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ROLE of NLR PROTEINS NLRP3 AND NLRX1 in ORAL BACTERIA INFECTED GINGIVAL EPITHELIAL CELLS

  • Author(s): Hung, Shu-Chen
  • Advisor(s): Jensen, Kirk
  • Ojcius, David
  • et al.
Abstract

Periodontitis, a chronic inflammatory disease results from both dysbiotic microbiota and dysregulated innate immune responses in the periodontal tissue, can affect up to 90% of the population in industrialized countries and developing countries globally. However, how dysbiosis triggers dysregulated inflammatory responses remains unclear. Many NOD-like receptors (NLRs) have been shown to contribute to the dysregulated inflammation in periodontitis. Thus, we investigated the role of NLRs in periodontitis in molecular level.

NLRs play an important role in regulation of host innate immunity, yet their role in periodontitis remains to be defined. NLRP3, one of the well-studied NLR family members, can form a multiprotein complex called inflammasome and has been shown to be one of the dysregulated inflammatory responses in periodontitis. We have previously reported that infection of gingival epithelial cells (GECs) with periopathogen Porphyromonas gingivalis, keystone pathogens for periodontitis, requires an exogenous danger signal such as extracellular ATP (eATP) to activate NLRP3 inflammasome and caspase-1, thereby inducing secretion of interleukin (IL)-1β. Stimulation with eATP also stimulates production of reactive oxygen species (ROS) in GECs. However, the mechanism by which ROS is generated in response to eATP, and the role that different purinergic receptors may play in inflammasome activation, is still unclear. In this study, we began with revealing that the purinergic receptor P2X4 is assembled with the receptor P2X7 and its associated pore, pannexin-1. eATP induces ROS production through a complex consisting of the P2X4, P2X7, and pannexin-1. P2X7−mediated ROS production can activate the NLRP3 inflammasome and caspase-1 by oxidizing endogenous ligands for NLRP3. Furthermore, separate depletion or inhibition of P2X4, P2X7, or pannexin-1 complex blocks IL-1β secretion in P. gingivalis-infected GECs following eATP treatment. However, activation via P2X4 alone induces ROS generation but not inflammasome activation. These results suggest that ROS is generated through stimulation of a P2X4/P2X7/pannexin-1 complex, and reveal an unexpected role for P2X4, which acts as a positive regulator of inflammasome activation during microbial infection.

NLRX1, a new member of the NLR family that localizes to mitochondria, has been shown to modulate mitochondrial ROS (mROS) generation. mROS activates the NLRP3 inflammasome upon eATP stimulation or microbial infection, yet the role of NLRX1 in NLRP3 inflammasome activation has not been examined. In this study, we further revealed the mechanism by which NLRX1 positively regulates eATP-elicited NLRP3 inflammasome activation through mROS in GECs. Fluorescence microscopy showed that depletion of NLRX1 by shRNA attenuates eATP-induced mROS generation and redistribution of the NLRP3 inflammasome adaptor protein, ASC. Furthermore, we have shown that another periopathogen Fusobacterium nucleatum infection activates NLRP3 inflammasome in GECs. Depletion of NLRX1 inhibits F. nucleatum infection-activated caspase-1, suggesting that it also inhibits the NLRP3 inflammasome. Therefore, we propose that NLRX1 may promote F. nucleatum-caused dysregulated pro-inflammatory responses in periodontitis. On the other hand, the mechanism by which F. nucleatum infection-induces IL-8 expression is still unclear. We showed that NLRX1 also acts as a negative regulator in NF-κB signaling to modulate IL-8 expression. Thus, NLRX1 stimulates detection of the pathogen F. nucleatum via the inflammasome, while dampening cytokine production. We expect that commensals should not activate the inflammasome, but NLRX1 should still decrease their ability to stimulate inflammation (to prevent hyper-regulated cytokine expression). Consequently, we conclude that NLRX1 may act as a potential switch in regards to the virulence of F. nucleatum in healthy or diseased oral cavity.

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