Heterozygous Mutation in IκBNS Leads to Reduced Levels of Natural IgM Antibodies and Impaired Responses to T-Independent Type 2 Antigens

Mice deficient in central components of classical NF-κB signaling have low levels of circulating natural IgM antibodies and fail to respond to immunization with T-independent type 2 (TI-2) antigens. A plausible explanation for these defects is the severely reduced numbers of B-1 and marginal zone B (MZB) cells in such mice. By using an ethyl-N-nitrosourea mutagenesis screen, we identified a role for the atypical IκB protein IκBNS in humoral immunity. IκBNS-deficient mice lack B-1 cells and have severely reduced numbers of MZB cells, and thus resemble several other strains with defects in classical NF-κB signaling. We analyzed mice heterozygous for the identified IκBNS mutation and demonstrate that these mice have an intermediary phenotype in terms of levels of circulating IgM antibodies and responses to TI-2 antigens. However, in contrast to mice that are homozygous for the IκBNS mutation, the heterozygous mice had normal frequencies of B-1 and MZB cells. These results suggest that there is a requirement for IκBNS expression from two functional alleles for maintaining normal levels of circulating natural IgM antibodies and responses to TI-2 antigens.

Innate-like B cells play significant roles in the early defense against pathogens and, at steady state, anti-inflammatory mediators, such as IL-10, and polyreactive IgM antibodies are secreted by these cell subsets to help maintain homeostasis. There is a clear division of labor between the different innate-like B cells. B-1a cells are thought to secrete most of the natural polyreactive antibodies found in the serum (1). Innate response activator B cells exert protective effects against sepsis by secreting GM-CSF (2). Marginal zone B (MZB) cells, through their location at the interface between the blood and the immune system, help initiate responses against blood-borne antigens [reviewed in Ref. (3)]. B cells regulate inflammation through various mechanisms, including production of anti-inflammatory cytokines, such as IL-10 and IL-35 (4,5). The main IL-10 producing innate-like B cells, collectively named B10, are found within the MZB (5) and B-1a cell subsets (4,6). Innate-like B cells are also pivotal for the response against viral and bacterial infections (1). Particularly, the innate-like B cell subsets B-1b and MZB cells are the predominant responding B cells to T-independent type 2 (TI-2) antigens found on the surface of a number of pathogens, such as Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitides (3,7,8).
In addition to their distinct roles in homeostasis and response to different stimuli, the innate-like B cell subsets also have distinct developmental pathways. B-1 cells are generated readily from the yolk salk, paraaortic splanchnopleura, and liver during early fetal development (9,10), while these organs are less effective at generating follicular B cells. In contrast, hematopoietic stem cells from adult bone marrow predominantly generate follicular B and MZB cells (9), collectively referred to as B-2 cells. Immature B cells mature in the spleen and undergo selection at various transitional stages before becoming naive B cells (11). B-2 cells are continuously replenished from the adult bone marrow and diverge into follicular B cells and MZB cells at the transitional B cell stage (12,13). B-1 cells may develop from a separate progenitor population (14) and mature via a phenotypically distinct B-1 transitional B cell intermediate, which is found at high frequencies in the spleen of neonatal mice (15). The different B cell subsets require distinct stimuli for development and maintenance. For example, MZB cells are dependent on Notch signaling, and therefore mice with impaired Notch2 completely lack MZB cells. However, Notch signaling is not required for B-1 or follicular B cell development (12). The distinct B cell subsets also show different requirements for NF-κB signaling (16).
Classical NF-κB signaling is required for the generation of B-1 cells, particularly the B-1a subset, which is absent in a number of mouse strains where this pathway has been ablated [reviewed in Ref. (22)]. Reduction in MZB cell numbers is also seen in the absence of classical NF-κB signaling, while follicular B cells are less affected (23,24). Although relatively little is known about the function of atypical IκB proteins in B cell development, roles for BCL-3 and IκBNS have recently been demonstrated. BCL-3 deficiency leads to increased numbers of MZB cells (25), while decreased B-1 and MZB cellularity was observed upon overexpression of BCL-3 (26). Absence of functional IκBNS leads to reductions in B-1b and MZB cell frequencies (27,28) and complete absence of B-1a cells, while follicular B cell frequencies are intact (15,28). In terms of B cell lymphopoiesis, IκBNS-deficient mice thus resemble other mouse strains with impaired classical NF-κB signaling. In addition to the role of classical NF-κB signaling in B cell development, it is also required for normal function of mature B cells. B cells from p50, BCL10, and CARMA1-deficient mice display reduced proliferation and antibody production to anti-IgM, anti-CD40, or LPS compared to wild-type (wt) cells in vitro (29)(30)(31).
Mice with impaired classical NF-κB signaling have reduced levels of circulating natural IgM and IgG3 antibodies and fail to mount antibody responses to TI-2 antigens in vivo. We previously described that mice lacking functional IκBNS due to a mutation in the nfkbid gene (bumble mice) also display reduced IgM and IgG3 levels and fail to respond to immunization with NP-ficoll (27,28). Whether the impaired antibody response in mice deficient in classical NF-κB pathway signaling is a consequence of the reduced numbers of B-1 and MZB cells or is due to defects in B cell function remains unknown. Here, we demonstrate that the lack of IκBNS even at the heterozygous state (IκBNS + /bmb) led to severely reduced antibody responses against TI-2 antigens, suggesting haploinsufficiency for IκBNS in the response to such antigens. Interestingly, unlike homozygous bumble mice, the heterozygous mice displayed apparently normal frequencies and numbers of MZB and B-1 cells. This indicates that the reduced responses to TI-2 antigens in these mice are due to a direct requirement of IκBNS in response to B cell receptor engagement rather than a secondary effect due to lack of responding cells.

