CELL ACTIVATION MICRORNAS IN DRUG- SPECIFIC CD4+ T

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UP-REGULATION OF T - CELL ACTIVATION MICRORNAS IN DRUGSPECIFIC CD4+ T - CELLS FROM HYPERSENSITIVE PATIENTS Alejandra Monroy-Arreola, Noe Duran-Figueroa, Silvia Mendez-Flores, Judith Dominguez-Cherit, Joel Watkinson, Agustin Badillo-Corona, Paul Whitaker, Dean Naisbitt, and Luis Castrejon Flores Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.7b00330 • Publication Date (Web): 12 Apr 2018 Downloaded from http://pubs.acs.org on April 13, 2018

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Chemical Research in Toxicology

UP-REGULATION OF T - CELL ACTIVATION MICRORNAS IN DRUGSPECIFIC CD4+ T - CELLS FROM HYPERSENSITIVE PATIENTS.

Authors Alejandra Monroy-Arreola†, Noé V Durán-Figueroa†, Silvia Méndez-Flores‡, Judith Domínguez-Cherit‡, Joel Watkinson§, Jesús A Badillo-Corona†, Paul Whitaker║, Dean J Naisbitt§, José L Castrejón-Flores†*.

Institutional affiliations †

Instituto Politécnico

Nacional,

Unidad

Profesional

Interdisciplinaria

de

Biotecnología, 07340, México. ‡

Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, 14080,

México. §

MRC Centre for Drug Safety Science, University of Liverpool, L69 3GE, UK.

║

St James Hospital, Leeds, LS9 7TF, UK.

Corresponding author Dr. Jose L Castrejon Flores Instituto Politécnico Nacional; Adress: Av. Acueducto de Guadalupe, Barrio la Laguna Ticoman, Mexico City, Mexico; Phone: + 52 55 5729 6000 Ext: 56339; email: [email protected]

ACS Paragon Plus Environment

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TABLE OF CONTENTS

(IFN-γ, IL-22)

+ (Piperacillin / Carbamazepine)

APC

+

CD4 T Cells


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ABSTRACT

Dysregulation in the expression of microRNAs (miRNAs), single stranded RNAs which regulate gene expression, has been associated with diseases such as Stevens-Johnson syndrome (SJS) / toxic epidermal necrolysis (TEN), although their cellular origin has not been explored. Thus, the focus of this work was to study expression patterns of reported miRNAs involved in T - cell activation following drug-specific stimulation in peripheral blood mononuclear cells (PBMCs) and drug specific CD4+ T - cell clones (TCC) from patients with different cutaneous manifestations of delayed-type drug hypersensitivity reactions. CD4+ T - cells from hypersensitive patients were stimulated to proliferate, secreted cytokines (IFN-γ and IL-22), cytolytic molecules (Granzyme B) and up-regulate miRNAs 24 to 48 h after drug exposure. Carbamazepinespecific CD4+ T - cells that proliferated to the greatest extent and secreted the highest levels of IFN-γ showed an up-regulation of miR-18a and miR-155. In contrast, piperacillinspecific CD4+ T - cells displaying high expression of miR-9 and miR-21 showed an association with the extent of proliferation, but not IFN-γ secretion.

MiR-155

up-regulation

was

detected

in

PBMCs

from

all

hypersensitive patients 24 h after drug treatment, while miR-18a and miR-21 expression was up-regulated after 48 h. These findings demonstrate that miRNAs are expressed during drug specific CD4+ T - cell activation and shows a new regulation path for drug hypersensitivity reactions.


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INTRODUCTION

Adverse drug reactions (ADR) account for almost 6% of all hospital admissions causing approximatively 42,000 deaths every year in the European Union with an estimated economic loss of around 79 billion euros.1,2 Drug hypersensitivity reactions, although rare, are one of the most serious forms of ADRs. Recent analysis of computational integrative data has estimated that a single drug could directly modify 25 transition immune states. The authors hypothesize that some of these changes may lead to human iatrogenic diseases that cannot currently be predicted during drug development.3 The presence of drug-specific T - cells in the peripheral blood and inflamed tissue of hypersensitivity patients has been widely documented.4-6 Several factors including genetic background, co-morbidities at the time of the reaction, metabolism and reactivity of the drug, individually or together, contribute to the generation of the drug-specific T - cell response. Despite the complexity of the major histocompatibility complex (MHC)

– drugs – T - cell receptor (TCR)

interaction, it has been possible to elucidate three basic mechanisms of drug specific T - cell activation through in vitro experiments using PBMCs from hypersensitive

patients:

(1)

the

hapten/pro-hapten

pathway;

