Old age and male sex are important risk factors for COVID-19 severity. Several studies have investigated whether immune responses during SARS-CoV-2 infection are influenced by demographic factors such as age and sex [18, 19]. Although they identified immune profiles associated with severe COVID-19, they could not assess whether these were secondarily induced by the disease, or due to a-priori immune differences between different groups. In the present study, we show that the immune characteristics associated with severe COVID-19, such as specific changes in cell populations and circulating inflammatory proteins, are already present in healthy elderly and men. Interestingly, while the season did not impact the immune response to SARS-CoV-2 stimulation
in the entire group, there was a clear difference in the responses between the young and old. Young individuals, but not the elderly, improve their IFN responses to SARS-CoV-2 during the summer. COVID-19 progression to a severe clinical picture is related to the depletion of several immune cell types, including naïve CD4+ and CD8+, and CD56 high NK cells. Our study demonstrates the age-related decline of these cells in healthy individuals even before the infection, which likely contributes to their incapacity to eliminate the virus. These data are supported by studies suggesting that some of these cell types are scarcer in uninfected elderly and males [20, 21]. Naïve B lymphocyte numbers were also reduced with advanced age, which would undermine the development of adaptive immunity and antibody production upon infection . The aging process does not only alter cell numbers, but also the functions ) . All these might cumulatively disrupt the response against SARS-CoV-2 infection.
We showed that one of the striking differences in immune cell types between males and females was the number of CD4+ T cells. Although we cannot rule out that significant differences in cell populations might not determine the disease severity, SARS-CoV-2-specific CD4+ T cells were strongly linked with milder COVID-19, unlike antibodies and CD8+ T cell numbers . Fast induction of CD4+ T cells was related to a milder disease, while defects in inducing SARS-CoV-2-specific CD4+ T cells were associated with severe or fatal COVID-19. An exaggerated systemic inflammation has been associated with severe COVID-19, mirrored by high circulating concentrations of pro-inflammatory mediators . Among those, IL-8, IL-18, and MCP-1 have been frequently reported, and the first two characterize the immune response of men with a severe outcome . Notably, their concentrations are already higher in both men and the elderly in our healthy cohorts. Another protein displaying the same pattern is HGF, acting on epithelial and T cells promoting migration , which has higher concentrations in the circulation of severe patients . The circulating HGF concentrations are higher in men, but also increases with age in women.
Chemokines are critical inflammatory mediators, and MCP-2, CCL3, CCL4, CCL19, and CXCL10 are all more abundant in males. ENRAGE (S100A12), produced by neutrophils and monocytes, is also higher in healthy males than females. Monocytes expressing high S100A12 and IL-8 are linked to COVID-19 severity . Additionally, anti-inflammatory proteins IL-10 and PD-L1 are elevated in healthy males and severe COVID-19. Initially considered a negative feedback mechanism for infection-induced inflammation, there are arguments suggesting these proteins as biomarkers of immune exhaustion, which is likely to play a substantial role in the pathophysiology of COVID-19 [29, 30]. Notably, the association of early IL-10 production with COVID-19 severity supports this idea . Severity markers increasing with old age, but not affected by sex, include IL-6 and OPG. IL-6 secreted by hyperactive monocytes contributes to low HLA-DR expression and lymphopenia in severe COVID- 19 . TNF-family cytokine receptor OPG, abundant in ICU patients, increases with old age in our healthy cohorts . High OPG concentrations in females are likely due to estrogen's effects promoting OPG expression to inhibit bone resorption . Another TNF-family member, TRANCE (RANKL), which is lower in severe COVID-19 cases, also declines with advancing age in healthy individuals. T cells are one of the primary TRANCE sources, which might explain its scarcity in the elderly and severe COVID-19 patients with lymphopenia . Of note, concentrations of circulating IL-7 and IFNγ, which are increased in severe COVID-19, are similar in men and women. Therefore, T cell numbers being higher in women is unlikely due to IL-7- induced lymphopoiesis, whereas higher T cell numbers do not necessarily lead to more circulating IFNγ. An overview of the age- and sex-dependent immune profiles in healthy individuals potentially predisposing to severe COVID-19 upon infection is provided in Figure 7. Environmental factors are also known to affect immune responses.
Certain infections such as influenza follow a seasonal pattern , and the evolution of the pandemic last year also suggested273that COVID-19 incidence might follow a seasonal variation . Therefore, we questioned whether this might be due to seasonal changes in the immune response to the virus. In the entire group, we found no clear seasonal response against SARS-CoV-2 , although this could be due to the limited sample size. Interestingly, young individuals improve their IFN response to SARS-CoV-2 during the summer, while the elderly do not. Further research with larger cohorts is required to validate seasonality's full impact on anti-SARS-CoV-2 host defense. Our results indicate that the immune response upon in vitro SARS-CoV-2 stimulation varies depending on age and sex. The response of the elderly is characterized by low IFNγ and elevated IL- 1RA production. IFNγ is crucial for an effective response of T- and NK cells to viral infections, and its deficiency is associated with severe COVID-19 . Depleted NK and T cell pools might explain why the elderly areless capable of producing IFNγ upon SARS-CoV-2 stimulation. Poor IFNγ response, especially in the fall, might put the elderly at higher risk for severe COVID-19. Men and women produce comparable amounts of IFNγ, although the T cell numbers are higher in women. Other roles of T cells besides IFN production may contribute to the better prognosis of women with COVID-19. One important point is that these defects are present in the whole population, not at the individual level: while the elderly as a group have lower immune responses, there are certainly aged individuals who have effective immune reaction. This inter-individual variability could be the reason why some 290elderly or some men have good responses against SARS-CoV-2 and develop only mild disease. In COVID-19, an overproduction of pro-inflammatory cytokines contributes to the pathophysiology late in the disease . On the other hand, cytokines such as IL-1β might also be crucial for an early anti-viral response. The deficiency of IL-1β or its receptor causes higher viral load and mortality in murine models . Furthermore, genetic variants in IL1B contribute to influenza susceptibility in humans . We observed higher IL-1β production in women in response to SARS-CoV-2 in vitro, arguing that an initial potent anti-viral defense is essential to prevent severe disease. Moreover, IL- 1RA is an antagonist of IL-1 bioactivity, helping prevent excessive inflammation . However, early IL-1RA production in patients is associated with COVID-19 severity , while our finding of higher IL-1RA production in the elderly suggests that IL-1RA might be hindering their ability to mount an optimal immune response against SARS-CoV-2. Alternatively, high IL-1RA production with increasing age might mirror the general inflammatory profile of elderly individuals. In conclusion, our findings shed light on the immunological factors that might explain why men and the elderly have a higher risk of developing severe COVID-19. These results also emphasize the importance of the IL1β/IL-1RA axis and IFNγ in anti-SARS-CoV-2 response. We propose that intrinsically different immune characteristics, including plasma inflammatory mediators and immune cell populations, in healthy people would influence their immune response upon SARS-CoV-2 infection and the severity of the disease. Results of this study inform prophylactic and therapeutic efforts.