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Innate Cytokine Responses in Porcine Macrophage Populations: Evidence for Differential Recognition of Double-Stranded RNA

Crystal L. Loving^*,, Susan L. Brockmeier, Wenjun Ma^,, Juergen A. Richt and Randy E. Sacco^1,*,

^* Immunobiology Graduate Program, Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011; and Respiratory Diseases Research Unit and Virus and Prion Diseases Research Unit, U.S. Department of Agriculture/Agricultural Research Service, National Animal Disease Center, Ames, IA 50010

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Pulmonary airways are vulnerable to infection because of exposure to Ag during respiration. The innate, antiviral response must be activated rapidly after pathogen recognition, and alveolar macrophages (AM) play a role in this response. TLR3 and protein kinase R (PKR) recognize dsRNA, a replication intermediate of RNA viruses, and initiate transcription of IFN-. In this study, synthetic dsRNA poly(I:C) was used to investigate innate responses of porcine AM compared with responses of peritoneal macrophages (PM). Poly(I:C) triggered IFN- in AM and PM, but levels in AM were higher. In contrast, mRNA levels of IFN-stimulated genes, Mx and PKR, were greater in PM than AM. Low levels of Mx and PKR transcription in AM were not due to deficient type I IFN receptor signaling, as exogenous IFN- induced nuclear translocation of phosphorylated STAT1. To investigate the differential mechanism by which IFN- transcription is activated in AM and PM, 2-aminopurine (2-AP) was used to block dsRNA-mediated activation of PKR. IFN-, Mx, and PKR mRNA levels in AM after poly(I:C) treatment were unaffected by 2-AP; conversely, transcription of IFN-, Mx, or PKR remained at baseline levels in PM. Phosphorylated PKR was detected in PM, but not AM, after poly(I:C) treatment. In addition to IFN- gene induction, mRNA levels of TNF- and RANTES were higher in AM than PM after poly(I:C) stimulation. In summary, differential dsRNA-induced cytokine expression patterns between AM and PM provide evidence that dsRNA recognition and subsequent signaling is likely mediated via TLR3 in AM and PKR in PM.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
As sentinels to host defense in the lung, alveolar macrophages (AM)² are relatively susceptible to infection because of continuous Ag exposure during respiration. AM are present in noninflammed alveoli and are likely the first cell encountered in the lumen of the lower respiratory tract. Cytokines and chemokines produced by activated AM have an important role in providing early protection against pathogen invasion and recruiting effector cells to sites of infection. Furthermore, AM can serve as APCs or antimicrobial effector cells.

The dsRNA produced during viral replication is a pathogen-associated molecular pattern (PAMP) of viruses and is recognized by host expressed pattern recognition receptors, e.g., TLRs. Type I IFNs (such as IFN- and IFN-) are critical for innate control of virus replication as well as activation of the adaptive immune response (1). Upon viral invasion, IFN- is synthesized, secreted, and signals through the type I IFN receptor (IFNAR), inducing transcription of several antiviral mediators, including protein kinase R (PKR, dsRNA-dependent protein kinase) and Mx (myxovirus resistant, IFN-inducible GTPase).

IFN- production is induced after dsRNA binds to TLR3 in the host cell (2). TLR3 uses the adaptor proteins MyD88 and Toll/IL-1R domain-containing adapter-inducing IFN- (TRIF) protein (also referred to as TICAM-1) for transduction of activation signals (3). MyD88-dependent signaling involves NF-B activation for the transcription of proinflammatory cytokines, such as TNF- (4). MyD88-independent pathway of TLR3 signaling uses TRIF, which activates IFN regulatory factor (IRF)-3, IRF-7 (5, 6), and NF-B (4). IRF-3 activates transcription of IFN- and RANTES, while IRF-3 and IRF-7 are involved in IFN- transcription (7, 8, 9, 10).

PKR is a serine/threonine kinase constitutively expressed in the cytoplasm, but can be up-regulated in an IFN-dependent manner. PKR can induce the type I IFN response by phosphorylating IB and subsequently activating the NF-B transcription factor (11, 12) or act as an antiviral mediator by limiting viral protein translation (13). Thus, PKR can induce the production of IFN- or act as mediator of the type I IFN response (13, 14, 15). Deciphering the role of PKR in the host immune response has been accomplished using a chemical compound, 2-aminopurine (2-AP). 2-AP inhibits serine/threonine kinase activity and has been shown to specifically inhibit PKR function (16, 17, 18, 19).

