Interferon response factors 3 and 7 protect against Chikungunya virus hemorrhagic fever and shock

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Interferon response factors 3 and 7 protect against Chikungunya virus hemorrhagic fever and shock

Penny A Rudd, Jane Wilson, Joy Gardner, Thibaut Larcher, Candice Babarit, Thuy T Le, Itaru Anraku, Yutaro Kumagai, Yueh-Ming Loo, Michael Gale Jr,

et al.

To cite this version:

Penny A Rudd, Jane Wilson, Joy Gardner, Thibaut Larcher, Candice Babarit, et al.. Interferon

response factors 3 and 7 protect against Chikungunya virus hemorrhagic fever and shock. Journal of

Virology, American Society for Microbiology, 2012, 86, pp.9888-9898. �10.1128/JVI.00956-12�. �hal-

01191139�

Version postprint

1

Interferon Response Factors 3 and 7 Protect against Chikungunya Virus

1

Hemorrhagic Fever and Shock

2

3

Running title; IRF3/7 prevent chikungunya virus hemorrhagic shock

4

5

Penny A Rudd,

^1,2

Jane Wilson,

^1,3

Joy Gardner,

¹

Thibaut Larcher,

⁴

Candice Babarit,

⁴

Thuy T Le,

¹ 6

Itaru Anraku,

¹

Yutaro Kumagai,

⁵

Yueh-Ming Loo,

⁶

Michael Gale Jr,

⁶

Shizuo Akira,

⁵

Alexander A.

7

Khromykh,

²

Andreas Suhrbier.

^1,2,#

8 9

1

Queensland Institute of Medical Research, Brisbane, Qld. 4029, Australia

10

2

Australian Infectious Diseases Research Centre, School of Chemistry & Molecular Biosciences,

11

University of Queensland, Brisbane, Qld. 4072, Australia

12

3

School of Medicine, University of Queensland, Brisbane, Qld. 4072, Australia

13

4

Institut National de Recherche Agronomique, Unité Mixte de Recherche 703, Ecole Nationale

14

Vétérinaire, Nantes, France

15

5

Laboratory of Host Defense, WPI Immunology Frontier Research Center, Osaka University, Osaka

16

565-0871, Japan

17

6

Department of Immunology, University of Washington School of Medicine, Seattle, WA 98195,

18

USA.

19 20

#

Corresponding author; Dr A Suhrbier, Queensland Institute of Medical Research, Post Office

21

Royal Brisbane Hospital, Qld., 4029, Australia; Tel: +61-7-33620415. Fax: +61-7-33620107. E-

22

mail: andreasS@qimr.edu.au

23

24

Word count abstract; 175

25

Word count text; 5610

26

27

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

J. Virol. doi:10.1128/JVI.00956-12

JVI Accepts, published online ahead of print on 3 July 2012

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2 28

ABSTRACT

29

Chikungunya virus (CHIKV) infections can produce severe disease and mortality. Here we show

30

that CHIKV infection of adult mice deficient in interferon response factors 3 and 7 (IRF3/7

^-/-

) was

31

lethal. Mortality was associated with undetectable serum IFNα/β, §50 and §10 fold increases in

32

IFNγ and TNF, respectively, increased virus replication, edema, vasculitis, hemorrhage, fever

33

followed by hypothermia, oliguria, thrombocytopenia, and raised hematocrits. These features are

34

consistent with hemorrhagic shock and were also evident in infected IFNα/β-receptor deficient

35

mice. In situ hybridization suggested CHIKV infection of endothelium, fibroblasts, skeletal muscle,

36

mononuclear cells, chondrocytes and keratinocytes in IRF3/7

^-/-

mice; all but the latter two stained

37

positive in wild-type mice. Vaccination protected IRF3/7

^-/-

mice, suggesting defective antibody

38

responses were not responsible for mortality. IPS-1- and TRIF-dependent pathways were primarily

39

responsible for IFNα/β induction, with IRF7 up-regulated >100 fold in infected wild-type mice.

40

These studies suggest that inadequate IFNα/β responses following virus infection can be sufficient

41

to induce hemorrhagic fever and shock, a finding with implications for understanding severe

42

CHIKV disease, and dengue hemorrhagic fever / dengue shock syndrome.

43 44

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INTRODUCTION

46

Chikungunya virus (CHIKV) is a mosquito-borne, single-stranded positive sense RNA virus (genus

47

alphavirus) that has caused sporadic outbreaks of predominantly rheumatic disease, primarily in

48

Africa and Asia (57). CHIKV was often confused with dengue because of similarities in clinical

49

presentation (5); however, this continues to be an issue only in the absence of appropriate

50

serological and/or molecular diagnosis (2). The largest documented outbreak of CHIKV disease

51

ever recorded occurred during 2004-2011, starting in Kenya, spreading across the Indian Ocean

52

Islands to India and South-East Asia, and reaching

New Caledonia in 2011

. Over 260,000 cases

53

(about one-third of the population) were reported in Reunion Island (France) and an estimated 1.4-

54

6.5 million cases occurred in India. The first autochthonous CHIKV infections in Europe were seen

55

in Italy in 2007 and France in 2010. Due to international travel, imported cases were reported in

56

nearly 40 countries including Europe, Japan and the USA. Although Aedes aegypti is the classical

57

vector for CHIKV, the recent outbreak was associated with the emergence of a new clade of

58

CHIKV viruses, which were efficiently transmitted by Aedes albopictus mosquitoes, a vector that

59

has seen a dramatic global expansion in its geographic distribution. The recent CHIKV outbreak

60

was also associated with severe disease manifestations, and some mortality. At present, no licensed

61

vaccine or particularly effective drug is available for human use for any alphavirus

,

although

62

analgesics and non-steroidal anti-inflammatory drugs can provide relief from rheumatic symptoms

63

(53, 57).

