Integrated analysis of dysregulated microRNA and mRNA expression in intestinal epithelial cells following ethanol intoxication and burn injury.

Gut barrier dysfunction is often implicated in pathology following alcohol intoxication and burn injury. MicroRNAs (miRNAs) are negative regulators of gene expression that play a central role in gut homeostasis, although their role after alcohol and burn injury is poorly understood. We performed an integrated analysis of miRNA and RNA sequencing data to identify a network of interactions within small intestinal epithelial cells (IECs) which could promote gut barrier disruption. Mice were gavaged with ~ 2.9 g/kg ethanol and four hours later given a ~ 12.5% TBSA full thickness scald injury. One day later, IECs were harvested and total RNA extracted for RNA-seq and miRNA-seq. RNA sequencing showed 712 differentially expressed genes (DEGs) (padj < 0.05) in IECs following alcohol and burn injury. Furthermore, miRNA sequencing revealed 17 differentially expressed miRNAs (DEMs) (padj < 0.1). Utilizing the miRNet, miRDB and TargetScan databases, we identified both validated and predicted miRNA gene targets. Integration of small RNA sequencing data with mRNA sequencing results identified correlated changes in miRNA and target expression. Upregulated miRNAs were associated with decreased proliferation (miR-98-3p and miR-381-3p) and cellular adhesion (miR-29a-3p, miR-429-3p and miR3535), while downregulated miRNAs were connected to upregulation of apoptosis (Let-7d-5p and miR-130b-5p) and metabolism (miR-674-3p and miR-185-5p). Overall, these findings suggest that alcohol and burn injury significantly alters the mRNA and miRNA expression profile of IECs and reveals numerous miRNA-mRNA interactions that regulate critical pathways for gut barrier function after alcohol and burn injury.

Bacterially induced activation of interleukin-18 in porcine intestinal mucosa.

Interleukin (IL)-18 is a cytokine with structural and functional properties similar to IL-1beta and IL-12, respectively. It is activated by caspase-1 cleavage, like IL-1beta, and induces interferon (IFN)-gamma, like IL-12. In order to study the role of IL-18 in the immune response to infectious diseases of mucosal surfaces we cloned and expressed porcine IL-18 and developed antibodies to the protein. Porcine IL-18 retains the caspase-1 cleavage site present in other mammalian IL-18 proteins, but has two potential N-linked glycosylation sites not found in those proteins. Porcine interleukin-18 mRNA and protein are expressed in immune tissues including lymph nodes and gut associated lymphoid tissues. Specific cell types containing IL-18 include lung and splenic macrophages, nonadherent spleen cells and intestinal epithelial cells. Although IL-18 transcription is moderately induced by lipopolysaccharide, the magnitude and total expression level are small compared to those of interleukin-1beta. In vivo and ex vivo infection of intestinal mucosa with Salmonella choleraesuis resulted in a decrease in size of IL-18, consistent with cleavage of the preprotein by caspase-1. Thus, IL-18 is present in mucosal tissues where it could play a role in the immune response to invading pathogens.

Differential regulation of macrophage interleukin-1 (IL-1), IL-12, and CD80-CD86 by two bacterial toxins.

The ability of innate immune cells to differentially respond to various bacterial components provides a mechanism by which the acquired immune response may be tailored to specific pathogens. The response of innate immune cells to bacterial components provides regulatory signals to cognate immune cells. These signals include secreted cytokines and costimulatory molecules, and to a large extent they determine the quantitative and qualitative nature of the immune response. In order to determine if innate immune cells can differentially respond to bacterial components, we compared the responses of macrophages to two bacterially derived molecules, cholera toxin (CT) and lipopolysaccharide (LPS). We found that CT and LPS differentially regulated the expression of interleukin-12 (IL-12) and CD80-CD86 but not that of IL-1beta. LPS and CT each induced IL-1beta expression in macrophages, while only LPS induced IL-12 and only CT induced CD80-CD86. These differences were markedly potentiated in gamma interferon (IFN-gamma)-treated macrophages, in which LPS potently induced IL-12 and CD80-CD86 expression. In contrast, IFN-gamma treatment had no effect on the expression of IL-1beta. These results define a molecular basis for the differential pathogenicities of bacterial toxins and are relevant to the design of vaccine adjuvants able to selectively induce desired types of immunity.

