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The Journal of Immunology, 2006, 177: 7203-7210.
Copyright © 2006 by The American Association of Immunologists, Inc.

Bruton’s Tyrosine Kinase Is Required for TLR-Induced IL-10 Production1

Nathan W. Schmidt2, Vivian T. Thieu2, Brandon A. Mann, Ayele-Nati N. Ahyi and Mark H. Kaplan3

Department of Pediatrics and Department of Microbiology and Immunology, H. B. Wells Center for Pediatric Research and Walther Oncology Center, Indiana University School of Medicine, and Walther Cancer Institute, Indianapolis, IN 46202


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Bruton’s tyrosine kinase (Btk) is a critical signaling mediator downstream of the B cell Ag receptor. X-linked agammaglobulinemia is caused by mutations in Btk resulting in multiple defects in B cell development and function, and recurrent bacterial infections. Recent evidence has also supported a role for Btk in TLR signaling. We demonstrate that Btk is activated by TLR4 in primary macrophages and is required for normal TLR-induced IL-10 production in multiple macrophage populations. Btk-deficient bone marrow-derived macrophages secrete decreased levels of IL-10 in response to multiple TLR ligands, compared with wild-type (WT) cells. Similarly, Btk-deficient peritoneal and splenic macrophages secrete decreased IL-10 levels compared with WT cultures. This phenotype correlates with Btk-dependent induction of NF-{kappa}B and AP-1 DNA binding activity, and altered commensal bacteria populations. Decreased IL-10 production may be responsible for increased IL-6 because blocking IL-10 in WT cultures increased IL-6 production, and supplementation of IL-10 to Btk-deficient cultures decreased IL-6 production. Similarly, injection of IL-10 in vivo with LPS decreases the elevated IL-6 serum levels during endotoxemia in Btk-deficient mice. These data further support a role for Btk in regulating TLR-induced cytokine production from APCs and provide downstream targets for analysis of Btk function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Bruton’s tyrosine kinase (Btk),4 a member of the Tec family of tyrosine kinases, is involved in B cell development and function (1). Btk deficiency results in the human condition X-linked agammaglobulinemia (XLA) and a similar xid in mice (1). In the absence of Btk activity, there is a block in B cell development resulting in reduced mature B cells, peritoneal B1 cells, and decreased serum IgM and IgG3 (1). The few B cells that reach maturation have impaired Ag receptor signaling and are unable to respond to type II T-independent Ags (1). Immune defects in XLA patients render them susceptible to recurrent bacterial infections, despite the ability of Btk-deficient B cells to respond to T-dependent Ags (2). This suggests that Btk is involved in additional aspects of the immune response to bacterial infections.

Recent evidence has supported a role for Btk in the innate immune system. Btk is phosphorylated following stimulation of human monocyte cell lines and primary human monocytes with LPS, and can interact with multiple components of TLR pathways including the Toll/IL-1R domain of MyD88, Mal, IL-1R-associated kinase 1, and with the TLRs themselves (3, 4). Btk phosphorylates Mal, resulting in Mal degradation (5, 6). This effect may be responsible for the requirement of Btk in TLR4-stimulated phosphorylation of the NF-{kappa}B subunit p65 on serine 536, which is critical for the transactivation activity of NF-{kappa}B (4, 6, 7). Based on experiments with XLA patient samples and xid mice, Btk-dependent signaling is required for normal production of TNF-{alpha} and IL-1β, although xid cells also show increased levels of IL-12 compared with wild-type (WT) cells (3, 8, 9). It is still unclear whether Btk affects the production of other cytokines or results in altered signaling apart from effects on NF-{kappa}B activation.

