyte cluster upon H. pylori infection (online supplemental figure 3J), in contrast to the small intestinal goblet cell cluster, where cell dynamics seem to be restricted to the goblet cell cluster itself. When assessing the expression of Atoh1, which is known to drive terminal differentiation into the secretory lineage,28 in stem cells of both small intestine and colon, a signficiantly lower expression was found upon H. pylori infection (online supplemental figure 3K). These findings indicate a skewed differentiation of stem cells rather into unspecialised colonozytes than into goblet cells.
Together, these results indicate that H. pylori induces carcinogenic signalling pathways and has a detrimental impact on mucus-producing goblet cells in the small intestine and colon of WT and Apc mutant mice.
H. pylori infection favours the presence of mucus-degrading microbiota and shapes a pro-inflammatory and pro-carcinogenic microbiota signature
Microbiota alterations and aberrant presence of certain bacterial species in the small intestine have been related to the development of CRC.29 This could be an additional mechanism by which H. pylori contributes to intestinal carcinogenesis, since H. pylori infection has been shown to alter microbiota signatures.30 Therefore, we assessed to which extent H. pylori infection influenced small intestinal and colonic microbial composition by performing 16S RNA sequencing (dataset).14 While we found significantly increased abundance of Helicobacter spp in the stomach of 24 weeks infected mice, we did not detect H. pylori in intestine and colon (online supplemental figure 4A). When comparing the microbiota in caecum and colon of infected and non-infected mice via taxonomic profiling, we observed apparent changes at phylum level upon H. pylori infection (figure 4A). Furthermore, we found signs of decreased α-diversity in small intestine upon H. pylori infection (online supplemental figure 4B) as well as significantly different β-diversity in caecum, stool and small intestine between non-infected and infected mice (online supplemental figure 4C). Differential abundance testing revealed Akkermansia spp to be enriched in 24 weeks infected WT mice (online supplemental figure 4D, figure 4B). When exploring the data for further species sharing the mucus-degrading characteristics of Akkermansia spp, we found an increase in Ruminococcus spp (figure 4B and online supplemental figure 4D). The abundance of both species was also higher in Apc mutant mice (online supplemental figure 1A) upon H. pylori infection (figure 4C,D).
To determine the functional effects of H. pylori-induced microbiota signatures independent of mutant APC, we performed a stool transfer experiment from infected and non-infected specific pathogen-free (SPF) mice into germ-free WT mice (online supplemental figure 4E). This revealed an increased T-cell infiltration into intestinal and colonic epithelia (figure 4E and online supplemental figure 4F), a reduction of Foxp3+ Treg cells (figure 4F) and lower amounts of RORγt+ Treg cells (figure 4G), which are known to be microbiota-induced,31 in WT stool recipients from H. pylori-infected mice. Furthermore, we observed enhanced STAT3 signalling in intestines of WT mice that received stool from H. pylori-infected mice (figure 4H and online supplemental figure 4G). In order to ultimately assess the contribution of H. pylori-induced changes in microbiota to intestinal carcinogenesis, we performed a further stool transfer experiment from SPF non-infected and H. pylori-infected Apc +/1638N mice and WT littermates into germ-free Apc +/1638N mice (online supplemental figure 4H). Higher tumour numbers in stool recipients from H. pylori-infected mice were found, which was already evident in WT mice and further enhanced in an Apc +/1638N background (online supplemental figure 4I).
Together, H. pylori alters the microbiota of the lower GI tract, favours mucus-degrading microbiota in both WT and Apc mutant mice and induces a pro-inflammatory and pro-carcinogenic microbiota signature.
