IDO1 promotes CSFV replication by mediating tryptophan metabolism to inhibit NF-κB signaling

ABSTRACT

Tryptophan metabolism plays a crucial role in facilitating various cellular processes essential for maintaining normal cellular function. Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the conversion of tryptophan (Trp) into kynurenine (Kyn), thereby initiating the degradation of Trp. The resulting Kyn metabolites have been implicated in the modulation of immune responses. Currently, the role of IDO1-mediated tryptophan metabolism in the process of viral infection remains relatively unknown. In this study, we discovered that classical swine fever virus (CSFV) infection of PK-15 cells can induce the expression of IDO1, thereby promoting tryptophan metabolism. IDO1 can negatively regulate the NF-κB signaling by mediating tryptophan metabolism, thereby facilitating CSFV replication. We found that silencing the IDO1 gene enhances the expression of IFN-α, IFN-β, and IL-6 by activating the NF-κB signaling pathway. Furthermore, our observations indicate that both silencing the IDO1 gene and administering exogenous tryptophan can inhibit CSFV replication by counteracting the cellular autophagy induced by Rapamycin. This study reveals a novel mechanism of IDO1-mediated tryptophan metabolism in CSFV infection, providing new insights and a theoretical basis for the treatment and control of CSFV.

IMPORTANCE

It is well known that due to the widespread use of vaccines, the prevalence of classical swine fever (CSF) is shifting towards atypical and invisible infections. CSF can disrupt host metabolism, leading to persistent immune suppression in the host and causing significant harm when co-infected with other diseases. Changes in the host’s metabolic profiles, such as increased catabolic metabolism of amino acids and the production of immunoregulatory metabolites and their derivatives, can also influence virus replication. Mammals utilize various pathways to modulate immune responses through amino acid utilization, including increased catabolic metabolism of amino acids and the production of immunoregulatory metabolites and their derivatives, thereby limiting viral replication. Therefore, this study proposes that targeting the modulation of tryptophan metabolism may represent an effective approach to control the progression of CSF.

INTRODUCTION

Classical swine fever (CSF), caused by the classical swine fever virus (CSFV), is a severe and highly contagious viral disease in pigs. Clinical manifestations of CSF include high fever, hemorrhage, and seizures, leading to significant economic losses in the global swine industry (1–3). CSFV belongs to the Pestivirus genus in the Flaviviridae family and is a single-stranded RNA virus with a genome length of 12.3 kb (4, 5). The CSFV genome contains a long open reading frame that encodes a polyprotein of 3,989 amino acids, which is processed to generate four structural proteins (C, Erns, E1, and E2) and eight non-structural proteins (NPro, P7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (6, 7).

Many viruses have the ability to hijack host metabolism and utilize host energy and metabolites to meet their own energy and biosynthetic demands. Numerous studies have indicated that CSFV can sustain its replication by altering cellular metabolism. For instance, CSFV modulates glycolysis by hijacking lactate dehydrogenase B and regulates NF-κB signaling through mitochondrial autophagy (8). Additionally, CSFV can enhance the entry of free fatty acids into mitochondria for β-oxidation and ATP production to acquire energy (9). However, there is still limited research on the interplay between CSFV infection and amino acid metabolism.

L-Trp, an indispensable amino acid for mammals, plays a pivotal role in multiple facets, encompassing the regulation of animal mood, maintenance of body immunity, as well as cell activation and proliferation (10). Upon entry into the mammalian organism, L-Trp primarily undergoes metabolism via the kynurenine (Kyn) pathway, wherein IDO1 serves as the principal rate-limiting enzyme (11). The metabolic processes of tryptophan not only supply the essential nutrients for normal cellular activities but also assume an irreplaceable role in governing immunity and inflammation regulation (12). When the organism encounters certain stimuli like viral infection, IDO1 expression is markedly upregulated, leading to the depletion of L-Trp within the cytoplasm and the generation of numerous metabolites, including Kyn and Kynurenic acid (13). Among these metabolites, Kyn is capable of binding to and activating an immunosuppressive transcription factor, the aromatic hydrocarbon receptor (AhR), thereby perpetuating the state of immunosuppression and attenuating the host’s immune response (14). Kyn was shown to be a pro-inflammatory metabolite. In cultured mouse astrocytes in vitro, Kyn treatment upregulates NLRP2 transcription through an NF-κB-dependent pathway. Intraperitoneal injection of Kyn activates NLRP2 inflammasome in mouse hippocampal astrocytes (15). Furthermore, according to the analysis of transcriptomic data from IDO1-deficient mice, the lack of IDO1 significantly downregulated pathways involved in TLR and NF-κB signaling (16). And IDO1 inhibition could significantly promote vascular inflammation in mice (17).

The transcription factor Nuclear Factor Kappa B (NF-κB) plays a crucial role in promoting the production of various cellular factors, including type I interferons, which are closely associated with inflammatory responses and innate immunity (18, 19). Previous studies have demonstrated that CSFV infection can inhibit the NF-κB signaling pathway to enhance its own replication (20, 21). However, the reciprocal regulation between tryptophan metabolism and the NF-κB pathway, as well as their respective roles in CSFV infection, remains poorly elucidated.

Amino acid metabolism serves as a key regulator of various cellular processes in addition to its role in immune responses (22). Mammalian target of rapamycin (mTOR) acts as a central sensor for multiple autophagy signals, including inflammation and amino acid starvation. Deprivation of amino acids leads to the inhibition of mTOR activation, thereby alleviating its suppression on downstream autophagic processes (23, 24). In addition, the autophagic process plays an indispensable role in the infection process of CSFV. Previous studies conducted in our laboratory have demonstrated that autophagy significantly enhances the in vitro replication of CSFV (25, 26).

Therefore, in this study, we investigated the relationship between CSFV and host IDO1-mediated tryptophan metabolism. Our results indicate that CSFV can promote tryptophan metabolism by activating the expression of IDO1, and the activation of IDO1 can inhibit the NF-κB pathway by mediating tryptophan metabolism, thereby favoring viral replication. Furthermore, silencing of IDO1 gene function can enhance the production of type I interferon and IL-6 by activating NF-κB. Furthermore, our results reveal that both silencing of IDO1 gene function and exogenous supplementation of tryptophan can effectively suppress CSFV replication by antagonizing RAPA-induced cellular autophagy.

