THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 28, pp. 23769 –23778, July 6, 2012
`© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
`
`Tyrosine Kinase Btk Is Required for NK Cell Activation□S
`
`Received for publication, April 16, 2012, and in revised form, May 7, 2012 Published, JBC Papers in Press, May 15, 2012, DOI 10.1074/jbc.M112.372425
`Yan Bao‡§1,2, Jian Zheng¶1, Chaofeng Han§1, Jing Jin§, Huanxing Han‡, Yinping Liu¶, Yu-Lung Lau¶, Wenwei Tu¶3,
`and Xuetao Cao§
`From the ‡Translational Medicine Center, Changzheng Hospital, and §National Key Laboratory of Medical Immunology and
`Institute of Immunology, Second Military Medical University, Shanghai 200433 and the ¶Department of Paediatrics and Adolescent
`Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong SAR, China
`Background: Whether Btk is involved in the regulation of NK cell innate function remains unknown.
`Results: Btk⫺/⫺ murine and human NK cells have decreased innate immune responses to the TLR3 ligand.
`Conclusion: Btk is required for activation of NK cells.
`Significance: Our results provide mechanistic insight into TLR3-triggered NK cell activation and make Btk a considerable
`target of the anti-inflammatory drug.
`
`Bruton tyrosine kinase (Btk) is not only critical for B cell
`development and differentiation but is also involved in the reg-
`ulation of Toll-like receptor-triggered innate response of
`macrophages. However, whether Btk is involved in the regula-
`tion of natural killer (NK) cell
`innate function remains
`unknown. Here, we show that Btk expression is up-regulated
`during maturation and activation of mouse NK cells. Murine
`Btkⴚ/ⴚ NK cells have decreased innate immune responses to the
`TLR3 ligand, with reduced expressions of IFN-␥, perforin, and
`granzyme-B and decreased cytotoxic activity. Furthermore, Btk
`is found to promote TLR3-triggered NK cell activation mainly
`by activating the NF-␬B pathway. Poly(I:C)-induced NK cell-
`mediated acute hepatitis was observed to be attenuated in
`Btkⴚ/ⴚ mice or the mice with in vivo administration of the Btk
`inhibitor. Correspondingly, liver damage was aggravated in
`Btkⴚ/ⴚ mice after the adoptive transfer of Btkⴙ/ⴙ NK cells, fur-
`ther indicating that Btk-mediated NK cell activation contrib-
`utes to TLR3-triggered acute liver injury. Importantly, reduced
`TLR3-triggered activation of human NK cells was observed in
`Btk-deficient patients with X-linked agammaglobulinemia, as
`evidenced by the reduced IFN-␥, CD69, and CD107a expression
`and cytotoxic activity. These results indicate that Btk is required
`for activation of NK cells, thus providing insight into the physi-
`ological significance of Btk in the regulation of immune cell
`functions and innate inflammatory response.
`
`Bruton tyrosine kinase (Btk),4 a cytoplasmic tyrosine kinase
`and the most studied member of Tec family kinases,
`is
`expressed in almost all hematopoietic lineages except T cells
`
`□S This article contains supplemental Figs. S1–S3.
`1 These authors contributed equally to this work.
`2 To whom correspondence may be addressed: 7th Floor, Library Bldg., Sec-
`ond Military Medical University, 800 Xiangyin Rd., Shanghai 200433, China.
`Tel.: 86-21-81871910; Fax: 86-21-81871909; E-mail: baoyan@yahoo.cn.
`3 To whom correspondence may be addressed: Rm. L7-58, 7/F Laboratory
`Block, Faculty of Medicine Bldg., 21 Sassoon Rd., Hong Kong, China. Tel.:
`852-2819-9354; Fax: 852-2819-8142; E-mail: wwtu@hku.hk.
`4 The abbreviations used are: Btk, Bruton tyrosine kinase; TLR, Toll-like recep-
`tor; XLA, X-linked agammaglobulinemia; XCI, X chromosome inactivation;
`PMA, phorbol 12-myristate 13-acetate; NK, natural killer; ALT, alanine ami-
`notransferase; AST, aspartate aminotransferase.
`This is an Open Access article under the CC BY license.
