key: cord-0281671-a36c0b23
authors: Chen, Y.; Bai, T.; Beck, S.; Stanelle-Bertram, S.; Chen, T.; Dong, J.; Yang, J.; Wang, L.; Wang, D.; Shu, Y.; Gabriel, G.
title: Low testosterone and high cytokine levels correlate with lethal H7N9 infections in men: a retrospective cohort study from 98 H7N9 patients
date: 2020-05-11
journal: nan
DOI: 10.1101/2020.05.07.20093914
sha: d4b0a5b06fc978e667006a4b0a9b5cb00c8c1498
doc_id: 281671
cord_uid: a36c0b23

Background. Human infections with avian influenza A (H7N9) virus emerged in East China in March 2013. In contrast to seasonal influenza A viruses, H7N9 infections showed a strong sex bias. Over the five epidemic waves in China, ~70% of all H7N9 cases were observed in men. Thus, in this human cohort, we retrospectively analyzed sex hormones as well as inflammatory cytokine and chemokine levels in men and women infected with avian H7N9 influenza. Methods. We systematically analyzed the underlying correlation based on established human cohorts of two age groups (18-49 years and [≥]50 years) including laboratory-confirmed H7N9 cases as well as seasonal influenza cases, H7N9 close contacts and poultry workers as controls in dependency on sex. The level of testosterone, estradiol and cytokines/chemokines were measured in all study participants. We compared the levels of sex hormones, cytokines/chemokines by sex and disease outcome. Findings. We included H7N9 cases (n=98), close contacts (n=71), poultry workers (n=108) and mild seasonal influenza cases (n=53) in this study. Samples were collected between 2014 and 2017. All control groups showed similar median age within H7N9 cases except for the seasonal influenza group with a younger median age. In H7N9 infected men, testosterone levels were strongly reduced compared to male H7N9 virus-negative close contacts or males with seasonal influenza. Low testosterone levels in H7N9 infected men correlated with high inflammatory cytokine levels, e.g. IL-6, and lethal outcome in those 18-49 years of age. No significant differences were detected in estradiol levels in H7N9 infected men. In H7N9 infected women ([≥] 50 years), estradiol levels were significantly elevated compared to H7N9 virus-negative close contacts. However, increased estradiol levels did not significantly correlate with lethal outcome in women albeit a slight tendency towards poor outcome could be detected. Interpretation. This study provides evidence that low testosterone levels in H7N9 influenza infected men correlate with inflammatory cytokine/chemokine responses and lethal outcome. Thus, treatment of H7N9 influenza virus infected patients should consider sex-specific mitigation strategies.

in dependency on sex. The level of testosterone, estradiol and cytokines/chemokines were 38 measured in all study participants. We compared the levels of sex hormones, 39 cytokines/chemokines by sex and disease outcome. 40 41 Findings We included H7N9 cases (n=98), close contacts (n=71), poultry workers (n=108) 42 and mild seasonal influenza cases (n=53) in this study. Samples were collected between 2014 43 and 2017. All control groups showed similar median age within H7N9 cases except for the 44 seasonal influenza group with a younger median age. In H7N9 infected men, testosterone levels 45 were strongly reduced compared to male H7N9 virus-negative close contacts or males with 46 seasonal influenza. Low testosterone levels in H7N9 infected men correlated with high 47

Introduction 67 68 In early 2013, human infections with avian influenza A (H7N9) virus were first reported in 69 China 1 . Since then, the H7N9 avian influenza A virus caused five epidemic waves from 2013 70 to 2017. Exposure at live poultry markets was considered a major risk factor for human 71 infections with H7N9 virus. A primary epidemiological study revealed that 71% of all H7N9 72 cases were males in the first wave 2 . The high degree of sex bias in men consistently accounted 73 with similar proportions (68%-71%) during the entire five H7N9 epidemic waves 3 . Currently, 74 very little knowledge exists on the complex interplay of biological sex and gender specific 75 behavior as contributors to influenza disease outcome. The World Health Organization (WHO) 76

Western Pacific team proposed gender-associated practices and norms, such as more frequent 77 exposure of men to birds, may pose one of the reasons for sex-specific H7N9 incidence 4 . 78

