3 4 · 50 and Strongyloides stercolaris affect people worldwide, especially in developing countries...

IL-25 promotes efficient protective immunity against T. spiralis infection 1

by enhancing antigen-specific IL-9 response 2

Running Title: The role of IL-25 during T. spiralis infection 3

Pornpimon Angkasekwinai1#, Potjanee Srimanote2, Yui-Hsi Wang3, Anek Pootong1, 4

Yuwaporn Sakolvaree4, Kovit Pattanapanyasat5, Wanpen Chaicumpa4, Sansanee Chaiyaroj6, 5

and Chen Dong7 6

1Department of Medical Technology, Faculty of Allied Health Sciences, Thammasat 7

University, Pathumthani 12120,Thailand 8

2Graduate Program, Faculty of Allied Health Sciences, Thammasat University, 9

Pathumthani 12120, Thailand, 10

3Division of Allergy and Immunology, University of Cincinnati, Cincinnati Children’s 11

Hospital Medical Center, Cincinnati, OH 45229, USA. 12

4Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, 13

Bangkok, Thailand 14

5Center of Excellence for Flow Cytometry, Office for Research and Development, Faculty of 15

Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand 16

6Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand 17

7Department of Immunology, University of Texas and MD Anderson Cancer Center, 18

Houston, Texas 77030, USA. 19

#Correspondence: Pornpimon Angkasekwinai 20

Faculty of Allied Health Sciences, Thammasat University, Email: [email protected] 21

Keywords: IL-9, IL-25, T cell responses, T. spiralis infection 22 23

Copyright © 2013, American Society for Microbiology. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.00646-13 IAI Accepts, published online ahead of print on 29 July 2013

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Abstract 24 25

Mammalian hosts often develop distinct immune response against the diverse parasitic 26

helminthes that have evolved for immune evasion. IL-25, an IL-17 cytokine family member, 27

plays a key role in initiating the protective immunity against several parasitic helminthes; 28

however, the involvements and underlying mechanisms by which IL-25 mediates immune 29

response against T. spiralis infection have not been investigated. Herein, we showed that IL-30

25 functions in promoting protective immunity against T. spiralis infection. Mice treated with 31

IL-25 exhibited less worm burdens and fewer numbers of muscle larvae in the later stage of 32

T. spiralis infection. In contrast, mice treated with neutralizing antibody against IL-25 failed 33

to expel T. spiralis effectively. During T. spiralis infection, intestinal IL-25 expression was 34

rapidly elevated before the onset of IL-4 and IL-9 induction. While antigen specific Th2 and 35

Th9 immune responses were both developed during T. spiralis infection, antigen-specific Th9 36

response appeared to be transiently induced in the early stage of infection. Mice transferred 37

with antigen-specific T cells deficient of IL-9 were less effective in worm clearance than 38

those transferred with wild type T cells. The strength of antigen-specific Th9 immune 39

response against T. spiralis could be enhanced or attenuated after treatments with IL-25 or 40

neutralizing antibody against IL-25, respectively, correlating positively with the levels of 41

intestinal mastocytosis and the expression of IL-9-regulated genes, including mast cell- and 42

paneth cell-specific genes. Thus, our study demonstrates that intestinal IL-25 promotes 43

protective immunity against T. spiralis infection by inducing antigen specific Th9 immune 44

response. 45

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Introduction 48

Gastrointestinal roundworm parasites such as Trichuris muris, Trichinella spiralis, 49

and Strongyloides stercolaris affect people worldwide, especially in developing countries (1). 50

Each of these parasites resides in a distinct anatomical compartment of the host, which 51

launches a protective immune response against the invading parasite (2). Trichinella spiralis 52

is known to be a food-borne, zoonotic parasite that infects the small intestine. Following 53

parasite infection, encysted first-stage larvae mature into adults in the small intestine, where 54

they reside and reproduce within the intestinal epithelial cells (1). Female adult worms will 55

produce larvae which then migrate to muscle. The host protective mechanism in 56

gastrointestinal helminth infection is known to be mediated by Th2 immune responses (3). 57

Although most components of the Th2 immune response exhibit stereotypical activation 58

against these intestinal helminth parasites, certain effector molecules are capable of mediating 59

specific protective effects against a particular parasite (1). 60

IL-25 (IL-17E), a cytokine of the IL-17 family is involved in the initiation of type-2 61

immune responses (4-6). Several experimental evidences indicate that IL-25 is derived from 62

epithelial cells and plays important roles in mucosal immunity (5, 7). IL-25 is known to 63

mediate host protective immunity to several intestinal helminthes. During Trichuris muris 64

infection, IL-25 could promote Th2 cytokine-dependent immune response and goblet cell 65

hyperplasia, while limited pro-inflammatory cytokine production and chronic intestinal 66

inflammation (7). Other studies demonstrated that IL-25-deficient mice had impaired Th2 67

protective immunity and diminished intestinal smooth muscle and epithelial responses to N. 68

brasiliensis infection, thereby resulting in the failure to expel Nippostrongylus brasiliensis 69

efficiently (8), (9). Whether IL-25 also plays a critical role in the rapid expulsion of T. 70

spiralis has not been addressed. 71

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Cytokines secreted by Th2 cells, such as IL-4 and IL-13 but not IL-5, are known to be 72

effective against tissue-dwelling intestinal nematode parasites, including T. spiralis. IL-9, a 73

known Th2-associated cytokine, enhances biological function of IL-4 in accelerating worm 74

expulsion (10). In the intestinal mucosa, IL-9 modulates epithelial cell function by up-75

regulating the expression of several innate immunity mediators, including Paneth cell marker 76

angiogenin 4, cryptdins, and phospholipase A2 genes, (11). IL-9-mediated worm expulsion 77

is correlated with mast cell expansion and secretion of specific proteases such as mouse mast 78

cell protease 1 (mMCPT-1) (1, 12-13). Accumulating studies suggest that IL-9 may be a 79

pivotal cytokine to mediate effective expulsion of T. spiralis, possibly via triggering 80

epithelial cell response and amplification of intestinal mastocytosis and mMCPT-1 release (2, 81

12, 14). Indeed, IL-9 transgenic mice exhibited elevated intestinal mastocytosis and had 82

increased levels of serum mMCPT-1, which were associated with the rapid expulsion of T. 83

spiralis from the gut (14). Mice that lack mast cells failed to expel worms during T. spiralis 84

infection (12, 15). These lines of evidence support a role for IL-9 as a specific effector 85

molecule against T. spiralis infection. 86

Recent studies demonstrate that IL-9 can be produced by a specialized population of 87

T cells, termed Th9 cells (16-17). It was suggested that Th9 cells are distinct from the Th2 88

cell lineage and mainly function in mucosal immunity (16). TGF-β and IL-4 potentiated the 89

differentiation of Th9 cells from naïve CD4+ T cells by enhancing IL-9 production from 90

activated T cells in vitro (16-17). Inhibition of Th9 cell development by blocking TGF-β 91

signaling resulted in the diminished immune response to T. muris, indicating the importance 92

of Th9 cells in the protective immunity to helminth parasites (16). Other factors, such as IL-1 93

