. 2017 Apr 1;198(7):2865-2875.
Epub 2017 Feb 20.
Local TNFR1 Signaling Licenses Murine Neutrophils for Increased TLR-Dependent Cytokine and Eicosanoid Production
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Local TNFR1 Signaling Licenses Murine Neutrophils for Increased TLR-Dependent Cytokine and Eicosanoid Production
Free PMC article
Neutrophils are generally the first immune cells recruited during the development of sterile or microbial inflammation. As these cells express many innate immune receptors with the potential to directly recognize microbial or endogenous signals, we set out to assess whether their functions are locally influenced by the signals present at the onset of inflammation. Using a mouse model of peritonitis, we demonstrate that neutrophils elicited in the presence of C-type lectin receptor ligands have an increased ability to produce cytokines, chemokines, and lipid mediators in response to subsequent TLR stimulation. Importantly, we found that licensing of cytokine production was mediated by paracrine TNF-α-TNFR1 signaling rather than direct ligand sensing, suggesting a form of quorum sensing among neutrophils. Mechanistically, licensing was largely imparted by changes in the posttranscriptional regulation of inflammatory cytokines, whereas production of IL-10 was regulated at the transcriptional level. Altogether, our data suggest that neutrophils rapidly adapt their functions to the local inflammatory milieu. These phenotypic changes may promote rapid neutrophil recruitment in the presence of pathogens but limit inflammation in their absence.
Copyright © 2017 by The American Association of Immunologists, Inc.
Conflict of interest statement
The authors have no conflicting financial interests.
Figure 1. Neutrophil cytokine production is licensed locally by microbial ligands
A. Representative luminescence after PMA stimulation in the presence of luminol (upper panel) of neutrophils purified from the peritoneal cavity of mice injected with uric acid (blue) or zymosan (red), or as a control, from the bone marrow of uninjected mice (black). Average bacterial killing by neutrophils purified as above, or in the absence of any neutrophils (lower panel). B. Representative flow cytometry plots gated on neutrophils in the peritoneal exudate of mice injected with uric acid or zymosan and left unstimulated (grey) or restimulated with Pam3CSK4 (red). Percentage of TNF-positive neutrophils are indicated in the lower left panel. Geometric mean fluorescence intensities (gMFI) for TNFα of neutrophils or inflammatory monocytes in the peritoneal exudate of mice injected with uric acid or zymosan, after Pam3CSK4 restimulation, are indicated in the lower right panel. C. Representative flow cytometry plot of a mixed culture of cells harvested from mice injected with uric acid (blue) and zymosan (red) after Pam3CSK4 restimulation. D–F. Representative flow cytometry plots (D) and percentage of TNF-positive neutrophils or gMFI for TNFα (E, F) from neutrophils gated out of total bone marrow cells (D–E) or Percoll-purified neutrophils (F) after stimulation with zymosan or not (1
st stim, + and − respectively), followed by restimulation with Pam3CSK4 or not (2 nd stim, + and − respectively). G. Supernatants from purified neutrophils (as in F) stimulated with media alone, Curdlan or TDB and restimulated with or without Pam3CSK4 were analyzed by Luminex for the indicated cytokines. Data were representative of 2 (A, C, G), 6 (B) or 10 (D) experiments. Dots plots (B, E, F) are pooled from 6–10 mice from 6 experiments. *, p<0.05;**, p<0.01;***, p<0.001
Figure 2. Licensing of highly purified neutrophils
Representative flow cytometry plots of total bone marrow (input) or FACS-purified neutrophils showing purity (top) and intracellular TNFα levels (bottom) after Pam3CSK4 restimulation (red) or no stimulation (grey). Numbers indicate the percentage of neutrophils (top) or percentage of TNFα-positive cells (bottom). Data are representative of 2 independent experiments. Neutrophils were sorted on a MoFlo (Beckman Coulter) either on the basic of FSC-SSC in the absence of antibodies (columns 1 and 2) or on the basis of Ly6G expression (column 3).
Figure 3. Neutrophil licensing is mediated by a paracrine signal
A. Representative flow cytometry plots gated on neutrophils from the indicated mice after in vitro stimulation of bone marrow cells with TDB followed by Pam3CSK4. B–C. Representative flow cytometry plots gated on neutrophils harvested from chimeras generated by transfer of CD45.1 WT cells (red), CD45.2
Fcer1g cells (FcRγ-deficient, blue) or a 1:1 mix of those cells on WT or −/− Fcer1g hosts. Mice were injected with TDB, and peritoneal exudates were restimulated with Pam3CSK4. C. Quantification of the gMFI in the samples described above D. Congenically marked cells from the indicated mice were mixed at a 1:1 or 1:9 ratio before restimulation as above. Representative overlays of CD45.1 WT cells (red) and CD45.2 −/− Fcer1g neutrophils (blue) are shown. Data presented are representative of 3 independent experiments with 3 mice per group. −/−
Figure 4. Identification of TNFα as the licensing factor
A–B. Representative flow cytometry plots of bone marrow neutrophils incubated with curdlan, 0.22um-filtered curdlan (A, top) or 0.22um-filtered supernatants of bone marrow cells stimulated with curdlan or not (A, bottom) or 0.22um-filtered supernatants of RAW 264.7 cells stimulated with curdlan or not (B, top). Supernatants of RAW 264.7 cells stimulated with curdlan were also treated with proteinase K and incubated for 5 min at 95°C before incubation with neutrophils (B, bottom). Data are representative of three independent experiments. C. 2D gel electrophoresis of total supernatants from RAW 264.7 cells (green) or RAW 264.7 cells stimulated with curdlan (red). D. Supernatants from RAW 264.7 cells stimulated with curdlan were fractionated by anion exchange chromatography, and naïve neutrophils were incubated with fractions, indicated by the NaCl at which the fraction eluted. Total protein in the fraction (in A
260.mL, in blue) and gMFI for TNFα in the neutrophils incubated with that fraction and restimulated with Pam (left panel). Representative plots of neutrophils incubated with a 1:10 dilution of the flow-through (=0mM NaCl) or the indicated fractions and restimulated with Pam3CSK4 (right panel). E. 2D gel electrophoresis of proteins present in the 250mM NaCl fraction obtained as in D.F. (Top) Representative flow cytometry plots of neutrophils gated from bone marrow cells left untreated or incubated with 50ng/mL recombinant TNFα, then restimulated with Pam3CSK4 (red) or not (solid grey). (Bottom) Percentage of TNFα+ neutrophils and gMFI for TNFα in neutrophils pretreated with the indicated concentrations of TNFα, then restimulated with Pam3CSK4. Dotted lines represent these values for neutrophils in the absence of recombinant TNFα. G. Representative flow cytometry plots gated on neutrophils from the indicated mice after in vitro stimulation of bone marrow cells with zymosan or 50ng/mL recombinant TNFα or in the absence of pre-stimulation (ø), followed by restimulation with Pam3CSK4. Data are representative of 3 independent experiments.
