Regulation of neonatal IgA production by the maternal microbiota.

Infants are prone to enteric infections due to an underdeveloped immune system. The maternal microbiota, through shaping the neonatal microbiota, helps establish a strong immune system in infants. We and others have observed the phenomenon of enhanced early neonatal immunoglobulin A (IgA) production in preweaning immunocompetent mice nursed by immunodeficient dams. Here, we show that this enhancement of IgA in neonates results from maternally derived microbiota. In addition, we have found that the neonatal IgA production can be induced by Lactobacillus reuteri, which is enriched in the milk of immunodeficient dams. Moreover, we show that while the production of neonatal IgA is dependent on neonatal T cells, the immunodeficient maternal microbiota-mediated enhancement of neonatal IgA has a T cell-independent component. Indeed, this enhancement may be dependent on type 3 innate lymphoid cells in the neonatal small intestinal lamina propria. Interestingly, maternal microbiota-induced neonatal IgA does not cross-react with common enteric pathogens. Future investigations will determine the functional consequences of having this extra IgA.

Fabrication and characterization of PLGA nanoparticles encapsulating large CRISPR-Cas9 plasmid.

BACKGROUND: The clustered regularly interspaced short palindromic repeats (CRISPR) and Cas9 protein system is a revolutionary tool for gene therapy. Despite promising reports of the utility of CRISPR-Cas9 for in vivo gene editing, a principal problem in implementing this new process is delivery of high molecular weight DNA into cells. RESULTS: Using poly(lactic-co-glycolic acid) (PLGA), a nanoparticle carrier was designed to deliver a model CRISPR-Cas9 plasmid into primary bone marrow derived macrophages. The engineered PLGA-based carriers were approximately 160 nm and fluorescently labeled by encapsulation of the fluorophore 6,13-bis(triisopropylsilylethynyl) pentacene (TIPS pentacene). An amine-end capped PLGA encapsulated 1.6 wt% DNA, with an encapsulation efficiency of 80%. Release studies revealed that most of the DNA was released within the first 24 h and corresponded to ~ 2-3 plasmid copies released per nanoparticle. In vitro experiments conducted with murine bone marrow derived macrophages demonstrated that after 24 h of treatment with the PLGA-encapsulated CRISPR plasmids, the majority of cells were positive for TIPS pentacene and the protein Cas9 was detectable within the cells. CONCLUSIONS: In this work, plasmids for the CRISPR-Cas9 system were encapsulated in nanoparticles comprised of PLGA and were shown to induce expression of bacterial Cas9 in murine bone marrow derived macrophages in vitro. These results suggest that this nanoparticle-based plasmid delivery method can be effective for future in vivo applications of the CRISPR-Cas9 system.

Pulmonary Exposure to Magnéli Phase Titanium Suboxides Results in Significant Macrophage Abnormalities and Decreased Lung Function.

Coal is one of the most abundant and economic sources for global energy production. However, the burning of coal is widely recognized as a significant contributor to atmospheric particulate matter linked to deleterious respiratory impacts. Recently, we have discovered that burning coal generates large quantities of otherwise rare Magnéli phase titanium suboxides from TiO2 minerals naturally present in coal. These nanoscale Magnéli phases are biologically active without photostimulation and toxic to airway epithelial cells in vitro and to zebrafish in vivo. Here, we sought to determine the clinical and physiological impact of pulmonary exposure to Magnéli phases using mice as mammalian model organisms. Mice were exposed to the most frequently found Magnéli phases, Ti6O11, at 100 parts per million (ppm) via intratracheal administration. Local and systemic titanium concentrations, lung pathology, and changes in airway mechanics were assessed. Additional mechanistic studies were conducted with primary bone marrow derived macrophages. Our results indicate that macrophages are the cell type most impacted by exposure to these nanoscale particles. Following phagocytosis, macrophages fail to properly eliminate Magnéli phases, resulting in increased oxidative stress, mitochondrial dysfunction, and ultimately apoptosis. In the lungs, these nanoparticles become concentrated in macrophages, resulting in a feedback loop of reactive oxygen species production, cell death, and the initiation of gene expression profiles consistent with lung injury within 6 weeks of exposure. Chronic exposure and accumulation of Magnéli phases ultimately results in significantly reduced lung function impacting airway resistance, compliance, and elastance. Together, these studies demonstrate that Magnéli phases are toxic in the mammalian airway and are likely a significant nanoscale environmental pollutant, especially in geographic regions where coal combustion is a major contributor to atmospheric particulate matter.

