Neutrophils trap and kill bacteria by forming highly decondensed chromatin structures, termed neutrophil extracellular traps (NETs). We previously reported that histone hypercitrullination catalyzed by peptidylarginine deiminase 4 (PAD4) correlates with chromatin decondensation during NET formation. However, the role of PAD4 in NET-mediated bacterial trapping and killing has not been tested. Here, we use PAD4 knockout mice to show that PAD4 is essential for NET-mediated antibacterial function. Unlike PAD4+/+ neutrophils, PAD4−/− neutrophils cannot form NETs after stimulation with chemokines or incubation with bacteria, and are deficient in bacterial killing by NETs. In a mouse infectious disease model of necrotizing fasciitis, PAD4−/− mice are more susceptible to bacterial infection than PAD4+/+ mice due to a lack of NET formation. Moreover, we found that citrullination decreased the bacterial killing activity of histones and nucleosomes, which suggests that PAD4 mainly plays a role in chromatin decondensation to form NETs instead of increasing histone-mediated bacterial killing. Our results define a role for histone hypercitrullination in innate immunity during bacterial infection.

SUMMARY Dietary soluble fibers are fermented by gut bacteria into short-chain fatty acids (SCFA), which are considered broadly health-promoting. Accordingly, consumption of such fibers ameliorates metabolic syndrome. However, incorporating soluble fiber inulin, but not insoluble fiber, into a compositionally defined diet, induced icteric hepatocellular carcinoma (HCC). Such HCC was microbiota-dependent and observed in multiple strains of dysbiotic mice but not in germ-free nor antibiotics-treated mice. Furthermore, consumption of an inulin-enriched high-fat diet induced both dysbiosis and HCC in wild-type (WT) mice. Inulin-induced HCC progressed via early onset of cholestasis, hepatocyte death, followed by neutrophilic inflammation in liver. Pharmacologic inhibition of fermentation or depletion of fermenting bacteria markedly reduced intestinal SCFA and prevented HCC. Intervening with cholestyramine to prevent reabsorption of bile acids also conferred protection against such HCC. Thus, its benefits notwithstanding, enrichment of foods with fermentable fiber should be approached with great caution as it may increase risk of HCC.

The tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) has been studied for chronic disease preventive effects, and is marketed as part of many dietary supplements. However, case reports have associated the use of green tea-based supplements with liver toxicity. We studied the hepatotoxic effects of high dose EGCG in male CF-1 mice. A single dose of EGCG (1500 mg/kg, i.g.) increased plasma alanine aminotransferase (ALT) by 138-fold and reduced survival by 85%. Once-daily dosing with EGCG increased hepatotoxic response. Plasma ALT levels were increased 184-fold following two once-daily doses of 750 mg/kg, i.g. EGCG. Moderate to severe hepatic necrosis was observed following treatment with EGCG. EGCG hepatotoxicity was associated with oxidative stress including increased hepatic lipid peroxidation (5-fold increase), plasma 8-isoprostane (9.5-fold increase) and increased hepatic metallothionein and γ-histone 2AX protein expression. EGCG also increased plasma interleukin-6 and monocyte chemoattractant protein 1. Our results indicate that higher bolus doses of EGCG are hepatotoxic to mice. Further studies on the dose-dependent hepatotoxic effects of EGCG and the underlying mechanisms are important given the increasing use of green tea dietary supplements, which may deliver much higher plasma and tissue concentrations of EGCG than tea beverages.

