IL-23 orchestrates mucosal responses to serotype ... · interferon (Ifn)-γ, interleukin (Il)-22...

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IL-23 orchestrates mucosal responses to Salmonella enterica serotype Typhimurium in the intestine Running title: IL-23 orchestrates mucosal responses to Salmonella Ivan Godinez 1 , Manuela Raffatellu 1 , Hiutung Chu 1 , Tatiane A. Paixão 1,2 , Takeshi Haneda 1 , Renato L. Santos 2 , Charles L. Bevins 1 , Renée M. Tsolis 1 and Andreas J. Bäumler 1, * 1 Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, One Shields Ave., Davis, CA 2 Departamento de Clínica e Cirurgia Veterinárias, Escola de Veterinária, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil * Correspondence: E-mail: [email protected] Fax: 530-754-7240 Phone: 530-754-7225 ACCEPTED Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. Infect. Immun. doi:10.1128/IAI.00933-08 IAI Accepts, published online ahead of print on 27 October 2008 on August 27, 2019 by guest http://iai.asm.org/ Downloaded from

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IL-23 orchestrates mucosal responses to Salmonella enterica serotype

Typhimurium in the intestine

Running title: IL-23 orchestrates mucosal responses to Salmonella

Ivan Godinez1, Manuela Raffatellu1, Hiutung Chu1, Tatiane A. Paixão1,2, Takeshi

Haneda1, Renato L. Santos2, Charles L. Bevins1, Renée M. Tsolis1 and Andreas J.

Bäumler1,*

1 Department of Medical Microbiology and Immunology, School of Medicine, University

of California at Davis, One Shields Ave., Davis, CA

2 Departamento de Clínica e Cirurgia Veterinárias, Escola de Veterinária, Universidade

Federal de Minas Gerais, Belo Horizonte, MG, Brasil

* Correspondence: E-mail: [email protected]

Fax: 530-754-7240

Phone: 530-754-7225

ACCEPTED

Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.00933-08 IAI Accepts, published online ahead of print on 27 October 2008

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ABSTRACT

Salmonella enterica serotype Typhimurium causes an acute inflammatory reaction in the

cecum of streptomycin pre-treated mice that involves T cell-dependent induction of

interferon (Ifn)-γ, interleukin (Il)-22 and Il-17 expression. Here we investigated the role of

IL-23 in initiating these inflammatory responses using the streptomycin pre-treated

mouse model. Compared to wild type mice, expression of Il-17 was abrogated, Il-22

expression was markedly reduced but Ifn-γ expression was normal in the cecum of IL-23

p19 deficient mice during serotype Typhimurium infection. IL-23 p19 deficient mice also

exhibited a markedly reduced expression of regenerating islet-derived 3 gamma

(Reg3g), keratinocyte-derived cytokine (Kc), and reduced neutrophil recruitment into the

cecal mucosa during infection. Analysis of CD3+ lymphocytes in the intestinal mucosa by

flow cytometry revealed that αβ T cells were the predominant cell type expressing the IL-

23 receptor in naïve mice. However, a marked increase in the number of IL-23 receptor

expressing γδ T cells was observed in the lamina propria during serotype Typhimurium

infection. Compared to wild type mice, γδ T cell receptor deficient mice exhibited blunted

expression of Il-17 during serotype Typhimurium infection while Ifn-γ expression was

normal. These data suggested that γδ T cells are a significant, but not the sole source of

IL-17 in the acutely inflamed cecal mucosa of mice. Collectively our results point to IL-23

as an important player in initiating a T cell-dependent amplification of inflammatory

responses in the intestinal mucosa during serotype Typhimurium infection.

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INTRODUCTION

Salmonella enterica serotype Typhimurium elicits an acute inflammatory

response in the intestinal mucosa of humans that can be modeled using streptomycin

pre-treated mice (2). This inflammatory reaction is initiated by direct contact of serotype

Typhimurium with host cells, such as epithelial cells, macrophages or dendritic cells,

followed by an amplification of inflammatory responses in tissue (31). Responses that

are effectively amplified in tissue give rise to the most prominent changes in gene

expression observed in the intestinal mucosa during serotype Typhimurium infection,

including markedly increased mRNA levels of Ifn-γ, Il-17, and Il-22 (9, 24, 25). T cells

play an important role in amplifying inflammatory responses in the cecal mucosa,

because depletion of CD3+ cells causes in a dramatic reduction in cecal inflammation

and neutrophil recruitment (9). T cell depletion also results in a markedly blunted

induction of Ifn-γ, Il-17, and Il-22 in the intestinal mucosa during serotype Typhimurium

infection (9, 25). IL-17 and IL-22 help to orchestrate intestinal inflammation by inducing

the production of neutrophil chemoattractants (e.g. KC or IL-8), dendritic cell

chemoattractants (e.g. CCL20), and antimicrobials (e.g. Lipocalin-2 and iNOS) in the

mucosa (9, 25, 42). However, the mechanisms by which T cell-dependent amplification

of responses to serotype Typhimurium infection is initiated in the intestinal mucosa have

not been explored experimentally.

In other models of infection, cytokines released by macrophages or dendritic

cells have been implicated in stimulating cytokine production by T cells. For example,

detection of bacterial flagellin by cytosolic pattern recognition receptors in macrophages

activates caspase 1, resulting in the release of mature IL-18 (6, 20, 21, 34). IL-18 can

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stimulate antigen experienced T cells to rapidly secrete IFN-γ during bacterial infection

by an antigen-independent mechanism, thereby significantly amplifying early effector

responses in vivo (32). In a mouse model of Klebsiella pneumoniae lung infection,

bacterial stimulation of Toll-like receptor 4 on dendritic cells results in IL-23 production

(13). IL-23 in turn triggers the rapid production of IL-17 and IL-22 by T cells (1, 12),

which is required for efficient neutrophil recruitment in this model (39, 40). IL-23 has also

been implicated in enhancing inflammatory responses elicited by other bacterial

pathogens, including Citrobacter rodentium, Pseudomonas aeruginosa, Mycoplasma

pneumoniae and Mycobacterium bovis (5, 36, 38, 42). The goal of this study was to

determine whether IL-23 contributes to an amplification of inflammatory responses in the

cecal mucosa during serotype Typhimurium infection of streptomycin pre-treated mice.

