Top 20 up regulated genes in spleen of <i>Npc1^−/−</i> mice across three age groups (20–71 days).

<p>Genes marked in bold are related to innate immunity and the genes marked in bold and also underlined are innate immunity genes catalogued by InnateDB.</p

Blood cell parameters and hematological analyses in <i>Npc1^−/−</i> mice.

<p>Blood (∼20µl) was collected from female <i>Npc1^+/−</i> (n = 2, age 63 and 66 days) and <i>Npc1^−/−</i> mice (n = 2, age 63 and 66 days) by cheek bleed. Blood cell parameters were analyzed by Hemavet 950. Values represent mean±SEM. Abbreviations are, WBC = White Blood Cells, NE = Neutrophils, LY = Lymphocytes, MO = Monocytes, EO = Eosinophils, BA = Basophils, RBC = Red Blood Cells, Hb =  Hemoglobin, HCT = Hematocrit, MCV = Mean Corpuscular Volume, MCH = Mean Corpuscular Hemoglobin, MCHC =  Mean Corpuscular Hemoglobin Concentration, RDW = Red Cell Distribution width, PLT  =  Platelet, and MPV = Mean Platelet Volume. K/µl stands for 1000/µl.</p

Genomic Expression Analyses Reveal Lysosomal, Innate Immunity Proteins, as Disease Correlates in Murine Models of a Lysosomal Storage Disorder

<div><p>Niemann-Pick Type C (NPC) disease is a rare, genetic, lysosomal disorder with progressive neurodegeneration. Poor understanding of the pathophysiology and a lack of blood-based diagnostic markers are major hurdles in the treatment and management of NPC and several additional, neurological lysosomal disorders. To identify disease severity correlates, we undertook whole genome expression profiling of sentinel organs, brain, liver, and spleen of <em>Balb/c Npc1^−/−</em> mice relative to <em>Npc1^+/−</em> at an asymptomatic stage, as well as early- and late-symptomatic stages. Unexpectedly, we found prominent up regulation of innate immunity genes with age-dependent change in their expression, in all three organs. We shortlisted a set of 12 secretory genes whose expression steadily increased with age in both brain and liver, as potential plasma correlates of neurological and/or liver disease. Ten were innate immune genes with eight ascribed to lysosomes. Several are known to be elevated in diseased organs of murine models of other lysosomal diseases including Gaucher’s disease, Sandhoff disease and MPSIIIB. We validated the top candidate lysozyme, in the plasma of <em>Npc1^−/−</em> as well as <em>Balb/c Npc1^nmf164</em> mice (bearing a point mutation closer to human disease mutants) and show its reduction in response to an emerging therapeutic. We further established elevation of innate immunity in <em>Npc1^−/−</em> mice through multiple functional assays including inhibition of bacterial infection as well as cellular analysis and immunohistochemistry. These data revealed neutrophil elevation in the <em>Npc1</em>^−/− spleen and liver (where large foci were detected proximal to damaged tissue). Together our results yield a set of lysosomal, secretory innate immunity genes that have potential to be developed as pan or specific plasma markers for neurological diseases associated with lysosomal storage and where diagnosis is a major problem. Further, the accumulation of neutrophils in diseased organs (hitherto not associated with NPC) suggests their role in pathophysiology and disease exacerbation.</p> </div

Increased neutrophils in spleen of <i>Npc1</i>^−/− mice. (A)

<p>Flow cytometric analysis of innate immune cells in spleen of <i>Npc1^+/−</i> and <i>Npc1^−/−</i> mice. Splenocytes from un-infected <i>Npc1^+/−</i> and <i>Npc1^−/−</i> female littermates (age 6–8 weeks) mice were isolated and stained with anti-CD335 for NK cells, anti-CD11c for dendritic cells (DC), anti-F4/80 and CD11b for monocytes and macrophages (Mo/MO), anti-Gr-1 and CD11b for neutrophils. The data represent the mean from two independent experiments with a total of 6 mice (3 each experiment). Error bars show the mean±SD. Gating parameters are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048273#pone.0048273.s003" target="_blank">Figure S3</a>. <b>(B)</b> Flow cytometric analysis of innate immune cells in the spleen of <i>S. typhimurium</i> infected <i>Npc1^+/−</i> and <i>Npc1^−/−</i> mice. Mice at 6–8 weeks were infected with <i>S. typhimurium</i> intraperitoneally (see Materials and Methods) and splenocytes were prepared at 48 hpi. Innate immune cells were analyzed as described in A. The data represent the mean from three independent experiments with a total of 6 mice (2 each experiment). Error bars indicate the mean±SD. Statistical significance was determined using Student’s <i>t</i> test. Gating parameters are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048273#pone.0048273.s003" target="_blank">Figure S3</a>.</p

Top 20 up regulated genes in liver of <i>Npc1^−/−</i> mice across three age groups (20–71 days).

