Nitric Oxide Synthase Induction TLR Regulation of SPSB1 Controls ...

The Journal of Immunology

TLR Regulation of SPSB1 Controls Inducible Nitric Oxide Synthase Induction Rowena S. Lewis,* Tatiana B. Kolesnik,* Zhihe Kuang,* Akshay A. D’Cruz,*,† Marnie E. Blewitt,* Seth L. Masters,* Andrew Low,* Tracy Willson,* Raymond S. Norton,*,1 and Sandra E. Nicholson*,† The mammalian innate immune system has evolved to recognize foreign molecules derived from pathogens via the TLRs. TLR3 and TLR4 can signal via the TIR domain-containing adapter inducing IFN-b (TRIF), which results in the transcription of a small array of genes, including IFN-b. Inducible NO synthase (iNOS), which catalyzes the production of NO, is induced by a range of stimuli, including cytokines and microbes. NO is a potent source of reactive nitrogen species that play an important role in killing intracellular pathogens and forms a crucial component of host defense. We have recently identified iNOS as a target of the mammalian SPSB2 protein. The SOCS box is a peptide motif, which, in conjunction with elongins B and C, recruits cullin-5 and Rbx-2 to form an active E3 ubiquitin ligase complex. In this study, we show that SPSB1 is the only SPSB family member to be regulated by the same TLR pathways that induce iNOS expression and characterize the interaction between SPSB1 and iNOS. Through the use of SPSB1 transgenic mouse macrophages and short hairpin RNA knockdown of SPSB1, we show that SPSB1 controls both the induction of iNOS and the subsequent production of NO downstream of TLR3 and TLR4. Further, we demonstrate that regulation of iNOS by SPSB1 is dependent on the proteasome. These results suggest that SPSB1 acts through a negative-feedback loop that, together with SPSB2, controls the extent of iNOS induction and NO production. The Journal of Immunology, 2011, 187: 3798–3805.

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he mammalian innate immune system has evolved to recognize foreign molecules derived from pathogens, such as LPS, bacterial lipopeptides, CpG motifs, and dsRNA. This sensing occurs via TLRs of which there are 10 in humans (TLR 1 to 10) and 11 in mice (TLR 1 to 7, TLR9, and TLR 11 to 13) (1, 2). All TLRs, with the exception of TLR3, are able to trigger the MyD88 pathway, which results in activation of NF-kb and MAPKs via the IL-1R–associated kinase complex. This then leads to the synthesis of inflammatory cytokines including IL-1b, IL-6, IL-8, IL-12, and TNF-a (3–5). TLR3 and TLR4 can signal via TIR domain-containing adapter inducing IFN-b (TRIF) (6–8), activation of which results in the induction of a smaller array of genes including IFN-b (3–5). The cytokines induced by the TLRs include IFN a and b, IL-1b, IL-6, and TNF-a, of which IFN-a/b and TNF-a subsequently induce iNOS. In addition, iNOS ex*Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia; and †Department of Medical Biology, University of Melbourne, Parkville, Victoria 3052, Australia 1 Current address: Monash Institute of Pharmaceutical Sciences, Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.

Received for publication September 7, 2010. Accepted for publication August 2, 2011. This work was supported in part by the National Health and Medical Research Council, Australia (Program Grant 461219, Project Grants 461233 and 637384, fellowships to R.S.N. and S.E.N., and Independent Research Institute Infrastructure Support Scheme Grant 361646) and a Victorian State Government Operational Infrastructure Support grant. Address correspondence and reprint requests to Dr. Sandra E. Nicholson, Inflammation Division, Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia. E-mail address: [email protected] Abbreviations used in this article: BMDM, bone marrow-derived macrophage; iNOS, inducible NO synthase; M-CSF, macrophage colony stimulating factor; poly-IC, polyinosinic-polycytidylic acid; qPCR, quantitative PCR; shRNA, short hairpin RNA; TRIF, TIR domain-containing adapter inducing IFN-b. Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002993