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MATERIALS AND METHODS

Bacterial strains and culture conditions. Serotype Typhimurium strain IR715

is a fully virulent, nalidixic acid resistant derivative of isolate ATCC14028 and was used

in all experiments (33). Bacteria were cultured aerobically at 37°C in Luria-Bertani (LB)

broth.

Animal experiments. Mice deficient in p19 (IL-23 p19-/- mice) were generated by

breeding B6.129S5-ll23p19tm1Lex mice with C57BL/6 mice under specific pathogen-free

conditions in a barrier facility. IL-23 p19-/- mice and wild-type littermates were bred and

genotyped at the Mutant Mouse Regional Resource Center at the University of

California, Davis. Mice deficient for Tcd, the gene encoding the δ T cell receptor chain,

were obtained from Jackson laboratory (B6.129P2-Tcrdtm1Mom/J).

To study inflammation in the cecum, streptomycin-pretreated mice were orally

infected with serotype Typhimurium as described previously (2). In brief, mice were

inoculated with streptomycin (0.1 ml of a 200mg/ml solution in sterile water)

intragastrically. IL-23 p19-/- mice (N = 16) and wild-type littermates (N = 9) were

inoculated intragastrically 24 hours later with bacteria (0.1ml containing approximately

5x108 colony forming units [CFU]). As a control, IL-23 p19-/- mice (N = 6) and wild-type

littermates (N = 8) were inoculated with 0.1ml of sterile LB broth (mock infection). Trd -/-

(N = 6) and wild-type C57/B6 (N = 5) mice were infected as described above with

serotype Typhimurium. As a control, wild-type (N = 6) and Trd -/- mice (N = 4) were

inoculated with 0.1ml LB broth (mock infection). At 48 hours after infection, mice were

euthanized and samples of the cecum collected for isolation of mRNA and for

histopathological analysis. For bacteriologic analysis, cecal contents and Peyer’s

patches were homogenized and serial 10-fold dilutions spread on agar plates containing

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the appropriate antibiotics. For isolation of intra-epithelial and lamina propria

lymphocytes from infected mice, three groups of two 8-10 week old mice (C57BL/6,

Jackson lab) were inoculated with streptomycin (0.1 ml of a 200mg/ml solution in sterile

water) intragastrically. Twenty-four hours later, mice were inoculated with bacteria (0.1ml

containing approximately 5x108 CFU). IEL and LPL were isolated 48 hours post

infection.

Real-time PCR. For analysis of changes in gene expression after serotype

Typhimurium infection in the mouse cecum, tissue was collected and immediately snap-

frozen in liquid nitrogen at the site of surgery, and store at -80○C until processing. RNA

was then extracted from snap-frozen tissue with TriReagent (Molecular Research

Center) according to instruction by the manufacturer. Next, 1µg from each sample was

reverse transcribed in 50 µl volume (Taqman reverse transcription reagent; Applied

Biosystems) and 4 µl of cDNA was used for each real-time reaction. Real-time PCR was

performed using SYBR Green (Applied Biosystems) and the 7900HT Fast Real-Time

PCR System. The data were analyzed using a comparative threshold cycle method

(Applied Biosystems). Increases in cytokine expression in infected mice were calculated

relative to the average level of the respective cytokine in 8 mock-infected wild type mice.

A list of genes analyzed in this study with the respective primers is provided in Table 1.

For analysis of absolute copy number expression for Il-17 and Reg3g, real-time

PCR was performed using 1µl of cDNA (as described above) for each reaction in a

temperature cycler equipped with a fluorescence detection monitor (LightCycler, Roche

Diagnostics, Mannheim, Germany). Thus, cDNA corresponding to 20ng RNA served as

a template in a 10µl reaction containing 4mM MgCl2, 0.5µM of each primer and 1X

LightCycler-Fast Start DNA Master SYBR Green I mix (Roche Diagnostics). A negative

control reaction without cDNA template was included with each set of reaction to check

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for possible contamination. The PCR conditions were: initial denaturation at 95 °C for 10

min, followed by 45 cycles with each cycle consisting of denaturation, 95 °C for 15 s;

annealing at 60 °C for 5 s; and extension at 72 °C for 10 s. The cycle-to-cycle

fluorescence emission was monitored at 530 nm and analyzed using LightCycler

Software (Roche Diagnostics). Gene-specific plasmid standards were included with

every set of reactions and standard curves generated for each gene product was used to

quantify expression of Il-17 and Reg3g. All reactions were run in duplicate and inter-

sample variation was <10%.

Histopathology. Tissue samples were fixed in formalin, processed according to

standard procedures for paraffin embedding, sectioned at 5 µm, and stained with

hematoxylin and eosin. A veterinary pathologist scored inflammatory changes using a

blind-sample analysis. Neutrophil counts were determined per high (x400)-magnification

microscopy, and numbers were averaged from 10 microscopic fields for each animal.

Isolation of intestinal lymphocytes. Intra-epithelial lymphocytes (IEL) and

lamina propria lymphocytes (LPL) were isolated from C57BL/6 mice by standard

procedures (4, 19, 30). Briefly, three groups of two naïve mice and three groups of two

serotype Typhimurium infected mice were euthanized and organs from each group of

mice were combined. Intestines were removed beginning from the duodenum and

ending at the proximal colon. Intestines were dissected by removing the remaining

mesentery and vasculature. Each segment was then opened longitudinally, the luminal

content was removed, and the tissue was cut into approximately 5 mm sections with a

scalpel. The sections were subsequently washed in cold 1x HBSS (Gibco catalog no.

