Table 1.
Sex differences for septic WT mice at 24-hour timepoint.
Table 2.
Sex differences for septic MMP7KO mice.
Fig 1.
Severity of illness, hypothermia, bacterial burden, and weight loss over the course of a 24-hour septic insult [cecal slurry (CS; 1.6mg/g) + hyperoxia (HO; 95% O2)] in MMP7KO mice and their wild-type littermates.
Septic mice (in both WT and MMP7KO) showed significantly greater illness severity (A) and loss of body temperature (B) at all timepoints after 4 hours post-CS+HO, as well as spleen bacterial burden (C), peritoneal bacterial burden (D), and lung bacterial burden (E) at 24-hours post CS+HO compared to control mice. WT septic mice also showed significantly greater weight loss compared to WT control mice at all 12-hours and 24-hours post CS+HO (F). MMP7KO septic mice had significantly greater weight loss at 24-hours post CS+HO and numerically greater weight loss compared to MMP7KO control mice. No differences were observed across the genotypes for septic mice any of the measured outcomes. N = 9-29. [Statistical analysis: Repeated Two-way ANOVA (A, B, F); Kruskal-Wallis test with a Dunn’s multiple comparisons test (C-E)]. Each point represents either the combined median of male and female animals (A, B, F) or an individual male (solid circle) or female (open circle) animal (D-F). Error bars indicate interquartile range, while horizontal lines represent statistical comparisons between septic WT (black) or MMP7KO (pink) and their respective controls (A, B, F). while horizontal line indicates combined median of male and female animals (C-E). Control = 5% dextrose + room air at 21% O2.
Fig 2.
Systemic Inflammation associated with a 24-hour septic insult [cecal slurry (CS; 1.6mg/g) + hyperoxia (HO; 95% O2)] in MMP7KO mice and their wild-type littermates.
Septic WT mice had significantly higher peritoneal wash inflammatory cell counts (A), peritoneal wash neutrophil counts (B), and plasma concentrations of concentrations of TNF-α (C), IL-1β (D), IL-6 (E), CXCL-1 (F), IL-12p70 (G), IFN-γ (H), and IL-10 (I) compared to their control mice. Similar significant increases were observed for septic MMP7KO mice compared to their controls, except for plasma IL-12p70 (G), IFN-γ(H). In septic MMP7KO mice plasma IL-10 (I; p =0.0811) trended towards an increased concentration compared to MMP7KO control mice. No differences were observed between MMP7KO and WT mice in either treatment group. N = 6-19. [Statistical analysis: Kruskal-Wallis test with a Dunn’s multiple comparisons test]. Each point represents an individual male (solid circle) or female (open circle) animal. Horizontal line indicates combined median of male and female animals. Control = 5% dextrose + room air at 21% O2. TNF-α = tumor necrosis factor-α, IL-12p70 = interleukin-12p70, IL-6 = interleukin-6, IL-1β = interleukin-1β, and CXCL-1 = C-X-C motif chemokine ligand 1, IFN-γ = interferon gamma, IL-10 = interleukin-10.
Fig 3.
Lung inflammation induced by a septic insult [cecal slurry (CS; 1.6mg/g) + hyperoxia (HO; 95% O2)] in female and male MMP7KO mice compared to their wild-type littermates.
Septic mice (in both WT and MMP7KO) had significantly higher BAL total cell counts (A), and BAL concentrations of TNF-α (B), IL-1β (C), and CXCL-1 (D), and IL-10 (E), as well as lung tissue IL-1β (F) and CXCL-1 (G) mRNA expression compared to their control mice. Septic WT mice also had significantly higher BAL concentrations of IL-6 (H), as well as lung tissue IFN-γ (I) and IL-6 mRNA (J) compared to WT controls, with BAL IL-12p70 (K) being numerical higher. Septic MMP7KO mice had numerically higher BAL IL-6 concentrations as well a lung tissue CXCL-1 and IL-6 mRNA compared to MMP7KO control mice, but did not differ with regards to lung tissue IFN-γmRNA expression or BAL concentrations of IL-12p70. Septic mice did not differ from control mice across either genotype for BAL IFN-γ (L) or BAL neutrophils (M). No differences between MMP7KO and WT genotypes were observed for any outcomes of lung inflammation in either treatment group. N = 6-19. [Statistical analysis: Kruskal-Wallis test with a Dunn’s multiple comparisons test]. Each point represents an individual male (solid circle) or female (open circle) animal. Horizontal line indicates combined median of male and female animals. Control = 5% dextrose + room air at 21% O2, BAL= bronchoalveolar lavage, TNF-α = tumor necrosis factor-α, IL-12p70 = interleukin-12p70, IL-6 = interleukin-6, IL-1β = interleukin-1β, and CXCL-1 = C-X-C motif chemokine ligand 1, IFN-γ = interferon gamma, IL-10 = interleukin-10.
