Abstract
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.
Highlights
Visceral leishmaniasis (VL), caused by Leishmania donovani, results in almost 0.5–1 million new infection cases worldwide [1]
Computational analysis as well as immune precipitation based experiments authenticated that L. donovani induces super enhancer complex mediated transcriptional upregulation of miR146a-5p
bone marrow derived macrophages (BMDMs) were infected with virulent L. donovani promastigotes at increasing multiplicity of infection ratios (MOIs) ranging from 1:0.5 to 1:20 and times of infection
Summary
Visceral leishmaniasis (VL), caused by Leishmania donovani, results in almost 0.5–1 million new infection cases worldwide [1]. Lack of a successful vaccine, toxicity of available anti-leishmanial therapeutics, emergence of resistant parasite strains, HIV/VL co-infection and development of post kala-azar dermal leishmaniasis (PKDL) are causing therapeutic failure cases in VL [2]. L. donovani establishes an immune-supressive infection by colonizing within mammalian macrophages and trans-differentiating from promastigote to amastigote stage[4]. Bone marrow derived macrophages (BMDMs), polarized by Th2 cytokines like interleukin 4 (IL4) or interleukin 13 (IL13) are termed as M2 macrophages [7]. L. donovani skews Thelper (Th) response towards the Th2 type which involves induction of macrophage arginase 1 (Arg1) and IL10, and prevents pro-inflammatory cytokines and nitric oxide (NO) generation [6]. Besides Arg, M2 macrophages are characterized by enrichment of surface markers like C-C Motif Chemokine Receptor 7 (CCR7), chitinase 3 like-3 (YM-1), resistin-like molecule alpha(FIZZ1) as well as transcription factors like phosphorylated signal transducer and activator of transcription 6 (pSTAT6), CCAAT/enhancer-binding protein beta (c/EBPβ) etc.[7,8,9,10,11]
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