Transcription Profiling of NOD-like Receptors in the Human Cornea with Disease

<p><i>Purpose</i>: To investigate the expression of nucleotide-binding oligomerization domain-like receptors (NLRs) in human corneas with disease and corneal cells.</p> <p><i>Methods</i>: The expression of NOD1, NOD2, NLRP1, and NLRP3 was analyzed using real-time RT-PCR in (1) corneas with active infection, history of herpetic stromal keratitis (HSK), chronic allograft rejection, and limbal stem cell deficiency (LSCD), and (2) human corneal cells after lipopolysaccharide (LPS) stimulation. Healthy corneas and cells without LPS served as controls.</p> <p><i>Results</i>: The mRNA levels of <i>NOD2</i> and <i>NLRP3</i> were increased in corneas with infection and HSK. Conversely, the levels of <i>NOD1, NOD2, NLRP1</i>, and <i>NLRP3</i> transcripts were decreased in corneas with LSCD. In corneas with rejection, the expression of <i>NOD1</i> and <i>NLRP1</i> was downregulated. Corneal endothelial cells upregulated the expression of <i>NOD2</i> and <i>NLRP3</i> upon LPS.</p> <p><i>Conclusions</i>: The changes in the NLR expression may reflect different susceptibility to infectious and non-infectious injuries in corneas with various diseases.</p

Microarray-based analysis of gene expression profiles in peripheral blood of patients with acute primary angle closure

<p><i>Background</i>: We investigated the expression of molecules in peripheral blood mononuclear cells (PBMCs) and plasma of patients with acute primary angle closure (APAC).</p> <p><i>Materials and methods</i>: Peripheral blood was collected from patients with APAC (<i>n</i> = 10) and age-matched controls (<i>n</i> = 5). The gene transcription profile was analyzed in PBMCs using microarrays and validated by real-time reverse transcription polymerase chain reaction (RT-PCR). The levels of secreted proteins were evaluated in plasma by ELISA.</p> <p><i>Results</i>: 347 gene transcripts were up-regulated by 2-fold or more, and 696 transcripts down-regulated 2-fold or more in PBMCs from patients compared to controls. The most highly up-regulated gene was thrombospondin-1 (<i>TSP-1</i>, 8.66-fold increase), and the most down-regulated gene was prostaglandin-endoperoxide synthase 2 (<i>PTGS2</i>, 9.09-fold decrease). Real-time RT-PCR assay confirmed the increase of <i>TSP-1</i> and the decrease of <i>PTGS2</i> in PBMCs of patients. ELISA revealed that the levels of <i>TSP-1</i> and active transforming growth factor (TGF)-β1 that is activated by <i>TSP-1</i> were elevated in plasma of patients, while the level of prostaglandin E2 (<i>PGE2</i>) that is converted by <i>PTGS2</i> was reduced. The plasma level of <i>TSP-1</i> was positively correlated with that of active <i>TGF-β1</i>.</p> <p><i>Conclusions</i>: Our data suggest that the molecular network including <i>TSP-1, TGF-β1</i>, and <i>PGE2</i> might be involved in the pathogenesis of APAC and PACG.</p

Effects of subconjunctival administration of anti-high mobility group box 1 on dry eye in a mouse model of Sjӧgren’s syndrome

<div><p>Purpose</p><p>Extracellular high mobility group box 1 (HMGB1) acts as a damage associated molecular pattern molecule through the Toll-like receptor to promote autoreactive B cell activation, which may be involved in the pathogenesis of Sjӧgren’s syndrome. The aim of this study was to investigate the effect of subconjunctival administration of anti-HMGB1 on dry eye in a mouse model of Sjӧgren’s syndrome.</p><p>Methods</p><p>Ten weeks-old NOD.B10.<i>H2</i>^<i>b</i> mice were subconjunctivally injected with 0.02 to 2 μg of anti-HMGB1 antibodies or PBS twice a week for two consecutive weeks. Tear volume and corneal staining scores were measured and compared between before- and after-treatment. Goblet cell density was counted in PAS stained forniceal conjunctiva and inflammatory foci score (>50 cells/focus) was measured in extraorbital glands. Flow cytometry was performed to evaluate the changes in BrdU^<b>+</b> cells, IL-17-, IL-10-, or IFNγ-secreting cells, functional B cells, and IL-22 secreting innate lymphoid cells (ILC3s) in cervical lymph nodes. The level of IL-22 in intraorbital glands was measured by ELISA.</p><p>Results</p><p>Injection of 2 μg or 0.02 μg anti-HMGB1 attenuated corneal epithelial erosions and increased tear secretion (p<0.05). Goblet cell density was increased in 0.2 μg and 2 μg anti-HMGB1-treated-mice with marginal significance. The inflammatory foci score, and the number of BrdU^<b>+</b> cells, IL-17-, IL-10-, IFNγ-secreting cells, and functional B cells did not significantly change following anti-HMGB1 treatment. Surprisingly, the percentage of ILC3s was significantly increased in the draining lymph nodes (p<0.05), and the expression of IL-22 was significantly increased in the intraorbital glands (p<0.05) after administration of 2 μg anti-HMGB1.</p><p>Conclusion</p><p>This study shows that subconjunctival administration of anti-HMGB1 attenuates clinical manifestations of dry eye. The improvement of dry eye may involve an increase of ILC3s, rather than modulation of B or plasma cells, as shown using a mouse model of Sjӧgren’s syndrome.</p></div