(18), IL-33 (19), and IL-25 (20) were also shown to enhance IL-9 production by Th9 cells. In 94

the absence of IL-25, allergic asthma was alleviated in association with reduced Th2 95

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cytokines and IL-9 (20). Notably, IL-25 potentiated the effect of TGF-β and IL-4 in 96

promoting Th9 differentiation in vitro. Whether IL-25 plays a critical role in regulating the 97

kinetics and function of antigen specific IL-9-producing T cell response that has the potential 98

to mediate protective immunity against T. spiralis infection in vivo remains unclear. 99

In this study, we demonstrate that IL-25 mediates protective immune response to T. 100

spiralis by enhancing antigen-specific Th9 cell function. Following T. spiralis infection, IL-101

25 mRNA and protein were induced before the expression of IL-9 in the intestine. Indeed, the 102

antigen-specific Th9 response occurred transiently in the early-stage and appeared to be 103

important for mediating an effective worm clearance. We also showed that exogenous IL-25 104

treatments enhanced antigen-specific IL-9 production which was associated with the 105

increased worm expulsion in the intestine and muscle, while IL-25 blockade reduced antigen-106

specific IL-9 response and worm expulsion. These changes of antigen-specific Th9 response 107

mediated by IL-25 treatments or blockade correlated with the alteration of mast cell number 108

and the expression levels of IL-9-regulated genes, including mast cell protease-1 and paneth 109

cell marker Cryptdin and Ang4 in the intestine. By contrast, IL-25 treatments failed to 110

modulate the expression of these IL-9-regulated genes in IL-9-deficient mice during T. 111

spiralis infection. These results suggest that IL-25 mediates effective immune response to 112

expel T. spiralis infection through the induction of antigen-specific Th9 immune response. 113

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Materials and Methods 119

Animals 120

C57Bl/6 mice and BALB/C mice were obtained from The National Laboratory Animal 121

Center, Mahidol University. Female 6-8 weeks old mice were used for experiments. IL-9-122

deficient mice in BALB/C background were kindly provided by Dr. Andrew McKenzie 123

(Medical Research Council Laboratory of Molecular Biology, Cambridge, U.K.). All animal 124

studies were approved by the Thammasat University Animal Care and Use Committee. 125

Monoclonal antibodies and flow cytometry 126

Recombinant mouse IL-25–Ig protein was prepared as we previously reported (5). Anti–IL-127

25 mAbs were generated as previously described (5). PerCP-conjugated anti-CD4 (GK1.5), 128

PE-conjugated-anti-IL-4 (11B11), PE-conjugated-anti-IL-17 (TC11-18H10) antibodies were 129

from BD Pharmingen. APC-conjugated anti-IL-9 (RM9A4) antibody was from BioLegend. 130

Cells were analyzed using a FACSCalibur cytometer (BD Biosciences). 131

Parasite infection and worm expulsion 132

T. spiralis (ISS62) (21) originated from the outbreak in the Mae Hong Son Province in 1986 133

was obtained from the Department of Parasitology, Faculty of Medicine, Khon Kaen 134

University and was maintained through infection in ICR mice (22). The larvae were obtained 135

from infected mice after 30 day post infection by homogenizing muscle with pepsin-HCl 136

digestion. C57Bl/6 or BABL/C or IL-9-deficient mice were infected orally with 400 T. 137

spiralis larvae and sacrificed at various time points post infection. Anti-IL-25 antibody (100 138

μg/mice) (5) or control rat IgG (100 μg/mice) or IL-25Ig (5 μg/mice) (5) were given 139

intraperitoneally at day 0, 1, and 3 after infection. For worm burden analysis, small intestines 140

of infected mice at day 7 and day 14 post infection were removed, open longitudinally, and 141

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incubated in Hanks’ Balanced Salt Solution (HBSS) at 37°C for 3 hr. Following incubation, 142

intestines were agitated and worms were then counted using inverted microscope. Muscle 143

larvae burdens were assessed 30 days post infection in whole carcasses as described 144

previously (23). 145

Histology 146

Intestinal tissue samples (jejunum) were taken 10 cm from the pylorus and were fixed in 10% 147

buffered formalin, and subsequently dehydrated in ethanol and embedded in paraffin wax. 148

Sections were stained with Leder stain for mast cells. Numbers of mast cells were expressed 149

per villus crypt unit (VCU). 150

Evaluation of cytokine production 151

Mesenteric lymph nodes or spleens were harvested from mice infected with T. spiralis at 152

various time points after infection or from naïve mice (23). Single-cell suspensions were 153

prepared and further subjected to intracellular cytokine analysis and ELISA (5, 20). For 154

intracellular cytokine analysis, cells were restimulated with 500 ng/ml ionomycin and 50 155

ng/ml PMA in the presence of GolgiStop (BD Biosciences) for 5 hours (5, 20). Cells were 156

permeabilized with a Cytofix/Cytoperm kit (BD Biosciences) and analyzed for the expression 157

of IL-9, IL-4, and IL-17. For ELISA, single-cell suspensions were stimulated with or without 158

T. spiralis extract antigens (50 μg/ml). Following 3-day incubation at 37°C with 5% CO2, 159

culture supernatants were collected and analyzed for the production of cytokines. For 160

restimulation experiments, single-cell suspensions were cultured with antigen for 7 days, 161

followed by purifying for CD4+ cells using anti-CD4-conjugated microbeads and separating 162

out the CD4+ cell population by MACS according to the manufacturer’s instruction 163

(Miltenyi-Biotec). CD4+ cells were restimulated with anti-CD3 overnight, and culture 164

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supernatants were collected and analyzed for cytokine production by ELISA. The antibody 165

pairs for IL-4, IL-9, and IL-17 were obtained from BD Pharmingen, and assays were 166

performed according to the manufacturer's instructions. For quantitative measurement of 167

intestinal IL-25, the intestines (jejunum) were excised and homogenized in cold PBS (24), 168

and the resulting supernatants were measured for IL-25 using the IL-25 ELISA kit from R&D 169

Systems. 170

Real-time RT-PCR analysis 171

The small intestines (jejunum) were removed from naïve or T. spiralis-infected mice and 172

homogenized in TRIzol reagent (Invitrogen). Total RNA extracted using TRIzol reagent was 173

used to generate cDNA using oligo-dT, random hexamers, and MMLV reverse transcriptase 174

(Invitrogen) (5, 20). For quantitation of cytokines, cDNA samples were amplified in IQTM 175

SYRB® Green Supermix (Biorad Laboratories). The data were normalized to actin 176

expression (Actb). The primer pairs for analysis of cytokines (20) and for Mcpt1, Mcpt2, 177

Ang4, and Cryptdins were used as previously described (11). 178

Cell transfer experiment 179

Mesenteric lymph node cells were obtained on day 7 and 14 after infection with 400 T. 180

spiralis larvae. Single cell suspensions were prepared and cultured with T. spiralis antigen 181