64

IFNα/β and anti-viral antibodies have been shown to be important for protection against

65

infection, disease and/or mortality caused by CHIKV and other alphaviruses (15, 53, 57). CHIKV

66

infection of IFNα/β-receptor-deficient mice results in death, with IFNα/β receptor expression on

67

non-haematopoietic cells required for protection (51). In addition, CHIKV infection does not appear

68

to stimulate significant IFNα/β production in haematopoietic cells, with IFNα/β being largely

69

produced by infected non-hematopoietic cells (51). At least three host sensor pathways have been

70

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implicated in the production of IFNα/β during CHIKV infection; these involve (i) the RIG-I-like

71

receptors, retinoic acid-inducible gene I (RIG-I) and melanoma-differentiation-associated gene 5

72

(Mda5), signaling via interferon-β promoter stimulator 1 (IPS-1; also known as MAVS, VISA and

73

Cardif), (ii) Toll like receptor 3 (TLR3) signaling via TIR domain-containing adaptor inducing

74

interferon-ȕ (TRIF), and (iii) Toll like receptor 7 (TLR7) signaling via myeloid differentiation

75

primary response gene 88 (MyD88) (51, 53, 66).

76

Downstream of RIG-I/Mda5/IPS-1, TLR3/TRIF and TLR7/MyD88 lie interferon regulatory

77

factor 3 (IRF3) and interferon regulatory factor 7 (IRF7), two key transcription factors involved in

78

the induction of IFNα/β (53), with IRF7 up regulation important for induction of IFNαs and the

79

positive feedback that produces robust IFNα/β responses (33, 48, 49). (In myeloid cells IRF3/7-

80

independent IFNα/β production has also been described (10)). The central role of IRF3 and/or IRF7

81

has been illustrated for a number of viruses by the use of IRF7

^-/-

and/or IRF3

^-/-

mice. For instance,

82

IRF7

^-/-

or IRF3

^-/-

mice infected with encephalomyocarditis virus showed significantly higher levels

83

of mortality compared with wild-type (WT) mice (22). In contrast, infection with herpes simplex

84

virus infection was lethal in IRF7

^-/-

, but not IRF3

^-/-

mice (22). Increased morality was also seen after

85

infection of IRF7

^-/-

or IRF3

^-/-

mice with the virulent West Nile virus strain (NY99), whereas

86

infection with the naturally attenuated West Nile strain (Kunjin) was only universally lethal in

87

IRF3/7

^-/-

double knockout mice (9). IRF7 has been shown to be important for optimum production

88

of IFNα/β in murine embryonic fibroblasts (MEFs) after infection with a number of viruses (22),

89

and is critical for IFNα/β production by plasmacytoid dendritic cells (12). IRF3 has been shown to

90

be important for optimum IFNα/β production by non-hematopoietic and hematopoietic cells in

91

response to certain virus infections (22), and IRF3-dependent apoptotic signaling can also

92

contribute significantly to the host’’s protection from viral infection (4).

93

We have recently developed an adult WT mouse model of CHIKV infection and rheumatic

94

disease (15). Given the importance of IFNα/β in protection against CHIKV infection (53) and the

95

ability of CHIKV to inhibit IFNα/β receptor signaling (14), we used this model to determine the

96

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relative importance of IRF3 and IRF7 in CHIKV infection and disease using IRF3

^-/-

, IRF7

^-/-

and

97

IRF3/7

^-/-

mice. Infection in IRF3/7

^-/-

mice was lethal and, interestingly, death was associated with

98

hemorrhagic shock.

99 100

MATERIALS AND METHODS

101

Mice. IRF3

^-/-

and IRF7

^-/-

mice were generated by Dr T. Taniguchi (University of Tokyo) (22,

102

49). These mice and IRF3/7

^-/-

mice (10) were provided by Dr M.S. Diamond (Washington

103

University School of Medicine, St Louis). IPS

^-/-

knockout mice on a C57Bl/6 background were

104

created using conventional methods (25) (see Supplemental Material 1). TRIF

^-/-

and MyD88

^-/-

mice

105

have been described previously (68). All mice were on a C57BL/6 background. WT mice were

106

purchase from Animal Resources Centre (Canning Vale, WA, Australia).

107

Mouse infection and monitoring. Mice (6-12 weeks) were inoculated with CHIKV (LR2006-

108

OPY1) and virus titers and foot swelling determined as described (15). Mice were inoculated with

109

CHIKV (10

⁴

log

10

50% cell culture infectivity dose (CCID

50

) in 40 µl RPMI 1640 (supplemented

110

with 2% fetal calf serum) by shallow s.c. injection into the top, towards the lateral side, of each hind

111

foot in the metatarsal region, injecting toward the ankle. Arthritis (foot swelling) was monitored by

112

measuring the height and width of the metatarsal area of the hind feet using digital calipers and is

113

presented as a group average of the percentage increase in foot height x width for each foot

114

compared with the same foot on day 0. The virus preparations had undetectable mycoplasma and

115

endotoxin contamination as measured by sensitive bioassays (23, 27). Animal work was conducted

116

in accordance with good animal practice (NHMRC, Australia), and was approved by the QIMR

117

animal ethics committee.

