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in chromosome Y regulates susceptibility to influenza A 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 , ranging from to , are more encephalomyelitis (EAE) in SJL/J mice, which are controlled by susceptible than 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 burden in males is due to virus B3 virus (CVB3) (26) and susceptibility to CVB3-induced , which drives the development of secondary male 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 -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 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 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 , 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 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 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, , and 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- 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 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 (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. We found that 32 genes showed sig- and ChrYLEWES. nificantly higher expression in ChrYPWD lungs relative to B6 lungs, whereas 98 genes showed lower expression (Fig. 3 A and B). SJL 129S1 RF MA LEWES Strikingly, whereas the majority (20 of 32) of the genes showing B6, ChrY , ChrY , ChrY , ChrY , and ChrY PWD consomic strains did not exhibit a significant difference in survival increased expression in ChrY lungs represented coding genes, from B6 (designated B6-like); however, ChrYA/J, ChrYSWR, only a minority of the of the genes with decreased expression were BUB PWD ST coding (14 of 98) (Fig. 3B). Instead, the majority (63 of 98) of ChrY , ChrY ,andChrY exhibited significantly lower PWD survival compared with B6 [designated ChrYST(ST)-like]. genes showing decreased expression in ChrY lungs were To test whether or not the effect of ChrY on IAV suscepti- classified as small , suggesting that the presence of a het- bility in males correlates with weight loss, we regressed the strain erologous ChrY may regulate the expression of small RNAs as distribution pattern (SDP) for percent survival against the SDP part of its mechanism regulating global gene expression. In this for mean maximum percent weight loss. Although a significant regard, we previously showed that ChrY can regulate global gene effect of ChrY on mean maximum percent weight loss was de- expression, including the expression of splice variants and miRNA, tected (F = 2.0, P = 0.03), mean maximum % weight loss was not in a cell type-specific and background-specific fashion (27). Importantly, several genes exhibiting higher expression in significantly associated with survival (F = 0.7, P = 0.4; r = −0.3, PWD P = SI Appendix ChrY lungs were of immunologic relevance: two genes 0.4) ( , Fig. S1). Similarly, mean % weight loss γδ was not correlated with survival among the B6-like (F = 0.1, coding for the T-cell receptor (TCR) and several genes P = r = P = F = P = r = − encoding Ig heavy and chains, suggesting potential differ- 0.8; 0.2, 0.8) or ST-like ( 2.0, 0.3; 0.6, γδ P = 0.3) strains. Taken together, these data establish that genetic ences in recruitment and/or activation of T cells and B cells (Fig. 3C). The former is of particular interest, given the reported variation in ChrY influences survival of male mice following – γδ primary infection with IAV, and does so independent of association between IL-17 producing lung T cells and acute weight loss. To assess the impact of ChrY genetic variation on the sex dif- ference in susceptibility to IAV, we pooled the male consomic 100 survival data into two groups, B6-like and ST-like (based on sig- nificance of difference in survival compared with B6; see above), 80 and compared these data with survival data for B6 male and fe- χ2 = B6 M male mice (Fig. 2). Compared with B6 females, both B6 ( 8.2, B6 M-like df = 1, P = 0.004) and B6-like males (χ2 = 11.2, df = 1, P = 0.0008) 60 ** were significantly less susceptible to IAV infection, as expected; *** however, ST-like males were not significantly different from B6 40 females (χ2 = 1.1, df = 1, P = 0.5). The sex difference seen be- % Survival B6 F (30) ns tween B6 and B6-like males vs. B6 females, whereby females 20 ST M-like display greater susceptibility to IAV, is consistent with the pre- viously reported sex difference between B6 males and females (2, 0 28), and our results suggest that this sex difference can be over- 0 5 10 15 20 ridden by ChrY variants in the ST-like group. Taken together, these data establish that genetic variation in ChrY influences the Day Post-Infection survival of male mice following primary infection with IAV, and Fig. 2. ChrY polymorphism significantly impacts the sex difference in survival thus contributes to the associated sex differences. following primary infection with IAV. Male (M) and (F) B6 and ChrY Previous work using the four core genotypes model on the B6 consomic mice were challenged with IAV as in Fig. 1. The survival data for background [B6-XX, B6-XY, B6.Cg-Srydl1Rlb, and B6.Cg- dl1Rlb female B6 mice were compared with that of B6 males, the pooled survival data Tg(Sry)2EiSry ] has shown that the com- for B6-like males, and ST-like males. The significance of differences in survival plement does not impact the susceptibility to IAV (29). However, compared with B6 females was determined using the log-rank (Mantel–Cox) the effect of natural genetic variation in ChrY on susceptibility to test and is indicated as follows: **P ≤ 0.01; ***P ≤ 0.001; ns, not significant.