RESULTS

CSFV promotes tryptophan metabolism through activation of IDO1

Our previous metabolomics study revealed alterations in L-Trp levels upon CSFV infection in porcine PK-15 cells (27). To further elucidate the alterations in tryptophan metabolism following CSFV infection, we infected PK-15 cells with CSFV and measured the levels of L-Trp and its metabolite L-Kyn in the cell culture supernatant. Our results showed that compared to the uninfected group, CSFV infection led to a decrease in L-Trp levels and an increase in L-Kyn levels in the cell culture supernatant (Fig. 1A and B). IDO1 exhibits widespread expression in various mammalian tissues and organs, serving as the principal rate-limiting enzyme for catalyzing the metabolic conversion of Trp along the kynurenine pathway outside the liver. Therefore, we examined the impact of CSFV infection on the expression of IDO1 in PK-15 cells. Our results demonstrated that CSFV infection upregulated both mRNA and protein expression of IDO1 in PK-15 cells (Fig. 1C and D). To investigate whether the promotion of tryptophan metabolism during CSFV infection is mediated by the activation of IDO1, we examined the effects of IDO1 gene silencing on the levels of L-Trp and its metabolite L-Kyn during CSFV infection. The results showed that IDO1 gene silencing alleviated the decrease in L-Trp levels and the increase in L-Kyn levels induced by CSFV infection (Fig. 1E and F). These findings suggest that CSFV infection can promote tryptophan metabolism through the in vitro activation of IDO1.

Fig 1.

CSFV promotes tryptophan metabolism through activation of IDO1. (A) PK-15 cells were infected with CSFV (MOI = 1), and the concentration of L-Trp in the culture supernatant was determined by enzyme-linked immunosorbent assay at 48 h. (B) PK-15 cells were infected with CSFV (MOI = 1), and the concentration of L-Kyn in the culture supernatant was determined by enzyme-linked immunosorbent assay at 48 h. (**P < 0.01 and ***P < 0.001 calculated using t-test). (C) IDO1 mRNA relative levels in PK-15 cells were analyzed by quantitative real-time PCR (qRT-PCR). (D) Western blot showing IDO1 protein expression (*P < 0.05, **P < 0.01, and ***P < 0.001 calculated using two-way ANOVA). (E) PK-15 cells were transfected with IDO1-interfering RNA before infection with CSFV (MOI = 1), and the concentration of L-Trp in the culture supernatant was determined by enzyme-linked immunosorbent assay at 48 h. (F) PK-15 cells were transfected with IDO1-interfering RNA before infection with CSFV (MOI = 1), and the concentration of L-Kyn in the culture supernatant was determined by enzyme-linked immunosorbent assay at 48 h. (**P < 0.01 calculated using t-test). (G) PK-15 cells were infected with CSFV (MOI = 1), and the concentration of L-Trp in the culture supernatant was determined by enzyme-linked immunosorbent assay at 48 h. (H) PK-15 cells were infected with CSFV (MOI = 1), and the concentration of L-Kyn in the culture supernatant was determined by enzyme-linked immunosorbent assay at 48 h (*P < 0.05 and **P < 0.01 calculated using two-way ANOVA. ns, not significant).

Additionally, to determine whether the addition of L-TRP can elevate the levels of L-KYN in the culture medium, and to investigate the potential existence of a negative feedback regulatory mechanism between L-KYN and L-TRP, we supplemented standard DMEM culture medium with 1 mM L-Kyn and 1.6 mM L-Trp, respectively. Subsequently, we assessed the levels of L-TRP and L-KYN in the culture supernatant individually. The results indicate that, compared to the control group, L-Kyn does not modulate the levels of L-Trp in the culture supernatant, whereas L-Trp increases the levels of L-Kyn in the culture supernatant (Fig. 1G and H).

Trp inhibits CSFV replication, and Kyn promotes CSFV replication

Research has shown that during viral myocarditis infection, IDO1 can inhibit the production of type I interferon by promoting tryptophan metabolism via the kynurenine pathway, thereby facilitating viral replication in mouse hosts (28).

We aimed to investigate the roles of tryptophan and kynurenine in CSFV infection. PK-15 cells were cultured in a standard DMEM medium supplemented with various concentrations of L-Trp. Cell viability was assessed using the CCK-8 assay to determine the optimal concentration range of L-Trp suitable for addition to PK-15 cells, which was found to be 1.6 mM (Fig. 2A). Subsequently, we added 1.6 mM L-Trp and 1 mM L-Kyn separately to standard DMEM medium to observe their effects on CSFV proliferation. The results indicated that compared to the control group, CSFV proliferation decreased significantly upon L-Trp treatment (Fig. 2B through D). Conversely, the addition of L-Kyn significantly increased CSFV proliferation levels (Fig. 2E through G).

Fig 2.

Trp inhibits CSFV replication and kyn promotes CSFV replication. (A) The effect of different Trp concentration on PK-15 cell viability was detected by CCK8 method using a normal cell-based DMEM medium as a control group. (****P < 0.0001 calculated using t-test. ns, not significant). (B) PK-15 cells were infected with CSFV (MOI = 1), and 1.6 mM L-Trp was added to the cell culture medium. CSFV NS5B copy numbers were determined by qRT-PCR. (C) CSFV virus titers in the supernatant were determined as 50% tissue culture infective doses (TCID50)/mL. (D) Western blot for CSFV E2 protein expression, and the relative protein levels were estimated by histograms representing density readings of the gel bands with Image J, and the ratios were calculated relative to Tubulin control. (E) PK-15 cells were infected with CSFV (MOI = 1), and 1.0 mM L-Kyn was added to the cell culture medium. CSFV NS5B copy numbers were determined by qRT-PCR. (F) CSFV virus titers in the supernatant were determined as 50% tissue culture infective doses (TCID50)/mL. (G) Western blot for CSFV E2 protein expression, and the relative protein levels were estimated by histograms representing density readings of the gel bands with Image J, and the ratios were calculated relative to Tubulin control (**P < 0.01, ***P < 0. 01, and ****P < 0.0001 calculated using two-way ANOVA).

IDO1 facilitates CSFV replication through mediation of tryptophan metabolism

Investigating the role of IDO1 in CSFV infection, we utilized strategies involving both silencing and overexpression of IDO1 to evaluate its impact on CSFV proliferation. Our results demonstrated that functional silencing of the IDO1 gene led to a significant decrease in CSFV proliferation levels (Fig. 3A through C), whereas IDO1 overexpression resulted in a notable increase in CSFV proliferation levels (Fig. 3D through F).

Fig 3.

