1. Immunology and Inflammation
  2. Neuroscience
Download icon

Neutrophils promote CXCR3-dependent itch in the development of atopic dermatitis

  1. Carolyn M Walsh
  2. Rose Z Hill
  3. Jamie Schwendinger-Schreck
  4. Jacques Deguine
  5. Emily C Brock
  6. Natalie Kucirek
  7. Ziad Rifi
  8. Jessica Wei
  9. Karsten Gronert
  10. Rachel B Brem
  11. Gregory M Barton  Is a corresponding author
  12. Diana M Bautista  Is a corresponding author
  1. University of California, Berkeley, United States
  2. Buck Institute for Research on Aging, United States
Research Article
Cite this article as: eLife 2019;8:e48448 doi: 10.7554/eLife.48448
5 figures, 2 tables and 5 additional files

Figures

Figure 1 with 10 supplements
The MC903 model parallels the progression of human atopic disease and suggests a temporal sequence of AD pathogenesis.

(A) Exact permutation test (10,000 iterations, see Materials and methods) for significance of mean absolute log2 fold change in gene expression at Day 8 (MC903 vs. ethanol) of custom-defined groups of genes for indicated categories (see Figure 1—source data 1). (B) Log2 fold change in gene expression (MC903 vs. ethanol) in mouse skin at indicated time points for key immune and mouse/human AD genes that were significantly differentially expressed for at least one time point in the MC903 model. Only genes from our initial list (see Materials and methods) differentially expressed at corrected p<0.05 and changing >2 fold between treatments for at least one condition are shown. Green bars = increased expression in MC903 relative to ethanol; magenta = decreased expression. Exact values and corrected p-values are reported in Figure 1—source data 2 and Source Data 1 Supplemental Data, respectively. D1 = 6 hr post-treatment; D2 = Day 2; D5 = Day 5; D8 = Day 8. (C) Scratching behavior of mice treated with MC903 or ethanol for indicated length of time (two-way ANOVA: ****pinteraction <0.0001, F(2,409) = 13.25; Sidak’s multiple comparisons: pday 3 = 0.1309, n = 62,51 mice; *pday 5 = 0.0171, n = 69,56 mice; ****pday 8 < 0.0001, n = 92,85 mice). Exact values displayed in Figure 1—source data 3. (D) Log2 fold change in gene expression of neutrophil chemoattractants (upper), Th2 cytokines (middle) and T cell chemoattractants (lower, from RNA-seq data). (E) Neutrophil counts in MC903- and ethanol-treated skin at indicated time points (two-way ANOVA: **ptreatment = 0.0023, F(1,102) = 9.82; Sidak’s multiple comparisons: pday 2 > 0.999, n = 4,4 mice; pday 3 = 0.9801, n = 5,5 mice; ***pday 5 = 0.0003, n = 6,8 mice; ***pday 8 = 0.0001, n = 40,38 mice). (F) Basophil counts in MC903- and ethanol-treated skin at indicated time points (two-way ANOVA: **ptreatment = 0.0051, F(1,102) = 8.17; Sidak’s multiple comparisons: pday 2 > 0.999, n = 4,4 mice; pday 3 = 0.8850, n = 5,5 mice; pday 5 = 0.0606, n = 6,8 mice; ****pday 8 < 0.0001, n = 40,38 mice). (G) CD4+ T cell counts in MC903- and ethanol-treated skin at indicated time points (two-way ANOVA: **ptime = 0.0042, F(1,44) = 9.10; pday 3 = 0.9998, n = 8,6 mice; pday 5 = 0.2223, n = 9,8 mice; **pday 8 = 0.0021, n = 11,8 mice). Day 8 immune cell infiltrate represented as % of CD45+ cells in Figure 1—figure supplement 2A–B (see Supplementary file 3 for all experimental conditions). Exact values displayed in Figure 1—source data 4 and representative FACS plots for myeloid and T cell gating shown in Figure 1—figure supplement 3A and Figure 1—figure supplement 4A. For Figure 4E–G, data from mice receiving i.p. injection of PBS (see Figure 4) in addition to MC903 or EtOH are also included. (H) (Upper and Lower) Representative maximum intensity Z-projections from immunohistochemistry (IHC) of whole-mount mouse skin on Day 2 of the MC903 model. Skin was stained with neuronal marker beta-tubulin III (BTIII; green). Hair follicle autofluorescence is visible in the magenta channel. Images were acquired on a confocal using a 20x water objective. (I) Quantification of innervation (see Materials and methods) of mouse skin as determined from BTIII staining (*p=0.012; two-tailed t-test (t = 3.114; df = 9); n = 7,4 images each from two mice per treatment). Day 1 IHC results as follows: 31.78 ± 18.39% (MC903) and 31.51 ± 16.43% (EtOH); p=0.988; two-tailed unpaired t-test; n = 6 images each from two mice per treatment. Exact values are reported in Figure 1—source data 5. (J) Quantification of CGRP+ nerve fibers (see Materials and methods) in skin (**p=0.0083; two-tailed t-test (t = 2.868; df = 25); n = 15, 12 images from three mice per treatment). Exact values are reported in Figure 1—source data 5. Representative images in Figure 1—figure supplement 9A.

Figure 1—source data 1

Values displayed in the bar plot shown in Figure 1A.

https://cdn.elifesciences.org/articles/48448/elife-48448-fig1-data1-v3.csv
Figure 1—source data 2

Values displayed in the heat map shown in Figure 1B.

https://cdn.elifesciences.org/articles/48448/elife-48448-fig1-data2-v3.csv
Figure 1—source data 3

Values displayed in the bar plot shown in Figure 1C.

https://cdn.elifesciences.org/articles/48448/elife-48448-fig1-data3-v3.csv
Figure 1—source data 4

Values displayed in the bar plots shown in Figure 1E–G and Figure 1—figure supplement 5A–C.

https://cdn.elifesciences.org/articles/48448/elife-48448-fig1-data4-v3.csv
Figure 1—source data 5

Values displayed in the bar plots shown in Figure 1I and Figure 1J.

https://cdn.elifesciences.org/articles/48448/elife-48448-fig1-data5-v3.csv
Figure 1—source data 6

Values displayed in the heat map shown in Figure 1—figure supplement 1A.

https://cdn.elifesciences.org/articles/48448/elife-48448-fig1-data6-v3.csv
Figure 1—source data 7

Values displayed in the heat map shown in Figure 1—figure supplement 6A.

https://cdn.elifesciences.org/articles/48448/elife-48448-fig1-data7-v3.csv
Figure 1—source data 8

Values displayed in the heat map shown in Figure 1—figure supplement 7A.

https://cdn.elifesciences.org/articles/48448/elife-48448-fig1-data8-v3.csv
Figure 1—source data 9

Values displayed in the bar plot shown in Figure 1—figure supplement 10A.

https://cdn.elifesciences.org/articles/48448/elife-48448-fig1-data9-v3.csv
Figure 1—figure supplement 1
Expression of mouse and human itch genes.

