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. Author manuscript; available in PMC: 2018 Dec 1.
Published in final edited form as: Virology. 2017 Sep 28;512:144–150. doi: 10.1016/j.virol.2017.09.007

Protective role of Indoleamine 2,3 dioxygenase in Respiratory Syncytial Virus associated immune response in airway epithelial cells

Devi Rajan 1, Raghavan Chinnadurai 2, Evan O Keefe 1, Seyhan Boyoglu-Barnum 3, Sean O Todd 1, Tina V Hartert 4, Jacques Galipeau 2, Larry J Anderson 1,*
PMCID: PMC5653408  NIHMSID: NIHMS909373  PMID: 28963880

Abstract

RSV is a major cause of severe lower respiratory infection in infants and young children. With no vaccine yet available, it is important to clarify mechanisms of disease pathogenesis. Since indoleamine-2,3-dioxygenase (IDO) is an immunomodulatory enzyme and is upregulated with RSV infection, we studied it in vivo during infection of BALB/c mice and in vitro in A549 cells. RSV infection upregulated IDO transcripts in vivo and in vitro. IDO siRNA decreased IDO transcripts ~2 fold compared to control siRNA after RSV infection but this decrease did not affect RSV replication. In the presence of IFN-γ, siRNA-induced decrease in IDO expression was associated with an increase in virus replication and increased levels of IL-6, IL-8, CXCL10 and CCL4. Thus, our results show IDO is upregulated with RSV infection and this upregulation likely participates with IFN-γ in inhibition of virus replication and suppression of some host cell responses to infection.

Keywords: Respiratory Syncytial Virus, IDO, Cytokine, Chemokine, Tryptophan

Introduction

Respiratory syncytial virus (RSV) belongs to the genus Pneumovirus in the family Paramyxoviridae. It is an enveloped negative sense single stranded RNA virus. RSV is a major cause of severe lower respiratory infection including bronchiolitis and pneumonia in infants and young children. Most infants are infected before 1 year of age, and almost everyone gets infected by 2 years of age. In addition, RSV causes respiratory disease in persons with compromised cardiac, pulmonary, or immune systems and in the elderly [1,2,3,4]. It is estimated that RSV infection leads to as many as 170,000 hospitalizations and 2.1 million outpatient visits each year in the United States among children younger than 5 years old [3,5] and 177,000 hospitalizations and 14,000 deaths among adults older than 65 years [4]. RSV infection in infancy has also been associated with later development of reactive airway disease and asthma. The extent of RSV disease has made it a high priority for vaccine develop but neither a vaccine or highly effective treatment is yet available [3,4,5,6,7,8,9,10].

Understanding the pathogenesis of RSV disease provides the foundation for developing new approaches to vaccines and anti-viral drugs. Recent reports show that RSV induces Indoleamine-2,3-dioxygenase (IDO) in dendritic cells (DCs) [11] and Mesenchymal Stromal Cells (MSCs) [12] and there is a direct correlation between RSV replication and IDO expression [11]. Since IDO is an immunomodulatory enzyme that has been shown to affect a number of immune responses including some linked to RSV [11,12], it might participate in the pathogenesis of RSV disease. IDO catalyzes the degradation of the essential amino acid L-tryptophan and generates a family of catabolites known as kynurenines. It is expressed in various cell types including activated macrophages and other immunoregulatory cells [13] and reported to play a role in a variety of pathophysiological processes such as antimicrobial and antitumor defense, neuropathology, immunoregulation, and antioxidant activity [14,15,16,17,18,19,20,21,22,23]. For example, IDO has been reported to cause apoptosis of Th-1 but not Th-2 lymphocytes [24,25] and Th2- type responses have been associated with RSV disease. A number of viruses, bacteria, parasites, and various cytokines including IFN-γ induce IDO. Given the earlier studies of RSV and IDO, RSV’s immune regulatory effects, and the breadth of IDOs immune regulatory effects, we sought to investigate IDO in RSV infection in the BALB/c mice and human airway epithelial cells (A549). Airway epithelial cells are the primary site for RSV human infection.

