SARS-CoV-2 induced CYP19A1 expression in thelung correlates with increased aromatization of
testosterone-to-estradiol in male golden hamsters
Stephanie Stanelle-Bertram
Heinrich Pette Institute Hamburg
Ber n Schaumburg
Leibniz Institute for Experimental Virology
Nancy Mounogou Kouassi
Heinrich Pette Institute Hamburg
Sebastian Beck
Heinrich Pette Institute Hamburg
Martin Zickler
Leibniz Institute for Experimental Virology
Georg Beythien
Universtity of Veterinary Medicine, HannoverKathrin Becker
University of Veterinary Mediicine Hannover
Tian Bai
Leibniz Institute for Experimental Virology
Hanna Jania
Leibniz Institute for Experimental Virology
Zacharias Müller
Leibniz Institute for Experimental Virology
Vinicius Pinho dos Reis
Leibniz Institute for Experimental Virology
Vanessa Krump-Buzumkic
University for Veterinary Medicine Hannover
Alan Kadek
Leibniz Institute for Experimental Virology
Charlotte Uetrecht
Leibniz Institute for Experimental Virology
Maria Schroeder
University Hospital Hamburg-Eppendorf
Dominik Jarczak

University Hospital Hamburg-Eppendorf
Axel Nierhaus
University Hospital Hamburg-Eppendorf
Stefan Kluge
University Hospital Hamburg-Eppendorf
Pietro Scaturro
Heinrich Pette Institute, Leibniz Institute for Experimental Virology https://orcid.org/0000-0001-90983087
Wolfgang Baumgärtner
University of Veterinary Medicine Hannover https://orcid.org/0000-0001-8151-5644
Karin Klingel
University Hospital Tuebingen
Gabriel Guelsah (  guelsah.gabriel@leibniz-hpi.de )Leibniz Institute for Experimental Virology

Biological Sciences - Article
Keywords: Androgens, Sex Bias, Dysregulation of Sex Hormones, Abundant CYP19A1 Expression, Sexspeci c Disease Outcome, Complex Metabolic Cross Talk
Posted Date: November 21st, 2020
DOI: https://doi.org/10.21203/rs.3.rs-107474/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License.
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SARS-CoV-2 infection induced CYP19A1 expression in the lungcorrelates with increased aromatization of testosterone-toestradiol in male golden hamsters

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Stephanie Stanelle-Bertram1#, Berfin Schaumburg1#, Nancy Mounogou Kouassi1, SebastianBeck1, Martin Zickler1, Georg Beythien2, Kathrin Becker2, Tian Bai1, Hanna Jania1, Zacharias
Müller1, Vinicius Pinho dos Reis1, Vanessa Krupp-Buzumkic3, Alan Kadek4,5, Charlotte
Uetrecht4,5, Maria Schroeder6, Dominik Jarczak6, Axel Nierhaus6, Stefan Kluge6, Pietro
Scaturro7, Wolfgang Baumgärtner2, Karin Klingel8 and Gülsah Gabriel1,3

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1

Department for Viral Zoonoses-One Health, Heinrich Pette Institute, Leibniz Institute forExperimental Virology, Hamburg, Germany; 2Department of Pathology, University of Veterinary
Medicine Hannover, Hannover, Germany; 3Institute for Virology, University for Veterinary Medicine
Hannover, Hannover, Germany; 4Junior Research Group for Dynamics of Viral Structures, Heinrich
Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany; 5European XFEL
GmbH, Schenefeld, Germany; 6Department of Intensive Care Medicine, University Medical Center
Hamburg-Eppendorf, Hamburg, Germany; 7Junior Research Group for Systems Arbovirology,
Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany; 8Institute for
Pathology and Neuropathology, University Hospital Tuebingen, Germany;#

shared first authorshipcorrespondence to: guelsah.gabriel@leibniz-hpi.de

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Summary29

SARS-CoV-2 infection is associated with increased morbidities in men compared to women.

30

Androgens are believed to play an important role in SARS-CoV-2 pathogenesis in men due to31

the postulated androgen-dependency of ACE2 and TMPRSS2. However, it is yet unclear

32

whether the sex bias is mediated by SARS-CoV-2 infection itself or by other confounding

33

factors. Here, using the golden hamster model, we show that SARS-CoV-2 infection attacks

34

reproductive organs, causes massive dysregulation of sex hormones and induces elevated35transcription of the androgen-to-estrogen converting enzyme aromatase CYP19A1 in the lung.

36

In male hamsters, SARS-CoV-2 infection causes severely depleted testosterone and highly37

elevated estradiol levels. In female hamsters, SARS-CoV-2 infection causes reduced estradiol

38levels. Hormonal dysregulation in infected animals is followed by severe weight loss compared39

to control groups treated with poly(I:C) or PBS. Lungs of SARS-CoV-2 infected animals

40

present abundant CYP19A1 expression in the endothelium and in macrophages, particularly in41

males. Prominent CYP19A1 expression in endothelial cells and macrophages was verified in42lung sections of deceased Covid-19 males compared to females. Our results demonstrate that43

SARS-CoV-2 infection leads to massive dysregulation of sex hormones, which may increase

44

the risk for sex-specific disease outcome particularly in combination with comorbidities. These45

findings provide insights into the complex metabolic cross talk between SARS-CoV-2 infection46

and sex hormones.

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Introduction

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51

SARS-CoV-2 was first detected in humans in Wuhan, China in November 2019 and was

52

declared a pandemic by the World Health Organization on 11th March 2020. Since, then ~46

53

million people were diagnosed with SARS-CoV-2 and ~1.1 million died worldwide (as of 1st

54

November 2020). There is accumulating evidence that men are more vulnerable to severe

55

SARS-CoV-2 caused disease (Covid-19) outcome compared to women across countries 1. A

56

cohort study from the UK with 17 million patients enrolled, identified that being male and older

57

age belongs to the highest risk factors 2. However, the increased Covid-19 case fatality rate in58

males is not reflected by increased SARS-CoV-2 male incidence 3-5. This further highlights the59

existence of factors that predispose men to an elevated risk to die of Covid-19 compared to60

women 6. Another population-based study analyzing data obtained from ~194 million men and

61~201 million women across England and Wales, France, Germany, Italy, Netherlands, Portugal,

62

Korea and Spain revealed that the overall male-to-female sex ratio increased in combination

63

with increasing age 7. Factors that mediate sex disparity in Covid-19 outcome might include a64complex interaction of biological sex differences (e.g. chromosomes, sex steroids) or gender65

aspects (e.g. social behavior) 8. Sex differences of genetic origin are constant throughout life,

66

while sex differences originating from hormonal effects may alter with increasing age.

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68

To identify the impact of biological sex on SARS-CoV-2 disease outcome, we analyzed the69

dynamics of sex hormones, sex hormone converting enzymes, viral loads and expression of70

viral entry factors (ACE2, TMPRSS2) in combination with unbiased lung proteome analysis

71

using the golden hamster model 9,10 reported to reflect major clinical findings, such as systemic72

spread.

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Results

74
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Respiratory SARS-CoV-2 infection disseminates to the reproductive organs of male and76

female hamsters

77

In order to assess potential sex differences in SARS-CoV-2 pathogenesis, we intranasally78

infected male and female hamsters. As controls, animals were intranasally treated with PBS or79

poly(I:C) as an immune stimulant. Both sexes underwent weight loss upon SARS-CoV-2

80

infection with a peak drop of ~20% at day 6 p.i. compared to control groups (Figure 1a and b).

81

Both, male and female hamsters recovered reaching their initial weight on day 14 p.i.. No82

significant difference was observed in weight loss or recovery time between male and female

83

animals (Figure 1c). SARS-CoV-2 replicated to high titers in the lungs of male and female

84

hamsters causing pneumonia with multifocal moderate infiltrates of mononuclear cells and85

neutrophils unlike PBS or poly(I:C) controls (Figure 1d, e; Extended Data Figure 1).

86

Interestingly, replicating SARS-CoV-2 was also detected in the plasma and the gonads (Figure871f-o). In the plasma, replicating virus was detected in 4 out of 5 male animals and in 3 out of 5

88

female animals (Figure 1f). In the testis, 1 out of 5 animals showed replicating virus and 3 out

89

of 5 were viral RNA (vRNA) positive without major pathological changes (Figure 1g-i). In the90

ovaries, 4 out of 5 animals were virus positive and 2 out of 5 animals presented vRNA with no91

severe pathologies (Figure 1j-l). In the light of the infected ovaries, we also assessed whether

92

SARS-CoV-2 could also infect the uterus and found that 2 out of 5 animals were virus positive93

and 3 out of 5 animals were vRNA positive again without causing major pathological changes

94(Figure 1m-o).

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96

These findings show that SARS-CoV-2 replicates in the organs of the reproductive system of97

hamsters. No significant difference is detected in weight loss or survival upon SARS-CoV-2

98

infection between the herein used young and healthy male and female animals without existing

99

comorbidities reflecting clinical reports 2,7.

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101

SARS-CoV-2 infection mediates elevated macrophage inflammatory protein and reduced

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vascular endothelial growth factor levels in the lungs of male and female hamsters

103

We then studied sex-dependent local and systemic innate immune responses of SARS-CoV-2

104

infected hamsters compared to PBS and poly(I:C) treated control animals at various time points

105

after infection. We analyzed a panel of 13 different cytokines and chemokines (eotaxin, MIP-

106

1α, MIP-1β, IFN-γ, IL-2, IL-6, IL-10, IL-12, IL-13, IL-1β, MCP-1, TNF-α and VEGF). In the4

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lung, on day 1 p.i., infected groups did not show significant cytokine/chemokine induction

108

except for poly(I:C) treated males and females for MIP-1α and MIP-1β (Extended Figure Data

1092a-k). On day 3 p.i. in the lung, IL-6, IL-1ra, Eotaxin and MIP-1α levels were significantly

110

increased in infected male and female animals as compared to uninfected control groups

111(Figure 2a-e). IFN-γ levels were higher in female SARS-CoV-2 infected animals compared to112

males, albeit the intrinsic IFN-γ levels were already elevated in the lungs of PBS treated groups

113(Figure 2f). Interestingly, VEGF was the only cytokine within the panel assessed that was

114

significantly reduced in SARS-CoV-2 infected male and female hamsters compared to115

uninfected controls (Figure 2g). All other cytokines and chemokines did not show any

116

significant alterations between groups and sexes, albeit IL-10 and IL-12 levels seemed to be

117

slightly reduced in infected female hamsters as compared to the respective PBS or poly(I:C)

118

controls (Figure 2h-o). On day 6 p.i., most altered cytokines and chemokines in the infected119groups were again comparable to the uninfected controls in both sexes (Extended Data Figure1202l-s). However, eotaxin and MIP-1α levels were still highly elevated in the lungs of SARS-

121

CoV-2 infected male and female hamsters (Extended Data Figure 2l, m). MIP-1β levels that122

were unaltered on day 3 p.i., showed high levels on day 6 p.i. in infected hamsters. VEGF levels

123

remained strongly reduced in the lungs of SARS-CoV-2 infected male and female animals even124

on day 6 p.i. (Extended Data Figure 2o). In the plasma, however, most cytokines and125

chemokines assessed did not show major differences among all groups with the exception of126

slightly reduced eotaxin levels at day 6 p.i. in infected females (Extended Data Figure 3).

127
128

These findings show that SARS-CoV-2 infection mediates inflammatory cytokine responses

129

mainly occur in the lungs of male and female hamsters. In particular, a strong induction of130

macrophage secreted inflammatory chemokines MIP-1α and MIP-1β levels correlates with131

suppressed VEGF levels that is involved in vascular wound repair processes.

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133

SARS-CoV-2 infection mediates reduced testosterone and increased estradiol levels in the134

plasma of male hamsters

135

Infectious SARS-CoV-2 particles were detected in the gonads of infected animals, where major

136

sex hormones are produced and then secreted into the blood system. Thus, any changes in137

systemic sex hormone responses would have implications on immune responses given the138

presence of androgen and/or estrogen receptors on the surface of most immune cells 11.

139

Therefore, we measured the major sex hormones in the plasma of SARS-CoV-2 infected140

animals in comparison to uninfected controls treated with PBS or poly(I:C) at multiple time5

141

points after infection to allow identification of potential dynamic processes. Testosterone levels

142in the plasma of SARS-CoV-2 infected male hamsters were significantly reduced on day 3 p.i.,

143

started to recover on day 6 p.i. and fully recovered on day 14p.i. to comparable levels of

144

uninfected control groups treated with PBS or poly(I:C) (Figure 3a). Testosterone levels in the145

plasma of SARS-CoV-2 infected female hamsters were similarly low among all infected and146

uninfected groups and likely not allowing the detection of potential alterations (Figure 3b).