To further define a role for Btk in TLR-induced responses, we compared in vitro WT and Btk-deficient macrophage responses and in vivo responses to bacterial products. Btk-deficient bone marrow-derived macrophages (BMDM) demonstrated decreased IL-10 production following stimulation with multiple TLR ligands. The decreased IL-10 production in Btk-deficient BMDM correlates with altered activation of LPS-induced signaling pathways. Altered IL-10 may be responsible for increased IL-6 production in vivo and in vitro. Thus, Btk regulates the balance of pro- and anti-inflammatory cytokine production, suggesting it is a critical molecule in regulating innate immune responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Btk-deficient mice on the C57BL/6 background (The Jackson Laboratory) were maintained in the Indiana University Laboratory Animal Research Center. Control C57BL/6 mice were purchased from Harlan Bioproducts. WT and IgM-deficient (10) C57BL/6 mice were from The Jackson Laboratory.

Generation of BMDM and isolation of tissue macrophages

Generation of BMDM have been previously described (11). Briefly, femur bone marrow cells were plated out 2–3 x 105 cell/ml overnight at 37°C, and then stimulated the following morning with 10 ng/ml murine M-CSF (PeproTech). The cells were cultured an additional 7–8 days at 37°C. To isolate peritoneal cavity cells, peritoneal exudate cells were incubated for 3 h at 37°C in the supplemented BMDM medium. Nonadherent cells were washed away and adherent cells were scrapped off petri dishes and used as peritoneal macrophages. Splenic macrophages were isolated using anti-mouse CD11b microbeads (Miltenyi Biotec) according to manufacturer’s recommendations.

Cellular phenotypic analysis by flow cytometry

Cells (1 x 106 per sample) were washed in PBS with 2% BSA and 0.1% NaN3 (FACS buffer). Cells were first incubated with anti-Fc{gamma}R Ab, clone 2.4G2 (BD Pharmingen) for 10 min. Cells were then stained with CD11b-FITC, F4/80-PE, TLR4-MD2-PE, CD86-PE, or MHC class II (MHC II)-PE Abs (eBioscience) for 15 min. All staining was done at 4°C followed by one wash with FACS buffer. Cells were then fixed in FACS buffer that contained 0.5% formaldehyde.

RNA purification, cDNA synthesis, and quantitative RT-PCR

WT and Btk-deficient BMDM were stimulated with IFN-{gamma} (50 ng/ml) for 4 h and total RNA was isolated with TRIzol reagent (Invitrogen Life Technologies). Reverse transcriptase reactions were done using the Cloned AMV First-Strand cDNA Synthesis kit (Invitrogen Life Technologies). Quantitative RT-PCR was performed by the comparative threshold cycle ({Delta}CT) method and normalized to β2-microglobulin using the 7500 Real-Time PCR instrument (Applied Biosystems). Relative expression of the indicated genes is expressed as the fold induction over inactivated WT cells. The TaqMan primers for CXCL9, CXCL10, suppressor of cytokine signaling 1 (SOCS1), SOCS3, and β2-microglobulin are from Applied Biosystems.

Analysis of cytokine production by ELISA

Macrophages (5 x 105/ml) were incubated in the absence or presence of 100 ng/ml LPS from Salmonella typhosa (Sigma-Aldrich), 10 µg/ml peptidoglycan, 50 µg/ml poly(I:C), or 5 µg/ml Escherichia coli DNA (CpG DNA) (Sigma-Aldrich). Cell supernatants were collected after 24 h. Cytokine production was assayed using ELISA with anti-IL-6 and -IL-10 (BD Pharmingen) Abs. Presence of cytokine was assessed by measuring color change after the addition of ELISA substrate buffer (10% diethanolamine, 0.5 mM MgCl2, 0.02% NaN3 (pH 9.8)) supplemented with 5 mg/ml Sigma 104 phosphatase substrate (Sigma-Aldrich).

EMSA

Nuclear extracts were prepared from stimulated cells using the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Pierce). NF-{kappa}B and AP-1 DNA binding activity were assayed using the Gel Shift Assay Systems (Promega). A 4% nondenaturing acrylamide gel was used to separate protein bound DNA sequences and the unbound DNA sequences. The gel was then dried onto filter paper and exposed to x-ray film (Pierce) to detect protein-bound DNA.

