Sex-Based Differences in Susceptibility to Severe Acute Respiratory Syndrome Coronavirus Infection

Abstract

Pathogenic human coronaviruses (CoVs), such as the severe acute respiratory syndrome (SARS)-CoV and the Middle East respiratory syndrome-CoV, cause acute respiratory illness. Epidemiological data from the 2002-2003 SARS epidemic and recent Middle East respiratory syndrome outbreak indicate that there may be sex-dependent differences in disease outcomes. To investigate these differences, we infected male and female mice of different age groups with SARS-CoV and analyzed their susceptibility to the infection. Our results showed that male mice were more susceptible to SARS-CoV infection compared with age-matched females. The degree of sex bias to SARS-CoV infection increased with advancing age, such that middle-aged mice showed much more pronounced differences compared with young mice. Enhanced susceptibility of male mice to SARS-CoV was associated with elevated virus titers, enhanced vascular leakage, and alveolar edema. These changes were accompanied by increased accumulation of inflammatory monocyte macrophages and neutrophils in the lungs of male mice, and depletion of inflammatory monocyte macrophages partially protected these mice from lethal SARS. Moreover, the sex-specific differences were independent of T and B cell responses. Furthermore, ovariectomy or treating female mice with an estrogen receptor antagonist increased mortality, indicating a protective effect for estrogen receptor signaling in mice infected with SARS-CoV. Together, these data suggest that sex differences in the susceptibility to SARS-CoV in mice parallel those observed in patients and also identify estrogen receptor signaling as critical for protection in females.

Figure 1. Male mice are more susceptible to MA15 infection than female mice

(A) 9 month male and female mice were infected with 1250 PFU, 5000 PFU and 10⁴ PFU of MA15 and survival was monitored for 12 days. (B) 2 and 9 month-old B6 male and female mice were infected with 5000 PFU and 18-20 months B6 mice were challenged with 1000 PFU MA15. Survival was monitored for 12 dpi. (C) 2, 5 and 9 month-old BALB/c were infected with 10⁴ and 50 PFU, respectively, of MA15 and were monitored for morbidity and mortality. A and B, Data are derived from 2 independent experiments with of 4-5 mice/group/experiment. C, Data are derived from 2-3 independent experiments with 3-5 mice/group/experiment (left and center panels) and one experiment with 4-5 mice/group (right panel). Statistical significance was determined using the log-rank test with 95% CI. *P<0.05, **P<0.01, ***P < 0.001.

Figure 2. Virus titers and lung pathology in MA15 infected mice

9 month male and female mice were infected with 5000 PFU of MA15 and lungs were analyzed for titer (A), viral antigen staining in the lungs at different times p.i. (B), gross pathology and vascular leakage in lungs of naïve and MA15 infected male and female mice on day 4 p.i. (C) and histology in naïve and MA15 infected male and female mice on day 4 p.i. (D). Lung inflammation and edema scores were determined at day 4 p.i. (E). These data are derived from 4-5 mice per group. (A) Data are representative of two independent experiments. Statistical significance was determined as described in Materials and Methods. *P<0.05.

Figure 3. Increased IMM accumulation in the lungs of MA15-infected male mice

Mice were infected with 5000 PFU of MA15 and analyzed for IMMs (A, B) or neutrophils (C, D) in the lungs on days 0, 1 and 3 p.i. Representative FACS plots show percentage of IMMs (A) and neutrophils (D) in the lings. Bar graphs show percentage and total number of IMMs (B-C) and neutrophils (E-F) at different time p.i. Data are representative of 2 independent experiments with 4 mice/group/experiment. Statistical significance was determined as described in Materials and Methods. *P<0.05, **P<0.01, ***P < 0.001.

Figure 4. Enhanced proinflammatory cytokines/chemokines in the lungs of MA15 challenged male mice

8-9 month-old male and female mice were infected with 5000 PFU of MA15. A) mRNA levels of antiviral and proinflammatory cytokines/chemokines in the lungs were measured at different times p.i. (B-C) FACS plots and bar graphs show percentage and number of cytokine producing IMMs in the lungs on day 3 p.i. following 7 hr ex-vivo incubation in the presence of Brefeldin A in male and female mice. (D) Nine-month old female and male mice were treated with control Ig or MC21 antibody (anti-CCR2, depletes IMMs) at -6hr and day 1. These mice were infected with 4000 PFU of MA15 and survival was recorded. (A) Data were obtained from 4-5 mice/group. (B-D) Data represent 2 independent experiments with 4-5 mice/group/experiment. Statistical significance was determined as described in Materials and Methods. *P<0.05, **P<0.01, ***P < 0.001.

Figure 5. Enhanced susceptibility of male mice to MA15 infection is independent of T and B cell response

7-8 month old male and female RAG1^-/- mice were challenged with 1750 PFU of MA15 and monitored for morbidity and mortality for 14 days. Data are derived from 7-8 mice/group. Statistical significance for survival curves was determined using the log-rank test with 95% CI. *P<0.05, **P<0.01 ***, P < 0.001

Figure 6. Estrogen receptor signaling protects female mice from lethal MA15 infection

9-10 month-old male and female and their gonadectomized counterparts mice were infected with 5000 PFU of MA15. A) Nine-month old gonadectomized (n=8) or non-gonadectomized (n=8), and (B) control (n=6) or flutamide treated (n=7) male mice were monitored for disease severity. (C) Percentage weight loss and survival among control (n=12) or ovariectomized female (n=12) mice. (D) Female mice treated with vehicle (corn oil) (N=9), or Tamoxifen (1 mg/mouse, n=9-10) or ICI 182, 780 (1 mg/mouse, n=10) in 100μl corn oil were infected with 5000 PFU of MA15 and monitored for morbidity and mortality. Data are derived from 2-3 independent experiments with 3-5 mice/group/experiment. Statistical significance for survival curves was determined using the log-rank test with 95% CI. *P<0.05, **P<0.01, ***P < 0.001

Figure 7. Virus titer and IMM accumulation in ovariectomized female mice

Nine-month old control or ovariectomized mice were infected with 5000 PFU of MA15 and virus titers and inflammatory cell accumulation were analyzed. (A) Lung virus titers in 9-month old male and female mice at days 2 and 4 p.i. (B, C) IMM and neutrophil accumulation in the lungs at day 2 p.i. Data are derived from 2 independent experiments with 3-4 mice/group/experiment (A) or are representative of 2 independent experiments with 3-4 mice/group/experiment (B-C). Statistical significance was determined as described in Materials and Methods. *P<0.05, **P<0.01, ***P < 0.001.