Mahira Zaheer

Aqueous Tear Deficiency Increases Conjunctival Interferon-γ (IFN-γ) Expression and Goblet Cell Loss

PURPOSE To investigate the hypothesis that increased interferon-γ (IFN-γ) expression is associated with conjunctival goblet cell loss in subjects with tear dysfunction. METHODS Goblet cell density (GCD) was measured in impression cytology from the temporal bulbar conjunctiva, and gene expression was measured in cytology samples from the nasal bulbar conjunctiva obtained from 68 subjects, including normal control, meibomian gland disease (MGD), non-Sjögren syndrome (non-SSATD)-, and Sjögren syndrome (SSATD)-associated aqueous tear deficiency. Gene expression was evaluated by real-time PCR. Tear meniscus height (TMH) was measured by optical coherence tomography. Fluorescein and lissamine green dye staining evaluated corneal and conjunctival disease, respectively. Between-group mean differences and correlation coefficients were calculated. RESULTS Compared to control, IFN-γ expression was significantly higher in both ATD groups, and its receptor was higher in SSATD. Expression of IL-13 and its receptor was similar in all groups. Goblet cell density was lower in the SSATD group; expression of MUC5AC mucin was lower and cornified envelope precursor small proline-rich region (SPRR)-2G higher in both ATD groups. Interferon-γ transcript number was inversely correlated with GCD (r = -0.37, P < 0.04) and TMH (r = -0.37, P = 0.02), and directly correlated with lissamine green staining (r = 0.51, P < 0.001) and SPRR-2G expression (r = 0.32, P < 0.05). CONCLUSIONS Interferon-γ expression in the conjunctiva was higher in aqueous deficiency and correlated with goblet cell loss and severity of conjunctival disease. These results support findings of animal and culture studies showing that IFN-γ reduces conjunctival goblet cell number and mucin production.

Age-related spontaneous lacrimal keratoconjunctivitis is accompanied by dysfunctional T regulatory cells.

In both humans and animal models, the development of Sjögren syndrome (SS) and non-SS keratoconjunctivitis sicca (KCS) increases with age. Here, we investigated the ocular surface and lacrimal gland (LG) phenotype of NOD.B10.H2b mice at 7–14, 45–50, and 96–100 weeks. Aged mice develop increased corneal permeability, CD4+ T-cell infiltration, and conjunctival goblet cell loss. Aged mice have LG atrophy with increased lymphocyte infiltration and inflammatory cytokine levels. An increase in the frequency of CD4+Foxp3+ T regulatory cells (Tregs) was observed with age in the cervical lymph node (CLN), spleen, and LG. These CD4+CD25+ cells lose suppressive ability, while maintaining expression of Foxp3 (forkhead box P3) and producing interleukin-17 (IL-17) and interferon-γ (IFN-γ). An increase of Foxp3+IL-17+ or Foxp3+IFN-γ+ cells was observed in the LG and LG-draining CLN. In adoptive transfer experiments, recipients of either purified Tregs or purified T effector cells from aged donors developed lacrimal keratoconjunctivitis, whereas recipients of young Tregs or young T effector cells failed to develop disease. Overall, these results suggest inflammatory cytokine-producing CD4+Foxp3+ cells participate in the pathogenesis of age-related ocular surface disease.

Interferon-gamma deficiency protects against aging-related goblet cell loss

Aging is a well-recognized risk factor for dry eye. Interferon-gamma (IFN-γ) has been implicated in conjunctival keratinization and goblet cell loss in dry eye. We investigated the role of IFN-γ in age-related dry eye by evaluating young (8 weeks) and aged (15 months; 15M) C57BL/6 (B6) and IFN-γKO mice. Age effects on the conjunctiva and cornea epithelium were assessed with PAS staining and corneal staining, respectively. Expression of T cell-related cytokines (IL-17A, IFN-γ), chemokines (CXCL10 and CCL20), in the ocular surface epithelium was evaluated by real time PCR. A significant decrease in filled goblet cells was noted in 15M B6 mice and this was significantly lower than age and sex-matched IFN-γKO mice. Aged male B6 had significantly higher IFN-γ, and CXCL10 mRNA in their conjunctiva than female B6 mice. Aged IFN-γKO females had significantly higher IL-17A mRNA in conjunctiva than IFN-γKO males and B6 mice. Corneal barrier dysfunction was observed in 15M female B6 and aged IFN-γKO mice of both sexes; however it was significantly higher in IFN-γKO compared to B6 mice. While there was a significant increase in IL 17A, and CCL20 in corneas of aged female B6 and IFN-γKO mice compared to males, these changes were more evident in aged female IFN-γKO group. Partial resistance of IFN-γKO mice to aging-induced goblet cell loss indicates IFN-γ is involved in the age-related decline in conjunctival goblet cells. Increased corneal IL-17A expression paralleled corneal barrier disruption in aging female of both strains. IFN-γ appears to suppress IL-17A on the ocular surface.

