yamaguchi et al, jvi · 2015. 12. 14. · yamaguchi et al, jvi 3 38 introduction 39 respiratory...

Yamaguchi et al, JVI

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Pre-exposure to CpG protects against the delayed effects of neonatal RSV infection 1

Running title: Protective effect of CpG on neonatal lungs 2

Yuko Yamaguchi1, James A. Harker1, Belinda Wang1, Peter J. Openshaw1, John S. Tregoning1, * and 3 Fiona J. Culley1,*. 4

1 Department of Respiratory Medicine, The Centre for Respiratory Infection and the MRC & Asthma 5 UK Centre in Allergic Mechanisms of Asthma, National Heart and Lung Institute, Imperial College 6 London, St. Mary’s Campus, London W2 1PG, UK. 7

* Contributed equally. 8

Corresponding authors: Dr John Tregoning, Mucosal Infection & Immunity, Section of Infectious 9 Diseases, Imperial College London, St Mary’s Campus, London, W2 1PG, UK. Phone: +44 20 7594 10 3176 Fax: +44 20 7594 2538, e-mail: [email protected] and Dr Fiona Culley, Department 11 of Respiratory Medicine, National Heart and Lung Institute, Imperial College London, St. Mary’s 12 Campus, London W2 1PG, UK. Tel: +44 20 7594 3855; fax: +44 207 262 8913; E-mail address: 13 [email protected] 14

Current addresses: YY, The Jenner Institute Laboratories, University of Oxford; BW, School of 15

Veterinary Medicine and Science, University of Nottingham; JAH, Department of Biological Sciences, 16

University of California San Diego, La Jolla, CA, USA; JST, Mucosal Infection and Immunity Group, 17

Section of Infectious Diseases, Imperial College London, UK. 18

Abstract word count: 190 19

Body text word count: 2,833 20

mailto:[email protected]

mailto:[email protected]


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Abstract 21

Severe respiratory viral infection in early life is associated with recurrent wheeze and asthma in later 22

childhood. Neonatal immune responses tend to be skewed towards T helper 2 (Th2) responses, 23

which may contribute to the development of a pathogenic recall response to respiratory infection. 24

Since neonatal Th2 skewing can be modified by stimulation with TLR ligands, we investigated the 25

effect of exposure to CpG oligodeoxynucleotides (TLR9 ligand) prior to neonatal RSV infection in 26

mice. CpG pre-exposure was protective against enhanced disease during secondary adult RSV 27

challenge, with a reduction in viral load and an increase in Th1 responses. A similar Th1 switch and 28

reduction in disease was observed if CpG was administered in the interval between neonatal 29

infection and challenge. In neonates, CpG pre-treatment led to a transient increase in expression of 30

MHCII and CD80 on CD11c positive cells and IFNγ production by NK cells after RSV infection, 31

suggesting that the protective effects may be mediated by APC and NK cells. We conclude that the 32

adverse effects of early life respiratory viral infection on later lung health might be mitigated by 33

conditions that promote TLR activation in the infant lung. 34

35

Key Words: Innate, Neonatal, TLR, respiratory tract infection, RSV, mouse 36

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3

Introduction 38

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection during infancy 39

and childhood, infecting about 90% of infants by two years of age (32). The problems associated 40

with RSV infection not only occur during the acute phase of infection but also occur in later life, 41

increasing the risk of wheeze for more than a decade after primary infection (28,30). It is not known 42

whether this is an effect of severe RSV infection on lung development, or if there are pre-existing 43

factors for wheezy lungs that makes these children more susceptible to infantile RSV infection. Some 44

of the genetic polymorphisms associated with the risk of severe RSV bronchiolitis are also risk factors 45

for allergy and asthma (32). However, the association between infantile bronchiolitis and asthma is 46

comparable in groups with different family histories of asthma suggesting a negative effect of severe 47