<p>Genes marked in bold are related to innate immunity and the genes marked in bold and also underlined are innate immunity genes catalogued by InnateDB.</p

Attenuated proliferation of <i>S. typhimurium</i> in (A) spleen and (B) liver of <i>Npc1^−/−</i> mice.

<p><i>Npc1</i>^+/+, <i>Npc1^+/− and Npc1</i>^−/−, mice (age 6–8 weeks) were infected with <i>S. typhimurium</i> (1×10⁴ CFU) by i.p injection. At 48 hpi, mice were sacrificed, organs isolated and bacterial CFU were determined. The data obtained from 3 independent experiments are shown. n = 10 for <i>Npc1</i>^+/+ and <i>Npc1</i>^−/− and n = 8 for <i>Npc1</i>^+/−. Error bar show the mean±SEM. Student’s <i>t</i> test was carried out to determine the statistical significance.</p

List of 12 potential biomarker genes.

<p>Genes marked in bold code for secretory lysosomal proteins.</p

Enrichment of top 10 biofunctions pathways in brain, liver and spleen of <i>Npc1</i>^−/− mice. (A)

<p>The top 10 biofunctions pathways derived from IPA analyses of differentially expressed genes in the brain of <i>Npc1</i>^−/− mice and ranked by ‘p values’ (lowest to highest) are shown. The numbers along each bar represent the total number of differentially expressed genes (both up and down regulated) categorized in each biofunction (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048273#pone.0048273.s007" target="_blank">Table S4</a>). A total of 53 genes (45 up and 8 down regulated) associated with immune response were enriched in the brain of <i>Npc1</i>^−/− across all time points (B) Bar diagram shows the top 10 biofunctions enriched in the liver of <i>Npc1</i>^−/− compared to <i>Npc1</i>^+/−. A total of 209 genes (159 up and 50 down regulated) associated with the immune response were enriched in the liver of <i>Npc1</i>^−/− across all time points (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048273#pone.0048273.s007" target="_blank">Table S4</a>). (C) Bar diagram shows the top 10 biofunctions enriched in the spleen of <i>Npc1</i>^−/− compared to <i>Npc1</i>^+/−. A total of 58 genes (49 up and 9 down regulated) associated with the immune response were enriched in the spleen of <i>Npc1</i>^−/− across all time points (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048273#pone.0048273.s007" target="_blank">Table S4</a>).</p

Top 20 up regulated genes in brain of <i>Npc1^−/−</i> mice across the life span (20–84 days).

<p>Genes marked in bold are related to innate immunity and the genes marked in bold and also underlined are innate immunity genes catalogued by InnateDB.</p

Age-dependent weight loss and plasma lysozyme activity in <i>Npc1^nmf164</i> mice.

<p><b>(A)</b> Weight curves obtained for female animals of the following genotypes. <i>Npc1^nmf164</i> WT, <i>Npc1</i>^+/+ (n = 5); <i>Npc1^nmf164</i> heterozygotes, <i>Npc1</i>^+/− (n = 9); <i>Npc1^nmf164</i> homozygotes, <i>Npc1</i>^−/− mice (n = 5). Homozygote mutant mice started to lose weight from week 12: typically, they survive 17–18 weeks. Mean values ±SD are shown. <b>(B)</b> Lysozyme activity in the plasma of <i>Npc1^nmf164</i> WT, <i>Npc1</i>^+/+; <i>Npc1^nmf164</i> heterozygotes, <i>Npc1</i>^+/−; <i>Npc1^nmf164</i> homozygotes, <i>Npc1</i>^−/−. ‘n’ denotes the number of mice used per group. Error bars show the mean±SD. ‘NS’ indicates not significant <b>(C)</b> Scatter plot of the plasma lysozyme activity of untreated <i>Npc1^nmf164</i> (age 42–49 days) and HPβCD or vehicle treated female mice (age 50–55 days). Median values are indicated by horizontal bars. ‘NS’ indicates not significant. Statistical significance was determined using Student’s <i>t</i> test.</p