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.     

Secreted IL‐1α promotes T‐cell activation and expansion of CD11b+Gr1+ cells in carbon tetrachloride‐induced liver injury in mice

Interleukin‐1α is mainly expressed on the cell membrane, but can also be secreted during inflammation. The roles of secreted and membrane IL‐1α in acute liver inflammation are still not known. Here, we examined the functions of secreted and membrane IL‐1α in a mouse model of carbon tetrachloride‐induced acute liver injury. We show that secreted IL‐1α aggravates liver damage and membrane IL‐1α slightly protects mice from liver injury. Further studies showed that secreted IL‐1α promotes T‐cell activation. It also increased the expansion of CD11b⁺Gr1⁺ myeloid cells, which may serve as a negative regulator of acute liver inflammation. Moreover, secreted IL‐1α induced IL‐6 production from hepatocytes. IL‐6 neutralization reduced the proliferation of CD11b⁺Gr1⁺ myeloid cells in vivo. CCL2 and CXCL5 expression was increased by secreted IL‐1α in vitro and in vivo. Antagonists of the chemokine receptors for CCL2 and CXCL5 significantly reduced the migration of CD11b⁺Gr1⁺ myeloid cells. These results demonstrate that secreted and membrane IL‐1α play different roles in acute liver injury. Secreted IL‐1α could promote T‐cell activation and the recruitment and expansion of CD11b⁺Gr1⁺ myeloid cells through induction of CCL2, CXCL5, and IL‐6. The controlled release of IL‐1α could be a critical regulator during acute liver inflammation.     

IFN‐γ regulates survival and function of tumor‐induced CD11b+Gr‐1high myeloid derived suppressor cells by modulating the anti‐apoptotic molecule Bcl2a1

Myeloid derived suppressor cells (MDSCs) play a critical role in suppression of immune responses in cancer and inflammation. Here, we describe how regulation of Bcl2a1 by cytokines controls the suppressor function of CD11b⁺Gr‐1^high granulocytic MDSCs. Coculture of CD11b⁺Gr‐1^high granulocytic MDSCs with antigen‐stimulated T cells and simultaneous blockade of IFN‐γ by the use of anti‐IFN‐γ blocking antibody, IFN‐γ^?/? effector T cells, IFN‐γR^?/? MDSCs or STAT1^?/? MDSCs led to upregulation of Bcl2a1 in CD11b⁺Gr‐1^high cells, improved survival, and enhanced their suppressor function. Molecular studies revealed that GM‐CSF released by antigen‐stimulated CD8⁺ T cells induced Bcl2a1 upregulation, which was repressed in the presence of IFN‐γ by a direct interaction of phosphorylated STAT‐1 with the Bcl2a1 promotor. Bcl2a1 overexpressing granulocytic MDSCs demonstrated prolonged survival and enhanced suppressor function in vitro. Our data suggest that IFN‐γ/ STAT1‐dependent regulation of Bcl2a1 regulates survival and thereby suppressor function of granulocytic MDSCs.     

c‐Rel but not NF‐κB1 is important for T regulatory cell development

Regulatory T (Treg) cells are crucial for maintaining peripheral tolerance and controlling T‐cell responses. The generation of Treg in the thymus requires TCR triggering and CD28 costimulation. Engagement of these receptors induces a number of signalling pathways, including the activation of NF‐κB via PKCθ and the Bcl‐10/CARMA1/MALT complex. Previous studies have shown that PKCθ, Bcl‐10 and CARMA1 are important for Treg development. It is unclear, however, whether different members of the NF‐κB family contribute to Treg development or homeostasis. In this study, we show that Treg numbers are reduced in the absence of c‐Rel but not NF‐κB1 (p50). Furthermore, using mixed bone marrow chimeras from WT and KO animals, we demonstrate that the requirement for PKCθ, Bcl‐10 and c‐Rel is T‐cell intrinsic, and cannot be rescued by the presence of WT cells. Therefore, c‐Rel and NF‐κB1 have differential roles in Treg development.     

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.     