(10 μg/ml). After 7 days, cells were then washed and resuspended in PBS. Culture cells 182

were then pooled and enriched for CD4+ cells by using anti-CD4 microbeads (L3T4), 183

followed by positive magnetic beads separation (Miltenyi Biotec). We also tested for the 184

induction of IL-9-producing T cells by staining the restimulated cultured cells before 185

performing transfer experiment. CD4+ enriched cells from mice infected for 7 days (Th2+Th9 186

cells) or those infected for 14 days (Th2 cells) were intravenously injected into syngeneic 187

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recipient mice with 2x107 cells, 24 hours before oral infected with 400 T. spiralis larvae. 188

Infected mice were killed on day 6 after infect with T. spiralis and the intestines were 189

harvested for the analysis of cytokine gene expression, worm burdens, and mast cell numbers. 190

For some adoptive transfer experiment, IL-9-deficient mice were transferred with antigen-191

specific T cells prepared from IL-9-deficient mice or wild type (BALB/C) mice after 7 day 192

post infection as described above. Mice were then orally infected with 400 T. spiralis larvae 193

on the next day and analyzed for antigen-specific cytokine production in mesenteric lymph 194

nodes. Worm burdens were counted on day 6 after T. spiralis infection. 195

Statistical Analysis 196

Each experiment was conducted two or three times. Data are presented as mean value + SD. 197

Data were analyzed using the Student’s t test or one-way ANOVA with Turkey’s post hoc 198

analysis. Statistical analysis was performed with GraphPad Prism 5 software. A value of p < 199

0.05 was considered significant. 200

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Results 202

203

IL-25 is involved in the host protective immune responses against T. spiralis infection 204

To test whether IL-25 is involved in the host protective immune responses to T. 205

spiralis infection, we assessed worm burdens in the intestines of T. spiralis-infected mice 206

following IL-25 cytokine treatments. Mice were treated with IL-25 fusion protein at day 0, 1, 207

and 3 following T. spiralis infection and the numbers of worms present in the intestine of 208

infected mice were determined at 7 and 14 days post infection. Compared to untreated mice, 209

mice treated with IL-25 significantly reduced worm burdens in the intestine at 7 days post 210

infection (Figure 1A). At 14 days post infection, a few worms remained in untreated mice; 211

however, IL-25 treatments resulted in the complete worm expulsion in infected mice (Figure 212

1A). To investigate the biological significance of the rapid expulsion observed in the IL-25-213

treated mice, muscle larvae burdens were assessed at 30 days post infection. As expected, 214

mice treated with IL-25 fusion proteins had a 3-fold reduction of muscle larvae number than 215

those in mice without treatments (Figure 1B). 216

To further examine the protective role of IL-25 in mediating immune responses 217

against T. spiralis infection, mice were treated with IL-25 neutralizing antibody or control rat 218

IgG antibody at day 0, 1, and 3 following T. spiralis infection and their numbers of intestinal 219

adult worm and muscle larvae were counted and compared. We found that the administration 220

of IL-25 neutralizing antibody in T. spiralis-infected mice resulted in a 2-fold increase of 221

worm burden (Figure 1C) (p < 0.05, compared with those in rat IgG antibody-treated group). 222

Compared to the control antibody-treated group at 14 days post infection, mice treated with 223

IL-25 neutralizing antibody were less competent to expel worms efficiently (Figure 1C). 224

Moreover, muscle larvae depositions in mice treated with IL-25 neutralizing antibody were 225

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significantly increased compared to those in rat IgG-treated mice at 30 days post infection 226

(Figure 1D), supporting the finding of the delayed worm expulsion rate in the intestines. 227

These results demonstrate that IL-25 plays important roles in mediating the host protective 228

immunity against T. spiralis infection. 229

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Temporal IL-25 expression precedes the intestinal IL-9 induction during T. spiralis 231

infection 232

To begin to address the mechanisms underlying IL-25-mediated protective immune 233

response against T. spiralis, we set out to examine intestinal IL-25 expression during 234

helminth infection. RNA samples isolated from the small intestines of mice sacrificed on 1, 235

7, 14, 30 day post infection with T. spiralis were reversed transcribed and served as template 236

for assessments of targeted gene expression using quantitative real-time PCR analyses. 237

Notably, intestinal Il25 expression was significantly elevated (> 25 fold) in mice infected 238

with T. spiralis on day 1 post infection compared to that in naïve mice (Figure 2A). The 239

helminth-induced intestinal Il25 expression declined gradually and was still detectable at day 240

30 post infection (Figure 2A). Consistent with these data, the kinetics of intestinal IL-25 241

protein secretion measured by ELISA peaked at day 1 post infection, but was normalized at 242

day 14 post infection (Fig. 2B). In addition to cytokine IL-25, we also observed significantly 243

elevated expression of IL-25’s cognate receptor Il17rb (> 6 fold) at day 7 post infection. One 244

day post infection, we observed a trend of increase of intestinal Tgfb (> 3 fold) and Il33 (> 2 245

fold) transcript expression (though this was not statistically significant) and the expression of 246

these genes remained elevated at day 7, 14 and 30 post infection (Figure 2A). Since TGF-β, 247

IL-25, and IL-33 cytokines were shown to promote the induction of IL-9-producing T cells 248

(16, 19-20), we examined the expression of Il9 as well as Il4 transcripts during T. spiralis 249

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infection. In contrast to the early induction of Il25, Il33, and Tgfb, the expression of intestinal 250

Il9 and Il4 transcripts were not induced at day 1, but increased significantly at day 7 post 251

infection (Figure 2A). These results reveal that T. spiralis infection triggers a rapid intestinal 252

IL-25 production that precedes the inductions of Il4 and Il9 gene expression in mice. 253

254

T. spiralis infection induces transient antigen-specific IL-9-producing T cell response 255

Previous studies suggest that the induction of IL-9-producing T cell response is 256

essential for the host to mount the protective immunity against helminth parasite infection. 257

(16). Having observed elevated intestinal IL-9 expression, we next address whether IL-9-258

producing T cell response are involved in the protective immunity against T. spiralis 259

infection. Indeed, a significant induction of both IL-4- and IL-9-producing CD4+ T cells in 260

the mesenteric lymph nodes were detected 7 days after T. spiralis infection (Figure 3A). 261

Notably, we also observed an increase in a population of non-CD4 cells producing IL-9 262

following T. spiralis infection (Figure 3A). To examine the duration of the induced IL-4 and 263

IL-9-producing T cell response against parasitic antigens, immune cells isolated from 264

splenocytes or mesenteric lymph nodes of mice infected with T. spiralis for 7 days, 14 days, 265

and 30 days were stimulated ex vivo with T. spiralis extract for 3 days before collecting 266

culture supernatants for the measurements of their cytokine production by ELISA. Compared 267

to naïve mice, we detected a greater induction of antigen-specific IL-9 and IL-4 production in 268

mice after 7 days of infection (Figure 3B). While antigen-specific IL-4 production in 269

mesenteric lymph nodes sustained on day 14 post-infection, antigen-specific IL-9 production 270

declined after 7 days of infection (Figure 3B). Notably, the appearance of IL-4- or IL-9-271

producing CD4+ T cell response in mesenteric lymph nodes corresponded to the temporal 272

expression pattern of intestinal Il9 and Il4 transcripts shown in Figure 2. Since Th17 cells 273

