key: cord-0284198-lhu0z6qf authors: Wang, Chang; Lashua, Lauren P.; Carter, Chalise E.; Johnson, Scott K.; Wang, Minghui; Ross, Ted M.; Ghedin, Elodie; Zhang, Bin; Forst, Christian V. title: Sex disparities in influenza: a multiscale network analysis date: 2021-03-26 journal: bioRxiv DOI: 10.1101/2021.03.25.437108 sha: 41da19ecb4716e50f1e1c3cb6b4ce2402eeed213 doc_id: 284198 cord_uid: lhu0z6qf Sex differences in the pathogenesis of infectious diseases due to differential immune responses between females and males have been well documented for multiple pathogens. However, the molecular mechanism underlying the observed sex differences in influenza virus infection remains poorly understood. In this study, we used a network-based approach to characterize the blood transcriptome collected over the course of infection with influenza A virus from female and male ferrets to dissect sex-biased gene expression. We identified significant differences in the temporal dynamics and regulation of immune responses between females and males. Our results elucidate sex-differentiated pathways involved in the unfolded protein response (UPR), lipid metabolism, and inflammatory responses, including a female-biased IRE1/XBP1 activation and male-biased crosstalk between metabolic reprogramming and IL-1 and AP-1 pathways. Overall, our study provides molecular insights into sex differences in transcriptional regulation of immune responses and contributes to a better understanding of sex bias in influenza pathogenesis. Sex-related differences shaped by multidimensional biological characteristics that define 58 females and males exert considerable influence on the pathogenesis of various diseases [1], 59 including autoimmune diseases [2] , cancers [3] [4] [5] , and infectious diseases caused by diverse 60 pathogens [6] [7] [8] . Each infectious disease exhibits a distinct pattern of sex bias in the prevalence, 61 intensity, and outcome of infections [6, 9] , as well as in the responses to antiviral drugs and 62 vaccines [10, 11] . For example, females have a higher fatality rate following exposure to 63 influenza A viruses (IAV) [12] and more robust antibody responses and adverse reactions after 64 vaccination than males [13] [14] [15] , while accumulating evidence suggests a male bias in 65 mortality [16, 17] . Thus an in-depth understanding of sex differences in the pathogenesis of 66 diseases and consideration in the rational design of prophylactic and therapeutic strategies 67 represent an important step towards personalized medicine. The observed sex differences in disease pathogenesis have been primarily attributed to the 70 differences in the innate and adaptive immune responses between females and males. In both 71 arms of immunity, the sexes differ in multiple aspects, including the detection of pathogen 72 nucleic acids by pattern recognition receptors (PRRs) such as the Toll-like receptors (TLRs), the 73 number and activity of immune cells, and the production of cytokines and chemokines (reviewed 74 in [1, 18] ). These intricate differences in immune functions between sexes have a powerful 75 impact on infectious disease pathogenesis. For example, an augmented response to pathogens 76 in females allows better control and clearance of pathogens while promoting increased 77 immunopathology [6, 7, 10] . During influenza infections, female mice exhibited a more robust 78 induction of pro-inflammatory cytokines and chemokines in their lungs, including TNF-, IFN-, 79 IL-6, and CCL2, accompanied by greater weight loss, hypothermia, and mortality than male 80 mice [19] . The etiology underlying sex differences in immunity involves intrinsic and extrinsic factors that 83 exert a combinatorial effect on the immune system's functioning. Although the microbiome [20-84 23] and nutritional status [24] [25] [26] [27] [28] [29] have been implicated in modulating immune responses, sex 85 hormones and genetic mediators are the most widely appreciated factors shaping differential 86 immunity between females and males. In addition to the profound effects of sex hormones that 87 have been extensively demonstrated, genetic differences attributed to immune-related genes 88 and microRNAs (miRNAs) that are located on the sex chromosomes also play an important role 89 in determining the distinct immune responses between sexes, especially in prepubertal children, 90 postmenopausal females and age-matched males (reviewed in [1, 6, 7, 18, [30] [31] [32] [33] ). However, 91 the pathways and cellular responses that mediate the differences in response to influenza 92 infection have not been fully elucidated. Moreover, the confounding effects of hormone and 93 genetic factors in intact animals and human populations impose a challenge in resolving the 94 mechanism of sex differences in influenza pathogenesis. To address the molecular mechanisms underlying sex differences in influenza infection 97 responses, we assessed the transcriptomic dynamics of blood cell responses in neutered 98 female and male ferrets throughout the infection. We detected a temporal shift in mounting 99 immune responses against viral infection between females and males. Using a multiscale 100 network-based approach, we further identified pathways and cellular processes commonly 101 induced in both sexes and those uniquely regulated in females or males. Our data revealed 102 gene regulatory pathways