Sympathetic nerve aggravates autoimmune skin disease via NE–adrenergic receptor axis: Neuroimmune cross-talk insights from vitiligo

Abstract

The dysregulation of cross-talk between the sympathetic nervous system (SNS) and the immune system is closely linked to the development of autoimmune diseases. This study used vitiligo as a model to elucidate the role of the SNS in autoimmune skin diseases. Evidence from clinical and animal studies confirmed abnormal activation of the SNS in vitiligo. Chemical sympathectomy improved the disease phenotype, reduced pathogenic cytokine levels, and inhibited the infiltration and effector functions of CD8⁺ T cells. Mechanistic investigations showed that norepinephrine (NE) released by the SNS interacts with adrenergic receptors to drive fibroblasts and keratinocytes to secrete chemokines CXCL9/10 and inflammatory mediators IL-6/15, establishing a pathological immune microenvironment conducive to the recruitment and activation of CD8⁺ T cells. Adrenergic receptor antagonists could reverse this abnormal immune microenvironment. This study elucidated the pathogenic mechanism of the SNS in vitiligo via the NE–adrenergic receptor–fibroblast/keratinocyte pathway, providing a theoretical foundation for neuroimmune therapy.

Sympathetic nerves play an important role in autoimmune skin diseases through the norepinephrine–adrenergic receptor axis.

INTRODUCTION

The sympathetic nervous system (SNS), a key component of the autonomic nervous system, is primarily responsible for responding to stress and emergencies (1). The “fight or flight” response mediated by it has become a classic physiological regulatory paradigm (1). Traditionally, the function of the SNS has been focused on coping with external threats and modulating physiological responses (1). However, in recent years, growing evidence indicates extensive cross-talk between the SNS and the immune system (2). SNS regulates the function of immune cells by releasing neurotransmitters, such as norepinephrine (NE), which act on adrenergic receptors (3). These discoveries have expanded our understanding of neuroimmune regulation and highlighted the multifaceted roles of the SNS in maintaining internal balance and addressing challenges from both internal and external environments.

The incidence of autoimmune diseases has risen notably, which has become an important public health issue worldwide (4). The core pathogenesis of autoimmune diseases is the disorder of self-antigen recognition by the immune system, leading to aberrant attacks of immune cells on self-tissues (5, 6). It involves key links such as imbalanced activation of T/B cells, abnormal antigen presentation, and dysregulation of the cytokine network (5). As a critical component of neuroimmune cross-talk, dysfunction of the SNS is closely associated with immune regulatory disorders in autoimmune diseases (7, 8). Studies have shown that the SNS can directly or indirectly participate in the pathological processes of autoimmune diseases by regulating the activation and proliferation of immune cells and the secretion of cytokines (9–11).

As the largest neuro-endocrine-immune organ in the human body, the skin’s complex neural innervation network and abundant immune cells form a unique neuroimmune microenvironment (12). Clinical observations have revealed that patients with immune-mediated skin diseases such as vitiligo and atopic dermatitis often exhibit abnormal SNS function (13, 14). Sympathetic nerve excitation induced by mental stress significantly exacerbates disease progression (15). NE secreted by the sympathetic nerve is the primary source of NE in the skin (16). Meanwhile, skin cells widely express adrenergic receptors, providing targets for the immunomodulatory effects of SNS signals (17). However, current research on the regulatory mechanisms of SNS in autoimmune skin diseases still has crucial evidence gaps, and its specific patterns and molecular mechanisms in disease development remain incompletely elucidated.

Vitiligo is an autoimmune skin disease characterized by the destruction of epidermal melanocytes, with typical clinical manifestations of white depigmented lesions on the skin (18). Vitiligo is primarily classified into segmental vitiligo and nonsegmental vitiligo (19). Previous studies have shown that the pathogenesis of segmental vitiligo is closely related to neurological factors (20). However, the association between nonsegmental vitiligo, which accounts for more than 85% of patients with vitiligo, and the nervous system has not been fully elucidated (18). Current studies indicate that the core pathogenic mechanism involves self-reactive CD8⁺ T cell–mediated killing of melanocytes (21). Nonimmune components in the skin, such as fibroblasts and keratinocytes, mediate abnormal local skin immune microenvironments by regulating chemokine networks, including CXCL9 and CXCL10, which synergistically promote the recruitment and activation of CD8⁺ T cells (22, 23). Infiltrating CD8⁺ T cells in the skin secrete effector molecules, such as interferon-γ (IFN-γ) and Granzyme B, under the action of multiple cytokines to exert their function of killing melanocytes (22). Notably, clinical observations have found significantly elevated levels of SNS-related neurotransmitters such as NE and catecholamines in the skin and plasma of patients with nonsegmental vitiligo, suggesting an intrinsic association between SNS dysfunction and disease development (24, 25).

Here, using vitiligo (referring to nonsegmental throughout this study) as a disease model, we systematically explored the role and mechanism of SNS in autoimmune skin diseases. The study first analyzed clinical samples from patients with vitiligo to detect the expression of SNS-related markers and established a clinical evidence chain. Subsequently, CD8⁺ T cell–mediated vitiligo mouse models were used, combined with methods such as chemical sympathectomy, to monitor the driving effects of altered SNS activity on disease progression. Meanwhile, single-cell RNA sequencing (scRNA-seq) data and in vitro cell experiments were integrated to systematically analyze the molecular mechanisms by which SNS regulated vitiligo development. Through the integration of multidimensional evidence from clinical, animal, and cellular studies, this study revealed the role of the SNS in autoimmune skin diseases, providing a theoretical basis for the development of targeted intervention strategies.