(A) Log2 fold change in gene expression (MC903 vs. ethanol) in mouse skin at indicated time points for genes implicated in mouse or human acute or chronic itch that were significantly differentially expressed for at least one time point in the MC903 model. Green bars = increased expression in MC903 relative to ethanol; magenta = decreased expression. Exact values and corrected p-values are reported in Figure 1—source data 6 and Source Data 1 Supplemental Data, respectively.

Figure 1—figure supplement 2
Immune cells represented as % of CD45+ cells.

(A) Number of CD45+ cells in MC903-treated skin on days 2–8 of the model. (B) Skin-infiltrating immune cell subtypes on days 2–8 of the MC903 model shown in Figure 1, represented as % of CD45+ cells. CD4+ T cell measurements were acquired using a separate staining panel from different animals than the myeloid cell measurements (see Materials and methods) and were not included. See Supplementary file 3 for % of CD45+ cell measurements for all flow cytometry experiments. Error bars represent mean ± SEM.

Figure 1—figure supplement 3
Myeloid and granulocyte gating strategy.

(A-C) Representative FACS plots of cells isolated from MC903-treated cheek skin showing gating strategy for neutrophils (A), inflammatory monocytes (A), mast cells (B), basophils (B), and eosinophils (C) as shown in Figure 1E–F and Figure 1—figure supplement 5.

Figure 1—figure supplement 4
T cell gating strategy.

(A) Representative FACS plots of cells isolated from MC903-treated cheek skin showing gating strategy for CD4+ T cells as shown in Figure 1G.

Figure 1—figure supplement 5
Immune cell counts in MC903-treated skin.

(A) Inflammatory monocyte counts in MC903- and ethanol-treated skin at indicated time points (two-way ANOVA: ptreatment = 0.0662, F(1,102) = 3.44; n = 4,4,5,5,6,8,40,38 mice). (B) Mast cell counts in MC903- and ethanol-treated skin at indicated time points (two-way ANOVA: **ptreatment = 0.0024, F(1,102) = 9.69; Sidak’s multiple comparisons: pday 2 > 0.999, n = 4,4 mice; pday 3 = 0.3019, n = 5,5 mice; pday 5 = 0.0586, n = 6,8 mice; ****pday 8 < 0.0001, n = 40,38 mice). (C) Eosinophil counts in MC903- and ethanol-treated skin at indicated time points (two-way ANOVA: ptime = 0.0471, F(3,102) = 2.74; Sidak’s multiple comparisons: pday 2 > 0.999, n = 4,4 mice; pday 3 = 0.3596, n = 5,5 mice; pday 5 = 0.9998, n = 6,8 mice; **pday 8 = 0.0020, n = 40,38 mice). Data from mice receiving i.p. injection of PBS (see Figure 4) in addition to MC903 or EtOH are also included. Exact values displayed in Figure 1—source data 4.

Figure 1—figure supplement 6
Protease receptor activation triggers rapid upregulation of neutrophil chemoattractant genes in human keratinocytes.

(A) Heat map showing log2 fold change in gene expression in cultured human keratinocytes 3 hr after SLIGRL treatment (100 µM; bottom; see Figure 1—source data 7) compared to vehicle controls, as measured by RNA-seq. Genes are sorted by descending corrected p-value; only significantly differentially expressed (p<0.05) are displayed. Exact values and corrected p-values are reported in Figure 1—source data 7 and Source Data 1 Supplemental Data, respectively.

Figure 1—figure supplement 7
Expression of neuronal genes and axon guidance molecules in skin.

(A) Log2 fold change in gene expression (MC903 vs. EtOH) in mouse skin at indicated time points for markers of locally translated sensory neuronal transcripts or genes implicated in neurite remodeling and/or axon guidance that were significantly differentially expressed for at least one time point in the MC903 model. Green bars = increased expression in MC903 relative to ethanol; magenta = decreased expression. Exact values and corrected p-values are reported in Figure 1—source data 8 and Source Data 1 Supplemental Data, respectively.

Figure 1—figure supplement 8
Method of image quantification for whole mount skin.

(A) Representative maximum intensity z-projection of beta tubulin III staining in cheek skin. (B) Binary image after edge-detection. (C) % Area innervated was calculated from the percentage of the image area which was occupied by the regions of interest (ROIs) outlined in red.

Figure 1—figure supplement 9
Peptidergic fibers display hyperinnervation in MC903-treated skin.

(A) Representative maximum intensity Z-projections from immunohistochemistry (IHC) of whole-mount mouse skin on day 2 of the MC903 model. Skin was stained with peptidergic neuronal marker Calcitonin related-gene peptide (CGRP; white). Images were acquired on a confocal microscope using a 20x water objective.

Figure 1—figure supplement 10
Inflammatory lipids in MC903-treated skin.

(A) Quantification of indicated lipids from 6 mm biopsy punches of cheek skin of MC903- and EtOH-treated mice (at day 8) by LC-MS/MS (**p=0.006 (t = 4.148,df = 6), *p=0.024 (t = 3.003,df = 6), ***p=0.0007 (t = 6.392,df = 6), *p=0.022 (t = 3.058,df = 6); two-tailed unpaired t-tests; n = 4 mice per group, see Figure 1—source data 8).

