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Inflammation characterized by the expression and release of cytokines and chemokines is implicated in the development and progression of atherosclerosis. Oxidatively modified low density lipoproteins, central to the formation of atherosclerotic plaques, have been reported to signal through Toll-like receptors (TLRs), TLR4 and TLR2, in concert with scavenger receptors to regulate the inflammatory microenvironment in atherosclerosis. This study evaluates the role of low density lipoproteins (LDL) and oxidatively modified LDL (oxmLDL) in the expression and release of proinflammatory mediators IκBζ, IL-6, IL-1β, TNFα, and IL-8 in human monocytes and macrophages. Although standard LDL preparations induced IκBζ along with IL-6 and IL-8 production, this inflammatory effect was eliminated when LDL was isolated under endotoxin-restricted conditions. However, when added with TLR4 and TLR2 ligands, this low endotoxin preparation of oxmLDL suppressed the expression and release of IL-1β, IL-6, and TNFα but surprisingly spared IL-8 production. The suppressive effect of oxmLDL was specific to monocytes as it did not inhibit LPS-induced proinflammatory cytokines in human macrophages. Thus, TLR ligand contamination of LDL/oxmLDL preparations can complicate interpretations of inflammatory responses to these modified lipoproteins. In contrast to providing a proinflammatory function, oxmLDL suppresses the expression and release of selected proinflammatory mediators.

Burkholderia cenocepacia is an opportunistic pathogen that causes chronic infection and induces progressive respiratory inflammation in cystic fibrosis patients. Recognition of bacteria by mononuclear cells generally results in the activation of caspase-1 and processing of IL-1β, a major proinflammatory cytokine. In this study, we report that human pyrin is required to detect intracellular B. cenocepacia leading to IL-1β processing and release. This inflammatory response involves the host adapter molecule ASC and the bacterial type VI secretion system (T6SS). Human monocytes and THP-1 cells stably expressing either small interfering RNA against pyrin or YFP–pyrin and ASC (YFP–ASC) were infected with B. cenocepacia and analyzed for inflammasome activation. B. cenocepacia efficiently activates the inflammasome and IL-1β release in monocytes and THP-1. Suppression of pyrin levels in monocytes and THP-1 cells reduced caspase-1 activation and IL-1β release in response to B. cenocepacia challenge. In contrast, overexpression of pyrin or ASC induced a robust IL-1β response to B. cenocepacia, which correlated with enhanced host cell death. Inflammasome activation was significantly reduced in cells infected with T6SS-defective mutants of B. cenocepacia, suggesting that the inflammatory reaction is likely induced by an as yet uncharacterized effector(s) of the T6SS. Together, we show for the first time, to our knowledge, that in human mononuclear cells infected with B. cenocepacia, pyrin associates with caspase-1 and ASC forming an inflammasome that upregulates mononuclear cell IL-1β processing and release.

Obligate intracellular pathogens such as Leishmania specifically target host phagocytes for survival and replication. Phosphoinositide 3-kinase γ (PI3Kγ), a member of the class I PI3Ks that is highly expressed by leukocytes, controls cell migration by initiating actin polymerization and cytoskeletal reorganization, which are processes also critical for phagocytosis. In this study, we demonstrate that class IB PI3K, PI3Kγ, plays a critical role in pathogenesis of chronic cutaneous leishmaniasis caused by L. mexicana . Using the isoform-selective PI3Kγ inhibitor, AS-605240 and PI3Kγ gene-deficient mice, we show that selective blockade or deficiency of PI3Kγ significantly enhances resistance against L. mexicana that is associated with a significant suppression of parasite entry into phagocytes and reduction in recruitment of host phagocytes as well as regulatory T cells to the site of infection. Furthermore, we demonstrate that AS-605240 is as effective as the standard antileishmanial drug sodium stibogluconate in treatment of cutaneous leishmaniasis caused by L. mexicana . These findings reveal a unique role for PI3Kγ in Leishmania invasion and establishment of chronic infection, and demonstrate that therapeutic targeting of host pathways involved in establishment of infection may be a viable strategy for treating infections caused by obligate intracellular pathogens such as Leishmania .

Pneumococcal lung infections represent a major cause of death worldwide. Single nucleotide polymorphisms (SNPs) in the NFKBIZ gene, encoding the transcription factor IκBζ, are associated with increased susceptibility to invasive pneumococcal disease. We hence analyzed how IκBζ might regulate inflammatory responses to pneumococcal infection. We first demonstrate that IκBζ is expressed in human blood monocytes but not in bronchial epithelial cells, in response to wild type pneumococcal strain D39. D39 transiently induced IκBζ in a dose dependent manner, with subsequent induction of downstream molecules involved in host defense. Of these molecules, IκBζ knockdown reduced the expression of IL-6 and GMCSF. Furthermore, IκBζ overexpression increased the activity of IL-6 and GMCSF promoters, supporting the knockdown findings. Pneumococci lacking either pneumolysin or capsule still induced IκBζ. While inhibition of TLR1/TLR2 blocked D39 induced IκBζ expression, TLR4 inhibition did not. Blockade of p38 MAP kinase and NFκB suppressed D39 induced IκBζ. Overall, our data demonstrates that IκBζ regulates monocyte inflammatory responses to Streptococcus pneumoniae by promoting the production of IL-6 and GMCSF.

Background: Zinc deficiency is a cause of immune dysfunction and infection. Previous human studies have shown that the activation of the acute phase response alters zinc metabolism. Whether the alteration in zinc metabolism is predictive of disease severity in the setting of critical illness is unclear. Objective: We sought to determine whether differences occur in zinc metabolism at the onset of critical illness between infected (septic) and noninfected subjects. Design: We conducted this prospective study in an adult medical intensive care unit (MICU) at a tertiary care hospital. Subjects were enrolled within 24 h of intensive care unit admission. Subjects who did not meet sepsis criteria were considered for the critically ill control (CIC) arm. After patient consent, blood was immediately collected to measure plasma zinc and cytokine concentrations and zinc transporter gene expression in peripheral blood monocytes. Clinical data during the MICU stay were also recorded. Results: A total of 56 patients were evaluated (22 septic, 22 CIC, and 12 healthy subjects). Plasma zinc concentrations were below normal in CIC patients and further reduced in the septic cohort (57.2 6 18.2 compared with 45.5 6 18.1 lg/dL). Cytokine concentrations increased with decreasing plasma zinc concentrations (P = 0.05). SLC39A8 gene expression was highest in patients with the lowest plasma zinc concentrations and the highest severity of illness. Conclusions: The alteration of zinc metabolism was more pronounced in septic patients than in noninfected critically ill patients. Specifically, sepsis was associated with lower plasma zinc concentrations and higher SLC39A8 mRNA expression, which correlated with an increased severity of illness, including cardiovascular dysfunction. Am J Clin Nutr doi: 10.3945/ajcn.110.008417.

Interferon-γ (IFN-γ) production by natural killer (NK) cells and cytotoxic lymphocytes is a key component of innate and adaptive immune responses. Because inhibitor of κB-ζ (IκBζ), a Toll-like receptor (TLR)/interleukin-1 receptor (IL-1R) inducible transcription factor, regulates IFN-γ production in KG-1 cells, we tested IκBζ's role in the classic lymphocyte pathway of IL-12/IL-18–induced IFN-γ. Upon stimulation with IL-12/IL-18, monocyte-depleted human peripheral blood lymphocytes expressed the 79-kDa form of IκBζ and released IFN-γ. CD56+ NK cells were shown to be the IκBζ-producing lymphocyte subpopulation, which also released abundant IFN-γ in response to IL-12/IL-18. Importantly, IκBζ was undetectable in CD56− lymphocytes where IFN-γ release was 10-fold lower. In addition, small interfering RNA knockdown of IκBζ suppressed IFN-γ expression in CD56+ cells. The association of IκBζ with the IFN-γ promoter was documented by chromatin immunoprecipitation. IFN-γ promoter activity from IκBζ overexpression was confirmed by luciferase reporter assay. Finally, IκBζ coprecipitated with p65 and p50 NF-κB in NK cells in response to IL-12/IL-18, suggesting that IκBζ's effects on IFN-γ promoter activity are coregulated by NF-κB. These results suggest that IκBζ functions as an important regulator of IFN-γ in human NK cells, further expanding the class of IκBζ-modulated genes.

Burkholderia cenocepacia infections in CF patients involve heightened inflammation, fatal sepsis, and high antibiotic resistance. Proinflammatory IL-1β secretion is important in airway inflammation and tissue damage. However, little is known about this pathway in macrophages upon B. cenocepacia infection. We report here that murine macrophages infected with B. cenocepacia K56-2 produce proinflammatory cytokine IL-1β in a TLR4 and caspase-1-mediated manner. We also determined that the OPS (O antigen) of B. cenocepacia LPS contributes to IL-1β production and pyroptotic cell death. Furthermore, we showed that the malfunction of the CFTR channel augmented IL-1β production upon B. cenocepacia infection of murine macrophages. Taken together, we identified eukaryotic and bacterial factors that contribute to inflammation during B. cenocepacia infection, which may aid in the design of novel approaches to control pulmonary inflammation.