RSV infection on the developing lung, leading to post bronchiolitic wheeze. 48

The neonatal immune response (1) and in particular the immune response in the lung (25) is Th2 49

skewed. We have developed a mouse model of the delayed sequelae of early life RSV infection (4) 50

and using this model, we have previously shown that altering the T helper balance away from Th2 51

using recombinant viruses that express cytokines can reduce the disease outcome (14). One factor 52

influencing neonatal Th2 skewing is APC immaturity, there are fewer APC in neonatal lungs (25), they 53

are severely deficient in MHC class II and co-stimulatory molecule expression (including CD40, CD80 54

and CD86) and exhibit reduced IL-12 production (10). The insufficient IL-12 responses and the low 55

MHC-peptide density (favouring priming of Th2 type responses (21)) may limit the Th1 response in 56

neonates and hence lead to Th2-skewing (2). 57

Exposure to ligands of the Toll like receptor family (TLR) of pattern recognition receptors can induce 58

accelerated maturation of APC switching the response away from Th2 towards a protective Th1 59

response (25), which can be protective against the development of allergy (20). Evidence from 60

cohorts of farm children have shown that increased exposure to LPS is a protective factor against 61

allergy (8). Other TLR can also have a protective effect against the development of asthma, 62


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polymorphisms in the TLR9 promoter region, which may affect expression levels for example by 63

altering NF-kB binding (23), have been associated with wheeze in later life (9,29) and levels of CpG 64

oligodeoxynucleotides are greater in dust from rural homes (26). In a murine model, CpG treatment 65

protects neonatal mice from the development of allergy (6). 66

We wished to determine whether TLR ligands have a similar protective effect against the 67

development of the sequelae of neonatal RSV infection by altering the phenotype of the cellular 68

immune response. Intranasal exposure to CpG prior to neonatal RSV infection reduced the disease 69

on subsequent adult re-infection. The reduction in disease was associated with a switch to Th1 type 70

responses and reduced viral load, linked to APC maturation induced by exposure to TLR ligands. 71


5

Methods 72

Mice and Virus. Time mated pregnant BALB/c (Harlan, Carthorpe, UK) were purchased at <14d 73

gestation and pups were weaned at 3wk old. BALB/c mice were infected i.n. with 4x104 PFU/gram 74

body weight RSV A2 at 4 days (neonatal ~ 105 PFU in 20μl) or 4-6 weeks of age (adults ~ 5 x 105 PFU 75

in 100μl) under isoflurane anaesthesia. Mice were re-infected i.n. at 8wk, with 106 PFU in 100µl. RSV 76

A2 strain was grown in HEp-2 cells and viral titre determined by plaque assay. Where stated mice 77

were treated with 10 μg unmethylated or methylated CpG ODN 1826 in 20 μl PBS (5’-78

TCCATGACGTTCCTGACGTT where Cs are methylated in control CpG). RSV viral load was assessed by 79

RT-PCR for the L gene using 900 nM forward primer (5’-GAACTCAGTGTAGGTAGAATGTTTGCA-3’), 80

300 nM reverse primer (5’-TTCAGCTATCATTTTCTCTGCCAAT-3’), and 100 nM probe (5’-FAM-81

TTTGAACCTGTCTGAACATTCCCGGTT-TAMRA-3’) and normalised against 18s rRNA levels. 82

RSV-specific antibody quantification. Serum antibody was assessed by ELISA as described previously 83

(13). Antigen was prepared by infecting HEp-2 cells with RSV at 1 PFU/cell. Microtiter plates were 84

coated overnight with either RSV or HEp-2 antigen. After blocking with 1% BSA for 1h, dilutions of 85

test samples were added for a further 1h. Bound antibody was detected using peroxidase-86

conjugated rabbit anti-mouse Ig (Dako) and o-phenylenediamine as a substrate. RSV-specific 87

antibody was determined by subtracting the RSV absorbance from the HEp-2 absorbance for the 88

same sample. Specific isotypes were measured following the same protocol, changing the primary 89

antibody. 90

Cell Preparation and flow cytometry. After infection, animals were culled using i.p. pentobarbitone. 91

