Characteristics of women according to the presence of dry eye.

<p>Characteristics of women according to the presence of dry eye.</p

Characteristics of men according to the presence of dry eye.

<p>Characteristics of men according to the presence of dry eye.</p

Association between dry eye and depression in the overall participants and by sex.

<p>Association between dry eye and depression in the overall participants and by sex.</p

Change in CD45⁺ cells in dry eye-induced lacrimal glands.

<p>During 10 days of DE induction, (A) LGs were obtained on Days 0, 2, 4, 6, and 10 for IHC staining of CD45⁺ cells. (B) The fold change of CD45⁺ cells was measured at Days 0, 2, 4, 6, and 10. (C) During DE induction, several groups of mice were administered anti-Dll4 Ab and GSI to inhibit the NOTCH1-DLL4 axis. Anti-IgG Ab was administered as a control for the anti-Dll4 Ab group, and DMSO was administered as a negative control. LGs were obtained at Day 10 of DE induction and were prepared for IHC staining of CD45⁺ cells. (D) The actual percentage of CD45⁺ cells was calculated. (E) Flow cytometry for CD45⁺ cells was performed for each condition according to the manufacturer’s protocol. (F) DE was induced for 10 days in B6 and HIF-1CKOmice. LGs were obtained and prepared for IHC staining. (G) The actual percentage of CD45⁺ cells was calculated for non-DE HIF-1α CKO mice and the DE HIF-1α CKO mice. (H) Flow cytometry was performed for the two groups. Student’s t-test for statistical analysis: *p<0.05, **p<0.01, †p<0.001. Error bars indicate standard deviation. (CTL = normal control; DE = dry eye; α-Dll4 = anti-Dll4 antibody; α-IgG = anti-IgG antibody; GSI = γ-secretase inhibitor; DMSO = dissolved dimethyl sulfoxide; HIF-1α CKO = HIF-1α conditional knockout).</p

Change in NOTCH signaling in dry eye-induced lacrimal glands.

<p>After C57BL/6 mice were housed in a CEC with scopolamine administration for 10 days, LGs were obtained and prepared for qRT-PCR, and (A) mRNA levels of NOTCHs, DLLs, and JAGs were measured. (B) The change in the mRNA level of NOTCH1/DLL4 during DE induction was measured (C) During DE induction, each LG samples were prepared for immunoblot for NOTCH1 and DLL4 at Day 2, Day 4, Day 6, Day 8, and Day 10. Densitometry for protein concentreation quantification was done by using ImageJ software. Student’s t-test for statistical analysis: *p<0.05, **p<0.01, †p<0.001. Error bars indicate standard deviation. (CTL = normal control; DE = dry eye).</p

Change in NOTCH signaling and LYVE-1 expression in dry eye-induced HIF-1α conditional knockout mice.

<p>WT B6 and HIF-1α CKO mice were housed in a CEC with scopolamine administration for 10 days. LGs were obtained and prepared for qRT-PCR and (A) The mRNA level of NOTCH1, DLL4, LYVE-1, and Podoplanin at Day 10 was measured. (B) Immunoblot and densitometry for NOTCH1 and LYVE-1 were measured at Day 10. (C) Immunofluorescence staining of LYVE-1 was performed for DE-induced WT B6 mice and DE-induced HIF-1α CKO mice at Day 10. Student’s t-test for statistical analysis: *p<0.05, **p<0.01. Error bars indicate standard deviation. (WT = wild-type; DE = dry eye; HIF-1α CKO = HIF-1α conditional knockout).</p

Change in LYVE-1, VEGF-C, VEGF-D, and VEGFR3 expression after inhibition of the NOTCH1-Dll4 axis by intraperitoneal injection of monoclonal anti-Dll4 antibody and γ-secretase inhibitor.

