Immunology and Inflammation

Role of mast cells in endomicrobial sepsis revisited: Mast cell-deficient mice show normal immunological protection

Thorsten B Feyerabend author has email address
Fabienne Schochter
Alpaslan Tasdogan
Maren Bechberger
Sébastien Boutin
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
Pharmacy Department, University Hospital Heidelberg, Heidelberg, Germany
Department of Infectious Diseases, University Hospital Heidelberg, Heidelberg, Germany
Institute for Medical Microbiology, University Hospital Schleswig-Holstein, Lübeck, Germany

https://doi.org/10.7554/eLife.106789.2

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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.
(A). 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 x 10⁸) bacteria (A), or high dose (14 x 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-Cpa3^Cre/+) 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 following saline (NaCl) control injection, or one or two hours after low dose bacterial challenge (3-6 x 10⁸ bacteria). Boxes indicate the 25th to 75th percentiles, center lines the median, whiskers the minimum to maximum values, and each dot represents an individual mouse. Statistical analyses were performed on log(Y+1)-transformed values. Ordinary two-way ANOVA with genotype and time as factors revealed no significant effects of genotype or time x genotype interaction for any of the cytokines, only significant effects of time were observed (F and P values in Table S1). Sample sizes were n = 6 per genotype at 1 h, n = 4 per genotype at 2 h, and for the NaCl group: n = 5 (+/+), n = 4 (Cre/+), and n = 3 (W/Wv), except for IL-10 where +/+ mice were n = 4. (G-H). Absolute numbers of (G) neutrophils (Gr1⁺ CD11b⁺) and (H) macrophages (Gr1⁻ CD11b⁺ F4/80⁺) in the peritoneal cavity of mice treated as in (C-F). Statistical analyses were performed on log(Y)-transformed values using ordinary two-way ANOVA with genotype and time as factors. Significant effects of time, but no effects of genotype or time x genotype interaction, were observed. (F and P values in Table S1). Sample sizes were for NaCl: n = 5 (+/+), n = 3 (Cre/+), and n = 4 (W/Wv); at 1h: n = 6 (+/+), n = 6 (Cre/+), and n = 4 (W/Wv); and at 2h n = 4 (+/+), n = 4 (Cre/+), and n = 3 (W/Wv).

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 x 10⁸) isolated from the cecum of Kit^+/+ or Kit^W/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-Whitney test.

Cecal microbiota composition in Kit^W/Wv and Cpa3^Cre/+ mice compared with their respective littermates.
(A) Community composition of bacterial orders in cecal samples from Kit^W/Wv (outer circle) and Kit^+/+ (inner circle) mice shown as mean relative abundances (n = 6 per group). (B) Relative abundance of bacterial families in individual mice shown as grouped bar plots organized by family. Bars represent data of individual Kit^+/+ (green) and Kit^W/Wv (blue) animals. Statistical analysis was performed using ALDeX2. (C) Community composition of bacterial orders in cecal samples from Cpa3^Cre/+ (outer circle) and Cpa3^+/+ (inner circle) mice shown as mean relative abundances (n = 5 per group). (D) Relative abundance of bacterial families in individual mice shown as grouped bar plots organized by family. Bars represent data of individual Cpa3^+/+ (black) and Cpa3^Cre/+ (red) animals. Statistical analysis was performed using ALDeX2. Taxa with a relative abundance >0.1% in at least one sample within each panel are shown individually; all other taxa were summed up in “Others”.

Co-housing of Kit^W/Wv with Kit^+/+ mice normalizes the susceptibility of Kit^W/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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