(2)

the

pharmacological interaction or P-I theory; and (3) the altered self-repertoire pathway.7,8 Furthermore, the ability of drug-specific T - cells to damage target tissue such as liver and skin has been described.9,10 Despite this, little is known about the role of post-transcriptional regulators, particularly miRNAs, in the pathogenesis of drug hypersensitivity. MiRNAs are single stranded 22 nucleotides RNAs, that regulate gene expression by binding to the 3’ untranslated region (UTR) of their target promoting translational repression or


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the direct degradation of the mRNA. Up-regulation of a specific miRNA promotes higher inhibition of the target gene which manifest in phenotypic and physiological changes in the cell.11 Over a hundred miRNAs have been shown to be expressed by immune cells and have an impact in basic immunological process. Despite the wide variety of miRNAs showing an altered expression, only a few have been studied into detail. These include miR-9, miR-17≈19 cluster, miR-21, miR-146a, miR-155, miR-181a, and miR-214. For the most of these, their target genes have been validated and their function has been evaluated during T - cell activation by functional assays12-18.

Regarding drug hypersensitivity reactions, microarray

analysis have been made using biopsies of patients with mild and severe cutaneous reactions, and some of the miRNAs, selected in our study, showed an altered expression. In particular, a direct correlation between miR-18a expression and the severity of the reaction was observed. Moreover, bioinformatics and functional analysis demonstrated that miR-18a targets the BCL2L10 gene, a negative regulator of apoptosis. Interestingly, the same miRNA is up-regulated during CD4+ T - cell activation and has a similar effect on CD4+ T - cells, but only when it is overexpressed alone.13, 19 The aim of this study was to determine the changes in the expression of miRNAs previous shown to be involved in CD4+ T - cell activation (miR-9, miR18a, miR-19b, miR-21, miR-155 and miR-214) using drug-specific CD4+ T - cell clones and PBMCs obtained from drug hypersensitive patients. Our results show a polarized pattern of miRNA up-regulation during CD4+ T - cell activation that correlates with the degree of proliferation.


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EXPERIMENTAL PROCEDURES

Cell Culture All cells were cultured in RPMI-1640 medium (Caisson, USA) supplemented with 10% of inactivated human AB serum (MP Biomedicals, USA), 2 mmol/L HEPES buffer (Caisson), 25 µg/mL transferrin, 100 mM L-glutamine (SigmaAldrich), 100 µg/mL streptomycin and 100 U/mL penicillin (Sigma-Aldrich). For TCC, the culture media was enriched with 100 U/mL human recombinant IL-2 (Peprotech, USA). PBMCs or TCC were cultured for 24 or 48 h with medium alone, piperacillin (PIP; 2 mmol/L), carbamazepine (CBZ; 50 µg/mL) or sulfamethoxazole (SMX; 200 µg/mL). Cyclosporine A (CSA; 1 µg/mL) was used in certain experiments. TCC were washed and cultured with medium only to remove any trace of IL-2 prior to addition of the drugs. All reagents were purchased from Sigma-Aldrich (USA) unless stated otherwise. PBMC isolation Eight hypersensitive patients and three tolerant patients were recruited for blood donation after informed consent. The protocols for this study were approved by the Local Research Ethics Committee in Mexico and the UK. PBMCs were isolated from fresh blood using Lymphoprep (Stemcell Technologies, UK) density gradient separation media. Generation of TCC TCC were generated from PBMCs of piperacillin and carbamazepine hypersensitive patients after treatment with the drugs for 2 weeks to enrich the drug-responsive T - cell population. These drugs were selected to include hapten and PI pathways of T - cell activation in our study as in vitro piperacillin and carbamazepine-specific T - cells are activated via hapten and PI pathways,


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respectively. A total of 288, 96 and 29 CD4+ T - cells were plated in a 96 well plate resulting in a cell density of 3 cells/well, 1 cell/well and 0.3 cells/well, respectively. T - cells cultured with allogenic irradiated PBMCs (5x105 cells/well) as feeder cells and the mitogen PHA (5 µg/mL). Cells were fed every two days with medium containing IL-2 and restimulated with PHA after 14 days. Lymphocyte Transformation Test PBMCs from hypersensitive patients and tolerant donors were incubated with sulfamethoxazole (100–400 µg/mL), lamotrigine (10-80 µg/mL), carbamazepine (10 - 50 µg/mL) or piperacillin (0.5 – 4 mmol/L) for 5 days. [3H]-thymidine was added for the final 16 h of the assay to measure proliferative responses. Tetanus toxoid (5 µg/mL) was used as a positive control. Proliferation and cytokine release from drug-specific TCC TCC were cultured with piperacillin (2 mmol/L) or carbamazepine (50 µg/mL) for 48 h and proliferation and cytokine release (IFN-y, IL-22 and Granzyme B) were measured using [3H]-thymidine and ELISpot (Mabtech, Sweden), respectively. PHA (10 µg/mL) was used as positive control. RNA extraction and qRT-PCR analysis Total cellular RNA was extracted using TRI-Reagent (Sigma, USA) following the manufacturer’s instructions. Briefly, RNA was re-suspended in water and quantified using a NanoDrop 2000/2000c UV-Spectrophotometer (Thermo Scientific, USA). Using 100 ng of total RNA, miRNAs were reverse transcribed using the Universal cDNA synthesis kit II (Exiqon, Denmark). The cDNA was mixed with ExiLENT SYBR Green Master Mix (Exiqon) and with specific primer sets for miR-9, miR-18a, miR-19b, miR-21, miR-146, miR-155 and miR-214. Quantitative PCR was performed using a ViiA 7 Real-Time PCR System