Figure 2 with 5 supplements
Neutrophils are necessary and sufficient for itch behaviors.

(A) Scratching behavior of uninjected and PBS-injected mice (combined) and aGr1-injected mice treated with MC903 or ethanol for indicated length of time (two-way ANOVA: ****pinteraction <0.0001, F(4,447) = 7.16; Tukey’s multiple comparisons: pday 3 MC903 vs. EtOH = 0.1111 n = 62,51,17 mice; *pday 5 MC903 vs. EtOH = 0.0154, pday 5 MC903 vs. aGr1 = 0.9854, pday 5 aGr1 vs. EtOH = 0.2267, n = 69,56,17 mice; ****pday 8 MC903 vs. EtOH <0.0001, ***pday 8 MC903 vs. aGr1 = 0.0007, pday 8 aGr1 vs. EtOH = 0.1543, n = 92,85,17 mice). (B) Neutrophil count from cheek skin of uninjected/PBS-injected MC903- and ethanol-treated, and aGr1-injected MC903-treated mice on day 8 (one-way ANOVA: ****p<0.0001, F(2,92) = 10.59; Tukey’s multiple comparisons: ****pMC903 vs. EtOH <0.00001, n = 40,38 mice; *pMC903 vs. aGr1 MC903 = 0.0109, n = 40,17 mice; paGr1 vs. EtOH = 0.8859, n = 38,17 mice). (C) Basophil count from cheek skin of uninjected/PBS-injected MC903- and ethanol-treated, and aGr1-injected MC903-treated mice on day 8 (one-way ANOVA: ****p=0.0001, F(2,92) = 14.61; Tukey’s multiple comparisons: pMC903 vs. aGr1 MC903 = 0.3217, n = 40,17 mice, ****pMC903 vs. EtOH <0.0001, n = 40,38 mice, *paGr1 MC903 vs. EtOH = 0.0204, n = 17,38 mice). (D) CD4+ T cell count from cheek skin of PBS-injected MC903- and ethanol-treated, and aGr1-injected MC903-treated mice on day 8 (two-way ANOVA: **ptreatment = 0.0035, F(1,35) = 9.82; Holm-Sidak multiple comparisons for PBS versus aGr1: pMC903 = 0.8878, n = 9,11 mice; pEtOH = 0.5201, n = 8,9 mice). Control MC903 and EtOH data from Figure 2B–C are also displayed in Figure 1. Exact values displayed for Figure 2A–D in Figure 2—source data 1. (E) Scratching behavior of mice immediately after injection of 1 µg CXCL1 or PBS (s.c. cheek). For neutrophil-depletion experiments, mice received 250 µg anti-Gr1 (aGr1) 20 hr prior to cheek injection of CXCL1 or PBS (one-way ANOVA: ****p<0.0001, F(4,88) = 75.53; Tukey’s multiple comparisons: *pCXCL1 vs. PBS = 0.0126, n = 36,31 mice; paGr1-CXCL1 vs. aGr1-PBS > 0.9999, n = 10,10 mice; paGr1-CXCL1 vs. PBS = 0.9986, n = 10,31 mice). Exact values displayed in Figure 2—source data 2. (F) Scratching behavior of WT and TSLPR KO (TSLPR KO) mice treated with MC903 or ethanol for indicated length of time (two-way ANOVA: ****pinteraction <0.0001, F(9,657) = 4.93; Tukey’s multiple comparisons: ****pday 8 WT MC903 vs. EtOH <0.0001, *pday 8 WT MC903 vs. KO MC903 = 0.0194, **pday 8 KO MC903 vs. KO EtOH = 0.0039, n = 92,85,36,26 mice; ****pday 12 WT MC903 vs. EtOH <0.0001, **pday 12 WT MC903 vs. KO MC903 = 0.0028, pday 12 KO MC903 vs. KO EtOH = 0.7061, n = 26,26,27,23 mice). (G) Neutrophil count from cheek skin of wild-type MC903- and ethanol-treated, and TSLPR KO MC903-treated mice on day 5 (two-way ANOVA: **pgenotype = 0.0025, F(2,125) = 6.28; Tukey’s multiple comparisons: ****pday 5 WT MC903 vs. WT EtOH <0.0001, n = 6,8 mice; pday 5 WT MC903 vs. KO MC903 = 0.2198, n = 6,6 mice; *pday 5 WT EtOH vs. KO MC903 = 0.0212, n = 8,6 mice). (H) Basophil count from cheek skin of wild-type MC903- and ethanol-treated, and TSLPR KO MC903-treated mice on day 8 (two-way ANOVA: **pgenotype = 0.0003, F(2,117) = 8.87; Tukey’s multiple comparisons: ****pday 8 WT MC903 vs. WT EtOH <0.0001, n = 40,38 mice; ****pday 8 WT MC903 vs. KO MC903 <0.0001, n = 40,15 mice; pday 8 WT EtOH vs. KO MC903 = 0.9519, n = 38,15 mice). See also Figure 2—figure supplement 5A. For Figure 2G–H, data from days 3, 5, and 8 are presented in Figure 2—source data 3. (I) CD4+ T cell count from cheek skin of wild-type MC903- and ethanol-treated, and TSLPR KO MC903-treated mice on day 8 (one-way ANOVA: **p=0.0053, F(2,24) = 6.564; Tukey’s multiple comparisons: *pWT MC903 vs. WT EtOH = 0.0163, n = 11,8 mice; *p MC903 vs. KO MC903 = 0.0130, n = 11,8 mice; pWT EtOH vs. KO MC903 = 0.9953, n = 8,8 mice). Wild-type MC903 and EtOH data from 2 F-H are also displayed in Figure 1. Exact values for Figure 2F–I displayed in Figure 2—source data 3. (J) Neutrophil count from cheek skin of wild-type MC903- and ethanol-treated mice on day 12 of the MC903 model. MC903-treated animals received daily i.p. injections of 250 µg aGr1 antibody or PBS (250 µL) on days 8–11 of the model (one-way ANOVA: *p=0.01, F(2,13) = 6.69; Tukey’s multiple comparisons: *pMC903-PBS vs. EtOH = 0.0141, n = 6,5 mice; *pMC903-PBS vs. MC903-aGr1 = 0.10330, n = 6,5 mice; pMC903-aGr1 vs. EtOH = 0.9005, n = 5,5 mice). (K) Time spent scratching over a thirty minute interval for wild-type MC903- and ethanol-treated mice on day 12 of the MC903 model. MC903-treated animals received daily i.p. injections of 250 µg aGr1 antibody or PBS (250 µL) on days 8–11 of the model (one-way ANOVA: ****p<0.0001, F(2,26) = 53.1; Tukey’s multiple comparisons: ****pMC903-PBS vs. EtOH <0.0001, n = 12,5 mice; ****pMC903-PBS vs. MC903-aGr1 < 0.0001, n = 12,12 mice; pMC903-aGr1 vs. EtOH = 0.3734, n = 12,5 mice). Values from bar plots are reported in Figure 2—source data 5.