pression in response to LPS and polyinosinic-polycytidylic acid (poly-IC) can occur directly through NF-kb, without the need for cytokine stimulation. There are three isoforms of NO synthases, neuronal NO synthase (NOS1), inducible NO synthase (iNOS, or NOS2), and endothelial NO synthase (NOS3), all of which catalyze the production of NO from L-arginine, oxygen, and NADPH (9–12). NO is a powerful source of the reactive nitrogen species that play an important role in the killing of intracellular pathogens and, as such, forms a crucial component of the host defense system (13–15). Given that NO is a potent effector molecule, its production needs to be tightly controlled. Accordingly, the activity of iNOS is regulated at the transcriptional, translational, and posttranslational levels by ubiquitination and proteasomal degradation, as shown in primary bronchial epithelial cells and several cell lines (16–18). We have recently identified SPSB2 as the adapter protein in an E3 ubiquitin ligase complex that ubiquitinates iNOS, targeting it for proteasomal degradation (19). The SOCS box is a sequence motif, first identified in SOCS proteins, which, in conjunction with elonginBC, recruits cullin-5 and Rbx-2 to form an active E3 complex (20, 21). Ubiquitination involves the transfer of a ubiquitin molecule via an E1–E2–E3 enzyme cascade (22), where the E3 ligase functions as both a substrate recognition molecule and a catalyst for the transfer of ubiquitin to a lysine in the substrate protein (23). The SPSB proteins (SPSB1 to SPSB4) encompass a central SPRY domain involved in protein–protein interactions and a C-terminal SOCS box (24, 25). Several SPSB SPRY domain structures have been solved recently (26–28), and the SPSB1, SPSB2, and SPSB4 SPRY domains have been shown to interact with a peptide motif identified in their target proteins via an extended interface on the SPSB SPRY domain that is created by five variable loops (26, 28). When overexpressed, SPSB1, SPSB2, and SPSB4 (but not SPSB3) can interact with a DINNN motif in the N-terminal region of iNOS (19).


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Although we have shown that SPSB2 targets iNOS for ubiquitination, resulting in its proteasomal degradation and thereby regulating NO production (19), it was unclear whether other SPSB family members also had a role in regulating iNOS. In this study, we extend our earlier studies and characterize the interaction between SPSB1 and iNOS to demonstrate that SPSB1 is an important regulator of iNOS during the early induction phase. SPSB1 is unique among its family members in that its expression is regulated by the same TLR pathways that induce iNOS expression, suggesting a negative-feedback loop that, together with SPSB2, controls the extent of iNOS and NO production.

Materials and Methods Mice pUBc constructs containing the SPSB1 (Spsb1) coding region with (Spsb1T/+) and without the SOCS box (Spsb1DSBT/+) were generated to express SPSB1 with an N-terminal Flag epitope under the ubiquitin C promoter. Transgenic constructs were injected into C57BL/6 fertilized eggs followed by implantation into pseudopregnant C57BL/6 females. Progeny were screened by Southern blot for germline transmission of the transgene. Protein expression was confirmed by immunoprecipitation and Western blot with anti-Flag Abs. SPSB2-null mice (Spsb22/2) and mice expressing SPSB2 transgenes, either with (Spsb2T/+) or without (Spsb2DSBT/+) the SOCS box, have been described previously (19, 29). All mice were bred in pathogen-free facilities at the Walter and Eliza Hall Institute of Medical Research. Ethics approval was obtained from the Animal Ethics Committee at the Walter and Eliza Hall Institute of Medical Research.

Cytokines and Abs

(Complete Cocktail tablets; Roche), 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 1 mM NaF. Proteins were immunoprecipitated using anti-Flag Ab conjugated to Sepharose (M2; Eastman Kodak, Rochester, NY) for 3 h at 4˚C. N-terminal Flag epitopes were then detected by Western blot. For iNOS ubiquitination, cells were lysed as above, with the addition of 10 mM N-ethylmaleimide to the lysis buffer, and immunoprecipitated overnight using rabbit polyclonal anti-iNOS Ab at 4˚C, followed by the addition of protein A–Sepharose for 2 h. Ubiquitinated iNOS proteins were then detected by Western blot.

Western blot analysis BMDM were plated at 1 3 106 cells/well, stimulated with 100 ng/ml LPS, 25 mg/ml poly-IC or 20 ng/ml LPS, and 10 ng/ml IFN-g for the times indicated and subsequently lysed in KALB lysis buffer (32) containing protease inhibitors (Complete Cocktail tablets; Roche), 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 1 mM NaF. Alternatively, for iNOS clearance assays, BMDM were incubated with 20 ng/ml LPS, 25 mg/ ml poly-IC or 20 ng/ml LPS, and 10 ng/ml IFN-g overnight, washed, replenished with fresh medium, and lysed at the indicated times. Proteins were separated by SDS-PAGE under reducing conditions and electrophoretically transferred to either Osmonics polyvinyl difluoride membranes (Millipore) or Amersham nitrocellulose (GE Healthcare, Little Chalfont, U.K.). Membranes were blocked overnight in 10% w/v skim milk and incubated with primary Ab for 2 h. For detection of ubiquitinated proteins, nitrocellulose membranes were first treated with 0.5% v/v glutaraldehyde/0.1 M potassium phosphate, pH 7, before blocking overnight in 5% w/v BSA (Sigma-Aldrich). Ab binding was visualized with either peroxidase-conjugated sheep antimouse Ig Ab (GE Healthcare) or peroxidase-conjugated sheep anti-rabbit Ig Ab (Chemicon, Billerica, MA) and the Amersham ECL system (GL Healthcare) or the Immobilon Western detection reagents (Millipore). To reblot, the membrane was first stripped of Abs in 0.1 M glycine, pH 2.9.

