Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SLC15A2 and SLC15A4 Mediate the Transport of Bacterially-Derived Di/Tripeptides to Enhance the NOD-Dependent Immune Response in Mouse Bone Marrow-Derived Macrophages

doi: 10.4049/jimmunol.1800210

Figure Lengend Snippet: POT transporter expression in mouse bone marrow. Gene expression of Pept1 (A), protein expression of PEPT1 (B), gene expression of Pept2 (C), gene expression of Pht1 (D), gene expression of Pht2 (E), and protein expression of PEPT2 (F) in wildtype, Pht1 knockout (KO) and Pept2 knockout (KO) mice. The positive control was small intestine for PEPT1 and kidney for PEPT2. Quantification of protein (i.e., transporter/β-actin ratio) is shown below the figure. One representative example is shown of three individual immunoblots.

Article Snippet: Chemicals and materials The Lineage Cell Depletion Kit and CD11b MicroBeads were from Miltenyi Biotec Inc (Auburn, CA); recombinant mouse M-CSF (1.5 ng/mL) and Quantikine ® ELISA mouse IL-6 and TNF-α kits were from R&D systems, Inc. (Minneapolis, MN); MDP, MDP control (L,L-isomer), iE-DAP, Tri-DAP, MDP-rhodamine and LPS-EB Ultrapure were from InvivoGen (San Diego, CA); DAPI was from Molecular Probes (Eugene, OR); [ 3 H]GlySar (2.8Ci/mmol), [ 3 H]L-histidine (30 Ci/mmol) and [ 14 C]mannitol (53 mCi/mmol) were from Moravek Chemical Inc. (Brea, CA); GlySar, GlyPro, glycine and L-histidine were from Sigma-Aldrich (St. Louis, MO); rabbit anti-mouse PEPT1 and PEPT2 antisera were from Lampire Biological Laboratories (Pipersville, PA) ( 37 ); mouse anti-GFP, mouse β-actin monoclonal, goat anti-mouse IgG-HRP, and goat anti-rabbit IgG-HRP antibodies were from Bio-Rad (Hercules, CA); Oregon Green 488, goat anti-rabbit IgG, ProLong Diamond Antifade Mountant with DAPI, RMPI 1640 media and FBS were from Thermo Fisher Scientific (Waltham, MA); mouse Pept1, Pept2, Pht1, Pht2 and Gapdh primers for quantitative real-time PCR were from Integrated DNA Technologies (Coralville, Iowa).

Techniques: Expressing, Knock-Out, Positive Control, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SLC15A2 and SLC15A4 Mediate the Transport of Bacterially-Derived Di/Tripeptides to Enhance the NOD-Dependent Immune Response in Mouse Bone Marrow-Derived Macrophages

doi: 10.4049/jimmunol.1800210

Figure Lengend Snippet: Protein expression of PEPT2 in cell subtypes of mouse bone marrow. PEPT2 protein was examined in (A) Lin− (includes hematopoietic and mesenchymal stem cells, and their progenitors) and Lin+ (includes T and B cells, macrophages, monocytes, granulocytes, erythrocytes and their committed precursors) preparations, and (B) residential macrophages and bone marrow-derived macrophages (BMDMs) isolated from wildtype mice. The positive control was wildtype mouse kidney for panel A and bone marrow for panel B (which was validated in Fig. 1F). Quantification of protein (i.e., transporter/β-actin ratio) is shown below each figure. One representative example is shown of three individual immunoblots.

Article Snippet: Chemicals and materials The Lineage Cell Depletion Kit and CD11b MicroBeads were from Miltenyi Biotec Inc (Auburn, CA); recombinant mouse M-CSF (1.5 ng/mL) and Quantikine ® ELISA mouse IL-6 and TNF-α kits were from R&D systems, Inc. (Minneapolis, MN); MDP, MDP control (L,L-isomer), iE-DAP, Tri-DAP, MDP-rhodamine and LPS-EB Ultrapure were from InvivoGen (San Diego, CA); DAPI was from Molecular Probes (Eugene, OR); [ 3 H]GlySar (2.8Ci/mmol), [ 3 H]L-histidine (30 Ci/mmol) and [ 14 C]mannitol (53 mCi/mmol) were from Moravek Chemical Inc. (Brea, CA); GlySar, GlyPro, glycine and L-histidine were from Sigma-Aldrich (St. Louis, MO); rabbit anti-mouse PEPT1 and PEPT2 antisera were from Lampire Biological Laboratories (Pipersville, PA) ( 37 ); mouse anti-GFP, mouse β-actin monoclonal, goat anti-mouse IgG-HRP, and goat anti-rabbit IgG-HRP antibodies were from Bio-Rad (Hercules, CA); Oregon Green 488, goat anti-rabbit IgG, ProLong Diamond Antifade Mountant with DAPI, RMPI 1640 media and FBS were from Thermo Fisher Scientific (Waltham, MA); mouse Pept1, Pept2, Pht1, Pht2 and Gapdh primers for quantitative real-time PCR were from Integrated DNA Technologies (Coralville, Iowa).

