Fig 1.
Mitochondrial ROS are produced by PMNs in response to Streptococcus pneumoniae infection.
WT (C57BL/6) bone marrow-derived PMNs were infected with S. pneumoniae TIGR4 at various MOIs for 10 minutes and mitochondrial ROS measured using MitoSOX. (A) Gating strategy. (B) The geometric Mean Fluorescent Intensity (MFI) or (C) % of mitochondrial ROS producing cells were determined by flow cytometry. (D) PMNs were treated with vehicle control or the mitochondrial ROS scavenger MitoTEMPO and then mock treated (uninfected) or infected with S. pneumoniae (+Sp) TIGR4 at a MOI of 50. Cells were monitored for intracellular ROS production over the course of 60 minutes using chemiluminescent luminol. (B-D) Representative data shown are from 1 out of 5 separate experiments in which n = 3 technical replicates were used per condition. Line and Bar graphs represent the mean +/-SD. (B and C) * 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) * indicates significant differences between infected groups +/- MitoTEMPO as determined by 2-way ANOVA followed by Tukey’s multiple comparisons test.
Fig 2.
Bacterial factors required for mitochondrial ROS production by PMNs.
(A) WT (C57BL/6) bone-marrow derived PMNs were mock-treated (Uninfected) or infected with Live (Live Sp), heat-killed (HK Sp) or formalin fixed (Formalin-fixed Sp) S. pneumoniae TIGR4 at a MOI of 10 for 10 minutes and mitochondrial ROS was measured using MitoSOX. Data are representative of 1 out of 3 separate experiments in which n = 3 technical replicates were used per condition. (B) WT PMNs were infected with GFP-expressing S. pneumoniae TIGR4 at a MOI of 10 for 10 minutes. GFP positive vs. negative populations were gated on and compared for production of mitochondrial ROS using MitoSOX by flow cytometry. Data are representative of 1 of 5 experiments in which n = 3 technical replicates were used per condition. (C) WT PMNs were seeded in 24-well plates and either directly infected with S. pneumoniae TIGR4 at a MOI of 10, or were separated from the bacteria by a trans-well. The % of MitoSOX+ cells were determined using flow cytometry. Data are representative from 1 of 3 separate experiments in which n = 3 technical replicates were used per condition. (D) PMNs were either directly infected with S. pneumoniae TIGR4 at a MOI of 10, or treated with bacterial supernatant. The % of MitoSOX+ cells were determined using flow cytometry. Data are representative from 1 of 4 separate experiments in which n = 3 technical replicates were used per condition. (E) WT PMNs were mock-treated (Uninfected) or infected at a MOI of 10 for 10 minutes with either wild type S. pneumoniae TIGR4 (+TIGR4), a pneumolysin deletion mutant (+ΔPLY), a capsular deletion mutant (+ΔCPS) or bacteria lacking superoxide dismutase (+ΔsodA). The % of MitoSOX+ cells were determined by flow cytometry. Data are representative of 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 indicated groups as measured by one-way ANOVA followed by Tukey’s multiple comparison test. n.s. indicates not significant.
Fig 3.
MyD88 signaling required for mitochondrial ROS production by infected PMNs.
Bone-marrow-derived PMNs were isolated from C57BL10 (WT) (open bars), MyD88+/- (dark bars), and MyD88-/- (grey bars) mice. PMNs were infected with S. pneumoniae TIGR4 (+Sp) at a MOI of 10 or mock-treated (Uninfected) for 10 minutes. (A) The % of MitoSOX+ cells were determined using flow cytometry. Data are representative from 1 of 4 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 indicated groups as measured by one-way ANOVA followed by Tukey’s multiple comparison test. (B) 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 four separate experiments (n = 4 mice/ group) are shown. $ indicates significantly different from 1 as measured by one-sample t-test.
Fig 4.
Mitochondrial ROS are required for the ability of PMNs to kill S. pneumoniae.
(A) WT (C57BL/6) bone-marrow derived PMNs were treated with vehicle control (VC), the mitochondrial ROS scavenger MitoTEMPO or Euk134 or the NADPH oxidase inhibitor DPI and then infected with S. pneumoniae TIGR4. (B) WT (C57BL/6) bone-marrow derived PMNs were treated with vehicle control (VC) or MitoTEMPO and infected with S. pneumoniae D39, 19F, or 23F. The percentage of bacterial killing was determined with respect to no PMN controls under the same treatment conditions. Data are pooled from (A) n = 5 and (B) n = 3 separate experiments. Bar graphs represent the mean +/-SD. * indicates significant differences between the indicated groups as determined by (A) one-way ANOVA followed by Tukey’s multiple comparisons test or (B) unpaired Student’s t-test. n.s. indicates not significant.
Fig 5.
A2B adenosine receptor signaling blunts mitochondrial ROS production.
(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.
Fig 6.
A2B adenosine receptor signaling impairs PMN antibacterial function.
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.
Fig 7.
A2BR impairs host resistance against pulmonary infection with S. pneumoniae.
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).
Fig 8.
Mitochondrial ROS are produced by PMNs following pulmonary challenge.
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 Materials and Methods). (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.
Fig 9.
Mitochondrial ROS are produced in circulating PMNs in response to systemic infection.
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 Materials and Methods). (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.
Fig 10.
A2BR impairs host resistance against systemic infection with S. pneumoniae.
(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.
Fig 11.
Mitochondrial ROS are required for the antimicrobial activity of human PMNs.
PMNs were isolated from the blood of young healthy donors and (A) infected with S. pneumoniae TIGR4 at the indicated MOIs or mock-treated (uninfected) for 10 minutes. The % of MitoSOX+ cells were determined using flow cytometry. Data shown are from 4 separate donors where each condition was tested in triplicates per donor. Bar graphs represent the mean +/-SD. * indicates significant differences from uninfected controls as determined by one-way ANOVA followed by Dunnett’s multiple comparisons test. (B) PMNs were pre-treated with vehicle control (VC), the general ROS scavenger Ascorbic Acid, the mitochondrial ROS scavenger MitoTEMPO, the NADPH oxidase inhibitor Diphenyleneiodonium chloride (DPI) or the A2B Agonist (BAY 60–6583) and then infected with S. pneumoniae TIGR4. For each donor, the average percent bacterial killing compared to a no PMN control was calculated from triplicate wells per condition. Data from 4 donors are shown. Bar graphs represent the mean +/-SD. * indicates significant differences from VC treated PMNs as determined by one-way ANOVA followed by Dunnett’s multiple comparisons test.
Fig 12.
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.