Molecular Mechanisms of Multi-Organ Failure in COVID-19 and Potential of Stem Cell Therapy

As the number of confirmed cases and deaths occurring from Coronavirus disease 2019 (COVID-19) surges worldwide, health experts are striving hard to fully comprehend the extent of damage caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although COVID-19 primarily manifests itself in the form of severe respiratory distress, it is also known to cause systemic damage to almost all major organs and organ systems within the body. In this review, we discuss the molecular mechanisms leading to multi-organ failure seen in COVID-19 patients. We also examine the potential of stem cell therapy in treating COVID-19 multi-organ failure cases.


Introduction
The first outbreak of COVID-19 was reported in Wuhan, China, in December 2019. Subsequently, the outbreak spread globally and was declared a pandemic by the World Health Organization in March 2020 [1]. As of October 2021, over 236 million cases have been reported, with 4.8 million deaths worldwide [2]. The mortality rate of COVID-19 is around 2%, and the disease's spread is exceedingly high [3,4]. A significant proportion of patients display mild symptoms or are asymptomatic [5]. However, the remainder of the cases are severe, especially in the aged and immune-compromised population [6]. Patients with existing comorbidities such as cardiovascular disease, diabetes mellitus, hypertension, renal dysfunction, liver damage, and cancers exhibit poor prognoses [7]. Death is ultimately caused due to acute respiratory distress syndrome (ARDS), septic shock, cardiac damage, renal dysfunction, and multi-organ failure [8,9].
The host immune response is thought to play a vital role in the pathogenesis and clinical expression of COVID-19. While immune suppression is a risk factor for infection, the immune system's hyperactivation in response to infection can cause severe complications and organ damage [10]. Though vaccines are in various development stages, testing, and distribution, there are no robust therapeutics for either treatment or symptomatic management of the disease yet. Another alarming fact is that many recovered patients will suffer from lasting effects and disability due to COVID-19 [11]. Therefore, alternative therapeutic modalities need to be studied and developed for managing severe outcomes of the disease. The US Food and Drug Administration (FDA) is currently exploring various single-agent and combination treatments for the disease. These include antivirals, cell and gene therapies, immunomodulators, and neutralizing antibodies [12].
Stem cell-based regenerative medicine is one such field that might hold the key to reviving tissue damage caused by COVID-19. Stem cells play a significant role in Figure 1. SARS-CoV-2 infection of lungs leading to cytokine storm resulting in multi-organ failure. 1 Lung cells infected by the coronavirus, 2 Macrophages recognize the virus and release cytokines, 3 Cytokines attract additional immune cells, such as lymphocytes and monocytes, and they generate more cytokines, causing a storm-like cycle of inflammation that damages lung cells. 4 Damage can also occur because of fibrin production (clot formation), 5 Blood vessels surrounding the lungs are weakened, allowing fluid to leak into the lung cavities, resulting in respiratory failure, 6. Due to the heightened coagulation state, blood that is meant to flow to other organs such as the heart, liver, and kidneys will be obstructed, causing these organs to fail and lead to multiorgan failure.

Immunological Complications
Cytokines are proteins that recruit immune cells to the site of infection. A moderate immune response leads to a rise in proinflammatory cytokines such as tumor necrosis factoralpha (TNF-α), interleukin 6 (IL-6), and interleukin 1 (IL-1), and a host of lymphocytes and T cells. However, in a severe immune reaction, we see a sudden extreme induction of these proinflammatory cytokines, also known as the cytokine storm. Cytokine storms lead to widespread inflammation in the body [27]. As a result, vascular membranes become highly permeable, giving rise to fluid movement from blood vessels into organ tissue [28]. The resulting effect is an organ/tissue reaching the brink of failure due to the lack of blood and oxygen. Anti-inflammatory therapies targeting the cytokine storm are suggested to decrease the mortality of COVID-19 patients. Downstream signaling pathways such as JAK/STAT, NF-κB, and NLRP3 as well as cytokine targets such as IL-6, IL-1 β, IFN-γ, TNF-α, IL-12/23, IL-17A, GM-CSF (granulocyte-macrophage colony-stimulating factor) are being explored for treating COVID-19 induced cytokine storm [29].

