1) Which is the most correct description of amplicon sequencing (mentioned multiple times in this review paper)? a. Sequencing a specific marker (locus) for a single microbial species b. Sequencing t
Review ArticleArchaea: forgotten players in the microbiome
Corinna Bang* and Ruth A. Schmitz
Institute for General Microbiology, University of Kiel (CAU), Kiel, Germany
Correspondence:Ruth A. Schmitz ([email protected])
Archaea, the third domain of life containing unique membrane composition and highly
diverse cell wall structures, were only recognized 40 years ago. Initially identified in
extreme environments, they are currently recognized as organisms ubiquitously present
in most, if not all, microbiomes associated with eukaryotic hosts. However, they have
been mostly overseen in microbiome studies due to the lack of standardized detection
protocols and to the fact that no archaeal pathogen is currently known. Recent years
clearly showed that (i) archaea are part of the microbiomes associated with plants,
animals and humans, (ii) form biofilms and (iii) interact and activate the human immune
system. Future studies will not only define the host-associated diversity of archaea
(referred to as‘archaeome’) but also contribute to our understanding of the comprehen-
sive metabolic interplay between archaea and bacteria and the long-term gain insights
into their role in human health and their potential role(s) during disease development.
Introduction
Archaea belong to the third domain of life [1,2] and their phylogenetic distance to bacteria is reflected
by both genetic and structural differences. In contrast to their bacterial counterparts, archaea have not
only a unique and unusual membrane composition, but also higher diversity of different cell wall
structures, e.g. surface layer proteins and heteropolysaccharides, and also for some methanoarchaea,
pseudomurein [3–5]. In addition, many proteins involved in transcription and protein synthesis are
more similar to those found in Eukarya, e.g. RNA polymerase, ribosomal proteins and elongation
factors [6]. During the past 40 years, these properties were mainly thought to be unique adaptions to
the broad variety of extreme environments, in which archaea have been predominantly found [7].
However, in recent years, several studies discovered numerous archaeal species living under non-
extreme conditions, e.g. also in the marine environment (reviewed in refs [8,9]), and particularly, they
were found as part of the microbiome of multicellular host organisms ( plants and animals) (reviewed
in ref. [10], see also Mahnert et al. in this issue [61]). Although bacteria represent the major part of
complex microbial consortia that are associated with animals and plants, recently adapted molecular
tools revealed the additional presence of archaeal species within these communities. To this end, the
actual numbers of archaea in reported microbiomes might still be underestimated depending on the
methods used.
Overall, due to the rapid technological advancement of high-throughput amplicon-based sequen-
cing techniques and metagenome high-throughput sequencing (e.g. using the Illumina technique), the
number of microbiome research studies has increased enormously during the last decade. While the
use of bacteria-specific primers targeting various variable regions of the 16S rRNA gene (e.g. variable
regions 1–2or3–4) is well established and used in most projects and all sequencing facilities, the
development of primer sets targeting other host-associated microorganisms, such as archaea, is still
not methodologically sound. Archaea are often ignored in medical-based microbiome studies, most
probably due to the absence of a known pathogen. In addition, methodological problems, such as inef-
ficient DNA extraction, unsuitable primer choice or incompleteness of 16S rRNA gene reference data-
bases as well as much lower numbers of complete archaeal genomes, heavily impede data accuracy on
the‘archaeome’[11,12]. Consequently, research on the complexity of the archaeome is still in its
infancy and knowledge on archaeal diversity, particularly within the human microbiome, is mainly
*Present address: Institute of
Clinical Molecular Biology,
University of Kiel (CAU), Kiel,
Germany.
Version of Record published:
22 November 2018 Received: 28 May 2018
Revised: 1 October 2018
Accepted: 4 October 2018
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based onfindings by chance based on 16S rRNA amplicon-sequencing of bacteria and more recently on meta-
genomic approaches. However, several studies described suitable techniques for specific detection of archaeal
microbiome members during the last few years [12,13]. Of particular importance are the studies of Koskinen
et al. [12] and Pausan et al. [14], in which amplicon-based next-generation sequencing methods for archaea
associated with the human body were established and evaluated with samples from different human body sites
as well as quantification of archaea by qPCR. During these studies, an astonishing high number of archaeal
members within the human microbiome were discovered—and even more impressive, body site-specific
archaeal colonization was demonstrated (Figure 1). However, other studies developing archaea-specific
amplicon-based next-generation sequencing (NGS) methods also enrolled the need for habitat-dependent
adjustment; e.g. archaea-specific primers used for biogas reactors in a study by Fischer et al. [15] failed to
amplify archaea associated with humans (unpublished data).
