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

Exploring The Multiple Functions Of Neutrophils

Neutrophils are one of the most effective antimicrobial cells in the innate immune system. They arrive at a site of infection or inflammation in response to a chemotactic gradient. At this site, they phagocytose invading pathogens and aid in their destruction through both oxidative and non-oxidative mechanisms. This is known to be mediated through the release of toxic granule contents and antimicrobial effector proteins. After their antimicrobial activity is perfo rmed, neutrophils undergo a programmed cell death process known as apoptosis and are quickly cleared from sites of inflammation by phagocytes. As such, neutrophils are believed to be fast acting, short-living effector cells that perform antimicrobial activities with limited influence on ensuing immune responses. Nevertheless, neutrophils are increasingly being shown to be regulators of adaptive immune responses. I show that neutrophils can act as professional antigen presenting cells capable of processing and presenting antigen to T cells. I also show that acquisition of antigen presentation ability is T cell-contact dependent and that the resulting T cell response is skewed towards a Th1 and Th17 phenotype. Neutrophils also have the ability to undergo a cell death process termed NETosis in which they extrude extracellular DNA that can trap and kill pathogens. I show that both mouse and human neutrophils undergo NETosis in response to the protozoan parasite Toxoplasma gondii and that the resulting entrapment leads to parasite killing. NETosis in response to Toxoplasma is parasiteinvasion independent and is mediated partly by signaling through the ERK pathway. The in vivo role of neutrophils during a Toxoplasma infection was also addressed. I show that neutrophil depleted and infected mice succumb to infection with highly dysregulated pulmonary immune responses. Depletion of neutrophils led to an increased phagocyte inflammatory state that resulted in an increase in T cell activity and cytokine production leading to pathology and ultimately death of the host. Data presented in this thesis challenge the traditional view of neutrophils and portray them more as immunoregulatory cells that can influence, shape, and regulate an immune response well beyond the stages of innate immunity and even beyond their own death

Toxoplasma gondii triggers phosphorylation and nuclear translocation of dendritic cell STAT1 while simultaneously blocking IFNγ-induced STAT1 transcriptional activity.

The protozoan Toxoplasma gondii actively modulates cytokine-induced JAK/STAT signaling pathways to facilitate survival within the host, including blocking IFNγ-mediated STAT1-dependent proinflammatory gene expression. We sought to further characterize inhibition of STAT1 signaling in infected murine dendritic cells (DC) because this cell type has not previously been examined, yet is known to serve as an early target of in vivo infection. Unexpectedly, we discovered that T. gondii infection alone induced sustained STAT1 phosphorylation and nuclear translocation in DC in a parasite strain-independent manner. Maintenance of STAT1 phosphorylation required active invasion but intracellular parasite replication was dispensable. The parasite rhoptry protein ROP16, recently shown to mediate STAT3 and STAT6 phosphorylation, was not required for STAT1 phosphorylation. In combination with IFNγ, T. gondii induced synergistic STAT1 phosphorylation and binding of aberrant STAT1-containing complexes to IFNγ consensus sequence oligonucleotides. Despite these findings, parasite infection blocked STAT1 binding to the native promoters of the IFNγ-inducible genes Irf-1 and Lrg47, along with subsequent gene expression. These results reinforce the importance of parasite-mediated blockade of IFNγ responses in dendritic cells, while simultaneously showing that T. gondii alone induces STAT1 phosphorylation

<i>Toxoplasma</i> induces <i>in vitro</i> STAT1 binding activity in BMDC.

<p>Nuclear extracts were prepared from cells infected with the RH parasite strain (Tg, <i>Toxoplasma gondii</i>; 3∶1 parasites to cells) or treated with IFNγ (100 ng/ml) for 6 hours. The <i>in vitro</i> binding activity of nuclear STAT1 to solid phase IFNγ-activated sequence (GAS) oligonucleotides was assessed using an ELISA-based method. Binding activity is expressed as fold increase over cells cultured in medium alone (Med, value of 1). +cOligo, addition of soluble competitive oligonucleotides; +mOligo, addition of mutated non-competitive oligonucleotides. The experiment was repeated three times with similar results. *, p<0.05 comparing nuclear extracts alone with nuclear extracts plus cOligo.</p

<i>Toxoplasma</i> blocks IFNγ-driven STAT1-dependent gene induction.

