PD-L1 inhibits acute and chronic pain by suppressing nociceptive neuron activity via PD-1

Abstract

Programmed cell death ligand-1 (PD-L1) is typically produced by cancer cells and suppresses immunity through the receptor PD-1 expressed on T cells. However, the role of PD-L1 and PD-1 in regulating pain and neuronal function is unclear. Here we report that both melanoma and normal neural tissues including dorsal root ganglion (DRG) produce PD-L1 that can potently inhibit acute and chronic pain. Intraplantar injection of PD-L1 evoked analgesia in naive mice via PD-1, whereas PD-L1 neutralization or PD-1 blockade induced mechanical allodynia. Mice lacking Pd1 (Pdcd1) exhibited thermal and mechanical hypersensitivity. PD-1 activation in DRG nociceptive neurons by PD-L1 induced phosphorylation of the tyrosine phosphatase SHP-1, inhibited sodium channels and caused hyperpolarization through activation of TREK2 K⁺ channels. PD-L1 also potently suppressed nociceptive neuron excitability in human DRGs. Notably, blocking PD-L1 or PD-1 elicited spontaneous pain and allodynia in melanoma-bearing mice. Our findings identify a previously unrecognized role of PD-L1 as an endogenous pain inhibitor and a neuromodulator.

Figure 1. Exogenous PD-L1 inhibits formalin-induced inflammatory pain and increases pain threshold in naïve mice

(a) Formalin-induced Phase-I and Phase-II inflammatory pain, as measured by duration of spontaneous pain behavior (flinching/licking) in every 5 min, is reduced by intraplantar (i.pl.) pretreatment of PD-L1 (1–10 µg). *P<0.05, vs. vehicle (PBS), One-Way ANOVA, n = 7–10 mice/group. PD-L1 was administered 30 min prior to the formalin injection. (b) Basal mechanical pain assessed in von Frey test in naive mice. Notice an increase in paw withdrawal threshold after PD-L1 injection (1 and 5 µg, i.pl.). *P<0.05, vs. human IgG, repeated measures Two-Way ANOVA, n = 5 mice/group. Arrow indicates drug injection. Data are mean ± s.e.m.

Figure 2. Endogenous PD-L1 regulates pain sensitivity in naive mice via PD-1

(a) ELISA analysis showing endogenous expression of PD-L1 in non-malignant tissues of naïve mice and melanoma tissue removed from a mouse hindpaw 4 w after melanoma cell inoculation. Note that PD-L1 is widely expressed in various non-malignant tissues. n = 3 mice/group. (b) Inhibition of endogenous PD-L1 and PD-1 induces mechanical allodynia in naïve mice. PD-L1 was neutralized with soluble PD-1 (sPD-1, 5 µg, i.pl.), and PD-1 was blocked by monoclonal antibodies RMP1-14 (mouse anti-PD-1 antibody, 5 µg, i.pl.) and Nivolumab (human anti-PD-1 antibody, 10 µg, i.pl.). *P<0.05, vs. human IgG, repeated measures Two-Way ANOVA, n = 5 mice/group. Arrow indicates drug injection. (c,d) Reduced mechanical and thermal pain threshold in Pd1^−/− mice, as shown in von Frey test (c) and hot plate test (d). *P<0.05, Two-tailed student t-test, n = 6 mice/group. Data are mean ± s.e.m.

Figure 3. PD-1 is expressed by mouse DRG neurons and nerve axons

(a–d) In situ hybridization (ISH) images showing Pd1 mRNA expression in DRG of wild-type (WT) not Pd1 knockout (Pd1^−/−) mice. (a) Low magnification image of ISH with anti-sense probe showing Pd1 mRNA in DRG neurons of WT mice. Scale, 50 µm. (b) High magnification image of double ISH (red) and Nissl staining (green) in DRG sections. Scale, 20 µm. (c) ISH image showing loss of Pd1 mRNA expression in DRG neurons in Pd1^−/− mice. Scale, 50 µm. (d) ISH image of sense control probe. Scale, 50 µm. (e) Left, image of immunostaining showing broad PD-1 expression in mouse DRG neurons. Middle, PD-1 expression is lost in Pd1^−/− mice. Right, absence of PD-1 immunostaining by the treatment of a blocking peptide. Blue DAPI staining shows all the cell nuclei in DRG sections. Scale, 50 µm. (f) Size frequency distribution of PD-1-positive and total neurons in mouse DRGs. A total of 1555 neurons from 4 WT mice were analyzed. (g,h) Double staining of PD-1 and NF200 in DRG (g) and sciatic nerve (h) sections of mice. Note that PD-1 expression in both NF200-positive and NF200-negative DRG neurons and sciatic nerve axons. Scales, 50 µm. (i) Double immunostaining of PD-1 and CGRP in mouse sciatic nerve. PD-1 is present in axons co-expressing CGRP. Scale, 50 µm. Arrows in g–i indicate the double-labeled neurons and axons.

