Eosinophil expression of Triggering receptor expressed on myeloid cells-1 (TREM-1) restricts type 2 lung inflammation.

Asthma affects 25 million Americans and recent advances in treatment are effective for only a portion of severe asthma patients. Triggering Receptor Expressed on Myeloid cells 1 (TREM-1), an innate receptor that canonically amplifies inflammatory signaling in neutrophils and monocytes, plays a central role in regulating lung inflammation. It is unknown how TREM-1 contributes to allergic asthma pathology. Utilizing a murine model of asthma, flow cytometry revealed TREM-1+ eosinophils in the lung tissue and airway during allergic airway inflammation. TREM-1 expression was restricted to recruited, inflammatory eosinophils. Expression was induced on bone marrow derived eosinophils by incubation with IL-33, LPS, or GM-CSF. Compared to TREM-1- airway eosinophils, TREM-1+ eosinophils were enriched for pro-inflammatory gene sets including migration, respiratory burst, and cytokine production. Unexpectedly, eosinophil-specific ablation of TREM-1 exacerbated airway IL-5 production, airway MUC5AC production, and lung tissue eosinophil accumulation. Further investigation of transcriptional data revealed apoptosis and superoxide generation related gene sets were enriched in TREM-1+ eosinophils. Consistent with these findings, Annexin V and Caspase 3/7 staining demonstrated higher rates of apoptosis among TREM-1+ eosinophils compared to TREM-1- eosinophils in the inflammatory airway. In vitro, Trem1/3-/- bone marrow derived eosinophils consumed less oxygen than WT in response to PMA, suggesting that TREM-1 promotes superoxide generation in eosinophils. These data reveal protein level expression of TREM-1 by eosinophils, define a population of TREM-1+ inflammatory eosinophils, and demonstrate that eosinophil TREM-1 restricts key features of type 2 lung inflammation.

Vitamin D-mediated effects on airway innate immunity in vitro.

INTRODUCTION: Vitamin D supplementation has been suggested to enhance immunity during respiratory infection season. We tested the effect of active vitamin D (calcitriol) supplementation on key airway innate immune mechanisms in vitro. METHODS: Primary human airway epithelial cells (hAECs) grown at the air liquid interface were supplemented with 10-7 M calcitriol for 24 hours (or a time course) and their antimicrobial airway surface liquid (ASL) was tested for pH, viscoscity, and antibacterial and antiviral properties. We also tested hAEC ciliary beat frequency (CBF). Next, we assessed alterations to hAEC gene expression using RNA sequencing, and based on results, we measured neutrophil migration across hAECs. RESULTS: Calcitriol supplementation enhanced ASL bacterial killing of Staphylococcus aureus (p = 0.02) but did not enhance its antiviral activity against 229E-CoV. It had no effect on ASL pH or viscosity at three timepoints. Lastly, it did not affect hAEC CBF or neutrophil migration, although there was a trend of enhanced migration in the presence of a neutrophil chemokine (p = 0.09). Supplementation significantly altered hAEC gene expression, primarily of AMP-related genes including CAMP and TREM1. CONCLUSION: While vitamin D supplementation did not have effects on many airway innate immune mechanisms, it may provide a useful tool to resolve respiratory bacterial infections.

TREM-1 is required for enhanced OpZ-induced superoxide generation following priming.

Inflammatory agents, microbial products, or stromal factors pre-activate or prime neutrophils to respond to activating stimuli in a rapid and aggressive manner. Primed neutrophils exhibit enhanced chemotaxis, phagocytosis, and respiratory burst when stimulated by secondary activating stimuli. We previously reported that Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) mediates neutrophil effector functions such as increased superoxide generation, transepithelial migration, and chemotaxis. However, it is unclear whether TREM-1 is required for the process of priming itself or for primed responses to subsequent stimulation. To investigate this, we utilized in vitro and in vivo differentiated neutrophils that were primed with TNF-α and then stimulated with the particulate agonist, opsonized zymosan (OpZ). Bone marrow progenitors isolated from WT and Trem-1-/- mice were transduced with estrogen regulated Homeobox8 (ER-Hoxb8) fusion transcription factor and differentiated in vitro into neutrophils following estrogen depletion. The resulting neutrophils expressed high levels of TREM-1 and resembled mature in vivo differentiated neutrophils. The effects of priming on phagocytosis and oxidative burst were determined. Phagocytosis did not require TREM-1 and was not altered by priming. In contrast, priming significantly enhanced OpZ-induced oxygen consumption and superoxide production in WT but not Trem-1-/- neutrophils indicating that TREM-1 is required for primed oxidative burst. TREM-1-dependent effects were not mediated during the process of priming itself as priming enhanced degranulation, ICAM-1 shedding, and IL-1ß release to the same extent in WT and Trem-1-/- neutrophils. Thus, TREM-1 plays a critical role in primed phagocytic respiratory burst and mediates its effects following priming.

