nihms594078.pdf

Innate immune recognition of the microbiota promotes host-

microbial symbiosis

Hiutung Chu

and

Sarkis K. Mazmanian

Division of Biology, California Institute of Technology, Pasadena CA 91125 USA

Abstract

Pattern recognition receptors (PRRs) are traditionally known to recognize microbial molecules

during infection to initiate inflammatory responses. However, ligands for PRRs are not exclusive

to pathogens, and are abundantly produced by the resident microbiota during normal colonization.

Mechanism(s) that underlie this paradox have remained unclear. Recent studies reveal that gut

bacterial ligands from the microbiota signal through PRRs to promote host tissue and immune

development, and protection from disease. Furthermore, evidence from both invertebrate and

vertebrate models reveals that innate immune receptors are required to promote long-term

colonization by the microbiota. This emerging perspective challenges current paradigms in

immunology, and suggests that PRRs may have evolved, in part, to mediate the bidirectional

crosstalk between microbial symbionts and their hosts.

Conventional wisdom suggests that the immune system evolved to combat infection, and

that distinguishing between self and non-self molecules is a basic feature of innate

immunity. As proposed by Charles Janeway, the recognition of microbial molecules, termed

pathogen-associated molecular patterns (PAMPs), is critical in selectively driving immune

responses to infectious agents

. Studies identifying and characterizing host receptors that

recognize specific PAMPs, named pattern recognition receptors (PRRs), have provided

evidence that PRR signaling is critical in coordinating immune responses and protection

against pathogens (see reviews

2-5

). However, this view has been challenged by the

emerging appreciation that animals harbor a diverse and complex symbiotic microbiota

6-10

which normally does not trigger inflammation. PAMPs, by definition, are universally

conserved structures that are generally invariant and essential in all microorganisms. Thus,

PAMP expression is not limited to pathogens, but also common to the microbiota. As such,

it has been proposed that these molecules be renamed microbe-associated molecular patterns

(MAMPs)

. Furthermore, host PRRs are constantly exposed to MAMPs, in the absence of

infection. These molecules are largely provided by the commensal microbiota that colonize

our skin and mucosal surfaces. Despite the continuous presence of numerous MAMPs,

commensal microbes usually do not elicit inflammatory responses, but rather, may

contribute to various aspects of host development and enhanced immune function

Surprisingly, this beneficial influence is mediated, in part, by commensal stimulation of host

PRRs

Correspondences to: Sarkis K Mazmanian (sarkis@caltech.edu).

NIH Public Access

Author Manuscript

Nat Immunol

. Author manuscript; available in PMC 2014 July 24.

Published in final edited form as:

Nat Immunol

. 2013 July ; 14(7): 668–675. doi:10.1038/ni.2635.

NIH-PA Author Manuscript

How these molecules and receptors are able to achieve such divergent and opposing

responses between pathogens and symbiosis is a frontier in our understating of innate

immunity. It has been proposed that the context in which the host receives MAMP

stimulation dictates the quality of the immune response. During infection, MAMP signals

are received in the presence of other cues, such as cell damage caused by infection

and/or

cytosolic detection of MAMPs

, resulting in inflammation. During symbiosis, not only

does the microbiota generally not harm host cells and MAMPs are sensed in the absence of

exposed self-antigens, but it appears that some MAMPs directly promote beneficial

outcomes. This review will focus on how MAMP recognition by PRRs under

steady-state

conditions promotes immune development, protection from disease and maintains

homeostasis. The concepts presented here collectively demonstrate that PRRs may have

evolved in both the invertebrate and vertebrate immune systems to communicate with

commensals and maintain beneficial, symbiotic coexistence with the microbiota.

Pattern recognition in Drosophila promotes homeostasis

Extensive work using

Drosophila

as a model system has highlighted the important functions

of PRRs in host defense, as well as homeostasis. Toll, the founding example of a PRR, was

initially discovered in

Drosophila melanogaster

(the fruit fly)

. However, the realization

that

Drosophila

Toll does not directly recognize MAMPs, unlike the mammalian TLR

signaling pathway, left the open question of how a suite of bacterial ligands are recognized.