`JULY 6, 2012 • VOLUME 287 • NUMBER 28
`
`(1–3). Btk has been shown to be crucial for B cell development,
`differentiation and function because of its predominant expres-
`sion in different developmental stages of B lymphocytes, from
`hematopoietic stem cells, common lymphoid progenitor, to
`pre-B, pro-B, immature, and mature B cells (4). For example,
`Btk deficiency is associated with lack of circulating mature B
`cells and low reactivity to TI antigens of B cells, revealing a
`profound block in central B cell development and responsive-
`ness of B cells (5, 6). Moreover, individuals with mutation of the
`gene encoding Btk, diagnosed as X-linked agammaglobuline-
`mia (XLA), suffer from the disabled generation of all classes of
`immunoglobulins and therefore fail to mount effective humoral
`immune responses (7). The mutation of the Btk gene in mice
`leads to X-linked immunodeficiency, with a condition similar
`to XLA (8).
`Although a series of defective functions of B cells have been
`ascertained in Btk-deficient models, there are also many
`reports regarding the defects of other cell types, such as macro-
`phages, platelets, and osteoclasts in Btk-deficient mice (8–11).
`Btk-deficient macrophages show the enhanced susceptibility to
`apoptotic death upon exposure to the microbial and immune
`inflammatory signals such as LPS and IFN-␥in vitro. In mixed
`bone marrow chimeras, Btk deficiency primarily leads to loss of
`peripheral macrophages without affecting bone marrow devel-
`opment, suggesting a role of Btk in inflammation-induced
`macrophage apoptosis and subsequently regulating macro-
`phage life span (8). Furthermore, specific Btk inhibition abol-
`ishes Fc␥RIII-induced TNF-␣, IL-1␤, and IL-6 production and
`thus suppresses myeloid cell-mediated arthritis (9). Moreover,
`once Btk-mediated signal is inhibited, thrombin-induced plate-
`let aggregation is abolished due to failure of microtubular
`polymerization but maintains their depolymerization (10).
`However, whether Btk is involved in the regulation of NK cell
`function remains unknown. Therefore, the physiological and
`pathophysiological significance of Btk expression in different
`cell types of the hematopoietic system, such as NK cells, needs
`to be further investigated.
`Because both Toll-like receptors (TLRs) and Btk are consti-
`tutively expressed in B cells and myeloid cells, the cooperation
`of Btk and TLRs has attracted much attention recently. In B1
`cells, TLR9 signaling is suppressed by dephosphorylation of
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`Btk Enhances NK Cell Activation
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`Btk, leading to attenuated activation of nuclear factor-␬B (NF-
`␬B) p65RelA and consequently blocking the excessive produc-
`tion of autoantibody (12). Btk-dependent induction of heme
`oxygenase-1 (HO-1) gene expression is observed in the macro-
`phages upon stimulation with ligands of TLR2, TLR6, TLR7,
`and TLR9 (13). Btk⫺/⫺ mice also show much more susceptibil-
`ity to the infection of Listeria monocytogenes, which are recog-
`nized and cause downstream effects through TLR2 (14). These
`findings suggest that Btk is required for major TLR pathways
`but exert different functions in TLR-mediated responses. Our
`recent work shows that Btk participates in intracellular MHC
`class II molecule-mediated full activation of the TLR pathway
`by interacting with MyD88 and TRIF, promoting TLR-trig-
`gered production of proinflammatory cytokines and type I
`interferon in macrophages and dendritic cells (15). Therefore,
`Btk could cross-talk with TLR2, TLR3, TLR4, TLR6, TLR7, and
`TLR9 pathways in B cells or myeloid cells. However, whether
`Btk can cooperate with TLRs in other kinds of innate cells, such
`as NK cells, is still unknown.
`NK cells are major effectors of the innate immune system,
`serving as the first line defense against infection, the regulator
`of immune response, and the monitoring of malignant cells
`(16). Indeed, the roles of NK cells are diverse, and their func-
`tions are controlled by the balance of a series of activating and
`inhibitory receptors (17, 18). Besides IL-2, IL-18, IL-15, NKAT,
`and STAT4, which have been previously confirmed to regulate
`cytotoxicity and production of TNF-␣and IFN-␥of NK cells,
`more regulators have been identified to control NK cell func-
`tions. For example, activating transcription factor 3 (ATF3)
`decreases the transcription and secretion of IFN-␥in NK cells
`through interacting with a cis-regulatory element of the IFN-␥
`gene (19). Transcription factor Hlx expression in activated NK
`cells temporally controls and limits the monokine-induced pro-
`duction of IFN-␥, partially through the targeted depletion of
`STAT4 (20). EWS/FLI1-activated transcript 2 (EAT-2)A and
`EAT-2B are recently known to act as positive regulators of sig-
`naling lymphocyte activation molecule family receptor-specific
`NK cell cytotoxicity by promoting phosphorylation of Vav-1,
`which is known to be implicated in NK cell killing (17), yet
`PRDM1/Blimp-1 has the capacity to coordinately suppress the
`release of IFN-␥, TNF-␣, and TNF-␤by NK cells through direct
`binding to multiple conserved regulatory regions at the IFNG
`and TNF loci (21). It is known that activation of protein-ty-
`rosine kinases is involved in TLR signaling and immune cell
`activation. Whether tyrosine kinase Btk is involved in the reg-
`ulation of NK cell activation after challenge with ligands of
`TLRs needs to be identified.
