Microisolater cages at the University of Maryland Baltimore animal facilities. Mice were euthanized for tissue collection by CO2 asphyxiation followed by thoracotomy.T Cell Co-culture ExperimentsCoculture experiments were performed by plating 16105 BMDCs per well in 90 well U-bottom plates and stimulating with 10 mg/mL OVA peptide. CD4+ T cells were isolated from spleens from 6?4 week old C57BL/6 OT-II Foxp3-GFP mice using the CD4+ MagCellect Isolation Kit (R D Systems) according to the manufacturer’s instructions. T cells were added 56105 cells per well to the BMDC in 96 well plates in the presence of either Treg promoting conditions (20 ng/mL TGF-b (R D Systems) 25 U of mIL-2 (E-bioscience, San Diego, CA), or TH17 promoting conditions (2 ng/mL TGF-b (R D Systems) 20 ng/mL mIL-6 (Gemini Bio-products, Sacramento, CA). Alternatively, 16105 BMDCs were plated in 90 well U-bottom plates and stimulated with media alone or 10 mg/mL H. pylori SS1 antigen lysate. CD4+ T cells were isolated from spleens from 6?4 week old C57BL/6 mice infected with H. pylori SS1 and 56105 T cells were added to the wells in the absence of any additional stimulation.Bacterial Strains and InfectionE. coli K12 was purchased from ATCC (#29425) (Manassas, VA) and grown on LB plates supplemented with POR 8 amphotericin B (2.5 mg/ml). The mouse-adapted H. pylori Sydney Strain 1 (SS1) [38]and strain 26695 (ATCC #700392) were grown on Columbia agar (Difco, Detroit, MI) supplemented with7 horse blood and antibiotics at 37uC. For inoculation of mice, bacteria were transferred to 10 ml Brucella broth (Difco) supplemented with 10 fetal bovine serum (Invitrogen, Carlsbad, CA) and amphotericin B (2.5 mg/ml). Liquid cultures were established in T25 flasks and maintained at 37uC with 10 CO2. Infections with H. pylori SS1 were performed by delivering 16107 CFU in 0.5 ml Brucella broth by oral gavage using a 20 G feeding needleTable 1. H. pylori associated gene expression changes.Gene Name Antimicrobial peptides Elastase 2, neutrophil (Ela2) Cathelicidin antimicrobial peptide (CAMP) Lipocalin 2 (Lcn2) Anti-inflammatory molecules Zinc finger CCCH type containing 12A (Zc3h12a) Acyloxyacyl hydrolase (Aoah) Interleukin-1 receptor-associated kinase 3 (Irak3/IRAK-M) Nuclear factor of kappa light polypeptide gene enhancer in 23148522 B-cells inhibitor, zeta (Nfkbiz/IkB-f) Tribbles homolog 3 (Drosophila) (Trib3) Vanin 3 (Vnn3) Trafficking Molecules Vesicle transport through interaction with t-SNAREs homolog 1A (yeast) (Vti1a) doi:10.1371/journal.pone.0066914.tFold Change (H. pylori vs. Media alone)24.76 23.27 2.1.46 1.53 2.21 2.71 3.98 4.1.The Role of IRAK-M in H. pylori ImmunityFlow Cytometry AnalysisT-cells were stained with anti-CD4-APC and anti-IL17A-PE (eBioscience). BMDCs were stained with anti-MHCII-Pacific Blue, anti-PD-L1 PE, anti-CD40 PE-Cy5, anti-CD86 PE-Cy5 (eBioscience). All cells were analysed using a LSRII flow cytometer (BD Biosciences, San Hose, CA). Data were analyzed by FlowJo7 software (Tree Star, Ashland, OR).HDAC-IN-3 Adoptive Transfer ExperimentsCD4+ T cells were isolated from the spleens of FoxP3-GFP mice using the MagCellect Mouse CD4+ T cell isolation kit (R D Systems) and sorted for GFP negative cells using a BD FACSAria flow cytometer. A total of 26106 CD4+, GFP2 cells were transferred into WT and IRAK-M2/2 recipients by tail vein injection. Animals were infected with SS1 on day 3 and animals were harvested at 8 weeks for analysis. RNA was isolated from gastric tissue using the R.Microisolater cages