Department of Immunology

Fabienne Mackay

Immunology outside the square:Are immune functions influenced by otherbiological systems in the body such as the

neurological functions?

IMM3042

Developing Immunology of the future

Thinking outside of the box

The immune system

1- CNS

2- endocrine system

3- metabolism

4-TraditionalChinese medicine

5- Epigenetics (mirRNA,methylation)

6-cancers

Fabienne MackayDepartment of Immunology

Psychological stress and immunedefences

The adaptive immune response Antigen

APC such as a DC

T cells

AgLPS

“Mature” DC

RecirculationThrough lymphoid tissue

Th1Th2

•Cell-mediated immunity•Defense againstintracellular pathogens

•Allergy•Defense againstextracellular pathogens

BCD4

T

B cell follicleT cell area

Effector cells

BTc

The T helper paradigm

Th1cell

IL-12

IFNγ

IFNγ

NaïveCD4+

T cell

Autoimmunity DTH responses

Th2 cell

IL-4

IL-4

Allergy, asthma IL-4

Th17cell

TGF-βIL-23, IL-6

AutoimmunityInflammationIL-17

IL-12 and the regulation of Th1/Th2

T cellIL-12

IFNγGM-CSFTNFIL-8 Proliferation

Th2

Macrophages, neutrophils, DCs

Th1

B cell

IgE IgA

IFNγ IgG2a

An example of interaction between the neuronal and immunesystems

A fundamental bimodal role for neuropeptide Y1 receptor in the immune system and amodulator of cytokine production.

Stress

Neuropeptide Y

Immune cells

Neuropeptide Y

• Present in the peripheral, central and sympatheticnervous systems• Also been found in nonsympathetic neurons in severalorgans• Highly conserved• Family of hormones with peptide YY (PYY) andpancreatic polypeptide (PP)

NPY mRNA distribution in themouse brain

NPY

PYY

PP

Y P S K PD N P G E D A P A E D MA R Y Y S A L RH Y I N L I T RQ R Y

Y P I K P E A P G E D AS P E E L N R Y Y AS L RH Y L N L V T RQ R Y

A P L E P V Y P G D N A T P E QMAQ Y A AD L R R Y I N M L T R P R Y

Neuropeptide Y receptors

• 5 receptors: Y1, Y2, Y4, Y5, y6

• 7 transmembrane GPCRs of Rhodopsin-like superfamily

• NPY and PYY identical affinity for all Y receptors, PP prefers Y4

• Act in an inhibitory fashion, eg inhibition of cAMP accumulation

• NPY induces proliferation via Y1 / MAPK pathway

Seizure Protection

Heart RateNPY

Pain

Nerve Regeneration

Memory

Anxiety

Bone Homeostasis

Thermogenesis

Alcohol ToleranceMetabolic Rate

Blood Pressure

Aggression

Reproduction

Feeding Behavior

Circadian Rhythm

Immune System

Expression of NPY receptor Y1 by immune cellsY1 receptor expressionin leukocytes

Y1Rβ-tubulin

Human leukocytes, cell-sorted

brainCD4 CD8 NK B Mφ PMN

Y1R18S

Rat PBMCY1RGAPDH

Mouse lymph-node cells

brainLN T

Bedoui et al.

Mouse leukocytes, cell-sorted

+ spl B CD4 CD8 Mφ DC NK MC Y1-/-

MφWheway, 2005

Y1R

Phenotype of Y1-/- mice

⇔ Food intake

⇑ Body weight

⇑ Adiposity

⇑ Nociception (perception of pain)

⇑ Aggressive behaviour

⇑ Alcohol consumption

⇔Heart rate

⇑Fertility

⇑Neurogenesis

Immune responses?

Metabolic

Behavioural

Neurological

Physiological

Immune phenotype of Y1-/- mice

***

Basal serum antibody

0

5

10

15

Y1

WT

IgG1 IgG2a IgG2b IgG3 IgA IgM

Titre

(log

2)

**

0

5

10

15

Y1

WTWT Y1-/-

0

5

10

15

Y1

WT

• Smaller spleens•⇓ mature B cells in spleen,

mesenteric and peripheral LN andperitoneal lavage•Lymphocyte survival normal invitro