LPS from Salmonella minnesota was obtained from Alexis Biochemicals (San Diego, CA), poly-IC potassium salt from Sigma-Aldrich (St. Louis, MO), IFN-a and IFN-b from PBL IFN source (Piscataway, NJ), human recombinant TGF-b from R&D Systems (Minneapolis, MN), and Pam3Cys-Ser-Lys4 from Invivogen (San Diego, CA). Mouse monoclonal antiiNOS Ab was obtained from BD Biosciences (Franklin Lakes, NJ; 610329) and used for Western blot. Rabbit polyclonal anti-iNOS Ab was obtained from Millipore (Temecula, CA; 06-573) and was used for immunoprecipitation. Rabbit polyclonal anti-ERK Ab was obtained from Cell Signaling Technologies (Danvers, MA), and mouse monoclonal anti-ubiquitin Ab P4D1 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA; sc-8017). Rat anti-Flag Ab was a kind gift from Drs. D. Huang and L. O’Reilly (Walter and Eliza Hall Institute of Medical Research).

NO production by macrophages in response to LPS or poly-IC was determined by measuring nitrite (NO22), a stable and nonvolatile breakdown product of NO, in culture supernatants using the Griess reaction (33). BMDM (1.0 3 106 per well) were stimulated with 20 ng/ml LPS or 25 mg/ ml poly-IC for the times indicated. Culture supernatants (100 ml) were mixed with 10 ml 1% sulfanilamide, incubated at room temperature for 10 min, and then mixed with 10 ml 0.1% N-1-naphthylethylenediamine dihydrochloride in 2.5% polyphosphoric acid and further incubated at room temperature for 5 min. Absorbance was measured at 550 nm, and nitrite concentration was determined by comparison with sodium nitrite standards.

Real-time quantitative PCR

Short hairpin RNA-mediated knockdown of SPSB1

RNA was extracted using the RNeasy kit from Qiagen (Hilden, Germany) and cDNA synthesized using Superscript II reverse transcriptase from Invitrogen (Carlsbad, CA). Real-time quantitative PCR (qPCR) was performed as previously described (30) using FastStart universal SYBR Green master mix (Roche, Mannheim, Germany)

Murine bone marrow-derived macrophages (BMDM) were derived by culture of whole bone marrow in DMEM supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin, 10% FBS (Sigma-Aldrich), and 20% L-cell conditioned medium as a source of macrophage colony stimulating factor (M-CSF) (31) and maintained at 37˚C in a humidified atmosphere with 10% CO2. FACS analysis confirmed that .95% of adherent cells were positive for CD11b expression (Mac-1) after 6 d in culture.

Oligonucleotides targeting Spsb1 (59-CCAGATGCAGAGAATAAACTA-39) were designed as described previously (34). shRNAmir constructs were created by annealing long oligonucleotides encoding the forward and reverse strands of the short hairpin RNA (shRNA) in 53 annealing buffer (0.5 M potassium acetate, 0.01 M magnesium acetate, and 0.15 M HEPES pH 7.4) for 5 min at 95˚C, followed by incubation for 10 min at 80˚C and a 5- to 7-h ramp from 80 to 4˚C (reducing by 0.5˚C every 2.5 min). Annealed oligonucleotides were subsequently subcloned into the LMP vector by virtue of the XhoI and EcoRI overhangs (35, 36). Non-sense shRNAmir control constructs in the LMP vector have been previously described (37). To generate retrovirus, 293T cells were transfected as described previously (37). The medium was replaced with DMEM containing 10% FBS and 20% L-cell conditioned medium 24 h posttransfection and viral supernatants harvested the following day. Total bone marrow was collected and RBCs removed by washing in red cell removal buffer (154.4 mM NH4Cl, 0.1 mM EDTA, 12 mM NaHCO3). Retroviral supernatants were applied to culture dishes pretreated with 32 mg/ml RetroNectin (Takara Biosciences, Shiga, Japan) and centrifuged for 1 h at 4000 3 g at 4˚C. Bone marrow cells were transduced by culturing on the viral-loaded RetroNectin-coated plates in the presence of 4 mg/ml polybrene for 24 h. Cells were replated in fresh media and incubated for 48 h, after which 2 mg/ml puromycin was added to select for infected cells. Six days postinfection, adherent macrophages were harvested and plated for subsequent experiments. qPCR was used to confirm knockdown of Spsb1 mRNA.

