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. 2024 Dec 23;8(1):136–145. doi: 10.1021/acsptsci.4c00496

Lipopolysaccharide-Neutralizing Peptide Modulates P2X7 Receptor-Mediated Interleukin-1β Release

Jonas Engelhardt †, Anna Klawonn †, Ann-Kathrin Dobbelstein †, Aliaa Abdelrahman ‡, Johannes Oldenburg §, Klaus Brandenburg ∥, Christa E Müller ‡, Günther Weindl †,*
PMCID: PMC11729421  PMID: 39816791

Abstract

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Lipopolysaccharide (LPS)-neutralizing peptides are emerging as new potential therapeutic modalities to treat sepsis and skin infections. Purinergic ligand-gated ion channels (P2X receptors) play a critical role in various biological processes, including inflammation. Recent drug development efforts have significantly focused on the modulation of P2X receptors. Here, we investigated the effects of the synthetic LPS-neutralizing peptide Pep19–2.5 on human P2X receptors in cells of the innate immune system. Pep19–2.5 concentration-dependently triggered Ca2+ influx, interleukin (IL)-1β, and lactate dehydrogenase (LDH) release in Toll-like receptor-stimulated human macrophages and monocytes. Ca2+ influx was mediated at least partially by P2X7 receptors, and IL-1β and LDH release by P2X7 receptors, respectively. Confocal microscopy confirmed the colocalization of Pep19–2.5 with P2X7 receptors. Pep19–2.5-induced IL-1β release in primed cells was dependent on K+ efflux, caspase-1, and the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 inflammasome. In the presence of the P2X7 receptor agonist 2′(3′)-O-(4-benzoylbenzoyl)adenosine-5′-triphosphate, Pep19–2.5 reduced IL-1β and LDH release. In 1321N1, astrocytoma cells stably transfected with human P2X receptors, Pep19–2.5 potently modulated P2X7 and P2X4 receptors (IC50 values of 0.346 and 0.146 μM, respectively) but showed less (P2X1, P2X3) or no activity (P2X2) at other P2X receptor subtypes. Our findings underline the potential of LPS-neutralizing peptides as modulators of P2X receptors, thus expanding their applicability beyond the treatment of sepsis to the treatment of inflammatory diseases.

Keywords: synthetic anti-LPS peptides, inflammation, P2X7 receptors, IL-1beta, NLRP3 inflammasome

Sepsis describes a life-threatening systemic inflammation facilitated by pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs).1 PAMPs, such as lipopolysaccharide (LPS), can activate Toll-like receptors (TLRs) and promote the expression of nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 (NLRP3) associated proteins, cytokines, and cytokine precursors like pro-interleukin (IL)-1β. The expression is mediated through nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in cells of the innate immune system, a process also known as priming.2,3 Stimulation of primed cells by DAMPs, such as adenosine triphosphate (ATP), leads to activation of the NLRP3 inflammasome.4,5 High extracellular concentrations of ATP activate P2X7 receptors, which trigger K+ efflux thereby promoting NLRP3 assembly and facilitating caspase-1 activation. Caspase-1 then processes pro-IL-1β into bioactive IL-1β, one of the most potent proinflammatory cytokines. ATP is released following intracellular LPS sensing by human caspase-4 and −5, or mouse caspase-11,6 and at high concentrations during cell death.7

P2X receptors represent an important class of ligand-gated ion channels permeable to sodium (Na+), potassium (K+) and calcium (Ca2+) ions.8 Inhibiting P2X receptors holds promise as a therapeutic strategy for a wide range of diseases. Antagonists for P2X1, P2X2, P2X3, and P2X4 receptors are being explored for their potential in neuroprotection, pain management, chronic cough treatment and inflammatory diseases, respectively.9 Among the P2X receptor family members, the P2X7 receptor is the most extensively studied. It plays a major role in inflammatory and immune responses, extending beyond NLRP3 inflammasome activation.4,10 For instance, the P2X7 receptor promotes the release of several cytokines in addition to IL-1β, and is involved in cytotoxicity and host–pathogen interactions.