Immunoparalysis defined by prolonged monocyte human leukocyte antigen DR depression is associated with adverse outcomes in adult severe sepsis and can be reversed with granulocyte macrophage colony-stimulating factor (GM-CSF). We hypothesized that immunoparalysis defined by whole-blood ex vivo lipopolysaccharide-induced tumor necrosis factor-alpha (TNFα) response200 pg/mL beyond day 3 of multiple organ dysfunction syndrome (MODS) is similarly associated with nosocomial infection in children and can be reversed with GM-CSF.In study period 1, we performed a multicenter cohort trial of transplant and nontransplant multiple organ dysfunction syndrome (MODS) patients (≥2 organ failure). In study period 2, we performed an open-label randomized trial of GM-CSF therapy for nonneutropenic, nontransplant, severe MODS patients (≥3 organ failure) with TNFα response160 pg/mL.Immunoparalysis was observed in 34% of MODS patients (n = 70) and was associated with increased nosocomial infection (relative risk [RR] 3.3, 95% confidence interval [1.8-6.0] p0.05) and mortality (RR 5.8 [2.1-16] p0.05). TNFα response200 pg/mL throughout 7 days after positive culture was associated with persistent nosocomial infection, whereas recovery above 200 pg/mL was associated with resolution of infection (p0.05). In study period 2, GM-CSF therapy facilitated rapid recovery of TNFα response to200 pg/mL by 7 days (p0.05) and prevented nosocomial infection (no infections in seven patients versus eight infections in seven patients) (p0.05).Similar to in adults, immunoparalysis is a potentially reversible risk factor for development of nosocomial infection in pediatric MODS. Whole-blood ex vivo TNFα response is a promising biomarker for monitoring this condition.

Macrophages mediate crucial innate immune responses via caspase-1-dependent processing and secretion of interleukin 1β (IL-1β) and IL-18. Although infection with wild-type Salmonella typhimurium is lethal to mice, we show here that a strain that persistently expresses flagellin was cleared by the cytosolic flagellin-detection pathway through the activation of caspase-1 by the NLRC4 inflammasome; however, this clearance was independent of IL-1β and IL-18. Instead, caspase-1-induced pyroptotic cell death released bacteria from macrophages and exposed the bacteria to uptake and killing by reactive oxygen species in neutrophils. Similarly, activation of caspase-1 cleared unmanipulated Legionella pneumophila and Burkholderia thailandensis by cytokine-independent mechanisms. This demonstrates that activation of caspase-1 clears intracellular bacteria in vivo independently of IL-1β and IL-18 and establishes pyroptosis as an efficient mechanism of bacterial clearance by the innate immune system.

The ability of Legionella pneumophila to cause pneumonia is determined by its capability to evade the immune system and grow within human monocytes and their derived macrophages. Human monocytes efficiently activate caspase-1 in response to Salmonella but not to L. pneumophila. The molecular mechanism for the lack of inflammasome activation during L. pneumophila infection is unknown. Evaluation of the expression of several inflammasome components in human monocytes during L. pneumophila infection revealed that the expression of the apoptosis-associated speck-like protein (ASC) and the NOD-like receptor NLRC4 are significantly down-regulated in human monocytes. Exogenous expression of ASC maintained the protein level constant during L. pneumophila infection and conveyed caspase-1 activation and restricted the growth of the pathogen. Further depletion of ASC with siRNA was accompanied with improved NF-κB activation and enhanced L. pneumophila growth. Therefore, our data demonstrate that L. pneumophila manipulates ASC levels to evade inflammasome activation and grow in human monocytes. By targeting ASC, L. pneumophila modulates the inflammasome, the apoptosome, and NF-κB pathway simultaneously.

Endotoxin administration recapitulates many of the host responses to sepsis. Inhibitors of the cysteine protease caspase 1 have long been sought as a therapeutic because mice lacking caspase 1 are resistant to LPS-induced endotoxic shock. According to current thinking, caspase 1-mediated shock requires the proinflammatory caspase 1 substrates IL-1β and IL-18. We show, however, that mice lacking both IL-1β and IL-18 are normally susceptible to LPS-induced splenocyte apoptosis and endotoxic shock. This finding indicates the existence of another caspase 1-dependent mediator of endotoxemia. Reduced serum high mobility group box 1 (HMGB1) levels in caspase 1-deficient mice correlated with their resistance to LPS. A critical role for HMGB1 in endotoxemia was confirmed when mice deficient for IL-1β and IL-18 were protected from a lethal dose of LPS by pretreatment with HMGB1-neutralizing Abs. We found that HMGB1 secretion from LPS-primed macrophages required the inflammasome components apoptotic speck protein containing a caspase activation and recruitment domain (ASC), caspase 1 and Nalp3, whereas HMGB1 secretion from macrophages infected in vitro with Salmonella typhimurium was dependent on caspase 1 and Ipaf. Thus, HMGB1 secretion, which is critical for endotoxemia, occurs downstream of inflammasome assembly and caspase 1 activation.

Background: Although smoking rates continue to decline in US adults, the number of electronic cigarette (e-cig) users is rapidly growing, especially among youth. Exposure to cigarette smoke is known to result in lung cancer and chronic obstructive pulmonary disease, is associated with inflammatory and immune responses generally, and effects gene expression related these and other pathways. However, the effects of e-cigs on the gene expression and inflammation in the bronchial epithelium are largely unknown. We conducted a cross-sectional study to evaluate the effects of e-cig use on gene expression in the bronchial epithelium, in comparison to effects on never smokers and cigarette smokers. Methods: Epithelia brushings were obtained by bronchoscopy from never smokers (n=42), e-cig users (n=14), and current smokers (n=16) (overall, age 21-30, 78% European-American, 54% male). RNA was extracted and profiled for the whole genome transcriptome (Affymetrix Human Transcriptome Array 2.0). Between group differences were determined with the t-test. False Discovery Rate (FDR) < 0.05 was considered significant. Functional and network analyses were performed using Ingenuity Pathway Analysis (IPA) software. Results: The average number of cigarettes smoked per day in the smoker group was 16, ranging from 10 to 20 cigarettes/day. In the e-cig user group, the average number of puffs inhaled per day was 173, ranging from 20 to 600, and the average volume of e-liquid per day was 8 ml, ranging from 2 to 20 ml. Of the 14 e-cig users, eleven identified themselves as former smokers (average 24 months since last cigarette), while three were never-smokers. In microarray analysis, comparing never smoker and smoker groups, there were 2,536 differentially expressed genes (DEGs; 1,235 up-regulated genes, 1,301 down-regulated genes in smokers) and 69 DEGs comparing never smoker and e-cig user groups (59 up-regulated genes, 10 down-regulated genes in e-cig users). In addition, there were 108 DEGs between e-cig user and smoker groups (83 up-regulated genes, 25 down-regulated genes in smokers). Several well-known smoking-related genes such as CYP1B1, AKR1B10, ALDH3A1, CYP2A13, and CX3CL1 were significantly decreased in their expression for e-cig users compared to smokers. Unsupervised hierarchical clustering revealed that the expression profiles of never smokers and e-cig users are more similar to each other compared to profiles of smokers. Pathway analysis of smoking-related genes revealed that NRF2-mediated oxidative stress was the most significant canonical pathway (P = 7.88E-11). Conclusions: Consistent with the hypothesis that e-cigs are less harmful than smoking, bronchial epithelium gene expression profiles of never smokers and e-cig users are more similar, while smokers exhibit distinct profiles. Associations of different nicotine products are apparent in gene expression profiles. Citation Format: Daniel Y. Weng, Min-Ae Song, Theodore M. Brasky, Joseph P. McElroy, Ewy Mathe, Jo L. Freudenheim, Mark D. Wewers, Peter G. Shields. The effects of electronic cigarette exposure on genome-wide expression in human bronchial epithelium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3233.

