Enhanced B Cell Receptor Signaling Partially Compensates for Impaired Toll-like Receptor 4 Responses in LPS-Stimulated IκBNS-Deficient B Cells

Lipopolysaccharide (LPS) stimulates dual receptor signaling by bridging the B cell receptor and Toll-like receptor 4 (BCR/TLR4). B cells from IκBNS-deficient bumble mice treated with LPS display reduced proliferative capacity and impaired plasma cell differentiation. To improve our understanding of the regulatory role of IκBNS in B cell activation and differentiation, we investigated the BCR and TLR4 signaling pathways separately by using dimeric anti-IgM Fab (F(ab’)2) or lipid A, respectively. IκBNS-deficient B cells exhibited reduced survival and defective proliferative capacity in response to lipid A compared to B cells from wildtype (wt) control mice. In contrast, anti-IgM stimulation of bumble B cells resulted in enhanced viability and increased differentiation into CD138+ cells compared to control B cells. Anti-IgM-stimulated IκBNS-deficient B cells also showed enhanced cycle progression with increased levels of c-Myc and cyclin D2, and augmented levels of pCD79a, pSyk, and pERK compared to control B cells. These results suggest that IκBNS acts as a negative regulator of BCR signaling and a positive regulator of TLR4 signaling in mouse B cells.


Introduction
Polyclonal B cell stimulation leads to proliferation and differentiation of activated B cells into antibody-secreting plasma cells (PCs). Depending on the type of antigen, the response can be T cell-independent (TI) or T cell-dependent (TD). TD responses to protein-based antigens require cooperation of CD4 + T cells, while TI antigens can stimulate antibody production in the absence of cognate T cell help. TI antigens are classified as TI-1 and TI-2 types. TI-2 antigens, typically polysaccharides and polymers, contain highly repetitive epitopes that induce a strong immune response by the cross-linking of multiple B cell receptors (BCR). TI-1 antigens, such as lipopolysaccharide (LPS), are polyclonal B cell activators that can stimulate B cells directly through co-engagement of Toll-like receptors (TLRs) [1,2].
LPS, a widely used mouse B cell mitogen, is the major outer membrane component of Gram-negative bacteria, such as Escherichia coli, Salmonella enterica, and Haemophilus influenzae [3]. It has an evolutionarily conserved structure composed of three covalently linked, conformationally distinct parts: a hydrophobic lipid A backbone, a hydrophilic core oligosaccharide, and distal polysaccharide chains (O-antigen) [4]. The lipid A moiety is responsible for the endotoxic activity and immunostimulatory effects of LPS [5].
The innate immune system of mammals recognizes the lipid A motif through two receptor complexes, TLR4/MD-2 [6][7][8] and CD180 (RP105)/MD-1 [9,10], while the repetitive polysaccharide region of LPS cross-links BCRs. Thus, the optimal immune responses to LPS requires a concomitant engagement of both BCR and TLR4 [11]. Stimulation of these two receptors initiates signal transduction cascades that induce the translocation of nuclear factor kappa B (NF-κB) factors from their sequestered state in the cytoplasm to the nucleus, regulating the transcription of target genes [12][13][14].
We previously described a mouse strain that lacks the atypical IκB protein, IκBNS, due to a non-sense mutation in the nfkbid gene (bumble mice) [18]. IκBNS was reported to bind to p50 homodimers, p52, and the Rel proteins [17,19]. Bumble (henceforward referred to as bmb) mice exhibit reduced frequencies of splenic marginal zone (MZ) B cells and peritoneal B-1 cells, and show an impaired humoral immune response to TI antigens [18,20], which are related to metabolically dysregulated and diminished PC differentiation [21][22][23].
In the present study, we used bmb mice to investigate the regulatory role of IκBNS in TI B cell activation in vitro. Specifically, we investigated BCR and TLR4 stimulation separately using dimeric anti-IgM Fab (F(ab') 2 ) (hereinafter anti-IgM) or the lipid A component of LPS, respectively. We demonstrate that bmb B cells displayed impaired survival and altered proliferative capacity upon lipid A stimulation compared to B cells from wt mice. In contrast, in the absence of IκBNS, anti-IgM stimulation resulted in enhanced B cell survival, which was associated with increased blast formation and a higher proportion of B cells that differentiated into CD138 + plasma cells. Furthermore, in response to anti-IgM stimulation, bmb B cells displayed accelerated proliferation and cell cycle progression with a partial G2 arrest, which correlated with elevated c-Myc and cyclin D2 levels and enhanced phosphorylation of early downstream components of BCR signaling, such as CD79, ERK, and Syk. These results offer an enhanced understanding of the role of IκBNS in LPS-induced B cell activation.

Mice
IκBNS-deficient bumble mice, generated by ENU mutagenesis of C57BL/6J mice, and B cell specific IκBNS conditional knock-out mice have been described previously [18,24]. Mice were bred and maintained at Karolinska Institutet animal research facility KM-A under ethical permit number 20513-2020 (ethical statement approval date: 11 February 2021) (Jordbruksverket). Studies were conducted with the approval of the Committee for Animal Ethics (Stockholms Norra Djurförsöksetiska nämnd), and all experimental procedures were performed in accordance with institutionally approved protocols. Genotyping of the IκBNS conditional knock-out offspring was carried out by PCR analysis of genomic DNA, as described previously [24]. 8-14-week-old, age-and sex-matched mice were used for the experiments.