Figure 2—figure supplement 1
aGr1 treatment preferentially depletes neutrophils.

(A) Representative flow cytometry plots of cells collected from blood of mice injected with PBS or aGr1 (250 μg, i.p.) once-daily for five days concurrent with daily MC903 topical treatment. Shown are CD45.2+CD11b+ cells, plotted by Ly6G and Ly6C signal, with neutrophil (Neuts.) and inflammatory monocyte (IMs) populations indicated. Neutrophils were defined as Cd11b+Ly6G+Ly6Cmid/high and IMs were defined as Cd11b+Ly6G-Ly6Chigh (see Materials and methods). (B) Representative flow cytometry plot as in A, depicting neutrophil and IM populations from blood collected on day 8. (C) (Left) Neutrophil counts in blood shown as % of Cd11b+ cells from aGr1/MC903 (black triangles) and PBS/MC903 (gray circles)-treated animals on days 3, 5, and 8 of the model (two-way repeated measures ANOVA: ****ptreatment <0.0001, F(1,31) = 299.5; Sidak’s multiple comparisons: ****pday 3 < 0.0001; ****pday 5 < 0.0001; ****pday 8 < 0.0001, n = 16,17 mice). (Right) Inflammatory monocyte counts in blood shown as % of Cd11b+ cells from aGr1/MC903 and PBS/MC903-treated animals on days 3, 5, and 8 of the model (two-way repeated measures ANOVA: *ptreatment = 0.0468, F(1,31) = 4.287; Sidak’s multiple comparisons: **pday 3 = 0.0015; pday 5 = 0.1918; pday 8 = 0.2013, n = 16,17 mice). Exact values displayed in Figure 2—source data 4.

Figure 2—figure supplement 2
CXCL1 rapidly and selectively recruits neutrophils to skin.

(A) Representative flow cytometry plots of cells from cheek skin of mice injected with PBS or CXCL1 (1 μg in 20 µL, s.c.). Shown are CD45.2+CD11b+ cells, plotted by Ly6G and Ly6C signal, with neutrophil and inflammatory monocyte (IMs) populations indicated. (B) Neutrophil count from cheek skin of mice 5, 15, and 30 min after injection of CXCL1 or PBS (two-way ANOVA: *pinteraction = 0.0239, F(2,21) = 4.48; Sidak’s multiple comparisons: p5 min >0.9999, n = 4,5 mice; *pday 15 min = 0.0141, n = 4,4 mice; **pday 30 min = 0.0031, n = 3,7 mice). Exact values displayed in Figure 2—source data 2. (C) Blood neutrophils as % of Cd11b+ cells approximately 20 hr after injection of 250 µg aGr1 (n = 15 mice). Mice assayed for CXCL1-evoked itch behavior immediately preceding blood isolation (see Figure 2E). Exact values displayed in Figure 2—source data 2. See Figure 2—figure supplement 1C for representative blood neutrophil measurements from PBS-injected animals.

Figure 2—figure supplement 3
Neutrophil depletion does not affect chloroquine-evoked itch.

(A) Scratching behavior of mice immediately after injection of chloroquine (CQ) or PBS (s.c. cheek). For neutrophil-depletion experiments, mice received 250 µg anti-Gr1 (aGr1) 20 hr prior to cheek injection of CQ or PBS (two-tailed t-test: ****p<0.0001 (t = 10.58, df = 14); n = 6,10 mice). Exact values displayed in Figure 2—source data 2.

Figure 2—figure supplement 4
Loss of TSLPR reduces skin basophil and mast cell numbers in the first week of AD development.

(A) Basophil count from cheek skin of wild-type MC903- and ethanol-treated, and TSLPR KO MC903-treated mice after 3 or 5 days of treatment (two-way ANOVA: ***ptime = 0.0003, F(2,117) = 8.87; Tukey’s multiple comparisons: pday3 WT MC903 vs. KO MC903 = 0.6540, n = 3,5 mice; *pday 5 WT MC903 vs. KO MC903 = 0.1023, n = 6,6 mice; pday 5 WT EtOH vs. KO MC903 = 0.9077, n = 8,6 mice; pday 5 WT MC903 vs. WT EtOH = 0.0264, n = 6,8 mice). (B) Mast cell count from cheek skin of wild-type MC903- and ethanol-treated, and TSLPR KO MC903-treated mice after 3, 5, or 8 days of treatment (two-way ANOVA: *pgenotype = 0.0384, F(2,117) = 3.35; Tukey’s multiple comparisons: pday 3 WT MC903 vs. KO MC903 = 0.4133, n = 3,5 mice; pday 5 WT MC903 vs. KO MC903 = 0.9882, n = 6,6 mice; *pday 5 WT MC903 vs. WT EtOH = 0.0440, n = 6,5 mice; *pday 5 KO MC903 vs. WT EtOH = 0.0294, n = 6,5 mice; *pday 8 WT MC903 vs. KO MC903 = 0.0188, n = 40,15 mice; ****pday 8 WT MC903 vs. WT EtOH < 0.0001, n = 40,38 mice; pday 8 WT EtOH vs. KO MC903 = 0.7810, n = 38,15 mice). Data from days 3, 5, and 8 are presented in Figure 2—source data 3.