Cells were harvested as described previously (33). Prior to staining cells were blocked with CD16/32. 92

For surface staining antibodies against the surface markers CD3, CD4, CD8, CD11c, MHCII, CD80, 93

CD86 (BD) were added in 1:100 dilution and live/ dead discrimination was performed using 7AAD, 94

percentages were based on live cells. For intracellular staining, cells were stimulated for 4h at 37°C 95

in the presence of 10 μg/ml Brefeldin A, 100g/ml PMA and 10g/ml ionomycin. Cells were 96


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permeabilised with 0.5% saponin and stained with directly conjugated anti-IFNγ or anti-IL4. Samples 97

were run on an LSR (BD) and analysed using Winlist (Verity). 98

IFNγ Cytokine ELISA. Cytokine levels were assessed in or lung mash supernatants by ELISA using a 99

pair of capture and biotinylated detection antibodies (Cytokine: BD or Chemokine: R&D systems) 100

following the manufacturer’s instructions. Mediator concentrations were quantified by comparison 101

to recombinant cytokine standards. 102

Statistical analysis. Results are expressed as mean S.E.M.; statistical significance was calculated by 103

ANOVA followed by Tukey tests when there were greater than 3 groups and t tests for the 104

comparison of 2 groups using GraphPad Prism software. All calculations were performed using 105

GraphPad Prism software. 106


7

Results 107

CpG pre-treatment reduces the delayed sequelae of neonatal RSV infection. 108

Neonatal RSV infection primes for a long-lasting propensity to enhanced disease during adult re-109

infection with RSV (33). To test the effect of TLR ligands on this response, neonatal mice were pre-110

treated with intranasal CpG (using 10μg CpG ODN 1826) or PBS on day 3 of life prior to RSV infection 111

on day 4 of life. This was followed by adult RSV re-infection at 8 weeks of age. Daily weight changes 112

were monitored during re-infection and presented as percentages of the weight on day 0 (Figure 113

1A). During adult re-infection, mice infected with RSV as neonates (nnRSV) started losing weight on 114

day 1, peaking at day 6. In contrast, the onset of weight loss was delayed in the CpG pre-treated 115

group (nnCpG-RSV) and they lost weight on days 3 and 4 only. The weight loss in CpG pre-treated 116

mice peaked at about 10 % of their original weight and they fully recovered by day 7. There were 117

significant differences between the nnCpG-RSV and nnRSV group from day 3 to 7 (p<0.01 from day 3 118

to 5, p<0.001 on day 6 and 7). CpG pre-treated mice had fewer cells recruited to the lungs than 119

nnRSV mice (Figure 1B). The CpG pre-treated group had a significantly lower viral titre compared to 120

the nnRSV group (Figure 1C, p<0.05). These results demonstrate that neonatal CpG pre-treatment 121

provides a level of protection from the delayed sequelae of neonatal RSV infection. 122

The protective effect of CpG pre-treatment could have been due to any synthetic DNA fragments 123

with or without unmethylated CpG motifs, thus neonatal mice were treated with methylated 124

(mCpG) or unmethylated CpG oligodeoxynucleotides of exactly the same sequence. Only the 125

unmethylated CpG pre-treated group were protected from further weight loss while the other 126

groups continued losing weight until the day 4 peak (Figure 1D, p<0.05). From these studies we 127

observe that CpG pre-treatment is protective against disease on adult RSV re-challenge. 128

129

CpG pre-treatment increases Th1 responses on rechallenge. 130


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We reasoned that CpG pre-treated mice might show improved humoral immunity, providing better 131

protection from rechallenge infection and lower viral titres in than untreated nnRSV mice. However, 132

there was no difference in anti-RSV serum antibody and the ability of the sera to neutralise virus 133

four days after RSV re-challenge between groups with or without CpG pre-treatment (data not 134

shown). 135

Since levels of total anti-RSV antibodies are similar in the neonatally primed groups, a reduction of 136

the viral titre in CpG pre-treated mice could instead be caused by strong antiviral cellular responses. 137

