Crucial Role of Linear Ubiquitin Chain Assembly Complex–Mediated Inhibition of Programmed Cell Death in TLR4-Mediated B Cell Responses and B1b Cell Development

Toll-like receptors recognize specific patterns of microbial components known as pathogen-associated molecular patterns (1). Upon recognition of pathogen-associated molecular patterns, TLRs activate multiple signaling cascades, including the NF-κB and MAP kinase pathways, culminating in expression of a variety of proinflammatory molecules that orchestrate innate immune responses to pathogens (1). TLRs also play important roles in initiating acquired immunity because TLR stimulation induces maturation of dendritic cells, thereby increasing expression of costimulatory molecules and MHC–antigen complexes (2). In addition, TLR stimulation directly activates B lymphocytes to induce proliferation and differentiation into Ab-producing cells (3, 4).

TLR signaling must be tightly regulated because uncontrolled activation may result in life-threatening diseases, such as chronic inflammation and autoimmune diseases (5). Recent studies show that the ubiquitin system plays critical roles in regulating TLR signaling (6, 7). The ubiquitin system is a posttranslational modification system that controls protein function (8). In many cases, the ubiquitin molecule is conjugated to target proteins to form polyubiquitin chains. These polyubiquitin chains are synthesized by serial conjugation of the C-terminal glycine residue in one ubiquitin molecule to one of seven lysine residues in another ubiquitin molecule (9). We identified a novel ubiquitin ligase complex that generates N-terminally linked linear polyubiquitin chains, which we named the linear ubiquitin chain assembly complex (LUBAC) (10). The LUBAC comprises one catalytic subunit, HOIP, and two accessary subunits, HOIL-1L and SHARPIN (11). We and others showed that LUBAC-catalyzed linear ubiquitination is involved in activation of the canonical NF-κB pathway and in inhibition of cell death in response to TNF-α stimulation (12–15). When we analyzed the roles of LUBAC in B cells, we found that it plays important roles in Ab responses to thymus-dependent (TD) and type II thymus-independent (TI-II) Ags by inducing canonical NF-κB and ERK activation via CD40 and TACI, respectively, and that it is essential for B1 cell development (16). In addition, we showed that LUBAC is involved in TLR-stimulated activation of the canonical NF-κB and ERK pathways (16). However, the precise roles of LUBAC in TLR-mediated B cell responses remain unclear.

In this study, we investigated the function of LUBAC in TLR-mediated B cell responses and found that LUBAC plays critical roles in B cell responses induced by TLR4 stimulation through the inhibition of programmed cell death. In addition, we discovered that LUBAC-mediated inhibition of necroptosis is crucial for B1b cell development and/or maintenance.

HOIP conditional Δlinear mice (HOIP^FL mice), in which LUBAC E3 activity can be deleted in a Cre recombinase–dependent manner, were reported previously (16). B cell–specific Cre recombinase transgenic mice (mb1-cre mice) were kindly provided by Dr. M. Reth (Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany) (17). TRIF-deficient mice were obtained from Oriental BioService (Kyoto, Japan) (18). All mice were bred and maintained under specific pathogen-free conditions. All animal protocols were approved by Kyoto University.

Plasmids expressing hCas9 and single-guide RNA (sgRNA) targeted to the mouse RIP3 (pX330–mRIP3-1 and pX330–mRIP3-2) gene were prepared by ligating oligonucleotides into the BbsI site of the pX330 plasmids (19). The sgRNA sequences were as follows: 5′-AACCCGAGTGCCCTCGGCCC-3′ (mRIP3-1) and 5′-AGGTCCCGGTGCAGGAGCGG-3′ (mRIP3-2). Fertilized oocytes obtained from breeding HOIP^fl/fl males with HOIP^fl/fl females were microinjected with 1–2 pl of pX330–mRIP3-1 and pX330–mRIP3-2 (5 ng/μl). The genomic region around sgRNA targets was amplified by PCR using primer set mRIP3-f1 (5′-AGAGTGTGGT CACGAGCCTGAGTG-3′) and mRIP3-r1 (5′-TCATGTGTGTAGTCTTGTAT TCAG-3′) and then sequenced. Mice harboring a 7-bp deletion (mRIP3-1) or 5-bp deletion (mRIP3-2) were selected for further analyses.

