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Reprint requests Address requests for reprints to: Prof Dr Thomas F. Meyer, Department of Molecular Biology, Max Planck Institute for Infection Biology, Charitéplatz 1, 10117 Berlin, Germany. fax: +49 30 28 460 401.
Despite inducing an inflammatory response, Helicobacter pylori can persist in the gastric mucosa for decades. H pylori expression of cholesterol-α-glucosyltransferase (encoded by cgt) is required for gastric colonization and T-cell activation. We investigated how cgt affects gastric epithelial cells and the host immune response.
MKN45 gastric epithelial cells, AGS cells, and human primary gastric epithelial cells (obtained from patients undergoing gastrectomy or sleeve resection or gastric antral organoids) were incubated with interferon gamma (IFNG) or interferon beta (IFNB) and exposed to H pylori, including cagPAI and cgt mutant strains. Some cells were incubated with methyl-β-cyclodextrin (to deplete cholesterol from membranes) or myriocin and zaragozic acid to prevent biosynthesis of sphingolipids and cholesterol and analyzed by immunoblot, immunofluorescence, and reverse transcription quantitative polymerase chain reaction analyses. We compared gene expression patterns among primary human gastric cells, uninfected or infected with H pylori P12 wt or P12Δcgt, using microarray analysis. Mice with disruption of the IFNG receptor 1 (Ifngr1–/– mice) and C57BL6 (control) mice were infected with PMSS1 (wild-type) or PMSS1Δcgt H pylori; gastric tissues were collected and analyzed by reverse transcription quantitative polymerase chain reaction or confocal microscopy.
In primary gastric cells and cell lines, infection with H pylori, but not cgt mutants, blocked IFNG-induced signaling via JAK and STAT. Cells infected with H pylori were depleted of cholesterol, which reduced IFNG signaling by disrupting lipid rafts, leading to reduced phosphorylation (activation) of JAK and STAT1. H pylori infection of cells also blocked signaling by IFNB, interleukin 6 (IL6), and IL22 and reduced activation of genes regulated by these signaling pathways, including cytokines that regulate T-cell function (MIG and IP10) and anti-microbial peptides such as human β-defensin 3 (hBD3). We found that this mechanism allows H pylori to persist in proximity to infected cells while inducing inflammation only in the neighboring, non-infected epithelium. Stomach tissues from mice infected with PMSS1 had increased levels of IFNG, but did not express higher levels of interferon-response genes. Expression of the IFNG-response gene IRF1 was substantially higher in PMSS1Δcgt-infected mice than PMSS1-infected mice. Ifngr1–/– mice were colonized by PMSS1 to a greater extent than control mice.
H pylori expression of cgt reduces cholesterol levels in infected gastric epithelial cells and thereby blocks IFNG signaling, allowing the bacteria to escape the host inflammatory response. These findings provide insight into the mechanisms by which H pylori might promote gastric carcinogenesis (persisting despite constant inflammation) and ineffectiveness of T-cell–based vaccines against H pylori.
For reasons not well-understood, Helicobacter pylori can persist life-long in the stomach despite causing a strong inflammatory response, increasing the risk for serious sequelae such as ulcers and cancer.
By extracting cholesterol from host cells, H pylori blocks the assembly of IFN and other cytokine receptors, rendering the infected mucosa unable to respond properly to inflammatory signals from stroma and immune cells.
Bacterial mutants for the cgt gene, which is required for cholesterol extraction, are unable to establish an in vivo infection, preventing a detailed analysis of its effects in the animal model.
This study provides important insight into the pitfalls of past vaccination approaches for H pylori. It also explains how cholesterol rich diet could negatively influence the inflammatory condition of the infected stomach mucosa.
About half the world’s population is chronically infected with the gram-negative bacterium Helicobacter pylori, which is implicated in severe gastric disease, including peptic ulcer and adenocarcinoma.
Yet, H pylori is able to escape full elimination by host immunity through an unknown mechanism, resulting in a severe chronic inflammatory condition that represents a crucial aspect of its pathogenesis.
Gastric epithelial cells display receptors for type I (α/β) and type II (γ) interferons, the subunits of which (IFNAR1/IFNAR2 and IFNGR1/IFNGR2, respectively) are assembled in specialized cholesterol-rich membrane microdomains, known as lipid rafts.
triggers JAK (Janus Kinase) and STAT (signaling transducer and activator of transcription) signaling via STAT1/2 phosphorylation and nuclear translocation to promote downstream expression of genes involved in inflammation and defense, including interferon regulatory factors (IRF), which further amplify the IFN response through positive feedback via STATs.