Antimicrobial peptides (AMPs) have therapeutic potential in the treatment of sepsis, skin infections, and wounds by direct antimicrobial effects and modulating the host immune response.11,12 AMPs include LPS-neutralizing peptides such as LL-37, polymyxin B (PMB), and the synthetic peptide Pep19–2.5. The endogenous peptide LL-37 demonstrates broad-spectrum antimicrobial and immunomodulatory effects;13 however, it promotes inflammation at high concentrations.14 PMB is a potent peptide antibiotic drug but with significant risks of nephrotoxicity and neurotoxicity.15 Pep19–2.5 shows selective anti-inflammatory properties with low cytotoxicity, offering a potentially safer alternative.16,17 Pep19–2.5 dampens inflammatory responses induced by LPS and outer membrane vesicles in vitro18−22 and prevents lethal sepsis in vivo.23,24 Pep19–2.5 neutralizes LPS through direct interaction, binding to LPS via physicochemical interactions with its negative charges and lipid A moiety.25 Furthermore, Pep19–2.5 demonstrates immunomodulatory effects independent of its LPS-neutralizing activity, accelerates wound healing in mice, and promotes keratinocyte migration via P2X7 receptor activation.20,26

LL-37 and PMB also modulate P2X7 receptors, IL-1β secretion and NLRP3 inflammasome activation.27−30 Previously, we showed that P2X7 receptors are involved in the internalization of Pep19–2.5 in macrophages, and stimulation of TLR-primed macrophages with Pep19–2.5 led to P2X7 receptor-dependent IL-1β release.22 However, the mechanisms underlying the P2X7 receptor-mediated immunomodulatory effects of Pep19–2.5 remain elusive.

In this work, we investigated whether Pep19–2.5 modulates P2X7 receptors in human innate immune cells in a manner similar to that previously reported for LL-37 or PMB. We report mechanistic insights into Pep19–2.5-induced IL-1β release and highlight previously unrecognized modulatory effects of Pep19–2.5 on different P2X receptor subtypes and P2X7-induced IL-1β release.

Results

Pep19–2.5 Triggers Intracellular Ca2+ Increase and Promotes the Release of IL-1β and LDH

Previously, we have shown that Pep19–2.5 increases cytosolic calcium in keratinocytes.26 In THP-1 macrophages, Pep19–2.5 induced a robust and concentration-dependent increase in intracellular Ca2+ starting at 4 μM (Figure 1A). The maximum response was observed at the highest peptide concentration tested (18 μM). The endogenous peptide LL-37 showed similar kinetics of Ca2+ influx in human macrophages in a P2X7 receptor-dependent manner.31

Figure 1.

Figure 1

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Pep19–2.5 triggers intracellular Ca2+ increase and promotes IL-1β and LDH release. (A) THP-1 macrophages were loaded with Fluo-4 AM and probenecid. Pep19–2.5 was added at increasing concentrations. Arrows indicate the addition of control or stimuli. Results are expressed as % increase over control. Mean, n = 4. (B) THP-1 macrophages and (C) primary monocytes and were stimulated with Pam3CSK4 (1 μg/mL). After 3 h, cells were incubated with increasing concentrations of Pep19–2.5 for 3 h. Supernatants were assayed for IL-1β release by (B, C) ELISA and (C) LDH release. Mean ± SEM, n = 3–4.

IL-1β and LDH release is strongly associated with P2X7 receptor activation.4,32,33 We selected the TLR2/1 ligand Pam3CSK4 as the priming stimulus instead of LPS as it does not interact with Pep19–2.5.17 Stimulation of Pam3CSK4-primed macrophages and primary monocytes with Pep19–2.5 resulted in a concentration-dependent release of IL-1β and LDH. In THP-1 macrophages, maximum IL-1β release occurred at a peptide concentration of 18 μM (Figure 1B). In primary monocytes, maximum IL-1β release was observed at 11 μM, while maximum LDH release was noted at 15 μM (Figure 1C).

Pep19–2.5-Induced Ca2+ Increase and Release of IL-1β and LDH is P2X7 Receptor-Dependent

To strengthen our hypothesis of P2X receptor involvement, we analyzed Ca2+ influx in the presence of the P2X receptor inhibitor pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS).34,35 Addition of PPADS completely blocked Pep19–2.5-induced intracellular Ca2+ increase (Figure 2A). To assess whether the observed Ca2+ influx depends at least partially on the P2X7 receptor, we transfected HEK293T cells with the human P2X7 receptor or control vector. BzATP, a potent P2X7 receptor agonist,36 and Pep19–2.5 induced a sustained increase in intracellular Ca2+ only in cells expressing the human P2X7 receptor (Figure 2B). The calcium ionophore calcimycin was used as a control and triggered Ca2+ release in HEK293T cells independently of the P2X7 receptor.

