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. 2022 Jun 29;13:912748. doi: 10.3389/fimmu.2022.912748

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Copyright © 2022 Kumar, Rojas and Edgerton

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Sap6 mediated PAR2 activation results in decreased epithelial barrier integrity and increases C. albicans invasion. (A) Changes in TEER value in OECs treated with rSap6 (0.1-10µM), PAR2-ANT+rSap6 and PAR2-AP were calculated over 48 h using an EVOM2 epithelial volt ohmmeter. Measured TEER values were normalized to untreated OECs. There was a dose dependent reduction of TEER value in OECs treated with rSap6 (0.1-10µM) with 10 μM rSap6 causing a 78% reduction in TEER similar to PAR2-AP treated cells. OECs pre-treated with PAR2-ANT before Sap6 treatment did not show any significant change in TEER. (B) OEC levels of E-cadherin and occludin proteins were examined by immunoblot and densitometry following 24 h treatment of cells with PAR2-AP, rSap6 or PAR2-ANT+rSap6 using E-cadherin or occludin antibodies. OEC treated with either rSap6 or PAR2-AP showed significant reduced levels of both E-cadherin and occludin compared to OEC control, while pretreatment with PAR2-ANT before Sap6 restored levels of both E-cadherin and occludin to control. Significance was calculated using two-way ANOV compared to untreated OECs. P values are p ≤ 0.01 (**), p ≤ 0.001 (***). (C) Cell surface expression of E-cadherin was visualized microscopically following rSap6 or PAR2-AP treatment, then OECs were fixed, permeabilized and stained with E-cadherin (green) and Hoestch (blue). Untreated OECs showed high expression of surface E-cadherin, whereas treatment with rSap6 or PAR2-AP reduced E-cadherin expression that was blocked by pretreatment with PAR2-ANT. Scale bar indicates 10 μm. (D) Candida albicans cells (1 × 10⁵) were added to OEC monolayers pretreated with PAR2-AP, rSap6, RGD+rSap6 or PAR2-ANT+rSap6. After 4.5 h, epithelial cells were permeabilized and C albicans cells were stained with anti-Candida antibody and Alexa Fluor 488 to visualize non-invading cells (green). Invasion was quantitated as percentage fold change in invasion comparing to untreated OECs. OECs treated with rSap6 or PAR2-AP had higher invasion while rSap6 (Inact) did not change invasion levels of C albicans. Pre-treatment of OECs with PAR2-ANT or RGD+rSap6 resulted in reduced hyphal invasion. Scale bar indicates 20 µm.