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Shunbin Xu, Chithra Muraleedharan; The miR-183/96/182 Cluster Regulates Cytokine Production By Macrophages (MΦ) In Response To Lipopolysacchride (LPS). Invest. Ophthalmol. Vis. Sci. 2017;58(8):5775.
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Previously, we showed that the miR-183/96/182 cluster is expressed in innate immune cells, including MΦ and polymorphonuclear neutrophils (PMN). Inactivation of this cluster in mice led to decreased pro-inflammatory chemotactic cytokines (e.g., MCP1, MIP2 and IL-1β) in the cornea after Peudomonas aeruginosa (PA) infection and an overall decreased severity of PA keratitis. PMN from knockout (ko) mice and MΦ with knockdown of the cluster had enhanced phagocytic and killing abilities. These data suggest that this cluster modulates corneal response to PA infection through its regulation on innate immune cells. We hypothesize that miR-183/96/182 has significant roles in cytokine production of MΦ in response to PA infection. This research is to start to test this hypothesis.
MΦ cell line, Raw264.7 cells (4 X105 cells/well. ATCC) were treated with PA (strain 19660, 1X107 CFU/well, MOI 25) for 3, 6 and 16 hours (hrs), or with LPS (Sigma), a major component of PA, at 0.5, 100 and 1000 ng/ml for 6 and 16 hrs. Total RNA was harvested for qRT-PCR, and supernatant of the media for ELISA assays. To understand its role in chemokine production in MΦ, we knocked down the function of the cluster by transfection of anti-miR-183 and anti-miR-182 (anti-miR-183/-182. 10 nM each) in Raw cells, since miR-96 is barely expressed and has little functional significance in MΦ. 48 hrs after transfection, cells were treated with LPS (100 ng/ml) for 6 hrs followed by harvesting total RNA for qRT-PCR and supernatant of the media for ELISA assays.
1) PA or LPS treatment significantly induced production of MCP1, MIP2 and IL-1β in a time- and dosage-dependent manner. Significant and non-saturated responses were observed at 6 hrs; 100 ng/ml of LPS induced a near maximal response.2) In anti-miR-183/182 transfected cells, miR-183 and miR-182 were down-regulated, while the expression of miR-96 was unaffected. ELISA assays showed that knockdown of miR-183/-182 resulted in a significant decrease of LPS-induced MCP1, MIP2 and IL-1β production.
Our data suggest that miR-183/96/182 cluster directly regulates chemokine production by MΦ in response to LPS, which contributes to its modulation of the overall corneal response to PA infection. Further studies will uncover the underlying molecular mechanism and the potential of miR-183/96/182 as a therapeutic target for treatment of PA infection.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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