The recent emergence of highly pathogenic influenza (strain H5N1) and SARS coronavirus heightens the need for studies investigating the activation and regulation of pulmonary antiviral immune responses, particularly in AM. Increased cytokine production from H5N1-infected AM may play a role in disease severity (20), but AM are critical for controlling influenza viral replication (21). SARS coronavirus infection of M induces chemokine but not IFN- expression, indicating a dysregulation of the antiviral response (22). Unlike mice, pigs are naturally susceptible to influenza viruses and coronavirus (porcine respiratory coronavirus), making the pig a useful model for investigating respiratory viral infection in the natural host. Moreover, unique differences in type I IFN responses between mice and humans have been described (23, 24), indicating the need for additional animal infection models to thoroughly understand type I IFN activation and regulation during viral infections.

In this study, we analyzed the induction of the type I IFN system by porcine AM and PM in response to the synthetic dsRNA molecule polyriboinosinic:polyribocytidylic (poly(I:C)) acid. We provide evidence that recognition and subsequent signaling following exposure of PM to dsRNA occurred through PKR, whereas the dsRNA response in AM likely involved TLR3. In addition, we show that AM regulation of the type I IFN response in pigs differs from that previously described in mice, pointing to interesting species-specific differences in the regulation of innate, antiviral immune responses.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Animals

Conventionally raised, 3- to 6-wk-old pigs of either gender were maintained in isolation rooms at the National Animal Disease Center (Ames, IA) according to facility animal care and use guidelines. Animals received i.m. antibiotic ceftiofur 1 ml/day for 3 days (Excenel; 50 mg/ml), beginning the day pigs were transported to the facility. For the collection of lavage fluid, pigs were anesthetized with a combination of ketamine and xylazine given i.m. and subsequently euthanized with an overdose of pentobarbital given i.v. Three to six animals per experiment were used and the number of replicates is noted in each figure.

M isolation and culture

The peritoneal cavity was washed three times with 250 ml of 0.01 M PBS (pH 7.4) (using a total volume of 750 ml) and 500 ml was recovered. Lungs were lavaged with 300 ml of PBS and 200 ml was recovered. Collected lavage was centrifuged at 400 x g for 15 min, cells washed once with PBS, and resuspended in supplemented medium (RPMI 1640, 5% swine sera, 5 mM HEPES, 1 mM L-glutamine, antibiotic-antimycotic, and 50 µg/ml gentamicin (Invitrogen Life Technologies)). Cells were cultured in 150 x 15-mm petri dishes for 2 h at 37°C. Nonadherent cells were removed and adherent M harvested with a cell scraper, collected, washed once, and counted on a hemacytometer. M were seeded at 3.5 x 10⁵ cells per well in a 48-well flat-bottom plate with a final volume of 500 µl for cytokine studies (RT-PCR and bioassay) or at 2 x 10⁶ cells per well in a 24-well plate with a final volume of 750 µl for Western blot studies. M were stimulated for the indicated times with 50 µg/ml poly(I:C) (Amersham Biosciences) and/or 200 U of recombinant porcine IFN- (rpIFN-; R&D Systems). Where indicated, cells were pretreated with 5 mM 2-AP (Sigma-Aldrich) for 45 min. Nonstimulated cells were included at each time point as controls.

RNA preparation and real-time RT-PCR

At each indicated time point, supernatant was collected and stored at –80°C. M were lysed on the culture plate with RLT buffer, first step for RNA isolation (RNeasy Mini Isolation kit; Qiagen). RNA was isolated, DNase-treated (RNase-free DNase; Qiagen), and cDNA was synthesized using random hexamers according to manufacturer’s recommendations (Invitrogen Life Technologies). SYBR Green-based real-time PCR was conducted for various mRNA targets according to manufacturer’s recommendations (SYBR Green Master mix; Applied Biosystems). Briefly, SYBR Green Master mix, primers, and template were mixed in a 20-µl reaction and cycled as follows: 95°C for 15 min; 95°C for 15 s followed by 50°C for 1 min (45 cycles), and a final dissociation step. All samples were run in duplicate. Levels of mRNA were calculated using the threshold cycle 2^–Ct method, which expresses mRNA in treated cells relative to nonstimulated cells after normalizing to -actin (25). Primers were designed using Primer Express, purchase from Integrated DNA Technologies, and used at a final concentration of 600 nM. Primer pair efficiency was confirmed using the method described by Livak and Schmittgen (25). PCR products were <100 bp in size and primers were as follows: -actin forward 5'-CTCCTTCCTGGGCATGGA-3', reverse 5'-CGCACTTCATGATCGAGTTGA-3'; IFN- forward 5'-TGCAACCACCACAATTCC-3', reverse 5'-CTGAGAATGCCGAAGATCTG-3'; IFN- forward 5'-GCCTCCTGCACCAGTTCTACA-3', reverse 5'-TGCATGACACAGGCTTCCA-3'; Mx-1 forward 5'-TAGGCAATCAGCCATACG-3', reverse 5'-GTTGATGGTCTCCTGCTTAC-3'; PKR forward 5'-GAGAAGGTAGAGCGTGAAG-3', reverse 5'-CCAGCAACCGTAGTAGAG-3'; RANTES forward 5'-CTGCTTTGCCTACCTCTC-3', reverse 5'-CTTGCTGCTGGTGTAGAA-3'; and TNF- forward 5'-CCACGTTGTAGCCAATGTC-3', reverse 5'-CTGGGAGTAGATGAGGTACAG-3'. Primer pair efficiency was confirmed using the method described by Livak and Schmittgen (25).

