In vitro activation of monocytes from pregnant women by different types of bacterial lipopolysaccharides leads different immunological pathways

Recent studies show a relationship between complications during pregnancy and
the presence of an maternal infection during pregnancy, like for instance the
association between bacterial vaginosis and preterm birth. There is growing
evidence that maternal periodontal disease during pregnancy is a risk factor
for preterm birth or preeclampsia. Periodontal disease is a severe and
destructive infection of the periodontium (tissues surrounding the teeth),
caused mainly by gramnegative micro-organisms. Althought more than 500 species
have been identified in the oral cavity, only a small group of periodontopathic
micro-organisms (like Fusobacterium nucleatum, Tannerella forsythensis,
Prevotella intermedia, Treponema denticola, Micromonas micros en Porphyromonas
gingivalis) seems to play a role in the development of periodontal disease.
Gramnegative micro-organisms contain cell wall components like
lipopolysaccharides (LPS) which trigger the immune system to produce
proinflammatory cytokines, like IL-1 and TNF-alfa. Especially P. gingivalis
seems to be capable of producing large amounts of LPS. The role of LPS in the
immune response during pregnancy is subject of interest. Conducted studies show
that extreme low-dose infusion of E. coli LPS into pregnant rats leads to a
preeclampsia-like syndrome, characterized by hypertension and proteinuria. This
syndrome is pregnancy-specific: identically treated non-pregnant rats don't
develop these symptoms. This shows that pregnant rats are more susceptible to
LPS than non-pregnant rats. In our recently conducted study *The effect of
Porphyromonas gingivalis lipopolysaccharides in pregnant rats: a pilot study*we
investigated the possible role of P. gingivalis-LPS in the pathogenesis of
preeclampsia. The results show that LPS of P. gingivalis does lead to
growth-restriction of the placenta and the fetus, and also leads to
hypertension, but doesn't induce a preeclampsia-like syndrome in the pregnant
rat. LPS of different types of micro-organisms seem to have different effects
on pregnancy. Whether or not pregnant rats are more susceptible for P.
gingivalis-LPS compared with non-pregnant rats is subject of further
investigation.
The immune system recognizes LPS of different micro-organisms through pattern
recognition receptors (toll-like receptors) on the cell wall of monocytes. At
present, 13 different types of toll-like receptors have been identified, of
which TLR-4 and TLR-2 have been studied most frequently. TLR-4 recognizes LPS
of gramnegative micro-organisms, like E. coli. TLR-2 mainly recognizes
peptidoglycan of grampositive micro-organisms. Recent studies however show that
LPS of the gramnegative bacteria P. gingivalis activates TLR-2 but not TLR-4.
This might be explained by the fact that the chemical structure of P.
gingivalis lipid A differs from the lipid A produced by bacteria such as E.
coli. Activation of a specific toll-like receptor will trigger the monocyte to
a modulated immune response. Activation of TLR-4 leads to the production of Th1
cytokines like IL-1, IL-6 en TNF-alfa, which stimulates the cellular immune
response. Activation of TLR-2 leads to the production of Th2 cytokines, like
PGE2, IL-4, IL-5, IL-10 and IL-13, which stimulates the humoral immune
response. Succesful pregnancy induces an immune bias toward a Th2-immune
response. It has been postulated that Th1-immunity is not compatible with
pregnancy. A pathological pregnancy, like preeclampsia, is associated with a
Th1-Th2 shift, toward Th1-immunity. The mechanisms responsible for this
pathological shift in cytokine production in preeclamptic pregnancies are not
understood. It is possible that activation of different toll-like receptors
(induced by a mixed infection, like periodontal disease) initiates different
kinds of immunological pathways. The results of our animal experiments suggest
that LPS of gramnegative bacteria which activates TLR-4 is capable of inducing
preeclampsia-like symptoms (hypertension and proteinuria), while LPS of
gramnegative bacteria which activates TLR-2 induces growth-restriction and
hypertension, but not proteinuria.
The aim of this project is to test if the expression of TLR-4 and TLR-2 on
monocytes changes during pregnancy. We also want to study whether different
types of LPS (of different types of bacteria, like E. coli and P. gingivalis)
trigger the monocytes to produce Th1 or Th2 cytokines. Since pregnant women are
more susceptibility to LPS, we also would like to study if the cytokine
production of monocytes changes during pregnancy after stimulation with
different types of LPS.

Objective of this study is to investigate cytokine production and expression of
toll-like receptors on monocytes in blood samples from pregnant and
non-pregnant women using sonicates from different types of oral pathogenic
micro-organisms and isolated LPS.

Two blood samples, drawn from the antecubital vein, will be taken from pregnant
patients during routine vein puncture at 30 weeks of pregnancy. Two blood
samples will be taken from age-matched controls during the follicular fase of
their menstrual cycle. The expression of TLR-2 and TLR-4 on the monocyte in
unstimulated whole blood samples of both groups (pregnant and non-pregnant)will
be measured using fluorescent antibodies. The total amount of monocytes
expressing these TLR's and the density of expression will be measured using
flow cytometry. Blood samples will be stimulated in vitro with sonicates from
different oral periodontopathic micro-organisms and different doses of LPS of
E. coli and P. gingivalis to determine to cytokine production of pregnant and
non-pregnant monocytes. The production of different cytokines (like IL-1 beta,
IL-6, IL-10, IL-13, IL-18 and TNF-alfa) will be measured after stimulation
(flow cytometry/ELISA). We will also investigate the recently discovered, new
interleukins IL-23 and IL-29 (of the IL-12 family) which are associated with a
Th1-immune response and a disturbed placental implantation.

Two extra blood samples, drawn from the antecubital vein, will be taken from
pregnant patients during routine vein puncture at 30 weeks of pregnancy. Two
blood samples will be taken from controls during the follicular fase of their
menstrual cycle. In order to exclude patients with severe periodontal disease
and patients with oral pathogenic micro-organisms, all participants will be
submitted to a quick periodontal screening (DPSI-screening) and a
microbiological screening. No risks are to be expected.