The effector mechanisms controlling colonization by H pylori remain sparsely understood. Epithelial cells can produce antimicrobial peptides (AMP), such as hBD3 (human β-defensin 3), which effectively kills H pylori
However, how hBD3 remains blocked despite IFNG and IL22 stimulation is unclear.
H pylori, which is auxotroph for cholesterol, extracts the lipid from host membranes to incorporate it into its outer membrane as an α-glucosylated derivative, using the enzyme Cgt encoded by the gene HP0421 (cgt).
Here, we provide insight into the underlying mechanism by showing that the IFN response is subverted by H pylori in gastric epithelial cells. This is caused by Cgt-dependent cholesterol depletion, resulting in the destruction of lipid rafts, failure of IFN receptor subunit assembly and, ultimately, lack of downstream signaling. Similarly, Cgt blocks IL6 and IL22 signaling. Our data provide evidence for the highly effective destruction of the responsiveness of H pylori-infected epithelium, even in the presence of strong cytokine signals from the adjacent micro-environment, thus impairing mucosal defense.
Materials and Methods
Human gastric tissue specimens were obtained from individuals undergoing gastrectomy or sleeve resection, under the ethics approval by the Charité Ethics Committee (EA1/058/11 and EA1/129/12). Animal experiments were performed in mice maintained under pathogen-free conditions based on approval by the Ethics Committee for Animal Experimentation of the State of Berlin (G0205/12).
Cell Culture Infection and Treatment
Bacteria were collected from plates and resuspended in RPMI 1640 (serum-free). All cells were grown to 60% confluency, washed twice with phosphate-buffered saline, and serum starved with RPMI 1640 for 16 hours prior to experiments. Infection was carried out under serum-starved conditions at 37 °C, 5% CO2 for the indicated times at multiplicity of infection (MOI) 20 or 50. Treatment with methyl-β-cyclodextrin (mβCD; Sigma, St. Louis, MO) was carried out for 5 hours at indicated concentrations. Biosynthesis of sphingolipids and cholesterol was inhibited by treating cells with myriocin (50 μmol/L; Sigma) for 72 hours and zaragozic acid (50 μmol/L, Cayman Chemical: Ann Arbor, MI) for the last 18 hours. In selected experiments, bacteria were treated with water-soluble cholesterol (Sigma) at 1 mg/mL for 1 hour prior to infection. Alternatively, polyethylene glycol (PEG)-cholesterol (10 mg/mL; Sigma) was added to cultures during the last hour of infection, while mock-infected cells were treated with an equal volume containing cholesterol. After infection, recombinant human IFNG (R&D Systems: Minneapolis, MN; 10 ng/mL), IFNB (PBL Assay Science, Piscataway, NJ; 2300 U/mL), IL6 (Peprotech, Rocky Hill, NJ; 25 ng/mL) or IL22 (Peprotech; 50 ng/mL) were added to selected wells and maintained for indicated times until the end of the experiment.
Cells from organoids or freshly isolated glands were seeded in trans-well inserts (Millipore (Merck): Darmstadt, GE; PIHP01250) in 24-well plates and wells filled with 400 μL primary cell culture medium. Once cells had formed a confluent monolayer, medium on top of the cells was removed to start air-liquid interface (ALI) culture. Cultures were kept at 37°C, 5% CO2 in a humidified incubator. Ten days later cells were infected by placing 50 μL of bacteria in phosphate-buffered saline (MOI 100) on top of the filters for 3 days.
Microarray data have been deposited in the Gene Expression Omnibus (GEO; www.ncbi.nlm.nih.gov/geo/) of the National Center for Biotechnology Information under GEO accession number GSE76589.