14185) containing 0.015M HEPES (Gibco catalog no.15630) a total of six times to

remove mucus and remaining fecal matter.

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For IEL isolation, tissue was placed and stirred for 15 minutes at room

temperature in pre-warmed (37°C) 1x HBSS containing 10% FBS (Gibco catalog no.

10082), 0.015M HEPES, and 5mM EDTA and stirred for 15 minutes 37°C followed by

three 15-minute washes with buffer adjusted to room temperature. The supernatant from

each wash was pooled and poured through a nylon wool column to enrich for T cells and

remove mucus. The resulting cell suspension was used to analyze IEL.

To isolate LPL, the tissue remaining after IEL isolation was stirred in pre-warmed

(37°C) 1x RPMI (Sigma R1145) containing 10% FBS, penicillin/streptomycin (Gibco

catalog no.15240-062) and 0.015M HEPES, and 1.6mg/mL collagenase (Sigma-Aldrich

C6885) for 45 minutes in a 37°C incubator. The resulting cell suspension was washed

twice with 1x HBSS containing 0.015M HEPES, enriched for T cells using a nylon wool

column and used to analyze LPL.

Flow Cytometry. The IEL and LPL cell suspensions containing approximately

4x106 cells each were resuspended in cold PBS and stained with Aqua LIVE/DEAD cell

discriminator (Invitrogen #L34597) as per manufacturer protocol. Cells were then stained

for one hour in the dark at 4°C with optimized concentrations of anti-CD3 Alexa750-APC

(eBioscience clone 17A2), anti-CD8 Alexa700 (eBioscience clone 53-6.7), anti-CD4

Pacific Blue (eBioscience clone RM4-5), anti-TCR GD R-PE (BD Pharmingen clone

GL3), and biotinylated polyclonal anti-IL-23R (R&D systems BAF1686). Cells were

washed twice with PBS containing 1% bovine serum albumin (FACS buffer). Cells were

then stained for one hour with streptavidin conjugated Qdot 605 (Invitrogen Q10101MP).

Stained cells were washed once in FACS buffer and subsequently fixed in 4% formalin

for one hour. Cells were then washed once and resuspended in FACS buffer and

analyzed using an LSR II (Becton-Dickinson, San Jose, CA) flow cytometer. Data were

analyzed using Flowjo software (Treestar, inc. Ashland, OR). Gates were set on singlets

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then on live lymphocytes. Subsequent gates were based on Fluorescence-Minus-One

and unstained controls.

Statistical analysis. Fold changes in mRNA levels measured by real-time PCR

underwent logarithmic transformation, and percentage values underwent angular

transformation prior to analysis by Student’s t test.

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RESULTS

IL-23 is required for induction of Il-17, Il-22 and Reg3g expression, but not for Ifn-γγγγ

expression, in the cecal mucosa during serotype Typhimurium infection

We have recently shown that Ifn-γ, Il–17 and Il–22 are among the genes whose

transcript levels are increased most prominently in the cecum of streptomycin pre-

treated mice during serotype Typhimurium infection (9). To study the contribution of IL-

23 in triggering cytokine production in the cecal mucosa, we compared the mRNA levels

of Ifn-γ, Il–17 and Il–22 in IL-23 deficient mice and their wild-type littermates in response

to inoculation with serotype Typhimurium or sterile LB broth (mock infection). IL-23 is a

heterodimer composed of p19 and p40. The p40 subunit is shared with IL-12, a

heterodimer of p40 and p35. We used IL-23p19–/– mice to determine the role of IL-23 in

amplifying inflammatory responses in the intestine. We recently established the time

course of cytokine production in the streptomycin pre-treated mouse model, which

shows that pro-inflammatory cytokines are strongly induced in the cecal mucosa by 48

hours after serotype Typhimurium infection (9). We therefore chose the 48-hour time

point for experiments described in this study.

We compared mRNA levels of cytokines to levels detected in mock-infected wild

type mice (Figure 1). As expected, Il-23p19 mRNA was detected neither in mock-

infected nor in serotype Typhimurium-infected IL-23p19–/– mice (Figure 1A). Compared

to mock-infected wild type mice, Il-23p19 mRNA levels were increased approximately 10

fold in wild type mice infected with serotype Typhimurium. Importantly, while Il–17 mRNA

levels were markedly increased in serotype Typhimurium-infected wild type mice, no

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induction of Il–17 expression was observed in IL-23p19–/– mice (Figure 1B). These data

suggested that induction of Il–17 expression in the cecal mucosa of mice 48 hours after

serotype Typhimurium infection was fully dependent on the presence of IL-23. While Il–

22 mRNA levels were increased in response to serotype Typhimurium infection in both

wild type mice and IL-23p19–/– mice, induction was significantly greater (p<0.001) in wild

type mice (Figure 1C). These data suggested that IL-23 dependent mechanisms

contributed to an induction of Il–22 expression in the cecal mucosa of mice. However,

the increase in Il-22 mRNA levels observed in serotype Typhimurium-infected IL-23p19–

/– mice suggested that IL-23-independent mechanisms also contributed to IL-22

production in vivo. Serotype Typhimurium infection induced Ifn-γ mRNA to similar levels

in both wild type mice and IL-23p19–/– mice (Figure 1D), indicating that IFN-γ production

is induced by IL-23-independent mechanisms in the cecal mucosa. The lower degree of

Il–17 and Il–22 expression in the ceca in IL-23p19–/– mice was not due to differences in

bacterial load, because similar bacterial numbers were recovered from intestinal

contents and intestinal tissue of serotype Typhimurium-infected wild type mice and IL-

23p19–/– mice (Figure 1E).

Next, we determined the absolute number of Il-17 transcripts using quantitative

real-time PCR (Figure 2). In wild type mice, the absolute number of Il-17 transcripts was

markedly increased during serotype Typhimurium infection compared to mock infection.