There was significantly more IFNγ in the airways on day 4 post infection in the CpG pre-treatment 138

group (Figure 2A, p<0.05) and a decrease in IL-5 levels on d7 (Figure 2B). There was also a significant 139

NK cell recruitment to the lungs on day 4 post infection (Figure 2C, p<0.01). There was an increase in 140

CD4 cell number (Figure 2D) and the CpG pre-treated group had significantly more IFNγ producing 141

CD4+ T cells (p<0.05, Figure 2E) and significantly fewer IL-4 producing CD4+ T cells than the RSV group 142

(p<0.05, Figure 2F). There were also significantly more CD8+ T cells in the CpG pre-treated mice on 143

day 7 (p<0.01, Figure 2G). Supporting a shift to a Th1 phenotype was a reduction in eosinophil 144

recruitment (p<0.05, Figure 2H) and a switch of IgG isotype to IgG2a (p<0.001, Figure 2I, J and K). 145

These results suggest that CpG pre-treatment alters the responses increasing Th1 responses, 146

improving viral clearance during re-infection and reducing disease. 147

CpG has been shown to have a protective effect as immunotherapy against allergic sensitisation (15). 148

We wished to determine whether the protective effect of CpG only occurred prior to neonatal 149

infection, or if it could modulate the recall immune response after it had developed. To test this we 150

administered CpG intranasally 4 weeks after neonatal RSV infection and then re-infected mice as 151

adults. CpG treated mice lost significantly less weight than untreated mice (p<0.05, Figure 3A). The 152

reduced illness may be explained by boosting the amount of RSV-specific serum antibody at 4-153

weeks-old,but the amount of total antibody was not altered by interval CpG treatment (data not 154

depicted), and there was no difference in IgG1 (Figure 3B) or IgG2a (Figure 3C) levels, suggesting that 155


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these are set at the time of the initial infection. There was a significant increase in IFNγ (p<0.01, 156

Figure 3D) and a decrease in both IL-5 (p<0.05, Figure 3E) and eosinophil number (Figure 3F) in the 157

airways on d4 post infection. But CpG did not significantly alter the numbers of NK (Figure 3G), CD4 158

(Figure 3H) or CD8 (Figure 3I) cells recruited to the lungs post infection. Therefore, the pathogenic 159

cellular recall response following neonatal RSV response is not permanently imprinted and can be 160

switched by later exposure to CpG. 161

Effect of CpG on antigen presenting cells in the neonatal lung 162

To further examine the mechanism by which CpG exposure was protective against secondary 163

challenge with RSV, we focussed on the role of antigen presenting cells (known to be highly TLR 164

responsive). It has been observed that dendritic cells (DCs) in the neonatal lung have a Th2 bias, but 165

that this can be modified by exposure to LPS or BCG (25). To determine if a similar effect occurs with 166

CpG, we compared phenotypes of CD11c+ APC in adult and neonatal mice, to give a general view of 167

what is occurring to lung DCs in response to RSV vs RSV+CpG. 7-day-old neonatal mice and 8-week-168

old adult mice were infected with RSV (4 x 104 pfu per gram weight) and compared to age matched 169

PBS treated controls. Four mice were sacrificed from each group on day 4, 7 and 11 after infection 170

and lung samples were collected for further analysis. 171

Significantly fewer cells were recruited to the lungs of neonatal mice than adult mice following 172

primary infection (p<0.001, data not depicted). To measure numbers of APC, cells from the lung 173

were stained for the surface marker CD11c, MHC class II and the marker of activation CD86, cells 174

were scored as a percentage of total live (7-AAD negative) cells. There were no significant 175

differences in the percentage or number of activated CD11c+ APC in the RSV infected neonates 176

compare to their age-matched PBS controls at any time point (Figure 4A). However, in adult mice 177

both the percentage and the number of activated CD11c+ APC increased after RSV infection 178

compared to the PBS group and the neonatal RSV group, peaking on day 11 (Figure 4A, p<0.001). 179


