The Role of JAK-3 in Regulating TLR-Mediated Inflammatory Production in Innate Immune Cells

This information is current as Huizhi Wang, Jonathan Brown, Shegan Gao, Shuang Liang, of September 28, 2021. Ravi Jotwani, Huaxin Zhou, Jill Suttles, David A. Scott and Richard J. Lamont J Immunol 2013; 191:1164-1174; Prepublished online 24 June 2013;

doi: 10.4049/jimmunol.1203084 Downloaded from http://www.jimmunol.org/content/191/3/1164

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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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Role of JAK-3 in Regulating TLR-Mediated Inflammatory Cytokine Production in Innate Immune Cells

Huizhi Wang,* Jonathan Brown,† Shegan Gao,‡ Shuang Liang,* Ravi Jotwani,* Huaxin Zhou,* Jill Suttles,*,† David A. Scott,*,† and Richard J. Lamont*,†

The role of JAK-3 in TLR-mediated innate immune responses is poorly understood, although the suppressive function of JAK3 inhibition in adaptive immune response has been well studied. In this study, we found that JAK3 inhibition enhanced TLR-mediated immune responses by differentially regulating pro- and anti- inflammatory cytokine production in innate immune cells. Specifically, JAK3 inhibition by pharmacological inhibitors or specific small interfering RNA or JAK3 gene knockout resulted in an increase in TLR-mediated production of proinflammatory while concurrently decreasing the production of IL-10. Inhibition of JAK3 suppressed phosphorylation of PI3K downstream effectors including Akt, mammalian target of rapamycin complex 1, glycogen synthase kinase 3b (GSK3b), and CREB. Constitutive activation of Akt or inhibition of GSK3b abrogated the capability of JAK3 Downloaded from inhibition to enhance proinflammatory cytokines and suppress IL-10 production. In contrast, inhibition of PI3K enhanced this regulatory ability of JAK3 in LPS-stimulated monocytes. At the transcriptional level, JAK3 knockout lead to the increased phosphorylation of STATs that could be attenuated by neutralization of de novo inflammatory cytokines. JAK3 inhibition exhibited a GSK3 activity-dependent ability to enhance phosphorylation levels and DNA binding of NF-kB p65. Moreover, JAK3 inhibition correlated with an increased CD4+ T cell response. Additionally, higher neutrophil infiltration, IL-17 expression, and intestinal epithelium erosion were observed in JAK3 knockout mice. These findings demonstrate the negative regulatory function http://www.jimmunol.org/ of JAK3 and elucidate the signaling pathway by which JAK3 differentially regulates TLR-mediated inflammatory cytokine produc- tion in innate immune cells. The Journal of Immunology, 2013, 191: 1164–1174.

oll-like receptor–mediated inflammatory cytokine pro- of TIRAP-MyD88 leads to the activation of IL-1R–associated duction plays a critical role in the activation of innate and kinase 4, in turn activating TNFR-associated factor 6 and, ultimately, T acquired immune responses. The magnitude and quality leading to the activation of the NF-kB pathway that mediates the of cytokines must be fine-tuned to achieve the effective clearance production of pro- and anti- inflammatory cytokines. In contrast, of pathogens while limiting the amount of inflammation to avoid the recruitment of Toll/IL-1R domain–containing adapter-inducing by guest on September 28, 2021 toxicity and collateral tissue damage (1–3). Thus, the mechanisms IFN-b–related adaptor molecule and TRIF mediates a signaling modulating TLR-mediated cytokine production have attracted con- cascade involving the activation of two noncanonical IkBkinases, siderable attention. TLR engagements initiate several signaling TANK-binding kinase-1 and IkB kinase-ε,aswellasthephospho- pathways through various combinations of the adapter molecules specific translational modificationofthetranscription factors MyD88, TIRAP, Toll/IL-1R domain–containing adapter-inducing NF-kB, activating -2/c-Jun, and IFN regulatory IFN-b (TRIF), Toll/IL-1R domain–containing adapter-inducing factor 3 that subsequently induce type I IFN and an antiviral re- IFN-b–related adaptor molecule, and SARM (4). TLR4 signal- sponse (5, 6). SARM acts as a negative regulator of TLR signaling ing is the most complex, as all five adapters are involved. Activation by directly suppressing the function of TRIF, TRIF-dependent IFN regulatory factor 7, and NF-kB activities (7, 8). Although a sig- nificant body of literature exists on TLR signaling, many key *Oral Health and Systemic Disease Research Group, University of Louisville School elements involved in the regulation of TLR signaling remain un- of Dentistry, Louisville, KY 40202; †Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, KY 40202; and known, namely the role of upstream tyrosine kinases on TLR ‡Department of Oncology, Cancer Institute, First Affiliated Hospital of Henan Uni- functions and how they ultimately shape cytokine profiles upon versity of Science and Technology, Luoyang 471500, China pathogen-associated molecular pattern recognition. Received for publication November 6, 2012. Accepted for publication May 24, 2013. Tyrosine phosphorylation is one of the earliest events noted after This work was supported by Grant R01 DE017921 (to R.J.L.) from the National TLR ligation, and tyrosine kinases are involved in the regulation of Institute of Dental Research. TLR-mediated inflammation in at least two ways. Tyrosine kinases H.W. and R.J.L. designed research, analyzed and interpreted data, performed re- can either act as components of TLR initiated signaling pathways search, and wrote the manuscript. J.B., S.G., R.J., and H.Z. performed research. S.L. and D.A.S. performed research, analyzed and interpreted data, and wrote the man- and thus directly control cytokine production or they can be in- uscript. J.S. designed research and analyzed and interpreted data. volved in the signaling stimulated by TLR-induced cytokines (9, 10). Address correspondence and reprint requests to Dr. Richard J. Lamont or Dr. Huizhi JAK is an important tyrosine kinase family that includes JAK1, Wang, University of Louisville School of Dentistry, 501 South Preston Street, Baxter JAK2, JAK3, and Tyk2 (9, 11). Past studies have showed that defects I Room 221B, Louisville, KY 40202. E-mail addresses: [email protected] (R.J.L.) or [email protected] (H.W.) of JAK3 can result in some of the commonest inherited severe Abbreviations used in this article: BMDM, bone marrow–derived macrophage; immunodeficiencies (12, 13). Although the functional role of JAK3 GSK3b, glycogen synthase kinase 3b; mTORC1, mammalian target of rapamycin in adaptive immune responses has been well investigated, the impact complex 1; PMN, polymorphonuclear neutrophil; siRNA, small interfering RNA; of JAK3 on TLR-mediated innate immune responses is controver- TRIF, Toll/IL-1R domain–containing adapter-inducing IFN-b; WT, wild-type. sial, and the underlying mechanisms remain unclear (14–17). In this Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 regard, Yamaoka et al. (14) initially reported that JAK3 knockout www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203084 The Journal of Immunology 1165 enhanced the production of IL-12 and IL-10 in LPS-stimulated Transfection, cytokine detection, and Western blot bone marrow–derived dendritic cells, which subsequently resulted Transfection of human monocytes was carried out by electroporation using in exaggerated Th1 differentiation. In contrast, Kim et al. (17) a Nucleofector device (Amaxa) according to the manufacturer’s protocols. showed that JAK3 knockout in macrophages significantly reduced Briefly, purified 4 3 106 monocytes were resuspended in 100 ml Nucle- TLR4-mediated IL-10 production, which resulted in enhanced ofector solution (Human Monocytes Nucleofector ; Amaxa) along with IL-1b–converting enzyme activity that consequently augmented 2 mg GFP-coding plasmid (pCMV-GFP) and 2 mg siRNA duplexes or ectopic plasmids for each target. Immediately after electroporation, 400 ml the production of IL-1b. Thus, these studies showed differing effects prewarmed M-199 containing 10% FCS were added to cells that were then of JAK3 on TLR4-mediated pro- and anti-inflammatory cytokine transferred into culture plates containing prewarmed M-199 with 10% production, and the function and role of JAK3 remains to be FCS. At 48 h posttransfection, cells were exposed to LPS (1 mg/ml) with determined. or without inhibitor. Cell lysates were prepared as previously described (20, 21). Images were acquired using the Kodak Image Station 4000MM In this study, we report that JAK3 negatively regulates TLR4- system (Eastman Kodak, New Haven, CT). For siRNA studies, the levels of mediated inflammatory responses through differentially regulat- total JAK3 were assessed by Western blot at 3 d postransfection. For experi- ing pro- and anti-inflammatory cytokine production. Inhibition ments using inhibitors for PI3K (LY294002; 25 mM) or GSK3 (SB216763; of JAK3 attenuated TLR4-mediated activation of PI3K and then 10 mM), control cells were pretreated for 2 h with 0.01% DMSO (organic suppressed the phosphorylation of Akt and its downstream con- solvent control) prior to LPS (1 mg/ml) stimulation. For siRNA studies, transfected cells were stimulated with LPS (1 mg/ml) 3 d posttransfection, stitutive active kinase, glycogen synthase kinase 3b (GSK3b), and cell-free supernatants were assayed for cytokine levels by ELISA which augmented proinflammatory cytokine level and repressed (eBioscience) 20 h after the addition of LPS. IL-10 production via enhancing NF-kB activity and decreasing

