Colitis
Seven-week-old C57BL/6 female mice with initial body weight of 19 ± 1 g
were used in this study. To prepare DSS-induced colitis mouse model, 2%
DSS (molecular weight 36,000–50,000 Da; MP Biomedicals LLC, Solon, OH,
USA) dissolved in autoclaved drinking water was provided to micead libitum for 5 days. Mice were then provided with normal
drinking water until the end of the experiment. Each dose of LMT503
(Korean patent 10-2022-0125359, Lmito Therapeutics, Sungnam, Republic of
Korea) or tofacitinib (Selleck Chemicals, Houston, TX) in 0.5% methyl
cellulose/0.2% Tween80 (Sigma-Aldrich, St. Louis, MO, USA) was used to
treat mice by oral gavage once every day starting from 5 days after DSS
treatment. During the whole period, body weight and disease activity
index (body weight loss, rectal bleeding, and diarrhea) were monitored
daily and scored as described previously (Kim et al., 2020). At day 10,
colon lengths were measured, and colon histology studies were proceeded.
To determined bacterial translocation, the spleen and mesenteric lymph
node (MLN) isolated from mice at day 10 were homogenized and serially
diluted. Serial dilutions were plated onto LB agar plates and incubated
at 37°C overnight. CFU/g was then determined. For mice
CX3CR1+ macrophage depletion,
CX3CR1-DTR mice received i.p. injection of 200 ng
diphtheria toxin at day 1, day 4, and day 7 of DSS treatment.
Salmonella Typhimurium Challenge
Mice were challenged orally with 106 CFU of wild-type
invasive Salmonella Typhimurium strain UK-1 at day 13 of DSS
treatment. At 3 days after UK-1 challenge, mice were sacrificed and
their spleens, Peyer’s Patches and mesenteric lymph nodes were isolated
to determine Salmonella Typhimurium strain UK-1 CFU. Tissue was
homogenized and serially diluted. Serial dilutions were plated onto XLD
agar plates with a selective growth medium for Salmonella (BD
Biosciences, Franklin Lakes, NJ, USA). Red colonies with a black center
were determined as Salmonella Typhimurium strain UK-1.