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It is known that CpG treatment causes the recruitment and maturation of splenic APC in neonates 180

following subcutaneous immunisation (11). To test whether CpG altered APC activation in the lungs 181

of neonatal mice after viral infection, 6 day old mice were infected with RSV, with or without 182

intranasal CpG pre-treatment. CpG pre-treatment had no effect on lung viral load during primary 183

infection (Figure 4B). The number of CD11c+ cells in neonatal lungs on day 1 and 4 was not altered by 184

CpG pre-treatment or RSV infection (Figure 4C). However the CpG-RSV treated mice showed a 185

significant increase in the expression levels of MHC class II molecules on CD11c+ cells, compared to 186

the naïve as well as RSV infection-only groups (Figure 4D, p<0.01). The expression of CD80 on CD11c+ 187

cells was also significantly increased by pre-treatment compared to the naïve group (Figure 4E 188

p<0.05). There was a 1.75-fold increase in the percentages of lung CD11c+ cells expressing both MHC 189

class II and CD80 between the CpG-RSV and RSV-only groups (21.4 ± 2.7% compared to 12.3 ± 2.3% 190

of the lung cells, respectively, 10.9 ± 1.5% for the naïve group). These differences were observed 191

only at the day 1 time point and were not sustained until day 4. CpG pre-treatment had no effect on 192

CD4 or CD8 cell number in the lungs (data not depicted), however there was an increase in the 193

number of DX5+ NK cells (Figure 4F) and a significant increase in the number of IFNγ secreting NK 194

cells on day 1 post infection (p<0.05, Figure 4G). The data show that CpG pre-treatment changed the 195

APC phenotype in the lungs at a very early time point which may switch the response away from 196

Th2. 197


11

Discussion 198

We show that CpG exposure prior to neonatal RSV infection led to increased protection against adult 199

re-infection, and to reduced disease on adult re-exposure to RSV. Similar effects were also seen if 200

CpG was delivered intranasally after the complete resolution of primary infection, in the period 201

between neonatal challenge and adult re-infection. These effects were associated with a notable 202

increase in Th1 responses in the lung, probably due to an increase in APC activation caused by the 203

CpG exposure. 204

We and others have shown that cellular immune responses to RSV infection can be both protective 205

and pathogenic (24). The ideal protective response reduces viral load without excess local 206

inflammation, whereas the adverse pathogenic responses causes inflammation without effectively 207

controlling viral load. For example, in adult RSV infection, CCL3 depletion increased pro-208

inflammatory RSV-specific cells but reduced the total number of cells (35). The data from our 209

previous studies suggest that neonatal RSV infection induces hyper-inflammatory cellular responses 210

during re-infection and reducing this cellular infiltrate can reduce disease (33), but the interaction 211

with APC was critical in determining outcome (34). The current study shows that the response 212

following neonatal infection can be switched from pathogenic to protective using TLR ligands. These 213

and other studies suggest that the context of the initial exposure to virus or viral antigen determines 214

the outcome of subsequent exposures and parallels can be drawn with the development of asthma 215

and allergy in early life (7). But these responses are relatively plastic, for example the incidence of 216

post bronchiolitic wheeze decreases with age. This may reflect subsequent exposures to TLR ligands 217

modulating the immune response and our interval CpG treatment experiment support this idea. 218

The T helper balance of the response to RSV is important in determining whether it will be 219

pathogenic or protective. Previously we have observed a reduction in weight loss on adult re-220

infection following the use of a recombinant virus expressing IFNγ (14) and the addition of 221

recombinant IFNγ has been shown to have a protective effect (18). However there were differences 222


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between CpG and IFNγ treatment, whilst both treatments suppressed Th2-type responses during 223

rechallenge infection only CpG treatment enhanced Th1 responses. Furthermore only CpG 224

treatment reduced the viral titre on re-infection. CpG has previously been shown to alter the Th2 225

skewing of neonatal APC (16). It is of note that strongly skewed Th1 responses can also be 226

pathogenic, when IFNγ expressing RSV was used in adult mice, it led to increased disease on 227

rechallenge (12), therefore a balanced cellular response is desirable. 228

Our data suggest that deficient activation of neonatal APC is involved in the delayed sequelae of RSV 229

infection, neonatal CD11c+ cells failed to up-regulate MHC class II and co-stimulatory molecules on 230