`In this study, we found Btk expression is up-regulated during
`the maturation and activation of murine NK cells. Btk⫺/⫺ NK
`cells, from both mice and human, show decreased expression of
`IFN-␥ and reduced cytotoxicity in response to TLR3 ligand.
`Poly(I:C)-induced acute hepatitis is observed to be attenuated
`once the Btk gene is mutant or the Btk inhibitor is administered
`in vivo in mice, which can be reversed by adoptive transfer of
`Btk⫹/⫹ NK cells. In addition, Btk is found to promote TLR3-
`triggered NK cell activation mainly through the increased acti-
`vation of the NF-␬B pathway. Our study demonstrated for the
`first time that Btk is required for TLR3-triggered activation of
`
`NK cells, thus contributing to the better understanding of the
`physiological significance of Btk in the regulation of NK cell
`functions.
`
`EXPERIMENTAL PROCEDURES
`Mice—C57BL/6 mice (Joint Venture Sipper BK Experimen-
`tal Animals Co., Shanghai, China) and Btk⫺/⫺ mice (The Jack-
`son Laboratory, Bar Harbor, ME) were maintained in a specific
`pathogen-free facility and used at 6–10 weeks of age. All animal
`experiments were performed in accordance with the Guide for
`the Care and Use of Laboratory Animals from the National
`Institutes of Health, with the approval of the Scientific Investi-
`gation Board of the Second Military Medical University,
`Shanghai.
`XLA Patients—Five XLA patients (Btk⫺/⫺) and seven age-
`matched normal controls were recruited from Hong Kong, and
`the research protocol was approved by the Institutional Review
`Board of the University of Hong Kong/Hospital Authority
`Hong Kong West Cluster.
`Purification of NK Cells from Mice and Patients—For isola-
`tion of mouse splenic NK cells, anti-CD3 and anti-DX5
`microbeads were used as recommended by the manufacturer
`(Miltenyi). To purify mouse NK cells at different developmental
`stages, bone marrow lymphocytes or
`splenocytes
`from
`C57BL/6 mice were incubated with monoclonal antibodies (BD
`Biosciences), and then NK precursors (CD3⫺ CD14⫺ CD19⫺
`NK1.1⫺ CD122⫹ NKG2D⫹), immature NK (CD3⫺ NK1.1⫹
`DX5⫺), and mature NK (CD3⫺ NK1.1⫹ DX5⫹) were sorted
`with fluorescence-activated Dako MoFloTM XDP to a purity of
`⬎98% as described previously (18). For isolation of primary
`human NK cells, peripheral blood mononuclear cells were iso-
`lated from whole-blood samples of XLA patients (Btk⫺/⫺) and
`age-matched normal controls by Ficoll-Hypaque (GE Health-
`care) gradient centrifugation. NK cells were magnetically sepa-
`rated from peripheral blood mononuclear cells with CD56
`microbeads (Miltenyi Biotec). The purity of isolated CD56⫹
`CD3⫺ NK cells was consistently⬎97%, as determined by flow
`cytometry.
`Activation of NK Cells—Mouse splenic NK cells were puri-
`fied and then stimulated with poly(I:C) (20 ␮g/ml), PMA (50
`ng/ml)/ionomycin (500 ng/ml), or IL-12 (20 ng/ml)/IL-18 (5
`ng/ml), respectively, for the time indicated and then were col-
`lected for the phenotypic and functional analysis. Human NK
`cells were cultured in RPMI 1640 medium supplemented with
`10% FBS (Invitrogen) at 1 ⫻ 106/ml with a supplement of
`poly(I:C) (Sigma) (final concentration 40 ␮g/ml) or an equal
`volume of PBS overnight in 37 °C, 5% CO2. For intracellular
`cytokine analysis, brefeldin A was added at a final concentra-
`tion of 10 ␮g/ml at 4 h before the end of NK cell incubation as
`we described previously (22), and NK cells were washed with
`PBS twice before phenotype and function analysis to remove
`resident poly(I:C).