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(Thermo Scientific, USA). The results of the qRT-PCR were calculated as relative expression (RE) by comparing the expression of drug treated cells with non-treated using U6 as a reference gene. All qPCRs were performed in triplicate and values higher than 1 were considered as an up-regulation in the expression. Statistical Analysis Statistical analysis was performed using student’s t test and Mann Whitney Rank Sum test. The correlation value R was calculated using the logarithm for exponential correlation. Ethics Committee All procedures were followed in accordance with ethical standards of the responsible committee on human experimentation, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, approval number: 912, México and MRC Centre for Drug Safety Science, University of Liverpool, approval number: 12/NW/0525, UK.


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RESULTS

Activation of CD4+ T - cells with piperacillin and carbamazepine. A total of 11 CD4+ T - cell clones from carbamazepine and piperacillin patients were used. IFN-γ / IL-22 and IFN-γ / granzyme-B were selected as relevant ELISpot readouts for piperacillin and carbamazepine, respectively, based on recent investigations.20 All clones were stimulated to proliferate and secrete cytokines/cytolytic molecules after drug-treatment (Table 1 and figure 1). All TCC secreted IFN-γ following drug treatment. Four out of five piperacillinspecific TCC secreted IL-22, while all of the carbamazepine-specific TCC secreted Granzyme-B. Drug-specific CD4+ T - cell clones up-regulate particular sets of miRNAs. Expression of miRNAs in activated TCC was measured by qRT-PCR at 24 h when incubated with antigen presenting cells (APC) in the presence or absence of the relevant drugs. A drug-specific increase in miR-155 expression was observed with all clones. MiR-18, miR-21 and miR-9 showed up regulation in 10, 7 and 6 of the piperacillin- and carbamazepine-clones (Figure 2, A and B). Only two carbamazepine TCC showed up-regulation of miR-19b and miR-146, whereas piperacillin TCC did not up-regulate these miRNA (data not shown). Increased expression of mir-214 was not detected. A strong positive correlation was observed between the extent of proliferation and miR-155 (R2 = 0.85) and miR-18a (R2 =0.74) expression in carbamazepine clones (Table 1). A similar relationship was not observed with the piperacillin clones with the same miRNAs; however, a moderate but positive correlation between the extent of piperacillin-specific proliferation and miR-9 expression (R2 = 0.51) was determined.


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To confirm that miRNAs were expressed by CD4+ T - cells during drug challenge, we incubated APC and TCC in the absence of drug, and the expression of the miRNAs was measured a different time points. As expected, the RE values were below one in most cases, though some TCC showed a slightly higher value, but when the same TCC was incubated in the presence of the drug a positive three to ten fold change was seen (Table 1, Figure 2 A and B). Moreover, to rule out any change in miRNA expression because of a nonspecific interaction of the drugs and other immune cell receptors, TCC and APC were incubated alone in the presence of the drug, and no change of miRNAs expression was observed (Figure 2 D). Finally, to demonstrate that miRNAs up-regulation was the result of the initial signals provided by the drug to the TCR, we incubated the best drug responding TCC (clones that expressed all miRNAs with drug-treatment) with a non-toxic concentration of cyclosporine-A. A significant decrease in expression of all the miRNAs, below the reference value, was observed with cyclosporine-A. Figure 2 C compares the response of clones to drug in the presence and absence of cyclosporine-A. Thus, our results demonstrate that miRNA up-regulation is the result of a drug specific CD4+ T - cell activation. Time-dependent

upregulation

miRNA

in

drug-stimulated

PBMC

from

hypersensitive patients To investigate if the miRNAs up-regulated in TCC are also up-regulated in PBMC of hypersensitive patients, we focused on miR-18a, miR-21 and miR155.