CREB activity, respectively. Moreover, JAK3 inhibition resulted Immunohistochemistry Downloaded from in the increase of CD4+ T cell responses in vitro along with en- Intestinal tissue samples from WT and JAK3 knockout mice were fixed hanced polymorphonuclear neutrophil (PMN) infiltration, IL-17 in 4% formaldehyde, embedded in OCT compound, and stored at 280˚C. expression, as well as erosion of the intestinal epithelium. Col- Serial sections (8-mm thick) were stained for neutrophils and/or IL-17 using single or double immunofluorescence. For staining, slides were lectively, these results identify the negative regulatory role of JAK3 rehydrated, blocked, and incubated for 1 h at room temperature with pri- in TLR4-mediated inflammatory responses and characterize the role mary FITC-conjugated Abs to mouse Ly6G, a specific neutrophil marker

of PI3K–Akt–GSK3b signaling pathway in JAK3-mediated control (FITC-conjugate; LifeSpan BioSciences) or with unconjugated human/mouse http://www.jimmunol.org/ of inflammatory responses. IL-17A (Santa Cruz Biotechnology) Abs. Alexa Fluor 594–conjugated goat anti-rabbit IgG (Molecular Probes) was used as a secondary Ab for IL-17 staining. The specificity of staining was confirmed by using appro- priate FITC-conjugated isotype controls or normal rabbit IgG followed by Materials and Methods Alexa Fluor 594-goat anti-rabbit IgG. Images were captured using a laser- Mice and reagents scanning confocal microscope (Olympus FV1000; Olympus). tm1Ljb B6129SF2 and JAK3 homozygous mutant mice (129S4-Jak3 /J) were NF-kB p65 and CREB nuclear binding assay purchased from The Jackson Laboratory. Mice were housed in a specific pathogen-free facility at the University of Louisville School of Medicine, Nuclear lysates were obtained from human monocytes using a nuclear/

