Journal: Scientific Reports

Article Title: Vasoactive intestinal peptide blockade suppresses tumor growth by regulating macrophage polarization and function in CT26 tumor-bearing mice

doi: 10.1038/s41598-023-28073-6

Figure Lengend Snippet: Conditioned medium from CT26 cells induced M2 macrophage polarization and VPAC expression in RAW264.7 cells. Real-time PCR analyses of M1 macrophage markers TNF-α , iNOS , and CXCL10 ( A ), M2 macrophage markers Mrc-1 , IL-1rn , and CCL-22 ( B ), immune checkpoint markers SIRP-α , PD-1 , and PD-L1 ( C ), and VIP receptors Vipr1 and Vipr2 ( D ) in RAW264.7 cells at day 4 after incubation with 20% conditioned medium derived from CT26 with DMEM complete medium (CT26-CM) or 20% RPMI medium with DMEM complete medium (control); n = 5–6/group. Representative western blotting images showing protein expression levels of VPAC1 and VPAC2 in RAW264.7 cells at day 4 after incubation with CT26-CM or control ( E , left). Western blotting analysis of VPAC1 and VPAC2 protein expression in RAW264.7 cells at day 4 after incubation with control medium or CT26-CM (E, right); n = 5/group. The full-length blots of ( E ) are included in Supplemental Fig. . *p < 0.05 vs control, **p < 0.01 vs control.

Article Snippet: In a separate experiment, [Lys15, Arg16, Leu27] VIP (1–7)/GRF (8–27) (Phoenix Pharmaceuticals)—a selective VPAC1 agonist —or BAY 55-9837 (Tocris Bioscience, Minneapolis, MN)—a selective VPAC2 agonist—was added daily at a dose of 100 nM . mRNA extraction was performed on day 3 or day 4 after CT26-CM incubation, depending on the experiment.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Incubation, Derivative Assay, Control, Western Blot

Journal: Scientific Reports

Article Title: Vasoactive intestinal peptide blockade suppresses tumor growth by regulating macrophage polarization and function in CT26 tumor-bearing mice

doi: 10.1038/s41598-023-28073-6

Figure Lengend Snippet: Knockdown or activation of VPAC2 disturbed macrophage polarization in RAW264.7 cells incubated with CT26-CM. Effect of Vipr1 ( A ) or Virp2 ( B ) silencing on gene expression of M1 and M2 macrophage markers in RAW264.7 cells at day 4 after incubation with CT26-CM. Negative control siRNA was used as control; n = 6–9/group. Effect of [Lys15, Arg16, Leu27] VIP (1–7)/GRF (8–27), a VPAC1 agonist, ( C ) or BAY 55–9837, a VPAC2 agonist, ( D ) on the mRNA expression of M1 and M2 macrophage markers in RAW264.7 cells at day 3 after incubation with CT26-CM. PBS was used as vehicle; n = 6–10/group. *p < 0.05 vs Negative control siRNA or vehicle.

Article Snippet: In a separate experiment, [Lys15, Arg16, Leu27] VIP (1–7)/GRF (8–27) (Phoenix Pharmaceuticals)—a selective VPAC1 agonist —or BAY 55-9837 (Tocris Bioscience, Minneapolis, MN)—a selective VPAC2 agonist—was added daily at a dose of 100 nM . mRNA extraction was performed on day 3 or day 4 after CT26-CM incubation, depending on the experiment.

Techniques: Knockdown, Activation Assay, Incubation, Gene Expression, Negative Control, Control, Expressing

Journal: Scientific Reports

Article Title: Vasoactive intestinal peptide blockade suppresses tumor growth by regulating macrophage polarization and function in CT26 tumor-bearing mice

doi: 10.1038/s41598-023-28073-6

Figure Lengend Snippet: CD68 macrophages were highly expressed in tumor stroma and co-expressed with VPAC2, but not VPAC1, in human colorectal cancer specimens. Representative images of CD68 (green) and VPAC1 (red; A , B ), and CD68 (green) and VPAC2 (red; C , D ) staining in the tumor stroma of human colorectal cancer specimens. B and D were enlarged images from white square region indicated in ( A ) and ( C ), respectively. All images represented cancer lesions. Nuclei were stained by DAPI (blue). White arrow indicated double positive cells. Scale bar 100 µm.

