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.

Pollen/TLR4 Innate Immunity Signaling Initiates IL-33/ST2/Th2 Pathways in Allergic Inflammation.

Innate immunity has been extended to respond environmental pathogen other than microbial components. Here we explore a novel pollen/TLR4 innate immunity in allergic inflammation. In experimental allergic conjunctivitis induced by short ragweed (SRW) pollen, typical allergic signs, stimulated IL-33/ST2 signaling and overproduced Th2 cytokine were observed in ocular surface, cervical lymph nodes and isolated CD4+ T cells of BALB/c mice. These clinical, cellular and molecular changes were significantly reduced/eliminated in TLR4 deficient (Tlr4-d) or MyD88 knockout (MyD88-/-) mice. Aqueous SRW extract (SRWe) directly stimulated IL-33 mRNA and protein expression by corneal epithelium and conjunctiva in wild type, but not in Tlr4-d or MyD88-/- mice with topical challenge. Furthermore, SRWe-stimulated IL-33 production was blocked by TLR4 antibody and NF-kB inhibitor in mouse and human corneal epithelial cells. These findings for the first time uncovered a novel mechanism by which SRW pollen initiates TLR4-dependent IL-33/ST2 signaling that triggers Th2-dominant allergic inflammation.

Protective Effects of L-Carnitine Against Oxidative Injury by Hyperosmolarity in Human Corneal Epithelial Cells.

PURPOSE: L-carnitine suppresses inflammatory responses in human corneal epithelial cells (HCECs) exposed to hyperosmotic stress. In this study, we determined if L-carnitine induces this protective effect through suppression of reactive oxygen species (ROS)-induced oxidative damage in HCECs. METHODS: Primary HCECs were established from donor limbal explants. A hyperosmolarity dry-eye model was used in which HCECs are cultured in 450 mOsM medium with or without L-carnitine for up to 48 hours. Production of reactive oxygen species (ROS), oxidative damage markers, oxygenases and antioxidative enzymes were analyzed by 2',7'-dichlorofluorescein diacetate (DCFDA) kit, semiquantitative PCR, immunofluorescence, and/or Western blotting. RESULTS: Reactive oxygen species production increased in HCECs upon substitution of the isotonic medium with the hypertonic medium. L-carnitine supplementation partially suppressed this response. Hyperosmolarity increased cytotoxic membrane lipid peroxidation levels; namely, malondialdehyde (MDA) and hydroxynonenal (HNE), as well as mitochondria DNA release along with an increase in 8-OHdG and aconitase-2. Interestingly, these oxidative markers were significantly decreased by coculture with L-carnitine. Hyperosmotic stress also increased the mRNA expression and/or protein production of heme oxygenase-1 (HMOX1) and cyclooxygenase-2 (COX2), but inhibited the levels of antioxidant enzymes, superoxide dismutase-1 (SOD1), glutathione peroxidase-1 (GPX1), and peroxiredoxin-4 (PRDX4). However, L-carnitine partially reversed this altered imbalance between oxygenases and antioxidant enzymes induced by hyperosmolarity. CONCLUSIONS: Our findings demonstrate for the first time that L-carnitine protects HCECs from oxidative stress by lessening the declines in antioxidant enzymes and suppressing ROS production. Such suppression reduces membrane lipid oxidative damage markers and mitochondrial DNA damage.

Effects of azithromycin on gene expression profiles of proinflammatory and anti-inflammatory mediators in the eyelid margin and conjunctiva of patients with meibomian gland disease.