Material and Methods

Animal studies

Animal studies were performed according to a protocol approved by the Emory University (Atlanta, GA) Institutional Animal Care and Use Committee. Four- to six-week-old, specific-pathogen-free female BALB/c mice (Charles River Laboratory, Wilmington, MA) were housed in microisolator cages, fed sterilized water and food ad libitum and challenged intranasally with 1 × 106 PFU of RSV A2 or r19F in serum-free minimal essential medium (MEM) (volume, 50 μl). Lung specimens were collected at different days post infection and stored in RNA lyzol at −80° freezer until use. Before use, lung specimens were thawed and homogenized using minibead beater (Biospec Products, Bartlesville, OK).

Human airway epithelial cells and viruses

A549 cells were obtained from American Type Culture Collection (ATCC) and grown in minimum essential medium (MEM) supplemented with 10% fetal calf serum (FCS), 1mM L-Glutamine and 1× penicillin streptomycin. RSV A2 strain was provided by A.G.P.Oomens [26] and A2 recombinant 19F strain (r19F) by M Moore [27]. The RSV A2 strain is widely used in RSV studies. We included the r19F strain because it induces different features of disease in mice that might be related to IDO [27]. For virus preparation, HEp-2 cells were infected with 0.01MOI of RSV A2 and five days later, media was collected and centrifuged briefly to remove the cellular debris, and the clarified supernatant was purified by centrifugation through a 20% sucrose cushion at 14,000g for 2 hours. The infectivity titer was determined in 96 well flat bottom tissue culture plates with ~5000 HEp-2 cells/well and 10-fold serial dilutions of virus in 8 replicates. The infected cells were incubated at 370C for 5 days and replication of virus determined by an RSV enzyme-linked immunosorbent assay (ELISA) and tissue culture infectivity dose (TCID50) calculated by the Reed Muench method [28].

RT-PCR

Supernatant and cell pellet collected from infected AECs were briefly centrifuged to remove the cell debris. Total RNA was extracted from supernatant and cells using Qiagen RNEasy mini kit according to manufacturer’s instructions. RSV RNA was assayed by a real-time RT-PCR assay using AgPath-ID™ One-Step RT-PCR Reagents and the Applied Biosystems 7500 Fast Real-Time PCR System (Life Technologies Corporation, Carlsbad, CA). The primers and probes for the RSV matrix (M) gene (forward primer, 5′-GGC AAA TAT GGA AAC ATA CGT GAA-3′; reverse primer, 5′-TCT TTT TCT AGG ACA TTG TAY TGA ACA G-3′; probe, 5′-6-carboxyfluorescein (FAM)-TGT CCG TCT TCT ACG CCC TCG TC- black hole quencher 1 (BHQ-1)-3′) were obtained from Integrated DNA Technologies (IDT) (Coralville, IA). [29]. Infected AECs were lysed and total RNA was extracted using an RNeasy plus mini kit (QIAGEN). Normalized RNA was used to convert cDNA using Quantitect reverse transcription kit (QIAGEN). SYBR green (Perfecta Sybr green fast mix, Quanta Biosciences) real-time PCR was performed with primer pairs for IDO and other tryptophan enzymes [30]. For animal studies, RNA was extracted from lung homogenates and RSV RNA was assayed by a real-time RT-PCR.

SiRNA Knock down

A549 cells were seeded in 96-well plate at a concentration of 5000 cells/well 1 day prior to transfection with ON-TARGETplus Human IDO1 (3620) siRNA-SMARTpool and ON-TARGETplus Non-targeting pool from Dharmacon GE. During transfection, the cells were conditioned with serum-free 10 mM HEPES containing α-MEM for 30 min. 2 μl of 100 μM siRNA solution and 4 μl DharmaFECT 1 reagent was added to 250 μl α-MEM containing 10 mM HEPES. The siRNA solution and the DharmaFECT reagent were mixed and incubated at room temperature for 30 min. 50 μl of the siRNA transfection mixture was added to each well. The cells were then incubated for 5 h followed by replacement of the transfection medium with A549 culture medium.

