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INNATE IMMUNITY OF FISH (OVERVIEW) Bergljót Magnadóttir Institute for Experimental Pathology, University of Iceland, Keldur v. Vesturlandsveg, IS-112 Reykjavík, Iceland [email protected] http://www.hi.is/gadus Noffi Turku Finland May 2004 plenary talk

INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

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Page 1: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

INNATE IMMUNITY OF FISH

(OVERVIEW)

Bergljót Magnadóttir

Institute for Experimental Pathology, University of Iceland,

Keldur v. Vesturlandsveg, IS-112 Reykjavík, Iceland

[email protected]

http://www.hi.is/gadus

Noffi Turku Finland May 2004 plenary talk

Page 2: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

• “The biological wisdom that instructs the

acquired immunity”

• To improve the general well being of fish in

aquaculture and protect fish larvae before their

defence system is fully competent

The innate immune system

– a renewed interest

Page 3: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

1. The different roles of the innate immune system

2. The innate immune system’s recognition of non-self

3. The main parameters of the innate immune system

4. Modification and manipulation of the innate immune system

5. The ontogeny of the innate immune system

Overview

Examples from studies of cod

Page 4: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

Innate elimination of infection

• Of primary importance for disease

resistance in fish

– First line of defence

– Instantaneous

– Relatively temperature independent

• The acquired response is limited in fish, slow and

temperature dependent

1. The different roles of the innate immune system

Page 5: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

• Secretion of cytokines by macrophages and

natural killer cells/non-specific cytotoxic cells

• Activation of the alternative and lectin

complement pathways

• Preferential uptake by lectin receptors on

APC’s (“signal two”)

• LPS induced receptors (TLR’s) on APC’s

Innate communication with the

acquired system

1. The different roles of the innate immune system

Page 6: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

• Maintaining cellular and molecular

equilibrium by feedback inhibition and

clearing away apoptotic and necrotic cells

and cell debris

Homeostasis

1. The different roles of the innate immune system

Page 7: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

1. The tasks of the innate immune system

2. The innate immune system’s recognition of

non-self

3. The main parameters of the innate immune

system

4. Modification and manipulation of the innate

immune system

5. The ontogeny of the innate immune system

Overview

Page 8: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

• Pattern recognition receptors/proteins

– Limited number of germline-encoded

molecules common to most invertebrates and

vertebrates

• Lectins, enzymes, complement proteins etc.

The innate recogniton of non-self

2. The innate recognition of non-self

Page 9: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

• Associated with foreign/pathogenic agents

– e.g. bacterial-, viral- and fungal- polysaccarides,

peptidoglycans, lipopolysaccarides (LPS), b1,3-

glucan, bacterial DNA, viral dsRNA

• Associated with host’s own danger signals

– The host’s nucleic acids, heat shock proteins,

chaperons and high mannose (oligomannose)

glycoproteins

The molecular patterns recognized

2. The innate recognition of non-self

Page 10: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

1. The tasks of the innate immune system

2. The innate immune system’s recognition of

non-self

3. The main parameters of the innate immune

system

4. Modification and manipulation of the innate

immune system

5. The ontogeny of the innate immune system

Overview

Page 11: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

• Physical parameters

– Mucus, scales, epidermis

• Cellular parameters

– Phagocytes (granulocytes (neutrohils) and

monocytes/macrophages), non-specific

cytotoxic cells (NCC), epithelial cells,

dendritic cells

• Humoral parameters

The main parameters of the innate

immune system

3. The main parameters of the innate immune system

Page 12: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

Humoral innate parameters

• Growth inhibitors

– Iron binding (transferrin), interferon

• Protease inhibitors

– a2-Macroglobulin

• Lytic enzymes

– Lysozyme, chitinase, ACP

• Agglutinins/precipitins/opsonins (Lectins)