Sulfamethoxazole,

lamotrigine,

piperacillin

and

carbamazepine

hypersensitive patients presenting with a range of cutaneous manifestations, varying in severity from maculopapular exanthema to toxic epidermal


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necrolysis, were included in the study (Table 2). PBMC from all patients, but one, were stimulated to proliferate in the presence of the culprit drug (stimulation index of 2 or higher; Table 2). Because of the variety of skin manifestations, which might affect the number of blood circulating drug-specific T - cells and their phenotype/function21, miRNA expression was assessed at two-time points, 24 and 48 h. PBMCs from all patients, including the one with negative lymphocyte transformation test showed upregulation of miRNAs after drug treatment (Figure 3). As seen with the TCC, no increase in PBMCs miRNA was observed over the duration of the assay in the absence of the drug in the PBMCS (Figure 4). The time in which the different miRNAs were expressed with drug treatment differed. A higher level of miR-18a was detected in most patients after 48 h, while miR-21 was expressed earlier, at 24 h. In contrast, miR-155 was detected at a similar level at both time-points, irrespectively of the severity of the skin reaction. Drug-stimulated PBMCs from the SJS/TEN patients showed high levels of all the miRNAs analyzed. Figure 4 compares the lymphocyte transformation test results of individual hypersensitive and tolerant patients with the upregulation of miR-18a, miR-21 and miR-155. All patients showed a positive expression of at least 1, in most cases 2, miRNAs. In some cases, drug-treated samples displayed 5-20 times higher miRNAs expression than basal levels. Up-regulation of miRNAs was seen with drug-treated PBMCs from piperacillin hypersensitive patients; however, a direct correlation between miRNAs up-regulation and proliferation was not always observed. Table 3 summarizes the relative expression values of all the patients included in the cohort.


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When the expression of miRNAs was assessed in piperacillin tolerant or other tolerant controls, the presence of the culprit drugs did not up-regulate the expression of any of the miRNAs tested (Figure 4).


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DISCUSSION

A great deal of attention has focused on the changes in gene expression, particularly miRNAs dysregulated during T - cell activation, since they play an important role in the phenotype of the activated cells and neighbouring cells. Tcell activation is paramount for the initiation of drug hypersensitivity reactions; however, until now, the regulation of miRNA expression following drug-specific T - cell activation has not been addressed. Here, we have shown an upregulation in the expression of several miRNAs, previously studied during T cell activation, in drug-specific CD4+ T - cell clones and PBMCs from patients with different forms of cutaneous hypersensitivity. Two sets of TCC were analysed to explore the different patterns of miRNAs expressed during drug specific CD4+ T - cell activation. Piperacillin-specific and carbamazepine-specific TCC were used because, at least in vitro, the drugs activate T - cell receptors via hapten and PI pathways, respectively. With carbamazepine, the kinetics of T - cell activation are too rapid for antigen processing to be a prerequisite. Furthermore, inhibition of antigen processing does not alter the strength of the carbamazepine-specific T-cell response. After drug-specific CD4+ T - cell activation, most of the selected miRNAs were upregulated, and in certain circumstances their expression directly correlated with the strength of the drug-specific CD4+ T - cell proliferative response. Thus, by directly comparing miRNA up-regulation and CD4+ T - cell proliferation, it was possible to establish the polarized pattern of miRNAs. Strongly proliferating carbamazepine-specific TCC showed the highest levels of miR-18a and miR155 up-regulation, while miR-9 up-regulation was seen with the TCC proliferating strongly to piperacillin. MiR-19b and miR-146 up-regulation was not


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consistently expressed, only two of the CBZ clones showed positive values, whereas miR-214 was not up-regulated with any of the TCC. It is important to emphasize that although this pattern of miRNAs expression seems to be characteristic for the CD4+ T - cells, further investigation is needed to explore the miRNAs pattern of in drug specific CD8+ T - cell clones. Previous microarray studies have found a significant change in the expression of several miRNAs in biopsies and plasma of patients with SJS/TEN, where miR-18a, a member of the miR-17 ≈ 92 cluster family, showed the highest upregulation.19 Mechanistic studies using a miR-18a mimic transfected into keratinocytes revealed that the miRNA was involved in the induction of apoptosis in vitro. Interestingly, the same effect was seen in CD4+ T - cells when the miRNA was artificially and individually transduced during CD4+ T - cell activation. Therefore, it is possible that the apoptotic effect seen in drug activated CD4+ T - cells is not evident because of the expression of other members of the cluster or the expression of miR-9, miR-21 and miR-155, which collectively promote proliferation. Therefore, if the effects of miR-18a are not seen directly in the drug activated CD4+ T - cells, it is possible that the miRNA could target a different cell type. Nonetheless, our results showed that under certain circumstances

drug-specific

activation might

contribute to the

overexpression of miR-18a. MiRNA communication between different cell types has been demonstrated in cutaneous contact hypersensitivity reactions where exosomes containing miR150 travel in plasma delivering a suppressive effect on the inflammatory response.22 Thus, it is necessary to determine the expression levels of miR-18a in microvesicles secreted during drug challenge of different T - cells repertoires,


