2022-Differential cytokine profiles produced by anti-epileptic drug re-exposure of peripheral blood mononuclear cells derived from severe anti-epileptic drug patients and non- allergic controls

Cytokine 157 (2022) 155951 Contents lists available at ScienceDirect Cytokine journal homepage: www.elsevier.com/locate/cytokine Differential cytokine profiles produced by anti-epileptic drug re-exposure of peripheral blood mononuclear cells derived from severe anti-epileptic drug patients and non-allergic controls Yuttana Srinoulprasert a,*, Pongsathorn Kumkamthornkul a, Papapit Tuchinda b, Sattawut Wongwiangjunt c, Sith Sathornsumetee c, Kowit Jongjaroenprasert d, Kanokvalai Kulthanan b a Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand b Department of Dermatology, Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand c Department of Internal Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand d Adverse Drug Reaction Unit, Pharmacy Department, Siriraj Hospital, Mahidol University, Thailand ARTICLE INFO ABSTRACT Keywords: Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) and drug reactions with eosinophilia and Anti-epileptic drug hypersensitivity systemic symptoms (DRESS) are the most common severe cutaneous adverse drug reactions (SCARs). Anti- Cytokines epileptic drugs are one of the most common drugs causing SCARs. Cytokine profiles of SCARs during culprit DRESS drug exposure have never been characterized. This study aimed to identify cytokine patterns between SCARs and SJS/TEN non-SCARs in epilepsy patients and the patterns of DRESS and SJS/TEN. Epilepsy patients that showed allergic Epilepsy responses to anti-epileptic drugs that manifested as SJS/TEN or DRESS were recruited. Epilepsy patients with no drug allergy symptoms and healthy people were also recruited as control groups. Peripheral blood mononuclear cells (PBMCs) were isolated and co-cultured with assigned anti-epileptic drugs according to the lymphocyte transformation test (LTT). LTT and measurement of cytokine levels in supernatants were performed on day six of cell cultivation. This study identified different cytokine expression patterns between SCAR and non-SCAR in epilepsy patients. Significant levels of IL-10, IL-12, IL-17, and GM-CSF were detected in non-SCAR epilepsy. However, the levels of IL-2, IL-5, IL-13, and IFN-gamma were significantly higher in supernatants of PBMCs of DRESS cultivated with AEDs relative to those of SJS/TEN. These cytokine levels were positively correlated with the cell proliferation index. Production of IL-5 and IL-13 was a unique characteristic of DRESS PBMCs. This study was the first to demonstrate distinct differences in cytokine levels between SCAR and non-SCAR PBMCs in ep­ ilepsy, which could help explain the immune-pathomechanism of drug hypersensitivity in SCARs. Different patterns of cytokine production and cell proliferation between DRESS and SJS/TEN in AED hypersensitivity were also demonstrated. Production of IL-5 and IL-13 might be a promising marker to define drug hypersensitivity in DRESS. 1. Introduction non-immediate reaction, the immunopathomechanisms of which are described by type I and type II-IV hypersensitivity reactions, respec­ Immune-mediated type B adverse drug reactions (ADR), alterna­ tively. Among non-immediate DHR, severe cutaneous adverse drug re­ tively termed drug hypersensitivity reactions (DHR), are considered to actions (SCARs), consisting of Stevens-Johnson syndrome (SJS)/toxic be a serious public health problem due their life-threatening nature. This epidermal necrolysis (TEN), acute generalized exanthematous pustulosis type of ADR is a major contributor to hospitalization and mortality (AGEP), and drug reactions with eosinophilia and systemic symptoms throughout the world. DHR can manifest itself as either an immediate or (DRESS), are of the greatest concern because their morbidity and * Corresponding author at: Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, 2 Wanglang Rd., Bangkok-noi, Bangkok 10700, Thailand. E-mail address: [email protected] (Y. Srinoulprasert). https://doi.org/10.1016/j.cyto.2022.155951 Received 3 February 2022; Received in revised form 1 June 2022; Accepted 20 June 2022 Available online 27 June 2022 1043-4666/© 2022 Elsevier Ltd. All rights reserved.

