Salmonella typhimurium

Intravitreal injection was used to locally administer 1 µg LPS (Salmonella typhimurium; Sigma) in order to induce ocular inflammation as described previously [13 (link)]. Briefly, mice anesthetized with intraperitoneal ketamine (100 mg/kg)/xylazine (10 mg/kg) injection received 1 µL contralateral injections of either saline (1× PBS) vehicle or LPS (diluted in 1X PBS vehicle), via a 10 µL glass syringe (Hamilton) with a 33-gauge needle, into the vitreous chamber of each eye. To ensure minimal damage to ocular tissues, the eye was gently proptosed and held in place by clasping the surrounding eyelids during injections. Whole retinal tissue was harvested, using the “Winkling” method, 24 h after immune induction for downstream assessment of innate immune infiltration using flow cytometry [35 (link),36 (link),37 (link)].

Flagellin and LPS-specific serum IgG levels were quantified by ELISA. Microtiter plates were coated overnight with purified flagellin from Salmonella Typhimurium (Sigma, 100 ng per well) or LPS (from E. coli 0128: B12, Sigma, 2 μg per well) diluted in carbonate–bicarbonate buffer. Serum samples diluted 1:200 were then applied. After incubation and washing, wells were incubated with HRP-linked anti-mouse IgG (1:1000, SouthernBiotech, 1015-05). Quantification was performed using the colorimetric peroxidase substrate tetramethylbenzidine and optical density was read at 450 nm (Versamax microplate reader). Data are reported as optical density corrected by subtracting background (determined by readings in samples lacking serum).

The fecal load of LPS and flagellin were quantified using HEK-Blue-mTLR4 and HEK- Blue-mTL5 cells, respectively (Invivogen, San Diego, CA, USA). The previously processed fecal supernatant was applied to the mammalian cells and incubated for 24 h at 37 °C. Cell culture supernatants were applied to QUANTI-Blue medium (Invivogen) and alkaline phosphatase activity was measured at 620 nm after 30 min. Purified LPS from E. coli (Sigma) and flagellin from Salmonella typhimurium (Sigma) were used for standard curve determination.

Rats were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg body weight) and azepromazine (2.5 mg/kg body weight) before any treatment, and were randomly assigned to four different experimental groups.
Endotoxin-induced uveitis was induced by footpad injections of 200 µg LPS (100 µg each footpad) from Salmonella typhimurium (Sigma-Aldrich, St. Louis, MO, USA), diluted in 0.2 ml saline. Control animals received the same amount of saline without LPS [25] .
Immediately after the LPS injection, rats were i.v. injected with Daclizumab (5 mg/kg body weight) diluted in saline, or the same amount of saline without Daclizumab. Table 1 summarizes the different treatments and the number of rats used.

All experiments were conducted according to the Association for Research in Vision and Ophthalmology (ARVO) statement on the use of animals. Ethics approval for this study was obtained from the Animal Ethics Committee of the Chinese University of Hong Kong. Female Sprague-Dawley rats (about 250 g, 6–8 weeks old) were obtained from the Laboratory Animal Service Centre of Chinese University of Hong Kong. All animals were housed at 25°C with 12/12 hour light-dark cycles and were allowed to access food and water freely. After overnight fasting and body weights were recorded, EIU was induced by injection of lipopolysaccharide (LPS; Salmonella typhimurium; Sigma Chemicals, St. Louis, MO) in sterile saline at 1 mg/kg into a footpad of the rats as previous study (2 (link)). In brief, the rats were randomly divided into three groups (n=6): i) control group - footpad injected with saline and oral feeding with water; ii) LPS group - footpad injected with 1 mg/kg LPS and oral feeding with water; iii) GTE group – footpad injected with 1 mg/kg LPS and oral feeding with 550 mg/kg GTE 2 hours later. Twenty-four hours after LPS injection, rats were anesthetized and terminated by taking blood through heart puncture and cervical dislocation. Plasma and retina samples were collected for metabolomics analysis.

B-1a cells were isolated from the peritoneal cavity and cultivated under limiting dilution conditions in the presence of 30 μg/ml of LPS (Salmonella typhimurium, Sigma-Aldrich, St. Louis, MO, USA), using 3 × 10³ cells of the S17 stroma cell line as feeder cells per culture as feeder cells [29 (link)]. Variable numbers of B-1a cells from each mouse strain in 22 replicates for each cell concentration (10,000; 3,000; 1,000; 300; 100; 18, 6, 2, and 0.66) were cultured to determine the frequency of B-1a clones secreting IgM binding to dsDNA. The growth of dsDNA IgM secreting B cell clones was evaluated by ELISA for the presence of IgM anti-dsDNA in the culture supernatant by ELISA from cells derived from each mouse strain. The percentage of negative cultures was plotted against the B cell number per well, and frequencies of dsDNA specific cells were calculated according to Poisson’s distribution [30 (link)].