Owing to the recent discoveries within thefield of archaeome research, which are based not only on the
overall presence of archaea as part of (most) microbial communities associated with eukaryotic hosts, but also
on the evaluation of their molecular interaction with their eukaryotic hosts, this review aims to summarize the
current knowledge on archaea as the forgotten players in microbiome research.
Occurrence of archaea as part of the microbiome
Although a relevant fraction of the microbial biomass on Earth (in the ocean and on land) is constituted by
archaea, their presence as part of naturally occurring microbial consortia associated with eukaryotic hosts has
been often ignored and thus underestimated for a long time. This fact might be mainly due to the complex cul-
tivation of most archaeal strains as well as their sophisticated handling under standard laboratory conditions.
However, the rapid advancement of archaea-specific new-generation sequencing methodologies enabled the
discovery of numerous unknown archaeal species associated with every ecosystem studied so far (reviewed in
ref. [10]). This fact is not surprising considering the enormous metabolic capabilities within the domain of
Archaea that comprise phototrophy, organotrophy and lithotrophy [7], and the fact that naturally most micro-
organisms are present and grow in multispecies consortia that enable syntrophic and often symbiotic relation-
ships between all community members.
With respect to their overall abundance in and on eukaryotes, archaeal species are associated with the micro-
biome of plants and of animals, including humans. Today, the‘metaorganism’concept is widely accepted con-
sidering each multicellular organism as a macroscopic host and its associated microorganisms and where the
microbiota is crucially involved in the development, growth,fitness/productivity, adaptation, diversification and
health of their eukaryotic host [16]. Composition of this microbiota depends not only on the genotype and diet
of the host [17] but also on the environment [18]. Particularly in higher eukaryotes, this microbiota is specific
for the diverse body parts [19]. These microbiome characteristics are true not only for bacteria, viruses and
fungi, but also for archaea (archaeome).
Plants
Diverse plant organs carry a unique and highly diverse archaeome; however, information on specific interac-
tions between archaea and plants is generally scarce. One of the most prominent examples of plant-associated
archaea are methanogens that form part of the anaerobic rhizosphere of rice growing in low-oxygen wetland
plants [20]. Particularly, Methanocellales (Rice Cluster 1) and Methanosaetaceae thereby contribute to 10% of
the worldwide methane release from rice plant photosynthates [21]. Additional community members of low-
oxygen roots and rhizospheres of plants are ammonium-oxidizing archaea, which are found in high abun-
dances and prevent ammonia loss in such environments [22–24]. Furthermore, there is some evidence that
archaea as part of the plant microbiome might be involved in the protection against stress and secondary
metabolite production [25]. There is more evidence to suggest important ecological roles of archaea associated
with plants; future research is required to explore the nature of these interactions.
