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Supplementary Figure from Saliva and Lung Microbiome Associations with Electronic Cigarette Use and Smoking

The microbiome has increasingly been linked to cancer. Little is known about the lung and oral cavity microbiomes in smokers, and even less for electronic cigarette (EC) users, compared with never-smokers. In a cross-sectional study (n = 28) of smokers, EC users, and never-smokers, bronchoalveolar lavage and saliva samples underwent metatranscriptome profiling to examine associations with lung and oral microbiomes. Pairwise comparisons assessed differentially abundant bacteria species. Total bacterial load was similar between groups, with no differences in bacterial diversity across lung microbiomes. In lungs, 44 bacteria species differed significantly (FDR < 0.1) between smokers/never-smokers, with most decreased in smokers. Twelve species differed between smokers/EC users, all decreased in smokers of which Neisseria sp. KEM232 and Curvibacter sp. AEP1-3 were observed. Among the top five decreased species in both comparisons, Neisseria elongata, Neisseria sicca, and Haemophilus parainfluenzae were observed. In the oral microbiome, 152 species were differentially abundant for smokers/never-smokers, and 17 between smokers/electronic cigarette users, but only 21 species were differentially abundant in both the lung and oral cavity. EC use is not associated with changes in the lung microbiome compared with never-smokers, indicating EC toxicity does not affect microbiota. Statistically different bacteria in smokers compared with EC users and never-smokers were almost all decreased, potentially due to toxic effects of cigarette smoke. The low numbers of overlapping oral and lung microbes suggest that the oral microbiome is not a surrogate for analyzing smoking-related effects in the lung.Prevention Relevance:The microbiome affects cancer and other disease risk. The effects of e-cig usage on the lung microbiome are essentially unknown. Given the importance of lung microbiome dysbiosis populated by oral species which have been observed to drive lung cancer progression, it is important to study effects of e-cig use on microbiome.

The microbiome has increasingly been linked to cancer. Little is known about the lung and oral cavity microbiomes in smokers, and even less for electronic cigarette (EC) users, compared with never-smokers. In a cross-sectional study (n = 28) of smokers, EC users, and never-smokers, bronchoalveolar lavage and saliva samples underwent metatranscriptome profiling to examine associations with lung and oral microbiomes. Pairwise comparisons assessed differentially abundant bacteria species. Total bacterial load was similar between groups, with no differences in bacterial diversity across lung microbiomes. In lungs, 44 bacteria species differed significantly (FDR < 0.1) between smokers/never-smokers, with most decreased in smokers. Twelve species differed between smokers/EC users, all decreased in smokers of which Neisseria sp. KEM232 and Curvibacter sp. AEP1-3 were observed. Among the top five decreased species in both comparisons, Neisseria elongata, Neisseria sicca, and Haemophilus parainfluenzae were observed. In the oral microbiome, 152 species were differentially abundant for smokers/never-smokers, and 17 between smokers/electronic cigarette users, but only 21 species were differentially abundant in both the lung and oral cavity. EC use is not associated with changes in the lung microbiome compared with never-smokers, indicating EC toxicity does not affect microbiota. Statistically different bacteria in smokers compared with EC users and never-smokers were almost all decreased, potentially due to toxic effects of cigarette smoke. The low numbers of overlapping oral and lung microbes suggest that the oral microbiome is not a surrogate for analyzing smoking-related effects in the lung. Prevention Relevance: The microbiome affects cancer and other disease risk. The effects of e-cig usage on the lung microbiome are essentially unknown. Given the importance of lung microbiome dysbiosis populated by oral species which have been observed to drive lung cancer progression, it is important to study effects of e-cig use on microbiome.

Mechanical ventilation generates injurious forces that exacerbate lung injury. These forces disrupt lung barrier integrity, trigger proinflammatory mediator release, and differentially regulate genes and non-coding oligonucleotides including microRNAs. In this study, we identify miR-146a as a mechanosensitive microRNA in alveolar macrophages that has therapeutic potential to mitigate lung injury during mechanical ventilation. We use humanized in-vitro systems, mouse models, and biospecimens from patients to elucidate the expression dynamics of miR-146a needed to decrease lung injury during mechanical ventilation. We find that the endogenous increase in miR-146a following injurious ventilation is not sufficient to prevent lung injury. However, when miR-146a is highly overexpressed using a nanoparticle delivery platform it is sufficient to prevent injury. These data indicate that the endogenous increase in microRNA-146a during mechanical ventilation is a compensatory response that partially limits injury and that nanoparticle delivery of miR-146a is an effective strategy for mitigating lung injury during mechanical ventilation., There are no targeted pharmacologic therapies to treat lung injury during mechanical ventilation (MV). Here the authors identify a mechanosensitive microRNA (miR-146a) in alveolar macrophages during MV and increase miR-146a to supraphysiological levels in these cells to mitigate ventilator induced lung injury.

There is a strong link between cigarette smoking and pulmonary complications among people living with HIV. However, the effects of smoking on the local lung immune environment in this population remain unclear. Bronchoalveolar lavage and saliva were collected from HIV-infected smokers involved in a prospective study investigating alveolar macrophage expression of host defense molecules. Salivary cotinine concentrations were inversely related to expression of the immune cell receptor nucleotide-binding oligomerization domain-2 and the cathelicidin antimicrobial peptide LL-37. The negative correlation between salivary cotinine and LL-37 was particularly strong. Our study provides insight into how nicotine may adversely affect lung innate immunity in HIV.

Background: Nicotine-containing electronic cigarette (e-cig) use has become widespread. However, understanding the biological impact of e-cigs compared with smoking on the lung is needed. There are major gaps in knowledge for chronic effects and for an etiology to recent acute lung toxicity leading to death among vapers. Methods: We conducted bronchoscopies in a cross-sectional study of 73 subjects (42 never-smokers, 15 e-cig users, and 16 smokers). Using bronchoalveolar lavage and brushings, we examined lung inflammation by cell counts, cytokines, genome-wide gene expression, and DNA methylation. Results: There were statistically significant differences among never-smokers, e-cig users, and smokers for inflammatory cell counts and cytokines (FDR q < 0.1). The e-cig users had values intermediate between smokers and never-smokers, with levels for most of the biomarkers more similar to never-smokers. For differential gene expression and DNA methylation, e-cig users also more like never-smokers; many of these genes corresponded to smoking-related pathways, including those for xenobiotic metabolism, aryl hydrocarbon receptor signaling, and oxidative stress. Differentially methylated genes were correlated with changes in gene expression, providing evidence for biological effects of the methylation associations. Conclusions: These data indicate that e-cigs are associated with less toxicity than cigarettes for smoking-related pathways. What is unknown may be unique effects for e-cigs not measured herein, and a comparison of smokers completely switching to e-cigs compared with former smokers. Clinical trials for smokers switching to e-cigs who undergo serial bronchoscopy and larger cross-sectional studies of former smokers with and without e-cig use, and for e-cigs who relapse back to smoking, are needed. Impact: These data can be used for product regulation and for informing tobacco users considering or using e-cigs. What is unknown may be unique effects for e-cigs not measured herein, and clinical trials with serial bronchoscopy underway can demonstrate a direct relationship for changes in lung biomarkers.

Apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD) (ASC) is a 22 kDa protein that functions as the central adaptor for inflammasome assembly. ASC forms insoluble specks in monocytes undergoing pyroptosis, and the polymerization of ASC provides a template of CARDs that leads to proximity-mediated autoactivation of caspase-1 in canonical inflammasomes. However, specks are insoluble protein complexes, and solubility is typically important for protein function. Therefore, we sought to define whether ASC specks comprise active inflammasome complexes or are simply the end stage of exhausted ASC polymers. Using a THP-1 cell-lysing model of caspase-1 activation that is ASC dependent, we compared caspase-1 activation induced by preassembled insoluble ASC specks and soluble monomeric forms of ASC. Unexpectedly, after controlling for the concentration dependence of ASC oligomerization, we found that only insoluble forms of ASC promoted caspase-1 autocatalysis. This link to insolubility was recapitulated with recombinant ASC. We show that purified recombinant ASC spontaneously precipitated and was functional, whereas the maltose-binding protein-ASC fusion to ASC (promoting enhanced solubility) was inactive until induced to insolubility by binding to amylose beads. This functional link to insolubility also held true for the Y146A mutation of the CARD of ASC, which avoids insolubility and caspase-1 activation. Thus, we conclude that the role of ASC insolubility in inflammasome function is inextricably linked to its pyrin domain-mediated and CARD-mediated polymerizations. These findings will support future studies into the molecular mechanisms controlling ASC solubility.

Mycobacterium tuberculosis (M.tb) is deposited into the alveolus where it first encounters the alveolar lining fluid (ALF) prior contacts host cells. We demonstrated that M.tb-exposure to human ALF alters its cell surface, driving better M.tb infection control by professional phagocytes. Contrary to these findings, our results with non-professional phagocytes alveolar epithelial cells (ATs) define two distinct subsets of human ALFs; where M.tb exposure to Low (L)-ALF or High(H)-ALF results in low or high intracellular bacterial growth rates in ATs, respectively. H-ALF exposed-M.tb growth within ATs was independent of M.tb-uptake, M.tb-trafficking, and M.tb-infection induced cytotoxicity; however, it was associated with enhanced bacterial replication within LAMP-1+/ABCA1+ compartments. H-ALF exposed-M.tb infection of ATs decreased AT immune mediator production, decreased AT surface adhesion expression, and downregulated macrophage inflammatory responses. Composition analysis of H-ALF vs. L-ALF showed H-ALF with higher protein tyrosine nitration and less functional ALF-innate proteins important in M.tb pathogenesis. Replenishment of H-ALF with functional ALF-innate proteins reversed the H-ALF-M.tb growth rate to the levels observed for L-ALF-M.tb. These results indicate that dysfunctionality of innate proteins in the H-ALF phenotype promotes M.tb replication within ATs, while limiting inflammation and phagocyte activation, thus potentiating ATs as a reservoir for M.tb replication and survival.

Background An outbreak of E-cigarette or Vaping Product Use-Associated Lung Injury (EVALI) with significant morbidity and mortality was reported in 2019. While most patients with EVALI report vaping tetrahydrocannabinol (THC) oils contaminated with vitamin E acetate, a subset report only vaping with nicotine-containing electronic cigarettes (e-cigs). Whether or not e-cigs cause EVALI, the outbreak highlights the need for identifying long term health effects of e-cigs. EVALI pathology includes alveolar damage, pneumonitis and/or organizing pneumonia, often with lipid-laden macrophages (LLM). We assessed LLM in the lungs of healthy smokers, e-cig users, and never-smokers as a potential marker of e-cig toxicity and EVALI. Methods A cross-sectional study using bronchoscopy was conducted in healthy smokers, e-cig users, and never-smokers (n = 64). LLM, inflammatory cell counts, and cytokines were determined in bronchial alveolar fluids (BAL). E-cig users included both never-smokers and former light smokers. Findings High LLM was found in the lungs of almost all smokers and half of the e-cig users, but not those of never-smokers. LLM were not related to THC exposure or smoking history. LLM were significantly associated with inflammatory cytokines IL-4 and IL-10 in e-cig users, but not smoking-related cytokines. Interpretation This is the first report of lung LLM comparing apparently healthy smokers, e-cig users, and never-smokers. LLM are not a specific marker for EVALI given the frequent positivity in smokers; whether LLMs are a marker of lung inflammation in some e-cig users requires further study. Funding The National Cancer Institute, the National Heart, Lung, and Blood Institute, the Food and Drug Administration Center for Tobacco Products, the National Center For Advancing Translational Sciences, and Pelotonia Intramural Research Funds

Inflammasome activation is regulated in part by the posttranslational modification of inflammasome proteins. Tyrosine phosphorylation is one possible modification. Having previously shown that the protein tyrosine kinase (PTK) inhibitor AG126 greatly inhibits inflammasome activation, we sought to uncover the target kinase. To do this, we screened a commercial tyrosine kinase library for inhibition of inflammasome-dependent IL-18/IL-1β release and pyroptosis. THP-1 cells (human monocyte cell line) were incubated with PTK inhibitors (0.1, 1, and 10 μM) before stimulation with LPS followed by ATP. The PTK inhibitors DCC-2036 (Rebastinib) and GZD824, specific for Bcr-Abl kinase, showed the most severe reduction of IL-18 and lactate dehydrogenase release at all concentrations used. The suggested kinase target, cAbl kinase, was then deleted in THP-1 cells by CRISPR/Cas9 editing and then tested for its role in inflammasome function and potential to phosphorylate the inflammasome adaptor ASC. The cABL knockout not only significantly inhibited inflammasome function but also decreased release of phosphorylated ASC after LPS/ATP stimulation. One predicted target of cAbl kinase is tyrosine 146 in ASC. Complementation of ASC knockout THP-1 cells with mutated Y146A ASC significantly abrogated inflammasome activation and ASC oligomerization as compared with wild-type ASC complementation. Thus, these findings support cAbl kinase as a positive regulator of inflammasome activity and pyroptosis, likely via phosphorylation of ASC.