Article Snippet: In a separate experiment, [Lys15, Arg16, Leu27] VIP (1–7)/GRF (8–27) (Phoenix Pharmaceuticals)—a selective VPAC1 agonist —or BAY 55-9837 (Tocris Bioscience, Minneapolis, MN)—a selective VPAC2 agonist—was added daily at a dose of 100 nM . mRNA extraction was performed on day 3 or day 4 after CT26-CM incubation, depending on the experiment.

Techniques: Staining

Journal: Scientific Reports

Article Title: Vasoactive intestinal peptide blockade suppresses tumor growth by regulating macrophage polarization and function in CT26 tumor-bearing mice

doi: 10.1038/s41598-023-28073-6

Figure Lengend Snippet: List of antibodies and their application in the present study.

Article Snippet: In a separate experiment, [Lys15, Arg16, Leu27] VIP (1–7)/GRF (8–27) (Phoenix Pharmaceuticals)—a selective VPAC1 agonist —or BAY 55-9837 (Tocris Bioscience, Minneapolis, MN)—a selective VPAC2 agonist—was added daily at a dose of 100 nM . mRNA extraction was performed on day 3 or day 4 after CT26-CM incubation, depending on the experiment.

Techniques: Western Blot

Journal: PLOS Pathogens

Article Title: Mitochondrial ROS production by neutrophils is required for host antimicrobial function against Streptococcus pneumoniae and is controlled by A2B adenosine receptor signaling

doi: 10.1371/journal.ppat.1010700

Figure Lengend Snippet: (A) A2BR -/- (grey) and WT (black) bone-marrow derived PMNs were mock treated (uninfected) or infected with S . pneumoniae (+Sp) TIGR4 at a multiplicity of infection (MOI) of 50 in the presence or absence of MitoTEMPO. Cells were monitored for intracellular ROS production over 60 minutes using luminol. Data are representative from 1 of 3 experiments in which n = 3 technical replicates were used per condition. * indicates significant differences between infected A2BR -/- and WT controls as well as infected A2BR -/- +/- MitoTEMPO treatment as determined by 2-way ANOVA followed by Tukey’s multiple comparisons test. Line graphs represent the mean +/-SD. (B-C) WT (Black) and A2BR -/- (light grey) marrow-derived PMNs were infected with S . pneumoniae TIGR4 at the indicated MOIs for 10 minutes. +MitoTEMPO were treated with the drug prior to infection at an MOI of 50. (B) The % of MitoSOX+ cells as well as (C) the amount of MitoSOX produced (geometric MFI) were determined using flow cytometry. (B-C) Representative data shown are from 1 out of 8 separate experiments in which n = 3 technical replicates were used per condition. Bar graphs represent the mean +/-SD. * indicates significant differences from uninfected controls and # indicates significant differences between the indicated groups as measured by one-way ANOVA followed by Tukey’s multiple comparison test. (D) Fold increases in MitoSOX+ cells upon bacterial infection were calculated by dividing the values of infected conditions by uninfected controls for each mouse strain. Data pooled from eight separate experiments (n = 8 mice/ group) are shown. $ indicates significantly different from 1 as measured by one-sample t-test and * indicates significant differences between the indicated groups as measured by Student’s t-test. (E) WT PMNs were treated with vehicle control (VC) or the A2BR Agonist BAY60-6583 for 30 minutes. Cells were then mock-treated (Uninfected) or infected with S . pneumoniae TIGR4 at a MOI of 10 for 10 minutes. The % of mitochondrial ROS producing cells were determined by flow cytometry. Representative data shown are from 1 out of 5 separate experiments in which n = 3 technical replicates were used per condition. Bar graphs represent the mean +/-SD. * indicates significant differences from uninfected controls and # indicates significant differences between the indicated groups as measured by one-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: For in vitro assays, the effect of A2BR was assessed using A2BR agonist BAY 60–6583 (Tocris) used at 285.7nM, a concentration corresponding to the 2xKi for the receptor.