Y. Srinoulprasert et al. Cytokine 157 (2022) 155951 mortality rates are high [1]. The mechanisms of SCAR have been pro­ (GE Healthcare Biosciences) within 4 h after collection. After washing, posed to involve type IV hypersensitivity reactions, which are mediated viability of PBMCs was estimated by trypan blue exclusion and their by drug-specific T lymphocytes leading to tissue/organ damage [2]. viability >95% was accepted to perform further experiment. The PBMCs were counted and resuspended in R9 culture medium at a cell density of According to a previous report, anti-epileptic drugs (AEDs) are one of 2 × 106 cells per ml. LTT was performed according to the protocol the most common drugs causing SCARs [3]. The incidence of SCARs published by Pichler and colleagues [23,24]. Phenytoin, carbamazepine, induced by AEDs varies from 5 to 78% depending on the type of SCARs, lamotrigine, and valproic acid were purchased as pure substances from and the SCARs can be initiated 1–12 weeks after drug administration Sigma Corporation, USA. The drugs were freshly prepared in appro­ [4]. Phenytoin (PHE) and carbamazepine (CBZ) are aromatic AEDs priate solvents and finally diluted in RPMI-1640 to reach three different commonly associated with SCARs with prevalence risks of 13% and non-toxic concentrations. The PBMCs from each subject were incubated 11%, respectively [5–8]. Valproic acid (VPA), a non-aromatic AED, with the assigned drug (three concentrations) in quintuplicate and could provoke SCARs; however, its occurrence is rare at 1% [5,9,10]. incubated at 37 ◦C under 5% CO2 for 6 days. The culprit drugs and their Although VPA has been safer for epilepsy treatment compared to aro­ concentrations used in this study was shown in Supplementary Table 1. matic AEDs, VPA co-treatment with other AEDs, such as lamotrigine Phytohemagglutinin (PHA) and tetanus toxoid were used as a positive (LTG), can induce hypersensitivity reactions [9]. stimulating control, and R9 culture medium was used as a negative control. Cell proliferation was assessed in terms of 3H-thymidine Many reports have demonstrated that AEDs, especially PHE, CBZ, incorporation that was measured as counts per minute (cpm) on a beta and VPA, can enhance the levels of IL-1, 2, and 6 in sera of both healthy counter. The results were calculated as the stimulation index (SI), which people and epilepsy patients who did not develop adverse reactions is ratio between cpm of PBMCs cultured with culprit drug and cpm of [11–13]. By contrast, cytokine profiles in patients suffering from SCARs PBMCs cultured without culprit drug. were different and depended on the SCARs manifestation. Recall drug exposure can activate culprit drug-specific T cells to elicit immune re­ 2.3. Cytokine detection sponses by releasing a variety of cytokines leading to drug hypersensi­ tivity reactions. Orchestration of the cytokine profile might define the Prior to adding 3H thymidine, 20 micro-liters of supernatant from pattern of skin manifestations [14–16]. In DRESS, exposure to AEDs, each of the quintuplicates were collected and pooled. The pooled su­ such as CBZ, PHE, and LTG for 2–12 weeks, induced drug-specific T cells pernatants were split into three aliquots and frozen at − 80 ◦C. Each to secrete high levels of IL-5 and IFN-γ [16,17]. Additionally, increasing aliquot was thawed and subjected to cytokine profiling using multiplex levels of IL-2 and IFN-γ induced by PHE or CBZ in DRESS were also cytokine detection (Luminex). Th1/Th2 profile Luminex plus IL-6 and reported [13,16,18,19]. Studies measuring cytokine production in sera IL-17 kits were purchased from BioRAD®. Multiplex cytokine detection and blister fluid of SJS/TEN patients revealed increased levels of IL-6, IL- was performed following the manufacturer’s instructions. In brief, the 10, IFN-γ and TNF-α [20,21]. Secretion levels of IL-2, IL-5, and IFN-γ supernatants were incubated with bioplex beads in a round-bottomed