IL-15 exhibits pleiotropic effects on NK and CD8+ T cells and contributes to host protection or immunopathology during infection. Although both type I IFNs and IFN-γ upregulate IL-15 expression, their effects on IL-15 upregulation and underlying mechanisms have not been compared comprehensively. In addition, little is known about trans-presentation of IL-15 by epithelial cells to lymphocytes. In this study, we analyzed the expression of IL-15 and IL-15Rα in the human hepatocyte-derived Huh-7 cell line after stimulation with IFN-α, IFN-β, or IFN-γ using RT-PCR, flow cytometry, and confocal microscopy. We also performed knockdown experiments to investigate the signaling pathway involved in IL-15 upregulation. IFN-γ more potently upregulated IL-15 expression in Huh-7 cells than IFN-α and IFN-β. Knockdown experiments revealed that IFN-γ– and IFN-β–induced IL-15 expression relied on IFN regulatory factor 1 (IRF1), which is upregulated by STAT1 and IFN-stimulated gene factor 3, respectively. Inhibitor of κB kinase α/β was also involved in IFN-γ–induced upregulation of IL-15. Furthermore, human NK cells were activated by coculture with IFN-γ–treated Huh-7 cells, which was abrogated by knocking down IL-15Rα in IFN-γ–treated Huh-7 cells, indicating that IFN-γ–induced IL-15 on Huh-7 cells activates NK cells via trans-presentation. In summary, our data demonstrate that IFN-γ potently elicits IL-15 trans-presentation by epithelial cells via IRF1. These data also suggest that the IFN-γ–IRF1–IL-15 axis may be a regulatory target for the treatment of diseases with IL-15 dysregulation.
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Interleukin-15 is a member of the four α-helix bundle cytokine family (1). IL-15 plays a pivotal role in the development, proliferation, and activation of NK and CD8+ T cells (1, 2), contributing to host protection and the elimination of virus-infected cells (3). In contrast, dysregulated IL-15 induces bystander activation of CD8+ T cells, and these IL-15–activated CD8+ T cells contribute to immunopathological tissue injury during acute viral hepatitis and celiac disease (3–6). Thus, tight regulation of IL-15 expression is important for maintaining immune homeostasis.
The IL-15 receptor complex is composed of the common γ-chain (CD132), the IL-2/IL-15 receptor β-chain (IL-2/15Rβ), and IL-15 receptor α-subunit (IL-15Rα). IL-15 primarily delivers its signals in a cell contact–dependent manner through the trans-presentation of membrane-bound IL-15–IL-15Rα complexes to neighboring cells expressing IL-2/IL-15Rβ and the common γ-chain (7). Signaling by cis-presentation or soluble IL-15–IL-15Rα complexes has also been proposed (8–11). When human CD8+ T cells are stimulated in vitro with recombinant IL-15, cis-presentation mediates the IL-15 responses (10). In addition, the presence of soluble IL-15–IL-15Rα complexes in the serum of patients with melanoma and polyinosinic:polycytidylic acid– or IFN-α–stimulated mice suggests the possibility of signaling by soluble IL-15–IL-15Rα complexes (9, 11, 12). However, the trans-presentation of membrane-bound IL-15–IL-15Rα complexes is considered a major mechanism of action of IL-15 with strong potency (3, 11).
Although IL-15 transcripts are expressed ubiquitously in various cell types (12–18), IL-15 protein expression is tightly regulated, and the secreted form is hardly detected (2, 19, 20). IL-15 protein production has been studied primarily in myeloid cells, including dendritic cells (DCs) and inflammatory monocytes, and these cells are considered to be the main sources of IL-15 during infections (20–22). Recent studies reported that epithelial cells, including enterocytes, bronchial epithelial cells, and hepatocytes, can be cellular sources of IL-15 (4, 23, 24). However, the regulation of IL-15 expression in epithelial cells remains largely elusive.