IDO1 facilitates CSFV replication through the mediation of tryptophan metabolism. (A) PK-15 cells were transfected with IDO1 interfering RNA before infection with CSFV (MOI = 1). CSFV NS5B copy numbers were determined by qRT-PCR. (B) CSFV virus titers in the supernatant were determined as 50% tissue culture infective doses (TCID50)/mL. (C) Western blot for CSFV E2 protein expression, and the relative protein levels were estimated by histograms representing density readings of the gel bands with Image J, and the ratios were calculated relative to Tubulin control. (D) PK-15 cells were transfected with pCAGGS-IDO1 plasmid before infection with CSFV (MOI = 1). CSFV NS5B copy numbers were determined by qRT-PCR. (E) CSFV virus titers in the supernatant were determined as 50% tissue culture infective doses (TCID50)/mL. (F) Western blot for CSFV E2 protein expression, and the relative protein levels were estimated by histograms representing density readings of the gel bands with Image J, and the ratios were calculated relative to Tubulin control. (G) PK-15 cells were transfected with IDO1 interfering RNA prior to infection with CSFV (MOI = 1), and an additional 1.6 mM L-Trp or 1 mM L-Kyn was added to the medium. CSFV NS5B copy numbers were determined by qRT-PCR at 24 h. (H) Western blot for CSFV E2 protein expression, and the relative protein levels were estimated by histograms representing density readings of the gel bands with Image J, and the ratios were calculated relative to Tubulin control. (**P < 0.01, ***P < 0.001, and ****P < 0.0001 calculated using two-way ANOVA. ns, not significant).

To further elucidate whether IDO1 mediates CSFV replication through its enzymatic activity, we silenced the expression of IDO1 and supplemented the cell culture medium with either 1.6 mM L-Trp or 1 mM L-Kyn and subsequently assessed CSFV replication. The results indicated that silencing of the IDO1 gene attenuated the effect of L-Trp on CSFV replication, while the addition of L-Kyn alleviated the inhibitory effect of IDO1 gene silencing on CSFV replication (Fig. 3G and H). These findings suggest that IDO1 promotes CSFV replication by participating in the regulation of tryptophan metabolism.

Given that IDO1 can regulate tryptophan metabolism by degrading L-Trp, and L-Kyn is an important biologically active downstream metabolite, we aimed to evaluate whether IDO1 modulates CSFV replication through this pathway. We silenced the expression of IDO1 and supplemented the cell culture medium with either 1.6 mM L-Trp or 1 mM L-Kyn and then assessed CSFV replication. The results showed that functional silencing of the IDO1 gene attenuated the inhibitory effect of exogenous L-Trp on CSFV replication. Conversely, IDO1 gene silencing did not diminish the stimulatory effect of exogenous L-Kyn on CSFV replication (Fig. 3G and H). These data indicate that IDO1 regulates CSFV replication through downstream metabolites rather than IDO1 itself.

IDO1 promotes CSFV replication by inhibiting NF-κB signaling

Numerous studies have demonstrated that IDO1 participates in various immune processes, including the regulation of innate immunity and autoimmunity. The NF-κB signaling facilitates the release of various cellular factors downstream, including type I interferon, thereby participating in the activation of innate immune responses (29–31). Therefore, we employed IDO1 silencing and IDO1 overexpression approaches to assess their impact on NF-κB pathway proteins. The results revealed that, compared to the control group, IDO1 gene silencing significantly upregulated the expression of NF-κB pathway proteins, including P65, p-P65, IκBα, and p-IκBα proteins (Fig. 4A). Conversely, overexpression of IDO1 led to a significant decrease in the expression of P65, p-P65, IκBα, and p-IκBα proteins (Fig. 4B). Next, we employed confocal microscopy to observe the nuclear translocation of P65. The results revealed that, compared to the control group, IDO1 gene silencing led to an increased presence of red fluorescence in the cell nucleus, indicating enhanced nuclear translocation of P65. These findings collectively suggest that IDO1 can suppress NF-κB signal transduction (Fig. 4C and D).

Fig 4.

IDO1 promotes CSFV replication by inhibiting the NF-κB signaling. (A and B) Western blot for P65, p-P65, IκBα, p-IκBα, and IDO1 expression in IDO1 overexpression or gene silencing PK-15 cells. The relative levels of proteins were estimated by histograms representing the density reading of the gel bands with Image J, and the ratios were calculated relative to tubulin control. (C) Laser confocal detection of P65 nuclear translocation in IDO1 gene silencing PK-15 cells at 24 h. Indirect immunofluorescence detection of P65 (red) was performed with rabbit anti-P65 antibody, and cell nuclei were indicated by DAPI (blue) staining. (D) PK-15 cells were transfected with IDO1 interfering RNA before infection with CSFV (MOI = 1); Laser confocal detection of P65 nuclear translocation in PK-15 cells at 24 h. Indirect immunofluorescence detection of P65 (red) was performed with rabbit anti-P65 antibody, indirect immunofluorescence detection of E2 (green) was performed with mouse anti-E2 antibody, and cell nuclei were indicated by DAPI (blue) staining. (E) PK-15 cells were treated with BAY for 2 h before transfection with IDO1-interfering RNA and then infected with CSFV (MOI = 1), and CSFV NS5B copy numbers were determined by qRT-PCR. (F) Western blot for P65, p-P65, IκBα, p-IκBα, and CSFV E2 protein expression, and the relative protein levels were estimated by histograms representing density readings of the gel bands with Image J, and the ratios were calculated relative to Tubulin control (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 calculated using two-way ANOVA. ns, not significant).

To investigate whether IDO1 can promote CSFV replication by inhibiting NF-κB signaling, we silenced the expression of IDO1 and treated the cells with the specific NF-κB inhibitor, BAY 11-7082. We then assessed the replication of CSFV. The results showed that BAY 11-7082 alleviated the inhibitory effect of IDO1 gene silencing on CSFV replication (Fig. 4E and F), indicating that IDO1 can promote CSFV replication by suppressing NF-κB signaling transduction.

Trp inhibits CSFV replication by activating NF-κB signaling

Compared to the normal physiological state, the metabolic profile of tryptophan undergoes substantial alterations during inflammatory conditions, underscoring the potential pivotal role of tryptophan metabolism in inflammatory responses (32). To investigate the impact of Trp on NF-κB signaling, we supplemented the cell culture medium with an exogenous addition of 1.6 mM L-Trp and assessed its influence on the protein expression of the NF-κB. The results revealed a significant increase in the expression levels of NF-κB pathway proteins, including P65, p-P65, IκBα, and p-IκBα, in the cells treated with L-Trp compared to the control group (Fig. 5A). Confocal microscopy analysis revealed an increased intensity of red fluorescence in the cell nucleus upon treatment with L-Trp, indicating enhanced nuclear translocation of P65 (Fig. 5B and C). These findings collectively suggest that exogenous L-Trp can activate the NF-κB signaling. After treating PK-15 cells with BAY, the addition of L-Trp to the culture medium was performed to evaluate the replication status of CSFV. The results revealed that BAY alleviated the inhibitory effect of L-Trp on CSFV (Fig. 5D and E), indicating that Trp can suppress CSFV replication by activating the NF-κB signaling.

Fig 5.