147

Estradiol levels were significantly elevated in SARS-CoV-2 infected male hamsters on day 3

148

p.i. with still high levels on day 6 p.i. and fully recovered levels on day 14 p.i. compared to149

uninfected control groups (Figure 3c). Estradiol levels in infected female hamsters on the other150

hand were significantly reduced on day 3 p.i. compared to uninfected controls, while on day 6

151

and 14 p.i. estradiol levels were again comparable among all groups (Figure 3d). Cortisol levels

152in the plasma of SARS-CoV-2 infected male and female hamsters were elevated on day 3 and153

6 p.i. normalizing back on day 14 p.i. likely due to infection stress (Figure 3e, f). Progesterone

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levels were slightly elevated on day 3 but not 6 or 14 days p.i. in SARS-CoV-2 infected male155

and female hamsters unlike their respective negative controls (Figure 3g, h).

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Importantly, we included additional control groups to see whether any respiratory viral158

infection would mediate reduced testosterone and increased estradiol levels in the plasma.

159

Therefore, we used a 2009 pandemic H1N1 influenza A virus (pH1N1) isolate and an avian

160

H7N9 influenza A virus isolated from a patient. Both, pH1N1 and H7N9 influenza virus

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infections mediated low testosterone levels upon infection but did not alter estradiol levels in162

male mice in line with previous reports 12. Thus, the shift in sex hormones from testosterone-

163

to-estradiol detected seems to be specific for SARS-CoV-2 infection in male animals

164(Extended Data Figure 4).

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These findings show that SARS-CoV-2 infection mediates severe dysregulation of sex

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hormones. In infected males, reduced testosterone levels are paralleled with increased estradiol

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levels unlike in infected females.

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SARS-CoV-2 infection in the lung correlates with reduced ACE2 and TMPRSS2171

transcription

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Both SARS-CoV-2 entry factors, ACE2 and TMPRSS2 possess postulated androgen receptors

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13,14

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CoV-2 disease outcome. Therefore, we measured the transcription levels of ACE2 andgiving rise to the discussion whether testosterone levels in males might determine SARS-

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TMPRSS2 as well as controls including DPP4, ACE and the androgen receptors AR and ZIP9.

176

In the lung, ACE2 mRNA levels in SARS-CoV-2 infected male and female animals were177

unaffected on day 1 p.i., became significantly reduced on day 3 p.i. and were further reduced

178

on day 6 p.i. (Figure 3i-k). Transcription levels of ACE, a homologue of ACE2, in the lung,179

were not affected (Figure 3l). Interestingly, ACE2 transcription was not impaired in the testis

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or in the ovaries (Figure 3m and n). In the lung, a similar trend was detected with DPP4, the181

MERS-CoV receptor, and TMPRSS2 resulting in significantly reduced DPP4 and TMPRSS2

182

levels (Figure 3o-q). In contrast, transcription levels of the internal (AR) or membrane-bound183(ZIP9) androgen receptors were not changed (Figure 3r and s).

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185

These findings show that reduced transcription levels of ACE2 and TMPRSS2 correlate with186

reduced testosterone levels on day 3 p.i. However, transcriptional levels still remain reduced on

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day 6 p.i., albeit testosterone levels already start to recover on day 6 p.i.. This observation is in

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line with previous reports on ACE2 and TMPRSS2 downregulation in SARS-CoV infected

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cells 15-17. These data might suggest that the transcription of SARS-CoV-2 entry factors might190

not be exclusively controlled by testosterone.

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SARS-CoV-2 infection in the lung is not associated with major differences between sexes

193

Data obtained so far indicate that cytokine and chemokine induction upon SARS-CoV-2

194

infection mainly occurs in the lung. Thus, we wanted to identify potential cellular factors that195

might mediate severe lung pathology in infected male and female animals. Therefore, we used

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an unbiased proteomic approach. Herein, protein levels were quantified in the lungs of hamsters

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infected with SARS-CoV-2 or control groups treated with PBS or poly(I:C), respectively.

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Expression of various cellular proteins changed in SARS-CoV-2 infected animals compared to199

uninfected control groups (Extended Data Figure 5 and 6). However, only three cellular

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proteins were detected that significantly differ between males and females upon SARS-CoV-2

201

infection (Figure 4). In infected females, the Golgi SNARE protein and the Ig-like domain-202

containing protein were expressed to significantly higher levels compared to infected males.

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Interestingly, serum amyloid A-5 protein was expressed to significantly higher levels in

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infected males compared to females. Serum amyloid A protein is an acute-phase protein, that205

is stimulated by pro-inflammatory cytokines (IL-6, IL-2, TNF-α, IFN-γ and TGF) and released

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in response to general inflammation 18. The identification of limited factors that are significantly

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altered between males and females upon SARS-CoV-2 infection suggests that other or208

additional factors might be responsible for the increased vulnerability of males to Covid-19.
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SARS-CoV-2 infection induces increased aromatase CYP19A1 transcription in the lung210

but not in the gonads

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Since data obtained from the unbiased proteomics analysis revealed no major differences in

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altered protein levels between males and females, we focused on the dysregulation of sex

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hormones and its potential underlying mechanisms. Reduced testosterone and elevated estradiol

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levels in males indicate altered aromatization. The conversion of testosterone-to-estradiol

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occurs via the aromatase CYP19A1, which is expressed in a wide range of tissues (including216

gonads and the lung) but also in endothelial cells and in the vascular wall in humans and various

217

mammalian species 19-22. The aromatase CYP19A1 gene contains predicted binding sites for218

key transcription factors, such as estrogen receptor, progesterone receptor, androgen receptor,

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C/EBP-α (CCAAT-enhancer binding protein α; mainly involved in myeloid differentiation),

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SF-1 (steroidogenic factor-1; involved in sex determination by controlling activity of genes

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related to the reproductive glands or gonads and adrenal glands), NFAT-1/-2 (a regulator of T-

222

cell response), IRF-1,-3 (controls expression of various cytokines), C-ets-1 (controls the223

expression of cytokine and chemokine genes), FOXP3 (involved in regulatory T-cell function),

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STAT1,-3,-4,-6 (regulators of various aspects of cellular immunity) and NF-κB (regulates

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genes responsible for innate and adaptive immune response) 23-27 indicating a key role in226

reproduction and immunity (Figure 5a). Thus, we first assessed whether SARS-CoV-2

227

infection mediated induction of cytokines and chemokines in the lung may alter aromatase

228

CYP19A1 transcription. Since the CYP19A1 gene of the golden hamster is not fully sequenced,229

we first sequenced regions of the gene and designed specific primers (data not shown). Lack of

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availability of full genome sequences of the golden hamster model might reflect one limitation

231of the proteome analysis. Interestingly, we could detect significantly elevated aromatase

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CYP19A1 mRNA levels starting on day 3 p.i. in SARS-CoV-2 infected males and females,

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albeit levels were slightly higher in males as compared to the control groups treated with PBS

234

or poly(I:C) (Figure 5b and c). CYP19A1 mRNA levels remained elevated, particularly in235

males, on day 6 p.i. (Figure 5d). To exclude that also other sex hormone converting enzymes

236

might be affected upon SARS-CoV-2 infection in the lung, we assessed mRNA levels of the 5-237

α reductase as a control, which converts testosterone-to-dihydrotestosterone. Transcription

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levels of 5-α reductase were not affected in the lungs of SARS-CoV-2 infected male or female239

hamsters compared to the PBS and poly(I:C) control groups (Figure 5e). Since the CYP19A1

240

aromatase is also expressed in the gonads, we analyzed its expression in the testis and ovaries

241

of infected versus uninfected animals. CYP19A1 aromatase mRNA levels were not altered in

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the testis or ovaries upon SARS-CoV-2 infection as compared to the control treatments (Figure
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5f and g). This suggests that CYP19A1 aromatase is dysregulated in the lung upon SARS-CoV-244

2 infection, particularly in males. Since estradiol plays a key role in endothelial function and245

angiogenesis, we wanted to know whether endothelial activation might be detected in the lung.

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Therefore, we measured levels of endothelial lipase, which is secreted by vascular endothelial

247

cells and is involved in metabolic disorders 28,29. Herein, we detected significantly elevated248

endothelial lipase levels in males, albeit a non-significant increase was also detected in females

249

upon SARS-CoV-2 infection on days 3 and 6 p.i. compared to uninfected controls treated either

250

with PBS or poly(I:C) (Figure 5h and i).

251
252

These findings suggest that transcription of the CYP19A1 aromatase gene is specifically up-

253

regulated in the lungs of SARS-CoV-2 infected animals correlating with endothelial activation,

254

particularly in males.

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Aromatase CYP19A protein is abundantly expressed in perivascular and peribronchiolar

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infiltrates of male hamsters infected with SARS-CoV-2

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We then wanted to identify, which cell types in the lung express the CYP19A aromatase.

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Therefore, we stained serial lung sections of male and female hamsters either infected with

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SARS-CoV-2 or uninfected controls treated with PBS or poly(I:C), respectively. CYP19A

261

aromatase expression was directly compared to serial sections stained with markers for cell262

types known to express aromatase, such as the endothelium (CD31, vWF) and smooth muscle

263

cells (SMA). In addition, we used markers to detect immune cells (CD204, CD3) and viral NP

264(Figure 6; Extended Data Figure 7). To assess expression levels in different tissues, the265

aromatase immunoreactivity was scored semiquantitatively in the bronchiolar epithelium, the266

endothelium and the peribronchiolar as well as perivascular regions of the lung sections. All

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groups showed CYP19A1 aromatase expression in endothelial cells, bronchiolar epithelium as

268

well as perivascular connective tissue, whereas peribronchiolar expression was only present in269

poly(I:C) treated and in infected animals (Figure 6). Male PBS treated controls showed an

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overall low immunoreactivity of CYP19A aromatase in bronchiolar epithelium, endothelial

271

cells and perivascular connective tissue (Figure 6a). Female PBS treated control animals

272

showed only very low reactivity in respective tissues (Figure 6b). Poly(I:C) treated controls273

and infected animals showed an especially high level of immunoreactivity in perivascular274

connective tissue, beside the additional expression of CYP19A aromatase in peribronchiolar

275

tissue (Figure 6). The perivascular as well as the peribronchiolar tissue was both infiltrated by276

moderate amounts of macrophages (CD204-positive) and low amounts of T-lymphocytes
9277(CD3-positive). In both sexes, expression was considerably higher in infected animals than in278

poly(I:C) controls. Male individuals showed substantially higher expression than females

279(Figure 6).

280
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Thus, histological findings in the golden hamster model indicate that CYP19A1 protein is282

expressed to higher amounts in the male compared to the female lung upon SARS-CoV-2

283

infection.

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Aromatase CYP19A1 protein is abundantly expressed in endothelial cells and286

macrophages of fatal male Covid-19 cases287

To see whether increased CYP19A1 aromatase expression detected in the hamster model can

288

be translated into the clinical setting, we stained lung sections obtained from seven deceased

289

Covid-19 patients (four males and three females) as well as negative controls against the290

CYP19A1 aromatase. All Covid-19 patients presented CYP19A1 positive cells in their lungs

291(Figure 7a-h). CYP19A was expressed in endothelial cells and particularly in macrophages

292around major areas of inflammation. Interestingly, a significantly higher expression of293

CYP19A1 was observed in macrophages and endothelial cells in all male compared to female

294

Covid-19 patients or control patients without lung disease. In a semiquantitative score judging

295

the intensity of signals in CYP19A positive cells (0-3) we found in males (n=4, 3 +/- 0, in

296

females n=3, 2 +/- 0 and in control patients (n=3, 1 +/- 0).

297
298

To assess whether CYP19A1 induction is mediated by viral infection directly, we infected ex299

vivo human lung cultures (Calu-3) with SARS-CoV-2 and pH1N1 influenza as a control. We

300

could detect significantly elevated CYP19A1 transcription levels upon SARS-CoV-2 infection

301

unlike H1N1 infection or PBS treated controls (Figure 7i).

302
303

These findings suggest that SARS-CoV-2 infection specifically induces elevated CYP19A1

304

transcription in the lung. Histological findings from Covid-19 cases further show that305

CYP19A1 protein is most abundantly expressed in macrophages of deceased men compared to306

women.

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Discussion309

Our results in the golden hamster model revealed key differences in sex hormone levels upon

310

SARS-CoV-2 infection that might play an important role in sex-specific disease outcome,

311

particularly in combination with other comorbidities. First, SARS-CoV-2 replicates in

312

reproductive organs likely leading to impaired sex hormone production, which might affect313

paracrine functions (Extended Data Figure 8). Second, we found that in males unlike in314

females, systemic reduction of testosterone levels is accompanied by elevated estradiol levels,

315

which might additionally affect endocrine functions of the sex hormones. Control male animals

316

infected with H1N1 and H7N9 influenza A virus presented reduced testosterone levels as shown

317

before but not elevated estradiol levels 12. This is in line with findings from human cohorts,

318

where H1N1 and H7N9 influenza A virus infection in men correlated with low testosterone but

319

not elevated estradiol levels 30. This is also in agreement with our previous report that critically320

ill male Covid-19 patients present low testosterone and high estradiol levels 31. In contrast to321

the findings here in the hamster model, critically ill female patients presented not reduced but

322

elevated estradiol levels combined with elevated testosterone levels 31. However, it should be323

considered that 10 out of 11 critically ill Covid-19 women analyzed in the above mentioned

324

study 31 were postmenopausal in contrast to the young female animals used in this study.

