Association of miR-155, miR-187 and Inammatory Cytokines Il-10 and TNF-α in Chronic Opiate Abusers

The current study determined levels of inammatory cytokines TNF-α, IL-6, IL-10 and immune-regulatory miR-155 and miR-187 expressions in chronic opiate abuse. Adults (n = 48), meeting the 5th Edition of the DSM criteria regarding opioid use disorder, and healthy controls (n = 46) were included in the study. Serum samples were analysed for inammatory cytokines IL-10, IL-6, TNF-α using ELISA and PBMCs processed for miRNA expression using SybrGreen chemistry. Cases showed signicantly raised IL-10 and TNF-α and reduced IL-6. Using RNU6 for normalization, dose-dependent corresponding upregulation of miR-155-5p and downregulation of miR-187-5p were evident at opiate dose > 1500 gm/day, with a corresponding increase of TNF-α and IL-10. MiR-155 showed a signicant negative correlation with IL-6 and TNF-α; miR187 showed a signicant positive association with TNF-α at > 1000 g/day consumption. Therefore, increasing consumption of opium probably enhances inammation leading to immunomodulation and aberrant expression of has-miR-155-5p and has-miR-187-5p in opiate abusers.


Introduction
Opium is the second most commonly abused substance after tobacco in developing countries of the Middle East and Asian regions. In India, the number of opiate abusers is increasing every year with a great number residing in Western Rajasthan [1]. Opioids can interfere with the immune system by participating in the function of the immune cells and causing modulation of the innate and acquired immune responses. The traditional notion of opioids is immunosuppressive [2]. However, recent studies indicate that the role of opioid receptors working through various mechanisms. Since the immune system plays a critical role in various physiological and pathophysiological processes, including in ammation, tumour growth and metastasis and drug abuse, therefore it is important to elucidate the relationship between opioids and immune function [2]. Cytokines are an important weapon in the armoury of in ammatory reactions. They are produced by the immune cells and play a signi cant role in regulating the activity of the immune system. Cytokine production is stimulated by several stimulants (e.g. levetiracetam, topiramate, and carbamazepine) [3]; and opium derivatives have also been shown to affect cytokine production [4]. Furthermore, there are con icting reports on the levels of pro-and anti-in ammatory cytokines in opiate addiction [5,6]. IL-10 acts as an anti-in ammatory cytokine, with reports of its higher levels in opium abusers as compared to the control group [5] and IL-10 negatively regulates acute and chronic in ammation by blocking or reducing the output of pro-in ammatory cytokines from immune cells, including TNF-α, IL-6, and IL-12 has been reported. IL-10 suppresses cytokines primarily at the level of gene transcription but also in uences post-transcriptional events [7]. MicroRNAs (miRNAs) are evolutionarily conserved small, noncoding RNAs that post-transcriptionally regulate gene expression by targeting speci c messenger RNAs (mRNAs) for degradation or translational repression. Emerging evidence suggests that miRNAs play a key role in the regulation of immunological functions including innate and adaptive immune responses, development and differentiation of immune cells, and prevention of autoimmunity [8]. Studies are now revealing that miRNAs modulate the concentration of key target proteins over a narrow range in a dose-dependent manner, i.e. the cellular concentration of a miRNA dictates the protein output of its target genes and is therefore of key importance in miRNA-mediated control. Small changes in the concentration of key cellular proteins being affected by miRNA control, can have signi cant biological consequences, in line with many examples of pathogenic hemizygous null mutations in man and mouse [9]. Therefore, miRNAs play a crucial role in the regulation of the immune system.
There are no studies exploring the roles of microRNA in opiate dependents, however, microarray analysis in varied drug addictions have revealed altered expression of miRNA regulating both innate and adaptive immune systems like miR-155, miR-187, miR-190, and miR184 [10]. Besides, Rossato et al. (2012) [11] showed in human monocytes that miR187 is involved in the physiological regulation of IL-10-driven antiin ammatory responses and demonstrated miRNA-mediated strategies controlling cytokine expression in human monocytes activated by a TLR4 agonist (LPS). Similarly, miR155 is a signi cant player in both innate and adaptive immunity and is upregulated in macrophages in alcohol abuse [12].
Although there is an elevated incidence of in ammatory disorders in drug abusers (alcohol, opioid) [13], the role of miRNA in the immune regulation of drug abusers and the functions of most miRNAs concerning drug abuse and immune diseases is still not clearly understood [14].
No studies have explored the dynamics of pro and anti-in ammatory cytokines in response to changing dose of stimulant, i.e. opiate and expression of their corresponding miRNAs, i.e. IL-10 for miR187 and TNF-α for miR155. We hypothesize that chronic opiate addiction triggers immune modulation leading to altered levels of IL-10, IL-6 and TNF-α and their targeted hsa-miR-155-5p and hsa-miR-187-5p. This will help understand the roles of these miRNAs concerning IL-10, IL-6 and TNF-α in opiate addiction. Since immunomodulation forms the basis of several disease conditions, this may go a long way in understanding immune diseases in such patients.