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at different time points. Furthermore, it will be important to evaluate their effect on keratinocytes. Interestingly, carbamazepine CD4+ T - cells showed the highest expression of miR-18a, and carbamazepine is associated with a high incidence of SJS/TEN.23 Thus, it is possible that the activation of TCC with carbamazepine is related to the over expression of miR-18a, and together with other factors such as genetic background be a predisposing element for the development of severe bullous reactions. MiR-155 was also highly expressed in TCCs, and besides its effect on proliferation, it plays an important role in the inflammatory response. Functional studies using miR-155-/- mice show a tendency to generate lower numbers of Th1 and Th17 cells during autoimmune diseases, and in vitro studies with CD4+ T - cells from miR-155-/- mice secreted lower levels of IFN-γ when compared with the same cells from the WT animals.24 Thus, we compared the selective expression of MiR-155 with the secretion of IFN-γ after drug treatment. The level of expression with carbamazepine-specific TCC correlated with the level of IFN-γ secretion, although not in a linear manner, in four of the six clones tested, but this pattern was only seen in two of the five piperacillin TCC. IL-2 is necessary for TCC survival, and its presence does not affect the expression of miR-21 and miR-155. In contrast, it might affect the expression of miR-9 which targets genes related to IL-2 expression.12 Thus, to avoid any interference of IL-2 in our studies, the TCC were deprived of the growth factor 48 h before stimulating the clones with either piperacillin or carbamazepine. To demonstrate that miRNA up-regulation was the result of drug-specific CD4+ Tcell activation and downstream signalling an extra experiment was conducted where cyclosporine-A was added to the assay. The drugs did not increase


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miRNA expression in CD4+ T - cell clones in the absence of drug or in the presence of cyclosporine-A. In the next component of the study, we evaluated miRNA expression in activated

PBMCs

from

patients

with

different

forms

of

cutaneous

hypersensitivity associated with the antibiotics sulfamethoxazole and piperacillin and the anticonvulsants lamotrigine and carbamazepine. MiR-18a, miR-21, and miR-155 were studied, because they showed the higher levels of expression in the experiments among different TCC. Given the low frequencies of drugspecific T-cells in peripheral blood and the heterogeneity of their patient cohort , we decided to analyse the expression of miRNAs at two time points 24 and 48 h. PBMCs from patients were stimulated to proliferate in vitro in the presence of the culprit drug. Similarly, upregulation of all three miRNA was detected. MiR21 expression was upregulated to the greatest extent in most patients at 24 h, while miR-18a showed higher expression after 48 h. MiR-155 expression was elevated at both time points. The expression of this miRNA was drug- and timedependent supporting again that this miRNA is expressed after drug challenge. Thus, it might be possible to use miR-155 as an early diagnostic biomarker of drug hypersensitivity reactions. To conclude, our results clearly show a change in the levels of expression of miRNAs in activated CD4+ T - cells from hypersensitive patients after drug challenge. These results contribute to the current understanding of the role of miRNAs in cutaneous reactions by demonstrating that they could originate from drug-specific T - cells during activation. However, how these miRNAs expressed in drug specific T - cells end up in the plasma or skin altering cellular and tissue physiology in drug hypersensitive patients’ needs to be further


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investigated. Importantly our research opens a new field to investigate the use of miRNAs as biomarkers for in vitro diagnosis of drug hypersensitivity reactions.


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FUNDING INFORMATION

This work was supported by a grant from the Mexican National Council for Science and Technology (CONACyT) FOSSIS-201184 hold it by JLCF and FOSSIS-201388 hold it by NVDF. AMA is a PhD student supported by a PhD scholarship funded by the Mexican National Council for Science and Technology.


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CONFLICT OF INTEREST

The authors declare no conflict of interest.


