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Purpose: To identify single nucleotide polymorphisms (SNPs) in the brain-derived neurotrophic factor (BDNF), vitamin D receptor (VDR), and DNASE1 genes that may be associated with dry eye disease (DED), and determine whether this association varies by the presence of depression., Methods: A case-control study was performed with 64 DED cases and 51 controls. We collected 2 mL of saliva following a routine eye exam. Genotyping was performed using both custom and predesigned TaqMan SNP genotyping assays for 12 hypothesized SNPs. Genotype and allele frequencies of cases and controls were evaluated. Odds ratios were calculated for allele frequencies. Stratified analysis was performed to determine if the association between SNPs and DED varied by depression status., Results: A total of 18% of cases had the minor allele A of Val66Met (rs6265) SNP in the BDNF gene compared with 9% of the controls (P = 0.05). Odds ratio was 2.22. Two SNPs (Fokl-rs2228570 and Apal-rs7975232) in the VDR genes also varied between DED cases and controls. Cases were 1.72 and 1.66 times more likely to have the minor allele A in rs2228570 and rs7975232, respectively, than controls (P = 0.06 for both). While not statistically significant, among patients with depression, DED cases were 3.93 times more likely to have the minor allele A of the Val66Met SNP compared to controls., Conclusions: This pilot study showed that Val66Met in the BDNF gene and two SNPs, Fokl and Apal, in the VDR gene may potentially be associated with DED. Additionally, the association between DED and Val66Met may vary by depression status.

Purpose: To determine the abundance of extracellular DNA (eDNA) in tear fluid of patients with dry eye disease (DED) and to report clinical outcomes after DNase I eyedrops use to reduce excessive tear fluid eDNA., Methods: Tear fluid was collected from healthy control subjects and patients with DED. The eDNA abundance was determined with the PicoGreen dye assay. The DED symptoms and clinical signs were recorded and correlated with eDNA abundance. Two patients with DED having excessive eDNA in tear fluid were treated with DNase I eyedrops., Results: The PicoGreen dye assay measures tear fluid eDNA abundance after a 2-minute incubation time. With longer incubations, admixed cells also contribute to eDNA measurements. The mean (SE) eDNA abundance in healthy control subjects' tear fluid was 1.4 (0.2) μg/mL. The mean (SE) eDNA abundance in tear fluid of patients with nonautoimmune DED, autoimmune DED, and graft versus host disease was significantly higher: the values were 2.9 (0.6), 5.2 (1.2), and 9.1 (2.3) μg/mL, respectively (P < 0.05). In most of these patients, the PicoGreen dye kinetic assay of tear fluid showed an increase in fluorescence signal due to the presence of viable cells in tear fluid. Tear fluid eDNA had the best correlation with corneal Rose Bengal staining (r = 0.55). Treatment of patients having DED with DNase I eyedrops reduced eDNA abundance, abrogated signal increase, and improved comfort., Conclusions: Excessive eDNA is present in tear fluid of patients with dry eyes. A novel therapeutic approach for managing DED may be to measure eDNA abundance in tear fluid with the PicoGreen dye assay and reduce excessive amounts with DNase I eyedrops.

Purpose: We characterized fluorescent bone marrow cells (YFP(+) BMCs) in the thy1-YFP mouse and determine if they promote trigeminal ganglion (TG) cell neurite growth., Methods: Excimer laser annular keratectomy was performed in thy1-YFP mice, and corneas were imaged. BMCs were harvested from femur and tibia, and the expression of surface markers on YFP(+) BMCs was analyzed by flow cytometry. The immunosuppressive action of BMCs (YFP(+) and YFP(-)) was evaluated in an allogenic mixed lymphocyte reaction (MLR). Neurotrophic action of BMCs (YFP(+) and YFP(-)) was determined in compartmental and transwell cultures of dissociated TG cells., Results: Following annular keratectomy, YFP(+) BMCs infiltrated the cornea. YFP(+) BMCs shared surface markers (CD11b+Gr1+Ly6C+Ly6G-F4/80(low)) with monocytic myeloid-derived suppressor cells (MDSCs), had similar morphology, and suppressed T-cell proliferation in allogenic MLR in a dose-dependent manner. YFP(+) BMCs, but not YFP(-) BMCs, significantly increased growth of TG neurites in vitro. When cultured in a transwell with TG neurites, YFP(+) BMCs expressed neurotrophins and secreted nerve growth factor (NGF) in conditioned medium. YFP(+) BMCs that infiltrated the cornea maintained their phenotype and actions (neuronal and immune)., Conclusions: YFP(+) BMCs in thy1-YFP mice have immunophenotypic features of MDSCs. They secrete NGF and promote neuroregeneration. Their immunosuppressive and neurotrophic actions are preserved after corneal infiltration. These findings increase our understanding of the beneficial roles played by leukocyte trafficking in the cornea and may lead to therapeutic strategies that use NGF-secreting myeloid cells to repair diseased or injured neurons.

Purpose: We determined whether nucleases are deficient in the tear fluid of dry eye disease (DED) patients, and whether this causes extracellular DNA (eDNA) and neutrophil extracellular trap (NET) accumulation in the precorneal tear film, thus causing ocular surface inflammation., Methods: Exfoliated cells adhered to Schirmer test strips were collected on glass slides, and immunofluorescence confocal microscopy was used to evaluate neutrophils, eDNA, NETs, and their molecular components. Similar experiments were performed with mucoid films collected from the inferior conjunctival fornix or bulbar conjunctiva. We used quantitative PCR to evaluate eDNA signaling pathways and inflammatory cytokine expression. We also determined the amount of ocular surface eDNA and evaluated tear fluid nuclease activity., Results: eDNA, NETs, and neutrophils were present on the ocular surface in DED patients and abundant in mucoid films. NETs consisted of eDNA, histones, cathelicidin, and neutrophil elastase. Tear fluid nuclease activity was decreased significantly in DED patients, whereas the amount of eDNA on the ocular surface was increased significantly. Expression of genes downstream of eDNA signaling, such as TLR9, MyD88, and type I interferon, as well as the inflammatory cytokines interleukin-6 and tumor necrosis factor-α, was significantly increased in DED patients., Conclusions: Extracellular DNA production and clearance mechanisms are dysregulated in DED. Nuclease deficiency in tear fluid allows eDNA and NETs to accumulate in precorneal tear film, and results in ocular surface inflammation. These findings point to novel therapeutic interventions in severe DED based on clearance of eDNA, NETs, and other molecular components from the ocular surface.