Cytokine assays

Multiplex luminex assays were performed from the collected supernatant according to the manufacturer’s instructions for IFN–γ, IL–12 (p40/p70) IL–13, RANTES, MIP–1α, MIG, MIP– 1β, IL–1β, IL–2, IL–4, IL–5, IL–6, IL–2R, MCP–1, Eotaxin, IL–8, IL–10, IL–15, IL–17, IL– 1RA, GM–CSF,, TNF–α, IL–7, IP–10, IFN–α (Human cytokine 30-plex panel, Life technologies) using Luminex × MAP technology.

Statistical Analysis

Specimens were tested in duplicate and experiments were performed at least two times. Statistical analysis was performed using GraphPad Prism 5.0 software. P < .05 was considered statistically significant based on Mann Whitney 2-tailed Student t tests.

Results and Discussion

To determine whether RSV induces IDO, A549 cells were seeded in 24 well plate and RSV A2 was inoculated at a concentration of 0.02, 0.2 and 2 MOI. Three days post infection, the supernatant and cell pellet were harvested and RSV replication was determined in the supernatant. RT-PCR results show an increase in viral RNA with increasing inoculum of A2 (Fig 1A). IDO mRNA levels were measured in infected A549 cell pellets by quantitative SYBR Green real-time PCR. Our results show an increase in IDO expression that corresponded to the level of RSV replication in A549 cells (Fig 1B). Next, to determine if the induction of IDO might contribute to strain differences in disease, we also infected A549 cells with RSV r19F. r19F unlike A2 induces lung mucous secretion and increased airway resistance, two features of RSV disease in humans, in mice [27]. Fig 1C shows that r19F also induces IDO in A549 cells in a fashion similar to A2. To determine if RSV infection in vivo is associated with induction of IDO, we tested lung homogenates of BALB/c mice three, five and eight days after RSV challenge. Lung specimens were thawed and homogenized using minibead beater (Biospec Products, Bartlesville, OK) before use. IDO PCR showed that both A2 and r19F induced IDO to similar levels in mouse lung homogenate eight days post challenge (Fig 1D) but not at three and five days post challenge (data not shown).

Fig 1. RSV induces IDO in vitro and in vivo.

Fig 1

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(A) The relative amount of viral RNA detected in supernatants of A549 cells infected with RSV A2 in real time (RT) PCR and represented as inverse cycle threshold (Ct) values. Ct levels reflect the number of cycles required to exceed the background level; inverse Ct levels (1/Ct) are proportional to the amount of target nucleic acid in the sample. RT-PCR underwent 40 cycles of amplification. The data are represented as average ± SD from a representative experiment. (B) A549 cells were infected with indicated MOI of RSV A2 for 72 h. RNA was extracted from the cells and the expression levels of IDO mRNA were quantitated by quantitative SYBR Green real-time PCR. GAPDH mRNA levels were used as internal controls. The ΔΔCt method was applied to calculate the fold change. (C) IDO expression using two different RSV strains is shown. (D) IDO expression in lung tissue homogenate of BALB/c mice challenged with RSV A2 or 19F is shown.

Next, to determine if RSV A2 modulates other tryptophan catabolites in kynurenine pathway (Fig 2A), we assessed levels of mRNA associated with various tryptophan degrading enzymes including Kynurenine Amino Transferases (KAT1, 2, 3 and 4,) 3-Hydroxyanthranilate 3,4-dioxygenase (HAAO), Kynureninase (KYNU), Kynurenine-3-MonoOxygenase (KMO), Quinolinate Phospho Ribosyl Transferase (QPRT), Tryptophan 2,3-Dioxygenase (TDO) by real time PCR. We looked at these enzymes since it is known that these enzymes catalyze different steps of kynurenine pathway and produce metabolites collectively referred to as kynurenine metabolites with various effects including regulation of immune responses [31]. RT PCR results at day three post A2 virus infection of the AECs showed that A2 induced IDO while downregulating KAT1 (kynurenine aminotransferases) and QPRT (quinolinate phosphoribosyl transferase) dose dependently as seen by the increase of CT values normalized to GAPDH internal control CT values (Fig 2B). These enzymes act downstream of IDO in the kynurenine enzyme pathway. Kynurenine formed from the first step is a branch point with different outcomes (Fig 2A). Interestingly, other enzymes including KAT2, 3, 4, HAAO, KYNU, KMO and TDO were not affected by RSV infection. Thus, our result indicates that RSV upregulates IDO which might lead to kynurenine accumulation. The down regulation of the other two enzymes would decrease catabolism of kynurenine to kynurenine metabolites and presumably would also lead to accumulation of kynurenine.