Figure 2.

Figure 2

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Pep19–2.5-induced intracellular Ca2+ increase is partially P2X receptor-dependent. (A) THP-1 macrophages were loaded with Fluo-4 AM and probenecid. Afterward, cells were incubated with or without 100 μM PPADS. Pep19–2.5 was added at increasing concentrations. Arrows indicate the addition of control or stimuli. Results are expressed as % increase over control. Mean, n = 3. (B) HEK293T cells were transfected with a plasmid encoding the human P2X7 receptor or an empty control vector and loaded with Fluo-4 AM and probenecid. Subsequently, calcimycin (10 μM), BzATP (150 μM) or Pep19–2.5 was added. Arrows indicate the addition of control or stimuli. Baseline was subtracted. Results are expressed as % increase over control. Mean, n = 2.

We then investigated the underlying mechanism of IL-1β and LDH release in macrophages and primary monocytes. K+ efflux is the common denominator leading to NLRP3 inflammasome activation and subsequently to the release of IL-1β.37 Intracellular K+ was measured using Ion Potassium Green (IPG)-2, a K+-sensitive dye. Incubation of THP-1 macrophages with Pep19–2.5 resulted in a significant reduction in IPG-2 positive cells, indicating a decrease in intracellular K+ levels (Figure 3A). Furthermore, inhibiting NLRP3 inflammasome activation by adding extracellular K+38 led to a significant decrease in Pep19–2.5-induced IL-1β release (Figure 3B).

Figure 3.

Figure 3

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Pep19–2.5-induced IL-1β and LDH release is P2X7 receptor-dependent. (A) THP-1 macrophages were loaded with IPG-2 AM and probenecid. Afterward, cells were incubated with or without 300 μM oxATP for 30 min and with 18 μM Pep19–2.5 for 15 min. Cells were analyzed by flow cytometry. The histograms are representative of four independent experiments. Bar charts show mean + SEM, n = 4. One-way ANOVA followed by Tukey’s post-test. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001. (B) THP-1 macrophages were stimulated with Pam3CSK4 (1 μg/mL). After 3 h, potassium chloride (75 mM) was added together with 18 μM Pep19–2.5 for 3 h in THP-1 macrophages. Supernatants were assayed for IL-1β release by ELISA. Mean + SEM, n = 3. One-sample t-test against 100%. **P ≤ 0.01, ****P ≤ 0.0001. (C) THP-1 macrophages and (D) primary monocytes were stimulated with Pam3CSK4 (1 μg/mL). After 3 h, inhibitors of NLRP3 (10 μM MCC950), caspase-1 (10 μM Ac-YVAD-cmk), pan-caspase (40 μM Z-VAD-FMK), P2X receptors (100 μM PPADS) and P2X7 (1 μM A-804598; 300 μM oxATP) were added 1 h before incubation with (C) 18 μM and (D) 11 μM Pep19–2.5 for 3 h. Supernatants were assayed for IL-1β release by ELISA and (D) LDH release. Mean + SEM, n = 3–9. One-sample t-test against 100% or one-way ANOVA followed by Tukey’s post-test. ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (E) THP-1 macrophages were stimulated with 11 μM NBD-labeled D-Pep19–2.5 for 30 min and analyzed by confocal immunofluorescence microscopy. Immunofluorescence staining shows Pep19–2.5 (green) and P2X7 receptor expression (red). Cell nuclei were stained with Hoechst (blue). Arrows indicate colocalization of Pep19–2.5 and P2X7 receptors. Scale bar, 10 μm. Pictures are representative of three independent experiments.

Next, we used multiple inhibitors targeting known IL-1β releasing pathways. The NLRP3 inflammasome inhibitor MCC95039 reduced IL-1β release in both THP-1 macrophages (Figure 3C) and primary monocytes (Figure 3D), although only weak inhibition was observed in macrophages. Caspase-1 inhibitor Ac-YVAD-cmk40,41 and pan-caspase inhibitor Z-VAD-fmk42 both reduced IL-1β release. However, only partial inhibition was observed again in THP-1 macrophages. Consistent with the data obtained by analyzing Ca2+ influx, the P2X antagonist PPADS blocked IL-1β release in macrophages and primary monocytes. The reversible P2X7 receptor antagonist A-80459843 did not reduce IL-1β release while complete inhibition was observed in the presence of oxATP, an irreversible P2X7 antagonist44 (Figure 3C,D). LDH release was inhibited only by PPADS and oxATP, while the other inhibitors tested did not reduce LDH release (Figure 3D). OxATP also partially restored Pep19–2.5-induced K+ efflux (Figure 3A).

