Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Mar 20;10(3):e0120284.
doi: 10.1371/journal.pone.0120284. eCollection 2015.

Immunomodulatory cross-talk between conjunctival goblet cells and dendritic cells

Laura Contreras-Ruiz  1 , Sharmila Masli  1
Affiliations

Immunomodulatory cross-talk between conjunctival goblet cells and dendritic cells

Laura Contreras-Ruiz et al. PLoS One. .

Abstract

Goblet cells are secretory epithelial cells of mucosal tissues that confer protection from environmental agents or pathogens via expression and secretion of soluble mucins. Loss of these cells is associated with several chronic inflammatory disorders of the mucosa. Although demonstrated to transfer antigens from the luminal surface to stromal cells in the intestinal mucosa, it is not known if goblet cells contribute to the regulation of an immune response. In this study we report that similar to intestinal and respiratory mucosal epithelia, mouse ocular surface epithelia predominantly express the TGF-ß2 isoform. Specifically, we demonstrate the ability of goblet cells to express TGF-ß2 and increase it in response to Toll-Like Receptor 4 mediated stimulus in cultures. Goblet cells not only express TGF-ß2, 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-ß2, 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-ß2 in ocular mucosa and implicates their immunomodulatory function in maintaining mucosal immune homeostasis.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Conjunctival epithelium predominantly expresses TGF-ß2 and molecules associated with its activation, TSP-1 and CD36.
Expression of TGF-ß1 and -ß2 isoforms in conjunctiva was assessed using (a) immunofluorescence and (b) real time PCR. Both isoforms were detected in conjunctival epithelium, including goblet cell rich areas, with a significantly higher expression of TGF-ß2. Results are presented as relative expression to that of the housekeeping gene GAPDH. (c) Immunostaining for TSP-1 and its receptor CD36 was detected in WT mouse conjunctival epithelium. Nuclei were counterstained with DAPI (blue). Magnification = x40. (*P
Fig 2
Fig 2. Cultured conjunctival goblet cells express TGF-ß2, TSP-1 and CD36.
(a) The expression of TGF-ß1 and TGF-ß2 isoforms in primary cultures of conjunctival goblet cells was assessed using flow cytometry. Representative histograms from 3 independent experiments: unstained cells (empty histogram), isotype controls (grey histogram), and cultured goblet cells (solid histogram). The mean fluorescence Intensity (MFI) of TGF-ß2 staining was significantly increased as compared to TGF-ß1. (b) Expression of TGF-ß1 and -ß2 isoforms was evaluated in cultured goblet cells using real time PCR. Results are presented as relative expression to that of the housekeeping gene GAPDH. (*P
Fig 3
Fig 3. Stimulation of cultured goblet cells with LPS induces increased expression, secretion and activation of TGF-ß2.
(a) Expression of TGF-ß1 and -ß2 in LPS-exposed goblet cells was determined using real time PCR. Results are presented as relative expression to that of the housekeeping gene GAPDH. (b) Total TGF-ß and (c) active TGF-ß2 secreted by LPS-exposed goblet cells were measured using MFB-F11 reporter cells as described in methods. Changes in the levels relative to those secreted by untreated cells are presented. (*P
Fig 4
Fig 4. Thrombospondin-1, CD36 and TGF-ß2 colocalize on the goblet cell surface.
Flow cytometry analysis of the surface staining of CD36, TSP-1 and TGF-ß2 on cultured goblet cells derived from WT (a), TSP-1 −/− (b) or CD36 −/− (c) mice. Membrane CD36+, TSP-1+ and TGF-ß2+ goblet cells were detected in WT cultures, while no membrane bound TGF-ß2 was detectable in goblet cells derived from either TSP-1 −/− or CD36 −/− mice. Data are representative of 2 independent experiments.
Fig 5
Fig 5. Goblet cells deficient in TSP-1 or CD36 fail to release active TGF-ß2.
Activation of TGF-ß2 by LPS-exposed WT, TSP-1 deficient or CD36 −/− cultured conjunctival goblet cells was assessed using MFB-F11 reporter cell line. Unlike WT, both TSP-1 and CD36 deficient goblet cells failed to increase their active TGF-ß2 in response to LPS exposure in culture. Changes in the levels relative to the untreated goblet cells are presented. (*P
Fig 6
Fig 6. Goblet cell derived TGF-ß2 contributes to the tolerogenic phenotype of dendritic cells.
(a) Cultured WT goblet cells were grown to confluence on transwell polyester membranes. Multilayered growth was confirmed by H&E staining. WT BMDCs were cultured in the lower chamber. (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. Surface MHC class II staining was assessed by flow cytometry on WT (c) or TSP-1 deficient (d) BMDCs co-cultured with indicated goblet cells. Results are presented as relative expression to that of the housekeeping gene GAPDH, and as mean fluorescence intensity (MFI). (*P
Fig 7
Fig 7. Immunomodulatory function of conjunctival goblet cells in ocular surface mucosa.
In response to an LPS-mediated stimulus, conjunctival goblet cells increase expression of TGF-ß2, and activate it in a TSP-1 dependent manner via a TSP-1 receptor, CD36. Such goblet cell derived active TGF-ß2 modulates the phenotype of surrounding dendritic cells toward an immature or tolerogenic state, decreasing the expression of activation markers, such as MHC class II, CD80, CD86 and CD40.
See this image and copyright information in PMC

References

    1. Knop N, Knop E. Conjunctiva-associated lymphoid tissue in the human eye. Invest Ophthalmol Vis Sci 2000; 41: 1270–1279. - PubMed
    1. Reinoso R, Martin-Sanz R, Martino M, Mateo ME, Blanco-Salado R, Calonge M, et al. Topographical distribution and characterization of epithelial cells and intraepithelial lymphocytes in the human ocular mucosa. Mucosal Immunol 2012; 5: 455–467. 10.1038/mi.2012.27 - DOI - PubMed
    1. Egan RM, Yorkey C, Black R, Loh WK, Stevens JL, Storozynsky E, et al. In vivo behavior of peptide-specific T cells during mucosal tolerance induction: antigen introduced through the mucosa of the conjunctiva elicits prolonged antigen-specific T cell priming followed by anergy. J Immunol 2000; 164: 4543–4550. - PubMed
    1. Mantelli F, Argueso P. Functions of ocular surface mucins in health and disease. Curr Opin Allergy Clin Immunol 2008; 8: 477–483. 10.1097/ACI.0b013e32830e6b04 - DOI - PMC - PubMed
    1. Gipson IK, Inatomi T. Cellular origin of mucins of the ocular surface tear film. Adv Exp Med Biol 1998; 438: 221–227. - PubMed

Publication types

MeSH terms