1. Volume 139, Issue 5, Pages 1686-1698.e6 (November 2010)
Polyamines Impair Immunity to Helicobacter pylori by Inhibiting L-Arginine Uptake Required for Nitric Oxide Production 
Rupesh Chaturvedi, Mohammad Asim, Svea Hoge, Nuruddeen D. Lewis, Kshipra Singh, Daniel P. Barry, Thibaut de Sablet, M. Blanca Piazuelo, Aditya R. Sarvaria, Yulan Cheng, Ellen I. Closs, Robert A. Casero, Alain P. Gobert, Keith T. Wilson 
Gastroenterology 
Volume 139, Issue 5, Pages e6 (November 2010)
DOI: /j.gastro
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2. Figure 1
Induction of CAT2 and L-Arg uptake in RAW macrophages and effect of spermine and L-lysine. Macrophages were incubated with H pylori lysate (HPL) at a multiplicity of infection of 100. The mRNA expression for CAT1 and CAT2 was assessed at 6 hours after HPL stimulation ± the inhibitor (Inhib) spermine (Spm; 12.5 μmol/L) or L-lysine (Lys; 20 mmol/L) by (A) real-time PCR and (B) semiquantitative RT-PCR. (C) Western blot analysis for CAT2 (80 kilodaltons), iNOS (130 kilodaltons), and β-actin (42 kilodaltons) 24 hours after stimulation. (D) L-Arg transport assessed as uptake of 14C-L-Arg into cells at the times indicated. (E and F) NO levels measured as concentration of NO2−, at 24 hours after stimulation. (F) L-Arg was added to L-Arg-free, serum-free medium ± Spm. (A–F) Data are from 3 experiments performed in duplicate. (A and E) **P < .01 versus control; (D) *P < .05, **P < .01 versus control, HPL + Spm, and HPL + Lys; (E and F) §§P < .01, §§§P < .001 versus HPL alone.
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3. Figure 2
Effect of transfection of CAT2 siRNA on H pylori–stimulated iNOS expression and NO production. RAW cells were transiently transfected with either scrambled siRNA or siRNA to CAT2. (A) mRNA expression assessed by RT-PCR at 6 hours after stimulation with H pylori lysate (HPL). (B) Western blot analysis at 24 hours after stimulation. (A and B) n = 3. (C) L-Arg uptake and (D) NO2− levels, measured 24 hours after stimulation. **P < .01 versus control macrophages transfected with scrambled siRNA; §§P < .01 versus HPL-stimulated macrophages transfected with scrambled siRNA (n = 6). Scr, scrambled.
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4. Figure 3
Transfection of ODC siRNA enhances L-Arg uptake and iNOS protein expression in RAW macrophages. (A) Real-time PCR for ODC with stimulation with lysates of C rodentium (C rod), C jejuni (C jej), and H pylori lysate (HPL) all at a multiplicity of infection of 100. (B) Top panel, RT-PCR for CAT2; bottom panel, Western blot analysis for CAT2 and β-actin. (C) L-Arg transport. Scr, scrambled. *P < .05, **P < .01 versus control macrophages transfected with scrambled siRNA; §§P < .01 versus HPL-stimulated macrophages transfected with scrambled siRNA. (D) Western blot analysis for iNOS and β-actin. (E) Densitometry of iNOS Western blots, standardized to β-actin. (F) NO2− levels measured at 24 hours after stimulation. (E and F) **P < .01; ***P < .001 versus macrophages transfected with scrambled siRNA. n = 3–4 for all experiments.
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5. Figure 4
Induction of CAT2 in mouse and human H pylori gastritis tissues. (A) RT-PCR for CAT2 in mouse tissues from uninfected and H pylori–infected mice at 4-months postinoculation. (B) Real-time PCR for CAT2 in mouse tissues, n = 4 control and 9 infected. **P < .01 versus uninfected tissues. (C) CAT2 expression by real-time PCR in human tissues from uninfected patients with normal histology (n = 4), H pylori–negative gastritis (n = 4), and H pylori–positive gastritis (n = 6). **P < .01 versus normal; §§P < .01 versus H pylori–negative gastritis tissues. (D) Immunofluorescent detection of CAT2 that localizes to macrophages. Mouse tissues were stained for CAT2 detected with fluorescein isothiocyanate (FITC; green) and for the macrophage marker F4/80, detected with tetramethyl rhodamine isothiocyanate (red). Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). Co-localization is shown in merged images by yellow color. Magnification for each image is 200×, plus 600× for inset. (E) Immunohistochemistry for CAT2 in human biopsies, magnification is 200× in left and center panels, and 600× in right panel.
