Candida albicans-derived mannoproteins activate NF-κB in reporter cells expressing TLR4, MD2 and CD14.

The ability of soluble C. albicans 20A (serotype A) mannoprotein (CMP) to serve as a ligand for toll-like receptor 4 (TLR4) and its co-receptors was examined using commercially available and stably-transfected HEK293 cells that express human TLR4, MD2 and CD14, but not MR. These TLR4 reporter cells also express an NF-κB-dependent, secreted embryonic alkaline phosphatase (SEAP) reporter gene. TLR4-reporter cells exhibited a dose-dependent SEAP response to both LPS and CMP, wherein peak activation was achieved after stimulation with 40-50 μg/mL of CMP. Incubation on polymyxin B resin had no effect on CMP's ligand activity, but neutralized LPS-spiked controls. HEK293 Null cells lacking TLR4 and possessing the same SEAP reporter failed to respond to LPS or CMP, but produced SEAP when activated with TNFα. Reporter cell NF-κB responses were accompanied by transcription of IL-8, TNFα, and COX-2 genes. Celecoxib inhibited LPS-, CMP-, and TNFα-dependent NF-κB responses; whereas, indomethacin had limited effect on LPS and CMP responses. SEAP production in response to C. albicans A9 mnn4Δ mutant CMP, lacking phosphomannosylations on N-linked glycans, was significantly greater (p ≤ 0.005) than SEAP responses to CMP derived from parental A9 (both serotype B). These data confirm that engineered human cells expressing TLR4, MD2 and CD14 can respond to CMP with NF-κB activation and the response can be influenced by variations in CMP-mannosylation. Future characterizations of CMPs from other sources and their application in this model may provide further insight into variations observed with TLR4 dependent innate immune responses targeting different C. albicans strains

CMP-induced TLR4-signaling activates <i>TNF</i>α and <i>IL-8</i> mRNA expression.

<p><i>TNFα</i> (squares) and <i>IL-8</i> (triangles) transcript levels were assessed by quantitative PCR. (A) HEK-TLR4 reporter cells (filled symbols) and HEK-Null reporter cells (open symbols) cells were stimulated with 12.5 ng/mL LPS for the indicated times prior to RNA isolation and qPCR analyses. (B) HEK-TLR4 reporter cells were treated with different doses of CMP (1 mg/mL, solid line; 100 μg/mL, dashed line) for the indicated times (0–4 h). Significance is represented by the asterisk, p ≤ 0.05.</p

Celecoxib inhibits CMP activation of TLR4 in a dose-dependent manner.

<p>HEK-TLR4 cells were pre-treated with with 0 μM (filled circles), 10 μM (open triangles), 30 μM (open diamonds), or 100 μM (open circles) celecoxib for one hour prior to treatment with increasing concentrations of CMP as indicated. SEAP activity was measured the next day to evaluate cellular activation. CMP-induced SEAP activity was significantly reduced by celecoxib treatment (open symbols). Significance between the collective regressions is represented by the asterisks p ≤ 0.005.</p

PAGE and Western blot analysis of CMP preparations.

<p>CMPs were combined with SDS sample buffer containing DTT to yield 1 mg/mL samples: 10ul of each sample was applied per well in 2–8% Tris acetate polyacrylamide gels and electrophoresed with a Tris/Tricine SDS running buffer. Western blot was performed in a standard discontinuous Tris/CAPS buffer. Gel lanes are: Lane A, molecular weight markers; Lane B, polysaccharide-stained CMP; Lane C, silver-stained CMP. Duplicate gels were also blotted to nitrocellulose. Lane D & E, 2 independent CMP samples probed with polyclonal rabbit anti-mannan antibody (Dako); Lane F & G, repeated 2 independent CMP samples probed with polyclonal rabbit anti-<i>C</i>. <i>albicans</i> antibody (Thermo Fisher). Chemiluminescent visualization of primary antibody binding used a goat anti-rabbit-peroxidase conjugate and a Super Signal West Pico Substrate Kit. These images are representative of 2 independent preps, wherein D and F represent a single CMP sample that displayed more activity than the E and G sample.</p

CMPs derived from <i>C</i>. <i>albicans</i> A9 and A9 <i>mnn</i>4<i>Δ</i> mutant strains both stimulate HEK-TLR4 cells.

