Capillary-based, multifunctional manipulation of particles and fluids via focused surface acoustic waves.

Surface acoustic wave (SAW)-enabled acoustofluidic technologies have recently atttracted increasing attention for applications in biology, chemistry, biophysics, and medicine. Most SAW acoustofluidic devices generate acoustic energy which is then transmitted into custom microfabricated polymer-based channels. There are limited studies on delivering this acoustic energy into convenient commercially-available glass tubes for manipulating particles and fluids. Herein, we have constructed a capillary-based SAW acoustofluidic device for multifunctional fluidic and particle manipulation. This device integrates a converging interdigitated transducer to generate focused SAWs on a piezoelectric chip, as well as a glass capillary that transports particles and fluids. To understand the actuation mechanisms underlying this device, we performed finite element simulations by considering piezoelectric, solid mechanic, and pressure acoustic physics. This experimental study shows that the capillary-based SAW acoustofluidic device can perform multiple functions including enriching particles, patterning particles, transporting particles and fluids, as well as generating droplets with controlled sizes. Given the usefulness of these functions, we expect that this acoustofluidic device can be useful in applications such as pharmaceutical manufacturing, biofabrication, and bioanalysis.

Eosinophil Specialization Is Regulated by Exposure to the Esophageal Epithelial Microenvironment.

Distinct subsets of eosinophils are reported in inflammatory and healthy tissues, yet the functions of uniquely specialized eosinophils and the signals that elicit them, particularly in eosinophilic esophagitis (EoE), are not well understood. Herein, we report an ex-vivo system wherein freshly isolated human eosinophils were cocultured with esophageal epithelial cells and disease-relevant pro-inflammatory (IL-13) or pro-fibrotic (TGF-ß) cytokines. Compared with untreated cocultures, IL-13 increased expression of CD69 on eosinophils, whereas TGF-ß increased expression of CD81, CD62L, and CD25. Eosinophils from IL-13-treated cocultures demonstrated increased secretion of GRO-α, IL-8, and M-CSF and also generated increased extracellular peroxidase activity following activation. Eosinophils from TGF-ß-treated cocultures secreted increased IL-6 and exhibited increased chemotactic response to CCL11 compared with eosinophils from untreated or IL-13-treated coculture conditions. When eosinophils from TGF-ß-treated cocultures were cultured with fibroblasts, they upregulated SERPINE1 expression and fibronectin secretion by fibroblasts compared with eosinophils that were cultured with GM-CSF, alone. Translational studies revealed that CD62L was heterogeneously expressed by eosinophils in patient biopsies. Our results demonstrate that disease-relevant pro-inflammatory and pro-fibrotic signals present in the esophagus of EoE patients cause distinct profiles of eosinophil activation and gene expression.

Acoustic separation and concentration of exosomes for nucleotide detection: ASCENDx.

Efficient isolation and analysis of exosomal biomarkers hold transformative potential in biomedical applications. However, current methods are prone to contamination and require costly consumables, expensive equipment, and skilled personnel. Here, we introduce an innovative spaceship-like disc that allows Acoustic Separation and Concentration of Exosomes and Nucleotide Detection: ASCENDx. We created ASCENDx to use acoustically driven disc rotation on a spinning droplet to generate swift separation and concentration of exosomes from patient plasma samples. Integrated plasmonic nanostars on the ASCENDx disc enable label-free detection of enriched exosomes via surface-enhanced Raman scattering. Direct detection of circulating exosomal microRNA biomarkers from patient plasma samples by the ASCENDx platform facilitated a diagnostic assay for colorectal cancer with 95.8% sensitivity and 100% specificity. ASCENDx overcomes existing limitations in exosome-based molecular diagnostics and holds a powerful position for future biomedical research, precision medicine, and point-of-care medical diagnostics.

Aryl hydrocarbon receptor and IL-13 signaling crosstalk in human keratinocytes and atopic dermatitis.

