In this study, we determined the polarization of macrophages using six surface markers of polarization, including CD40, CD80, CD16, CD163, CD206, and CD200R. CD40 and CD80 were considered biomarkers for the M1 macrophages as they are highly expressed in M1 macrophages but not in M2 macrophages [28]. CD16 and CD163 were regarded as IL10 M2 biomarkers [29], while CD206 and CD200R have been wildly used as IL4 M2 biomarkers [33]. Compared with cells from healthy donors, cells from rheumatoid arthritis patients expressed higher levels of CD40 and CD80, yet, they had lower levels of CD16, CD163, CD206, and CD200R. Our study demonstrated that compared with macrophages from healthy donors, macrophages from rheumatoid arthritis patients have a higher proportion of M1 polarization cells, but had a lower proportion of M2 polarization cells. We suggested the higher level of pro-inflammatory M1 polarization of cells was associated with the inflammatory in rheumatoid arthritis patients, on the other hand, the lower levels of anti-inflammatory M2 biomarkers in cells from rheumatoid arthritis patients indicated that these cells had a lower level of inflammatory.
We also explored the role of the CDKN2B-AS1/MIR497/TXNIP axis in rheumatoid macrophages. Surprisingly, in macrophages from healthy donors, there was no correlation among CDKN2B-AS1, MIR497, and TXNIP mRNA levels. These results suggested that the CDKN2B-AS1/MIR497/TXNIP axis might not play role in macrophages from healthy donors. However, in macrophages from rheumatoid arthritis patients, CDKN2B-AS1 was significantly negatively correlated with MIR497, CDKN2B-AS1 was significantly positively correlated with TXNIP, and MIR497 was significantly negatively correlated with TXNIP. These results indicated that function of the CDKN2B-AS1/MIR497/TXNIP axis was activated only in rheumatoid macrophages but not in macrophages from healthy donors. We suggested that when the CDKN2B-AS1, MIR497, and TXNIP were at a normal level, they were not able to significantly interacted with each other. Only when they were at an abnormal level could they regulated the downstream targets. This mechanism might also involve some unidentified feedback routes that can be explored in the future.
To validate the regulation of the CDKN2B-AS1/MIR497/TXNIP axis in the polarization of macrophages, we interfered with the expression and function of the axis from upstream to downstream. The role of the CDKN2B-AS1/MIR497/TXNIP axis in macrophages was summarized in Fig. 7A. Our study demonstrated that CDKN2B-AS1 regulated TXNIP through MIR497. The complementary binding sites of the CDKN2B-AS1/MIR497/TXNIP axis were shown in Fig. 7B. Notably, because the complementary binding sites only have seven complementary base pairs, the CDKN2B-AS1/MIR497/TXNIP axis might only have relatively weak interaction, which might account for the deregulation of this axis in health donor cells. The sponging of MIR497 by CDKN2B-AS1 can inhibit the level of endogenous MIR497. Most of the miRNAs play roles in cells by their inhibition toward their target mRNA [17]. In this study, as MIR497 directly suppressed the translation of TXNIP mRNA to TXNIP protein, the CDKN2B-AS1/MIR497/TXNIP axis regulated the level of TXNIP. Our study also revealed a novel regulation role of TXNIP in the polarization of macrophages. We suggested that TXNIP protein can facilitate the M1 pro-inflammatory polarization but inhibited the M2 anti-inflammatory polarization in macrophages, thereby positively regulated the inflammation in rheumatoid arthritis.
However, the monocytes in this study were in vitro differentiated, which were different from the macrophages obtained from a patient that might be affected by the internal environment of the patient. Yet, this study was focusing on the gene expression and the roles of the CDKN2B-AS1/MIR497/TXNIP axis. We think the effect of the genotype of the cells was more critical than the internal environment that induced the differentiation of cells. We presumed that the cells from patients have a genotype that results in the onset of rheumatoid arthritis and the genotype would not be affected by the internal environment of the patients. Thus, we only studied in vitro differentiated cells. In addition, the mechanisms of TXNIP regulating the polarization of macrophages have not been studied in this study. One potential mechanism involved might be the ion channel regulation in macrophages. Ion channels have been found to be critical in cell biology [34–37], some macrophages expressed functional ion channels [38]. TXNIP has been found to be involved in some ion channel regulations [39]. Furthermore, regarding the clinical treatment of rheumatoid arthritis, many anesthetics have been applied in therapy to relieve rheumatoid arthritis pain [40, 41]. These drugs usually have multiple ion channel targets [42, 43]. Many anesthetics can also have multiple ion channel targets[42] and regulate the immune system [44, 45] that might affect rheumatoid arthritis. Whether the use of these drugs impacts macrophages in rheumatoid arthritis is not clear. In addition, the potential role of the CDKN2B-AS1/MIR497/TXNIP axis in other bone diseases is also interesting. Studies have found that TXNIP is closely associated with bone metabolism [46–48]. Rheumatoid arthritis and osteoporosis were two of the most common bone diseases and might share common pathogenic mechanisms [49, 50]. Many pathways have been proposed to regulated osteoporosis [51–54], but whether osteoporosis can be regulated by the CDKN2B-AS1/MIR497/TXNIP axis can also be explored in the future.
To conclude, through studying macrophages from human blood samples and a cultured macrophage cell line, we demonstrated that elevated CDKN2B-AS1 in macrophages promoted the M1 polarization and inhibited the M2 polarization of macrophage by MIR497-mediated TXNIP regulation. Our results revealed a novel role of the CDKN2B-AS1/MIR497/TXNIP axis in the polarization of rheumatoid macrophages and shed light on molecular mechanisms of rheumatoid macrophage polarization. This study also provided potential pharmacological targets for rheumatoid arthritis therapy.