Interruption of the CXCL13/CXCR5 Chemokine Axis Enhances Plasma IgM Levels and Attenuates Atherosclerosis DevelopmentFunding The authors' research is supported by grant NHS2005B294 from the Dutch Heart Foundation to E.A.L.B.; by the Alexander von Humboldt Foundation, a grant from the Interdisciplinary Center for Clinical Research within the faculty of Medicine at the RWTH Aachen University, the DZHK (German Centre for Cardiovascular Research) and the BMBF (German Ministry of Education and Research), and NWO-ZonMw Veni (91619053) to E.P.C.v.d.V; by the DFG (SFB1123 TP A1) and by the European Research Council (AdG°692511) to C.W.
03 October 2019
18 October 2019
14 December 2019 (online)
Atherosclerosis is a chronic inflammatory disease typified by the subendothelial accumulation of lipids and inflammatory cells. With disease progression, lesions can develop into fibroatheromatous lesions, prone to rupture causing thrombus formation, which is the main cause of cardiovascular complications like myocardial infarction and stroke. Chemokines play an important role in the development and progression of atherosclerosis. In the past decade, multiple other functions, beyond the classical chemotaxis, have been reported like cell survival and angiogenesis, and platelet activation.   
CXCL13 is a homeostatic chemokine, highly expressed by stromal tissue and follicular dendritic cells, where it is instrumental in the guidance of lymphocytes to secondary lymphoid organs.   Its only known receptor, CXCR5, is also implicated in lymphocyte homing. The CXCL13-CXCR5 chemokine axis has been associated with various inflammatory conditions.   Surprisingly, we could show that CXCL13, although being a homeostatic chemokine, is prominently expressed in human atherosclerotic lesions and is increased in plasma of patients with carotid atherosclerosis. Together, these data infer a potential causative role for the CXCL13-CXCR5 chemokine axis in atherosclerosis.
In this study, we aimed to address the impact of defective hematopoietic CXCL13-CXCR5 chemokine signaling on atherosclerosis formation in mice. Hereto, we generated LDLr−/− chimeras (female, 12 weeks) with hematopoietic deficiency of either Cxcl13 or Cxcr5 by bone marrow transplantation. Mice transplanted with C57Bl/6J bone marrow (wild-type [WT]) were used as controls. All animal work was approved by the regulatory authority of Maastricht University and performed in compliance with the Dutch government guidelines. After bone marrow reconstitution, mice were placed on a Western type diet (WD; 0.25% cholesterol) for 4 weeks, after which a semiconstrictive carotid artery collar was placed bilaterally to accelerate atherogenesis and mice were left for an additional 8 weeks on WD ([Fig. 1A]). The right common carotid artery was paraformaldehyde fixed and paraffin-sectioned (4 μm; at 100 μm intervals), after which they were stained by hematoxylin/eosin and analyzed for lesion size and composition (Macrophages: Mac-3, BD-Pharmingen; Collagen content: Sirius Red staining).
Hematopoietic deficiency of Cxcr5 did neither affect body weight (20.0 g ± 0.4 vs. 20.4 g ± 0.9 for WT mice) nor plasma cholesterol levels (1067 pg/mL ± 67.8 vs. 1085 pg/mL ± 89.6 for WT mice). However, atherosclerotic lesion development in the carotid artery was significantly reduced in hematopoietic Cxcr5 −/− mice, compared with WT controls ([Fig. 1B]). While plaque macrophage content did not differ ([Fig. 1C]), lesional collagen content was decreased ([Fig. 1D]) as was necrotic area ([Fig. 1E]). These data clearly point to a causal role of hematopoietic CXCR5 in atherosclerosis development.
As CXCL13 is expressed both by stromal cells and various leukocyte subsets, we addressed the question whether the reduced atherogenic response in Cxcr5 −/− mice was driven by hematopoietic or by stromal CXCL13. Hematopoietic Cxcl13 deficient mice showed no difference in body weight (21.3 g ± 0.7 vs. 20.4 g ± 0.9 for WT mice) and plasma cholesterol levels (895.2 pg/mL ± 73.7 vs. 1085 pg/mL ± 89.6 for WT mice). Similar to hematopoietic Cxcr5 −/− deficiency, that of Cxcl13 also resulted in a significantly reduced lesion burden ([Fig. 1F]). Both lesional macrophage and collagen content was unchanged in hematopoietic Cxcl13 deficient mice ([Fig. 1G, H]). Similar to Cxcr5 −/− chimeras, hematopoietic Cxcl13 deficiency resulted in a striking decrease in necrotic core size ([Fig. 1I]).
