Andy Wullaert

NF-κB in the regulation of epithelial homeostasis and inflammation

The IκB kinase/NF-κB signaling pathway has been implicated in the pathogenesis of several inflammatory diseases. Increased activation of NF-κB is often detected in both immune and non-immune cells in tissues affected by chronic inflammation, where it is believed to exert detrimental functions by inducing the expression of proinflammatory mediators that orchestrate and sustain the inflammatory response and cause tissue damage. Thus, increased NF-κB activation is considered an important pathogenic factor in many acute and chronic inflammatory disorders, raising hopes that NF-κB inhibitors could be effective for the treatment of inflammatory diseases. However, ample evidence has accumulated that NF-κB inhibition can also be harmful for the organism, and in some cases trigger the development of inflammation and disease. These findings suggested that NF-κB signaling has important functions for the maintenance of physiological immune homeostasis and for the prevention of inflammatory diseases in many tissues. This beneficial function of NF-κB has been predominantly observed in epithelial cells, indicating that NF-κB signaling has a particularly important role for the maintenance of immune homeostasis in epithelial tissues. It seems therefore that NF-κB displays two faces in chronic inflammation: on the one hand increased and sustained NF-κB activation induces inflammation and tissue damage, but on the other hand inhibition of NF-κB signaling can also disturb immune homeostasis, triggering inflammation and disease. Here, we discuss the mechanisms that control these apparently opposing functions of NF-κB signaling, focusing particularly on the role of NF-κB in the regulation of immune homeostasis and inflammation in the intestine and the skin.

The Pseudomonas aeruginosa Type III secretion system plays a dual role in the regulation of caspase‐1 mediated IL‐1β maturation

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that forms a serious problem for immunocompromised patients and also the leading cause of mortality in cystic fibrosis. The overall importance of a functional Type III secretion system (T3SS) in P. aeru inosa virulence has been well established, but the underlying mechanisms are still unclear. Using in vitro infected macrophages as w as a murine model of acute lung infection, we show that the Caspase‐1 mediated maturation and secretion of IL‐1β needs a translocation competent T3SS and Flagellin, but not the Type III effector proteins ExoS, ExoT and ExoY. However, ExoS was found to negative regulate the P. aeruginosa induced IL‐1β maturation by a mechanism that is dependent on its ADP ribosyltransferase activity. Moreov ExoS deficiency also switched the mode of macrophage death from apoptosis to pro‐inflammatory pyroptosis. Altogether, these da demonstrate a dual role for the P. aeruginosa T3SS in the regulation of Caspase‐1 mediated IL‐1β production and provide new insigh into the mechanisms of immune evasion by this pathogen.

LIND/ABIN-3 Is a Novel Lipopolysaccharide-inducible Inhibitor of NF-κB Activation

Recognition of lipopolysaccharide (LPS) by Toll-like receptor (TLR)4 initiates an intracellular signaling pathway leading to the activation of nuclear factor-κB (NF-κB). Although LPS-induced activation of NF-κB is critical to the induction of an efficient immune response, excessive or prolonged signaling from TLR4 can be harmful to the host. Therefore, the NF-κB signal transduction pathway demands tight regulation. In the present study, we describe the human protein Listeria INDuced (LIND) as a novel A20-binding inhibitor of NF-κB activation (ABIN) that is related to ABIN-1 and -2 and, therefore, is further referred to as ABIN-3. Similar to the other ABINs, ABIN-3 binds to A20 and inhibits NF-κB activation induced by tumor necrosis factor, interleukin-1, and 12-O-tetradecanoylphorbol-13-acetate. However, unlike the other ABINs, constitutive expression of ABIN-3 could not be detected in different human cells. Treatment of human monocytic cells with LPS strongly induced ABIN-3 mRNA and protein expression, suggesting a role for ABIN-3 in the LPS/TLR4 pathway. Indeed, ABIN-3 overexpression was found to inhibit NF-κB-dependent gene expression in response to LPS/TLR4 at a level downstream of TRAF6 and upstream of IKKβ. NF-κB inhibition was mediated by the ABIN-homology domain 2 and was independent of A20 binding. Moreover, in vivo adenoviral gene transfer of ABIN-3 in mice reduced LPS-induced NF-κB activity in the liver, thereby partially protecting mice against LPS/d-(+)-galactosamine-inducedmortality. Taken together, these results implicate ABIN-3 as a novel negative feedback regulator of LPS-induced NF-κB activation.