Mice
Mice were housed and bred at the animal research facility, MTC, Karolinska Institutet. Bumble mice, generated by N-ethyl-Nnitrosourea (ENU) mutagenesis of C57BL/6J mice, and their wt C57BL/6J counterparts were described previously (28). Animal studies were conducted with Committee for Animal Ethics (Stockholms Norra Djurförsöksetiska nämnd) approval.

cell Preparation
Splenocytes were prepared as a single cell suspension using a 70-μm cell strainer. Peritoneal cells were isolated by flushing with cold PBS/1% FBS (5-10 ml). Peritoneal cells were discarded if contaminated with blood. Cell suspensions were diluted in RPMI-1640 supplemented with 2 mM l-glutamine, penicillin (100 IU)-streptomycin (100 μg/ml), and 10% fetal bovine serum (complete RPMI). Splenocyte cell suspensions were washed once in Ca 2+ -free Mg 2+ -free PBS and treated with red blood cell lysis buffer before further processing. B cells were isolated from the spleen using the B cell isolation kit and from the peritoneal cavity using the Pan-B cell isolation kit (Stemcell Technologies). The purity of the isolated populations was ≥90%.

adoptive Transfer
Isolated peritoneal B cells (2 × 10 6 cells) from wt or heterozygous mice were mixed with 50 μg NP (40)-ficoll and injected i.p. into bumble mice. Splenic B cells (30 × 10 6 cells) were injected i.v. elisa ELISA was performed by coating ELISA plates (Nunc) with polysaccharide antigens: 500 ng/well of NP (25) conjugated with BSA (Biosearch Technologies) or the pneumococcal polysaccharide antigens Type 1 161-X™ or Type 3 169-X™ (both ATCC) or 1:100 dilution of Pneumovax (corresponding to 50 ng/well of each antigen contained in the vaccine). To measure total IgM and IgG3 levels, plates were coated with unconjugated anti-IgM or anti-IgG3 (Southern Biotech). Plates were incubated overnight (4°C). Following washing (PBS + 2% Tween20) and blocking for 1 h with PBS containing 2% dry milk, serum was added in threefold serial dilutions in blocking buffer and incubated for 1.5 h at room temperature (RT) before addition of secondary antibody HRP-coupled anti-IgM or IgG3 (Southern Biotech). The assay was developed with TMB substrate (KPL) followed by 1M H2SO4 and the OD was read at 450 nm using an Asys Expert 96 ELISA reader (Biochrom).

elispot assay for Detection of antibodysecreting cells
Detection of total IgM and NP-specific IgM producing cells was performed using enzyme-linked immunosorbent spot (ELISpot) assay. MultiScreen-IP filter plates (Millipore) were pretreated with 70% ethanol and washed in sterile PBS. Plates were coated with 5 μg/ml anti-mouse IgM (Southern Biotech) or 5 μg/ml of NP (25), conjugated with BSA (Biosearch Technologies), diluted in PBS, and incubated overnight at 4°C. The following day, plates were washed in sterile PBS, blocked in complete RPMI medium with 50-μM 2-mercaptoethanol and 10-mM HEPES for 1 h at 37°C, and the indicated cell numbers added in triplicate. Plates were incubated for 17 h at 37°C in 5% CO2. Cells were then removed by washing in PBS and 0.1 μg/well of biotinylated anti-mouse IgM (Mabtech) diluted in PBS was added to the wells. After 2 h of incubation at RT, plates were washed and developed with 100 μl of 5-bromo-4-chloro-3-indolyl phosphate/NBT-plus substrate (Mabtech). The reaction was stopped when distinct spots could be observed, by rinsing the plates extensively in tap water. Spots were counted by ELISpot reader (CTL) and analyzed using the Biospot suite (CTL).