Changes in percentage of adaptive immune cells in draining lymph nodes of NOD.B10.<i>H2</i>^<i>b</i> mice after anti-HMGB1 treatment.

<p>(A) BrdU staining showing no significant proliferative changes in CD3^<b>+</b> T cells or B220^<b>+</b> B cells. (B) Percentage of Th17 (CD3^<b>+</b>CD4^<b>+</b>IL-17^<b>hi</b>) cells and Tc17 (CD3^<b>+</b>CD8^<b>+</b>IL-17^<b>hi</b>) cells were not altered after treatment. (C) IFNγ-secreting T cells (CD3^<b>+</b>CD4^<b>+</b>IFNγ^<b>hi</b> or CD3^<b>+</b>CD8^<b>+</b>IFNγ^<b>hi</b>) were not affected by treatment. Data are presented as mean ± standard error. (Th17, T helper cells secreting IL-17; Tc17, cytotoxic T cells secreting IL-17).</p

Functional characteristics of innate lymphoid cells (ILCs).

<p>Functional characteristics of innate lymphoid cells (ILCs).</p

Changes in innate lymphoid cells (ILCs) in draining lymph nodes and changes in IL-22 expression in intraorbital glands after anti-HMGB1 treatment.

<p>(A) Fold changes in ILC3 percentage (CD3^<b>-</b>B220^<b>-</b>CD45^<b>+</b>IL-22^<b>hi</b> cells; NCR^<b>+</b> or NCR^<b>-</b> ILC3) showing a significant increase following 2 μg anti-HMGB1 treatment compared with control (Kruskal-Wallis test, PBS vs. 2μg anti-HMGB1, *p = 0.025). Fold increase in NCR^<b>-</b> ILC3 percentage (CD3^<b>-</b>B220^<b>-</b>CD45^<b>+</b>NKp46^<b>-</b>IL-22^<b>hi</b> cells)(Kruskal-Wallis test, PBS vs. 2 μg anti-HMGB1, **p = 0.0142). (B) Increased IL-22 levels after 2 μg anti-HMGB1 treatment (Kruskal-Wallis test, PBS vs. 2 μg anti-HMGB1, *p = 0.025). (C) No change in percentage of CD3^<b>+</b>IL-22^<b>hi</b> cells (Th22 cells or γδ T cells) in draining lymph nodes. Data are presented as mean ± standard error. (D) Representative images of NCR^<b>-</b> ILC3s (CD3^<b>-</b>B220^<b>-</b>CD45^<b>+</b>NKp46^<b>-</b>IL-22^<b>hi</b>cells) in PBS- and 2 μg anti-HMGB1-treated groups. NCR, natural cytotoxicity receptor.</p

Changes in functional B cells in cervical lymph nodes, anti-SSA antibodies in serum, and inflammation foci scores in extra orbital lacrimal glands in NOD.B10.<i>H2</i>^<i>b</i> mice after anti-HMGB1 treatment.

<p>(A) No significant change in percentage of plasma cells (CD3^<b>-</b>B220^<b>+</b>CD138^<b>+</b> cells). (B) No change in IL-10-secreting B regulatory cells (CD3^<b>-</b>CD19^<b>+</b>B220^<b>+</b>IL-10^<b>hi</b>). (C) No change in the level of anti-SSA (RO 60) antibodies after treatment. (D) No significant changes in inflammatory foci scores (> 50 lymphocytes/focus) among all groups. Data are presented as mean ± standard error.</p

Intracellular and extracellular roles of HMGB1 protein.