Figure 2—figure supplement 5
Neutrophils robustly infiltrate the skin in the DNFB mouse model of atopic dermatitis.

(A) Neutrophil count from ear skin of wild-type DNFB- and vehicle-treated mice 24 hr after challenge with DNFB or vehicle performed five days after initial DNFB sensitization on shaved rostral back skin (***p=0.0004; two-tailed t-test (t = 4.290; df = 18); n = 10 mice per group). Values from bar plot is reported in Figure 2—source data 6.

Figure 3 with 1 supplement
The MC903 model induces rapid and robust changes in neuronal tissue.

(A) Exact permutation test (10,000 iterations, see Materials and methods) for significance of mean absolute log2 fold change in gene expression at Day 8 (MC903 vs. ethanol) of custom-defined groups of genes for indicated categories (see Figure 3—source data 1). (B) Log2 fold change in gene expression (MC903 vs. ethanol) in mouse trigeminal ganglia (TG) at indicated time points for all genes which were significantly differentially expressed for at least one time point in the MC903 model. Green bars = increased expression in MC903 relative to ethanol; magenta = decreased expression. Exact values and corrected p-values are reported in Figure 3—source data 2 and Source Data 1 Supplemental Data, respectively. (C) Representative composite images showing immune cells (CD45, green), and sensory neurons (Prph, magenta) with DAPI (blue) in sectioned trigeminal ganglia from mice treated with Vehicle or MC903 for five days on the cheek. (D) Quantification of images examining average number of CD45+ cells per section and average ratio of CD45:Peripherin cells per section after five days of treatment (p=0.562 (t = 0.6318, df = 4), 0.542 (t = 0.6660, df = 4); two-tailed unpaired t-tests, n = 33–159 fields of view (images) each of both trigeminal ganglia from three mice per condition treated bilaterally). (E) Representative composite images showing immune cells (CD45, green), and sensory neurons (Peripherin (Prph), magenta) with DAPI (blue) in sectioned trigeminal ganglia from mice treated with Vehicle or MC903 for eight days on the cheek. (F) Quantification of images examining average number of CD45+ cells per section and average ratio of CD45:Peripherin cells per section after eight days of treatment (**p=0.0019 (t = 5.977,df = 5), **p=0.0093 (t = 4.107,df = 4); two-tailed unpaired t-tests; n = 42–172 fields of view (images) each of both trigeminal ganglia from 3 EtOH or 4 MC903 animals treated bilaterally). Scale bar = 100 µm. Images were acquired on a fluorescence microscope using a 10x air objective. Values from bar plots and all TG IHC data are available in Figure 3—source data 3. (G) Log2 fold change in gene expression (MC903 vs. ethanol) in mouse spinal cord on day 8 showing selected differentially expressed genes (padjusted < 0.05). Exact values and corrected p-values are reported in Source Data 1 Supplemental Data.

Figure 3—source data 1

Values displayed in the bar plot shown in Figure 3A.

https://cdn.elifesciences.org/articles/48448/elife-48448-fig3-data1-v3.csv
Figure 3—source data 2

Values displayed in the heat map shown in Figure 3B.

https://cdn.elifesciences.org/articles/48448/elife-48448-fig3-data2-v3.csv
Figure 3—source data 3

Quantification of all IHC samples from trigeminal ganglia, and Values displayed in the bar plots shown in Figure 3D,F.

https://cdn.elifesciences.org/articles/48448/elife-48448-fig3-data3-v3.csv
Figure 3—figure supplement 1
Method of image quantification for sectioned trigeminal ganglia.

(A) Representative composite image showing CD45 (green), Peripherin (magenta), and DAPI (blue). (B) Single-channel CD45 image with automated min/max intensity thresholding. (C) Resultant binary image generated from (B). (D) Cells were counted as the number of regions of interest (ROIs) outlined in blue.

Figure 4 with 1 supplement
Neutrophils are required for induction of the itch-inducing chemokine CXCL10.

(A) Log2 fold change (Day 8 MC903 vs. EtOH) of Th2 genes in skin from uninjected wild-type, aGR1-treated, and TSLPR KO animals. (B) Log2 fold change (Day 8 MC903 vs. EtOH) of chemokine genes in skin from uninjected wild-type, aGr1-treated, and TSLPR KO animals. (C) Log2 fold change (Day 8 MC903 vs. EtOH) of activity-induced genes in trigeminal ganglia from uninjected wild-type, aGr1-treated, and TSLPR KO animals. (D) Log2 fold change (Day 8 MC903 vs. EtOH) of Lcn2 and activity-induced genes in spinal cord from uninjected and aGr1-treated wild-type mice on day 8. For Figure 4A–D, exact values and corrected p-values are reported in Source Data 1 Supplemental Data. (E) Quantification of innervation (see Materials and methods) of MC903 and EtOH-treated mouse skin as determined from BTIII staining (p=0.8985; two-tailed t-test (t = 0.1294; df = 18); n = 9,11 images each from two mice per treatment. Exact values are reported in Figure 4—source data 1. (F) CXCL10 levels in skin homogenate as measured by ELISA on day 8 of the MC903 model for uninjected animals (left; *p=0.029 (t = 2.715, df = 7); two-tailed t-test; n = 4,5 animals), animals which received aGr1 for 8 days (middle; p=0.43 (t = 0.815, df = 11); two-tailed t-test; n = 6,6 animals), and TSLPR KO animals (right; *p=0.0357 (t = 2.696, df = 6); two-tailed t-test; n = 4,4 animals. Skin homogenates were isolated on separate days and so uninjected, WT samples were not compared to aGr1-treated samples or to TSLPR KO samples. (G) (Left) Time spent scratching over a thirty minute interval on days 5, 8, and 12 of the MC903 model, one hour after mice were injected with either 3.31 mM of the CXCR3 antagonist AMG 487 or vehicle (20% HPCD in PBS; 50 µL s.c. in rostral back); (two-way ANOVA: ****ptreatment <0.0001, F(1,67) = 50.64; Tukey’s multiple comparisons: *pday 5 = 0.0216, n = 8,10 mice; ***pday 8 = 0.0007, n = 18,21 mice; ****pday 12 < 0.0001, n = 8,8 mice). (Right) Time spent scratching over a thirty minute interval one hour after mice were injected with either 3.31 mM of the CXCR3 antagonist AMG 487 or vehicle (20% HPCD in PBS; 50 µL s.c. in rostral back), and immediately after mice were injected with 50 mM chloroquine (20 µL i.d., cheek). p=0.92 (t = 0.0964, df = 8); two-tailed t-test; n = 5,5 mice. Values from bar plots in Figure 4F–G are displayed in Figure 4—source data 2.

