Journal: Microbiology Spectrum

Article Title: Spleen tyrosine kinase inhibitors disrupt human neutrophil swarming and antifungal functions

doi: 10.1128/spectrum.02549-21

Figure Lengend Snippet: Fungal growth in the presence of neutrophil swarms and SYK inhibititors. Treatment of human neutrophils with SYK inhibitors results in significant loss of fungal restriction. Human neutrophils were treated with the indicated SYK inhibitor or with an appropriate vehicle control before being added to wells. ( A ) The area covered by C. albicans after 16 h, normalized to the growth of C. albicans alone for the same time, was quantified. N ≥ 414 swarms across six independent donors. ( B ) The time it took for C. albicans yeast to germinate into hyphae and for those hyphae to escape the neutrophil swarm area were quantified and plotted. N ≥ 80 swarms across five independent donors. ( C ) The average fluorescent intensity of C. albicans (which expresses a far-red fluorescent protein) was quantified over the time-lapse. The approximate slope of the fluorescent intensity plot for each time point was found to determine the rate of fungal growth. N = 16 swarms from a representative donor. Error bars represent the standard deviation for panel A or the standard error for panel B. **** P ≤ 0.0001.

Article Snippet: The SYK inhibitors R406, RO9021, and PRT-060318 2HCL (Selleck Chemicals) were used for SYK inhibition ( ).

Techniques: Control, Standard Deviation

Journal: Microbiology Spectrum

Article Title: Spleen tyrosine kinase inhibitors disrupt human neutrophil swarming and antifungal functions

doi: 10.1128/spectrum.02549-21

Figure Lengend Snippet: Neutrophil swarming function is disrupted by SYK inhibition. ( A ) Disruption of SYK signaling inhibits neutrophil swarming and NET release. Human neutrophils were treated with the indicated SYK inhibitor or appropriate vehicle control. Neutrophils were stained with Hoechst and the average fluorescent intensity was quantified at the target as an indicator of neutrophils’ swarm intensity. ( B ) The slope of these intensity plots was determined over the first hour. ( C ) The average fluorescent intensity of sytox green staining at the target was quantified over the time-lapse as an indicator of DNA release. ( D ) The rate of DNA release between 5 and 13 h is reduced in the presence of SYK inhibitors. N = 16 swarms from a representative donor for all panels. Error bars represent the standard deviation for panels B and D or the standard error for panels A and C. **** P ≤ 0.0001.

Article Snippet: The SYK inhibitors R406, RO9021, and PRT-060318 2HCL (Selleck Chemicals) were used for SYK inhibition ( ).

Techniques: Inhibition, Disruption, Control, Staining, Standard Deviation

Journal: Microbiology Spectrum

Article Title: Spleen tyrosine kinase inhibitors disrupt human neutrophil swarming and antifungal functions

doi: 10.1128/spectrum.02549-21

Figure Lengend Snippet: Cytokine treatment improves neutrophil function during SYK inhibition. Treatment with GM-CSF or GCSF can partially rescue fungal restriction during SYK inhibition. ( A ) Neutrophils were treated with the indicated inhibitor or inhibitor plus cytokine. The area covered by C. albicans after 16 h, normalized to the growth of C. albicans alone for the same time period, was quantified for PRT treatment. N ≥ 382 across at least three independent donors. ( B ) The area covered by C. albicans after 16 h, normalized to the growth of C. albicans alone for the same time period, was quantified for R406 treatment. N ≥ 383 across at least three independent donors. ( C ) The area covered by C. albicans after 16 h, normalized to the growth of C. albicans alone for the same time period, was quantified for RO9021 treatment. N ≥ 478 across at least three independent donors. ( D ) The rate of DNA release (fluorescent intensity of Sytox) and neutrophil accumulation at the swarming target (MFI Hoechst) was quantified for PRT, R406, RO9021, and cytokine priming. N = 16 swarms from a representative donor. Error bars represent standard deviation for panels A, C, and E or standard error for panel D. **** P ≤ 0.0001.

