**3. Discussion**

#### *3.1. COVID-19 Pathophysiology and Cytokine Storm*

Cytokine storm is seen in a variety of syndromes, including macrophage activation syndrome (MAS), hemophagocytic lymphohistiocytosis (HLH), and chimeric antigen receptor (CAR) T-cell therapy (used to treat lymphomas and leukemias) associated with hyperinflammation (known as cytokine release syndrome) [3,34–38]. MAS is a rare and potentially life-threatening condition entailing a cytokine storm in which macrophage and lymphocyte interactions are dysregulated, thus leading to cytokine

release [39]. HLH is a critical condition marked by hyperinflammation, hemophagocytosis, and histiocyte proliferation, and is classified as either familial HLH or secondary HLH [40]. One potential mechanism for the cytokine storm in HLH disorders may be an increase in cytokines, including TNF α, IL-1, interferon gamma (IFN-γ), IL-6, sTNFRs, and soluble IL-2R (sIL-2R), as a consequence of dysregulated interactions between lymphocytes and macrophages [39]. Patients with COVID-19 exhibit clinical attributes similar to secondary HLH: hyperferritinemia, cytopenia, and ARDS [12].

The inflammatory cytokine storm observed in COVID-19 shares features with MAS and HLH, but has some distinguishing characteristics [3]. In COVID-19, compared with MAS, the increases in ferritin are not as high, and organ damage is primarily restricted to the lungs [38]. The clinical attributes of patients with COVID-19 also mirror those in cytokine release syndrome, which arise as a consequence of CAR-T cell therapies for certain lymphomas [20]. Importantly, not all patients with COVID-19 undergo cytokine storm; this phenomenon only occurs in a certain subset of severe cases [12]. The classic clinical picture of a patient with cytokine storm involves rapid respiratory deterioration [41,42]. Our patient began to decline and demonstrated symptoms of respiratory distress (labored breathing) on the first day of admission, and had reportedly been experiencing symptoms for 7 days prior.

Cytokine storminstigates a robustimmune-driven attack on the body, leading to ARDS (andmulti-organ failure) and ultimately causing morbidity in severe COVID-19 [43,44]. The pathophysiology of cytokine storm is not fully understood, but is currently thought to be based on the mechanisms of inflammatory disorders, including MAS and cytokine release syndrome. Initially, SARS-CoV-2 binds angiotensin converting enzyme 2 (ACE-2) receptors and then invades the respiratory epithelium [37,45]. Dendritic cells and alveolar macrophages are activated because of the presence of SARS-CoV-2 and subsequently release IL-6, which is also secreted by the respiratory epithelium [37,45]. A cascade of the cytokines IL-1B, IL-12, and TNFα results, and their secretion induces WBCs to release cytokines, thus e ffectively perpetuating an inflammatory cycle [12,45]. These cytokines also enter the circulation and cause systemic multi-system pathology [46].

The robust hyperinflammatory cytokine response induces the apoptosis of endothelial and epithelial cells in the lungs, resulting in tissue injury, leakage and edema, and ARDS [46,47]. As immune cells destroy alveolar tissue, permeability increases, thus resulting in fluid entry into the alveoli; less oxygen enters the blood, because alveolar type I cells are diminished, and a loss of surfactant leads to alveolar collapse. These responses together impair normal gas exchange [3,48]. Additionally, cytokines cause vasodilation, thereby contributing to a build-up of fluid in the alveoli, diluting surfactant, and causing alveolar collapse; as alveolar type I and alveolar type II cells are destroyed, the alveoli collapse, and ARDS ensues [48].

An increased synthesis of collagen and TGFα and deposition of fibrin are also observed in the development of ARDS [3]. Features of ARDS additionally include alveolar exudate, edema, and cellular infiltration, thus resulting in damaged alveoli and limited gas exchange [21]. Xu et al. [44] have described post-mortem biopsies of COVID-19 patients with ARDS in China that showed classic ARDS-related features, such as pneumocyte desquamation, the formation of hyaline membranes, and pulmonary edema. Additionally, the presence of lymphocyte-dominated mononuclear inflammatory infiltrate in the interstitium was observed bilaterally [44]. Because of the high numbers of mononuclear lymphocytes, these T-cells have been thought to potentially enter the pulmonary circulation and trigger an inflammatory storm [21].

