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A membrane-tethering pepducin derived from formyl peptide receptor 3 shows strong therapeutic effects against sepsis
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
A membrane-tethering pepducin derived from formyl peptide receptor 3 shows strong therapeutic effects against sepsis Ha Young Lee a, **, Hyung Sik Kim a, Yu Sun Jeong a, Ji Cheol Kim a, Yong-Soo Bae a, b, You Hwan Jo c, Joon Seong Park d, Yoe-Sik Bae a, b, * a
Department of Biological Sciences, Sungkyunkwan University, Suwon, 16419, Republic of Korea Science Research Center (SRC) for Immune Research on Non-Lymphoid Organ (CIRNO), Sungkyunkwan University, Suwon, 16419, Republic of Korea Department of Emergency Medicine, Seoul National University Bundang Hospital, Seongnam, 13620, Republic of Korea d Department of Hematology-Oncology, Ajou University School of Medicine, Suwon, 16499, Republic of Korea b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 December 2019 Accepted 11 January 2020 Available online xxx
Formyl peptide receptors (FPRs) are G protein-coupled receptors mainly expressed in inflammatory myeloid cells. Previous reports demonstrated that human neutrophils express only FPR1 and FPR2 but not FPR3. Here, we found that FPR3 is expressed in sepsis patient derived neutrophils and Fpr3 is expressed in the mouse neutrophils. To test the role of Fpr3 in neutrophil activity, we synthesized Fpr3 pepducins and successfully developed an agonistic pepducin that stimulates Fpr3, eliciting calcium increase and chemotactic migration of neutrophils. We also found that administration of an Fpr3 pepducin in an experimental mouse sepsis model significantly increased the survival rate. The pepducin markedly inhibited lung injury, splenocyte apoptosis, and inflammatory cytokine production. Bacterial counts were significantly decreased by the pepducin in septic mice. Based on these results, we suggest that FPR3 can be regarded as a new target to control sepsis, and the newly generated Fpr3-based pepducin can be used for the development of anti-septic agents. © 2020 Elsevier Inc. All rights reserved.
Keywords: Formyl peptide receptor 3 Neutrophil Pepducin Sepsis
1. Introduction Formyl peptide receptors (FPRs) are classical chemoattractant receptors and members of G-protein coupled receptor (GPCR) family [1]. Three FPR members exist in humans (FPR1, FPR2 and FPR3) and eight in mice [Fpr1, Fpr2, Fpr3 (Fpr-rs1) and Fpr-rs3 to 7] [2]. Mouse Fpr1 and Fpr2 are known as orthologues of human FPR1 and FPR2, respectively. Recently, Fpr-rs1 has been named Fpr3 and reported to share a specific pathogen ligand with human FPR3 and also described as an orthologue of FPR3 [3]. The FPRs are mainly expressed in innate immune cells including neutrophils and monocytes/macrophages [4e6]. FPRs play important roles in chemotactic cell migration and mediate diverse biological activities [2,4,7,8]. FPRs have been suggested as putative targets against several diseases including sepsis [9,10]. In particular, we previously demonstrated the therapeutic effects of an FPR agonist (WKYMVm)
against experimental polymicrobial sepsis [10]. Unlike other GPCRs, FPRs are known to have variety of ligands [2,9]. Recently, pepducins have been developed and used as agonists or antagonists of GPCRs [11,12]. We previously reported human FPR2 pepducins, and mouse Fpr1 and Fpr2 pepducins have also been reported by another group [13e15]. Despite the numerous studies on FPRs, most studies have focused on FPR1 and FPR2. This is probably because FPR3 ligands are relatively unknown compared to FPR1 and FPR2. Therefore, it is very important to find and develop FPR3-specific ligands for the study of (patho)physiological function of FPR3. Here, we found that neutrophils from sepsis patients express FPR3 and mouse neutrophils express Fpr3 mRNA. We investigated the function of Fpr3 in neutrophil by synthesizing Fpr3-based pepducins. We also examined the effects of Fpr3 pepducins against an experimental polymicrobial sepsis.
2. Materials and methods * Corresponding author. Department of Biological Sciences, Sungkyunkwan University, Suwon 16419, Republic of Korea. ** Corresponding author. E-mail addresses: [email protected] (H.Y. Lee), [email protected] (Y.-S. Bae).
2.1. Materials Pepducins were synthesized from Anygen (Gwangju, Korea).
https://doi.org/10.1016/j.bbrc.2020.01.058 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: H.Y. Lee et al., A membrane-tethering pepducin derived from formyl peptide receptor 3 shows strong therapeutic effects against sepsis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.058
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Fura-2/AM was purchased from Molecular Probes (Eugene, OR, USA). Fluorescence conjugated anti-FPR3 antibody was purchased from R&D systems (Minneapolis, MN, USA). Antibodies used for Western blotting were purchased from SantaCruz (Dallas, TX, USA). Cytochrome c, cytochalasin B, G418, type I collagen and LPS were purchased from Sigma-Aldrich (St. Louis, MO, USA). Boyden chambers were purchased from Neuroprobe, Inc. (Gaithersburg, MD, USA). Percoll was purchased from GE Healthcare (Chicago, IL, USA). ACK lysis buffer was purchased from Gibco (Gaithersburg, MD, USA). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA). 2.2. FACS analysis Surface expression was analyzed by FACSCanto II flow cytometer (BD Bioscience, San Joes, CA) using fluorescence conjugated specific antibodies. 2.3. Isolation of human and mouse neutrophils Peripheral blood was collected from healthy donors or sepsis patients. These experiments were approved by the Seoul National University Bundang Hospital’s Institutional Review Board for ethics and Ajou University Hospital’s Institutional Review Board for ethics. Peripheral neutrophils were isolated from peripheral blood as described previously [16]. C57BL/6 mice (male) were purchased from Orient Bio (Seongnam, Korea). All animal experiments were performed in accordance with the guidelines of the Korean Food and Drug Administration. All experiments involving animals received the approval of the Institutional Review Committee for Animal Care and Use at Sungkyunkwan University (Suwon, Korea). Mouse bone marrow neutrophils were isolated from the femurs and tibias of mice as described previously [17]. Isolated cells were then stained with anti-Ly6G (1A8) antibody and found to be over 95% Ly6G-positive. 2.4. RT-PCR analysis Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) and cDNA was obtained using the Maxime RT Premix Kit (iNtRON, Seongnam, Korea). Primers were synthesized according to a previous report [18]. Target genes were amplified by RT-PCR using the Maxime PCR PreMix Kit (iNtRON).
for HEK293 cells) at 37 C (90 min for mouse neutrophils, 6 h for HEK293 cells). Migrated cells were stained with hematoxylin (Sigma-Aldrich) and stained cells were counted using a light microscope. 2.8. Measurement of intracellular calcium concentration Intracellular calcium concentration was measured using Grynkiewcz’s method with Fura-2/AM as described previously [19]. 2.9. Measurement of superoxide anion generation Superoxide anion generation was determined by measuring cytochrome c reduction using a microtiter 96-well plate ELISA reader (EL312e; Bio-Tek Instruments, Winooski, VT) as previously described [20]. 2.10. Mice and cecal ligation and puncture (CLP) experimental sepsis model The experimental CLP sepsis model was conducted as described previously [21]. 2.11. Tissue histology The mice were euthanized 24 h after CLP, and their lungs and livers were fixed, sectioned, and stained with hematoxylin and eosin for morphological analysis. 2.12. Measurement of colony forming units (CFUs) in CLP mouse model At 24 h after CLP, peritoneal lavage fluids were collected and cultured overnight on blood-agar base plates (Trypticase™ Soy Agar Deeps; BD Biosciences, San Jose, CA) at 37 C. The numbers of CFUs were counted as described previously [21]. 2.13. TUNEL assay The mice were euthanized 24 h after CLP, and their spleens were isolated and fixed. The TUNEL assay was performed on paraffin tissue sections using a standard histological protocol as described previously [21].