Cell extracts and Western blotting

M were seeded at 2 x 10⁶ cells per well in a 24-well plate with a final volume of 750 µl. At each indicated time point, supernatant and cells were collected and centrifuged at 300 x g for 5 min to pellet cells. Cytoplasmic and nuclear fractions were collected according to manufacturer’s recommendations (NE-PER Extract; Pierce) and stored at –80°C.

For Western blot studies, 10 µg of lysate was mixed with Laemmli sample buffer, heated at 95°C for 5 min, and run into a 12% SDS-PAGE gel under denaturing and reducing conditions. Proteins were transferred to 0.45-µm nitrocellulose using Tris-glycine transfer buffer (Invitrogen Life Technologies). After membranes were incubated in Superblock (Pierce) for 1 h at room temperature, primary Ab was added and membranes incubated overnight at 4°C on an orbital shaker. Membranes were washed six times with PBS/0.05% Tween 20 and incubated with appropriate HRP-labeled secondary Ab for 2 h at room temperature. Membranes were washed as described and incubated for 2 min with substrate (SuperSignal PicoWest; Pierce). Signals were detected using radiographic film (X-OMAT MR; Kodak). For reprobing, membranes were incubated for 20 min at 37°C in stripping buffer (62.5 mM Tris, 2% SDS, and 2% 2-ME) and then washed four times with PBS/0.05% Tween 20. Abs used were: anti-STAT1, anti-phospho-STAT1 (Tyr⁷⁰¹), anti-PKR, anti-phospho-PKR (Thr⁴⁵¹), all from Cell Signaling Technology, and anti--actin (Abcam).

Type I IFN assays

Levels of type I IFN secreted by stimulated AM and PM were measured as previously described (26, 27), with some modifications. Serial 10-fold dilutions of supernatant collected from nonstimulated and stimulated AM and PM were transferred to individual wells of a 96-well plate containing confluent PK-15 cells. After 1 h, supernatant was removed and PK-15 cells were infected with recombinant vesicular stomatitis virus (VSV) expressing GFP (VSV-GFP) at a multiplicity of infection of 0.1. At 14 h postinfection, GFP was visualized by fluorescence microscopy. As VSV is highly sensitive to type I IFN, the presence of IFN in the culture supernatant was determined by decreased GFP signal, indicating the ability to inhibit VSV-GFP replication. Samples from stimulated and nonstimulated cells were compared. As a control, the ability of porcine IFN- to inhibit VSV-GFP replication was confirmed using a commercially available rIFN- (PBL Biomedical). In addition, IFN- levels were quantified by ELISA using paired anti-porcine IFN- mAb (clones F17 and K9) and rpIFN- as a standard (PBL Biomedical).

Statistical analysis

Student’s t test was used for statistical analysis using Prism software. Values of p < 0.05 are noted.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Type I IFN transcription is significantly greater in porcine AM than PM after poly(I:C) stimulation