were employed as markers for the identification of culprit drugs in the plate at room temperature (RT) for thirty minutes while the plate was SJS/TEN [16,22]. shaken at 800 round per minute (rpm). After incubation, three washes were performed on the plate. Antibodies for detection were added to The expression of these mediators could contribute to skin tissue each well and incubated at room temperature for 30 min on an 800-rpm damage in SCARs patients. However, whether similar patterns of cyto­ shaker at RT. The plate was then washed three times before streptavi­ kine expression occur in healthy people has never been evaluated. din–phycoerythrin solution was added and incubated at RT for 10 min. Therefore, a comparison of the cytokine patterns derived from PBMCs of After the final washing, assay buffer was added to each well, and the affected epilepsy patients and healthy controls (non-epilepsy and epi­ plate was analyzed using a Bio-plex reader. lepsy) upon AED exposure in vitro was conducted to investigate systemic immune reactions. 2.4. Analysis and statistical analysis 2. Materials and methods SI values of LTT were used to identify associations with the levels of cytokine production. It is known that PBMCs induced with culprit drugs 2.1. Volunteer recruitment did not release cytokines in a dose-dependent manner. Our preliminary analysis of cytokine levels from PBMCs stimulated with three different Two groups of patients were recruited in this study: (1) a responders concentrations of anti-epileptic drugs found that the results obtained (R) group, consisting of patients exposed to anti-epileptic drugs that from each individual drug concentration were comparable among three clinically expressed either DRESS or SJS/TEN, and (2) an exposed non- drug concentrations used in this study. In addition, when average responder (ENR) group, consisting of patients exposed to anti-epileptic cytokine levels obtained from all three drug concentrations were used in drugs without any clinical manifestations of a cutaneous drug reac­ the analysis, the results were similar to those obtained from single drug tion. Subjects in the responder group were diagnosed according to concentration analysis. Therefore, we decided to express our data using clinical criteria, which were confirmed by dermatologists. Healthy average cytokine levels throughout this study. Where appropriate, people were recruited as a naïve control. All donors were recruited ac­ parametric or non-parametric analyses were used to evaluate the sig­ cording to ethical procedures and gave informed consent. Causality nificance of correlations found between the cytokine levels from each assessment was performed in every case for the responder group. During group. The significance of correlations between SI values and cytokine recruitment, each donor was confirmed to have no inflammatory con­ levels was determined using the Spearman’s correlation test. All the dition (i.e., fever, clinical illness, or less than 4 weeks after discharge graphs, calculations, and statistical analyses were performed using from hospital). In the case of the responder group, blood samples were GraphPad Prism software version 9.0 for Mac/Windows (GraphPad obtained at least 4 weeks (12 weeks for DRESS), but not more than 5 Software, San Diego, CA, USA). Results were considered statistically years, after the subjects had fully recovered from severe cutaneous re­ significant when the p-value was <0.05. actions. This study was approved by the Siriraj institutional review board, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand (COA NO:Si 557/2013). 2.2. Isolation of PBMCs and lymphocyte transformation test (LTT) Peripheral mononuclear cells (PBMCs) were isolated from heparin­ ized blood by density gradient centrifugation using Ficoll-Paque PLUS 2

