The regulation of the PD-1/PD-L1 pathway in imiquimod-induced chronic psoriasis itch and itch sensitization in mouse

Abstract

PD-1/PD-L1 inhibitors have been demonstrated to induce itch in both humans and experimental animals. However, whether the PD-1/PD-L1 pathway is involved in the regulation of chronic psoriatic itch remains unclear. This study aimed to investigate the role of the PD-1/PD-L1 pathway in imiquimod-induced chronic psoriatic itch. The intradermal injection of PD-L1 in the nape of neck significantly alleviated chronic psoriatic itch in imiquimod-treated skin. Additionally, we observed that spontaneous scratching behavior induced by imiquimod disappeared on day 21. Still, intradermal injection of PD-1/PD-L1 inhibitors could induce more spontaneous scratching for over a month, indicating that imiquimod-treated skin remained in an itch sensitization state after the spontaneous scratching behavior disappeared. During this period, there was a significant increase in PD-1 receptor expression in both the imiquimod-treated skin and the spinal dorsal horn in mice, accompanied by significant activation of microglia in the spinal dorsal horn. These findings suggest the potential involvement of the peripheral and central PD-1/PD-L1 pathways in regulating chronic itch and itch sensitization induced by imiquimod.

Introduction

The Programmed Cell Death Protein 1 (PD-1) and Programmed Death-Ligand 1 (PD-L1) pathway is a crucial immune regulatory mechanism.^1,2 Binding between PD-1 and PD-L1 triggers signaling pathways that inhibit the activity of T cells, suppressing their ability to attack tumor cells. ³ Clinically, PD-1/PD-L1 inhibitors have been extensively utilized for the treatment of various tumors, exhibiting remarkable therapeutic efficacy.^4,5 Recent studies shown that the expression of the PD-1 and PD-L1 in both the central and peripheral nervous systems in various related diseases.^6,7 PD-L1 functions to inhibit acute and chronic pain by suppressing the activity of nociceptive neurons through PD-1. ⁷ In an acute migraine model, the expression of PD-L1 and PD-1 in trigeminal ganglia may exert a pain-inhibitory effect. ⁸ Our previous studies have demonstrated that intradermal administration of PD-1/PD-L1 inhibitors induces notable scratching behavior in mice. ⁹ Additionally, a considerable number of patients undergoing clinical treatment with PD-1/PD-L1 inhibitors have reported adverse itch reactions, with some even discontinuing immune therapy due to severe itch caused by PD-1/PD-L1 inhibitors.^10,11 These findings underscore the significant involvement of the PD-1/PD-L1 pathway in the transmission of pain and itch. However, the current understanding of PD-1/PD-L1 pathway-mediated itch, particularly its mechanisms in chronic itch, remains insufficient.

Psoriasis is a chronic inflammatory skin condition affecting 2%–3% of the global population. ¹² Itch is a prevalent symptom in 60%–90% of individuals with psoriasis. ¹³ It has been confirmed that the expression of PD-1 receptors is elevated in the skin with psoriasis in mouse, and PD-L1 treatment can alleviate psoriatic skin symptoms.^14,15 However, there is limited research on PD-1/PD-L1 pathway in regulating the chronic psoriasis itch. Additionally, psoriasis is among the skin-related adverse reactions linked to PD-1/PD-L1 inhibitors.^16,17 Psoriatic plaques and itch induced by PD-1/PD-L1 inhibitors mostly occur in patients who had a pre-existing diagnosis or history of psoriasis before immunotherapy.^18,19 This suggests a sensitization to itch in individuals with psoriasis or a history thereof, where PD-1/PD-L1 inhibitors seem to act as triggers for itch sensitization. However, the precise mechanisms behind this itch sensitization remain unclear. Furthermore, PD-1 in microglia has an inhibitory effect on immune cells in the spinal dorsal horn. ²⁰ Enhanced microgliosis in the spinal dorsal horn is induced by chronic morphine treatment in PD-1-deficient mice. ²¹ Similarly, the activation of microglia in the spinal dorsal horn also plays a significant role in the chronic itch model induced by imiquimod.^22,23 Nevertheless, whether microglia in the spinal dorsal horn are involved in itch sensitization in psoriasis remains unknown.