D. melanogaster

has 13 peptidoglycan recognition protein (PGRP) genes that are

alternatively spliced into 19 different proteins, one of the largest repertoires of PGRPs

currently known for any organism

. The role of

Drosophila

PGRPs as PRRs was

discovered during the identification of upstream receptors that activate the signal

transduction pathways, Toll and Imd (immune-deficiency)

18, 19

, which show high

similarities with the mammalian interleukin-1 (IL-1)-TLR and tumor necrosis factor (TNF)

pathways

. However, Toll does not function as a pattern recognition receptor as it does not

directly recognize MAMPs

. Instead, PGRP-SA and PGRP-SD form a complex upstream

of the Toll pathway

18, 19

. Upon peptidoglycan recognition (from Gram-positive bacteria),

PGRP-SA triggers a protease cascade, resulting in Toll dimerization, leading to activation of

the transcription factor NF-

B and production of antimicrobial peptides (AMPs)

Additionally, fungi and Gram-negative bacteria trigger the Imd pathway via PGRP-LC,

mediating innate immune responses

19, 22, 23

. Upon Imd pathway activation, NF-

signaling results in the production of AMPs, targeting invasive pathogens during infection

(Fig. 1a,b).

Intriguingly, in addition to regulating AMP production in response to infection, the Imd

pathway also shapes the response to commensal microbiota. The Imd pathway regulator,

Caudal, is important in shaping the composition of gut microbiota

. Caudal suppresses

Imd-dependent expression of AMPs by blocking AMP gene promotors. Additional negative

regulators of Imd, such as Pirk (Fig. 1b), sequester PGRP-LC in the cytoplasm to prevent

exposure to peptidoglycan and activation of the Imd pathway, which is critical in regulating

commensal populations, maintaining intestinal homeostasis and restraining overactive

immune responses

25, 26

Chu and Mazmanian

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In addition to sensing bacterial ligands and activating an inflammatory response, a subset of

Drosophila

PGRPs have amidase activity and participate in hydrolyzing pro-inflammatory

peptidoglycan into non-immunostimulatory fragments

27, 28

(Fig. 1b). These catalytic

PGRPs, such as PGRP-LB and PGRP-SC, serve to limit the availability of peptidoglycan

released from symbiotic gut bacteria, preventing over-activation of the Imd pathway and

dampening the immune response. In fact, deletion of PGRP-LB and PGRP-SC leads to a

ten-fold increase in AMPs compared to wild-type flies

, revealing the importance of

amidase PRGPs in detoxifying the biological activity of peptidoglycan and controlling an

overactive systemic immune response. While these studies highlight a role of PGRPs in

maintaining gut homeostasis by controlling the immune responses to commensal bacteria,

future studies are necessary to reveal how this pathway discriminates between symbiotic and

pathogenic bacteria. What is clear, however, is that sensing of bacterial ligands in

Drosophila

is critical for host-microbial symbiosis in the absence of infection.

Hydra TLR signaling promotes bacterial colonization

As a member of the second oldest phyla, Cnidaria,

Hydra

spp. serve as a simple model

system to study the evolutionary origins of commensalism (Fig. 1c). The intimate

relationship between

Hydra

and specific bacterial species reveal that the primitive immune

system of

Hydra

is able to recognize its symbionts, promoting a highly evolved partnership

with its host

Hydra

lacks migratory phagocytic cells and hemolymph, rather relying on

PRRs and the production of antimicrobial peptides by epithelial cells for immune

protection

. In the absence of conventional TLRs, analysis of the

Hydra magnipapillata

genome identified two genes with a Toll-interleukin-1 receptor (TIR) domain and

transmembrane domain (HyTRR-1 and -2), each lacking leucine-rich repeats (LRR) in the

extracellular region, which is typical of classical TLRs

. Two additional genes were

identified encoding for transmembrane proteins with TLR-related LRRs in the extracellular

region (HyLRR-1 and -2). Signaling by the HyLRR-2 and HyTRR-1 complex following

microbial pattern recognition recruits the adaptor protein, MyD88 and triggers the

production of AMPs, such as periculin-1

(Fig. 1d). In addition to bactericidial activity,

maternal expression of periculin-1 is involved in shaping the microbiome of the embryo