H pylori Blocks JAK/STAT Signaling upon IFNG Treatment
To assess the influence of H pylori on the response to IFNG, we infected MKN45 gastric epithelial cells with strains P12 and P1 for 6 or 24 hours, followed by treatment with IFNG for 30 minutes. Immunoblotting revealed STAT1 phosphorylation in response to IFNG treatment in non-infected cells, cells infected with heat-killed bacteria, and cells infected for 6 hours (Figure 1A). Surprisingly, after prolonged infection (24 hours) IFNG stimulation failed to activate STAT1, irrespective of the H pylori strain (Figure 1A). To analyze the dynamics of this phenotype, we performed a time course experiment, showing that STAT1 phosphorylation was diminished after 16 hours and completely blocked from 24 until 96 hours of infection with P12 (Supplementary Figure 1A). To investigate the underlying mechanisms, we thus chose the 24 hour time point of infection as a reference. In AGS cells, too, 24 hours of infection inhibited the response to IFNG (Supplementary Figure 1B). MOI 10 was sufficient to partially block IFNG signaling in MKN45 cells within 24 hours and a complete block was observed with MOI 50 (Supplementary Figure 1C), which was therefore chosen for further experiments.
Upon IFNG stimulation, an activated IFNGR complex phosphorylates JAK1 and JAK2 kinases, which in turn phosphorylate STAT1. After 24 hours of infection with P12 or P1, neither JAK1 nor JAK2 was phosphorylated any more in MKN45 cells (Figure 1B and 1C, middle panel; Supplementary Figure 1D). Notably, JAK1 (Figure 1B) and JAK2 (data not shown) were activated in non-infected MKN45 cells even without IFNG stimulation; however, this was also diminished after infection. Infection conditions did not compromise cell viability (Supplementary Figure 1E).
Cgt is Required for Inactivation of IFNG-JAK/STAT1 Pathway
To identify the bacterial factor involved in the block of IFNG signaling, we infected MKN45 cells with H pylori wild type and mutant strains. As a recent report linked CagA translocation to STAT1 dephosphorylation via SHP-2 activation,
we tested deletion mutants for cagPAI, which encodes the entire type IV secretion system, cagA and cgt. While the cagA and cagPAI mutant strains still inhibited JAK/STAT1 signaling upon IFNG stimulation, the cgt mutant did not (Figure 1D and Supplementary Figure 1B). Immunofluorescence analysis showed that P12Δcgt (Figure 1C and Supplementary Figure 1D) did not block JAK2 activation upon IFNG treatment, despite adhering to epithelial cells at levels comparable to wild type. Similarly, STAT1 signaling upon IFNG treatment was still inhibited in a Cgt-dependent manner at 48 h.p.i. (Supplementary Figure 1F). Although genetic complementation of the cgt-mutant strain only partially reconstituted Cgt expression (Supplementary Figure 1G), it was enough to restore the ability to impair JAK/STAT1 activation (Figure 1E) in MKN45 cells. Finally, we evaluated the consequences of disrupted JAK/STAT1 signaling by analyzing expression of downstream genes after IFN treatment, by infecting MKN45 cells for 24 hours with P12 wild type or P12Δcgt prior to treatment with IFNG for 2.5 or 5 hours, followed by reverse transcription–quantitative polymerase chain reaction (RT-qPCR). Expression of IRF1, as well as the T-cell attractant chemokines MIG and IFNG-inducible protein 10 (IP)-10 (encoded by CXCL9 and CXCL10 genes, respectively), was strongly up-regulated in non-infected cells upon IFNG treatment (Supplementary Figure 1H). Cells infected with P12Δcgt also responded to IFNG, but after infection with wt P12 the response was significantly reduced. Accordingly, Cgt activity blocks the JAK/STAT1 pathway, as well as transcription of downstream genes involved in amplification of the IFNG response.
Inhibition of IFNG Response is Linked to Host Cholesterol Depletion, Lipid Raft Disruption, and IFNGR Assembly
Cholesterol acquisition by H pylori has been linked to the destruction of lipid rafts in epithelial cells.
we observed cholesterol depletion in cells infected with wild-type H pylori (Supplementary Figure 2A). To connect this to the block of the IFNG response, we treated cells with mβCD, which depletes cholesterol from eukaryotic membranes. Treatment did not affect cellular viability (Supplementary Figure 2B) but impaired both JAK1 and STAT1 phosphorylation (Figure 2A). We also inhibited the biosynthesis of sphingolipids and cholesterol by combined treatment with myriocin and zaragozic acid to disrupt lipid raft function.