Figure 4. PD-L1 suppresses neuronal excitability in mouse DRG neurons via PD-1

(a–f) Patch clamp recordings in dissociated (a–d) and whole-mount (e,f) mouse DRG neurons with small diameters (<25 µm). (a) Left, traces of action potentials (AP) showing an inhibitory effect of PD-L1 (10 ng/ml) in WT neurons. Current injection for AP induction starts from +10 pA and increases 10 pA per step. Right, rheobase change in WT and Pd1^−/− mice. n = 6 neurons/2 mice. (b) PD-L1 induces hyperpolarization of the resting membrane potential (RMP). Right, change of RMP in WT and Pd1^−/− mice. n = 6 neurons/2 mice. Note that PD-L1 fails to suppress action potential (a) and alter RMP (b) in Pd1^−/− mice. (c,d) Altered RMP and increased excitability in DRG neurons of Pd1^−/− mice. (c) RMP in WT and Pd1^−/− mice. *P<0.05, paired two-tailed t-test, n = 30 neurons/2 mice. (d) Number of action potentials evoked by current injection in WT and Pd1^−/− mice. *P<0.05, Two-Way ANOVA followed Bonferroni’s post-hoc test, n = 30 neurons/2 mice. (e) Whole-mount DRG recording showing increased action potential firing in small-sized DRG neurons after perfusion of sPD-1 (30 ng/ml). Left, traces of evoked action potential before and after sPD-1 perfusion. Right, action potential frequency following sPD-1 perfusion. *P<0.05, paired two-tailed Student’s t-test, n = 11 neurons/3 mice. (f) Whole-mount DRG recording showing increased action potential firing in small-sized neurons following Nivolumab incubation (2 h, 300 ng/ml). Left, traces of evoked action potential in neurons incubated with control (artificial CSF), human IgG and Nivolumab. Right, frequency of action potentials showing the effects of human IgG and Nivolumab. *P<0.05, vs. control and human IgG, One-Way ANOVA, followed by Bonferroni’s post-hoc test, n = 8–18 neurons/3 mice. Data are mean ± s.e.m.

Figure 5. PD-L1 inhibits neuronal hyperexcitability and neuropathic pain after nerve injury

(a,b) PD-L1 blocks the CCI-induced increases in action potential frequency in small-diameter neurons of whole-mount DRG. (a) Traces of action potentials 4 d after chronic constriction injury (CCI) and the effects of PD-L1 (1 and 10 ng/ml). (b) Frequency of action potentials. *P<0.05, vs. sham control, ^#P<0.05, vs. control (no treatment), One-Way ANOVA, n = 6–9 neurons/group. (c,d) Intrathecal PD-L1 inhibits CCI-induced mechanical allodynia (c) and thermal hyperalgesia (d). *P < 0.05, vs. vehicle, repeated measures Two-Way ANOVA, n = 5 mice/group. Arrow indicates drug injection. (e) Randall-Selitto test showing increased baseline mechanical pain threshold after intrathecal PD-L1 injection in naïve mice. *P < 0.05, vs. vehicle, ^#P < 0.05, vs. baseline (BL), repeated measures Two-Way ANOVA, n = 5 mice/group. Arrow indicates drug injection. Data are mean ± s.e.m.

Figure 6. PD-L1 modulates neuronal excitability and pain via SHP-1

(a) Intrathecal PD-L1 (i.t. 1 µg, 30 min) increased phosphorylation of SHP-1 (pSHP-1) in mouse DRG neurons. Left, images of pSHP-1 immunostaining in vehicle and PD-L1 treated group. Scale, 50 µm. Middle, enlarged images from the boxes. Scale, 50 µm. Right, intensity of immunofluorescence of pSHP-1⁺ neurons. *P<0.05, Two-tailed t-test, n = 4 mice/group. (b) Paw withdrawal frequency to a 0.6 g filament in naïve mice and the effects of i.pl. SSG (SHP-1 inhibitor), PD-L1, and PD-L1 plus SSG in naïve mice. Note that PD-L1 induced analgesia is abolished by SSG. *P<0.05, vs. vehicle (PBS), ^#P<0.05, vs. PD-L1, n.s., no significance, One-Way ANOVA, n = 5 mice/group. (c) Inhibition of transient sodium currents by PD-L1 (10 ng/ml) in dissociated DRG neurons and the effect of SSG (11 µM). Left, traces of sodium currents. Right, time course of relative sodium currents. *P<0.05, Two-Way repeated measures ANOVA, n = 6–9 neurons/2 mice. (d) Regulation of RMP by PD-L1 (10 ng/ml) and its blockade SSG (11 µM) in dissociated DRG neurons. *P<0.05, two-tailed Student’s t-test, n = 6–8 neurons/2 mice. (e) PD-L1 increases TREK2 activity via SHP-1 in CHO cells. Left, traces of TREK2-induced outward currents and the effects of PD-L1 and SSG. Right, quantification of outward currents and RMP changes. *P<0.05, two-tailed Student’s t-test, n = 6–8 cells/2 cultures. Data are mean ± s.e.m.