ARDS and severe illness in patients with COVID-19 usually develop 1–2 weeks after the onset of symptoms [49]. Our understanding of ARDS in COVID-19 is still evolving. It has been suggested that COVID-19 may be characterized by a unique form of ARDS, marked by a feature not seen in classic ARDS: the seemingly preserved compliance and respiratory mechanics relative to the high degree of hypoxemia [50]. Figure 3 depicts the alveolar changes that occur in severe COVID-19.

**Figure 3.** Alveolar changes due to cytokine syndrome-induced acute respiratory distress syndrome (ARDS) in severe coronavirus disease 2019 (COVID-19), according to current understanding [3,48,50,51]. This model is based in part on hypotheses (because our understanding of ARDS remains incomplete), along with reported pathological findings in COVID-19-related ARDS from post-mortem biopsies. Many of the features are hallmark characteristics of conventional ARDS. Nonetheless, COVID-19 is increasingly believed to display an atypical form of ARDS [50].

#### *3.2. Diagnostic and Prognostic Roles of Serum Markers for Severe COVID-19 and Cytokine Storm*

The onset of the hyperinflammation in cytokine storm may be evidenced by coagulopathy, cytopenia, tissue damage, the inflammation of liver tissue, and the activation of macrophages and hepatocytes [38]. Therefore, laboratory findings potentially suggesting the onset of cytokine storm in patients with COVID-19 may include increased levels of acute phase markers (such as D-dimer, CRP, LDH, ferritin, and troponin), low platelets, decreased fibrinogen, lymphopenia, thrombocytopenia, increased LDH, and transaminitis (elevated AST and ALT) [31–33,38]. Lymphopenia is a commonly reported feature of COVID-19 cytokine storm, and given that the cytokine storm must be orchestrated by other leukocytes (not T cells), an elevated WBC count is also common, suggesting that lymphopenia with leukocytosis may be a key feature in the differential diagnosis of COVID-19 [51]. In our patient, lymphopenia was observed on day 1, and on day 3 the patient developed leukocytosis. Elevated CRP and ferritin levels are key in the diagnosis of MAS and HLH. Recent studies have suggested a similar marker profile in severe COVID-19. Interestingly, cytokine storm in COVID-19 differs from that associated with other viruses as the increase in ferritin in COVID-19 is relatively modest [38]. Ferritin has been suggested as a prognostic indicator for cytokine storm in COVID-19 [38,52].

Serum inflammatory biomarkers may have a role in assessing disease progression, since a poor prognosis in COVID-19 appears to be correlated with abnormal serum markers and clinical attributes of cytokine storm [38]. A recent retrospective observational study of 21 patients in China comparing the attributes of moderate versus severe COVID-19 found that severe cases are more often typified by hypoalbuminemia and lymphopenia, with relatively high levels of ALT, LDH, and CRP, and particularly high levels of TNF-<sup>α</sup>, IL-2R, IL-6, and IL-10 [53]. One recent meta-analysis of 21 COVID-19-related studies on 3377 patients has shown that the levels of interleukin 10 (IL-10), IL-6, and ferritin strongly correlate with disease severity [30]. The study also showed that patients who experienced severe disease and even mortality had thrombocytopenia, lymphocytopenia, and higher WBC counts compared with those with moderate disease and disease resolution [30]. Mehta et al. [12] have suggested that inflammatory biomarkers, including ESR, decreased platelets, and increased ferritin, should be used by clinicians to risk-stratify patients who might benefit from immunomodulating treatments (such as IL-6 inhibitors). One recent study of 343 hospitalized patients with COVID-19 in Wuhan showed that D-dimer levels above 2.0 μg/mL on admission were predictive of mortality, thereby suggesting a potential prognostic role of D-dimer [54].

