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We previously identified that autoreactive B cells from BXD2 mice can be targeted by IL-17, leading to upregulation of the expression of regulators of G-protein signaling (Rgs) genes that facilitated the development of spontaneous germinal centers. Little is known about the signaling pathway used by IL-17 to upregulate RGS. In the current study, we found that IL-17 rapidly activates the canonical NF-κB signaling pathway and that BXD2 B cells exhibit higher basal and activated phosphorylated p65 levels than B6 or BXD2-Il17ra−/− B cells. Inhibition of p65 phosphorylation down-regulated RGS16 expression and abrogated the IL-17-induced chemotactic arrest of B cells in response to CXCL12. Knockdown of TNFR-associated factor 6 or NF-κB activator 1 in 70Z/3 pre-B cells led to decreased Rgs16 expression, indicating that both of these two genes are involved in IL-17-mediated activation of NF-κB signaling in B cells. These findings identify the signaling pathway regulated by IL-17 to contribute to the development of spontaneous germinal centers in autoimmune BXD2 mice.
Interleukin-17 has been shown to play a major role, especially in enhancing chemotaxis as related to the innate immune response. Chemokines upregulated by IL-17 include CXCL1, CXCL2 (1), CXCL5(2), CXCL8 (IL-8) (3), CXCL9, CXCL10 (4), CCL2, and CCL20 (5). IL-17 upregulates additional pro-inflammatory mediators, including IL-6, TNF-α, G-CSF, GM-CSF, C-reactive protein, matrix metalloproteases, and anti-microbial proteins, and is especially potent in expansion and recruitment of neutrophils (6).
The importance of IL-17 regulation on B cells has been shown recently to be associated with the development of spontaneous germinal centers (GCs) in autoimmune BXD2 mice (7). The BXD2 recombinant inbred mouse strain was generated by inbreeding the intercross progeny of C57BL/6J and DBA/2J mice for more than 20 generations (8, 9). These mice develop high titers of pathogenic autoantibodies and a spontaneous erosive arthritis that progresses as the mice age. BXD2 mice also exhibit spontaneous development of GCs in the spleen, and we have established that this formation of GCs plays a critical role in the production of pathogenic autoantibodies (7).
IL-17 signaling has been shown to be mediated through NF-κB activator 1 (ACT1) nd TNFR-associated factor 6 (TRAF6) (10, 11). The IL-17R family cytoplasmic tails have been shown to have homology with IL-1/TLR/IL-1R domain now referred to as the SEFIR. The SEFIR domain in IL-17RA is essential for IL-17 signaling (10, 12, 13). The IL-1/TLR domain contains a protein-protein interaction motif found in TLRs (IL-1Rs) and also is found in ACT1, an activator of NF-κB that had been linked to the B cell activation factor from the TNF family (BAFF) and CD40L signaling (11, 14). ACT1 also contains a TRAF6 binding motif and IL-17 activation of the NF-κB and MAPK pathways requires TRAF6 to induce IL-6 (10).
The chemokine receptors CXCR4 and CXCR5 and their respective ligands CXCL12 and CXCL13 facilitate recruitment of lymphocytes in lymphoid follicles to create GCs (15). The high levels of IL-17 that are characteristic of the BXD2 mice (7) are associated with the upregulation in the expression of the regulators of G-protein signaling (Rgs) 16 and Rgs13 genes in B cells. This increased expression of the Rgs16 and Rgs13 genes reduces the B cell chemotactic responses to the CXCL12 and CXCL13 chemokines, thereby promoting the retention of the B cells within the GCs and providing an optimal microenvironment for the generation of pathogenic autoantibodies (7). The associated reduction in the B cell chemotactic responses are absent in BXD2-Il17ra−/− mice, suggesting that one important IL-17/IL-17RA signaling function in B cells is to mediate the upregulation of Rgs genes.