Fig 4.
Alveolar-capillary barrier disruption induced by a septic insult [cecal slurry (CS; 1.6mg/g) + hyperoxia (HO; 95% O2)] in MMP7KO mice and their wild-type littermates.
Compared to control mice, septic WT and MMP7KO mice showed significant increases in lung wet-to-dry weight ratios (A). Septic WT mice also showed significantly higher BAL protein compared to WT controls, while BAL protein in septic MMP7KO mice was not different compared to MMP7KO control mice (B). Septic MMP7KO mice also showed numerically higher BAL IgM compared to MMP7KO controls, while septic WT mice did not differ compared to control WT mice (C). No differences were observed between WT and MMP7KO mice in either treatment group. N = 6-21. [Statistical analysis: Kruskal-Wallis test with a Dunn’s multiple comparisons test]. Each point represents an individual male (solid circle) or female (open circle) animal. Horizontal line indicates combined median of male and female animals. Control = 5% dextrose + room air at 21% O2, BAL= bronchoalveolar lavage, IgM = immunoglobulin M.
Fig 5.
Histological Lung Injury induced by a septic insult [cecal slurry (CS; 1.6mg/g) + hyperoxia (HO; 95% O2)] in MMP7KO mice and their wild-type littermates.
Representative whole lung sections in mice used for histology scoring (A). Lungs were stained with H&E and 20x images of the entire section scanned by the Vanderbilt University Medical Center Digital Histology Shared Resource using the Leica SCN400 Slide Scanner. Septic mice (WT and MMP7KO) had numerically higher total lung injury scores compared to their respective controls (B). No differences were observed between WT and MMP7KO mice in either treatment group. Individual parameters of lung histology injury score are presented as a table (C). N = 3-10. [Statistical analysis: (B) Kruskal-Wallis test with a Dunn’s multiple comparisons test; (C) Mann-Whitney U test]. Each point represents an individual male (solid circle) or female (open circle) animal. Horizontal line indicates combined median of male and female animals. Control = 5% dextrose + room air at 21% O2. RBC = red blood cell, IQR = interquartile range.
Fig 6.
Kidney Dysfunction, Inflammation, and Injury induced by a septic insult [cecal slurry (CS; 1.6mg/g) + hyperoxia (HO; 95% O2)] in MMP7KO mice and their wild-type littermates.
For both genotypes, septic mice showed significantly higher plasma urea nitrogen concentrations (A), kidney tissue mRNA expression of IL-1β (B), IL-6 (C), CXCL-1 (D), and NGAL (D), as well as kidney tissue NGAL protein (F) compared to control mice. Septic WT mice also had significantly higher kidney tissue mRNA expression of KIM-1, while septic MMP7KO had numerically higher KIM-1 mRNA (H). However, septic mice did not differ from control for kidney tissue KIM-1 protein in either genotype (G). No differences were observed between WT and MMP7KO mice in either treatment group. Representative western blots are displayed for NGAL (E) and KIM-1 (H) relative to β-actin. N = 6-20. [Statistical analysis: Kruskal-Wallis test with a Dunn’s multiple comparisons test]. Each point represents an individual male (solid circle) or female (open circle) animal. Horizontal line indicates combined median of male and female animals. Control = 5% dextrose + room air at 21% O2. IL-1β = interleukin-1β, IL-6 = interleukin-6, and CXCL-1 = C-X-C motif chemokine ligand 1, NGAL = neutrophil gelatinase-associated lipocalin, KIM-1 = kidney injury marker-1.
Fig 7.
Lung and Kidney Matrix Metalloproteinase Expression induced by a septic insult [cecal slurry (CS; 1.6mg/g) + hyperoxia (HO; 95% O2)] in MMP7KO mice and their wild-type littermates.
WT septic mice had significantly higher lung MMP9 (A) and MMP2 (C), as well as kidney MMP9 (E), but not kidney MMP2 (F) mRNA expression compared to WT control mice. Septic MMP7KO mice had significantly higher lung MMP9 and numerically higher kidney MMP9 compared to control MMP7KO mice. MMP7KO septic mice did not differ with regards to MMP9 or MMP2 in either organ. No differences were observed between genotypes for control mice. N = 6-21. [Statistical analysis: Kruskal-Wallis test with a Dunn’s multiple comparisons test]. Each point represents an individual male (solid circle) or female (open circle) animal. Horizontal line indicates combined median of male and female animals. Control = 5% dextrose + room air at 21% O2. MMP9 = matrix metalloproteinase-9, MMP2 = matrix metalloproteinase-2.