Western analysis

BMDM were stimulated with 100 ng/ml LPS from S. typhosa for the indicated amount of time. Cells were either washed in ice-cold PBS and lysed in lysis buffer plus protease and phosphatase inhibitors to generate whole-cell extracts or to generate nuclear extracts as described for EMSA. Lysates were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Schleicher and Schuell). Proteins were detected using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences). Western analysis was performed using anti-phospho- and -total-Btk, p38 MAPK, ERK1/2, and JNK Abs (Cell Signaling Technology), Abs to MAPK phosphatase-1 (MKP-1), RelA, RelB, c-Fos, and c-Jun (Santa Cruz Biotechnology), and anti-actin Ab (Oncogene Research Products).

Fecal bacteria populations

Fresh stool samples were collected from mice and diluted in PBS (0.1 g of feces:100 µl of PBS). Samples were plated out on brain-heart infusion with sheep blood agar plates (Remel) and grown in anaerobic or aerobic conditions for 3 or 2 days, respectively, at 37°C. Samples were additionally plated out at several dilutions on selective medium that allowed the growth of specific aerobic bacteria populations. Selective media for bacterial species were as follows: de Man-Rogosa-Sharpe (lactobacilli species), Columbia colistin, nalidixic acid with 5% sheep blood (Gram-positive cocci), MacConkey (Gram-negative bacilli), Bile Esculin Azide (enterococci and group D streptococci), and eosin-methylene blue (Gram-negative enterics) (Remel). After 2 days at 37°C, colonies were counted from each plate. Total CFU per gram of stool were calculated.

Endotoxemia

Mice were weighed and then injected in the peritoneal cavity with 20 mg/kg E. coli 055:B5 LPS (Sigma-Aldrich) dissolved in PBS. Btk-deficient mice were injected with either LPS alone, or LPS in combination with 10 µg of IL-10. Ten hours after injection, mice were sacrificed and serum was harvested for analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Btk is required for TLR-induced IL-10 production from macrophage populations

Btk has been implicated in TLR-induced signaling pathways. To expand on these observations, we wanted to test whether Btk was required for TLR-induced responses in macrophage populations. We first determined that Btk is not required for primary BMDM development. Development of the macrophage phenotype was similar in WT and Btk-deficient cultures as assessed by staining for CD11b, F4/80, TLR4-MD2, MHC II, and CD86 expression, cell morphology and uptake of FITC-dextran (Fig. 1, A and B, and data not shown). We also tested IFN-{gamma}-induced gene expression in these populations. IFN-{gamma}-induced expression of CXCL9 and CXCL10 was indistinguishable between WT and Btk-deficient cells (Fig. 1C). Although basal levels of SOCS1 and SOCS3 were modestly higher in Btk-deficient cells, the fold induction by IFN-{gamma} was similar between WT and Btk-deficient cells. Thus, Btk is not required for the development or cytokine responses of BMDM.


Figure 1
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  		FIGURE 1. BMDM development in the absence of Btk. A, BMDM were incubated with Abs that recognized the cell surface proteins CD11b and F4/80 and analyzed by flow cytometry. Data are representative of two experiments. B, BMDM were gated on CD11b and analyzed for expression of TLR4-MD2, MHC II, and CD86. Numbers represent the average mean fluorescence intensity from two experiments. C, BMDM were incubated with IFN-{gamma} for 4 h. RNA was isolated and real-time RT-PCR was performed. Data represent the average fold induction ± SEM from three independent experiments.

 
We next examined in vivo-differentiated macrophage populations. Splenic macrophage numbers and phenotype (expression of CD11b, F4/80, TLR4-MD2, and CD86) were similar in WT and Btk-deficient mice (Fig. 2A). Btk-deficient splenic macrophages had a slight increase in MHC II expression (Fig. 2A). Btk-deficient peritoneal macrophages comprised a much larger proportion of the peritoneal lavage population than the WT population, largely due to the absence of B cells in the Btk-deficient peritoneum (Fig. 2B) (2, 12). However, WT and Btk-deficient peritoneal macrophages had similar expression of CD11b, F4/80, and TLR4-MD2 (Fig. 2B). Thus, Btk deficiency does not affect the development of these macrophage populations in vivo.


