Tlr2 ko mice

PGRN KO mice, TLR2 KO mice, TLR4 KO mice, TLR7 KO mice, and IFNAR KO mice raised on the C57BL/6 background were purchased from the Jackson Laboratory (Bar Harbor, ME). All mice were genotyped by PCR using genomic tail DNA. WT C57BL/6 mice were purchased from Chongqing Medical University. All mice were housed in the specific pathogen-free animal facility at Chongqing Medical University. In all experiments described here, sex- and age-matched mice were used.

Male C57BL/6 and TLR2KO mice were purchased from Jackson Laboratories (Bar Harbor, Maine, USA). The animal studies were approved by the Institutional Animal Care and Use Committee at the University of Minnesota (Protocol No. 1203A11091). All procedures were conducted in line with the guidelines set forth by the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Typically, 8–10 week old animals were used for our studies. IEC-6 cell lines were obtained from American Type Culture Collection and cultured as recommended by the supplier. IEC-6 cells are rodent small intestinal epithelial cell lines that have been used to study the intestinal barrier and integrity in previous publications42 (link).

The main mouse vaccine validation protocol is identical to the NIH mouse model and is illustrated in Fig. 7 and in the first half of Fig. 7a (up to 60 days). Two types of mouse vaccine models are shown in Fig. 7. Figure 7a tested wild-type C57Bl/6 mice and matching TLR-2-KO mice from Jackson Inc. Figure 8a used only wt-C57Bl/6 mice. Age- and sex-matched mice (6–8 weeks) were tested naïve or vaccines given s.c. to the hind legs of mice (estimated to contain 1 × 10⁶ CFUs through plating on 7H11 agar). After vaccination, at time points indicated, mice were aerosol challenged with approximately 100 CFU of virulent Mtb Erdman using a Glas-Col (Indiana, USA) aerosol apparatus. Four weeks after challenge or at indicated times, organs were harvested for CFUs as described before. Significance of differences in the CFU counts was calculated using two-way ANOVA as outlined below. Five mice per vaccine strain were used and three–four individual mice per time point for flow-cytometry analysis. Challenge Mtb was differentiated from BCG vaccine by culturing organ homogenates in 7H11 agar containing Thiophene-2- carboxylic acid hydrazide (10 µg/mL), which inhibits BCG, but not wild-type Mtb.