Circulating levels of a soluble type I IFNR are elevated in diseases, such as chronic inflammation, infections, and cancer, but whether it functions as an antagonist, agonist, or transporter is unknown. In this study, we elucidate the in vivo importance of the soluble type I IFNAR, soluble (s)IFNAR2a, which is generated by alternative splicing of the Ifnar2 gene. A transgenic mouse model was established to mimic the 10–15-fold elevated expression of sIFNAR2a observed in some human diseases. We generated transgenic mouse lines, designated SolOX, in which the transgene mRNA and protein-expression patterns mirrored the expression patterns of the endogenous gene. SolOX were demonstrated to be more susceptible to LPS-mediated septic shock, a disease model in which type I IFN plays a crucial role. This effect was independent of “classical” proinflammatory cytokines, such as TNF-α and IL-6, whose levels were unchanged. Because the increased levels of sIFNAR2a did not affect the kinetics of the increased interferonemia, this soluble receptor does not potentiate its ligand signaling by improving IFN pharmacokinetics. Mechanistically, increased levels of sIFNAR2a are likely to facilitate IFN signaling, as demonstrated in spleen cells overexpressing sIFNAR2a, which displayed quicker, higher, and more sustained activation of STAT1 and STAT3. Thus, the soluble IFNR is an important agonist of endogenous IFN actions in pathophysiological processes and also is likely to modulate the therapeutic efficacy of clinically administered IFNs.

This article is featured in In This Issue, p.3999

Fine-tuning the actions of cytokines, such as IFNs, is critical to their effective modulation of physiological processes, disease pathogenesis, and their therapeutic application without adverse consequences. Type I IFNs are immunoregulatory cytokines with potent activities that impact on many diseases, including cancer, autoimmunity, and particularly, microbial infection. In recent years there has been an explosion in our understanding of how microbial pathogens are detected and the innate immune response activated by pattern recognition receptors, such as TLRs, Nod like-receptors, and RIG-I–like helicases (1). Activation of signaling cascades by these pattern recognition receptors leads to the production of chemokines, inflammatory mediators (e.g., NO), and potent proinflammatory cytokines, such as TNF-α, IL-6, and IFNs, which are necessary to successfully resolve infections. However, excessive production of these proinflammatory mediators can contribute to chronic disease and exaggerated inflammatory responses, resulting in organ damage, hypotension, and hypoperfusion and leading to lethal septic shock (2). The type I IFNs primarily activated by IRF transcription factors are a significant component of TLR4 signaling and play a critical role in mediating the lethal effects of septic shock (35).

Type I IFNs are a multigene family consisting of IFN-α, IFN-β, and other subtypes (δ, ε, ω, κ, τ, ξ), some of which show tissue-specific expression (6). However, all are classified as type I IFNs by their ability to exert their biological effects through binding a receptor complex consisting of at least two transmembrane chains, IFNAR1 and IFNAR2, followed by activation of the intracellular Jak/STAT pathway and others, such as the MAPK pathway and the PI3K pathway (7, 8). Differential splicing of the Ifnar2 gene generates a long transmembrane IFNAR2 (tmIFNAR2; IFNAR2c) and a soluble (s)IFNAR2a isoform (912). tmIFNAR2c is ubiquitously expressed and binds IFN ligands with high (nanomolar) affinity relative to the low-affinity binding of the cognate IFNAR1 chain (13). Although the function of transmembrane IFNARs has been extensively studied, the role of the sIFNAR2a isoform has been less well characterized.

Soluble forms of many cytokine receptors and adhesion molecules are critical regulators of inflammation and immunity and provide mechanisms for modulating cytokine responses (14, 15). Because of their ligand-binding ability, soluble cytokine receptors can function as antagonists by binding to the ligand and competing with the transmembrane form of the receptor (16). Alternatively, they can act as carrier proteins for the ligand to protect it from proteolysis, improve stability, modulate tissue distribution, or decrease clearance (17, 18). Soluble receptors also can function as agonists by modulating ligand pharmacokinetics or potentiating signaling by a mechanism known as trans-signaling: a ligand binds its high-affinity soluble receptor and then interacts with a transmembrane low-affinity receptor chain to transduce a biological signal (19, 20). IL-6 trans-signaling is a well-characterized example of this mechanism (1921).

sIFNAR2a were first identified in human urine (22). Subsequently, mouse Ifnar2 was cloned, and a mechanism of alternative splicing, similar to that in humans, was demonstrated to generate a soluble receptor isoform in the mouse (12). Previous experiments demonstrated that recombinant murine sIFNAR2a and the extracellular domain of tmIFNAR2 can both bind IFN with nanomolar affinity (13, 2327). Subsequently, we demonstrated that sIFNAR2a can modulate IFN actions in vitro as either an agonist or an antagonist, analogous to other soluble cytokine receptors (24). However, there was no direct evidence for an in vivo function. The sIfnar2a transcript was found to be abundantly expressed in most tissues, often at >10-fold higher levels than the tmIfnar2 transcript (24), and it appeared to be regulated independently of the transmembrane isoform. sIFNAR2a was detected in mouse body fluids using specific Abs (12, 24). Furthermore, increased levels of serum sIFNAR2a were reported in patients with chronic viral infections (e.g., HCV or HIV), inflammatory diseases (e.g., systemic lupus erythematosus), and cancers (e.g., hairy cell leukemia, chronic myelogenous leukemia, various adenocarcinomas, and renal cell carcinoma) (22, 2832). These findings imply an in vivo function for sIFNAR2a. Although most reports have speculated an inhibitory function, definitive evidence of its in vivo action was lacking.

Given the potent antiviral, immunoregulatory, and antiproliferative actions of IFNs, increased sIFNAR2a in the aforementioned diseases could modulate disease pathogenesis regulated by endogenous IFNs. sIFNAR2a also may influence the efficacy of IFN administered for clinical purposes in chronic viral infections or multiple sclerosis, for example. To determine the in vivo role of the soluble type I IFNR and, in particular, to model the human disease status of overexpressed sIFNAR2a, we generated a transgenic mouse model that overexpresses the soluble receptor under the control of its endogenous promoter with the aim of replicating temporal and tissue-specific regulation of expression. We termed this mouse line SolOX (soluble IFNAR2a overexpressing). Because the IFN pathway is important to the outcome of TLR4-mediated signaling (33, 34), we used the TLR4-mediated LPS-induced septic shock model to determine the effect of sIFNAR2a overexpression on disease outcomes. We demonstrated that excess sIFNAR2a exacerbates LPS-induced IFN-β effects independently of the MyD88-dependent pathway, which initiates production of proinflammatory cytokines, such as IL-6 and TNF-α. Importantly, our investigation of IFN signaling in SolOX mice demonstrates that sIFNAR2a does not block, but rather amplifies IFN responses in vivo by a direct agonistic action on signal transduction without impacting on serum IFN kinetics. In this study we demonstrate the in vivo actions of sIFNAR with implications for chronic diseases in which elevated levels of soluble receptor have been reported and for modulation of exogenous, therapeutic IFN.