3492 | www.pnas.org/cgi/doi/10.1073/pnas.1620889114 Krementsov et al. Downloaded by guest on September 26, 2021 + lungs can be accounted for largely by an increase in Vγ4 and ABPWD C γ − γ − γδ γ + C D B6 ChrY Cyp2e1 V 1 V 4 T cells (most likely V 6 ) (Fig. 4 and ). We next PWD γδ Coding Down in Y Ighv6-6 examined cytokine production by lung T cells using in- Up in YPWD Ighv1-78 miRNA Car3 tracellular and flow cytometry, and found a strong in- PWD Igkv12-89 – γδ Non-coding crease in IL-17 producing T cells in ChrY lungs compared Igkv4-61 γ Small RNA Cfd with B6, whereas no significant difference in IFN -producing cells Ighv1-50 was observed (Fig. 4 E and F). Interestingly, no significant dif- Ribosomal Ighv5-16 Igkv4-74 ferences in IL-17 or IFNγ production by αβ T cells were observed 0 20406080 Igkv4-86 G H Number of genes Ighv1-72 (Fig. 4 and ). Taken together, our results demonstrate that PWD Igkv4-51 increased mortality in ChrY mice is associated with a strongly Ighv1-62-3 augmented presence of proinflammatory IL-17–producing γδ T Igkv10-95 + − − Tcrg-C3 γ γ γ Cidec cells belonging to the V 4 and V 1 V 4 subsets. Ighv1-67 Ighv5-9 Adipoq ChrY-Dependent Differences in Adult Serum Testosterone Levels Trgj1 and Prenatal Exposure to Testosterone Do Not Correlate with Gm4832 Serpina3f Susceptibility to IAV Infection. The current paradigm is that the Gm21738 Gm10721 sex differences in IAV infection is believed to be primarily a Gm10719 function of the cell-extrinsic immunostimulatory effects of 17β- Gm10720 Gm10718 estradiol and immunosuppressive effects of testosterone (T) on Ide the immune system (36). To test whether the effect of ChrY on Gm10717 Gm11168 IAV susceptibility in males is related to ChrY-dependent dif- Gm17535 Gm10715 ferences in adult serum T, we regressed the SDP for T levels in Gm10800 -8 0 10 naïve adult mice measured in our previous studies (26, 27) against the SDP for survival following primary infection. Al- Fig. 3. ChrY modulates the transcriptional profile in the IAV-infected lung. though a significant effect of ChrY on adult serum T levels was Male B6 and ChrYPWD mice were infected with 5,000 EIU of PR8. On day 6 detected (F = 3.2, P = 0.001) (26, 27), T levels were not signif- postinfection, whole-lung RNA was extracted and subjected to transcriptional icantly associated with survival (F = 2.8, P = 0.1; r = 0.5, P = 0.1) profiling and differential expression analysis, as described in Materials and (SI Appendix, Fig. S3), suggesting that ChrY-dependent differ- Methods.(A) Heatmap of gene expression (displayedrelativetotherowmean) PWD ences in adult serum T levels are unlikely to be major drivers of of all genes exhibiting differential expression in B6.ChrY compared with B6 differential susceptibility to IAV in ChrY consomic mice, al- lungs (jfold changej>2; P < 0.05, ANOVA). (B) Major categories of differentially – expressed genes. (C)Heatmapofexpression(displayedasfoldchangeinB6. though a differential effect of ChrY on T levels and/or ChrY T PWD interactions (19) during IAV infection cannot be completely ChrY lungs relative to B6) of all coding genes exhibiting significant differ- P = ential expression. Genes of interest are highlighted in bold or italic type. ruled out. The data are marginally suggestive (at 0.1) of a possible positive relationship between T levels and survival post- IAV challenge, which is in agreement with work by the Klein lung injury caused by IAV infection (32). To confirm the tran- group (29, 