RSV infection. Delayed maturation of neonatal APC may be a mechanism to reduce harmful 231

inflammatory responses to self or benign environmental antigens in early life (2). It has been shown 232

that neonatal APC are deficient in a number of key signalling molecules including IRF3 (3) and IRF7 233

(5). However some TLR ligands can activate neonatal APC including R848 acting on TLR7/8 (19), LPS 234

acting on TLR4 (25) and CpG acting on TLR9 (31). One question is why RSV infection does not induce 235

a similar maturation of neonatal APC, given that RSV has been shown to interact with TLR3 (27) and 236

TLR4 (17). One possibility is that the virally encoded genes suppress the innate response and it has 237

been recently demonstrated that the RSV NS1 protein suppresses Th1 type responses (22), in the 238

context of the pre-skewed lung environment in early life this suppression may be particularly potent. 239

In conclusion, we show that prior exposure to the TLR9 ligand CpG is protective against the delayed 240

effects of neonatal RSV infection. These delayed effects may model some of the features of post 241

bronchiolitic wheeze and viral exacerbations of asthma and therefore TLR ligand administration may 242

have therapeutic potential in prevention of respiratory morbidity in childhood. These results may 243

also go some way to explain how exposure to bacterial products in early life can protect against the 244

development of asthma. 245

Acknowledgements This work was supported by Wellcome Trust Programme Grant 071381/Z/03/Z 246

(UK) and an MRC New Investigator Research Grant (G10001763). 247


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29. Smit, L. A. M., V. r. Siroux, E. Bouzigon, M. P. Oryszczyn, M. Lathrop, F. Demenais, F. 339 Kauffmann, and B. H. a. A. E. C. G. on behalf of the Epidemiological Study on the Genetics 340 and Environment of Asthma. 2009. CD14 and Toll-like Receptor Gene Polymorphisms, Country 341 Living, and Asthma in Adults. American Journal of Respiratory and Critical Care Medicine 342 179:363-368. 343

30. Stein, R. T., D. Sherrill, W. J. Morgan, C. J. Holberg, M. Halonen, L. M. Taussig, A. L. Wright, 344 and F. D. Martinez. 1999. Respiratory syncytial virus in early life and risk of wheeze and allergy 345 by age 13 years. Lancet 354:541-545. 346

31. Sun, C. M., E. Deriaud, C. LeClerc, and R. Lo-Man. 2005. Upon TLR9 signaling, CD5+ B cells 347 control the IL-12-dependent Th1-priming capacity of neonatal DCs. Immunity 22:467-477. 348

32. Tregoning, J. S. and J. Schwarze. 2010. Respiratory viral infections in infancy: causes, clinical 349 symptoms, virology, and immunology. Clin.Microbiol.Rev. 23:74-98. 350

33. Tregoning, J. S., Y. Yamaguchi, J. Harker, B. Wang, and P. J. Openshaw. 2008. The role of T 351 cells in the enhancement of RSV infection severity during adult re-infection of neonatally 352 sensitized mice. J.Virol. 82:4115-4124. 353

34. Tregoning, J. S., Y. Yamaguchi, B. Wang, D. Mihm, J. A. Harker, E. S. Bushell, M. Zheng, G. 354 Liao, G. Peltz, and P. J. Openshaw. 2010. Genetic susceptibility to the delayed sequelae of 355 neonatal respiratory syncytial virus infection is MHC dependent. J.Immunol. 185:5384-5391. 356

35. Tregoning, J. S., P. K. Pribul, A. M. J. Pennycook, T. Hussell, B. Wang, N. Lukacs, J. Schwarze, 357

F. J. Culley, and P. J. M. Openshaw. 2010. The Chemokine MIP1/CCL3 Determines Pathology 358 in Primary RSV Infection by Regulating the Balance of T Cell Populations in the Murine Lung. 359 PLoS ONE 5:e9381. 360

