1. Volume 19, Issue 8, Pages 1572-1585 (May 2017)
Chemokine Signaling and the Regulation of Bidirectional Leukocyte Migration in Interstitial Tissues 
Davalyn Powell, Sebastien Tauzin, Laurel E. Hind, Qing Deng, David J. Beebe, Anna Huttenlocher 
Cell Reports 
Volume 19, Issue 8, Pages (May 2017)
DOI: /j.celrep
Copyright © 2017 The Author(s) Terms and Conditions

2. Cell Reports 2017 19, 1572-1585DOI: (10.1016/j.celrep.2017.04.078)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1
Cxcr1 Is Required for Neutrophil Recruitment to Tissue Wounds, whereas Cxcr2 Is Required for Neutrophil Resolution
(A) Sudan black staining of 3 days post-fertilization (dpf) larvae at 1, 3, and 6 hr post-wound (hpw) reveals Cxcr1−/− larvae recruit less neutrophils to tail-transection compared to Cxcr1+/− control.
(B) Quantification of neutrophils caudal to the notochord (n = 16 heterozygote [het] and 19 mutant larvae [1 hpw], 18 het and 18 mutant [3 hpw], and 25 het and 21 mutant [6 hpw]).
(C) Quantification of total neutrophils in 3 dpf Cxcr1+/− and Cxcr1−/− larvae (n = 19 het and 23 mutant).
(D) Sudan black staining of 3 dpf larvae at 3 and 6 hpw show more neutrophils at the wound microenvironment in Cxcr2−/− embryos than WT.
(E) Quantification of neutrophils caudal to the notochord in Cxcr2−/− and WT (n = 70 WT and 46 mutant [3 hpw] and 83 WT and 54 mutant [6 hpw]).
(F) Total neutrophil number is higher in Cxcr2−/− than WT (n = 48 WT and 40 mutant larvae).
Error bars represent SE. ∗p < 0.05.
Cell Reports  , DOI: ( /j.celrep )
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4. Figure 2
Cxcr2 Is Required for Neutrophil Response to Cxcl8a and Pseudomonas aeruginosa Infection
(A) Representative imaging of the otic vesicle (white dotted outline) 2 hr after injection with supernatant from HEK cells overexpressing zebrafish Cxcl8a or control supernatant. Supernatant was injected into the otic vesicle of 3 dpf Tg(mpx:Dendra) WT or Cxcr2−/− larvae (neutrophils in green).
(B) Number of neutrophils in the otic vesicle of Cxcl8a or control supernatant injected larvae (n = 59 WT and 58 mutant [Ctrl] and 56 WT and 54 mutant [zCxcl8a]).
(C) Neutrophil fold change response to Cxcl8a over control supernatant.
(D) Representative imaging of the otic vesicle (white dotted outline) 2 hr after injection with P. aeruginosa in WT or Cxcr2 mutant Tg(mpx:Dendra) larvae.
(E) Number of neutrophils in the otic vesicle of P. aeruginosa or vehicle-injected larvae (n = 62 WT and 73 mutant [PBS] and 76 WT and 76 mutant [P. aeruginosa]).
(F) Neutrophil fold change response to P. aeruginosa over PBS control. ∗p < 0.05.
Error bars represent SE.
Cell Reports  , DOI: ( /j.celrep )
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5. Figure 3 Neutrophil Reverse Migration Is Impaired in cxcr2 Mutants
(A) Time-lapse imaging of Tg(mpx:Dendra) WT and Cxcr2−/− tail fins after wounding.
(B) Quantification of average neutrophil presence at wound normalized to the average initial neutrophil recruitment to wound at 30 min post-wound (n = 11 WT and 14 mutant; one representative experiment). WT larvae exhibit neutrophil recruitment to wound peaking at 2 hpw with resolution back to 30 min post-wound levels by 6 hpw, whereas Cxcr2−/− larvae maintain high levels of neutrophil infiltration in the wound microenvironment at 6 hpw.
(C) Schematic of photoconversion of Cxcr2−/− neutrophils at wound. Tg(mpx:Dendra) Cxcr2−/− larvae were wounded at 3 dpf. Photoconversion of Dendra+ neutrophils within the wounded tail fin (white dotted outlines in D) was performed at 3 hpw, and neutrophil reverse migration was assessed at 6 hpw.
(D) Neutrophils pre- and post-photoconversion at 3 hpw and at 6 hpw were imaged in WT and Cxcr2−/−.
(E) Quantification of photoconverted neutrophils in the wound microenvironment (magenta cells in dotted boxes, D; n = 25 WT and 30 mutants). cxcr2−/− wounds contained a higher level of photoconverted cells at 3 and 6 hpw.
(F) Percent of photoconverted cells that remained at the wound at 6 hpw was calculated. Cxcr2−/− neutrophils remain at the wound at a higher frequency than WT neutrophils. ∗p < 0.05.
Error bars represent SE.
