Modulation of Oxidative Stress and Inflammation in the Aged Lacrimal Gland.

Inflammation and oxidative stress accompany aging. This study investigated the interplay between oxidative stress and inflammation in the lacrimal gland. C57BL/6 mice were used at 2 to 3, 12, and 24 months of age. Nuclear factor erythroid derived-2-related factor 2 (Nrf2)-/- and corresponding wild-type mice were used at 2 to 3 and 12 to 13 months of age. A separate group of 15.5 to 17 months of age C57BL/6 mice received a diet containing an Nrf2 inducer (Oltipraz) for 8 weeks. Aged C57BL/6 lacrimal glands showed significantly greater lymphocytic infiltration, higher levels of MHC II, IFN-γ, IL-1ß, TNF-α, and cathepsin S (Ctss) mRNA transcripts, and greater nitrotyrosine and 4-hydroxynonenal protein. Young Nrf2-/- mice showed an increase in IL-1ß, IFN-γ, MHC II, and Ctss mRNA transcripts compared with young wild-type mice and greater age-related changes at 12 to 13 months of age. Oltipraz diet significantly decreased nitrotyrosine and 4-hydroxynonenal and decreased the expression of IL-1ß and TNF-α mRNA transcripts, while decreasing the frequency of CD45+CD4+ cells in lacrimal glands and significantly increasing conjunctival goblet cell density compared with a standard diet. The findings provide novel insight into the development of chronic, low-grade inflammation and oxidative stress in age-related dry eye. New therapies targeting oxidative stress pathways will be valuable in treating age-related dry eye.

Rapamycin Eyedrops Increased CD4+Foxp3+ Cells and Prevented Goblet Cell Loss in the Aged Ocular Surface.

Dry eye disease (DED), one of the most prevalent conditions among the elderly, is a chronic inflammatory disorder that disrupts tear film stability and causes ocular surface damage. Aged C57BL/6J mice spontaneously develop DED. Rapamycin is a potent immunosuppressant that prolongs the lifespan of several species. Here, we compared the effects of daily instillation of eyedrops containing rapamycin or empty micelles for three months on the aged mice. Tear cytokine/chemokine profile showed a pronounced increase in vascular endothelial cell growth factor-A (VEGF-A) and a trend towards decreased concentration of Interferon gamma (IFN)-γ in rapamycin-treated groups. A significant decrease in inflammatory markers in the lacrimal gland was also evident (IFN-γ, IL-12, CIITA and Ctss); this was accompanied by slightly diminished Unc-51 Like Autophagy Activating Kinase 1 (ULK1) transcripts. In the lacrimal gland and draining lymph nodes, we also observed a significant increase in the CD45+CD4+Foxp3+ cells in the rapamycin-treated mice. More importantly, rapamycin eyedrops increased conjunctival goblet cell density and area compared to the empty micelles. Taken together, evidence from these studies indicates that topical rapamycin has therapeutic efficacy for age-associated ocular surface inflammation and goblet cell loss and opens the venue for new investigations on its role in the aging process of the eye.

Goblet cell-produced retinoic acid suppresses CD86 expression and IL-12 production in bone marrow-derived cells.

Conjunctival goblet cell loss in ocular surface diseases is accompanied by increased number of interleukin-12 (IL-12)-producing antigen-presenting cells (APCs) and increased interferon-γ (IFN-γ) expression. This study tested the hypothesis that mouse conjunctival goblet cells produce biologically active retinoic acid (RA) that suppresses CD86 expression and IL-12 production by myeloid cells. We found that conditioned media from cultured conjunctival goblet cells (CjCM) suppressed stimulated CD86 expression, NF-κB p65 activation and IL-12 and IFN-γ production in unstimulated and lipopolysaccharide-stimulated cultured bone marrow-derived cells (BMDCs) containing a mixed population of APCs. Goblet cell-conditioned, ovalbumin-loaded APCs suppressed IFN-γ production and increased IL-13 production in co-cultured OTII cells. The goblet cell suppressive activity is due in part to their ability to synthesize RA from retinol. Conjunctival goblet cells had greater expression of aldehyde dehydrogenases Aldh1a1 and a3 and ALDEFLUOR activity than cornea epithelium lacking goblet cells. The conditioning activity was lost in goblet cells treated with an ALDH inhibitor, and a retinoid receptor alpha antagonist blocked the suppressive effects of CjCM on IL-12 production. Similar to RA, CjCM increased expression of suppressor of cytokine signaling 3 (SOCS3) in BMDCs. SOCS3 silencing reversed the IL-12-suppressive effects of CjCM. Our findings indicate that conjunctival goblet cells are capable of synthesizing RA from retinol secreted by the lacrimal gland into tears that can condition APCs. Evidence suggests goblet cell RA may function in maintaining conjunctival immune tolerance and loss of conjunctival goblet cells may contribute to increased Th1 priming in dry eye.

