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Blood First Edition Paper, prepublished online December 23, 2016; DOI 10.1182/blood-2016-06-722538
Reduction of aGvHD using chicken antibodies directed against intestinal
pathogens in a murine model
Abdellatif Bouazzaoui1,2,3, Elisabeth Huber4, Alexander Dan5, Faisal A. Al-Allaf2,3,
Jochen Pfirstinger1, Günter Sprotte6, Josef Köstler7, Andreas Hiergeist7, Andre
Gessner7, Wolfgang Herr1, Ernst Holler1
1
Medical Clinic 3 – Hematology/Oncology, University Hospital Regensburg,
Regensburg, Germany
2
Science and technology unit, 3Department of Medical Genetics, Umm AlQura
University, Mecca, Saudi Arabia
4
Department of Pathology, University Hospital Regensburg, Regensburg
5
IgNova GmbH, Oberursel, Germany
6
Department of Anesthesiology, University Hospital Würzburg, Würzburg, Germany
7
Department of Clinical Microbiology and Hygiene, University Hospital Regensburg,
Regensburg, Germany
Running title: Hens IgY antibodies against E. coli reduce aGVHD
Key words: stem cell transplantation, graft-versus-host disease, Immunoglobulin Yolk
(IgY), hen antibody, cytokine, bone marrow transplantation, murine
Word counts:
Abstract:
Tables:
Supplementary Figures:
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References: 23
1
Copyright © 2016 American Society of Hematology
Figures: 2
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Corresponding author:
1. Prof. Ernst Holler
Medical Clinic 3 – Hematology/Oncology
University of Regensburg Medical Center
Franz-Josef-Strauss Allee 11
D-93053 Regensburg, Germany
Phone: +49-941-944-5531, Fax: +49-941-944-5543, E-mail: [email protected]
2. Dr. Abdellatif Bouazzaoui
Medical Clinic 3 – Hematology/Oncology
University of Regensburg Medical Center
Franz-Josef-Strauss Allee 11
D-93053 Regensburg, Germany
Phone: +49-941-944-5589, Fax: +49-941-944-5147,
E-mail: [email protected] or [email protected]
2
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Allogeneic bone marrow transplantation (alloBMT) is a unique curative therapy for
diverse diseases. However, its use is limited by the development of severe treatmentrelated complications, most importantly, the occurrence of acute graft-versus-hostdisease (aGVHD). Despite the use of modern immunosuppressive drugs, aGVHD
remains the major cause of morbidity and mortality.1-3 Acute GVHD manifestation
depends on the degree of human leukocyte antigen (HLA) differences between
recipients and donors, the T cell fraction in the graft, patient’s age, and
prophylaxis/therapy regimens.1,2,4 In addition, GVHD development is influenced by
microbiota in the gut.5-7 Beelen et al. demonstrated that mice raised in germ-free
conditions did not develop GVHD, whereas control animals raised in conventional
conditions died early after transplantation.5 Other studies presented evidence for an
association between the GVHD outcome and elimination of Lactobacillales before
BMT or reintroduction of probiotic Lactobacillus after BMT.4,8,9 In light of these
results, we hypothesized that orally applied antibodies produced in hens to capture
and eliminate bacteria and/or bacterial products in the gut might influence the
bacterial composition and lead to improvement of GVHD outcome.
Using a haploidentical murine model, B6D2F1 mice conditioned with total body
irradiation (TBI) received bone marrow cells (BM) and splenocytes (SC) from either
syngeneic (Syn=B6D2F1) or allogeneic (Allo=C57BL/6) donors. Starting 2 days
before transplantation (day -2) through day 28 after transplantation, animals received
feed pellets containing IgY (Immunoglobulin Yolk) antibodies from hens immunized
with heat inactivated Escherichia coli (E. coli), Clostridium perfringens and
Salmonella typhimurium (IgNova GmbH, Oberursel, Germany). In an alternative
protocol animals received identical pellets from day -2 to day 15 for subsequent stool
3
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analyses. Thereafter, severity of aGVHD, microbial composition and cytokine levels
were analyzed and compared to control animals receiving feed pellets without IgY.
