Daniel P. Barry

L-arginine Supplementation Improves Responses to Injury and Inflammation in Dextran Sulfate Sodium Colitis

Inflammatory bowel disease (IBD), consisting of Crohns disease and ulcerative colitis (UC), results in substantial morbidity and is difficult to treat. New strategies for adjunct therapies are needed. One candidate is the semi-essential amino acid, L-arginine (L-Arg), a complementary medicine purported to be an enhancer of immunity and vitality in the lay media. Using dextran sulfate sodium (DSS) as a murine colonic injury and repair model with similarities to human UC, we assessed the effect of L-Arg, as DSS induced increases in colonic expression of the y + cationic amino acid transporter 2 (CAT2) and L-Arg uptake. L-Arg supplementation improved the clinical parameters of survival, body weight loss, and colon weight, and reduced colonic permeability and the number of myeloperoxidase-positive neutrophils in DSS colitis. Luminex-based multi-analyte profiling demonstrated that there was a marked reduction in proinflammatory cytokine and chemokine expression with L-Arg treatment. Genomic analysis by microarray demonstrated that DSS-treated mice supplemented with L-Arg clustered more closely with mice not exposed to DSS than to those receiving DSS alone, and revealed that multiple genes that were upregulated or downregulated with DSS alone exhibited normalization of expression with L-Arg supplementation. Additionally, L-Arg treatment of mice with DSS colitis resulted in increased ex vivo migration of colonic epithelial cells, suggestive of increased capacity for wound repair. Because CAT2 induction was sustained during L-Arg treatment and inducible nitric oxide (NO) synthase (iNOS) requires uptake of L-Arg for generation of NO, we tested the effect of L-Arg in iNOS−/− mice and found that its benefits in DSS colitis were eliminated. These preclinical studies indicate that L-Arg supplementation could be a potential therapy for IBD, and that one mechanism of action may be functional enhancement of iNOS activity.

Polyamines Impair Immunity to Helicobacter pylori by Inhibiting L-Arginine Uptake Required for Nitric Oxide Production

BACKGROUND & AIMS Helicobacter pylori-induced immune responses fail to eradicate the bacterium. Nitric oxide (NO) can kill H pylori. However, translation of inducible NO synthase (iNOS) and NO generation by H pylori-stimulated macrophages is inhibited by the polyamine spermine derived from ornithine decarboxylase (ODC), and is dependent on availability of the iNOS substrate L-arginine (L-Arg). We determined if spermine inhibits iNOS-mediated immunity by reducing L-Arg uptake into macrophages. METHODS Levels of the inducible cationic amino acid transporter (CAT)2, ODC, and iNOS were measured in macrophages and H pylori gastritis tissues. L-Arg uptake, iNOS expression, and NO levels were assessed in cells with small interfering RNA knockdown of CAT2 or ODC, and in gastric macrophages. The ODC inhibitor, α-difluoromethylornithine, was administered to H pylori-infected mice for 4 months after inoculation. RESULTS H pylori induced CAT2 and uptake of L-Arg in RAW 264.7 or primary macrophages. Addition of spermine or knockdown of CAT2 inhibited L-Arg uptake, NO production, and iNOS protein levels, whereas knockdown of ODC had the opposite effect. CAT2 and ODC were increased in mouse and human H pylori gastritis tissues and localized to macrophages. Gastric macrophages from H pylori-infected mice showed increased ODC expression, and attenuated iNOS and NO levels upon ex vivo H pylori stimulation versus cells from uninfected mice. α-Difluoromethylornithine treatment of infected mice restored L-Arg uptake, iNOS protein expression, and NO production in gastric macrophages, and significantly reduced both H pylori colonization levels and gastritis severity. CONCLUSIONS Up-regulation of ODC in gastric macrophages impairs host defense against H pylori by suppressing iNOS-derived NO production.

Arginase II Restricts Host Defense to Helicobacter pylori by Attenuating Inducible Nitric Oxide Synthase Translation in Macrophages

