Richard L. Ferrero

The innate immune protein NLRC5 plays a key role in cancer immune surveillance. Reduced NLRC5 expression is associated with a poor prognosis for many types of cancers. Previously, we showed that mice with a myeloid‐specific deletion of Nlrc5 ( Nlrc5 ^mø‐KO ) develop gastric lymphoid lesions to Helicobacter infection resembling early‐stage marginal zone lymphoma. We hypothesized that NLRC5 deficiency may promote a tumor‐permissive microenvironment mediated by tumor‐associated macrophages (TAMs). Consistent with this hypothesis, splenic macrophages from Helicobacter ‐infected Nlrc5 ^mø‐KO mice had upregulated expression of genes encoding the TAM receptor tyrosine kinases, Axl and Mertk. The levels of AXL and MERTK gene expression and MERTK phosphorylation were increased in NLRC5 ^−/− THP‐1 macrophages when compared with WT cells. In response to Helicobacter stimulation, Nlrc5 ^−/− macrophages had significantly elevated anti‐inflammatory responses (IL‐10, TGF‐β, Socs1 , Socs3 ) compared with WT cells. Importantly, Nlrc5 ^−/− macrophages showed enhanced efferocytosis and reduced antigen presentation to CD8 ⁺ T cells. Pretreatment of macrophages with AXL and MERTK inhibitors (R428, UNC2025) resulted in reduced efferocytosis and phosphorylation of downstream signaling molecules, STAT3 and ERK1/2. We propose that defective NLRC5 signaling in macrophages leads to tumor‐permissive responses, thereby promoting the development of gastric lymphoid neogenesis to Helicobacter infection.

Nucleomodulins are a family of bacterial virulence proteins that traffic to the nucleus of host cells to disrupt nuclear processes and, in some cases, promote carcinogenesis. The mechanisms by which nucleomodulins are secreted and injected into host cells are not entirely clear. We hypothesised that bacterial extracellular vesicles (EVs), also known as outer membrane vesicles, may represent a novel mechanism for nucleomodulin delivery to host cells. To address this, we studied the role of EVs in the secretion of Helicobacter pylori tumour necrosis factor‐α‐inducing protein (Tipα), a protein that was reported to undergo nuclear trafficking and to promote carcinogenesis. Importantly, we showed that most Tipα present in H. pylori culture supernatants is associated with EVs and, moreover, is encapsulated within these particles in its biologically active dimeric form. Confocal microscopy and cell fractionation studies demonstrated that EVs carry Tipα to the nuclear compartment of host cells and can bind host DNA. EVs from isogenic tipA mutants induced greater pro‐inflammatory cytokine responses in host cells than EVs from wild‐type bacteria; however, this effect was dependent on the EV isolation method. We propose that EVs represent a new mechanism by which bacteria secrete and deliver nucleomodulins to their target cells, resulting in altered immune responses and possibly carcinogenesis.

Helicobacter pylori infects approximately half of the world's population, resulting in lifelong gastric infections. To promote lifelong colonization, H. pylori modulates host immunity via unknown mechanisms. Here we show that H. pylori can harness the pan-immunosuppressive cytokine interleukin-37 (IL-37) to facilitate pathogenesis, enhance colonization and prevent the development of innate, cellular and humoral immunity. We show that H. pylori increased the production of IL-37 in human gastric biopsies, and IL-37 secretion by gastric epithelial cells and human gastric mucosoids. We found that H. pylori induced IL-37 secretion by epithelial cells via activation of host pathogen recognition receptors TLR2, TLR4 and NOD1, in addition to the H. pylori-encoded cag pathogenicity island, revealing that H. pylori utilises multiple mechanisms to induce IL-37 production during infection. Once produced, IL-37 attenuated TLR2, TLR4 and NOD1-mediated activation and TLR-mediated IL-8 responses to H. pylori infection. Using transgenic mice expressing human IL-37, we found that IL-37 promoted immunosuppression by significantly increasing H. pylori colonization, limiting gastric inflammation, and reducing H. pylori-specific antibody responses. Furthermore, we identified the multiple mechanisms whereby IL-37 functions to impair the development of adaptive immunity. IL-37 abolished human T and B cell responses by impairing their activation, migration, and preventing immune synapse formation. Moreover, IL-37 inhibited proliferation of gastric-derived H. pylori-specific T cells isolated from H. pylori-infected patients, revealing a mechanism whereby IL-37 functions to prevent pathogen-specific cellular immune responses. Collectively, our findings reveal that H. pylori induces production of the pan-immunosuppressive cytokine IL-37 to enhance colonization, modulate gastritis and suppress innate, cellular and humoral immunity to ultimately promote pathogenesis in the host. These findings advance our understanding of H. pylori-mediated disease and identify gastric IL-37 as a therapeutic target to combat H. pylori infection and associated diseases.

