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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. [ABSTRACT FROM AUTHOR]

IκB proteins regulate the inhibition and activation of NF-κB transcription factor complexes. While classical IκB proteins keep NF-κB complexes inactive in the cytoplasm, atypical IκB proteins act on activated NF-κB complexes located in the nucleus. Most of the knowledge regarding the function of IκB proteins has been collected in vitro, while far less is known regarding their impact on activation and regulation of immune responses during in vivo infections. Combining in vivo Listeria monocytogenes (Lm) infection with comparative ex vivo transcriptional profiling of the hepatic response to the pathogen we observed that in contrast to wild type mice that mounted a robust inflammatory response, IκBNS-deficiency was generally associated with a transcriptional repression of innate immune responses. Whole tissue transcriptomics revealed a pronounced IκBNS-dependent reduction of myeloid cell-associated transcripts in the liver together with an exceptionally high Nfkbid promoter activity uncovered in Ly6Chigh inflammatory monocytes prompted us to further characterize the specific contribution of IκBNS in the inflammatory response of monocytes to the infectious agent. Indeed, Ly6Chigh monocytes primed during Lm infection in the absence of IκBNS displayed a blunted response compared to wild type-derived Ly6Chigh monocytes as evidenced by the reduced early expression of hallmark transcripts of monocyte-driven inflammation such as Il6, Nos2 and Il1β. Strikingly, altered monocyte activation in IκBNS-deficient mice was associated with an exceptional resistance against Lm infection and protection was associated with a strong reduction in immunopathology in Lm target organs. Of note, mice lacking IκBNS exclusively in myeloid cells failed to resist Lm infection, indicating that the observed effect was not monocyte intrinsic but monocyte extrinsic. While serum cytokine-profiling did not discover obvious differences between wild type and IκBNS-/- mice for most of the analyzed mediators, IL-10 was virtually undetectable in IκBNS-deficient mice, both in the steady state and following Lm infection. Together, we show here a crucial role for IκBNS during Lm infection with IκBNS-deficient mice showing an overall blunted pro-inflammatory immune response attributed to a reduced pro-inflammatory signature in Ly6Chigh monocytes. Reduced immunopathology and complete protection of mice against an otherwise fatal Lm infection identified IκBNS as molecular driver of inflammation in listeriosis.

Mice lacking the atypical inhibitory kappa B (IκB) protein, IκBNS, a regulator of the NF-κB pathway encoded by the nfkbid gene, display impaired antibody responses to both T cell-independent (TI) and T cell-dependent (TD) antigens. To better understand the basis of these defects, we crossed mice carrying floxed nfkbid alleles with mice expressing Cre under the transcriptional control of the Cd79a gene to create mice that lacked IκBNS expression only in B cells. Analyses of these conditional knock-out mice revealed intact CD4+ and CD8+ T cell populations, including preserved frequencies of FoxP3+ regulatory T cells, which are known to be reduced in IκBNS knock-out mice. Like IκBNS knock-out mice, mice with conditional IκBNS ablation in B cells displayed defective IgM responses to TI antigens and a severe reduction in peritoneal B-1a cells. However, in contrast to mice lacking IκBNS altogether, the conditional IκBNS knock-out mice responded well to TD antigens compared to the control mice, with potent IgG responses following immunization with the viral antigen, rSFV-βGal or the widely used hapten-protein model antigen, NP-CGG. Furthermore, B cell intrinsic IκBNS expression was dispensable for germinal center (GC) formation and T follicular helper cell responses to NP-CGG immunization. The results presented here suggest that the defect in antibody responses to TD antigens observed in IκBNS knock-out mice results from a B cell extrinsic defect.

