Neutrophil swarms require LTB4 and integrins at sites of cell death in vivo

Abstract

Neutrophil recruitment from blood to extravascular sites of sterile or infectious tissue damage is a hallmark of early innate immune responses, and the molecular events leading to cell exit from the bloodstream have been well defined1,2. Once outside the vessel, individual neutrophils often show extremely coordinated chemotaxis and cluster formation reminiscent of the swarming behaviour of insects3,4,5,6,7,8,9,10,11. The molecular players that direct this response at the single-cell and population levels within the complexity of an inflamed tissue are unknown. Using two-photon intravital microscopy in mouse models of sterile injury and infection, we show a critical role for intercellular signal relay among neutrophils mediated by the lipid leukotriene B4, which acutely amplifies local cell death signals to enhance the radius of highly directed interstitial neutrophil recruitment. Integrin receptors are dispensable for long-distance migration12, but have a previously unappreciated role in maintaining dense cellular clusters when congregating neutrophils rearrange the collagenous fibre network of the dermis to form a collagen-free zone at the wound centre. In this newly formed environment, integrins, in concert with neutrophil-derived leukotriene B4 and other chemoattractants, promote local neutrophil interaction while forming a tight wound seal. This wound seal has borders that cease to grow in kinetic concert with late recruitment of monocytes and macrophages at the edge of the displaced collagen fibres. Together, these data provide an initial molecular map of the factors that contribute to neutrophil swarming in the extravascular space of a damaged tissue. They reveal how local events are propagated over large-range distances, and how auto-signalling produces coordinated, self-organized neutrophil-swarming behaviour that isolates the wound or infectious site from surrounding viable tissue.

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Figure 1: Neutrophil extravascular swarming dynamics.
Figure 2: LTB4 promotes neutrophil recruitment from distant sites.
Figure 3: Integrin and GPCR signalling at the neutrophil cluster.
Figure 4: LTB4 requirement for swarming in infected lymph nodes.

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Acknowledgements

We thank L. Birnbaumer, R. Fässler, E. Tuomanen, P. M. Murphy, S. Akira, S. Monkley, D. Critchley, R. Wedlich-Söldner and M. Sixt for providing mice for this study, J.G. Egen and J. Tang for assistance with imaging, M. Parsek for providing P. aeruginosa–GFP, J.H. Kehrl, P.M. Murphy, R. Varma and members of the Germain laboratory for discussions. This work was supported by the Intramural Research Program of National Institute of Allergy and Infectious Diseases and National Cancer Institute, National Institutes of Health. T.L. was supported by a Human Frontiers Science Program Long-Term Fellowship. W.K. is presently a member of the Deutsche Forschungsgemeinschaft (DFG)-funded excellence cluster ImmunoSensation, Bonn, Germany.

Author information

Affiliations

Authors

Contributions

T.L. and R.N.G. designed the experiments, interpreted the data and wrote the paper. P.V.A., J.M.W. and C.A.P. contributed to data interpretation and experimental design, as well as the development of the final version of the paper. T.L. performed all skin-imaging experiments. W.K. performed intravital imaging of infected lymph nodes. B.R.A. developed the software for graphical display of imaging data. T.L., B.R.A. and P.V.A. conducted quantitative analysis of the data.

Corresponding authors

Correspondence to Tim Lämmermann or Ronald N. Germain.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-16, a Supplementary Table, Supplementary Notes 1-2 and additional references. (PDF 12491 kb)