<p>BMDC were infected with the RH strain of <i>T. gondii</i> (Tg, ratio of 3 parasites/cell), treated with treated IFNγ (100 ng/ml), or pre-infected for 2 hours with RH prior to addition of IFNγ (Tg+IFNγ). At the indicated time points post cytokine treatment, total RNA was harvested and reverse-transcribed to cDNA prior to qPCR amplification of the IFNγ-responsive, STAT1-dependent genes <i>Irf-1</i> (A) and <i>Lrg-47</i> (B). Fold change in gene expression is expressed relative to BMDC in medium alone. Samples were normalized to the house-keeping gene GAPDH. To assess native chromatin binding, ChIP was performed using an anti-STAT1α antibody followed by qPCR amplification with primers specific for the <i>Irf-1</i> promoter (C). Experimental conditions are replicated as in (A and B), with a time point of 2 hours shown. In (C), fold change in promoter binding is expressed relative to untreated cells (Med). Samples were normalized to input chromatin. Experiments were repeated at least three times with similar results.</p

STAT1 phosphorylation is confined to infected cells.

<p>(A) BMDC were left in medium alone (Med), infected with type I (RH), type II (PTG) or type III (M774.1) strains of <i>Toxoplasma</i> (3∶1 ratio of parasites to cells) or treated with murine IFNγ (100 ng/ml). After 22 hours supernatants were collected (22 hr sup), centrifuged and filtered to remove debris and parasites, and subsequently transferred to additional untreated/uninfected BMDC for an additional 20 hours (B). For all samples (A and B), cytoplasmic (C) and nuclear (N) fractions were prepared and subjected to immunoblot analysis with phospho-Tyr701-STAT1 (pY-STAT1). PARP and Rab5a served as cytoplasmic and nuclear loading controls, respectively. (C – E) Cells were infected with the RH strain at a ratio of 0.5 parasites/cell for 20 hours, then subjected to intracellular staining for <i>Toxoplasma</i> (anti-p30/SAG-1) and pY-STAT1 prior to flow cytometric analysis. BMDC were gated on uninfected (D) and infected (E) populations to assess pY-STAT1 expression (blue lines) relative to staining with an isotype control antibody (red lines). All experiments were repeated at least twice with similar results.</p

<i>Toxoplasma</i> induces STAT1 phosphorylation and nuclear translocation in BMDC.

<p>(A) BMDC were left in medium alone (Med), infected with type I (RH), II (PTG) or III (M774.1) strains of <i>Toxoplasma</i> (3∶1 ratio of parasites to cells), or treated with murine IFNγ (100 ng/ml), prior to fractionation into cytoplasmic (C) and nuclear (N) extracts at the time points indicated. Samples were subjected to immunoblot analysis for phospho-Tyr701-STAT1 (pY-STAT1) and phospho-Ser-STAT1 (pS-STAT1). PARP and Rab5a served as loading controls for nuclear and cytoplasmic fractions, respectively. (B) Cells were infected with live parasites of the three strains as in (A) or exposed to heat-inactivated (HI) tachyzoites for six hours. Cytoplasmic and nuclear fractions were collected and immunoblot analyis was performed as in (A). (C) RH parasites were pre-treated for 10 min on ice with 1 μM cytochalasin D (CytD) prior to infection in the continued presence of the drug. Cells treated with the solvent DMSO alone served as controls. Cells were fractionated after six hours and subjected to immunoblot analysis for pY-STAT1. (D and E) BMDC were treated with IFNγ or infected with RH in comparison with either the <i>cps1-1</i> replication-deficient strain (D) or the ΔROP16 strain (E). Samples were fractionated after 6 and 20 hours and subjected to immunoblot analysis for pY-STAT1. All experiments were repeated at least three times with similar results.</p

Hepatitis susceptible mouse strains display increased liver leukocyte CD44 expression in response to LD feeding.

<p>Hepatic leukocyte preparations were gated on forward and side-scatter and CD44 levels of B6 (A), BALB/c (B) and AKR (C) mice were examined by flow cytometry. Solid lines represent mice on LD and shaded lines represent mice on SD.</p