Functional selective FPR1 signaling in favor of an activation of the neutrophil superoxide generating NOX2 complex

Abstract

The formyl peptide receptors FPR1 and FPR2 are abundantly expressed by neutrophils, in which they regulate proinflammatory tissue recruitment of inflammatory cells, the production of reactive oxygen species (ROS), and resolution of inflammatory reactions. The unique dual functionality of the FPRs makes them attractive targets to develop FPR-based therapeutics as novel anti-inflammatory treatments. The small compound RE-04-001 has earlier been identified as an inducer of ROS in differentiated HL60 cells but the precise target and the mechanism of action of the compound was has until now not been elucidated. In this study, we reveal that RE-04-001 specifically targets and activates FPR1, and the concentrations needed to activate the neutrophil NADPH-oxidase was very low (EC₅₀ ∼1 nM). RE-04-001 was also found to be a neutrophil chemoattractant, but when compared to the prototype FPR1 agonist N-formyl-Met-Leu-Phe (fMLF), the concentrations required were comparably high, suggesting that signaling downstream of the RE-04-001-activated-FPR1 is functionally selective. In addition, the RE-04-001-induced response was strongly biased toward the PLC-PIP₂ -Ca²⁺ pathway and ERK1/2 activation but away from β-arrestin recruitment. Compared to the peptide agonist fMLF, RE-04-001 is more resistant to inactivation by the MPO-H₂ O₂ -halide system. In summary, this study describes RE-04-001 as a novel small molecule agonist specific for FPR1, which displays a biased signaling profile that leads to a functional selective activating of human neutrophils. RE-04-001 is, therefore, a useful tool, not only for further mechanistic studies of the regulatory role of FPR1 in inflammation in vitro and in vivo, but also for developing FPR1-specific drug therapeutics.

Keywords: Biased signaling; Chemotaxis; Formyl peptide receptors; NADPH-oxidase; Neutrophils; Small compounds.

FIGURE 1

The novel small compound RE‐04‐001 (for short—RE—in figures and legends) activates neutrophil‐like HL60 cells. (A) A sensitive technique to measure superoxide production was used to determine the ability of RE to activate neutrophil‐like HL60 cells (10⁵ cells). Cells were pre‐incubated at 37°C for 5 min before agonist stimulation (indicated by arrows) with RE (100 nM, solid line), the formyl peptide receptor (FPR)1 agonist fMLF (100 nM, dashed line) and the FPR2 agonist WKYMVM (100 nM, dotted line). A representative experiment out of three independent experiments is shown. Inset: The peak O₂ ⁻ production induced by RE (triangles) and the two established FPR agonists fMLF (closed circles) and WKYMVM (open circles) from three independent experiments are shown. (B) The activity of DMSO (vehicle control, dashed lines) on its own in activating neutrophil‐like HL60 cells was measured by its ability to trigger a release of superoxide. Inset: a transient rise in intracellular Ca²⁺ ([Ca²⁺]_i). As a positive control, fMLF (100 nM and 10 nM, respectively) was used in parallel to trigger the oxidase release and a transient rise in [Ca²⁺]_i (solid lines)

FIGURE 2

The small compound RE triggers formyl peptide receptor (FPR)1‐mediated intracellular rise of Ca²⁺ independent of Gαq protein activation in human neutrophils. Prior reports show that FPRs primarily use a Gαi rather that a Gαq containing G protein to induce a transient rise of intracellular Ca²⁺ ([Ca²⁺]_i) in human neutrophils. To investigate the RE receptor preference and G protein coupling the transient rise in [Ca²⁺]_i was determined in human neutrophils. (A)–(B) The transient rise of [Ca²⁺]_i in neutrophils was induced by different concentrations of RE (10 nM to 0.01 nM), fMLF (10 to 0.1 nM) and WKYMVM (1 nM). (C) Effect of the FPR1 antagonist cyclosporin H (CysH, 1 µM, middle panel) or the FPR2 antagonist PBP₁₀ (1 µM, right panel) on the transient rise of [Ca²⁺]_i induced by RE (0.1 nM), fMLF (1 nM), and WKYMVM (20 nM). Control cells received no antagonist (left panel). Agonist addition was indicated by arrows. (D) Neutrophils received YM‐254890 (a selective Gαq inhibitor; 200 nM) 5 min before stimulation with fMLF (10 nM), RE (0.1 nM), or PAF (0.5 nM). Control cells received no YM‐254890. (A)–(D) Representative traces from three independent experiments are shown