A strong type I IFN response to dsRNA is critical for the production of downstream antiviral mediators. TLR3 and PKR recognize dsRNA and initiate the transcription of IFN- (28). Accordingly, we investigated type I IFN gene transcription (IFN- and IFN-) following stimulation of AM and PM with poly(I:C), a synthetic dsRNA molecule. In addition, rpIFN- was used to determine whether exogenous IFN altered type I IFN responsiveness to poly(I:C). Transcription of IFN- and IFN- after poly(I:C) stimulation was greater in AM than in PM at both the 12 h (Fig. 1A) and 24 h (Fig. 1B) time points. The addition of rpIFN-, in the presence or absence of poly(I:C), did not significantly alter transcription of either type I IFN gene in AM or PM. In response to poly(I:C), IFN- mRNA levels were highest at 12 h in both AM and PM, with mRNA levels decreasing by 24 h after stimulation. However, mRNA levels of IFN- were nearly 100-fold higher in AM in comparison to PM 12 h after poly(I:C) stimulation (Fig. 1A). By 24 h after stimulation, PM IFN- and IFN- mRNA levels were similar to baseline, or that of mock-treated cells. Yet, mRNA levels of both IFN- and IFN- remained elevated in AM at the 24-h time point (Fig. 1B). Overall, AM responded to poly(I:C) stimulation with higher levels of type I IFN gene transcription (IFN- and IFN-) than PM and the addition of exogenous IFN- did not alter the response.

View larger version (29K): [in this window] [in a new window]   FIGURE 1. Poly(I:C) induced IFN- and IFN- transcription in AM and PM. Type I IFN (IFN- and IFN-) mRNA levels were measured by real-time PCR in AM and PM. At 12 h (A) and 24 h (B) after stimulation with poly(I:C), rpIFN-, or poly(I:C) plus rpIFN- (I:C + ). Data are expressed as mean ± SEM fold increase in gene expression relative to mock-treated cells. Data are representative of three independent experiments.

Transcription of IFN-dependent antiviral mediators, PKR and Mx, is greater in PM than AM after poly(I:C) or rpIFN- stimulation

Type I IFN proteins (IFN- and IFN-) function by inducing the expression of several antiviral mediators, such as Mx and PKR, within the host cell. These antiviral proteins inhibit viral protein translation and viral assembly and consequently, control virion production (29). Both M populations responded to poly(I:C) with an increase in transcription of IFN- and IFN- (Fig. 1). We therefore wanted to determine whether poly(I:C) treatment resulted in transcription of IFN-dependent antiviral mediators, Mx and PKR. AM and PM transcription of Mx and PKR was examined after poly(I:C) stimulation. In parallel, cells were stimulated with rIFN- to investigate transcriptional changes induced by exogenous type I IFN.

Both M populations responded to poly(I:C) with increased transcription of Mx and PKR, with the greatest transcriptional changes observed at 24 h (Fig. 2B). After 12 h of poly(I:C) treatment (Fig. 2A), AM exhibited a 2-fold increase in Mx mRNA, whereas PKR mRNA levels remained near baseline (expression level of 1). PM transcription of Mx and PKR after poly(I:C) stimulation was higher, with a 6-fold increase in Mx mRNA and 4-fold increase in PKR mRNA levels after 12 h. At 24 h, Mx and PKR mRNA levels remained elevated, with higher expression levels observed for both cell types. At 24 h poststimulation, a 12-fold increase in Mx mRNA levels and 6-fold increase in PKR mRNA levels was observed in PM. It is interesting to note that poly(I:C) stimulation of PM resulted in a significantly greater increase in Mx and PKR transcription (Fig. 2) than AM, even though AM showed a greater induction of IFN- and IFN- mRNA (Fig. 1) after poly(I:C) stimulation.

View larger version (30K): [in this window] [in a new window]   FIGURE 2. Differential transcription of ISGs, Mx and PKR, in AM and PM. Levels of mRNA were measured by real-time PCR in AM and PM 12 h (A) and 24 h (B) after stimulation with poly(I:C), rpIFN-, or poly(I:C) plus rpIFN- (I:C + ). Data are expressed as mean ± SEM fold increase in gene expression relative to mock-treated cells. Data are representative of three independent experiments.

AM phosphorylate STAT1 in response to rIFN- indicating a functional IFNAR

One explanation for the low levels of Mx and PKR transcription after poly(I:C) or rpIFN- stimulation of AM would be the absence of or defective signaling via the IFNAR. IFN- or IFN- protein binding to IFNAR induces receptor rearrangement and subsequent autophosphorylation of Jak. STAT1, after phosphorylated by Jak, binds to phosphorylated STAT2 and translocates into the nucleus. A heterotrimer of STAT1:STAT2:IRF-9 binds to DNA at the IFN-stimulated response element, a conserved sequence in the promoter region of several genes, including Mx and PKR (30). Overall, STAT1 is critical for coordination of the IFN- signal; therefore, we examined STAT1 phosphorylation and subsequent translocation into the nucleus in AM after poly(I:C), rpIFN-, or poly(I:C) plus rpIFN- stimulation. Phosphorylated STAT1 was detected in both the cytoplasmic and nuclear fractions of AM (Fig. 3) and PM (data not shown) lysates 2 h after rpIFN- stimulation. STAT1 protein was detected in the cytoplasmic fraction of poly(I:C)-treated AM, but phosphorylated STAT1 was not detected in the cytoplasm or nuclear fractions. If rpIFN- was added together with poly(I:C), phosphorylated STAT1 could be detected in the nuclear fraction, but to a lesser extent than that observed using rpIFN- only. Overall, AM responded to rpIFN- with the phosphorylation and nuclear translocation of STAT1 indicating functional IFNAR.