`Quantitative PCR—Total RNA was prepared using the
`RNeasy kit (Qiagen, Valencia, CA). cDNA was generated with a
`cDNA archive kit (Applied Biosystems, Foster City, CA). Real
`time PCR was performed on a Light Cycler 1.5 PCR system
`(Roche Diagnostics) using SYBR Green real time PCR master
`mix reagents (Applied Biosystems, Foster City, CA) specific for
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`mouse Btk and the internal control ␤-actin (15). Data were
`normalized to ␤-actin using the 2⫺⌬⌬Ct method.
`Flow Cytometry—The following monoclonal antibodies were
`used in this study: anti-hCD56 (HCD56), anti-hCD3 (HIT3a),
`anti-hCD69 (FN50), anti-hCD107a (H4A3), anti-hIFN-␥(B27),
`anti-mCD3 (17A2), anti-mCD14 (Sa14-2), anti-mCD19 (6D5),
`anti-mNK1.1 (PK136), anti-mCD122 (5H4), anti-mNKG2D
`(C7), anti-mDX5 (DX5), anti-mCD69 (H1.2F3), anti-mLy-49A
`(YE1/48.10.6), and anti-mIFN-␥ (XMG1.2) (Biolegend); anti-
`mLy-49C/I (5E6), anti-hNKG2D (1D11), anti-hNKp44 (p44–
`8.1), anti-hNKp46 (9E2), anti-h-granzyme-B (GB11), and anti-
`perforin (G9) (Pharmingen); anti-mperforin (eBioOMAK-D);
`and anti-mGranzyme-B (16G6), anti-mLy-49D (eBio4E5), and
`anti-mNKG2A/C/E (20d5) (eBioscience).
`For surface staining, NK cells were stained with specific anti-
`bodies. For intracellular IFN-␥, perforin, and granzyme-B
`staining, cells were fixed, permeabilized, and then labeled with
`the antibodies indicated above. To avoid nonspecific staining,
`cells were incubated with FcR blocking reagent (Miltenyi Bio-
`tec) prior to the specific staining. All data were acquired on an
`LSR II (BD Biosciences) and analyzed by FlowJo software (Tree-
`Star) as described previously (14, 23).
`Immunoblot Assay—Cells were lysed with RIPA buffer (Cell
`Signaling Technology, Beverly, MA) supplemented with prote-
`ase inhibitor mixture. Protein concentrations of the extracts
`were measured with BCA assay (Pierce). The immunoblot assay
`was performed as described previously (15).
`Cytotoxicity Assay of NK Cells—For detection of mouse NK
`cell cytotoxicity, splenic NK cells were prepared and incubated
`at different E:T ratios with Yac-1 cells for 4 h at 37 °C. Then the
`cells were collected, stained with anti-NK1.1, stained with 7-a-
`mino-actinomycin D after washing, and analyzed by FACS. Live
`target cells were assayed by gating on the NK1.1⫺ 7-AAD⫺
`population. Cytotoxicity (%) ⫽ (NK1.1⫺ cells ⫺ NK1.1⫺7-
`AAD⫺ cells)/(NK1.1⫺ cells) ⫻ 100. For detection of human NK
`cell cytotoxicity, a live/dead cell-mediated cytotoxicity kit
`(Invitrogen) was used. Briefly, K562 target cells were stained
`with 3,3-dioctadecyloxacarbocyanine perchlorate and then
`cocultured with NK cells at an effector cell/target cell (E/T)
`ratio of 10:1 in the presence of propidium iodide for 2 h. After
`incubation, cytotoxicity was analyzed by flow cytometry and
`calculated as the percentage of DiO⫹PI⫹ cells in the total num-
`ber of DiO⫹ cells as we described before (24).
`Chemical Inhibition of Btk in Vitro and in Vivo—Purified
`murine NK cells were pretreated with LFM-A13 (100 ␮M) for 30
`min for in vitro inhibition of Btk. For in vivo experiments, WT
`mice were given 50 mg/kg LFM-A13 4h before poly(I:C) injec-
`tion, and we then analyzed their serum ALT, AST, and IFN-␥at
`the indicated times.
`Poly(I:C)-induced Acute Liver Injury—Mice were injected
`with poly(I:C), and liver histology, serology, and survival were
`assessed as we described previously (18).
