Functional immunomics: Microarray analysis of IgG autoantibody repertoires predicts the future response of NOD mice to an inducer of accelerated diabetes

One's present repertoire of antibodies encodes the history of one's past immunological experience. Can the present autoantibody repertoire be consulted to predict resistance or susceptibility to the future development of an autoimmune disease? Here we developed an antigen microarray chip and used bioinformatic analysis to study a model of type 1 diabetes developing in non-obese diabetic (NOD) male mice in which the disease was accelerated and synchronized by exposing the mice to cyclophosphamide at 4 weeks of age. We obtained sera from 19 individual mice, treated the mice to induce cyclophosphamide-accelerated diabetes (CAD), and found, as expected, that 9 mice became severely diabetic while 10 mice permanently resisted diabetes. We again obtained serum from each mouse afterCAD induction. We then analyzed the patterns of antibodies in the individualmice to 266 different antigens spotted on the antigen chip. We identified a select panel of 27 different antigens (10% of the array) that revealed a pattern of IgG antibody reactivity in the pre-CAD serathat discriminated between the mice resistant or susceptible to CAD with 100% sensitivity and 82% specificity (p=0.017). Surprisingly, the set of IgG antibodies that was informative before CAD induction did not separate the resistant and susceptible groups after the onset of CAD; new antigens became criticalfor post-CAD repertoire discrimination. Thus, at least for a model disease, present antibody repertoires can predict future disease; predictive and diagnostic repertoires can differ; and decisive information about immune system behavior can be mined by bioinformatic technology. Repertoires matter.Comment: See Advanced Publication on the PNAS website for final versio

Microbially mediated reduction of FeIII and AsV in Cambodian sediments amended with 13C-labelled hexadecane and kerogen

Microbial activity is generally accepted to play a critical role, with the aid of suitable organic carbon substrates, in the mobilisation of arsenic from sediments into shallow reducing groundwaters. The nature of the organic matter in natural aquifers driving the reduction of AsV to AsIII is of particular importance but is poorly understood. In this study, sediments from an arsenic rich aquifer in Cambodia were amended with two 13C-labelled organic substrates. 13C-hexadecane was used as a model for potentially bioavailable long chain n-alkanes and a 13C-kerogen analogue as a proxy for non-extractable organic matter. During anaerobic incubation for 8 weeks, significant FeIII reduction and AsIII mobilisation were observed in the biotic microcosms only, suggesting that these processes were microbially driven. Microcosms amended with 13C-hexadecane exhibited a similar extent of FeIII reduction to the non-amended microcosms, but marginally higher AsIII release. Moreover, gas chromatography–mass spectrometry analysis showed that 65 % of the added 13C-hexadecane was degraded during the 8-week incubation. The degradation of 13C-hexadecane was microbially driven, as confirmed by DNA stable isotope probing (DNA-SIP). Amendment with 13C-kerogen did not enhance FeIII reduction or AsIII mobilisation, and microbial degradation of kerogen could not be confirmed conclusively by DNA-SIP fractionation or 13C incorporation in the phospholipid fatty acids. These data are, therefore, consistent with the utilisation of long chain n-alkanes (but not kerogen) as electron donors for anaerobic processes, potentially including FeIII and AsV reduction in the subsurface

Monocyte accumulation in mouse atherogenesis is progressive and proportional to extent of disease

Monocytes participate importantly in the pathogenesis of atherosclerosis, but their spatial and temporal recruitment from circulation remains uncertain. This study tests the hypothesis that monocyte accumulation in atheroma correlates with the extent of disease by using a sensitive and simple quantitative assay that allows tracking of highly enriched populations of blood monocytes. A two-step isolation method yielded viable and functionally intact highly enriched peripheral blood monocyte populations (>90%). Recipient mice received syngeneic monocytes labeled in two ways: by transgenically expressing EGFP or with a radioactive tracer [(111)In]oxine. After 5 days, more labeled cells accumulated in the aorta, principally the aortic root and ascending aorta, of 10-wk-old ApoE(−/−) compared with 10-wk-old C57BL/6 mice (223 ± 3 vs. 87 ± 22 cells per aorta). Considerably more monocytes accumulated in 20-wk-old ApoE(−/−) mice on either chow (314 ± 41 cells) or high-cholesterol diet (395 ± 53 cells). Fifty-week-old ApoE(−/−) mice accumulated even more monocytes in the aortic root, ascending aorta, and thoracic aorta after both chow (503 ± 67 cells) or high-cholesterol diet (648 ± 81 cells). Labeled monocyte content in the aorta consistently correlated with lesion surface area. These data indicate that monocytes accumulate continuously during atheroma formation, accumulation increases in proportion to lesion size, and recruitment is augmented with hypercholesterolemia. These results provide insights into mechanisms of atherogenesis and have implications for the duration of therapies directed at leukocyte recruitment