that were differentially regulated between sexes and likely involved in 103 marked sexual differences in influenza virus-induced pathogenesis, providing molecular and 104 functional insights for functional evaluation and development of therapeutic strategies. An integrated network approach for systematic characterization of the immune response 108 to influenza infection in females and males. To evaluate the genetic factors mediated sex differences in the immune response to influenza 110 virus infection, we conducted a study with neutered female and male ferrets. We examined the 111 transcriptional response in their blood cells over the course of the infection (Fig. 1a) . Briefly, To systematically characterize sex differences in the immune response to influenza virus 124 infection, we used a co-expression network-based approach that integrated transcriptional 125 patterns with physiological traits and enabled the identification of sex-specific gene expression 126 signatures (Fig. 1b) . Specifically, differential expression analysis was carried out within each females and males (Tables S1 and S2), the most significant overlaps between any two given 148 sets were observed at 3 and 5 dpi within or between sexes, followed by those detected between 149 3-5 dpi in each sex and 1 dpi in females or 8 dpi in males ( Fig. 2a and Fig. S2 ). It is noteworthy 150 that DEGs at 14 dpi in females did not share significant overlaps with any other sets (Fig. 2a) both sexes, including the innate immune response and cytokine signaling -particularly type I ( / ) and II ( ) interferon (IFN) signaling -hemostasis, the urokinase-type plasminogen 159 activator (uPA) and its receptor (uPAR) mediated signaling, DNA replication and the cell cycle 160 (Fig. 2b) . These processes exhibited differential temporal kinetics of activation, with IFN 161 signaling being initiated immediately upon infection. However, cell cycle-related processes were 162 revealed later in the infection (Fig. 2b) . We also detected enrichment of down-regulated genes To further investigate the temporal dynamics of infection-responsive processes in both sexes, we examined the fold change (FC) of DEGs associated with the enriched MSigDB gene sets 169 over time. We found more robust activation of type I and II IFN signaling pathways in females 170 than males, as many genes were immediately and more strongly activated at 1 dpi in females 171 ( Fig. 3a-3b) . A temporal shift in the expression kinetics between sexes was also evident in 172 platelet activation, signaling, and aggregation, which were important in maintaining hemostasis 173 and modulating inflammation [36] . Many genes involved in platelet activity were immediately 174 activated at 1 dpi and substantially subsided by 8 dpi in females, in contrast to an attenuated 175 activation at 1 dpi and continuous expression beyond 8 dpi in males (Fig. 3c) , further 176 suggesting a more rapid response in females than males. Interestingly, we also detected a DEG, 177 GLIPR1L2 (GLI pathogenesis-related 1 like 2), with extreme temporal shift as manifested by 178 inverse alteration kinetics between the sexes (r = -0.9993, p = 0.0238). GLIPR1L2 was down-179 regulated at 1-3 dpi and returned to baseline expression by 5 dpi in the females but was 180 continuously down-regulated from 3 dpi onwards in males (Fig. 3d) . Although the function of 181 GLIPR1L2 in viral infections remains unclear, it has been shown to reside in the same gene 182 cluster with GLIPR1 (GLI pathogenesis-related protein 1) and GLIPR1L1 and be targeted by the 183 tumor suppressor p53 during cancer development and progression [37] . Taken together, these results indicate temporal differences in the immune response dynamics between the sexes, 185 marked by a prompt response in females compared with a lagged response in males. Co-expression network analysis identifies shared and sex-unique expression patterns in 188 the immune response. Although differential expression analysis enabled a global overview of the immune response in inflammation, immune cell migration, hemostasis, and apoptosis induction and clearance 203 exerted by various blood cells (Fig. 4c) , revealing more specific processes than those 204 associated with DEGs globally. Some modules were also positively associated either with viral 205 load in one or both sexes or with weight loss in males ( Fig. 4c and Fig. S4 ). For example, viral 206 load-related modules in both females and males were consistently enriched in gene set 207 signatures of IFN signaling, primarily type I IFN signaling. In contrast, viral load-related modules 208 in females alone were associated with specific inflammation-related pathways, lysosome-related 209 processes, the unfolded protein response (UPR), and hemostasis. Those only in males were associated with apoptosis-related pathways ( Fig. 4c and Fig. S4 ). Although we did not detect 211 any weight loss-related modules in females, those in males exhibited signatures of various 212 generic processes and pathways, including transcription, translation and its regulation, the 213 AHSP pathway, nuclear -catenin pathway, 4-1BB dependent immune response pathway, 214 stathmin pathway, the primary immunodeficiency pathway, porphyrin metabolism, and 215 generation of second messengers ( Fig. 4c and Fig. S4) , suggesting a complex relationship 216 between weight loss and underlying molecular