Flow cytometry
Cells were incubated with Fc block (anti-CD16/32, BD) and stained with fluorochrome-conjugated monoclonal antibodies in PBS/2% FBS using the following antibodies:  In an ENU mutagenesis screen for antibody response defects, we identified a role of IκBNS for B cell development and function. We observed that mice homozygous for a specific mutation in nfkbid lacked responses to the TI-2 antigen NP-ficoll and had reduced levels of circulating IgM and IgG3 antibodies (28). These mice, named bumble, were found to have a T → G transversion in the conserved donor splice site in the fourth intron of nfkbid, the gene encoding IκBNS. This mutation is predicted to prevent splicing of the fourth intron from the nfkbid transcript, resulting in a premature stop codon after exon 4. Such a transcript would likely be targeted for nonsense-mediated decay or encode only 65 of 327 amino acids (aa) encoded by full-length nfkbid, and therefore not be expected to retain any function. The phenotype for bumble mice was copied in mice with a targeted mutation in the nfkbid gene (28) and was similar in IκBNS knockout mice (27). Here, we report that mice heterozygous for the bumble mutation displayed reduced NP-specific IgM and IgG3 antibody responses after NP-ficoll immunization ( Figure 1A). Furthermore, when analyzing isolated splenocytes for NP-specific IgM antibody-secreting cells (ASC), we observed significantly (p < 0.001) lower numbers in bumble heterozygous compared to wt mice ( Figure 1B). To test if the TI-2 antigen response defect in heterozygous bumble mice was due to a B cell intrinsic defect, we transferred isolated splenic and peritoneal B cells to bumble mice and immunized them with NP-ficoll. Bumble mice that had not received isolated B cells did not respond to immunization, whereas bumble mice that had received wt B cells did. Bumble mice that had received B cells from heterozygous bumble mice had an intermediate response to NP-ficoll immunization, suggesting that the defective antibody response to TI-2 antigens in heterozygous bumble mice is due to a B cell intrinsic defect (Figure 1C). Similarly to bumble homozygotes, mice with the heterozygous bumble mutation displayed significantly reduced total serum levels of IgM and IgG3  (a,B) or two (c) independent experiments. Graphs display mean + SD. Statistically significant differences between bumble, bumble heterozygotes, and wt mice are indicated by *p < 0.05, **p < 0.01, and ***p < 0.001 by Mann-Whitney test. Frontiers in Immunology | www.frontiersin.org antibodies (p < 0.05) ( Figure 1D). Thus, relative to homozygous bumble mice and wt mice, heterozygous bumble mice presented with an intermediary phenotype in terms of response to NP-ficoll and levels of circulating IgM and IgG3 antibodies.

