Jungsoo Joo

Olfm4 deletion enhances defense against Staphylococcus aureus in chronic granulomatous disease

Chronic granulomatous disease (CGD) patients have recurrent life-threatening bacterial and fungal infections. Olfactomedin 4 (OLFM4) is a neutrophil granule protein that negatively regulates host defense against bacterial infection. The goal of this study was to evaluate the impact of Olfm4 deletion on host defense against Staphylococcus aureus and Aspergillus fumigatus in a murine X-linked gp91phox-deficiency CGD model. We found that intracellular killing and in vivo clearance of S. aureus, as well as resistance to S. aureus sepsis, were significantly increased in gp91phox and Olfm4 double-deficient mice compared with CGD mice. The activities of cathepsin C and its downstream proteases (neutrophil elastase and cathepsin G) and serum levels of IL-1β, IL-6, IL-12p40, CXCL2, G-CSF, and GM-CSF in Olfm4-deficient as well as gp91phox and Olfm4 double-deficient mice were significantly higher than those in WT and CGD mice after challenge with S. aureus. We did not observe enhanced defense against A. fumigatus in Olfm4-deficient mice using a lung infection model. These results show that Olfm4 deletion can successfully enhance immune defense against S. aureus, but not A. fumigatus, in CGD mice. These data suggest that OLFM4 may be an important target in CGD patients for the augmentation of host defense against bacterial infection.

Prevalence of Helicobacter pylori Infection and its Relation with Body Mass Index in a Chinese Population

Helicobacter pylori infection is highly prevalent worldwide. The association between obesity and H. pylori infection is controversial in the literature. This study aims to investigate the prevalence of H. pylori infection and its relation with body mass index (BMI) in a Chinese population.

Differences in the Phenotype, Cytokine Gene Expression Profiles, and In Vivo Alloreactivity of T Cells Mobilized with Plerixafor Compared with G-CSF

Plerixafor (Mozobil) is a CXCR4 antagonist that rapidly mobilizes CD34+ cells into circulation. Recently, plerixafor has been used as a single agent to mobilize peripheral blood stem cells for allogeneic hematopoietic cell transplantation. Although G-CSF mobilization is known to alter the phenotype and cytokine polarization of transplanted T cells, the effects of plerixafor mobilization on T cells have not been well characterized. In this study, we show that alterations in the T cell phenotype and cytokine gene expression profiles characteristic of G-CSF mobilization do not occur after mobilization with plerixafor. Compared with nonmobilized T cells, plerixafor-mobilized T cells had similar phenotype, mixed lymphocyte reactivity, and Foxp3 gene expression levels in CD4+ T cells, and did not undergo a change in expression levels of 84 genes associated with Th1/Th2/Th3 pathways. In contrast with plerixafor, G-CSF mobilization decreased CD62L expression on both CD4 and CD8+ T cells and altered expression levels of 16 cytokine-associated genes in CD3+ T cells. To assess the clinical relevance of these findings, we explored a murine model of graft-versus-host disease in which transplant recipients received plerixafor or G-CSF mobilized allograft from MHC-matched, minor histocompatibility–mismatched donors; recipients of plerixafor mobilized peripheral blood stem cells had a significantly higher incidence of skin graft-versus-host disease compared with mice receiving G-CSF mobilized transplants (100 versus 50%, respectively, p = 0.02). These preclinical data show plerixafor, in contrast with G-CSF, does not alter the phenotype and cytokine polarization of T cells, which raises the possibility that T cell–mediated immune sequelae of allogeneic transplantation in humans may differ when donor allografts are mobilized with plerixafor compared with G-CSF.