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.

Immune phenotype of the CD4+ T cells in the aged lymphoid organs and lacrimal glands.

Aging is associated with a massive infiltration of T lymphocytes in the lacrimal gland. Here, we aimed to characterize the immune phenotype of aged CD4+ T cells in this tissue as compared with lymphoid organs. To perform this, we sorted regulatory T cells (Tregs, CD4+CD25+GITR+) and non-Tregs (CD4+CD25negGITRneg) in lymphoid organs from female C57BL/6J mice and subjected these cells to an immunology NanoString® panel. These results were confirmed by flow cytometry, live imaging, and tissue immunostaining in the lacrimal gland. Importantly, effector T helper 1 (Th1) genes were highly upregulated on aged Tregs, including the master regulator Tbx21. Among the non-Tregs, we also found a significant increase in the levels of EOMESmed/high, TbetnegIFN-γ+, and CD62L+CD44negCD4+ T cells with aging, which are associated with cell exhaustion, immunopathology, and the generation of tertiary lymphoid tissue. At the functional level, aged Tregs from lymphoid organs are less able to decrease proliferation and IFN-γ production of T responders at any age. More importantly, human lacrimal glands (age range 55-81 years) also showed the presence of CD4+Foxp3+ cells. Further studies are needed to propose potential molecular targets to avoid immune-mediated lacrimal gland dysfunction with aging.

Calcineurin Inhibitor Voclosporin Preserves Corneal Barrier and Conjunctival Goblet Cells in Experimental Dry Eye.

Objective: The purpose of this study was to evaluate the potential of voclosporin (VOS) in preventing goblet cell (GC) loss and modulating interferon-gamma (IFN-γ) producing CD4+ T cells in the mouse desiccating stress (DS) dry eye model. Methods: Mice were subjected to DS and treated topically with vehicle, VOS, or cyclosporine A as a treatment control. Corneal barrier function was evaluated after 5 and conjunctival GC density after 10 days of desiccation. CD4+ T cells were isolated from ocular surface draining lymph nodes of dry eye donor mice and adoptively transferred into immune deficient RAG1-/- mice from which tears and conjunctiva were collected for the evaluation of inflammatory cytokines/chemokines and GC density. Results: Compared to the vehicle-treated group, VOS was significantly better in preserving corneal barrier function and preventing DS-induced conjunctival GC loss. CD4+ T cells from VOS treated dry eye donors caused less conjunctival GC loss than vehicle and suppressed expression of IFN-γ signature genes to a similar extent and transforming growth factor-beta to a greater extent than cyclosporine in adoptive transfer recipients. Conclusion: These findings suggest that VOS preserves corneal barrier function and conjunctival GCs and suppresses IFN-γ producing CD4+ T cells in experimental dry eye.

Age-related Autoimmune Changes in Lacrimal Glands.

Aging is a complex process associated with dysregulation of the immune system and low levels of inflammation, often associated with the onset of many pathologies. The lacrimal gland (LG) plays a vital role in the maintenance of ocular physiology and changes related to aging directly affect eye diseases. The dysregulation of the immune system in aging leads to quantitative and qualitative changes in antibodies and cytokines. While there is a gradual decline of the immune system, there is an increase in autoimmunity, with a reciprocal pathway between low levels of inflammation and aging mechanisms. Elderly C57BL/6J mice spontaneously show LGs infiltration that is characterized by Th1 but not Th17 cells. The aging of the LG is related to functional alterations, reduced innervation and decreased secretory activities. Lymphocytic infiltration, destruction, and atrophy of glandular parenchyma, ductal dilatation, and secretion of inflammatory mediators modify the volume and composition of tears. Oxidative stress, the capacity to metabolize and eliminate toxic substances decreased in aging, is also associated with the reduction of LG functionality and the pathogenesis of autoimmune diseases. Although further studies are required for a better understanding of autoimmunity and aging of the LG, we described anatomic and immunology aspects that have been described so far.

