Molecular Phenotyping of Immune Cells from Young NOD Mice Reveals Abnormal Metabolic Pathways in the Early Induction Phase of Autoimmune Diabetes

<div><p>Islet leukocytic infiltration (insulitis) is first obvious at around 4 weeks of age in the NOD mouse – a model for human type 1 diabetes (T1D). The molecular events that lead to insulitis and initiate autoimmune diabetes are poorly understood. Since TID is caused by numerous genes, we hypothesized that multiple molecular pathways are altered and interact to initiate this disease. We evaluated the molecular phenotype (mRNA and protein expression) and molecular networks of <em>ex vivo</em> unfractionated spleen leukocytes from 2 and 4 week-old NOD mice in comparison to two control strains. Analysis of the global gene expression profiles and hierarchical clustering revealed that the majority (∼90%) of the differentially expressed genes in NOD mice were repressed. Furthermore, analysis using a modern suite of multiple bioinformatics approaches identified abnormal molecular pathways that can be divided broadly into 2 categories: metabolic pathways, which were predominant at 2 weeks, and immune response pathways, which were predominant at 4 weeks. Network analysis by Ingenuity pathway analysis identified key genes/molecules that may play a role in regulating these pathways. These included five that were common to both ages (TNF, HNF4A, IL15, Progesterone, and YWHAZ), and others that were unique to 2 weeks (e.g. MYC/MYCN, TGFB1, and IL2) and to 4 weeks (e.g. IFNG, beta-estradiol, p53, NFKB, AKT, PRKCA, IL12, and HLA-C). Based on the literature, genes that may play a role in regulating metabolic pathways at 2 weeks include Myc and HNF4A, and at 4 weeks, beta-estradiol, p53, Akt, HNF4A and AR. Our data suggest that abnormalities in regulation of metabolic pathways in the immune cells of young NOD mice lead to abnormalities in the immune response pathways and as such may play a role in the initiation of autoimmune diabetes. Thus, targeting metabolism may provide novel approaches to preventing and/or treating autoimmune diabetes.</p> </div

Transcripts with highly significant expression differences in spleen leukocytes of 4 week old NOD mice compared to two control strains.

<p>Statistical analysis of mRNA expression data by 1-way-ANOVA (Benjamini-Hochberg, p<0.005) followed by hierarchical clustering identified 76 probe sets (representing 72 different genes) that were differentially expressed in spleen leukocytes of 4 week old NOD mice compared to two control strains (NON and C57BL/6); H, indicates 8 genes (∼11%) with significantly higher expression in NOD relative to controls; the rest, 68 (∼89%) had significantly lower expression in NOD. Fold change (FC) was calculated by ratio of means of expression in NOD mice versus controls; FC of genes of lower expression in NOD are indicated by negative values. We assigned the genes to selected functional categories based on information obtained by searches of public databases. Other genes under “Other” include: Fcrla, Otu1, Ankrd16, Treml2, Plekha2, Fam3c, 0610039P13Rik, Fam65b, Pyhin1, <b>5031439G07Rik, C9orf21, LOC434484 (H)</b>; Unknown genes include: <b>D17H6S56E-5, A630001G21Rik</b>, A530030E21Rik. Genes highlighted with <b>bold font</b> were differentially expressed at both 2 and 4 weeks.</p

Enriched KEGG Pathways for the lists of differentially expressed proteins in spleen leukocytes from 2 week and 4 week old NOD mice compared to two control strains.

<p>One-way ANOVA (p<0.05) of protein expression data followed by hierarchical clustering and protein identification revealed 9 and 7 proteins whose expression was altered in spleen leukocytes from NOD mice compared to two control strains (NON and C57BL/6) at 2 weeks and 4 weeks of age, respectively. The two lists of differentially expressed proteins were combined and protein identifiers (IDs) were converted to gene IDs. Gene IDs were uploaded and analyzed in WebGestalt (<a href="http://bioinfo.vanderbilt.edu/webgestalt" target="_blank">http://bioinfo.vanderbilt.edu/webgestalt</a>) for enriched KEGG pathways using the hypergeometric test (p<0.01, Benjamini-Hochberg). Pathways represented by ≥2 proteins are shown.</p

Proteome network created by Ingenuity Pathway Analysis from the dataset of 4 week-old mice.

<p>A single network was generated from the list of proteins that were differentially expressed in 4 week-old NOD mice compared to both control strains, NON and C57BL/6. The protein spots from which these proteins were identified were selected from the hierarchical cluster of 26 protein spots that had significant expression differences between strains at 4 weeks. The gene names derived from our uploaded lists (focus genes) are represented by gray icons. White icons represent genes (or endogenous chemicals) derived solely from the IPA knowledge base and that could be algorithmically connected to the focus genes.</p

Enriched molecular function/cellular component Gene Ontology (GO) categories for the lists of differentially expressed transcripts in spleen leukocytes from 2 week and 4 week old NOD mice compared to two control strains.

