nihms371633.pdf

Intestinal Microbes Affect Phenotypes and Functions of

Invariant Natural Killer T cells in Mice

Gerhard Wingender

*,†

Dariusz Stepniak

Philippe Krebs

‡,

Lin Lin

Sara McBride

Wei

Jonathan Braun

Sarkis K. Mazmanian

, and

Mitchell Kronenberg

*,1

Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, 9420

Athena Circle, La Jolla, CA 92037, USA

†

Institute for Molecular Medicine, University of Bonn,

Bonn, Germany

‡

Department of Genetics, The Scripps Research Institute, 10550 North Torrey

Pines Road, La Jolla, CA 92037, USA

Division of Experimental Pathology, Institute of Pathology,

University of Bern, Bern, Switzerland

Department of Pathology and Laboratory Medicine,

University of California Los Angeles, 10833 Le Conte Ave; CHS 13-222, Los Angeles, CA 90095

Division of Biology, California Institute of Technology, 1200 E. California Bl., Pasadena, CA

91125, USA

Abstract

Background & Aims—

Invariant natural killer T (

NKT) cells undergo canonical, V

14–J

rearrangement of the T-cell receptor (TCR) in mice; this form of the TCR recognizes glycolipids

presented by CD1d.

NKT cells mediate many different immune reactions. Their constitutive

activated and memory phenotype and rapid initiation of effector functions after stimulation

indicate previous antigen-specific stimulation. However, little is known about this process. We

investigated whether symbiotic microbes can determine the activated phenotype and function of

NKT cells.

Methods—

We analyzed the numbers, phenotypes, and functions of

NKT cells in germ-free

mice, germ-free mice reconstituted with specified bacteria, and mice housed in specific pathogen-

free (SPF) environments.

Results—

SPF mice, obtained from different vendors, have different intestinal microbiota.

NKT

cells isolated from these mice differed in TCR V

7 frequency and cytokine response to antigen,

which depended on the environment.

NKT cells isolated from germ-free mice had a less mature

phenotype and were hypo-responsive to activation with the antigen

-galactosylceramide. Intra-

gastric exposure of germ-free mice to

Sphingomonas

bacteria, which carry

NKT cell antigens,

fully established phenotypic maturity of

NKT cells. In contrast, reconstitution with

Escherichia

coli

, which lack specific antigens for

NKT cells, did not affect the phenotype of

NKT cells. The

Author for correspondence: La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, San Diego, CA, 92037, Tel: (858)

752- 6540, Fax: (858) 752-6990, mitch@liai.org, URL: http://www.liai.org.

Competing Interest

The authors have no competing interests regarding this work.

Author contributions

G.W. and M.K. designed research, G.W., D.S., P.K., L.L. B.W. and S.M. conducted experiments and acquired data, G.W. and M.K

wrote the manuscript, S.K.M., J.B. and M.K. obtained funding. All authors approved the manuscript.

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NIH Public Access

Author Manuscript

Gastroenterology

. Author manuscript; available in PMC 2013 August 01.

Published in final edited form as:

Gastroenterology

. 2012 August ; 143(2): 418–428. doi:10.1053/j.gastro.2012.04.017.

NIH-PA Author Manuscript

effects of intestinal microbes on

NKT cell responsiveness did not require toll-like receptor

signals, which can activate

NKT cells independently of TCR stimulation.

Conclusions—

Intestinal microbes can affect

NKT cell phenotypes and functions in mice.

Keywords

GalCer; T-cell activation; mucosa; TLR

Introduction

Invariant natural killer T (

NKT) cells are a unique subset of T lymphocytes characterized

by the expression of an invariant TCR rearrangement, V

14-J

18 in mice (V

NKT

cells) and an orthologous V

24-J

18 (V

) in humans, and the recognition of antigens

presented by CD1d, a non-polymorphic MHC class I-like antigen-presenting molecule

1–4

CD1d binds lipid structures, and one of the best-studied

NKT cell antigens is

galactosylceramide (

GalCer), a synthetic version of a glycolipid originally isolated from a

marine sponge

NKT cells express surface molecules characteristic of antigen-experienced lymphocytes,

and antigenic stimulation leads to the rapid induction of effector functions by

NKT cells

such as the production of T

1- and T

2 cytokines and potent cytotoxicity

1–4

. As a

consequence of their vigorous early response,

NKT cells have been implicated in diverse

immune reactions, including the pathogenesis of inflammatory diseases of the liver,

pancreas and intestine. Similar data in human patients are relatively sparse, still they suggest

comparable roles for

NKT cells in different contexts. In the case of inflammatory bowel

disease, most of the findings are consistent with a protective role for

NKT cells during T

mediated diseases and a deleterious one in T

2 diseases

5,6

. The fact that

NKT cells can

cause either beneficial or detrimental effects in different disease models illustrates their

dichotomous function and their ability to polarize the ensuing immune response in either a

