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Role of Translocator 18 KDa Ligands in the Activation of Leukotriene B4 Activated G-Protein Coupled Receptor and Toll Like Receptor-4 Pathways in Neutrophils

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Role of Translocator 18 KDa Ligands in the Activation of Leukotriene B4 Activated G-Protein Coupled Receptor and Toll Like Receptor-4 Pathways in Neutrophils

Léonard de Vinci Kanda Kupa et al. Front Pharmacol.

Abstract

TSPO (Translocator 18 KDa; tryptophan-rich sensory protein oxygen sensor) is a constitutive outer mitochondrial membrane protein overexpressed in inflammatory cells during local or systemic processes. Despite its expression is characterized, role of TSPO in inflammation remains elusive. For this study, we investigated the role of TSPO ligands on neutrophil functions elicited by two different inflammatory pathways. Peritoneal neutrophils were isolated from male Balb-C mice, treated with TSPO ligand diazepam, Ro5-4864 or PK11195 (1,100 or 1000 nM; 2 h) and further stimulated with lipopolysaccharide from Escherichia coli (LPS), a binding for Toll-Like Receptor-4 (TLR4), or leukotriene B4 (LTB4), a G-protein coupled receptor (GPCR) ligand. LPS treatment did not lead to overexpression of TSPO on neutrophils, and pre-treatment with any TSPO ligand did not alter cytokine expression, adhesion molecule expression, or the production of reactive oxygen and nitrogen species caused by LPS stimulation. Conversely, all TSPO ligands impaired LTB4's actions, as visualized by reductions in L-selectin shedding, β2 integrin overexpression, neutrophil chemotaxis, and actin filament assembly. TSPO ligands showed distinct intracellular effects on LTB4-induced neutrophil locomotion, with diazepam enhancing cofilin but not modifying Arp2/3 expression, and Ro5-4864 and PK11195 reducing both cofilin and Arp2/3 expression. Taken together, our data exclude a direct role of TSPO ligands in TLR4-elicited pathways, and indicate that TSPO activation inhibits GPCR inflammatory pathways in neutrophils, with a relevant role in neutrophil influx into inflammatory sites.

Keywords: Arp2/3; LPS; LTB4; TSPO modulation; cofilin; cytokines; neutrophils chemotaxis.