and the University of Louisville Institutional Animal Care and Use Com- cytosolic isolation kit purchased from Active Motif. Nuclear lysates were by guest on September 28, 2021 mittee approved all animal protocols. Pam3CSK4, Flagellin, and ultra-pure analyzed for DNA binding levels of NF-kB p65 and phosphorylated CREB LPS from Escherichia coli 0111:B4 were purchased from Invivogen. (S133) using TransAM NF-kB p65 and CREB Transcription Factor ELISA Phosphorylated and total JAK3 Abs were purchased from Assay Biotec. Kits (Active Motif) and performed according to the manufacturer’s protocol. Isotype-matched control Ab (IgG1) and functional-grade neutralizing anti- human IL-10 (clone JES3-9D7) mAb were purchased from eBioscience. Statistical analysis All other Abs were obtained from Cell Signaling Technology. The GSK3 inhibitor SB216763 (Toris) has been characterized and shown to be spe- Statistical significance between groups was evaluated by the ANOVA and cific for GSK3 without discernible effects on a panel of 24 other kinases the Tukey multiple comparison test using the InStat program (GraphPad, (18). JAK3 inhibitor WHIP-154 and T-1377 (CP-690550) were purchased San Diego, CA). Differences between groups were considered significant at , from Tocris and LC Laboratories, respectively. PI3K inhibitor LY294002 the level of p 0.05. was purchased from LC Laboratories. Nontargeting pools of small inter- fering RNA (siRNA) and a mixture of four prevalidated siRNA duplexes Results specific for GSK3b or JAK3 (ON TARGET-plus) were purchased from Pharmacological inhibition of JAK3 enhances TLR-mediated Dharmacon. All ectopically expressed plasmids were purchased from Addgene. TransAM CREB and NF-kB p65 transcription factor ELISA kits proinflammatory cytokine production while suppressing the were purchased from Active Motif. The NF-kB p65 (Ser529/536) inhibitory production of IL-10 peptide set was purchased from Imgenex. Cytokine ELISA kits were pur- chased from eBioscience. Although JAK3 has been reported to be involved in TLR-mediated innate immune responses, the impact of JAK3 activity on TLR- Cell preparation mediated inflammatory cytokine production is controversial, and at times, opposing results have been reported (14, 16, 17, 22). To PBMCs were obtained from the venous blood of healthy donors as per determine the effects of JAK3 inhibition on TLR-mediated in- protocols approved by the University of Louisville, Institutional Review Board, Human Subjects Protection Program, study number 12.0373. Monocytes flammatory cytokine production by innate immune cells, purified were isolated by negative selection using the human monocyte isolation kit human monocytes were used to determine the production of TLR- II from Miltenyi Biotec. The purity of monocytes was routinely .90%, mediated inflammatory cytokine production in the presence and as determined by flow cytometry using an FITC-labeled anti-CD14 Ab. absence of the JAK3 inhibitor T-1377 (CP-690550). Because the + 2 + Memory CD4 T cells (CD45RA CD45RO ) were purified from PBMCs IC s of this inhibitor on JAK2 and JAK3 are 20 and 1 nM, re- by negative selection using kits from Miltenyi Biotec. Bone marrow cells 50 from wild-type (WT) or JAK3 knockout mice were isolated by flushing spectively (23), a serial titration of T-1377 was used to minimize femurs and tibiae with HBSS supplemented with 2% heat-inactivated FBS. the influence of JAK2 on the regulatory ability of JAK3 in LPS- The cells were then passed through a 70-mm cell strainer. Bone marrow– stimulated human monocytes. As shown in Fig. 1A–D, JAK3 in- derived macrophages (BMDM) were generated as previously described hibition with T-1377 at concentrations of 1 and 10 nM significantly (19). Cells were cultured in RPMI 1640 medium supplemented with 10% enhanced the production of IL-12, TNF-a, and IL-6 in LPS- FBS (R10), 50 mM2-ME,1mMsodiumpyruvate,2mML-glutamine, 20 mM HEPES, 50 U/ml penicillin, and 50 mg/ml streptomycin (RPMI stimulated human monocytes, while suppressing the production complete). of the prototypical anti-inflammatory cytokine IL-10. Moreover, 1166 Jak3 REGULATES TLR-MEDIATED INFLAMMATORY CYTOKINES

FIGURE 1. JAK3 inhibitors differentially regulate TLR4-mediated inflammatory cytokine production in human monocytes. Purified human monocytes were pretreated with different JAK3 inhibitors (T-1377 or WHIP-154 [10 mM]) or IL-10–neutralizing Ab (5 mg/ ml) using rat IgG1 as an isotype control for 2 h and then stimulated with LPS (1 mg/ml), Pam3CSK4 (500 Downloaded from ng/ml), or Flagellin (5 mg/ml). After 24 h of stimula- tion, the cell-free supernatants were collected and the levels of IL-12p40 (A), IL-6 (B), TNF-a (C), and IL-10 (D) were determined by ELISA. For (A)–(D), lower concentration of T-1377 (,10 nM) enhanced the pro- duction of IL-12p40 (A), IL-6 (B), and TNF-a (C), D while suppressing IL-10 levels ( ) produced by LPS- http://www.jimmunol.org/ stimulated human monocytes. JAK3 inhibition by 1 nM T-1377 also differentially regulated production of IL-12 and IL-10 in TLR2- or TLR5-stimulated mono- cytes (E, F). With different doses of LPS, 1 nM T-1377 exhibited a similar regulatory effect on the production of IL-12 and IL-10 (G). In the context of IL-10 neu- tralization, JAK3 inhibition (1 nM T-1377) remains capable of enhancing LPS-mediated proinflammatory cytokine production (H). A second JAK3 inhibitor WHIP-154 (10 mM) showed the same capability to by guest on September 28, 2021 increase or decrease the production of IL-12p40, IL-6, TNF-a, and IL-10 (I), respectively. Data represent the arithmetic mean 6 SD of three biological replicates. *p , 0.05, **p , 0.01, ***p , 0.001.

