Acid exposure disrupts mucus secretion and impairs mucociliary transport in neonatal piglet airways.

Tenacious mucus produced by tracheal and bronchial submucosal glands is a defining feature of several airway diseases, including cystic fibrosis (CF). Airway acidification as a driving force of CF airway pathology has been controversial. Here we tested the hypothesis that transient airway acidification produces pathologic mucus and impairs mucociliary transport. We studied pigs challenged with intra-airway acid. Acid had a minimal effect on mucus properties under basal conditions. However, cholinergic stimulation in acid-challenged pigs revealed retention of mucin 5B (MUC5B) in the submucosal glands, decreased concentrations of MUC5B in the lung lavage fluid, and airway obstruction. To more closely mimic a CF-like environment, we also examined mucus secretion and transport following cholinergic stimulation under diminished bicarbonate and chloride transport conditions ex vivo. Under these conditions, airways from acid-challenged pigs displayed extensive mucus films and decreased mucociliary transport. Pretreatment with diminazene aceturate, a small molecule with ability to inhibit acid detection through blockade of the acid-sensing ion channel (ASIC) at the doses provided, did not prevent acid-induced pathologic mucus or transport defects but did mitigate airway obstruction. These findings suggest that transient airway acidification early in life has significant impacts on mucus secretion and transport properties. Furthermore, they highlight diminazene aceturate as an agent that might be beneficial in alleviating airway obstruction.

Airway cholinergic history modifies mucus secretion properties to subsequent cholinergic challenge in diminished chloride and bicarbonate conditions.

NEW FINDINGS: What is the central question of this study? What is the impact of airway cholinergic history on the properties of airway mucus secretion in a cystic fibrosis-like environment? What is the main finding and its importance? Prior cholinergic challenge slightly modifies the characteristics of mucus secretion in response to a second cholinergic challenge in a diminished bicarbonate and chloride transport environment. Such modifications might lead to retention of mucus on the airway surface, thereby potentiating exacerbations of airway disease. ABSTRACT: Viral infections precipitate exacerbations in many airway diseases, including asthma and cystic fibrosis. Although viral infections increase cholinergic transmission, few studies have examined how cholinergic history modifies subsequent cholinergic responses in the airway. In our previous work, we found that airway resistance in response to a second cholinergic challenge was increased in young pigs with a history of airway cholinergic stimulation. Given that mucus secretion is regulated by the cholinergic nervous system and that abnormal airway mucus contributes to exacerbations of airway disease, we hypothesized that prior cholinergic challenge would also modify subsequent mucus responses to a secondary cholinergic challenge. Using our established cholinergic challenge-rechallenge model in pigs, we atomized the cholinergic agonist bethanechol or saline control to pig airways. Forty-eight hours later, we removed tracheas and measured mucus secretion properties in response to a second cholinergic stimulation. The second cholinergic stimulation was conducted in conditions of diminished chloride and bicarbonate transport to mimic a cystic fibrosis-like environment. In pigs previously challenged with bethanechol, a second cholinergic stimulation produced a mild increase in sheet-like mucus films; these films were scarcely observed in animals originally challenged with saline control. The subtle increase in mucus films was not associated with changes in mucociliary transport. These data suggest that prior cholinergic history might modify mucus secretion characteristics with subsequent stimulation in certain environmental conditions or disease states. Such modifications and/or more repetitive stimulation might lead to retention of mucus on the airway surface, thereby potentiating exacerbations of airway disease.

Sex-specific airway hyperreactivity and sex-specific transcriptome remodeling in neonatal piglets challenged with intra-airway acid.