Background: Alteration of the oral microbiome (microbial dysbiosis) with cigarette smoking is well established. However, the effect of electronic cigarettes (e-cigs) use on the oral microbiome is unknown, although there are emerging data that e-cigs induce microbial changes similar to smoking. In smoking-related diseases, such as chronic obstructive pulmonary disease, there are changes in the oral microbiome and in the expression of genes involved in inflammatory pathways. Similar to the oral microbiome, it is feasible that smoking tobacco and e-cig use could also affect the lung microbiome. To the best of our knowledge, there is only one published study investigating smoking tobacco effects on the oral and lung microbiome. No published studies have evaluated concurrent effects of e-cigs in the oral and lung microbiome. Aims: We hypothesize that microbial dysbiosis and expression of inflammatory cytokines in the oral cavity and lung will differ between smokers and nonsmokers, and that e-cig users will have microbial dysbiosis more similar to smokers. To accomplish this, we propose 1) to examine the association of oral and lung microbiome in nonsmokers, smokers and e-cig users, 2) to determine if the oral microbiome and the lung microbiome differ among these groups, and 3) to determine correlation of the microbiota with host expression of inflammation-related genes. Methods: A cross-sectional study using bronchoscopy and oral rinse collection of 10 never-smokers, 8 cigarette smokers, and 10 e-cig users was conducted. For each study participant, RNA was extracted from saliva and bronchoalveolar lavage (BAL) samples for total transcriptome analysis using RNA-seq; facilitating this approach allows measurement of bacterial communities and human inflammatory cytokine expression in the same assay. To determine microbial dysbiosis by smoking status, the Mann Whitney U-test and Kruskal-Wallis H-test were used with Bonferroni correction for multiple comparisons. Both effect size (fold change >1.5) and adjusted p-value cutoffs ( Results: In preliminary analyses we identified 2,257 bacterial strains in saliva samples and 1592 in BAL samples. We found a lack of concordance of highly abundant bacteria in the oral cavity and lungs. The top twenty expressed human genes were associated with RNA splicing, RNA elongation and miRNAs. Comparisons of microbial dysbiosis by smoking status are currently under way. Conclusion: The composition of the microbiome for saliva is different from that of BAL. Comparison of the metatranscriptome and transcriptome between the lung and oral cavity, as well as between smokers, nonsmokers and e-cigarette users, will allow us to observe how e-cig use compares with cigarette smoking and never smoking in terms of microbial dysbiosis and inflammatory cytokines. Citation Format: Kevin L. Ying, Min-Ae Song, Daniel Y. Weng, Quentin A. Nickerson, Joseph P. McElroy, David Frankhouser, Pearlly S. Yan, Ralf Bundschuh, Theodore M. Brasky, Mark D. Wewers, Ewy Mathé, Jo L. Freudenheim, Peter G. Shields. Using oral and lung microbiome to assess microbial dysbiosis and inflammatory response to electronic cigarettes and to cigarettes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1231.

Background: The use of electronic cigarettes (e-cigs) is increasing rapidly among both adults and youth, including among both smokers and never-smokers, and is thought to be less harmful than cigarettes. However, biological changes including inflammation have not been studied in target organs such as the lung, particularly for e-cigs; further, it is not known if changes are reversible in smokers who switch to e-cigs. Methods: We conducted a cross-sectional bronchoscopy study of e-cig users (n=13), cigarette smokers (n=16), and never-smokers (n=40), age 21-30, to assess inflammatory cell counts and cytokines in bronchoalveolar lavages (BAL) fluids. BAL total cell counts were measured using the Countess Automated Cell Counter. Differential cells were performed on stained cytospins by a blinded clinical histopathologist. Smoking-associated inflammatory cytokines were measured using a V-PLEX Plus Pro-inflammatory Panel. Non-parametric Mann-Whitney and Kruskal-Wallis tests were used for cell counts. Log10 transformed cytokines were used for 1-way ANOVA. Results: Smokers averaged 16 cigarettes/day (SD: 4.7) for 7 years (SD: 4.1). E-cig users vaped an average e-liquid nicotine content of 12 mg/ml (SD: 7.6) and 8 ml/day (SD: 3.9), for an average of 3 years (SD: 0.9); mean time since smoking among e-cig users was 24 months (SD: 16) (2 were never-smokers). Smokers compared to never-smokers had significantly increased total cell counts, macrophages and neutrophils, and decreased lymphocytes (7.15E-05 Discussion: Inflammation differed for never-smokers and smokers, with markers of inflammation intermediate for e-cig users, consistent with the hypothesis that e-cigs are less harmful. Study of whether e-cig use is more harmful than never-use and never-smoking is warranted. Because this cross-sectional study provides only indirect evidence of causation, additional observation studies and randomized trials are needed to understand changes in inflammation for e-cig users, both former- never-smokers. Citation Format: Min-Ae Song, Theodore M. Brasky, Jo L. Freudenheim, Joseph P. McElroy, Daniel Y. Weng, Kevin L. Ying, Quentin A. Nickerson, Sarah A. Reisinger, Mark D. Wewers, Peter G. Shields. Electronic cigarettes and inflammation in the human lung [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3237.

Background: The use of electronic cigarettes (e-cigs) among smokers and never-smokers is increasing, and is considered to be less harmful than cigarettes. However, this has not been studied in target organs for smoking-related diseases, especially the lung. Altered DNA methylation is seen in smokers and contributes to lung carcinogenesis. However, it is not known how e-cigs affect methylation in the lung or whether smoking-induced DNA methylation changes can be reversed in former smokers who switch to e-cigs. Methods: We conducted a cross-sectional bronchoscopy study of e-cig users (n=12), cigarette smokers (n=10), and never-smokers (n=10) (age 21-30, 84% European-American, 66% male). Bisulfite-converted DNA extracted from lung epithelium collected by bronchial brushing was analyzed for 865,859 CpGs with the llumina Infinium Methylation EPIC Chip in a single batch. Probes were filtered out if on sex chromosomes, cross-reactive, or SNP associated, leaving 735,317 CpGs for analysis. One-way analysis of covariance using M-values was used to identify CpGs differentially methylated for the three groups. False Discovery Rate (FDR) of q Results: Smokers averaged 18 cigarettes/day (SD: 4.2) and 6 years of smoking (SD: 4.5). E-cig users vaped an average e-liquid nicotine content of 10 mg/mL (SD: 11) and 8 mL/day (SD: 4), for an average of three years (SD: 1). Mean time since smoking among the e-cig users was 31 months (SD: 15); 3 were never-smokers. We identified 517 differentially methylated CpGs among the three groups (FDR q Discussion: DNA methylation differed for never-smokers and smokers, with e-cig users' methylation intermediate, consistent with the hypothesis that e-cigs are less harmful than smoking; whether e-cig use is more harmful than never-smoking needs to be studied. Because this study is cross-sectional, association, and not causation, is indicated. Additional observational studies and randomized trials are warranted to understand biologic changes in the lung for smokers and never-smokers using e-cigs. Citation Format: Min-Ae Song, Dominic J. Smiraglia, Theodore M. Brasky, Daniel Y. Weng, Joseph P. McElroy, Sarah A. Reisinger, Kevin L. Ying, Quentin A. Nickerson, Mark D. Wewers, Peter G. Shields, Jo L. Freudenheim. Lung epithelium DNA methylation: Electronic cigarette users, smokers, and never-smokers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3236.

Among human natural killer (NK) cell intermediates in secondary lymphoid tissue (SLT), stage 3 CD34(-)CD117(+)CD161(+)CD94(-) immature NK (iNK) cells uniquely express aryl hydrocarbon receptor (AHR) and interleukin-22 (IL-22), supporting a role in mucosal immunity. The mechanisms controlling proliferation and differentiation of these cells are unknown. Here we demonstrate that the IL-1 receptor IL-1R1 was selectively expressed by a subpopulation of iNK cells that localized proximal to IL-1beta-producing conventional dendritic cells (cDCs) within SLT. IL-1R1(hi) iNK cells required continuous exposure to IL-1beta to retain AHR and IL-22 expression, and they proliferate in direct response to cDC-derived IL-15 and IL-1beta. In the absence of IL-1beta, a substantially greater fraction of IL-1R1(hi) iNK cells differentiated to stage 4 NK cells and acquired the ability to kill and secrete IFN-gamma. Thus, cDC-derived IL-1beta preserves and expands IL-1R1(hi)IL-22(+)AHR(+) iNK cells, potentially influencing human mucosal innate immunity during infection.

Rationale Inflammasome assembly and activation is a complex process regulated, in part, by posttranslational modification of inflammasome proteins. ASC is a universal adaptor in virtually all inflammasome platforms and its modification is critical in regulation of inflammasome function. In particular, PTK inhibitor AG126 greatly inhibits inflammasome activation. Methods We generated stable THP-1 cells with either overexpression of ASC (YFP-ASC) or knock down of ASC (siRNA and CRISPR) followed by ASC knock in. To test the role of ASC phosphorylation in inflammasome assembly and activity, we mutated the highly conserved tyrosine 146 to Y146A. Cells were stimulated with LPS or live bacteria and inflammasome activity was measured by fluorescent microscopy, ELISA, and immunoblotting. Results Upon inflammasome activation ASC is released from the cell together with IL-1β, IL-18 and other inflammasome proteins. Inhibition of select tyrosine kinases severely reduces inflammasome activation, pyroptosis and ASC release. Elimination of the potential phosphorylation site Y146 dramatically changes visible ASC complex formation – from a single round speck to a filamentous structure. Cells with the Y146A ASC mutation show significantly abrogated inflammasome activation as compared to a wild type. Screening of a PTK inhibitor library (Selleckchem) linked several tyrosine kinases to regulating ASC phosphorylation and inflammasome activity. Conclusion ASC phosphorylation at Y146 by tyrosine kinases is an important regulator of inflammasome activity and pyroptosis.

