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Suppression of Toll-like receptor-mediated innate immune responses at the ocular surface by the membrane-associated mucins MUC1 and MUC16.

Menon BB, Kaiser-Marko C, Spurr-Michaud S, Tisdale AS, Gipson IK - Mucosal Immunol (2015)

Bottom Line: Using an in vitro model of corneal epithelial cells that are cultured to express MAMs, we show that reduced expression of either MUC1 or MUC16 correlates with increased message and secreted protein levels of the proinflammatory cytokines interleukin (IL)-6, IL-8, and tumor necrosis factor-α (TNF-α) following exposure of cells to the TLR2 and TLR5 agonists, heat-killed Listeria monocytogenes and flagellin, respectively.As mice express Muc1 (but not Muc16) in the corneal epithelium, a Muc1(-/-) mouse model was used to extend in vitro findings.Indeed, IL-6 and TNF-α message levels were increased in the corneal epithelium of Muc1(-/-) mice, in comparison with wild-type mice, following exposure of enucleated eyes to the TLR2 and TLR5 agonists.

View Article: PubMed Central - PubMed

Affiliation: Schepens Eye Research Institute, Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
Membrane-associated mucins (MAMs) expressed on the ocular surface epithelium form a dense glycocalyx that is hypothesized to protect the cornea and conjunctiva from external insult. In this study, the hypothesis that the MAMs MUC1 and MUC16, expressed on the apical surface of the corneal epithelium, suppress Toll-like receptor (TLR)-mediated innate immune responses was tested. Using an in vitro model of corneal epithelial cells that are cultured to express MAMs, we show that reduced expression of either MUC1 or MUC16 correlates with increased message and secreted protein levels of the proinflammatory cytokines interleukin (IL)-6, IL-8, and tumor necrosis factor-α (TNF-α) following exposure of cells to the TLR2 and TLR5 agonists, heat-killed Listeria monocytogenes and flagellin, respectively. As mice express Muc1 (but not Muc16) in the corneal epithelium, a Muc1(-/-) mouse model was used to extend in vitro findings. Indeed, IL-6 and TNF-α message levels were increased in the corneal epithelium of Muc1(-/-) mice, in comparison with wild-type mice, following exposure of enucleated eyes to the TLR2 and TLR5 agonists. Our results suggest that the MAMs MUC1 and MUC16 contribute to the maintenance of immune homeostasis at the ocular surface by limiting TLR-mediated innate immune responses.

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Related in: MedlinePlus

Expression of TLRs by differentiated corneal epithelial cellsRT-PCR analysis indicates that differentiated HCLE-NT cells express TLRs 2, 5, 6, and 10. An identical expression profile was observed in native corneal epithelial cells. In the figure, the panel labeled ‘Control’ included cDNA from a human monocytic cell line as template in the reactions. DNA molecular weight standards in kilobasepairs are indicated on the left of the gel.
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Figure 1: Expression of TLRs by differentiated corneal epithelial cellsRT-PCR analysis indicates that differentiated HCLE-NT cells express TLRs 2, 5, 6, and 10. An identical expression profile was observed in native corneal epithelial cells. In the figure, the panel labeled ‘Control’ included cDNA from a human monocytic cell line as template in the reactions. DNA molecular weight standards in kilobasepairs are indicated on the left of the gel.

Mentions: As a model system in this study, cultured HCLE cells that 1) have been stratified and allowed to differentiate for optimal MAM expression and 2) mimic several characteristics of the native corneal epithelium upon stratification were used.28 As a first step, the spectrum of TLRs expressed by differentiated HCLE cells was determined. By performing RT-PCR analyses, HCLE-NT cells were found to express TLRs 2, 5, 6, and 10 (Figure 1). A very faint expression of TLR4 was also observed. Importantly, an identical TLR expression profile was observed when cDNA derived from fresh human corneal epithelium, removed at the time of surgery, was used as template (Figure 1). HCLE-scrMUC1, HCLE-shMUC1, HCLE-scrMUC16, and HCLE-shMUC16 were also found to mimic the TLR expression profile of HCLE-NT cells (data not shown). Since TLR2 and TLR5 transcripts were found to be highly expressed in HCLE cells (Figure 1), agonists specific to these receptors were chosen for subsequent experiments. The reason for not choosing agonists to TLR6 and TLR10 was because these receptors are functional only upon association with TLR2 and neither ligand specificity nor function of TLR10 has been defined.44, 45


Suppression of Toll-like receptor-mediated innate immune responses at the ocular surface by the membrane-associated mucins MUC1 and MUC16.

