Early window of diabetes determinism in NOD mice, dependent on the complement receptor CRIg, identified by noninvasive imaging - PubMed (original) (raw)

Early window of diabetes determinism in NOD mice, dependent on the complement receptor CRIg, identified by noninvasive imaging

Wenxian Fu et al. Nat Immunol. 2012.

Abstract

All juvenile mice of the nonobese diabetic (NOD) strain develop insulitis, but there is considerable variation in their progression to diabetes. Here we used a strategy based on magnetic resonance imaging (MRI) of magnetic nanoparticles to noninvasively visualize local effects of pancreatic-islet inflammation to predict the onset of diabetes in NOD mice. MRI signals acquired during a narrow early time window allowed us to sort mice into groups that would progress to clinical disease or not and to estimate the time to diabetes development. We exploited this approach to identify previously unknown molecular and cellular elements correlated with disease protection, including the complement receptor of the immunoglobulin superfamily (CRIg), which marked a subset of macrophages associated with diabetes resistance. Administration of a fusion of CRIg and the Fc portion of immunoglobulin resulted in lower MRI signals and diabetes incidence. In addition to identifying regulators of disease progression, we show here that diabetes is set at an early age in NOD mice.

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Figures

Figure 1

Figure 1

Prediction of T1D in NOD mice by MRI-MNP: cross-sectional cohort. (a) MNP-MRI methodology. Top left: A representative T2-weighted MRI image (coronal view). M, muscle; P, pancreas; Sp, spleen; St, stomach. Top right: Pancreas and muscle T2 values were calculated using T2 Fit-map plug-ins from OsiriX software. A screenshot of one such Fit-map is shown. Te, echo time. Bottom: Pancreatic and muscle T2 values measured at the three standard time-points (pre-MNP, post-MNP, 24hr post-MNP) for a representative mouse. Probe accumulation value was calculated as ln(T2pre-MNP/T224hr post-MNP) (b) A cohort of 22 female NOD mice 10–17 weeks of age was injected with MNP; MRI scans were performed and analyzed as illustrated in panel A and detailed in Experimental Procedures. Animals were followed until 40 weeks of age, and were placed retrospectively into “pre-diabetic” and “non-diabetic” groups that did or did not develop clinical diabetes, respectively. *_P_=0.02; **_P_=0.0023. P value was determined by the Student’s t test (Prism5, Graphpad Inc.) (c) Correlation between baseline T2 and probe accumulation values. R2=0.8397, P<0.0001. P value was determined by the F test (Prism5, Graphpad Inc.). Throughout the figure: ms, microsecond; AU, arbitrary units; NS, not significant.

Figure 2

Figure 2

Prediction of T1D in NOD mice: longitudinal cohorts. Cohorts of 3-week-old female NOD mice were MNP injected and imaged as per Figure. 1, and then were re-injected and re-imaged every 3–4 weeks until 18 weeks of age. Individuals were followed until 40 weeks, permitting retroactive assignment into groups that did (pre-diabetic) or did not (non-diabetic) develop clinical diabetes. (a) Pancreatic T2pre-MNP values over time. *_P_=0.0194; **_P_=0.0006. (b) Pancreatic probe accumulation values. *_P_=0.0037; **_P_=0.0001. (c) Correlation between pancreatic probe accumulation values measured at 10 weeks and the time-to-onset of diabetes (n=13). R2=0.5557, _P_=0.0034. (d,e) Comparison of success of MNP-MRI (left) and insulin autoantibody (IAA) (right) assays in T1D prediction. A small cohort of 10-week-old mice was assayed by both of these methods, and was then followed for diabetes development until 40 weeks of age. (d) 10 week values for individual mice. *_P_=0.1167; **_P_=0.0031. (e) Correlation between values at 10 weeks and time to diabetes onset. Left, R2=0.7073, _P_=0.0359. Right, R2=0.3301, _P_=0.0645. Each symbol represents one mouse; bars indicate the group mean. P values were determined by Student’s t test (a,b,d) or F test (c,e). ms, microsecond; AU, arbitrary units; NS, not significant.

