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).