The RNA-binding protein tristetraprolin schedules apoptosis of pathogen-engaged neutrophils during bacterial infection
Florian Ebner, … , Michael Sixt, Pavel Kovarik
Florian Ebner, … , Michael Sixt, Pavel Kovarik
Published May 15, 2017
Citation Information: J Clin Invest. 2017;127(6):2051-2065. https://doi.org/10.1172/JCI80631.
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Research Article Immunology Infectious disease

The RNA-binding protein tristetraprolin schedules apoptosis of pathogen-engaged neutrophils during bacterial infection

Abstract

Protective responses against pathogens require a rapid mobilization of resting neutrophils and the timely removal of activated ones. Neutrophils are exceptionally short-lived leukocytes, yet it remains unclear whether the lifespan of pathogen-engaged neutrophils is regulated differently from that in the circulating steady-state pool. Here, we have found that under homeostatic conditions, the mRNA-destabilizing protein tristetraprolin (TTP) regulates apoptosis and the numbers of activated infiltrating murine neutrophils but not neutrophil cellularity. Activated TTP-deficient neutrophils exhibited decreased apoptosis and enhanced accumulation at the infection site. In the context of myeloid-specific deletion of Ttp, the potentiation of neutrophil deployment protected mice against lethal soft tissue infection with Streptococcus pyogenes and prevented bacterial dissemination. Neutrophil transcriptome analysis revealed that decreased apoptosis of TTP-deficient neutrophils was specifically associated with elevated expression of myeloid cell leukemia 1 (Mcl1) but not other antiapoptotic B cell leukemia/lymphoma 2 (Bcl2) family members. Higher Mcl1 expression resulted from stabilization of Mcl1 mRNA in the absence of TTP. The low apoptosis rate of infiltrating TTP-deficient neutrophils was comparable to that of transgenic Mcl1-overexpressing neutrophils. Our study demonstrates that posttranscriptional gene regulation by TTP schedules the termination of the antimicrobial engagement of neutrophils. The balancing role of TTP comes at the cost of an increased risk of bacterial infections.

Authors

Florian Ebner, Vitaly Sedlyarov, Saren Tasciyan, Masa Ivin, Franz Kratochvill, Nina Gratz, Lukas Kenner, Andreas Villunger, Michael Sixt, Pavel Kovarik

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Figure 5

TTP promotes neutrophil apoptosis in a cell-intrinsic manner.

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TTP promotes neutrophil apoptosis in a cell-intrinsic manner. (A) Apopto...
(A) Apoptosis was analyzed in peritoneal neutrophils elicited using 2 × 108 CFU HK S. pyogenes 16 hours prior to flushing of the peritoneal cavity. Representative flow plots show annexin V and live/dead staining of Ly6G+Ly6C+ neutrophils. The threshold for annexin V+ signal was based on fluorescence minus one (FMO) staining without annexin V. Numbers represent the percentage of cells in the respective quadrants. Dot plot for 3 pooled experiments (n = 13 TTPfl/fl; n = 17 TTPΔM) depicts the percentages of early apoptotic neutrophils (living annexin V+). (B) Neutrophils, elicited as in A, were analyzed for mitochondrial superoxide production (n = 9 TTPfl/fl; n = 8 TTPΔM), polarization of mitochondrial membrane (n = 7 TTPfl/fl; n = 8 TTPΔM), and active caspase-3 (n = 8 TTPfl/fl; n = 8 TTPΔM). (C) TTPfl/fl and TTPΔM BMDMs were incubated with pHrodo-stained nonapoptotic or apoptotic TTPfl/fl and TTPΔM neutrophils at a BMDM/neutrophil ratio of 1:2. CytD-pretreated BMDMs served as a control for adherent neutrophils. Bar graph shows the percentage of pHrodo+F4/80+ BMDMs (n = 3). Error bars indicate the mean ± SD. (D) Casein-elicited neutrophils from TTPfl/fl and TTPΔM mice were differently labeled, mixed at a 1:1 ratio, and injected i.p. into 4 recipient TTPfl/fl animals, in which peritonitis was induced 3 hours earlier using HK S. pyogenes. Representative flow plots 18 hours after transplantation and dot plots show retrieved transplanted neutrophils and the percentage of annexin V+ cells therein. (E) Neutrophil migration through a collagen matrix was followed by live imaging. Box plots show the average speed and displacement length of TTPfl/fl and TTPΔM neutrophils in untreated conditions or in a CXCL1 gradient. Error bars in A, B, and D represent the mean. Statistical analysis was determined by unpaired (A–D) or paired (E) Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001.