Regulation of Indoleamine-3,5-dioxygenase 1 (IDO1) in Lung Epithelial Cells by Coronaviruses (SARS-CoV) and Cytokine Signaling

Interferons (IFNs) and proinflammatory cytokines play an important role in the innate immune response to respiratory viruses, including coronaviruses (SARS-CoV). Metabolic profiling in the serum samples of coronavirus disease-19 (COVID-19) patients revealed altered cholesterol and tryptophan metabolism. Indoleamine-3,5-dioxygenase (IDO1) is the key enzyme involved in the tryptophan catabolism and induced by interferons and inflammatory cytokines. The regulation of IDO1 in immune cells and cancer was extensively studied. In this study, IDO1 regulation in human lung epithelial cells by coronaviruses and respiratory viruses as well as inflammatory cytokines was investigated. SARS-CoV-2 was a potent inducer of IFN – regulated metabolic enzymes such as IDO1, Cholesterol-25-hydroxylase (CH25H), Spermidine acetyltransferase (SAT1), and Sterile alpha motif and histidine/aspartic acid domain-containing protein (SAMHD1) at RNA levels in Calu-3 cells. Reconstitution of A549 lung epithelial cells with Angiotensin-converting enzyme 2 (ACE2) was necessary and sufficient to induce IDO1 at RNA levels. Influenza A virus (IAV) suppressed IDO1 RNA levels in a non-structural protein (NS1)-dependent manner in NHBE cells. In contrast, IDO1 RNA levels were dramatically induced in the lungs of mice infected with a reconstructed 1918 H1N1 influenza virus. Treatment of A549 cells with either type I or type II interferon induced IDO1 RNA levels. Furthermore, IDO1 levels were significantly higher in the lung tissues of COVID-19 patients in comparison with healthy controls. A mix of proinflammatory cytokines dramatically induced IDO1 and chemokine RNA levels in lung epithelial cells in a cell culture model, simulating the gene expression pattern in the lung tissue samples of COVID-19 patients. Furthermore, hypertonic saline solution (HTS) dramatically abrogated the gene expression induced by cytokine mix in human lung cells. The IDO1 protein interaction network included transcription factors STAT1 and STAT3. These studies suggest that IDO1 inhibition may be a potential therapeutic target in the treatment of viral and inflammatory diseases. SARS-CoV-2 infection and in COVID-19 patients (25,26). In this study, regulation of IDO1in human lung epithelial cells in response to respiratory viruses, interferon, and proinflammatory cytokines was investigated.


Introduction
SARS-CoV-2 infection and severe COVID-19 is the leading cause of death in the intensive care units (ICU) of the critical care hospitals around the world (1,2). A variety of high-throughput studies in the last two years have provided novel insights in the area of coronavirus-host interactions including cytokine mediated signal transduction pathways, gene expression, and identification of potential drug targets (3)(4)(5)(6)(7)(8).
Gene expression profiling by microarrays and RNA-seq enabled the global monitoring of cell-, tissue-, and disease-specific gene expression patterns in the human lung cells in response to SARS-CoV-2 and in COVID-19 patients (3,4,7,8). These methods provide simultaneous quantitative analysis of differential expression levels of thousands of genes on a single platform. Multiplex antibody assays provided important information on the temporal regulation of cytokine levels, including type I and type II interferon (IFN), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta 1(Il-1β), and interleukin 6 (IL-6) in the bronchoalveolar lavage fluid (BALF) of COVID-19 patients (9). Multilevel proteomics revealed the function of SARS-CoV-2 encoded viral proteins and their interactions with the host genome and global alteration of signaling pathways such as Transforming growth factor-beta (TGF-β) and autophagy in A549 human lung cells reconstituted with viral entry receptor ACE2 (10). Furthermore, metabolic studies revealed high levels of tryptophan catabolic product kynurenine in the serum of COVID-19 patients (11,12). Tryptophan is an essential amino acid for cell survival and the intracellular enzyme that breaks down tryptophan to kynurenine is known as Indoleamine-3,5-dioxygenase or IDO1 (13). Constitutive low expression of IDO1 was reported in multiple tissues including peripheral blood and enhanced expression in several cancer cells (14). The role of IDO1 in the context of immune responses and tumor cells was well established (13,14). Tumor cells up-regulate IDO1 to suppress T cell activity by two mechanisms-directly starving T cells of essential tryptophan and triggering T regulatory (Treg) cell development to suppress immune responses. Inhibition of IDO1 can reduce the number of regulatory T cells and restore T-cell function (15). Proinflammatory cytokines such as TNF-α, IL-1, IL-6, and interferon (IFN) treatment induced IDO1 RNA and enzyme levels (16)(17)(18)(19). A healthy microbiome in the human gut and tryptophan metabolism is required for neurotransmitter serotonin and neuropeptide melatonin synthesis. A diet low in tryptophan for 8 weeks in aged mice altered the gut microbiota composition, promoted inflammatory cytokine synthesis resulting in systemic inflammation (20). IDO1 was induced in multiple cell types in response to type I (IFN-α/β) and type II interferon (IFN-γ).
However, IFN-γ was more potent than IFN-α/β in the induction of IDO1 in most cell types antimicrobial effector mechanism that limits pathogen proliferation (22). The products of tryptophan catabolism by IDO1 such as 3-hydroxykynurenine and reactive oxygen species (ROS) produced by the action of proinflammatory cytokines promoted apoptosis in human retinal epithelial cells (17). Products of the kynurenine pathway stimulated by interferon treatment including quinolinic acid and picolinic acid were reported to have antiviral properties (23,24). Modulating the cell metabolism to their advantage is a key aspect of virus-host interactions. Transcriptional induction of the host catabolic enzymes by interferon depletes the essential components of cell survival such as the amino acid, lipid, nucleotide, and polyamine reserves to limit pathogen proliferation. Previous studies have shown the critical role of type I interferon signaling and transcription factors in response to SARS-CoV-2 infection and in 26). In this study, regulation of IDO1in human lung epithelial cells in response to respiratory viruses, interferon, and proinflammatory cytokines was investigated.

