key: cord-0283831-q71jn9jj authors: Regis, E.; Fontanella, S.; Lin, L.; Howard, R.; Haider, S.; Curtin, J. A.; Edwards, M. R.; Rattray, M.; Simpson, A.; Custovic, A.; Johnston, S. L. title: Sex differences in innate anti-viral immune responses to respiratory viruses date: 2020-09-18 journal: nan DOI: 10.1101/2020.09.18.20195784 sha: 87dd33f7320b0d1e8a10ce72584512526fc8e72e doc_id: 283831 cord_uid: q71jn9jj Males have excess morbidity and mortality from respiratory viral infections and especially so in COVID-19. The mechanisms explaining this excess in disease burden in males are unknown. Innate immune responses are likely critical in protection against a novel virus like SARS-CoV-2. We hypothesised that innate immune responses may be deficient in males relative to females. To test this we stimulated peripheral blood mononuclear cells (PBMCs) from participants in a population-based birth cohort with three respiratory viruses (rhinoviruses-RV-A16 and RV-A1, and respiratory syncytial virus-RSV) and two viral mimics (R848 and CpG-A, to mimic responses to SARS-CoV-2). We measured interferon (IFN) and IFN-induced chemokine responses and investigated sex differences in virus-induced responses. IFN-, IFN-{beta} and IFN-{gamma} responses to RV-A16 were deficient in males compared to females, fold-inductions being 1.92-fold (P<0.001), 2.04-fold (P<0.001) and 1.77-fold (P=0.003) lower in males than females, respectively. Similar significant deficiencies in innate immune responses were observed in males for eleven other cytokine-stimulus pairs. Responses in males were greater than those in females in only one of 35 cytokine-stimulus pairs investigated. Review of healthcare records revealed that 12.1% of males but only 6.6% of females were admitted to hospital with respiratory infections in the first year of life (P=0.017). Impaired innate anti-viral immunity in males likely results in high morbidity and mortality from respiratory viruses including COVID-19. Males may preferentially benefit from therapies that boost innate anti-viral immune responses. Males have excess morbidity and mortality from respiratory viral infections and especially so in COVID-26 19. The mechanisms explaining this excess in disease burden in males are unknown. Innate immune 27 responses are likely critical in protection against a novel virus like SARS-CoV-2. We hypothesised that 28 innate immune responses may be deficient in males relative to females. To test this we stimulated 29 peripheral blood mononuclear cells (PBMCs) from participants in a population-based birth cohort with 30 three respiratory viruses (rhinoviruses-RV-A16 and RV-A1, and respiratory syncytial virus-RSV) and two 31 viral mimics (R848 and CpG-A, to mimic responses to SARS-CoV-2). We measured interferon (IFN) and confirmed COVID-19 cases admitted to ICU by the 10 th April 2020 were male, and 27.5% female(10). A 69 recent large UK study of 20,133 patients admitted to hospital with COVID-19, reported more men were 70 admitted than women (men 60%, n=12 068; women 40%, n=8065)(11). An even larger study of primary 71 care records of 17,278,392 adults linked to 10,926 COVID-19-related deaths reported COVID-19-related 72 death was associated with: being male with a hazard ratio of 1.59(12). COVID-19 case identification in 73 population screening also reports a male preponderance, with more males than females testing positive 74 in both targeted testing (16.7% vs 11.0%) and in population screening (0.9% vs 0.6%)(13). The mechanisms explaining excess COVID-19 mortality, ICU and hospital admissions and case 76 identification in males are unknown (14) . Understanding how sexes differ in their responses to respiratory 77 viruses is critically important for the development of treatment and preventative strategies, which may 78 differ for males and females. Innate immune responses, mediated by anti-viral interferon (IFN) production 79 by virus-infected cells will be critical in protection against SARS-CoV-2, a new virus that humans have 80 never previously encountered. We have previously reported that deficient IFN-α(15), IFN-β(15, 16) and . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 18, 2020. . https://doi.org/10.1101/2020.09.18.20195784 doi: medRxiv preprint IFN-γ(17, 18) production by virus-infected cells from people with asthma is implicated in their increased 82 susceptibility to respiratory virus infections(19). However, little is known about the variation within the 83 human immune system in relation to the patterns of response to viruses at a population level. We 84 hypothesised that the adverse outcomes for males reported in COVID-19 may be related to deficient 85 innate immune responses to viruses in males relative to females. To investigate this, we stimulated 86 peripheral blood mononuclear cells (PBMCs) from male and female participants in a population-based 87 birth cohort with three common respiratory viruses and two viral mimics (R848 and CpG-A, to mimic 88 responses to SARS-CoV-2) and measured IFN and IFN-induced chemokine responses in supernatants. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 18, 2020. features between participants included in this analysis (n=345) and those who were not (n=406), either in 96 the whole population or stratified by sex (Table S1 ). The demographic and clinical characteristics of the 345 subjects included in this analysis are shown in 98 Table 1 . There were no significant differences between sexes in birth weight, relevant environmental 99 exposures (including position in sibship, pet ownership and tobacco smoke exposure) or common 100 respiratory diseases, such as wheezing and asthma. No significant differences were observed in cell 101 viability between sexes (Fig. S1 ). There was a significant induction of all IFNs and IFN-induced chemokines in response to all viral stimuli 104 compared to medium control (Figures 2 and S2 ). Cytokine responses to viral stimuli were not normally 105 distributed (Shapiro-Wilk test, Table S2 ). The most potent inducers of IFN-α were CpG-A, RSV and RV-106 A16, with median concentrations of 184.7pg/mL, 108.3pg/mL and 34.0pg/mL respectively compared to 107 0.0pg/mL in medium control (all P<0.001, Figure 2 ). RV-A1 and R848 also induced IFN-α, but to lesser 108 degrees (Fig. 2) . Induction of IFN-β followed a very similar pattern to that of IFN-α, but with lower 109 concentrations. The most potent inducers of IFN-γ were R848, RV-A16 and RSV, with median concentrations