The alternative NF-kappaB signalling pathway is a prerequisite for an appropriate immune response against lymphocytic choriomeningitis virus infection.

Two major nuclear factor-kappaB (NF-kappaB) signalling pathways are involved in the regulation of the immune response. While the classical NF-kappaB pathway is responsible for regulation of genes encoding components of the innate immune response, the alternative NF-kappaB signalling pathway mediates processes of the adaptive immune system. To evaluate the role of the NF-kappaB signalling pathways in the control of viral infection, we have used lymphocytic choriomeningitis virus (LCMV) infection of mice, which is known to be an excellent model for studying antiviral immune responses. Via the use of mice that were deficient in NF-kappaB subunits from either the classical (p50(-/-) mice) or the alternative NF-kappaB pathway (p52(-/-) mice), we were able to demonstrate that the alternative NF-kappaB pathway is required for the T-cell-mediated immune response against LCMV. Mice that were deficient in the alternative NF-kappaB pathway subunit p52 showed an impaired T-cell response against LCMV infection. Furthermore, these mice also showed an impaired T-cell-dependent humoral immune response against vesicular stomatitis virus (VSV) infection. Adoptive transfer experiments revealed that impaired priming, but not the T-cell response itself, was responsible for the defective cellular immune response against LCMV infection. Our data demonstrate that a functional alternative NF-kappaB signalling pathway is required to assure an adequate immune response after viral infection.

Rel/NF-kappaB family member RelA regulates NK1.1- to NK1.1+ transition as well as IL-15-induced expansion of NKT cells.

Development of NKT cells was shown to depend on lymphotoxin (LT) and IL-15 signaling pathways as well as on cytokine receptor common gamma chain. After positive selection, NKT-cell precursors transit through progressive maturation stages including proliferative expansion within the NK1.1(-) window. The transcription factors that integrate different signaling pathways into different stages of NKT-cell development are not well characterized. Here, we show that the Rel/NF-kappaB family member RelA regulates the NK1.1(-) to NK1.1(+) transition during NKT-cell development. RelA is also required for both IL-15- and IL-7-induced proliferation of CD44(hi)NK1.1(-) NKT-cell precursors. Activation of the invariant NKT-cell receptor induces both IL-15 receptor alpha and gamma chains' expression in an NF-kappaB-dependent manner, suggesting a molecular mechanism by which NF-kappaB regulates NKT-cell development. NF-kappaB also regulates TCR-induced expression of LT-alpha and LT-beta within NKT cells. In contrast to previous reports, however, we show that LT signaling is dispensable for thymic NKT-cell development but is essential for their colonization of peripheral organs such as liver.

Sepsis causes neuroinflammation and concomitant decrease of cerebral metabolism.

BACKGROUND: Septic encephalopathy is a severe brain dysfunction caused by systemic inflammation in the absence of direct brain infection. Changes in cerebral blood flow, release of inflammatory molecules and metabolic alterations contribute to neuronal dysfunction and cell death. METHODS: To investigate the relation of electrophysiological, metabolic and morphological changes caused by SE, we simultaneously assessed systemic circulation, regional cerebral blood flow and cortical electroencephalography in rats exposed to bacterial lipopolysaccharide. Additionally, cerebral glucose uptake, astro- and microglial activation as well as changes of inflammatory gene transcription were examined by small animal PET using [18F]FDG, immunohistochemistry, and real time PCR. RESULTS: While the systemic hemodynamic did not change significantly, regional cerebral blood flow was decreased in the cortex paralleled by a decrease of alpha activity of the electroencephalography. Cerebral glucose uptake was reduced in all analyzed neocortical areas, but preserved in the caudate nucleus, the hippocampus and the thalamus. Sepsis enhanced the transcription of several pro- and anti-inflammatory cytokines and chemokines including tumor necrosis factor alpha, interleukin-1 beta, transforming growth factor beta, and monocot chemoattractant protein 1 in the cerebrum. Regional analysis of different brain regions revealed an increase in ED1-positive microglia in the cortex, while total and neuronal cell counts decreased in the cortex and the hippocampus. CONCLUSION: Together, the present study highlights the complexity of sepsis induced early impairment of neuronal metabolism and activity. Since our model uses techniques that determine parameters relevant to the clinical setting, it might be a useful tool to develop brain specific therapeutic strategies for human septic encephalopathy.

NF-kappaB is a negative regulator of IL-1beta secretion as revealed by genetic and pharmacological inhibition of IKKbeta.

