Myd88 mice

C57BL/6J, TLR2^−/−, and MyD88^−/− mice were purchased from Jackson Laboratory (Bar Harbor, ME) and bred in‐house and maintained at the University of Vermont animals care facility under protocols approved by Institutional Animal Care and Use Committee. Endotoxin free LPS (Escherichia coli serotype O), Pam2Csk4, Zy, ZD, Curdlan, and WGP were purchased from Invivogen (San Diego, CA). 2‐deoxy‐glucose (2DG) was purchased from Sigma (St. Louis, MO). Antibodies for flow cytometry: 7‐Aminoactinomycin D (7‐AAD), anti‐CD11c (clone N418), anti‐CD86 (clone GL‐1), and anti‐CD40 (clone 3/23) antibodies were purchased from BD Biosciences (San Jose, CA) and Biolegend (San Diego, CA). For Western blot analysis, all antibodies were from Cell Signaling (Danvers, MA) (phosph‐Akt T308 [clone D25E6]), pan‐Akt (clone C67E7), cleaved caspase‐1 (clone E2G2I), and cleaved IL‐1β (clone E7V2A) except for β‐actin (clone 643802), which was purchased from Biolegend. PI3K (20 uM), Syk (10 uM), and TBK1 (5 uM) pharmacologic inhibitors (LY294002, PRT062607, and BX‐795 respectively) were purchased from Selleckchem (Houston, TX).

Wild‐type (WT) C57BL/6 mice and TLR4^‐/‐ mice on the C57BL/6 background were ordered from GemPharmatech. The Myd88^‐/‐ mice (Stock NO: 009088) on C57BL/6 background were obtained from Jackson Laboratory. All mice were kept in standard animal facilities with 12/12 h of light/dark cycles and were free to water and diet. The experiments associated with mice were approved by the Ethics Committee of Cangzhou Central Hospital. The age of all mice used in this study was 6–8 weeks. Experimental mice were killed by CO₂ asphyxiation to achieve euthanasia.

All animal experiments were performed according to the NIH Guidelines for Care and Use of Animals with protocol approval from the Montana State University (Protocol number 2011-44) and Washington State University (Protocol number 00453-004) Institutional Animal Care and Use Committees. Male C57Bl/6, C3H/HeJ, NOD2^−/−, and MyD88^−/− mice were obtained from Jackson Laboratories. Animals were co-housed according to treatment group under SPF conditions, with ad libitum access to food and water. All experiments were performed when animals were eight weeks of age and all groups contained five mice/group. GRA treatments and rotavirus infections were performed as previously described [12] (link). Briefly, mice were administered 50 mg/kg of GRA by oral gavage on day one and then a second time on day three. No adverse effects were observed in GRA treated mice. In experiments where groups included rotavirus infected mice, 10⁵ shedding dose 50 (SD₅₀) of murine rotavirus strain EW was administered by oral gavage on day two. EW does not cause diarrhea or other illness in mice >15 days of age, and infection is measured by antigen shedding in feces. Mice were sacrificed on day eleven by overdose CO₂ inhalation.

WT C57BL/6J mice, BALB/cByJ mice, and Myd88^–/– mice were purchased from The Jackson Laboratory. Tlr9^–/– and Tlr9^loxp/loxp (Flox) mice (33 (link)) on the C57BL/6J background were from Mark J. Shlomchik’s laboratory (Department of immunology, University of Pittsburgh). Ccl19-cre mice (34 (link)) were obtained from Burkhard Ludwig (Institute of Immunobiology, Kantonsspital St. Gallen, St. Gallen, Switzerland). FRC-Tlr9^–/– mice were generated with the Cre-loxP technique and were identified by PCR-based genotyping, using multiple primer pairs, as described previously (17 (link)). These animals were bred in our facilities. All mice were maintained under pathogen-free conditions. Mice were randomly assigned to different experimental or control groups at between 6 and 8 weeks of age. Upon arrival, all mice were acclimated in or animal facility for 3 or more days prior to any experiments.

TLR2^−/−,TLR4^−/−, and MyD88^−/−mice (C57BL/6J background) were from The Jackson Laboratory (Bar Harbor, ME). Specific pathogen-free wild-type (WT) C57BL/6J mice were purchased from the Model Animal Research Center of Nanjing University. Kunming mice (a kind of Swiss Webster mice) were from the Experimental Animal Center of the Third Military Medical University. P. yoelii (Plasmodium yoelii) yoelii 265BY, the uncloned parasite, is isolated in 1969 in Center Africa Republic48 (link), and maintained by passage between Kunming mouse and mosquito. All methods were carried out in accordance with the approved Guide for the Care and Use of Laboratory Animals of the Third Military Medical University. All experimental protocols were approved by the Animal Institute of Third Military Medical University.

Dectin-1^−/− and MyD88^−/− mice were purchased from The Jackson Laboratory; TLR2^−/− were provided by Shizuo Akira (Osaka University, Osaka, Japan). All knockout mice have a C57BL/6 background and were maintained at the animal production service facilities (SCSIE, University of Valencia). Wild-type C57BL/6 mice (Envigo) were used as controls; wild-type CD45.1-positive allotype mice (B6.SJL-Ptprc^a Pepc^b/BoyCrl strain, also known as C57BL/6-Ly5.1 [Charles River Laboratories]) and the transgenic mice (B6.Cg-Tg[CAG-DsRed*MST]1Nagy/J strain, also known as DsRed.T3 [The Jackson Laboratory]) were used for in vivo transplantation assays and/or for the in vitro coculture assays. Experiments were conducted with 8- to 12-week-old mice (regardless of sex). Experiments were approved by the Committee on the Ethics of Animal Experiments of the University of Valencia (permit numbers 2017/VSC/PEA/00207 and 2019/VSC/PEA/0030) and performed according to Spanish law under Reales Decretos 1201/2005 and 53/2013. All efforts were made to minimize suffering.