The Calcium Channel Inhibitor Nimodipine Shapes the Uveitogenic T Cells and Protects Mice from Experimental Autoimmune Uveitis through the p38–MAPK Signaling Pathway

Autoimmune uveitis (AU) is one of the sight-threatening ocular inflammatory disorders characterized by massive retinal vascular leakage, inflamed lesions, and macular edema (1, 2). Timely suppression of inflammation and prevention of recurrence is essential to avoid irreversible tissue damage and permanent vision loss. The proliferation, activation, and differentiation of the effector T cells (T_effs) contribute to ocular impairments by secreting inflammatory cytokines such as IL-17A, IFN-γ, and TNF-α (3, 4). Regulation of the T cells is a crucial therapeutic strategy for AU. Calcium is one of the most important second messengers maintaining the physiological and biochemical processes, such as muscle contraction and neurotransmitter release, in many types of cells. Recently, lymphocytes, including T cells, have been demonstrated to express Ca²⁺ permeable channels and transporters, and the activation of TCRs followed by calcium entry via calcium channels is important for energy metabolism, proliferation, activation, and differentiation of T cells (5). The altered intracellular Ca²⁺ concentration regulates T cells function and is related to various autoimmune or inflammatory diseases (6). Targeted modulation of Ca²⁺ channels or Ca²⁺-conducted TCR-activation signals might have an anti-inflammatory effect.

As an l-type (long-lasting) voltage-activated Ca2⁺ (CaV) channels antagonist, nimodipine can inhibit contractions of vascular smooth muscle and often can be used to lower blood pressure with favorable clinical tolerability and safety. Studies have reported that nimodipine demonstrates positive effects in CNS diseases of patients and animal models. It has been approved for the prevention of vasospasms in subarachnoid hemorrhage, neuroprotection, and remyelination in multiple sclerosis (7–10). The protective properties of nimodipine are presumed to be associated with the modulation of calcium homeostasis and prevention of intracellular calcium overload. However, the underlying molecular mechanism is still elusive. In vitro studies suggest that l-type calcium channels played a fundamental role in the induction and/or proliferation of reactive astrocytes and the inhibition of microglial activation (11, 12). However, whether nimodipine could regulate lymphocytes, especially T cells remains to be uncovered.

As described above, calcium channels may be implicated in signal transduction of lymphocytes and play a potential role in shaping the immune response. Based on this report, we assessed the potential influence of l-type Ca²⁺ channels inhibitor nimodipine therapy on autoimmune inflammation in experimental AU (EAU) mice.

Female wild type C57BL/6J mice (6–8 wk old) were purchased from Guangzhou Animal Testing Center and maintained in specific pathogen–free conditions with a 12 h light–dark cycle with stable temperature (23 ± 2°C) and humidity (55 ± 10%). All animal experiments were performed according to a protocol approved by the Institutional Animal Care and Use Committee of Zhongshan Ophthalmic Center, Sun Yat-sen University, and all procedures were performed in compliance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.

Human IRBP_1–20 (GPTHLFQPSLVLDMAKVLLD, Shanghai Shengong, Shanghai, China) was emulsified with CFA containing 5 mg/ml heat-denatured Mycobacterium tuberculosis (Chondrex, Seattle, WA) in a 1:1 volume ratio (v/v). C57BL/6J mice were immunized via s.c. injection of 200 µl of emulsion (IRBP_1–20 200 µg/mouse) at the base of the tail and two thighs; 200 ng of pertussis toxin (List Biological Laboratories, Campbell, CA) was injected i.p. in each mouse on days 0 and 2 after immunization.

T cells isolated from the draining lymph nodes (DLNs) and spleen of EAU mice (day 14) were stimulated by IRBP_1–20 (20 μg/ml) for 72 h. The cells were washed with PBS three times. Then, C57BL/6J mice were injected with IRBP_1–20–specific CD4⁺ T cells (20 million living cells/mice) through the tail vein.