– C-type lectins, pentraxins

• Natural antibodies

– Anti-TNP/DNP antibody activity

• Cytokines and chemokines

• Antibacterial peptides

3. The main parameters of the innate immune system

Page 13: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

Humoral innate parameters

• Growth inhibitors

– Iron binding (transferrin), interferon

• Protease inhibitors

– a2-Macroglobulin

• Lytic enzymes

– Lysozyme, chitinase, ACP

• Lectins

– C-type lectins and pentraxins

• Natural antibodies

– Anti-TNP/DNP antibody activity

• Cytokones and chemokines

• Antibacterial peptides

3. The main parameters of the innate immune system

Page 14: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

• Lysozyme

• Chitinase

• Cathepsins

• Haemolytic activity

– alternative complement pathway

Lytic enzymes

3. The main parameters of the innate immune system

Page 15: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

The haemolytic activity of serum from wild and

cultured cod sampled before 2000

Inactivation temperature The effect of EDTA

After ~ 2000: No or very low, heat sensitive spontanesous

haemolytic activity in serum from cultured cod

3. The main parameters of the innate immune system

Page 16: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

Haemolytic activity and ApoLP A-I

ApoLP AI

Serum/SDS-PAGE

HA+ HA -

•PEG precipitation

•Q-Sepharose HP

•Superose 6/Superdex 200

•MonoQ

•SDS-PAGE

Isolation of C3 from HA - serum

3. The main parameters of the innate immune system

Page 17: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

Haemolytic activity and ApoLP A-I

• Cod serum treated

with human ApoLP

A-I:

– 5.5 mg ApoLP A-I /

ml serum for 2 h at

4°C

– Reduced the SH50%

activity by about 40%

3. The main parameters of the innate immune system

Page 18: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

In situ hybridization detection of apolipoprotein A-I in

43 days old cod larvae

3. The main parameters of the innate immune system

brain

spinal

cord

control

control

Page 19: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

• The results suggest that ApoLP A-I may act

as an inhibitor of the spontaneous

haemolytic activity by hydrophobic

association with C3 under certain conditions

• also protecting certain organs like the

brain

3. The main parameters of the innate immune system

Page 20: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

• C-type lectins

– Mannan binding lectin (MBL)

– Ficolin-like lectin in cod serum with affinity

for mannose and N-acetyl glucosamine

• Pentraxins

– C-reactive protein (CRP) in cod

– Serum amyloid P (SAP)

Lectins

3. The main parameters of the innate immune system

Page 21: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

• A defensive role (opsinin) as well as an

acute phase protein in fish

• Binds bacterial glycans

(phosphorylcholine) and LPS

• Binds to receptors on phagocytes

C-reactive protein

3. The main parameters of the innate immune system

Page 22: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

CRP in cod serum

Concentration in normal

serum 300 – 600 µg/ml

PNGase has no effect

3. The main parameters of the innate immune system

Page 23: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

• Produced in the absence of gene-rearrangement

• Produced without specific antigen stimulation

• Show high anti-hapten (TNP/DNP) activity

• High levels in many fish species including cod

• Involved in innate defence against bacteria and

viruses in fish

• Can inhibit specific antibody response in fish

Natural antibodies

3. The main parameters of the innate immune system

Page 24: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

Natural antibodies of cod

The effect of increasing size/age and

temperature

The effect of increasing temperature

The effect of adjuvant injectionThe effect of bacterial dosage

0

6500 cfu/ml

3. The main parameters of the innate immune system

Page 25: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

1. The tasks of the innate immune system

2. The innate immune system’s recognition of

non-self

3. The main parameters of the innate immune

system

4. Modification and manipulation of the innate

immune system

5. The ontogeny of the innate immune system

Overview

Page 26: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

• Genetic traits

• Seasonal factors

• Temperature

• Pollution

• Stress

• Diseases

• Vaccination

• Food additives

• Immunostimulants

• Probiotics

Modifiers of innate immunity in fish

4. The modification and manipulation of the innate immune system

Page 27: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

• Immunostimulants

– Used to enhance the activity of innate

parameters and improve the quality and

survival of fish and fish larvae in

aquaculture

– Agents used that activate PRR’s

• Like b1,3 glucans, peptidoglycans of bacteria,

LPS, fungal & lichen derivatives, chitosan,

synthetic dinucleotides etc.