1- or T

2 - direction

. In contrast to this diversity in the functional outcome, a protective

role of

NKT cells has almost uniformly been reported both in animal models and in human

patients with type I diabetes

8,9

In addition to

GalCer, glycolipid antigens known to stimulate the majority of

NKT cell

have been reported in two types of bacteria. One type is

Sphingomonas/Sphingobium

species, which have glycosphingolipids similar to the original sponge antigen

10,11

. The

second type,

Borrelia burgdorferi

, is the causative agent of Lyme disease

. Several

additional pathogens have been reported to have glycolipid antigens that activate

NKT

cells, include

Leishmania donovani

and

Helicobacter pylori

13–15

, but in such cases it may be

only a subset of the cells that are stimulated. More generally, the distribution and prevalence

NKT cell antigens in the microbiota and in the wider environment, as well as their role in

NKT cell function under non-inflammatory conditions remain to be determined.

The constitutively activated phenotype of

NKT cells has been attributed to the presence of

self-agonist glycolipid ligands that drive the selection of these cells and stimulate them

continually in the periphery. While there is some evidence for this, we set out to determine

the role of intestinal bacteria in shaping the phenotype and function of

NKT cells. Our

hypothesis was substantiated by the previous finding that ribosomal DNA sequences from

Sphingobium yanoikuyae

and related species are found in the mouse intestine

16,17

suggesting these could be commensal organisms. Furthermore, sequences from the related

bacteria

Novosphingobium aromaticivorans

have been found in the human intestine

. In

addition, we showed previously that intragastric challenge with

S. yanoikuyae

stimulated

peripheral

NKT cells

. This indicated that gut-derived

NKT cell antigens are capable of

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activating peripheral

NKT cells. Here we show that intestinal bacteria can modulate the

phenotype, TCR V

-usage and the immune responses of

NKT cells, and that antigens from

commensals that engage the semi-invariant TCR are a likely contributing factor.

Material and Methods

Mice and cell lines

Mice were housed under SPF conditions at the animal facilities of the La Jolla Institute for

Allergy and Immunology (La Jolla, CA), the The Scripps Research Institute (La Jolla, CA)

and the Department of Pathology and Laboratory Medicine (Los Angeles, CA) or housed

under germ-free conditions at the California Institute of Technology (Pasadena, CA) in

accordance with the Institutional Animal Care Committee guidelines. C57BL/6 mice were

purchased from the Jackson Laboratory (Bar Harbor, ME) or from Taconic Farms (Hudson,

NY); Swiss Webster germ-free and SPF housed animals from Taconic Farms; and

B6.129S1-

Il12b

tm1Jm

/J (

IL-12p40

−/−

) from Jackson Laboratory. MyD88 and TRIF (

Lps2

)

double deficient mice

and restricted flora (RF) mice have been described previously

20,21

Sphingobium yanoikuyae

and

Escherichia coli

were purchased from American Type Culture

Collection (Manassas, VA). The T cell lymphoma RMA was virally transfected to stably

express CD1d as previously described

, resulting in the line RMA-CD1d.

Reagents and monoclonal antibodies

-galactosylceramide (

GalCer) was obtained from the Kirin Pharmaceutical Research

Corporation (Gunma, Japan). CFDA-SE was obtained from Invitrogen (Carlsbad, CA).

Monoclonal antibodies (mAbs) against the following mouse antigens were used in this

study:

-integrin (M293), CCR9 (9B1, eBioCW-1.2), CD1d (1B1), CD3

(145.2C11,

17A2), CD4 (GK1.5, RM4-5), CD5 (53-7.3), CD8

(53-6.7, 5H10), CD19 (1D3, 6D5),

CD25 (PC61.5), CD44 (IM7), CD45R (B220, RA3-6B2), CD69 (H1.2F3), CD103 (2E7),

CD122 (TM-b1), CD127 (A7R34), TCR

(H57-597), NK1.1 (PK136), V

2 (B20.6), V

(TR310), GM-CSF (MP1-22E9), IL-2 (JES6-5H4), IL-4 (11B11), IL-13 (eBio13A), IFN

(XMG1.2) and TNF

(MP6-XT22). Antibodies were purchased from BD Biosciences (San

Diego, CA), BioLegend (San Diego, CA), eBioscience (San Diego, CA) or Invitrogen.