Various stimuli induce IL-15 upregulation (8, 22, 25–27). In vivo or in vitro treatment with polyinosinic:polycytidylic acid or LPS increases the expression of IL-15 and IL-15Rα in DCs (11, 25), which is partially mediated by type I IFNs (25). Type I IFNs, including IFN-α and IFN-β, also directly upregulate IL-15 mRNA and protein expression (25, 28). Previous studies have shown that type I IFNs are critically required for IL-15 production during infection with pathogens, such as Candida and varicella zoster virus (22, 26). In addition to type I IFNs, IFN-γ, the sole type II IFN, upregulates expression of IL-15 and IL-15Rα in monocytes and endothelial cells (29–31). However, the difference between type I IFNs and IFN-γ in the effect on IL-15 upregulation has not been comprehensively investigated. Furthermore, little is known about the signaling pathways involved in types I and II IFN-induced IL-15 upregulation.
In the present study, we compared the effects of type I IFNs and IFN-γ on IL-15 expression in epithelial cells, particularly liver-derived cells, and investigated the signaling pathways involved in IFN-induced IL-15 upregulation. We found that IFN-γ is more potent in IL-15 upregulation than type I IFNs. In addition, IFN-β and IFN-γ activated distinct signaling pathways, leading to upregulation of IL-15 and IL-15Rα expression. We also assessed whether IFN-γ–induced IL-15 expression in epithelial cells is functionally active by analyzing its effect on NK cells.
Materials and Methods
Cell culture, IFN treatment, and siRNA transfection
Huh-7, HeLa, and A549 cells were cultured in DMEM supplemented with 5% FBS (Corning) and penicillin/streptomycin. Wild-type (WT), STAT1-null, and STAT2-null human telomerase reverse transcriptase human mammary epithelial (hTERT-HME1) cells were kindly provided by Dr. George R. Stark and Dr. HyeonJoo Cheon (Lerner Research Institute, Cleveland Clinic, Cleveland, OH) and cultured in mammary epithelial growth medium (CC-3150; Lonza). Normal human primary bronchial epithelial cells (PCS-300-010; American Type Culture Collection) were thawed and cultured in 12-well plates according to the manufacturer’s instructions.
Recombinant human IFN-α (11100-1; PBL Assay Science), IFN-β (300-02BC; PeproTech), IFN-γ (300-02; PeproTech), IFN-λ1 (1598-IL; R&D Systems), and IFN-λ3 (5259-IL; R&D Systems) were used at the indicated concentrations. To knock down specific gene expression, small interfering RNAs (siRNAs) were transfected into Huh-7 cells using Lipofectamine RNAiMAX (Invitrogen) by reverse protocol. Briefly, in 1 well of a 24-well plate, 6 pmol of siRNAs and 1 μl of Lipofectamine were diluted in 100 μl of Opti-MEM I (Life Technologies) to form complexes, and then 5 × 104 trypsinized Huh-7 cells were added. Twelve-well plates were used for flow cytometry and coculture experiments, and the amount of reagent was doubled. On day 3 after transfection, the number of cells transfected with specific siRNA was ∼70–100% of the number of control siRNA-transfected cells. The siRNAs were siIL15RA (3601-1, 3601-2) and siSTAT1 (6772-1) from Bioneer (Republic of Korea); siSTAT2 (sc-29492) and siIRF9 (sc-38013) from Santa Cruz Biotechnology; and siIRF1 (s7501) from Invitrogen.
Surface IL-15 staining
To stain the surface IL-15 in adherent cell lines, cells were detached from the culture dishes using Accutase Cell Dissociation Reagent (Life Technologies). After washing with FACS buffer (PBS supplemented with 1% FBS, 0.05% sodium azide, and 2 mM EDTA), ∼3 × 105 cells were resuspended in 50 μl FACS buffer. Cell suspensions were stained with anti–IL-15 biotinylated Abs (BAM247; R&D Systems) at a concentration of 0.2 μg/ml, followed by allophycocyanin-conjugated streptavidin (BioLegend). Dead cells were excluded using the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen).
Reverse transcription quantitative PCR (qPCR)
For total RNA extraction, Huh-7 cells were subjected to cell lysis on a 24-well plate, and the Ribospin RNA purification kit (GeneAll) was applied. cDNAs were synthesized using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo). qPCR was run in the QuantStudio 1 system with the following TaqMan assays (Applied Biosystems): IL-15 (Hs01003716_m1), IL-15RA (Hs00542604_m1), STAT2 (Hs01013123_m1), IFN regulatory factor 9 (IRF9; Hs00196051_m1), IRF1 (Hs00971965_m1), and GAPDH (Hs02786624_g1). STAT1 (ID: 4150) and hypoxanthine phosphoribosyltransferase (HPRT; ID: 3591) primer/probe sequences were obtained from the RTPrimerDB database (32). The amount of specific mRNA in each sample was calculated by QuantStudio software (version 1.5.0; Applied Biosystems), normalized to housekeeping genes such as HPRT or GAPDH (Supplemental Fig. 1B), and expressed as the fold change over mRNA levels in negative controls.