In this study, we further explored the role of the PD-1/PD-L1 pathway in chronic psoriatic itch induced by imiquimod in mouse. Additionally, we observed that spontaneous scratching behavior induced by imiquimod treatment disappeared on day 21, but the imiquimod-treated skin remained in a itch sensitization state more than a month, indicating that intradermal injection of PD-1/PD-L1 inhibitors could induce more spontaneous scratching. During this period, there was a significant increase in PD-1 receptor expression in both the imiquimod-treated skin and the spinal dorsal horn of mice, accompanied by significant activation of microglia in the spinal dorsal horn. These findings collectively suggest the potential involvement of the peripheral and central PD-1/PD-L1 pathways in regulating chronic itch and itch sensitization induced by imiquimod.

Materials and methods

Animals

Adult male and female C57BL/6J mice (12 weeks-old) were purchased from the Experimental Animal Center of Anhui Medical University. The animals were housed with free access to food and water under controlled conditions (20°C–24°C, humidity 60%–65%, and a 12:12 h light:dark cycle) for at least 3 days before the experiments were conducted. The nape of the neck was shaved 3 days before the experiment was conducted. All the animal procedures were approved by the Animal Care and Use Committee of Anhui Medical University.

Drug administration

Primary antibodies for ionized calcium-binding adapter molecule 1 (Iba-1; 1:100, ab178847) and recombinant mouse PD-L1 (1 in 50 µL of saline, ab130039) were purchased from Abcam (San Francisco, CA, USA). The small molecule PD-1/PD-L1 inhibitors, BMS-1 (50 μg in 50 µL of saline, HY-19991) and BMS-202 (50 μg in 50 µL of saline, HY-19745) and PLX5622 in AIN-76A Diet (contains 1200 mg of PLX5622 per Kg of diet, HY-114153C) was purchased from MedChemExpress (Shanghai, China). Furthermore, the primary antibodies for PD-1 (1:200, 18106-1-AP) were purchased from Proteintech (Wuhan, China).

Imiquimod-induced chronic psoriasis itch

As previously described,^9,22 a chronic psoriasis itch model was constructed by painting 62.5 mg of 5% imiquimod cream (Med-Shine, China) on the shaved nape of neck skin (2.5 × 2 cm) daily for 5 consecutive days. The control mice were treated with a vehicle control cream (Vaseline Lanette cream), and spontaneous scratching behaviors were recorded using a video camera. The scratching behavior was video recorded for 1 h on day 0, 3, 6, 9, 12, 15,18 and 21 and scratches were counted blindly.

Behavioral tests

Scratching behaviors experiments were performed as previously described. ²⁴ The mice were placed individually in separate plastic chambers (10 cm × 10 cm × 13 cm) and allowed to acclimatize for 30 min. In the mouse model of acute itch, the scratching behavior was video recorded for 30 min after the intradermal injection of BMS-1 or BMS-202 in the nape of the neck. Intradermal injection of PD-L1 was administered in an experimental setup to observe PD-L1 effect on imiquimod-induced chronic psoriasis itch. Behavioral videos were recorded within 5 h post intradermal injection of PD-L1 on day 6 following 5 days of imiquimod treatment. The total number of scratches was counted blindly.

Haematoxylin and eosin (HE) staining and immunohistochemical (IHC)

Briefly, as previously described, ⁹ all the animals were deeply anesthetized with isoflurane. After heart perfusion with PBS followed by 4% paraformaldehyde, the imiquimod-treated skin and spinal cord (C3–C4) was extracted and post-fixed in 4% paraformaldehyde overnight. The paraffin-embedded was cut into sections (4 μm) and washed with distilled water after dewaxing. The paraffin sections were stained with hematoxylin and eosin, dehydrated through a graded series of ethyl alcohol and xylene. To detect the expression of PD-1, the primary antibody of rabbit anti-PD-1 (1:200), goat anti-rabbit secondary antibodies and diaminobenzidine for chromogenic reaction were applied. To detect the expression of Iba-1, the primary antibody of rabbit anti-Iba-1 (1:500) and fluorescein isothiocyanate-conjugated secondary antibodies were applied. The sections were scanned using a Panoramic Midi II scanner (3DHistech, Budapest, Hungary). Each group included 4-5 mice, and 4 non-adjacent skin or spinal cord sections were randomly selected for each mouse. The intensity of IBA-1 and PD-1 was analyzed using Image J software.

