NF‐κB1, NF‐κB2 and c‐Rel differentially regulate susceptibility to colitis‐associated adenoma development in C57BL/6 mice

NF‐κB signalling is an important factor in the development of inflammation‐associated cancers. Mouse models of Helicobacter‐induced gastric cancer and colitis‐associated colorectal cancer have demonstrated that classical NF‐κB signalling is an important regulator of these processes. In the stomach, it has also been demonstrated that signalling involving specific NF‐κB proteins, including NF‐κB1/p50, NF‐κB2/p52, and c‐Rel, differentially regulate the development of gastric pre‐neoplasia. To investigate the effect of NF‐κB subunit loss on colitis‐associated carcinogenesis, we administered azoxymethane followed by pulsed dextran sodium sulphate to C57BL/6, Nfkb1^?/?, Nfkb2^?/?, and c‐Rel^?/?mice. Animals lacking the c‐Rel subunit were more susceptible to colitis‐associated cancer than wild‐type mice, developing 3.5 times more colonic polyps per animal than wild‐type mice. Nfkb2^?/? mice were resistant to colitis‐associated cancer, developing fewer polyps per colon than wild‐type mice (median 1 compared to 4). To investigate the mechanisms underlying these trends, azoxymethane and dextran sodium sulphate were administered separately to mice of each genotype. Nfkb2^?/? mice developed fewer clinical signs of colitis and exhibited less severe colitis and an attenuated cytokine response compared with all other groups following DSS administration. Azoxymethane administration did not fully suppress colonic epithelial mitosis in c‐Rel^?/? mice and less colonic epithelial apoptosis was also observed in this genotype compared to wild‐type counterparts. These observations demonstrate different functions of specific NF‐κB subunits in this model of colitis‐associated carcinogenesis. NF‐κB2/p52 is necessary for the development of colitis, whilst c‐Rel‐mediated signalling regulates colonic epithelial cell turnover following DNA damage. © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Interaction of Bordetella pertussis filamentous hemagglutinin with human TLR2: identification of the TLR2‐binding domain

Filamentous hemagglutinin (FHA) is a major adhesion and virulence factor of Bordetella pertussis and also a main component of acellular pertussis vaccines. Interaction of FHA with different receptors on human epithelial and immune cells facilitates entrance and colonization of bacteria as well as immunomodulation of the host immune response. Three overlapping segments of the FHA gene were cloned in a prokaryotic expression vector and the recombinant proteins were purified. These recombinant fragments along with the native FHA protein were employed to assess their potential Toll‐like receptor (TLR) stimulatory effects and to localize the TLR binding region. TLR stimulation was monitored by applying HEK293‐Blue cell lines cotransfected with TLR2, 4, or 5 and a NF‐κB reporter gene. Culture supernatants were checked for secretion of the reporter gene product and IL‐8 as indicators of TLR stimulation. Native FHA was found to strongly stimulate TLR2, but not TLR4 or TLR5 transfected cells. Among recombinant FHA fragments only the fragment spanning amino acid residues 1544–1917 was able to exhibit the TLR2 stimulating property of FHA. Interaction of FHA with TLR2 suggests its involvement in induction of the innate immune system against Bordetella pertussis. The TLR2‐binding domain of FHA may contribute to immunoprotection against pertussis infection.     

Brain‐Derived Neurotrophic Factor Enhances Bcl‐xL Expression Through Protein Kinase Casein Kinase 2‐Activated and Nuclear Factor Kappa B‐Mediated Pathway in Rat Hippocampus

Brain‐derived neurotrophic factor (BDNF) was shown to produce its neuroprotective effect through extracellular signal‐regulated kinase 1/2 (ERK1/2) and phosphatidylinositol‐3 kinase (PI3‐K) signaling. But whether other pathways also mediate the neuroprotective effect of BDNF is less known. In this study, we found that direct administration of BDNF to rat hippocampal CA1 area dose‐dependently increased the mRNA and protein levels of Bcl‐xL. BDNF also increased protein kinase casein kinase II (CK2) activity and NF‐κB phosphorylation at Ser529 dose‐dependently. Further, transfection of the wild‐type CK2α DNA to CA1 neurons increased nuclear factor kappa B (NF‐κB) phosphorylation and Bcl‐xL mRNA expression, whereas transfection of CK2α156A, the catalytically inactive mutant of CK2α, decreased these measures. Moreover, transfection of CK2α small interfering RNA (siRNA) blocked the enhancing effect of BDNF on NF‐κB phosphorylation and Bcl‐xL expression. These results were further confirmed by treatment of 4,5,6,7‐tetrabromobenzotriazole (TBB), a specific CK2 inhibitor. Transfection of NF‐κBS529A, the dominant negative mutant of NF‐κB, prevented the enhancing effect of BDNF on Bcl‐xL expression. More importantly, BDNF activation of CK2 is not affected by co‐administration of the ERK1/2 inhibitor, PD98059, and the PI3‐K inhibitor, LY294002. These results demonstrate a novel BDNF signaling pathway and provide an alternative therapeutic strategy for the protective effect of BDNF on hippocampal neurons in vivo.     