Figure 2
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  		FIGURE 2. Splenic and peritoneal macrophage development in the absence of Btk. A, Flow cytometric analysis of CD11b and F4/80 expression on spleen cells from WT and Btk-deficient mice. Numbers represent the average of percentages from three mice in each quadrant. CD11b+ spleen cells were analyzed for expression of TLR4-MD2, MHC II, and CD86. Numbers represent the average mean fluorescence intensity (MFI) from three experiments. B, Flow cytometric analysis of CD11b and F4/80 expression on WT and Btk-deficient peritoneal lavage cells. Numbers represent the average of percentages from three mice in each quadrant. CD11b+ peritoneal lavage cells were gated on and analyzed for expression of TLR4-MD2. Numbers represent the average MFI from three experiments.

 
To identify downstream targets of Btk-dependent signaling, we examined TLR ligand-induced cytokine production in cultures of BMDM from WT and Btk-deficient mice. Btk-deficient BMDM stimulated with multiple TLR ligands (peptidoglycan, TLR2; poly(I:C), TLR3; LPS, TLR4; CpG DNA, TLR9) generated decreased levels of IL-10, compared with WT BMDM (Fig. 3A). Analysis of peritoneal and splenic macrophages revealed a similar decrease in LPS-induced IL-10 production compared with WT cells (Fig. 3, B and C). Because Btk has been shown to be important for LPS-induced IL-10 production from dendritic cells (13), IL-10 appears to be a Btk-dependent TLR-induced target in multiple cell types.


Figure 3
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  		FIGURE 3. Decreased IL-10 production in Btk-deficient macrophages. A, BMDM were incubated in the absence or presence of 10 µg/ml peptidoglycan, 50 µg/ml poly(I:C), 100 ng/ml LPS, or 5 µg/ml CpG DNA. Cell supernatants were harvested after 24 h for analysis of IL-10 using ELISA. Data are representative of at least three experiments and shown as the mean ± SEM. B and C, Peritoneal (B) and splenic (C) macrophages were incubated in the absence or presence of 100 ng/ml LPS. Cell supernatants were harvested and analyzed as in A. Data are representative of three experiments and shown as the mean ± SEM.

 
Multiple TLR-induced signaling pathways are altered by the absence of Btk in BMDM

LPS stimulation results in Btk phosphorylation in human monocytes and monocyte cell lines (3, 4, 9). Culture extracts from BMDM were examined for phosphorylated Btk in the absence of stimulation and at 5 and 10 min after LPS stimulation. The data show that LPS induces a transient phosphorylation of Btk (Fig. 4A). To further define the downstream effectors of Btk signaling, we next analyzed DNA binding activity of NF-{kappa}B and AP-1. EMSA analysis indicated decreased induction of NF-{kappa}B and AP-1 DNA binding activity in the Btk-deficient BMDM stimulated with either LPS or E. coli DNA (CpG DNA) compared with induction in WT cells (Fig. 4B). Decreased TLR-induced NF-{kappa}B activity is consistent with two previous reports (4, 7). To define the binding complex components that are affected by Btk deficiency, we performed immunoblot analysis of nuclear extracts from resting and TLR-stimulated cells. We observed decreased nuclear localization of RelA in response to TLR ligands in Btk-deficient cells, compared with WT cells (Fig. 4C). Although RelB nuclear localization was not significantly induced by TLR stimulation, levels present in the nucleus were also decreased in Btk-deficient cells. Nuclear levels of c-Jun and c-Fos were also decreased in Btk-deficient cells (Fig. 4C). Because cytoplasmic expression of these proteins was not altered (data not shown), these results suggest that Btk is required for TLR-induced nuclear localization of these transcription factors.


