Global Autorecognition and Activation of Complement by Mannan-Binding Lectin in a Mouse Model of Type 1 Diabetes

Abstract

Increasing evidence links mannan-binding lectin (MBL) to late vascular complications of diabetes. MBL is a complement-activating pattern recognition molecule of the innate immune system that can mediate an inflammation response through activation of the lectin pathway. In two recent animal studies, we have shown that autoreactivity of MBL is increased in the kidney in diabetic nephropathy. We hypothesize that long-term exposure to uncontrolled high blood glucose in diabetes may mediate formation of neoepitopes in several tissues and that MBL is able to recognize these structures and thus activate the lectin pathway. To test this hypothesis, we induced diabetes by injection of low-dose streptozotocin in MBL double-knockout (MBL/DKO) mice. Development of diabetes was followed by measurements of blood glucose and urine albumin-to-creatinine ratio. Fluorophore-labelled recombinant MBL was injected intravenously in diabetic and nondiabetic mice followed by ex vivo imaging of several organs. We observed that MBL accumulated in the heart, liver, brain, lung, pancreas, and intestines of diabetic mice. We furthermore detected increased systemic complement activation after administration of MBL, thus indicating MBL-mediated systemic complement activation in these animals. These new findings indicate a global role of MBL during late diabetes-mediated vascular complications in various tissues.

Figure 1

Measurement of blood glucose and weight in diabetic and control mice. (a) Blood glucose measurement of diabetic and nondiabetic mice, (b) change in blood glucose from start to end of the study for diabetic (start: n = 16, end: n = 12) and nondiabetic (start: n = 13, end: n = 12) mice. (c) difference in body weight during the study, (d) difference in body weight from start to end of the study for diabetic (start: n = 16, end: n = 12) and nondiabetic (start: n = 13, end: n = 12) mice. Student's t-test was used to test the difference between groups, which was indicated in the figure when significant. ^∗∗p < 0.01. Data is given in mean with standard deviations (SDs).

Figure 2

Organ-to-body weight ratio of diabetic and control mice. (a) Weight ratio measurements at the end of the study of diabetic (n = 12) and nondiabetic (n = 12) mice in the (a) kidney, (b) heart, (c) liver, (d) brain, (e) spleen, and (f) lung. Student's t-test was used to test the difference between groups, which was indicated in the figure when significant. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. Data is given in mean with standard deviations (SDs).

Figure 3

Albumin-to-creatinine ratio (AcR) in diabetic and nondiabetic mice. AcR measurements of diabetic (n = 12) and nondiabetic (n = 12) mice at the end of the study. Student's t-test was used to test the difference between groups. ^∗∗∗∗p < 0.0001. Data is given in mean with standard deviations (SDs).

Figure 4

Test for preserved functionality of labelled rMBL. (a) Unlabelled and labelled rMBL were incubated on mannan-coated plates in calcium- or EDTA-containing buffers. Values measured in calcium buffer were defined as 100%, and subsequent samples were calculated according to these. (b) Unlabelled and labelled rMBL were incubated on mannan-coated plates in the presence of either mannose or galactose. Values acquired in the presence of galactose were defined as 100%, and subsequent samples were calculated according to these. Representative of two individual experiments.

Figure 5

Ex vivo imaging and region of interest (ROI) quantification of selected organs from diabetic and nondiabetic mice injected with either AF680-labelled rMBL or PBS. (a) Heart, (b) liver (right lobe), (c) brain, (d) spleen, (e) lung (superior left lobe), (f) part of the pancreas, (g) part of the small intestines, (h) and part of the large intestines. Left side, top: CCD photo, bottom: fluorescent image. Scale bar is given in radiant efficiency (photons/sec/cm²/sr)/(μW/cm²). Two-way ANOVA was used to test the difference between groups (P_int). Data is expressed in mean with standard deviations (SDs). Images are presented as CCD camera photo and fluorescence images. The experiment was repeated twice, and the data is based on 6 individual mice in each group (n = 24 total). Data was obtained with similar results.

Figure 6

Circulating C3a as a measurement of complement activation. The difference in change of C3a plasma levels from 0 h to 24 h between the nondiabetic (a) and the diabetic (b) groups was tested by Student's t-test on delta values and indicated in the figure when significant. ^∗∗p < 0.01. Data is given in mean with standard deviations (SDs).