Huh-7 cells were stained with PKH26 (Sigma-Aldrich) according to the manufacturer’s protocol to visualize cell membranes, stained with anti–IL-15 Abs as in the flow cytometric analysis, and then attached to slide glasses using a Cytospin centrifuge. These slides were fixed with 1% formaldehyde solution and stained with DAPI before being mounted. The slides were observed under an LSM780 confocal microscope (Carl Zeiss). Images were collected in the 1024 × 1024–pixel format with an oil-immersion objective (100×) and processed by ZEN 3.1 software (Carl Zeiss).
Western blot analysis
Cell lysates were prepared using radioimmunoprecipitation assay lysis buffer supplemented with protease and phosphatase inhibitors. Proteins were separated by electrophoresis in 4–20% gradient gels and transferred to PVDF membranes. The membranes were blocked and incubated with primary Abs overnight at 4°C, followed by incubation with HRP-conjugated secondary Abs. SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) was used for chemiluminescence detection of proteins. The primary Abs were STAT1 (610120; BD Biosciences), STAT2 (72604; Cell Signaling Technology), IRF9 (76684; Cell Signaling Technology), IRF1 (8478; Cell Signaling Technology), and β-actin (ab8227; Abcam).
Human samples, lymphocyte isolation, and NK cell sorting
Peripheral blood was obtained from six healthy donors. This study was reviewed and approved by the institutional review board of Korea Advanced Institute of Science and Technology (Daejeon, Republic of Korea; KH2018-118) and conducted according to the principles of the Declaration of Helsinki. Informed consent was obtained from all study participants. PBMCs were isolated by density gradient centrifugation using Lymphocyte Separation Medium (Corning). After isolation, the cells were cryopreserved in FBS with 10% DMSO (Sigma-Aldrich) until use. In some experiments, NK cells were enriched from PBMCs using the human NK cell isolation kit (130-092-657; Miltenyi Biotec) and MACS according to the manufacturer’s instructions. The purity of NK cells was >90%.
On day 0, 1 × 105 Huh-7 cells were transfected with 20 nM of siControl or siIL15RA in 12-well plates. On day 1, the transfection media were replaced with fresh media with or without 20 ng/ml IFN-γ. On day 2, Huh-7 cells were washed twice with fresh media to remove residual IFN-γ. Freshly isolated or thawed PBMC/NK cell suspension (5 × 104 cells) was added to the Huh-7 cells and incubated for 24 h before analysis. In some experiments, IL-15 blocking Ab (MAB2471; R&D Systems) was added to Huh-7 cells at 10 μg/ml 30 min before adding PBMCs.
Multicolor flow cytometry
In coculture experiments, PBMCs or sorted NK cells were stained with fluorochrome-conjugated Abs for specific markers at room temperature for 10 min. The Abs used in the experiments were BV510-conjugated anti-CD3 (UCHT1), BV786-conjugated anti-CD56 (NCAM16.2), BV650-conjugated anti-CD69 (FN50), BB515-conjugated anti-CD25 (2A3), PE-conjugated anti-NKG2D (1D11), and BV421 or Alexa Fluor 647–conjugated anti-NKp30 (p30-15; all from BD Biosciences). Dead cells were excluded using LIVE/DEAD near-infrared fluorescent reactive dye (Invitrogen). Multicolor flow cytometry was performed using an LSR II or Celesta instrument (BD Biosciences), and the data were analyzed by FlowJo version 10.6.2 software (BD Biosciences).
Statistical analyses were performed in Prism 8 software (GraphPad Software). The following statistical methods were used to test for significance: two-way ANOVA followed by Tukey’s multiple comparison analysis (Fig. 1A, 1B) or Sidak’s multiple comparison analysis (Fig. 1F, 1G); unpaired t test (Figs. 2A, 3A, 4); one-way ANOVA followed by Dunnett’s multiple comparison analysis (Figs. 2C, 2D, 3C, 3D); or the Wilcoxon signed-rank test (Fig. 5). In all analyses, p < 0.05 was considered significant.
