Neutrophil Inflammatory Response Is Downregulated by Uptake of Superparamagnetic Iron Oxide Nanoparticle Therapeutics

Abstract

Superparamagnetic iron oxide nanoparticles (SPION) are employed as diagnostics and therapeutics following intravenous delivery for the treatment of iron deficiency anemia (IDA) in adult patients with chronic kidney failure. Neutrophils are the first defense against blood borne foreign insult and recruit to vascular sites of inflammation via a sequential process that is characterized by adhesive capture, rolling, and shear resistant arrest. A primary chemotactic agonist presented on the glycocalyx of inflamed endothelium is IL-8, which upon binding to its cognate membrane receptor (CXCR1/2) activates a suite of responses in neutrophils. An early response is degranulation with accompanying upregulation of β2-integrin (CD11/CD18) and shedding of L-selectin (CD62L) receptors, which exert differential effects on the efficiency of endothelial recruitment. Feraheme is an FDA approved SPION treatment for IDA, but its effect on the innate immune response of neutrophils during inflammation has not been reported. Here, we studied the immunomodulatory effects of Feraheme on neutrophils freshly isolated from healthy human subjects and stimulated in suspension or on inflammatory mimetic substrates with IL-8. Cells treated with Feraheme exhibited reduced sensitivity to stimulation with IL-8, indicated by reduced upregulation of membrane CD11b/CD18 receptors, high affinity (HA) CD18, and shedding of CD62L. Feraheme also inhibited N-formyl-Met-Leu-Phe (fMLP) induced reactive oxygen species production. Neutrophil rolling, arrest, and migration was assessed in vascular mimetic microfluidic channels coated with E-selectin and ICAM-1 to simulate inflamed endothelium. Neutrophils exposed to Feraheme rolled faster on E-selectin and arrested less frequently on ICAM-1, in a manner dependent upon SPION concentration. Subsequent neutrophil shape change, and migration were also significantly inhibited in the presence of Feraheme. Lastly, Feraheme accelerated clearance of cytosolic calcium flux following IL-8 stimulation. We conclude that uptake of Feraheme by neutrophils inhibits chemotactic activation and downregulates normal rolling to arrest under shear flow. The mechanism involves increased calcium clearance following chemotactic activation, which may diminish the efficiency of recruitment from the circulation at vascular sites of inflammation.

Keywords: immunosupression; inflammation; innate immunity; iron oxide; mechanosignaling; nanoparticle; neutrophil degranulation; neutrophil recruitment.

Figure 1

Effect of Feraheme concentration on integrin and selectin expression following chemotactic stimulation of neutrophils. Isolated human neutrophils were incubated with 1 nM IL-8 and Feraheme magnetic nanoparticles (MNP) for 25 min and cell surface expression of (A) CD62L, (B) CD11b, and (C) HA CD18 was assessed by flow cytometry. Representative histograms depict fluorescent antibody detection for each adhesion receptor at baseline receptor expression and following IL-8 stimulation in presence and absence of MNP. Bivariate data are presented as the percent shedding from unstimulated baseline mean ± SEM for CD62L and percent of unstimulated baseline expression mean ± SEM for CD11b and HA CD18 (n ≥ 4 donors) with experimental replicates averaged for each donor. Paired T-test was performed comparing the average value at each concentration to the 0 mg/ml Feraheme condition of the same donor * and ** denote p value ≤.05 and ≤ 0.01, respectively.

Figure 2

Feraheme alters adhesion receptor expression over a dose range in stimulation with IL-8. Isolated human neutrophils were incubated with IL-8 and Feraheme MNP for 25 min and cell surface expression of (A) CD62L, (B) CD11b, (C) CD18, and (D) HA CD18 was assessed by flow cytometry. The data are presented as the percent of maximum receptor expression mean ± SEM (n ≥ 5 donors) with experimental replicates averaged for each donor. Paired T-tests were performed comparing the average of the 0 mg/ml to the 4 mg/ml conditions for the same donor *, **, and *** denote p value ≤.05, ≤ 0.01, and ≤ 0.001, respectively.

Figure 3

Feraheme alters CXCR expression in neutrophils. Isolated human neutrophils were incubated with IL-8 or vehicle and Feraheme for 25 min and cell surface expression of (A) CXCR1 and (B) CXCR2 was assessed by flow cytometry. The data are presented as mean ± SEM (n ≥4 donors) with experimental duplicates averaged for each donor. Paired T-tests were performed comparing the average value for the 4 mg/ml to the 0 mg/ml Feraheme conditions of the same donor * and *** denote p value ≤.05 and ≤ 0.001, respectively. Paired T-tests were performed comparing the average value for a condition to the 0 nM IL-8 condition of the same donor $ denotes p value ≤.05.

Figure 4

Feraheme inhibits ROS production in fMLP stimulated neutrophils. Isolated human neutrophils stained with 2 µM DHR 123 and treated with or without Feraheme were preincubated with 1 nM IL-8 or vehicle control for 10 min before addition of 1 µM fMLP or vehicle control for 5 min. The data are presented as mean ± SEM (n ≥ 4 donors) with experimental duplicates averaged for each donor. Paired ratio T-test was performed comparing the average value for an experimental condition to the 0 mg/ml Feraheme condition of the same donor. ** denotes a p value ≤ 0.01.