It has been shown that NF-κB is involved in the mediation of IL-17 downstream signaling in various mammalian cell types, such as myofibroblasts (16), intestinal epithelia cells (17), and articular chondrocytes (18). IL-17 has been shown to use the NF-κB pathway to promote the survival and differentiation of B cells from human lupus patients (19). It is not known whether the IL-17-mediated RGS response also is dependent on the NF-κB signaling pathway. In this study, we show that in autoimmune B cells of BXD2 mice, the IL-17-mediated upregulation in RGS16 expression was associated with rapid phosphorylation and degradation of IκBα as well as phosphorylation of p65 (P-p65) and its translocation to the nucleus. Inhibition of phosphorylation of Ser276 on p65 with a specific membrane-permeable peptide inhibitor blocked IL-17-induced upregulation of RGS16 expression, and thus the IL-17-induced inhibition of chemotaxis of B cells in responses to CXCL12. In 70Z/3 pre-B cells, knockdown of Traf6 or TNFR-associated factor 3 interacting protein 2 (Traf3ip2), the gene encoding ACT1, by small interfering RNA (siRNA) also blocked the increase in P-p65 and Rgs16 expression levels by IL-17 signaling. Together, this finding extends our previously described model in which IL-17 can inhibit B cell chemotactic responses to CXCL12 (7) via its activation of the SEFIR and NF-κB signaling pathway, leading to upregulation of RGS16.
C57BL/6 and BXD2 recombinant inbred mice were obtained from The Jackson Laboratory (Bar Harbor, ME). B6-Il17ra−/− mice, obtained from Amgen (Thousand Oaks, CA), were backcrossed with the BXD2 mice for seven generations (7). All of the mice were cared for according to the Institutional Animal Care and Use Committee guidelines.
B cells were enriched from single-cell suspensions of spleens from 8- to 12-wk-old mice using magnetic anti-CD19 microbeads and an AutoMACS magnetic cell sorter (Miltenyi Biotech, Auburn, CA). The B cell preparations were 98% pure as confirmed by FACS.
The 70Z/3 cell line was obtained from the American Type Culture Collection (Manassas, VA). Abs against p65 (C22B4), P-p65 (93H1), transcription factor RelB (C1E4), IκBα (44D4), phospho-IκBα (14D4), phospho-p38 MAPK (28B10), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (197G2) rabbit mAb, phospho-C/EBPβ (Thr235) (Cell Signaling Technology, Beverly, MA), p50 (H119), p52 (K27), SP1 transcription factor (1C6), C/EBPβ (C-19), C/EBPδ (C-22) (Santa Cruz Biotechnology, Santa Cruz, CA), RGS16 (Prosci, Poway, CA), and GAPDH (R&D Systems, Minneapolis, MN) were used to detect their respective Ags.
RNA was isolated by TRIzol reagent (Invitrogen, Carlsbad, CA) and converted to cDNA by a First Strand cDNA Synthesis kit (Fermentas, Burlington, Ontario, Canada). Real-time PCR reactions were performed in triplicate using the SYBR Green PCR Master Mix (Bio-Rad, Hercules, CA) in an iQ™5 Multicolor Real-Time PCR Detection System (Bio-Rad) with the following primers: Il17ra, GGTGGCGGTTTTCCTTCAG (F), GTGGTTTGGGTCCCCATCA (R); Il17rc, GCGGTATTTGACTGTTTCG (F), TGCTCCTCAGAGACATCC (R); Rgs16, GGTACTTGCTACTCGCTTTTCC (F), CAGCCGCGTCTTGAACTCT (R); Rgs13, TGCAGAGATGAATCTAAGAGGCT (F), GGTTTCACATGCCATCCAGAAT (R); Rgs1, TTTTCTGCTAGCCCAAAGGA (F), TGGTTGGCAAGGAGTTTTTC (R); Traf6, GCGAGAGATTCTTTCCCTGAC (F), TGTATTAACCTGGCACTTCTGG (R); Traf3ip, TTTAGAAGGCACCCTGAAC (F), CCGTAAGTGTGAACCGATG (R); Gapdh, AGGTCGGTGTGAACGGATTTG (F), TGTAGACCATGTAGTTGAGGTCA (R).
