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Dillon Hawley, Claire Kublin, Audrey Michel, Lisa Clapisson, Driss Zoukhri; RNAseq Profiling and Deep Immuno-phenotyping of Regenerating Lacrimal Glands. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1409.
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© ARVO (1962-2015); The Authors (2016-present)
The murine lacrimal gland (LG) is capable of repair following experimentally induced injury. The repair starts 2-3 days following injury and is usually completed by the 5th or 7th day. The purpose of the present studies was to use RNAseq technology and CyTOF deep immuno-phenotyping to identify mediators of LG repair.
The LGs of BALB/c mice were injected (2 ml) with either saline (control) or interleukin 1 alpha (1 mg). LGs were harvested 1, 2, 3, 4, 5, 7, or 14 days following injury. For RNAseq 500 ng of extracted RNA was used to construct libraries for sequencing on a HiSeq 2500 (Illumina, San Diego, CA). STAR aligner was used for genome alignment followed by RSEM and DESeq2 for gene expression quantification and downstream differential expression analysis. For immuno-phenotyping, single cells were isolated from LGs using collagenase II (264 U/mL) prior to staining with an optimized cocktail of 30 metal-conjugated antibodies and acquisition on a CyTOF II (Fluidigm, San Francisco, CA).
Of a total of 43,346 Coding DNA Sequences analyzed, the expression of only 2,188 was statistically significantly different compared to saline injected LGs. The majority of the genes differentially expressed at day 1 and 2 were associated with inflammation. Between day 4 and 5 (tissue repair phase), there was selective upregulation of extracellular matrix (ECM) macro-components (collagens, elastin, tenascins, etc.), enzymes involved in synthesis of ECM components (lysyl oxidase) and ECM remodeling enzymes (MMPs, ADAMs, and ADAMTS). Immuno-phenotyping identified neutrophils and monocytes as the major infiltrating cell populations at days 1, 2, and 3, while pDCs, NK, B, and T cell populations showed no major changes compared to uninjured LGs.
The innate immune system, neutrophils and monocytes, are identified as key mediators of the inflammatory response in acute LG injury. Following this inflammatory response, the LG undergoes dramatic histological alterations during regeneration, although, the expression of only a few genes seems to be differentially altered during 3 and 7 days post injury.
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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