Y. Srinoulprasert et al. Cytokine 157 (2022) 155951 3. Results 5.1, and 5.2, respectively, when compared to unstimulated conditions (without AEDs). The former cytokine profile may reveal Th2 predomi­ 3.1. Demographic data of volunteers nant response corresponding to type IVb hypersensitivity mechanism of DRESS. In contrast, the latter cytokine profile may imply Th1 cytotoxic Twenty epilepsy patients (five males and 15 females with a median predominant response related to type IVc hypersensitivity mechanism of age of 40 years), suffering from AED-induced SCARs and 20 epilepsy SJS/TEN. A comparison of the levels of cytokine production from PBMCs patients (12 males and eight females with a median age of 41.5 years), of naïve, ENR, and patients (DRESS and SJS/TEN) under AED stimula­ who had no SCARs upon AED treatment were recruited as the case tion is shown in Fig. 1. The levels of IL-2, IL-4, IL-10, IL-12, IL-17, IFN-γ, group. Healthy people (seven males and 13 females with a median age of TNF-α, and GM-CSF derived from ENR PBMCs were significantly higher 26 years), who had no history of anticonvulsant drug exposure, were than those of naïve PBMCs. Interestingly, significantly higher IL-5 and recruited as the control group. The median age among all groups in this IL-13 levels were observed for DRESS’s PBMCs compared that in ENR’s study was not statistically different. The clinical features and findings of and naïve PBMCs. IL-2 and IFN-γ levels derived from DRESS PBMCs these patients are summarized in Table 1. Peripheral blood mononuclear were significantly higher than those from ENR cells. However, the levels cells (PBMCs) from some volunteers were incubated with more than one of IL-4, IL-10, IL-12, IL-17, GM-CSF, and TNF-α derived from DRESS AED. SCARs cases in this study were composed of DRESS (11/20) and PBMCs were significantly lower than those from ENR PBMCs, as SJS/TEN (9/20). All case and control patients had a normal immune demonstrated in Fig. 1 and Supplementary Figure 6.1. In the case of SJS/ response to PHA and tetanus toxin (data not shown). AEDs used in this TEN, the levels of all cytokines, except for IL-17 and GM-CSF, were study were phenytoin, valproic acid, carbamazepine, lamotrigine, and equivalent to cytokine levels released from naïve PBMCs. Surprisingly, phenobarbital. the levels of IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17, GM-CSF, IFN- γ, and TNF-α from SJS/TEN PBMCs were significantly lower than those Values corresponding to the list of culprit drugs indicate the numbers from ENR and DRESS cells, as shown in Fig. 1 and Supplementary of LTT experiments performed, which did not equal to the numbers of Figure 6.2. cases. DRESS; drug reaction with eosinophilia and systemic symptoms, SJS; Stevens-Johnson syndrome/toxic epidermal necrolysis Levels of multiple cytokines in supernatants of PBMCs derived from healthy individuals (N), exposed non-responders (ENR), and responders 3.2. Differential cytokine profile of PBMCs upon AED re-exposure (DRESS and SJS/TEN), cultured in the presence of culprit drugs. Lumi­ nex kits were used to determine the levels of IL-2, IL-4, IL-5, IL-10, IL-12, The Th1/Th2 cytokine profile and the levels of two inflammatory IL-13, IL-17, GM-CSF, IFN-γ and TNF-α as described in the methods cytokines (IL-6 and IL-17) were used to study the immunological section. *p values <0.05 were statistically significant, NS stands for non- response to AED exposure, as shown in Fig. 1 and Supplementary significance. Figure 7. Many cytokines (IL-2, IL-4, IL-10, IL-12, IL-17, GM-CSF, IFN-g, and TNF-a) were produced at significantly higher levels in ENR PBMCs 3.3. Cytokine and proliferation responses of PBMCs during AED re- re-exposed with AEDs than in healthy controls. As demonstrated in exposure Supplementary Figure 1, the basal levels of nearly all cytokines (except IL-6 and IL-13) produced by ENR PBMCs were significantly higher than T cell responses contribute to type IV hypersensitivity. In addition to those produced by healthy individuals and patients. The increased changes in cytokine