(b) After 24 h of co-culture, BMDCs were harvested and the expression of MHC class II, CD80, CD86 and CD40 was analyzed by RT-PCR. express TGF-?2, but are also able to activate it in a thrombospondin-1 (TSP-1) dependent manner via their cell surface receptor CD36. Furthermore, goblet cell derived soluble factors that possibly include TGF-?2, alter dendritic cell (DC) phenotype to a tolerogenic type by downregulating DC expression of MHC class II and co-stimulatory molecules CD80, CD86 and CD40. Thus our study demonstrates goblet cells as a cellular source of active TGF-?2 in ocular mucosa and implicates their immunomodulatory function in maintaining mucosal immune homeostasis. Introduction The mucin secreting conjunctival epithelium forms a mucosal barrier between subepithelial immune cells and the environment. Similar to other mucosal surfaces the conjunctiva is endowed with its local lymphoid tissue, conjunctiva associated lymphoid tissue (CALT), comprised of cells capable of mounting innate and adaptive immune responses [1,2]. At Nelfinavir Mesylate steady state immunologic tolerance is induced against harmless antigens and commensal bacteria, while inflammatory immune response is mounted against pathogens to prevent Nelfinavir Mesylate infections [3]. Mechanisms underlying such homeostatic balance between tolerance and immunity at the ocular surface have not been fully explored. By secreting mucins conjunctival epithelial cells, including goblet cells, are known to aid in the elimination of offending environmental agents [4]. The importance of goblet cells in particular in maintaining ocular surface homeostasis is well established [5]. Loss of these cells is a common feature in several inflammatory diseases of the ocular surface, including Stevens-Johnson syndrome, ocular mucous membrane pemphigoid, alkali burn, neutrophilic keratitis, graft-versus-host-disease, and Sj?grens syndrome [6,7,8]. In addition to protecting the ocular surface via mucin secretion, goblet cells have been shown to contribute to the innate immune response by secreting mature IL-1? via activation of the NLRP3 inflammasome [9]. However, unlike other mucosal surfaces contribution of conjunctival epithelial cells in priming the adaptive immune response has remained unaddressed. The strategic location of goblet cells in the conjunctiva allows them direct contact with environmental agents and the conjunctival stroma that harbors dendritic cells (DCs). Dendritic cells in the conjunctiva are detected in organized follicles of CALT and diffusely distributed through the stroma along with intraepithelial lymphocytes [2,10,11]. Both CD11b+ and CD103+ subsets of CD11c+ dendritic cells are reported in murine conjunctiva and are known to contribute significantly to local immune responses [12]. Topically delivered soluble Nelfinavir Mesylate antigen on the ocular surface is detectable as associated with CD11c+ dendritic cells in the draining cervical lymph nodes [13]. Such dendritic cells capable of priming host adaptive immune responses are located in close proximity to mucin secreting goblet cells of the conjunctiva [11]. The structural location of goblet cells at the interface of the external environment and stromal immune cells makes them promising candidates for modulating the mucosal environment and as a consequence DC function and dependent immune responses. In this study, taking advantage of now feasible primary Rabbit Polyclonal to CCT7 culture of murine conjunctival goblet cells, we investigate their potential role in modulating adaptive immune response. Although conjunctiva, as other mucosal surfaces, is a TGF-? rich environment [14], it is not known if goblet cells serve as a cellular source of this immunomodulatory cytokine. Recently predominant expression of the TGF-?2 isoform was reported in human conjunctival epithelial cells during chronic ocular surface inflammation [15]. In this study we evaluated if normal mouse conjunctiva, and specifically goblet cells, predominantly express this isoform and if its expression is modulated via toll-like receptor (TLR) stimulation. Moreover, it has been reported that due to an absence of the integrin binding RGD sequence in the LAP of the TGF-?2 isoform, an integrin independent mechanism activates the latent form of TGF-?2 [16]. Thrombospondin-1 (TSP-1), an extracellular matrix protein expressed by many ocular cell types, represents one such mechanism and has been shown to efficiently activate TGF-?2 [17]. Therefore we determined if goblet cells activate their endogenous TGF-?2, if any, in a TSP-1 dependent manner. In addition, to determine the immunomodulatory capacity of goblet cells we evaluated the effect of goblet cell derived soluble factors on dendritic cell phenotype. The literature to date is limited in regard to goblet cells as sources of active TGF-?, and to the best of our knowledge, no published report exists on the effect of goblet cells on adaptive immune response at the ocular surface. Our results demonstrate for the first time that conjunctival goblet cells can not only express and secrete an immunomodulatory cytokine, TGF-?2, but can also activate it in a TSP-1 dependent manner via its receptor, CD36. Our results thus determine an as yet unfamiliar immunomodulatory function of conjunctival goblet cells based on their.