Figure 4
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  		FIGURE 4. Multiple TLR-induced signaling pathways are altered in the absence of Btk. A, BMDM were incubated in the absence or presence of 100 ng/ml LPS for 0, 5, and 10 min. Cell lysates were analyzed for Btk phosphorylation by immunoblotting with an anti-phosphorylated Btk and anti-Btk Ab. Numbers below the blot represent the band intensities of p-Btk over total Btk. Data are representative of two experiments. B, BMDM were stimulated with 100 ng/ml LPS or 5 µg/ml CpG DNA for 4 h. Nuclear extracts were incubated with 32P-labeled consensus DNA binding sequences for NF-{kappa}B or AP-1. Relative binding is calculated by dividing the band intensity over protein loading control. Data are representative of two experiments. C, Nuclear cell extracts from B were immunoblotted for the indicated proteins. D, BMDM were stimulated with 100 ng/ml LPS for the indicated amount of time. Whole-cell extracts (WCE) were immunoblotted with anti-MKP-1. Membranes were stripped and probed with anti-actin. Numbers below blot represent band intensities of MKP1 over actin. Data are representative of two experiments. E, WCE from cells stimulated as in C were analyzed by immunoblot with an anti-phosphorylated-p38 Ab, anti-phosphorylated-ERK1/2, or anti-phosphorylated-JNK/stress-activated protein kinase (SAPK) Ab. Membranes were stripped and reprobed with anti-p38, anti-ERK1/2, or anti-JNK/SAPK Abs. Data are representative of four experiments. F, Densitometry was performed on data in E by dividing band intensities from phosphorylated proteins over total proteins.

 
MKP-1, a dual-specificity phosphatase that dephosphorylates p38, ERK1/2, and JNK (14, 15), has recently been shown to regulate TLR-induced IL-10 production (16, 17, 18). Moreover, MKP-1 expression is induced by LPS or peptidoglycan in macrophages (16, 18, 19, 20, 21). To determine whether MKP-1 is a Btk pathway target and whether it might be involved in the altered production of IL-10, we examined induction of MKP-1. In WT BMDM, LPS induced MKP-1 expression by 45 min and induction peaked by 60 min (Fig. 4D). There was decreased MKP-1 induction in Btk-deficient BMDM stimulated with LPS compared with WT BMDM (Fig. 4D). To define the effects of decreased MKP-1 induction, we next examined the activation of the MAPKs p38, ERK1/2, and JNK. Although LPS stimulation resulted in a similar amount of p38 and JNK phosphorylation in WT and Btk-deficient BMDM, there was enhanced phosphorylation of ERK1/2 in the Btk-deficient BMDM compared with WT BMDM (Fig. 4, E and F). In contrast to our data, MKP-1-deficient mice demonstrated that p38 MAPK is the primary target of MKP-1, and that the absence of MKP-1 results in increased IL-10 production. This suggests that, although Btk does regulate MKP-1 expression and MAPK activity, these events are not responsible for Btk-dependent IL-10 production.

Altered commensal aerobic bacteria homeostasis in Btk-deficient mice

Mice deficient in IL-10 have intestinal inflammatory disease (22). Moreover, TLR recognition of commensal bacteria is critical in regulating intestinal epithelial homeostasis (23) and mice deficient in IL-10 have altered immune responses to commensal bacteria (24). We speculated that the decreased IL-10 production in Btk-deficient mice might result in intestinal inflammation or alterations in commensal bacteria populations. Histological analysis of colon and cecum segments from Btk-deficient mice revealed no significant inflammation and no differences in intestinal mucus production between WT and Btk-deficient tissues (data not shown). Analysis of fresh stool samples from WT and Btk-deficient mice revealed a significant decrease in aerobic bacteria but not anaerobic bacteria (Fig. 5A). We then examined specific populations of aerobic bacteria and noted a significant decrease in the enterococci and group D streptococci populations in the Btk-deficient mice, compared with WT (Fig. 5B). Because it is possible the decrease in commensal bacteria in the Btk-deficient mice is a result of the B cell defect observed in these mice, rather than TLR signaling defects, we also analyzed fecal bacteria populations from IgM-deficient (µMT) mice, which lack mature B cells (10). Examination of the fecal bacteria in IgM-deficient mice revealed no differences vs WT mice (Fig. 5C). This suggests the decrease in bacterial populations in Btk-deficient mice is due to the role of Btk in the innate immune system and not a result of defects in B cell development and function.


