Andrew Stent

The MUC1 mucin protects against Helicobacter pylori pathogenesis in mice by regulation of the NLRP3 inflammasome

Objectives The mucin MUC1, best known for providing an epithelial barrier, is an important protective host factor in both humans and mice during Helicobacter pylori pathogenesis. This study aimed to identify the long-term consequences of MUC1 deficiency on H. pylori pathogenesis and the mechanism by which MUC1 protects against H. pylori gastritis. Design Wildtype and Muc1−/− mice were infected for up to 9 months, and the gastric pathology, immunological response and epigenetic changes assessed. The effects of MUC1 on the inflammasome, a potent inflammatory pathway, were examined in macrophages and H. pylori-infected mice deficient in both MUC1 and inflammasome components. Results Muc1−/− mice began to die 6 months after challenge, indicating Muc1 deficiency made H. pylori a lethal infection. Surprisingly, chimaeric mouse infections revealed MUC1 expression by haematopoietic-derived immune cells limits H. pylori-induced gastritis. Gastritis in infected Muc1−/− mice was associated with elevated interleukin (IL)-1β and epigenetic changes in their gastric mucosa similar to those in transgenic mice overexpressing gastric IL-1β, implicating MUC1 regulation of an inflammasome. In support of this, infected Muc1−/−Casp1−/− mice did not develop severe gastritis. Further, MUC1 regulated Nlrp3 expression via an nuclear factor (NF)-κB-dependent pathway and reduced NF-κB pathway activation via inhibition of IRAK4 phosphorylation. The importance of this regulation was proven using Muc1−/−Nlrp3−/− mice, which did not develop severe gastritis. Conclusions MUC1 is an important, previously unidentified negative regulator of the NLRP3 inflammasome. H. pylori activation of the NLRP3 inflammasome is normally tightly regulated by MUC1, and loss of this critical regulation results in the development of severe pathology.

Helicobacter pylori thiolperoxidase as a protective antigen in single- and multi-component vaccines

Helicobacter pylori is an important pathogen of the human stomach, and the development of a protective vaccine has been an enticing goal for many years. The H. pylori antioxidant enzymes superoxide dismutase (SOD) and catalase (KatA) have been shown to be protective as vaccine antigens in mice, demonstrating that the organisms antioxidant enzyme system is a fruitful target for vaccine development. The research described here demonstrates that an additional antioxidant enzyme, thiolperoxidase (Tpx), is effective as a prophylactic vaccine antigen via both systemic and mucosal routes. The functional relationship between SOD, KatA and Tpx also provided an opportunity to investigate synergistic or additive effects when the three antigens were used in combination. Although the antigens still provided equivalent protection when administered in combination, no additional protection was observed. Moreover a decrease in antibody titres to the individual antigens was observed when delivered in combination via the nasal route, though not when injected subcutaneously. The findings of this paper demonstrate that the antioxidant system of H. pylori presents a particularly rich resource for vaccine development.

Evaluation of superoxide dismutase from Helicobacter pylori as a protective vaccine antigen

Helicobacter pylori, the major cause of gastric cancer, have mechanisms that allow colonization of the inhospitable gastric mucosa, including enzymes such as superoxide dismutase (SOD) which protect against reactive oxygen species. As SOD is essential for in vivo colonization, we theorized it might constitute a viable vaccine target. H. pylori SOD was expressed in E. coli and a purified recombinant protein used to vaccinate mice, prior to live H. pylori challenge. Partial protective immunity was induced, similar to that commonly observed with other antigens tested previously. This suggests SOD may have utility in a combination vaccine comprising several protective antigens.