processes. Moreover, enrichment of blood cell- AHSP pathway, and translocation of ZAP-70 to the Immunological synapse, which were 238 generally suppressed in both sexes (Fig. 4) , similar to those observed in DEGs. We conducted 239 pairwise comparisons between each module in the female and male networks with Fisher's 240 Exact Test (FET) to determine the similarity of modules and their key regulators (Table S9) . We 241 identified significant overlaps between the modules associated with those shared signatures in 242 both sexes (Table S9) . However, the key regulators in modules with matched signatures shared 243 limited overlap ( Fig. S5 and Table S9 ). These results suggest similar transcriptional programs 244 in both sexes upon infection are coordinated by different regulators in females and males. To further pinpoint the sex-specific gene co-expression patterns that implicated distinct 247 functional signatures, we next focused on the modules associated with sex-unique gene set 248 signatures and determined the uniqueness of their module memberships in both sexes. We did 249 a module member similarity test (i.e., FET) along with a differential gene correlation analysis (Table S10) . We detected one 253 female module, M139, which had a female-unique composition determined by FET (Table S9) , and that was associated with the activation of chaperone genes by XBP1(s) (Fig. 4c and Table 255 S7). We also identified two male modules, M152 and M569, which had male-unique regulatory 256 relationships identified with DGCA between module counterparts (Table S10) and were 257 associated with the AP-1 (activator protein-1) and IL-1 pathways, respectively ( Fig. 4c and 258 Table S8 ). The female-specific module M139 contained XBP1 (X-box binding protein 1) as its 259 key regulator (Fig. 5a) . The majority of the genes within this module exhibited a rapid and 260 strong induction immediately following infection at 1 dpi in females (Fig. S6a) compared with a 261 milder induction in males (Fig. 5b) mediators. By characterizing the blood transcriptome differences, we detected a temporal 301 difference in the dynamics of immune responses between sexes, manifested by a more rapid 302 activation in females than males upon infection. The immediate response in females is likely 303 attributed to germline-encoded bias in innate immune sensing, as TLR7 that is located on the X 304 chromosome and encodes an innate PRR recognizing single-stranded RNA can not only have a 305 higher expression level in females than males due to X-inactivation escape [57] , but also induce 306 more robust IFN-production in peripheral blood mononuclear cells (PBMCs) isolated from 307 women than those from men in vitro [58] . Moreover, we found limited overlap in the key 308 regulators modulating commonly altered processes upon infection between sexes, suggesting 309 differential regulation of various aspects of immune responses in females and males. This Given the time series information for each different dataset, we used two distinct methods to 447 identify differentially expressed genes. We used a one-way ANOVA-like approach as 448 implemented in the Limma-package [77] to identify differentially expressed genes across the 449 time series-termed significant response genes. Significance is defined based on a false 450 discovery rate (FDR) of 5% or less. DEGs is used as a generic term for differentially expressed 451 genes, including SRGs. Statistical significance of intersections between any two given DEG sets The colors indicate the comparison within each sex (i.e., pink for females and blue for males). Detailed information about DEGs in each module over time can be found in Sex differences in immune responses Epidemiology and estimated population burden of selected 631 autoimmune diseases in the United States Sex disparities in cancer incidence by period and age Sex disparities in cancer mortality and survival Sex Differences in Cancer: Epidemiology SeXX matters in infectious disease pathogenesis Sex differences in immune responses to infectious diseases Child mortality estimation: Estimating sex differences in childhood 644 mortality since the 1970s How sex and age affect immune responses, susceptibility to 646 infections, and response to vaccination Sex influences immune responses to viruses, and efficacy of prophylaxis and 648 treatments for viral diseases The intersection of sex and gender in the treatment of 650 influenza. Current Opinion in Virology Antibody responses and cross protection against lethal influenza A 653 viruses differ between the sexes in C57BL/6 mice. Vaccine Biological sex affects vaccine efficacy and protection against influenza 655 in mice Age-associated changes in the impact of sex steroids on influenza 657 vaccine responses in males and females Considering how biological sex impacts immune responses and 659 COVID-19 outcomes Sex differences in immune responses that underlie COVID-19 661 disease outcomes Sex Differences in the Immune Response Gender Differences in Infection and Treatments for Infectious Diseases Elevated 17beta-estradiol protects females from influenza A virus 666 pathogenesis by suppressing inflammatory responses Sex differences in the gut microbiome drive hormone-dependent 669 regulation of autoimmunity Gender bias in autoimmunity is influenced by microbiota Sex steroids mediate bidirectional interactions between 673 hosts and microbes. Hormones and Behavior The microgenderome revealed: Sex differences in bidirectional 675 interactions between the microbiota, hormones, immunity and disease susceptibility. 