Acute airway acidification is a potent stimulus of sensory nerves and occurs commonly with gastroesophageal reflux disease, cystic fibrosis, and asthma. In infants and adults, airway acidification can acutely precipitate asthma-like symptoms, and treatment-resistant asthma can be associated with gastroesophageal reflux disease. Airway protective behaviors, such as mucus secretion and airway smooth muscle contraction, are often exaggerated in asthma. These behaviors are manifested through activation of neural circuits. In some populations, the neural response to acid might be particularly important. For example, the immune response in infants is relatively immature compared with adults. Infants also have a high frequency of gastroesophageal reflux. Thus, in the current study, we compared the transcriptomes of an airway-nervous system circuit (e.g., tracheal epithelia, nodose ganglia, and brain stem) in neonatal piglets challenged with intra-airway acid. We hypothesized that the identification of parallel changes in the transcriptomes of two neutrally connected tissues might reveal the circuit response, and, hence, molecules important for the manifestation of asthma-like features. Intra-airway acid induced airway hyperreactivity and airway obstruction in male piglets. In contrast, female piglets displayed airway obstruction without airway hyperreactivity. Pairwise comparisons revealed parallel changes in genes directly implicated in airway hyperreactivity ( scn10a) in male acid-challenged piglets, whereas acid-challenged females exhibited parallel changes in genes associated with mild asthma ( stat 1 and isg15). These findings reveal sex-specific responses to acute airway acidification and highlight distinct molecules within a neural circuit that might be critical for the manifestation of asthma-like symptoms in pediatric populations.

Strategies for measuring airway mucus and mucins.

Mucus secretion and mucociliary transport are essential defense mechanisms of the airways. Deviations in mucus composition and secretion can impede mucociliary transport and elicit airway obstruction. As such, mucus abnormalities are hallmark features of many respiratory diseases, including asthma, cystic fibrosis and chronic obstructive pulmonary disease (COPD). Studying mucus composition and its physical properties has therefore been of significant interest both clinically and scientifically. Yet, measuring mucus production, output, composition and transport presents several challenges. Here we summarize and discuss the advantages and limitations of several techniques from five broadly characterized strategies used to measure mucus secretion, composition and mucociliary transport, with an emphasis on the gel-forming mucins. Further, we summarize advances in the field, as well as suggest potential areas of improvement moving forward.

A novel kinase function of a nucleoside-diphosphate-kinase homologue in Porphyromonas gingivalis is critical in subversion of host cell apoptosis by targeting heat-shock protein 27.

We have previously shown that a homologue of a conserved nucleoside-diphosphate-kinase (Ndk) family of multifunctional enzymes and secreted molecule in Porphyromonas gingivalis can modulate select host molecular pathways including downregulation of reactive-oxygen-species generation to promote bacterial survival in human gingival epithelial cells (GECs). In this study, we describe a novel kinase function for bacterial effector, P. gingivalis-Ndk, in abrogating epithelial cell death by phosphorylating heat-shock protein 27 (HSP27) in GECs. Infection by P. gingivalis was recently suggested to increase phosphorylation of HSP27 in cancer-epithelial cells; however, the mechanism and biological significance of antiapoptotic phospho-HSP27 during infection has never been characterised. Interestingly, using glutathione S-transferase-rNdk pull-down analysed by mass spectrometry, we identified HSP27 in GECs as a strong binder of P. gingivalis-Ndk and further verified using confocal microscopy and ELISA. Therefore, we hypothesised P. gingivalis-Ndk can phosphorylate HSP27 for inhibition of apoptosis in GECs. We further employed P. gingivalis-Ndk protein constructs and an isogenic P. gingivalis-ndk-deficient-mutant strain for functional examination. P. gingivalis-infected GECs displayed significantly increased phospho-HSP27 compared with ndk-deficient-strain during 24 hr infection. Phospho-HSP27 was significantly increased by transfection of GFP-tagged-Ndk into uninfected-GECs, and in vitro phosphorylation assays revealed direct phosphorylation of HSP27 at serines 78 and 82 by P. gingivalis-Ndk. Depletion of HSP27 via siRNA significantly reversed resistance against staurosporine-mediated-apoptosis during infection. Transfection of recombinant P. gingivalis-Ndk protein into GECs substantially decreased staurosporine-induced-apoptosis. Finally, ndk-deficient-mutant strain was unable to inhibit staurosporine-induced Cytochrome C release/Caspase-9 activation. Thus, we show for the first time the phosphorylation of HSP27 by a bacterial effector-P. gingivalis-Ndk-and a novel function of Ndks that is directly involved in inhibition of host cell apoptosis and the subsequent bacterial survival.