High-frequency irreversible electroporation is an effective tumor ablation strategy that induces immunologic cell death and promotes systemic anti-tumor immunity.

BACKGROUND: Despite promising treatments for breast cancer, mortality rates remain high and treatments for metastatic disease are limited. High-frequency irreversible electroporation (H-FIRE) is a novel tumor ablation technique that utilizes high-frequency bipolar electric pulses to destabilize cancer cell membranes and induce cell death. However, there is currently a paucity of data pertaining to immune system activation following H-FIRE and other electroporation based tumor ablation techniques. METHODS: Here, we utilized the mouse 4T1 mammary tumor model to evaluate H-FIRE treatment parameters on cancer progression and immune system activation in vitro and in vivo. FINDINGS: H-FIRE effectively ablates the primary tumor and induces a pro-inflammatory shift in the tumor microenvironment. We further show that local treatment with H-FIRE significantly reduces 4T1 metastases. H-FIRE kills 4T1 cells through non-thermal mechanisms associated with necrosis and pyroptosis resulting in damage associated molecular pattern signaling in vitro and in vivo. Our data indicate that the level of tumor ablation correlates with increased activation of cellular immunity. Likewise, we show that the decrease in metastatic lesions is dependent on the intact immune system and H-FIRE generates 4T1 neoantigens that engage the adaptive immune system to significantly attenuate tumor progression. INTERPRETATION: Cell death and tumor ablation following H-FIRE treatment activates the local innate immune system, which shifts the tumor microenvironment from an anti-inflammatory state to a pro-inflammatory state. The non-thermal damage to the cancer cells and increased innate immune system stimulation improves antigen presentation, resulting in the engagement of the adaptive immune system and improved systemic anti-tumor immunity.

Using Klebsiella pneumoniae to Model Acute Lung Inflammation in Mice.

Lung infections caused by bacteria can induce a spectrum of immune responses, which is in part determined by the level of exposure and the degree of the host response. The host response involves pattern recognition receptors (PRRs) which sense pathogen and damage associated molecular patterns. Therefore, models of acute lung inflammation are necessary for further understanding the role of the innate immune system during bacterial infection in humans. Mice are a widely used model organism for studying important aspects of human lung pathogenesis, including acute and chronic inflammatory diseases. Klebsiella pneumoniae is a gram-negative bacterium that is commonly associated with respiratory infections, especially in a hospital setting. In this protocol, we describe a model of bacteria-mediated lung inflammation using K. pneumoniae. After a single intratracheal administration of K. pneumoniae, mice showed a strong level of Th1-mediated immune activation in the lungs. The model described here, while optimized for K. pneumonia, can be performed using other bacteria, fungi, and viruses as well.

Maternal Influence and Murine Housing Confound Impact of NLRP1 Inflammasome on Microbiome Composition.

The NLRP1 inflammasome attenuates inflammatory bowel disease (IBD) progression and colitis-associated tumorigenesis. A possible mechanism postulates that the lack of the NLRP1 inflammasome creates permissive niches in the gut for pathogenic bacteria to flourish, causing dysbiosis and increased IBD susceptibility. To evaluate this hypothesis, we characterized the gut microbiome of wild-type, Nlrp1b-/-, and Asc-/- mice under naïve conditions by sequencing the V3 region of the 16s rRNA gene. For both genetically modified mouse lines, the microbiome composition reflected overrepresentation of bacteria associated with dysbiosis relative to wild-type animals. Measurement of short- and medium-chain fatty acids by mass spectrometry further revealed significant differences between genotypes. However, prior to concluding that the NLRP1 inflammasome plays a role in regulating the composition of the microbiome, we evaluated two additional strategies for cohousing wild-type and Nlrp1b-/- mice: breeding homozygous parents and cohousing at weaning, and breeding from heterozygous parents and cohousing littermates. We found that maternal influence was the greater predictor of microbiome composition rather than genotype. With the rise in microbiome research across disciplines, our study should be viewed as a cautionary example that illustrates the importance of careful breeding and housing strategies when evaluating host-microbiome interactions.

