Negatively Regulates TLR-Mediated Activation Pathways Young Jun Kang, Brenda Kusler, Motoyuki Otsuka, Michael Hughes, Nobutaka Suzuki, Shinobu Suzuki, Wen-Chen Yeh, This information is current as Shizuo Akira, Jiahuai Han and Patricia P. Jones of September 26, 2021. J Immunol 2007; 179:4598-4607; ; doi: 10.4049/jimmunol.179.7.4598 http://www.jimmunol.org/content/179/7/4598 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Calcineurin Negatively Regulates TLR-Mediated Activation Pathways1

Young Jun Kang,2,3* Brenda Kusler,* Motoyuki Otsuka,† Michael Hughes,* Nobutaka Suzuki,‡ Shinobu Suzuki,‡ Wen-Chen Yeh,‡ Shizuo Akira,§ Jiahuai Han,† and Patricia P. Jones3*

In innate immunity, microbial components stimulate macrophages to produce antimicrobial substances, cytokines, other proin- flammatory mediators, and IFNs via TLRs, which trigger signaling pathways activating NF-␬B, MAPKs, and IFN response factors. We show in this study that, in contrast to its activating role in T cells, in macrophages the protein phosphatase calcineurin negatively regulates NF-␬B, MAPKs, and IFN response factor activation by inhibiting the TLR-mediated signaling pathways. Evidence for this novel role for calcineurin was provided by the findings that these signaling pathways are activated when calcineurin is inhibited either by the inhibitors cyclosporin A or FK506 or by small interfering RNA-targeting calcineurin, and that activation of these pathways by TLR ligands is inhibited by the overexpression of a constitutively active form of calcineurin. Downloaded from We further found that I␬B-␣ degradation, MAPK activation, and TNF-␣ production by FK506 were reduced in macrophages from mice deficient in MyD88, Toll/IL-1R domain-containing adaptor-inducing IFN-␤ (TRIF), TLR2, or TLR4, whereas macrophages from TLR3-deficient or TLR9 mutant mice showed the same responses to FK506 as those of wild-type cells. Biochemical studies indicate that calcineurin interacts with MyD88, TRIF, TLR2, and TLR4, but not with TLR3 or TLR9. Collectively, these results suggest that calcineurin negatively regulates TLR-mediated activation pathways in macrophages by inhibiting the adaptor proteins MyD88 and TRIF, and a subset of TLRs. The Journal of Immunology, 2007, 179: 4598–4607. http://www.jimmunol.org/

n innate immunity, microbial components such as LPS, li- TLR10 has been identified in humans, but its ligand specificity is poprotein, peptidoglycan, unmethylated CpG DNA motifs, unknown (3, 12). TLR11, which is only expressed in mice, but not I flagellin, and viral dsRNA stimulate macrophages to produce in humans, is reported to be important in the immune response proinflammatory cytokines such as TNF-␣, IL-1, IL-6, and IL-12, against the uropathogenic bacteria (13). TLRs are expressed extra- as well as chemokines, inducible NO synthase, other antimicrobial or intracellularly. Although some TLRs (TLRs 1, 2, 4, 5, and 6) are responses, and coreceptor molecules. The interactions of the mi- expressed on the cell surface, others (TLRs 3, 7, 8, and 9) are crobial components with TLRs lead to the activation of the tran- found in intracellular compartments such as endosomes (14). Al- by guest on September 26, 2021 scription factor NF-␬B (1–3), which is required for the induction though the receptor for each ligand is specific, the downstream of these effector functions. The TLR-activated signaling pathway events are mediated by two shared signaling pathways. The first, is evolutionarily conserved, because activation of antimicrobial which is MyD88 dependent, is activated by all TLRs, except gene expression in insects by bacteria or fungi occurs via a ho- TLR3, and also by IL-1R (15, 16). Activated receptors induce the mologous pathway triggered by Toll receptors that leads to the formation of a complex of the adaptor protein MyD88 (17), which activation of the dorsal/rel family of NF-␬B homologues (4). recruits IL-1R-associated kinase (IRAK)4-4, which in turn binds Bacterial components activate specific TLRs on the plasma and activates IRAK-1, which then undergoes autophosphorylation membrane, as follows: lipid A component of bacterial endotoxin (18, 19). Following activation, phosphorylated IRAK-1 binds LPS activates TLR4 (5); lipoprotein and peptidoglycan activate TNFR-associated factor (TRAF)6 (20, 21), which in turn forms a TLR2 (6); bacterial CpG DNA activates TLR9 (7); flagellin acti- complex at the plasma membrane with TGF-␤-activated kinase vates TLR5 (8); viral dsRNA activates TLR3 (9); and ssRNA and (TAK)1, TAK1-binding protein (TAB)1, and TAB2, which in- synthetic nucleotide derivatives activate TLR7 and 8 (10, 11). duces the phosphorylation of TAB2 and TAK1. Activated TAK1 phosphorylates and activates the I␬B kinases (IKKs). The NF-␬B p50:p65 complex is normally sequestered in an inactive form in *Department of Biological Sciences, Stanford University, Stanford, CA 94305; †De- ␬ partment of Immunology, The Scripps Research Institute, La Jolla, CA 92037; ‡Ad- the cytoplasm through interaction with I Bs. Activated IKK phos- vanced Medical Discovery Institute, University Health Network and Department of phorylates I␬Bs, which are then ubiquitinated and degraded by Medical Biophysics, University of Toronto, Toronto, Ontario, Canada; and §Depart- proteasomes (22–24). The free NF-␬B complex translocates to the ment of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan nucleus and activates the transcription of target genes. TAK1 also Received for publication March 29, 2007. Accepted for publication July 11, 2007. activates pathways leading to the activation of ERK, JNK, and p38 MAPK pathways (25), which activate downstream transcription 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 factors that contribute to inflammatory gene expression. with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by funds from Stanford University (to P.P.J.). 2 Current address: Department of Immunology, The Scripps Research Institute, 10550 4 Abbreviations used in this paper: IRAK, IL-1R-associated kinase; AKAP, A-kinase North Torrey Pines Road, La Jolla, CA 92037. anchoring protein; CsA, cyclosporin A; IKK, I␬B kinase; IP-10, IFN-␥-inducible 3 Address correspondence and reprint requests to Dr. Young Jun Kang, 10550 North protein-10; IRF, IFN response factor; siRNA, small interfering RNA; TAB, TGF-␤- Torrey Pines Road, IMM-32, Department of Immunology, The Scripps Research activated kinase 1-binding protein; TAK, TGF-␤-activated kinase; TRAF, TNFR- Institute, La Jolla, CA 92037; E-mail address: [email protected] or Dr. Patricia P. associated factor; TRIF, Toll/IL-1R domain-containing adaptor-inducing IFN-␤. Jones, 371 Serra Mall, Gilbert Building, Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020; E-mail address: [email protected] Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org The Journal of Immunology 4599