IFN-γ induces IL-15 upregulation more potently than IFN-α and IFN-β in epithelial cells
First, we compared the effect of type I IFNs and IFN-γ on IL-15 expression. We treated the hepatocyte-derived Huh-7 cells with IFN-α, -β, and -γ at various concentrations and analyzed surface IL-15 protein expression by flow cytometry. We found that IFN-γ more strongly upregulated IL-15 than IFN-α and IFN-β (Fig. 1A). When we analyzed the transcript levels of IL-15 and IL-15Rα, similar findings were observed (Fig. 1B). However, IFN-λs did not upregulate surface IL-15 expression (Supplemental Fig. 1A). A stronger effect of IFN-γ on IL-15 induction was also observed in experiments using A549, a lung-derived cell line, and HeLa cells, a uterine cervix–derived cell line (Fig. 1C). Moreover, in normal human primary bronchial epithelial cells, IFN-γ effectively increased IL-15 expression, but IFN-β did not (Fig. 1D). Confocal microscopy confirmed that IFN-induced IL-15 was located on the surface of Huh-7 cells (Fig. 1E).
Next, we studied the kinetics of IFN-induced IL-15 upregulation by comparing the expression level of IL-15 and IL-15Rα transcripts 6 and 16 h after stimulation. IL-15 mRNA expression after IFN-α, -β, or -γ stimulation significantly decreased 16 h after stimulation compared with 6 h after stimulation. Notably, IL-15Rα mRNA expression after IFN-γ stimulation significantly increased 16 h after stimulation, whereas the expression after IFN-α or IFN-β stimulation significantly decreased (Fig. 1F). These results indicate that, compared with IFN-α and IFN-β, IFN-γ exhibits more long-lasting effects on IL-15Rα upregulation. To identify the role of IL-15Rα in surface IL-15 expression, we knocked down IL-15Rα expression using siRNA (Supplemental Fig. 1B). Surface IL-15 protein expression was significantly decreased by IL-15Rα knockdown (Fig. 1G), indicating that IL-15Rα substantially contributes to IL-15 expression on the cell surface.
Taken together, these findings indicate that IFN-γ induces IL-15 upregulation in epithelial cells more potently than IFN-α and IFN-β.
IFN-β upregulates IL-15 expression via the IFN-stimulated gene factor 3 (ISGF3)/IRF1 pathway
Binding of IFNs to their receptors delivers intracellular signals through activation of the JAK/STAT pathways and IRFs. Typically, type I IFN signaling triggers the formation of ISGF3, which consists of STAT1, STAT2, and IRF9. In turn, ISGF3 induces the transcription of IFN-stimulated genes, including antiviral proteins and IRF1 (33, 34). When we stimulated WT, STAT1-null, and STAT2-null hTERT-HME1 cells with IFN-β, surface IL-15 expression was significantly upregulated in WT hTERT-HME1 cells, but not in STAT1-null and STAT2-null hTERT-HME1 cells (Fig. 2A), indicating that both STAT1 and STAT2 are required for IFN-β–induced IL-15 upregulation.
We also investigated IFN-β–induced upregulation of surface IL-15 expression in Huh-7 cells after knockdown of the expression of ISGF3 components (STAT1, STAT2, and IRF9) or IRF1 using siRNAs. IFN-β–induced upregulation of surface IL-15 expression was partially abrogated by knocking down ISGF3 and IRF1 (Fig. 2B), indicating that both ISGF3 and IRF1 are required for IFN-β–induced surface IL-15 upregulation. Next, we examined IFN-β–induced expression of IL-15 and IL-15Rα mRNA after knockdown of the expression of ISGF3 components or IRF1 using siRNAs (Supplemental Fig. 2A). The IFN-β–induced IL-15 mRNA upregulation was significantly decreased by knocking down STAT2, IRF9, STAT1/STAT2, or STAT1/STAT2/IRF9 (Fig. 2C) and tended to be decreased by IRF1 knockdown, but the difference was not significant. The IFN-β–induced IL-15Rα mRNA upregulation was significantly reduced by knocking down STAT2, IRF9, STAT1/STAT2, STAT1/STAT2/IRF9, or IRF1 (Fig. 2D). These results confirm the role of ISGF3 and IRF1 in the IFN-β–induced upregulation of IL-15 expression.