Objectives: The key to further improving outcomes in sepsis lies in understanding and abrogating the dysfunctional immune response that leads to organ failure. Activation of gasdermin-D, a pore-forming protein within the inflammasome cascade, has recently been recognized as the critical step in pyroptosis and organ dysfunction. In this study, we sought to investigate the presence of gasdermin-D in critically ill subjects. Design, Setting, and Patients: Prospective pilot study comparing microparticulate active gasdermin-D levels in critically ill patients admitted to the medical ICU at The Ohio State University Medical Center to healthy donors and clinical outcomes. Interventions: None. Measurements and Main Results: Plasma was collected from subjects upon consent and microparticles were isolated by ultracentrifugation. Proteins of interest were identified by immunoblot analysis of microparticle lysates. Quantification was accomplished by densitometry using ImageJ software (National Institutes of Health, Bethesda, MD). Investigators were then unblinded and compared microparticulate active gasdermin-D levels to physician adjudicated clinical diagnoses and outcomes. No appreciable levels of active gasdermin-D were observed in microparticles from healthy volunteers and nonseptic critically ill patients. However, elevated levels of gasdermin-D were noted in microparticles from the septic cohort of critically ill patients. Furthermore, a significant positive correlation by linear regression was noted when microparticulate active gasdermin-D levels were compared with microparticulate levels of CD63, an exosomal marker, CD14, a monocyte marker, and CD69, a marker of monocyte activation (R2 = 0.37, p = 0.0011, R2 = 0.85, p < 0.0001, and R2 = 0.43, p = 0.0003, respectively). Conclusions: This is the first study to demonstrate circulating active gasdermin-D in septic patients in the intensive care setting. Our findings also suggest that active gasdermin-D in septic patients is encapsulated in exosomes derived from activated monocytes. Further characterization in the clinical setting is warranted.

During mechanical ventilation, injurious biophysical forces exacerbate lung injury. These forces disrupt alveolar capillary barrier integrity, trigger proinflammatory mediator release, and differentially regulate genes and non-coding oligonucleotides such as microRNAs. In this study, we identify miR-146a as a mechanosensitive microRNA in alveolar macrophages that has therapeutic potential to mitigate lung injury during mechanical ventilation. We used humanized in-vitro systems, mouse models, and biospecimens from mechanically ventilated patients to elucidate the expression dynamics of miR-146a that might be required to decrease lung injury during mechanical ventilation. We found that the endogenous increase in miR-146a following injurious was relatively modest and not sufficient to prevent lung injury. However, when miR-146a was highly overexpressed using a nanoparticle-based delivery platform in vivo, it was sufficient to prevent lung injury. These data indicate that the endogenous increase in microRNA-146a during MV is a compensatory response that only partially limits VILI and that nanoparticle delivery approaches that significantly over-express microRNA-146a in AMs is an effective strategy for mitigating VILI.

Current tuberculosis (TB) treatments include chemotherapy and preventative vaccination with Mycobacterium bovis Bacillus Calmette-Guerin (BCG). In humans, however, BCG vaccination fails to fully protect against pulmonary TB. Few studies have considered the impact of the human lung mucosa (alveolar lining fluid (ALF)), which modifies the Mycobacterium tuberculosis (M.tb) cell wall, revealing alternate antigenic epitopes on the bacterium surface that alter its pathogenicity. We hypothesized that ALF-induced modification of BCG would induce better protection against aerosol infection with M.tb. Here we vaccinated mice with ALF-exposed BCG, mimicking the mycobacterial cell surface properties that would be present in the lung during M.tb infection. ALF-exposed BCG-vaccinated mice were more effective at reducing M.tb bacterial burden in the lung and spleen, and had reduced lung inflammation at late stages of M.tb infection. Improved BCG efficacy was associated with increased numbers of memory CD8+ T cells, and CD8+ T cells with the potential to produce interferon-γ in the lung in response to M.tb challenge. Depletion studies confirmed an essential role for CD8+ T cells in controlling M.tb bacterial burden. We conclude that ALF modifications to the M.tb cell wall in vivo are relevant in the context of vaccine design.

TOPIC: Critical Care TYPE: Original Investigations PURPOSE: Gasdermin-D (GSDMD) is a pore-forming molecule that serves as the final effector of pyroptosis (a form of inflammatory cell death) following activation of the cellular inflammasome in monocytes. Disulfiram (DSM), a drug used in the treatment of alcohol abuse, has recently been shown to inhibit GSDMD via disruption of pore formation after its cleavage by the inflammatory caspases (1, 4, and 5). The aim of this study was to elucidate the inhibitory effects of DSM on GSDMD-mediated pyroptosis and explore its role as a potential therapeutic agent in sepsis. METHODS: A series of experiments was conducted using THP1 cells (a human monocytic cell line) or purified human monocytes from the blood of healthy donors. Samples were pretreated with DSM in various concentrations 30 minutes prior to inflammasome activation with bacterial lipopolysaccharide (LPS). Cells were then assessed under light microscopy for swelling and fluorescent microscopy for the formation of ASC specks (activated oligomers of the adaptor protein required for caspase activation). Cell death was assessed using a lactate dehydrogenase (LDH) cytotoxicity assay. Interleukin-1-beta (IL1s) release was measured by ELISA. GSDMD cleavage, pro-IL1s synthesis, and caspase-1 cleavage were assessed using immunoblots. RESULTS: Unexpectedly, low dose DSM accentuated inflammasome activation. Dramatic inflammasome induction was observed at a concentration of 10uM, a dose associated with near peak inhibition in the prior study. However, higher doses of 50 to 100 uM suppressed pyroptosis as evidenced by loss of cell swelling and ASC speck formation, as well as LDH and IL-1s release. The DSM effect occurred downstream of caspase-1 activation as evidenced by preserved intracellular pro-IL1s synthesis and caspase-1 cleavage in the presence of DSM. Contrary to the prior report, GSDMD cleavage appeared to be inhibited at these higher doses. CONCLUSIONS: DSM unexpectedly exhibits upstream activation of the inflammasome at low doses but downstream inhibition at high doses. CLINICAL IMPLICATIONS: Although DSM's inhibitory effects on inflammasome function make it a promising agent for the future treatment of acute inflammatory conditions such as sepsis and covid-19, our results indicate that further study is needed to better characterize its possible activating effects at lower concentrations. DISCLOSURES: Collaboration via BARDA grant funding relationship with Beckman Coulter, Inc. Please note: 9/19/2019-8/31/2014 Added 04/27/2021 by Elliott Crouser, source=Web Response, value=Grant/Research Support No relevant relationships by Gregory Eisinger, source=Web Response No relevant relationships by Mikhail Gavrilin, source=Web Response No relevant relationships by Wissam Osman, source=Web Response No relevant relationships by Evan Prather, source=Web Response No relevant relationships by Mark Wewers, source=Web Response