Introduction: Atopic dermatitis (AD) is an allergic skin disease mediated by skin barrier impairment and IL-13-driven immune response. Activation of the aryl hydrocarbon receptor (AHR) has shown promise in early clinical trials for AD; however, the mechanism by which AHR partially ameliorates AD is not well known. Methods: Gene expression data from human biopsies were analyzed, and compared to gene expression from RNA-sequencing in our in-vitro HaCaT cell model system. Western blot, ELISA qRT-PCR were used to further explore the relationship between AHR and IL-13 signaling in HaCaT cells. Results: The AHR target gene CYP1A1 was decreased in lesional skin compared with healthy control skin (p = 4.30 × 10-9). Single-cell RNA sequencing (scRNAseq) demonstrated increased AHR expression (p < 1.0 × 10-4) and decreased CYP1A1 expression in lesional AD keratinocytes compared with healthy control keratinocytes (p < 0.001). Activation of AHR by AHR agonists in HaCaT cells reversed IL-13-dependent gene expression of several key genes in AD pathogenesis, most notably the eosinophil chemoattractant CCL26 (eotaxin-3). Differentially expressed genes in keratinocytes of patients with AD substantially overlapped with genes regulated by AHR agonists from HaCaT cells by RNAseq, but in reverse direction. Mechanistically, there was evidence for direct transcriptional effects of AHR; AHR binding motifs were identified in the differentially expressed genes from lesional AD keratinocytes compared to control keratinocytes, and AHR activation did not modify IL-13-dependent signal transducer and activator of transcription 6 (STAT6) translocation to the nucleus. Discussion: Together, these data suggest that the AHR pathway is dysregulated in AD and that AHR modulates IL-13 downstream signaling in keratinocytes through genome-wide, transcriptional regulatory effects.

A TLR4/TRAF6-dependent signaling pathway mediates NCoR coactivator complex formation for inflammatory gene activation.

The nuclear receptor corepressor (NCoR) forms a complex with histone deacetylase 3 (HDAC3) that mediates repressive functions of unliganded nuclear receptors and other transcriptional repressors by deacetylation of histone substrates. Recent studies provide evidence that NCoR/HDAC3 complexes can also exert coactivator functions in brown adipocytes by deacetylating and activating PPARγ coactivator 1α (PGC1α) and that signaling via receptor activator of nuclear factor kappa-B (RANK) promotes the formation of a stable NCoR/HDAC3/PGC1ß complex that coactivates nuclear factor kappa-B (NFκB)- and activator protein 1 (AP-1)-dependent genes required for osteoclast differentiation. Here, we demonstrate that activation of Toll-like receptor (TLR) 4, but not TLR3, the interleukin 4 (IL4) receptor nor the Type I interferon receptor, also promotes assembly of an NCoR/HDAC3/PGC1ß coactivator complex. Receptor-specific utilization of TNF receptor-associated factor 6 (TRAF6) and downstream activation of extracellular signal-regulated kinase 1 (ERK1) and TANK-binding kinase 1 (TBK1) accounts for the common ability of RANK and TLR4 to drive assembly of an NCoR/HDAC3/PGC1ß complex in macrophages. ERK1, the p65 component of NFκB, and the p300 histone acetyltransferase (HAT) are also components of the induced complex and are associated with local histone acetylation and transcriptional activation of TLR4-dependent enhancers and promoters. These observations identify a TLR4/TRAF6-dependent signaling pathway that converts NCoR from a corepressor of nuclear receptors to a coactivator of NFκB and AP-1 that may be relevant to functions of NCoR in other developmental and homeostatic processes.

Kruppel-like factor 2+ CD4 T cells avert microbiota-induced intestinal inflammation.

Intestinal colonization by antigenically foreign microbes necessitates expanded peripheral immune tolerance. Here we show commensal microbiota prime expansion of CD4 T cells unified by the Kruppel-like factor 2 (KLF2) transcriptional regulator and an essential role for KLF2+ CD4 cells in averting microbiota-driven intestinal inflammation. CD4 cells with commensal specificity in secondary lymphoid organs and intestinal tissues are enriched for KLF2 expression, and distinct from FOXP3+ regulatory T cells or other differentiation lineages. Mice with conditional KLF2 deficiency in T cells develop spontaneous rectal prolapse and intestinal inflammation, phenotypes overturned by eliminating microbiota or reconstituting with donor KLF2+ cells. Activated KLF2+ cells selectively produce IL-10, and eliminating IL-10 overrides their suppressive function in vitro and protection against intestinal inflammation in vivo. Together with reduced KLF2+ CD4 cell accumulation in Crohn's disease, a necessity for the KLF2+ subpopulation of T regulatory type 1 (Tr1) cells in sustaining commensal tolerance is demonstrated.