Figure 4—figure supplement 1
MC903-dependent gene expression changes in aGr1-treated and TSLPR KO animals.

(A) Heat map showing log2 fold change in gene expression (Day 8 MC903 vs. EtOH) for itch-associated genes in wild-type, aGr1-treated, and TSLPR KO skin. Green bars = increased expression in MC903 relative to ethanol; magenta = decreased expression. Exact values and corrected p-values are reported in Figure 4—source data 3 and Source Data 1 Supplemental Data, respectively. (B) Heat map showing log2 fold change in gene expression (Day 8 MC903 vs. EtOH) for wild-type, aGr1-treated, and TSLPR KO mouse trigeminal ganglia (TG) at indicated time points for all genes which were significantly differentially expressed for at least one time point in the MC903 model (See Figure 2D). Green bars = increased expression in MC903 relative to ethanol; magenta = decreased expression. Exact values and corrected p-values are reported in Figure 4—source data 4 and Source Data 1 Supplemental Data, respectively.

Model of early AD pathogenesis.

(A) AD induction first results in increased protease expression and barrier dysfunction, which drives production of the cytokines TSLP and CXCL1 via PAR2 activation within keratinocytes. CXCL1 can recruit neutrophils via its receptor CXCR2. Neutrophils may evoke itch by multiple pathways, including degranulation and release of proteases and histamine, production of sensitizing lipids such as PGE2 and LTB4, (Hashimoto et al., 2018) and induction of CXCL10 expression, which can activate sensory neurons via CXCR3. TSLP activates a number of immune cells to elicit IL-4 production, including basophils, which results in increased IL-4, recruitment of CD4+ T cells, (Oetjen et al., 2017) and sensitization of neurons to promote itch later in the model.