Regulatory T cells (Treg cells) act as a major rheostat regulating the strength of immune responses, enabling tolerance of harmless foreign antigens, and preventing the development of pathogenic immune responses in various disease settings such as cancer and autoimmunity. Treg cells are present in all lymphoid and non-lymphoid tissues, and the latter often fulfill important tasks required for the physiology of their host organ. The activation of NF-κB transcription factors is a central pathway for the reprogramming of gene expression in response to inflammatory but also homeostatic cues. Genetic mouse models have revealed essential functions for NF-κB transcription factors in modulating Treg development and function, with some of these mechanistic insights confirmed by recent studies analyzing Treg cells from patients harboring point mutations in the genes encoding NF-κB proteins. Molecular insights into the NF-κB pathway in Treg cells hold substantial promise for novel therapeutic strategies to manipulate dysfunctional or inadequate cell numbers of immunosuppressive Treg cells in autoimmunity or cancer. Here, we provide an overview of the manifold roles that NF-κB factors exert in Treg cells. [ABSTRACT FROM AUTHOR]

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.

The transcriptional factor NF-κB is a nuclear factor involved in both physiological and pathological processes. This factor can control the transcription of more than 400 genes, including cytokines, chemokines, and their modulators, immune and non-immune receptors, proteins involved in antigen presentation and cell adhesion, acute phase and stress response proteins, regulators of apoptosis, growth factors, other transcription factors and their regulators, as well as different enzymes; all these molecules control several biological processes. NF-κB is a tightly regulated molecule that has also been related to apoptosis, cell proliferation, inflammation, and the control of innate and adaptive immune responses during onset of labor, in which it has a crucial role; thus, early activation of this factor may have an adverse effect, by inducing premature termination of pregnancy, with bad outcomes for the mother and the fetus, including product loss. Reviews compiling the different activities of NF-κB have been reported. However, an update regarding NF-κB regulation during pregnancy is lacking. In this work, we aimed to describe the state of the art around NF-κB activity, its regulatory role in pregnancy, and the effect of its dysregulation due to invasion by pathogens like Trichomonas vaginalis and Toxoplasma gondii as examples.

The transcriptional factor NF-κB is a nuclear factor involved in both physiological and pathological processes. This factor can control the transcription of more than 400 genes, including cytokines, chemokines, and their modulators, immune and non-immune receptors, proteins involved in antigen presentation and cell adhesion, acute phase and stress response proteins, regulators of apoptosis, growth factors, other transcription factors and their regulators, as well as different enzymes; all these molecules control several biological processes. NF-κB is a tightly regulated molecule that has also been related to apoptosis, cell proliferation, inflammation, and the control of innate and adaptive immune responses during onset of labor, in which it has a crucial role; thus, early activation of this factor may have an adverse effect, by inducing premature termination of pregnancy, with bad outcomes for the mother and the fetus, including product loss. Reviews compiling the different activities of NF-κB have been reported. However, an update regarding NF-κB regulation during pregnancy is lacking. In this work, we aimed to describe the state of the art around NF-κB activity, its regulatory role in pregnancy, and the effect of its dysregulation due to invasion by pathogens like Trichomonas vaginalis and Toxoplasma gondii as examples. [ABSTRACT FROM AUTHOR]

Acquisition of effector functions in T cells is guided by transcription factors, including NF‐κB, that itself is tightly controlled by inhibitory proteins. The atypical NF‐κB inhibitor, IκBNS, is involved in the development of Th1, Th17, and regulatory T (Treg) cells. However, it remained unclear to which extend IκBNS contributed to the acquisition of effector function in T cells specifically responding to a pathogen during in vivo infection. Tracking of adoptively transferred T cells in Listeria monocytogenes infected mice antigen‐specific activation of CD4+ T cells following in vivo pathogen encounter to strongly rely on IκBNS. While IκBNS was largely dispensable for the acquisition of cytotoxic effector function in CD8+ T cells, IκBNS‐deficient Th1 effector cells exhibited significantly reduced proliferation, marked changes in the pattern of activation marker expression, and reduced production of the Th1‐cell cytokines IFN‐γ, IL‐2, and TNF‐α. Complementary in vitro analyses using cells from novel reporter and inducible knockout mice revealed that IκBNS predominantly affects the early phase of Th1‐cell differentiation while its function in terminally differentiated cells appears to be negligible. Our data suggest IκBNS as a potential target to modulate specifically CD4+ T‐cell responses. [ABSTRACT FROM AUTHOR]

