Comprehensive Assessment of the Intrinsic Pancreatic Microbiome.

OBJECTIVE: We sought to comprehensively profile tissue and cyst fluid in patients with benign, precancerous, and cancerous conditions of the pancreas to characterize the intrinsic pancreatic microbiome. SUMMARY BACKGROUND DATA: Small studies in pancreatic ductal adenocarcinoma (PDAC) and intraductal papillary mucinous neoplasm (IPMN) have suggested that intra-pancreatic microbial dysbiosis may drive malignant transformation. METHODS: Pancreatic samples were collected at the time of resection from 109 patients. Samples included tumor tissue (control, n=20; IPMN, n=20; PDAC, n=19) and pancreatic cyst fluid (IPMN, n=30; SCA, n=10; MCN, n=10). Assessment of bacterial DNA by quantitative PCR and 16S ribosomal RNA gene sequencing was performed. Downstream analyses determined the relative abundances of individual taxa between groups and compared intergroup diversity. Whole-genome sequencing data from 140 patients with PDAC in the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium (CPTAC) were analyzed to validate findings. RESULTS: Sequencing of pancreatic tissue yielded few microbial reads regardless of diagnosis, and analysis of pancreatic tissue showed no difference in the abundance and composition of bacterial taxa between normal pancreas, IPMN, or PDAC groups. Low-grade dysplasia (LGD) and high-grade dysplasia (HGD) IPMN were characterized by low bacterial abundances with no difference in tissue composition and a slight increase in Pseudomonas and Sediminibacterium in HGD cyst fluid. Decontamination analysis using the CPTAC database confirmed a low-biomass, low-diversity intrinsic pancreatic microbiome that did not differ by pathology. CONCLUSIONS: Our analysis of the pancreatic microbiome demonstrated very low intrinsic biomass that is relatively conserved across diverse neoplastic conditions and thus unlikely to drive malignant transformation.

Multidimensional Immunophenotyping of Intraductal Papillary Mucinous Neoplasms Reveals Novel T Cell and Macrophage Signature.

BACKGROUND: Intraductal papillary mucinous neoplasms (IPMN) are the only radiographically identifiable precursor to pancreatic adenocarcinoma, yet little is known about how these lesions progress to cancer. Inflammation has been associated with dysplastic progression; however, the cause and composition of this inflammation remains poorly characterized. We sought to comprehensively profile immune cell infiltration using parallel spatial transcriptomic and flow cytometric techniques. METHODS: Twelve patients with resected IPMN exhibiting both high-grade dysplasia (HGD) and low-grade dysplasia (LGD) were selected for spatial transcriptomics (NanoString GeoMx). Immune (CD45+), epithelial (PanCK+), and stromal (SMA+) compartments were analyzed separately using the GeoMx NGS Pipeline. An additional 11 patients resected for IPMN of varying degrees of dysplasia underwent immunophenotyping using flow cytometry (DURAClone IM). RESULTS: Spatial transcriptomics revealed that T cells represent the dominant immune cell within IPMN stroma, which was confirmed by flow cytometry (56%). Spatial profiling found that the T-cell infiltrate was significantly higher in regions of LGD compared with HGD (62% vs. 50%, p = 0.038). Macrophages were the only other immune cell type with > 10% abundance, yet conversely, were generally more abundant in regions of HGD compared to LGD (19% vs. 11%, p = 0.058). Correspondingly, immune cells within regions of HGD demonstrated transcriptional upregulation of genes associated with macrophage activity including secretion (CXCL1) and phagocytosis (C1QA, C1S, C4B). CONCLUSIONS: IPMN immune infiltrate is primarily composed of T cells and macrophages. Regions of HGD appear to be relatively deplete of T cells and show a trend toward macrophage enrichment compared with regions of LGD.

In vivo assessment of respiratory burst inhibition by xenobiotic exposure using larval zebrafish.

Currently, assessment of the potential immunotoxicity of a given agent involves a tiered approach for hazard identification and mechanistic studies, including observational studies, evaluation of immune function, and measurement of susceptibility to infectious and neoplastic diseases. These studies generally use costly low-throughput mammalian models. Zebrafish, however, offer an excellent alternative due to their rapid development, ease of maintenance, and homology to mammalian immune system function and development. Larval zebrafish also are a convenient model to study the innate immune system with no interference from the adaptive immune system. In this study, a respiratory burst assay (RBA) was utilized to measure reactive oxygen species (ROS) production after developmental xenobiotic exposure. Embryos were exposed to non-teratogenic doses of chemicals and at 96 h post-fertilization, the ability to produce ROS was measured. Using the RBA, 12 compounds with varying immune-suppressive properties were screened. Seven compounds neither suppressed nor enhanced the respiratory burst; five reproducibly suppressed global ROS production, but with varying potencies: benzo[a]pyrene, 17ß-estradiol, lead acetate, methoxychlor, and phenanthrene. These five compounds have all previously been reported as immunosuppressive in mammalian innate immunity assays. To evaluate whether the suppression of ROS by these compounds was a result of decreased immune cell numbers, flow cytometry with transgenic zebrafish larvae was used to count the numbers of neutrophils and macrophages after chemical exposure. With this assay, benzo[a]pyrene was found to be the only chemical that induced a change in the number of immune cells by increasing macrophage but not neutrophil numbers. Taken together, this work demonstrates the utility of zebrafish larvae as a vertebrate model for identifying compounds that impact innate immune function at non-teratogenic levels and validates measuring ROS production and phagocyte numbers as metrics for monitoring how xenobiotic exposure alters the innate immune system.