RESULTS

Increased sympathetic innervation in vitiligo

To analyze the association between the SNS and vitiligo, this study detected the distribution of sympathetic nerves in the skin through immunofluorescence staining of the sympathetic nerve marker tyrosine hydroxylase (TH) and evaluated sympathetic nerve activation by measuring the expression level of NE, a key neurotransmitter of SNS activation. Results showed that compared with the normal control (NC) skin from the same anatomical site, the density of TH⁺ sympathetic nerve fibers in the perilesional skin of patients with vitiligo was significantly increased (Fig. 1, A and B). In addition, immunofluorescence staining further revealed that sympathetic nerve fibers and CD8⁺ T cells are in close spatial proximity in the perilesional skin of patients with vitiligo (Fig. 1A), suggesting a potential regulatory role of sympathetic nerves on CD8⁺ T cells in vitiligo.

Fig. 1. Sympathetic nerve density and transmitter NE were increased in vitiligo.

(A) Representative immunofluorescence images of TH (a sympathetic nerve marker, red) and CD8 (green) expression in the skin of normal control (NC) and perilesional skin of patients with vitiligo from the same anatomical site. Nuclei were counterstained with DAPI (blue) in the immunofluorescence images. CD8⁺ T cells are shown by white arrows. Scale bars, 100 μm. (B) Quantification of TH⁺ fiber density in the skin of NC (n =3) and patients with vitiligo (n =3) in (A). (C) The serum levels of norepinephrine (NE) in NC (n =21) and patients with vitiligo (n =42). (D) The correlation between serum levels of NE and body surface area (BSA) was analyzed using the Spearman correlation test in patients with vitiligo (n =42). (E) The correlation between serum levels of NE and disease duration was analyzed using the Spearman correlation test in patients with vitiligo (n =42). (F) The serum levels of NE in NC mice and vitiligo mice (n =4 per group). (G) The serum levels of NE in the tail skin of NC mice and vitiligo mice (n =4 per group). Error bars represent mean ± SD from three independent experiments. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001; ns, not significant.

Enzyme-linked immunosorbent assay (ELISA) experiments showed that serum NE levels in patients with vitiligo (n = 42) were significantly higher than those in NC (n =21) (Fig. 1C). Furthermore, we evaluated the relationship between SNS activity and the severity and duration of vitiligo. Correlation analysis showed that serum NE levels in patients with vitiligo were positively correlated with the body surface area score and disease duration (Fig. 1, D and E). The NE levels in serum and skin tissue also showed consistent elevation in vitiligo mice (Fig. 1, F and G). These results indicated that the activation of SNS promoted the development of vitiligo, and its activation degree was positively correlated with the severity and course of the disease.

Chemical sympathectomy attenuated depigmentation in vitiligo mice

We further established an autoimmune-induced CD8⁺ T cell–mediated vitiligo mouse model. In brief, this model was induced by intradermal inoculation of B16F10 melanoma cells and depletion of regulatory T (T_reg) cells in C57BL/6 mice, which activates endogenous autoreactive CD8⁺ T cells through shared antigens between melanoma cells and melanocytes, thereby destroying epidermal melanocytes and triggering vitiligo (Fig. 2A). Starting from the second week postmodeling, chemical sympathectomy was performed via intraperitoneal injection of 6-hydroxydopamine (6-OHDA; a neurotoxin selectively depleting peripheral sympathetic neurons) three times per week until week 12, after which the mice were euthanized for analysis (Fig. 2A). Compared with vehicle-treated mice, the 6-OHDA–treated mice showed a significant reduction in the density of sympathetic nerve fibers in the skin and down-regulated mRNA expression of TH (Fig. 2, B and C). The significant decrease of NE levels in serum and skin in the 6-OHDA–treated mice further confirmed the effectiveness of chemical sympathectomy (Fig. 2, D and E). Phenotypic analysis showed that 6-OHDA–treated vitiligo mice exhibited attenuated vitiligo-like phenotypes, characterized by reduced depigmentation in the tail skin (Fig. 2, F and G). Moreover, 6-OHDA–treated vitiligo mice showed slower disease progression compared with mice exposed to vehicle administration, as evidenced by the degree of depigmentation in the dorsal and ventral sides of the mouse tail skin (Fig. 2, G and H). In conclusion, chemical sympathectomy alleviated the severity and progression of vitiligo.

Fig. 2. Chemical sympathectomy inhibited pigmentation loss in vitiligo mice.

(A) Schematic illustration of the establishment of the vitiligo mouse model and the injection of 6-OHDA. (B) Immunofluorescence and statistical analysis of TH (red) expression in the skin of NC mice and vitiligo mice (n =4 per group). Nuclei were counterstained with DAPI (blue) in the immunofluorescence images. Scale bar, 100 μm. (C) qRT-PCR analysis of mRNA levels of TH in the tail skin of mice in the vehicle and 6-OHDA groups at week 12 (n =4 per group). (D and E) The levels of NE in the serum and tail skin of mice at week 12 (n =4 per group). (F) Representative photos of pigmentation in the tail skin of mice from the vehicle and 6-OHDA groups at week 12. Scale bars, 750 μm. (G) Representative tail images and ImageJ analysis of mice in NC, vehicle, and 6-OHDA groups at week 12 (*dorsal skin, #ventral skin). (H) The tail skin pigmentation percentages of mice in the three groups were calculated over six consecutive weeks (from week 6 to week 12) using ImageJ software (n =4 per group). All vehicle mice were only treated with 0.1% ascorbic acid in 0.9% sterile NaCl. Error bars represent mean ± SD from three independent experiments. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001; ns, not significant.

Chemical sympathectomy inhibited the infiltration and effector functions of CD8⁺ T cells

Next, we investigated the impact of chemical sympathectomy on key immune cell populations in vitiligo. Absolute counting via flow cytometry revealed a significant and consistent reduction in the absolute numbers of CD8⁺ T cells across the tail skin, inguinal lymph nodes (the primary draining lymph nodes for tail skin), and the spleen of vitiligo mice following chemical sympathectomy (fig. S1, A to D). In contrast, no significant differences were observed in the absolute counts of dendritic cells (DCs) or T_reg cells between the two groups (fig. S1, A to D). Furthermore, whole-mount staining of the tail skin demonstrated that, compared with the vehicle-treated mice, 6-OHDA–treated mice exhibited reduced infiltration of CD8⁺ T cells in lesional areas, accompanied by an increased number of melanocytes (Fig. 3A). Collectively, these data reveal that chemical sympathectomy reduces the abundance and local cutaneous infiltration of CD8⁺ T cells in vitiligo.