Immunoprecipitation

Statistical analysis

Primer sequences GAPDH: forward 59-TTGTCAAGCTCATTTCCTGGT-39, reverse 59-TTACTCCTTGGAGGCCATGTA-39; SPSB1: forward 59-CGGGGACTCAAGGGTAAAA-39, reverse 59-AGGGGCTCAGGATCAAGTC-39; SPSB2: forward 59-AAGAAGAGTGGAGGAACCACAAT-39, reverse 59-CAAAGGCAGAGTGGATATTTGAC-39; SPSB3: forward 59-GCAGCTCTAACTGGGCTATGACTC-39, reverse 59-ACAGGCACAGCACTGGGGATGGATG-39; SPSB4: forward 59-GAGTGCTGTGTGGGGTCA-39, reverse 59-AGGGCTGAGCGGATGGAT-39.

Generation of macrophages

+/+

T/+

T/+

T/+

BMDM from Spsb1 , Spsb1 , Spsb1DSB , and Spsb2 transgenic mice were lysed in KALB lysis buffer (32) containing protease inhibitors

NO production

Statistical analysis of NO and qPCR assays was performed using an unpaired t test with a 95% confidence level.

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Results SPSB1 expression is regulated by IFN-a/b and TGF-b Although our previous studies had demonstrated that SPSB2 regulates iNOS by ubiquitination and proteasomal degradation, we wished to determine whether other SPSB family members might also play a role in this process. As an initial step, we investigated whether SPSB proteins were transcriptionally regulated in response to LPS and poly-IC, receptor ligands that act through the TLR pathways to induce iNOS. BMDM from C57BL/6 mice were incubated with 20 ng/ml LPS, 10 mg/ml poly-IC, or 10 ng/ml Pam3CSK4. In response to the endotoxin LPS, only SPSB1 mRNA was transiently induced at 4 h with expression decreasing at 6 and 8 h, with no observed induction of Spsb2, Spsb4 (Fig. 1A), or Spsb3 (data not shown). Similarly, Spsb1 was the only Spsb induced in response to poly-IC treatment, with expression again peaking at 4 h (Fig. 1B). In contrast, none of the Spsb genes were expressed in response to Pam3CSK4 (Fig. 1B), suggesting that the induction may be TRIF dependent. To delineate further the TLR pathway regulating Spsb1, we used BMDM from C57BL/ 6, TRIF-deficient (Trif2/2 ), or MyD88-deficient (MyD882/2 ) mice stimulated with either 20 ng/ml LPS or 10 mg/ml poly-IC and analyzed the expression of Spsb1. As shown previously, Spsb1 expression was induced in wild-type macrophages at 4 h in response to both stimuli (Fig. 1C, 1D). Notably, Spsb1 expression was induced in response to LPS in the MyD882/2 but not in the Trif2/2 macrophages in response to either LPS or poly-IC (Fig. 1C, 1D), demonstrating that transcription of Spsb1 is activated downstream of both TLR3 and TLR4 and is dependent on the adapter TRIF. Given the delayed kinetics of Spsb1 induction and that induction was TRIF dependent, the data suggest that Spsb1 may be induced in response to IFN-b. BMDM were incubated with either 1000 U/ml IFN-a or IFN-b and Spsb expression analyzed by qPCR. Spsb1 mRNA was rapidly induced in response to both IFN-a and IFN-b

FIGURE 1. TLR regulation of Spsb genes. BMDM were generated from C57BL/6 mice and incubated in medium containing M-CSF (L-cell conditioned medium) and either 10 ng/ml LPS (A), 10 mg/ml poly-IC or 10 ng/ml Pam3CSK4 (B), 1000 U/ml IFN-a or 100 U/ml IFN-b (E), or TGF-b (F). Alternatively, BMDM were derived from C57BL/6, Trif2/2, or MyD88 2/2 mice and incubated in medium containing M-CSF and 10 ng/ ml LPS (C) or 10 mg/ml poly-IC (D) over an 8-h period. Total RNA was extracted, and SPSB mRNA levels were analyzed by qPCR (normalized against GAPDH). All bars represent means and SDs for samples derived from three individual mice.