118

Clinical measurements. Body temperature was measured using a digital thermometer (Digitech,

119

Rydalmere, Australia) and a 2 mm thermocouple bead probe, which was lightly pressed for §30

120

secs into the pit of the rear leg of the restrained mouse with the leg folded over the probe. Urine

121

output following scruff-induced urination was measured by collecting and weighing the urine

122

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produced within §1 min of the mouse being restrained. Blood platelet counts were determined using

123

a hemocytometer and heparinized blood diluted 1/20 in phosphate buffered 1% ammonium oxalate

124

solution. Hematocrits were measured using standard hematocrit tubes (Beckton Dickinson

,

North

125

Ryde, NSW, Australia) and were expressed as the percent difference from control uninfected mice.

126

Cytokine/chemokine analyses. Serum cytokine/chemokine protein levels were analyzed by

127

using the BD Cytokine Bead Array Bioanalyzer system (Becton Dickinson, Franklin Lakes, NJ)

128

according to the manufacturer’’s instructions. Bioactive IFNα/β was measured by a cytopathic effect

129

inhibition bioassay using Semliki Forest virus infection of L-929 cells as described previously (15).

130

Histology. Tissues were fixed in 10% neutral buffered formalin, feet were decalcified (15%

131

EDTA in 0.1% phosphate buffer over 10 days), tissue was embedded in paraffin wax, and 6 µm-

132

thick sections were cut and stained with hematoxylin-eosin. Slides were scanned using Aperio Scan

133

Scope XT digital slide scanner (Aperio, Vista, CA, USA).

134

In situ hybridization. A 450-bp digoxigenin-labeled CHIKV probe sequence (GenBank:

135

DQ443544.2, nucleotides 7371-7818) was hybridized to paraffin sections and detected with anti-

136

digoxigenin antibody conjugated with alkaline phosphatase. The corresponding region of RRV was

137

used as a negative control. For details see Supplemental Material 1.

138

Real time quantitative RT-PCR. RT-PCR was performed essentially as described (15). For

139

details see Supplemental Material 1.

140

Infection of MEFs. IRF3

^-/-

, IRF7

^-/-

and IRF3/7

^-/-

MEFs were seeded (2 x10

⁵

per well) in 12 well

141

plates, treated with the indicated amount of recombinant mouse IFNα (Hycult Biotech, Uden, The

142

Netherlands) overnight and then infected with CHIKV (MOI=0.1) for 2 h. The cells were washed

143

and cultured, with supernatant assayed for virus titers 24 h later.

144

Statistics. Analysis was performed using IBM SPSS Statistics (version 19). The t test was used

145

if the difference in the variances was <4, skewness was <-2, and kurtosis was <2; otherwise, the

146

non-parametric Mann-Whitney U test was used. For survival analysis the log rank statistic was

147

used.

148

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RESULTS

149

Chikungunya virus infection of IRF3

^-/-

, IRF7

^-/-

and IRF3/7

^-/-

mice. To determine the

150

requirement for IRF3 and IRF7 for survival from CHIKV infection, IRF3

^-/-

, IRF7

^-/-

and IRF3/7

^-/- 151

mice were infected with a Reunion Island isolate of CHIKV (15). All IRF3/7

^-/-

mice died between

152

days 4-6 post infection, whereas all IRF3

^-/-

, IRF7

^-/-

and WT mice survived (Fig. 1A). Mortality in

153

the IRF3/7

^-/-

mice was associated with a significant increase in viremia, which was clearly evident

154

from day 3 onwards and was §4 logs higher than that seen in WT mice on day 5 (Fig. 1B). The

155

viremia in IRF7

^-/-

mice was §1 log higher than that seen in WT mice on days 3 and 4 post infection

156

(Fig. 1B), but this only approached significance on day 3 (p=0.053, Mann Whitney U test). The

157

viremia in IRF3

^-/-

mice was not significantly different from that seen in WT mice (Fig. 1B). Viral

158

titers in several organs were also substantially higher in IRF3/7

^-/-

mice, often 6-8 logs higher than

159

those seen in WT mice (Fig. 1C). Although virus was found in brain (Fig. 1C), no overt

160

neurological symptoms (gait, paralysis) were evident (data not shown). Viral titers in the feet were

161

significantly higher (§3 logs, p=0.01) in IRF3/7

^-/-

mice compared with WT mice on day 6 (Fig. 1D).

162

These experiments clearly show that either IRF3 or IRF7 is required for survival following CHIKV

163

infection, with IRF3/7

^-/-

mice showing significantly higher viremia, tissue titers and disease, and

164

mortality occurring within several days of infection. Infection with an Asian isolate of CHIKV (15)

165

was also lethal in IRF3/7

^-/-

mice (data not shown).

166

Foot swelling after CHIKV infection. We have previously shown that WT mice produce a

167

measurable foot swelling and arthritis day 6-7 following CHIKV inoculation into feet (15). Similar

168

infection of IRF3

^-/-

, IRF7

^-/-

and IRF3/7

^-/-

mice also resulted in foot swelling, but swelling occurred

169

much earlier (day 2-4) and was slightly more pronounced in IRF3

^-/-

mice, substantially more

170

pronounced in IRF7

^-/-

mice, and even more pronounced in IRF3/7

^-/-

mice (Fig. 1E, F).

171

Injection of heat inactivated CHIKV (60°C for 30 mins) into IRF3/7

^-/-

or WT mice did not result

172

in any significant swelling (data not shown). Although subcutaneous (s.c.) (base of tail) inoculation

173

of CHIKV into WT mice did not result in foot swelling (15), this route of infection did result in

174

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some foot swelling in IRF3/7

^-/-

mice (data not shown). Mortality in IRF3/7

^-/-

mice was unchanged

175

when virus was inoculated s.c. (data not shown).

