MINI REVIEW
published: 12 September 2019
doi: 10.3389/fimmu.2019.02174
Antibiotic Treatment Protocols and
Germ-Free Mouse Models in
Vascular Research
Franziska Bayer 1†, Stefanie Ascher 1†, Giulia Pontarollo 1 and Christoph Reinhardt 1,2*
1 Center for Thrombosis and Hemostasis (CTH), University Medical Center Mainz, Johannes Gutenberg University Mainz,
Mainz, Germany, 2 German Center for Cardiovascular Research (DZHK), Partner Site RheinMain, Mainz, Germany
The gut microbiota influence host vascular physiology locally in the intestine, but also
evoke remote effects that impact distant organ functions. Amongst others, themicrobiota
affect intestinal vascular remodeling, lymphatic development, cardiac output and vascular
function, myelopoiesis, prothrombotic platelet function, and immunovigilance of the
host. Experimentally, host-microbiota interactions are investigated by working with
Edited by:
animals devoid of symbiotic bacteria, i.e., by the decimation of gut commensals by
Benoît Ho-Tin-Noé,
Institut National de la Santé et de la antibiotic administration, or by taking advantage of germ-free mouse isolator technology.
Recherche Médicale Remarkably, some of the vascular effects that were unraveled following antibiotic
(INSERM), France
treatment were not observed in the germ-free animal models and vice versa. In this
Reviewed by:
Karen Ho, review, we will dissect the manifold influences that antibiotics have on the cardiovascular
Northwestern University, United States system and their effects on thromboinflammation.
Dalil Hannani,
UMR5525 Techniques de l’Ingénierie Keywords: platelets, germ-free mouse models, antibiotics, thrombosis, microbiota, vascular function
Médicale et de la Complexité
Informatique, Mathématiques et
Applications, INTRODUCTION
Grenoble (TIMC-IMAG), France
*Correspondence: During the past decade, microbiome research has started to explore how the densely colonized
Christoph Reinhardt gut resident ecosystem (microbiota) affects the host’s vascular physiology (1, 2). This symbiotic
christoph.reinhardt@ microbial community, whose composition is highly dependent on nutrition, interferes with host
unimedizin-mainz.de metabolism and constitutes a chronic inflammatory stimulus. Nowadays, the various influences
†These authors have contributed of the gut microbiome on vascular inflammatory phenotypes, such as atherosclerosis, myocardial
equally to this work infarction, arterial thrombosis, and stroke, are being increasingly recognized (3–6). Moreover,
recent research with germ-free (GF) mouse isolator technology revealed a reduced tendency of
Specialty section: arterial thrombus formation in different carotid artery mouse thrombosis models, arguing for a
This article was submitted to contribution of the gut microbiota to thromboinflammation (7–9).
Molecular Innate Immunity, Indeed, mounting evidence is linking the gut microbiota to the onset of cardiovascular disease
a section of the journal and arterial thrombosis (10–12). Among microbial-associated molecular patterns (MAMPs), the
Frontiers in Immunology
microbiota-derived choline-metabolite trimethylamine (TMA) that is produced by gut bacterial
Received: 03 June 2019 TMA-lyases (cutC) and targets the liver, is just emerging as a risk factor for thrombotic
Accepted: 28 August 2019 manifestations (8, 13–15). Through the action of flavin-containingmonoxygenase-3 (FMO3), TMA
Published: 12 September 2019
is oxidized into trimethylamine N-oxide (TMAO) (16), a metabolite that was reported to relieve
Citation: agonist-induced platelet activation (7). Remarkably, both elevated TMAO plasma levels and the
Bayer F, Ascher S, Pontarollo G and activation of Toll-like receptor (TLR) signaling by MAMPs were shown to accelerate atherogenesis
Reinhardt C (2019) Antibiotic
in the apolipoprotein E (Apoe)-deficient mice, which are currently used as an animal model of
Treatment Protocols and Germ-Free
Mouse Models in Vascular Research. atherosclerosis (17, 18). Clearly, the influences of the microbiota on atherosclerosis are not limited
Front. Immunol. 10:2174. to the TMAO pathway and pattern-recognition receptors, as additional microbiota-derived factors,
doi: 10.3389/fimmu.2019.02174 such as short chain fatty acids (SCFA), were recently identified (19).