IKKbeta-dependent NF-kappaB activation plays a key role in innate immunity and inflammation, and inhibition of IKKbeta has been considered as a likely anti-inflammatory therapy. Surprisingly, however, mice with a targeted IKKbeta deletion in myeloid cells are more susceptible to endotoxin-induced shock than control mice. Increased endotoxin susceptibility is associated with elevated plasma IL-1beta as a result of increased pro-IL-1beta processing, which was also seen upon bacterial infection. In macrophages enhanced pro-IL-1beta processing depends on caspase-1, whose activation is inhibited by NF-kappaB-dependent gene products. In neutrophils, however, IL-1beta secretion is caspase-1 independent and depends on serine proteases, whose activity is also inhibited by NF-kappaB gene products. Prolonged pharmacologic inhibition of IKKbeta also augments IL-1beta secretion upon endotoxin challenge. These results unravel an unanticipated role for IKKbeta-dependent NF-kappaB signaling in the negative control of IL-1beta production and highlight potential complications of long-term IKKbeta inhibition.

Crosstalk between keratinocytes and adaptive immune cells in an IkappaBalpha protein-mediated inflammatory disease of the skin.

Inflammatory diseases at epithelial borders develop from aberrant interactions between resident cells of the tissue and invading immunocytes. Here, we unraveled basic functions of epithelial cells and immune cells and the sequence of their interactions in an inflammatory skin disease. Ubiquitous deficiency of the IkappaBalpha protein (Ikba(Delta)(/Delta)) as well as concomitant deletion of Ikba specifically in keratinocytes and T cells (Ikba(K5Delta/K5Delta lckDelta/lckDelta)) resulted in an inflammatory skin phenotype that involved the epithelial compartment and depended on the presence of lymphocytes as well as tumor necrosis factor and lymphotoxin signaling. In contrast, mice with selective ablation of Ikba in keratinocytes or lymphocytes showed inflammation limited to the dermal compartment or a normal skin phenotype, respectively. Targeted deletion of RelA from epidermal keratinocytes completely rescued the inflammatory skin phenotype of Ikba(Delta)(/Delta) mice. This finding emphasizes the important role of aberrant NF-kappaB activation in both keratinocytes and lymphocytes in the development of the observed inflammatory skin changes.

Alteration of NF-kappaB activity leads to mitochondrial apoptosis after infection with pathological prion protein.

Nuclear factor kappa B (NF-kappaB) is a key regulator of the immune response, but in almost the same manner it is involved in induction of inflammation, proliferation and regulation of apoptosis. In the central nervous system activated NF-kappaB plays a neuroprotective role. While in some neurodegenerative disorders the role of NF-kappaB is well characterized, there is poor knowledge on the role of NF-kappaB in prion disease. We found binding but no transcriptional activity of the transcription factor in vitro. Characterizing the mechanism of cell death after infection with pathological prion protein increased caspase-9 and caspase-3 activity was detected and the lack of NF-kappaB activity resulted in the inability to activate target genes that usually play an important role in neuroprotection. Additionally, we investigated the role of NF-kappaB after prion infection of Nfkb1(-/-), Nfkb2(-/-) and Bcl3(-/-) mice and central nervous system-specific p65-deleted mice revealing an accelerated prion disease in NF-kappaB2- and Bcl-3-deficient mice, which is in line with a reduced neuroprotective activity in prion infection. Based on our findings, we propose a model whereby the alteration of NF-kappaB activity at the early stages of infection with pathological prion protein leads to neuronal cell death mediated by mitochondrial apoptosis.

Genetic inactivation of RelA/p65 sensitizes adult mouse hepatocytes to TNF-induced apoptosis in vivo and in vitro.

BACKGROUND & AIMS: The transcription factor nuclear factor (NF)-kappaB plays a critical role in mediating survival of hepatocytes in response to tumor necrosis factor (TNF)-alpha during development because mice deficient for the NF-kappaB subunit RelA/p65 die in utero because of TNF-induced liver apoptosis. For the adult liver, conflicting concepts exist as to whether soluble TNF can trigger apoptosis when NF-kappaB activation is impaired. By creating a mouse model in which the transactivating NF-kappaB subunit RelA/p65 can be genetically inactivated in hepatocytes using the Cre/lox system, we sought to clarify the role of NF-kappaB in TNF-mediated hepatocyte apoptosis. METHODS: Deletion of RelA/p65 in the liver was achieved using an inducible conditional knockout system (rela(F/F)MxCre mice) or, hepatocyte-specifically, using a developmental conditional knockout system (rela(F/F)AlbCre mice). RESULTS: Disruption of RelA/p65 rendered mice sensitive to lethal liver injury upon TNF administration. Primary RelA/p65-deficient hepatocytes showed no NF-kappaB activation and undergo rapid apoptosis after TNF treatment. In contrast, hepatocytes deficient for I kappa B-kinase beta (IKK beta), displayed residual NF-kappaB activity and consecutively only mild apoptosis in response to TNF. TNF-induced apoptosis in RelA/p65-deficient hepatocytes was accompanied by prolonged activation of c-jun activating kinase (JNK) and rapid, largely proteasome-independent elimination of the long splice form of the antiapoptotic cellular FLICE inhibitor protein (c-FLIP(L)). Gene silencing of caspase-8, caspase-inhibitors, inhibition of JNK, or administration of antioxidants inhibited apoptosis and elimination of c-FLIP(L). CONCLUSIONS: RelA/p65 is essential for TNF-induced NF-kappaB activation in adult hepatocytes. Genetic deletion of a functional RelA/p65 sensitizes these cells to apoptosis in response to soluble TNF in vivo and in vitro.