The cytoplasmic and nuclear extracts from B cells were prepared with NE-PER® Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL). Equal amounts of protein extracts were electrophoresed on 8–10% SDS polyacrylamide gels, and transferred onto polyvinylidene difluoride membranes. Anti-rabbit HRP-conjugated Ab (Cell Signaling Technology) was used at a 1:3000 dilution, and anti-mouse HRP-conjugated Ab (Santa Cruz Biotechnology) was used at a 1:5000 dilution. HRP Abs were detected using the chemiluminescence reagent (Pierce). The scanned figures were visualized and quantified using ImageJ software.
Cytospin preparations of splenic B cells were fixed and permeabilized with BD Cytofix/Cytoperm™ solution (BD Biosciences, San Jose, CA), then sequentially stained with 1:100 dilution of P-p65 rabbit mAb (93H1, Cell Signaling Technology) and 1:200 dilution of FITC-conjugated donkey anti-rabbit Ab (BD Biosciences). The slides were viewed with a DM IRBE inverted Nomarski/epifluorescence microscope (Leica Microsystems, Deerfield, IL) outfitted with TCS NT laser confocal optics (Leica Microsystems).
Whole spleen cells from B6, BXD2 and BXD2-Il17ra−/− mice were stained with biotin-peanut agglutinin (PNA) (Vector Laboratory, Burlingame, CA), CD19-Alexa Fluor 700 (eBioscience, San Diego, CA) and IL17RA-PE (eBioscience). PNA+ cells were revealed by a secondary allophycocyanin-streptavidin conjugate. The purified splenic B cells from B6 and BXD2 mice were stained in the cold with biotin-PNA (Vector Laboratory) and PE-anti-Fas (Biolegend, San Diego, CA). The B cells were quickly warmed to 37°C and stimulated with IL-17 (30 ng/ml) for 5 min. The B cells then were cooled quickly, fixed and permeabilized with BD Cytofix/Cytoperm™ solution (BD Biosciences), then sequentially stained with 1:100 dilution of P-p65 rabbit mAb (93H1, Cell Signaling Technology) and 1:200 dilution of FITC-conjugated donkey anti-rabbit Ab (BD Biosciences). An isotype control rabbit IgG was used for gating of P-p65-positive cells. The stained whole spleen cells or purified B cells were analyzed on a LSRII FACS analyzer (BD Biosciences).
The NF-κB p65 (Ser276) inhibitory peptide (DRQIKIWFQNRRMKWK-KQLRRPSDRELSE) contains a protein transduction (PTD) sequence (DRQIKIWFQNRRMKWKK) derived from antennapedia and a Ser276 site (boldface) that is phosphorylated during NF-κB activation thereby blocking p65-Ser276 phosphorylation (IMGENEX, San Diego, CA). The control peptide consists of the PTD sequence (IMGENEX).
The purified spleen B cells from BXD2 mice or 70Z/3 pre-B cells were loaded into the upper well insert of a Transwell system (Costar, Cambridge, MA), and the chemokine CXCL12 (100 ng/ml) was added to the bottom chamber. After incubation for 2 h, the cells that remained in the inserts or that had migrated to the lower chamber were counted. The chemotaxis index was calculated by dividing the number of cells that migrated in response to the chemokine by the number of cells that migrated in the absence of chemokine (7).
siRNAs for Traf3ip2 (AAAGCAUUAGGUAAACUUGGGUCUG, Invitrogen), Traf6 (AAAGUUCACAAAUUUCACCACCUCC, Invitrogen) and control scrambled siRNAs (Invitrogen) were transfected into 70Z/3 cells at a final concentration of 100 nM using BLOCK-iT™ Transfection Kit (Invitrogen). The transfection efficiency in 70Z/3 is >80%, as determined by the BLOCK-iT™ Fluorescent Oligo. After transfection for 24 h, cells were stimulated with IL-17 (30 ng/ml) for 4h, and harvested for quantitative real-time PCR or for cell migration analysis as described earlier.