Neuropeptides in asthma, chronic obstructive pulmonary disease and cystic fibrosis.

The nervous system mediates key airway protective behaviors, including cough, mucus secretion, and airway smooth muscle contraction. Thus, its involvement and potential involvement in several airway diseases has become increasingly recognized. In the current review, we focus on the contribution of select neuropeptides in three distinct airway diseases: asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. We present data on some well-studied neuropeptides, as well as call attention to a few that have not received much consideration. Because mucus hypersecretion and mucus obstruction are common features of many airway diseases, we place special emphasis on the contribution of neuropeptides to mucus secretion. Finally, we highlight evidence implicating involvement of neuropeptides in mucus phenotypes in asthma, COPD and cystic fibrosis, as well as bring to light knowledge that is still lacking in the field.

Solitary Cholinergic Stimulation Induces Airway Hyperreactivity and Transcription of Distinct Pro-inflammatory Pathways.

Airway hyperreactivity is a hallmark feature of asthma and can be precipitated by airway insults, such as ozone exposure or viral infection. A proposed mechanism linking airway insults to airway hyperreactivity is augmented cholinergic transmission. In the current study, we tested the hypothesis that acute potentiation of cholinergic transmission is sufficient to induce airway hyperreactivity. We atomized the cholinergic agonist bethanechol to neonatal piglets and forty-eight hours later measured airway resistance. Bethanechol-treated piglets displayed increased airway resistance in response to intravenous methacholine compared to saline-treated controls. In the absence of an airway insult, we expected to find no evidence of airway inflammation; however, transcripts for several asthma-associated cytokines, including IL17A, IL1A, and IL8, were elevated in the tracheas of bethanechol-treated piglets. In the lungs, prior bethanechol treatment increased transcripts for IFNγ and its downstream target CXCL10. These findings suggest that augmented cholinergic transmission is sufficient to induce airway hyperreactivity, and raise the possibility that cholinergic-mediated regulation of pro-inflammatory pathways might contribute.

Epigenetic small-molecule screen for inhibition and reversal of acinar ductal metaplasia in mouse pancreatic organoids.

Background: Acinar ductal metaplasia (ADM) is among the earliest initiating events in pancreatic ductal adenocarcinoma (PDAC) development. Methods: We developed a novel morphology-based screen using organoids from wildtype and p48Cre/+ (Cre) mice to discover epigenetic modulators that inhibit or reverse pancreatic ADM more effectively than the broad-spectrum HDAC inhibitor trichostatin A (TSA). Results: Of the 144 compounds screened, nine hits and two additional natural product HDAC inhibitors were validated by dose-response analysis. The class I HDAC inhibitors apicidin and FK228, and the histone methyltransferase inhibitor chaetocin demonstrated pronounced ADM inhibition and reversal without inducing significant cytotoxicity at 1 µM. Thioester prodrug class I HDAC inhibitor largazole attenuated ADM while its disulfide homodimer was effective in both ADM inhibition and reversal. Prioritized compounds were validated for ADM reversal in p48Cre/+; LSL-KrasG12D/+ (KC) mouse organoids using both morphological and molecular endpoints. Molecular index analysis of ADM reversal in KC mouse organoids demonstrated improved activity compared to TSA. Improved prodrug stability translated into a stronger phenotypic and molecular response. RNA-sequencing indicated that angiotensinogen was the top inhibited pathway during ADM reversal. Conclusion: Our findings demonstrate a unique epigenetic mechanism and suggest that the phenotypic screen developed here may be applied to discover potential treatments for PDAC.