After transplantation we determined the presence of IgY antibodies in stool and
serum samples. IgY antibodies were present in stool samples (Figure 1A), but not in
the serum (data not shown). One week after BMT, syngeneic recipients
demonstrated minor weight changes due to radiation toxicity-related tissue damage
but continuously recovered, and all animals survived until day 28 (Figure 1B). In
contrast, allogeneic recipients developed severe clinical symptoms within the first
week after transplantation. However, treatment of allogeneic recipients with IgY
antibodies resulted in reduced weight loss and decreased clinical GVHD scores when
compared to control animals. Furthermore, only 12.5% of the animals receiving IgY
feed pellets died within 4 weeks after transplantation (Figure 1B) compared to 40% in
the control group (p=0.045). On day 28 after alloBMT, animals receiving IgY
antibodies exhibited significantly reduced TNF serum levels (1.86±0.13 vs.
4.04±0.63pg/mg protein, p<0.01) (Figure 1C). Similarly, levels of IL2 and IL6 were
significantly reduced in treated animals (0.02±0.003 vs. 0.05±0.01pg/mg protein,
p<0.05 and 0.3±0.1 vs. 0.78±0.2pg/mg protein, p<0.05, respectively).However, the
difference in IFNγ levels did not reach statistical significance (0.41±0.07 vs. 0.7±0.14
pg/mg protein, p=0.073). Serum analysis on day 7 did not reveal a significant
difference in relevant cytokine levels (data not shown).
The pathology score, performed 28 days after transplantation, showed less organ
injury in syngeneic animals compared to allogeneic animals, indicating the inferior
effect of the conditioning regimen on organ damage.10 In contrast, allogeneic mice
displayed a high pathology score in liver, lung, small intestine, colon, and skin,
4
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whereby the difference between IgY-fed animals and controls was significant for the
colon only (4.07±0.57 vs. 5.58±0.51, p<0.05).The impact of IgY on colon pathology is
not unexpected because the colon hosts more than 1500 bacterial species11,12 and
the association between the gut microbiome and GVHD.4,5 In this context we
analyzed the stool microflora at day 15 and detected a significantly reduced bacterial
diversity in syngeneic mice compared to allogeneic mice (Sup. Figure 1, syngeneic
vs. allogeneic controls, 0.87±0.02 vs. 0.94±0.01, p=0.019 and syngeneic vs.
allogeneic IgY, 0.8±0.05 vs. 0.89±0.01, p=0.038, respectively). Interestingly, there
was also a significant difference between allogeneic controls and allogeneic IgY-fed
mice (0.94±0.01 vs. 0.89±0.01, p=0.009). Moreover, the bacterial load differed
between allogeneic controls and allogeneic IgY-fed mice, reaching significance at
day 15 (4.08±0.54x109vs.1.1±0.56x109, p=0.04).
Previous studies demonstrated extensive microbial change in consequence of a
decreased production of antimicrobial peptides due to the loss of Paneth cells during
aGVHD development.9,13,14 The loss of Paneth cells may account for the higher
bacterial load in allogeneic animals compared to syngeneic controls. Analyzing the
bacterial composition, we identified bacteria from10 different phylums and 19
classes, whereby only 5 phylums (Firmicutes, Bacteroidetes, Actinobacteria,
Proteobacteria, and Verrucomicrobia) containing 8 classes presented more than 98%
of the whole microbiota (Sup. Figure 2-3). Using ANOVA, we found significant
differences in 4 classes (Sup. Figure 3). Additional Mann Whitney U test analysis
between allogeneic animals receiving IgY pellets and controls showed similar levels
at day 28 (data not shown), but at day 15 after transplantation (Figure 2A) we found a
significant increase of Lacotobacillus reuteri (21.95±4.18% vs. 0.85±0.3%) and
another
uncultured
bacteriumfrom
the
5
phylum
Firmicutes
(4.18±0.08%
vs.
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0.17±0.05%) in allogeneic IgY animals versus controls. These results are in line with
a previous study demonstrating the important role of Lactobacillales in the GVHD
outcome.8,9 Contrary to the increase of L. reuteri and uncultured bacterium from the
phylum Firmicutes, we found a decreased level of E. coli (0.05±0.02% vs.