NF-κB is the best known mediator of IL-17-associated cellular responses (16–18). It is constitutively activated in many autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus and type1 diabetes (20). We found that the B cells from the spleens of autoimmune BXD2 mice exhibited significantly higher levels of basal P-p65 as compared with those of the B cells from the spleens of the parental B6 mice (Fig. 1A, Supplemental Fig 1), and that the levels of P-p65 in the B cells from the spleens of these mice were enhanced by stimulation with IL-17 (30 ng/ml, Fig. 1A, Supplemental Fig 1). The levels of P-p65 in the spleens of the BXD2-Il17ra−/− mice were markedly lower and were not responsive to IL-17 treatment (Fig. 1A, Supplemental Fig 1).
BXD2 mice display high levels of serum IL-17 and IL-17 expression in the splenic CD4 T cells (7), which may account for higher levels of IL-17 signaling. To determine whether B cells of B6 mice could exhibit an equivalent response to IL-17 at higher levels of IL-17 or at longer incubation times, B cells from BXD2 and B6 mice were analyzed after incubation with IL-17 (150 ng/ml) for 5 min or after 15 and 30 min with 30 ng/ml IL17 (Fig. 1B). Higher levels of IL-17 were not able to significantly elevate P-p65 in B cells of B6 mice compared with those of the control with no IL-17. There was a markedly lower P-p65 induction by IL-17 in B6 B cells at 15 and 30 min with 30 ng/ml IL17 compared with that of BXD2 B cells (Fig. 1B).
There is an ~5-fold increase in the Il17ra mRNA in total spleen B cells of BXD2 mice compared with those of B6 mice (7). B cells from BXD2 contain a dramatically increased population of Fas+PNA+ GC B cells compared with those of B cells from B6 mice (21). We therefore asked whether increased IL-17R and IL-17 stimulation of P-p65 in B cells from BXD2 mice was limited to GC B cells, or all of the B cells exhibited higher responses to IL-17. Responses of B cell subpopulations were analyzed by intracellular staining and FACS analysis of IL-17-stimulated B cells (Fig. 1C, 1D). There were increased P-p65 levels in response to IL-17 in both GC and non-GC B cells of BXD2 mice compared with those of the corresponding B cells from B6 mice. Consistent with this result, both non-GC (Fas−PNA−) and GC (Fas+PNA+) B cells exhibited higher levels of Il17ra expression (Fig. 1E). Increased expression of IL-17RA on the surfaces of B cells from BXD2 mice also was confirmed by FACS analysis (Fig. 1F). B cells from BXD2 mice also exhibited higher levels of Il-17rc (Supplemental Fig 2). There are also higher levels of Il17rc in BXD2-Il17ra−/− mice. Together, these results suggest that higher levels of Il17ra expression and higher IL-17-stimulated P-p65 are intrinsic properties of B cells from BXD2 mice.
The addition of IL-17 not only upregulated the levels of P-p65 in the B cells from the spleens of BXD2 mice, but also promoted its translocation to the nucleus. In the absence of IL-17 stimulation, the levels of P-p65 in the cytoplasm were considerably higher in BXD2 B cells than in B6 B cells (Fig. 2, upper panels). Within 5 minutes after addition of IL-17, the levels of P-p65 in the cytoplasm of BXD2 B cells were increased in most of these cells (Fig. 2A, middle panels), compared those of similarly treated B cells from the B6 control mice (Fig. 2B, middle panels). There was a dramatic reduction of the levels of P-p65 in the nuclei of B cells from BXD2 mice at 15 minutes after the addition of the IL-17, but high levels of P-p65 persisted in the cytoplasm (Fig. 2A, lower panels). The upregulation of P-p65 observed in almost all B cells of BXD2 mice further supports that this increased response to IL-17 is not due to an increased population of GC B cells compared with that of normal B6 mice.
