Neutralizing Anti-IL20 Antibody Treatment Significantly Modulates Low Grade Inflammation without Affecting HbA1c in Type 2 Diabetic db/db Mice.

Low grade inflammation is present in pre-clinical and human type 2 diabetes. In this process, several cytokines like IL-1β and inflammatory cells like macrophages are activated and demonstrated to participate to the disease initiation and progression. IL-20 is a cytokine known to play non-redundant roles in progression of several inflammatory diseases. To address the therapeutic effect of inhibiting the IL-20 pathway in diabetes, diabetic db/db mice were treated with neutralizing anti-IL20 antibodies in vivo and both metabolic and inflammatory parameters were followed. Diabetic islets expressed the IL-20 cytokine and all IL-20 receptor components in elevated levels compared to resting non-diabetic islets. Islets were responsive to ex vivo IL-20 stimulation measured as SOCS induction and KC and IL-6 production. Neutralizing anti-IL20 treatment in vivo had no effect on HbA1c or weight although the slope of blood glucose increase was lowered. In contrast, anti-IL20 treatment significantly reduced the systemic low-grade inflammation and modulated the local pancreatic immunity. Significant reduction of the systemic IL-1β and MCP-1 was demonstrated upon anti-IL20 treatment which was orchestrated with a reduced RANTES, IL-16 and IL-2 but increased TIMP-1, MCP-1 and IL-6 protein expression locally in the pancreas. Interestingly, anti-IL20 treatment induced an expansion of the myeloid suppressor CD11bGr1int macrophage while reducing the number of CD8 T cells. Taken together, anti-IL20 treatment showed moderate effects on metabolic parameters, but significantly altered the low grade local and systemic inflammation. Hence, future combination therapies with anti-IL20 may provide beneficial therapeutic effects in type 2 diabetes through a reduction of inflammation

Plasma cytokine signature.

<p>At termination of the experiment, plasma was generated from the mice and analyzed for IL-1β (A), CCL2 (B), TNFα (C), IL-17 (D), IL-6 (E), CXCL10 (F) and KC (G). Graph shows individual values from each mouse (each group had 12 mice) and the corresponding geometic mean value. Statistical evaluation is performed with 1-way ANOVA.</p

Heat map analysis and the relative expression of proteins in pancreas at the termination of the experiment.

<p>Heat map analysis showing the proteins up (green) and down (red) regulated by anti-IL20 treatment arranged in order of fold modulation (increased on top and decreased on bottom) compared for individual anti-IL20 antibody treated mice compared to vehicle treated mice. Four pancreatic tissues were evaluated in each group. Statistical evaluation is performed with 1-way ANOVA.</p

Proteins regulated in the diabetic pancreatic tissue after anti-IL20 antibody treatment compared to vehicle treatment.

<p>The proteins demonstrating most pronounced inhibition by anti-IL20 antibody treatment RANTES (A), IL-16 (B), IL-2 (C), CXCL9 (D). The proteins demonstrating most pronounced induction by anti-IL20 antibody treatment TIMP-1 (E), MCP-1 (F), IL-6 (G) and CXCL13 (H). Four pancreatic tissues were evaluated in each group. Statistical evaluation is performed with 1-way ANOVA.</p

Metabolic effect and oral glucose test after anti-IL20 treatment.

<p>One week post last treatment with anti-IL-20, the mice was terminated. During the experiment and at time of termination HbA1c (A), blood glucose (B) and weight (C) was determined. Oral glucose evaluation was conducted one day before termination of the experiment (D). Area under the curve oral glucose test was calculated (E). Plasma level of insulin was measured during the oral glucose tolerance test (F). Each group contained 12 animals. Statistical evaluation comparing the start value in each group with the termination value as well as the termination value between the groups was performed with one way ANOVA in A-C. Linear regression analysis was performed to determine if the two groups showed a difference in the slope of the parameter evaluated during the time of the experiment A-C. Student’s T-test was used to determine statistical significance in E.</p

Liraglutide Reduces Both Atherosclerosis and Kidney Inflammation in Moderately Uremic LDLr-/- Mice

