Germinal Center-Derived Antibodies Promote Atherosclerosis Plaque Size and Stability.

BACKGROUND: Atherosclerosis progression is modulated by interactions with the adaptive immune system. Humoral immunity can help protect against atherosclerosis formation; however, the existence, origin, and function of putative atherogenic antibodies are controversial. How such atherosclerosis-promoting antibodies could affect the specific composition and stability of plaques, as well as the vasculature generally, remains unknown. METHODS: We addressed the overall contribution of antibodies to atherosclerosis plaque formation, composition, and stability in vivo (1) with mice that displayed a general loss of antibodies, (2) with mice that had selectively ablated germinal center-derived IgG production, or (3) through interruption of T-B-cell interactions and further studied the effects of antibody deficiency on the aorta by transcriptomics. RESULTS: Here, we demonstrate that atherosclerosis-prone mice with attenuated plasma cell function manifest reduced plaque burden, indicating that antibodies promote atherosclerotic lesion size. However, the composition of the plaque was altered in antibody-deficient mice, with an increase in lipid content and decreases in smooth muscle cells and macrophages, resulting in an experimentally validated vulnerable plaque phenotype. Furthermore, IgG antibodies enhanced smooth muscle cell proliferation in vitro in an Fc receptor-dependent manner, and antibody-deficient mice had decreased neointimal hyperplasia formation in vivo. These IgG antibodies were shown to be derived from germinal centers, and mice genetically deficient for germinal center formation had strongly reduced atherosclerosis plaque formation. mRNA sequencing of aortas revealed that antibodies are required for the sufficient expression of multiple signal-induced and growth-promoting transcription factors and that aortas undergo large-scale metabolic reprograming in their absence. Using an elastase model, we demonstrated that absence of IgG results in an increased severity of aneurysm formation. CONCLUSIONS: We propose that germinal center-derived IgG antibodies promote the size and stability of atherosclerosis plaques, through promoting arterial smooth muscle cell proliferation and maintaining the molecular identity of the aorta. These results could have implications for therapies that target B cells or B-T-cell interactions because the loss of humoral immunity leads to a smaller but less stable plaque phenotype.

Green listed-a CRISPR screen tool.

Motivation: Genome editing using versions of the bacterial CRISPR/Cas9 system can be used to probe the function of selected genes in any organism. Green Listed is a web-based tool that rapidly designs custom CRISPR screens targeting sets of genes defined by the user. It could thus be used to design screens targeting for example all genes differentially expressed during a specific stimuli or all genes related to a specific pathway or function, as well as to generate targeted secondary screens following a large-scale screen. Availability and Implementation: The software, including a demo function as well as explanatory texts and videos, is available through greenlisted.cmm.ki.se . Contact: fredrik.wermeling@ki.se.

Proteins linked to extinction in contextual fear conditioning in the C57BL/6J mouse.

Studying fear extinction is a major topic in neuroscience. No information on systematic studies on the linkage of contextual fear conditioning (cFC) with hippocampal protein levels is available and we were therefore interested in protein differences between animals with poor and good extinction. cFC was carried out in C57BL/6J mice, hippocampi were taken and proteins were run on two-dimensional gel electrophoresis with subsequent quantification of protein spots. In-gel digestion with trypsin and identification by ion trap MS/MS (high-capacity ion trap) was used for the identification of significantly different hippocampal proteins between mice with good and poor performance of extinction. Signaling protein ras-related protein rab-7A and septin 8 levels were significantly higher in hippocampus of poor extinguishers, whereas ubiquitin carboxyterminal hydrolase isozyme L1 showed higher levels in animals with good extinction performance. A series of additional proteins showed significantly different levels between groups but the abovementioned were confirmed by immunoblotting. The abovementioned proteins have never been reported to be linked to extinction, memory, or learning and herein evidence for the involvement of several proteins in extinction mechanism as well as probably representing pharmaceutical targets is provided. Moreover, it is intriguing to demonstrate the differences between good and poor extinction performance at the protein level.

Differences in hippocampal protein levels between C57Bl/6J, PWD/PhJ, and Apodemus sylvaticus are paralleled by differences in spatial memory.

