Lampe1: An ENU-Germline Mutation Causing Spontaneous Hepatosteatosis Identified through Targeted Exon-Enrichment and Next-Generation Sequencing

Using a small scale ENU mutagenesis approach we identified a recessive germline mutant, designated Lampe1 that exhibited growth retardation and spontaneous hepatosteatosis. Low resolution mapping based on 20 intercrossed Lampe1 mice revealed linkage to a ∼14 Mb interval on the distal site of chromosome 11 containing a total of 285 genes. Exons and 50 bp flanking sequences within the critical region were enriched with sequence capture microarrays and subsequently analyzed by next-generation sequencing. Using this approach 98.1 percent of the targeted DNA was covered with a depth of 10 or more reads per nucleotide and 3 homozygote mutations were identified. Two mutations represented intronic nucleotide changes whereas one mutation affected a splice donor site in intron 11–12 of Palmitoyl Acetyl-coenzyme A oxygenase-1 (Acox1), causing skipping of exon 12. Phenotyping of Acox1Lampe1 mutants revealed a progression from hepatosteatosis to steatohepatitis, and ultimately hepatocellular carcinoma. The current approach provides a highly efficient and affordable method to identify causative mutations induced by ENU mutagenesis and animal models relevant to human pathology

Regulation of Inflammation by IL-17A and IL-17F Modulates Non-Alcoholic Fatty Liver Disease Pathogenesis.

Non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease worldwide. While it is well-accepted that inflammation is central to NAFLD pathogenesis, the immune pathway(s) orchestrating disease progression are poorly defined. Notably, IL-17RA signaling, via IL-17A, plays an important role in obesity-driven NAFLD pathogenesis. However, the role of the IL-17F, another IL-17RA ligand, in NAFLD pathogenesis has not been examined. Further, the cell types expressing IL-17RA and producing IL-17RA ligands in the pathogenesis of NAFLD have not been defined. Here, IL-17RA-/-, IL-17A-/-, IL-17F-/- and wild-type (WT) mice were fed either standard chow diet or methionine and choline deficient diet (MCDD)--a diet known to induce steatosis and hepatic inflammation through beta-oxidation dysfunction--and hepatic inflammation and NAFLD progression were subsequently quantified. MCDD feeding augmented hepatic IL-17RA expression and significantly increased hepatic infiltration of macrophages and IL-17A and IL-17F producing CD4+ and CD8+ T cells in WT mice. In contrast, IL-17RA-/-, IL-17A-/-, and IL-17F-/- mice, despite increased steatosis, exhibited significant protection from hepatocellular damage compared to WT controls. Protection from hepatocellular damage correlated with decreased levels of hepatic T-cell and macrophage infiltration and decreased expression of inflammatory mediators associated with NAFLD. In sum, our results indicate that the IL-17 axis also plays a role in a MCDD-induced model of NAFLD pathogenesis. Further, we show for the first time that IL-17F, and not only IL-17A, plays an important role in NAFLD driven inflammation

IL-17 axis deficiency does not modulate systemic effects of MCDD feeding.

<p>8 week old male IL-17RA^-/-, IL-17A^-/-, and IL-17F^-/- mice on a C57BL/6 background and WT controls (<i>n</i> = 4-8/condition) were placed on MCDD or chow diet for 4 weeks. (<b>A</b>) Weight loss as percent of starting body weight. (<b>B</b>) Total daily food intake. (<b>C</b>) Serum triglyceride levels. (<b>D</b>) Serum cholesterol levels. (<b>E</b>) Liver weight as a percentage of total body weight. (<b>F</b>) Hepatic mitochondrial oxygen consumption rate in mice fed chow. (<b>G</b>) Hepatic mitochondrial oxygen consumption rate in mice fed MCDD. (<b>H</b>) Hepatic SREBP-1c mRNA expression. (<b>I</b>) Hepatic PPARα mRNA expression. (<b>J</b>) Hepatic Lipe mRNA expression. (<b>K</b>) Hepatic KLF15 mRNA expression. AU = Arbitrary Units, as compared to beta actin. Data represents means + SE; a representative of two separate experiments. Student <i>t</i> test *<i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001.</p

IL-17 axis deficiency augments hepatic steatosis, but protects from hepatocellular damage.