`Adoptive Transfer of WT NK Cells into Btk⫺/⫺ Mice—Splenic
`NK cells from WT C57BL/6 mice were purified by magnetic
`activated cell sorting, and then intravenously injected into
`Btk⫺/⫺ mice (5 ⫻ 106). After poly(I:C) was administrated to
`induce acute hepatitis, liver injury was assessed and evaluated
`as described previously (18).
`
`Btk Enhances NK Cell Activation
`
`FIGURE 1. Btk expression is up-regulated during maturation and activa-
`tion of murine NK cells. A and B, splenic NK cells were purified by magnetic
`activated cell sorting from C57BL/6 mice. Quantitative RT-PCR analyses of Btk
`(A) and Western blot analysis of p-Btk (B) were performed 8 h or 20 minafter in
`vitro stimulation with PMA/ionomycin, poly(I:C), or IL-12/IL-18, respectively.
`C, expression of Btk protein was analyzed in different developmental stages
`of NK cells. Data are means ⫾ S.E. of triplicate experiments. **, p ⬍ 0.01.
`
`Statistics—Data are expressed as means ⫾ S.E. Statistical
`analysis was performed by Student’s paired t test or two-way
`analysis of variance with a multiple-comparison test using
`Prism 5 (GraphPad Software). A value of p ⬍ 0.05 was consid-
`ered statistically significant.
`
`RESULTS
`Btk Expression Increases Along with Differentiation and Acti-
`vation of Murine NK Cells—To investigate whether Btk is
`involved in the regulation of NK cell function, we analyzed the
`expression of Btk in mature NK cells stimulated with PMA/
`ionomycin, poly(I:C), or IL-12/IL-18 (Fig. 1, A and B). Both
`mRNA expression and phosphorylated protein levels of Btk
`were significantly increased in NK cells after the stimulations.
`The expression of Btk protein gradually increased during the
`maturation of NK cells from small levels in NK cell precursors
`(Fig. 1C). These results suggest that Btk may be involved in the
`development and differentiation of NK cells.
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`Btk Enhances NK Cell Activation
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`FIGURE 2. Btk deficiency leads to the impaired activation of murine NK cells in response to TLR3 ligand. A, CD69 expression of splenic WT or Btk⫺/⫺ NK
`cells with or without stimulation of poly(I:C). B, Ly49A, Ly49C/I, Ly49D, NKG2A/C/E, and NKG2D expression on splenic WT and Btk⫺/⫺ NK cells with or without
`stimulation of poly(I:C). C, intracellular IFN-␥, perforin, and granzyme-B expression of WT and Btk⫺/⫺ NK cells. D, cytotoxicity of WT and Btk⫺/⫺ NK cells against
`YAC-1 cells. Data are mean ⫾ S.D. of triplicate experiments. All experiments above were performed four times with similar results. Data are means ⫾ S.E. of
`triplicate experiments.
`
`Btk Deficiency Leads to the Decreased Activation of NK Cells
`in Both Mice and Humans—By in vitro stimulation with poly(I:
`C), we found that NK cells isolated from Btk⫺/⫺ mice (Btk⫺/⫺
`NK cells) had a lower CD69 expression compared with WT
`(Btk⫹/⫹) NK cells (Fig. 2A). The expressions of Ly49A, Ly49C/I,
`Ly49D, NKG2A/C/E, and NKG2D also decreased on unstimu-
`lated Btk⫺/⫺ NK cells, compared with that in Btk⫹/⫹ NK cells
`(Fig. 2B). However, poly(I:C) stimulation significantly up-regu-
`lated the expression of NKG2D and Ly49D on mature Btk⫹/⫹
`NK cells, although no differences in expression of NKG2D and
`Ly49D were observed on Btk⫺/⫺ NK cells even after poly(I:C)
`treatment (Fig. 2B). Furthermore, intracellular expression of
`IFN-␥, perforin, and granzyme-B in Btk⫺/⫺ NK cells was signif-
`icantly decreased in response to the stimulation with poly(I:C)
`(Fig. 2C). Consequently, the cytotoxic activity of TLR3-acti-
`vated Btk⫺/⫺ NK cells was significantly reduced, compared
`with that in Btk⫹/⫹ NK cells (Fig. 2D).
`We further investigated the function of primary NK cells
`isolated from XLA patients with poly(I:C) stimulation in vitro.
`Similarly, these human Btk⫺/⫺ NK cells also exhibited the
`decreased expression of intracellular IFN-␥, CD69, CD107a,
`and reduced cytotoxic activity compared with normal NK cells
`(Fig. 3). Interestingly, expression of NKG2D, NKp44, NKp46,
`perforin, and granzyme-B in human Btk⫺/⫺ NK cells was com-
`parable with that in normal control cells regardless of whether
`it was stimulated with poly(I:C) or not (supplemental Fig. S1).
