NOD2 dependent neutrophil recruitment is required ...

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post intradermal injection with L3 larvae. As control group served C57BL/6 mice injected with PBS. Three mice per group. Lta. 2,2325. 0,2656. 0,3326. Map3k7.
NOD2 dependent neutrophil recruitment is required for early protective immune responses against infectious Litomosoides sigmodontis L3 larvae

Jesuthas Ajendra1,*, Sabine Specht1,*, Sebastian Ziewer1, Andrea Schiefer1, Kenneth Pfarr1,3, Marijo Parčina1, Thomas A. Kufer2, Achim Hoerauf1, 3, #, Marc P. Hübner1, # 1

Institute of Medical Microbiology, Immunology and Parasitology, University Hospital of Bonn, Bonn, Germany 2 Institute of Nutritional Medicine, University Hohenheim, Stuttgart, Germany 3 German Center for Infection Research (DZIF), partner site Bonn-Cologne, Bonn, Germany

*, #contributed equally

a

b

20µm

c

d

Suppl. Figure 1. NOD2-/- mice have reduced neutrophil elastase activity in the skin tissue after intradermal L3 injection. Representative histological sections of skin tissue 3h post (a) PBS treatment and (b) L3 injection in WT animals as well as (c) PBS treated and (d) L3 injected Nod2-/animals. Arrows in (b) indicate neutrophils with high elastase activity.

Siglec F

c

c-Kit

0.2

-/-

2 D

O

N

D

O

2

W

L3

S -/-

T

2 D O N

D O

ns

*

N

f

Granzyme B 0.10

PB

S

-/-

PB

S -/-

2

W

N

O N

e

IL-6 0.6

PB

L3 T

T

2

W

-/-

PB

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L3

0.00

N

D

O

2

D

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-/-

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PB

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L3

S PB T W

d

0.02

L3

0.0

0

0.04

L3

1

0.4

ns

*

T

2

iNOS 0.06

fold change

0.6

fold change

3

fold change

b

*

W

a

TSLP *

0.06

*

ns

0.2

fold change

fold change

fold change

0.08 0.4

0.06 0.04

0.04

0.02

0.02

L3

D O N

D 2 N O

2

-/-

-/-

PB

S

L3 T W

PB W

T

-/-

2 D O N

D 2 N O

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L3

S PB

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L3

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N O

D 2

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-/-

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S PB T W

0.00 L3

0.00 L3

0.0

Suppl. Figure 2. NOD2-/- mice have a reduced inflammatory response in the skin tissue after intradermal L3 injection. Gene expression analysis by qRT-PCR of skin tissue 3h post L3 injection in NOD2-/- and WT animals for (a) Siglec F, (b) c-Kit, (c) iNOS, (d) IL-6, (e) Granzyme B, and (f) TSLP. n=6-10 for TSLP, IL-6 and c-Kit; n=3-6 for Siglec F, iNOS and Granzyme B. Shown are means ±SD. Data was tested for statistical significance by Kruskal-Wallis test followed by Dunn‘s post test.

Neutrophils

F4/80 neg. c-Kit neg.

WT L3s

F4/80 neg. c-Kit neg.

F4/80 neg. c-Kit neg.

CD11b FITC

SSC

F4/80 APC

2.9% of total

Neutrophils

NOD2-/- L3s 1.0% of total

Neutrophils

WT PBS 0.9% of total

FSC

c-Kit PE

Gr1 PE-Cy7

Suppl. Figure 3. Gating strategy to identify Gr1+CD11b+ neutrophils from skin. The neutrophil population was identified based on FSC and SSC characteristics and determined to be c-Kit and F4/80 negative and Gr1 and CD11b positive. Shown are representative figures of samples from skin tissue of L3 or PBS injected WT and NOD2-/- mice.

