Cell Host & Microbe, Volume 27 Supplemental Information Club Cell TRPV4 Serves as a Damage Sensor Driving Lung Allergic Inflammation Darin L. Wiesner, Richard M. Merkhofer, Carole Ober, Gregory C. Kujoth, Mengyao Niu, Nancy P. Keller, James E. Gern, Rebecca A. Brockman-Schneider, Michael D. Evans, Daniel J. Jackson, Thomas Warner, Nizar N. Jarjour, Stephane J. Esnault, Michael B. Feldman, Matthew Freeman, Hongmei Mou, Jatin M. Vyas, and Bruce S. Klein
Transcript
Cell Host & Microbe, Volume 27
Supplemental Information
Club Cell TRPV4 Serves as a Damage Sensor
Driving Lung Allergic Inflammation
Darin L. Wiesner, Richard M. Merkhofer, Carole Ober, Gregory C. Kujoth, MengyaoNiu, Nancy P. Keller, James E. Gern, Rebecca A. Brockman-Schneider, Michael D.Evans, Daniel J. Jackson, Thomas Warner, Nizar N. Jarjour, Stephane J.Esnault, Michael B. Feldman, Matthew Freeman, Hongmei Mou, Jatin M.Vyas, and Bruce S. Klein
INVENTORY OF SUPPLEMENTAL MATERIALS - Wiesner et. al.
Supplemental Figure 2. Multiplexing antibodies to identify 17 pulmonary leukocyte subsets with 12 color flow cytometry.
Supplemental Figure 3. CCR2, but not CCL2, is required for allergic inflammation.
Supplemental Figure 4. Protease activated receptors 1 and 2 do not detect Alp1.
Supplemental Figure 5. Alkaline protease 1 does not cause inflammation via release of C3a from Complement C3.
Supplemental Figure 6. Fungal protease does not interact with the coagulation pathway.
Supplemental Figure 7. Calcineurin and IKK2 are dispensable in type-2 alveolar cells, pulmonary neuroendocrine cells, and ciliated cells for the allergic response to protease.
2. Supplemental Experimental Procedures - none
3. Supplemental References - none
Ladd
er
rAlp1
Untran
sform
ed
A B
Prot
ease
Act
ivity
(Flu
ores
cenc
e U
nits
)
Protein (mg/mL)
Active Alp1
Alp1
Heat-inactive
Untransformed
C Recombinant Alp1
Sigma Proteinase
Protein (mg/mL)101.250.15
SigmaRecombinant
Protein (mg/mL)101.250.15
D
Prot
ease
Act
ivity
(Flu
ores
cenc
e U
nits
)
GAT
A3+
Th
Cel
ls (#
/Lun
gs)
MoD
C(#
/Lun
gs)
Eos
(#/L
ungs
)
NormalrAlp1
Sigma
NormalrAlp1
Sigma
NormalrAlp1
Sigma
E
Cel
ls (#
/Lun
gs)
F
GMMDMEM
Alp1
Af293 G
MM
Af293 D
MEM
CEA10 G
MM
CEA10 DMEM
GMMDMEM
Alp1
Af293 G
MM
Af293 D
MEM
CEA10 G
MM
CEA10 DMEM
GMMDMEM
Alp1
Af293 G
MM
Af293 D
MEM
CEA10 G
MM
CEA10 DMEM
MoDCGATA3+ Th Cells Eosinophils
Supplemental Figure 1 (relates to Fig 1). Recombinant Aspergillus alkaline protease 1. A) Coomassie stained gel of culture supernate from Pichia pastoris transformed with Alp1 or untransformed. rAlp1 is a ~33kDa protein. B) Protease activity assay (FITC-Casein) of titrations of concentrate of culture supernate from rAlp1 transformed (active or boiled) and untransformed P. pastoris. C) 2-fold serial dilutions of Sigma-Aldrich fungal proteinase or rAlp1 were separated on a polyacrylamide gel and stained with Comassie. D) A fluorometic enzyme activity assay of 2-fold serial dilutions of Sigma-Aldrich fungal proteinase and rAlp1. Blue arrows indicate the concentration of protease that was administered to mice in E). Cellular response a 15 dpi with Sigma-Aldrich proteinase or rAlp1. F) Cellular response to Alp1, culture filtrates, and media controls 7 dpi. Abbreviations: rAlp1 = recombinant alkaline protease 1. dpi = days post inoculation. These are representative data from two or more independent experiments.