Myriocin and zaragozic acid treatment indeed significantly blocked the induction of IRF1 expression in response to IFNG (Supplementary Figure 2C). In addition, when we coated H pylori with water-soluble cholesterol before infection to abolish cholesterol transfer from host cells, they did not block the cellular response to IFNG treatment (Figure 2B). Cholesterol coating also restored the capacity of wt H pylori-infected cells to up-regulate IRF1 in response to IFNG, at levels comparable to non-infected cells (Supplementary Figure 2D). Cholesterol coating did not affect bacterial viability (Supplementary Figure 2E). To control for any effects on the initial host-pathogen interplay (eg, by masking bacterial adhesins), we repeated the experiment by adding PEG-cholesterol to the medium during the last hour of infection. This partially rescued the capacity of infected cells to respond to IFNG treatment, further highlighting the importance of cholesterol as a mediator of inflammation in response to H pylori (Figure 2C).
The IFNGR subunits 1 and 2 need to merge at cholesterol-rich micro-domains
We investigated the impact of H pylori on the integrity of lipid rafts by assessing potential alterations in the assembly of functional IFNGR. According to immunofluorescence analysis, surface accumulation of glycosphingolipid ganglioside GM1, a constituent marker of lipid rafts, is lost after infection with P12 wt and P12ΔcagA (Figure 2D, quantification of the relative membrane GM1 signal in Supplementary Figure 2F). In contrast, infection with P12Δcgt did not notably alter GM1 distribution (Figure 2D and Supplementary Figure 2F). In addition, we performed a membrane fractionation to separate lipid rafts as detergent-resistant membranes. In control cells, fractions containing cholesterol-rich micro-domains, identified by the presence of raft markers GM1 or caveolin, partitioned in top fractions (Supplementary Figure 2G). Combining these fractions for Western blot analysis showed that GM1 as well as IFNGR1 were lost in detergent-resistant membranes upon infection with wild-type P12 but not P12Δcgt, regardless of IFNG treatment (Figure 2E). In total lysates, IFNGR1 and IFNGR2 were detected in all conditions (Supplementary Figure 2H). Finally, we performed immunoprecipitation of IFNGR subunit 2 to test co-precipitation of IFNGR1. Immunoprecipitation specificity was controlled for by absence of the transferrin receptor CD71, a protein not associated with lipid rafts or the IFNGR complex (Supplementary Figure 2I). In line with recent findings,
MKN45 cells showed constitutive oligomerization of IFNGR subunits even in the absence of IFNG (Figure 2F). Despite this, prolonged wt H pylori infection but not P12Δcgt abolished assembly of receptor subunits. Coating H pylori with exogenous cholesterol prior to infection restored assembly of IFNGR subunits (Figure 2G). Together, these data suggest that subversion of JAK/STAT1 signaling by H pylori takes place at the very top of the pathway, by preventing assembly of IFNG receptor subunits through lipid raft destruction.
H pylori Blocks the IFNG Response In Vivo in a Cgt-Dependent Manner
Reportedly, H pylori cgt mutants are unable to colonize mice.
we investigated whether the IFNG response is linked to bacterial clearance. After 3 days of infection PMSS1Δcgt was already undetectable in wild-type mice (Figure 3A). Mice infected with PMSS1 or PMSS1Δcgt presented similar IFNG levels in the stomach; however, the induction of the IFNG downstream response gene IRF1 was substantially higher in PMSS1Δcgt-infected mice (Figure 3B). Next, we infected mice for 2 weeks with PMSS1 to allow a stronger Th1 response to develop. We observed a corpus-predominant gland occupation by H pylori in all analyzed mice (Figure 3C). Notably, the IFNG expression (Figure 3D, left) was higher compared with the less colonized antrum. However, IRF1 expression remained at similar levels (Figure 3D, right). Infection with wild-type H pylori thus leads to increased levels of IFNG, but fails to increase expression of interferon response genes, consistent with the notion of a Cgt-dependent block. Finally, although we reasoned that H pylori PMSS1Δcgt might be able to infect Ifngr1-knock out mice (Figure 3A, right), this was not the case. Knockout mice did show increased colonization by wild-type PMSS1, indicating that additional defense-related pathways apart from IFNG might contribute to the clearance of PMSS1Δcgt.
Cholesterol Depletion by H pylori Inhibits Type I IFN, IL6, and IL22 Signaling and Downstream hBD3 Response
Lipid raft integrity is also known to be required for type I IFNs and IL6 signaling.