View larger version (12K): [in this window] [in a new window]   FIGURE 3. Phosphorylation of STAT1 (pY-STAT-1) after stimulation with rpIFN- in AM. Cells were mock-treated or stimulated with rpIFN-, poly(I:C), or poly(I:C) plus rpIFN- (I:C + ) for 2 h. Equal amounts of cytoplasmic (C) and nuclear (N) lysates were probed using anti-STAT1 and anti-phosphorylated STAT1. Extracts from HeLa cells treated with rIFN- were used as a positive control (+). Data are representative of three animals and two separate experiments.

AM produce bioactive IFN- in response to poly(I:C) stimulation

After poly(I:C) stimulation, AM transcribed both IFN- and IFN- mRNA; however, the IFN-stimulated genes (ISG) Mx and PKR typically induced by IFN- protein, did not increase as significantly as that observed in PM. AM did respond to the addition of rpIFN- with the nuclear translocation of phosphorylated STAT1, indicating an intact receptor and signaling machinery (Fig. 3). To determine whether the IFN- and IFN- mRNA detected in AM after poly(I:C) treatment was translated into biologically active protein, a type I IFN bioassay was used. AM and PM were exposed to poly(I:C) for 2 h before the supernatant was replaced; cells were washed once to ensure that poly(I:C) did not remain in the supernatant, and subsequently replenished with fresh medium. After 12 or 24 h, supernatant was collected and assayed for bioactive type I IFN using type I IFN-sensitive VSV-GFP. Fig. 4 shows that supernatants from poly(I:C) stimulated, but not mock-treated, AM and PM contained biologically active type I IFN protein. VSV-GFP replication was greatest in mock-treated cells, and AM supernatants inhibited VSV-GFP replication to a greater extent than PM supernatants. Similar to transcriptional observations (Fig. 1), AM likely produce more bioactive type I IFN after poly(I:C) stimulation than PM, indicated by the lower GFP signal observed for AM when compared with PM (Fig. 4). Furthermore, porcine IFN- ELISA results showed that AM secreted more IFN- (18.7 ng/ml) than PM (3.8 ng/ml) 24 h after poly(I:C) stimulation (Fig. 4B). These data indicate the lack of Mx and PKR transcription after poly(I:C) stimulation in AM was not due to a lack of type I IFN protein production. Interestingly, these results differ from a previous study in mice showing that murine AM transcribe IFN- after poly(I:C) stimulation, but do not secrete IFN- protein (31).

View larger version (29K): [in this window] [in a new window]   FIGURE 4. Production of bioactive type I IFN by AM and PM 12 h (A) and 24 h (B) after stimulation with poly(I:C). Supernatants collected from stimulated AM or PM were incubated on PK-15 cells for 1 h. GFP-labeled VSV was added to each well and fluorescence measured after 12 h. Bioactive type I IFN inhibits GFP-VSV replication, indicated by low levels of GFP fluorescence.

2-AP inhibits poly(I:C) induced transcription of type I IFN and ISG in PM but not AM

To investigate the differential mechanism by which IFN- and IFN- mRNA transcription is activated in AM and PM, 2-AP was used to block PKR activation. PKR, like TLR3, is a cellular sensor for dsRNA and is able to induce IFN- transcription via the NF-B pathway (11, 12). Thus, PKR is not only an effector molecule in the antiviral immune response, but is also capable of initiating the type I IFN system (15). 2-AP has been shown to act as a serine/threonine kinase inhibitor and to specifically block activation of PKR (19). To determine whether activation of PKR may play a role in IFN- and IFN- transcription, AM and PM were treated with 5 mM 2-AP for 45 min before stimulation with poly(I:C), rpIFN-, or poly(I:C) plus IFN-. AM levels of IFN- and IFN- mRNA transcript after poly(I:C) treatment did not change significantly when pretreated with 2-AP (Fig. 5). Conversely, IFN- and IFN- mRNA levels were significantly lower in PM pretreated with 2-AP after poly(I:C) stimulation. In addition, Mx and PKR mRNA levels were significantly lower in 2-AP pretreated PM. Mx and PKR transcription in AM was not up-regulated in response to poly(I:C) (Fig. 2A), nor did levels change with 2-AP pretreatment (Fig. 5). Overall, 2-AP inhibited activation of the type I IFN response in PM after poly(I:C) stimulation, but not in AM, indicating a different mechanism of type I IFN activation between these two M populations.