Frontiers in Immunology | www.frontiersin.org 1 September 2019 | Volume 10 | Article 2174
Bayer et al. Germ-Free Mice in Vascular Research
Intriguingly, current research, investigating the impact of influences, affecting vascular physiology, cardiovascular disease
the gut microbiota on vascular phenotypes, has revealed development and thromboinflammation.
discrepancies between broad-spectrum antibiotic treatment
protocols and investigations on GF mouse models. To give an DIFFERENCES AND SIMILARITIES OF
example, antibiotic treatment of (Apoe)-deficient mice starting
GERM-FREE AND ANTIBIOTIC-TREATED
at weaning (4 weeks of age) resulted in a reduced development
of aortic root lesions at 20 weeks of age, when the mice MICE
were fed with a 1.0% choline-rich chow diet (17). Likewise,
Kasahara et al. reported reduced aortic root plaque areas together In 1885, Louis Pasteur claimed that a life without microbial
with reduced macrophage and CD4+ cell infiltration of the associates is not possible (27). Several years later, in the
aortic sinus, when studying GF Apoe-deficient mice kept on an 1940s, the first colonies of GF rodents were established.
irradiated chow diet and analyzed at 20 weeks of age (20). To Since its beginning, the usage of this technology became a
complicate the picture, the aortic root lesion size of GF Apoe- valuable model to understand how the microbiota impacts
deficient mice kept for 12 weeks on 1.2% choline-rich chow host physiology and disease processes (28). Gnotobiotic animals
diet starting at the age of 8 weeks was not different compared such as mice colonized with Altered Schaedler Flora (a defined
to conventionally raised (CONV-R) control mice (21). In the bacterial community) or germ-free (axenic) mice, lacking all
study conducted by Wright et al. no differences in aortic root microorganisms, are animal models characterized by a defined
lesion size was reported when GF and CONV-R Apoe-deficient colonization status (29). In particular, GF mice are bred and
mice were fed with a Western diet at weaning until 22 weeks kept for their whole lifetime in sterile isolators to prevent their
of age (22). Finally, similar to the study of Lindskog Jonsson exposure to microorganisms. These animals are a biological
et al., Stepankova et al. reported increased atherosclerotic lesion model system to either study the complete absence of microbes,
sizes in the thoracic aortas of GF Apoe-deficient mice after or to investigate the effects of colonization with selected
feeding with a low cholesterol diet for 3–4 months (3, 21). and known microbial species (e.g., in the mono-association
These seemingly controversial results could be explained by experiment) (30). However, the GF mouse model is a labor-
apparently minor differences in the experimental protocols, e.g., intensive technology, which requires permanent controls for the
the mouse line studied, the various diets used, the feeding time hygiene status of the isolators and special facilities (30, 31).
scheme applied, or the normalization of the measured lesion As an alternative and more basic method for the depletion
sizes. On the other hand, it is becoming increasingly clear that of microbiota, administration of broad-spectrum antibiotics is
the interpretation of data collected after microbiota decimation commonly used. In contrast to GF mice, antibiotic application
experiments by antibiotics should be considered with caution, does not lead to the depletion of all microbes, but can
given the experimental variables and the several side effects of selectively deplete different members of the gut microbiota
antibiotic treatment protocols. It should also be kept in mind that (32). Furthermore, to prevent dehydration during treatment,
antibiotic decimation of the microbiota using broad-spectrum Reikvam et al. recommended to gavage mice instead of delivering
antibiotics represents a selective pressure that favors overgrowth the antibiotics in the drinking water, or to combine both
of resistant bacterial taxa (23), which could in principle be applications (33).
causative at least for some of the described outcomes. Importantly, decimation of themicrobiota affects the anatomy
Moreover, recent research has implicated the intestinal and function of various organs such as liver and gut (34, 35)
microbiota in arterial thrombosis and it has been proposed (summarized in Figure 1). One of the most evident anatomical
that selective inhibition of the TMA-generating gut microbial changes is the enlarged cecum, observed both in GF and
enzymes could lower thrombotic risk (24). While TMAO has antibiotic-treated mice (30, 33). Furthermore, GF mice present
been shown to facilitate agonist-induced platelet activation elongated villus structures, a reduced villus width, and poorly
(7), it was demonstrated that microbiota-derived TLR-ligands developed capillary networks in small intestinal villi (1, 36, 37). In
promote the activation of hepatic endothelial cells, triggering addition to altered organ morphology and physiology, immune
von Willebrand factor (VWF) synthesis and release (8, 15, cell populations are influenced by antibiotic treatment (38–40).