Rationale Apoptosis-associated speck-like protein containing a CARD (ASC) serves as an adaptor to link NOD-like receptors to caspase-1 during inflammasome activation. The oligomerization of ASC into a large speck-like complex appears to be crucial to this process. Furthermore, extracellular ASC specks may propagate an inflammatory response. Although extremely high levels of ASC exist in BALF, little is known about whether BALF ASC exists in the soluble form or as part of a speck complex. Methods Subjects (normal volunteers and patients with pneumonia) underwent IRB approved BAL. BALF was removed from cells by centrifugation at 400g and ASC was quantified by ELISA. Additionally, BALF was spun at 16,000g to pellet ASC specks and separate them from soluble ASC. Samples were crosslinked with DSS, separated on a denaturing gel, and immunoblotted with an anti-ASC antibody to visualize ASC oligomers. Results Extracellular levels of ASC were very high in the BALF when measured by ELISA and immunoblot. However, although pelletable ASC oligomers could easily be observed in stimulated THP-1 cell supernatants, no oligomers of ASC were found in BALF samples from healthy donors or pneumonia patients, and negligible amounts of ASC pelleted down when BALF was spun at high speed. Conclusion In contrast to inflammasome activated THP-1 cells, extracellular ASC in BALF exists primarily in the soluble non-oligomeric state. This observation held true for healthy donors and donors with lung infections.

IL-6 is a pleiotropic cytokine implicated in the pathogenesis of disorders such as sepsis and cancer. We noted that human monocytes are excellent producers of IL-6 as compared with monocyte-derived macrophages. Because macrophages from molecule containing ankyrin repeats induced by LPS (MAIL) knockout animals have suppressed IL-6 production, we hypothesized that regulation of MAIL is key to IL-6 production in humans and may explain the differences between human monocytes and macrophages. To test this hypothesis fresh human monocytes and monocyte-derived macrophages were compared for MAIL expression in response to LPS. LPS-induced monocyte MAIL expression was highly inducible and transient. Importantly for our hypothesis MAIL protein expression was suppressed during differentiation of monocytes to macrophages. Of note, the human MAIL protein detected was the 80 kDa MAIL-L form and human MAIL showed nuclear localization. Human MAIL-L bound to p50 subunit of the NF-κB and increased IL-6 luciferase promoter activity in a cEBPβ, NF-κB, and AP-1-dependent fashion. Like the differences in MAIL induction, monocytes produced 6-fold more IL-6 compared with macrophages (81.7 ± 29.7 vs 12.6 ± 6.8 ng/ml). Furthermore, suppression of MAIL by small interfering RNA decreased the production of IL-6 significantly in both THP-1 cells and in primary monocytes. Costimulation of monocytes with LPS and muramyl dipeptide induced an enhanced IL-6 response that was suppressed by siMAIL. Our data suggests that MAIL is a key regulator of IL-6 production in human monocytes and plays an important role in both TLR and NOD-like receptor ligand induced inflammation.

Relative to monocytes, human macrophages are deficient in their ability to process and release IL-1β. In an effort to explain this difference, we used a model of IL-1β processing and release that is dependent upon bacterial escape into the cytosol. Fresh human blood monocytes were compared with monocyte-derived macrophages (MDM) for their IL-1β release in response to challenge with Francisella novicida. Although both cell types produced similar levels of IL-1β mRNA and intracellular pro-IL-1β, only monocytes readily released processed mature IL-1β. Baseline mRNA expression profiling of candidate genes revealed a remarkable deficiency in the pyrin gene, MEFV, expression in MDM compared with monocytes. Immunoblots confirmed a corresponding deficit in MDM pyrin protein. To determine whether pyrin levels were responsible for the monocyte/MDM difference in mature IL-1β release, pyrin expression was knocked down by nucleofecting small interfering RNA against pyrin into monocytes or stably transducing small interfering RNA against pyrin into the monocyte cell line, THP-1. Pyrin knockdown was associated with a significant drop in IL-1β release in both cell types. Importantly, M-CSF treatment of MDM restored pyrin levels and IL-1β release. Similarly, the stable expression of pyrin in PMA-stimulated THP-1-derived macrophages induces caspase-1 activation, associated with increased IL-1β release after infection with F. novicida. In summary, intracellular pyrin levels positively regulate MDM IL-1β responsiveness to Francisella challenge.

Rationale: Little is known about the genetic regulation of granulomatous inflammation in sarcoidosis. Objectives: To determine if tissue gene array analysis would identify novel genes engaged in inflammation and lung remodeling in patients with sarcoidosis. Methods: Gene expression analysis was performed on tissues obtained from patients with sarcoidosis at the time of diagnosis (untreated) (n = 6) compared with normal lung tissue (n = 6). Expression of select genes was further confirmed in lung tissue from a second series of patients with sarcoidosis and disease-free control subjects (n = 11 per group) by semi-quantitative RT-PCR. Interactive gene networks were identified in patients with sarcoidosis using Ingenuity Pathway Analysis (Ingenuity Systems, Inc., Redwood, CA) software. The expression of proteins corresponding to selected overexpressed genes was determined using fluorokine multiplex analysis, and immunohistochemistry. Selected genes and proteins were then analyzed in bronchoalveolar lavage fluid in an independent series of patients with sarcoidosis (n = 36) and control subjects (n = 12). Measurements and Main Results: A gene network engaged in Th1-type responses was most significantly overexpressed in the sarcoidosis lung tissues, including genes not previously reported in the context of sarcoidosis (e.g., IL-7). MMP-12 and ADAMDEC1 transcripts were most highly expressed (> 25-fold) in sarcoidosis lung tissues, corresponding with increased protein expression by immunohistochemistry. MMP-12 and ADAMDEC1 gene and protein expression were increased in bronchoalveolar lavage samples from patients with sarcoidosis, correlating with disease severity. Conclusions: Tissue gene expression analyses provide novel insights into the pathogenesis of pulmonary sarcoidosis. MMP-12 and ADAMDEC1 emerge as likely mediators of lung damage and/or remodeling and may serve as markers of disease activity.

Macrophages are unique innate immune cells that play an integral role in the defense of the host by virtue of their ability to recognize, engulf, and kill pathogens while sending out danger signals via cytokines to recruit and activate inflammatory cells. It is becoming increasingly clear that purinergic signaling events are essential components of the macrophage response to pathogen challenges and disorders such as sepsis may be, at least in part, regulated by these important sensors. The activation of the P2X(7) receptor is a powerful event in the regulation of the caspase-1 inflammasome. We provide evidence that the inflammasome activation requires "priming" of macrophages prior to ATP activation of the P2X(7)R. Inhibition of the inflammasome activation by the tyrosine kinase inhibitor, AG126, suggests regulation by phosphorylation. Finally, the P2X(7)R may also be activated by other elements of the host response such as the antimicrobial peptide LL-37, which adds a new, physiologically relevant agonist to the P2X(7)R pathway. Therapeutic approaches to inflammation and sepsis will certainly be enhanced by an increased understanding of how purinergic receptors modulate the inflammasomes.

Although much is known about classic IFNgamma inducers, little is known about the IFNgamma inducing capability of inflammasome-activated monocytes. In this study, supernatants from LPS/ATP-stimulated human monocytes were analyzed for their ability to induce IFNgamma production by KG-1 cells. Unexpectedly, monocyte-derived IFN inducing activity was detected, but it was completely inhibited by IL-1beta, not IL-18 blockade. Moreover, size-fractionation of the monocyte conditioned media dramatically reduced the IFNgamma inducing activity of IL-1beta, suggesting that IL-1beta requires a cofactor to induce IFNgamma production in KG-1 cells. Because TNFalpha is known to synergize with IL-1beta for various gene products, it was studied as the putative IL-1beta synergizing factor. Although recombinant TNFalpha (rTNFalpha) alone had no IFNgamma inducing activity, neutralization of TNFalpha in the monocyte conditioned media inhibited the IFNgamma inducing activity. Furthermore, rTNFalpha restored the IFNgamma inducing activity of the size-fractionated IL-1beta. Finally, rTNFalpha synergized with rIL-1beta, as well as with rIL-1alpha and rIL-18, for KG-1 IFNgamma release. These studies demonstrate a synergistic role between TNFalpha and IL-1 family members in the induction of IFNgamma production and give caution to interpretations of KG-1 functional assays designed to detect functional IL-18.