In contrast, mock-infected and serotype Typhimurium IL-23p19–/– mice had almost

identical Il-17 transcript levels (Figure 2A). These data further supported the idea that

increases in Il–17 transcript levels were entirely IL-23-dependent. We also determined

absolute transcript levels of Reg3g, a gene encoding an antimicrobial, whose production

is induced by IL-22 in the cecal mucosa of mice during inflammation (42). During

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serotype Typhimurium infection, Reg3g was produced at high levels in the cecal mucosa

of wild type mice, averaging 11,000 copies/ng RNA compared to 470 copies/ng in mock-

infected mice (Figure 2B). Induction of Reg3g was largely IL-23 dependent, since IL-

23p19–/– mice exhibited markedly reduced transcript levels during serotype Typhimurium

infection. In summary, our results supported the idea that IL-23 helps to amplify

inflammatory responses in the cecal mucosa by inducing expression of IL-17 and by

contributing to a full induction of IL-22 expression during serotype Typhimurium infection.

IL-23 contributes to neutrophil recruitment in the cecal mucosa during serotype

Typhimurium infection

Since IL-17 and IL-22 play a major role in orchestrating inflammatory responses

in the intestinal mucosa of mice (25, 42), we investigated the consequences of the IL-

17/IL-22 deficiency in IL-23p19–/– mice (Figure 3). As these cytokines orchestrate a

mucosal inflammatory response resulting in neutrophil influx at the site of infection, we

hypothesized that p19 deficient mice would exhibit reduced expression of neutrophil

chemoattractants and reduced neutrophil influx in the cecal mucosa after infection with

serotype Typhimurium. We first investigated expression of the neutrophil

chemoattractant KC in the cecum of wild type mice and IL-23p19–/– mice 48 hours after

infection with serotype Typhimurium. Compared to mock-infected wild type mice, Kc

mRNA levels were markedly elevated (approximately 200 fold) in serotype Typhimurium-

infected wild type mice (Figure 3A). Induction of Kc expression was notably blunted in

the ceca of serotype Typhimurium-infected IL-23p19–/–mice compared to serotype

Typhimurium-infected wild type mice (P < 0.001). These data suggested that IL-23 is

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required for the full induction of neutrophil chemoattractants in the cecal mucosa during

serotype Typhimurium infection.

Next, we quantified neutrophil recruitment in the cecal mucosa by determining

counts per microscopic field at high magnification. Few neutrophils were detected in the

cecal mucosa of mock-infected mice while infection with serotype Typhimurium was

accompanied by marked neutrophil recruitment. However, there were significantly (P =

0.02) less neutrophils per field in serotype Typhimurium-infected ceca of IL-23p19–/–

mice compared serotype Typhimurium-infected ceca of wild type mice (Figure 3B).

These results were in good agreement with the lower Kc expression observed in

serotype Typhimurium-infected IL-23p19–/–mice (Figure 3A). The severity of

inflammatory changes was reduced in serotype Typhimurium-infected IL-23p19–/–mice

(Figure 3C) compared to serotype Typhimurium-infected wild type mice (Figure 3D).

However, compared to mock-infected mice (Figure 3E and 3F), serotype Typhimurium

infection was associated with marked inflammatory changes (Figure 3C and 3D). In

summary, our data suggested that IL-23 contributed to the recruitment of neutrophils into

the cecal mucosa during serotype Typhimurium infection.

The IL-23 receptor (IL-23R) is expressed by a subset of intestinal T cells

We have recently shown that depletion of T cells results in a marked reduction in

the expression levels of IL-17 and IL-22 in the cecum of streptomycin pre-treated mice

during serotype Typhimurium infection (9). These data suggest that the IL-17/IL-22

deficiency observed in IL-23p19–/– mice (Figure 1) could be explained by hypothesizing

that IL-23 stimulates a subset of intestinal T cells to produce IL-17 and IL-22. This

hypothesis would predict that a subset of intestinal T cells expresses the receptor for IL-

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23. To test this prediction, we isolated intra-epithelial lymphocytes (Figure 4A) and

lamina propria lymphocytes (Figure 4B) from the intestine of mice and analyzed

expression of surface markers by flow cytometry. Intra-epithelial CD3+ cells were divided

into cells expressing the γδ T cell receptor (γδ T cells) and γδ T cell receptor negative

cells (representing αβ T cells) (Figure 4C). Finally, αβ T cell subsets were defined based

on expression of CD4 and CD8 (Figure 4D). The same procedure was applied to lamina

propria CD3+ cells (Figure 4E and F).

Approximately 40% of the intra-epithelial CD3+ cells expressed the γδ T cell

receptor in naïve mice (i.e. CD8+ γδ+ T cells and CD8- γδ

+ T cells constituted

approximately 40% of intra-epithelial CD3+ T cells) (Figure 5A). In contrast, γδ T cells

were only a minor population (approximately 10%) of the lamina propria CD3+ cell

population, which was dominated by CD4+ T cells. That is, approximately 50% of CD3+

cells in the lamina propria were CD4+ CD8- γδ- T cells (Figure 5B). These data were

consistent with previous studies on the composition of intestinal intra-epithelial and

lamina propria CD3+ cell populations in the mouse (8). No significant differences in the

relative proportions of T cell subsets were observed during analysis of tissue collected

from naïve mice compared to tissue collected from serotype Typhimurium-infected mice

(Figure 5).

We next investigated expression of the IL-23 receptor by T cell subsets isolated

from the intestinal mucosa (Figure 6). In naïve mice, the overall fraction of CD3+ intra-

epithelial lymphocytes or CD3+ lamina propria lymphocytes that expressed the IL-23

receptor was approximately 10%. In the intra-epithelial CD3+ lymphocyte population, the

majority of cells expressing the IL-23 receptor were CD4-CD8- γδ- cells, regardless of

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whether tissue had been collected from naïve mice or from serotype Typhimurium-

infected mice (Figure 6A). Previous studies suggest that CD4-CD8- γδ- cells in the gut

mucosa of mice comprise natural killer T (NKT) cells and CD4-CD8- T cells (15). In the

lamina propria CD3+ lymphocyte population of naïve mice, the majority of cells

expressing the IL-23 receptor were either CD4-CD8- γδ- cells or CD4+CD8- γδ- cells

(potentially representing TH17 cells) (Figure 6B).