Nimodipine (chemical abstract service number [CAS]: 66085-59-4, Selleck Chemicals, Houston, TX) was dissolved in the vehicle solution (2% dimethyl sulfoxide, 5% Tween 80, 10% polyethene glycol, and PBS) and kept in the dark. Mice were injected with nimodipine daily (10 mg/kg), as previously reported, starting on the same day of immunization till sacrifice. The vehicle solution without nimodipine was injected as a control group (vehicle) with the same protocol.

The photos of the fundus (fundus camera: Phoenix Technology, Campbell, CA) were taken for evaluating retinal inflammation on day 21. Eyes were dissected, fixed, and stained with H&E to evaluate the pathological score. The clinical and pathological score was graded on a scale from 0 to 4 according to the previously described criteria (13).

For in vitro assays, the cells were isolated from the DLNs and spleen of C57BL/6J mice on day 21 after immunization. Intraocular infiltrated cells were isolated from the eyes of EAU mice as described previously (14). The ocular tissues were minced and dispersed in RPMI-1640 medium containing collagenase D (1 mg/ml, Roche, Basel, Switzerland) and DNase I (100 µg/ml, Sigma-Aldrich) and incubated for 15 min at 37°C. The samples were then filtered through a 70-µm disposable sieve, washed, and resuspended in a culture medium. Flow cytometry was used for the analysis of naive CD4⁺ T (CD4⁺CD62L⁺CD44⁻), Th1 (CD4⁺IFN-γ⁺), Th17 (CD4⁺IL-17A⁺), and regulatory T cells (T_regs) (CD4⁺CD25⁺Foxp3⁺).

The T cell recall assays were performed on day 21 after immunization. The cells were seeded into 96-well plates at a density of 2 × 10⁵ cells per well and cultured in complete RPMI-1640 medium (with 10% FBS, 1% penicillin–streptomycin, 1% sodium pyruvate, and 0.1% β-mercaptoethanol) supplemented with 20 µg/ml hIRBP_1–20 at 37°C and 5% CO₂ and treated with nimodipine (CAS no. 66085-59-4, Selleck Chemicals) at different concentrations (0, 10, 20, 40, or 80 µM). After 72 h, the cells were harvested for intracellular cytokine analysis by flow cytometry. The IFN-γ, IL-17A, and TNF-α concentrations in the cell culture supernatants were determined using ELISA kits (Invitrogen, Carlsbad, CA).

Naive CD4⁺ T cells were isolated from DLNs and spleen with EasySepMouse Naive CD4⁺ T Cell Isolation Kit (STEMCELL Technologies, Vancouver, BC, Canada) and then cultured in 96-well plates at a density of 2 × 10⁵ cells/well adding 1 × 10⁵/well anti-CD3/CD28 beads (Invitrogen), both with and without nimodipine (0, 10, 20, or 40 µM). The cultures were also supplemented with Th1 differentiation condition (ImmunoCult mouse Th1 differentiation supplement, STEMCELL Technologies, Vancouver, BC, Canada) for Th1 conversion. The cells were harvested for flow cytometry assay of CD69 expression after 6 h culture and intracellular cytokine of Th1 cells after 4 d, respectively.

The cells were first labeled with CFSE (BioLegend, San Diego, CA), and then the CD3⁺ T cells were obtained using Dynabeads Untouched Mouse T Cells Kit (Invitrogen). The labeled CD3⁺ T cells were cultured in 96-well plates at a density of 2 × 10⁵ cells per well for 4 d, adding 1 × 10⁵/well anti-CD3/CD28 beads both with and without nimodipine (0, 10, 20, or 40 µM).

The identification of reactive oxygen species (ROS) by using oxidized 2-7-dichlorodihydro-fluoresceindiacetate by flow cytometry was performed as previously reported (15). The cells were harvested and incubated with 2-7-dichlorodihydro-fluoresceindiacetate for 20–30 min. The cells were washed with PBS three times before the assay. The accumulation of dichlorofluorescein in cells was measured by flow cytometry.

