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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
http://www.hi.is/gadus
Noffi Turku Finland May 2004 plenary talk
• “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
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
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
• 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
• 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
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
• 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
• 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
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
• 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
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
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
• Lysozyme
• Chitinase
• Cathepsins
• Haemolytic activity
– alternative complement pathway
Lytic enzymes
3. The main parameters of the innate immune system
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
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
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
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
• 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
• 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
• 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
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
• 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
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
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
• 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
• 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
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
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
• 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
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
The ontogeny of innate parameters in cod
Poster
5. The ontogeny of the innate immune system
Innate parameters not enhanced by
immunostimulators?
• 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
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
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)
Photograph by Valdimar Gunnarson, Hafró