Techniques: Expressing, Derivative Assay, Isolation, Positive Control, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SLC15A2 and SLC15A4 Mediate the Transport of Bacterially-Derived Di/Tripeptides to Enhance the NOD-Dependent Immune Response in Mouse Bone Marrow-Derived Macrophages

doi: 10.4049/jimmunol.1800210

Figure Lengend Snippet: GlySar and MDP-rhodamine uptake studies in BMDMs of wildtype and Pept2 knockout mouse. (A) effect of potential inhibitors on the uptake of 1.0 μM [3H]GlySar where GlyPro or glycine were present at 5 mM, and MDP (L,D-isomer), MDP control (L,L-isomer), r-iE-DAP or tri-DAP were present at 1 mM. GlySar uptakes were corrected for nonspecific binding using [14C]mannitol, and represented as % control, relative to wildtype mice. Data are expressed as mean ± SE (n=6) in which each experiment was performed in triplicate. Treatment groups with the same letter were not statistically different, as determined by ANOVA and Tukey’s test. (B) uptake of MDP-rhodamine in wildtype and Pept2 knockout mice. MDP-rhodamine (red) was marked by arrows and the nuclei (blue) were stained by DAPI (40× magnification). Density measurements are shown in the right-hand panel (LabWorks Image Acquisition and Analysis Software v4.5, UVP Inc, Upland, CA), and expressed as mean ± SE (n=10 cells). Statistical differences between the two genotypes were determined by an unpaired (two-sample) t-test. ***p ≤ 0.001.

Article Snippet: Chemicals and materials The Lineage Cell Depletion Kit and CD11b MicroBeads were from Miltenyi Biotec Inc (Auburn, CA); recombinant mouse M-CSF (1.5 ng/mL) and Quantikine ® ELISA mouse IL-6 and TNF-α kits were from R&D systems, Inc. (Minneapolis, MN); MDP, MDP control (L,L-isomer), iE-DAP, Tri-DAP, MDP-rhodamine and LPS-EB Ultrapure were from InvivoGen (San Diego, CA); DAPI was from Molecular Probes (Eugene, OR); [ 3 H]GlySar (2.8Ci/mmol), [ 3 H]L-histidine (30 Ci/mmol) and [ 14 C]mannitol (53 mCi/mmol) were from Moravek Chemical Inc. (Brea, CA); GlySar, GlyPro, glycine and L-histidine were from Sigma-Aldrich (St. Louis, MO); rabbit anti-mouse PEPT1 and PEPT2 antisera were from Lampire Biological Laboratories (Pipersville, PA) ( 37 ); mouse anti-GFP, mouse β-actin monoclonal, goat anti-mouse IgG-HRP, and goat anti-rabbit IgG-HRP antibodies were from Bio-Rad (Hercules, CA); Oregon Green 488, goat anti-rabbit IgG, ProLong Diamond Antifade Mountant with DAPI, RMPI 1640 media and FBS were from Thermo Fisher Scientific (Waltham, MA); mouse Pept1, Pept2, Pht1, Pht2 and Gapdh primers for quantitative real-time PCR were from Integrated DNA Technologies (Coralville, Iowa).

Techniques: Knock-Out, Binding Assay, Staining, Software

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SLC15A2 and SLC15A4 Mediate the Transport of Bacterially-Derived Di/Tripeptides to Enhance the NOD-Dependent Immune Response in Mouse Bone Marrow-Derived Macrophages

doi: 10.4049/jimmunol.1800210

Figure Lengend Snippet: Effect of PEPT2 on the expression of IL-6 and TNF-α in BMDMs of wildtype and Pept2 knockout mice. BMDMs were treated for 24 hr with 0–10 ng/mL LPS plus 10 μg/mL MDP (L,D-isomer; A and B), tri-DAP (C and D), or 10 ng/mL LPS plus 10 μg/mL MDP control (L,L-isomer; E and F), after which cytokine concentrations in the cell culture media were measured. Data are expressed as mean ± SE (n=4) in which each experiment was performed in triplicate. Statistical differences between the two genotypes were determined by an unpaired (two-sample) t-test. *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001.