Fig. 3. Chemical sympathectomy reduced the number of CD8⁺ T cells and inhibited the effector function of CD8⁺ T cells in vitiligo mice.

(A) Representative whole-mount immunofluorescent staining images (top row) and corresponding density images (bottom row) of melanocytes (red) and CD8⁺ T cells (green) in the epidermis of mouse tail skin from NC, vehicle, and 6-OHDA groups. Nuclei were counterstained with DAPI (blue). Scale bar, 200 μm. Statistical analysis of melanocyte and CD8⁺ T cell numbers is shown (n =3 per group). (B to D) Flow cytometry and statistical analysis of IFN-γ⁺ CD8⁺ T cells in the tail epidermis, dermis, and lymph nodes of mice in the NC, vehicle, and 6-OHDA groups (n =3 per group). (E to G) Flow cytometry and statistical analysis of Granzyme B⁺ CD8⁺ T cells in the tail epidermis, dermis, and lymph nodes of mice in the NC, vehicle, and 6-OHDA groups (n =3 per group). All vehicle mice were only treated with 0.1% ascorbic acid in 0.9% sterile NaCl. Error bars represent mean ± SD from three independent experiments. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001; ns, not significant.

Given the pivotal role of CD8⁺ T cells in the pathogenesis of vitiligo, we further examined alterations in their effector functions following chemical sympathectomy. CD8⁺ T cells are involved in the destruction of melanocytes by secreting effector molecules such as IFN-γ and Granzyme B (22). We observed that the capacity of CD8⁺ T cells to secrete IFN-γ and Granzyme B was significantly impaired in both the skin and inguinal lymph nodes of 6-OHDA–treated mice compared with vehicle-treated controls (Fig. 3, B to G). These findings suggest that chemical sympathectomy effectively alleviated the immunopathological damage in vitiligo by suppressing the recruitment and effector functions of CD8⁺ T cells.

Sympathetic nerve regulation of CD8⁺ T cells depended on NE secretion

The reduced levels of NE and the impaired number and effector functions of CD8⁺ T cells after sympathetic nerve ablation suggest that the regulation of CD8⁺ T cells by sympathetic nerves may rely on NE secretion. To validate this hypothesis, we established a vitiligo mouse model with NE supplementation based on 6-OHDA treatment (6-OHDA + NE group) (Fig. 4A). ELISA showed that the levels of NE in the serum and tail skin of the 6-OHDA + NE group were higher than those in the 6-OHDA group, indicating that exogenous NE effectively reversed the neurotransmitter depletion caused by sympathetic nerve ablation (Fig. 4, B and C). Quantitative analysis of pigmentation in the dorsal and ventral regions of the tail skin revealed that vitiligo mice in the 6-OHDA + NE group exhibited a significant increase in depigmentation compared to those in the 6-OHDA group (Fig. 4, D and E). Whole-mount staining of the tail skin demonstrated that vitiligo mice in the 6-OHDA + NE group had more CD8⁺ T cells and fewer melanocytes than those in the 6-OHDA group (Fig. 4, F and G). To further examine the number and effector function of CD8⁺ T cells, flow cytometry analysis was performed on the epidermis, dermis, and inguinal lymph nodes. Results showed that the 6-OHDA + NE group mice had more CD8⁺ T cell infiltration in the epidermis, dermis, and inguinal lymph nodes than the 6-OHDA group mice (Fig. 5, A and B, and fig. S2A). In addition, the production of IFN-γ and Granzyme B by CD8⁺ T cells in the epidermis, dermis, and inguinal lymph nodes was increased in the 6-OHDA + NE group mice, confirming the restoration of their cytotoxic function (Fig. 5, C to F, and fig. S2, B and C). These results suggested that NE supplementation rescued the reduced CD8⁺ T cell infiltration and weakened cytotoxic function caused by sympathetic nerve ablation in vitiligo mice. Thus, NE was a key mediator through which the SNS regulated the number and function of CD8⁺ T cells.

Fig. 4. Supplementation with NE rescued the disease phenotype of vitiligo mice.

(A) Establishment and administration of the vitiligo mouse model. (B and C) The levels of NE in the serum and tail skin of mice in the vehicle, 6-OHDA, and 6-OHDA + NE groups were determined by ELISA at week 12 (n =4 per group). (D) Representative tail images and ImageJ analysis of mice in the vehicle, 6-OHDA, and 6-OHDA + NE groups at week 12. (E) The tail skin pigmentation percentages of mice in the three groups in six consecutive weeks (from week 6 to week 12) were calculated using ImageJ software (n =4 per group). (F) Representative whole-mount immunofluorescent staining images (top row) and corresponding density images (bottom row) of melanocytes (red) and CD8⁺ T cells (green) in the epidermis of mouse tail skin from the vehicle, 6-OHDA, and 6-OHDA + NE groups. Nuclei were counterstained with DAPI (blue). Scale bar, 200 μm. (G) Statistical analysis of melanocytes and CD8⁺ T cells in the tail epidermis of mice in the vehicle, 6-OHDA, and 6-OHDA + NE groups (n =4 per group). All vehicle mice were only treated with 0.1% ascorbic acid in 0.9% sterile NaCl. Error bars represent mean ± SD from three independent experiments. ^**P < 0.01 and ^***P < 0.001.

Fig. 5. Supplementation with NE rescued the number and effector function of skin CD8⁺ T cells in vitiligo mice.