37) demonstrating that T is protective in IAV scriptional profiling results, we isolated cells from infected lungs infection. of B6 and ChrYPWD mice at day 6 after PR8 infection. Flow Exposure to different levels of androgens during development cytometry analysis identified a twofold increase in the number plays an important role in programming many adult phenotypes and proportion of γδ T cells in ChrYPWD lungs compared with and provides a proxy for determining differential exposure to B6, which was not observed for αβ T cells (Fig. 4 A and B). androgen levels during development (38–41). Therefore, to ex- Despite the increase in Ig gene expression in ChrYPWD lungs amine whether the effect of ChrY on IAV susceptibility is re- compared with B6 (Fig. 3C), we did not observe a significant lated to developmental differences in ChrY-dependent T levels, difference in the number or proportion of B cells in the lung we compared the SDPs for IAV survival with the 5-wk-old INFLAMMATION

(Fig. 4 A and B). weight-adjusted anogenital distance index (AGDI) (42), as de- IMMUNOLOGY AND Lung γδ T cells are divided into several major subsets based on scribed previously (21). Although a significant effect of ChrY on + + + + + their TCR use: Vγ1 ,Vγ4 , and Vγ6 (33). The Vγ4 and Vγ6 AGDI was detected (F = 3.8, P < 0.0001), this was not signifi- subsets are of particular interest, given their propensity to pro- cantly associated with IAV susceptibility (F = 1.6, P = 0.2; r = duce IL-17 and promote inflammation in the lung and other 0.4, P = 0.2) (SI Appendix, Fig. S4), suggesting that differential + + organs (34, 35). Analysis of the Vγ1 and Vγ4 subsets in the susceptibility to IAV is unlikely to be a function of ChrY- infected lung revealed that the increase in γδ T cells in ChrYPWD dependent differences in T exposure during development.

Fig. 4. ChrY regulates the frequency of γδ T cells in A B C D ) ) 3 the IAV-infected lung, and their production of IL-17. 40 6 10 80 300 **** ** 35 8 **** **** PWD 30 6 Male B6 and ChrY mice were infected with 5,000 60 25 4 20 200 2 EIU of PR8. On day 6 postinfection, lung leukocytes 15 8 **** 0.8 **** 40 were isolated, counted, and subjected to flow 6 0.6 100 4 0.4 20 2 0.2 % Positive Cells cytometry analysis, as described in Materials and % Positive Cells 0 0.0 0 0 Cell Number (x10 + + + Cell Number (x10 + + + + - - + + - - + Methods.(A and B) Proportion (A) and frequency CD19 TCRβ TCRγδ CD19 TCRβ TCRγδ Vγ1 Vγ1 Vγ4 Vγ4 Vγ1 Vγ1 Vγ4 Vγ4 (B) of cells expressing the indicated markers (CD19, E F G H ) β γδ + 10 3 40 10 4 TCR , and TCR ) that were gated on live CD45 cells. **** + − − 8 ** 8 TCRγδ cells were also pregated on CD19 TCRβ 30 3 − 6 6 20 2 CD11b cells. (C and D) Proportion (C) and frequency 4 4 10 1 (D) of cells expressing the indicated markers (Vγ1 and 2 2 + + − − % Positive Cells % Positive Cells γ γδ β % Positive Cells 0 0 0 0 V 4), gated on live TCR CD45 CD19 TCR + + Cell Number (x10 + + + + + + − IFNγ IL-17 IFNγ IL-17 IFNγ IL-17 IFNγ IL-17 CD11b cells. (E and F) Proportion (E) and frequency + − − (F) of cells positive for the indicated intracellular cytokines (IFNγ and IL-17) gated on live TCRγδ TCRβ CD11b cells. (G and H) Proportion of cells positive for the indicated cytokines gated on live TCRβ+ CD4+ (G) and TCRβ+ CD8+ (H) cells. The significance of observed differences was determined using two-way ANOVA, followed by post hoc comparisons using Sidak’s correction for multiple comparisons. **P < 0.01; ****P < 0.0001.