production, a proliferation of antigen-specific T amounts of cytokines secreted by ENR PBMCs may indicate Th1/Th2 cells could also play a role. In this study, LTT was employed to study the cytokine responses of memory T cells re-exposed to AEDs. Nevertheless, proliferative response of AED-specific T cells. Association between AEDs had no effect in inducing PBMCs from healthy and ENR partici­ cytokine responses and proliferation of T cells in allergic patients was pants to produce significant levels of all cytokines, as shown in Sup­ evaluated. As shown in Fig. 2, the range of the SI of DRESS PBMCs was plementary Figures 2 and 3. To investigate the responder group in more higher than that of SJS/TEN PBMCs (Fig. 3). Fig. 2c and 2f, respectively, detail, the levels of cytokine production from DRESS and SJS/TEN pa­ reveal a strong correlation (r > 0.80) between the degrees of SI and the tients’ PBMCs re-stimulated with AEDs were also determined. Fig. 1 and levels of IL-5 and IL-13 in DRESS. Proliferation and levels of IL-2, IL-4, Supplementary Figure 4 revealed that AEDs significantly increased the GM-CSF, IFN-g, and TNF-a were shown to have a moderate correlation production of IL-2, IL-5, IL-13, IFN-γ, and TNF-α in DRESS PBMCs. (r = 0.5–0.7) as depicted in Fig. 2a, 2b, 2 h, 2i, and 2j, respectively. Increased IL-5 and IL-13 production by DRESS PBMCs may correlate Statistically, these correlations were significant. However, a significant with eosinophilic manifestations in patients. AEDs exposure led to correlation was not found between other cytokines and proliferation insignificant increases in the levels of IL-4, IL-6, IL-10, IL-12, IL-17, and (Fig. 2d, 2e, and 2 g). Even though there was no significant correlation GM-CSF in DRESS PBMCs and those of IL-2, IL-12, IL-17, and GM-CSF between cytokine production and SI in the SJS/TEN groups, as shown in produced by SJS/TEN PBMCs, as shown in Supplementary Figures 4, Fig. 3, their lower levels of proliferation (SI < 80) followed the trend of lower cytokine production, as indicated in Fig. 1. Table 1 Healthy Exposed- non Responders Characteristics of volunteers. controls responders Levels of IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17, GM-CSF, IFN-α 20 and TNF-α, produced from PBMCs of DRESS patients were further Number of volunteers 20 20 40 analyzed to detect any correlation with cell proliferation (Stimulation Median (years) 26 41.5 5/15 Index; SI). Dashed lines represent the 95% confidence interval best-fit Sex (Male/Female) 7/13 12/8 lines. r represents the correlation statistic, and p represents the p- Number of reactions value. A p-value < 0.05 was considered to be significant. incubated with defined drug 20 10 13 Phenytoin 20 72 Levels of IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17, GM-CSF, IFN-γ, Valproic acid 11 34 and TNF-α,produced from PBMCs of SJS/TEN patients were further Carbamazepine 6 30 analyzed to detect any correlation with cell proliferation (Stimulation Lamotrigine 0 01 Index; SI). Dashed lines represent the 95% confidence interval best-fit Phenobarbital lines. r represents the correlation statistic, and p represents the p- Types of SCARs – – 11 value. A p-value < 0.05 was considered to be significant. SJS/TEN – –9 DRESS 4. Discussion Many medications have been reported to cause SCARs, and AEDs are 3

Y. Srinoulprasert et al. Cytokine 157 (2022) 155951 Fig. 1. Comparison of cytokine levels produced from PBMCs derived from healthy, exposed non-responder individuals, DRESS and SJS/TEN patients cultured with culprit drugs in LTT assays. 4