Single-cell suspensions prepared from the spleen and peritoneal cavity were stained with fluorochrome-conjugated Abs. The (4-hydroxy-3-nitrophenyl)acetyl (NP)–binding B cells were detected using (4-hydroxy-5-indo-3-nitrophenyl)acetyl (NIP)-BSA-PE. All samples were acquired using a FACSCanto II (BD Biosciences), and the results were analyzed using FlowJo software (Tree Star).

Mice were injected i.p. with 20 μg of NP (0.15)–LPS (Biosearch Technologies) and bled via the tail vein both before and after immunization. Serum levels of anti-NP Abs were determined by ELISA.

Splenic B cells were negatively selected using anti-CD43 and anti-CD5 microbeads and a MACS Separation Column (Miltenyi Biotec). Purity was around 95%. Purified splenic B cells were cultured in DMEM supplemented with 10% FCS, 50 μM 2-ME, 10 mM HEPES-KOH (pH 7.4), 100 U/ml penicillin, and 100 μg/ml streptomycin, and then stimulated with LPS (Sigma-Aldrich) or CpG DNA (InvivoGen) at the indicated concentrations for the indicated periods. For CFSE labeling, cells were incubated for 10 min at 37°C in RPMI 1640 medium containing 5 μM CFSE, followed by washing with RPMI 1640 medium containing 10% FCS. Labeled cells were cultured and exposed to various stimuli. After 48 or 72 h, cell proliferation was measured by flow cytometry. To analyze cell viability, splenic B cells were cultured with 20 μg/ml LPS, 10 μg/ml lipid A, or 100 nM CpG DNA for 24, 48, or 72 h or with 10 μg/ml polyinosinic:polycytidylic acid [poly(I:C)] for 4, 8, 12, or 16 h. Dead cells were labeled with TO-PRO-3 (Life Technologies), and the proportion of live cells was expressed as the percentage of TO-PRO-3–negative cells. To analyze Ab production in vitro, splenic B cells were cultured with 20 μg/ml LPS or 100 nM CpG DNA for 7 d. Ig concentrations in culture supernatants were measured by ELISA.

Ninety-six–well ELISA plates (Thermo Fisher Scientific) were coated with 10 μg/ml NP-BSA (Biosearch Technologies) to measure anti-NP Abs and 1 μg/ml anti-mouse IgM, IgG2b, IgG2c, and IgG3 Abs (SouthernBiotech) to measure total Igs and then were blocked with 1% BSA in 1× PBS. Appropriately diluted serum or culture supernatants were incubated at room temperature for 60 min or at 4°C overnight, and then HRP-conjugated anti-mouse IgM, IgG2b, IgG2c, or IgG3 Abs (SouthernBiotech) were added to the wells. BD OptEIA (BD Biosciences) was used as the substrate, and absorbance at 450 nm was measured using a microplate reader (Molecular Devices).

Whole cell lysates were prepared as previously described, separated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes (Merck Millipore) (16).

