4 DISCUSSION
The present study highlights an impairment of mitochondrial function in
leukocytes from COVID-19 patients, expressed as a decrease in
mitochondria membrane potential, associated by increase in ROS
production that induces morphological changes and cell death in
leukocytes. Our results confirm the pro-oxidant and cytotoxic profile of
SARS-CoV-2 in leukocytes and reveal a modulatory action of cellular and
organic damage events as part of an integral lesion response.
Summarizing these findings showed that SARS-CoV-2 infection increases
the levels of oxidative stress markers such as peroxide and nitric
oxide, as well as markers of organic damage such as protein
carbonylation. These results were associated with an increase in the
inhibition of mitochondrial function that leads to both morphological
and functional cell injury and therefore culminate in cell death of
leukocytes involving an immunosuppression event which contributes to
generalized tissue injury in COVID-19 patients.
The relevance of these findings is considerable since it represents, to
our knowledge, one of the first reports available in the literature that
describes the ability of mitochondrial inhibition by SARS-CoV-2
infection in response to inflammation-modulation to evoke an integral
oxidative response in leukocytes, thus contrasting the cytotoxic profile
of the virus with respect to endothelial and alveolar cell in COVID-19
disease.13 It has been determined that viral
stimulation in COVID-19 is prone to elicit intensive immunological
reactions, cytokine storm and immune-cell
infiltration.4 However, some immunocytes can produce
numerous ROS including peroxide, nitric oxide and hydroxyl radical, as
reported in other virus studies.14 ROS is important
for regulating immunological responses, but excessive ROS will induce
the oxidize proteins, lipids, DNA leading to destruction not only
virus-infected cells but also normal cells in lung, endothelial tissue
and even the immune cells themselves.15 Therefore, it
could be established that modulation of the cellular redox state in
early stages is very important to mitigate the cytotoxic events caused
by COVID-19 infection.
Most studies have tried to show physio-pathological mechanisms, where
inflammation and oxidative stress as a result of inflammation have been
implicated in the pathogenesis of COVID-19.1,3,5 Thus,
a high number of leukocytes are involved in the inflammatory process and
an elevated level of interleukins has been detected in the plasma of
COVID-19 patients,16 that promote degranulation of
polymorphonuclear cells and the production of ROS which promotes
oxidative stress inducing cellular and tissue
damage.17 This oxidative stress is also active
epithelial and endothelial cells to generate chemotactic molecules that
recruit neutrophils, monocytes and lymphocytes which potentiate
inflammation and oxidative stress and therefore tissue
injury.7,17,18 During viral infection, circulating
neutrophils increases free radical release, lipid peroxidation and
reduce nitric oxide, which is an endothelial vasodilator. Moreover,
oxidative stress affect repair mechanisms and the immune system
function, which is one of the main events of the inflammatory response,
increasing cytotoxic processes such as mitochondrial inhibition and
accelerated apoptosis, that can be related to the severity and
progression of COVID-19 disease.19 Studies indicate
that COVID-19 infection is capable of producing an excessive immune
reaction in the host, generating an leukocytes activation where
monocytes larger than normal can be seen.20 Therefore,
COVID-19 infection leads to excessive activation of monocytes /
macrophages with the development of a cytokine storm and, consequently,
leads to the appearance of acute respiratory distress syndrome
(ARDS).21 From these reported studies and the found
findings, it is suggested that the intervention in modulating the immune
response be in early stages, to prevent leukocyte over-activation and
cellular toxic effects.
On the other hand, mitochondria play pivotal roles in cell homeostasis
of leukocytes as well as other cells. Accordingly, the increased energy
expenditure secondary to a cytokine storm can lead to a non-adaptive
state, overwhelming the metabolic reserve capacity of mitochondria both
from cells infected with COVID-19 and those that respond to infection.
As a normal body function against pathogens, mitochondria also produce
ROS, however, excessive ROS production can be damaging in a similar way
to the infection generated by coronavirus, thus inducing a mitochondrial
dysfunction which potentiates cellular damage.22Together, the combination of impaired respiration, diminished ATP
production, increased ROS, and reduced detoxification capacity with
dysregulated immune functions seems likely to play a pivotal role in the
increased inflammation and severity of COVID-19.22,23
It has been reported that the depletion of cellular adenosine
triphosphate (ATP) can lead to cellular dysfunction induced by
COVID-1924 and the immune cells are not an exception.