Rationale: Monocytes are central to the initiation of the inflammatory response in sepsis, with caspase-1 activation playing a key role. Monocyte deactivation during sepsis has been linked to poor outcomes. Objectives: Given the importance of caspase-1 in the immune response, we investigated whether monocytes from patients early in septic shock demonstrate alterations in mRNAs for caspase-1–related molecules. Methods: Patients with septic shock (n = 26; age >18 years), critically ill intensive care unit patients (n = 20), and healthy volunteers (n = 22) were enrolled in a prospective cohort study in a university intensive care unit. Demographic, biological, physiologic, and plasma cytokine measurements were obtained. Monocytes were assayed for ex vivo tumor necrosis factor-α production, and fresh monocyte mRNA was analyzed by quantitative reverse-transcription polymerase chain reaction for Toll-like receptors, NOD-LRR proteins, cytokines, and nuclear factor-κB–related genes. Measurements and Main Results: Relative copy numbers for the inflammasome mRNAs for ASC, caspase-1, NALP1, and Pypaf-7 were significantly lower in patients with septic shock compared with critically ill control subjects. NALP1 mRNA levels were linked to survival in patients with sepsis (P = 0.0068) and correlated with SAPS II scores (r = −0.63). Conclusions: These data suggest that monocyte deactivation occurs during the earliest stages of the systemic inflammatory response and that changes in inflammasome mRNA expression are part of this process.

LPS stimulates monocytes/macrophages through the activation of signaling events that modulate the production of inflammatory cytokines. Apigenin, a flavonoid abundantly found in fruits and vegetables, exhibits anti-proliferative and anti-inflammatory activities through poorly defined mechanisms. In this study, we demonstrate that apigenin inhibits the production of proinflammatory cytokines IL-1β, IL-8, and TNF in LPS-stimulated human monocytes and mouse macrophages. The inhibitory effect on proinflammatory cytokine production persists even when apigenin is administered after LPS stimulation. Transient transfection experiments using NF-κB reporter constructs indicated that apigenin inhibits the transcriptional activity of NF-κB in LPS-stimulated mouse macrophages. The classical proteasome-dependent degradation of the NF-κB inhibitor IκBα was observed in apigenin LPS-stimulated human monocytes. Using EMSA, we found that apigenin does not alter NF-κB-DNA binding activity in human monocytes. Instead we show that apigenin, as part of a non-canonical pathway, regulates NF-κB activity through hypophosphorylation of Ser536 in the p65 subunit and the inactivation of the IKK complex stimulated by LPS. The decreased phosphorylation on Ser536 observed in LPS-stimulated mouse macrophages treated with apigenin was overcome by the over-expression of IKKβ. In addition, our studies indicate that apigenin inhibits in vivo LPS-induced TNF and the mortality induced by lethal doses of LPS. Collectively, these findings suggest a molecular mechanism by which apigenin suppresses inflammation and modulates the immune response in vivo.

Human lungs continuously handle various pollutants, microbes, and allergens. The pulmonary innate immune response eliminates most of these foreign particles while maintaining a sterile environment within the lung. This response is tightly regulated in order to minimize inflammation and protect the host. This review focuses on the major pulmonary innate immune components that respond to infectious agents, including alveolar macrophages and their major receptors and surfactant, and its ability to regulate the host response to certain infectious pathogens. Finally, potential therapeutic applications relevant to these innate immune determinants will be discussed.

Rationale: Caspase-1 processes interleukin 1β (IL-1β) and IL-18 but may also contribute to apoptosis. In this context, caspase-1 knockout mice have been shown to be protected from endotoxin-induced mortality, whereas IL-1β knockout mice are not protected. Objectives: We therefore sought to delineate the mechanisms responsible for the differential responses between caspase-1 and IL-1β knockout mice. Methods: Caspase-1 knockout, IL-1β knockout, and IL-1β/IL-18 double knockout mice were compared with wild-type mice for survival after intraperitoneal challenge with live Escherichia coli. Measurements and Main Results: Caspase-1 knockout animals were protected from bacterial challenge, whereas wild-type, IL-1β knockout, and IL-1β/IL-18 double knockout animals were not. Wild-type animals and both IL-1β knockout and IL-1β/IL-18 double knockout mice demonstrated significant splenic B lymphocyte apoptosis, which was absent in the caspase-1 knockout mice. Importantly, IL-1β/IL-18 double knockout mice were protected from splenic cell apoptosis and sepsis-induced mortality by the caspase inhibitor zVAD-fmk. Furthermore, wild-type but not caspase-1 knockout splenic B lymphocytes induced peritoneal macrophages to assume an inhibitory phenotype. Conclusion: Taken together, these findings suggest that caspase-1 is important in the host response to sepsis at least in part via its ability to regulate sepsis-induced splenic cell apoptosis.

Adiponectin (Ad), a member of the adipocytokine family, has been reported to possess antiinflammatory properties. We investigated the effects of full-length human Ad (hAd) on phorbol 12-myristate 13-acetate (PMA)-induced O2-* generation by human neutrophils. hAd, even at the lowest tested concentration of 0.001 microg/ml, after 30-min pretreatment of cells, significantly inhibited O2-* generation by neutrophils stimulated with PMA (100 nM). However, no relation between the dose of hAd and extent of inhibition of PMA-induced O2-* generation was observed with increasing the concentration of hAd up to 1 microg/ml. hAd also significantly inhibited neutrophil O2-* generation stimulated by N-formyl-methionyl-leucyl-phenylalanine (100 microM) and diacylglycerol (500 nM), as well as the PMA-induced neutrophil nitroblue tetrazolium reduction and H2O2 formation. Pretreatment of neutrophils with pronase-digested hAd failed to inhibit the PMA-induced O2-* generation. For the first time, this study revealed that Ad inhibited O2-* generation by neutrophils, possibly through regulation of NADPH oxidase.

Receptor interacting protein-2 (RIP2) is a caspase recruitment domain (CARD)-containing kinase that interacts with caspase-1 and plays an important role in NF-κB activation. Apoptosis-associated speck-like protein containing a CARD (ASC) is a PYRIN and CARD-containing molecule, important in the induction of apoptosis and caspase-1 activation. Although RIP2 has also been linked to caspase-1 activation, RIP2 knockout animals fail to show a defect in caspase-1-mediated processing of proIL-1β to its active form. Therefore, RIP2 function in binding to caspase-1 remains poorly understood. We hypothesized that caspase-1 may serve as a scaffolding molecule that promotes RIP2 interaction with IκB kinase-γ thus inducing NF-κB activation. We further hypothesized that ASC, which also interacts with caspase-1 via its CARD, may interfere with the caspase-1 RIP2 interaction. In HEK293 cells, ASC induced prominent activation of caspase-1 and proIL-1β processing. RIP2 transient transfection induced transcription of an NF-κB reporter gene. This RIP2-induced NF-κB activity and caspase-1 binding was inhibited in a dose-dependent fashion by ASC. Consistent with a role for caspase-1 as a scaffold for RIP2, caspase-1 knockout macrophages were suppressed in their ability to activate NF-κB, and septic caspase-1 knockout animals produced less IL-6, a functional marker of NF-κB activity. Lastly, THP-1 cells treated with small interfering RNA for ASC decreased their caspase-1 activity while enhancing their NF-κB signal. These data suggest that ASC may direct caspase-1 away from RIP2-mediated NF-κB activation, toward caspase-1-mediated processing of proIL-1β by interfering with the RIP2 caspase-1 interaction.

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.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).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.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.

Background Plasma viral load and blood CD4 counts are accepted indicators of severity of illness in patients with HIV-1. Lung CD4 counts have not been evaluated in asymptomatic HIV-1 patients as indicators of disease severity. Objective To determine if lung lymphocyte counts in asymptomatic subjects with HIV compare with plasma viral loads and blood CD4 counts in predicting survival. Design Retrospective, cross-sectional analysis. Setting Midwestern urban community, December 1996 to August 1998. Participants HIV-seropositive subjects (n = 95) without AIDS-related pulmonary complications. Measurements Plasma viral load, blood hemoglobin and blood lymphocyte subtypes, lung lymphocyte subtypes from BAL, body mass index, and mortality. Results Eight of the 95 subjects (8.4%) had died at the 4-year follow-up. Lung CD4 counts were significantly related to mortality by univariable analysis (2.5 × 10 3 /mL vs 0.9 × 10 3 /mL, median values for survivors vs nonsurvivors, respectively, p = 0.010). Modeling using exact methods further showed lung CD4 counts to be a significant predictor of survival after individually adjusting for potential confounders, including plasma viral load and blood CD4 count. Conclusions Lung CD4 counts in patients with HIV-1 infection may provide an independent predictor of survival.

Antimicrobial peptides form an important component of the innate immune system. The cathelicidin family, a key member of the antimicrobial peptide defenses, has been highly conserved throughout evolution. Though widespread in mammals, there is currently only one identified human example, hCAP-18/LL-37. The cathelicidins have been found to have multiple functions, in addition to their known antimicrobial and lipopolysaccharide-neutralizing effects. As a result, they profoundly affect both innate and adaptive immunity. Currently, antimicrobial peptides are being evaluated as therapeutic drugs in disease states as diverse as oral mucositis, cystic fibrosis, and septic shock. One such peptide, the cathelicidin hCAP-18/LL-37, is reviewed in detail in the context of its role in lung physiology and defense.