`To define the role of Btk in development and survival of NK
`cells, we also compared the percentages of NK cells in mono-
`nuclear cells between WT and Btk⫺/⫺ mice, and we found that
`
`significantly less NK cells were contained in spleen, liver, and
`peripheral blood in Btk⫺/⫺ mice (supplemental Fig. S2). How-
`ever, no difference was found in the absolute number of NK
`cells in peripheral blood between the XLA patients and healthy
`controls, although there was a trend for a decrease in the per-
`centage of NK cells in lymphocytes in XLA patients (supple-
`mental Fig. S3). These data demonstrated that Btk is required
`for TLR3-triggered NK cell activation in both mice and human,
`although its role might be different in the development and
`survival of NK cells in mice and human.
`TLR-triggered NK Cell Activation Is Suppressed by Btk
`Inhibitor—We then applied the chemical inhibitor of Btk,
`LFM-A13, to further evaluate the role of Btk in TLR3-triggered
`murine NK cell activation. As shown in Fig. 4A, expression of
`CD69 was decreased in TLR3-triggered NK cells after LFM-
`A13 treatment. Similarly, inhibition of Btk by LFM-A13 down-
`regulated the intracellular expressions of IFN-␥, perforin, and
`granzyme-B in TLR3-triggered NK cells (Fig. 4B). Consistently,
`LFM-A13-pretreated NK cells exhibited significantly de-
`creased cytotoxicity (Fig. 4C). These data further confirmed
`that the inducible Btk is an important positive regulator in
`TLR3-triggered NK cell activation.
`Btk Promotes TLR3-triggered NK Cell Activation Mainly
`through NF-␬B Pathway—To illustrate the mechanism under-
`lying the positive regulation of TLR3-triggered NK cell activa-
`tion by Btk, we investigated the status of MAPK and NF-␬B
`pathways in WT and Btk⫺/⫺ NK cells. As shown in Fig. 5A,
`poly(I:C) stimulation failed to induce efficient phosphorylation
`of IKK␣/␤and degradation of I␬B␣in Btk⫺/⫺ NK cells, result-
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`Btk Enhances NK Cell Activation
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`FIGURE 4. Suppression of TLR3-triggered NK cell activation by chemical
`inhibitor of Btk. CD69 (A), IFN-␥, perforin, granzyme-B expression (B), and
`cytotoxicity (C) of poly(I:C)-stimulated WT NK cells with or without pretreat-
`ment of Btk inhibitor LFM-A13 were analyzed. The data shown represent the
`means ⫾ S.E., **, p ⬍ 0.01.
`
`cells, in which NF-␬B activation was already blunted. Inhibition
`of ERK and JNK did not significantly affect IFN-␥production in
`both WT and Btk⫺/⫺ NK cells. These results indicated that Btk
`promotes TLR3-triggered NK cell activation, at least in part,
`through increased activation of the NF-␬B pathway.
`Btk Contributes to the Poly(I:C)-induced NK Cell-mediated
`Acute Liver Injury—We further investigated the role of Btk in
`NK cell activation in vivo based on poly(I:C)-induced NK cell-
`mediated liver hepatitis model (18). After being challenged with
`poly(I:C), ALT and AST levels in the sera of WT mice increased
`significantly and reached the peak at 20 h after challenge,
`whereas IFN-␥ production peaked at 8 h (Fig. 6, A and B). In
`Btk⫺/⫺ mice, ALT and AST levels in the sera were significantly
`lower than the levels in WT mice, whereas IFN-␥production
`hardly increased at all (Fig. 6C). Crucially, Btk⫺/⫺ mice
`achieved prolonged survival after being administered a lethal
`dose of poly(I:C) (Fig. 6D). Correspondingly, fewer necrotic
`areas and fewer infiltrated lymphocytes were observed in
`Btk⫺/⫺ mice after poly(I:C) challenge by histological analysis
`(Fig. 6E). These data indicated that Btk might promote poly(I:
`C)-induced NK cell-mediated acute liver injury in mice.