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6. Figure 5
Induction of ODC in mouse and human H pylori gastritis tissues. At 4 months after inoculation in mice, ODC was assessed by (A) RT-PCR, (B) real-time PCR, and (C) enzyme activity. (B and C) **P < .01 versus uninfected. (B) n = 4 uninfected and 9 infected and (C) n = 5 uninfected and 5 infected. In human beings, ODC was assessed by (D) RT-PCR, (E) real-time PCR, and (F) immunohistochemistry. (E) **P < .01 for H pylori–positive gastritis (n = 6) versus uninfected normal tissues (n = 4); §§P < .01 versus H pylori–negative gastritis tissues (n = 4). (F) Magnifications are 200× for both normal and H pylori–positive gastritis, and 600× in the inset.
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7. Figure 6
Deficient iNOS protein expression and NO production in gastric macrophages from H pylori–infected mice. Mice were inoculated with H pylori or broth control, and after 4 months gastric macrophages were isolated by positive selection. Cells from both uninfected (light bars) and H pylori–infected (black bars) mice then were treated ex vivo with vehicle control (Ctrl) or H pylori lysate (HPL) for 24 hours. (A–C) Real-time PCR analysis of CAT2, ODC, and iNOS mRNA levels, respectively. (D) iNOS protein levels determined by flow cytometry; data are composite results in relative fluorescence units. (E) Representative histogram tracings for the conditions shown in panelD. (A–D) *P < .05, **P < .01 versus unstimulated control cells from uninfected mice; #P < .05, ##P < .01 for HPL-stimulated cells from infected mice versus unstimulated cells from infected mice; §P < .05 for HPL-stimulated cells from infected mice versus HPL-stimulated cells from uninfected mice (n = 6). (F) NO2− levels (left panel) and L-Arg uptake (right panel). Spermine (Spm; 12.5 μmol/L) was added at the start of the ex vivo stimulation. *P < .05, **P < .01 versus unstimulated control cells from uninfected mice; §§P < .01 for HPL-stimulated cells from infected mice versus HPL-stimulated cells from uninfected mice; ##P < .01 for stimulation with HPL + Spm versus HPL only in cells from uninfected mice (n = 6).
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8. Figure 7
Inhibition of polyamine synthesis restores L-Arg uptake and iNOS-derived NO production and reduces H pylori colonization and gastritis. Mice were infected with H pylori or broth control and received DFMO (1% wt/vol) in the drinking water or water alone for 4 months, beginning the day after inoculation with H pylori. (A) Polyamine levels in stomach tissues. (B–D) Gastric macrophages were isolated from the groups indicated. (B) L-Arg uptake, (C) iNOS protein expression by flow cytometry, and (D) NO2− levels were assessed. (A–D) *P < .05, **P < .01, ***P < .001 versus uninfected control; §§P < .01, §§§P < .001 versus H pylori–infected without DFMO. (A) n = 6 mice per group, (B) n = 6–13 mice per group; each symbol represents a different mouse. (E) H pylori colonization and level of gastritis. Left panel, colonization by serial dilution and culture; center panel, quantitative PCR for ureB; right panel, histology scores for gastritis severity. Histologic scores in uninfected mice were 0.8 ± 0.3 (n = 14) and 0.7 ± 0.3 (n = 5), in mice receiving water alone or DFMO, respectively. §§P < .01, §§§P < .001 versus HP without DFMO. (F) Representative H&E staining of gastritis tissues. Arrowheads indicate crypt abscesses.