CTV Labeling
To monitor cell division, purified B cells were labeled with 2.5 µM CellTrace Violet (CTV) dye (Thermo Fisher Scientific, Waltham, MA, USA) in PBS at 37 • C for 20 min according to the manufacturer's protocol prior to stimulation. Cell divisions were analyzed with the proliferation platform of FlowJo v10.7.2 software (Tree Star, Ashland, OR, USA).

Cell Cycle Analysis
For cell cycle analysis at the end of the culture period, the stimulated B cells were harvested and counted. Cell numbers and viability were determined with trypan blue exclusion using a Countess II automated cell counter (Thermo Fisher Scientific, Waltham, MA, USA). After counting, cells were washed, then resuspended in 0.5 mL of ice-cold PBS. To the cell suspension, 1 mL of ice-cold absolute ethanol was added dropwise while the tube was gently vortexed. Cells were kept on ice for at least 30 min, then stored at −20 • C until further procedures. For propidium iodide (PI) staining, ethanol-fixed cells were washed twice with ice-cold PBS and then, based on the cell numbers, were resuspended and incubated in PI staining solution (500 µL/1 M cells; 20 µg/mL propidium iodide (#P4170, Sigma-Aldrich, St. Louis, MO, USA), 50 µg/mL RNAse A (#R6513, Sigma-Aldrich, St. Louis, MO, USA), and 0.1% Triton X-100 in PBS) for 30 min at 37 • C. Alternatively, stimulated B cells were pulsed with 10 µM thymidine analog 5-Bromo-2deoxyuridine (BrdU) (#B5002, Sigma-Aldrich, St. Louis, MO, USA) for the last 1 h of culture. After harvesting, counting, and fixing (as described above), DNA were denatured with 2 M HCl/0.5% Triton X-100 for 30 min at room temperature (RT) following neutralization with 0.1 M Sodium Tetraborate (Na 2 B 4 O 7 ), pH 8.5 for 15 min at RT. Cells were then stained with FITC-conjugated anti-BrdU antibody (#556028, BD Biosciences, Franklin Lakes, NJ, USA) in PBS/0.5% BSA/0.5% Tween 20 for 30 min at RT. After washing in PBS/0.5% BSA, stained cells were incubated with PI solution for 1 h at 37 • C [25,26]. To distinguish cells in G0 stage, intracellular staining of Ki-67 (Alexa Fluor 488-conjugated anti-mouse Ki-67 (#16A8, BioLegend, San Diego, CA, USA)) was performed using the BD transcription factor buffer set (BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer's protocol. To discriminate mitotic cells in the G2/M phase, Alexa Fluor 488-conjugated anti-pHH3 (Ser10) (#D2C8, Cell Signaling Technology, Beverly, MA, USA) was used, together with PI [27]. Cell acquisition was performed on a BD FACSCelesta instrument (BD Biosciences, Franklin Lakes, NJ, USA).

Seahorse Mitochondrial Stress Test
To measure mitochondrial respiration of FoB cells in real time, a Seahorse XFe96 Extracellular Flux Analyzer with a Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies, Santa Clara, CA, USA) was used [22]. After 3 days of BCR stimulation, FoB cells were harvested, counted using trypan blue exclusion, and washed with Seahorse XF RPMI assay medium. After this, 2 × 10 5 cells/well were seeded in 40 µL assay medium on a poly-Dlysine-coated (Sigma-Aldrich, St. Louis, MO, USA) XF 96-well cell culture microplate. The plate was centrifuged at 300×g for 1 sec without break, rotated, and centrifugation was repeated. After this, 140 µL of assay medium was added per well, and the plate was rested for 40 min in a 37 • C, non-CO 2 incubator. During the assay, the following compounds (final concentrations) were injected sequentially into the wells: oligomycin (1.264 µM) (Sigma-Aldrich, St. Louis, MO, USA; #O4876), FCCP (2 µM) (Sigma-Aldrich, St. Louis, MO, USA; #C2920), and rotenone, together with antimycin A (both 0.5 µM; Sigma-Aldrich, St. Louis, MO, USA; #R8875 and #A8674, respectively). Oxygen consumption rate (OCR) was measured three times, every three minutes from each well, before and after each injection. OCR values were normalized to protein concentrations (determined by BCA assay (Thermo Fisher Scientific, Waltham, MA, USA)) and analyzed using the Wave Desktop software 2.6.1 (Agilent Technologies, Santa Clara, CA, USA).

