Kirk J. Maurer

Toxoplasma gondii Rhoptry Kinase ROP16 Activates STAT3 and STAT6 Resulting in Cytokine Inhibition and Arginase-1-Dependent Growth Control

The ROP16 kinase of Toxoplasma gondii is injected into the host cell cytosol where it activates signal transducer and activator of transcription (STAT)-3 and STAT6. Here, we generated a ROP16 deletion mutant on a Type I parasite strain background, as well as a control complementation mutant with restored ROP16 expression. We investigated the biological role of the ROP16 molecule during T. gondii infection. Infection of mouse bone marrow-derived macrophages with rop16-deleted (ΔROP16) parasites resulted in increased amounts of IL-12p40 production relative to the ROP16-positive RH parental strain. High level IL-12p40 production in ΔROP16 infection was dependent on the host cell adaptor molecule MyD88, but surprisingly was independent of any previously recognized T. gondii triggered pathway linking to MyD88 (TLR2, TLR4, TLR9, TLR11, IL-1ß and IL-18). In addition, ROP16 was found to mediate the suppressive effects of Toxoplasma on LPS-induced cytokine synthesis in macrophages and on IFN-γ-induced nitric oxide production by astrocytes and microglial cells. Furthermore, ROP16 triggered synthesis of host cell arginase-1 in a STAT6-dependent manner. In fibroblasts and macrophages, failure to induce arginase-1 by ΔROP16 tachyzoites resulted in resistance to starvation conditions of limiting arginine, an essential amino acid for replication and virulence of this parasite. ΔROP16 tachyzoites that failed to induce host cell arginase-1 displayed increased replication and dissemination during in vivo infection. We conclude that encounter between Toxoplasma ROP16 and the host cell STAT signaling cascade has pleiotropic downstream effects that act in multiple and complex ways to direct the course of infection.

Roles of Infection, Inflammation, and the Immune System in Cholesterol Gallstone Formation

Cholesterol gallstone formation is a complex process mediated by genetic and environmental factors. Until recently, the role of the immune system in the pathogenesis of cholesterol gallstones was not considered a valid topic of research interest. This review collates and interprets an extensive body of basic literature, some of which is not customarily considered to be related to cholelithogenesis, describing the multiple facets of the immune system that appear to be involved in cholesterol cholelithogenesis. A thorough understanding of the immune interactions with biliary lipids and cholecystocytes should modify current views of the pathogenesis of cholesterol gallstones, promote further research on the pathways involved, and lead to novel diagnostic tools, treatments, and preventive measures.

The DNA damage checkpoint protein ATM promotes hepatocellular apoptosis and fibrosis in a mouse model of non-alcoholic fatty liver disease

Steatoapoptosis is a hallmark of non-alcoholic fatty liver disease (NAFLD) and is an important factor in liver disease progression. We hypothesized that increased reactive oxygen species resulting from excess dietary fat contribute to liver disease by causing DNA damage and apoptotic cell death, and tested this by investigating the effects of feeding mice high fat or standard diets for 8 weeks. High fat diet feeding resulted in increased hepatic H2O2, superoxide production, and expression of oxidative stress response genes, confirming that the high fat diet induced hepatic oxidative stress. High fat diet feeding also increased hepatic steatosis, hepatitis and DNA damage as exemplified by an increase in the percentage of 8-hydroxyguanosine (8-OHG) positive hepatocytes in high fat diet fed mice. Consistent with reports that the DNA damage checkpoint kinase Ataxia Telangiectasia Mutated (ATM) is activated by oxidative stress, ATM phosphorylation was induced in the livers of wild type mice following high fat diet feeding. We therefore examined the effects of high fat diet feeding in Atm-deficient mice. The prevalence of apoptosis and expression of the pro-apoptotic factor PUMA were significantly reduced in Atm-deficient mice fed the high fat diet when compared with wild type controls. Furthermore, high fat diet fed Atm−/− mice had significantly less hepatic fibrosis than Atm+/+ or Atm+/− mice fed the same diet. Together, these data demonstrate a prominent role for the ATM pathway in the response to hepatic fat accumulation and link ATM activation to fatty liver-induced steatoapoptosis and fibrosis, key features of NAFLD progression.

