Miriam Koch

CCL17-expressing dendritic cells drive atherosclerosis by restraining regulatory T cell homeostasis in mice

Immune mechanisms are known to control the pathogenesis of atherosclerosis. However, the exact role of DCs, which are essential for priming of immune responses, remains elusive. We have shown here that the DC-derived chemokine CCL17 is present in advanced human and mouse atherosclerosis and that CCL17+ DCs accumulate in atherosclerotic lesions. In atherosclerosis-prone mice, Ccl17 deficiency entailed a reduction of atherosclerosis, which was dependent on Tregs. Expression of CCL17 by DCs limited the expansion of Tregs by restricting their maintenance and precipitated atherosclerosis in a mechanism conferred by T cells. Conversely, a blocking antibody specific for CCL17 expanded Tregs and reduced atheroprogression. Our data identify DC-derived CCL17 as a central regulator of Treg homeostasis, implicate DCs and their effector functions in atherogenesis, and suggest that CCL17 might be a target for vascular therapy.

A dual function for chaperones SSB–RAC and the NAC nascent polypeptide–associated complex on ribosomes

In addition to assisting with protein folding, SSB and NAC also regulate ribosome biogenesis (see also companion paper from Albanèse et al. in this issue).

CD8+ T Cells Regulate Monopoiesis and Circulating Ly6C-high Monocyte Levels in Atherosclerosis in Mice.

RATIONALE Proinflammatory adaptive immune responses are recognized as major drivers of atherosclerotic lesion formation. Although CD8(+) T cells have recently been proposed as a proatherogenic cell subset, their full scope of actions remains to be elucidated. OBJECTIVE We here addressed the contribution of CD8(+) T cells to monocyte trafficking in atherosclerosis. METHOD AND RESULTS We observed that CD8(+) T cells express proinflammatory cytokines (interferon-γ, tumor necrosis factor-α, and interleukin-12) within atherosclerotic lesions and spleens of high-fat diet-fed low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice. Antibody-mediated CD8(+) T-cell depletion in high-fat diet-fed Ldlr(-/-) mice decreased atherosclerotic plaque formation, associated with decreased macrophage accumulation within lesions. Despite a reduction in vascular chemokine (CC-motif) ligand 2 and chemokine (CXC-motif) ligand 1 expression, CD8(+) T-cell depletion did not directly affect monocyte recruitment to inflamed vessels. However, CD8(+) T-cell depletion decreased chemokine (CC-motif) ligand serum concentrations and circulating Ly6C(high) monocyte counts. We further evidenced that CD8(+) T-cell depletion decreased levels of mature monocytes and myeloid granulocyte-monocyte progenitors in the bone marrow and spleen of hypercholesterolemic mice, effects that were partially reproduced by interferon-γ neutralization, showing a role for interferon-γ. CONCLUSIONS These data suggest that CD8(+) T cells promote atherosclerosis by controlling monopoiesis and circulating monocyte levels, which ultimately contributes to plaque macrophage burden without affecting direct monocyte recruitment, identifying this cell subset as a critical regulator of proatherogenic innate immune cell responses in atherosclerosis.

Programmed Cell Death-1 Deficiency Exacerbates T Cell Activation and Atherogenesis despite Expansion of Regulatory T Cells in Atherosclerosis-Prone Mice

T cell activation represents a double-edged sword in atherogenesis, as it promotes both pro-inflammatory T cell activation and atheroprotective Foxp3+ regulatory T cell (Treg) responses. Here, we investigated the role of the co-inhibitory receptor programmed cell death-1 (PD-1) in T cell activation and CD4+ T cell polarization towards pro-atherogenic or atheroprotective responses in mice. Mice deficient for both low density lipoprotein receptor and PD-1 (Ldlr−/−Pd1−/−) displayed striking increases in systemic CD4+ and CD8+ T cell activation after 9 weeks of high fat diet feeding, associated with an expansion of both pro-atherogenic IFNγ-secreting T helper 1 cells and atheroprotective Foxp3+ Tregs. Importantly, PD-1 deficiency did not affect Treg suppressive function in vitro. Notably, PD-1 deficiency exacerbated atherosclerotic lesion growth and entailed a massive infiltration of T cells in atherosclerotic lesions. In addition, aggravated hypercholesterolemia was observed in Ldlr−/−Pd1−/− mice. In conclusion, we here demonstrate that although disruption of PD-1 signaling enhances both pro- and anti-atherogenic T cell responses in Ldlr−/− mice, pro-inflammatory T cell activation prevails and enhances dyslipidemia, vascular inflammation and atherosclerosis.