TREM-1 regulates neutrophil chemotaxis by promoting NOX-dependent superoxide production.

Neutrophil migration across tissue barriers to the site of injury involves integration of complex danger signals and is critical for host survival. Numerous studies demonstrate that these environmental signals fundamentally alter the responses of extravasated or "primed" neutrophils. Triggering receptor expressed on myeloid cells 1 (TREM-1) plays a central role in modulating inflammatory signaling and neutrophil migration into the alveolar airspace. Using a genetic approach, we examined the role of TREM-1 in extravasated neutrophil function. Neutrophil migration in response to chemoattractants is dependent upon multiple factors, including reactive oxygen species (ROS) generated either extracellularly by epithelial cells or intracellularly by NADPH oxidase (NOX). We, therefore, questioned whether ROS were responsible for TREM-1-mediated regulation of migration. Thioglycollate-elicited peritoneal neutrophils isolated from wild-type (WT) and TREM-1-deficient mice were stimulated with soluble and particulate agonists. Using electron paramagnetic resonance spectroscopy, we demonstrated that NOX2-dependent superoxide production is impaired in TREM-1-deficient neutrophils. Consistent with these findings, we confirmed with Clark electrode that TREM-1-deficient neutrophils consume less oxygen. Next, we demonstrated that TREM-1 deficient neutrophils have impaired directional migration to fMLP and zymosan-activated serum as compared to WT neutrophils and that deletion or inhibition of NOX2 in WT but not TREM-1-deficient neutrophils significantly impaired direction sensing. Finally, TREM-1 deficiency resulted in decreased protein kinase B (AKT) activation. Thus, TREM-1 regulates neutrophil migratory properties, in part, by promoting AKT activation and NOX2-dependent superoxide production. These findings provide the first mechanistic evidence as to how TREM-1 regulates neutrophil migration.

Identification of a Novel Splice Variant Isoform of TREM-1 in Human Neutrophil Granules.

Triggering receptor expressed on myeloid cells-1 (TREM-1) is critical for inflammatory signal amplification. Humans have two forms of TREM-1: a membrane receptor, associated with the adaptor DAP12, and a soluble receptor detected at times of infection. The membrane receptor isoform acts synergistically with the TLR pathway to promote cytokine secretion and neutrophil migration, whereas the soluble receptor functions as a counterregulatory molecule. In multiple models of sepsis, exogenous administration of soluble forms of TREM-1 attenuates inflammation and markedly improves survival. Despite intense interest in soluble TREM-1, both as a clinical predictor of survival and as a therapeutic tool, the origin of native soluble TREM-1 remains controversial. Using human neutrophils, we identified a 15-kDa TREM-1 isoform in primary (azurophilic) and secondary (specific) granules. Mass spectrometric analysis, ELISA, and immunoblot confirm that the 15-kDa protein is a novel splice variant form of TREM-1 (TREM-1sv). Neutrophil stimulation with Pseudomonas aeruginosa, LPS, or PAM(3)Cys4 resulted in degranulation and release of TREM-1sv. The addition of exogenous TREM-1sv inhibited TREM-1 receptor-mediated proinflammatory cytokine production. Thus, these data reveal that TREM-1 isoforms simultaneously activate and inhibit inflammation via the canonical membrane TREM-1 molecule and this newly discovered granular isoform, TREM-1sv.

Characterization of a novel mouse model with genetic deletion of CD177.

Neutrophils play an essential role in the innate immune response to infection. Neutrophils migrate from the vasculature into the tissue in response to infection. Recently, a neutrophil cell surface receptor, CD177, was shown to help mediate neutrophil migration across the endothelium through interactions with PECAM1. We examined a publicly available gene array dataset of CD177 expression from human neutrophils following pulmonary endotoxin instillation. Among all 22,214 genes examined, CD177 mRNA was the most upregulated following endotoxin exposure. The high level of CD177 expression is also maintained in airspace neutrophils, suggesting a potential involvement of CD177 in neutrophil infiltration under infectious diseases. To determine the role of CD177 in neutrophils in vivo, we constructed a CD177-genetic knockout mouse model. The mice with homozygous deletion of CD177 have no discernible phenotype and no significant change in immune cells, other than decreased neutrophil counts in peripheral blood. We examined the role of CD177 in neutrophil accumulation using a skin infection model with Staphylococcus aureus. CD177 deletion reduced neutrophil counts in inflammatory skin caused by S. aureus. Mechanistically we found that CD177 deletion in mouse neutrophils has no significant impact in CXCL1/KC- or fMLP-induced migration, but led to significant cell death. Herein we established a novel genetic mouse model to study the role of CD177 and found that CD177 plays an important role in neutrophils.