However, a follow-up study suggested increased risk in older men is not due to higher exposure 79 time to live poultry market 5 . 80 81 Sex hormones, particularly testosterone and estradiol are important biological factors that 82 affect sex differences in immune responses [6] [7] [8] [9] . In mouse models, low concentrations of 83 estradiol in female mice was associated with enhanced pro-inflammatory responses and H1N1 84 influenza pathogenesis 10 . In contrast, high estradiol levels protected female mice from lethal 85 influenza infection by dampening inflammatory cytokine responses 10 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. difference of sex hormones and cytokines/chemokines between survival and dead H7N9 cases 160 in each age group was analyzed using the unpaired, two-tailed Student's t-test (non-parametric). 161

Association between sex hormones and cytokine/chemokines were determined using linear 162 regression and correlation analysis (Pearson). Since the results of cytokine/chemokines 163 exhibited deviated scale than testosterone, all values in the statistical analysis and figures were 164 used after Ln-transformation in comparison of cytokines/chemokines in all groups and linear 165 regression with testosterone. Statistical significance was defined as p < 0·05 (* p < 0·05, 166 ** p < 0·01, *** p < 0·001). 167 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 11, 2020. . https://doi.org/10.1101/2020.05.07.20093914 doi: medRxiv preprint

The study funders had no role in study design, data collection, data analysis, data interpretation, 169 or writing of the report. The corresponding authors had full access to all the data in the study 170 and had final responsibility for the decision to submit for publication. 171 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. all control groups, the median age is similar in close contacts and poultry workers compared to 184 H7N9 cases in both younger and older age groups (Table 1) . However, the age of seasonal 185 influenza cases in younger age groups was lower compared to H7N9 cases. Consistent with 186 H7N9 cases, the proportion of males in all control groups is higher than females, except for the 187 seasonal influenza cases with a similar proportion in both sexes. 188 189 Low testosterone levels in H7N9 infected men are associated with lethal outcome 190

In order to assess the role of sex hormones in the observed sex bias of H7N9 cases, we first 191 measured testosterone concentrations in all cohorts. Testosterone levels were strongly reduced 192 in H7N9 infected men of both age groups assessed compared to virus negative H7N9 close 193 contacts, poultry workers and those with seasonal influenza ( Figure 1A ). Low testosterone 194 levels strongly correlated with lethal outcome in H7N9 infected men in the age group of 18-49 195 years olds ( Figure 1B ). No significant differences in testosterone levels were observed in H7N9 196 infected women compared to their close contacts and poultry workers. However, testosterone 197 levels were significantly increased in those infected with seasonal influenza compared to all 198 other female groups ( Figure 1C ). However, no significant impact of testosterone levels was 199 observed regarding H7N9 disease outcome in women ( Figure 1D (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 11, 2020. . https://doi.org/10.1101/2020.05.07.20093914 doi: medRxiv preprint Conversely, we measured estradiol levels as a predominant female hormone in all cohorts. 207

Seasonal influenza infected men of 18-49 years of age presented slightly but significantly 208 increased estradiol levels compared to poultry workers, H7N9 cases and their H7N9 virus-209 negative close contacts (Figure 2A ). No differences were detected among all groups of ≥50 210 years of age ( Figure 2A) . However, estradiol levels were not associated with disease outcome 211 in H7N9 infected men ( Figure 2B ). In H7N9 infected women of ≥50 years of age, estradiol 212 levels were significantly elevated compared H7N9 virus-negative close contacts ( Figure 2C ). 213

Increased estradiol levels, however, were not significantly associated with disease outcome in 214 females but a tendency towards an increased risk for lethal outcome may be speculated among 215 both age groups ( Figure 2D ). These findings suggest that estradiol does not significantly affect 216 disease outcome in H7N9 infected men. 217

