*3.1. Inhibition of DC Function*

APCs play a key role in the initialization of adaptive immunity via promoting T cell differentiation. APCs capture antigens from the external environment via endocytosis or phagocytosis and degrade them into peptide fragments and binding with MHC II molecules. Antigen peptide–MHCII complex-loaded APCs then contact with naïve T cells via TCRs on the surface and initiate T cell polarization. DCs are the most important professional APCs [28,29]. This function of APCs is regulated by pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRR) [28,30,31]. HO-1 is constitutively expressed in immature DCs (iDCs) such as human monocyte-derived iDCs, freshly isolated rat splenic DC subsets, and rat bone marrow-derived iDCs, and is downregulated during DC maturation [32]. Signaling through PAMPs and its receptor can also regulate HO-1 expression in APCs [28,32,33]. In a mouse model of allergic airway inflammation, reinfusion of DCs highly expressing HO-1 significantly alleviated allergic airway inflammation [34], suggesting a regulatory effect of HO-1 on the antigen-presentation function

of DCs. HO-1 can regulate DCs through multiple modes, such as effects on maturation, antigen presentation, and release of cytokines and extracellular vesicles (EVs). EVs are membranous structures loaded with various proteins, lipids, and nucleic acids and play important role in cell–cell communication.

First, HO-1 can inhibit DC maturation. Inhibition of HO-1 in DCs promotes the maturation of DCs [32,35,36], whereas over-expression of HO-1 was shown to inhibit maturation of bone marrow-derived DCs presenting a tolerance phenotype, as well as the presentation of exogenous soluble antigen to naïve T cells [34,37–41], which further affect the polarization of Naïve T cells towards Th1, Th2, and Th17 cells subsets. The function of APCs is regulated by PAMPs, HO-1 and its end-product CO can inhibit DC maturation by interfering with PAMPs and receptor binding. For example, CO can modify the natural conformation of toll-like receptor 4 to reduce DC maturation [42,43]. Thus, similar impairments of key steps required for correct conformational assembly of this complex on the surface of APCs are likely to reduce DC sensitivity to LPS stimulation by interfering with LPS recognition. In addition, upregulation of HO-1 activity renders DCs insensitive to LPS-induced activation of the p38 mitogen-activated protein kinase/cAMPresponse element-binding protein/activating transcription factor 1 signaling pathway [38]. Importantly, all of the above factors could influence the LPS-induced maturation of DCs through effects on APCs.

Second, HO-1 and CO can inhibit antigen presentation to regulate DCs. In the process of antigen presentation, APCs first capture antigen components and endocytose them to form early endosomes, late endosomes, and fuse with proteasome/MHC molecules containing endosomes; then, they can fuse with lysosomes to form an MHC peptide complex. HO-1 and CO not only reduce the capability of APCs to identify PAMPs but also impair fusion between late endosomes and lysosomes [40], reduce mitochondrial membrane potential and ATP production in DCs, impairing cargo transport and endosometo-lysosome fusion [39]. Disrupting fusion between antigen-containing late endosomes and lysosomes further blocks antigen transport by preventing the formation of MHC-II-peptide fragments in lysosomes, thus inhibiting the presentation of soluble antigens by DCs.

Third, HO-1 can regulate patterns of cytokines released by DCs. DCs highly expressing HO-1 secrete high levels of interleukin 10 (IL-10) and TGF-β, and low levels of IL-12 and IL-23, yielding a microenvironment conducive to the differentiation of naïve T cells into regulatory T cells (Tregs) rather than Th2 or Th17 cells [32,34]. In addition, overexpression of HO-1 in DCs can inhibit DCs maturation as we discussed above and direct naïve T cells polarization towards Treg subtypes [34]. The absence of HO-1 in APCs abolished the suppressive activity of Treg cells on effector T cells, indicating that HO-1 activity in APCs is important for the inhibitory function of Tregs [44]. This evidence indicates that the regulatory role of HO-1 on Tregs partly via APCs inhibitory manner.

Finally, HO-1 can regulate immune responses by inhibiting the release of EVs from DCs. DC-derived EVs lead to allergic airway inflammation by presenting allergens and directly contacting CD4<sup>+</sup> T cells. Our previous study found that stimulating DCs with dust mite extract expressing MHC II resulted in the concentrated release of EVs, which induced Th2 cell differentiation in vitro. In an animal model of asthma, concentrated EVs were produced following house dust mite stimulation of the airway, indicating typical allergic airway inflammation. In hemin-induced EV-sensitized mice, allergic airway inflammation was significantly alleviated; EOS infiltration and mucus secretion were reduced in the airway; levels of IL-4, IL-5, and IL-13 were decreased in the lung; numbers of Th2 cells in the mediastinum lymph node (MLN) were decreased; numbers of Treg cells in MLN were increased; and numbers of Th17 cells were reduced. These results suggest that the anti-inflammatory effects of EVs are executed through regulation of Th17/Treg balance and inhibition of Th2 and Th17 cell proliferation [45].

