The Role of TNF-α in Mice with Type 1- and 2- Diabetes

Background: Previously, we have demonstrated that short-term treatment of new onset diabetic Non-obese diabetic (NOD) mice, mice that are afflicted with both type 1 (T1D) and type 2 (T2D) diabetes with either Power Mix (PM) regimen or alpha1 antitrypsin (AAT) permanently restores euglycemia, immune tolerance to self-islets and normal insulin signaling. Methodology and Principal Findings: To search for relevant therapeutic targets, we have applied genome wide transcriptional profiling and systems biology oriented bioinformatics analysis to examine the impact of the PM and AAT regimens upon pancreatic lymph node (PLN) and fat, a crucial tissue for insulin dependent glucose disposal, in new onset diabetic non-obese diabetic (NOD) mice. Systems biology analysis identified tumor necrosis factor alpha (TNF-$\alpha$) as the top focus gene hub, as determined by the highest degree of connectivity, in both tissues. In PLNs and fat, TNF-$\alpha$ interacted with 53% and 32% of genes, respectively, associated with reversal of diabetes by previous treatments and was thereby selected as a therapeutic target. Short-term anti-TNF-$\alpha$ treatment ablated a T cell-rich islet-invasive and beta cell-destructive process, thereby enhancing beta cell viability. Indeed anti-TNF-$\alpha$ treatment induces immune tolerance selective to syngeneic beta cells. In addition to these curative effects on T1D anti-TNF-e33254 treatment restored in vivo insulin signaling resulting in restoration of insulin sensitivity. Conclusions: In short, our molecular analysis suggested that PM and AAT both may act in part by quenching a detrimental TNF-$\alpha$ dependent effect in both fat and PLNs. Indeed, short-term anti-TNF-$\alpha$ mAb treatment restored enduring euglycemia, self-tolerance, and normal insulin signaling

List of fold changes of the top 30 genes out of 238 genes counter-regulated by AAT and Power Mix treatment in fat.

<p>List of fold changes of the top 30 genes out of 238 genes counter-regulated by AAT and Power Mix treatment in fat.</p

Counter-regulated network signature in PLNs.

<p>A) Interactive Network of top 20 focus gene hubs. B) Ranked list of top 20 focus gene hubs on the basis of degree of connectivity. A merged network was generated from the top 7 networks of AAT treatment counter-regulated genes in PLNs. The Ingenuity pathways analysis (IPA) tool was used to generate and merge the significantly effected networks from the AAT treatment counter-regulated genes. The focus gene hubs were ranked in the merged network on the basis of degree of connectivity.</p

Immunohistochemical staining for CD3 and insulin in NOD pancreatic islets from different experimental groups.

<p>Representative frozen sections from a 3 week old female pre diabetic NOD mouse without insulitis (A, CD3; B, insulin), a newly diagnosed diabetic female mouse (C, CD3; D, insulin) and a diabetic NOD female mouse treated with anti-TNF-α (E, CD3; F, insulin). Overall the figures document increasing cellular infiltration by CD3+ T cells and the associated loss of insulin positive cells in diabetic (C, D), as compared to prediabetic, hosts (A, B). After anti-TNF-α treatment, intra-islet infiltration by CD3+ T cells is abolished with the appearance of a circumferential non-invasive CD3+ T cell process (E). After treatment many islets are found with normal insulin+ beta cells (F).</p

Anti-TNF-α treatment restores insulin signaling in new onset diabetic NOD mice.

<p>A) Compares tyrosyl phosphorylation of the insulin receptor in normal controls vs. newly diabetic and anti-TNF-α treated NOD mice. B) Compares tyrosyl phosphorylation of IRS-1 in normal controls vs. newly diabetic and anti-TNF-α treated NOD mice. The left side shows representative immunoblots. The right side shows quantitative analysis of the immunoblots. Mice (n = 6–8/group) were fasted overnight and injected with human insulin (20 units/kg body weight i.p.) to acutely stimulate insulin signaling. Mice were sacrificed 10 minutes later. Fat tissue obtained (50 days post-treatment) was dissected and frozen in liquid nitrogen before immunoblotting analysis of insulin signaling proteins among i) normal non-diabetic NOD mice; ii) newly diagnosed diabetic NOD mice treated with conventional insulin therapy; iii) anti-TNF-α treated NOD mice at 50 days post- treatment.</p

Counter-regulated network signature in fat.

<p>A) Interactive Network of top 20 focus gene hubs. B) Ranked list of top 20 focus gene hubs on the basis of degree of connectivity. The interactive network based analysis on the 238 fat counter-regulated genes identified 5 significant networks (Score≥40) related to cell cycle, lipid metabolism and carbohydrate metabolism. The merged network was generated from the top 3 networks of genes counter-regulated in fat by AAT and PM treatment. The focus gene hubs were ranked in the merged network on the basis of degree of connectivity.</p

Short-term treatment of Diabetic NOD mice with anti-TNF-α therapy restores immune tolerance to beta cells.

<p>NOD-sp spontaneous new onset diabetic NOD mice;</p>*<p>NOD-sp/stz, a streptozotocin induced diabetic state was induced in NOD recipients. Spontaneously diabetic NOD mice (<b>Groups C, D</b>) were previously restored to a euglycemic state by anti-TNF-α treatment. These mice remained euglycemic 240–300 days following the cessation of treatment.</p><p>Syngeneic NOD.SCID islet (<b>Groups A, C</b>) or allogeneic C57BL/6 (<b>Groups B, D</b>) islet grafts were transplanted into NOD recipients.</p

Counter-regulation of gene expression by AAT or PM as determined by DNA microarrays of normal (NOR), diabetic (DIA) and AAT or PM treated PLNs and fat tissues from NOD mice.

<p>The genes were identified in a supervised analysis using an absolute fold change of 2 and P value<0.05. <b>A</b>) Hierarchical cluster analysis of top transcripts that are differentially expressed in PLNs extracted from diabetic mice vs. normal non-diabetic control mice. <b>B</b>) Cluster analysis illustrating the transcripts that are significantly counter-regulated by AAT treatment in PLNs. The columns represent the samples and rows represent the genes. Gene expression is shown with pseudocolor scale (−1 to 1) with red denoting high gene expression levels and green denoting low gene expression levels of genes.</p