*2.6. Correlation between CD11c Expression Levels and Plasma Levels of Inflammatory Mediators in RA Patients*

Because CD11c is a marker for classically activated macrophages (M1 macrophages) [24,25], which produce proinflammatory cytokines [26], we examined the correlation between CD11c expression and plasma levels of inflammatory mediators in RA patients. As shown in Figure S3, CD11c expression levels were positively correlated with plasma levels of IL-6 (*r* = 0.2928, *p* = 0.0352) or IL-8 (*r* = 0.2917, *p* = 0.0359). There was no significant correlation between CD11c expression levels and other inflammatory mediators.

## *2.7. The E*ff*ects of L5 on Cytokine Expression in THP-1 Cells*

Because of the significant association of L5 with the expression of CD11c, which affects secretion of inflammatory mediators, we examined whether L5 had an effect on the expression of proinflammatory cytokines, such as IL-6, IL-8, and TNF-α, in THP-1 cells and THP-1 cell-derived macrophages. The results showed that L5 upregulated the expression of IL-6, IL-8, and TNF-α in both monocytes and macrophages (Figure 3A–F). Moreover, the levels of IL-6 and IL-8 were significantly higher in monocytes or macrophages treated with high-dose L5 (relative of control group, mean ± SEM, 13.6 ± 2.5 folds, 81.1 ± 7.6 folds; and 365.9 ± 73.5 folds, 9.9 ± 1.6 folds, respectively) than in those treated with high-dose L1 (1.4 ± 0.2 folds, 2.1 ± 0.6 folds; and 0.9 ± 0.4 folds, 2.4 ± 1.1 folds, respectively, all *p* < 0.05, Figure 3A,B,D,E). Monocytes and macrophages treated with high-dose L5 (relative of control group, mean ± SEM, 6.82 ± 0.82 folds, 19.45 ± 3.93 folds) also induced significantly higher TNF-α expression than that seen in untreated cells (all *p* < 0.05, Figure 3C,F).

#### *2.8. Proposed Model for the Potential Role of L5 in RA-Related Atherogenesis*

L5 induces the expression of CD11c, which has been reported to be associated with vascular plaque formation [23,27] and as a marker of M1 macrophages with secretion of inflammatory cytokines [26]. L5 also upregulates the expression of IL-6 and IL-8 on both monocytes and macrophages. We have previously shown that L5 induces the expression of lectin-like oxidized LDL receptor-1 (LOX-1) [15], a receptor that is involved in a variety of atherogenic responses including foam cell formation [16]. The combination of foam cell accumulation and increased cytokine activity in the microenvironment synergistically promotes plaque formation (Figure 4).

**Figure 3.** Effects of L5 on the expression of cytokines in monocytes and macrophages. The differences in the mRNA expression levels are shown for IL-6 (**A**), IL-8 (**B**), and (**C**) tumor necrosis factor-α (TNF-α) in THP-1 cells (human monocytic cell line) treated with different doses of L1 and L5. The differences in the mRNA expression levels are shown for IL-6 (**D**), IL-8 (**E**), and (**F**) TNF-α in THP-1 cells-derived macrophages treated with different doses of L1 and L5. Data are the mean ± SEM for three independent experiments. \* *p* < 0.05.

**Figure 4.** Proposed model for the potential role of L5 in RA-related atherogenesis. L5 induces the expression of CD11c, which is associated with vascular plaque formation [23,27] and is a marker for M1 macrophages that secrete proinflammatory cytokines [26]. L5 also upregulates the expression of inflammatory cytokines, interleukin (IL)-6 and IL-8, in both monocytes and macrophages. In addition, L5 induces LOX-1 expression and promotes foam cell formation by uptake of L5. The elevated levels of inflammatory cytokines and macrophage foam cell formation may contribute to vascular plaque formation in RA-related atherogenesis.