361 362

363


16

Figure Legends 364

Figure 1. Pre-treatment with unmethylated CpG reduces the delayed effects of neonatal RSV 365

infection. Neonatal mice were treated with 10μg CpG (day 3 of life) one day prior to RSV infection 366

(day 4 of life), 8 weeks later mice were re-infected with RSV. Weight loss (A), lung cell number (B) 367

and lung viral load at day 4 (C) were measured after infection. The effect of pre-treatment with 368

methylated and unmethylated CpG on weight loss during secondary infection was compared (D). n = 369

5 mice per group per timepoint. Mean ± SEM. * p<0.05, ** p<0.01. 370

Figure 2. Treatment with CpG prior to neonatal RSV increases anti-viral cellular immunity. On day 3 371

of life Neonatal mice were treated with 10μg CpG (•, black bars) or PBS (○, white bars) one day prior 372

to RSV infection (day 4 of life), 8 weeks later mice were re-infected with RSV. IFNγ (A) and IL-5 (B) 373

measured in BAL by ELISA. Lung DX5+ NK cells (C), CD4 T cells (D), IFNγ (E) and IL-4 (F) producing CD4 374

T cells CD8 T cells (G) and airway eosinophilia (H) were measured by flow cytometry on day 4 and/ or 375

day 7. RSV specific IgG1 (I), IgG2a (J) and the ratio (K) were measured in serum on day 4 post 376

challenge by ELISA. Data representative of 2 experiments, bars represent n=4 mice per group +/- 377

SEM. *p<0.05, **p<0.01 378

Figure 3. Interval CpG application abrogates neonatal disease. 4 day old BALB/c mice were 379

infected i.n. 105 PFU RSV or sham infected with PBS. 4 weeks later mice were given i.n. CpG 380

(open symbols and bars) or PBS (filled symbols and bars), 8 weeks later mice were 381

challenged with 106 PFU of RSV. Weight was monitored following infection (A). RSV specific 382

IgG1 (B) and IgG2a (C) subtypes were measured by ELISA. Airway IFNγ (D), IL-5 (E) and 383

eosinophilia (F) on day 4 after infection. Lung NK (G), CD4 cells (H) and CD8 cells (I) were 384

measured on day 4 and 7 post infection. Data representative of 2 independent repeats, n = 5 385

mice per group per timepoint. Mean ± SEM. * p<0.05, ** p<0.01. 386

387


17

Figure 4. CpG pre-treatment increases APC activation. Neonatal or adult mice were infected with 388

RSV intranasally or left uninfected. At d 0, 4, 7 and 11 post infection, lungs were removed and the 389

number of CD11c+ cells expressing MHCII and CD86 characterised by flow cytometry (A), *** 390

p<0.001 comparing Ad RSV with NN RSV, ### p<0.001 comparing Ad RSV with Ad PBS. Neonatal mice 391

were treated with 10ug CpG one day prior to RSV infection. Lungs were removed and viral load (B) 392

measured on d4 post infection and CD11c+ cell number (C) and MFI of MHCII (D) and CD80 (E) 393

measured by FACS on d1 and d4 post infection. Numbers of DX5+ NK cells (F) and IFNγ producing NK 394

cells (F) were also evaluated in the lungs by FACS. Bars/ points represent mean +/- SEM of n=4 mice, 395

* p<0.05, ** p<0.01. 396

397


18

Figure 1

Figure 2

0 1 2 3 4 5 6 760

70

80

90

100

110

nnCpG-RSV

nnRSV

day post re-infection

% d

ay 0

weig

ht

**** *** ***

***

nnRSV nnCpG-RSV

103

104

105

106

**

RS

V L

gene c

opy n

um

ber


tota

l lu

ng c

ells

(x1

06)