Screening Helicobacter pylori genes induced during infection of mouse stomachs

AIM To investigate the effect of in vivo environment on gene expression in Helicobacter pylori (H. pylori) as it relates to its survival in the host. METHODS In vivo expression technology (IVET) systems are used to identify microbial virulence genes. We modified the IVET-transcriptional fusion vector, pIVET8, which uses antibiotic resistance as the basis for selection of candidate genes in host tissues to develop two unique IVET-promoter-screening vectors, pIVET11 and pIVET12. Our novel IVET systems were developed by the fusion of random Sau3A DNA fragments of H. pylori and a tandem-reporter system of chloramphenicol acetyltransferase and beta-galactosidase. Additionally, each vector contains a kanamycin resistance gene. We used a mouse macrophage cell line, RAW 264.7 and mice, as selective media to identify specific genes that H. pylori expresses in vivo. Gene expression studies were conducted by infecting RAW 264.7 cells with H. pylori. This was followed by real time polymerase chain reaction (PCR) analysis to determine the relative expression levels of in vivo induced genes. RESULTS In this study, we have identified 31 in vivo induced (ivi) genes in the initial screens. These 31 genes belong to several functional gene families, including several well-known virulence factors that are expressed by the bacterium in infected mouse stomachs. Virulence factors, vacA and cagA, were found in this screen and are known to play important roles in H. pylori infection, colonization and pathogenesis. Their detection validates the efficacy of these screening systems. Some of the identified ivi genes have already been implicated to play an important role in the pathogenesis of H. pylori and other bacterial pathogens such as Escherichia coli and Vibrio cholerae. Transcription profiles of all ivi genes were confirmed by real time PCR analysis of H. pylori RNA isolated from H. pylori infected RAW 264.7 macrophages. We compared the expression profile of H. pylori and RAW 264.7 coculture with that of H. pylori only. Some genes such as cagA, vacA, lpxC, murI, tlpC, trxB, sodB, tnpB, pgi, rbfA and infB showed a 2-20 fold upregulation. Statistically significant upregulation was obtained for all the above mentioned genes (P < 0.05). tlpC, cagA, vacA, sodB, rbfA, infB, tnpB, lpxC and murI were also significantly upregulated (P < 0.01). These data suggest a strong correlation between results obtained in vitro in the macrophage cell line and in the intact animal. CONCLUSION The positive identification of these genes demonstrates that our IVET systems are powerful tools for studying H. pylori gene expression in the host environment.

Genetic organization and conjugal plasmid DNA transfer of pHP69, a plasmid from a Korean isolate of Helicobacter pylori

We isolated pHP69, a 9,153 bp plasmid from Helicobacterpylori with a 33.98% (G+C) content. We identified 11 open reading frames (ORFs), including replication initiation protein A (repA), fic (cAMP-induced filamentation protein), mccC, mccB, mobA, mobD, mobB, and mobC, as well as four 22 bp tandem repeat sequences. The nucleic acid and predicted amino acid sequences of these ORFs exhibited significant homology to those of other H. pylori plasmids. pHP69 repA encodes a replication initiation protein and its amino acid sequence is similar to those of replicase proteins from theta-type plasmids. pHP69 contains two types of repeat sequences (R1 and R2), a MOBHEN family mobilization region comprising mobC, mobA, mobB, and mobD, and genes encoding microcin B and C. Among the 36 H. pylori strains containing plasmids, mobA or mccBC are present in 12 or 6, respectively and 3 contain both genes. To examine intrinsic capability of H. pylori for conjugative plasmid transfer, a shuttle vector pBHP69KH containing pHP69 and replication origin of pBR322 was constructed. It was shown that this vector could stably replicate and be mobilized among clinical H. pylori strains and demonstrated to gene transfer by natural plasmid.

Cutting Edge: Expression of IRF8 in Gastric Epithelial Cells Confers Protective Innate Immunity against Helicobacter pylori Infection.

IFN regulatory factor 8 (IRF8) is expressed in many types of blood cells and plays critical roles in cellular differentiation and function. However, the role of IRF8 in nonhematopoietic systems remains poorly understood. In this study, we provide evidence that IRF8 is a transcriptional modulator of the gastric mucosa necessary for limiting Helicobacter pylori colonization. H. pylori infection significantly upregulated expression of IRF8, which, in turn, promoted IFN-γ expression by gastric epithelial cells. Mice deficient in IRF8 exhibited increased H. pylori colonization and aborted induction of mucosal IFN-γ. Genome-wide analyses of IFN-γ–treated gastric epithelial cells by chromatin immunoprecipitation sequencing and RNA sequencing led to the identification of IRF8 target genes, with many belonging to the IFN-regulated gene family that was observed previously in immune cells. Our results identify the IRF8–IFN-γ circuit as a novel gastric innate immune mechanism in the host defense against infection with H. pylori.

Investigating the Role of Helicobacter pylori PriA Protein

In bacteria, PriA protein, a conserved DEXH‐type DNA helicase, plays a central role in replication restart at stalled replication forks. Its unique DNA binding property allows it to recognize and stabilize stalled forks and the structures derived from them. PriA plays a very critical role in replication fork stabilization and DNA repair in E. coli and N. gonorrhoeae. In our in vivo expression technology screen, priA gene was induced in vivo when Helicobacter pylori infects mouse stomach.

