Immunomodulatory effects of Inulin-type fructans in a hepatitis B vaccination study

Beneficial health effects of dietary fibers have gained considerable attention
by the scientific community during recent years. Fibers can be found in many
products such as cereals, bread, pasta, vegetables as well as in sugar-, and
fat replacers. High fiber intake is associated with lower mortality in humans
suffering from circulatory, digestive, and non-cardiovascular noncancerous
inflammatory diseases (Jacobs et al. 2007,Park et al. 2011,Chuang et al. 2012).
These associations are similar for men and women and are observed in most
countries even after careful adjustment for potentially confounding lifestyle
and dietary differences (Landberg. 2012). However, all these studies have
applied mixtures of fibers rather than specific types of molecules. Therefore,
it is currently still unknown which type of fiber(s) are responsible for these
effects. There is an urgent need for studies addressing specific fibers
(Landberg. 2012) to obtain a better understanding of what mechanisms and which
components underlie beneficial health effects. There are different types of
dietary fibers. In this study Inulin type fructans (ITFs) and resistant starch
corn (RSC) will be studied.
Inulin-type fructans (ITFs) are a specific type of dietary fiber,
isolated from chicory root and sugar beet root. Upon supplementation in food,
ITFs have been reported to enhance immunity in the gut and to lower frequency
of infections and to alleviate symptoms of colitis (Leenen and Dieleman. 2007)
and even allergy (Schouten et al. 2011). These ITFs can be found in many food
products in which sugar beet and chicory has been applied. Before the
beneficial effects were discovered, ITFs were already widely and safely used in
various products as sugar-, and fat replacers, and applied as texturizers in
*light* food products (Hidaka et al. 1991).
ITFs are marketed as so-called prebiotics, meaning that ITFs support the
function and growth of microorganisms in the intestine such as gut lactobacilli
(Leenen and Dieleman. 2007,Casellas et al. 2007) and bifidobacteria (Leenen and
Dieleman. 2007,Lindsay et al. 2006). The beneficial effects of these fibers are
thought to derive from the growth and activity of these bacteria, as well as
from the increases in their fermentation products, such as Short Chain Fatty
Acids (SCFAs) (Meijer et al. 2010,Vinolo et al. 2011). These bacteria are by
this mechanism considered to induce immune effects, such as increasing the
number of IgA-producing plasma cells (Guilliano et al. 2001,Woof. 2002),
increasing or improving phagocytosis (Nagl et al. 2002), or increasing the
proportion of T lymphocytes and Natural Killer cells (Reid et al. 2003,Ouwehand
et al. 2002).
However, our recent research showed that ITFs can do more, i.e. they can
directly affect immune cells. Specific ITFs can bind and stimulate specific
pattern recognition receptors (PRR) in humans. PRRs are the sensors of the
human immune system and determine whether an immune response will occur. The
best characterized family of PRRs is that of the Toll-like receptors. We found
on human reporter cell lines that ITFs stimulate specific Toll-like receptors
and in particular TLR2. This stimulation of TLR signaling is considered to be
important, as TLR signaling in gut dendritic cells enhances antigen uptake and
presentation (Veldhoen et al. 2008) and therefore stimulates immunity. The
effects of ITFs were, however, dependent on the molecular weight of the ITFs
applied. Low molecular weight ITFs were found to have a more regulatory effect
on human leukocytes (isolated from blood) while high molecular weight ITFs
activate the immune response. We especially found a difference in the
IL-10/IL-12 ratio, which is considered to be regulatory when high and
stimulating when low. These data not only suggest that ITFs can directly affect