Figure 7. PD-L1 suppresses action potential firing and sodium currents and regulates resting membrane potentials in human DRG neurons

(a) PD-1 immunostaining in a human DRG section. Blue DAPI staining labels all nuclei of cells in DRG. Scale, 50 µm. (b,c) In vitro patch-clamp recording in dissociated small-diameter human DRG neurons (30–50 µm). (b) Suppression of evoked action potential firing by PD-L1. Insert shows a human DRG neuron with a recording pipette. Scale, 25 µm. Blue and red arrows show the shift of RMP after the PD-L1 treatment. (c) Percentage change of action potential frequency (left) and rheobase change (right) following PD-L1 perfusion (10 ng/ml). *P<0.05, vs. vehicle, Two-tailed Student’s t-test, n = 7–10 neurons/3 donors. (d) Reduction of RMP after PD-L1 perfusion. Right, quantification of RMP change. *P<0.05, vs. vehicle, Two-tailed Student’s t-test, n = 13 and 17 neurons/3 donors. (e) Inhibited of transient sodium currents in dissociated human DRG neurons by PD-L1 (10 ng/ml) and the effect of SSG (11 µM). Left, traces of sodium currents. Right, time course of relative sodium currents showing time-dependent inhibition by PD-L1. *P<0.05, Two-Way repeated measures ANOVA, n = 5–8 neurons/2 donors. Data are mean ± s.e.m.

Figure 8. Blocking of PD-L1 or PD-1 signaling induces spontaneous pain and allodynia in a mouse melanoma model

(a) Tumor growth after melanoma cell inoculation (MCI) in a hindpaw. Left, images of ipsilateral hindpaw (red arrow) and contralateral hindpaw and an isolated melanoma (top) at MCI-4w. Scales, 5 mm. Right, time course of tumor growth after MCI, revealed by hindpaw volume change. BL, baseline. *P<0.05, vs. BL, One-Way ANOVA, n = 25 mice/group. (b) Serum PD-L1 levels in sham control mice and melanoma-bearing mice (MCI-4w). *P<0.05, two-tailed Student’s t-test. n= 6 mice/group. (c,d) Time course of mechanical pain (c) and spontaneous pain (duration of licking/flinching, d) after MCI. Note that tumor growth is not associated with the development of mechanical allodynia and spontaneous pain. n = 21 and 25 mice/group. (e) Induction of spontaneous pain by soluble PD-1 (sPD-1) following i.pl. injection at MCI-4w. Note a rapid onset of spontaneous pain by sPD-1 within 30 min. *P<0.05, compared with vehicle, two-tailed Student’s t-test. n= 6 and 7 mic/group. (f) Induction of ongoing pain (CPP) in melanoma-bearing mice by sPD-1 (i.pl.). Left, paradigm for assessing CPP in a two-chamber test. Right, difference in time spent in drug-paired compartment between the pre-conditioning and post-conditioning phases. *P<0.05, two-tailed Student’s t-test, n= 7–8 mice/group. (g,h) Induction of mechanical allodynia (g, n=11 mice/group) and spontaneous pain (h, n=9 mice/group) by peri sciatic injection of PD-1-targeting siRNA (2 µg) but not by control non-targeting siRNA (NT, 2 µg), given at MCI-4w. *P<0.05, repeated measures Two-Way ANOVA (g) and two-tailed Student’s t-test (h). (i,j) Intravenous Nivolumab (3 and 10 mg/kg), given at MCI-4w (indicated with an arrow), induces mechanical allodynia (i, n=4–6 mice/group) and spontaneous pain 3 h after the injection (j, n=6 mice/group). *P < 0.05, compared with control human IgG4, repeated measures Two-Way ANOVA (i) and two-tailed Student’s t-test (j). (k,l) Intravenous Nivolumab (10 mg/kg, MCI-4w) increases spontaneous firing of afferent fibers in the sciatic nerve 3 h after the injection. (k) Traces of discharges in melanoma-bearing mice treated with Nivolumab and human IgG4 control. (l) Number of spikes in 2 hours after the treatment. *P<0.05, two-tailed student’s t-test, n = 5 mice/group. Data are expressed as mean ± s.e.m.