TLQP-21 is a low potency partial C3aR activator on human primary macrophages

Abstract

TLQP-21 is a 21-amino acid neuropeptide derived from the VGF precursor protein. TLQP-21 is expressed in the nervous system and neuroendocrine glands, and demonstrates pleiotropic roles including regulating metabolism, nociception and microglial functions. Several possible receptors for TLQP-21 have been identified, with complement C3a receptor (C3aR) being the most commonly reported. However, few studies have characterised the activity of TLQP-21 in immune cells, which represent the major cell type expressing C3aR. In this study, we therefore aimed to define the activity of both human and mouse TLQP-21 on cell signalling in primary human and mouse macrophages. We first confirmed that TLQP-21 induced ERK signalling in CHO cells overexpressing human C3aR, and did not activate human C5aR1 or C5aR2. TLQP-21 mediated ERK signalling was also observed in primary human macrophages. However, the potency for human TLQP-21 was 135,000-fold lower relative to C3a, and only reached 45% at the highest dose tested (10 μM). Unlike in humans, mouse TLQP-21 potently triggered ERK signalling in murine macrophages, reaching near full activation, but at ~10-fold reduced potency compared to C3a. We further confirmed the C3aR dependency of the TLQP-21 activities. Our results reveal significant discrepancy in TLQP-21 C3aR activity between human and murine receptors, with mouse TLQP-21 being consistently more potent than the human counterpart in both systems. Considering the supraphysiological concentrations of hTLQP-21 needed to only partially activate macrophages, it is likely that the actions of TLQP-21, at least in these immune cells, may not be mediated by C3aR in humans.

Keywords: C3aR; C5aR1; C5aR2; Complement C3a; TLQP-21.

Figure 1

hTLQP-2 and mTLQP-21 activate ERK signalling in CHO-K1 and CHO-C3aR cells. hTLQP-21, mTLQP-21 and plasma-derived human C3a were tested in (A) non-transfected CHO-K1 or (B) CHO cells stably expressing human C3aR. CHO cells were serum-starved overnight and then stimulated with various ligands for 10 min before being lysed. The phospho-ERK1/2 content in the lysate was measured and expressed as fold-baseline before being combined. The maximum relative pERK1/2 activity induced by each ligand is shown in (C). Data represent mean ± S.E.M. of triplicate measurements from 3-4 independent experiments (n = 3-4). Two-way ANOVA with Dunnett’s post hoc analysis. *P < 0.05, ***P < 0.001, ****P < 0.0001. Ligand treated versus medium treated cells for each cell line.

Figure 2

hTLQP-2 and mTLQP-21 do not activate human C5aR1 or C5aR2. (A) CHO cells stably expressing human C5aR1 were serum-starved overnight and then stimulated with various ligands for 10 min before being lysed. The phospho-ERK1/2 content in the lysate was measured and expressed as counts/s. (B) HEK293 cells were transiently transfected using C5aR2-Venus and β-arrestin 2-nanoluc BRET pairs for 24 hours, and seeded overnight. Filtered light emissions between 460-485 nm (nanoluc) and 520-545 nm (Venus) were continually monitored for 90 min, with respective ligands (mTLQP-21 and hTLQP-21, 10 μM; hC5a, 100 nM) added at the 0 min time point. Data are expressed as ligand-induced BRET (Venus/nanoLuc emission) ratios. Data represent mean ± S.E.M. of triplicate measurements from a single experiment.

Figure 3

Agonistic and antagonistic activities of hTLQP-21 and mTLQP-21 in human monocyte-derived macrophages. Serum-starved HMDMs (50,000/well) were (A) stimulated with medium or respective ligands at the indicated concentrations for 10 min and then lysed, or (B) pre-treated with 5 µM SB290157 for 30 min prior to stimulation. (C) HMDMs were pre-treated with 10 μM hTLQP-21 or mTLQP-21 for 30 min before being stimulated with respective concentrations of human C3a. (D) HMDMs were pre-treated with 10 nM hC3a, 10 μM hTLQP-21 or mTLQP-21 for 30 min before being stimulated with the same concentrations of the above ligands for 10 min and then lysed. (E) HMDMs were pre-treated with 10 μM hTLQP-21 or mTLQP-21 for 30 min before being stimulated with respective concentrations of human C5a for 10 min and then lysed. The phospho-ERK1/2 content in the cell lysate was measured and normalised to the maximum hC3a/hC5a-induced levels before being combined. Data represent mean ± S.E.M. of triplicate measurements using cells from 4 independent donors (n = 4). Two-way ANOVA with Dunnett’s post hoc analysis. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Ligand pre-treated versus vehicle treated donor-matched cells.

Figure 4

TLQP-21 triggers ERK signalling through C3aR in murine bone marrow-derive macrophages. BMDMs (90,000/well) from wildtype mice (A) or with C3aR knockout mice (B), were serum-starved overnight and then stimulated with respective ligands at the indicated concentrations for 10 min. The phospho-ERK1/2 content in the cell lysate was measured and normalised to the medium only-treated levels before being combined. Data represent mean ± S.E.M. of triplicate measurements using cells from 3 mice (n = 3).