2.5. Establishment of Fpr3-expressing HEK293 stable cell lines 2.14. Cytokine assay in vivo and in vitro HEK293 cells were transfected with the vector or Fpr3 cDNA, using the Lipofectamine 2000 reagent. Vector- or Fpr3-expressing HEK293 stable cell lines were selected to have 500 mg/ml G418 antibiotic resistance. 2.6. Western blot analysis The cells were lysed in lysis buffer as described previously [16]. Extracted proteins were separated by SDS-PAGE, and transferred onto a nitrocellulose membrane. Membranes were then incubated with specific antibodies. Enhanced chemiluminescence was used to detect protein levels with antigen-antibody complexes.
To measure the production of CLP-induced cytokines in serum or peritoneal lavage fluids, mice were subjected to CLP surgery and pepeducins were injected at 2 h and 14 h later. The serum or peritoneal lavage fluids were collected 24 h after surgery. Isolated mouse neutrophils (3 106 cells/0.3 ml) were pre-incubated with mF3pal_14 for 30 min, and then stimulated with absence or 100 ng/ ml LPS for 24 h. After collecting cell-free supernatants, serum or peritoneal lavage fluids, the levels of cytokine were measured by ELISA according to the manufacturer’s instructions (e-Bioscience, San Diego, CA). 2.15. Statistical analysis
2.7. Chemotaxis assay Chemotaxis assays were performed using multiwell chambers as described previously [16]. Briefly, isolated mouse neutrophils or HEK293 cells were applied to polycarbonate filters (3 mm pore size for mouse neutrophils, type I collagen pre-coated 10 mm pore size
The results were evaluated with GraphPad prism software (GraphPad Software, La Jolla, CA). Statistical analysis was performed by the Student’s t-test. All results are expressed as the mean ± S.E. Survival data were analyzed using the long-rank test. A p-value < 0.05 was considered statistically significant.
Please cite this article as: H.Y. Lee et al., A membrane-tethering pepducin derived from formyl peptide receptor 3 shows strong therapeutic effects against sepsis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.058
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3. Results 3.1. Neutrophils from sepsis patients express FPR3 Through flow cytometry analysis, we compared the expression pattern of FPR3 in neutrophils isolated from healthy donors and sepsis patients. FPR3 expression was only apparent in the neutrophils of sepsis patients but not of healthy donors (Fig. 1A left). Although FPR3 expression in the neutrophils of sepsis patients is variable depending on the individual, sepsis patient-derived neutrophils showed significantly increased FPR3 expression (Fig. 1A right).
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anti-Myc antibody (Fig. 1D, inset). Functional activity of transfected Fpr3 was confirmed by chemotactic migration with FPR ligands. Fpr3-expressing HEK293 cells specifically migrated upon stimulation with F2L, an FPR3 agonist, but not vector-expressing HEK293 cells (Fig. 1D). We examined the effects of three Fpr3 pepducins on cell migration activity in vector- or Fpr3-expressing HEK293 cells. mF3pal_9, mF3pal_14 and mF3pal_16 significantly increased chemotactic cell migration of Fpr3-expressing HEK293 cells, but not vector-expressing HEK293 cells (Fig. 1E). These results indicate that the synthesized Fpr3 pepducins act on Fpr3.
3.3. Fpr3 regulates mouse neutrophil activity 3.2. Pepducins stimulating mouse Fpr3 were developed A previous report demonstrated that mouse neutrophils express Fpr3 and the expression is enhanced by LPS stimulation [3]. In this study, we also observed that mouse neutrophils express Fpr3 mRNA as well as Fpr1 and Fpr2 (Fig. 1B). To investigate the function of Fpr3 in neutrophil, we synthesized three types of Fpr3 pepducins, mF3pal_9, mF3pal_14 and mF3pal_16, as shown in Fig. 1C. To investigate whether these pepducins act on Fpr3, we generated stable Fpr3-expressing HEK293 cells by transfecting the cells with Myc-tagged Fpr3 cDNA. Fpr3 expression in the established Fpr3/ HEK293 cell line was confirmed by Western blot analysis using
Intracellular Ca2þ plays an important role in the modulation of neutrophil activity [22]. To investigate the function of Fpr3 in neutrophil, we measured intracellular Ca2þ modulation by treating three Fpr3 pepducins in mouse neutrophil. Stimulation of mouse neutrophils with the three pepducins strongly elicited intracellular calcium increase in a concentration-dependent manner, showing maximal activity at 5 mM (mF3pal_14 and mF3pal_16) or 10 mM (mF3pal_9) (Fig. 2A). Since chemotactic migration is an important functional activity of neutrophils in response to FPR family agonists [2], we tested the effects of the three pepducins on mouse neutrophil chemotaxis. The three pepducins significantly
Fig. 1. Pepducins stimulating Fpr3 were developed. (A) Neutrophils isolated from healthy donors or sepsis patients were stained with anti-FPR3 antibody. FPR3 expression was determined by flow cytometry analysis. (B) Expression of Fpr members of mouse neutrophils were analyzed by RT-PCR. (C) Sequence of Fpr3-based pepducins. (D) HEK293 cells were transfected with Myc-tagged Fpr3 cDNA using Lipofectamine 2000. Transfected cells were then selected by 500 mg/ml G418 containing culture medium. Fpr3 expression in HEK293 cells was confirmed by Western blot assay (D, inset). Transfected cells were used for chemotaxis assay upon treatment with 1 mM fMLF, 1 mM MMK1 and 5 mM F2L. (E) Vector- or Fpr3-expressing HEK293 cells were added to the upper wells of type I collagen pre-coated 10 mm pore size chemotaxis chamber in the presence of 0, 0.1, 1 and 5 mM of mF3pal_9, mF3pla_14, and mF3pal_16 for 6 h. Migrated cells were stained with hematoxylin and stained cells were counted using a light microscope (D, E). Data shown are representative of three independent experiments (A left, B and D inset). Data is presented as the mean ± SE of 6 (healthy donors) or 11 (sepsis patients) (A right). Data are mean ± SE of three independent experiments (D, E). ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001 by t-test compared with not treat (NT) control. ##p < 0.01 by t-test compared with vector/HEK293 control.