41586_2013_BFnature12175_MOESM16_ESM.mov

Three hours after induction of a 15 s ear skin trauma, endogenous blood-circulating inflammatory cells had entered into the extravascular space of the ear dermis of a DsRed+/+Cx3cr1gfp/gfpTyrc-2J/c-2J mouse before laser-induced focal tissue damage was induced (center). This representative video shows the immediate response of fast-migrating neutrophils (small red cells) towards a focal tissue damage site, while monocytes (green) migrate with slower speeds and follow the developing neutrophil cluster with delay. Accumulating, small DsRed-positive cells were identified as Ly6G-positive neutrophils in Supplementary Fig. 3a differing from other static stromal elements (vessels, hair follicles, skin-resident cells) that are also pseudo-colored in red. Graphic analysis of this video is presented in Fig. 1b and Supplementary Fig. 3a. Similar neutrophil and monocyte kinetics were observed in DsRed+/+Cx3cr1gfp/+Tyrc-2J/c-2J mice. Two-photon intravital microscopy (x, y, z = 246µm, 246µm, 26µm; merge of z-stack), time-lapse over 2 h (10 frames/s). (MOV 10371 kb)

Differential kinetics of neutrophil and monocyte directed migration towards a focal tissue damage site

Three hours after induction of a 15 s ear skin trauma, endogenous blood-circulating inflammatory cells had entered into the extravascular space of the ear dermis of a DsRed+/+Cx3cr1gfp/gfpTyrc-2J/c-2J mouse before laser-induced focal tissue damage was induced (center). This representative video shows the immediate response of fast-migrating neutrophils (small red cells) towards a focal tissue damage site, while monocytes (green) migrate with slower speeds and follow the developing neutrophil cluster with delay. Accumulating, small DsRed-positive cells were identified as Ly6G-positive neutrophils in Supplementary Fig. 3a differing from other static stromal elements (vessels, hair follicles, skin-resident cells) that are also pseudo-colored in red. Graphic analysis of this video is presented in Fig. 1b and Supplementary Fig. 3a. Similar neutrophil and monocyte kinetics were observed in DsRed+/+Cx3cr1gfp/+Tyrc-2J/c-2J mice. Two-photon intravital microscopy (x, y, z = 246µm, 246µm, 26µm; merge of z-stack), time-lapse over 2 h (10 frames/s). (MOV 10371 kb)

41586_2013_BFnature12175_MOESM17_ESM.mov

Bone marrow-derived neutrophils from C57BL/6 mice were CMFDA-labeled and injected intradermally into the ventral ear skin of a Tyrc-2J/c-2J mouse 3 h before laser-induced focal tissue damage. This representative video shows the biphasic chemotactic response of neutrophils (pseudo-colored in green) sensing the focal tissue damage (autofluorescence, green) (left) within the fibrous collagenous connective tissue of the ear skin dermis (visualized by second harmonic generation, white) (right). Graphic analysis of this video is presented in Fig. 1c. Two-photon intravital microscopy (x, y, z = 322µm, 351µm, 16µm; merge of z-stack), time-lapse over 49 min 30 s (10 frames/s). (MOV 4553 kb)

Biphasic neutrophil swarming response to focal tissue damage

Bone marrow-derived neutrophils from C57BL/6 mice were CMFDA-labeled and injected intradermally into the ventral ear skin of a Tyrc-2J/c-2J mouse 3 h before laser-induced focal tissue damage. This representative video shows the biphasic chemotactic response of neutrophils (pseudo-colored in green) sensing the focal tissue damage (autofluorescence, green) (left) within the fibrous collagenous connective tissue of the ear skin dermis (visualized by second harmonic generation, white) (right). Graphic analysis of this video is presented in Fig. 1c. Two-photon intravital microscopy (x, y, z = 322µm, 351µm, 16µm; merge of z-stack), time-lapse over 49 min 30 s (10 frames/s). (MOV 4553 kb)