Consistent with the results described above, within 5 min after addition of IL-17, the levels of P-p65 in total and cytoplasmic extracts of BXD2 B cells increased (Fig. 3A–3D). There was also a dramatic increase in the levels of P-p65 in nuclear extract (Fig. 3E, 3F). The levels of P-p65 in the B cell nuclear extract from BXD2 mice returned to baseline levels at 15 min after the addition of IL-17 (Fig. 3E, 3F), but high levels of P-p65 persisted in the cytoplasmic extract (Fig. 3C, 3D).
In most cell types, NF-κB is retained usually in the cytoplasm of the unstimulated cells by IκB family proteins. Upon stimulation, the IκB kinase (IKK) complex is activated, resulting in the phosphorylation of IκBs (22, 23). The phosphorylated IκBs are ubiquitinated and subsequently degraded, which will release transcription factor NF-κB (22, 23). In this study, we also found that IL-17 stimulation was associated with a significant increase in the levels of phosphorylated IκBα in the cytoplasm at the 5 min time point. Consistent with this, the total levels of IκBα in the cytoplasm decreased at the 5 min time point (Fig 3C, 3D). The levels of p50, a product of p105, were not elevated in the nuclear extracts of the BXD2 B cells with IL-17 stimulation (Fig. 3A, 3E).
Activation of p38 MAP kinase (MAPK), p44/42 MAPK (Erk1/2), and C/EBPβ also has been shown as a downstream signaling pathway of IL-17 (10, 12, 24). There was significantly increased induction of phospho-p38 MAPK and phospho-p44/42 MAPK (Erk1/2) in BXD2 B cells up to 30 min after IL-17 stimulation (Fig. 3A, 3B), but we did not find the increased induction of phospho-C/EBPβ (Fig. 3A, 3B) or increased nuclear levels of C/EBPβ or C/EBPδ (Supplemental Fig. 3). We also did not find any increase in the formation of p52, the noncanonical NF-κB subunit, or translocation of p52 or transcription factor RelB to the nucleus (Supplemental Fig. 3). Taken together, IL-17 strongly activates the canonical NF-κB signaling pathway in autoimmune BXD2 B cells.
We have found previously that after 4 h of culture with IL-17, there was equivalent upregulation of Rgs16 and Rgs13 in B cells from the spleens of BXD2 and B6 mice (7). Consistent with these previously published data, we found that treatment of the B cells from BXD2 mice with IL-17 resulted in a significant increase in the expression of Rgs16 mRNA, as determined by quantitative real-time PCR, within 2 h of IL-17 treatment (p < 0.01) (Fig. 4A). Similarly, treatment of B6 B cells with IL-17 enhanced the levels of Rgs16 mRNA, but the magnitude of the response was lower and the response was delayed in that it peaked at 4 h after addition of IL-17 (Fig. 4A, left panel). It has been reported that Rgs1 (25) and Rgs13 (26, 27) are highly expressed in GC B cells and some B cell lines. Although we found that Rgs13 expression was upregulated significantly in the IL-17-treated B cells (p < 0.05) (Fig. 4B), the relative magnitude of the response was lower than that observed for Rgs16. However, consistent with the results of Rgs16, the peak response occurred more rapidly in BXD2 B cells than in B6 B cells. Rgs1 was not upregulated in B cells after IL-17 stimulation (Fig. 4C), which is consistent with the previously published data (7).
To determine whether IL-17 signaling through the canonical NF-κB pathway is required for upregulation of RGS16 at the protein level, the BXD2 B cells were preincubated with a membrane-permeable inhibitory peptide that specifically blocks the phosphorylation of Ser276 of p65 during NF-κB activation (28). We found that pretreatment with this inhibitor blocked the IL-17-induced RGS16 in the B cells of BXD2 mice (Fig. 5A, 5B). To further determine whether the down-regulation of RGS16 induced by this peptide inhibitor affected the IL-17-induced migration toward the CXCL12 chemokine, the BXD2 B cells were pretreated with the NF-κB p65-Ser276 inhibitory peptide (inhibitor) or control peptide PTD prior to culture for 4 hours in the presence or absence of IL-17. Analysis of the migration toward CXCL12 using a chemotaxis chamber confirmed our previous finding that IL-17 treatment resulted in a significant reduction in the migration activity of BXD2 B cells in response to CXCL12 (p < 0.05) (Fig. 5C) (7). Furthermore, pre-incubation of the BXD2 B cells with the NF-κB p65-Ser276 inhibitory peptide prevented the inhibitory effect of IL-17 on the chemotactic response of the BXD2 splenic B cells to CXCL12 (p < 0.01) (Fig. 5C). Our data indicate that in the B cells of autoimmune BXD2 mice, IL-17 activates the canonical NF-κB signaling pathway, and that the activation of the canonical NF-κB signaling pathway is associated with the upregulation of RGS16 expression and the physiologically important response of these B cells to the CXCL12 chemokine.