325

Current studies further highlight that the menstrual status in women may affect Covid-19

326

outcome suggesting that more investigation is needed in pre- and postmenopausal females to327

understand the impact of female hormones on female pathogenesis 32. Third, we show that328

transcription and expression of the CYP19A1 aromatase, which is responsible for the329

conversion of testosterone-to-estradiol is elevated in the lungs of SARS-CoV-2 infected

330

animals, particularly in males. Indeed, previous reports also showed that CYP19A1 aromatase331

is detected to higher levels in male animals and humans compared to their female counterparts

332

33

333

significantly increased levels of endothelial lipase, which is an endothelial activation marker

334

involved in metabolic disorders 28,29. Histochemical analysis revealed that CYP19A1 is335

expressed to particular high levels in the perivascular region, including macrophages of infected336

male compared to female hamsters. It was proposed that the immunological hyper-response,337

characterized by widespread endothelial damage, complement-induced blood clotting and338

systemic microangiopathy play critical roles in Covid-19 exacerbation 34. Endothelial activation339

and the complement system are highly debated as playing important roles in severe SARS-340

CoV-2 disease outcome in humans 35. CYP19A1 was shown before to be highly expressed in

341

macrophages and proposed to be sufficient for aromatization of estradiol levels from. Elevated CYP19A1 levels in the lung of SARS-CoV-2 infected male hamsters correlate with

11

342

testosterone to promote cell proliferation, auto-regulate cytokine production and thus fuel343

inflammation processes 36. Macrophages express androgen and estrogen receptors that may

344

regulate their activation and thus contribute to sex-specific immune responses 37. CYP19A1

345

plays a key role in breast cancer and many clinical trials have shown the efficacy of aromatase346

inhibitors in the management of breast cancer 38. Herein, particularly estradiol is a known

347

promoter of vascular inflammation and inhibition of the aromatase was shown to reduce

348

vascularization and formation of metastasis. In line with this observation, it was shown in349

murine models that mice lacking the CYP19A1 gene are not able to fully repair lung damage

350

compared to mice expressing a functional CYP19A1 gene 39. Finally, CYP19A1 expression351

was detected in endothelial cells and macrophages of seven deceased Covid-19 patients.

352

CYP19A1 expression was more pronounced in male than female patients. Macrophages and353

the macrophage activation syndrome were repeatedly proposed to play a key role in lung354

inflammation and endothelial damage in Covid-19 patients 40,41.

355
356

Collectively, our findings suggest that increased expression of CYP19A1 in the lung upon

357

SARS-CoV-2 infection promotes elevated aromatization and thus likely mediates reduced358

systemic testosterone and elevated estradiol levels in males. Active SARS-CoV-2 replication359

in the testis additionally impairs testosterone production in males. Thus, lack of systemic and360

local testosterone levels in males might result in lack of induction of androgen receptor

361

containing immune cells required to combat viral infection. An additional increase in systemic362

and local estradiol levels in males might additionally fuel up inflammatory processes via

363

estrogen receptor containing immune cells and their activation, e.g. macrophages (Extended

364

Figure 9). It should be further noted that increasing age in men as well as comorbidities with365

endothelial involvement, such as cardiovascular diseases, adipositas and type II diabetes are

366

associated with hypogonadism (reduced testosterone levels). In cardiovascular diseases 42,367

adipositas 43,44 and type II diabetes 45 low testosterone levels may be combined with high368

estradiol levels due to increased aromatase activity. Thus, it is tempting to speculate whether

369

multiple testosterone level reducing and/or estradiol level increasing hits might contribute to370

severe and even fatal Covid-19 outcome in men. Future studies are required to understand the371

impact of sex hormones on SARS-CoV-2 pathogenesis in comorbidity models.

372
373

12

374

Materials and Methods

375
376

Viruses

377

The

378

PRJEB41216 and sample ERS5312751) was isolated by inoculation of VeroE6 cells with 200379µl of a human nasopharyngeal swab of a confirmed patient with Covid-19 in Hamburg and380

propagated for three serial passages in VeroE6 cells. VeroE6 were cultivated in DMEM (Sigma-

381

Aldrich GmbH) with 2 % fetal bovine serum, 1 % penicillin-streptomycin and 1 % L-glutamine382

at 37°C for virus propagation. The H7N9 avian influenza A virus (A/Anhui/1/13) and 2009383

pandemic H1N1 influenza A virus (A/Hamburg/NY1580/09; pH1N1) were grown and titrated

384

in MDCK II cells. MDCK II cells were cultivated in MEM (Sigma-Aldrich GmbH) with 0,2 %385

bovine serum albumin (BSA), 1 % penicillin-streptomycin and 1 % L-glutamine at 37°C. All

386

cells were tested negative for Mycoplasma sp. by PCR. All infection experiments with SARS-

387

CoV-2 and H7N9 influenza A virus were performed in the biosafety level 3 (BSL-3) laboratory388

at the Heinrich Pette Institute, Leibniz Institute for Experimental Virology in Hamburg,

389

Germany.

SARS-CoV-2

strain(SARS-CoV-2/Germany/Hamburg/01/2020;

ENA

study

390
391

Animal experiments

392

Male and female golden hamsters (8-10 weeks old) were purchased from Janvier and male

393

C57BL/6 mice (8 weeks old) were purchased from Envigo. All animals were kept under394

standard housing conditions (21 ± 2 °C, 40–50% humidity, food and water ad libitum) with a395

12:12 light–dark cycle at the Heinrich Pette Institute, Leibniz Institute for Experimental

396

Virology in Hamburg, Germany. For SARS-CoV-2 infection, hamsters were anaesthetized with

397

150 mg/kg ketamine and 10 mg kg-1 xylazine by intraperitoneal injection. The animals were398

intranasally inoculated with 105 plaque forming units (p.f.u.) SARS-CoV-2, mock infected with

399

PBS or were administered with 1 mg kg-1 poly(I:C). Body weight was monitored daily up to 14

400days post infection (d p.i.). On day 1, 3 and 6 p.i., five animals per group were euthanized by401

intraperitoneal injection of an overdosis of pentobarbital. Blood was drawn by cardiac puncture402

and collected in EDTA tubes. Blood was centrifuged for 10 min at 2,000 g and 4 °C, and the403

plasma was stored at −80 °C. Lungs and gonads were homogenized in 1x PBS and stored at -404

80 °C. For histopathological examination, lungs and gonads were fixed by immersion in 10%405

neutral-buffered formalin and embedded in paraffin. For total RNA isolation, lungs and gonads

406

were incubated in RNAprotect Tissue Reagent (QIAGEN), for at least 24 hours at 4 °C and407

stored at –80 °C.
13

408

For influenza A virus infection, mice were anaesthetized with 100 mg kg-1 ketamine and 10 mg

409

kg-1 xylazine by intraperitoneal injection. The animals were intranasally inoculated with 105

410

p.f.u. H7N9 avian influenza A virus (A/Anhui/1/13), 104 p.f.u. pH1N1 influenza A virus

411(A/Hamburg/NY1580/09) or mock infected with PBS. Body weight was monitored daily. On

412

day 3 p.i., four or five animals per group were anesthetized with isoflurane and blood was drawn

413

by the retrobulbar route and collected in EDTA tubes. Blood was centrifuged for 10 min at

414

2,000 g and 4 °C, and the plasma was stored at -80 °C.

415

All animal experiments were performed in strict accordance with the guidelines of German

416

animal protection law and were approved by the relevant German authority (Behörde für417

Gesundheit und Verbraucherschutz; protocols N 32/2020 and 01/15).

418
419

Histology and immunhistochemistry

420

Tissues were routinely embedded in paraffin and evaluated via light microscopy of hematoxylin

421

and eosin (HE) stained slides. Immunohistochemical detection of SARS-CoV-2 nucleoprotein

422(NP) aromatase, cluster of differentiation (CD204), von-Willebrandt-factor (vWF), cluster of423

differentiation 31 (CD31), cluster of differentiation 3 (CD3) and smooth muscle actin (SMA)424

in the golden hamster lungs was performed using the EnVision+ System (Dako Agilent

425

Pathology Solutions). Serial sections of tissue were dewaxed and rehydrated in isopropanol and426

96% ethanol followed by blockage of endogenous peroxidase by incubation in 85% ethanol427with 0.5% H2O2 for 30 min at room temperature. Antigen retrieval was performed by incubation428

in citrate (10 mM citric acid, 0.05% Tween 20) or citrate-Na2H2EDTA buffer (10 mM citric429

acid, 2 mM Na2H2EDTA, 0.05% Tween 20) for 20 min in a microwave at 800 W, followed by430

20 min at room temperature. Sections were afterwards transferred to Shandon Coverplates™

431(Thermo Electron GmbH) and stained with either polyclonal antibodies directed against432

aromatase (Abcam, ab18995, 1:500), CD31 (Acris, AP15436PU, 1:100), CD204 (Abnova

433

Corporation, MAB1710, 1:500) and von-Willebrandt-Factor (Dako Agilent Pathology

434

Solutions, 20051017, 1:500) or monoclonal antibodies against smooth muscle actin (Dako435

Agilent Pathology Solutions, 20071964, 1:100), CD3 (Dako Agilent Pathology Solutions,

436

A0452, 1:100) and SARS-CoV-2 NP (Sino Biological, 40143-MM05, 1:32,000) over night at

437

4°C. Antibodies were diluted in PBS containing 1% BSA with and addition of 0,3% Triton X-

438

100 for the aromatase, smooth muscle actin and SARS-CoV-2 NP stainings. Sections were439

subsequently rinsed, and the peroxidase-labeled polymer was applied as secondary antibody for440

30 minutes. Visualization of the reaction was accomplished by incubation in chromogen 3,3-441

diaminobenzidine tetrahydrochloride (DAB, 0.05%) and 0.03% H2O2 in 1x PBS for 5 min and
14

442

afterwards counterstained with Mayer’s hematoxylin for 1 min. For negative controls, the443

primary antibody was replaced by either rabbit normal serum (1:3,000) or ascites fluid from444

Balb/c mice (1:1,000). The serial stainings were afterwards analyzed by light microscopy.

445
446

Aromatase immunoreactivity was scored semiquantitativley in the bronchiolar epithelium, the447

endothelium and the peribronchiolar as well as perivascular regions of the lungs. Signal

448

intensity in respective regions was very low (0,5), low (1), low to moderate (2), moderate (3),449

moderate to high (4), or high (5). Signal distribution was assessed as: focal/oligofocal (≤3 foci;4501), multifocal (>3 foci; 2) and diffuse (3). For better compararive interpretation a final score for451

each region and animal was calculated as product of signal intensity times signal distribution.

452
453

For immunohistochemical detection of aromatase in lungs of deceased Covid-19 patients lung454

tissue probes were fixed in 4% phosphate-buffered formaldehyde and embedded in paraffin.

455

Immunohistochemical analysis was performed by using the polyclonal antibody directed456

against aromatase (Abcam, ab18995, 1:400) on an automated immunostainer following the457

manufacturer's protocol (Benchmark; Ventana Medical Systems, Tucson, AZ) and using the458

ultraView detection system (Ventana) and diaminobenzidine as substrate. Tissue sections were459

counterstained with hematoxylin. For all deceased patients, informed consent had been obtained

460

from the next of kin 46.

461
462

Determination of viral titers and viral RNA levels

463

Homogenization of organs was performed in 1 ml 1x PBS with 5 sterile, stainless steel beads464(Ø 2 mm, Retsch) at 30 Hz for 10 min in the mixer mill MM400 (Retsch). The plaque assays

465

were performed on VeroE6 cell monolayers and stained with crystal violet after 72 hours. The

466

tissue homogenisates or plasma were titrated on VeroE6 cells in 10-fold serial dilutions for 30

467

min at 37°C and overlaid with MEM (Sigma-Aldrich) supplemented with 0,2 % BSA, 1% L-

468

glutamine, 1% penicillin-streptomycin, 1 µg ml-1 L-1-tosylamido-2-phenylethyl chloromethyl469

ketone (TPCK) treated trypsin (Sigma-Aldrich) and 1,25% Avicel. After 72 hours p.i., cells

470

were fixed with 4% paraformaldehyde and the plaques were visualized by crystal violet

471

staining.

472

Viral RNA was isolated from plasma or homogenized organs using the QIAamp Viral RNA

473

Mini Kit (QIAGEN) according to the manufacturer’s instructions. SARS-CoV-2 RNA levels

474

were then determined by quantitative reverse transcription real-time PCR (qRT-PCR) using the475

RealStar® SARS-CoV-2 RT-PCR Kit RUO (altona Diagnostics). An internal control provided
15

476

by the kit was used as a sample preparation control as well as an extended dry spin step for 10477

min at 17000g at room temperature.