Statistical analysis

The data are expressed as the mean ± SEM. The behavioral data were analyzed using t-tests, one-way analysis of variance (ANOVA) tests, or two-way ANOVA (Bonferroni post hoc) tests by GraphPad Prism 8. The results with p < .05 were considered to be statistically significant.

Results

The PD-1/PD-L1 pathway mediates imiquimod-induced chronic psoriasis itch in skin

In our previous research, ⁹ we observed a significant increase in PD-1 receptor expression in the spinal dorsal horn of mice treated with Imiquimod. Additionally, intrathecal injection of PD-L1 notably reduced the imiquimod-induced chronic psoriatic itch, indicating the potential involvement of the central PD-1/PD-L1 pathway in imiquimod-induced chronic psoriasis itch. In this study, we further explored the role of the peripheral PD-1/PD-L1 pathway in imiquimod-induced chronic psoriasis itch. The results demonstrate that intradermal injection of PD-L1 (5 and 10 μg/50 μL) in imiquimod-treated skin significantly reduced spontaneous scratching behavior in mice induced by Imiquimod (Figure 1(a)). IHC analysis further revealed a significant increase in PD-1 receptor expression in Imiquimod-treated skin (Figure 1(b) and (c)).

Figure 1.

The PD-1/PD-L1 pathway mediates Imiquimod-induced chronic psoriasis itch in skin. Intradermal injection of PD-L1 (5 and 10 μg/50 μL; (a) in imiquimod-treated skin significantly reduces spontaneous scratching behavior in mice on day 6 (^&p < .05, ^&& and **p < .01, ***p < .001 when compared with the vehicle group, n = 5 mice per group). IHC analysis revealed a significant increase in PD-1 receptor (b), (c) expression in imiquimod-treated skin on day 6 (****p < .0001 when compared with the vehicle group, n = 5 per group). All data were shown as mean ± SEM and were evaluated using t-test or two-way ANOVA (Bonferroni post hoc).

Figure 2.

The progression of itch and skin symptoms in mouse model of imiquimod-induced psoriasis. 5% imiquimod cream on the shaved nape of neck skin (2.5 × 2 cm) daily for 5 consecutive days to eliciting chronic psoriasis itch. (a), (b) HE staining results revealed that the skin thickness in imiquimod-treated skin had essentially returned to normal on day 15 (****p < .0001 when compared with the vehicle group, n = 5 mice per group). (c) Spontaneous scratching behavior in the mice persisted until day 21 before completely disappearance (**p < .01, ***p < .001, ****p < .0001 when compared with the vehicle group, n = 5-6 mice per group). All data were shown as mean ± SEM and were evaluated using two-way ANOVA (Bonferroni post hoc).

The progression of itch and skin symptoms in mouse model of imiquimod-induced psoriasis

To investigate the possibility of itch sensitization following the disappearance of itch symptoms in the mouse model of imiquimod-induced psoriasis, we initially observed the progression of itch and skin symptoms in the imiquimod-induced psoriasis. HE staining results revealed that the skin thickness in imiquimod-treated skin had essentially returned to normal by day 15 (Figure 2(a) and (b)). However, spontaneous scratching behavior in the mice persisted until day 21 before completely disappearance (Figure 2(c)).

The PD-1/PD-L1 inhibitor induces increased spontaneous scratching in the mouse model of imiquimod-induced psoriasis

Following the disappearance of spontaneous scratching symptoms in the mouse model of imiquimod-induced psoriasis, we administered intradermal injections of PD-1/PD-L1 small-molecule inhibitors, BMS-1 and BMS-202 (Figure 3(a)). The results revealed that on days 21, 28, 35, 42, and 49, both BMS-1 and BMS-202 induced increased spontaneous scratching in the imiquimod-treated mice (Figure 3(b) and (c)). Additionally, we assessed the expression of PD-1 receptors in the skin tissues of mice on days 6, 21, 28, 35, 42, and 49 post imiquimod treatment using IHC. The findings indicated that at days 21, 28, 35, 42, and 49, there was a sustained significant elevation in PD-1 expression in the mouse skin tissues (Figure 3(d) and (e)).