Positive manipulation of the innate

immune system

4. The modification and manipulation of the innate immune system

Page 28: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

Immunostimulants tested on cod larvae

• LPS from E.coli & Asa +

• Lichenan (beta glucan) 0

• Thamnolan (heteroglycan) +

• Polymannuronic acid 0

• Laminarin 0

• Levamisol -

• Vitamin C 0

• Cod liver oil 0

• Yeast DNA (+)

• Chitosan (+)

• LPS from E.coli & Asa

• Polymannuronic acid

• Chitosan

S. Guðmundsdóttir Oral 17

4. The modification and manipulation of the innate immune system

The effect on survival The effect on disease resistance

Page 29: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

1. The tasks of the innate immune system

2. The innate immune system’s recognition of

non-self

3. The main parameters of the innate immune

system

4. Modification and manipulation of the innate

immune system

5. The ontogeny of the innate immune system

Overview

Page 30: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

• Parameters studied:

– The first appearance of phagocytes and

phagocytic activity

– The complement components (C3)

– Other humoral parameters

Innate parameters in ontogeny

5. The ontogeny of the innate immune system

Page 31: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

The FISHAID project*

• Carp shows early appearance of innate (1 d.p.f.) and specific (4 d.p.f.) parameters. Innate parameters are enhanced by immunostimulation

• Sea bass shows relatively late appearance of complement (15 d.p.h.) and specific (IgM, 50 d.p.h.) parameters. Innate parameters are not enhanced by immunostimulation

• Wolffish shows a relatively quick appearance of innate and specific components (1 week p.h.) and has maternal C3. Innate parameters are enhanced by immunostimulation

• Halibut shows early appearance of C3 (5 d.p.h.) and specific components at 20 d.p.h.

• Cod . . .

5. The ontogeny of the innate immune system

*Co-ordinator R. Dalmo, Tromsø

Page 32: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

The ontogeny of innate parameters in cod

Poster

5. The ontogeny of the innate immune system

Innate parameters not enhanced by

immunostimulators?

Page 33: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

• The innate system is of vital importance to fish disease defence - being fast and highly active

• Its recogntion of non-self/danger signal is based on widely distributed but structurally limited patterns

• Innate parameters are influenced by a variety of factors

• Manipulation of innate parameters is problematic

• Innate parameters are generally active before or soon after hatching

Summary

Page 34: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

Some points to consider

• The communication between the innate and the acquired immune systems in fish is interesting in view of the long period between infection and specific resonse (up to 12 weeks)

• Immunostimulants sometimes inhibit specific immune parameters – is the use of immunostimulants in larvae culture a good idea?

• Studies of the ontogeny of the innate system calls for the application of genetic and proteomic methods

Page 35: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

ThanksThe NCM-project 1996-1999:

Lars Pilstöm & Trond Jørgensen

Uppsala, Sweden Tromsø, Norway

The FISAID project 2000-2004:

Roy Dalmo, Tromsø, Norway, co-

ordinator

Giuseppe Scapigliati, Viterbo, Italy

Jan Rombout, Wageningen, Holland

Ian Bricknell & Tim Bowden, Aberdeen,

Scotland

The Fish group at Keldur

Helga, Sigríður, Gísli, Bryndís,

Slavko, Bergljót, Sigurður,

Bjarnheiður + Sigrún and Árni

(inserted)

Page 36: INNATE IMMUNITY OF FISH (OVERVIEW) Noffi 2004.pdf · 1. The different roles of the innate immune system 2. The innate immune system’s recognition of non-self 3. The main parameters

Photograph by Valdimar Gunnarson, Hafró