TGF‐β induces the differentiation of human CXCL13‐producing CD4+ T cells

In the ectopic lymphoid‐like structures present in chronic inflammatory conditions such as rheumatoid arthritis, a subset of human effector memory CD4⁺ T cells that lacks features of follicular helper T (Tfh) cells produces CXCL13. Here, we report that TGF‐β induces the differentiation of human CXCL13‐producing CD4⁺ T cells from naïve CD4⁺ T cells. The TGF‐β‐induced CXCL13‐producing CD4⁺ T cells do not express CXCR5, B‐cell lymphoma 6 (BCL6), and other Tfh‐cell markers. Furthermore, expression levels of CD25 (IL‐2Rα) in CXCL13‐producing CD4⁺ T cells are significantly lower than those in FoxP3⁺ in vitro induced Treg cells. Consistent with this, neutralization of IL‐2 and knockdown of STAT5 clearly upregulate CXCL13 production by CD4⁺ T cells, while downregulating the expression of FoxP3. Furthermore, overexpression of FoxP3 in naïve CD4⁺ T cells downregulates CXCL13 production, and knockdown of FoxP3 fails to inhibit the differentiation of CXCL13‐producing CD4⁺ T cells. As reported in rheumatoid arthritis, proinflammatory cytokines enhance secondary CXCL13 production from reactivated CXCL13‐producing CD4⁺ T cells. Our findings demonstrate that CXCL13‐producing CD4⁺ T cells lacking Tfh‐cell features differentiate via TGF‐β signaling but not via FoxP3, and exert their function in IL‐2‐limited but TGF‐β‐rich and proinflammatory cytokine‐rich inflammatory conditions.     

IL‐15 modulates the balance between Bcl‐2 and Bim via a Jak3/1‐PI3K‐Akt‐ERK pathway to promote CD8αα+ intestinal intraepithelial lymphocyte survival

IL‐15 is an essential survival factor for CD8αα⁺ intestinal intraepithelial lymphocytes (iIELs) in vitro and in vivo. However, the IL‐15‐induced survival signals in primary CD8αα⁺ iIELs remains elusive. Although Bcl‐2 level in CD8αα⁺ iIELs positively correlates with IL‐15Rα expression in the intestinal epithelial cells, overexpression of Bcl‐2 only moderately restores CD8αα⁺ γδ iIELs in Il15^?/? mice. Here, we found that IL‐15 promptly activated a Jak3‐Jak1‐PI3K‐Akt pathway that led to the upregulation of Bcl‐2 and Mcl‐1. This pathway also induced a delayed but sustained ERK1/2 activation, which not only was necessary for the maintenance of Bcl‐2 but also resulted in the phosphorylation of extra‐long Bim at Ser⁶⁵. The latter event facilitated the dissociation of Bim from Bcl‐2 without affecting Bim abundance in IL‐15‐treated CD8αα⁺ iIELs. Using an adoptive cell transfer approach, we found that either overexpression of Bcl‐2 or removal of Bim from CD8αα⁺ iIELs promoted their survival in Il15ra^?/? mice. Taken together, IL‐15 promotes CD8αα⁺ iIEL survival by both increasing Bcl‐2 levels and dissociating Bim from Bcl‐2 through activation of a Jak3‐Jak1‐PI3K‐Akt‐ERK1/2 pathway, which differs from a previously reported IL‐15‐induced survival signal.     

Genetic ablation of PI3Kγ results in defective IL‐17RA signalling in T lymphocytes and increased IL‐17 levels

The signalling molecule PI3Kγ has been reported to play a key role in the immune system and the inflammatory response. In particular, it facilitates the migration of haemato‐poietic cells to the site of inflammation. In this study, we reveal a novel role for PI3Kγ in the regulation of the pro‐inflammatory cytokine IL‐17. Loss of PI3Kγ or expression of a catalytically inactive mutant of PI3Kγ in mice led to increased IL‐17 production both in vitro and in vivo in response to various stimuli. The kinetic profile was unaltered from WT cells, with no effect on proliferation or other cytokines. Elevated levels of IL‐17 were not due to an aberrant expansion of IL‐17‐producing cells. Furthermore, we also identified an increase in IL‐17RA expression on PI3Kγ^?/? CD4⁺ T cells, yet these cells exhibited impaired PI3K‐dependent signalling in response to IL‐17A, and subsequent NF‐κB phosphorylation. In vivo, instillation of recombinant IL‐17 into the airways of mice lacking PI3Kγ signalling also resulted in reduced phosphorylation of Akt. Cell influx in response to IL‐17 was also reduced in PI3Kγ^?/? lungs. These data demonstrate PI3Kγ‐dependent signalling downstream of IL‐17RA, which plays a pivotal role in regulating IL‐17 production in T cells.     

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.