<p>The A9 <i>mnn</i>4 <i>Δ</i> mutant CMP lacks phosphodiester-linked extensions on the <i>N</i>-linked glycans and was found to be superior to CMP from the A9 parent strain as a TLR4 agonist. In this experiment SEAP conversion was stopped at 45 min. Peak LPS stimulation corresponded to mean absorbance of 0.11 +/- 0.004 absorbance units (positive control). The negative control wells received only additional media. All data were converted to percentage of LPS control by dividing the mean net absorbance of CMP treated cells by the mean net absorbance of LPS treated cells. SEM was converted to % SEM in the same manner. Significance that is represented by the asterisks was p ≤ 0.005.</p

Celecoxib inhibits TNFα-induced activation of HEK-TLR4 cells in a dose-dependent manner.

<p>HEK-TLR4 cells were treated with varying concentrations of celecoxib for one hour after which cells were stimulated with TNFα (2μg/well). After overnight incubation, SEAP activity was measured to evaluate cellular activation. Significance is represented by the asterisks, p ≤ 0.0005.</p

Celecoxib-treatments below 100mM are not cytotoxic to HEK-TLR4 cells.

<p>The HEK reporter cells were cultured overnight in dilutions of celecoxib (triangles) or Triton X-100 (control; squares). The X axis shows the final concentrations of celecoxib in the culture media and the numerical labels above the square data labels show the mM concentrations of Triton X-100 used in the positive cytotoxic controls. Cell viability was tested using XTT + menadione. Substrate (XTT) conversion was monitored and measured as OD 490 during a 12 h incubation. Net absorbance was adjusted by subtraction of media only (acellular) absorbance and % cytotoxicity was computed as an absorbance quotient using treated cell XTT conversion/non treated cell XTT conversion x 100%. Computed SEM were negligible and while shown in the graph, they are hidden by the symbols.</p

Indomethacin inhibits TLR4 activation only at high concentrations.

<p>HEK-TLR4 cells were pre-treated with a range of indomethacin concentrations (0–160 μM) for 1 h. After pre-treatment, cells were subjected to stimulation with media (filled circles), 12.5 ng/mL LPS (open squares) or 40 μg/mL CMP (open triangles). Ligand concentrations were selected after determining the lowest concentration required to achieve near-maximal SEAP activity. SEAP activity was measured after overnight incubation to evaluate cellular activation. The asterisk represents the significance (p ≤ 0.05) wherein the variance in mean SEAP activity of media plus indomethacin is compared with CMP or LPS treatments in the presence of 160 μM indomethacin.</p

LPS- and CMP-mediated activation of TLR4 signaling induces <i>COX-2</i> expression.

<p>(A) HEK-TLR4 reporter cells (filled squares) and HEK-Null reporter cells (gray squares) were stimulated with 12.5 ng/mL LPS over 4 hours. Additionally, HEK-TLR4 cells were stimulated with 1 mg/mL CMP for 4 hours (B). Total RNA was isolated and subjected to quantitative PCR analysis. <i>COX-2</i> expression in individual samples was normalized to expression of <i>β-actin</i> and overall expression is relative to cells treated with media alone. Significance is represented by the asterisk, p ≤ 0.05.</p

Polymyxin B depletion of LPS from spiked-positive control samples abolishes SEAP responses.

<p>To demonstrate the effectiveness of LPS depletion, samples of LPS-spiked media (0.16–10 ng/ml) were incubated twice on polymyxin columns with gentle rocking at room temperature. After elution from the columns, the effluents were applied to TLR4-reporter cells (as performed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189939#pone.0189939.g002" target="_blank">Fig 2</a>) and the stimulation was assessed with QuantiBlue. The gray bar represents the mean background of SEAP activity +/- SEM). The % net change in O.D. (HEK treatment O.D. minus background O.D.) produced by polymyxin depyrogenation of 10 ng/ml LPS was determined to be 89%. Significance is represented by the asterisks, p ≤ 0.005.</p