Fig 2. RSV induce and inhibit tryptophan degrading enzyme expression in A549 cells.

Fig 2

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(A) Steps in Kynurenine pathway (modified from Ref 30). (B) Representative heat map with CT values (red = high expression, blue = low expression). A549 cells were infected with indicated MOI of RSV A2 for 72 h. RNA was extracted from the cells and the expression levels of different tryptophan degrading enzyme mRNA were quantitated by quantitative SYBR Green real-time PCR.

To better define the role of IDO on the RSV infection of AEC, we used siRNA to knock down IDO expression. In these experiments, we transfected A549 cells with IDO siRNA or Control siRNA using Dharmafect 1 (Dharmacon, Lafayette, CO) transfection reagent. Transfected cells were incubated for 5 h followed by replacement of the transfection medium with A549 culture medium. 24 hours post-transfection, the cells were infected with 1MOI of RSV for 2 hours followed by addition of fresh A549 medium. Three days post infection, cells and supernatants were harvested, and IDO mRNA levels measured. We found that RSV infection of A549 cells transfected with IDO siRNA, produced significantly less IDO (~2 fold) than cells transfected with control siRNA (Fig 3A) but had similar levels of virus replication as indicated by levels of RSV RNA detected by RT PCR of cell supernatants (Fig 3B).

Fig 3. IDO do not modulate RSV replication in resting cells but restricts replication in IFN-γ treated A549 cells.

Fig 3

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(A) A549 cells were transfected with control and IDO siRNA for 12 hours followed by infection with 1MOI of RSV A2. RNA was extracted from the cells 72h post infection and the expression level of IDO mRNA was quantitated by quantitative SYBR Green real-time PCR. GAPDH mRNA levels were used as internal controls. The ΔΔCt method was applied to calculate the fold change. (B) The relative amount of viral RNA was detected in supernatants of transfected/infected A549 cells in real time (RT) PCR and represented as inverse cycle threshold (Ct) values. The data are represented as average ± SD from two independent experiments that were performed in duplicates. (C) A549 cells were treated with 25ng/ml IFN-γ followed by inoculation with 1MOI of RSV A2. RNA was extracted from the supernatant and the relative amount of viral RNA was detected in real time (RT) PCR. (D) IDO mRNA expression was detected from the cells that were harvested three days post infection. (E) RT PCR showing viral RNA in control and IDO knock out A549 cells that were treated with IFN-γ. Cumulative data are shown from two independent experiments that were performed in duplicates.

It has been previously reported that IFN-γ inhibits virus replication and inflammatory responses [32,33]. Specifically, exposure of A549 to IFN-γ for 48 h prior to RSV infection reduced viral titers at early time points [34]. This reduction was associated with the upregulation of antiviral proteins, such as IFIT1and Mx1 [34]. High levels of IFN-γ exposure prior to RSV infection have also been shown to inhibit RSV replication in adult BALB/c mice [35]. Though IFN-γ is largely considered a proinflammatory cytokine [36] it has also been reported to have anti-inflammatory effects [37,38,39]. Since IFN-γ is also a potent inducer of IDO [40,41,42], we wondered if some of the IFN-γ effects on RSV infection might be mediated by induction of IDO. To investigate this possibility, we treated cells with IFN-γ and looked at IDO expression and virus replication. Fig 3C shows that as expected IFN-γ treatment decreased virus replication in A549 cells (P<0.05). Interestingly, significantly higher levels (~6 fold) of IDO were induced by infection in IFN-γ treated compared to untreated cells (Fig 3D) suggesting IFN-γ facilitated upregulation of IDO by RSV. To confirm IDO’s role in IFN-γ’s effects on RSV infected A549 cells, we studied cells transfected with control siRNA and IDO siRNA. In these experiments, cells were transfected as noted above and 24 hours later infected with RSV. Two hours later fresh media containing 25ng/ml IFN-γ was added to the transfected/infected cells and the supernatant was harvested at 3 days post infection. RSV RNA was determined in the collected supernatant by performing RT-PCR and cytokine and chemokine levels were analyzed by multiplex luminex assay. RT-PCR results shows an increase (P<0.05) in virus RNA in IDO knock down cells when compared to the control knock down cells after IFN-γ treatment (Fig 3E). This shows that IDO contributes to the restriction of RSV replication associated with IFN-γ treatment.