Therefore, an early assessment of these biomarkers may be critical, since rapidly identifying an emerging cytokine storm and targeting immune dysregulation in patients with COVID-19 before the rapid progression to ARDS has the potential to avoid the need for mechanical ventilation, given the low rates of survival among patients with COVID-19 who are placed on ventilators [22,38]. Experience in MAS and cytokine release syndrome has shown that intervening at an early stage of disease may be important for avoiding irreversible damage to tissue [38].

#### *3.3. The Role of IL-6 in Cytokine Storm and COVID-19*

IL-6 is a pleiotropic cytokine in the glycoprotein-130 (gp130) family of cytokines [55,56]. It has a myriad of physiological functions, including the production of acute phase reactants, stimulation of immunoglobulin production by activated B cells, regulation of bone homeostasis, lipid oxidation, glucose metabolism, and regulation of energy expenditure and appetite [56–59]. The mechanistic and multifunctional complexity of IL-6 is underscored by its anti-inflammatory e ffects (production of acute phase reactants from liver and epithelial cell regeneration), in addition to its pro-inflammatory properties (inflammatory cell recruitment, disrupted di fferentiation of regulatory T-cells, and inhibition of inflammatory cell apoptosis) [37,60]. Therefore, the role of IL-6 in COVID-19 is complex and not fully elucidated [3]. For instance, theoretically, the high IL-6 levels in COVID-19 pneumonia have been suggested to have beneficial or deleterious e ffects, since IL-6 in other infections can enhance viral replication or suppression in experimental models [3,61]. However, emphasis has been placed on its mainly pro-inflammatory nature in COVID-19, as confirmed by clinical reports on patients with COVID-19.

IL-6 plays a major role in various inflammatory and autoimmune disorders [62]. Importantly, an excessive generation of IL-6 during infections and tissue injury is believed to be responsible for cytokine release syndrome [59]. IL-6 dysregulation leads to the activation of complement and coagulation, inducing vascular leakage [24,63–65]. The activation of IL-6 is thought to be the key feature of the progression of COVID-19 pneumonia to ARDS and hyperinflammation [45].

IL-6 peak levels have been associated with pulmonary disease progression in COVID-19 [66]. Importantly, on day 2, our patient showed elevated IL-6 levels. In a recent retrospective cohort study of 201 patients in Wuhan, China, Wu et al. [67] reported a statistically significant correlation between IL-6 and mortality.

Interestingly, a role of IL-6 as a prognostic marker for COVID-19 has also been suggested. One recent meta-analysis of studies involving a total of 264 patients with COVID-19 in China reported that patients with severe COVID-19 have higher IL-6/IFN-γ ratios than those with more moderate conditions, thus suggesting that the IL-6 and IFN-γ levels may serve as a possible prognostic tool in managing patients with COVID-19 [68].

Figure 4 illustrates the proposed inflammatory sequence of the cytokine storm observed in COVID-19 and the key role played by IL-6.

**Figure 4.** Proposed model of the cytokine storm in severe COVID-19, revealing the mechanistic complexity of the cytokine cascade and subsequent pathology [3,21,37,45,46,69]. The model is primarily based on current knowledge of cytokine storm, as seen in macrophage activation syndrome (MAS), hemophagocytic lymphohistiocytosis (HLH), and chimeric antigen receptor (CAR) T-cell therapy. Notably, cytokine storm in both MAS and HLH displays increased levels of cytokines interleukin (IL)-1, IL-2, IL-18, macrophage colony stimulating factor (M-CSF), interferon-γ, and tumor necrosis factor (TNF) α, in addition to IL-6, although definitive causality due to all these cytokines has not been determined [23].