176

Loss of IFNα/β, and elevated IFNγ, MCP-1, IL-6 and TNF in CHIKV infected IRF3/7

^-/- 177

mice. High levels of serum IFNα/β (as measured by bioassay) could be detected in WT mice after

178

CHIKV infection (Fig. 2A, IFNα/β), as reported previously (15). IRF3

^-/-

mice showed serum

179

IFNα/β levels comparable with those seen in WT mice. However, only low levels of serum IFNα/β

180

were detected in IRF7

^-/-

mice on day 2, and serum IFNα/β was below detection in IRF3/7

^-/-

mice

181

(Fig. 2A, IFNα/β). Real time quantative RT PCR of feet day 2 post infection paralleled these

182

findings, with IFNα mRNA levels in WT>IRF3

^-/-

>IRF7

^-/-

>IRF3/7

^-/-

mice and IFNβ mRNA levels

183

in WT>IRF3

^-/-

=IRF7

^-/-

>IRF3/7

^-/-

mice, and only low levels of IFNβ mRNA induced in IRF3/7

^-/- 184

mice (Fig. 2B).

185

These observations suggest IRF7 is the main transcription factor involved in IFNα/β production

186

after CHIKV infection, an observation also reported for other viral infections (9, 22). The small

187

amount of IFNα/β seen in IRF7

^-/-

mice appeared sufficient to contain the viremia (Fig. 1B) and

188

prevent mortality (Fig. 1A), perhaps consistent with the high sensitivity of alphaviruses (36),

189

including CHIKV, to IFNα/β (55) (see also Fig. 6).

190

The serum levels of inflammatory cytokine/chemokines were also measured in CHIKV infected

191

mice. IRF3/7

^-/-

mice showed significantly elevated levels of IFNγ, monocyte chemoattractant

192

protein-1 (MCP-1, also known as CCL2), interleukin-6 (IL-6) (both peaking on day 2) and tumor

193

necrosis factor (TNF) (elevated from day 2 to 6) when compared with WT mice (Fig. 2A). On day 2

194

post infection, serum from IRF3/7

^-/-

mice contained >50 fold more IFNγ, and §10 fold more MCP-

195

1, IL-6 and TNF than WT mice (Fig. 2A). In IRF3

^-/-

and IRF7

^-/-

mice, the levels of these mediators

196

were not significantly different from those in WT mice (Fig. 2A). Serum levels of IL-12 and IL-10

197

were not significantly changed, and IL-1β was not detected for any mouse strain (data not shown).

198

The absence of detectable serum IFNα/β in IRF3/7

^-/-

mice thus appeared to correlate with very high

199

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levels of IFNγ, and high levels of other inflammatory mediators.

200

Mortality in IRF3/7

^-/-

mice was associated with severe edema and hemorrhage. To gain

201

insights into the mechanisms responsible for mortality in IRF3/7

^-/-

mice, histological analysis of a

202

number of tissues was undertaken on day 5 post infection. Foot swelling was associated with severe

203

generalized edema and multifocal severe hemorrhage, with the most severe lesions observed deep in

204

the dermis and subcutaneous tissues (Fig. 3A-C). (Skin and subcutaneous tissue from uninfected

205

IRF3/7

^-/-

mice is shown in Fig. 3D). Some hemorrhage was also evident in IRF7

^-/-

mice (data not

206

shown), but was not seen in IRF3

^-/-

mice (data not shown) or in WT mice on day 5 post infection

207

(Supplementary Material 2A, B) or day 7 post infection (15). Edema was marked in IRF7

^-/-

mice,

208

but less severe that in IRF3/7

^-/-

mice (data not shown). Edema was also present in IRF3

^-/-

mice (data

209

not shown) and WT mice day 5 post infection (Supplementary Material 2A) and day 7 post

210

infection (15).

211

IRF3/7

^-/-

mice showed mutifocal marked vasculitis characterized by fibrinoid necrosis of the

212

vascular wall, with karyorrhexis of neutrophil nuclei, indicative of intramural leukocytoclasis

213

(degenerate leukocytes inside the vascular wall) and perivascular fibrin exudation and extravasated

214

red blood cells (Fig. 3C). Such lesions were mild or absent in WT mice day 5 post infection

215

(Supplementary Material 2C) and day 7 post infection (15). The mortality seen in CHIKV infected

216

IRF3/7

^-/-

mice (Fig. 1A) thus appeared to be associated with severe edema, hemorrhage and

217

necrotizing vasculitis.

218

Scattered foci of marked skin necrosis with bullae formation were seen in the epidermis of

219

IRF3/7

^-/-

mice (Fig. 3E), with such lesions rare or mild in IRF7

^-/-

mice, and not observed in IRF3

^-/- 220

mice (data not shown) or WT mice (Supplementary Material 2E). (Epidermis from uninfected

221

IRF3/7

^-/-

mice is shown in Fig. 3F). Exudative arthritis was evident with inflammatory cells and

222

fibrin present in joint synovia of IRF3/7

^-/-

mice (Fig. 3G). Such lesions were not observed in IRF3

^-/- 223

mice (data not shown) or WT mice day 5 post infection (Supplementary Material 2D), but are

224

present in WT mice day 7 post infection (15). Joint tissue from uninfected IRF3/7

^-/-

mice is shown

225

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10

in Fig. 3H. Liver, spleen, muscle, lymph nodes, kidney, lung, intestine and brain from IRF3/7

^-/- 226

mice 5 days post infection were also examined. Apart from some lung consolidation, minimal

227

leukocytic perivascular cuffs and slight pericapillary edema in the brain, and mild edema (see

228

below) and mild focal hemorrhage in muscle tissues, no other significant lesions were seen (data not

229

shown).

