Title

Porphyromonas gingivalis’ Nucleoside Diphosphate Kinase Contributes to IL-1β Production and Secretion in Macrophages

ORCiD

Cassio Almeida-da-Silva: 0000-0001-9173-7208

Document Type

Conference Presentation

Conference Title

12th World Congress on Inflammation

Location

Boston, MA

Conference Dates

August 8-12, 2015

Date of Presentation

8-12-2015

Abstract

Porphyromonas gingivalis (Pg) are Gram-negative bacteria associated with periodontitis. One of Pg’s virulence factors is a homolog of nucleoside disphosphate kinase (NDK) enzyme, which is secreted by Pg. NDK cleaves extracellular ATP (eATP), suppressing ATP-induced P2 9 7-dependent apoptosis and ATP-induced reactive oxygen species (ROS) production via P2X7-NADPH-oxidase, and contributes to Pgpersistence in gingival epithelial cells. Our group showed that ATP via P2X7 controls infections with different kind of intracellular parasites such as Leishmania amazonensis and Toxoplasma gondii via IL-1b production. We also showed that IL-1b secretion induced by P2X7 signaling in macrophages is impaired by Pg’s fimbriae. Here, we investigated the role of Pg’s NDK in infection in vitro. To analyze whether IL-1b secretion is modulated by Pg’s NDK, we infected bone marrow derived macrophages (BMDM) with wild-type Pg or NDKdeficient (Pg D ndk) (MOI of 100) for 6, 18 or 24 h, and treated the cells or not with 5 mM ATP during the last 30 min. BMDM infected with Pg D ndk showed less IL-1b secretion by ELISA than wild-type Pg at all times tested. IL-1b secretion was dependent on P2X7 signaling since there was no secretion in BMDM from P2X7-/- mice infected with Pg. To verify immature pro-IL-1b production, we analyzed IL1-b in BMDM infected with Pg by Western blotting. By densitometry, we quantified 46 % less pro-IL-1b in cellular extracts and 46 % less IL-1b in supernatants of BMDM infected with Pg D ndk than wild-type Pg. Next, we tested the invasion capacity of Pg strains using the antibiotic protection assay. Pg strains infected at similar levels after 2 h of infection. Further, to verify whether Pg’s NDK hydrolyzes eATP, we quantified this nucleotide in BMDM supernatants after Pg infection. We detected more ATP in BMDM supernatants infected with Pg D ndk than wild-type Pg. Recently, adenosine (Ado) was shown to potentiate IL-1b secretion in macrophages treated with LPS/ATP. Therefore, we evaluated IL-1b release in BMDM supernatants after infection with Pg and treatment with ATP, Ado or its pan-antagonist receptor (CGS15943). Ado potentiated IL-1b secretion induced by Pg/ATP, and this enhancement was abolished by CGS15943. Together, these results indicate that Pg’s NDK contributes to IL-1b production and secretion in BMDM, and that the mechanism might be due to Ado production and signaling via Ado receptor after ATP cleavage by Pg’s NDK and other BMDM ectonucleotidases.

Comments

Financial support CAPES, CNPq, FAPERJ, National Institutes of Health.

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