`To further confirm the contribution of Btk to the pathology
`of TLR-3-triggered liver damage, we applied LFM-A13 to
`inhibit the activity of Btk in vivo. As shown in Fig. 7, after intra-
`
`FIGURE 3. Impaired activation and cytotoxic activity of Btkⴚ/ⴚ human NK
`cells after poly(I:C) stimulation. Highly purified human NK cells from XLA
`patients (Btk⫺/⫺) and age-matched normal controls were stimulated with
`poly(I:C) overnight and examined for the expressions of surface activation
`marker CD69 (A) and intracellular IFN-␥(B) by flow cytometry. C and D, treated
`NK cells were cocultured with K562 at E/T ratio of 10:1 in the presence of
`anti-CD107a antibody for 2 h. The surface expression of CD107a on NK cells
`(C) and specific lysis of target K562 cells (D) were then analyzed by flow cytom-
`etry. XLA patients (n ⫽ 5) and age-matched normal controls (n ⫽ 7) are
`shown. The data shown are represent for Mean⫾S.E., *, p ⬍ 0.05; **, p ⬍ 0.01.
`
`ing in the reduced phosphorylation of p65 and IRF3. Moreover,
`pretreatment with Btk inhibitor LFM-A13 suppressed TLR3-
`triggered activation of NF-␬B in NK cells (Fig. 5B). Similarly the
`phosphorylations of ERK and JNK were also reduced in either
`Btk-deficient or LFM-A13-pretreated NK cells (Fig. 5, A and B).
`To investigate which signal pathway is responsible for the
`attenuated function of Btk⫺/⫺ NK cells, we pretreated WT and
`Btk⫺/⫺ NK cells with specific inhibitors for NF-␬B, ERK, and
`JNK before poly(I:C) stimulation (Fig. 5C). The TLR3-induced
`IFN-␥production was significantly decreased by NF-␬B inhib-
`itor (pyrrolidine dithiocarbamate), although not in Btk⫺/⫺ NK
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`Btk Enhances NK Cell Activation
`
`FIGURE 5. Btk positively regulates TLR3-triggered NK cell functions by activating NF-␬B pathway. Splenic NK cells were purified by magnetic activated
`cell sorting from WT or Btk⫺/⫺ mice. A, Western blot analysis of MAPK and NF-␬B pathways in TLR3-triggered Btk⫺/⫺ and WT NK cells. B, MAPK and NF-␬B
`pathways were analyzed in TLR3-triggered WT NK cells with or without LFM-A13 pretreatment. C, after pretreatment with pyrrolidine dithiocarbamate (PDTC)
`(NF-␬B inhibitor, 50 ␮M), SP600125 (JNK inhibitor, 10 ␮M), or PD98059 (ERK inhibitor, 10 ␮M) for 30 min, intracellular IFN-␥expression of poly(I:C)-stimulated WT
`and Btk⫺/⫺ NK cells was then analyzed.
`
`peritoneal administration with LFM-A13, serum levels of ALT,
`AST, and IFN-␥in poly(I:C)-treated WT mice were markedly
`reduced. Histological analysis also showed attenuated liver
`damage, marked as less necrosis and fewer lymphocytes infil-
`tration, in mice treated with LFM-A13 (Fig. 7D). These data
`emphasized the crucial role of Btk in TLR3-triggered NK cell
`activation in vivo.
`Replacement of Btk⫺/⫺ NK Cells with WT NK Cells Rescues
`Poly(I:C)-induced Acute Liver Hepatitis in Mice—To determine
`whether the impaired NK cell function resulted in the attenu-
`ated experimental liver damage in Btk⫺/⫺ mice observed above,
`we depleted NK cells in Btk⫺/⫺ mice by PK136 monoclonal
`antibody and then transferred the purified WT NK cells 2 days
`later. One day post-cell transfer, poly(I:C) was administered. As
`shown in Fig. 8, the NK-deleted Btk⫺/⫺ mice showed signifi-
`cantly declined levels of serum ALT, AST, and IFN-␥. In con-
`trast, their serum ALT, AST, and IFN-␥ levels dramatically
`increased after the infusion of WT NK cells. Moreover, more
`severe liver tissue damage was observed in Btk⫺/⫺ mice receiv-
`ing poly(I:C) administration after the transfer of WT NK cells
`(Fig. 8D). These results demonstrated that Btk-mediated NK
`cell activation is responsible for the pathogenesis of poly(I:C)-
`induced acute liver hepatitis, which further confirmed the
`indispensable role of Btk in TLR3-triggered NK cell activation.