Protease-activated receptor 1 suppresses Helicobacter pylori gastritis via the inhibition of macrophage cytokine secretion and interferon regulatory factor 5

Chronic gastritis from Helicobacter pylori infection is a major factor in the development of gastric adenocarcinoma. Factors that regulate gastritis severity are important in determining which individuals are susceptible to H. pylori–associated disease. Although protease-activated receptor 1 (PAR1) has been identified as one such host factor, its mechanism of action is unknown. Using chimeric mice, we demonstrated that PAR1-mediated protection against H. pylori gastritis requires bone marrow–derived cells. Analyses of the gastric mucosa revealed that PAR1 suppresses cellular infiltration and both T helper type 1 (Th1) and T helper type 17 (Th17) responses to infection. Moreover, PAR1 expression was associated with reduced vaccine-mediated protection against H. pylori. Analyses of H. pylori–stimulated macrophages revealed that PAR1 activation suppressed secretion of interleukin (IL)-12 and IL-23, key drivers of Th1 and Th17 immunity, respectively. Furthermore, PAR1 suppressed interferon regulatory factor 5 (IRF5), an important transcription factor for IL-12 and IL-23, both in the infected mucosa and following bacterial stimulation. PAR1 suppression of IRF5 and IL-12/23 secretion by macrophages provides a novel mechanism by which the host suppresses the mucosal Th1 and Th17 response to H. pylori infection. Dysregulation of this process is likely an important factor in the susceptibility of some individuals to H. pylori–associated disease.

Helicobacter pylori defense against oxidative attack

Helicobacter pylori is a microaerophilic, gram-negative pathogen of the human stomach. Despite the chronic active gastritis that develops following colonization, H. pylori is able to persist unharmed in the stomach for decades. Much of the damage caused by gastric inflammation results from the accumulation of reactive oxygen/nitrogen species within the stomach environment, which can induce oxidative damage in a wide range of biological molecules. Without appropriate defenses, this oxidative damage would be able to rapidly kill nearby H. pylori, but the organism employs a range of measures, including antioxidant enzymes, biological repair systems, and inhibitors of oxidant generation, to counter the attack. Despite the variety of measures employed to defend against oxidative injury, these processes are intimately interdependent, and any deficiency within the antioxidant system is generally sufficient to cause substantial impairment of H. pylori viability and persistence. This review provides an overview of the development of oxidative stress during H. pylori gastritis and examines the methods the organism uses to survive the resultant damage.

Mucosal-Associated Invariant T Cells Augment Immunopathology and Gastritis in Chronic Helicobacter pylori Infection

Mucosal-associated invariant T (MAIT) cells produce inflammatory cytokines and cytotoxic granzymes in response to by-products of microbial riboflavin synthesis. Although MAIT cells are protective against some pathogens, we reasoned that they might contribute to pathology in chronic bacterial infection. We observed MAIT cells in proximity to Helicobacter pylori bacteria in human gastric tissue, and so, using MR1-tetramers, we examined whether MAIT cells contribute to chronic gastritis in a mouse H. pylori SS1 infection model. Following infection, MAIT cells accumulated to high numbers in the gastric mucosa of wild-type C57BL/6 mice, and this was even more pronounced in MAIT TCR transgenic mice or in C57BL/6 mice where MAIT cells were preprimed by Ag exposure or prior infection. Gastric MAIT cells possessed an effector memory Tc1/Tc17 phenotype, and were associated with accelerated gastritis characterized by augmented recruitment of neutrophils, macrophages, dendritic cells, eosinophils, and non-MAIT T cells and by marked gastric atrophy. Similarly treated MR1−/− mice, which lack MAIT cells, showed significantly less gastric pathology. Thus, we demonstrate the pathogenic potential of MAIT cells in Helicobacter-associated immunopathology, with implications for other chronic bacterial infections.

Recombinant Herpesvirus Glycoprotein G Improves the Protective Immune Response to Helicobacter pylori Vaccination in a Mouse Model of Disease

