key: cord-0933697-wcmmt5bl
authors: Jayaramayya, Kaavya; Mahalaxmi, Iyer; Subramaniam, Mohana Devi; Raj, Neethu; Dayem, Ahmed Abdal; Lim, Kyung Min; Kim, Se Jong; An, Jong Yub; Lee, Yoonjoo; Choi, Yujin; Kirubhakaran, Arthi; Cho, Ssang-Goo; Vellingiri, Balachandar
title: Immunomodulatory effect of mesenchymal stem cells and mesenchymal stem-cell-derived exosomes for COVID-19 treatmen
date: 2020-08-31
journal: BMB Rep
DOI: 10.5483/bmbrep.2020.53.8.121
sha: 1c0eb34294a2bc585b79d669cb0c70dbb0b0f167
doc_id: 933697
cord_uid: wcmmt5bl

The world has witnessed unimaginable damage from the coronavirus disease-19 (COVID-19) pandemic. Because the pandemic is growing rapidly, it is important to consider diverse treatment options to effectively treat people worldwide. Since the immune system is at the hub of the infection, it is essential to regulate the dynamic balance in order to prevent the overexaggerated immune responses that subsequently result in multiorgan damage. The use of stem cells as treatment options has gained tremend-ous momentum in the past decade. The revolutionary mea-sures in science have brought to the world mesenchymal stem cells (MSCs) and MSC-derived exosomes (MSC-Exo) as thera-peutic opportunities for various diseases. The MSCs and MSC-Exos have immunomodulatory functions; they can be used as therapy to strike a balance in the immune cells of patients with COVID-19. In this review, we discuss the basics of the cyto-kine storm in COVID-19, MSCs, and MSC-derived exosomes and the potential and stem-cell-based ongoing clinical trials for COVID-19.

The world has been facing a dreadful situation due to the spread of the Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) (1). However, neither confirmed effective antiviral medications nor vaccines are available to deal with this emergency (2) . Many reports have suggested that it is the cytokine storm in COVID-19 that leads to acute respiratory distress syndrome (ARDS) (3) . The cytokine storm in COVID-19 refers to the fact that a variety of cytokines are rapidly produced after viral infections (4) . In addition, such a cytokine storm induces hypoxia, and direct viral infection can cause cellular damage. Multiorgan damage and injury have been concomitant with COVID-19, and can be observed more in patients with a more severe form of the disease (5) .

Stem cells are specialized cells that can renew themselves by means of cell division and can differentiate into multilineage cells. Mesenchymal stem cell (MSCs) have immunomodulatory features and secrete cytokines and immune receptors that regulate the microenvironment in the host tissue (6) . In addition, it has been observed that the crucial role of MSCs in therapy has been mediated by exosomes released by the MSCs. These exosomes have exhibited immunomodulatory, antiviral, anti-fibrotic, and tissue-repair-related functions in vivo; similar effects have been observed in vitro (6) .

The dynamic equilibrium maintained by innate and adaptive immunity is essential for impeding the progression of COVID-19 (7). In patients infected with SARS-CoV-2, the plasma levels of IL-1, IL-1RA, IL-7, IL-8, IL-10, IFN-, monocyte chemoattractant peptide (MCP)-1, macrophage inflammatory protein (MIP)-1A, MIP-1B, G-CSF, and TNF- are significantly higher than in controls. The levels of these factors are also increased in patients who were admitted to ICUs (8). Similarly, reductions in the levels of T cells and NK cells have been observed in COVID-19 patients (9). The loss of such cells can impair the immune system (10). The levels of the helper T cells, cytotoxic suppressive T cells, and regulatory T cells are much lower in 19 . When SARS-CoV-2 binds the cell, the ACE2 receptors become occupied. This increases AngII which results in lung fibrosis, inflammation, and damage. The infected cell also undergoes cell death as a result of the viral infection. Macrophages engulf the dead cells and release DAMPSs, which bind the TLR and activated NF- by means of MyD88. Activated NF- binding activates the inflammasome. Binding of the virus to the receptor also upregulates IL-6 and TNF-alpha, further activating NF-. Increase in ATP binds the-P2X7 receptor, which in turn increases Ca2+, which causes lysosomal damage and further activation of the inflammasome. Continuous activation of the inflammasome produces the cytokine storm, resulting in multiorgan damage. patients with COVID-19 than in their healthy and less severe counterparts. The decrease in the regulatory T cells may hamper their ability to inhibit the chronic inflammation (11). Interestingly, a remarkable increase is observed in the naïve T cells, where as the memory T cells are reduced in infected patients (10). The reduced expression of memory cells may be a plausible explanation for the increased rates of reinfection by SARS-CoV-2.

