**1. Introduction**

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019 in Wuhan, China. The World Health Organization (WHO) declared coronavirus disease 2019 (COVID-19) a pandemic health emergency as of 31 January 2020. The treatment goal in COVID-19 patients is to prevent or to decrease the strong virus induced inflammatory stimuli associated with a wide spectrum of poor prognosis clinical manifestations [1]. Heme oxygenase 1 (HO-1) is a microsomal enzyme with a primary antioxidant and antiinflammatory role involved in heme degradation, generating carbon monoxide (CO), biliverdin (BV), and free iron (Fe2+) [2]. Hence, HO-1 induction is a useful approach for inflammatory diseases treatment [3–6]. Additionally, HO-1 displays antiviral properties against a wide range of viruses [7]. Hemin, a previously Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved drug for acute intermittent porphyria treatment [8,9], is a well known inducer of HO-1 that increases its plasma concentration in humans. Thus, hemin rises as a promising drug candidate against the replication of different viruses, including SARS-CoV-2. In this review, we summarize

**Citation:** Toro, A.; Ruiz, M.S.; Lage-Vickers, S.; Sanchis, P.; Sabater, A.; Pascual, G.; Seniuk, R.; Cascardo, F.; Ledesma-Bazan, S.; Vilicich, F.; et al. A Journey into the Clinical Relevance of Heme Oxygenase 1 for Human Inflammatory Disease and Viral Clearance: Why Does It Matter on the COVID-19 Scene? *Antioxidants* **2022**, *11*, 276. https://doi.org/10.3390/ antiox11020276

Academic Editors: Elias Lianos and Maria G. Detsika

Received: 7 January 2022 Accepted: 26 January 2022 Published: 29 January 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the current research on the protective role of HO-1 in inflammatory diseases and in a diverse range of viral infections, positioning this protein as a potential therapeutic target to ameliorate COVID-19's clinical manifestations.

#### **2. Re-Emergence of the Coronavirus Disease**

Coronaviruses (CoVs) are a large family of positive sense, single stranded RNA (+ssRNA) viruses that infect humans, other mammals, and birds, causing respiratory, enteric, hepatic, and neurologic diseases [10,11]. CoVs first became renowned in 2002–2003 during an outbreak of a virus with zoonotic origin; the severe acute respiratory syndrome coronavirus (SARS-CoV) originated in China, with 8096 cases and 774 deaths reported between 2002 and 2003 [12], and a case–fatality ratio of 7.2% [13]. In 2012, the Middle East respiratory syndrome coronavirus (MERS-CoV), another virus with zoonotic origin, emerged in Saudi Arabia and caused 927 fatalities among 2581 registered cases [14,15].

By the end of 2019, the Wuhan Health Commission from China reported a number of pneumonia cases of unknown cause and varying severity in the city of Wuhan, China. High throughput sequencing allowed the quick identification of a novel CoV belonging to the beta-coronavirus family, which was named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as the causative agent of Coronavirus disease 2019 (COVID-19) [16]. This pathogen rapidly spread globally via travel related cases; constituting a pandemic and setting an immense challenge for public health [16]. Unlike SARS-CoV and MERS-CoV, SARS-CoV-2 can be transmitted among people before the onset of symptoms or from asymptomatic individuals [17], limiting effective control of the spread. As of 30 December 2021, the WHO reports 281,808,270 confirmed cases and 5,411,759 deaths worldwide [15].

Although COVID-19 is primarily a respiratory disease, SARS-CoV-2 has the capacity to infect a broad range of cell types in different organs and systems, including the central nervous system [18]. SARS-CoV-2 infection begins in the proximal airways and could trigger severe and sometimes fatal symptoms when reaching the distal lung [19]. Among the severe respiratory diseases caused by SARS-CoV-2, acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) [20,21] are complications mainly caused by an exacerbated immune response in elders and patients with comorbidities [22]. Cytokine storm contributes to ALI and the development of ARDS in patients with severe pneumonia caused by SARS-CoV-2, as well as SARS-CoV and MERS-CoV [23,24]. Surprisingly, in addition to the respiratory symptoms, patients may suffer from cardiac, hematological, neurological, hepatic, gastrointestinal and kidney complications [25]. COVID-19 may result in long term sequelae characterized by organ injuries that cannot be completely reversed. Several patients, even those with mild cases, may develop lasting symptoms that can have disabling consequences [26,27].