There is considerable debate whether peroxisome proliferatoractivated receptor B/D (PPARB/D) ligands potentiate or suppress colon carcinogenesis. Whereas administration of a PPARB ligand causes increased small intestinal tumorigenesis in Apc min/+ mice, PPARB-null (Pparb À/À ) mice exhibit increased colon polyp multiplicity in colon cancer bioassays, suggesting that ligand activation of this receptor will inhibit colon carcinogenesis. This hypothesis was examined by treating wild-type (Pparb +/+ ) and Pparb À/À with azoxymethane, coupled with a highly specific PPARB ligand, GW0742. Ligand activation of PPARB in Pparb +/+ mice caused an increase in the expression of mRNA encoding adipocyte differentiationrelated protein, fatty acid-binding protein, and cathepsin E. These findings are indicative of colonocyte differentiation, which was confirmed by immunohistochemical analysis. No PPARB-dependent differences in replicative DNA synthesis or expression of phosphatase and tensin homologue, phosphoinositide-dependent kinase, integrin-linked kinase, or phosphoAkt were detected in ligand-treated mouse colonic epithelial cells although increased apoptosis was found in GW0742-treated Pparb +/+ mice. Consistent with increased colonocyte differentiation and apoptosis, inhibition of colon polyp multiplicity was also found in ligand-treated Pparb +/+ mice, and all of these effects were not found in Pparb À/À mice. In contrast to previous reports suggesting that activation of PPARB potentiates intestinal tumorigenesis, here we show that ligand activation of PPARB attenuates chemically induced colon carcinogenesis and that PPARB-dependent induction of cathepsin E could explain the reported disparity in the literature about the effect of ligand activation of PPARB in the intestine. (Cancer Res 2006; 66(8): 4394-401)

In host–pathogen arms races, increases in host resistance prompt counteradaptation by pathogens, but the nature of that counteradaptation is seldom directly observed outside of laboratory models. The best-documented field example is the coevolution of myxoma virus (MYXV) in European rabbits. To understand how MYXV in Australia has continued to evolve in wild rabbits under intense selection for genetic resistance to myxomatosis, we compared the phenotypes of the progenitor MYXV and viral isolates from the 1950s and the 1990s in laboratory rabbits with no resistance. Strikingly, and unlike their 1950s counterparts, most virus isolates from the 1990s induced a highly lethal immune collapse syndrome similar to septic shock. Thus, the next step in this canonical case of coevolution after a species jump has been further escalation by the virus in the face of widespread host resistance.

The role of peroxisome proliferator-activated receptor-␤ (PPAR␤) in the molecular regulation of skin carcinogenesis was examined. Increased caspase-3 activity associated with apoptosis was found in the skin of wildtype mice after tumor promotion with 12-O-tetradecanoylphorbol-13-acetate, and this effect was diminished in PPAR␤-null mice. The onset of tumor formation, tumor size, and tumor multiplicity induced from a two-stage carcinogen bioassay (7,12-dimethylbenz[a]anthracene/ 12-O-tetradecanoylphorbol-13-acetate) were significantly enhanced in PPAR␤-null mice compared with wild-type mice. To begin to characterize the molecular changes underlying this PPAR␤-dependent phenotype, microarray analysis was performed and a number of differentially regulated gene products were identified including ubiquitin C. Subsequent promoter analysis, reporter gene assays, site-directed mutagenesis, and electrophoretic mobility shift assays provide evidence that PPAR␤ regulates ubiquitin C expression, and that ubiquitination of proteins is influenced by PPAR␤. These results strongly suggest that activation of PPAR␤-dependent target genes provides a novel strategy to inhibit tumor promotion and carcinogenesis.Keratinocytes, the major cell type of the epidermis, provide a protective epidermal barrier against the external environment. The regulation of proliferation and the unique process of differentiation in keratinocytes are essential in maintaining epidermal function (1). Keratinocyte proliferation and differentiation are regulated by various biological factors including cytokines and extraneous signals through different signal transduction pathways. For example, activation of transcription factors such as activator protein 1 (AP-1), 1 and signal transducer and activator of transcription, can regulate the expression of genes that in turn influence cell proliferation and differentiation. AP-1 is a major regulator of keratinocyte function, and is activated by phosphorylation via several mechanisms including the protein kinase C and mitogen-activated protein kinase pathways. Alterations in these pathways induced by chemical toxicants can cause disruption of keratinocyte cell cycle control, which could lead to skin cancer (1, 2).Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors and members of the nuclear hormone receptor superfamily (3-5). PPARs modulate target gene expression in response to ligand activation after heterodimerization with retinoid X receptor and binding to peroxisome proliferator-responsive elements. Three different PPAR isoforms, each encoded by distinct genes, have been identified and designated PPAR␣, PPAR␤ (also referred to as PPAR␦), and PPAR␥. PPARs exhibit relatively unique tissue distribution and appear to have distinct physiological functions (3-5). Extensive studies have been performed examining the biological role of PPAR␣, and many of these were facilitated by the use of the PPAR␣-null mouse. Through these studies, it was shown that PPAR␣ mediates the pleiotro...

