Prophylactic Injection of Recombinant Alpha-Enolase Reduces Arthritis Severity in the Collagen-Induced Arthritis Mice Model

Abstract

Objective: To evaluate the ability of the glycolytic enzyme alpha-enolase (ENO1) or its immunodominant peptide (pEP1) to reduce the severity of CIA in DBA/1 mice when injected in a prophylactic way.

Methods: Mice were treated with mouse ENO1 or pEP1 one day prior to collagen II immunization. Clinical assessment was evaluated using 4 parameters (global and articular scores, ankle thickness and weight). Titers of serum anti-ENO1, anti-cyclic citrullinated peptides (anti-CCP) and anti-CII (total IgG and IgG1/IgG2a isotypes) antibodies were measured by ELISA at different time-points. Disease activity was assessed by histological analysis of both anterior and hind paws at the end of experimentation.

Results: Prophylactic injection of 100 μg of ENO1 reduced severity of CIA. Serum levels of anti-CII antibodies were reduced in ENO1-treated mice. Concordantly, ENO1-treated mice joints presented less severe histological signs of arthritis. ENO1 did not induce a shift toward a Th2 response since IgG1/IgG2a ratio of anti-CII antibodies remained unchanged and IL-4 serum levels were similar to those measured in the control group.

Conclusions: Pre-immunization with ENO1 or its immunodominant peptide pEP1 reduces CIA severity at the clinical, immunological and histological levels. Effects of pEP1 were less pronounced. This immunomodulatory effect is associated with a reduction in anti-CII antibodies production but is not due to a Th1/Th2 shift.

Fig 1. Prophylactic injection of recombinant ENO1 reduces arthritis severity in the collagen-induced arthritis mouse model.

100μg CII was injected to DBA/1 mice at day 0 in CFA, a boost was performed 21 days later in IFA. Recombinant enolase (ENO1) or control (BSA) was injected subcutaneously one day before the first immunization with CII. For ENO1, two doses were tested (10μg = ENO1 10μg (n = 10) and 100μg = ENO1 100μg (n = 10)). For control mice (n = 10), BSA (100 μg) was injected in the same buffer as the one used for ENO1 buffer. Clinical status was evaluated by global score (A), articular score (B), weight variation (C) and ankle thickness (D). Bars show the mean ± SEM; * = p < 0.05; ** = p < 0.01 by 2way ANOVA and Bonferroni post-test.

Fig 2. Prophylactic injection of the immunodominant epitope of ENO1 from P. gingivalis (pEP1) reduces arthritis severity in the collagen-induced arthritis mouse model.

100μg CII was injected to DBA/1 mice at day 0 in CFA, a boost was performed 21 days later in iFA. pEP1 or its control (peptide of 19 aminoacid with random sequence) were injected intraperitoneally one day before the first immunization with CII. Two doses were tested (10μg = pEP1 10μg (n = 10) and 100μg = pEP1 100μg (n = 10)). Clinical status was evaluated by global score (A), articular score (B), weight variation (C) and ankle thickness (D). Bars show mean ± SEM; * = p < 0.05, by 2way ANOVA and Bonferroni post-test.

Fig 3. Antibody production after prophylactic injection of ENO1 or pEP1 in CIA mice model.

Serum levels of anti-ENO1 (A), anti-pEP1 (C) and anti-collagen II (B and D) antibodies in control or treated mice were measured by ELISA. AU is considered as Arbitrary Units. Bars represent the mean ± SEM; * = p < 0.05; ** = p < 0.01, by 2way ANOVA and Bonferroni post-test (A and B); * = p < 0.05, by Mann-Withney (C and D).

Fig 4. Antibody production after prophylactic injection of ENO1 or pEP1 in CIA mice model.

Serum levels of anti-CII IgG1 and IgG2a isotypes were measured by ELISA and IgG1/IgG2a ratio was calculated (A and B). Serum was obtained at different time points after the first CII immunization. Bars represent the mean ± SEM. Statistical analysis used are 2way ANOVA (A) or Mann-Withney (B).

Fig 5. No difference in serum levels of IL-4 and IL-6 in ENO1 or pEP1 treated mice compared to control mice.

Serum levels of IL-4 (A and C) and IL-6 (B and D) in ENO1 treated mice (A-B) and pEP1 treated mice (C-D), compared to control mice, were measured by Luminex. Bars represent the mean ± SEM. Statistical analysis used are 2way ANOVA (A and B) or Mann-Withney (C and D).

Fig 6. Prophylactic injection of ENO1 but not pEP1 reduces arthritis and joint destruction in the CIA mouse model.

Histological analyses were performed on CIA mice receiving prophylactic injection of ENO1 (A) or pEP1 (B) and were compared to those carried out in controls. Both hind and anterior paws were dissected and fixed for 48h in 10% phosphate-buffered formaldehyde 80 days after CII immunization. Different scores were assessed: inflammation (Infl), synovitis (Syno), cartilage resorption (Cart Res) and bone erosion (Bone Ero). In C, anterior paws of control mice (left) and anterior paws of CIA mice who received ENO1 100μg in a prophylactic way (right) were represented. 1: Synovial tissue hypertrophy; 2: Newly formed cartilage; 3: Fibrin deposit; 4: Cartilage resorption. Histograms show mean ± SEM; * = p < 0.05; ** = p < 0.01 by t-tests.