The COVID-19 era is far from being constrained, and the emergence of new viral variants causing future outbreaks remains a threat. As of December 2021, the WHO has defined five variants of concern (Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529)), as well as two variants of interest (Lambda (C.37) and Mu (B.1.621)) [15]. It has been reported that some variants, such as the recently described Omicron [28], have higher transmission rates and are able to escape neutralizing antibodies generated by natural infection and vaccination, or therapeutic antibodies [29–32]. Incomplete knowledge on the pathogenesis of SARS-CoV-2 and the diverse array of symptoms and manifestations of COVID-19 pose a great challenge for the development of effective treatments that should mainly focus on both, decreasing viral replication and modulating the immune response.

#### **3. Cytokine Storm and Inflammation**

Inflammation involves defense mechanisms against infection or injury. It is responsible for activating both innate and adaptive immune responses [33,34]. During infections, innate cells recognize pathogen associated molecular patterns (PAMPs) from the invading agent. In the case of inflammation triggered by tissue damage, trauma or ischemia, innate cells recognize host specific molecules that are released during cell injury or necrotic death, defined as damage associated molecular patterns, such as nucleic acids and adenosine triphosphate (ATP) [33]. During the early stages of inflammation, innate immune cells and endothelial cells (EC) release a diverse set of cytokines: chemotactic cytokines, such as monocyte chemotactic protein-3 (MCP-3) and interferon (IFN) γ-induced protein 10 (IP-10), and recruit other immune cells to the site of infection or inflammation. Proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-1β [35,36], are also released and trigger the activation of inflammatory pathways, including the mitogen activated protein kinase (MAPK), nuclear factor kappa-B (NF-κB), and Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways [34]. Some pathogenic viruses (i.e., highly virulent subtypes of influenza) and bacteria (i.e., *Francisella tularensis*) can induce the acute dysregulated production of inflammatory cytokines, known as "cytokine storm" or hypercytokinemia [37]. The hypercytokinemia and exacerbated secondary events, such as coagulation, eventually result in widespread necrosis, organ failure and death [33,38].

Once SARS-CoV-2 infects target cells, innate immune cells are recruited to the infection site, where they release cytokines and initiate the activation cascade of adaptive B and T cell immune responses [39]. In most cases, the immune system is able to eliminate virus infected cells and resolve the immune response. However, in some patients, this process is dysfunctional, impairing the effective clearance of infected cells, and causing severe damage to the host [40].

#### **4. The Lead Role of Interferons upon Viral Infection**

During viral infections, pattern recognition receptors are stimulated to produce IFN by the innate immune cells. IFNs are crucial for the induction of an antiviral state via autocrine and paracrine signaling. There are three types of IFNs: type I, type II and type III. Type I (IFNα, IFNβ, IFNω, IFNτ, IFNε) and Type III (IFNλ1, IFNλ2/3, IFNλ4) share similar dynamics after binding to its receptor, as cross-phosphorylation between JAK1 and tyrosine kinase 2 (TYK2) occurs [41]. Subsequently, a docking site for STAT1 is exposed, STAT1 is phosphorylated, translocates to the nucleus, and induces the transcription of interferon stimulated genes (ISGs). The IFNs biological effects are wide, including immuno-regulation, antiviral, anti-angiogenic, and pro-apoptotic functions [42]. However, many pathogens have evolved to elude the action mechanisms of these powerful cytokines [43–45].