<p>One-way ANOVA (p<0.05, Benjamini-Hochberg) of mRNA expression data followed by hierarchical clustering identified 187 and 327 probe sets whose expression was altered in spleen leukocytes from NOD mice compared to two control strains (NON and C57BL/6) at 2 weeks and 4 weeks, respectively. The lists of differentially expressed genes were analyzed in WebGestalt (<a href="http://bioinfo.vanderbilt.edu/webgestalt" target="_blank">http://bioinfo.vanderbilt.edu/webgestalt</a>) for enriched GO categories using hypergeometric test (p<0.01, Benjamini-Hochberg); dashes (-) indicate that the corresponding categories were not significantly enriched at the respective ages. Categories represented by ≥5 genes are shown. <b>Bold text</b> indicates categories that were common to both age groups.</p

Hierarchical clustering of 175 genes with highly significant expression differences between strains at 2 weeks.

<p>The list was generated by one-way ANOVA of 24,959 probe sets (p<0.005, Benjamini-Hochberg multiple test correction). A total of 51 genes were differentially expressed in NOD mice relative to both control strains (NON and C57BL/6, C57): 45 were of lower expression (L) and 6 were of higher expression (H) in NOD mice. The color intensity of the rectangles representing each gene for each sample indicates the degree of increase (red) or decrease (blue) of the gene expression signal relative to the mean signal intensity (yellow).</p

Enriched biological process Gene ontology (GO) categories for the lists of differentially expressed transcripts in spleen leukocytes from 2 and 4 week old NOD mice compared to two control strains.

<p>One-way ANOVA (p<0.05, Benjamini-Hochberg) of mRNA expression data followed by hierarchical clustering identified 187 and 327 probe sets whose expression was altered in spleen leukocytes from NOD mice compared to two control strains (NON and C57BL/6) at 2 weeks and 4 weeks, respectively. The lists of differentially expressed genes were analyzed in WebGestalt (<a href="http://bioinfo.vanderbilt.edu/webgestalt" target="_blank">http://bioinfo.vanderbilt.edu/webgestalt</a>) for enriched GO categories using hypergeometric test (p<0.01, Benjamini-Hochberg); dashes (-) indicate that the corresponding categories were not significantly enriched at the respective ages. Categories represented by ≥5 genes are shown. <b>Bold text</b> indicates categories that were common to both age groups.</p

Transcripts significantly differentially expressed in spleen leukocytes between 2 weeks and 4 weeks of age in NOD mice but not in two control strains.

<p>Statistical analysis of mRNA expression data by unpaired t-Test (p<0.001) followed by Venn diagram analysis identified 66 probe sets (representing 65 different genes) that were uniquely differentially expressed in spleen leukocytes of NOD mice between 2 and 4 weeks of age in comparison to two control strains, NON and C57BL/6. Fold change (FC) was calculated by ratio of means of expression at 2 weeks versus 4 weeks; FC of genes of lower expression at 4 weeks (compared to 2 weeks) are indicated by negative values. Genes with FC<1.5 (and their FC values) included: Syt11 (1.47), Snap47 (1.46), Cklf (−1.45), Cd274 (1.43), Heatr7a (1.42), Fam76b (−1.41), Npsr1 (1.39), Nrn1 (1.38), Eef2k (1.24), Tomm34 (1.21), and IL17rb (1.2). Expression of approximately 62% (40) of the genes was upregulated at 4 weeks compared to expression at 2 weeks of age.</p

Functional categories associated with the major Ingenuity Pathway Networks of differentially expressed transcripts/proteins in spleen leukocytes from 2 week and 4 week old NOD mice.

<p>One-way ANOVA of expression data sets followed by hierarchical clustering identified transcripts/proteins differentially expressed in spleen leukocytes from NOD mice compared to two control strains (NON and C57BL/6). The lists of differentially expressed genes were analyzed by Ingenuity Pathway Analysis (<a href="http://www.ingenuity.com" target="_blank">www.ingenuity.com</a>).</p>a<p>Genes that were present in the uploaded lists.</p

Hierarchical clustering of 189 genes with highly significant expression differences between strains at 4 weeks.

<p>The list was generated by one-way ANOVA of 24,959 probe sets (p<0.005, Benjamini-Hochberg multiple test correction). A total of 76 genes were differentially expressed in NOD mice relative to both control strains (NON and C57BL/6): 67 were of lower expression (L) and 9 were of higher expression (H) in NOD mice. The color intensity of the rectangles representing each gene for each sample indicates the degree of increase (red) or decrease (blue) of the gene expression signal relative to the mean signal intensity (yellow).</p