Please cite this article as: H.Y. Lee et al., A membrane-tethering pepducin derived from formyl peptide receptor 3 shows strong therapeutic effects against sepsis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.058
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Fig. 2. Fpr3 regulates mouse neutrophil activity. (A) Mouse neutrophils were treated with several concentrations (0.1, 1, 5 and 10 mM) of mF3pal_9, mF3pal_14 and mF3pal_16. Relative cytosolic Ca2þ concentrations are expressed as fluorescence ratios (340:380 nm). (B) Mouse neutrophils were added to the upper wells of 3 mm pore size chemotaxis chambers in the presence of 0, 0.1, 1, 5, and 10 mM of mF3pal_9, mF3pal_14 or mF3pal_16 for 90 min. Migrated cells were stained with hematoxylin and stained cells were counted using a light microscope. (C) Mouse neutrophils were stimulated with 10 mM of mF3pal_9, mF3pal_14 and mF3pal_16 for 5 min. Superoxide anion production was determined by measuring cytochrome c reduction. Data shown are representative of at least three independent experiments (A). Data are mean ± SE of three independent experiments (B, C). ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001 by t-test compared with the vehicle treated control.
stimulated mouse neutrophil chemotaxis, showing concentrationdependency (Fig. 2B). The results indicate that the three Fpr3 pepducins are stimuli that cause mouse neutrophil activation, leading to calcium increase and chemotactic migration. Neutrophils are well known innate immune cells and play an important role against infection through reactive oxygen species (ROS) generation [23]. We also examined whether these pepducins could affect on ROS generation in mouse neutrophils. Isolated mouse neutrophils were stimulated with 10 mM of pepducins for 5 min, mF3pal_14 and mF3pal_16 were significantly increase the ROS generation, but not mF3pal_9 (Fig. 2C). 3.4. mF3pal_14 administration shows therapeutic effects against sepsis The regulation of neutrophil activity is known to be important in sepsis pathology. In particular, anti-bacterial activity through ROS generation is helpful to alleviate the symptoms of sepsis. Based on the results of Fpr3 pepducins-induced regulation of neutrophil
activation (Fig. 2), we investigated whether Fpr3 pepducins could have a therapeutic effect in sepsis using mF3pal_14 and mF3pal_16 which had effects on cell migration, intracellular Ca2þ modulation and ROS generation. To investigate the effects of Fpr3 pepducins against sepsis, we used the CLP mouse sepsis model. After CLP surgery, the two types of Fpr3 pepducin (mF3pal_14 and mF3pal_16) or a vehicle were injected four times at 12 h intervals. The survival rate of vehicle administered CLP mice decreased to 20% in 3 days (data not shown). However, mF3pal_14 administered CLP mice showed a markedly increased survival rate (Fig. 3A). We did not detect any therapeutic effects of mF3pal_16 against CLP sepsis (data not shown). To further investigate the effects of mF3pal_14, we synthesized a scrambled sequence pepducin of mF3pal_14 (scmF3pal_14; pal_VRLPRSSNVFAKKH) as a control pepducin. Several dosages of mF3pal_14 (1, 2, 4, and 8 mg/kg) or 8 mg/kg of scmF3pal_14 were administered four times at 12 h intervals post CLP surgery. mF3pal_14 strongly increased the survival rate in a dosage-dependent manner, showing a maximal survival rate at 4 mg/kg (Fig. 3A). Previous studies report that mortality in sepsis is
Please cite this article as: H.Y. Lee et al., A membrane-tethering pepducin derived from formyl peptide receptor 3 shows strong therapeutic effects against sepsis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.058
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Fig. 3. mF3pal_14 has therapeutic effects against CLP. (A) Different dosages of mF3pal_14 (1, 2, 4, and 8 mg/kg) or scmF3pal_14 (8 mg/kg) were i.p. injected four times into CLP mice at 2, 14, 26, and 38 h after CLP. Survival was monitored for 10 days after CLP. (BeE) mF3pal_14 (4 mg/kg, i.p.) or scmF3pal_14 (4 mg/kg i.p.) was administered 2 and 14 h after CLP, and the mice were sacrificed 24 h after surgery. (B) Lung and liver tissue sections were stained with H&E (original magnification 200). (C, D) Spleen tissue sections were used for a TUNEL assay (original magnification 200) (C), and TUNEL-positive cells were counted (D). (E) Peritoneal lavage fluids collected 24 h after CLP were cultured overnight on blood-agar base plates at 37 C; the number of CFUs was then counted. The data are representative of ten mice per group (A) and five mice per group (B, C). Scale bar; 100 mm (B, C). Data are mean ± SE of three independent experiments (D, E). *p < 0.05, **p < 0.01, ***p < 0.001 by log-rank test compared with scrambled pepducin treated group (A). **p < 0.01 by t-test compared with scrambled pepducin treated control (D).
closely associated with increase of lung inflammation [21]. Administration of mF3pal_14 alleviated lung inflammation by CLP (Fig. 3B). Liver damage was also moderately alleviated in mF3pal_14 injected mice (Fig. 3B). Immune cell apoptosis in the spleen plays an important role in the pathology of CLP sepsis [24]. Splenocyte apoptosis was measured from mF3pal_14 or scmF3pal_14 administered CLP mice. A TUNEL assay showed that splenocyte apoptosis was significantly reduced by mF3pal_14 injection (Fig. 3C and D). Bacterial burden positively correlates with the increased mortality of CLP mice [24]. Bacterial colony counts were dramatically decreased by mF3pal_14 administration (Fig. 3E). 3.5. mF3pal_14 administration inhibits CLP-induced proinflammatory cytokine production
cytokine levels in CLP mice. Increased levels of TNF-a, IL-6 and IL-1b production in peritoneal fluid and serum in CLP mice were significantly decreased by mF3pal_14 administration (Fig. 4A and B). We then examined the direct effects of mF3pal_14 on inflammatory cytokine production in mouse neutrophils. Mouse neutrophils were stimulated with several concentration of mF3pal_14 (0, 1, 5 and 10 mM), but mF3pal_14 alone did not increased pro-inflammatory cytokines production (Fig. 4C). And then, we examined the effect of mF3pal_14 on LPS-induced pro-inflammatory cytokine production in mouse neutrophil. Stimulation of mouse neutrophils with LPS strongly increased the production of TNF-a and IL-6 (Fig. 4C). Addition of mF3pal_14 (0, 1, 5 and 10 mM) prior to LPS stimulation significantly decreased the production of these two inflammatory cytokines in a dose-dependent manner (Fig. 4C). 4. Discussion
Proinflammatory cytokines exacerbate the inflammatory responses by CLP [25]. Since mF3pal_14 elicited strong therapeutic effects against sepsis, we examined the effects of the pepducin on
Unlike other FPR family members, the functional role of FPR3 in the innate immune response is largely unknown because of the
Please cite this article as: H.Y. Lee et al., A membrane-tethering pepducin derived from formyl peptide receptor 3 shows strong therapeutic effects against sepsis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.058
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Fig. 4. mF3pal_14 inhibits inflammatory cytokine production in vivo and in vitro. (A, B) mF3pal_14 (4 mg/kg) or scmF3pal_14 (4 mg/kg) was i.p. injected two times into CLP mice, at 2 and 14 h after CLP, and the mice were sacrificed 24 h after surgery. Peritoneal fluid (A) and serum (B) were collected at 24 h after CLP. (C) Mouse neutrophils were preincubated with 0, 1, 5 and 10 mM of mF3pal_14 for 30 min and then stimulated with absence or 100 ng/ml LPS for 24 h. (AeC) Cytokine levels were measured by ELISA. Data are presented as mean ± SE (n ¼ 3) (AeC). *p < 0.05, ***p < 0.001 by t-test compared with scrambled pepducin treated control (A, B). ns: not significant, **p < 0.01, ***p < 0.001 by t-test compared with -LPS control, #p < 0.05, ##p < 0.01, ###p < 0.001; t-test compared with -mF3pal_14 treated control (C).
