In this study, we analyzed the relationship between Gal-3 and innate immunity cytokine profile (TNF-α, IL-1β, IL-10, IL-12), functional phenotype of lymphocytes, monocytes and dendritic cells, clinical, biochemical, and radiographic outcomes with disease severity. Patients with COVID-19 were classified into the mild, moderate, severe and critical group. We found correlation between gender and age with disease severity. Most patients in critical group were male (71.4%) and older (over 68 years), meaning older male patients are more prone to develop the most severe stage of COVID-19 with worse clinical symptoms. Our results are in line with the studies of Statsenko et al., Liu et al. and Meng et al. where authors suggested that age and gender corelates with disease severity confirming that increased risk of developing the most severe form of COVID-19 is often in elderly and male patients12,13,14. Frequency of fever, fatigue, dyspnea, chest pain, auscultatory attenuated breathing sound, crackles, whistling was significantly higher in moderate, severe and critical groups compared with the mild one (Table 1).
As disease progresses, we noted increased values of white blood cell count, neutrophil count, values of urea, glycemia, creatinine, BILD, BILT, AST, ALT, CK, LDH, D-dimer, CRP, PCT, Ferritin, as well as decreased values of lymphocyte and monocyte count, albumin, Sa02 and p02. The same findings were reported in studies of Rathores et al. and Bairwa15,16.
COVID-19 can affect lungs, heart, kidneys, gastrointestinal tract, and brain by specific host defense responses associated with inflammatory activity and coagulopathy2,17,18 . In patients with severe and critical features occurs uncontrolled immune-inflammatory response with rapid production of cytokines of IL-1 family, TNFα, IL-6, granulocyte-colony stimulating factor and several chemokines19. Significantly higher sera levels of PCT were noted in severe and critical group and might be marker of cytokine storm or multiple infections20. As the clinical picture of the patients deteriorated, the damage to the lung tissue was bigger. These results suggest a correlation between CXR findings and disease severity and indicate significant difference between CXR findings in defined groups.
We measured serum values of proinflammatory cytokines IL-1β, TNF-α and IL-12 and anti-inflammatory IL-10. During COVID-19 progression, abnormal levels of IL-1β, TNF-α, IL-2, IL-7, IL-12, macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), and others can be detected in patient’s blood21,22. IL-10 is anti-inflammatory cytokine important for immune response suppression and tissue damage restriction. Several studies confirmed dramatically increment of IL-10 in COVID-19 patients23,24. Possible explanation is that parallel with rising of proinflammatory cytokines, IL-10 increases in order to limit inflammation25. Our results showed significantly higher level of IL-1β, TNF-α, IL-12 and IL-10 in patients with stage IV of COVID-19 in comparison to milder forms of the disease (Fig. 1). These results are in line with previous studies confirming growing level of inflammation as COVID-19 progresses. The almost unchanged ratio between IL-10 and proinflammatory cytokines during COVID-19 progression (Fig. 1) points on similar dynamics of all cytokine’s growth.
Flow cytometry analyses revealed higher percentages of TNF-α+T cells, IL1-β producing dendritic cells and IL1-β+ and TNF-α producing monocytes in the peripheral blood of patients in the stage IV. (Fig. 2). Previous study confirmed that cytokine release syndrome is in positive correlation with the degree of COVID-19 severity, which is depicted by higher production of proinflammatory cytokines26. It has been shown that TNF-α can directly propagate production of other proinflammatory cytokines such as IL-6 and IL-1β27. In line with these confirmations is our result showing predomination of TNF-α producing T cells, IL1-β producing dendritic cells and IL1-β+ and TNF-α producing monocytes in the most severe stage of the disease (Fig. 2). Higher numbers of TNF-α/IL1-β producing T cells/dendritic cells/ monocytes represent most likely source of increased systemic TNF-α and IL1-β. CCR5 is a protein expressed constitutively on many immune and non-immune cells involved in different immune processes. Our analyses showed significantly higher expression of CCR5 on T cells in stage IV compared to milder forms of COVID-19 (Fig. 2). During COVID-19, infected airway epithelial cells increase production of CCL5 that functions as chemotactic molecule by binding to CCR528. So, higher expression of CCR5 on T cells can enable linking of CCL5 to CCR5 stimulating migration of T lymphocytes in patient’s lungs and promoting inflammation and more severe form of disease. This result explains reduced lymphocyte count in patients with more severe COVID-19 (Table 2).