SPSB1 IS AN INDUCIBLE REGULATOR OF iNOS EXPRESSION within 2 h (Fig. 1E), confirming that Spsb1 is a type I IFN signature gene. iNOS has previously been shown to be reduced at both the mRNA and protein levels by TGF-b (38–40); we therefore explored whether this could be mediated by TGF-b induction of SPSB proteins. Addition of TGF-b resulted in induction of Spsb1 at 2 h, peaking at 4 h, and was followed by a reduction in expression at 6 h. Conversely, there was no induction of other Spsb family members (Fig. 1F and data not shown). Treatment with medium alone resulted in a modest induction of Spsb1, presumably due to the presence of small amounts of cytokines such as TGF-b in the FCS. Forced expression of SPSB1 can reduce iNOS levels We have shown previously that SPSB1 interacts with iNOS when overexpressed in 293T cells (19). We have also demonstrated that forced expression of SPSB2 reduces the expression of iNOS after LPS stimulation (19). In the current experiments, we have repeated the LPS induction using SPSB2 transgenic macrophages as a positive control. To determine the functional consequences of the SPSB1 interaction with iNOS, we analyzed the kinetics and magnitude of iNOS expression in BMDM overexpressing SPSB1. BMDM from mice expressing Flag-tagged Spsb1 (Spsb1T/+), Spsb1 lacking the SOCS box (Spsb1DSB T/+), or Spsb2 (Spsb2T/+) transgene or from wild-type (Spsb1+/+) littermates were stimulated with 100 ng/ml LPS or 25 mg/ml poly-IC for various times, then lysed, and iNOS expression was analyzed by Western blot. In Spsb1+/+ and Spsb1DSBT/+ BMDM, iNOS expression was detected at 6 and 5 h with LPS and poly-IC treatment, respectively, and continued until 32 h (Fig. 2A, 2B). The early kinetics of iNOS induction remained unchanged in BMDM overexpressing either Spsb1T/+ or Spsb2T/+, although iNOS expression in Spsb1T/+ and Spsb2T/+ BMDM was lost within 12–24 h of treatment compared with 32 h in the Spsb1+/+ cells (Fig. 2A, 2B, top and bottom panels). iNOS expression in BMDM derived from mice


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FIGURE 2. Forced expression of SPSB proteins inhibits iNOS expression. A and B, BMDM from either Spsb1+/+, Spsb1T/+, Spsb1DSBT/+, or Spsb2T/+ mice were incubated with 100 ng/ml LPS (A) or 25 mg/ml poly-IC (B) for the times indicated. C, Alternatively, BMDM were incubated with or without (2) 20 ng/ml LPS and 10 ng/ml IFN-g, washed, replenished with fresh medium with (+) or without (2) the proteasomal inhibitor MG-132 (10 mM), and lysed at the indicated times postwash. Lysates were then separated by SDS-PAGE and analyzed by Western blot using anti-iNOS Abs (A–C, upper panels). Equivalent protein loading was confirmed by stripping and reprobing membranes with anti-ERK Abs (A, B, lower panels) or anti-tubulin Abs (C, lower panel). Western blots shown in A and B are representative of three independent experiments. D, Expression of Flag-tagged SPSB transgenes in LPS-treated BMDM from Spsb1+/+, Spsb1T/+, Spsb1DSBT/+, or Spsb2T/+ mice was confirmed by anti-Flag immunoprecipitation and Western blot (upper panel). iNOS expression was confirmed by Western blot with anti-iNOS Abs (middle panel, arrow indicates iNOS), and equivalent loading was analyzed by Western blot with anti-ERK Abs (lower panel). E, BMDM were incubated with 20 ng/ml LPS and 10 ng/ml IFN-g overnight followed by 10 mM MG-132 (+) for 4 h and lysed or were washed and replenished with fresh medium with (+) or without (2) MG-132 and lysed at 6 and 8 h postwash. iNOS was then immunoprecipitated followed by Western blot with anti-ubiquitin Abs. Immunoprecipitates (middle panel) and lysates (bottom panel) were probed with anti-iNOS Abs. F, BMDM from Spsb1+/+, Spsb1T/+, Spsb1DSBT/+, or Spsb2T/+ mice were cultured in medium containing either 100 ng/ml LPS or 25 mg/ml poly-IC. Culture supernatants were assayed for nitrite by the Griess assay at 16, 24, and 32 h. Bars represent means and SDs for samples derived from three individual mice and at 24 h are representative of four independent experiments. *p , 0.05.

expressing the Spsb1DSB T/+ was comparable with that of cells from wild-type mice (Fig. 2A, 2B, middle panels), suggesting that SPSB1 regulation of iNOS is a SOCS box-dependent mechanism. To determine if regulation of iNOS by SPSB1 was dependent on the proteasome, BMDM from Spsb1T/+ or wild-type controls

treated with the proteasomal inhibitor MG-132 were analyzed for iNOS expression. Overnight treatment of macrophages with LPS and IFN-g resulted in robust expression of iNOS, which was consistently reduced in cells expressing either the Spsb1 or Spsb2 transgene (Fig. 2C, 2E, bottom panels). Upon removal of the