Since IFN-γ also effects the cytokine/chemokine response to RSV infection, we next looked at IDOs effect on a variety of chemokines and cytokines in RSV infected cells. It has previously been reported that pro-inflammatory cytokines such as IL-6 and TNF-α are present in the bronchoalveolar lavage (BAL) fluid from infants with RSV bronchiolitis [43] as well as CXCL10 (IP-10), CXCL8 (IL-8), CCL2 (MCP1), CCL3 (MIP-1α), and CCL5 (RANTES)[44].

As noted in earlier studies, a number of chemokines and cytokines were induced by RSV infection of airway epithelial cells (Table 1) including IL-1β, IL-6, IL-12, RANTES, CCL3, CCL4, MCP-1, IL-15, IFN-γ, IL-1RA, CXCL10 and IL-2R. Most of the RSV-specific increases in cytokines and chemokines were unaffected by IDO siRNA. However, RSV-specific levels of CXCL10 (P<0.05) increased in IDO siRNA treated cells (Table 1). The addition of IFN-γ increased levels of IL-6, CCL4, MCP-1, IL-15, IL1RA, CXCL10, IL2R, MIG and IL-8. Presence of IDO depressed this increase for IL-6 (p=0.0009), IL-8 (p=0.0009), CCL4 (p=0.002) and CXCL10 (p=0.04) (Fig. 4A, B, C and D). RSV infection of human respiratory epithelial cell lines is known to induce transcription-dependent secretion of the C-X-C chemokine IL-8 [45,46] and of the C-C chemokine RANTES, but not that of other C-C chemokines, monocyte chemotactic protein-1 or -3 or CCL4 [47,48].

Table 1.

Cytokine response upon exposure of RSV to IDO and Control knock down A549 Cells with or without IFN-γ treatment