In transgenic polyps that overexpress periculin-1a, the bacterial community structure is

dramatically different, with a decrease in

-Proteobacteria and an increase in

Proteobacteria. Additionally, a significantly lower bacterial load was observed in transgenic

polyps compared to control polyps, indicating that periculin-1a plays a role in both

controlling and shaping bacterial colonization during

Hydra

development.

In addition to defense against pathogenic microbes, MyD88 signaling also contributes to

host-mediated colonization by commensal bacteria. Using a transgenic MyD88 knockdown

approach, the function of PRR signaling in

Hydra

was examined. While the overall

composition of the bacterial microbiota remained unchanged, MyD88-mediated PRR

signaling promotes reestablishment of bacterial homeostasis

. Thus studies in

Hydra

, a

primitive organism, reveal that recognition of commensal bacteria appears to be an ancient

function of innate immune signaling, suggesting that PRRs have evolved to mediate host-

microbe communication.

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PRR signaling maintains squid-

Vibrio

symbiosis

The relationship between the Hawaiian bobtail squid,

Euprymna scolopes

, and the Gram-

negative bacteria,

Vibrio fisheri

, is one of the most extensively studied examples of host-

microbial symbiosis to date

34-36

. The bioluminescent bacterium, found in seawater,

colonizes the crypts of the light organ in the juvenile host as a mono-specific

symbiont

(Fig. 1e). The light organ is continually exposed to numerous microbes in the

surrounding seawater, similar to the mammalian intestinal tract. Yet the squid is able to

selectively and exclusively harbor a single species of bacteria. This intimate symbiosis

allows the nocturnal squid to utilize light produced by

V. fisheri

as a counter-illumination

camouflage strategy, a clever adaptation to avoid detection by predators swimming below.

A series of elegant studies have shown that shortly after hatching of the juvenile squid, the

symbiont promotes colonization and beneficial co-existence with its host. MAMP signaling

mediates this specificity and initiates the colonization process, promoting host light organ

development

38, 39

(Fig. 1f). During the initial interaction with the squid, the bacterial

symbiont induces a sequence of events to promote colonization. The juvenile light organ has

a pair of ciliated appendages, which secrete mucus upon exposure to the bacterial trigger, a

peptidoglycan fragment known as tracheal cytotoxin (TCT)

. This mucus not only serves

as a chemoattractant for

V. fisheri

, but also a substrate for growth and aggregation to seed

the founding microbial population of the light organ. Following the initial colonization, the

symbiont then induces a second sequence of events driving the maturation of the squid light

organ by directing a switch from a permissive to a restrictive state to ensure

V. fisheri

colonization dominance. The bacterial triggers that drive this switch, lipopolysaccharide

(LPS) and TCT, are necessary to induce morphogenesis of the light organ via apoptosis and

regression of epithelial cells, leading to a loss of ciliated appendages and preventing entry of

other bacteria into the developed light organ

(Fig. 1e, f). Therefore, the squid utilizes

MAMPs as morphogens that direct normal developmental programs.