#### *3.4. IL-6 Signal Transduction Pathway*

IL-6 signaling occurs through a complex sequence of induction. There are two forms of the IL-6 receptor: a membrane-bound IL-6 receptor (IL-6R) and a soluble IL-6 receptor (sIL-6R) [70] IL-6 signaling primarily occurs through two modes: the classic signaling pathway mediated by membrane-bound IL-6R and the trans-signaling pathway mediated by sIL-6R [56,59]. Membrane-bound IL-6R is mainly found on hepatocytes, certain epithelial cells, megakaryocytes, and some groups of leukocytes; thus, the classic signaling pathway only occurs in these locations [55,71]. In contrast, the trans-signaling pathway can occur in all cells of the body, because it primarily requires the presence of the gp130 receptor protein, which is expressed in all cells [60].

In the classic signaling pathway, IL-6 binds membrane-bound IL-6R on the membrane of target cells and then interacts with the gp130 receptor to form a complex [55,70]. The activation of gp130 leads to the downstream activation of the Janus activated kinase-signal transducer and activator of transcription (JAK/STAT) pathway by influencing JAK (which is constitutively bound to the gp130 cytoplasmic domain), thereby inducing STAT3 phosphorylation (activation of STAT3) [56,70]. Therefore, IL-6s interaction with its receptor ultimately leads to the activation of STAT3. The JAK/STAT3 complex translocates to the nucleus, where it activates transcription and consequently leads to the expression of acute phase proteins, which are a main feature of inflammation; their induction may have homeostatic effects [56,60].

This JAK/STAT pathway is regulated through negative feedback by suppressors of cytokine synthesis (SOCS-1 and SOCS-3) [56,59]. SOCS-1 binds activated JAK and SOCS-3 binds phosphorylated gp130, thus halting JAK activation [56]. Notably, IL-6R signaling also activates the JAK-SHP2 mitogen activated protein kinase (MAP-kinase) pathway, in addition to the JAK-STAT signaling pathway, thereby resulting in the activation of several transcription factors [56,59]. In the trans-signaling pathway, the binding of IL-6 to sIL-6R in cells that lack surface expression of the IL-6 receptor results in coupling with gp130, which in turn leads to signal transduction [56,70]. This pathway is hypothesized to be pro-inflammatory [60].

#### *3.5. Potential Therapeutic Role of IL-6 Receptor Inhibition in COVID-19*

IL-6 receptor inhibition has promise as a therapeutic strategy because it results in a blockade of signal transduction and gene expression [70]. IL-6 and its interactions with the IL-6 receptor stimulate the release of various acute phase proteins (including fibrinogen, CRP, hepcidin, and serum amyloid A) and promote inflammation. Therefore, the blockade of this interaction can be used to attenuate systemic inflammation in immune disorders such as Castleman disease and rheumatoid arthritis [59,72].

For example, two IL-6 receptor inhibitors used to treat rheumatoid arthritis are tocilizumab and sarilumab [33]. Tocilizumab is a humanized monoclonal antibody (IgG1k subclass) that acts against both IL-6R and sIL-6R receptors and is traditionally used to treat rheumatoid arthritis, systemic juvenile idiopathic arthritis, Castleman disease, and other autoimmune disorders [37,73–75]. The U.S. FDA cleared tocilizumab in 2017 for the treatment of cytokine release syndrome resulting from CAR-T cell therapy [37]. Figure 5 depicts the IL-6 signaling pathway and the role of tocilizumab in IL-6 receptor antagonism.

**Figure 5.** IL-6 classic and trans-signaling pathways, with a model of tocilizumab-mediated therapeutic receptor antagonism [21,55,56,60,70,76]. The diagram displays the normal sequences of the classic and trans-signaling pathways. Tocilizumab acts on both pathways (on the IL-6 receptor in the classic pathway and the soluble IL-6 receptor in the trans-signaling pathway). Note: IL-6 also uses trans presentation (in which IL-6 and membrane-bound IL-6R on dendritic cells are presented to gp130-expressing T-cells in proximity) as the third mode of signal transduction, but this pathway is not shown here [56,75].