Chemical Genetics in C. elegans Identifies Anticancer Mycotoxins Chaetocin and Chetomin as Potent Inducers of a Nuclear Metal Homeostasis Response.

C. elegans numr-1/2 (nuclear-localized metal-responsive) is an identical gene pair encoding a nuclear protein previously shown to be activated by cadmium and disruption of the integrator RNA metabolism complex. We took a chemical genetic approach to further characterize regulation of this novel metal response by screening 41,716 compounds and extracts for numr-1p::GFP activation. The most potent activator was chaetocin, a fungal 3,6-epidithiodiketopiperazine (ETP) with promising anticancer activity. Chaetocin activates numr-1/2 strongly in the alimentary canal but is distinct from metal exposure, because it represses canonical cadmium-responsive metallothionine genes. Chaetocin has diverse targets in cancer cells including thioredoxin reductase, histone lysine methyltransferase, and acetyltransferase p300/CBP; further work is needed to identify the mechanism in C. elegans as genetic disruption and RNAi screening of homologues did not induce numr-1/2 in the alimentary canal and chaetocin did not affect markers of integrator dysfunction. We demonstrate that disulfides in chaetocin and chetomin, a dimeric ETP analog, are required to induce numr-1/2. ETP monomer gliotoxin, despite possessing a disulfide linkage, had almost no effect on numr-1/2, suggesting a dimer requirement. Chetomin inhibits C. elegans growth at low micromolar levels, and loss of numr-1/2 increases sensitivity; C. elegans and Chaetomiaceae fungi inhabit similar environments raising the possibility that numr-1/2 functions as a defense mechanism. There is no direct orthologue of numr-1/2 in humans, but RNaseq suggests that chaetocin affects expression of cellular processes linked to stress response and metal homeostasis in colorectal cancer cells. Our results reveal interactions between metal response gene regulation and ETPs and identify a potential mechanism of resistance to this versatile class of preclinical compounds.

Targeted and functional genomics approaches to the mechanism of action of lagunamide D, a mitochondrial cytotoxin from marine cyanobacteria.

Lagunamide D, a cyanobacterial cyclodepsipeptide, exhibits potent antiproliferative activity against HCT116 colorectal cancer cells (IC50 5.1 nM), which were used to probe the mechanism of action. Measurements of metabolic activity, mitochondrial membrane potential, caspase 3/7 activity and cell viability indicate the rapid action of lagunamide D on mitochondrial function and downstream cytotoxic effects in HCT116 cells. Lagunamide D preferentially targets the G1 cell cycle population and arrests cells in G2/M phase at high concentration (32 nM). Transcriptomics and subsequent Ingenuity Pathway Analysis identified networks related to mitochondrial functions. Lagunamide D induced mitochondrial network redistribution at 10 nM, suggesting a mechanism shared with the structurally related aurilide family, previously reported to target mitochondrial prohibitin 1 (PHB1). Knockdown and chemical inhibition of ATP1A1 sensitized the cells to lagunamide D, as also known for aurilide B. We interrogated potential mechanisms behind this synergistic effect between lagunamide D and ATP1A1 knockdown by using pharmacological inhibitors and extended the functional analysis to a global level by performing a chemogenomic screen with a siRNA library targeting the human druggable genome, revealing targets that modulate susceptibility to lagunamide D. In addition to mitochondrial targets, the screen revealed hits involved in the ubiquitin/proteasome pathway, suggesting lagunamide D might exert its effects by additionally affecting proteostasis. Our analysis illuminated cellular processes of lagunamide D that can be modulated in parallel to mitochondrial functions. The identification of potential synergistic drug combinations that can alleviate undesirable toxicity may open possibilities to resurrect this class of compounds for anticancer therapy.

Epigenetic Small-Molecule Screen for Inhibition and Reversal of Acinar Ductal Metaplasia in Mouse Pancreatic Organoids.