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ABREVIATIONS LIST

miRNAs

microRNAs

PBMCs

Peripheral Blood Mononuclear cells

PHA

Phytohemagglutinin

TCC

Drug specific CD4+ T cell clones

APC

Antigen presenting cells

IFN-γ

Interferon gamma

IL-2

Interleukin 2

IL-22

Interleukin 22

ADR

Adverse drug reactions

MHC

Major Histocompatibility Complex

TCR

T-cell receptor

UTR

Untranslated Region

MPE

Maculopapular exanthema

SJS

Stevens-Johnson syndrome

TEN

Toxic Epidermal Necrolysis

CBZ

Carbamazepine

PIP

Piperacillin

SMX

Sulfamethoxazole

LMTG

Lamotrigine

CSA

Cyclosporine-A

SPF

Spot forming unit

RE

Relative expression

qRT-PCR

Real-Time Quantitative Reverse Transcription PCR

SI

Stimulation index

CPM

counts per minute


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promoting Th1 responses and preventing inducible Treg differentiation. Blood 118, 5487-5497. (14) Smigielska-Czepiel, K., van den Berg, A., Jellema, P., Slezak-Prochazka, I., Maat, H., van den Bos, H., van der Lei, R. J., Kluiver, J., Brouwer, E., Boots, A. M. H., and Kroesen, B.-J. (2013) Dual Role of miR-21 in CD4+ T-Cells: Activation-Induced miR-21 Supports Survival of Memory T-Cells and Regulates CCR7 Expression in Naive T-Cells. PLOS ONE 8, e76217. (15) Curtale, G., Citarella, F., Carissimi, C., Goldoni, M., Carucci, N., Fulci, V., Franceschini, D., Meloni, F., Barnaba, V., and Macino, G. (2010) An emerging player in the adaptive immune response: microRNA-146a is a modulator of IL-2 expression and activation-induced cell death in T lymphocytes. Blood 115, 265-273. (16) Gracias, D. T., Stelekati, E., Hope, J. L., Boesteanu, A. C., Doering, T., Norton, J., Mueller, Y. M., Fraietta, J. A., Wherry, E. J., Turner, M., and Katsikis, P. D. (2013) MicroRNA-155 controls CD8(+) T cell responses by regulating interferon signaling. Nature immunology 14, 593-602. (17) Li, Q. J., Chau, J., Ebert, P. J., Sylvester, G., Min, H., Liu, G., Braich, R., Manoharan, M., Soutschek, J., Skare, P., Klein, L. O., Davis, M. M., and Chen, C. Z. (2007) miR-181a is an intrinsic modulator of T cell sensitivity and selection. Cell 129, 147-161. (18) Jindra, P. T., Bagley, J., Godwin, J. G., and Iacomini, J. (2010) Costimulationdependent expression of microRNA-214 increases the ability of T cells to proliferate by targeting Pten. J Immunol 185, 990-997. (19) Ichihara, A., Wang, Z., Jinnin, M., Izuno, Y., Shimozono, N., Yamane, K., Fujisawa, A., Moriya, C., Fukushima, S., Inoue, Y., and Ihn, H. (2014) Upregulation of miR-18a-5p contributes to epidermal necrolysis in severe drug eruptions. J Allergy Clin Immunol 133, 1065-1074. (20) Sullivan, A., Wang, E., Farrell, J., Whitaker, P., Faulkner, L., Peckham, D., Park, B. K., and Naisbitt, D. J. (2018) beta-Lactam hypersensitivity involves expansion of circulating and skin-resident TH22 cells. J Allergy Clin Immunol. 141, 235-249. (21) S. J. Posadas and W. J. Pichler (2007) Delayed drug hypersensitivity reactions – new concepts. Allergy 37, 989-999. (22) Bryniarski, K., Ptak, W., Jayakumar, A., Pullmann, K., Caplan, M. J., Chairoungdua, A., Lu, J., Adams, B. D., Sikora, E., Nazimek, K., Marquez, S., Kleinstein, S. H., Sangwung, P., Iwakiri, Y., Delgato, E., Redegeld, F., Blokhuis, B. R., Wojcikowski, J., Daniel, A. W., Groot Kormelink, T., and Askenase, P. W. (2013) Antigen-specific, antibody-coated, exosome-like nanovesicles deliver suppressor T-cell microRNA-150 to effector T cells to inhibit contact sensitivity. J Allergy Clin Immunol 132, 170-181. (23) Naisbitt, D. J., Britschgi, M., Wong, G., Farrell, J., Depta, J. P. H., Chadwick, D. W., Pichler, W. J., Pirmohamed, M., and Park, B. K. (2003) Hypersensitivity Reactions


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to Carbamazepine: Characterization of the Specificity, Phenotype, and Cytokine Profile of Drug-Specific T Cell Clones. Molecular Pharmacology 63, 732. (24) O'Connell, R. M., Kahn, D., Gibson, W. S., Round, J. L., Scholz, R. L., Chaudhuri, A. A., Kahn, M. E., Rao, D. S., and Baltimore, D. (2010) MicroRNA-155 promotes autoimmune inflammation by enhancing inflammatory T cell development. Immunity 33, 607-619.


