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.