In the U.S., tocilizumab is sold under the trade name Actemra, whereas in Europe, its trade name is RoActemra [73]. Tocilizumab has an additional use in treating the cytokine release syndrome that specifically results from CAR T-cell therapy used in B-cell malignancies [33,34]. Therefore, tocilizumab and sarilumab have been suggested to have therapeutic potential in patients with COVID-19 when there is suspicion of a cytokine storm according to elevated acute phase reactant markers (e.g., ferritin, D-dimer, CRP, and LDH), as was the case in our patient [31–33].

Accordingly, on day 1 of admission, we initiated an intravenous (IV) administration of tocilizumab. For the treatment for CAR-T therapy-initiated cytokine release syndrome, a conventional IV infusion of tocilizumab (alone or with corticosteroids) is indicated with a weight-based dose at 8 mg/kg for patients weighing at least 30 kg, which corresponded to a dose of 1005 mg in our patient [77]. We administered two smaller doses (500 mg each) with a 12 hour interval not based on weight, given the experimental nature of its use in COVID-19 and to mitigate any adverse effects. The National Health Commission of China's official guidelines in the "Chinese Clinical Guidance for COVID-19 Pneumonia Diagnosis and Treatment" sugges<sup>t</sup> using tocilizumab in patients who display widespread lung lesions and disease in severe stages with elevated IL-6 levels; its use is contraindicated in patients with ongoing infections, particularly tuberculosis [25]. The first dose has been suggested to be introduced at 4–8 mg/kg (but the suggested dose is 400 mg diluted to 100 mL in addition to 0.9% normal saline with more than 1 hour of infusion time) [25]. These guidelines also recommend that if patients have a suboptimal response after the first dose, a second administration with the same dose can be performed after 12 hours, with a maximum of two administrations [25]. Additionally, it has been recommend that a single dose does not exceed 800 mg [25]. Therefore, the dosing regimen for our patient was similar to this recommended dosing protocol, but with a slightly higher dose per administration.

Importantly, IL-6 inhibitors attenuate the immune response, increasing the risk of opportunistic infections, leukopenia, and liver injury [73,78]. Some known adverse effects of tocilizumab include liver disease, allergic reactions, anaphylaxis, stomach and abdominal pain, skin and soft tissue infections, neutropenia, and hypercholesterolemia [73,79]. Reactivation of tuberculosis can occur, although this has been reported to be less common than that after treatment with tumor necrosis factor inhibitors [73]. Additionally, a possible association between tocilizumab and osteonecrosis of the jaw has been reported [80]. However, our patient did not exhibit any noticeable symptoms due to tocilizumab. Notably, IL-6 inhibitors may be cost-prohibitive [81]. Clinicians should weigh the benefits and limitations of tocilizumab therapy compared with other currently used drugs for the managemen<sup>t</sup> of COVID-19 (Table 1).


*TMID*

**2020**, *5*, 112

#### *3.6. Evidence and Reports of Tocilizumab Use for COVID-19*

Our experience at a small-sized urban community hospital mirrors the positive results reported in other cases in the literature. The first report that sparked considerable attention in China described 21 patients with severe COVID-19 at two di fferent Chinese hospitals who received one administration of tocilizumab in conjunction with standard therapy; all patients reported dramatic clinical improvements [90]. The patients received a single 400 mg tocilizumab dose and standard recommended therapy with lopinavir, methylprednisolone, oxygen treatment, and symptom relief medications. The fever declined in several days, and marked improvement was observed in all patients. Lymphocyte and CRP levels decreased after tocilizumab therapy. Fifteen patients showed a decreased oxygen intake, and one patient no longer needed oxygen therapy. Twenty patients were discharged (average of 13.5 days after tocilizumab therapy), and one was removed from the ICU [21]. No adverse e ffects were reported. As a result, the National Health Commission of China o fficially added tocilizumab to its guidelines in the "Chinese Clinical Guidance for COVID-19 Pneumonia Diagnosis and Treatment" [21,25].

Luo et al. [13] conducted a retrospective study of 15 patients with COVID-19 in Wuhan, China. The study assessed 15 patients with COVID-19 (12 males and three females with a median age of 73 years) treated with tocilizumab (eight patients were given a combination with methylprednisolone). Ten patients were administered a single dose and two were given double doses of tocilizumab. Serum acute phase reactants CRP and IL-6 were measured before and after therapy. Two patients were "moderately ill," and the rest were in a serious or critical condition. Ten patients had at least one co-morbidity. The CRP levels decreased in all patients. The IL-6 levels decreased in most (11) patients. Mortality was seen in three patients and disease exacerbation occurred in two patients. The remaining ten patients showed stabilization. A total of four critically ill patients were given a single dose; three patients died and one experienced disease aggravation. The authors concluded that tocilizumab may have benefits. The study further suggested that a single dose of tocilizumab (even when used with glucocorticoids) may not be su fficient for improvement in critical patients, whereas repeated doses may result in improvement. The limitations of the study were its retrospective and observational nature, small sample size, and non-randomized sample, with a substantial number of patients with comorbidities. Doses of tocilizumab varied among patients, some of whom received a double dose and approximately half of whom received methylprednisolone in addition to tocilizumab.

Other reports have described patients treated with tocilizumab with hydroxychloroquine without other major drugs, similar to our patient. De Luna et al. [91] have reported the case of a 45-year-old patient with COVID-19 in France who had sickle cell anemia with acute chest syndrome and pneumonia, and who was administered tocilizumab (in addition to hydroxychloroquine) and showed subsequent improvement. He presented on day 1 with an oxygen saturation of 91%, and then deteriorated to 80% oxygen saturation on day 2, at which time he was administered IV tocilizumab (8 mg/kg dose), in addition to the ongoing hydroxychloroquine (200 mg every 8 hours) and supplemental oxygen. On day 3, he showed improvement. In our patient, tocilizumab was administered in conjunction with ongoing hydroxychloroquine and other standard medications on day 1, followed by a second dose 12 hours later on the morning of day 2. Our patient also showed major improvement after tocilizumab administration, although we administered two doses rather than one.

Fontana et al. [92] have also described the successful use of tocilizumab in conjunction with hydroxychloroquine (with other immunosuppressive drugs) in a 61-year-old man with a previous kidney transplant for ESRD from chronic interstitial nephritis and an extensive past medical history (nodal marginal zone lymphoma, pulmonary embolism, Parkinson's disease, and neurogenic bladder). The patient was admitted for a continual fever and shivering, and over the course of the hospital stay, was diagnosed with COVID-19. His arterial pO2 declined to 57 mmHg, and low-flowing oxygen via a nasal canula was started. Hydroxychloroquine (200 mg, twice per day) was administered, and the ongoing cyclosporine dose was decreased by half. Two days later, because of the lack of improvement, cyclosporine was withdrawn, and the methylprednisolone dose was increased to 16 mg daily. After 324 mg subcutaneous administration of tocilizumab, the patient's fever resolved, the arterial pO2 improved progressively, and oxygen therapy was discontinued. The IL-6 levels increased 6 days after tocilizumab administration (to 619.11 pg/mL), probably because of receptor inhibition as opposed to a lack of drug e fficacy. The patient was discharged on day 22 without a fever and with 95% peripheral oxygen saturation on ambient air.

Importantly, this is the only case in the literature showing possible adverse e ffects of tocilizumab administration: leukopenia, neutropenia, and pseudomonas infection. However, these were treated and adequately managed. IV immunoglobulins (IVIG) were administered to alleviate ensuing leukopenia with neutropenia, which was thought to have arisen from tocilizumab administration. The patient's leukocyte count increased. Meropenem was re-introduced after a urine culture showing multi-drug resistant *Pseudomonas aeruginosa*, which might have resulted from tocilizumab treatment, given that secondary bacterial infections are a known adverse e ffect. Furthermore, the patient had several comorbidities and, as a transplant patient, was already being treated with cyclosporine. Therefore, the role of tocilizumab in his outcome is di fficult to determine. Additionally, because he had also been treated with hydroxychloroquine, IVIG, methylprednisolone, and azithromycin, the role of any individual drug cannot be established.