In vitro and in vivo bioactivity of single-chain interleukin-12.

Interleukin-12 is a heterodimeric cytokine with potent immunoregulatory properties, making it a potential vaccine adjuvant and an immune response modulator. The study of its function is confounded by its heterodimeric structure. In order to facilitate the study of interleukin-12 in both in vitro and in vivo models, we constructed a single-chain porcine interleukin-12 gene and expressed the recombinant protein in Pichia pastoris. Single-chain porcine interleukin-12 was bioactive in vitro on both human and porcine cells as measured by its ability to induce proliferation of lymphoblasts and interferon-gamma secretion by lymph node cells. In contrast, the p40 subunit of porcine interleukin-12 alone did not induce proliferation or inhibit the activity of the single-chain porcine interkeukin-12. The in vivo bioactivity of single-chain porcine interleukin-12 was demonstrated in an oral immunization model where it increased antigen-specific IgA and IgG in jejunal mucus. These results indicate that binding of interleukin-12 to its receptor and transduction of intracellular signals requires both p40 and p35 subunits. The bioactivity of interleukin-12 expressed as a single polypeptide will facilitate its in vivo delivery and study of its structure and function.

Efficient assembly of rat hepatocyte spheroids for tissue engineering applications.

Freshly harvested primary rat hepatocytes cultivated as multicellular aggregates, or spheroids, have been observed to exhibit enhanced liver-specific function and differentiated morphology compared to cells cultured as monolayers. An efficient method of forming spheroids in spinner vessels is described. Within 24 h after inoculation, greater than 80% of inoculated cells formed spheroids. This efficiency was significantly greater than that reported previously for formation in stationary petri dishes. With a high specific oxygen uptake rate of 2.0 x 10(-9) mmol O(2)/cell/h, the oxygen supply is critical and should be monitored for successful formation. Throughout a 6-day culture period, spheroids assembled in spinner cultures maintained a high viability and produced albumin and urea at constant rates. Transmission electron microscopy indicated extensive cell-cell contacts and tight junctions between cells within spheroids. Microvilli-lined bile canaliculus-like channels were observed in the interior of spheroids and appeared to access the exterior through pores at the outer surface. Spheroids from spinner cultures exhibited at least the level of liver-specific activity as well as similar morphology and ultrastructure compared to spheroids formed in stationary petri dishes. Hepatocytes cultured as spheroids are potentially useful three-dimensional cell systems for application in a bioartificial liver device and for studying xenobiotic drug metabolism. (c) 1996 John Wiley & Sons, Inc.

Experimental autoimmune myocarditis in A/J mice is an interleukin-4-dependent disease with a Th2 phenotype.

Myocarditis in humans is often associated with an autoimmune process in which cardiac myosin (CM) is a major autoantigen. Experimental autoimmune myocarditis (EAM) is induced in mice by immunization with CM. We found that EAM in A/J mice exhibits a Th2-like phenotype demonstrated by the histological picture of the heart lesions (eosinophils and giant cells) and by the humoral response (association of IgG1 response with disease and up-regulation of total IgE). Blocking interleukin (IL)-4 with anti-IL-4 monoclonal antibody (mAb) reduced the severity of EAM. This reduction in severity was associated with a shift from a Th2-like to a Th1-like phenotype represented by a reduction in CM-specific IgG1; an increase in CM-specific IgG2a; an abrogation of total IgE response; a decrease in IL-4, IL-5, and IL-13; as well as a dramatic increase in interferon (IFN)-gamma production in vitro. Based on the latter finding, we hypothesized that IFN-gamma limits disease. Indeed, IFN-gamma blockade with a mAb exacerbated disease. The ameliorating effect of IL-4 blockade was abrogated by co-administration of anti-IFN-gamma mAb. Thus, EAM represents a model of an organ-specific autoimmune disease associated with a Th2 phenotype, in which IL-4 promotes the disease and IFN-gamma limits it. Suppression of IFN-gamma represents at least one of the mechanisms by which IL-4 promotes EAM.