(A and B) Flow cytometry and statistical analysis of CD8⁺ CD45⁺ T cells in the mouse tail epidermis and dermis of vehicle, 6-OHDA, and 6-OHDA + NE groups (n =3 per group). (C and D) Flow cytometry and statistical analysis of IFN-γ⁺ CD8⁺ T cells in the mouse tail epidermis and dermis of vehicle, 6-OHDA, and 6-OHDA + NE groups (n =3 per group). (E and F) Flow cytometry and statistical analysis of Granzyme B⁺ CD8⁺ T cells in the mouse tail epidermis and dermis of vehicle, 6-OHDA, and 6-OHDA + NE groups (n =3 per group). (G) The secretion levels of CXCL9, CXCL10, IL-6, and IL-15 in the tail skin of mice in the vehicle, 6-OHDA, and 6-OHDA + NE groups were detected by ELISA (n =4 per group). All vehicle mice were only treated with 0.1% ascorbic acid in 0.9% sterile NaCl. Error bars represent mean ± SD from three independent experiments. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001.

Next, we sought to determine the target cells regulated by sympathetic nerves in vitiligo. We analyzed the expression levels of adrenergic receptors in various cell populations in the skin through information from the scRNA-seq database. The results showed that CD8⁺ T cells mainly expressed β2-adrenergic receptors (ADRB2) (fig. S3A). Quantitative real-time polymerase chain reaction (qRT-PCR) revealed decreased ADRB2 expression on CD8⁺ T cells in the peripheral blood of patients with vitiligo compared with NC (fig. S3B). However, NE treatment did not affect the number or effector function of CD8⁺ T cells (fig. S3, C to E). These results suggested that the regulation of CD8⁺ T cells by sympathetic nerves through NE in vitiligo might involve other mechanisms.

Sympathetic nerves reestablished the cutaneous immune microenvironment through the NE–adrenergic receptor axis

The pathogenesis of vitiligo involves a complex immune regulatory network. In addition to CD8⁺ T cells, cutaneous stromal cells (such as keratinocytes and fibroblasts) play a key role in establishing a proinflammatory immune microenvironment that promotes disease progression by secreting important chemokines (e.g., CXCL9 and CXCL10) and inflammatory cytokines [e.g., interleukin-6 (IL-6) and IL-15]. Therefore, we investigated the effect of the sympathetic nerve on the local skin immune microenvironment in vitiligo. qRT-PCR and ELISA analysis showed that compared with the vehicle-treated group, the mRNA and protein levels of chemokines (CXCL9/10) and proinflammatory cytokines (IL-6/15) in the skin of vitiligo mice in the 6-OHDA–treated group were significantly decreased, while exogenous supplementation of NE effectively reversed those effects (fig. S2D and Fig. 5G). It is suggested that sympathetic nerves might participate in the construction of the cutaneous proinflammatory immune microenvironment by regulating the function of skin stromal cells.

Skin scRNA-seq analysis revealed that fibroblasts highly expressed α2A-adrenergic receptors (ADRA2A), while keratinocytes highly expressed ADRB2 (fig. S3A). This indicates that sympathetic nerves may regulate fibroblasts and keratinocytes through the NE–adrenergic receptor axis to influence the skin immune microenvironment. Volcano plots and bubble plots further showed that the expression levels of ADRA2A in fibroblasts and ADRB2 in keratinocytes in the skin of patients with vitiligo were higher than those in healthy controls (Figs. 6, A and B, and 7, A and B).

Fig. 6. Sympathetic nerves modulated the proinflammatory response of fibroblasts through the NE-ADRA2A axis.

(A) Volcano plots illustrated the expression of adrenergic receptors on fibroblasts in human skin, including ADRA1A, ADRA1B, ADRA1D, ADRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, and ADRB3. (B) Bubble map illustrated the up-regulation of ADRA2A expression on fibroblasts in the lesional skin of patients with vitiligo and the normal skin of healthy controls. (C) Representative immunofluorescence images of fibroblasts, ADRA2A, CXCL9, CXCL10, and CD8⁺ T cells from the normal skin of the healthy control and the lesional skin of the patient with vitiligo (n =3 per group). Nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. (D) Immunofluorescence analysis of ADRA2A (red) expression in BJ cells after treatment with 5 μM NE for 48 hours. Cell nuclei were stained with DAPI (blue). Scale bar, 50 μm. (E) Secretion levels of CXCL9, CXCL10, IL-6, and IL-15 in BJ cells were detected by ELISA with treatment with aposcopolamine (Apos) or NE (n =3 per group). (F) Secretion levels of CXCL9, CXCL10, IL-6, and IL-15 in BJ cells with ADRA2A siRNA or control siRNA were detected by ELISA (n =3 per group). Error bars represent mean ± SD from three independent experiments. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001; ns, not significant.

Fig. 7. Sympathetic nerves modulated the proinflammatory response of keratinocytes through the NE-ADRB2 axis.

(A) Volcano plots illustrated the expression of adrenergic receptors on keratinocytes in human skin, including ADRA1A, ADRA1B, ADRA1D, ADRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, and ADRB3. (B) Bubble map illustrated the up-regulation of ADRB2 expression on keratinocytes in the lesional skin of patients with vitiligo and the normal skin of healthy controls. (C) Representative immunofluorescence images of keratinocytes, ADRB2, CXCL9, CXCL10, and CD8⁺ T cells from the normal skin of the healthy control and the lesional skin of the patient with vitiligo (n =3 per group). Nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. (D) Immunofluorescence analysis of ADRB2 (red) expression in keratinocytes after treatment with 5 μM NE for 48 hours. Cell nuclei were stained with DAPI (blue). Scale bar, 50 μm. (E) Secretion levels of CXCL9, CXCL10, IL-6, and IL-15 in keratinocytes were detected by ELISA with treatment with NE or ICI (n =3 per group). (F) Secretion levels of CXCL9, CXCL10, IL-6, and IL-15 in keratinocytes with ADRB2 siRNA or control siRNA were detected by ELISA (n =3 per group). Error bars represent mean ± SD from three independent experiments. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001; ns, not significant.

