*4.3. HO-1 Inhibits MC Function*

MCs are important effector cells in asthma. Sensitized MCs can be activated and degranulated to release various preformed mediators and pre-synthesized mediators, such as proteases, cytokines, chemokines, and arachidonic acid metabolites. MCs can de novo synthesize lipid mediators by enzymes located in the plasma membrane and synthesize mRNAs encoding cytokines and chemokines. MCs can also regulate inflammation via the secretion of exosomes containing regulatory molecules [61–63]. MCs participate in asthmatic inflammation which is characterized by inflammatory cell infiltration, microvascular leakage, airway hyperresponsiveness, bronchoconstriction by degranulation, and the secretion of various mediators [64–66]. Moreover, MCs participate in allergic inflammation via regulating T cells, DCs, and other inflammatory cells, which results in further chemotaxis, infiltration, and activation of EOS, neutrophils, and other inflammatory cells in the airway [61,67]. Recent studies revealed that MCs can regulate Th17-mediated autoimmune diseases by inducing Tregs [64].

HO-1 has important regulatory effects on MC function. HO-1 is expressed in MC [5] and MC cell lines [68], and can be induced during MCs degranulation [5,68] and upregulation of HO-1 decreased MCs degranulation induced by complex 48/80 and leukocyte adhesion to blood vessels [5]. Upregulation of HO-1 during MCs degranulation was related to cellular oxidative stress since upregulation of HO-1 was inhibited by antioxidant N-acetyl-L-cysteine [68]. HO-1 and its end-products, BR and BV, can inhibit adhesion and degranulation of MCs [68]. HO-1 can also inhibit the production of inflammatory mediators in MCs by selectively inhibiting the DNA-binding activity of the AP-1 transcription factor [69].

In addition, HO-1 regulates MC-mediated immune regulation. Co-culture of MCs and DCs led to a significant release of tumor necrosis factor-α (TNF-α), IL-6, and interferon (IFN), which promoted DC maturation. Upregulation of HO-1 in MCs before co-culture with DCs inhibited expression of costimulatory molecules on DCs and inhibited DCs maturation [70]. In contrast, downregulation of HO-1 expression promoted MC degranulation, DC costimulatory molecule expression, and DC maturation. These findings suggest that upregulation of HO-1 in MCs stabilizes the MC membrane and prevents its degranulation, thereby maintaining the DCs in an immature state to ultimately alleviate the immune response [39].

#### *4.4. HO-1 Regulated Inflammation by Inhibiting NLRP3 Inflammasomes*

Factors such as environmental irritants and respiratory infections are common triggers of asthma exacerbation and neutrophilic airway inflammation responds to these situations. Signaling through inflammasome activation plays a key role in neutrophilic airway inflammation. The inflammasome is an intracellular protein complex and activated by ligation of PAMPs and its receptor. Upon ligand sensing, inflammasome components assemble and self-oligomerize, followed by autoactivation of caspase-1 and leading to cleave pro-IL-1β and pro-IL-18 to IL-1β and IL-18, T helper 17 activation, IL-8/IL-6 overproduction, thus initiating or aggravating neutrophilic airway inflammation. Inflammasomes also involved in caspase-1-mediated pyroptosis [71,72]. Among the known inflammasomes, nucleotidebinding domain and leucine-rich repeat protein 3 (NLRP3) are crucially involved in the pathogenesis of asthmatic airway inflammation [73].