Article Snippet: The SYK inhibitors R406, RO9021, and PRT-060318 2HCL (Selleck Chemicals) were used for SYK inhibition ( ).

Techniques: Inhibition, Standard Deviation

Journal: Blood Advances

Article Title: Platelet spleen tyrosine kinase is a key regulator of anti-PF4 antibody–induced immunothrombosis ∗ †

doi: 10.1182/bloodadvances.2024014167

Figure Lengend Snippet: VITT Abs induce procoagulant PLTs and increased TG via SYK-dependent signaling pathways. (A-B) PLTs were incubated with isolated IgG (A) or (B) serum from patients with VITT or HCs in the presence of exogenous PF4 (10 μg/mL) and tested for changes in the expression levels of P-selectin (CD62p) and PS with FC. As indicated, PLTs were preincubated with SYK inhibitors R406 (5 μM), PRT-060318 (1 μM), entospletinib (5 μM), and lanraplenib (5 μM), respectively. The number of patients tested is reported in each graph. Unpaired or paired t test is shown. (C) Representative TG curve induced on PLTs after incubation with IgGs from 1 patient with VITT in the presence of vehicle (red line), R406 (5 μM, blue solid line) or lanraplenib (5 μM, dashed blue line). Each curve represents the amounts of generated thrombin over time. (D) Data were quantified as peak thrombin generated (nM) using Thrombinoscope software and GraphPad Prism, version 10.1.0. Violin plots showing the distribution of the values were generated using GraphPad Prism, version 10.1.0. Paired t test is shown. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001. N, number of patients tested; ns, nonsignificant; PS, phosphatidylserine.

Article Snippet: To investigate the contribution of PLT SYK to Ab-induced PLT alterations, WB samples or PLTs were pretreated with different SYK inhibitors including R406, the active metabolite of the clinically used SYK inhibitor fostamatinib, PRT-060318 (PRT-318), which was reported to inhibit thrombus formation in an in vivo model of HIT as well as the selective second- and next-generation inhibitors entospletinib (GS-9973) and lanraplenib, respectively, (GS-9876; all from Selleck, Houston, TX) for 30 minutes at room temperature (RT) before the incubation with VITT IgGs (Tübingen) or VITT positive plasmas (Sydney), respectively.

Techniques: Protein-Protein interactions, Incubation, Isolation, Expressing, Generated, Software

Journal: Blood Advances

Article Title: Platelet spleen tyrosine kinase is a key regulator of anti-PF4 antibody–induced immunothrombosis ∗ †

doi: 10.1182/bloodadvances.2024014167

Figure Lengend Snippet: SYK inhibition significantly reduces VITT plasma–induced procoagulant PLT response in WB. WB from healthy individuals was pretreated with SYK inhibitors R406 (5 μM), PRT-060318 (1 μM), entospletinib (5 μM), and lanraplenib (5 μM), or vehicle control (dimethyl sulfoxide [DMSO]) for 15 minutes before exposure to PLT agonist SFLLRN (5 μM) and HC or VITT plasma. Procoagulant PLTs were enumerated by FC. One-way analysis of variance with the Dunnett's multiple comparison test was performed. The number of patients tested is reported in each graph. ∗∗∗ P < .001;∗∗∗∗ P < .0001. N, number of patients tested.

Article Snippet: To investigate the contribution of PLT SYK to Ab-induced PLT alterations, WB samples or PLTs were pretreated with different SYK inhibitors including R406, the active metabolite of the clinically used SYK inhibitor fostamatinib, PRT-060318 (PRT-318), which was reported to inhibit thrombus formation in an in vivo model of HIT as well as the selective second- and next-generation inhibitors entospletinib (GS-9973) and lanraplenib, respectively, (GS-9876; all from Selleck, Houston, TX) for 30 minutes at room temperature (RT) before the incubation with VITT IgGs (Tübingen) or VITT positive plasmas (Sydney), respectively.

