C3h hen mice

The mouse oral squamous cell carcinoma cell line Sq-1979 was obtained from RIKEN BRC (Ibaraki, Japan) and cultured in Minimum Essential Medium Eagle (Sigma-Aldrich, MO, USA) supplemented with 10% fetal bovine serum (Hyclone Laboratories, UT, U.S.A.) and Penicillin–Streptomycin Solution (FUJIFILM Wako Pure Chemical, Osaka, Japan), and kept at 37 °C in 5% CO₂/95% air. Female C3H/HeN mice purchased from Jackson Laboratory Japan (Yokohama, Japan) were housed in pathogen-free conditions. IL-12p40 knockout (KO) mice were generated and provided by Jackson Laboratory Japan on a C3H/HeN background. IL-12p40 gene knockout was confirmed by polymerase chain reaction (PCR)-based genotyping. All mouse experimental protocols were approved by the Animal Ethics Committee of Zeria Pharmaceutical Co., Ltd.

To identify lncRNA transcripts expressed during R. conorii infection, we employed an established mouse model of infection (26 (link)). C3H/HeN mice were obtained from the Jackson Laboratory and housed in an ABSL3 laboratory suite. Following acclimatization, the animals were infected with a high dose of R. conorii (2.25 × 10⁵ pfu/mouse) administered through the tail vein injection. The control group of animals received identical volume of saline intravenously (26 (link)). The animals were then monitored at least once daily for overt signs of disease (ruffled fur, hunched posture, and photophobia) and the body weights were recorded. On day 3 post-infection, mice were euthanized and the lungs were removed aseptically. The tissues thus collected were either snap-frozen or stored at −20°C in RNAlater™ solution. All the animal procedures were performed in accordance to the National Institutes of Health Guide for the Care and use of Laboratory Animals, and were maintained by the approval of Institutional Animal Care and Use Committee at the University of Texas Medical Branch (UTMB) (protocol #1109042). The University has a file with the Office of Laboratory Animal Welfare and an approved Assurance Statement (#A3314-01). Use of any cell line in this study was exempt by Institutional Review Board (IRB), and approved by Institutional Biosafety committee (IBC), UTMB.

Ixodes ricinus larvae and nymphs were obtained from the breeding facility of the Institute of Parasitology, Biology Centre, Czech Academy of Sciences. Ticks were maintained in wet chambers with a humidity of about 95%, temperature of 24°C, and day/night period set to 15/9 h. To prepare both infected and uninfected I. ricinus nymphs, the larvae were fed on either infected or uninfected mice and allowed to molt to nymphs, and after 4 to 6 weeks they were used for further experiments. Inbred, pathogen-free C3H/HeN mice (The Jackson Laboratory, Bar Harbor, ME) were used for the pathogen transmission experiments.

C57BL/6 J and C3H/HeN mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and Taconic Biosciences (Germantown, NY) and maintained under specific pathogen-free conditions in The Scripps Research Institute vivarium and Niigata University animal facility. All male mice used in the experiments were 4–12 weeks in age. Animals were to be excluded from analysis only if they displayed obvious illness or death; these conditions were not observed and no animals were excluded. No randomization of the allocation of animals to experimental groups was performed.

Four-week-old female C3H/HeN mice (Charles River, Wilmington, MA) were used for all experiments. Mice were infected by intradermal injection as previously described [31] (link) with ∼10⁴B. burgdorferi ML23ΔdbpBA/vector, or derivatives expressing dbpA_N40-D10/E9, dbpA_VS461, dbpA_PBr, or dbpA_VS461ΔC11. For the mice sacrificed at 3 days post-infection, the skin at the inoculation site was collected. For the mice sacrificed at 7, 14, 21, or 28 days post-infection, skin at the inoculation site, the tibiotarsal joint, knee joint, bladder, heart, and ear were collected. For infections of mice defective in adaptive immunity, four-week-old C3H-SCID mice (Jackson Lab, Bar Harbor, ME) were infected as described above for C3H/HeN mice. In C3H-SCID mice, a dose of 10³ resulted in a 30- to 236-fold difference in bacterial load of B. burgdorferi strain ML23 and ML23ΔdbpBA/vector, whereas a dose of 10⁴ resulted in indistinguishable colonization by two strains. Hence to maximize the chances of revealing differences in colonization due to the production of the DbpA variants, the lower (i.e. 10³) dose was used in this study. All SCID mice were sacrificed on 28 days post-infection, and skin at the inoculation site, the tibiotarsal joint, knee joint, bladder, heart, and ear were collected.