230

Paucity of cellular infiltrates and more endomysial edema in CHIKV infected IRF3/7

^-/- 231

mice. Only minimal cellular infiltrates were apparent in the swollen feet of IRF3/7

^-/-

mice (Fig. 3A-

232

C). This was clearly evident in skeletal muscle of IRF3/7

^-/-

mice (compare muscle from uninfected

233

(Fig. 3I with infected (Fig. 3J) mice) and contrasted with the abundant infiltrates seen in IRF7

^-/- 234

(Fig. 3K), IRF3

^-/-

(Fig. 3L) and WT mice day 5 post infection (Supplementary Material 2F, G) and

235

day 7 post infection (15). Muscle fiber necrosis was also evident in the latter three mice (Fig. 3K,L)

236

(Supplementary Material 2F) (15) and is likely due to infiltrating macrophages (32).

237

Muscle tissue from CHIKV infected IRF3/7

^-/-

mice showed the occasional fragmented myocyte

238

(Fig. 3J, arrow) and substantial endomysial edema (increased fluid between myocytes) (Fig. 3,

239

compare I with J), which was less severe in IRF7

^-/-

mice (Fig. 3K) and not apparent in muscle from

240

IRF3

^-/-

mice (Fig. 3L), or WT mice (Supplementary Material 2F, G) (15).

241

Tissue localization of virus replication by in situ hybridization. The cells infected by CHIKV

242

in adult WT animals have not been extensively characterized, although infection of

243

monocyte/macrophages (21, 28), muscle satellite cells (40), and fibroblasts (53) have been reported

244

in primates. In situ hybridization of WT feet day 3 post infection, illustrated that CHIKV RNA

245

could be detected in the cytoplasm of the following; (i) cells lining blood vessels, with morphology

246

and location consistent with endothelial cells (Fig. 4A), (ii) skeletal muscle cells (Fig. 4B), (iii) rare

247

cells in the dermis with morphology consistent with fibroblasts and macrophages (Fig. 4C), (iv)

248

cells in the synovial membrane (Fig. 4D) and (v) cells of the periosteum (Fig 4E). In IRF3/7

^-/-

mice

249

3 days post infection, cells lining blood vessels and muscle fibers were also stained (Fig. 4F, G).

250

Positive cells in the dermis were also found (most with fibroblastic and monocytic morphology),

251

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but were considerably more numerous in IRF3/7

^-/-

mice (Fig. 4H). Some adnexal sebaceous glands

252

also stained positive in the dermis (Fig. 4H, inset). A large number of positive staining cells (with

253

location and morphology consistent with chondrocytes) were also seen in the articular cartilage of

254

IRF3/7

^-/-

mice (Fig. 4I), with such staining not evident in WT mice (data not shown). Positive cells

255

were not detected in the synovial membrane of IRF3/7

^-/-

mice (data not shown).

256

In WT mice day 5.5, cells lining the blood vessels continued to stain positive (Fig. 4J). However,

257

staining was no longer evident in muscle (Fig. 4K). Positively staining cells with fibroblast and

258

monocytic morphology continued to be present in the dermis (Fig. 4L), and the latter were

259

occasionally present in blood vessels (Fig. 4M). Cells with macrophage and fibroblast morphology

260

were also occasionally present in connective tissue (data not shown). No staining was seen in the

261

epidermis (Fig. 4N). In IRF3/7

^-/-

mice, cells lining blood vessels (Fig. 4O), muscle cells (Fig. 4P),

262

dermal cells with fibroblast morphology (Fig. 4Q), and cells in connective tissue with monocytic

263

morphology (Fig. 4R) continued to be stained. The epidermis also stained positive in IRF3/7

^-/-

mice

264

in several areas (Fig. 4S), suggesting infection of keratinocytes. No such staining was observed in

265

WT mice (data not shown).

266

No staining was obtained using (i) tissue from uninfected mice, (ii) antisense probes coding for

267

the same region of Ross River virus (RRV), or (iii) CHIKV sense probes (data not shown). Minus

268

strand RNA, which would be detected by sense probes, is substantially more short-lived and less

269

abundant than positive strand RNA in alphavirus infections.

270

The pattern and the type of cells infected in IRF3/7

^-/-

mice thus appeared to be distinct from

271

those infected in WT mice. In IRF3/7

^-/-

mice articular cartilage cells and more dermal fibroblasts

272

appeared to be infected day 3, and on day 5.5 there was ongoing skeletal muscle infection and

273

infection of epidermis. Although hemorrhage was readily detected in IRF3/7

^-/-

(but not in WT)

274

mice, infection of blood vessel lining cells did not appear to be any more extensive in IRF3/7

^-/- 275

compared with WT mice (data not shown and Fig. 4, compare A and F, and J and O).

276 277

Version postprint

12

Clinical signs in CHIKV infected IRF3/7

^-/-

mice were consistent with hemorrhagic shock.

278

On day 2 after infection of IRF3/7

^-/-

mice, a significant fever concomitant with the peak viremia

279

was observed (a concomitance also seen in CHIKV patients (57)), with a dramatic drop in body

280

temperature seen on days 3 and 4 (Fig. 5A). Hypothermia is indicative of hypovolemic shock, and a

281

sudden change from fever to hypothermia is also a predictor of impending dengue shock syndrome

282

(DSS) (44). WT mice infected with CHIKV did not develop a detectable fever, nor did they show a

283

significant drop in body temperature on day 4/5 post infection, although a slight dip was evident on

284

day 3 (Fig. 5A).

285

The urine output in IRF3/7

^-/-

mice 4-5 days post infection was dramatically reduced (oliguria)

286

compared with WT mice (Fig. 5B). This is again a feature of hypovolemic shock (67) and is

287

observed in DSS (61).

