The role and host-directed targeting of long non-coding RNAs in macrophage polarization during Mycobacterium tuberculosis infection

In 2020, the World Health Organization (WHO) reported 1.5 million tuberculosis (TB)- associated deaths and an incidence of 10 million new cases. The causative, Mycobacterium tuberculosis (Mtb), evades host immune responses by skewing macrophage polarization towards a less microbicidal alternative state to avoid classical effector killing functions. However, the molecular details underlying these evasion mechanisms remain incomplete and current therapy is challenged with drug resistance. Host-directed therapy (HDT) has recently gained attention, with long non-coding RNAs (lncRNAs) as potential targets due to their emerging roles in pathogenic immune responses. We previously performed cap analysis gene expression (CAGE) transcriptomics on IFN-γ stimulated (classically activated) and IL-4/IL-13 stimulated (alternatively activated) mouse macrophages, identifying 151 differentially expressed lncRNAs following Mtb infection. We validated the top 11 differentially expressed lncRNAs and two were chosen for this study, lncRNA-125, whose expression was regulated at different levels unstimulated and in response to IFN-γ and IL-4/IL-13, and lncRNA-612 whose expression was only induced by IFN-γ stimulation. Interestingly, the expression of lncRNA125 and lncRNA-612 was downregulated following Mtb infection. Therefore, this study aimed at functionally validating these lncRNAs in unstimulated, IFN-γ and IL-4/IL-13 stimulated and/or Mtb-infected mouse and human macrophages by a loss-of-function approach using chemically engineered antisense oligonucleotides (gapmeRs). Knockdown of lncRNA-125 by gapmeRs reduced Mtb growth and anti-inflammatory cytokine production mediated by increased apoptosis, nitrite and pro-inflammatory cytokine production in IL-4/IL-13 prestimulated mouse macrophages. Whereas knockdown of lncRNA-125 in IFN-γ pre-stimulated mouse macrophages favoured Mtb growth and anti-inflammatory cytokine production, with reduction of apoptosis, nitrite and pro-inflammatory cytokine production. Therefore, indicating that lncRNA-125 regulates macrophage polarization during Mtb infection. Knockdown of lncRNA-125 in human macrophages resulted in reduced Mtb growth and increased proinflammatory cytokine production in unstimulated, IFN-γ and IL-4/IL-13 pre-stimulated BMDMs infected with Mtb. Comparatively, gapmeR knockdown of lncRNA-612 reduced Mtb growth and increased pro-inflammatory cytokine production in IFN-γ pre-stimulated mouse and human macrophages. In mouse macrophages, these responses were mediated by increased apoptosis and nitrite production, with reduced anti-inflammatory cytokine production. Overall, these findings highlight lncRNAs as novel host factors to be further investigated as targets for TB diagnostics and adjunctive HDTs.

Grantor

Department of Clinical Laboratory Sciences

Year dc:date.issued

2022

Author dc:creator

• Pillay, Shandré

Advisors dc:contributor.advisor

• Brombacher, Frank
• Guler, Reto
• Tamgue, Ousman

• clinical laboratory sciences

Handle dc:identifier.uri

http://hdl.handle.net/11427/36719

OAI identifier oai:identifier

oai:open.uct.ac.za:11427/36719