DISCUSSION
In our study demonstrated that components of innate immune system during COVID-19 are well preserved. No siginificant defect in ROS production and apoptosis in monocytes and neutrophils. However, there was significant decrease in absolute numbers of all lymphocyte subsets in particular B cell and CD4+ T cells, but also NK and CD8+ T cells which is associated with disease severity. Activation marker CD69 on CD8+ and CD4+T lymphocytes was found to be increased, but not CD25. Innate-like cytotoxic and NK like T cells and CD3+HLA-DR+ and CD8+CD28- regulatory T cell subsets were increased in COVID patients. In addition, increased lymphocyte apoptosis was seen which may be responsible for development of lymphopenia.
The success for the immune system is a complex process that includes of innate at first followed by adaptive immune system activation in which controls innate system not to harm own cells with sustained reactions while producing specific, focused responses for eradicating the microorganism in the shortest time. Therefore, proper immune response against microorganisms including viruses depends on a fine tuned balance between innate and adaptive immune systems (7, 8, 24).
As a part of innate immune system cells, monocytes, as antigen-presenting cells, constitutively express Major Histocompatibility Complex (MHC) Antigen Class II, HLA-DR. This expression is a requirement for the communication between innate and adaptive immune systems. The peptides that were processed from microorganisms are presented by antigen-presenting cells to the T cells in the context of MHC molecules to activate adaptive T lymphocytes. The decrease in the expression of HLA-DR on monocytes is generally accepted as a marker of immune paralysis (20). In our study, we found decreased expression of HLA-DR on monocytes in all clinical courses of the patients with COVID-19 in comparison to healthy control. However, since we do not have a diseased control group, we did not have an idea of monocyte HLA-DR expression in viral diseases. A recent study showed that COVID-19 patients exhibited a less pronounced decrease in HLA-DR expression on monocytes in comparison to bacterial septic shock patients (13). On the other hand, CD16+ monocytes are a well-defined group known as intermediate and/or non-classical monocytes. They constitute 2-8% and 2-11% respectively of circulating monocytes under normal conditions (43). While CD16- monocytes secrete high levels of IL-6, CD16+ monocytes release high levels of IL-1β and TNF and are considered as pro-inflammatory monocytes (6). In our group of patients, we demonstrated more than 50% of monocytes of COVID-19 patients were CD16+ proinflammatory monocytes indicating that monocytes of COVID-19 patients actively contributed to the inflammatory process.
Neutrophils have critical roles in acute inflammation. Various neutrophil subgroups has also been defined according to their surface molecule profiles (25). One of the neutrophil surface molecules, CD10 (CALLA), is a neutral endopeptidase and cuts inflammatory peptides such as substance-P, met-enkephalin, fMLP. Those peptides play role in acute inflammation (26). Mature neutrophils are CD10 positive. Each has different effects on T lymphocytes. CD10 positive neutrophils inhibit interferon-γ release and proliferation of T lymphocytes, while CD10 negatives help T cell survival (27). CD16 is Fc gamma receptor III and is a necessary molecule for neutrophil phagocytosis (28). Following activation and apoptosis, neutrophil CD16 is cut by ADAM17 metalloprotease and it sheds from membrane(29). Thus, CD16 shedding is a marker for neutrophil activation and apoptosis. In our group of patients, CD10 and CD16 positive mature neutrophils were slightly decreased in the severe group. This reduction can be evaluated as leaving their place to immature neutrophils, in line with the severity of inflammation. Our finding that CD16 expression is not different from controls as a percentage can be considered as an indication that COVID-19 disease does not cause neutrophil apoptosis and neutrophils remain competent. Supporting this hypothesis, no defect in ROS production of neutrophils and monocytes has been found. Our finding that early, late apoptosis and cell death in monocytes, especially in neutrophils, are not different from controls. Together with our all data on the innate cells, we consider that COVID-19 does not target the monocytes and neutrophils of innate immune system cells.
The percentage of NK cells was found to be normal even in the severe group of patients in contrast to the previous study (30, 31). A decrease was observed in the number of NK cells, but this reduction was not as excessive as in B cells). Future studies on direct cytotoxicity or antibody-mediated cytotoxicity of NK cells will help us to interpret this issue better.
