Molecular mechanisms of inflammation-induced cancer: T-cells as targets for prevention and cure of Heliobacter-related gastric preneoplasia

Gastric adenocarcinoma is one of the most common causes of cancer-related deaths worldwide and develops as a consequence of chronic gastric inflammation caused by persistent infection with the bacterium Helicobacter pylori. In humans as well as in experimentally infected mice, gastric carcinogenesis progresses through a series of preneoplastic lesions which manifest histologically as chronic atrophic gastritis, epithelial hyperplasia, intestinal metaplasia and dysplasia. Gastric tumorigenesis is preceded by excessive local and systemic immune responses against Helicobacter pylori. It has become increasingly obvious that host genetic predisposition combined with the virulence properties of the colonizing Helicobacter strain determine the severity of disease outcome. Bacterial eradication shows beneficial effects in a subset of patients with atrophic gastritis, but fails to reverse intestinal metaplasia and to prevent malignant transformation. We and others have shown recently that Helicobacter-induced stomach-infiltrating pathogenic CD4+ T-effector cells and their cytokines constitute central mediators of gastric preneoplasia. Therefore, the aim of this study was to identify therapeutic targets that modulate the Helicobacter-specific T-cell response and thus can be exploited to prevent the progression of Helicobacter-induced premalignant lesions. We utilized a C57BL/6 mouse model of experimental Helicobacter felis infection in which the development of gastric preneoplasia is reminiscent of the human setting. In this model, infected mice develop chronic atrophic gastritis accompanied by epithelial hyperplasia and intestinal metaplasia as early as three months post infection. I chose to focus on two molecular players known to play crucial roles at the interface of inflammation and cancer, the cyclooxygenases (COX) and poly(ADP-ribose) polymerases (PARPs). Both enzymes are obvious candidates to assess in the context of Helicobacter-induced gastric preneoplasia for the following reasons: COX-2 and its main product PGE2 function as potent immunosuppressants and modulators of inflammatory responses on the one hand, and possess tumor promoting properties on the other. The process catalyzed by PARPs, the poly(ADP-ribosyl)ation of specific acceptor proteins, is an important post-transcriptional modification involved in the regulation of pro-inflammatory genes, and also participates in the DNA damage response after genotoxic damage. We utilized pharmacological as well as genetic approaches to manipulate these key players at the interface of inflammation and cancer and to elucidate their contribution to Helicobacter-induced preneoplasia. The pharmacological inhibition of COX-2 enzymatic activity during Helicobacter infection in mice led to significantly accelerated and aggravated disease symptoms characterized by more severe gastritis and epithelial pathology as well as elevated IFN-γ levels. In line with these findings, the administration of synthetic PGE2 to infected mice completely suppressed and even reversed pre-existing Helicobacter-associated precancerous lesions, including intestinal metaplasia. The protective effect of PGE2 treatment was accompanied by decreased gastric IFN-γ levels, reduced gastric neutrophil and T-cell infiltration and a reduction of T-cellular Th-1 differentation in the draining mesenteric lymph nodes. Similarly, systemic administration of the inhibitor of poly(ADP- ribosyl)ation, PJ34, reversed pre-existing metaplastic lesions and provided sustainable cure if the treatment was combined with bacterial eradication therapy. The protective effect of both treatments was found to be due to the potent suppression of pathogenic T-cell responses. Data obtained from complementary ex vivo and in vitro experiments indicate that PGE2 and PJ34 treatments abolish T-cellular proliferation, activation-dependent cytokine secretion and migration of CD4+ T-effector cells. This effect was accompanied and probably caused by the transcriptional suppression of the genes encoding IFN-γ and IL-2, the latter being a cytokine essential for continuous effector and regulatory T-cell stimulation and perpetuation of adaptive immune responses. Another focus of this study was to elucidate Helicobacter pylori’s intrinsic genotoxic propterties and to thus determine a possible direct contribution of the bacteria to gastric