To further investigate the link between Pep19–2.5 and the P2X7 receptor, we performed immunofluorescence staining of the P2X7 receptor, as previously demonstrated for LL-37.45 Colocalization between Pep19–2.5 and the P2X7 receptor was observed (Figure 3E).

Pep19–2.5 Reduces BzATP-Induced IL-1β and LDH Release

The P2X7 receptor agonist BzATP induces IL-1β-release in TLR-primed cells.46,47 Incubation of Pam3CSK4-primed THP-1 macrophages (Figure 4A) or primary monocytes (Figure 4B) with BzATP induced a robust IL-1β release. Pep19–2.5 reduced BzATP-induced IL-1β release in THP-1 macrophages (Figure 4A) and primary monocytes (Figure 4B). BzATP-induced LDH release was also reduced (Figure 4B). The strongest reduction was observed at the highest peptide concentration tested (18 μM). IL-1β release was reduced to 64% in THP-1 macrophages and to 55% in primary monocytes. LDH release was reduced to 60% in primary monocytes. Incubation of Pep19–2.5 together with BzATP reduced intracellular Ca2+ increase compared to BzATP alone (Figure 4C).

Figure 4.

Figure 4

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Pep19–2.5 reduces BzATP-induced LDH and IL-1β release. (A) THP-1 macrophages and (B) primary monocytes were stimulated with Pam3CSK4 (1 μg/mL). After 3 h, cells were incubated with BzATP (300 μM) or different concentrations of Pep19–2.5 together with BzATP (300 μM) for 3 h. Supernatants were assayed for IL-1β release by (A, B) ELISA and (B) LDH release. Mean ± SEM, n = 3–4. Two-sample t-test. *P ≤ 0.05, **P ≤ 0.01. (C) THP-1 macrophages were loaded with Fluo-4 AM and probenecid. BzATP (300 μM) without or with Pep19–2.5 (7 μM) was added. Arrows indicate the addition of control or stimuli. Baseline was subtracted. Results are expressed as % increase over control. Mean, n = 2.

Pep19–2.5 Potently Modulates P2X7 and P2X4 Receptors

To differentiate the modulatory activity of Pep19–2.5 on different P2X receptor subtypes, we measured intracellular Ca2+ increase in 1321N1 astrocytoma cells stably expressing human P2X1, P2X2, P2X3, P2X4, or P2X7 receptors.48,49 In the presence of Pep19–2.5, P2X receptors were activated with ATP except for P2X7-transfected cells, for which the more potent agonist BzATP was used. Pep19–2.5 potently reduced activation of P2X7 and P2X4 receptors, but showed less (P2X1, P2X3) or no activity at P2X2 receptors (IC50 > 10 μM) (Figure 5 and Table 1). IC50 values in the nanomolar range were determined for P2X7 (0.346 μM) and P2X4 (0.146 μM) receptors, while IC50 values in the micromolar range were determined for P2X1 (4.23 μM) and P2X3 (10.1 μM) receptors. Percent reduction at 10 μM of Pep19–2.5 observed for P2X7 and P2X4 receptors were 87 and 83% respectively.

Figure 5.

Figure 5

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Pep19–2.5 modulates activation of multiple P2X receptor subtypes. Concentration-dependent modulatory effect of Pep19–2.5 on Ca2+ influx induced by ATP or BzATP at 1321N1 cells expressing hP2X1, hP2X2, hP2X3, hP2X4, or hP2X7 receptors. Curves are mean ± SEM of four separate experiments performed in duplicate.

Table 1. Potency of Pep19-2.5 in Ca2+ Influx Assays at P2X Receptors Stably Expressed in 1321N1 Astrocytoma Cellsa.

  Pep19–2.5, IC50 ± SEM (μM) (% reduction at 10 μM ± SEM)
P2X1R 4.23 ± 0.77 (82 ± 6)
P2X2R >10 (37 ± 13)
P2X3R 10.1 ± 0.7 (60 ± 6)
P2X4R 0.146 ± 0.036 (83 ± 4)
P2X7R 0.346 ± 0.046 (87 ± 9)
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a

Data are mean ± SEM of four separate experiments performed in duplicate.