immune responses, but also that high and low molecular weight ITFs may have
different in vivo effects. These findings made us decide to perform a pilot
phenotyping study of human immune cells when the diet was supplemented with 8
grams of low molecular weight ITFs. This was done as part of an educational
training in which third year life science students were trained in
immunological techniques with their own blood. We found even after a short term
intake of not more than 7 days and in a very small study, that the frequency
of T helper 1 (Th1) cells, as well that of regulatory T cells both showed
increased trends. This prompted us to verify these data in a larger study.
The other fiber supplement that will be studied in this research is RSC. From
earlier experiments, which are performed in UMCG and the university of
Wageningen related to Th1 or Th2 immune skewing. RSC showed to be a strong
Th1-stimulating fiber (see C2 Samenvatting amendement for more details).
Resistant starch is starch that is not digested in the small intestine, and
enters the large bowel. Resistant starch is a type of dietary fiber that is
found in many different (plant) food products like beans, whole vegetables,
whole grains and rice. There are four types of resistant starch: RS1, RS2, RS3
and RS4. Ingestion of resistance starch may be useful in management of the
metabolic (glycaemic) control and reduction of appetite (Robertson 2012,
Bodinham 2010, Bodinham 2014). Also a role in the reduction of inflammation has
been suggested (Higgins 2013, Moreau 2003). The fermentation of resistant
starch predominately takes place in the proximal colon. The fermentation of
resistant starch results in the production of short chain fatty acids (SCFAs)
(Cummings 1987). Until now, only little research has been performed evaluating
the potential immunomodulatory capacity of resistant starch (Vos 2007). The
choice of the fiber RSC in these vaccination protocol increases the window to
observe fiber mediated vaccination efficacy enhancement. Considering the data
of these experiments performed in the UMCG and the university of Wageningen
related to Th1 and Th2 immune skewing, the above mentioned RSC seems to be a
well argumented choice to improve Th1 responses.
To be able to show more robust effects of ITF or RSC intake, we will
include a vaccination study in the present protocol. Vaccination efficacy
studies are often applied to demonstrate stimulation of immunity by food
components (Olivares et al. 2007,Soh et al. 2010) and is therefore an accepted
method to study effects of food components. As we found that ITFs stimulate
TLR2 which is involved in enhancing antigen-uptake (Blander and Medzhitov.
2006,Underhill et al. 1999), and as we found a trend in increased Th1 and
regulatory T cells we expect that ITFs may be effective in boosting vaccination
responses. We also expect differences in efficacy with the different molecular
weight as they induce different responses on human leukocytes. For the reasons
described above, we also expect that RSC may boost vaccination response in a
Th1-dependent manner.
A vaccination protocol that might benefit from a stimulation of the
immune system is hepatitis B vaccination. This vaccination protocol is applied
at the UMCG as a mandatory vaccination for third year bachelor-students of
Life-sciences and Medicine. At the moment of their study, students of
Life-sciences and Medicine start research training at the UMCG in which they
might have to handle human material. Hepatitis B vaccination usually has a low
efficacy, since most people have to undergo three shots of vaccination before
they reach protective immunoglobulin titers. Especially the first shot is
characterized by low efficacy. This, however, is an advantage for our study as
we may be able to increase the efficacy of the first vaccination.