Techniques: Derivative Assay, Infection, Produced, Flow Cytometry, Comparison, Control

Journal: PLOS Pathogens

Article Title: Mitochondrial ROS production by neutrophils is required for host antimicrobial function against Streptococcus pneumoniae and is controlled by A2B adenosine receptor signaling

doi: 10.1371/journal.ppat.1010700

Figure Lengend Snippet: WT (C57BL/6) (black) or A2BR -/- (light grey) bone-marrow derived PMNs were treated with vehicle control (VC), the mitochondrial ROS scavenger MitoTEMPO, or the A2BR agonist BAY 60–6583 and then infected with S . pneumoniae TIGR4. The percentage of bacterial killing was determined with respect to no PMN controls under the same treatment conditions. Data are pooled from n = 5 separate experiments. Bar graphs represent the mean +/-SD. * indicates significant differences from VC treated controls for each mouse strain and # indicates significant differences between indicated VC treated WT vs A2BR -/- groups as measured by one-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: For in vitro assays, the effect of A2BR was assessed using A2BR agonist BAY 60–6583 (Tocris) used at 285.7nM, a concentration corresponding to the 2xKi for the receptor.

Techniques: Derivative Assay, Control, Infection, Comparison

Journal: PLOS Pathogens

Article Title: Mitochondrial ROS production by neutrophils is required for host antimicrobial function against Streptococcus pneumoniae and is controlled by A2B adenosine receptor signaling

doi: 10.1371/journal.ppat.1010700

Figure Lengend Snippet: A2BR -/- (light grey) and wild type (WT) C57BL/6 (black) mice were infected with S . pneumoniae TIGR4 intra-tracheally. One set of mice were harvested at the indicated hours post infection for enumeration of bacterial numbers in the lungs (A) and blood (B) by plating on blood agar plates. Data are pooled from three separate experiments and each dot represents one individual mouse. * indicates significant differences as measured by unpaired Student’s t-test. The percentages indicate the fraction of mice that became bacteremic and # indicates significant differences between the mouse groups in the incidence of bacteremia as measured by Fisher’s exact test. (C-D) Another set of mice were monitored for clinical signs and symptoms of diseases (clinical score) (C) and survival (D) over time. Data are pooled from three separate experiments with n = 11 mice per group. * indicates significant differences as measured by Mann-Whitney test (C) and Log-Rank Mantle Cox test (D).

Article Snippet: For in vitro assays, the effect of A2BR was assessed using A2BR agonist BAY 60–6583 (Tocris) used at 285.7nM, a concentration corresponding to the 2xKi for the receptor.

Techniques: Infection, MANN-WHITNEY

Journal: PLOS Pathogens

Article Title: Mitochondrial ROS production by neutrophils is required for host antimicrobial function against Streptococcus pneumoniae and is controlled by A2B adenosine receptor signaling

doi: 10.1371/journal.ppat.1010700

Figure Lengend Snippet: A2BR -/- (light grey) mice were infected with S . pneumoniae TIGR4 intra-tracheally. At 18 hours following challenge, the number of mitochondrial ROS producing PMNs (Ly6G+) in the lungs (B) and circulation (C) were determined by flow cytometry using MitoSOX (see ). (A) Gating strategy is shown including controls (FMO indicates absence of MitSOX dye only). (B-C) Data are pooled from two separate experiments and each dot represents an individual mouse. (C) * indicates significant differences from uninfected controls for each mouse strain and # indicates significant differences between indicated groups as determined by one-way ANOVA followed by Tukey’s multiple comparisons test.