Article Snippet: Chemicals and materials The Lineage Cell Depletion Kit and CD11b MicroBeads were from Miltenyi Biotec Inc (Auburn, CA); recombinant mouse M-CSF (1.5 ng/mL) and Quantikine ® ELISA mouse IL-6 and TNF-α kits were from R&D systems, Inc. (Minneapolis, MN); MDP, MDP control (L,L-isomer), iE-DAP, Tri-DAP, MDP-rhodamine and LPS-EB Ultrapure were from InvivoGen (San Diego, CA); DAPI was from Molecular Probes (Eugene, OR); [ 3 H]GlySar (2.8Ci/mmol), [ 3 H]L-histidine (30 Ci/mmol) and [ 14 C]mannitol (53 mCi/mmol) were from Moravek Chemical Inc. (Brea, CA); GlySar, GlyPro, glycine and L-histidine were from Sigma-Aldrich (St. Louis, MO); rabbit anti-mouse PEPT1 and PEPT2 antisera were from Lampire Biological Laboratories (Pipersville, PA) ( 37 ); mouse anti-GFP, mouse β-actin monoclonal, goat anti-mouse IgG-HRP, and goat anti-rabbit IgG-HRP antibodies were from Bio-Rad (Hercules, CA); Oregon Green 488, goat anti-rabbit IgG, ProLong Diamond Antifade Mountant with DAPI, RMPI 1640 media and FBS were from Thermo Fisher Scientific (Waltham, MA); mouse Pept1, Pept2, Pht1, Pht2 and Gapdh primers for quantitative real-time PCR were from Integrated DNA Technologies (Coralville, Iowa).

Techniques: Expressing, Knock-Out, Cell Culture

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SLC15A2 and SLC15A4 Mediate the Transport of Bacterially-Derived Di/Tripeptides to Enhance the NOD-Dependent Immune Response in Mouse Bone Marrow-Derived Macrophages

doi: 10.4049/jimmunol.1800210

Figure Lengend Snippet: Effect of PHT1 on the LPS-MDP stimulated immune response, and uptake studies of MDP or L-histidine. (A) BMDMs from wildtype and Pht1 knockout mice were treated for 24 hr with 5 ng/mL LPS plus 10 μg/mL MDP, after which IL-6 and TNF-α concentrations in the cell culture media were measured. Data are expressed as mean ± SE (n=6) in which each experiment was performed in triplicate. Statistical differences between the two genotypes were determined by an unpaired (two-sample) t-test. ***p ≤ 0.001. (B) BMDMs from wildtype mice were treated for 24 hr with 5 ng/mL LPS alone, and in the presence of 1.0 μM bafilomycin A1 (BAF), 10 μg/mL MDP, or 10 μg/mL MDP plus 1 μM BAF. BMDMs were pretreated with BAF for 30 min prior to being co-incubated with MDP and/or LPS. IL-6 concentrations in the cell culture media were then measured. Data are expressed as mean ± SE (n=3) in which each experiment was performed in triplicate. Treatment groups with the same letter were not statistically different, as determined by ANOVA and Tukey’s test. (C) uptake of MDP-rhodamine in BMDMs prepared from wildtype and Pht1 knockout mice. MDP-rhodamine (red) was marked by arrows and the nuclei (blue) were stained by DAPI (100× magnification). Particle density is shown in the right-hand panel and expressed as mean ± SE (n=6–8 cells). Statistical differences between the two genotypes were determined by an unpaired (two-sample) t-test. ***p ≤ 0.001. (D) effect of potential inhibitors on the uptake of 1.0 μM [3H]histidine into endosome-enriched liver preparations from wildtype and Pht1 knockout mice. Endosomes were pretreated with MDP (1.0 mM) or BAF (1.0 μM) for 30 min prior to experimentation. Data are expressed as mean ± SE (n=3) in which each experiment was performed in triplicate. Treatment groups with the same letter were not statistically different, as determined by ANOVA and Tukey’s test. (E) fusion protein of GFP-mPHT1 (90 kd) was detected with an anti-GFP monoclonal antibody in the endosome of pcDNA3.1-mPht1/CT-GFP transfected HEK293 cells, but not in pcDNA3.1-CT-GFP transfected HEK293 (mock) cells. (F) expression of PEPT2 protein in mouse liver endosomes. One representative example is shown of three individual immunoblots.

