Neutrophil-specific deletion of Syk kinase results in reduced host defense to bacterial infection

Abstract

Leukocyte-specific CD18 integrins are critical in mediating cell recruitment and activation during host defense responses to bacterial infection. The signaling pathways downstream of CD18 integrins are dependent on the spleen tyrosine kinase, Syk. To investigate the role integrin signaling plays in host defense, we examined the responses of Syk-deficient neutrophils to bacterial challenge with serum-opsonized Staphylococcus aureus and Escherichia coli. Syk-conditional knockout mice lacking this kinase specifically in myeloid cells or just neutrophils were also used to investigate host responses in vivo. Syk-deficient neutrophils manifested impaired exocytosis of secondary and tertiary granules, reduced cytokine release, and very poor activation of the NADPH oxidase in response to serum-opsonized S aureus and E coli. These functional defects correlated with impaired activation of c-Cbl, Pyk2, Erk1/2, and p38 kinases. Bacterial phagocytosis, neutrophil extracellular trap formation, and killing were also reduced in Syk-deficient cells, with a more profound effect after S aureus challenge. In vivo, loss of Syk in myeloid cells or specifically in neutrophils resulted in reduced clearance of S aureus after subcutaneous or intraperitoneal infection, despite normal recruitment of inflammatory cells. These results indicate that loss of Syk kinase-mediated integrin signaling impairs leukocyte activation, leading to reduced host defense responses.

Figure 1

Impaired degranulation in syk^−/− neutrophils in response to bacteria. (A) Flow cytometric analysis of CD11b and CD62L levels on WT or syk^−/− neutrophils stimulated for 60 minutes with rag1^−/− serum-opsonized (op) S aureus (MOI = 5) or E coli (MOI = 10). Neutrophils of the indicated genotype were quantified for CD11b (MFI) after stimulation with (B) S aureus or (C) E coli; percentage CD62L⁻ (shed) after stimulation with (D,F) S aureus, (E,G) E coli, or (H-I) bacteria opsonized with heat-inactivated (HI op) serum. (J) Release of gelatinase granules by WT or syk^−/− neutrophils stimulated in suspension with S aureus or E coli for 60 minutes and analyzed by gelatinase zymogram. (K) Quantitation of gelatinase zymogram (MOI = 1). (L) Lactoferrin release (MOI = 1). Error bars represent SD. *P < .05, **P < .01, ^#P < .001 compared with WT by 2-way analysis of variance (ANOVA) with Bonferroni posttests. All data are representative of 3 or more independent experiments.

Figure 2

Syk-deficient neutrophils have altered cytokine profiles after stimulation with bacteria. (A) WT (white bars) or syk^−/− (black bars) neutrophils were stimulated with rag1^−/− serum-opsonized S aureus or E coli (MOI = 5) for 16 hours. Culture supernatants or cell pellets lysed in low detergent lysis buffer were analyzed for TNF-α, MIP1α, MIP2, KC, IL-1β, IP-10, or MCP1 by multiplex bead array (Milliplex). Data are the mean of 2 independent experiments, each with an N = 2 or N = 3. (B) WT, syk^−/−, CD11b^−/−, or CD18^−/− neutrophils were stimulated with rag1^−/− serum-opsonized S aureus or E coli (MOI = 2) for 16 hours and assessed for (B) TNF-α secretion into the supernatant or (C) TNF-α synthesis and storage in cell lysates by plate-based ELISA, and represent at least 3 independent experiments. Data are presented as mean ± SD. *P < .05, **P < .01, # P < .001 compared with WT by 2-way ANOVA with Bonferroni posttests.

Figure 3

Syk-deficient neutrophils display reduced integrin-mediated superoxide production in response to bacteria. (A) Flow cytometric detection of superoxide by fluorescence conversion of APF in WT or syk^−/− neutrophils after stimulation with APC-labeled S aureus (MOI = 5). (B) WT, syk^−/−, CD11b^−/−, or CD18^−/− neutrophils were plated in microtiter wells containing S aureus (MOI = 5) or (C) E coli (MOI = 10), in cytochrome c media. Production of superoxides (SOx) by respiratory burst was measured as reduction of cytochrome c. Data are representative of at least 3 independent experiments and are mean ± SD. (D) Western blot analysis of p40^phox phosphorylation (Thr 154) after priming of neutrophils for 30 minutes with 50 ng/mL TNF-α and, where indicated, 30 minutes with S aureus (Sa) MOI = 20, E coli (Ec) MOI = 40, or 10nM PMA. (E) Quantitation of phospho-p40^phox normalized to actin. (F-G) Intracellular activation of elastase was detected by flow cytometry after cleavage of ElastoLux. Neutrophils of the indicated genotype were stimulated with (F) S aureus or (G) E coli (MOI = 5) for the indicated time points. Data are representative of 3 independent experiments and are mean ± SD. *P < .05, **P < .01, ^#P < .001 compared with WT by ANOVA.