carcinogenesis. We investigated the potential of Helicobacter pylori to directly damage host cellular DNA and thereby contribute to the acquisition of tumor-promoting mutations. To this end, various cell lines and primary cells were infected with H. pylori and the induction of DNA double strand breaks (DSB) and subsequent DNA damage signaling was investigated. Pulse field gel electrophoresis revealed that H. pylori infection causes DSB in an infection dose-dependent manner. The appearance of DSB depended on contact of live bacteria with the host cell, but occurred independently of the H. pylori virulence factors VacA and the Cag pathogenicity island and was not observed upon infection with E. coli. H. pylori infection-induced DSB resulted in a DNA damage response evident as phosphorylation of H2AX (γH2AX) and foci formation of 53BP1 and MDC1 proteins at sites of DSB. DSB were efficiently repaired by the host cells, but only after antibiotic eradication of H. pylori. The data suggest an active role of Helicobacter in the process of host cellular transformation and may involve a specific bacterial factor delivered upon host-pathogen contact. Taken together, the data obtained in the course of my PhD thesis show that Helicobacter possesses genotoxic properties and that pharmacological targeting of CD4+ T-cells inhibits and reverses pre-existing precancerous lesions in a mouse model of Helicobacter-induced gastric preneoplasia. The results thus provide insights into new therapeutic targets for the treatment of patients that are refractory to Helicobacter eradication therapy or present with untreatable gastric cancer precursor lesions. Das Magenkarzinom zählt zu den häufigsten tödlichen Krebserkrankungen weltweit. Die Entstehung basiert auf einer chronischen Entzündung der Magenmukosa, verursacht durch eine persistierende Infektion mit Helicobacter pylori. Bei Menschen wie bei Mäusen verläuft die gastrale Karzinogenese ausgehend von präneoplastischen Krebsvorstufen. Histologisch manifestieren sich diese Vorstufen als atrophische Gastritits, epitheliale Hyperplasie, intestinale Metaplasie und Dysplasie. Die gastrale Krebsentstehung ist somit ein immunpathologischer Prozess, welcher durch eine übertrieben starke Immunantwort gegen das magenkolonisierende Bakterium Helicobacter pylori verursacht wird. Die Virulenz des Helicobacter-Stammes sowie die genetische Prädisposition des Wirtes bestimmen den Schweregrad der fortschreitenden Erkrankung. Bei Personen mit bestimmten gastralen Präkanzerosen kann durch die Eradikation des Keimes ein Rückgang der Erkrankung herbeigeführt werden. Intestinale Metaplasien und deren Weiterentwicklung zu einer malignen Erkrankung hingegen können dadurch nicht verhindert werden. Daten, die in unserm Labor generiert wurden zeigen, dass pathogene T-Zellen und deren Zytokine für die Entstehung von Magenkrebs und dessen Vorstufen mitverantwortlich sind. Das Ziel der vorliegenden Arbeit war es, mögliche verantwortliche Faktoren und gegen dieselben gerichtete Wirkstoffe zu identifizieren, welche pathogene T-Zell Antworten hemmen und dadurch das Fortschreiten von gutartigen Krebsvorstufen zu bösartigen Läsionen unterbinden können. Zudem untersuchten wir, ob H. pylori selbst die DNS der infizierten Wirtszellen schädigen und somit selbst kanzerogen wirken kann. Für die Studien infizierten wir C57BL/6 Mäuse mit Helicobacter felis, einem nahen Verwandten des human-pathogenen H. pylori. Die Entstehung der Präkanzerosen in unserem Modell ist mit derjenigen im Menschen vergleichbar. Infizierte Mäuse entwickeln nach drei Monaten verschiedene Krebsvorstufen in der Magenmukosa: Gastritis, Hyperplasie des Epithels und intestinale Metaplasie. Wir untersuchten die Funktion von zwei wichtigen Komponenten, die einerseits während der Entzündungsreaktion und andererseits auch in bestehenden Tumoren eine wichtige Rolle spielen und somit als Beschleuniger der Krebsentwicklung fungieren könnten: die Cyclooxygenasen (insbesondere COX-2) und die Poly(ADP-ribose)polymerasen (PARPs). Das von der COX-2 synthetisierte PGE2 hemmt die Immunantwort, koordiniert die Entzündungsreaktion und besitzt andererseits aber auch krebsunterstützende Funktionen. Eine ähnlich wichtige Rolle spielt die post-transkriptionelle Poly(ADP-ribosyl)ierung von Proteinen, die in der Entzündungsreaktion mitwirken. Den Beitrag dieser Proteine zur Helicobacter-induzierten Krebsentstehung untersuchten wir, indem wir die Funktion der beiden Vorgänge pharmakologisch unterdrückten. Eine