How are the symbiotic bacterial cues sensed by

E. scolopes

? PGRPs were implicated in

mediating responses to MAMPs during symbiosis

. To date, five EsPGRP have been

identified in the bobtail squid

39, 42

, although only EsPGRP-1 and EsPGRP-2 have been

extensively characterized, as both are expressed in the juvenile light organ. EsPGRP-1

expression is localized within the nucleus of the ciliated epithelium of the light organ, and

upon symbiont colonization, the bacterial signal TCT directs the loss of EsPGRP-1,

inducing the initiation of the apoptotic pathway

(Fig. 1f). Apoptosis of the ciliated

epithelium and the light organ appendage marks the completion of development and

colonization of the squid light organ. Alternatively, EsPGRP-2 possesses N-acetyl-muramyl-

L-alanine amidase activity, hydrolyzing peptidoglycan for degradation, diminishing its

toxicity and pro-inflammatory properties

(Fig. 1f). This activity is particularly important

V. fisheri

colonization occurs in high numbers and peptidoglycan is continually shed in

the light organ, posing the risk of inflammatory distress. Similar to

Drosophila

, the

EsPGRP-2 amidase that degrades peptidoglycan into non-immunostimulatory fragments

implicates its role in attenuating inflammatory responses, a prerequisite for symbiosis. Thus

V. fisheri

has evolved to co-opt innate immunity to promote colonization of the squid,

conferring benefits to both partners during mutualism. In conclusion, the collective data

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from the invertebrate models reveal PRRs are instrumental in host defense against infection,

the recent discovery of their role during symbiosis reveals the dual function of innate

immunity in responding to both pathogens and the commensal microbiota.

Maintaining tolerance in zebrafish

The zebrafish (Fig. 2a) has gained considerable attention as a model vertebrate system

due to ease of genetic manipulation

46, 47

, the ability to study germ-free conditions

, and

resemblance to mammalian physiology. Specifically, the conserved PRR system is highly

similar to other vertebrates, and the zebrafish model has proven to be a useful tool to

examine host-microbe interactions

. A recent study revealed a role for the zebrafish

intestinal alkaline phosphatase (IAP) to dephosphorylate the immunostimulatory LPS,

thereby modulating intestinal inflammation in response to commensal microbes, the primary

source of LPS in the gastrointestinal tract

(Fig. 2b). LPS is a major component of the

outer membrane of Gram-negative bacteria, found in both pathogenic and commensal

bacteria alike. However, LPS recognition by TLR4 results in induction of signaling cascades

that lead to activation of NF-

B and the production of pro-inflammatory cytokines. Upon

bacterial colonization (or administration of exogenous LPS), IAP expression and activity is

induced in a MyD88-dependent fashion

. TLR orthologs have been identified in the

zebrafish genome, with several TLR4 orthologs reported

and hypothesized to recognize

LPS (however, it is important to note that several studies have reported a lack of LPS

responsiveness in some zebrafish TLR4 paralogs

52, 53

). IAP deficiency results in excessive

intestinal neutrophil infiltration, a process involving functional MyD88 and TNF. Thus,

commensal-derived LPS signaling via TLR4-MyD88 leads to upregulation of IAP and

detoxification of LPS, and prevents inflammatory responses to the resident microbiota.

Additionally, following the initial discovery in zebrafish, IAP has been shown to be

involved in the maintenance of homeostasis in the bobtail squid

and mice

55, 56

. The

identification of IAP revealed yet another mechanism in which PRR signaling is crucial in

preventing uncontrolled inflammation in response to commensal microbes, thus promoting

colonization by the microbiota.

PRR signaling promotes intestinal homeostasis in mice

The mammalian intestine is home to an abundant and complex consortium of bacteria that

orchestrate important immune and metabolic functions within the host. Several studies in the

murine model (Fig. 2c) have shown that the composition of the microbiome, as well as the

spatial location of gut bacteria within the intestine, dictates the balance of tolerogenic (Fig.

2d) versus pro-inflammatory (Fig. 2e) immune responses in the gut. PRR signaling in mice

appears to play a key role in both directing the spatial segregation of the microbiota, as well

as shaping the composition of the commensal microbiota.

