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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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Expression of proinflammatory cytokines in HCLE-NT, HCLE-scrMUC1, HCLE-shMUC1, HCLE-scrMUC16, and HCLE-shMUC16 following exposure to TLR2 and TLR5 agonists[A, B] qRT-PCR analysis indicates that the message levels of the proinflammatory cytokines IL-6, IL-8, and TNF-α are increased in HCLE-shMUC1 and HCLE-shMUC16 cells in comparison to HCLE-NT, HCLE-scrMUC1, and HCLE-scrMUC16 cells following exposure of these cells to the TLR2 and TLR5 agonists, HKLM and flagellin, respectively. GAPDH was used as endogenous control in the qRT-PCRs. [C, D] Results from Luminex assays indicate that HCLE-shMUC1 and HCLE-shMUC16 cells secrete increased levels of IL-6, IL-8 and TNF-α following a 12 h exposure of the cells to HKLM and flagellin. Data shown are from experiments performed in biological triplicates. One way ANOVA was done to determine overall significance between groups. For internal comparisons, unpaired t-tests with Bonferroni correction were performed. *p<0.025 was considered significant.
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Figure 3: Expression of proinflammatory cytokines in HCLE-NT, HCLE-scrMUC1, HCLE-shMUC1, HCLE-scrMUC16, and HCLE-shMUC16 following exposure to TLR2 and TLR5 agonists[A, B] qRT-PCR analysis indicates that the message levels of the proinflammatory cytokines IL-6, IL-8, and TNF-α are increased in HCLE-shMUC1 and HCLE-shMUC16 cells in comparison to HCLE-NT, HCLE-scrMUC1, and HCLE-scrMUC16 cells following exposure of these cells to the TLR2 and TLR5 agonists, HKLM and flagellin, respectively. GAPDH was used as endogenous control in the qRT-PCRs. [C, D] Results from Luminex assays indicate that HCLE-shMUC1 and HCLE-shMUC16 cells secrete increased levels of IL-6, IL-8 and TNF-α following a 12 h exposure of the cells to HKLM and flagellin. Data shown are from experiments performed in biological triplicates. One way ANOVA was done to determine overall significance between groups. For internal comparisons, unpaired t-tests with Bonferroni correction were performed. *p<0.025 was considered significant.

Mentions: To test the hypothesis that the MAMs MUC1 and MUC16 suppress TLR-mediated innate immune responses, the responsiveness of differentiated MUC1- and MUC16-knockdown HCLE cells to agonists specific to TLR2 and TLR5 was investigated. As outcomes of TLR2 and TLR5 signaling, the levels of the proinflammatory cytokines IL-6, IL-8, and TNF-α in the different cell lines was measured by qRT-PCR. Following exposure of HCLE-NT, HCLE-scrMUC1, HCLE-shMUC1, HCLE-scrMUC16, and HCLE-shMUC16 to the agonists for 4 h, the message levels of IL-6, IL-8, and TNF-α were several fold higher (ranging from 2-fold to 6-fold) in HCLE-shMUC1 and HCLE-shMUC16 cells in comparison to the controls (Figure 3A, B). Under conditions where HCLE cells were not exposed to any agonist, the message levels of IL-6, IL-8, and TNF-α remained comparable among the different cell lines (Supplementary Figure S1) indicating that these cytokines are not constitutively overexpressed in the knockdown cells. Also, the levels of IL-6, IL-8, and TNF-α secreted into the culture medium by the different cells were measured following a 12 h agonist exposure. HCLE-shMUC1 and HCLE-shMUC16 cells were found to secrete significantly higher levels of IL-6, IL-8, and TNF-α in comparison to control cells (Figure 3C, D).


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 proinflammatory cytokines in HCLE-NT, HCLE-scrMUC1, HCLE-shMUC1, HCLE-scrMUC16, and HCLE-shMUC16 following exposure to TLR2 and TLR5 agonists[A, B] qRT-PCR analysis indicates that the message levels of the proinflammatory cytokines IL-6, IL-8, and TNF-α are increased in HCLE-shMUC1 and HCLE-shMUC16 cells in comparison to HCLE-NT, HCLE-scrMUC1, and HCLE-scrMUC16 cells following exposure of these cells to the TLR2 and TLR5 agonists, HKLM and flagellin, respectively. GAPDH was used as endogenous control in the qRT-PCRs. [C, D] Results from Luminex assays indicate that HCLE-shMUC1 and HCLE-shMUC16 cells secrete increased levels of IL-6, IL-8 and TNF-α following a 12 h exposure of the cells to HKLM and flagellin. Data shown are from experiments performed in biological triplicates. One way ANOVA was done to determine overall significance between groups. For internal comparisons, unpaired t-tests with Bonferroni correction were performed. *p<0.025 was considered significant.
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Figure 3: Expression of proinflammatory cytokines in HCLE-NT, HCLE-scrMUC1, HCLE-shMUC1, HCLE-scrMUC16, and HCLE-shMUC16 following exposure to TLR2 and TLR5 agonists[A, B] qRT-PCR analysis indicates that the message levels of the proinflammatory cytokines IL-6, IL-8, and TNF-α are increased in HCLE-shMUC1 and HCLE-shMUC16 cells in comparison to HCLE-NT, HCLE-scrMUC1, and HCLE-scrMUC16 cells following exposure of these cells to the TLR2 and TLR5 agonists, HKLM and flagellin, respectively. GAPDH was used as endogenous control in the qRT-PCRs. [C, D] Results from Luminex assays indicate that HCLE-shMUC1 and HCLE-shMUC16 cells secrete increased levels of IL-6, IL-8 and TNF-α following a 12 h exposure of the cells to HKLM and flagellin. Data shown are from experiments performed in biological triplicates. One way ANOVA was done to determine overall significance between groups. For internal comparisons, unpaired t-tests with Bonferroni correction were performed. *p<0.025 was considered significant.
Mentions: To test the hypothesis that the MAMs MUC1 and MUC16 suppress TLR-mediated innate immune responses, the responsiveness of differentiated MUC1- and MUC16-knockdown HCLE cells to agonists specific to TLR2 and TLR5 was investigated. As outcomes of TLR2 and TLR5 signaling, the levels of the proinflammatory cytokines IL-6, IL-8, and TNF-α in the different cell lines was measured by qRT-PCR. Following exposure of HCLE-NT, HCLE-scrMUC1, HCLE-shMUC1, HCLE-scrMUC16, and HCLE-shMUC16 to the agonists for 4 h, the message levels of IL-6, IL-8, and TNF-α were several fold higher (ranging from 2-fold to 6-fold) in HCLE-shMUC1 and HCLE-shMUC16 cells in comparison to the controls (Figure 3A, B). Under conditions where HCLE cells were not exposed to any agonist, the message levels of IL-6, IL-8, and TNF-α remained comparable among the different cell lines (Supplementary Figure S1) indicating that these cytokines are not constitutively overexpressed in the knockdown cells. Also, the levels of IL-6, IL-8, and TNF-α secreted into the culture medium by the different cells were measured following a 12 h agonist exposure. HCLE-shMUC1 and HCLE-shMUC16 cells were found to secrete significantly higher levels of IL-6, IL-8, and TNF-α in comparison to control cells (Figure 3C, D).

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