1

Mouse Models of Inflammatory Lung Diseases

Zhou Zhu, MD, Ph.D.

Division of Allergy and Clinical Immunology Johns Hopkins University School of Medicine

Outlines

• Basic lung structure, function, and inflammatory lung diseases

• Methods, techniques, and approaches to studying lung diseases

• Mouse models of inflammatory lung diseases

From Wikipedia

Lung: Basic Structure

Anatomy

2

Lung: Basic Structure

H&E (100x)Lung Histology

AirwaysBloodVesselAlveoli

Lung Function and Diseases

• Gas exchange: O2 from atmosphere to bloodstream and CO2

from bloodstream to atmosphere

• Diseases• COPD• Asthma• Pneumonia• Pulmonary fibrosis• Sarcoidosis• Cystic fibrosis• Lung cancer• …

Studying the Lung: Methods and Techniques

• Pulmonary Function Test (PFT)• Airway resistance• Lung compliance

• Bronchoalveolar Lavage (BAL)• Inflammatory cells in the airway: total and differential• Mediators: cytokines, chemokines

• Lung tissues • Histology: H&E, Alcian Blue, PAS, Trichrome• Proteins• RNA• Immunohistochemistry (IHC)• FACS analysis of cell populations• Culture of specific cell types

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Studying the Lung: Approaches

• Stimulation• Allergens• Cigarette smoke

• Transgenics• Inflammatory cytokines

• Knock-out & knock-in • Cytokines & receptors• Siglecs

• Mutant strains• Phosphatases

Day 0 Day 18

Immunization with OVAi.p. injection

50 µµµµg OVA / 1 mg alum in 200 µµµµL

Day 7

PFT & Sacrifice

Day 14 Day 15

Intranasal50 µg OVA

Day 16

Allergen Induced Allergic Asthma

Protocol for ovalbumin (OVA) induced allergic asthma

OVA Induced Airway Inflammation

PBS OVA

BAL Cells

Macrophages Eosinophils

4

OVA Induced Airway Inflammation

PBS/WT

OVA/WT

0

10

20

30

40

BA

L To

tal c

ell n

umbe

r (X

104 )

Mac Neu EosLym

0

20

40

60

80

100

120 PBS/WT

OVA/WTce

ll d

iffer

entia

tio

n(%

)

Total Cell Counts Differential Cell Counts

OVA Induced Lung Inflammation

PBS OVA

H&E (100x)

OVA Induced Lung Inflammation

PBS OVA

H&E (400x)

5

OVA Induced Lung Inflammation

PBS OVA

Alcian Blue (100x)

OVA Induced Lung Inflammation

PBS OVA

Alcian Blue (400x)

OVA Induced Lung Inflammation

PBS OVA

Trichrome (100x)

6

OVA Induced Lung Inflammation

PBS OVA

Trichrome (400x)

Elastase Induced Emphysema

Protocol for elastase induced emphysema

Day 0

Porcine Pancreatic Elastase

1u/mouse, I.T. once

28

Sacrifice &

Collect BAL, lung tissue

1 2 3 7 14 21

Elastase Induced Airway Inflammation

PBS

BAL Cells

Day 2 Day 21

7

Elastase Induced Emphysema

Lung Histology (H&E 20x)

Day 3 Day 21

SHP-1

Src Homology 2 domain-containing protein tyrosinePhosphatase-1

Encoded by thehcp genePrimarily expressed in hematopoietic-derived cellsNegative regulator of a variety of growth factor/cytokinesignaling pathwaysActivated SHP-1→ binds to its target proteins→ dephos-phorylates the phospho-tyrosine residues→ terminates thesignaling

SHP-1 Regulation of Signaling

Inactive

SH2

SH2 N

C

SH2

SH2

N

C

ITIM

ITIM

Subs

Active

P

P Subs

8

SHP-1 Deficiency in Mice

� Motheaten mice (me/me) – a natural mutant strain

� Viable motheaten mice (mev/mev) – another spontaneous mutant strain

⇒ The mutations cause aberrant splicing of the SHP-1 transcript, resulting in diminished production of functional SHP-1 protein

� Phenotypeinflammatory lesions of the skin, increased neutrophils in the peripheral blood, glomerularnephritis, abnormal lymphoid tissues, pneumonitis with macrophage infiltration in the alveoli, increased production of immunoglobulins, depressed immune responses, profound NK-cell deficiency, and a shortened life span (average 22 days and 61 days, respectively)

IL-1

3Rα1

α1

α1α1

IL-4

Rαα αα

IL-4/IL-13

Tyk-2

IL-4/IL-13Responsive GenesS

TAT-

6

IRS-1/2Cell Growth& Survival

Jak-1

γγ γγc

IL-4

Rαα αα

IL-4

PI3K

IL-4/IL-13 Signaling Systems

Type I Type II

SHP-1SHIP-1SOCS

Time (min): 0 3 5 10 30 5 10 30

TNF-αααα IL-13

p- SHP-1

SHP-1

A B C

IHC: Control IgG αααα-SHP-1 αααα-SHP-1

MLE12

Mouse Lung

SHP-1 Expression and Activation in Lung Epithelial Cells

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Hypothesis

SHP-1 is an important regulator of • Immunological homeostasis of the lung• Allergic inflammatory responses