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SPSB1 IS AN INDUCIBLE REGULATOR OF iNOS EXPRESSION stimulus and washing of the cells, iNOS was further reduced in Spsb1T/+ macrophages (8, 12 h postwash; Fig. 2C), but this was reversed with MG-132 treatment, indicating that iNOS clearance was mediated by proteasomal degradation (Fig. 2C, top panel, 2E, bottom panel). The SOCS box recruits an E3 ubiquitin ligase complex, and it is the polyubiquitination of substrates that targets them for degradation by the proteasome. Consistent with this, macrophages expressing either the Spsb1 or Spsb2 transgene showed enhanced ubiquitination of iNOS at 6 and 8 h postwash in the presence of MG-132 (Fig. 2E). Macrophage expression of the Spsb transgenes was confirmed by anti-Flag immunoprecipitation and Western blot analysis after overnight stimulation with 20 ng/ml LPS (Fig. 2D, top panel). As expected, the expression of Spsb1 and Spsb2 transgenes inversely correlates with the level of iNOS expression (Fig. 2D, middle panel). Notably, the Spsb1 transgene was expressed at significantly lower levels than the Spsb2 transgene in BMDM, suggesting that Spsb1 may be a more potent inhibitor of iNOS, although it is possible that both transgenes are expressed above a threshold level required to degrade this substrate. BMDM from Spsb1+/+, Spsb1T/+, Spsb1DSBT/+, and Spsb2T/+ were incubated with 20 ng/ml LPS or 25 mg/ml poly-IC and the culture supernatant analyzed for NO production using the Griess assay. At 16 and 24 h poststimulus, NO production in Spsb1T/+ BMDM was significantly reduced in response to both LPS and poly-IC compared with Spsb1+/+ cells. NO was also significantly reduced at 32 h in response to LPS, whereas NO production by Spsb1DSBT/+ macrophages was comparable with that of wild-type controls at all time points (Fig. 2F). Spsb1 regulates the induction of iNOS To determine the physiological relevance of the SPSB1–iNOS interaction, we used viral-mediated shRNA technology to reduce Spsb1 expression in BMDM. Retroviral shRNA constructs were designed to target Spsb1 or express a nonspecific shRNA sequence with no mammalian target (non-sense). To confirm that the shRNA construct was able to effectively knock down Spsb1 expression, BMDM transduced with either a non-sense control shRNA virus or Spsb1 shRNA virus were incubated with or without 10 ng/ml LPS for 4 h followed by qPCR analysis for Spsb1 mRNA levels. Spsb1 expression was significantly reduced in BMDM transduced with Spsb1 shRNA virus compared with that in BMDM infected with non-sense control shRNA virus (Fig. 3A). To analyze the kinetics of iNOS induction in BMDM with reduced Spsb1 expression, transduced cells were stimulated with either 100 ng/ml LPS or 25 mg/ml poly-IC for various times, then lysed, and iNOS expression was analyzed by Western blot. In BMDM transduced with non-sense shRNA virus, iNOS expression was detected at 8 h. In BMDM transduced with Spsb1 shRNA virus, expression of iNOS was detected earlier at 6 h and was generally enhanced in response to both stimuli (Fig. 3B). Once the stimulus was removed, the kinetics of iNOS clearance remained essentially unchanged, although an increased amount of iNOS

FIGURE 3. shRNA-mediated knockdown of Spsb1 results in enhanced and earlier expression of iNOS. A, BMDM from C57BL/6 mice were infected with either non-sense control shRNA or Spsb1 shRNA and incubated with or without 10 ng/ml LPS for 4 h, lysed, and analyzed for expression of Spsb1 via qPCR. B, BMDM from C57BL/6 mice were infected with either non-sense control shRNA or Spsb1 shRNA and incubated with 100 ng/ml LPS or 25 mg/ml poly-IC for the times indicated. C, Alternatively, BMDM were incubated with 20 ng/ml LPS or 25 mg/ml poly-IC overnight, washed, replenished with fresh medium, and lysed at the indicated times. Lysates were then analyzed by Western blot using antiiNOS Abs (B, C, upper panels). Equivalent protein loading was confirmed

by stripping and reprobing membranes with anti-ERK Abs (B, C, lower panels). Western blots shown are representative of two independent experiments. D and E, BMDM from C57BL/6 mice were infected with either non-sense control shRNA or Spsb1 shRNA and cultured in medium containing either 100 ng/ml LPS (D) or 25 mg/ml poly-IC (E). Culture supernatants were assayed for nitrite by the Griess assay at 16, 24, and 32 h. All bars represent means and SDs (n = 3). Each replicate represents independently infected pools of cells from different mice and at 24 h are representative of three independent experiments. *p , 0.05.