Importantly, in serotype Typhimurium-infected tissue, we observed a marked

increase in the lamina propria CD3+ lymphocyte population of CD8- γδ+ cells expressing

the IL-23 receptor (Figure 6C). This notable increase in IL-23 receptor expressing γδ T

cells during serotype Typhimurium infection raised the overall fraction of CD3+ lamina

propria lymphocytes that expressed the receptor for IL-23 above 20%. In summary, our

results show that a fraction (10-20%) of CD3+ lymphocytes in the intestinal mucosa of

mice express the receptor for IL-23. Furthermore, 48 hours after serotype Typhimurium

infection, we observed a marked increase in IL-23 receptor expressing γδ T cells.

γδγδγδγδ T cells contribute to Il-17 expression in the inflamed cecal mucosa of mice

Since an increase in IL-23 receptor expressing γδ T cells was the only notable

change in mucosal T cell populations observed during serotype Typhimurium infection

(Figure 6B and C), we investigated whether γδ T cells contribute to cytokine production

in the inflamed murine cecum. To this end, we compared the mRNA levels of Ifn-γ and

Il–17 in γδ T cell receptor deficient (Trd-/-) mice and wild-type controls (C57BL/6 mice) in

response to inoculation with serotype Typhimurium or sterile LB broth (mock infection)

(Figure 7). There was a compensatory increase in Il-23p19 mRNA expression in

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serotype Typhimurium-infected γδ T cell receptor deficient mice (Figure 7A). Il–17 mRNA

levels induced during serotype Typhimurium infection were significantly lower in γδ T cell

receptor deficient mice than in wild type (P < 0.05) (Figure 7B). In contrast, serotype

Typhimurium infection induced Ifn-γ mRNA (Figure 7C) and Il-22 mRNA (Figure 7D) to

similar levels in both wild type mice and γδ T cell receptor deficient mice. Similar

bacterial numbers were recovered from intestinal contents of infected mice (Figure 7E).

These data suggested that γδ T cells contributed to Il-17 expression in the inflamed cecal

mucosa. However, unlike IL-23p19–/– mice, γδ T cell receptor deficient mice still exhibited

increased Il–17 mRNA levels in response to serotype Typhimurium infection, suggesting

that γδ T cells are not the sole source of IL-17 in the inflamed cecum.

The absolute number of Il-17 transcripts in the cecal mucosa was quantified

using real-time PCR (Figure 8). Mice with γδ T cell deficiency induced Il-17 expression in

response to S. Typhimurium infection (P = 0.003), but transcript levels were markedly

reduced compared to those measured in wild type mice infected with serotype

Typhimruium (P = 0.03) (Figure 8A). These data further supported the idea that γδ T

cells contribute Il–17 expression in the cecal mucosa. Although absolute transcript levels

of Reg3g were reduced (Figure 8B) and lower numbers of neutrophils were observed in

cecal tissue of γδ T cell receptor deficient mice during serotype Typhimurim infection

(Figure 8C), these differences were not statistically significant. Our data suggest that the

partial inhibition of Il-17 expression (Figure 7B and 8A), which was accompanied by

normal expression of Il-22 (Figure 7D), was not sufficient to significantly reduce Reg3g

expression or neutrophil recruitment in γδ T cell receptor deficient mice (Figure 8B and

8C). Thus, while γδ T cells contribute to Il-17 expression, our data suggest that there

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must be additional cellular sources to fully account for the increased IL-17 and IL-22

production in the inflamed cecum of the mouse.

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DISCUSSION

Acute intestinal inflammation characterized by a massive neutrophil influx is a

hallmark of serotype Typhimurium infection (27, 35, 41). However, the precise

mechanisms by which this host response is orchestrated in tissue have not been fully

worked out. We have recently shown that depletion of CD3+ lymphocytes markedly

reduces the ability of mice to recruit neutrophils into the cecal mucosa and to produce

KC, IFN-γ, IL-22 and IL-17, which are among the most prominently induced cytokines in

serotype Typhimurium-infected tissue (9). These data suggest that T cells are an

important component of mechanisms that help to amplify inflammatory responses to

serotype Typhimurium infection in the intestinal mucosa. However, little is known about

how serotype Typhimurium infection initiates these T cell-dependent amplification

mechanisms. Here we show that diverse subsets of T cells in the intestinal mucosa

expresses the receptor for IL-23, a cytokine important for initiating the production of KC,

RegIIIγ, IL-22 and IL-17 in response to serotype Typhimurium infection. IFN-γ production

was not affected by IL-23 deficiency, suggesting that the early expression of this

important cytokine by T cells (i.e. at 2 days after infection) is triggered through other

pathways, perhaps involving IL-12 or IL-18 production. For example, IL-18 has recently

been implicated in amplifying inflammatory responses early after serotype Typhimurium

infection in the spleen of mice by triggering IFN-γ production in antigen experienced CD4

T cells by an antigen-independent mechanism (32).

