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\title{SARS-CoV-2 induces mitochondrial dysfunction and cell death by oxidative
stress in leukocytes of COVID-19 patients}



\author[1]{Joel De la Cruz-Enriquez}%
\author[1]{Emmanuel Rojas-Morales }%
\author[1]{Maria Gema Ruiz-Garcia}%
\author[1]{Julio Tobon-Velasco}%
\author[1]{Jose Jimenez}%
\affil[1]{Centro Medico Nacional de Biologia Molecular}%


\vspace{-1em}



  
  \date{May 18, 2024}


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\selectlanguage{english}
\begin{abstract}
Background: From sepsis to COVID-19-induced multi-organ failure,
inflammation and immune system activation play an important role. It has
been argued that inflammation and over-activation of the immune system
could be mediating a pro-oxidant microenvironment that can induce
cytotoxic effects that potentiate tissue damage favoring organic
deterioration. Aims: To investigate whether induction of oxidative
stress by COVID-19 infection could inhibit mitochondrial function and
cause cellular damage in leukocytes. Methods: We evaluated plasma levels
of nitric oxide, hydrogen peroxide and protein carbonylation using
spectrophotometry, in addition to evaluating mitochondrial function and
cell death by fluorescence microscopy and leukocyte morphology, in
COVID-19 patients at two time points: viremia and severe sepsis with
multi-organ failure. Results: COVID-19 induces increased oxidative
stress markers that activate cellular damage processes. In the viremia
stage, was observe with an increase in peroxide (28.9\%), nitric oxide
(370.3\%) and carbonylated proteins (61.8\%), which was correlated with
an increase in inhibition of mitochondrial function (66\%), early
apoptosis (212\%) necrosis (405\%), and leukocytes-reactivity. The
severe sepsis stage with multi-organ failure also showed a further
increase in levels of peroxide (46.4\%) with a slight decrease in nitric
oxide (216.2\%) but with more carbonylated proteins (102\%), regarding
what was observe in viremia. This oxidative process was correlate with
less inhibition of mitochondrial function (32.4\%) and an increase in
late apoptosis (463\%), and morphology changes evidencing damage in the
leukocytes. Conclusion: SARS-CoV-2 induced damage promotes levels of
oxidative stress markers and mitochondrial dysfunction that potentiate
morphological changes and cell death in leukocytes. These cellular
effects could be integrating into the physiopathology of COVID- 19.
These processes explain the rapid changes in the immune system, and that
present an initial over-activation and early massive death due to
SARS-CoV-2 infection, promoting endothelial-alveolar damage that would
cause multi-organ failure.%
\end{abstract}%



\sloppy


\textbf{WHAT'S KNOWN? (what is already known about this subject?)}

Current research indicates that SARS-CoV-2 infection induces various
processes of cellular damage mediated by inflammation derived from the
activity of the immune system, and that these events potentiate cellular
damage in various tissues such as the endothelium, lungs, heart, etc. In
severe cases, it has been indicated that patients have leukopenia,
however, the effect of the infection on the cells of the immune system
and in the early stages of the disease has not been evaluated.

\textbf{WHAT'S NEW? (what does this study contribute to the
literature?)}

Our results show the effects of SARS-CoV-2 infection on mitochondrial
function and cell damage in early stages, specifically in leukocytes as
a fundamental population that must mediate the damage induced by the
infection, and that responds to the inductive effect. of oxidative
stress. So it proposes a fundamental condition in the damage induced by
the infection.

\textbf{1 INTRODUCTION}

COVID-19 is a viral infection induced by the coronavirus of severe
respiratory syndrome (SARS-CoV-2), that represents a serious health
problem worldwide; due to its association with various
pathophysiological processes such as: inflammation, increased
proinflammatory cytokines, and cell death, which could be closely
related to redox imbalance or oxidative stress.\textsuperscript{1} The
high risk of severity and mortality from SARS-CoV-2 infection or
COVID-19 disease has been associated with several clinical indicators
such as: lymphocytopenia, increased D-dimer, increased serum ferritin,
and high titers IL-6.\textsuperscript{1,2}

The inflammatory response induced by SARS-CoV-2 can generate an effect
of cellular oxidative stress, which induces an increase in circulatory
inflammatory mediators, including cytokines, produced through redox
pathway activation in patients with sepsis.\textsuperscript{3,4}Because,
oxidative stress and inhibition of mitochondrial activity may be an
alternative to explain the tissue injury associated with
COVID-19-induced damage to endothelial, alveolar, and cardiac cells,
among other.\textsuperscript{5,6} We consider it vitally important that
health professionals can analyze and understand a little more about
these pathophysiological mechanisms, to evaluate and design new
protocols that allow a better prognosis for patients.

