Morphine-induced exacerbation of activated immune response is mediated by miR-155

According to data from 2018, 128 people died in United states after opioid overdose every day, while in Oklahoma, 43% of drug overdose deaths involved opioid abuse contributing to a major health problem of epidemic proportions. Among opioid use disorders, chronic morphine use and abuse has been documented to severely compromise the immune system and hence, increase the risk of opportunistic infections like tuberculosis, HIV infections and pneumonia in opioid addicts. Morphine-induced immune modulation has been shown to be either indirect (ultimately affecting CNS) or direct through opioid receptor on immune cells. Also, many studies have also identified changes in expression profiles of specific miRNAs after chronic morphine abuse. In the current study, we are targeting regulation of OPRM1 gene encoding mu opioid receptor by miR-155 through transcription factor PU.1 binding to distal promoter of OPRM1 in morphine-induced immunosuppression.

Aim: We are establishing miR-155 mediated regulation of transcription factor PU.1 ultimately regulating OPRM1 gene expression in LPS-induced immune cells activation.

Method: We used WT and mu opioid receptor knockout (MORKO) mice and splenic macrophage cell line J774.1 to study morphine-induced immunosuppression. Blood samples from mice were used for miRNA array study to select miR-155 as a target miRNA. J774.1 cells were activated by LPS treatment (100ng/ml) and morphine sulfate (1uM) was used to suppress LPS-induced activation. RNA and proteins were extracted from these cells to evaluate the expression of mu opioid receptor and miR-155 expression. We used techniques like PCR, quantitative PCR, western blotting in this study.

Results and Discussion: miRNA-155 was confirmed as a target microRNA from miRNA array. This was further confirmed in splenic macrophages rather than T cells, thymus cells or bone marrow cells in mice tissues. For in vitro studies, we selected splenic macrophage cell line J774.1 and it showed the similar results compared to splenic macrophages. We further confirmed that miR-155 targets transcription factors PU.1 and C/EBPb. Distal promoter of OPRM1 has PU.1 binding site which is involved in negative regulation of OPRM1 gene expression. For future directions, we are targeting miR-155 overexpression, antagomir assay against miR-155, luciferase assay and chromatin immunoprecipitation experiments to further confirm LPS-induced, PU.1-mediated regulation of OPRM1 gene.