JAK3 inhibition with T-1377 at concentrations of 1 nM also dif- (Fig. 1H). In addition, a second JAK3 inhibitor, WHIP-154, was used ferentially regulated TLR2- and TLR5-mediated production of IL- toconfirmtheregulatoryfunctionofJAK3inTLR4-mediated 12 and IL-10 in human monocytes (Fig. 1E, 1F). We also tested cytokine production. Similar to T-1377, WHIP-154 pretreatment the cellular responses with different LPS doses in the context of enhanced TLR4-mediated proinflammatory cytokine production JAK3 inhibition and found JAK3 inhibition exhibits a significantly and concurrently reduced the level of IL-10 (Fig. 1I). regulatory ability over different doses of LPS-mediated inflam- matory cytokine production (Fig. 1G). To determine the influence siRNA knockdown or genetic deletion of JAK3 results in of de novo–synthesized IL-10 on JAK3-mediated inhibition, neu- elevated proinflammatory cytokine production and suppressed tralizing Ab was used to block IL-10 and then the regulatory effect IL-10 production upon TLR4 stimulation of JAK3 inhibition on LPS stimulation was assessed. We found As both T-1377 (CP-690550) and WHIP-154 can have activity JAK3 inhibition remained capable of enhancing the production of against JAK2 (23), to isolate the specific influences of JAK3 inhibi- proinflammatory cytokine in the context of IL-10 blockade, indicat- tion on TLR4-mediated inflammatory cytokine production, we used ing JAK3 directly affects TLR4-mediated inflammatory responses prevalidated siRNA to knockdown JAK3 in human monocytes. As The Journal of Immunology 1167 shown in Fig. 2A, JAK3 silencing by specific siRNA resulted in a ability of the PI3K–Akt pathway to suppress the levels of proin- substantial decrease of total JAK3 in human monocytes, as compared flammatory cytokines while augmenting anti-inflammatory cyto- with control. To exclude the possible off-target effect of JAK3 kine production upon TLR stimulation (28). Because we have siRNA, we tested the expression of total JAK2 and found no observed the differential regulatory ability of JAK3 inhibition on substantial change of JAK2 in JAK3 siRNA-treated monocytes. TLR4-mediated inflammatory cytokines, and previous reports have Inhibition of JAK3 significantly enhanced TLR4-mediated IL-12, shown the possible links between the JAK3 and PI3K pathways TNF-a, and IL-6 production and suppressed IL-10 level in LPS- (22, 29–31), we next sought to determine whether JAK3 inhibition stimulated human monocytes (Fig. 2B). These results corroborated regulates the TLR4-mediated inflammatory response through PI3K the cytokine data obtained with the pharmacological inhibitors. and its downstream signaling components such as Akt, mammalian Because neither pharmacological inhibition nor siRNA can com- target of rapamycin complex 1 (mTORC1), GSK3b, and CREB. pletely ablate JAK3 without effects on other isoforms of JAKs, As shown in Fig. 3A and 3B, compared with the control group, we next used JAK3-knockout macrophages to determine the effect T-1377–treated monocytes exhibited reduced phosphorylation of of JAK3 in TLR4-mediated inflammatory cytokine production. As mTORC1, Akt, GSK3b, and CREB over time upon LPS stimu- compared with WT control, JAK3 knockout resulted in significantly lation. In contrast, inhibition of Akt or GSK3b has no discernible increased IL-12, TNF-a, and IL-6 while concurrently suppressing effect on the phosphorylated levels of JAK3, compared with cells the production of IL-10 in LPS-stimulated macrophages (Fig. 2C– stimulated with LPS alone (Fig. 3C, 3D). We next used JAK3 F). Taken together, these data demonstrate that JAK3 inhibition knockout BMDM to confirm the functional role of JAK3. As shown leads to elevated proinflammatory cytokine production and sup- in Fig. 3E and 3F, compared with WT control, JAK3-knockout pressed IL-10 production upon TLR4 stimulation. BMDM exhibited substantially decreased phosphorylation of Downloaded from mTORC1, Akt, GSK3b, and CREB over time upon LPS stimu- The effects of JAK3 inhibition on the activation of the PI3K– lation. Because numerous studies have shown JAK-STATs play a Akt pathway and its downstream constitutive active kinase critical role in the cytokine-mediated signaling pathway (32–35), b GSK3 in LPS-stimulated innate immune cells we next examined whether JAK3 knockout–modified autocrine The PI3K pathway has been demonstrated to modulate TLR4- inflammatory cytokine production would subsequently affect STAT

mediated inflammatory immune responses by differentially regu- phosphorylation. JAK3-knockout cells and WT controls were http://www.jimmunol.org/ lating the levels of pro- and anti-inflammatory cytokines (24–27). stimulated with LPS at different time points, and then tyrosine Our recent studies found that a downstream constitutive active phosphorylation of STAT3, STAT4, STAT5, and STAT6 was kinase of the PI3K–Akt pathway, GSK3b, is responsible for the examined. As shown in Fig. 3G and 3H, JAK3 knockout potently by guest on September 28, 2021

FIGURE 2. JAK3 deficiency enhances the produc- tion of IL-12, TNF-a, and IL-6 while decreasing IL- 10 levels in TLR4-stimulated cells. Purified human monocytes were pretreated with nontarget or JAK3- specific siRNA for 72 h and then stimulated with LPS for 24 h. Whole-cell lysates and cell-free supernatants were collected to determine the transfection efficiency and cytokine levels, respectively. (A) siRNA-mediated knockdown of JAK3 protein levels was assessed by Western blot. Total JAK2 levels were also tested to exclude an off-target effect of JAK2. The ratio of total-JAK3 or JAK2 to total b-actin was determined by densitometry. (B) siRNA silencing-mediated JAK3 inhibition enhances the production of IL-12, TNF-a, and IL-6 while decreasing IL-10 levels in LPS-stimu- lated cells. For (C)–(F), WT and JAK3-knockout (KO) BMDM were generated and stimulated with LPS for 24 h. Cell-free supernatants were collected and assayed for cytokine levels by ELISA. JAK3 knockout en- hanced TLR4-induced production of IL-12 (C), IL-6 (D), TNF-a (E) and reduced IL-10 levels (F) in LPS- stimulated BMDM. Data represent the arithmetic mean 6 SD of three biological replicates. *p , 0.05, **p , 0.01, ***p , 0.001. m, murine. 1168 Jak3 REGULATES TLR-MEDIATED INFLAMMATORY CYTOKINES

FIGURE 3. JAK3 inhibition suppresses TLR4-mediated activity of the PI3K path- way while enhancing the phosphorylation levels of STATs. Purified human monocytes were pretreated with JAK3 inhibitor (1 nM T-1377) for 2 h and then stimulated with LPS over a 4-h time course. (A) Total cell lysates were probed for the levels of phos- phorylated mTORC1, Akt, GSK3b, and CREB by Western blot, and the ratio of phospho- to total proteins was determined by densitometry (B). (C) Purified human monocytes were stimulated with LPS in the presence or absence of PI3K or GSK3 in- hibitor. The levels of phosphorylated JAK3

were analyzed by Western blot and the ratio Downloaded from to total JAK3 was determined by densi- tometry (D). From (E)–(H), WT and JAK3- knockout (KO) BMDM were treated with LPS for up to 4 h, total cell lysates were collected at the given time points, and probed for the levels of phosphorylated mTORC1,