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TABLES Table 1 Table 1. Proliferation, IFN-y secretion and miRNAs expression by drug specific CD4+ T - cell clones obtained from piperacillin and carbamazepine hypersensitive patients. MiR-9 TCC

SI

IFN-γ A

B

No drug

MiR-18

Drug C

MiR-21

MiR-155

No drug

Drug

No drug

Drug

No drug

Drug

0.02

84.03 ± 12.7

0.39

8.29 ± 1.43

0.73

871.6 ± 47.08

PIP #1 10.83 PIP #2 16.93

+++ +++

0.37

6.17 ± 1.68

0.6

3.86 ± 0.89

0.57

5.87 ± 2.87

0.69

7.79 ± 2.64

0.01

2.68 ± 1.00

PIP #3

8.77

+++

1.23

8.02 ± 2.72

1.56

1.04 ± 0.01

3.64

32.16 ± 4.05

1.01

47.77 ± 13.49

PIP #4

3.54

+++

0.38

1.29 ± 0.08

2.03

114.5 ± 0.44

1.6

1.48 ± 0.82

1.29

3.5 ± 0.95

0.67

1.61 ± 0.35

1.47

0.66 ± 0.02

1.59

9.18 ± 1.04

PIP #5

1.60

++

1.36

0.77 ± 0.01

CBZ #7

18.00

+++

0.57

2.58 ± 0.35

0.54

438.8 ± 46.3

0.77

4.66 ± 0.46

1.25

CBZ #8

1.74

+

0.22

3.47 ± 0.91

0.19

2.8 ± 0.62

1.24

14.14 ± 0.00

0.21

2.16 ± 0.56

CBZ #9

4.17

+++

1.09

0.28 ± 0.02

0.38

1.48 ± 0.33

0.24

0.51 ± 0.43

0.90

12.14 ± 3.38

CBZ #10 8.93

++

1.39

1.4 ± 0.31

2.46

14.91 ± 3.33

0.67

2.89 ± 0.46

1.88

24.65 ± 6.33

0.41

6.53 ± 0.91

0.92

20.55 ± 4.66

1.19

21.83 ± 1.37

0.86

1.49 ± 0.08

0.65

0.00 ± 0.00

1.59

2.61 ± 0.12

CBZ #11 13.89

+++

0.67

0.36 ± 0.14

CBZ #12 1.70

+++

0.2

0.01 ± 0.00

105.9 ± 7.4

A Data presented as stimulation index, SI, calculated as cpm in drug incubation/ cpm in control incubations. B Spot forming units, SFU, after Piperacillin (PIP; 2 mmol/L) and Carbamazepine (CBZ; 50 µg/mL) treatment: +, 10-30; ++, 30-100; +++ >100. C Relative expression, RE. All values are the mean of a triplicate ± standard deviation


24

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Table 2 Table 2. Patients’ clinical information and lymphocyte transformation test results. Time taking the Co-morbilities drug (days)

Patient

Sex

Age

Drug

Skin

SMX1

F

60

MPE

10

RI

5.0

SMX2

M

35

Sulfamethoxazole / Trimethoprim

MPE

9

RI

3.0

LMTG1

M

32

TEN

7

MDD

2.0

SJS

12

MDD

2.4

SJS

7

MDD

2.1

MPE

7

CF

3.6

MPE

8

CF

1.2

MPE

11

CF

28.0

Lamotrigine LMTG2

M

40

CBZ1

F

47

PIP1

F

33

PIP2

F

29

PIP3

M

28

Carbamazepine

Piperacillin

SI

Male (M) or female (F) hypersensitive patients to sulfamethoxazole (SMX 1-2), lamotrigine (LMTG 1-2), carbamazepine (CBZ 1) and piperacillin (PIP 1-3) with different skin clinical manifestations including maculopapular exanthema (MPE), toxic epidermal necrolysis (TEN) and Stevens-Johnson syndrome (SJS) were enrolled in the study. Patients were diagnosis with respiratory infection (RI), major depressive disorder (MDD) or Cystic fibrosis (CF). Proliferation was measured as stimulation index (SI).


25


Page 26 of 32

Table 3 Table 3. Time dependent miRNAs in vitro up-regulation of PBMCs challenge with different drugs and obtained from patients with mild and severe forms of hypersensitivity. .

