Additional file 1: Figure S1. of The role of C1q in recognition of apoptotic epithelial cells and inflammatory cytokine production by phagocytes during Helicobacter pylori infection

C1q protein inhibits THP-1 macrophage H. pylori stimulated inflammatory cytokine release. THP-1 macrophages were washed and resuspended in serum free medium (Xvivo 10 + L-glut) and pretreated with C1q (0–80 μg/ml) for 30 min before stimulation with H. pylori (MOI 100) for 24 h. Supernatants were collected and TNF-α and IL-6 were measured by specific ELISA. The average and individual values of 2 replicates from n = 2 independent experiments are shown. (PPTX 55 kb

Additional file 1: Figure S1. of The role of C1q in recognition of apoptotic epithelial cells and inflammatory cytokine production by phagocytes during Helicobacter pylori infection

C1q protein inhibits THP-1 macrophage H. pylori stimulated inflammatory cytokine release. THP-1 macrophages were washed and resuspended in serum free medium (Xvivo 10 + L-glut) and pretreated with C1q (0–80 μg/ml) for 30 min before stimulation with H. pylori (MOI 100) for 24 h. Supernatants were collected and TNF-α and IL-6 were measured by specific ELISA. The average and individual values of 2 replicates from n = 2 independent experiments are shown. (PPTX 55 kb

Functional stability of GM1-NPs in the presence of intestinal luminal factors.

<p>(A) Intensity-weighted size distribution of GM1-NPs incubated 30 min in distilled water or diluted porcine bile solution (1:16 dilution in water). (B) Fluorescence imaging of DiD-labeled GM1-NPs absorbed with FITC-CTB and incubated for 30 min in 1:16 diluted porcine bile solution. (C) GM1-NPs were loaded with FITC-CTB for 30 min, washed, and resuspended in luminal fluid from the small intestine of normal adult mice, or PBS as a control. After incubation for 24 h at 37°C, particle-bound and free FITC-CTB were separated by dialysis, and bound FITC-CTB was measured by fluorescence spectroscopy and related to the initial amount bound (mean ± SD, n = 3). Background readings were obtained with free FITC-CTB without GM1-NPs. (D) Fecal homogenates from mice were mixed 1:1 with GM1-NPs or PEG-NPs in culture media, and incubated for 1 h at 37°C, after which CT (10 ng/mL) was added for an additional 1 h before addition to HCA7 monolayers. After 2 h, cAMP levels in the supernatants were determined by ELISA (mean ± SD, n = 3; *p<0.05 vs PEG-NPs).</p

Model of therapeutic effect of GM1-NPs.

<p>GM1-coated nanoparticles act as decoys to absorb CT produced by <i>V</i>. <i>cholerae</i> before it can bind to epithelial cells to stimulate cAMP production and epithelial chloride secretion, and inhibit sodium absorption.</p

<i>In vivo</i> efficacy of GM1-NPs against CT and live <i>V</i>. <i>cholerae</i>.

<p>(A) Ligated intestinal loops were prepared in the distal small intestine of adult C57BL/6 mice, and injected with PBS as a control, or with 2.5 μg CT, without and with prior addition of GM1-NPs or control PEG-NPs. Fluid accumulation in the loops was determined after 4 h, and related to loop length (each point represents one animal, horizontal lines are geometric means; *p<0.05 vs PEG-NPs). (B) Loops were injected with PBS as a control, or live <i>V</i>. <i>cholerae</i> with GM1-NPs or control PEG-NPs. Fluid accumulation was determined after 16 h (each point represents one animal, horizontal lines are geometric means; *p<0.05 vs PEG-NPs). (C) Images of representative intestinal loops. (D) cAMP was measured in the luminal fluid collected from loops after injection of live <i>V</i>. <i>cholera</i>e with GM1-NPs or control PEG-NPs (n = 3; mean ± SD; *p<0.05 vs PEG-NPs).</p

Preparation and physical characterization of GM1-coated nanoparticles.