Discussion

P2X receptors, particularly P2X7, are in the focus of current drug development efforts. The P2X7 receptor mediates various functions, including cell death, infection, and inflammation.4 Here, we propose the synthetic LPS-neutralizing peptide Pep19–2.5 (Aspidasept) as a novel P2X receptor modulator.

Pep19–2.5 concentration-dependently induced intracellular Ca2+ increase in human macrophages. The intracellular Ca2+ increase was suppressed by PPADS suggesting that Pep19–2.5 activates P2X receptors in a nonselective manner. This is supported by results obtained in human keratinocytes.26 The observation that Pep19–2.5 induces Ca2+ responses in cells transfected with the human P2X7 receptor, compared to mock-transfected cells, suggests the involvement of the P2X7 receptor. The endogenous LPS-neutralizing peptide LL-37 activates P2X7 receptors and induces Ca2+ influx in macrophages.29,31 Since LL-37 facilitates IL-1β release,28 we hypothesized a similar activity for Pep19–2.5. Indeed, stimulation of primed human macrophages or monocytes with Pep19–2.5 led to concentration-dependent IL-1β and LDH release. IL-1β release in both cell types was NLRP3- and caspase-1-dependent, although the effects were more pronounced in human primary monocytes. Inhibition of P2X receptors completely blocked Pep19–2.5-induced IL-1β release, indicating the involvement of the P2X7 receptor, a key activator of IL-1β release via a mechanism dependent on K+ efflux, the NLRP3 inflammasome, and caspase-1.4,37 The weak reduction of P2X7 mediated IL-1β release after NLRP3 inhibition in human THP-1 macrophages, contrasting the strong inhibition observed in human primary monocytes, is consistent with our previous findings.50 LDH release could result from NLRP3 inflammasome-mediated pyroptosis.51 However, our findings indicate that Pep19–2.5-induced LDH release is possibly dependent on the P2X7 receptor. This is in line with results obtained with the P2X receptor agonist ATP where LDH release was inhibited by blocking the P2X7 receptor but not NLRP3.33

Different hypotheses have been proposed that describe the interaction between the P2X7 receptor and LPS-neutralizing peptides. Since IL-1β release induced by LL-37 is blocked by inhibitors that also prevent P2X7 receptor activation by ATP, it has been suggested that LL-37 interacts with the P2X7 receptor at the ATP-binding site. However, no direct interaction has been reported.28 Furthermore, it was shown that the C-terminal domain of the P2X7 receptor is not required for LL-37 activity and binding.29 We hypothesize a similar mode of interaction between Pep19–2.5 and the P2X7 receptor. Pep19–2.5-induced intracellular Ca2+ increase as well as IL-1β and LDH release was completely blocked by PPADS. PPADS acts by sterically blocking agonist access to the ATP binding site.35 The allosteric and noncompetitive inhibitor A80459843 did not reduce Pep19–2.5-induced IL-1β and LDH release. This suggests that Pep19–2.5 interacts with the P2X7 receptor at the ATP binding site. However, Pep19–2.5 likely interacts with the P2X7 receptor via a different mechanism compared to ATP. This is supported by the findings with the P2X7 antagonist oxATP,44 which significantly reduced LL-37-28 and Pep19–2.5-induced IL-1β release. Although PPADS and oxATP are commonly used to inhibit P2X or P2X7 receptors, both inhibitors have off-target effects.52,53 Therefore, the inhibition of Pep19–2.5-induced Ca2+ increase, as well as its effects on IL-1β and LDH release, may involve receptor-independent mechanisms. Indeed, membrane interactions near the P2X7 receptor have been proposed as a possible explanation for the modulatory effects of LL-37.29 It is likely that Pep19–2.5 modulates the P2X7 receptor through a similar mechanism. The interaction between Pep19–2.5 and P2X7 receptors is further evidenced by the results obtained in our transfection experiments and by colocalization microscopy. Colocalization between LL-37 and the P2X7 receptor has previously been reported,45 supporting our observation. In contrast to the results reported for LL-37 and observed for Pep19–2.5, PMB does not induce IL-1β release in TLR-primed cells.27 We hypothesize that PMB and Pep19–2.5 differentially interact with the P2X7 receptor.