HO-1/CO has a potential regulatory role in inflammasome signaling. Li and colleges [74] demonstrate that the induction of HO-1 by hemin inhibited LPS-induced production of IL-1β, inhibited NLRP3 inflammasome activation in human gingival epithelial cells in vivo. Luo and colleges [75] also demonstrated that upregulation of HO-1 by hemin inhibited LPS-induced NLRP3 inflammasome activation, reducing IL-1β and IL-18 production in sepsis-induced acute lung injury. On the contrary, inhibition of HO-1 activity reversed the above results. On the other hand, CO can inhibit LPS and ATP-induced caspase-1 activation and production of IL-1β and IL-18 in bone marrow-derived macrophages. Treatment of CO-releasing molecule-2 (CORM-2) both inhibited NLRP3 inflammasome activation in LPS-induced acute lung injury and ER stress-induced inflammation [76]. CORM2 also can inhibit caspase-1activiation [77–79] and thioredoxin-interacting protein (TXNIP)–NLRP3 complex formation [77,78]. Our previous study showed that HO-1 products, CO and BR, inhibited the NLRP3–RXR axis and NLRP3 inflammasome-mediated apoptosis of AECs, which inhibits subsequent production of IL-25, IL-33, thymic stromal lymphopoietin, and other pro-Th2 epithelial-derived cytokines. In addition, we found that HO-1 bound to the NACHT domain of NLRP3 and RXRα/RXRβ subunits, suggesting that the non-enzymatic action of HO-1 may be involved in the regulation of NLRP3 inflammasomes [80].

#### *4.5. HO-1 Promotes Polarization of Macrophages to M2 Phenotype*

Macrophages are one of the important inflammatory cells involved in the pathogenesis of asthma [81]. Macrophages are divided into M1 and M2 subpopulations according to their responses to environmental or inflammatory stimuli. M1 macrophages respond to proinflammatory cytokines, such as IFN-γ and TNF-α, and promote a local Th1 environment. M2 macrophages (alternatively referred to as activated macrophages) respond to IL-4 and IL-13, promote the production of the anti-inflammatory cytokine IL-10, and regulate Th2 immune responses [82–84]. HO-1 is considered a regulator of immune responses because it can promote the polarization of M2 macrophages. Indeed, HO-1 is highly expressed in M2 macrophage subsets, and its elevation in response to multiple stimuli can drive phenotypic transfer to M2 macrophages [85–87]. HO-1-knockout bone marrow macrophages (mHO-1-KO) exposed to LPS (M1-inducer) or IL-4 (M2-inducer) exhibited an enhanced M1 phenotype and inhibited M2 phenotype. In contrast, promotion of the M2 phenotype was observed in HO-1-overexpressing (HO-1-Tg) mice [88]. Collectively, these studies support the hypothesis that HO-1 promotes the M2 phenotype. However, there is a lack of studies demonstrating the role of HO-1 on the regulation balance between M1 and M2 subpopulations in clinical studies or animal models of asthma, and further studies are required.

#### **5. Subcellular Localization and Anti-Inflammatory Mechanism of HO-1**

It is well accepted for a long time that the biological functions of HO-1 are related to its enzymatic products CO, BR/BV, and ferritin. A recent study revealed that HO-1 exerts its function via interaction between other cellular proteins in an activity-independent manner, which is referred as "non-canonical effects of HO-1" in published studies. Those different fashions may in part relate to specific subcellular locations of HO-1.

HO-1 was initially identified in the endoplasmic reticulum (ER). HO-1 is mainly located in the ER under physiological conditions. Recently, more specific subcellular locations of HO-1 have been revealed. HO-1 was identified in mitochondria, plasma membrane, the caveolae, and the nucleus after various stimuli (e.g., LPS, or hypoxia) [89]. Varying the subcellular localization of HO-1 protein has various effects on its cell-protective functions which may depend on the structural integrity of the HO-1 protein. Except for the nucleus, HO-1 is fixed on the membrane by a transmembrane sequence (TMS) located at the carboxyl terminal of its protein structure, and the rest of the protein structure faces the cytoplasm and colocalizes with cytochrome P450 reductase (CPR) and BVR to facilitate heme degradation [90]. Cleavage of the TMS enables HO-1 relocation. On the other hand, CPR can stabilize the HO-1 protein structure to prevent its relocation and maintain its enzymatic activity by promoting oligomerization [91]. HO-1 locates in the mitochondria, vacuole, and plasma membrane and maintains full protein structure and enzymatic activity and coexists with biliverdin reductase [92,93], indicating that its function is achieved mainly through enzymatic activity. The functional significance of HO-1 in different cellular compartments remains unclear. In caveolae, HO-1 activity is negatively modulated by caveolin-1 (CAV1). It could serve as a brake on HO-1 function and provide a possible approach for the active extracellular transfer of HO-1 [94]. In mitochondria, HO-1 induces increased ROS, which appears important for its regulation of mitochondrial heme content, and plays an important role in apoptosis [89,92].