Statistics
Differences between groups were analyzed by unpaired t test or Mann-Whitney t-test using GraphPad Prism v8. Statistical significance is indicated with ns (non-significant) for p > 0.05, * for p ≤ 0.05, ** for p ≤ 0.01, *** for p ≤ 0.001, and **** for p ≤ 0.0001. [29] and exerts pro-survival activity on both immature and mature B cells [30,31]. LPS stimulates dual BCR and TLR4 signaling by engaging the BCR through its polysaccharide and TLR4 through its lipid A component [11]. The nuclear IκBNS regulator is downstream of both the BCR and TLR4 pathways, modulating the NF-κB response ( Figure 1A). In previous studies, we showed that B cells from IκBNS-deficient bmb mice display reduced capacity to proliferate and differentiate in response to LPS in vitro and in vivo [22,23]. However, the respective roles of the two pathways were not investigated.

LPS induces proliferation and differentiation of mouse B cells into antibody-secreting plasma cells (PCs)
Here, to improve our understanding of the role of IκBNS in LPS-stimulated B cells, we used lipid A to stimulate TLR4 in the absence of BCR engagement and examined survival, proliferation, and the capacity of the B cells to differentiate into PCs in vitro. Following three days of culture in the presence of either LPS or lipid A, we found significantly fewer viable cells in the IκBNS-deficient B cell cultures compared to control cultures, and the decline was more pronounced following lipid A stimulation ( Figure 1B).
Following mitogenic stimulation, B cells enlarge in size, which can be monitored by flow cytometry as an increased forward (FSC) vs. side scattered (SSC) profile (Supplementary Figure S1A). Following three days stimulation with either LPS or lipid A, we observed significantly reduced frequencies of live blasted cells in bmb B cell cultures compared to controls (85% blasted cells in wt vs. 70-80% in bmb). The defect was more striking in the case of lipid A stimulation, because only 15-25% of the viable bmb cells showed blasted phenotype in contrast to the 60-70% of wt cells ( Figure 1C).
Flow cytometric analysis of viability using a fixable live/dead dye showed a modest, but significant, reduction in the frequencies of live cells (viability marker low ) (10-15% reduction) in LPS-stimulated bmb cultures, compared to in wt cultures, with a greater decline (40-50%) after lipid A stimulation ( Figure 1D).  Representative overlayed histograms showing CTV dilution, corresponding graphs depicting the frequency of cells per each division from wt (grey) and bmb (blue) mice. (E) Representative FACS plots showing CD138 B220 co-staining for discriminating B220 + CD138 + plasma blasts (PB) and B220 low/− CD138 + plasma cells (PC). Bar graphs showing the percentages of PB and PC populations in wt (grey) and bmb (blue) cell cultures. (F) Histogram overlays and bar graphs representing the expression levels of TLR4/MD-2, CD180 (RP105), and MD-1 in freshly isolated wt (grey) and bmb (blue) B cells. FMO staining of wt (light grey) and bmb (light blue) B cells used as controls. Data were plotted as mean ± SD. Results are representative of four independent experiments with 3-4 mice for each genotype. Statistical significance was determined by Mann-Whitney t-test; ns (non-significant) for p > 0.05, * for p ≤ 0.05. Figure 1A was created with BioRender.com. Proliferation of LPS-or lipid A-stimulated B cells was traced by the dilution of the fluorescent division tracking dye, CellTrace Violet (CTV). As reported previously [23], the absence of IκBNS resulted in an altered proliferative capacity to LPS, with higher frequencies of cells in the non-divided fraction and the early-mid divisions (divisions 3-5), with only a small proportion of cells reaching the last division (division 7). The frequency of viable cells was markedly reduced in lipid A-stimulated bmb cell cultures, compared to in control cultures. The frequencies of cells in the non-divided fraction and in the earlymid divisions (divisions 1-4) were significantly higher in bmb cultures, with only a small portion reaching the later divisions (beyond 5) compared to control cultures ( Figure 1D). The reduced viability and proliferative response to lipid A was irrespective of the lipid A concentration used for stimulation (Supplementary Figure S1B).
To compare the effect of IκBNS for LPS-or lipid A-stimulated PC differentiation, we analyzed cells by flow cytometry after three days in vitro stimulation. Dual staining of B220 and the plasma blast (PB)/PC marker, CD138 (syndecan-1), showed that both stimuli promoted PC differentiation of wt B cells, while bmb B cells failed to generate PCs in response to either TLR4 ligand. In agreement with our previous findings [22], the frequency of PBs was greater in LPS-stimulated bmb cultures than in control cultures, while after lipid A treatment, there was no significant difference in the frequency of PBs between the wt and bmb B cell cultures ( Figure 1E). B cells recognize LPS and lipid A by two receptor complexes, the TLR4/myeloid differentiation factor-2 (MD-2) and CD180 (RP105)/MD-1 [7]. To determine whether the reduced responsiveness of bmb B cells to LPS and lipid A resulted from differences in cell surface levels of these receptors, we analyzed their expression by flow cytometry on freshly isolated B cells. The expression of both the TLR4/MD-2 complex and CD180 were comparable in wt and bmb B cells, while MD-1 level was slightly elevated in bmb B cells compared to in control cells ( Figure 1F). This experiment showed that the reduced responsiveness of bmb B cells to TLR4 stimulation in terms of viability, blasting, proliferation, and PC differentiation was not the result of reduced receptor expression.