TRAF6 and ACT1 have been reported to be involved in upstream signaling of IL-17 in many cell types (13, 29). Both of them are expressed at equivalent levels in B cells from BXD2 mice compared with those in B6 and BXD2 Il17ra−/− B cells (Supplemental Fig 4). To determine whether TRAF6 or ACT1 was required for IL-17R signaling in B cells, we used siRNA for Traf6 or Traf3ip2, the gene encoding ACT1, to knock down these genes in the 70Z/3 pre-B cell line. Comparison between the 70Z/3 pre-B cell line and B cells from the spleens of BXD2 mice indicated that 70Z/3 cells would be an appropriate model for BXD2 B cells. 70Z/3 cells exhibited comparable expression levels of Il17ra (data not shown).
Treatment of 70Z/3 cells with IL-17 resulted in increased levels of P-p65 from 5–60 min after IL-17 stimulation, although the peak time of induction differed somewhat from that for BXD2 B cells (Fig. 6A, 6B). Consistent with the results from BXD2 B cells, treatment of 70Z/3 B cells with IL-17 also led to significantly increased expression of RGS16 from 1–10 h following stimulation (Fig. 6C, 6D). We also confirmed that upregulation of RGS16 in the 70Z/3 cells was dependent upon P-p65 as determined by pretreatment with the NF-κB p65-Ser276 inhibitory peptide (Fig. 6E, 6F). These results are consistent with the report by Li et al. (30) that after LPS stimulation, the expression of Rgs16 is dependent on the canonical NF-κB pathway in the 70Z/3 pre-B cell line.
Treatment of 70Z/3 cells with siRNA for Traf6 or Traf3ip2 resulted in ~67% and 66% reductions in the respective mRNA levels at 24 h after transfection compared with those for the scrambled siRNA results, respectively (Fig. 7A). Treatment of 70Z/3 cells with siRNA for Traf6 or Traf3ip2 also resulted in a significant decrease in the P-p65 activation level (Fig. 7B, 7C).
On treatment of the 70Z/3 cells with IL-17 (30 ng/ml) for 4 h after siRNA transfection, we found that pre-treatment with siRNA for Traf6 resulted in ~57% reduction in expression of Rgs16 (p < 0.05) and that pre-treatment with siRNA for Traf3ip2 resulted in an ~60% reduction in expression of Rgs16 (p < 0.05) (Fig. 7D), compared with that for the scrambled siRNA. That is to say, a block in expression of either of these two genes resulted in a dramatic reduction in the IL-17-stimulated expression of Rgs16. Chemotaxis of B cells in response to CXCL12 was reduced significantly in 70Z/3 cells pretreated with scrambled siRNA after culture with IL-17. This decreased chemotaxis was strongly reversed by treatment with Traf6 siRNA (Fig. 7E) and was restored partially by treatment with Traf3ip2 siRNA (Fig. 7E). These results indicate that IL-17/IL-17RA signaling in B cells requires both TRAF6 and ACT1 and signals through the canonical NF-κB pathway to upregulate RGS16 expression, which inhibits chemotaxis in response to CXCL12.
