**3. The pathophysiology of the SARS-CoV-2 infection: what we know**

#### **3.1 Cytokine storm**

The story of the cytokine 'breeze' transformation to the 'storm' starts with the infection of the cell through receptor−ligand interactions which activates massive numbers of leucocytes, particularly B cells, T cells, natural killer cells, monocytes, dendritic cells and macrophages. The release of inflammatory cytokines from these cells attract and activate more white blood cells. Cytokine breeze starts locally post-primary infection with appearing classical signs of inflammation including, calour (heat), dolour (pain), rubor (redness), tumour (swelling or oedema) and loss of function. At the beginning, the localized response works for eliminating the trigger. The host response involving the increase in blood flow, facilitation of leucocyte extravasation and delivery of plasma proteins to the site of injury, increase in body temperature and pain triggering spreads throughout the body via systemic circulation. These responses along with the host repair processes results in either gradually restored organ function or recovery happening by fibrosis which may lead organ dysfunction [51, 52].

Fibroblasts proliferate and invade the intra-alveolar zone constructing fibroblast foci. This seems as the beginning of the pulmonary fibrosis pathogenesis [53]. Lung sections from two patients with early-phase COVID-19 pneumonia demonstrated the characteristics similar to this initiation step of fibrosis [54]. Fibroblast foci were observed in the airways, besides the edema, type II pneumocyte hyperplasia with

infiltration of inflammatory cellular and multinucleated cells. Some reactive epithelial hyperplasia areas are also abundant alveolar macrophages [54]. The SARS-CoV-2 caused progressing injury of the alveolar zone, apears to establish a pro-inflammatory microenvironment triggering this aberrant response with partial replacement of normal tissue by fibrous tissue. Since the severe and critical COVID-19 presentations show strong involvement of inflammatory components and possibly loss of resident stem cell stocks, the research is focused in investigating the role of cellular therapy using immune response-suppressing MSCs for COVID-19 therapy [55].

In COVID-19, the extend of the cytokine release can be the ground for the diversity of the clinical manifestations. The term 'cytokine breeze' meaning a mild/ nonlethal cytokine release response to infection includes the symptoms of increased local temperature (heat), myalgia, arthralgia, nausea, rash, depression, and other mild flu-like symptoms. The compensatory repair process in the body is launched for the reparation of the organs and tissues affected. The term 'cytokine strom' is used to describe the similar sudden and uncontrolled cytokine releases observed in autoimmune, hemophagocytic lymphohistiocytosis, sepsis, cancers, acute immunotherapy responses, and infectious diseases [56, 57].

All these cytokine strom ailments were not only observed in SARS-Cov-2, but also previously reported in SARS-Cov-1 and MERS-Cov cohorts [58, 59]. Hyperinflammation, though, is characteristic for SARS-Cov-2 which is a unique immunological feature of COVID-19. The data reported from recovered and seriously ill patients suggests that there is a significant relationship between severe inflammation and mortality. The main components of the cytokine strom are the critical pro-inflammatory immune elements in the inflammation site [51]. Once the immune system is activated by infection, drug or any stimulus, the cytokines (IFN, IL, chemokines, CSF, TNF, etc.) are released in high levels into the circulation leading to deleterious and diffuse impact on multiple organs.

At the moment, the factors responsible for triggering the inflammatory sequence resulting in cytokine strom are still ambiguous. It is attributed to an imbalance in immune-system regulation resulting from increasing immune cell activation via TLR or other mechanism and decreasing in anti-inflammatory response.

Altough the local and systemic cytokine responses of host to theinfection are essential parts of the host's initial response to infection, a cytokine strom, due to the harmful effects on the host, almost always is a pathological process [31]. Normally, to keep the pathogen under control, the cytokines released from natural killer (NK) cells and macrophages, activated T cells, and humoral immunity work to resolve the inflammation, along with the antibody-dependent cell-mediated cytotoxicity [60]. When looked in some more detail, epithelial cells produce local cytokines like IFN-/ and IL-1 which can protect neighbouring cells by stimulating IFNstimulated gene expression. This also activates the immune competent cells such as NK cells. In turn, the lytic potential of NK cells increased and IFN- secretion is potentiated [61]. IFN- actives the resident macrophages which amplifies TLRmediated stimulation, specifically induce the high NK cells release [62]. On one side, the IL-12 acts to increase NK IFN- secretion, on the other side, increased levels of IL-6 also may limit the immune response by its effects on the cytotoxic activity of NK cells via the down-regulation of intracellular perforin and granzyme B levels [63]. The disease does not regress but progress further, the activities of the T cells and humoral responses causes additional cytokine responses. This process, like pouring petrol on fire, results in greater or sustained antigen release and added TLR ligands from viral-induced cytotoxicity [64]. Concurrently, an insufficient negative feedback mechanism by IL-10 and IL-4 would be expected to increase the severity of cytokine responses toward a cytokine storm. The exacerbated fire of the lethal cytokine storm reveals widespread alveolar damage characterized by

