Live Imaging of the Skin in 3D Prof. Kenji Kabashima · Ag presentation MemoryT cells (Th1/Tc1...

Live Imaging of the Skin in 3D Prof. Kenji Kabashima

1The screen versions of these slides have full details of copyright and acknowledgements

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Prof. Kenji Kabashima MD PhDAssociate Professor

Department of DermatologyKyoto University Graduate School of Medicine

Kyoto, Japan

Skin Immune StatusLive Imaging of the Skin in 3D

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Contents

1. Immune response in the skin

2. Live imaging of the skin in 3D

3. Immune response to haptens

4. Immune response to protein antigens

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Skin as an immune organ

Epidermis

Dermis

Immunohistochemistry (S100)

1,000 Langerhans cells/mm2



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External stimuli to the skin

• Physical stress

• Dryness

• Ultraviolet light exposure

• Bacteria, fungus, virus, parasites

• Haptens, metals, chemicals

• Protein antigens

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Atopic dermatitis(Mite, dust, pollen etc.)

Variety of immune responses to antigens

Urticaria(Egg, fish etc.)

Contact dermatitis(metal, urushiol etc.)

Psoriasis vulgaris(Self antigen/DNA?)

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Th2

Atopic dermatitis, Urticaria

IL‐4IL‐5IL‐13IL‐25

CD4+ T cell subsets in skin diseases

Th17

Psoriasis

IL‐17A/FIL‐21IL‐22

Treg

Immune suppression,Tolerance

TGF‐βIL‐10IL‐35

Th1

Contact dermatitis

IFN‐γ

Naïve CD4 T cells



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How are the varietyof immune responses induced?

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Histology: 2D Cell culture

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Skin: 3D

Shimizu’s Textbook of Dermatology



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Contents





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Two photon microscopy

Twophoton

Singlephoton

by Brad Amos MRC, Cambridge

Excitation with long pulse laser

(800‐1,000 nm)

500 μm in depth

Pinpoint excitation

Low phototoxicity

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Langerhans cells in 2D



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Langerhans cells in 3D

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3D imaging of the skinBM‐derived cellsBlood vesselsCollagen fibers

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Mt. Fuji at the sunrise or sunset?

Mt. Fuji at the sunset



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Visualization of the skin with 3D + time axis

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Langerhans cell mobility

Steady states Inflammation

Langerin‐EGFP mice (kindly provided by Dr. Malissen)Red: isolectin for keratinocyte

18 Langerin‐EGFP mice

Langerhans cell migration from the epidermis

LCs in the dermis

Epidermis

Dermis



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Contents





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Irritation dermatitis• Ag‐independent: cement, diaper, etc. in human/PMA, croton oil in mice.

Mediated by mast cells etc. (not by T cells)

Allergic contact dermatitis• Ag‐specific: metals (ex. Ni, Cr), haptens (ex. Urushiol), etc.

Mediated by Tc1/Th1‐> classic DTH

Contact dermatitis

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Visualization of irritation dermatitis

20h later

Label CD4 T cells: CFSE (green)CD8 T cells: TRITC (red)

Purify and stimulate CD4 and CD8 T cells with anti CD3 Abunder Th1 skewing condition

PMA

No treatment

Intravenous injection



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In the steady state

CD8+ T cells

CD4+ T cells

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PMA application

CD8+ T cells

CD4+ T cells

In irritation dermatitis

20 hr after PMALive imaging for 1 hr

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T cells migrate smoothly in the skin during irritation dermatitis

The mean velocity of T cells in the skin is about

6 μm/min

Egawa G et al., J. Invest. Dermatol. 2011 Apr; 131(4): 977‐9



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Allergic contact dermatitis

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Cutaneous DC migration

and maturation

Sensitization (~5d)

Schema of allergic contact dermatitis

Antigen (metals, haptens) exposure

KeratinocytesTNF‐αIL‐1α Cutaneous

dendritic cells (DCs)

Draining LNs Naïve T cells

Cutaneous DCsAg presentation

Memory T cells (Th1/Tc1 cells)

Elicitation (24~48 h)

Antigen re‐exposure

Effector T cell (Th1/Tc1) accumulation in the skin

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Contact hypersensitivity (CHS) model