Hematological Complications
Lymphopenia, or reduction of white blood cells in the blood, is a characteristic clinical hallmark of COVID-19 infection. Typically, CD4, CD8, T, and NK cell counts are significantly decreased. Other observed effects are abnormalities of granulocytes and monocytes along with hypercoagulability leading to thrombocytopenia. An enhanced threat of disseminated intravascular coagulation, specifically, the propensity of clot formation throughout the body, is reported in patients [30,31]. Increased D-dimer concentrations in the non-survivors COVID-19 patients were typical [32,33]. Treatment approaches targeting thrombin, coagulation factor Xa, and thrombin receptor [34] should be considered to reduce SARS-CoV-2 micro thrombosis.

Respiratory Complications
Injury to the lungs in COVID-19 may occur through direct or indirect mechanisms. Various cell types in the airways and lungs exhibit the ACE2 receptor. These include type II alveolar cells, ciliated epithelial cells, and pulmonary vascular endothelium. SARS-CoV-2 can infect these cell types and cause cell death directly. Another mechanism involves activating angiotensin-II, resulting in increased vascular leakiness and pulmonary edema leading to pneumonia [35]. The immune system also plays a critical role in the clinical manifestation of COVID-19 fibroproliferative lung disease [36]. The release of proinflammatory cytokines and activation of macrophages and dendritic cells triggers cell death of infected cells [37,38]. Another factor responsible for pulmonary failure is microthrombi formation in the vasculature of the lungs [39]. Lung biopsies of COVID-19 patients showed an activated complement system in the alveolar epithelial cells with acute and chronic inflammation [38].

Cardiac Complications
Patients with cardiac complications caused due to COVID-19 are termed as suffering from Acute COVID-19 Cardiovascular Syndrome (ACovCS) [40]. There are two different mechanisms for an individual to develop ACovCS. A hypoxia-induced myocardial injury can occur due to a cytokine storm's precipitation, as discussed before. This is accompanied by intracellular acidosis and increased oxidative stress. On the other hand, myocarditis can occur through ACE2 facilitated direct infection of cardiac myocytes resulting in arrhythmias or cardiac arrest [41,42]. However, it is important to note that in a majority of individuals COVID-19 related myocarditis did not accompany immune cell infiltration pointing to cell death from obstructed blood flow likely due to constricted pericytes or clumping of red blood cells [43]. The histopathological analysis reported fibrosis and myocyte hypertrophy in most COVID-19 patients [44]. Thus, patients with existing cardiovascular disease and hypertension are at heightened risk for mortality due to ischemia and myocardial necrosis factors. Comorbidities such as obesity and diabetes have also been found to indirectly cause adverse cardiac complications. For example, obesity arising out of COVID-19 quarantining is linked with stress, causing a persistent inflammatory state that leads to the deposition of atherosclerotic plaques, rendering obese individuals more susceptible to cardiovascular events [45,46]. A study reported a greater incidence of diabetes in patients with COVID-19 associated cardiac injury [47].

Renal Complications
Patients with renal complications often suffer from acute kidney injury (AKI) and proteinuria. Lymphopenia, macrophage activation syndrome, hypercoagulability, and cytokine storm, among other factors, can promote AKI. Other potential mechanisms can include sepsis, endothelial impairment, and rhabdomyolysis. Furthermore, the microthrombi formation may lead to acute ischemic injury and hypoxia, resulting in renal tubular necrosis [48][49][50]. Various reports confirmed that the development of AKI significantly increased the mortality rate in COVID-19 patients. SARS-CoV-2 infection increased blood urea nitrogen (BUN) and serum creatinine levels, leading to renal failure in many patients [48]. Autopsies of COVID-19 patients detected fibrin thrombi, indicating severe endothelial damage in the glomerular region [51].

Hepatic Complications
Studies have observed that the severity of COVID-19 infection corresponds to the severity of hepatic complications in patients. Very high levels of liver enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were found in the blood of patients with severe COVID-19 infections [52]. Cytotoxic T cell activation and dysregulated immune responses increased liver biomarkers and COVID-19 severity [53]. One mechanism for viral entry to the liver might be through the ACE2 receptors found on cells lining the bile duct. Other liver injury mechanisms are thought to be linked to oxygen deprivation, the toxicity of antiviral/antimalarial treatments, and inflammatory cells' passage to the liver [54,55].

