Streptococcus pyogenes NAD+-Glycohydrolase Reduces Skeletal Muscle ßNAD+ Levels Independently of Streptolysin O.

Necrotizing soft tissue infections caused by Streptococcus pyogenes (group A streptococcus [GAS]) are characterized by rapid and extensive necrosis of fascia and muscle. Molecular epidemiological studies have demonstrated a positive correlation between GAS isolates that cause invasive infections and the production of S. pyogenes NAD+-glycohydrolase (SPN), an NADase secreted by GAS, but the effect of SPN on muscle cells has not been described. Thus, using standard ßNAD+ and ATP quantification assays, we investigated the effects of SPN on cultured human skeletal muscle cell (SkMC) ßNAD+ and ATP with and without streptolysin O (SLO)-a secreted cholesterol-dependent cytolysin known to act synergistically with SPN. We found that culture supernatants from GAS strains producing SLO and SPN depleted intracellular ßNAD+ and ATP, while exotoxins from a GAS strain producing SLO and an enzymatically-inactive form of SPN had no effect on ßNAD+ or ATP. Addition of purified, enzymatically-active SPN to NADase-negative culture supernatants or sterile media reconstituted ßNAD+ depletion but had no effect ATP levels. Further, SPN-mediated ßNAD+ depletion could be augmented by SLO or the homologous cholesterol-dependent cytolysin, perfringolysin O (PFO). Remarkably, SPN-mediated ßNAD+ depletion was SkMC-specific, as purified SPN had minimal effect on epithelial cell ßNAD+. Taken together, this study identifies a previously unrecognized role for SPN as a major disruptor of skeletal muscle ßNAD+. Such activity could contribute to the rapid and widespread myonecrosis characteristic of severe GAS soft tissue infections.

Enrichment of Antigen-Specific Class-Switched B Cells from Individuals Naturally Immunized by Infection with Group A Streptococcus.

The low frequency of circulating antigen-specific memory B cells is a considerable obstacle in the discovery and development of human monoclonal antibodies for therapeutic application. Here, we evaluate two solid-phase isolation methods to enrich the number of antigen-specific B cells from individuals naturally immunized against streptolysin O (SLO), a key virulence factor and known immunogen of group A streptococcus (GAS). Class-switched B cells obtained from individuals with a history of GAS infection were separated from peripheral blood mononuclear cells (PBMCs) by immunomagnetic methods. SLO-specific B cells were further enriched directly by binding to SLO monomers and captured by streptavidin-coated magnetic microbeads or indirectly by binding a fluorescently labeled SLO-streptavidin tetramer and captured by anti-fluorophore immunomagnetic microbeads. SLO-bound B cells were quantitated by flow cytometry and/or expanded in batch culture to determine IgG specificity. From individuals who have suffered a GAS infection ≥2 years prior, only the direct method enriched SLO-specific B cells, as determined by flow cytometry. Likewise, in batch culture, B cells isolated by the direct method resulted in an average of 375-fold enrichment in anti-SLO IgG, while no enrichment was observed for B cells isolated by the indirect method. The direct method established here provides a simple approach to increase low-frequency antigen-specific B cell populations supporting many downstream applications, such as immortalization of B cells, cloning of immunoglobulin genes, or purification of antibodies from supernatant for future study. Overall, this process is efficient, is inexpensive, and can be applied to many naturally immunogenic antigens.IMPORTANCE Bacteria called group A streptococci can cause a variety of skin and soft tissue infections ranging from mild pharyngitis ("strep throat") to deadly necrotizing fasciitis (sometimes called "flesh-eating" disease). In each case, the development of disease and the degree of tissue damage are mediated by toxins released from the bacteria during infection. Consequently, novel therapies aimed at clearing bacterial toxins are greatly needed. One promising new treatment is the utilization of monoclonal antibodies delivered as an immunotherapeutic for toxin neutralization. However, current methods of antibody development are laborious and costly. Here, we report a method to enrich and increase the detection of highly desirable antigen-specific memory B cells from individuals previously exposed to GAS using a cost-effective and less-time-intensive strategy. We envision that this method will be incorporated into many applications supporting the development of immunotherapeutics.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) increases necroinflammation and hepatic stellate cell activation but does not exacerbate experimental liver fibrosis in mice.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant and high-affinity ligand for the aryl hydrocarbon receptor (AhR). Increasing evidence indicates that AhR signaling contributes to wound healing, which involves the coordinated deposition and remodeling of the extracellular matrix. In the liver, wound healing is attributed to the activation of hepatic stellate cells (HSCs), which mediate fibrogenesis through the production of soluble mediators and collagen type I. We recently reported that TCDD treatment increases the activation of human HSCs in vitro. The goal of this study was to determine how TCDD impacts HSC activation in vivo using a mouse model of experimental liver fibrosis. To elicit fibrosis, C57BL6/male mice were treated twice weekly for 8weeks with 0.5ml/kg carbon tetrachloride (CCl4). TCDD (20µg/kg) or peanut oil (vehicle) was administered once a week during the last 2weeks. Results indicate that TCDD increased liver-body-weight ratios, serum alanine aminotransferase activity, and hepatic necroinflammation in CCl4-treated mice. Likewise, TCDD treatment increased mRNA expression of HSC activation and fibrogenesis genes, namely α-smooth muscle actin, desmin, delta-like homolog-1, TGF-ß1, and collagen type I. However, TCDD treatment did not exacerbate fibrosis, nor did it increase the collagen content of the liver. Instead, TCDD increased hepatic collagenase activity and increased expression of matrix metalloproteinase (MMP)-13 and the matrix regulatory proteins, TIMP-1 and PAI-1. These results support the conclusion that TCDD increases CCl4-induced liver damage and exacerbates HSC activation, yet collagen deposition and the development of fibrosis may be limited by TCDD-mediated changes in extracellular matrix remodeling.