41586_2013_BFnature12175_MOESM18_ESM.mov

Neutrophils from C57BL/6 mice were CMFDA-labeled and injected together with propidium iodide (PI) intradermally into the ventral ear skin of a Tyrc-2J/c-2J mouse 2-4 h before laser-induced focal tissue damage. This video shows a single early-recruited neutrophil at the damage site that loses its intracellular dye (green) while its nucleus becomes PI-positive (red) as an indicator of cell death/lysis. Exactly at that time, neutrophils from distant sites increase in speed and directionality and migrate towards the dying neutrophil (left; with motion paths over the last 5 min as white dragon tails, right). At the beginning of the video, some skin-resident cells are PI-positive as a consequence of the intradermal injection and/or unspecific dye uptake. Graphic analysis of this video is presented in Fig. 2a and Supplementary Fig. 4. Two-photon intravital microscopy (x, y, z = 116µm, 155µm, 22µm; merge of z-stack), time-lapse over 21 min 30 s (10 frames/s). (MOV 1557 kb)

Cell death/lysis of few neutrophils leads to amplified recruitment of neutrophils from distant sites

Neutrophils from C57BL/6 mice were CMFDA-labeled and injected together with propidium iodide (PI) intradermally into the ventral ear skin of a Tyrc-2J/c-2J mouse 2-4 h before laser-induced focal tissue damage. This video shows a single early-recruited neutrophil at the damage site that loses its intracellular dye (green) while its nucleus becomes PI-positive (red) as an indicator of cell death/lysis. Exactly at that time, neutrophils from distant sites increase in speed and directionality and migrate towards the dying neutrophil (left; with motion paths over the last 5 min as white dragon tails, right). At the beginning of the video, some skin-resident cells are PI-positive as a consequence of the intradermal injection and/or unspecific dye uptake. Graphic analysis of this video is presented in Fig. 2a and Supplementary Fig. 4. Two-photon intravital microscopy (x, y, z = 116µm, 155µm, 22µm; merge of z-stack), time-lapse over 21 min 30 s (10 frames/s). (MOV 1557 kb)

41586_2013_BFnature12175_MOESM19_ESM.mov

Various GPCR-deficient (Cxcr2-/-, upper row; Fpr2-/-, middle row; Fpr1-/-, lower row) and control neutrophils were differentially dye-labeled and injected intradermally in a 1:1 ratio into the ventral ear skin of Tyrc-2J/c-2J mice 2-4 h before laser-induced focal tissue damage. This video shows representative experiments of GPCR-deficient (pseudo-colored in yellow) and control neutrophils (pseudo-colored in turquois) migrating side-by-side towards a damage site (left panels) with motion paths over the last 5 min as dragon tails in the corresponding pseudo-color (WT, middle; KO, right panels). Graphic analysis of several experiments is presented in Supplementary Fig. 8 and did not reveal any differences between knockout and control neutrophil dynamics. Two-photon intravital microscopy (x, y, z = 105µm, 147µm, 14-22µm; merge of z-stack), time-lapse over 29 min 30 s (10 frames/s). (MOV 2681 kb)

Cxcr2-/-, Fpr1-/-, and Fpr2-/- neutrophils show unaltered interstitial chemotaxis to sites of focal tissue damage

Various GPCR-deficient (Cxcr2-/-, upper row; Fpr2-/-, middle row; Fpr1-/-, lower row) and control neutrophils were differentially dye-labeled and injected intradermally in a 1:1 ratio into the ventral ear skin of Tyrc-2J/c-2J mice 2-4 h before laser-induced focal tissue damage. This video shows representative experiments of GPCR-deficient (pseudo-colored in yellow) and control neutrophils (pseudo-colored in turquois) migrating side-by-side towards a damage site (left panels) with motion paths over the last 5 min as dragon tails in the corresponding pseudo-color (WT, middle; KO, right panels). Graphic analysis of several experiments is presented in Supplementary Fig. 8 and did not reveal any differences between knockout and control neutrophil dynamics. Two-photon intravital microscopy (x, y, z = 105µm, 147µm, 14-22µm; merge of z-stack), time-lapse over 29 min 30 s (10 frames/s). (MOV 2681 kb)