Animals
Most of the multicellular organisms studied to date were shown to be inhabited not only by trillions of bacteria
and fungi, but also by archaea. The vast majority of archaea that were initially identified within animals belong
to the phylum Euryarchaeota, with a particular high percentage of anaerobically growing methanogens found
in their digestive tracts. Interestingly, the overwhelming majority of methanoarchaea in the gastrointestinal tract
of most animals are related to theMethanobrevibactergenus [26]. This genus was shown to enhance the
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efficiency of bacterial polysaccharide fermentation through the process of methanogenesis, since it is able to
consume several fermentation products of primary and secondary fermenters such as methanol, hydrogen and
carbon dioxide [27,28].Methanobrevibacterspecies are thus highlyflexible in forming syntrophic interactions
with a broad range of ( fermenting) bacteria—generating and releasing methane into the surrounding. Since
methane is considered to be a climate relevant gas ( greenhouse gas) with one of the highest global-warming
potential [29], the substantial methane release originating from livestock is an environmental concern and is
frequently considered and discussed [30]. This is mainly because methane is predominantly produced within
the rumen of ruminants before it is released via eructation and breathing [31]. On the one hand, methane yield
could be highly influenced by diet composition. On the other hand, substances inhibiting methanogenesis are
under development these days. One example for this is the recently found 3-nitrooxypropanol, which specific-
ally targets the key enzyme of methanogenesis (methyl-coenzyme M reductase) and thereby reduces enteric
methane emissions from livestock without apparent negative side effects for the animals [32]. Besides
Methanobrevibacterspecies, 16S rRNA amplicon-based sequence analysis demonstrated the occurrence of
Methanosphaera,Methanomicroccous,Methanobacterium,Methanomicrobium,Methanomassiliicoccusand
Methanosarcinaspecies in the rumen of cattle, yaks, sheep, reindeers, goats, water buffalos and deer (reviewed
in ref. [10]). In fact, archaeal diversity was also shown for other mammals including horses, pigs and kangar-
oos, though again almost only methanogens were detected therein. Also for non-mammals, a huge diversity of
archaea was demonstrated by 16S rRNA gene-based analysis—besides birds and reptiles, methanogenic
archaea and some crenarchaeal species were found in termites and shrimps (reviewed in ref. [10]). Since all
these studies have been performed based on 16S rRNA gene sequence analysis using mostly universal prokary-
otic primers, the archaeal diversity in the gastrointestinal tract (GIT) of animals might be much higher. For
example, a recent study focused on archaeon-specific primers to unravel the archaeal diversity in great apes and
found at least 200 archaeal operational taxonomic units (OTUs) in the GIT of orangutans, gorillas, bonobos
Figure 1. The human archaeome.
Timeline overview of the discovery of archaeal species found as a part of the human microbiome at different sites. Since the
first discovery of archaeal species in the early 1980s by cultivation, further detection took almost 30 years. Though already in
the late 2010s, some uncultivated species were detected by metagenome and amplicon-sequencing, only the development of
optimized NGS methods in 2017 was able to unravel a niche differentiation of human-associated archaeal communities
including not only methanogenic archaea, but also Thaumarchaeota as well as archaeal species from the DPANN
superphylum.
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and chimpanzees with the highest diversity observed for orangutans [13]. Phylogenetic diversity analysis of the
species revealed numerous OTUs out of three major groups: Methanobacteriales, Methanomassiliicoccales and
Thaumarchaeota. Most interestingly, the authors showed that overall the archaeal diversity was at least twofold
less in humans when compared with their closest relatives—the great apes. However, this study clearly
demonstrated that several archaeal species are conserved across all great ape species, implying a continual
essential function of archaea in the breakdown of dietary compounds throughout hominoid evolution [13].
Humans
Alreadyintheearly1980s,thefirst archaeal member has been shown to be part of the human intestine—
Methanobrevibacter smithii[33]. Although only few years later, an additional archaeal strain could be
isolated from stool samples (Methanosphaera stadtmaniae[34]), it took over 30 years to develop
archaea-specific detection methods in order to unravel their overall high diversity in and on humans
(Figure 2)[12]. In the meantime, the application of universal primer pairs and the development of metagen-
ome analysis revealed the existence of several further archaeal strains—but with overall low reproducibility
within different studies due to missing standardized methods (reviewed in refs [10,35]). However, over the
past few years, three studies using optimized DNA extraction and 16S rRNA gene sequencing demonstrated
overall massive and unexpected archaeal diversity not only in the intestine, but also on human skin, lungs
and nose [12–14]. But, Raymann et al. used primer pair Arch516F/Arch915R for exploring the archaeal
diversity in stool samples from great apes and humans [13]; Koskinen et al. and Pausan et al. focused on
nested-PCR approaches using various specific primer pairs for studying the human microbiome [12,14]. All
three studies developed specific computational analysis pipelines adapted to the poor deposition of archaeal
sequences in reference databases. With respect to the overall diversity in the intestine, these studies clearly
demonstrated thatM. smithiiandM. stadtmaniaeare only two exemplary species out of the diverse group of
theMethanobacteriales. In addition, they found a massive diversity ofMethanomassiliicoccalesstrains, a
group whosefirst memberMethanomassiliicoccus luminyensishas been initially found by cultivation in a
study only a few years ago [36,37]. However, only Koskinen et al. and Pausan et al. were able to describe add-
itional thaumarchaeal and archaeal signatures of the DPANN superphylum (
Diapherotrites, Parvarchaeota,
Aenigmarchaeota, Nanoarchaeota, Nanohaloarchaea) by their method in the intestine. This might be due not
only to the used method, but also to the sample selection. In contrast to the study of Raymann et al., who
used exclusively stool samples, the other two studies focused on biopsy samples of different GIT locations in
addition to stool samples.
