REVI E W Open Access
Role of intestinal microbiota and
metabolites on gut homeostasis and
human diseases
Lan Lin
1*
and Jianqiong Zhang
2*
Abstract
Background: A vast diversity of microbes colonizes in the human gastrointestinal tract, referred to intestinal
microbiota. Microbiota and products thereof are indispensable for shaping the development and function of host
innate immune system, thereby exerting multifaceted impacts in gut health.
Methods: This paper reviews the effects on immunity of gut microbe-derived nucleic acids, and gut microbial
metabolites, as well as the involvement of commensals in the gut homeostasis. We focus on the recent findings
with an intention to illuminate the mechanisms by which the microbiota and products thereof are interacting with
host immunity, as well as to scrutinize imbalanced gut microbiota (dysbiosis) which lead to autoimmune disorders
including inflammatory bowel disease (IBD), Type 1 diabetes (T1D) and systemic immune syndromes such as
rheumatoid arthritis (RA).
Results: In addition to their well-recognized benefits in the gut such as occupation of ecological niches and
competition with pathogens, commensal bacteria have been shown to strengthen the gut barrier and to exert
immunomodulatory actions within the gut and beyond. It has been realized that impaired intestinal microbiota not
only contribute to gut diseases but also are inextricably linked to metabolic disorders and even brain dysfunction.
Conclusions: A better understanding of the mutual interactions of the microbiota and host immune system, would
shed light on our endeavors of disease prevention and broaden th e path to our discovery of immune intervention
targets for disease treatment.
Keywords: Intestinal microbiota, Gut homeostasis, Immune responses, Regulatory T cells (Tregs), Dendritic cells
(DCs), Metabolic disorder
Background
Human gastrointestinal tract is known to host trillions
of microbes [1, 2], the number of which reaches approxi-
mately 10
14
cells in the entire gut of a healthy individual
[1]. Amongst these resident gut microbes, 4000 strains
are present constituting the intestinal microbiota [3].
Through co-evolution, the host has not only tolerated but
also evolved to necessitate the colonization by beneficial
microbes, terme d commensals, for m ultifaceted aspects
of immune development and function [4]. Defect s in
mucosal tolerance are believed to cause human disor-
ders including inflammatory bowel disease (IBD) exem-
plified by Crohn’s disease and ulcerative colitis [5].
As the first line defense of host against pathogens, innate
immune responses rely on a family of receptors known as
pattern recognition receptors (PRRs) including Toll-like re-
ceptors (TLRs), and nucleotide-binding oligomerization
domain-like (NOD-like) receptors. TLRs are key innate im-
mune receptors to perceive pathogen-associated molecular
patterns (PAMPs), which are specific pathogenic “molecular
signature” [6]. Subsequent to sensing microbial PAMPs,
TLRs enable the initiation of inflammatory responses
and eventually eliminate the pathogenic invaders. The
phenomenon that both commensals and pathogenic mi-
crobes can interact with host immune system through
* Correspondence: linl04@seu.edu.cn; zhjq@seu.edu.cn
1
Department of Bioengineering, Medical School, Southeast University,
Nanjing 210009, People’s Republic of China
2
Key Laboratory of Developmental Genes and Human Disease, Ministry of
Education, Department of Microbiology and Immunology, Medical School,
Southeast University, Nanjing 210009, People’s Republic of China
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Lin and Zhang BMC Immunology (2017) 18:2
DOI 10.1186/s12865-016-0187-3
similar conserved ligands —PAMPs, drives us to address
such question as to how host immune system differentiates
pathogens from commensals at the intestinal mucosal
interface exposed to continuous microbial stimuli.
Severe host tissue damage may be resulted from immune
hypersensitivity towards intestinal flora or dietary nutrients.
To circumvent this, the host implements a variety of regu-
latory mechanisms for organ homeostasis maintenance.
Regulatory T cells (Tregs) serve one such mechanism as
evidenced by the otherwise catastrophic consequences
under genetic/or physical ablation of the Treg population
[7]. Tregs are the specialized T cells with immunosuppres-
sive activity through an array of mechanisms that influence
both dendritic cells (DCs) and effector cells [8].