Pancreas-specific RelA/p65 truncation increases susceptibility of acini to inflammation-associated cell death following cerulein pancreatitis.

Activation of the transcription factor NF-kappaB/Rel has been shown to be involved in inflammatory disease. Here we studied the role of RelA/p65, the main transactivating subunit, during acute pancreatitis using a Cre-loxP strategy. Selective truncation of the rela gene in pancreatic exocrine cells led to both severe injury of the acinar cells and systemic complications including lung and liver damage. Our data demonstrated that expression and induction of the protective pancreas-specific acute phase protein pancreatitis-associated protein 1 (PAP1) depended on RelA/p65. Lentiviral gene transfer of PAP1 cDNA reduced the extent of necrosis and infiltration in the pancreata of mice with selective truncation of RelA/p65. These results provide in vivo evidence for RelA/p65 protection of acinar cell death via upregulation of PAP1. Moreover, our data underscore the pancreas-specific role of NF-kappaB/Rel and suggest multidimensional roles of NF-kappaB/Rel in different cells and contexts during inflammation.

Bim and Noxa are candidates to mediate the deleterious effect of the NF-kappa B subunit RelA in cerebral ischemia.

The transcription factor nuclear factor kappaB (NF-kappaB) is well known for its antiapoptotic action. However, in some disorders, such as cerebral ischemia, a proapoptotic function of NF-kappaB has been demonstrated. To analyze which subunit of NF-kappaB is functional in cerebral ischemia, we induced focal cerebral ischemia in mice with a germline deletion of the p52 or c-Rel gene or with a conditional deletion of RelA in the brain. Only RelA deficiency reduced infarct size. Interestingly, expression of the proapoptotic BH3 (Bcl-2 homology domain 3)-only genes Bim and Noxa in cerebral ischemia depended on RelA and the upstream kinase IKK (IkappaB kinase). RelA stimulated Bim and Noxa gene transcription in primary cortical neurons and bound to the promoter of both genes. Thus, the deleterious function in cerebral ischemia is specific for the NF-kappaB subunit RelA and may be mediated through Bim and Noxa.

Abnormal organogenesis of Peyer's patches in mice deficient for NF-kappaB1, NF-kappaB2, and Bcl-3.

BACKGROUND & AIMS: Nuclear factor (NF) kappaB1, NF-kappaB2, and Bcl-3 encode for proteins of the NF-kappaB/Rel/IkappaB families, known as regulators of innate and adoptive immune responses. Targeted disruption of these genes showed essential roles in lymphoid organ development and organization. METHODS: NF-kappaB1-, NF-kappaB2-, and Bcl-3-deficient mouse lines were established, and their role in organogenesis of Peyer's patches (PP) was investigated. RESULTS: Macroscopic inspection showed a reduced number and size of PP in Bcl-3(-/-) and NF-kappaB1(-/-) mice but failed to detect PP in NF-kappaB2(-/-) mice. Whole-mount in situ hybridization revealed the presence of interleukin-7 receptor-alpha spots in NF-kappaB2(-/-) mice, indicating no defect in PP organogenesis of NF-kappaB2(-/-) mice in principle. Immunostaining shows that residual lymphocytes mainly consist of T cells. B cells are substantially reduced and are accumulated as terminal extravasations. Organized follicular structures and follicular dendritic cell networks fail to form, and myeloid, but not lymphoid, dendritic cells are obviously reduced. Expression of the chemokines macrophage inflammatory protein-3alpha, B-lymphocyte chemoattractant, and thymus-expressed chemokine is impaired in epithelial cells and in the subendothelial dome area that is not well defined. A similar but less severe phenotype is seen in Bcl-3(-/-) mice, which also do not develop germinal centers. In contrast, in NF-kappaB1(-/-) mice, T-cell numbers are visibly reduced, and no alteration could be observed in the B-cell and dendritic-cell populations. CONCLUSIONS: These data show that all 3 genes are crucial for PP development but contribute differently to PP organogenesis.