Fewer igM antibody-secreting cells in heterozygous Bumble Mice
We next asked if the reduced IgM levels in heterozygous bumble mice were due to fewer IgM-secreting B cells or if less IgM was produced per cell. To assess this, we performed ELISpot for total IgM producing splenic and bone marrow B cells. The same numbers of cells were plated from wt, heterozygous, and homozygous bumble mice. As expected, no IgM ASCs were detected from homozygous bumble mice. The fraction of splenic B cells spontaneously secreting IgM in heterozygous bumble mice was slightly lower than in wt mice, although this difference was not statistically significant (p = 0.23) (Figure 2A). The frequency of IgM ASCs in bone marrow was significantly lower in heterozygous bumble mice than in wt mice (p < 0.01). As an indication of the amount of IgM produced per IgM ASC, we evaluated the mean spot size. We observed no measurable differences in mean spot size between heterozygous and wt mice ( Figure 2B). These data indicate that the reduced levels of natural IgM antibodies in the serum is due to fewer IgM ASCs in the heterozygous bumble rather than less IgM secreted per ASC.
heterozygous Bumble Mice have normal Frequencies of the Major B cell subsets The main responding B cell subsets against TI-2 antigens are B-1b cells and MZB cells (3,7,8), while B-1a cells are believed to produce most of the natural IgM antibodies found in the serum at steady state (1). Homozygous bumble mice had normal numbers of follicular B cells, but completely lacked B-1a cells, and displayed a severe reduction in the frequencies of B-1b and MZB cells (15,28). Transfer of wt peritoneal cells to bumble mice completely restored serum natural IgM levels and partly restored the antibody response to immunization with NP-ficoll (15). This suggested that the lack of B-1b cells in homozygous bumble mice formed the basis for the impaired response to T-independent antigens. To investigate if heterozygous bumble mice have defects in B cell development, we used the following strategy to phenotypically distinguish the major B cell subsets. B220 is expressed by all B-2 cells (MZB and follicular B cells), but at lower levels on most B-1 cells. CD11b and CD43 are expressed by B-1 cells, and CD5 is expressed by the B-1a cell subset. CD23 is expressed by B-2 cells, but not MZB cells, while MZB cells express high levels of CD21. When analyzing the different B cell subsets in heterozygous bumble mice, we found that neither overall splenic B cell nor MZB cell numbers were significantly different between wt mice and heterozygous bumble mice. In contrast, mice homozygous for the bumble mutation had significantly reduced MZB cell numbers as expected (p < 0.01) ( Figure 3A). The increased surface IgM level seen in homozygous bumble mice (28) was not observed in the heterozygous state ( Figure 3B). Furthermore, homozygous bumble mice have decreased frequencies of transitional T3 B cells (15), while in heterozygous mice, the T3 B cell frequencies were similar to those of wt mice ( Figure 3C). No differences in bone marrow B cell progenitor populations were observed between wt, homozygous, and heterozygous bumble mice ( Figure 3D).
As we reported previously, homozygous bumble mice completely lacked B-1a cells and had significantly reduced B-1b cell frequencies (p < 0.01) (15). In contrast, peritoneal B-1a and B-1b cell frequencies were similar in wt and heterozygous bumble mice ( Figure 4A). Similarly, bone marrow B-1a cell and spleen B-1a cell frequencies were indifferent between heterozygous bumble and wt mice (Figures 4B,C). Overall, these data indicate that the development and maintenance of all the major B cell subsets occur normally in heterozygous bumble mice.

impaired antibody response to the Ti-2 Vaccine Pneumovax in heterozygous Bumble Mice
We next investigated if the impaired response to TI-2 antigens in heterozygous bumble mice would extend to the clinically relevant human polysaccharide vaccine Pneumovax. To this end, wt, heterozygous, and homozygous bumble mice were immunized with Pneumovax intraperitoneally. By 6 days post-immunization, wt mice mounted a strong antibody response against the Pneumovax vaccine antigens. No response was observed in homozygous bumble mice, while heterozygous bumble mice displayed an intermediary response (Figure 5A). We also investigated the Pneumovax-elicited responses to the polysaccharide antigens type 1 161-X ( Figure 5B) and type 3 169-X (Figure 5C). Similarly to total Pneumovax-specific antibodies, the responses against both polysaccharides were significantly diminished in heterozygous bumble mice compared to in wt mice (p < 0.01).

DiscUssiOn
The data presented here demonstrate that a heterozygous mutation in IκBNS causes a significant impairment of B cell function.
Mice that express one wt allele and one allele containing the bumble mutation in nfkbid had diminished antibody responses to T-independent type II antigens and reduced levels of circulating natural IgM and IgG3 antibodies. The bumble mutation causes a premature stop codon after exon 4 in the nfkbid gene and the resulting transcript encodes only 65 of the 327 aa of the full-length IκBNS (28). The resulting mRNA transcript is likely targeted for degradation by nonsense-mediated decay. Thus, the spontaneous secretion of natural antibodies and responses to TI-2 antigens in bumble heterozygotes are likely impaired as a result of haploinsufficiency for the nfkbid gene. Previous work has shown that p50 −/− , BCL10 −/− , and IκBα hypermorphic mice fail to respond to immunization with TI-2 antigens (32,33), illustrating that this defect is a general feature of deficiency in the classical NF-κB signaling pathway. Similar to other proteins of the classical NF-κB pathway, IκBNS also plays an important role for maintaining normal levels of serum natural antibodies and for the antibody response against TI-2 antigens (27,28). Notably, the major B cell subsets responsible for natural antibody production and response to TI-2 antigens, B-1 and MZB cells are lacking in mice with impaired classical NF-κB signaling, which is also similar for mice with non-functional IκBNS (15,27,28). It was therefore interesting that heterozygous bumble mice had decreased serum IgM and IgG3 levels and defective responses to TI-2 antigens, despite having apparently normal numbers and frequencies of B-1 and MZB cells. This suggested that the lack of specific B cell subsets is not the sole reason for the reduced levels of natural antibodies and response to TI-2 antigens in IκBNSdeficient mice. Rather, this indicated a more direct role of IκBNS for these aspects of immunity downstream of B cell receptor signaling. Particularly, the lack of response to TI-2 antigens of heterozygous bumble mice may be due to a B cell intrinsic defect in activation or ASC differentiation, although it is also possible that the BCR repertoire may be altered when IκBNS is only expressed from one allele. We did not observe any reduction in B-1a, MZB, or follicular B cell numbers in heterozygous bumble mice, suggesting that one functioning allele of nfkbid is enough to facilitate normal B cell development. However, we cannot rule out the possibility that a modest impairment of B cell development is masked by homeostatic proliferative mechanisms or altered bone marrow output to control distribution of immune cells (34).
There are only few reports of heterozygous mutations in the NF-κB pathways leading to loss of B cell function. Hypermorphic heterozygous mutations in IκBα cause impaired antibody responses (33) and CARMA1-deficient mice lacking CARD (ΔCARD) showed defective B cell proliferation at the heterozygous state (35). Since ΔCARD was found to act as a dominant-negative inhibitor of TCR-induced NF-κB activation, the impaired B cell function in heterozygous ΔCARD-deficient mice may be due to the truncated CARMA1 protein interfering with function of the wt protein (36). Protein kinase C-β (PKCβ) initiates a phosphorylation cascade that activates the CBM complex downstream BCR signaling. Heterozygous missense mutations in the gene   16-week-old mice with four to seven mice per group, and results are representative of at least two independent experiments. Graphs display mean + SD. Statistically significant differences between bumble, bumble heterozygotes, and wt mice are indicated by *p < 0.05 and **p < 0.01 by Mann-Whitney test.