Next, we assessed the cytokine and chemokine responses in H7N9 infected patients compared 220 to their close contacts, poultry workers or those infected with seasonal influenza. In general, 221 H7N9 infection resulted in highly increased cytokine and chemokine levels compared to their 222 H7N9-negative close contacts or poultry workers ( Figure S1 and S2). Seasonal influenza virus 223 infected patients also presented elevated cytokine and chemokine levels but in general less than 224 those infected with H7N9 ( Figure S1 men correlated with elevated IL-7 and MIP-1ß levels ( Figure S3B and S3C). These findings 238 suggest that differential cytokines are associated with disease outcome in males and females. 239 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. We could show that (1) high cytokine levels and (2) low testosterone levels correlate with lethal 241 outcome in H7N9 infected men. In order to assess potential mechanistic links between 242 individual cytokines and testosterone, we compared their individual levels in the same patient 243 with disease outcome. In H7N9 infected men (18-49 years old), among all assessed cytokines, 244 particularly IL-6, IL-8, IL-10, IL-1RA and GM-CSF levels were negatively associated with 245 testosterone concentrations in H7N9 survivors with the exception of TNF-α. Thus, a significant 246 number of H7N9 surviving men had high testosterone and low cytokine levels ( Figure 4A-E) . 247

In contrast, all men who succumbed to H7N9 influenza presented low testosterone and high 248 cytokine levels. In H7N9 infected men ≥50 years, a similar correlation was observed for the 249 same cytokines as described for the 18-49 years olds (Figure 4 G-L). However, death was not 250 significantly associated with low testosterone and high cytokine levels. In H7N9 infected 251 women, positive linear association between cytokine and testosterone levels was observed 252 unlike the observations in men ( Figure S4 ). These findings, suggest that in men, expression 253 levels of cytokines, such as IL-6, increases with reducing testosterone levels suggestive of 254 potentially interlinked mechanisms. 255 256 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. old and those ≥50 years of age) from laboratory-confirmed H7N9 (n=98) and seasonal 270 influenza (n=53)cases, epidemiological linked poultry workers (n=108) and close contacts of 271 H7N9 cases that were virus-negative (n=71). We systematically analyzed the association of the 272 main estrogen (estradiol) and androgen (testosterone) levels and correlated to cytokine and 273 chemokine levels known to be hallmarks of influenza disease outcome in both sexes. 274

We observed a strong suppression of testosterone levels in H7N9 infected men in both age 276 groups (18-49 years and ≥50 years). One limitation of this study is that we have no information 277 regarding potential comorbidities in these groups, such as diabetes type II or obesity, which 278 are known to lower testosterone levels in men 17-20 . However, our data is in line with data 279 reported in murine models, where infection of healthy male mice with influenza A virus was 280 shown to reduce testosterone levels by independent laboratories 10,12 . Interestingly, treatment of 281 female and male mice with testosterone protected animals from lethal outcome 12 . However, 282 testosterone treatment of elderly male mice was less effective 21 . These data strongly hint 283 towards an active reduction of testosterone levels by influenza A virus infection by a yet 284 unknown mechanism. 285 286 For chronic infections, such as HCV or HIV, it was repeatedly shown that these infections 287 cause low testosterone levels likely due to constant inflammation in infected individuals 22, 23 . 288

In line with chronic conditions, such as type II diabetes, it is believed that chronic inflammatory 289 diseases can cause low testosterone levels. Since testosterone plays key roles in not only 290 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 11, 2020. . 12 fertility but also innate and adaptive immunity, one would hypothesize that all these 291 "testosterone level" decreasing hits may increase vulnerability towards infections in general. 292 293 Albeit the underlying mechanism causing testosterone deficiency is not known, there is 294 increasing evidence that testosterone levels may modulate inflammatory cytokines 24-27 . It 295 would be interesting to see whether other acute infections, such as the current pandemic Severe 296 acute respiratory syndrome coronavirus 2(SARS-CoV-2), may also cause low testosterone 297 levels. 298 299 However, despite the well-documented impact of elevated cytokines to disease severity and 300 H7N9 disease outcome 28,29 , no studies considered sex differences in inflammatory responses 301 and the role of sex hormones until now. In our study, elevated pro-inflammatory cytokines and 302 chemokines, such as TNF-α, IL-8, IFN-α2, IFN-γ, IL-6, MCP-1, IP10, and anti-inflammatory 303 cytokine IL-10 were detected in both male and female H7N9 cases without major statistically 304 significant differences. However, testosterone levels were negatively associated with specific 305 cytokines and chemokines, such as IL-6, and survival rates in H7N9 infected men. This is 306 particularly interesting, since high IL-6 levels were repeatedly reported to be a poor prognostic 307 marker for severe infections, including coronavirus disease 2019 30 . Clearly, future 308 investigations are required to unravel the underlying mechanism of testosterone deficiency in 309 H7N9 infected men. Moreover, future studies are needed to understand whether this is a 310 specific mechanism or whether low testosterone levels can also be detected in other acute viral 311 infections. Increasing knowledge on the regulation of sex hormone levels will provide an 312 important basis to understand diseases with sex disparity and to develop sex specific therapies 313 against viral infections. 314 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. We thank Chinese National Influenza Surveillance Network and all staff at local CDCs in 330