Total RNA was isolated using an RNeasy Mini Kit (Qiagen). DNase-treated RNA (20 ng) was reverse transcribed to cDNA using the ReverTra Ace qPCR RT kit (Toyobo). Real-time PCR was performed with Power SYBR Green PCR Master Mix (Applied Biosystems) in an ABI ViiA7 Real-Time PCR system (Applied Biosystems). The primers for each gene were as follows: Nfkbia, forward 5′-GCCAGGAATTGCTGAGGCACTT-3′, reverse 5′-GTCTGCGTCAAGACTGCTACAC-3′; Bcl2l1, forward 5′-GGTGAGTCGGATTGCAAGTT-3′, reverse 5′-GCTGCATTGTTCCCGTAGAG-3′; Bcl2a1a, forward 5′-TCCACAAGAGCAGATTGCCCTG-3′, reverse 5′-GCCAGCCAGATTTGGGTTCAAAC-3′; Cflar, forward 5′-GCTCTACAGAGTGAGGCGGTTT-3′, reverse 5′-CACCAATCTCCATCAGCAGGAC-3′; Egr1, forward 5′-AGCGAACAACCCTATGAGCACC-3′, reverse 5′-ATGGGAGGCAACCGAGTCGTTT-3′; Egr2, forward 5′-CCTTTGACCAGATGAACGGAGTG-3′, reverse 5′-CTGGTTTCTAGGTGCAGAGATGG-3′; β-actin, forward 5′-CATTGCTGACAGGATGCAGAAGG-3′, reverse 5′-TGCTGGAAGGTGGACAGTGAGG-3′. All gene expression data were normalized to that of β-actin.

The following Abs were used for flow cytometry analysis: FITC–anti-IgM (II/41), PE–anti-CD138 (281-2), PE–anti-CD5 (53-7.3), BV421–anti-CD5 (53-7.3), and biotin–anti-IgG3 (R40-82) (all from BD Biosciences); PE–anti-CD23 (B3B4), APC–anti-AA4.1 (AA4.1), APC–anti-CD19 (eBio 1D3), and APC streptavidin (all from eBioscience); and PerCP–anti-B220 (RA3-6B2) and PE/Cy7-CD19 (6D5) (BioLegend). NIP-BSA-PE was used to detect NP-binding cells. The following Abs were used for immunoblot analysis: phospho-IκBα, cleaved caspase-8, caspase-3, and RIP1 (Cell Signaling Technology); RelA, c-Rel, IκBα, and tubulin (Santa Cruz Biotechnology); β-actin (Sigma-Aldrich); lamin B1 (Abcam), and caspase-8 (Enzo Life Sciences); and RIP3 (Novus Biologicals) and H2B (Merck Millipore). The following reagents were used for stimulations: LPS from Escherichia coli 055:B5 (Sigma-Aldrich); lipid A, diphosphoryl from Salmonella enterica Re 595 (Re mutant) (Sigma-Aldrich); poly(I:C) high molecular weight (InvivoGen); and class B CpG oligonucleotide ODN1826 (InvivoGen).

Cells were stimulated with 20 μg/ml LPS or 100 nM CpG DNA for 24 h and then pulsed with 10 μM EdU for an additional 16 h. Cells were then stained with the Click-iT Plus Flow Cytometry Assay kit (Thermo Fisher Scientific) and FxCycle Violet (Thermo Fisher Scientific) and analyzed by flow cytometry.

Statistical analyses were performed using a two-tailed unpaired Student t test. A p value < 0.05 was deemed significant. All analyses were performed using StatPlus (AnalystSoft).

We previously showed that loss of the ligase activity of LUBAC impairs Ab responses of B cells to TD and TI-II Ags by suppressing CD40- and TACI-mediated activation of the canonical NF-κB and ERK pathways, respectively (16). We also reported that loss of LUBAC ligase activity attenuated TLR-stimulated activation of the canonical NF-κB and ERK pathways in B cells, albeit less prominently than CD40-mediated activation (16). However, the role of LUBAC in TLR-induced B cell responses in vivo is unclear. Because TLR-mediated signaling is important for Ab responses to TI-I Ags, we dissected the Ab response to TI-I Ags in B-HOIP^Δlinear mice in which LUBAC ligase activity is deleted specifically from B cells. Immunization of B-HOIP^Δlinear mice with NP-LPS, in which an NP hapten is conjugated to a TLR4 ligand (LPS), led to a severe reduction in production of NP-specific IgM, IgG2b, IgG2c, and IgG3 Abs (Fig. 1A). NP-binding IgM-positive B cells expanded in control mice 4 d after immunization, whereas no expansion of NP-binding IgM-positive B cells was observed, although the number of NP-binding IgM-positive B cells was comparable between control and B-HOIP^Δlinear mice immunized with PBS (Fig. 1B). In addition, loss of LUBAC activity attenuated generation of IgM⁺CD138⁺ plasmablasts and IgG3⁺ B cells 4 d after immunization (Fig. 1C, 1D). These results demonstrate that the defective Ab response to NP-LPS in B-HOIP^Δlinear mice is due to a failure of B cells to expand and differentiate into plasma cells and indicate that LUBAC plays important roles in TLR4-mediated B cell responses in vivo.