limitation of known ligands of FPR3. Although F2L has been reported to be a ligand for FPR3, F2L is also known to act on FPR1 and FPR2 [26]. GPCR signaling is mediated by the binding of G protein(s) to the intracellular regions of each receptor, especially the intracellular loop 3 region [15]. Several previous studies have been conducted to identify novel GPCR ligands that can act intracellularly by synthesizing pepducins based on the intracellular loop 3 sequences of GPCRs [11,12,27]. We also previously synthesized pepducins for FPR2 and reported them as novel FPR2 intracellular agonists or antagonists [13,14]. The lack of known FPR3-selective ligands led us to develop pepducins based on the intracellular loop 3 region of Fpr3. Involvement of Fpr3 with the Fpr3 pepducins
was confirmed by using the Fpr3/HEK293 cell line (Fig. 1E). Regarding the functional role of Fpr3 in mouse neutrophils, we found that activation of Fpr3 in mouse neutrophils by the three pepducins mF3pal_9, mF3pal_14 and mF3pal_16 elicited chemotactic migration and intracellular calcium increase in mouse neutrophils (Fig. 2A and B). Our results suggest that Fpr3 expressed in neutrophils may regulate neutrophil activity leading to neutrophil trafficking. To explore immunological role of Fpr3 in pathogenic condition, we investigated the effects of Fpr3-based pepducins against experimental sepsis and found that mF3pal_14 increased the survival rate of CLP mouse by strongly reducing liver and lung damage
Please cite this article as: H.Y. Lee et al., A membrane-tethering pepducin derived from formyl peptide receptor 3 shows strong therapeutic effects against sepsis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.058
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and splenocyte apoptosis (Fig. 3AeD). mF3pal_14 also enhanced bactericidal activity in CLP mice (Fig. 3E). It is known that bactericidal activity is mediated by generation of ROS such as superoxide anion [24,28]. As shown in Fig. 2C, mF3pal_14 significantly increased superoxide anion generation in isolated mouse neutrophils. These results suggest that mF3pal_14 administration increases survival rate by stimulating bacterial killing activity via superoxide anion generation in mouse neutrophils. Moreover, mF3pal_14 significantly decreased the levels of inflammatory cytokines increased by CLP surgery (Fig. 4A and B). Since mF3pal_14 inhibited the increase of inflammatory cytokines by LPS in mouse neutrophils in vitro (Fig. 4C), the result suggests that mF3pal_14induced therapeutic effects against sepsis would be mediated by inhibition of inflammatory cytokine production through neutrophil Fpr3. In this study, we found that the three Fpr3 pepducins stimulates intracellular Ca2þ increase and cell migration in mouse neutrophils, however, superoxide anion generation was limited to mF3pal_14 and mF3pal_16 but not to mF3pal_9 (Fig. 2AeC). Since previous reports demonstrated that FPR2 mediates different signal transduction and cellular responses showing ligand-selective manner [16,29,30], our results suggest that Fpr3 also can be differentially modulated by different ligands. We also found that only mF3pal_14 strongly increased the survival rate, while mF3pal_16 failed to increase the survival rate. At this point it is unclear on the mechanism involved in the differential effects of the two pepducins in sepsis. Since we found that only mF3pal_16 but not mF3pal_14 strongly stimulated the migration of other immune cells such as macrophage (data not shown), it would be necessary to examine the roles of other immune cells on the regulation of sepsis pathology by the two pepducins. Previous studies demonstrated that FPR family members play crucial roles in the regulation of the innate immune response through neutrophils, which express FPR1 and FPR2. Neutrophils were known not to express FPR3 on the cell surface, based on the lack of surface staining using an anti-FPR3 antibody in human neutrophils [31]. In this study, we found that neutrophils isolated from sepsis patients express surface FPR3 (Fig. 1A). Taken together, our results suggest that Fpr3 could be considered as a new target for the control of polymicrobial sepsis, and mF3pal_14 could be used as a material to develop therapeutic agents against sepsis. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT and future Planning (NRF2016R1A2B4013128, NRF-2017R1A5A1014560, NRF2018R1A2B3003868). References [1] E.R. Prossnitz, R.D. Ye, The N-formyl peptide receptor: a model for the study of chemoattractant receptor structure and function, Pharmacol. Ther. 74 (1997) 73e102. [2] R.D. Ye, F. Boulary, J.M. Wang, et al., International union of basic and clinical pharmacology. LXXIII. Nomenclature for the formyl peptide receptor (FPR)
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Tsien, A new generation of Ca2þ indicators with greatly improved fluorescence properties, J. Biol. Chem. 260 (1985) 3440e3450. [20] Y.S. Bae, H. Bae, Y. Kim, et al., Identification of novel chemoattractant peptides for human leukocytes, Blood 92 (2001) 2854e2862. [21] S.D. Kim, H.Y. Lee, J.W. Shim, et al., Activation of CXCR2 by extracellular matrix degradation product acetylated Pro-Gly-Pro has therapeutic effects against sepsis, Am. J. Respir. Crit. Care Med. 184 (2011) 243e251. [22] U.Y. Schaff, I. Yamayoshi, T. Tse, et al., Calcium flux in neutrophils synchronizes beta2 integrin adhesive and signaling events that guide inflammatory recruitment, Ann. Biomed. Eng. 36 (2008) 632e646. [23] S. Brechard, E.J. Tschirhart, Regulation of superoxide production in neutrophils: role of calcium influx, J. Leukoc. Biol. 84 (2008) 1223e1237. [24] D.J. Steams-Kurosawa, M.F. Osuchowski, C. Valentine, et al., The pathogenesis of sepsis, Annu. Rev. Pathol. 6 (2011) 19e48. [25] M. Bosmann, P.A. Ward, The inflammatory response in sepsis, Trends Immunol. 34 (2013) 129e136. [26] H.Y. Lee, S.Y. Lee, E.H. Shin, et al., F2L, a peptide derived from heme-binding protein, inhibits formyl peptide receptor-mediated signaling, Biochem. Biophys. Res. Commun. 10 (2007) 985e990. [27] K. O’Callaghan, A. Kuliopulos, L. Covic, Turning receptors on and off with intracellular pepducins: new insights into G-protein-coupled receptor drug development, J. Biol. Chem. 287 (2012) 12787e12796. [28] T.N. Mayadas, X. Cullere, C.A. Lowell, The multifaceted functions of neutrophils, Annu. Rev. Pathol. 9 (2014) 181e218. [29] H.Y. Lee, S.D. Kim, J.W. Shim, et al., Serum amyloid A induces CCL2 production via formyl peptide receptor-like 1-mediated signaling in human monocytes, J. Immunol. 18 (2008) 4332e4339. [30] H.Y. Lee, S.H. Jo, C. Lee, et al., Differential production of leukotriene B4 or prostaglandin E2 by WKYMVm or serum amyloid A via formyl peptide receptor-like 1, Biochem. 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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