In critically ill COVID-19 patients, a hyperinflammation state prevails. In March 2020, a retrospective study on 150 patients from Wuhan, China, found elevated levels of IL-6 and C-reactive protein (CRP) in SARS-CoV-2 infected patients that died compared with discharged patients [46]. An independent report based on 50 COVID-19 patients with moderate to severe disease, identified IP-10, MCP-3, and IL-1 receptor antagonist (IL-1ra) as independent predictors for disease severity [47]. A longitudinal analysis showcased IL-18 and IFN-α as top biomarkers for predicting mortality. Consequently, higher counts of inflammatory monocytes, plasmablast like neutrophils and eosinophils have been described in patients with severe disease [39].

Blanco-Melo et al. reported an impairment in the response of type I and type III IFNs against SARS-CoV-2 infection [40]. In contrast, a recent study found that severe COVID-19 cases showed an exacerbated expression of type I IFNs, which could lead to augmented inflammation [48]. Several clinical trials evaluating IFNs have been carried out in COVID-19 patients. Two different studies showed a reduction in the mortality rate after IFNβ-1a and IFNβ-1b treatment [49,50]. Another study, using IFNα-2b, reported a decrease in detectable SARS-CoV-2 in the upper respiratory tract associated with lower inflammatory cytokines levels, such as IL-6 and CRP [51]. In addition, peginterferon λ treatment was associated with a reduction in viral RNA [52]. Furthermore, there are several ongoing clinical trials, using recombinant human IFNs or IFNs combined with other drugs [53–55]. These evidences highlight IFNs as potential targets for COVID-19 treatment. In the next section we will focus on the stimulation of IFN pathway by HO-1 induction.

#### **5. Understanding the Protective Role of Heme Oxygenase 1**

Heme oxygenases (HO) are metabolic enzymes that partake in the degradation of the heme group [2]. To date, three isoforms of this protein have been found: HO-1, which can be induced by external factors (such as hypoxia, oxidative stress, heat shock, reactive oxygen species (ROS), among others) [56]; HO-2, a constitutively expressed isoform; and HO-3, a nonfunctional isoform in humans [57].

In particular, HO-1, encoded by the *HMOX1* gene, is involved in the maintenance of cellular homeostasis, exerting a cytoprotective role by its anti-inflammatory, anti-oxidative and anti-apoptotic functions, as revealed in a human case of genetic HO-1 deficiency [58]. This enzyme participates not only in normal physiological processes, but also performs a protective role in inflammatory physiopathological conditions, such as kidney disease [59], cancer [60,61], cardiovascular disease [62], asthma [63] and inflammatory bowel diseases [4,64].

HO-1 is expressed in most cell types and tissues; however, its capacity to counteract inflammation seems to be critically dependent on its specific functions in myeloid cells and in EC [65]. In myeloid cells, HO-1 acts as a key regulator of the TLR4/TLR3/IRF3 induced production of IFN-β and primary IRF3 target genes in macrophages [66] and modulates maturation and specific functions of dendritic cells [67,68]. Moreover, HO-1 over-expression in macrophages negatively regulates the expression of diverse proinflammatory molecules and increases the expression of anti-inflammatory cytokines [69–71]. Among HO-1 effects on EC, it is significant to mention its ability to inhibit the expression of pro-inflammatory genes related to EC activation, such as the TNF-α-induced adhesion molecules, E-selectin and VCAM-1, via a mechanism associated with the inhibition of NF-κB activation [72].

HO-1 cleaves the heme group generating BV, CO and Fe2+. Heme is usually bound to a myriad of proteins and it is involved in several homeostatic functions [56]. However, elevated concentrations of heme can cause cell damage because it is a pro-oxidant molecule. It can diffuse through cell membranes and deliver a redox active iron, producing ROS [73]. Excessive amounts of these molecules are toxic and induce oxidative stress that, in turn, generates DNA and protein damage, aggregation and lipid peroxidation, triggering cells permeability and driving cell lysis and death [73].