Utilizing the Lung as a Model to Study Nanoparticle-Based Drug Delivery Systems.

Intranasal administration is a highly effective route for drug delivery and biodistribution studies. Indeed, this route of delivery has become the method of choice to distribute diverse pharmacological agents both locally and systemically. In the majority of preclinical animal models and in human patients, intranasal administration is the preferred method to deliver therapeutic agents to the airways and lungs. However, issues with drug stability and controlled release in the respiratory tract are common problems with many therapeutic agents. Nanoparticle delivery via intranasal administration has tremendous potential to circumvent these common issues. Over the past 30 years nanoparticles have gained increased interest as therapeutic delivery vehicles and as tools for improved bioimaging. Integral to the success of nanoparticles in drug delivery and biodistribution is the utilization of mouse models to characterize therapeutic strategies under physiologically relevant in situ conditions. Here, we describe a model of nanoparticle administration to the lungs utilizing intranasal administration and discuss a variety of highly useful techniques to evaluate nanoparticle up-take, biodistribution, and immune response. While these protocols have been optimized for intranasal administration of common fluorescently labeled nanoparticles, they can be applied to any nanoparticle or drug delivery system of interest targeting the lungs and airways.

Photo-triggered release of 5-fluorouracil from a MOF drug delivery vehicle.

A nano metal-organic-framework (nanoMOF) was employed as a first-of-its kind drug delivery vehicle (DDV) for the photo-controlled release of therapeutics with simultaneous breakdown of the carrier into small molecules.

Modulating inflammation through the negative regulation of NF-κB signaling.

Immune system activation is essential to thwart the invasion of pathogens and respond appropriately to tissue damage. However, uncontrolled inflammation can result in extensive collateral damage underlying a diverse range of auto-inflammatory, hyper-inflammatory, and neoplastic diseases. The NF-κB signaling pathway lies at the heart of the immune system and functions as a master regulator of gene transcription. Thus, this signaling cascade is heavily targeted by mechanisms designed to attenuate overzealous inflammation and promote resolution. Mechanisms associated with the negative regulation of NF-κB signaling are currently under intense investigation and have yet to be fully elucidated. Here, we provide an overview of mechanisms that negatively regulate NF-κB signaling through either attenuation of signal transduction, inhibition of posttranscriptional signaling, or interference with posttranslational modifications of key pathway components. While the regulators discussed for each group are far from comprehensive, they exemplify common mechanistic approaches that inhibit this critical biochemical signaling cascade. Despite their diversity, a commonality among these regulators is their selection of specific targets at key inflection points in the pathway, such as TNF-receptor-associated factor family members or essential kinases. A better understanding of these negative regulatory mechanisms will be essential to gain greater insight related to the maintenance of immune system homeostasis and inflammation resolution. These processes are vital elements of disease pathology and have important implications for targeted therapeutic strategies.

Noncanonical NF-κB signaling and the essential kinase NIK modulate crucial features associated with eosinophilic esophagitis pathogenesis.