The gastrointestinal surface is coated with a layer of mucus

, largely limiting bacterial

access to the epithelium that separates the trillions of microorganisms in the gut lumen from

host tissues

(Fig. 3a). Additionally, secretory immunoglobulin A (IgA) maintains barrier

functions of the epithelium

59, 60

. This physical separation of microbiota and intestinal gut

mucosa is mediated by MyD88 signaling in intestinal epithelial cells

. In the absence of

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MyD88, commensal bacteria gain close proximity to the intestinal surface, resulting in a

100-fold increase in mucosal-associated bacteria compared to wild-type animals. Similar to

invertebrate models, MyD88 signaling results in production of AMPs in vertebrate systems

as well

. MyD88 signaling controls the production of several AMPs by specialized

intestinal epithelial cells termed Paneth cells, such as RegIII

, a C-type lectin that targets

Gram-positive bacteria (Fig. 3a)

61-63

. RegIII

-deficient animals exhibit a defect in the

spatial segregation of mucosa-associated bacteria with the microbiota penetrating the mucus

layer and making intimate contact with host tissue, a phenotype similar to intestinal

epithelial cell-specific MyD88-deficient mice

. The increased bacterial burden at the

intestinal surface of the small intestine results in immune activation, with elevated IgA and

1 responses. Upon depletion of the microbiota with antibiotic treatment, the elevated IgA

and T

1 responses in Myd88-deficeint mice were diminished. This suggests that commensal

microbiota drives MyD88 signaling via TLR stimulation under steady-state conditions,

inducing the expression of AMPs, such as RegIII

, to restrict bacterial colonization on

intestinal surfaces and limit an immune response to resident bacteria.

In addition to controlling the spatial segregation of intestinal bacteria, PRRs play a key role

in shaping the composition of the microbiota. The impact of this process is apparent in

studies of inflammatory bowel disease (IBD). Crohn's disease, a form of IBD, involves an

overactive immune response and impaired barrier function in the gut. Genome-wide

association studies have connected polymorphisms in the Nod2 innate immune receptor to

increased susceptibility to Crohn's disease

64, 65

. Nod2 is a member of the NLR family of

cytoplasmic proteins. It recognizes a derivative of peptidoglycan, muramyl dipeptide

(MDP), found in both Gram-positive and negative bacteria. Signaling of Nod2 requires the

adaptor protein Rip2k, which activates downstream signaling cascades involving NF-

Nod2-deficiency has been linked to impaired expression of Paneth cell

-defensins, a family

of AMPs

66, 67

. Accordingly, Nod2 was found to be required for the regulation of

commensal microbiota in the terminal ileum, where Nod2 expression is mainly localized

An increase in

Bacteroides

and

Firmicutes

was observed in Nod2-deficient animals

compared to wild-type littermates, revealing that Nod2 is involved not only in shaping the

composition of the microbiota, but also in restricting bacterial numbers in the ileal crypts.

Notably, Nod2-deficient mice are impaired in the ability to clear the pathobiont

Helicobacter hepaticus

. Similarly, Rip2k-deficient animals exhibit the same phenotype, and

Rip2k was recently identified as a Crohn's susceptibility gene

, corroborating this innate

immune signaling pathway in shaping the commensal microbiota.

Consistent with the role of Nod2 in promoting host-microbial symbiosis, the PGRP family is

involved in the regulation of commensal microbiota in mice. Mammals have four PGRPs

(PGRP-1 – 4), where PGRP-1, -3, and -4 are directly bactericidal, and PGRP-2 is an

amidase that hydrolyzes peptidoglycan – all of which are found at varying levels of

expression in the colon

. Animals deficient in any one of the PGRPs harbor a microbiota

that promote increased sensitivity to dextran sulfate sodium (DSS)-induced colitis, a mouse

model of IBD

. Indeed, germ-free animals inoculated with stool from PGRP-deficient

donor mice are more sensitive to DSS colitis compared to animals that received stool from

wild-type mice and display increased mortality, weight loss and colitis scores. Thus,