Evaluation of total keratometry and its accuracy for intraocular lens power calculation in eyes after corneal refractive surgery.

PURPOSE: To compare the accuracy of total keratometry (TK) and standard keratometry (K) from a swept-source optical coherence tomography biometer for intraocular lens (IOL) power calculation in eyes with previous corneal refractive surgery. SETTING: Cullen Eye Institute, Baylor College of Medicine, Houston, Texas, USA. DESIGN: Retrospective case series. METHODS: The differences between the TK and K and their association with K were assessed. For IOL power calculation, combinations of 1) K with Haigis, Haigis-L, and Barrett True-K, and 2) TK with Haigis (Haigis-TK) were used. The mean absolute error (MAE) and the percentages of eyes within prediction errors of ± 0.50 diopters (D), ± 1.00 D, and ± 2.00 D were calculated. RESULTS: The study comprised 129 eyes. For Haigis, Haigis-L, Barrett True-K, and Haigis-TK, respectively, the MAEs were 0.72 D, 0.61 D, 0.54 D, and 0.50 D in the myopic laser in situ keratomileusis (LASIK)/photorefractive keratectomy (PRK) group, and 0.74 D, 0.68 D, 0.71 D, and 0.70 D in hyperopic LASIK/PRK group. For the radial keratotomy (RK) eyes, the MAEs were 0.66 D, 0.71 D, and 0.72 D for the Haigis, Barrett True-K, and Haigis-TK formulas, respectively. In the myopic LASIK/PRK group, the Barrett True-K and Haigis-TK produced significantly lower MAEs than did Haigis (P < .05). In the hyperopic LASIK/PRK and RK groups, there were no significant differences between the formulas in MAEs and percentages of eyes within the above prediction errors. CONCLUSIONS: The performance of the combination of Haigis and TK in refractive prediction was comparable with Haigis-L and Barrett True-K in eyes with previous corneal refractive surgery.

Short ragweed pollen promotes M2 macrophage polarization via TSLP/TSLPR/OX40L signaling in allergic inflammation.

This study was to explore the role and mechanism of macrophages in pollen-triggered allergic inflammation. A murine model of short ragweed (SRW) pollen-induced experimental allergic conjunctivitis (EAC), and bone marrow (BM)-macrophages cultures were used. Typical allergic manifestations and TSLP-stimulated Th2 hyperresponse were observed in ocular surface of EAC model in wild-type (WT) mice induced by SRW. The M2 phenotype markers, Arg1, Ym1 and FIZZ1, were highly expressed by conjunctiva and draining cervical lymph nodes (CLNs) of WT-EAC mice when compared with controls, as evaluated by RT-qPCR and Immunofluorescent double staining with macrophage marker F4/80. The stimulated expression of TSLPR and OX40L by macrophage was detected in conjunctiva and CLNs by RT-qPCR, double staining, and flow cytometry. M2 macrophages were found to produce TARC and MDC. In contrast, EAC model with TSLPR-/- mice did not show allergic signs and any increase of Th2 cytokines (IL-4, IL-5 and IL-13) and M2 markers. In vitro cultures confirmed that SRW extract stimulates expression of TSLPR, OX40L, TARC, MDC, and three M2 markers by BM-macrophages from WT mice, but not from TSLPR-/- mice. These findings demonstrate that SRW pollen primes macrophage polarization toward to M2 phenotype via TSLP/TSLPR/OX40L signaling to amplify allergic inflammation.

Age-associated antigen-presenting cell alterations promote dry-eye inducing Th1 cells.