Trp inhibits CSFV replication by activating the NF-κB signaling. (A) PK-15 cells were infected with CSFV (MOI = 1), and 1.6 mM L-Trp was added to the cell culture medium. Western blot for P65, p-P65, IκBα, and p-IκBα expression in PK-15 cells. The relative protein levels were estimated by histograms representing density readings of the gel bands with Image J, and the ratios were calculated relative to Tubulin control. (B and C) Laser confocal detection of P65 nuclear translocation in PK-15 cells at 24 h. (D) PK-15 cells were treated with BAY for 2 h, and 1.6 mM L-Trp was added to the cell culture medium, and CSFV NS5B copy numbers were determined by qRT-PCR. (E) Western blot for P65, p-P65, IκBα, p-IκBα, and CSFV E2 protein expression, and the relative protein levels were estimated by histograms representing density readings of the gel bands with Image J, and the ratios were calculated relative to Tubulin control (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 calculated using two-way ANOVA. ns, not significant).

Kyn promotes CSFV replication by inhibiting NF-κB signaling

To investigate the influence of Kyn on NF-κB signaling, we supplemented the cell culture medium with an exogenous addition of 1.0 mM L-Kyn and assessed its influence on the protein expression of the NF-κB signaling. The results revealed a significant decrease in the expression levels of NF-κB pathway proteins, including P65, p-P65, IκBα, and p-IκBα, in the cells treated with L-Kyn compared to the control group (Fig. 6A). Additionally, BAY alleviated the promotive effect of L-Kyn on CSFV (Fig. 6B and C). These findings collectively suggest that Kyn can enhance CSFV replication by inhibiting the NF-κB signaling.

Fig 6.

Kyn promotes CSFV replication by inhibiting the NF-κB signaling. (A) PK-15 cells were infected with CSFV (MOI = 1), and 1.0 mM L-Kyn was added to the cell culture medium. Western blot for P65, p-P65, IκBα, and p-IκBα expression in PK-15 cells. The relative protein levels were estimated by histograms representing density readings of the gel bands with Image J, and the ratios were calculated relative to Tubulin control. (B) PK-15 cells were treated with BAY for 2 h, and 1.0 mM L-Kyn was added to the cell culture medium, and CSFV NS5B copy numbers were determined by qRT-PCR. (C) Western blot for P65, p-P65, IκBα, p-IκBα, and CSFV E2 protein expression, and the relative protein levels were estimated by histograms representing density readings of the gel bands with Image J, and the ratios were calculated relative to Tubulin control (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 calculated using two-way ANOVA. ns, not significant).

L-Kyn is essential for IDO1-mediated NF-κB

We perturbed the expression of IDO1 and supplemented the cell culture medium with 1 mM L-Kyn. Subsequently, we assessed the expression of NF-κB signaling-related proteins. The results indicated that L-Kyn effectively attenuated the enhancement of NF-κB signaling pathway-related protein expression caused by IDO1 gene silencing (Fig. 7). Furthermore, L-Kyn significantly mitigated the enhancement of P65 nuclear translocation induced by IDO1 gene silencing (Fig. 7B). Even under CSFV infection conditions, L-Kyn exhibited the capability to inhibit the NF-κB signaling activation caused by IDO1 gene silencing (Fig. 7C and D). These data support the notion that IDO1 regulates NF-κB activation levels by modulating L-Kyn levels rather than IDO1 itself. In summary, these findings suggest that IDO1 suppresses the NF-κB signaling pathway by regulating the levels of downstream metabolite L-Kyn, thereby promoting CSFV replication.

Fig 7.

L-Kyn is essential for IDO1-mediated NF-κB. (A) Relative expression of P65, p-P65, IκBα, and p-IκBα proteins detected by immunoblotting. The relative protein levels were estimated by histograms representing density readings of the gel bands with Image J, and the ratios were calculated relative to Tubulin control. (B) After transfection with IDO1-interfering RNA, cells were cultured with an additional 1 mM L-Kyn in the medium, and P65 nuclear translocation was detected by laser confocal at 24 h. (C) Relative expression of P65, p-P65, IκBα, p-IκBα, and E2 proteins detected by immunoblotting. The relative protein levels were estimated by histograms representing density readings of the gel bands with Image J, and the ratios were calculated relative to Tubulin control (*P < 0.05, **P < 0.01, and ****P < 0.0001 calculated using two-way ANOVA. ns, not significant). (D) PK-15 cells were transfected with IDO1 interfering RNA before infection with CSFV (MOI = 1), cells were cultured with an additional 1 mM L-Kyn in the medium, and P65 nuclear translocation was detected by laser confocal at 24 h.

IDO1-mediated suppression of NF-κB inhibits the expression of innate immune factors IL-6, IFN-α, and IFN-β

The innate immune system plays a crucial role in immediate defense against microbial pathogens. The expression of innate immune factors is tightly regulated to maintain immune homeostasis.

To investigate the impact of IDO1 on the expression of innate immune factors, we examined the relative expression of innate immune factors IL-6, IFN-α, and IFN-β after overexpressing IDO1. Results showed that compared to the control group, the relative expression levels of IL-6, IFN-α, and IFN-β significantly decreased after IDO1 overexpression (Fig. 8A). Enzyme-linked immunosorbent assay was employed to measure the levels of innate immune factors IL-6, IFN-α, and IFN-β in the culture supernatant. The results demonstrated that compared to the control group, the levels of IL-6, IFN-α, and IFN-β in the culture supernatant significantly decreased after IDO1 overexpression (Fig. 8B).

Fig 8.

IDO1-mediated suppression of NF-κB inhibits the expression of innate immune factors IL-6, IFN-α, and IFN-β. (A) The relative expression of cytokines IL-6, IFN-α, and IFN-β was determined by qRT-PCR. (B) The concentration of cytokines IL-6, IFN-α, and IFN-β were determined by enzyme-linked immunosorbent assay. (C) PK-15 cells were treated with BAY for 2 h before transfection with IDO1-interfering RNA, and the relative expression of cytokines IL-6, IFN-α, and IFN-β was detected at 24 h using qRT-PCR. (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 calculated using two-way ANOVA).

To investigate whether IDO1 reduces the expression of innate immune factors by inhibiting NF-κB, we interfered with the expression of IDO1 and treated PK-15 cells with BAY. Results showed that BAY effectively attenuated the activating effect of IDO1 gene silencing on the relative expression of innate immune factors IL-6, IFN-α, and IFN-β (Fig. 8C). These findings indicate that IDO1 can reduce the relative expression of innate immune factors IL-6, IFN-α, and IFN-β by inhibiting NF-κB signaling transduction.