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mammalian PGRPs are important in shaping a homeostatic commensal microbiota and

preventing intestinal inflammation

The studies we outline involving MyD88-mediated induction of RegIII

, Nod2 and PGRPs

highlight the role of innate immune signaling in shaping the composition and localization of

the mammalian microbiota. It is important to emphasize that while this may be the result of

bidirectional crosstalk between symbiotic gut bacteria and the host, there is clearly a role for

pathogen detection by these immune regulators. Future studies aimed at understanding how

innate immunity shapes the microbiome, and whether gut bacteria actively signal through

PRRs to coordinate the immune response, will be critical. As such, the identification of

Nod1 as a receptor utilized by the microbiota to induce isolated lymphoid follicles (ILFs) in

the gut may provide a glimpse into a process where specific gut bacteria educate the

development of the immune system via PRRs

TLRs mediate host-protective immune responses

Several studies have identified a role for TLRs in mediating non-inflammatory immune

responses to the microbiota, challenging the paradigm that PRRs have evolved solely to

recognize and respond to pathogens. MyD88-deficient mice are more susceptible to DSS

colitis, suggesting that commensal bacteria may be directly recognized by TLRs under

steady-state conditions to mediate host-protective responses

. To corroborate this notion,

depletion of gut bacteria with antibiotics results in increased susceptibility to DSS;

remarkably, oral feeding of LPS and lipoteichoic acid (LTA) corrects this predisposition to

colitis, revealing that TLR ligands have beneficial effects on the host

. As DSS induces

intestinal injury, these findings suggest that TLR signaling by the microbiota leads to

maintenance of intestinal epithelial homeostasis in the absence of enteric pathogens.

Peptidoglycan, LPS and other widely conserved bacterial patterns mediate host-microbial

interactions that span from

Hydra

to mice. It appears, however, that species-specific TLR

ligands have also been evolved by the microbiota. The human commensal

Bacteroides

fragilis

produces polysaccharide A (PSA) that directs the maturation of the mammalian

immune system, specifically promoting the development and function of CD4

T cells

One outcome of this process is the activation of Foxp3

regulatory T (T

reg

) cells to produce

interleukin 10 (IL-10), which shapes a toloregenic immune response that is protective in

animal models of IBD and multiple sclerosis (MS)

76-78

(Fig. 3). Remarkably, PSA is a

unique TLR2 ligand found (to our knowledge) only in the human microbiome, which

orchestrates anti-inflammatory immune responses that ameliorate immune-mediated

diseases. TLR2-deficient mice are not protected by PSA against colitis

. PSA is delivered

by outer membrane vesicles (OMVs) that bud from the surface of

B. fragilis

and are

internalized by intestinal dendritic cells (DCs)

. TLR2-deficient DCs do not promote

Foxp3

reg

responses and IL-10 production

in vitro

, demonstrating that specific gut

bacterial molecules have evolved to promote benefits to the host via PRR signaling. The

concept of interkingdom communication via innate immune receptors was recently extended

Bifidobacterium breve

, a probiotic that signals through TLR2 to induce another subset of

regulatory T cells termed Tr1 cells

. While the bacterial ligand remains unknown,

B. breve

prevents intestinal inflammation by activating IL-10-producing Tr1 cells in the gut. The

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demonstration that

B. fragilis

and

B. breve

mediate beneficial adaptive immune responses

via TLR signaling reveals a deep co-evolutionary symbiosis founded on a “molecular

dialogue” using the PRR system.

PRRs on lymphocytes promote intestinal homeostasis

It is worth mentioning that the PRRs discussed up to this point are expressed among

epithelial cells and myeloid cells, which comes to no surprise, as PRRs are classic innate

immune signaling receptors (and invertebrates lack an adaptive immune system). However,

several studies have highlighted the importance of TLR-MyD88 signaling among

lymphocytes. In B cell-specific MyD88-deficient mice, bacteria disseminate to systemic

sites, such as liver or lung, following DSS-induced colonic damage – but not in epithelial or