Background: Acinar ductal metaplasia (ADM) is among the earliest initiating events in pancreatic ductal adenocarcinoma (PDAC) development. Methods: We developed a novel morphology-based screen using organoids from wildtype and p48 Cre/+ (Cre) mice to discover epigenetic modulators that inhibit or reverse pancreatic ADM more effectively than the broad-spectrum HDAC inhibitor trichostatin A (TSA). Results: Of the 144 compounds screened, nine hits and two additional natural product HDAC inhibitors were validated by dose-response analysis. The class I HDAC inhibitors apicidin and FK228, and the histone methyltransferase inhibitor chaetocin demonstrated pronounced ADM inhibition and reversal without inducing significant cytotoxicity at 1 µM. Thioester prodrug class I HDAC inhibitor largazole attenuated ADM while its disulfide homodimer was effective in both ADM inhibition and reversal. Prioritized compounds were validated for ADM reversal in p48 Cre/+ ;LSL-Kras G12D/+ (KC) mouse organoids using both morphological and molecular endpoints. Molecular index analysis of ADM reversal in KC mouse organoids demonstrated improved activity compared to TSA. Improved prodrug stability translated into a stronger phenotypic and molecular response. RNA-sequencing indicated that angiotensinogen was the top inhibited pathway during ADM reversal. Conclusion: Our findings demonstrate a unique epigenetic mechanism and suggest that the phenotypic screen developed here may be applied to discover potential treatments for PDAC.

Inflammasome Activation in Gingival Epithelial Cells.

Inflammasomes are multiprotein complexes that assemble in host cells upon recognition of infection or danger via pattern recognition receptors and/or danger recognition receptors. The assembly of inflammasomes results in the activation of caspase-1 and is followed by the enzymatic maturation and secretion of inflammatory cytokines like interleukin 1ß (IL-1ß) and IL-18.In the oral cavity, gingival epithelial cells (GECs) line the mucosa and have a barrier role for invading pathogens. In these cells, the NLRP3 inflammasome is the best studied and has been shown to play a role in the inflammatory immune response against a variety of oral pathogens. As such, in order to study gingivitis and other oral pathologic inflammatory conditions, studying the activation of inflammasomes is important. The simplest way to detect inflammasome activation is to detect the activated caspase-1, as well as the secretion of mature IL-1ß and IL-18, via ELISA, Western blot, and immunofluorescence techniques.Here we describe the detection of NLRP3 inflammasome activation by the oral pathogen Porphyromonas gingivalis in human GECs via these three methods and mention other methods and techniques that we have successfully used together with these in our quest to understand the host-pathogen interaction.

An epigenetic small molecule screen to target abnormal nuclear morphology in human cells.

Irregular nuclear shapes are a hallmark of human cancers. Recent studies suggest that alterations to chromatin regulators may cause irregular nuclear morphologies. Here we screened an epigenetic small molecule library consisting of 145 compounds against chromatin regulators for their ability to revert abnormal nuclear shapes that were induced by gene knockdown in noncancerous MCF10A human mammary breast epithelial cells. We leveraged a previously validated quantitative Fourier approach to quantify the elliptical Fourier coefficient (EFC ratio) as a measure of nuclear irregularities, which allowed us to perform rigorous statistical analyses of screening data. Top hit compounds fell into three major mode of action categories, targeting three separate epigenetic modulation routes: 1) histone deacetylase inhibitors, 2) bromodomain and extraterminal domain protein inhibitors, and 3) methyl-transferase inhibitors. Some of the top hit compounds were also efficacious in reverting nuclear irregularities in MDA-MB-231 triple negative breast cancer cells and in PANC-1 pancreatic cancer cells in a cell-type-dependent manner. Regularization of nuclear shapes was compound-specific, cell-type specific, and dependent on the specific molecular perturbation that induced nuclear irregularities. Our approach of targeting nuclear abnormalities may be potentially useful in screening new types of cancer therapies targeted toward chromatin structure.