Two important questions arise from the results of our study. First, which intestinal

T cell subsets contribute to IL-17 and IL-22 production during serotype Typhimurium

infection. In the lung mucosa, γδ T cells have been implicated as an important source of

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IL-17 during M. tuberculosis and M. bovis infection (18, 22, 36). Similarly, injection of

Escherichia coli into the peritoneal cavity of naive mice triggers IL-23 production in a

TLR4 signaling-dependent manner and the resulting IL-17 production originates largely

from γδ T cells (28). Our results suggest that γδ T cells are also one of the cellular

sources of IL-17 in the serotype Typhimurium infected mouse. However, γδ T cell

deficient mice were still able to produce Il-17 mRNA, albeit at reduced levels, during

serotype Typhimurium infection, suggesting that additional cell types contributed to

production of this cytokine in the inflamed cecum. In addition to γδ T cells, the receptor

for IL-23 was expressed predominantly by CD3+CD4-CD8- γδ

- cells (NKT cells and/or

CD4-CD8- T cells) in the intestinal epithelium and by CD4+ T cells and CD3+CD4-CD8- γδ

-

cells in the lamina propria. Each of these cell types has been implicated as a source of

IL-17 production in different animal models of inflammation (3)(13)(17)(16)(23)(25). A

distinct subset of CD4- NKT cells produces IL-17, contributing to infiltration of neutrophils

in a galactosylceramide-induced model of airway inflammation (17). NKT cells

constitutively express IL-23R and rapidly produce IL-17 upon stimulation with IL-23 (23).

IL-17 mRNA has been shown to be specifically expressed by a subset of murine CD4-

CD8- T cells (16). In contrast, IL-17 is mainly derived from CD4+ T cells during M.

pneumoniae lung infection (38). Both CD4+ T cells and CD8+ T cells are a source of IL-

17 during infection of mice with H. pylori (3) or K. pneumoniae (13). Finally, depletion of

memory CD4+ T cells by simian immunodeficiency virus blunts IL-17 responses elicited

early (i.e. 5 hours) after serotype Typhimurium infection of the ileal mucosa in rhesus

macaques (25), pointing to an innate induction of these T cell responses. Thus, TH17

cells contribute to IL-17 production in the ileal mucosa of a relevant animal species.

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The second question arising from this study relates to the mechanisms that

initiate IL-23 production during serotype Typhimurium infection. Electron microscopic

analysis of serotype Typhimurium infection shows that all bacteria detected in the

intestinal mucosa have an intracellular location, either within mononuclear phagocytes

(macrophages and/or dendritic cells) or within neutrophils (7, 26). Since only a very

small fraction of cells in infected tissue contain bacteria, the sum-total capacity for

cytokine production by these cells may be limited in scope. However, macrophages and

dendritic cells infected with serotype Typhimurium are a potential source of IL-23 and

our data suggest that this cytokine helps to amplify a subset of inflammatory responses

in tissue. During K. pneumoniae infection, release of IL-23 by dendritic cells in the lung

mucosa is triggered through stimulation of TLR4 by lipopolysaccharide (13). Similarly,

production of IL-23 by murine bone marrow-derived dendritic cells in response to S.

enterica serotype Enteritidis infection is TLR4-dependent (29). However,

CD11c+CX3CR1+ mucosal dendritic cells do not generate MyD88-dependent responses

in the ceca of serotype Typhimurium infected mice (10), suggesting that the signals

produced at mucosal sites are not mimicked adequately by bone marrow-derived cells. A

recent finding that points to macrophages as possible sources of IL-23 is the observation

that the inflamed human intestine contains a unique subset of CD14+ intestinal

macrophages, which produces larger amounts of IL-23 than the resident CD14-

macrophages (14). Alternatively, serotype Typhimurium may stimulate mucosal dendritic

cells or mucosal macrophages to produce IL-23 through MyD88-independent

mechanisms, which have been proposed to contribute to cecal inflammation (11). One

possible MyD88-independent mechanism leading to IL-23 production by dendritic cells is

the activation of the intracellular bacterial sensor NOD2. IL-23 produced by this NOD2-

dependent, MyD88-independent mechanism results in IL-17 production in human

memory T cells (37). However, additional work is needed to understand the precise

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mechanisms by which the IL-23/IL-17 axis is triggered in the intestinal mucosa during

serotype Typhimurium infection.

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ACKNOWLEDGEMENTS

We would like to thank Sebastian Winter, Maria Winter, and Sean-Paul Nuccio

for their help with animal experiments. We would also like to thank Monica Macal and

Carol Oxford for their input in designing flow cytometry panels.

This investigation was conducted in a facility constructed with support from

Research Facilities Improvement Program Grant Number C06 RR12088-01 from the

National Center for Research Resources, National Institutes of Health. Work in AJB's

laboratory was supported by Public Health Service grants AI040124, AI044170 and

AI079173. TAP and RLS are recipient of fellowships from CNPq (Conselho Nacional de

Desenvolvimento Científico e Tecnológico, Brasília, Brazil). I.G. was supported by Public

Health Service grant AI060555.

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FIGURE LEGENDS

Figure 1: Cytokine expression elicited by serotype Typhimurium in streptomycin pre-

treated wild type mice (black bars) and streptomycin pre-treated IL-23 deficient mice

(gray bars) 48 hours after infection measured by quantitative real-time PCR. (A-D) Bars

represent fold changes in mRNA levels of Il-23 (A), Il-17 (B), Il-22 (C) and Ifn-γ (D)

compared to mRNA levels detected in a group of mock-infected wild type mice (N = 8).

Data are shown as geometric means of fold-changes ± standard error determined for

RNA from individual mice. (E) Average bacterial numbers (CFU) recovered 48 hours

after serotype Typhimurium infection from colon contents or Peyer’s patch tissue of wild

type mice (black bars) or IL-23 deficient mice (gray bars). Statistical significance of

differences is indicated by P values above brackets. NS, not significant.

Figure 2: Absolute transcript levels of Il-17 (A) and Reg3g (B) in IL-23 p19 deficient

mice (IL-23p19-/-, gray bars) or wild type littermates (black bars) determined by

quantitative real-time PCR 48 hours after mock infection or infection with serotype

Typhimurium. Data represent mean mRNA copy numbers per 20ng of RNA ± standard

error. Statistically significant differences are indicated by P values.