Day 0: sensitizationHapten (such as DNFB) on the abdomen

Day 5: elicitationHapten (such as DNFB) on the ears‐> Measure ear thickness change

Mouse CHS = human contact dermatitis



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Single hapten elicitation induces CHS (Th1/Tc1 mediated DTH responses)

• Evaluating the extant of skin inflammation can be achieved

by measuring ear swelling

• Peak response is around 12‐24 hours after elicitation,

with IFN‐γ production in the skin

Kitagaki H. et al., J. Immunol. 159: 2484, 1997

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CHS model

With DNFB

ElicitationSensitization

DNFB‐sensitized T cells (green)

TNCB‐sensitized T cells (red)

Adoptive transfer

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Live imaging of T cellsin the elicitation phase of CHS

DNFB‐T cells

TNCB‐T cells

Egawa and Kabashima et al.,J. Invest. Dermatol. (2011)

24 after challenge,movie for 60 min



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Cognate antigen‐dependent T cell mobility in the elicitation of CHS

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DNFB‐T cell

CD11c+ dendritic cells

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Day 5: elicitationHaptenon the ears

Repeated application of hapten on the ears 3 times per wk for 4 wk

Kitagaki H. JID/JI 1995,1997

Chronic CHS model

Day 0: sensitizationHaptenon the abdomen



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Repeated hapten elicitation induces atopic dermatitis‐like skin lesions (ITH, Th2)

• In contrast to single elicitation, chronic hapten exposure

induced immediate type hypersensitivity

• Th1 cytokine IFN‐γ was not detected in the skin,but Th2 cytokine IL‐4 was detected

• Elevated serum IgE

• Eosinophil infiltration in the skin

→ Atopic dermatitis‐like skin lesions

→ Hapten‐Atopy Hypothesis


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What is the mechanismof shifting from Th1 to Th2?

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Accumulation of basophils in LNs

Otsuka A. et al., Nat Commun. 2013

Basophils: CD49b+ FcεRI+ IgE+ CD200R+ c‐Kit‐



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Mast cells Basophils

• Express the αβγ2 form of FcεRI on their surface

• Secrete chemical mediators upon crosslinking of IgE with antigens

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Basophils in LNs express MHC class II

Otsuka A. et al., Nat Commun. 2013

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Basophils exhibit low phagocytotic activity but express IL‐4

DQ‐OVA shows fluorescenceafter phagocytosis



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Th2 shifting is impaired by depletion of basophils using Bas TRECK mice

Otsuka A. et al., Nat. Commun. 2013

Th2‐type Ig

Th1‐type Ig

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Basophil

Naïve T

MHC class II IL‐4

Th2 skewing → Atopic dermatitis‐like skin lesion

IL‐4

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Alopecia areata

• 2% life time occurrence

• Treatment: Topical/systemic steroid



43 SADBE (squaric acid dibutylester) treatment: chronic CHS

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Repeated hapten application‐induced Th2

• IL‐10, which is a suppressive cytokine produced

by regulatory T‐cells, is detected by hapten exposure, especially by repeated hapten exposure

• This finding suggests that regulatory T‐cells accumulating into the skin may modulate the cutaneous immune responses


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Tregs localize in skin in the steady states

Human: Dr. Kupper et al. reported that 10‐20% of T cells in human skin are Tregs (JI 2006)

Collaboration with Dr. Shohei Hori at RIKEN



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Number of Tregs increases under cutaneous immune responses

Tomura et al., JCI 2010Honda et al., JACI 2010

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Role of Tregs in CHS

Foxp3‐hCD52 mouse: Treg depletion with anti‐hCD52 neutralizing Ab

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Tregs terminate CHS

→ Deplete Tregs

Tomura et al., J. Clin. Invest. 2010



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Contact dermatitis (DTH); Th1

Single hapten exposure

Diversity of immune responses to hapten

Repeated exposure

Induce atopic dermatitis (ITH); Th2

By basophils

Terminate DTH

By Tregs

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Contents





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Epi‐dermis

Hapten (FITC)

Dermis

Protein Ag (FITC‐OVA)