25). In mouse models, both pathways promote carotid artery Because the GF animal model and antibiotic decimation of the
thrombosis and thrombus growth was reduced in the GF microbiota may result in different vascular phenotypes with
mouse model, linking the gut microbiota mechanistically to respect to anatomy and function (8, 17, 41, 42), it is important
arterial thrombosis (7, 8, 26). In the literature, in addition to be aware of the limitations of these experimental models that
to atherosclerotic phenotypes, there are numerous other enable the exploration of microbiota-host interactions.
examples on microbiota-dependent vascular phenotypes that
differ between gnotobiotic mouse models and depletion of the ANTIBIOTICS AND THEIR INFLUENCE ON
gut microbiota with antibiotics. MYELOID CELL FUNCTION
Here, we provide an overview on GF mouse isolator
technology and antibiotic treatment protocols for microbiota In addition to the resulting selection of resistant bacterial
depletion that are widely used to study vascular phenotypes. taxa (23), antibiotics severely influence the development of
We will explain the limitations of these mouse models, but the myeloid cell lineage (40), which may greatly hamper the
also describe the recently gained insights on microbiota-driven interpretation of results on vascular physiology. It is firmly
Frontiers in Immunology | www.frontiersin.org 2 September 2019 | Volume 10 | Article 2174
Bayer et al. Germ-Free Mice in Vascular Research
FIGURE 1 | Differences and similarities of germ-free (axenic) and broad-spectrum antibiotic-treated mice (decimation of microbes). While both techniques result in the
aberrant enlargement of the cecum, GF mice are less susceptible for colonic inflammation, and present elongated villus structures compared to antibiotic treated
animals. In both the animal models, absence of gut microbiota alters protein expression levels in the liver. In the bone marrow, the two mouse models present both
reduced granulocytes, monocytes and progenitor cells, but higher T cell levels. On the other hand, while B cells in GF animals are increased, in antibiotic-treated
counterparts they were reported to be decreased. Both the complete absence and the decimation of the gut microbiota influences vascular physiology and have an
effect on vascular disease. Neointimal hyperplasia, a proliferation and inflammation response to arterial injury, was increased in antibiotic treated rats. Antibiotic
treatment leads to a diminished development of aortic root lesion. Additionally, germ-free mice were protected from cardiac inflammation and arterial thrombus growth.
established that long-term treatment with beta-lactam antibiotics microbiota-induced sensing of peptidoglycan of nucleotide-
results in an inhibition of granulopoiesis (43). In this clinical binding oligomerization domain-containing protein-1 (NOD1),
study, the presence of granulocyte precursors with the lack of and by the production of interleukin-17A (IL-17A) in the
well-differentiated myeloid cells was observed in human bone ileum (44). The requirement of NOD1 sensing in mesenchymal
marrow aspirates, discussed by the authors as a myelotoxic stromal cells in the reduction of immune cells survival was
effect of beta-lactam antibiotics. Most important, in bone confirmed in a subsequent study with GF mice, showing
marrow cultures, Neftel et al. described a direct inhibitory that these models presented an overall reduced numbers of
effect of beta-lactam antibiotics on in vitro granulopoiesis. In hematopoietic stem and multipotent progenitor cells (39). By
addition, thrombocytopenia caused by beta-lactam antibiotic using the same antibiotic cocktail over 14 days and an alternative
treatment was remarked. Likewise, reduced neutrophil counts antibiotic cocktail containing ciprofloxacin and metronidazole,
were reported in the bone marrow of long-term antibiotics Josefsdottir et al. demonstrated that not only myeloid cell
treated mice, which was explained by neutrophil aging promoted numbers were dramatically reduced, but also the peripheral
by MAMPs derived from the gut microbiota (40). blood counts of various lymphocyte subsets along with the counts
In contrast, Hergott et al. demonstrated that antibiotic of hematopoietic progenitors were diminished (B-cells, CD4+
treatment accelerates the turnover of circulating neutrophils T-cells, CD8+ T-cells) (38). Interestingly, it was demonstrated
and inflammatory monocytes, which showed decreased cell that the cell cycle activity in hematopoietic stem cells and
counts in the blood, the bone marrow and the spleen (44). myeloid progenitors was increased in the antibiotic treated
In this work, it was described that long-term antibiotic group, excluding a direct myelotoxic effect of the antibiotics used
treatment (i.e. 3–4 weeks) with the frequently used cocktail by murine bone marrow culture experiments. Of note, there was
of neomycin, vancomycin, metronidazole, and ampicillin for no antibiotic effect on the hematopoietic progenitor population
microbiota decimation was associated with reduced survival of and on granulocytes in signal transducer and activator of
neutrophils and inflammatory monocytes, which was confirmed transcription 1 (Stat1)-deficient mice, indicating that upstream
in the GF mouse model. This phenotype was explained by pathways of this transcription factor may play a critical role.