The murine sIfnar2a-transgenic minigene construct was produced by fusing the 8.5-kb genomic region immediately 5′ of the putative transcription start site, exon 1 and intron 1 of the Ifnar2 gene with sIfnar2a cDNA starting from exon 2 to its 3′ end. The 8.5-kb 5′ flanking region, exon 1 and 8 kb of intron 1 of Ifnar2, was cloned from genomic DNA into a pBluescript vector (Stratagene, La Jolla, CA). sIfnar2a cDNA (RefSeq accession number NM_001110498), consisting of 165–1031 bp of sequence region with a stop codon and polyadenylation signal previously described (12) and the remainder of intron 1, was PCR amplified and subcloned into pBluescript using the EcoRV and ClaI sites. A secondary PCR reaction using splicing by overlap extension (35) was used to fuse the fragments containing exon 1, 5′ intron 1 with 3′ intron 1 and sIfnar2a. These two intron 1–containing fragments were then PCR amplified and cloned in-frame into pGEM-T (Promega, Madison, WI); the resultant fragment was excised and directionally cloned into the pBluescript vector containing the 5′ flanking region to produce the sIfnar2a minigene construct (Fig. 1A). All plasmids were checked for correct sequences by restriction digestion and sequencing. The 20.5-kb minigene DNA was excised from the vector using SalI, purified by dialysis, microinjected into pronuclei of fertilized eggs of F1 mice, and transferred into pseudopregnant recipient females.

FIGURE 1.

Generation and characterization of SolOX mice. (A) Schematic diagram of the Ifnar2 genomic locus and the “minigene” transgenic construct used to generate soluble Ifnar2a-transgenic mice. Restriction enzyme sites used in minigene construction and Southern blotting are also shown. The black boxes labeled E1–E9 represent exons, and the arrows labeled B114 and MIR2-19 designate the oligonucleotide primers used to screen by PCR for the presence of the transgene. (B) PCR genotyping of mice. Mice were genotyped as transgenic or wild-type based on a PCR assay of genomic DNA extracted from mouse tail clippings, using a forward primer hybridizing to exon 3 (B114) and a reverse primer hybridizing to sIfnar2a specific exon 7′ (MIR2-19). This PCR reaction only amplified a 682-bp fragment from the minigene integrated into transgenic genomic DNA and not the 8-kb region from the endogenous Ifnar2 gene under the conditions used for PCR. The presence of the transgene in three founder mouse lines and its absence in DNA from wild-type and Ifnar2−/− mice demonstrate the specificity of the screening assay. (C) Transgene copy number was determined by quantitative Southern blot analysis. Southern blots were hybridized with a 32P-labeled DraI and MspI DNA fragment encoding the sIfnar2a minigene. Standards generated by serially diluting known amounts (1–100 copies) of the enzyme-digested transgene fragment are shown by bands on the left side of the blot, whereas DraI and MspI enzyme-digested genomic DNA from three sIfnar2a-transgenic mice (Tg3, Tg7, and Tg6) and two wild-type (WT) mice are presented on the right side of the blot.

FIGURE 1.

Generation and characterization of SolOX mice. (A) Schematic diagram of the Ifnar2 genomic locus and the “minigene” transgenic construct used to generate soluble Ifnar2a-transgenic mice. Restriction enzyme sites used in minigene construction and Southern blotting are also shown. The black boxes labeled E1–E9 represent exons, and the arrows labeled B114 and MIR2-19 designate the oligonucleotide primers used to screen by PCR for the presence of the transgene. (B) PCR genotyping of mice. Mice were genotyped as transgenic or wild-type based on a PCR assay of genomic DNA extracted from mouse tail clippings, using a forward primer hybridizing to exon 3 (B114) and a reverse primer hybridizing to sIfnar2a specific exon 7′ (MIR2-19). This PCR reaction only amplified a 682-bp fragment from the minigene integrated into transgenic genomic DNA and not the 8-kb region from the endogenous Ifnar2 gene under the conditions used for PCR. The presence of the transgene in three founder mouse lines and its absence in DNA from wild-type and Ifnar2−/− mice demonstrate the specificity of the screening assay. (C) Transgene copy number was determined by quantitative Southern blot analysis. Southern blots were hybridized with a 32P-labeled DraI and MspI DNA fragment encoding the sIfnar2a minigene. Standards generated by serially diluting known amounts (1–100 copies) of the enzyme-digested transgene fragment are shown by bands on the left side of the blot, whereas DraI and MspI enzyme-digested genomic DNA from three sIfnar2a-transgenic mice (Tg3, Tg7, and Tg6) and two wild-type (WT) mice are presented on the right side of the blot.

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Ten sIfnar2a-transgenic founders were identified by primary screens using Southern blots of genomic DNA from F1 mice (Monash Animal Research Platform). Mouse plasma was obtained by orbital bleeds using heparinized capillary tubes or by cardiac puncture. Plasma sIFNAR2a levels were analyzed by SDS-PAGE and chemifluorescence-based immunoblotting. This procedure identified three transgenic founders expressing higher than wild-type levels of sIFNAR2a. These transgenic mice were designated SolOX mice. Founder males were bred with F1 (C57BL/6 × CBA) females, and three transgenic lines were established. Six- to ten-week-old, age-matched mice were used in all experiments. Wild-type littermates were used as controls for sIfnar2a-transgenic experiments, and C57BL/6 wild-type mice were used as controls for Ifnar1−/− (36) and Ifnar2−/− (37) mouse experiments. All mouse experiments were conducted with approval from the Monash University animal ethics committee.