Not4‐dependent translational repression is important for cellular protein homeostasis in yeast

Translation of aberrant or problematic mRNAs can cause ribosome stalling which leads to the production of truncated or defective proteins. Therefore, cells evolved cotranslational quality control mechanisms that eliminate these transcripts and target arrested nascent polypeptides for proteasomal degradation. Here we show that Not4, which is part of the multifunctional Ccr4–Not complex in yeast, associates with polysomes and contributes to the negative regulation of protein synthesis. Not4 is involved in translational repression of transcripts that cause transient ribosome stalling. The absence of Not4 affected global translational repression upon nutrient withdrawal, enhanced the expression of arrested nascent polypeptides and caused constitutive protein folding stress and aggregation. Similar defects were observed in cells with impaired mRNA decapping protein function and in cells lacking the mRNA decapping activator and translational repressor Dhh1. The results suggest a role for Not4 together with components of the decapping machinery in the regulation of protein expression on the mRNA level and emphasize the importance of translational repression for the maintenance of proteome integrity.

Deficiency of HIF1α in Antigen-Presenting Cells Aggravates Atherosclerosis and Type 1 T-Helper Cell Responses in Mice

Objective—Although immune responses drive the pathogenesis of atherosclerosis, mechanisms that control antigen-presenting cell (APC)–mediated immune activation in atherosclerosis remain elusive. We here investigated the function of hypoxia-inducible factor (HIF)-1&agr; in APCs in atherosclerosis. Approach and Results—We found upregulated HIF1&agr; expression in CD11c+ APCs within atherosclerotic plaques of low-density lipoprotein receptor–deficient (Ldlr−/−) mice. Conditional deletion of Hif1a in CD11c+ APCs in high-fat diet–fed Ldlr−/− mice accelerated atherosclerotic plaque formation and increased lesional T-cell infiltrates, revealing a protective role of this transcription factor. HIF1&agr; directly controls Signal Transducers and Activators of Transcription 3 (Stat3), and a reduced STAT3 expression was found in HIF1&agr;-deficient APCs and aortic tissue, together with an upregulated interleukin-12 expression and expansion of type 1 T-helper (Th1) cells. Overexpression of STAT3 in Hif1a-deficient APCs in bone marrow reversed enhanced atherosclerotic lesion formation and reduced Th1 cell expansion in chimeric Ldlr−/− mice. Notably, deletion of Hif1a in LysM+ bone marrow cells in Ldlr−/− mice did not affect lesion formation or T-cell activation. In human atherosclerotic lesions, HIF1&agr;, STAT3, and interleukin-12 protein were found to colocalize with APCs. Conclusions—Our findings identify HIF1&agr; to antagonize APC activation and Th1 T cell polarization during atherogenesis in Ldlr−/− mice and to attenuate the progression of atherosclerosis. These data substantiate the critical role of APCs in controlling immune mechanisms that drive atherosclerotic lesion development.

Dendritic cell subset distributions in the aorta in healthy and atherosclerotic mice.

Dendritic cells (DCs) can be sub-divided into various subsets that play specialized roles in priming of adaptive immune responses. Atherosclerosis is regarded as a chronic inflammatory disease of the vessel wall and DCs can be found in non-inflamed and diseased arteries. We here performed a systematic analyses of DCs subsets during atherogenesis. Our data indicate that distinct DC subsets can be localized in the vessel wall. In C57BL/6 and low density lipoprotein receptor-deficient (Ldlr −/−) mice, CD11c+ MHCII+ DCs could be discriminated into CD103− CD11b+F4/80+, CD11b+F4/80− and CD11b−F4/80− DCs and CD103+ CD11b−F4/80− DCs. Except for CD103− CD11b− F4/80− DCs, these subsets expanded in high fat diet-fed Ldlr −/− mice. Signal-regulatory protein (Sirp)-α was detected on aortic macrophages, CD11b+ DCs, and partially on CD103− CD11b− F4/80− but not on CD103+ DCs. Notably, in FMS-like tyrosine kinase 3-ligand-deficient (Flt3l −/−) mice, a specific loss of CD103+ DCs but also CD103− CD11b+ F4/80− DCs was evidenced. Aortic CD103+ and CD11b+ F4/80− CD103− DCs may thus belong to conventional rather than monocyte-derived DCs, given their dependence on Flt3L-signalling. CD64, postulated to distinguish macrophages from DCs, could not be detected on DC subsets under physiological conditions, but appeared in a fraction of CD103− CD11b+ F4/80− and CD11b+ F4/80+ cells in atherosclerotic Ldlr −/− mice. The emergence of CD64 expression in atherosclerosis may indicate that CD11b+ F4/80− DCs similar to CD11b+ F4/80+ DCs are at least in part derived from immigrated monocytes during atherosclerotic lesion formation. Our data advance our knowledge about the presence of distinct DC subsets and their accumulation characteristics in atherosclerosis, and may help to assist in future studies aiming at specific DC-based therapeutic strategies for the treatment of chronic vascular inflammation.