A membrane-tethering pepducin derived from formyl peptide receptor 3 shows strong therapeutic effects against sepsis Ha Young Lee a, **, Hyung Sik Kim a, Yu Sun Jeong a, Ji Cheol Kim a, Yong-Soo Bae a, b, You Hwan Jo c, Joon Seong Park d, Yoe-Sik Bae a, b, * a
Department of Biological Sciences, Sungkyunkwan University, Suwon, 16419, Republic of Korea Science Research Center (SRC) for Immune Research on Non-Lymphoid Organ (CIRNO), Sungkyunkwan University, Suwon, 16419, Republic of Korea Department of Emergency Medicine, Seoul National University Bundang Hospital, Seongnam, 13620, Republic of Korea d Department of Hematology-Oncology, Ajou University School of Medicine, Suwon, 16499, Republic of Korea b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 December 2019 Accepted 11 January 2020 Available online xxx
Formyl peptide receptors (FPRs) are G protein-coupled receptors mainly expressed in inflammatory myeloid cells. Previous reports demonstrated that human neutrophils express only FPR1 and FPR2 but not FPR3. Here, we found that FPR3 is expressed in sepsis patient derived neutrophils and Fpr3 is expressed in the mouse neutrophils. To test the role of Fpr3 in neutrophil activity, we synthesized Fpr3 pepducins and successfully developed an agonistic pepducin that stimulates Fpr3, eliciting calcium increase and chemotactic migration of neutrophils. We also found that administration of an Fpr3 pepducin in an experimental mouse sepsis model significantly increased the survival rate. The pepducin markedly inhibited lung injury, splenocyte apoptosis, and inflammatory cytokine production. Bacterial counts were significantly decreased by the pepducin in septic mice. Based on these results, we suggest that FPR3 can be regarded as a new target to control sepsis, and the newly generated Fpr3-based pepducin can be used for the development of anti-septic agents. © 2020 Elsevier Inc. All rights reserved.
Keywords: Formyl peptide receptor 3 Neutrophil Pepducin Sepsis
1. Introduction Formyl peptide receptors (FPRs) are classical chemoattractant receptors and members of G-protein coupled receptor (GPCR) family [1]. Three FPR members exist in humans (FPR1, FPR2 and FPR3) and eight in mice [Fpr1, Fpr2, Fpr3 (Fpr-rs1) and Fpr-rs3 to 7] [2]. Mouse Fpr1 and Fpr2 are known as orthologues of human FPR1 and FPR2, respectively. Recently, Fpr-rs1 has been named Fpr3 and reported to share a specific pathogen ligand with human FPR3 and also described as an orthologue of FPR3 [3]. The FPRs are mainly expressed in innate immune cells including neutrophils and monocytes/macrophages [4e6]. FPRs play important roles in chemotactic cell migration and mediate diverse biological activities [2,4,7,8]. FPRs have been suggested as putative targets against several diseases including sepsis [9,10]. In particular, we previously demonstrated the therapeutic effects of an FPR agonist (WKYMVm)
against experimental polymicrobial sepsis [10]. Unlike other GPCRs, FPRs are known to have variety of ligands [2,9]. Recently, pepducins have been developed and used as agonists or antagonists of GPCRs [11,12]. We previously reported human FPR2 pepducins, and mouse Fpr1 and Fpr2 pepducins have also been reported by another group [13e15]. Despite the numerous studies on FPRs, most studies have focused on FPR1 and FPR2. This is probably because FPR3 ligands are relatively unknown compared to FPR1 and FPR2. Therefore, it is very important to find and develop FPR3-specific ligands for the study of (patho)physiological function of FPR3. Here, we found that neutrophils from sepsis patients express FPR3 and mouse neutrophils express Fpr3 mRNA. We investigated the function of Fpr3 in neutrophil by synthesizing Fpr3-based pepducins. We also examined the effects of Fpr3 pepducins against an experimental polymicrobial sepsis.
2. Materials and methods * Corresponding author. Department of Biological Sciences, Sungkyunkwan University, Suwon 16419, Republic of Korea. ** Corresponding author. E-mail addresses: [email protected] (H.Y. Lee), [email protected] (Y.-S. Bae).
2.1. Materials Pepducins were synthesized from Anygen (Gwangju, Korea).
https://doi.org/10.1016/j.bbrc.2020.01.058 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: H.Y. Lee et al., A membrane-tethering pepducin derived from formyl peptide receptor 3 shows strong therapeutic effects against sepsis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.058
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Fura-2/AM was purchased from Molecular Probes (Eugene, OR, USA). Fluorescence conjugated anti-FPR3 antibody was purchased from R&D systems (Minneapolis, MN, USA). Antibodies used for Western blotting were purchased from SantaCruz (Dallas, TX, USA). Cytochrome c, cytochalasin B, G418, type I collagen and LPS were purchased from Sigma-Aldrich (St. Louis, MO, USA). Boyden chambers were purchased from Neuroprobe, Inc. (Gaithersburg, MD, USA). Percoll was purchased from GE Healthcare (Chicago, IL, USA). ACK lysis buffer was purchased from Gibco (Gaithersburg, MD, USA). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA). 2.2. FACS analysis Surface expression was analyzed by FACSCanto II flow cytometer (BD Bioscience, San Joes, CA) using fluorescence conjugated specific antibodies. 2.3. Isolation of human and mouse neutrophils Peripheral blood was collected from healthy donors or sepsis patients. These experiments were approved by the Seoul National University Bundang Hospital’s Institutional Review Board for ethics and Ajou University Hospital’s Institutional Review Board for ethics. Peripheral neutrophils were isolated from peripheral blood as described previously [16]. C57BL/6 mice (male) were purchased from Orient Bio (Seongnam, Korea). All animal experiments were performed in accordance with the guidelines of the Korean Food and Drug Administration. All experiments involving animals received the approval of the Institutional Review Committee for Animal Care and Use at Sungkyunkwan University (Suwon, Korea). Mouse bone marrow neutrophils were isolated from the femurs and tibias of mice as described previously [17]. Isolated cells were then stained with anti-Ly6G (1A8) antibody and found to be over 95% Ly6G-positive. 2.4. RT-PCR analysis Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) and cDNA was obtained using the Maxime RT Premix Kit (iNtRON, Seongnam, Korea). Primers were synthesized according to a previous report [18]. Target genes were amplified by RT-PCR using the Maxime PCR PreMix Kit (iNtRON).
