Damage associated molecular pattern molecule-induced microRNAs (DAMPmiRs) in human peripheral blood mononuclear cells
Sebnem Unlu1, Siuwah Tang, Ena Wang
1Department of Surgery, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America. unlus@upmc.edu
Abstract:
Endogenous damage associated molecular pattern molecules (DAMPs) released from necrotic, damaged or stressed cells are associated with an inflammatory response. Whether the microRNA (miR) expression signature of this response is different from that of a pathogen associated molecular pattern (PAMP)-stimulated inflammatory response is unknown. We report here that miR-34c and miR-214 are significantly expressed in fresh human peripheral blood mononuclear cells (PBMCs) exposed to DAMP-containing freeze-thaw lysates, or to conditioned media from serum-starved and glucose-deprived cells (p<6×10(-4) and p<3.7×10(-3)), respectively. Interestingly, only miR-34c expression was differentially expressed in PBMCs exposed to freeze-thaw lysates or conditioned media from wildtype High Mobility Group B1 (HMGB1(+/+)) mouse embryonic fibroblast (MEF) cells, when compared to cultures exposed to lysates or conditioned media from HMGB1(-/-) MEFs. miR-155 expression in these cultures was negligible, but was significantly expressed in PBMCs stimulated with Lipopolysaccahride (LPS) or most other Toll-like receptor (TLR) ligands, making it the prototypic "PAMPmiR". Exposure to a damaged human colorectal carcinoma cell line lysate (HCT116) similarly resulted in increased miR-34c and miR-214 levels. When PBMCs were pre-transfected with anti-miR-34c and then exposed to lysate, expression levels of IKKγ mRNA, a putative target of miR-34c, increased, while protein levels of IKKγ in cultures transfected with a pre-miR-34c were abrogated. Levels of miR-34c expression (as well as pro-inflammatory cytokines, IL-1β and TNFα) decreased when PBMC cultures were briefly pre-incubated with the K(+) channel (inflammasome) inhibitor, glybenclamide, suggesting that inflammasome activation is upstream of miR-34c expression in response to DAMPs. Our findings demonstrate that a specific microRNA expression signature is associated with the inflammatory response to damaged/injured cells and carries implications for many acute and chronic inflammatory disorders.
Insights
Specific microRNAs, miR-34c and miR-214, are upregulated in response to cellular damage signals (DAMPs). This contrasts with pathogen signals (PAMPs), highlighting a distinct microRNA signature for DAMP-induced inflammation.
Area of Science:
- Immunology
- Molecular Biology
- Cellular Biology
Background:
- Endogenous damage-associated molecular patterns (DAMPs) trigger inflammatory responses.
- The microRNA (miR) expression profile distinguishing DAMP-stimulated from pathogen-associated molecular pattern (PAMP)-stimulated inflammation remains unclear.
Purpose of the Study:
- To investigate the specific microRNA signature associated with DAMP-induced inflammation.
- To compare the microRNA response to DAMPs versus PAMPs.
- To explore the regulatory mechanisms of microRNA involvement in DAMP responses.
Main Methods:
- Human peripheral blood mononuclear cells (PBMCs) were exposed to DAMP sources (freeze-thaw lysates, conditioned media from stressed cells, cancer cell lysates).
- Differential microRNA expression analysis was performed.
- Specific microRNAs (miR-34c, miR-214, miR-155) and their targets (IKKγ) were analyzed using transfection and Western blotting.
- The role of inflammasome activation was assessed using the inhibitor glybenclamide.
Main Results:
- miR-34c and miR-214 were significantly upregulated in PBMCs exposed to DAMPs.
- miR-155 was identified as a prototypic 'PAMPmiR', upregulated by LPS and TLR ligands but not DAMPs.
- miR-34c regulates IKKγ expression, and its upregulation is dependent on inflammasome activation.
- DAMP-induced inflammation involves a distinct microRNA signature, including miR-34c and miR-214.
Conclusions:
- A specific microRNA expression signature, characterized by miR-34c and miR-214, is associated with the inflammatory response to damaged cells (DAMPs).
- This finding differentiates DAMP-mediated inflammation from PAMP-mediated inflammation.
- The results have implications for understanding and potentially treating acute and chronic inflammatory disorders.
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