View larger version (10K): [in this window] [in a new window]   FIGURE 5. Role of PKR in AM and PM type I IFN responses to dsRNA. Transcription of type I IFN and ISGs in AM and PM stimulated with poly(I:C) for 12 h with or without 2-AP pretreatment (45 min). 2-AP blocks PKR activation and was used to determine the role of PKR in differential type I IFN response between M populations. Data are expressed as mean ± SEM fold increase in gene expression relative to mock-treated cell. Data are representative of three independent experiments.

Addition of rpIFN- to PM and AM pretreated with 2-AP recovers Mx and PKR transcription

Pretreatment of PM with 2-AP blocked the transcription of both IFN- and IFN- mRNA as well as downstream ISGs, Mx and PKR (Fig. 5), in response to poly(I:C). We sought to determine whether the loss of Mx and PKR transcription in poly(I:C)-stimulated 2-AP pretreated PM resulted from low levels of IFN-. Thus, 2-AP-treated PM were exposed to rpIFN- to determine whether exogenous IFN- could recover Mx and PKR transcription. As shown in Fig. 6A, transcription of Mx and PKR was recovered in 2-AP-pretreated PM with the addition of rpIFN-. Recovery of Mx and PKR mRNA expression was also detected in 2-AP pretreated PM after poly(I:C) plus rpIFN- stimulation. Poly(I:C) treatment of PM for 24 h (without 2-AP) induced a 17-fold increase in Mx mRNA levels and a 10-fold increase in PKR mRNA levels (Fig. 2B), which was not significantly different from mRNA levels of both ISGs observed after poly(I:C) plus rpIFN- stimulation after 2-AP pretreatment (Fig. 6B). The addition of rpIFN- to poly(I:C)-stimulated PM pretreated with 2-AP did not alter IFN- or IFN- transcription from that observed for cells not pretreated with 2-AP (data not shown). These data indicate that the lack of Mx and PKR transcription in poly(I:C) stimulated PM pretreated with 2-AP was the result of 2-AP-mediated inhibition of IFN- and IFN- production.

View larger version (27K): [in this window] [in a new window]   FIGURE 6. Transcription of the ISGs Mx and PKR in AM and PM stimulated with rpIFN- (A) or poly(I:C) + rpIFN- (B) for 24 h with () or without () 2-AP pretreatment (45 min). Data are expressed as mean ± SEM fold increase in gene expression relative to mock-treated cells. Data are representative of three independent experiments.

PKR protein levels and phosphorylation increase in poly(I:C)-treated PM but not AM

IFN- and IFN- transcription induced after poly(I:C) stimulation was inhibited by 2-AP pretreatment of PM, but not AM (Fig. 5), suggesting that PKR is likely involved in activation of the type I IFN response in PM but not AM. To determine whether PKR activation was involved in the differential response of AM and PM to poly(I:C) stimulation, the phosphorylation of PKR after poly(I:C) stimulation was examined (Fig. 7). PKR protein levels were increased in poly(I:C)-stimulated PM but not AM, and phosphorylated PKR was detected only in PM. These data indicate that PKR protein in PM was up-regulated and phosphorylated following stimulation with poly(I:C), whereas such changes were not observed in poly(I:C)-stimulated AM.

View larger version (43K): [in this window] [in a new window]   FIGURE 7. Role of PKR in M type I IFN responses. M were mock-treated or stimulated with rpIFN-, poly(I:C), or poly(I:C) plus rpIFN- for 18 h. Top, Equal amounts of cytoplasmic extracts were probed using anti-phosphorylated PKR (p-PKR), anti-PKR, or anti--actin. Modulation of PKR and phosphorylated PKR as determined by the ratio of target protein to -actin is shown (bottom). Data are representative of three animals and two separate experiments.