for HEK293 cells) at 37 C (90 min for mouse neutrophils, 6 h for HEK293 cells). Migrated cells were stained with hematoxylin (Sigma-Aldrich) and stained cells were counted using a light microscope. 2.8. Measurement of intracellular calcium concentration Intracellular calcium concentration was measured using Grynkiewcz’s method with Fura-2/AM as described previously [19]. 2.9. Measurement of superoxide anion generation Superoxide anion generation was determined by measuring cytochrome c reduction using a microtiter 96-well plate ELISA reader (EL312e; Bio-Tek Instruments, Winooski, VT) as previously described [20]. 2.10. Mice and cecal ligation and puncture (CLP) experimental sepsis model The experimental CLP sepsis model was conducted as described previously [21]. 2.11. Tissue histology The mice were euthanized 24 h after CLP, and their lungs and livers were fixed, sectioned, and stained with hematoxylin and eosin for morphological analysis. 2.12. Measurement of colony forming units (CFUs) in CLP mouse model At 24 h after CLP, peritoneal lavage fluids were collected and cultured overnight on blood-agar base plates (Trypticase™ Soy Agar Deeps; BD Biosciences, San Jose, CA) at 37 C. The numbers of CFUs were counted as described previously [21]. 2.13. TUNEL assay The mice were euthanized 24 h after CLP, and their spleens were isolated and fixed. The TUNEL assay was performed on paraffin tissue sections using a standard histological protocol as described previously [21].
2.5. Establishment of Fpr3-expressing HEK293 stable cell lines 2.14. Cytokine assay in vivo and in vitro HEK293 cells were transfected with the vector or Fpr3 cDNA, using the Lipofectamine 2000 reagent. Vector- or Fpr3-expressing HEK293 stable cell lines were selected to have 500 mg/ml G418 antibiotic resistance. 2.6. Western blot analysis The cells were lysed in lysis buffer as described previously [16]. Extracted proteins were separated by SDS-PAGE, and transferred onto a nitrocellulose membrane. Membranes were then incubated with specific antibodies. Enhanced chemiluminescence was used to detect protein levels with antigen-antibody complexes.
To measure the production of CLP-induced cytokines in serum or peritoneal lavage fluids, mice were subjected to CLP surgery and pepeducins were injected at 2 h and 14 h later. The serum or peritoneal lavage fluids were collected 24 h after surgery. Isolated mouse neutrophils (3 106 cells/0.3 ml) were pre-incubated with mF3pal_14 for 30 min, and then stimulated with absence or 100 ng/ ml LPS for 24 h. After collecting cell-free supernatants, serum or peritoneal lavage fluids, the levels of cytokine were measured by ELISA according to the manufacturer’s instructions (e-Bioscience, San Diego, CA). 2.15. Statistical analysis
2.7. Chemotaxis assay Chemotaxis assays were performed using multiwell chambers as described previously [16]. Briefly, isolated mouse neutrophils or HEK293 cells were applied to polycarbonate filters (3 mm pore size for mouse neutrophils, type I collagen pre-coated 10 mm pore size
The results were evaluated with GraphPad prism software (GraphPad Software, La Jolla, CA). Statistical analysis was performed by the Student’s t-test. All results are expressed as the mean ± S.E. Survival data were analyzed using the long-rank test. A p-value < 0.05 was considered statistically significant.
Please cite this article as: H.Y. Lee et al., A membrane-tethering pepducin derived from formyl peptide receptor 3 shows strong therapeutic effects against sepsis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.058
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3. Results 3.1. Neutrophils from sepsis patients express FPR3 Through flow cytometry analysis, we compared the expression pattern of FPR3 in neutrophils isolated from healthy donors and sepsis patients. FPR3 expression was only apparent in the neutrophils of sepsis patients but not of healthy donors (Fig. 1A left). Although FPR3 expression in the neutrophils of sepsis patients is variable depending on the individual, sepsis patient-derived neutrophils showed significantly increased FPR3 expression (Fig. 1A right).
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anti-Myc antibody (Fig. 1D, inset). Functional activity of transfected Fpr3 was confirmed by chemotactic migration with FPR ligands. Fpr3-expressing HEK293 cells specifically migrated upon stimulation with F2L, an FPR3 agonist, but not vector-expressing HEK293 cells (Fig. 1D). We examined the effects of three Fpr3 pepducins on cell migration activity in vector- or Fpr3-expressing HEK293 cells. mF3pal_9, mF3pal_14 and mF3pal_16 significantly increased chemotactic cell migration of Fpr3-expressing HEK293 cells, but not vector-expressing HEK293 cells (Fig. 1E). These results indicate that the synthesized Fpr3 pepducins act on Fpr3.
3.3. Fpr3 regulates mouse neutrophil activity 3.2. Pepducins stimulating mouse Fpr3 were developed A previous report demonstrated that mouse neutrophils express Fpr3 and the expression is enhanced by LPS stimulation [3]. In this study, we also observed that mouse neutrophils express Fpr3 mRNA as well as Fpr1 and Fpr2 (Fig. 1B). To investigate the function of Fpr3 in neutrophil, we synthesized three types of Fpr3 pepducins, mF3pal_9, mF3pal_14 and mF3pal_16, as shown in Fig. 1C. To investigate whether these pepducins act on Fpr3, we generated stable Fpr3-expressing HEK293 cells by transfecting the cells with Myc-tagged Fpr3 cDNA. Fpr3 expression in the established Fpr3/ HEK293 cell line was confirmed by Western blot analysis using
Intracellular Ca2þ plays an important role in the modulation of neutrophil activity [22]. To investigate the function of Fpr3 in neutrophil, we measured intracellular Ca2þ modulation by treating three Fpr3 pepducins in mouse neutrophil. Stimulation of mouse neutrophils with the three pepducins strongly elicited intracellular calcium increase in a concentration-dependent manner, showing maximal activity at 5 mM (mF3pal_14 and mF3pal_16) or 10 mM (mF3pal_9) (Fig. 2A). Since chemotactic migration is an important functional activity of neutrophils in response to FPR family agonists [2], we tested the effects of the three pepducins on mouse neutrophil chemotaxis. The three pepducins significantly
Fig. 1. Pepducins stimulating Fpr3 were developed. (A) Neutrophils isolated from healthy donors or sepsis patients were stained with anti-FPR3 antibody. FPR3 expression was determined by flow cytometry analysis. (B) Expression of Fpr members of mouse neutrophils were analyzed by RT-PCR. (C) Sequence of Fpr3-based pepducins. (D) HEK293 cells were transfected with Myc-tagged Fpr3 cDNA using Lipofectamine 2000. Transfected cells were then selected by 500 mg/ml G418 containing culture medium. Fpr3 expression in HEK293 cells was confirmed by Western blot assay (D, inset). Transfected cells were used for chemotaxis assay upon treatment with 1 mM fMLF, 1 mM MMK1 and 5 mM F2L. (E) Vector- or Fpr3-expressing HEK293 cells were added to the upper wells of type I collagen pre-coated 10 mm pore size chemotaxis chamber in the presence of 0, 0.1, 1 and 5 mM of mF3pal_9, mF3pla_14, and mF3pal_16 for 6 h. Migrated cells were stained with hematoxylin and stained cells were counted using a light microscope (D, E). Data shown are representative of three independent experiments (A left, B and D inset). Data is presented as the mean ± SE of 6 (healthy donors) or 11 (sepsis patients) (A right). Data are mean ± SE of three independent experiments (D, E). ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001 by t-test compared with not treat (NT) control. ##p < 0.01 by t-test compared with vector/HEK293 control.