CXCR3-Dependent CD4+ T Cells Are Required to Activate Inflammatory Monocytes for Defense against Intestinal Infection

Chemokines and their receptors play a critical role in orchestrating immunity to microbial pathogens, including the orally acquired Th1-inducing protozoan parasite Toxoplasma gondii. Chemokine receptor CXCR3 is associated with Th1 responses, and here we use bicistronic CXCR3-eGFP knock-in reporter mice to demonstrate upregulation of this chemokine receptor on CD4+ and CD8+ T lymphocytes during Toxoplasma infection. We show a critical role for CXCR3 in resistance to the parasite in the intestinal mucosa. Absence of the receptor in Cxcr3−/− mice resulted in selective loss of ability to control T. gondii specifically in the lamina propria compartment. CD4+ T cells were impaired both in their recruitment to the intestinal lamina propria and in their ability to secrete IFN-γ upon stimulation. Local recruitment of CD11b+Ly6C/G+ inflammatory monocytes, recently reported to be major anti-Toxoplasma effectors in the intestine, was not impacted by loss of CXCR3. However, inflammatory monocyte activation status, as measured by dual production of TNF-α and IL-12, was severely impaired in Cxcr3−/− mice. Strikingly, adoptive transfer of wild-type but not Ifnγ−/− CD4+ T lymphocytes into Cxcr3−/− animals prior to infection corrected the defect in inflammatory macrophage activation, simultaneously reversing the susceptibility phenotype of the knockout animals. Our results establish a central role for CXCR3 in coordinating innate and adaptive immunity, ensuring generation of Th1 effectors and their trafficking to the frontline of infection to program microbial killing by inflammatory monocytes.

Synergy Between Intraepithelial Lymphocytes and Lamina Propria T Cells Drives Intestinal Inflammation During Infection

Oral infection of C57BL/6 mice with Toxoplasma gondii triggers severe necrosis in the ileum within 7–10 days of infection. Lesion development is mediated by Th-1 cytokines, CD4+ T cells, and subepithelial bacterial translocation. As such, these features share similarity to Crohns disease. Recently, we uncovered a role for intraepithelial lymphocytes (IELs) in mediating pathology after Toxoplasma infection. We show here that αβ and not γδ T-cell IELs mediate intestinal damage. By adoptive transfer of mucosal T cells into naive Rag1−/− mice, we demonstrate that IELs do not function alone to cause inflammatory lesions, but act with CD4+ T lymphocytes from the lamina propria (LP). Furthermore, recipient mice pretreated with broad-spectrum antibiotics to eliminate intestinal flora resisted intestinal disease after transfer of IELs and LP lymphocytes. Our data provide valuable new insights into the mechanisms of intestinal inflammation, findings that have important implications for understanding human inflammatory bowel disease.

CCR2 and CD44 Promote Inflammatory Cell Recruitment during Fatty Liver Formation in a Lithogenic Diet Fed Mouse Model

Non-alcoholic fatty liver disease (NAFLD) is a common disease with a spectrum of presentations. The current study utilized a lithogenic diet model of NAFLD. The diet was fed to mice that are either resistant (AKR) or susceptible (BALB/c and C57BL/6) to hepatitis followed by molecular and flow cytometric analysis. Following this, a similar approach was taken in congenic mice with specific mutations in immunological genes. The initial study identified a significant and profound increase in multiple ligands for the chemokine receptor CCR2 and an increase in CD44 expression in susceptible C57BL/6 (B6) but not resistant AKR mice. Ccr2−/− mice were completely protected from hepatitis and Cd44−/− mice were partially protected. Despite protection from inflammation, both strains displayed similar histological steatosis scores and significant increases in serum liver enzymes. CD45+CD44+ cells bound to hyaluronic acid (HA) in diet fed B6 mice but not Cd44−/− or Ccr2−/− mice. Ccr2−/− mice displayed a diminished HA binding phenotype most notably in monocytes, and CD8+ T-cells. In conclusion, this study demonstrates that absence of CCR2 completely and CD44 partially reduces hepatic leukocyte recruitment. These data also provide evidence that there are multiple redundant CCR2 ligands produced during hepatic lipid accumulation and describes the induction of a strong HA binding phenotype in response to LD feeding in some subsets of leukocytes from susceptible strains.

Helicobacter hepaticus urease is not required for intestinal colonization but promotes hepatic inflammation in male A/JCr mice.