Figure 4

Syk is required for normal phosphorylation events after bacterial stimulation. Western blot analysis of (A) c-Cbl or (B) Pyk2 phosphorylation after stimulation of WT or syk^−/− neutrophils with S aureus (Sa) or E coli (Ec) for 10 minutes. For quantitation, c-Cbl and p-c-Cbl were detected on separate gels and were each normalized to actin; p-Pyk2 was normalized to total Pyk2 detected on the same gel. (C-D) Flow cytometric histograms of intracellular (C) p-Erk1/2 at 10 minutes in response to S aureus or at 15 minutes in response to E coli or (D) p-p38 at 20 minutes in response to S aureus or E coli. Histograms of unstimulated WT (black dash) or syk^−/− (filled gray) and stimulated WT (black) or syk^−/− (gray) neutrophils are shown. Time course quantitation is of the MFI. Error bars represent ± SD. *P < .05, **P < .01, #P < .001 by 2-way ANOVA with Bonferroni posttests. All data are representative of 3 or more independent experiments.

Figure 5

Signaling through Erk and p38 is required for neutrophil degranulation events. (A) WT neutrophils pretreated with dimethyl sulfoxide vehicle, 20μM PD98059, 3μM SB203580, or both compounds were stimulated for 60 minutes with S aureus (MOI = 5) or E coli (MOI = 10), then analyzed by flow cytometry for CD11b and CD62L. Quantitation of (B) CD11b MFI ± SD, and (C) percentage of CD62L⁻ mean ± SD. Data are ± SD and are representative of 3 or more independent experiments. (D) WT neutrophils pretreated as in panel A were plated in microtiter wells containing S aureus (MOI = 5) or E coli (MOI = 10), in cytochrome c media, and assessed for superoxide (SOx) production as in Figure 3. Data are representative of at least 3 independent experiments. Error bars represent ± SD. **P < .01, ^#P < .001, by 2-way ANOVA with Bonferroni posttests.

Figure 6

Syk is required for optimal phagocytosis and killing of rag1^−/− serum-opsonized bacteria. (A) Phagocytosis was determined by flow cytometric analysis of FITC-labeled S aureus or E coli by WT, syk^−/−, CD11b^−/−, or CD18^−/− neutrophils. (B) Intracellular bacterial killing capacity assessed by viable S aureus or E coli colony-forming units (CFUs). Data are the mean (n = 3 replicates) of the percentage remaining CFU relative to the addition of gentamicin (set as time = 0 minutes). (C) Cytospins of TNF-α primed neutrophils stimulated for 2 hours with S aureus or E coli and stained with 4,6-diamidino-2-phenylindole (DNA/red), antihistone antibody (green), and phalloidin (actin/blue). White arrowheads indicate cells classified as NETs. (D) NETs quantified by both loss of nuclear structure and exclusion of the actin cytoskeleton, expressed as a percentage of total neutrophils scored. Error bars represent ± SD. Data are representative of 3 independent experiments. *P < .05, **P < .01, #P < .001 by ANOVA.

Figure 7

Neutrophil-specific deletion of Syk results in a defective host response to S aureus. Analysis of syk deletion by flow cytometry of Syk protein in (A) neutrophils (Ly6G⁺CD11b⁺) and (B) monocytes (Ly6G⁻CD11b⁺) from the bone marrow (BM), peripheral blood (PB), and air pouch lavage (AP). Syk^KO cells from syk^−/− chimeras or syk ^f/fVav1cre^Tg mice are negative controls (gray histograms) relative to syk^f/−LysMcre^Tg/+, syk^f/−Mrp8cre^Tg (dashed black lines), or syk^f/− (solid black line) mice. Histograms are representative of at least 4 animals. (C) CFU of S aureus and neutrophil infiltrate and (D) CFU of E coli and neutrophil infiltrate in the air pouch lavage of syk^f/+LysMcre^Tg/+ and syk^f/−LysMcre^Tg/+ mice 24 hours after the initiation of infection. (E) CFU of S aureus and neutrophil infiltrate and (F) CFU of E coli and neutrophil infiltrate in the air pouch lavage of syk^f/f, syk^f/−, and syk^f/−Mrp8cre^Tg mice 24 hours after the initiation of infection. Data represent at least 3 independent experiments, with at least 3 animals per group. The difference in S aureus counts in the air pouch is statistically significant; *P < .05 by Mann-Whitney U test. (G) Analysis of TNF-α and MIP-1α levels in S aureus–infected air pouches from syk^f/+LysMcre^Tg/+ and syk^f/−LysMcre^Tg/+ mice. *P < .05, unpaired t test.