T-bet deficiency reduces atherosclerosis and alters plaque antigen-specific immune responses

The influence of the immune system on atherosclerosis involves both helper T (Th) cell and antibody responses to plaque antigens. These responses may have proatherogenic and protective effects. T-bet is a transcription factor required for Th1 differentiation and regulates the balance between Th1 and Th2 responses in inflammatory diseases. To clarify how helper T cell subset differentiation influences atherosclerosis, we compared lesion development and immune responses to plaque antigens in low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice with or without functional T-bet genes. Atherosclerosis was significantly reduced in T-bet-deficient Ldlr(-/-) mice compared with Ldlr(-/-) controls, and the lesions that did develop in the absence of T-bet had less smooth muscle cell content. Furthermore, T-bet deficiency caused a Th2 switch in the response to the atherosclerosis-associated antigen heat shock protein-60, and a change in T-dependent isotypes of oxidized LDL-specific antibodies. Of particular significance, T-bet deficiency caused a >250% increase in the titer of E06 antibodies, which are known to be atheroprotective and whose production by B-1 B cells is enhanced by IL-5. These findings establish that T cell subset differentiation influences both T cell and antibody responses that modulate atherosclerosis, and validate the therapeutic goal of skewing T responses to atherosclerosis-associated antigens

Immune dysregulation accelerates atherosclerosis and modulates plaque composition in systemic lupus erythematosus

Patients with systemic lupus erythematosus (SLE) have accelerated atherosclerosis. The underlying mechanisms are poorly understood, and investigations have been hampered by the absence of animal models that reflect the human condition of generalized atherosclerosis and lupus. We addressed this problem by transferring lupus susceptibility to low-density lipoprotein (LDL) receptor-deficient (LDLr(−/−)) mice, an established model of atherosclerosis, creating radiation chimeras with NZM2410-derived, lupus-susceptible, B6.Sle1.2.3 congenic or C57BL/6 control donors (LDLr.Sle and LDLr.B6, respectively). LDLr.Sle mice developed a lupus-like disease characterized by production of double-stranded DNA autoantibodies and renal disease. When fed a Western-type diet, LDLr.Sle chimeras had increased mortality and atherosclerotic lesions. The plaques of LDLr.Sle mice were highly inflammatory and contained more CD3(+) T cells than controls. LDLr.Sle mice also had increased activation of CD4(+) T and B cells and significantly higher antibody to oxidized LDL and cardiolipin. Collectively, these studies demonstrate that the lupus-susceptible immune system enhances atherogenesis and modulates plaque composition

Bispecific Abs against modified protein and DNA with oxidized lipids

4-Hydroxy-2-nonenal (HNE), a racemic mixture of 4R- and 4S-enantiomers, is a major product of lipid peroxidation and is believed to be largely responsible for the cytopathological effects observed during oxidative stress. HNE reacts with histidine to form a stable HNE-histidine Michael addition-type adduct possessing three chiral centers in the cyclic hemiacetal structure. We have previously raised the mAbs, anti-R mAb 310 and anti-S mAb S412, that enantioselectively recognized the R-HNE-histidine and R-HNE-histidine adducts, respectively, and demonstrated the presence of both epitopes in vivo. In the present study, to further investigate the anti-HNE immune response, we analyzed the variable genes and primary structure of these Abs and found that the sequence of R310 was highly homologous to anti-DNA autoantibodies, the hallmark of systemic lupus erythematosus. An x-ray crystallographic analysis of the R310 Fab fragment showed that the R-HNE-histidine adduct binds to a hydrophobic pocket in the antigen-binding site. Despite the structural identity to the anti-DNA autoantibodies, however, R310 showed only a slight crossreactivity with the native double-stranded DNA, whereas the Ab immunoreactivity was dramatically enhanced by the treatment of the DNA with 4-oxo-2-nonenal (ONE), an analog of HNE. Moreover, the 7-(2-oxo-heptyl)-substituted 1,N(2)-etheno-type ONE-2′-deoxynucleoside adducts were identified as alternative epitopes of R310. Molecular mimicry between the R-HNE-histidine configurational isomers and the ONE-DNA base adducts is proposed for the dual crossreactivity