Krementsov et al. PNAS | March 28, 2017 | vol. 114 | no. 13 | 3493 Downloaded by guest on September 26, 2021 Differential Susceptibility to IAV Does Not Correlate with Sry EAE (Sly, Ssty1, Ssty2, Sry,andRbmy), CVB3-induced AI myo- Defining Mus mus musculus and Mus mus domesticus A and B Type carditis (Sly and Rbmy), and survival following CVB3 infection ChrYs. Sry (sex-determining region of ChrY) expression alleles, (Ssty1, Ssty2,andRbmy). In this regard, a comparison of the SDPs which when inherited by B6 mice heterozygous for the t-complex for survival following primary infection with IAV and that for encoded TOrleans on Chr17, lead to varying degrees of CVB3 (F = 2.3, P = 0.2; r = −0.5, P = 0.2) (SI Appendix,Fig.S6) sex reversal (43–45). We have previously shown that neither further supports the concept that the ChrY genetic variation un- survival of male CVB3-infected B6-ChrY mice nor the suscep- derlying the phenotypic differences in survival following primary tibility to EAE correlate with the distribution of these Sry ex- infection with IAV and CVB3 is distinct. pression alleles (26, 27). Furthermore, we found that Sry Taken together, these data suggest that unidentified ChrY expression alleles do not influence the level of adult T pro- genetic variation that is unrelated and not linked to CNV in duction in the B6-ChrY consomic strains (26). To examine ChrY multicopy genes controls survival following IAV infection, whether Sry alleles influence susceptibility to IAV, we compared and modifies the sex differences in IAV pathogenesis. In this the SDP of Sry alleles with the SDP for survival following pri- regard, it has been suggested that the widely expressed single- mary infection with IAV. The results revealed that Sry alleles, copy ChrY genes function as regulators of gene expression, which define M. m. musculus and M. m. domesticus A and B type translation, and protein stability, and as such likely play essential ChrYs (43–45), do not correlate with susceptibility to IAV (r = roles in male viability, development, and sex differences in health −0.3, P = 0.3) (SI Appendix, Fig. S5). and disease (22). However, directly testing the hypothesis that polymorphism in one or more of these genes controls the sus- Copy Number Variation in ChrY Multicopy Genes Does Not Correlate ceptibility to IAV and the sex differences will require sequencing with Survival of Male Mice Following Primary Infection with IAV. The the ∼3.5-Mb short arm of ChrY, the gene rich region encoding long arm of murine ChrY encodes several multicopy gene fam- these genes (47), across each of the strains used in this study, as ilies, which exhibit significant strain-specific copy number vari- was recently done for the male-specific region of ChrY in the rat ation (CNV), and the physiological roles of which are only where MSY genetic variation was reported to alter a broad range beginning to be understood (20, 21). Importantly, our studies in of inbred rat phenotypes (48). EAE and CVB3-induced autoimmune myocarditis revealed a Notwithstanding, it is worth noting that a spontaneous in strong association between CNV in multicopy ChrY genes and the long arm of ChrY has been shown to influence lymphocyte susceptibility to these autoimmune diseases (27, 46). Based on development (49), raising the possibility that the genome-wide these results, we hypothesized that CNV in multicopy ChrY regulatory activity of ChrY also may involve changes in the spatial genes may also control survival following primary infection with positioning of X-linked and autosomal genes associated with tran- IAV. Unexpectedly, CNV in any