In other case reports, patients were also treated with several drugs in conjunction with tocilizumab, and they often had distinct comorbidities. Zhang et al. [93] have described the case of a 60-year-old male COVID-19 patient in Wuhan, China, with multiple myeloma who received tocilizumab and showed clinical improvement. He initially received moxifloxacin, umifenovir, and chemotherapy. He was later given methylprednisolone after experiencing chest tightness and shortness of breath. On day 9, he was intravenously administered one dose of tocilizumab. On day 12, he no longer felt chest tightness. The IL-6 levels decreased overall, with a slight momentary rebound elevation. On day 19, his CT scan showed a diminished classic ground-glass appearance. He was successfully discharged.

Other studies have reported patients who were also administered two or more doses of tocilizumab and improved. Michot et al. [94] have described the case of a 42-year-old male with metastatic sarcamatoid clear cell renal carcinoma who developed COVID-19 and showed improvement after two doses of tocilizumab. During the hospital stay, he received lopinavir-ritonavir on day 7 for 5 days. On day 8, he experienced sudden dyspnea and a decrease in oxygen saturation. The tocilizumab was administered in two doses of 8 mg/kg with an 8-hour interval in between. The patient showed clinical improvement, his fever regressed, and he had decreased oxygen requirements. His CRP levels dramatically decreased. Importantly, these positive results must be viewed by considering that the patient already had an immunosuppressed status due to cancer, and that because he received lopinavir-ritonavir, any benefit from this drug alone or the combination remains unclear.

Cellina et al. [95] have characterized the case of 64-year-old man with no comorbidities who developed COVID-19 and experienced dyspnea and decreased oxygen saturation at 90% on day 6 of his hospital stay. On day 7, he was started on assisted ventilation, and tocilizumab was administered in two doses of 8 mg/kg, with 12 hours between doses (days 7 and 8). On day 9, the CRP and WBC count declined, and his condition improved. He was weaned o ff ventilatory support. On day 14, his CT revealed improvements.

Similarly, Di Giambenedetto et al. [26] have reported the successful resolution of clinical symptoms in three male hospitalized patients with COVID-19 (71, 45, and 53 years of age) after treatment with tocilizumab. Clinical improvement was seen in all three patients. In one patient (71-year-old male) who was given two doses of tocilizumab, there was a resolution of fever, the oxygen saturation improved, and the CRP returned to a normal range. In the second patient (45-year-old male) treated with two doses of tocilizumab in addition to antiviral treatment, there was clinical improvement, fever resolution, and a decrease in CRP levels after tocilizumab infusion. In the third patient (53-year-old) treated with three doses of tocilizumab, dyspnea resolved, the oxygen saturation improved, and the CRP levels decreased.

In one investigation by Jacobs et al. [96], over the course of 24 days, 32 patients with COVID-19 were placed on extracorporeal membrane oxygen (ECMO), followed by the administration of adjunctive drugs. Five survived, and two of the five survivors were treated with tocilizumab or sarilumab. However, the clinical details of these patients and the specifics of drug administration have not been described. This study had a small sample size and was observational and retrospective. Additionally, other factors might have contributed to the outcomes (e.g., the use of ECMO).

In another observational study, Giamarellos-Bourboulis et al. [97] administered tocilizumab to six patients who were part of a group of 54 patients with COVID-19 under study for immune dysregulation and immune responses. The plasma of these patients was also studied. The absolute lymphocyte levels in the six patients decreased after tocilizumab therapy. The introduction of tocilizumab in plasma-enriched cell medium partially restored HLA-DR expression on cells. Importantly, IL-6 is believed to be the cause of decreased HLA-DR on CD14 monocytes [97]. Severe respiratory failure is associated with a significant decrease in HLA-DR expression on CD14 monocytes [97]. Therefore, the investigators suggested that tocilizumab partially relieves the immune dysregulation seen in COVID-19.