Study participants and inclusion and exclusion criteria
The study was conducted in the Department of Biochemistry and Psychiatry of a tertiary care centre for a duration of one and half year. Opiate dependent males were recruited from the outpatient departments of the de-addiction clinic of the Department of Psychiatry, and a non-government organization (NGO), Manklaav drug de-addiction and rehabilitation centre of the city reporting for de-addiction. The study was planned as a case-control study and was initiated after the Institutional Ethics Committee (IEC) approval.
A total of 48 opiate abusers were recruited after informed consent. Adults, who met the Fifth Edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) criteria of the American Psychiatric Association regarding opioid use disorder (n = 48), and healthy controls (Control Group or CG, n = 46).
The inclusion criteria were to include male patients of the age group 18-50 years, who were chronic opiate abusers (not abusing other substances like cocaine, marijuana or amphetamine), for a minimum of one year. Female opiate abusers do not report to the de-addiction clinics due to the patriarchal social structure of the region. A convenience sampling method was employed at the de-addiction clinic. All opiatedependent subjects consumed opium orally as pure opium (available in packets of 50 gms). The daily dose consumed was reported by the attendants of the patients. A urine sample of the study participants was collected for the screening of opiate; those having a value >300 ng/ml was considered positive and were recruited to the study. Urinary Opiate was analysed using the Enzyme Immunoassay kit of Randox and the cut-off was taken as mentioned in the kit insert. The cases recruited showed no evidence of withdrawal symptoms (such as insomnia, nausea, vomiting, diarrhea, agitation, headaches, hallucinations and blurred vision) at the time of sampling. Multi-drug abuse was excluded and cases with only oral opium abuse were included. History of any other drug consumption which affects the immune response was taken to exclude those participants. Patients with any co-morbidity (Diabetes mellitus, Hypertension, Hypothyroidism, Coronary artery disease, Rheumatic disorders, HIV, HCV and other in ammatory disorders as well as smoking) were also excluded from the study.
The controls were individuals working in our healthcare setup, such as students and hospital staff who did not have any history of drug abuse or smoking and did not have any in ammatory disorder (rheumatoid arthritis, diabetes and asthma based on history and physical examination), and had no psychiatric ailment.

Sample collection, RNA isolation, and reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR)
A venous blood sample collected on the day of their admission, with the last opiate dose taken within 24 hours of the sample collection. The venous blood sample of the study subjects was collected in erythrocyte lysis buffer (Qiagen). The PBMCs cell lysate was processed for extraction of miRNA (Qiagen miRNAeasy mini kit). The quantity and quality of the extracted miRNA were assessed using a Nanoquant plate (BioTek) at 260 nm and 280 nm, and those with a ratio greater than 1.8 were processed further for cDNA synthesis using cDNA kit speci c for hsa-miR-155-5p and has-miR-187-5p (miScript II RT Kit by Qiagen). The cDNA samples were further analysed using the qPCR CFX96 of Bio-Rad using SybrGreen chemistry (Qiagen miScript SYBR Green PCR Kit). The PCR data was analysed using the CFX96 analysis software and expressed as delta Cq values. The fold change expression was analysed after normalization using RNU6.