#### *Cellular Therapy as Promising Choice of Treatment for COVID-19 DOI: http://dx.doi.org/10.5772/intechopen.96900*

hyaline membrane formation and infiltration of interstitial lymphocytes [65, 66]. In COVID-19 disease, a cytokine storm is demonstrated frequently in patients with severe-to-critical symptoms; concurrently the lymphocytes and NK cell counts are sharply reduced with elevations in levels of D-dimer, C-reactive protein (CRP), ferritin, and procalcitonin which are the inflammation biomarkers [67].

As the reported evidence regarding the immunological response to SARS-CoV-2 is quite limited, we are able to compile and interpret the relevant information from the published information. After the host is invaded by the virus, host innate immune system through pattern recognition receptors (PRRs) including C-type lectin-like receptors, Toll-like receptor (TLR), NOD-like receptor (NLR), and RIG-I like receptor (RLR), is the first to pick out [68]. The inflammatory factors' expression, dendritic cells' maturation, and type I interferons (IFNs) synthesis are promoted by the virus for basically two main purposes: limiting the spread of the virus, and phagocytosis of the viral antigens [68]. Whilst the escape of the virus from the immune responses is facilitated by the N protein of the virus [69], a strong troop of the adaptive immune response joins the combat against the virus, with its elements of T lymphocytes including CD4+ and CD8+ T cells. CD4+ T cells stimulate B cells to produce virus-specific antibodies, and CD8+ T cells directly kill virus-infected cells. T helper cells produce proinflammatory cytokines to enhance the antiinflammatory process. Paradoxically, SARS-CoV-2 induces the apoptosis of the T cells, hence inhibit their function. The major role of humoral immunity over its complements such as C3a and C5a and antibodies cannot be overlooked in the fight against the virus [70, 71]. Here comes another paradox where the immune system overreaction of the generates a large amount of free radicals locally causing severe damages to the lungs and other organs, even multi-organ failure and even death [62, 72].

In severe cases, it has been reported that SARS-CoV-2 affects heart, kidney, liver, GI-system, resulting in multiple organ dysfunction and in some cases even death [73]. One study supports that the novel virus also could potentially infect the enterocytes through a ACE2 enzyme; as ACE2 is highly expressed on enterocytes may help to explain why diarrhea occurs with acute infection as well as the fecal shedding observed [74]. Since the ACE-2 receptors are also expressed on other tissues like kidney, liver, heart and digestive system organs; thus, explaining the rapid progression towards systemic inflammatory conditions as observed in critically ill patients [75]. Hence, it is worth to consider that the infection spreading in broader scale would have impact the inflammatory cascade sources in a number of tissues in several organs, besides the lung.

### **4. The treatment options in SARS-CoV-2: what to use**

Based on evidence from laboratory, animal, and clinical studies, the WHO recommends the drugs for treatment of COVID-19 includes Remdesivir, Lopinavir/Ritonavir, Lopinavir/Ritonavir with interferon beta-1a, chloroquine, and hydroxychloroquine [76].

Remdesivir is a monophosphoramide prodrug that causes premature termination of viral RNA replication. It was developed against Ebola, MERS-CoV, and SARS-CoV, before the COVID-19 pandemic shook the globe. Potent interference of remdesivir with the NSP12 polymeras3e of SARS-CoV-2 was shown in vitro despite intact ExoN proofreading activity [73, 77]. It is suggested that when the baricitinib which is an inflammatory drug used in combination with anti-viral drugs like Remidesivir, increases the potential of the drug to reduce viral infection [78, 79].

The Lopinavir/Ritonavir drugis a protease inhibitors combination. It is usually used to treat HIV infection; from the laboratory experiments, it is evident that

these drugs could be used to treat the COVID-19 infections [80]. The lopinavir and ritonavir are used as a regimen single-agent or combination with either ribavirin or interferon-α [81]. It is also reported that the interferon beta-1a, which is used to treat multiple sclerosis, can also be used as a remedial approach for COVID-19 disease [73].