Transepithelial migration of neutrophils into the lung requires TREM-1.

Acute respiratory infections are responsible for more than 4 million deaths each year. Neutrophils play an essential role in the innate immune response to lung infection. These cells have an armamentarium of pattern recognition molecules and antimicrobial agents that identify and eliminate pathogens. In the setting of infection, neutrophil triggering receptor expressed on myeloid cells 1 (TREM-1) amplifies inflammatory signaling. Here we demonstrate for the first time that TREM-1 also plays an important role in transepithelial migration of neutrophils into the airspace. We developed a TREM-1/3-deficient mouse model of pneumonia and found that absence of TREM-1/3 markedly increased mortality following Pseudomonas aeruginosa challenge. Unexpectedly, TREM-1/3 deficiency resulted in increased local and systemic cytokine production. TREM-1/3-deficient neutrophils demonstrated intact bacterial killing, phagocytosis, and chemotaxis; however, histologic examination of TREM-1/3-deficient lungs revealed decreased neutrophil infiltration of the airways. TREM-1/3-deficient neutrophils effectively migrated across primary endothelial cell monolayers but failed to migrate across primary airway epithelia grown at the air-liquid interface. These data define a new function for TREM-1 in neutrophil migration across airway epithelial cells and suggest that it amplifies inflammation through targeted neutrophil migration into the lung.

Hepatitis C virus infection and hepatic stellate cell activation downregulate miR-29: miR-29 overexpression reduces hepatitis C viral abundance in culture.

BACKGROUND: Chronic hepatitis C virus (HCV)-induced liver fibrosis involves upregulation of transforming growth factor (TGF)-ß and subsequent hepatic stellate cell (HSC) activation. MicroRNAs (miRNAs) regulate HCV infection and HSC activation. METHODS: TaqMan miRNA profiling identified 12 miRNA families differentially expressed between chronically HCV-infected human livers and uninfected controls. To identify pathways affected by miRNAs, we developed a new algorithm (pathway analysis of conserved targets), based on the probability of conserved targeting. RESULTS: This analysis suggested a role for miR-29 during HCV infection. Of interest, miR-29 was downregulated in most HCV-infected patients. miR-29 regulates expression of extracellular matrix proteins. In culture, HCV infection downregulated miR-29, and miR-29 overexpression reduced HCV RNA abundance. miR-29 also appears to play a role in HSCs. Hepatocytes and HSCs contribute similar amounts of miR-29 to whole liver. Both activation of primary HSCs and TGF-ß treatment of immortalized HSCs downregulated miR-29. miR-29 overexpression in LX-2 cells decreased collagen expression and modestly decreased proliferation. miR-29 downregulation by HCV may derepress extracellular matrix synthesis during HSC activation. CONCLUSIONS: HCV infection downregulates miR-29 in hepatocytes and may potentiate collagen synthesis by reducing miR-29 levels in activated HSCs. Treatment with miR-29 mimics in vivo might inhibit HCV while reducing fibrosis.

Correlation between microRNA expression levels and clinical parameters associated with chronic hepatitis C viral infection in humans.

MicroRNAs (miRNAs) are small RNAs that regulate gene expression pathways. Previous studies have shown interactions between hepatitis C virus (HCV) and host miRNAs. We measured miR-122 and miR-21 levels in HCV-infected human liver biopsies relative to uninfected human livers and correlated these with clinical patient data. miR-122 is required for HCV replication in vitro, and miR-21 is involved in cellular proliferation and tumorigenesis. We found that miR-21 expression correlated with viral load, fibrosis and serum liver transaminase levels. miR-122 expression inversely correlated with fibrosis, liver transaminase levels and patient age. miR-21 was induced ∼twofold, and miR-122 was downregulated on infection of cultured cells with the HCV J6/JFH infectious clone, thus establishing a link to HCV. To further examine the relationship between fibrosis and the levels of miR-21 and miR-122, we measured their expression levels in a mouse carbon tetrachloride fibrosis model. As in the HCV-infected patient samples, fibrotic stage positively correlated with miR-21 and negatively correlated with miR-122 levels. Transforming growth factor ß (TGF-ß) is a critical mediator of fibrogenesis. We identified SMAD7 as a novel miR-21 target. SMAD7 is a negative regulator of TGF-ß signaling, and its expression is induced by TGF-ß. To confirm the relationship between miR-21 and the TGF-ß signaling pathway, we measured the effect of miR-21 on a TGF-ß-responsive reporter. We found that miR-21 enhanced TGF-ß signaling, further supporting a relationship between miR-21 and fibrosis. We suggest a model in which miR-21 targeting of SMAD7 could increase TGF-ß signaling, leading to increased fibrogenesis.