at the University of Maryland Baltimore animal facilities. Mice were euthanized for tissue collection by CO2 asphyxiation followed by thoracotomy.T Cell Co-culture ExperimentsCoculture experiments were performed by plating 16105 BMDCs per well in 90 well U-bottom plates and stimulating with 10 mg/mL OVA peptide. CD4+ T cells were isolated from spleens from 6?4 week old C57BL/6 OT-II Foxp3-GFP mice using the CD4+ MagCellect Isolation Kit (R D Systems) according to the manufacturer’s instructions. T cells were added 56105 cells per well to the BMDC in 96 well plates in the presence of either Treg promoting conditions (20 ng/mL TGF-b (R D Systems) 25 U of mIL-2 (E-bioscience, San Diego, CA), or TH17 promoting conditions (2 ng/mL TGF-b (R D Systems) 20 ng/mL mIL-6 (Gemini Bio-products, Sacramento, CA). Alternatively, 16105 BMDCs were plated in 90 well U-bottom plates and stimulated with media alone or 10 mg/mL H. pylori SS1 antigen lysate. CD4+ T cells were isolated from spleens from 6?4 week old C57BL/6 mice infected with H. pylori SS1 and 56105 T cells were added to the wells in the absence of any additional stimulation.Bacterial Strains and InfectionE. coli K12 was purchased from ATCC (#29425) (Manassas, VA) and grown on LB plates supplemented with amphotericin B (2.5 mg/ml). The mouse-adapted H. pylori Sydney Strain 1 (SS1) [38]and strain 26695 (ATCC #700392) were grown on Columbia agar (Difco, Detroit, MI) supplemented with7 horse blood and antibiotics at 37uC. For inoculation of mice, bacteria were transferred to 10 ml Brucella broth (Difco) supplemented with 10 fetal bovine serum (Invitrogen, Carlsbad, CA) and amphotericin B (2.5 mg/ml). Liquid cultures were established in T25 flasks and maintained at 37uC with 10 CO2. Infections with H. pylori SS1 were performed by delivering 16107 CFU in 0.5 ml Brucella broth by oral gavage using a 20 G feeding needleTable 1. H. pylori associated gene expression changes.Gene Name Antimicrobial peptides Elastase 2, neutrophil (Ela2) Cathelicidin antimicrobial peptide (CAMP) Lipocalin 2 (Lcn2) Anti-inflammatory molecules Zinc finger CCCH type containing 12A (Zc3h12a) Acyloxyacyl hydrolase (Aoah) Interleukin-1 receptor-associated kinase 3 (Irak3/IRAK-M) Nuclear factor of kappa light polypeptide gene enhancer in 23148522 B-cells inhibitor, zeta (Nfkbiz/IkB-f) Tribbles homolog 3 (Drosophila) (Trib3) Vanin 3 (Vnn3) Trafficking Molecules Vesicle transport through interaction with t-SNAREs homolog 1A (yeast) (Vti1a) doi:10.1371/journal.pone.0066914.tFold Change (H. pylori vs. Media alone)24.76 23.27 2.1.46 1.53 2.21 2.71 3.98 4.1.The Role of IRAK-M in H. pylori ImmunityFlow Cytometry AnalysisT-cells were stained with anti-CD4-APC and anti-IL17A-PE (eBioscience). BMDCs were stained with anti-MHCII-Pacific Blue, anti-PD-L1 PE, anti-CD40 PE-Cy5, anti-CD86 PE-Cy5 (eBioscience). All cells were analysed using a LSRII flow cytometer (BD Biosciences, San Hose, CA). Data were analyzed by FlowJo7 software (Tree Star, Ashland, OR).Adoptive Transfer ExperimentsCD4+ T cells were isolated from the spleens of FoxP3-GFP mice using the MagCellect Mouse CD4+ T cell isolation kit (R D Systems) and sorted for GFP negative cells using a BD FACSAria flow cytometer. A total of 26106 CD4+, GFP2 cells were transferred into WT and IRAK-M2/2 recipients by tail vein injection. Animals were infected with SS1 on day 3 and animals were harvested at 8 weeks for analysis. RNA was isolated from gastric tissue using the R.
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