Neurological Complications
ACE 2 receptors responsible for attachment and subsequent internalization of SARS-CoV-2 are also found in glial cells in the brain and spinal neurons. Hence, the virus can damage the neuronal tissue and result in hypoxic brain injury and immune-mediated damage to the Central Nervous system (CNS) [56].
A study conducted by the National Hospital, Queen Square, London, and University College London Hospital detailed clinical and paraclinical data on neurological disorders observed during and after the COVID-19 infection. Patients displayed a wide range of CNS and Peripheral Nervous System (PNS) complications together with neuroinflammation. These pathological manifestations can be a direct effect of the virus on the nervous system, para or post-infectious immune-mediated disease, and neurological complications of the systemic impacts of COVID-19 infection. Out of 43 COVID-19 patients, ten patients above the age of 50 suffered septic encephalopathy and presented symptoms such as confusion, psychosis, seizures, etc. Complications such as difficulty speaking, deteriorating vision, cognitive abilities, and Acute Disseminated Encephalomyelitis (ADEM) with hemorrhagic transformations were reported in critically ill patients [57]. Alteration in mental status was frequent in patients with severe infection, especially in those requiring intensive care management. However, this was documented more in older groups, which might be suffering from latent neurocognitive degenerative disease or multiple medical comorbidities, often associated with sepsis and hypoxia. MRI diagnosis in severe cases showed abnormalities in the brain's temporal lobe with hemorrhagic lesions. Furthermore, features like vascular and ADEM-like pathology, with macrophages and axonal injury, were reported.
Further, seizures were reported with viral encephalitis and subsequent activation of neuro-inflammatory pathways in critically ill patients. Lymphocytic panencephalitis, meningitis, and brainstem inflammatory change with neuronal loss were observed in post-mortem reports [58]. A recent report on neurological complications of children of age < 18 years described a distinct neurological syndrome associated with lesions in the corpus callosum's splenium. These patients were previously healthy but had the onset of neurological symptoms after the COVID-19 infection. Symptoms observed were encephalopathy, headaches, brainstem-cerebellar signs, muscle weakness, and reduced reflexes [59].
To summarize, the neurological manifestations of COVID-19 can be divided into direct and indirect effects. Symptoms like anosmia, hypogeusia meningitis, encephalitis, cerebral vasculitis, and myalgia result from a direct viral invasion of the CNS and PNS. Encephalopathy arising out of hypoxia, cytokine storm, and hypercoagulable state leading to stroke are some of the indirect manifestations seen in the CNS [60]. Another critical factor that needs to be considered is the impairment of the blood-brain barrier (BBB) due to endothelial dysfunction. This may give rise to the virus's invasion and inflammatory cells into the CNS, leading to further neurological manifestations [54,61].

Stem Cell Therapy
Stem cells are undifferentiated or partially differentiated cells in the body that can differentiate into various cell types and proliferate indefinitely (self-renewal). Their primary function is to serve as a reserve for the body [62]. Stem cells can be of embryonic or adult origin. Based on their functionality, stem cells can be grouped into three categories, pluripotent, hematopoietic, and mesenchymal types [63][64][65]. Pluripotent stem cells can differentiate and mature into any of the three fundamental groups of cells important in human developmental biology. Embryonic stem cells are used for in vitro fertilization purposes [66]. Hematopoietic stem cells can differentiate into various types of blood cells. They are obtained from bone marrow or umbilical cord blood and are used in bone marrow transplants. However, both these types of stem cells are currently not used to treat COVID-19. Lastly, MSCs are non-hematopoietic cells that can be differentiated into skeletal tissue such as muscle, bone, cartilage, fat, etc. These cells have immunomodulatory capabilities and have been approved as treatments for a host of autoimmune diseases [67,68]. Additionally, the therapeutic effects of stem cells were recently ascribed to their ability to replace damaged cells. However, we now know that stem cells' pro-regenerative quality is also due to paracrine functions and the ability to release microvesicles. Microvesicles are known to contain growth factors, bioactive lipids, anti-apoptotic factors which enhance cell function and stimulate angiogenesis in damaged tissues. They are also known to transfer proteins, mRNA, and microRNA between cells [69].
MSCs can be isolated from bone marrow, placenta, umbilical cord blood, and adipose tissue of the same individual (autologous) or another individual (allogeneic) [70]. These are then cultured in vitro and can be injected back into the diseased body. Once inside, they secrete a host of anti-inflammatory mediators that can accelerate tissue repair and revival [71]. MSCs are most likely to have favorable effects when treating acute inflammatory conditions [72]. Conditions frequently encountered in COVID-19 patients, such as cytokine storm, ARDS, and sepsis, are likely to be prime candidates for MSC-based therapy.