SARS-CoV-2 binds to the Angiotensin-converting enzyme 2 (ACE2) receptor and enters the host cell (1) . During infection, the innate and adaptive immune systems work together to inactivate the virus. Since leukocytes and neutrophils are present in higher concentrations in COVID-19 individuals, these immune cells may result in the cytokine storm (10). After viral entry, the virus induces pyroptosis and cell death. The dead cells recruit macrophages to the site of injury that phagocytose them. The phagocytes then express damage-associated molecular patterns (DAMPs), which bind to the toll-like receptors (TLR) and induce nuclear factor kappa B (NF-) signalling by means of the MyD88 pathway. NF- enters the nucleus and catalyzes the transcription of pro-IL-1 and procaspase-1. When additional signals are detected, the pro-IL-1 and procaspase 1 are cleaved into IL-1 and caspase 1 (12). The activated NOD-, LRR-and pyrin domain-containing protein 3 (NLRP3) recruits the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and pro-caspase-1 to form the NLRP3 inflammasome (13). In addition, the phagocytosis releases ATP, which binds to the P2X purinoceptor 7 (P2RX7) and activates the inflammasome (14). The increased calcium levels caused by the viral proteins results in lysosomal damage, thereby releasing cathepsins that activate the inflammasome (15). Further, the binding of SARS-CoV-2 to the ACE2 reduces the available ACE2 receptors on the cell surface. This increases the levels of Angiotensin II (AngII) in the extracellular space, because ACE2 converts AngI and AngII into Ang 1-9 and Ang1-7, respectively. AngII increases the levels of TNF- and IL-6 in the cell that upregulates NF-, activating the inflammasome (12). The continuous activation of the inflammasome results in a cytokine storm, which recruits more immune cells, necrosis, and cell death. This inflammasome pathway further causes tissue injury in various organs ( Fig. 1 ).

MSCs are predominantly isolated from the bone marrow, adipose tissue, dental pulp, umbilical cord, Wharton's jelly, placenta, synovial fluid, endometrium, and peripheral blood. These cells exhibit different cell-surface markers and can be used for a variety of treatment options (Table 1) . MSCs can undergo in vitro amplification and self-renewal, and have low immunogenicity and immune-modulatory functions; the latter have attracted attention in clinical trials (16). MSCs have been widely used in various cellular therapies, such as pre-clinical studies, as well as in some clinical trials, because of their high safety and efficacy (17, 18). MSCs can exert immune-modulatory effects in the host cells of both the innate and the http://bmbreports.org 

It has currently become apparent that MSCs induce therapeutic characteristics by a paracrine pathway by releasing bioactive substances known as secretomes (25). MSC-secretomes are made of soluble proteins, including cytokines, chemokines, growth factors, and extracellular vesicles (EVs), which include microvesicles and exosomes (26). Stem cells release these secretomes by common secretory mechanisms. When the culture medium or secretome are injected into the patients, the neighboring cells assimilate the molecules by paracrine signalling (27). The exosomes themselves contain numerous bioactive molecules, which include microRNAs (miRNA), transfer RNAs (tRNA), long noncoding RNAs (lncRNA), growth factors, proteins, and lipids. The lipid content of the exosomes provide an added advantage by aiding in the infusion of the exosomes with the plasma membrane of the neighboring cells (28). The molecules involved in regulation of cell growth, proliferation, survival, and immune responses are released by exosomes, are elaborately illustrated in Fig. 2 . Upon internalization of the molecules in the secretome, the neighboring cells modulate various downstream pathways, including immunomodulation, suppression of apoptosis, prevention of fibrosis, and remodelling of the injured tissues (25). http://bmbreports.org BMB Reports 

In COVID-19, multiorgan damage has been seen in manyinfected individuals. MSC-Exos is known to alleviate lung injury in asthmatic models and ARDS (40, 41) . MSC-Exos may also be useful in the treatment of cardiovascular (42) and renal problems (43) . Hence, they can be used to treat organ damage associated with COVID-19. Similarly, MSC-EVs have also exhibited inhibitory activity on the hemagglutination of avian, swine, and human influenza viruses (44) . Likewise, MSC-Exos lowered the death rate in H7N9 patients without any toxic effects during follow-up examinations (45) . In addition, these exosomes consist of adhesion molecules that accurately guide them to the injured site. The usage of the exosomes may be preferred to the MSCs, since they can easily cross the bloodbrain barrier, are inexpensive, and cannot undergo independent self-renewal, hence preventing adverse consequences, such as tumor formation. In this pandemic situation, MSC-Exos may be considered as a good treatment option to alleviate the effect of SARS-CoV-2 infection.