Immunofluorescence costaining confirmed substantially higher expression of ADRA2A in fibroblasts and ADRB2 in keratinocytes within vitiligo skin than in healthy skin (Figs. 6C and 7C). Of particular importance, ADRA2A⁺ fibroblasts and ADRB2⁺ keratinocytes exhibited prominent spatial co-localization with CXCL9, CXCL10, and infiltrating CD8⁺ T cells (Figs. 6C and 7C). These in vivo findings strongly confirm that ADRA2A-expressing fibroblasts and ADRB2-expressing keratinocytes are integral components of the pathological immune microenvironment in vitiligo. In vitro experiments further elucidated the molecular effects of NE on these stromal cells. NE stimulation up-regulated ADRA2A expression in human skin fibroblast cells (BJ cells) and ADRB2 expression in human immortalized keratinocytes (HaCaT cells) (Figs. 6D and 7D and figs. S4A and S5A), while simultaneously driving the release of downstream inflammatory mediators. Specifically, in BJ cells, NE markedly enhanced the transcription and secretion of CXCL9, CXCL10, and IL-6; these effects were reversed by the ADRA2A inhibitor aposcopolamine or ADRA2A small interfering RNA (siRNA) (fig. S4, B and C, and Fig. 6, E and F). However, NE stimulation had no significant effect on the transcription and secretion levels of IL-15 in BJ cells (fig. S4, B and C, and Fig. 6, E and F). In HaCaT cells, NE stimulation promoted increased transcription and secretion levels of CXCL9, CXCL10, IL-6, and IL-15, and these effects were reversed by the ADRB2 inhibitor ICI 118,551 hydrochloride (ICI) or ADRB2 siRNA (fig. S5, B and C, and Fig. 7, E and F). Collectively, sympathetic nerves activated fibroblasts and keratinocytes through the NE-ADRA2A/ADRB2 axis, drove the formation of a proinflammatory microenvironment, and thereby promoted vitiligo progression (Fig. 8).

Fig. 8. Schematic diagram of sympathetic nerves in vitiligo.

Sympathetic nerves contribute to vitiligo by promoting the proinflammatory effects of fibroblasts and keratinocytes through the secretion of NE, which in turn recruit CD8⁺ T cells to kill melanocytes.

DISCUSSION

Our study revealed an SNS-driven mechanism underlying vitiligo progression, specifically connecting sympathetic innervation to nonsegmental vitiligo, a subtype previously considered devoid of neural involvement. Moreover, we identified that sympathetic signaling modulates the local immune microenvironment to drive autoimmune skin pathogenesis. We first confirmed the abnormal activation of the SNS in patients with vitiligo. Chemical sympathectomy notably alleviated depigmentation in vitiligo mice and inhibited the infiltration and effector function of CD8⁺ T cells by decreasing NE levels. Further investigations revealed that the SNS activated fibroblasts and keratinocytes through the NE–adrenergic receptor axis, promoting their secretion of chemokines (CXCL9/10) and proinflammatory mediators (IL-6/15), thus establishing an immune microenvironment conducive to the recruitment and activation of CD8⁺ T cells. Although previous studies have suggested that the SNS may be involved in the development of autoimmune skin diseases, the mechanisms and molecular pathways remain unclear (26–28). This study elucidated the mechanism by which the SNS promotes vitiligo progression through the “neurotransmitter-stromal cell-CD8⁺ T cell” pathway, providing further insights into the role of the SNS in autoimmune skin diseases.

As a frontier organ for neuroimmune interaction, the skin’s unique innervation pattern provides an anatomical basis for immune regulation (12). Nerves in the skin are divided into sensory nerves and sympathetic nerves, each with distinct functions (10). The role of sensory nerves in regulating immune responses in the skin by releasing neuropeptides, such as substance P and calcitonin gene–related peptide (CGRP), has been confirmed (10, 29). For example, CGRP secreted by sensory nerves can enhance the antigen-presenting capacity of dermal cDC1 cells and initiate and amplify the autoimmune CD8⁺ T cell response against melanocytes to drive the progression of vitiligo (30). CGRP secreted by sensory nerves can exacerbate inflammatory responses in rosacea by activating dermal γδ T cells (31). Sympathetic nerves primarily regulate physiological functions such as blood flow and sweat gland secretion in the skin, and participate in immune regulation by releasing catecholamine neurotransmitters like NE (3). While previous studies have predominantly focused on sensory nerve–mediated skin immune regulation, our study reveals that sympathetic nerves also play a nonnegligible role in autoimmune skin diseases. Sympathetic nerves can promote the progression of vitiligo by remodeling the skin immune microenvironment and regulating the functions of both nonimmune and immune cells. Notably, a previous study showed that chemical sympathectomy did not alter the progression of vitiligo (32). Unlike our research on intervening in SNS activities during the initiation phase of vitiligo, a previous study performed chemical sympathectomy before modeling (32). This early ablation strategy might have failed to reflect the driving role of the SNS in the early stage of vitiligo. In addition, the maintenance of the sympathetic nerve ablation state may be constrained by a temporal window, as regeneration of sympathetic fibers or recovery of NE levels could lead to deviations in subsequent effect detection. Our findings highlight the critical role of the SNS in vitiligo progression and provide a perspective for deepening the understanding of neuroimmune cross-talk in autoimmune skin diseases.