Please cite this article as: H.Y. Lee et al., A membrane-tethering pepducin derived from formyl peptide receptor 3 shows strong therapeutic effects against sepsis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.058
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Fig. 2. Fpr3 regulates mouse neutrophil activity. (A) Mouse neutrophils were treated with several concentrations (0.1, 1, 5 and 10 mM) of mF3pal_9, mF3pal_14 and mF3pal_16. Relative cytosolic Ca2þ concentrations are expressed as fluorescence ratios (340:380 nm). (B) Mouse neutrophils were added to the upper wells of 3 mm pore size chemotaxis chambers in the presence of 0, 0.1, 1, 5, and 10 mM of mF3pal_9, mF3pal_14 or mF3pal_16 for 90 min. Migrated cells were stained with hematoxylin and stained cells were counted using a light microscope. (C) Mouse neutrophils were stimulated with 10 mM of mF3pal_9, mF3pal_14 and mF3pal_16 for 5 min. Superoxide anion production was determined by measuring cytochrome c reduction. Data shown are representative of at least three independent experiments (A). Data are mean ± SE of three independent experiments (B, C). ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001 by t-test compared with the vehicle treated control.
stimulated mouse neutrophil chemotaxis, showing concentrationdependency (Fig. 2B). The results indicate that the three Fpr3 pepducins are stimuli that cause mouse neutrophil activation, leading to calcium increase and chemotactic migration. Neutrophils are well known innate immune cells and play an important role against infection through reactive oxygen species (ROS) generation [23]. We also examined whether these pepducins could affect on ROS generation in mouse neutrophils. Isolated mouse neutrophils were stimulated with 10 mM of pepducins for 5 min, mF3pal_14 and mF3pal_16 were significantly increase the ROS generation, but not mF3pal_9 (Fig. 2C). 3.4. mF3pal_14 administration shows therapeutic effects against sepsis The regulation of neutrophil activity is known to be important in sepsis pathology. In particular, anti-bacterial activity through ROS generation is helpful to alleviate the symptoms of sepsis. Based on the results of Fpr3 pepducins-induced regulation of neutrophil
activation (Fig. 2), we investigated whether Fpr3 pepducins could have a therapeutic effect in sepsis using mF3pal_14 and mF3pal_16 which had effects on cell migration, intracellular Ca2þ modulation and ROS generation. To investigate the effects of Fpr3 pepducins against sepsis, we used the CLP mouse sepsis model. After CLP surgery, the two types of Fpr3 pepducin (mF3pal_14 and mF3pal_16) or a vehicle were injected four times at 12 h intervals. The survival rate of vehicle administered CLP mice decreased to 20% in 3 days (data not shown). However, mF3pal_14 administered CLP mice showed a markedly increased survival rate (Fig. 3A). We did not detect any therapeutic effects of mF3pal_16 against CLP sepsis (data not shown). To further investigate the effects of mF3pal_14, we synthesized a scrambled sequence pepducin of mF3pal_14 (scmF3pal_14; pal_VRLPRSSNVFAKKH) as a control pepducin. Several dosages of mF3pal_14 (1, 2, 4, and 8 mg/kg) or 8 mg/kg of scmF3pal_14 were administered four times at 12 h intervals post CLP surgery. mF3pal_14 strongly increased the survival rate in a dosage-dependent manner, showing a maximal survival rate at 4 mg/kg (Fig. 3A). Previous studies report that mortality in sepsis is
Please cite this article as: H.Y. Lee et al., A membrane-tethering pepducin derived from formyl peptide receptor 3 shows strong therapeutic effects against sepsis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.058
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Fig. 3. mF3pal_14 has therapeutic effects against CLP. (A) Different dosages of mF3pal_14 (1, 2, 4, and 8 mg/kg) or scmF3pal_14 (8 mg/kg) were i.p. injected four times into CLP mice at 2, 14, 26, and 38 h after CLP. Survival was monitored for 10 days after CLP. (BeE) mF3pal_14 (4 mg/kg, i.p.) or scmF3pal_14 (4 mg/kg i.p.) was administered 2 and 14 h after CLP, and the mice were sacrificed 24 h after surgery. (B) Lung and liver tissue sections were stained with H&E (original magnification 200). (C, D) Spleen tissue sections were used for a TUNEL assay (original magnification 200) (C), and TUNEL-positive cells were counted (D). (E) Peritoneal lavage fluids collected 24 h after CLP were cultured overnight on blood-agar base plates at 37 C; the number of CFUs was then counted. The data are representative of ten mice per group (A) and five mice per group (B, C). Scale bar; 100 mm (B, C). Data are mean ± SE of three independent experiments (D, E). *p < 0.05, **p < 0.01, ***p < 0.001 by log-rank test compared with scrambled pepducin treated group (A). **p < 0.01 by t-test compared with scrambled pepducin treated control (D).
