Potential strategy for RA prevention：reduced DNA fragment accumulation by upregulating TREX1

Potential strategy for RA prevention：reduced DNA fragment accumulation by upregulating TREX1

Background

Rheumatoid arthritis (RA) is a chronic and systemic autoimmune disease with poorly understood pathogenesis. Several factors such as age, smoking history, poor body mass index (BMI), immunogenicity and gene polymorphisms have been shown to be potentially associated with the pathogenesis of RA and its refractoriness. As an inevitable process in life, aging is a major contributor to the increasing risk of various diseases.

Metabolic DNA damage and sustained activation of the immune system are important biological activities that drive cellular senescence. During cellular senescence, DNA fragments accumulate in blood and enter the circulation to become circulating free DNA (cfDNA). Of note, DNA fragmentation is also known to be a biological feature of many diseases, such as trauma, sepsis, aseptic inflammation, myocardial infarction, stroke, transplant rejection, diabetes mellitus, sickle cell disease, and others. Both Aicardi-Goutières syndrome and familial frostbite lupus share a common phenotypic feature - abnormal activation of the immune system caused by the inability to clear self DNA or self RNA fragments in a timely manner. After treatment with disease-modifying antirheumatic drugs (DMARDs), DNA fragmentation was significantly reduced in the plasma of RA patients, which illustrated that DNA fragmentation may be associated with RA pathogenesis. Based on the hypothesis that the increased incidence of RA in the middle-aged and elderly patients may be related to age-related accumulation of DNA fragments, clarifying the source of DNA fragments in the plasma of these patients and investigating how to improve the efficiency of the clearance mechanism may open new avenues for the prevention and treatment strategies for RA.

To validate that DNA fragmentation is one of the factors contributing to the development of rheumatoid arthritis, Weidan Luo's research team from Biophysics Laboratory for Innovative Drug Discovery, State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Macao, China, did a series of studies and published an article entitled “Self-DNA accumulation as a risk factor for accelerating the pathogenesis of rheumatoid arthritis in elderly individuals” in Research Square. The article calls that the DNA fragments are the effective makers of inflammation-releasing mediators, whereas the TREX1 gene is a potent degrader of DNA fragments and suggests that TREX1 is a key gene regulating cellular immunity and aging. This conclusion provides us with a new idea to prevent rheumatoid arthritis by rapidly clearing excess DNA fragments from the body to ensure that senescent cells remain healthy.

3'->5' DNA exonuclease TREX1 significantly downregulated in RA patients

To investigate whether accumulation of DNA fragments could stimulate an aberrant immune response, the researchers first determined the gene expression levels of TREX1 (a DNA fragment clearance enzyme) and cGAS (a DNA fragment sensor) in peripheral blood samples from healthy volunteers, rheumatoid arthritis (RA) patients, and osteoarthritis (OA) patients. The results in Fig.1A and Fig.1B show that the gene expression level of TREX1 but not cGAS was significantly decreased in RA patients compared with healthy controls and OA patients, suggesting that DNA fragment accumulation is closely involved in human RA pathogenesis. Fig. 1C demonstrates that transcript levels of both TREX1 and cGAS were high in blood samples from healthy aged rats (>12 months old) compared with young control rats (4 weeks old). Moreover, overexpression of TREX1, which can clear excessive DNA fragments in aged rats, can prevent aberrant immune responses and maintain the health status of animals.

Figure 1

Role of TREX1 and cGAS signaling pathways in DNA fragment-induced release of proinflammatory cytokines

Transfection of DNA fragments into RA-FLSs (rheumatoid arthritis fibroblast-like synoviocytes) stimulated the expression of the DNA fragment clearance enzyme TREX1 and cGAS-STING, and upregulated the expression of proinflammatory cytokines (TNF-α, IL-1β, IL-2, IL-6, IL-8, IL-25 and IFN-β), and all were dose-dependent (Fig. 1D&E). However, the increased expression of TREX1, cGAS-STING, and proinflammatory cytokines occurred only within the first 24 hours of transfection with DNA fragments, and the expression of these activated genes declined after 48 to 72 hours of transfection. Thus, the researchers speculate that DNA fragment-induced TREX1 upregulation may contribute to the clearance of DNA fragments that accumulate in the cytoplasm, reducing cGAS-STING and proinflammatory cytokine levels.

Moreover, Western blot results also confirmed that TREX1 and cGAS protein expression was time - and dose-dependent with the introduction of DNA fragments into RA-FLSs (Fig. 2A). To reveal the roles of TREX1 and cGAS-STING signaling in DNA fragment-induced inflammatory responses, TREX1 and cGAS were silenced by siRNA in RA-FLSs before transfection of DNA fragments, and cGAS knockout RA-FLSs were constructed. As shown in Fig. 2B, silencing TREX1 expression activated cGAS-STING signaling, which further promoted proinflammatory cytokine release in RA-FLSs which was transfected with DNA fragments, whereas knockout of cGAS significantly downregulated cGAS-STING signaling and inhibited the release of DNA fragment-induced proinflammatory cytokine. These results confirm that DNA fragments activate inflammatory responses and that both TREX1 and cGAS play regulatory roles in DNA fragment-induced inflammatory responses.

