A novel Siglec-F+ neutrophil subset in the mouse nasal mucosa exhibits an activated phenotype and is increased in an allergic rhinitis model.

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Figure legends

Fig. 1.

Two neutrophil subsets are observed in the nasal mucosa. (A) Expression of Ly-6G,

Siglec-F, and CD11c in CD45+ cells in the nasal mucosa from PBS- or OVA-challenged mice.

Gate I, SSChiLy-6GloSiglec-F+CD11clo cells (eosinophils); gate II, Ly-6GhiSiglec-F−CD11c−

cells (conventional Siglec-F− neutrophils); and gate III, Ly-6GhiSiglec-F+CD11c+ cells

(Siglec-F+ neutrophils). (B) May–Grünwald–Giemsa staining of sorted cells in gates I, II, and

III shown in (A). Scale bar, 10 µm. (C) Expression of Ly-6G, Siglec-F, and CD11c in CD45+

cells in the blood from PBS- or OVA-challenged mice. (D) May–Grünwald–Giemsa staining

of sorted cells in gates I and II shown in (C). Scale bar, 10 µm. (E) Expression of Siglec-F and

CD11c in Ly-6Ghi neutrophils in the NALT, CLN, and spleen from PBS- or OVA-challenged

mice. (F) The number of eosinophils (I), Siglec-F− neutrophils (II), and Siglec-F+ neutrophils

(III) in the nasal mucosa from PBS- or OVA-challenged mice. Data (mean ± SEM) represent

the results from three mice per group. *p < 0.05, **p < 0.01.

Fig. 2.

Siglec-F+ neutrophils are located in the nasal lamina propria. Immunofluorescence

staining of nasal mucosa sections (A) and NALT and spleen sections (B) from PBS- or

OVA-challenged mice. Frozen sections were stained for Ly-6G (red), Siglec-F (green), and

DAPI (blue). Arrowheads indicate cells co-expressing Ly-6G and Siglec-F. Squares in the

images on the left in each panel correspond to the images on the right. Scale bars, 50 µm (left

images) and 10 µm (right images).

Fig. 3.

Siglec-F+ neutrophils exhibit an activated phenotype. (A) Expression of

16

representative adhesion molecules in eosinophils (I), Siglec-F− neutrophils (II), and Siglec-F+

neutrophils (III) in the nasal mucosa from PBS- or OVA-challenged mice. Representative

staining results with mAbs to indicated adhesion molecules (open graphs) and isotype

controls (shaded graphs) are shown. (B) Quantitative analysis of expression of representative

adhesion molecules. Data (mean ± SEM) are expressed as the mean fluorescence intensity

(MFI) and represent the results from three mice per group. *p < 0.05, **p < 0.01, ***p <

0.001. (C) Expression of representative chemokine receptors in eosinophils (I), Siglec-F−

neutrophils (II), and Siglec-F+ neutrophils (III) in the nasal mucosa from PBS- or

OVA-challenged mice. Representative staining results with mAbs to indicated chemokine

receptors (open graphs) and isotype controls (shaded graphs) are shown.

Fig. 4.

Siglec-F+ neutrophils exhibit enhanced effector functions. (A) ROS production in

eosinophils (I), Siglec-F− neutrophils (II), and Siglec-F+ neutrophils (III) in the nasal mucosa

from OVA-challenged mice. Data (mean ± SEM) are expressed as the MFI increase calculated

by subtracting the average of MFI of unstimulated cells from the MFI of LPS-stimulated cells

and represent the results from four mice. (B) Phagocytic activity of eosinophils (I), Siglec-F−

neutrophils (II), and Siglec-F+ neutrophils (III) in the nasal mucosa from PBS- or

OVA-challenged mice. Representative flow cytometry histograms of E. coli uptake are shown.

(C) Quantitative analysis of phagocytic activity. Data (mean ± SEM) are expressed as the MFI

and represent the results from three mice per group. *p < 0.05. (D) Expression of Siglec-F,

CD11c, and CD54 in Siglec-F– neutrophils stimulated with or without LPS. Data (mean ±

SEM) are expressed as the MFI and represent the results from three mice per group. *p < 0.05,

**p < 0.01, ***p < 0.001.

17

Figure 1

Nasal mucosa

SSC

II

60.2%

CD11c

III

0.06%

PBS

8.6%

70.4%

II

95.5%

3.4%

Siglec-F

OVA

Ly-6G

5.5%

III

0.9%

3.8%

SSC

74.4%

II

91.0%

CD11c

NALT

PBS

III

2.6%

CLN

Spleen

III

4.0%

III

0.05%

II

94.7%

II

75.7%

OVA

Siglec-F

III

0.0%

II

93.4%

III

1.8%

II

85.1%

III

0.11%

II

82.5%

II

93.2%

CD11c

Nasal mucosa

Cell number

400000

II

OVA

98.5%

Blood

III

20.6%

Siglec-F

OVA

Ly-6G

52.3%

II

PBS

II

73.6%

7.0%

**

II

200000

III

20000

300000

150000

15000

200000

100000

10000

100000

50000

5000

PBS OVA

PBS

PBS

75.9%

12.3%

III

14.2%

27.3%

PBS OVA

PBS OVA

OVA

III

Ly-6G/Siglec-F

DIC

Ly-6G/Siglec-F

Ly-6G/Siglec-F

NALT

DIC

DIC

PBS

Ly-6G/Siglec-F

Ly-6G/Siglec-F

DIC

Nasal mucosa

DIC

OVA

Ly-6G/Siglec-F

PBS

DIC

OVA

Figure 2

Spleen

Figure 3

CD11a

PBS

CD11b

OVA

PBS

CD18

OVA

PBS

OVA

PBS

CD54

OVA

PBS

OVA

CD54

***

***

***

II

III

PBS

CD62L

CD29

CD49d

PBS

OVA

OVA

II

III

CD11a

MFI

20000

CD11b

***

CD18

40000

15000

30000

10000

20000

5000

10000

10000

***

1500

8000

1000

6000

4000

II

III

CD49d

MFI

3000

**

6000

500

2000

II

III

CD29

II

III

II

III

CD62L

4000

PBS

OVA

***

3000

4000

2000

2000

2000

1000

1000

II

III

CCR1

PBS

II

III

OVA

II

III

CCR2

PBS

II

III

CCR3

OVA

PBS

OVA

CXCR2

PBS

OVA

Figure 4

3000

MFI

2000

1000

-1000

-2000

II

III

37ºC

PBS

On ice

OVA

PBS

OVA

II

III

MFI

40000

30000

20000

20000

PBS

OVA

15000

10000

5000

II

Siglec-F

1500

on ice

37ºC

on ice

37ºC

37ºC

on ice

III

CD11c

800

CD54

2500

**

2000

400

MFI

1000

MFI

MFI

600

1500

1000

500

200

500

***

LPS−

LPS+

Supplementary Figure

Fig. S1. Siglec-F+ neutrophils are barely observed in the small intestine. Expression of

Ly-6G, Siglec-F, and CD11c in CD45+ cells in the small intestine from PBS- or

OVA-challenged mice is shown.

...