Article Snippet: For in vitro assays, the effect of A2BR was assessed using A2BR agonist BAY 60–6583 (Tocris) used at 285.7nM, a concentration corresponding to the 2xKi for the receptor.

Techniques: Infection, Flow Cytometry

Journal: PLOS Pathogens

Article Title: Mitochondrial ROS production by neutrophils is required for host antimicrobial function against Streptococcus pneumoniae and is controlled by A2B adenosine receptor signaling

doi: 10.1371/journal.ppat.1010700

Figure Lengend Snippet: A2BR -/- (light grey) mice were infected with S . pneumoniae TIGR4 intra-peritoneally. At 6 and 18 hours following challenge, the number (B) of mitochondrial ROS producing PMNs (Ly6G+) and (C) the amount of mitochondrial ROS produced (MFI MitoSOX) by PMNs in the circulation were determined by flow cytometry using MitoSOX (see ). (A) Gating strategy is shown including controls (FMO indicates absence of MitSOX dye only). (B-C) Data are pooled from four separate experiments and each dot represents an individual mouse. * indicates significant differences from uninfected controls for each mouse strain as determined by Kruskal-Wallis test.

Article Snippet: For in vitro assays, the effect of A2BR was assessed using A2BR agonist BAY 60–6583 (Tocris) used at 285.7nM, a concentration corresponding to the 2xKi for the receptor.

Techniques: Infection, Produced, Flow Cytometry

Journal: PLOS Pathogens

Article Title: Mitochondrial ROS production by neutrophils is required for host antimicrobial function against Streptococcus pneumoniae and is controlled by A2B adenosine receptor signaling

doi: 10.1371/journal.ppat.1010700

Figure Lengend Snippet: (A-B) WT C57BL/6 mice treated with vehicle control or the mitochondrial ROS scavenger MitoTEMPO were infected with S . pneumoniae D39 intra-peritoneally. (A) survival as well as (B) bacteremia was followed over time. Data shown are from two separate experiments with n = 5 mice per group. * indicates significant differences as measured by Log-Rank Mantle Cox test (A) and Mann-Whitney test (B). (C-D) A2BR -/- and WT C57BL/6 mice treated with vehicle control or the mitochondrial ROS scavenger MitoTEMPO were infected with S . pneumoniae TIGR4 intra-peritoneally. (C) survival as well as (D) bacteremia and clearance of infection (E) was followed over time. Data shown are from three separate experiments with n = 12 mice per group. * indicates significant differences as measured by Log-Rank Mantle Cox test (C) and Kruskal Wallis test (D). $ indicates significant differences between the mouse groups in the clearance of bacteremia as measured by Fisher’s exact test (E). n.s. indicates not significant.

Article Snippet: For in vitro assays, the effect of A2BR was assessed using A2BR agonist BAY 60–6583 (Tocris) used at 285.7nM, a concentration corresponding to the 2xKi for the receptor.

Techniques: Control, Infection, MANN-WHITNEY

Journal: PLOS Pathogens

Article Title: Mitochondrial ROS production by neutrophils is required for host antimicrobial function against Streptococcus pneumoniae and is controlled by A2B adenosine receptor signaling

doi: 10.1371/journal.ppat.1010700

Figure Lengend Snippet: S . pneumoniae induces mitochondrial ROS production by PMNs in a manner partially dependent on MyD88. This response is regulated by A2B receptor signaling and is required for the antimicrobial activity of PMNs. Images were created with Biorender.com .

Article Snippet: For in vitro assays, the effect of A2BR was assessed using A2BR agonist BAY 60–6583 (Tocris) used at 285.7nM, a concentration corresponding to the 2xKi for the receptor.

Techniques: Activity Assay