Several studies highlight heme catabolism end products as potential therapeutic targets in vascular disease, based on their anti-inflammatory and antiproliferative functions [74]. BV and its reduced form, bilirubin (BR), are powerful antioxidants that are able to scavenge ROS and counteract the oxidative stress. BV and BR are critical for the regulation of inflammation by exerting immunosuppressive effects [75], as they have been reported to have potent anti-inflammatory activity against insulin resistance by reducing visceral obesity and adipose tissue inflammation [76].

In addition, CO is considered an anti-apoptotic [77], antiproliferative and antiinflammatory factor [78]. CO contributes to blood vessel development [79] and promotes angiogenesis, a crucial process involved in tissue reparation after a pathological state [80]. Interestingly, CO also reduces inflammation and inhibits apoptosis by interacting with antigenpresenting cells and suppressing T cell proliferation [81]. Moreover, it has been reported that it downregulates proinflammatory cytokines via the p38/MAPK pathway in RAW 264.7 macrophages and C57BL/6 mice [70], by the c-Jun N-terminal kinase (JNK) pathway in a murine model of sepsis [82] and through the extracellular signal regulated kinase (ERK) signaling pathway in CD4+ T cells [81]. Further, HO-1/CO induced downregulation of the NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome activation has been demonstrated in different models of murine hepatic and lung inflammatory injury [83–85].

Moreover, the HO-1 mediated increase in Fe2+ concentration upregulates the expression of ferritin, an iron chelating protein [86]. Ferritin exerts antioxidative and cytoprotective effects [74], as this product scavenges redox active Fe2+, rendering it not harmful for cells and avoiding subsequent production of ROS via Fenton reaction. Fe2+ performs its function by inhibiting IL-2 and IgG production, and downregulating the MAPK and NF-κB signaling pathways [56,75,87].

#### *Therapeutic Potential of HO-1 Induction to Treat Chronic Inflammation*

As HO-1 and its reaction products exert protective anti-inflammatory effects in different preclinical models [3–5,88–90], the induction of the HO-1 system has emerged as a promising potential therapy for chronic inflammatory diseases. Most of the studied strategies are based on the use of the traditional pharmacological inducers: hemin [91,92], an FDA and EMA approved drug, and cobalt protoporphyrin IX (CoPP) [5,93,94]. In addition, many phytochemicals, such as quercetin, curcumin and resveratrol, are currently under investigation, due to their potential as HO-1 alternative inducers to counteract inflammation processes with lower cytotoxic secondary effects [95–97]. Alternatively, there are also a few approved drugs, such as 5-aminosalicylic acid (5-ASA), dimethyl fumarate (DMF), and 5-aminolevulinic acid (5-ALA), whose beneficial properties in inflammatory conditions are explained, at least in part, by their capacity to induce HO-1 [96,98,99] (Figure 1). Additionally, another effective option is the use of BV/BR based therapies, which have proven to be effective for these chronic pathologies [100–102] and/or the direct administration of CO via inhalation, CO-releasing molecules (CORMs) or hybrid carbon monoxide-releasing molecules (HYCOs). HYCOs are a type of compound where CORMs are combined with DMF, causing a powerful anti-inflammatory action due to its effect on the NRF2/HO-1 pathway [5,100,103–106] (Figure 1).

**Figure 1.** Inducers of HO-1. HO-1 degrades heme producing equimolar amounts of carbon monoxide (CO), biliverdin (BV) and Fe2+. HO-1's inducers are grouped into protoporphyrins, a type of porphyrins that forms heme; phytochemicals, natural antioxidants compounds contained in plants; and approved drugs, compounds that were previously approved by the FDA. CoPP: cobalt protoporphyrin IX; 5-ALA: 5-aminolevulinic acid; DMF: dimethyl fumarate; 5-ASA: 5-aminosalicylic acid; CORMs: CO-releasing molecules; HYCOs: Hybrid carbon monoxide-releasing molecules; BR: bilirubin. The images of HO-1 and CoPP were taken from RCSB PDB (PDB ID: 1N3U) and The National Center for Biotechnology Information [107,108].