Akt, GSK3b, CREB (E), STAT3, STAT4, http://www.jimmunol.org/ STAT5, and STAT6 (G) by Western blot and the ratio of phospho- to total protein deter- mined by densitometry (F, H). Anti–IL-12 and anti–IL-6 Ab cocktails were used to determine the effect of JAK3 inhibition on the tyrosine phosphorylation of STAT3, STAT4, STAT5, and STAT6 upon the neu- tralization of IL-12 and IL-6 signaling. WT and JAK3 knockout BMDM were pretreated by guest on September 28, 2021 with neutralizing anti–IL6 and anti–IL-12 mixture for 2 h and then stimulated with LPS. Whole-cell lysates were probed for the levels of phosphorylated STATs and the ratio of phospho- to total STATs determined by densitometry (I, J). Data are representa- tive of three to five biological replicates.

increased the levels of tyrosine phosphorylation of STAT3, STAT4, JAK3 differentially regulates LPS-induced cytokine production STAT5, and STAT6 at late time points upon LPS stimulation, in a GSK3b-dependent manner as compared with WT control. This result is consistent with and The PI3K pathway has been shown to play a critical regulatory role expands upon an earlier study showing JAK3 inhibition resulted in TLR4-mediated inflammatory cytokine production via phos- in enhanced STAT3 phosphorylation (35). Our previous study has phorylating and then inhibiting the activity of a downstream kinase, shown that TLR-induced phosphorylation of STATs was mainly GSK3b (28). Phosphorylation and inactivation of GSK3 is ef- caused by de novo–synthesized cytokines (36). Hence, we next fectuated by Akt after activation of PI3K. Because our data have investigated the effect of the neutralizing Abs against inflamma- shown that inhibition of JAK3 differentially regulated LPS-induced tory cytokines (including IL-6 and IL-12) on STAT phosphorylation cytokine production and decreased the LPS-induced phosphory- b in the context of JAK3 inhibition. Compared with control cells, lation of Akt and GSK3 , we next investigated the functional role of this pathway in JAK3-mediated regulation of inflammatory neutralization of inflammatory cytokines clearly ameliorated the cytokine production. As shown in Fig. 4A, inhibition of PI3K with ability of JAK3 inhibition to enhance tyrosine phosphorylation of the pharmacological inhibitor LY294002 significantly enhanced STATs (Fig. 3I, 3J). These data verified our findings that JAK3 the ability of JAK3 inhibition to augment proinflammatory cytokine inhibition was capable of differentially regulating inflammatory production and suppress IL-10 levels in LPS-stimulated monocytes. cytokines, and these de novo–synthesized cytokines contribute We next asked whether overexpression of ectopic constitutively to the subsequent tyrosine phosphorylation of STATs in TLR4- active Akt would attenuate the hyperinflammatory response induced stimulated cells. by JAK3 inhibition. As compared with cells expressing empty vector, The Journal of Immunology 1169

FIGURE 4. The ability of JAK3 inhibition to reg- ulate TLR4-mediated inflammatory cytokine produc- tion is dependent on the activity of PI3K and GSK3b. The levels of IL-12, TNF-a,IL-6,andIL-10were assayed by ELISA in LPS-stimulated human mono- cytes. The regulatory effect of JAK3 inhibition was significantly enhanced by PI3K inhibition (LY294002) A ( ) and abrogated by constitutive expression of Akt Downloaded from (B, C). Inhibition of GSK3 mediated by pharmaco- logical inhibitor (D)orspecificsiRNA(E, F)attenu- ated the regulatory ability of JAK3 on LPS-mediated inflammatory cytokine production in human mono- cytes. (G) Hemagglutinin (HA) expression levels were detected by Western blot 48 h posttransfection in nontransfected monocytes and monocytes transfected http://www.jimmunol.org/ to confirm the transfection efficiency of kinase-dead (K85A) plasmid encoding GSK3b.(H) Monocytes transfected with kinase-dead (K85A) plasmid exhibi- ted a loss in the phosphorylation levels of the GSK3- specific substrate glycogen synthase (S640/641). (I) As compared with monocytes transfected with empty vector control, the kinase-dead (K85A) GSK3b mutant abrogated the ability of JAK3 inhibition to increase the production of TNF-a, IL-12, and IL-6 and de- creased the IL-10 level in LPS-stimulated cells. Data by guest on September 28, 2021 represent the arithmetic mean 6 SD of three biolog- ical replicates. *p , 0.05, **p , 0.01, ***p , 0.001.

expression of constitutively active Akt resulted in significantly significantly abrogated JAK3 inhibition-mediated regulation of lower production of IL-12, TNF-a, and IL-6 in LPS-stimulated inflammatory cytokines production (Fig. 4D). Similarly, siRNA- monocytes (Fig. 4B, 4C). Moreover, overexpression of constitu- mediated GSK3b silencing (Fig. 4E) also significantly attenuated tively active Akt also abrogated the effect of JAK3 inhibition upon the capability of JAK3 to differentially regulate LPS-induced cy- LPS stimulation (Fig. 4C). It suggested that the regulatory ability tokine production, as compared with nonspecific siRNA-treated hu- of JAK3 is dependent on the activity of PI3K. Because GSK3b has man monocytes (Fig. 4F). As confirmatory evidence, we found been shown to play a critical role in TLR4-mediated inflammatory TLR4 stimulation of monocytes expressing a kinase dead mutant cytokine production and we have observed that JAK3 inhibition encoding GSK3b (K85A) significantly (p , 0.05) attenuated the attenuated the phosphorylation of GSK3b upon LPS stimulation production of IL-12, IL-6, and TNF-a and enhanced IL-10 levels (Fig. 3A), we next determined whether GSK3b activity impacts upon JAK3 inhibitor pretreatment (Fig. 4G–I). Taken together, these the JAK3 inhibition-mediated cytokine production. After pretreat- results demonstrated that the ability of JAK3 inhibition to differ- ing JAK3-inactivated monocytes with GSK3 inhibitor (SB216763) entially regulate LPS-induced inflammatory cytokine production is and then stimulating with LPS, we found GSK3 inactivation regulated by PI3K and its downstream kinases Akt and GSK3b. 1170 Jak3 REGULATES TLR-MEDIATED INFLAMMATORY CYTOKINES

