Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation

Abstract

Signaling cascades converting the recognition of pathogens to efficient inflammatory responses by neutrophils are critical for host survival. SKAP2, an adaptor protein, is required for reactive oxygen species (ROS) generation following neutrophil stimulation by integrins, formyl peptide receptors, and for host defense against the Gram-negative bacterial pathogens, Klebsiella pneumoniae and Yersinia pseudotuberculosis. Using neutrophils from murine HoxB8-immortalized progenitors, we show that SKAP2 in neutrophils is crucial for maximal ROS response to purified C-type lectin receptor agonists and to the fungal pathogens, Candida glabrata and Candida albicans, and for robust killing of C. glabrata. Inside-out signaling to integrin and Syk phosphorylation occurred independently of SKAP2 after Candida infection. However, Pyk2, ERK1/2, and p38 phosphorylation were significantly reduced after infection with C. glabrata and K. pneumoniae in Skap2-/- neutrophils. These data demonstrate the importance of SKAP2 in ROS generation and host defense beyond antibacterial immunity to include CLRs and Candida species.

Keywords: Biological sciences; Immunology; Microbiology; cell biology.

Graphical abstract

Figure 1

Transferred Skap2−/− DIV neutrophils fail to control K. pneumoniae infection in neutrophil-depleted mice despite normal C-type lectin receptor expression (A) C57BL/6J (B6) wild-type mice were treated with PBS (control, black circles) or α-Ly6G (1A8, triangles) antibody intraperitoneally. 16 hours post-antibody injection, α-Ly6G-treated mice were intravenously injected with sterile PBS containing no cells (black triangles), B6 wild-type (blue triangles) and Skap2−/− (red triangles) DIV neutrophils (see STAR Methods). All B6 mice were then infected retropharyngeally with 5 × 10³ cfu K. pneumoniae. Lungs were harvested after 24 hours and plated for bacterial burden (log (cfu/g lungs)). Data were compiled from n = 3 independent experiments with 1–3 mice each group. (B–I) BALB/c wild-type (WT) or Skap2−/− mice were infected intranasally with K. pneumoniae, and lungs were harvested after 24 hours. Cells were stained with CD11b and Ly6G, fixed with 4% formaldehyde, and stained with α-Dectin-1, α-Mincle, or α-Dectin-2 along with their respective secondary fluorescent-conjugated antibody (see STAR Methods). Neutrophils (CD11b⁺ Ly6G⁺) were further gated to analyze expression of Dectin-1, Mincle, and Dectin-2. CD11b + Ly6G + cells were assessed for CLRs and CD11b integrin expression, and presented as (B, D, F, and H) percent of cells, or (C, E, G, and I) mean fluorescent intensity (MFI). Data are compiled from n = 2 independent experiments with 3 mice each group. Center bars are geometric means, and each dot represents a mouse. Dotted lines indicate (B, D, F, and H) average level in PBS-treated lungs or (C, E, G, nd I) MFI of sample stained with fluorescent-conjugated secondary antibody only. Significance was assessed using (A) one-way ANOVA with Sidak's, or (B–I) two-tail unpaired Student’s t test. See also Figures S1 and S2.

Figure 2

TDB, furfurman, and curdlan-induced ROS requires SKAP2 (A and B) The respiratory burst of BALB/c wild-type (WT) and Skap2−/− DIV neutrophils plated on immobilized TDB, an agonist for mincle-containing dimers, furfurman (Fur), an agonist for Dectin-2, or curdlan (Cur), an agonist to Dectin-1. The total concentration of superoxide produced after 60 min was calculated by the sum of the area under the curves and presented as (A) total superoxide (values were normalized to unstimulated samples of each respective genotype), or (B) as percentage of PMA-induced ROS. Data are presented as mean ± SEM compiled from n = 8–9 independent experiments from two different HoxB8-GMP cell lines per each genetic background (WT or Skap2−/−). (C–H) DIV neutrophils were stained with CD11b and Ly6G, fixed with 4% formaldehyde, and stained with α-Mincle, α-Dectin-2, or α-Dectin-1. Surface receptor levels are reported as mean fluorescence intensity (MFI) with mean ± SEM from n = 5–7 independent experiments from two different HoxB8-GMP cell lines. Significance was assessed using (A and B) one-way ANOVA with Sidak's, or (D, F, and H) two-tail unpaired Student’s t test. See also Figure S3.