`
`DISCUSSION
`In this report, we provide the functional description of Btk in
`NK cell activation. We show that Btk accumulates during mat-
`uration and activation of NK cells and acts as a positive regula-
`tor of NK cell activation by up-regulating expression of IFN-␥,
`granzyme-B, and perforin and enhancing cytotoxicity of NK
`cells in mice. The data strongly suggested Btk is responsible for
`poly(I:C)-induced NK cell activation, giving rise to the suscep-
`tibility of TLR3-triggered liver hepatitis. The observation that
`decreased TLR3-triggered activation of human NK cells from
`XLA patients is observed with the reduced IFN-␥, CD69, and
`CD107a expression and cytotoxicity, our results demonstrate
`that Btk is required for TLR3-triggered NK cell activation.
`NK cells are important innate immune cells but they also
`bridge and influence adaptive immunity (25), whose activation
`relies on germ line-encoded receptors. Activated NK cells not
`only utilize polarized exocytosis of secretory lysosomes to kill
`infected or neoplastic cells (26, 27), but they also regulate and
`maintain the balance between positive and negative immune
`responses through secretion of cytokines and chemokines and
`killing of autologous immune cells, including activated T cells
`and dendritic cells (28, 29). Considering their enigmatic roles in
`different physiological processes, it is urgent to find critical reg-
`ulators of NK cell function and to clarify their underlying mech-
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`Btk Enhances NK Cell Activation
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`FIGURE 6. Btk deficiency attenuates poly(I:C)-induced acute liver injury in vivo. WT and Btk⫺/⫺ mice were injected with poly(I:C) (20 ␮g/g body weight)
`intraperitoneally. Serum ALT (A), AST (B), IFN-␥(C) were assayed at the indicated times. Data are means ⫾ S.E. of triplicate experiments. **, p ⬍ 0.01. D, survival
`of WT and Btk⫺/⫺ mice was observed after injection of high dose of poly(I:C) (30 ␮g/g body weight). E, 18 h later, after fixed and embedded, sliced liver sections
`were stained with hematoxylin-eosin. Arrows indicated the liver necrosis and infiltration of lymphocytes.
`
`anisms. On the basis of the observation of rapid Btk phosphor-
`ylation in NK cells after poly(I:C), PMA/ionomycin, or IL-12/
`IL-18 stimulation, we further
`investigated whether Btk
`regulates the activation of NK cells and their underlying mech-
`anisms. As expected, Btk⫺/⫺ NK cells exert decreased poly(I:C)
`up-regulated WT NK cells, along with the decreased expression
`of IFN-␥, perforin, and granzyme-B and consequently reduced
`cytotoxicity, which confirmed that Btk is a positive regulator of
`NK cell function upon poly(I:C) stimulation. Indeed, we also
`found Btk can also amplify the function of IL-12/IL-18-stimu-
`
`lated NK cells (data not shown). However, even though Btk
`deficiency led to a significantly reduced killing capacity of
`human NK cells with the decreased CD69, CD107a, and IFN-␥
`expression after poly(I:C) stimulation, NKG2D, NKp44, and
`NKp46 expression remained stable. Additionally, no statistical
`quantitative difference of NK cell number was observed in the
`peripheral blood between XLA patients and normal controls.
`These data suggested that although human and mice share
`essential developmental characteristics, some differences still
`exist. For example, X chromosome inactivation (XCI) allows
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`JULY 6, 2012 • VOLUME 287 • NUMBER 28
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`Btk Enhances NK Cell Activation
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`FIGURE 7. TLR3-triggered liver hepatitis is attenuated by inhibition of Btk in vivo. WT mice were administered with LFM-A13 (50 mg/kg) and then poly(I:C)
`(20 ␮g/g) intraperitoneally. Sera were collected for assays of ALT (A) and AST (B) 20 h later, and serum IFN-␥was examined 8 h after administration (C). Data are
`means ⫾ S.E. of triplicate experiments. *, p ⬍ 0.05; **, p ⬍ 0.01. D, 18 h later, liver necrosis and infiltration of lymphocytes were observed after hematoxylin-eosin
`staining.
`dosage compensation of the expression from the sex chromo-
`some in mammalian female cells (30). To clarify the corre-
`sponding mechanism, human embryonic stem cells and mouse
`embryonic stem cells were used and were supposed to shed
`light on the XCI process in early embryogenesis, but studies of
`embryonic stem cells from these two species largely indicated
`inconsistency in the status of XCI (31). Human embryonic stem
`cells showed the various states of XCI, which were proved to be
`caused by epigenic manipulations involving XIST (the crucial
`gene of XCI initiation) promoter methylation/demethylation as
`well as in vitro culture conditions (32). These findings suggest
`possible explanations for the partially inconsistent observa-
`tions in Btk⫺/⫺ NK cells of human and mouse, and epigenic
`regu