Blockierung der COX-2 und folglich auch der PGE2 Synthese während der Infektion führte zu einem signifikant beschleunigten Krankheitsverlauf und zu verstärkten Krankheitsmerkmalen im Bereich der Magenmukosa der infizierten Mäuse. Zusätzlich konnten wir hohe IFN-γ Konzentrationen und eine geringere Anzahl Bakterien in der Magenmukosa nachweisen. Übereinstimmend mit dieser Beobachtung bewirkte eine Verabreichung von exogenem PGE2 eine markante Regredienz der Gastritis und unterdrückte nicht nur die Entstehung von Helicobacter-assoziierten Präkanzerosen, sondern bewirkte auch einen Rückgang der intestinalen Metaplasie. Dies ging mit tiefen IFN-γ Konzentrationen und hoher bakterieller Kolonisierung im Magen einher. Zusammenfassend zeigen die erhobenen Daten, dass die Verabreichung von exogenem, synthetischen PGE2 an infizierte Mäuse nicht nur die Entstehung von Präkanzerosen unterdrückte, sondern es zeigt sich auch, dass bestehende präkanzeröse Läsionen einschliesslich der intestinalen Metaplasien geheilt werden konnten und die Magenschleimhaut sich in der Folge normalisierte. Diese Resultate sprechen für eine immunsuppressive Rolle von PGE2, was dazu führt, dass keine Immunzellen in die Magenschleimhaut einwandern können um die Helicobacter-Infektion zu bekämpfen, was wiederum eine hohe Helicobacter-Kolonisierung zur Folge hat. Einen ähnlichen Effekt zeigte die Behandlung von infizierten Mäusen mit PJ34. In behandelten Mäusen konnte ebenfalls die Entstehung von Helicobacter-induzierten Krebsvorstufen verhindert werden und auch bereits bestehende Veränderungen der Magenschleimhaut konnten geheilt werden. Unsere in vitro Untersuchungen haben weiterhin gezeigt, dass eine Behandlung mit PGE2 und PJ34 die Aktivierung von T-Zellen hemmt und damit die entzündungsinduzierte Zytokinsekretion und die Migration von CD4+ Effektor-T-Zellen zum Infektfokus verhindert. Zudem führte die Behandlung zu einer Blockierung der Transkription von IFN-γ und IL-2. IFN-γ spielt eine wichtige Rolle bei der Bekämpfung der Kolonisierung, führt aber gleichzeitig auch zur Entstehung von Veränderungen in der Magenschleimhaut. IL-2 ist ein essentielles Zytokin, das für die Aktivierung und die Proliferation von regulatorischen und Effektor-T-Zellen sowie für die Aufrechterhaltung der erworbenen Immunantwort verantwortlich ist. Dies erklärt auch den lindernden Effekt von PGE2 und PJ34. Mit andern Worten, die beiden Substanzen verhindern die Entstehung einer Entzündungsreaktion im Magen, indem sie Proliferation und die Sekretion von Effektorzytokinen durch T-Zellen unterdrücken. Neben den immunpathologischen Ursachen der gastralen Karzinogenese untersuchten wir auch, ob Helicobacter pylori die DNS der Wirtszelle direkt schädigen kann und dadurch die Tumorentstehung provozieren könnte. Dazu infizierten wir verschiedene Zelllinien mit H. pylori und untersuchten dann die Intaktheit der Wirts-DNS und ob dabei DNS-Doppelstrangbrüche (DSB) entstanden sind. Ausserdem untersuchten wir, ob die betroffenen Zellen auf die DNA Schäden mit der Aktivierung assozierter Signalkaskaden reagierten. Wir konnten feststellen, dass die Helicobacter-Infektion tatsächlich DNS Schäden induziert. Darüber hinaus haben wir gezeigt, dass die DSB durch wirtseigene Reparaturmechanismen repariert werden konnten, aber nur nachdem die Bakterien vorher abgetötet worden waren. Die Voraussetzung für die Entstehung von DSB war der Kontakt der lebenden Bakterien mit den Zellen, wobei die DSB aber unabhängig von den bekannten H. pylori- Virulenzfaktoren VacA und CagPAI hervorgerufen wurden. Des weiteren induzierte die bakterielle Infektion DSB-abhängige Signalkaskaden wie die Phosphorylierung von H2AX (γH2AX) und die Fokusbildung der 53BP1 und MDC1 Proteine. Unsere Daten deuten darauf hin, dass H. pylori die Wirts-DNA direkt schädigt, also auch ohne Einwirkung von Entzündungszellen und so möglicherweise eine Wirtszellentransformation bewirken oder fördern kann. Zusammengefasst zeigen unsere Resultate also auf, dass H. pylori genotoxisches Potenzial besitzt und dass spezifische Therapien, welche die pathogene T-Zell-Immunantwort hemmen nicht nur die von H. pylori induzierten Präkanzerosen unterdrückten, sondern dass bereits bestehende Läsionen sich zurückbilden konnten. Diese gewonnenen Erkenntnisse dienen somit als Grundlage für die Entwicklung entsprechender Medikamente zur Behandlung von resistenten Helicobacter-Infektionen und Präkanzerosen, die das Entstehen eines Magenkrebses in Zukunft verhindern sollen