JAK3 inhibition modifies TLR4-mediated DNA binding activity JAK3 inhibition positively regulates T cell responses by of NF-kB and CREB and consequently regulates inflammatory controlling inflammatory cytokine production cytokine induction Several previous studies have shown that low doses of JAK3 in- We next examined whether JAK3 inhibition of Akt and GSK3b hibitor enhanced T cell responses, whereas many others reported could affect downstream signaling molecules/transcription factors that JAK3 inhibition suppresses adaptive immune responses by involved in the control of LPS-mediated inflammatory cytokine disrupting autocrine cytokine signaling of T and B cells (16, 37– production. Our previous study (28) demonstrated that GSK3 in- 39). Because we observed pharmacological inhibition, siRNA, activation differentially regulates TLR-mediated inflammatory or genetic deletion of JAK3 enhanced proinflammatory cytokine cytokine production. GSK3 increases the phosphorylation of production in innate immune cells, we next examined whether the CREB that displaces NF-kB from CBP, leading to diminished regulatory function of JAK3 inhibition in innate immune cells also proinflammatory cytokine production (28). We therefore tested impacted subsequent T cell responses. To this end, we cultured whether JAK3 inhibition could affect LPS-mediated DNA binding autologous memory CD4+ T cells with supernatants isolated from activity of NF-kB and CREB and whether this effect was de- human monocytes under different stimulation scenarios. We ob- pendent on the activity of GSK3. As expected, compared with served that supernatant from monocytes stimulated with LPS LPS simulation alone, JAK3 inhibition enhanced phosphorylation alone had the ability to enhance CD4+ memory T cell responses by of NF-kB p65 (S536) (Fig. 5A, 5B) and its DNA binding activity augmenting IFN-g and IL-17 production (Fig. 6A, 6B). Moreover, (Fig. 5C) but reduced that of CREB in LPS-stimulated monocytes using supernatants from monocyte pretreatment with JAK3 in- (Fig. 5D). We also observed that inhibition of GSK3 was able to hibitor and LPS reinforced the enhancement of IFN-g and IL-17, reverse the effect of JAK3 inhibition of NF-kB and CREB DNA whereas supernatant from monocyte pretreated with JAK3 inhib- Downloaded from binding (Fig. 5C, 5D). Because our previous studies have shown itor alone reduced the production of IFN-g and IL-17 (Fig. 6A, 6B). that GSK3 inhibition suppresses proinflammatory cytokine pro- These results indicate that JAK3 inhibition in innate immune cells duction by LPS-stimulated cells via its capacity to reduce the can positively regulate subsequent T cells responses by enhancing association of CBP with NF-kB (28), we predicted TLR4-mediated the production of IFN-g and IL-17. activity of NF-kB would affect the capability of JAK3 inhibition to enhance the production of inflammatory cytokine production JAK3 knockout mice display high levels of intestinal http://www.jimmunol.org/ in LPS-stimulated monocytes. To test this hypothesis, we used inflammation and PMN infiltration NF-kB P65 inhibitory peptide and observed that inhibition of It has been reported that the intestinal lamina propria is the largest NF-kB p65 prevented the ability of T-1377 to increase IL-12 and reservoir of CD4+ and that either IL-6 and/or IL-17 decrease IL-10 production in LPS-stimulated monocytes, as com- can play a crucial role in the initiation and progression of intestine pared with control (Fig. 5E, 5F). bowel diseases (40–44). In the current study, we observed in vitro by guest on September 28, 2021

FIGURE 5. JAK3 inhibition affects the phosphory- lation levels and DNA binding activity of NF-kB and CREB in monocytes. Purified human monocytes were pretreated with JAK3 inhibitor (T-1377; 10 nM), con- trol peptide, or NF-kB inhibitory peptide and then stimulated with LPS. Whole-cell lysates, cytosoplas- mic, and nuclear cell fractions were collected after stimulation with LPS at the time points indicated. (A) JAK3 inhibition increased the phosphorylation of NF-kB (S536) upon LPS stimulation. (B) Densitometer scans of phospho–NF-kB (S536) and total b-actin were performed and recorded as the ratio of phospho–NF-kB (S536)/total b-actin. NF-kB and CREB DNA binding assays were performed to measure the levels of total NF-kBp65 (C)(10mg nuclear lysate) and CREB (S133) (D)(20mg nuclear lysate) in monocytes stimulated with LPS for 4 h in the presence and absence of the JAK3 inhibitor (T-1377), GSK3 inhibitor (SB216763), or both. For (E) and (F), the functional effect of JAK3 inhibition on NF-kB P65 activity in LPS-stimulated monocytes was assessed by pretreatment of cells with a control peptide or NF-kB inhibitor peptide (100 mM) followed by stimulation with LPS for 20 h. The levels of IL-12p40 (E) and IL-10 (F) were determined by ELISA. For (C)–(F), data represent the arithmetic mean 6 SD of three biological replicates. *p , 0.05, ***p , 0.001. The Journal of Immunology 1171 Downloaded from