4 70

2

4

6

8

nnCpG-RSV

nnRSV

0 1 2 3 4 5 6 790

95

100

105

CpGmCpG

RSV


% d

ay 0

weig

ht

*

A B C D

4 70

2

4

6


lung C

D4+

cells

(x1

05)

nnRSV nnCpG-RSV0.0

0.5

1.0

1.5

*

d7 I

L-4

+ C

D4

+ce

lls (

x10

5)

nnRSV nnCpG-RSV0.0

0.5

1.0

1.5

2.0

d7 I

FN+

CD

4+

cells

(x1

05)

*

103 104 105 1060.00

0.25

0.50

0.75

1.00

nnCpG-RSV

nnRSV

sera dilution

d4 a

nti-

RS

V I

gG

1 (

A490)


air

way I

FN

(ng/m

l)

4 70

2

4

6*

nnRSV

nnCpG-RSV

d7 a

irw

ay I

L-5

(pg/m

l)

nnRSV nnCpG-RSV0

10

20

30

4 70.0

0.5

1.0

1.5

2.0

**


lung D

X5+

cells

(x1

05)

4 70

2

4

6

8**


lung C

D8+

cells

(x1

05)

nnRSV nnCpG-RSV0.0

0.5

1.0

1.5

2.0 *

d4 a

irw

ay e

osin

ophils

(x1

04)

A B C

D E F G

H I

103 104 105 1060.00

0.25

0.50

0.75

1.00

sera dilution

d4 a

nti-

RS

V I

gG

2a (

A490)

nnRSV nnCpG-RSV0

2

4

6

8

10

IgG

2a/I

gG

1 o

n d

4

***

J K


19

Figure 3

RSV-PBS-RSV RSV-CpG-RSV0

10

20

30

40

50

60

70 *

Air

way I

L-5

(pg/m

l)

Sera Dilution

Anti-R

SV

IgG

1 (

A490)

101 102 103 1040.0

0.5

1.0

1.5

2.0 nnRSV-PBS-RSV

nnRSV-CpG-RSV

0 1 2 3 4 5 6 760

70

80

90

100

110

nnRSV-PBS-RSV

nnRSV-CpG-RSV


% o

rigin

al w

eig

ht

******

**

***

4 70

5

10

15


NK

cells

(%

)

nnRSV-PBS-RSV

nnRSV-CpG-RSV

4 70

5

10

15


CD

4+ T

cells

% lungs

RSV-PBS-RSV RSV-CpG-RSV0.0

0.5

1.0

1.5

2.0

2.5**

Air

way I

FN

(ng/m

l)

A B

D E

RSV-PBS-RSV RSV-CpG-RSV0

2

4

6

8

10

BA

L e

osin

ophils

(x1

04)

G H

4 70

5

10

15

20


CD

8+ T

cells

(%

)

I

Sera Dilution

Anti-R

SV

IgG

2a (

A490)

101 102 103 1040.0

0.5

1.0

1.5

2.0C

F


20

Figure 4

0 1 2 3 4 5 6 7 8 9 10 110

5

10

15

day post infection

CD

11c

+ c

ells

exp

ress

ing

MH

C c

lass

II

and C

D86 (

10

5)

***

***###

Ad RSV

NN RSV

Ad PBS

NNPBS

PBS-RSV CpG-RSV PBS-PBS101

102

103

104

105

106

ND

RS

V L

gene c

opy n

um

ber

day post infection

CD

11c+

cells

(x1

05)

1 40

1

2

3

4 PBS-RSV

CpG-RSV

PBS-PBS

day post infection

MH

C c

lass I

I (M

FI)

1 40

200

400

600

800

1000

****

day post infection

CD

80 (

MF

I)

1 40

50

100

150

*

1 40

2

4

6

8

10

day post infection

DX

5+

cells

(x1

05)

1 40

2

4

6

8

10

day post infection

IFN

g+

DX

5+

cells

(x

10

3) *

A B C D

E F G

yamaguchi et al, jvi · 2015. 12. 14. · yamaguchi et al, jvi 3 38 introduction 39 respiratory...

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