Figure 3

TDB, furfurman, and curdlan-stimulated neutrophil adhesion are SKAP2-dependent (A–J) Firmly-adherent WT (A–E) and Skap2−/−(F–J) DIV neutrophils stimulated with immobilized TDB (B, G), furfurman (C-H), or curdlan (D, I) for 1 hour were imaged by bright-field microscopy. Control cells were plated onto surface coated with 10% FBS (Unstim) (A, F), or IgG immune complexes (IC) (E, J) for 1 hour. Original magnification was 20× (n = 3–4 independent experiments in technical duplicates). (K and L) Images were blinded prior to counting the number of rounded and spread cells, which are presented as the percentage of (K) total cells counted (values were normalized to unstimulated samples of each respective genotype), or (L) of IC-induced adhered neutrophils. Significance was assessed using (K) one-way, or (L) two-way ANOVA with Sidak’s post-test.

Figure 4

SKAP2 is not required for CLR-induced integrin activation The assessment of high affinity integrin conformation was measured by the binding of soluble Alexa 488-conjugated fibrinogen to WT and Skap2−/− neutrophils stimulated with TDB, furfurman (Fur), and curdlan (Cur). Neutrophils were stimulated for 20 min at 37°C with 5% CO₂. Alexa 488-conjugated fibrinogen was then added to each well, and the cells were incubated for another 15 min at 37°C with 5% CO₂. Some cells were left untreated/unstimulated (PBS), treated with 2.5mM Mn²⁺ as a positive control for the last 15 min of incubation, or treated with 2.5mM EDTA for 30 min prior to the addition of fibrinogen to the plate as negative control. Cell suspensions were collected, fixed with 1% formaldehyde, and analyzed by flow cytometry. (A–F) Representative histograms gated on live cells. (G) The quantification of the percentage of fibrinogen-bound cells by DIV neutrophils (normalized to EDTA-treated level, divided by % of fibrinogen-bound by Mn²⁺-treated cells). (H) Level of fibrinogen-bound cells by Skap2−/− DIV neutrophils as fold-change to WT level. The data are presented as the mean ± SEM from n = 4–5 independent experiments in technical duplicate. Significance was assessed using two-way ANOVA with Sidak’s post-test.

Figure 5

SKAP2 is required for maximal C. glabrata and C. albicans-induced ROS production, and neutrophil killing (A–C) The respiratory burst of WT and Skap2−/− DIV neutrophils following C. glabrata and C. albicans stimulation (MOI 1) using isoluminol chemiluminescence. (A and B) A representative experiment performed in technical triplicate with data presented as mean ± SEM. Values shown are with background subtraction of the unstimulated samples plated on FBS-coated surface. (C) The total concentration of superoxide produced after 45 min was calculated by the sum of the area under the curves and presented as the percentage of PMA-induced ROS. DIV data are presented as mean ± SEM compiled from n = 6 independent experiments in technical triplicates. Significance was assessed using two-way ANOVA with (A and B) Tukey’s post-test or (C) Sidak’s post-test. (D–F) WT and Skap2−/− DIV neutrophils were co-incubated with yeasts (C. albicans for 2 hr, and C. glabrata for 18 hr) at MOI 0.2. Neutrophils were lysed and PrestoBlue dye was then added to measure the yeast viability by fluorescent readings. Percent of yeast killing was calculated as follows: 100 – (100∗viability of yeast plus neutrophils/viability of yeast only). (D) Data are a representative in technical triplicate, and significance was assessed using two-way ANOVA with Sidak’s. (E and F) The average yeast killing of technical replicates was calculated from each independent experiment. The Skap2−/− values were divided by WT values in each respective experiment. The WT level of yeast killing in each experiment was set to 100 as indicated by the dotted line. The percent of yeast killing by WT DIV neutrophils was divided by the average of the 5–6 experiments. Data were compiled from n = 5–6 independent experiments and presented as mean ± SEM with each experiment represented by a different color. Significance was assessed using paired Student’s t-test.

