Immunology and zebrafish: Spawning new models of human disease

- Vertebrate (like mammals)

- Evolutionary closer to human than fruit fly en worm

- Small fish (3-4 cm)

- Use relatively little space (economic benefit)

- slender and torpedo-shaped

– pink at their belly

– tend to chase the females before  they breed

- Fat when filled with eggs

- No pink belly

mice 99,7%

zebrafish 93%

    - AB zebrafishline (Oregon)

- Large clutches (200-300 eggs per week)

- Embryo’s transparent

-  Follow development in vivo

-  Develop within 24 hours (21 days of mice)

-  Fish eggs pose no ethical problems and laboratory animal certificate

Zebrafish are hard to inbreed. When tey are sibelings enough DNA is shared.

The modification let to a pointmutation. Comparing several genomes, they learn where the mutation takes place.

They take the male's sperm, which causes fenotype mutations in the offspring. Then they go back to the sperm DNA and look at the pointmutation causing the change in fenotype

It's a known  peace of DNA, so it's easier to find it back in the genome. Virussen work the same

• Macrophages (24 hpf)

• Granulocytes (48 hpf)

• NK cells

• Complement

• Lectins

• Toll-like receptors

• Cytokines (IL-10, IFNγ, TNFα, TGFβ)

zebrafish feed themselves after 6 days, and then they could as official testinganimals. Before that time, they can be compared with babies. Babies also don't have a adaptive immunsystem when they are born.

Evolutionary it came from fish. the Gnathostomes developed T-cell receptors and Ig (antibodies). The agnathans developed variable lymphocyte receptors.

• B cells

• antibodies (IgD, IgM, IgZ)

• T cells

• Major Histocompatibility Complex (MHC)

• Dendritic Cells

IgG

Same: Platelets/trombocytes, neutrophil, eosinophil, monocyte and lymphocyt

Different: Erythrocytes

The acute-phase response 

The host–pathogen interaction

Chemotaxis to sites of injury

Escherichia coli

Salmonella typhimurium

Mycobacterium marinum

• non‐pathogenic

• infection cleared within a day

Salmonella typhimurium

• replicates in leukocytes

• spreads through blood vessels

• acute and lethal infection

• chronic infection

• similar to human tuberculosis

• replicates inside granulomas

Tuberculosis caused by mycobacterium marinum. In humans Mycobacterium tuberculosis -> 

bacteria phagocytosed by alveolar macrophages -> local proinflammation reaction leads to granuloma or tubercle -> Granuloma: 'containment' phase: No obvious signs of disease But no extinction either -> Formation of caseous centre granuloma -> Viable infectious bacilli in airways

- M. marinum phagocytosed by macrophages

- Infected macrophages extravasate and cluster

chart/ understand immune response and indentify key players in mycobacterial infection

different M. marium strains induce an acute (Mma 5) or chronic infection (Mma 11)

1) E11 and Mma20 partially overlapping host responses

2) Major contribution innate component immune system in response to M. marinum infection

Common 963 up-regulated genes:

-immunity related transcription factor genes: cebpa, cebpb, debpd, hhex, rel, spi1

-toll like receptor signaling genes: tlr1, tlr2, tlr5a, tlr5b, tlr8b, tlr22, myd88, tirap, irak4

-cytokine and chemokine genes: il1b, il10, tnfa, tnfb and cxcr4b, cxcr3.2

-complement component genes: c3b, c6, cfb

-matrix metalloproteinases: mmp9, mmp13, mmp14a

Several genes involved in WNT signaling up- or downregulated

• Transcription factor genes (23 up)

• Hematopoiesis linked genes (melk and spry4)

• Coronin (coro1a)

• Defense response genes (hamp1, lyz, tlr1, tlr2, tirap, sarm1)

• Protease genes (nots)

• Metalloprotease gene (adam8 (CD156))

• Few cytokine genes up (il12a, tnfa, tnfb)

– Several others in adults (il1b, il10, cxcl12a)

MMPs:

-endopeptidases

-catalyze turnover extracellullar matrix

-leukocyte recruitment

-cytokine and chemokine processing (pro-IL1β and pro-TNFα)

IFNγ knock down zebrafish have increased M. marinum infection

– zebrafish genes / cell types involved in infections

– Bacterial genes involved in infections

– Embryo model

– Transgenic fish

– Fluorescent bacteria/ cells (for example cancer  cells)