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Moesin and myosin phosphatase confine neutrophil orientation in a chemotactic gradient.

Liu X, Yang T, Suzuki K, Tsukita S, Ishii M, Zhou S, Wang G, Cao L, Qian F, Taylor S, Oh MJ, Levitan I, Ye RD, Carnegie GK, Zhao Y, Malik AB, Xu J - J. Exp. Med. (2015)

Bottom Line: Neutrophils respond to invading bacteria by adopting a polarized morphology, migrating in the correct direction, and engulfing the bacteria.Attractant-induced activation of myosin phosphatase deactivated moesin at the prospective leading edge to break symmetry and establish polarity.Subsequent translocation of moesin to the trailing edge confined the formation of a prominent pseudopod directed toward pathogens and prevented secondary pseudopod formation in other directions.

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Affiliation: Department of Pharmacology and Department of Medicine, University of Illinois, Chicago, IL 60612.

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Moesin regulates neutrophil microbial killing and inflammation. (A and B) WT and moesin knockout (Msn−/Y) mice were i.t. injected with P. aeruginosa (2 × 105). 8 h later, lungs were isolated, and the number (A) and percentage (B) of surviving colonies derived from lysates were determined. (C) WT and Msn−/Y neutrophils were incubated with opsonized P. aeruginosa for the indicated times, and surviving colonies were determined. (A–C) **, P < 0.01 compared with WT (Student’s t test). (D–F) Microvascular injury was induced in a classical LSR by consecutive injections of 80 µg LPS and then 0.2 µg TNF or PBS as controls. (D) Macroscopic appearance of dorsal skin in WT and Msn−/Y mice after LSR. Bar, 5 mm. (E) The degree of hemorrhage in the WT and Msn−/Y mice in D was estimated based on densitometry analysis of skin samples receiving either LPS or PBS injection. Results are shown as the ratio of the value with LPS versus the value with PBS. ***, P < 0.001 (Student’s t test). (F) Tissue MPO activities in skins were measured and normalized by tissue weight. Data are presented as V-Max value/g tissue. **, P < 0.01 (Student’s t test). (G) WT or Msn−/Y mice were i.p. injected with 10 nM fMLF, and neutrophil emigration into the peritoneal cavity was assessed after 4 h. For all groups, n = 3–4 mice. *, P < 0.05; **, P < 0.01 (Student’s t test). (H) WT and Msn−/Y neutrophils were exposed to a 10-nM fMIFL gradient, and CI was calculated by the ratio of net migration in the correct direction to the total migration length. ***, P < 0.001 compared with WT (Student’s t test). Data are representative of (D) or are compiled from (A–C and E–H) three independent experiments (mean and SEM in A–C and E–H).
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fig1: Moesin regulates neutrophil microbial killing and inflammation. (A and B) WT and moesin knockout (Msn−/Y) mice were i.t. injected with P. aeruginosa (2 × 105). 8 h later, lungs were isolated, and the number (A) and percentage (B) of surviving colonies derived from lysates were determined. (C) WT and Msn−/Y neutrophils were incubated with opsonized P. aeruginosa for the indicated times, and surviving colonies were determined. (A–C) **, P < 0.01 compared with WT (Student’s t test). (D–F) Microvascular injury was induced in a classical LSR by consecutive injections of 80 µg LPS and then 0.2 µg TNF or PBS as controls. (D) Macroscopic appearance of dorsal skin in WT and Msn−/Y mice after LSR. Bar, 5 mm. (E) The degree of hemorrhage in the WT and Msn−/Y mice in D was estimated based on densitometry analysis of skin samples receiving either LPS or PBS injection. Results are shown as the ratio of the value with LPS versus the value with PBS. ***, P < 0.001 (Student’s t test). (F) Tissue MPO activities in skins were measured and normalized by tissue weight. Data are presented as V-Max value/g tissue. **, P < 0.01 (Student’s t test). (G) WT or Msn−/Y mice were i.p. injected with 10 nM fMLF, and neutrophil emigration into the peritoneal cavity was assessed after 4 h. For all groups, n = 3–4 mice. *, P < 0.05; **, P < 0.01 (Student’s t test). (H) WT and Msn−/Y neutrophils were exposed to a 10-nM fMIFL gradient, and CI was calculated by the ratio of net migration in the correct direction to the total migration length. ***, P < 0.001 compared with WT (Student’s t test). Data are representative of (D) or are compiled from (A–C and E–H) three independent experiments (mean and SEM in A–C and E–H).

Mentions: To address the role of moesin in neutrophil-mediated microbial killing and inflammation, we monitored the killing of bacteria in mouse lungs after inducing pneumonia using Pseudomonas aeruginosa strain 103 (PA103) through intratracheal (i.t.) injection. We observed an augmented load of P. aeruginosa in moesin knockout (Msn−/Y) lungs compared with WT lungs (Fig. 1 A; P < 0.01). Approximately 88% of the total 2 × 105 bacteria injected were killed in WT lungs, whereas only ∼26% were killed in Msn−/Y lungs (Fig. 1 B). To assess direct microbial killing by neutrophils, we isolated Msn−/Y neutrophils and performed bacterial killing in vitro. Compared with WT neutrophils, Msn−/Y neutrophils showed a significantly reduced microbial killing ability (Fig. 1 C; P < 0.01).