Y. Srinoulprasert et al. Cytokine 157 (2022) 155951 Fig. 1. (continued). among the most common SCARs causative drugs [24–29]. Three percent Interestingly, the elevated basal levels of cytokines in ENR volun­ of epilepsy patients have cutaneous reactions, and the incidence of ep­ teers might be related to epilepsy patients’ immunological dysfunction, ilepsy patients suffering from SCARs could be up to 4 of every 10,000 the effect of AEDs, or both, as summarized by Godhwani and Bahna. patients [30]. AED hypersensitivity can be potentially dangerous and They reported that high levels of IL-2, IL-6, and TNF-α were detected in life-threatening. The mortality rate of SCARs among epilepsy patients seizure patients even though they did not take any AEDs. An increase in can reach up to 50% [4]. The varying cytokine expression patterns many cytokines (such as IL-2, IL-6, IL-8, and fibroblast growth factors) produced according to epileptic status, AED exposure, and SCARs have induced by treatment with the first-generation AEDs was also reported been reviewed elsewhere [8,31]. Comparison of the cytokine patterns [8]. Nevertheless, our findings might explain why pathology did not among epilepsy patients (SCARs and non-SCARs) and healthy controls develop in the ENR groups. High levels of IL-10 produced by ENR PBMCs upon AED exposure has never been reported. could suppress immune responses as it acts as an immunoregulator. Increases in IL-12, IL-17, IFN-γ, and TNF-α may also enhance the Th1 Basal levels of almost all cytokines produced from PBMCs from response, thereby counteracting the Th2 response (IL-4, IL-5, and IL-13) healthy and ENR individuals in our investigation (excluding IL-6 and in DRESS and Tc (cytotoxicity) in SJS/TEN [31,36]. The cytokine release GM-CSF) were in the same range as those found in the healthy sera pattern from ENR PBMCs re-exposed to AEDs was similar to the Th0-like [32,33]. High levels of IL-6 could be observed due to spontaneous pro­ cytokine pattern released from allergen-specific T cells of non-allergic duction in the ex vivo cultivation [34]. Surprisingly, the high levels of people revealed by Kailaanmaki and colleagues [37]. They reported IL-6 and GM-CSF production that were observed in vitro in this research that allergen-specific T cell clones existed in non-allergic subjects. These might be due to a lack of regulatory factors (such as gamma-globulin clones expressed a majority of Th1/Th0 cytokines. These findings are with metal complex or other regulatory factors) that normally control consistent with this report and other studies showing that CD4+ T cells the production of these cytokines in vivo, as revealed by Cheknev et al. can produce both Th1 and Th2 cytokine subsets (Th0 functional CD4+ T [35]. Despite this, the levels of all cytokines in the supernatants of cells) [38,39]. This phenomenon may explain why antigen-specific T healthy (non-AED-exposure) and ENR (AED-exposure) PBMCs culti­ cells do not provoke hypersensitivity in non-allergic people [37–41]. vated with AEDs were comparable to the baseline levels in supernatants from cultures without AEDs. This result revealed that AEDs had no effect Significant increases in IL-2, IL-5, IL-13, IFN-γ, and TNF-α production on the immunological response in control groups. from DRESS PBMCs compared to healthy and ENR PBMCs indicate a role 5

Y. Srinoulprasert et al. Cytokine 157 (2022) 155951 Fig. 2. Correlation between levels of cytokine production and proliferation of PBMCs from epilepsy patients who have suffered from DRESS. for these cytokines in the immunopathogenesis of DRESS. This finding finding was consistent with the findings of a study by Porebski and was consistent with the finding of Lochmatter and colleagues [16]. colleagues demonstrating a strong correlation between lymphocyte Notably, the levels of these cytokines were correlated with the stimu­ proliferation and IFN-γ production in patients who were allergic to CBZ lation index, suggesting that proliferation and cytokine production (r = 0.71, p < 0.0001) [22]. However, our study did not find signifi­ might be positively associated, as shown in Fig. 2a, 2c, 2f, 2i, and 2j. Our cantly increased production of cytokines from PBMCs of the SJS/TEN 6

