Chronic opiate abuse is capable of significant immunomodulation and possibly forms the basis of various diseases associated with opiate abuse . Regulation of inflammatory 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 inflammatory 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 . The current study for the first time shows the effect of chronic opiate abuse, as well as its dose on the expression of inflammatory cytokines and their associated miRNAs (Tables 1 & 3). We have reported earlier in a prospective clinical study a strong association of low-grade inflammation with dependence years in pure opiate abusers . 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 significantly raised levels of TNF-α suggest a pro-inflammatory status.
The hsa-miR-155-5p is a pleiotropic molecule that regulates both central and peripheral inflammation  (Qayum et al., 2016). It has been reported to be induced by both IL-10  and TNF-α . We observed chronic opiate abuse induces an inflammatory response, wherein as the dose increases, the anti-inflammatory response of IL-10 is over-ridden as observed by its non-significant increase and a significant 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). 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 pro-inflammatory 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 . 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-inflammation in epilepsy in both human and animal model . A similar axis may be responsible for the dose-dependent immunomodulation to enhance a pro-inflammatory cytokine response of chronic opium abusers.
IL-10 negatively regulates acute and chronic inflammation by blocking or reducing the output from immune cells of pro-inflammatory cytokines, including TNF-α, IL-6, and IL-12.  Since the amplitude and duration of the inflammatory 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 non-significant 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). Thus suggesting an anti-inflammatory 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-significant an increasing level of IL-10. An up-regulation of miR-187 at higher doses was still not sufficient to inhibit the expression of TNF-α and IL-6 mRNA. Thus, showing that a compensatory increase of pro-inflammatory 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-inflammatory properties of hsa-miR-155-5p, TNF-α translation is enhanced by the presence of hsa-miR-155-5p via increasing mRNA stability . Therefore, our reports of a dose-dependent significant 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 inflammatory effect of chronic exposure to opiates. Further, a Pearson’s correlation analysis (Table 5) showed miR-155-5p to be significantly negatively associated with IL-6 and TNF-α and miR-187-5p showed significantly positively associated with TNF-α at >1000 gm/day consumption. Also, linear regression analysis showed TNF-α to be an independent significant 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 inflammation. 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 inflammatory environment and thus showing a state of low-grade inflammation 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-inflammatory 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.