Aryl Hydrocarbon Receptor Activation by TCDD Modulates Expression of Extracellular Matrix Remodeling Genes during Experimental Liver Fibrosis.

The aryl hydrocarbon receptor (AhR) is a soluble, ligand-activated transcription factor that mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Increasing evidence implicates the AhR in regulating extracellular matrix (ECM) homeostasis. We recently reported that TCDD increased necroinflammation and myofibroblast activation during liver injury elicited by carbon tetrachloride (CCl4). However, TCDD did not increase collagen deposition or exacerbate fibrosis in CCl4-treated mice, which raises the possibility that TCDD may enhance ECM turnover. The goal of this study was to determine how TCDD impacts ECM remodeling gene expression in the liver. Male C57BL/6 mice were treated for 8 weeks with 0.5 mL/kg CCl4, and TCDD (20 µg/kg) was administered during the last two weeks. Results indicate that TCDD increased mRNA levels of procollagen types I, III, IV, and VI and the collagen processing molecules HSP47 and lysyl oxidase. TCDD also increased gelatinase activity and mRNA levels of matrix metalloproteinase- (MMP-) 3, MMP-8, MMP-9, and MMP-13. Furthermore, TCDD modulated expression of genes in the plasminogen activator/plasmin system, which regulates MMP activation, and it also increased TIMP1 gene expression. These findings support the notion that AhR activation by TCDD dysregulates ECM remodeling gene expression and may facilitate ECM metabolism despite increased liver injury.

Monocyte chemoattractant protein-1 is not required for liver regeneration after partial hepatectomy.