Poly(I:C) induces the transcription of immediate-early genes, TNF- and RANTES, in AM but not PM

An important function of M is early production of cytokines and chemokines after PAMP recognition to recruit effector cells to sites of infection. TLR3 recognizes dsRNA and signals for the transcription of IFN- as well as immediate-early genes, such as TNF- and RANTES, via the MyD88-dependent and -independent pathways, respectively (32). TNF- mRNA transcription is controlled by MyD88-dependent activation of the NF-B signaling pathway (4). Conversely, RANTES transcription is MyD88-independent and relies on the TLR3-TRIF-IRF-3 pathway (8). To determine whether the observed differences in the type I IFN activation of AM and PM extended to signaling pathways controlling immediate-early genes, dsRNA induced transcription of TNF- and RANTES was examined. TNF- and RANTES mRNA levels were significantly greater in AM than PM after poly(I:C) stimulation (Fig. 8A). Induction of both TNF- and RANTES in AM was dependent on the presence of poly(I:C), as rpIFN- did not alter transcription of either gene. 2-AP pretreatment of AM did not inhibit the production of TNF- or RANTES in response to poly(I:C), suggesting that PKR is not involved in TNF- or RANTES induction (Fig. 8B). Overall, these data indicate that AM likely recognize and respond to dsRNA via TLR3 and PM uses PKR for dsRNA-induced responses.

View larger version (38K): [in this window] [in a new window]   FIGURE 8. Differential transcription of MyD88-dependent and -independent genes TNF- and RANTES between AM and PM 12 h (A) after stimulation with poly(I:C), rpIFN-, or poly(I:C) plus rpIFN-. B, Pretreatment with 2-AP in poly(I:C)-stimulated AM and PM does not affect MyD88-dependent or -independent gene transcription (TNF- and RANTES). Data are expressed as the mean ± SEM fold increase relative to mock-treated cells. Data are representative of three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

One important finding presented in this study is the differential mechanism by which different species regulate AM responsiveness to dsRNA. The lung is a particularly vulnerable organ because of constant Ag exposure during respiration. Pulmonary immune responses must be able to efficiently eliminate or control infectious agents, yet limited in magnitude to avoid uncontrolled inflammation that would compromise pulmonary function. A recent report investigating murine AM responses to dsRNA demonstrated that IFN- mRNA is transcribed but not secreted after poly(I:C) stimulation (31). In addition, it is unlikely that murine AM secreted IFN- after poly(I:C) stimulation because STAT1 was not phosphorylated (31). Thus, the murine AM type I IFN response after dsRNA stimulation is considerably different from porcine AM response, as porcine AM secreted bioactive type I IFN after poly(I:C) stimulation (Fig. 4). Nonetheless, porcine AM also appeared to regulate the type I IFN response, but instead of limiting IFN- production (31), porcine AM may control transcription of ISGs (e.g., Mx and PKR) in response to IFN-. IFN- protein binds IFNAR, and receptor-associated kinases Jak1 and Tyk2 induce phosphorylation of the transcription factors STAT1 and STAT2. Once phosphorylated, STAT1 can form a homodimer or a heterodimer with STAT2 (30) and bind to specific DNA promoter sequences to induce the transcription of various IFN-dependent genes (29). STAT1 homodimers bind to the IFN--activated sequence element, whereas STAT1:STAT2 heterodimer (with IRF-9) binds to the IFN-stimulated response element (30). Phosphorylated STAT1 was detected in porcine AM stimulated with rpIFN- for at least 2 h, indicating an intact IFNAR. Yet, poly(I:C) stimulation of AM did not induce phosphorylated STAT1 and detection of phosphorylated STAT1 was reduced if AM were stimulated with both poly(I:C) plus rpIFN- (Fig. 3), suggesting the activation of a phosphorylated STAT1 inhibitory response after poly(I:C) stimulation. Suppressors of cytokine signaling (SOCS) are a family of regulatory proteins (SOCS-1 through SOCS-9) that can inhibit cytokine signaling transduction pathways, including STAT1-dependent signaling (34, 35). SOCS proteins are up-regulated by cytokines (e.g., IFN-) and by TLR agonists (36). SOCS-1 and SOCS-3 have been shown to inhibit IFN-induced expression of antiviral proteins including Mx (37). It is possible that porcine AM constitutively express SOCS proteins, thus, regulating responsiveness to IFN-. In addition, poly(I:C) signaling via TLR3 in AM may enhance the production of inhibitory proteins, subsequently inhibiting the transcription of ISG to poly(I:C) induced IFN-. Taken together, both murine and porcine AM have unique mechanisms for tightly regulating type I IFN responses.

