July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
The miR-183/96/182 Cluster Modulates Pseudomonas aeruginosa-induced Keratitis Through Regulation of Innate Immune Cells
Author Affiliations & Notes
  • Shunbin Xu
    Ophthalmology/Anatomy & Cell Biology, Wanye State University, Detroit, Michigan, United States
  • Linda D Hazlett
    Anatomy & Cell Biology, Wayne State University School of Medicine, Detroit, Michigan, United States
  • Chithra Muraleedharan
    Ophthalmology/Anatomy & Cell Biology, Wanye State University, Detroit, Michigan, United States
  • Sharon A McClellan
    Anatomy & Cell Biology, Wayne State University School of Medicine, Detroit, Michigan, United States
  • Sandamali Amarasingha Ekanayaka
    Anatomy & Cell Biology, Wayne State University School of Medicine, Detroit, Michigan, United States
  • Ronald P Barrett
    Anatomy & Cell Biology, Wayne State University School of Medicine, Detroit, Michigan, United States
  • Footnotes
    Commercial Relationships   Shunbin Xu, Wayne State University (P); Linda Hazlett, Wayne State University (P); Chithra Muraleedharan, None; Sharon McClellan, None; Sandamali Amarasingha Ekanayaka, None; Ronald Barrett, None
  • Footnotes
    Support  This work was supported by grants R01 EY02605902 (SX), R01EY016058 (LDH), and P30EY004068 (LDH) from the National Eye Institute (NEI), National Institutes of Health (NIH) and by a Research to Prevent Blindness (RPB) unrestricted grant to the Department of Ophthalmology, Kresge Eye Institute. LDH is a recipient of an Alcon Research Institute Award.
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 1545. doi:https://doi.org/
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    • Get Citation

      Shunbin Xu, Linda D Hazlett, Chithra Muraleedharan, Sharon A McClellan, Sandamali Amarasingha Ekanayaka, Ronald P Barrett; The miR-183/96/182 Cluster Modulates Pseudomonas aeruginosa-induced Keratitis Through Regulation of Innate Immune Cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):1545. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : The miR-183/96/182 cluster (miR-183/96/182) plays an important role in Pseudomonas aeruginosa (PA)-induced keratitis. Inactivation of miR-183/96/182 in mice resulted in decreased pro-inflammatory cytokines in the infected cornea, increased phagocytosis and intracellular killing of PA by macrophages (MΦ) and polymorphonuclear neutrophils (PMN), and ultimately, decreased severity of keratitis. The current study dissects the underlying molecular mechanisms in these events.

Methods : Mouse MΦ-like Raw264.7 cells (ATCC) were transfected with anti-miR-183/96/182 (Exiqon). Thioglycollate or casein-elicited peritoneal MΦ or PMN were isolated from miR-183/96/182 knockout (miR-183C-/-. ko) and wild-type (wt) mice, and stimulated with lipopolysaccharide (LPS) (100 ng/ml. Sigma) or PA (MOI 25, Strain 19660, ATCC) for 6 hours. The supernatant was collected for ELISA assays (R&D Systems); cells were harvested to prepare RNA for quantitative RT-PCR, or protein analyses by Western blot or ELISA. DNAX Activating Protein 12 kDa (DAP12) is a predicted target of miR-183. DAP12 ko mice (kindly provided by Dr. Lewis L. Lanier of UCSF) were bred with miR-183C+/- to produce miR-183/96/182 ko mice on the background of DAP12+/- (miR-183C-/-; DAP12+/-) or DAP12+/+ (miR-183C-/-;DAP12+/+), which were then subjected to PA infection as we described previously.

Results : Knockdown (KD) of miR-183/96/182 in Raw cells resulted in decreased pro-inflammatory cytokines upon LPS and/or PA stimulation. Peritoneal MΦ from ko mice showed an increased basal level of pro-inflammatory cytokines; however, a decreased induction in response to LPS and PA stimulation. PMN from ko mice showed increased reactive oxygen species (ROS) production. KD or over-expression of miR-183/96/182 in Raw cells resulted in increased or decreased expression of DAP12, respectively, suggesting DAP12 is targeted by miR-183/96/182. Decreased dosage of DAP12 in miR-183C-/-;DAP12+/- mice led to increased severity of PA keratitis compared to miR-183C-/-;DAP12+/+ mice, similar to the response of miR-183/96/182 wt mice.

Conclusions : miR-183/96/182 regulates pro-inflammatory cytokine expression in MΦ and ROS production in PMN, which may contribute to a decreased inflammatory response and increased intracellular bacterial killing by PMN in the ko mice. DAP12 potentially mediates, at least partially, the functions of miR-183/96/182 in PA keratitis.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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