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. 2017 Nov 3;292(44):18312-18324.
doi: 10.1074/jbc.M117.802629. Epub 2017 Sep 25.

Neutrophil microparticle production and inflammasome activation by hyperglycemia due to cytoskeletal instability

Stephen R Thom  1 , Veena M Bhopale  2 , Kevin Yu  2 , Weiliang Huang  3 , Maureen A Kane  3 , David J Margolis  4
Affiliations

Neutrophil microparticle production and inflammasome activation by hyperglycemia due to cytoskeletal instability

Stephen R Thom et al. J Biol Chem. .

Abstract

Microparticles are lipid bilayer-enclosed vesicles produced by cells under oxidative stress. MP production is elevated in patients with diabetes, but the underlying cellular mechanisms are poorly understood. We hypothesized that raising glucose above the physiological level of 5.5 mm would stimulate leukocytes to produce MPs and activate the nucleotide-binding domain, leucine-rich repeat pyrin domain-containing 3 (NLRP3) inflammasome. We found that when incubated in buffer with up to 20 mm glucose, human and murine neutrophils, but not monocytes, generate progressively more MPs with high interleukin (IL)-1β content. Enhanced MP production required generation of reactive chemical species by mitochondria, NADPH oxidase, and type 2 nitric-oxide synthase (NOS-2) and resulted in S-nitrosylation of actin. Depleting cells of capon (C-terminal PDZ ligand of neuronal nitric-oxide synthase protein), apoptosis-associated speck-like protein containing C-terminal caspase recruitment domain (ASC), or pro-IL-1β prevented the hyperglycemia-induced enhancement of reactive species production, MP generation, and IL-1β synthesis. Additional components required for these responses included inositol 1,3,5-triphosphate receptors, PKC, and enhancement of filamentous-actin turnover. Numerous proteins become localized to short filamentous actin in response to S-nitrosylation, including vasodilator-stimulated phosphoprotein, focal adhesion kinase, the membrane phospholipid translocation enzymes flippase and floppase, capon, NLRP3, and ASC. We conclude that an interdependent oxidative stress response to hyperglycemia perturbs neutrophil cytoskeletal stability leading to MP production and IL-1β synthesis.