Impaired classical NF‐κB pathway signaling causes reduced antibody responses to T‐independent (TI) antigens. We investigated the potential reasons for defective TI responses in mice lacking the atypical inhibitory kappa B (IκB) protein of the NF‐κB pathway, IκBNS. Analyses of the plasma cell compartment in vitro and in vivo after challenge with lipopolysaccharide (LPS) showed significant decreases in the frequencies of plasma cells in the absence of IκBNS. In vitro activation of B cells via the B cell receptor or via Toll‐like receptor 4 revealed that early activation events were unaffected in IκBNS‐deficient B cells, while proliferation was reduced compared to in similarly stimulated wildtype (wt) B cells. IκBNS‐deficient B cells also displayed impaired upregulation of the transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI), which is essential for TI responses, and decreased sensitivity to TACI ligands upon stimulation. Furthermore, IκBNS‐deficient B cells, in contrast to wt B cells, displayed altered expression of IRF4, Blimp‐1 and Pax5 upon LPS‐induced differentiation, indicating impaired transcriptional regulation of plasma cell generation. [ABSTRACT FROM AUTHOR]

IκBNS is a nuclear IκB protein which negatively regulates nuclear factor-κB activity. We demonstrated that IκBNS deficiency accelerates atherosclerosis in LDL receptor-deficient (LDLr−/−) mice via increased interleukin (IL)-6 production by macrophages. Previous studies showed that the increase in IL-6 might contribute to the development of atherosclerotic lesions. However, whether an anti-mouse IL-6 receptor antibody (MR16-1) can protect atherosclerotic lesions in atherogenic mice remains to be elucidated. We investigated atherosclerotic lesions in LDLr−/− and IκBNS−/−/LDLr−/− mice after 16 weeks consumption of a high-fat diet. All mice received intraperitoneal injections of MR16-1 or phosphate-buffered saline (PBS) (control) once a week during a high-fat diet consumption. Treatment of MR16-1 yielded no adverse systemic effects, and we detected no significant differences in serum cholesterol levels in either group. The atherosclerotic lesions were significantly increased in IκBNS−/−/LDLr−/− compared with LDLr−/− mice (p

Background In allergic asthma, environmental allergens including house dust mite ( HDM) trigger pattern recognition receptors and activate downstream signaling pathways including NF-κB pathways not only in immune cells but also in airway epithelial cells. Recent studies have shown that NF-κB activation is regulated positively or negatively depending on the cellular context by Iκ BNS (encoded by the gene Nfkbid), one of atypical IκB proteins, in the nucleus. Therefore, we hypothesized that Iκ BNS expressed in immune cells or epithelial cells is involved in the regulation of asthmatic responses. Aim To determine the roles of Iκ BNS in HDM-induced asthmatic responses. Methods Roles of Iκ BNS in HDM-induced airway inflammation and airway hyper-responsiveness ( AHR) were examined by using Iκ BNS-deficient ( Nfkbid −/−) mice. Roles of Iκ BNS expressed in hematopoietic cells and nonhematopoietic cells were separately evaluated by bone marrow chimeric mice. Roles of Iκ BNS expressed in murine tracheal epithelial cells (m TECs) were examined by air-liquid interface culture. Results House dust mite-induced airway inflammation and AHR were exacerbated in mice lacking Iκ BNS in hematopoietic cells. In contrast, HDM-induced airway inflammation was exacerbated, but AHR was attenuated in mice lacking Iκ BNS in nonhematopoietic cells. The induction of Muc5ac, a representative mucin in asthmatic airways, was reduced in Nfkbid −/− m TEC, whereas the induction of Spdef, a master regulator of goblet cell metaplasia, was not impaired in Nfkbid −/− m TEC. Moreover, Iκ BNS bound to and activated the MUC5 AC distal promoter in epithelial cells. Conclusion Iκ BNS is involved in inducing Muc5ac expression in lung epithelial cells and causing AHR in HDM-induced asthma models. [ABSTRACT FROM AUTHOR]

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 ENU 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.

