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

Abstract

Polymorphonuclear cells (PMNs) control Streptococcus pneumoniae (pneumococcus) infection through various antimicrobial activities. We previously found that reactive oxygen species (ROS) were required for optimal antibacterial function, however, the NADPH oxidase is known to be dispensable for the ability of PMNs to kill pneumococci. In this study, we explored the role of ROS produced by the mitochondria in PMN antimicrobial defense against pneumococci. We found that the mitochondria are an important source of overall intracellular ROS produced by murine PMNs in response to infection. We investigated the host and bacterial factors involved and found that mitochondrial ROS (MitROS) are produced independent of bacterial capsule or pneumolysin but presence of live bacteria that are in direct contact with PMNs enhanced the response. We further found that MyD88-/- PMNs produced less MitROS in response to pneumococcal infection suggesting that released bacterial products acting as TLR ligands are sufficient for inducing MitROS production in PMNs. To test the role of MitROS in PMN function, we used an opsonophagocytic killing assay and found that MitROS were required for the ability of PMNs to kill pneumococci. We then investigated the role of MitROS in host resistance and found that MitROS are produced by PMNs in response to pneumococcal infection. Importantly, treatment of mice with a MitROS scavenger prior to systemic challenge resulted in reduced survival of infected hosts. In exploring host pathways that control MitROS, we focused on extracellular adenosine, which is known to control PMN anti-pneumococcal activity, and found that signaling through the A2B adenosine receptor inhibits MitROS production by PMNs. A2BR-/- mice produced more MitROS and were significantly more resistant to infection. Finally, we verified the clinical relevance of our findings using human PMNs. In summary, we identified a novel pathway that controls MitROS production by PMNs, shaping host resistance against S. pneumoniae.

Neutrophil-driven cardiac damage during invasiveStreptococcus pneumoniaeinfection is regulated by CD73

“…Streptococcus pneumoniae serotypes 4 (TIGR4) and 2 (D39) were used in this study [55]. Bacteria were grown in Todd-Hewitt broth (BD Biosciences) supplemented with Oxyrase and 0.5% yeast extract at 37°C/5% CO 2 .…”

“…Flow cytometry was used to determine the numbers of PMNs, the expression of EAD-pathway components on the PMNs and cardiomyocytes, and measurement of total ROS produced by the PMNs isolated from the blood and heart of infected animals euthanized at 6-, 12- and 24hpi. Blood (300μl per mouse) was first collected by portal vein snips using 50mM EDTA/PBS as an anticoagulant [56] followed by the perfusion and harvesting of the heart tissue, as explained above. The hearts were longitudinally cut and one half homogenized to determine bacterial numbers [19] while the other half minced into pieces and digested for 20 minutes with RPMI 1640 media with 10% FBS, 1 mg/ml Type II collagenase (Worthington), and 50 U/ml Deoxyribonuclease I (Worthington) at 37°C/ 5% CO2 to get a single cell suspension.…”

“…For examining total ROS production by PMNs in vivo , mice were euthanized at 6-, 12- and 24hpi following invasive pneumococcal pneumonia. The heart tissue were then minced into pieces and digested for 20 minutes with RPMI 1640 supplemented with 10% FBS, 1 mg/mL Type II collagenase (Worthington), and 50 U/mL Deoxyribonuclease I (Worthington) at 37°C/ 5% CO2, as previously described [56]. Single-cell suspensions were resuspended in FACS buffer (HBSS/ 1% FBS), treated with Fc block and surface stained with for Ly6G and DCFDA dye (2’,7’ –dichlorofluorescin diacetate) (total ROS) for 30 minutes at 37°C.…”

“…Flow cytometry was used to determine the numbers of PMNs, the expression of EAD-pathway components on the PMNs and cardiomyocytes, and measurement of total ROS produced by the PMNs isolated from the blood and heart of infected animals euthanized at 6-, 12-and 24hpi. Blood (300μl per mouse) was first collected by portal vein snips using 50mM EDTA/PBS as an anticoagulant [56] followed by the perfusion and harvesting of the heart tissue, as explained above.…”

Streptococcus pneumoniaeinfection of lung epithelial cells induces internalization of surface GPI-anchored proteins through pneumolysin-mediated activation of host Rho GTPases

“…Murine infection studies: Mice anesthetized under isoflurane treatment were intratracheally (it) instilled with S. pneumoniae TIGR4 at a dose of 2 x 10 6 CFU, as described previously [34,102].…”