Aging is a significant risk factor for dry eye. Here we used a murine aging model to investigate the effects of aging on antigen presenting cells (APCs) and generation of pathogenic T helper (Th)-1 cells. Our results showed that APCs from aged mice accumulate at the conjunctiva, have higher levels of co-activation marker CD86 and lower aldehyde dehydrogenase activity. Using topical ovalbumin peptide as a surrogate antigen, we observed an increased number of antigen-loaded APCs in the draining cervical lymph nodes in the aged group and loss of tight junction protein occludin in the conjunctiva. Aged cervical lymph nodes APCs showed a greater generation of Th1 cells than young APCs in antigen-presentation assays in vitro. Aged lacrimal glands, and draining nodes showed an accumulation of IFN-γ producing CD4+T cells, while Th-17 cells were present only in aged draining nodes. There was also an age-related increase in CD4+CXCR3+IFN-γ+ cells in the conjunctiva, nodes, and lacrimal glands while CD4+CCR6+IL-17A+ cells increased in the draining nodes of aged mice. Adoptive transfer of aged CD4+CXCR3+ cells into young, naive immunodeficient recipients caused greater goblet cell loss than young CD4+CXCR3+ donor cells. Our results demonstrate that age-associated changes in APCs are critical for the pathogenesis of age-related dry eye.

Impact of omalizumab in children from a middle-income country with severe therapy-resistant asthma: A real-life study.

BACKGROUND: Severe asthma in children is a global health problem. Severe therapy-resistant asthma (STRA) in children is a major clinical challenge due to persistent symptoms despite high doses of corticosteroids and results in high public health costs. Omalizumab (anti-IgE monoclonal antibody) has been described as an effective add-on therapy in these patients. The characteristics of children with STRA from low- and middle-income countries have scarcely been reported, and no real-life study has been published on the effects of omalizumab in this group of patients. The aim of our study is to report the first clinical real-life experiences with omalizumab in Brazilian children with STRA. METHODS: Children (6-18 years old) from a referral center who were diagnosed with STRA were included in this retrospective study based on our clinical databases. The included children had undergone at least 6 months of omalizumab treatment and fulfilled the following initial criteria: 1) >6 years old; 2) a positive skin-prick test for at least one aeroallergen; and 3) a serum total IgE level between 30 and 1500 IU/mL. Clinical and lung function variables were analyzed before and after treatment. RESULTS: Fourteen children (mean age: 11.9 years; percentage female: 72%) were included in this study. Omalizumab treatment significantly increased control of the disease according to a standardized questionnaire administered at every visit (P < 0.0001), ceased hospitalizations in 70% (P = 0.02) of patients, and allowed 8/9 (89%) patients to be weaned off oral steroids (P = 0.004). CONCLUSIONS: In this retrospective report, the use of omalizumab in Brazilian children with STRA significantly improved disease control, decreased hospitalizations, and allowed suspension of continuous oral corticosteroids.

Effect of isolation rearing on the expression of AMPA glutamate receptors in the hippocampal formation.

Reduced glutamatergic signaling may contribute to cognitive dysfunction in schizophrenia. Glutamatergic synapses might be the site of primary abnormalities in this disorder with the dopaminergic changes being secondary to altered glutamatergic transmission. Isolation rearing of rats from weaning has been used as an experimental model for affective disorders like schizophrenia. In this immunohistochemistry study we evaluate the changes in the expression of GluR1 and GluR2 AMPA receptors in the hippocampus, amygdala and entorhinal cortex induced by isolation rearing. Two groups of Wistar rats (grouped and isolated, n = 6/each) were used. Isolation rearing induced a significant decrease in GluR1- and GluR2-immunopositive cells in the hippocampus. For GluR1 the reduction was 31% in the hilus of dentate gyrus (p = 0.02) and 47% in CA3 (p = 0.002). For GluR2 the reduction was 52% in the hilus of dentate gyrus (p < 0.0001) and 29% in CA1 (p = 0.002). Isolation rearing induced a non-significant decrease in GluR1-immunopositive cells in the basolateral amygdala (p = 0.066) while no alteration was found in the lateral nucleus (p = 0.657). For GluR2 no changes were induced by isolation in both nuclei of the amygdala. In the entorhinal cortex no apparent difference was seen in GluR1- or GluR2-immunopositive cells when isolated reared rats were compared to grouped rats. The results suggest that isolation rearing from weaning induces changes on the expression of AMPA glutamate receptors in the hippocampus similar to those reported for postmortem human brains with schizophrenia. These findings also contribute to additional evidence for using isolation rearing of rats from weaning as an animal model for schizophrenia.