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Danger signal adenosine via adenosine 2a receptor stimulates growth of Porphyromonas gingivalis in primary gingival epithelial cells.

ABSTRACT: Extracellular signaling during inflammation and chronic diseases involves molecules referred to as 'Danger Signals' (DS), including the small molecule adenosine. We demonstrate that primary gingival epithelial cells (GEC) express a family of G-protein coupled receptors known as adenosine receptors, including the high-affinity receptors A1 and A2a and low-affinity receptors A2b and A3. Treatment of Porphyromonas gingivalis-infected GEC with the A2a receptor-specific agonist CGS-21680 resulted in elevated intracellular bacterial replication as determined by fluorescence microscopy and antibiotic protection assay. Additionally, A2a receptor antagonism and knockdown via RNA interference significantly reduced metabolically active intracellular P. gingivalis. Furthermore, analysis of anti-inflammatory mediator cyclic AMP (cAMP) following A2a receptor selective agonist CGS-21680 stimulation induced significantly higher levels of cAMP during P. gingivalis infection, indicating that adenosine signaling may attenuate inflammatory processes associated with bacterial infection. This study reveals that the GEC express functional A2a receptor and P. gingivalis may use the A2a receptor coupled DS adenosine signaling as a means to establish successful persistence in the oral mucosa, possibly via downregulation of the pro-inflammatory response.

SUBMITTER: Spooner R

PROVIDER: S-EPMC3960722 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Danger signal adenosine via adenosine 2a receptor stimulates growth of Porphyromonas gingivalis in primary gingival epithelial cells.

Spooner R R DeGuzman J J Lee K L KL Yilmaz O O

Molecular oral microbiology 20140212 2

Extracellular signaling during inflammation and chronic diseases involves molecules referred to as 'Danger Signals' (DS), including the small molecule adenosine. We demonstrate that primary gingival epithelial cells (GEC) express a family of G-protein coupled receptors known as adenosine receptors, including the high-affinity receptors A1 and A2a and low-affinity receptors A2b and A3. Treatment of Porphyromonas gingivalis-infected GEC with the A2a receptor-specific agonist CGS-21680 resulted in ...[more]

PMID: 24517244

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Project description:Porphyromonas gingivalis is a pathogen in severe periodontal disease. Able to exploit an intracellular lifestyle within primary gingival epithelial cells (GECs), a reservoir of P. gingivalis can persist within the gingival epithelia. This process is facilitated by manipulation of the host cell signal transduction cascades which can impact cell cycle, cell death and cytokine responses. Using microarrays, we investigated the ability of P. gingivalis 33277 to regulate microRNA (miRNA) expression in GECs. One of several miRNAs differentially regulated by GECs in the presence of P. gingivalis was miR-203, which was upregulated 4-fold compared with uninfected controls. Differential regulation of miR-203 was confirmed by qRT-PCR. Putative targets of miR-203, suppressors of cytokine signaling (SOCS) 3 and 6, were evaluated by qRT-PCR. SOCS3 and SOCS6 mRNA levels were reduced >5-fold and >2-fold, respectively, in P. gingivalis-infected GECs compared with controls. Silencing miR-203 using a si-RNA construct reversed the inhibition of SOCS3 expression. A dual luciferase assay confirmed binding of miR-203 to the putative target binding site of SOCS3 3’ UTR. Western blot analysis demonstrated that activation of Stat3, a downstream target of SOCS, was diminished following miR-203 silencing. This study shows that induction of miRNAs by P. gingivalis can modulate important host signaling responses. MicroRNAs differentially expressed in human gingival epithelial cells in response to P. gingivalis infection were identified. P. gingivalis were reacted with primary gingival epithelial cells at an MOI of 100 for 6 hours at 37C in 5% CO2. Co-cultures were carried out in triplicate. The GECs were lysed with Trizol (Invitrogen) prior to RNA extraction and total RNA isolation. The microarray assay was performed using a service provider (LC Sciences). The assay started from 4 to 8 µg total RNA sample which was size fractionated using a YM-100 Microcon centrifugal filter (Millipore) and the small RNAs (< 300 nt) isolated were 3’-extended with a poly(A) tail using poly(A) polymerase. An oligonucleotide tag was then ligated to the poly(A) tail for later fluorescent dye staining. Hybridization was performed overnight on a µParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies). The hybridization melting temperatures were balanced by chemical modifications of the detection probes. Hybridization used 100 uL 6xSSPE buffer (0.90 M NaCl, 60 mM Na2HPO4, 6 mM EDTA, pH 6.8) containing 25% formamide at 34 °C. After RNA hybridization, tag-conjugating Cy3 dyes were circulated through the microfluidic chip for dye staining. Fluorescence images were collected using a laser scanner (GenePix 4000B, Molecular Device) and digitized using Array-Pro image analysis software (Media Cybernetics). Microarray data were analyzed by first subtracting the background and then normalizing the signals using a LOWESS filter (Locally-weighted Regression). For a transcript to be listed as detectable it had a signal intensity higher than 3×(background standard deviation) and spot coefficient of variance (CV) < 0.5. CV was calculated by (standard deviation)/(signal intensity). When repeating probes are present on an array, a transcript is listed as detectable only if the signals from at least 50% of the repeating probes are above detection level. t-Test is performed between “control” and “test” sample groups. T-values are calculated for each miRNA, and p-values are computed from the theoretical t-distribution. miRNAs with p-values below a critical p-value (typically 0.01) are selected for cluster analysis. The clustering is done using hierarchical method and is performed with average linkage and Euclidean distance metric. This experiment includes a total of 6 samples which are divided in to 2 groups, each with 3 biological repeats: 3 untreated normal samples as controls and 3 infected samples as the treated condition.

Dataset Information

Danger signal adenosine via adenosine 2a receptor stimulates growth of Porphyromonas gingivalis in primary gingival epithelial cells.

Publications

Danger signal adenosine via adenosine 2a receptor stimulates growth of Porphyromonas gingivalis in primary gingival epithelial cells.

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