As detected earlier by Moissl-Eichinger and her collaborators, several thaumarchaeal species were shown
associated with the human skin [12,38]. Most interestingly, Woesearchaeota, Aenigmarchaeota and unclassified
species from the DPANN superphylum were also identified in skin samples. Furthermore, Koskinen et al. and
Pausan et al. explored the archaeal diversity of nose and lung samples [12,14]. Interestingly, the nose was
shown to carry a huge abundance of methanoarchaea comparable to that of the intestine, though
Thaumarchaeota were found in higher fractions. In contrast, the lung samples carried roughly no methanoarch-
aea, small fractions of Thaumarchaeota, but vast abundances of Woesearchaeota belonging to the DPANN
superphylum as well as unclassified members of this superphylum. In summary, an archaea-specific landscape
of four different human body sites could be demonstrated by these studies, which was not previously recog-
nized and which will change current research on the human-associated archaea (archaeome) as part of the
human microbiome (Figure 2, see also Mahnert et al. in this issue [61]).
With respect to the overall functional role of these human-associated archaeal strains, more detailed studies
dealing with metabolic-association pathways between detected archaea and bacteria are urgently needed.
Indeed, it is well known that methanoarchaea in the intestine can form syntrophic relationships with primary
and secondary fermenters resulting in lower hydrogen partial pressures potentially influencing the overall com-
munity structure present in the GIT [28,39]. For thaumarchaeal species, ammonia oxidation has been specu-
lated as a very important metabolic process. However, to date, information on metabolic capabilities of
Woesearchaeota and other species belonging to the DPANN superphylum is rare and thus needs to be uncov-
ered to identify their overall importance as part of the human microbiota.
Biofilms
In nature, microorganisms are predominantly surface-associated together with other microbial strains in mixed
consortia or biofilms. For archaea in general, biofilm formation has been shown for various species (reviewed
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in ref. [40]), particularly in extreme environments such as deep lakes, aquifers and/or hydrothermal vents. Even
for the well-studied mucosa-associated strainsM. smithiiandM. stadtmaniae, it was not only shown that their
genetic information potentially allows adhesion to surfaces [39,41], but that they are indeed able to generate bio-
films on abiotic surfaces [42](Figure 2). It is thus hypothesized that methanoarchaea in the GIT might also
occur in microbial biofilms settled on the mucosal surface. The huge diversity of archaeal strains on biopsies of
the human intestine found in the study of Koskinen et al. [12] might also argue for this hypothesis. Indeed,
strong adhesion ofM. stadtmaniaeon human immune cells and on epithelial cell lines was frequently observed
during stimulation experiments in our group. It has also been noted that, possibly caused by disposal of exopoly-
saccharides,M. stadtmaniaecells easily aggregate in biofilms (Figure 2). Further evidence comes from studies
elucidating oral biofilms, in whichMethanobrevibacter oralishas been observed in at least every second patient
suffering from periodontal disease [43]. In addition, species of theMethanomassiliicoccusgenus were found to
be part of these subgingival plaques [44]. Biofilm formation appears also most probably for thaumarchaeal
species on the human skin and for Woesearchaeota in the human lung, though this has to be confirmed in the
near future from ongoing studies. One recent study unraveled metabolic potentials of 19 Woesearchaeotal organ-
isms in distinct biotopes [45]. By applying additional co-occurrence analysis, the authors demonstrated the exist-
ence of potential consortia between Woesearchaeota and anaerobic methanoarchaea in anoxic biotopes and
hypothesized a syntrophic metabolic model for a Woesearchaeota-methanogens consortium [45]. Since
Woesearchaeotal species have been found alongside methanoarchaea in GIT biopsy samples [12] such a consor-
tium is plausible, though this warrants future studies.
As a part of the eukaryotic-associated microbiota, archaeal strains are exposed to various defense systems of
their hosts. Onefirst line of epithelial defense is the secretion of antimicrobial agents such as antimicrobial
Figure 2. Biofilm formation of human mucosa-associated archaeal strains.