The second pathway, which is MyD88 independent, is triggered University, Stanford, CA). FLAG-tagged human TRIF was cloned into by TLR4 (which also activates the MyD88-dependent pathway) pcDNA expression vector (Invitrogen Life Technologies). The pEGFP-C1 vector was purchased from BD Clontech; pNF-␬B-luciferase vector was and TLR3, leading to the activation of the transcription factor IFN ␤ ␤ purchased from Stratagene; pSV- -galactosidase vector was purchased response factor (IRF)3 and IFN- production (9, 26, 27), which from Promega. Control and CnA (the catalytic subunit of calcineurin, ␣ contributes to innate antiviral responses. The Toll/IL-1R domain- isoform)-targeting siRNA were purchased from Santa Cruz Biotechnology. containing adaptor-inducing IFN-␤ (TRIF) was identified as an Calcineurin inhibitor FK506 was provided by Fujisawa Pharmaceutical adaptor for TLR3 and TLR4 for the MyD88-independent pathway or purchased from LC Laboratories. LPS (Escherichia coli O111:B4) was obtained from Sigma-Aldrich and List Biological Laboratories; soluble (28, 29). peptidoglycan (Staphylococcus aureus) was purchased from Fluka; cal- Calcineurin is a serine/threonine phosphatase that consists of a cineurin inhibitor CsA and poly(I:C) were purchased from Sigma-Aldrich. catalytic subunit, CnA, and a regulatory subunit, CnB. It partici- Mouse rTNF-␣ was purchased from Roche Molecular Biochemicals; hu- pates in a variety of biological responses, including lymphocyte man rIL-1␤ was purchased from R&D Systems; and unmethylated CpG DNA and Pam CSK were purchased from InvivoGen. FK506, cyclospor- activation, neuronal development, muscle remodeling, memory, 3 4 ine, and all TLR ligands were tested for contamination of endotoxin by and heart valve morphogenesis (30). In T lymphocytes, calcineurin Limulus amebocyte lysate end-point test and found to have no or insignif- positively regulates the activation of NF-AT. Following binding of icant levels. the TCR to peptide:MHC complexes, the increase in intracellular Abs to p65, p50, I␬B-␣,I␬B-␤, IKK-␣, MEK-1, phospho-ERK-1, free Ca2ϩ leads to calcineurin activation through the binding of ERK-1, JNK, p38, IRAK-1, IRF3, calcineurin (CN-A), TLR4, and MyD88 2ϩ were purchased from Santa Cruz Biotechnology. Anti-IKK-␤ Ab was pur- Ca and calmodulin. Calcineurin dephosphorylates the inactive chased from Upstate Biotechnology; Abs to phopho-JNK, phospho-p38, cytosolic form of the transcription factor NF-ATc, which then and phospho-MEK-1/2 were purchased from Cell Signaling Technology; translocates to the nucleus and binds to the promoter/enhancer re- anti-GADPH Ab was purchased from Chemicon International. Abs to Downloaded from gion of critical response genes such as IL-2. In B cells, AU-1 and GFP were purchased from Covance, and FLAG-M2 Ab was calcineurin activity is required for immune responses in vivo (31). purchased from Sigma-Aldrich. Calcineurin is the target of the immunosuppressive drugs FK506 Reporter gene assays () and cyclosporin A (CsA); these drugs block cal- cineurin activity by forming inhibitory complexes with FK506- Transfected cells were lysed and assayed for luciferase activity using the Luciferase Assay Kit, according to the manufacturer’s protocol (Promega). binding proteins and , respectively. http://www.jimmunol.org/ Transfection efficiency was monitored by assaying ␤-galactosidase activity Information about effects of FK506 or CsA on the activation of by using ␤-Galactosidase Assay Kit (Roche Molecular Biochemicals). NF-␬B has been limited and unclear. These immunosuppressive ␬ drugs have been reported to reduce the activation of NF- BinT Preparation of nuclear extracts and EMSA cells (32, 33). However, we have shown (34) that FK506 and CsA activate NF-␬B and induce cytokine expression in nonactivated Nuclear extracts were prepared, as previously described (34). Radiolabeling of NF-␬B oligonucleotides with [␥-32P]ATP, EMSA, and supershift assays was macrophages and several other nonlymphoid cell types. This study performed, according to the manufacturer’s protocols (Promega). examines the mechanism by which calcineurin inhibitors activate TLR signaling and the target of negative regulation by calcineurin Preparation of cell lysates and Western blot in the TLR activation pathways. We demonstrate that inhibition of by guest on September 26, 2021 Preparation of cell lysates and Western blot analysis were performed, as calcineurin activates both the MyD88-dependent and the MyD88- previously described (35). independent pathways, reflecting calcineurin’s inhibitory effects on receptor-proximal signaling events through its interactions with IKK in vitro kinase assay certain TLRs and the adaptors MyD88 and TRIF. IKK in vitro kinase assays were performed, as described (36). Briefly, cells were washed twice with ice-cold PBS and lysed in lysis buffer, and 300 ␮g Materials and Methods of cell lysate was incubated with 4 ␮g of anti-IKK-␣ or IKK-␤ Abs at 4°C Mice, cell culture, and transfections for 2 h, and 30 ␮l of protein G-agarose beads (Santa Cruz Biotechnology) were added and incubated at 4°C for an additional 3 h. The immunopre- Thioglycolate-elicited peritoneal macrophages from wild-type mice, mice cipitate was washed three times with lysis buffer and twice with kinase deficient in MyD88, TRIF, TLR2, TLR3, or TLR4, or from TLR9 mutant buffer containing 25 mM Tris-Cl (pH 7.5), 5 mM ␤-glycerophosphate, 2 TLR9CpG1/CpG1 mice (provided to J. Han by B. Beutler, The Scripps Re- mM DTT, 0.1 mM sodium orthovanadate, 10 mM MgCl , 1 mM PMSF, 1 search Institute, La Jolla, CA) were aseptically harvested. The protocol for 2 ␮g/ml leupeptin, 2 ␮g/ml aprotinin, and 5 ␮g/ml pepstatin. The immuno- the use of animals was approved by the Institutional Animal Care and Use precipitate was incubated in 20 ␮l of kinase buffer supplemented with 5 Committees at Stanford University and The Scripps Research Institute. ␮Ci of [␥-32P]ATP and 5 ␮g of GST-I␬B-␣ (1–54) at 30°C for 30 min, and The mouse macrophage cell line RAW 264.7 was maintained in com- the reaction was terminated by adding 4ϫ SDS sample buffer. The samples plete RPMI 1640 medium (supplemented with 10% FBS and penicillin- were separated by SDS-PAGE on 10% acrylamide gels. The gel was cut in streptomycin). For the luciferase reporter gene assay, cells were transiently half, as follows: the upper part (above molecular mass of 47.5 kDa) was transfected with the corresponding expression vectors using DEAE-dextran, transferred to polyvinylidene difluoride membrane for the Western blot, following the manufacturer’s instructions (Promega). For the transfection of and the lower part was dried and subjected to autoradiography. small interfering (siRNA), 40 nM control or targeting siRNA was transfect- ected into RAW 264.7 cells using Lipofectamine 2000 reagent (Invitrogen Life Technologies). The human embryonic kidney cell line 293T was maintained in Native PAGE for analysis of IRF3 activation complete DMEM (supplemented with 10% FBS and penicillin-streptomycin), Native PAGE was performed, as described (37). and transiently transfected using Lipofectamine 2000. Plasmids and reagents RNA preparation and RT-PCR analysis Constitutively active calcineurin expression vector pEGFP-C1-(⌬CnA- Total RNA was isolated using RNeasy miniprep kit (Qiagen), and RT-PCR (⌬CaM-⌬AI)) was obtained from M. Kubo (Science University of Tokyo, was performed. The primer sets used were the following: for mouse IFN-␤, Tokyo, Japan). Wild-type and dominant-negative forms of MyD88 expres- forward, CCACAGCCCTCTCCATCAACTATAAGC, and reverse, AGC sion vectors were obtained from A. Mantovani (Mario Negri Institute, TCTTCAACTGGAGAGCAGTTGAGG; for mouse IFN-␥-inducible Milan, Italy). TLR4 and TLR3 expression vectors were obtained from R. protein-10 (IP-10), forward, CATCAGCACCATGAACCCAAGC, and Medzhitov (Yale University, New Haven, CT). Wild-type IRAK-1, wild- reverse, CCTCACTCCAGTTAAGGAGCC; for the mouse housekeep- type IRAK-4, and two different forms of dominant-negative IRAK-4 (N- ing gene GAPDH, forward, TCGTGGAGTCTACTGGCGT, and re- IRAK-4) (aa 1–191) and IRAK-4 KK213AA expression vectors were verse, GCCTGCTTCACCACCTTCT. The PCR products were analyzed cloned into pFLAG-C expression vector (obtained from S. Her, Stanford on a 1.2% agarose gel. 4600 NEGATIVE REGULATION OF TLR SIGNALING BY CALCINEURIN Downloaded from