Figure 5

Feraheme alters the kinetics of neutrophil rolling and arrest in microfluidic flow channels. (A) Neutrophil rolling velocity over an E-selectin substrate treated with vehicle or Feraheme (1 mg/ml, 4 mg/ml). (B) Cumulative rolling velocities of neutrophils over an E-selectin substrate treated with vehicle or Feraheme (1 mg/ml, 4 mg/ml). (C) Mean neutrophil rolling velocity over an E-selectin+ ICAM-1 substrate treated with Feraheme (1mg/ml, 4 mg/ml) and/or IL-8 (0.5 nM). (D) Cumulative rolling velocities of neutrophils over an E-selectin + ICAM-1 substrate treated with vehicle or Feraheme (1 mg/ml, 4 mg/ml) and/or IL-8 (0.5 nM). (E) Neutrophil rolling to arrest and migration over an E-selectin+ ICAM-1 substrate treated with Feraheme (1 mg/ml, 4 mg/ml) and/or IL-8 (0.5 nM). * and ^$ denote T-Test p-values of ≤.05 as compared to the 0 mg/ml MNP condition for arrest and migration, respectively. (F) Neutrophil time to arrest over an E-selectin+ ICAM-1 substrate treated with Feraheme (1 mg/ml, 4 mg/ml) and/or IL-8 (0.5 nM). For B, D, and F, * and **** denote T-Test p-values of ≤.05 and ≤ 0.0001, respectively. Data in B, D, E, and F are presented in mean ± SEM (n ≥ 4 donors) with at least 15 cells for each donor per condition.

Figure 6

Feraheme antagonism of E-selectin ligand bond formation. (A) Neutrophil rolling on a substrate of E-selectin in the presence of vehicle control or Feraheme MNP (4 mg/ml) was dynamically imaged using qDF to detect L-selectin (AF488 anti-human DREG55) and PSGL-1 (PE anti-human PL-1) engagement in the plane of adhesive contact. (B) L-selectin and (C) PSGL-1 receptor cluster area and frequency were determined. (D) L-selectin and (E) PSGL-1 receptor density was determined and reported as mean ± SEM (n = 3 donors) with at least 10 cells for each donor per condition. * and **** denote T-test p-values of ≤.05 and ≤ 0.0001, respectively, compared with the 0 mg/ml condition.

Figure 7

Feraheme (4 mg/ml) accelerates clearance of cytosolic calcium following IL-8 stimulated calcium flux. (A) Diagram depicts the pathway of Ca2+ mediated neutrophil activation and antagonism by CGS 21680 via the Adenosine A_2A receptor. (B) Kinetics of cytosolic calcium clearance following maximal release due to 1 nM IL-8 at t = 0 s. (C) Duration for Ca2+ clearance to reach 20% of maximum value. (D) Fold change between maximal calcium flux and baseline level in untreated cells. Data are presented in mean ± SEM (n = 3 donors) with experimental replicates for each donor. Paired T-tests were performed comparing experimental conditions to the NO MNP condition of the same donor *, and ** denote p value ≤.05, and ≤ 0.01, respectively.

Figure 8

Feraheme MNP (4 mg/ml) accelerated clearance of IL-8 stimulated cytosolic Ca2+ is independent of the Adenosine A_2A receptor. The Adenosine A_2A receptor antagonist ZM (2.5 µM) was applied for 5 min in calcium buffer. (A) Diagram depicts calcium flux in cells preincubated with ZM before treatment with the Adenosine A_2A receptor agonist CGS (B) Kinetics of cytosolic calcium resequestration from maximal flux induced by IL-8 (1 nM) at t=0 sec. (C) Time from maximal calcium flux until calcium levels reach 20 percent of max value. (D) Fold change between maximal calcium flux and untreated cells. Data are presented in mean ± SEM (n=3 donors) with experimental replicates for each donor. Paired T-tests were performed comparing experimental conditions to the NO MNP condition of the same donor. * denotes a p value ≤.05.

Figure 9

Schematic depicting the proposed mechanism of Ca2+ sequestration and inhibition of downstream response by Feraheme. Feraheme may inhibit neutrophil function through activation of endo-membrane Ca2+-ATPases leading to accelerated clearance of cytosolic calcium inhibiting intracellular signaling. CXCR1/2 ligation by endogenous receptor ligands (IL-8, fMLP) leads to dissociation of Gα from Gβγ subunits of G proteins activating PLCβ2/3 which splits phosphatidylinositol 4,5 biphosphate (PIP₂). PIP₂ splits releasing inositol-1,4,5 triphosphate (IP₃) that binds to IP₃ receptor (IP₃R) on the surface of the endoplasmic reticulum inducing calcium flux which signals for downstream effector functions (integrin activation, degranulation, and reactive oxygen species production). We propose that Feraheme MNP are being endocytosed by neutrophils which through an unknown intermediary lead to activation of endo-membrane Ca2+-ATPases that sequester calcium and inhibit calcium signaling of functional responses down stream of GPCR.