Thrombus formation following atherosclerotic plaque rupture, resulting in obstruction of the arterial lumen, is the most common underlying cause of cardiovascular events, such as stroke and myocardial infarction [1]. Vulnerable or rupture-prone plaques are often rich in lipids and inflammatory cells, and covered by a thin fibrous cap.

Co-stimulatory molecules, part of the immune checkpoint family, have been revealed as essential drivers of atherosclerotic plaque progression and are thus emerging as strong drug target candidates for stabilization of vulnerable plaques [2-4]. Tumor necrosis factor superfamily member 14 (TNFSF14), also known as LIGHT and CD258, is expressed mainly by lymphocytes and antigen-presenting cells [5,6]. LIGHT has been implicated as a driver of disease progression in several inflammatory diseases, often featuring fibrotic and/or autoimmune elements, including rheumatoid arthritis [7], asthma [8], atopic dermatitis [9], and systemic sclerosis [10]. In atherosclerotic mouse models, LIGHT deficiency has been reported to both aggravate and ameliorate atherosclerosis through differing effects on inflammation and immune cell homeostasis [11,12]. Little is known about the potential involvement of LIGHT signaling in human cardiovascular disease (CVD), and further insight is vital for future evaluation of LIGHT as a potential therapeutic target.

While LIGHT is reportedly expressed in foam cell-rich regions of human atherosclerotic plaques [13,14], it is not known whether LIGHT has a role in promoting a high-risk plaque phenotype. To address whether LIGHT levels are associated with features of atherosclerotic plaque vulnerability, we have analyzed LIGHT expression in relation to plaque stability markers in human carotid endarterectomy plaques.

Detailed methods are provided in Supplementary Methods.

Here, we show that LIGHT levels are higher in carotid endarterectomy plaques from symptomatic, compared to asymptomatic patients, and associated with markers of an inflammatory plaque environment with active ECM turnover (Figure 3).

Much of the current interest in the potential therapeutic value of interfering with co-stimulatory signaling pathways to stabilize and/or combat progression of atherosclerosis has been generated through reports by us and others identifying co-stimulatory molecules as having essential roles in atherosclerotic CVD [31]. While in experimental models, co-stimulatory molecule triggering has most often been described as atherogenic, in the case of LIGHT, reports from murine atherosclerosis models have yielded contrasting results. Though inactivation of the LIGHT/lymphotoxin beta receptor (LTβR) axis ameliorates inflammation and macrophage proliferation associated with atherosclerosis burden in the presence of insulin resistance and metabolic syndrome [12], LIGHT deficiency has also been found to aggravate atherosclerosis by promoting plaque apoptosis as well as Th17 (over Treg) cell populations [11]. Contrastingly, overexpression of LIGHT by T-cells was found to exacerbate hypercholesterolemia [32], which also suggests aggravated atherogenesis, though it should be noted that the latter two studies were carried out in different murine models (apolipoprotein E and low-density lipoprotein receptor deficiency, respectively). Considering these data, exploring LIGHT expression in relation to plaque vulnerability parameters in human plaques is an important next step in investigating the potential role of this molecule as a key player in atherosclerosis.

Though increased levels of circulating LIGHT have been reported after stroke [33], as well as in patients diagnosed with stable and unstable angina [34,35], in our endarterectomy cohort, we did not find any association between circulating LIGHT levels and either preoperative cerebrovascular symptoms or post-operative cardiovascular events or mortality. Surprisingly, there was instead a tendency towards negative association with cardiovascular mortality; however, it did not hold up to adjusting for possible confounders. Moreover, though associated with a rupture-prone plaque phenotype at the time of endarterectomy, plaque levels of LIGHT did not show potential as a marker for future cardiovascular survival. These results were further corroborated by Mendelian randomization using three large cohorts (SCALLOP [Systematic and Combined Analysis of Olink Proteins] [36], deCODE [37], and UK Biobank Pharma Proteomics Project [UKB-PPP] [38]), which did not demonstrate a strong causal relationship between plasma LIGHT levels and cardiovascular outcomes. Though a weak causal effect on LAS was identified using the deCODE cohort, it could not be validated in the other two examined datasets, suggesting limited generalizability. These results also raise the possibility that LIGHT-upregulation in vulnerable plaques may be induced as part of a process not actively driving plaque destabilization but rather serve a reparative function. Moreover, the role of LIGHT in plaques may be linked to the activity of other biological factors, such as MMP-9 degradation of the cap or low plaque content of reparative elements such as TGF-β2, which is consistent with our observed association of plaque LIGHT content with higher MMP-9 and lower TGF-β2 levels.