Helicobacter pylori is a major causative agent in several upper gastroduodenal tract diseases, including gastric cancer. The development of methods to genetically manipulate H. pylori by natural transformation has allowed a greater understanding of its biology and role in these diseases. Nevertheless, the transformation methods used for H. pylori are time-consuming, requiring growth of these fastidious and slow-growing bacteria from −80°C stocks. The aim of the study was to develop a more rapid and convenient method for generating H. pylori mutants. We describe here a method in which competent H. pylori bacteria can be stored at −80°C and used in transformations on the day of resuscitation, similar to methods routinely used for Escherichia coli . This means that transformation can be performed at will and that transformants can be obtained within days, rather than weeks. Furthermore, we show that bacteria remain competent for at least six months storage at −80°C and that the method is applicable to strains with varying levels of natural competence. Transformation efficiencies of the bacteria varied between 10 ¹ and 10 ⁶ transformants/total colony-forming units/µg donor DNA, depending on the strain. We suggest that this improved method will facilitate studies on H. pylori and, moreover, may be applicable to other naturally transformable pathogens with fastidious growth requirements and requiring ultra-low temperature refrigeration for long-term preservation. IMPORTANCE: Genetic manipulation is an important tool in the study of pathogenic bacteria and their interactions with the host. Many pathogenic bacteria are naturally transformable; however, transformation experiments can be impeded by the slow-growing and fastidious nature of some species. One such bacterium is Helicobacter pylori , which requires resuscitation from −80°C and multiple subcultures prior to transformation. The method described in the current study uses a simple modification of a conventional method of natural transformation. Using this method, competent H. pylori bacteria can be stored for long periods (at least six months) and resuscitated as needed for use in experiments. The method circumvents the need for multiple and lengthy subcultures prior to transformation, nor does it involve costly materials, complicated procedures, or sophisticated equipment. Thus, we describe a simple, inexpensive, and time-efficient method that may have broader applications for use with other fastidious bacteria.

Cytolethal distending toxins (CDTs) are released by Gram-negative pathogens into the extracellular medium as free toxin or associated with extracellular vesicles (EVs), commonly known as outer membrane vesicles (OMVs). CDT production by the gastrointestinal pathogen Campylobacter jejuni has been implicated in colorectal tumorigenesis. Despite CDT being a major virulence factor for C. jejuni , little is known about the EV-associated form of this toxin. To address this point, C. jejuni mutants lacking each of the three CDT subunits (A, B, and C) were generated. C. jejuni cdtA, cdtB, and cdtC bacteria released EVs in similar numbers and sizes to wild-type bacteria, ranging from 5 to 530 nm (mean ± SEM = 118 ±6.9 nm). As the CdtAC subunits mediate toxin binding to host cells, we performed “surface shearing” experiments, in which EVs were treated with proteinase K and incubated with host cells. These experiments indicated that CDT subunits are internal to EVs and that surface proteins are probably not involved in EV-host cell interactions. Furthermore, glycan array studies demonstrated that EVs bind complex host cell glycans and share receptor binding specificities with C. jejuni bacteria for fucosyl GM1 ganglioside, P1 blood group antigen, sialyl, and sulfated Lewis ^x . Finally, we show that EVs from C. jejuni WT but not mutant bacteria induce cell cycle arrest in epithelial cells. In conclusion, we propose that EVs are an important mechanism for CDT release by C. jejuni and are likely to play a significant role in toxin delivery to host cells. IMPORTANCE: Campylobacter jejuni is the leading cause of foodborne gastroenteritis in humans worldwide and a significant cause of childhood mortality due to diarrheal disease in developing countries. A major factor by which C. jejuni causes disease is a toxin, called cytolethal distending toxin (CDT). The biology of this toxin, however, is poorly understood. In this study, we report that C. jejuni CDT is protected within membrane blebs, known as extracellular vesicles (EVs), released by the bacterium. We showed that proteins on the surfaces of EVs are not required for EV uptake by host cells. Furthermore, we identified several sugar receptors that may be required for EV binding to host cells. By studying the EV-associated form of C. jejuni CDT, we will gain a greater understanding of how C. jejuni intoxicates host cells and how EV-associated CDT may be used in various therapeutic applications, including as anti-tumor therapies.

The interleukin-1 family members, IL-1β and IL-18, are processed into their biologically active forms by multi-protein complexes, known as inflammasomes. Although the inflammasome pathways that mediate IL-1β processing in myeloid cells have been defined, those involved in IL-18 processing, particularly in non-myeloid cells, are still not well understood. Here we report that the host defence molecule NOD1 regulates IL-18 processing in mouse epithelial cells in response to the mucosal pathogen, Helicobacter pylori. Specifically, NOD1 in epithelial cells mediates IL-18 processing and maturation via interactions with caspase-1, instead of the canonical inflammasome pathway involving RIPK2, NF-κB, NLRP3 and ASC. NOD1 activation and IL-18 then help maintain epithelial homoeostasis to mediate protection against pre-neoplastic changes induced by gastric H. pylori infection in vivo. Our findings thus demonstrate a function for NOD1 in epithelial cell production of bioactive IL-18 and protection against H. pylori-induced pathology.