Trp promotes the expression of innate immune factors IL-6, IFN-α, and IFN-β, and Kyn inhibits their expression

To investigate the effects of L-Trp and L-Kyn on the expression of innate immune factors, we supplemented the cell culture medium with 1.6 mM L-Trp and evaluated the relative expression of innate immune factors IL-6, IFN-α, and IFN-β. The results demonstrated that exogenous L-Trp increased the relative expression levels of innate immune cytokines IL-6, IFN-α, and IFN-β in PK-15 cells compared to the control group (Fig. 9A). Additionally, enzyme-linked immunosorbent assay was used to measure the levels of innate immune factors IL-6, IFN-α, and IFN-β in the culture supernatant, revealing that exogenous L-Trp increased the levels of IL-6, IFN-α, and IFN-β in the culture supernatant compared to the control group (Fig. 9B).

Fig 9.

Trp promotes the expression of innate immune factors IL-6, IFN-α, and IFN-β and kyn inhibits their expression. (A and C) The relative expression of cytokines IL-6, IFN-α, and IFN-β was determined by qRT-PCR. (B and D) The concentration of cytokines IL-6, IFN-α, and IFN-β was determined by enzyme-linked immunosorbent assay (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 calculated using two-way ANOVA).

Conversely, exogenous L-Kyn reduced the relative expression levels of innate immune cytokines IL-6, IFN-α, and IFN-β (Fig. 9C), as well as decreased the levels of IL-6, IFN-α, and IFN-β in the culture supernatant (Fig. 9D), compared to the control group.

Silencing the IDO1 gene inhibits cellular autophagy by promoting mTOR phosphorylation

In addition to its role in modulating NF-κB signaling, the signals of amino acid abundance or deficiency play a crucial role in regulating the dynamics of the amino acid sensor mTOR. The activation or inhibition of mTOR, in turn, exerts control over cellular autophagy, which represents an essential mechanism for cell survival (33).

To investigate the role of IDO1 in cellular autophagy, we employed both IDO1 gene silencing and IDO1 overexpression approaches to assess their effects on mTOR phosphorylation levels and the expression of autophagy-related proteins. Our study results revealed that in PK-15 cells, functional silencing of the IDO1 gene led to enhanced mTOR phosphorylation levels, accompanied by a decrease in the expression of autophagy proteins ATG5, Beclin1, and LC3II. Additionally, the expression levels of the autophagic flux marker protein P62 were elevated (Fig. 10A). Conversely, overexpression of IDO1 in PK-15 cells resulted in decreased mTOR phosphorylation levels, accompanied by an increase in the expression of autophagy proteins ATG5, Beclin1, and LC3II. Furthermore, a decrease in the expression levels of the autophagic flux marker protein P62 was observed (Fig. 10B). Changes in autophagic flux were examined using confocal microscopy. PK-15 cells were transfected with the autophagy dual fluorescence reporter plasmid mRFP-GFP-LC3. Under acidic conditions with a low pH in autolysosomes, the GFP signal is quenched, while the RFP signal remains fluorescent. Thus, the differential fluorescence of mRFP and GFP within the lysosomes can indicate the occurrence of cellular autophagy. The results revealed that compared to the control group, CSFV infection led to autophagy activation, as evidenced by a higher proportion of merged images displaying red fluorescence, while silencing of IDO1 gene function resulted in a restoration of the proportion of merged images displaying red fluorescence to a lower level (Fig. 10C). Conversely, IDO1 overexpression led to a higher proportion of merged images showing red fluorescence (Fig. 10D). To further elucidate the impact of IDO1 on cellular autophagy, transmission electron microscopy was employed to examine the occurrence of autophagy in cells overexpressing IDO1. The electron micrographs revealed a significant increase in the number of autolysosomes in cells overexpressing IDO1 (Fig. 10E through G). These findings indicate that IDO1 can enhance cellular autophagy.

Fig 10.

Silencing the IDO1 gene inhibits cellular autophagy by promoting mTOR phosphorylation. (A and B) Relative expression of p-mTOR, mTOR, ATG5, Beclin1, P62, and LC3II proteins detected by immunoblotting. The relative protein levels were estimated by histograms representing density readings of the gel bands with Image J, and the ratios were calculated relative to Tubulin control (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 calculated using two-way ANOVA). (C and D) Laser confocal detection of mRFP-EGFP-LC3 fluorescence in PK-15 cells at 24 h. RFP was shown as red light, and GFP was shown as green light. (E and F) Electron microscopy images revealed the autolysosomes ultrastructure in IDO1 overexpressing cells. PK-15 cells were transfected with pCAGGS-HA (left) or pCAGGS-IDO1 (right) for 48 h and analyzed by electron microscopy. In the magnified image, a significant increase in the number of autolysosomes was observed in IDO1 overexpressing cells. Scale bar: 2 µm. (G) Quantification of the autolysosomes per 4 cell image (**P < 0.01 calculated using one-way ANOVA). (H) PK-15 cells were treated with RAPA for 2 h before transfection with IDO1-interfering RNA, and the relative expression of p-mTOR, mTOR, P62, and LC3II proteins was detected at 24 h by immunoblotting. The relative protein levels were estimated by histograms representing density readings of the gel bands with Image J, and the ratios were calculated relative to Tubulin control (*P < 0.05, ***P < 0.001, and ****P < 0.0001 calculated using two-way ANOVA. ns, not significant).

To investigate whether silencing the IDO1 gene can suppress cellular autophagy by promoting mTOR phosphorylation, we transfected cells with siIDO1 and treated PK-15 cells with the mTOR-specific inhibitor rapamycin. We then assessed the expression levels of p-mTOR, mTOR, LC3II, and P62 proteins. The results showed that rapamycin mitigated mTOR phosphorylation and the inhibitory effect of IDO1 gene silencing on cellular autophagy (Fig. 10H). These findings suggest that silencing the IDO1 gene can suppress cellular autophagy by promoting mTOR phosphorylation.

Trp inhibits cellular autophagy by promoting mTOR phosphorylation

To investigate the role of Trp in cellular autophagy, we supplemented the cell culture medium with 1.6 mM L-Trp and examined its effects on mTOR phosphorylation levels and the expression of autophagy-related proteins. The results demonstrated that L-Trp increased the expression of p-mTOR protein and decreased the expression of autophagy-related proteins ATG5, Beclin1, and LC3II in PK-15 cells. Concurrently, the expression level of the autophagic flux marker protein P62 was elevated (Fig. 11A). Changes in autophagic flux were assessed using confocal microscopy, and the results indicated that compared to the control group, CSFV infection-induced autophagy activation, with a higher proportion of merged images displaying red fluorescence, whereas the proportion of merged images displaying red fluorescence returned to a lower level after L-Trp treatment (Fig. 11B). The occurrence of cellular autophagy following exogenous L-Trp supplementation was examined using transmission electron microscopy, showing a reduction in the number of autolysosomes (Fig. 11C through E). These findings collectively indicate that Trp can inhibit cellular autophagy.

Fig 11.