FIGURE 6. Modulation of inflammatory cytokines by JAK3 in human FIGURE 7. JAK3 knockout aggravated the erosion of intestinal epi- + monocytes enhanced Th1 and Th17 cytokine production by memory CD4 thelium and enhanced the infiltration of PMN and expression of IL-17. (A) + T cells. Human monocytes and autologous memory CD4 T cells were PMN infiltration and IL-17 expression were assessed by staining sections http://www.jimmunol.org/ isolated from PBMC. Monocytes were stimulated with LPS in the presence of intestinal tissue with FITC-conjugated anti-Ly6G (a specific neutrophil and absence of JAK3 inhibitor (T-1377) for 24 h. Cell-free supernatant marker) and Alexa Fluor 594–conjugated IL-17 (original magnification isolated from nonstimulated monocytes, LPS-stimulated monocytes, or 340). Scale bars, 50 mm. (B) H&E staining of serial sections of small monocytes stimulated with LPS in the presence of JAK3 inhibitor were intestine from WT mice and JAK3 knockout (2/2) mice showing the in- + transferred to culture with autologous memory CD4 T cells plated in 96- tegrity of intestinal epithelium (original magnification 320). well plates (2 3 105/well) precoated with or without anti-CD3 (1 mg/ml). After 96 h of coculture, cell-free supernatants were collected and assayed for IFN-g (A) or IL-17 (B) by ELISA. Data represent the arithmetic mean of JAK3 diminished the phosphorylation level of GSK3b, result- 6 , , , SD of three biological replicates. *p 0.05, **p 0.01, ***p 0.001. ing in enhanced GSK3b activity and subsequent elevated TLR4- mediated inflammatory responses. This finding identified GSK3b by guest on September 28, 2021 as a downstream signaling target of JAK3 in TLR4-stimulated in- that JAK3 inhibition enhanced proinflammatory cytokine pro- nate immune cells. By analyzing downstream transcription factors duction including IL-6 and IL-17. Moreover, compared with WT of GSK3b, we observed that JAK3 inhibition enhanced the DNA mice, .90% knockout mice exhibited some symptoms of intestine binding activity of NF-kB and attenuated that of CREB upon bowel diseases including lower body weight and severe rectal LPS stimulation, and this effect was dependent on the activity prolapses after 20 wk (data not shown). These observations sug- of GSK3b. Moreover, the functional role of NF-kB was verified gest that JAK3 inhibition may enhance intestinal microbiota- by the finding that inhibitory peptide-mediated blocking of NF-kB induced inflammatory responses. In an effort to investigate this attenuated the ability of JAK3 inhibition to enhance TLR4-mediated possibility, we assessed the presence of IL-17 and PMN infiltra- proinflammatory cytokine production. Additionally, we found JAK3 tion in the lamina propria of the intestine of JAK3-deficienct mice. inhibition was capable of increasing CD4+ T cell responses in vitro We discovered a high degree of PMN infiltration and a mild in- and enhancing PMN infiltration, as well as IL-17 expression in crease in IL-17 expression in the lamina propria of JAK3 knockout intestine tissues. Collectively, these findings establish a negative mice, as compared with WT mice (Fig. 7A). Moreover, obvious regulatory role for JAK3 in TLR4-mediated proinflammatory damage in the intestinal epithelial layer was also found in JAK3- responses and characterize the involvement of the PI3K–Akt– knockout mice (Fig. 7B). Considering previous studies have GSK3b signaling pathway in JAK3-mediated control of inflam- demonstrated the functional role of PMN infiltration and IL-17 matory responses. expression in the initiation and development of intestine bowel JAK3 has been demonstrated to be involved in cytokine signaling diseases (40, 45), these data indicated that JAK3 may partici- through coupling with the gc receptor and subsequently to play a pate in regulation of the initiation and development of intestine critical role in the development, proliferation, and differentiation inflammation. of B cells and T cells (38, 46). Despite numerous studies noting the immunosuppressive effect of JAK3 inhibition in the treatment Discussion of organ transplantation and several other inflammatory diseases In the current study, we demonstrated the negative regulatory role (47–49), there is no consistent evidence for a role of JAK3 inhi- of JAK3 in TLR4-mediated inflammatory responses and charac- bition in TLRs mediated inflammatory responses. Yamaoka et al. terized the cell-signaling pathway by which JAK3 inhibition (14) reported that a genetic deficiency of JAK3 or its gc chain enhanced the production of proinflammatory cytokines while receptor resulted in the elevation of inflammatory cytokines in- concurrently suppressing the anti-inflammatory cytokine IL-10 cluding IL-12 and IL-10 in LPS-stimulated mice bone marrow– in innate immune cells. We found inhibition of JAK3 attenuated derived dendritic cells, whereas other groups have shown JAK3 TLR4-mediated activity of PI3K and then suppressed the phos- inhibition or knockout reduced the production of IL-10 in TLR4- phorylation of Akt and its downstream kinase GSK3b. Inhibition mediated innate immune cells (17). In our current study, using 1172 Jak3 REGULATES TLR-MEDIATED INFLAMMATORY CYTOKINES primary human monocytes and gene knockout JAK3 macro- exhibits its regulatory role through this pathway. Our current data phages, we found JAK3 inhibition or genetic deficiency resulted in support this concept, because either pharmacological inhibition or enhanced production of TNF-a, IL-6, and IL-12 but reduced IL- ectopic expression of kinase dead GSK3b abrogated the differential 10 production. The discrepancy in IL-10 production among the regulatory ability of JAK3 inhibition upon LPS stimulation. studies is likely due to several factors such as different cell types, Considering the similarity of JAK3 and mTORC1 inhibitors on different maturation stage of dendritic cells, and the influence of suppressing adaptive immune responses while enhancing TLR4- JAK3 deficiency on other signaling pathways. Our current findings mediated proinflammatory cytokine levels, our study suggested highlight the functional role of JAK3 in innate immune cells and this could be a common feature for molecules that possess the ca- elucidate the signaling pathway by which JAK3 negatively reg- pability to suppress adaptive immune responses while concurrently ulates TLR-mediated inflammatory cytokine production. enhancing the immune responses of innate immune cells. At the The PI3K–Akt pathway has been demonstrated to negatively transcriptional level, we found that JAK3 inhibition differentially regulate TLR mediated-inflammatory responses (28). However, how altered the TLR4-mediated DNA binding activity of CREB and TLRs initiate the activation of PI3K pathway and the possible NF-kB. Furthermore, peptide-mediated inhibition of NF-kB sup- role of tyrosine kinases like JAKs in this process are less known. pressed proinflammatory cytokine production in JAK3 inhibited Previous studies have demonstrated that activation of PI3K occurs cells stimulated with LPS. These observations suggest that JAK3 via its regulatory subunit p85 binding to a phosphotyrosine residue differentially regulates TLR4-mediated inflammatory responses (YXXM) present within activated cellular receptors (50). Upon through modifying the activity of NF-kB and CREB. Taken together, TLR stimulation, PI3K was activated through binding with the our current study characterized the signaling pathway, PI3K–Akt–