According to tissue damage, accumulation of inflammatory cells
associated with endothelial tissue has been reported, as well as
apoptotic bodies in alveolar cells and others.\textsuperscript{7} Thus,
it is postulated the accumulation of mononuclear cells in the lung and
in the small pulmonary vessels induce congestion and therefore
endothelial tissue damage associated with the
lung.\textsuperscript{8}The endothelial dysfunction is a main
determinant of microvascular dysfunction, which is associated with the
processes of inflammation and oxidative stress, that potentiate tissue
edema associated with a pro-coagulant state.\textsuperscript{6,9}

The SARS-CoV-2 induce an explosion of inflammatory cytokines, reactive
oxygen species (ROS), and cell death-induced by this cell events is a
cause which can result in significant endothelial and multi-organ
damage, so the regulation of oxidative stress is
essential.\textsuperscript{3,4,7} Thus, modulation of oxidative stress
may be able to prevent the cellular and tissue injury and development
severe disease symptoms in coronavirus patients by reduce the
immuno-pathology of coronavirus infection on patients' health after the
active phase of the infection is over, so that, if we could reduce or
modulate cellular toxic effects, it would make COVID-19 disease more
controllable.\textsuperscript{9,11}

Therefore, this study aimed to evaluate the association between
different biomarkers of oxidative stress with parameters of
mitochondrial and cellular function to evaluate the relationship between
oxidative stress and leukocyte function in patients with COVID-19. This
process contributes to the understanding of the possible link between
oxidative stress and the risk of pathogenesis, severity and mortality in
patients affected by SARS-CoV-2 infection.

\textbf{2 MATERIALS AND METHODS}

\textbf{2.1 Study and subjects} . Patients with laboratory confirmed
COVID-19 infection, diagnosed using quantitative reverse
transcriptase--polymerase chain reaction (qRT-PCR), and clinical
symptomatology of COVID-19 (history of fever, any respiratory symptom
and chest radiographs confirmed pulmonary lesions (for moderate and
severe cases), were included. The baseline characteristics of the
patients for their classification are shown in Table 1.

The patients were classified into two groups according to disease
severity considering the following parameters. I) Viremia: Patients with
fever (\textgreater{}38 \textsuperscript{o}C), d-dimer (0.6-0.9 mg/ml),
lactate (<\selectlanguage{english}2mmol/L), leukopenia, anosmia, myalgia, arthralgia, fatigue,
cough, sore throat, runny nose, and sneezing. II) Severe sepsis with
multi-organ dysfunction syndrome (MODS): Patients with d-dimer
(>\selectlanguage{english}10mg/ml), creatinine (\textgreater{}1.5 mg/dl), procalcitonin (+),
lactate (>\selectlanguage{english}2mmol/L), hypotension, electrolyte imbalance and who had any
of the following were considered in critical condition: acute
respiratory distress syndrome (ARDS), respiratory failure requiring
mechanical ventilation, may have shock, encephalopathy, myocardial
injury, heart failure, coagulation dysfunction and acute kidney injury
or failure of other organs requiring admission to the intensive care
unit.

In addition, patients with respiratory problems similar to COVID-19
disease but with a negative confirmatory test, were included as damage
control.

\textbf{2.2 Biochemistry analysis}

\textbf{2.2.1 Peroxide assay.} The peroxide content was determined in
plasma by means of a quantitative colorimetric assay, according to the
kit indications ''QuantiChrom\textsuperscript{TM} Peroxide Assay Kit
(DIOX-250) - BioAssay Systems'', at the end of the process the optical
density at 570 nm was quantified in ELISA plate reading equipment
(Chromate), reporting values of ng/ml.

\textbf{2.2.2} \textbf{Nitric oxide assay.} The nitric oxide content was
determined in plasma by means of a quantitative colorimetric assay,
according to the kit indications ''QuantiChrom\textsuperscript{TM}
Nitric Oxide Assay Kit (D2NO-100) - BioAssay Systems'', at the end of
the process the optical density was quantified at 570 nm, in an ELISA
plate reading equipment (Chromate), reporting in \selectlanguage{greek}µ\selectlanguage{english}M values.