In congruence, we observed that H pylori P12 also inhibited IFNB-induced STAT1 signaling in MKN45 cells, and this effect was dependent on the presence of cgt but not cagA or cagPAI (Figure 4A). Also, mβCD treatment inhibited IFNB signaling in a dose-dependent manner (Supplementary Figure 3A). Similarly, wild-type infection and mβCD treatment, but not infection with the cgt mutant, prevented STAT3 phosphorylation upon IL6 treatment (Figure 4B). IL22 is a crucial cytokine for mediating the epithelial defense against mucosal pathogens. Binding to receptors in epithelial cells triggers STAT3 activation, inducing expression of antimicrobial factors.
Again, 24-hour infection with wt P12 but not a P12Δcgt mutant strain inhibited IL22 signaling transduction in epithelial cells (Figure 4C); the block was also observed upon mβCD treatment (Figure 4C). Genetic rescue of the cgt mutant partially restored the bacterial capacity to block IL22 signaling (Supplementary Figure 3B). Because IL22 signaling has not previously been reported to depend on cholesterol, we demonstrated restoration of IL22 signaling by adding PEG-cholesterol to MKN45 cells before IL22 stimulation (Supplementary Figure 3C).
Because IFNG and IL22 are reported to induce epithelial expression of hBD3,
we tested the impact of infection on the cytokine-induced hBD3 response. Infection of MKN45 cells for 24 hours with P12Δcgt but not P12 wt, followed by treatment with IFNG for 5 hours, induced a significant increase of hBD3 transcription (Figure 4D, left), which was even more dramatic after 24-hour IFNG treatment (Figure 4D, right). In contrast, the increase over time in non-infected and P12 wt-infected cells was minimal. Similarly, IL22 treatment also induced hBD3 expression in P12Δcgt-infected cells, albeit to a lower extent, following a similar time course as IFNG (Figure 4E). In summary, H pylori blocks the production of hBD3 in epithelial cells stimulated with IFNG or IL22. Interestingly, co-stimulation with a cgt-deficient strain induced substantially higher hBD3 expression compared with cytokine treatment alone. Overall, these data confirm that cholesterol depletion by H pylori not only inhibits the response to IFNG, but also type I IFNs, IL6, and IL22.
Inhibition of JAK/STAT Signaling in Human Primary Gastric Epithelial Cells
to validate our observations during authentic host-pathogen interaction. Cells derived from primary gastric antral organoids were seeded on plastic and infected with H pylori under serum starvation before IFNG or IFNB treatment for 30 minutes. Similar to results obtained with MKN45, H pylori P12, but not P12Δcgt, prevented JAK1/STAT1 phosphorylation (Figure 5A). These differences were also observed at higher MOI and in cells isolated from the corpus region (Supplementary Figure 4A), independent of donors (Supplementary Figure 4B). In contrast to cancer cell lines, primary gastric epithelial cells did not exhibit constitutive JAK1 activation (Figure 5A). Block of IFNG signaling was observed at 24 hours post-infection, but not at earlier time points (Supplementary Figure 4C) or with a low MOI (Supplementary Figure 4D). Cholesterol-coated bacteria, however, failed to block JAK/STAT1 signaling (Figure 5B). Further, block of IFN signaling, as determined by the up-regulation of the type II IFN-activated gene CXCL9 in response to IFNG stimulation, was observed in H pylori P12 wt, P12ΔcagA, P12ΔcagPAI, or P12ΔvacA, but not in P12Δcgt-infected cells (Supplementary Figure 4E). Moreover, H pylori P12 wt, but not P12Δcgt, inhibited STAT3 phosphorylation upon IL22 treatment at 24 hours post-infection (Figure 5C). Genetic rescue of P12Δcgt restored its capacity to block the response to IFNG and IL22 in primary epithelial cells (Supplementary Figure 4F and G).