Statistical analysis
The statistical analysis was performed using SPSS21 for correlation analysis of biochemical parameters and PCR data. The continuous variables were analysed using descriptive statistics. Comparison between groups was carried out by one-way analysis of variance (ANOVA), and linear regression analysis was used to analyse the effects of variable opium consumption.

Results
The current case-control study demonstrated in chronic opiate abusers signi cantly raised IL-10, and TNF-α, a signi cantly reduced IL-6 ( Table 1) as compared with healthy controls. Further, one way ANOVA for in ammatory cytokines showed a non-signi cant difference across the groups when categorised according to dose of opiate (oral consumption of opium), although the rise in TNF-α was much greater than the rise in anti-in ammatory IL-10 ( Table 2). An analysis of fold change expression of hsa-miR155-5p and hsa-miR-187-5p showed downregulation (Table 3). This may be responsible for the higher expression of IL-10 and TNF-α in the cases as compared to controls. However, one-way ANOVA for miRNA expression with different amount of opium consumption (Table 3) showed that with an increasing dose, there was upregulation of miR-155-5p at a consumption of 750-1000 gm opium and >1500 gm opium, which possibly is due to the effect of raised TNF-α. At an opium consumption <1000 gm/day both the miRNAs showed no signi cant association with the cytokines (Table 4). Pearson's correlation analysis showed a signi cantly negative association of miR-155-5p with IL-6 and TNF-α and a positive association with IL-10 at opium consumption greater than 1000 gm/day. For miR-187-5p, there was a signi cantly positive association with TNF-α at a daily consumption >1000 gm/day (Table 5).