ɣδ T cell
ILC Th cell
MAIT cell
CCR6
IL-3
3RCD8
CD4
TCRɣδ
TCRβ
CD19
CD90
.2
CD11c
CD64
MHCII
CD11
c
ILC ɣδ T
Th cell
CD8MAIT
B cell
Neu.
Alv. Mac.
Eos.
CD103 cDC
MoDC
CD11bcDC
Ly6G
Sigl
ecF
CD11b
CD10
3CD
64
CD11b
Ly6C
CD11b
NK1
.1
NK Cell
Monocyte
A BSeparateCombined
Leuk
ocyt
es (#
/Lun
gs)
C Naïve
Protease
Supplemental Figure 2 (relates to Fig 2). Multiplexing antibodies to identify 17 pulmonary leukocyte subsets with 12 color flow cytometry. A) Leukocytes harvested from lungs of mice 15 dpi with rAlp1 and stained with the following antibodies: FITC - Siglec F/TCRɣδ; PercpCy5.5 - B220; PE - CD64/IL-33Rα; PECy7 - CD11c/TCRβ; BUV395 - Ly6G/CD4; BV421 - CD103/NK1.1; BV510 - Ly6C; BV650 - CD11b/CD8; BV785 -CD90.2; APC -CCR6; AF700 – MHCII; APCe780 - Dead/IV. B) Comparison of lymphocytes and myeloid cells analyzed by two methods. C) Cellular response in control and protease treated mice 15 dpi. Abbreviations: Alv Mac = Alveolar macrophages, CCR = C-C chemokine receptor, cDC = conventional dendritic cell, Eos = Eosinophils, IL = Interleukin, ILC = Innate Lymphoid Cell, Neu = MAIT = Mucosal-associated invariant T cells, Mo-DC = monocyte-dendritic cells, NK = Natural killer, TCR = T cell receptor, Th = Helper T cell. These are representative data from two or more independent experiments.
Sigl
ecF
Liposome
CD11c
Clodrosome
Alv.
Mac
. (#/
Lung
)Lip
osom
eClod
rosom
e
C
Mo-
DC
(#/L
ungs
)WT (O
+P)
WT (Ova
)CCR2 K
O (O+P
)A
GAT
A3+
Th2
Cel
ls (#
/Lun
gs)
Eos
(#/L
ungs
)
WT (O+P
)
WT (Ova
)CCR2 K
O (O+P
)
WT (O+P
)
WT (Ova
)CCR2 K
O (O+P
)
* * *
*
Mo-
DC
(#/L
ungs
)Wild
Type
Naive
CCL2 KO
B
GAT
A3+
Th2
Cel
ls (#
/Lun
gs)
Eos
(#/L
ungs
)
Wild Typ
e
Naive
CCL2 KO
Wild Typ
e
Naive
CCL2 KO
* * *
D AcetylatedTubulin
SurfactantProtein C CGRP
Supplemental Figure 3 (relates to Fig 3). CCR2, but not CCL2, is required for allergic inflammation. Mo-DC, IL-33R+ Th cell, and eosinophil response to rAlp1 by A) CCR2-/- or B) CCL2-/- mice 15 dpi. C) Alveolar macrophages from the lungs of clodrosome- and control-treated mice. D) Confocal images of lung sections from mice 6 hours post-protease treatment. Green dye indicates CCL2 expression and red dye indicates epithelial cell subset (acetylated tubulin = ciliated cell; surfactant protein C = type-2 alveolar cell; CGRP = pulmonary neuroendocrine cell). 100X objective. Statistics: unpaired, non-parametric T test; * P < 0.05. Abbreviations: Alv. Mac. = Alveolar macrophage, CCL = C-C chemokine ligand, CCR = C-C chemokine receptor, CGRP = Calcitonin gene-related peptide, dpi = days post-inoculation, Eos = Eosinophils, IL = Interleukin, Mo-DC = monocyte-dendritic cells, Th = T Helper cell. These are representative data from two or more independent experiments.