Structures of static biofilms (incubation time of 3 days) formed byMethanosphaera stadtmaniae(A1–A3) and
Methanobrevibacter smithii(B1–B3). Analysis followed using either a confocal laser scanning microscope (ZEISS LSM 700)
after staining with the Live/Dead viability Kit showing live cells stained with Syto9 ( green) and dead cells with propidium iodide
(red) (A1andB1, site view 2D;A2andB2, 3D) or a scanning electron microscope (Hitachi S-4800) (A3andB3). Scale bars
represent 5mm(A3andB3).
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peptides (AMPs) [46]. Susceptibilities of various mucosa-associated methanoarchaea to several AMPs were
determined during the last years and differed markedly among the tested strains [47,48]. Most
mucosa-associated methanoarchaeal strains belong to the order Methanobacteriales, whose members have a
pseudomurein-containing cell wall and distinct lipid compositions [49]. Interestingly, these strains were more
resistant against the lytic effects of human-derived AMPs than, for example, members of the Methanosarcinales
or Methanomassiliicoccales. However, differences were also found within this order withM. stadtmaniaebeing
the most resistant member to these lytic effects. During these studies, it became evident that particularly this
strain might have evolved various resistance strategies response to AMPs—with biofilm formation and
enhanced heteropolysaccharide production in detail. As mentioned before,M. stadtmaniaeis considered to
form biofilms on gut epithelial cells. Since the concentration of AMPs close to the epithelia is thought to be
higher compared with the concentration in the lumen,M. stadtmaniaemight be exposed to higher concentra-
tions of human AMPs and has thus possibly evolved additional genomic and morphological adaptations.
Interestingly, mucosa-associated microorganisms are currently thought to be much more important regard-
ing the molecular interaction with the host’s immune system, since they are closer to the epithelium compared
with microorganisms in the lumen [50]. Thus, such an interaction between archaea and their human host is
most likely and will be discussed in the following section.
Interaction with the human immune system
Owing to the lack of recognized pathogens within the domain Archaea, their immunological potential was not
questioned for many years. However, during the last years, a few studies demonstrated activation of humane
immune cells and pro-inflammatory cytokine responses by peripheral blood mononuclear cells and by
monocyte-derived dendritic cells (moDCs) particularly resulting after inoculation with the gut-associated
archaeonM. stadtmaniae[51,52]. This process was shown to be initiated after phagocytosis and endosomal
lysis ofM. stadtmaniaeby human immune cells. Following increased phosphorylation of mitogen-activated
protein kinases (MAPKs) and transcription factors, high release of pro-inflammatory cytokines (including
interleukins and interferons) was observed [51,53]. Although both studies showed recognition and not only
innate, but also adaptive immune response by human immune cells in response to this archaeon, no specific
receptor involved was identified. Since archaea in general lack common bacterial-associated molecular patterns
such as lipopolysaccharides, peptidoglycan or bacterial-likeflagellin, the question for an archaea-associated
molecular pattern and the receptor involved in recognition emerged. In 2017, our collaborators and we demon-
strated that the purified RNA ofM. stadtmaniaeserves as a potent immune stimulator in immune cells.
Moreover, this study identified Toll-like receptors (TLRs) 7 and 8 as the respective involved pattern-recognition
receptors by the use of human myeloid cells and respective mutants [53]. Thus, for thefirst time, specific rec-
ognition of and response to an archaeon by human cells at the molecular level was proved (summarized in
Figure 3). Additionally, this molecular interaction was shown to result in the triggering of the NLRP3 inflam-
masome via a TLR8-dependent pathway, which resembles characteristics of both so far known inflammasome
activation pathways and thus might represent an archaea-specific one.
Still, the question remains unclear if (or if not) archaea might be involved in the development of diseases.
Several studies proposed an involvement of methanoarchaea in gastrointestinal diseases such as colon
cancer, obesity, anorexia and inflammatory bowel disease (IBD) (reviewed in ref. [35]). However, most of
these studies lack appropriate methods for the quantification of archaea and thus reported controversial
results of overall archaeal abundances in studied patient groups—generally failing to conclude any associ-
ation to disease development. On the other hand, the study of Blais-Lecours et al. [54] appropriately deter-
mined increased the abundance ofM. stadtmaniaein patients suffering from IBD, although a relatively
small patient size group was examined. In relationship to the observed high pro-inflammatory potential and
inflammasome activation that was observed for this strain, a potential involvement in the development and
manifestation of IBD should not be ruled out. In addition, strong B- and T-cell responses within the drain-
ing lymph nodes that further result in adaptive immune responses such as selective immunoglobulin secre-
tion were obtained and strongly argue for systemic immune responses that might occur ifM. stadtmaniae
enters the bloodstream [51,52,54].