Background: Electronic cigarettes (e-cigs) are one of the most popular tobacco products in the US. Little is known regarding their pulmonary effects. E-cigs induce similar oxidant reactivity as cigarette smoke and promote oxidative damage/inflammation in airway cells. Given that mtDNA is more prone to oxidative stress than nuclear DNA because of a less effective proofreading system, mtDNA alterations may be important indicators of e-cigs' toxic effects. Clinically, mitochondrial DNA alteration is an emerging biomarker of respiratory diseases. Methods: We compared mtDNA copy number (mtDNA-CN) from lung brushings in a cross-sectional bronchoscopy study of healthy young adults, e-cig users (EC)(n=15), non-smokers, non-EC users (NS)(n=43), and smokers (SM)(n=26). We examined associations of mtDNA-CN with immune response (differential cell counts and cytokines in bronchoalveolar lavage), DNA methylation and gene expression brushings. Associations for: 1) EC vs NS vs SM, and 2) tobacco product users (EC+SM) vs NS for MtDNA-CN with immune response, methylation, and expression were made using linear regression. Further, significant features by group interactions were followed up by within-group tests. False Discovery Rate (FDR) at 0.1 was considered significant. Ingenuity pathway analysis was used to identify the most significantly enriched pathways/molecular functions/diseases. Results: MtDNA-CN was not significantly different among the three groups (P=0.06). MtDNA-CN was higher in SM than NS (P=0.02), and in tobacco product users than NS (P=0.02); EC mtDNA-CN tended to be intermediate between the 2 other groups. There were significantly positive associations of IL-2 and IL-4 with mtDNA-CN in EC, but not in SM or NS (Interaction FDR=0.06 for both). We found 147 transcripts (60 genes) and 1,153 CpGs (713 genes) to be significantly associated with mtDNA-CN in all three groups. The most common canonical pathway of the signatures for both expression and methylation were immune responses. The top molecular and cellular functions for both included cell death and survival. Ten transcripts (LINC01184, SNU13, RPL35A, COLCA1, HLA-DRB1, LOC105379655, TRIM9, TCIRG1, CLPB, MIR2114) and 3,929 CpGs (top: ULK4, STARD13, HLCS, FLT1, TMEM91, CYP2J2) were associated with mtDNA-CN in E-cig users only. Some of these genes are known to play a role in lung diseases, including cancer. For the signatures associated in all groups, we found many more significant signatures (236 vs 147 transcripts and 40,830 vs 1,153 CpGs) in the two group vs. three group comparisons, respectively. Conclusion: While the sample size was small, this study is the first to suggest that mtDNA-CN is a site of pulmonary toxic effects. We found associations of mtDNA-CN with inflammatory markers among EC users, and with a number of biological signatures, particularly genes related to immune response, in the lungs of EC, SM, and NS, but differently by groups for some. Citation Format: Kellie M. Mori, Joseph P. McElroy, Daniel Y. Weng, Sangwoon Chung, Sarah A. Reisinger, Kevin L. Ying, Quentin A. Nickerson, Theodore M. Brasky, Mark D. Wewers, Jo L. Freudenheim, Peter G. Shields, Min-Ae Song. Lung mitochondrial DNA copy number variations: E-cig users, smokers, and never-smokers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 751.

During Gram-negative bacterial infections, excessive LPS induces inflammation and sepsis via action on immune cells. However, the bulk of LPS can be cleared from circulation by the liver. Liver clearance is thought to be a slow process mediated exclusively by phagocytic resident macrophages, Kupffer cells (KC). However, we discovered that LPS disappears rapidly from the circulation, with a half-life of 2–4 min in mice, and liver eliminates about three quarters of LPS from blood circulation. Using microscopic techniques, we found that ∼75% of fluor-tagged LPS in liver became associated with liver sinusoidal endothelial cells (LSEC) and only ∼25% with KC. Notably, the ratio of LSEC-KC–associated LPS remained unchanged 45 min after infusion, indicating that LSEC independently processes the LPS. Most interestingly, results of kinetic analysis of LPS bioactivity, using modified limulus amebocyte lysate assay, suggest that recombinant factor C, an LPS binding protein, competitively inhibits high-density lipoprotein (HDL)–mediated LPS association with LSEC early in the process. Supporting the previous notion, 3 min postinfusion, 75% of infused fluorescently tagged LPS-HDL complex associates with LSEC, suggesting that HDL facilitates LPS clearance. These results lead us to propose a new paradigm of LSEC and HDL in clearing LPS with a potential to avoid inflammation during sepsis.

Immunosuppression is a major complication of alcoholism that contributes to increased rates of opportunistic infections and sepsis in alcoholics. The NLRP3 inflammasome, a multiprotein intracellular pattern recognition receptor complex that facilitates the cleavage and secretion of the proinflammatory cytokines IL-1β and IL-18, can be inhibited by ethanol, and we sought to better understand the mechanism through which this occurs and whether chemically similar molecules exert comparable effects. We show that ethanol can specifically inhibit activation of the NLRP3 inflammasome, resulting in attenuated IL-1β and caspase-1 cleavage and secretion, as well as diminished apoptosis-associated speck-like protein containing a CARD (ASC) speck formation, without affecting potassium efflux, in a mouse macrophage cell line (J774), mouse bone marrow–derived dendritic cells, mouse neutrophils, and human PBMCs. The inhibitory effects on the Nlrp3 inflammasome were independent of γ-aminobutyric acid A receptor activation or N-methyl-d-asparate receptor inhibition but were associated with decreased oxidant production. Ethanol treatment markedly decreased cellular tyrosine phosphorylation, whereas administration of the tyrosine phosphatase inhibitor sodium orthovanadate prior to ethanol restored tyrosine phosphorylation and IL-1β secretion subsequent to ATP stimulation. Furthermore, sodium orthovanadate–induced phosphorylation of ASC Y144, necessary and sufficient for Nlrp3 inflammasome activation, and secretion of phosphorylated ASC were inhibited by ethanol. Finally, multiple alcohol-containing organic compounds exerted inhibitory effects on the Nlrp3 inflammasome, whereas 2-methylbutane (isopentane), the analogous alkane of the potent inhibitor isoamyl alcohol (isopentanol), did not. Our results demonstrate that ethanol antagonizes the NLRP3 inflammasome at an apical event in its activation through the stimulation of protein tyrosine phosphatases, an effect shared by other short-chain alcohols.