478
479

Measurement of hormone and cytokine levels

480

Hormone levels were measured in homogenized testis and ovaries as well as in plasma samples

481

using a custom-made MILLIPLEX MAP Multi-Species Hormone Magnetic Bead Panel

482(Merck), analyzing testosterone, cortisol and progesterone according to the manufacurer’s

483

instructions in a Bio-Plex 200 System with high-throughput fluidics (HTF; Bio-Rad). Estradiol

484

levels (Calbiotech) in homogenized testis and ovaries as well as plasma samples were analyzed

485

by ELISA following the manufacturer’s instructions. ELISAs were measured on an Infinite M

486

Nano ELISA microplate reader (Tecan). Female hamsters in diestrus were excluded from487

estradiol anaylsis.

488

A panel of 13 cytokines and chemokines (eotaxin, macrophage inflammatory protein 1α and 1β

489(MIP-1α, -1β), interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-10

490(IL-10), interleukin-12 (IL-12(p70)), interleukin-13 (IL-13), interleukin-1β (IL-1β), monocyte491

chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), and vascular endothelial492

growth factor (VEGF)) was measured in homogenized lungs and plasma using a custom-made493

Bio-Plex Pro™ Mouse Cytokine multiplex (Bio-Rad) according to the manufacturer’s

494

instructions in a Bio-Plex 200 System with high-throughput fluidics (HTF; Bio-Rad).

495
496

Infection of Calu-3 cells with SARS-CoV-2

497

Calu-3 cells (ATCC) were seeded in 6-well plates. After 24 hours Calu-3 cells were infected498with SARS-CoV-2 or pH1N1 at a multiplicity of infection (MOI) of 1 in 500 µl DMEM with499

1% P/S, 1% L-glutamine and 2% FCS or DMEM with 1% penicillin-streptomycin, 1% L-

500

glutamine and 0.4% BSA, respectively, for 45 min at 37° C. After washing twice with 1x PBS,

501

2 ml of infection medium DMEM with 1 % penicillin-streptomycin, 1 % L-glutamine and 2 %502

FCS or DMEM with containing 1 % penicillin-streptomycin, 1 % L-glutamine, 0.1 % FCS and503

1 µg/ml TPCK-treated Trypsin (Sigma-Aldrich GmbH)504

incubated for 24 hours at 37 °C.

were added and the cells were

505
506

Determination of cytokine, receptor and protease expression levels by real-time507

quantitative PCR (RT-qPCR)

508

RNAprotect-fixed lungs were homogenized in 700 µl lysis buffer RL with 5 sterile, stainless509

steel beads (Ø 2 mm, Retch) at 30 Hz for 10 min in the mixer mill MM400 (Retsch). Calu-3
16

510

cells were lysed in 1 ml TRIZOL (Invitrogen) per well, incubated for 5 min at RT. The cell511

lysate were resuspended and transferred into a fresh tube. 250 µl chloroform were added to each

512

sample, vortexted for 30 sec and subsequently centrifuged for 15 min at 13,000 g. Total RNA

513

was isolated from homogenized tissues and the aqueous phase of the lysed Calu-3 cells using514

the innuPREP RNA Mini Kit 2.0 (Analytik Jena) according to the manufacturer’s instructions

515

with an additional DNase I treatment using the RNase-free DNase Set (QIAGEN). The RNA516

was eluted in RNase-free water and mixed with 1 U µl-1 RiboLock RNase inhibitor (Thermo

517

Fisher Scientific). The cDNA synthesis was performed using random nonamer primers (Gene518

Link, pd(N)9, final concentration: 5 μM), SuperScript III Reverse Transcriptase (Thermo Fisher

519

Scientific) according to the manufacturer´s instructions and 2 µg total RNA. The cDNA was

520

generated using the GeneAmp PCR System 9700 (Applied Biosystems; cycle: 25 °C for 5 min,521

50 °C for 60 min, 70 °C for 15 min, 4 °C hold). Reactions were set up with PCR grade Water

522(Roche) in LightCycler® 480 Multiwell Plate 96 Reaction Plates (Roche). Briefly, 2 µl of

523

cDNA template were added to 10 µl FastStart Essential DNA Green Master (Roche) and 300

524

nM of forward and reverse primer, respectively. RT-qPCR runs were conducted using

525

LightCycler® 96 Real-Time PCR System (Roche) with endpoint fluorescence detection: 10526

min at 95 °C and 45 amplification cycles (15 s at 95 °C, 10 s at 65 °C and 20 s at 72 °C).

527

Analysis was performed in duplicates for each gene. Negative controls and sample without

528

reverse transcriptase were included to detect contaminations. Relative expression values were529

determined using a modified e–ΔΔCt method. Ct values of each samples were normalized to Ct

530

values of the reference gene of the respective sample and the average of the ΔCt value (n = 2

531

technical replicates) was determined. Hprt1 was used as a reference gene for analyses of mRNA

532

expression levels in the lung and Ywhaz was used reference gene for analyses of mRNA

533

expression levels in the gonads. The relative 2-ΔCt expression values of the biological replicates534

are presented. The following primer sequences were used for RT-qPCR: Hprt1 (Hypoxanthin535

phosphoribosyltransferase 1) forward 5’-TCCCAGCGTCGTGATTAGTG-3’, reverse 5’-

536

GTGATGGCCTCCCATCTCTT-3’; Ywhaz forward 5’-AGCCTTTGTCTGCCTCACTC-3’,

537

reverse538

TCACCTCTGGTCTTCTGGACT-3’, reverse 5’-TCTGGACCCTTTACCTCTTGTT-3’ IL-

539

1rα

540

TAGCCGGCAATGAGTTGGTT-3’; Eotaxin forward 5’- AGAGAGCCTGAGACCAACAC-

541

3’,

542

ATGGAGGGGACCTCGTCTTT-3’, reverse 5’-GATGGCCTGGTTGTCCTTCA-3’; VEGF

543

forward 5’-CTCCACCATGCCAAGTGGTC-3’, reverse 5’-TCTCATCGGGGTACTCCTGG-

5’forward
reverseAAGTTAACTGGCTCTACTCCCC-3’,

IL-6

5’-ATGGAAATCTGCAGGGGACG-3’,
5’-AACTGGGATAGAGCCTGGGTG-3’;

IFN-γ

forwardreverseforward

5’-

5’5’-

17

544

3’;

ACE2

forward5’-CAGAGGGAGCAGATGGCTAC-3’,545

CCCACATGTCACCAAGCAAA-3’; ACE forward 5’-TCGCCCATCTTAACGGCTAC-3’;

546

reverse547

ACCACCACACTGATCACAGC-3’, reverse 5’-TAGAGGTCGATGTTGCCTGC-3’; DPP4

548

forward549

ACCCATGCCACATCACACAA-3’; AR forward 5’-GGCCTTACCTGGATTCCGC-3’,

550

reverse551

CCCTTGTACTGGGCTTCGTT-3’,

552

CYP19A1

553

TCTTTCAAGTCCTTGGCGGAT-3’;

554

ACCCGGGGAAACAGGATACA-3’, reverse 5’- GAACAGGGCAAACACTCCAC-3’;

555

endothelial

556

TGAGGTCTTCCGTCAAGTGC-3’; ribosomal protein L32 (RPL32; human) forward 5’-

557

GAAGTTCCTGGTCCACAACG-3’,

558

3’;.CYP19A1 aromatase (human) forward 5’-CGGCCTTGTTCGTATGGTCA-3’, reverse 5’-

559

CAGAAGGGTCAACACGTCCA-3’;.

5’-ATCCAGGTTGATGTGCTGGG-3’;

TMPRSS2

5’-AAGGCAGGAGCTGTGAATCC-3’,

5’-AGAGCATCCTGGAGTTGACG-3’;

aromataselipaseforwardforwardreverseZIP9reverseforwardreverse(Slc39a9)5’-5’-CTGCTCCCAAAGCTACACCA-3’;
reductase

35’-GAAACCCAGCGCAAACTGAC-3’,

reverse5’-

5’-

forward5’-ATGCGGCACATCATGCTGAA-3’,
5-α

5’-

reverseforwardreverse5’5’5’-

5’-GCGATCTCGGCACAGTAAG-

560
561

Proteomic analysis

562

Sample preparation. Lungs homogenates were incubated with 4% SDC, boiled 10 min at 98 °C,

563

and sonicated (4 °C for 15 min). Fifty micrograms of cleared protein lysates were564

reduced/alkylated (10 mM TCEP, 40 mM chloroacetamide) for 1h, and digested overnight with

565

LysC and Trypsin (1:50 w/w proteases:substrate ratio). Peptides were purified on stage tips

566

with two SDB-RPS Empore filter discs (3M, Maplewood, MN, USA) and resuspended in 0.3%

567

ACN/0.3%TFA to a final concentration of 250 ng/µl.

568

UHPLC and timsTOFpro settings. Samples were analyzed on a nanoElute (framework569

V1.1.0.63; Bruker, Germany) coupled to a timsTOF Pro (Bruker) equipped with a CaptiveSpray570

source. Peptides (250 ng) were first injected onto a trap column (Acclaim PepMap 100 C18,571

5mm X 300 μm, 5 μm particles; Thermo-Fisher, Germany) and next separated on a 25cm X

572

75μm analytical column, 1.6μm C18 beads with a packed emitter tip (Aurora Series with CSI,

573

IonOpticks, Australia). The column temperature was maintained at 50°C using an integrated

574

column oven. The column was equilibrated using 5 column volumes before loading sample in

575

100% buffer A (97.9% LC-MS grade water, 2% ACN, 0.1% FA). Samples were separated at576

400nl/min using a linear gradient from 2% to 25% buffer B (99.9% ACN, 0.1% FA) over 90min

577

before ramping to 37% (10min), and 95% of buffer B (10min) and sustained for 10min (total
18

578

separation method time 120min). The timsTOF Pro (Bruker) was operated in positive ion data579

dependent acquisition PASEF mode using Compass Hystar 5.1.8.1. Settings as follows: Mass

580

Range 100 to 1700m/z, 1/K0 Start 0.6 V.s/cm2 End 1.6 V./cm2, Ramp time 100ms, Lock Duty

581

Cycle to 100%, Capillary Voltage 1500V, Dry Gas 3 l/min, Dry Temp 180°C, PASEF settings:

582

10 MS/MS scans (total cycle time 1.16sec), charge range 0-5, active exclusion for 0.4 min,583

Scheduling Target intensity 20000, Intensity threshold 2500, variable CID collision energy 20-584

59 eV.

585

Raw data processing and analysis. Raw MS data were processed with the MaxQuant software

586

package, version 1.6.17 using the built-in Andromeda search engine and the label-free

587

quantitation algorithm as described previously47. Additionally, the intensity-based absolute

588

quantification (iBAQ) algorithm and Match Between Runs option were used. Data were589

searched against the Mesocricetus auratus proteome (UniprotKB, release 2020_06) containing

590

forward and reverse (decoy) sequences concatenated with the SARS-CoV-2 polyprotein with

591

the

592

carbamidomethylation was set as fixed and methionine oxidation and N-acetylation as variable

593

modifications. Search peptide tolerance was set at 70ppm and main search set at 30ppm (other594

settings left as default). Experiment type was set as TIMS-DDA with no modification to default

595

settings. Search results were filtered with a false discovery rate (FDR) of 0.01 for peptide and596

protein identifications. The Perseus software package, version 1.6.10.4 was used to further

597

process the data. Protein tables were filtered to eliminate the identifications from the reverse

598

decoy database and common contaminants. In analyzing MS data, only proteins identified on599

the basis of at least one peptide and a minimum of three quantitation events in at least one

600

experimental group were considered. IBAQ protein intensity values were normalized against

601

the median intensity of each sample (using only peptides with recorded intensity values across602

all samples and biological replicas), log-transformed and missing values filled by imputation

603

with random numbers drawn from a normal distribution calculated for each sample.

604

Significantly regulated proteins were determined by multiple equal variance t-tests with605

permutation-based false discovery rate statistics. We performed 250 permutations and the FDR

606

threshold was set at 0.05. The parameter S0 was set at 0.1 to separate background from607

specifically enriched interactors. Results were plotted as Volcano plot and heat map using the608

Perseus software package 48.

individual

viral

open

reading

framesannotatedmanually.

In

MaxQuant,

609
610
611
19

612

Data availability

613

The mass-spectrometry-based proteomics will be uploaded in public repositories. Unique614

dataset identifiers are pending.

615
616

Data analysis

617

All data were analyzed with the Prism software (GraphPad, 8.4.3) using Kruskal-Wallis one-618

way analysis of variance (ANOVA) followed by Dunn’s multiple comparisons test or unpaired

619

two-tailed Student’s t-test as indicated in the respective legends. Statistical significance was620

defined as p < 0.05 (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). Additionally, ELISA data were621

evaluated using the “Four Parameter Logistic Curve” online data analysis tool, MyAssays Ltd.,622

25 th October 2012, http://www.myassays.com/four-parameter-logistic-curve.assay. Detection

623

of outliers were performed using Grubbs’ test in GraphPad QuickCalcs Outlier Calculator.