GalCer-loaded CD1d tetramers were produced as described

Cell Preparation,

in vivo

challenge and flow cytometry

Single-cell suspensions from liver, spleen, thymus and intestine were prepared as

described

24,25

In vivo

cytotoxicity assays and cell staining for flow cytometry were

performed as reported previously

NKT cells were activated in vivo by i.v. injection of

GalCer and analyzed 90min later. Bacterial suspensions were gavaged using a

20Gx1.5 feeding needles.

Statistical analysis

Results are expressed as mean ± standard error of the mean (SEM). Comparisons were

drawn using a two-tailed Student t-test or ANOVA test. p-values <0.05 were considered

significant and are indicated with *p<0.05, **p≤0.01 and ***p≤0.001. Each experiment was

repeated at least twice, and background values were subtracted.

Results

Distribution and phenotype of intestinal iNKT cells

We examined the frequency of

NKT cells in different sites in the intestine, because there

have been conflicting data on the frequency and distribution of these cells in the gut

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mucosa

. We analyzed

NKT cells in the LPL and IEL compartments of the small (SI) and

large intestines (LI).

NKT cells could readily be detected in LPL and IEL from both, small

and large intestine of specific-pathogen-free (SPF) C57BL/6J mice (Figure 1A). The signal

was specific for

NKT cells, as indicated by the low background when utilizing unloaded

CD1d tetramers (Figure 1A). We consistently observed a higher frequency of

NKT cells in

the small intestine than in the large intestine; and a higher frequency in the LPL than in IEL

(Figure 1B). It is notable that the frequency of

NKT cells in the SI-LPL was comparable to

the value in the spleen. The majority of intestinal

NKT cells were CD4

and NK1.1

, with

the exception of decreased NK1.1 expression by LI-IEL

NKT cells (Figure 1C and

Supplemental figure 1A). Most intestinal

NKT cells were CD69

, CD44

and CD122

similar to their splenic counterparts (data not shown). Furthermore, only a small fraction of

intestinal

NKT cells expressed CD103 (Supplemental figure 1B, 1C), and intestinal

NKT

cells also were mostly negative for the

-integrin (data not shown). In contrast, we detected

expression of CCR9 on

NKT cells derived from the LPL (Figure 1D).

Environmental influences on the responsiveness and V

-usage of iNKT cells

It has been reported that the housing conditions provided by the commercial vendors at

Taconic Farms (Tac) and the Jackson Laboratory (Jax), and the consequent difference in the

intestinal microbiota, can impact the composition and function of conventional, CD4

lymphocytes in the intestine

. To test if such differences could influence the responsiveness

of peripheral

NKT cells, we directly compared their phenotype and function in SPF C57BL/

6 animals from both vendors. The percentage of

NKT cells in the thymus, spleen and liver

of Tac mice tended to be lower than in Jax mice, a difference which was statistically

significant in all experiments however only in the spleen (Figure 2A). Primary V

NKT

cells use commonly three V

chains paired with the invariant TCR

-chain. V

8.1/2 is most

abundant, comprising approximately 55% of the total, with the other principal ones being

7 (14%) and V

2 (7%)

1,26

. The analysis of the V

-usage of the

NKT cells from Tac and

Jax C57BL/6 mice revealed a significantly higher frequency of V

NKT cells in the

thymus, spleen and liver of Tac mice (Figure 2B). No difference, however, was observed for

7 usage of

NKT cells in either SI-IEL or SI-LPL (Supplemental figure 2A), or for the

frequency of V

NKT cells in any of the organs analyzed (Supplemental figure 2A, 2B).

The decrease of V

NKT cells in Jax mice was balanced out by a correlative increase of

NKT cells expressing V

8 (data not shown). Furthermore, we observed no significant

differences between Tac and Jax

NKT cells in the surface expression of NK1.1, CD4,

CD25, CD44, CD69 and CD122 in any tissue analyzed (data not shown). However, we

noted in the SI-LPL, but not in any other organ analyzed, a significantly increased frequency

of CD127

CD4

−

NKT cells in the Tac compared to the Jax derived mice (Figure 2D, 2E

and data not shown). We also assessed the effector functions of the

NKT cells following

activation with

GalCer. The frequency of cytokine-producing

NKT cells tended to be

lower in Tac mice (Figure 2C). This difference, however, was statistically significant only

for TNF

in all four experiments, while significance for differences in IL-4 and IFN

was

not consistently observed. The lower TNF

production of Tac

NKT cells could not be

explained by the difference in the V

-usage, as the cytokine production in the Tac

NKT

cells was reduced irrespective of the V

expressed (Supplemental figure 2C).