FIGURE 2. Knockdown of calcineurin enhances the activation of TLR- mediated signaling pathway. RAW 264.7 cells were transfected with con- trol or calcineurin-targeting siRNA, and incubated for 3 days, and cells

were cultured 4 h before treatment. A, Cell lysates were prepared and http://www.jimmunol.org/ analyzed by immunoblot using anti-calcineurin or GAPDH Abs. B, Cells were treated with medium (None), LPS (0.1 ␮g/ml), peptidoglycan (PGN, 10 ␮g/ml), poly(I:C) (20 ␮g/ml), or CpG DNA (2.5 ␮g/ml) for 4 h, and nuclear extracts were prepared for EMSA. To provide a comparison with FIGURE 1. Calcineurin inhibitors activate TLR signaling. A, RAW effects of FK506 on levels of activated NF-␬B, nontransfected cells were 264.7 cells were stimulated by LPS (1 ␮g/ml), FK506 (FK, 10 ␮g/ml, stimulated with FK506 for 4 h. The x-ray films of the EMSAs from non- obtained from Fujisawa), or CsA (50 ␮g/ml) for 4 h. Nuclear extracts were transfected FK506-treated or siRNA-transfected cells were exposed for 4 h, prepared, and NF-␬B DNA-binding activity was determined by EMSA. whereas the EMSA film from siRNA-transfected nonstimulated cells was Supershift assays were performed by incubating LPS-stimulated nuclear exposed for 16 h. CЈ, Cold competition of FK506-treated cell nuclear ex- extract with anti-p50 or anti-p65 Abs. B, RAW 264.7 cells were stimulated tract using unlabeled NF-␬B probe. C, Cells transfected with control or by guest on September 26, 2021 by LPS, FK506, or CsA for the indicated time period, and cell lysates were calcineurin siRNA were treated with medium (None), poly(I:C), LPS, or prepared and subjected to SDS-PAGE, followed by Western blot with in- CpG DNA for 24 h. Culture supernatants were assayed for TNF-␣ by dicated Abs. C, IKK in vitro kinase assay. RAW 264.7 cells were stimu- ELISA. D, Cells transfected with control or calcineurin siRNA were lated by LPS or FK506 for the indicated time period, and cell lysates were treated with medium, LPS, or poly(I:C) for 6 h, and RNA samples were prepared and subjected to kinase assay using GST-I␬〉-␣ (1–54) as a sub- prepared for RT-PCR using IFN-␤ or GAPDH primers. E, The same as C, strate. Kinase activity (KA) was visualized by autoradiography, and en- except cells were treated with medium or FK506 (10 ␮g/ml). Data are p Ͻ 0.01 vs ,ءء p Ͻ 0.05, and ,ء .zyme immunoprecipitation was confirmed by immunoblotting with the cor- shown as means Ϯ SD of triplicates responding anti-IKK-␣ or IKK-␤ Abs (IB). D, Peritoneal macrophages control. Results shown are representative of two to three independent were stimulated with 0, 1, or 10 ␮g/ml FK506 and 0, 0.01, or 0.1 ␮g/ml experiments. LPS for 24 h. Culture supernatants were collected to measure TNF-␣ levels by ELISA. E, RAW 264.7 cells were stimulated with medium (None), LPS ␮ ␮ ␮ (0.1 g/ml), poly(I:C) (20 g/ml), or FK506 (10 g/ml), and harvested for1hat4°C, and further incubated with protein G-agarose for2hat4°C. after3hofincubation, and total RNA was prepared and subjected to The immunoprecipitate was washed and subjected to SDS-PAGE and RT-PCR using IFN-␤ (ifn-b) and IP-10 (ip-10) primers. GAPDH (gapdh) immunoblotting. primers were used as the internal control. F, RAW 264.7 cells were stim- ulated with medium (None), FK506 (10 ␮g/ml), poly(I:C) (20 ␮g/ml), or Statistical analysis ␮ LPS (0.1 g/ml), and incubated for 6 h, and cell lysates were subjected to Statistical significance was analyzed by Student’s t test. native PAGE and immunoblotting using anti-IRF3 Ab. G, RAW 264.7 cells were stimulated by LPS, or the indicated amount of FK506, and Results incubated for 30 min. Cell lysates were subjected to SDS-PAGE and im- Calcineurin inhibitors activate NF-␬B through the munoblotting with indicated Abs. Results shown are representative of two ␬ to four separate experiments. phosphorylation and degradation of I Bs induced by activated I␬B kinases Our previous studies showed that treatment with FK506 activates Coimmunoprecipitation and immunoblotting NF-␬B in mouse peritoneal macrophages, mouse myelomonocytic For analysis of interactions between calcineurin and MyD88, TRIF, TLR2, cell line WEHI-3, and L cell fibroblasts (34). To investigate the TLR3, TLR4, and TLR9, 293T cells were transfected with expression vec- mechanism of calcineurin regulation of the NF-␬B activation path- tors and harvested after 24 h, and cell lysates were immunoprecipitated way, cells of the mouse macrophage cell line RAW 264.7 were using Abs and analyzed by SDS-PAGE and immunoblotting. treated with LPS, FK506, or CsA for 4 h, and nuclear extracts were For the analysis of the dissociation of calcineurin from TLR4 and MyD88, RAW 264.7 cells were treated with FK506 (10 ␮g/ml) for 0 or 30 prepared for EMSA. Treatment with LPS or calcineurin inhibitors min, and cell lysates were subjected to immunoprecipitation using anti- activated p50:p65 NF-␬B complexes and induced the degradation