A randomised controlled trial (ChiCTR 2000029308) aimed to evaluate the efficiency and safety of lopinavir and ritonavir in severe COVID-19 patients, comparing lopinavir-ritonavir (n: 99) to standard care (n: 100). There was a significant difference in the time to clinical improvement between the two groups on day 14, whereas this difference was not statistically significant on day 28. The decrease of 5.8% in mortality at 28 days and the length of stay in the ICU reduced as five days in the lopinavir-ritonavir treatment [82].

Spike protein from virus binds to ACE2 or CD147 on the host cell, mediating viral invasion and dissemination of virus among other cells [55, 83]. In addition to ACE2, it has recently been demonstrated that S protein of novel virus also binds to CD147. Meplazumab which is an anti-CD147 humanized antibody, co-immunoprecipitation, ELISA, and immuno-electron microscope were handled to demonstrate the new CD147 path of viral invasion. This importanty evidence has been providing a key target for the development and administration of specific anti-SARS-CoV-2 medicines [84].

ACE Inhibitor and Angiotensin Receptor-1 Blocker are also medications used for the curative purposes of COVID-19. As already mentioned, SARS-CoV-2 enters the type II pneumocytes via the ACE2 receptor. Functionally ACE2 receptor has a mutual physiological action to ACE1, it converts the angiotensin II back into angiotensin I. Thus, patients taking receptor blocker will have an increased plasma angiotensin II. On the contrary, patients taking inhibitor will have low angiotensin II levels [85, 86]. Its effect in the alveolar tissue is still unknown. Discontinuation of ACEi or ARBs is not recommended yet as hypertension is an acute risk of discontinuation and can exacerbate the clinical course and increase mortality of COVID-19 if infected by SARS-CoV-2. Although chloroquine is an anti-malarial medication, it can inhibit pH-dependent stages of replication in viruses, as well as having immunomodulation which is dependent on the suppression of cytokines (IL-6 and TNF-α) production and dissemination. Secondary COVID-19 rates can be minimized with pre- and post-exposure prophylaxis in an individual with document exposure to SARS-CoV-2. Therefore, hydroxychloroquine has been hypothesized to be an adequate chemoprophylaxis candidate to reduce secondary COVID-19 [87].

WHO recommends to continue the use of ibuprofen as antipyretic agent, yet the first-line antipyretic remains to be acetaminophen.

The use of systemic corticosteroids in the management of ARDS secondary to viral pneumonia is debatable. The rationale behind this that the corticosteroids prolong the viral shedding time and maintain a systemic anti-inflammatory condition. This will minimize the precipitation of ARDS, dyspnea, and severe pneumonia.

The systemic corticosteroid usage in the management of ARDS developed due to viral pneumonia is still under discussion. The aim of this medication use is that corticosteroids prolong the viral shedding time and maintain a systemic antiinflammatory state that will minimize the precipitation of ARDS, dyspnea, and severe pneumonia [76].

Considerable amount of protection is provided by the convalescent plasma collected from donors who have survived an infectious disease by developing antibodies is considered to provide a great degree of protection for recipients affected by the emerging virus [88]. Convalescent plasma is an old tool that has been successfully used to treat numerous infectious diseases, including the 2003 SARS-CoV-1 epidemic, 2009–2010 H1N1 influenza virus pandemic, and 2012 MERS-CoV epidemic [88–91] for which there is no effective treatment.

Based on the clinical effectiveness of convalescent plasma, such as signs of improvement approximately 1 week after convalescent plasma transfusion, effectively neutralizing SARS-CoV-2, leading to impeded inflammatory responses and improved symptom conditions without severe adverse events the FDA has granted clinical permission for applying convalescent plasma to the treatment of critically ill COVID-19 patients [92]. Antibiotics with immunomodulatory actions are used in therapy with antiviral drugs and to avoid secondary infections, such as bacterial and fungal infections in patients. Besides their antimicrobial function, antibiotics such as Azithromycin show immunomodulatory properties, which can reduce inflammatory macrophage polarization and inhibit NF-κB signaling pathways, minimizing the hyperinflammation damage. Since the beginning, antibiotics have been used with good results in mortality reduction and shortening of intubation time in COVID-19 disease [93, 94].

The expressive number of deaths and confirmed cases of SARS-CoV-2 call for an urgent demand of effective and available drugs for COVID-19 treatment. Currently, multiple avenues for therapies are being explored.