Although a major function of B cells is to mediate humoral immunity by producing antigen-specific antibodies, a specific subset of B cells is important for immune suppression, which is mainly mediated by the secretion of the anti-inflammatory cytokine interleukin-10 (IL-10). However, the mechanism by which IL-10 is induced in B cells has not been fully elucidated. Here, we report that IκBNS, an inducible nuclear IκB protein, is important for Toll-like receptor (TLR)-mediated IL-10 production in B cells. Studies using IκBNS knockout mice revealed that the number of IL-10-producing B cells is reduced in IκBNS-/- spleens and that the TLR-mediated induction of cytoplasmic IL-10-positive cells and IL-10 secretion in B cells are impaired in the absence of IκBNS. The impairment of IL-10 production by a lack of IκBNS was not observed in TLR-triggered macrophages or T-cell-receptor-stimulated CD4+ CD25+ T cells. In addition, IκBNS-deficient B cells showed reduced expression of Prdm1 and Irf4 and failed to generate IL-10+ CD138+ plasmablasts. These results suggest that IκBNS is selectively required for IL-10 production in B cells responding to TLR signals, so defining an additional role for IκBNS in the control of the B-cell-mediated immune responses. [ABSTRACT FROM AUTHOR]

NF-κB transcription factors play essential roles in hematopoiesis. In this review, we summarize the requirements of different components of the NF-κB pathway for B-1 cell development and maintenance. The B-1 cell developmental steps are also reviewed, with particular emphasis on stages where NF-κB signaling may be critical. [ABSTRACT FROM AUTHOR]

The dynamic regulation of NF-κB activity in the uterus maintains a favorable environment of cytokines necessary to prepare for pregnancy throughout the estrous cycle. Recently, the mechanisms that directly regulate the NF-κB transcriptional activity in different tissues are of growing interest. IκBNS and BCL-3 are negative nuclear regulators of NF-κB activity that regulate IL-6 and TNF-α transcription, respectively. Both cytokines have been described as important factors in the remodeling of uterus for blastocyst implantation. In this work we analyzed in ICR mice the mRNA expression and protein production profile of IL-6, TNF-α, and their correspondent negative transcription regulators IκBNS or BCL-3 using real-time PCR, western blot and immunochemistry. We found that the expression of TNF-α and IL-6 was oscillatory along the estrous cycle, and its low expression coincided with the presence of BCL-3 and IκBNS, and vice versa, when the presence of the regulators was subtle, the expression of TNF-α and IL-6 was exacerbated. When we compared the production of TNF-α and IL-6 in the different estrous stages relating with diestrus we found that at estrus there is an important increase of the cytokines ( p < 0.05) decreasing at metestrus to reach the basal expression at diestrus. In the immunochemistry analysis we found that at diestrus BCL-3 is distributed all over the tissue with a barely detected TNF-α, but on the contrary, at estrus the expression of BCL-3 is not detected with TNF-α clearly observable along the tissue; the same phenomenon occur in the analysis of IκBNS and IL-6. With that evidence we suggest that the expression of TNF-α and IL-6 might be regulated through NF-κB nuclear regulators BCL-3 and IκBNS in the uterus of mice as has been demonstrated in other systems. [ABSTRACT FROM AUTHOR]