Discrimination of cell-intrinsic and environment-dependent effects of natural genetic variation on Kupffer cell epigenomes and transcriptomes.

Noncoding genetic variation drives phenotypic diversity, but underlying mechanisms and affected cell types are incompletely understood. Here, investigation of effects of natural genetic variation on the epigenomes and transcriptomes of Kupffer cells derived from inbred mouse strains identified strain-specific environmental factors influencing Kupffer cell phenotypes, including leptin signaling in Kupffer cells from a steatohepatitis-resistant strain. Cell-autonomous and non-cell-autonomous effects of genetic variation were resolved by analysis of F1 hybrid mice and cells engrafted into an immunodeficient host. During homeostasis, non-cell-autonomous trans effects of genetic variation dominated control of Kupffer cells, while strain-specific responses to acute lipopolysaccharide injection were dominated by actions of cis-acting effects modifying response elements for lineage-determining and signal-dependent transcription factors. These findings demonstrate that epigenetic landscapes report on trans effects of genetic variation and serve as a resource for deeper analyses into genetic control of transcription in Kupffer cells and macrophages in vitro.

Characterization of Head Acceleration Exposure During Youth Football Practice Drills.

Many head acceleration events (HAEs) observed in youth football emanate from a practice environment. This study aimed to evaluate HAEs in youth football practice drills using a mouthpiece-based sensor, differentiating between inertial and direct HAEs. Head acceleration data were collected from athletes participating on 2 youth football teams (ages 11-13 y) using an instrumented mouthpiece-based sensor during all practice sessions in a single season. Video was recorded and analyzed to verify and assign HAEs to specific practice drill characteristics, including drill intensity, drill classification, and drill type. HAEs were quantified in terms of HAEs per athlete per minute and peak linear and rotational acceleration and rotational velocity. Mixed-effects models were used to evaluate the differences in kinematics, and generalized linear models were used to assess differences in HAE frequency between drill categories. A total of 3237 HAEs were verified and evaluated from 29 football athletes enrolled in this study. Head kinematics varied significantly between drill categorizations. HAEs collected at higher intensities resulted in significantly greater kinematics than lower-intensity drills. The results of this study add to the growing body of evidence informing evidence-based strategies to reduce head impact exposure and concussion risk in youth football practices.

Expansion of interferon inducible gene pool via USP18 inhibition promotes cancer cell pyroptosis.

While immunotherapy has emerged as a breakthrough cancer therapy, it is only effective in some patients, indicating the need of alternative therapeutic strategies. Induction of cancer immunogenic cell death (ICD) is one promising way to elicit potent adaptive immune responses against tumor-associated antigens. Type I interferon (IFN) is well known to play important roles in different aspects of immune responses, including modulating ICD in anti-tumor action. However, how to expand IFN effect in promoting ICD responses has not been addressed. Here we show that depletion of ubiquitin specific protease 18 (USP18), a negative regulator of IFN signaling, selectively induces cancer cell ICD. Lower USP18 expression correlates with better survival across human selected cancer types and delays cancer progression in mouse models. Mechanistically, nuclear USP18 controls the enhancer landscape of cancer cells and diminishes STAT2-mediated transcription complex binding to IFN-responsive elements. Consequently, USP18 suppression not only enhances expression of canonical IFN-stimulated genes (ISGs), but also activates the expression of a set of atypical ISGs and NF-κB target genes, including genes such as Polo like kinase 2 (PLK2), that induce cancer pyroptosis. These findings may support the use of targeting USP18 as a potential cancer immunotherapy.

