Unlocking bat immunology: establishment of Pteropus alecto bone marrow-derived dendritic cells and macrophages

Bats carry and shed many emerging infectious disease agents including Ebola virus and SARS-like Coronaviruses, yet they rarely display clinical symptoms of infection. Bat epithelial or fibroblast cell lines were previously established to study the bat immune response against viral infection. However, the lack of professional immune cells such as dendritic cells (DC) and macrophages has greatly limited the significance of current investigations. Using Pteropus alecto (P. alecto) GM-CSF plus IL4, FLT3L and CSF-1, we successfully generated bat bone marrow-derived DC and macrophages. Cells with the phenotype, morphology and functional features of monocyte-derived DC, bona fide DC or macrophages were obtained in GM-CSF/IL4, FLT3L or CSF-1 cultures, respectively. The successful generation of the first bat bone marrow-derived immune cells paves the way to unlocking the immune mechanisms that confer host resilience to pathogens in bats

Loss of gastrokine-2 drives premalignant gastric inflammation and tumor progression

Chronic mucosal inflammation is associated with a greater risk of gastric cancer (GC) and, therefore, requires tight control by suppressive counter mechanisms. Gastrokine-2 (GKN2) belongs to a family of secreted proteins expressed within normal gastric mucosal cells. GKN2 expression is frequently lost during GC progression, suggesting an inhibitory role; however, a causal link remains unsubstantiated. Here, we developed Gkn2 knockout and transgenic overexpressing mice to investigate the functional impact of GKN2 loss in GC pathogenesis. In mouse models of GC, decreased GKN2 expression correlated with gastric pathology that paralleled human GC progression. At baseline, Gkn2 knockout mice exhibited defective gastric epithelial differentiation but not malignant progression. Conversely, Gkn2 knockout in the IL-11/STAT3-dependent gp130[superscript F/F] GC model caused tumorigenesis of the proximal stomach. Additionally, gastric immunopathology was accelerated in Helicobacter pylori–infected Gkn2 knockout mice and was associated with augmented T helper cell type 1 (Th1) but not Th17 immunity. Heightened Th1 responses in Gkn2 knockout mice were linked to deregulated mucosal innate immunity and impaired myeloid-derived suppressor cell activation. Finally, transgenic overexpression of human gastrokines (GKNs) attenuated gastric tumor growth in gp130[superscript F/F] mice. Together, these results reveal an antiinflammatory role for GKN2, provide in vivo evidence that links GKN2 loss to GC pathogenesis, and suggest GKN restoration as a strategy to restrain GC progression

The regulatory effects of protease activated receptor 1 on Helicobacter pylori-induced inflammation

© 2010 Dr. Yok Teng ChionhHelicobacter pylori, one of the world’s most prevalent pathogens, infects the gastric mucosa of approximately half the human population. These infections cause a wide range of pathologies from asymptomatic gastritis to gastric cancer due to host genetic variation and a multitude of diverse factors that govern host-pathogen interactions. One host factor hypothesised to be the key regulator of H. pylori-induced gastritis is G protein-coupled Protease-Activated Receptor 1 (PAR1). PAR1, is the downstream signaller of thrombin, a crucial protease generated during inflammation and the main effector of the blood coagulation cascade. It is also known that H. pylori infected patients have higher PAR1 expression in the stomach compared to healthy individuals. Given this knowledge, this thesis was directed towards obtaining a greater understanding of the role of PAR1 in H. pylori-induced gastritis. The role of PAR1 in H. pylori inflammation was examined using PAR1-/- and wild-type mice. H. pylori infection of PAR1-/- mice induced significantly more severe gastritis compared to wild-type controls. H. pylori infected PAR1-/- mice also expressed more pro-inflammatory cytokines (MIP-2, IFNγ and IL-17) in the gastric tissues. These results demonstrated that PAR1 has a protective function against H. pylori induced gastritis. H. pylori stimulation of primary gastric epithelial cells deficient in PAR1 induced increased levels of NF-κB and the pro-inflammatory cytokine, MIP-2 production which is consistent with observations made in vivo. This study suggested that gastric epithelial cells could contribute to the regulation of inflammation. Subsequently, through the use of bone marrow chimeras, it was demonstrated that haematopoietic cells and not epithelial cells play a major role in PAR1 regulation of gastritis. To investigate if PAR1 has a role protective immunity against H. pylori, PAR1-/- and wild-type mice were vaccinated with formalin fixed H. pylori. Immunisation of PAR1-/- and wild-type mice induced a significant increase in antibody titres (including IgA, IgG1 and IgG2 subclasses) in both PAR1-/- and wild-type mice. However, vaccination of PAR1-/- and wild-type mice produced similar reductions in H. pylori colonisation, suggesting that PAR1 does not play an important role in vaccine-mediated protection against this infection. To further indentify the cells types involved in regulating H. pylori-induced inflammation, splenocytes, macrophages and dendritic cells were obtained from PAR1-/- and wild-type mice. No differences were observed in MIP-2 production from PAR1-/- and wild-type macrophages and dendritic cells suggesting that macrophages and dendritic cells are not involved in the PAR1 regulatory process. In contrast, splenocytes stimulated by PAR1 activating peptide and H. pylori expressed significantly less MIP-2 and IFNγ than splenocytes stimulated by H. pylori alone supporting the in vivo findings. Furthermore, it was also observed that PAR1 expression plays a positive role in the regulation of PAR2 expression in splenocytes. PAR2 unlike PAR1 is activated by trypsin and has a pro-inflammatory role in H. pylori infections, as PAR2-/- mice develop reduced severity of H. pylori-induced gastritis. This suggested while PAR2 has an opposing role to PAR1, during inflammation, the expression PAR1 and PAR2 may work in a positive feedback loop mechanism. In summary, this thesis demonstrated that PAR1 is an important host factor in H. pylori pathogenesis. This effect is mediated by haematopoietic cells, where PAR1 modulates pro-inflammatory cytokines production. In vitro studies supported this novel in vivo finding and also demonstrated that PAR1 can modulate PAR2, presumably via a feedback loop mechanism. Further investigations into the specific cell(s) involved in the protective effect of PAR1 in H. pylori-induced gastritis would prove invaluable to identify potential signalling mechanisms of PAR1 which would be useful in the treatment of H. pylori pathogenesis and other inflammatory diseases