FIGURE 3

The small compound RE induces formyl peptide receptor (FPR)1‐mediated NADPH‐oxidase activation independent of Gαq protein activation from human neutrophils. Prior reports show that FPR1 agonist activate the neutrophil NADPH‐oxidase and the response involves a Gαi containing G protein. To investigate the RE receptor preference and G protein coupling the release of superoxide anions (O₂ ⁻) was determined in human neutrophils. (A) Neutrophils were stimulated with RE (10 nM, solid line), fMLF (100 nM, dotted line), or WKYMVM (100 nM, dashed line). One representative trace of O₂ ⁻ production out of three independent experiments is shown. (B) Dose–response of RE and fMLF. The EC₅₀‐values and 95% confidence interval (CI) were determined based on the peak O₂ ⁻ response (n = 3). (C) Effect of CysH (1 µM, black bars) or PBP₁₀ (1 µM, gray bars) pre‐incubated with neutrophils for 5 min before activation with RE (10 nM), fMLF (100 nM), or WKYMVM (100 nM). The data are presented as percent of remaining NADPH‐oxidase activity in the presence of antagonists as compared to the responses from control cells (mean ± sd, n = 3). One‐way ANOVA followed by Dunnett's post‐hoc test was used to calculate significance. (D) Neutrophils were first stimulated with RE‐04‐001 (10 nM, arrow to the left) and then further challenged a second stimulation with WKYMVM (100 nM), fMLF (100 nM), or RE (10 nM) as indicated. (E) Naïve or TNFα (37°C, 20 min) primed neutrophils were challenged with RE (10 nM). One representative trace out of three independent experiments is shown. Inset: Comparison between the peak O₂ ⁻ responses released from naïve (black bars) and TNFα primed cells (gray bars) stimulated with RE (10 nM), fMLF (100 nM), or WKYMVM (100 nM). Data are presented as mean ± sd (n = 3) and paired t‐test was used to calculate the TNFα priming effect. (F) Comparison between the peak O₂ ⁻ responses released by neutrophils pretreated with or without the YM‐254890 (200 nM) for 5 min before activation with RE (10 nM), fMLF (100 nM), WKYMVM (100 nM), or PAF (100 nM). Data are presented as percent of remaining NADPH‐oxidase activity in the presence of YM‐254890, compared to the responses from control cells (mean ± sd, n = 3). Paired t‐test was used to calculate the effect of YM‐254890

FIGURE 4

Neutrophil chemotaxis induced by fMLF and RE. Neutrophil migration toward RE and fMLF placed in the bottom wells was determined. (A) Representative micrographs (10× magnification) of neutrophils migrated into the lower compartment containing buffer (spontaneous migration), fMLF (10 nM), or RE (50 nM). (B) Quantification of neutrophil migration toward fMLF (10 nM), WKYMVM (30 nM), or different concentrations of RE by analyzing the amount of myeloperoxidase (MPO) in cells recovered from the lower compartments after 90 min of migration period. Data are presented as chemotaxis index (AU, MPO activity of cells recovered from the bottom wells after migration in absorbance unit at 450 nm) from three independent experiments (mean + sd, n = 3)