Trp inhibits cellular autophagy by promoting mTOR phosphorylation. (A) Relative expression of p-mTOR, ATG5, Beclin1, P62, and LC3II proteins detected by immunoblotting. The relative protein levels were estimated by histograms representing density readings of the gel bands with Image J, and the ratios were calculated relative to Tubulin control (**P < 0.01, ***P < 0.001, and ****P < 0.0001 calculated using two-way ANOVA. ns, not significant). (B) Laser confocal detection of mRFP-EGFP-LC3 fluorescence in PK-15 cells at 24 h. RFP was shown as a red light and GFP was shown as a green light. (C and D) Electron microscopy images revealed the autolysosomes ultrastructure in PK-15 cells. In the magnified images, a decrease in the number of autolysosomes was observed as a result of L-Trp treatment. Scale bar: 2 µm. (E) Quantification of the autolysosomes per 4 cell image (**P < 0.01 calculated using one-way ANOVA). (F) PK-15 cells were treated with RAPA for 2 h, and 1.6 mM L-Trp was added to the cell culture medium, and the relative expression of p-mTOR, P62, and LC3 proteins was detected at 24 h by immunoblotting. The relative protein levels were estimated by histograms representing density readings of the gel bands with Image J, and the ratios were calculated relative to Tubulin control (**P < 0.01, and ****P < 0.0001 calculated using two-way ANOVA. ns, not significant).

To investigate whether Trp can inhibit cellular autophagy by promoting mTOR phosphorylation, PK-15 cells were treated with Rapamycin followed by supplementation of 1.6 mM L-Trp to the cell culture medium. The protein expression levels of p-mTOR, LC3II, and P62 were examined. The results showed that Rapamycin could alleviate the activation of p-mTOR and the inhibitory effect on cellular autophagy caused by L-Trp (Fig. 11F). These findings suggest that L-Trp can inhibit cellular autophagy by promoting mTOR phosphorylation.

Silencing of the IDO1 gene and exogenous Trp can inhibit CSFV replication by antagonizing RAPA-induced cellular autophagy

Studies have demonstrated that CSFV infection can induce autophagy activation, which significantly enhances the in vitro replication of CSFV (25). To further investigate the mechanism by which IDO1 influences CSFV replication, PK-15 cells were transfected with siIDO1 and subsequently treated with Rapamycin to examine the effect on CSFV replication. The results demonstrated that Rapamycin could alleviate the inhibitory effect of siIDO1 gene silencing on CSFV replication (Fig. 12A and B). These findings suggest that IDO1 gene silencing can inhibit CSFV replication by antagonizing Rapamycin-induced cellular autophagy. PK-15 cells were treated with Rapamycin, followed by the addition of 1.6 mM L-Trp to the cell culture medium, and the replication of CSFV was examined. The results indicated that Rapamycin could alleviate the inhibitory effect of L-Trp on CSFV replication. These findings suggest that L-Trp can inhibit CSFV replication by counteracting the cellular autophagy induced by Rapamycin (Fig. 12C and D).

Fig 12.

Silencing of the IDO1 gene and exogenous Trp can inhibit CSFV replication by antagonizing RAPA-induced cellular autophagy. (A) PK-15 cells were treated with RAPA for 2 h before transfection with IDO1-interfering RNA, and CSFV NS5B copy numbers were determined by qRT-PCR. (B) Western blot for CSFV E2 protein expression, and the relative protein levels were estimated by histograms representing density readings of the gel bands with Image J, and the ratios were calculated relative to Tubulin control (**P < 0.01 and ****P < 0.0001 calculated using two-way ANOVA). (C) PK-15 cells were treated with RAPA for 2 h, and 1.6 mM L-Trp was added to the cell culture medium, and CSFV NS5B copy numbers were determined by qRT-PCR. (D) Western blot for CSFV E2 protein expression, and the relative protein levels were estimated by histograms representing density readings of the gel bands with Image J, and the ratios were calculated relative to Tubulin control (**P < 0.01, ***P < 0.001, and ****P < 0.0001 calculated using two-way ANOVA. ns, not significant).

DISCUSSION

There exist numerous intricate reciprocal regulatory mechanisms between viral infection and host metabolism, Viruses have the ability to hijack host metabolism in order to suppress host immune responses or acquire sufficient resources and energy, thereby promoting their own replication (34). Conversely, alterations in host metabolism can impact viral replication and propagation by modulating host immune responses. Research has demonstrated that inhibiting Trp catabolic metabolism in mice during Zika virus infection can significantly enhance the levels of chemokines CCL1 and CXCL-1 in the mouse brain, resulting in reduced Zika virus-induced neuronal death and degeneration, thereby promoting viral replication (35). Trp, an essential amino acid in mammals, not only participates in the synthesis of macromolecules such as proteins and nucleic acids but also plays a regulatory role in the innate immune response. Research indicates that the metabolic product of Trp, Kyn, can bind to the AhR and activate it, leading to the binding of AhR to the xenobiotic response elements (XRE) or dioxin response elements in the IL-6 promoter. This interaction promotes the production of IL-6 (36).

CSFV not only induces sustained immunosuppression in pigs but also frequently leads to secondary infections by other pathogens, posing a significant and severe threat. Moreover, our understanding of the mechanisms underlying the immunosuppression caused by the interplay between CSFV and host metabolism remains incomplete. In this context, the relationship between tryptophan metabolism and CSFV infection deserves careful attention. In this study, we observed that CSFV infection promotes tryptophan metabolism in PK-15 cells by activating the expression of IDO1 (Fig. 1). However, the mechanism by which CSFV activates the expression of IDO1 remains unclear. We speculate that this may be attributed to the activation of IFN-γ induced by CSFV infection, leading to enhanced expression of IDO1 (37, 38). Our subsequent data suggest that IDO1 facilitates CSFV replication by mediating tryptophan metabolism (Fig. 2 and 3). Interestingly, Trp and Kyn exert opposing effects on the in vitro proliferation of CSFV, leading us to speculate that the inhibitory effect of Trp on CSFV replication is independent of IDO1.

In response to various pathogen invasions, including viruses, mammals activate innate immune responses to combat infections through direct actions on invading pathogens or by inducing local inflammation. The activation of NF-κB is generally regarded as a protective response in the host against pathogen invasion. Research has shown that during CSFV infection, the activation of NF-κB promotes the production of type I interferon in the host, thereby participating in antiviral responses. Throughout evolution, the metabolic pathways of animals intertwine and merge with these immune regulatory activities, resulting in complex interactions. For example, during SEV infection, serine metabolism can counteract the virus-induced production of IFN-β, thereby facilitating viral replication (39). In this study, we demonstrated that silencing the IDO1 gene function promotes the activation of the NF-κB signaling pathway, leading to a significant increase in nuclear translocation of P65 and subsequent inhibition of CSFV replication (Fig. 4). Furthermore, our findings revealed that L-Trp can inhibit CSFV replication by activating NF-κB (Fig. 5), whereas L-Kyn can promote CSFV replication by inhibiting NF-κB (Fig. 6). Further results of the study showed that IDO1 can effectively suppress NF-κB signaling through mediating tryptophan metabolism, thereby promoting CSFV replication (Fig. 7). Additionally, our results revealed the suppression of NF-κB signaling activation by CSFV infection. However, the underlying reasons for this inhibitory effect of CSFV on NF-κB activation remain unknown. We speculate that the interaction between NS3 and NS5B proteins of CSFV and specific host proteins may contribute to this suppression of NF-κB signaling. Furthermore, during the evolutionary process, CSFV might have developed various other immune evasion mechanisms, yet the precise regulatory mechanisms behind these effects require further investigation.