closely associated with increase of lung inflammation [21]. Administration of mF3pal_14 alleviated lung inflammation by CLP (Fig. 3B). Liver damage was also moderately alleviated in mF3pal_14 injected mice (Fig. 3B). Immune cell apoptosis in the spleen plays an important role in the pathology of CLP sepsis [24]. Splenocyte apoptosis was measured from mF3pal_14 or scmF3pal_14 administered CLP mice. A TUNEL assay showed that splenocyte apoptosis was significantly reduced by mF3pal_14 injection (Fig. 3C and D). Bacterial burden positively correlates with the increased mortality of CLP mice [24]. Bacterial colony counts were dramatically decreased by mF3pal_14 administration (Fig. 3E). 3.5. mF3pal_14 administration inhibits CLP-induced proinflammatory cytokine production
cytokine levels in CLP mice. Increased levels of TNF-a, IL-6 and IL-1b production in peritoneal fluid and serum in CLP mice were significantly decreased by mF3pal_14 administration (Fig. 4A and B). We then examined the direct effects of mF3pal_14 on inflammatory cytokine production in mouse neutrophils. Mouse neutrophils were stimulated with several concentration of mF3pal_14 (0, 1, 5 and 10 mM), but mF3pal_14 alone did not increased pro-inflammatory cytokines production (Fig. 4C). And then, we examined the effect of mF3pal_14 on LPS-induced pro-inflammatory cytokine production in mouse neutrophil. Stimulation of mouse neutrophils with LPS strongly increased the production of TNF-a and IL-6 (Fig. 4C). Addition of mF3pal_14 (0, 1, 5 and 10 mM) prior to LPS stimulation significantly decreased the production of these two inflammatory cytokines in a dose-dependent manner (Fig. 4C). 4. Discussion
Proinflammatory cytokines exacerbate the inflammatory responses by CLP [25]. Since mF3pal_14 elicited strong therapeutic effects against sepsis, we examined the effects of the pepducin on
Unlike other FPR family members, the functional role of FPR3 in the innate immune response is largely unknown because of the
Please cite this article as: H.Y. Lee et al., A membrane-tethering pepducin derived from formyl peptide receptor 3 shows strong therapeutic effects against sepsis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.058
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Fig. 4. mF3pal_14 inhibits inflammatory cytokine production in vivo and in vitro. (A, B) mF3pal_14 (4 mg/kg) or scmF3pal_14 (4 mg/kg) was i.p. injected two times into CLP mice, at 2 and 14 h after CLP, and the mice were sacrificed 24 h after surgery. Peritoneal fluid (A) and serum (B) were collected at 24 h after CLP. (C) Mouse neutrophils were preincubated with 0, 1, 5 and 10 mM of mF3pal_14 for 30 min and then stimulated with absence or 100 ng/ml LPS for 24 h. (AeC) Cytokine levels were measured by ELISA. Data are presented as mean ± SE (n ¼ 3) (AeC). *p < 0.05, ***p < 0.001 by t-test compared with scrambled pepducin treated control (A, B). ns: not significant, **p < 0.01, ***p < 0.001 by t-test compared with -LPS control, #p < 0.05, ##p < 0.01, ###p < 0.001; t-test compared with -mF3pal_14 treated control (C).
limitation of known ligands of FPR3. Although F2L has been reported to be a ligand for FPR3, F2L is also known to act on FPR1 and FPR2 [26]. GPCR signaling is mediated by the binding of G protein(s) to the intracellular regions of each receptor, especially the intracellular loop 3 region [15]. Several previous studies have been conducted to identify novel GPCR ligands that can act intracellularly by synthesizing pepducins based on the intracellular loop 3 sequences of GPCRs [11,12,27]. We also previously synthesized pepducins for FPR2 and reported them as novel FPR2 intracellular agonists or antagonists [13,14]. The lack of known FPR3-selective ligands led us to develop pepducins based on the intracellular loop 3 region of Fpr3. Involvement of Fpr3 with the Fpr3 pepducins
was confirmed by using the Fpr3/HEK293 cell line (Fig. 1E). Regarding the functional role of Fpr3 in mouse neutrophils, we found that activation of Fpr3 in mouse neutrophils by the three pepducins mF3pal_9, mF3pal_14 and mF3pal_16 elicited chemotactic migration and intracellular calcium increase in mouse neutrophils (Fig. 2A and B). Our results suggest that Fpr3 expressed in neutrophils may regulate neutrophil activity leading to neutrophil trafficking. To explore immunological role of Fpr3 in pathogenic condition, we investigated the effects of Fpr3-based pepducins against experimental sepsis and found that mF3pal_14 increased the survival rate of CLP mouse by strongly reducing liver and lung damage
Please cite this article as: H.Y. Lee et al., A membrane-tethering pepducin derived from formyl peptide receptor 3 shows strong therapeutic effects against sepsis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.058
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and splenocyte apoptosis (Fig. 3AeD). mF3pal_14 also enhanced bactericidal activity in CLP mice (Fig. 3E). It is known that bactericidal activity is mediated by generation of ROS such as superoxide anion [24,28]. As shown in Fig. 2C, mF3pal_14 significantly increased superoxide anion generation in isolated mouse neutrophils. These results suggest that mF3pal_14 administration increases survival rate by stimulating bacterial killing activity via superoxide anion generation in mouse neutrophils. Moreover, mF3pal_14 significantly decreased the levels of inflammatory cytokines increased by CLP surgery (Fig. 4A and B). Since mF3pal_14 inhibited the increase of inflammatory cytokines by LPS in mouse neutrophils in vitro (Fig. 4C), the result suggests that mF3pal_14induced therapeutic effects against sepsis would be mediated by inhibition of inflammatory cytokine production through neutrophil Fpr3. In this study, we found that the three Fpr3 pepducins stimulates intracellular Ca2þ increase and cell migration in mouse neutrophils, however, superoxide anion generation was limited to mF3pal_14 and mF3pal_16 but not to mF3pal_9 (Fig. 2AeC). Since previous reports demonstrated that FPR2 mediates different signal transduction and cellular responses showing ligand-selective manner [16,29,30], our results suggest that Fpr3 also can be differentially modulated by different ligands. We also found that only mF3pal_14 strongly increased the survival rate, while mF3pal_16 failed to increase the survival rate. At this point it is unclear on the mechanism involved in the differential effects of the two pepducins in sepsis. Since we found that only mF3pal_16 but not mF3pal_14 strongly stimulated the migration of other immune cells such as macrophage (data not shown), it would be necessary to examine the roles of other immune cells on the regulation of sepsis pathology by the two pepducins. Previous studies demonstrated that FPR family members play crucial roles in the regulation of the innate immune response through neutrophils, which express FPR1 and FPR2. Neutrophils were known not to express FPR3 on the cell surface, based on the lack of surface staining using an anti-FPR3 antibody in human neutrophils [31]. In this study, we found that neutrophils isolated from sepsis patients express surface FPR3 (Fig. 1A). Taken together, our results suggest that Fpr3 could be considered as a new target for the control of polymicrobial sepsis, and mF3pal_14 could be used as a material to develop therapeutic agents against sepsis. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT and future Planning (NRF2016R1A2B4013128, NRF-2017R1A5A1014560, NRF2018R1A2B3003868). References [1] E.R. Prossnitz, R.D. Ye, The N-formyl peptide receptor: a model for the study of chemoattractant receptor structure and function, Pharmacol. Ther. 74 (1997) 73e102. [2] R.D. Ye, F. Boulary, J.M. Wang, et al., International union of basic and clinical pharmacology. LXXIII. Nomenclature for the formyl peptide receptor (FPR)
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Please cite this article as: H.Y. Lee et al., A membrane-tethering pepducin derived from formyl peptide receptor 3 shows strong therapeutic effects against sepsis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.058