Nuclear factor-kappa B plays a central role in tumour necrosis factor-mediated liver disease

Deregulation of the apoptotic program is considered an important cause in liver disease. It became clear that the cytokine tumour necrosis factor (TNF) is of specific interest in this context. Therefore, from a clinical point of view, therapeutic control of TNF-receptor signalling pathways is highly desirable. These TNF-initiated signalling pathways result in a direct apoptotic response as well as potent activation of proinflammatory gene expression via activation of the transcription factor nuclear factor-kappa B (NF-kappaB). Since the latter pathway contributes to a series of liver pathologies, inhibition of hepatic NF-kappaB activation was viewed as a potential therapy for liver injury. However, the more recent finding that NF-kappaB activation in hepatocytes is anti-apoptotic shows that NF-kappaB signalling represents a problematic therapeutic target. Here we review the role of TNF and NF-kappaB in liver pathophysiology, and the underlying mechanisms of hepatocyte sensitisation to TNF toxicity in vivo. Based on this knowledge, we suggest some potential strategies for the treatment of TNF-mediated liver disease.

NEMO Prevents RIP Kinase 1-Mediated Epithelial Cell Death and Chronic Intestinal Inflammation by NF-κB-Dependent and -Independent Functions

Summary Intestinal epithelial cells (IECs) regulate gut immune homeostasis, and impaired epithelial responses are implicated in the pathogenesis of inflammatory bowel diseases (IBD). IEC-specific ablation of nuclear factor κB (NF-κB) essential modulator (NEMO) caused Paneth cell apoptosis and impaired antimicrobial factor expression in the ileum, as well as colonocyte apoptosis and microbiota-driven chronic inflammation in the colon. Combined RelA, c-Rel, and RelB deficiency in IECs caused Paneth cell apoptosis but not colitis, suggesting that NEMO prevents colon inflammation by NF-κB-independent functions. Inhibition of receptor-interacting protein kinase 1 (RIPK1) kinase activity or combined deficiency of Fas-associated via death domain protein (FADD) and RIPK3 prevented epithelial cell death, Paneth cell loss, and colitis development in mice with epithelial NEMO deficiency. Therefore, NEMO prevents intestinal inflammation by inhibiting RIPK1 kinase activity-mediated IEC death, suggesting that RIPK1 inhibitors could be effective in the treatment of colitis in patients with NEMO mutations and possibly in IBD.

Adenoviral gene transfer of ABIN‐1 protects mice from TNF/galactosamine‐induced acute liver failure and lethality

Tumor necrosis factor (TNF) is a proinflammatory cytokine that plays a central role in acute and chronic hepatitis B and C infection and alcoholic liver disease as well as fulminant liver failure. TNF‐induced liver failure is characterized by parenchymal cell apoptosis and inflammation leading to liver cell necrosis. The transcription factor NF‐κB is believed to mediate at least part of the proinflammatory effects of TNF, and is therefore a favorite drug target. However, NF‐κB also suppresses TNF‐mediated hepatocyte apoptosis, implicating a potential cytotoxic effect of NF‐κB inhibitors in the liver. This dual function of NF‐κB emphasizes the need for therapeutics that can inhibit both TNF‐induced NF‐κB activation and cell death. Here we describe that adenoviral expression of the NF‐κB inhibitory protein ABIN‐1, but not an IκBα superrepressor (IκBαs), completely prevents lethality in the TNF/D‐(+)‐galactosamine–induced model of liver failure. Protection was associated with a significant decrease in TNF‐induced leukocyte infiltration as well as hepatocyte apoptosis. The differential effects of ABIN‐1 and IκBαs suggest a role for an NF‐κB independent function of ABIN‐1. Indeed, ABIN‐1 was found to prevent not only NF‐κB activation, but also apoptosis of cultured hepatocytes in response to TNF, explaining its protective effect against TNF‐induced liver failure. In conclusion, ABIN‐1 has a dual NF‐κB inhibitory and anti‐apoptotic activity in the liver, which might be of considerable interest for the treatment of inflammatory liver diseases. (HEPATOLOGY 2005;42:381–389.)

Nlrp6- and ASC-Dependent Inflammasomes Do Not Shape the Commensal Gut Microbiota Composition

&NA; The gut microbiota regulate susceptibility to multiple human diseases. The Nlrp6‐ASC inflammasome is widely regarded as a hallmark host innate immune axis that shapes the gut microbiota composition. This notion stems from studies reporting dysbiosis in mice lacking these inflammasome components when compared with non‐littermate wild‐type animals. Here, we describe microbial analyses in inflammasome‐deficient mice while minimizing non‐genetic confounders using littermate‐controlled Nlrp6‐deficient mice and ex‐germ‐free littermate‐controlled ASC‐deficient mice that were all allowed to shape their gut microbiota naturally after birth. Careful microbial phylogenetic analyses of these cohorts failed to reveal regulation of the gut microbiota composition by the Nlrp6‐ and ASC‐dependent inflammasomes. Our results obtained in two geographically separated animal facilities dismiss a generalizable impact of Nlrp6‐ and ASC‐dependent inflammasomes on the composition of the commensal gut microbiota and highlight the necessity for littermate‐controlled experimental design in assessing the influence of host immunity on gut microbial ecology. HighlightsNon‐littermate controls fail to define host genetic impacts on the gut microbiotaLittermate separation does not reveal ASC‐Nlrp6 impacts on the gut microbiotaLifetime littermate separation does not reveal Nlrp6 impacts on DSS colitis &NA; Inflammasomes were proposed to shape gut ecology based on dysbiosis in mutant mice versus non‐littermate wild‐types. Mamantopoulos et al. show that inflammasomes do not affect gut microbiota composition when controlling for non‐genetic confounders. This finding dismisses the suggested role for inflammasomes in controlling host health through regulation of intestinal ecology.