We observed that Pep19–2.5 reduced LDH and IL-1β release in Pam3CSK4-primed THP-1 macrophages and primary human monocytes when stimulated with the potent P2X7 ligand BzATP. The effect is independent of TLR signaling, as Pep19–2.5 was added simultaneously with BzATP after the priming step, and it does not interact with TLR2/1 ligand Pam3CSK4.17 The reduction of BzATP-induced Ca2+ influx in THP-1 macrophages supports our hypothesis of an effect on receptor activation. LL-37 also interferes with P2X7-mediated IL-1β release in primary human monocytes. In the presence of LL-37, ATP-induced IL-1β release was partially reduced,28 further highlighting the similarities between LL-37 and Pep19–2.5. In contrast, PMB has been reported to potentiate ATP-dependent IL-1β and LDH release.27 This finding supports a different mode of action for Pep19–2.5 compared to PMB.

Finally, we characterized the modulatory effects of Pep19–2.5 on different P2X receptor subtypes. Pep19–2.5 most potently attenuated P2X4 and P2X7 receptor signaling, making Pep19–2.5 the first LPS-neutralizing compound that shows dual P2X4 and P2X7 receptor modulation. P2X4 receptors are able to potentiate P2X7 receptor-induced NLRP3 inflammasome activation,54 therefore Pep19–2.5 might directly and indirectly modulate P2X7 receptor-induced IL-1β release.

Conclusions

Taken together with the highly potent LPS-neutralizing and wound healing effects of Pep19–2.5 reported earlier,20,22,26 our findings strengthen the potential of Pep19–2.5 as a novel anti-infective and immunomodulatory drug. Attenuation of P2X7 receptor activation reduces IL-1β release and oxidative stress induced by LPS,55−57 a key mediator in sepsis caused by Gram-negative bacteria.58 Consequently, reducing P2X7-mediated effects may help prevent immune overactivation when Pep19–2.5 is used as a treatment for sepsis. Our findings highlight the significance of Pep19–2.5′s signaling functions in human innate immune responses beyond its LPS-neutralizing activity.

Methods

Peptides

Pep19–2.5 (GCKKYRRFRWKFKGKFWFWG) was purchased from Bachem (Bubendorf, Switzerland) and was produced under GMP conditions. The fluorescently labeled NBD-D-Pep19–2.5 was synthesized as previously described.22 The purity of all peptides was higher than 95% as determined by HPLC and mass spectrometry.

Cell Culture

THP-1 cells (ACC 16, DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany) were cultured in RPMI 1640 (11530586, Thermo Fisher Scientific, Darmstadt, Germany) containing 100 U/mL penicillin, 100 μg/mL streptomycin (P4333), 2 mM l-glutamine (G7513, both from Sigma-Aldrich, Taufkirchen, Germany) and 10% heat-inactivated fetal bovine serum (FBS; S0615, Sigma-Aldrich) (growth medium) at a density of (2–8) × 105 cells/mL. Cells were used from passage 4–25 and maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. THP-1 cells were regularly tested negative for mycoplasma contamination (VenorGeM Classic Mycoplasma PCR detection kit, 11–8100, Minerva Biolabs, Berlin, Germany). To generate THP-1-derived macrophages, THP-1 monocytes were seeded into 24-well plates at a density of 4 × 105 cells/well in growth medium including 25 ng/mL PMA (phorbol 12-myristate 13-acetate; tlrl-pma, Invivogen, Toulouse, France). After 48 h, adherent cells were carefully washed with PBS (phosphate buffered saline; P04–53500, PAN-Biotech, Aidenbach, Germany) and rested in PMA-free growth medium for 24 h.

Peripheral blood mononuclear cells (PBMCs) were isolated from buffy-coat donations (Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn) by density gradient centrifugation using Biocoll separation media (BS L6115, Bio&Sell, Nuremberg, Germany). PBMCs were seeded into 24-well plates at a density of 5 × 106 cells/well in RPMI 1640 growth medium and incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air for 1 h. Monocytes were enriched by plastic adherence. PBMCs were washed three times with PBS and nonadherent cells discarded. The studies with human blood were approved by the ethics committee of the University Clinic Bonn (315/22) and written informed consent was obtained from all healthy donors.

HEK293T cells (ACC 635, DSMZ-German Collection of Microorganisms and Cell Cultures GmbH) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, P04–03500, PAN-Biotech) containing 4.5 g/L glucose, 2 mM l-glutamine and 10% (v/v) heat-inactivated FBS (HEK293T growth medium).

