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.     

NF‐κB‐inducing kinase is a key regulator of inflammation‐induced and tumour‐associated angiogenesis

Angiogenesis is essential during development and in pathological conditions such as chronic inflammation and cancer progression. Inhibition of angiogenesis by targeting vascular endothelial growth factor (VEGF) blocks disease progression, but most patients eventually develop resistance which may result from compensatory signalling pathways. In endothelial cells (ECs), expression of the pro‐angiogenic chemokine CXCL12 is regulated by non‐canonical nuclear factor (NF)‐κB signalling. Here, we report that NF‐κB‐inducing kinase (NIK) and subsequent non‐canonical NF‐κB signalling regulate both inflammation‐induced and tumour‐associated angiogenesis. NIK is highly expressed in endothelial cells (ECs) in tumour tissues and inflamed rheumatoid arthritis synovial tissue. Furthermore, non‐canonical NF‐κB signalling in human microvascular ECs significantly enhanced vascular tube formation, which was completely blocked by siRNA targeting NIK. Interestingly, Nik^?/? mice exhibited normal angiogenesis during development and unaltered TNFα‐ or VEGF‐induced angiogenic responses, whereas angiogenesis induced by non‐canonical NF‐κB stimuli was significantly reduced. In addition, angiogenesis in experimental arthritis and a murine tumour model was severely impaired in these mice. These studies provide evidence for a role of non‐canonical NF‐κB signalling in pathological angiogenesis, and identify NIK as a potential therapeutic target in chronic inflammatory diseases and tumour neoangiogenesis. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Selective blockade of endothelial NF‐κB pathway differentially affects systemic inflammation and multiple organ dysfunction and injury in septic mice

Endothelium has long been considered both a source and a target of systemic inflammation. However, to what extent endothelial activation contributes to systemic inflammation remains unclear. This study addresses the relative contribution of endothelial activation to systemic inflammation and multiple organ dysfunction and injury (MOD/I) in an E. coli peritonitis model of sepsis. We prevented endothelial activation using transgenic (TG) mice that conditionally overexpress a mutant I‐κBα, a NF‐κB inhibitor, selectively on endothelium. TG mice and their transgene negative littermates (WT) were injected with saline or E. coli (10⁸ CFU per mouse). At 7 h after E. coli infection, markers of systemic inflammation, endothelial activation, and MOD/I were assessed. WT‐E. coli mice showed significantly increased serum levels of TNF‐α, IL‐1β, IFN‐γ, IL‐6, KC, and MCP‐1; tissue levels of TNF‐α, IL‐6, KC, MCP‐1, ICAM‐1, and VCAM‐1; endothelial leakage index in heart, lungs, liver, and kidney; significantly increased serum levels of AST, ALT, BUN, and creatinine; and increased mortality. Blockade of NF‐κB‐mediated endothelial activation in TG mice had no effects on serum levels of TNF‐α, IL‐1β, IFN‐γ, IL‐6, KC, and MCP‐1 (markers of systemic inflammation), and tissue levels of TNF‐α, IL‐6, KC, and MCP‐1, but significantly reduced tissue levels of ICAM‐1 and VCAM‐1 (markers of endothelial inflammation and activation) in those four organs. TG‐E. coli mice displayed reversed endothelial leakage index; reduced serum levels of AST, ALT, BUN, and creatinine; and improved survival. Our data demonstrate that endothelial NF‐κB‐driven inflammatory response contributes minimally to systemic inflammation, but plays a pivotal role in septic MOD/I, suggesting that endothelium is mainly a target rather than a source of systemic inflammation. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

ΔNp63 isoforms differentially regulate gene expression in squamous cell carcinoma: identification of Cox‐2 as a novel p63 target

The p53 homologue p63 produces six different isoforms that are important in development of epithelial tissues and squamous cell carcinoma of the head and neck (SCCHN). In SCCHN, the expression of p63 isoforms is highly complex, with over‐expression of ΔNp63 and p63β isoforms in many tumours. To date, little is known about the functions of different ΔNp63 isoforms and elucidating the distinctive properties of ΔNp63 isoforms will help to clarify how they influence tumour biology. By gene expression profiling of SCCHN cells over‐expressing the ΔNp63 isoforms we identified different effects of the three isoforms, with ΔNp63β being more effective at gene induction than ΔNp63α and ΔNp63γ, whereas ΔNp63γ was most effective at repressing gene expression. Thus, tumours expressing even low levels of ΔNp63β or ΔNp63γ may have distinct clinicopathological characteristics important for metastasis and therapeutic response. Induction of cyclooxygenase‐2 (Cox‐2) was shown by each isoform and data were confirmed by independent quantitative RT–PCR and western blotting. No direct binding of ΔNp63 to the Cox‐2 promoter could be seen, neither could any evidence for Cox‐2 induction as a consequence of activated NF‐κB pathway responses be found. As Cox‐2 is known to inhibit radiotherapy responses in SCCHN patients, data indicate an additional mechanism through which ΔNp63 acts to promote cell survival and influence therapeutic response of SCCHN. MIAME‐compliant data have been deposited in the MIAME Express database (Accession No. E‐MEXP‐1842). Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Inhibition of R5‐tropic HIV type‐1 replication in CD4+ natural killer T cells by γδ T lymphocytes