<p>In the resting state, HMGB1 localizes to the nucleus, where it causes DNA bending and enhances the interaction of other proteins with DNA and their transcriptional activities. However, under conditions such as sterile injury or infection, HMGB1 is released either actively or passively into the extracellular space where it has distinctly different roles as a cytokine. The disulfide-bonded form of HMGB1, the usual form of extracellular HMGB1, elicits an inflammatory response, including dendritic cell maturation or autoreactive B cell activation, through specific receptors such as RAGE, TLR2, TLR4, or TLR9. Meanwhile, cysteine all-reduced HMGB1 does not have proinflammatory properties but behaves as a chemotactic cytokine through CXCR4. However, the cysteine all-oxidized form of HMGB1, which is produced during apoptotic cell death, loses the ability to induce inflammation and chemotaxis and gives rise to tolerance. HMGB1 = high mobility group box 1; HMGB1-SS = disulfide-bonded form of HMGB1; HMGB1-all Cys-SH = cysteine all-reduced HMGB1 (cysteines at positions 23, 45, and 106 of HMGB1 express a thiol group); HMGB1-Cys106-SO3H = cysteine 106-oxidized HMGB1 (cysteine at position 106 of HMGB1 expresses a sulfonic acid group); HMGB1-all Cys-SO3H = cysteine all-oxidized HMGB1 (cysteines at positions 23, 45, and 106 of HMGB1 express a sulfonic acid group); NA = nucleic acid; IL-1 = interleukin-1; LPS = lipopolysaccharide; CXCL12 = C-X-C motif chemokine ligand 12; RAGE = receptor for advanced glycation end products; TLR2 = toll-like receptor type 2; IL-1R = interleukin-1 receptor; TLR4 = toll-like receptor type 4; MD2 = lymphocyte antigen 96; CXCR4 = C-X-C motif chemokine receptor type 4; TLR9 = toll-like receptor type 9.</p

Stanniocalcin-1 Protects Retinal Ganglion Cells by Inhibiting Apoptosis and Oxidative Damage

<div><p>Optic neuropathy including glaucoma is one of the leading causes of irreversible vision loss, and there are currently no effective therapies. The hallmark of pathophysiology of optic neuropathy is oxidative stress and apoptotic death of retinal ganglion cells (RGCs), a population of neurons in the central nervous system with their soma in the inner retina and axons in the optic nerve. We here tested that an anti-apoptotic protein stanniocalcin-1 (STC-1) can prevent loss of RGCs in the rat retina with optic nerve transection (ONT) and in cultures of RGC-5 cells with CoCl₂ injury. We found that intravitreal injection of STC-1 increased the number of RGCs in the retina at days 7 and 14 after ONT, and decreased apoptosis and oxidative damage. In cultures, treatment with STC-1 dose-dependently increased cell viability, and decreased apoptosis and levels of reactive oxygen species in RGC-5 cells that were exposed to CoCl₂. The expression of HIF-1α that was up-regulated by injury was significantly suppressed in the retina and in RGC-5 cells by STC-1 treatment. The results suggested that intravitreal injection of STC-1 might be a useful therapy for optic nerve diseases in which RGCs undergo apoptosis through oxidative stress.</p></div

Ocular staining score and tear secretion in NOD.B10 mice after anti-HMGB1 treatment.

<p>(A) Decreased ocular staining score in 2 μg anti-HMGB1-treated mice (Wilcoxon matched-pairs signed rank test, 10 weeks vs. 11 weeks,* p = 0.0156; 10 weeks vs. 12 weeks, * p = 0.0156) and in 0.02 μg anti-HMGB1-treated mice (Wilcoxon matched-pairs signed rank test, 10 weeks vs. 12 weeks, ** p = 0.0313) compared to the baseline score. (B) Reduced tear secretion in PBS-treated control mice (Wilcoxon matched-pairs signed rank test, 10 weeks vs. 12 weeks, ^ςp = 0.002). Increased tear secretion in 0.02 μg anti-HMGB1-treated mice (Wilcoxon matched-pairs signed rank test, 10 weeks vs. 11 weeks, ^ζp = 0.0273; 10 weeks vs. 12 weeks, ^ζζζp = 0.0078) and in 2 μg anti-HMGB1-treated mice (Wilcoxon matched-pairs signed rank test, 10 weeks vs. 11 weeks, ^ξp = 0.0005; 10 weeks vs. 12 weeks, ^ξξp = 0.0342) compared to the pre-treatment baseline. (C) Representative images of ocular staining scoring after treatment at 12 weeks. Data are presented as mean ± standard error.</p