288

A marked thrombocytopenia was observed in IRF3/7

^-/-

mice on days 4-5 post infection, with

289

mice showing a mean §70% drop in platelet counts to <300 x 10

³

/µl (Fig. 5C). In humans the

290

normal range for platelet counts is 150 to 400 x 10

³

/µl, much lower than the §1000 x 10

³

/µl seen in

291

C57BL/6 mice (Fig. 5C). In humans a 70% drop would result in platelet counts of 45-120 x 10

³

/µl.

292

Thrombocytopenia with platelet counts of <100 x 10

³

/µl represents one of the current criteria for the

293

diagnosis of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) (56).

294

Measurement of the hematocrit in IRF3/7

^-/-

mice showed a mean 15.8% (range 7-24%) increase

295

on day 5 post infection (Fig. 5D), whereas infected WT mice showed no significant change from

296

uninfected controls (Fig. 5D). Elevated hematocrits are indicative of hemoconcentration and plasma

297

leakage. Hematocrit increases of >20% are a diagnostic criterion for DHF/DSS (61).

298

The clinical features preceding mortality in CHIKV infected IRF3/7

^-/-

mice, taken together with

299

the histology (which showed widespread edema), strongly suggest that death in these animals was

300

due to hypovolemic shock.

301

IFNα/β receptor deficient (IFNAR

^-/-

) mice. CHIKV infection of IFNAR

^-/-

mice gave similar

302

results to IRF3/7

^-/-

mice (Supplemental Material 3), suggesting loss of IFNα/β (rather than other

303

Version postprint

13

IRF3/7-dependent) responses predispose to hemorrhagic shock.

304

IRF3/7

^-/-

mice could be protected by vaccination. Vaccination with inactivated CHIKV fully

305

protected IRF3/7

^-/-

mice against viremia and disease, suggesting that mortality of IRF3/7

^-/-

mice

306

after CHIKV infection was not due to a defect in antibody responses (Supplemental Material 4).

307

Prophylactic IFNα treatment of IRF3/7

^-/-

mice failed to prevent mortality. IFNα at 10

³

IU

308

(i.v.) significantly reduced the viremia and prevented disease in adult WT mice if given before

309

CHIKV challenge (15). Injection (i.v.) of a 10 fold higher dose (10

⁴

IU) into IRF3/7

^-/-

mice prior to

310

infection was only able to delay mortality by 24-48 h; although this was significant, all mice

311

ultimately died (Fig. 6A). Foot swelling was also delayed by §24 h, but was also not prevented

312

(data not shown).

313

Adoptive transfer of WT splenocytes or WT splenic CD11b

⁺

cells into IRF3/7

^-/-

mice delayed,

314

but was unable to prevent, mortality (data not shown), consistent with the observation that

315

hematopoietic cells do not produce sufficient IFNα/β to protect mice against CHIKV infection (51).

316

IFNα treatment did not effectively inhibit CHIKV replication in IRF3/7

^-/-

MEFs. The

317

inability of prophylactic IFNα treatment to prevent CHIKV-mediated mortality in IRF3/7

^-/-

mice

318

(Fig. 6A) is perhaps surprising. We thus investigated the ability of IFNα to reduce CHIKV

319

replication in IRF3/7

^-/-

cells in vitro, by treating MEFs from IRF3/7

^-/-

mice with a range of IFNα

320

concentrations. The cells were then infected with CHIKV and virus production measured. WT

321

MEFs produced significantly lower titers of CHIKV after treatment with IFNα concentrations •10

322

IU/ml (Fig 6B, t-test, WT 0 vs 10-1000 IU/ml, p<0.006). In contrast, there was no significant effect

323

on CHIKV titers in IRF3/7

^-/-

MEFs until 1000 IU/ml of IFNα were used (Fig 6B, t-test, IRF3/7

^-/-

0

324

vs 1000 IU/ml, p=0.028). IRF7

^-/-

MEFs showed an intermediate phenotype requiring treatment with

325

100 IU/ml IFNα before significant reductions in CHIKV titers were seen (Fig 6B, Mann Whitney U

326

test, IRF7

^-/-

0 vs 100 IU/ml, p=0.037). CHIKV titers in IRF3

^-/-

MEFs were not significantly different

327

from WT MEFs. IRF3/7

^-/-

MEFs thus needed to be treated with 100x more, and IRF7

^-/-

with 10x

328

more IFNα before significant reductions in CHIKV titers were achieved.

329

Version postprint

14

IRF7 mRNA was up-regulated >100 fold in WT mice after CHIKV infection. Up regulation

330

of IRF7 (but not IRF3) is important for the positive feedback and induction of IFNαs that generates

331

a robust IFNα/β response following viral infection in MEFs (33, 48, 49). The likely importance of

332

IRF7 for optimal IFNα/β production after CHIKV infection was also suggested by the low level of

333

serum IFNα/β production in IRF7

^-/-

mice after CHIKV infection (Fig. 2A). Quantative RT-PCR

334

analysis of feet from CHIKV infected WT mice illustrated that IRF7 mRNA, but not IRF3 mRNA,

335

was up-regulated >100 fold after CHIKV infection (Fig. 6C). (IFNα treatment also up-regulated

336

IRF7 mRNA expression in MEFs - data not shown). Up regulation of IRF7 in non-hematopoietic

337

cells (51) following CHIKV infection thus likely promotes IFNα/β production and protection

338

against exacerbated disease. These data also suggest that an important activity of prophylactic IFNα

339

treatment against CHIKV in WT mice is up regulation of IRF7, rather than the induction of an anti-

340

viral state.