Figure 3.

The PD-1/PD-L1 inhibitor induces increased spontaneous scratching in the mouse model of imiquimod-induced psoriasis. Following the disappearance of itch symptoms, we administered intradermal injections of PD-1/PD-L1 small-molecule inhibitors, BMS-1 and BMS-202 in imiquimod-treated skin. (a) A timeline of experiments. The results revealed that on days 21, 28, 35, 42, and 49, both BMS-1 (b) and BMS-202 (c) induced increased spontaneous scratching in imiquimod-treated mice (*p < .05, **p < .01, ***p < .001 when compared with the vehicle group, n = 6 mice per group). (d), (e) We assessed the expression of PD-1 receptors in the skin tissues of mice on days 6, 21, 28, 35, 42, and 49 post imiquimod treatment using IHC. The findings indicated that at days 21, 28, 35, 42, and 49, there was a sustained significant elevation in PD-1 expression in the mouse skin tissues (*p < .05, ***p < .001, ****p < .0001 when compared with the vehicle group, n = 5 per group). All data were shown as mean ± SEM and were evaluated using one or two-way ANOVA (Bonferroni post hoc).

The expression of Iba-1 and PD-1 in the spinal cord dorsal horn markedly increased and persisted after the disappearance of skin and itch symptoms in the mouse model of imiquimod-induced psoriasis

The activation of spinal cord dorsal horn microglial has been demonstrated to mediate imiquimod-induced chronic psoriasis itch. Additionally, PD-1 expression was significantly elevated in the spinal cord dorsal horn of the mouse model of imiquimod-induced psoriasis. In order to investigate the roles of microglial and PD-1 in itch sensitization in the mouse model of imiquimod-induced psoriasis, we continuously monitored the expression of microglial cell markers Iba-1 and PD-1 in the spinal cord dorsal horn. The results revealed a significant and sustained elevation of Iba-1 (Figure 4(a) and (b)) and PD-1 (Figure 4(c) and (d)) expression in the spinal cord dorsal horn on days 21, 28, 35, 42, and 49, even during a period after the disappearance of skin symptoms and spontaneous scratching behavior.

Figure 4.

The expression of Iba-1 and PD-1 in the spinal cord dorsal horn markedly increased and persisted after the disappearance of skin and itch symptoms in the mouse model of imiquimod-induced psoriasis. In order to investigate the roles of microglial and PD-1 in itch sensitization in the mouse model of imiquimod-induced psoriasis, we continuously monitored the expression of microglial cell markers Iba-1 and PD-1 in the spinal cord dorsal horn. The results revealed a significant and sustained elevation of Iba-1 (a), (b) and PD-1 (c), (d) expression in the spinal cord dorsal horn on days 21, 28, 35, 42, and 49, even during a period after the disappearance of skin symptoms and spontaneous scratching behavior. All data were shown as mean ± SEM and were evaluated using one-way ANOVA (Bonferroni post hoc; *p < .05, **p < .01, ***p < .001 when compared with the vehicle group, n = 3-4 per group).

Microglial depletion alleviates the itch sensation elicited by the PD-1/PD-L1 inhibitor in the mouse model of imiquimod-induced psoriasis

PLX5622 is a highly selective brain penetrant and orally active colony-stimulating factor 1 receptor inhibitor, which was reported to specifically eliminate microglia in spinal cord. ²⁵ To clarify whether itch sensation elicited by the PD-1/PD-L1 inhibitor was related to microglia activation in the mouse model of imiquimod-induced psoriasis, we fed mice with a PLX5622 diet for 14 days to deplete microglia from day 6. Result shown that, Iba1-marked microglia decreased in spinal cord dorsal horn of PLX5622-treated mice on day 21 (Figure 5(a) and (b)). Furthermore, intradermal injection of BMS-1 or BMS-202 significantly reduced spontaneous scratching in PLX5622-treated mice (Figure 5(c)), suggesting the potential involvement of spinal microglia in regulating itch sensitization in the mouse model of imiquimod-induced psoriasis.

Figure 5.