Study objectives: Evidence exists that HIV-seropositive individuals may be at increased risk for the development of precocious pulmonary emphysema. HIV infection is also associated with antioxidant deficiency in both the serum and lungs, and it is therefore possible that increased oxidant stress may contribute to parenchymal lung injury occurring in the setting of HIV. We sought to determine the regional distribution of emphysema and regional distribution of glutathione (GSH) concentrations among HIV-seropositive subjects with emphysema. Design: Cross-sectional evaluation of a prospective, longitudinal study. Setting: University teaching hospital. Subjects/measurements: HIV-seropositive subjects without AIDS-related pulmonary complications participating in a descriptive study of lung biology in HIV-seropositive individuals. Emphysema scoring and evaluation of emphysema lobar distribution was performed among 40 subjects with emphysema. Eleven subjects underwent BAL of the right middle lobe (RML) and right upper lobe (RUL) with measurement of epithelial lining fluid (ELF) GSH in each lobe. Results: We found that the mean emphysema scores were much higher in the upper lobes compared to the rest of the lung. Mean GSH levels were significantly greater in the RUL compared to the RML. The regional differences were present in both smokers and nonsmokers. Conclusions: We conclude that in the setting of HIV, emphysema is more prominent and lung GSH concentrations are higher in the upper lobes. We hypothesize that the increased GSH may represent a compensatory response to increased oxidant stress in the upper lobes. (CHEST 2004; 126:1439–1442)

LPS stimulates monocytes/macrophages through TLR4, resulting in the activation of a series of signaling events that potentiate the production of inflammatory mediators. Recent reports indicated that the inflammatory response to LPS is diminished by PI3K, through the activation of the serine/threonine kinase Akt. SHIP is an inositol phosphatase that can reverse the activation events initiated by PI3K, including the activation of Akt. However, it is not known whether SHIP is involved in TLR4 signaling. In this study, we demonstrate that LPS stimulation of Raw 264.7 mouse macrophage cells induces the association of SHIP with lipid rafts, along with IL-1R-associated kinase. In addition, SHIP is tyrosine phosphorylated upon LPS stimulation. Transient transfection experiments analyzing the function of SHIP indicated that overexpression of a wild-type SHIP, but not the SHIP Src homology 2 domain-lacking catalytic activity, up-regulates NF-κB-dependent gene transcription in response to LPS stimulation. These results suggest that SHIP positively regulates LPS-induced activation of Raw 264.7 cells. To test the validity of these observations in primary macrophages, LPS-induced events were compared in bone marrow macrophages derived from SHIP+/+ and SHIP−/− mice. Results indicated that LPS-induced MAPK phosphorylation is enhanced in SHIP+/+ cells, whereas Akt phosphorylation is enhanced in SHIP−/− cells compared with SHIP+/+ cells. Finally, LPS-induced TNF-α and IL-6 production was significantly lower in SHIP−/− bone marrow-derived macrophages. These results are the first to demonstrate a role for SHIP in TLR4 signaling, and propose that SHIP is a positive regulator of LPS-induced inflammation.

ProIL-1β processing by IL-1β-converting enzyme (ICE) and the subsequent release of mature IL-1β are highly regulated events in the monocyte/macrophage response to pathogens. This process occurs in a controlled way through the activation of the constitutively expressed 45-kDa ICE precursor (proICE). To characterize the signaling pathways involved in ICE regulation in human monocytes/macrophages, we analyzed ICE activation in the presence of specific inhibitors of classic signaling pathways. Although LPS-induced ICE activity was not significantly affected by interruption of extracellular signal-regulated kinase, p38 kinase, or phosphoinositol 3-kinase, Janus kinase 3 (JAK3) inhibition produced a significant dose-dependent enhancement of LPS-induced ICE activity. Support for the inhibitory role of JAK3 was shown by the fact that IL-4 (which uses JAK1 and JAK3 signaling) suppressed LPS-induced ICE activity and by the finding that JAK3 knockout macrophages have increased LPS-induced ICE activation. To understand how JAK3 down-regulates LPS-induced ICE activity in monocytes, we hypothesized that JAK3 signaling enhances IL-10 production. In support of this model we show that LPS-induced IL-10 expression was synchronous with ICE deactivation, IL-4 induced the release of IL-10, exogenous IL-10 suppressed LPS-induced ICE activity, a neutralizing IL-10 Ab increased LPS-induced ICE activity, and, finally, JAK3 knockout macrophages displayed significantly reduced LPS-induced IL-10 production. These findings support a model in which JAK3 signaling enhances IL-10 production leading to down-regulation of ICE activation and suppression of IL-1β processing and release.

The release of IL-1β is a tightly controlled process that requires induced synthesis of the precursor pro-IL-1β and a second stimulus that initiates cleavage and secretion of mature IL-1β. Although ATP as a second stimulus potently promotes IL-1β maturation and release via P2X7 receptor activation, millimolar ATP concentrations are needed. The human cathelicidin-derived peptide LL37 is a potent antimicrobial peptide produced predominantly by neutrophils and epithelial cells. In this study, we report that LL37 stimulation of LPS-primed monocytes leads to maturation and release of IL-1β via the P2X7 receptor. LL37 induces a transient release of ATP, membrane permeability, caspase-1 activation, and IL-1β release without cell cytotoxicity. IL-1β release and cell permeability are suppressed by pretreatment with the P2X7 inhibitors oxidized ATP, KN04, and KN62. In the presence of apyrase, which hydrolyzes ATP to AMP, the effect of LL37 was not altered, indicating that LL37 rather than autocrine ATP is responsible for the activation of the P2X7 receptor. We conclude that endogenous LL37 may promote IL-1β processing and release via direct activation of P2X7 receptors.

Objective Macrophages are an important reservoir for the HIV and contribute to innate lung defense by their ability to phagocytose, digest, and process invading pathogens. We hypothesized that HIV-1 infection may lead to a defect in the phagocytic activity of alveolar macrophages. Design In order to test this hypothesis, the phagocytic activity of alveolar macrophages from asymptomatic HIV-1 seropositive subjects was compared to healthy seronegative control subjects. Macrophages from one cohort were fed with Escherichia coli and from another cohort with opsonized sheep RBCs (SRBCs), and the phagocytic index was determined at different time intervals. Setting A tertiary-care, urban, university-based referral center. Participants Asymptomatic HIV-1 seropositive subjects and healthy seropositive control subjects recruited from local community. Results No differences were found in the phagocytic activity between alveolar macrophages from the first cohort of eight seropositive and nine seronegative subjects. Although not statistically significant, there was a trend toward a lower phagocytic activity of HIV-positive smokers compared to HIV-positive nonsmokers. Opsonized phagocytic capacity (using opsonized SRBCs) was further analyzed in a second set of five HIV-positive subjects and five healthy control subjects. Whereas HIV status did not affect opsonized SRBC uptake, a history of smoking was associated with a statistically significant depression in phagocytic index. Conclusions Although there is no significant impairment of phagocytic capacity in HIV-positive subjects compared to HIV-negative control subjects, cigarette smoking produces a significant depression in phagocytic activity that is amplified in HIV-positive smokers.

Objective This study was designed to determine the role played by the mitochondrial permeability transition in the pathogenesis of mitochondrial damage and dysfunction in a representative systemic organ during the acute phase of endotoxemia. Design A well-established, normotensive feline model was employed to determine whether pretreatment with cyclosporine A, a potent inhibitor of the mitochondrial permeability transition, normalizes mitochondrial ultrastructural injury and dysfunction in the liver during acute endotoxemia. Setting The Ohio State University Medical Center research laboratory. Subjects Random source, adult, male conditioned cats. Interventions Hemodynamic resuscitation and maintenance of acid-base balance and tissue oxygen availability were provided, as needed, to minimize the potentially confounding effects of tissue hypoxia and/or acidosis on the experimental results. Treatment groups received isotonic saline vehicle (control; n = 6), lipopolysaccharide (3.0 mg/kg, intravenously; n = 8), or cyclosporine A (6.0 mg/kg, intravenously; n = 6) or tacrolimus (FK506, 0.1 mg/kg, intravenously; n = 4) followed in 30 mins by lipopolysaccharide (3.0 mg/kg, intravenously). Liver samples were obtained 4 hrs posttreatment, and mitochondrial ultrastructure, function, and cytochrome c, Bax, and ceramide contents were assessed. Measurements and main results As expected, significant mitochondrial injury was apparent in the liver 4 hrs after lipopolysaccharide treatment, despite maintenance of regional tissue oxygen availability. Namely, mitochondria demonstrated high-amplitude swelling and exhibited altered respiratory function. Cyclosporine A pretreatment attenuated lipopolysaccharide-induced mitochondrial ultrastructural abnormalities and normalized mitochondrial respiratory control, reflecting protection against inner mitochondrial membrane damage. However, an abnormal permeability of outer mitochondrial membranes to cytochrome c was observed in all lipopolysaccharide-treated groups and was associated with increased mitochondrial concentrations of Bax and ceramide. Conclusions These studies confirm that liver mitochondria are early targets of injury during endotoxemia and that inner and outer mitochondrial membrane damage occurs through different mechanisms. Inner mitochondrial membrane damage appears to relate to the mitochondrial permeability transition, whereas outer mitochondrial membrane damage can occur independent of the mitochondrial permeability transition. Preliminary evidence suggests that Bax may participate in lipopolysaccharide-induced outer mitochondrial membrane damage, but further investigations are needed to confirm this.