However, one patient described in the literature by Ferrey et al. [98] remained in a critical condition after tocilizumab therapy. He was a 56-year-old male with end-stage renal disease dependent on hemodialysis and with hypertension, who developed severe COVID-19. He developed ARDS and was intubated. He was given hydroxychloroquine in conjunction with standard drugs for ARDS and septic shock. On day 6, he received tocilizumab. The authors report that he remained in a critical condition at the time of the case report (his care was ongoing). Importantly, the patient had other notable comorbidities, which may have influenced his outcome.

Interestingly, the experience reported for one patient with COVID-19 by Minhai et al. [99] may even sugges<sup>t</sup> a potential prophylactic role of tocilizumab in preventing severe COVID-19. The authors report the case of a 57-year-old female patient in Switzerland who experienced systemic sclerosis, insulin-dependent type 2 diabetes mellitus, and obesity, and who was already being administered tocilizumab (8 mg/kg every 4 weeks IV) before presenting to the hospital 4 weeks after the last tocilizumab dose with symptoms of COVID-19. She was quarantined at home and monitored, and she received no drug administration (her scheduled tocilizumab infusion for systemic sclerosis was postponed). Ultimately, she only developed a mild form of COVID-19 and subsequently recovered. The authors postulate that the pre-COVID-19 administration of tocilizumab might have led to a mild form of the disease and might possibly have prevented severe COVID-19, particularly given the patient's several comorbidities, which placed her at high risk of developing severe COVID-19. Therefore, in contrast to other patients, this patient had received tocilizumab before SARS-CoV-2 infection. Nonetheless, a definitive role of tocilizumab in preventing severe COVID-19 in this patient cannot be established. Notably, she was already immunosuppressed because of her comorbidities.

The relative sudden recovery in nearly all of these reported cases after tocilizumab administration during respiratory deterioration suggests that tocilizumab may have a therapeutic benefit. Only a certain subset of patients might strongly benefit from tocilizumab. Interestingly, only one case showed any adverse e ffect of tocilizumab, thus suggesting the relative safety of this treatment, although potential adverse e ffects might still occur. Moreover, tocilizumab might have an optimal therapeutic effect when used concomitantly with other drugs. Future studies should explore which patients are most likely to benefit from tocilizumab, determine whether its sole use or combined therapy yields the most optimal e ffect, and identify the most e ffective dosing regimens.

In addition, only one case involved a patient placed on mechanical ventilation and subsequently administered tocilizumab for successful recovery; all other patients were administered the drug at a time when their respiratory function was declining, but they had not ye<sup>t</sup> been placed on mechanical ventilation, thus suggesting that tocilizumab might work most e ffectively at the time of emergence of a cytokine storm. Precise identification of the optimal time period in which to administer tocilizumab is critical, and further investigations may shed light on the most suitable time to use the drug. The case described by Minhai et al. [99] suggests the need for studies examining the potential role of tocilizumab

in preventing severe COVID-19. A summary of existing cases in the literature that describe tocilizumab use in patients with COVID-19 is shown in Table 1.

Our case report has some notable limitations. The patient was concomitantly treated with several drugs (including hydroxychloroquine and azithromycin) that might have influenced his clinical course. Therefore, we cannot definitely ascertain whether any one drug agen<sup>t</sup> or the combination played a role in his recovery. Additionally, because most patients with SARS-CoV-2 infection recover (the case-fatality rate is low, at an estimated 2–3%), determining whether the introduction of tocilizumab might be solely responsible for lung recovery is challenging.<sup>6</sup> However, given that the patient was declining with deteriorating respiratory symptoms, and that sharp recovery and relief was observed after tocilizumab administration, tocilizumab may have had a role in the patient's clinical improvement.