Cell Stimulation

Primary monocytes and THP-1 macrophages were preincubated with Pam3CSK4 (tlrl-pms-1, Invivogen) or RPMI 1640 for 3 h. Afterward, cells were stimulated for additional 3 h with Pep19–2.5 (Bachem, Bubendorf, Switzerland), BzATP (2′/3′-O-(4-benzoylbenzoyl)adenosine-5′-triphosphate; NU-1620-25, Jena Bioscience, Jena, Germany) or a combination thereof. In selected experiments, cells were preincubated with the noncompetitive P2X7 receptor antagonist A804598 (14473, Tocris Bioscience, Bristol United Kingdom), irreversible P2X7 receptor antagonist oxATP (oxidized ATP) (505758, Merck, Darmstadt, Germany), P2X receptor antagonist PPADS (0625, Tocris Bioscience), NLRP3 inhibitor MCC950 (5479, Tocris Bioscience), caspase-1 inhibitor Ac-YVAD-cmk (10014, Biomol, Hamburg, Germany) or pan-caspase inhibitor Z-VAD-fmk (tlrl-vad, Invivogen) 1 h before stimulation with Pep19–2.5. To examine the dependency of K+ efflux, Pam3CSK4-primed THP-1 macrophages were stimulated in the presence of potassium chloride (75 mM, 6781.3, Carl Roth, Karlsruhe, Germany).

Transfection of HEK293T Cells

HEK293T cells were transfected with pUNO1-hP2RX7 (puno1-hp2rx7) or pUNO1-mcs (puno1-mcs, both from Invivogen) plasmids. Briefly, cells were seeded into poly-l-lysine (P6282–5MG, Sigma-Aldrich)-coated 6-well plates in DMEM (P04–03500, PAN-Biotech) containing 4.5 g/L glucose, 5% (v/v) heat-inactivated FBS (S0615), 2 mM l-glutamine (G7513, all from Sigma-Aldrich) (transfection medium) at a density of 6.25 × 105 cells/well and kept at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. After 24 h, the supernatant was discarded and the cells were incubated for 4 h with fresh transfection medium. Linear polyethylenimine (PEI; 24765 (100), Polysciences, Warrington, USA) was then added at a 1:3 plasmid/PEI (w:w) ratio containing 3 μg plasmid per well. After 24 h, the medium was replaced with fresh transfection medium, and the plate was incubated for another 24 h.

Calcium Assays

In THP-1 macrophages and transfected HEK293T cells, intracellular Ca2+ increase was measured using the Ca2+-sensitive dye Fluo4-AM (20551, AAT Bioquest, Pleasanton, USA). THP-1 monocytes were seeded at a density of 1 × 105 cells/well in 96 well plates and differentiated into macrophages as described above. Transfected HEK293T cells were also seeded at a density of 1 × 105 cells/well. Three μM Fluo4-AM, 1 mM probenecid (Sigma-Aldrich) and 0.05% pluronic acid (20052, AAT Bioquest) in HEPES-buffered HBSS (dye loading buffer) was added, and the cells were incubated at room temperature for 1 h. Cells were then incubated with or without PPADS in HEPES-buffered HBSS for 30 min. Calcimycin, also known as A23187 (C7522, Sigma-Aldrich), BzATP or Pep19–2.5 was added at the indicated time point. Fluorescence intensity was recorded over time (Flexstation, Molecular Devices, Sunnyvale, USA; excitation/emission = 490:525 nm).

The modulatory potency of Pep19–2.5 on human P2X receptor ion channels was determined by assessing its ability to reduce agonist-induced Ca2+ influx utilizing 1321N1 astrocytoma cells stably transfected with one of the human P2X receptor subtypes, P2X1, P2X2, P2X3, P2X4, or P2X7. The P2X7 receptor was activated by BzATP (1000 nM), while all other investigated P2X receptor subtypes were activated by ATP (100, 1200, 120, and 180 nM for P2X1, P2X2, P2X3 and P2X4; respectively) as previously described.48,49 An ATP or BzATP concentration which caused ∼80% of the maximal effect was used for receptor stimulation.

Potassium Assay

K+ efflux was measured using the K+-sensitive dye IPG2-AM (ION Biosciences, San Marcos, USA). THP-1 macrophages were generated as described above and detached using TrypLE Express (12604013, Thermo Fisher Scientific). 4.4 μM IPG2-AM, 1 mM probenecid and 0.05% pluronic acid in HEPES-buffered HBSS (Dye loading buffer) was added, and cells were incubated at 37 °C for 1 h. Cells were incubated with or without oxATP in RPMI for 30 min. Subsequently, Pep19–2.5 was added for 15 min. Cells were analyzed by flow cytometry (CytoFLEX, Beckman Coulter, Krefeld, Germany; excitation/emission = 488:525 nm) using FlowJo v10.8 (BD Biosciences, Heidelberg, Germany).