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were recently found to be associated with muscle contraction during T. spiralis infection (25), 274

we investigated the appearance of these cells during infection. Compared to naïve mice, mice 275

infected with T. spiralis also displayed enhanced antigen-specific IL-17 production on day 7 276

post infection (Figure 1B). To investigate whether IL-9 and IL-4 producing CD4+ T cells 277

can be expanded ex vivo in the presence of T. spiralis extract antigen, mesenteric lymph node 278

cells collected from mice at day 7 and 14 post infection were cultured with T. spiralis extract 279

antigens for 7 days before restimulation for intracellular cytokine analysis. Notably, the 280

frequencies of IL-9 producing (10%-15%) and IL-4 producing (8%-11%) CD4+ T cells from 281

mesenteric lymph node of mice 7-day after infection were significantly increased ex vivo in 282

the presence of T. spiralis extract antigens (Figure 3C). While the frequency of IL-4-283

producing CD4+ T cells (7%-12%) that could be activated by T. spiralis extract antigens and 284

expanded ex vivo remained constant in mesenteric lymph node of mice 14 days post 285

infection, the frequency of antigen specific IL-9-producing CD4+ T cells (1%-3%) in these 286

infected mice declined (Figure 3C). Notably, these IL-9-producing CD4+T cells did not 287

produce IL-4, IL-5 and IL-17 concomitantly (Figure 3C and Supplementary Figure 1). Our 288

data suggest that both antigen-specific Th2 (IL-4-producing CD4+ T cells) and Th9 (IL-9-289

producing CD4+ T cells) responses occur concurrently during T. spiralis infection; however 290

antigen specific Th9 immune response may be induced transiently at the early stage of T. 291

spiralis infection. 292

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Antigen-specific Th9 immune responses facilitate the expulsion of T. spiralis 294

To examine whether antigen-specific Th9 immune response can facilitate the 295

expulsion of T. spiralis, we compared worm burdens in mice that were adoptively transferred 296

with both antigen-specific Th9 and Th2 cells or with antigen specific Th2 cells 24 hours 297

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before infection with T. spiralis larvae. As shown in Figure 4A, mice received enriched 298

antigen-specific Th9 and Th2 cells that were generated from mesenteric lymph node of mice 299

infected for 7 days (Supplementary figure 2), exhibited high level of intestinal Il9 expression 300

compared to those in mice receiving enriched antigen specific Th2 cells (Figure 4A). We did 301

not see the induction of IFN-γ and IL-17 expression in mice transferred with cells from mice 302

infected for 7 or 14 days. Compared to mice without transfer, both groups of infected mice 303

that were adoptively transferred with antigen specific Th9 plus Th2 cells or antigen specific 304

Th2 cells only, had reduced worm burdens. However, the infected mice that were received 305

antigen specific Th9 plus Th2 cells were more competent to expel worm than those received 306

only antigen-specific Th2 cells (Figure 4B). 307

To provide a direct evidence that IL-9 derived from antigen specific CD4+ T cells is 308

involved in T. spiralis worm clearance, we reconstituted IL-9-deficient mice with antigen-309

specific CD4+ T cells from wild type mice or mice deficient of IL-9, and then assessed and 310

compared their antigen-specific cytokine production and worm burden after 6 day post T. 311

spiralis infection. Indeed, we could detect antigen-specific IL-9 and IL-4 production by 312

mesenteric lymph node cells from IL-9-deficient mice after the reconstitution with wild type 313

antigen-specific CD4+ T cells, but only antigen-specific IL-4 production after reconstitution 314

with IL-9-deficient antigen-specific CD4+ T cells (Figure 4C). Notably, these IL-9-deficient 315

mice reconstituted with wild type antigen-specific CD4+ T cells had fewer worm burdens 316

than those reconstituted with IL-9-deficient antigen-specific CD4+ T cells (Figure 4D). 317

Collectively, our results demonstrate that the infected mice may be more competent to expel 318

T. spiralis after the acquisition of antigen specific Th9 cells. 319

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Antigen-specific Th9 immune response to T. spiralis is regulated by IL-25 321

We previously showed that IL-25 promotes the induction of IL-9 in allergic asthma 322

(20). The finding that IL-25 expression preceded the induction of IL-9 in the intestine of T. 323

spiralis-infected mice prompted us to test whether the modulation of IL-25 activity can alter 324

T. spiralis-induced Th9 immune response. At day 7 post-infection, mesenteric lymph node 325

cells isolated from mice treated with IL-25 fusion protein or IL-25 neutralizing antibody or 326

untreated mice were activated with T. spiralis antigens for 3 days before collection of 327

supernatants for the analyses of antigen-specific cytokine production by ELISA. We found 328

that antigen-specific T cells in T. spiralis-infected mice treated with IL-25 secreted 329

significant higher amounts of Th2 cytokines IL-4, IL-13, and IL-9 than those of infected mice 330

without IL-25 treatments, while no significant change in IFN-γ and IL-17 production was 331

observed after IL-25 treatments (Figure 5A). By contrast, anti-IL-25 neutralizing antibody 332

treatments significantly attenuated the induction of antigen-specific IL-9 and other Th2 333

cytokines production in the mesenteric lymph node of infected mice (Figure 5B) (p < 0.05 for 334

IL-4, IL-5, IL-9, and IL-13, compared with those in rat IgG antibody-treated group). Notably, 335

IL-25 blockade resulted in the increase of antigen specific IFN-γ production, but not antigen 336

specific IL-17 production (Figure 5B). Our data thus suggest that IL-25 promotes protective 337

immune response against T. spiralis helminth infection through the induction of antigen-338

specific Th2 and Th9 immune response. 339

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IL-25 regulates IL-9-mediated effector function during T. spiralis infection 344

Previous studies indicate that IL-9 can regulate several innate immune cells in the 345

intestinal mucosa, including epithelial cells (goblet and paneth cells) and mast cells (11). IL-346

9-promoted T. spiralis expulsion was found to be associated with the presence of mast cells 347

and the expression of mouse mast cell proteases (12, 14-15). These studies led us to 348

hypothesize that IL-25-induced Th9 immune response can trigger IL-9-mediated effector 349

function that lead to the efficient expulsion of intestinal T. spiralis. To begin to test this 350

hypothesis, we first examined the effect of IL-25 on modulating intestinal Il9 expression in 351

mice infected with T. spiralis. At day 7 post-infection, the expression of Il9 and other Th2 352

cytokine genes (Il4, Il5, Il3, and Il10) in the intestines of mice after IL-25 treatments at day 0, 353

1, and 3 following T. spiralis infection were significantly elevated (Figure 6A). By contrast, 354

IL-25 blockade using IL-25 neutralizing antibody resulted in a significant reduction of T. 355

spiralis-induced expressions of intestinal Il9 and other Th2 cytokine genes (Il4, Il5, Il3, and 356