In immunoblot analysis, we found that knocking down ISGF3 components reduced the IFN-β–induced IRF1 upregulation (Fig. 2E), indicating that ISGF3 is required for IRF1 upregulation. In summary, IFN-β upregulates IL-15 expression via the ISGF3/IRF1 pathway in epithelial cells.
IFN-γ upregulates IL-15 expression via the STAT1/IRF1 pathway
Next, we investigated the IFN-γ signaling pathway involved in upregulation of IL-15 expression. IFN-γ signaling induces the expression of downstream genes, including IRF1, via the formation of STAT1 homodimers (33, 35). We found that IFN-γ upregulated surface IL-15 expression in WT and STAT2-null hTERT-HME1 cells, but not in STAT1-null hTERT-HME1 cells (Fig. 3A), indicating that STAT1, but not STAT2, is critically required for IFN-γ–induced IL-15 upregulation.
IFN-γ–induced upregulation of surface IL-15 expression was also examined in Huh-7 cells after knockdown of the expression of ISGF3 components or IRF1 using siRNAs. IFN-γ–induced upregulation of surface IL-15 expression was abrogated by knocking down IRF1 (Fig. 3B), but it was not changed by knocking down ISGF3 components. These data indicate that IRF1, but not ISGF3, is required for IFN-γ–induced surface IL-15 upregulation. We also examined IFN-γ–induced expression of IL-15 and IL-15Rα mRNA after knockdown of the expression of ISGF3 components or IRF1 using siRNAs (Supplemental Fig. 2B). The IFN-γ–induced IL-15 mRNA upregulation tended to be decreased by IRF1 knockdown, but the difference was not significant (Fig. 3C). The IFN-γ–induced upregulation of IL-15Rα mRNA was significantly abrogated by knocking down IRF1 (Fig. 3D). These results confirm the role of IRF1 in the IFN-γ–induced upregulation of IL-15 protein expression. In immunoblot analysis, we found that knockdown of STAT1 reduced IFN-γ–induced IRF1 upregulation (Fig. 3E), indicating that STAT1 is required for IRF1 upregulation.
Taken together, the results indicate that IFN-γ upregulates IL-15 expression in a STAT1- and IRF1-dependent manner, but ISGF3 is not required, indicating that IRF1 is upregulated by STAT1 homodimer in IFN-γ–treated cells and that IFN-γ upregulates IL-15 expression via the STAT1/IRF1 pathway.
Inhibitor of κB kinase α/β (IKKα/β) partially mediates IFN-γ–induced upregulation of IL-15
Previous studies have reported an additional IFN-γ signaling pathway that involves IKK (36, 37). Therefore, we examined whether IKK also participates in IL-15 expression after IFN-γ stimulation. To disrupt IKK signaling, we used an IKKα/β inhibitor, IKK16, at a concentration of 1 μM, at which cell viability was not compromised (Fig. 4A). We cultured Huh-7 cells with or without 1 μM IKK16, stimulated the cells with IFN-γ, and analyzed the surface expression of IL-15. IKK16 significantly decreased the surface expression of IL-15 protein in IFN-γ–treated Huh-7 cells (Fig. 4B). We also examined the transcript levels of IL-15 and IL-15Rα. IKK16 significantly decreased IL-15Rα mRNA expression (Fig. 4C), assaying CXCL10 mRNA expression as a positive control (36). Collectively, these data imply that IKKα/β partially mediates IFN-γ–induced IL-15 expression.
