Infections with parasitic helminths are counted as neglected tropical
diseases; they infect millions of people worldwide, causing high morbidity
and economic loss. Many parasites establish long lasting infections in the host
by blocking immune recognition, activation and effector pathways. To allow
in depth research on their modes of immune evasion, several mouse models
for parasitic helminth infections have been established. Heligmosomoides
polygyrus for example is a gastrointestinal nematode of rodents exhibiting a
wide spectrum of immunomodulatory effects, mediated in part by soluble
molecules released by adult worms in vitro, the excretory/secretory products
(HES). HES is a potent inhibitor of dendritic cell (DC) activation by Toll-like
receptor (TLR) ligands, completely abolishing LPS induced IL-12 production
and reducing the upregulation of cell surface activation markers. As of now,
neither the modulatory molecule nor its mechanism of action are known.
Here, the effect of HES on TLR ligand induced DC maturation was
characterized in considerably more detail compared to previous publications.
It could be shown to inhibit DC maturation induced by various TLR ligands,
on both protein and mRNA levels. These effects were comparable in both
C57BL/6 and BALB/c derived cells; in contrast to this HES differentially
affected alternative activation of BMDC from these two mouse strains.
Although for most of the experiments GM-CSF differentiated BMDC were
used, HES also inhibited LPS induced activation of splenic CD11c+ cells as
well as the activation of all three populations described in Flt3-L differentiated
BMDC - pDCs, CD11b+ cDCs and CD24+ cDCs. Furthermore, it could
be shown here that HES also inhibits LPS induced maturation in human
monocyte derived DCs.
In the search for the component in HES responsible for its inhibition
of TLR ligand induced DC maturation, exosome depleted HES rather than
exosomes was inhibitory, and the effect was heat labile. This lead to the
conclusion that the modulatory molecule has a protein component which
is indispensable for its effect; following this reasoning HES was subjected
to fractionation, with subsequent analysis of the fraction protein contents
by mass spectrometry. The top nine candidate proteins were expressed
recombinantly; however, the recombinants were not able to inhibit LPS
induced DC activation.
In parallel, experiments to elucidate the mechanism by which HES
inhibits TLR ligand induced DC maturation were performed. This led to the
conclusion that HES induces changes in the cells that, while not affecting the
induction of signalling downstream of TLRs, do impair its maintenance.
As a complement to these experiments, the transcriptomes of LPS and
LPS+HES treated cells eight hours after LPS stimulation were compared.
This revealed that transcripts encoding a number of transcription factors
inducing the expression of activation markers after TLR ligation were reduced
upon treatment of cells with HES, as were the transcript levels of IRAK2,
a kinase necessary for persistent signalling. In addition, HES increased
the transcript levels for several factors known to negatively regulate DC
maturation, including ATF3.
Furthermore, this analysis revealed changes in transcript levels of factors
like HIF-1a, indicating an even greater reliance on aerobic glycolysis if cells
were treated with HES, in addition to hints at increased ER and oxidative
stress.
In conclusion, this work narrows down the list of potential DC modulators
in HES, gives a first insight into changes in DC metabolism induced by HES
and sheds light on the role of a number of signalling pathways with important
roles in DC activation as targets of DC inhibition by HES