Il10) (Figure 7A) (p < 0.05, compared with those in rat IgG antibody-treated group). We did 357

not detect a significant induction of IFN-γ gene expression in the intestine of T. spiralis-358

infected mice after either IL-25 treatments or IL-25 blockade (Figure 6A, 7A). Notably, the 359

elevation of intestinal Il9 gene expression after IL-25 treatments were positively correlated 360

with the numbers of intestinal mast cells (Figure 6B). Moreover, IL-25 treatments induced 361

significant increase in the expression of IL-9-regulated genes, such as mouse mast cell 362

protease Mcpt1 and Mcpt2, as well as Cryptdins and Ang4 that participate in mast cell and 363

paneth cell responses, respectively in the intestine (Figure 6C). By contrast, treatments with 364

IL-25 neutralizing antibody attenuated T. spiralis-induced intestinal mast cell recruitments 365

and expression of Mcpt1, Mcpt2, cryptdins and Ang4 transcripts compared to those in 366

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infected mice received control rat IgG antibody (Figure 7B and C). Thus, our data suggest 367

that IL-25 may promote the induction of IL-9-mediated immune response that triggers 368

intestinal mastocytosis and paneth cells, resulting in effective T. spiralis expulsion. 369

Next, we examine whether IL-25 can enhance IL-9-mediated effector functions that 370

promote effective protective immunity against T. spiralis infection in vivo. While wild type 371

mice treated with IL-25 were more competent in expelling worm than those without 372

treatments, IL-9-deficient mice failed to respond to IL-25 treatments and remain ineffective 373

in worm expulsion (Fig. 8A). As expected, the enhanced immune response in expelling worm 374

infection in wild type mice after IL-25 treatments was correlated with increased expression of 375

IL-9-regulated genes, mcpt1, mcpt2 and cryptdins (Fig. 8B). Furthermore, the failure to 376

mount enhanced immune response against T. spiralis infection in IL-9-deficient mice after 377

IL-25 treatments coincided with the findings that the expression of IL-9-regulated genes, 378

including; mcpt1, mcpt2 and cryptdins in these mice remained unchanged (Fig. 8B). 379

Interestingly, while the significantly increased intestinal Il5 expression induced by IL-25 380

treatments were comparable in both wild type and IL-9-deficient mice (>200-folds), the 381

increased intestinal Il13 expression was less pronounced in mice deficient of IL-9 than those 382

in wild type mice after IL-25 treatments (Fig. 8B). These data indicate that IL-25-regulated 383

IL-9 effector function plays important roles in immunity to T. spiralis infection. 384

385

386

Discussion 387

IL-25 is an important cytokine in the initiation of type-2 immune responses (4). There 388

is strong evidence supporting a crucial role of IL-25 in mediating the protective immunity to 389

gastrointestinal helminthes, such as Nippostrongylus braziliensis (8-9) and Trichuris muris 390

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(7); however the involvement of this cytokine in driving the immune response against 391

Trichinella spiralis infection has not been addressed. In this study, we showed that IL-25 392

enhanced effective protective immunity to T. spiralis infection. Following T. spiralis 393

infection, the expression of intestinal IL-25 was up-regulated and preceded IL-9 expression. 394

IL-25-mediated host protective immune responses to T. spiralis was associated with the 395

induction of antigen-specific Th9 and Th2 immune response. Treatments with exogenous IL-396

25 induced the increased antigen-specific IL-9 production, expression of Mcpt1, Mcpt2, 397

Cryptdins, and Ang4 transcripts, and intestinal mastocytosis, which resulted in the enhanced 398

worm expulsion . In contrast, IL-25 blockade resulted in an inefficacy in worm expulsion, 399

correlating with the reduced intestinal IL-9 expression, mast cell number, as well as mast 400

cell- and paneth cell-specific gene expression. These findings substantiate the function of IL-401

25 in evoking protective immunity against T. spiralis infection by regulating IL-9 effector 402

function. 403

During T. spiralis infection, we showed that IL-25 expression was increased in the 404

intestine on day 1 after infection, suggesting that IL-25 may function in the early stage of T. 405

spiralis infection. The kinetics of intestinal IL-25 expression during T. spiralis infection 406

appeared to be different from that of mice infected with N. braziliensis which was peaked at 407

day 9 post-infection (9). The difference in the kinetics of IL-25 expression may be due to the 408

distinct life cycles of these parasites. During T. spiralis infection, parasitic larvae initiate the 409

process by penetrating the columnar epithelium of the small intestine and the larvae rapidly 410

develop into adult. Previous studies indicate that epithelial cells of lung and intestine are the 411

major IL-25 producers (5, 7, 9). T. spiralis might induce IL-25 expression, while penetrating 412

into the epithelial layer. Thus, early induction of intestinal IL-25 may be a critical step in 413

initiating the protective immunity against T. spiralis infection. 414

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In addition to IL-25, intestinal Il4 and Il9 expression were also induced 7 days post 415

infection. Unlike intestinal Il4 expression that was sustained from day 7 to day 14 post 416

infection, intestinal Il9 expression was transiently induced, peaked at day 7 post infection, 417

and then declined at day 14 post infection. Our finding of a transient Il9 induction in vivo is 418

coincided with a recent study showing that IL-9 was produced but declined rapidly during in 419

vitro differentiation of naïve T cells with TGF-β and IL-4(26). It is possible that the strong 420

signals from the antigens or environmental stimuli induced by the parasite in the early stage 421

of infection are required for the induction and maintenance of Il9 expression and that the 422

absence of these signals in subsequent stages of infection results in the decline in its 423

expresssion. Correlating with the IL-9 expression pattern in the intestine, we showed that 424

antigen-specific Th9 response in mesenteric lymph nodes declined rapidly after 7 day post 425

infection. Mice transferred with antigen-specific T cell deficient in IL-9 were less effective in 426

T. spiralis worm clearance than those receiving wild type antigen-specific T cells, suggesting 427

that the combination of antigen-specific Th9 and Th2 response may be required for effective 428

clearance of T. spiralis in the intestine. Interestingly, we also observed increased frequencies 429

of IL-9 production by CD4- cells in some experiments. Whether these non-T/non-B IL-9 430

producers during T. spiralis infection are the recently described type 2 innate lymphoid cells 431

(ILC2) remains to be investigated (27). 432

IL-9 production can be regulated by several cytokines (16-20); however, the 433

regulation of Th9 cell differentiation during helminth infection is less clear. In vitro, TGF-β 434

and IL-4 were the major stimulators for Th9 cell induction (16-17). The absence of TGF-β 435

signaling resulted in an impaired IL-9 production that correlated with increased worm burden 436

(16). Furthermore, our previous findings demonstrated that IL-25 can promote Th9 cell 437

differentiation (20). IL-25 blockade resulted in alleviated allergic asthma that was coincided 438

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with reduced Il9 expression in the lung (20). In this study, we show that T. spiralis infection 439

triggers early induction of Il25 as well as Tgfb and Il4 expression that may be essential for the 440

optimal generation of Th9 cells in vivo. Indeed, when IL-25 production was abrogated during 441

