Expression and Function of Granzymes A and B in Escherichia coli Peritonitis and Sepsis

Abstract

Escherichia (E.) coli is the most common causative pathogen in peritonitis, the second most common cause of sepsis. Granzymes (gzms) are serine proteases traditionally implicated in cytotoxicity and, more recently, in the inflammatory response. We here sought to investigate the role of gzms in the host response to E. coli-induced peritonitis and sepsis in vivo. For this purpose, we used a murine model of E. coli intraperitoneal infection, resembling the clinical condition commonly associated with septic peritonitis by this bacterium, in wild-type and gzmA-deficient (gzmA-/- ), gzmB-/- , and gzmAxB-/- mice. GzmA and gzmB were predominantly expressed by natural killer cells, and during abdominal sepsis, the percentage of these cells expressing gzms in peritoneal lavage fluid decreased, while the amount of expression in the gzm+ cells increased. Deficiency of gzmA and/or gzmB was associated with increased bacterial loads, especially in the case of gzmB at the primary site of infection at late stage sepsis. While gzm deficiency did not impact neutrophil recruitment into the abdominal cavity, it was accompanied by enhanced nucleosome release at the primary site of infection, earlier hepatic necrosis, and more renal dysfunction. These results suggest that gzms influence bacterial growth and the host inflammatory response during abdominal sepsis caused by E. coli.

Figures

Figure 1

Lymphocyte source of intracellular granzymes A and B in peritoneal lavage fluid (PLF) and blood of wild-type mice before and after intraperitoneal infection with E. coli. Mice were infected intraperitoneally with 1.3∗104 CFU E. coli and sacrificed before and at 6, 14, and 20 h after infection. To show the lymphocyte source of each granzyme, the total gzmA- or gzmB-positive cells were selected and, the lymphocyte subsets to which those gzm-expressing cells belong, identified. (a) and (c) show the percentage of the total gzmA and (b) and (d) of gzmB expressed by different lymphocyte subsets from PLF ((a) and (b)) and blood ((c) and (d)) are shown. Data are box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile, and largest observation. N = 5-6 at each time point.

Figure 2

Granzyme A and B expression in NK cells from wild-type mice before and after intraperitoneal infection with E. coli. Mice were infected intraperitoneally with 1.3∗104 CFU E. coli and sacrificed before and at 6, 14, and 20 h after infection. Percentage and median fluorescence intensity (MFI) of intracellular gzmA (a, b) and gzmB (c, d) in NK cells from peritoneal lavage fluid (PLF) and blood are shown. Data are box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile, and largest observation. N = 5-6 at each time point. ∗P < 0.05, ∗∗P < 0.01 determined by Mann–Whitney U test.

Figure 3

Bacterial loads in peritoneal lavage fluid (PLF), blood, liver, and lung of wild-type, gzmA−/−, gzmB−/−, and gzmAxB−/− mice during E. coli peritonitis. Mice were infected intraperitoneally with 1.3∗104 CFU E. coli and sacrificed at 6, 14, and 20 h after infection. Data are box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile, and largest observation. N = 7-8 per group at each time point. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 by Mann–Whitney U test. Dotted line represents the lower detection limit of CFU.

Figure 4

Cytokine and chemokine levels as well as neutrophil counts in peritoneal lavage fluid (PLF) of wild-type, gzmA−/−, gzmB−/−, and gzmAxB−/− mice during E. coli peritonitis. Mice were infected intraperitoneally with 1.3∗104 CFU E. coli and sacrificed at 6, 14, and 20 h after infection. Data are box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile, and largest observation. N = 7-8 per group at each time point. ∗P < 0.05 and ∗∗P < 0.01 determined by Mann–Whitney U test. Dotted lines represent lower detection limits. NA: not available.

Figure 5

Histopathology of the liver and lung from wild-type, gzmA−/−, gzmB−/−, and gzmAxB−/− mice during E. coli peritonitis. Mice were infected intraperitoneally with 1.3∗104 CFU E. coli and sacrificed at 6, 14, and 20 h after infection. Pictures of hematoxylin-eosin staining of representative tissue samples are shown; original magnification 10x; inset shows a 40x magnification of a thrombus (arrow); scale bars: 200 μm. For each organ slide (1 per mouse), the whole section was examined and scored. (a), (b), (c), and (d) show, respectively, wild-type and gmzA−/−, gzmB−/−, and gzmAxB−/− mice ((b), (c), and (d) presenting necrotic areas) livers 14 h after infection. Necrosis (expressed as percentage in (i)) and thrombi (j) were found at 14 and 20 h in the liver. (e), (f), (g), and (h) show, respectively, wild-type and gmzA−/−, gzmB−/−, and gzmAxB−/− mice lungs 20 h after infection. Semiquantitative histology total score of lung slides are represented (k). Data are box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile, and largest observation. N = 7-8 per group at each time point. ∗P < 0.05 determined by Mann–Whitney U test.