Because Ag-specific B cells in B-HOIP^Δlinear mice failed to expand upon immunization with a TI-I Ag, we examined the involvement of LUBAC-mediated linear ubiquitination in TLR-mediated cell proliferation using splenic B cells from B-HOIP^Δlinear mice. As expected from the results shown above, LPS-induced proliferation of B cells was severely impaired by the lack of ubiquitin ligase activity (Fig. 2A). However, to our surprise, proliferation of HOIP^Δlinear B cells was only marginally attenuated after stimulation with TLR9 for 48 or 72 h (when compared with those from littermate controls) (Fig. 2A). Next, we examined induction of DNA synthesis 40 h after stimulation with LPS or CpG DNA by analyzing incorporation of EdU into genomic DNA. Loss of LUBAC activity led to a marginal decrease in EdU incorporation by B cells stimulated with CpG DNA (Fig. 2B); however, EdU incorporation by LPS-stimulated HOIP^Δlinear B cells was severely impaired. Moreover, LPS led to a specific increase in the number of HOIP^Δlinear B cells with reduced DNA content, suggesting that LPS stimulation induced massive HOIP^Δlinear B cell death (Fig. 2B). Next, we examined the effects of TLR4 or TLR9 stimulation on survival of B cells and found that TLR4 stimulation caused a marked reduction in the number of HOIP^Δlinear B cells, whereas TLR9 stimulation increased the survival of HOIP^Δlinear B cells (levels were comparable with those of control B cells) (Fig. 2C). Because LPS could also activate TLR2, we stimulated HOIP^Δlinear B cells with lipid A, a TLR4-specific ligand. Lipid A stimulation induced death of HOIP^Δlinear B cells, as was the case with LPS stimulation (Fig. 2C), which confirmed that TLR4 induced cell death of HOIP^Δlinear B cells. These results indicate that stimulation with TLR4 and TLR9 have a differential effect on the proliferation and survival of HOIP^Δlinear B cells. We also investigated whether deletion of LUBAC had effects on differentiation of B cells into Ab-secreting cells via in vitro LPS and CpG DNA stimulation. Because Ab production by LPS stimulation was more severely impaired than that by CpG DNA stimulation, loss of LUBAC activity also affected plasma cell differentiation induced by LPS or CpG DNA differentially (Fig. 2D). We previously reported that TLR4- and TLR9-stimulated canonical NF-κB and ERK activation was similarly impaired in HOIP^Δlinear B cells (16). Therefore, we re-evaluated NF-κB and ERK activation induced by TLR4 and TLR9 signaling in HOIP^Δlinear B cells. TLR4-mediated nuclear translocation of RelA and c-Rel, phosphorylation and degradation of IκBα, and induction of mRNAs encoding NF-κB target genes (IκBα, Bcl-xL, and A1a) were reduced to levels comparable to those in B cells induced by TLR9 stimulation by the loss of linear ubiquitination activity (Fig. 3A–C). This was also the case with respect to induction of mRNAs encoding ERK targets Egr1 and Egr2 (Fig. 3D), which confirmed our previous observation that loss of LUBAC ligase activity affects TLR4- and TLR9-induced NF-κB and ERK activation almost equally (16). Collectively, these results suggest that ablation of LUBAC ligase activity attenuates immediate responses, such as canonical NF-κB and ERK activation elicited by both TLR4 and TLR9, to a similar extent but that responses over longer time periods, including cell proliferation and the differentiation into Ab-secreting cells, were much more heavily attenuated after TLR4 than after TLR9 stimulation. Also, LUBAC ligase activity is required to inhibit TLR4-mediated B cell death.