Contaminants within cell culture media often co‐isolate with eukaryotic extracellular vesicles (EVs) thus affecting their biological properties. It has yet to be investigated if this is also true for bacterial EVs (BEVs), especially for organisms grown in complex culture media containing animal‐derived products. To address this question, we isolated BEVs from the fastidious bacterium Helicobacter pylori grown in either standard Brain Heart Infusion (BHI) medium or BHI depleted of animal‐derived products (D‐BHI). We show that BEVs prepared from bacteria grown in D‐BHI medium have similar morphologies, size ranges and yields to those prepared from standard medium. Similarly, no differences were found in the ability of H. pylori BEVs to induce IL‐8 responses in epithelial cells. However, H. pylori BEVs prepared from D‐BHI medium were of higher purity than those prepared from standard medium. Importantly, proteomic analyses detected 3.4‐fold more H. pylori proteins and 10‐fold fewer bovine‐derived proteins in BEV samples prepared from D‐BHI rather than the standard method. Fifty‐seven H. pylori proteins were uniquely detected in BEV samples prepared from D‐BHI. In conclusion, we have described an improved method for BEV isolation. Furthermore, we demonstrate how animal‐derived products in bacteriological culture media may adversely affect proteomic analyses of BEVs.

Helicobacter pylori (H. pylori) infection can trigger chronic gastric inflammation perpetuated by overactivation of the innate immune system, leading to a cascade of precancerous lesions culminating in gastric cancer. However, key regulators of innate immunity that promote H. pylori–induced gastric pathology remain ill‐defined. The innate immune cytosolic DNA sensor absent in melanoma 2 (AIM2) contributes to the pathogenesis of numerous autoimmune and chronic inflammatory diseases, as well as cancers including gastric cancer. We therefore investigated whether AIM2 contributed to the pathogenesis of Helicobacter‐induced gastric disease. Here, we reveal that AIM2 messenger RNA and protein expression levels are elevated in H. pylori–positive versus H. pylori–negative human gastric biopsies. Similarly, chronic Helicobacter felis infection in wild‐type mice augmented Aim2 gene expression levels compared with uninfected controls. Notably, gastric inflammation and hyperplasia were less severe in H. felis–infected Aim2^−/− versus wild‐type mice, evidenced by reductions in gastric immune cell infiltrates, mucosal thickness and proinflammatory cytokine and chemokine release. In addition, H. felis–driven proliferation and apoptosis in both gastric epithelial and immune cells were largely attenuated in Aim2^−/− stomachs. These observations in Aim2^−/− mouse stomachs correlated with decreased levels of inflammasome activity (caspase‐1 cleavage) and the mature inflammasome effector cytokine, interleukin‐1β. Taken together, this work uncovers a pathogenic role for the AIM2 inflammasome in Helicobacter‐induced gastric disease, and furthers our understanding of the host immune response to a common pathogen and the complex and varying roles of AIM2 at different stages of cancerous and precancerous gastric disease.

Mucosa‐associated lymphoid tissue (MALT) lymphoma is a B‐cell tumour that develops over many decades in the stomachs of individuals with chronic Helicobacter pylori infection. We developed a new mouse model of human gastric MALT lymphoma in which mice with a myeloid‐specific deletion of the innate immune molecule, Nlrc5, develop precursor B‐cell lesions to MALT lymphoma at only 3 months post‐Helicobacter infection versus 9–24 months in existing models. The gastric B‐cell lesions in the Nlrc5 knockout mice had the histopathological features of the human disease, notably lymphoepithelial‐like lesions, centrocyte‐like cells, and were infiltrated by dendritic cells (DCs), macrophages, and T‐cells (CD4⁺, CD8⁺ and Foxp3⁺). Mouse and human gastric tissues contained immune cells expressing immune checkpoint receptor programmed death 1 (PD‐1) and its ligand PD‐L1, indicating an immunosuppressive tissue microenvironment. We next determined whether CD40L, overexpressed in a range of B‐cell malignancies, may be a potential drug target for the treatment of gastric MALT lymphoma. Importantly, we showed that the administration of anti‐CD40L antibody either coincident with or after establishment of Helicobacter infection prevented gastric B‐cell lesions in mice, when compared with the control antibody treatment. Mice administered the CD40L antibody also had significantly reduced numbers of gastric DCs, CD8⁺ and Foxp3⁺ T‐cells, as well as decreased gastric expression of B‐cell lymphoma genes. These findings validate the potential of CD40L as a therapeutic target in the treatment of human gastric B‐cell MALT lymphoma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.