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Bayer et al. Germ-Free Mice in Vascular Research
While the obvious question for the fundamental differences platelets are incorporated (55, 56). A more recent publication
between antibiotic treatment and GF housing state with revealed the underlying mechanism for vancomycin-induced
regard to hematopoietic functions is apparent, these important thrombocytopenia. Towhid et al. described platelet apoptosis
studies clearly demonstrate that antibiotic intervention has paralleled by mitochondria depolarization, activation of caspase-
manifold effects on innate immune cell types, which may 3, cell membrane scrambling and ceramide formation. Further
lead to inconclusive mechanistic data in hematologic research. tests revealed that Ca2+ is necessary for vancomycin to
Therefore, gnotobiotic models based on GF knock-out mouse cause these effects. They hypothesized that the cell scrambling
strains are essential to control for the validity of results obtained induces an accelerated clearance of platelets from the blood,
by antibiotic treatment protocols. resulting in the observed thrombocytopenia. Importantly, all
the described side effects are induced by exploiting vancomycin
concentrations normally reached during standard protocols of
ANTIBIOTICS AND THEIR INFLUENCE ON
antibiotic treatments (57).
CLOTTING, PLATELET FUNCTION AND Penicillin antibiotics may also have an effect on platelets.
THE VASCULATURE Clinically relevant concentrations of penicillin G and
carbenicillin seem to have a global effect on platelet membrane
Antibiotic Treatment Interferes With receptors, as platelets become less responsive to physiologic
Vitamin K Metabolism and Clotting Factors agonists and fail to aggregate with bovine factor VIIIa stimulation
The lipophilic essential vitamin K has two sources: it can be (45). Subsequently, Pastakia et al. have shown that penicillin
found in green leafed plants as phylloquinone (vitamin K1) and G inhibits thrombin-induced upregulation of GPIb-IX levels
as the microbial metabolite menaquinone (vitamin K2), which on the platelet surface (58, 59). In contrast, other beta-lactam
is produced by several bacterial species (45–48). Synthesis of antibiotics (ceftriaxone, ceftazidime, and aztreonam) did not
clotting factors II, VII, IX, and X and their post-translational inhibit platelet aggregation (60). On the contrary, ceftriaxone
modifications in the liver are vitamin K-dependent (45, 46). The (and to a lesser extent aztreonam) was shown to enhance
correlation between antibiotic treatment and increased bleeding platelet aggregation, but the underlying molecular mechanisms
risk due to vitamin K deficiency was already described in remain unclear. Another antibiotic presenting inhibitory effect
1952 by Dam et al. (47). Over the years, more case reports on in vitro platelet aggregation, even after oral treatment, is
of hypoprothrombinemic bleeding were published, involving metronidazole (61).
several classes of antibiotics (46, 48–50). The underlying mechanisms for thrombocytopenia (i.e., a
In this context, since 1980s, N-methylthiotetrazole circulating platelet count inferior to 150,000/mm3) are increased
(NMTT) cephalosporins (for example cefoperazone, cefotetan, platelet consumption/destruction or reduced platelet production.
moxalactam, and cefamandole) administration has been Linezolid, an antibiotic commonly used to treat vancomycin-
frequently reported to result in impaired hemostasis (45, 46, 50– resistant enterococci and methicillin-resistant Staphylococcus
52). Here, several mechanisms and speculations are discussed aureus infections, is known to induce myelosuppression, but is
in the literature, including: (a), interference of NMTT-side more frequently associated with thrombo- than pancytopenia
chain with vitamin K metabolism (45), (b), indirect inhibition (62). It was described that linezolid has no direct toxic effects
of vitamin K-dependent blood coagulation (50, 53), and on platelets and it does not affect the differentiation of
(c), reduction of menaquinone producing bacteria (50, 53). hematopoietic stem cells, but via phosphorylation of an enzyme
A more recent publication by Fotouhie et al. supports in relevant for platelet release, it induces thrombocytopenia (62).