DCs, constituting the firs t point of contact between
gut commensals and mammalian immune system [9],
are central to harmonizing the host tolerance (to self-
antigens) with host immunity (to pathogens) in the per-
ipheral lymphoid tissues [10]. DCs are able to present
innocuous self and non-self antigens in a manner that
promotes tolerance [8]. The predominant mechanism by
which DCs induce and maintain peripheral tolerance
involves the gener ation of Tregs from naïve T cells, the
expansion of pre-existing Tregs, the production of IL-10
and other immunomodulatory cytokines, and the pro-
motion of T cell anergy or depletion [11, 12].
Immature DCs (iDCs), present in all peripheral tissues,
are capable of acquiring antigenic material from their
microenvironment, but are poorly immunogenic (also
called tolergenic). The pathogenic microbial signals can
be sensed by iDCs for propelling their conversion into
mature DCs, which, present within secondary lymphoid
organs, could obtain the capacity of promoting T cell
immunity but lose the capacity of antigen upta ke [13].
In short, DCs are able to trigger seemingly opposite
states —— immunity and tolerance depending on differ-
ent microenvironment conditions [13]. Intestinal DCs,
together with macrophages and epithelial cells, may
serve as sentinels in the microbial milieu of intestine.
The exceptional characteristic of intes tinal microenvir-
onment necessitates host immune system not only to
avoid the hyper-immune reactivity to the gut lumen
laden with commensals and dietary components etc,
but also to retain t he capacity of fighting pathogenic
microbes.
Extensive studies in germ-free (GF) mice, in the past
decades, have demonstrated an indispensable role of
microbiota in shaping host intestine immune system
[14]. In contrast to conven tionally raised mice, GF mice
have hypopla stic Peyer’s patches, decreased numbers in
IgA-secreting plasma cells and lamina propria CD4+ T
cells, relatively structureless se condary lymphoid tissues
(i.e. spleen and peripheral lymph nodes) and other im-
munologic defects. Inoculation of a healthy murine
commensal microbiota into GF mice has been found to
reverse these immunologic deficiencies [14]. In addition
to immunostimulatory effects as afore-described, certain
members of intestine microbiota may exert immuno-
modulatory actions that involve reversible alterations in
differentiation/or effector function of host immune cell
subsets , exemplified by segmented filamentous bacteria
(SFB), Bacteroides fragilis, Clostridia XIVa and IV.
This a spe ct will be re viewed in details in the Se ction
of “ Commensals and gut homeostasis”.Furthermore,
compelling evidence with microbiota-derived metabo-
lites, mainly referring to small-molecule constituents
such as short-cha in fatty acids (S CFAs) and quorum
sensing signals, has established the import ance of
chemical signaling in communicating microbial rich-
ness and composition with host. And microbial me-
tabolites can be sensed by host immune system in
addition to PAMPs , which in turn influences host
immune responses. Butyrate, a kind of microbiota-
originated SCFAs containing four carbons, has been
recently reported to have immunomodulatory effects
on intestinal macrophages and thereby conferring
them hyporesponsive to commensal microbiota resid-
ing in the colon [15]. Notwithstanding, the underlying
mechanisms as to how intestinal microbiota, as a
whole, educates host immune system within the gut
and beyond, as well as the ide ntification of bacterial
spec ies-specific contributi on during the microbiota-
host immunity interaction still await to be elucidated.
As a paradigm of b acterial strain-spe cific mole cules,
butyrate acts as HDAC inhibitors and ligands for G-
protein-coupled re ceptors (GP CRs) and is considered
as a crucial signaling molecule affecting host immune
responses [16].
Majority of human lymphoid tissu e is located within
the lining of the major tracts that are predominant
entry sites of microbes into host, referring to respira-
tory, gastrointestinal (GI) and genitourinary tracts,
which are collectively termed the mucosa-a ssociated
lymphoid tissues. The intestinal mucosa appears to be
the largest surface within human body facing enor-
mous amount s of microbial antigens either resident
or ingested. This re view summari zes the recent ad-
vances in the field of microbiota and their products
interacting with the GI mucosal immune system. We
aim to p rovide an update into the research progress
relevant to the possible con tributions of microbiota
and their product s to the intestinal homeostas is main-
tenance, which, hopefully, would facilitate the virtual
discovery and insightful design of promising thera-
peutic target s for treatment of human disorders in as -
sociation with intestinal dysbiosis and autoimmunity,
such as type 1 diabetes ( T1D), systemic immune syn-
dromes (i.e. IBD etc.) and even colorectal cancer.