Immunomodulatory and Regenerative Effects of MSCs
MSCs are referred to as "guardians of inflammation" because of their immunomodulatory effect through the secretion of cytokines, chemokines, growth factors, exosomes, etc. MSCs regulate the inflammatory microenvironment through cell-to-cell contact and the secretion of regulatory molecules. These affect the activation, maturation, proliferation, differentiation, and effector functions of various immune cells involved in innate and adaptive immunity. Innate immunity is mediated through NK cells, macrophages, neutrophils. On the other hand, adaptive immunity is facilitated by T cells and B cells ( Figure 2). Dendritic cells (DC) act as the connecting link between innate and adaptive immunity.
NK cells secrete cytokines like IFN-γ and exhibit cytotoxic functions in response to viral infection. MSCs can reduce the proliferation of NK cells, inhibiting their cytotoxic functions through key mediators such prostaglandin E2 (PGE2), indolamine 2,3-dioxygenase (IDO), and human leukocyte antigen G5 (HLA-G5) [73]. Macrophages play a vital role in innate immunity by engulfing foreign agents or aberrant cells. There are mainly two forms of activated macrophages grouped into M1 and M2. M1 exhibits a proinflammatory response in contrast to M2, which displays an anti-inflammatory response. PGE2 secreted by MSC influences the macrophage transition from proinflammatory M1 into an anti-inflammatory M2. M2 macrophage expresses high levels of anti-inflammatory cytokines, reduces levels of TNF-α, and IL-12 with higher phagocytic activity [74]. In the inflammatory process, neutrophils generate more reactive oxygen species (ROS) and reduce antioxidant levels. MSCs secrete IL-6, which reduces ROS levels without affecting the phagocytic activity of neutrophils [75]. T-cells, once activated, proliferate and secrete inflammatory cytokines and chemokines. MSCs facilitate their immunomodulatory activity by recruiting local helper (Th) and effector T cells in the inflammatory environment via Chemokine (C-X-C motif) ligands-CXCL9 and CXCL10. B-cells are vital for humoral immunity and secrete antibodies when stimulated. MSCs inhibit B cell activation, proliferation, and differentiation during inflammation through contact inhibition [76][77][78].
MSCs not only play a role in immune regulation, but also the regeneration and reconstruction of tissue. MSCs have differentiation properties conducive to tissue regeneration and can secrete hepatocyte growth factor, vascular endothelial growth factor, and keratinocyte growth factor. These functions can promote the regeneration of type II alveolar epithelial cells [79]. This shows a potential use for MSCs in recovery for severe COVID-19 cases wherein alveolar injury has occurred. It is suggested that MSCs suppress the over-activated inflammatory response, promote recovery of lung function, and potentially influence the progress of pulmonary fibrosis. MSCs have already been shown to significantly contribute to the recovery of patients from severe COVID-19 in Phase 1 clinical trial [14]. A larger phase 2/3 trial is in progress, with 100 participants recruited to evaluate the safety and efficacy of human umbilical cord-derived MSCs as a treatment for severe COVID-19 cases [80]. Additionally, MSCs and other stem cell types and derivatives have been indicated to be capable of promoting regeneration in other tissues, such as vascular [81], renal [82], hepatic [83], and neurological [84]. Through their immune regulatory functions, and role in contributing to tissue repair MSCs represent a promising area for the treatment of COVID-19 [81].