Besides potentially direct involvement in inflammatory diseases, methanogenic archaea have shown to
support growth of facultative pathogenic bacteria. In the oral cavity, as one example,M. oraliswas identified in
many studies dealing with periodontal and endodontic diseases [55–57]. Owing to its increased abundance and
its metabolic capabilities, it was hypothesized that this methanoarchaeon is able to enhance growth of
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potentially pathogenic anaerobic bacteria by significantly lowering the redox potential in the microhabitats
[55,57]. Arguing in this direction, archaea might represent metabolic key species within microbial consortia
inhabiting the human body ecosystem [58,59] and thus might indeed have the potential to contribute to
disease development. However, this hypothesis urgently warrants future studies that include newly established
methods and optimized protocols.
Conclusions and outlook
With the fast development of high-throughput sequencing analysis during the last decades, our knowledge on
the bacteriome that is involved in health and disease of all multicellular organisms has increased rapidly.
However, due to the lack of standardized protocols for the detection of further members of the complete
microbiome, such as fungi, archaea and viruses, the overall interactions between microbes among each other
and interactions between their hosts remain largely unclear. Complete understanding of effectiveness and inter-
play of present microorganisms is though urgently needed to unravel mechanisms, which are important for
prevention of disease development. Thus, future studies should additionally use established workflows for 16S
rRNA gene-based high-throughput analysis specific for archaea besides the already well-established ones for
bacteria. Results from these studies will not only gain insights into the potential roles of archaea during disease
development, but might also contribute to our understanding of the comprehensive metabolic interplay
between archaea and bacteria, which is certainly an unanswered question important for overall human health
and development.
Summary
Archaea are present in most, if not all, microbiomes associated with eukaryotic hosts—
plants animals and humans
Archaea have been mostly overseen in previous microbiome studies due to the lack of stan-
dardized detection protocols and due to the fact that no archaeal pathogen is currently
known.
Figure 3. Molecular cross-talk between archaea and human immune cells.
Phagocytosis ofM. stadtmaniaeleads to the activation of innate and adaptive immune responses. (A) moDCs were stimulated
withM. stadtmaniaefor a period of 4 h in order to determine phagocytosis. Formed phagolysosomes in moDCs were stained
with LysoTracker Red DND-99 during the time of incubation, and cells were labeled with Hoechst for DAPI staining. Scale bar
indicates 10μm. (B) Schematic simplification of immune cell activation. After phagocytosis ofM. stadtmaniae, phagolysosomes
are formed in order to degrade archaeal cells. Subsequently, delivered RNA derived from archaea is recognized by Toll-like
receptors 7 and 8 in the endosomes leading to intracellular signaling cascades. Thefinal activation of MAPKs and transcription
factorsfinally result in the release of pro-inflammatory cytokines, antimicrobial peptides and the expression of modulatory
surface molecules in order to active adaptive immune responses.
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Archaea interact with their human host and activate the human immune system.
Future studies on the host-associated archaeomes will allow to gain insights into their role in
human health and their potential role(s) during disease development.
In the future, interactions between bacteria, archaea, virus and fungi as well as interactions
between their hosts have to be studied to reach a complete understanding of the comprehen-
sive metabolic interplay between microorganisms and hosts.
Complete understanding of effectiveness and interplay of present microorganisms is urgently
needed to unravel mechanisms, which are important for the prevention of disease
development.
Abbreviations
AMPs, antimicrobial peptides; GIT, gastrointestinal tract; IBD, inflammatory bowel disease; MAPKs,
mitogen-activated protein kinases; TLRs, Toll-like receptors; moDCs, monocyte-derived dendritic cells; NGS,
next-generation sequencing; OTUs, operational taxonomic units.
Funding
This work was supported by the DFG (German Research Foundation) [HH2758/4-2, SCHM1051/11-2].
Competing Interests
The Authors declare that there are no competing interests associated with the manuscript.
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