CCR6 selectively promotes monocyte mediated inflammation and atherogenesis in mice

The chemokine receptor CCR6 is expressed by various cell subsets implicated in atherogenesis, such as monocytes, Th17 and regulatory T cells. In order to further define the role of CCR6 in atherosclerosis, CCR6-deficient (Ccr6-/-) mice were crossed with low-density lipoprotein receptor-deficient (Ldlr-/-) mice to generate atherosclerosis-prone mice deficient in CCR6. Compared to Ldlr-/- controls, atherosclerotic burden in the aortic sinus and aorta were reduced in Ccr6-/-Ldlr-/- mice fed a high fat diet, associated with a profound depression in lesional macrophage accumulation. Local and systemic distributions of T cells, including frequencies of Th1, Th17 and regulatory T cells were unaltered. In contrast, circulating counts of both Gr-1(high) and Gr1(low) monocytes were reduced in Ccr6-/-Ldlr-/- mice. Moreover, CCR6 was revealed to promote monocyte adhesion to inflamed endothelium in vitro and leukocyte adhesion to carotid arteries in vivo. Finally, CCR6 selectively recruited monocytes but not T cells in an acute inflammatory air pouch model. We here show that CCR6 functions on multiple levels and regulates the mobilisation, adhesion and recruitment of monocytes/macrophages to the inflamed vessel, thereby promoting atherosclerosis, but is dispensable for hypercholesterolaemia-associated adaptive immune priming. Targeting CCR6 or its ligand CCL20 may therefore be a promising therapeutic strategy to alleviate atherosclerosis.

Directed PCR-free engineering of highly repetitive DNA sequences

BackgroundHighly repetitive nucleotide sequences are commonly found in nature e.g. in telomeres, microsatellite DNA, polyadenine (poly(A)) tails of eukaryotic messenger RNA as well as in several inherited human disorders linked to trinucleotide repeat expansions in the genome. Therefore, studying repetitive sequences is of biological, biotechnological and medical relevance. However, cloning of such repetitive DNA sequences is challenging because specific PCR-based amplification is hampered by the lack of unique primer binding sites resulting in unspecific products.ResultsFor the PCR-free generation of repetitive DNA sequences we used antiparallel oligonucleotides flanked by restriction sites of Type IIS endonucleases. The arrangement of recognition sites allowed for stepwise and seamless elongation of repetitive sequences. This facilitated the assembly of repetitive DNA segments and open reading frames encoding polypeptides with periodic amino acid sequences of any desired length. By this strategy we cloned a series of polyglutamine encoding sequences as well as highly repetitive polyadenine tracts. Such repetitive sequences can be used for diverse biotechnological applications. As an example, the polyglutamine sequences were expressed as His6-SUMO fusion proteins in Escherichia coli cells to study their aggregation behavior in vitro. The His6-SUMO moiety enabled affinity purification of the polyglutamine proteins, increased their solubility, and allowed controlled induction of the aggregation process. We successfully purified the fusions proteins and provide an example for their applicability in filter retardation assays.ConclusionOur seamless cloning strategy is PCR-free and allows the directed and efficient generation of highly repetitive DNA sequences of defined lengths by simple standard cloning procedures.

Coagulation factor XII induces pro-inflammatory cytokine responses in macrophages and promotes atherosclerosis in mice

Atherosclerosis is considered a chronic inflammatory disease of the vessel wall. Coagulation pathways and immune responses contribute to disease development. The role of coagulation factor XII (FXII) in vascular inflammation, however, remains controversial. We here investigated the function of FXII in atherosclerosis using apolipoprotein E and FXII-deficient (F12-/-Apoe-/-) mice. Compared to F12+/+Apoe-/- controls, atherosclerotic lesion formation was reduced in F12-/-Apoe-/- mice. This was associated with a decrease in serum interleukin (IL)-1β and IL-12 levels and reduced expression of pro-inflammatory cytokines in the aorta in atherosclerotic F12-/-Apoe-/- mice, as well as diminished Th1-cell differentiation in the aorta, blood, and lymphoid organs. No changes in circulating bradykinin, thrombin-antithrombin-complexes or plasminogen were observed. Mechanistically, activated FXII (FXIIa) was revealed to directly induce bone marrow-derived macrophages to secrete pro-inflammatory cytokines, including tumour necrosis factor-α, IL-1β, IL-12, and IL-6. Exposure of bone marrow-derived antigen presenting cells to FXIIa similarly induced pro-inflammatory cytokines, and an enhanced capacity to trigger antigen-specific interferon γ-production in CD4+ T cells. Notably, bone-marrow derived macrophages were capable of directly activating FXII. Moreover, the induction of cytokine expression by FXIIa in macrophages occurred independently of FXII protease enzymatic activity and was decreased upon phospholipase C treatment, suggesting urokinase-type plasminogen activator receptor (uPAR) to confer FXIIa-induced cell signalling. These data reveal FXII to play an important role in atherosclerotic lesion formation by functioning as a strong inducer of pro-inflammatory cytokines in antigen-presenting cells. Targeting of FXII may thus be a promising approach for treating cardiovascular disease.