Gene expression datasets and Bioinformatics
Human tissue gene expression data of IDO1 was retrieved from the GTEx database. Lung cell atlas data were downloaded from Human Protein Atlas. Microarray data in GEO datasets (GSE147507) (GSE47960) (GSE156295), (GSE70445), and GEO profiles were retrieved from the Pubmed (NIH) resources. Geo datasets were analyzed with the GeoR2R method (NCBI). Gene expression in human lung cell lines infected with respiratory viruses and COVID-19 patients was retrieved from Immgen RNA seq SKYLINE resources. Additional gene expression datasets were downloaded from the H2V database, Immgen browsers, Coronascape, and Signaling Pathway Project. Cluster analysis was performed using gene expression software tools (27). Protein-protein interactions were visualized in the STRING database (28). Gene ontology (GO) and signaling pathway analysis was done in Metascape (29). Interferon-related data was retrieved from WWW.Interferome.org. Gene-specific information was retrieved from standard bioinformatics websites.

Profiling cell-specific expression of IDO1 in the human lung
IDO1 was expressed at low levels in several tissues and up-regulated in many cancer cells (13,14).
Tissue distribution analysis in the GTEx database of human tissues revealed that IDO1 was highly expressed in the lung. colon and esophagus mucosa ( Figure 1). These organs are in direct contact with respiratory or food-borne pathogens. IDO1 is predominantly localized in the cytoplasm. The other intracellular locations of IDO1 includes the nucleoplasm, vesicles and mitochondria. The human lung is a complex organ, composed of more than 30 cell types including type I (AT1) and type II (AT2) epithelial cells, club cells, ciliated cells, endothelial cells, and immune cells. The Human Protein Atlas (HPA) database contains detailed information on tissue and cell RNA expression levels of genes. Cell-specific RNA and quantitative expression analysis in the HPA human lung cell atlas revealed that IDO1 was highly expressed in the macrophage c-2, club c-7, and endothelial c-9 cell population. AT2 cell expression levels of IDO1 in c-1 and c-6 were very low and undetectable, respectively ( Figure 2). The localization of IDO1 in club cells (also known as Clara cells) may be of functional importance as they are involved in the protection of airway tissue from the toxic chemicals that reach the lung via inhalation.
Club cells are essential for the integrity and regeneration of airway epithelium and express several enzymes involved in drug and xenobiotic metabolism such as cytochrome P450, monooxygenases and glutathione peroxidases (30). IDO2 catalyzes the same reaction as IDO1 but with much less efficiency than IDO1 and approximately 50% of caucasians have genetic polymorphisms that abolish its expression (31). IDO2 expression was detectable in mac c-2 and T-cell c-3 cell population. Furthermore, Secretoglobin (SCGB1A1) representing a cell-specific marker was highly expressed in club cells ( Figure   2). Heat map representation of gene expression profile of IDO1 along with AT2 , AT1, club, ciliate cellspecific markers were shown for a comparison purpose ( Figure 3).