T. spiralis infection, we detected increased worm burden that associated with a reduction of 442

the frequency of antigen specific Th9 and Th2 cells. Thus, early induction of IL-25 after T. 443

spiralis infection may evoke protective immunity against parasite through promoting the 444

induction of Th9 and Th2 immune response. Indeed, we demonstrated that the enhanced 445

worm clearance driven by IL-25-induced Th9 immune response occurred only in wild type 446

mice, not IL-9-deficient mice, suggesting that IL-9 function participates in IL-25-enhanced 447

protective immunity to T. spiralis. Notably, we observed that IL-9-deficient mice were 448

competent in T. spiralis worm clearance. Consistent with our findings, neutralization of IL-9 449

using anti-IL-9 antibody had no significant effect on worm expulsion, while overexpression 450

IL-9 or exogenous IL-9 treatments in mice resulted in an accelerating worm expulsion in T. 451

spiralis infection (10, 14, 28). It is likely that the up-regulation of IL-9 expression induced 452

by IL-25 may be important for the optimal induction of IL-4 and IL-13. Our results thus 453

could not rule out the possibility that other Th2 cytokines may participate in the regulation of 454

IL-25-induced protective immunity to T. spiralis. Previous studies showed that IL-33 can 455

initiate IL-9 protein secretion in vitro in human CD4+ T cells (19). However, we observed 456

that IL-33 expression was not significantly induced by T. spiralis infection. 457

Helminthes genera and species possess distinct features that stimulate immune 458

responses; therefore, a host may deploy differing sets of defense mechanisms against these 459

separate parasites. The principal function of cytokine IL-9 in the intestine is to regulate 460

innate immune cells, including mast cell and epithelial cells. In small intestine, IL-9 461

administration not only induced mast cell-specific genes, but also up-regulated innate 462

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immunity genes, including paneth cell markers such as angiogenin 4, cryptdins, and 463

phospholipase A2 genes (11). Mast cells seem to be important for T. spiralis worm 464

expulsion, while they have few involvements in N. braziliensis expulsion (1, 15, 29). The 465

numbers of Paneth was found to be increase in the epithelial monolayers of T. spiralis-466

infected mice. Enhanced secretion of Paneth cell products such as cryptdins and other 467

antimicrobial proteins shown to be regulated by mucosal T cells is expected to contribute to 468

immunity against T. spiralis infection (30). Our finding that IL-9 is important for IL-25-469

enhancing protective immunity against T. spiralis infection prompted us to investigate the 470

numbers of mast cells and the expression of mast cell protease and paneth cell specific genes 471

in the intestines following IL-25 cytokine or antibody treatment. The modulation of IL-25 472

production during T. spiralis infection could alter intestinal expression of mast cell protease 473

genes (Mcpt1, Mcpt2) and paneth cell-specific gene (Cryptdins and Ang4), which was 474

correlated positively with the changes of antigen specific Th9 response, thus linking the role 475

of IL-25 in regulating IL-9-mediated effector function. Indeed, our finding that IL-25 476

treatments failed to induce the increased expression of those IL-9 targeted genes in mice 477

deficient of IL-9 further substantiates the role of IL-25/IL-9 axis in promoting function of 478

mast cells and paneth cells that leads to protective immunity against T. spiralis infection. In 479

contrast to our findings, the number of mast cells and level of mouse mast cell protease-1 480

were not changed in the gut of IL-25-deficient mice infected with T. muris (7). It has been 481

reported that mucosal mast cell response was not required for protection against infection 482

with T. muris (31). Indeed, the specific intestinal habitats of parasites may influence types of 483

effector immune responses. T. muris eggs were hatched in the ileum of the small intestine 484

and the larvae then migrate to the cecum where they invade the mucosal epithelial cells at the 485

crest of the crypt, while T. spiralis larvae migrate to small intestinal sites at the base of villi 486

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where they reside in a syncytium of epithelial cells (2). Together, our findings and other 487

studies highlight the view that cytokines, such as IL-25 may play a crucial role in mediating 488

effective protective immune response against distinct type of helminth infection. 489

In conclusion, we provide in vivo evidences that IL-25 promotes protective immunity 490

against T. spiralis infection with a distinct pathway by inducing Th9 cell response that drive 491

intestinal mastocytosis and paneth cell function. Future investigations on understanding the 492

roles of type 2 innate lymphoid cells (ILC2), as well as Th9 and Th2 cells, in contributing to 493

effective immune responses against parasite infection may provide novel insights in 494

designing better approaches to prevent parasitic infection. 495

496

Acknowledgements 497

We thank Dr. Andrew McKenzie (Medical Research Council Laboratory of Molecular 498

Biology, Cambridge, U.K.) for IL-9-deficient mice, Dr. Wanchai Maleewong and Dr. 499

Pewpan Maleewong (Department of Parasitology, Faculty of Medicine, Khon Kaen 500

University) for T. spiralis strain information. the Faculty of Allied Health Sciences, 501

Thammasat University for the support, Pattra Moonjit and the Faculty of Veterinary 502

Medicine, Kasetsart University (Kamphaeng Saen Campus) for their help in histology. This 503

work was supported by the Research Grant for New Scholar (co-funded by the Thailand 504

Research Fund (TRF) and Commission on Higher Education, MRG5380229), the 505

Coordinating Center for Thai Government Science and Technology Scholarship Students of 506

the National Science and Technology Development Agency (CSTS, NSTDA), the National 507

Research University Project of Thailand, Office of the Higher Education Commission. 508

The authors declare no conflicting financial interests. 509

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510

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614

Figure Legends 615

Figure 1. IL-25 mediates protective immunity to T. spiralis infection. 616

(A-B) C57Bl/6 mice were administered with or without IL-25Ig intraperitoneally at day 0, 1, 617

and 3 following T. spiralis infection. (A) At day 7 and day 14 post-infection, whole 618

intestines were harvested and analyzed for adult worms in the intestine. (B) At day 30 post-619

infection, whole carcasses of infected mice from different groups were analyzed for muscle 620

larvae burden. (C-D) C57Bl/6 mice were administered with control rat IgG or anti-IL-25 621

neutralizing antibody intraperitoneally at day 0, 1, and 3 following T. spiralis infection. (C) 622

Adult worms in small intestine were counted at day 7 and day 14 post-infection, (D) Muscle 623

larvae were analyzed in mice sacrificed at day 30 post-infection. Graphs depict mean±SD 624

and are a representative of three independent experiments with three to four mice per group. 625

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Significance was determined by Student’s t test * p < 0.05 (compared with data in control 626

treated group). 627

Figure 2. Il25 expression is up-regulated transiently and precedes intestinal Il9 628

induction in T. spiralis-infected mice. 629

C57Bl/6 mice were infected with T. spiralis for 1, 7, 14, or 30 days. Small intestines 630

(jejunum) were harvested from naïve mice or mice infected at the indicated time points. (A) 631

Total RNA was isolated and subjected to cDNA synthesis and subsequent real-time PCR 632

analysis of cytokine gene expression. Data are expressed as fold induction over actin (Actb) 633

expression, with the mRNA levels in the naive group set as 1. (B) The small intestines 634