Monocytes are important components of the innate immune response. The number of circulating monocytes is controlled in part by apoptosis. We have previously shown that monocyte apoptosis requires the activation of caspase-3, a central component of the apoptotic machinery, and that several stimulatory signals, including endotoxin (lipopolysaccharide [LPS]), induce monocyte survival, by the inhibition of caspase-3. We hypothesized that the Th2 anti-inflammatory cytokine, interleukin (IL)-4, may also influence monocyte life span by modulating the apoptotic cascade and the kinases known to be activated by LPS. Here, we show that the IL-4-dependent killing of LPS-treated monocytes reactivates the apoptotic cascade blocked by endotoxin, evidenced by the activity of the effector caspase-3 and the upstream caspases-8 and -9. IL-4 did not affect the activity of caspase-3 or the fragmentation of DNA in nonstimulated monocytes, suggesting that the induction of the apoptotic cascade by IL-4 is specific for stimulated monocytes. In addition, we show that the ability of IL-4 to induce apoptosis is associated with the dephosphorylation of the extracellular signal-regulated kinase, but not with changes in TLR4 expression. Together, these findings suggest a molecular mechanism by which the anti-inflammatory cytokine IL-4 modulates the life span of monocytes at least in part by an extracellular signal-regulated kinase-dependent pathway.

Study objectives Recent evidence suggests that chronic or latent viral infection may be an important predisposing factor in the development of COPD among smokers. As such, understanding if HIV-seropositive smokers are at heightened risk for respiratory symptoms may have relevance with regard to COPD pathogenesis. The current study was done to systematically identify the prevalence of respiratory symptoms among an HIV-seropositive population and to identify associated clinical features. Design and setting Cross-sectional analysis at an academic medical center. Participants Three hundred twenty-seven HIV-seropositive individuals without a history of AIDS-related pulmonary complications. Fifty-two HIV-negative individuals with a similar age and smoking history served as a comparison group. Measurements Administration of the American Thoracic Society Division of Lung Diseases respiratory questionnaire, and pulmonary function studies. Results Respiratory symptoms including dyspnea (41.6% vs 7.7%), cough (40% vs 25%), and phlegm production (41.9% vs 23.1%) were extremely common in the HIV-group and significantly more common than in the HIV-negative group. Current or prior cigarette smoking was the most important predictor of respiratory symptoms among the HIV-seropositive group. The use of the antiretroviral agent lamivudine was associated with a significant reduction in dyspnea. Conclusion HIV-seropositive individuals are at increased risk for the development of respiratory symptoms even prior to the onset of AIDS-related pulmonary complications. This may reflect a heightened susceptibility to the effects of cigarette smoking.

The acute respiratory distress syndrome (ARDS) remains a major cause of morbidity and mortality. Enhanced fibrosis and elevated procollagen III levels have been linked to increased mortality. We hypothesized that transforming growth factor (TGF)-beta 1 may play an important role in ARDS, given its role in stimulating fibrosis. Using reverse transcriptase in situ polymerase chain reaction (RT in situ PCR) and immunohistochemistry, we analyzed lung tissue from four fibroproliferative ARDS cases and control subjects. We also compared active TGF-beta 1 levels in the bronchoalveolar lavage (BAL) fluid of 13 de novo ARDS cases, and 7 normal control subjects. RT in situ PCR showed TGF-beta 1 mRNA expression in fibroproliferative ARDS cases. Immunohistochemistry confirmed protein expression in these samples. Controls were negative for both techniques. In the newly enrolled ARDS cases, TGF-beta 1 levels, as measured by luciferase assay, were elevated in the 11 of 13 samples, averaging 98 +/- 40 pg/mg protein. Controls had no detectable TGF-beta 1 activity. These data suggest that activation of TGF-beta 1 may be important in the early phases of acute lung injury in addition to driving fibroproliferation. These data may lead to new therapeutic approaches in ARDS through more targeted inhibition of fibrosis.

Human monocytes/macrophages express three classes of receptors for IgG: FcgammaRI, FcgammaRII, and FcgammaRIII. The expression and function of these receptors has been extensively studied with the exception of one, FcgammaRIIb. While the mRNA for FcgammaRIIb has been detected in human monocytes, the protein has remained elusive. Studies in mouse models indicated that the macrophage FcgammaRIIb serves to down-regulate FcgammaR-mediated phagocytosis and immune complex-induced inflammation. FcgammaRIIb has also been shown to modulate the action of cytotoxic antibodies against tumors in mouse models. Hence, an understanding of how FcgammaRIIb expression is regulated is of great importance. Here we demonstrate for the first time FcgammaRIIb protein expression and function in human monocytes. We also report that the expression of FcgammaRIIb is highly up-regulated by interleukin-4, a Th2 cytokine, and that the up-regulation of FcgammaRIIb results in a decrease in the phagocytic efficiency of interleukin-4-treated THP-1 cells. Furthermore co-clustering FcgammaRIIb with FcgammaRIIa resulted in enhanced phosphorylation of the inositol phosphatase SHIP, association of SHIP with Shc, and phosphorylation of additional proteins around 120 and 60-65 kDa, with a concomitant attenuation of Akt activation. We, therefore, propose that FcgammaRIIb serves to inhibit FcgammaRI/IIa-mediated macrophage activation using SHIP as its effector.

The link between smoking tobacco and changes in the oral microbiome in response to tobacco smoking are well established. It is not known if there are changes in response to electronic cigarettes (e-cig). These changes in the microbiome are associated with increased numbers of disease causing pathogens. Currently there are no published studies that have investigated the relationship of smoking tobacco on both the oral and lung microbiome. There is insufficient evidence showing whether changes in oral cavity and lung microbiome are also seen in e-cig users. We will study the oral cavity and lung of non-smokers, smokers and e-cig users to examine concordance between oral cavity and the lungs as well as comparing the three groups, examining the microbiomes and expression of inflammatory markers. We hypothesize that microbial dysbiosis and expression of inflammatory cytokines will differ for smokers and non-smokers; and that e-cig users will have microbial dysbiosis similar to cigarette smokers. A cross-sectional study is being conducted on three groups, 1) never-smokers, 2) cigarette smokers, and 3) e-cig users. For each study participant, saliva and bronchoalveolar lavage (BAL) are being collected to measure microbiome. RNA is extracted from saliva and BAL samples for total transcriptome analysis using RNA-seq. This analysis will detect human and bacterial reads thereby allowing observations of bacterial communities as well as human inflammatory cytokine response to bacterial presence. 85% to 98% of BAL sample reads aligned to the human genome compared to less than 50% from saliva samples. The alignment results allow us to deduce that the majority of reads from BAL samples are human and that the majority of the reads in saliva samples are bacterial. Preliminary results show detection of human RNA expression and of bacterial reads are present in both saliva and BAL samples. More samples are being processed and the comparison of BAL and saliva samples between the three groups will be discussed. Citation Format: Kevin Ying, Min-Ae Song, Daniel Y. Weng, Quentin Nickerson, David Frankhouser, Pearlly S. Yan, Ralf Bundschuh, Mark D. Wewers, Ewy Mathé, Jo L. Freudenheim, Peter G. Shields. Assessing microbial dysbiosis of electronic cigarettes and cigarette smokers using oral and lung microbiome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 246. doi:10.1158/1538-7445.AM2017-246

The purpose of this study was to elucidate the mechanism of the airborne poultry dust (particulate matter, PM)-induced respiratory tract inflammation, a common symptom in agricultural respiratory diseases. The study was based on the hypothesis that poultry PM would induce the release of inflammatory cytokine interleukin-8 (IL-8) by respiratory epithelial cells under the upstream regulation by cytosolic phospholipase A2 (cPLA2) activation and subsequent formation of cyclooxygenase (COX)- and lipoxygenase (LOX)-catalyzed arachidonic acid (AA) metabolites (eicosanoids). Human lung epithelial cells (A549) in culture were treated with the poultry PM (0.1-1.0 mg) for different lengths of time, following which PLA2 activity, release of eicosanoids and secretion of IL-8 in cells were determined. Poultry PM (1.0 mg/ml) caused a significant activation of PLA2 in a time-dependent manner (15-60 min), which was significantly attenuated by the calcium-chelating agents, cPLA2-specific inhibitor (AACOCF3) and antioxidant (vitamin C) in A549 cells. Poultry PM also significantly induced the release of COX- and LOX-catalyzed eicosanoids (prostaglandins, thromboxane A2 and leukotrienes B4 and C4) and upstream activation of AA LOX in the cells. Poultry PM also significantly induced release of IL-8 by the cells in a dose- and time-dependent manner, which was significantly attenuated by the calcium chelating agents, antioxidants and COX- and LOX-specific inhibitors. The current study for the first time revealed that the poultry PM-induced IL-8 release from the respiratory epithelial cells was regulated upstream by reactive oxygen species, cPLA2-, COX- and LOX-derived eicosanoid lipid signal mediators.

