Bordetella pertussis toxin ptx

Peptide SGIPYIISYLHPGNTILHVD representing residues 161–180 of IRBP was purchased from China Peptide Co., Ltd (Shanghai, China). Bordetella pertussis toxin (PTx) and Complete Freund Adjuvant (CFA) were purchased from Sigma-Aldrich (St. Louis, USA).

EAU was induced as described previously [29 (link)]. IL-27Rα^−/− and WT littermates were immunized subcutaneously with 200 μL emulsion of 200 μg human IRBP_1–20 (GPTHLFQPSLVLDMAKVLLD, Hanhong, China) in an equal volume of complete Freund’s adjuvant (CFA, Sigma-Aldrich, St. Louis, MO, USA) containing 2.5 mg/mL Mycobacterium tuberculosis strain 37RA (Sigma-Aldrich, St. Louis, MO, USA). In addition, these mice also received 0.5 μg of Bordetella pertussis toxin (PTX, Sigma-Aldrich, St. Louis, MO, USA) intraperitoneally on the day of immunization and the next day of immunization.

Eight-week-old pathogen-free C57BL/6 males were allowed to adapt to laboratory environment week before the experiment began (n = 48). Experimental autoimmune encephalomyelitis (EAE) was induced by subcutaneous injection in the rear flanks with 200 μg of myelin oligodendrocyte glycoprotein MOG_35–55 (purity > 95%) per mouse emulsified in complete Freund’s adjuvant (CFA) containing 300 μg of Mycobacterium tuberculosis H37RA strain. Immediately afterwards and again 2 days later, the animals received an intraperitoneal injection of 400 ng of Bordetella pertussis toxin (PTX; Sigma, St. Louis, MO, USA) in 100 μL of PBS, pH 7.2. Mice were orally infected with 300 L3 of H. polygyrus 21 days postimmunization. Clinical signs and ascending paralysis in EAE were assessed as described before [13 (link)]. Four experimental groups of mice were formed. The first group was immunized with EAE (EAE), the second group of mice was infected with H. polygyrus (HP), and the third group was immunized with EAE and infected with H. polygyrus (HP EAE). The control group consisted of uninfected, untreated mice (CTR). The mice were euthanized and samples were obtained six days after infection, when the parasite was in its fourth larval stage.

EAE was induced in 10–12-week-old female nTNFR1KO and nTNFR2KO mice, and their respective TNFR1ff and TNFR2ff littermate controls as previously described [31 (link)]. EAE was induced by subcutaneous (s.c.) tail base injection of 37 μg of rat myelin OLG glycoprotein 35-55 peptide (MOG) dissolved in 100 μl of saline and emulsified in 100 μl complete Freund’s adjuvant (CFA) (Sigma-Aldrich) supplemented with additional 400 μg of H37Ra Mycobacterium tuberculosis (Sigma-Aldrich) (MOG/CFA). Mice also received an i.p. injection of 200 ng of Bordetella pertussis toxin (PTx) (Sigma-Aldrich) on days 0 and 2. Mice were assessed daily for clinical signs as previously described [31 (link)]. Briefly, mice were examined using an EAE scoring scale as follows: grade 0, no clinical symptom; grade 1, limp tail; grade 2, hindlimb weakness; grade 3, hindlimb paralysis; grade 4, forelimb and hindlimb paralysis; grade 5, moribund or dead. Animals with a score of 4 or above were euthanized. Mice were allowed free access to food and water throughout the experiment.

To induce EAU, C57BL/6 mice were immunized with a 200 µL emulsion of 150 µg human IRBP_1–20 (GPTHLFQPSLVLDMAKVLLD, Hanhong, China) in an equal volume of complete Freund’s adjuvant (CFA, Sigma-Aldrich, St. Louis, MO, USA) containing 2.5 mg/mL Mycobacterium tuberculosis (strain 37RA) (Sigma-Aldrich, St. Louis, MO, USA) and 0.5 µg of Bordetella pertussis toxin (PTX, Sigma-Aldrich, St. Louis, MO, USA), as described in a previous study [36 (link)].
For treatment, EAU mice were intravitreally injected with mouse-recombinant IL-24 (200 ng in 2 µL) (R&D Systems, Minneapolis, MN, USA) into the left eye at the onset of disease. As a negative control, 2 µL of PBS was injected into the right eye. Eyes were examined by funduscopy at regular intervals using the Micron-IV retinal imaging system for small animals (Phoenix Research Laboratories, Pleasanton, CA, USA) and scored on a scale of 0–4, as described in previous study [37 (link)]. Experiments were terminated on day 6–8 after disease onset for eye collection.