SHP-1 Deficiency and Phenotype

mev/+: Phenotypically Normalmev/mev: Inflammatory phenotype

Pulmonary Inflammation

0

20

40

60

80

100

Total Mac Eos Lym Neu

WT

mev*

*

*

* *

WT mev

10x 10x

40x 100x

mev mev

10

Mucus Hyperproduction

PAS Alcian Blue

WT

mev

A. B.

MUC5AC

WT mev

Immunoblot

RT-PCR

ββββ -Actin

MUC5AC

WT mev

Pulmonary Fibrosis

WT mev

WT mev

TGF-ββββ1ββββ-Actin

Trichrome RT-PCR

*

*

0

50

100

150

200

250

300

350

400

450

Total Activated

BA

L T

GF

- ββ ββ1

(pg/

ml) WT

mev

ELISA

RL

(cm

H2O

/ml/s

)

0

2

4

6

8

10

12

14

0 2 4 6 8 10 12 14

* * **

WT (n=4)

mev (n=9)

Methacholine (mg/ml)(FlexiVent System)

Airway Resistance and Hyperresponsiveness

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Upregulation of Th2 Cytokines

WT

meV

0

10

20

30

40

50

60

70

IL-4 IL-5 IL-13 IFN-γγγγ

**

*

WT

ββββ -Actin

IL-13

IL-4

IFN- γγγγ

IL-5

mevRT-PCR ELISA

mev/STAT6KOmev/IL-13KOmev/IL-4KO

Role of Th2 Cytokines in Pulmonary Inflammation

Macrophage Eosinophil Neutrophil Lymphocyte

0

10

20

30

40

50

60

70

80

90

SHP-1 +/+ +/+ -/- -/-IL-13 +/+ -/- +/+ -/-

*

* * *

Diff

eren

tial x

104

0

20

40

60

80

100

120

SHP-1 +/+ +/+ -/- -/-IL-13 +/+ -/- +/+ -/-

*

Tot

al C

ells

x10

4

mev/IL-13KO

IL-13KO

mev

WT

H&E BAL Cellularity

0

30

60

90

120

150

180

Macrophage Eosinophi l Neutrophi l Lymphocyte

SHP-1 +/+ +/+ -/- -/-STAT-6 +/+ -/- +/+ -/-

Diff

ere

ntia

l x10

4

* * *

SHP-1 +/+ +/+ -/- -/-STAT-6 +/+ -/- +/+ -/-

Tot

al C

ells

x10

4

0

50

100

150

200

250

300

*

mev/STAT6KO

STAT6KO

mev

WT

H&E BAL Cellularity

mev/STAT6KOmev/IL-13KO

Increased Mast Cells in the Lung

x100 x100

WT mev

12

Histamine Content and Release

Total Spontaneous

His

tam

ine

(ng/

mL

)

0.0

0.1

0.2

0.3

0.4

0.5WT

mev

*

*

0.0

0.1

0.2

0.3

0.4

0.5

Total SpontaneousPMA/Ionomycin

His

tam

ine

(ng/

mL

)

*

*

*

Spleen(1x106 cells)

Lung(1x106 cells)

Cytokine Production by Splenocytes

IL-13

0

5

10

15

20

25

30

35

WT mev

(pg

/m

L)

0

10

20

30

40

50

60

70

80

90

100

WT mev

IL-4

0

50

100

150

200

250

300

350

400

WT mev

MIP-1αααα

0

20

40

60

80

100

120

WT mev

IP-10

0

1

2

3

4

5

6

7

8

9

10

WT mev

IFN-γγγγ

0

20

40

60

80

100

120

140

WT mev

IL-12

(pg

/m

L)

Medium

Anti-FcεεεεRIαααα

Medium

Anti-FcεεεεRIαααα

Hexosaminidase Content and Release by BMMC

% Hexosaminidase Released from BMMC

0

50

100

150

200

250

300

350

400

Total Blank PMA/ Ionomycin

Hex

osa

min

idas

e (%

) WT

mev

13

C57BL/6 mev

KW-sh mev x KW-sh

Mast Cells and Lung Inflammation

Crossbreeding to mast cell deficient Kw-sh mice

mev mice:• Spontaneous and progressive pulmonary inflammation

rich in macrophages, lymphocytes, and eosinophils

↑ Th2 cytokines IL-4, IL-5, and IL-13

↑ Th2 chemokines Eotaxin and MCP-1

• Mucus hyperplasia ↑ Mucin genes

• Subepithelial and parenchymal fibrosis ↑ TGF-β

• Increased airway resistance and AHR

• Mast cell-dependent

Summary