Figure 3: Neutrophil recruitment into the cecal mucosa. (A) Expression of the neutrophil

chemoattractant Kc elicited by serotype Typhimurium in streptomycin pre-treated wild

type mice (black bars) and streptomycin pre-treated IL-23 deficient mice (gray bars) 48

hours after infection measured by quantitative real-time PCR. Bars represent fold

changes in mRNA levels compared to mRNA levels detected in a group of mock-infected

wild type mice (N = 8). Data are shown as geometric means of fold-changes ± standard

error determined for RNA from individual mice. (B) The numbers of neutrophils per

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microscopic field were determined by a veterinary pathologist during a blinded

examination of slides from the cecal mucosa. Data represent means±standard error.

Statistical significance of differences is indicated by P values. (C to F) Histopathological

appearance of the murine cecum of serotype Typhimurium-infected IL-23 deficient mice

(C), serotype Typhimurium-infected wild type mice (D), mock-infected IL-23 deficient

mice (E) or mock infected wild type mice (F). All images were taken from hematoxylin

and eosin stained cecal sections at the same magnification (100x).

Figure 4: Isolation of T cells from the intestinal epithelium and lamina propria. Total

number of live CD3+ cells present in preparations of intra-epithelial lymphocytes (A) and

lamina propria lymphocytes (B) from naïve mice (N = 6, gray bars) or serotype

Typhimurium infected mice (N = 6, black bars). (C) Representative example of the

gating strategy used to define γδ+ and γδ- T cell populations among live intra-epithelial

lymphocytes. (D) Representative example of the gating strategy used to separate intra-

epithelial γδ- T cells into different subsets. (E) Representative example of the gating

strategy used to define γδ+ and γδ- T cell populations among live lamina propria

lymphocytes. (F) Representative example of the gating strategy used to separate lamina

propria γδ- T cells into different subsets. (C-F) Axis represent the fluorescence intensity

produced by fluorescent antibody conjugates recognizing the γδ T cell receptor (γδTCR),

CD3, CD4 or CD8.

Figure 5: Characterization of T cell subsets in the intestine of naïve mice (N = 6, gray

bars) or serotype Typhimurium-infected mice (N = 6, black bars). (A) T cell subsets in

the intra-epithelial lymphocyte population are shown as percentage of the total number

of intra-epithelial T cells (CD3+ intra-epithelial lymphocytes). (B) T cell subsets in the

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lamina propria lymphocyte population are shown as percentage of the total number of

lamina propria T cells (CD3+ lamina propria lymphocytes). Data are shown as

mean±standard error.

Figure 6: Expression of IL-23R by intra-epithelial T cells (A) and lamina propria T cells

(B) in the intestine of naïve mice (N = 6, gray bars) or serotype Typhimurium-infected

mice (N = 6, black bars). (A) IL-23R expressing cells expressing the indicated markers

(CD4, CD8 and/or γδ TCR) are shown as a percentage of the total number of intra-

epithelial T cells (CD3+ intra-epithelial lymphocytes). (B) IL-23R expressing cells

expressing the indicated markers (CD4, CD8 and/or γδ TCR) are shown as a percentage

of the total number of lamina propria T cells (CD3+ lamina propria lymphocytes). Data

are shown as mean±standard error. Statistical significance of differences is indicated by

P values. (C) Representative example of IL-23 receptor expression by CD8- γδ+ lamina

propria T cells pooled from the intestine of two naïve mice (left panel) or two serotype

Typhimurium infected mice (right panel).

Figure 7: Cytokine expression elicited by serotype Typhimurium in streptomycin pre-

treated wild type mice (C57BL/6, black bars) or streptomycin pre-treated T cell receptor

δ chain deficient mice (Trd-/-, gray bars) 48 hours after infection measured by quantitative

real-time PCR. (A-D) Bars represent fold changes in mRNA levels of Il-23 (A), Il-17 (B),

Ifn-γ (C) and Il-22 (D) compared to mRNA levels detected in a group of mock-infected

wild type mice (N = 8). Data are shown as geometric means of fold-changes ± standard

error determined for RNA from individual mice. (E) Average bacterial numbers (CFU)

recovered 48 hours after serotype Typhimurium infection from colon contents or Peyer’s

patch tissue of wild type mice (black bars) or γδ T cell receptor deficient mice (gray bars).

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Statistical significance of differences is indicated by P values above brackets. NS, not

significant.

Figure 8: Absolute transcript levels of Il-17 (A) and Reg3g (B) in γδ T cell receptor

deficient mice (Trd-/-, gray bars) or wild type mice (C57BL/6, black bars) determined by

quantitative real-time PCR 48 hours after mock infection or infection with serotype

Typhimurium. Data represent mean mRNA copy numbers per 20ng of RNA ± standard

error. (C) Numbers of neutrophils per microscopic field were determined by a veterinary

pathologist during a blinded examination of slides from the cecal mucosa. Data

represent means±standard error. Statistically significant differences are indicated by P

values above brackets. NS, not significant.

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Table 1: Primers for real-time PCR.

Gene Primer pairs

Gapdh 5’-TGTAGACCATGTAGTTGAGGTCA-3’

5’-AGGTCGGTGTGAACGGATTTG-3’

Il23 p19 5’-TGTGCCTAGGAGTAGCAGTCCTGA-3’

5’-TTGGCGGATCCTTTGCAAGCAGAA-3’

Il-17

(relative)

5’-GCTCCAGAAGGCCCTCAGA-3’

5’-AGCTTTCCCTCCGCATTGA-3’

Il-22 5’-GGCCAGCCTTGCAGATAACA-3’

5’-GCTGATGTGACAGGAGCTGA -3’

Kc 5’-TGCACCCAAACCGAAGTCAT-3’

5’-TTGTCAGAAGCCAGCGTTCAC-3’

Ifn-γ 5’- TCAAGTGGCATAGATGTGGAAGAA-3’

5’-TGGCTCTGCAGGATTTTCATG-3’

Reg3g 5’-CCTCAGGACATCTTGTGTC-3’

5’-TCCACCTCTGTTGGGTTCA-3’

Il-17

(absolute)

5’-AACCCCCACGTTTCTCAGCAAAC-3’