It has been reported that CSFV infection can activate autophagy by inhibiting phosphorylation of mTORC1, which favors its own replication (40). Cellular autophagy is regulated by various signals, including amino acid deprivation, and plays an indispensable role during CSFV infection. CSFV infection can enhance autophagy to suppress the RIG-I-IRF3 signaling axis and JAK-STAT signaling pathway, thereby blocking the production of type I interferon and inhibiting cell apoptosis caused by CSFV infection (41). CSFV infection can also induce autophagy mediated by endoplasmic reticulum stress and activate complete autophagy through the PERK and IRE1 pathways to sustain its persistent infection (42). Therefore, we investigated the impact of IDO1 and tryptophan on mTOR-mediated autophagy and whether this influence plays a role in CSFV infection. Our results showed that overexpression of IDO1 led to decreased expression of p-mTOR, increased expression of LC3II, and an observed increase in the number of autolysosomes under electron microscopy (Fig. 10). Subsequently, we examined the effect of Rapamycin on autophagy and CSFV replication in IDO1 gene silencing conditions (Fig. 12). The results revealed that IDO1 gene silencing could inhibit CSFV replication by antagonizing Rapamycin-induced cellular autophagy. Further investigation revealed that L-Trp similarly inhibited CSFV replication by counteracting Rapamycin-induced cellular autophagy (Fig. 11 and 12).

In summary, our findings demonstrate that activation of the metabolic enzyme IDO1 can inhibit the NF-κB signaling through mediating tryptophan metabolism, thereby favoring viral replication. Additionally, our results reveal that both IDO1 gene silencing and exogenous Trp can inhibit CSFV replication by antagonizing Rapamycin-induced cellular autophagy. Although the incidence of CSFV has decreased in intensity, it still maintains a high level of mixed infection and latent infection, causing sustained immune suppression in the host and posing a potential significant threat. Our study, focusing on the regulatory mechanisms of immune response through Trp metabolism, provides the first insights into the impact of CSFV infection on IDO1-mediated tryptophan metabolism and the mechanisms regulated by tryptophan metabolism. This provides a theoretical basis for further control of CSFV development.

MATERIALS AND METHODS

Cell and virus

The swine kidney cell line PK-15 (ATCC, CCL-33) cells were grown in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, 11995500) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, 10099). Cells were cultured at 37°C in a 5% CO2 incubator. The CSFV strain (Shimen) used in the study was propagated in PK-15 cells and was isolated in our laboratory from a typical symptom of the swine. Viral titers are expressed as 50% tissue culture infective doses (TCID50)/0.1 mL based on PK-15 cell assays. For the comparison of viral replication in cells, virus infections were performed at a MOI of 1. For other experiments, viral stocks were prepared at a MOI of 1. PK-15 cells were infected at varying MOIs according to the requirements of different experiments. Mock infection comprised treatment with phosphate-buffered saline (PBS; Thermo Fisher Scientific, 10010023). After 1 h, the inoculum was removed by aspiration. The cells were then washed with PBS and cultured in a complete medium at 37°C for various times until harvesting

Reagents and antibodies

The chemical reagents used in this study are as follows: dimethyl sulfoxide (Sigma-Aldrich, V900090), BAY (Selleck Chemical, 11-7082, S2913), Rapamycin (Cell Signaling Technology, 9904, S7711), L-TRP (Selleck Chemical, S3987), L-KYN (Selleck Chemical, S5839).

The primary antibodies used in this study were as follows: rabbit monoclonal anti-IDO1 (Biorbyt, AS1466), mouse monoclonal anti-CSFV E2 (Jai Balajee Trading Company, 9011), mouse monoclonal anti-TUBA (Beyotime, AT819), rabbit monoclonal anti-HA (Earthox, E022050-1), rabbit monoclonal anti-P65 (Beyotime, AF0246), rabbit monoclonal anti-p-P65 (Affinity, AF2006), rabbit monoclonal anti-p-IKBIA (Beyotime, AF5851), mouse monoclonal anti-IKBIA (Cell Signaling Technology, 112B2), rabbit monoclonal anti-p-mTOR (Abcam, ab109268), rabbit monoclonal anti-SQSTIM1/p62 (Cell Signaling Technology, 39749), rabbit monoclonal anti-Beclin1 (Cell Signaling Technology, 3495), rabbit monoclonal anti-Atg5 (Cell Signaling Technology, D5F5U), rabbit polyclonal anti-MAP1LC3B (Cell Signaling Technology, 2775).

DNA constructs and RNA interference

The full-length swine IDO1 gene (GenBank, HM209418.1) was amplified by PCR and cloned into the pCAGGS-HA vector using EcoRI (Takara, 1040S) and KpnI (Takara, 1068A) to generate pCAGGS-IDO1. siRNAs against IDO1 were synthesized by Sangon Biotech.

The IDO1 -specific siRNAs were: 5′-CCAGGAAAUGAGAACCUAUTT-3′ (siIDO1-1) and 5′-AUAGGUUCUCAUUUCCUGGTT-3′ (siIDO1-3). PK-15 cells were grown to 60% confluence in 6-well cell culture plates and were transfected with siRNAs and using Lipofectamine 3000 reagent (ThermoFisher, L3000015) according to the manufacturer’s instructions. Briefly, 1 µg of 50 nM siRNA and 2 µL P3000 was diluted in 50 µL of serum-free OptiMEM (ThermoFisher Scientific, company, 22600050), and 3 µL Lipofectamine 3000 was also diluted in 50 µL of serum-free OptiMEM. The dilutions were mixed thoroughly and incubated at 25°C for 15 min. The mixture was then pipetted into the OptiMEM and further cultured at 37°C for 24 h. Following CSFV infection, the cells were incubated in fresh medium at 37°C for 48 h. The protein targeted for knockdown was evaluated by western blotting.