•T cell development in thymus

normal

JW281003PLN.020JW281003PLN.020

100

101

102

103

104

CD44

100

101

102

103

104

CD44

JW281003PLN.024JW281003PLN.024

100

101

102

103

104

CD44

100

101

102

103

104

CD44

JW281003PLN.020JW281003PLN.020

100

101

102

103

104

CD44

100

101

102

103

104

CD44

JW281003PLN.024JW281003PLN.024

100

101

102

103

104

CD44

100

101

102

103

104

CD44

0

500

1000

1500

CD4 CD8

Y1-/-

WT

0

50

100

150

200

CD4 CD8

Y1-/-

WT

Altered T cell phenotype in Y1-/- mice

Naive

Effector

Y1+/+ Y1-/-

CD44

CD62

L

CD4

CD8

51.5

12.4

80.8

3.5

23.6

10.0

65.5

2.6**

*

*

*

Y1+/+

Y1-/-

Y1+/+

Y1-/-

N.B. Percentage of total CD4 or CD8 shown

cells

(x10

3 )ce

lls (x

10 3 )

The Delayed-Type Hypersensitivity (DTH) protocol

• Classic T helper 1 (Th1) mediated model

•Day 0 •Day 7

Sensitisation(mBSA/CFA)

Footpad re-challenge(mBSA)

•Day 8 •Day 9

Measurement offootpad swelling

• Experimental protocol:

•Day 14

Culled for serumantibody ELISA

0

25

50

75

100

Column 5

Column 4

Y1

WT

Reduced Th1 responses in Y1-/- mice

0

2500

5000

7500

10000

Y1-/-

WT

Column 7

WT

DTH footpad swelling

WT Y1-/-

WT

Y1-/-

**

IL-4IFN-γ

WT Y1-/- WT

pg/m

l cyt

okin

e

*

Y1-/-

% in

crea

se in

sw

ellin

g

Conflicting observations

T cells T cells?Y1-/-Y1+/+

NPYOr Y1 agonist

Inhibition of Th1 responsesInhibition of EAE

Inhibition of Th1 responses

-How can we reconcile the idea that Y1 is a negative regulator of Th1 T cellfunction with the fact that Y1-/- mice are protected against Th1-mediateddisorders?

Others have shown:Bedoui et al. 2004

JI, Trends Immunol.Vol 10, pp 508-512

We are showing this:

A possible explanation

Antigen

APC such as a DC

T cells

AgLPS

“Mature” DC

RecirculationThrough lymphoid tissue

Th1Th2

•Cell-mediated immunity•Defense againstintracellular pathogens

•Allergy•Defense againstextracellular pathogens

BCD4

T

B cell follicleT cell area

Effector cells

BTc

Y1 signalling

+-

Step 1Step 2

Using Y1 agonists

Antigen

APC such as a DC

T cells

AgLPS

“Mature” DC

RecirculationThrough lymphoid tissue

Th1Th2

•Cell-mediated immunity•Defense againstintracellular pathogens

•Allergy•Defense againstextracellular pathogens

BCD4

T

B cell follicleT cell area

Effector cells

BTc

Y1 signalling

+-

Step 1Step 2

No Th1

Using Y1-/- mice

Antigen

APC such as a DC

T cells

AgLPS

“Mature” DC

RecirculationThrough lymphoid tissue

Th1Th2

•Cell-mediated immunity•Defense againstintracellular pathogens

•Allergy•Defense againstextracellular pathogens

BCD4

T

B cell follicleT cell area

Effector cells

BTc

Y1 signalling

+-

Step 1Step 2

No Th1

To prove this we had to test Y1-/- T cells and APCseparately

T cell responses in Y1-/- mice

0

50

100

150

200

250

0 2.5 5 7.5 10 12.5

11/4/05 aCD3 proliferation exp

Y1

WT

[3HT

]thym

idin

e up

take

(cpm

x10

3)

• Y1-/- T cells are hyper responsive to αCD3 stimulation and undergo more divisionin vitro

αCD3 response

αCD3 (µg/ml)

*

***

WT

Y1-/-

Cell division

WT

Y1-/-

CFSEce

ll nu

mbe

r

Model of autoimmune colitis

•Rag1-/- mice are recipients – noT or B cells•Naïve CD4 T cells(CD4+CD45RBhi) are donor cells•In the lymphopenic environment,T cells proliferate and areactivated by an unknown gutantigen resulting in colitis•Mice develop colitis after 21-28days

CD4+CD45RBhi transfermodel

Y1-/- T cells hyperproliferate in lymphopenichost resulting in severe colitis

-20

-15

-10

-5

0

5

0 10 20 30

Data out to day 28

Column 9

Column 7

% w

eigh

t cha

nge

Days

***

WT donor Y1-/- donor

CD4+ CD45RBhi transfer colitis model in RAG1-/- mice

***

**

0

250

500

750

1000

IFNg

IFNg Y1

IFNg WT

0

25

50

75

100

CD4 T

CD4 Y1

CD4 T

pg/m

l IFN

-γ

CD4+

in s

plee

n (x

10 3 )

*****

WT donor Y1-/- donor WT donor Y1-/- donor

F

Y1+/+ T cells ⇒ RAG-/-

Y1-/- T cells ⇒ RAG-/-

Y1+/+ T cells ⇒ RAG-/-

Day12

Day28

What about the function ofY1-/- APCs ?