41586_2013_BFnature12175_MOESM20_ESM.mov

Ltb4r1-/- and control neutrophils were differentially dye-labeled and injected intradermally in a 1:1 ratio into the ventral ear skin of a Tyrc-2J/c-2J mouse 3 h before laser-induced, 25 µm-sized focal tissue damage. This representative video shows Ltb4r1-/- (pseudo-colored in yellow) and control neutrophils (pseudo-colored in turquois) migrating side-by-side towards the damage site (left) with motion paths over the last 10 min as dragon tails in the corresponding pseudo-color (middle and right). Graphic analysis of several experiments is presented in Fig. 2b and Supplementary Fig. 9 and revealed impaired recruitment of Ltb4r1-/- neutrophils with both chemotactic index and velocity reduced at later time points. Two-photon intravital microscopy (x, y, z = 167µm, 209µm, 18µm; merge of z-stack), time-lapse over 30 min (10 frames/s). (MOV 2640 kb)

Leukotriene B4 optimizes neutrophil interstitial recruitment to sites of focal damage

Ltb4r1-/- and control neutrophils were differentially dye-labeled and injected intradermally in a 1:1 ratio into the ventral ear skin of a Tyrc-2J/c-2J mouse 3 h before laser-induced, 25 µm-sized focal tissue damage. This representative video shows Ltb4r1-/- (pseudo-colored in yellow) and control neutrophils (pseudo-colored in turquois) migrating side-by-side towards the damage site (left) with motion paths over the last 10 min as dragon tails in the corresponding pseudo-color (middle and right). Graphic analysis of several experiments is presented in Fig. 2b and Supplementary Fig. 9 and revealed impaired recruitment of Ltb4r1-/- neutrophils with both chemotactic index and velocity reduced at later time points. Two-photon intravital microscopy (x, y, z = 167µm, 209µm, 18µm; merge of z-stack), time-lapse over 30 min (10 frames/s). (MOV 2640 kb)

41586_2013_BFnature12175_MOESM21_ESM.mov

Ltb4r1-/- and control neutrophils were differentially dye-labeled and injected intradermally in a 1:1 ratio into the ventral ear skin of a Tyrc-2J/c-2J mouse 3 h before laser-induced, 10 µm-sized focal tissue damage. This representative video shows Ltb4r1-/- (pseudo-colored in yellow) and control neutrophils (pseudo-colored in turquois) migrating side-by-side towards the damage site (left) with motion paths over the last 10 min as dragon tails in the corresponding pseudo-color (middle and right). Graphic analysis of this experiment is presented in Fig. 2c and Supplementary Fig. 10 and revealed that Ltb4r1-/- neutrophils are poorly recruited from distant tissue sites. After the experiment, non-motile neutrophils responded with directed migration to a second laser damage (that was set closer to these cells) confirming viability and responsiveness of these cells (not shown). Two-photon intravital microscopy (x, y, z = 167µm, 302µm, 20µm; merge of z-stack), time-lapse over 30 min (10 frames/s). (MOV 2713 kb)

Leukotriene B4 optimizes neutrophil interstitial recruitment from distant tissue sites

Ltb4r1-/- and control neutrophils were differentially dye-labeled and injected intradermally in a 1:1 ratio into the ventral ear skin of a Tyrc-2J/c-2J mouse 3 h before laser-induced, 10 µm-sized focal tissue damage. This representative video shows Ltb4r1-/- (pseudo-colored in yellow) and control neutrophils (pseudo-colored in turquois) migrating side-by-side towards the damage site (left) with motion paths over the last 10 min as dragon tails in the corresponding pseudo-color (middle and right). Graphic analysis of this experiment is presented in Fig. 2c and Supplementary Fig. 10 and revealed that Ltb4r1-/- neutrophils are poorly recruited from distant tissue sites. After the experiment, non-motile neutrophils responded with directed migration to a second laser damage (that was set closer to these cells) confirming viability and responsiveness of these cells (not shown). Two-photon intravital microscopy (x, y, z = 167µm, 302µm, 20µm; merge of z-stack), time-lapse over 30 min (10 frames/s). (MOV 2713 kb)