Regulation of IDO1 expression by coronaviruses
Most of the gene expression studies on SARS-CoV-2 were carried out in well-established human lung cell lines such as Calu-3, A549, and NHBE. Basal expression of IDO1 was very low in these cell lines.
Infection of Calu-3 cells with SARS-CoV-2 dramatically induced IDO1 RNA levels within 12 hours and further enhanced by 24 hours ( Figure 4A). Mock-infection for 12 or 24 hours served as controls. In contrast, SARS-CoV-1 induction of IDO1 RNA levels was much lower at 12 and 24 hours in Calu-3 cells. Interferon-stimulated genes (ISG) play a major role in antiviral response and metabolic reprogramming of the cells and are essential to limit the pathogen spread (32). Some of the well-known metabolic effectors of interferon signaling including Cholesterol-25-hydroxylase (CH25H) involved in cholesterol metabolism, Spermidine/spermine acetyltransferase 1 (SAT1) involved in polyamine regulation, Sterile alpha motif and histidine/aspartic acid domain-containing protein 1 (SAMHD1) involved in innate immunity and IDO1 involved in tryptophan metabolism were induced by SARS-CoV-2 in Calu-3 cells within 24 hours ( Figure 4B). CH25H represses the cholesterol synthesis by converting cholesterol to 7-hydroxy cholesterol. SAT1 and IDO1 are rate-limiting enzymes in the catabolism of polyamines and tryptophan, respectively. SAMHD1 has deoxynucleoside triphosphate (dNTPase) activity and reduces cellular dNTP levels to restrict viral replication. These enzymes deplete the metabolic building blocks that are required for optimal viral replication and multiplication (32). SARS-C0V-2 was more potent than SARS-CoV-1 in the induction of several interferon-responsive genes (25).
In contrast, SAMHD1 was equally induced by SARS-CoV1 and SARS-C0V-2 ( Figure 4B). The temporal induction of IDO1 was correlated with IFN-beta (IFNB1), STAT1, STAT2, and IRF9 at RNA levels ( Figure 5A). These results are consistent with the interpretation that IFN-β production by viral

Regulation of IDO1 expression by influenza and other respiratory viruses
Influenza virus is a major respiratory pathogen that often causes significant mortality and morbidity, especially among young children and geriatric patients (36). Influenza is generally limited to the upper respiratory tract, but when the lower respiratory tract becomes involved, significant lung damage was observed. This may occur with pandemic strains (such as 1918 H1N1 virus that killed more than 50 million worldwide) or pathogenic influenza strains (such as H5N1). Highly pathogenic strains of influenza such as the reconstructed 1918 influenza virus (H1N1) induced a dramatic increase in inflammatory cytokines, influx of neutrophils and macrophages resulting in inflammation, tissue injury and death in mice (37,38). In contrast, seasonal non-pandemic strains of influenza inhibit type I interferon and inflammatory responses. In these strains, the virus encodes a non-structural protein (NS1) that is a potent inhibitor of Interferon regulatory factor 3 (IRF3) and type I interferon production (39).
Furthermore, NS1 inhibits STAT1 activation resulting in the attenuation of interferon-stimulated gene expression (40). Induction of IDO1 expression in NHBE bronchial epithelial cells was abrogated by wild-type influenza A virus (IAV). In contrast, IAV infection with a deleted NS1 (IAVNS1) resulted in the induction of IDO1 expression in NHBE cells ( Figure 7A). This induction of IDO1 was correlated with the induction of IFN-beta (IFNB1) and interferon-inducible transcription factor RNA of STAT1, STAT2, IRF9 and IRF7 ( Figure 7B). Furthermore, IAV mutant virus with NS1 deletion also induced metabolic gene expression of CH25H and SAMHD1 in NHBE cells ( Figure 7B). Infection of mice with the reconstructed 1918 influenza virus dramatically increased the cytokines TNF-α and IFN-γ, and antiviral and inflammatory gene expression (37). These include well-known interferon-stimulated genes (ISG) such as Guanylate binding protein (Gbp2) Interferon induced protein with tetratricopeptide 2 (Ifit2) and Ido1 ( Figure 7C). Respiratory syncytial virus (RSV) infections are common in children and not a concern for adults or pose a pandemic threat. RSV infection also induced interferon-regulated metabolic enzymes IDO1 and SAMHD1 expression in A549 cells ( Figure 8A and 8B). These studies suggest that multiple factors such as virus strains, host-encoded factors (ACE2), virus-encoded factors (NS1), and cell type play an important role in determining viral pathogenesis. A snapshot summary of IDO1 regulation by influenza and coronaviruses was shown (Figure 9). The red and blue ovals represented induction and suppression in separate microarray experiments, respectively. SAMHD1 was induced by cytokines such as IL-6, IFN-α/β, IFN-γ, TLR-ligands such as LPS, and respiratory viruses ( Figure 10).
These results are consistent with SAMHD1's role in controlling antiviral and inflammatory gene expression (41,42). Furthermore, Cholesterol-25-hydroxylase and Spermidine acetyltransferase were also regulated by TLR-ligands and respiratory viruses (Supplementary data). and Nod-like receptors (NLR) to sense molecular signatures associated with pathogens (43). These receptors and associated adaptor molecules trigger kinase cascades leading to the activation of transcription factors such as nuclear factor-kB (NF-kB) and Interferon regulatory factors (IRF3, IRF7) resulting in the production of antiviral cytokines such as type-I Interferons (IFN) and proinflammatory cytokines such as Tumor necrosis Factor-α (TNF-α), Interleukin-1β (IL-1β) and Interleukin-6 (IL-6).