Of late, stem-cell-based studies in the treatment of COVID-19

have been gaining momentum. The efficiency and safety of usage of exosomes that had been obtained from BM-MSCs was recently tested on 24 SARS-CoV-2 patients (46) . These patients exhibited moderate to severe ARDS. When the exosomes were introduced into the patients, there were no side effects, and patients improved in clinical status and oxygenation (46) . In a similar study, patients treated with MSCs showed a remarkable improvement in pulmonary function, higher levels of peripheral lymphocytes, and a reduction in the cells that trigger the cytokine storm. Interestingly, the MSCs did not exhibit ACE2 or TMPRSS2 expression, showing that they may not be infected with COVID-19 (47) . Several clinical trials are in the pipeline for usage of stem cells for the treatment of COVID-19 (Table 2 ). Wharton's jelly-derived MSCs (WJ-MSCs), which have been used in various studies based on stem-cell therapy and trials, are in progress for their usage for COVID-19 treatment (48) . Moreover, adipose tissue-derived AD-MSCs have been used in a few studies in various doses and protocols for COVID-19 therapy (49) . Likewise, a novel trial includes inhalation of MSC-Exos for alleviation of symptoms (50) . In addition, MSCs from dental pulp (51) and olfactory mucosa http://bmbreports.org (52) were administered in various doses. MSCs in the clinical trials are predominantly administered intravenously; i.v. injection and, in some studies, MSCs have been given as adjuvant therapy in addition to drugs like oseltamivir, hormones, hydroxychloroquine, and azithromycin (53, 54) . These trials reveal promising new routes for the battle against COVID-19 (55-94).

Stem cells have been studied extensively for their ability to regenerate and for the treatment of various diseases. Recently, we devised an improved protocol for the isolation of urinederived stem cells and their further differentiation into immune cells (95) . Moreover, our research group promoted the hematopoietic differentiation of hiPSCs using a novel small molecule (96) . At the advent of COVID-19, it has become mandatory to discover therapeutic strategies that are easily reproducible and cost effective. Drugs currently available for the treatment of COVID-19 include ones that target viral replication. These drugs include camo-stat mesylate, which is involved in the inhibition of viral fusion to the cell membrane, and favipiravir and remdesivir, which are anti-viral drugs. However, because the cytokine storm is found predominantly in COVID-19 patients, it is essential to consider drugs that inhibit viral replication while treating the cytokine storm. Hence, MSC-Exos may be appropriate therapeutic agents for COVID-19 (97) . MSCs can be more advantageous than other anti-inflammatory agents, because they can provide immunomodulatory effects based on the host cells. In addition to these effects, MSCs can prevent fibrosis of tissues, enable reversal of lung dysfunction, and aid in the regeneration of damaged tissue, which can be significantly beneficial for COVID-19-associated organ damage (98, 99) . Because the healing properties of the MSCs can be primarily attributed to the secretomes or exosomes, using them may be more effective than using MSCs themselves. Exosomes can be mass-produced, administered systematically with minimaltoxicity, and be able to reach the cell targets more efficiently. In addition to their inherent immunomodulatory potential, the MSC-Exos can also be used as a drug-delivery system (100) . MSC-Exos can be modified in vivo to release exosomes that have a higher immunomodulatory potential (101) and can be cultured using various cytokines to exhibit an anti-inflammatory state (102) . Although MSC-Exos appear to be promising therapeutic agents for COVID-19, more experimental research is necessary for them to be used clinically. Moreover, it is essential to optimize the protocols for storage and isolation of MSC-Exos for the treatment of COVID-19. It is also imperative to do experiments to understand the underlying mechanisms of COVID-19 in order to optimize MSC-Exo therapy for treatment (97) . Further, it is also essential to find the optimum dosage, route of administration, and treatment schedule for MSC-Exos. Hence, since MSCs are more widely studied in these aspects than are MSC-Exos, they are predominantly preferred in clinical trials for COVID-19 (103) .

COVID-19 has invoked frenzy in individuals worldwide. The unceasing increase of infection and death has halted the lives of the citizens of countries everywhere. Hence, it is important to discover novel therapeutic platforms and productive measures without further delay (104) . The therapies produced must be easily reproducible and available in large quantities so that enough bioactive molecules will be available for all individuals who have succumbed to COVID- 19 

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The author Dr. VB would like to thank Bharathiar University for providing the necessary infrastructure facility and 

The authors have no conflicting interest.