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or
reference
IdentifiersAdditional
information
Strain,
strain background
(Mus musculus)
C57BL/6; WT; wild-typeThe Jackson LaboratoryJackson Stock #: 000664; RRID:IMSR_JAX:000664
Strain, strain background(Mus musculus)C57BL/6; WT; wild-typeCharles River LaboratoriesRRID:IMSR_CRL:27; Charles River strain code #: 027;
Strain, strain background(Mus musculus)Crlf2tm1Jni; TSLPR KOPMID: 14993294RRID:MGI:3039553; MGI Cat# 3039553Obtained from the laboratory of Steven F. Ziegler (Ben Aroya Research Institute)
AntibodyPurified anti-mouse Ly-6G/Gr-1 antibody. Low endotoxin, no azide, in PBS; anti-GR1 (RB6-8C5); aGr1UCSF CoreUCSF Core Cat# AM051Obtained from the laboratory of Daniel Portnoy (UC Berkeley)
AntibodyLEAF Purified anti-mouse Ly-6G/Ly-6C (Gr-1); antibody; RB6-8C5; aGr1BiolegendRRID:AB_313379; BioLegend Cat# 108414
AntibodyAnti-β-tubulin III (Rabbit polyclonal; 1:1000)AbcamRRID:AB_444319; Cat # ab18207
AntibodyAnti-CGRP (Rabbit polyclonal; 1:1000)ImmunostarRRID:AB_572217; Cat # 24112
AntibodyAnti-Peripherin (Chicken polyclonal; 1:1000)AbcamRRID:AB_777207; Cat # ab39374
AntibodyGoat Anti-Mouse
IgG H and L Alexa Fluor 488 (Goat polyclonal; 1:1000)
AbcamRRID:AB_2688012; Cat # ab150117
AntibodyGoat anti-Chicken IgY (H+L) Secondary Antibody, Alexa Fluor 488 (Goat polyclonal; 1:1000)ThermoFisher ScientificRRID:AB_2534096; Cat # A-11039
AntibodyGoat Anti-Chicken IgG (H+L) Secondary Antibody, Alexa Fluor 594 (Goat polyclonal; 1:1000)ThermoFisher ScientificRRID:AB_2534099; Cat # A11042
AntibodyGoat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 594 (Goat polyclonal; 1:1000)InvitrogenRRID:AB_2556545; Cat # R37117
Commercial assay or kitPromocell Keratinocyte Growth Medium 2PromocellCat # C-20011
Cell line (human)Normal Human Epidermal Keratinocytes (NHEK), single juvenile donor, cryopreservedPromocellCat # C-12001
OtherLiberase TM Research Grade; LiberaseRocheCat # 5401119001
OtherDnase I from bovine pancreasSigmaCat # 11284932001
OtherAmbionª DNase I (RNase-free); DNAseAmbionCat # AM2222
Peptide, recombinant proteinSLIGRL-NH2;
SLIGRL
TocrisCas 171436-38-7; Cat #1468
Commercial assay or kitQiagen RNeasy
mini kit
QiagenCat # 74104
Commercial assay or kitRNAzol RTSigma-AldrichCat # R4533-50ML
Chemical compound, drug(2-Hydroxypropyl)-
β-cyclodextrin; HPCD
Sigma-AldrichCas 128446-35-5; Cat # H107
Chemical compound, drugMethyl alcohol; Methanol; MeOHSigma-AldrichCas 67-56-1;
Cat # 34860
Chemical compound, drugEthanol, Absolute (200 Proof), Molecular Biology Grade, Fisher BioReagents; Absolute Ethanol, Molecular-Biology grade; Ethanol; EtOHFischer ScientificCas 64-17-5; Cat # BP2818100
Chemical compound, drugMC903; CalcipotriolTocrisCas 112965-21-6; Cat # 2700
Chemical compound, drug(±)-AMG 487; AMGTocrisCas 947536-03-0; Cat # 4487
Chemical
compound, drug
Chloroquine diphosphate; ChloroquineSigma-AldrichCAS 50-63-5; Cat # C6628
Chemical
compound, drug
Dimethyl sulfoxide; DMSOSigma-AldrichCat # 8418–100 mL
Chemical compound, drugFormaldehyde, 16%, methanol free, Ultra Pure; Paraformaldehyde; PFAPolysciences, Inc.Cat # 18814–10
Chemical compound, drugTissue Tek Optimal cutting temperature compound (OCT)Sakura Finetek USACat # 4583
Chemical compound, drugTriton X-100 solution; Triton X-100BioUltraCAS 9002-93-1; Cat # 93443
Chemical
compound, drug
Phosphate-buffered saline (PBS), pH 7.4; PBSGibcoCat # 10010023
Chemical compound, drugBenzyl benzoateSigma-AldrichCAS 120-51-4; Cat # B6630
Chemical compound, drugBenzyl alcoholSigma-AldrichCAS 100-51-6; Cat # 305197
Chemical
compound, drug
SucroseSigma-AldrichCAS 57-50-1; Cat # S0389
Chemical compound, drugLIVE/DEAD Fixable Aqua Dead Cell Stain Kit, for405 nm excitation; AquaThermoFisher ScientificCat # L34957
Chemical compound, drugIsofluranePiramalCAS 26675-46-7
Chemical compound, drug4',6-Diamidino-2-Phenylindole, Dihydrochloride; DAPIThermoFisher ScientificCAS 28718-90-3; Cat # 1306
Chemical compound, drug4',6-Diamidino-2-Phenylindole, Dihydrochloride; DAPI LIVE/DEADInvitrogenCat # L34961
Chemical compound, drugFluoromount-GThermoFisher ScientificCat # 00-4958-02
AntibodyGoat Anti-Mouse IgG - H and L - Fab Fragment Polyclonal Antibody, Unconjugated, Abcam; F(ab) anti-mouse IgG (Goat polyclonal; 1:200)AbcamRRID:AB_955960; Cat # ab6668
AntibodyAnti-Mouse CD45.2 Purified 100 ug antibody, Thermo Fisher Scientific; Mouse anti-CD45.2 (Mouse monoclonal; 1:1000)eBioscienceRRID:AB_467261; Cat # 14-0454-82
AntibodyPurified anti-mouse CD16/32 antibody. Low endotoxin, no azide, in PBS; Rat anti-Mouse CD16/32 (2.4G2) (Rat monoclonal; 1:1000)UCSF CoreUCSF Core Cat# AM004
Commercial assay or kitDuoSet ELISA Ancillary Reagent Kit 2R and D SystemsCat # DY008
Commercial
assay or kit
Mouse CXCl10 DuoSet ELISAR and D SystemsCat # DY466
Commercial assay or kitPierce BCA Protein Assay Kit - Reducing Agent
Compatible
ThermoFisher ScientificCat # 23250
Chemical compound, drug2-Amino-2-(hydroxymethyl)−1,3-propanediol; Trizma base, TRIS, TRIS baseSigma-AldrichCas 77-86-1; Cat # T4661
Chemical compound, drugEthylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid; EGTASigma-AldrichCas 67-42-5; Cat # E3889
Chemical compound, drugEthylenedinitrilo)tetraacetic acid; EDTASigma-AldrichCas 60-00-4; Cat # E9884
Commercial assay or kitPhosSTOP inhibitorRocheCat # 4906845001
Chemical compound, drugSodium deoxycholate,≥97% (titration); Sodium deoxycholateSigma-AldrichCas 302-95-4; Cat # D6750
Chemical compound, drugPhenylmethylsulfonyl fluoride; PMSFSigma-AldrichCas 329-98-6; Cat # 10837091001
Chemical
compound, drug
1-Fluoro-2,4,-dinitrobenzene; DNFBSigmaCas 70-34-8; Cat # D1529
Commercial assay or kitcOmplete protease inhibitor cocktailRocheCat # 11697498001
OtherAdvanced RPMI Medium 1640; RPMIGibcoCat # 12633012
OtherFetal Bovine Serum; FBS; FCSHyCloneCat # 30396.03
Othersodium pyruvate 100 mMGibcoCat # 11360070