Techniques: Inhibition, Clinical Proteomics, Control, Comparison

Journal: Blood Advances

Article Title: Platelet spleen tyrosine kinase is a key regulator of anti-PF4 antibody–induced immunothrombosis ∗ †

doi: 10.1182/bloodadvances.2024014167

Figure Lengend Snippet: WB SYK inhibition prevents from VITT Ab–mediated multicellular thrombus formation ex vivo. (A-B) Whole blood from healthy individuals was incubated with IgG from HCs or patients with VITT in the presence of exogenous PF4 (10 μg/mL) and SYK inhibitors R406 or lanraplenib (both 5 μM) or vehicle control before recalcification and perfusion through microfluidic channels at a venous shear rate of 250 sec –1 (10 dyne/cm 2 ) for 25 minutes. (A) After perfusion, images were acquired at magnification ×40. Scale bar, 20 μm. (B) Violin plots showing the percentage of total surface area coverage (% SAC) by DiOC 6 (nonprocoagulant PLTs), annexin-V (procoagulant PS), fibrin, count of Hoechst-positive–labeled cells (leukocytes); and cumulative total % SAC with DiOC 6 , PS, and fibrin–labeled thrombus captured in the microfluidic channel. The number of patients tested is reported in each graph. Unpaired or paired t test is shown. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001. N, number of patients tested; ns, nonsignificant.

Article Snippet: To investigate the contribution of PLT SYK to Ab-induced PLT alterations, WB samples or PLTs were pretreated with different SYK inhibitors including R406, the active metabolite of the clinically used SYK inhibitor fostamatinib, PRT-060318 (PRT-318), which was reported to inhibit thrombus formation in an in vivo model of HIT as well as the selective second- and next-generation inhibitors entospletinib (GS-9973) and lanraplenib, respectively, (GS-9876; all from Selleck, Houston, TX) for 30 minutes at room temperature (RT) before the incubation with VITT IgGs (Tübingen) or VITT positive plasmas (Sydney), respectively.

Techniques: Inhibition, Ex Vivo, Incubation, Control, Shear, Labeling

Journal: Blood Advances

Article Title: Platelet spleen tyrosine kinase is a key regulator of anti-PF4 antibody–induced immunothrombosis ∗ †

doi: 10.1182/bloodadvances.2024014167

Figure Lengend Snippet: VITT Ab–induced procoagulant PLT-mediated thrombus formation can be prevented via the inhibition of SYK in PLTs. (A-B) PLTs from healthy individuals were incubated with IgG from patients with VITT in the presence of exogenous PF4 (10 μg/mL) and indicated SYK inhibitors or vehicle control (upper panel) before reconstitution into autologous PLT-depleted WB and perfusion through microfluidic channels at a venous shear rate of 250 sec –1 (10 dyne) for 25 minutes. (A) After perfusion, images were acquired at magnification ×40. Scale bar, 20 μm. (B) Violin plots showing the percentage of total (% SAC) by DiOC 6 , PS, fibrin(ogen), count of Hoechst-positive–labeled cells; and cumulative total % SAC of DiOC 6 , PS, and fibrin(ogen)-labeled thrombus captured in the microfluidic channel. Unpaired or paired t test is shown. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001. N, number of patients tested; ns, nonsignificant.

Article Snippet: To investigate the contribution of PLT SYK to Ab-induced PLT alterations, WB samples or PLTs were pretreated with different SYK inhibitors including R406, the active metabolite of the clinically used SYK inhibitor fostamatinib, PRT-060318 (PRT-318), which was reported to inhibit thrombus formation in an in vivo model of HIT as well as the selective second- and next-generation inhibitors entospletinib (GS-9973) and lanraplenib, respectively, (GS-9876; all from Selleck, Houston, TX) for 30 minutes at room temperature (RT) before the incubation with VITT IgGs (Tübingen) or VITT positive plasmas (Sydney), respectively.

Techniques: Inhibition, Incubation, Control, Shear, Labeling