DC-specific MyD88-deficient animals

. Further, it has recently been appreciated that T

cell subsets express functional TLRs

. Transfer of MyD88-deficient T cells into RAG-

deficient animals results in reduced intestinal inflammation

. Conversely, while classically

thought to promote immunity, it now appears that TLR signaling by T cells can restrain

inflammatory responses. For example, treatment of CD4

T cell subsets with a TLR4

agonist increases suppressive activity and enhances protection from colitis

. Therefore,

TLRs represent a dynamic signaling system that trigger various immune outcomes, and TLR

signaling directly by adaptive immune cells mediates reactions in the absence of innate

immune cells

86-88

Did gut bacteria evolve to induce anti-inflammatory responses through TLR signaling solely

to improve host health, or are there direct benefits to the microbe? PSA from

B. fragilis

enhances the anti-inflammatory function of T

reg

cells by signaling directly through TLR2 on

CD4

T cells

. Modulation of T

reg

activity achieves long-term colonization of the gut by

B. fragilis

by suppressing T

17 responses directed toward the microbe. Therefore, PSA is

distinct from TLR2 ligands of pathogens, which elicit inflammation, and sensing of gut

bacterial molecules to mediate colonization further supports a role for TLR signaling in

promoting host-microbe symbiosis. These findings imply that the host is not ‘hard-wired’ to

distinguish symbionts from enteric pathogens, and specific microbial ligands have evolved

to actively allow mutualism between mammals and beneficial bacteria of the microbiota.

Recognition of commensals promotes extra-intestinal immune function

While the beneficial effects of the resident microbes have been extensively studied within

the gut, a more diverse role for the microbiota in modulating systemic immunity is

emerging. With such abundant numbers of microbes inhabiting the mammalian gut, one can

imagine the high concentrations of MAMPs that may circulate to systemic sites.

Peptidoglycan from the gut microbiota is translocated into sera and bone marrow, priming

systemic innate immunity by enhancing neutrophil function

(Fig. 3b). Specifically,

peptidoglycan recognition is mediated via Nod1, which recognizes meso-diaminopimelic

acid (meso-DAP) containing cell wall fragments found on Gram-negative bacteria.

Treatment of mice with broad-spectrum antibiotics to deplete the microbiota, and thus

circulating peptodoglycan, leads to a significant reduction in neutrophil killing of both

Streptococcus pneumoniae

and

Staphylococcus aureus

. This defect in neutrophil function

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is rescued by administration of meso-DAP, which restores the innate immune response after

microbiota depletion. Bone marrow derived neutrophils from Nod1-deficient mice are also

defective in

S. pneumoniae

and

S. aureus

killing

. Hence, microbial products derived from

the gut microbiota serve to prime systemic immune responses via Nod1 signaling to rapidly

respond to pathogenic microorganisms.

The range of influence by PRR of commensals extends to protection against allergic

diseases

. Further support of systemic influences by the gut microbiota is revealed with

pattern recognition of commensals by B cells, which promotes systemic immune

development, resulting in decreased allergic inflammation

. Antibiotic-treated and germ-

free animals display increased serum IgE and basophil frequencies, implicating a role for the

microbiota in regulating T

2 responses and allergic inflammation

. B cell-intrinsic

MyD88 signaling is critical in controlling steady-state IgE levels in the serum, as well as

circulating basophil populations

(Fig. 3c). MyD88 deficiency in B cells results in higher

levels of serum IgE and increased basophil surface bound IgE frequencies

Mechanistically, MAMPs derived from commensal bacteria were found to limit basophil

proliferation and development in the bone marrow

. Thus, commensal-derived signals are

able to direct basophil development from bone marrow precursors via MyD88 signaling,

linking the gut microbiota to the regulation of hematopoiesis.