PKC,ERK/p38 MAP kinases and NF‐κB targeted signalling play a role in the expression and release of IL‐1β and CXCL8 in Porphyromonas gingivalis‐infected THP1 cells

Porphyromonas gingivalis is a keystone pathogen in periodontitis and is gaining importance in cardiovascular pathogenesis. Protease‐activated receptors (PARs), toll‐like receptors (TLRs) and nucleotide‐binding oligomerization domain (NOD) on monocytes recognize the structural components on P. gingivalis, inducing inflammatory intermediates. Here, we elucidate the modulation of PARs, TLRs, NODs, and the role of MAPK and NF‐κB in IL‐1β and CXCL8 release. THP1 cells were stimulated with P. gingivalis wild‐type W50 and its isogenic gingipain mutants: Rgp mutant E8 and Kgp mutant K1A. We observed modulation of PARs, TLRs, NOD, IL‐1β and CXCL8 expression by P. gingivalis. Gingipains hydrolyse IL‐1β and CXCL8, which is more evident for IL‐1β accumulation at 24 h. Inhibition of PKC (protein kinase C), p38 and ERK (extracellular signal‐regulated kinases) partially reduced P. gingivalis‐induced IL‐1β at 6 h, whereas PKC and ERK reduced CXCL8 at both 6 and 24 h. Following NF‐κB inhibition, P. gingivalis‐induced IL‐1β and CXCL8 were completely suppressed to basal levels. Overall, TLRs, PARs and NOD possibly act in synergy with PKC, MAPK ERK/p38 and NF‐κB in P. gingivalis‐induced IL‐1β and CXCL8 release from THP1 cells. These pro‐inflammatory cytokines could affect leucocytes in circulation and exacerbate other vascular inflammatory conditions such as atherosclerosis.     

Synergistic regulation of Pseudomonas aeruginosa‐induced cytokine production in human monocytes by mannose receptor and TLR2

The immune response to pathogen is regulated by a combination of specific PRR, which are involved in pathogen recognition. Pseudomonas aeruginosa, a bacterium that causes life‐threatening disease in immuno‐compromised host, is recognized by distinct members of the TLR family. We have previously shown that viable P. aeruginosa bacteria are recognized by human monocytes mainly through TLR2. Using ligand‐specific blocking antibodies, we herein show that the mannose receptor (MR), a phagocytic receptor for unopsonized P. aeruginosa bacteria, contributes equally to TLR2 in proinflammatory cytokine production by human monocytes in response to P. aeruginosa infection. Synergy of both receptors totally controls the immune response. Viable P. aeruginosa bacteria activate NF‐κB and MAPK pathways and enhance TLR2‐mediated signaling in MR‐transfected human embryonic kidney 293 cells. Moreover, MR follows the same kinetics and colocalizes with TLR2 in the endosome during in vivo infection of human macrophages with P. aeruginosa. The studies provide the first demonstration of a significant role for MR, synergistic with TLR2, in activating a proinflammatory response to P. aeruginosa infection.     

NF‐κB2/p100 deficiency impairs immune responses to T‐cell‐independent type 2 antigens