3803 was again observed in Spsb1 knockdown BMDM compared with that of the non-sense shRNA control after overnight stimulation (Fig. 3C). Using a Griess assay, NO production was measured in response to 20 ng/ml LPS (Fig. 3D) and 25 mg/ml poly-IC (Fig. 3E) in Spsb1 knockdown and control BMDM. At 16, 24, and 32 h, there was a significant increase in NO production in Spsb1 knockdown BMDM in response to LPS and a significant increase at 24 and 32 h in response to poly-IC. Similar results were also observed at 48 h (data not shown). Spsb1 and Spsb2 coordinate to regulate iNOS expression We have shown recently that Spsb2 regulates iNOS expression via proteasomal degradation and that removal of Spsb2 leads to prolonged iNOS expression and enhanced NO production (19). We therefore wanted to determine if Spsb1 and Spsb2 coordinate to regulate iNOS expression. Spsb22/2 BMDM transduced with either a non-sense control shRNA or Spsb1 shRNA virus were incubated with or without 10 ng/ml LPS for 4 h followed by qPCR analysis for Spsb1 mRNA levels. Spsb1 expression was significantly reduced in BMDM transduced with Spsb1 shRNA virus compared with that in BMDM transduced with non-sense control shRNA virus (Fig. 4A). To analyze iNOS induction, BMDM were stimulated with 100 ng/ml LPS or 25 mg/ml poly-IC for various times, lysed, and iNOS expression analyzed by Western blot. Spsb22/2 BMDM transduced with non-sense shRNA virus showed an induction of iNOS at 6 h posttreatment, which continued throughout the time course for both LPS and poly-IC. iNOS expression was enhanced in Spsb22/2 BMDM transduced with Spsb1 shRNA virus, and iNOS was again observed at an earlier time point (5 h) (Fig. 4B). In a clearance assay, iNOS expression in Spsb22/2 BMDM transduced with non-sense shRNA is prolonged as we have seen previously (19), although after transduction with Spsb1 shRNA virus, there is an additional accumulation of iNOS protein (Fig. 4C). At 8, 16, and 24 h, there was a modest increase in NO production in Spsb1 knockdown Spsb22/2 BMDM compared with that of controls in response to LPS and poly-IC (Fig. 4D, 4E). At 48 h, similar amounts of NO were observed in both non-sense and Spsb1 shRNA virus-transduced Spsb22/2 BMDM stimulated with LPS and poly-IC (data not shown). As the amount of NO produced by Spsb22/2 macrophages is already elevated, one possible explanation for this effect could be saturation of the medium, resulting in negative feedback to limit NO production. Alternatively, the supply of arginine could be exhausted.

Discussion

FIGURE 4. Spsb1 and Spsb2 coordinate to regulate iNOS expression. A, BMDM from Spsb22/2 mice were infected with either non-sense control shRNA or Spsb1 shRNA and incubated with or without 10 ng/ml LPS for 4 h, lysed, and analyzed for expression of Spsb1 via qPCR. B, BMDM from Spsb22/2 mice were infected with either non-sense control shRNA or Spsb1 shRNA, incubated with 100 ng/ml LPS or 25 mg/ml poly-IC, and lysed at the indicated times. Lysates were analyzed by Western blot using anti-iNOS Abs (upper panels). Equivalent protein loading was confirmed by stripping and reprobing membranes with anti-ERK Abs (lower panels). Western blots shown are representative of two independent experiments. C, Alternatively, Spsb22/2 BMDM were incubated with 20 ng/ml LPS or 25 mg/ml poly-IC overnight, washed, replenished with fresh medium, and lysed at the indicated times. Lysates were analyzed as in B. D and E, BMDM from Spsb22/2 mice were infected with either non-sense control

NO produced by iNOS in response to infection is a critical defense used by the host to kill various pathogens. However, as excessive NO can harm the host, NO production and its removal postinfection must be tightly controlled. In this study, we have identified SPSB1 as a negative regulator of iNOS expression and consequently NO production. We demonstrate that Spsb1 expression is regulated by the pro- and anti-inflammatory cytokines IFN-a/b and TGF-b, respectively, and that expression of SPSB1 under a constitutive promoter restricts iNOS expression and increases the clearance of

shRNA or Spsb1 shRNA and cultured in medium containing either 100 ng/ ml LPS (D) or 25 mg/ml poly-IC (E). Culture supernatants were assayed for nitrite by the Griess assay at 6, 16, and 32 h. All bars represent means and SDs (n = 3). Each replicate represents independently infected pools of cells from different mice and at 24 h are representative of two independent experiments. *p , 0.05.