Disruption of Trim9 function abrogates macrophage motility in vivo.

The vertebrate immune response comprises multiple molecular and cellular components that interface to provide defense against pathogens. Because of the dynamic complexity of the immune system and its interdependent innate and adaptive functionality, an understanding of the whole-organism response to pathogen exposure remains unresolved. Zebrafish larvae provide a unique model for overcoming this obstacle, because larvae are protected against pathogens while lacking a functional adaptive immune system during the first few weeks of life. Zebrafish larvae were exposed to immune agonists for various lengths of time, and a microarray transcriptome analysis was executed. This strategy identified known immune response genes, as well as genes with unknown immune function, including the E3 ubiquitin ligase tripartite motif-9 (Trim9). Although trim9 expression was originally described as "brain specific," its expression has been reported in stimulated human Mϕs. In this study, we found elevated levels of trim9 transcripts in vivo in zebrafish Mϕs after immune stimulation. Trim9 has been implicated in axonal migration, and we therefore investigated the impact of Trim9 disruption on Mϕ motility and found that Mϕ chemotaxis and cellular architecture are subsequently impaired in vivo. These results demonstrate that Trim9 mediates cellular movement and migration in Mϕs as well as neurons.

Neutrophil transfer of miR-223 to lung epithelial cells dampens acute lung injury in mice.

Intercellular transfer of microRNAs can mediate communication between critical effector cells. We hypothesized that transfer of neutrophil-derived microRNAs to pulmonary epithelial cells could alter mucosal gene expression during acute lung injury. Pulmonary-epithelial microRNA profiling during coculture of alveolar epithelial cells with polymorphonuclear neutrophils (PMNs) revealed a selective increase in lung epithelial cell expression of microRNA-223 (miR-223). Analysis of PMN-derived supernatants showed activation-dependent release of miR-223 and subsequent transfer to alveolar epithelial cells during coculture in vitro or after ventilator-induced acute lung injury in mice. Genetic studies indicated that miR-223 deficiency was associated with severe lung inflammation, whereas pulmonary overexpression of miR-223 in mice resulted in protection during acute lung injury induced by mechanical ventilation or by infection with Staphylococcus aureus Studies of putative miR-223 gene targets implicated repression of poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) in the miR-223-dependent attenuation of lung inflammation. Together, these findings suggest that intercellular transfer of miR-223 from neutrophils to pulmonary epithelial cells may dampen acute lung injury through repression of PARP-1.

Mucins and Their Sugars. Critical Mediators of Hyperreactivity and Inflammation.

Excessive mucus causes severe airflow obstruction in fatal asthma. It is also present in mild to moderate disease, but is poorly understood and treated. Mucus overproduction is associated with dysregulated expression of the mucins MUC5AC and MUC5B. Whereas increased MUC5AC is a consistent finding, MUC5B varies-remaining stably produced in some patients but strongly repressed in others (>90%). Patients with lower MUC5B display worsened asthma phenotypes including airway hyperreactivity (AHR) to methacholine (MCh) and eosinophilic inflammation. To better understand the roles of mucins in asthma, we generated Muc5ac and Muc5b knockout ((-/-)) mice. AHR to MCh was abolished in antigen-challenged Muc5ac(-/-) mice, due to prevention of heterogeneous mucous plugging that occurred in allergic wild-type mice during MCh-induced bronchoconstriction. Thus, in addition to the established role of smooth muscle-mediated airway narrowing, Muc5ac is an essential noncontractile AHR component. We also found that, unlike Muc5ac(-/-) mice, Muc5b-deficient mice were not protected from asthma phenotypes. Furthermore, whereas inflammation was unaffected by Muc5ac deficiency, it was exaggerated in the absence of Muc5b. On the basis of these differential effects, we are now determining how asthma phenotypes are regulated by mucin isoform specificity. Glycosylation is dramatically different: Muc5ac is heavily fucosylated whereas Muc5b is mainly sialylated. Fucosylation increases mucus viscoelasticity, and FUT2, the enzyme that catalyzes mucin α1,2-fucosylation, is associated with severe asthma exacerbation risk. Sialylation is required for binding to siglec (sialic acid-binding immunoglobulin-like lectin) receptors on leukocytes. Eosinophils express Siglec-F (mouse) or Siglec-8 (human). Engagement by sialoside ligands induces eosinophil apoptosis, and Muc5b via sialylated termini that require the α2,3-sialyltransferase ST3Gal3 for synthesis binds Siglec-F and induces apoptosis in mouse eosinophils. Because Muc5b is required for host defense in mouse lungs, inhibiting MUC5AC while preserving or enhancing MUC5B functions may be effective for treating asthma.