Inhibition of NLRP3 Inflammasome Pathway by Butyrate Improves Corneal Wound Healing in Corneal Alkali Burn

Epithelial cells are involved in the regulation of innate and adaptive immunity in response to different stresses. The purpose of this study was to investigate if alkali-injured corneal epithelia activate innate immunity through the nucleotide-binding oligomerization domain-containing protein (NOD)-like receptor family pyrin domain containing 3 (NLRP3) inflammasome pathway. A unilateral alkali burn (AB) was created in the central cornea of C57BL/6 mice. Mice received either no topical treatment or topical treatment with sodium butyrate (NaB), β-hydroxybutyric acid (HBA), dexamethasone (Dex), or vehicle (balanced salt solution, BSS) quater in die (QID) for two or five days (d). We evaluated the expression of inflammasome components including NLRP3, apoptosis-associated speck-like protein (ASC), and caspase-1, as well as the downstream cytokine interleukin (IL)-1β. We found elevation of NLRP3 and IL-1β messenger RNA (mRNA) transcripts, as well as levels of inflammasome component proteins in the alkali-injured corneas compared to naïve corneas. Treatment with NLRP3 inhibitors using NaB and HBA preserved corneal clarity and decreased NLRP3, caspase-1, and IL-1β mRNA transcripts, as well as NLRP3 protein expression on post-injury compared to BSS-treated corneas. These findings identified a novel innate immune signaling pathway activated by AB. Blocking the NLRP3 pathway in AB mouse model decreases inflammation, resulting in greater corneal clarity. These results provide a mechanistic basis for optimizing therapeutic intervention in alkali injured eyes.

Sjögren-Like Lacrimal Keratoconjunctivitis in Germ-Free Mice

Commensal bacteria play an important role in the formation of the immune system but their role in the maintenance of immune homeostasis at the ocular surface and lacrimal gland remains poorly understood. This study investigated the eye and lacrimal gland phenotype in germ-free and conventional C57BL/6J mice. Our results showed that germ-free mice had significantly greater corneal barrier disruption, greater goblet cell loss, and greater total inflammatory cell and CD4+ T cell infiltration within the lacrimal gland compared to the conventionally housed group. A greater frequency of CD4+IFN-γ+ cells was observed in germ-free lacrimal glands. Females exhibited a more severe phenotype compared to males. Adoptive transfer of CD4+ T cells isolated from female germ-free mice into RAG1KO mice transferred Sjögren-like lacrimal keratoconjunctivitis. Fecal microbiota transplant from conventional mice reverted dry eye phenotype in germ-free mice and decreased CD4+IFN-γ+ cells to levels similar to conventional C57BL/6J mice. These findings indicate that germ-free mice have a spontaneous lacrimal keratoconjunctivitis similar to that observed in Sjögren syndrome patients and demonstrate that commensal bacteria function in maintaining immune homeostasis on the ocular surface. Thus, manipulation of intestinal commensal bacteria has the potential to become a novel therapeutic approach to treat Sjögren Syndrome.

Protective role of commensal bacteria in Sjögren Syndrome

CD25 knock-out (CD25KO) mice spontaneously develop Sjögren Syndrome (SS)-like inflammation. We investigated the role of commensal bacteria by comparing CD25KO mice housed in conventional or germ-free conditions. Germ-free CD25KO mice have greater corneal barrier dysfunction, lower goblet cell density, increased total lymphocytic infiltration score, increased expression of IFN-γ, IL-12 and higher a frequency of CD4+IFN-γ+ cells than conventional mice. CD4+ T cells isolated from female germ-free CD25KO mice adoptively transferred to naive immunodeficient RAG1KO recipients caused more severe Sjögren-like disease than CD4+ T cells transferred from conventional CD25KO mice. Fecal transplant in germ-free CD25KO mice reversed the spontaneous dry eye phenotype and decreased the generation of pathogenic CD4+IFN-γ+ cells. Our studies indicate that lack of commensal bacteria accelerates the onset and severity of dacryoadenitis and generates autoreactive CD4+T cells with greater pathogenicity in the CD25KO model, suggesting that the commensal bacteria or their metabolites products have immunoregulatory properties that protect exocrine glands in the CD25KO SS model.