Unlike HO-1 in the ER, mitochondria, and caveolae, nuclear HO-1(NHO-1) exists in a truncated form (28 kDa) in the COOH terminal with a lack of enzyme activity to degrade heme [89]. Therefore, nuclear HO-1 may exert its roles independent of enzyme activity [89,95]. HO-1 in the ER will be truncated and translocate to the nucleus under pathological conditions or external stimuli, leading to cellular stress [94]. NHO-1 has been reported as a regulator of nuclear transcription factor activities such as NF-κB, AP-1, and Nrf2 [96], the latter of which regulates the antioxidant response. By regulating gene expression levels in the nucleus, HO-1 provides resistance to redox stimulation that can protect cells from dysfunction or death, making it an important part of signaling involved in the cellular response to oxidative stress. As the DNA-binding motif of typical transcription factors is not detected in the HO-1 protein structure and direct recruitment of HO-1 to DNA has not been observed, nuclear HO-1 is unlikely to regulate gene expression by directly binding DNA. It is speculated that HO-1 acts as a transcriptional coregulatory protein that binds transcription factors or complexes to regulate their DNA-binding affinity, thus indirectly regulating transcription of key response genes [89].

Our previous study investigated the non-enzymatic anti-inflammatory effects of HO-1. We found that HO-1 inhibited Th17 cell differentiation mainly by inhibiting IL-6-induced STAT3 phosphorylation and therefore inhibited activation of the STAT3/RORγT signaling pathway. Co-IP results indicated that endogenous HO-1 directly bound STAT3 rather than Jak1, JAK2, or SOCS3, suggesting that HO-1 inhibited STAT3 phosphorylation by interacting with STAT3. This result is consistent with the finding of Elguero and colleagues. In their study, they demonstrated that HO-1 and STAT3 bind to each other and therefore inhibit the phosphorylation of STAT3 and subsequent nuclear translocation of pSTAT3 in prostate cancer cells [97]. Our study demonstrated that co-transfection of 293T cells with plasmids containing HO-1 and STAT3 domains revealed that HO-1 bound all regions of the STAT3 protein except the helical domains, especially the transcriptional activation region (AA 689–770). Further experiments confirmed that HO-1 directly bound STAT3, in particular, the transcriptional activation domain-containing Tyr705 in the STAT3 protein. However, whether HO-1 plays an indirect regulatory role through intermediate proteins (for example, whether HO-1 leads to increased dephosphorylation of STAT3 by promoting SHP-1 activation) or inhibits phosphorylation by directly binding STAT3 requires further study [98].

#### **6. Potential Clinical Application of HO-1**

Results from basic research demonstrated that HO-1 alleviated allergic airway inflammation by its anti-inflammatory, antioxidative stress, and immune regulation properties. Moreover, how to apply those results to clinic use has attracted the interest of researchers. Currently, research focused on the value of HO-1 in both diagnostic and therapeutic applications. In view of diagnosis, clinical studies revealed that exhaled CO elevated in asthma patients and the levels of exhaled CO were associated with the severity of asthma and disease exacerbation [99–101], indicating that exhaled CO has potential value in asthma management as a noninvasive tool. On the other hand, therapeutic applications of drugs targeting HO-1 activity and expression have attracted significant attention, including pharmacologic modulators of HO-1, gene therapy, and enzymatic byproducts of HO-1 such as CO and CO-releasing molecules (CORM) [3,102–104]. Despite the achievement of attempts in animal studies with promising results, pharmacologic use of HO-1 targeted drugs still faces great challenges, especially safety and efficacy.