of the ChrY multicopy genes scription, that is, movement toward or away from transcriptional did not correlate with differences in survival following primary centers (50). Our findings demonstrate that ChrY variants regulate infection with IAV (Table 1). the expression of small RNAs with striking directionality (Fig. 3C), To test whether the lack of an association between CNV and IAV further strengthening the concept of epigenetic or structural regu- pathogenesis is a general feature of viral infections, we examined the lation of genome-wide gene expression by ChrY. In addition, and relationship of CNV in ChrY multicopy genes (46) with survival consistent with structural regulation, none of the differentially following primary infection with 50 pfu of CVB3 (26). Regression expressed genes in our analysis were found to reside on ChrY. and correlation analysis comparing the SDP of survival with CNV at The primary mechanisms through which IAV infection leads Rbm31y (RNA binding motif 31, Y-linked), Rbmy (RNA binding to death focus on the relative contribution of direct viral cyto- motif protein, ), Sly (Sycp3 like Y-linked), Srsy (serine- pathicity vs. immunopathologically mediated damage, leading to rich, secreted, Y-linked), Ssty1 (spermiogenesis-specific transcript on loss of homeostasis and respiratory function. The direct cyto- the Y 1), and Ssty2 revealed that CVB3 survival is in fact associated pathicity model posits that although the innate immune system with CNV at Ssty1 (F = 15.5, P = 0.002; r = −0.8, P = 0.005), Ssty2 reaches a high level of activation (51–54), it nevertheless is in- (F = 7.4, P = 0.02; r = −0.6, P = 0.02), and Rbmy (F = 6.9, P = capable of containing the pathogen before viral cytopathicity 0.02; r = 0.6, P = 0.02) (Table 1). causes loss of lung homeostasis (55, 56). In this view, the in- The lack of an association between CNV in ChrY multicopy flammatory response is a correlate of the damaging infection and genes and susceptibility to IAV suggests that the genetic variation not directly immunopathologic. In contrast, the immunopathol- underlying the phenotypic differences in survival following primary ogy model posits that lethality is immunopathologic in nature, infection with IAV is distinct from that controlling susceptibility to with lung function dysregulated largely through the damaging effects of leukocytes on epithelial and endothelial cells (57, 58). Our finding that differential susceptibility to IAV infection be- Table 1. Results of regression and correlation analysis of % tween B6 and B6-ChrYPWD mice is associated with a strongly survival following IAV and CVB3 infection with CNV in multicopy augmented presence of proinflammatory IL-17–producing γδ T + − − ChrY genes cells belonging to the Vγ4 and Vγ1 Vγ4 subsets, and not with differences in viral replication, is consistent with the role of Multicopy gene IAV CVB3 ChrY genetic variation in mediating its effects via immuno- Sly F = 0.02 (0.9) F = 1.8 (0.2) pathologic damage rather than viral cytopathicity. Additional r = 0.05 (0.9) r = 0.4 (0.2) studies are needed to address whether this mechanism is com- Ssty1 F = 0.8 (0.4) F = 15.5 (0.002) mon to all ST-like ChrY consomic male mice. Interestingly, our r = 0.3 (0.4) r = −0.8 (0.002) finding that differential survival was not associated with differ- Ssty2 F = 1.6 (0.2) F = 7.4 (0.02) ences in the number of neutrophils in BAL fluid suggests that r = 0.4 (0.2) r = −0.6 (0.02) ChrY genetic variation controlling IAV-induced lethality does so Srsy F = 0.01 (0.9) F = 0.01 (1.0) independently of the recently described chemokine-driven feed- r = 0.03 (0.9) r = 0.02 (1.0) forward circuit involving proinflammatory neutrophils (59). With respect to the role of human ChrY genetic variation in Rbmy F = 0.02 (0.9) F = 6.9 (0.02) viral infections, an association between ChrY I and r = −0.05 (0.9) r = 0.6 (0.02) = = AIDS progression in HIV-infected men has been identified. Rbmy31 r 0.3 (0.5) r 0.3 (0.3) Among European Americans, men inheriting ChrY haplogroup I The significance of associations was determined by linear regression (F) show accelerated progression to AIDS and related death, as well and the Pearson product-moment correlation test, except for Rbmy31, which as delayed HIV-1 viral suppression during HAART therapy, was determined using the Spearman rank-order correlation test. Numbers in compared with other ChrY (60). Interestingly, a parentheses are the associated P values. genetic evaluation of ChrY haplogroup I has not identified the

3494 | www.pnas.org/cgi/doi/10.1073/pnas.1620889114 Krementsov et al. Downloaded by guest on September 26, 2021 particular genetic variant associated with AIDS progression (61). and 5% (vol/vol) FCS before being passed through a 100-μm-pore filter. Cells Taken together, these observations and our present results were counted as above, and 2 million cells were used for multicolor surface suggest that genetic variation in ChrY plays a critical role in staining and flow cytometry, using fluorophore-conjugated antibodies to regulating infectious disease pathogenesis in mammals. Clearly, detect the following antigens: CD45, CD19, CD11b, TCRβ, -TCRγδ,Vγ1, only when we have obtained a detailed understanding of the and Vγ4 (Biolegend). Dead cells were excluded using the Live-Dead UV-Blue sex-specific regulatory in infectious disease patho- staining ( Technologies). genesis will we be able to fully appreciate the mechanisms For intracellular cytokine staining, another 2 million cells were stimulated underlying sex differences in health and disease, and move with 5 ng/mL PMA and 250 ng/mL ionomycin (Sigma-Aldrich) in the presence forward with modeling sex-specific therapeutic interventions of Golgi Plug reagent (BD Biosciences) for 4 h. Cells were then labeled with the UV-Blue Live-Dead fixable stain and then labeled for the following based on targeting cell-extrinsic and/or cell-intrinsic sex-specific γδ β pathways. surface antigens: CD11b, CD4, CD8, pan-TCR , and TCR . Cells were then fixed with 1% paraformaldehyde (Sigma-Aldrich), permeabilized with PBS – γ Materials and Methods containing 0.2% saponin, and labeled with anti IL-17A and anti-IFN anti- bodies (Biolegend). Labeled cells were analyzed on an LSRII flow cytometer Mice. All of the mice used in this study were bred and maintained in the (BD Biosciences). facility at the University of Vermont. The B6-ChrY consomic panel of mice used in this study was purchased from The Jackson Laboratory. The Transcriptional Profiling of PR8-Infected Lungs. Whole-lung RNA was isolated number of backcrosses performed before cryopreservation is indicated next as described above. RNA integrity and quality were assayed using an Agilent to each strain when this information was available on The Jackson Laboratory 2100 Bioanalyzer. Based on RNA quality, five RNA samples per group (B6 and website. Males from each consomic strain were backcrossed to female C57BL/ B6.ChrYPWD) were selected for microarray analysis. Each sample represented 6J mice for an additional five generations before experimentation: C57BL/6J an individual mouse. Microarray analysis was performed using the Clariom D (B6), C57BL/6J-ChrY129S1/SvImJ/NaJ (B6-ChrY129), C57BL/6J-ChrYA/J/NaJ (B6-ChrYA/J), platform with a WT PLUS Target Prep Kit (Affymetrix) at the University of C57BL/6JEi-ChrYBUB/BnJ/EiJ (B6-ChrYBUB) N22, C57BL/6JEi-ChrYLEWES/EiJ (B6- Vermont’s Vermont Network Microarray Facility, as follows. RNA ChrYLEWES) N33, C57BL/6-ChrYPWD/Ph/ForeJ (B6-ChrYPWD) N14, C57BL/6JEi- (100 ng) was used to synthesize cDNA through a First-Strand reverse-tran- ChrYRF/J/EiJ (B6-ChrYRF) N22, C57BL/6JEi-ChrYSJL/J/EiJ (B6-ChrYSJL)N20, scription reaction and Second-Strand RNA degradation reaction, using re- C57BL/6JEi-ChrYST/bJ/EiJ (B6-ChrYST)N20,B6Ei.MA-AChrYMA/MyJ/EiJ (B6- agents from the GeneChip WT PLUS Reagent Kit (Affymetrix). cRNA was ChrYMA) N19, B6Ei.SWR-AChrYSWR/J/EiJ (B6-ChrYSWR) N20. then synthesized through an overnight (16-h) reaction, which uses a T7 RNA polymerase. The cRNA was purified using an Affymetrix and Infection. The H3 variant of CVB3 was made from an infectious magnetic bead protocol. Sample concentrations were determined using a cDNA clone as described previously (62). Mice were infected with 50 pfu of 40 μg/mL/A260 constant on a Nanodrop 1000 spectrophotometer. virus i.p. in 0.5 mL of PBS (26). μ Mouse-adapted Puerto Rico A/PR/8/34 H1N1 (PR8) virus was used to infect Approximately 15 g of cRNA was then used to synthesize sense, single- mice. Male and female mice were anesthetized using isoflurane and infected strand cDNA using reagents from the GeneChip WT PLUS Reagent Kit. Any 3 remaining cRNA template was hydrolyzed with RNase H. Single-stranded intranasally with 5 × 10 EIU (∼1LD50) of PR8 in 0.05 mL of PBS under brief isoflurane anesthesia. Mice were monitored daily for weight loss and other cDNA was purified using an Affymetrix magnetic bead protocol. Sample μ clinical signs of illness. that became grossly moribund were euthanized. concentrations were determined using a 33 g/mL/A260 constant on a Nanodrop 1000 spectrophotometer. Approximately 5.5 μg of single- stranded cDNA was fragmented using uracil DNA-glycosylase (10 U/μL) and BAL Fluid Collection, Lung RNA Analysis, and Lung Leukocyte Isolation and PWD 3 apurinic/apyrimidinic endonuclease 1 (1,000 U/μL), provided in the GeneChip Characterization. Male B6 and B6-ChrY mice were infected with 5 × 10 WT PLUS Reagent Kit. Samples were then labeled with biotin using terminal EIU of PR8. On day 7 postinfection, animals were euthanized by pentobar- μ bital injection. For collection of BAL fluid, 1 mL of PBS was slowly instilled deoxynucleotidyl transferase (30 U/ L), also provided in the GeneChip WT into the lung intratracheally using a cannula, and fluid was collected by PLUS Reagent Kit. The efficiency of the fragmentation and labeling reac- aspiration. BAL fluid cells were pelleted by centrifugation and then stained tions was verified using NeutrAvidin (10 mg/mL) with a gel-shift assay. with propidium iodide (1 μg/mL) for dead cell exclusion. Live cells were Samples were combined with a hybridization mix, injected into mouse counted with a MACSQuant VYB flow cytometer (Miltenyi Biotec), and 2.5 × Clariom D arrays, and placed in an Affymetrix GeneChip Hybridization Oven 104 cells per mouse were cytospun (Cytospin Cytocentrifuge; Thermo Fisher 645 at 45 °C and 60 rpm for 16.5 h overnight. Arrays were stained using an Scientific) onto glass slides and then stained with the Hema-3 kit (Bio- Affymetrix GeneChip Fluidics Station 450 and scanned with an Affymetrix chemical Sciences) in accordance with the manufacturer’s instructions. GeneChip Scanner 3000 7G. INFLAMMATION

