In vivo role of leukocyte ADAM17 in the inflammatory and host responses during E. coli-mediated peritonitis

Abstract

Inflammation is the body's initial response to infection, which is harmful when excessive, as exemplified in sepsis inflammatory syndromes. Ectodomain shedding by the membrane metalloprotease ADAM17 is an emerging regulator of inflammation, as it directs the activity of various inflammatory modulators. At this time, however, little is known about the in vivo function of ADAM17. Here, we show that ADAM17-deficient leukocytes afforded mice a survival benefit following Escherichia coli-mediated peritoneal sepsis, which was associated with a reduction in systemic proinflammatory cytokine levels and bacterial burden. A more rapid yet transitory neutrophil infiltration into the peritoneal cavity of conditional ADAM17 knockout mice was observed when compared with control mice, suggesting a mechanism for their enhanced clearance of bacteria. Preventing the shedding of L-selectin augments neutrophil recruitment, and we show that L-selectin shedding by peritoneal neutrophils in conditional ADAM17 knockout mice was impaired. Moreover, their peritoneal TNF-alpha levels were markedly lower than control mice following E. coli challenge. These events indicate key molecular processes involved in the altered time course of neutrophil recruitment in conditional ADAM17 knockout mice. Overall, our study provides novel in vivo evidence of the instrumental role of ADAM17 in modulating inflammation and host resistance during Gram-negative bacterial infection.

Figure 1.

Resistance of (Vav1-cre)Adam17^flox/ΔZn mice to E. coli-mediated peritoneal sepsis. (A) (Vav1-cre)Adam17^flox/ΔZn (ADAM17 KO) and Adam17^flox/ΔZn (Control) mice were injected i.p. with 1.5 × 10⁸ E. coli 0111:B4 and monitored for survival for up to 7 days. All deaths occurred within 72 h following infection. The y-axis indicates percent survival, and the x-axis indicates time after infection. A total of 22 mice in each group from four independent experiments were infected. P = 0.0002, log-rank test. (B) (Vav1-cre)Adam17^flox/ΔZn and control mice (age- and sex-matched) were injected i.p. with 5 × 10⁷ E. coli 0111:B4. Four and 24 h after E. coli challenge, bacterial levels in the peritoneal cavity were determined. Results are expressed as mean ± sd of four mice in each group at each time-point. Data are representative of three independent experiments.

Figure 2.

Systemic inflammation is reduced in (Vav1-cre)Adam17^flox/ΔZn mice following E. coli challenge. (Vav1-cre)Adam17^flox/ΔZn and Adam17^flox/ΔZn mice (age- and sex-matched) were injected i.p. with 5 × 10⁷ E. coli 0111:B4. Four hours later, plasma levels of the indicated cytokines were quantified by ELISA. Results are expressed as mean ± sd of four mice in each group. Data are representative of three independent experiments. Plasma levels of the cytokines in both groups of mice prior to infection were below the detection sensitivity of the ELISA (data not shown).

Figure 3.

Neutrophil recruitment in (Vav1-cre)Adam17^flox/ΔZn mice during E. coli-mediated peritonitis. (A) Peripheral blood samples from uninfected (Vav1-cre)Adam17^flox/ΔZn and Adam17^flox/ΔZn mice were analyzed for neutrophil counts. Results are expressed as mean ± sd of 10 mice in each group. (B) (Vav1-cre)Adam17^flox/ΔZn and control mice (age- and sex-matched) were injected i.p. with 5 × 10⁷ E. coli 0111:B4 (B–D). Four and 24 h after E. coli challenge, neutrophil levels in the peritoneal cavity of both groups of mice were determined. Results are expressed as mean ± sd of four mice in each group at each time-point. Data are representative of three independent experiments. (Vav1-cre)Adam17^flox/ΔZn and control mice had few if any peritoneal neutrophils prior to infection (data not shown). (C) Relative surface expression levels of L-selectin and LFA-1 were determined on peritoneal neutrophils (Ly-6G⁺, F4/80⁻) from (Vav1-cre)Adam17^flox/ΔZn and control mice 4 h after E. coli challenge. The dashed lines indicate staining of neutrophils from (Vav1-cre)Adam17^flox/ΔZn mice by an isotype-matched negative control mAb for the L-selectin or the LFA-1 mAb. The staining intensity of leukocytes from control mice by the same isotype-matched negative control mAb was comparable (data not shown). The x-axis = Log 10 fluorescence. Data are representative of three independent experiments. (D) Four hours after E. coli challenge, the levels of TNF-α in the peritoneal lavage fluid of (Vav1-cre)Adam17^flox/ΔZn and control mice were analyzed by ELISA. Results are expressed as mean ± sd of 10 mice in each group from three independent experiments.