Role of Heterozygous IκBNS Mutation
Frontiers in Immunology | www.frontiersin.org encoding PKCβ were found to lead to impaired antipolysaccharide antibody responses and reduced natural antibody levels, despite apparently normal B cell development (37). Several clinical cases with mutations in NF-κB proteins have demonstrated important roles of both the classical and alternative NF-κB signaling pathways for B cell development and function in humans. Hypermorphic mutations in the gene encoding IκBα lead to impaired phosphorylation-driven degradation of the mutant protein and thereby reduced NF-κB signaling (38). Hypermorphic heterozygous mutations in IκBα cause ectodermal dysplasia with immunodeficiency as evidenced by recurrent severe infections (38,39). These patients have increased numbers of B and T cells, but display both B and T cell functional defects (39). Notably, the patient symptoms and lymphocyte functional defects could to a large extent be reproduced when introducing one of the hypermorphic IκBα mutations (S32I) to mice (33). Similar symptoms to IκBα hypermorphs are evident in patients with NEMO deficiency and B cells from these patients do not respond to CD40 ligation (40). More recently, several patients with mutations in the CBM complex have been described. Combined immunodeficiency (CID) resulting from impaired classical NF-κB signaling due to CARMA1 deficiency was associated with hypogammaglobulinaemia, impaired BAFF-R expression and a block of B cell maturation at the transitional B cell stage (41,42). Human MALT1 deficiency, also manifested by CID, was associated with lack of MZB cells and failure to respond to Pneumovax vaccination (43,44), a phenotype that is very similar to that seen in the corresponding mouse model (45). Rapid advances  16-week-old mice with five mice per immunized group, and results are representative of two independent experiments. Graphs display mean + SD. Statistically significant differences between bumble, bumble heterozygotes, and wt mice are indicated by **p < 0.01 by Mann-Whitney test.
in identifying genes underlying human immunodeficiencies will reveal if also IκBNS plays a role in this disease group.
So far, there are few reports of heterozygous mutations in the NF-κB pathway leading to immunodeficiency in humans. Heterozygous gain-of-function mutations in the gene encoding CARMA1 were shown to result in constitutive NF-κB activity and are manifested by lymphocytosis but impaired memory B cells and low TI-2 responses (46,47). It is interesting to speculate if heterozygous mutations in genes encoding components of BCR signaling, including classical NF-κB pathway mediators, may contribute to the observed variability in antipolysaccharide immune responses in the human population (48,49). We describe here that a heterozygous mutation in the nfkbid gene encoding the atypical IκB protein IκBNS led to reduced steady state IgM and IgG3 antibody levels and impaired response to vaccination with TI-2 antigens in mice. Heterozygous mutations in genes of the NF-κB pathway could potentially lead to haploinsufficiency for B cell function, resulting in lower antibody responses to vaccination and increased susceptibility to infection, which should be considered in future studies.    (B) Representative staining of wild type bone marrow for progenitor and mature B cell populations. Pro B (B220 + CD43 + CD19 + CD93 + ), pre B (B220 + CD43 -IgM -CD93 + ), immature B (B220 + CD43 -IgM + CD93 + ), and mature B cells (B220 + CD43 -IgM + CD93 -).