The polymeric mucin Muc5ac is required for allergic airway hyperreactivity.

In asthma, airflow obstruction is thought to result primarily from inflammation-triggered airway smooth muscle (ASM) contraction. However, anti-inflammatory and smooth muscle-relaxing treatments are often temporary or ineffective. Overproduction of the mucin MUC5AC is an additional disease feature that, while strongly associated pathologically, is poorly understood functionally. Here we show that Muc5ac is a central effector of allergic inflammation that is required for airway hyperreactivity (AHR) to methacholine (MCh). In mice bred on two well-characterized strain backgrounds (C57BL/6 and BALB/c) and exposed to two separate allergic stimuli (ovalbumin and Aspergillus extract), genetic removal of Muc5ac abolishes AHR. Residual MCh responses are identical to unchallenged controls, and although inflammation remains intact, heterogeneous mucous occlusion decreases by 74%. Thus, whereas inflammatory effects on ASM alone are insufficient for AHR, Muc5ac-mediated plugging is an essential mechanism. Inhibiting MUC5AC may be effective for treating asthma and other lung diseases where it is also overproduced.

Antiinflammatory and Antimicrobial Effects of Thiocyanate in a Cystic Fibrosis Mouse Model.

Thiocyanate (SCN) is used by the innate immune system, but less is known about its impact on inflammation and oxidative stress. Granulocytes oxidize SCN to evolve the bactericidal hypothiocyanous acid, which we previously demonstrated is metabolized by mammalian, but not bacterial, thioredoxin reductase (TrxR). There is also evidence that SCN is dysregulated in cystic fibrosis (CF), a disease marked by chronic infection and airway inflammation. To investigate antiinflammatory effects of SCN, we administered nebulized SCN or saline to ß epithelial sodium channel (ßENaC) mice, a phenotypic CF model. SCN significantly decreased airway neutrophil infiltrate and restored the redox ratio of glutathione in lung tissue and airway epithelial lining fluid to levels comparable to wild type. Furthermore, in Pseudomonas aeruginosa-infected ßENaC and wild-type mice, SCN decreased inflammation, proinflammatory cytokines, and bacterial load. SCN also decreased airway neutrophil chemokine keratinocyte chemoattractant (also known as C-X-C motif chemokine ligand 1) and glutathione sulfonamide, a biomarker of granulocyte oxidative activity, in uninfected ßENaC mice. Lung tissue TrxR activity and expression increased in inflamed lung tissue, providing in vivo evidence for the link between hypothiocyanous acid metabolism by TrxR and the promotion of selective biocide of pathogens. SCN treatment both suppressed inflammation and improved host defense, suggesting that nebulized SCN may have important therapeutic utility in diseases of both chronic airway inflammation and persistent bacterial infection, such as CF.

Muc5b is required for airway defence.

Respiratory surfaces are exposed to billions of particulates and pathogens daily. A protective mucus barrier traps and eliminates them through mucociliary clearance (MCC). However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus. Genetic variants are linked to diverse lung diseases, but specific roles for MUC5AC and MUC5B in MCC, and the lasting effects of their inhibition, are unknown. Here we show that mouse Muc5b (but not Muc5ac) is required for MCC, for controlling infections in the airways and middle ear, and for maintaining immune homeostasis in mouse lungs, whereas Muc5ac is dispensable. Muc5b deficiency caused materials to accumulate in upper and lower airways. This defect led to chronic infection by multiple bacterial species, including Staphylococcus aureus, and to inflammation that failed to resolve normally. Apoptotic macrophages accumulated, phagocytosis was impaired, and interleukin-23 (IL-23) production was reduced in Muc5b(-/-) mice. By contrast, in mice that transgenically overexpress Muc5b, macrophage functions improved. Existing dogma defines mucous phenotypes in asthma and chronic obstructive pulmonary disease (COPD) as driven by increased MUC5AC, with MUC5B levels either unaffected or increased in expectorated sputum. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%. By distinguishing a specific role for Muc5b in MCC, and by determining its impact on bacterial infections and inflammation in mice, our results provide a refined framework for designing targeted therapies to control mucin secretion and restore MCC.