IκBNS-Deficient B Cells Display Improved Survival, Proliferation, and Differentiation in Response to BCR Stimulation
Bmb follicular B (FoB) cells display elevated surface IgM BCR expression (Supplementary Figure S2 and [18,32]). However, the potential effect of this on B cell activation and proliferation was previously not studied in detail. To explore this, we stimulated wt and bmb B cells with dimeric anti-IgM Fab (F(ab') 2 ) (hereinafter anti-IgM) [33] and determined cell viability and proliferation by flow cytometry. Following a three-day stimulation protocol, we observed increased cell numbers and viability in bmb B cell cultures compared to in control cultures (Figure 2A,B). The enhanced survival in the absence of IκBNS was associated with the augmented frequency, size, and granularity of live blasted cells, as shown by the FSC/SSC profiles ( Figure 2C). Notably, a large proportion (40-50%) of the blasted bmb cells was CD138 + , while only 15-25% of wt cells expressed CD138 ( Figure 2D). The proliferative capacity of the IκBNS-deficient B cells was also altered upon BCR stimulation, with a greater proportion of bmb B cells reaching division 2, but with very few cells reaching the last division, compared to control B cells ( Figure 2E). To assess whether the strength of BCR engagement influenced IκBNS-deficient B cell proliferation, we stimulated the B cells with different doses of anti-IgM ranging from 1 to 50 µg/mL for three days. We found that an increasing anti-IgM dose resulted in enhanced B cell expansion, irrespective of IκBNS, with higher counted live cell numbers in bmb compared to in wt cultures up until 10 µg/mL anti-IgM. Notably, IκBNS-deficient B cells exhibited enhanced spontaneous cell death, as the frequency of live cells was significantly lower in the non-stimulated bmb cell cultures, compared to in wt cells (Supplementary Figure S3A). Similar to LPS or lipid A stimulation, IκBNS-defective B cells activated with 5 µg/mL or higher doses of anti-IgM showed altered proliferation, as the frequencies of cells were significantly lower in the non-divided fraction and the earlier divisions (division 1, or division 2 in the case of the highest anti-IgM concentration). At 5-50 µg/mL anti-IgM concentrations, we observed remarkably more bmb cells in division 3, while the cell frequencies reduced beyond the 4th divisions and were lower than in wt cultures (Supplementary Figure S3A). We next examined whether the duration of BCR ligation enhanced the proliferation rate of IκBNS-deficient B cells. After 5 days of anti-IgM stimulation, despite increased viability, bmb B cells displayed defective proliferation with a significantly smaller proportion of cells progressing beyond division 4, while wt cells reached 7 divisions (Supplementary Figure S3B). B cell activation is associated with the upregulation of co-stimulation molecules. We found that 24 h of anti-IgM stimulation increased CD86 surface expression in both bmb and wt B cells, although the levels were significantly lower in bmb cells. Stimulation for 48 h resulted in a further increase of surface CD86 in wt cells, while this was not observed in bmb cells. There was no further upregulation of CD86 after 72 h of stimulation in either of the cultures ( Figure 2F). Collectively, these results demonstrate that B cells lacking IκBNS displayed enhanced early responses to anti-IgM in terms of viability and blast formation, while activation and proliferation were perturbed.

Accelerated Cell Cycle Progression of BCR-Stimulated IκBNS-Deficient B Cells Is Associated with a Partial Block in G2
The observed perturbation in bmb B cell proliferation upon BCR ligation prompted us to investigate cell cycle progression following anti-IgM stimulation. First, we examined the expression of the proliferation-associated nuclear protein, Ki-67, which is present during all active phases of the cell cycle (G1, S, G2, and M), but is absent from quiescent (G0) cells [34]. Following 48 and 72 h BCR stimulation, we found elevated frequencies of Ki-67 + cells in bmb cultures compared to in wt ( Figure 3A). To discriminate the different cell cycle phases (Sub-G0/G1, G0/G1, S, G2/M), we applied bromodeoxyuridine (BrdU) to mark the cells in S phase in combination with propidium iodide (PI) staining, as an indicator of DNA content, and analyzed B cell cultures from bmb and wt mice at 48 and 72 h following anti-IgM stimulation. Cells with lower DNA content (< 2N; Sub-G0/G1) were defined as apoptotic cells. The percentage of the sub-G0/G1 population in bmb cultures was significantly reduced at both time points, in comparison with the percentage in wt cultures (Supplementary Figure S4). After excluding the sub-G0/G1 population, we evaluated the proportions of each cell cycle stage in the live populations.
IκBNS-deficient bmb B cells entered the cell cycle at an elevated rate compared to control cells, as demonstrated by a significant decrease in cell frequencies in the G0/G1 phase (BrdU negative cells with a 2N DNA content), associated with increased frequencies in both BrdU positive cells, marking S phase, and BrdU negative cells with 4N DNA content, as G2/M phase after 72 h anti-IgM stimulation. At the 48-h time point, we found lower frequencies of bmb cells in the G2/M stage, compared to wt control cells ( Figure 3B). To further distinguish the G2 and M phases, we stained cells for both DNA content (PI) and histone H3 phosphorylated at serine 10 (pHH3), which marks cells in mitosis. At 72 h post-stimuli, we found a reduced proportion of pHH3 positive, mitotic (M) cells within the G2/M population in bmb cell culture compared to the proportion in wt cells. Therefore, bmb B cells appeared to accumulate in the G2 phase, with fewer cells entering the mitotic stage ( Figure 3C).
Cell cycle progression is a tightly regulated process, controlled by multiple proteins (cyclins, cyclin-dependent kinases). The NF-κB target transcription factor, c-Myc, plays a crucial role in BCR-triggered cell proliferation by controlling cell cycle entry into the G1 phase, or the transition from the G1 to the S phase via cyclin D2 induction (rev. in [35]). Time course analysis of c-Myc and cyclin D2 expression levels by Western blot showed significantly higher levels of c-Myc protein in bmb B cells compared to in wt cells, after both 48 and 72 h BCR stimulation, with a more prominent difference at 72 h. Cyclin D2 levels were comparable in both strains at 48 h, while after 72 h of anti-IgM treatment cyclin D2 was slightly downregulated in wt but remained high in bmb B cells ( Figure 3D). Taken together, these results show that anti-IgM-stimulated bmb B cells exhibited an accelerated cell cycle entry, which was associated with elevated c-Myc and cyclin D2 expression compared to control B cells. However, the proliferating bmb cells were unable to complete the cycling program, because a higher proportion of cells were found in G2, and lower proportion of cells were found in the M stage, compared to similarly stimulated wt B cells.

