Porphyromonas gingivalis Cell Wall Components Induce Programmed Death Ligand 1 (PD-L1) Expression on Human Oral Carcinoma Cells by a Receptor-Interacting Protein Kinase 2 (RIP2)-Dependent Mechanism

Abstract

Programmed death-ligand 1 (PD-L1/B7-H1) serves as a cosignaling molecule in cell-mediated immune responses and contributes to chronicity of inflammation and the escape of tumor cells from immunosurveillance. Here, we investigated the molecular mechanisms leading to PD-L1 upregulation in human oral carcinoma cells and in primary human gingival keratinocytes in response to infection with Porphyromonas gingivalis (P. gingivalis), a keystone pathogen for the development of periodontitis. The bacterial cell wall component peptidoglycan uses bacterial outer membrane vesicles to be taken up by cells. Internalized peptidoglycan triggers cytosolic receptors to induce PD-L1 expression in a myeloid differentiation primary response 88 (Myd88)-independent and receptor-interacting serine/threonine-protein kinase 2 (RIP2)-dependent fashion. Interference with the kinase activity of RIP2 or mitogen-activated protein (MAP) kinases interferes with inducible PD-L1 expression.

Keywords: B7-H1; PD-L1; Porphyromonas gingivalis; immune evasion; immune suppression; signaling pathway.

FIG 1

PD-L1 induction by P. gingivalis. Upper: viable and heat-killed bacteria were added to SCC-25 cells in the indicated MOIs. After 1 day, cells were harvested and equal amounts of protein contained in cell lysates were used for Western blotting using the indicated antibodies, including β-Actin as a loading control. Lower: triplicates of the Western blots were used for protein quantification using the Image J software. Protein amounts in untreated cells were arbitrarily set as 1; the error bars show standard deviations.

FIG 2

Expression of PD-L1: P. gingivalis extracts were fractionated into cytosolic and PDG-containing total membrane (TM) fractions. (A) P. gingivalis was extracted to generate cytosolic (Cyt) and TM fractions. These fractions were separated together with a PDG control by SDS-PAGE and the gel was stained with Coomassie blue. The positions of molecular weight markers are shown; the position of PDG is indicated by a box. (B) The TM fraction and PDG in the indicated concentrations were used to treat SCC-25 cells for 1 day, followed by analysis of PD-L1 expression using Western blot (upper) and a quantitative analysis of three Western blots (lower). (C) Upper: primary human gingival keratinocytes (PHGKs) were incubated with P. gingivalis PDG at the indicated concentrations for 1 day. Cells were harvested and equal amounts of protein contained in cell lysates were used for Western blotting using PD-L1 antibodies. Lower: the results from three Western blots were quantified by Image J. PD-L1 protein expression from unstimulated cells was arbitrarily set as 1; the error bars show standard deviations; *, P < 0.05.

FIG 3

Prepared muropeptides treated with mutanolysin from Streptomyces globisporus ATCC 21553 were used to analyze P. gingivalis-derived peptidoglycan using MALDI-TOF mass spectrometry. (A) Overview of the spectrum indicating two clusters of signals (regions 1 and 2). (B and C) Both clusters are composed of repeating motifs. The annotated mass differences of 42 Da show the presence of acetyl residues, whereas the mass differences of 57 Da indicate glycine-repeating units. Besides these clusters, no significant signals were detectable, indicating that the preparation was pure.

FIG 4

OMVs and PDG from P. gingivalis strain W83 trigger PD-L1 expression. (A) SCC-25 cells were treated with the indicated concentrations of OMVs for 1 day and PD-L1 expression was determined by Western blotting (upper) and shown as the quantification of three independent experiments (lower). Triplicates of the Western blots were used for protein quantification using the Image J software. Protein amounts in untreated cells were arbitrarily set as 1; the error bars show standard deviations. (B) The P. gingivalis TM fraction was labeled with a fluorochrome ester and added to the cells for the indicated periods. Flow cytometry was used to determine the fluorescence uptake. Data represent the mean ± SD of 5 independent experiments. The results are shown as box plots. The boxes represent the 2nd and 3rd quartiles as the middle 50% of the scores. The line that divides the boxes displays the median. The lower and upper whiskers indicate the 1st and 4th quartiles. The crosses mark the mean values. (C) Cells were incubated with suramin at the indicated concentrations and infected with P. gingivalis (MOI 100) for 1 day and the PD-L1 expression was investigated using flow cytometry. The quantity of the PD-L1 expression is described as mean fluorescence intensity (MFI), provided in arbitrary units. All investigations were performed in three different independent experiments. The results (MFI of P. gingivalis infected against noninfected cells) were analyzed using independent two-sample Student’s t tests. The character of the evaluation was explorative. The probability of error was set to 5% and shown as P values; n = 3, *, P < 0.05; ‡, P < 0.01.

FIG 5

Myd88-independent PD-L1 induction by P. gingivalis. (A) SCC-25 cells were transfected with a plasmid encoding a Myd88-specific Cas9 enzyme cleaving in the first exon and individual cell clones were tested for expression of Myd88 and Cas9 by Western blotting as shown. (B) The indicated control and Myd88-deficient SCC-25 cells were stimulated with P. gingivalis TM and PD-L1 expression was analyzed by Western blotting.

FIG 6

RIP2-dependent PD-L1 induction by P. gingivalis. (A) SCC-25 cells were transfected with a plasmid encoding a RIP2-specific Cas9 enzyme cleaving in the promoter just upstream from the transcription start site and individual cell clones were tested for expression of RIP2 by Western blotting as shown. (B) Cells with wild-type and reduced levels of RIP2 were treated for 1 day with the indicated concentrations of P. gingivalis TM and analyzed by Western blotting for PD-L1 expression as shown. (C) The experiment was done as described for panel B except that C12-iE-DAP was used as a stimulating agent.

FIG 7

P. gingivalis-induced expression of PD-L1 depends on the kinase activity of RIP2 and MAPKs. (A) SCC-25 cells were incubated with the RIP2 inhibitor gefitinib and stimulated with P. gingivalis TM for 4 and 24 h as shown, followed by the analysis of PD-L1 expression by Western blotting. (B) SCC-25 cells were incubated with the indicated MAPK and NOD inhibitors as shown and stimulated with the P. gingivalis TM fraction. PD-L1 protein expression was scored by immunoblotting and the results from three Western blots were quantified by Image J. Maximal PD-L1 protein expression from stimulated cells was arbitrarily set as 100%; the error bars show standard deviations. Triplicates of the Western blots were used for protein quantification using the Image J software; the error bars show standard deviations. (C) PHGKs were stimulated with the P. gingivalis TM fraction in the presence of the indicated inhibitors for 24 h. The analysis of PD-L1 expression was revealed by Western blotting; the results from three Western blots were quantified by Image J. Maximal PD-L1 protein expression from stimulated cells was arbitrarily set as 100%; the error bars show standard deviations.