To determine if the observed differences were acquired and did not stem from minor

variations due to genetic drift, we co-housed newborn offsprings of Tac and Jax mice, to

allow the environmental factors, including the intestinal microbiota, to equalize. When

analyzed side-by-side after eight to ten weeks later, we did not find difference in

NKT cell

frequency, nor in V

-usage and function (Figure 2A–C). Therefore, these data clearly

demonstrate that differences in the environment of Tac- and Jax-derived animals can

modulate the frequency, V

-usage and cytokine production of

NKT cells.

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iNKT cells from germ-free mice are hyporesponsive

To determine more directly if the normal gut microbiota affects the development and

function of peripheral

NKT cells, we compared

NKT cells derived from Swiss Webster

animals raised in germ-free (GF) conditions with those from mice raised in SPF conditions.

Although relative

NKT cell numbers recovered from GF and SPF animals did not differ

significantly (data not shown), we observed that unstimulated

NKT cells from the spleen,

liver and thymus of GF mice uniformly expressed lower levels of the activation markers

CD69, CD25 and CD5 (Figure 3A and supplemental figure 3A). When GF and SPF animals

were challenged with the potent

NKT cell antigen

GalCer, the difference in CD69

expression between GF and SPF mice was even more pronounced (Figure 3B), suggesting

that

NKT cells from GF animals respond less vigorously. Importantly, cytokine production

NKT cells from GF animals, as measured by intracellular cytokine staining, was

significantly lower compared to their SPF counterparts (Figure 3B). We also observed a

similar difference after stimulating splenocytes from GF and SPF mice with

GalCer in

vitro (Supplemental figure 3B).

NKT cells are not highly dependent on co-stimulation for

activation

and the expression level of CD1d on antigen presenting cells (APCs) was

comparable in SPF and GF animals (data not shown). Nonetheless, it was possible that

differences in the maturation state of APC caused the reduced responses of

NKT cells from

GF mice. To avoid the influence of endogenous APCs, we stimulated splenocytes from GF-

and SPF-raised animals with

GalCer-loaded, CD1d transfected RMA lymphoma cells

vitro

. In this experimental set-up and similar to the previous results,

NKT cells derived

from GF animals produced significantly less cytokines than cells from SPF animals (Figure

3C). These data demonstrate that, independently of any putative effect on APCs,

NKT cells

from GF mice respond less vigorously to antigen stimulation than

NKT cells from SPF

animals.

As Swiss Webster mice are not fully inbred, we aimed to confirm that

NKT cells from GF

mice are hyporesponsive by testing GF animals on the C57BL/6 background. Similar to

their Swiss Webster counterparts, splenic

NKT cells from C57BL/6 GF animals showed a

significant impairment in antigen-stimulated cytokine production (Figure 3D) and up-

regulation of CD69 expression (Supplemental figure 3C).

Apart from cytokine production, activated

NKT cells display potent cytotoxic activity. To

test if the presence of the intestinal microbiota affects the cytotoxic potential of

NKT cells,

we injected GF- and SPF-housed Swiss Webster animals with CFSE-labeled B cells loaded

in vitro

with

GalCer, and measured cytotoxicity

in vivo

four hours later

. The

GalCer

specific

in vivo

cytotoxicity in GF mice was significantly lower than that observed in SPF

animals (Figure 3E), indicating that the microbiota is also important for the development

and/or maintenance of the cytotoxic capability of

NKT cells. Altogether these data

demonstrate that

NKT cells from GF animals are hypo-responsive to antigen stimulation in

a cell-intrinsic fashion.

Furthermore, we found a significantly higher frequency of intestinal

NKT cells in GF than

in SPF Swiss Webster mice in all four intestinal compartments (Figure 3F), suggesting that

the homing/expansion of

NKT cells to the intestine does not require the gut microbiota to

the same extent as for

αβ

T cells

. The analysis of the V

-usage of the

NKT cells from GF

and SPF C57BL/6 mice revealed a significantly lower frequency of V

NKT cells in the

thymus and spleen of GF mice (Supplemental figure 3D). Similar to other organs analyzed

(Figure 3A), in GF mice the expression of CD69 was lower on intestinal

NKT cells than in

SPF mice (Supplemental figure 3E and data not shown). Furthermore, while no differences

for the expression of CD103,

-integrin and CCR9 on intestinal

NKT cells from GF

compared to SPF mice were observed (Supplemental figure 3F and data not shown), the

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frequency of CD127

CD4

−

NKT cells in the small intestine was lower in the GF animals

(Figure 3F).