Inhibition of ADP ribosylation prevents and cures helicobacter-induced gastric preneoplasia

Gastric adenocarcinoma develops as a consequence of chronic inflammation of the stomach lining that is caused by persistent infection with the bacterium Helicobacter pylori. Gastric carcinogenesis progresses through a sequence of preneoplastic lesions that manifest histologically as atrophic gastritis, intestinal metaplasia, and dysplasia. We show here in several preclinical models of Helicobacter-induced atrophic gastritis, epithelial hyperplasia, and metaplasia that the inhibition of ADP ribosylation by the small-molecule inhibitor PJ34 not only prevents the formation of gastric cancer precursor lesions, but also efficiently reverses preexisting lesions. PJ34 exerts its chemopreventive and therapeutic effects by impairing Helicobacter-specific T-cell priming and T(H)1 polarization in the gut-draining mesenteric lymph nodes. The subsequent infiltration of pathogenic T cells into the gastric mucosa and the ensuing gastric T cell-driven immunopathology are prevented efficiently by PJ34. Our data indicate that PJ34 directly suppresses T-cell effector functions by blocking the IFN-gamma production of mesenteric lymph node T cells ex vivo. Upon exposure to PJ34, purified T cells failed to synthesize ADP-ribose polymers and to activate the transcription of genes encoding IFN-gamma, interleukin 2, and the interleukin 2 receptor alpha chain in response to stimuli such as CD3/CD28 cross-linking or phorbol 12-myristate 13-acetate/ionomycin. The immunosuppressive and chemoprotective effects of PJ34 therefore result from impaired T-cell activation and T(H)1 polarization, and lead to the protection from preneoplastic gastric immunopathology. In conclusion, ADP-ribosylating enzymes constitute novel targets for the treatment of Helicobacter-associated gastric lesions predisposing infected individuals to gastric cancer and may also hold promise for the treatment of other T cell-driven chronic inflammatory conditions and autoimmune pathologies

Development of the chronic fatigue syndrome in severely fatigued employees: predictors of outcome in the Maastricht cohort study

Study objective: To identify risk factors of the development of the chronic fatigue syndrome (CFS), the persistence or recurrence of fatigue, or recovery from fatigue in a large sample of fatigued employees. Design: Analyses were based on the Maastricht cohort study (MCS), a prospective population based cohort study among more than 12 000 employees. Multiple regression models were used to identify predictors of CFS-like caseness (meeting research criteria for CFS), non-CFS fatigue caseness, or no fatigue caseness. Setting: The working population in the Netherlands. Participants: 1143 employees with medically unexplained fatigue were followed up prospectively for 44 months. Main results: At 44 month follow up, 8% of the employees were CFS-like cases (none of who reported to have received a CFS diagnosis), 40% were non-CFS fatigue cases, and 52% were no longer fatigue cases. Factors that predicted CFS-like caseness compared with non-CFS fatigue caseness were high age, exhaustion, female sex, low education, and visits to the general practitioner. Factors that predicted CFS-like caseness compared with no fatigue caseness were fatigue, exhaustion, low education, visits to the GP and occupational physician, and bad self rated health. Factors that predicted non-CFS fatigue caseness compared with no fatigue caseness were fatigue, low self perceived activity, exhaustion, anxious mood, and bad self rated health. Conclusions: Unexplained fatigue among employees in some instances is a precursor of the development of CFS. The prognostic role of self rated health suggests that prevention and treatment of chronic fatigue should be aimed at changing the perception of health or illness. Less clear is the role of health care seeking or receiving a CFS diagnosis