341

The role of TRIF, IPS-1 and MyD88. IRF3 and IRF7 lie downstream of RIG-I/Mda5/IPS-1,

342

TLR3/TRIF and TLR7/MyD88 pathways, and although signaling via these pathways during

343

CHIKV infections has been reported (51, 53, 66), the relative importance of these pathways for

344

controlling infection, preventing disease and inducing IFNα/β in vivo has not been investigated.

345

CHIKV infection of TRIF

^-/-

, IPS-1

^-/-

and MyD88

^-/-

mice resulted in significantly higher mean

346

viremias on days 3 and 4, days 4 and 5, and day 4 post infection, respectively, when compared with

347

WT mice (Fig. 7A, indicated by *). Foot swelling was significantly more pronounced in TRIF

^-/-

and

348

IPS-1

^-/-

mice, beginning earlier, peaking higher and lasting longer (particularly in IPS

^-/-

mice) than

349

in WT mice (Fig. 7B, *). There was no significant difference in foot swelling in MyD88

^-/-

mice. For

350

serum IFNα/β levels, WT > MyD88

^-/-

> TRIF

^-/-

> IPS-1

^-/-

mice, a ranking that generally follows the

351

viremia and foot swelling data (when considering area under the curves). These data suggest that

352

RIG-I/Mda5 signaling via IPS-1 is the most important for IFNα/β production in response to

353

CHIKV infection, followed by the TLR3/TRIF

^-/-

pathway, with the MyD88

^-/-

-dependent pathway

354

the least important.

355

Version postprint

15

DISCUSSION

356

Here we show that IRF3 or IRF7 are critical for survival after CHIKV infection consistent with

357

recent studies (50). CHIKV infection of IRF3/7

^-/-

mice resulted in no detectable serum IFNα/β,

358

large increases in virus titers, high levels of serum IFNγ, TNF, IL-6 and MCP-1, with animals

359

ultimately dying of hemorrhagic shock. Mortality did not appear to be due to defective antibody

360

responses, as vaccination fully protected IRF3/7

^-/-

mice. Prophylactic IFNα treatment had only

361

limited effect against CHIKV infection both in vivo (in IRF3/7

^-/-

mice) and in vitro (in IRF3/7

^-/- 362

MEFs), with up regulation of IRF7 likely required for optimal IFNα/β production (33, 48, 49). Our

363

studies suggest that CHIKV infection leads to IFNα/β production primarily via the RIG-

364

I/Mda5/IPS-1 and TLR3/TRIF

^-/-

pathways, with downstream signaling involving IRF7 (and to a

365

lesser extent IRF3).

366

The disease manifestations in CHIKV infected IRF3/7

^-/-

mice included fever and edema, features

367

well described for human CHIKV disease (57). A skin rash is also common in CHIKV disease

368

patients (57); whether this is due to infection of keratinocytes is unclear (41), with the current study

369

only able to detect epidermal infection in IRF3/7

^-/-

and not WT mice. Herein we also provide the

370

first evidence that CHIKV can infect mature skeletal muscle cells. So far only CHIKV infection of

371

human muscle satellite cells has been reported (40), although RRV has been shown to infect

372

skeletal muscle in young mice (34). Myocyte infection may in part explain the myalgia, a common

373

manifestation of human CHIKV disease (57). Infection of chondrocytes by CHIKV has not

374

previously been reported, although infection of periosteum has been shown for Sindbis virus (18)

375

and RRV in young mice (34), with such infections likely to contribute to rheumatic disease. CHIKV

376

infection of monocytes/macrophages, fibroblasts and endothelial cells in vivo have been reported

377

previously (19, 28). The more severe manifestations seen in IRF3/7

^-/-

(and IFNAR

^-/-

) mice,

378

hemorrhage, thrombocytopenia and hypovolemic shock, have also been reported for severe CHIKV

379

infections in human neonates and children (16, 47), with such manifestations occasionally

380

associated with mortality in neonates (16, 37, 45, 47, 57). Human neonates have defective innate

381

Version postprint

16

antiviral responses, including defective IRF7-mediated responses (12, 31). Hemorrhagic fever

382

associated with CHIKV infections has also been reported in children (13, 24, 45) and some adults

383

(39, 47). CHIKV infection of IRF3/7

^-/-

(and IFNAR

^-/-

) mice thus recapitulates many of the features

384

seen during severe human disease. These findings thus suggest that a paucity of IFNα/β responses

385

may predispose to severe CHIKV disease in humans.

386

The parallels between the pathological and clinical signs preceding mortality in CHIKV infected

387

IRF3/7

^-/-

and (IFNAR

^-/-

) mice and those seen in humans with DHF/DSS are striking. Oliguria,

388

increased hematocrit, fever followed by hypothermia, edema, hemorrhage, and thrombocytopenia

389

are all features of DHF/DSS. Elevated levels of IFNγ, TNF, IL-6 and MCP-1 (CCL2) are also key

390

players in DHF/DSS (30, 42). As IRF3/7

^-/-

and (IFNAR

^-/-

) mice lack IFNα/β responses, an

391

important driver of DHF/DSS may also be inadequate IFNα/β responses. This concept has been

392

proposed previously based on the ability of antibody-dependent enhancement to suppress IFNα/β

393

responses (17, 58). The idea is also supported by an array analysis of DSS patients, which showed

394

that type 1 IFN-induced gene transcripts were less abundant in DSS patients (54). In addition, IRF7

395

has also been identified as a key transcription factor mediating the early response to dengue (20).

396

(Conceivably, inhibition of IFNα/β receptor signaling by dengue (35) is also involved, with CHIKV

397

reported to have similar activity (14)).