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Figure 2

Joint injection of DNA fragments induced arthritis in TREX1 conditional knockout (TREX1^Cre) rats

To elucidate the role of TREX1 in the pathogenic mechanism of DNA fragmentation in adjuvant-induced arthritis (AIA) model, the researchers established a Cre-loxP-mediated TREX1 conditional knockout in SD rats (TREX1^Cre) by CRISPR/Cas-mediated genome editing, in which Cre adeno-associated virus was provided by Ubigene. One of the results in Fig. 3A-C shows after injection of 200ug DNA fragments into the hind limb joint cavity, severe hind paw swelling, erythema, and joint rigidity were observed in both TREX1Cre/+ AIA rats and healthy control TREX1Cre/+ rats. Moreover, TREX1 knockout or downregulation may increase the severity of arthritis in control and AIA rats with hindlimb joint injection of DNA fragments. These findings suggest that TREX1 may have a beneficial therapeutic effect on RA by clearing cfDNA from joint tissues.

Figure 3

DNA fragments promoted T-cell activation and reduced Treg cell populations in TREX1^Cre rats

To examine the effects of DNA fragments on autoimmune reactions in rats, sonicated rat muscle DNA fragments were injected via the tail vein into both healthy control TREX1^Cre/+ rats and AIA model TREX1^Cre/+ rats. Consistent with the above experimental results, both the healthy control TREX1^Cre/+ rats and the AIA model TREX1^Cre/+ rats injected with the DNA fragment showed more severe joint swelling in the hind limbs, significantly higher arthritis scores, and more severe bone destruction. The results of flow cytometry analysis showed that the AIA TREX1^Cre/+ model rats had significantly increased numbers of CD8 + T lymphocytes but decreased numbers of Treg cells. After intravenous injection of DNA fragments, both AIA model TREX1^Cre/+ (Cre H/L) rats and healthy control TREX1^Cre/+ (Cre H/L) rats showed further increased numbers of CD8 + T lymphocytes and decreased numbers of Treg cells compared with healthy control TREX1 wild-type rats.

Figure 4

Adeno-associated virus (AAV)- mediated overexpression of TREX1 suppressed synovial inflammation in AIA rats

To further investigate the role of TREX1 in the pathogenesis of AIA in rats, adeno-associated virus expressing TREX1 (AAV-TREX1) was injected into the joint cavity (2.5×10¹⁰ PFU or 2.5×10⁸ PFU) or tail vein (1.0×10¹¹ PFU or 1.0×10⁹ PFU) of rats, of which the TREX1 expressing adeno-associated virus was provided by Ubigene. The hind paws of AAV-EGFP injected AIA rats (control) developed severe swelling, erythema, and joint rigidity. But after injection of AAV-TREX1 into the joint cavity and tail vein, AIA rats showed significant reductions in both the arthritis score and the hind paw volume, suggesting that the severity of AIA was markedly reduced in rats with TREX1 overexpressed. So, it indicated that TREX1 may play an inhibitory role in the development of arthritis in AIA rats by clearing DNA fragments.

 Ubigene provided the Cre and TREX1 expressing adeno-associated virus packaging service for this study, assisted in TREX1 knockout rat construction and TREX1 overexpression to verify the regulatory role of TREX1 in DNA fragment mediated inflammatory response. In addition to adeno-associated virus, Ubigene also provides lentivirus and adenovirus packaging services with Ubigene exclusive developed formula which can ensure strong virus viability and high titer. And the service turnaround is fast to 3 weeks, click for more service details > >

Figure 5

Aging-related cellular senescence and arthritis led to c-Jun /c-Fos transcription factor expression imbalance and TREX1 expression suppression

To further demonstrate the role of E2F1 in TREX1 expression, the researchers showed that E2F1 was overexpressed in RA-FLSs in a dose-dependent manner and that the transcript levels of TREX1 and DP-1 (important activators of E2F transcription factors) were significantly and gradually increased. In parallel, UV-mediated DNA damage and direct DNA fragment challenge induced the expression of E2F1, DP-1, c-jun, and c-Fos, while TREX1 was upregulated in RA-FLSs in a time-dependent manner. However, the presence of E2F1 siRNA significantly suppressed the high TREX1 expression, indicating that E2F1 is a key transcription factor for TREX1. To further elucidate the regulatory role of TREX1 in aging and RA, the researchers measured the expression of TREX1 and components of the AP-1 complex (c-Jun/c-Fos) in both early- and late-passage RA-FLSs transfected with DNA fragments. TREX1 expression was significantly lower in late-passage RA-FLSs than in early-passage RA-FLSs, regardless of successful transfection with DNA fragments. siRNA knockdown of c-Fos significantly inhibited DNA fragment mediated TREX1 expression in early-passage RA-FLSs. Both in aging-related cellular senescence and under arthritic conditions, the accumulation of cfDNA can be promoted by the suppression of TREX1 transcription factors such as E2F1 and AP-1 thereby activating the autoimmune responses.

Figure 6

In summary, this study illustrates for the first time: ①DNA fragments may be potential immunogenic factors predisposing to RA; ②Downregulation of TREX1 will promote the release of proinflammatory factors and may be involved in the accumulation of DNA fragments in RA patients, thereby activating the cGAS/STING signaling pathway to further exacerbate the inflammatory response. In addition to this, the study links autoimmune tissue inflammation to aging, delineating the efficient clearance of DNA fragments as an important strategy for the treatment and prevention of RA and aging-related diseases.