YXXM motif of the adaptor molecules such as MyD88 or the GSK3b, that JAK3 depends upon to affect TLR4-mediated in- Downloaded from intracellular component of TLR receptors such as TLR2/3 (51, flammatory cytokine production. 52). Several lines of evidence showed JAK family members are Although numerous reports have shown JAK3 inhibition sup- involved in the regulation of TLR signaling (53, 54). However, no presses inflammation responses in several autoimmune diseases study to date has identified how JAKs are involved in the signaling (47, 49), whether this is truly attributable to inhibition of JAK3 has events of TLRs, if JAKs are involved in the tyrosine phosphory- not yet been established. It has been reported that the IC50 value of

lation of TLRs, or how they may act in a synergistic, independent, the most widely investigated JAK3 inhibitor, T-1377 (CP-690550), http://www.jimmunol.org/ or opposing manner in innate immune cells. In this study, we dem- is 1 nM for JAK3 but 20 nM for JAK2 in lymphocytes (23). onstrated JAK3 inhibition suppressed activity of the PI3K–Akt– Additionally, as accurate concentrations of inhibitors are difficult GSK3b pathway, which enhances the production of proinflammatory to achieve in vivo, it is unlikely that all of the inflammatory sup- cytokines while decreasing IL-10 levels in innate immune cells. Our pressive effects of T-1377 can be attributed to JAK3 inhibition. unpublished data showed that JAK3 inhibition attenuated tyrosine Because several recent studies (56–58) have established a sup- phosphorylation of TLR4 and JAK3 at early time points, which pressive function for JAK2 inhibition in TLR-mediated immune indicated JAK3-mediated tyrosine phosphorylation is involved in responses, the suppressive effect of T-1377 on inflammatory TLR-initiated signaling events. Although the connectivity between responses might be mainly due to JAK2, not JAK3, inhibition. A JAK3 and TLR4 is still not known, TLR4 receptor stimulation recent study by Yoshida et al. (16) substantiated this possibility, by guest on September 28, 2021 induces a conformational change, which may bring JAK3 into showing that a low dose of T-1377 enhanced the inflammatory close proximity, resulting in activation via trans phosphorylation. responses and accelerated the onset of experimental autoimmune Upon TLR-mediated phosphorylation, activated JAK3 could phos- encephalomyelitis. A very recent published abstract also reported phorylate tyrosine residues within the cytoplasmic domains of as- that specific inhibition of JAK1 and JAK2 is more effective than sociated receptor or adaptor molecules that allow for the recruitment inhibition of JAKs in protecting mice from acute graft-versus-host of downstream signaling molecules. disease by significantly decreasing alloreactive CD4+ T cells (59). Mapping of the PI3K pathway by several groups revealed that These observations indicated JAK2 and JAK3 might have distinct the ability of the PI3K pathway to dictate regulation of pro- and anti- regulatory function in inflammatory responses. Our current study inflammatory cytokines largely depended upon the inactivation of demonstrated JAK3 inhibition, either by titrated concentrations of the serine/threonine kinase GSK3b (19, 20, 28). Several groups pharmacological inhibitor or genetic knockdown, had an ability have shown that JAK3 activation is involved in the activation of the to enhance the proinflammatory cytokine production and related PI3K pathway following various stimuli (22, 29–31). In particular, T cell responses. As a negative regulator of inflammation, JAK3 inhibition of JAK3 was shown to downregulate PI3K activation and knockout also resulted in the high expression of IL-17 and infil- Akt phosphorylation in mast cells in the present of Ag stimulation tration of PMN in the lamina propria of the distal intestine. These (30). However, it was unclear if JAK3 was required for the TLR- results identified the negative regulatory role of JAK3 in TLR- mediated action of the PI3K pathway. Our present study has pro- induced inflammatory cytokine production. vided evidence for the first time, to our knowledge, demonstrating Whereas numerous studies have reported that pharmacological the PI3K–Akt–GSK3b pathway is responsible for JAK3-mediated inhibition of JAK3 was capable of suppressing the inflammatory differential regulation of inflammatory cytokine production by LPS- immune response by disrupting the development of lymphocytes, stimulated innate immune cells. Moreover, we observed several the effect of JAK3 on innate immune cells and the cellular mech- signaling molecules, such as mTORC1 and P70S6K, involved in anism responsible for this effect have remained obscure. As it is well JAK3-mediated regulation of cytokine production (data not shown). known that the innate immune responses prime the adaptive immune This finding is consistent with our previous published results on the cells to enhance the host defense ability, elucidating the indirect regulatory effect of the PI3K–mTORC1 pathway and its downstream influence of JAK3 inhibition on the responses of autologous CD4+ serine/threonine kinases P70S6K and GSK3b in TLR4-mediated T cells will be helpful to comprehensively assess the regulatory human monocytes (36, 55). Because our previous publication has effect of JAK3 inhibition on TLR-mediated immune responses. demonstrated that mTORC1 inhibition led to differential cytokine The work presented in this study demonstrated that JAK3 inhibition production (36), and because inhibition of JAK3 resulted in de- enhanced TLR4-mediated proinflammatory cytokine production phosphorylation of mTORC1 and its downstream signaling mol- while concurrently reducing the IL-10 levels in innate immune cells. ecules P70S6K and GSK3b, it is reasonable to predict that JAK3 We also discovered that the PI3K–Akt–GSK3b signaling pathway The Journal of Immunology 1173 is used by JAK3 to differentially regulate TLR4-mediated cyto- 22. Hirata, N., Y. Yanagawa, K. Iwabuchi, and K. Onoe´. 2009. Selective regulation of interleukin-10 production via pathway in murine conventional kine production. JAK3 inhibition induced enhancement of proin- dendritic cells. Cell. 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