IκB proteins regulate the inhibition and activation of NF-κB transcription factor complexes. While classical IκB proteins keep NF-κB complexes inactive in the cytoplasm, atypical IκB proteins act on activated NF-κB complexes located in the nucleus. Most of the knowledge regarding the function of IκB proteins has been collected in vitro , while far less is known regarding their impact on activation and regulation of immune responses during in vivo infections. Combining in vivo Listeria monocytogenes (Lm) infection with comparative ex vivo transcriptional profiling of the hepatic response to the pathogen we observed that in contrast to wild type mice that mounted a robust inflammatory response, IκB NS -deficiency was generally associated with a transcriptional repression of innate immune responses. Whole tissue transcriptomics revealed a pronounced IκB NS -dependent reduction of myeloid cell-associated transcripts in the liver together with an exceptionally high Nfkbid promoter activity uncovered in Ly6C high inflammatory monocytes prompted us to further characterize the specific contribution of IκB NS in the inflammatory response of monocytes to the infectious agent. Indeed, Ly6C high monocytes primed during Lm infection in the absence of IκB NS displayed a blunted response compared to wild type-derived Ly6C high monocytes as evidenced by the reduced early expression of hallmark transcripts of monocyte-driven inflammation such as Il6 , Nos2 and Il1β . Strikingly, altered monocyte activation in IκB NS -deficient mice was associated with an exceptional resistance against Lm infection and protection was associated with a strong reduction in immunopathology in Lm target organs. Of note, mice lacking IκB NS exclusively in myeloid cells failed to resist Lm infection, indicating that the observed effect was not monocyte intrinsic but monocyte extrinsic. While serum cytokine-profiling did not discover obvious differences between wild type and IκB NS -/- mice for most of the analyzed mediators, IL-10 was virtually undetectable in IκB NS -deficient mice, both in the steady state and following Lm infection. Together, we show here a crucial role for IκB NS during Lm infection with IκB NS -deficient mice showing an overall blunted pro-inflammatory immune response attributed to a reduced pro-inflammatory signature in Ly6C high monocytes. Reduced immunopathology and complete protection of mice against an otherwise fatal Lm infection identified IκB NS as molecular driver of inflammation in listeriosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Frentzel, Jeron, Pausder, Kershaw, Volckmar, Schmitz and Bruder.)

Highlights: [•] mIκBNS is degraded rapidly by proteasome without ubiquitylation. [•] N-terminal PEST sequence is responsible for the unstable nature of mIκBNS. [•] PEST sequence is not critical for nuclear localization of mIκBNS. [•] There is single bona fide NLS at the C-terminus of mIκBNS. [Copyright &y& Elsevier]

Mice lacking the atypical inhibitory kappa B (IκB) protein, IκBNS, a regulator of the NF-κB pathway encoded by the nfkbid gene, display impaired antibody responses to both T cell-independent (TI) and T cell-dependent (TD) antigens. To better understand the basis of these defects, we crossed mice carrying floxed nfkbid alleles with mice expressing Cre under the transcriptional control of the Cd79a gene to create mice that lacked IκBNS expression only in B cells. Analyses of these conditional knock-out mice revealed intact CD4 + and CD8 + T cell populations, including preserved frequencies of FoxP3 + regulatory T cells, which are known to be reduced in IκBNS knock-out mice. Like IκBNS knock-out mice, mice with conditional IκBNS ablation in B cells displayed defective IgM responses to TI antigens and a severe reduction in peritoneal B-1a cells. However, in contrast to mice lacking IκBNS altogether, the conditional IκBNS knock-out mice responded well to TD antigens compared to the control mice, with potent IgG responses following immunization with the viral antigen, rSFV-βGal or the widely used hapten-protein model antigen, NP-CGG. Furthermore, B cell intrinsic IκBNS expression was dispensable for germinal center (GC) formation and T follicular helper cell responses to NP-CGG immunization. The results presented here suggest that the defect in antibody responses to TD antigens observed in IκBNS knock-out mice results from a B cell extrinsic defect., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Khoenkhoen, Ádori, Solís-Sayago, Soulier, Russell, Beutler, Pedersen and Karlsson Hedestam.)