624
625

20

626

Author contributions

627

GG designed the study and wrote the manuscript. SSB and BS performed all animal

628

experiments, analyzed the data and revised the manuscript. NMK, SB, MZ, TB, HJ, ZM, VPdR

629

and VKB assisted with the animal experiments and performed molecular analysis. GB, KB and630

WB performed histopathological analysis of the animal organs. AK, CU and PS performed

631

proteomic experiments and analysis. MS, DJ, AN and SK provided clinical samples from

632

Covid-19 patients for SARS-CoV-2 virus isolation. KK analyzed the lungs of fatal Covid-19

633

cases. All authors approved the manuscript. We thank Martin Müller for technical assistance.

634

We thank staff of the Core Facility Small Animal Models of the Heinrich Pette Institute, Leibniz

635

Institute of Experimental Virology, particularly Ursula Müller and Oliver Strauch for their636

excellent support with the golden hamster model.

637
638

Acknowledgment

639

This study was supported by a rapid response grant from the federal ministry of health (BMG)640

to G.G. Further support was provided by a BMBF (Federal Ministry of Education and Research)

641

project entitled RAPID (Risk assessment in re-pandemic respiratory infectious diseases),

642

01KI1723G and by the Ministry of Science and Culture of Lower Saxony in Germany (14 -643

76103-184 CORONA-15/20) to W.B.. KK receives funding of the Ministry of Science,

644

Research and the Arts Baden-Württemberg and the Deutsche Herzstiftung for research on

645

SARS-CoV-2. AK acknowledges funding through Alexander von Humboldt foundation

646

through a postdoctoral fellowship. AK and CU are furthermore supported through a rapid

647

response grant from the federal ministry of health (BMG) and MS SPIDOC within the European

648

Union’s Horizon 2020 research and innovation program under grant agreement No. 801406.

649
650

Competing interest statement651

The authors declare no competing interests.

21

652

Figure legends

653
654

Figure 1. SARS-CoV-2 pathogenesis in male and female golden hamsters. a, b, Weight loss655

of male (a) and female (b) golden hamsters infected with SARS-CoV-2 (105 p.f.u. ml-1; male,656

n = 9; female n = 10), Poly(I:C) (n = 9) or PBS (n = 10). Statistical significance was assessed657

by Kruskal-Wallis one-way ANOVA (*p < 0.05, **p<0.01, ***p<0.001). c, Area under the658

curve (AUC) for body weight of SARS-CoV-2 infected male and female golden hamsters. d,659

Viral lung titers of SARS-CoV-2 infected golden hamsters (3d p.i., n = 5). e, Haematoxylin and660

eosin (HE) and SARS-CoV-2 nucleoprotein (NP) staining in lungs of infected golden hamsters

661(3 dpi; representative pictures are shown, n = 5). f, Viral plasma titers of SARS-CoV-2 infected662

golden hamsters (3d p.i., n = 5). g, h, Viral titers (g) and viral RNA levels (h) in testis of male663

golden hamsters (3d p.i., n = 5). i, HE staining of testis from male golden hamsters (3d p.i., n =6645). j, k, Viral titers (j) and viral RNA levels (k) in ovary of female golden hamsters (3d p.i., n665= 5). l, HE staining of ovary from female golden hamsters (3d p.i., n = 5). m, n, Viral titers (m)666

and viral RNA levels (n) in uteri of female golden hamsters (3d p.i., n = 5). o, HE staining in

667

uteri of female Syrian golden hamsters (3d p.i., n = 5). a-d,f,g,h,j,k,m,n, Values are shown as

668

means, error bars as SD.

669
670

Figure 2. Immune response in the lungs of SARS-CoV-2 infected male and female golden

671

hamsters. a, IL-6 mRNA expression levels (n = 5; male Poly(I:C): n = 4). b, IL-1ra mRNA

672

expression levels (n = 5; male Poly(I:C): n = 4). c, d, Eotaxin mRNA (c) and protein (d)673expression levels (n = 5; male Poly(I:C): n = 4). e, MIP-1α protein expression levels (n = 5). f,

674

IFN-γ mRNA expression levels (n = 5; male Poly(I:C): n = 4). g, VEGF protein expression

675

levels (n = 4; SARS-CoV-2: n = 3). h, IL-10 protein expression levels (n = 5). i, IL-12 protein

676

expression levels (n = 5). j, IL-1β protein expression levels (n = 5). k, IL-2 protein expression

677

levels (n = 5). l, IL-13 protein expression levels (n = 5). m, MIP-1b protein expression levels

678(n = 5). n, MCP-1 protein expression levels (n = 5). o, TNF-α protein expression levels (n = 5).

679

Cytokine and chemokine levels were measured in lung homogenates at 3 d p.i.. Values are

680

shown as means; error bars as SD; detection limits are indicated as dotted lines. Statistical

681

significance was assessed by Kruskal-Wallis one-way ANOVA and by unpaired Student’s t-

682

test (*p < 0.05, **p<0.01).

683
684

Figure 3. Sex hormones and SARS-CoV-2 entry receptors in infected male and female

685

golden hamsters. a, b, Testosterone levels in plasma of male (a) and female (b) golden
22

686

hamsters. c, d, Estradiol levels in plasma of male (c) and female (d) golden hamsters. e, f,

687

Cortisol levels in plasma of male (e) and female (f) golden hamsters. g, h, Progesterone levels

688

in plasma of male (g) and female (h) golden hamsters. Values are shown as means; error bars

689

are shown as SD (n = 5; estradiol male3d p.i.: n = 10; estradiol male 14d p.i. SARS-CoV-2: n690= 4; estradiol female 3d p.i. Poly(I:C) and SARS-CoV-2: each n = 8). i-k, ACE2 mRNA

691

expression levels at day 1 (i), 3 (j) and 6 (k) p.i. (n = 5; male Poly(I:C) 1d p.i.: n = 4; male and692

female Poly(I:C) 3d p.i.: n = 4) in the lungs of infected golden hamsters. l, ACE mRNA

693

expression levels (n = 5; male Poly(I:C): n = 4) in the lungs of infected golden hamsters. m, n,694

ACE2 mRNA expression levels in testis of male (m) and ovary of female (n) golden hamsters

695

at day 3 p.i. (n = 5; male SARS-CoV-2: n = 4). o, DPP4 mRNA expression levels (n = 5; male696

Poly(I:C): n = 4) in the lungs of infected golden hamsters. p, q, TMPRSS2 mRNA expression

697

levels at day 3 (p) and 6 (q) p.i. in the lungs of infected golden hamsters. r, AR mRNA

698

expression levels in the lungs of infected golden hamsters (n = 5; Poly(I:C): n = 4). s, ZIP9699mRNA expression levels in the lungs of infected golden hamsters (n = 5; male Poly(I:C): n =7004). The mRNA expression levels are presented as negative exponents of ∆ct values; values are

701

shown as means; error bars are shown as SD. Statistical significance was assessed by Kruskal-

702

Wallis one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001).

703
704

Figure 4. Proteomic analysis of the lungs of SARS-CoV-2 infected male and female golden

705

hamsters. a, Volcano plot displaying host proteins up- or down-regulated in male versus female706

golden hamsters upon SARS-CoV-2 infection (orange and green dots, respectively).

707

Significantly-regulated proteins are separated from background proteins by a hyperbolic curve

708(dotted grey line). b, Heatmap of iBAQ intensities for significantly regulated proteins

709(Student’s t-test, FDR 0.05, p < 0.05) in the total proteome across experimental groups and710

biological replicates.

711
712

Figure 5. CYP19A1 and endothelial activation in the lungs of SARS-CoV-2 infected male713

and female golden hamsters. a, Schematic overview of binding sites for transcription factors

714

in the four promoter regions (P1, P2, P3, and P4) of the aromatase gene CYP19A1. b-d,

715

Aromatase mRNA expression levels at day 1 (b), 3 (c) and 6 (d) p.i. (n = 5; male Poly(I:C): n716= 4). e, 5α-Reductase mRNA expression levels (n = 5; male Poly(I:C): n = 4). f, g, Aromatase

717mRNA expression levels in testis of male (f) and ovary of female (g) golden hamsters at 3d p.i.

718

(n = 5; male SARS-CoV-2: n = 4). h, i, Endothelial lipase mRNA expression levels at day 3 (h)719

and 6 (i) p.i. (n = 5; male Poly(I:C) 3d p.i.: n = 4). The mRNA expression levels are presented
23

720

as negative exponents of ct values; values are shown as means; error bars are shown as SD,

721

detection limits are indicated as dotted lines. Statistical significance was assessed by Kruskal-

722

Wallis one-way ANOVA (*p < 0.05, **p < 0.01).

723
724

Figure 6. CYP19A1 protein expression in the lungs of SARS-CoV-2 infected male and725

female golden hamsters. a, b, Immunohistochemistry of serial sections from lungs of male (a)726

and female (b) golden hamsters for aromatase, SARS-CoV-2 NP, macrophages (CD204),727

endothelial cells (vWF and CD31), T cells (CD3) and smooth muscle (SMA). Bronchus (B),728

vessel (V), alveola (A), stars indicate perivascular connective tissue and arrowheads indicate

729

macrophages. Representative pictures are shown. c-f Semiquantitative scoring of the of730

aromatase CYP19A1 immunoreactivity in different lung regions of male and female golden731

hamsters at day 3 p.i. (n = 5). Immunoreactivitiy was examined by light microscopy in the732

endothelium (c), the bronchiolar epithelium (d), the perivascular (e) and peribronchiolar region

733(f) in the lungs of infected hamsters. Signal intensity in respective regions was very low (0,5),734

low (1), low to moderate (2), moderate (3), moderate to high (4), or high (5). Signal distribution735

was assessed as: focal/oligofocal (≤3 foci; 1), multifocal (>3 foci; 2) and diffuse (3). For better

736

comparative interpretation a final score for each region and animal was calculated as product737

of signal intensity times signal distribution. Statistical significance was assessed by Kruskal-

738

Wallis one-way ANOVA and by unpaired Student’s t-test (*p < 0.05, **p<0.01).

739
740

Figure 7. CYP19A1 protein expression in the lungs of fatal Covid-19 cases.

741

Immunohistochemistry of lungs from male (a-d) and female (f-h) patients with Covid-19 or742

controls (e; representative picture of n = 3 are shown) stained for aromatase. The intensity of743

staining was significantly higher in endothelial cells and especially in macrophages in male (a-744d) compared to female (f-h) Covid-19 patients and control patients (e). Stars indicate

745

endothelial cells and arrowheads indicate macrophages. Scale bar, 100 µm.

746
747

Extended Data Figure 1. Viral load in SARS-CoV-2 infected male and female golden

748

hamsters. a, Viral lung titers of SARS-CoV-2 infected golden hamsters at 6d p.i. (n = 5). b,

749

Haematoxylin and eosin (HE) and SARS-CoV-2 nucleoprotein (NP) staining in lungs of750

infected golden hamsters at 6d p.i. (representative pictures are shown, n = 5).

751
752

Extended Data Figure 2. Immune response in the lungs of SARS-CoV-2 infected male and753

female golden hamsters. a-k, Cytokine and chemokine expression levels at day 1 p.i. for
24

754

eotaxin (a), MIP-1α (b), IFN-γ (c), VEGF (d), IL-10 (e), IL-12 (f), IL-1β (g), IL-2 (h), IL-13755(i), MIP-1β (j) and TNF-α (k). l-s, Cytokine and chemokine expression levels at day 6 p.i. for756

eotaxin (l), MIP-1α (m), IFN-γ (n), VEGF (o), IL-12 (p), IL-2 (q), MIP-1β (r) and TNF-α (s).

757

Cytokine and chemokine levels were measured in lung homogenisates (n = 5). Values are

758

shown as means; error bars as SD; detection limits are indicated as dotted lines. Statistical759

significance was assessed by Kruskal-Wallis one-way ANOVA and by unpaired Student’s t-

760

test (*p < 0.05, **p<0.01).

761
762

Extended Data Figure 3. Immune response in the plasma of SARS-CoV-2 infected male763

and female golden hamsters. a-k, Cytokine and chemokine expression levels at day 1 p.i. for764

eotaxin (a), MIP-1α (b), IFN-γ (c), VEGF (d), IL-10 (e), IL-12 (f), IL-1β (g), IL-2 (h), IL-13765(i), MCP-1 (j) and TNF-α (k). l-s, Cytokine and chemokine expression levels at day 3 p.i. for766

eotaxin (l), MIP-1α (m), VEGF (n), IL-1β (o), IL-2 (p), IL-13 (q), MCP-1 (r) and TNF-α (s).

767

t-z2, Cytokine and chemokine expression levels at day 6 p.i. for eotaxin (t), MIP-1α (u), IFN-

768

γ (v), VEGF (w), IL-1β (x), IL-2 (y), MCP-1 (z1) and TNF-α (z2).Cytokine and chemokine

769

levels were measured in plasma (n = 5). Values are shown as means; error bars as SD; detection

770

limits are indicated as dotted lines. Statistical significance was assessed by Kruskal-Wallis one-771

way ANOVA and by unpaired Student’s t-test (*p < 0.05).

