Download Helminth infections: The structure trans

Helminth infections: The structure transformation of lymph nodes in infected
mice
Corinne Aus der Au1, Camille J. Gschwend2, Lalit K. Dubey3 and Nicola L. Harris3
1
PMS Kreuzlingen; 2Gymnasium Campus Muristalden, Bern; 3Global Health Institute, EPFL Lausanne, School of Life
Science
Helminths are parasitic worms, which are a major health problem in developing countries,
especially for school and pre-school aged children. Over 1 billion people are suffering from
helminth infections.
Introduction
Many people are suffering from worm infections and it’s a global health issue. Effective control of
many internal helminth infections in humans and animals is still largely a future aspiration. In
humans, these pathogens remain a major problem in areas of poverty, poor sanitation, overcrowding and malnutrition. Immunological control of helminths via vaccination requires
a thorough knowledge of the host–parasite interactions, the immune responses
involved in protection and the biology of
the parasite itself.
To reach this goal it’s necessary to understand how worm infections change the immune response and how this host parasite
interaction leads to better immune response.
Figure 1: life cycle of an HP worm
To understand the biology of infection, it
is important to know the life cycle of the worms. In present research work we focus on a helminth
parasite namely: Heligmosomoides Polygyrus (HP), which naturally infect mice in nature. An infected mouse segregates the eggs of the worm, which goes into the soil and grow to a 3rd larval
stage called L3 stage. These L3 can infect other mice in which these L3 larvae can grow in to
sexually matured adult (L5) which mate inside host and release new eggs and this cycle continues
(Figure 1).
Lymph nodes have a well-defined lymphoid architecture which contain B-cells and T-cells and
are primary site for induction of an efficient immune response. During helminth infection an antibodies producing B-cells with help from T-cells play an important protective role. After an infection the T-cell get activated and produce IL-4 molecules, which binds to a specific receptor
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namely IL4R-α and further helps B-cells to produce antibodies to fight against the infection. IL4 and IL4R-α is a critical link between interdependence of B and T cells in protective immunity.
In our laboratory we investigated the structure of mesenteric lymph node during HP infection. We
compared the lymph node of normal mice (called as wild type) vs. mice having mutation in IL4Rα (called as knock out mice).
With the help of histology followed by fluorescence microscopy it is possible to study the T- and
the B-cells along with lymphatic structure in lymph node. We utilized these methods to study the
number and location of T-cell, B-cell follicles and lymphatic in different stadiums of illness. To
find out how the IL-4 receptors influences the lymph node structure specially T cells, B-cells and
lymphatic we used lymph nodes from mice lacking IL4R-α.
Material and Methods
To count the B-cells on a lymph node slide we had to prepare the slides in many different steps.
This procedure is called histology. We worked with Heligmosomoides polygyrus (HP) worms,
which only infect mice. But they are comparable with worms which can infect human beings.
Our slides with an already prepared mLN (mesenteric lymph node) have dried at room temperature
over night before used them for the histological processing. First we put them into acetone for 10
to 20 minutes at -20° C to fix them. Then they had to dry at room temperature for 15 minutes. We
defined the area of the lymph node with a hydrophobic pen. After the rehydration in PBS (phosphate buffered saline) we made the blocking in a Phosphate buffered saline (PBS) with 1% BSA
(bovine serum albumin) and 1% mouse serum for 60 minutes at room temperature. This solution
blocks all unspecific sites. After each step we washed the slides in PBS three times for 5 minutes
to remove unbound stains. To mark the B-cells, T-cells and lymphatic we needed two different
antibodies and a fluorochrom label. First we added three different primary antibodies (Rat Anti
Mouse B220, American Hamster Anti Mouse CD3, Rabbit Anti Mouse Lyve-1) and incubated it
over night at 4° C in a humidified chamber. After another washing we added the secondary antibodies, connected to the first
ones (Donkey Anti Rat IqG
Alexa.488, Goat Anti American Hamster Alexa.594, Donkey Anti Rabbit Alexa.647).
Image 3: Histology of a B-cell
The fluorochrome is already
bound on the secondary antibodies and helps to give the
cells different colors. (Figure2) We left the slights incubating for 45 to 60 minutes at
room temperature. The next
step after the washing was the
adding of the DAPI which Figure 2: Histology
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binds to nuclear DNA and help us to visualize the nucleus at the microscope. After 15 minutes we
washed the lymph nodes for a last time with PBS and mounted them in mounting medium, using
a coverslip and sealed them with nail vanish at the frame. Then we just had to wait until the nail
vanish had dried out and we went imaging the slides with the microscope. We scanned the B-cells,
T-cells and lymphatic in different colors, to have separate look at them at the analysis.
On the Wacom tablet we identified the B-cell follicles and used an to count them and collect the
number. The results gave us the data about the changing of B-cell follicles in the course of disease.
We had also a closer look at the lymphatic in connection with the B-cells. Another data, we get
was about the position of the B-cell follicles at the different stadiums of infection.
Results
Lymph node of a normal mouse (no genetic mutation)
On figure 3 you can see the lymph nodes of a wild type mouse (without mutation) uninfected, 12
days and 21 day after the infection (dpi = days post infection).
Lymph node of a mutated mouse (no IL-4-Rα)
Scale bar = 1’000 μm
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Figure 4 shows the lymph node of the mutated mice (without IL-4-Rα). As in fig. 3 there are
three stages of infection (non-infected, 12 and 21 dpi).
B-cell follicles counting
Number of B-cell follicles
****
100
Count
80
****
Number of stars indicate the signification of the difference (ns = not
significant).
****
***
60
ns
40
20
0
WT KO WT
Naive
KO WT KO
12 dpi HP 21 dpi HP
Discussion
The cellular structure of the lymph node dramatically changes during the course of infection. The
data shows that the B-cell follicles accrete in normal mice during the helminth infection.
Wild type (Non-mutated) mice have higher number of B-cell follicles in comparison with knock
out mice (no IL-4-Rα). This leads us to the fact that this receptor has a huge influence on the Bcells growth and thereby modulating a protective immune response.
Overall, our data suggest that during the infection lymph node undergoes profound structural
changes where B-cell and lymphatic’s reorganized along with T-cells.
Conclusion
Intestinal helminth infection influences the structure of lymph node. These changes are mainly an
increase in number of B-cell follicles and inwards growth of lymphatics. The mice having mutation
in IL-4-Rα have lesser number of B-cell follicles compared to non-mutated mice.
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Gratitude
I would like to thank Dr. Lalit Kumar Dubey who showed us the whole lab, who had enough
patience to answer our question again and again and who explained us every thing without leaving
us all the time.
A great thank goes to Prof Nicola L. Harris who made our stay possible and who welcomed us
really warmly.
I would also like to thank the whole Harris lab members who helped us with problems and received
us warmly as a guests.
In last I sincerely thank the whole Schweizer Jugend forscht foundation for organizing this week
and supporting our interest to get an insight of research labs.
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