Male sex is a risk factor for acute COVID-19 respiratory tract infection
A sex imbalance in the hospitalization and mortality from COVID-19 patients has been reported worldwide [10,11,12,13,14]. We examined the sex (self-identified) distribution in our hospitalized patients that were graded into three groups (mild, moderate and severe) [8]. There were more males than females in the mild and severe group (Fig. 1A, B). We also analyzed the male/female ratio by age in 10-year increments. In most age groups, the ratios were above one, indicating more male vs. female patients (Fig. 1C). Interestingly, when analyzed based on ethnicity, the male/female ratios were above one in almost all ethnicities except for African Americans (Fig. 1D). The overall mortality rate was higher in male vs. female patients (Fig. 1E).
Inflammatory mediator levels in plasma after acute COVID-19 respiratory tract infection
Previous studies suggested that the severity of COVID-19 is associated with an increased level of inflammatory mediators including cytokines and chemokines [15, 16]. Here, we examined 48 mediators (Additional file 2) in Cohort 1 patients, among which 38 were detectable by our Multiplex ELISA assay. We presented the data of TNF-α, IL-6, IL-8, Eotaxin, IL-10, MCP-3, MDC (CCL22), GROα, IP-10, sCD40L, MIP-1β, and Fractalkine only, as differences between groups were found. However, data of other mediators (EGF, FGF-2, Flt-3 ligand, G-CSF, GM-CSF, IFNα2, IFNγ, IL-1α, IL-1β, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-7, IL-9, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, IL-17E/IL-25, IL-17F, IL-18, IL-22, IL-27, MCP-1, M-CSF, MIG, MIP-1α, PDGF-AA, PDGF-AB/BB, RANTES, TGFα, TNFβ, and VEGF-A) showed no group differences (data not shown). Overall (no disease severity grouping; Fig. 2A–J), IL-6 levels were significantly higher in men vs. women in the COVID groups (Fig. 2B). When compared with controls, Eotaxin in men, and MCP-3 in women were significantly lower (Fig. 2C, E), and IL-10 in women was higher (Fig. 2D), a control vs. COVID-19 difference that was not seen in the counterpart sex, respectively. No sex differences were found in levels of MDC (CCL22), GROα, IP-10, and sCD40L, although males and females exhibited synchronous changes compared to controls (Fig. 2F–I). For samples from Cohort 2 patients, three pro- (IL-6, IL-8, TNF-α) and three anti-inflammatory mediators (IL-10, TGF-β1, CD200) were assayed by conventional ELISA. Sex differences were only seen in IL-10 levels, which were significantly higher in females vs. males at 14 days (Fig. 2J). Together with the data from the cohort 1 patients (Fig. 2D), this suggests that a more robust anti-inflammatory response was seen in female vs. male COVID-19 patients.
Sex differences in the levels of inflammatory markers are dependent on the severity of COVID-19 illness
The symptoms of COVID-19 are variable, ranging from mild fever to severe illness and intensive care unit (ICU) admission. The severity of the disease may be responsible for the cytokine profiles. We examined if the sex differences seen in the above overall data (Fig. 2) remained in patients with different disease severity (mild, moderate and severe). Interestingly, we found that the most dramatic sex differences were seen in the severe groups, e.g., males in the severe group had significantly higher levels of IL-6, IL-8, and MCP-1 than females (Fig. 3B, C, L), a sex difference not seen in mild or moderate group. Males with severe disease also had significantly higher levels of GROα, sCD40L, and MIP-1β than control, mild, or moderate male groups, which was not seen in females (Fig. 3G, I, J). Of note, compared to controls, the anti-inflammatory cytokine, IL-10, significantly increased in females with moderate and severe disease, but was not significantly elevated until the severe stage in males (Fig. 3E). For TNF-α, Eotaxin, MDC (CCL22), IP-10, and Fractalkine, the data revealed no sex differences. Taken together, male COVID-19 patients with severe illness have an exacerbated immune response compared to females with severe disease.
Sex differences in circulating immune cells
To further evaluate the immune response to COVID-19, we performed flow cytometry on fresh blood samples to examine leukocytes from Cohort 2 patients at three time points (24 h, 7 days, and 14 days) since hospital admission. A panel of antibodies to detect neutrophils (Table 1), monocytes (Table 2), and lymphocytic T and B cells (Table 3) was used to identify these leukocyte subsets. Active neutrophils were gated as CD11b+DEspR+ [17,18,19] (Fig. 4A), monocytes as CD11b+CD45+CD14+CD24− (Fig. 4B), T cells as CD45+CD3+ and B cells as CD45+CD19+ (Fig. 4C). Interestingly, sex differences were found in both myeloid cells (neutrophils/monocytes) and lymphocytes, but exhibited different patterns by sex. Male patients had more neutrophils and monocytes at each time point (all significant except a trend at 24 h for monocytes) than female patients (Fig. 4D, E). However, there was a significant elevation in B cells in females vs. males at both 7 days and 14 days (Fig. 4F). No sex difference was seen in T cells (Fig. 4G).
Pneumonia and other organ involvement
After infection with COVID-19, patients often have signs of other organ involvement or comorbidities in addition to pneumonia. We reviewed the hospitalization history of our patients and analyzed organ involvement. Figure 5A shows the percentage of pneumonia, sepsis, acute kidney injury (AKI), deep vein thrombosis (DVT), and myocardial infarction in all patients. Overall, more male patients develop these complications than females, with significant differences in pneumonia and AKI. Figure 5B and C shows the percentages of these diseases in Cohort 1 and Cohort 2 patients, respectively. In males with moderate disease, the percentage of pneumonia was significantly higher than that of females. Prevalence of sepsis showed an increasing trend across the severity of COVID-19 infection in males (p = 0.012) but not in females.