C
B
Mass (M/z)
Arbi
trary
Uni
ts
ProteaseTrypsin
SLIGR RLETQPPITGK
Mouse PAR2 – GRNNSKGR38½SLIGRLETQPPITGKGVPV
NoEnzyme
LETQPPITGKIL
-33R
α+Th
2 C
ells
(#/L
ung)
Wild Typ
ePAR2 K
O
Naïve
Wild Typ
ePAR2 K
O
Naïve
Mo-
DC
(#/L
ung)
Wild Typ
ePAR2 K
O
Naïve
Mouse PAR1 – RTDATVNPR41½SFFLRNPSENTFELVPLGDEEA
Mass (M/z)
ProteaseThrombinNo EnzymeAr
bitra
ry U
nits
1
2
1345
IL-3
3Rα+
Th2
Cel
ls (#
/Lun
g)
Mo-
DC
(#/L
ung)
D
Wild Typ
ePAR1 K
O
Naïve
Wild Typ
ePAR1 K
O
Naïve
Wild Typ
ePAR1 K
O
Naïve
Eos
(#/L
ung)
Eos
(#/L
ung)
n.s. n.s. n.s.
n.s. n.s. n.s.
Supplemental Figure 4 (relates to Fig 4). Protease activated receptors 1 and 2 do not detect Alp1. A) A peptide surrogate for the cleavage domain of PAR1 was incubated with fungal protease or thrombin control, and the cleavage products were measured by mass spectrometry. 1=RTDATVNPR; 2=SFFLRNPSENTFELVPLGDEE; 3=LRNPSENTFELVP or RNPSENTFELVPL; 4=LRNPSENTFELVPL; 5=FLRNPSENTFELVPL. B) Same as A) with PAR2 peptide. Mo-DC, Th2 cells, and eosinophils from lung of naïve or protease-treated wild type and C) PAR1-/- or D) PAR2-/- mice. Statistics: unpaired, non-parametric T test; * P < 0.05. Abbreviations: dpi = days post-inoculation, Eos = Eosinophils, IL = Interleukin, Mo-DC = monocyte-dendritic cells, PAR = Protease activated receptor, Th = T Helper cell. These are representative data from two or more independent experiments.
B
D
IL-3
3Rα
Th2
Cel
ls (#
/Lun
g)
Eos
(#/L
ung)
Mo-
DC
(#/L
ung)
Wild Typ
e
Naïve
C3 KO
Wild Typ
e
Naïve
C3 KO
Wild Typ
e
Naïve
C3 KO
Mass (M/z)
C3 + ProteaseC3a
Arbi
trary
Uni
ts
C
C3C3a
C3 + P
~9 kDa
A
Merge
C3aRDAPI
Scgb1a1
9080 8931
n.s. n.s. n.s.
Supplemental Figure 5 (relates to Fig 4). Alkaline protease 1 does not cause inflammation via release of C3a from Complement C3. A) C3aR expression by club cells (Scgb1a1+) in naive mice. B) Western blot of purified C3a, C3, and C3 incubated with Alp1. C) MALDI-TOF analysis of B). D) Cellular immune response in the lungs of wild type or C3-/- mice 15 dpi. Statistics: unpaired, non-parametric T test; * P < 0.05. Abbreviations: C = complement, dpi = days post-inoculation, Eos = Eosinophils, IL = Interleukin, Mo-DC = monocyte-dendritic cells, Th = T Helper cell. These are representative data from two or more independent experiments.
E
Human Fibrinogen
Thrombin
Protease
C
D
Mass (M/z)
Arbi
trary
Uni
ts
ProteaseThrombinNo Enzyme
Fib AFib B
Eos
(#/L
ung)
Mo-
DC
(#/L
ung)
B
050
020
00
ProteaseThrombin
- - + +50
00-
Protea
se
Naïve
Protea
se +
Hirudin
(5U)
Eos
(#/L
ung)
Mo-
DC
(#/L
ung)
F + P
F only
F + P +
H
F + P
F only
F + P +
H
Th2
Cel
ls (#
/Lun
g)
MoD
C(#
/Lun
g)
Eos
(#/L
ung)
F
Naïve
MyD88
KO
Wild Typ
eTLR
2347
9 KO
Naïve
MyD88
KO
Wild Typ
eTLR
2347
9 KO
Naïve
MyD88
KO
Wild Typ
eTLR
2347
9 KO
A
Eos
(#/L
ung)
Mo-
DC
(#/L
ung)
ILC
2 (#
/Lun
g)Naïv
eMyD
88 KO
Wild Typ
e
n.s. n.s.