The SNS regulates immune functions by releasing catecholamine neurotransmitters such as NE, which bind to adrenergic receptors (including α and β adrenergic receptors) on cell surfaces (33). Previous studies have shown that NE secreted by sympathetic nerves acts on α-adrenergic receptors on macrophages to enhance their production of nitric oxide and inducible nitric oxide synthase, thereby exacerbating rheumatoid arthritis (34). The SNS regulates the balance of T cell subsets via the NE-ADRB2 axis, inhibiting excessive immune responses in immune thrombocytopenia (35). Our study found that fibroblasts and keratinocytes were skin cell types with high adrenergic receptor expression, and this expression was enhanced in vitiligo lesions. NE stimulation upregulated the expression of ADRA2A in fibroblasts and ADRB2 in keratinocytes, driving their secretion of chemokines and proinflammatory cytokines. Administration of corresponding adrenergic receptor antagonists or siRNA effectively reversed these effects. These findings strongly confirmed that the SNS promoted vitiligo progression by secreting NE, which bound to adrenergic receptors on fibroblasts and keratinocytes.

Mounting evidence indicates that the regulation of immune functions by the SNS is of remarkable complexity. In this study, we found that NE released by the SNS does not directly act on the core immune cells of vitiligo, CD8⁺ T cells. Instead, it promoted the secretion of chemokine CXCL9/10 and proinflammatory mediator IL-6/15 by acting on fibroblasts and keratinocytes. This formed an indirect regulatory pathway of “neurotransmitter–stromal cell–immune cell” that influenced CD8⁺ T cell functions and drives vitiligo progression. This indirect regulatory paradigm is also observed in other diseases. For example, the SNS promotes atherosclerosis by releasing NE to induce vascular endothelial cells to secrete CXCL1 and CCL7, driving neutrophil and monocyte recruitment (36). In obesity, the SNS activates β-adrenergic receptors on adipose mesenchymal cells via NE, promoting the secretion of glial-derived neurotrophic factor and enhancing the lipolytic functions of ILC2 (11). These cross-disease evidences suggest that SNS-mediated indirect immune regulation is one of the important patterns of neuroimmune cross-talk. Moreover, adrenergic receptors are widely expressed on various immune cells, enabling the SNS to directly regulate immune cell functions by binding NE to these receptors (33). For instance, in psoriasis, NE released by the SNS binds to ADRB1 on γδ T cells, activating p38 mitogen-activated protein kinase and nuclear factor κ light chain enhancer of activated B cells (NF-κB) pathways to promote IL-17 secretion and exacerbate inflammation (28). Crucially, our study revealed that SNS targeted the key stromal cells (fibroblasts and keratinocytes) to establish a sustained proinflammatory immune microenvironment, thus driving the progression of vitiligo. This finding not only deepened our understanding of SNS function in skin immune diseases but also provided important evidence for targeted intervention in the skin microenvironment.

The activation and effector functions of CD8⁺ T cells are regulated by multiple cytokines (37). Among these, the CXCL9/CXCL10-CXCR3 axis mediates the recruitment of CD8⁺ T cells into the skin, whereas IL-6 and IL-15 promote the differentiation of CD8⁺ T cells into cytotoxic T lymphocytes (38–42). The expression of these chemokines and proinflammatory cytokines is highly dependent on the activation of Janus kinase–signal transducer and activator of transcription (JAK-STAT) and NF-κB signaling pathways (43–45). Previous studies have demonstrated that NE could modulate the transcriptional activity of both JAK-STAT and NF-κB pathways through adrenergic receptors on vascular smooth muscle cells or astrocytes (46–48). We thus speculate that the NE secreted by SNS might play an important role in vitiligo by activating the JAK-STAT and NF-κB pathways. Notably, the expression of IL-15 in fibroblasts is not regulated by NE, suggesting molecular specificity in the response of different stromal cells to NE. Previous studies have shown that adrenergic receptors are involved in regulating the proliferation, differentiation, and migration of keratinocytes and fibroblasts (49, 50). This study further suggested that the SNS endowed keratinocytes and fibroblasts with a proinflammatory function through adrenergic receptors, enhancing our understanding of their regulatory mechanisms.

To date, there are still huge challenges in the treatment of vitiligo. Although glucocorticoids, calcineurin inhibitors, and JAK inhibitors are widely used, the prognosis of vitiligo remains unsatisfactory. This study confirmed that adrenergic receptor antagonists can reverse the abnormal inflammatory cascade in the skin microenvironment of vitiligo and attenuate the effector functions of CD8⁺ T cells, suggesting that targeting adrenergic receptors may be a potential strategy for vitiligo therapy. Previous studies have shown that inhibiting ADRB1 in T helper cell 17 (T_H17) cells alleviates disease phenotypes and inflammatory infiltration in psoriasis mice (28). ADRB2 antagonists can effectively delay the onset of rheumatoid arthritis and reduce the severity of joint damage by blocking the inhibitory effect of NE on T_H1 cells development (51, 52). Multiple adrenergic receptor antagonists are already widely used in clinical practice. For example, the β-blocker propranolol is used in the treatment of infantile hemangiomas, hypertension, and anxiety (53–55). The safety data accumulated from long-term clinical use provide critical references for exploratory clinical studies on vitiligo treatment. The rapid clinical translation pathway based on existing drugs is expected to break through the current treatment difficulties of vitiligo.

Methods for sympathectomy include local surgical sympathectomy and chemical sympathectomy (56, 57). Chemical sympathectomy is widely used because of its advantages of convenience and safety (58). Given the specific characteristics of the vitiligo mouse model, in this study, we achieved ideal sympathetic nerve ablation in the skin through intraperitoneal injection of 6-OHDA. However, this study cannot rule out the possible impact of systemic administration on the systemic immune system.

In conclusion, this study, through clinical and experimental evidence, reveals the mechanism by which the SNS drives vitiligo progression via the NE–adrenergic receptor–fibroblast/keratinocyte axis. This discovery not only deepens our understanding of neuroimmune cross-talk but also provides potential targets for the treatment of autoimmune diseases. The development of therapeutic strategies targeting adrenergic receptors holds promising hope for the treatment of autoimmune diseases.