Increased Mitochondrial Respiration in BCR-Stimulated IκBNS-Deficient B Cells
Cell growth and proliferation is an energy-consuming process, which requires increased mitochondrial ATP production to support the energetic demand of the cells [36][37][38][39][40]. In a recent study, we showed that the absence of IκBNS in LPS-stimulated B cells resulted in elevated oxidative phosphorylation (OXPHOS) [22]. To investigate mitochondrial function in BCR-stimulated, proliferating bmb B cells, we measured the mitochondrial oxygen consumption rates (OCR) by Seahorse analyzer. Because bmb mice have similar FoB cell numbers as wt mice but reduced marginal zone (MZ) B cell frequencies [18], we used flow cytometry-sorted FoB cells to investigate mitochondrial respiration. Following 72 h of anti-IgM stimulation, bmb FoB cells showed significantly increased basal, maximal oxygen consumption rate (OCR), spare respiratory capacity, and elevated ATP-coupled respiration/OCR, compared to wt FoB cells (Figure 4). These results demonstrate that the loss of IκBNS results in enhanced mitochondrial activity in anti-IgM-stimulated B cells.
D2 was slightly downregulated in wt but remained high in bmb B cells ( Figure 3D). Taken together, these results show that anti-IgM-stimulated bmb B cells exhibited an accelerated cell cycle entry, which was associated with elevated c-Myc and cyclin D2 expression compared to control B cells. However, the proliferating bmb cells were unable to complete the cycling program, because a higher proportion of cells were found in G2, and lower proportion of cells were found in the M stage, compared to similarly stimulated wt B cells.  analysis of c-Myc, cyclin D2, and β-actin levels was performed using whole cell extracts of anti-IgM F(ab') 2 -stimulated wt or bmb B cells, as indicated. Results are representative of two independent experiments with 3 or 4 mice. Data were plotted as mean ± SD, and statistical significance was determined by Mann-Whitney t-test; ns (non-significant) for p > 0.05, * for p ≤ 0.05, ** for p ≤ 0.01.
creased mitochondrial ATP production to support the energetic demand of the cells [36][37][38][39][40]. In a recent study, we showed that the absence of IκBNS in LPS-stimulated B cells resulted in elevated oxidative phosphorylation (OXPHOS) [22]. To investigate mitochondrial function in BCR-stimulated, proliferating bmb B cells, we measured the mitochondrial oxygen consumption rates (OCR) by Seahorse analyzer. Because bmb mice have similar FoB cell numbers as wt mice but reduced marginal zone (MZ) B cell frequencies [18], we used flow cytometry-sorted FoB cells to investigate mitochondrial respiration. Following 72 h of anti-IgM stimulation, bmb FoB cells showed significantly increased basal, maximal oxygen consumption rate (OCR), spare respiratory capacity, and elevated ATP-coupled respiration/OCR, compared to wt FoB cells (Figure 4). These results demonstrate that the loss of IκBNS results in enhanced mitochondrial activity in anti-IgM-stimulated B cells.  We next sought to explore whether the enhanced survival and accelerated cell cycle in the absence of IκBNS was the result of increased BCR signaling. We performed intracellular FACS analysis for early phosphorylation events triggered by BCR cross-linking, such as pCD79a, pSyk, and pERK. We found higher pCD79 levels in unstimulated bmb compared to control cells, while the basal levels of pSyk and pERK in unstimulated wt and bmb B cells were similar. Following anti-IgM treatment, the levels of all examined phosphoproteins were elevated compared to unstimulated cells, and they were significantly higher in bmb B cells compared to in wt cells ( Figure 5). These results suggest that IκBNS regulates early molecular events downstream of BCR signaling.