Breaking down the complex pathophysiology of eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a chronic and progressive immune-mediated disease of the esophagus associated with antigen-driven type 2 inflammation and symptoms of esophageal dysfunction. Our understanding of EoE pathophysiology has evolved since its initial recognition more than 20 years ago and has translated into diagnostic and novel therapeutic approaches that are affecting patient care. The mechanisms underlying disease development and progression are influenced by diverse factors, such as genetics, age, allergic comorbidities, and allergen exposures. Central to EoE pathophysiology is a dysregulated feed-forward cycle that develops between the esophageal epithelium and the immune system. Allergen-induced, type 2-biased immune activation by the esophageal epithelium propagates a cycle of impaired mucosal barrier integrity and allergic inflammation, eventually leading to tissue remodeling and progressive organ dysfunction. Herein, we review the current understanding of fundamental pathophysiological mechanisms contributing to EoE pathogenesis.

Characterizing Exposure to Head Acceleration Events in Youth Football Using an Instrumented Mouthpiece.

Understanding characteristics of head acceleration events (HAEs) in youth football is vital in developing strategies to improve athlete safety. This study aimed to characterize HAEs in youth football using an instrumented mouthpiece. Youth football athletes (ages 11-13) participating on two teams were enrolled in this study for one season. Each athlete was instrumented with a mouthpiece-based sensor throughout the season. HAEs were verified on film to ensure that mouthpiece-based sensors triggered during contact. The number of HAEs, peak resultant linear and rotational accelerations, and peak resultant rotational velocity were quantified. Mixed effects models were used to evaluate differences in mean kinematic metrics among all HAEs for session type, athlete position, and contact surface. A total of 5,292 HAEs were collected and evaluated from 30 athletes. The median (95th percentile) peak resultant linear acceleration, rotational acceleration, and rotational velocity was 9.5 g (27.0 g), 666.4 rad s-2 (1863.3 rad s-2), and 8.5 rad s-1 (17.4 rad s-1), respectively. Athletes experienced six (22) HAEs per athlete per session (i.e., practice, game). Competition had a significantly higher mean number of HAEs per athlete per session and mean peak rotational acceleration. Peak resultant rotational kinematics varied significantly among athlete positions. Direct head impacts had higher mean kinematics compared to indirect HAEs, from body collisions. The results of this study demonstrate that session type, athlete position, and contact surface (i.e., direct, indirect) may influence HAE exposure in youth football.

Integrating basic and applied research to estimate carnivore abundance.

A clear connection between basic research and applied management is often missing or difficult to discern. We present a case study of integration of basic research with applied management for estimating abundance of gray wolves (Canis lupus) in Montana, USA. Estimating wolf abundance is a key component of wolf management but is costly and time intensive as wolf populations continue to grow. We developed a multimodel approach using an occupancy model, mechanistic territory model, and empirical group size model to improve abundance estimates while reducing monitoring effort. Whereas field-based wolf counts generally rely on costly, difficult-to-collect monitoring data, especially for larger areas or population sizes, our approach efficiently uses readily available wolf observation data and introduces models focused on biological mechanisms underlying territorial and social behavior. In a three-part process, the occupancy model first estimates the extent of wolf distribution in Montana, based on environmental covariates and wolf observations. The spatially explicit mechanistic territory model predicts territory sizes using simple behavioral rules and data on prey resources, terrain ruggedness, and human density. Together, these models predict the number of packs. An empirical pack size model based on 14 years of data demonstrates that pack sizes are positively related to local densities of packs, and negatively related to terrain ruggedness, local mortalities, and intensity of harvest management. Total abundance estimates for given areas are derived by combining estimated numbers of packs and pack sizes. We estimated the Montana wolf population to be smallest in the first year of our study, with 91 packs and 654 wolves in 2007, followed by a population peak in 2011 with 1252 wolves. The population declined ~6% thereafter, coincident with implementation of legal harvest in Montana. Recent numbers have largely stabilized at an average of 191 packs and 1141 wolves from 2016 to 2020. This new approach accounts for biologically based, spatially explicit predictions of behavior to provide more accurate estimates of carnivore abundance at finer spatial scales. By integrating basic and applied research, our approach can therefore better inform decision-making and meet management needs.