Background: Little is known about the microbiomes of the lung and oral cavity with electronic cigarette (e-cig) use and how they compare to those of smokers and never-smokers. E-cigs have been promoted as a safe alternative to smoking cigarettes. Given the recent outbreak of E-cigarette or Vaping product use Associated Lung Injury (EVALI) there is an urgent need for understanding the biological effects of e-cig use on the lung and oral cavity, including effects on the microbiome. Previous studies have been limited to 16S-rRNA sequencing which were used to detect bacteria genera. In this study, we used metatranscriptome profiling to study differentially abundant bacteria species in the oral and lung microbiome of never-smokers, smokers, and e-cig users. Methods: A cross-sectional study of 10 never-smokers, 8 cigarette smokers, and 10 e-cig users was conducted, with saliva and bronchoalveolar lavage (BAL) collected for each study participant. RNA was extracted from saliva and BAL samples for total transcriptome RNA-seq analysis. Sequences were aligned with bowtie2 v.2.2.8 to the human genome (hg19) and non-aligned reads were aligned and annotated using NCBI metagenomes database and Kraken v.1. Differences in the microbiome by smoking status were determined by pairwise comparisons using limma-voom with FDR q-value cutoffs Results: The distribution of richness and evenness of bacterial communities measured by Shannon diversity in our metatranscriptome data did not significantly differ between the three smoking status groups. When comparing levels of bacteria species between groups in the saliva, 234 were differentially abundant between smokers and never-smokers, and 39 were differentially abundant between smokers and e-cig users. In the lung, 87 bacterial species were differentially abundant between smokers and never-smokers and 36 were differentially abundant between smokers and e-cig users. Notably, no bacteria species were differentially abundant when comparing e-cig users and never-smokers in both the saliva and lung samples. There are 50 bacterial species found to be differentially abundant in both the lung and saliva samples, 47 of which are decreased in smokers. These 47 bacteria species included common commensal oral microbiome species such as Haemophilus parainfluenzae, Capnocytophaga gingivalis and Neisseria species. The 3 species that were increased in smokers were Lactobacillus species. Conclusion: Our findings suggests that smoking cigarettes may alter populations of common commensal species in both the oral and lung microbiome. The lack of differentially abundant bacterial species between electronic cigarette users and never-smokers indicates that e-cigs may alter bacterial species to a lesser extent than smoking. Citation Format: Kevin L. Ying, Min-Ae Song, Daniel Y. Weng, Quentin A. Nickerson, Joseph P. McElory, Theodore M. Brasky, Noah B. Whiteman, Mark D. Wewers, Jo L. Freudenheim, Ewy A. Mathé, Peter G. Shields. Lung and salivary microbiome in electronic cigarette users, never-smokers, and smokers: A pilot cross-sectional study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1163.

Introduction: Lipid laden macrophages (LLM) from lung bronchoscopies have been identified as characteristic for some patients with e-cigarette (e-cig) or vaping product use associated lung injury (EVALI). While vaping with THC oils containing vitamin E acetate is thought to have caused the outbreak, there is a subset of the EVALI cases who reported vaping only with nicotine-containing e-cigs. A recent animal study showed that propylene glycol (PG) and vegetable glycerin altered lipid homeostasis and induced LLM. In addition to the associations with EVALI, understanding the health effects of vaping with e-cigs in general needs additional investigation. Objective: We examined alterations in alveolar lipid homeostasis comparing e-cig users to cigarette smokers and never-smokers. Further, we determined the association of LLM with inflammatory cytokines. Methods: LLMs, inflammatory cell counts, and cytokines were determined in bronchial alveolar fluids (BAL) from a cross-sectional study of 62 volunteer subjects, age 21-45. Participants who were e-cig users were either never-smokers or former smokers. Cigarette smoking and e-cig use were confirmed by biomarkers of exposure, including lung and urine cotinine and 3-hydroxycotinine and urine anatabine, nicotelline, and PG. Results: High LLM counts were found in the lungs of almost all smokers and about half of the e-cig users, but not in any never-smokers (P0.05). LLMs were not related to THC exposure. Discussion: This is the first report comparing LLMs in the lungs of e-cig users with smokers and never-smokers. LLMs were found in lungs of both smokers and a portion of e-cig users. Given their presence among smokers, they are not likely to be useful as a marker identifying those with EVALI. However, LLMs may be one of the markers for specific disease risks associated with e-cig usage, but not smoking. Citation Format: Min-Ae Song, Peter G. Shields, Jo L. Freudenheim, Theodore M. Brasky, Joseph P. McElroy, Joseph P. McElroy, Sarah A. Reisinger, Daniel Y. Weng, Rongqin Ren, Thomas Eissenberg, Mark D. Wewers, Konstantin Shilo. The association of alveolar lipid laden macrophages with inflammatory cytokines in electronic cigarette users [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-159.

Background: Little is known about the microbiomes of the lung and oral cavity with electronic cigarette (e-cig) use and how they compare to those of smokers and never-smokers. E-cigs have been promoted as a safe alternative to smoking cigarettes. Given the recent outbreak of E-cigarette or Vaping product use Associated Lung Injury (EVALI), there is an urgent need for understanding the biologic effects of e-cig use on the lung and oral cavity, including effects on the microbiome. Previous studies have been limited to 16S-rRNA sequencing, which was used to detect bacteria genera. In this study, we used metatranscriptome profiling to study differentially abundant bacteria species in the oral and lung microbiome of never-smokers, smokers, and e-cig users. Methods: A cross-sectional study of 10 never-smokers, 8 cigarette smokers, and 10 e-cig users was conducted, with saliva and bronchoalveolar lavage (BAL) collected for each study participant. RNA was extracted from saliva and BAL samples for total transcriptome RNA-seq analysis. Sequences were aligned with bowtie2 v.2.2.8 to the human genome (hg19) and nonaligned reads were aligned and annotated using NCBI metagenomes database and Kraken v.1. Differences in the microbiome by smoking status were determined by pairwise comparisons using limma-voom with FDR q-value cutoffs Results: The distribution of richness and evenness of bacterial communities measured by Shannon diversity in our metatranscriptome data did not significantly differ between the three smoking status groups. When comparing levels of bacteria species between groups in the saliva, 234 were differentially abundant between smokers and never-smokers, and 39 were differentially abundant between smokers and e-cig users. In the lung, 87 bacterial species were differentially abundant between smokers and never-smokers and 36 were differentially abundant between smokers and e-cig users. Notably, no bacteria species were differentially abundant when comparing e-cig users and never-smokers in both the saliva and lung samples. There are 50 bacterial species found to be differentially abundant in both the lung and saliva samples, 47 of which are decreased in smokers. These 47 bacteria species included common commensal oral microbiome species such as Haemophilus parainfluenzae, Capnocytophaga gingivalis, and Neisseria species. The 3 species that were increased in smokers were Lactobacillus species. Conclusion: Our findings suggests that smoking cigarettes may alter populations of common commensal species in both the oral and lung microbiome. The lack of differentially abundant bacterial species between electronic cigarette users and never-smokers indicates that e-cigs may alter bacterial species to a lesser extent than smoking. Citation Format: Kevin L. Ying, Min-Ae Song, Daniel Y. Weng, Quentin A. Nickerson, Joseph P. McElroy, Theodore M. Brasky, Noah B. Whiteman, Mark D. Wewers, Jo L. Freudenheim, Ewy A. Mathe, Peter G. Shields. Lung and salivary microbiome in electronic cigarette users, never-smokers, and smokers: A pilot cross-sectional study [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr A34.