The sIfnar2a minigene construct and genomic DNA extracted from wild-type or transgenic mouse tail snips and the sIfnar2a minigene construct were digested with DraI and MspI. Known amounts of the digested minigene were serially diluted to generate a standard curve representing a range of 1–100 gene copies. DNA digests were electrophoresed through 1% agarose gels and then transferred onto Gene Screen Plus (PerkinElmer, Glen Waverley, VIC, Australia) nylon membrane using 0.4 M NaOH capillary transfer. Membranes were soaked in 2× SSC and dried. The blots were hybridized with the 32P-labeled DraI– and MspI-digested sIfnar2a minigene probe and visualized on a Fuji FLA-2000 phosphorimager (Fujifilm, Tokyo, Japan). Densitometry was performed using Image Gauge v3.46 software (Fujifilm), and the number of transgene copies integrated into the genome of each mouse line was determined by comparing the band intensity of the genomic samples to that of the standards.

Genomic DNA was extracted from tail clippings, and oligonucleotide primers B114 (5′-GCTCTAGATCAAAACAATAGTGCAAATTTTTA-3′) and MIR2-19 (5′-CTAGAGACTATCACACCGTC-3′), complementary to regions in exon 4 and exon 7′ of the Ifnar2 gene, respectively, were used to amplify a 682-bp region of the minigene integrated in the transgenic mice. Detection of the endogenous gene would necessitate amplification of an 8-kb fragment across introns 4, 5, and 6 that was not generated with the conditions used.

Poly(A)+ mRNA from wild-type and SolOX-transgenic mouse tissues was prepared as previously described (38). Poly(A)+ mRNA was hybridized on Northern blots, as previously described, using a 32P-labeled full-length Ifnar2 cDNA probe (12). The blots also were probed with a 32P-labeled 1.2-kb Gapdh cDNA probe to determine equal loading of the mRNA. Northern blots were exposed to either film (Eastman Kodak, Rochester, NY) or a Phosphor screen, and quantitative analysis was performed using an FLA-2000 phosphorimager and Image Gauge v3.46 software (both from Fujifilm).

Age-matched SolOX-transgenic and wild-type littermates were injected i.p. with either 50 or 100 μg repurified Escherichia coli K-235 bacterial LPS (Sigma-Aldrich, Castle Hill, NSW, Australia) per 1 g body weight. The LPS was repurified using a previously described method (39). The viability of mice was examined every hour, or animals were killed at regular intervals (0, 1, 1.5, 2, 3, 4, 5, 6, 12, 24 h postinjection), and plasma and organs were collected. Organ samples were frozen in liquid nitrogen for RNA isolation, homogenized at 4°C for immunoblotting, fixed in formalin for histological processing, or processed for flow cytometry.

Mice were treated with i.p. injection of 50 μg/mouse CpG oligonucleotides 2216 (Type A; Innaxon Biosciences, Tewkesbury, U.K.) in Dulbecco’s PBS, and peritoneal exudate cells and spleens were collected after 1 or 3 h. Peritoneal exudate cells were obtained by flushing the peritoneal cavity with ice-cold Dulbecco’s PBS, as described previously (40), and single-cell suspensions of spleen cells were obtained before fixation and permeabilization for intracellular staining of phosphorylated STATs and analysis by flow cytometry, as described below.

Groups of mice were injected with LPS and killed at intervals, described above, over a time course of 12 h. Plasma samples were collected by cardiac puncture, and serum was assayed for TNF-α, IL-6, and IFN-β by ELISA. IL-6 (clone MP5-20F3, clone MP5-32C11) and TNF-α (clone G281-2626, clone MP6-XT3) capture and biotinylated detection Abs were purchased from BD Biosciences (North Ryde, NSW, Australia); IFN-β capture (clone 7F-D3) and detection Abs were from Seikagaku (Tokyo, Japan) and PBL Biomedical (Piscataway, NJ), respectively. Capture Abs were diluted in binding buffer (0.1 M Na2H2PO4) and coated overnight in 96-well Immunosorp plates (Nunc, Roskilde, Denmark). The plates were blocked in BSA, washed in PBS/Tween, and incubated with samples and standards (rIL-6, rTNF-α from BD Biosciences; rIFN-β from Sigma-Aldrich). Plates were washed with PBS and 0.1% (v/v) Tween 20, incubated with HRP-conjugated streptavidin, and color developed using 1 mM ABTS substrate. Absorbance was measured at 405 nm using the FLUOstar OPTIMA imaging system (BMG Labtech, Mornington, VIC, Australia). Standard curves were plotted using recombinant cytokine standards (rIL-6, rTNF-α, and rIFN-β), and sample concentrations were obtained by comparison.

Serum NO levels were assessed by measuring nitrite (NO2) using the Griess assay system kit (Promega), according to the manufacturer’s instructions. Absorbance at 520 nm was measured using the FLUOstar OPTIMA imaging system (BMG Labtech).

Murine L929 cells were cultured in RPMI 1640 supplemented with 10% FCS and 1% penicillin/streptomycin (Life Technologies, Melbourne, VIC, Australia). The antiviral activity of IFN preparations and serum samples was determined by a cytopathic effect reduction antiviral bioassay using L929 cells grown in 3% FCS and Semliki forest virus (37). National Institutes of Health international reference IFN preparation and rIFN-α4 (a gift from D. Gewert, BioLauncher, Cambridge, UK) were used as standards, as previously described (37).

Wild-type serum was analyzed by immunoblotting using a rabbit polyclonal anti-IFNAR2 primary Ab (24) and an anti-rabbit alkaline phosphatase–conjugated secondary Ab, together with ECF chemifluorescent reagent (GE Healthcare, Rydalmere, NSW, Australia). This method enabled direct visualization and quantitation of serum sIFNAR2a levels using an FLA-2000 phosphorimager. A standard curve consisting of known dilutions of serum was used to determine the linear range of the signal and the sensitivity of the assay. All transgenic serum sIFNAR2a levels were expressed relative to the wild-type serum sIFNAR2a level.