Microglial depletion alleviates the itch sensation elicited by the PD-1/PD-L1 inhibitor in the mouse model of imiquimod-induced psoriasis. To clarify whether itch sensation elicited by the PD-1/PD-L1 inhibitor was related to microglia activation in the mouse model of imiquimod-induced psoriasis, we fed mice with a PLX5622 diet for 14 days to deplete microglia from day 6. (a), (b) Iba1-marked microglia decreased in spinal cord dorsal horn of PLX5622-treated mice on day 21 (**p < .01 when compared with the vehicle group, n = 4 per group). (c) Intradermal injection of BMS-1 or BMS-202 significantly reduced spontaneous scratching in PLX5622-treated mice (*p < .05, **p < .01 when compared with the vehicle group, n = 6 per group).

Discussion

Chronic itch is a common symptom of psoriasis that significantly impacts the quality of life for patients. The PD-1/PD-L1 pathway contributes to itch, but its involvement in chronic psoriasis itch remains uncertain. ⁹ In this study, we investigated the role of the PD-1/PD-L1 pathway in the chronic psoriasis itch and itch sensitization induced by imiquimod. Initially, we conducted immunohistochemical analysis to detect PD-1 expression in imiquimod-treated skin, revealing a significant increase in PD-1 expression. Intradermal injection of recombinant mouse PD-L1 protein markedly reduced spontaneous scratching induced by imiquimod in mice. The recombinant mouse PD-L1 protein, with a molecular weight of 28 kDa, exhibits restricted permeability across the blood–brain barrier. This suggests that PD-L1 may alleviate imiquimod-induced chronic psoriasis itch by binding to peripheral PD-1. Peripheral PD-1 expression is widespread, notably in the peripheral nervous system, including peripheral nociceptors, axons, and DRG neurons.^7,8,26 This suggests that intradermal PD-L1 injection might inhibit imiquimod-induced chronic psoriasis itch by blocking PD-1 receptors on nociceptors. Additionally, as a chronic immune-inflammatory disease, psoriasis involves various cytokines in its pathogenesis, potentially contributing to itch regulation.^27–30 The PD-1/PD-L1 pathway is well-known for crucial role in the immune system and involvement in modulation of cytokine functions.^31–33 Intradermal injection of PD-L1 might also exert an anti-itch effect by influencing cytokine functionalities. This indicates that the PD-1/PD-L1 pathway potentially modulates imiquimod-induced chronic psoriasis itch through the neuroimmune pathway.