TLR4 or MyD88 Abs. Briefly, cell lysates were incubated with 5 ␮gofAb of I␬B-␣ and I␬B-␤ (Fig. 1, A and B). These results confirm in this The Journal of Immunology 4601 Downloaded from http://www.jimmunol.org/ FIGURE 3. Calcineurin inhibits the IL-1R/TLR-mediated activation of NF-␬B. A, RAW 264.7 cells were transfected with pNF-␬B-luc (1.5 ␮g), pSV-␤-gal (1.5 ␮g), and 4.5 ␮g of control or constitutively active calcineurin (⌬CnA) expression vectors. After 48 h, cells were stimulated by medium ␮ ␮ ␮ ␮ ␤ ␣ (None), Pam3CSK4 (Pam3, 200 ng/ml), poly(I:C) (20 g/ml), LPS (0.1 g/ml), CpG DNA (2.5 g/ml), FK506 (10 g/ml), IL-1 (20 ng/ml), or TNF- (10 ng/ml). Cell lysates were prepared and subjected to luciferase and ␤-galactosidase assays. After normalization with ␤-galactosidase activity, fold activation was calculated as the fold increase in luciferase activity compared with the unstimulated cells. B, 293T cells were transfected with IRAK-1- FLAG, MyD88-AU-1, and GFP-⌬CnA vectors. Cell lysates were analyzed by immunoblotting using anti-IRAK-1 Ab to examine the phosphorylation of transfected IRAK-1, and also probed with anti-AU-1 or GFP Abs. pEGFP-C1 vector was cotransfected as the control. C, RAW 264.7 cells were cotrans- fected with pNF-␬B-luc (1.5 ␮g), pSV-␤-gal (1.5 ␮g), and control, dn TRAF6, dn IRAK-1, dn MyD88, N-IRAK-1, or KA-IRAK-4 expression vectors (4.5 ␮g each). After 48 h, cells were stimulated by LPS (1 ␮g/ml) or FK506 (10 ␮g/ml) and incubated for 6 h. Cell lysates were prepared for luciferase and Ϫ Ϫ ␤-galactosidase assays. D, Wild-type or MyD88 / peritoneal macrophages were treated with medium (None), CpG DNA (2.5 ␮g/ml), poly(I:C) (25 by guest on September 26, 2021 Indicates ,ء .␮g/ml), or FK506 (indicated amount in ␮g/ml) for 4 h. Nuclear extracts were prepared, and DNA-binding activity was analyzed by EMSA nonspecific binding. Results shown are representative of three to four independent experiments.

cell line that calcineurin-specific inhibitors FK506 and CsA acti- ␮g/ml). FK506 did not significantly increase production of TNF-␣ at vate NF-␬B, consistent with the inhibition by calcineurin of the the higher concentration of LPS (0.1 ␮g/ml), probably due to the NF-␬B activation pathway. higher potency of LPS at this concentration. Thus, inhibiting cal- The degradation of phosphorylated I␬B-␣ and the activation of cineurin adds to the signaling induced by LPS that leads to TNF-␣ NF-␬B induced by LPS or calcineurin inhibitors were inhibited by production. the inclusion of the proteasome inhibitor N-acetyl-Leu-Leu-Nle- ␤ CHO (data not shown), reflecting the role of proteasomes in Calcineurin inhibitor induces the IRF3 activation and IFN- pI␬B-␣ degradation. To test whether FK506 treatment activates expression IKK-␣ and/or -␤, in vitro kinase assays were performed in RAW Whether FK506 activates the MyD88-independent pathway lead- 264.7 cells. The kinase activities of both IKK-␣ and IKK-␤ were ing to IFN-␤ expression was also investigated. RT-PCR analysis induced by LPS and also by FK506 (Fig. 1C). These results are con- showed that LPS, poly(I:C), and FK506 all induce the expression sistent with induction of I␬〉 degradation and NF-␬B activation and of IFN-␤ and chemokine IP-10 (Fig. 1E). To confirm the activation indicate that the activation of NF-␬B by calcineurin inhibitors is me- of the MyD88-independent pathway, lysates from RAW 264.7 diated by IKK complex activation, leading to the phosphorylation and cells treated with all three stimulants were tested for the presence degradation of I␬Bs. of the activated dimeric form of IRF3. Anti-IRF3 immunoblot re- Production of the inflammatory cytokine TNF-␣ was examined. vealed increased dimeric IRF3 in lysates from cells treated with Peritoneal macrophages were treated with several concentrations FK506 as well as LPS and poly(I:C) (Fig. 1F), suggesting that of FK506 for 24 h, and culture supernatants were collected to calcineurin also negatively regulates the IFN signaling pathway. measure TNF-␣ levels by ELISA (Fig. 1D). Treatment with FK506 or LPS induced the production of TNF-␣, consistent with Calcineurin inhibitors also activate TLR-mediated the activation of NF-␬B, a critical transcription factor for the ex- MAPK pathways pression of TNF-␣ in macrophages. We further examined whether Because the TLR signaling cascade also leads to the activation of FK506 augments the production of TNF-␣ when added with LPS the ERK, p38, and JNK MAPK pathways (25), whether FK506 (Fig. 1D). Stimulation of macrophages by LPS induced production activates MAPK pathways was examined. Treatment of RAW of TNF-␣ in a dose-dependent manner, and FK506 augmented the 264.7 cells with either LPS or FK506 induced the phosphorylation production of TNF-␣ at a lower dose of LPS treatment (i.e., 0.01 of the upstream kinase MEK-1/2 and of the ERK, JNK, and p38 4602 NEGATIVE REGULATION OF TLR SIGNALING BY CALCINEURIN

