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.

Objectives: Bacterial components are used to improve immune responses in patients with respiratory infections. Pharmacological formulations of bacterial components include a mixture of bacterial antigens, some of which are complete inactivated bacteria, that is, named bacterial suspensions; while others are fragments of bacteria, which are presented as bacterial lysates. Although bacterial lysates have been broadly used as immune-stimulators, the biological support for the therapeutic effectiveness of bacterial suspension has not yet been studied. Thus, the aim of our study was to investigate the immunological activity induced by bacterial suspension. Methods: This work was an exploratory translational study. Peripheral blood mononuclear cells were obtained from healthy donors and cultured in time–dose dependent assays with a commercial bacterial suspension. Flow cytometry was used for phenotypic analysis and for determining soluble cytokines in culture supernatants. Results: We observed that bacterial suspension activates B cells in a dose-dependent manner. Peripheral blood mononuclear cells were able to secrete IL-6 and IL-10 after 24 h of bacterial suspension stimulation. TLR2 expression was observed mainly on CD19+ CD38Lo B cells after 72 h of culture; remarkably, most of the TLR2+ CD19+ cells were also IL-10+. Conclusion: Our findings suggest that bacterial suspension induces the activation of B cell subsets as well as the secretion of IL-6 and IL-10. Expression of TLR2 on CD19+ cells could act as an activation loop of IL-10+ B regulatory cells. The clinical implications of these findings are discussed at the end of this article.

Bacterial lysates (BL) are used to improve immune response in patients with respiratory infections. BL are constituted by a mixture of bacterial antigens derived from different bacterial species; species included in these lysates are 14 different bacteria: S. pneumoniae, K. pneumoniae, B. catarrahalis, S. aureus, H. influenza, S. alpha, and beta, S. faecalis, S. epidermidis, B. pertussis, Proteus sp., Pseudomonas sp., E. coli, and C. pseudodiphteriae (BL-14) It has been suggested that BL activate innate response through TLR, however if BL-14 are able to induce B cell activation is not known and this was the aim of our study. Peripheral mononuclear blood cells (PBMC) were obtained from healthy donors and cultured with/without BL-14 (IPI ASAC Pharma) at different concentrations, after stimulation with BL-14, supernatant were collected and IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ, and IL-17A (BD Biosciences), were measured with cytometric bead arrays. PBMC were harvested at 24, 48 and 72 h, labelled against anti- CD19, CD38, CD69 or TLR2 (BD Biosciences), and analysed by flow cytometry. We observed dose-dependent increased activation of CD19+. After 72 h we observed a diminished frequency of B cells precursors and expansion of transitional B cells, these cells were TLR2+ cells. A significant increased concentration of IL-10 and IL-6 was observed at 24h, and 48 h after BL-14 stimulation. BL induces expansion of transitional B cells and IL-10 production through TLR2 recognition