Mouse Anti-Chick Type I Collagen IgG Antibody Assay Kit, TMB

Epstein-Barr virus (EBV) belongs to the  Herpesviridae family  and is considered to be one of the most prevalent viruses affecting humans. It is estimated that more than 90% of the world’s population is seropositive for EBV. After infection, EBV establishes a latency period in B lymphocytes, allowing for subsequent reactivation and recurrent infection. During these frequent recurrences, EBV antigens are shed including its DNA . In childhood, EBV infection can be asymptomatic or lead to mild illness. However, if the infection is acquired during adolescence, it results in infectious mononucleosis (IM).

EBV has been associated with lymphoproliferative malignancies such as Burkitt’s lymphoma, epithelial carcinomas such as nasopharyngeal carcinoma, and human immunodeficiency virus (HIV)-related diseases such as hairy leukoplakia. Additionally, EBV has been associated with an increased risk of developing autoimmune diseases such as multiple sclerosis (MS), systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA) ( 6Trusted Source ) .

Rheumatoid arthritis (RA) is an inflammatory disease that primarily affects joints, leading to cartilage breakdown and erosion, systemic complications, weakness, and early death ( 7Trusted Source )  .

•  This disease affects an estimated 1% of the global population and is twice as common in women as in men. Although the cause of RA is unknown, a number of risk factors have been identified, including environmental stimuli and genetic factors (  9Trusted Source  ). Infectious agents are considered the undisputed frontrunner among environmental triggers . In particular, EBV has been identified as the most common potential environmental trigger for RA. Studies have shown increased viral loads and increases in EBV-related autoreactive antibody levels in RA patients compared to healthy controls. EBV DNA/RNA has been identified in peripheral blood mononuclear cells (PBMC), synovial fluids and synovial membranes in RA patients.
•  Furthermore, antibodies against Epstein-Barr core antigen 1 (EBNA-1) and viral capsid antigen (VCA) were detected at higher levels in sera and synovium of patients with RA compared to non-RA controls. This leads to decreased control of the virus, sustained exposure to EBV antigens, and chronic inflammatory responses.
• Although IL-17A plays an important role in bacterial and fungal infections, when produced in excess, it is implicated in the pathogenesis of autoimmune diseases.
•  Several studies have shown a central role for IL-17A in inducing RA in humans. In patients with RA, high titers of IL-17A and its receptors have been detected in tissue extracts and synovial fluid.
• IL-17A plays a role in neutrophil differentiation, activation and release of cytokines from neutrophils, fibroblasts and monocytes, and induction of matrix metalloproteases (MMP). Cells that produce both IL-17A and IFNϒ have been implicated in autoimmunity and are found at high levels in inflamed human and murine tissues. Therefore, IL-17A plays an essential role in the pathogenesis of RA by mediating pannus growth, matrix turnover, cartilage destruction, and osteoclastogenesis.

Like other members of the  Herpesviridae  family, EBV DNA is rich in unmethylated CpG motifs. Studies have shown that these CpG motifs, present in the genomes of bacteria and some viruses, such as  herpes simplex virus 1  (HSV-1), can activate the immune system that promotes Th1-like responses. Previous research by our group showed that EBV DNA elicits proinflammatory responses, including the production of IL-17A cytokines, in mice and elicits immune responses in flies. Our group also showed that increased EBV DNA viral loads in patients with rheumatoid arthritis (RA) corresponded to higher serum levels of IL-17A. However, this correlation was not found in non-RA controls. Therefore, with the present study, we intended to investigate the influence of EBV DNA on disease processes and development. This was done by assessing the effect of its DNA on the occurrence and severity of arthritis in a mouse model of RA and determining the involvement of Th17 in the pro-inflammatory response.

Materials and methods

mice

The mouse model of arthritis used was collagen-induced arthritis (CIA) in C57BL/6J mice  (  30-32  )  . Female 12-week-old mice were obtained from the American University of Beirut (AUB) Animal Care Facility and treated according to Institutional Animal Care and Use Committee (IACUC) guidelines. They were housed in non-individually ventilated (non-IVC) cages in the same room. They had access to unlimited water and food. Mice were tagged with random ear tags and assigned to different groups; this was used for randomized allocation purposes.

