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. 2020 Apr 30;9:e56656. doi: 10.7554/eLife.56656

Figure 5. K. pneumoniae-stimulated ROS production requires SKAP2-dependent activation of tyrosine kinases.

(A–C) Respiratory burst of WT and Skap2-/- DIV neutrophils infected with Kp using isoluminol-chemiluminsence. Unstimulated (unstim), Kp-infected, and 100 nM PMA-treated cells were seeded on FBS-coated wells. (A) Representative experiment performed in triplicate of ROS (RLU) production following K. pneumoniae stimulation. (B) Total ROS (total RLU) produced after 30 min of stimulation was calculated as the total area under the curve shown in (A) of 4 independent experiments performed in technical triplicate. (C) WT and Skap2-/- neutrophils incubated with GFP-expressing K. pneumoniae (Kp-GFP) at MOI 40 for 15 or 30 min, stained with DAPI, and analyzed by flow cytometry for GFP-associated neutrophils. (D) Schematic of potential K. pneumoniae-activated signaling pathways tested by inhibitors. (E) Respiratory burst of WT DIV neutrophils untreated or treated with inhibitors using isoluminol-chemiluminescence assay. DIV neutrophils were pre-treated with DMSO, PP2 (iSFKs), R406 (iSyk), Ibrutinib (iBtk), Go 6083 (iPKC), U73122 (iPLCγ), or U73134 (nPLCγ/non-inhibitory analog of PLCγ inhibitor) for 10 min at 37°C and then infected with MOI 2.5 of K. pneumoniae or treated with 100 nM PMA and measured for 30 min. Total RLU was calculated as area under the curve. Data are a representative figure from 3 independent experiments showing mean ± SD performed in technical triplicate. Significance was assessed using one-way ANOVA with Sidak’s post-test. (F–L) WT and Skap2-/- neutrophils were infected with Kp for 10 or 15 min or stimulated with IC for 10 min at 37°C. Lysates were analyzed by western blot for pSFKs (Y416), pSyk (Y352), pPyk2 (Y402), and RhoGDI. Blots were then stripped and re-probed for total Syk or Pyk2. Data are compiled from 3 independent experiments. (B–C) Data are compiled from 2 to 4 independent experiments performed in technical triplicate. Statistics represent mean ± SEM. (F, H, K) Representative blot shown. (G, I, L) Solid symbols indicate values of blot shown. Bars indicate mean. Significance was assessed using one-way ANOVA (B) with Sidak’s post-test between WT and Skap-2-/-, or (G, I, L) between time points within each genotype, or two-way ANOVA with Sidak’s post-test between WT and Skap2-/- within the same point.

Figure 5.

Figure 5—figure supplement 1. ROS restricts K. pneumoniae infection in lungs and is induced by K. pneumoniae after infection of BM neutrophils.

Figure 5—figure supplement 1.

(A) Bacterial burden of wild-type C57BL/6 and Cybb-/- mice retropharyngeally infected with 5 × 103 cfu K. pneumoniae for 24 hr. Data are compiled from 2 independent experiments with 3–4 mice/genotype/experiment. Each dot represents a mouse and bars represent geometric means. Significance was assessed using two-tailed unpaired Student’s t test. (B–C) Respiratory burst of WT and Skap2-/- BM neutrophils with K. pneumoniae at the indicated MOI using an isoluminol-chemiluminesence assay. (B) Representative experiment of ROS (RLU) production following stimulation with K. pneumoniae, or PMA done in technical duplicate. (C) Total ROS (total RLU) produced after 30 min of stimulation from one experiment shown as mean ± SD. (D) Respiratory burst of WT and Skap2-/- BM neutrophils with Y. pseudotuberculosis ΔyscF at MOI 10 using an isoluminol-chemiluminesence assay.