H. pylori-induced colorectal carcinogenesis is prevented by eradication
The interplay between a pro-inflammatory response and activation of pro-carcinogenic signalling, accompanied by alterations in microbiota characterised H. pylori-driven intestinal tumourigenesis. To dissect the contribution of inflammation in the absence of microbiota, we infected Apc +/1638N mice under germ-free conditions (online supplemental figure 5A,B). We observed similar immune alterations as in SPF mice, namely increased CD3+ T cell infiltration and reduction of Treg cells in small intestine and colon (figure 5A, online supplemental figure 5E,B). In contrast, germ-free mice barely showed activation of STAT3 signalling, and no reduction of goblet cells upon H. pylori infection (online supplemental figure 5C–E). Importantly, we did not observe significant differences in tumour number between control and H. pylori-infected germ-free Apc +/1638N mice (figure 5C), which, in combination with our findings from stool transfer experiments (figure 4 and online supplemental figure S4), indicates a strong contribution of H. pylori-induced changes in microbiota to the tumour phenotype, and suggests, that H. pylori-induced carcinogenesis in the small intestine and colon is a multifactorial process involving the interplay of the pro-inflammatory immune response, alterations in microbiota and pro-carcinogenic signalling. Therefore, we next sought to determine whether eradication of H. pylori infection could abrogate the carcinogenic process, by treating the mice with a triple therapy regimen consisting of clarithromycin, metronidazole and omeprazole,32 reflecting the ‘Italian triple therapy’ regimen also used in infected patients to eradicate H. pylori (figure 5D and online supplemental figure 5F). Importantly, we found that after antibiotic eradication, tumour burden was at the same levels as in uninfected controls (figure 5E). To delineate that the underlying changes in the intestinal immune response were directly induced by H. pylori infection and independent of mutated Apc, we analysed the effect of H. pylori eradication on C57Bl/6 mice (figure 5D and online supplemental figure 5F), and found a lower CD3+ T cell infiltration in the stomach (online supplemental figure 5G), small intestine and colon (figure 5F, online supplemental figure 5E) compared with infected mice at 4 and 12 weeks posteradication. The percentage of Treg cells was initially reduced in eradicated mice 4 weeks after treatment, while 12 weeks post-treatment, the percentage of FoxP3+ T cells was observed to recover (figure 5G). A specific IL-17A/CD4+ T cell response was observed in infected mice, which was initially lost after eradication therapy, but then reappeared after longer recovery time (figure 5H), supporting the specificity of the response to H. pylori, based on the given antigen encounter and response also in eradicated mice. Importantly, the clearance of infection resulted in normalisation of STAT3 activation and the number of PAS-positive cells (figure 5I and online supplemental figure 5H), confirming that H. pylori is specifically responsible for these changes.
In summary, our results demonstrate that H. pylori directly enhances colon carcinogenesis by shaping intestinal and colonic immune responses and inducing profound changes in intestinal/colonic microbiota and epithelial homeostasis. Eradication of H. pylori infection prevents its tumour-promoting effects also in the colon, providing a possible additional strategy to reduce CRC burden.
H. pylori alters colonic homeostasis in human
Our mouse models showed that H. pylori affects intestinal and colonic homeostasis at different cellular and molecular levels, which can ultimately enhance tumour development. To determine whether these effects were also observed in humans, we analysed immune signatures in a cohort of 154 human colon tissue samples (online supplemental table 2). Based on immune responses and histology of the stomach, we stratified samples according to H. pylori status into currently (actively) infected and eradicated patients. We found that H. pylori-actively infected as well as eradicated individuals showed higher infiltration of CD3+ T cells in the colon compared with uninfected subjects (figure 6A). Using endoscopy-derived colon biopsies, we further characterised T cell responses by flow cytometry, which revealed tendencies towards more CD3+ T cells in the colonic mucosa of currently infected patients (online supplemental figure 6A). In contrast, CD4+ and CD8+ subsets were not affected by H. pylori status (online supplemental figure 6B). Notably, the number of FoxP3+ cells in the colonic mucosa was lowest in the currently infected group, whereas eradicated patients seem to level with negative controls (figure 6C). The overall loss of Tregs was confirmed via ChipCytometry (figure 6B), which additionally showed that intraepithelial localisation of Tregs is almost lost in colon samples from H. pylori-infected individuals (figure 6B and online supplemental figure 6C).