Overexpression of Substance P in pig airways increases MUC5AC through an NF-kß pathway.

Substance P (SP) is a tachykinin that regulates airway mucous secretion in both health and disease. Our study aimed to determine whether overexpression of SP without pre-existing inflammation was sufficient to induce changes in mucin secretion and transport in small airways. Utilizing porcine precision-cut lung slices, we measured the impact of AAV-mediated overexpression of SP on airway physiology ex vivo. Immunofluorescence signal intensity for MUC5AC was significantly increased in SP-overexpressed precision-cut lung slices compared to GFP controls. No difference in MUC5B signal intensity between treatments was detected. SP-overexpressed precision-cut lung slices also exhibited decreased IL10 mRNA, an important inhibitor of mucous cell metaplasia. Overt deficits in mucociliary transport were not noted, though a trend for decreased mean transport speed was detected in methacholine-challenged airways overexpressing SP compared to GFP controls. Pharmacologic inhibition of the NF-kß pathway abrogated the effects of overexpression of SP on both MUC5AC and IL10. Collectively, these data suggest that overexpression of SP in the absence of existing inflammation increases MUC5AC via activation of the NF-kß pathway. Thus, these data further highlight SP as a key driver of abnormal mucous secretion and underscore NF-kß signaling as a pathway of potential therapeutic intervention.

Long-term culturing of porcine nodose ganglia.

BACKGROUND: Neuronal cell cultures are widely used in the field of neuroscience. Cell dissociation allows for the isolation of a desired cell type, yet the neuronal complexity that distinguishes the nervous system is often lost as a result. Thus, culturing neural tissues in ex vivo format provides a physiological context that more closely resembles the in vivo environment. NEW METHOD: We developed a simple method to culture nodose ganglia neurons from neonatal pigs long-term in ex vivo format using an in-house media formulation derived from commercially available components. RESULTS: Ganglia were cultured for six and twelve months. mRNA expression of nestin was stable across time. Vasoactive intestinal peptide and tachykinin showed statistically insignificant increases and decreases in mRNA expression, respectively. mRNA expression of glia fibrillary acidic protein decreased, whereas myelin basic protein showed no statistically significant differences, over time. Immunofluorescence studies of sectioned ganglia demonstrated neurofilament-positive cell bodies, glia fibrillary acidic protein and myelin basic protein at all time points. A significant decrease in cell nuclei density and fragmented DNA were noted. COMPARISON WITH EXISTING METHOD(S): There are currently no methods that describe short-term or long-term culturing of porcine nodose ganglia. Further, the media formulation we developed is new and not previously reported. CONCLUSIONS: The simple procedure we developed for culturing nodose ganglia will enable both short-term and long-term investigations aimed at understanding peripheral ganglia in vitro. It is also possible that the methods described herein can be applied to other models, different developmental stages, and potentially other neural tissues.

In Situ Intraepithelial Localizations of Opportunistic Pathogens, Porphyromonas gingivalis and Filifactor alocis, in Human Gingiva.