#### **7. Conclusions**

During asthmatic airway inflammation, HO-1 regulates differentiation of Th1/Th2/Th17 cell subsets by inhibiting the functions of APCs (such as DCs and BAs), promotes Treg function, and suppresses allergic airway inflammation characterized by Th2-dominant EOS infiltration and neutrophil infiltration-dominant Th17 immune response. HO-1 directly inhibits airway inflammation by inhibiting BA, MCs, and certain functions during the inflammatory effective stage (summarized in Figure 1). HO-1 not only exerts antiinflammatory and immunomodulatory effects through its enzymatic products CO and BR/BV but also regulates the transcription of key genes by acting as a transcription coregulatory protein with transcription factors or complexes in the nucleus (summarized in Figure 2). However, studies evaluating the non-enzymatic immunoregulatory mechanism of HO-1 are limited, which represents an important direction for future research.

**Figure 1.** Multi-target effect of HO-1 and its immune regulation role in allergic airway inflammation. 1, HO-1 regulates APCs function and inhibits allergic airway inflammation at the initial stage: a, HO-1 and its end-product CO inhibit DC maturation by interfering with PAMPs and receptor binding, inhibit antigen presentation by impair fusion between late endosomes and lysosome, inhibit release of EVs from DCs and promote Treg polarization; b, BAs assist DCs participate in Th2 cells polarization by secreting "early" IL-4; BAs initiate Th2 polarization independently as APCs or BAs obtain MHC II-peptide complex from DCs through trogocytosis and subsequently initiate Th2 polarization; HO-1

inhibits BAs participate in Th2 cell differentiation by inhibits BAs activation, soluble antigen uptaken, expression of costimulatory molecules and secrete "early IL-4"; 2, HO-1 inhibits allergic airway inflammation at effective stage: a, HO-1 and its end-product inhibit CD4<sup>+</sup> T cell proliferation and function directly or via promotion of Treg; b, HO-1 inhibits Th17 cell-mediated neutrophilic airway inflammation by inhibiting Th17 cell polarization and IL-17A releasing; c, HO-1 and its end-products suppresses mast cell degranulation and releasing of inflammatory mediators; d, HO-1 and its endproduct inhibit NLRP3 inflammasome activation and subsequently inhibit IL-1β and IL-18 mediated airway inflammation.

**Figure 2.** Subcellular localization and roles of HO-1. **1**. HO-1 locates in endoplasmic reticulum (ER), mitochondria, plasma membrane, and the caveolae. HO-1 is fixed on the membrane by a transmembrane sequence (TMS) and stabilized by cytochrome P450 reductase (CPR) to prevent relocation. It remains full protein structure and enzymatic activity and its function is achieved mainly through enzymatic activity. Its enzymatic products CO, BV, and BR have been shown to have anti-inflammatory, antioxidant, cell cycle regulation properties; **2**. HO-1 will be truncated and relocated under pathological conditions or external stimuli which leads to cellular stress. Truncated HO-1 lacks enzyme activity to degrade heme and may act as a chaperone to regulate the activity of signaling pathway proteins or as a transcription factor regulator.

**Author Contributions:** W.Z. drafted the manuscript; Z.X. edited and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by grants from the National Natural Science Foundation of China (81970021, 82170035, 82170025), the Shanghai Municipal Science and Technology Commission Foundation (17ZR1418000), the Shanghai Municipal Health Commission (202040164) and Innovative Research Team of High-level Local Universities in Shanghai.

**Conflicts of Interest:** The authors declare no competing financial interest.

#### **References**