41586_2013_BFnature12175_MOESM22_ESM.mov

Ltb4r1-/- and control neutrophils were differentially dye-labeled and injected intradermally in a 1:1 ratio into the ventral ear skin of a leukotriene-deficient Alox5-/-Tyrc-2J/c-2J mouse 3 h before laser-induced focal tissue damage. This representative video shows Ltb4r1-/- (pseudo-colored in yellow) and control neutrophils (pseudo-colored in turquois) migrating side-by-side towards the damage site (left) with motion paths over the last 10 min as dragon tails in the corresponding pseudo-color (middle and right). Graphic analysis of several experiments is presented in Fig. 2d and Supplementary Fig. 11 and revealed that neutrophil-derived LTB4 improves the interstitial recruitment response of the neutrophil population. Two-photon intravital microscopy (x, y, z = 171µm, 165µm, 16µm; merge of z-stack), time-lapse over 34 min 30 s (10 frames/s). (MOV 3084 kb)

Neutrophil-derived LTB4 optimizes neutrophil interstitial recruitment to sites of focal damage

Ltb4r1-/- and control neutrophils were differentially dye-labeled and injected intradermally in a 1:1 ratio into the ventral ear skin of a leukotriene-deficient Alox5-/-Tyrc-2J/c-2J mouse 3 h before laser-induced focal tissue damage. This representative video shows Ltb4r1-/- (pseudo-colored in yellow) and control neutrophils (pseudo-colored in turquois) migrating side-by-side towards the damage site (left) with motion paths over the last 10 min as dragon tails in the corresponding pseudo-color (middle and right). Graphic analysis of several experiments is presented in Fig. 2d and Supplementary Fig. 11 and revealed that neutrophil-derived LTB4 improves the interstitial recruitment response of the neutrophil population. Two-photon intravital microscopy (x, y, z = 171µm, 165µm, 16µm; merge of z-stack), time-lapse over 34 min 30 s (10 frames/s). (MOV 3084 kb)

41586_2013_BFnature12175_MOESM23_ESM.mov

Talin-deficient (Tln1-/-) and control neutrophils were differentially dye labeled and injected intradermally in a 1:1 ratio into the ventral ear skin of Tyrc-2J/c-2J mice 2-3 h before laser-induced focal tissue damage was performed. This video shows two representative experiments of Tln1-/- and control neutrophils migrating side-by-side towards a damage site. In upper panels, Tln1-/- neutrophils were labeled with CMTPX (pseudo-colored in red) and control neutrophils with CMFDA (pseudo-colored in green). In lower panels, cell dyes were switched. Cell migration in relation to the fibrous collagenous connective tissue of the ear skin dermis was visualized by second harmonic generation (white; middle column) and motion paths over the last 10 min are presented as yellow dragon tails. Graphic analysis of several experiments is presented in Supplementary Fig. 13 and shows that active integrins are dispensable for interstitial neutrophil recruitment. Two-photon intravital microscopy (x, y, z = 149µm, 159µm, 16-20µm; merge of z-stack), time-lapse over 26 min 30 s (10 frames/s). (MOV 2428 kb)

Interstitial neutrophil recruitment does not require high-affinity integrin-mediated adhesion

Talin-deficient (Tln1-/-) and control neutrophils were differentially dye labeled and injected intradermally in a 1:1 ratio into the ventral ear skin of Tyrc-2J/c-2J mice 2-3 h before laser-induced focal tissue damage was performed. This video shows two representative experiments of Tln1-/- and control neutrophils migrating side-by-side towards a damage site. In upper panels, Tln1-/- neutrophils were labeled with CMTPX (pseudo-colored in red) and control neutrophils with CMFDA (pseudo-colored in green). In lower panels, cell dyes were switched. Cell migration in relation to the fibrous collagenous connective tissue of the ear skin dermis was visualized by second harmonic generation (white; middle column) and motion paths over the last 10 min are presented as yellow dragon tails. Graphic analysis of several experiments is presented in Supplementary Fig. 13 and shows that active integrins are dispensable for interstitial neutrophil recruitment. Two-photon intravital microscopy (x, y, z = 149µm, 159µm, 16-20µm; merge of z-stack), time-lapse over 26 min 30 s (10 frames/s). (MOV 2428 kb)