Discussion
Chronic opiate abuse is capable of signi cant immunomodulation and possibly forms the basis of various diseases associated with opiate abuse [15]. Regulation of in ammatory responses is ensured by coordinated control of gene expression of participating immune system proteins and cells. One group of gene expression regulators, functions of which have recently been started to be uncovered concerning any type of in ammatory condition, is a class of short single-stranded RNA molecules termed microRNAs (miRNAs). miRNAs function together with partner proteins and mainly cause gene silencing through degradation of target mRNAs or inhibition of translation [16]. The current study for the rst time shows the effect of chronic opiate abuse, as well as its dose on the expression of in ammatory cytokines and their associated miRNAs (Tables 1 & 3). We have reported earlier in a prospective clinical study a strong association of low-grade in ammation with dependence years in pure opiate abusers [1]. The current study reports a dose-dependent increase in the serum levels of TNF-α, expression hsa-miR-155-5p, and hsa-miR-187-5p with increasing per day consumption. The signi cantly raised levels of TNF-α suggest a pro-in ammatory status.
The hsa-miR-155-5p is a pleiotropic molecule that regulates both central and peripheral in ammation [17] (Qayum et al., 2016). It has been reported to be induced by both IL-10 [16] and TNF-α [18]. We observed chronic opiate abuse induces an in ammatory response, wherein as the dose increases, the antiin ammatory response of IL-10 is over-ridden as observed by its non-signi cant increase and a signi cant increase of TNF-α in cases as compared to controls.
At lower doses of opium, there is downregulation of miR-155-5p and a rise in expression of IL-10, as seen in Tables 2 & 3. At lower doses of opium, miR-187-5p gradually upregulated and possibly induced IL-10, as also observed by Rossato et al. (2012). [11] However, at opium consumption greater than 1500gm/day, there is upregulation of hsa-miR-155-5p which targets and inhibits IL-10 expression, thus showing a proin ammatory status acquisition with a very high dose of opium.
A higher expression of hsa-miR-155-5p causes inhibition of IL-10 and certain transcription factors like SHIP-1, SOCS-1 and BCL6 in macrophages and this relieves the inhibition on the expression of TNF-α [19]. Similar observations have been made in the current study since, with increased consumption of opium (750-1000 gm/day and >1500 gm/day), there was up-regulation of miR-155-5p and an increased level of TNF-α. Also, miR-155-5p and TNF-α axis exists in an animal model of temporal lobe epilepsy and is responsible for neuro-in ammation in epilepsy in both human and animal model [20]. A similar axis may be responsible for the dose-dependent immunomodulation to enhance a pro-in ammatory cytokine response of chronic opium abusers.
IL-10 negatively regulates acute and chronic in ammation by blocking or reducing the output from immune cells of pro-in ammatory cytokines, including TNF-α, IL-6, and IL-12. [10] Since the amplitude and duration of the in ammatory response are regulated precisely by IL-10, we analysed the plasma levels of IL-10 at various doses of opium abuse and it was observed that there was a gradual but nonsigni cant increase in the plasma levels of IL-10 with increasing dose of opium (Table 2). Further, Rossato et al. (2012) reported in an in vitro model, induction of the miR-187 in an IL-10 dependent manner, which further causes a transcriptional inhibition of mRNA of TNFα and IL-6 (via Ikβζ). [11] Thus suggesting an anti-in ammatory role of IL-10 and hsa-miR-187-5p. Correspondingly in the current study, with increasing dose, there was a downregulation of hsa-miR187-5p but non-signi cant an increasing level of IL-10. An up-regulation of miR-187 at higher doses was still not su cient to inhibit the expression of TNF-α and IL-6 mRNA. Thus, showing that a compensatory increase of pro-in ammatory cytokines may occur in response to rising IL-10.
At the cellular level, the hsa-miR-155-5p expression is strongly induced by different TLR ligands including LPS and consistent with the pro-in ammatory properties of hsa-miR-155-5p, TNF-α translation is enhanced by the presence of hsa-miR-155-5p via increasing mRNA stability [21]. Therefore, our reports of a dose-dependent signi cant increase in fold change expression of hsa-miR-155-5p (Table 3), and TNF-α levels are possibly due to chronic high levels of opium inducing the overexpression of hsa-miR-155-5p.
Clinical or recreational use/abuse of morphine has also been shown to be associated with sustained immune activation and promotes sepsis and septic shock, thus reinforcing the in ammatory effect of chronic exposure to opiates [22]. Further, a Pearson's correlation analysis (Table 5) showed miR-155-5p to be signi cantly negatively associated with IL-6 and TNF-α and miR-187-5p showed signi cantly positively associated with TNF-α at >1000 gm/day consumption. Also, linear regression analysis showed TNF-α to be an independent signi cant predictor of miR-187 expression (Table 6). Since miR187 regulates TNF-α expression via IL-10, the down-regulation of miR187 promotes the expression of TNF-α and in ammation. Also, TNF-α regulates the expression of miR-155-5p via IL-6, thus leading to an upregulation of miR-155-5p. Overall, with increasing opium consumption, there is a co-dependent regulatory network of cytokines and miRNAs which develop an in ammatory environment and thus showing a state of low-grade in ammation to be present in opiate addicts.
The small sample size is a limitation for the study and further larger prospective studies involving human participants along with the route of opiate abuse would be crucial to unravel the mechanistic details of altered dynamics of pro-and anti-in ammatory miRNAs. Another limitation is that a strict control on the dose of oral opium was reported by the attendants of the abusers; a better dose and response effect can be validated by using in-vitro models like cell lines or use of animal models.

Conclusion
The study results show that chronic opium abuse causes altered expression of hsa-miR187-5p, hsa-miR155-5p and may-be responsible for increasing serum TNF-α, lowering of IL-10. The altered dynamics of the two miRNA are affected by the daily consumption of opium, leading to the altered immune response observed with chronic opium abuse. Further, the characterization of these miRNAs in in-vitro models can explain the mechanistic details of immunomodulation observed in our study. We conclude that increasing consumption of opium enhanced in ammation by up-regulation of miR-155-5p and relative down-regulation of miR-187-5p, inhibiting IL-10, promoting IL-6 and relieving inhibition on TNF-α. Thus, opium dose may be responsible for the immunomodulation in opium abusers, leading to the aberrant expression of miRNA.
Future studies exploring the effect of chronic opiate dependence in association with duration of dependence can prove to be bene cial for better management of these patients.

Declarations
Con ict of Interest Statement: The authors declare no con ict of interests.     Only those cases for which dose was > = 1000 gm/day were considered for this model, which was signi cant at p < 0.020