The present study has 1 primary and 1 secondary objective:

1: The primary objective of the present study is to analyse the effects of
supplementation with ITFs of different chain lengths and RSC on hepatitis B
vaccination efficacy. The main parameter to study will be hepatitis B
vaccine-induced antibody titers.

2: To study underlying and concomitant mechanisms, modification of various
immune cell populations will also be evaluated. To evaluate whether the effect
of ITFs and RSC is induced by changing the microbiota, changes in microbiota
and levels of IgA in the feces will be analysed.

The format of the present intervention study is a double blind randomized
placebo-controlled trial. Male and female subjects will be recruited from a
population of students working in the University Medical Center Groningen
(UMCG), which require a hepatitis B vaccination in order to safely work with
patient material during their bachelor. The study population will consist of
individuals in the ages ranging from 18 to 35 years. Equal numbers of subjects
will be at random allocated into the following groups: I) receiving 8g/d of low
molecular weight ITFs (Frutafit®CLR), II) receiving 8g/d high molecular weight
ITFs (Frutafit®TEX!), or III) receiving 8g/d of RSC, IV) receiving 8g/d of
fructose, which serves as a placebo and is accepted by the scientific community
as an appropriate control. All supplements will be dissolved in 50 ml of
fiber-free lemonade before administration to mask any taste effects and to
standardize the way of intake. In each group, the subjects will receive a
vaccination against hepatitis B on day 7 of supplementation, and
supplementation of fibers will continue for the following 7 days (thus 14
consecutive days in total), since several dietary intervention studies around
vaccination protocols have shown that the period around the vaccination is
crucial in the development of the antibody response and this period is most
important when attempting to boost the response. The study subjects are asked
to fill in a nutrition diary and to refrain from intake of pre-, and probiotic
nutritional supplements outside of the specified study supplements.

Although our primary outcome is antibody titers following ITF or placebo
intake, we will also evaluate the effects of ITF intervention on immune cell
populations. Therefore, baseline blood samples will be collected from all
subjects before the start of supplementation (day 0), and blood samples will be
collected at day 7 (prior to vaccination), 14, 21, and 35 (on each time point
10 ml of heparinized blood will be collected per subject). Blood sampling will
be performed by trained professionals from the department of Laboratory
Medicine of the UMCG. To study peripheral immune cell populations, leukocytes
in whole blood will be stained for relevant cell markers to distinguish
different populations using flow cytometry.

T cell responses are expected to arise from day 4 post-vaccination onward.
Therefore T-cell responses will be analyzed in the blood samples collected from
each vaccinated subject at days 0, 7, and 14. Antibody titers will be analyzed
in the blood samples collected from each vaccinated subject at days 21 and 35;
this will be 14 and 28 days post-vaccination, to monitor titer amounts in time.
Antibody levels in blood samples will be analyzed using antibody-specific ELISA
microtiter plates.

Finally, microbiota composition and the short chain free fatty acid (SCFA)
profile (which are products produced by bacteria) as well as levels of fecal
IgA will be analyzed to evaluate effects of ITFs on microbiota. For these
parameters, fecal samples will be collected weekly from day 0 (baseline
microbiota and IgA) to day 35.

The subjects in this study will receive dietary fiber supplements (low
molecular weight ITF, high molecular weight ITF or RSC) or fructose as a
placebo that are safe, food grade compounds, which do not have any negative
side effects. 8g of fibers or placebo per day, dissolved in 50ml of fiber-free
lemonade (per day) will be given to healthy subjects, who then drink this
solution. The used concentration is in accordance to literature an effective
dose to stimulate the immune system (Gibson et al. 1995,Langlands et al. 2004).
An educational assignment in which students consumed this amount to study their
changes in immune cell populations demonstrated that the subjects did not have
difficulties consuming this amount of fibers.

Collection of fecal samples does not carry any risks for the participants.
Blood sampling will be performed by trained professionals from the department
of Laboratory Medicine of the UMCG and will comprise 1 tube containing a volume
of 10 ml of blood per person per sampling time point, in total 50ml of blood
will be drawn, spread over a time period of 35 days. Blood will be drawn from
the median cubital vein on the inside of the elbow. This may cause a small
hematoma (*blauwe plek*) at the site of sampling, however in general when
pressure is applied to the sampling site just after taking the sample this does
not occur. The hepatitis B vaccination which is included in this study takes
place in a safe and approved vaccination program which has been running for
several years. Since this study population would already receive this
vaccination for the safety of their student career while working in the UMCG it
poses no extra burden on these individuals. The availability of the vaccination
does not rely in any way on participation in this study. There are no benefits
for the individuals participating in this study. The benefits of this study are
represented by a gain in knowledge on the immunomodulatory effects of ITFs and
RSC, and in the case that supplementation boosts the production of
vaccine-specific antibodies, intake can be considered beneficial during
hepatitis B vaccination programs. Furthermore, knowledge can be gained on the
underlying effects such as shifts in immune cell populations and IgA
production. By studying microbiota and SCFA profiles, the induction of
bacterial strains can be evaluated and this can generate new hypotheses on the
relation between these bacteria and the observed immunological benefits.