Figure 3

Figure 3

Gene-expression profiling of pancreas-resident hematopoietic cells as a function of MRI signal. 10-week-old NOD females were MRI-scanned, the pancreas was excised and its cells dispersed, and gene expression profiling was performed on purified CD45+ cells. Gene expression values were correlated to the T2 values across all mice. (a) Normalized expression profiles of genes whose expression was most positively or negatively correlated with T2 values (each line represents an individual gene), super-imposed on the T2pre-MNP values for the mice (blue bars). (b) Distribution of expression/T2pre-MNP correlation coefficients for all genes (blue histogram). The red dotted line shows the distribution of correlation coefficients observed in a randomized dataset. (c) Cell-type-distribution analysis. Normalized expression values of the 100 genes most negatively correlated to T2 were examined for a panel of immune cells – data retrieved from the ImmGen database . T.4Nve.Sp, T.8Nve.Sp, spleen naïve CD4 and CD8+ T cells; T.4FP3+25+.Sp, spleen natural Treg cells; B.T1.Sp, B.T2.Sp, B.T3.Sp, spleen transitional B cells (stages 1, 2, 3); NK, natural killer cells; DC.4+.Sp, DC.8+.Sp, DC.4-8-11b+.Sp, DC.4-8-11b-.Sp, spleen CD4+, CD8+ CD4/8-double negative CD11b+ and CD4/8/11b-triple negative DCs; Mo.6C+II-.Bl, blood Ly6C+ MHC II− monocytes; MF, macrophages; Lu, lung, RP, spleen red pulp; MF.II-480hi.PC, peritoneal MHC II−F4/80hi macrophages.

Figure 4

Figure 4

Myeloid-lineage cell subsets infiltrating the pancreas at 10 weeks. Twenty female NOD mice were MRI-scanned at 10 weeks of age, whereupon they were euthanized, the pancreas isolated and dispersed, and cells analyzed by flow cytometry after staining with antibodies recognizing: CD45, CD11b, Ly6C, CD11c, and Gr-1. (a) Myeloid cell gating strategy: cells were pre-gated as CD45+DAPI−, and were secondarily separated according to display of CD11b and Ly6C; the Ly6C− subset was further split according to expression of Gr1 and CD11c. R1, Ly6Chi monocytes; R2, Ly6Clo monocytes; R3, MFs; R5, CD11chi DCs; R6, CD11clo pre-DCs; R7, neutrophils , . (b) Correlation between the percentage of CD11b+ in CD45+ cells and the pancreatic T2pre-MNP value. R2=0.2392, _P_=0.0287. (c) Correlation between the fraction of CD45+ cells comprised by each subset of myeloid cells and the pancreatic T2pre-MNP value. The R1–R3 and R5–R7 populations were those delineated in panel A. Solid rectangles highlight those plots with statistically significant P values, determined by F test.

Figure 5

Figure 5

Enrichment of CRIg+ tissue macrophages in pancreas of NOD mice protected from diabetes. (a) Genes associated with diabetes protection, i.e., strongly anti-correlated with pancreatic T2pre-MNP values fall into distinct functional classes. Gray bars represent the correlation coefficient between transcript level and pancreatic T2pre-MNP signal. (b) Correlation between pancreatic T2pre-MNP values (dotted line) and Crig transcript levels (solid line). Determined from the same microarray dataset as in Fig. 3. (c) Crig expression in different cell types isolated from the pancreas of 10-week-old NOD mice was quantified by quantitative RT-PCR. (d) CRIg expression on pancreatic myeloid-lineage cells from 10-week-old NOD mice. R1–R4 correspond to the regions gated in the left panel. (e,f) Strong negative correlation between the fraction of CRIg+ macrophages in the pancreatic lesion and the T2pre-MNP signal from a cohort of 10-week-old NOD females independent of those used for Fig. 4. (e) The value at the upper right of each panel represents the fraction of cells that were CRIg+ in the CD45+DAPI− CD11b+F4/80+ gate, ie macrophages. CRIg-positivity was set by comparison with an isotype-matched control mAb. (f) The correlation between MRI signals and % of CRIg+ macrophages in the pancreas for this cohort. R2=0.9212, _P_=0.0096.

Figure 6

Figure 6

Inhibition of pancreatic MRI-MNP signal and protection from diabetes after CRIg-Fc treatment of NOD female mice. (a) Experimental design: 3-week-old female NOD mice were ip-injected with CRIg-Fc or control-Fc twice a week until 10 weeks of age. They were MRI-MNP screened at 7–8 weeks and followed for diabetes development until 32 weeks. (b) Probe accumulation (left) and pancreatic T2 pre-MNP values (right) and in CRIg-Fc treated mice (_n_=10) compared with control-Fc treated mice (_n_=9). *_P_=0.0012; **_P_=0.0001. (c) Diabetes development in the same cohort. *_P_=0.0196. P value was determined by the Gehan-Breslow-Wilcoxon test (Prism5, Graphpad Inc.).

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