398

The very high levels of proinflammatory mediators, particularly IFNγ and TNF, seen in CHIKV-

399

infected IRF3/7

^-/-

and IFNAR

^-/-

mice are likely important contributors to the vascular leakage and

400

shock (42). However, the paucity of IFNα/β responsesѽ rather than the elevated viral load, may be

401

the main factor responsible for the high levels of these cytokines. IFNα/β has been shown to inhibit

402

IFNγ production by NK and T cells (7, 38), and in mice with high Trypanosome cruzi burdens,

403

IFNα/β has also been shown to suppress IFNγ production (6). IFNα/β can also suppress the

404

responses to IFNγ by down regulating the IFNγ receptor (43). The increase in IFNγ levels in

405

CHIKV infected IRF3/7

^-/-

and IFNAR

^-/-

mice are probably responsible for increased TNF

406

Version postprint

17

production by macrophages (43). These observations support the view that IFNα/β plays a critical

407

role in suppressing excessive IFNγ-mediated immune pathology during acute infection.

408

For both dengue and CHIKV infections it remains unclear what cells are producing and

409

responding to protective IFNα/β in vivo. Dengue virus can infect monocytes/macrophages (17),

410

fibroblasts (26), endothelial cells (11), skeletal muscle (46) and keratinocytes (59). Herein we

411

provide evidence that all these cell types are also infected by CHIKV in vivo. Although

412

hematopoietic cells (eg. plasmacytoid dendritic cells) are often believed to be the main producers of

413

IFNα/β in vivo, hematopoietic cells do not appear to be involved in production of protective

414

IFNα/β during CHIKV infection (51). The importance of IPS-1 for IFNα/β production, supports

415

the view that non-hematopoietic cells (51) directly infected by CHIKV are an important source of

416

protective IFNα/β in vivo. Although detection of viral dsRNA from alphavirus infected cells by

417

hematopoietic cell TLR3 is well established (10, 52), TLR3 is also expressed and/or can be up

418

regulated on endothelial cells (70), dermal fibroblasts (1) and keratinocytes (69). All these cell types

419

also express and/or can up regulate IRF7 and produce IFNα/β (3, 33, 48, 63). Detection of

420

alphaviral single-stranded RNA by TLR7 has not yet been formally demonstrated, but might be

421

assumed given its role in infection with other single-stranded RNA viruses (62). However, the

422

minor phenotypes seen in MyD88

^-/-

mice, suggests the TLR7/MyD88

^-/-

pathway plays a relatively

423

less important role during CHIKV infection and disease.

424

The importance of IPS-1 in non-hematopoietic cells for protective responses to CHIKV infection

425

was recently reported (50), and is consistent with the findings presented herein. MyD88 was

426

previously reported to be important for preventing CHIKV dissemination, with 0.5-1 log higher

427

viraemia (on day 2) and tissue viral titres (on day 3) observed in MyD88

^-/-

mice compared with WT

428

mice (51). We did not analyze tissue titres in MyD88

^-/-

mice, but saw a §1.7 log higher serum

429

viremia in MyD88

^-/-

mice compared with WT on day 4. The results are thus broadly comparable,

430

with differences likely attributable to differences in the infection models (eg virus preparation,

431

detection and route of inoculation).

432

Version postprint

18

A striking feature of CHIKV infection in IRF3/7

^-/-

mice is the paucity of infiltrating cells

433

compared with IRF3

^-/-

, IRF7

^-/-

and WT mice. Infiltrates in WT mice primarily contain monocytes

434

and macrophages (15), with infiltration of these cells largely dependent on chemokine-(C-C motif)

435

receptor 2 (CCR2), the receptor for MCP-1/CCL2 (Suan Poo in prep.). Despite the paucity of

436

IFNα/β (8), MCP-1 was very efficiently produced in CHIKV infected IRF3/7

^-/-

mice, illustrating

437

that loss of MCP-1 was not responsible. Two factors may explain the lack of infiltrating cells in

438

CHIKV-infected IRF3/7

^-/-

mice. Firstly, IFNα/β appears to be required for differentiation of resting

439

Ly6C

^lo

monocytes into inflammatory Ly6C

^hi

monocytes, with only the latter able to migrate in

440

response to MCP-1/CCR2 (29). Secondly, high levels of IFNγ and TNF have been shown to down

441

regulate expression of CCR2 in monocytes (60, 65), with TNF also shown to down regulate CCR2

442

expression in dendritic cells (64).

443

In summary, the studies described herein highlight the critical role of IRF7 (and to lesser extent

444

IRF3) in the production of protective IFNα/β via IPS-1- and TRIF-dependent (and to lesser extent

445

MyD88- dependent) pathways. The studies also illustrate the importance of IFNα/β responses in

446

protection against virus-induced hemorrhage and shock, with a compromised IFNα/β response

447

associated with high levels of IFNγ and TNF. By analogy, this work suggests that a paucity of

448

IFNα/β may also play an important role in DHF/DSS.

449 450 451

ACKNOWLEDGEMENTS

452

We thank Clay Winterford (Histotechnology Facility) and animal house staff (QIMR) for

453

excellent support, and Dr M. S. Diamond for supply of knockout mice.

454

This work was funded by the NHMRC, Australia. Equipment was funded by the Queensland

455

Tropical Health Alliance, and a donation from Prof Ed Westaway, Royal Australian Air Force

456

Association. A.S. and A.A.K. are research fellows with the NHMRC, and P.A.R. is a postdoctoral

457

fellow with the Canadian Institutes of Health Research.

458

Version postprint

19

The funders had no role in study design, data collection and analysis, decision to publish, or

459

preparation of the manuscript.

460 461 462

Version postprint

20

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