• IκBNS-deficient B cells display increased Blimp-1 expression and mTORC1 activation after LPS stimulation, but fail to form fully functional plasma cells. • B cell oxygen consumption is higher in absence of IκBNS. • Mitochondrial mass and ROS production are increased in IκBNS-deficient B cells. Mutations causing loss of the NF-κB regulator IκBNS, result in impaired development of innate-like B cells and defective plasma cell (PC) differentiation. Since productive PC differentiation requires B cell metabolic reprogramming, we sought to investigate processes important for this transition using the bumble mouse strain, deficient for IκBNS. We report that LPS-activated bumble B cells exhibited elevated mTOR activation levels, mitochondrial accumulation, increased OXPHOS and mROS production, along with a reduced capacity for autophagy, compared to wildtype B cells. Overall, our results demonstrate that PC differentiation in the absence of IκBNS is characterized by excessive activation during early rounds of B cell division, increased mitochondrial metabolism and decreased autophagic capacity, thus improving our understanding of the role of IκBNS in PC differentiation. [ABSTRACT FROM AUTHOR]

Aims IκBNS regulates a subset of Toll-like receptor (TLR)-dependent genes including interleukin-6 (IL-6) by inhibiting nuclear factor-κB (NF-κB). IL-6 is an inflammatory biomarker for cardiovascular diseases. The aim of this study was to determine whether IκBNS changes arterial inflammation and intimal hyperplasia after vascular injury. Methods and results We investigated neointimal formation in IκBNS-deficient (IκBNS−/−; C57BL/6 background) and wild-type (IκBNS+/+) mice 2 weeks after cuff injury. The mean intimal area and the intima/media ratio of IκBNS−/− mice increased 89% (8066 ± 1141 vs. 4267 ± 1095 μm2; P = 0.027) and 100% (0.72 ± 0.13 vs. 0.36 ± 0.09; P = 0.032) compared with IκBNS+/+ mice. We observed significant up-regulation of TLR4 in injured arteries of IκBNS−/− mice. NF-κB activity in the intima of IκBNS−/− mice was 5.1-fold higher (P = 0.008) compared with IκBNS+/+ mice at 7 days post-injury. IL-6 mRNA levels in injured arteries of IκBNS−/− mice were 1.8-fold higher (P = 0.002) compared with those of IκBNS+/+ mice at 3 days post-injury. Vascular smooth muscle cells from IκBNS−/− mice showed a significant increase in cell migration compared with those from IκBNS+/+ mice after IL-6 stimulation in the scratch-wound healing assay. Furthermore, anti-mouse IL-6 receptor antibody (MR16-1) significantly reduced intimal hyperplasia compared with control IgG injection in IκBNS−/− mice. These findings suggest that IL-6 participates in the development of neointimal hyperplasia after vascular injury in IκBNS−/− mice. Conclusion IκBNS down-regulates TLR4 expression, NF-κB activity, and IL-6 production after vascular injury. IκBNS might suppress intimal hyperplasia caused by vascular inflammation such as atherosclerosis, and restenosis after angioplasty. [ABSTRACT FROM PUBLISHER]