Menon BB, Kaiser-Marko C, Spurr-Michaud S, Tisdale AS, Gipson IK - Mucosal Immunol (2015)

Expression of TLRs by differentiated corneal epithelial cellsRT-PCR analysis indicates that differentiated HCLE-NT cells express TLRs 2, 5, 6, and 10. An identical expression profile was observed in native corneal epithelial cells. In the figure, the panel labeled ‘Control’ included cDNA from a human monocytic cell line as template in the reactions. DNA molecular weight standards in kilobasepairs are indicated on the left of the gel.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4495011&req=5

Figure 1: Expression of TLRs by differentiated corneal epithelial cellsRT-PCR analysis indicates that differentiated HCLE-NT cells express TLRs 2, 5, 6, and 10. An identical expression profile was observed in native corneal epithelial cells. In the figure, the panel labeled ‘Control’ included cDNA from a human monocytic cell line as template in the reactions. DNA molecular weight standards in kilobasepairs are indicated on the left of the gel.
Mentions: As a model system in this study, cultured HCLE cells that 1) have been stratified and allowed to differentiate for optimal MAM expression and 2) mimic several characteristics of the native corneal epithelium upon stratification were used.28 As a first step, the spectrum of TLRs expressed by differentiated HCLE cells was determined. By performing RT-PCR analyses, HCLE-NT cells were found to express TLRs 2, 5, 6, and 10 (Figure 1). A very faint expression of TLR4 was also observed. Importantly, an identical TLR expression profile was observed when cDNA derived from fresh human corneal epithelium, removed at the time of surgery, was used as template (Figure 1). HCLE-scrMUC1, HCLE-shMUC1, HCLE-scrMUC16, and HCLE-shMUC16 were also found to mimic the TLR expression profile of HCLE-NT cells (data not shown). Since TLR2 and TLR5 transcripts were found to be highly expressed in HCLE cells (Figure 1), agonists specific to these receptors were chosen for subsequent experiments. The reason for not choosing agonists to TLR6 and TLR10 was because these receptors are functional only upon association with TLR2 and neither ligand specificity nor function of TLR10 has been defined.44, 45

Bottom Line: Using an in vitro model of corneal epithelial cells that are cultured to express MAMs, we show that reduced expression of either MUC1 or MUC16 correlates with increased message and secreted protein levels of the proinflammatory cytokines interleukin (IL)-6, IL-8, and tumor necrosis factor-α (TNF-α) following exposure of cells to the TLR2 and TLR5 agonists, heat-killed Listeria monocytogenes and flagellin, respectively.As mice express Muc1 (but not Muc16) in the corneal epithelium, a Muc1(-/-) mouse model was used to extend in vitro findings.Indeed, IL-6 and TNF-α message levels were increased in the corneal epithelium of Muc1(-/-) mice, in comparison with wild-type mice, following exposure of enucleated eyes to the TLR2 and TLR5 agonists.

View Article: PubMed Central - PubMed

Affiliation: Schepens Eye Research Institute, Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
Membrane-associated mucins (MAMs) expressed on the ocular surface epithelium form a dense glycocalyx that is hypothesized to protect the cornea and conjunctiva from external insult. In this study, the hypothesis that the MAMs MUC1 and MUC16, expressed on the apical surface of the corneal epithelium, suppress Toll-like receptor (TLR)-mediated innate immune responses was tested. Using an in vitro model of corneal epithelial cells that are cultured to express MAMs, we show that reduced expression of either MUC1 or MUC16 correlates with increased message and secreted protein levels of the proinflammatory cytokines interleukin (IL)-6, IL-8, and tumor necrosis factor-α (TNF-α) following exposure of cells to the TLR2 and TLR5 agonists, heat-killed Listeria monocytogenes and flagellin, respectively. As mice express Muc1 (but not Muc16) in the corneal epithelium, a Muc1(-/-) mouse model was used to extend in vitro findings. Indeed, IL-6 and TNF-α message levels were increased in the corneal epithelium of Muc1(-/-) mice, in comparison with wild-type mice, following exposure of enucleated eyes to the TLR2 and TLR5 agonists. Our results suggest that the MAMs MUC1 and MUC16 contribute to the maintenance of immune homeostasis at the ocular surface by limiting TLR-mediated innate immune responses.

Show MeSH
Related in: MedlinePlus