To assess the global cellular response to infection with H pylori P12 wt or P12Δcgt, we performed microarray analysis with human primary gastric cells infected with P12 wt or P12Δcgt. Gene Ontology term enrichment analysis revealed responsiveness for genes involved in ‘response to external stimuli,’ ‘signal transduction,’ and ‘immune response,’ which was similar for both wt and P12Δcgt-infected cells (Figure 5D, and data not shown). Many of the pro-inflammatory genes up-regulated upon infection with either strain are related to NF-κB signaling (Figure 5E). Therefore, while H pylori effectively blocks the response to IFNG, IFNB, and IL22 in normal human gastric epithelial cells, initial sensing of the pathogen by NF-κB
H pylori Generates Micro-Niches of Diminished Inflammatory Response
To spatially resolve the effects H pylori exerts, we examined the resulting cellular phenotypes over longer periods of time using a novel infection model of human gastric primary cells in air-liquid interphase culture.
This model enables longer infection times and resembles the in vivo situation more closely because it features greater cell type diversity, a protective layer of mucin, and cell polarization (Supplementary Figure 5A). After 3 days of infection with P12 wt at MOI 100, cells were treated with IFNG for 30 minutes and analyzed by immunofluorescence. Interestingly, IFNG treatment led to nuclear translocation of phospho-STAT1 (Figure 6A, middle panel). Micro-colonies of spiral-shaped bacteria (red) formed infection foci (Figure 6B and Supplementary Figures 5A and B) correlating with areas of reduced phospho-STAT1 exhibition (Figure 6A, bottom panel). At lower magnification, areas of reduced STAT1 activation appeared to clearly correlate with infected areas, while non-infected areas of the same filter responded normally to IFNG (Figure 6B). Next, we quantified the number of phospho-STAT1–positive cells in infected compared with non- or less well-infected microscopic fields of the same filter. The vast majority of infected areas displayed phospho-STAT1 levels below the activation threshold, while non-infected fields exhibited a robust IFNG response (Supplementary Figure 5C). Infection with the P12Δcgt mutant at MOI 200 led to similar colonization densities, yet the infected monolayer exhibited full STAT1 phosphorylation upon IFNG similar to non-infected cells (Supplementary Figure 5D). Similarly, H pylori infection also hampered STAT3 phosphorylation upon IL22 treatment in infected areas (Figure 6C). Thus, using an advanced epithelial cell culture model, we demonstrate that H pylori prevents infected cells from responding to IFNG or IL22. This favors the formation of micro-colonies at micro-niches devoid of STAT1 or STAT3 activation. Although non-infected sites of the same culture retain full responsiveness, this does not halt micro-colony formation at protected sites.
A hallmark of gastric infections with H pylori is the strong NF-κB–driven response of the epithelium. This initiates a chronic inflammatory condition fueled by the recruitment of immune cells, which produce IFNs
As we report here, H pylori evolved a powerful means to prevent infected epithelial cells from responding to this cytokine burst – shutting down bactericidal activity right at the site of infection. This intriguing phenomenon, exerted by the bacterial enzyme Cgt, is in line with previous observations that point to an unexplained inhibition of IFNG-induced nuclear translocation of STAT1 during H pylori infection.
However, IFNG and IL17 merely control, rather than clear, established infections. Similarly, IL22 induces the production of relevant antimicrobial factors in gastric epithelial cells, but surprisingly IL22 knockout and wt mice show similar H pylori colonization rates.
Although this deficit of T-cell maturation can be rescued by vaccination, vaccine-driven T-cell activation normally achieves only a reduction of the bacterial load concomitant with an increased inflammation.
This paradox points to a block of the T-cell–mediated immunity at the effector side of host epithelial defense, which can be explained by the action of Cgt. Because Cgt acts only on infected cells, this gives rise to an intriguing scenario: while inflammation prevails in the infected tissue, Cgt generates protected islands of diminished defense, promoting pathogen survival. Indeed, this notion is corroborated by observations with chronically infected mice challenged with a secondary H pylori infection
: newly incoming bacteria were unable to colonize infected mice except for a few glands already occupied by the primary infection, pointing toward an overall anti-microbial environment in the infected stomach. Together these observations suggest that H pylori forms protected micro-niches surrounded by an otherwise inflamed and microbicidal milieu (Figure 7).
Amongst the T-cell chemotactic factors affected by Cgt are MIG and IP-10, which contribute to protection against H pylori.
Homeostasis of the mucosal colonization by H pylori, avoiding an excessive bacterial load, is thought to be achieved through secretion of a variety of innate factors, including defensins, mucins, and oxidative metabolites.
We have previously observed that EGFR-dependent induction of hBD3 is down-regulated upon translocation of H pylori CagA via the activation of SHP-214 and CagA-dependent activation of SHP-2 also interferes with IFNG signaling.