Bacterial products promote iNKT cell responsiveness in a TLR independent fashion

Bacterial products, via TLR signaling and induction of IL-12 and other cytokines by APCs,

can activate

NKT cells even in the absence of a microbial antigen that engages their TCR.

In order to establish if this alternative route of stimulation plays a role in shaping the

NKT

cell antigen responsiveness to intestinal microbiota, we utilized MyD88 and TRIF double-

deficient animals, which cannot respond to TLR ligands

. We did not detect any

phenotypic differences between C57BL/6 control and MyD88

−/−

Trif

Lps2/Lps2

mice (Figure

4A). Activation of

NKT cells from MyD88

−/−

Trif

Lps2/Lps2

animals with

GalCer caused

phenotypic changes that were also indistinguishable from the controls (Figure 4A).

Furthermore, we did not observe differences in

GalCer-induced cytokine production by

NKT cells (Figure 4B). Similarly, analysis of IL-12

−/−

animals showed no phenotypic or

functional differences with

NKT cells from wild-type animals (Figure 4C, 4D). Consistent

with these data, the frequency and phenotype of intestinal

NKT cells in the IL-12

−/−

animals were similar to C57BL/6 control mice (Supplemental figure 4). These data suggest

that the pathways of

NKT cell stimulation that depend on TLR stimulation of APCs cannot

account for the hyporesponsive phenotype and function of

NKT cells in GF mice.

Bacterial reconstitution corrects the hyporesponsive phenotype of iNKT cells

We then tested if the hyporesponsive phenotype of

NKT cells in GF animals could be

reversed. To this end, we co-housed GF with SPF animals for four weeks under SPF

conditions. After this time, we found that the phenotype of

NKT cells from SPF mice was

indistinguishable from the ones of previously GF mice (Figure 5A).

Next we reconstituted GF animals by gavage with live bacteria, either with the

Sphingomonas/Sphingobium

species

S. yanoikuyae

, which have

NKT cell antigens

, or

with

E. coli

, which are devoid of such antigens (data not shown). Analysis of CD69

expression on

NKT cells showed that reconstitution with

S. yanoikuyae

was sufficient to

normalize the hypo-responsive phenotype of

NKT cells from GF mice (Figure 5B, 5C). In

contrast, reconstitution of the GF animals with

E. coli

bacteria did not cause such a change

in the

NKT cell phenotype (Figure 5B, 5C). These data suggest that intestinal-derived

NKT

cell specific antigens from microbes are necessary to render peripheral

NKT cells fully

mature and ready to respond. Similar to C57BL/6 GF (Supplemental figure 3D)

NKT cells

from SW-GF animals displayed a lower frequency of V

cells and this frequency

normalized following reconstitution with

S. yanoikuyae

, but not with

E. coli

(Figure 5D),

suggesting antigen driven proliferation of

NKT cells.

To analyze the effect of a limited set of intestinal organisms on the responsiveness of

NKT

cells we also tested mice bearing a restricted flora (RF)

. RF mice carry an altered and

reduced microbiota, including different fungal and bacterial species as compared to SPF

mice

20,21

. The bacterial microbioata of RF mice is enriched for

Firmicutes spp

and devoid

Sphingomonas/Sphingobium spp

. Although

NKT cell numbers are reduced in RF

mice

, their response to

GalCer can be measured.

NKT cells derived from RF mice

displayed a higher frequency of V

NKT cells in the spleen (Supplemental figure 5).

Under resting conditions splenic

NKT cells from RF mice expressed lower CD69 levels and

displayed a lower up-regulation of this marker following

GalCer stimulation (Figure 5E).

Furthermore, fewer

NKT cells produced cytokines in the RF mice compared to the SPF

controls (Figure 5F), recapitulating the data we obtained in the GF animals.

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Discussion

Here we report a detailed record of the distribution and phenotype of

NKT cells in the

intestine. Furthermore, we show that bacterial products from the intestinal microbiota

contribute to the full responsiveness of peripheral V

NKT cells and can modulate their

phenotype and TCR V

-usage.