772
773

Extended Data Figure 4. Testosterone and estradiol levels in influenza A virus infected774

male mice. a, b, Testosterone (a) and estradiol (b) levels in plasma of H7N9 influenza A virus

775

infected male mice. c, d, Testosterone (a) and estradiol (b) levels in plasma of pH1N1 influenza

776

A virus infected male mice. Values are shown as means; error bars are shown as SD (n = 5;

777

testosterone pH1N1: n = 4). Statistical significance was assessed by unpaired Student’s t-test

778(**p < 0.01).

779
780

Extended Data Figure 5. Proteomic profile in the lungs of SARS-CoV-2 infected male781

golden hamsters. Heatmap of iBAQ intensities for significantly regulated proteins in SARS-

782

CoV-2 infected male golden hamsters compared to PBS control group (Student’s t-test, FDR

783

0.05, p < 0.05) in the total proteome across experimental groups and biological replicates.

784

Female and male animals are indicated in light pink and blue, respectively. Each column

785

represent an individual hamster. Gray color represents missing values (not determined (ND)).

786

25

787

Extended Data Figure 6. Proteomic profile in the lungs of SARS-CoV-2 infected female

788

golden hamsters. Heatmap of iBAQ intensities for significantly regulated proteins in SARS-

789

CoV-2 infected male hamsters compared to PBS control group (Student’s t-test, FDR 0.05, p <7900.05) in the total proteome across experimental groups and biological replicates. Female and791

male animals are indicated in light pink and blue, respectively. Each column represent an

792

individual hamster. Gray color represents missing values (not determined (ND)).

793
794

Extended Data Figure 7. CYP19A1 protein expression in the lungs of SARS-CoV-2

795

infected male and female golden hamsters. a, b, Immunohistochemistry of serial sections

796

from lungs of male (a) and female (b) golden hamsters for aromatase, SARS-CoV-2 NP,

797

macrophages (CD204), endothelial cells (vWF and CD31), T cells (CD3) and smooth muscle

798(SMA). Inlets from these overview pictures are shown in Figure 6.

799
800

Extended Data Figure 8. Hormone levels in the gonads of SARS-CoV-2 infected male and801

female golden hamsters.

802

a, b, Testosterone levels in testis of male (a) and ovary of female (b) golden hamsters. c, d,803

Estradiol levels in testis of male (c) and ovary of female (d) golden hamsters. e, f, Cortisol

804levels in testis of male (e) and ovary of female (f) golden hamsters. g, h, Progesterone levels in

805

testis of male (g) and ovary of female (h) golden hamsters. Values are shown as means; error

806

bars are shown as SD (n = 5; male SARS-CoV-2 14d p.i.: n = 4; estradiol female PBS and807

Poly(I:C): each n = 4). Statistical significance was assessed by Kruskal-Wallis one-way

808

ANOVA (*p < 0.05, **p < 0.01).

809
810

Extended Data Figure 9. Model of CYP19A mediated lung pathology in SARS-CoV-2

811

infection. We propose that the lung plays a key role in the aromatization of testosterone-to-812

estradiol. Since CYP19A1 is expressed to higher levels in males than females, the conversion

813

of testosterone-to-estradiol is enhanced in males. Elevated estradiol levels may fulfill their814

endocrine and paracrine functions. Since most immune cells, e.g. macrophages possess815

androgen and estrogen receptors their activation will be dependent on the availability of the816

respective sex hormone. Particularly estradiol plays an important role in endothelial

817

inflammation. Thus, males might be more prone to CYP19A1 driven endothelial inflammation.

818

26

819

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942

29

150

**

50

0.05

e

SARS-CoV-2

4.5

Poly(I:C)

*

1.5
1.0

2 -ΔCt

600
400

100

SARS-CoV-2

PBS

hPoly(I:C)

SARS-CoV-2

IL-10
25

*

20

1000

0.5

150

500

15
10
5

PBS

i

Poly(I:C)

0.0

SARS-CoV-2

PBS

j

IL-12

Poly(I:C)

SARS-CoV-2

PBS

k

IL-1β

300

8

ng ml -1

100

4
2

0

0
PBS

m

Poly(I:C)

SARS-CoV-2

Poly(I:C)ng ml -1

1500

ng ml -1

300

100
Poly(I:C)

SARS-CoV-2

PBS

SARS-CoV-2

Poly(I:C)

SARS-CoV-2

Male
Female

1000
500

0
PBS

Poly(I:C)

TNF-α

150

200

60

0
PBS

2000

0

IL-13

30

o

MCP-1

SARS-CoV-2

90

50

SARS-CoV-2

Poly(I:C)

120

100

400

50

l

IL-2

200

100

PBS

SARS-CoV-2

0
PBS

n

MIP-1βPoly(I:C)150

6

ng ml -1

*

200

0

0

ng ml -1

0

ng ml -1

Poly(I:C)

VEGF
*
**

1500

*

*

0
PBS

g

IFN-γ
*

*

50

SARS-CoV-2

200

ng ml -1

2

0
PBS

f

MIP-1α****800

ng ml -1

Poly(I:C)

*

200

1

0
PBS

*

ng ml -1

0.00

Eotaxin
250

3

100

**

0.10

*

4

**
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2 -ΔCt2 -ΔCt0.15

dEotaxinng ml -1

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PBS

Poly(I:C)

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Figure 2

4

4

3

2

pg ml -1

4

ng ml -1

3
2

4

6

8

10

12

2

14

4

6

8

d p.i.

e

f

4

6

8

10

12

14

ng ml -1

20

*
2

g

6

15

40

10

*

20

8

10

12

14

d p.i.

hPlasma (Progesterone)

60

*

4

d p.i.

Plasma (Progesterone)
25

*

40

9

0
2

14

Plasma (Cortisol)

60

20

5

15

*

10
5

0

0
4

6

8

10

12

14

0
2

4

6

d p.i.

i

8

10

12

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2

4

d p.i.

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j

0.20

ACE2 (3d p.i.)
**

0.15

0.15

*
2 -ΔCt

10

12

14

2

Poly(I:C)m

0.00

SARS-CoV-2

ACE2 (Testis)PBSnPoly(I:C)

ACE2 (6d p.i.)l

ACE2 (Ovary)

o

-02

15

-ΔCt

3x10

2

2x10 -03

2 -ΔCt-03

3x10

2x10 -02

00

0

TMPRSS2 (6d p.i.)r

0.5

2 -ΔCt2 -ΔCt*0.0
Poly(I:C)

)

V-2

*
*

1.0

0.0
Poly(I:C)PBS

SARS-CoV-2

Poly(I:C)

SARS-CoV-2

-Co

(I:C

**

SARS-CoV-2

ZIP9 (3d p.i.)

2.0

10

1.5

8

1.0
0.5

PBS

SARS-CoV-2

TMPRSS2 (3d p.i.)
2.0*

Poly(I:C)

0.5

s

AR (3d p.i.)

*
*

1.0

p
1.5

PBS

2 -ΔCt

*

PBS

SA

Po

Po
ly

PB
S

ly(
SA I:C)
RS
-Co
V-2

5

PB
S

1x10 -02

1.5

**

0

SARS-CoV-210

1x10 -03q

Poly(I:C)

DPP4 (3d p.i.)
20

40
20

PBS

4x10 -02

14

ACE (3d p.i.)

**
**

0.10

4x10 -03

12

60

0.00

SARS-CoV-2

10

PBS
Poly(I:C)
FemaleSARS-CoV-2

2 -ΔCtPBS

8PBS
Poly(I:C)
Male
SARS-CoV-2

0.05

0.00

6

d p.i.

0.05

0.05

4

d p.i.

0.15

2 -ΔCt2 -ΔCt

8

0.20

0.10

0.10

k

6

2 -ΔCt2

2 -ΔCt12

d p.i.

Plasma (Cortisol)
** *

80

10

ng ml -1

2

2

0

0

0

***

1

1

*

Plasma (Estradiol)
40
20
18

RS

ng ml -1

5

3dPlasma (Estradiol)

51

ng ml -1c

Plasma (Testosterone)pg ml -1b

Plasma (Testosterone)ng ml -1a

MaleFemale

6
4
2

0.0

0
PBS

Poly(I:C)

SARS-CoV-2

PBS

Poly(I:C)

SARS-CoV-2

Figure 3

4.5

a
28 kDaGolgi SNARE protein

Serum amyloid
A-5 protein

0

0.5

1

p-value [Welch’s t-test; -Log 10]
1.5 2
2.5 3
3.5

4

Ig-like domaincontaining protein

SARS-CoV-2
Nucleoprotein

-8

b

PBS

-6

-2
0
2
Fold change [Log 2 ][Male: SARS-CoV-2/Female: SARS-CoV-2]

Poly(I:C) SARS-CoV-2

-4

PBS

4

Poly(I:C) SARS-CoV-2

Serum amyloid A-5 protein
28 kDa Golgi SNARE protein
SARS-CoV-2 Nucleoprotein
Ig-like domain-containing protein
9

11

13

15

17

ND

iBAQ Intensities [Log 2 ]Figure 4400
200
0

0

e

Poly(I:C)f

IL-10 (1d p.i.)ng ml -1

100

4

SARS-CoV-2

PBS

j

IL-13 (1d p.i.)

150

ng ml -1

80

40

m

Poly(I:C)

800

n

400
200

Poly(I:C)q

Poly(I:C)

500

PBS

o

IFN-γ (6d p.i.)

2000

20

1500

15
10

2

Poly(I:C)

Eotaxin (6d p.i.)
**
*

150
100

150
100

PBS

p

Poly(I:C)

SARS-CoV-2

IL-12 (6d p.i.)

**

100
50
0

PBS

SARS-CoV-2

MIP-1β (6d p.i.)
*
*

200

ng ml -1

*

3

**

0

250

4

SARS-CoV-2

150

500

r

*

Poly(I:C)0

SARS-CoV-2

1000

PBS

5

Poly(I:C)

VEGF (6d p.i.)

25

SARS-CoV-2

IL-2 (6d p.i.)

l
250

1000

SARS-CoV-2

0
PBS

PBS

SARS-CoV-2

200

5

0

0
Poly(I:C)50
PBS

ng ml -1

600

4
2

0

SARS-CoV-2

MIP-1α (6d p.i.)**2

TNF-α (1d p.i.)

0
PBS

SARS-CoV-2

6

k

**

Poly(I:C)

IL-2 (1d p.i.)

*

PBS

50

0

h

1500

100

20

PBS

8

ng ml -1

ng ml -1

200

0

SARS-CoV-2

0

SARS-CoV-2

MIP-1β (1d p.i.)

100

60

Poly(I:C)ng ml -1iPoly(I:C)

Poly(I:C)

IL-1β (1d p.i.)
6

0
PBS

ng ml -1

PBS

150

0

1000
500

g

50

5

10

SARS-CoV-2s

Poly(I:C)

SARS-CoV-2

TNF-α (6d p.i.)*

1500

ng ml -1

ng ml -1

15
10

Poly(I:C)

IL-12 (1d p.i.)

20

1500

20

0
PBS

SARS-CoV-2

ng ml -1

PBS

ng ml -1**

VEGF (1d p.i.)
2000

ng ml -1

50

30

ng ml -1

100dng ml -1

600

IFN-γ (1d p.i)ng ml -1

150c

MIP-1α (1d p.i.)****ng ml -1b

Eotaxin (1d p.i.)ng ml -1

ng ml -1aPBS

Poly(I:C)

SARS-CoV-2

Male
Female

1000
500

50

1

0

0
PBS

Poly(I:C)

SARS-CoV-2

PBS

Poly(I:C)

SARS-CoV-20
PBS

Poly(I:C)

SARS-CoV-2

Extended Data Figure 2

b

MIP-1α (1d p.i.)100IL-10 (1d p.i.)f

SARS-CoV-2

IL-12 (1d p.i.)

PBS

g

40

300

80

0

i

Poly(I:C)

SARS-CoV-2

IL-13 (1d p.i.)jPoly(I:C)

MCP-1 (1d p.i.)kng ml -1

ng ml -1m

Poly(I:C)0

SARS-CoV-2

MIP-1α (3d p.i.)PBSnPoly(I:C)

50
0

PBS

q

Poly(I:C)PBS

SARS-CoV-2

IL-13 (3d p.i.)rPoly(I:C)

500

v

40

ng ml -1

20

IFN-γ (6d p.i.)20

z1

Poly(I:C)

100

SARS-CoV-2

MCP-1 (6d p.i.)

PBS

Poly(I:C)

SARS-CoV-2

z2

SARS-CoV-2

IL-1β (6d p.i.)

60
40

Poly(I:C)

SARS-CoV-20
PBS

Poly(I:C)

SARS-CoV-2

TNF-α (6d p.i.)

1500

1000

1000

500

Poly(I:C)

20
PBS

1500

Male
Female

500
0

0

0

x
80

PBS

ng ml -1

40

PBS

SARS-CoV-2

150

SARS-CoV-2

Eotaxin (6d p.i.)*0Poly(I:C)

200

100

SARS-CoV-2

100

100

150

Poly(I:C)

150

200

0

60

t

250

0

80

PBS

50

VEGF (6d p.i.)0IL-2 (6d p.i.)