Thus, CagA acts synergistically with Cgt in preventing IFN signaling and hBD3 synthesis, with Cgt expressed also in strains that lack the cagPAI T4SS. Our results, however, show a minimal contribution of CagA in blocking JAK/STAT signaling, possibly because of variations in the active motifs of CagA proteins in our European strain as compared with Asian strains,
Here, using H pylori, we achieve striking mechanistic insights into microbial cholesterol depletion from host cells, an apparently common, yet little appreciated virulence strategy. Accordingly, cholesterol depletion prevents the partitioning of IFNGR1 to lipid rafts and association with IFNGR2 subunits.
In MKN45 cells, we observed an association of IFNGR2 and IFNGR1 subunits even in the absence of infection, consistent with the abnormal, constitutive JAK1/2 activation. However, this association collapsed upon H pylori infection, rendering these cells unresponsive, independently of the presence of interferons.
Our in vivo experiments confirmed a Cgt-dependent block of the IFNG response, with the results obtained in Ifngr1 knockout mice indicating that additional pathways contribute to preventing colonization with the PMSS1Δcgt mutant. This finding is consistent with the inhibitory action of Cgt on multiple lipid raft-dependent pathways, including IL6 and IFNB,
and IL22, in addition to IFNG. Vice versa, Cgt may also cause receptor activation such as for EGFR and TGFBR, where cholesterol depletion increases the number of molecules available for ligand binding.
Apart from this, many signaling routes, particularly those involving the pro-inflammatory pathway NF-κB, appear to function normally in H pylori-infected cells, even at a time when cholesterol depletion has progressed.
We have substantiated our findings in primary epithelial cells from different donors. Importantly, the use of cells from human gastric organoids allowed us to address biological questions in a mutation-free background.
little data related to H pylori are available yet. Here, we also utilized an advanced ALI model, which offers several improvements: (i) cultures grow under the influence of Wnt to maintain stemness; (ii) cells differentiate toward a diversity of cells, including pit, neck, chief, parietal, and neuroendocrine cells; (iii) cell polarization and secretion of a protective mucus layer supports an authentic equilibrium between cells and bacteria, enabling long-term infection.
Accordingly, cholesterol supply in the context of H pylori infection enhances JAK/STAT signaling together with the release of antibacterial effectors. Such increased Th1 responses, however, are also thought to promote preneoplastic lesions.
This notion, in turn, is consistent with the situation in patients suffering from increased blood cholesterol levels, especially in the form of low-density lipoprotein-cholesterol, who often exhibit severe H pylori-induced gastritis.
Accordingly, a low-cholesterol diet may reduce the pathology of H pylori gastric infections. This places Cgt function, cholesterol metabolism, and inflammation at the crossroads of gastric pathogenesis and cancer.
The authors thank Jörg Angermann, Kirstin Hoffmann, Stefanie Mülllerke, and Ina Wagner for technical assistance, Robert Hurwitz for generating the Cgt antibody, Toni Aebischer, Bianca Bauer, Michael Fehlings, June Ghosh-Guha, and Eric Perret for fruitful discussions, and Rike Zietlow for expert editing of the manuscript.
Author contributions P.M., E.P., and L.P. designed and performed the experiments and analyzed the data; V.D. and M.K. performed the in vivo experiments; H.-J.M. analyzed the microarray data; P.S., F.B., M.S., A.I.M., M.K, and T.F.M. provided experimental guidance during the study; P.M., L.P., and T.F.M. wrote the manuscript; T.F.M. conceived the study and provided conceptual guidance.
Conflicts of interest The authors disclose no conflicts.
Grant support P.M. was supported by the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme FP7/2007-2013/ under REA grant agreement no. 316682; E.P. and L.P. received support from the Deutsche Forschungsgemeinschaft through grant SFB633 to T.F.M.; M.S. was funded as a clinician scientist by the Berlin Institute of Health (BIH). The funders played no role in the design of the study or the collection, analysis, and interpretation of the data.
Transcript profiling Microarray data have been deposited in the Gene Expression Omnibus (GEO; www.ncbi.nlm.nih.gov/geo/) of the National Center for Biotechnology Information and can be accessed with the GEO accession number GSE76589.
Author names in bold designate shared co-first authorship.