Quantitative real-time PCR

For targeted gene expression analysis, total RNA was prepared using a total RNA Kit I (Omega, R6834-01). Complementary DNA (cDNA) was synthesized using PrimeScript RT Master Mix (Takara, RR036A). Real-time qPCR was performed using SYBR Premix Ex Taq II (Takara, RR820A) using an iQ5 iCycler detection system (Bio-Rad, USA). Relative mRNA expression was assessed using the 2-ΔΔCt method and normalized to the housekeeping gene β-ACTIN. Primer sequences targeting the β-ACTIN gene were β-ACTIN 1: 5ʹ-GGCACCACACCTTCTACAACGAG-3ʹ; β-ACTIN 2: 5ʹ-TCATCTTCTCACGGTTGGCTTTGG-3ʹ. For CSFV genomic copies detection, viral RNA was extracted using a MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0 (Takara, 9766) and reverse-transcribed using PrimeScript RT Master Mix (Perfect Real Time; Takara, RR036A). The resulting cDNA was then amplified using SYBR Premix Ex Taq (Tli RNaseH Plus; Takara, RR420B) and an iQ5 iCycler detection system (Bio-Rad, USA). Primer sequences targeting the CSFV NS5B gene were CSFV1: 5ʹ-CCTGAGGACCAAACACATGTTG-3ʹ; CSFV2: 5ʹ-TGGTGGAAGTTGGTTGTGTCTG-3ʹ. Viral copy number was calculated using a standard curve from a recombinant plasmid containing the CSFV NS5B gene.

Immunoblotting

After treatment, cells were washed with cold PBS and incubated on ice with RIPA lysis buffer (Beyotime, P0013B) supplemented with 1 mM PMSF for 10 min. Cell lysates were then clarified by centrifugation at 15,000 × g for 20 min at 4°C. The protein concentration was determined using a BCA protein assay kit (Beyotime, P0012). Equal amounts of protein samples (20 µg) were diluted in 5 × SDS PAGE loading buffer and boiled for 5 min. Proteins were separated by 12.5% SDS-PAGE and electrotransferred onto polyvinylidene fluoride membranes (Beyotime, FFP30). After blocking with PBS containing 2% nonfat milk powder and 0.05% Tween 20 (Sigma-Aldrich, P2287) for 2 h at 25°C, the membrane was incubated with specific primary antibodies overnight at 4°C and then with the corresponding HRP-conjugated secondary antibodies at 37°C for 2 h at appropriate dilutions. The protein bands were visualized using an ECL Plus kit (Beyotime, P0018). Images of protein blots were obtained from a CanoScan LiDE 100 scanner (Canon, Japan).

Confocal immunofluorescence microscopy

Cells were grown in 35 mm petri dishes (NEST, GBD-35-20) with a glass bottom. When needed, the indicated plasmid DNA (mRFP-EGFP-LC3) was transfected with siIDO1 or no-target siRNA for analyzing the effect of siIDO1-induced autophagy was used for transfection.

Cells were washed with PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich, P6148) for 30 min at room temperature; they were then permeabilized with 0.2% triton X-100 (Sigma-Aldrich, T8787) for 10 min. The cells were blocked in PBS containing 5% bovine serum albumin (Beyotime, ST023) for 30 min. Next, the cells were stained with the indicated primary antibody of rabbit monoclonal antibody (anti-p-P65;1:200) in PBS buffer at 37°C, followed by a 1 h incubation

in PBS containing goat anti-rabbit secondary antibodies conjugated to FITC and TRITC at a dilution of 1:200. Wherever indicated, nuclei were stained with DAPI (Beyotime, C1002). The fluorescence signals were visualized with a TCS SP2 confocal fluorescence microscope (Leica TCS SP8).

Tryptophan and kynurenine measurement

Tryptophan levels in cell supernatant were assessed by spectrophotometric assays. In brief, 100 µL of cultured medium was mixed and incubated with 200 µL ice-cold 1 M perchloric acid (Sigma-Aldrich, 7601-90-3) on ice. After centrifugation at 4,000 × g for 15 min, the supernatant was neutralized to pH 7.0 by adding the same volume of 0.7 M tripotassium phosphate solution (Sigma-Aldrich, 529567 M). The neutralized supernatant (10 µL) was applied to spectrophotometric tryptophan assays using a Thermo Max microplate reader (Molecular Devices, USA). Tryptophan levels were estimated from a standard tryptophan calibration curve prepared under the same conditions. Levels of IL-6 in the supernatant were detected using ELISA kits (MM Biology, MM-0418O2) following the manufacturer’s instructions. Levels of IFN-α in the supernatant were detected using ELISA kits (MM Biology, MM-0378O1) following the manufacturer’s instructions. Levels of IFN-β in the supernatant were detected using ELISA kits (MM Biology, MM-0368O1) following the manufacturer’s instructions. Levels of L-Trp in the supernatant were detected using ELISA kits (ML Biology, ML-077292) following the manufacturer’s instructions. Levels of L-KYN in the supernatant were detected using ELISA kits (MB Biology,MB-10082A) following the manufacturer’s instructions. The samples were quantified using a microplate reader (Bio-Rad, USA).

Cell viability assay

Cell viability was determined using Cell Counting Kit-8 (CCK- 8, Beyotime, C0038) according to the manufacturer’s protocol. Briefly, approximately 1 × 104 cells (PK-15) per well were seeded in 96-well culture plates and cultured for 24 h at 37°C in a CO2 incubator. The cells were transfected with siIDO1 or siNC, using Lipofectamine 3000 reagent. After 48 h, cells were then cultured with 100 µL of fresh medium supplemented with 10 µL of CCK-8 solution, and the plates were further incubated for 4 h at 37°C. Subsequently, the optical density was measured at 450 nm using a microplate reader (Bio-Rad, USA)

Electron microscopy

To detect the effect of overexpressing IDO1 on cellular autophagy, after transfecting PK-15 cells grown in 10 cm dishes with pCAGGS-IDO1 and pCAGGS-HA, they were washed twice with PBS and fixed with 2.5% glutaraldehyde diluted in PBS at 4°C for 30 min. The cells were then collected in 1.5 mL microcentrifuge tubes and further fixed overnight. Cell pellets were dehydrated with an acetone series and embedded in epoxy resin. Next, ultrathin sections were prepared and observed by JEM-2010 HR transmission electron microscopy (JEOL).

Statistical analysis

Statistical analyses were performed using GraphPad Prism software version 8 (GraphPad, LaJolla, CA, USA). The data were expressed as arithmetic means ± standard deviation. The statistical differences between independent groups were assessed by multiple t test. P values less than 0.05 were regarded as statistically significant.

TCID50 assay

PK-15 cells were seeded in 96-well plates and then infected with serial 10-fold dilutions of supernatants in eight replicates. The plates were incubated for 72–96 h before virus titers were calculated.

DATA AVAILABILITY

It may be obtained from the corresponding author upon reasonable request.

ETHICS APPROVAL

The research was conducted in a biosafety level 2 laboratory throughout this study.