Interestingly, B1 cells were significantly enriched in blood of both Cxcr5 −/− and Cxcl13 −/− mice, which was, for Cxcl13 −/− mice, accompanied by an additional significant increase of B2 cells ([Fig. 1J]). This aligned with a reduced B1 contents in the lymph nodes ([Fig. 1K]), while B1 and B2 contents in the spleen ([Fig. 1M]) were not affected by interruption of the CXCL13-CXCR5 chemokine axis (data not shown); nor were B cell progenitors in bone marrow affected by either of the deficiencies ([Fig. 1L]). B1 and B2 were reported to exert divergent, even opposite, effects on atherosclerosis, at which B1 cells were shown to attenuate atherosclerosis, while B2 cells appear to aggravate disease. Although the exact role of B1b cells in atherosclerosis has for long been unclear, it was recently shown that they are, similarly to B1a cells, atheroprotective. The exact mechanism for their protective activity seems to differ from that of B1a cells, though for both subsets it is mainly mediated by the production and release of immunoglobulin M (IgM).  In support of this notion, levels of total ([Fig. 1N]; ProcartaPlex Immunassays, Invitrogen) as well as atheroprotective oxidized low-density lipoprotein-specific IgM ([Fig. 1O]) in serum were indeed sharply elevated in both Cxcr5 −/− and Cxcl13 −/− mice compared with WT mice.
These results clearly demonstrate that the homeostatic CXCL13-CXCR5 chemokine axis plays a causal role in atherosclerosis development, most likely by modulation of B1 cell distribution patterns to influence atheroprotective IgM production and secretion by these cells. Hematopoietic Cxcr5 −/− and Cxcl13 −/− also impacted on lesion composition, with a striking decrease in necrotic core content, potentially reflecting the delayed lesion development in Cxcl13/Cxcr5 deficient mice, carrying less advanced lesions. As our model reflects early stages of lesion development, we were unable to explore whether the antiapoptotic properties of CXCL13 could potentially result in beneficial plaque stabilizing effects within unstable lesions. Though this aspect regarding unstable lesions remains to be elucidated, the observed effects of CXCL13 deficiency on early stage atherosclerotic lesions align very well to the observations made by Kyaw et al, demonstrating that splenectomy reduced peritoneal B1a content and plasma IgM levels, leading to increased atherosclerotic lesion size and necrotic core expansion.
To further substantiate the relevance of the CXCL13-CXCR5 axis to human atherosclerosis, on top of already published associations, we examined the association of genetic variants at the CXCR5 and CXCL13 loci with atherosclerosis-related clinical outcomes. Applying expressive quantitative trait loci analysis in the Genotype-Tissue Expression data set, we identified the G-allele at rs77564610 which is associated with higher CXCR5 expression in whole blood (normalized effect size, +0.530; p = 1.9 × 10−6) and with increased risk of (subsequent) myocardial infarction (odds ratio [OR], 2.85; p = 1.1 × 10−3) in the cohort of the UK Biobank (n = 452,264). Moreover, we scrutinized common variants at the CXCR5 locus (±50 Kb) in the UK Biobank cohort and identified rs187248852 (minor allele frequency [MAF] = 0.4%) and rs73575424 (MAF = 10.6%) to be associated with ischemic stroke (OR, 4.87; p = 8.9 × 10−6) and myocardial infarction (OR, 1.10; p = 5.1 × 10−5), respectively. Finally, analysis performed on the CADISP cohort (n = 9,814)  yielded suggestive evidence of association with stroke for multiple single-nucleotide polymorphisms in the CXCL13 locus (p < 1.4 × 10−4), although most of them are intron variants ([Fig. 1P]). Together with evidence showing higher CXCL13 in patients with atherosclerosis, our analyses imply the involvement of CXCR5/CXCL13 axis in human atherosclerosis.
In conclusion, we are the first to describe a proatherogenic role of the CXCL13-CXCR5 dyad in atherosclerosis, an effect that is likely attributable to its impact on B1 cells and their IgM production. However, while we did not observe any difference in splenic B2 cell levels (Follicular/Marginal zone), a role of these cells, via T cell interaction, cannot be fully excluded at this point. Identifying this chemokine axis as novel regulator of atherosclerosis development, this study creates new opportunities for chemokine targeting therapy in atherosclerosis.
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