*n.s. n.s.
n.s. n.s. n.s.
n.s.n.s.
G
Mo-
DC
(#/L
ung)
Th2
Cel
ls (#
/Lun
g)
Eos
(#/L
ung) Alp1
PapainSubtilase
0d 3d 15d 0d 3d 15d 0d 3d 15d
Th2
Cel
ls (#
/Lun
g) PapainH
MoD
C(#
/Lun
g)Scg
b1a1
DTA
Naive
Wild Typ
e
n.s.
Scgb1
a1 DTA
Naive
Wild Typ
e
n.s.
Eos
(#/L
ung)
Scgb1
a1 DTA
Naive
Wild Typ
e
n.s. I Subtilase
MoD
C(#
/Lun
g)Scg
b1a1
DTA
Naive
Wild Typ
e
n.s.
Th2
Cel
ls (#
/Lun
g)Scg
b1a1
DTA
Naive
Wild Typ
e
n.s.
Eos
(#/L
ung)
Scgb1
a1 DTA
Naive
Wild Typ
e
n.s.
Supplemental Figure 6 (relates to Fig 4). Fungal protease does not interact with the coagulation pathway. A) Cellular response 3 dpi in mice treated with PBS, thrombin, protease, or thrombin + protease. B) Mo-DC and eosinophils from lungs of mice 3 days post treatment with PBS or protease in the presence and absence of a thrombin inhibitor (hirudin). C) Human fibrinogen incubated for 15 minutes with thrombin or rAlp1. Samples were tilted to assess clotting of the solution. D) Mass spectrometry analysis of cleavage products (fibrinopeptide A or B) from clotting assay in panel C. E) Mo-DC and Eosinophils from the lungs of mice treated with fibrinogen, fibrinogen + heat-inactivated protease, or fibrinogen + functional protease. F) Cellular response to rAlp1 15 dpi in the indicated knockout mice. G) Cellular response to rAlp1 (25� µg), papain (50� µg), or subtilase (12.5� µg). Cellur response 15 dpi with H) papain or I) subtilase in Scgb1a1-creERT2 x LSL-DTA mice. Statistics: unpaired, Mann-Whitney U test with Bonferroni adjustment for multiple comparisons; * P < 0.05. Abbreviations: dpi = days post-inoculation, Eos = Eosinophils, IL = Interleukin, ILC = Innate lymphoid cells, F+P+H = Fibrinogen + Protease + Heat-inactivation, Mo-DC = monocyte-dendritic cells, Th = T Helper cell, TLR = toll-like receptor. These are representative data from two or more independent experiments.
A
Mo-
DC
(#/L
ung)
No Cre
Naïve
Ascl1
Foxj1
Sftpc
BCNB1f/f IKK2f/f
Gat
a3+
Th2
Cel
ls (#
/Lun
g)Eo
sino
phils
(#
/Lun
g)
Mo-
DC
(#/L
ung)
Gat
a3+
Th2
Cel
ls (#
/Lun
g)Eo
sino
phils
(#
/Lun
g)
No Cre
Naïve
Ascl1
Foxj1
Sftpc
Protease Protease
Supplemental Figure 7 (relates to Fig 5). Calcineurin and IKK2 are dispensable in type-2 alveolar cells, pulmonary neuroendocrine cells, and ciliated cells for the allergic response to protease. Mice were challenged with rAlp1 and lungs harvested 15 dpi. The cellular immune respone was quantified in mice with epithelial subset-specific deletion of A) calcineurin or B) IKK2. Statistics: One-way ANOVA. Abbreviations: Ascl1 = Achaete-scute homolog 1, dpi = days post-inoculation, Foxj1 = forkhead box J1, Mo-DC = monocyte-dendritic cells, Scgb1a1 = Secretoglobin, Th = T Helper. Compiled data from at least two independent experiments.