Dermis

FITC‐OVAClaudin‐1DAPI

Tight junction

Epidermis

<1,000 Da (MW)Metal (Ni, Cr, Co), Urushiol, Preservatives, Most drugs

5,000‐150,000 Da (MW)Protein: Mite, House dust, Pollen, Animal hair

Kaplan DH. Immunity 2005Kissenpfenig Immunity 2005

Honda T. JACI 2010

Langerhans cells are dispensable for CHS



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Cutaneous dendritic cells

Epidermis

Dermis

Dermal dendritic cells

Immunohistochemistry: S100

Langerhans cells

1,000 LCs/mm2

53 Kubo et al., 2009 J .Exp. Med. 206: 2937‐46

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Keratinocytes modulate function of Langerhans cells

1. Allergens

IL‐1αIL‐6TNF‐αPGE2TSLP

etc…

2. Keratinocyte release

Epidermis

LC

Dermis dDC



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• TSLP (thymic stromal lymphopoietin) → Th2 induction (atopic dermatitis)

• TSLPR expression: dendritic cells, mast cells, T cells, basophils

TSLP and Th2 induction

1. Allergens


etc…


Epidermis

LC

Dermis dDC

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Normal skin

AD

Soumelis et al., Nat Immunol 2002 2: 673‐80

TSLP Langerin

TSLP Langerin

• TSLP is induced by scratch/protein antigen with protease activityMoniaga CS, Kabashima et al., Am. J. Pathol. 2013 182: 841‐851


etc…


TSLP and Th2 induction (2)

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Symptoms of AD are attenuated by LC depletion

LCs: + ‐ + Patch: OVA OVA Saline

Nakajima S. et al., JACI 2012. 129: 1048‐55

Saline OVA

Clinical score

Saline OVA

Specific IgElevel



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TSLPRs on LCs are essential for IgE induction

Specific IgElevel

Clinical score

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Langerhans cells

Protein antigen exposureScratch

TSLP TSLP receptor

Keratinocytes

Draining LNsNaïve T cells

Th2 induction

IgE induction

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Two types of atopic dermatitis

Tokura Y. J Dermatol Sci 2010; 58: 1–7Kabashima K. J Dermatol Sci 2013; 70: 3‐11



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Dermal DCs

Small molecules

Metal

Th1

Intrinsic AD

Dermal DCs

Langerhans cells

Draining LNs

Proteins

Th2

Extrinsic AD

Large molecules

TSLP‐TSLP receptor

Haptens

Th1

AD‐like dermatitis (Extrinsic/Intrinsic?)

Th2

BasoChronic exposure

Kabashima K. J Dermatol Sci 2013; 70: 3‐11

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Contents

• Immune response in the skin

• Live imaging of the skin in 3D

• Immune response to haptens

• Immune response to protein antigens

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Dept. Dermatology, Kyoto Univ.• All lab members• Yoshiki Miyachi

AK project, Kyoto Univ.• Satoshi Matsuoka• Michio Tomura• Takeshi Watanabe

Dept. of Pharmacology, Kyoto Univ.• Shuh Narumiya

Dept. of Physiology, Kyoto Univ.• Kazuhiro Iwai

Dept. of Microbiology, Kyoto Univ.• Masao Mitsuyama, Shunsuke Sakai

Kyoto University Graduate School of Biostudies • Kayo Inaba

Tokyo Univ.• Susumu Nakae, Makoto Arita

RIKEN・RCAI• Masato Kubo, Takaharu Okada

Dept of Dermatology, UOEH• All lab members

Dept of Dermatology, Hamamatsu Univ.• Yoshiki Tokura, Junichi Sakabe

Kumamoto Univ.• Yukihiko Sugimoto

INSERM CIML• Bernard Malissen, Sandrine Henri

University of Minnesota Dept. Immunol.• Daniel Kaplan, Botond Igyarto

Keio Univ.• Masa Amagai, Keisuke Nagao, Hiroshii Kawasaki• Jun Kudoh

UCSF Dept. of Immunol.• Jason Cyster, Chris Allen

Chang Gung University in Taiwan• WenHung Chung

Osaka Univ. IFREC• Masaru Ishi

Tokyo Technical Univ.• Tamio Sakamot

Tokyo Metropolitan Institute of Medical Sciences• Makoto Murakami, Tetsuya Hirabayashi

Rockefeller University, Mount Sinai University• James Krueger, Emma Guttman‐Yassky

Acknowledgements

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Live Imaging of the Skin in 3D Prof. Kenji Kabashima · Ag presentation MemoryT cells (Th1/Tc1...

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