Regulation of IDO1 expression by interferons and proinflammatory cytokine signaling
Multiplex cytokine assays revealed inflammatory cytokines and chemokines in the bronchoalveolar lavage fluid of COVID-19 patients (9). Natural killer (NK) cells produce type-II interferon known as IFN-γ (34). These cytokines activate transcription factors of STAT, IRF. NF-KB, and AP1 families and stimulate expression of a wide variety of genes encoding antiviral proteins and chemokines in lung epithelial cells resulting in the activation of key regulators of innate immunity (33). In type I Interferon (IFN-α/β) signaling STAT1, STAT2, and IRF-9 transcription factor complexes (ISGF3) bind to interferon-stimulated response element (ISRE) in the gene promoter region to regulate gene expression.
In contrast, STAT1 homodimers are predominantly activated and bind to gamma-activated sequence (GAS) in the gene promoter region to regulate IFN-γ−mediated gene expression. Furthermore, inducible transcription factor activation at RNA level enforces temporal regulation in both type-I and type-II IFN signaling (26). Interrogation of microarray datasets revealed that IFN-α/β treatment induced IDO1 and interferon-induced transcription factor at the RNA levels in 8 hours in A549 and HTBE human lung epithelial cells ( Figure 11A and 11B). Furthermore, IFN-α induced IDO1 RNA by 8 to 10-fold whereas IFN-γ dramatically up-regulated by several hundred-fold within 12 to 24 hours in A549 cells ( Figure 11C and 11D). Differential regulation of IDO1 by type-I and type-II interferons in multiple cell lines was reported in the Interferome database. Similarly, low and high induction of IDO1 by TLR ligands such as bacterial LPS and PAM3CSK4 was observed (Supplementary data). These studies reveal differential intensity of signaling mediating a dynamic range of IDO1 RNA fold-induction. A major limitation of these studies is the use of transformed or cancer cell lines. Immgen data on the effect of interferon treatment of primary immune cells such as dendritic cells and macrophages revealed that IFN-γ was more potent than IFN-α in the induction of IDO1 RNA levels within 2 hours (data not shown). Cross-talk between cytokine signal transduction pathways enables the fine-tuning of the host response to different pathogens. There are two major outcomes in a cross-talk between the signal transduction pathwayseither transcriptional synergy or a functional antagonism (44,45). For example, in mouse bone marrowderived macrophages, LPS and IFN-γ synergistically induced IDO1 RNA levels ( Figure 12A). In contrast, hypertonic saline solution (HTS) abrogated the induction of chemokines and inflammatory markers by a cytokine mixture of TNF-α, IL-1β, and IFN-γ in human lung epithelial cells (46). HTS also abrogated IDO1 induction by proinflammatory cytokine mix in these cells ( Figure 12B). These studies suggest that IDO1 RNA levels were differentially regulated in response to type I and type II interferons and inflammatory cytokines in human lung epithelial cells. Transcription factor binding site analysis in the promoter region of human IDO1 gene revealed GAS and ISRE elements as well as CCAAT enhancer-binding protein (CEBP-β) sequences located within the 1.3 Kb upstream of the transcription start site ( Figure 12C). Nuclear content of STAT1 and IRF1 was significantly increased following cotreatment of IFN-γ and TNF-α, compared with treatment with either cytokine in Hela cells. TNF-α alone or in combination with IFN-γ induced C/EBP-β in Hela cells (47,48). Gene promoter-luciferase reporter assays and electrophoretic mobility shift assay (EMSA) confirmed the role of these regulatory elements in mediating the synergistic activation of human IDO1 in response to IFN-γ and TNF-α (47,48).