To detect in vivo IFN signaling, spleens were harvested from LPS-treated and untreated mice. Lysates were prepared with a Potter-Elvehjem homogenizer using equal weight/volume of phosphoprotein lysis buffer (50 mM Tris [pH 7.4], 1% (v/v) Nonidet P-40, 10% (v/v) glycerol, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF, 1 mM DTT, 4.5 mM sodium pyrophosphate and protease inhibitor mixture [Sigma-Aldrich]). Samples were sonicated on ice for 20 s. The phosphorylation state of signaling molecules was analyzed by immunoblotting using anti-STAT Abs (Cell Signaling Technologies, Arundel, QLD, Australia). Equal amounts of protein or equal volumes of mouse serum were loaded, and proteins were resolved by 8, 10, or 15% SDS-PAGE. Proteins were transferred to Immobilon-P or Immobilon-FL (polyvinylidene difluoride) membranes (Merck Millipore, Kilsyth, VIC, Australia) using a semidry apparatus (Bio-Rad, Gladesville, NSW, Australia), according to the manufacturer’s instructions. Membranes were incubated with an anti-mouse IFNAR2 Ab (1:2000, for tissue and serum) (24) or STAT-1, STAT-1P, STAT-3, or STAT-3P (1:1000; for spleen lysates) (Cell Signaling Technologies), and the signal was detected using an appropriate (HRP- or alkaline phosphatase–conjugated or fluorescence-tagged) secondary Ab (Dako, Noble Park, VIC, Australia). Blots were visualized using an FLA-2000 phosphorimager and Image Gauge software (both from Fujifilm) or an Odyssey scanner and Odyssey image software (both from LI-COR Biosciences, Lincoln, NE).

Single-cell suspensions from the spleen were prepared (41). Cells were rested at 37°C for 1 h prior to stimulation with IFN-β (Sigma-Aldrich) at 1000 IU/ml for 0, 15, 30, 60, or 120 min. Immediately following stimulations, cells were fixed in 1% buffered formalin/PBS for 10 min at 37°C. Fixation was stopped by addition of 1 ml ice-cold PBS. Cells were permeabilized in 1 ml 90% methanol and stored at −80°C until staining for flow cytometry, at which point frozen cells were washed in flow cytometry buffer (0.5% BSA, 100 mM NaF, 1 mM NaV, 10 mM β-glycerophosphate, and 4.5 mM Na pyrophosphate in PBS) on ice. Staining for levels of tyrosine phosphorylated STAT1 and STAT3 was performed, per the manufacturer’s protocol (BD Biosciences), using Alexa Fluor 647–conjugated anti-Phospho-STAT1 (pY701) or anti-Phospho-STAT3 (pY705). Data were collected on a BD FACSCanto II and analyzed using FlowJo software. Gates were determined using Fluorescence Minus One controls. Data are expressed as mean fluorescence intensity.

Wild-type and SolOX-H mice were injected with 50 μg repurified LPS/1 g body weight, and spleens were harvested 6 or 24 h later to determine populations undergoing apoptosis. Single-cell suspensions of spleen from LPS-treated and untreated, wild-type and SolOX-H mice were prepared in PBS. For assessment of total spleen cell apoptosis, cells were blocked with anti-CD16/32 Ab (eBioscience), labeled using an Annexin V–FITC Apoptosis Detection Kit II (BD Pharmingen), acquired within 1 h of labeling by flow cytometry (FACSCanto; BD Immunocytometry Systems), and analyzed using FlowJo software.

Mice were sacrificed, and peripheral blood was collected by cardiac puncture. Hematopoietic organs, such as thymus, spleen, and bone marrow, were assessed for total organ cellularity by making single-cell suspensions. Viable cells were counted in a hemocytometer using trypan blue exclusion. FACS analysis (FACStar plus cell sorter; BD Biosciences) was used to determine the proportions of T cell and B cell lineage markers (CD4, CD8, CD3, B220, IgM, IgD) and macrophage, granulocyte, and neutrophil markers (Mac-1, GR-1), as previously described (42). Activation status was determined by levels of CD69 (eBioscience) on CD4+ T cells and by levels of CD80 (B7-1) and CD86 (B7-2; both from eBioscience) on B220+ B cells in the spleen. Anti-mouse IFNAR1 (MAR1-5A3; BioLegend) and anti-mouse IFN-αβ R2 (R&D Systems) Abs were used to determine the surface expression of Ifnar1 and Ifnar2 on spleen cells. Analysis gates were determined using Fluorophore Minus One gating controls or matched isotype controls, as indicated.

Spleens were snap-frozen and homogenized before RNA isolation using an RNeasy Kit (QIAGEN), as described previously (6, 40). Relative expression was determined using the ΔΔCt method, and genes were normalized to 18S rRNA. Cxcl9 was measured using a premade TaqMan gene expression assay, and Cxcl10 (forward primer 5′-AGAGCAGCACTTGGGTTC-3′, reverse primer 5′-ACGGCAGCACTTGGGTTC-3′) and Ccl5 (forward primer 5′-ATATGGCTCGGACACCACTC-3′, reverse primer 5′-GTGACAAACACGACTGCAAGA-3′) were measured using SYBR reagents (Applied Biosystems).

All data are reported as mean ± SEM. Data were analyzed by ANOVA using the Student t test. The Kaplan–Meier method was used to analyze survival. The log-rank test was used to compare survival curves. Differences were considered significant when p < 0.05. All analyses were done using GraphPad Prism software (GraphPad, La Jolla, CA).

Following pronuclear embryo injection of the soluble Ifnar2 minigene construct (Fig. 1A), 10 “founder” mice were identified as carrying the transgene by PCR genotyping, which identified the 682-bp transgene band (three are shown in Fig. 1B). The larger 8-kb fragment, which would be generated from the endogenous gene (Fig. 1A), was not amplified using the conditions described. The transgenic lines appeared to contain between one and five copies of the transgene (Fig. 1C).

Serum sIFNAR2a levels in the 10 transgenic founder lines were quantified by phosphor image analyses of Western blots (Fig. 2A) and expressed relative to wild-type levels (Fig. 2A). Three transgenic lines with 13-, 4-, or 2-fold sIFNAR2a serum levels relative to wild-type levels were selected for further analysis and were designated SolOX-H, SolOX-L1, and SolOX-L2, respectively (Fig. 2A).

FIGURE 2.