(jejunum) of naïve and infected mice were homogenized in cold PBS and supernatant was 635

analyzed for IL-25 content by ELISA. Graphs depict mean + SD and are representative of at 636

least two independent experiments with three to four mice per group; Significance was 637

determined by one-way ANOVA with Tukey’s post hoc analysis (* p < 0.05). 638

Figure 3. The kinetics of antigen-specific IL-9- and IL-4-producing T cell response 639

during infection with T. spiralis. 640

(A) C57Bl/6 mice were infected with T. spiralis for 7 days. Mesenteric lymph nodes from 641

naïve mice and infected mice were harvested, and analyzed for surface CD4 staining and 642

intracellular cytokine staining of IL-4 and IL-9. The results were presented as the percentage 643

of the cells and the total cell number (p < 0.05, compared with the number in naïve mice). 644

(B) C57Bl/6 mice were infected with T. spiralis. At the indicated time points [days post 645

infection (dpi)], mesenteric lymph nodes or spleens from naïve and infected mice were 646

harvested and restimulated with or without T. spiralis extract antigen (concentration of 50 647

μg/ml) for 3 days. The cytokine levels of culture supernatants were determined by enzyme-648

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linked immunosorbent assay (ELISA). (C) Mesenteric lymph node cells from naïve mice or 649

mice infected with T. spiralis for 7 or 14 days were cultured with T. spiralis extract antigen 650

(50 μg/ml) for 7 days and then were enriched for CD4+ T cells by MACS. Enriched CD4+ 651

cells were then restimulated and analyzed for intracellular cytokine staining (plots are gated 652

on CD4+ cells). The results were also presented as the percentage of the cells. Graphs depict 653

mean + SD and are representative of three experiments with three to four mice per group; 654

Significance was determined by Student’s t test (A, C) or one-way ANOVA with Tukey’s 655

post hoc analysis (B) * p < 0.05. 656

Figure 4. Antigen-specific Th9 cells enhance effective worm expulsion. 657

C57Bl/6 mice were infected with T. spiralis. At day 7 or day 14 post infection, mice were 658

sacrificed and their mesenteric lymph nodes were harvested and cultured with T. spiralis 659

extract antigen. After 7 days of culture, cells of 7-day or 14- day-infected mice were then 660

collected and enriched for CD4+ cells. Both antigen-specific Th2 and Th9 cells (2x107 cells) 661

obtained from 7-day-infected mice or antigen-specific Th2 cells obtained from 14-day-662

infected mice were transferred into C57Bl/6 mice. After 24 hours, the recipient mice were 663

then infected with T. spiralis. Six days post infection, mice were sacrificed and analyzed for 664

cytokine gene expression (A) and worm burden (B). IL-9-deficient mice were intravenously 665

transferred with antigen-specific CD4+ T cells prepared as above from wild type or IL-9-666

deficient mice, and transferred mice were then infected with T. spiralis. After 6 days post 667

infection, mice were then analyzed for antigen-specific cytokine by ELISA (C) and worm 668

burden (D). Graphs depict mean + SD and are representative of at least two independent 669

experiments with three to four mice per group; Significance was determined by one-way 670

ANOVA with Tukey’s post hoc analysis * p < 0.05. 671

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672

673

Figure 5. Antigen-specific IL-9 response to T. spiralis is regulated by IL-25. 674

C57Bl/6 mice were administered (A) with or without IL-25Ig or (B) with rat IgG antibody or 675

IL-25-neutralizing antibody intraperitoneally at day 0, 1, and 3 following T. spiralis infection. 676

At day 7 post-infection, Mesenteric lymph node cells were harvested and single cell 677

suspensions were then cultured with or without T. spiralis extract antigen (50 μg/ml). After 678

three days, supernatant was collected and analyzed for T. spiralis-specific cytokine 679

production by ELISA. Graphs depict mean±SD and are a representative of at least three 680

independent experiments with three to four mice per group. Significance was determined by 681

one-way ANOVA with Tukey’s post hoc analysis * p < 0.05. 682

Figure 6. Exogenous IL-25 treatment during T. spiralis infection enhances intestinal IL-683

9, mast cell and paneth cell-specific gene expression. 684

C57Bl/6 mice were administered with or without IL-25Ig intraperitoneally at day 0, 1, and 3 685

following T. spiralis infection. At day 7 post-infection, small intestines (jejunum) were 686

harvested and subjected for (A) RNA extraction, followed by cDNA synthesis and cytokine 687

gene expression by real-time PCR analysis. Data are expressed as fold induction over actin 688

(Actb) expression, with the mRNA levels in the naive group set as 1. (B) Small intestines 689

(jejunum) were fixed with 10% formalin buffer and subjected for histological analysis of 690

mast cells by leder staining. Numbers of mast cells were expressed per villus crypt unit 691

(VCU). (C) cDNA was analyzed for the expression of mouse mast cell protease 1 (Mcpt1), 692

mouse mast cell protease 2 (Mcpt2), paneth cell marker Crypdins and Ang4 by real-time 693

PCR. Graphs depict mean±SD and are a representative of at least two independent 694

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31

experiments with three to four mice per group. Significance was determined by one-way 695


Figure 7. IL-25 blockade during T. spiralis infection reduces intestinal IL-9, mast cell 697

and paneth cell-specific gene expression. 698

C57Bl/6 mice were administered with rat IgG (control) or anti-IL-25 neutralizing antibody 699

intraperitoneally at day 0, 1, and 3 after T. spiralis infection. At day 7 post infection, the 700

small intestines (jejunum) were removed and assessed for (A) cytokine gene expression by 701

real-time RT-PCR analysis. Data are expressed as fold induction over actin (Actb) expression, 702

with the mRNA levels in the naive group set as 1. (B) Jejunum tissue was fixed in 10% 703

formalin buffer and subjected for leader staining. Numbers of mast cells were expressed per 704

villus crypt unit (VCU). (C) cDNA was analyzed for mast cell and paneth cell-specific gene 705

expression by real-time PCR. Graphs depict mean + SD and are representative of at least 706

three independent experiments with three to four mice per group; Significance was 707

determined by one-way ANOVA with Tukey’s post hoc analysis * p < 0.05. 708

Figure 8. IL-9 is required for IL-25-enhanced T. spiralis worm clearance. 709

IL-9-deficient or wild type mice were administered with or without IL-25Ig intraperitoneally 710

at day 0, 1, and 3 following T. spiralis infection. At day 7 post-infection, small intestines 711

were harvested and subjected for worm burden (A) or gene expression analysis by real-time 712

PCR (B). Graphs depict mean±SD and are a representative of at least two independent 713

experiments with three to four mice per group. Significance was determined by one-way 714