\textbf{2.2.3 Oxidized proteins assay.} Protein carbonyl content in
plasma was determined as previously described.\textsuperscript{12} The
plasma was incubated with DNPH (2,4-Dinitrophenylhydrazine; 10 mM) and
HCl (2N) and finally with guanidine hydrochloride (6M). Assessment of
carbonyl formation was done on the basis of formation of protein
hydrazone by reaction with DNPH. The absorbance was measured at 370 nm.
Result was expressed as nmol carbonyl per mg protein.

\textbf{2.3 Mitochondrial function, cell death and morphology by
microscopy}

\textbf{2.3.1 Fluorescence assessment for Mitochondrial activity} .

Staining against Tetramethylrhodamine methyl-ester (TMRM - was used to
assess mitochondrial membrane potential, \selectlanguage{greek}ΔΨ\selectlanguage{english}m) and Hoechst were assessed
as a reliable marker of mitochondrial function and chromatin
condensation respectively. Buffy coat was first rinsed in PBS (pH=7.4)
for 5 min and then incubated with TMRM (1:200) in PBS for 30 min at
37\textsuperscript{o}C. Thereafter, the mixture was washed and incubated
with Hoechst (1:500) in PBS for 5 min at room temperature, and rinsed in
PBS (pH 7.4) for 5 min per step. The mixture was then mounted with
Vectashield and the image acquisition and processing were made with a
microscope (Iroscope) fitted with a digital camera and imaging system
using top view acquisition software. Fluorescent photomicrographs were
obtained and the number of reactive cells to TMRM and Hoechst per field
was determined in ten fields per sample, the results are shown by
measurement of the densitometry (relative units) or the number of cells.

\textbf{2.3.2 Fluorescence assessment for Apoptosis} . Staining against
annexin-V and propidium iodide were assessed as a reliable marker of
apoptosis and necrosis respectively. Buffy coat was first rinsed in
annexin-buffer for 5 min and then incubated with annexin-V/FITC (1:200)
in annexin-buffer for 20 min at RT. Thereafter, the mixture was washed
and incubated with propidium iodide (1:400) for 5 min at RT, and rinsed
in PBS (pH 7.4) for 5 min per step. The mixture was then mounted with
Vectashield and the image acquisition and processing were made with a
microscope (Iroscope) fitted with a digital camera and imaging system
using top view acquisition software. Fluorescent photomicrographs were
obtained and the number of reactive cells to annexin-V, PI or
annexin-V/PI per field was determined in ten fields per sample, the
results are shown by counting the number of positive cells.

\textbf{2.3.3 White blood cells morphology}

A peripheral blood film (made from a drop of blood from an EDTA
anticoagulated tube or skin puncture) is stained with Wright stain and
microscopically examined using scanning (10x), and oil (100x)
objectives. The WBC morphology was carefully evaluated using a 100x
microscope objective.

\textbf{2.4 Statistic analyses.} Results were expressed as mean values \selectlanguage{ngerman}±
SEM. All data were statistically analyzed using one-way analysis of
variance (ANOVA) for repeated measures, followed by post hoc
Bonferroni's test. All analytical procedures were performed using the
scientific statistic software GraphPad Prism 5 (GraphPad Scientific, San
Diego, CA, USA). Differences of P\selectlanguage{english}<0.05 were considered as statistically
significant.

\textbf{3 RESULTS}

\textbf{3.1 SARS-CoV-2 induces changes in oxidative stress markers in
blood samples of COVID-19 patients.}

Different markers of oxidative damage, including hydrogen peroxide and
nitric oxide formation and also some major toxic side effect were
analyzed such as protein carbonyl levels, were determined in the plasma
of COVID-19-patients. The amount of
H\textsubscript{2}O\textsubscript{2}was assessed as an index of
oxidative stress total, are shown in Fig. 1A. In control patients,
baseline H\textsubscript{2}O\textsubscript{2}levels was 712.7 \selectlanguage{ngerman}± 186.2
ng/mL, while in plasma from COVID-19-patients there were less increases
in H\textsubscript{2}O\textsubscript{2}formation of 28.9\% in the
viremia group and signi\selectlanguage{english}ficant increases of 46.4\% in the MODS group
(p\textless{}0.05), above control (919.3+-151.6 and 1044+-125.4;
respectively). On the other hand, the peroxide levels in the
non-positive MODS COVID-19 group were similar to the control group (11\%
above control).