The peripheral venous blood (10 ml) of five patients with active Behcet uveitis were collected into a heparinized tube. The PBMCs were isolated immediately by density gradient centrifugation (Ficoll-Hypaque; Pharmacia Biotech, Shanghai, China). Written informed consent was collected from each patient, the study was performed in accordance with the tenets of the Declaration of Helsinki, and institutional review board approval was obtained. Fresh PBMCs (2 × 10⁵ cells per well) were added to 96-well plates and incubated with LPS (100 ng/ml) and nimodipine at concentrations of 0, 20, 40, and 80 µM for 72 h. The inflammatory cells were analyzed by flow cytometry.

The CD3⁺ T cells were isolated from DLNs of EAU mice both with and without nimodipine treatment (n = 3, each group). The total RNAs were isolated with Direct-zol RNA MicroPrep kits (Zymo Research, R2062) processed. The total RNA was processed by the Yale Center for Genome Analysis using the Ribo-Zero rRNA Removal Kit, and the libraries were constructed and subjected to standard Illumina HiSeq2000 sequencing and obtained >40 million reads for each sample.

The transcripts were analyzed by R (4.0.3). The R package limma was employed to identify the differentially expressed genes (DEGs) with a fold change >2 and p value <0.05. The statistical significance was determined following the Hochberg–Benjamini method.

The genes up- or downregulated were assigned biological functions according to the Database for Annotation, Visualization, and Integrated Discovery. The enrichment score >1.0 and p value <0.05 were set for the identification of gene ontology (GO) terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) was applied for pathway enrichment analyses, and p < 0.05 was set as the cutoff value.

The gene set variation analyses (GSVA) were performed for the analysis of the gene set enrichment, and the cutoff value was set at enrichment score change >1.0 and p value <0.05. The R package GSVA was employed for GSVA.

The cells were incubated with Fc block (clone 2.4G2, Bio X Cell) and stained with the following Abs from BioLegend: anti-CD45 (BV510), anti-CD3 (BV421), anti-CD4 (Percp-cy5.5), anti-CD25 (PE-cy7), anti-CD44 (allophycocyanin), anti-CD62L (FITC), and anti-CD69 (PE). For intracellular cytokine staining, the cells were pulsed with PMA (50 ng/ml; Sigma), ionomycin (500 ng/ml; Sigma), and brefeldin A (1 µg/ml; Sigma) for 4 h. After further fixation and permeabilization, the intracellular cytokine staining for IFN-γ (BV786), IL-17A (BV650), and Foxp3 (FITC) was performed. For p-p38 (PE) staining, the cells were fixed with 4% paraformaldehyde and permeabilized with methanol at −20°C. The stained cells were collected on an LSR Fortessa (BD Biosciences). Data were analyzed using FlowJo software 10.0 (Tree Star, Ashland, OR).

The retinal tissues were isolated from the EAU treated both with and without nimodipine. The total RNA was isolated using the RNeasy Mini Kit (QIAGEN, Valencia, CA), and cDNA was synthesized using the PrimeScript RT Master Mix (Perfect Real Time, Takara Bio, Shiga, Japan). Real-time quantitative PCR was performed using SYBR Premix Ex Taq II (Takara Bio), according to the manufacturer’s instructions. The relative expression levels of IL-17A and IFN-γ were analyzed by the ΔΔCT method.

The Student t test (for parametric data) or Mann–Whitney U test (for nonparametric data) was used for the two-group comparisons. A p value < 0.05 was considered statistically significant. Data are displayed as mean ± SD. The experiments were repeated no fewer than three times.

To determine the therapeutic effect of nimodipine on EAU, nimodipine (10 mg/kg) or vehicle were i.p. injected into the mice daily following immunization. No abnormality such as weight loss, lethargy, or depilation was observed in any of the mice. On day 21, the fundus was photographed to evaluate the clinical score of retinal inflammation. The EAU mice treated with vehicle developed severe uveitis with higher clinical scores evident as the swollen retina, tortuous and dilated vessels, and patchy inflammatory infiltration. The EAU mice treated with nimodipine exhibited an almost normal retina (Fig. 1A). The eyeball sections were prepared for pathological evaluation. Histopathological manifestations were consistent with clinical changes, and nimodipine treatment decreased the retina folds, retinal lesions, and inflammatory cell infiltration (Fig. 1B).