We identified a significant increase in CD3-CD8+CD56+innate lymphoid cells and CD3+CD56+NK like innate T cells in all COVID-19 patients. CD3-CD8+CD56+, innate lymphoid cells similar to NK cells are potent cytotoxic and have strong capacity to release IFN-γ (32-35). We suggest that the increase of CD3-CD8+CD56+cells with their possible capacities of both cytotoxicity and releasing IFN-γ might demonstrate the cytotoxic/killer cells that try to respond rapidly to SARS-CoV-2 in COVID-19 patients. Increased expression of CD56 which is an adhesion molecule, may provide a privilege to these CD8+ cells to rapidly navigate between the periphery and the inflammation site. It might be considered that the decreased number of NK cells could be compensated by increased innate lymphoid cells.
In our group of patients, we have found that the absolute number of B lymphocytes was extremely reduced. Obviously, we should not expect proper immunoglobulin secretion from plasma cells in COVID-19 with that low number of B lymphocytes. It has already been shown that there was no peripheral B cell memory in Severe Acute Respiratory syndrom in six years follow-up (13, 36). In our study, we considered that this decrease might have been related to plasma cell differentiation. It has been known that plasmablasts were increased in the peripheral blood during viral infection (37, 38). However, we demonstrated that no plasmablast has found in the periphery. A second reason might be the migration of B lymphocytes to the inflammation site. But, histopathological studies did not support this hypothesis. Furthermore, it has been shown that there are no lymphocytes in lymph node and spleen (39) Another possibility is that, as a member of the adaptive immune system, B lymphocytes may be one of the targets that directly destroyed by SARS-CoV-2. As a part of imbalanced immune behavior, imbalanced cytokine release caused by SARS-CoV-2 may not permit proper involvement of B lymphocytes. More studies on B lymphocytes are require.
The significant increase in the percentages of CD3+and CD8+ T lymphocytes with their normal ranged absolute numbers in the mild patients can be considered as a sign of a strong immune response at the beginning. In the mild group, while the absolute number of CD4+ T cells are still within normal limits, this decrease in both NK and B cell populations suggests that a careful phenotypic follow-up can be used to predict disease progression from the beginning. While the decrease in the absolute numbers of NK and B cell remained the same throughout the disease progression in all patient groups, a sharper decrease in CD4 and CD8 T lymphocytes in the moderate group is noteworthy, and this decline continues as the disease becomes severe. Even that decrease in the number of CD4+ T cells in the severe group approaches the decrease in B lymphocytes.
One of the regulatory T cell populations, CD3+CD4-CD8-T cells constitute 1% of peripheral T cells (40). It has been previously shown that they have a strong suppressive effect on CD4+ and CD8+ cells, (21). Our finding that increased CD3+CD4-CD8 T cells especially in moderate and severe groups suggests that the activation control of the adaptive immune system is strong in COVID-19 even if it is not required. HLA-DR expression on T lymphocyte has long been known to down-regulate T cells that have already activated and to represent an important homeostatic regulatory mechanism(41). CD8+CD28- T cells represent another group of regulatory T cells that inhibit CD4+ T cell proliferation (22, 23). In our study, the increase of CD3+ HLA-DR+ and CD8+ CD28- T lymphocytes also indicate the presence of a tight adaptive immune down-regulation in COVID-19.
Our finding that early apoptosis or apoptotic activation in lymphocytes, which we measured with the conversion of a non-fluorescent substrate into fluorescence when is cut by active Caspase 3, indicates that the virus leads a process that initiates caspase 3 activation in lymphocytes. Indeed, a recent study demonstrated that ORF-3a protein of SARS-Cov-2 induced apoptosis (42). Our finding that folds increase upon stimulus in caspase 3 activation was low in the patient group in contrast to controls suggests that COVID-19 patients have highly activated lymphocytes before PMA stimulation. Caspase-3, which was already active at the basal level, cannot be activated further by stimulation. But even with this tired activation level, it is able to double the total lymphocyte death rate compared to the controls. This data can be interpreted as an indication that SARS-Cov-2 is destructive for peripheral lymphocytes.
In conclusion, this study, in which we examine partially the phenotypic and functional characteristics of the innate and adaptive immune system cells, shows that the innate immune system functions in COVID-19 are complete and competent, and the remaining a low number of lymphocytes is half-dead trying to respond to viral infection with a suppressive profile in COVID-19. It supports our hypothesis that innate-adaptive immune system communication is impaired in COVID-19. In this context, it will be very important to focus on additional functional studies related to apoptotic targets in explaining immune pathogenesis.