Acquisition of effector functions in T cells is guided by transcription factors, including NF-κB, that itself is tightly controlled by inhibitory proteins. The atypical NF-κB inhibitor, IκB NS, is involved in the development of Th1, Th17, and regulatory T (Treg) cells. However, it remained unclear to which extend IκB NS contributed to the acquisition of effector function in T cells specifically responding to a pathogen during in vivo infection. Tracking of adoptively transferred T cells in Listeria monocytogenes infected mice antigen-specific activation of CD4 + T cells following in vivo pathogen encounter to strongly rely on IκB NS . While IκB NS was largely dispensable for the acquisition of cytotoxic effector function in CD8 + T cells, IκB NS -deficient Th1 effector cells exhibited significantly reduced proliferation, marked changes in the pattern of activation marker expression, and reduced production of the Th1-cell cytokines IFN-γ, IL-2, and TNF-α. Complementary in vitro analyses using cells from novel reporter and inducible knockout mice revealed that IκB NS predominantly affects the early phase of Th1-cell differentiation while its function in terminally differentiated cells appears to be negligible. Our data suggest IκB NS as a potential target to modulate specifically CD4 + T-cell responses., (© 2019 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: IκBNS, a nuclear IκB protein, regulates a subset of Toll-like receptor (TLR) dependent genes. A cholate-containing high-fat diet (HFD(CA(+))) induces TLR4 mediated early inflammatory response. The present study aims to clarify that the lack of IκBNS promotes atherogenesis in low-density lipoprotein receptor-deficient (LDLr -/- ) mice fed HFD(CA(+)) compared with those fed a cholate-free HFD (HFD(CA(-)))., Methods and Results: Mice that lacked IκBNS (IκBNS -/- ) were crossed with LDLr -/- mice and formation of atherosclerotic lesions was analyzed after 6-week consumption of HFD(CA(+)) or HFD(CA(-)). IκBNS -/- /LDLr -/- mice fed HFD(CA(+)) (IκBNS -/- /LDLr -/- (CA(+))) showed a 3.5-fold increase of atherosclerotic lesion size in the aorta compared with LDLr -/- (CA(+)) mice ( p  < 0.01), whereas there was no difference between LDLr -/- (CA(-)) and IκBNS -/- /LDLr -/- (CA(-)) mice. Immunohistochemical analysis of the aortic root revealed that HFD(CA(+)) significantly increased Mac-3 (macrophage)-positive area by 1.5-fold ( p  < 0.01) and TLR4, interleukin-6 (IL-6) expression by 1.7-fold ( p  < 0.05) and 1.5-fold ( p  < 0.05), respectively, in IκBNS -/- /LDLr -/- (CA(+)) compared with LDLr -/- --(CA(+)) mice. Furthermore, active STAT3 (pSTAT3)-positive cells were significantly increased by 1.7-fold in the lesions of IκBNS -/- /LDLr -/- (CA(+)) compared with LDLr -/- (CA(+)) mice ( p  < 0.01). These findings suggest that IκBNS deficiency and HFD(CA(+)) promote atherogenesis in LDLr -/- mice via TLR4/IL-6/STAT3 pathway. Finally, we showed that the monocytes from peripheral blood of IκBNS -/- /LDLr -/- (CA(+)) mice were found to contain the highest proportion of Ly6C hi monocytes among the four groups, suggesting that lack of IκBNS enhanced inflammation in response to HFD(CA(+)) feeding., Conclusions: The present study is the first to demonstrate that the activation of innate immune system using HFD(CA(+)) induced significant inflammation and atherogenesis in IκBNS -/- /LDLr -/- compared with LDLr -/- mice.