Lung endothelial cell apoptosis and injury occur throughout all stages of acute lung injury/acute respiratory distress syndrome and impact disease progression. Caspases 1, 4, and 5 are essential for completion of the apoptotic program known as pyroptosis that also involves proinflammatory cytokines. Because gasdermin D (GSDMD) mediates pyroptotic death and is essential for pore formation, we hypothesized that it might direct caspase 1-encapsulated microparticle (MP) release and mediate endothelial cell death. Our present work provides evidence that GSDMD is released by LPS-stimulated THP-1 monocytic cells, where it is packaged into microparticles together with active caspase 1. Furthermore, only MP released from stimulated monocytic cells that contain both cleaved GSDMD and active caspase 1 induce endothelial cell apoptosis. MPs pretreated with caspase 1 inhibitor Y-VAD or pan-caspase inhibitor Z-VAD do not contain cleaved GSDMD. MPs from caspase 1-knockout cells are also deficient in p30 active GSDMD, further confirming that caspase 1 regulates GSDMD function. Although control MPs contained cleaved GSDMD without caspase 1, these fractions were unable to induce cell death, suggesting that encapsulation of both caspase 1 and GSDMD is essential for cell death induction. Release of microparticulate active caspase 1 was abrogated in GSDMD knockout cells, although cytosolic caspase 1 activation was not impaired. Last, higher concentrations of microparticulate GSDMD were detected in the plasma of septic patients with acute respiratory distress syndrome than in that of healthy donors. Taken together, these findings suggest that GSDMD regulates the release of microparticulate active caspase 1 from monocytes essential for induction of cell death and thereby may play a critical role in sepsis-induced endothelial cell injury.

Although the study of pathogen sensing by host defense systems continues to uncover a role for inflammasome components specific to particular pathogens, gaps remain in our knowledge. After internalization, Francisella escapes from the phagosome in mononuclear cells and is thought to be detected by intracellular pathogen-response-receptors pyrin and Aim2 in human and murine models, respectively. However, it remains controversial as to the role of pyrin in detecting Francisella. Our current work aims to study the contribution of inflammasome sensor, Pyrin in regulating microparticulate caspase-1/GSDM-D activation by Francisella. Our findings suggest that NLRP3 is central to the activation/release of active caspase-1/GSDM-D encapsulated in microparticles (MP) by Francisella. We also provide evidence that this regulation is independent of pyrin, implicated in sensing cytosolic Francisella in NLRP3-/- conditions where endogenous Pyrin is present. Absence of NLRP3 completely abrogated Francisella mediated MP caspase-1/GSDM-D activation and release both before and after internalization of the pathogen. However, deletion of pyrin not only enhanced both LPS and Francisella mediated MP active caspase-1/GSDM-D release, but pyrin overexpression resulted in a reduction of inflammasome activation and release; suggesting an inhibitory role of pyrin in LPS and Francisella mediated MP responses. This NLRP3 dependence and inhibitory effect of pyrin correlated with cytokine release as well. These observations also correlated with MPs ability to induce cell death; as LPS and Francisella-induced MPs from pyrin-deficient cells were more potent than wild-type monocytes whereas, NLRP3-/- MPs failed to induce cell death. Taken together, we report that NLPR3 not only mediates Francisella induced cytokine responses, but is also critical for cytokine-independent microparticle-induced inflammasome activation and endothelial cell injury independent of pyrin.

Asthma is a chronic lung disease characterized by inflammation centered upon bronchial epithelium. House dust mite is one of the most common respiratory allergens that trigger exacerbations of asthma. IκBζ (gene NFKBIZ) is a recently recognized member of the NF-κB family that can be induced in mononuclear phagocytes and lung epithelial cells and has been shown to play a prominent role in epithelial cell function. We therefore analyzed the role of IκBζ in regulating lung epithelial cell cytokine responses to house dust mite mix (HDM). We found that human bronchial epithelial cells express IκBζ and release IL-6 and granulocyte macrophage colony-stimulating factor (GMCSF) when cocultured with human monocytes and HDM. This response is blocked in the presence of IL-1 receptor antagonist (IL-1Ra), indicating that it is IL-1 mediated. Neither HDM-stimulated macrophages nor dendritic cells release IL-1β and subsequently induce cytokine release from the bronchial epithelial cells. Rhodobacter sphaeroides LPS (RS-LPS), a TLR4 antagonist, blocks the ability of HDM to induce IκBζ and release GMCSF from epithelial cells cocultured with monocytes. Additionally, human bronchial epithelial cells show no induction of IκBζ or cytokine responses to direct HDM stimulation. Finally, NFKBIZ small interfering RNA-mediated knockdown in the bronchial epithelial cells suppresses the release of IL-1-induced IL-6 and GMCSF. Our findings indicate a possible role for monocyte recruitment and lung epithelial cell IκBζ in mediating asthma associated inflammation. Thus, IκBζ, IL-1Ra, and RS-LPS deserve future study as potential modulators of house dust mite-induced asthma.