<p>While C57BL/6 mice were housed in a CEC with scopolamine administration for 10 days, several groups of mice were administered anti-Dll4 Ab and GSI for inhibiting the NOTCH1-DLL4 axis. Anti-IgG Ab was administered as a control for the anti-Dll4 antibody group, and DMSO was administered as a negative control. LGs were obtained after 10 days of DE induction and were prepared for qRT-PCR, immunoblot, and immunostaining. (A) The mRNA levels of LYVE-1, VEGF-C, VEGF-D, and VEGFR3 were measured using qPCR for each group. (B) Immunoblot and densitometry of LYVE-1 were measured for each group. (C) Immunofluorescence staining of LYVE-1 was performed for each group. Student’s t-test for statistical analysis: *p<0.05, **p<0.01. Error bars indicate standard deviation. (CTL = normal control; DE = dry eye; α-Dll4 = anti-Dll4 antibody; α-IgG = anti-IgG antibody; GSI = γ-secretase inhibitor; DMSO = dissolved dimethyl sulfoxide).</p

Dry eye stress activates NOTCH1-Dll4 axis and HIF-1α during lymphatic vessel formation of dry eye-induced lacrimal glands.

<p>DE stress activates Dll4/Notch pathway and HIF-1α in LGs. HIF-1α upregulates Dll4/Notch pathway and promotes lymphangiogenesis. Activation of Dll4/Notch pathway and HIF-1α results in increase of VEGF-C, VEGF-D, and VEGFR3, which results in lymphangiogenesis in LGs. (DE = dry eye).</p

Change in LYVE-1, PECAM, VEGF-C, VEGF-D, and VEGFR3 expression in dry eye-induced lacrimal glands.

<p>During 10 days of DE induction, LGs were obtained and prepared for qRT-PCR and (A) the mRNA level of LYVE-1 and PECAM was measuredat Day 2, Day 4, Day 6, Day 8, and Day 10 of DE induction. (B) The mRNA levels of VEGF-C, VEGF-D, and VEGFR3 were measured at Day 10. (C) Immunofluorescence staining of LYVE-1 for DE and control group was performed at Day 10. Student’s t-test for statistical analysis: *p<0.05, **p<0.01. Error bars indicate standard deviation. (CTL = normal control; DE = dry eye).</p

Langerhans cells prevent subbasal nerve damage and upregulate neurotrophic factors in dry eye disease

<div><p>The functional role of Langerhans cells (LCs) in ocular surface inflammation and nerve damage in dry eye (DE) disease has yet to be determined. This study was performed to investigate this relationship through both clinical study on DE patients and in vivo mouse models with induced DE disease. In a cross-sectional case-control study (54 eyes of DE patients; 34 eyes of control patients), average cell density, area, and process length of LCs were measured using confocal microscopy. Data were analyzed to determine whether changes in LCs are correlated with subbasal nerve plexus (SNP) parameters (nerve density, beading, and tortuosity). In DE patients, SNP density marginally decreased and nerve beading and tortuosity were significantly increased compared to the control group. The total number of LCs significantly increased in DE patients, and some LCs with elongated processes were found to be attached to nerve fibers. Interestingly, nerve loss and deformation were correlated with inactivation of LCs. In an <i>in vivo</i> experiment to elucidate the role of LCs in ocular surface inflammation and corneal nerve loss, we used a genetically modified mouse model (CD207-DTR) that reduced the population of CD207 (Langerin) expressing cells by injection of diphtheria toxin. In CD207-depleted mice with DE disease (CD207-dDTR+DE), corneal nerves in the central region were significantly decreased, an effect that was not observed in wild-type (WT)+DE mice. In CD207-dDTR+DE mice, infiltration of CD4+, CD19+, CD45+, and CD11b+ cells into the ocular surface was increased, as confirmed by flow cytometry. Increased IL-17 and IFN-γ mRNA levels, and decreased expression of neurotrophic factors and neurotransmitters, were also found in the CD207-dDTR+DE mice. These data support a functional role for LCs in negatively regulating ocular surface inflammation and exhibiting a neuroprotective function in DE disease.</p></div