NKT cells from specific-pathogen-free (SPF) mice derived

from different vendors differed in the frequency of

NKT cells, the proportion that expressed

7, and in their cytokine response after antigen stimulation. Additionally,

NKT cells

derived from GF animals displayed a less mature phenotype and were hypo-responsive to

antigen-specific activation, as measured by up-regulation of activation markers and the

production of cytokines. These effects on the acute, antigen-specific response of

NKT cells

in GF mice could be reversed days after oral exposure to bacteria expressing

NKT cell

antigens. Furthermore, full

NKT cell maturation and the constitutive activation state of

these cells did not require TLR-mediated signals. Together these findings suggest that

antigens from the microbiota that engage the semi-invariant TCR likely are responsible for

the effects observed.

In light of these findings, we were surprised that

NKT cells were increased in GF mice in

the lamina propria and epithelium of the small and large intestines, although like their

counterparts in the spleen and liver, the intestinal

KT cells from GF mice expressed lower

amounts of the activation antigen CD69. These data are consistent with a recent report

showing that

NKT cells are increased in the colon of GF mice due to increased production

of the chemokine CXCL16 (Olszak et al.,

Science

in press,

DOI: 10.1126/science.1219328

Interestingly, colonization of neonatal mice with intestinal flora prevented both the

increased accumulation of

NKT cells in the intestine and the contribution of these cells to

inflammation in the intestine and the lung (Olszak et al.,

Science

in press,

DOI: 10.1126/

science.1219328

), providing additional evidence for the modulation of

NKT cell function as

well as number by intestinal microbes.

The frequency and distribution of

NKT cells in intestinal tissue has not been fully

characterized, despite the role of

NKT cells in several models of inflammatory bowel

disease and intestinal infections

5,6

. Many of the studies reporting the presence of NKT cells

in the intestine relied on the co-expression of the TCR/CD3

complex and NK cell

receptors

5,6

, which does not allow for the unequivocal identification of

NKT cells. Using

CD1d tetramers loaded with

GalCer, however,

NKT cells have been reported in LPL

30,31

and in SI-IEL, where 80% of them were NK1.1

neg26

. A later report, however, did not detect

NKT cells in SI-IEL

. Here we report on the presence of

NKT cells in IEL and LPL of

both small and large intestine. We detected a higher relative percentage of

NKT cells in the

small rather than the large intestine, and also generally more in the LPL than in the IEL

compartments. The frequencies in LI-LPL were comparable to those in the lymph node, and

for the SI-LPL to those in the spleen. These data demonstrate that

NKT cells constitute a

significant lymphocyte population within the lamina propria.

It has been reported that

NKT cells can influence the microbial colonization and the

composition of intestinal bacteria

. Here we provide evidence that this influence is mutual.

NKT cells derived from SPF animals from two different vendors, Taconic Farms and

Jackson Laboratory, showed differences in the frequency of

NKT cells, V

7-usage and

cytokine production. These differences were dependent on the environment, as CD1d

expression was not different between the two strains and co-housing of the offspring

diminished them. Interestingly, although

NKT cells expressing V

7 have a lower avidity

for

GalCer

, it has been inferred that they have a higher avidity for the endogenous

selecting antigen(s)

. The environment-dependent increase in V

NKT cells in the Tac

C57BL/6 mice could therefore be due to differences in intestinal

NKT cell antigens.

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However, in preliminary experiments we could not recover detectable antigenic

NKT cell

activity in the intestinal contents from SPF mice (data not shown). While a difference in the

intestinal microbiota is likely responsible, especially considering the known differences

between Jax and Tac mice

, further experiments are required to determine the parameters in

the environment that are responsible for the differences in

NKT cells between mice from

the two vendors.

The finding that

NKT cells from GF Swiss Webster mice were hypo-responsive are in

contrast to those by Park et al.

, where no impairment of the

NKT cell response of GF

animals was detected. Several technical differences, however, set our study apart from the

previous one, including: (a) the use of CD1d/

GalCer-tetramers to unequivocally detect

NKT cells, in contrast to measuring NK1.1

TCR

cells, the only tools available at

that time; (b) the quantitative analysis of activation marker expression levels by determining

the mean fluorescence intensity, rather than expression by NKT cells

per se

; and finally (c)

the analysis of the

NKT cell cytokine response on a single cell level, rather than analysis of

cytokine mRNA from total splenocytes. However, despite the marked differences we

observed in

NKT cells from GF mice, we should not overlook the significant phenotypic

and functional overlap they have with

NKT cells from SPF mice, including an expanded

population, increased activation marker expression compared to naïve T lymphocytes, and

the ability of some of the cells, albeit a reduced percentage, to produce effector cytokines

rapidly.