Figure 5
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  		FIGURE 5. Decreased aerobic bacteria in Btk-deficient mice. Fresh stool samples were collected from mice and diluted in PBS. A, Diluted stool samples from WT and Btk–/– mice were plated on brain-heart infusion with sheep blood agar and grown in anaerobic or aerobic conditions at 37°C. Each point represents data from a single mouse determined by bacterial growth over several sample dilutions. B, Diluted stool samples from WT and Btk–/– mice were incubated on plates that selectively allowed the growth of specific bacteria as described in Materials and Methods. C, Diluted stool samples from WT and IgM–/– mice were plated out as described above. After 2 days of incubation at 37°C, colonies were counted on each plate. Asterisks indicate a significant change vs the bacteria population from WT mice (p < 0.05) using Tukey’s t test.

 
Increased IL-6 in Btk-deficient mice is due to decreased IL-10 production

Supernatants from LPS-stimulated macrophage populations in Fig. 3 were tested for levels of other cytokines using ELISA. The one other cytokine that was consistently affected by Btk deficiency was IL-6. Btk-deficient bone marrow-derived, peritoneal and splenic macrophages produced increased levels of IL-6, compared with WT cultures (Fig. 6).


Figure 6
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  		FIGURE 6. Increased IL-6 production from Btk-deficient macrophages. Bone marrow-derived (A), peritoneal (B), or splenic (C) macrophages were incubated in the absence or presence of 100 ng/ml LPS. Cell supernatants were harvested after 24 h for analysis of IL-6 using ELISA. Data are representative of at least three experiments and shown as the mean ± SEM.

 
We speculated that the observed increases in IL-6 production could be a result of defective IL-10 production, because it is documented that IL-10-deficiency results in increased IL-6 production in vivo (25, 26). To test this, we incubated LPS stimulated wild-type BMDM with anti-IL-10 or an isotype control. Although the isotype control did not alter IL-6 production by these cultured cells, anti-IL-10 did increase the amount of IL-6 generated in these cultures (Fig. 7A). Conversely, addition of IL-10 to Btk-deficient BMDM cultures dramatically decreased the levels of IL-6 produced by these cells (Fig. 7B). To examine this phenomenon in vivo, we determined serum cytokine levels following in vivo injection of LPS. Using a sublethal injection of LPS, we analyzed serum cytokine levels 10 h after injection. Despite higher doses of circulating LPS in Btk-deficient mice (27), there were decreased levels of IL-10 in the serum from Btk-deficient mice compared with serum from WT mice (Fig. 7C). Decreased IL-10 corresponded to increased circulating levels of IL-6 in serum from Btk-deficient mice, compared with serum from LPS-injected wild-type mice (Fig. 7D). Coinjection of IL-10 and LPS into Btk-deficient mice resulted in lower circulating IL-6 levels compared with mice injected with LPS alone (Fig. 7D), demonstrating that IL-10 can decrease IL-6 levels in vivo and that increased IL-6 levels are not an intrinsic result of Btk deficiency. Although these data do not unequivocally show that decreased IL-10 is responsible for increased IL-6 in Btk-deficient mice, they do indicate that Btk is an important regulator of in vivo inflammatory responses.