TLR4 transmits signals via not only MyD88 but also via TRIF, whereas TLR9 transduces signals only via MyD88. Because a recent report showed that LUBAC regulates cell death induced by TLR3, which uses only TRIF (1, 20), we tried to examine whether LUBAC controls TLR3-TRIF–mediated cell death by stimulating HOIP^Δlinear B cells with poly(I:C), a ligand of TLR3. We could confirm that poly(I:C) stimulation induced death of HOIP^Δlinear B cells more extensively than control B cells in a TRIF-dependent manner (Fig. 4A). We then examined involvement of TRIF in cell death of HOIP^Δlinear B cells induced by TLR4 ligands. As shown in Fig. 4A, ablation of TRIF in HOIP^Δlinear B cells completely suppressed not only LPS– but also lipid A–mediated reduction in the number of HOIP^Δlinear B cells (Fig. 4A). CFSE labeling experiments revealed that loss of TRIF restored LPS-induced cell proliferation of HOIP^Δlinear B cells only partially; this was also the case for LPS-induced DNA synthesis (Fig. 4B, 4C). To better understand the mechanism underlying partial recovery of LPS-induced proliferation of HOIP^Δlinear B cells induced by loss of TRIF, we examined the effect of TRIF ablation on TLR4-induced cell survival, proliferation, and DNA synthesis in B cells (Supplemental Fig. 1). Loss of TRIF did not affect LPS-induced survival of B cells. However, LPS-induced proliferation and DNA synthesis were partially suppressed by TRIF deficiency, and suppression was further potentiated by loss of LUBAC ligase activity, which underscored the partial recovery of LPS-induced proliferation of HOIP^Δlinear B cells after deletion of TRIF. Ablation of TRIF did not restore TLR4-induced plasma cell differentiation, as Ab responses to NP-LPS were not recovered by deletion of TRIF (Fig. 4D). This might be because TRIF in itself is required for Ab responses to NP-LPS (Fig. 4D), as in the case of LPS-induced proliferation. Collectively, these data demonstrate that TRIF is responsible for LPS-induced death of HOIP^Δlinear B cells, that LUBAC is necessary to inhibit TRIF-mediated cell death, and that both LUBAC and TRIF are required to induce TLR4-mediated proliferation and Ab responses to NP-LPS.

Caspases play critical roles in inducing programmed cell death (21). Caspase-8 forms a complex with TRIF and is involved in TRIF-mediated signaling pathways (22, 23). We next asked whether caspases in HOIP^Δlinear B cells are activated by LPS stimulation. LPS induced processing of caspase-8 and caspase-3 to their active forms in HOIP^Δlinear B cells but not in control B cells (Fig. 5A). We also detected the cleaved form of RIP1, which is a substrate of caspase-8, in LPS-stimulated HOIP^Δlinear B cells (Fig. 5A). These results suggest that TLR4 stimulation activates caspase-8 in B cells when LUBAC activity is lost. We also examined involvement of TRIF in caspase activation by deleting TRIF from HOIP^Δlinear B cells. We found that loss of TRIF suppressed TLR4-mediated activation of caspase-8 and caspase-3 (Fig. 5B), indicating that LUBAC plays a crucial role in inhibiting TRIF-mediated activation of caspase-8 upon TLR4 signaling. NF-κB suppresses caspase-8 activity by inducing c-FLIP, a negative regulator of caspase-8 (24). Therefore, we analyzed LPS-mediated induction of c-FLIP mRNA and found that induction of c-FLIP mRNA was reduced in HOIP^Δlinear B cells (Supplemental Fig. 2A). Cycloheximide, a translational inhibitor, can negate the NF-κB pathway because it suppresses de novo synthesis of proteins, including NF-κB–inducible proteins. Because LUBAC is involved in suppression of cell death in an NF-κB–independent fashion (14, 25), we evaluated LPS-induced cell death in HOIP^Δlinear B cells in the presence of cycloheximide. The number of HOIP^Δlinear B cells fell more rapidly than that of control B cells in response to LPS stimulation, even when protein synthesis was suppressed (Supplemental Fig. 2B). These results suggest that LUBAC inhibits LPS- and TRIF-mediated cell death of B cells through the new synthesis of NF-κB target genes and the direct regulation of the mediators of cell death.