#### *3.2. Inhibition of BA Function*

In addition to DCs, BAs are an important APC for initiating allergic inflammation. Although DCs have historically been considered an important APC for initiating T cell immune responses and forming memory immune cells, they cannot secrete IL-4 and independently initiate Th2 immune response. Recently, the role of BAs in Th2 immune responses and allergic diseases has attracted increased attention. We and others have confirmed that BAs with antigen-presentation functions express costimulatory molecules and secrete "early IL-4". Moreover, BAs can promote Th2 cell differentiation without exogenous IL-4 in vitro [46–49]. Currently, BAs are considered to both assist APCs (such as DCs) in the initiation of directional differentiation of Th2 cells by secreting Th2 cytokines (such as IL-4) and independently initiate Th2 immune responses as APCs [50]. BAs can also obtain MHC II–peptide complexes from DCs through trogocytosis to exert APC function [47]. Furthermore, our previous study demonstrated HO-1 expression in BAs by immunohistochemistry. Overexpression of HO-1 significantly inhibited the expression of activation marker CD200R and costimulatory factors, inhibited IL-4 release stimulated by DNP-OVA/anti-DNP-IgE, inhibited DQ-OVA up-taken both in the lung-derived BAs from asthma animal models and in cultured bone marrow-derived BAs, and subsequently, inhibited polarization of naïve T cells into Th2 cells in vitro and inhibited OVA-induced allergic airway inflammation and the Th2 immune response.

#### **4. HO-1 Inhibits Inflammation during the Effective Stage**

### *4.1. HO-1 Promotes Treg Cell Function and Inhibits Th2- and Th17 Cell-Mediated Inflammation*

The imbalance of the Th cell subgroup plays an important role in the pathogenesis of asthma. HO-1 inhibits Th cell functions via different mechanisms. Firstly, CO, which is one of the end-products of HO-1, can inhibit the proliferation of CD4+ T cells by blocking TCR-dependent IL-2 production [51]. Another end-product, BR, can inhibit CD4<sup>+</sup> T cells by inducing apoptosis, suppressing co-stimulatory molecule expression in CD4+ T cells, and inhibiting CD4+ cell proliferation [52].

Secondly, HO-1 can regulate the balance of the Th cell subgroup via Tregs. Tregs are important immune cells to maintain immune homeostasis. Tregs inhibit effector T cells proliferation and function via interactions with negative costimulatory molecules, secrete suppressive cytokines IL-10, and competition for IL-2 [53], and subsequently exert inhibitory effects on Th1, Th2, and Th17 cell-mediated inflammation [54–56]. HO-1 promotes Tregs function, which is regarded as an important mechanism for its immunomodulatory function. HO-1 expression is significantly different between CD4+ CD25<sup>+</sup> Treg cells and CD4+ CD25<sup>+</sup> T lymphocytes [7], and is consistent with Foxp3 expression in these two cell types. Transfection of Foxp3 into Jurkat T cells significantly upregulated the expression of HO-1 and inhibited their proliferation and cytokine production in a cell contact-dependent manner. Treatment of freshly isolated CD4<sup>+</sup> CD25high from the spleen with hemin or transfected with an HO-1 expression vector (pcDNA3HO-1) in vitro not only significantly enhanced Foxp3 expression and IL-10 secretion but also enhanced its ability to inhibit effector T cell proliferation. The regulatory role of HO-1 was significantly inhibited by the addition of an HO-1 activity inhibitor [11,57]. In an animal model of asthmatic allergic airway inflammation, overexpression of HO-1 induced by hemin enhanced proportions and functions of CD4<sup>+</sup> CD25<sup>+</sup> Treg cells [10,11] and alleviated OVA-induced allergic airway inflammation. On the contrary, inhibition of HO-1 activity with tin-protoporphyrin reversed the above effects of HO-1 [10,11]. These in vivo and in vitro studies show that HO-1 plays an important role in regulating Treg function, However, the direct role of HO-1 in regulating Treg function is challenged since HO-1-deficient mice not only exhibited a significantly higher proportion of Foxp3-expressing cells among total CD4<sup>+</sup> and CD4+ CD25+ cells in comparison to wild type mice but also displayed a similar inhibitory role in suppressing the proliferation of effector T cells in vitro. In the same study, HO-1-deficient APCs abolished the suppressive activity of Treg cells [44], indicating that HO-1 may regulate CD4+ CD25+ Treg cells by indirectly promoting Treg differentiation through inhibition of DC maturation. Considering Tregs have inhibitory effects on T cell subsets of Th1, Th2, and Th17 cells, we speculate that HO-1 enhances Treg function and therefore regulates the balance of Th1, Th2, and Th17 cells.

### *4.2. HO-1 Inhibits Th17 Cell-Mediated Inflammation*

Th17 cells, an important T cell subset in asthma, play key roles in refractory asthma and neutrophil-dominant asthma types by promoting neutrophil growth, development, and chemotactic aggregation in the airway. Th17 cells achieve these effects by secreting cytokines and are essential for inducing neutrophil infiltration-dominant asthma [26,58,59]. Overexpression of HO-1 inhibited the differentiation of naïve T cells into Th17 cells, as well as the secretion of IL-17A in vitro [60]. In an animal model of non-eosinophilic asthma, upregulation of HO-1 expression significantly reduced the proportion of Th17 cells, promoted IL-10 expression, reconstructed the balance of Th17/Treg cells in vivo, and subsequently inhibited Th17 cell-mediated neutrophilic airway inflammation. In contrast, inhibition of HO-1 activity reversed the inhibitory effect of HO-1 on neutrophil airway inflammation and activation of the Th17 cell signaling pathway [12].