Cytospins were examined on a light using 10× and 40× objec- Raw intensity CEL files were imported into Expression Console software IMMUNOLOGY AND tives, and differential cell counting was performed by a blinded observer (Affymetrix), and CHP files were generated for gene-level analysis. CHP files who identified cell types by morphology in 10 different randomly selected were imported into Transcriptome Analysis Console software (Affymetrix), fields per slide. BAL fluid cytokine (IL-6, IFNγ, TNFα, IL-4, GM-CSF, and IL-17) and gene level differential expression analysis was performed using the production was examined by ELISA using capture and detection antibody default one-way between-subject unpaired ANOVA settings (jfold changej>2; pairs, as described previously (63). The latter three cytokines were below the P < 0.05, ANOVA) to identify genes exhibiting significantly different expres- limit of detection. sion between B6 and B6.ChrYPWD lungs. Array results have been deposited into After BAL fluid collection, lungs were used for RNA extraction, as follows. the Gene Expression Omnibus system. Lungs were removed and placed into tubes containing 2.3-mm zirconia/silica beads (BioSpec) and lysis Buffer RLT (RNEasy Miniprep Kit; Qiagen). Lungs Statistical Analysis. Statistical analyses were performed using GraphPad were homogenized using a BeadBeater homogenizer (BioSpec), and RNA was Prism version 6.07 and SAS version 9.4 (SAS Institute). The specific tests used extracted in accordance with the manufacturer’s protocol. Influenza viral to assess the significance of the observed differences are detailed in the load was determined by measuring the RNA expression of the viral PA gene figure legends. A P value of ≤0.05 was considered to indicate statistical by qRT-PCR, which has been shown to correspond to viral titers by plaque significance. assays (64). The following primers were used: PR8-PA forward, GAGCC- TATGTGGATGGATTC; PR8-PA reverse, TGCAGTTCTGCCAGTACTTG. Data ACKNOWLEDGMENTS. We thank Dr. Laura Haynes (University of Connect- were normalized to the expression of the housekeeping gene, B2m. SYBR icut School of Medicine) for providing the PR8 influenza virus. Flow Green-based qRT-PCR was performed as described previously (65). cytometry was conducted at the Harry Hood Bassett Flow Cytometry and In a second series of experiments, animals were infected and euthanized as Cell Sorting Facility, University of Vermont College of Medicine. This work above. To isolate lung leukocytes, lungs were inflated with 1 mL of enzymatic was supported by NIH Grants NS061014, AI041747, NS060901, NS036526, digestion buffer (DMEM; HyClone), 1 mg/mL collagenase type IV (Life and NS069628 (to C.T.); AI119974 (to J.E.B.); and P20GM103496 (to O.D.). The research performed at the Vermont Genetics Network microarray facility Technologies), and 0.2 mg/mL DNase (Sigma-Aldrich), after which they were was supported by an Institutional Development Award from the National dissected away from the trachea and finely minced with scissors. Lung tissue Institute of General Medical Sciences (NIGMS) of the NIH under Grant was resuspended in enzymatic digestion buffer and shaken at 200 rpm at P20GM103449, and research performed at the Flow Cytometry and Cell 37 °C for 20 min. The samples were triturated twice with a 16-gauge needle, Sorting Facility was supported by NIGMS Grant P20GM103496. The contents with another intermediate shaking step of 20 min. Red blood cells were of this paper are solely the responsibility of the authors and do not lysed using Gey’s solution and the digested tissue was washed with 5 mL PBS necessarily represent the official views of the NIGMS or the NIH.

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