Exploiting dynamic enhancer landscapes to decode macrophage and microglia phenotypes in health and disease.

The development and functional potential of metazoan cells is dependent on combinatorial roles of transcriptional enhancers and promoters. Macrophages provide exceptionally powerful model systems for investigation of mechanisms underlying the activation of cell-specific enhancers that drive transitions in cell fate and cell state. Here, we review recent advances that have expanded appreciation of the diversity of macrophage phenotypes in health and disease, emphasizing studies of liver, adipose tissue, and brain macrophages as paradigms for other tissue macrophages and cell types. Studies of normal tissue-resident macrophages and macrophages associated with cirrhosis, obese adipose tissue, and neurodegenerative disease illustrate the major roles of tissue environment in remodeling enhancer landscapes to specify the development and functions of distinct macrophage phenotypes. We discuss the utility of quantitative analysis of environment-dependent changes in enhancer activity states as an approach to discovery of regulatory transcription factors and upstream signaling pathways.

Purification of mouse hepatic non-parenchymal cells or nuclei for use in ChIP-seq and other next-generation sequencing approaches.

Significant advancements in understanding disease mechanisms can occur through combined analysis of next-generation sequencing datasets generated using purified cell populations. Here, we detail our optimized protocol for purification of mouse hepatic macrophages (or other liver non-parenchymal populations) suitable for use in various next-generation sequencing protocols. An alternative framework is described for sorting pre-fixed hepatic nuclei populations. This strategy has the advantage of rapidly preserving the nuclei and can facilitate success with ChIP-seq for more challenging molecules. For complete details on the use and execution of these protocols, please refer to Muse et al. (2018), Sakai et al. (2019), and Seidman et al. (2020).

An optimized protocol for rapid, sensitive and robust on-bead ChIP-seq from primary cells.

Integrative analysis of next-generation sequencing data can help understand disease mechanisms. Specifically, ChIP-seq can illuminate where transcription regulators bind to regulate transcription. A major obstacle to performing this assay on primary cells is the low numbers obtained from tissues. The extensively validated ChIP-seq protocol presented here uses small volumes and single-pot on-bead library preparation to generate diverse high-quality ChIP-seq data. This protocol allows for medium-to-high-throughput ChIP-seq of low-abundance cells and can also be applied to other mammalian cells. For complete details on the use and execution of this protocol, please refer to Brigidi et al. (2019), Carlin et al. (2018), Heinz et al. (2018), Nott et al. (2019), Sakai et al. (2019), and Seidman et al. (2020).

Evidence of economical territory selection in a cooperative carnivore.

As an outcome of natural selection, animals are probably adapted to select territories economically by maximizing benefits and minimizing costs of territory ownership. Theory and empirical precedent indicate that a primary benefit of many territories is exclusive access to food resources, and primary costs of defending and using space are associated with competition, travel and mortality risk. A recently developed mechanistic model for economical territory selection provided numerous empirically testable predictions. We tested these predictions using location data from grey wolves (Canis lupus) in Montana, USA. As predicted, territories were smaller in areas with greater densities of prey, competitors and low-use roads, and for groups of greater size. Territory size increased before decreasing curvilinearly with greater terrain ruggedness and harvest mortalities. Our study provides evidence for the economical selection of territories as a causal mechanism underlying ecological patterns observed in a cooperative carnivore. Results demonstrate how a wide range of environmental and social conditions will influence economical behaviour and resulting space use. We expect similar responses would be observed in numerous territorial species. A mechanistic approach enables understanding how and why animals select particular territories. This knowledge can be used to enhance conservation efforts and more successfully predict effects of conservation actions.

Heterogeneity of HSCs in a Mouse Model of NASH.