Patient

Skin

SI

SMX1 SMX2 LMTG1 LMTG2 CBZ1 PIP1 PIP2 PIP3

MPE MPE TEN SJS SJS MPE MPE MPE

4.0 3.0 2.0 2.4 2.1 3.6 0.9 28.0

miR-18 24 h 71.30* 7.81 1.26 0.02 0.03 1.29 0.14 1.42

± 1.21 ± 2.25 ± 0.36 ± 0.00 ± 0.01 ± 1.31 ± 0.11 ± 0.36

miR-21 48 h

0.20 0.06 1.38 0.78 0.01 2.83 1.52 0.34

24 h ± 4.71

± 0.62

76.53 5.51 49.13 0.68 0.77 3.07 12.75

± 0.13

1.76

± 0.94

± 0.02 ± 0.09 ± 0.34 ± 0.34 ± 0.00 ± 0.25

± 0.69 ± 4.34 ± 0.58 ± 0.19 ± 0.75 ± 0.85

miR-155 48 h

0.37 0.95 26.76 22.97 0.22 8.81 12.88 0.57

± 0.04 ± 0.23 ± 10.79 ± 6.42 ± 0.03 ± 1.37 ± 2.55 ± 0.31

24 h 5.64 0.04 1.26 17.9 4.6 1.12 0.03 1.58

± 1.19 ± 0.01 ± 0.22 ± 2.41 ± 0.12 ± 0.32 ± 0.03 ± 0.11

48 h 0.76 0.40 8.01 525.24 0.45 14.83 5.12 1.58

Hypersensitive patients to sulfamethoxazole (SMX 1-2), lamotrigine (LMTG 1-2), carbamazepine (CBZ 1) and piperacillin (PIP 1-3) with different skin clinical manifestations including maculopapular exanthema (MPE), toxic epidermal necrolysis (TEN) and Stevens-Johnson syndrome (SJS) were enrolled in the study. Proliferation was measured as stimulation index (SI), and miRNAs values (*) are shown as relative expression (RE) and are the mean of a triplicate ± standard deviation.


26

± 0.05 ± 0.23 ± 0.81 ± 12.69 ± 0.08 ± 1.81 ± 1.79 ± 0.40

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FIGURES FIGURE 1

Figure 1. Cytokine secretion from activated drug specific CD4+ T - cells. A) IFN-γ secretion from piperacillin specific CD4+ T - cell clones and B) carbamazepine specific CD4+ T - cell clones incubated for 48 h with 2 mmol/L and 50 µg/mL of piperacillin and carbamazepine, respectively; C) IL-22 secretion from piperacillin specific CD4+ T - cell clones incubated for 48 h with 2 mmol/L piperacillin; D) Granzyme B secretion from carbamazepine specific CD4+ T - cell clones incubated 48 h with 50 µg/mL of carbamazepine. The number in the images correspond to the number of spot forming units (SFU) where a value above 30 SFU is considered positive.


27

Chemical Research in Toxicology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

1

Page 28 of 32

FIGURE 2

2

28 ACS Paragon Plus Environment

Page 29 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3

Figure 2. Upregulation of miR-9, miR-18a, miR-21, miR-155 is the result of the

4

interaction between APC, drugs and CD4+ drug specific T - cells. A) MiRNAs

5

expression in piperacillin specific TCC and B) Carbamazepine TCC in the

6

presence or absent of the culprit drug. C) Cyclosporine (1 µg/mL) blocks the up-

7

regulation of miR-9, miR-18a, miR-21 and miR-155 in two carbamazepine

8

specific TCC incubated with APC and the drug for 24 h. D) The presence of

9

APCs, the drugs and TCCs are necessary for miRNAs up-regulation. Values

10

are shown as relative expression and are the mean of a triplicate ± standard

11

deviation. Statistical analysis was performed between relative expression of

12

TCC cultured with medium alone and TCC cultured with drug. **P ≤ 0.05. n.s.,

13

Not significant.

14


29


15

Page 30 of 32

FIGURE 3

16 17

Figure 3. Time dependent expression of miRNAs in PBCMs of drug

18

hypersensitive patients. Relative expression of miR-18a, miR-21 and miR-155

19

from eight hypersensitive patients to sulfamethoxazole (SMX1-2), lamotrigine

20

(LMTG1-2), piperacillin (PIP1-3) or carbamazepine (CBZ1) with different clinical

21

manifestations. PBMCs were incubated in the presence of culprit drug for 24

22

and 48 h prior analysis. Values are shown as relative expression and are the

23

mean of a triplicate ± standard deviation

24


30

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25


FIGURE 4

26 27

31 ACS Paragon Plus Environment


Page 32 of 32

28

Figure 4. Maximal miRNAs up-regulation can be correlated with the degree of

29

proliferation in the presence of the culprits’ drugs. Relative expression of A)

30

miR-18a, B) miR-21 and C) miR-155, indicated with the bars, in drug-treated

31

PBMCs, shows that a positive expression of miRNAs correlates with a positive

32

proliferation (SI), indicated with symbols, in patients to sulfamethoxazole

33

(SMX1-2), lamotrigine (LMTG1-2), piperacillin (PIP1-3) or carbamazepine

34

(CBZ1) with different clinical cutaneous manifestations. Importantly, drug

35

treatment of PBMCs obtained from tolerant patients (Tol) or hypersensitivity

36

patients in the absence of the drug (*) did not up-regulate miRNAs or stimulate

37

proliferation.

38


32

CELL ACTIVATION MICRORNAS IN DRUG- SPECIFIC CD4+ T

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