ELISA

Cell culture supernatants were collected and analyzed for IL-1β release using a commercially available ELISA kit (88-7261-88, Thermo Fisher Scientific).

LDH Release

LDH assay was performed according to the manufacturer’s instructions (CyQUANT LDH Cytotoxicity Assay, Thermo Fisher Scientific). The percentage of LDH release was calculated compared to 100% cell lysis control.

Immunofluorescence

THP-1 macrophages were seeded on 8-well chamber slides (Millicell EZ Slides, 8-well Glass, Merck). After stimulation with 11 μM NBD-labeled D-Pep19–2.5 for 30 min at 37 °C, cells were fixed with ROTIHistofix 4% (P087.4, Carl Roth, Karlsruhe, Germany) for 10 min. Afterward, cells were washed with PBS and 0.1% Tween 20 (9005-64-5, Th. Geyer, Renningen, Germany) three times for 5 min, blocked with 10% normal goat serum (5425, Cell Signaling Technology) for 30 min at room temperature and incubated with primary antibody, rabbit-anti-P2X7 receptor (extracellular) (1:100; APR-004, Alomone Labs, Jerusalem, Israel), overnight at 4 °C. After washing, Cy5-conjugated antirabbit antibody (1:100, 111-175-144, Dianova, Hamburg, Germany) was applied for 1 h at room temperature. The cells were stained with Hoechst 33342 (62249, Thermo Fisher Scientific) for 5 min at room temperature. Cells were mounted in ProLong Glass Antifade Mountant (P36982, Thermo Fisher Scientific). Images were taken using a confocal laser microscope (Nikon Ti-E with A1 confocal scanner, Nikon, Düsseldorf, Germany) and processed using NIS-Elements 5.21.00 software (Nikon).

Statistical Analysis

Data are expressed as means or means + SEM. For multiple comparisons, statistically significant differences were determined by one-way ANOVA followed by a Tukey’s post-test. For single comparisons significant differences were determined by an unpaired two-sample t-test. For studies of relative inhibitory effects, BzATP- or Pep19–2.5-induced IL-1β release was set to 100%. All other values were calculated accordingly. Statistical differences were assessed by one-sample t-test against 100%. Differences were considered significant at *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Curve fits were done by four parameter nonlinear regression. IC50 values were determined for the antagonistic potency of Pep19–2.5 on human P2X ion channels. For inhibition of BzATP-induced IL-1β secretion in THP-1 macrophages constraints for top and bottom were set to 100 and 0%, respectively. Statistical analysis was performed using GraphPad Prism 9.5.1 (GraphPad Software Inc., San Diego, California).

Acknowledgments

Anika Püsche is acknowledged for expert technical assistance. The graphical abstract was created in BioRender. Weindl, G. (2024) https://BioRender.com/f72v704

Glossary

Abbreviations

AMP

antimicrobial peptide

ATP

adenosine 5′-triphosphate

BzATP

2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate

DAMP

damage-associated molecular pattern

IL

interleukin

LDH

lactate dehydrogenase

LPS

lipopolysaccharide

NF-κB

nuclear factor kappa-light-chain-enhancer of activated B cells

NLRP3

nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3

oxATP

oxidized ATP

PPADS

pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid

PAMP

pathogen-associated molecular pattern

PMB

polymyxin B

TLR

toll-like receptor

Author Contributions

J.E., A.K., A.D., and A.A. performed the experiments. J.E., A.K., A.D., A.A., C.E.M., and G.W. analyzed the data. K.O. and K.B. contributed reagents/materials. G.W. conceived the study. J.E. and G.W. wrote the manuscript with contributions of all other authors. All authors have given their approval to the final version of the manuscript.

C.E.M. and G.W. gratefully acknowledge funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—GRK2873 (494832089).

The authors declare the following competing financial interest(s): K.B. and G.W. are shareholders of Brandenburg Antiinfektiva GmbH, a company that develops Pep19-2.5. All other authors declare that they have no competing interests.

Special Issue

Published as part of ACS Pharmacology & Translational Sciencespecial issue “Purinergic Signaling”.

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