A hypothesis has been proposed, stating that ATP depletion can lead to
induce the cytotoxic mechanisms of COVID-19 and promote suppression of
the immune system. Adequate levels of ATP have been established to be
essential for maintaining active JAK/STAT cell signaling pathways that
are involved in INF-1 function, as well as preventing cytokine storm by
modulating the function of lysosomal TLR7. These events can potentially
make recruited immune cells more prone to early exhaustion against
COVID-19 if ATP levels decrease.24,25
In the study reported by Varga et al.,8 they observed
the presence of viral elements within endothelial cells and an
accumulation of inflammatory cells, with evidence of endothelial and
inflammatory cell death. In addition, they report that induction of
apoptosis and pyroptosis might have an important role in endothelial
cell injury in patients with COVID-19. And that damage is most likely
induced by over-activation of the phenotype changes of the immune
system. Several current reports emphasize the occurrence of lymphopenia
with drastically reduced numbers of both CD4 and CD8 T-cells in moderate
and severe COVID-19 cases. The extent of lymphopenia-seemingly
correlates with COVID-19-associated disease severity and
mortality.19,26 Furthermore, damage accumulation and a
poor DNA repair system in immune cells have been reported. The
overexpression of oxidative stress seen with a viral infection, along
with attenuated DNA repair capacity, could accelerate genome instability
and apoptosis in infected and non-infected cells.23
During the SARS-CoV-2 pandemic, quantitative hematologic abnormalities
have been reported in COVID-19 patients. Most of these common
hematological findings include lymphocytopenia, neutrophilia,
eosinopenia, mild thrombocytopenia (35%) or, less frequently,
thrombocytosis.27 However, similarly, morphological
changes in circulating cell lines have been reported like those observed
in this work.
An increase in reactive lymphocytes, sometimes called activated
lymphocytes or virocytes, have been reported in viral diseases. Showing
morphological and functional differences with respect to normal
leukocytes, this because they are the result of a polyclonal immune
response produced by antigenic stimulation derived from various
factors.28 Reactive lymphocytes are normally found in
2% in a healthy adult, we observed reactive lymphocytes, whose
percentage was 10%. These lymphocytes had a low cytoplasm in addition
to a marked basophilia. Indicating an over activation to counteract the
SARS CoV-2 infection. However, some studies show that the hematological
line of lymphocytes is reduced in covid-19 patients, even decreasing in
advanced stages.29,30 Therefore, it is considered a
poor prognostic factor. Monocytes such as macrophages have been
described to express ACE2 receptors, this characteristic makes them
vulnerable to infection with SARS-CoV-2, leading to activation and
transcription of proinflammatory genes.20,31 This
activation and fight of monocytes with SARS-CoV-2 shows morphological
changes in the monocytes, showing vacuoles in their cytoplasm and in the
nucleus, deformity of the membrane. Regarding neutrophils, toxic
granulation has been observed to be dense lysosomes with a high content
of peroxidases, alkaline phosphatase, and acid phosphatase. These
abnormally stained azurophil granules can be lysed, which is
morphologically evidenced as cytoplasmic vacuolization. According to
recent studies, such quantitative and qualitative abnormalities can be
related to cytokine storm and hyperinflammation, which is a fundamental
pathogenic factor in the evolution of the COVID-19
disease.27,32 Moreover, the inflammatory response and
viral effects on leukocytes could be responsible for these morphological
changes, which can be easily identified in leukocytes and can be
monitored with a Wright stain.
Finally, modulation of oxidative stress and inflammation can decrease
the rate of progression of cellular and tissue damage, preventing the
development of local and systemic complications of the disease.
Likewise, proper management of inflammation and mitochondrial
dysfunction can decrease the extent of tissue injury and therefore
improve recovery conditions and quality of life. Finally, oxidative
stress modulation, as well as inflammation and leukocyte mitochondrial
dysfunction form an essential part of comprehensive treatment from early
stages of SARS-CoV-2 infection. Therefore, studies should be carried out
to clarify the processes that lead to the premature death of leukocytes
and that therefore generate an effect similar to immunosuppression, and
that this entails should be avoided with earlier treatment.
Establishing the processes of cellular dysfunction during the
pathophysiological evolution of COVID-19 disease is essential. The
results obtained show how the mitochondrial activity decreases
throughout the infection caused by the SARS-CoV-2 virus promoted by
oxidative stress, and that they modify the mechanisms of cellular
adaptation, losing the ability to regulate the immune system, and the
endothelial damage, giving a synergistic effect on the sustained
inflammatory response (Fig. 6).
Therefore, it is plausible to believe that a mechanistic possibility for
our model could imply interaction between the generation of oxidative
stress and mitochondrial dysfunction, said mitochondrial inhibition
would cause an increase in the production of free radicals and
cytokines, which would cause a cellular change, increasing cellular
damage processes such as protein carbonylation and oxidation of the
macromolecules that lead to cytotoxic damage and culminate in cell death
(Fig. 7).