Formation of the splenic marginal zone (MZ) depends on the alternative NF‐κB signaling pathway. Recently, we reported that unrestricted activation of this pathway in NF‐κB2/p100‐deficient (p100^?/?) knock‐in mice alters the phenotype of MZ stroma and B cells. Here, we show that lack of the p100 inhibitor resulted in an expansion of both MZ B and peritoneal B‐1 cells. However, these cells failed to generate proliferating blasts in response to T‐cell‐independent type 2 (TI‐2) Ags, correlating with dampened IgM and absent IgG3 responses. This phenotype was in part due to increased activity of the NF‐κB subunit RelB. Moreover, p100^?/?→B6 BM chimeras were more susceptible to infection by encapsulated Streptococcus pneumoniae bacteria, pathogens that induce TI‐2 responses. In contrast to the TI‐2 defect, p100 deficiency did not impair immune responses to the TI‐1 Ag LPS and p100^?/? MZ B cells showed normal Ag transportation into B‐cell follicles. Furthermore, p100^?/? MZ B and B‐1 cells failed to respond to TI‐2 Ags in the presence of WT accessory cells. Thus, NF‐κB2/p100 deficiency caused a predominant B‐cell‐intrinsic TI‐2 defect that could largely be attributed to impaired proliferation of plasmablasts. Importantly, p100 was also necessary for efficient defense against clinically relevant TI‐2 pathogens.     

Hepatocyte‐specific activation of NF‐κB does not aggravate chemical hepatocarcinogenesis in transgenic mice

The NF‐κB signalling pathway plays important roles in liver organogenesis and carcinogenesis. Mouse embryos deficient in IKKβ die in mid‐gestation, due to excessive apoptosis of hepatoblasts. Although activation of the NF‐κB signalling pathway has been demonstrated in human hepatocellular carcinoma, the role of NF‐κB is controversial. Here, we have generated transgenic mice in which a constitutively active form of IKKβ was expressed in a hepatocyte‐specific manner. Using electrophoretic mobility shift assay, we documented increased NF‐κB activities and up‐regulated levels of NF‐κB downstream target genes, Bcl‐xL and STAT5, in the transgenic mouse livers. These results confirmed that the NF‐κB pathway was activated in the livers of the transgenic mice. However, there was no significant difference in tumour formation between transgenic and wild‐type mice up to an age of 50 weeks. When we treated the transgenic mice with the chemical carcinogen diethylnitrosamine (DEN), we observed no significant differences in the incidence and size of liver tumours formed in these mice with and without DEN treatment at 35 weeks of age, suggesting that the activated NF‐κB pathway in the livers of the transgenic mice did not enhance hepatocarcinogenesis. Interestingly, some of the transient transgenic embryos (E12.5) had abnormal excessive accumulation of nucleated red blood cells in their developing livers. In summary, NF‐κB activation in hepatocytes did not significantly affect chemical hepatocarcinogenesis. In addition, the TTR/IKKCA transgenic mice may serve as a useful model for studying the role of NF‐κB activation in hepatocarcinogenesis as well as inflammatory and metabolic diseases. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Down‐regulation of miR‐301a suppresses pro‐inflammatory cytokines in Toll‐like receptor‐triggered macrophages

In many types of tumours, especially pancreatic adenocarcinoma, miR‐301a is over‐expressed. This over‐expression results in negative regulation of the target gene of miR‐301a, the nuclear factor‐κB (NF‐κB) repressing factor (NKRF), increasing the activation of NF‐κB and production of NF‐κB‐responsive pro‐inflammatory cytokines such as interleukin‐8, interferon‐β, nitric oxide synthase 2A and cytochrome oxidase subunit 2 (COX‐2). However, in immune cells, mechanisms that regulate miR‐301a have not been reported. Similar to tumour cells, Toll‐like receptor (TLR) ‐activated macrophages produce NF‐κB‐responsive pro‐inflammatory cytokines. Therefore, it is of considerable interest to determine whether miR‐301a regulates the secretion of cytokines by immune cells. In the present study, we demonstrate that the expression of miR‐301a was decreased in TLR‐triggered macrophages. Through targeting NKRF, miR‐301a affected the activity of NF‐κB and the expression of pro‐inflammatory genes downstream of NF‐κB such as COX‐2, prostaglandin E₂ and interleukin‐6. In addition, when lipopolysaccharide‐treated macrophages were simultaneously stimulated with trichostatin A, an inhibitor of histone deacetylases, the expression of miR‐301a increased, whereas NKRF and pro‐inflammatory cytokine expression decreased. However, further investigation revealed that there was no correlation between the induction of miR‐301a and the inhibitory effect of trichostatin A on lipopolysaccharide‐induced gene expression in macrophages. In summary, our study indicates a new mechanism by which miR‐301a regulates inflammatory cytokine expression in macrophages, which may clarify the regulatory role of microRNAs in immune‐mediated inflammatory responses.     