part hypothesis (c), stating that several risk factors have to Although some influences of antibiotics on platelet functions
concur for the appearance of clinical symptoms (52). The were identified, the information on effects of antibiotic treatment
following risk factors were identified: insufficient dietary on the coagulation system remains sparse.
intake of phylloquinone, modification of normal gut microbial
communities via antibiotics, malabsorption of vitamin K, or
chronic liver disease (48). Hence, antibiotic treatment protocols Antibiotic Treatment and Vasomodulatory
may interfere with the synthesis of vitamin K-dependent blood Effects
clotting factors. Since the 1950s, aminoglycoside antibiotics are known to have
hypotensive effects and a negative inotropic effect on the heart
Antibiotic Treatment and Platelets (63). Gentamicin was reported to cause hypotension resulting
Other reasons for impaired hemostasis and thrombus formation from vasodilatation and relaxation of smooth muscle cells, thus
following antibiotics intake are thrombocytopenia or impaired yielding reduced vascular resistance, together with decreased
platelet function. In the early 1970s, the antibiotic ristocetin cardiac contraction force and bradycardia. The proposed
got withdrawn from clinical use because it induced platelet mechanism is an impaired Ca2+ influx. Neomycin, gentamicin
aggregation (54). As a result, many research groups then and, to a lesser extent, streptomycin, and kanamycin were
replaced ristocetin administration with vancomycin, as the demonstrated to have vasorelaxant effects on the cerebral arteries
two molecules share many chemical properties. However, even of dogs (64). All four antibiotics inhibited vasoconstriction after
if vancomycin itself is not able to directly trigger platelet administering depolarizing concentrations of potassium chloride
aggregation, it was reported to induce precipitates in which (64). Two mechanisms were proposed: (a), the inhibition of
Frontiers in Immunology | www.frontiersin.org 4 September 2019 | Volume 10 | Article 2174
Bayer et al. Germ-Free Mice in Vascular Research
phospholipase C (an enzyme that catalyzes the production of antibiotic treatment regimen exists (68) and residual surviving
the second messenger molecule inositol trisphosphate, which microbiota differs dependent on the animal facility and
drives the release of Ca2+ from the sarcoplasmatic reticulum) housing conditions, the reproducibility of this experimental
and (b), direct interference with Ca2+ influx by blocking L- procedure is at stake (69, 70). Therefore, all the functional
type voltage-dependent Ca2+ channels (64). Belus et al. revealed studies on the gut microbiota exploiting antibiotic treatment
the mechanism for the aminoglycoside antibiotics neomycin, should at least mention the limitations of this technique
gentamicin, and streptomycin negative inotropic effect. The and point out what kind of controls were included. In
resting and transient intracellular Ca2+ levels of rat ventricular contrast, the GF mouse model remains the state-of-the-art
myocytes are decreased, leading to reduced contractility approach for studying host-microbe and microbe-microbe
(65). Furthermore, high dosages of vancomycin (glycopeptide interactions, since mono-colonization, minimal microbial
antibiotic) and tobramycin (aminoglycoside antibiotic) have consortia or humanized microbial consortia are standardized
relaxing effects on the vascular smooth muscles (66). Not only experimental approaches (71). Therefore, if GF mouse
aminoglycoside antibiotics, but also beta-lactam antibiotics seem technology or gnotobiotic animal models are available and
to have an influence on Ca2+-influx in the vascular endothelium applicable, the use of antibiotic treatment should certainly
(60). This indicates that vascular function is significantly affected be reconsidered.
by the administration of several different antibiotics.
AUTHOR CONTRIBUTIONS
CONCLUSIONS
All authors listed have made a substantial, direct and intellectual
GF mouse models and antibiotic treatment protocols are contribution to the work, and approved it for publication.
frequently used as comparable methods to investigate
the effect of gut microbiota in triggering inflammatory FUNDING
vascular phenotypes. In times of spreading antimicrobial
resistance, every unnecessary use of antibiotics should be This work was supported by the BMBF (01EO1503), by a
carefully weighed up with the expected findings (67). As DFG Individual Grant to CR (RE 3450/5-2), a project grant
pointed out in this review, antibiotic treatment results in from the Boehringer Ingelheim Stiftung (Novel and neglected
significant changes of host physiology, but the underlying cardiovascular risk factors) to CR, and by an intramural Stufe 1
mechanisms remain often unclear. Since no standardized project grant (Inneruniversitäre Forschungsförderung).
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