Lin and Zhang BMC Immunology (2017) 18:2 Page 2 of 25
Review
Effects of gut microbe-derived nucleic acids on immunity
TLR9 senses unmethylated cytidine-phosphate-guanosine
(CpG) motifs of DNA
Host cells can initiate innate immune signaling upon
recognition of PAMPs (viz. conserved structures in
pathogenic microbes), of which nucleic acids are key
structures. The receptors for foreign nucleic acids in-
volve members of TLRs including TLR3, TLR7, TLR8,
and TLR9, and intracellular DNA sensors [17]. The endo-
somal localizations of TLR3 [activated by double-stranded
(ds) RNA], TLR7 and 8 [activated by single-stranded (ss)
RNA], TLR9 [activated by CpG motifs within ssDNA]
reflect the protective mechanism whereby unwanted inter-
actions of TLRs with self-nucleic acids could be cir-
cumvented. Another protective mechanism may involve
modifications of mammalian nucleic acids [18]. Detec-
tion of intracellular p athogens is achie ved by those
endosomally-expressed TLR3, and TLRs 7–9, eventu-
ally leading to the clearance of pathogens.
Among those TLRs in association with intracellular
invaders, TLR9 and signaling thereof are more exten-
sively investigated than others. Unmethylated CpG dinu-
cleotides that are enriched in prokaryotic DNAs of
intestinal flora, can be sensed by TLR9. Constitutive gut
flora DNA sensing is found to modulate the equilibrium
between regulatory and effector T cells in the murine GI
tract, suggesting the gut flora DNA as an immunological
adjuvant [19]. Moreover, unmethylated CpG has been
reported of immunostimulatory effect in mice and other
mammals, as well a s in-vitro human cell lines [20, 21].
Bacterial DNA and synthetic oligonucleotiodes (ODN),
which contain unmethylated CpG in common, are able
to activate the innate and adaptive immune system via
plasmacytoid dendritic cells (pDCs) and macrophages in
mammals [22].
Upon CpG stimulation, a signaling cascade is elicited
that leads to the production of proinflammatory cyto-
kines and type I IFNs [23, 24], the latter being predom-
inantly secreted by pDC. These soluble components
coordinate early innate and sequential adaptive immune
responses [24]. The tissue specificity and cellular pattern
of TLR expression are believed to vary with different
species, e ven in mammals. For instance, murine TLR9 is
expressed not only in pDC and B cells as human TLR9,
but also in macrophages and myeloid DCs as well [21].
Thus one should be cautious with predicting the effects
of TLR9 activation on humans by extrapolating from
murine data.
TLR9 signaling and autoimmunity
Several lines of evidence have revealed inappropriate
activations of TLR7, TLR8, and TLR9 in systemic lupus
erythematosus (SLE) and several other autoimmune
diseases. T and B cells specific for self-antigens can be
detected in healthy individuals but do not suffice to
provoke the development of autoimmune diseases. In
contrast, SLE individuals are reported to suffer from im-
paired clearance of apoptotic cells and increased circu-
lating levels of nucleosomes [18]. CpG motifs derived
from apoptotic debris could activate TLR9, notably
under the circumstance that they are converted into im-
mune complexes with pre-existing auto-antibodies,
followed by B cells stimulation through both TLR9 and
B-cell receptor, which in turn leads to autoimmunity
and systemic autoimmune disease [25]. In such SLE
individuals, host DNA/antibody complexes trigger and
sustain a pD C- and B cell-mediated immune response
[26, 27], which indicates self-DNA as damage-associated
molecular pattern (DAMP) modulating self-destructive
chronic immune activation [28].
Studies have characterized several proteins as inter-
mediate cofactors (chaperones) to initiate the TLR9 acti-
vation upon perception of CpG, which include human
cathelicidin LL-37 and the high mobility group box
(HMGB). Cathelicidin LL-37, a cationic peptide with
wide-spectrum antimicrobial activities, is chemotactic
for neutrophils, ma st cells, monocytes, and T cells [29].
In psoriasis patients LL-37 may serve as a converter of
self-DNA into pathogenic ligand due to its binding to
self-DNA. The resultant LL37-DNA comple x is found to
promote the endocytosis pathway and to sustain TLR9
activation by modifying the interaction with DNA [30].
Accordingly, LL37 facilitates TLR9 activation of self-
DNA and synthetic CpG DNA. CpG islands under
studyweredemonstratedtobeimmunostimulatory
when coupled with human cathelicidin L L-37, strongly
suggesting the critical role of LL-37 in the immunosti-
mulatory effe cts of CpG motif-containing mtDNA
fragments [24].