Helicobacter pylori infection of the stomach causes peptic ulcer disease and gastric cancer. Despite eliciting a vigorous immune response, the bacterium persists for the life of the host. An important antimicrobial mechanism is the production of NO derived from inducible NO synthase (iNOS). We have reported that macrophages can kill H. pylori in vitro by an NO-dependent mechanism, but supraphysiologic levels of the iNOS substrate l-arginine are required. Because H. pylori induces arginase activity in macrophages, we determined if this restricts NO generation by reducing l-arginine availability. Inhibition of arginase with S-(2-boronoethyl)-l-cysteine (BEC) significantly enhanced NO generation in H. pylori-stimulated RAW 264.7 macrophages by enhancing iNOS protein translation but not iNOS mRNA levels. This effect resulted in increased killing of H. pylori that was attenuated with an NO scavenger. In contrast, inhibition of arginase in macrophages activated by the colitis-inducing bacterium Citrobacter rodentium increased NO without affecting iNOS levels. H. pylori upregulated levels of arginase II (Arg2) mRNA and protein, which localized to mitochondria, whereas arginase I was not induced. Increased iNOS protein and NO levels were also demonstrated by small interfering RNA knockdown of Arg2 and in peritoneal macrophages from C57BL/6 Arg2−/− mice. In H. pylori-infected mice, treatment with BEC or deletion of Arg2 increased iNOS protein levels and NO generation in gastric macrophages, but treatment of Arg2−/− mice with BEC had no additional effect. These studies implicate Arg2 in the immune evasion of H. pylori by causing intracellular depletion of l-arginine and thus reduction of NO-dependent bactericidal activity.

The Apolipoprotein E-Mimetic Peptide COG112 Inhibits NF-κB Signaling, Proinflammatory Cytokine Expression, and Disease Activity in Murine Models of Colitis

Inflammatory bowel disease (IBD), consisting of Crohns disease and ulcerative colitis, is a source of substantial morbidity and remains difficult to treat. New strategies for beneficial anti-inflammatory therapies would be highly desirable. Apolipoprotein (apo) E has immunomodulatory effects and synthetically derived apoE-mimetic peptides are beneficial in models of sepsis and neuroinflammation. We have reported that the antennapedia-linked apoE-mimetic peptide COG112 inhibits the inflammatory response to the colitis-inducing pathogen Citrobacter rodentium in vitro by inhibiting NF-κB activation. We now determined the effect of COG112 in mouse models of colitis. Using C. rodentium as an infection model, and dextran sulfate sodium (DSS) as an injury model, mice were treated with COG112 by intraperitoneal injection. With C. rodentium, COG112 improved the clinical parameters of survival, body weight, colon weight, and histologic injury. With DSS, COG112 ameliorated the loss of body weight, reduction in colon length, and histologic injury, whether administered concurrently with induction of colitis, during induction plus recovery, or only during the recovery phase of disease. In both colitis models, COG112 inhibited colon tissue inducible nitric-oxide synthase (iNOS), KC, TNF-α, IFN-γ, and IL-17 mRNA expression, and reduced nuclear translocation of NF-κB, as determined by immunoblot and immunofluorescence confocal microscopy. IκB kinase (IKK) activity was also reduced, which is necessary for activation of the canonical NF-κB pathway. Isolated colonic epithelial cells exhibited marked attenuation of expression of iNOS and the CXC chemokines KC and MIP-2. These studies indicate that apoE-mimetic peptides such as COG112 are novel potential therapies for IBD.

Helicobacter pylori Induces ERK-dependent Formation of a Phospho-c-Fos·c-Jun Activator Protein-1 Complex That Causes Apoptosis in Macrophages

Macrophages are essential components of innate immunity, and apoptosis of these cells impairs mucosal defense to microbes. Helicobacter pylori is a gastric pathogen that infects half of the world population and causes peptic ulcer disease and gastric cancer. The host inflammatory response fails to eradicate the organism. We have reported that H. pylori induces apoptosis of macrophages by generation of polyamines from ornithine decarboxylase (ODC), which is dependent on c-Myc as a transcriptional enhancer. We have now demonstrated that expression of c-Myc requires phosphorylation and nuclear translocation of ERK, which results in phosphorylation of c-Fos and formation of a specific activator protein (AP)-1 complex. Electromobility shift assay and immunoprecipitation revealed a previously unrecognized complex of phospho-c-Fos (pc-Fos) and c-Jun in the nucleus. Fluorescence resonance energy transfer demonstrated the interaction of pc-Fos and c-Jun. The capacity of this AP-1 complex to bind to putative AP-1 sequences was demonstrated by oligonucleotide pulldown and fluorescence polarization. Binding of the pc-Fos·c-Jun complex to the c-Myc promoter was demonstrated by chromatin immunoprecipitation. A dominant-negative c-Fos inhibited H. pylori-induced expression of c-Myc and ODC and apoptosis. H. pylori infection of mice induced a rapid infiltration of macrophages into the stomach. Concomitant apoptosis depleted these cells, and this was associated with formation of a pc-Fos·c-Jun complex. Treatment of mice with an inhibitor of ERK phosphorylation attenuated phosphorylation of c-Fos, expression of ODC, and apoptosis in gastric macrophages. A unique AP-1 complex in gastric macrophages contributes to the immune escape of H. pylori.