We next focused on the epithelial compartment and, in concordance with our findings in mice and our eradication experiments, found a higher number of pSTAT3-positive epithelial cells and a concomitant loss of mucus-producing cells in the colon of currently infected subjects, which was attenuated in eradicated patients (figure 6A).
Finally, we assessed microbial changes in stool of patients and found a difference in β-diversity between actively H. pylori-infected and H. pylori-negative patients (p=0.062), but not between H. pylori-eradicated and H. pylori-negative patients (p=0.552) (figure 6D). In contrast, we neither detected significant changes in α-diversity (online supplemental figure 6D) nor in Firmicutes-to-Bacteroidetes ratio (online supplemental figure 6E) between the three groups. Comparative microbiome profiling revealed Prevotellaceae and Peptostreptococcales, which have been associated with CRC, to be differentially abundant in H. pylori-positive patients (online supplemental figure 6F,G).
These results confirm that the immune and epithelial signatures identified in mouse models upon H. pylori infection are also observed in humans, and are accompanied by changes in microbiota compositions, which can further contribute to colon carcinogenesis. Furthermore, the attenuated phenotype observed in H. pylori-eradicated patients further supports H. pylori status as an independent risk factor for CRC and simultaneously offers an option for CRC prevention for those patients at risk.
Although selectively colonising the stomach, chronic H. pylori infection is associated with several extragastric diseases.33 Epidemiological data indicate an association between H. pylori infection and a higher risk and aggressiveness of CRC, with an OR of 1.9,34 an OR higher than for most other known risk factors, such as smoking, alcohol and body mass index.34 However, these epidemiological data have not yet been confirmed experimentally, and a molecular mechanism by which H. pylori may promote CRC remained elusive. We employed Apc mutant mouse lines (Apc +/min and Apc +/1638N) as surrogate models for human CRC, and observed a nearly twofold increase in tumour numbers in mice infected with H. pylori, which coincides with the OR observed in epidemiological studies. Remarkably, this increase was observed in the small intestine, where both models usually show most tumours, and was especially evident in the colon. This prompted us to decipher the potential mechanisms driving H. pylori-induced carcinogenesis in the small intestine and colon.
The effects of H. pylori infection on other organs are best understood for the lung, where chronic H. pylori infection imposes a regulatory immune signature that protects from asthma disease.35 In contrast to these observations, we observed an H. pylori antigen-specific pro-inflammatory Th17-mediated response in the small intestine and colon, which was not balanced by an increase in Treg cells, as it occurs in the stomach or lung. The immune response mounted towards H. pylori originates from Peyer’s patches in the gut,2 which may explain why H. pylori-specific T cells also homed to intestinal and colonic mucosal sites. Interestingly, IL-17 was found to be increased in H. pylori-positive patients with gastritis and gastric cancer,36 and in CRC, where Th17 signatures, including RORC, IL17, IL23 and STAT3, were linked to poorer prognosis.37 Still, it was surprising to observe a loss of intestinal Treg cells, which also contrasts the balanced immune response usually observed in the stomach upon H. pylori infection. Additionally, we found that in the lower GI tract, Tregs were reprogrammed to upregulate Th17 differentiation markers. Murine and human studies have demonstrated that Treg cells can be reprogrammed to a distinct population, Foxp3+/IL17+ T cells, phenotypically and functionally resembling Th17 cells.38 Particularly in CRC, the presence of Foxp3+/IL17+ T cells has been reported to be increased in the mucosa and peripheral blood of patients with chronic colitis as well as in colorectal tumours.39 Foxp3+/IL17+ cells were shown to promote the development of tumour-initiating cells by increasing the expression of several CRC-associated markers such as CD44 and epithelial cell adhesion molecule EPCAM in bone marrow-derived mononuclear cells.40 Thus, the pro-inflammatory Th17 response elicited by H. pylori, especially the differentiation of Treg cells to a Th17 phenotype, may constitute one of the major mechanisms enhancing tumour development. This is in line with literature showing that altered T cell homeostasis is a key event during colorectal carcinogenesis, driving tumour development and progression, and determining treatment response of patients with CRC.41