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9. Supplementary Figure 1
Induction of CAT2 and L-Arg uptake in murine peritoneal macrophages, and effect of spermine and L-lysine. Cells were elicited by intraperitoneal injection of Bio-Gel beads, and cells were harvested 5 days later. Macrophages were plated and stimulated with H pylori lysates at a multiplicity of infection (MOI) of 100. (A) The mRNA expression for CAT1 and CAT2 was assessed at 6 hours after H pylori lysate (HPL) stimulation ± spermine (Spm; 12.5 mmol/L) or L-lysine (Lys; 20 mmol/L) by real-time PCR. (B) L-Arg transport assessed as uptake of 14C-L-Arg into cells at the times indicated. (A and B) Data are the mean ± standard error (n = 3 experiments in duplicate). **P < .01 versus control; §P < .05 for HPL + Spm versus HPL alone.
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10. Supplementary Figure 2
Effect of inhibition of proteosomal degradation on iNOS protein levels in RAW macrophages transfected with CAT2 siRNA. RAW cells were stimulated with H pylori lysate (HPL) in the absence and presence of the proteosomal inhibitor, lactacystin (10 μmol/L), for the times indicated. (A) Western blot analysis for iNOS and β-actin in RAW cells transfected with scrambled (Scr) siRNA. (B) Western blot analysis for iNOS and β-actin in RAW cells transfected with CAT2 siRNA. Similar results were obtained in 2 experiments.
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11. Supplementary Figure 3
Effect of transfection of ODC siRNA on L-Arg uptake, and NO production in RAW macrophages activated with live H pylori. (A) Cells transfected with scrambled siRNA or ODC siRNA were co-cultured with live H pylori (multiplicity of infection [MOI], 10) for the times indicated. L-Arg transport was measured as in Figure 1 for the conditions marked. *P < .05, **P < .01 versus H pylori–stimulated macrophages transfected with scrambled siRNA. L-Arg transport in unstimulated RAW cells transfected with scrambled siRNA was 1.9 ± 0.2 nmol 14C-L-Arg • min−1 • mg protein−1 and in unstimulated RAW cells transfected with CAT2 siRNA was 2.2 ± 0.8 nmol 14C-L-Arg • min−1 • mg protein−1. (B) NO2− levels measured at 24 hours after co-culture with live H pylori (MOI, 10). *P < .05; ***P < .001 versus macrophages transfected with scrambled siRNA. (C) Live H pylori (MOI, 100) was placed above Transwell filter supports (0.4-mm pore size), and NO2− levels were measured after 24 hours. **P < .01 versus macrophages transfected with scrambled siRNA. (A–C) Data are from 2 separate experiments performed in duplicate.
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12. Supplementary Figure 4
Effect of transfection of ODC siRNA on L-Arg uptake, and NO production in RAW macrophages activated with C jejuni or C rodentium. Cells were transfected with scrambled or ODC siRNA and activated with lysates of (A and B) C jejuni and (C and D) C rodentium for the times indicated at a multiplicity of infection (MOI) of 100. (A and C) L-Arg transport, measured as in Figure 1 and Supplementary Figure 1 for the conditions marked. (B and D) NO2− levels. L-Arg uptake levels in unstimulated RAW cells transfected with scrambled (Scr) siRNA and ODC siRNA were as listed in Supplementary Figure 3. (A–D) Data are from 2 separate experiments performed in duplicate.
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13. Supplementary Figure 5
Summary of alterations in host responses in macrophages owing to the effects of ODC-derived spermine on L-Arg uptake. CAT2 is the primary transporter of L-Arg into macrophages activated by H pylori. Extracellular L-Arg must be transported into stimulated macrophages to facilitate iNOS translation that is required to generate sufficient quantities of NO to kill extracellular H pylori. L-lysine acts as a competitive inhibitor that blocks L-Arg uptake and NO production in H pylori–stimulated macrophages. The induction of ODC by H pylori results in the generation of the polyamines putrescine, spermidine, and spermine. Spermine can be back-converted to spermidine, or when produced in large quantities by ODC it blocks the uptake of L-Arg by CAT2, without altering CAT2 mRNA or protein expression. Once in the cell, L-Arg also is used by arginase II to produce L-ornithine, which is used as the substrate for ODC to generate more polyamines. Inhibition of ODC with DFMO blocks the overproduction of polyamines, and, thus, restores iNOS synthesis, NO production, and antimicrobial defense against H pylori.
Gastroenterology  , e6DOI: ( /j.gastro )
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