We next examined whether LPS-induced cell death of HOIP^Δlinear B cells can be prevented by inhibiting caspases. To this end, we added a pan-caspase inhibitor, Z-VAD-FMK, to the cell culture and found that inhibiting caspases only slightly rescued survival of LPS-stimulated HOIP^Δlinear B cells (Fig. 6A). Because inhibition of caspase-8 in the cell-extrinsic apoptosis pathway results in necroptosis (rather than apoptosis) via formation of necrosomes, which comprise RIP1 and RIP3 (26), it seemed plausible that Z-VAD-FMK failed to prevent HOIP^Δlinear B cells from LPS-induced death because of the induction of necroptosis. Therefore, we asked whether inhibiting both apoptosis and necroptosis effectively restores survival of LPS-stimulated HOIP^Δlinear B cells. Suppressing necroptosis can be achieved by deleting RIP3 (26); however, it is almost impossible to obtain HOIP and RIP3 double-mutant mice simply by crossing HOIP^FL mice with RIP3-deficient mice because both the HOIP and RIP3 genes reside on the same chromosome, and the distance between the two genes is only around 200 kbp. Therefore, we introduced a RIP3 null allele into HOIP^FL mice using the CRISPR/Cas9 system and obtained two independent lines of RIP3-deficient HOIP^FL mice (Supplemental Fig. 3). We crossed these HOIP^FL RIP3^−/− mice with mb1-cre mice to obtain HOIP^Δlinear B cells lacking RIP3. LPS-induced cell death of HOIP^Δlinear B cells was not suppressed by ablating RIP3 alone (Fig. 6B), indicating that necroptosis is not a major cause of cell death in LPS-stimulated HOIP^Δlinear B cells. However, TLR4-induced death of HOIP^Δlinear B cells lacking RIP3 was strongly inhibited by treatment with Z-VAD-FMK (Fig. 6B). In addition, TLR4-mediated cell proliferation and DNA synthesis was restored, although not completely (Fig. 6B, 6C). Loss of RIP3 failed to restore Ab responses to NP-LPS in mice lacking LUBAC activity in B cells, although before immunization the serum concentration of IgM Ab specific for NP was significantly higher in B-HOIP^Δlinear/RIP3 knockout (KO) mice than in B-HOIP^Δlinear mice (Fig. 6E). When stimulated with LPS in vitro, we have observed that Z-VAD-FMK considerably restored Ab production by HOIP^Δlinear/RIP3 KO B cells, indicating that plasma cell differentiation was partially recovered by inhibition of both apoptosis and necroptosis (Fig. 6F). These results clearly show that HOIP^Δlinear B cells die via caspase-mediated apoptosis in response to LPS and that LUBAC plays a crucial role in inhibiting apoptosis during TLR4-mediated B cell responses.

We previously reported that development of follicular and marginal zone B cells in B-HOIP^Δlinear mice is normal, whereas the number of B1 cells (both B1a and B1b cells) is severely reduced (16). However, the exact roles of LUBAC in B1 cell development are unclear. Because the anti–cell death function of LUBAC has not been evaluated in B cells, we examined development and/or maintenance of B cells in B-HOIP^Δlinear mice lacking RIP3. The population of mature B cells in the spleen of B-HOIP^Δlinear mice was not affected by loss of RIP3 (Fig. 7A). However, the number of both B1a (CD5⁺, CD23⁻) and B1b (CD5⁻, CD23⁻) cells increased after RIP3 deletion; in particular, the number of B1b cells was restored to levels observed in control mice (Fig. 7B, 7D), suggesting involvement of LUBAC-mediated inhibition of necroptosis in regulating development and/or maintenance of B1 cells, especially B1b cells. Because LUBAC suppresses TRIF-mediated cell death, we examined the effects of TRIF ablation on the number of peritoneal B1 cells (Fig. 7C, 7D). Loss of TRIF, however, did not affect the number of B1a or B1b cells in B-HOIP^Δlinear mice. Collectively, these results show that LUBAC regulates B1 cell development and/or maintenance by inhibiting necroptosis induced by a receptor other than TLR3 or TLR4.