An understanding of the signaling pathways associated with stimulation of IL-17R in B cells should shed light on the circumstances that promote B cell retention in the GCs and the subsequent development of pathogenic autoantibodies. We previously showed that IL-17 (20 ng/ml) stimulation of purified B cells from B6 or BXD2 mice resulted in ~10-fold upregulation of Rgs16 and a lesser increase in Rgs13 levels at 4 h after stimulation. There was equivalent upregulation of Rgs16 levels in BXD2 compared with those in B6 mice (7). In the present experiment, we found that Rgs16 is the major RGS that is upregulated in BXD2 B cells compared with those in B6 B cells. We also found that the kinetics of upregulation Rgs mRNA is faster in BXD2 B cells compared with that in B6 B cells. Upregulation of Rgs16 levels in BXD2 B cells peaks at 2 h and is higher than the upregulation of Rgs16 levels in B6 B cells, which peaks at 4 h. IL-17-induced upregulation of Rgs13 also peaked at 2 h in BXD2 B cells and 4 h in B6 B cells. The present study therefore clearly showed that there was more rapid and greater upregulation of both Rgs13 and Rgs16 levels in BXD2 compared with those in B6 B cells.
A question is whether IL-17 acts on a small population of B cells, such as GC B cells in BXD2 mice, or on a larger population of B cells in B6 and BXD2 mice. The present experiments show that Il17ra expression is greatly increased on all B cells of BXD2 mice compared with that of B6 B cells. The level of stimulation indicated by P-p65 is upregulated significantly on all B cells of BXD2 mice compared with that off B cells from B6 mice. There is no significant greater increase in P-p65 signaling in GC B cells in BXD2 mice compared with that of non-GC B cells. These results indicate that IL-17RA expression and signaling, although higher on B cells of BXD2 mice, are features of all of the B cells and are not restricted to a subpopulation of B cells. However, because the majority of IL-17 producing CD4 T cells locate in the vicinity of a GC (7), this may be one factor that promotes the migration arrest of B cells in the GC area in the spleen of BXD2 mice in vivo.
IL-17R signaling occurs through homodimers or heterodimers of IL-17A and IL-17F, which interact through heterodimers of IL-17RA and IL-17RC, usually at a 2:1 ratio. Downstream signaling pathways have been characterized to use the NF-κB, MAPK and C/EBP pathways. IL-17 was shown to induce NF-κB signaling in mouse fibroblasts and human macrophages (31, 32). IL-17 also was shown to activate the NF-κB subunits p50 and p65 in intestinal epithelial cells (17). This activation appears to require TRAF6 and IκBα kinase, IKKα. IL-17 induced signal transduction resulted in regulation of three different families of MAPKs in IEC-6 cells (17). These can be linked to the NF-κB activation pathways. TRAF proteins also can activate JNK and NF-κB. Therefore, activation of ERK, JNK/stress-activated protein kinase, and p38 MAPKs by IL-17 in IEC-6 cells may be linked to the NF-κB activation pathway. Because the NF-κB pathway appeared to be most critical, it was studied in B cells in the current study.
Our results show that there is rapid upregulation of the P-p65 canonical pathway in B cells of B6 and BXD2 mice. Upregulation of P-p65 is increased greatly in B cells of BXD2 mice compared with those of B6 mice, which may be in part due to increased expression of Il17ra on the surfaces of both GC and non-GC B cells of BXD2 mice. Cytoplasmic and nuclear staining also demonstrates that almost all off the B cells in BXD2 mice exhibit upregulation of P-p65 first in the cytoplasm and then in the nucleus at 5 min after IL-17 stimulation. There was nearly complete downregulation of P-p65 and NF-κB in the nuclei of BXD2 B cells at 15 min, but P-p65 levels remained high in the cytoplasm and in cytoplasmic extracts from BXD2 mice. B cells from B6 mice exhibited a lesser degree of upregulation of P-p65 in both the cytoplasm and the nucleus in response to IL-17 stimulation. Also, at 5 min after IL-17 stimulation, P-IκBα levels increased in the cytoplasm. Activation of P-p65 was associated with induction of p38 MAPK and p44/42 MAPK (Erk1/2) as has been reported previously in other cells (17), but C/EBPβ was not upregulated in B cells. Similar to cytoplasmic levels of P-p65, which persists for 1 h after IL-17 stimulation, P-p38 MAPK and P-p44/42 MAPK (Erk1/2) remained high in total lysate for 30–60 min after IL-17 stimulation. However, levels of P-p65 in the nucleus were rapidly regulated at 5 min and were down-regulated at 15 min as confirmed by confocal microscopy and analysis of nuclear extract. These results suggest that NF-κB target genes are quickly modulated after IL-17 activation in B cells of BXD2 mice.