Figure 5. Consequences and kinetics of TNFα-mediated licensing
A. Top. Representative flow cytometry plots gated on neutrophils from a mixed culture of congenically marked WT (red) and TNFR1-KO (blue) bone marrow cells. Cells were stimulated with the indicated ligands, followed by restimulation with Pam3CSK4. Bottom. Geometric mean fluorescence intensity for TNFα in a mixed culture (empty columns) or individual cultures (full boxes) of WT (red) and TNFR1-KO (blue) neutrophils treated as above. B. Supernatants from purified neutrophils, stimulated with 10 ng/mL recombinant TNFα or not and restimulated with Pam3CSK4 or not, were analyzed by Luminex for the indicated cytokines. Data for A, B are representative of 4 independent experiments. C. Purified neutrophils, stimulated with 10 ng/mL recombinant TNFα or not and restimulated Pam3CSK4, fMLP or not, were lysed in methanol for analysis of lipid mediator production by LC-MS. Data are representative of two independent experiments performed in duplicate. D. Left. Geometric MFI for TNFα in neutrophils gated from bone marrow cells and treated with the indicated ligands (recombinant TNFα : black, zymosan : red, TDB : blue) for 10min to 16h. All cells were then restimulated with Pam3CSK4 for 6 hours. Right. Representative flow cytometry plots of neutrophils treated with the indicated ligands (recombinant TNFα : left, zymosan : center, TDB : right) or left untreated (solid grey). Colors indicate the duration of treatment before Pam3CSK4 restimulation. E. Geometric TNFα MFI and representative plots in neutrophils gated from bone marrow cells treated with recombinant TNFα for 12h, then washed and rested for 10 min to 4h, before restimulation with Pam3CSK4. 0 min (=no rest) positive controls were kept in the presence of rTNFα during restimulation. Data for D, E are representative of two independent experiments with duplicates at each timepoint.
Figure 6. TNFR1-mediated licensing prolongs zymosan-induced inflammation
A.B. Absolute counts (A) and TNFα gMFI (B) for neutrophils present in the peritoneal exudates of WT or TNFR1-deficient mice injected with zymosan for 16 or 42h. Data were obtained in two independent experiments with 3 mice/group. *, P<0.05 C. Relative survival of bone-marrow neutrophils cultured for 24h in the presence of the indicated concentrations of recombinant TNFα. Survival is calculated as a percentage of the number of live neutrophils harvested in media alone.
Figure 7. Mechanistic changes induced by neutrophil licensing
A. Representative flow cytometry plots of neutrophils treated with recombinant TNFα (red) of left untreated (solid grey) for 12 hours before restimulation with the indicated ligands for TLR receptors. Numbers display the percentage of TNFα positive neutrophils. B. Representative flow cytometry plots of neutrophils (or for the rightmost plot, inflammatory monocytes) gated from bone marrow cells left untreated (black, green) or incubated with 10ng/mL recombinant TNFα (blue, red), then restimulated Pam3CSK4 (green, red) or not (black, blue), depicting levels of IkBα, phospho-Erk and phospho-p38. C. qPCR analysis of purified neutrophils left untreated or incubated with 10 ng/mL recombinant TNFα, then restimulated with Pam3CSK4 or not, for the indicated genes. Values plotted represent fold change over untreated neutrophils (no TNF, no Pam3CSK4). Numbers represent the fold change between unlicensed and licensed neutrophils after Pam3CSK4 restimulation. D. Left. Bone marrow cells were cultured in the presence of OP-puromycin with or without cyclohexamide (CHX). Cells were then stained to label the OP group with Alexa 488 and gated on monocytes (grey, blue) or neutrophils (black, red). Right. Neutrophils were treated as in left after treatment with TNFα (grey, green) or in the absence of TNFα (black, red). Data are representative of 3 (A, B, D) or 4 (C) independent experiments.
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Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural
Cytokines / biosynthesis*
Eicosanoids / biosynthesis*
Neutrophil Infiltration / immunology
Neutrophils / immunology*
Polymerase Chain Reaction
Receptors, Tumor Necrosis Factor, Type I / immunology*
Signal Transduction / immunology*
Toll-Like Receptors / immunology
Receptors, Tumor Necrosis Factor, Type I