716

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0

30

60

90

120

150

Rat IgG Anti-IL-25

Worm

num

ber/

inte

stine

Day 7

Figure 1

A

D

B

C

0

5000

10000

15000

20000

25000

30000

Rat IgG Anti-IL-25

Muscle

larv

ae/m

ouse

0

5000

10000

15000

None IL-25Ig M

uscle

larv

ae/m

ouse

*

*

*

*

0

10

20

30

40

50

Rat IgG Anti-IL-25

Worm

num

ber/

inte

stine Day 14

0

20

40

60

80

100

None IL-25Ig

Worm

num

ber/

inte

stine

Day 7

0

5

10

15

20

None IL-25Ig

Worm

num

ber/

inte

stine

Day 14

*

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Figure 2

days post infection

0

1

2

3

4

Naïve 1 7 14 30

Fold

Induction

days post infection

Il33

0

10

20

30

40

Naïve 1 7 14 30

Fold

Induction

Il25

A

0

2

4

6

8

Naïve 1 7 14 30

Fold

Induction

Il9

days post infection

0

20

40

60

80

Naïve 1 7 14 30

Fold

Induction

Il4

days post infection

0

2

4

6

8

Naïve 1 7 14 30

Fold

Induction

days post infection

Il17rb

0

1

2

3

4

5

Naïve 1 7 14 30

Fold

Induction

Tgfb

days post infection

days post infection

B

0

20

40

60

80

100

Naïve 1 7 14 30

IL-2

5 (

pg/m

l)

*

* *

* *

*

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0

0.1

0.2

0.3

0.4

0.5

IL-4

(ng/m

l)

Figure 3

Naïve mice

T.spiralis-

infected mice

A

0.06% 0.08%

0.1% 0.32%

0.05% 0.06%

0.25% 0.35%

IL-4

CD4

IL-9

Day 7

C

1.6% 0.9%

9%

Day 14

2.6% 0.2%

2.9%

13% 1.3%

8.6%

14.6% 1.2%

1.4%

IL-9

IL-9

IL-4 IL-17

0

5

10

15

20

IL-9+ IL-4+ IL-9+IL-4+

Perc

enta

ge

Day 14

Day 7 *

0

1

2

3

4

5

Naïve Infected

cells (

x10

4)

IL-9+CD4+

0

1

2

3

4

5

Naïve Infected

cells (

x10

4)

IL-4+CD4+

* *

0 0.1 0.2 0.3 0.4 0.5

Naïve Infected

IL-9+CD4+

0

0.1

0.2

0.3

0.4

0.5

Naïve Infected

IL-4+CD4+

Perc

enta

ge

Perc

enta

ge

Spleen Mesenteric lymph node

dpi

B

* *

0

0.5

1

1.5

IL-9

(ng/m

l)

0

0.1

0.2

0.3

IL-4

(ng/m

l)

0

0.2

0.4

0.6

0.8

1

IL-1

7 (

ng/m

l)

0

0.2

0.4

0.6

0.8

1

IL-1

7 (

ng/m

l)

dpi

dpi

dpi

dpi

dpi

*

*

*

*

*

*

* *

+ T. spiralis antigen None

* *

0

0.5

1

1.5

2

IL-9

(ng/m

l)

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Figure 4

A

0

100

200

300

Fold

Induction

Il13

0

1

2

3

4

5

Fold

Induction

Il17

0

200

400

600

800

Fold

Induction

Il9

0

20

40

60

80

100

Fold

Induction

Il4

0

0.5

1

1.5

2

Fold

Induction

Ifng

D

* * *

* * *

0

20

40

60

80

100

Worm

burd

en/s

mall

inte

stine

B

*

C

* *

*

0

0.2

0.4

0.6

0.8

1

ng/m

l

IL-4

* *

0

30

60

90

120

150

Worm

num

ber/

inte

stine

* *

0

0.2

0.4

0.6

0.8

1

ng/m

l

IL-9

* *

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0

5

10

15

20

IL-1

3 (

ng/m

l)

0

0.05

0.1

0.15

0.2

IL-4

(ng/m

l)

0

0.1

0.2

0.3

0.4

0.5

IL-4

(ng/m

l)

A

Figure 5

*

B

*

0 2 4 6 8

10

IL-5

(ng/m

l) *

0

1

2

3

4

5

IL-9

(ng/m

l)

* *

0

0.5

1

1.5

2

IL-1

7 (

ng/m

l)

0

5

10

15

20

IL-1

3 (

ng/m

l)

* *

0

2

4

6

8

IFN

- (

ng/m

l)

*

0

5

10

15

20

25

IFN

- (

ng/m

l)

0

0.5

1

1.5

2

IL-1

7 (

ng/m

l)

0

2

4

6

8

10

IL-5

(ng/m

l)

* *

* *

0

1

2

3

4

5

IL-9

(ng/m

l)

* *

* *

+ T. spiralis antigen None

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Figure 6

0

0.5

1

1.5

2

Naïve None IL-25Ig

Fold

Induction

Ifng

0

50

100

150

Naïve None IL-25Ig

Fold

Induction

Il5

* *

0

100

200

300

400

500

Naïve None IL-25Ig

Fold

Induction

Il4 A

* *

0

50

100

150

200

Naïve None IL-25Ig

Fold

Induction

Il9

* *

0

2

4

6

8

Naïve None IL-25Ig

Fold

Induction

Il10

*

0

100

200

300

400

500

Naïve None IL-25Ig

Fold

Induction

Il13

* *

0

25

50

75

100

Naïve None IL-25Ig

Fold

Induction

Mcpt1

*

C

*

0

25

50

75

100

Naïve None IL-25Ig

Fold

Induction

Mcpt2 * *

0

1

2

3

Naïve None IL-25Ig

mast cell c

ount/

VC

U

B

* *

0

5

10

15

20

Naïve None IL-25Ig

Fold

Induction

Cryptdins

* *

0

10

20

30

40

50

Naïve None IL-25Ig

Fold

Induction

Ang4

* *

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Figure 7

0

50

100

150

200

250

Naïve Rat IgG Anti-IL-25

Fold

Induction

Il4

0

10

20

30

40

50


Fold

Induction

Il5

A

*

* * * *

0

50

100

150

200


Fold

Induction

Il9 * *

0

1

2

3


Fold

Induction

Il10

*

0

50

100

150


Fold

Induction

Il13

0

0.5

1

1.5

2


Fold

Induction

Ifng

*

0

0.5

1

1.5

2

mast cell c

ount/

VC

U

B

* *

C

0

10

20

30

40

Fold

Induction

Mcpt1

0

10

20

30

40

Fold

Induction

Mcpt2

* * * *

0

2

4

6

8

10

Fold

Induction

Cryptdins

* *

0

5

10

15

20

Fold

Induction

Ang4

* *

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0

20

40

60

80

100

WT IL-9KO

Worm

num

ber/

inte

stine

None IL-25

Figure 8

*

0

20

40

60

WT IL-9KO

Fold

Induction

Mcpt1 *

0

25

50

75

100

WT IL-9KO

Fold

Induction

Cryptdin

*

0

20

40

60

WT IL-9KO

Fold

Induction

Mcpt2 *

0

10

20

30

WT IL-9KO

Fold

Induction

Il9

*

0

50

100

150

WT IL-9KO

Fold

Induction

Il13 *

0

100

200

300

400

500

WT IL-9KO

Fold

Induction

Il5

* *

B

A

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3 4 · 50 and Strongyloides stercolaris affect people worldwide, especially in developing countries...

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Transcript of 3 4 · 50 and Strongyloides stercolaris affect people worldwide, especially in developing countries...