The T_effs, especially Th17 and Th1 cells, contribute to the autoimmune inflammatory damage. Therefore, the effect of nimodipine on the frequency of T_effs in the uveitic eyes was investigated. Nimodipine prevented the CD4⁺ T cells infiltration into the eyes of EAU mice and decreased the frequencies of Th17 and Th1 significantly (Fig. 1C–E). In the meantime, the inflammatory genes in the eyes were measured using quantitative PCR. Significantly decreased mRNA expressions of IL-17A and IFN-γ were detected in the nimodipine-treated group compared with the vehicle group (Fig. 1F, 1G). Our results demonstrated that nimodipine protects the EAU mice from retinal inflammation by inhibiting local inflammatory cells infiltration and inflammatory gene expression.

Naive T cells can be activated and differentiated into T_effs and T_regs under different polarizing conditions. T_effs, including Th1 and Th17 cells, produce proinflammatory cytokines to initiate tissue inflammation and contribute to damage. T_regs, also known as suppressor T cells, can suppress T_effs to maintain tolerance and prevent autoimmune disease. To explore the effect of nimodipine on the systemic immune response, the effect of nimodipine on the frequency of naive T cells, T_effs, and T_regs were investigated in DLNs of EAU. The cells were collected from the EAU mice treated both with and without nimodipine on day 21 after immunization and analyzed by flow cytometry. Compared with the EAU mice treated with vehicle, cells from EAU mice treated with nimodipine exhibited less activation, manifesting as increased naive CD4⁺ T cells (CD4⁺CD62⁺CD44⁻) and decreased memory CD4⁺ T cells (CD4⁺CD62⁻CD44⁺) (Fig. 2A). Reduced Th17/Th1 and upregulated T_regs were detected in the nimodipine-treated group (Fig. 2B, 2C). These results indicated that nimodipine suppresses the systemic immunological response by downregulating the CD4⁺ T activation, decreasing Th1 and Th17 cells, and potentiating the T_reg.

To address whether nimodipine could shape IRBP_1–20–specific T_eff in vitro, we collected lymphocytes from the DLNs of EAU mice and stimulated them with IRBP_1–20 in the presence of nimodipine at different concentrations (0, 20, 40, and 80 µM). Three days later, we analyzed the populations of IRBP_1–20–stimulated CD4⁺ T cells by flow cytometry and the secretion of cytokines in the supernatants by ELISA. The flow cytometry results showed that cell viabilities were similar at the concentration of 0, 20, and 40 µM, whereas at 80 µM, nimodipine decreased the viability of the cells greatly (Fig. 3A). Therefore, we chose 20 and 40 µM as the optimal concentrations for in vitro studies.

Compared with the vehicle group, the proportions of Th17 and Th1 cells were notably lowered by nimodipine in a dose-dependent manner (Fig. 3B, 3C). The reduced release of IL-17A and IFN-γ were also detected in the supernatants of nimodipine-treated cells (Fig. 3E, 3F). Although the expression of TNF-α was also reduced by nimodipine in a dose-dependent manner, there was no statistical difference between the nimodipine and vehicle groups (Fig. 3D, 3G). These results demonstrated that nimodipine could suppress IRBP_1–20–specific immunological responses, which was confirmed by diminished T_eff differentiation and inflammatory cytokine release.

