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

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.

Going ballistic: Leishmania nuclear subversion of host cell plasticity

“…There is good evidence that Leishmania acts on both of these TFs, likely establishing a mixed macrophage polarization profile. Leishmania survival has been shown to depend on STAT6 expression, which is induced in an miR146a-5p-dependent fashion during infection and enhances the expression of the M2 marker protein ARG1, an arginase that catalyzes the conversion of L-arginine into ornithine, known to promote Leishmania replication and survival [11,12]. In contrast, Leishmania infection interferes with NF-κB expression and function, and thus the antimicrobial M1 polarization profile at multiple levels (Figure 3), by inducing the formation of response on cellular metabolisms, and vice versa, the role of metabolic processes and individual metabolites in shaping the function of immune cells.…”

Leishmania survives by exporting miR-146a from infected to resident cells to subjugate inflammation

“…Inhibition of miR-146a lowered parasite burden in infected Balb/C mice as well as anti-inflammatory environment (M2 phenotype). Interestingly, this up-regulation of miR-146a during Ld infection is regulated by Super Enhancer components like BET bromodomain 4 (BRD4) and P300 ( Das et al, 2021 ).…”

miR-294 and miR-410 Negatively Regulate Tnfa, Arginine Transporter Cat1/2, and Nos2 mRNAs in Murine Macrophages Infected with Leishmania amazonensis

“…In L. major infection of human macrophages, hypoxia-inducible factor-1 (HIF-1α) regulates miR-210 levels, linking HIF-1α to susceptibility in other species, such as L. donovani and L. amazonensis [40,41]. miR-146a overexpression downregulates M1 markers such as Tnfa and Nos2, increasing Arg1 and IL-10 in L. donovani-infected BMDM [42]. Let-7e interferes in TLR, NFkB, IRF, and MAPK signaling in B6-BMDM infected with L. amazonensis [28].…”