Cytokine/
Chemokine		 −IFNγ +IFNγ
Control KO IDO KO Control KO IDO KO
0 0.2 2 0 0.2 2 0 0.2 2 0 0.2 2
IL-1b 8±3 8±3 23±11 8±3 8±3 23±5 8±2 11±6 38±5 6±3 17±9 42±7
IL-10 4±1 4±1 4±1 4±1 4±1 4±1 4±1 4±1 3±2 4±1 4±1 4±2
IL-13 11±5 12±6 12±6 11±4 10±4 13±5 12±2 11±3 14±5 11±3 12±2 16±3
IL-6 4±1 10±2 80±33 5±1 8±1 112±4 13±2 24±8 286±97 9±1 119±19 1445±253
IL-12 10±0 13±7 80±65 10±0 14±7 87±61 10±2 18±6 95±63 10±1 17±9 93±54
RANTES 20±7 197±51 1748±911 20±7 206±77 1760±716 9±8 337±215 1958±882 20±7 394±258 1855±898
Eotaxin 3±2 3±2 4±2 3±2 2±2 5±2 2±2 2±1 6±3 3±2 3±2 6±4
IL-17 26±1 20±12 14±14 26±1 26±1 14±14 26±1 26±1 14±14 26±1 26±1 20±12
CCL3 24±1 25±9 125±36 18±6 29±7 140±33 23±7 34±12 134±49 22±7 35±20 185±95
GM-CSF 4±2 4±2 5±2 4±3 4±3 4±2 4±2 4±2 5±2 4±2 4±2 3±3
CCL4 10±0 10±0 87±36 10±0 13±7 101±6 11±3 29±22 132±44 10±0 42±39 338±100
MCP-1 450±22 466±36 797±285 481±98 420±60 893±188 1561±930 1560±1035 3635±2829 1462±963 1774±1523 6422±5863
IL-15 52±8 32±19 144±68 45±11 49±7 140±26 47±13 62±27 284±147 49±7 72±35 338±142
IL-5 5±4 5±3 4±3 4±4 5±3 5±3 5±2 5±3 6±2 5±3 5±2 7±1
IFN-γ 3±2 11±4 107±62 7±3 10±5 114±53 1538±994 1534±825 2586±1941 1332±730 1408±1057 1990±1701
IFN-α 31±25 30±19 78±43 31±25 27±18 78±34 35±21 45±9 91±28 33±16 45±6 100±26
IL-1RA 61±11 172±140 1777±1986 70±48 128±88 1637±1854 61±11 136±97 2313±2548 79±42 143±107 2316±2559
TNF-α 6±3 6±3 17±12 5±3 6±3 16±6 5±3 5±2 23±9 6±3 7±1 30±6
IL-2 13±0 13±0 11±2 13±0 13±0 10±3 13±0 10±6 12±1 13±0 13±0 12±5
IL-7 17±7 17±7 17±7 17±7 17±7 17±7 17±7 17±7 23±9 17±7 17±7 39±18
CXCL1 4±1 23±8 279±12 4±1 23±1 426±19 31±12 340±26 3553±7 46±28 458±39 5303±1
0 7 1 2 8 48 9 761
IL-2R 29±3 23±15 192±63 29±3 27±5 209±76 29±3 67±63 256±79 29±3 88±73 265±102
MIG 34±24 34±24 34±24 34±24 34±24 34±24 34±24 34±24 206±170 34±24 34±24 237±177
IL-4 32±2 32±2 43±11 31±5 32±2 42±9 32±2 32±2 45±12 32±2 32±2 46±14
IL-8 1012±542 980±504 1441±1031 1045±404 838±246 1437±336 404±117 351±30 1126±524 452±88 667±24 6255±314
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Grey highlight Indicates significantly higher from control (P<0.05)

Fig 4. A, B, C, D.

Fig 4

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Levels of cytokines and chemokines (pg/ml) in the supernatant of control and IDO knock down A549 cells and RSV infection with or without IFN-γ treatment. Data are average ±SD from duplicate infection from two independent experiments.

Our results show that IDO is induced by RSV infection in vivo in BALB/c mice and in vitro in the human AEC line, A549. More importantly, it appears to contribute to IFN-γ associated effects on the response of A549 cells to RSV infection. We found that IDO contributed to INF-γ’s inhibition of virus replication and decreased INF-γ induced upregulation of IL-6, IL-8, CCL4 and CXCL10. IDO also decreased infection induced CXCL10 levels not associated with IFN-γ. Altogether, our results suggest that IDO and IFN-γ are linked in their effects on virus replication and host cell response to infection with IDO contributing to IFN-γ associated inhibition of virus replication and inhibiting INF-γ induction of some inflammatory cytokine/chemokine responses.

Highlights.

  • IDO is induced by RSV infection in vivo in BALB/c mice and in vitro in the human AEC line, A549.

  • IDO siRNA decreased IDO transcripts ~2 fold compared to control siRNA after RSV infection but this decrease did not affect RSV replication in A549 cells.

  • In the presence of IFN-γ, the siRNA induced decrease in IDO expression was associated with an increase in virus replication as well as increased levels of IL-6, IL-8, CXCL10 and CCL4.

Acknowledgments

Funding information

This work was supported by NIH 1U19AI095227 and support from the Immunology Core of Emory+Children’s Pediatric Research Center.

Footnotes

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Conflict of interest

The authors declare that there are no conflicts of interest.

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