NF-κB is essential for upregulation of RGS16. B cells incubated with a specific decoy peptide inhibitor of P-p65 that contained a PTD and could permeate into primary B cells of BXD2 mice resulted in nearly complete block of upregulation of RGS16 levels after IL-17 treatment. Treatment of primary B cells from BXD2 mice with IL-17 exhibited a decreased response to CXCL12. Treatment of primary B cells with the P-p65 decoy peptide was highly effective in blocking this IL-17-induced inhibition of chemotaxis to CXCL12. Together, these results indicate that IL-17 specifically signals through the classical NF-κB P-p65 pathway to induce expression of RGS16.
To determine whether TRAF6 and ACT1 are required for IL-17R signaling in B cells, we used a 70Z/3 pre-B cell line. This cell line expresses IL-17R and also signals upregulation of RGS16 to the canonical NF-κB pathway, because pretreatment with the P-p65 peptide inhibitor blocked IL-17 upregulation of RGS16. 70Z/3 B cells transfected with siRNA for Traf6 or Traf3ip2 exhibited significant inhibition of P-p65 signaling. siRNA for Traf6 or Traf3ip2 also significantly blocked IL-17 induction of RGS16. Correspondingly, there was normalization of the chemotactic response to CXCL12 after IL-17 treatment in B cells transfected with siRNA for either Traf6 or Traf3ip2. These results indicate that IL-17 signaling in B cells is similar to mechanisms in previously described in non-B cells and require TRAF6 and ACT1 to signal NF-κB (13, 29).
Finally, the results show that IL-17 induces a relatively larger increase in Rgs16 level (10 fold) compared with that for Rgs13 (2- to 3- fold). This is consistent with previous findings that NF-κB is a known transcription factor for RGS16 (30). However, RGS13 levels are constitutively higher in B cells and IL-17 leads to a larger absolute increase in the RGS13 level compared with that in untreated B cells. We propose that although RGS13 and RGS16 both regulate B cell chemotaxis and GC formation in response to IL-17, RGS16 upregulation, compared with that for RGS13, may provide a more finely tuned response to IL-17 to regulate the spontaneous GC response in BXD2 mice.
This work was supported by grants from the American College of Rheumatology Research and Education Foundation Within Our Reach: Finding a Cure for Rheumatoid Arthritis campaign (to J.D.M.), Alliance for Lupus Research Target Identification in Lupus program (to J.D.M.), Veterans Administration Merit Review (1I01BX000600–01 to J.D.M.), Daiichi-Sankyo (to J.D.M.), National Institutes of Health (1AI 071110–01A1 and ARRA 3RO1AI71110–02S1 to J.D.M. and RR-020136 and AI 083539Z to L.E.S.), Arthritis Foundation, and Lupus Research Institute (to H.-C.H.). Imaging of the translocation of NF-κB signaling was carried out by the Molecular Pathology and Human Cell/Tissue Core, Mucosal HIV and Immunobiology Center, University of Alabama at Birmingham, which is supported by Grant DK 064400 (to L.E.S.).
We thank Enid Keyser of the Arthritis and Musculoskeletal Disease Center Analytic and Preparative Cytometry Facility for operating theFACS instrument. We thank Dr. Fiona Hunterfor expert review of the manuscript and Carol Humber for excellentsecretarial assistance. We thank Dr. Sarah L. Gaffen for critical discussion and technical assistance for the analysis of C/EBP by western blot. Il17ra−/− mouse is a generous gift from Amgen Inc.
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