In regard to NO production are shown in Fig. 1B, baseline levels in
control group were found around 34.91+- 4.463 \selectlanguage{greek}µ\selectlanguage{english}mol of
NO\textsubscript{2}/mg protein. The COVID-19-induced increases in
nitrites accumulation were found around 370 \% in viremia
(p\textless{}0.01) and 216 \% in sepsis whit MODS (p\textless{}0.05),
above control (164.2\selectlanguage{ngerman}± 12.60 and 110.4± 13.39; respectively). In
addition, non-positive MODS COVID-19 group showing higher levels of
nitric oxide (p\textless{}0.01).

The levels of protein oxidation (expressed as the formation of protein
carbonyl as a marker of proteostasis-loss) from all experimental groups
are shown in Fig. 1C. Basal levels were 3.238±0.2746 nmol/mg protein.
COVID-19 produced changes in protein carbonyl when compared with control
group values at any of all times tested in 61.8\% in the viremia group
and 102\% in the MODS group; statistical significance was set at
p\textless{}0.05 (5.240±0.4537) and p\textless{}0.01 (6.542±0.3925)
respectively. The MODS not COVID-19 group, showed the highest protein
carbonyl levels (p\textless{}0.01). Whereas Fig. 1D presents the
correlation between peroxide levels and proteins carbonyl content found
in blood of COVID-19-patients. Noteworthy a correlation between
parameters was found of r = 0.88

\textbf{3.2 SARS-CoV-2 induces inhibition of mitochondrial function and
nuclear condensation in leukocytes of COVID-19 patients.}

Mitochondrial function is assessed by staining with TMRM and were
contrasted with nuclei stained with Hoechst in leukocytes. TMRM
fluorescence was diminished in COVID-19 patients (viremia and severe
sepsis with MODS), the former indicating a reduction in \selectlanguage{greek}ΔΨ\selectlanguage{english}m, are show in
Fig. 2. The basal levels of mitochondrial function were 70780\selectlanguage{ngerman}±5132 in
relative units. Regarding mitochondrial function in the viremia group a
44\% function was observed (31171±4449), while the MODS group presented
a percentage of 67.5\% (47785±4005) compared to the control,
representing an inhibition of 66\% (p\textless{}0.01) and 32.4\%
(p\textless{}0.05) respectively. Regarding the non-COVID MODS group, a
significant inhibition of mitochondrial function of 23.2\% was observed
compared to the control (Figure 2A).

In regard to nuclear condensation by count of cells with the highest
staining for Hoechst are show in Fig. 3. In the control group, an
average number of 11.6 cells with the highest fluorescents was observed,
while the COVID groups (viremia and MODS) showed an increase in nuclear
condensation of 31 (165.6\%) and 63 (442.7\%) cells respectively
(p\textless{}0.01). While the MODS group not positive for COVID also
showed an increase in the number of cells with nuclear condensation
(26.3 cells; 126.7\% above of control) (Fig. 3A).

On the other hand, the graphs of correlation between peroxide levels and
mitochondrial function are presented (Fig. 2B) and the correlation of
peroxide levels and nuclear condensation (Fig. 3B), determined in the
leukocytes of patients with COVID -19. It should be noted that a
correlation was found between the parameters of r = 0.84 (Fig. 2B) and r
= 0.90 (Fig. 3B).

\textbf{3.3 SARS-CoV-2 induces cell death and morphological changes in
leukocytes of COVID-19 patients.}

We analyzed cell death levels by annexin-V and propidium iodide
reactivity as a phenotypic marker for apoptosis and necrosis. Single
annexin-V expression shows early apoptosis, while the presence of
annexin-V and propidium iodide results in late apoptosis, and only
staining with propidium iodide establishes necrosis.

According to cell death (Fig. 4), an increase in both early apoptosis
(243.1\%, Fig. 4A) and late apoptosis (466.7\%, Fig. 4B) was observed in
the viremia-COVID-19 group, respect to control; in contrast, the
MODS-COVID-19 group, showed a greater increase in late apoptosis
(1363\%, p\textless{}0.001) compared to the early apoptosis (147.6\%,
p\textless{}0.01), was presented reflecting rapid changes in cellular
damage processes. Also, the MODS-COVID-19 negative group showed an
increase in early and late apoptosis processes (130\% and 496\%,
respectively) compared to the control group.