TLR9 recognizes not only CpG motifs “embedded” in
bacterial DNA but also similar motifs in verte brate
DNA, pinpointing that the same receptor perceives
PAMP and DAMP, which complies with the notion that
the immune system is more concerned with entities that
do damage than those that are foreign [31]. It also indi-
cates that similarities exist between pathogen-induced
responses and non-infectious inflammatory responses
[32]. CpG motifs in prokaryotic DNA are known to be
20 times more enriched than those in mammalian DNA;
and even found in the mammalian genomic DNA, they
are specifically methylated . MtDNA is predominantly
unmethylated in view of its prokaryotic origin based on
endosymbiosis theory [33]. Once eukaryotic cells undergo
apoptosis, necrosis, necroptosis and cell death in associ-
ation with autophagy, mtDNA is released acting as
mtDAMP. On the other hand, neutrophils, basophils and
eosinophils, upon stimulation, can release extracellular
Lin and Zhang BMC Immunology (2017) 18:2 Page 3 of 25
traps of mtDNA or genomic DNA. These traps contain
such antimicrobial peptides as cathelicidins and cell-
specific proteases. A growing body of evidence has re-
vealed that elevated levels of circulating mtDNA may
cause systemic inflammatory response syndrome in
trauma patients and also act as a trigger of neurodegener-
ation [34, 35]. The pDC may be stimulated by an influx of
neutrophils releasing extracellular traps of DNA [36], and
are subsequently recruited to the colorectum and gut
mucosa [37, 38]. Accordingly, fragmented mtDNA bearing
CpG motif may contribute to driving a Th1 polarization in
autoimmune disorder and chronic viral diseases [24].
TLR9 signaling and gut cancinoma
CpG-mediated TLR9 activation may serve as a new
therapeutic target for several cancerous conditions. The
potentials of TLR9 agonists (synthetic CpG ODN) in
therapeutic applications for infectious diseases, cancer
and asthma/allergy have been reviewed elsewhere [21].
Recent studies have determined the association of
TLR9 polymorphisms with human susceptibility to gas-
tric carcinoma and its prognosis in Chinese population
[39]. The work by Wang et al strongly suggests that
TLR9-1486C carriers are associated with an increased
risk and poor prognosis of gastric carcinoma in human
[39]. Another inde pendent group ha s shown the cell-
invasion-inducing potential of short DNA sequenc es and
bacterial DNAs in tested cell lines including human
MDA-MB-231 breast cancer, OE33 esophageal ad-
enocarcinoma, AGS gastric adenocarcinoma and Caco-2
colon carcino ma [40]. An array of DNA ligands was
investigated including short DNA sequences such as
CpG-ODN M362, 9-mer (hairpin), human telomeric se-
quence h-Tel22 G-quadruplex, and bacterial DNAs de-
rived from Escherichia coli and Helicob acter pylori [40].
DNA-induced invasion was shown to be suppressed by a
broad-spectrum matrix metalloproteinase (MMP) inhibi-
tor and in part by chloroquine, suggestive of its medi-
ation through en dosomal signaling, TLR9 and MMP
activation. This notion is reminiscent of the association
of MMP overexpression with brea st cancer brain metas-
tasis [41]. The work by Kauppila et al. strongly suggests
that bacterial DNAs could act as endogenous and
invasion-triggering TLR9 ligands and thereby accelerat-
ing local progression and metastasis of carcinoma in the
digestive tract [40].
Immunmodulatory effects of gut microbiota-derived DNA
It awaits elucidating how commensals communicate
with host cells to ensure immune homeostasis. As widely
known, commensals contain abundant oligodeoxynu-
cleotides with CpG motifs (CpG-ODN), the latter of
which has been shown to co-stimulate T cells analogous
to that achieved by CD28 stimulation, irrespective of
antigen-presenting cells (APCs). The inherent attribute
of CpG-ODN towards T cells may con tribute to the
adjuvanticity potency of microbital DNA and CpG-ODN
on T-cell-mediated immune responses [42].
Recent work with gut commensals demonstrated gut-
floral-derived DNA (gfDNA) as an intrinsic adjuvant to
prime intestinal immune responses, in which TLR9 signal-
ing is involved [19]. TLR9 signaling was found to lower
the activation threshold by negative and positive expan-
sions of Treg and Teff (effector T) cells, respectively, in
the gut, and was liable to development of protective re-
sponses upon oral infection. Thus gfDNA is strongly
suggested to be a natural adjuvant for initiating protective
immune responses via modulation of Treg/Teff cell ratio
at sites of mucosal challenge, which offers promising
therapeutic strategy against oral infection [19].