Mo1791 MyD88 Has a Key Role for OLFM4, a Novel Anti-Inflammatory Mediator in H. pylori Infection

G A A b st ra ct s an important role in the homeostasis of PP in response to the microbiota. However, we do not know the respective roles of Nod2 in the epithelial and immune compartments of PP. Methods: To investigate the part of Nod2 in epithelial and immune compartments within PP, chimeric mice were developed by reconstituting sublethally irradiated wild-type (WT) mice with bone marrow (BM) stem cells from Nod2 Knockout mice (KO) and vice versa. Three months following BM transplantation, paracellular permeability (FITC 4kD dextran flux) and E. coli translocation were assessed in Ussing chamber. The number of immune cells and cellular composition from PP of chimeric mice were determined by flow cytometry. Results: An excess of permeability and E. coli translocation was observed in PP of KO mice reconstituted by BM of KO mice (KO→KO) compared to WT mice reconstituted by BM WT mice (WT→WT). Permeability and E. coli translocation were also higher in PP from chimeric mice that did not express Nod2 in the immune compartment but expressed it in epithelial cells (KO→WT). These excesses of permeability and translocation in PP were comparable to Nod2 Knockout mice (KO→KO). Conversely, permeability and E. coli translocation from chimeric mice expressing Nod2 in immune cells but not in epithelial cells (WT→KO) were similar to controls. An excess of CD3+ T-cells but a decreased number of dendritic cells were observed in the PP from chimeric mice that did not express Nod2 in immune cells (KO→KO and KO→WT). Conclusion: Nod2 deficiency in the immune compartment is sufficient to alter the cellular composition of PP and the functionality of the follicle associated epithelium. These results highlight the importance of Nod2 in the dialogue between the immune and epithelial cells and may contribute to understand CD mechanisms.

Su1933 Investigating the Role of Helicobacter pylori Pria Protein

G A A b st ra ct s suppresses the development of MGC. To clarify the molecular changes due to H. pylori eradication, in this study, we evaluated differences in molecular alterations related to carcinogenesis in background mucosa of GC between H. pylori positive and negative patients. Materials and Methods: Seventy-seven consecutive patients who underwent ER for GC were enrolled. H. pylori status was analyzed in each patient by two methods: Giemsa staining and serum H. pylori-IgG antibody. Biopsy specimens were obtained from all cases, and DNA was extracted from intestinal metaplasia (IM), a precancerous lesion, via laser capture microdissection. Microsatellite instability (MSI) was evaluated at five loci based on the Bethesda panel; promoter methylation at hMLH1, E-cadherin, p16, and APC was assessed using methylation-specific polymerase chain reaction. Reactivity of monoclonal antibody for colonic phenotype (mAb Das-1) to IM was also evaluated by an immunoperoxidase assay. Results: H. pylori status was negative in 32 of 77 participants (41.6%). The incidence of MSI and hypermethylation at hMLH1, E-cadherin, p16, and APC genes in IM for H. pylori positive and negative patients, respectively, were 47.6%, 23.7%, 10.5% and 56.1% and 36.7%, 6.7%, 3.4%, 38.7% and 35.5%. There were no significant differences in molecular alterations between H. pylori positive and negative cases. mAb Das-1 reactivity to IM was 70.5% in H. pylori positive and 71.0% in H. pylori negative patients, demonstrating no significant difference. Conclusions: Early GC occurs frequently even in H. pylori negative patients. There were no significant differences in molecular alterations in IM between H. pylori positive and negative patients, suggesting that H. pylori eradication may not change the course of molecular alterations in background mucosa once GC has occured in the stomach.

Sa1758 NLRP3 Inflammasome Is Activated in Mice by Helicobacter pylori Infection

Background and Aim:Murine norovirus (MNV) infection was shown to induce histopathological changes in the intestine of conventional housed mice or lethal systemic infection in immunocompromised mice. The mechanism which triggered the inflammation is not well understood. In the present study, the influence of MNV infection on histological and immunological characteristics of mucosal inflammation in germfree and Schaedler flora colonized IL10-deficient (Il10-/-) mouse model was examined. Methods: Germfree (GF) C57Bl/6J-Il10-/mice and GF mice colonized with Schaedler Flora were monitored after MNV infection for structural and functional intestinal barrier changes using histology, RTqPCR, ELISA and TUNEL assay. Results: No inflammatory lesions were observed in GF B6Il10-/and MNV-infected mice whereas in MNV-infected GF mice colonized with Schaedler Flora inflammatory lesions were detected comparable to SPF mice infected with MNV. Interferon inducible genes were increased in the small intestine of GFmice 48h after infection. However, four weeks after infection GF mice showed no increased IFNgamma production, whereas in Schaedler Flora colonized mice IFNgamma production was increased similar to SPF infected mice. Although there is no reduction of gene expression of tight junction molecules and an enhanced rate of epithelial apoptosis 48h after infection of GF mice the expression level changed within 4 weeks in MNV infected Schaedler Flora. Conclusions: MNV induces inflammation and epithelial barrier disruption that depends on the presence of the bacteria. Thus, MNV and enteric microbiota together represent a potent colitogenic stimulus and inflammatory trigger.