Finally, in the necrotic death process (Fig. 4C) it is observed that the
viremia-COVID-19 group presents higher levels (3.8 folds more,
p\textless{}0.01) than the control; while the MODS-COVID-19 group
presented lower levels compared to the viremia group, but higher than
the control (1.6 folds higher, p\textless{}0.05). Regarding the negative
MODS COVID group, there was also a slight increase in necrosis (0.5
folds), although it was not significant compared to the control.

In regard to morphological changes in leukocytes are show in Fig. 5. It
is observed that infection by SARS-CoV-2 induces modifications in the
morphology of leukocytes, generating the presence of cellular structures
that indicate changes in their functions. In Figure 5A, Band neutrophils
with coarsely clumped chromatin and cytotoxic granulation, observed in
greater quantity in the MODS stage. 5B and 5C, Hypersegmented
neutrophils with vacuoles, in addition, loss of membrane integrity is
observed, these cells were found in the sepsis stage. 5D, Band
neutrophils with cytotoxic granulation and vacuoles, observed at all
stages. 5E, monocytes with both cytoplasmic and nuclear digestive
vacuoles are observed, indicating increased phagocytic activity. These
monocytes were observed in greater quantity in COVID-19 patients in MODS
stage and in smaller quantity in the sepsis stage; 5F, vacuolated
neutrophils, these neutrophils present vacuoles in the cytoplasm and
were observed in all stages of the disease. 5G: Leukocytes with membrane
rupture. In the MODS-COVID-19 stage, leukocytes with a completely
ruptured membrane were observed, indicating death from cell lysis. in
5H: Hypersegmented neutrophils with cytotoxic granulation are observed,
granulations indicate a systemic inflammatory process in addition to a
viral or bacterial infectious process, granulations were observed in all
stages of the disease, however hypersegmentation was observed at the
stage of MODS. 5I, reactive lymphocytes (hyperbasophilic) are observed,
which presents a scarce cytoplasm in addition to a marked basophilia,
smaller in size as a mature lymphocyte and observed at all stages of
SARS-CoV-2 infection.

\textbf{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.\textsuperscript{13} It has been determined that viral
stimulation in COVID-19 is prone to elicit intensive immunological
reactions, cytokine storm and immune-cell
infiltration.\textsuperscript{4} However, some immunocytes can produce
numerous ROS including peroxide, nitric oxide and hydroxyl radical, as
reported in other virus studies.\textsuperscript{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.\textsuperscript{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.\textsuperscript{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,\textsuperscript{16} that promote degranulation of
polymorphonuclear cells and the production of ROS which promotes
oxidative stress inducing cellular and tissue
damage.\textsuperscript{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.\textsuperscript{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.\textsuperscript{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.\textsuperscript{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).\textsuperscript{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.\textsuperscript{22}Together, 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.\textsuperscript{22,23}

It has been reported that the depletion of cellular adenosine
triphosphate (ATP) can lead to cellular dysfunction induced by
COVID-19\textsuperscript{24} 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.\textsuperscript{24,25}

In the study reported by Varga et al.,\textsuperscript{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.\textsuperscript{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.\textsuperscript{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.\textsuperscript{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.\textsuperscript{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.\textsuperscript{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.\textsuperscript{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.\textsuperscript{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).

\textbf{5 CONCLUSIONS}

The evidence gathered in this study suggests that SARS-CoV-2 infection
induces an oxidative response that potentiates cell damage evidenced by
inhibition of mitochondrial function, morphological changes and cell
death in leucocytes. The signaling associated with inflammation and
oxidative stress in COVID-19 disease establishes a paradigm of
comprehensive injury that must be consider in physiopathology. These
processes explain the rapid changes in the immune system, and that
present an initial over-activation and early massive death due to
SARS-CoV-2 infection, promoting endothelial-alveolar damage that would
cause multi-organ failure. From this, a relevant approach is shown to
develop therapeutic strategies based on this and other early processes
related to the pathogenic characteristics of the disease, although more
research is needed to detail the mechanisms of cytotoxicity, it is
essential to start establishing early actions in the therapy of this
disease.

\textbf{Acknowledgments:} Thanks to MD. Rosa Elena Zamudio for provide
the patient's blood samples with COVID-19.

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