IκBNS is a nuclear IκB protein which negatively regulates nuclear factor-κB activity. We demonstrated that IκBNS deficiency accelerates atherosclerosis in LDL receptor-deficient (LDLr -/- ) mice via increased interleukin (IL)-6 production by macrophages. Previous studies showed that the increase in IL-6 might contribute to the development of atherosclerotic lesions. However, whether an anti-mouse IL-6 receptor antibody (MR16-1) can protect atherosclerotic lesions in atherogenic mice remains to be elucidated. We investigated atherosclerotic lesions in LDLr -/- and IκBNS -/- /LDLr -/- mice after 16 weeks consumption of a high-fat diet. All mice received intraperitoneal injections of MR16-1 or phosphate-buffered saline (PBS) (control) once a week during a high-fat diet consumption. Treatment of MR16-1 yielded no adverse systemic effects, and we detected no significant differences in serum cholesterol levels in either group. The atherosclerotic lesions were significantly increased in IκBNS -/- /LDLr -/- compared with LDLr -/- mice ( p  < 0.01) under treatment of PBS. However, MR16-1 treatment abolished the significant difference of atherosclerotic lesions between IκBNS -/- /LDLr -/- and LDLr -/- mice. Interestingly, MR16-1 also significantly decreased atherosclerotic lesions in LDLr -/- mice compared with PBS treatment ( p  < 0.05). Immunostaining revealed percent phospho-STAT3-positive cell were significantly decreased in the atherosclerotic lesions of MR16-1 treated both IκBNS -/- /LDLr -/- and LDLr -/- mice compared with PBS-treated mice, indicating MR16-1 could suppress atherosclerotic lesions via the inhibition of IL-6-STAT3 signaling pathway. This study highlights the potential therapeutic benefit of anti-IL-6 therapy in preventing atherogenesis induced by dyslipidemia and/or inflammation.

The nuclear protein IκBζ, comprising the N-terminal trans-activation domain and the C-terminal ankyrin repeat (ANK) domain composed of seven ANK motifs, activates transcription of a subset of nuclear factor-κB (NF-κB)-dependent innate immune genes such as Lcn2 encoding the antibacterial protein lipocalin-2. Lcn2 activation requires formation of a complex containing IκBζ and NF-κB p50, a transcription factor that harbors the DNA-binding Rel homology region but lacks a trans-activation domain, on the promoter with the canonical NF-κB-binding site (κB site) and its downstream cytosine-rich element. Here we show that IκBζ productively interacts with p50 via Asp-451 in the N terminus of ANK1, a residue that is evolutionarily conserved among IκBζ and the related nuclear IκB proteins Bcl-3 and IκBNS Threonine substitution for Asp-451 abrogates direct association with the κB-site-binding protein p50, complex formation with the Lcn2 promoter DNA, and activation of Lcn2 transcription. The basic residues Lys-717 and Lys-719 in the C-terminal region of ANK7 contribute to IκBζ binding to the Lcn2 promoter, probably via interaction with the cytosine-rich element required for Lcn2 activation; glutamate substitution for both lysines results in a loss of transcriptionally active complex formation without affecting direct contact of IκBζ with p50. Both termini of the ANK domain in Bcl-3 and IκBNS function in a manner similar to that of IκBζ to interact with promoter DNA, indicating a common mechanism in which the nuclear IκBs form a regulatory complex with NF-κB and promoter DNA via the invariant aspartate in ANK1 and the conserved basic residues in ANK7., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Although a major function of B cells is to mediate humoral immunity by producing antigen-specific antibodies, a specific subset of B cells is important for immune suppression, which is mainly mediated by the secretion of the anti-inflammatory cytokine interleukin-10 (IL-10). However, the mechanism by which IL-10 is induced in B cells has not been fully elucidated. Here, we report that IκBNS , an inducible nuclear IκB protein, is important for Toll-like receptor (TLR)-mediated IL-10 production in B cells. Studies using IκB(NS) knockout mice revealed that the number of IL-10-producing B cells is reduced in IκB(NS)(-/-) spleens and that the TLR-mediated induction of cytoplasmic IL-10-positive cells and IL-10 secretion in B cells are impaired in the absence of IκB(NS). The impairment of IL-10 production by a lack of IκB(NS) was not observed in TLR-triggered macrophages or T-cell-receptor-stimulated CD4(+) CD25(+) T cells. In addition, IκB(NS)-deficient B cells showed reduced expression of Prdm1 and Irf4 and failed to generate IL-10(+) CD138(+) plasmablasts. These results suggest that IκB(NS) is selectively required for IL-10 production in B cells responding to TLR signals, so defining an additional role for IκB(NS) in the control of the B-cell-mediated immune responses., (© 2016 John Wiley & Sons Ltd.)

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.