As different studies confirmed that Gal-3 can act as stimulative or inhibiting molecule, the next goal of our study was analysis of Gal-3 in COVID-19 patients29,30. Significantly higher level of Gal-3 was detected in sera of patients in stage IV in comparison to patients in other stages of disease (Fig. 1). This result is in line with studies of Kazancioglu et al. and Cervantes-Alvarez et al. showing higher levels of Gal-3 in the patients with severe COVID-1910,31. We further investigate Gal-3 expression in T cells from peripheral blood. Flow cytometry analyses showed that patients in stage IV of COVID-19 had significantly higher percentage of Gal-3+ T cells compared to patients with milder disease (Fig. 2). Increased production of Gal-3 in T cells may be the source of elevated systemic Gal-3 in patients with severe form of COVID-19. Previous study showed that Gal-3 placed in the serum or on the cell membrane can increase inflammation via stimulation of migration and infiltration of neutrophils and other proinflammatory cells and massive production of different proinflammatory cytokines to the infected site32,33. It is possible that after migration to lung via CCR5-CCL5 interaction, T cells by expressing Gal-3 amplify airway inflammation via attracting immune cells and stimulating production of proinflammatory cytokines. After being released from the cell, Gal-3 can link to receptors on innate immune cells and act as alarmin by stimulating production of TNF-α, IL-1β, IL-6, IL-1234. These potential actions of Gal-3 are substantiated by increased systemic values of TNF-α, IL-1β, and IL-12 (Fig. 1) as well as strong positive correlation that is measured between Gal-3 and IL-1β and moderate positive correlation between Gal-3 and TNF-α and IL-12 (Table 3). As part of innate immunity, inflammasomes are receptors and sensors that can activate caspase-1 and facilitate inflammation in response to microorganisms35. Recent study showed that during COVID-19, as a response to the presence of Corona virus, human macrophages induce inflammasomes activity, that is followed by secretion of IL-1β and IL-18 and the extension of inflammation in lungs36. Moreover, some studies explained that in different diseases Gal3 can stimulate the function of inflammasome thus inducing proinflammatory process37. According to these data, it is possible that besides direct effect of Corona virus on the function of inflammasome, indirectly gal-3 can also potenatiate inflammation via inflammasome activity. Interestingly, elevated systemic values of Gal-3 were detected in patients in stage IV of COVID-19. This group is dominated by older male patients. As it is already known that Gal-3 levels in sera increase with age and have been associated with different diseases very frequent in the elderly population such as cardiovascular disease38, it appears that elevated Gal-3 may be due to aging itself.
Gal-3 significantly correlated with several biomarkers and clinical parameters (Table 4). Moderate positive correlation detected between Gal-3 and D dimer, CXR findings and urea. Moderate negative correlation noted between Gal-3 and p02, Sa02, lymphocyte and monocyte percentage (Table 4). All these biomarkers and parameters important for monitoring of COVID-19 patients correlate with Gal-3 and point on potentially important pathophysiological role of Gal-3 in COVID-19.
Our results revealed that Gal-3 could predict critical stage of COVID-19. According to our findings, systemic Gal-3 could be a valuable marker for COVID-19 severity.
We found higher systemic values of Gal-3, IL-10 and proinflammatory cytokines in patients with critically COVID-19. The increment of systemic Gal-3 is followed by increased expression of Gal-3 and chemokine CCR5 in T cells, increased production of TNF-α and IL1-β from PBMCs. Systemic values of Gal-3 strongly correlate with proinflammatory cytokines and clinical parameters of disease severity.
Taking all these in account we believe that Gal-3 may facilitate acquired proinflammatory immune response, and with intense innate pro-inflammatory immune response leads to severe inflammation in the lungs and poor outcome, which makes it a promising therapeutic target.