Our data obtained from MyD88

−/−

Trif

Lps2/Lps2

and IL-12

−/−

mice indicated that TLR-

ligands from the intestinal contents are not required for the full maturation of peripheral

NKT cells. These data do not exclude a potential role for other sensing molecules, like RIG-

I-like receptors (RLRs), and NOD-like receptors (NLRs)

, but their role in

NKT cell

activation are currently unknown. Therefore, the indirect or cytokine-mediated pathway for

NKT cell activation is likely not responsible for the homeostatic maintenance of the highly

activated and responsive state of these cells in SPF mice. Furthermore, reconstitution of GF

mice by oral administration of

S. yanoikuyae

, which contain relatively high affinity

glycosphingolipid antigens for the

NKT cell TCR, could recover the full phenotypic

maturity of these cells. In the absence of an isogenic

S. yanoikuyae

strain, we carried out

reconstitution with

E. coli

, a bacterium believed to lack antigens for the

NKT cell TCR.

Administration of

E. coli

did not normalize the phenotype of

NKT cells. These data

demonstrate the importance of intestinal bacterial products for facilitating the full degree of

NKT cell responsiveness, and they suggest that antigens for the semi-invariant TCR are

responsible.

NKT cell antigens have so far only been identified from a few bacterial sources

3,4

, the

distribution of

NKT cell antigens in the microbiota, and more generally in the environment,

remains incompletely characterized. We previously demonstrated specific

NKT cell

antigens in

Sphingobium yanoikuyae

. Such

Sphingomonas/Sphingobium

species are

ubiquitously present in water and soil

and are commensal species in the gut

16,17

Therefore we cannot exclude that a similar bacteria is a likely source of the intestinal

NKT

cell antigens. Still, the

Sphingomonas yanoikuyae

species were not reported to substantially

differ between Tac and Jax C57BL/6 animals

. In this context the observation is of interest

that mice bearing a restricted flora (RF) were not able to support full reactivity of

NKT

cells. RF mice lack

Sphingomonas/Sphingobium

species

, but also numerous other bacteria

species normally present in SPF mice

20,21

. We expect, however, that additional bacteria,

many of them non-infectious, contain

NKT cell antigens. For example, patients with

primary biliary cirrhosis (PBC) expressed antibodies against enzymes from the

Sphingomonas/Sphingobium

species

Novosphingobium aromaticivorans

aromaticivorans

was detected in the gut of PBC patients

and the activation of

NKT cells

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N. aromaticivorans-

derived antigens was linked to disease progression

37,38

. These data

demonstrated that commensal bacteria expressing

NKT cell antigens can contribute to

NKT cell-mediated inflammation. Together with our data, these findings suggest that the

composition of the intestinal microbiota may be an important exacerbating or causative

factor in other autoimmune diseases, with a possible contribution of

NKT cells.

The body exchanges substances with the environment via the mucosal surfaces of the lung

and the intestine. We recently demonstrated that

NKT cell antigens are present in house

dust and that the adjuvant effect they exerted during airway inflammation is dependent on

NKT cells

. Here we show that materials from the intestinal microbiota, likely

NKT cell

antigens, modulate the phenotype and function of peripheral

NKT cells. Together these

reports demonstrate that

NKT cells are sensitive in responding to the environment and that

antigens recognized by these cells are far more prevalent than previously anticipated.

Importantly, our findings indicate that the composition of the intestinal microbiota

influences the cytokine responsiveness of

NKT cells. It is thus conceivable that such

modulation not only could pertain to the magnitude of the antigen-induced cytokine

response, but also its polarization. Given the important role

NKT cells play in numerous

infectious and autoimmune diseases, our findings imply that the intestinal microbiota-

mediated modulation of

NKT cells could significantly affect the outcome of these diseases.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

This work was funded by NIH grants RO1 AI45053 and R37 AI71922 (M.K.), DK46763 (J.B., M.K.), DK078938

(S.K.M.) and an Outgoing International Fellowship by the Marie Curie Actions (G.W.). The authors wish to thank

Olga Turovskaya, Archana Khurana, Christopher Lena and the Department of Laboratory Animal Care at the La

Jolla Institute for Allergy & Immunology for excellent technical assistance. We are grateful to the scientific

contributions of Hilde Cheroutre, Maureen Bower, Mushtaq Husain, Yunji Park, Niranjana Nagarajan, Anup Datta

and Dirk Warnecke.