Overexpression of sIFNAR2s in SolOX mice. (A) Graph of serum sIFNAR2a levels in the 10 transgenic founder mice relative to wild-type serum sIFNAR2a levels. Three lines, expressing ∼2-fold (SolOX-L1), 4-fold (SolOX-L2), and 13-fold (SolOX-H) greater sIFNAR2a relative to wild-type serum levels, were identified for further studies. Inset, Representative Western blot of mouse serum probed with an anti-murine polyclonal IFNAR2 Ab. (B) Northern blot analysis of wild-type and SolOX-H mouse organs demonstrating increased sIfnar2a expression levels in organs from SolOX-H mice. Poly(A)+ mRNA was extracted from tissues and organs from wild-type and SolOX-H mice, and Northern blot analysis was performed using a 32P-labeled full-length Ifnar2 cDNA probe. Filters were washed and reprobed with a Gapdh probe. The band intensity was quantified using phosphorimager analysis. Results shown are representative of all three transgenic lines. Possibly as a result of the high levels of expression, a band representing an unprocessed transcript of 1.8 kb also was detected in all three SolOX founders. (C) Ifnar2 transcript levels in wild-type (n = 3) and SolOX-H (n = 3) mouse organs. Transcript levels relative to Gapdh were quantified by phosphorimager analysis of Northern blots. The band density of tmIfnar2 (black bars) and sIfnar2a (striped bars) is expressed in relative intensity units. Data are expressed as mean ± SEM. (D) Tissue sIFNAR2a protein levels in lungs and kidneys of SolOX-H and wild-type mice. Western blot analysis was performed with a rabbit anti-murine polyclonal IFNAR2 Ab (24).

FIGURE 2.

Overexpression of sIFNAR2s in SolOX mice. (A) Graph of serum sIFNAR2a levels in the 10 transgenic founder mice relative to wild-type serum sIFNAR2a levels. Three lines, expressing ∼2-fold (SolOX-L1), 4-fold (SolOX-L2), and 13-fold (SolOX-H) greater sIFNAR2a relative to wild-type serum levels, were identified for further studies. Inset, Representative Western blot of mouse serum probed with an anti-murine polyclonal IFNAR2 Ab. (B) Northern blot analysis of wild-type and SolOX-H mouse organs demonstrating increased sIfnar2a expression levels in organs from SolOX-H mice. Poly(A)+ mRNA was extracted from tissues and organs from wild-type and SolOX-H mice, and Northern blot analysis was performed using a 32P-labeled full-length Ifnar2 cDNA probe. Filters were washed and reprobed with a Gapdh probe. The band intensity was quantified using phosphorimager analysis. Results shown are representative of all three transgenic lines. Possibly as a result of the high levels of expression, a band representing an unprocessed transcript of 1.8 kb also was detected in all three SolOX founders. (C) Ifnar2 transcript levels in wild-type (n = 3) and SolOX-H (n = 3) mouse organs. Transcript levels relative to Gapdh were quantified by phosphorimager analysis of Northern blots. The band density of tmIfnar2 (black bars) and sIfnar2a (striped bars) is expressed in relative intensity units. Data are expressed as mean ± SEM. (D) Tissue sIFNAR2a protein levels in lungs and kidneys of SolOX-H and wild-type mice. Western blot analysis was performed with a rabbit anti-murine polyclonal IFNAR2 Ab (24).

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sIfnar2a expression also was examined by Northern blot analysis in a number of organs from wild-type and SolOX mice. As previously reported (43), relatively high sIfnar2a transcript levels were observed in liver and salivary gland of wild-type mice, with lower levels in kidney and testis (Fig. 2B). Increased expression of sIfnar2a transcripts was observed in these organs of all three independently derived transgenic mouse lines (SolOX-H as a representative sample is shown in Fig. 2B). Overall, the relative levels of sIfnar2a transcripts from SolOX animals mirrored the general expression patterns seen in wild-type animals (i.e., liver > salivary gland > kidney > testis) (Fig. 2C), corroborating the effectiveness of the minigene construct promoter and intron sequences used to drive the transgene in replicating the endogenous expression pattern. Importantly, there was no substantial difference in tmIfnar2 (Ifnar2c) transcript levels between SolOX and wild-type mice (Fig. 2B, 2C). Furthermore, there were no differences in the surface expression levels of IFNAR1 and IFNAR2 on spleen cells assessed by flow cytometry (data not shown). When sIFNAR2a protein levels were determined in mouse tissue by immunoblotting, high levels of sIFNAR2a were observed in SolOX tissue compared with wild-type tissue (Fig. 2D), consistent with the higher mRNA expression levels in these organs.

To investigate the role of sIFNAR2a in vivo, we set out to determine whether increased levels of sIFNAR2a in SolOX mice could modulate pathological effects in response to endotoxin, because TLR4-mediated IFN-β production is known to contribute to the pathological consequences of LPS (33).

To examine the systemic effects of sIFNAR2a on IFN-mediated LPS toxicity, we chose SolOX-H mice because the level of sIFNAR2a in serum had increased by a similar degree (10–15-fold) as reported in patients with chronic inflammatory diseases. We injected (i.p.) repurified LPS (50 or 100 μg/g of body weight) into age-matched wild-type and SolOX-H mice. At 72 h after injection of 100 μg/g of LPS, 36% of wild-type mice were alive (Fig. 3A). Surprisingly, only 10% of SolOX-H mice survived (Fig. 3A). SolOX-H mice were more susceptible to LPS-mediated septic shock compared with wild-type controls (p = 0.014, log-rank test), indicating that increased lethality occurred earlier. When a lower dose of LPS (50 μg/g) was used, a more pronounced difference was observed: within 20 h, SolOX-H mice showed 100% lethality compared with 42% for wild-type mice (Fig. 3B). Thus, at both doses of LPS, SolOX-H mice were more susceptible to LPS-induced septic shock. Experiments in SolOX-L mice at the higher dose of LPS showed no difference in susceptibility to LPS relative to wild-type mice (data not shown). This may indicate that a threshold-level increase in sIFNAR2a is necessary for a significant pathological outcome.

FIGURE 3.

Effect of sIFNAR2a on endotoxic shock in mice. (A) Evaluation of survival in response to LPS by SolOX-H mice relative to wild-type controls. A total of 100 μg/g body weight repurified LPS was injected i.p. (n = 20–25 mice). Decreased survival by SolOX-H mice relative to wild-type mice was observed (p = 0.0145, log-rank test). (B) Evaluation of survival in response to LPS in SolOX-H mice relative to wild-type controls. Repurified LPS was injected (50 μg/g) i.p. (n = 6–7). Decreased survival was observed in SolOX-H mice.