Alphaherpesviruses, which have co-evolved with their hosts for more than 200 million years, evade and subvert host immune responses, in part, by expression of immuno-modulatory molecules. Alphaherpesviruses express a single, broadly conserved chemokine decoy receptor, glycoprotein G (gG), which can bind multiple chemokine classes from multiple species, including human and mouse. Previously, we demonstrated that infection of chickens with an infectious laryngotracheitis virus (ILTV) mutant deficient in gG resulted in altered host immune responses compared to infection with wild-type virus. The ability of gG to disrupt the chemokine network has the potential to be used therapeutically. Here we investigated whether gG from ILTV or equine herpesvirus 1 (EHV-1) could modulate the protective immune response induced by the Helicobacter pylori vaccine antigen, catalase (KatA). Subcutaneous immunisation of mice with KatA together with EHV-1 gG, but not ILTV gG, induced significantly higher anti-KatA IgG than KatA alone. Importantly, subcutaneous or intranasal immunisation with KatA and EHV-1 gG both resulted in significantly lower colonization levels of H. pylori colonization following challenge, compared to mice vaccinated with KatA alone. Indeed, the lowest colonization levels were observed in mice vaccinated with KatA and EHV-1 gG, subcutaneously. In contrast, formulations containing ILTV gG did not affect H. pylori colonisation levels. The difference in efficacy between EHV-1 gG and ILTV gG may reflect the different spectrum of chemokines bound by the two proteins. Together, these data indicate that the immuno-modulatory properties of viral gGs could be harnessed for improving immune responses to vaccine antigens. Future studies should focus on the mechanism of action and whether gG may have other therapeutic applications.

Pathological and microbiological investigations into cases of bacterial chondronecrosis and osteomyelitis in broiler poultry

ABSTRACT Bacterial chondronecrosis and osteomyelitis (BCO) is increasingly recognized as a major cause of lameness in commercial broilers chickens worldwide, but the pathogenesis of the condition is incompletely understood. This was a longitudinal study of 20 commercial broiler farms in Victoria, Australia, to investigate the aetiology and pathology of BCO. Thorough postmortem examination was performed on culled and dead birds (n = 325) from 20 different flocks at either 1 week, 4 weeks or 5 weeks of age and samples were analysed by conventional bacteriology, molecular identification of infectious organisms detected, serology and histopathology. BCO occurs throughout the life of broiler flocks at a very high rate, with lesions detected in 28% (95% CI 23–34%) of the mortalities and culls. The condition occurs with similar prevalence in both the femur and tibiotarsus. BCO is an infectious process that appears to result from bacteraemia and haematological spread of bacterial pathogens, especially Escherichia coli, to the bones, with 65.3% bacterial isolates from histologically confirmed BCO identified as E. coli, 11.5% as Staphylococcus and the remainder composed of mixed infections or a range of other minor isolates. We observed that almost all E. coli isolated from cases of BCO are avian pathogenic E. coli, suggesting that preventative measures should be directed at this organism.

MAIT cells protect against pulmonary Legionella longbeachae infection

Mucosal associated invariant T (MAIT) cells recognise conserved microbial metabolites from riboflavin synthesis. Striking evolutionary conservation and pulmonary abundance implicate them in antibacterial host defence, yet their functions in protection against clinically important pathogens are unknown. Here we show that mouse Legionellalongbeachae infection induces MR1-dependent MAIT cell activation and rapid pulmonary accumulation of MAIT cells associated with immune protection detectable in immunocompetent host animals. MAIT cell protection is more evident in mice lacking CD4+ cells, and adoptive transfer of MAIT cells rescues immunodeficient Rag2−/−γC−/− mice from lethal Legionella infection. Protection is dependent on MR1, IFN-γ and GM-CSF, but not IL-17A, TNF or perforin, and enhanced protection is detected earlier after infection of mice antigen-primed to boost MAIT cell numbers before infection. Our findings define a function for MAIT cells in protection against a major human pathogen and indicate a potential role for vaccination to enhance MAIT cell immunity.Mucosal associated invariant T (MAIT) cells have been implicated in antibacterial responses. Here the authors show MAIT cells confer IFN-γ-mediated protection from lethal infection in a mouse model of Legionella infection, which can be enhanced by synthetic MR1 ligands.

Superoxide dismutase from Helicobacter pylori suppresses the production of pro-inflammatory cytokines during in vivo infection

Helicobacter pylori has undergone considerable adaptation to allow chronic persistence within the gastric environment. While H. pylori‐associated diseases are driven by an excessive inflammation, severe gastritis is detrimental to colonization by this pathogen. Hence, H. pylori has developed strategies to minimize the severity of gastritis it triggers in its host. Superoxide dismutase (SOD) is well known for its role in protecting against oxidative attack; less recognized is its ability to inhibit immunity, shown for SOD from mammalian sources and those of some bacterial species. This study examined whether H. pylori SOD (HpSOD) has the ability to inhibit the host immune response to these bacteria.