A new study published on the preprint server medRxiv* in June 2020 shows that immune cells, antibody levels, cytokine levels, and viral loads contribute to the sex difference seen in response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
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Male-Female Differences in COVID-19 Severity
The COVID-19 pandemic is caused by the SARS-CoV-2, which emerged in Wuhan, China, towards the end of 2019. Even as the number of cases throughout the world continues to grow, researchers discover that males are more prone to severe disease and death than females, in a ratio of 60:40 globally. A recent report in England showed that men were twice as likely to die of COVID-19 disease.
Many other infections are known to be more common in men than in women. For instance, hepatitis A and tuberculosis are both more common in men than in women, while viral loads in hepatitis C and HIV are higher in men. Also, immunization often results in a more robust immune response by women than by men.
Differences in Immune Response
The mechanism of this differential pathogenesis in the sexes remains unclear. The current study aimed to uncover the sex differences in the immune phenotype by four parameters: the viral load, the antibody levels, the level of cytokines, and the blood cell patterns and numbers.
The researchers enrolled COVID-19 patients admitted to the Yale New Haven Hospital between March 18th and May 9th, 2020, through the IMPACT study. There were 39 patients in cohort A, with 17 men and 22 women.
A second set of patients, cohort B, was also analyzed, including all patients from cohort A and another 54 patients who were not eligible for cohort A. The latter subset donated blood more than once as well as saliva and nasopharyngeal swabs, for a longitudinal follow-up.
Thus, there were altogether 93 patients from whom the investigators obtained fresh peripheral blood mononuclear cells (PBMCs), plasma, nasopharyngeal swabs, and saliva samples. A control group consisting of uninfected healthcare workers, was also tested.
The researchers carried out viral RNA evaluation of Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies directed explicitly against the S1 subunit of the spike protein and measurements of the cytokines and chemokines. The PBMCs were also stained and examined
During Early Infection, Innate Immunity Higher in Males
The researchers found that the median viral RNA titers in nasopharyngeal swabs and saliva were higher in males but not significantly. The same lack of significant difference was observed at baseline measurements of specific anti-SARS-CoV-2 antibodies, both IgM and IgG, in males and females.
The levels of innate immune cytokines and chemokines are reported to be higher in COVID-19 patients compared to controls. In Cohort A, interferon levels type I, II, and III were comparable, but in cohort B, the level of Interferon-alpha 2 (IFNA2) and Chemokine (C-C motif) ligand 8 (CCL8) was significantly higher in female patients compared to male patients. The researchers say, “These data indicated that innate inflammatory cytokines and chemokines are more robustly elevated early and throughout disease course in male patients over female patients.”
Non-Classical Monocytes Higher in Men Causing Neutrophil Recruitment
This was confirmed by the study of the immune cell phenotypes by flow cytometry. They found a significant reduction in T cell count and an increase in B cells in COVID-19 patients vs. controls in both sexes. Total monocytes were increased in both sexes, particularly a subset of intermediate monocytes in females particularly. In males, non-classical monocytes were increased.
This may mean that the progression from classical to non-classical monocytes is interrupted at the intermediate stage in female patients. Still, there is a more robust innate immune activation in males, causing a rise in the levels of innate immune chemokines and cytokines.
T Cell Activation Higher in Females
The T cell phenotype in COVID-19 showed no differences in CD4 and CD8 cells across patients or controls. Analysis of T cell subsets showed that CD38 and HLA-DR positive T cells were induced more robustly in female patients, as well as T cells with PD-1 and TIM-3 markers. Though both CD4 and CD8 T cells reflect these findings, the difference was more marked with the latter.
Intracellular cytokine staining showed that the levels of chemicals like IFNγ, Granzyme B, TNF, IL-6, and IL-2 were higher in patients of both sexes. Female COVID-19 patients showed a higher level of activation and terminally differentiated T cells than in males.
Age-Related to Lower T Cell Activation and Worsened Disease in Males
Males with worsening disease were significantly older and had a higher body mass index compared to those with stable disease. This was not observed in females with clinical deterioration. Activated T cells were also significantly lower in older men and those with worsening disease, and trends towards lower terminally differentiated T cells and IFNγ+ CD8 T cells at baseline.
Women showed no such difference between groups with stable and progressive disease. On the other hand, stable female patients had lower median viral RNA levels than those who were worsening or male patients.
T cell responses in women are typically robust compared to men, even in older women.
The Cytokine Storm and Female Patients
The excessive levels of pro-inflammatory cytokines in many patients with severe COVID-19 are often called the cytokine storm and may result in shock, organ injury, and multi-organ dysfunction. It is also thought to be the cause of massive lung injury. The present study also showed that Interleukin-8 (IL-8), Interleukin-18 (IL-8), and CCL5 (C-C Motif Chemokine Ligand 5) were higher in males than in females. IL-8 is linked to lymphopenia, a marker of progressive disease especially with the drop in T cells.
IL-8 also attracts neutrophils, which shows why increased neutrophils predict a worse outcome in COVID-19. Neutrophils are recruited by non-classical monocytes, which are increased robustly in male patients compared to female patients, partly as a result of higher CCL5 levels.
Anti-S IgG antibody titers were higher in women with stable disease compared to those with progressive disease or male patients at any stage. This indicates that the robust production of anti-S IgG antibodies is linked to their clinical stabilization.
In both sexes, higher levels of CXCL10 (IP-10) and M-CSF accompanied worsening disease. CXCL10 was observed to be higher in the latter category when only cohort A was analyzed by age groups. Some cytokines produced by innate immune cells, however, were specifically raised only in women with progressive disease, such as CCL5, TNFSF10 (TRAIL), and IL-15.
Disease Prognosis and T cell Activation
Overall, a higher innate immune activity in the form of increased TNFSF10 (TNF Superfamily Member 10) and Interleukin-15 (IL-15) was linked to worsening severity, but not the CD8 T cell count. Higher C-X-C motif chemokine 10 (CXCL10) corresponded to higher IFNγ+CD8 T cells in female patients (as did IL-15 levels), but the levels of CXCL10 were inversely related to anti-S IgG antibodies in females only.
On the contrary, males showed associations between more severe disease, older age, higher BMI, and low levels of activated CD8 T cells with poor Interferon‐gamma (IFN‐γ) production. The latter were predominantly in older males, an association absent in female patients.
Implications and Applications
The researchers sum up: “These differences seemed to highlight the differences between the sexes in the immune response against SARS-CoV-2 as well as the difference of the potential prognostic/predictive factors for clinical deterioration of COVID-19.”
The presence of estrogen response elements in the promoter regions of many antiviral and inflammatory genes may have a lot to do with the inherent difference in immune response between the sexes. The study suggests, “Vaccines and therapies to elevate T cell immune response to SARS-CoV-2 might be warranted for male patients, while female patients might benefit from therapies that dampen innate immune activation early during disease.”
medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.