Y. Srinoulprasert et al. Cytokine 157 (2022) 155951 Fig. 2. (continued). group, as the basal levels of almost all cytokines tested from the SJS/TEN TEN, indicating that they might help with future research into DRESS group were lower than the ENR and N groups. Aside from IL to 17 and etiology and other complications. Even though the baseline levels of GM-CSF, the levels of other cytokines produced by SJS/TEN’s PBMCs almost all cytokines produced by SJS/TEN PBMCs were lower than those were significantly lower than those produced by naïve PBMCs. One produced by PBMCs of healthy and DRESS, AEDs were able to cause an possible explanation for this finding could be due to immune disorders increase in several cytokines, including IL-2, IL-12, and GM-CSF, in the affecting both innate and adaptive immune reactions in SJS/TEN [5]. supernatant of SJS/TEN PBMCs cultivation. Although their increased production was not statistically significant enough to serve as SJS/TEN- Significant IL-2 production in PBMCs from DRESS but not SJS/TEN specific cytokine indicators, further research looking at Th1 responses in might contribute to lymphocyte proliferation, explaining why the LTT SJS/TEN and the effects of other culprit drugs are needed to clarify this for DRESS is more sensitive than the LTT for SJS/TEN [29]. Further­ issue. Releasing of granulysin and granzyme B from cytotoxic T cells more, considerable IL-4 production in DRESS, but not SJS/TEN, indi­ causing skin disorders was reported and proposed in the diagnostic and cated the presence of Th2-prone pathophysiology in DRESS. culprit drug identification [45,46]. Therefore, further research by our group will focus on the detection of both granulysin and granzyme B and The results of our study are evidence of different patterns of cytokine their use as diagnostic markers for culprit drug identification and production between DRESS and SJS/TEN upon antiepileptic drug re- prognostic makers in SJS/TEN patients. This may increase the sensitivity exposure. They also contribute basic knowledge concerning pathogen­ of LTT or even replace it. esis in DRESS and SJS/TEN. The relatively high levels of IL-5 and IL-13 are phenotype-specific and unique to DRESS [42]. This finding may One of this study’s limitations was the limited sample size for DRESS explain a cause of eosinophilic complications in DRESS patients, apart and SJS/TEN. More research with a more significant number of patients from dermatitis and hepatitis, such as eosinophilic myocarditis [43,44]. may be required. The pattern of cytokine production from DRESS and Both cytokines might serve as representative markers for diagnostic SJS/TEN in this study was derived from AED induction. Cytokine pat­ determination or prognostic parameters, which could be of clinical terns in DRESS and SJS/TEN PBMCs induced with other drugs remain benefit to DRESS patients. Production of IL-5 and IL-13 could be investigated. detected in supernatants of SJS/TEN’s PMBCs cultured with culprit drugs, which is consistent with results of previous studies [16,22,29]. 5. Disclosure However, their production levels were not high enough to be used for diagnostic or prognosis markers. Increased production of IL-2, IL-4, and The funding sponsors had no role in the design of the study, the IFN-γ from PBMCs of DRESS and SJS/TEN patients in our study was also collection, analysis, and interpretation of the data, the writing of the consistent with other findings [22,29]. Our results revealed that the manuscript, or the decision to publish the results. production of these cytokines from DRESS and SJS/TEN PBMCs over­ lapped, suggesting that it might not be phenotype specific. However, the levels of these cytokines were substantially higher in DRESS than in SJS/ 7

