Fusobacterium nucleatum infection of gingival epithelial cells leads to NLRP3 inflammasome-dependent secretion of IL-1ß and the danger signals ASC and HMGB1.

Fusobacterium nucleatum is an invasive anaerobic bacterium that is associated with periodontal disease. Previous studies have focused on virulence factors produced by F. nucleatum, but early recognition of the pathogen by the immune system remains poorly understood. Although an inflammasome in gingival epithelial cells (GECs) can be stimulated by danger-associated molecular patterns (DAMPs) (also known as danger signals) such as ATP, inflammasome activation by this periodontal pathogen has yet to be described in these cells. This study therefore examines the effects of F. nucleatum infection on pro-inflammatory cytokine expression and inflammasome activation in GECs. Our results indicate that infection induces translocation of NF-κB into the nucleus, resulting in cytokine gene expression. In addition, infection activates the NLRP3 inflammasome, which in turn activates caspase-1 and stimulates secretion of mature IL-1ß. Unlike other pathogens studied until now, F. nucleatum activates the inflammasome in GECs in the absence of exogenous DAMPs such as ATP. Finally, infection promotes release of other DAMPs that mediate inflammation, such as high-mobility group box 1 protein and apoptosis-associated speck-like protein, with a similar time-course as caspase-1 activation. Thus, F. nucleatum expresses the pathogen-associated molecular patterns necessary to activate NF-κB and also provides an endogenous DAMP to stimulate the inflammasome and further amplify inflammation through secretion of secondary DAMPs.

Pharmacological inhibition and reversal of pancreatic acinar ductal metaplasia.

Pancreatic acinar cells display a remarkable degree of plasticity and can dedifferentiate into ductal-like progenitor cells by a process known as acinar ductal metaplasia (ADM). ADM is believed to be one of the earliest precursor lesions toward the development of pancreatic ductal adenocarcinoma and maintaining the pancreatic acinar cell phenotype suppresses tumor formation. The effects of a novel pStat3 inhibitor (LLL12B) and the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) were investigated using 3-D cultures from p48Cre/+ and p48Cre/+LSL-KrasG12D/+ (KC) mice. LLL12B and TSA inhibited ADM in both KC and p48Cre/+ mouse pancreatic organoids. Furthermore, treatment with LLL12B or TSA on dedifferentiated acini from p48Cre/+ and KC mice that had undergone ADM produced morphologic and gene expression changes that suggest a reversal of ADM. Validation experiments using qRT-PCR (p48Cre/+ and KC) and RNA sequencing (KC) of the LLL12B and TSA treated cultures showed that the ADM reversal was more robust for the TSA treatments. Pathway analysis showed that TSA inhibited Spink1 and PI3K/AKT signaling during ADM reversal. The ability of TSA to reverse ADM was also observed in primary human acinar cultures. We report that pStat3 and HDAC inhibition can attenuate ADM in vitro and reverse ADM in the context of wild-type Kras. Our findings suggest that pharmacological inhibition or reversal of pancreatic ADM represents a potential therapeutic strategy for blocking aberrant ductal reprogramming of acinar cells.

P2X4 assembles with P2X7 and pannexin-1 in gingival epithelial cells and modulates ATP-induced reactive oxygen species production and inflammasome activation.

We have previously reported that Porphyromonas gingivalis infection of gingival epithelial cells (GEC) requires an exogenous danger signal such as ATP to activate an inflammasome and caspase-1, thereby inducing secretion of interleukin (IL)-1ß. Stimulation with extracellular ATP also stimulates production of reactive oxygen species (ROS) in GEC. However, the mechanism by which ROS is generated in response to ATP, and the role that different purinergic receptors may play in inflammasome activation, is still unclear. In this study, we revealed that the purinergic receptor P2X(4) is assembled with the receptor P2X(7) and its associated pore, pannexin-1. ATP induces ROS production through a complex consisting of the P2X(4), P2X(7), and pannexin-1. P2X(7)-mediated ROS production can activate the NLRP3 inflammasome and caspase-1. Furthermore, separate depletion or inhibition of P2X(4), P2X(7), or pannexin-1 complex blocks IL-1ß secretion in P. gingivalis-infected GEC following ATP treatment. However, activation via P2X(4) alone induces ROS generation but not inflammasome activation. These results suggest that ROS is generated through stimulation of a P2X(4)/P2X(7)/pannexin-1 complex, and reveal an unexpected role for P2X(4), which acts as a positive regulator of inflammasome activation during microbial infection.