MicroRNA-21 is upregulated during the proliferative phase of liver regeneration, targets Pellino-1, and inhibits NF-kappaB signaling.

During liver regeneration, normally quiescent liver cells reenter the cell cycle, nonparenchymal and parenchymal cells divide, and proper liver architecture is restored. The gene expression programs regulating these transitions are not completely understood. MicroRNAs are a newly discovered class of small regulatory RNAs that silence messenger RNAs by binding to their 3'-untranslated regions (UTRs). A number of microRNAs, including miR-21, have been shown to be involved in regulation of cell proliferation. We performed partial hepatectomies on mice and allowed the liver to regenerate for 1, 6, 12, 24, and 48 h and 4 and 7 days. We compared the expression of miR-21 in the posthepatectomy liver to the prehepatectomy liver by Northern blot and found that miR-21 was upregulated during the early stages of liver regeneration. NF-kappaB signaling is also activated very early during liver regeneration. It has been previously reported that NF-kappaB upregulates the miR-21 precursor transcript. The predicted miR-21 target, Pellino (Peli1), is a ubiquitin ligase involved in activating NF-kappaB signaling. We observed an inverse correlation between miR-21 and Peli1 mRNA levels during liver regeneration. miR-21 overexpression in cultured cells inhibited a Peli1 3'-UTR luciferase reporter. Using NF-kappaB reporter assays, we determined that miR-21 overexpression inhibits NF-kappaB signaling. In conclusion, miR-21 expression was upregulated during early stages of liver regeneration. Targeting of Peli1 by miR-21 could potentially provide the basis for a negative feedback cycle regulating NF-kappaB signaling.

Zinc-finger nucleases as a novel therapeutic strategy for targeting hepatitis B virus DNAs.

Hepatitis B virus (HBV) chronically infects 350-400 million people worldwide and causes >1 million deaths yearly. Current therapies prevent new viral genome formation, but do not target pre-existing viral genomic DNA, thus curing only approximately 1/2 of patients. We targeted HBV DNA for cleavage using zinc-finger nucleases (ZFNs), which cleave as dimers. Co-transfection of our ZFN pair with a target plasmid containing the HBV genome resulted in specific cleavage. After 3 days in culture, 26% of the target remained linear, whereas approximately10% was cleaved and misjoined tail-to-tail. Notably, ZFN treatment decreased levels of the hepatitis C virus pregenomic RNA by 29%. A portion of cleaved plasmids are repaired in cells, often with deletions and insertions. To track misrepair, we introduced an XbaI restriction site in the spacer between the ZFN sites. Targeted cleavage and misrepair destroys the XbaI site. After 3 days in culture, approximately 6% of plasmids were XbaI-resistant. Thirteen of 16 clones sequenced contained frameshift mutations that would lead to truncations of the viral core protein. These results demonstrate, for the first time, the possibility of targeting episomal viral DNA genomes using ZFNs.

Rational design leads to more potent RNA interference against hepatitis B virus: factors effecting silencing efficiency.

RNA interference (RNAi) can be an effective antiviral agent; however, overexpression of RNAi can be toxic through competition with the endogenous microRNA (miRNA) machinery. We used rational design to identify highly potent RNAi that is effective at nontoxic doses. A statistical analysis was conducted to pinpoint thermodynamic characteristics correlated with activity. Sequences were selected that conformed to a consensus internal stability profile (ISP) associated with active RNAi, and RNAi triggers were expressed in the context of an endogenous miRNA. These approaches yielded highly active hepatitis B virus (HBV) RNAi. A statistical analysis found a correlation between activity and nucleation by binding within the seed sequence to accessible regions in the target RNA. Guide strands were selected for favorable strand biasing, but increased strand biasing did not correlate with potency, suggesting a threshold effect. Exogenous short hairpin RNAs (shRNAs), but not miRNAs were previously reported to compete with miRNAs for the miRNA/RNAi machinery. In contrast, we show that exogenous Polymerase III- but not Polymerase II-driven miRNAs compete with exogenous miRNAs, at multiple steps in the miRNA pathway. Exogenous miRNAs also compete with endogenous miR-21. Thus, competition with endogenous miRNAs should be monitored even when using miRNA-based therapeutics. However, potent silencing was achieved at doses where competition was not observed.