Factor XII and uPAR upregulate neutrophil functions to influence wound healing
Evi X. Stavrou, Chao Fang, Kara L. Bane, Andy T. Long, Clément Naudin, Erdem Kucukal, Agharnan Gandhi, Adina Brett-Morris, Michele M. Mumaw, Sudeh Izadmehr, Alona Merkulova, Cindy C. Reynolds, Omar Alhalabi, Lalitha Nayak, Wen-Mei Yu, Cheng-Kui Qu, Howard J. Meyerson, George R. Dubyak, Umut A. Gurkan, Marvin T. Nieman, Anirban Sen Gupta, Thomas Renné, Alvin H. Schmaier
Evi X. Stavrou, Chao Fang, Kara L. Bane, Andy T. Long, Clément Naudin, Erdem Kucukal, Agharnan Gandhi, Adina Brett-Morris, Michele M. Mumaw, Sudeh Izadmehr, Alona Merkulova, Cindy C. Reynolds, Omar Alhalabi, Lalitha Nayak, Wen-Mei Yu, Cheng-Kui Qu, Howard J. Meyerson, George R. Dubyak, Umut A. Gurkan, Marvin T. Nieman, Anirban Sen Gupta, Thomas Renné, Alvin H. Schmaier
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Research Article Cell biology Inflammation

Factor XII and uPAR upregulate neutrophil functions to influence wound healing

Abstract

Coagulation factor XII (FXII) deficiency is associated with decreased neutrophil migration, but the mechanisms remain uncharacterized. Here, we examine how FXII contributes to the inflammatory response. In 2 models of sterile inflammation, FXII-deficient mice (F12–/–) had fewer neutrophils recruited than WT mice. We discovered that neutrophils produced a pool of FXII that is functionally distinct from hepatic-derived FXII and contributes to neutrophil trafficking at sites of inflammation. FXII signals in neutrophils through urokinase plasminogen activator receptor–mediated (uPAR-mediated) Akt2 phosphorylation at S474 (pAktS474). Downstream of pAkt2S474, FXII stimulation of neutrophils upregulated surface expression of αMβ2 integrin, increased intracellular calcium, and promoted extracellular DNA release. The sum of these activities contributed to neutrophil cell adhesion, migration, and release of neutrophil extracellular traps in a process called NETosis. Decreased neutrophil signaling in F12–/– mice resulted in less inflammation and faster wound healing. Targeting hepatic F12 with siRNA did not affect neutrophil migration, whereas WT BM transplanted into F12–/– hosts was sufficient to correct the neutrophil migration defect in F12–/– mice and restore wound inflammation. Importantly, these activities were a zymogen FXII function and independent of FXIIa and contact activation, highlighting that FXII has a sophisticated role in vivo that has not been previously appreciated.

Authors

Evi X. Stavrou, Chao Fang, Kara L. Bane, Andy T. Long, Clément Naudin, Erdem Kucukal, Agharnan Gandhi, Adina Brett-Morris, Michele M. Mumaw, Sudeh Izadmehr, Alona Merkulova, Cindy C. Reynolds, Omar Alhalabi, Lalitha Nayak, Wen-Mei Yu, Cheng-Kui Qu, Howard J. Meyerson, George R. Dubyak, Umut A. Gurkan, Marvin T. Nieman, Anirban Sen Gupta, Thomas Renné, Alvin H. Schmaier

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Figure 7

FXII signaling in neutrophils is a zymogen function.

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FXII signaling in neutrophils is a zymogen function. (A) WT neutrophils ...
(A) WT neutrophils were incubated in the absence (UT) or presence of 62 nM FXII and 10 μM Zn2+ for 1, 2, and 5 minutes. Western blot analysis for FXII was performed under reduced conditions using polyclonal anti-FXII antibody. Representative blot of n = 3. (B) Chromogenic assay of FXIIa activity. Neutrophils were allowed to adhere on a gelatin-coated 96-well plate for 10 minutes and subsequently incubated with fMLP, 62 nM FXII/10 μM Zn2+, or rising concentrations of FXIIa (0.62 nM, 6.2 nM, 62 nM)/10 μM Zn2+. S-2302 (200 μM) was added in each well, and changes in OD405 were continuously monitored on a microplate reader. Graph represents mean OD of a single experiment run in triplicate. (C) Left panel: cleavage of S-2302 is presented as change in mean OD (ΔOD) over the first 5 minutes of neutrophil incubation with media, fMLP, FXII/Zn2+, or rising concentrations of FXIIa/Zn2+. Note that 5 minutes is the time point used in pAkt2S474 signaling studies. Right panel: cleavage of S-2302 over 180 minutes. Graphs are presented in box-and-whiskers diagrams where minimum/maximum distribution and outliers are shown. Mean ΔOD, n = 4 individual experiments run in triplicate. (D) FXII immunoblotting of supernatant from HEK293 cells expressing WT FXII, FXII Locarno (FXII-R353P), and FXII double-mutant (FXII-D) (combined R353P and S544 substitutions). Mock: supernatant from nontransfected cells; Purified FXII: plasma-derived mouse FXII. (E) Neutrophils were treated with WT FXII/ Zn2+ or FXII-D variant and Zn2+. Lanes labeled Akti were pretreated with Akti XII (5 μM) for 30 minutes, followed by FXII/Zn2+ or FXII-D/Zn2+. Lysates were immunoblotted with antibodies against pAkt2S474. Media: conditioned media from transfected cells. (F) Percentage of pAkt2S474 (% RDU) in neutrophils. Mean ± SEM, n = 4 experiments. *P < 0.0001, 1-way ANOVA.