41586_2013_BFnature12175_MOESM24_ESM.mov

This video shows the collagenous fiber network (visualized by second harmonic generation) as neutrophils accumulate at the focal damage site in the dermis of a LysMgfp/+Tyrc-2J/c-2J mouse (Fig. 3a). Fiber bundles are displaced over time in the x-y-axis. Some disappearing fibers are also displaced in the z-axis out of the imaging volume (not shown). Two-photon intravital microscopy (x, y, z = 122µm, 49µm, 30µm; merge of z-stack), time-lapse over 32 min 30 s (10 frames/s). (MOV 1498 kb)

Congregating neutrophils displace fibrous collagen bundles from the wound center

This video shows the collagenous fiber network (visualized by second harmonic generation) as neutrophils accumulate at the focal damage site in the dermis of a LysMgfp/+Tyrc-2J/c-2J mouse (Fig. 3a). Fiber bundles are displaced over time in the x-y-axis. Some disappearing fibers are also displaced in the z-axis out of the imaging volume (not shown). Two-photon intravital microscopy (x, y, z = 122µm, 49µm, 30µm; merge of z-stack), time-lapse over 32 min 30 s (10 frames/s). (MOV 1498 kb)

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15 s ear skin trauma was induced in a DsRed+/+Tyrc-2J/c-2J mouse (for endogenous blood-circulating neutrophils to enter the extravascular space), 1 h later bone marrow-derived neutrophils from a Lifeact-GFP transgenic mouse were injected intradermally into the ventral ear skin followed 2 h later by laser-induced focal tissue damage. This representative video shows a zoom into the collagen-free wound center where both injected Lifeact-GFP neutrophils (pseudo-colored in green) and endogenous DsRed-positive neutrophils (pseudo-colored in red) migrate in close contact with each other. Lifeact-GFP outlines the actin cortex of neutrophils to demarcate the cell borders of injected individual cells. Two-photon intravital microscopy (x, y, z = 78µm, 78µm, 3µm; merge of z-stack), time-lapse over 34 min 28 s (10 frames/s). (MOV 12302 kb)

Neutrophils migrate in the collagen-free center in close contact with each other

15 s ear skin trauma was induced in a DsRed+/+Tyrc-2J/c-2J mouse (for endogenous blood-circulating neutrophils to enter the extravascular space), 1 h later bone marrow-derived neutrophils from a Lifeact-GFP transgenic mouse were injected intradermally into the ventral ear skin followed 2 h later by laser-induced focal tissue damage. This representative video shows a zoom into the collagen-free wound center where both injected Lifeact-GFP neutrophils (pseudo-colored in green) and endogenous DsRed-positive neutrophils (pseudo-colored in red) migrate in close contact with each other. Lifeact-GFP outlines the actin cortex of neutrophils to demarcate the cell borders of injected individual cells. Two-photon intravital microscopy (x, y, z = 78µm, 78µm, 3µm; merge of z-stack), time-lapse over 34 min 28 s (10 frames/s). (MOV 12302 kb)

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Talin-deficient (Tln1-/-) and control neutrophils were differentially dye-labeled and injected intradermally in a 1:1 ratio into the ventral ear skin of a Tyrc-2J/c-2J mouse 3 h before laser-induced focal tissue damage. This representative video shows Tln1-/- (pseudo-colored in green) and control neutrophils (pseudo-colored in red) accumulating at the damage site (left). Neutrophil movement in relation to collagen displacement is visualized by second harmonic generation (pseudo-colored in white) (middle, right). Lower panels show zoom-in on the margin of collagen displacement. The analysis of the clustering response is presented in Fig. 3b,c. Two-photon intravital microscopy (x, y, z = 368µm, 368µm, 10µm; merge of z-stack), time-lapse over 29 min 30 s (10 frames/s). (MOV 2618 kb)