MATERIALS AND METHODS

Patients and samples

Peripheral blood specimens and skin tissues were collected from patients diagnosed with vitiligo based on clinical manifestations, Wood’s lamp test, and histologic characteristics in the Department of Dermatology, Xijing Hospital of Fourth Military Medical University. Healthy controls were randomly recruited from the physical examination. Patients with any concomitant disease, topical therapy (corticosteroids, calcineurin inhibitors, and phototherapy), or systemic immunomodulatory drugs within 12 weeks were not enrolled in the study. Written informed consent was obtained from each patient and healthy control before enrollment. The clinical features of vitiligo patients and healthy controls are shown in table S1. The research was approved by the Ethics Committee of Xijing Hospital of Fourth Military Medical University (approval number: KY20172030-1) and was conducted by the Declaration of Helsinki.

Immunofluorescence

Deparaffinized 8-μm skin tissue sections were subjected to heat-mediated antigen retrieval with Tris-EDTA buffer (pH 9.0), followed by blocking with 5% goat serum for 1 hour at room temperature. Cultured cells on single-layer glass slides were washed with phosphate buffer solution [phosphate-buffered saline (PBS)] and fixed with 4% paraformaldehyde for 15 min, followed by blocking with 5% goat serum for 30 min. Samples were then incubated with primary antibodies (1:200) against TH (ab152, Abcam, UK), CD8 (ab237709, Abcam, UK), vimentin (ab8978, Abcam, UK), ADRA2A (14266-1-AP, Proteintech, USA), K14 (ab7800, Abcam, UK), ADRB2 (ab182136, Abcam, UK), CXCL9 (ab290643, Abcam, UK), or CXCL10 (10937-1-AP, Proteintech, USA) overnight at 4°C. After washing with PBS, sections or cells were incubated in secondary antibody for 1 hour at room temperature in the dark. After washing with PBS, the sections or cells were sealed with fluoroshield with 4′,6-diamidino-2-phenylindole (DAPI; F6057, Sigma-Aldrich, US). The image collection was performed using FV-1000/ES laser confocal microscopy (Olympus, Tokyo, Japan).

Cell culture and treatments

HaCaT cells were purchased from KeyGEN Biotech (KGG5572-1, Nanjing, China) and cultured in RPMI 1640 medium (Gibco, 11875093) with 10% fetal bovine serum (10099-141, Gibco, USA) and 0.1% penicillin-streptomycin stock solution. Cells were cultured in an atmosphere at 37°C under 5% CO₂. HaCaT cells were transfected with ADRB2 siRNA or control siRNA (Tsingke, Shanghai, China) with Lipofectamine 3000 (L3000015, Invitrogen, USA) following the manufacturer’s protocol. Treating HaCaT cells with 5 μM NE (HY-13715, MedChemExpress, Monmouth Junction, NJ, USA) alone or in combination with 1 μM ICI (HY-13951, MedChemExpress, Monmouth Junction, NJ, USA) for a duration of 48 hours. BJ cells (GNHu49, National Collection of Authenticated Cell Cultures, China) were cultured in Dulbecco’s Modified Eagle Medium (10566016, Gibco, USA) supplemented with 10% fetal bovine serum. Cells were cultured in a humidified atmosphere with 5% CO₂ at 37°C. siRNA targeting ADRA2A (Tsingke, Shanghai, China) was transfected into BJ cells by using Lipofectamine 3000 according to the manufacturer’s recommendations. Treating BJ with 5 μM NE alone or in combination with 10 nM aposcopolamine (HY-N8728, MedChemExpress, Monmouth Junction, NJ, USA) for a duration of 48 hours. For experiments, cultured supernatants were collected for ELISA, and the remaining cells were processed for RNA or protein at the indicated times.

ELISA analysis

ELISA analysis on serum samples, skin samples, and cell culture supernatants were performed using the NE ELISA Kit (E-EL-0047c, Elabscience, Wuhan, China), Human CXCL9 ELISA Kit (EHC114.96, Neobioscience Technology Co, Ltd., China), Human CXCL10 ELISA Kit (EHC157.96, Neobioscience Technology Co, Ltd., China), Human IL-6 ELISA Kit (EHC007.96, Neobioscience Technology Co, Ltd., China), Human IL-15 ELISA Kit (EHC013.96, Neobioscience Technology Co, Ltd., China), Mouse CXCL9 ELISA Kit (E-EL-M3077, Elabscience, Wuhan, China), Mouse CXCL10 ELISA Kit (E-EL-M0021, Elabscience, Wuhan, China), Mouse IL-6 ELISA Kit (EMC004.96, Neobioscience Technology Co, Ltd., China), and Mouse IL-15 ELISA Kit (EMC126.96, Neobioscience Technology Co, Ltd., China) following the manufacturer’s instructions. The absorbance at 450 nm was measured with a plate reader (Bio-Rad, Hercules, USA).

qRT-PCR

Total RNA was extracted using a TRIzol reagent (Invitrogen, 15596018). RNA was quantified and reverse transcribed into cDNA using a PrimeScript RT reagent kit (TaKaRa, AK4301). qRT-PCR was performed in a Bio-Rad Multicolor Real-time PCR Detection System (Bio-Rad, iQTM5) with SYBR Premix Ex Taq (TaKaRa, AKA1008) to determine mRNA expression. The primers used in this study are listed in table S2. Relative quantification was performed according to the ΔΔCT method, and results were expressed in the linear form using the formula 2^−ΔΔCT. β-Actin mRNA was used as an internal control.

Western blotting

After being washed with PBS, the cells were lysed in RIPA lysis buffer (P0013C, Beyotime, China) with protease inhibitor at 4°C for 20 min. The supernatant was collected by centrifugation to extract total cellular protein. Protein concentrations were detected using the BCA Protein Assay Kit (23227, Thermo Fisher Scientific, USA). Equal amounts of proteins were loaded and separated by 10% SDS–polyacrylamide gel electrophoresis (Bio-Rad, USA) and transferred to polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked with 5% nonfat milk at room temperature for 1 hour and then incubated with primary antibodies against ADRA2A (ab85570, Abcam, USA), ADRB2 (ab182136, Abcam, UK), and β-actin (8H10D10) (3700, Cell Signaling Technology, USA) at 4°C overnight. After washing with tris-buffered saline with Tween 20 three times, the membranes were incubated with corresponding secondary antibodies (Goat Anti-Rabbit IgG Antibody, Peroxidase Conjugated, AP132P, Sigma-Aldrich, USA; Goat Anti-Mouse IgG Antibody, Peroxidase Conjugated, AP124P, Sigma-Aldrich, USA) at room temperature for 1 hour. The protein bands were detected with the Western blotting detection system (871BRO7308, Bio-Rad, USA).