Dual Stimulation through BCR and TLR4 Has Synergistic Effect on Viability and Proliferating Capacity in IκBNS-Deficient B Cells
It was shown previously that simultaneous stimulation through BCR and TLR enhances B cell activation and proliferation additively [41,42]; however, a persistent signal via BCR can abolish the LPS-induced PC differentiation [43]. Comparing the effects of the single and combined stimulation on B cell viability and proliferation, we found that simultaneous engagement of BCR and TLR4 led to increased cell survival in both wt and bmb cultures, with an elevated synergistic effect in bmb B cell cultures. IκBNS-deficient B cells also showed increased proliferative ability (Figure 6A,B). Monitoring the expression of activation marker CD86 showed a synergic effect of the dual stimulation in both strains, as the frequency of CD86 + cell and MFI values for CD86 were significantly increased after anti-IgM plus lipid A treatment ( Figure 6C, Supplementary Figure S5). Thus, combined stimulation with lipid A fragment and anti-IgM led to increased viability and proliferating capacity of bmb B cells and elevated CD86 expression.
We next sought to explore whether the enhanced survival and accelerated cell cycle in the absence of IκBNS was the result of increased BCR signaling. We performed intracellular FACS analysis for early phosphorylation events triggered by BCR cross-linking, such as pCD79a, pSyk, and pERK. We found higher pCD79 levels in unstimulated bmb compared to control cells, while the basal levels of pSyk and pERK in unstimulated wt and bmb B cells were similar. Following anti-IgM treatment, the levels of all examined phosphoproteins were elevated compared to unstimulated cells, and they were significantly higher in bmb B cells compared to in wt cells ( Figure 5). These results suggest that IκBNS regulates early molecular events downstream of BCR signaling. Figure 5. Enhanced BCR signaling in IκBNS-deficient bumble B cells. B cells from wt and bumble (bmb) mice were stimulated with 25 μg/mL anti-IgM F(ab')2 for 5 min, and phosphoproteins were assessed by flow cytometry. Histogram overlays showing expression levels for pCD79α, pSyk, and pERK1/2 in non-stimulated wt (light grey), non-stimulated bmb (light blue), stimulated wt (dark grey), and stimulated bmb (dark blue) B cells. Adjacent graphs representing the corresponding geometric mean fluorescence intensity (gMFI) values. Graphs represent mean ± SD. Data are representative of two experiments with 3 or 4 mice of each strain. Statistical significance was determined by an unpaired t-test; ns (non-significant) for p > 0.05, ** for p ≤ 0.01, *** for p ≤ 0.001. Figure 5. Enhanced BCR signaling in IκBNS-deficient bumble B cells. B cells from wt and bumble (bmb) mice were stimulated with 25 µg/mL anti-IgM F(ab') 2 for 5 min, and phosphoproteins were assessed by flow cytometry. Histogram overlays showing expression levels for pCD79α, pSyk, and pERK1/2 in non-stimulated wt (light grey), non-stimulated bmb (light blue), stimulated wt (dark grey), and stimulated bmb (dark blue) B cells. Adjacent graphs representing the corresponding geometric mean fluorescence intensity (gMFI) values. Graphs represent mean ± SD. Data are representative of two experiments with 3 or 4 mice of each strain. Statistical significance was determined by an unpaired t-test; ns (non-significant) for p > 0.05, ** for p ≤ 0.01, *** for p ≤ 0.001.

Dual Stimulation through BCR and TLR4 Has Synergistic Effect on Viability and Proliferating Capacity in IκBNS-Deficient B Cells
To examine if increased responses to anti-IgM stimulation were a consequence of enhanced surface IgM expression in bmb FoBs [18,32], we used conditional knock-out mice deleted for nfkbid exclusively in the B cell lineage (referred to as nfkbid B− ). B cells in these mice express comparable levels of surface IgM as control nfkbid B+ B cells [24]. Following 72 h in vitro anti-IgM stimulation, in nfkbid B− B cell cultures we observed significantly higher live cell numbers compared to in control cultures ( Figure 7A). Consistent with this, discrimination of viable cells by flow cytometry showed an elevated frequency of live cells (viability dye low ) in the conditional knock-out cell culture ( Figure 7B). In contrast, TLR4 stimulation with LPS or lipid A resulted in significantly reduced B cell survival in the nfkbid B− cultures compared to in the controls, which was more remarkable in the case of lipid A (Figure 7A,B). Furthermore, in response to TLR4 activation, conditional knock-out B cells failed to differentiate into PCs, as B220 CD138 dual staining showed, while there was no reduction in the B220 + CD138 + PB population, a rather modest increase in the case of LPS stimulation ( Figure 7C). Upon anti-IgM stimulation, increased frequencies of CD138 + cells were generated in the nfkbid B− cultures compared to in the nfkbid B+ controls ( Figure 7D). Similar to IκBNS-deficient bmb B cells, conditional knock-out B cells displayed altered proliferative ability in response to all three stimuli ( Figure 7E). These results indicate that the elevated response of bmb B cells to anti-IgM is not due to the increased surface IgM expression. Therefore, we conclude that the reduced responsiveness to TLR4 stimulation, as well as the enhanced response to anti-IgM stimulation, are B cell intrinsic features.
via BCR can abolish the LPS-induced PC differentiation [43]. Comparing the effects of the single and combined stimulation on B cell viability and proliferation, we found that simultaneous engagement of BCR and TLR4 led to increased cell survival in both wt and bmb cultures, with an elevated synergistic effect in bmb B cell cultures. IκBNS-deficient B cells also showed increased proliferative ability (Figure 6A,B). Monitoring the expression of activation marker CD86 showed a synergic effect of the dual stimulation in both strains, as the frequency of CD86 + cell and MFI values for CD86 were significantly increased after anti-IgM plus lipid A treatment ( Figure 6C, Supplementary Figure S5). Thus, combined stimulation with lipid A fragment and anti-IgM led to increased viability and proliferating capacity of bmb B cells and elevated CD86 expression.