Nimodipine presented a prominent inhibitory effect on T_eff of EAU mice in vivo. To determine the clinical relevance of this finding, we extended the study from mouse cells to human cells. Therefore, we explored whether nimodipine has the potential of shaping human Th17 and Th1 cells, two major immunopathogenic lymphocyte populations in uveitis patients. Five active uveitis patients of Behcet disease (two females and three males) were enrolled in this study with a median age of 32 y old, ranging from 24 to 36. PBMCs from these patients were isolated and stimulated with LPS (100 ng/ml) in the presence of nimodipine at different dosages (0, 20, 40, and 80 µM) for 3 d. The percentages of viable cells were not greatly reduced by nimodipine at concentrations of 20 and 40 µM. However, nimodipine at a concentration of 80 µM increased cell death significantly. Therefore, nimodipine at concentrations of 20 and 40 µM was chosen for the subsequent experiments (Fig. 4A). The expressions of IL-17A, IFN-γ, and TNF-α of CD4⁺ T cells were measured by flow cytometry. The Th17 and Th1 cells were significantly suppressed by nimodipine at concentrations of 20 and 40 µM (Fig. 4B, 4C). The TNF-α expression was not greatly changed (Fig. 4D). Our results suggested that nimodipine can regulate human T cell pathogenicity, offering a potential choice for clinical therapy in future.

Recent advances have revealed that the fundamental processes in T cell biology, such as proliferation, TCR-mediated activation, and Th lineage differentiation, are closely linked to changes in the cellular metabolic programs (16). The dynamic regulation of energy metabolism plays an active role in shaping T cell responses. As both calcium and Ca²⁺ channels are involved in the cellular metabolism and TCR-activated signal (17), we explored whether nimodipine altered the energy metabolism and downstream TCR signal transduction events, such as proliferation, activation, and differentiation of T cells.

The CD3⁺ T cells labeled with CFSE were cultured with anti-CD3/CD28 beads in the presence of nimodipine (0, 10, 20, and 40 µM) for 4 d. The intracellular fluorescence of dichlorofluorescein and CFSE was detected by flow cytometry, which represented the ROS production (an indicator of cell energy metabolism) and proliferation, respectively. The results showed that the metabolism and proliferation of CD4⁺ T cells were reduced by nimodipine (20 and 40 μM) (Fig. 5A, 5B).

Further, the naive CD4⁺ T cells were isolated from DLNs and spleen and then cultured in a 96-well plate with anti-CD3/CD28 beads under Th1 cell differentiation condition with or without nimodipine. The expression of CD69 was assayed by flow cytometry after 6 h of culture. The upregulation of CD69 provided a quantitative measure of T cell activation and occurred within 6–12 h following anti-TCR stimulation. Treating cells with nimodipine resulted in a 20–30% reduction of CD69 in a dose-dependent manner (Fig. 5C). Meanwhile, the addition of nimodipine to the cultures significantly decreased the frequency of IFN-γ–expressing CD4⁺ T cells. Nimodipine inhibited the polarization of Th1 cells (Fig. 5D). Overall, nimodipine could regulate T cell biology, including energy metabolism, proliferation, activation, and differentiation.

Next, to explore the potential molecular mechanism involved in nimodipine-mediated immunoregulation, we performed the RNA sequencing of the isolated CD3⁺ T cells from DLNs of EAU mice both with and without nimodipine treatment (n = 3, each group). The data had been deposited in the National Center for Biotechnology Information Gene Expression Omnibus under accession code GSE183165, which can be accessed in https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE183165. We sought to determine the differences that existed in the T cells of EAU treated with nimodipine and those treated with vehicle. Abundant DEGs were detected between the two groups (Fig. 6A). Enrichment analyses including KEGG and GO showed significant differences in the p38–MAPK signaling pathway and T cell immune responses characterized by cells proliferation, activation, differentiation, and cytokines production. Both the p38–MAPK signaling pathway and T cell immune responses were negatively regulated by nimodipine in the EAU mice (Fig. 6B–D).

To further corroborate whether the p38–MAPK pathway was involved in the mechanism of nimodipine treatment on EAU, the cells were collected from the C57BL/6J mice without immunization and EAU mice both with and without nimodipine at day 21 after immunization and stained for CD4 and p-p38. The flow cytometry results showed that nimodipine treatment significantly downregulated the p38–MAPK signaling by inhibiting the p-p38 expression in the CD4⁺ T cells (Fig. 6E). The inhibition on p-p38 was also verified in vitro. The lymphocytes from DLNs of EAU mice were stimulated with IRBP_1–20 both with and without nimodipine at dosages of 20 and 40 µM for 3-d culture. Nimodipine (20 and 40 µM) treatment downregulated the p38–MAPK signaling in the IRBP_1–20–specific CD4⁺ T cells (Fig. 6F).