Change in gene expression in comparison to PBS treated wildtype controls Wildtype L3 Nod2-/- PBS Nod2-/- L3 Fold change Fold change Fold change Arg1 Ascc1 C3 Casp1 Ccl2 Ccl5 Ccl8 Ccl11 Ccl17 Ccl22 Ccl24 Ccr2 Ccr3 Ccr4 Ccr5 Cd14 Cd69 Cd207 Chi3l3 Chia Cma1 Cpa3 Cxcl2 Cxcl12 Cxcr1 Cxcr3 Cxcr4 Elane Epx Fcer1a Il11ra1 Gzmb Hdc Hrh1 Hrh2 Hrh3 Hrh4 Ifnb1 Ifng Il1b Il4 Il6 Il9 Il10 Il12a Il17a Il18 Il21 Il25 Il33 Il6ra Itgam Kit

0,4682 0,9968 0,615 0,8228 0,2923 1,4326 0,391 1,426 2,4714 1,1663 0,6236 1,3122 16,1336 4,3429 3,0356 2,0591 2,5883 1,2822 5,7305 11,7289 0,6935 0,3839 4,2732 0,4481 9,727 9,8856 1,2822 2,3817 11,6749 3,8247 0,5996 1,7355 2,8128 5,5098 16,8967 2,8719 8,9507 38,4612 22,5543 4,58 3,447 1,7395 9,0338 10,4252 13,6611 9,3093 1,2187 1,6533 4,6118 2,5468 0,5342 0,8003 1,1009

0,677 0,6631 0,6495 0,7617 0,4158 0,6116 0,2626 1,1953 1,1256 0,588 0,6173 0,3144 5,4289 0,1789 0,9378 1,29 0,5538 0,9709 0,7868 1,2403 0,5746 0,0638 0,6631 0,3747 0,4888 0,7547 0,8145 0,8126 2,2826 0,4255 0,3339 0,113 1,1925 0,859 2,115 0,6361 2,1896 4,4097 3,2132 1,3293 0,651 0,6088 0,4689 1,1413 1,1387 2,7524 1,4784 0,2177 0,2775 2,4748 0,6616 0,4026 0,1814

0,7766 0,5244 0,9991 0,5982 0,2336 0,6108 1,4359 0,4984 0,4319 0,2452 0,465 0,5391 8,8275 0,6622 2,5704 27,3854 0,9991 0,295 5,6516 0,5699 1,539 0,1867 67,8997 0,6122 7,7382 9,1177 2,1765 0,0674 0,175 1,0177 0,3865 0,0753 11,8378 1,5213 3,3219 0,1408 0,2876 1,1556 1,2215 70,6198 6,2854 0,2856 0,4319 0,9518 1,6725 8,0855 1,3336 0,0365 1,8817 6,3731 0,6179 1,4866 0,221

Lta Map3k7 Ncf2 Ncf4 Nfkb1 Nfkb2 Ngp Nlrp1a Nlrp3 Nod1 Nod2 Nos1 Nos2 Nr2c2 Prg2 Prkcd Ptgs2 Retnlb Retnlg Ripk2 Sell Siglec5 Sod2 Stat6 Tgfb1 Tlr2 Tlr4 Tlr6 Tnf Tslp Vegfa IL5 Mmp9 CCL3 CCL4 Actb B2m Gapdh

2,2325 0,314 1,1371 1,2443 0,8059 0,6637 4,5483 11,6479 9,5931 0,7196 0,3786 3,0497 3,8691 0,8096 5,035 1,7597 4,7196 7,9926 2,5292 1,456 7,9742 7,8281 0,6164 0,8343 0,5927 3,9504 3,5439 3,6689 1,9616 1,5461 2,29 2,7044 1,9525 2,1665 2,0734 0,2574 0,6871 0,943

0,2656 0,2233 0,6786 1,0526 0,9844 0,5131 0,3072 1,0238 0,9913 0,1708 0,4395 0,1518 0,324 0,2801 0,4933 0,1712 0,2995 0,2737 0,1769 0,0252 4,5652 1,4083 0,0092 0,6245 0,018 0,6662 0,6259 0,1518 0,2228 0,9913 1,6943 0,099 1,9106 1,5881 2,2099 0,3362 1,0575 0,7307

0,3326 0,3206 2,29 2,0782 0,8059 0,4715 0,5148 2,5645 5,8916 0,1783 0,0731 0,1471 0,2267 0,5172 0,1563 1,575 3,9504 0,2401 46,3769 0,0887 15,0185 0,8247 0,0521 0,0807 0,3176 2,3598 1,3585 3,3142 1,886 0,9584 2,9053 0,0258 13,6927 11,4876 21,1903 0,3865 0,7163 0,8718

Suppl. Table 1. Complete list of genes tested in PCR array analyzing skin tissue three hours post intradermal injection with L3 larvae. As control group served C57BL/6 mice injected with PBS. Three mice per group.