Mutations in the gene encoding APC are the most frequent driver mutations leading to sporadic CRC, together with mutations in TP53 and KRAS.42 43 Pro-inflammatory and proliferative signalling pathways such as STAT3, NF-κB and WNT signalling, activated by signals derived from epithelial and immune cells, drive chronic inflammation, a known mechanism contributing to CRC.24 44 CRC risk is markedly increased in patients with chronic inflammatory bowel disease, with the risk rising with the duration of disease, from 8.3% after 20 years, to 18.4% after 30 years.45 Mechanistically, besides immune signalling by Th17 cells, activation of pro-inflammatory signalling pathways as well as altered microbiota, contribute to the pathogenesis of colitis-associated cancer.46
The strong pro-inflammatory response induced locally by H. pylori in the small intestine was accompanied by the activation of NF-κB and STAT3 pathways. Activation of STAT3 signalling has been strongly related to tumour initiation and development and progression, while levels of activated STAT3 in the tissue correlate with tumour invasion, tumour, node, metastases stage and reduced overall survival of patients with CRC.47 In addition, the activation of epithelial STAT3 was reported to downregulate the expression of chemokines important for the recruitment of Treg cells in the intestine.25 Therfore, it is tempting to speculate that during H. pylori infection, activation of STAT3 in intestinal and colonic epithelial cells contributes to loss of Treg recruitment, thereby supporting malignant transformation of the intestinal tissue. This central role for STAT3 during carcinogenesis in the intestine is supported by the fact that depletion of STAT3 in Apc +/min mice led to a reduction in the incidence of early adenomas.48 Furthermore, in Apc +/min mice, tumour progression and metastasis were characterised by loss of STAT3 signalling in stem cells due to lower IL-22-receptor expression.48 49 We observed similar characteristics in our H. pylori-infected mice, indicative of more advanced tumourigenesis.
Importantly, we observed a reduction and normalisation of STAT3 levels after eradication of H. pylori, which resulted in a normalisation of tumour load. Notably, for eradication, a treatment regimen also applied in humans was used in order to be able to translate the results to humans.
The function of STAT3 as oncogene or tumour suppressor seems to be determined by the milieu eliciting its activation as well as the local gut microbiota,50 which is increasingly recognised as an important regulator of colonic cancer development.51 Interestingly, microbial induction of IL-17A production has been shown to endorse colon cancer initiation and progression in Apc +/min mice, which was mediated via STAT3 signalling.52 We thus hypothesised that alterations in microbiota compositions in the intestine and colon induced by H. pylori may also contribute to carcinogenesis.30 53 Indeed, when housing mice under germ-free conditions, activation of STAT3 and tumour development were lower upon H. pylori infection, but not completely normalised, indicating that microbiota alterations are involved in the phenotype but not exclusively responsible.
Such disturbances in gut microbiota communities have been shown to contribute to CRC development and progression.29 H. pylori is known to affect local gastric microbiota, and distant microbial populations in intestine and colon.30 54 It has been shown that inflammation-driven dysregulation of microbiota can promote colorectal tumour formation and progression55 and that in response to bacterial stimuli or pathogen-associated molecular receptors, pro-inflammatory pathways such as c-Jun/JNK and STAT3 signalling pathways are activated and accelerate intestinal tumour growth in Apc +/min mice.50 This was supported by our findings from transferring stool of H. pylori-infected SPF mice into germ-free mice, which led to lower abundance of FoxP3+ Treg cells and microbiota-induced (FoxP3+RORγt+) Treg cells as well as STAT3 induction in recipients of stool from H. pylori-infected mice, and eventually an accelerated tumour development in comparison to stool transferred from non-infected mice. Together, these data indicate that H. pylori-induced pro-inflammatory and pro-carcinogenic microbial signatures are involved in and indispensable to promote intestinal tumour growth.