•Y1-/- T cells are hyper-responsive and can differentiate into Th1 effector cells in the

presence of WT APCs

•Y1 receptor is a critical negative regulator of T cell activation

Reduction of DTH in Y1-/- mice is not due to an intrinsic T cell defect

100

101

102

103

104

OVA

Y1-/- DCs are functionally impaired

FITC- OVA

WT

0

500

1000

1500

2000

2500

Y1-/'-

WT

WT Y1-/-

% in

crea

se in

MFI

**

•Bone marrow derived DC (BMDDC) generated in presence of GM-CSF & IL-4

Y1-/-

Antigen uptake in vitro

[3HT

]thym

idin

e up

take

(cpm

x10

3)

0

100

200

300

stim only iDC LPS aCD40

Y1

WTWT

Y1-/-

***

***

anti-CD40

LPSiDCstim only

Allogenic MLR- Y1-/- DCsas stimulators

Y1-/- DCs are functionally impaired in vivo

0

25

50

75

100

DC DTH model data

Column 15

Column 14

WT/Y1 DC

WT/WT DC

DC induced antigen-specificT cell priming in vivo

% in

crea

se in

foot

pad

swel

ling

*

WTBMDDC

Y1-/-

BMDDC

Pulsed withmBSA

DC

Day 7Foot pad injection of mBSA

Swelling

-5

0

5

10

0 3 6 9 12

Days

RAG_Y1 RB colitis model

Y1 RAG

WT RAG

Rbhi colitis model in RAG1-/-Y1-/- mice

*

Y1-/- DCs are functionally impaired

WT/RAG

Y1-/-/RAG

% w

eigh

t cha

nge

Defective IL-12 production in Y1-/- mice

0

500

1000

1500

2000

2500

Column 5

Column 4

Y1-/-

WT

Homeostatic serumconcentration of IL-12

pg/m

l IL-

12 **

WT Y1-/-

resp WT 0

5

10

15

24 48 72

Time

DC prep# 7

Y1 LPS

WT LPS

Y1 con

WT con

0

5

10

15

20

25

24 48 72

Time

DC prep# 7

Y1 CD40

WT CD40

Y1 con

WT con

ng/m

l IL

-12

LPSanti-CD40

IL-12 production in stimulatedBMDDCs

WT Y1-/-

WT Y1-/-

Lethal irradiation

WT Y1-/-Inject BM cells fromdonor mouse

WT⇒Y1-/- and Y1-/-⇒WT

Y1-/- BMWT BM

WT Y1-/-

6 weeks reconstitution

DTH model

Chimeras

WT mouse withY1-/- ImmuneSystem

Y1-/- mouse withWT ImmuneSystem

Inhibition in Th1 models is immunecell intrinsic

0

20

40

60

80

100

Column 33

Column 32

Column 31

Column 30

Y1 (WT BM)

WT(Y1 BM)

Y1-/-

WT

Y1-/-

WT

WTY1-

/-

**

% in

crea

se in

sw

ellin

g

*

DTH

Y1-/-

WT

T cells

Y1 signalling

APC such as a DC

+-

Bimodal functions of Y1 receptor

-How this system fits with other known immuno-regulatory systems?

-What happens when exogenous levels of NPY rise ( e.g. stress)

Wheway et al. 2005, J. Exp. Med., 202:1527-1538

Bimodal functions of Y1 receptor

T cellAPC

Step 2Step 1

Y1

Activation

PresentationY1 agonist

OK

Y1

Stop

Y1 agonist

Y1

Y1-/- mice APC

Y1

T cell

StopStop

Activation

Presentation

Le Monde, FranceDecember 2005

The Australian, Dec 5th 2005 The Age, Dec 5th 2005

The Telegraph, Dec 5th 2005

Relevance in the clinic

-Y1 expression is reduced on T cells from patients with schizophrenia

-Well documented defects in Th1 responses inpatients with schizophrenia

-Schizophrenic patients have reduced numbersof circulating activated T cells

-T cells from schizophrenic patients are hyper-responsive ex-vivo but these patients havereduced in vivo T cell responses to tuberculin

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