Figure 2. Reduced inflammation in p38γ/δ KIKO mice in response to septic shock.

(A) Wild-type (WT) and p38γ/δKIKO mice were intravenously injected with 1 × 10⁵ CFU of C. albicans. Kidney fungal load was determined 3 days after infection. Each symbol represents an individual mouse. Figure shows mean ± standard error of the mean (SEM), ns not significant; *p ≤ 0.05, relative to WT kidney cells. Kidney cells were stained with (B) anti-CD45, (C) anti -Ly6G and -F4/80 antibodies and positive cells analysed by flow cytometry. CD45⁺ cells were gated and -F4/80⁺ and -Ly6G⁺ cells analysed by flow cytometry. Representative profiles are shown. Each symbol represents an individual mouse. Histograms shows mean ± SEM, ns not significant; *p ≤ 0.05, relative to WT kidney cells. (D) Mice were treated as in (A) and the mRNA levels of indicated genes in the kidney were measured by quantitative PCR (qPCR) 3 days after infection. Each symbol represents an individual mouse. Figure shows mean ± SEM (n = 4 mice/condition). ns, not significant, *p ≤ 0.05, **p ≤ 0.01. (E) WT (n = 31), p38γ/δKO (n = 18), and p38γ/δKIKO (n = 19) mice were injected with lipopolysaccharide (LPS) (50 μg/kg) and D-Gal (1 g/kg), and survival was monitored for up to 9 hr. Graph shows % survival at the indicated times. **p ≤0.01, ***p ≤ 0.001. (F) Serum from mice (E) was collected 2 hr after LPS/D-Gal injection, and TNFα, IL-6, and IL10 were measured in a Luminex cytokine assay. Each symbol represents an individual mouse. Figure shows mean ± SEM (n = 6–7 mice). ns, not significant; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. (G) Livers were collected 6 hr after LPS/D-Gal injection. Panels show haematoxylin and eosin (H&E) stained liver sections (left) and whole livers (right). (H) Serum ALT (alanine transaminase) and AST (aspartate aminotransferase) activity at 6 hr after LPS/D-Gal injection. Each symbol represents an individual mouse. Figure shows mean ± SEM (n = 5–7 mice). ns, not significant. (I) Apoptotic TUNEL positive (red) and total nuclei (Hoechst stained-blue) cells were counted using ImageJ programme and the percentage of apoptotic cells calculated. 25 sections per mouse were scored. Representative TUNEL stained liver sections are shown, and figure shows mean ± SEM (n = 6 mice). ns, not significant. Each symbol represents an individual mouse.

Figure 2—figure supplement 1. Characterization of immune cell populations of the p38γ/δKIKO mouse in basal conditions.

(A) In vitro bone marrow-derived macrophage (BMDM) development is not affected in p38γ/δKIKO mice. BMDM from wild-type (WT) and p38γ/δKIKO mice was stained with anti-F4/80 antibody, and analysed by flow cytometry. Results are representative plots (n = 3). (B) Bone marrow (BM) or spleen cell suspensions were stained with the indicated antibodies and analysed by flow cytometry. Representative flow cytometry profiles and analysis are shown. (C) Frequency and (D) total cell number of leucocytes (CD45⁺), myeloid cells (CD11b⁺), neutrophils (Ly6G⁺), and macrophages (F4/80⁺) in adult mouse BM or spleen. Percentage of myeloid cell population was determined relative to CD45⁺ cell and percentages of neutrophils and macrophages were determined relative to CD45⁺ CD11b⁺ cell. (E) Total cell number in BM or spleen of the indicated genotypes. (F) Weight of the spleen of the indicated genotypes. Each dot in C-F represents a single mouse (n = 4–5). ns, not significant; *p ≤ 0.05.