Abbreviations used in this paper

GalCer

-galactosylceramide

APC

antigen presenting cell

germ-free

invariant

IEL

intraepithelial lymphocytes

large intestine

LPL

lamina propria lymphocytes

mAb

monoclonal antibody

NKT

Natural Killer T

restricted flora

small intestine

SPF

specific pathogen free

T helper type 1

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T helper type 2

TLR

toll like receptor

invariant V

14 to J

18 TCR rearrangement

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Figure 1. Distribution and phenotype of intestinal

NKT cells

(A)

Lymphocytes from the indicated sites were incubated either with

GalCer loaded or

unloaded CD1d-tetramers, analyzed by flow cytometry and the frequencies of tetramer-

positive cells within live TCR

CD44

CD8

−

CD19

−

cells are shown.

(B, C)

Relative

percentage of

NKT cells within total live lymphocytes (B) and their expression of CD4 and

NK1.1 (C), from indicated sites. The graphs summarize data from 3–5 independent

experiments, with 6–9 samples per group.

(D)

Representative expression of CCR9 on

NKT

cells derived from the spleen (tinted, in both panels), IEL (dashed) or LPL (black line) from

the small or large intestine. The numbers in histograms denote the geometric mean values

for CCR9 on

NKT cells.

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Figure 2. Environmental influences on the responsiveness and V

-usage of

NKT cells

(A-C)

C57BL/6 animals, purchased from either Taconic Farms (Tac) or Jackson Laboratory

(Jax), were either analyzed within one week after delivery (top panels). Or alternatively,

new-born offspring from Tac or Jax mice were co-housed from 2–5 days after birth until

analysis 8–10 weeks later (lower panels). Relative frequency of

NKT cells (A) and their

7-usage (B) in indicated organs is shown. Production of indicated cytokines by splenic

NKT cells 90min after i.v. injection of

GalCer was analyzed by intracellular staining (C).

Representative data from four (top panels) or three (lower panels) independent experiments

are shown.

(D, E)

Frequency of CD127

CD4

−

NKT cells in indicated organs (D) or from

SI-LPL (E) from indicated mice. Representative data from three independent experiments

are shown.

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Figure 3.

NKT cells from germ-free animals are hyporesponsive

(A)

Expression of CD69, CD25 and CD5 by

NKT cells from indicated organs derived from

germ-free (GF) or specific-pathogen-free (SPF) housed Swiss Webster mice.

(B)

Expression

of CD69 (left panel) and indicated cytokines (right panel) by splenic

NKT cells from GF or

SPF housed Swiss Webster mice with or without

GalCer challenge in vivo (90min). The

expression of CD69 following

GalCer increased on SPF derived

NKT cells 1.9fold (MFI),

whereas the increase on GF derived

NKT cells was lower at 1.75fold

(SPF +/-

GalCer vs GF +/-

GalCer)

= 0.004).

(C)

Splenocytes from GF and SPF Swiss Webster

mice were co-cultured with

GalCer loaded RMA-CD1d cells for 4h and cytokine

production by

NKT cells was analyzed by intracellular staining.

(D)

GF or SPF housed

animals on the C57BL/6 background were injected with

GalCer and the cytokine

production by splenic

NKT cells was analyzed 90min later. The graph summarizes data

from two independent experiments, with 4–5 mice per group.

(E)

GalCer-specific

in vivo

cytotoxicity in spleen 4h after injection of B cell targets into GF or SPF housed Swiss

Webster mice. Representative data from two independent experiments are shown.

(F)

Relative percentage of

NKT cells within TCR

live lymphocytes (left) and of

CD127

CD4

−

NKT cells (right) from indicated organs of GF or SPF housed Swiss Webster

animals. The graphs summarize data from three independent experiments, with 5–8 mice per

group.

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Figure 4. Bacterial products promote

NKT cell responsiveness in a TLR independent fashion

(A, B)

C57BL/6J wild-type and MyD88

−/−

Trif

Lps2/Lps2

animals were either mock treated or

injected with

GalCer and 90min later the expression of indicated surface markers (A) and

cytokines (B) by splenic

NKT cells was analyzed.

(C, D)

C57BL/6J wild-type and IL-12

−/−

animals were either mock treated or injected with

GalCer and 90min later the expression

of indicated surface markers (C) and cytokines (D) by splenic

NKT cells was analyzed.

Representative data from two independent experiments are shown.

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