**6. HO-1 Mechanism of Action against Inflammatory Lung Diseases**

There is extensive literature about the role of HO-1 in lung diseases. This protein is expressed in type II pneumocytes and in alveolar macrophages and contributes to the protection of the lung tissue. The main HO-1 inducers in the lungs are pro-inflammatory cytokines, such as TNF-α and IL-6, the heme group and nitric oxide (NO), as well as hypoxia

and hyperoxia conditions [109] (Figure 2). There is sound evidence that states that HO-1 induction is a critical defense factor during acute and chronic lung processes [109–111].

**Figure 2.** HO-1 and inflammatory lung diseases. HO-1 is expressed in pulmonary cells and confers protection against inflammatory lung diseases such as acute respiratory distress syndrome (ARDS), acute lung injury (ALI), and SARS-CoV-2 infection. Schematic representation displaying HO-1's reaction and its products' protective effects in the lung tissue.

As mentioned earlier, during COVID-19 disease, the number of immune cells infiltrating lung tissues and the pro-inflammatory cytokines levels are augmented [112]. Consequently, anti-inflammatory proteins have a crucial role in halting the cytokine storm and the sequelae generated by viral infection [113].

In particular, ALI and ARDS are the most prevalent diseases emerging from an extended diversity of lung injuries [114,115]. ALI and ARDS are pathophysiologically characterized by lung damage, inflammatory infiltration, and an exacerbation of the host immune response [116]. Several reports indicate that ALI and ARDS might be explained by the presence of high ROS levels, where HO-1 acts as a protective factor against oxidative stress under pharmacological induction [117]. HO-1 induction by hemin shows a protective role against ventilator induced lung injury in rabbits with ALI/ARDS, increasing anti-inflammatory cytokine levels, such as IL-10, as well as decreasing the inflammatory infiltrate of immune system cells and the secretion of inflammatory cytokines, such as TNFα and IL-8 [118] (Figure 2). Furthermore, it has been found that HO-1 confers protection against ischemia-reperfusion injury (LIRI) [119].

HO-1 regulates diverse signaling pathways that are affected during pulmonary diseases. In rats, HO-1 inhibits the PERK/eIF2-α/ATF4/CHOP pathway, which is involved in the endoplasmic reticulum stress (ERS) characteristic in ALI, and also promotes the

decrease in intrapulmonary cell apoptosis [120]. It was also reported that the PI3K/Akt pathway attenuates oxidative damage during ALI/ARDS through HO-1 regulation [121]. In pathologies such as silicosis, characterized by excessive ROS production, lung injury is attenuated by HO-1 induction. The mechanism underlying this cytoprotective effect relies on the ERK pathway inhibition by HO-1, CO and BV [122].

Reaction products derived from the HO-1 mediated heme catalysis have protective roles in lung pathologies as well. CO is known to provide protection against ALI and ARDS by reducing cytokine and chemokine levels [105,117,123]. CO decreases EGR-1 (early growth response protein 1), a proinflammatory protein that regulates the expression of TNF-α and IL-2 [124], in mice lungs [123]. Fujita et al. demonstrated that *Hmox1* deficient mice had increased mortality after lung ischemia, an effect reverted by CO administration [125]. Furthermore, BV exerted antioxidative, anti-inflammatory and anti-apoptotic effects in a rat model of LIRI [126]. BV administration protected against hemorrhagic shock induced ALI through a decrease in the inflammatory infiltrate and proinflammatory cytokines levels [127].

#### **7. Unveiling How HO-1 Promotes Viral Clearance**

HO-1 has immunomodulatory properties on the innate immune response and there is compelling evidence suggesting that it also plays a central role in the modulation of adaptive immunity. HO-1 displays antiviral properties against a wide range of viruses [7] (Figure 3). Several reports have demonstrated that HO-1 induction is associated with the activation of the IFN pathway. However, the mechanism underlying the antiviral properties of HO-1 exerted by both its classical and noncanonical activities are yet to be fully elucidated.


**Figure 3.** HO-1's induction and its effect on different viral infections. Table containing previously reported studies about HO-1 involvement in influenza A virus (IAV), respiratory syncytial virus (RSV), human immunodeficiency virus (HIV), ebola virus (EBOV), dengue virus (DENV), zika virus (ZIKV), hepatitis C virus (HCV), hepatitis B virus (HBV), herpes simplex virus 2 (HSV-2), enterovirus 71 (EV71) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. The table includes the experimental model, HO-1 inducers, its mechanism of action, its effect and the study's PMID. CoPP: cobalt protoporphyrin IX, DMO-CAP: 6-demethoxy-4 -O-methylcapillarisin, ROS: Reactive oxygen species, IFN: interferon, MDM: monocyte derived macrophages, BV: Biliverdin, CO: carbon monoxide, CORM-2: CO-releasing molecule-2.

#### *7.1. Respiratory Viruses: IAV and RSV*

Influenza A virus (IAV) is a single stranded RNA virus whose infection remains a persistent global health threat with high morbidity and mortality [128]. An estimate of 4.0 to 8.8 deaths per 100,000 individuals with seasonal influenza associated respiratory occur annually (all types of influenza virus) [129]. Considering that the inhibition of virus induced ROS formation impairs IAV replication, proteins such as HO-1 are useful to counteract IAV infections in the host cell [130]. Wang et al. studied the effect of hemin in IAV infections and demonstrated that hemin attenuates the lymphocytopenia caused by IAV infection both in vitro and in vivo [131]. These results suggest that the anti-influenza effect of hemin may be mediated by HO-1's ability to regulate systemic and local inflammatory responses [131]. Furthermore, Hashiba et al. reported that HO-1 gene transfer is a potential strategy to treat lung injury caused by IAV [132] and Cummins et al. suggested that the therapeutic induction of HO-1 expression may represent a novel adjuvant to enhance influenza vaccine effectiveness [133]. It has also been reported that the HO-1 inducers rupestonic acid derivative YZH-106 and the flavonoid 6-demethoxy-4 -O-methylcapillarisin (DMO-CAP) inhibited IAV replication by activating the HO-1 mediated IFN response [134,135]. Additionally, Ma et al. evaluated the effect of CoPP in IAV infection, focusing on the IFN pathways. The authors demonstrated that HO-1 induction attenuates IAV replication, and the most intriguing finding was that the catalytic function of HO-1 was not essential for the anti-IAV effect of CoPP. Interestingly, they found that HO-1 interacts with IFN regulatory factor 3 (IRF3) promoting its phosphorylation and nuclear translocation, thus activating the IFN pathway. Consequently, CoPP treatment increased the expression of *IFITM3*, *PKR* and *OAS1*, three ISGs markedly involved in the anti-IAV response [128].

Respiratory syncytial virus (RSV) is an RNA virus of the Pneumoviridae family and the most common cause of lower respiratory tract infections in children worldwide [136]. It interacts with host cells' toll-like receptors in the primary airway epithelium, and promotes the expression and secretion of inflammatory cytokines [137], under the NF-κB pathway's regulation [138]. Similar to IAV, CoPP HO-1 induction inhibited RSV replication and viral particle production in lung adenocarcinoma (A549) and HEp-2 cells. Most importantly, in vivo assays in BALB/cJ mice treated prophylactically with CoPP also showed a reduction in viral replication and viral particle production, alongside a decrease in inflammatory cell infiltration, and inhibition of proinflammatory cytokine or chemokine secretion during RSV infection [139].

The mentioned reports suggest that HO-1 is involved in host cellular defense mechanisms against IAV and RSV infections. Of note, HO-1's antiviral effects are mediated by its classical and noncanonical functions.