Figure 7
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  		FIGURE 7. IL-10 regulates IL-6 in vitro and in vivo. A, C57BL/6 wild-type BMDM were incubated with 10 ng/ml LPS in the presence of an isotype Ab or 10 µg/ml anti-IL-10. Supernatants were harvested after 24 h to analyze IL-6 levels using ELISA. Data are shown as the mean ± SEM and are representative of three experiments. B, BMDM from C57BL/6 Btk-deficient mice were incubated with 10 ng/ml LPS in the presence or absence of 1 ng/ml IL-10. IL-6 levels were analyzed by ELISA. Data are shown as the mean ± SEM and are representative of three experiments. C, WT and Btk-deficient mice were injected with a sublethal dose of LPS. Mice were sacrificed, and serum was harvested 10 h after injection. Pooled serum samples were tested for IL-10 levels using ELISA. D, Serum samples from mice described in C and an additional group of Btk-deficient mice coinjected with LPS and 10 µg of IL-10 were tested for levels of IL-6 using ELISA. Data are the mean of four to six mice ± SEM. Groups were significantly different from each other based on ANOVA (p < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Although Btk is well characterized in B cell signaling and development, a growing body of evidence suggests that it is also involved in TLR signaling (3, 4, 5, 6, 7, 8, 9). In this report, we have demonstrated several novel aspects of the role of Btk in TLR signaling. First, we have shown that Btk is involved in responses to multiple TLR ligands. Second, we show that Btk regulates the production of TLR-induced IL-10 from multiple macrophage cell types. Third, in addition to NF-{kappa}B activation, Btk regulates the activation of AP-1 DNA binding activity, ERK activation, and the induction of MKP-1. Finally, we have shown that the decreased IL-10 levels may result in increased IL-6 production and a shift toward a more proinflammatory environment. Thus, these studies define important functions of Btk that may be critical in understanding innate immune responses and in treating clinical symptoms of XLA patients.

How Btk is involved in TLR signaling is only beginning to be understood. Recently, Btk was found to phosphorylate Mal, resulting in its ubiquitylation and degradation (5, 6). Although Mal activity appears to be restricted to TLR2 and TLR4 signaling (28, 29), there are clearly Btk-dependent functions of TLR3 and TLR9 signaling (Figs. 3 and 4), suggesting that Btk is involved in pathways that are independent of Mal. In addition to the role for a Btk-Mal pathway in NF-{kappa}B activity (4, 5, 6, 7), Mal may also be downstream of Btk in the induction of MKP-1 and in altered MAPK activity, because there was a defect in these events when Btk-deficient BMDM were stimulated with LPS but not with CpG (Fig. 4 and data not shown). Thus, Btk is required for Mal-dependent and -independent signals following activation by various TLR ligands.

Additional targets of Btk phosphorylation in TLR pathways are still unclear. Btk is necessary for phospholipase C{gamma}-2 activation following ligation of the BCR. However, we did not observe induction of phospholipase C{gamma}-2 phosphorylation following stimulation of WT or Btk-deficient BMDM with either LPS or CpG DNA (data not shown). It has recently been reported the adaptor molecules Dok-1 and Dok-2, which become tyrosine phosphorylated following TLR activation, can negatively regulate TLR4-induced ERK phosphorylation (30). The similarity to our observations in Btk-deficient macrophages (Fig. 4) suggests that the Dok proteins could be Btk targets. Additionally, mice deficient in glycogen synthase kinase-3β or TNFR-associated factor 3 have also recently been shown to have defects in TLR-induced IL-10 production (31, 32). These parallel data suggest Btk might also be involved in regulating the activity or recruitment of additional components to the TLR signaling complex. The downstream mediators of Btk-dependent IL-10 induction are also not clear, but may involve the defective induction of NF-{kappa}B and AP-1 DNA binding activity, both of which are known to regulate IL-10 (33, 34). Further experimentation will be required to resolve possible mechanisms.

Btk is required for TLR-induced IL-10 production in multiple cell types including macrophages and dendritic cells (Fig. 3) (13). The differences in reliance on Btk for IL-10 induction among these cells types may reflect the different maturation or activation status of these populations. Our data are similar to previous data showing decreased IL-10 production by adherent xid peritoneal cells, compared with control cells (35). This resulted in an increased phagocytic index and free radical production that could be decreased by addition of exogenous IL-10. In contrast, another report demonstrated increased IL-10 from Mycobacterium tuberculosis-infected xid mice, compared with infected WT mice (36). This discrepancy could be explained in a number of ways. First, the IL-10 expression seen in M. tuberculosis-infected lung macrophages may have been induced by stimuli other than TLR ligands that are not affected by Btk deficiency. Second, it is possible that lung macrophages are physiologically distinct from the other populations we have examined and do not require Btk for IL-10 induction. Further comparisons among these cell types will distinguish these possibilities.