FIGURE 3.

Effect of sIFNAR2a on endotoxic shock in mice. (A) Evaluation of survival in response to LPS by SolOX-H mice relative to wild-type controls. A total of 100 μg/g body weight repurified LPS was injected i.p. (n = 20–25 mice). Decreased survival by SolOX-H mice relative to wild-type mice was observed (p = 0.0145, log-rank test). (B) Evaluation of survival in response to LPS in SolOX-H mice relative to wild-type controls. Repurified LPS was injected (50 μg/g) i.p. (n = 6–7). Decreased survival was observed in SolOX-H mice.

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To determine whether sIFNAR2a-mediated signaling events broadly impacted on TLR signaling, we measured the levels of NF-κB–dependent cytokines TNF-α and IL-6, as well as serum nitrite, as an indicator for NO and IFN-β, after LPS administration (Fig. 4). Two distinct waves of IL-6 and TNF-α production were observed after LPS injection: an early peak (within 1.5–3 h) and a late peak (>12 h) were observed in both wild-type and SolOX mice (Fig. 4A, 4B). There were no significant differences in IL-6 and TNF-α levels between wild-type and SolOX mice (Fig. 4A, 4B). The secondary TNF-α response was much stronger than the initial response and may be responsible for some of the toxic effects of sepsis (Fig. 4B). Another key mediator in sepsis, inducible NO synthase, which is involved in NO production, is induced by proinflammatory cytokines, as well as by both type I and type II IFNs (44, 45). No difference in nitrite was observed between LPS-treated SolOX and wild-type mice (Fig. 4C).

FIGURE 4.

Increased sIFNAR2a expression does not affect NF-κB–mediated proinflammatory cytokine expression. (A) Serum IL-6 determined by ELISA in wild-type (n = 6) and SolOX-H (n = 6) mice determined 1.5–12 h postadministration of LPS. Data are mean ± SEM. (B) Serum TNF determined by ELISA in wild-type (n = 6) and SolOX-H (n = 6) mice after administration of LPS. Data are mean ± SEM. (C) Serum NO levels were determined by Griess assay at various times after administration of LPS (n = 6). Data are mean ± SEM. (D) Serum IFN levels were determined at 1, 3, 5, and 12 h post-LPS administration by cytopathic effect reduction antiviral bioassay (n = 6). Data are mean ± SEM. (E) Serum IFN-β levels, measured using an IFN-β–specific ELISA assay, at 0, 3, and 12 h post-LPS treatment (n = 3). Data are mean ± SEM.

FIGURE 4.

Increased sIFNAR2a expression does not affect NF-κB–mediated proinflammatory cytokine expression. (A) Serum IL-6 determined by ELISA in wild-type (n = 6) and SolOX-H (n = 6) mice determined 1.5–12 h postadministration of LPS. Data are mean ± SEM. (B) Serum TNF determined by ELISA in wild-type (n = 6) and SolOX-H (n = 6) mice after administration of LPS. Data are mean ± SEM. (C) Serum NO levels were determined by Griess assay at various times after administration of LPS (n = 6). Data are mean ± SEM. (D) Serum IFN levels were determined at 1, 3, 5, and 12 h post-LPS administration by cytopathic effect reduction antiviral bioassay (n = 6). Data are mean ± SEM. (E) Serum IFN-β levels, measured using an IFN-β–specific ELISA assay, at 0, 3, and 12 h post-LPS treatment (n = 3). Data are mean ± SEM.

Close modal

There were no differences observed between LPS-treated wild-type and SolOX mice with regard to LPS-induced IFN levels, as determined by an antiviral activity bioassay, or IFN-β levels, as determined by ELISA (Fig. 4D, 4E, respectively). Furthermore, no difference in the kinetics of increase or decrease in IFN production was observed. Therefore, the elevated levels of sIFNAR2a did not affect the kinetics of interferonemia. The concordance between the two methods of measuring IFN–bioassay and immunoassay–indicate that the presence of soluble receptor did not interfere with the bioassay. Overall, the increased levels of sIFNAR2a did not impact on early events of the primary TLR response, such as production of NF-κB–regulated proinflammatory cytokines or NO or IRF-mediated IFN-β production. This indicates that sIFNAR2a may modulate secondary TLR4-mediated IFN signals.

Binding of IFNs to the IFNAR complex leads to activation of the Jak/STAT signal-transduction pathway, resulting in tyrosine-phosphorylated STAT1- and STAT3-containing transcription factor complexes (46, 47) that mediate the biological effects attributed to IFN (48). To demonstrate that increased sIFNAR2a levels were potentiating IFN signaling, we analyzed an in vivo time course of STAT1 and STAT3 tyrosine phosphorylation in spleen cells from SolOX-H and wild-type mice after LPS treatment. Earlier and more sustained STAT1 phosphorylation was observed in SolOX-H mice compared with wild-type mice (Fig. 5A). In SolOX-H mice, peak levels of phospho- STAT1 were detected at 1 and 2 h after LPS treatment, as well as at reduced, but still relatively high, levels even after 6 h. In contrast, in wild-type mice STAT1 phosphorylation was barely detectable 1 h after treatment, peaked at 2 h, and decreased thereafter. This increase in STAT1 did not affect the gene expression of type I IFN–induced chemokines Cxcl9, Cxcl10, or Ccl5, which was comparable between genotypes upon LPS treatment (Supplemental Fig. 1). There were no differences in total STAT1 levels between SolOX-H and wild-type mice. To determine the type I IFN–dependent component in STAT1 phosphorylation, we analyzed STAT1 signaling in splenocytes from Ifnar1−/− mice after in vivo LPS treatment. STAT1 phosphorylation was not detected 1–3 h after LPS treatment in these knockout mice, indicating that early STAT1 tyrosine phosphorylation induced by LPS was entirely type I IFN dependent. However, STAT1 tyrosine phosphorylation was observed at 4 and 6 h after LPS treatment in Ifnar1−/− mice, indicating that, at this time, STAT phosphorylation was type I IFN independent. This late signaling was probably mediated by other LPS-induced cytokines, such as IL-6 or IFN-γ, which are known to activate these STATs. In summary, elevated serum sIFNAR2a resulted in enhanced LPS-induced, type I IFN STAT signaling (Fig. 6).

FIGURE 5.