Compared with PM, AM did not significantly increase transcription of ISGs, Mx and PKR, after stimulation with poly(I:C) or rpIFN-. Only when AM were pretreated with 2-AP did rpIFN- exposure result in enhanced expression of Mx and PKR. Interestingly, poly(I:C) could inhibit the effects of rpIFN- in 2-AP pretreated AM, as Mx and PKR transcription did not change significantly in 2-AP pretreated AM in the presence of poly(I:C) plus rpIFN- (Fig. 6). It has recently been demonstrated that NF-B negatively regulates type I IFN-induced gene expression, presumably by blocking the binding sites of various ISG promoters. NF-B (p50/p65) knockout cells inhibited influenza virus replication to a greater extent than wild-type cells, indicating that NF-B inhibits several functionally important type I IFN activities (38). Evidence suggests that IFN- activates NF-B transcription factors in a serine/threonine kinase-dependent manner (39). This may explain why Mx and PKR transcription occurred in rpIFN- stimulated AM only when pretreated with the serine/threonine kinase inhibitor, 2-AP. In such a scenario, 2-AP would inhibit a serine/threonine kinase involved in NF-B activation, and consequently, remove NF-B inhibition from the ISG promoter, resulting in enhanced Mx and PKR transcription in AM. Further studies are warranted to determine the mechanism involved in controlling AM type I IFN responses and identify proteins involved.

In addition to differences between porcine and murine M regulation of type I IFN responses to dsRNA, the current study highlights that M populations differentially respond to the same stimuli. Several pieces of data in the current study strongly suggest that porcine AM and PM recognition of dsRNA occurred through different dsRNA cellular sensors. The induction of type I IFN and RANTES gene transcription, which occurred in porcine AM, indicates the need for IRF-3 activation (8, 9). IRF-3 is activated by TRIF, an adaptor protein of TLR3 (10). Although dsRNA induced IFN- transcription in PM, RANTES transcription did not change in poly(I:C)-stimulated PM, indicating TLR-TRIF-IRF-3 is not likely involved in the PM response to poly(I:C). MyD88, another adaptor protein of TLR3, has been shown to induce NF-B activation for the subsequent transcription of IFN- and TNF- (4, 40). As PM did not show enhanced TNF- transcription in response to poly(I:C), the TLR3-MyD88-NF-B signaling pathway was not likely involved in PM response to poly(I:C). PKR can induce transcription of type I IFN after activation by dsRNA, and PKR is sensitive to the chemical compound 2-AP. PM, but not AM, IFN- transcription after poly(I:C) stimulation was sensitive to the effects of 2-AP. Taken together, TLR3 was likely involved in dsRNA-induced response elicited by AM, whereas PKR was involved in PM response.

Collectively, porcine AM and PM recognize the viral PAMP, dsRNA, through different cellular pattern recognition receptors, which results in differential transcription of IFN- and the proinflammatory cytokines TNF- and RANTES. Furthermore, results from the current study and other published reports indicate an interesting species-specific mechanism for regulating type I IFN responsiveness in AM. Regulation of the type I IFN response is likely specific to AM, as the lung requires judicious control of immune responses to prevent erroneous, uncontrolled activation that results in immunopathology. The recent emergence of highly pathogenic influenza and the critical role of AM for viral clearance exemplifies the need for understanding mechanisms of immune regulation in AM. Furthermore, pigs provide a useful model for investigating respiratory disease, as they are naturally susceptible to several respiratory pathogens, including influenza viruses.

	Acknowledgments

 
We thank Dr. John Hiscott (Montreal, Canada) for the gift of VSV-GFP virus. We thank Dr. Ray Waters for critical review of the manuscript, and Theresa Waters and Kim Driftmier for excellent technical support.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Address correspondence and reprint requests to Dr. Randy E. Sacco, Respiratory Diseases of Livestock Research Unit, National Animal Disease Center, U.S. Department of Agriculture/Agricultural Research Service, 2300 Dayton Road, Ames, IA 50010. E-mail address: rsacco{at}nadc.ars.usda.gov

² Abbreviations used in this paper: AM, alveolar macrophage; M, macrophage; PM, peritoneal M; PAMP, pathogen-associated molecular pattern; PKR, protein kinase R; VSV, vesicular stomatitis virus; IFNAR, type I IFN receptor; 2-AP, 2-aminopurine; ISG, IFN-stimulated gene; rpIFN, recombinant porcine IFN; IRF, IFN regulatory factor; TRIF, Toll/IL-1R domain-containing adapter-inducing IFN-; SOCS, suppressors of cytokine signaling.

Received for publication May 23, 2006. Accepted for publication September 29, 2006.

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