Porphyromonas gingivalis and Filifactor alocis are fastidious oral pathogens and etiological agents associated with chronic periodontitis. Although previous studies showed increased levels of the two obligate anaerobic species in periodontitis patients, methodologies for this knowledge were primarily limited to sampling subgingival plaque, saliva, or gingival crevicular fluid. To evaluate the extent to which P. gingivalis and F. alocis may invade the periodontal tissues, an in situ cross-sectional study was comparatively conducted on the gingival biopsy specimens of patients diagnosed with periodontal health or chronic periodontitis. Immunostained tissue sections for each organism were imaged by Super-Resolution Confocal Scanning Microscopy to determine the relative presence and localization of target bacterial species. Fluorescence-in-situ-hybridization (FISH) coupled with species specific 16S rRNA method was utilized to confirm whether detected bacteria were live within the tissue. In periodontitis, P. gingivalis and F. alocis revealed similarly concentrated localization near the basement membrane or external basal lamina of the gingival epithelium. The presence of both bacteria was significantly increased in periodontitis vs. healthy tissue. However, P. gingivalis was still detected to an extent in health tissue, while only minimal levels of F. alocis were spotted in health. Additionally, the micrographic analyses displayed heightened formation of epithelial microvasculature containing significantly co-localized and metabolically active dual species within periodontitis tissue. Thus, this study demonstrates, for the first-time, spatial arrangements of P. gingivalis and F. alocis in both single and co-localized forms within the complex fabric of human gingiva during health and disease. It also exhibits critical visualizations of co-invaded microvascularized epithelial layer of the tissue by metabolically active P. gingivalis and F. alocis from patients with severe periodontitis. These findings collectively uncover novel visual evidence of a potential starting point for systemic spread of opportunistic bacteria during their chronic colonization in gingival epithelium.

High-throughput gene screen reveals modulators of nuclear shape.

Irregular nuclear shapes characterized by blebs, lobules, micronuclei, or invaginations are hallmarks of many cancers and human pathologies. Despite the correlation between abnormal nuclear shape and human pathologies, the mechanism by which the cancer nucleus becomes misshapen is not fully understood. Motivated by recent evidence that modifying chromatin condensation can change nuclear morphology, we conducted a high-throughput RNAi screen to identify epigenetic regulators that are required to maintain normal nuclear shape in human breast epithelial MCF-10A cells. We silenced 608 genes in parallel using an epigenetics siRNA library and used an unbiased Fourier analysis approach to quantify nuclear contour irregularity from fluorescent images captured on a high-content microscope. Using this quantitative approach, which we validated with confocal microscopy, we significantly expand the list of epigenetic regulators that impact nuclear morphology.

Porphyromonas gingivalis traffics into endoplasmic reticulum-rich-autophagosomes for successful survival in human gingival epithelial cells.

Porphyromonas gingivalis, an opportunistic pathogen usurps gingival epithelial cells (GECs) as primary intracellular niche for its colonization in the oral mucosa. However, the precise characterization of the intracellular trafficking and fate of P. gingivalis in GECs remains incomplete. Therefore, we employed high-resolution three-dimensional-transmission-electron-microscopy to determine the subcellular location of P. gingivalis in human primary GECs upon invasion. Serial sections of infected-GECs and their tomographic reconstruction depicted ER-rich-double-membrane autophagosomal-vacuoles harboring P. gingivalis. Western-blotting and fluorescence confocal microscopy showed that P. gingivalis significantly induces LC3-lipidation in a time-dependent-manner and co-localizes with LC3, ER-lumen-protein Bip, or ER-tracker, which are major components of the phagophore membrane. Furthermore, GECs that were infected with FMN-green-fluorescent transformant-strain (PgFbFP) and selectively permeabilized by digitonin showed rapidly increasing large numbers of double-membrane-vacuolar-P. gingivalis over 24 hours of infection with a low-ratio of cytosolically free-bacteria. Moreover, inhibition of autophagy using 3-methyladenine or ATG5 siRNA significantly reduced the viability of intracellular P. gingivalis in GECs as determined by an antibiotic-protection-assay. Lysosomal marker, LAMP-1, showed a low-degree colocalization with P. gingivalis (∼20%). PgFbFP was used to investigate the fate of vacuolar- versus cytosolic-P. gingivalis by their association with ubiquitin-binding-adaptor-proteins, NDP52 and p62. Only cytosolic-P. gingivalis had a significant association with both markers, which suggests cytosolically-free bacteria are likely destined to the lysosomal-degradation pathway whereas the vacuolar-P. gingivalis survives. Therefore, the results reveal a novel mechanism for P. gingivalis survival in GECs by harnessing host autophagy machinery to establish a successful replicative niche and persistence in the oral mucosa.