5’-GGACCCCTTTACACCTTCTTTTCATTG -3’

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A

500

1000

1500

2000

2500

3000

3500B

IL-23p19–/– wild type

S. Typhimurium

infection

Mock

infection

IL-23p19–/–

Il–17

mRNA

(fold

increase)

P < 0.001

IL-23p19–/– wild type

S. Typhimurium

infection

Mock

infection

IL-23p19–/–

Ifn-γ

mRNA

(fold

increase)

500

1000

1500

2000

2500

3000

IL-23p19–/– wild type

S. Typhimurium

infection

Mock

infection

IL-23p19–/–

P < 0.001CIl–22

mRNA

(fold

increase)

200

400

600

800

1000

1200

IL-23p19–/– wild

type

S. Typhimurium

infection

Mock

infection

IL-23p19–/–

Il–23

mRNA

(fold

increase) 4

8

12

16

D

IL-23p19–/– wild

type

IL-23p19–/– wild

type

Peyer’s patchesColon contents

100

101

102

103

104

105

106

107

108E

CFU

Figure 1

NS

NS

NS

0

0

00

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Figure 2

0

100

200

300

400

500

600

700

800

IL-23p19–/– wild

type

IL-23p19–/– wild

type

S. Typhimurium

infection

Mock infection

AIl–17

mRNA

copy

number/

20ng

RNA

P < 0.001

IL-23p19–/– wild

type

IL-23p19–/– wild

type

S. Typhimurium

infection

Mock infection

BReg3g

mRNA

copy

number/

20ng

RNA

0

1x105

2x105

3x105

P < 0.001

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0

50

100

150

200

250

P = 0.001

IL-23p19–/– wild

type

S. Typhimurium

infection

Mock

infection

IL-23p19–/–

Kc

mRNA

(fold

increase)

ANeutrophils

(counts

per

microscopic

field)

B

IL-23p19–/– wild

type

Mock

infection

IL-23p19–/– wild

type

S. Typhimurium

infection

0

40

80

120 P = 0.02

Figure 3

C D

E F

Mock

infection

S. Typhimurium

infection

Wild typeIL-23p19–/–

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C

E

D

F

CD8

CD8

CD4

CD4

CD3

CD3

γδ

TCR

γδ

TCR

Lamina propria lymphocytes γδ- T cells

Intra-epithelial lymphocytes γδ- T cells

γδ+ T cells

γδ+ T cells

CD4-CD8- CD4-CD8+

CD4+CD8- CD4+CD8+

CD4-CD8- CD4-CD8+

CD4+CD8- CD4+CD8+

live

CD3+

lymphocytes/

106 events

A

Figure 4

B

0

10,000

20,000

30,000

40,000

50,000

0

20,000

40,000

60,000

80,000

naive S. Typhimurium

infection

Intra-epithelial lymphocytes

naive S. Typhimurium

infection

Lamina propria lymphocytes

live

CD3+

lymphocytes/

105 events

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0

10

20

30

40

50

CD4+CD8- CD4-CD8+ CD4-CD8- CD4+CD8+ CD8+ CD8-

0

10

20

30

40

50

60

70

γδ- T cells γδ+ T cells

% of

total

CD3+

cells

Intra-epithelial lymphocytes

naïve S. Typhimurium infection

CD4+CD8- CD4-CD8+ CD4-CD8- CD4+CD8+ CD8+ CD8-

γδ- T cells γδ+ T cells

% of

total

CD3+

cells

Lamina propria lymphocytes

naïve S. Typhimurium infection

A

B

Figure 5

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0

2

4

6

8

10

0

2

4

6

8

10

12

14

CD4+CD8- CD4-CD8+ CD4-CD8- CD4+CD8+ CD8+ CD8-

γδ- T cells γδ+ T cells

IL-23R+

cells as

% of total

CD3+ cells

Intra-epithelial lymphocytes

naïve S. Typhimurium infection

CD4+CD8- CD4-CD8+ CD4-CD8- CD4+CD8+ CD8+ CD8-

γδ- T cells γδ+ T cells

IL-23R+

cells as

% of total

CD3+ cells

Lamina propria lymphocytes

naïve S. Typhimurium infection

A

B P = 0.002

C

CD3

IL23R

S. Typhimurium infection

CD8-γδ+ lamina propria T cells

CD3

IL23R

Naïve

CD8-γδ+ lamina propria T cells

Figure 6

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0

200

400

600

800

1000

1200

1400B

Trd–/– wild type

S. Typhimurium

infection

Mock

infection

Trd–/–

Il–17

mRNA

(fold

increase)

P < 0.05A

Trd–/– wildtype

S. Typhimurium

infection

Mock

infection

Trd–/–

Il–23

mRNA

(fold

increase)

0

5

10

15

20

25

30

35

40

Figure 7

0

500

1000

1500

2000

Trd–/– wild type

S. Typhimurium

infection

Mock

infection

Trd–/–

Ifn-γ

mRNA

(fold

increase)

CNS

P < 0.05

Peyer’s patchesColon contents

Trd–/– wild type

Trd–/– wild type

NS

NS

100

101

102

103

104

105

106

107

CFU

E

0

200

400

600

800

1000

1200

1400Il-22

mRNA

(fold

increase)

DNS

Trd–/– wild

type

S. Typhimurium

infection

Mock

infection

Trd–/–

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100

200

300

400

500

600

Trd–/– wild

type

Trd–/– wild

type

S. Typhimurium

infection

Mock infection

B

Reg3g

mRNA

copy

number/

20ng RNA

P = 0.003

0

100

200

Neutrophils

(counts

per

microscopic

field)

C

Trd–/– wild

type

Trd–/– wild

type

S. Typhimurium

infection

Mock infection

0

1x106

2x106

Trd–/– wildtype

Trd–/– wildtype

S. Typhimurium

infection

Mock infection

AIl–17

mRNA

copy

number/

20ng RNA

NS

NS

Figure 8

P = 0.03

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