Another independent work with suppressive DNA
motifs of the commensal origin showed that these
oligonucleotides could contribute to the hierarchy of
commensal-derived signals and thereby facilitating the
maintenance of gut immune homeostasis [43]. Com-
mensal DNA w as pre viously demonstrated to promote
intestinal immunity. It has been unve iled that the
bacterial species-spe cific immunomodulatory capacity
of DNA is correlated with the frequency of motifs
exerting immunosuppressive action [43]. For instance,
DNAs of Lactobacillu s species, together with those of
various probiotics, are known to be enriched in sup-
pressive motifs capable of inhibiting DC activation
within lamina propria of intestine. In addition, im-
munosuppressive oligonucleotides could sustain Treg
cell convers ion during inflammation, and regulate
pathogen-triggered immunopathology and colitis. Collect-
ively, these data pinpoint the suppressive DNA motifs to
be a molecular ligand typical of commensals, supporting
the notion that a balance between stimulatory and re-
gulatory DNA motifs may contribute to the induction of
controlled immune responses in the GI tract, thereby
influencing the gut homeostasis maintenance [43]. The
above-mentioned findings suggested that the endogenous
regulatory DNA motifs abundant in specific commensal
bacteria could serve as the core of DNA-based vaccines of
therapeutic value.
Effects of gut microbial metabolites on immunity
Gut microbiota-released metabolites, which are interme-
diates and/or end products of dietary constituents by
commensal metabolism, may exert indispensable actions
on host immunity and health [44]. Some of anaerobic
gut microbes have the potential of converting dietary
carbohydrates into organic acids including lactate, and
short-chain fatty acids (SCFAs), the latter principally re-
ferring to acetate, propionate and butyrate. In mammals
butyrate serves as a predominant energy substrate for
Lin and Zhang BMC Immunology (2017) 18:2 Page 4 of 25
colonocytes and enterocytes [45, 46]. Propionate is
primarily absorbed by the liver while acetate is released
into peripheral tissues [46]. In human gut, bacteria of
the Bacteroidetes phylum secrete high levels of acetate
and propionate whereas those of the Firmicutes phylum
generate large amounts of butyrate [47]. Commensurate
with increasing interests of SCFAs pertinent to Bacteroi-
detes and Clostridia phylum in the human gut [48],
some other metabolites may serve as signaling molecules
for inter-bacterial communication and quorum sensing.
Among them are bacterial QS signals (also called autoin-
ducers, or pheromones) and poly-γ-glutamic acid, the
latter of which was recently characterized in Bacillus
subtilis. Significant progress has been made to broaden
our understanding about the modulatory effects of these
gut microbial metabolites on host immunity (Fig. 1).
Short-chain fatty acids (SCFAs)
A growing body of evidence has revealed SCFAs as key
metabolic and immune mediators [49, 50]. Distinct
bioactivities of SCFAs may be attributed to their rapid
absorption, with approximately only 5% being excreted
through faeces. For instance, apart from the predominant
energy source for the colonocytes, butyrate is found to be
anti-inflammatory mainly through the suppression of NF-
κB [51], be capable of altering the composition of the
mucus layer by inducing mucin synthesis [52–54] and of
exerting anti-cancer activities [55, 56]. Functional links are
thus proposed among the dietary components, the gut
microbiota composition and host immune homeostasis,
inferring that different dietary preference may, at least
partially, contribute to the racial and regional divergence
in human population susceptibility to autoimmune disor-
ders, inflammatory diseases and cancers.
Further studies with the experimental models of colitis
and arthritis, have demonstrated that SCFAs could bind the
GPR43 (G protein-coupled receptor 43, also known as free
fatty acid receptor 2, FF AR2) and thus repressing the
inflammation via interaction with FFAR2-expressing neu-
trophils [49, 57]. SCFAs, as endogenous ligands for the G-
protein-coupled receptors GPR41 (viz. FF AR3) and GPR43
(viz. FFA R2), have been illustrated to mediate an array of
Fig. 1 Gut microbial metabolites and host immune responses. CSF: Competence and sporulation factor; IECs: Intestinal epithelial cells. G
−
and G
+
indicate gram-negative and -positive bacteria, respectively
Lin and Zhang BMC Immunology (2017) 18:2 Page 5 of 25