LPS-induced IFN signaling. In vivo assessment of Jak/STAT signaling in response to LPS. Mice were injected i.p. with LPS; spleens were collected and lysates were prepared 0, 1, 2, 3, 4, and 6 h later. Lysates were examined by immunoblotting for total and tyrosine phosphorylated STAT1 and STAT3 levels. (A) LPS-induced STAT1 phosphorylation in SolOX-H, wild-type, and Ifnar1−/− mice (n = 3). (B) LPS-induced STAT3 phosphorylation in SolOX-H and wild-type mice (n = 3). (C) Increased sIFNAR2a in mice increases both the intensity and the duration of IFN-β–induced activation of STAT1 and STAT3 tyrosine phosphorylation in the spleen. Data are representative of two separate repeat experiments. (D) Immune cell subsets (T cell and B cell lineage markers (CD4, CD8, CD3, B220, IgM, and IgD) and macrophage, granulocyte, and neutrophil markers (Mac-1, GR-1) were analyzed by flow cytometry in spleen from SolOX-H mice (n = 4).

FIGURE 5.

LPS-induced IFN signaling. In vivo assessment of Jak/STAT signaling in response to LPS. Mice were injected i.p. with LPS; spleens were collected and lysates were prepared 0, 1, 2, 3, 4, and 6 h later. Lysates were examined by immunoblotting for total and tyrosine phosphorylated STAT1 and STAT3 levels. (A) LPS-induced STAT1 phosphorylation in SolOX-H, wild-type, and Ifnar1−/− mice (n = 3). (B) LPS-induced STAT3 phosphorylation in SolOX-H and wild-type mice (n = 3). (C) Increased sIFNAR2a in mice increases both the intensity and the duration of IFN-β–induced activation of STAT1 and STAT3 tyrosine phosphorylation in the spleen. Data are representative of two separate repeat experiments. (D) Immune cell subsets (T cell and B cell lineage markers (CD4, CD8, CD3, B220, IgM, and IgD) and macrophage, granulocyte, and neutrophil markers (Mac-1, GR-1) were analyzed by flow cytometry in spleen from SolOX-H mice (n = 4).

Close modal
FIGURE 6.

Increased sIFNAR2a levels in SolOX mice potentiate IFN-β–mediated toxic effects of LPS. TLR4 recognition of LPS and the resultant signaling via the MyD88-independent, Trif-dependent pathway lead to production of IFN-β, which, in turn, signals via the IFNAR complex. In SolOX mice, increased sIFNAR2a levels mediate rapid and intense IFN signaling, leading to potentiated LPS toxicity.

FIGURE 6.

Increased sIFNAR2a levels in SolOX mice potentiate IFN-β–mediated toxic effects of LPS. TLR4 recognition of LPS and the resultant signaling via the MyD88-independent, Trif-dependent pathway lead to production of IFN-β, which, in turn, signals via the IFNAR complex. In SolOX mice, increased sIFNAR2a levels mediate rapid and intense IFN signaling, leading to potentiated LPS toxicity.

Close modal

Similarly, earlier and stronger STAT3 phosphorylation was seen in SolOX-H mice compared with wild-type mice after LPS injection (Fig. 5B). In SolOX-H mice, STAT3 reached peak levels at 1 h post-LPS compared with 2 h in wild-type mice, and signaling was higher at 3 h in the former.

To examine the direct effect of IFN-β, we stimulated spleen cells in vitro with rIFN-β and observed increased levels of intracellular tyrosine-phosphorylated STAT1 and STAT3 in SolOX-H mice compared with wild-type mice (Fig. 5C). These data further strengthen the idea that the type I IFN response is potentiated in cells overexpressing sIFNAR2a (from SolOX-H mice) and acts to promote increased IFN signaling through the Jak/STAT pathway. There was no change noted in phosphorylated STAT5 levels in spleen cells following IFN-β stimulation (data not shown). Similarly, stimulation of thymocytes from SolOX-H mice also demonstrated elevated IFN-β–induced activation of STAT1 and STAT3 (data not shown). No phosphorylated STAT1/3/5 activity was detected by flow cytometry in spleen or thymic cells from IFNAR1−/− or IFNAR2−/− mice up to 2 h poststimulation with IFN-β (data not shown). In vitro stimulation of spleen and thymus cells with IFN-α also demonstrated similar results, with consistently elevated and prolonged IFN-α–induced phosphorylated STAT1 and STAT3 (data not shown). However, in vivo stimulation with CpG-ODN 2216 to specifically stimulate TLR9, which reportedly induces IFN-α production, did not reveal differences in phosphorylated STAT1 or STAT3 levels in peritoneal cells from wild-type and SolOX-H mice at 1 or 3 h postinjection (Supplemental Fig. 2), implying that the effect is TLR4/IFN-β specific. STAT1 or STAT3 activation could not be detected by flow cytometry in the spleen at 1 or 3 h post-i.p. CpG-ODN 2216 treatment (data not shown).

To ensure that the signaling defects in the LPS model were not due to a perturbation in cellular subsets of the spleen, immunophenotyping of mouse spleen was performed. The incidence of T cells (CD3, CD4, CD8), B cells (B220, IgM, IgD), granulocytes (Gr-1), and macrophages (Mac-1) was determined by flow cytometry using specific cell surface markers; it was similar in SolOX-H mice and their wild-type littermates (Fig. 5D). Furthermore, differences in signaling were not due to differences in the activation states (Supplemental Fig. 3) or apoptosis rates (Supplemental Fig. 4) of B or T cells in the spleens of mice following LPS treatment.

Although soluble cytokine receptors are known to have antagonist or agonist activities in many systems, no studies have identified the in vivo role of the soluble type I IFN receptor, despite its elevation in serum from patients with diseases known to be modulated by IFNs or who are treated with IFN therapy. We report and validate the first murine model, called SolOX-H, used to determine the in vivo effects of sIFNAR2 under endogenous control of expression. This novel murine model mimics the levels of soluble receptor reported in human diseases (2832), underscoring its value for relevant mechanistic studies. We demonstrate that sIFNAR2a-overexpressing SolOX-transgenic mice are more susceptible to TLR4-mediated septic shock than are wild-type mice. This effect is mediated independently of NF-κB–driven proinflammatory cytokines by directly exacerbating type I IFN signaling. Our study reinforces the contribution of IFNs to LPS toxicity and identifies a new in vivo amplifier of these responses, the sIFNAR2a protein, which circulates in human and mouse serum and other body fluids (22, 24).