To determine the effect of EBV DNA on the occurrence of arthritis in the RA mouse model

To assess whether EBV DNA affects the occurrence of arthritis in the CIA mouse model, female C57BL/6J mice were used. These mice were  divided into five groups as shown in FIG. Induction of arthritis was performed as previously described (  30  ,  33  ). The arthritis-inducing emulsion was prepared by mixing an equal volume of Type II chicken collagen (Chondrex, Inc., Redmond, Washington) with Freund’s Complete Adjuvant (CFA). For the preparation of CFA, heat-killed  Mycobacterium tuberculosis (Invivogen, Toulouse, France) was added to incomplete at a concentration of 3.3 mg/mlFreund’s adjuvant (IFA) (Chondrex, Inc, Redmond, Washington). Booster injections of chicken type II collagen were given 20 days after the initial collagen challenge; these were administered in a manner similar to the primary challenge but IFA was used instead of CFA. The emulsion injections were given subcutaneously in the tail. The volume of both the primary and booster injections of elicitor injected was 50 µl. C57BL/6J mice receiving 50 µl distilled water subcutaneously in the tail were included.

The study included an arthritis control group that received the inducing agent and the booster. One group was administered EBV DNA (Advanced Biotechnologies, Columbia, MD) 6 days before the first collagen challenge, while another group was administered EBV DNA 15 days after the first collagen challenge. The days selected for administration of EBV DNA were chosen based on our previous observation that the pro-autoimmune cytokine IL-17A peaked in mouse sera 6 days after administration of the DNA (  34). Therefore, in the current study, the DNA was administered such that the peak levels of IL-17A coincided with either collagen administration or the expected onset of symptoms, which occurs approximately 3 weeks after administration of the arthritis-inducing agent. Injections containing the viral DNA contained 144 x 10  ³  copies of EBV DNA in 100 µl distilled water and were administered intraperitoneally. Mice that received 144 x 10  ³  copies of EBV DNA intraperitoneally in 100 µl distilled water were also studied. In addition, a bacterial DNA-treated control group containing 27.2 ng of  Staphylococcus epidermidis was included – DNA obtained in 100 µl of water; this was administered 6 days before collagen (amount corresponds to 144 x 10  ³ copies of EBV DNA). The mice were then monitored for 70 days for the development of arthritis by examining the ankle macroscopically for redness and swelling.

The relative concentration of IgG antibodies against chicken collagen was detected by ELISA. Commercially available microtiter plates were coated with Type II chicken collagen (Chondrex, Inc, Redmond, Washington). This was done by diluting the collagen stock solution (4 mg/ml in 0.05 M acetic acid) using 1X phosphate buffered saline (PBS) to a final concentration of 10 µg/ml. The chicken collagen was then added to the different wells in 100 µl aliquots, sealed with an adhesive plastic cover and incubated overnight at 4°C. The collagen-coated plate was then washed three times with wash buffer (Abcam, Cambridge, UK). The plate was blocked with fetal bovine serum (FBS) for 30 minutes at room temperature. 

The sera of mice from the distilled water-treated group, the collagen-preserving group and EBV DNA 6 days before the collagen-treated group were diluted 1:1. 2 with FBS and transferred into their respective wells in 100 µl aliquots. The plate was then incubated for 2 hours at room temperature, after which it was washed three times using the wash buffer. Horseradish peroxidase (HRP)-conjugated goat IgG anti-mouse antibody (Pierce, Waltham, USA) was diluted to 1/2000 with 0.1 M Tris-buffered saline, pH 8, containing 25% FBS, and 100 µl was added to each well. The plate was incubated for 2 hours at room temperature, after which it was washed three times with the wash buffer. 3,3′,5.5“ -Tetramethylbenzidine (TMB) was then added to each well (100 µl) and incubated at room temperature in the dark for 10 minutes. A stop solution (Abcam) was then added to stop the reaction. The absorbances of the different samples were then measured with an ELISA reader at a wavelength of 450 nm.