0

200

w

50

y

20

250

PBS

50
Poly(I:C)30

SARS-CoV-2

0

SARS-CoV-2

10
PBS

ng ml -1

Poly(I:C)ng ml -1

ng ml -1

30

Poly(I:C)

500
PBS

SARS-CoV-2

10

1000

ng ml -1

MIP-1α (6d p.i.)4040Poly(I:C)

IL-2 (3d p.i.)

1500

0

SARS-CoV-2

p60

ng ml -1

ng ml -1

ng ml -1uPoly(I:C)

PBS

50

TNF-α (3d p.i.)

100
PBS

0

SARS-CoV-2

2000

1000

0

Poly(I:C)

80

s

400

200

100

PBS

1500

300

200

0

SARS-CoV-2

MCP-1 (3d p.i.)

500

Eotaxin (3d p.i.)

20

5
0

500

IL-1β (3d p.i.)ng ml -1

ng ml -1

ng ml -1

100

SARS-CoV-2

300

o

20

10

l

Poly(I:C)

400

PBS

150

15

PBS

0

SARS-CoV-2

VEGF (3d p.i.)

25

10

SARS-CoV-2

1000

200
PBS

20

1500

400

0

Poly(I:C)

TNF-α (1d p.i.)

600

100

IL-2 (1d p.i.)

0
PBS

SARS-CoV-2

800

200

SARS-CoV-2

30

0
PBS

300

Poly(I:C)

20

0
PBS

h

40

100

10

PBS

SARS-CoV-2

60

ng ml -1

ng ml -1

100

200

Poly(I:C)

IL-1β (1d p.i.)

400

20

0

0
Poly(I:C)

50

30

20

50
PBS

SARS-CoV-2

40

ng ml -1

Poly(I:C)ng ml -1

60

150

ng ml -1eng ml -1

200

0
PBS

ng ml -1

30

10

0

VEGF (1d p.i.)
80

20

100

d

ng ml -1

ng ml -1

ng ml -1

200

IFN-γ (1d p.i)
250

ng ml -1

*

300

c

40

ng ml -1

Eotaxin (1d p.i.)
400

ng ml -1aPBS

Poly(I:C)

SARS-CoV-2

PBS

Poly(I:C)

SARS-CoV-2

Extended Data Figure 3

aTestosterone

b

3

**

8

2

pg ml -1

ng ml -1

Estradiol
10

1

6
4
2
0

0
PBS

c

H7N9

Testosterone

PBS

d

H7N9

Estradiol
615

pg ml -1

ng ml -1

**
10

4

2

5

0

0
PBS

pH1N1

PBS

pH1N1

Extended Data Figure 4

PBS

Poly(I:C) SARS-CoV-2

PBS

Poly(I:C) SARS-CoV-2
Profilin (A0A1U7QJS1)
Protein S100 (A0A1U7R3M2)
Hemopexin (A0A1U7RF88)
Calgranulin-B (A0A1U7QW31)
GB1/RHD3-type G domain-containing protein (A0A3Q0D2X1)
MPN domain-containing protein (A0A1U7QEA5)
Molybdopterin synthase sulfur carrier subunit (A0A3Q0DAL0)COMM domain-containing protein 5 (A0A1U8CMH0)
Barrier-to-autointegration factor (A0A1U8C000)
Mid1-interacting protein 1 (A0A1U7QTL4)
Arrestin_C domain-containing protein (A0A1U8C875)
1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase (A0A1U7QYX8)
Succinate--CoA ligase [ADP-forming] subunit beta, mitochondrial (P86226)
Serine/arginine-rich splicing factor 2 (A0A1U7QAY0)
Myeloid-derived growth factor (A0A1U7QXU0)
Cytochrome c oxidase subunit (A0A1U7QYK6)
Caspase-6 (A0A1U7QNN7)Isoleucyl-tRNA synthetase (A0A3Q0DAV4)
Palmdelphin (A0A3Q0D2V8)
PDZ domain-containing protein (A0A1U7R1B2)
J domain-containing protein (A0A1U7R2H2)
rRNA/tRNA 2-O-methyltransferase fibrillarin-like protein 1 (A0A1U7QUH3)
FYN-binding protein isoform X1 (A0A3Q0CZ30)
Apoptosis-associated speck-like protein containing a CARD (A0A1U7QED4)
Chromatin complexes subunit BAP18 isoform X1 (A0A1U8BVE2)
Phosphatase and actin regulator (A0A1U7QS32)
IF rod domain-containing protein (A0A1U7RC15)
Peptidylprolyl isomerase (A0A1U8CT94)
28 kDa Golgi SNARE protein (A0A3Q0CFS4)
Spondin domain-containing protein (A0A3Q0CQ72)
Phosphatidylinositol-3,5-bisphosphate 3-phosphatase (A0A1U7QYI5)
RNA helicase (A0A1U7QZK9)
Ras-like protein family member 12 (A0A1U7QMZ5)
ANK_REP_REGION domain-containing protein (A0A3Q0D0G9)
Endo/exonuclease/phosphatase domain-containing protein (A0A1U7QF81)
Coiled-coil domain-containing protein 6 (A0A1U7Q6G6)Interferon-induced protein 44 (A0A1U8CGR8)
SAP domain-containing protein (A0A3Q0DA41)
PKS_ER domain-containing protein (A0A1U7QG90)
Alkaline phosphatase (W6E8V7)
VWFA domain-containing protein (A0A1U7Q573)
Non-specific serine/threonine protein kinase (A0A3Q0CIV2)
Serum amyloid A-5 protein (P81491)
SEC7 domain-containing protein (A0A1U7Q7K7)
Peptidase S1 domain-containing protein (A0A1U7QV66)
SAND domain-containing protein (A0A3Q0DBM1)
HSR domain-containing protein (A0A3Q0DAZ4)
Phospholipase B-like (A0A1U7QFP3)SERPIN domain-containing protein (Q60552)
CD177 antigen (A0A3Q0CFI0)
Interferon-induced protein with tetratricopeptide repeats 1-like (A0A3Q0CJD1)
C3/C5 convertase (A0A3Q0CE80)
TPR_REGION domain-containing protein (A0A1U8BJQ7)
TRAF-type domain-containing protein (A0A1U7QTR1)
GB1/RHD3-type G domain-containing protein (A0A1U8CFU7)
RING-type E3 ubiquitin transferase (A0A1U7QGE3)
N-myc-interactor (A0A1U7Q4X4)
Enoyl-CoA delta isomerase 1, mitochondrial (A0A1U8CFA4)
SWIB (A0A1U7Q9S2;A0A1U8CSW4;A0A1U7QB55)
UTP--glucose-1-phosphate uridylyltransferase (A0A1U7QBN3)
Aldedh domain-containing protein (A0A1U7RFZ4)
Thioredoxin domain-containing protein 12 (A0A1U7Q7V1)GATOR complex protein SEC13 (A0A1U7Q6W8)
OAS1_C domain-containing protein (A0A1U8CD91)
Ig-like domain-containing protein (A0A1U8CZF4;A0A1U8CTM6)
Clusterin (A0A1U8C2W4)
Myeloid cell nuclear differentiation antigen (A0A1U7R051)
Arginase (E7CXR7)
Ubiquitin-like protein ISG15 (A0A1U8CAF8)
Myxovirus resistance protein 2 (B2Z890)
Galectin-3-binding protein (P70117)
CAP_SARS-CoV-2 Nucleoprotein (P0DTC9)
Lipocln_cytosolic_FA-bd_dom domain-containing protein (A0A1U7QUU5)Interferon-induced protein with tetratricopeptide repeats 1-like (A0A1U8BQK4)
Uridine phosphorylase (A0A1U8CI56)
Xanthine dehydrogenase (A0A1U7QV01)
Lactotransferrin (A0A3Q0CY55)
CAP18_C domain-containing protein (A0A1U7QJA2)
C4a anaphylatoxin (A0A1U8CS54)SERPIN domain-containing protein (A0A1U8CDV4)
C3/C5 convertase (A0A1U7RGK9)
Peptidoglycan-recognition protein (A0A1U7QUI9)
Haptoglobin (O35086)

6

8

10

12

14

16

18

ND

iBAQ Intensities [Log 2 ]Extended Data Figure 5

PBS

Poly(I:C)

SARS-CoV-2

PBS

Poly(I:C)

SARS-CoV-2
Pregnancy zone protein-like (A0A1U8CUG0)
Hemopexin (A0A1U7RF88)
Calgranulin-B (A0A1U7QW31)
Selenium-binding protein 1 (A0A1U8CT86)
Glutathione transferase (A0A1U8BWE1)
Homeodomain-only protein (A0A1U7QKE3)
C3-beta-c (A0A3Q0D994)
Plasminogen (A0A3Q0CDV3)
C4a anaphylatoxin (A0A1U8CS54)
Carboxylic ester hydrolase (O35534)
Glyoxalase I (A0A1U7QQ12)
Tubulin polymerization-promoting protein family member 3 (A0A1U7QZG6)Glutathione S-transferase (P30116;A0A1U7Q2C9)Serum amyloid P-component (A0A1U7QG20;P07629)
Ig-like domain-containing protein (A0A1U8C678)
CAP_SARS-CoV-2 Nucleoprotein (P0DTC9)Arginase (E7CXR7)Interferon-induced protein with tetratricopeptide repeats 1-like (A0A1U8BQK4;A0A3Q0CJ11)
Myxovirus resistance protein 2 (B2Z890)
Histone H2A (A0A3Q0CL86;A0A3Q0D9T9)
Ras-related protein Rab-11A (A0A1U7QLB5;A0A1U7QY95)
Ribosomal_L23eN domain-containing protein (A0A1U7QE09)
1,4-beta-N-acetylmuramidase C (A0A1U8CNI4)
Inter-alpha-trypsin inhibitor heavy chain H4 isoform X1 (A0A3Q0DB97)
Zinc finger protein 14-like (A0A3Q0CGU2)
A2M_N_2 domain-containing protein (A0A3Q0DAY1)
Coagulation factor XII (A0A1U8C5I3)
Serum paraoxonase/arylesterase 1 (A0A1U7RA96)
SMB domain-containing protein (A0A1U7QY02)
C4b-binding protein alpha chain (A0A1U7QVF6)
Lactotransferrin (A0A3Q0CY55)
Uridine phosphorylase (A0A1U8CI56)
Xanthine dehydrogenase (A0A1U7QV01)
Ras-related C3 botulinum toxin substrate 2 (A0A1U7Q9G7)
Glycogenin-1 (A0A3Q0D5J5)
Septin-type G domain-containing protein (A0A3Q0CLX5)
Sulfurtransferase (A0A3Q0CF03)
Glutathione S-transferase (A0A1U7QX94)
D-dopachrome decarboxylase (A0A1U7QEV1)
C3/C5 convertase (A0A1U7RGK9)
Ceruloplasmin (A0A1U7R5I7)
Deoxynucleoside triphosphate triphosphohydrolase SAMHD1 (A0A3Q0DET6)SERPIN domain-containing protein (A0A1U8CDV4)
Beta-2-microglobulin (A0A1U7QLF7)
Haptoglobin (O35086)
C-type lectin domain-containing protein (A0A1U7QD88)
Protein LBH (A0A3Q0CHX8)
arf-GAP domain and FG repeat-containing protein 1 isoform X1 (A0A3Q0DA20)
Secernin-2 (A0A1U7QC71)
Cell adhesion molecule 1 isoform X1 (A0A1U7Q590)
Ubiquitin-like protein ISG15 (A0A1U8CAF8)
OAS1_C domain-containing protein (A0A1U8CD91)
Ig-like domain-containing protein (A0A1U8CZF4)Interferon-induced transmembrane protein 3 isoform X1 (A0A1U7Q534)
Neutrophil defensin 4 (P81466;P81468)
Galectin-3-binding protein (P70117;A0A1U7Q3N9)
Phospholipase B-like (A0A1U7QFP3)
Peptidase S1 domain-containing protein (A0A1U7QV66)
SAND domain-containing protein (A0A3Q0DBM1;A0A3Q0DB09)
HSR domain-containing protein (A0A3Q0DAZ4)Cathepsin X (A0A1U7QIB1)
WD_REPEATS_REGION domain-containing protein (A0A1U7RIJ7)
VIT domain-containing protein (A0A3Q0DHA7)
Angiotensin 1-10 (A0A1U7QEW9)
Ig-like domain-containing protein (A0A1U7R3R6)
Signal transducer and activator of transcription (Q4R4G1;Q1T7E9)
Myeloid cell nuclear differentiation antigen (A0A1U7R051)
E3 ubiquitin-protein ligase RNF114 (A0A1U7QWA5)
Granulins (A0A1U8BNJ0)
SelP_N domain-containing protein (A0A1U7RKG6)
Calcium-dependent serine proteinase precursor (P15156;A0A1U7Q699)
Macrophage-capping protein isoform X1 (A0A3Q0D411)
Autophagy-related protein 3 (A0A1U7QIR7)
F5/8 type C domain-containing protein (A0A3Q0CF57)
Clusterin (A0A1U8C2W4;P14683)
Inter-alpha-trypsin inhibitor heavy chain H3 (A0A1U7Q227)
Importin N-terminal domain-containing protein (A0A1U7R233)
Pulmonary surfactant-associated protein C (A0A3Q0CRM2)
J domain-containing protein (A0A1U7R189)
PP28 domain-containing protein (A0A1U7QSJ3)
Very long-chain specific acyl-CoA dehydrogenase, mitochondrial (A0A1U7QA30)
CD177 antigen (A0A3Q0CFI0)
Complement C1q subcomponent subunit A (A0A1U7R7E8)
Glyco_transf_64 domain-containing protein (A0A1U8C320)
Flavodoxin_2 domain-containing protein (A0A1U8BFD7)
N-acetyltransferase domain-containing protein (A0A1U7RKL2)
Complement component C6 (A0A3Q0CFM2)
Glycosyl-phosphatidylinositol-specific phospholipase D (A0A3Q0CE61)
Complement subcomponent C1r (A0A1U7Q6R5)Interleukin-1 (A0A1U7QQA5)
MIT domain-containing protein (A0A1U7QV86)
C3/C5 convertase (A0A3Q0CE80)
Interferon-induced protein with tetratricopeptide repeats 1-like (A0A3Q0CJD1)
CD5 antigen-like isoform X1 (A0A1U8C986)
Macrosialin (A0A1U7Q8F3)
TRAF-type domain-containing protein (A0A1U7QTR1)
RING-type E3 ubiquitin transferase (A0A1U7QGE3)
TPR_REGION domain-containing protein (A0A1U8BJQ7)
N-myc downstream-regulated gene 1 protein (A0A3Q0D3I4)
GB1/RHD3-type G domain-containing protein (A0A1U8CFU7)
N-myc-interactor (A0A1U7Q4X4)
TPR_REGION domain-containing protein (A0A1U7Q2K5)
SH3 domain-containing protein (A0A3Q0DBB3)
Aldedh domain-containing protein (A0A1U8C9A1)
Non-specific protein-tyrosine kinase (A0A3Q0DGC2)
CREB-regulated transcription coactivator 2 (A0A1U7R5W8)
Endo/exonuclease/phosphatase domain-containing protein (A0A1U7QF81)
SH3 domain-containing protein (A0A1U8BYD5)Acyl-coenzyme A synthetase ACSM5, mitochondrial (A0A1U8BSU1)
Late endosomal/lysosomal adaptor and MAPK and MTOR activator 1 (A0A1U7QSZ1)
2-5 oligoadenylate synthase (A0A1U7R9X5)
2-5 oligoadenylate synthase (A0A1U8C9R8)
Alkaline phosphatase (W6E8V7)
VWFA domain-containing protein (A0A1U7Q573)
Interferon-induced protein 44 (A0A1U8CGR8)
XAF1_C domain-containing protein (A0A3Q0CKW2)
Protein-tyrosine-phosphatase (A0A3Q0CQK5)LOW QUALITY PROTEIN: phytanoyl-CoA dioxygenase domain-containing protein 1 (A0A1U8CWU2)Heme oxygenase (A0A1U7R1W8)
FYN-binding protein isoform X1 (A0A3Q0CZ30)
Phosphatidylcholine-sterol acyltransferase (A0A1U7QCW4)Perilipin (A0A1U7RGF4)
Torsin (A0A3Q0DBQ7)
GRB2-related adapter protein 2 isoform X1 (A0A1U7Q9I3)
ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase (A0A1U7QJF0)
RNA helicase (A0A0G2YNC7)