Finally, PRR signaling in the gut appears to modulate immune responses at distant mucosal

surfaces. Antibiotic-treatment of mice diminishes adaptive immune responses to intranasal

infection with influenza virus

. Both CD4

T cell and cytotoxic CD8

T cell (CTL)

responses to influenza were significantly reduced in antibiotic-treated animals together with

virus-specific antibody titers, resulting in increased viral load in the respiratory tract

Administration of TLR ligands intrarectally restored T cell and antibody responses to

influenza in the lungs of these antibiotic-treated mice, suggesting that PRR stimulation in

the gastrointestinal tract is important in priming immunity at other mucosal surfaces

. The

intestinal microbiota was found to be critical in providing signals necessary for

inflammasome-dependent cytokine secretion in response to influenza infection

. Two

signals are necessary for the production (signal 1) and processing (signal 2) of IL-1

and

IL-18. Signal 1 is provided through TLR stimulation, resulting in the expression of pro-

IL-1

and pro-IL-18. Signal 2 is mediated through inflammasome activation, leading to

caspase-1 cleavage to mature IL-1

and IL-18. The commensal microbiota provides signal 1

during colonization, initially priming the immune response whereupon influenza infection

(signal 2) subsequently activates the inflammasome to help clear virus from the lungs

(Fig. 3d). These seminal examples of recognition of molecular patterns from commensal

bacteria elegantly illustrate that the microbiota co-opts PRRs to mediate beneficial

outcomes.

Do hosts distinguish beneficial from harmful bacteria via PRRs?

We have discussed several examples for how the innate immunity system responds to the

microbiota. However, a fundamental feature of the immune system is the critical distinction

between the resident microbiota and invading pathogens, and recent data are beginning to

suggest PRRs play a role on this process. The discovery of an intimate symbiosis with

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specific bacterial species indicates that the immune system of organisms ranging from

Hydra

to mammals is able to recognize its microbial partners, or alternatively, that

symbiotic bacteria actively promote a highly evolved associations with their hosts. In the

case of

B. fragilis

, PSA signals through TLR2 to promote its symbiotic association with

mucosal tissue

, a finding that implies the host is not ‘hard-wired’ to distinguish symbionts

from enteric pathogens, and that specific microbial ligands have evolved to actively allow

mutualism between mammals and beneficial bacteria of the microbiota. Examples revealing

that the microbiota protects the host from infectious agents via PRR signaling further

supports the notion that innate immunity is a mechanism of host-microbial communication.

As PRRs have historically been studied in the context of infectious agents, these findings

suggest that PRRs serve the dual function of sensing both pathogens and symbionts, with

very different consequences for both microbes (clearance vs. symbiosis) and hosts

(inflammation vs. immune homeostasis).

Conclusions

Microbes dominate as the most abundant life form on our planet, occupying almost every

terrestrial, aquatic and biological ecosystem. Throughout their lives, all metazoans

continuously encounter microorganisms that are essential for health or cause death. The

immune system is charged with the task of distinguishing beneficial microbes from

pathogens, much like it distinguishes self from non-self antigens, to coordinate appropriate

immune responses. As symbiotic microbes share similar molecular patterns with pathogens,

why don't we immunologically reject our microbiota during lifelong colonization? It was

initially suggested that symbiotic bacteria were simply ignored by the host

. Emerging

evidence, however, reveals that certain microbes directly engage the immune system and in

some cases actively shape beneficial host immune responses. Symbiosis is often achieved

through microbial molecules that are sensed by PRRs, the same innate immune system that

has been studied for years in responding to microbial infections. As the first eukaryotes

evolved in a world inhabited by bacteria, PRRs appear to facilitate a wide range of

interactions with this diverse microbial world. This new perspective suggests that simply

distinguishing self from non-self is insufficient to explain the basic functions of the innate

immune system, and future studies should consider how and why we tolerate our ‘microbial

self’.

Acknowledgments

We are grateful to A. Khosravi, S. W. McBride, G.Sharon, Y. Lee and M. Flajnik for thoughtful comments on the

manuscript, and apologize to our colleagues whose work could not be discussed due to space constraints. Work in

the laboratory of the authors is supported by grants from the Burroughs Wellcome Fund, Crohn's and Colitis

Foundation and the National Institutes of Health (DK078938).

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