### **3. Discussion**

The association between plasma L5 and the increased CVD risk in RA provides a new explanation for the paradoxically normal plasma LDL levels seen in these patients [15]. The present study was designed to explore the underlying molecular mechanisms of this unique clinical phenomenon. Here, we have shown that treatment with L5 upregulated foam cell formation in THP-1-derived-macrophages, whereas L1, even at high doses, exerted no effect. To identify the genetic variants attributable to L5-related atherosclerosis, we compared expression levels of candidate genes in RA patients with high L5% and normal L5%. We found a significant difference in the expression of *ITGAX*, the gene that encodes the integrin CD11c, which plays a pivotal role in vascular plaque formation [23]. L5 induced CD11c expression in THP-1 cells. Moreover, CD11c expression levels were positively correlated with plasma levels of IL-6 and IL-8. L5 also upregulated the expression of IL-6, IL-8, and TNF-α in in vitro assays. These findings suggest that L5 may contribute to atherogenesis by promoting foam cell formation, upregulating CD11c expression, and inducing the secretion of atherosclerosis-related mediators (Figure 4).

Dyslipidemia is a well-established traditional risk factor for atherosclerosis [6,7], and foam cell formation occurs in the early stage of atherogenesis in RA patients [28,29]. In the present study, we are the first to show significant enhancement of foam cell formation in macrophages treated with L5, which may be related to higher QRISK-2 scores in our patients with high L5% compared to those with normal L5%. Recent studies reveal that L5 promotes the differentiation of monocytes into macrophages in a dose-dependent manner [30], and L5 containing glycosylated apolipoprotein(apo) E may contribute to atherogenicity [31]. In addition, L5 containing apoCIII has been reported to induce monocytes adhesion with endothelial cells to contribute atherogenesis [32]. These findings support the finding that plasma L5% was significantly higher in RA patients with subclinical atherosclerosis than in those without [15], and support the link between L5 and the CVD risk [15,33,34]; L5 promotes foam cell formation.

The increased CVD risk in RA patients results from the intricate interactions among traditional CV risk factors, systemic inflammation, and genetic components [6,7,28,35]. In accordance with the findings that L5 is closely related to an increased CVD risk in autoimmune diseases [15,16], RA patients with high L5% in the current study had significantly higher levels of CD11c mRNA expression compared to

those with normal L5%. To validate L5's association with CD11c expression, we examined the effects of L5 on CD11c expression in THP-1 cells using flow cytometry analysis. The results showed that L5 enhanced CD11c expression, whereas L1 had no effect. The different effects may be related to the varied composition of apolipoprotein(apo) in L1, and L5:L1 contains apoB100, while L5 contains apoAI, apoE, and apoCIII [36]. Given the significant association of CD11c expression with vascular plaque formation in atherosclerosis in mice [23] or patients [27], this finding strongly supports the atherogenic role of L5 in RA and possibly other autoimmune diseases.

CD11c is a probable marker of proinflammatory M1 macrophages, which have the propensity to secrete inflammatory mediators [26,37] and thus promote atherogenesis [24,27]. Both IL-6 and IL-8 are well-established mediators of RA-related atherosclerosis [8–10], and we found that CD11c expression was positively correlated with increasing levels of IL-6 and IL-8. This prompted us to examine the effects of L5 on the expression of atherosclerosis-related mediators in monocytes and macrophages. Our finding that L5 upregulated the expression levels of IL-6, IL-8, and TNF-α in monocytes and macrophages further substantiated L5's atherogenic role through a mechanism mediated by CD11c.

Despite the novel findings in this pilot study, there were still some limitations. First, the sample size of RA patients in whom we could observe the emergence of CVD was small, which may reduce the statistical power. The effects of other medications, such as corticosteroids and disease-modifying anti-rheumatic drugs (DMARDs), should be considered because they may affect plasma levels of lipids and inflammatory mediators [38]. Finally, none of the enrolled patients in our study were in the early RA stage, which may limit the generalizability of these results to the whole population.