6

8

10

12

14

16

18

ND

iBAQ Intensities [Log 2 ]Extended Data Figure 6

5

20

30

4

15

20
10

2

0
2

3

4

5

6

1

2

d p.i.

4

5

6

1

2

3

4

5

d p.i.

d p.i.

g

6

3

100

*
1

2

3

*
4

d p.i.

5

6

5

2
1
0

0
1

2

3

4

d p.i.

5

6

1

2

3

4

5

6

d p.i.

hTestis (Progesterone)

Ovary (Progesterone)

10

50
30
25
20
15
10
5
0

ng ml -1

8

ng ml -1

3

Ovary (Cortisol)
4

10

0

0

0
1

Testis (Cortisol)
15

50

5

1

**

500
200
150

10

f

Ovary (Estradiol)

Testis (Estradiol)pg ml -1

Ovary (Testosterone)pg ml -1

Testis (Testosterone)
40

3

e

d

ng ml -1cng ml -1bng ml -1

ng ml -1

a

6
4
2
0
1

2

3

4

d p.i.

5

6

PBS
Poly(I:C)
Male
SARS-CoV-2

PBS
Poly(I:C)
FemaleSARS-CoV-2
1

2

3

4

5

6

d p.i.

Extended Data Figure 8

Extended Data Figure 9

Figures

Figure 1
SARS-CoV-2 pathogenesis in male and female golden hamsters. a, b, Weight loss of male (a) and female(b) golden hamsters infected with SARS-CoV-2 (105 p.f.u. ml-1; male, n = 9; female n = 10), Poly(I:C) (n =9) or PBS (n = 10). Statistical signi cance was assessed by Kruskal-Wallis one-way ANOVA (*p < 0.05,

**p<0.01, ***p<0.001). c, Area under the curve (AUC) for body weight of SARS-CoV-2 infected male andfemale golden hamsters. d, Viral lung titers of SARS-CoV-2 infected golden hamsters (3d p.i., n = 5). e,
Haematoxylin and eosin (HE) and SARS-CoV-2 nucleoprotein (NP) staining in lungs of infected golden
hamsters (3 dpi; representative pictures are shown, n = 5). f, Viral plasma titers of SARS-CoV-2 infected
golden hamsters (3d p.i., n = 5). g, h, Viral titers (g) and viral RNA levels (h) in testis of male golden
hamsters (3d p.i., n = 5). i, HE staining of testis from male golden hamsters (3d p.i., n = 5). j, k, Viral titers(j) and viral RNA levels (k) in ovary of female golden hamsters (3d p.i., n = 5). l, HE staining of ovary fromfemale golden hamsters (3d p.i., n = 5). m, n, Viral titers (m) and viral RNA levels (n) in uteri of female
golden hamsters (3d p.i., n = 5). o, HE staining in uteri of female Syrian golden hamsters (3d p.i., n = 5). ad,f,g,h,j,k,m,n, Values are shown as means, error bars as SD.

Figure 2

Immune response in the lungs of SARS-CoV-2 infected male and female golden hamsters. a, IL-6 mRNA
expression levels (n = 5; male Poly(I:C): n = 4). b, IL-1ra mRNA expression levels (n = 5; male Poly(I:C): n =4). c, d, Eotaxin mRNA (c) and protein (d) expression levels (n = 5; male Poly(I:C): n = 4). e, MIP-1α protein
expression levels (n = 5). f, IFN-γ mRNA expression levels (n = 5; male Poly(I:C): n = 4). g, VEGF protein
expression levels (n = 4; SARS-CoV-2: n = 3). h, IL-10 protein expression levels (n = 5). i, IL-12 protein
expression levels (n = 5). j, IL-1β protein expression levels (n = 5). k, IL-2 protein expression levels (n = 5). l,
IL-13 protein expression levels (n = 5). m, MIP-1b protein expression levels (n = 5). n, MCP-1 protein
expression levels (n = 5). o, TNF-α protein expression levels (n = 5). Cytokine and chemokine levels were
measured in lung homogenates at 3 d p.i.. Values are shown as means; error bars as SD; detection limits
are indicated as dotted lines. Statistical signi cance was assessed by Kruskal-Wallis one-way ANOVA
and by unpaired Student’s t test (*p < 0.05, **p<0.01).

Figure 3
Sex hormones and SARS-CoV-2 entry receptors in infected male and female golden hamsters. a, b,
Testosterone levels in plasma of male (a) and female (b) golden hamsters. c, d, Estradiol levels in plasmaof male (c) and female ( d) golden hamsters. e, f, Cortisol levels in plasma of male (e) and female (f)golden hamsters. g, h, Progesterone levels in plasma of male (g) and female (h) golden hamsters. Values
are shown as means; error bars are shown as SD (n = 5; estradiol male3d p.i.: n = 10; estradiol male 14dp.i. SARS-CoV-2: n = 4; estradiol female 3d p.i. Poly(I:C) and SARS-CoV-2: each n = 8). i-k, ACE2 mRNA

expression levels at day 1 (i), 3 (j) and 6 (k) p.i. (n = 5; male Poly(I:C) 1d p.i.: n = 4; male and female
Poly(I:C) 3d p.i.: n = 4) in the lungs of infected golden hamsters. l, ACE mRNA expression levels (n = 5;
male Poly(I:C): n = 4) in the lungs of infected golden hamsters. m, n, ACE2 mRNA expression levels in
testis of male (m) and ovary of female (n) golden hamsters at day 3 p.i. (n = 5; male SARS-CoV-2: n = 4).
o, DPP4 mRNA expression levels (n = 5; male Poly(I:C): n = 4) in the lungs of infected golden hamsters. p,
q, TMPRSS2 mRNA expression levels at day 3 (p) and 6 (q) p.i. in the lungs of infected golden hamsters.
r, AR mRNA expression levels in the lungs of infected golden hamsters (n = 5; Poly(I:C): n = 4). s, ZIP9mRNA expression levels in the lungs of infected golden hamsters (n = 5; male Poly(I:C): n = 4). The mRNA
expression levels are presented as negative exponents of Δct values; values are shown as means; error
bars are shown as SD. Statistical signi cance was assessed by Kruskal- Wallis one-way ANOVA (*p <0.05, **p < 0.01, ***p < 0.001).

Figure 4
Proteomic analysis of the lungs of SARS-CoV-2 infected male and female golden hamsters. a, Volcano
plot displaying host proteins up- or down-regulated in male versus female golden hamsters upon SARSCoV-2 infection (orange and green dots, respectively). Signi cantly-regulated proteins are separated frombackground proteins by a hyperbolic curve (dotted grey line). b, Heatmap of iBAQ intensities forsigni cantly regulated proteins (Student’s t-test, FDR 0.05, p < 0.05) in the total proteome acrossexperimental groups and biological replicates.

Figure 5
CYP19A1 and endothelial activation in the lungs of SARS-CoV-2 infected male and female golden
hamsters. a, Schematic overview of binding sites for transcription factors in the four promoter regions(P1, P2, P3, and P4) of the aromatase gene CYP19A1. b-d, Aromatase mRNA expression levels at day 1

(b), 3 (c) and 6 (d) p.i. (n = 5; male Poly(I:C): n = 4). e, 5α-Reductase mRNA expression levels (n = 5; malePoly(I:C): n = 4). f, g, Aromatase mRNA expression levels in testis of male (f) and ovary of female (g)golden hamsters at 3d p.i. (n = 5; male SARS-CoV-2: n = 4). h, i, Endothelial lipase mRNA expression levels
at day 3 (h) and 6 (i) p.i. (n = 5; male Poly(I:C) 3d p.i.: n = 4). The mRNA expression levels are presented
as negative exponents of ct values; values are shown as means; error bars are shown as SD, detection
limits are indicated as dotted lines. Statistical signi cance was assessed by Kruskal- Wallis one-way
ANOVA (*p < 0.05, **p < 0.01).

Figure 6
CYP19A1 protein expression in the lungs of SARS-CoV-2 infected male and female golden hamsters. a, b,
Immunohistochemistry of serial sections from lungs of male (a) and female (b) golden hamsters foraromatase, SARS-CoV-2 NP, macrophages (CD204), endothelial cells (vWF and CD31), T cells (CD3) andsmooth muscle (SMA). Bronchus (B), vessel (V), alveola (A), stars indicate perivascular connective tissue
and arrowheads indicate macrophages. Representative pictures are shown. c-f Semiquantitative scoringof the of aromatase CYP19A1 immunoreactivity in different lung regions of male and female golden
hamsters at day 3 p.i. (n = 5). Immunoreactivitiy was examined by light microscopy in the endothelium(c), the bronchiolar epithelium (d), the perivascular (e) and peribronchiolar region (f) in the lungs ofinfected hamsters. Signal intensity in respective regions was very low (0,5), low (1), low to moderate (2),moderate (3), moderate to high (4), or high (5). Signal distribution was assessed as: focal/oligofocal (≤3foci; 1), multifocal (>3 foci; 2) and diffuse (3). For better comparative interpretation a nal score for eachregion and animal was calculated as product of signal intensity times signal distribution. Statisticalsigni cance was assessed by Kruskal- Wallis one-way ANOVA and by unpaired Student’s t-test (*p < 0.05,**p<0.01).

Figure 7
CYP19A1 protein expression in the lungs of fatal Covid-19 cases. Immunohistochemistry of lungs frommale (a-d) and female (f-h) patients with Covid-19 or controls (e; representative picture of n = 3 areshown) stained for aromatase. The intensity of staining was signi cantly higher in endothelial cells andespecially in macrophages in male (a d) compared to female (f-h) Covid-19 patients and control patients(e). Stars indicate endothelial cells and arrowheads indicate macrophages. Scale bar, 100 μm.