Article Snippet: Chemicals and materials The Lineage Cell Depletion Kit and CD11b MicroBeads were from Miltenyi Biotec Inc (Auburn, CA); recombinant mouse M-CSF (1.5 ng/mL) and Quantikine ® ELISA mouse IL-6 and TNF-α kits were from R&D systems, Inc. (Minneapolis, MN); MDP, MDP control (L,L-isomer), iE-DAP, Tri-DAP, MDP-rhodamine and LPS-EB Ultrapure were from InvivoGen (San Diego, CA); DAPI was from Molecular Probes (Eugene, OR); [ 3 H]GlySar (2.8Ci/mmol), [ 3 H]L-histidine (30 Ci/mmol) and [ 14 C]mannitol (53 mCi/mmol) were from Moravek Chemical Inc. (Brea, CA); GlySar, GlyPro, glycine and L-histidine were from Sigma-Aldrich (St. Louis, MO); rabbit anti-mouse PEPT1 and PEPT2 antisera were from Lampire Biological Laboratories (Pipersville, PA) ( 37 ); mouse anti-GFP, mouse β-actin monoclonal, goat anti-mouse IgG-HRP, and goat anti-rabbit IgG-HRP antibodies were from Bio-Rad (Hercules, CA); Oregon Green 488, goat anti-rabbit IgG, ProLong Diamond Antifade Mountant with DAPI, RMPI 1640 media and FBS were from Thermo Fisher Scientific (Waltham, MA); mouse Pept1, Pept2, Pht1, Pht2 and Gapdh primers for quantitative real-time PCR were from Integrated DNA Technologies (Coralville, Iowa).

Techniques: Knock-Out, Cell Culture, Incubation, Staining, Transfection, Expressing, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SLC15A2 and SLC15A4 Mediate the Transport of Bacterially-Derived Di/Tripeptides to Enhance the NOD-Dependent Immune Response in Mouse Bone Marrow-Derived Macrophages

doi: 10.4049/jimmunol.1800210

Figure Lengend Snippet: Schematic depicting the PEPT2- and PHT1-mediated uptake of bacterially-derived products (e.g., MDP and tri-DAP) into the cytosol of macrophages, and their effect on NOD signaling and downstream production of proinflammatory cytokines.

Article Snippet: Chemicals and materials The Lineage Cell Depletion Kit and CD11b MicroBeads were from Miltenyi Biotec Inc (Auburn, CA); recombinant mouse M-CSF (1.5 ng/mL) and Quantikine ® ELISA mouse IL-6 and TNF-α kits were from R&D systems, Inc. (Minneapolis, MN); MDP, MDP control (L,L-isomer), iE-DAP, Tri-DAP, MDP-rhodamine and LPS-EB Ultrapure were from InvivoGen (San Diego, CA); DAPI was from Molecular Probes (Eugene, OR); [ 3 H]GlySar (2.8Ci/mmol), [ 3 H]L-histidine (30 Ci/mmol) and [ 14 C]mannitol (53 mCi/mmol) were from Moravek Chemical Inc. (Brea, CA); GlySar, GlyPro, glycine and L-histidine were from Sigma-Aldrich (St. Louis, MO); rabbit anti-mouse PEPT1 and PEPT2 antisera were from Lampire Biological Laboratories (Pipersville, PA) ( 37 ); mouse anti-GFP, mouse β-actin monoclonal, goat anti-mouse IgG-HRP, and goat anti-rabbit IgG-HRP antibodies were from Bio-Rad (Hercules, CA); Oregon Green 488, goat anti-rabbit IgG, ProLong Diamond Antifade Mountant with DAPI, RMPI 1640 media and FBS were from Thermo Fisher Scientific (Waltham, MA); mouse Pept1, Pept2, Pht1, Pht2 and Gapdh primers for quantitative real-time PCR were from Integrated DNA Technologies (Coralville, Iowa).

Techniques: Derivative Assay