IFN-γ–induced IL-15 on epithelial cells activates NK cells via trans-presentation
Finally, we examined whether IFN-γ–induced IL-15 on Huh-7 cells can activate NK cells. Control siRNA or IL-15Rα–specific siRNA-transfected Huh-7 cells were cultured with or without IFN-γ for 24 h and cocultured with PBMCs another 24 h, and then we analyzed the phenotype of NK cells (Fig. 5A). Coculture with IFN-γ–stimulated Huh-7 cells significantly increased the percentages of CD69+ or CD25+ activated cells among NK cells (Fig. 5B). However, the upregulation of CD69 and CD25 was abrogated when NK cells were cocultured with IL-15Rα siRNA-transfected Huh-7 cells (Fig. 5B), indicating that IL-15 expressed by IFN-γ–stimulated Huh-7 cells activates NK cells via trans-presentation. When we examined the expression of NK activating receptors on NK cells, both NKG2D and NKp30 were significantly upregulated by coculture with IFN-γ–stimulated Huh-7 cells (Fig. 5C). Similarly, IL-15Rα knockdown in IFN-γ–stimulated Huh-7 cells significantly abrogated the upregulation of NKG2D and NKp30 expression in NK cells (Fig. 5C). The same results were observed with magnetically sorted NK cells (Supplemental Fig. 3), though magnetically sorted NK cells exhibited relatively weaker activation than unsorted NK cells. Weaker activation of sorted NK cells may be explained by indirect effects of other immune cells in PBMCs, such as T cells. Taken together, these data indicate that membrane-bound IL-15/IL-15Rα directly activates NK cell via trans-presentation.
We also knocked down the expression of IRF1 or ISGF3 in Huh-7 cells and performed coculture experiments. We found that the knockdown of IRF1, but not ISGF3, in Huh-7 cells decreased the upregulation of CD69, CD25, NKG2D, and NKp30 in NK cells (Fig. 5D, 5E), indicating a significant role of IRF1 in IL-15 trans-presentation to NK cells by IFN-γ–stimulated Huh-7 cells (Fig. 6). In addition, we examined the effect of IKK16. When we pretreated Huh-7 cells with IKK16 and performed coculture experiments, the upregulation of CD69, but not NKGD2 or NKp30, was significantly decreased in cocultured NK cells (Supplemental Fig. 4A, 4B).
We further confirmed the ability of IFN-γ–induced IL-15 on Huh-7 cells to activate NK cells using IL-15 blocking Abs. Anti–IL-15 blocking Abs significantly abrogated the upregulation of CD69, CD25, NKG2D, and NKp30 expression on NK cells cocultured with IFN-γ–stimulated Huh-7 cells (Fig. 5F, 5G).
Given that IL-15 exhibits multiple functions and is frequently upregulated during infections, it is important to better understand the regulation of IL-15 expression. Although a series of studies have reported that both types I and II IFNs upregulate IL-15 expression, their effects on IL-15 upregulation have not been directly compared. In this study, we demonstrated that IFN-γ upregulates IL-15 expression in epithelial cells more potently than type I IFNs. In addition, IFN-β and IFN-γ activated distinct signaling pathways to upregulate IL-15 expression, though these pathways converged to IRF1. We also confirmed that the IL-15 expressed by epithelial cells in response to IFN-γ is functional by analyzing the effects on NK cells. Collectively, our findings suggest that IFN-γ may be a key driver of IL-15 expression during infections and other pathological conditions.
The coordination of epithelial cells and immune cells is essential for maintaining immune homeostasis in the mucosa (38–40). In particular, epithelial cells are involved in the activation of innate and adaptive immune cells by releasing various cytokines. Although IL-15 mRNA is ubiquitously expressed in diverse cell types, the expression of IL-15 protein has been studied primarily in myeloid cells, including DCs, macrophages, and monocytes (41). However, little is known about IL-15 expression by epithelial cells and its role in immune responses. In the present study, we showed that IFN-γ–stimulated epithelial cells express surface IL-15 and activate NK cells through trans-presentation of IL-15. Therefore, IL-15 expressed by epithelial cells may act as a critical player in regional immunity.
In the present study, we found that IRF1 is required for IL-15 expression induced by both IFN-β and IFN-γ (Fig. 6). Stronger upregulation of IL-15 by IFN-γ compared with IFN-β can be explained by a previous study that IRF1 expression was more long lasting in IFN-γ–treated cells than in IFN-β–treated cells (42). However, the mechanism of IRF1 upregulation was different between IFN-β and IFN-γ. Signal transduction by type I IFNs and IFN-γ is typically mediated by distinct complexes of IRF and STAT proteins, though cross-talk occurs (33). Type I IFN signaling triggers formation of the ISGF3 complex composed of STAT1, STAT2, and IRF9 (33, 34), and ISGF3 then activates IFN-stimulated genes, including antiviral proteins and IRFs (33). In contrast, IFN-γ signaling primarily relies on STAT1 homodimers (33, 35). As expected from the canonical signaling pathways of type I IFNs and IFN-γ, we found that ISGF3 knockdown reduces IRF1 upregulation by IFN-β. In contrast, STAT1 knockout abrogates surface IL-15 expression by IFN-γ. These findings indicate that ISGF3 and STAT1 primarily mediate IRF1 upregulation in IFN-β– and IFN-γ–treated cells, respectively (Fig. 6).
