Genetic Variation in Chromosome Y Regulates Susceptibility to Influenza a Virus Infection
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Genetic variation in chromosome Y regulates susceptibility to influenza A virus infection Dimitry N. Krementsova, Laure K. Casea, Oliver Dienzb, Abbas Razaa, Qian Fanga, Jennifer L. Athera, Matthew E. Poyntera, Jonathan E. Boysonb, Janice Y. Bunnc, and Cory Teuschera,d,1 aDepartment of Medicine, University of Vermont, Burlington, VT 05405; bDepartment of Surgery, University of Vermont, Burlington, VT 05405; cDepartment of Medical Biostatistics, University of Vermont, Burlington, VT 05405; and dDepartment of Pathology, University of Vermont, Burlington, VT 05405 Edited by Sabra Klein, Johns Hopkins University, Baltimore, MD, and accepted by Editorial Board Member Peter Palese January 23, 2017 (received for review December 20, 2016) Males of many species, ranging from humans to insects, are more encephalomyelitis (EAE) in SJL/J mice, which are controlled by susceptible than females to parasitic, fungal, bacterial, and viral genetic variation in ChrY (23–25). Moreover, with respect to infections. One mechanism that has been proposed to account for viral infections, we reported that genetic variation in ChrY this difference is the immunocompetence handicap model, which influences both survival following infection with Coxsackie- posits that the greater infectious disease burden in males is due to virus B3 virus (CVB3) (26) and susceptibility to CVB3-induced testosterone, which drives the development of secondary male sex autoimmune myocarditis (27). To address whether genetic vari- characteristics at the expense of suppressing immunity. However, ation in ChrY is capable of influencing susceptibility to IAV and emerging data suggest that cell-intrinsic (chromosome X and Y) the associated sex differences, we studied the susceptibility of a sex-specific factors also may contribute to the sex differences in panel of ChrY consomic strains on the C57BL/6J background infectious disease burden. Using a murine model of influenza A (B6-ChrY), and report that genetic variation in ChrY controls virus (IAV) infection and a panel of chromosome Y (ChrY) consomic susceptibility to IAV in male mice, as well as the observed sex differences. In addition, we show that ChrY variation controlling strains on the C57BL/6J background, we present data showing increased susceptibility to IAV is associated with augmented that genetic variation in ChrY influences IAV pathogenesis in pathogenic immune responses in the lung, including elicitation of males. Specific ChrY variants increase susceptibility to IAV in males proinflammatory IL-17–producing γδ T cells, without affecting and augment pathogenic immune responses in the lung, including viral replication. activation of proinflammatory IL-17–producing γδ T cells, without affecting viral replication. In addition, susceptibility to IAV segre- Results and Discussion gates independent of copy number variation in multicopy ChrY Genetic Variation in ChrY Controls Susceptibility to IAV in Males and gene families that influence susceptibility to other immunopatho- Influences Sex Differences in Susceptibility to IAV. To assess the role logical phenotypes, including survival after infection with coxsack- of ChrY genetic variation on susceptibility to IAV infection, we ievirus B3. These results demonstrate a critical role for genetic studied male mice from a panel of 11 B6-ChrY consomic strains variation in ChrY in regulating susceptibility to infectious disease. of mice using mouse-adapted Puerto Rico A/PR/8/34 H1N1 (PR8) IAV by challenging them with 5 × 103 egg infectious units influenza A virus | chromosome Y | sex | genetic variation | (EIU), equivalent to ∼1 B6-LD50. A significant overall effect of infectious disease strain on the survival of male mice was detected among the B6-ChrY consomic strains studied (χ2 = 49.5, df = 10, P < 0.0001) easonal infection with influenza A virus (IAV) is a significant (Fig. 1). When each B6-ChrY consomic line was compared with Sthreat to human health. IAV infects ∼15% of the world’s population annually, resulting in ∼1 million deaths (1). Throughout Significance history, multiple pandemics (three per century over the last 300 y) INFLAMMATION confirm the importance of IAV, and in the 20th century, three Including sex and gender into preclinical research is central to IMMUNOLOGY AND pandemics in particular have been exceptionally devastating. delineating the mechanisms contributing to sex differences in Epidemiologic evidence from both seasonal outbreaks and health and disease. The majority of sex differences in suscep- pandemics suggests that morbidity and mortality are significantly tibility to infectious diseases, such as influenza A virus (IAV), greater in women than in men (2). For example, a 2010 report are believed to be due to the influence of sex hormones on the from the World Health Organization concluded that the out- immune system; however, the idea that sex chromosomes also comes of pandemic IAV, as well as of avian