Y. Srinoulprasert et al. Cytokine 157 (2022) 155951 Fig. 3. Correlation between levels of cytokine production and proliferation of PBMCs from epilepsy patients who have suffered from SJS/TEN. 6. Data availability T. wrote the manuscript. The datasets generated and analyzed for the present study are Funding available from the corresponding author on request. Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Author contributions THAILAND Y.S. conceived and designed the experiments; P.K. performed the CRediT authorship contribution statement experiments; P.K and Y.S. analyzed the data; P.T., S.W., S.S., and K.K. Yuttana Srinoulprasert: Conceptualization, Methodology, Writing recruited patients; K.J. provided the ADR database, and Y.S., P.K., and P. 8

Y. Srinoulprasert et al. Cytokine 157 (2022) 155951 Fig. 3. (continued). – original draft, Writing – review & editing. Pongsathorn Kumkam­ org/10.1016/j.cyto.2022.155951. thornkul: Investigation, Data curation. Papapit Tuchinda: Resources, Validation, Writing – review & editing. Sattawut Wongwiangjunt: References Resources. Sith Sathornsumetee: Resources. Kowit Jongjar­ oenprasert: Resources. Kanokvalai Kulthanan: Resources, Writing – [1] M. Pirmohamed, P.S. Friedmann, M. Molokhia, Y.K. Loke, C. Smith, E. Phillips, review & editing. L. La Grenade, B. Carleton, M. Papaluca-Amati, P. Demoly, N.H. Shear, Phenotype standardization for immune-mediated drug-induced skin injury, Clin. Pharmacol. Declaration of Competing Interest Ther. 89 (6) (2011) 896–901. The authors declare that they have no known competing financial [2] W.J. Pichler, Delayed drug hypersensitivity reactions, Ann. Intern. Med. 139 (8) interests or personal relationships that could have appeared to influence (2003) 683–693. the work reported in this paper. [3] L.F. Li, C. Ma, Epidemiological study of severe cutaneous adverse drug reactions in Acknowledgments a city district of China, Clin. Exp. Dermatol. 31 (5) (2006) 642–647. We gratefully thank all the donors who participated in this project. [4] R. Mani, C. Monteleone, P.C. Schalock, T. Truong, X.B. Zhang, M.L. Wagner, Rashes The patients in this manuscript have given written informed consent to and other hypersensitivity reactions associated with antiepileptic drugs: A review the publication of their case details. Financial support for this study was of current literature, Seizure 71 (2019) 270–278. provided by the research fund of the Research Division, Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand. The Chalerm­ [5] B. Blaszczyk, W. Lason, S.J. Czuczwar, Antiepileptic drugs and adverse skin prakiat Fund supported P.T., S.W., S.S., K.K., and Y.S. We also thank Dr. reactions: An update, Pharmacol. Rep. 67 (3) (2015) 426–434. Kantima Kanchanapoomi for patient recruitment, Somkiat Ud-naen, and Thansuda Thansit for technical support. Last but not least, critical [6] J.A. Cramer, S. Mintzer, J. Wheless, R.H. Mattson, Adverse effects of antiepileptic reading and grammatical correction by Dr. James Dubbs were so drugs: a brief overview of important issues, Expert. Rev. Neurother. 10 (6) (2010) appreciated. 885–891. Appendix A. Supplementary material [7] N. Scheinfeld, Phenytoin in cutaneous medicine: its uses, mechanisms and side effects, Dermatol. Online. J. 9 (3) (2003) 6. Supplementary data to this article can be found online at https://doi. [8] N. Godhwani, S.L. Bahna, Antiepilepsy drugs and the immune system, Ann. Allergy. Asthma. Immunol. 117 (6) (2016) 634–640. [9] R.M. Nanau, M.G. Neuman, Adverse drug reactions induced by valproic acid, Clin. Biochem. 46 (15) (2013) 1323–1338. [10] M.G. Neuman, R.M. Nanau, T. Shekh-Ahmad, B. Yagen, M. Bialer, Valproic acid derivatives signal for apoptosis and repair in vitro, Clin. Biochem. 46 (15) (2013) 1532–1537. [11] T. Modeer, H. Domeij, I. Anduren, M. Mustafa, G. Brunius, Effect of phenytoin on the production of interleukin-6 and interleukin-8 in human gingival fibroblasts, J. Oral. Pathol. Med. 29 (10) (2000) 491–499. [12] I.S. Shiah, L.N. Yatham, C.B. Yeh, A.V. Ravindran, Effect of valproate on plasma levels of interleukin-6 in healthy male humans, Int. Clin. Psychopharmacol. 20 (6) (2005) 295–298. 9

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