China for their contribution on sample collection and diagnosis for H7N9 and seasonal 331 influenza A viruses. We would also like to thank staff at Sino-Japan Friendship Hospital for 332 the measurement of testosterone and estradiol. Finally, we thank the study participants and 333 their families. 334 335 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Table 1 . 346

Significant differences among all study groups were calculated using either one-way ANOVA 347 succumbed to the infection (survival and death, respectively). Samples were divided into two 369 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 11, 2020. . https://doi.org/10.1101/2020.05.07.20093914 doi: medRxiv preprint groups based on age: 18-49 and ≥50 years (yrs) old. The measurement was carried out using a 370 multiplex immunoassay. Analytes included were: G-CSF (granulocyte-colony stimulating 371 factor), GM-CSF (granulocyte-macrophage colony-stimulating factor), IL-8 (interleukin 8), 372 IL-10 (interleukin 10), IL-15 (interleukin 15), MCP-1/CCL2 (monocyte chemoattractant 373 protein 1), TNF-α (tumor necrosis factor alpha), eotaxin, MIP-1β/CCL4 (macrophage 374 inflammatory protein 1 beta), EGF (epidermal growth factor). Values are shown as means ± 375 SD. Statistical significant differences between groups were determined using the unpaired, 376 two-tailed Student's t-test and divided into: * p < 0·05, ** p < 0·01, *** p < 0·001. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 11, 2020. . https://doi.org/10.1101/2020.05.07.20093914 doi: medRxiv preprint Table  394 395 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 11, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 11, 2020. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 11, 2020. . https://doi.org/10.1101/2020.05.07.20093914 doi: medRxiv preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 11, 2020. . https://doi.org/10.1101/2020.05.07.20093914 doi: medRxiv preprint

Human infection with a novel avian-origin

Epidemiology of human infections with avian influenza 401

A(H7N9) virus in China

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A(H7N9) from poultry exposure

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Sex differences in immune responses

Sex, immunity and influenza

Sex Hormones and inmmunit of infection

Elevated 17β-estradiol protects females 417 from influenza a virus pathogenesis by suppressing inflammatory responses

Age and testosterone mediate influenza 420 pathogenesis in male mice

Influenza by Reducing the Pro-Inflammatory Cytokine Response in the Murine Lung

The Anti-Inflammatory Effects of Testosterone

World Health Organization. Human infection with avian influenza A(H7N9) virus

Isolation and characterization of H7N9 viruses from 433 live poultry markets -Implication of the source of current H7N9 infection in humans

Testosterone and estrogen 436 differently effect Th1 and Th2 cytokine release following trauma-haemorrhage

Testosterone level in men with type 2 diabetes 439 mellitus and related metabolic effects: A review of current evidence

Testosterone level and risk of type 2 442 diabetes in men: A systematic review and meta-analysis

Body mass index in relation to semen 445 quality and reproductive hormones among 1,558 Danish men

Testosterone treatment of aged male mice 448 improves some but not all aspects of age-associated increases in in fl uenza severity

Prevalence of testosterone deficiency in HIV-451 infected men under antiretroviral therapy

Testosterone in Men With Chronic Hepatitis 453

C Infection and After Hepatitis C Viral Clearance

Correlation between testosterone and the 455 inflammatory marker soluble interleukin-6 receptor in older men

Do Androgens Modulate the 458

Pathophysiological Pathways of Inflammation? Appraising the Contemporary

Androgen-androgen receptor 461 system improves chronic inflammatory conditions by suppressing monocyte 462 chemoattractant protein-1 gene expression in adipocytes via transcriptional regulation