H5N1, are generally may influence infectious disease susceptibility is being in- worse in females (3). However, in epidemiological studies in creasingly recognized. Our finding that genetic variation in which age and sex were included as covariates, the data indicate chromosome Y (ChrY) influences the survival of male mice that incidence and severity change as a function of age, with following IAV infection, and the associated sex differences in more males affected from birth through age 15 y and more fe- – IAV susceptibility, further establishes a critical role for ChrY in males affected across all postpubertal age ranges (4 17). Im- controlling the male-specific regulatory genome related to in- portantly, these data suggest that nonendocrine cell-intrinsic fectious disease susceptibility. (genetic) sex-specific factors may contribute to disease suscep- tibility in males. In this regard, the concept that genes on the X Author contributions: D.N.K., L.K.C., J.E.B., and C.T. designed research; D.N.K., L.K.C., O.D., and Y chromosomes (ChrX and ChrY) may influence sex dif- A.R., Q.F., J.L.A., and M.E.P. performed research; D.N.K., L.K.C., J.L.A., M.E.P., J.E.B., J.Y.B., ferences is being increasingly recognized (18–21), and it was and C.T. analyzed data; and D.N.K., J.E.B., and C.T. wrote the paper. recently suggested that the widely expressed ancestral single- The authors declare no conflict of interest. copy ChrY genes may function as dosage-sensitive regulators of This article is a PNAS Direct Submission. S.K. is a Guest Editor invited by the Editorial gene expression, translation, and protein stability, and as such may Board. play essential roles in male viability, development, and sex dif- Data deposition: The data reported in this paper have been deposited in the Gene Ex- ferences in health and disease (22). pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE94540). The age-by-sex interaction seen in IAV infection is strikingly 1To whom correspondence should be addressed. Email: [email protected]. reminiscent of the age-dependent sex differences in immune This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. responsiveness and susceptibility to experimental autoimmune 1073/pnas.1620889114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1620889114 PNAS | March 28, 2017 | vol. 114 | no. 13 | 3491–3496 Downloaded by guest on September 26, 2021 100 IAV was not addressed in this model, and our current findings show that pairing of different ChrY with B6-autosomes and B6- 80 ChrX can in fact influence IAV susceptibility. RF (15) B6-like B6 (45) SJL (9) 129S1 (16) ChrY Influences Pathogenic Inflammatory Responses in IAV-Infected 60 MA (19) LEWES (11) Lung. To understand the mechanisms underlying the increased susceptibility to IAV in ST-like ChrY consomic male mice, we 40 PWD % Survival A/J* (22) compared B6 mice and ChrY mice. The latter were selected ST-like because they exhibited the second-highest mortality overall, and 20 SWR** (12) BUB*** (14) because the PWD/PhJ strain is highly genetically divergent PWD** (10) PWD ST*** (14) compared with B6 (30, 31). Male B6 and ChrY mice were 0 0 5 10 15 20 infected with the same dose of IAV as in the mortality studies, Day Post-Infection followed by isolation of whole-lung RNA at the peak of viral replication, on day 6 postinfection, just before the onset of IAV- Fig. 1. ChrY polymorphism influences survival following primary infection induced mortality (Fig. 1). Viral replication was assessed using with IAV. Male mice from the indicated B6-ChrY consomic strains of mice quantitative RT-PCR (qRT-PCR) for viral RNA, and no differ- PWD were challenged with 5 × 103 EIU of PR8 virus. Data shown represent the ences between B6 and ChrY mice were found (SI Appendix, PWD combined results of two to three independent experiments per strain, with Fig. S2A), suggesting that the increased mortality in ChrY the total number of mice indicated in parentheses behind each strain name. mice is not due to enhanced viral replication. In addition, analysis Overall significance of differences in survival was determined using the log- of bronchoalveolar lavage (BAL) fluid did not reveal a significant rank (Mantel–Cox) test, with post hoc comparisons of each strain vs. B6 difference in the number and type of leukocytes between the two computed using the Dunnett–Hsu correction for multiple comparisons. A strains (SI Appendix,Fig.S2B and C), or in the production of the significant effect of ChrY on overall survival was detected (χ2 = 49.5, df = 10, proinflammatory cytokines IL-6, IFNγ, and TNFα (SI Appendix, P < 0.0001). The significance of the difference from B6 is indicated next to Fig. S2 D–F). ≤ ≤ ≤ each strain name, designated as follows: *P 0.05; **P 0.01; ***P 0.001. To obtain a global unbiased perspective of the pathogenic The following strains were not significantly different from B6, as indicated by RF SJL 129S1 MA events at the site of infection, we performed transcriptomic pro- a lack of a symbol next to the strain name: ChrY ,ChrY ,ChrY ,ChrY , filing of the infected lung.