Eosinophilic esophagitis (EoE) is an allergic disease of the esophagus driven by T cell and eosinophil responses to dietary allergens, resulting in chronic mucosal inflammation. Few spontaneous animal models of esophageal eosinophilia exist, with most studies relying on artificial sensitization procedures. NF-κB-inducing kinase (NIK; MAP3K14) is a key signaling molecule of the noncanonical NF-κB (NFKB1) pathway, an alternative signaling cascade producing chemokines involved in lymphoid stroma development and leukocyte trafficking. Nik-/- mice have been shown to develop a hypereosinophilic syndrome in peripheral blood and major filtering organs; however, the gastrointestinal mucosa of these mice has not been well characterized. We show that Nik-/- mice develop significant, localized eosinophilic esophagitis that mimics human EoE, including features such as severe eosinophil accumulation, degranulation, mucosal thickening, fibrosis and basal cell hyperplasia. The remainder of the GI tract, including the caudal stomach, small intestine and colon, in mice with active EoE are unaffected, also similar to human patients. Gene expression patterns in esophageal tissue of Nik-/- mice mimics human EoE, with thymic stromal lymphopoetin (TSLP) in particular also elevated at the protein level. In gene expression data sets from human biopsy specimens, we further show that many genes associated with noncanonical NF-κB signaling are significantly dysregulated in EoE patients, most notably a paradoxical upregulation of NIK itself with concurrent upregulation of powerful protein-level destabilizers of NIK. These findings suggest that Nik-/- mice could be useful as a spontaneous model of specific features of EoE and highlight a novel role for noncanonical NF-κB signaling in human patients.

TIPS pentacene loaded PEO-PDLLA core-shell nanoparticles have similar cellular uptake dynamics in M1 and M2 macrophages and in corresponding in vivo microenvironments.

Nanoparticle based drug delivery platforms have the potential to transform disease treatment paradigms and therapeutic strategies, especially in the context of pulmonary medicine. Once administered, nanoparticles disperse throughout the lung and many are phagocytosed by macrophages. However, there is a paucity of knowledge regarding cellular up-take dynamics of nanoparticles due largely to macrophage heterogeneity. To address this issue, we sought to better define nanoparticle up-take using polarized M1 and M2 macrophages and novel TIPS-pentacene loaded PEO-PDLLA nanoparticles. Our data reveal that primary macrophages polarized to either M1 or M2 phenotypes have similar levels of nanoparticle phagocytosis. Similarly, M1 and M2 polarized macrophages isolated from the lungs of mice following either acute (Th1) or allergic (Th2) airway inflammation also demonstrated equivalent levels of nanoparticle up-take. Together, these studies provide critical benchmark information pertaining to cellular up-take dynamics and biodistribution of nanoparticles in the context of clinically relevant inflammatory microenvironments.

Emerging Roles for Noncanonical NF-κB Signaling in the Modulation of Inflammatory Bowel Disease Pathobiology.

Crohn's disease and ulcerative colitis are common and debilitating manifestations of inflammatory bowel disease (IBD). IBD is characterized by a radical imbalance in the activation of proinflammatory and anti-inflammatory signaling pathways in the gut. These pathways are controlled by NF-κB, which is a master regulator of gene transcription. In IBD patients, NF-κB signaling is often dysregulated resulting in overzealous inflammation. NF-κB activation occurs through 2 distinct pathways, defined as either canonical or noncanonical. Canonical NF-κB pathway activation is well studied in IBD and is associated with the rapid, acute production of diverse proinflammatory mediators, such as COX-2, IL-1ß, and IL-6. In contrast to the canonical pathway, the noncanonical or "alternative" NF-κB signaling cascade is tightly regulated and is responsible for the production of highly specific chemokines that tend to be associated with less acute, chronic inflammation. There is a relative paucity of literature regarding all aspects of noncanonical NF-ĸB signaling. However, it is clear that this alternative signaling pathway plays a considerable role in maintaining immune system homeostasis and likely contributes significantly to the chronic inflammation underlying IBD. Noncanonical NF-κB signaling may represent a promising new direction in the search for therapeutic targets and biomarkers associated with IBD. However, significant mechanistic insight is still required to translate the current basic science findings into effective therapeutic strategies.

The Goldilocks Conundrum: NLR Inflammasome Modulation of Gastrointestinal Inflammation during Inflammatory Bowel Disease.