<p>(A) Schematic of GM1-NP fabrication. Poly(lactic-co-glycolic acid) (PLGA) dissolved in acetonitrile (CH₃CN) is added to an aqueous solution containing GM1. After acetonitrile evaporation, nanoparticles with a polymeric core and a lipid shell are formed. (B) Intensity-weighted size distribution of representative preparations of GM1-NPs and control PEG-NPs, and PLGA-NPs with a PLGA core but without a lipid shell. (C) Zeta potential of the indicated nanoparticle preparations (n = 3; mean ± SD; *p<0.05 vs. PLGA-NPs). (D) Nanoparticle size measurements over two weeks of incubation in distilled water or PBS (n = 3; mean ± SD). (E) Transmission electron micrograph of GM1-NPs.</p

Neutralization of CT activity with GM1-NPs.

<p>(A) A fixed concentration (10 ng/mL) of CT was combined with increasing amounts of the indicated nanoparticles, and the mixtures were added to confluent monolayers of human HCA7 intestinal epithelial cells. After 2 h, levels of secreted cAMP were determined in the supernatants by ELISA (n = 3; mean ± SD). (B) A fixed amount (1 μg/mL) of the indicated nanoparticles were combined with increasing concentrations of CT, the mixtures were added to HCA7 monolayers for 2 h, and levels of secreted cAMP levels were measured by ELISA (n = 3; mean ± SD). (C) GM1-NPs or control PEG-NPs were added to HCA7 monolayers, which were then infected for 2 h with live <i>V</i>. <i>cholerae</i> or left uninfected, and secreted cAMP was determined (n = 3; mean ± SD; *p<0.05 vs PEG-NPs).</p

APE1 regulates Nox1 expression.

<p>(A) Nox1 expression was examined in AGS pSIREN and shRNA cells infected with H. pylori for 1, 3 and 6h or left uninfected (0h). Corresponding densitometry results of three experiments are shown to the right of the western blot. (B) Immunofluorescence staining showing Rac1 and Nox1 after infection in AGS and shRNA cells. Scale bars indicate 10 μm. (C) Gene transcription levels of Nox1 (left panel) and APE1 (right panel) in gastric biopsies from H. pylori infected and uninfected patients. Each value was normalized to 18S and data were normalized to one of the uninfected samples. Levels of significance are indicated as follows: * p<0.05 and ** p<0.01.</p

H. pylori-induced ROS generation is mediated by Rac1.

<p>(A-C) ROS generation was measured by luminol oxidation in AGS cells (A), NCI-N87 cells (B) and primary gastric epithelial cells (C) after infection with H. pylori 26695 for 10, 30 or 60 min or left uninfected. (D) H. pylori-induced ROS generation (after 30 min) was measured in AGS cells after transfection with empty vector (pcDNA), constitutively active Rac1 (V12) or after treatment with Rac1 inhibitor (NSC23766). For A-C fold change of luminol oxidation compared to uninfected cells are shown as mean values (± SEM) of three independent experiments. (E-F) Rac1 activation was measured by a GTP pull down assay in AGS (E) and NCI-N87 (F) cells following infection with H. pylori at the indicated times. A representative western blot was selected from three independent experiments and densitometry results from multiple experiments are shown. Levels of significance are indicated as follows: * p<0.05, ** p<0.01 and *** p< 0.001.</p

APE1 inhibits H. pylori-induced ROS generation.

<p>ROS generation was measured by luminol oxidation (A) in WT AGS, control shRNA (pSIREN) or APE1 shRNA (shRNA) cells after infection with H. pylori for 30 min or left uninfected, (B) in infected cells that were either left untreated or treated with NSC23766 or DPI, (C) in shRNA cells either transfected with empty vector (pcDNA) or APE1; or treated with NAC or DPI before infection with H. pylori for 30 min or left uninfected and (D) in shRNA cells that were either transfected with pcDNA, APE1, V12 or both APE1 and V12 or treated with NSC23766 before infection with H. pylori or left uninfected. For A-D fold change of luminol oxidation compared to uninfected cells are shown as mean values (± SEM) of three independent experiments. (E) ROS generation was measured by confocal microscopy in AGS and shRNA cells after infection with H. pylori for 30 min or left uninfected. After infection ROS was detected using CM-H₂DCFDA and nuclei were stained with DAPI. Scale bar indicates 100 μm. The bar graph at the bottom shows quantification of multiple images from three independent experiments. Levels of significance are indicated as follows: * p<0.05, ** p<0.01 and *** p< 0.001.</p