Bispecific CD3/HER2 Targeting FynomAb Induces Redirected T Cell-Mediated Cytolysis with High Potency and Enhanced Tumor Selectivity

CD3 bispecific therapies retargeting T cells to tumors have recently demonstrated striking activity in patients. Several CD3 bispecific antibodies directed against various tumor targets are currently being investigated in the clinic across different tumors. One limitation of these therapies is the risk of target-related toxicity due to low-level expression of tumor antigen in normal tissue. In this study we have engineered a bispecific CD3/HER2 FynomAb, COVA420, which redirects T cells with high potency and selectivity to tumor cells with high HER2 expression in vitro and in vivo. COVA420 activity depends on high HER2 density as no activity was observed on cells with lower HER2 levels as found in human normal tissue. These results suggest that COVA420 may spare normal tissue expressing low levels of HER2 while still having uncompromised efficacy on tumor cells with high HER2 expression. This concept may be applied to other cancer antigens that otherwise cannot be targeted by T cell redirecting approaches, and may therefore expand the applicability of CD3 bispecific FynomAbs to a larger number of solid tumors

The BatR/BatS Two-Component Regulatory System Controls the Adaptive Response of Bartonella henselae during Human Endothelial Cell Infection ▿ † ‡

Here, we report the first comprehensive study of Bartonella henselae gene expression during infection of human endothelial cells. Expression of the main cluster of upregulated genes, comprising the VirB type IV secretion system and its secreted protein substrates, is shown to be under the positive control of the transcriptional regulator BatR. We demonstrate binding of BatR to the promoters of the virB operon and a substrate-encoding gene and provide biochemical evidence that BatR and BatS constitute a functional two-component regulatory system. Moreover, in contrast to the acid-inducible (pH 5.5) homologs ChvG/ChvI of Agrobacterium tumefaciens, BatR/BatS are optimally activated at the physiological pH of blood (pH 7.4). By conservation analysis of the BatR regulon, we show that BatR/BatS are uniquely adapted to upregulate a genus-specific virulence regulon during hemotropic infection in mammals. Thus, we propose that BatR/BatS two-component system homologs represent vertically inherited pH sensors that control the expression of horizontally transmitted gene sets critical for the diverse host-associated life styles of the alphaproteobacteria

Prostaglandin E2 prevents Helicobacter-induced gastric preneoplasia and facilitates persistent infection in a mouse model

BACKGROUND AND AIMS:: Persistent infection with the human pathogen Helicobacter pylori increases the risk of gastric cancer. In this study, we investigated the role of cyclooxygenase-2 (COX-2) and its main product, prostaglandin E(2) (PGE(2)), in the development of Helicobacter-induced gastritis and gastric cancer precursor lesions. METHODS:: We utilized mouse models of Helicobacter-induced gastric preneoplasia and vaccine-induced protection to study the effects of COX-2 inhibition and PGE(2) treatment on the induction of Helicobacter-specific immune responses and gastric premalignant immunopathology. RESULTS:: COX-2 and PGE(2) are up-regulated upon Helicobacter infection in cultured epithelial cells and in the gastric mucosa of infected mice. Inhibition of COX-2 activity with Celecoxib significantly accelerated early preneoplasia; conversely, systemic administration of synthetic PGE(2) prevented the development of premalignant pathology and completely reversed pre-existing lesions by suppressing IFN-gamma production in the infected stomachs. The protective effect of PGE(2) was accompanied by increased Helicobacter colonization in all models. All in vivo effects were attributed to immunosuppressive effects of PGE(2) on CD4(+) T-helper 1 (Th1) cells, which fail to migrate, proliferate and secrete cytokines when exposed to PGE(2)in vitro and in vivo. T-cell inhibition was found to be due to silencing of IL-2 gene transcription, and could be overcome by supplementation with recombinant IL-2 in vitro and in vivo. CONCLUSIONS:: COX-2-dependent production of PGE(2) has an important immunomodulatory role during Helicobacter infection, preventing excessive local immune responses and the associated immunopathology by inhibiting the effector functions of pathogenic Th1 cells