Our data revealed a distinct mucus-degrading microbiota signature associated with H. pylori infection in mice, namely enrichment with Akkermansia spp and Ruminococcus spp, while in human samples from H. pylori-infected patients, bacterial taxa associated with CRC, Prevotellaceae and Peptostreptococcales, were found.29 Although some studies established an inverse correlation between the presence of Akkermansia and GI diseases,56 Akkermansia has been reported to be increased in patients with CRC most likely due to the overexpression of certain mucins in the tumours.57 Notably, we also observed a general loss of goblet cells, which are important to produce mucins and antimicrobial peptides. This loss of goblet cells was also observed in clinical samples from H. pylori-infected patients undergoing colonoscopy. Thus, H. pylori infection disrupts, by two distinct mechanisms, intestinal mucus integrity essential to maintain a healthy barrier to impair bacterial penetration. In the absence of a sufficient regulatory T cell response—as observed here—which normally keeps inflammatory signals at bay, the carefully balanced homeostasis maintained in the gut by the interplay of a ‘healthy’ microbiome and an intact mucosa then fails to balance the pro-inflammatory signature elicited by H. pylori infection, enabling carcinogenesis. Eradication of H. pylori restored intestinal homeostasis with reappearance of goblet cells and normalised the intestinal immune signature, which then completely abrogated the tumour-promoting effect.
Importantly, when analysing colonic biopsies from H. pylori-infected patients, we could observe the very same alterations as seen in mice, with activation of pro-carcinogenic signalling pathways and a significant reduction in Treg cells, and an increase of CD3+ cells. The attenuated phenotype in eradicated patients highlight the clinical relevance of our findings and indicate that H. pylori infection is more than a mere risk factor for colon carcinogenesis, but actively promotes a pro-carcinogenic niche in the colon that may be prevented by eradication of H. pylori, which therefore could decrease the risk of CRC development in infected individuals. However, studies showing a correlation between H. pylori infection and CRC did not address the effect of antibiotic therapy. The inclusion of such cohorts in future studies is important to determine the impact of H. pylori eradication in CRC development.
In summary, our study provides solid experimental evidence that H. pylori infection accelerates intestinal and colonic tumour development, and offers insight into the underlying mechanisms. We suggest H. pylori screening and eradication as a potential measure for CRC prevention strategies.
Data availability statement
Data are available in a public, open access repository. Data are available on reasonable request. Raw single cell RNA sequencing and 16S rRNA sequencing data have been deposited with links to BioProject accession number PRJNA808836 in the NCBI BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA808836/).
Patient consent for publication
This study was approved by Klinikum rechts der Isar #322/18Klinikum Bayreuth #241_20Bc. Participants gave informed consent to participate in the study before taking part.
We thank members of the laboratory ‘Chronic inflammation and carcinogenesis’ for experimental help as well as critical discussion, with special thanks to Maximilian Koch, Karin Taxauer, Martin Skerhut and Teresa Burrell for experimental support. We thank Julia Horstmann and the team of the ColoBAC study at the Klinik und Poliklinik für Innere Medizin II, Klinikum rechts der Isar, for the supply of human biopsies. We thank Core Facility Microbiome of the ZIEL Institute for Food & Health, Technical University of Munich, for 16S rRNA sequencing services, as well as Dharmesh Singh and Nyssa Cullin. We thank Core Facility Gnotobiology of the ZIEL Institute for Food & Health, Technical University of Munich, for germ-free mice.
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
RM-L and MG contributed equally.
Contributors AR, RM-L and MG conceived the study. AR, AD and RM-L designed and analysed experiments. SJ contributed and provided code and support to single cell RNA sequencing and chip cytometry. VE and AW contributed to experiments. SJ and DHB provided methodological expertise. AD, SJ, RM-L and DHB contributed to data interpretation. MV, MM and MQ provided human biopsies. KPJ provided mouse models and critically revised the article. DH, DHB and LD critically revised the article. AR and RM-L wrote the article. AR, RM-L and MG revised the article. MG acquired the funding and is the guarantor of the article. All authors read and reviewed the article.
Funding This work was funded by the Deutsche Forschungsgemeinschaft (DFG (German Research Foundation)) SFB1371/1-395357507 (project P09 and project P04).
Competing interests None declared.
Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.
Provenance and peer review Not commissioned; externally peer reviewed.
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