In addition, as shown in (Fig. 6G), the flow cytometry results showed that nimodipine suppressed the CD69 expression of the naive CD4⁺ T cells under Th1 polarization condition at 6 h. To determine whether the p38–MAPK pathway signaling mediated the inhibition of nimodipine in T cell activation, asiatic acid (CAS no. 464-92-6, Selleck Chemicals), a reported p38 activator, was added into the culture system, and the CD69 expression was examined. Results showed that asiatic acid (20 µM) led to nimodipine’s inhibition on CD69. To sum up, nimodipine alleviated EAU by regulating T cells through p38–MAPK pathway signaling.

To further prove the inhibitory capacity of nimodipine on the pathogenicity of IRBP_1–20–specific T cells, we performed the adoptive transfer experiment. The spleens and DLNs of EAU mice were isolated to make single cells suspension, which was stimulated by IRBP_1–20 (20 μg/ml), separately, with a vehicle, nimodipine (20 µM), asiatic acid (20 µM), and both nimodipine (20 µM) and asiatic acid (20 µM) for 3 d. Equal numbers of IRBP_1–20–specific T cells pretreated with different conditions were adoptively transferred to the C57BL/6J mice through the tail vein. The mice were evaluated 2 wk later. The photos of the fundus showed mice receiving IRBP_1–20–specific T cells with vehicle, and those with asiatic acid alone developed severe ocular inflammatory features at day 14, including obvious chorioretinal lesions, significant vascular leakage, and vasculitis. However, nimodipine (20 µM)–treated IRBP_1–20–specific T cells failed to induce uveitis, and the protective effect was faded by asiatic acid at 20 µM concentration (Fig. 7A, 7B). IRBP_1–20–specific T cells pretreated with both nimodipine and asiatic acid induced a retinal inflammation with small retinal folds and slight cell infiltration. The results further revealed that p38–MAPK signaling was involved in the nimodipine-mediated effect on pathogenic T cells in EAU.

In the present research, we explored the effect of nimodipine on EAU, a classic animal model representative of human uveitis. We demonstrated that exogenous nimodipine treatment attenuated the severity of EAU mice. Histopathological analysis showed alleviated vasculitis and reduced inflammatory changes in the eye of nimodipine-treated EAU mice. Moreover, nimodipine was proved to be able to regulate T_eff/T_reg balance by suppressing the energy metabolism, proliferation, activation, and Th1 cell differentiation of the T cells. Further, the RNA sequencing and molecular mechanism studies established that nimodipine alleviates EAU by regulating T cells through the p38–MAPK pathway signaling.

Autoreactive CD4⁺ T cells play a major role in the initiation and orchestration of EAU (3, 4). The activated CD4⁺ T cells migrate from the peripheral circulation and lymphatic organs to the retina, where the cascade of inflammatory reactions is initiated by secreted cytokines and chemokines. To mount effective immune responses, stimulation of the TCRs by specific Ags can trigger calcium release and influx via Ca²⁺ channels, and then the intracellular messenger molecules, Ca²⁺ ions, transmit signals from the surface to the nucleus, which activates the transcription factors such as CREB, MEF, and the NF of activated T cell. These transcription factors are important for the cellular metabolism, proliferation, activation, and differentiation of T cells (5, 18, 19). Previous studies presented that the deletion of subunits β3 in mice led to a reduction in Ca²⁺ channels expression and partial inhibition of activation of the T cells. The β-subunit–deficient CD4⁺ T cells displayed defects in cytokine production (IL-2, IL-4, and IFN-γ) (20). Based on the above, it can be inferred those drugs contributing to the calcium homeostasis in the regulation of the functions of T cells may have therapeutic potential in autoimmune and inflammatory diseases.