There is a significant strain-dependent performance in the Morris water maze (MWM), a paradigm for the evaluation of spatial memory. In contrast, information on molecular differences that may be responsible for differences in spatial memory performance is limited. The aim of the study was therefore to investigate differences in hippocampal protein levels in three groups with different performance in the MWM. C57BL/6J (inbred laboratory strain), PWD/PhJ (inbred strain derived from wild animals of Mus musculus), and Apodemus sylvaticus (AS, genus Apodemus) mice were used for the experiments. Proteins from hippocampi, obtained from a behavioral study on these animals, were extracted and run on two-dimensional gel electrophoresis. Proteins spots were quantified, and spots with significantly different levels were identified by mass spectrometry using an ion trap. A series of 49 proteins from different pathways and cascades (signaling, neuronal network, protein synthesis, secretion and degradation, and antioxidant system; intermediary, fat, and carbohydrate metabolism) were significantly different among hippocampi at the stringent statistical level of P ≤ 0.001. These findings are paralleled by differences in the spatial navigation abilities between the strains within the species Mus musculus (C57BL/6 vs. PWD/PhJ) and between the genera Mus and Apodemus. As shown previously, AS learned the task in the MWM and showed good memory retention when tested at the probe trial (day 12), whereas C57BL/6J learned the task, but failed at the probe trial at day 12 as well as PWD/PhJ that failed to learn the task and failed at the probe trial at day 12. A list of above-mentioned proteins were different between PWD/PhJ with bad and AS with good memory retention and may therefore be related to performance in the MWM and thus to spatial memory formation. The experimental approach, however, does not allow discriminating between differences in protein levels a priori and different protein levels induced by the MWM testing. © 2010 Wiley-Liss, Inc.

Biased TCR gene usage in citrullinated Tenascin C specific T-cells in rheumatoid arthritis.

We aimed to search for common features in the autoreactive T cell receptor (TCR) repertoire in patients with rheumatoid arthritis (RA), focusing on the newly identified candidate antigen citrullinated Tenascin C (cit-TNC). Mononuclear cells from peripheral blood or synovial fluid of eight RA-patients positive for the RA-associated HLA-DRB1*04:01 allele were in-vitro cultured with recently identified citrullinated peptides from Tenascin C. Antigen-specific T cells were isolated using peptide-HLA tetramer staining and subsequently single-cell sequenced for paired alpha/beta TCR analyses by bioinformatic tools. TCRs were re-expressed for further studies of antigen-specificity and T cell responses. Autoreactive T cell lines could be grown out from both peripheral blood and synovial fluid. We demonstrate the feasibility of retrieving true autoreactive TCR sequences by validating antigen-specificity in T cell lines with re-expressed TCRs. One of the Tenascin C peptides, cit-TNC22, gave the most robust T cell responses including biased TCR gene usage patterns. The shared TCR-beta chain signature among the cit-TNC22-specific TCRs was evident in blood and synovial fluid of different patients. The identification of common elements in the autoreactive TCR repertoire gives promise to the possibility of both immune monitoring of the autoimmune components in RA and of future antigen- or TCR-targeted specific intervention in subsets of patients.

SNAP-23 and VAMP-3 contribute to the release of IL-6 and TNFα from a human synovial sarcoma cell line.

Fibroblast-like synoviocytes are important mediators of inflammatory joint damage in arthritis through the release of cytokines, but it is unknown whether their exocytosis from these particular cells is SNARE-dependent. Here, the complement of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in human synovial sarcoma cells (SW982) was examined with respect to the secretion of interleukin-6 (IL-6) and tumour necrosis factor α (TNFα), before and after knockdown of a synaptosome-associated protein of molecular mass 23 kDa (SNAP-23) or the vesicle-associated membrane protein 3 (VAMP-3). Wild-type SW982 cells expressed SNAP-23, VAMP-3, syntaxin isoforms 2-4 and synaptic vesicle protein 2C (SV2C). These cells showed Ca²âº-dependent secretion of IL-6 and TNFα when stimulated by interleukin-1ß (IL-1ß) or in combination with K⁺ depolarization. Specific knockdown of SNAP-23 or VAMP-3 decreased the exocytosis of IL-6 and TNFα; the reduced expression of SNAP-23 caused accumulation of SV2 in the peri-nuclear area. A monoclonal antibody specific for VAMP-3 precipitated SNAP-23 and syntaxin-2 (and syntaxin-3 to a lesser extent). The formation of SDS-resistant complexes by SNAP-23 and VAMP-3 was reduced upon knockdown of SNAP-23. Although the syntaxin isoforms 2, 3 and 4 are expressed in SW982 cells, knockdown of each did not affect the release of cytokines. Collectively, these results show that SNAP-23 and VAMP-3 participate in IL-1ß-induced Ca²âº-dependent release of IL-6 and TNFα from SW982 cells.