Active integrins are essential for sustained access to the neutrophil cluster in the collagen-free wound center

Talin-deficient (Tln1-/-) and control neutrophils were differentially dye-labeled and injected intradermally in a 1:1 ratio into the ventral ear skin of a Tyrc-2J/c-2J mouse 3 h before laser-induced focal tissue damage. This representative video shows Tln1-/- (pseudo-colored in green) and control neutrophils (pseudo-colored in red) accumulating at the damage site (left). Neutrophil movement in relation to collagen displacement is visualized by second harmonic generation (pseudo-colored in white) (middle, right). Lower panels show zoom-in on the margin of collagen displacement. The analysis of the clustering response is presented in Fig. 3b,c. Two-photon intravital microscopy (x, y, z = 368µm, 368µm, 10µm; merge of z-stack), time-lapse over 29 min 30 s (10 frames/s). (MOV 2618 kb)

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Itgb2-/- and control neutrophils were differentially dye-labeled and injected intradermally in a 1:1 ratio into the ventral ear skin of a Tyrc-2J/c-2J mouse 3 h before laser-induced focal tissue damage. This representative video shows Itgb2-/- (pseudo-colored in green) and control neutrophils (pseudo-colored in red) accumulating at the damage site (left). Neutrophil movement in relation to collagen displacement is visualized by second harmonic generation (pseudo-colored in white) (middle, right). The analysis of the clustering response is presented in Fig. 3c and Supplementary Fig. 15a. Two-photon intravital microscopy (x, y, z = 368µm, 368µm, 12µm; merge of z-stack), time-lapse over 29 min 30 s (10 frames/s). (MOV 2646 kb)

β2 integrins are essential for access of the neutrophil cluster in the collagen-free wound center

Itgb2-/- and control neutrophils were differentially dye-labeled and injected intradermally in a 1:1 ratio into the ventral ear skin of a Tyrc-2J/c-2J mouse 3 h before laser-induced focal tissue damage. This representative video shows Itgb2-/- (pseudo-colored in green) and control neutrophils (pseudo-colored in red) accumulating at the damage site (left). Neutrophil movement in relation to collagen displacement is visualized by second harmonic generation (pseudo-colored in white) (middle, right). The analysis of the clustering response is presented in Fig. 3c and Supplementary Fig. 15a. Two-photon intravital microscopy (x, y, z = 368µm, 368µm, 12µm; merge of z-stack), time-lapse over 29 min 30 s (10 frames/s). (MOV 2646 kb)

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Gnai isoform-deficient and control neutrophils were differentially dye-labeled and injected intradermally in a 1:1 ratio into the ventral ear skin of a Tyrc-2J/c-2J mouse 3 h before laser-induced focal tissue damage. This video shows representative experiments of Gnai2-/- (upper panels) or Gnai3-/- (lower panels) (both pseudo-colored in green) and control neutrophils (pseudo-colored in red) accumulating at the damage site. Neutrophil movement in relation to collagen displacement is visualized by second harmonic generation (pseudo-colored in white) (middle, right). The analysis of the clustering response is presented in Fig. 3f and Supplementary Fig. 15b. Two-photon intravital microscopy (x, y, z = 185µm, 185µm, 14µm; merge of z-stack), time-lapse over 56 min 30 s (10 frames/s). (MOV 4956 kb)