Nimodipine is an antagonist of the l-type Ca²⁺ channels, which are broadly expressed in the mouse and human cells including T cells. Several decades ago, studies demonstrated the potency of nimodipine in vasodilation and neuroprotection, thereby improving neurologic function because of intracranial hemorrhage and protecting the retina during retinal ischemia–reperfusion (8, 21–23). The majority of studies have focused on subarachnoid hemorrhage and cerebral vasospasm, but few studies exploited the anti-inflammatory properties, especially those of nimodipine, on the T cells. Sanz et al. (24) showed that nimodipine inhibited the Aβ-stimulated IL-1β synthesis and dose-dependent release from the primary microglia in Alzheimer disease. Zamora et al. (25) reported that the production of proinflammatory cytokines such as TNF-α, IL-1β, and TGF-β1 were significantly decreased in the brain by demyelination because of nimodipine treatment. A retrospective clinical study found that nimodipine could effectively reduce inflammatory cytokines in patients of hypertensive cerebral vasospasm compared with amlodipine (9). However, the regulation of T cell biology and the molecular mechanism of anti-inflammatory and immunoregulatory properties of nimodipine has not been well determined. To the best of our knowledge, this is the first study to attempt to explore the possibility of using the Ca²⁺ channel inhibitor, nimodipine, to alleviate EAU by regulating T cell biology. Because EAU is initiated and orchestrated by autoreactive CD4⁺ T cells, we focused on the CD4⁺ T cells in both in vivo and in vitro experiments. The analysis of lymphocytes in lymph nodes of EAU mice showed that nimodipine decreased the T_effs (both Th17 and Th1) differentiation, suppressed the activation of naive CD4⁺ T cells, and resumed the balance of T_eff/T_reg both locally and systemically protecting the mice from EAU. Wang et al. (26) reported that mice deficient for CaV3.1 were resistant to the induction of experimental autoimmune encephalomyelitis and showed reduced productions of the GM-CSF by the CNS-infiltrating Th1 and Th17 cells. Similarly, results from our study support these findings that the inhibition of Ca²⁺ channels by nimodipine could regulate the balance of T_eff/T_reg and protect the mice from uveitis.

In vitro, nimodipine reduced the IL-17A and IFN-γ expression in IRBP_1–20–specific CD4⁺ T cells of EAU mice. Interestingly, the inhibitory effect of nimodipine on CD4⁺ T cells was also proved in the LPS-stimulated PBMCs of uveitis patients, which laid a theoretical foundation for future clinical application. The subsequent study revealed that the inhibition of Ca²⁺ signals by nimodipine was accompanied by the reduced production of ROS of energy metabolism, proliferation, CD69 expression of early activation, and Th1 cell differentiation of T cells. The results were consistent with certain previous studies. The CaV1.4-deficient mice showed a reduction in the levels of mature thymocytes and peripheral CD4⁺ and CD8⁺ T cells, suggesting the role of CaV1.4 in promoting T cell survival and expansion (27). However, Ingwersen et al. (28) demonstrated that nimodipine improved the clinical outcome in experimental autoimmune encephalomyelitis but did not alter the lymphocyte subtypes (proportion of CD4⁺, CD8⁺, and CD19⁺ cells), proliferation, and T cell activation markers of lymphocytes in vitro. In their study, the proportion of CD4⁺ T cells was not altered by nimodipine, but the proportions of CD4⁺ T cell subsets, including Th1/Th17, T_reg, and naive T cells, had not been subdivided and analyzed. As for the activation pattern of lymphocytes, they used cells from immunized mice, which were already mostly activated. Research from Ingwersen et al. (28) demonstrated that nimodipine could not reverse the activated lymphocytes. In our study, we evaluated the effect of nimodipine on naive T cells polarization into activated T cells and proved nimodipine could inhibit the activation. Taken together, we and Ingwersen et al. (28) investigated different aspects of T cells influenced by nimodipine.