<p>8 week old male IL-17RA^-/-, IL-17A^-/-, and IL-17F^-/- mice on a C57BL/6 background and WT controls (<i>n</i> = 4-8/condition) were placed on MCDD or chow diet for 4 weeks. (<b>A</b>) Hepatic triglyceride levels. (<b>B</b>) Hepatic SCD-1 mRNA expression. (<b>C</b>) Serum alanine transaminase (ALT) levels. AU = Arbitrary units, as compared to beta actin. Data represent means + SE; a representative of two separate experiments. Student <i>t</i> test *<i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001.</p

MCDD-driven NAFLD alters hepatic levels of IL-17A and IL-17F production.

<p>8 week old male C57BL/6 mice (<i>n</i> = 4/condition) were placed on MCDD or chow diet for 4 weeks. (<b>A</b>) IL-17A and IL-17F production was determined in cells following the gating on: CD45⁺, TCRβ⁺, NK1.1^-, CD4⁺ or CD8⁺. (<b>B</b>) Percent of hepatic immune cells (CD45⁺) producing IL-17A. (<b>C</b>) Percent of hepatic immune cells (CD45⁺) producing IL-17F. (<b>D</b>) Percent of hepatic TCRβ⁺ cells (CD4⁺ or CD8⁺ T cells) producing IL-17A. (<b>E</b>) Percent of hepatic TCRβ⁺ cells (CD4⁺ or CD8⁺ T cells) producing IL-17F. (<b>F</b>) Mean fluorescent intensity of IL-17A in TCRβ⁺ cells (CD4⁺ or CD8⁺ T cells). (<b>G</b>) Mean fluorescent intensity of IL-17F in TCRβ⁺ cells (CD4⁺ or CD8⁺ T cells). (<b>H</b>) Percent of hepatic immune cells (CD45⁺) expressing GR-1 and CD11b. Data represent means + SE; a representative of two separate experiments. Student <i>t</i> test **<i>P</i> < 0.01.</p

The IL-17 axis regulates macrophage infiltration to the liver.

<p>8 week old male IL-17RA^-/-, IL-17A^-/-, and IL-17F^-/- mice on a C57BL/6 background and WT controls (<i>n</i> = 4-8/condition) were placed on MCDD or chow diet for 4 weeks. (<b>A</b>) Representative immunohistochemistry staining of CD68⁺ macrophages in the livers of WT mice fed chow and WT, IL-17RA^-/-, IL-17A^-/- and IL-17F^-/- mice on MCDD and average number of CD68⁺ cells per 20x field. (<b>B-D</b>) Hepatic mRNA expression: (<b>B</b>) CCL2; (<b>C</b>) CCL22; (<b>D</b>) TNFα. (<b>E</b>) Representative Sirius Red staining of the livers from WT, IL-17RA^-/-, IL-17A^-/-, and IL-17F^-/- mice fed MCDD. AU = Arbitrary units, as compared to beta actin. Data represents means + SE; a representative of two separate experiments. Student <i>t</i> test *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***P < 0.001.</p

Placenta growth factor augments airway hyperresponsiveness via leukotrienes and IL-13

Airway hyperresponsiveness (AHR) affects 55%-77% of children with sickle cell disease (SCD) and occurs even in the absence of asthma. While asthma increases SCD morbidity and mortality, the mechanisms underlying the high AHR prevalence in a hemoglobinopathy remain unknown. We hypothesized that placenta growth factor (PlGF), an erythroblast-secreted factor that is elevated in SCD, mediates AHR. In allergen-exposed mice, loss of Plgf dampened AHR, reduced inflammation and eosinophilia, and decreased expression of the Th2 cytokine IL-13 and the leukotriene-synthesizing enzymes 5-lipoxygenase and leukotriene-C4-synthase. Plgf-/- mice treated with leukotrienes phenocopied the WT response to allergen exposure; conversely, anti-PlGF Ab administration in WT animals blunted the AHR. Notably, Th2-mediated STAT6 activation further increased PlGF expression from lung epithelium, eosinophils, and macrophages, creating a PlGF/leukotriene/Th2-response positive feedback loop. Similarly, we found that the Th2 response in asthma patients is associated with increased expression of PlGF and its downstream genes in respiratory epithelial cells. In an SCD mouse model, we observed increased AHR and higher leukotriene levels that were abrogated by anti-PlGF Ab or the 5-lipoxygenase inhibitor zileuton. Overall, our findings indicate that PlGF exacerbates AHR and uniquely links the leukotriene and Th2 pathways in asthma. These data also suggest that zileuton and anti-PlGF Ab could be promising therapies to reduce pulmonary morbidity in SCD.status: publishe