Moesin and myosin phosphatase confine neutrophil orientation in a chemotactic gradient.

Liu X, Yang T, Suzuki K, Tsukita S, Ishii M, Zhou S, Wang G, Cao L, Qian F, Taylor S, Oh MJ, Levitan I, Ye RD, Carnegie GK, Zhao Y, Malik AB, Xu J - J. Exp. Med. (2015)

Moesin regulates neutrophil microbial killing and inflammation. (A and B) WT and moesin knockout (Msn−/Y) mice were i.t. injected with P. aeruginosa (2 × 105). 8 h later, lungs were isolated, and the number (A) and percentage (B) of surviving colonies derived from lysates were determined. (C) WT and Msn−/Y neutrophils were incubated with opsonized P. aeruginosa for the indicated times, and surviving colonies were determined. (A–C) **, P < 0.01 compared with WT (Student’s t test). (D–F) Microvascular injury was induced in a classical LSR by consecutive injections of 80 µg LPS and then 0.2 µg TNF or PBS as controls. (D) Macroscopic appearance of dorsal skin in WT and Msn−/Y mice after LSR. Bar, 5 mm. (E) The degree of hemorrhage in the WT and Msn−/Y mice in D was estimated based on densitometry analysis of skin samples receiving either LPS or PBS injection. Results are shown as the ratio of the value with LPS versus the value with PBS. ***, P < 0.001 (Student’s t test). (F) Tissue MPO activities in skins were measured and normalized by tissue weight. Data are presented as V-Max value/g tissue. **, P < 0.01 (Student’s t test). (G) WT or Msn−/Y mice were i.p. injected with 10 nM fMLF, and neutrophil emigration into the peritoneal cavity was assessed after 4 h. For all groups, n = 3–4 mice. *, P < 0.05; **, P < 0.01 (Student’s t test). (H) WT and Msn−/Y neutrophils were exposed to a 10-nM fMIFL gradient, and CI was calculated by the ratio of net migration in the correct direction to the total migration length. ***, P < 0.001 compared with WT (Student’s t test). Data are representative of (D) or are compiled from (A–C and E–H) three independent experiments (mean and SEM in A–C and E–H).
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fig1: Moesin regulates neutrophil microbial killing and inflammation. (A and B) WT and moesin knockout (Msn−/Y) mice were i.t. injected with P. aeruginosa (2 × 105). 8 h later, lungs were isolated, and the number (A) and percentage (B) of surviving colonies derived from lysates were determined. (C) WT and Msn−/Y neutrophils were incubated with opsonized P. aeruginosa for the indicated times, and surviving colonies were determined. (A–C) **, P < 0.01 compared with WT (Student’s t test). (D–F) Microvascular injury was induced in a classical LSR by consecutive injections of 80 µg LPS and then 0.2 µg TNF or PBS as controls. (D) Macroscopic appearance of dorsal skin in WT and Msn−/Y mice after LSR. Bar, 5 mm. (E) The degree of hemorrhage in the WT and Msn−/Y mice in D was estimated based on densitometry analysis of skin samples receiving either LPS or PBS injection. Results are shown as the ratio of the value with LPS versus the value with PBS. ***, P < 0.001 (Student’s t test). (F) Tissue MPO activities in skins were measured and normalized by tissue weight. Data are presented as V-Max value/g tissue. **, P < 0.01 (Student’s t test). (G) WT or Msn−/Y mice were i.p. injected with 10 nM fMLF, and neutrophil emigration into the peritoneal cavity was assessed after 4 h. For all groups, n = 3–4 mice. *, P < 0.05; **, P < 0.01 (Student’s t test). (H) WT and Msn−/Y neutrophils were exposed to a 10-nM fMIFL gradient, and CI was calculated by the ratio of net migration in the correct direction to the total migration length. ***, P < 0.001 compared with WT (Student’s t test). Data are representative of (D) or are compiled from (A–C and E–H) three independent experiments (mean and SEM in A–C and E–H).
Mentions: To address the role of moesin in neutrophil-mediated microbial killing and inflammation, we monitored the killing of bacteria in mouse lungs after inducing pneumonia using Pseudomonas aeruginosa strain 103 (PA103) through intratracheal (i.t.) injection. We observed an augmented load of P. aeruginosa in moesin knockout (Msn−/Y) lungs compared with WT lungs (Fig. 1 A; P < 0.01). Approximately 88% of the total 2 × 105 bacteria injected were killed in WT lungs, whereas only ∼26% were killed in Msn−/Y lungs (Fig. 1 B). To assess direct microbial killing by neutrophils, we isolated Msn−/Y neutrophils and performed bacterial killing in vitro. Compared with WT neutrophils, Msn−/Y neutrophils showed a significantly reduced microbial killing ability (Fig. 1 C; P < 0.01).

Bottom Line: Neutrophils respond to invading bacteria by adopting a polarized morphology, migrating in the correct direction, and engulfing the bacteria.Attractant-induced activation of myosin phosphatase deactivated moesin at the prospective leading edge to break symmetry and establish polarity.Subsequent translocation of moesin to the trailing edge confined the formation of a prominent pseudopod directed toward pathogens and prevented secondary pseudopod formation in other directions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Department of Medicine, University of Illinois, Chicago, IL 60612.

Show MeSH
Related in: MedlinePlus