Ocular allergic diseases are frequently seen in ophthalmological clinical practice. Immunological damage is mediated by a local Th2 inflammatory microenvironment, accompanied by changes in circulating cell subsets, with more effector cells and fewer T regulatory cells (Tregs). This study aimed to evaluate the involvement of toll-like receptor 4 (TLR4) and α-melanocyte stimulating hormone (α-MSH) in the immune regulation associated with perennial allergic conjunctivitis (PAC). We performed an Ag-specific stimulation during 72 h of culturing with or without lipopolysaccharide (LPS) or α-MSH in peripheral blood mononuclear cells (PBMC), analyzing the cell subsets and cytokines induced by the stimuli. We also determined α-MSH in tear samples from healthy donors (HD) or PAC patients. Our findings demonstrate an immunological dysregulation characterized by an increased frequency of CD4+TLR4+ in the PBMC of patients with PAC, compared to HD. Most of these CD4+TLR4+ cells were also CD25+, and when α-MSH was added to the culture, the percentage of CD4+CD25+FoxP3+ increased significantly, while the percentage of CD69+ cells and cytokines IL-4 and IL-6 were significantly decreased. In tears, we found an increased concentration of α-MSH in PAC patients, compared with HD. These findings indicate a novel mechanism involved in controlling ocular allergic diseases, in which α-MSH diminishes the concentration of IL-6 and IL-4, restoring the frequency of Tregs and down-regulating CD4 activation. Moreover, we demonstrated the involvement of CD4+TLR4+ cells as an effector cell subset in ocular allergy.

Introduction Allergic conjunctivitis (AC) is one of the most frequent ophthalmological diseases. AC is characterized by ocular surface inflammation and systemic immune dysregulation with low frequency of CD4+CD25+FOXP3+ regulatory T cells and higher frequency of circulating effector T cells. It has been reported that α-melanocyte stimulating hormone (α-MSH) constitutes one of the principal mechanisms of immune regulation in the anterior chamber of the eye and the ocular surface, promoting the differentiation of Tregs in a murine model of experimental autoimmune uveitis and suppressing autoreactive T cells in a murine model of experimental autoimmune encephalomyelitis. This work aimed to evaluate the involvement of α-MSH in Tregs induction in cells obtained from patients with AC. Methods Peripheral blood mononuclear cells (PBMC) were obtained from blood sampling after assent consent was obtained from pediatric patients sensitized to house dust mite (HDM) and diagnostic with AC. PBMC were cultured with HDM and/or α-MSH. After 72h of culture, PBMC were harvested and stained with anti-CD4-AmCyan, anti-CD25-PE, anti-FOXP3-FITC and anti-CD69-APC-Cy7 and analyzed by flow cytometry. IL-2, IL-4, IL-6, IL-10, IL-17, TNF-a and IFN-γ were determined by cytometric bead arrays. Results A significant increase in the frequency of CD4+CD25+FOXP3+ T cells and a significant decrease in the frequency of CD4+CD69+ cells were observed in HDM+α-MSH stimulated cells, when compared with HDM. Remarkably, IL-4 showed a significant decrease in cultured cells stimulated with HDM+α-MSH. Conclusion These results suggest that a-MSH inhibit the IL-4 production, reestablishing the development of Treg cells and control pathogenic CD4+CD69+ T cells.