BACKGROUND We have previously shown that critically ill children transfused with red blood cells (RBCs) of longer storage durations have more suppressed monocyte function after transfusion compared to children transfused with fresher RBCs and that older stored RBCs directly suppress monocyte function in vitro, through unknown mechanisms. We hypothesized that RBC-derived microvesicles (MVs) were responsible for monocyte suppression. STUDY DESIGN AND METHODS To determine the role of stored RBC unit–derived MVs, we cocultured monocytes with supernatants, isolated MVs, or supernatants that had been depleted of MVs from prestorage leukoreduced RBCs that had been stored for either 7 or 30 days. Isolated MVs were characterized by electron microscopy and flow cytometry. Monocyte function after coculture experiments was measured by cytokine production after stimulation with lipopolysaccharide (LPS). RESULTS Monocyte function was suppressed after exposure to supernatants from 30-day RBC units compared to monocytes cultured in medium alone (LPS-induced tumor necrosis factor-α production, 17,611 ± 3,426 vs. 37,486 ± 5,598 pg/mL; p = 0.02). Monocyte function was not suppressed after exposure to MV fractions. RBC supernatants that had been depleted of MVs remained immunosuppressive. Treating RBC supernatants with heat followed by RNase (to degrade protein-bound RNA) prevented RBC supernatant–induced monocyte suppression. CONCLUSION Our findings implicate soluble mediators of stored RBC-induced monocyte suppression outside of MV fractions and suggest that extracellular protein-bound RNAs (such as microRNA) may play a role in transfusion-related immunomodulation.

The use of electronic cigarettes (e-cigs) is increasing rapidly, but their effects on lung toxicity are largely unknown. Smoking is a well-established cause of lung cancer and respiratory disease, in part through inflammation. It is plausible that e-cig use might affect similar inflammatory pathways. E-cigs are used by some smokers as an aid for quitting or smoking reduction, and by never smokers (e.g., adolescents and young adults). The relative effects for impacting disease risk may differ for these groups. Cell culture and experimental animal data indicate that e-cigs have the potential for inducing inflammation, albeit much less than smoking. Human studies show that e-cig use in smokers is associated with substantial reductions in blood or urinary biomarkers of tobacco toxicants when completely switching and somewhat for dual use. However, the extent to which these biomarkers are surrogates for potential lung toxicity remains unclear. The FDA now has regulatory authority over e-cigs and can regulate product and e-liquid design features, such as nicotine content and delivery, voltage, e-liquid formulations, and flavors. All of these factors may impact pulmonary toxicity. This review summarizes current data on pulmonary inflammation related to both smoking and e-cig use, with a focus on human lung biomarkers. Cancer Epidemiol Biomarkers Prev; 26(8); 1175–91. ©2017 AACR.

Pseudomonas aeruginosa is a ubiquitous environmental organism and an opportunistic pathogen that causes chronic lung infections in the airways of cystic fibrosis (CF) patients as well as other immune-compromised individuals. During infection, P. aeruginosa enters the terminal bronchioles and alveoli and comes into contact with alveolar lining fluid (ALF), which contains homeostatic and antimicrobial hydrolytic activities, termed hydrolases. These hydrolases comprise an array of lipases, glycosidases, and proteases and thus, they have the potential to modify lipids, carbohydrates and proteins on the surface of invading microbes. Here we show that hydrolase levels between human ALF from healthy and CF patients differ. CF-ALF influences the P. aeruginosa cell wall by reducing the content of one of its major polysaccharides, Psl. This CF-ALF induced Psl reduction does not alter initial bacterial attachment to surfaces but reduces biofilm formation. Importantly, exposure of P. aeruginosa to CF-ALF drives the activation of neutrophils and triggers their oxidative response; thus, defining human CF-ALF as a new innate defense mechanism to control P. aeruginosa infection, but at the same time potentially adding to the chronic inflammatory state of the lung in CF patients.

Type III secretion systems (T3SS) are central virulence factors for many pathogenic Gram-negative bacteria, and secreted T3SS effectors can block key aspects of host cell signaling. To counter this, innate immune responses can also sense some T3SS components to initiate anti-bacterial mechanisms. The Yersinia pestis T3SS is particularly effective and sophisticated in manipulating the production of pro-inflammatory cytokines IL-1β and IL-18, which are typically processed into their mature forms by active caspase-1 following inflammasome formation. Some effectors, like Y. pestis YopM, may block inflammasome activation. Here we show that YopM prevents Y. pestis induced activation of the Pyrin inflammasome induced by the RhoA-inhibiting effector YopE, which is a GTPase activating protein. YopM blocks YopE-induced Pyrin-mediated caspase-1 dependent IL-1β/IL-18 production and cell death. We also detected YopM in a complex with Pyrin and kinases RSK1 and PKN1, putative negative regulators of Pyrin. In contrast to wild-type mice, Pyrin deficient mice were also highly susceptible to an attenuated Y. pestis strain lacking YopM, emphasizing the importance of inhibition of Pyrin in vivo. A complex interplay between the Y. pestis T3SS and IL-1β/IL-18 production is evident, involving at least four inflammasome pathways. The secreted effector YopJ triggers caspase-8- dependent IL-1β activation, even when YopM is present. Additionally, the presence of the T3SS needle/translocon activates NLRP3 and NLRC4-dependent IL-1β generation, which is blocked by YopK, but not by YopM. Taken together, the data suggest YopM specificity for obstructing the Pyrin pathway, as the effector does not appear to block Y. pestis-induced NLRP3, NLRC4 or caspase-8 dependent caspase-1 processing. Thus, we identify Y. pestis YopM as a microbial inhibitor of the Pyrin inflammasome. The fact that so many of the Y. pestis T3SS components are participating in regulation of IL-1β/IL-18 release suggests that these effects are essential for maximal control of innate immunity during plague., Author Summary Many pathogenic Gram-negative bacteria express type III secretion systems (T3SS) that translocate bacterial proteins into host cells with the potential of altering normal cell processes. Yersinia pestis, the causative agent of plague, harbors a T3SS which is particularly effective in suppressing innate immunity and release of pro-inflammatory cytokines IL-1β and IL-18, potent triggers of anti-bacterial responses. These cytokines are produced via processing by active caspase-1 in inflammasome complexes. Pyrin is an inflammasome component that recognizes alterations in certain host cell signals. Here we show that the T3SS effector protein YopM inhibits effector YopE-mediated Pyrin-induced caspase-1 activation, IL-1β, IL-18 and cell death triggered by Y. pestis. We also found that blocking the Pyrin pathway is important for disease development in a mouse model of bubonic plague. Thus, YopM is a microbial molecule blocking Pyrin inflammasomes.