In addition to the canonical signaling pathway of IFN-γ, we found that IKK partially mediates IFN-γ–induced IL-15 upregulation. An ancillary signaling pathway that involves IKK is essential for the induction of a subset of IFN-γ–stimulated genes, such as CXCL10 (37). A previous study also showed that IKKβ promotes the binding of IRF1 to the DNA binding site (36). Notably, we found that IKK inhibition decreases IL-15Rα transcription. Considering that IKKβ activates IRF1, IKKβ may mediate IL-15 expression by activating IRF1.
In the present study, we found that trans-presentation of IL-15 by IFN-γ–stimulated epithelial cells is needed for NK activation in coculture assays. Although previous studies have reported that soluble IL-15 exists in vivo (9, 27), the NK-activating effects were abrogated by knockdown of IL-15Rα expression in the present study. A recent study proposed three modes of IL-15 trans-presentation, including direct contact, shedding, and trans-endocytosis, with distinct functional outcomes (43). It would be interesting to investigate the action mode of trans-presentation of IFN-γ–induced IL-15 in further studies.
During viral infection, IFN-γ is primarily secreted by NK cells, Th1 CD4+ T cells, and cytotoxic CD8+ T cells in response to Ag or cytokine stimulation (35). Secreted IFN-γ may promote IL-15 production by epithelial cells via IRF1, which in turn activates lymphocytes via trans-presentation of IL-15. This plausible positive feedback may cause aberrant activation of NK or cytotoxic CD8+ T cells and subsequent immune-mediated tissue injury. We previously reported that IL-15 produced by hepatocytes from hepatitis A virus–infected liver activates bystander CD8+ T cells to exert innate-like cytotoxicity that correlates with liver injury (4). In addition, IRF1 has been reported to promote liver transplant ischemia/reperfusion injury via IL-15 production by hepatocytes (44). More recently, Sahoo et al. analyzed transcriptomic datasets generated from tissues autopsied from patients who died of coronavirus disease 2019 (COVID-19) and showed significant increases in the expression of IL-15 and IL-15RA in the lungs from patients with severe COVID-19 (24). Moreover, immunohistochemistry showed that lung epithelial cells from patients with COVID-19 express high levels of IL-15 and IL-15Rα (24). Further studies are needed to investigate the clinical implication of IFN-γ–upregulated IL-15 from epithelial cells in human diseases caused by pathogen infection.
In summary, our results demonstrate that IFN-γ potently induces IL-15 trans-presentation by epithelial cells via IRF1. These findings suggest a critical role of IFN-γ in IL-15 upregulation that is associated with tissue protection or immunopathological tissue injury in infection or inflammatory conditions. In this regard, the IFN-γ–IRF1–IL-15 axis may be a regulatory target for the treatment of diseases with IL-15 dysregulation.
We thank Dr. George Stark and Dr. HyeonJoo Cheon for the gift of the wild-type, STAT1-null, and STAT2-null hTERT-HME1 cells.
This work was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (NRF-2018R1A6A3A01010977) and by the Samsung Science and Technology Foundation under Project SSTF-BA1402-51.
T.-S.K. and E.-C.S. designed the study. T.-S.K. and M.-S.R. performed the experiments. T.-S.K., M.-S.R., and E.-C.S. analyzed the data and wrote the manuscript.
The online version of this article contains supplemental material.
Abbreviations used in this article
coronavirus disease 2019
human telomerase reverse transcriptase human mammary epithelial
- IKK, inhibitor of κB kinase; IL-2/15Rβ
IL-2/IL-15 receptor β-chain
IL-15 receptor α
inhibitor of κB kinase
IFN regulatory factor 1
IFN-stimulated gene factor 3
small interfering RNA
The authors have no financial conflicts of interest.