Suggesting a protective role for LIGHT in atherosclerotic plaque progression by coordinating local T-cell immunity, Hurtado-Genovés et al. [11] found lower TNFSF14 gene expression in atherosclerotic aortic wall biopsies from CAD patients to be associated with altered levels of T-cell differentiation markers and cell death, while anti-apoptotic and cytotoxic pathways were induced by LIGHT-ligation in vitro [11]. Beyond these studies, the role of LIGHT in plaque vulnerability has only been investigated in human atherosclerosis by immunohistochemical detection in macrophage-rich plaque regions [13,14] and through an observed tendency for greater LIGHT presence in inflammatory, compared to fibrous, plaque regions (though notably measured by Western blot in only two samples) [13].

Our finding of associations between the levels of LIGHT and inflammatory mediators and lipids in human carotid plaques may further suggest that effects of LIGHT on plaque vulnerability features differ with lymphocyte or myeloid cell stimulation. Also in line with this notion are the particularly strong correlations between plaque LIGHT content and pro-inflammatory cytokines expressed by several different cell types, such as the IL-1β/IL-6 axis and CCL2 (by myeloid and non-immune cells) and CCL5 (mainly by T-cells) [39,40]. These results align with previous reports of myeloid cell LIGHT signaling promoting pro-inflammatory and atherogenic features. These studies included observed LIGHT-upregulation in blood Ly6C^high macrophages (from ApoE^-/- mice) and following stimulation with oxLDL [35], and exogenous LIGHT in oxLDL-induced THP-1-derived macrophages [34]. In addition, contrary to reports on T-cells, rather than prevent cell death and cytotoxicity, LIGHT stimulation of macrophages seems to mediate vascular endothelial growth factor (VEGF)-induced apoptosis [41]. Collectively, studies so far thus indicate LIGHT triggering in plaques to have both atheroprotective and atherogenic functions depending on cellular context and future studies will be required to draw further conclusions on the net effects of LIGHT signaling in human atherosclerotic CVD.

The occurrence of plaque rupture is dictated by differing plaque ECM quantity, structure, and components, a balance that may be shifted by the presence of co-stimulatory signaling [1,2,42]. LIGHT has previously been found to promote SMC proliferation and drive a fibrous response in keratinocytes [43,44]. Though driving plaque fibrosis may be hypothesized as another potential atheroprotective role for LIGHT, our study found LIGHT to correlate mainly with ECM synthesis and degradation markers, as well as with (mesenchymal cell mitogen) PDGF, rather than with structural ECM components, such as collagen. This suggests LIGHT signaling to be associated with active tissue repair secondary to ECM degradation, as promoted by a highly inflamed plaque microenvironment rather than direct involvement in driving a fibrous response in plaques. The negative correlation between LIGHT and TGF-β2 is also in line with this notion.

The study has certain limitations. The reported results are mainly related to associations through which conclusions about a causal role in plaque destabilization cannot be drawn. Though MR analyses were performed, we are still limited by available expression data from carotid tissues, specifically plaque tissue. Our LIGHT measurements are limited to one Swedish cohort with advanced atherosclerosis, and generalization would require similar assessments in additional cohorts representing different ethnicities and stages of the disease. Furthermore, the sex of the included subjects is skewed towards men (68%), which broadly mirrors the sex ratios for carotid endarterectomies performed at the Skåne University Hospital but nonetheless limits our ability to stratify analyses by sex. However, we found no association between LIGHT plaque content and sex, making bias less likely. Finally, the proximity extension assay used to measure LIGHT content in plaques is semi-quantitative and, thus, unable to provide absolute plaque-concentration of the analyte. The assay can also not distinguish between soluble and membrane-bound LIGHT in plaque homogenates; however, both forms can induce signaling through herpesvirus entry mediator (HVEM) and LTβR.

The content of the co-stimulatory molecule LIGHT in atherosclerotic plaques not only correlates with markers of plaque destabilization, but is also significantly elevated in plaques from symptomatic patients, compared to those from asymptomatic patients, though a robust causal effect on CVD outcomes could not be shown. These results thus associate LIGHT content with a rupture-prone plaque phenotype, potentially upregulated as part of a reparative response, warranting further studies.