Bordetella pertussis toxin

Mouse MOG_35–55 (GL Biochem, Shanghai, China) was emulsified in complete Freud’s adjuvant (Sigma-Aldrich, St Louis, MO, USA) supplemented with Mycobacterium tuberculosis H37Ra (Difco Laboratories, Detroit, MI). Mice were injected s.c. with the MOG in the dorsal flank on day 0. The mice were also injected i.p. with 500 ng of purified Bordetella pertussis toxin (Sigma-Aldrich). The mice were then observed for clinical scores based on the following scale: 0, normal; 0.5, partially limp tail; 1, paralyzed tail; 2, loss in coordinated movement, hind limb paresis; 2.5, one hind limb paralyzed; 3, both hind limbs paralyzed; 3.5, hind limbs paralyzed, weakness in forelimbs; 4, forelimbs paralyzed; 5, moribund or dead. As required by animal ethics, mice were euthanized beyond a clinical score of 4.

Antigens and reference standards used to quantify antibody concentrations are listed in Table S1. Hep B surface antigen (adw) protein and rubella virus capsid full-length protein were purchased from AbCam (Cambridge, UK). Bordetella pertussis toxin, pertussis filamentous hemagglutinin (FHA), and Corynebacterium diphtheria toxin were purchased from Sigma-Aldrich (St. Louis, MO). Tetanus toxoid was purchased from Reagent Proteins (San Diego, CA). Influenza recombinant hemagglutinin (rHA) antigens were purchased from Protein Sciences (Meriden, CT). Pertussis fimbriae 2/3 and pertussis pertactin (69 kDa protein) were purchased from List Biological Labs (Campbell, CA).
The following World Health Organization (WHO) international reference standards were obtained from the National Institute of Biological Standards and Control (NIBSC, Potters Bar, UK) for calculating antigen-specific IgG concentrations: anti-hepatitis B surface antigen immunoglobulin, anti-rubella immunoglobulin, pertussis antiserum, tetanus immunoglobulin, and diphtheria antitoxin IgG (NIBSC code numbers: 07/164, RUBI-1–94, 06/140, TE-3, 10/262, respectively). A commercially available polyclonal IVIG preparation, Privigen, was purchased from CSL Behring (King of Prussia, PA) and used as positive control for influenza-specific antibody measurement.

Mice were immunized by subcutaneous administration of 100 μl methylated bovine serum albumin (mBSA; 1 mg/ml, Sigma-Aldrich) emulsified in complete Freund’s adjuvant (CFA, Sigma-Aldrich). Mice were injected i.p. with 200 ng of heat-inactivated Bordetella pertussis toxin (Sigma-Aldrich). The immune response was boosted after 7 days with identical administration of mBSA/CFA. Inflammatory arthritis was induced 21 days after the initial immunizations by intra-articular administration of 10 μl mBSA (10 mg/ml) into the right hind knee joint. For the WT-PBS group, 10 μl phosphate buffered saline (PBS) was injected into the knee joint. Swelling was monitored by measuring knee joint diameter using a POCO 2T micrometer (Kroeplin), and recorded as the difference between right (inflamed) and left (contralateral) diameters. Knee joints were harvested at day 15 post-AIA for histopathological assessment of disease severity (arthritic index).