Gαi2-, not Gαi3-dependent signals control neutrophil clustering

Gnai isoform-deficient and control neutrophils were differentially dye-labeled and injected intradermally in a 1:1 ratio into the ventral ear skin of a Tyrc-2J/c-2J mouse 3 h before laser-induced focal tissue damage. This video shows representative experiments of Gnai2-/- (upper panels) or Gnai3-/- (lower panels) (both pseudo-colored in green) and control neutrophils (pseudo-colored in red) accumulating at the damage site. Neutrophil movement in relation to collagen displacement is visualized by second harmonic generation (pseudo-colored in white) (middle, right). The analysis of the clustering response is presented in Fig. 3f and Supplementary Fig. 15b. Two-photon intravital microscopy (x, y, z = 185µm, 185µm, 14µm; merge of z-stack), time-lapse over 56 min 30 s (10 frames/s). (MOV 4956 kb)

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Ltb4r1-/- and control neutrophils were differentially dye-labeled and injected intradermally in a 1:1 ratio into the ventral ear skin of a Tyrc-2J/c-2J mouse (upper panels) or leukotriene-deficient Alox5-/-Tyrc-2J/c-2J mouse (lower panels) 3 h before laser-induced focal tissue damage. This video shows representative experiments of Ltb4r1-/- (pseudo-colored in green) and control neutrophils (pseudo-colored in red) accumulating at the damage site of the respective host. Neutrophil movement in relation to collagen displacement is visualized by second harmonic generation (pseudo-colored in white) (middle and right). The analysis of the clustering response is presented in Fig. 3d-f and Supplementary Fig. 15c and 16a. Two-photon intravital microscopy (x, y, z = 185µm, 185µm, 14µm; merge of z-stack), time-lapse over 30 min (10 frames/s). (MOV 2657 kb)

Neutrophil-derived LTB4 controls neutrophil clustering

Ltb4r1-/- and control neutrophils were differentially dye-labeled and injected intradermally in a 1:1 ratio into the ventral ear skin of a Tyrc-2J/c-2J mouse (upper panels) or leukotriene-deficient Alox5-/-Tyrc-2J/c-2J mouse (lower panels) 3 h before laser-induced focal tissue damage. This video shows representative experiments of Ltb4r1-/- (pseudo-colored in green) and control neutrophils (pseudo-colored in red) accumulating at the damage site of the respective host. Neutrophil movement in relation to collagen displacement is visualized by second harmonic generation (pseudo-colored in white) (middle and right). The analysis of the clustering response is presented in Fig. 3d-f and Supplementary Fig. 15c and 16a. Two-photon intravital microscopy (x, y, z = 185µm, 185µm, 14µm; merge of z-stack), time-lapse over 30 min (10 frames/s). (MOV 2657 kb)

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Mice were infected with P. aeruginosa-GFP in the footpad before 2P-IVM was performed on the draining popliteal lymph nodes when comparable neutrophil numbers were present in the subcapsular sinus at indicated times after infection (WT: 3 h, Ltb4r1-/-: 4.5 h). Neutrophil-GFP signal is pseudo-colored (heat map) to indicate neutrophil clusters (white) in WT-LysMgfp/+ mice (left) and Ltb4r1-/-LysMgfp/+ mice (right). The analysis of the clustering response is presented in Fig. 4a-c. Time-lapse over 39 min 20 s (10 frames/s). (MOV 2568 kb)

LTB4 requirement for swarming in infected lymph nodes

Mice were infected with P. aeruginosa-GFP in the footpad before 2P-IVM was performed on the draining popliteal lymph nodes when comparable neutrophil numbers were present in the subcapsular sinus at indicated times after infection (WT: 3 h, Ltb4r1-/-: 4.5 h). Neutrophil-GFP signal is pseudo-colored (heat map) to indicate neutrophil clusters (white) in WT-LysMgfp/+ mice (left) and Ltb4r1-/-LysMgfp/+ mice (right). The analysis of the clustering response is presented in Fig. 4a-c. Time-lapse over 39 min 20 s (10 frames/s). (MOV 2568 kb)

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Lämmermann, T., Afonso, P., Angermann, B. et al. Neutrophil swarms require LTB4 and integrins at sites of cell death in vivo . Nature 498, 371–375 (2013). https://doi.org/10.1038/nature12175

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