Itch sensitization denotes an augmented reactivity of sensory neurons associated with itch to standard or subthreshold afferent input, and the induction of excessive itch responses can be prompted by stimuli like mechanical or chemical factors. ³⁴ Peripheral itch sensitization mechanisms can be ascribed to an escalated excitability of sensory neurons, potentially stemming from heightened expression, sensitivity, and responsiveness of receptors, including PAR-2, cytokine receptors, TRPV1 and TRPA1.^34,35 Central itch sensitization mechanisms may manifest in both neurons and glial cells. ³⁶ In dorsal horn of the spinal cord, the heightened expression of GRPR receptors in neurons or aberrant regulation of inhibitory descending pathways could serve as contributing factors to the itch sensitization. ³⁷ Moreover, spinal cord glial cells, encompassing microglia and astrocytes, have the potential to induce itch sensitization by releasing inflammatory cytokines. ³⁸ In this study, we observed that chronic psoriatic itch induced spontaneous scratching virtually disappeared on day 21. During the 4 weeks following the cessation of spontaneous scratching behavior, we intradermally administered PD-1/PD-L1 inhibitors under imiquimod-treated skin. Mice exhibited increased spontaneous scratching during this period, indicating the manifestation of itch sensitization. It is important to note that the duration of itch sensitization in mice may extend beyond the observed 4 weeks, as we did not continue observation beyond this period. This phenomenon suggests that the use of PD-1/PD-L1 inhibitors in clinical cancer therapy might trigger more intense pruritic adverse reactions in patients with psoriasis or a recent history of psoriasis. To explore the mechanisms underlying itch sensitization, we assessed the expression of PD-1 receptors in the skin and spinal cord dorsal horn in mice. The results revealed that, 4 weeks after the spontaneous itch disappeared in the chronic psoriatic itch model induced by imiquimod, both the skin and spinal cord dorsal horn continued to exhibit elevated PD-1 expression. This implies the involvement of the PD-1/PD-L1 pathway in the itch sensitization of the chronic psoriatic model induced by imiquimod. PD-1 receptor, as a negative regulator in immune responses, prevents excessive activation of the immune system. ³⁹ Simultaneously, PD-1 expressed in neurons, upon binding with its ligand PD-L1, reduces neuronal excitability. ⁷ Psoriasis, characterized as an immune-mediated inflammatory disease, relies on immune system activation for the exacerbation of skin symptoms.^40,41 Elevated expression of PD-1 receptors in immune cells within skin tissue serves as a protective mechanism against immune cell activation, potentially functioning as the body’s self-defense mechanism against worsening psoriatic skin symptoms. Additionally, we speculate that the upregulation of PD-1 receptors may also occur at the nerve endings in the skin, where PD-1 receptors expressed on nociceptors play a role in countering the itch symptoms associated with psoriasis. Our previous experimental results also indicate that the expression of PD-1 receptors in the dorsal horn of the spinal gray matter increases. Intrathecal injection of PD-L1 can reduce spontaneous scratching induced by imiquimod treatment in mice, implying the involvement of PD-1 receptors in itch transmission in the spinal cord dorsal horn. However, after the disappearance of skin symptoms and itch in the mouse model of psoriasis, the expression of PD-1 receptors in the skin and spinal cord dorsal horn does not rapidly decrease. This may be a mechanism by which the body safeguards against the recurrence of psoriatic skin symptoms and itch. Nevertheless, interference with the binding of highly expressed PD-1 receptors and their ligand by PD-1/PD-L1 inhibitors induces more intense itch. This might explain why PD-1/PD-L1 inhibitors induce increased scratching behavior in imiquimod-treated mice. Furthermore, the expression of the microglial marker IBA-1 in the dorsal horn of the spinal cord dorsal horn remains elevated after the disappearance of itch symptoms in imiquimod-treated mice. Inhibiting the activation of microglial cells alleviates the excessive scratching induced by PD-1/PD-L1 inhibitors in imiquimod-treated mice. This suggests that the activation of microglial cells in the dorsal horn of the spinal gray matter may also be involved in PD-1/PD-L1 pathway-mediated itch sensitization in imiquimod-treated mice.

This phenomenon holds great significance for the clinical application of PD-1/PD-L1 inhibitors. On one hand, it suggests that patients with psoriasis or a history of psoriasis using PD-1/PD-L1 inhibitors may experience more severe adverse itching reactions. Additionally, in our previous research, we observed a similar occurrence in a belly subcutaneous melanoma model where subcutaneous injection of PD-1/PD-L1 inhibitors in the nape of neck induced more scratching behavior compared to control mice. This could be attributed to the upregulation of PD-1 receptor expression induced by tumors. Tumors not only suppress immune responses in the tumor microenvironment but also influence the release of specific cytokines, exerting inhibitory effects on the systemic immune system through peripheral blood circulation. Tumor resection completely resets the systemic immune landscape.^42,43 We hypothesize whether tumors can regulate the expression of PD-1 receptors on nerve endings and neurons by influencing the functionality of cytokines. In light of this, we raise the question of whether the impact of tumors on PD-1 receptor expression in the immune system is synchronous with the expression in the nervous system. We have previously successfully established a method for inducing acute itching through subcutaneous injection of PD-1/PD-L1 inhibitors. If the impact of tumors on the expression of PD-1 receptors in peripheral immune cells and on PD-1 receptors in peripheral nociceptors is synchronous, the observation of the severity of itch induced by subcutaneous injection of PD-1/PD-L1 inhibitors before patients undergo PD-1/PD-L1 inhibitor immunotherapy could be employed to predict the efficacy of future immunotherapy. If these hypotheses are validated, it would provide a convenient and quick method for predicting the effectiveness of PD-1/PD-L1 inhibitors.

In conclusion, our study emphasizes the role of the PD-1/PD-L1 pathway in imiquimod-induced chronic psoriatic itch and itch sensitization. This is highly significant for comprehending the involvement of the PD-1/PD-L1 pathway in itch and refining the application of PD-1/PD-L1 inhibitors in anti-tumor therapy.