Muc1 limits Helicobacter felis binding to gastric epithelial cells but does not limit colonization and gastric pathology following infection

The mucin Muc1 is constitutively expressed by the gastric mucosa and is likely the first point of direct contact between the host stomach and the adherent pathogens. The expression of Muc1 has been shown to limit colonization of mice by Helicobacter pylori, known to adhere to the gastric epithelium, as well as associated pathology. However, the potential role of this mucin against nonadherent Helicobacter has not been previously studied. We therefore examined the importance of Muc1 on the pathogenesis of Helicobacter felis, believed not to adhere to the murine mucosa

M-Cell Targeting of Whole Killed Bacteria Induces Protective Immunity against Gastrointestinal Pathogens▿

As the majority of human pathogens infect via a mucosal surface, delivery of killed vaccines by mucosal routes could potentially improve protection against many such organisms. Our ability to develop effective killed mucosal vaccines is inhibited by a lack of adjuvants that are safe and effective in humans. The Ulex europaeus agglutinin I (UEA-I) lectin specifically binds M cells lining the murine gastrointestinal tract. We explored the potential for M-cell-targeted vaccination of whole, killed Helicobacter pylori, the main causative agent of peptic ulcer disease and gastric cancer, and Campylobacter jejuni, the most common cause of diarrhea. Oral delivery of UEA-I-agglutinated H. pylori or C. jejuni induced a significant increase in both serum and intestinal antibody levels. This elevated response (i) required the use of whole bacteria, as it did not occur with lysate; (ii) was not mediated by formation of particulate clumps, as agglutination with a lectin with a different glycan specificity had no effect; and (iii) was not due to lectin-mediated, nonspecific immunostimulatory activity, as UEA-I codelivery with nonagglutinated bacteria did not enhance the response. Vaccination with UEA-I-agglutinated, killed whole H. pylori induced a protective response against subsequent live challenge that was as effective as that induced by cholera toxin adjuvant. Moreover, vaccination against C. jejuni by this approach resulted in complete protection against challenge in almost all animals. We believe that this is the first demonstration that targeting of whole killed bacteria to mucosal M cells can induce protective immunity without the addition of an immunostimulatory adjuvant

Muc1 mucin limits both Helicobacter pylori colonization of the murine gastric mucosa and associated gastritis

Background & Aims: The MUC1 mucin is expressed on the cell surface of epithelial cells fining the gastric mucosa. Epidemiologic studies suggest that functional allelic variations in the MUC1 gene may play a role in human susceptibility to Helicobacter pylori-associated pathologies, including gastric adenocarcinoma. We have evaluated the impact of Muc1 expression on the colonization and pathogenesis of gastric Helicobacter infections. Methods: Wild-type and Muc1-deficient mice were infected with H pylori and colonization and gastritis levels determined. Primary gastric cells were used to examine the impact of Muc1 expression on bacterial adherence. Results: Mice lacking Muc1 were colonized by 5-fold more H pylori within I day of infection, and this difference was maintained for at least 2 months postinfection. Mice heterozygous for the null Muc1 allele developed intermediate bacterial colonization. Although wild-type mice developed only a mild gastritis when infected for 2 months with H pylori, Muc1(-/-) mice developed an atrophic gastritis marked by loss of parietal. cells. We demonstrate H pylori adhesion to purified MUC1 and significantly increased adhesion to cultured murine Muc1 null gastric epithelial cells, suggesting that Muc1 acts as a decoy limiting binding to the cell surface. Conclusions: Muc1 provides a protective barrier, which limits both acute and chronic colonization by H pylori, as well as playing a major role in limiting the inflammation induced by Helicobacter infection. We propose that Muc1 restricts access of H pylori to the epithelial surface, hence reducing exposure of the host to proinflammatory bacterial products