Clinical Trials
The treatment potential of stem cell therapy has been widely explored in immunological, cardiovascular, renal, pulmonary, and hepatic diseases at the preclinical level. However, the current bulk of clinical data is insufficient to demonstrate the unequivocal efficacy of stem cell therapy in patients with complex diseases like organ failure. Patients with dysfunctional renal and hepatic organs are likely to need organ transplants toward their disease's end-stage. Kidney failure in chronic kidney disease (CKD) or AKI can be attributed to various complicating factors, including diabetes and heart disease. Similarly, liver failure may be caused by multiple factors such as cirrhosis, Hepatitis B or C, and hemochromatosis, etc. However, there are not enough kidney and liver donors available for the number of needing patients. In this backdrop, stem cell therapy has emerged as a promising option for these patients. Studies have shown minimal adverse events and, in some cases, even positive outcomes for patients undergoing stem cell treatments for liver conditions [85,86].
MSC therapies have been approved in several countries for treating several diseases due to their immuno-modulatory effects. They are potent candidates to treat severe cases of COVID-19 and have been used in clinical trials for therapeutic purposes [87]. A list of completed and active clinical trials using MSC therapy to treat COVID-19 associated conditions is provided in Tables 1 and 2, respectively. Similarly, we have outlined the available results of specific studies in Table 3. , and other MSCs, as well as exosomes from MSCs, are being tested in clinical trials. UC-MSCs are the most effective for treating COVID-19 patients due to their proliferative capability and immunomodulatory effects. In a recent clinical study, laboratory tests of C-reactive protein (CRP), alanine aminotransferase (ALT), creatinine, serum ferritin (SF), and platelets before and after the UC-MSCs treatment at days 0, 3, and 7 were recorded for both experimental and control group. In the UC-MSCs treatment group, there was a decline of IL-6 within three days after UC-MSCs infusion, which remained stable for the following four days. The partial pressure of arterial oxygen: percentage of inspired oxygen (PaO2/FiO2) ratio improved in most severe cases. Representative chest CT scan images showed controlled lung lesions within six days, which completely disappeared within two weeks of treatment. A reduced trend in the levels of proinflammatory cytokines was noted within 14 days [104].   In another study, a double-blind, phase 1/2a, randomized, controlled trial was performed in subjects with ARDS secondary to COVID-19. Twenty-four subjects (12 per group) were recruited for this study. At 28 days post the last infusion, patient survival was 91% and 42% in the UC-MSC and control groups, respectively (p = 0.015). No serious adverse events were observed related to UC-MSC infusions [137]. Although several studies have been completed, their results have not been declared. As such, several concerns regarding the safety and efficacy of MSCs treatment for COVID-19 associated lung disease are unanswered. For example, we do not know the best administration route, whether intravenous, intramuscular, or through the nasopharyngeal route. Apart from this, there is no consensus on critical factors such as MSC tissue of origin, type of culture environment, and dosing.

Future Directions
The field of stem cell therapy for treating COVID-19 is gaining a lot of traction. As of October 2021, 100 studies in various countries were displayed on the clinicaltrials.gov website. During a pandemic, as in the case of COVID-19, treatment options are often accelerated and provided emergency authorization to save as many lives as possible. However, it is difficult to ascertain the cause and effect of therapy and outcome based on observational studies alone. Therefore, it is crucial to conduct randomized, double-blinded, placebo-control trials to fully ascertain the efficacy of stem cell therapy as a viable option for COVID-19. Furthermore, it is essential to know the underlying molecular mechanisms through which stem cells fight disease. As such, future studies, both preclinical and clinical with mechanistic approaches, would propel the field further in the right direction.
SARS-CoV-2 attracts leukocytes to the site of infection, thereby initiating the immune response with cytokines' help. It is known that an increase in ROS accompanies an increase in the immune response. This causes oxidative stress, cell apoptosis, lipid peroxidation, and protein oxidation, further worsening the immune response [139,140]. We hypothesize that facilitating ROS elimination through a better understanding of antioxidant pathways may prevent the oxidative injury caused by SARS-CoV-2 infection. Many studies support the antioxidant and regenerative properties of MSCs [141,142]. A key transcriptional factor, nuclear factor erythroid 2-related factor 2 (NRF2), has been studied for its involvement in regulating antioxidant signals [143]. A recent study published by Olagnier et al. showed suppression of NRF2 in COVID-19 patients, thereby strengthening our hypothesis [144]. Therefore, further studies focusing on NRF2 as a molecular target for treating COVID-19 related complications can provide additional insights into the disease.

Conclusions
Patients with severe COVID-19 infection often develop multi-organ failure. The damage to organs and organ systems is either through direct infection or hampered physiological processes in response to the infection. It is crucial to consider the immune system as the focal point to understand better and integrate the other organs' complications. Given the immunomodulatory properties of stem cells, it is essential to conduct further research to study stem cell therapy's potential in alleviating COVID-19 multi-organ failure.
Author Contributions: A.B., conceived the study and prepared the drafting of the manuscript. S.R., B.N. and S.M. edited and revised the manuscript. L.C., assisted with the drafting and editing of the manuscript, oversaw the entire project, and provided funding support. All authors have read and agreed to the published version of the manuscript.