IMPORTANCE: Topical application of azithromycin suppresses expression of proinflammatory mediators while restoring transforming growth factor ß1 (TGF-ß1) levels as evaluated by eyelid margin and conjunctival impression cytology. OBJECTIVE: To explore the effects of azithromycin therapy on expression of proinflammatory and anti-inflammatory mediators in meibomian gland disease (MGD). DESIGN, SETTING, AND PARTICIPANTS: Case-control study performed in a clinic setting from August 17, 2010, to December 31, 2010. Sixteen patients with posterior blepharitis and conjunctival inflammation due to MGD were treated with azithromycin, 1%, drops for 4 weeks. Impression cytology of the lower eyelid margin and tarsal conjunctiva to measure cytokine expression by quantitative real-time polymerase chain reaction as well as tear collection to measure matrix metalloproteinase 9 (MMP-9) activity were performed once in 8 asymptomatic healthy control participants and 5 times in the 16 symptomatic patients (every 2 weeks for 8 weeks), before, during, and after azithromycin treatment. EXPOSURE: Azithromycin, 1%, drops for 4 weeks. MAIN OUTCOMES AND MEASURES: Cytokine expression in the eyelid margin and conjunctiva, and MMP-9 activity in tears. RESULTS: Compared with a 1-time measurement of 8 healthy participants, among 16 symptomatic patients, the mean (SD; 95% CI) fold change of expression of proinflammatory mediators interleukin 1ß (IL-1ß), IL-8, and MMP-9 increased to 13.26 (4.33; 11.14-15.38; P < .001), 9.38 (3.37; 7.73-11.03; P < .001), and 13.49 (4.92; 11.08-15.90; P < .001), respectively, in conjunctival cells and to 11.75 (3.96; 9.81-13.69; P < .001), 9.31 (3.28; 7.70-10.92; P < .001), and 11.52 (3.50; 9.81-13.24; P < .001), respectively, in the eyelid margin of patients with MGD. In contrast, the mean (SD; 96% CI) fold change of expression of TGF-ß1 messenger RNA (mRNA) decreased to 0.58 (0.25; 0.46-0.70; P = .02) and 0.63 (0.14; 0.56-0.70; P = .02) in conjunctival and eyelid margin cells, respectively, of patients with MGD. Azithromycin, 1%, caused a change in the expression pattern of these mediators toward normal levels during 4 weeks of treatment. Levels of IL-1ß, IL-8, and MMP-9 mRNA remained suppressed, although they rebounded toward pretreatment values 4 weeks after azithromycin withdrawal. Expression of TGF-ß1 increased during treatment and remained at levels similar to the healthy controls after drug withdrawal. Change in tear MMP-9 activity was similar to the pattern of MMP-9 transcripts. CONCLUSIONS AND RELEVANCE: While the study did not control for potential confounding factors over time independent of the intervention that may have contributed to the results, topical azithromycin suppressed expression of proinflammatory mediators and increased expression of TGF-ß1 to normal levels. Increased TGF-ß1 expression may contribute to the anti-inflammatory activity of azithromycin in MGD.

TSLP and downstream molecules in experimental mouse allergic conjunctivitis.

PURPOSE: To explore the potential role of thymic stromal lymphopoietin (TSLP) and its downstream molecules in the development of ocular allergic inflammation using a short ragweed (SRW)-induced mouse model of allergic conjunctivitis (AC). METHODS: BALB/c mice were topically challenged with SRW pollen after they were sensitized with SRW in the footpad. After the last SRW challenge, the corneal epithelium, conjunctiva, and cervical lymph nodes were harvested for total RNA extraction and gene expression by RT and real-time PCR, and whole eye globes were collected to make cryosections for immunohistochemical staining. RESULTS: Repeated topical challenges with SRW allergen generated typical signs of AC in mice. Compared with the untreated controls, TSLP mRNA expression and immunoreactivity were significantly increased in the corneal and conjunctival epithelia of SRW-induced AC mice. CD11c(+) and OX40L(+) immunoreactive cells largely infiltrated the conjunctiva with increased mRNA levels of CD11c, TSLPR, and OX40L detected in the corneal epithelium, conjunctiva, and cervical lymph nodes. CD4(+) Th2 cell infiltration was evidenced by increased levels of mRNA and immunoreactivity of CD4, IL-4, IL-5, and IL-13 in the ocular surface, mainly in the conjunctiva, accompanied by increased expression of OX40, STAT6, and GATA3, in AC mice. The maturation of immature DCs was observed with the use of TSLP containing conditioned media from corneal epithelial cultures exposed to polyI:C, which stimulates TSLP production. CONCLUSIONS: This study provides new findings regarding the role of local mucosal epithelial cells in the initiation of ocular allergic inflammation by producing a novel proallergic cytokine, TSLP, which activates dendritic cells to prime Th2 differentiation and allergic inflammation through the TSLP-TSLPR and OX40L-OX40 signaling pathway.