Discussion
The innate immune system has evolved to serve as the first line of defense against pathogens. This process involves the recognition of pathogen-associated molecular patterns (PAMPs) on pathogens by pattern recognition receptors (PRRs) expressed by host cells. LPS is a well-characterized PAMP in the outer membrane of Gram-negative bacteria, which induces B cell activation by the dual engagement of TLR4 and BCR, via the small lipid A moiety and the connected polysaccharide chain, respectively. Synergy of the two signaling pathways leads to the activation of NF-κB cascade, engendering a protective response against capsular bacteria [11].
In the present study, we investigated the role of IκBNS in BCR-and TLR4-induced B cell activation using the bmb mouse strain deficient for the NF-κB pathway regulator, IκBNS protein [18]. We applied monophosphoryl lipid A and bivalent anti-IgM F(ab') 2 fragment (anti-IgM) to study the two pathways separately.
We report that in the absence of functional IκBNS, B cells show reduced survival after lipid A or LPS stimulation in vitro. Viable cells exhibited altered cell proliferation, with increased cell frequencies in the early, and reduced frequencies in the late divisions. Similar to the LPS response [23], lipid A treatment did not promote PC differentiation of bmb B cells.
In contrast, anti-IgM stimulation induces elevated bmb cell survival, cell growth, and blast formation associated with a higher frequency of CD138 + B cells in comparison with wt cells.
Moreover, following BCR stimulation, bmb B cells showed a perturbed proliferation and accelerated cell cycle entry, accompanied by a partial block in the G2 phase. The enhanced proliferation is associated with elevated levels of c-Myc and cyclin D2.
Furthermore, anti-IgM-stimulated bmb B cells displayed increased phosphorylation of early BCR signaling molecules, such as CD79a, Syk, and ERK. Studies on IκBNS B cell conditional knock-out mice revealed that the enhanced BCR response is B cell intrinsic and not the consequence of elevated surface IgM expression that was found in bmb B cells.
Here, in agreement with our previous study [23], we showed that LPS-and lipid Astimulated bmb B cells exhibited an altered proliferation profile, with an initial acceleration followed by a decline, by which less cells achieve the later divisions.
TLR4 belongs to the family of PRRs characterized by two major domains: the extracellular leucine-rich repeat motif (LRR) and the intracellular Toll/interleukin 1 (IL-1) receptor (TIR) signaling domain [13].
Functional LPS receptor expression is essential for proper B cell activation and antibody production. Even though B cells express relatively low TLR4/MD-2 in comparison with macrophages, analysis of mutant mice demonstrate that this complex is indispensable for B-cell responses to LPS [7,56].
Besides TLR4, the TLR-like molecule CD180 (RP105) was reported to be implicated in LPS-triggered B cell activation [57]. Similar to TLR4, CD180 has a conserved extracellular leucine-rich repeat (LRR) domain; however, it lacks the intracellular Toll-IL-1R (TIR) signaling domain. The expression and function of CD180 are dependent on the co-expression of MD-2 homologue MD-1 protein [58,59]. The two complexes together are crucial for the appropriate LPS or lipid A-induced B cell responses.
In the current study, analysis of the expression levels of TLR4/MD-2 and CD180/MD-1 complexes on purified B cells from wt and bmb mice did not reveal reduced expression of the LPS receptors in bmb, which indicates that the proliferative defect in response to LPS or lipid A is not due to decreased receptor expression. However, normal receptor expression or proper physical interaction between receptor and ligand is not always manifested in an activation signal, as showed in lipid A-stimulated MyD88 −/− B cells, which failed to proliferate or up-regulate the activation marker CD86 [60]. Thus, to elucidate the survival and proliferation defect in LPS-/lipid A-stimulated bmb B cells, further evaluation of LPS/lipid A-triggered signaling pathways is needed.
Besides its potent mitogenic effect, LPS promotes the survival of immature and mature B cells [30,61] by preventing apoptosis through the NF-κB-dependent regulation of pro/antiapoptotic molecules [62,63]. LPS-or lipid A-stimulated bmb B cells, however, showed reduced viability, while BCR stimulation by anti-IgM triggered an elevated survival of bmb B cells, indicating that IκBNS has an opposite regulatory role during the TLR4-and BCR-induced cell survival, namely a positive in TLR4-and a negative in BCR-stimulated.
Upon antigen activation, B cells differentiate into antibody secreting PCs, which can be identified by the high surface expression of CD138 (syndecan-1) [64,65] in conjunction with low levels of B220 [66]. As we previously reported, LPS-stimulated bmb B cells failed to generate PCs, both in vitro and in vivo, as the PC differentiation was suspended in the premature CD138 + B220 + pre-PB, PB stages [22,23]. This was also the case with the TLR4 agonist, lipid A.