The apolipoprotein E-mimetic peptide COG112 inhibits the inflammatory response to citrobacter rodentium in colonic epithelial cells by preventing NF-κB activation

Inflammatory bowel disease arises from the interplay between luminal bacteria and the colonic mucosa. Targeted inhibition of pro-inflammatory pathways without global immunosuppression is highly desirable. Apolipoprotein (apo) E has immunomodulatory effects and synthetically derived apoE-mimetic peptides are beneficial in models of sepsis and neuroinflammation. Citrobacter rodentium is the rodent equivalent of enteropathogenic Escherichia coli, and it causes colitis in mice by colonizing the surface of colonic epithelial cells and inducing signaling events. We have reported that mice deficient in inducible nitric-oxide (NO) synthase (iNOS) have attenuated C. rodentium-induced colitis. We used young adult mouse colon (YAMC) cells that mimic primary colonic epithelial cells to study effects of an antennapedia-linked apoE-mimetic peptide, COG112, on C. rodentium-activated cells. COG112 significantly attenuated induction of NO production, and iNOS mRNA and protein expression, in a concentration-dependent manner. COG112 inhibited the C. rodentium-stimulated induction of iNOS and the CXC chemokines KC and MIP-2 to the same degree as the NF-κB inhibitors MG132 or BAY 11-7082, and there was no additive effect when COG112 and these inhibitors were combined. COG112 significantly reduced nuclear translocation of NF-κB, when assessed by electromobility shift assay, immunoblotting, and immunofluorescence for p65. This correlated with inhibition of both C. rodentium-stimulated IκB-α phosphorylation and degradation, and IκB kinase activity, which occurred by inhibition of IκB kinase complex formation rather than by a direct effect on the enzyme itself. These studies indicate that apoE-mimetic peptides may have novel therapeutic potential by inhibiting NF-κB-driven proinflammatory epithelial responses to pathogenic colonic bacteria.

Activation of EGFR and ERBB2 by Helicobacter pylori Results in Survival of Gastric Epithelial Cells With DNA Damage

BACKGROUND & AIMS The gastric cancer-causing pathogen Helicobacter pylori up-regulates spermine oxidase (SMOX) in gastric epithelial cells, causing oxidative stress-induced apoptosis and DNA damage. A subpopulation of SMOX(high) cells are resistant to apoptosis, despite their high levels of DNA damage. Because epidermal growth factor receptor (EGFR) activation can regulate apoptosis, we determined its role in SMOX-mediated effects. METHODS SMOX, apoptosis, and DNA damage were measured in gastric epithelial cells from H. pylori-infected Egfr(wa5) mice (which have attenuated EGFR activity), Egfr wild-type mice, or in infected cells incubated with EGFR inhibitors or deficient in EGFR. A phosphoproteomic analysis was performed. Two independent tissue microarrays containing each stage of disease, from gastritis to carcinoma, and gastric biopsy specimens from Colombian and Honduran cohorts were analyzed by immunohistochemistry. RESULTS SMOX expression and DNA damage were decreased, and apoptosis increased in H. pylori-infected Egfr(wa5) mice. H. pylori-infected cells with deletion or inhibition of EGFR had reduced levels of SMOX, DNA damage, and DNA damage(high) apoptosis(low) cells. Phosphoproteomic analysis showed increased EGFR and erythroblastic leukemia-associated viral oncogene B (ERBB)2 signaling. Immunoblot analysis showed the presence of a phosphorylated (p)EGFR-ERBB2 heterodimer and pERBB2; knockdown of ErbB2 facilitated apoptosis of DNA damage(high) apoptosis(low) cells. SMOX was increased in all stages of gastric disease, peaking in tissues with intestinal metaplasia, whereas pEGFR, pEGFR-ERBB2, and pERBB2 were increased predominantly in tissues showing gastritis or atrophic gastritis. Principal component analysis separated gastritis tissues from patients with cancer vs those without cancer. pEGFR, pEGFR-ERBB2, pERBB2, and SMOX were increased in gastric samples from patients whose disease progressed to intestinal metaplasia or dysplasia, compared with patients whose disease did not progress. CONCLUSIONS In an analysis of gastric tissues from mice and patients, we identified a molecular signature (based on levels of pEGFR, pERBB2, and SMOX) for the initiation of gastric carcinogenesis.