FIGURE 4. Targets of negative regulation by calcineurin. Peritoneal macrophages from wild- type, MyD88Ϫ/Ϫ, TRIFϪ/Ϫ, TLR2Ϫ/Ϫ, TLR3Ϫ/Ϫ, TLR4Ϫ/Ϫ, or TLR9 mutant TLR9CpG1/CpG1 mice were A, incubated with 10 ␮g/ml FK506 for the in- dicated time period and harvested for Western blot analysis using indicated Abs, or B, incubated with

medium (None), Pam3CSK4 (Pam3, 200 ng/ml), poly(I:C) (25 ␮g/ml), LPS (0.1 ␮g/ml), CpG DNA Downloaded from (2.5 ␮g/ml), or FK506 (10 ␮g/ml) for 24 h, as in- dicated in each figure. Culture supernatants were as- sayed for TNF-␣ by ELISA. Data are shown as p Ͻ ,ءء ;p Ͻ 0.01 ,ء .means Ϯ SD of triplicates p Ͻ 0.001 vs control. Results shown ,ءءء and ;0.005 are representative of two to three independent http://www.jimmunol.org/ experiments. by guest on September 26, 2021

kinases (Fig. 1G), and as will be shown below, FK506 also acti- also enhanced in the calcineurin-knockdown cells (Fig. 2, C and vated MAPKs in mouse peritoneal macrophages. These results D). As was true for the activation of NF-␬B itself, the low basal demonstrate that calcineurin inhibitors activate MAPK pathways level of TNF-␣ production was higher in the knockdown cells. In and indicate that calcineurin negatively regulates the common addition, levels of IFN-␤ and TNF following stimulation with TLR TLR-proximal step(s) leading to both NF-␬B and MAPK ligands LPS, poly(I:C), or CpG DNA were higher in calcineurin activation. siRNA-targeted cells. In contrast, cells in which calcineurin was knocked down by siRNA were not activated by FK506 (Fig. 2E). Knockdown of calcineurin enhances the activation of This confirms that FK506 activates cells by inhibiting calcineurin, TLR-mediated pathway not through nonspecific or TLR-activating effects. These results To confirm that calcineurin functions as an inhibitor of TLR sig- support the conclusion that calcineurin inhibits the activation of naling pathways, which become activated when calcineurin is not TLR signaling pathways in resting cells and that inhibiting cal- active, experiments were performed using siRNA to knockdown cineurin mimics and synergizes with TLR ligands in activating calcineurin levels. RAW 264.7 cells were transfected with control downstream signaling pathways. or calcineurin-targeting siRNA (targeting catalytic subunit of cal- ␬ cineurin A ␣ isoform) and cultured for 3 days. Knockdown of Calcineurin inhibits the TLR-mediated NF- B activation ␣ calcineurin was confirmed by immunoblot (Fig. 2A). Similar to the pathway, but not the TNF- receptor-mediated activation effects of inhibiting calcineurin activity with CsA or FK506, the pathway basal level of active NF-␬B was higher in the knockdown cells. As The finding that calcineurin inhibitors induce NF-␬B activation shown in Fig. 2B (top), higher levels of active NF-␬B were present indicates that in resting macrophages calcineurin inhibits the in nuclear extracts from unstimulated calcineurin siRNA-trans- NF-␬B activation pathway. Therefore, NF-␬B activation by spe- fected cells than from unstimulated control siRNA-transfected cific stimuli might be blocked or overcome by active calcineurin. cells. In addition, the level of active NF-␬B induced by LPS, pep- To test this, cells were transfected with a vector encoding a con- tidoglycan, or CpG DNA was higher in the siRNA-transfected stitutively active form of the calcineurin A subunit, whose regu- calcineurin-knockdown cells (Fig. 2B, bottom). Induction of IFN-␤ latory regions (calmodulin-binding and autoinhibitory regions) mRNA and TNF-␣ secretion in response to LPS or poly(I:C) was have been deleted (⌬CnA-(⌬CaM-⌬AI)) (38). To assay NF-␬B The Journal of Immunology 4603 activation, cells were cotransfected with a pNF-␬B-luc reporter vector and a ␤-galactosidase expression vector as the transfection control. Transfected cells were stimulated by FK506, various bac- terial components, poly(I:C), IL-1␤, or TNF-␣ (which binds to TNFR and activates NF-␬B through a distinct set of receptor-prox- imal signaling proteins that also lead to IKK activation). In control vector-transfected cells, the various bacterial components, FK506, poly(I:C), IL-1␤, and TNF-␣, all activated NF-␬B, but in consti- tutively active calcineurin vector-transfected cells, NF-␬B activa- tion by the bacterial components, FK506, poly(I:C), or IL-1␤, was inhibited (Fig. 3A). Importantly, however, NF-␬B activation by TNF-␣ was not affected (Fig. 3A). The TLR-specific microbial components and IL-1␤ all activated NF-␬B in a dose-dependent manner, and activation at all concentrations was inhibited by calcineurin (data not shown). These results indicate that cal- cineurin specifically inhibits the IL-1R/TLR-proximal part of this pathway that is not shared by the TNF-␣-mediated NF-␬B activa- tion pathway that is dependent on TRAF2 (39, 40) (i.e., upstream of the TRAF6/TAK1/TAB complex). Conversely, FK506 and CsA Downloaded from appear to activate NF-␬B by blocking calcineurin’s inhibition of the receptor-proximal segment of the IL-1R/TLR pathway. Calcineurin regulates receptor-proximal steps in TLR-mediated NF-␬B activation pathway