Prolonged administration of peroxisome proliferators to rodents typically leads to hepatocarcinogenesis. Peroxisome proliferator-activated receptor-alpha (PPARalpha) is required to mediate alterations in PPARalpha target gene expression, repress apoptosis, enhance replicative DNA synthesis, oxidative stress to DNA and hepatocarcinogenesis induced by the relatively specific PPARalpha agonist, Wy-14,643. Interestingly, administration of the less specific PPARalpha agonist, bezafibrate, leads to a modest induction of PPARalpha target genes in the absence of PPARalpha expression. In these studies, the role of PPARalpha in modulating hepatocarcinogenesis induced by long-term feeding of 0.5% bezafibrate was examined in wild-type (+/+) and PPARalpha-null (-/-) mice. The average liver weight was significantly higher in (+/+) and (-/-) mice fed bezafibrate than controls, but this effect was considerably less in (-/-) mice as compared with similarly treated (+/+) mice. Increased levels of mRNA encoding cell cycle regulatory proteins and DNA repair enzymes were found in (+/+) mice fed bezafibrate, and this effect was not found in (-/-) mice. In mice fed bezafibrate for 1 year, preneoplastic foci, adenomas and a hepatocellular carcinoma were found in (+/+) mice, while only a single microscopic adenoma was found in one (-/-) mouse. This effect was observed in both Sv/129 and C57BL/6N strains of mice, although only preneoplastic foci were observed in the latter strain. Interestingly, hepatic cholestasis was observed in 100% of the bezafibrate-fed (-/-) mice, and this was accompanied by significantly elevated hepatic expression of mRNA encoding bile salt export pump and lower expression of mRNA encoding cytochrome P450 7A1, consistent with enhanced activation of the bile acid receptor, farnesoid X receptor. Results from these studies demonstrate that the PPARalpha is required to mediate hepatocarcinogenesis induced by bezafibrate, and that PPARalpha protects against potential cholestasis.

The availability of relevant and useful animal models is critical for progress in the development of effective vaccines and therapeutics. The infection of rabbits and non-human primates with fully virulent Bacillus anthracis spores provides two excellent models of anthrax disease. However, the high cost of procuring and housing these animals and the specialized facilities required to deliver fully virulent spores limit their practical use in early stages of product development. Conversely, the small size and low cost associated with using mice makes this animal model more practical for conducting experiments in which large numbers of animals are required. In addition, the availability of knockout strains and well-characterized immunological reagents makes it possible to perform studies in mice that cannot be performed easily in other species. Although we, along with others, have used the mouse aerosol challenge model to examine the outcome of B. anthracis infection, a detailed characterization of the disease is lacking. The current study utilizes a murine aerosol challenge model to investigate disease progression, innate cytokine responses, and histological changes during the course of anthrax after challenge with aerosolized spores. Our results show that anthrax disease progression in a complement-deficient mouse after challenge with aerosolized Sterne spores is similar to that described for other species, including rabbits and non-human primates, challenged with fully virulent B. anthracis. Thus, the murine aerosol challenge model is both useful and relevant and provides a means to further investigate the host response and mechanisms of B. anthracis pathogenesis.