*2.3. The E*ff*ects of L5 on Macrophage Foam Cell Formation*

To investigate the potential effects of L5 on macrophage foam cell formation, we treated THP-1 cells with 10 ng/ml phorbol myristate acetate (PMA) for 48 h to stimulate differentiation into macrophages. Then, the monocyte-derived macrophages were stimulated with different doses of L5 (10, 25, or 50 μg/mL) or L1 (10, 25, or 50 μg/mL) at 37 ◦C for 48 h. As illustrated in Figure 1, the L5 induced foam cell formation, and a dose-dependent upregulation of foam cell formation of macrophages after L5 treatment (mean ± standard error of mean (SEM), 12.05 ± 2.35% in L5 (10 μg/mL); 50.13 ± 3.9% in L5 (25 μg/mL); 90.69 ± 1.82% in L5 (50 μg/mL), *p* < 0.05). The high-dose L5 also induced significantly more foam cell formation than high-dose L1 (11.00 ± 2.59%, *p* < 0.05).

**Figure 1.** Effects of L5 on macrophage foam cell formation. THP-1 cells were incubated with (**A**) high-dose L5 (50 μg/mL), (**B**) midden-dose L5 (25 μg/mL), (**C**) low-dose L5 (10 μg/mL), (**D**) control (phosphate buffered saline, (**E**) high-dose L1 (50 μg/mL), (**F**) midden-dose L1 (25 μg/mL), and (**G**) low-dose L1 (10 μg/mL) for 48 h. (**H**) Difference in the proportion of macrophage foam cell formation among the different groups. Data are presented as the mean ± SEM for three independent experiments. \* *p* < 0.05, determined by one-way ANOVA.

#### *2.4. Comparison of CD11c mRNA Expression Levels between RA Patients with High L5% and Normal L5%*

To identify the genes potentially involved in RA-related atherosclerosis, we examined the mRNA expression of 20 candidate genes. The results showed 10 differentially expressed genes in RA patients compared to healthy controls (Figure S1). Given the augmented effects of L5 on macrophage foam cell formation, we identified the candidate genes involved in L5-related atherosclerosis in RA patients by quantitative real-time polymerase chain reaction (qRT-PCR) assay. The results showed a significant difference in the expression levels of 10 candidate genes: *ABCA1, ACTR2, AFF4, CD11c, NPC1, PPFIA1, SMARCA2, WSB1, ZFAND6,* and *ZNF652*, between RA patients and healthy controls (Figure S1). Then, we examined the difference in the expression levels of these 10 genes between RA patients with high L5% and normal L5%. The results indicated a significant difference in the mRNA expression levels of only one gene, *ITGAX* (for CD11c), between the two groups (Figure S2).

In enrolled participants, we examined the difference in CD11c mRNA expression levels between RA patients (*n* = 93) and healthy controls (*n* = 41). The results showed significantly higher levels of CD11c expression in RA patients (relative of actin expression, mean ± SEM, 0.0545 ± 0.0059 folds) compared to healthy controls (0.0126 ± 0.0037 folds, *p* < 0.01, Figure 2A). Moreover, significantly higher levels of CD11c expression were observed in RA patients with high L5% (0.0752 ± 0.0139 folds) than in those with normal L5% (0.0446 ± 0.0054 folds, *p* < 0.05, Figure 2B). After exclusion of patients with cardiovascular events, we still revealed a significant difference in the CD11c expression levels between patients with high L5% (mean ± SEM, 0.0617 ± 0.0093 folds) and normal L5% (0.0404 ± 0.0051 folds, *p* < 0.05).

**Figure 2.** Comparison of CD11c (*ITGAX*) mRNA expression (**A**) between rheumatoid arthritis (RA) patients and healthy controls (HC), and (**B**) between RA patients with high L5% and normal L5%. (**C**) Representative histogram of the flow cytometric analysis of CD11c expression on THP-1 cells. (**D**) Bar graph showing the percent CD11c expression levels on THP-1 cells treated with different doses of L1 or L5 and fetal bovine serum (FBS)-treated control cells. Data are the mean ± SEM for three independent experiments. \* *p* < 0.05.