After ligand binding, the TLRs (except TLR3) recruit the adaptor http://www.jimmunol.org/ protein MyD88, which in turn recruits IRAK-4, which recruits and activates IRAK-1, resulting in its hyperphosphorylation and deg- FIGURE 5. Calcineurin is associated with MyD88, TRIF, TLR2, and radation (19, 41). We tested whether the activation and phosphor- TLR4, not with TLR3 and TLR9. A, 293T cells were transfected with ylation of IRAK-1 are inhibited by the expression of constitutively TLR4-FLAG, MyD88-AU-1, and GFP-⌬CnA or control GFP vectors. Cell active calcineurin. The 293T cells were transfected with MyD88- lysates were immunoprecipitated with anti-AU-1 Ab and subjected to im- AU-1, FLAG-IRAK-1, and constitutively active calcineurin ex- munoblotting with indicated Abs. *, IgH. B, 293T cells were transfected with FLAG-TRIF, FLAG-IRAK-4, and GFP-⌬CnA or control GFP vec- pression vectors. Immunoblots showed that the phosphorylation of tors. Cell lysates were immunoprecipitated with anti-FLAG Ab and subjected IRAK-1 following activation by MyD88 overexpression was in- to immunoblotting with indicated Abs. C, 293T cells were transfected with by guest on September 26, 2021 hibited by the overexpression of constitutively active calcineurin FLAG-tagged TLR 2, 3, 4, or 9, and GFP-⌬CnA expression vectors. Cell in a dose-dependent manner (Fig. 3B). It is unlikely that IRAK-1 lysates were immunoprecipitated with anti-FLAG Ab and subjected to immu- itself is the direct target of negative regulation by calcineurin, be- noblotting with indicated Abs. D, RAW 264.7 cells were untreated or treated cause calcineurin failed to dephosphorylate in vivo hyperphospho- with FK506 (10 ␮g/ml) for 30 min, and cell lysates were immunoprecipitated rylated IRAK-1 immunoprecipitated from lysates of LPS-activated with isotype or indicated Abs, and the immunoprecipitate was resolved in cells and because the association of IRAK-1 and calcineurin could SDS-PAGE, followed by immunoblotting using indicated Abs. Results shown not be detected (data not shown). are representative of two to four independent experiments. Upstream of the IKK complex, the proteins involved in the TLR-proximal signaling cascade include MyD88, IRAK-4, IRAK-1, and TRAF6. As one approach to testing whether these proteins participate in the activation of NF-␬B induced by FK506, Targets of negative regulation by calcineurin cells were transfected with vectors encoding dominant-negative or The targets of negative regulation by calcineurin were further in- mutant forms of these signaling proteins, and cotransfected with vestigated using peritoneal macrophages from mice deficient in the NF-␬B-luc reporter vector. In the cells transfected with any of MyD88, TRIF, TLR2, TLR3, or TLR4, or from TLR9 mutant these dominant-negative or mutant expression vectors, NF-␬B ac- mice. Compared with wild-type macrophages incubated with tivation by either LPS or FK506 was inhibited (Fig. 3C). These FK506, FK506-treated cells from mice deficient in TLR4, TLR2, results support the conclusion that calcineurin negatively regulates MyD88, or TRIF showed reduced/delayed degradation of I␬B-␣ the NF-␬B activation pathway at early, receptor-proximal step(s). and phosphorylation of p38, JNK, and ERK MAPKs. In contrast, Experiments were performed to determine which receptor-prox- cells from TLR3-deficient or TLR9 mutant mice responded nor- imal step or component is negatively regulated by calcineurin. The mally to FK506 (Fig. 4A). Levels of TNF-␣ secretion by FK506- dependence of FK506-induced NF-␬B activation on MyD88 was treated cells from various deficient/mutant mice paralleled the de- assessed by testing whether FK506 activates NF-␬B in macro- gree of I␬B-␣ degradation and of MAPK phosphorylation. phages from MyD88 knockout mice. In MyD88Ϫ/Ϫ cells, the level Induction of TNF-␣ secretion by FK506 was reduced in macro- of activation of NF-␬B by FK506 or by the MyD88-dependent phages from mice deficient in MyD88, TRIF, TLR2, or TLR4, ligand CpG DNA was reduced compared with the level in the whereas TNF-␣ secretion by TLR3-deficient or TLR9 mutant cells wild-type cells, but activation by the MyD88-independent ligand was the same as that of wild-type cells (Fig. 4B). Together with the poly(I:C) was not (Fig. 3D). Residual NF-␬B activation in finding that constitutively active calcineurin blocks activation by MyD88Ϫ/Ϫ cells by FK506 presumably reflects its activation of ligands for all TLR tested, including TLR3 and TLR9, these results the MyD88-independent pathway (see below). These results indi- strongly suggest that the receptor-proximal adaptor proteins cate that calcineurin acts on or above MyD88 to block downstream MyD88 and TRIF, and TLR2 and TLR4, but not TLR3 and TLR9, signaling events leading to NF-␬B activation. are targets of negative regulation by calcineurin. 4604 NEGATIVE REGULATION OF TLR SIGNALING BY CALCINEURIN

FIGURE 6. Model for the negative regu- lation of TLR signaling by calcineurin. A,In nonactivated (resting) macrophages, cal- cineurin interacts and negatively regulates the adaptor proteins MyD88 and TRIF, and TLRs expressed on the plasma membrane such as TLR2 and TLR4, not TLRs that are only expressed in endosomes (TLR3 and TLR9 have been tested), the initial compo- nents of the pathways leading to NF-␬B, MAPK, and IRF3 activation. B, Calcineurin inhibitors or ligand binding to the TLRs lead to the activation of NF-␬B and other tran- scription factors (TFs) via MyD88-depen- dent and MyD88-independent pathways. The filled and open symbols denote the in- active and active forms of the molecules, re- spectively. Endosomes are indicated as dou- ble-lined circles. See Discussion. Downloaded from