2.18±0.76%) and another uncultured bacterium from the phylum Bacteroidetes
(5.35±2.62% vs. 35.65±8.13%) in stool samples of allogeneic IgY-fed animals
compared to allogeneic controls. Interestingly, only E.coli showed a significant
difference between levels at day 15 and day 28 (0.05±0.02% vs. 1.63±0.09%,
p<0.05) in IgY-fed animals (Figure 2B). Our findings are in line with previous studies
demonstrating that administration of IgY leads to reduction of the viral load.15,16 The
exactmechanisms remain unknown so far, however, several mechanisms have been
proposed in earlier publications, including agglutination, opsonization, and toxin
neutralization.17,18 Furthermore, previous studies revealed a correlation between high
organ damage, population shift towards E. coli and the abundance of adherentinvasive E. coli in patients with Crohn’s disease compared with healthy subjects.19,20
Of further interest, stool samples of animals receiving IgY pellets presented high
levels of Akkermansia compared with controls. Recently, Li and coworkers confirmed
the anti-inflammatory role of this genus in atherosclerosis apolipoprotein E-deficient
mice (Apoe-/-)
21
whereas in another study, the authors showed that this genus is
pathogenic.22
In summary, the improvement of aGVHD outcome of animals treated with IgY may be
mediated by reducing pathogenic bacteria such as E. coli and by increasing the
amount of probiotic bacteria such as L. reuteri. This microbial shift may account for
the reduction of IDO, TLR2,4 and NOD2 expression and a subsequent decrease in
chemokine and cytokine expressionin the colon (data not shown).
6
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Conclusion: This study confirmed that oral immunoglobulin administration can be
considered as an attractive strategy for passive bacterial modification in the gut to
improve the GVHD outcome. Combination of this treatment strategy with other
approaches23 (antibiotics, probiotics, postbiotics and prebiotics approaches) in order
to manipulate the microbiota-host interactions can improve the GVHD outcome
significantly.
7
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Acknowledgement:
This work was supported by IgNova GmbH, Oberursel, Germany
Authorship Contributions:
A. B: designed experiments, performed experiments, analyzed data and wrote the
manuscript; E. H: performed experiments and analyzed data; J. K: performed
experiments; A. H: performed experiments, analyzed data and wrote the manuscript;
A. G: provided reagents and designed experiments; A. D; F. A. A; J. P; G. S; W. H:
provided reagents and wrote the manuscript; E. H: provided reagents, designed
experiments and wrote the manuscript
Conflict of Interest Disclosures:
The authors declare no conflict of interests
8
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Figure legends
Figure 1: IgY antibodies in the stool, survival rates, cytokine levels and
histopathologyscores
Lethally irradiated B6D2F1 mice received bone marrow cells supplemented with
splenocytes from either syngeneic (B6D2F1) or allogeneic (C57BL/6) donors.
Thereafter, animals received feed pellets with IgY or control pellets without IgY. (A)
On day 15 after transplantation, stool samples were isolated and the presence of IgY
antibodies was determined by ELISA. (B) Survival of syngeneic and allogeneic
recipients. (C) Serum cytokine levels were determined on day 28 after alloBMT using
cytokine bead assay. (D) Histopathology scores for small intestine, colon, liver, lung,
and skin at day 28 after BMT. Data are presented as mean ± SEM; *p<0.05,
**p<0.01.
Figure 2: Microbiota composition in stool samples
After transplantation, animals received feed pellets with hen antibodies (IgY) or
control pellets (without IgY). The microbiota composition was determined in stool
samples using new generation sequencing method. (A) Microbiome composition on
day 15 after transplantation. (B) Microbiome composition of allogeneic mice on day
15 and 28 after transplantation.Data are presented as mean ± SEM; *p<0.05,
**p<0.01.
12
Figure 1
Figure 2
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From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
Prepublished online December 23, 2016;
doi:10.1182/blood-2016-06-722538
Reduction of aGvHD using chicken antibodies directed against intestinal
pathogens in a murine model
Abdellatif Bouazzaoui, Elisabeth Huber, Alexander Dan, Faisal A. Al-Allaf, Jochen Pfirstinger, Günter
Sprotte, Josef Köstler, Andreas Hiergeist, Andre Gessner, Wolfgang Herr and Ernst Holler
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