Immune Evasion by Helicobacter pylori Is Mediated by Induction of Macrophage Arginase II

Helicobacter pylori infection persists for the life of the host due to the failure of the immune response to eradicate the bacterium. Determining how H. pylori escapes the immune response in its gastric niche is clinically important. We have demonstrated in vitro that macrophage NO production can kill H. pylori, but induction of macrophage arginase II (Arg2) inhibits inducible NO synthase (iNOS) translation, causes apoptosis, and restricts bacterial killing. Using a chronic H. pylori infection model, we determined whether Arg2 impairs host defense in vivo. In C57BL/6 mice, expression of Arg2, but not arginase I, was abundant and localized to gastric macrophages. Arg2−/− mice had increased histologic gastritis and decreased bacterial colonization compared with wild-type (WT) mice. Increased gastritis scores correlated with decreased colonization in individual Arg2−/− mice but not in WT mice. When mice infected with H. pylori were compared, Arg2−/− mice had more gastric macrophages, more of these cells were iNOS+, and these cells expressed higher levels of iNOS protein, as determined by flow cytometry and immunofluorescence microscopy. There was enhanced nitrotyrosine staining in infected Arg2−/− versus WT mice, indicating increased NO generation. Infected Arg2−/− mice exhibited decreased macrophage apoptosis, as well as enhanced IFN-γ, IL-17a, and IL-12p40 expression, and reduced IL-10 levels consistent with a more vigorous Th1/Th17 response. These studies demonstrate that Arg2 contributes to the immune evasion of H. pylori by limiting macrophage iNOS protein expression and NO production, mediating macrophage apoptosis, and restraining proinflammatory cytokine responses.

EGFR regulates macrophage activation and function in bacterial infection

EGFR signaling regulates macrophage function, but its role in bacterial infection has not been investigated. Here, we assessed the role of macrophage EGFR signaling during infection with Helicobacter pylori, a bacterial pathogen that causes persistent inflammation and gastric cancer. EGFR was phosphorylated in murine and human macrophages during H. pylori infection. In human gastric tissues, elevated levels of phosphorylated EGFR were observed throughout the histologic cascade from gastritis to carcinoma. Deleting Egfr in myeloid cells attenuated gastritis and increased H. pylori burden in infected mice. EGFR deficiency also led to a global defect in macrophage activation that was associated with decreased cytokine, chemokine, and NO production. We observed similar alterations in macrophage activation and disease phenotype in the Citrobacter rodentium model of murine infectious colitis. Mechanistically, EGFR signaling activated NF-κB and MAPK1/3 pathways to induce cytokine production and macrophage activation. Although deletion of Egfr had no effect on DC function, EGFR-deficient macrophages displayed impaired Th1 and Th17 adaptive immune responses to H. pylori, which contributed to decreased chronic inflammation in infected mice. Together, these results indicate that EGFR signaling is central to macrophage function in response to enteric bacterial pathogens and is a potential therapeutic target for infection-induced inflammation and associated carcinogenesis.

Heme Oxygenase-1 Dysregulates Macrophage Polarization and the Immune Response to Helicobacter pylori

Helicobacter pylori incites a futile inflammatory response, which is the key feature of its immunopathogenesis. This leads to the ability of this bacterial pathogen to survive in the stomach and cause peptic ulcers and gastric cancer. Myeloid cells recruited to the gastric mucosa during H. pylori infection have been directly implicated in the modulation of host defense against the bacterium and gastric inflammation. Heme oxygenase-1 (HO-1) is an inducible enzyme that exhibits anti-inflammatory functions. Our aim was to analyze the induction and role of HO-1 in macrophages during H. pylori infection. We now show that phosphorylation of the H. pylori virulence factor cytotoxin-associated gene A (CagA) in macrophages results in expression of hmox-1, the gene encoding HO-1, through p38/NF (erythroid-derived 2)-like 2 signaling. Blocking phagocytosis prevented CagA phosphorylation and HO-1 induction. The expression of HO-1 was also increased in gastric mononuclear cells of human patients and macrophages of mice infected with cagA+ H. pylori strains. Genetic ablation of hmox-1 in H. pylori–infected mice increased histologic gastritis, which was associated with enhanced M1/Th1/Th17 responses, decreased regulatory macrophage (Mreg) response, and reduced H. pylori colonization. Gastric macrophages of H. pylori–infected mice and macrophages infected in vitro with this bacterium showed an M1/Mreg mixed polarization type; deletion of hmox-1 or inhibition of HO-1 in macrophages caused an increased M1 and a decrease of Mreg phenotype. These data highlight a mechanism by which H. pylori impairs the immune response and favors its own survival via activation of macrophage HO-1.