Calcineurin associates with MyD88, TRIF, and TLRs mouse myelomonocytic cell line, primary mouse astrocytes, and To test whether calcineurin interacts with MyD88, TRIF, and the L929 fibroblast cell line, and more recently have extended TLRs, expression constructs encoding tagged TLR4, MyD88, and these findings to the mouse macrophage cell line RAW 264.7, constitutively active calcineurin were transiently expressed in mouse bone marrow-derived macrophages and dendritic cells and splenic dendritic cells, and the human monocytic cell lines U937 293T cells, and possible protein interactions were assessed by im- http://www.jimmunol.org/ munoprecipitation and immunoblotting. GFP-tagged constitutively and THP-1. Importantly, treatment of calcineurin inhibitors alone active calcineurin was detected in AU-1-tagged MyD88 immuno- did not induce the activation of NF-␬B in mouse T cells (34) or the precipitates from cells cotransfected either with MyD88 alone or human Jurkat T cell line (data not shown). In T cells, TNF-␣ is with MyD88 and TLR4 (Fig. 5A) (note that the amount of GFP- induced by activation of NF-AT (42), which is inhibited by cal- calcineurin in the immunoprecipitate is greater in cells cotrans- cineurin inhibitors, whereas in macrophages and dendritic cells it fected with FLAG-TLR4 (see below)). is induced by NF-␬B activation. As we reported earlier (34), Coimmunoprecipitation analysis was also used to test whether NF-␬B is activated by FK506 and CsA, but not by the immuno- calcineurin interacts with TLR2, TLR4, and TRIF, but not with suppressant rapamycin, which, like FK506 and cyclosporin, binds TLR3 and TLR9, as suggested by the functional studies with cells immunophilins and inhibits their peptidyl-prolyl activ- by guest on September 26, 2021 from defective/mutant mice. GFP-tagged calcineurin was coimmu- ity, but unlike FK506 and CsA, rapamycin does not inhibit cal- noprecipitated from lysates of transfected 293T cells with FLAG- cineurin. That FK506 activates macrophages by inhibiting cal- TRIF, but not with FLAG-IRAK-4, and with FLAG-TLR2 and cineurin and not by a nonspecific calcineurin-independent activity FLAG-TLR4, but not with FLAG-TLR3 or FLAG-TLR9 (Fig. 5, was shown by the failure of FK506 to activate cells in which B and C). These results support the findings that MyD88, TRIF, calcineurin was knocked down by siRNA. TLR2, and TLR4, but not TLR3 or TLR9, are targets of negative Because calcineurin is involved in embryonic development (30), regulation by calcineurin. The finding that calcineurin associates no calcineurin-negative mice have been generated. Calcineurin A␣ with TLR4 also explains the higher levels of calcineurin in MyD88 knockout mice show a variety of abnormalities and a shortened life immunoprecipitates when TLR4 was cotransfected (Fig. 5A). span (43), and retain partial phosphatase activity due to calcineurin Interaction of endogenous calcineurin with TLR4 and MyD88 A␤, resulting in the partial impairment of T cell functions (44, 45). was examined. Calcineurin was coimmunoprecipitated with TLR4 Calcineurin A␤-deficient mice fail to generate mature T cells (46). or MyD88 from untreated cell lysates, indicating the interaction of Therefore, calcineurin inhibitors FK506 and CsA have been an endogenous TLR4 and MyD88 with calcineurin (Fig. 5D). In con- important approach for dissecting the function of calcineurin in trast, little or no calcineurin was detected in the TLR4 or MyD88 innate immunity. The activation of NF-␬B by these calcineurin immunoprecipitates from FK506-treated cell lysates, suggesting that the calcineurin inhibitor FK506 induces the dissociation of inhibitors or by knocking down calcineurin with siRNA, together ␬ calcineurin from TLR4 and MyD88, which most likely leads to the with the inhibition of NF- B activation by expression of consti- activation of TLR signaling pathway. tutively active calcineurin, strongly suggests that calcineurin is a Together these findings demonstrate that calcineurin negatively negative regulator of antimicrobial and proinflammatory gene ac- regulates both MyD88-dependent and MyD88-independent TLR tivation in macrophages and other cell types that contribute to signaling pathways, and that calcineurin appears to act by inter- innate immunity. acting with TLR2 and TLR4 as well as with the receptor-proximal The findings reported in this study demonstrate that the mech- adaptor proteins MyD88 and TRIF. anisms of NF-␬B activation by calcineurin inhibitors are similar to those of the well-characterized NF-␬B activation pathways trig- Discussion gered by microbial components (schematized in Fig. 6). As with Ligand binding to the IL-1R and TLRs leads to the activation of stimulation by TLR ligands, the phosphorylation and degradation NF-␬B and MAPKs, critical events for the induction of antimicro- of I␬B-␣ were shown to be required for the activation of NF-␬Bby bial mediators, proinflammatory cytokines, and other effector func- calcineurin inhibitors. Although it was previously reported that tions. We previously reported (34) that calcineurin inhibitors ac- FK506 activates NF-␬B through the phosphorylation and degra- tivate NF-␬B in mouse peritoneal macrophages, the WEHI-3 dation of I␬B-␣ in L929 fibroblast cells (36), in that study IKK-␣ The Journal of Immunology 4605

or IKK-␤ activation was reported not to be involved. In our stud- the nucleus (34) (nuclear localization of NF-AT is dependent on its ies, FK506 activated both IKK-␣ and IKK-␤, leading to activation dephosphorylation by calcineurin). Exposing these cells to cal- of NF-␬B through the phosphorylation and proteasome-mediated cineurin inhibitors resulted in a decrease in NF-AT and a concom- degradation of I␬B proteins. itant increase in the activation and nuclear localization of NF-␬B. Calcineurin negatively regulates not only the MyD88-depen- We found that the free calcium level in resting macrophages is dent, but also the MyD88-independent pathways. Constitutively 130–150 nM (unpublished observation, T. Chan, P. Jones, R. active calcineurin inhibits NF-␬B activation by poly(I:C), whereas, Luik, and R. Lewis), and reports in the literature also indicate that conversely, FK506 activates IRF3 and induces IFN-␤ and IP-10 resting macrophages have higher levels of intracellular free cal- expression, and siRNA-mediated calcineurin knockdown increases cium (100–200 nM) than do resting T cells (47, 48). This appears IFN-␤ mRNA expression. That macrophages from TRIF-deficient to be sufficient to support a basal level of calcineurin activity in mice show delayed/reduced degradation of I␬B and activation of macrophages that normally keeps the signaling pathways off until MAPKs following FK506 treatment compared with cells from calcineurin activity is blocked or bypassed following activation by wild-type mice is also consistent with a role for calcineurin in IL-1R/TLR ligands. inhibiting the MyD88-independent signaling pathway. The mechanism(s) by which active calcineurin negatively reg- Results from a number of functional studies indicate that cal- ulates the activation of the IL-1R/TLR pathway at the level of the cineurin regulates early, receptor-proximal step(s) in the TLR sig- receptors and their proximal adaptor proteins is not known. Be- naling pathway. First, because overexpression of constitutively ac- cause calcineurin is a serine/threonine phosphatase, one possible ␬ tive calcineurin blocked the activation of NF- B in macrophages mechanism is that calcineurin removes a serine- or threonine-as-

