Protective effect of hydroxychloroquine on rheumatoid arthritis-associated atherosclerosis

Abstract

Background: Patients with rheumatoid arthritis (RA) have an increased risk for cardiovascular disease. We examined the effect of gut microbiota in a mouse model of RA that develops atherosclerosis.

Methods: We created three groups of K/BxN female mice that were positive for the anti-glucose-6-phosphate isomerase (GPI) antibody: control diet (CD), high fat diet (HFD), and HFD with hydroxychloroquine (HFD + HCQ). Serological tests were used to detect the serum levels of total cholesterol (TCHO), low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), anti-GPI antibody titers, and serum cytokines. Atherosclerotic plaque was determined by histological analysis, and gut microbiota were determined by 16sV4 sequencing.

Results: Relative to mice given the CD, those receiving the HFD had increased serum levels of LDL-C, TCHO, and TG, decreased serum levels of HDL-C, increased atherosclerotic lesions in the aortic root, and altered gut microbiota. Addition of HCQ to HFD decreased the serum levels of LDL-C, TCHO, and TG, increased serum levels of HDL-C, and decreased the atherosclerotic lesions in the aortic root. Mice receiving HFD + HCQ also had the greatest bacterial diversity among the three experimental groups. Moreover, HCQ treatment significantly increased the abundance of Akkermansia and Parabacteroides, and decreased the abundance of Clostridium sensu stricto cluster 1, and therefore may be responsible for the reduced RA-associated atherosclerosis and dyslipidemia.

Conclusion: Our mouse model of RA indicated that HFD increased ankle width and aggravated atherosclerosis and dyslipidemia, and that HCQ alleviated the dyslipidemia and atherosclerosis, but had no effect on ankle width.

Keywords: atherosclerosis; autoimmunity; hydroxychloroquine; intestinal microbiota; rheumatoid arthritis.

Figure 1

Ankle width and anti‐GPI antibody titers of K/BxN mice after treatment. Ankle widths of hind limbs (A) and anti‐GPI antibody titers (B) of K/BxN mice in the different treatment groups. Ankle widths were measured weekly from 8 to 24 wk, and serum anti‐GPI antibody titers were determined by ELISA after 10 wk of treatment. Here and below, each group had 4‐9 mice *Statistically significant difference between the HFD and CD groups (P < 0.05)

Figure 2

Splenic transitional T1 B and T2 B cells of total lymphocytes (%). Flow cytometry plots of splenic T1 B cells and T2 B cells of K/BxN mice in the different treatment groups after 16 wk (A) and quantification of these results (B, C). Cells were gated, and plotted onto forward scatter FCS‐A and SSC‐A. B cells were plotted onto B220 and side scatter (SSC), splenic transitional T1 and T2 B cells were plotted with B220, AA4.1, and CD23. B220⁺AA4.1⁺CD23^‐ T1 B cells and B220⁺AA4.1⁺CD23⁺ T2 B cells were then identified *Statistically significant difference between the HFD and CD groups (P < 0.05)

Figure 3

Detection of atherosclerosis index. Dyslipidemia (A, B, C, D) and atherosclerosis in the aortic root (E, F) of K/BxN mice in the different treatment groups after 10 wk of treatment. Magnification: 40× *Statistically significant difference between the HFD and CD groups (P < 0.05), and between the HFD and HFD + HCQ groups (P < 0.05) **Statistically significant difference between the HFD and CD groups (P < 0.01) ***Statistically significant difference between the HFD and CD groups (P < 0.001)

Figure 4

Intestinal microbiota of K/BxN mice in the different treatment groups after 10 wk of treatment. 16S V4 region rDNA gene sequences were analyzed for determination of shared and independent bacterial OTUs (Venn diagram, A), Simpson's index of community diversity (B), and abundance of dominant bacteria at the level of the phylum (C), family (D), genus (E), order (F), and class (G). Student's t test was used to compare the abundance of A muciniphila (P < 0.05), Clostridium sensu stricto‐1 (P < 0.05), Ruminiclostridium (P < 0.05) and Parabacteroides (P < 0.05) in the CD, HFD and HFD + HCQ groups (H‐J)