BACKGROUND: Liver regeneration following 70 % partial hepatectomy (PH) requires the coordinated expression of soluble mediators produced by macrophages. Monocyte chemoattractant protein-1 (MCP-1) is a potent stimulus of monocyte recruitment and macrophage activation. The goal of this study was to determine how MCP-1 contributes to liver regeneration. METHODS: PH was performed on anesthetized C57Bl/6 (wild type) and MCP-1 knockout mice, and macrophage-produced cytokines and hepatocyte proliferation were measured. RESULTS: In wild type mice, hepatic MCP-1 protein levels increased 4-6 h after PH, and elevated plasma MCP-1 levels were detected 12 h after PH. Hepatocyte proliferation was comparable in MCP-1 knockout and wild type mice, as was the expression of macrophage-derived cytokines, TNFα and IL-6, and levels of phosphorylated STAT3. The number of CCR2(+) cells in the liver was similar in MCP-1 knockout and wild type mice, which suggests that other chemokines may recruit CCR2(+) cells in the absence of MCP-1. Studies with CCR2 knockout mice revealed that hepatocyte proliferation was suppressed ~40 % compared to wild type mice 36 h after PH, but proliferation and liver-body-weight ratios were similar at 48 h. CONCLUSION: These findings suggest that MCP-1 is not required for PH-induced liver regeneration, yet the role of CCR2 warrants further study.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increases human hepatic stellate cell activation.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a halogenated aromatic hydrocarbon that elicits toxicity through the aryl hydrocarbon receptor (AhR). In the liver, gross markers of TCDD toxicity are attributed to AhR activation in parenchymal hepatocytes. However, less is known regarding the consequences of TCDD treatment on non-parenchymal cells in the liver. Hepatic stellate cells (HSCs) are non-parenchymal cells that store vitamin A when quiescent. Upon liver injury, activated HSCs lose this storage ability and instead function in the development and maintenance of inflammation and fibrosis through the production of pro-inflammatory mediators and collagen type I. Reports that TCDD exposure disrupts hepatic retinoid homeostasis and dysregulates extracellular matrix remodeling in the liver led us to speculate that TCDD treatment may disrupt HSC activity. The human HSC line LX-2 was used to test the hypothesis that TCDD treatment directly activates HSCs. Results indicate that exposure to 10nM TCDD almost completely inhibited lipid droplet storage in LX-2 cells cultured with retinol and palmitic acid. TCDD treatment also increased LX-2 cell proliferation, expression of α-smooth muscle actin, and production of monocyte chemoattractant protein-1 (MCP-1), all of which are characteristics of activated HSCs. However, TCDD treatment had no effect on Col1a1 mRNA levels in LX-2 cells stimulated with the potent profibrogenic mediator, transforming growth factor-ß. The TCDD-mediated increase in LX-2 cell proliferation, but not MCP-1 production, was abolished when phosphoinositide 3-kinase was inhibited. These results indicate that HSCs are susceptible to direct modulation by TCDD and that TCDD likely increases HSC activation through a multi-faceted mechanism.

Consequences of TCDD treatment on intra-hepatic lymphocytes during liver regeneration.

Increasing evidence demonstrates a physiological role for the aryl hydrocarbon receptor (AhR) in regulating hepatocyte cell cycle progression. Previous studies have used a murine model of liver regeneration to show that exposure to the potent exogenous AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), suppresses hepatocyte proliferation in vivo. Based on recent reports that natural killer (NK) cells negatively regulate liver regeneration, coupled with the well-established immunomodulatory effects of TCDD, it was hypothesized that alterations in lymphocyte activation contribute to the suppression of liver regeneration in TCDD-treated mice. To test this, mice were treated with TCDD (20 µg/kg) 1 day prior to 70% partial hepatectomy (PH), in which two-thirds of the liver was surgically resected. Lymphocytes were collected from the remnant liver and analyzed by flow cytometry. Whereas exposure to TCDD did not alter the number of NK cells or CD3(+) T-cells recovered from the regenerating liver, it reduced the percentage and number of intra-hepatic NKT cells 42 h after PH. With regard to lymphocyte activation, TCDD treatment transiently increased CD69 expression on NK and NKT cells 12 h after PH, but had no effect on intracellular levels of IFNγ in NK, NKT, or CD3(+) T-cells. To determine the relevance of NK cells to the suppression of liver regeneration by TCDD, mice were treated with anti-Asialo GM-1 (ASGM-1) antibody to deplete NK cells prior to TCDD treatment and PH, and hepatocyte proliferation was measured using bromodeoxyuridine incorporation. Exposure to TCDD was found to inhibit hepatocyte proliferation in the regenerating liver of NK cell-depleted mice and control mice to the same extent. Hence, it is unlikely that enhanced numbers or increased activation of NK cells contribute to the suppression of liver regeneration in TCDD-treated mice.

Regulation of hepatocyte fate by interferon-γ.

Interferon (IFN)-γ is a cytokine known for its immunomodulatory and anti-proliferative action. In the liver, IFN-γ can induce hepatocyte apoptosis or inhibit hepatocyte cell cycle progression. This article reviews recent mechanistic reports that describe how IFN-γ may direct the fate of hepatocytes either towards apoptosis or a cell cycle arrest. This review also describes a probable role for IFN-γ in modulating hepatocyte fate during liver regeneration, transplantation, hepatitis, fibrosis and hepatocellular carcinoma, and highlights promising areas of research that may lead to the development of IFN-γ as a therapy to enhance recovery from liver disease.