Figure 6

SFK-Syk-Btk are required for neutrophil ROS production following stimulation of TDB, curdlan, furfurman, C. albicans, and C. glabrata The respiratory burst of DIV (A, B, E, and F) or human (C and D) neutrophils treated with inhibitors using (A–D) a cytochrome C assay or (E and F) isoluminol chemiluminescence. (A–D) Neutrophils were pre-treated with DMSO (Veh), PP2 (iSFKs), R406 (iSyk), or Ibrutinib (iBtk) for 10 min at 37°C and then plated onto immobilized TDB, furfurman, or curdlan (cur), and measured for 60 min. (E and F) DIV neutrophils were pre-treated with inhibitors as in (A–D), and then infected with C. albicans or C. glabrata (MOI 1) and measured for 30 min. (E) A representative experiment is shown in technical triplicate with data presented as mean ± SEM. The total superoxide was calculated as area under the curve after subtracting the uninfected samples, and presented as (A and C) total production or (B) as percentage of PMA-induced ROS. (A–D and F) Data are compiled from (B–D) n = 4–7 or (F) n = 2 independent experiments performed in technical triplicate and presented as mean ± SEM with dots representing averages of independent experiments. Significance was assessed using one-way ANOVA with Sidak’s post-test.

Figure 7

SKAP2 is required for optimal C. glabrata-stimulated phosphorylation of Pyk2 but not Syk kinases WT and Skap2−/− neutrophils were (A–C and G–I) plated onto immobilized TDB, furfurman (Fur), or IgG immune complexes (IC), or (D–F and J–L) stimulated with MOI 2 of C. albicans and C. glabrata for 15 min at 37°C. Lysates were analyzed by Western blot for pSyk (Y352), pPyk2 (Y402), and RhoGDI, then stripped, and re-probed for respective total proteins. Quantification is shown in Tables S1–S4. Data were compiled from 3-5 independent experiments. (A, D, G, and J) Representative blot shown with arrows to indicate bands used for analysis.(B, E, H, and K) Fold change from each experiment with respect to unstimulated, symbols represent the value from each experiment, solid symbols indicating values of blot shown, bars indicate mean. Statistics represent mean ± SEM. (C, F, I, and L) The Skap2−/− values were divided by WT values in each respective experiment. The average WT level of CLR-stimulated phosphorylation was set to 1 as indicated by the dotted line. Data were compiled from n = 3–5 independent experiments with data presented as mean ± SEM. Significance was assessed using (B, E, and H) two-way ANOVA with Tukey's post-test or (K) one-way ANOVA within each group, or two-way ANOVA between WT and KO groups with Sidak’s post-test. See also Figure S4 and Tables S1–S4.

Figure 8

SKAP2 is required for optimal C. glabrata, and K. pneumoniae-induced ERK1/2 and p38 phosphorylation WT and Skap2−/− neutrophils were (A–F) plated onto immobilized TDB, furfurman (Fur), or IgG immune complexes (IC), or stimulated with (G–L) MOI 2 of C. albicans and C. glabrata for 15 min or (M-R) MOI 40 of K. pneumoniae for 10 or 15 min at 37°C. Lysates were analyzed by Western blot for pERK1/2, p38, and RhoGDI, then stripped, and re-probed for respective total proteins. Quantification was completed as described in Tables S5–S9. (A, D, G, J, M, and P)Representative blot shown with arrow to indicate bands used for analysis. (B, E, H, K, N, and Q) Fold change from each experiment with respect to unstimulated; symbols represent the value from each experiment; solid symbols indicating values of blot shown; bars indicate mean. Statistics represent mean ± SEM. (C, F, I, L, O, and R) The Skap2−/− values were divided by WT values in each respective experiment. The average WT level was set to 1 and is indicated by the dotted line. Data were compiled from n = 2–4 independent experiments. Significance was assessed using unpaired one-way ANOVA within each group, or two-way ANOVA between WT and KO groups with Sidak’s post-test except for (E) which used paired two-way ANOVA with Sidak’s post-test. See also Tables S5–S9.

Figure 9

SKAP2 functions prior to or after Syk activation after infection with different pathogens The binding of K. pneumoniae to surface receptors on neutrophils leads to the SKAP2-mediated phosphorylation of Syk and/or Pyk2 and requires the production of extracellular ROS. The binding of C. glabrata and C. albicans does not require SKAP2 for Syk phosphorylation; however, Pyk2 phosphorylation was dependent on SKAP2. Binding by the CLR agonist, furfurman and TBD induces Syk and Pyk phosphorylation independently of SKAP2. Likewise, SKAP2 contributes to extracellular ROS production and cell adhesion following the stimulation of integrins and fMLP (Nguyen et al., 2020; Boras et al., 2017; Shaban et al., 2020).