LUBAC is the only known ubiquitin ligase complex that generates linear polyubiquitin chains (11). We and others showed that LUBAC plays a role in activating the canonical NF-κB pathway and inhibiting TNF-α–induced cell death (12–15). The role of LUBAC during development and function of B and T lymphocytes has also been dissected (16, 27–30). In B cells, LUBAC plays crucial roles in TD and TI-II Ab responses by controlling CD40- and TACI-mediated activation of the canonical NF-κB and ERK pathways, respectively; it also regulates B1 cell development (16). However, the precise roles of LUBAC in TLR-elicited Ab responses have not been probed, although we found that LUBAC controls TLR-induced activation of the canonical NF-κB and ERK pathways in B cells (16). In this study, we investigated the function of LUBAC in TLR-mediated B cell responses.

Ab responses to NP-LPS were heavily impaired in B-HOIP^Δlinear mice; this was confirmed by the observation that HOIP^Δlinear B cells did not proliferate, but rather underwent cell death, in response to LPS. We also showed that LPS-induced death of HOIP^Δlinear B cells was mainly mediated through caspase-mediated apoptosis, which was itself induced by the TRIF pathway. This observation also explains why cell death of HOIP^Δlinear B cells was not induced by CpG DNA stimulation because TLR9 does not use TRIF for the signal transduction. LUBAC may suppress cell death induced by the TRIF signaling pathway via multiple mechanisms. It was shown that B cells lacking c-FLIP, a molecule that is induced by canonical NF-κB activation and that inhibits caspase-8, which can be activated by TRIF, underwent cell death by LPS stimulation (31). Thus, one possible mechanism is attenuated induction of c-FLIP because we found that induction of c-FLIP mRNA by LPS stimulation is lower in HOIP^Δlinear B cells than in control cells. However, induction of c-FLIP may not be the sole mechanism because HOIP^Δlinear B cells died more extensively than control B cells even in the presence of a protein synthesis inhibitor that inhibited c-FLIP induction. Another possible mechanism is inhibition of formation of the cell death–inducing protein complex by the linear ubiquitination activity of LUBAC because LUBAC-mediated conjugation of linear polyubiquitin chains to molecules within the TNFR1 signaling complex inhibits caspase-8 activation upon TNF signaling, and several molecules (including RIP1) are shared between the TNFR1 and TRIF signaling pathways (15, 32–35). It is noteworthy that LPS induced proliferation of B cells from HOIP^Δlinear/TRIF KO mice less effectively than those from HOIP^Δlinear/RIP3 KO mice treated with the caspase inhibitor, albeit less prominently than in control B cells (Fig. 6). Because TLR4 induces NF-κB activation independently of MyD88 (18, 36), we suspect that LUBAC is involved in cell proliferation as well as protection from cell death upon TRIF signaling. Although ablating TRIF did not rescue impaired TLR4-mediated proliferation because of the loss of LUBAC activity, loss of TRIF fully restored survival of HOIP^Δlinear B cells (Fig. 4). We observed that TLR4-mediated activation of the canonical NF-κB and PI3-kinase pathways, both positive regulators of B cell survival, was reduced but was still clearly induced in HOIP^Δlinear/TRIF KO B cells (data not shown). Thus, LUBAC plays crucial roles in proliferation and protection from death, but not in survival of B cells, in response to TLR4 signaling. We have also observed that LPS-mediated differentiation of HOIP^Δlinear B cells into plasma cells was partially rescued by inhibition of both apoptosis and necroptosis but not by ablation of either TRIF or RIP3, as was the case with the restoration of proliferation of HOIP^Δlinear B cells. TLR9-stimulated proliferation and plasma cell differentiation were also reduced in HOIP^Δlinear B cells. These results imply that proper proliferation is requisite for TLR-induced plasma cell differentiation. We have previously found that TLR4- and TLR9-mediated activation of canonical NF-κB and ERK pathways was similarly impaired in HOIP^Δlinear B cells. Thus, we think that LPS-induced plasma cell differentiation was only partially recovered by inhibition of both apoptosis and necroptosis because LUBAC activity is also required for activation of canonical NF-κB and ERK pathways. Our observations that HOIP^Δlinear B cells showed impaired responses to TLR9, which transmits signals only through MyD88, and that TLR4-mediated proliferation of HOIP^Δlinear/TRIF KO B cells was weaker than that of TRIF KO B cells clearly confirmed our previous finding that LUBAC is involved in MyD88-mediated activation of canonical NF-κB and ERK pathways (13, 16). Collectively, we conclude that LUBAC controls TLR-induced activation of B cell responses by inhibition of TRIF-mediated cell death as well as activation of canonical NF-κB and ERK pathways (Supplemental Fig. 4).