676 Seminars in Immunopathology Periconceptional maternal micronutrient supplementation is associated 678 with widespread gender related changes in the epigenome: a study of a unique resource 679 in the Gambia DNA methylation differences after exposure to prenatal famine are 681 common and timing-and sex-specific Reduced risk of neonatal respiratory infections among breastfed girls 684 but not boys Sex differences in the effects of maternal vitamin supplements on 686 mortality and morbidity among children born to HIV-infected women in Tanzania Effects of antenatal multiple micronutrient supplementation on 689 birthweight and gestational duration in Nepal: double-blind Sexual dimorphism in immune development and in response to 692 nutritional intervention in neonatal piglets The effects of hormones on sex differences in infection: From genes to 696 behavior The X chromosome and sex-specific effects in infectious disease 698 susceptibility The X chromosome and immune associated genes Modeling host responses in ferrets during A/California/07/2009 influenza 702 infection Multiscale Embedded Gene Co-expression Network Analysis Platelet activation and aggregation promote lung inflammation and 706 influenza virus pathogenesis Identification and characterization of RTVP1/GLIPR1-like genes, a novel 709 p53 target gene cluster DGCA: A comprehensive R package for Differential Gene 711 Roles of endoplasmic reticulum stress in immune responses The unfolded protein response: controlling cell fate decisions under ER stress 715 and beyond Fc epsilonRI, but not FcgammaR, signals induce prostaglandin D2 and 717 E2 production from basophils Critical Roles for PU.1, GATA1, and GATA2 in the expression of human 720 FcepsilonRI on mast cells: PU.1 and GATA1 transactivate FCER1A, and GATA2 721 transactivates FCER1A and MS4A2 Understanding Fc receptor 724 involvement in inflammatory diseases: From mechanisms to new therapeutic tools. 725 Frontiers in Immunology PGE2 deficiency predisposes to anaphylaxis by causing mast cell 727 hyperresponsiveness NAD+-linked 15-hydroxyprostaglandin dehydrogenase: Structure and 730 biological functions Long-chain acyl-CoA synthetase in fatty acid metabolism involved in liver 732 and other diseases: an update Long-chain acyl-CoA synthetase 1 role in sepsis and immunity: 735 Perspectives from a parallel review of public transcriptome datasets and of the literature. 736 Frontiers in Immunology Macrophages and lipid metabolism New insights on the role of lipid metabolism in the metabolic 740 reprogramming of macrophages Interleukin-18 amplifies macrophage polarization and morphological 742 alteration, leading to excessive angiogenesis Interleukin-18 in health and disease Tissue-specific sex differences in human gene expression Widespread sex differences in gene expression and splicing in the 748 adult human brain Sex differences in gene expression and regulatory networks 750 across 29 human tissues Sex Differences in the Blood Transcriptome Identify Robust Changes 752 in Immune Cell Proportions with Aging and Influenza Infection Sex differences in the human peripheral blood transcriptome Autoreactive B cell responses to RNA-related antigens due to TLR7 757 gene duplication TLR7 ligands induce higher IFN-alpha production in females. The 759 Journal of Immunology TLR activation of the transcription factor XBP1 regulates innate 761 immune responses in macrophages Plasma cell differentiation and the unfolded protein response 763 intersect at the transcription factor XBP-1 The roles of apoptosis, autophagy and unfolded protein response in 765 arbovirus, influenza virus, and HIV infections Influenza A viral replication is blocked by inhibition of the inositol-767 requiring enzyme 1 (IRE1) stress pathway Glycomic analysis of host response reveals high mannose as a key 770 mediator of influenza severity Sexual dimorphism of liver endoplasmic reticulum stress 772 susceptibility in prepubertal rats and the effect of sex steroid supplementation Long-chain acyl-CoA synthetase in fatty acid metabolism involved in liver 775 and other diseases: An update Fatty acid activation in carcinogenesis and cancer development: 778 Essential roles of long-chain acyl-CoA synthetases Lipid metabolism in regulation of macrophage functions AP-1: a double-edged sword in tumorigenesis AP-1 as a regulator of cell life and death AP-1 transcription factors as regulators of immune responses in 787 cancer Activator protein 1 (Fos/Jun) functions in inflammatory bone and skin 789 disease Transcriptional regulation in the immune 791 system: all roads lead to AP-1 Targeting inflammation by 793 modulating the Jun/AP-1 pathway. Annals of the Rheumatic Diseases The role of the FcεRI β-chain in allergic diseases. International Archives 796 of Allergy and Immunology Age-Related Pathology Associated with H1N1 A/California Influenza Virus Infection Bioconductor package for 800 differential expression analysis of digital gene expression data Linear models and empirical bayes methods for assessing differential 803 expression in microarray experiments Efficient test and visualization of multi-set 805 intersections Massive mining of publicly available RNA-seq data from human