Recent advances have revealed significant insight into inflammatory bowel disease (IBD) pathobiology. Ulcerative colitis and Crohn's disease, the chronic relapsing clinical manifestations of IBD, are complex disorders with genetic and environmental influences. These diseases are associated with the dysregulation of immune tolerance, excessive inflammation, and damage to the epithelial cell barrier. Increasing evidence indicates that pattern recognition receptors, including Toll-like receptors (TLRs) and nucleotide-binding domain and leucine-rich repeat-containing proteins (NLRs), function to maintain immune system homeostasis, modulate the gastrointestinal microbiome, and promote proper intestinal epithelial cell regeneration and repair. New insights have revealed that NLR family members are essential components in maintaining this immune system homeostasis. To date, the vast majority of studies associated with NLRs have focused on family members that form a multiprotein signaling platform called the inflammasome. These signaling complexes are responsible for the cleavage and activation of the potent pleotropic cytokines IL-1ß and IL-18, and they facilitate a unique form of cell death defined as pyroptosis. In this review, we summarize the current paradigms associated with NLR inflammasome maintenance of immune system homeostasis in the gastrointestinal system. New concepts related to canonical and noncanonical inflammasome signaling, as well as the implications of classical and alternative inflammasomes in IBD pathogenesis, are also reviewed.

The NLRP1 inflammasome attenuates colitis and colitis-associated tumorigenesis.

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins are a diverse family of pattern recognition receptors that are essential mediators of inflammation and host defense in the gastrointestinal system. Recent studies have identified a subgroup of inflammasome forming NLRs that modulate the mucosal immune response during inflammatory bowel disease (IBD) and colitis associated tumorigenesis. To better elucidate the contribution of NLR family members in IBD and cancer, we conducted a retrospective analysis of gene expression metadata from human patients. These data revealed that NLRP1, an inflammasome forming NLR, was significantly dysregulated in IBD and colon cancer. To better characterize the function of NLRP1 in disease pathogenesis, we used Nlrp1b(-/-) mice in colitis and colitis-associated cancer models. In this paper, we report that NLRP1 attenuates gastrointestinal inflammation and tumorigenesis. Nlrp1b(-/-) mice demonstrated significant increases in morbidity, inflammation, and tumorigenesis compared with wild-type animals. Similar to data previously reported for related inflammasome forming NLRs, the increased inflammation and tumor burden was correlated with attenuated levels of IL-1ß and IL-18. Further mechanistic studies using bone marrow reconstitution experiments revealed that the increased disease pathogenesis in the Nlrp1b(-/-) mice was associated with nonhematopoietic-derived cells and suggests that NLRP1 functions in the colon epithelial cell compartment to attenuate tumorigenesis. Taken together, these data identify NLRP1 as an essential mediator of the host immune response during IBD and cancer. These findings are consistent with a model whereby multiple NLR inflammasomes attenuate disease pathobiology through modulating IL-1ß and IL-18 levels in the colon.

Caspase-11 attenuates gastrointestinal inflammation and experimental colitis pathogenesis.

Nucleotide-binding domain and leucine-rich repeat containing protein inflammasome formation plays an essential role in modulating immune system homeostasis in the gut. Recently, a caspase-11 noncanonical inflammasome has been characterized and appears to modulate many biological functions that were previously considered to be solely dependent on caspase-1 and the canonical inflammasome. To better elucidate the function of this noncanonical inflammasome during inflammatory bowel disease, experimental colitis was induced in wild-type and Casp11(-/-) mice utilizing dextran sulfate sodium (DSS). Here, we report that caspase-11 attenuates acute experimental colitis pathogenesis. Casp11(-/-) mice showed significantly increased morbidity and colon inflammation following DSS exposure. Subsequent cytokine analysis revealed that IL-1ß and IL-18 levels in the colon were significantly reduced in the Casp11(-/-) mice compared with the wild-type animals. Additional mechanistic studies utilizing IL-1ß and IL-18 reconstitution revealed that Casp11(-/-) hypersensitivity was associated with the loss of both of these cytokines. Bone marrow reconstitution experiments further revealed that caspase-11 gene expression and function in both hematopoietic- and nonhematopoietic-derived cells contribute to disease attenuation. Interestingly, unlike caspase-1, caspase-11 does not appear to influence relapsing remitting disease progression or the development of colitis-associated tumorigenesis. Together, these data identify caspase-11 as a critical factor protecting the host during acute DSS-induced colonic injury and inflammation but not during chronic inflammation and tumorigenesis.