Srihari Narayanan

Interleukin-1 receptor-1-deficient mice show attenuated production of ocular surface inflammatory cytokines in experimental dry eye.

Purpose: To compare inflammatory cytokine and defensin expression in response to experimental dry eye (EDE) in interleukin-1 receptor-1 (IL-1R1)-deficient (KO) mice with age-matched wild-type mice (WT). Methods: EDE was induced by subcutaneous scopolamine injection, exposure to low humidity, and an air draft for 5 days in 4- to 6-week-old KO and WT mice. Expression of cytokines IL-1α, IL-1β, tumor necrosis factor (TNF)-α, IL-6, and mouse β-defensins (mBD)-1, mBD-2, and mBD-3 was evaluated by real-time polymerase chain reaction in scraped corneal epithelial cells and whole conjunctival tissues. A multiplex bead assay was performed to quantitate IL-1α, IL-2, IL-4, IL-10, interferon (IFN)-γ, and TNF-α levels in tear fluid, and an enzyme immunoassay was used to quantitate IL-1β levels in tear fluid. Results: EDE significantly increased RNA transcripts for IL-1α and β in the conjunctiva and for TNF-α in the corneal epithelium of WT mice. Levels of IL-1α, IL-1β, and IL-6 were significantly lower in the corneal epithelium and conjunctiva, and TNF-α was significantly lower in the cornea of KO mice after 5 days of EDE than WT mice. Tear fluid IL-1α concentration increased above baseline on days 2-4 of EDE in WT and KO mice. A similar pattern was observed for tear TNF-α. Tear IL-1β increased throughout the 5 days of EDE in WT and KO mice. IFN-γ, IL-2, IL-4, and IL-10 were undetectable in tear fluid of either strain before or after EDE. Corneal mBD-1 mRNA expression was unchanged and conjunctival mBD-1 transcripts decreased in WT and increased in KO mice with EDE. Untreated WT corneas, but not those of KO mice, expressed mBD-2 transcripts, whereas in the conjunctiva, mBD-2 increased in WT and decreased in KO mice with EDE. Corneal mBD-3 mRNA expression was undetected in WT mice, but increased after EDE in KO mice. Conjunctival mBD-3 transcripts were only detected in WT with EDE. Conclusions: These findings indicate that IL-1 signaling is responsible in part for the increased expression of inflammatory cytokines and the changes in mBDs by the ocular surface tissues in response to desiccating stress. These results show the important regulatory aspects of IL-1 on ocular surface epithelial inflammation.

In vitro activity of human β-defensin 2 against Pseudomonas aeruginosa in the presence of tear fluid

ABSTRACT Pseudomonas aeruginosa causes vision-threatening keratitis and is difficult to treat due to emerging resistance. Human β-defensin 2 (hBD-2) is an antimicrobial peptide expressed by ocular surface epithelia with broad-spectrum activity against various pathogens, including P. aeruginosa. The activity of hBD-2 against P. aeruginosa in the presence of human tears or NaCl was studied. In some experiments, tears were heat-inactivated, filtered, and separated into cationic/anionic fractions or mucin MUC5AC was removed by immunoprecipitation before use. Immunoprecipitation was performed to study the interaction between hBD-2 and MUC5AC. hBD-2 activity was reduced by 40 to 90% in the presence of 17.5 to 70% (vol/vol) tears. NaCl reduced hBD-2 activity, but at most it could account for only 36% of the inhibitory effect of tears. Heat inactivation and filtration attenuated the ability of tears to inhibit hBD-2 activity by 65 and 68%, respectively. Anionic tear fractions significantly reduced (86%) the activity of hBD-2, whereas only a 22% reduction was observed with the cationic fractions. In the absence of MUC5AC, the activity of hBD-2 was restored by 64%. Immunoprecipitation studies suggested that the loss of hBD-2 activity in tears is due to a direct binding interaction with MUC5AC. Our data showed that the antimicrobial activity of hBD-2 is sensitive to the presence of human tears and that this is partly due to the salt content and also the presence of MUC5AC. These data cast doubt on the effectiveness of hBD-2 as an antimicrobial peptide, and additional studies are required to conclusively elucidate its role in innate immunity at the ocular surface in vivo.