<div><p>Chronic kidney disease (CKD) leads to uremia. CKD is characterized by a gradual increase in kidney fibrosis and loss of kidney function, which is associated with a progressive increase in risk of atherosclerosis and cardiovascular death. To prevent progression of both kidney fibrosis and atherosclerosis in uremic settings, insight into new treatment options with effects on both parameters is warranted. The GLP-1 analogue liraglutide improves glucose homeostasis, and is approved for treatment of type 2 diabetes. Animal studies suggest that GLP-1 also dampens inflammation and atherosclerosis. Our aim was to examine effects of liraglutide on kidney fibrosis and atherosclerosis in a mouse model of moderate uremia (5/6 nephrectomy (NX)). Uremic (n = 29) and sham-operated (n = 14) atherosclerosis-prone low density lipoprotein receptor knockout mice were treated with liraglutide (1000 μg/kg, s.c. once daily) or vehicle for 13 weeks. As expected, uremia increased aortic atherosclerosis. In the remnant kidneys from NX mice, flow cytometry revealed an increase in the number of monocyte-like cells (CD68⁺F4/80^-), CD4⁺, and CD8⁺ T-cells, suggesting that moderate uremia induced kidney inflammation. Furthermore, markers of fibrosis (i.e. Col1a1 and Col3a1) were upregulated, and histological examinations showed increased glomerular diameter in NX mice. Importantly, liraglutide treatment attenuated atherosclerosis (~40%, p < 0.05) and reduced kidney inflammation in NX mice. There was no effect of liraglutide on expression of fibrosis markers and/or kidney histology. This study suggests that liraglutide has beneficial effects in a mouse model of moderate uremia by reducing atherosclerosis and attenuating kidney inflammation.</p></div

Liraglutide attenuates atherosclerosis in uremic LDLr-/- mice.

<p>Uremic LDLr-/- mice were treated with vehicle (NX; n = 14) or liraglutide (NX LIRA; n = 15) and sham operated control LDLr-/-mice were treated with vehicle (SHAM; n = 14). After 11 weeks of treatment with full dose (1000 μg/kg), atherosclerosis was quantified as the relative plaque area in % of the total aortic arch area in all mice (<b>A</b>) and in NX mice with urea levels >20 (<b>B</b>). Depicted values are mean±SEM. *p<0.05 as determined by 1-way ANOVA followed by Sidak’s multiple comparisons post-test. n = 14–15 mice per group.</p

Uremia increases glomerular size, but not cortical collagen content in LDLr-/- mice.

<p>Representative pictures of kidney sections from control (SHAM), uremic (NX) and liraglutide treated uremic (NX LIRA) mice stained with Massons trichrome (<b>A</b>). Scale bar = 200 μm for top row and 100 μm for bottom row. Glomerular diameters were measured (<b>B</b>) (25–49 glomeruli were assessed per kidney, n = 7–8 in each group) and collagen deposition in the kidney cortex was quantified (<b>C</b>) (n = 5–7 in each group) using the Visiopharm software. **p<0.01, as determined by 1-way ANOVA followed by Sidak’s multiple comparisons post-test.</p

Liraglutide attenuates NX mediated kidney inflammation in LDLr-/- mice.

<p>A-F: flow cytometry analysis of kidneys from control (SHAM; n = 7), uremic (NX; n = 7) or liraglutide treated uremic (NX LIRA; n = 7) LDLr-/- mice showing the number of CD4⁺ T-cells (<b>A</b>), CD8⁺ T-cells (<b>B</b>), monocyte-like (CD68⁺F4/80^-) and macrophage-like (CD68⁺F4/80⁺) cells (<b>C</b> and <b>D</b>) relative to kidney weight. On macrophage-like cells, median fluorescent intensity (MFI) for the M1 marker CD11c (<b>E</b>) or the M2 marker CD206 (<b>F</b>) was detected. Depicted values are mean±SEM. *p<0.05, **p<0.01, ***p<0.005 as determined by 1-way ANOVA followed by Sidak’s multiple comparisons post-test. n = 6 mice per group.</p