NLRX1 modulates differentially NLRP3 inflammasome activation and NF-κB signaling during Fusobacterium nucleatum infection.

NOD-like receptors (NLRs) play a large role in regulation of host innate immunity, yet their role in periodontitis remains to be defined. NLRX1, a member of the NLR family that localizes to mitochondria, enhances mitochondrial ROS (mROS) generation. mROS can activate the NLRP3 inflammasome, yet the role of NLRX1 in NLRP3 inflammasome activation has not been examined. In this study, we revealed the mechanism by which NLRX1 positively regulates ATP-induced NLRP3 inflammasome activation through mROS in gingival epithelial cells (GECs). We found that depletion of NLRX1 by shRNA attenuated ATP-induced mROS generation and redistribution of the NLRP3 inflammasome adaptor protein, ASC. Furthermore, depletion of NLRX1 inhibited Fusobacterium nucleatum infection-activated caspase-1, suggesting that it also inhibits the NLRP3 inflammasome. Conversely, NLRX1 also acted as a negative regulator of NF-κB signaling and 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, and NLRX1 should decrease their ability to stimulate expression of pro-inflammatory cytokines such as IL-8. Therefore, NLRX1 may act as a potential switch with regards to anti-microbial responses in healthy or diseased states in the oral cavity.

Opportunistic Pathogen Porphyromonas gingivalis Modulates Danger Signal ATP-Mediated Antibacterial NOX2 Pathways in Primary Epithelial Cells.

Porphyromonas gingivalis, a major opportunistic pathogen in the etiology of chronic periodontitis, successfully survives in human gingival epithelial cells (GECs). P. gingivalis abrogates the effects of a host danger molecule, extracellular ATP (eATP)/P2X7 signaling, such as the generation of reactive oxygen species (ROS) via the mitochondria and NADPH oxidases (NOX) from primary GECs. However, antimicrobial functions of ROS production are thoroughly investigated in myeloid-lineage immune cells and have not been well-understood in epithelial cells. Therefore, this study characterizes antibacterial NOX2 generated ROS and host downstream effects in P. gingivalis infected human primary GECs. We examined the expression of NOX isoforms in the GECs and demonstrate eATP stimulation increased the mRNA expression of NOX2 (p < 0.05). Specific peptide inhibition of NOX2 significantly reduced eATP-mediated ROS as detected by DCFDA probe. The results also showed P. gingivalis infection can temporally modulate NOX2 pathway by reorganizing the localization and activation of cytosolic molecules (p47phox, p67phox, and Rac1) during 24 h of infection. Investigation into downstream biocidal factors of NOX2 revealed an eATP-induced increase in hypochlorous acid (HOCl) in GECs detected by R19-S fluorescent probe, which is significantly reduced by a myeloperoxidase (MPO) inhibitor. MPO activity of the host cells was assayed and found to be positively affected by eATP treatment and/or infection. However, P. gingivalis significantly reduced the MPO product, bactericidal HOCl, in early times of infection upon eATP stimulation. Analysis of the intracellular levels of a major host-antioxidant, glutathione during early infection revealed a substantial decrease (p < 0.05) in reduced glutathione indicative of scavenging of HOCl by P. gingivalis infection and eATP treatment. Examination of the mRNA expression of key enzymes in the glutathione synthesis pathway displayed a marked increase (p < 0.05) in glutamate cysteine ligase (GCL) subunits GCLc and GCLm, glutathione synthetase, and glutathione reductase during the infection. These suggest P. gingivalis modulates the danger signal eATP-induced NOX2 signaling and also induces host glutathione synthesis to likely avoid HOCl mediated clearance. Thus, we characterize for the first time in epithelial cells, an eATP/NOX2-ROS-antibacterial pathway and demonstrate P. gingivalis can circumvent this important antimicrobial defense system potentially for successful persistence in human epithelial tissues.