Keywords: NLRP3; actin; inflammasome; interleukin 1 (IL-1); nitric oxide; nitrosylation.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Biochemical mechanism for high glucose–mediated MP production. Data suggest that the initial event triggering MP generation by elevated glucose is enhanced mitochondrial production of reactive species such as superoxide (O2˙̄) and H2O2. These act on endoplasmic reticulum IP3 receptors, followed by activation of protein kinase C isoforms that increase Nox activity. The data demonstrate an interactive role for agents generated by several enzymes. Therefore, we indicate that Nox-derived O2˙̄ feeds back to cause further mitochondrial oxidant production and also reacts with nitric oxide (•NO) and reactive agents produced by interactions involving these two radical species leading to formation of cytosolic SNO-actin. Actin turnover is enhanced by linkage of VASP to SNO-actin, and accelerated polymerization hastens linkage of Rac 1/2 and FAK that are depicted in the figure with dotted lines. FAK links NOS-2 with actin, enhancing its activity. There is also linkage of floppase and flippase to cytosolic actin, which may impact enzyme activity and are required for the membrane phospholipid changes ultimately required for formation. Apoptosis-associated Speck protein with CARD domain (ASC) also links to short-filamentous (F)-actin, and the assembled NLRP3 inflammasome, including pro-IL-1β, mediates caspase activation to produce active IL-1β. Activated caspase also feeds back to increase Nox activity. High glucose also triggers an increase in capon linkage to short F-actin, and this relationship is required for NOS-2 activation, which in turn is required for perpetuation of enhanced actin turnover via SNO-actin production.
Figure 2.
Figure 2.
MP production by human neutrophils. MP were counted in suspensions of neutrophils (5.5 × 105/ml in PBS containing 1 mm CaCl2, 1.5 mm MgCl2, and 5.5 to 20 mm glucose) and incubated for the indicated times. MPs were also isolated from suspensions after 2-h incubations, and content of IL-1β was measured. These values are shown in boxes to the right in the figure. All data shown are mean ± S.E., n = 4, *, p < 0.05.
Figure 3.
Figure 3.
MP production at 2 h by mouse (open squares) and human (closed circles) neutrophils. Cell suspensions of neutrophils (5.5 × 105/ml in PBS containing 1 mm CaCl2, 1.5 mm MgCl2, and 5.5 to 20 mm glucose) were incubated for 2 h, and individual data points as well as mean ± S.E. bars are shown. Viability of cell preparations at the end of the incubations are shown in boxes at the top of the figure as mean ± S.E.
Figure 4.
Figure 4.
Effect of incubation with siRNA to deplete UCP2 in neutrophils. As an example to demonstrate the efficacy of siRNA to deplete a specific protein in murine neutrophils, Western blot lysates from three different preparations are shown for cells incubated with a control siRNA or siRNA to UCP2. Cytosolic actin from the same lysates are also shown. Ratios shown at the bottom were calculated from protein band densities measured in each lysate set.
Figure 5.
Figure 5.
Reactive species production by neutrophils exposed to 5.5, 11, or 20 mm glucose. A, murine neutrophils (5.5 × 105/ml PBS containing 1 mm CaCl2, 1.5 mm MgCl2, and 5.5 mm glucose) were first incubated with 5 μm MitoSOX Red for 10 min, and then washed and resuspended in the buffer with different glucose concentrations as shown for up to 1 h. B, cells were incubated with the buffer shown for 2 h, and then 10 μm DCF-DA was added, and fluorescence was monitored. Note that when aliquots of cells were incubated in the different glucose concentrations for only 30 min or 1 h, DCF-DA was added, and the same fluorescence rates were measured as for incubations lasting 2 h. This method was chosen because, as discussed under “Experimental procedures,” DCF can autocatalyze its own oxidation (79). Values are mean ± S.E., n = 5 for each point. Using the same methods, effects of various inhibitors and siRNA depletion of cells are shown in Table 1.
Figure 6.
Figure 6.
S-Nitrosylated protein. Lysates of murine neutrophils incubated in buffer containing 5.5 or 20 mm glucose for 1 h were prepared according to the biotin-switch assay, and the entire blot is shown. + indicates that after 30 min of incubation, cells were exposed to UV light for 5 min and then left for the remainder of 1 h. Lower blot shows cytosolic actin in each homogenate to control for protein loading. Ratios reflect results from four replicate studies as mean ± S.E. for individual 43-kDa biotin band density/actin band density of cell lysates normalized to the mean ratio for the 5.5 mm glucose samples.
Figure 7.
Figure 7.
Nitrotyrosine in cell proteins. This is a representative Western blot of lysates from murine neutrophils incubated with 5.5, 11, or 20 mm glucose for 2 h that was probed with an anti-nitrotyrosine antibody.
Figure 8.
Figure 8.
Actin polymerization rate. Permeabilized murine neutrophil suspensions were processed as described under “Experimental procedures” and incubated in buffer containing 5.5 or 20 mm glucose for 1 h, and where indicated, the samples were incubated for 30 min, then exposed to UV light for 5 min, and left in the dark for the remainder of 1 h. Data are mean fluorescence/min ± S.E., n = 3, all groups. AU, arbitrary units.
Figure 9.
Figure 9.
Protein associations in the Triton-soluble short F-actin, G-actin, and Triton-insoluble actin fractions. Murine neutrophils were incubated with buffer containing 5.5 or 20 mm glucose for 1 h, and where indicated, the samples were exposed to UV light between 30 and 35 min of the 1-h incubation, prior to addition of DTSP, to cross-link the proteins. Samples were then fractioned based on Triton solubility (see “Experimental procedures”) and subjected to Western blotting. Representative blots among three replicate experiments are shown. After Western blotting, protein band densities were quantified and normalized to the actin band in each lysate. The ratio of each protein relative to actin was compared with that calculated for the 5.5 mm glucose neutrophils in each experiment. Therefore, data in the figure show the fold-change in band density normalized to the ratio observed in 5.5 mm glucose (control) cells for each actin fraction. Data are mean ± S.E. (n = 3); *, p < 0.05 versus cells incubated in 5.5 mm glucose and not exposed to UV light.
Figure 10.
Figure 10.
Confocal microscope images of neutrophils. Murine neutrophils were exposed to 5.5 or 20 mm glucose in buffer for 2 h, then fixed, permeabilized, and stained as described under “Experimental procedures.” Images are typical from at least three replicate trials using different neutrophil preparations.
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