The diagnosis and characteristics of moderate dry eye in non-contact lens wearers

Purpose. To identify and characterize moderate dry eye in non–contact lens wearers with a new scoring system-based dry eye questionnaire and to determine which objective tests better differentiate patients with moderate dry eye from healthy patients. Methods. Fifty-two healthy subjects (21 women and 31 men with a mean age of 27.8 ± 9.2 years) and 37 subjects with moderate dry eye (33 women and 4 men with a mean age of 36.4 ± 12.9 years) completed a 42-item dry eye questionnaire. Seventeen healthy subjects (11 women and 6 men with a mean age of 30.5 ± 9.7 years) and 28 subjects with moderate dry eye (24 women and 4 men with a mean age of 38.50 ± 3.8 years) underwent additional objective assessment of ocular surface health, tear osmolality, tear stability, and tear volume. Results. Subjects with moderate dry eye scored significantly higher (49.8 ± 20.3, P<0.0001) on the dry eye questionnaire than did normal subjects (11.7 ± 10.3). Ocular irritation symptoms worsened with progression of time of day in both groups of subjects. Internal reliability (0.95 Cronbach α) was excellent, and concurrent validity (Spearman ρ 0.507) was acceptable when compared to the McMonnies and Ho dry eye questionnaire. Significant differences in tear osmolality (P<0.00001), invasive tear breakup time (P<0.034), and corneal vital dye staining (P<0.0001) were detected between the two groups of subjects. A stepwise linear regression on objective clinical tests, however, did not account for 77% of the total variance in the questionnaire scores. Conclusions. A unique scoring system-based dry eye questionnaire was validated to separate non–contact lens wearers with moderate dry eye from healthy subjects. Objective tests of tear osmolality and stability and ocular surface integrity were better than other clinical measures at identifying differences between the two subject groups. The results strongly support the evidence that the diagnosis and treatment of moderate dry eye requires a detailed assessment of self-perceived symptoms and that objective clinical testing alone may be insufficient.

Effect of Hyperosmolality on β-Defensin Gene Expression by Human Corneal Epithelial Cells

Purpose: As human β-defensins (hBD) are important antimicrobial peptides at epithelial surfaces, including the ocular surface, we tested the effect of hyperosmolar conditions on the expression of these peptides by human corneal epithelial cells (HCECs). Methods: Simian virus 40-transformed HCECs (n = 5) or primary cultured HCECs (n = 5) were treated with serum-free media or serum-free hyperosmolar (400-500 mOsm/kg) media for 24 hours or serum-free 500 mOsm/kg media for 12 to 48 hours. The effect of hyperosmolality on interleukin-1β (IL-1β)-induced hBD-2 expression was also tested. IL-6 expression was studied as a marker of IL-1β function. Expression of hBD-1, -2, and -3 and IL-6 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). The levels of active IL-1β (culture supernatants and cell lysates) and pro-IL-1β (cell lysates) were detected by enzyme-linked immunosorbent assay. Results: HCECs constitutively expressed hBD-1 and -3 but not hBD-2. Hyperosmolar media had no effect on the basal expression of hBD-1 or -3 and did not induce the expression of hBD-2. Treatment with 500 mOsm/kg media for 24 hours decreased the ability of IL-1β to upregulate hBD-2 and IL-6 expression. Active or pro-IL-1β was not detected in any cell culture sample. Conclusion: Our results suggest that the hyperosmolar environment observed in diseases such as dry eye does not alter defensin expression. However, a hyperosmolar environment may influence cytokine function in ocular surface cells and thus affect their response to injury and inflammation.