FIGURE 5

The small compound RE potently triggers ERK1/2 phosphorylation but poorly recruits β‐arrestin. (A) Phosphorylation of ERK1/2 (pERK1/2) was determined in neutrophil lysates after stimulation with different concentrations of fMLF or RE as indicated for 2 min. Data are presented as percentage of phosphorylated ERK (% pERK) from two to three independent experiments that were run with duplicates (mean + sd). (B) Comparison of phosphorylated ERK induced by fMLF (2 nM) and RE (2 nM) and Student's paired t‐test was used to calculate statistics. *P < 0.05 (C) β‐arrestin recruitment was monitored in CHO cells over‐expressing FPR1 stimulated with 100 nM of fMLF or different concentrations of RE as indicated. Data are presented as percentage of the response induced by 100 nM fMLF (mean + sd, n = 3). Inset: FPR2 over‐expressing CHO cells were stimulated with the FPR2 agonist WKYMVM (100 nM) or RE (100 nM). Data are presented as percentage of the response induced by 100 nM WKYMVM. (D). Effect of RE on the fMLF response (10 nM) and WKYMVM response (25 nM) in FPR1 cells (black bars) and FPR2 cells (gray bars), respectively. Data are presented as percentage of remaining β‐arrestin recruitment in the presence of RE‐04‐001 as compared to the responses from control cells (mean + sd, n = 3)

FIGURE 6

The small compound RE activated FPR1 modulates other GPCR‐mediated neutrophil response. Receptor crosstalk was studied in the NADPH‐oxidase activation assay by measuring the superoxide anions (O₂ ⁻) production from cells desensitized with RE‐04‐001 and from naïve cells received no RE‐04‐001. (A) Crosstalk between RE activated FPR1 and IL8. Neutrophils were activated with RE‐04‐001 (10 nM, indicated by the first arrow) when the response had declined, the cells received a second dose of IL8 (100 ng/ml; indicated by the second arrow). Inset: Quantification of the second IL8 response in RE or fMLF pre‐activated cells from three independent experiments (mean + sd, n = 3). (B) A second dose of different concentrations of RE was added to cells pretreated with Cmp58 (1 µM), the FFAR2 allosteric modulator. Peak O₂ ⁻ production are shown from three independent experiments (mean + sd, n = 3). (C) RE (10 nM, indicated by the first arrow) activated cells received a second stimulation with PAF (100 nM; indicated by the second arrow). One representative experiment out of three independent experiments is shown. Inset: Quantification of the second PAF‐response from neutrophils prestimulated with either RE (10 nM) or fMLF (100 nM). Data are presented as percentage of control response (not pre‐activated) (mean + sd, n = 4). (D) Neutrophils were desensitized with RE (10 nM) to obtain FPR1_des cells before a second stimulation with PAF (100 nM). The FPR1 antagonist CysH was added just prior PAF stimulation (solid line) or cells received no addition before PAF stimulation (dashed line). Representative traces of O₂ ⁻ production is shown. Inset: Peak O₂ ⁻ production induced by PAF from naïve cells and FPR_des cells desensitized with RE or fMLF (100 nM) received with or without CysH are shown (mean + sd, n = 4). Paired Student's t‐test was used to calculate statistical significance between treated and control groups

FIGURE 7

Modulation of the small compound RE activity by latrunculin A and the myeloperoxidase (MPO)/H₂O₂ system. The NADPH‐oxidase activity of neutrophils was determined. (A) Naïve neutrophils and neutrophils pre‐incubated with the actin cytoskeleton‐disrupting drug latrunculin A (LA; 25 ng/ml, 5 min) were activated with RE (10 nM). One representative experiment of three independent experiments is shown. Inset: The peak NADPH‐oxidase activities induced in naïve or LA treated neutrophils by RE (10 nM) or fMLF (100 nM) are shown (mean + sd, n = 3). (B). Neutrophils activated by RE (10 nM, first arrow), when the response had declined, were reactivated with LA (25 ng/ml, second arrow). One representative experiment of three independent experiments is shown. Inset: The peak NADPH‐oxidase activity induced by RE (100 nM), or fMLF (100 nM) from naïve neutrophils and that during reactivation with LA are shown (mean + sd, n = 3). (C). Oxidization of the agonist toward the MPO (1 µg/ml) + H₂O₂ (10 µM) system. The remaining activity of agonist after oxidization was measured by their ability to trigger ROS production from neutrophils in comparison to the control response with agonists received no MPO‐H₂O₂ from three independent experiments (mean + sd, n = 3). The final concentrations of agonists used in the oxidase assay: fMLF (100 nM); WKYMVM (100 nM); RE (12.5 nM); Act‐389949 (ACT, 12.5 nM); and Cmp43 (250 nM). Paired t‐test was used to calculate the statistical significance of agonist treated with or without MPO‐H₂O₂