Visualization of Protein interaction network of IDO1 in STRING database
Protein interactions mediate post-translational modifications such as phosphorylation that activate or inhibit signaling and provide novel insights into the biological functions. These interactions can be visualized as a graph using the information in databases such as BIOGRID and STRING (28). Protein interactions of IDO1 interrogated in the STRING database revealed extensive connections to enzymes involved in tryptophan metabolism such as tryptophan 2,3-dioxygenase 2 (TDO2), neurotransmitter synthesis such as Monoamine oxidase A (MAOA), and Dopamine decarboxylase (DDC) as well as cytochrome P450 enzymes CYP1A1 and CYP1B1 involved in drug metabolism ( Figure 13A). MAOA is located in the outer mitochondrial membrane and catalyzes the oxidative deamination of biogenic and xenobiotic amines (49). DDC, also known as aromatic-L-amino acid decarboxylase catalyzes the decarboxylation of L3,4-dihydroxyphenylalanine (L-DOPA) to dopamine (50). Human protein atlas (HPA) cell-specific RNA data revealed the co-expression of MAOA, DDC, CYP1A1, CYP1B1 enzymes with IDO1 in club cells revealing a potential for functional interactions (data not shown). Furthermore, IDO1 interacts with Suppressors of cytokine signaling 3 (SOCS3), STAT1, and STAT3 ( Figure 13B).
These interactions may play a major role in the transcriptional regulation of interferon and interleukin signaling (51-53).

Regulation of IDO1 in Healthy and COVID-19 patients
The innate immune response to a pathogenic virus infection must be controlled precisely and any imbalance in cytokine response may result in lung injury and death (3,54). Previous studies have revealed differential regulation of several AT2-specific genes such as surfactant proteins (SFTPA1, SFTPA2, SFTPB, and SFTPC), and transport proteins (LAMP3 and SLC34A2) in the COVID-19 lung tissues (55).
Interrogation of microarray datasets revealed that AT1 markers such as Advanced glycosylation endproduct specific receptor (AGER) and Epithelial membrane protein 2 (EMP2), ciliate cell marker Sentan, ciliary apical structure protein (SNTN), and club cell marker Secretoglobin family 1 member1 (SCGB1A1) were differentially regulated in the lung tissue of healthy and COVID-19 patients ( Figure   14A). It is interesting to note that many of these proteins were identified as secreted proteins (surfactant proteins, SCGB1A1) or located in the plasma membrane (SLC34A2, LAMP3, EMP2, and AGER) suggesting the possibility of extracellular interactions between the proteins leading to altered biological functions in COVID-19 patients ( Figure 14B). Furthermore, RNA levels of IDO1 and enzymes highly expressed in the club cells such as Aldehyde dehydrogenase 3 family member A1 (ALDH3A1) and Glutathione peroxidase 2 (GPX2) were significantly increased in the lung tissue of COVID-19 patients in comparison with healthy controls ( Figure 14A). These results are consistent with the well-known role of club cells in drug metabolism and detoxification (30). A mix of proinflammatory cytokines such as IFNγ, TNF-α, and IL-1β induced a dramatic increase in cytokines and chemokines in the lung epithelial cells in cell-culture (46). The cytokine mix-induced gene expression patterns were similar to the transcriptomic changes in the lung tissue of COVID-19 patients ( Figure 15A and 15B

Conclusion
Metabolic profiling of serum samples from COVID-19 patients revealed alterations of cholesterol and tryptophan metabolic pathways (11,12). Interferon-stimulated genes (ISG) encoding catabolic enzymes such as IDO1, CH25H, SAT1, and SAMHD1 deplete the metabolic building blocks such as essential amino acid, lipid, polyamine, and nucleotide pools to restrict viral replication and multiplication (32). Furthermore, gene expression profiling of nasal swabs revealed that mild or recovered COVID -19 has a robust type I interferon response in the nasopharynx than critically ill patients (63). These results suggest that type I interferon signaling is important for innate immunity to SARS-CoV-2 infection and of diagnostic value for predicting the progression of the disease. However, the efficacy of antiviral drugs such as remidisvir, hydroxychloroquine, and interferon for the treatment of COVID-19 patients remains controversial (64). In contrast, treatment with anti-inflammatory dexamethasone has proven to be therapeutically beneficial to COVID-19 patients (65). The emergence of SARS-CoV-2 variants such as delta with increased viral replication and pathogenicity in the human populations is of major concern.

IDO1 was induced by interferons and SARS-CoV
There is a need to develop novel strategies to down-regulate the inflammatory response in COVID-19 patients. IDO1 inhibitors are in clinical trials either as monotherapy or in combination with other therapies for cancer treatment (66,67). Whether the IDO1 inhibitor treatment alone or in combination with dexamethasone to dampen inflammation has any beneficial effects in COVID-19 patients remains to be determined.