Although nimodipine can mitigate EAU, the underlying mechanisms remain to be fully elucidated. Then, we explored the mechanism involved in TCR-activated Ca²⁺-related signals in the T cells of EAU mice. The RNA sequencing and functional enrichment analysis of T cells hinted that nimodipine shaped the uveitogenic T cells through the p38–MAPK signaling pathway. The family of MAPKs, including p38, ERK, and JNK, regulate a large number of extracellular signals involving cytokine secretion, proliferation, survival, and apoptosis (29). The p38–MAPK signals have been reported to be potentiated in the mast cell–mediated inflammation (30) and the blockade of p38 attenuated IL-1β, TNF-α, and IL-6 in astrocytoma and microglial cells (31, 32). The p38–MAPK interacted with the l-type CaV channels in multiple ways. Liu et al. (33) reported that TGF-β1 regulated l-type CaV channels through a mechanism dependent on MEK, JNK1/2, and p38–MAPK signal pathways in the cortical neurons. The l-type CaV channels upregulated p38–MAPK activation in CXCL12-induced neuronal death (34). Nimodipine can protect the retina through the downregulation of p38–MAPK and caspase-3 expression in a mice retinal ischemia–reperfusion model (21). However, all of the above experiments were conducted using neurocytes. The alteration of p38–MAPK signals of T cells because of nimodipine has not been reported yet.

The p38 activation is tightly regulated by phosphorylation/dephosphorylation events. Thus, we assayed the expression of p-p38 by flow cytometry and found that cells isolated from EAU showed higher p38 phosphorylation compared with the cells from the nonimmunized mice, whereas the nimodipine treatment prevented the p38 from phosphorylation. This effect was also found in the cultured IRBP_1–20–specific CD4⁺ T cells in a dose-dependent manner. Moreover, the inhibition of nimodipine on the CD4⁺ T cells could be rescued by p38 activator asiatic acid. The adoptive transfer experiments demonstrated partial rescue of nimodipine by asiatic acid in the clinical score of recipient mice, presenting as mild inflammation of retina. The mild inflammatory changes like tortuous and dilated vessels could not be detected in histopathological examination occasionally, which contributed to no significant difference in pathological scores between nimodipine group and nimodipine plus asiatic acid group. To our knowledge, this is the first study to demonstrate that the nimodipine shaped the T cells to immune response and reduced inflammation through the p38–MAPK signaling pathway.

Nimodipine has been proven to dilate the cerebral blood vessels and increase the cerebral blood flow in animals and humans. Preliminary findings reveal its potential benefit for the treatment of a wide range of cerebrovascular disorders and an inhibitory effect on age-related neurocytes degeneration (7, 8, 22). As nimodipine is commonly used and well tolerated clinically, the successful application of nimodipine in the EAU mice may contribute to other autoimmune diseases. Several comorbidities such as hypertension, coronary heart disease, and cerebral disorders often concur in patients with autoimmune diseases, including multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, and uveitis, limiting the choice of medication and compromising the life quality. Thus, drugs like nimodipine that possess both anti-inflammatory and comorbidity alleviation properties may benefit these patients the most. However, for autoimmune diseases and patients without cardiovascular and cerebrovascular disorders, the indication of nimodipine is considered off label at present. Clinicians and patients should collaboratively assess the risks and benefits before initiating treatment. The efficacy and safety of nimodipine in the treatment of these patients needs clinical validation, which calls for randomized controlled trials.

In summary, our study was the first, to our knowledge, to demonstrate the therapeutic effects of nimodipine on experimental uveitis. Nimodipine alleviated the local and systemic inflammation significantly and shaped the uveitogenic T cells through the p38–MAPK signaling pathway. These results enriched our understanding of the pathogenic mechanism of EAU and expanded the potential clinical use of Ca²⁺ channel inhibitors. It provided a new promising option for the treatment of AU, further promoting the value of nimodipine in the therapy of other autoimmune diseases especially those with cardio–cerebrovascular comorbidities.

The authors have no financial conflicts of interest.