We have also observed that IL-6 production is increased in the absence of Btk (Figs. 6 and 7). This could be a result of increased MAPK activation (Fig. 4) or it could be indirect, with increased IL-6 levels due to the defective induction of IL-10 in Btk-deficient cells. In support of this, blocking IL-10 increases IL-6 from wild-type BMDM, and exogenous IL-10 can decrease IL-6 levels from both in vitro cultures and in the serum of LPS-injected Btk-deficient mice. The cellular source of IL-10 and IL-6 during endotoxemia is not entirely clear. However, given the tissue specificity of Btk, and the absence of IL-10-producing B cells in the absence of Btk (37), it is likely that macrophages contribute, at least partially, to the observed phenotype. This is also true of our analysis of differences in fecal bacterial species between WT and Btk-deficient mice. Our observations of increased IL-6 production in vivo and in vitro, concomitant with a decrease in IL-10, agree with studies showing that levels of IL-6 and other inflammatory cytokines are increased in IL-10-deficient mice (25, 26). IL-10 represses IL-6 production through Stat3- and/or NF-{kappa}B-dependent mechanisms (38, 39). In that respect, we have shown that TLR-induced NF-{kappa}B activity is decreased in Btk-deficient BMDM, and it was recently shown that Stat3 activation in Btk-deficient dendritic cells is decreased (Fig. 4) (13). Our results are different from recent reports of XLA patient cells where defects in IL-10 or IL-6 production were not observed (9, 40). This could be due to the types of cells being examined (PBMC vs bone-marrow derived, splenic or peritoneal macrophages), because we have seen some variations in the degree to which TLR responses in the three populations we have examined are affected by the loss of Btk (Figs. 3 and 6). It is also possible that there are different effects of distinct mutations of Btk.

The defects in APC (dendritic cell and macrophage) production of IL-10 could also have dramatic effects on the development of adaptive immunity in XLA patients. Decreased IL-10 production from Btk-deficient cells (Fig. 3 and Ref. 13), could play a role in altering Th1/Th2 balance, resulting in the previously described Th1 skewing in XLA patients (41, 42). Indeed, the ability of DC to produce IL-10 has recently been shown to be critical in determining IL-12 production from DC and whether T cells develop into Th1 or Th2 cells (41). Furthermore, the lack of IL-10 from Btk-deficient dendritic cells results in increased T cell activation, hyper-IgE levels and increased inflammation in vivo (13). Thus, by altering innate immune responses to TLR ligands, Btk may also indirectly regulate T cell effector functions.

One of the clinical phenotypes of patients diagnosed with XLA is recurring bacterial infections and increased occurrence of sepsis (43, 44, 45). Although the loss of mature B cells in XLA patients is certainly involved in the decreased clearance of bacteria, altered cytokine production, including decreased IL-10, and decreased NO production in xid or Btk-deficient mice could result in a defect in the innate immune response to bacteria (Figs. 1 and 3, and Refs. 3 and 8). This places Btk not only as a central regulator of B cell maturation and function but also in the development of innate and adaptive immune responses.


   Acknowledgments
 
We thank Dr. Cheong-Hee Chang for critical review of this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


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

1 This work was supported by U.S. Public Health Service Awards (to M.H.K.) from the National Institutes of Health. N.W.S. was a predoctoral fellow of the American Heart Association. V.T.T. was supported by Public Health Service Training Grant HL007910. Back

2 N.W.S. and V.T.T. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Mark H. Kaplan, Department of Pediatrics, and Microbiology and Immunology, H. B. Wells Center for Pediatric Research, Indiana University School of Medicine, 702 Barnhill Drive, RI 2600, Indianapolis, IN 46202. E-mail address: mkaplan2{at}iupui.edu Back

4 Abbreviations used in this paper: Btk, Bruton’s tyrosine kinase; XLA, X-linked agammaglobulinemia; BMDM, bone marrow-derived macrophage; SOCS, suppressor of cytokine signaling; MHC II, MHC class II; MKP-1, MAPK phosphatase-1; WT, wild type. Back

Received for publication March 31, 2006. Accepted for publication August 21, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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