3804 iNOS from BMDM in response to LPS and poly-IC. Conversely, we have shown that reducing SPSB1 levels via shRNA technology enhances iNOS induction. To date, there are only a handful of genes known to be induced by TRIF (41–44). Our results have uncovered a previously unrecognized downstream target of TRIF in Spsb1. Unlike the other SPSB family members, Spsb1 is induced after stimulation of TLR3 and TLR4 via the TRIF adapter and the production of IFN-b. IFN a and b, which are produced by many cell types and confer antiviral activity, shape the downstream events after pathogenic stimulation by inducing a range of genes, including iNOS, to help combat infection. iNOS regulates the expression of NO, which has been shown to inhibit the early stage of viral replication, thereby preventing viral spread and enhancing the clearance of virus from the host (45). In vitro experiments have suggested that NO does so by inhibiting viral RNA and DNA replication and protein synthesis (46–49). In this study, we demonstrate that IFN a and b induce expression of Spsb1, which helps to regulate iNOS induction and the subsequent production of NO in what appears to be a negative feedback loop. In confirmation of our results, a search of available microarray data using the Interferome program (http://www.interferome.org) also identifies SPSB1 as a type I IFN response gene. This positions SPSB1 as an important regulator of the antiviral host response. The lack of available Abs to SPSB1 has hampered confirmation of SPSB1 expression at the protein level. Spsb1 was induced by not only IFN a and b but also TGF-b. Pathogens can evade the host response by inducing TGF-b production, which in turn suppresses the killing activity of macrophages and enhances intracellular proliferation of the pathogen (38, 50–53). By inducing TGF-b, the subsequent induction of Spsb1 may be a mechanism by which pathogens subvert the host response to downregulate iNOS. Our results suggest that SPSB1 and SPSB2 coordinate to regulate iNOS expression. When SPSB1 is removed, iNOS expression is increased, consistent with the Spsb1 mRNA expression at 4 h and in contrast to our observations with SPSB2-deficient cells, where the early kinetics of induction remained unchanged (19). Knockdown of SPSB1 in SPSB2-deficient macrophages results in earlier detection of iNOS than with loss of SPSB1 or SPSB2 alone (5 versus 6 h; Figs. 3, 4) (19). Conversely, forced expression of SPSB1 enhances iNOS clearance yet somewhat surprisingly does not delay the timing of induction, perhaps because the low level of transgene does not exceed the expression of endogenous SPSB1 in the early phase of the response. Together with our demonstration that Spsb1 is the only Spsb gene induced after stimulation with pro- and anti-inflammatory cytokines, the SPSB1 knockdown data suggest that both Spsb1 and Spsb2 may act in concert to regulate the NO response (Fig. 5). Our previous studies have shown that Spsb2 mRNA is downregulated with LPS stimulation at 4 to 6 h, and this coincides with upregulation of iNOS. We can hypothesize that the transient expression of Spsb1 through TLR3 and TLR4 ensures that the potentially toxic NO response does not occur until the pathogenic stimulus has reached a particular duration of infection. When this critical point is reached, the expression of both SPSB1 and SPSB2 is decreased (19), and the high output of iNOS and NO can then kill the microorganism. Once the infection has been dealt with, the two proteins then synchronize to degrade iNOS and limit further cellular toxicity (Fig. 5). To our surprise, the Spsb1 transgene lacking a SOCS box (Spsb1DSBT/+) did not act as a dominant negative, perhaps because sufficient levels of endogenous SPSB proteins are available to mediate iNOS ubiquitination and degradation.

SPSB1 IS AN INDUCIBLE REGULATOR OF iNOS EXPRESSION

FIGURE 5. Schematic representation of NO regulation by SPSB proteins. Stimuli such as LPS and poly-IC trigger the activation of TLR3 and TLR4, which leads to TRIF activation, downstream type I IFN induction, and subsequent iNOS expression. Alternatively, LPS and poly-IC can directly switch on iNOS via NF-kb. The same TLR pathways can transiently induce Spsb1, which acts as a negative feedback mechanism to limit iNOS expression. SPSB1 and SPSB2 act in concert to control the expression of iNOS.

Induction of iNOS and downstream NO production have multiple roles, not only in fighting infectious disease but also in a range of autoimmune and inflammatory diseases (54–58). In the case of chronic infections, a potential therapeutic strategy to prolong iNOS expression and increase NO production could be to disrupt the SPSB–iNOS interaction. Given that SPSB1 and SPSB2 bind to the same sequence in iNOS (19), inhibitors that mimic the peptide to target the SPRY binding site have the added advantage of disrupting regulation by multiple SPSB proteins. By disrupting the SPSB–iNOS interaction, the effect of prolonging iNOS should be seen only in the target cell where iNOS is being produced in response to infection. This strategy would limit the toxicity associated with excessive systemic NO.

Acknowledgments We thank Sarah Freeman for technical assistance and Liana Mackiewicz, Merle Dayton, and Jaclyn Gilbert for excellent animal husbandry. We thank Prof. Paul Hertzog (Monash Institute of Medical Research) for the kind gift of rIFN-b. The TRIF- and MyD88-deficient mice were generously provided by Prof. Shizuo Akira (Osaka University, Japan). We are grateful to Prof. Nicos Nicola for critical reading of the manuscript and Dr. Ross Dickins for helpful discussions.

Disclosures The authors have no financial conflicts of interest.


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