Immunology and Inflammation

Susceptibility of Kit-mutant mice to sepsis caused by enteral dysbiosis, not mast cell deficiency

Thorsten B Feyerabend author has email address
Fabienne Schochter
Alpaslan Tasdogan
Hans-Reimer Rodewald author has email address

Division of Cellular Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany
Institute for Immunology, University of Ulm, Ulm, Germany
Gynecology and Obstetrics, University Clinics Ulm, Ulm, Germany
Department of Dermatology, University Hospital Essen and German Cancer Consortium (DKTK), Essen, Germany

https://doi.org/10.7554/eLife.106789.1

Open access
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Figures and data

Kit^W/Wv mutant mice, but not mast cell-deficient Cpa3^Cre/+ mice, are susceptible for cecal ligation and puncture sepsis.
Survival of B6-Cpa3^+/+ (100% of n = 10), B6-Cpa3^Cre/+ (100% of n = 11), and WBB6F1-Kit^W/Wv (25 % of n = 12) mice to mild (25 G needle) cecal ligation and puncture. WBB6F1-Kit^W/Wv mice vs. B6-Cpa3^+/+ (p < 0.0004).
(B). Survival of B6-Cpa3^+/+ (55 % of n = 49), B6-Cpa3^Cre/+ (42 % of n = 59), and WBB6F1-Kit^W/Wv (0 % of n = 3) mice under severe (22 G needle) cecal ligation and puncture. B6-Cpa3^+/+ vs. B6-Cpa3^Cre/ p = 0.2345. WBB6F1-Kit^W/Wv vs. B6-Cpa3^+/+ p < 0.0001.
(C). Survival of WBB6F1-Cpa3^+/+ (82.5 %, n = 63), WBB6F1-Cpa3^Cre/+ (74.2 %, n = 62), and WBB6F1-Kit^W/Wv (0 %, n = 24) mice to severe (22 G needle) cecal ligation and puncture. P-values for curve comparisons in A-C were calculated using the Mantel-Cox Log-rank test.
(D-F). Kinetics (in hours) of median serum concentrations for TNFα (D), IL-6 (E), and MCP-1 (F) in B6-Cpa3^+/+, B6-Cpa3^Cre/+, and WBB6F1-Kit^W/Wv mice after severe (22-G needle) cecal ligation and puncture (solid lines), or after sham operations (same procedure but without cecal ligation and puncture) (dashed lines).
(G-I). From the kinetic shown in D-F, box plots were drawn for the 24-h time point indicating serum concentrations for TNFα, IL-6, and MCP-1 in B6-Cpa3^+/+, B6-Cpa3^Cre/+ and WBB6F1-Kit^W/Wv mice. Each dot represents an individual mouse. The boxes extend from the 25th to 75th percentiles and the median is indicated. Whiskers range from minimum to maximum. P-values were calculated on log-transformed values by one-way ANOVA with Tukey’s correction for multiple comparisons.

Kit^W/Wv mice are as resistant to injection of cecal slurry as Kit^+/+ mice.
(A, B). Survival of B6-Cpa3^+/+, B6-Cpa3^Cre/+, and WBB6F1-Kit^W/Wv mice following injection of low dose (3 × 10⁸) bacteria (A), or high dose (14 × 10⁸) bacteria (B) from intestines of normal C57BL/6 mice. For both doses, the outcome for Kit^W/Wv mice was statistically comparable to that of Kit wild-type mice, and mast cell deficiency (B6-Cpa3+/+ versus B6-Cpa3Cre/+) also played no role in survival. Low dose survival: +/+ 100% of n = 12; Cre/+ 100% of n = 16; W/Wv 94% of n = 16 and p (+/+ vs W/Wv) = 0.3865; high dose survival: +/+ 10% of n = 10; Cre/+ 11% of n = 9; W/Wv 45% of n = 11 and p (+/+ vs W/Wv) = 0.1110. P-values of the survival curve comparisons were calculated using the Mantel-Cox Log-rank test. (C-F). Concentration and kinetic of inflammatory cytokine responses in peritoneal lavage fluid from B6-Cpa3^+/+, B6-Cpa3^Cre/+ and WBB6F1-Kit^W/Wv after saline (NaCl) control injections, or one or two hours after low dose (3-6 × 108 bacteria) i.p. injection. Boxes in the plots extend from the 25th to 75th percentiles and the median is indicated. Whiskers range from minimum to maximum. Each dot represents an individual mouse. For p values, we compared the 2-hour anti-bacterial response vs saline for each genotype, and the response at 2 hours between the genotypes. P-values were calculated by ordinary one-way Anova with Šídák correction for multiple comparison. Sample numbers were for 1h: n = 6; 2h: n = 4 (for all genotypes); NaCl: n = 3 (+/+), n = 2 (Cre/+), n = 1 (W/Wv).
Absolute numbers of neutrophils (Gr1⁺ CD11b⁺) in the peritoneal cavity of B6-Cpa3^+/+, B6-Cpa3^Cre/+ and WBB6F1-Kit^W/Wv after NaCl control injections, or one or two hours after low dose (3-6 × 10⁸) bacteria i.p. injection. Group sizes were for NaCl: n = 3 (+/+), n = 2 (Cre/+), n = 2 (W/Wv); for 1h: n = 6 (+/+), n = 6 (Cre/+), and n = 4 (W/Wv), and for 2h n = 4 (+/+), n = 4 (Cre/+), and n = 3 (W/Wv). P-values were calculated on log-transformed values by ordinary one-way Anova with Šídák correction for multiple comparison.
(H). Absolute numbers of macrophages (Gr1⁻ CD11b⁺ F4/80⁺) in the samples as in G. Sample size and calculation of P=values as in (G). 2h: not done (n.d.).

Kit^W/Wv mice harbor more pathogenic enteral bacteria than Kit^+/+ mice.
(A-C). Survival of Cpa3^+/+ (A), Cpa3^Cre/+ (B), and Kit^W/Wv (C) mice following injection of increasing doses of bacteria (2, 5, 10, 20 or 40 × 10⁸) isolated from the cecum of Kit^+/+ or KitW/Wv donor mice. All animals in this experiment were on the WBB6F1 strain background. Group sizes (n) are given in the figure. (D). Bacterial colony forming units were determined for cecal slurries of Kit^+/+ (n = 6) and Kit^W/Wv mice (n = 6) which included all cecal slurries used in (A-C). Bars graphs show the mean + SD, each dot represents counts from one individual mouse. P-values were calculated with the Mann-Whithney test.

Co-housing of Kit^W/Wv with Kit^+/+ mice normalizes the susceptibility of KitW/Wv mice to cecal ligation and puncture.
Kit^+/+ and Kit^W/Wv mice (WBB6F1 background) were housed together from weaning onwards until cecal ligation and puncture (22 G needle). This constant co-housing, increased the survival rate of Kit^W/Wv mice (n=38) and equalized it (p=0.3809) to the probability of survival of Kit^+/+ mice (n=25). P-values were calculated using the Mantel-Cox Log-rank test.

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