Sex differences in murine susceptibility to systemic viral infections

Abstract

Increased susceptibility to autoimmunity in females is often viewed as the consequence of enhanced immunoreactivity providing superior protection against infections. We paradoxically observed greater mortality in female compared to male mice during systemic viral infections with three large double-stranded DNA viruses (herpes simplex virus type I [HSV], murine cytomegalovirus [MCMV], and vaccinia virus [VV]). Indeed, female mice were 27-fold more susceptible to infection with HSV than male mice. Elimination of estrogen by ovariectomy in female mice or addition of estrogen to castrated male mice only partially eliminated the observed sex differences following HSV infection. However, the differences observed in survival between female and male mice were nearly abrogated in the absence of type I interferon receptor signaling and substantially mitigated in absence of DAP12 signaling. Interestingly, the sex-specific impact of type I interferon receptor and DAP12 signaling differentially influenced survival during systemic viral infections with type I interferon receptor signaling enhancing male survival and DAP12 signaling increasing the susceptibility of female mice. These results have potential implications for the sex disparities observed in human autoimmune disorders.

Figure 1. Female mice experience reater mortality during systemic viral infections than male mice

Survival curves are shown for female (black diamonds with solid line) and male (unfilled circles with dashed line) mice following infection over a range of inoculum doses of large double-stranded DNA viruses. A) Significantly inferior survival was observed in the female compared to the male mice following systemic infection with HSV. HSV survival curves represent cumulative results from 5 independent experiments with 5–10 mice/dose/experiment (n=198 females and n=186 males). Similar but smaller differences in male and female survival were observed following infection with MCMV (B) and vaccinia (C). MCMV survival curves are shown with cumulative results from 7 and 5 independent experiments with female (n=135) and male (n=127) mice, respectively. Vaccinia survival curves represent aggregated results from four independent experiments (n=157 females and n=106 males). D) Kaplan-Meier analysis is shown of the survival of female (black diamonds with solid line; n=50) and male (unfilled circles with dashed line; n=50) mice following i.p. infection with 3.16×10⁷ pfu of HSV. The data are aggregated from five independent experiments.

Figure 2. Serum inflammatory cytokine levels and viral titers following i.p. infection with HSV

A) No differences were observed in serum proinflammatory cytokine levels in female (black bars; n=3/time-point) and male (white bar; n=3/time-point) mice infected with 3.16×10⁷ pfu of HSV. B) No differences were observed in hepatic viral titers from female (black diamonds) and male (open circles) mice following i.p. infection with 3.16×10⁷ pfu of HSV (3–6 mice/time-point; data at day 1 p.i. are from two independent time-points). C) Significantly higher viral titers were observed in the brainstems of female (black diamonds) mice compared to male (open circles) mice on day 6 p.i. with 3.16×10⁷ pfu of HSV (3–4 mice/time-point). Level of detection of the assay is noted in B) and C) by a dotted line.

Figure 3. Sex differences in susceptibility to systemic HSV infection are only partially attributable to sex steroids

A) The superior survival of male mice is not compromised in the absence of androgens. Survival curves of wt (black circles with dashed line) and castrated (gray unfilled circles with dashed line) male mice are shown following infection with HSV (10 mice/group/inoculum dose). B) The absence of estrogens only partially eliminates the increased susceptibility of female mice during systemic HSV infection. Survival curves of wt (black diamonds) and ovariectomized (gray diamonds) female mice are shown following infection with HSV (10–11 mice/group/inoculum dose; representative of two independent experiments). C) Castrated male mice treated with estrogen (stripped black bar; n=11) experience increased mortality compared to either castrated (outlined dashed bar (n=10) or wt (outlined black bar; n=50, cumulative data from five experiments) male mice following infection with 3.16×10⁷ pfu/mouse HSV but are still substantially more resistant than female mice (filled black bar; n=50, cumulative data from five experiments). Survival data for ovarectomized female mice (gray filled bar; n=21, cumulative from two experiments) are shown for comparison.

Figure 4. Sex differences in susceptibility to systemic viral infections are nearly abrogated in the absence of type I interferon signaling

A) No differences were observed in serum IFNα levels in wt female (black diamonds; n=5–9/time-point) and male (open circles; n=6–9/time-point on days 0, 0.5, and 1 p.i. with n=3 at day 2 p.i.) mice infected with 3.16×10⁷ pfu of HSV. Gray bars represent median values for each time-point. Data are aggregated results from two independent experiments. B) Although the absence of type I interferon signaling results in significantly increased sensitivity to HSV infection, sex differences in mortality are nearly eliminated following systemic infection with all three viruses (HSV, MCMV, and VV) in the absence of type I interferon signaling. LD₅₀ values are shown for wt female (black bars), wt male (black outlined bars), IFNαβR-deficient female (solid grey bars), and IFNαβR-deficient male (outlined grey bars) mice following infection with HSV, MCMV, and vaccinia. C) The observed sex difference in survival following systemic HSV infection is amplified in the setting of IFNαβR-haploinsufficiency. HSV survival curves with IFNαβR+/− mice represent cumulative results from 4 independent experiments with female (diamonds with dashed line; n=61) and male (black unfilled circles with dashed line; n=43) IFNαβR+/− mice. Cumulative survival data for wt female (black diamonds with solid line) and male (black unfilled circles with solid line) mice from Fig. 1A are shown for comparison.

Figure 5. Sex differences in susceptibility to systemic viral infections are mitigated in the absence of DAP12 signaling

A) Sex differences in survival following systemic infection with HSV were significantly reduced in DAP12-signaling deficient mice. Cumulative survival data with female (diamonds with solid line; n=28) and male (circles with dashed line; n=34) DAP12KI mice from four independent experiments following infection with 3.16×10⁷ pfu of HSV demonstrate 71% survival of the female mice. Comparison with wt mice can be made by examining Fig. 1D where 18% of female wt mice survived. B) Brainstem titers are shown for female (black diamonds; n=4, 11, and 4 on days 5, 6, and 7 p.i., respectively) and male (open circles n=4 and 11 on days 5 and 6 p.i., respectively) DAP12KI mice following infection with 3.16×10⁷ pfu of HSV/mouse. Data represent cumulative results from three independent experiments. Dotted line indicates the level of detection in the assay. C) Increased female mortality was not ameliorated by depletion of pDCs prior to infection with 3.16×107 pfu of HSV. Aggregated results from two independent experiments are shown with BDCA2-DTR Tg females (diamonds with solid line; n=10) and males (circles with dashed line; n=10) treated with DT every 2 days to selectively deplete pDCs. D) No differences were observed in serum IFNα levels in DAP12KI female (black diamonds; n=7/time-point) and male (open circles; n=6–7/time-point on days 0 and 0.5 p.i.) mice infected with 3.16×107 pfu of HSV. Gray bars represent median values for each time-point. Data was combined from two independent experiments. E) No differences were observed in serum inflammatory cytokine levels in female (black bars; n=3) and male (white bar; n=3) DAP12KI mice on day 6 p.i. with 3.16×107 pfu of HSV.