Induction and administration of vitiligo in mice

All animal experiments were reviewed and approved by the Ethics Committee of Animal Care of the Fourth Military Medical University (approval number: KY20243154-1). As previously reported in the literature, 8-week-old female C57BL/6 mice were inoculated intradermally in the central dorsal skin with 2 × 10⁵ B16F10 cells on day 0 and then treated with anti-CD4 monoclonal antibody (Clone GK1.5, Bio X cell, USA) intraperitoneally on days 4 and 10 to eliminate T_reg cells (23). Primary tumors of the skin were surgically excised on day 12. For chemical sympathectomy, 6-OHDA (162957, Sigma-Aldrich) solution was freshly prepared by dissolving in 0.1% ascorbic acid (HY-B0166R, MedChemExpress, Monmouth Junction, NJ, USA) in 0.9% sterile NaCl. 6-OHDA (100 mg/kg; body weight) was injected intraperitoneally three times per week. For supplemental NE, NE solution was prepared by dissolving in 0.9% sterile NaCl. NE was injected intraperitoneally at 1.5 mg/kg (body weight) three times per week. Control animals were injected with an equal volume of vehicle (0.1% ascorbic acid in 0.9% sterile NaCl). The extent of depigmentation of mouse tails (ventral and dorsal) was objectively quantified every 2 weeks using ImageJ software, as previously described (images of pigmented areas were converted to black, and images of depigmented areas were converted to white) (59).

Mouse sample collection

Mice were anesthetized with isoflurane, blood was collected from the eye socket, and serum was separated by centrifugation. Tail skin was collected from anesthetized mice for whole-mount staining, qRT-PCR, ELISA, and flow cytometry, followed by gauze pressure dressing of the wound and application of styptic powder to stop bleeding. Inguinal lymph nodes and spleen were taken for flow cytometry from the euthanized mice.

Whole-mount staining and imaging of mouse tail epidermis

Hair was removed using depilatory cream, and the tail skin was obtained after removing the mouse’s tailbone. Tail skin was then collected, flattened, placed in 20 mM EDTA solution, and incubated at 37°C for 1.5 hours with shaking. The dermis and epidermis were then carefully separated, and sebaceous glands were removed from the epidermis under the stereomicroscope (M205 FA, Leica). The epidermis was then sequentially fixed in 4% paraformaldehyde for 8 min, washed in PBS for 3 × 15 min, permeabilized in 0.3% H₂O₂ for 20 min at −20°C, and washed in PBS for 3 × 15 min. Blocked for 2 hours in a solution of 1% bovine serum albumin, 2% donkey serum, and 0.3% Triton X-100 in PBS. Samples were then incubated with primary antibodies against CD8α (ab22378, 1:400, Abcam, UK), and Melan-A (ab210546, 1:400, Abcam, UK) overnight at 4°C. After being washed with PBS, samples were incubated in secondary antibody (Cy3-donkey anti-rat IgG, 712-165-150, 1:1000, Jackson ImmunoResearch, USA; Alexa Fluor 647-donkey anti-rabbit IgG, 711-605-152, 1:1000, Jackson ImmunoResearch, USA; DAPI, F6057, 1:1000, Sigma-Aldrich, USA) for 2 hours at room temperature in the dark. After washing with PBS, samples were sealed with 50% glycerol. Whole-mount samples were imaged as Z-stacks on a confocal microscope (LSM 880, Carl Zeiss, USA). Microscopy data were analyzed by Imaris software (Bitplane, Oxford, UK) and R Studio to quantify the cell distribution and density.

Flow cytometry

The tail skin, inguinal lymph nodes, and spleen of mice were obtained to prepare the single-cell suspension. Peripheral blood mononuclear cells derived from the patients’ peripheral blood were isolated using Ficoll-Hypaque density gradient centrifugation (Dakewei, Shenzhen, China). Cells were collected, washed, and then stained at 4°C in the dark with anti-mouse CD45 (FITC, BioLegend), anti-mouse CD8α (APC/Cy7, BioLegend), anti-mouse CD4 (PERCP, BioLegend), anti-mouse CD25 (APC, BioLegend), anti-mouse FOXP3 (PE, BioLegend), anti-mouse CD11c (APC, BioLegend), anti-mouse MHC-II (PE, BioLegend), anti-human CD8α (APC, BioLegend), IFN-γ (BV605, BioLegend), and Granzyme B (PE, BioLegend). Subsequently, the cells were washed, fixed, and analyzed using flow cytometry. The data were collected on the Cytomics FC 500 flow cytometer (Beckman Coulter, USA) and then analyzed with FlowJo version 10 software (Tree Star Inc.).

Statistical analysis

Statistical data analysis was performed using GraphPad Prism version 9.0 software (GraphPad Software). Each experiment was biologically repeated at least three times. All summary results are presented as mean ± SD. To calculate the P values, the Student t test was performed for two-group comparisons, and one-way analysis of variance (ANOVA) or two-way ANOVA was utilized for three or more groups. Associations between serum NE levels and clinical characteristics in patients with vitiligo were determined by Spearman correlation analysis. P values less than 0.05 were considered statistically significant (^*P < 0.05, ^**P < 0.01, and ^***P < 0.001).

Supplementary Materials

This PDF file includes:

Figs. S1 to S5

Tables S1 and S2