Background: Human monocyte inflammatory responses differ between virulent and attenuated Francisella infection. Results: A mixed infection model showed that the virulent F. tularensis Schu S4 can attenuate inflammatory cytokine responses to the less virulent F. novicida in human monocytes. Conclusion: F. tularensis dampens inflammatory response by an active process. Significance: This suppression may contribute to enhanced pathogenicity of F. tularensis. Francisella tularensis is a Gram-negative facultative bacterium that can cause the disease tularemia, even upon exposure to low numbers of bacteria. One critical characteristic of Francisella is its ability to dampen or subvert the host immune response. Previous work has shown that monocytes infected with highly virulent F. tularensis subsp. tularensis strain Schu S4 responded with a general pattern of quantitatively reduced pro-inflammatory signaling pathway genes and cytokine production in comparison to those infected with the less virulent related F. novicida. However, it has been unclear whether the virulent Schu S4 was merely evading or actively suppressing monocyte responses. By using mixed infection assays with F. tularensis and F. novicida, we show that F. tularensis actively suppresses monocyte pro-inflammatory responses. Additional experiments show that this suppression occurs in a dose-dependent manner and is dependent upon the viability of F. tularensis. Importantly, F. tularensis was able to suppress pro-inflammatory responses to earlier infections with F. novicida. These results lend support that F. tularensis actively dampens human monocyte responses and this likely contributes to its enhanced pathogenicity.

Caspase-1 activation is a central event in innate immune responses to many pathogenic infections and tissue damage. The NLRP3 inflammasome, a multiprotein scaffolding complex that assembles in response to two distinct steps, priming and activation, is required for caspase-1 activation. However, the detailed mechanisms of these steps remain poorly characterized. To investigate the process of LPS-mediated NLRP3 inflammasome priming, we used constitutively present pro–IL-18 as the caspase-1–specific substrate to allow study of the early events. We analyzed human monocyte caspase-1 activity in response to LPS priming, followed by activation with ATP. Within minutes of endotoxin priming, the NLRP3 inflammasome is licensed for ATP-induced release of processed IL-18, apoptosis-associated speck-forming complex containing CARD, and active caspase-1, independent of new mRNA or protein synthesis. Moreover, extracellular signal–regulated kinase 1 (ERK1) phosphorylation is central to the priming process. ERK inhibition and small interfering RNA–mediated ERK1 knockdown profoundly impair priming. In addition, proteasome inhibition prevents ERK phosphorylation and blocks priming. Scavenging reactive oxygen species with diphenylene iodonium also blocks both priming and ERK phosphorylation. These findings suggest that ERK1-mediated posttranslational modifications license the NLRP3 inflammasome to respond to the second signal ATP by inducing posttranslational events that are independent of new production of pro–IL-1β and NOD-like receptor components.

Recognition of microbial components via innate receptors including the C-type lectin receptor Dectin-1, together with the inflammatory environment, programs dendritic cells (DCs) to orchestrate the magnitude and type of adaptive immune responses. The exposure to β-glucan, a known Dectin-1 agonist and component of fungi, yeasts, and certain immune support supplements, activates DCs to induce T helper (Th)17 cells that are essential against fungal pathogens and extracellular bacteria but may trigger inflammatory pathology or autoimmune diseases. However, the exact mechanisms of DC programming by β-glucan have not yet been fully elucidated. Using a gene expression/perturbation approach, we demonstrate that in human DCs β-glucan transcriptionally activates via an interleukin (IL)-1- and inflammasome-mediated positive feedback late-induced genes that bridge innate and adaptive immunity. We report that in addition to its known ability to directly prime T cells toward the Th17 lineage, IL-1 by promoting the transcriptional cofactor inhibitor of κB-ζ (IκB-ζ) also programs β-glucan-exposed DCs to express cell adhesion and migration mediators, antimicrobial molecules, and Th17-polarizing factors. Interferon (IFN)-γ interferes with the IL-1/IκB-ζ axis in β-glucan-activated DCs and promotes T cell-mediated immune responses with increased release of IFN-γ and IL-22, and diminished production of IL-17. Thus, our results identify IL-1 and IFN-γ as regulators of DC programming by β-glucan. These molecular networks provide new insights into the regulation of the Th17 response as well as new targets for the modulation of immune responses to β-glucan-containing microorganisms.

Objective Immune dysregulation during sepsis is poorly understood, however, lymphocyte apoptosis has been shown to correlate with poor outcomes in septic patients. The inflammasome, a molecular complex which includes caspase-1, is essential to the innate immune response to infection and also important in sepsis induced apoptosis. Our group has recently demonstrated that endotoxin-stimulated monocytes release microvesicles (MVs) containing caspase-1 that are capable of inducing apoptosis. We sought to determine if MVs containing caspase-1 are being released into the blood during human sepsis and induce apoptosis.. Design Single-center cohort study Measurements 50 critically ill patients were screened within 24 hours of admission to the intensive care unit and classified as either a septic or a critically ill control. Circulatory MVs were isolated and analyzed for the presence of caspase-1 and the ability to induce lymphocyte apoptosis. Patients remaining in the ICU for 48 hours had repeated measurement of caspase-1 activity on ICU day 3. Main Results Septic patients had higher microvesicular caspase-1 activity 0.05 (0.04, 0.07) AFU versus 0.0 AFU (0, 0.02) (p

Natural killer (NK) cells are an early source of immunoregulatory cytokines during the innate immune response to viruses, bacteria, and parasites. NK cells provide requisite IFN-gamma to monocytes for the elimination of obligate intracellular pathogens. IL-1beta is a pro-inflammatory cytokine produced by monocytes (i.e. a monokine) during the early immune response to infection, but its role in promoting human NK cell IFN-gamma production is unknown. The current study examines the ability of the monokine IL-1beta, plus IL-12, to costimulate IFN-gamma production by resting CD56(bright) and CD56(dim) human NK cell subsets. CD56(bright) NK cells stimulated with IL-1beta plus IL-12 produced abundant IFN-gamma protein, while little IFN-gamma was produced in identical cultures of CD56(dim) cells. In addition, upon activation with IL-1beta, CD56(bright) NK cells exhibited considerably greater phosphorylation of extracellular signal-regulated kinases p42/44 as compared to CD56(dim) NK cells. Quantitative PCR analysis showed brisk induction of IFN-gamma gene expression following costimulation with IL-1beta plus IL-12 in CD56(bright) NK cells, but intracellular flow cytometry revealed that only a fraction (42+/-2.3%) of CD56(bright) NK cells account for this high IFN-gamma production. These data suggest that the monokine IL-1beta is a potent costimulus of IFN-gamma production by a subset of NK cells following infectious insult.

Interleukin (IL)-1beta and IL-18 are structurally similar proteins that require caspase-1 processing for activation. Both proteins are released from the cytosol by unknown pathway(s). To better characterize the release pathway(s) for IL-1beta and IL-18 we evaluated the role of lipopolysaccharide priming, of interleukin-1beta-converting enzyme (ICE) inhibition, of human purinergic receptor (P2X(7)) function, and of signaling pathways in human monocytes induced by ATP. Monocytes rapidly processed and released both IL-1beta and IL-18 after exogenous ATP. Despite its constitutive cytosolic presence, IL-18 required lipopolysaccharide priming for the ATP-induced release. Neither IL-1beta nor IL-18 release was prevented by ICE inhibition, and IL-18 release was not induced by ICE activation itself. Release of both cytokines was blocked completely by a P2X7 receptor antagonist, oxidized ATP, and partially by an antibody to P2X(7) receptor. In evaluating the signaling components involved in the ATP effect, we identified that the protein-tyrosine kinase inhibitor, AG126, produced a profound inhibition of both ICE activation as well as release of IL-1beta/IL-18. Taken together, these results suggest that, although synthesis of IL-1beta and IL-18 differ, ATP-mediated release of both cytokines requires a priming step but not proteolytically functional caspase-1.