The ligase activity of LUBAC is essential for development and/or maintenance of B1 cells (16). Because the canonical NF-κB pathway plays a role in B1 cell development in addition to BCR signaling (37, 38), we suspected that attenuated canonical NF-κB activation might underlie the defective development and/or maintenance of B1 cells in B-HOIP^Δlinear mice. Unexpectedly, we found that ablating RIP3 increased the number of peritoneal B1 cells in B-HOIP^Δlinear mice; this increase in cell number was more prominent for B1b than for B1a cells (Fig. 7). The major role of RIP3 is to induce necroptosis elicited by various stimuli (39). Thus, it seems plausible that LUBAC-mediated protection from necroptosis also plays crucial roles in development and/or maintenance of B1 cells, especially B1b cells, highlighting the importance of precise regulation of necroptosis for maintenance of B1 cell homeostasis under physiological conditions. Because ablation of TRIF did not rescue development of B1 cells in B-HOIP^Δlinear mice, TRIF is not involved in the induction of necroptosis that causes reduction of B1 cells. In contrast to B1b cells, the number of B1a cells in B-HOIP^Δlinear mice was increased only marginally by RIP3 deficiency, indicating that LUBAC regulates the development of B1a and B1b cells in a different manner. In the case of development and/or maintenance of B1b cells, LUBAC-mediated inhibition of RIP3-mediated necroptosis via some unknown receptor may play a key role. BCR signaling also plays a crucial role in B1 cell development; however, LUBAC activity was not necessary for BCR-mediated activation of the canonical NF-κB pathway in primary murine B cells (16). TRAF6, which is involved in canonical NF-κB activation, is also necessary for development of B1a cells, but not B1b cells, and LUBAC is involved in NF-κB activation mediated by various receptors, including those that use TRAF6 as a signal inducer (6, 40, 41). Thus, B1a cell development and/or maintenance might be regulated by a receptor that uses both TRAF6 and LUBAC to induce NF-κB and inhibit cell death, including necroptosis. Identification of such receptors involved in development and/or maintenance of B1a and B1b cells is of great import if we are to better understand B1 cell development and function.

In this study, we analyzed the roles of LUBAC in TLR signaling by using mouse B cells. However, our finding in this study might apply to human B cell biology because protective Ab responses to Haemophilus influenzae were impaired in patients with autoinflammatory disease caused by HOIP mutation, and TLR4 was shown to be required for immune responses to H. influenzae (42, 43). It is of great interest to clarify whether LUBAC plays similar roles in human B cells in the near future.

We thank Y. Sugahara and A. Himeno for technical assistance and Dr. M. Reth and Dr. T. Takemori for providing the mb1-cre mice and NIP-BAS-PE, respectively.

The authors have no financial conflicts of interest.