OtherN-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid; HEPES 1MGibcoCat # 15630080
OtherL-Glutamine 200 mMGibcoCat # 25030081
OtherPenicillin-Streptomycin (10,000 U/mL; Pen-StrepGibcoCat # 15140122
OtherCollagenase VIIISigma-AldrichCat # C2139-500MG
Commercial assay or kitInvitrogen
CountBright Absolute Counting Beads, for flow cytometry; Counting Beads
InvitrogenCat # C36950
AntibodyCD45 Monoclonal Antibody (30-F11), APC-eFluor 780, eBioscience(TM), Thermo Fisher Scientific; CD45-APC/eFluor 780 (30-F11) (Rat monoclonal; 1:200)eBioscienceRRID:AB_1548781; Cat # 47-0451-82
AntibodyCD11b Monoclonal Antibody (M1/70), PE-Cyanine7, eBioscience(TM), Thermo Fisher Scientific; CD11b-PE/Cy7 (M1/70) (Rat monoclonal; 1:200)BD BiosciencesRRID:AB_469588; Cat # 25-0112-82
AntibodyPE-Cyanine7 Anti-Human/Mouse CD45R (B220) (RA3-6B2) Antibody, Tonbo Biosciences; B220-PE/Cy7 (RA3-6B2) (Rat monoclonal; 1:200)Tonbo BiosciencesRRID:AB_2621849; Cat # 60–0452
AntibodyCD11c Monoclonal Antibody (N418), PE-Cyanine7, eBioscience(TM), Thermo Fisher Scientific; CD11c-PE/Cy7 (N418) (Armenian Hamster monoclonal; 1:200)eBioscienceRRID:AB_469590; Cat # 25-0114-82
AntibodyCD3e Monoclonal Antibody (145–2 C11), FITC, eBioscience(TM), Thermo Fisher Scientific; CD3-FITC (145–2 C11) (Armenian Hamster monoclonal; 1:200)eBioscienceRRID:AB_464882; Cat # 11-0031-82
AntibodyBrilliant Violet 785 anti-mouse CD8a antibody, BioLegend; CD8-BV785 (53–6.7) (Rat monoclonal; 1:200)BiolegendRRID:AB_1121880; Cat # 100749
AntibodyRat Anti-CD4 Monoclonal Antibody, Phycoerythrin Conjugated, Clone GK1.5, BD Biosciences; CD4-PE (GK1.5) (Rat monoclonal; 1:200)BD BiosciencesRRID:AB_395014; Cat # 553730
AntibodyAlexa Fluor 647 anti-mouse TCR γ/δ Antibody; gdTCR-AF647 (GL3) (Armenian Hamster monoclonal; 1:200)BiolegendRRID:AB_313826; Cat # 118133
AntibodyCD117 (c-Kit) Monoclonal Antibody (2B8), Biotin; c-Kit-Biotin (ACK2) (Rat monoclonal; 1:200)eBioscienceRRID:AB_466569; Cat # 13-1171-82
AntibodyFceR1 alpha Monoclonal Antibody (MAR-1), PE, eBioscience; FceRI-PE (MAR-1) (Armenian Hamster monoclonal; 1:200)eBioscienceRRID:AB_466028; Cat # 12-5898-82
AntibodyCD49b (Integrin alpha 2) Monoclonal Antibody (DX5), PE-Cyanine7, eBioscience;
CD49b-PE/Cy7 (DX5) (Rat monoclonal; 1:200)
eBioscienceRRID:AB_469667; Cat # 25-5971-82
AntibodyAnti-Siglec-F-APC, mouse (clone: REA798); SiglecF-APC; (Rat monoclonal; 1:200)Miltenyi BiotechRRID:AB_2653441; Cat # 130-112-333
OtherStreptavidin FITC; SA-FITCeBioscienceRRID:AB_11431787; Cat # 11-4317-87
AntibodyLy-6C Monoclonal Antibody (HK1.4), PerCP-Cyanine5.5, eBioscience; Ly6C-PerCP/Cy5.5 (HK1.4) (Rat monoclonal; 1:200)eBioscienceRRID:AB_1518762;
Cat # 45-5932-82
AntibodyvioletFluor 450 Anti-Human/Mouse CD11b (M1/70); CD11b-violet fluor 450 (M1/70) (Rat monoclonal; 1:200)Tonbo BiosciencesRRID:AB_2621936; Cat # 75–0112
AntibodyAF700 anti-mouse Ly-6G Antibody (1A8); Ly6G-AF700 (1A8) (Rat monoclonal; 1:200)BioLegendRRID:AB_1064045; Cat # 127621
AntibodyCD45.2 Monoclonal Antibody (104), APC-Cy7, eBioscience; CD45.2-APC/Cy7 (104) (Mouse monoclonal, 1:200)eBioscienceRRID:AB_1272175; Cat # 47-0454-82
Software, algorithmIgorPro version 6.3WaveMetricshttps://www.wavemetrics.com/order/order_igordownloads6.htm
Software, algorithmMicrosoft Excel 2011Microsofthttps://www.microsoft.com/en-us/store/d/excel-2016-for-mac/
Software, algorithmFIJINIHhttps://imagej.net/Fiji/Downloads
Software, algorithmGraphpad Prism 7Graphpadhttps://www.graphpad.com/scientific-software/prism/
Software, algorithmR-3.6.0The R Project for Statistical Computinghttps://cran.r-project.org/bin/macosx/
Software, algorithmPython 2.7Anacondahttps://www.anaconda.com/distribution/
Software, algorithmHTSeq 0.11.1Python Package Indexhttps://htseq.readthedocs.io/en/release_0.11.1/install.html
Software, algorithmTrimmomaticPMID: 24695404https://github.com/timflutre/trimmomatic
Software, algorithmTophat 2.1.1PMID: 19289445https://ccb.jhu.edu/software/tophat/
Software, algorithmEdgeRPMID: 19910308; PMID: 22287627https://bioconductor.org/packages/release/bioc/html/edgeR.html
Software, algorithmDESeqPMID: 20979621https://bioconductor.org/packages/release/bioc/html/DESeq.html
Software, algorithmFlowJo 10.4.2FlowJo; Treestarhttps://www.flowjo.com/solutions/flowjo/downloads
OtherBovine serum albumin, cold ethanol fraction, pH 5.2,≥96%; BSASigma-AldrichCAS 9048-46-8; Cat # A4503
OtherNGS; Goat serum; Normal goat serumAbcamCat # ab7481
Author response table 1
NeutrophilsBasophilsInfl. Monos.Mast Cells
INPUTDay 5Day 8Day 5Day 8Day 5Day 8Day 5Day 8
Effect size1.5180.8361.4331.0021.17110.82410.7361.0670
α error prob0.050.050.050.050.050.050.050.05
# groups22222222
sample size1478147814781478
OUTPUT
λ32.2654.61828.99102.3419.252.987.58331.54
Fcrit4.7473.9664.7473.9664.7473.9664.7473.966
Num. df11111111
Denom. df1276127612761276
Power0.99310.98610.9790.9990.7150.999

Additional files

Source data 1

The outputs of all DESeq differential expression analyses used to determine adjusted p value and log2 fold change for all RNA-seq experiments in the manuscript.

https://cdn.elifesciences.org/articles/48448/elife-48448-data1-v3.zip
Supplementary file 1

Number of mapped reads and sample information for all RNA-seq samples represented in the manuscript.

https://cdn.elifesciences.org/articles/48448/elife-48448-supp1-v3.csv
Supplementary file 2

Outputs of statistical tests performed on behavioral and flow cytometry data to determine whether select data sets could be combined.

https://cdn.elifesciences.org/articles/48448/elife-48448-supp2-v3.xlsx
Supplementary file 3

All flow cytometry data from Figures 12 represented as % of CD45+ cells.

https://cdn.elifesciences.org/articles/48448/elife-48448-supp3-v3.xlsx
Transparent reporting form
https://cdn.elifesciences.org/articles/48448/elife-48448-transrepform-v3.pdf

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)