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6. Figure 4 Cxcr2 Inhibitor SB225002 Blocks Neutrophil Reverse Migration
(A) Schematic of photoconversion of DMSO- or SB treated larvae. Tg(mpx:Dendra) were wounded with tail transection at 3 dpf. DMSO or SB was applied at 2 hpw. Photoconversion of neutrophils within the wounded fin (white dotted box in D) was performed at 3 hpw, and neutrophil reverse migration was assessed at 6 hpw.
(B) Sudan black staining at 6 hpw of WT larvae treated with DMSO or Cxcr2 inhibitor SB Larvae were wounded at 3 dpf and treated with drug or vehicle control at 2 hpw.
(C) Quantification of neutrophils caudal to the notochord at 6 hpw (n = 20 DMSO larvae and 18 SB225002).
(D) Neutrophils pre- and post-photoconversion at 3 hpw and at 6 hpw were imaged in DMSO- or SB treated larvae.
(E) Quantification of photoconverted neutrophils in the wound microenvironment (magenta cells in dotted boxes, D). SB treated larvae contained a higher level of photoconverted cells within the tail fin at 3 and 6 hpw (n = 21 larvae, each).
(F) Percent of photoconverted cells that remained at the wound at 6 hpw was calculated. SB treated neutrophils remain at the wound at a higher frequency than in DMSO control. ∗p < 0.05.
Error bars represent SE.
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7. Figure 5
Interstitial Behavior in the Wound Microenvironment Is Altered in cxcr2-Deficient Zebrafish
(A) Representative image of neutrophil tracks within the tail fin of WT or Cxcr2−/− larvae. Each color track represents an individual neutrophil (white dotted line traces wound edge).
(B) Representative tracks of neutrophil morphology at the wound in WT or Cxcr2−/− larvae. Individual neutrophils are color coded to represent duration at the wound (see legend), with each colored track representing one neutrophil over the duration of its time at the wound.
(C) Representative neutrophil tracks at the wound of WT or cxcr2 morphant larvae.
(D) Average meandering index of neutrophils at the wound in WT is greater than in Cxcr2−/− larvae (n = 20, each).
(E and F) Average neutrophil duration at the wound is greater (E) in Cxcr2−/− compared to WT and (F) in cxcr2 MO compared to WT (n = 20, each). ∗p < 0.05.
Error bars represent SE.
Cell Reports  , DOI: ( /j.celrep )
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8. Figure 6
Cxcl8a Is Required for Neutrophil Reverse Migration from the Wound
(A) Average number of neutrophils at the wound is compared in WT, cxcr1−/−, cxcr2−/−, and cxcl8a−/− larvae (n = 13–32 larvae per time point/condition).
(B) Sudan black staining of neutrophils at 3 and 6 hpw in WT and cxcl8a−/−.
(C) Quantification of neutrophils at the wound shows more neutrophils at 6 hpw in cxcl8a−/− compared to heterozygote siblings (n = 74 WT and 75 mutants [3 hpw] and 91 WT and 70 mutants [6 hpw]).
(D) Representative tracks of neutrophils at the wound in cxcl8a+/− and cxcl8a−/− larvae.
(E) Neutrophil duration at the wound is greater in cxcl8a−/− than in heterozygotes (n = 17 het and 15 mutant larvae).
(F) Meandering index is decreased in cxcl8a−/− compared to het siblings. ∗p < 0.05.
Error bars represent SE.
Cell Reports  , DOI: ( /j.celrep )
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9. Figure 7
Exposure to CXCL8 Inhibits Chemotaxis and Promotes Chemokinesis of Human Neutrophils
(A) Representative tracks of human neutrophils exposed to CXCL8 gradient or no gradient in a microfluidics device (n = 221, 262, and 90 cells, respectively). Pink tracks represent net migration toward source, and blue tracks represent migration away from the source. Black arrow indicates direction of gradient from low concentration to high when present.
(B–E) Quantification of chemotactic index of human neutrophils exposed to a low-concentration CXCL8 gradient (0.025 μM), high-concentration gradient (11.25 μM) without treatment, high-concentration gradient after pre-treatment with CXCL8, or with no gradient (n = 703, 1,210, 1,250, and 497 cells, respectively; B–E). Chemotactic index under high-concentration gradient is decreased when neutrophils are pre-exposed to CXCL8. (C) Percent of migration away from source under high concentration gradient is increased by pre-treatment with CXCL8. (D) Average velocity of neutrophils is increased with high-concentration gradient exposure. Average velocity of pre-treatment condition cells is increased compared to chemokinetic cells. (E) Total distance migrated is greater in pre-activated cells compared to chemokinetic cells.
(F) Model of neutrophil recruitment to infection, which requires Cxcr2; recruitment via Cxcr1 to wounded tissue, which produces Cxcl8 and other pro-inflammatory cues; and neutrophil resolution away from the wound, which is mediated by Cxcr2 sensing of Cxcl8 potentially produced by macrophages and other cells of the wound microenvironment. ∗p < 0.05.
Error bars represent SE.
Cell Reports  , DOI: ( /j.celrep )
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