After the development of highly active anti‐retroviral therapy, it became clear that the majority of emergent HIV‐1 is macrophage‐tropic and infects CD4⁺, CCR5‐expressing cells (R5‐tropic). There are three distinct cell populations, R5‐tropic, HIV‐1‐susceptible CD4⁺ cells: (i) natural killer T (NKT) cells, (ii) dendritic cells and macrophages, and (iii) tissue‐associated T cells residing primarily at mucosal surfaces. We have confirmed that CD4⁺ NKT cells derived from peripheral blood mononuclear cells (PBMCs) predominantly express CCR5 rather than CXCR4, whereas the reverse is true for CD4⁺ T cells derived from circulating PBMCs, and that R5‐tropic HIV‐1 expands efficiently in the CD4⁺ NKT cells. Moreover, when PBMCs depleted of CD8α⁺ cells were stimulated in the presence of α‐galactosylceramide (α‐GalCer) and R5‐tropic HIV‐1 [NL(AD8)], the production of HIV‐1 virions was not suppressed, whereas, similar to the untreated PBMCs, depletion of CD8β⁺ cells from PBMCs significantly inhibited virion production. These findings suggest that CD8αα⁺ but not CD8αβ⁺ cells may have the ability to inhibit R5‐tropic HIV‐1 replication in CD4⁺ NKT cells. Here, we show that co‐culturing R5‐tropic HIV‐1‐infected CD4⁺ NKT cells with CD8αα⁺ γδ T cells, in particular Vγ1Vδ1 cells, but not with CD8αα⁺ NKT cells or CD8αα⁺ dendritic cells, inhibits HIV‐1 replication mainly by secreting chemokines, such as macrophage inflammatory proteins 1α and 1β and RANTES. Collectively, these results indicate the importance of CD8αα⁺ γδ T cells in the control of R5‐tropic HIV‐1 replication and persistence in CD4⁺ NKT cells.     

5,7‐dihydroxy‐3,4,6‐trimethoxyflavone inhibits the inflammatory effects induced by Bacteroides fragilis enterotoxin via dissociating the complex of heat shock protein 90 and IκBα and IκB kinase‐γ in intestinal epithelial cell culture

Enterotoxin produced by enterotoxigenic Bacteroides fragilis (BFT) has been associated with mucosal inflammation and diarrhoeal diseases. In this study, the anti‐inflammatory molecular mechanism of 5,7‐dihydroxy‐3,4,6‐trimethoxyflavone (eupatilin) was characterized in an HT‐29 intestinal epithelial cell line stimulated with BFT. Pre‐treatment of HT‐29 cells with eupatilin decreased the production significantly of both interleukin (IL)‐8 and prostaglandin E₂ induced by BFT in a dose‐dependent manner. BFT‐activated nuclear factor‐kappaB (NF‐κB) signals in HT‐29 cells and pretreatment with eupatilin suppressed NF‐κB activation that resulted in the significant inhibition of IL‐8 and cyclo‐oxygenase‐2 expression. BFT‐induced phosphorylation of both IκBα and IκB kinase (IKK) signals was prevented in eupatilin‐pretreated HT‐29 cells. Transfection of siRNA for IKK‐α and IKK‐β decreased the production of IL‐8 and prostaglandin E₂; however, the transfection of IKK‐β siRNA showed a more significant reduction of BFT‐induced IκBα phosphorylation compared with that of IKK‐α siRNA. In addition, herbimycin A, a specific inhibitor of heat shock protein 90 (Hsp90), decreased the BFT‐induced activation of IKK and NF‐κB, suggesting that Hsp90 is associated with a pathway of IKK‐NF‐κB‐IL‐8/cyclo‐oxygenase‐2 gene signalling. Furthermore, eupatilin dissociated the complex between Hsp90 and IKK‐γ in BFT‐stimulated HT‐29 cells. These results suggest that eupatilin can suppress the NF‐κB signalling pathway by targeting the Hsp90‐IKK‐γ complex in intestinal epithelial cells and may attenuate BFT‐induced inflammatory responses.     

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.