␤ Downloaded from by microbial components, IL-1 , or FK506, but not by TNF, cal- sociated phosphate group that is essential for activation. No serine/ cineurin must negatively regulate the common portion of the IL- threonine phosphorylation of TLRs, MyD88, or TRIF has been ␬ 1R/TLR-mediated NF- B activation cascade not shared by the described. However, evidence has been published that TLR4 ac- TNF-mediated pathway, i.e., upstream of the TRAF6/TAK1/TAB tivation by LPS results in MyD88 tyrosine phosphorylation, al- complex (2). A similar conclusion can be drawn from the finding lowing recruitment of PI3K to MyD88 and activation of Akt, that calcineurin inhibitors also activate MAPK pathways, which which contributes to the activation of NF-␬B and expression of are also activated by TAK1. Third, FK506-induced activation of IL-1␤ (49). Kinase-mediated serine/threonine phosphorylation and http://www.jimmunol.org/ NF-␬B was inhibited by the expression of dominant-negative calcineurin-mediated dephosphorylation of the receptors and prox- forms of the receptor-proximal components MyD88, IRAK-4, and imal adaptors could play a role in regulating the activation status IRAK-1. Fourth, the phosphorylation of IRAK-1 induced by the of these proteins. overexpression of MyD88 was inhibited by the expression of con- Several cytoplasmic proteins that function as calcineurin inhib- stitutively active calcineurin. Finally, that calcineurin acts at re- itors have been identified, and in some cases they are components ceptor-proximal steps of the TLR activation pathway was most of complexes with receptors and other signaling components that convincingly demonstrated by the finding that activation by FK506 regulate the activation of signaling pathways. For example, in neu- of downstream signaling pathways leading to I␬B-␣ degradation

rons, calcineurin is associated with A-kinase anchoring protein by guest on September 26, 2021 and MAPK phosphorylation is delayed or impaired in macro- (AKAP), which inhibits its phosphatase activity. Protein kinases A phages from mice deficient in MyD88, TRIF, TLR2, or TLR4 and C are also associated with AKAP in these complexes (50, 51). compared with macrophages from wild-type mice. Interestingly, Thus, complexes of AKAP, calcineurin, and protein kinases con- FK506 activation of downstream pathways is not impaired in mac- rophages from TLR3-deficient or TLR9 mutant mice. stitute a signal regulation complex that may reversibly modulate These observations suggest that the targets of negative regula- the phosphorylation status and activity of signaling molecules. tion by calcineurin are the adaptor proteins MyD88 and TRIF, and Calcineurin inhibitors FK506 and CsA are potent immunosup- some TLRs as follows: TLR2 and TLR4 (which are expressed on pressants, reflecting calcineurin’s key role in T cell activation due the cell surface), but not TLR3 or TLR9 (which are not on the cell to its calcium flux-activated dephosphorylation and activation of surface, but in endosomal membranes). Whether this apparent NF-AT. Treatment with either inhibitor reduces immune-mediated specificity of calcineurin interaction reflects the cellular localiza- rejection following organ transplantation. However, they also tion of the TLR or other TLR differences, such as in their cyto- cause side effects, including nephrotoxicity, hypertension, and in- plasmic domains, remains to be determined. The ability of consti- creased risk of cardiovascular events (52, 53). The pathogenesis of tutively active calcineurin to inhibit NF-␬B activation by TLR3 nephrotoxicity and the other side effects induced by calcineurin and TLR9 ligands (poly(I:C) and CpG DNA, respectively) pre- inhibitors are complex and incompletely understood, but among sumably reflects inhibitory interactions of calcineurin with the the factors implicated are renal and systemic vasoconstriction, in- adaptors TRIF and MyD88. creased release of endothelin-1, and increased expression of TGF- Consistent with the functional evidence that calcineurin nega- ␤1. Endothelin-1 transcription is controlled by NF-␬B in vascular tively regulates receptor-proximal steps in the TLR activation endothelium (54), and endothelin-1 itself is proinflammatory, be- pathway, calcineurin was shown to associate both with TLR2 and cause it activates NF-␬B in macrophages (55). Interstitial fibrosis TLR4, but not with TLR3 or TLR9, and with the proximal adaptor caused by CsA is associated with increased expression of TGF-␤1 proteins MyD88 and TRIF. (56), and TGF-␤1 is responsive to induction by IL-1 in macro- Our findings (Ref. 34 and this study) that treatment of macro- phages, which activates NF-␬B and several other transcription fac- phages and some other cell types with calcineurin inhibitors acti- tors (57). IL-6 has been reported to contribute to nephrotoxicity vates NF-␬B and MAPK indicate that these resting cells have basal (58, 59), and FK506 induces IL-6 production in fibroblasts through levels of calcineurin activity that help to maintain the TLR-medi- the activation of NF-␬B (60). A role for FK506-induced NF-␬B ated NF-␬B and MAPK activation pathways in an “off” position activation in inducing nephrotoxicity was shown by a study in rats that is reversed by calcineurin inhibitors (Fig. 6). The presence of of chronic FK506 nephropathy, in which the NF-␬B inhibitor pyr- basal calcineurin activity is supported by nuclear localization stud- rolidine dithiocarbamate reduced FK506-induced monocyte/mac- ies with nonactivated mouse peritoneal macrophages and WEHI-3 rophage infiltration, tubular injury, and intestinal fibrosis, which myelomonocytic cells that showed detectable levels of NF-AT in are hallmarks of nephrotoxicity (61). 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