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Critical Role of Apoptotic Speck Protein Containing a Caspase Recruitment Domain (ASC) and NLRP3 in Causing Necrosis and ASC Speck Formation Induced by Porphyromonas gingivalis in Human Cells¹

Max Tze-Han Huang^*, Debra J. Taxman^*, Elizabeth A. Holley-Guthrie^*, Chris B. Moore^*, Stephen B. Willingham, Victoria Madden, Rebecca Keyser Parsons, Gerald L. Featherstone, Roland R. Arnold, Brian P. O'Connor^* and Jenny Pan-Yun Ting^2,*

^* Department of Microbiology and Immunology, Curriculum in Genetics and Molecular Biology, Lineberger Comprehensive Cancer Center, Department of Pathology, and School of Dentistry Diagnostic Sciences and General Dentistry, University of North Carolina, Chapel Hill, NC 27599

Periodontal disease is a chronic inflammatory disorder that leads to the destruction of tooth-supporting tissue and affects 10–20 million people in the U.S. alone. The oral pathogen Porphyromonas gingivalis causes inflammatory host response leading to periodontal and other secondary inflammatory diseases. To identify molecular components that control host response to P. gingivalis in humans, roles for the NLR (NBD-LRR) protein, NLRP3 (cryopyrin, NALP3), and its adaptor apoptotic speck protein containing a C-terminal caspase recruitment domain (ASC) were studied. P. gingivalis strain A7436 induces cell death in THP1 monocytic cells and in human primary peripheral blood macrophages. This process is ASC and NLRP3 dependent and can be replicated by P. gingivalis LPS and Escherichia coli. P. gingivalis-induced cell death is caspase and IL-1 independent and exhibits morphological features consistent with necrosis including loss of membrane integrity and release of cellular content. Intriguingly, P. gingivalis-induced cell death is accompanied by the formation of ASC aggregation specks, a process not previously described during microbial infection. ASC specks are observed in P. gingivalis-infected primary human mononuclear cells and are dependent on NLRP3. This work shows that P. gingivalis causes ASC- and NLRP3-dependent necrosis, accompanied by ASC speck formation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grants 1-R01-DE016326-01A1 and 1-U54-AI057157-03 (to J.P.-Y.T.).

² Address correspondence and reprint requests to Dr. Jenny Pan-Yun Ting, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599. E-mail address: panyun{at}med.unc.edu

³ Abbreviations used in this paper: NLR, nucleotide-binding domain leucine-rich repeat; LRR, leucine-rich repeat; NACHT, the NAIP, CIITA, HET-E, and TP1 domain; NALP3, the NACHT-, LRR-, PYD-containing protein; CARD, caspase recruitment domain; ASC, apoptotic speck protein containing a CARD; MOI, multiplicity of infection; Sh-, short hairpin-; PI, propidium iodide; TEM, transmission electron microscopy; PARP, poly(ADP-ribose) polymerase; Sh-CNTRL, control Sh-RNA with a mutated target ASC sequence; EV, empty vector.