To investigate the effects of sIFNAR2a overexpression in vivo, we chose a well-characterized IFN-regulated disease mouse model of LPS-induced septic shock (35, 49, 50). LPS-induced sepsis mediated via the TLR4 receptor has been clearly demonstrated by an array of gene-knockout mice in which key components of the TLR4-mediated, MyD88-independent, IFN-inducing and -signaling pathway have been targeted (Fig. 6), including the TLR adaptor molecule Trif (51), Irf3 (52), Ifnβ (4, 5), Tyk2 (4, 53), and Ifnar1 (34); all such mice survive lethal endotoxic shock induced by a high dose of LPS. In our experiments, SolOX-H mice expressing 13-fold greater sIFNAR2a serum levels were more susceptible to the toxic effects of LPS than were their wild-type littermates. Analysis of serum levels of proinflammatory cytokines with a known role in the pathogenesis of endotoxic shock, such as IL-6 and TNF-α, which are induced by the MyD88-dependent NF-κB pathway demonstrated that these cytokines were not affected in SolOX mice. Other mediators of inflammation, such as NO, also were not affected in SolOX mice. These results demonstrate that the enhanced sensitivity to LPS toxicity in SolOX-H mice is not due to increased proinflammatory cytokines or inflammatory mediators, despite reports that IFN signaling can regulate levels of TLRs themselves on peripheral blood leukocytes (54). Taken together, our data indicate that increased levels of sIFNAR2a potentiate toxic effects of LPS by directly regulating IFN-β signaling during sepsis.

To investigate the mechanism of sIFNAR2a exacerbation of LPS-induced IFN-β effects, we examined STAT tyrosine phosphorylation, a well-described early event in IFN signaling. In spleen cells from SolOX-H–transgenic mice, STAT1 and STAT3 phosphorylation was more rapid, intense, and sustained up to 6 h after in vivo LPS treatment compared with wild-type mice. Comparison with Ifnar1−/− mice allowed us to demonstrate that the early STAT phosphorylation observed 1–3 h post-LPS in SolOX-H mice was entirely dependent on type I IFN signaling and not other LPS-induced cytokines. Thus, our data clearly demonstrate that cells overexpressing sIFNAR2a amplify IFN-β signaling via STAT1 and STAT3, both of which have been implicated directly in mediating LPS toxicity (53, 55, 56).

If sIFNAR2a facilitates IFN signaling, how is this achieved? Some soluble receptors, such as sIL4R, act as carrier proteins and modulate pharmacokinetics of their ligand, thereby acting as in vivo agonists (17). Importantly, high levels of sIFNAR2a had no effect on the maximal levels of circulating IFN-β or on the rate of increase or decrease of interferonemia. However, we cannot exclude that differential effects of sIFNAR2a on IFN distribution or retention at local tissue sites remains one possible explanation for enhanced IFN signaling in SolOX mice.

An alternative explanation for the potentiated IFN signaling mediated by sIFNAR2a is a trans-signaling mechanism similar to that described for sIL-6Rα (19, 5759) and sIL-15R (60, 61). Indeed, our previous in vitro studies demonstrate that this can occur: Ifnar2−/− thymocytes treated with IFN and recombinant sIFNAR2a were observed to generate an antiproliferative response (24). The potentiated signaling seen in SolOX-H mice may occur as a result of the high concentrations of ligand-bound sIFNAR2a outcompeting the cell surface IFNAR2 chain and directly interacting with the IFNAR1 chain, thereby mediating a potentiated biological response (Fig. 6). Alternatively, unequal expression of trans-membrane IFNAR1 and IFNAR2 receptors, particularly when IFNAR1 is higher (62), may potentiate IFN effects by trans-signaling (Fig. 6). Because IFNAR1 has a higher affinity for ligand-bound sIFNAR2a relative to unbound IFN (13, 63), this may lead to faster receptor interactions and rapid activation of Jak/STAT signaling, as observed in our in vivo IFN-signaling studies.

IFN is used to treat patients with viral infections or cancers, and a proportion of these patient populations is unresponsive to IFN therapy. It has been hypothesized by some groups that increased sIFNAR2a is a factor responsible for resistance to IFN therapy. However, our studies directly show that increased levels of sIFNAR2a do not decrease IFN-mediated responses; in fact, the opposite occurs, and IFN activity is amplified. Indeed, our data support other studies in which the lack of response to IFN therapy did not correlate with high sIFNAR2a levels observed in patient serum (2831, 64). Therefore, the SolOX mouse line is a valuable new model with which to study the impact of pathological levels of endogenous sIFNAR2a on exogenously administered type I IFNs. Furthermore, we demonstrate that the regulatory sequences used in the minigene construct contain the elements necessary for endogenous regulation of expression; this mouse model also may be useful for examining tissue- and isoform-specific regulation of Ifnar2 expression in vivo.

To our knowledge, this is one of the first studies to demonstrate an in vivo biological role for sIFNAR2a. This preferentially expressed, circulating receptor clearly potentiates signal transduction by type I IFNs. Based on these studies, the elevated levels of sIFNAR2a in human diseases, such as chronic hepatitis and various cancers, assume new importance. The consequence of amplifying IFN’s effects on pathogenesis in these diseases should be re-evaluated, and there is obvious relevance for our finding to IFN therapy. It will be important to determine whether sIFNAR2a differentially affects the therapeutic index of type I IFN, namely, the activities beneficial to the patient versus dose-limiting toxicity.

We thank Lucas Law, Georgia Pavasovic, Ivan Bertoncello, and Bernadette Scott for technical assistance and Brendan Jenkins, Ashley Mansell, Rebecca Piganis, and Rebecca Smith for discussions and critical reading of the manuscript.

This work was supported by Australia’s National Health and Medical Research Council and the Victorian Government’s Operational Infrastructure Support Program. N.E.M. is an Australian Research Council Australian Research and Queen Elizabeth II Fellow. P.J.H. is an Australian National Health and Medical Research Council Senior Principal Research Fellow.

The online version of this article contains supplemental material.

Abbreviations used in this article:

s

soluble

SolOX

soluble IFNAR2 overexpressing

tmIFNAR2

transmembrane IFNAR2.

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The authors have no financial conflicts of interest.

Supplementary data