of 111 102.1pg/mL, 60.7pg/mL and 35.7pg/mL, respectively, compared to 0.2pg/mL in medium control (all 112 P<0.001, Figure 2 ). RV-A1 and CpG-A also induced IFN-γ, but to lesser degrees (Fig. 2) . The most potent inducers of the IFN-induced chemokine CXCL10/IP-10, were RV-A16, RSV and CpG-A, 114 with median concentrations of 1841.0pg/mL, 1515.2pg/mL and 1343.4pg/mL respectively, compared to 115 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 18, 2020. . https://doi.org/10.1101/2020.09.18.20195784 doi: medRxiv preprint 130.6pg/mL in medium control samples (all P<0.001, Figure 2 ). RV-A1 and R848 also induced CXCL-116 10/IP-10, but to lesser degrees. The IFN-induced chemokines CCL2/MCP1, CCL4/MIP-1β and CCL13/MCP4 were also all induced by all 118 viral stimuli, CCL2/MCP1 and CCL4/MIP-1β most potently by R848 and CCL13/MCP4 most potently by 119 RV-A1 (Fig. S2 ). Results of the comparisons between sexes are presented in Table 2 . The trend across all viral stimuli and 122 responses was remarkably consistent, as almost all of the IFNs and IFN-induced chemokines had higher 123 levels of induction in females compared to males: out of 35 stimulus-cytokine pairs, females exhibited 124 higher induction than males in 32 cases (Table 2) . The induction of IFN-a was significantly higher in females than in males for all five viral stimuli (with P-126 values ranging from <0.001 to 0.018, Table 2 ). After adjustment for multiple testing, these differences . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. anti-viral responses, we examined proportions of males and females whose innate anti-viral responses 149 were below certain lower thresholds. We restricted this analysis to IFN-α and low thresholds were defined were observed for response of IFN-α to RV-A1, and CpG-A (Fig. S4 ), but these did not reach statistical 154 significance. Having observed diminished innate immune responses to viral stimuli in males, and greater proportions of 157 males with weak innate immune responses, we investigated whether frequencies of early-life LRTIs were 158 also different between sexes in our cohort. We focussed on early life, as this is when innate immune 159 responses will be most important, as the very young will have had little opportunity to develop memory 160 responses. Results are presented in Table S3 . Among participants attending follow up at age 16 years 161 who had primary care records available for inspection (n=651), 12.1% of males and 6.6% of females were 162 admitted to hospital with LRTI in the first year of life (P=0.017). In the second year of life, 6.1% of males 163 but only 0.75% of females were admitted to hospital with LRTI (P <0.001). We observed a similar trend 164 for RSV-positive bronchiolitis, with 4.32% of males hospitalised, compared to 1.64% of females, but the 165 difference between the sexes did not reach formal statistical significance (P=0.067). 167 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Type I and II IFNs also cooperate and work together to activate macrophages, NK cells, dendritic cells . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 18, 2020. and with severe viral LRTIs in infants in our cohort, make it almost certain that the deficient IFN 218 responses we report in males will be at least in part responsible for the adverse outcomes to SARS-CoV-219 2 infection currently being reported in COVID-19 in males(2, 4-10, 12, 13). . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 18, 2020. . https://doi.org/10.1101/2020.09.18.20195784 doi: medRxiv preprint We were not able to assess the importance of induction of the type III IFNs, IFN-λs 1-3, which are very 248 important in innate antiviral responses against respiratory viruses(31, 33-35), as the type III IFNs were not 249 significantly induced by any infection/stimulus in the PBMCs that we studied (data available on request). This is likely because respiratory epithelial cells are the main source of type III IFNs, and the cells within Our findings also have important implications for a hotly debated topic, namely whether "man flu" actually 296 exists or not(51). Here we provide a biological basis to explain why males would be expected to suffer 297 more than females when infected by respiratory viruses. In conclusion, high morbidity and mortality from respiratory viruses including COVID-19 in males is likely 299 explained by impaired innate anti-viral immune in males compared to females. Males may preferentially 300 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted September 18, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted September 18, 2020. . https://doi.org/10.1101/2020.09.18.20195784 doi: medRxiv preprint Detailed methods are presented in the Supplementary Appendix. The study subjects were participants in the Manchester Asthma and Allergy Study, a population-based 306 birth cohort study(52). The study was approved by the research ethics committee; we obtained written 307 informed consent. PBMCs were collected from subjects at age 16 years and cryopreserved. We used 308 male or female sex as assigned at birth. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted September 18, 2020. The rest of the authors declare that they have no relevant conflicts of interest. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 18, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 18, 2020. . https://doi.org/10.1101/2020.09.18.20195784 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 18, 2020. . https://doi.org/10.1101/2020.09.18.20195784 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 18, 2020. . https://doi.org/10.1101/2020.09.18.20195784 doi: medRxiv preprint Lung infection--a public health priority A new coronavirus associated with human respiratory disease in China Sex influences immune responses to viruses, and efficacy of prophylaxis and 376 treatments for viral diseases Risk factors for severity and mortality in adult COVID-19 inpatients in Wuhan Clinical course and risk factors for mortality of adult inpatients with COVID-19 in 380 China: a retrospective cohort study Novel Coronavirus Pneumonia Emergency Response Epidemiology Characteristics of COVID-19 patients dying in Italy The number of deaths involving COVID-19 for females was lower than males in 387 all age groups Baseline Characteristics and Outcomes of 1591 Patients Infected With SARS-389 CoV-2 Admitted to ICUs of the Lombardy Region r. c. 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