3: Which infection model to use?

Question 1

Name 4 in vitro models?

Answer 1

Cell lines, primary cells, modified cell lines and organoids.

Question 2

What is the problem of studying M. tuberculosis?

Answer 2

It grows extremely slow, is highly infectious and in humans only 5% develop an active disease.

Question 3

What is the THP-1 cell line?

Answer 3

THP-1 is a monocytic cell line which differentiates into macrophages by culturing in PMA. THP-1 is isolated from a 1-year old boy with acute monocytic leukaemia. Cancer cell lines have chromosomal mutations, so that’s also the case for THP-1. So the THP-1 cell line is different from ‘normal’ cells. Especially they miss the mannose receptor gene, which is important in macrophages for taking up e.g. tuberculosis.

Question 4

What are immortalized cell lines?

Answer 4

Cells that can proliferate for an extended amount of time. They are infected by SV-40, express E6/E7 and telomerase. There will be chromosomal mutations, so they are different than ‘normal’ cells.

Question 5

What are primary cells and what is their disadvantage?

Answer 5

Primary cells: PBMCs. These cells do not have a mutated chromosome.
Disadvantage of primary cells is the availability, contamination, phenotypic and genetic variation. Because people differ (so PBMCs differ: batch to batch variation).

Question 6

How do you stimulate monocytes to differentiate into macrophages?

Answer 6

GM-CSF for M1 macrophages and M-CSF for M2 macrophages.

Question 7

What are modified (humanized) cell lines?

Answer 7

They are improved cells for when the host cells need specific human receptors or proteins for the model.

Question 8

What is an example of a modified cell line?

Answer 8

HBV needs the receptor NTCP on host cells to infect cells. Hepatocyte cell lines all turn down NTCP expression. Modifying HEPG2 with expression of NTCP will alow HBV infection.

Question 9

What are organoids?

Answer 9

Organoids (and organs-on-a-chip) are multiple cell types which mimic an organ. Interesting possibilities in theory, but as yet no proven added value.

Question 10

What questions can you answer using in vitro study of M. tuberculosis?

Answer 10

Phagocytosis (receptors), Phagosome maturation arrest, Phagosomal escape, Survival in the face of macrophage effectors, modulation of cell death pathways and immune sensing and signalling.

Question 11

What questions can not be answered using in vitro study of M. tuberculosis?

Answer 11

The formation and outcome of the granuloma. contribution of the immune system or other cell types, effects of treatment.

Question 12

What are the advantages of mice as an infection model?

Answer 12

Mice have different 100% inbred lines, which are identical and therefore reproducible results. They are genetically traceable, there are many drugs available that block pathways, and there is already a lot of knowledge and tools.

Question 13

Why are mice not a good model for TB?

Answer 13

Because M. tuberculosis is not a natural pathogen in mice, and the lung pathology is different than humans.

Question 14

What is the most accurate model for TB and why?

Answer 14

Non-human primates, mostly macaques. They are naturally susceptible to M. tuberculosis and their disease is similar to humans.

Question 15

What are humanized mouse models?

Answer 15

Humanized mouse model can be made by immune system replacement. Ethical issues (because use of human fetal liver), expensive.
Human mouse model can also be transgenic mice expressing crucial human genes.

Question 16

What are BLT mice?

Answer 16

Bone marrow/liver/thymus mice where there is co-xenotransplantation of autologous human fetal liver and thymic tissue, alongside CD34+ HS injection. The thymic tissue allows the formation of a ‘BLT organoid’, which supports T-cell populations and engraftment of all major haemapoetic lineages.

Question 17

What is an example of a humanized mouse model?

Answer 17

S. aureus virlence factors are human specific. They use the receptor hC5a, so when hC5aR knock-in mice are used, they are more sensitive to S. aureus.

Question 18

What are alternative infection models that are also used?

Answer 18

Dictyostelium discoidum, Wax moth larvae, Drosophila melanogaster (fruitfly), C. elegans, Danio rerio (zebrafish).

Question 19

Why would we use the zebrafish as an infection model?

Answer 19

Zebrafish as infection model is cheap and genetic screening is possible. The embryo development is transparent and externally, so you can follow it in vivo. They develop within 30 hours and lay 100-200 eggs per week. Fish embryos pose minor ethical issues (under a week are not seen as living).
The zebrafish embryo immune system is only innate. The adults have also adaptive immune system.

Question 20

Why do we use M. marinum in stead of M. tuberculosis in zebrafish?

Answer 20

M. tuberculosis does not grow at 28-30 degrees (temp. of the fish), the M. marinum does and is closely related to the M. tuberculosis.

Question 21

What breakthroughs of TB have been found using the M. marinum model?

Answer 21

Pathogenic mycobacteria escape the phagolysosome, the initiation of granuloma formation is not dependent on the adaptive immune system and Leukotriene B4/lipoxin balance is important for disease development. Zebrafish are also used for drug development.