TAK1 maintains the survival of immunoglobulin λ‐chain‐positive B cells

TAK1 (MAP3K7) mediation of the IκB kinase (IKK) complex?nuclear factor‐κB (NF‐κB) pathway is crucial for the activation of immune response and to perpetuate inflammation. Although progress has been made to understand TAK1 function in the B‐cell receptor (BCR) signaling, the physiological roles of TAK1 in B‐cell development, particularly in the bone marrow (BM), remain elusive. Previous studies suggested that the IKK complex is required for the development of immunoglobulin light chain λ‐positive B cells, but not for receptor editing. In contrast, NF‐κB activity is suggested to be involved in the regulation of receptor editing. Thus, NF‐κB signaling in early B‐cell development is yet to be fully characterized. Therefore, we addressed the role of TAK1 in early B‐cell development. TAK1‐deficient mice showed significant reduction of BM Igλ‐positive B‐cell numbers without any alteration in the BCR editing. Furthermore, the expression of survival factor Bcl‐2 was reduced in TAK1‐deficient BM B cells as assessed by microarray and quantitative PCR analyses. Ex vivo over‐expression of exogenous Bcl‐2 enhanced the survival of TAK1‐deficient Igλ‐positive B cells. TAK1–IKK–NF‐κB signaling contributes to the survival of λ‐chain‐positive B cells through NF‐κB‐dependent anti‐apoptotic Bcl‐2 expression.     

New evidence of connections between increased O‐GlcNAcylation and inflammasome in the oral mucosa of patients with oral lichen planus

Oral lichen planus (OLP) is considered a chronic inflammatory immune‐mediated disease of the oral mucosa. Immunopathogenesis of OLP is thought to be associated with cell‐mediated immune dysregulation. O‐GlcNAcylation is a form of reversible glycosylation. It has been demonstrated that O‐GlcNAcylation promoted nuclear factor kappa B (NF‐κB) signalling. Activation of NF‐кB can induce expression of nucleotide‐binding domain‐like receptor family pyrin domain containing 3 (NLRP3) inflammasome, which is a large intracellular multi‐protein complex involving an immune response. Dysregulated expression of the NLRP3 inflammasome was reported to be associated with autoinflammatory diseases. No integrative studies between O‐GlcNAcylation and NLRP3 inflammasome in OLP patients have been reported. The present study aimed to determine the immunohistochemical expression of O‐GlcNAcylation, NF‐κB signalling molecules and NLRP3 inflammasome in oral mucosae of OLP patients. Oral tissue samples were collected from 30 OLP patients and 30 healthy individuals. Immunohistochemical staining and analyses of immunostaining scores were performed to evaluate expression of O‐GlcNAcylation, NF‐κB signalling molecules and NLRP3 inflammasome. According to observations in this study, significantly higher levels of O‐GlcNAcylation, NF‐κB signalling molecules and NLRP3 inflammasome were demonstrated in OLP patients compared with control subjects (P < 0·001). Positive correlations among O‐GlcNAcylation, NF‐κB signalling molecules and NLRP3 inflammasome were also observed in OLP samples (P < 0·01). In conclusion, the present study provides supportive evidence that increased O‐GlcNAcylation is associated with increased expression of NLRP3 inflammasome via the NF‐κB signalling pathway. These findings provide a new perspective on immunopathogenesis of OLP in relation to autoinflammation.     

Suppression of oxLDL‐Induced MMP‐9 and EMMPRIN Expression by Berberine via Inhibition of NF‐κB Activation in Human THP‐1 Macrophages

Upregulation of matrix metalloproteinases (MMPs) and extracellular matrix metalloproteinase inducer (EMMPRIN) by macrophages leads to atherosclerotic plaque rupture by degradation of the extracellular matrix. NF‐κB activation regulates many key inflammatory genes linked to atherosclerosis. In the present study, the function of berberine, a natural extract from Rhizoma coptidis, on MMP‐9 and EMMPRIN expression, the role of NF‐κB activation in oxLDL‐stimulated macrophages, and the possible mechanism in which NF‐κB activation is involved were investigated. Berberine inhibited the expression of MMP‐9 and EMMPRIN at both mRNA and protein levels. The phosphorylation of IκB‐α and nuclear translocation of p65 protein were reduced by berberine, suggesting that NF‐κB activation was inhibited by berberine in oxLDL‐stimulated macrophages. Overall, berberine suppressed the expression of MMP‐9 and EMMPRIN by at least reducing partly the activity of NF‐κB in oxLDL‐induced macrophages. Anat Rec, 2012. © 2011 Wiley Periodicals, Inc.