Background: As electronic cigarette (e-cig) use increases in the US, investigation of their effects are critical. Alteration of the lung microbiome, oral microbiome, and inflammation are well established effects of cigarettes; however the effects of e-cigs are yet unknown. Individuals with smoking-related lung disease have alterations in their lung microbiome compared to healthy individuals and their lung microbiomes appear more similar to their oral microbiomes when compared to healthy individuals. To our knowledge, only one study has examined smoking tobacco’s concurrent effects in the oral and lung microbiome and none have examined e-cig use. Further, none have studied e-cigs’ effect on the lung microbiome and inflammation. We hypothesized that e-cig use would affect the lung microbiome, and that the effects are different from smokers and never-smokers; alteration of the lung microbiome will also affect inflammatory gene expression in the lungs. Methods: A cross-sectional study of bronchoscopy with bronchoalveolar lavage (BAL) of 10 never-smokers, 8 cigarette smokers, and 10 e-cig users was conducted. RNA was extracted from BAL samples for total transcriptome RNA-seq analysis, allowing measurement of the microbiome and human gene expression. Differences in the microbiome by smoking status were determined by the Kruskal-Wallis test. Pairwise Wilcoxon rank sum tests with Holm correction was used. Effect size (fold change >1.5) and adjusted P-value cutoffs ( Results: We identified 53 differentially-abundant bacterial species in BAL samples by smoking group. Among them, the majority were less abundant in the lung of smokers and ~20 are normally found in the oral microbiome. While there were significant differences in differentially-abundant microbes between e-cig users and smokers and between smokers and never-smokers, the microbiome of e-cig users did not differ from that of never-smokers. In preliminary analyses of gene expression, there were 2,400 differentially-expressed human genes among the three groups, of which 58 are inflammatory pathway genes. Conclusion: The majority of differentially-abundant microbes observed by smoking group are largely due to smokers. The microbiome of e-cig users is more similar to that of never-smokers. Interestingly, nearly half of microbes that are altered in the lung microbiome due to smoking use are bacterial species normally found in the oral microbiome. These findings suggest that the alterations in the oral microbiome associated with smoking cigarettes may also be reflected in the lung microbiome. Citation Format: Kevin L. Ying, Min-Ae Song, Daniel Y. Weng, Quentin A. Nickerson, Joseph P. McElroy, Theodore M. Brasky, Mark D. Wewers, Ewy Mathé, Jo L. Freudenheim, Peter G. Shields. Microbial and inflammatory response to electronic cigarette and cigarette use [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 664.

OBJECTIVES/SPECIFIC AIMS: Verbal communication is a critical component for professional development and leadership. Yet, many clinical translational scientists lack the skills in communication of their scientific work in a meaningful and exciting manner that conveys the potential impact of their work on human health to the lay public, stakeholders, and to other scientists in different fields. We hypothesized that formal communication training could improve information transfer by trainees that would enhance their career development. METHODS/STUDY POPULATION: We therefore formalized a program for the KL2 scholars at the Ohio State University Center for Clinical and Translational Science that provided training from communications experts to develop a short, concise, and relevant talk about their field of research to general audiences. The program was a hybrid of workshop and individualized training. It culminated in each of the six scholars presenting public talk at the OSU STEM research dissemination and outreach space, the STEAM Factory. The scholars were administered a survey to assess their knowledge of the concepts presented in the training prior to and following the receiving the treatment, as well as their overall assessment of the experience. RESULTS/ANTICIPATED RESULTS: The poster will present the positive results of this evaluation and the impact of the training on the KL2 scholars. DISCUSSION/SIGNIFICANCE OF IMPACT: The poster explain the training as a model that other CTSA KL2 programs could adapt for their trainees.

Burkholderia cenocepacia, the causative agent of cepacia syndrome, primarily affects cystic fibrosis patients, often leading to death. In the lung, epithelial cells serve as the initial barrier to airway infections, yet their responses to B. cenocepacia have not been fully investigated. Here, we examined the molecular responses of human airway epithelial cells to B. cenocepacia infection. Infection led to early signaling events such as activation of Erk, Akt, and NF-κB. Further, TNFα, IL-6, IL-8, and IL-1β were all significantly induced upon infection, but no IL-1β was detected in the supernatants. Because caspase-1 is required for IL-1β processing and release, we examined its expression in airway epithelial cells. Interestingly, little to no caspase-1 was detectable in airway epithelial cells. Transfection of caspase-1 into airway epithelial cells restored their ability to secrete IL-1β following B. cenocepacia infection, suggesting that a deficiency in caspase-1 is responsible, at least in part, for the attenuated IL-1β secretion.

Interleukin 1β (IL-1β) is critical for the in vivo survival, expansion and effector function of IL-17-producing helper T (T(H)17) cells during autoimmune responses, including experimental autoimmune encephalomyelitis (EAE). However, the spatiotemporal role and cellular source of IL-1β during EAE pathogenesis are poorly defined. In the present study, we uncovered a T cell-intrinsic inflammasome that drives IL-1β production during T(H)17-mediated EAE pathogenesis. Activation of T cell antigen receptors induced expression of pro-IL-1β, whereas ATP stimulation triggered T cell production of IL-1β via ASC-NLRP3-dependent caspase-8 activation. IL-1R was detected on T(H)17 cells but not on type 1 helper T (T(H)1) cells, and ATP-treated T(H)17 cells showed enhanced survival compared with ATP-treated T(H)1 cells, suggesting autocrine action of T(H)17-derived IL-1β. Together these data reveal a critical role for IL-1β produced by a T(H)17 cell-intrinsic ASC-NLRP3-caspase-8 inflammasome during inflammation of the central nervous system.

SummaryInflammasomes are multiprotein complexes that include members of the NLR (nucleotide-binding domain leucine-rich repeat containing) family and caspase-1. Once bacterial molecules are sensed within the macrophage, the inflammasome is assembled, mediating the activation of caspase-1. Caspase-11 mediates caspase-1 activation in response to lipopolysaccharide and bacterial toxins, and yet its role during bacterial infection is unknown. Here, we demonstrated that caspase-11 was dispensable for caspase-1 activation in response to Legionella, Salmonella, Francisella, and Listeria. We also determined that active mouse caspase-11 was required for restriction of L. pneumophila infection. Similarly, human caspase-4 and caspase-5, homologs of mouse caspase-11, cooperated to restrict L. pneumophila infection in human macrophages. Caspase-11 promoted the fusion of the L. pneumophila vacuole with lysosomes by modulating actin polymerization through cofilin. However, caspase-11 was dispensable for the fusion of lysosomes with phagosomes containing nonpathogenic bacteria, uncovering a fundamental difference in the trafficking of phagosomes according to their cargo.