Urease activity contributes to bacterial survival in the acidic environment of the stomach and is essential for persistent infection by known gastric helicobacters such as the human pathogen Helicobacter pylori. Several enterohepatic Helicobacter species (EHS) that primarily infect the less acidic intestine also have very active urease enzymes. The importance of urease and its contribution to pathogenesis for these EHS are poorly understood. In this study, we generated a urease-deficient, isogenic mutant (HhureNT9) of Helicobacter hepaticus 3B1 (Hh 3B1), an EHS that possesses a urease gene cluster similar to that of H. pylori. Lack of urease activity did not affect the level of cecal colonization by HhureNT9 compared to Hh 3B1 in male A/JCr mice (P=0.48) at 4 months post-inoculation (MPI). In contrast, there was no HhureNT9 detected in the livers of any infected mice, whereas all livers from the Hh 3B1-infected mice were PCR-positive for Hh 3B1. The mice infected with HhureNT9 developed significantly less severe hepatitis (P=0.017) and also produced significantly lower hepatic mRNA levels of proinflammatory cytokines IFN-gamma (P=0.0007) and TNF-alpha (P<0.0001) compared to the Hh 3B1-infected mice. The Hh 3B1-infected mice developed significantly higher total IgG, Th1-associated IgG2a and Th2-associated IgG1 responses to infection. These results indicate that H. hepaticus urease activity plays a crucial role in hepatic disease but is not required for cecal colonization by H. hepaticus.

An Analysis of the Role of the Indigenous Microbiota in Cholesterol Gallstone Pathogenesis

Background and Aims Cholesterol gallstone disease is a complex process involving both genetic and environmental variables. No information exists regarding what role if any the indigenous gastrointestinal microbiota may play in cholesterol gallstone pathogenesis and whether variations in the microbiota can alter cholesterol gallstone prevalence rates. Methods Genetically related substrains (BALB/cJ and BALB/cJBomTac) and (BALB/AnNTac and BALB/cByJ) of mice obtained from different vendors were compared for cholesterol gallstone prevalence after being fed a lithogenic diet for 8 weeks. The indigenous microbiome was altered in these substrains by oral gavage of fecal slurries as adults, by cross-fostering to mice with divergent flora at <1day of age or by rederiving into a germ-free state. Results Alterations in the indigenous microbiome altered significantly the accumulation of mucin gel and normalized gallbladder weight but did not alter cholesterol gallstone susceptibility in conventionally housed SPF mice. Germ-free rederivation rendered mice more susceptible to cholesterol gallstone formation. This susceptibility appeared to be largely due to alterations in gallbladder size and gallbladder wall inflammation. Colonization of germ-free mice with members of altered Schaedler flora normalized the gallstone phenotype to a level similar to conventionally housed mice. Conclusions These data demonstrate that alterations in the gastrointestinal microbiome may alter aspects of cholesterol gallstone pathogenesis and that in the appropriate circumstances these changes may impact cholesterol cholelithogenesis.

Seroprevalence of Alphaviruses in Chelonians from Southeastern Louisiana

ABSTRACT Eastern equine encephalitis virus, an alphavirus, is the causative agent of eastern equine encephalitis, an arthropod-borne disease endemic to North, Central, and South America. In addition to horses, humans, and birds, the eastern equine encephalitis virus can also infect reptiles. Although experimental studies have demonstrated that reptiles can remain viremic for longer periods of time than mammals and birds, as well as remain viremic through hibernation, the overall role of reptiles in the epidemiology for the virus is unclear. A cross-sectional study was performed to estimate the seroprevalence of eastern equine encephalitis virus in chelonians from southeastern Louisiana. Blood was collected from 117 chelonians in southeastern Louisiana in the summer of 2000 (n=76) and the spring of 2001 (n=41). Hemagglutination inhibition assay and plaque reduction neutralization test were utilized to determine exposure. Six (5%) of the 117 chelonians were seropositive for eastern equine encephalitis virus...

The Experimental Use of Syrian Hamsters

The Syrian hamster (Mesocricetus auratus) possesses a variety of unique features which make them desirable research models. Despite these features, experimental use of the hamster has declined over the past 20–30 years. The current chapter will focus on research and literature describing the experimental use of the hamster with a particular emphasis on research done since the previous edition of this text (1987–present).