BACKGROUND AND AIMS: In clinical and experimental NASH, the origin of the scar-forming myofibroblast is the HSC. We used foz/foz mice on a Western diet to characterize in detail the phenotypic changes of HSCs in a NASH model. APPROACH AND RESULTS: We examined the single-cell expression profiles (scRNA sequencing) of HSCs purified from the normal livers of foz/foz mice on a chow diet, in NASH with fibrosis of foz/foz mice on a Western diet, and in livers during regression of NASH after switching back to a chow diet. Selected genes were analyzed using immunohistochemistry, quantitative real-time PCR, and short hairpin RNA knockdown in primary mouse HSCs. Our analysis of the normal liver identified two distinct clusters of quiescent HSCs that correspond to their acinar position of either pericentral vein or periportal vein. The NASH livers had four distinct HSC clusters, including one representing the classic fibrogenic myofibroblast. The three other HSC clusters consisted of a proliferating cluster, an intermediate activated cluster, and an immune and inflammatory cluster. The livers with NASH regression had one cluster of inactivated HSCs, which was similar to, but distinct from, the quiescent HSCs. CONCLUSIONS: Analysis of single-cell RNA sequencing in combination with an interrogation of previous studies revealed an unanticipated heterogeneity of HSC phenotypes under normal and injured states.

Niche-Specific Reprogramming of Epigenetic Landscapes Drives Myeloid Cell Diversity in Nonalcoholic Steatohepatitis.

Tissue-resident and recruited macrophages contribute to both host defense and pathology. Multiple macrophage phenotypes are represented in diseased tissues, but we lack deep understanding of mechanisms controlling diversification. Here, we investigate origins and epigenetic trajectories of hepatic macrophages during diet-induced non-alcoholic steatohepatitis (NASH). The NASH diet induced significant changes in Kupffer cell enhancers and gene expression, resulting in partial loss of Kupffer cell identity, induction of Trem2 and Cd9 expression, and cell death. Kupffer cell loss was compensated by gain of adjacent monocyte-derived macrophages that exhibited convergent epigenomes, transcriptomes, and functions. NASH-induced changes in Kupffer cell enhancers were driven by AP-1 and EGR that reprogrammed LXR functions required for Kupffer cell identity and survival to instead drive a scar-associated macrophage phenotype. These findings reveal mechanisms by which disease-associated environmental signals instruct resident and recruited macrophages to acquire distinct gene expression programs and corresponding functions.

Epigenetic Regulation of Kupffer Cell Function in Health and Disease.

Kupffer cells, the resident macrophages of the liver, comprise the largest pool of tissue macrophages in the body. Within the liver sinusoids Kupffer cells perform functions common across many tissue macrophages including response to tissue damage and antigen presentation. They also engage in specialized activities including iron scavenging and the uptake of opsonized particles from the portal blood. Here, we review recent studies of the epigenetic pathways that establish Kupffer cell identity and function. We describe a model by which liver-environment specific signals induce lineage determining transcription factors necessary for differentiation of Kupffer cells from bone-marrow derived monocytes. We conclude by discussing how these lineage determining transcription factors (LDTFs) drive Kupffer cell behavior during both homeostasis and disease, with particular focus on the relevance of Kupffer cell LDTF pathways in the setting of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis.

Neutralization of Oxidized Phospholipids Ameliorates Non-alcoholic Steatohepatitis.

Oxidized phospholipids (OxPLs), which arise due to oxidative stress, are proinflammatory and proatherogenic, but their roles in non-alcoholic steatohepatitis (NASH) are unknown. Here, we show that OxPLs accumulate in human and mouse NASH. Using a transgenic mouse that expresses a functional single-chain variable fragment of E06, a natural antibody that neutralizes OxPLs, we demonstrate the causal role of OxPLs in NASH. Targeting OxPLs in hyperlipidemic Ldlr-/- mice improved multiple aspects of NASH, including steatosis, inflammation, fibrosis, hepatocyte death, and progression to hepatocellular carcinoma. Mechanistically, we found that OxPLs promote ROS accumulation to induce mitochondrial dysfunction in hepatocytes. Neutralizing OxPLs in AMLN-diet-fed Ldlr-/- mice reduced oxidative stress, improved hepatic and adipose-tissue mitochondrial function, and fatty-acid oxidation. These results suggest targeting OxPLs may be an effective therapeutic strategy for NASH.