Figures

GRAPHICAL ABSTRACT 1
GRAPHICAL ABSTRACT 1
Proposal mechanisms of TSPO ligands on GPCR and TLR-4 pathways in neutrophils. In the left: GPCR (G-protein coupled receptor) pathway regulation by TSPO ligands. Upon BLTR (Leukotriene B receptor) activation by LTB4 (leukotriene B4), the coupled G-protein dissociates in two subunits. The Gβγ subunit activates PI3K (phosphatidylinositol-3-kinase) which leads to an intracellular increase of PIP3 (Phosphatidylinositol-3,4,5-trisphosphate). The increase of PIP3 is associated with activation of ARP2/3 (Actin related proteins 2/3), which mediates actin polymerization. In the other hand, the G protein subunit Gα activates PLC (phospholipase C) close to the plasmatic membrane (PM). PLC hydrolyses the membrane PIP2 (Phosphatidylinositol-4,5-bisphosphate) to form IP3 (Inositol triphosphate) and DAG (diacylglycerol). IP3 leads to cofilin activation, while DAG recruits protein kinase C (PKC), which activates p47phox, a subunits of NADPH-oxidase allowing NADPH-oxidase activation and reactive oxygen species (ROS) production. Cofilin activation leads to actin depolymerization and ensures, together with actin polymerization, the actin turn over and chemotaxis. TSPO ligands inhibit both cofilin and ARP2/3 expression, impairing chemotaxis. In the right: TLR4 (Toll like receptor) pathway regulation by TSPO ligands. LPS (lipopolysaccharides) activates TLR4, which is coupled with Syk (Spleen tyrosine kinase), NOX4 (NADPH-oxidase – 4) and the Myeloid differentiation primary-response protein (MYD88). NOX4 act as a direct ROS producer while Syk acts amplifying PLC pathway by increasing intracellular DAG and ROS production via NADPH-oxidase activation. Upon TLR4 activation, MYD88 recruits interleukin receptor-associated kinase proteins (IRAKs) which activates TRAF6(TNF Receptor-associated factor 6) which in turn activates TAK1(TGFβ activated kinase). TAK1 activation leads to IKK (Inhibitor of NF-κB kinase) complex activation and to MAPKs (Mitogen activated proteins kinase) activation. Activated IKK complex leads to NF-κB (Nuclear factor kB) activation and release while MAPKs activation leads to phosphorylation of several transcriptional factors. Both NF-κB and phosphorylated transcriptional factors migrate to the nucleus for transcriptional regulation. Then inflammatory mediators such as cytokines and adhesion molecules are regulated. TSPO ligands did not show any regulation in this pathway.
FIGURE 1
FIGURE 1
Role of TSPO ligands in LPS effects on neutrophils. Mice peritoneal neutrophils were incubated with TSPO ligands (Diazepam [DZP], Ro5-4864 [RO] or PK11195 [PK]) for 2 h and further incubated with culture medium (basal) or LPS for 18 h to quantify TNF-α (A), IL-6 (B), IL-10 (C), and NO (G), or for 1 h to determine the expression of adhesion molecules L-selectin (D) and β2 integrin (E), ROS production (F), and TSPO expression (H). The data are expressed as mean ± SEM of cells collected from 4 animals in each group. P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001 vs. basal values. The data were statistically analyzed by ANOVA followed by Dunnet test.
FIGURE 2
FIGURE 2
Role of TSPO ligands in LPS effects on macrophages. Mice peritoneal macrophages were incubated with TSPO ligands (Diazepam [DZP], Ro5-4864 [RO] or PK11195 [PK]) for 2 h and further incubated with culture medium (basal) or LPS for 18 h to quantify TNF-α (A) and NO (C), or for 1 h to determine ROS production (B) and TSPO expression (D). The data are expressed as mean ± SEM of cells collected from 4 animals in each group. P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001 vs. basal values, and #P < 0.05 vs. respective value in RO group; ##P < 0.01 vs. respective value in R10 and vehicle groups. The data were statistically analyzed by ANOVA followed by Dunnet test.
FIGURE 3
FIGURE 3
Role of TSPO ligands in LTB4-induced expression of adhesion molecules by neutrophils. Mice peritoneal neutrophils were incubated with TSPO ligands (Diazepam [DZP], Ro5-4864 [RO] or PK11195 [PK]) for 2 h and further incubated with culture medium (basal) or LTB4 100 nM for 20 min to quantify L-selectin (A) and β2 integrin (B) expression. The data are expressed as mean ± SEM of cells collected from 4 animals in each group. P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001 vs. basal values. The data were statistically analyzed by ANOVA followed by Dunnet test.
FIGURE 4
FIGURE 4
Role of TSPO ligands in LTB4-induced neutrophil chemotaxis. Mice peritoneal neutrophils were incubated with TSPO ligands (Diazepam [A], Ro5-4864 [B] or PK11195 [C]) for 2 h and further incubated with culture medium (basal) or LTB4 (0.1–100 nM) for 2 h to quantify the number of migrated neutrophils. The data are expressed as mean ± SEM of cells collected from 6 animals in each group. #P < 0.05 ##P < 0.01 vs. medium in basal group; ∗∗P < 0.01; ∗∗∗P < 0.001 vs. respective medium values. The data were statistically analyzed by ANOVA followed by Dunnet test.
FIGURE 5
FIGURE 5
Role of TSPO ligands in LTB4-induced actin polymerization and cofilin and ArP2/3 expression by neutrophils. Mice peritoneal neutrophils were incubated with TSPO ligands (Diazepam [DZP], Ro5-4864 [RO] or PK11195 [PK]) for 2 h and further incubated with culture medium (basal) or LTB4 1000 nM for 1.5 min (90 s) to quantify actin polymerization (A), expression of cofilin (B), expression of ARP2/3 by confocal microscopy (C) and by western blot (D). The data are expressed as mean ± SEM. cells collected from 4 animals in each group for confocal microscopy and 4 western blot analysis using a pool of neutrophils obtained from 8 animals in each analysis. ##P < 0.01; ###P < 0.001 vs. respective medium; P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001 vs. LTB medium group; vs. respective basal value. P < 0.05; ∗∗P < 0.01 vs. medium control group and #P < 0.05 vs. medium stimulated group (For western blot analysis). The data were statistically analyzed by ANOVA followed by Dunnet test.
FIGURE 6
FIGURE 6
Representative images from confocal microscopy. Green represents cofilin expression, red represents actin polymerization and blue represents Arp2/3 expression.

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