101 Ornithine Decarboxylase Disrupts Host Immune Tolerance in Helicobacter pylori Infection by Attenuating Macrophage Production of TGF-β and Nitric Oxide

Abstract

Background: Helicobacter pylori (Hp) infection results in a Th1/Th17 polarized inflammatory response. We have reported that expression of ornithine decarboxylase (ODC), the ratelimiting enzyme for polyamine synthesis, is increased in macrophages (Macs) in mouse and human Hp gastritis tissues, and that inhibition of ODC reduces gastritis and Hp colonization in mice. Our aim was to determine if ODC impairs host tolerance induced by Macs and promotes STAT1-mediated Th1/Th17 adaptive immune responses to Hp. Methods: Wild type (WT) and ODC+/C57BL/6 mice were infected with Hp SS1 for 4 months. We measured in gastric tissues: Hp colonization by qPCR, histological inflammation (0-12), TGFβ by Western blotting, and cytokines by Luminex assay. TGFβ in gastric Macs (GMacs) was measured by qPCR and immunofluorescencemicroscopy. Splenic Macs (SMacs) were isolated and activated with French-pressed Hp lysates (HPL); levels of TGFβ mRNA and nitric oxide (NO) production were measured by qPCR and Griess assay, respectively. Splenocytes, or co-culture of splenocytes depleted of Macs (SMacs-) with SMacs were activated with HPL for 48 h ± anti-TGF-β neutralizing antibody or the iNOS inhibitor 1400W, and IFNγ, IL17, IL-10, pSTAT1, and pSTAT3 were analyzed in CD4+ cells by flow cytometry. Results: In Hp-infected ODC+/versus WT mice, there was decreased histological gastritis (from 5.8 ± 0.3 to 3.2 ± 0.2; p , 0.05) and Hp colonization was reduced by 2 log orders ( p , 0.05). In SMacs from ODC+/-vs. WT mice, ex vivo activation with HPL increased TGFβ mRNA levels by 10-fold (p , 0.01) and NO production (from 6.2 ± 0.4 to 15.3 ± 0.9 μM; p , 0.01), but there was no difference in IL-10 expression. In HPL-stimulated splenocytes, IFNγ+ and IL-17+cells were increased in WT mice, but IL-10+ cells were increased in ODC+/mice. Pretreatment of splenocytes with anti-TGFβ antibody or 1400W before ex vivo activation increased IFN-γ+ and IL-17+, but not IL-10+ cells in ODC+/mice (Table 1). When WT SMacs were co-cultured with WT or ODC+/SMacs-, upon ex vivo stimulation there were increased IFN-γ+ cells, but when ODC+/SMacs were co-cultured with WT or ODC+/SMacs-, this did not occur. Pretreatment with anti-TGFβ or 1400W in the co-culture of ODC+/-SMacs with WT or ODC+/SMacsincreased IFN-γ+ cells (Table 2). In stomach tissue lysates from ODC+/vs. WT mice, IFN-γ and IL-17 levels were decreased, while IL-10 was increased. TGF-β mRNA and protein expression was increased in stomach tissues and in GMacs of ODC+/mice. In these studies, the pSTAT1/pSTAT3 ratio was decreased when IFN-γ and IL-17 were decreased and IL-10 was increased. Conclusions: During Hp infection, ODC inhibits TGF-β and NO production by Macs that impairs host immune tolerance manifested as a skewed adaptive (Th1/Th17) response, inappropriate inflammation, and persistent Hp colonization. Table 1