Divalent anti-IgM F(ab') 2 fragment, which mimics antigen for BCR, promotes B cell activation, blast formation, and proliferation. However, proper PC differentiation and antibody synthesis requires the help of cognate, antigen-primed T cells [67,68].
Remarkably, we observed a perturbed proliferation pattern in the anti-IgM-stimulated bmb cultures, as in the LPS-or lipid A-treated cultures. Specifically, the elevated cell frequencies in mid divisions were accompanied by reduced cell frequencies in the later divisions. These results suggest a general B cell proliferation deficit in response to the examined TI stimuli, in the absence of IκBNS.
Furthermore, unexpectedly, after 3 days of anti-IgM activation, a remarkable proportion of the live bmb cells were found to express the PC marker CD138 ( Figure 2D). It has been proposed that CD138 (syndecan-1) promotes the survival of mature PCs [69], as well as multiple myeloma cells, by binding of anti-apoptotic factors, growth factors, and pro-survival cytokines, such as APRIL, or IL-6 through its heparan sulfate moieties [70,71]. In accordance with this, IgM-stimulated bmb cell cultures contained elevated numbers and frequencies of viable cells. Notably, the elevated anti-IgM response in the conditional knock-out B cells, which shows comparable surface IgM expression levels to the control B cells, indicates that the enhanced BCR-induced response in bmb B cells is not a consequence of elevated surface IgM expression on these cells.
The B cell proliferation defect in the single p50 and c-Rel mutant mice is associated with a block in the G1 phase [72], while in the double knock-out it was manifested as a failure of cell growth. This was due to impaired c-Myc expression, which normally is induced by NF-κB1/c-Rel dimers via a PI3K-dependent pathway [73]. Besides its function in cell size regulation, whose process is uncoupled from cell division, c-Myc has a crucial role in cell cycle control [35,[74][75][76].
Cell cycle is strictly regulated by the heterodimeric complexes of cycling-dependent kinases (CDKs), cyclins, and inhibitors of CDKs [77]. c-Myc plays a crucial role in the BCR-triggered cell proliferation by controlling cell cycle entry into the G1 phase [78][79][80], and the transition from the G1 to the S phase via the direct induction of cyclin D2 [81][82][83].
Although IκBNS is important in LPS- [23], or anti-IgM-induced B cell proliferation, its possible role in cell cycle progression has not been studied.
Notably, BCR-triggered bmb B cells exhibited increased cell size, which was associated with accelerated cell cycle entry and transition to the S phase, and a concurrent partial block in the G2 phase with reduced entry into mitosis. The latter is reflected by the decreased frequency of cells that reached later divisions. These features coincided with elevated c-Myc and cyclin D2 expression levels.
Akkaya et al. reported that BCR-stimulated mouse B lymphocytes rapidly increase their metabolic activity, including both oxidative phosphorylation and glycolysis compared to the non-activated state. However, in the absence of a costimulatory second signal that is provided by helper T cells, BCR-stimulated B cells progressively loose mitochondrial function and glycolytic capacity, leading to cell death [84]. Interestingly, anti-IgM-stimulated bmb B cells showed elevated OXPHOS and survival even after 3 days activation compared to similarly stimulated wt B cells. Further analyses are needed to resolve the detailed molecular mechanisms for this.
Over the years, we have studied the B cell response in IκBNS-deficient bumble mice with the objective to better understand how it regulates the NF-κB pathway and the differentiation of B cells in response to different stimuli. IκBNS remains a relatively understudied component of the NF-κB pathway, but we find it important as its loss in B cells clearly abrogates the T cell-independent antibody response [20,23]. We speculate that mutations in human nfkbid may underpin some common variable immunodeficiency syndrome (CVID) cases that remain unmapped, and we therefore believe that it is important to characterize its role in B cell development, activation, and differentiation in a model system as CVID patients often have defective responses to encapsulated bacteria [85].

Conclusions
In conclusion, our results demonstrate that the NF-κB regulator IκBNS plays an essential role in BCR-and TLR4-induced B cell survival, cell growth, cell cycle progression, and proliferation. Furthermore, our data suggest an opposing regulatory role for IκBNS in BCR and TLR4 pathways in terms of cell survival, and a complex and dual role during LPS-and anti-IgM-mediated proliferation and cell cycle regulation. These results broaden our knowledge of the role of IκBNS in the T-independent B cell responses.