Super enhancer-mediated transcription of miR146a-5p drives M2 polarization during Leishmania donovani infection.

The outcome of Leishmania donovani infection depends upon the dynamic interchanges between M1 and M2 macrophages. Information of the involvement of microRNAs (miRNAs) and epigenetic modifiers in regulating macrophage plasticity during L. donovani infection is still elusive. Differential expression analysis of polarization-regulating miRNAs, revealed significant enrichment of miR146a-5p during Leishmania donovani infection. A sustained enrichment of miR146a-5p was observed in both infected bone marrow derived macrophages (BMDMs) and BALB/c mice organs. We found involvement of miR146a-5p in phagocytosis and survivability of parasites. Moreover, miR146a-5pgot enriched in interleukin 4- stimulated BMDMs, indicating its possible involvement in M2 polarization. Upon transfecting BMDMs with miRVANA anti-146a oligos, M2 markers (CCR7, YM-1, FIZZ-1, arginase-1, IL10 and IL4) and transcription factors (p-STAT6 and c/EBPβ) got depleted with concomitant augmentation of M1-polarizing transcription factors (p-STAT1, AP1 and IRF-1), miR146a target genes (TRAF6 and IRAK1), M1 cytokines (IL12 and TNFα), iNOS, nitric oxide, and nuclear translocation of phospho p-65 subunit. Neutralization of intracellular mature miR146a-5p pool in infected BALB/c mice lower organ parasite burden and expressions of M2 markers and IL10 with enrichment of M1 markers like iNOS and IL12. Additionally, we explored the novel role of super enhancer (SE), a cis-acting regulatory component, to enrich miR146a-5p expression during infection. Enhanced expression and nuclear retention of SE components like BET bromodomain 4 (BRD4) and p300 were found in infected BMDMs. Upon silencing BRD4, expressions of miR146a-5p and M2 markers were down regulated and TRAF6, IRAK1 and iNOS levels increased. STRING V.11 based predication and immune precipitation confirmed the strong interaction amongst BRD4, p300 and RNA pol II (RpbI). Chromatin immune precipitation studies suggested the recruitment of BRD4 at the enhancer loci of miR146a-5p gene during infection. Altogether, our findings revealed a novel role of BRD4/p300-depdendent super-enhancer in regulating miR146a expression during L. donovani infection which in turn mediates M2 polarization and immune-suppression. Author summary: Visceral leishmaniasis (VL), caused by protozoan parasites Leishmania donovani, is the most severe form of leishmaniasis and is highly lethal if left untreated. Major obstacle for successful therapy of VL originates from the life-long immune-suppression triggered in the post kala-azar dermal leishmaniasis (PKDL) patients during infection. Identification of molecular principles behind such immune-suppression will add success in VL therapeutics. L. donovani hijacks the host macrophages and converts them from pro-inflammatory M1 to immune-suppressive M2 type, which allows successful infection establishment. Herein, we explored the indispensable role of miRNA-146a-5p in conversion of M1 to M2 type during infection. Both in vitro and in vivo miRNA silencing established miR146a-5p as an imperative negative regulator ofM1 polarization. Computational analysis as well as immune precipitation based experiments authenticated that L. donovani induces super enhancer complex mediated transcriptional upregulation of miR146a-5p. BET bromodomain protein 4 (BRD4) forms this SE complex along with p300 histone acetyl transferase and RNA pol II. Silencing of BRD4 significantly abrogated miR146a-5p mediated M2 polarization. In short, our current findings established a previously unrecognized role of BRD4-depdendent super enhancers in orchestrating persistent transcription of macrophage miR146a-5p which in turn promotes M2 polarization during L. donovani infection. [ABSTRACT FROM AUTHOR]