Bacteria regulate intestinal epithelial cell differentiation factors both in vitro and in vivo.

Background The human colon harbours a plethora of bacteria known to broadly impact on mucosal metabolism and function and thought to be involved in inflammatory bowel disease pathogenesis and colon cancer development. In this report, we investigated the effect of colonic bacteria on epithelial cell differentiation factors in vitro and in vivo. As key transcription factors we focused on Hes1, known to direct towards an absorptive cell fate, Hath1 and KLF4, which govern goblet cell. Methods Expression of the transcription factors Hes1, Hath1 and KLF4, the mucins Muc1 and Muc2 and the defensin HBD2 were measured by real-time PCR in LS174T cells following incubation with several heat-inactivated E. coli strains, including the probiotic E. coli Nissle 1917+/− flagellin, Lactobacilli and Bifidobacteria. For protein detection Western blot experiments and chamber-slide immunostaining were performed. Finally, mRNA and protein expression of these factors was evaluated in the colon of germfree vs. specific pathogen free vs. conventionalized mice and colonic goblet cells were counted. Results Expression of Hes1 and Hath1, and to a minor degree also of KLF4, was reduced by E. coli K-12 and E. coli Nissle 1917. In contrast, Muc1 and HBD2 expression were significantly enhanced, independent of the Notch signalling pathway. Probiotic E. coli Nissle 1917 regulated Hes1, Hath1, Muc1 and HBD2 through flagellin. In vivo experiments confirmed the observed in vitro effects of bacteria by a diminished colonic expression of Hath1 and KLF4 in specific pathogen free and conventionalized mice as compared to germ free mice whereas the number of goblet cells was unchanged in these mice. Conclusions Intestinal bacteria influence the intestinal epithelial differentiation factors Hes1, Hath1 and KLF4, as well as Muc1 and HBD2, in vitro and in vivo. The induction of Muc1 and HBD2 seems to be triggered directly by bacteria and not by Notch.

Overexpression of α1-acid glycoprotein in transgenic mice leads to sensitisation to acute colitis

Background: α1-Acid glycoprotein (α1-AGP) is an acute phase protein in most mammalian species whose concentration rises 2–5-fold during an acute phase reaction. Its serum concentration has often been used as a marker of disease, including inflammatory bowel disease (IBD). High α1-AGP levels were found to have a prognostic value for an increased risk of relapse in IBD. Aims: To investigate a possible role for increased serum levels of α1-AGP in the development of IBD. Methods: Dextran sodium sulphate (DSS) 2% was added to the drinking water of transgenic mice, overexpressing the rat α1-AGP gene, to induce acute colitis, thus mimicking the conditions of relapse. Clinical parameters, inflammatory parameters, and histological analyses on colon sections were performed. Results: Homozygous α1-AGP-transgenic mice started losing weight and showed rectal bleeding significantly earlier than heterozygous transgenic or wild-type mice. Survival time of homozygous transgenic mice was significantly shorter compared with heterozygous and wild-type mice. The higher susceptibility of homozygous α1-AGP-transgenic mice to DSS induced acute colitis was also reflected in higher local myeloperoxidase levels, higher inflammation scores of the colon, and higher systemic levels of interleukin 6 and serum amyloid P component. Local inflammatory parameters were also significantly different in heterozygous transgenic mice compared with wild-type mice, indicating a local dosage effect. In homozygous transgenic mice, significantly higher amounts of bacteria were found in organs but IgA levels were only slightly lower than those of control mice. Conclusion: Sufficiently high serum levels of α1-AGP result in a more aggressive development of acute colitis.

General Strategies in Inflammasome Biology.

The complementary actions of the innate and adaptive immune systems often provide effective host defense against microbial pathogens and harmful environmental agents. Germline-encoded pattern recognition receptors (PRRs) endow the innate immune system with the ability to detect and mount a rapid response against a given threat. Members of several intracellular PRR families, including the nucleotide-binding domain and leucine-rich repeat containing receptors (NLRs), the AIM2-like receptors (ALRs), and the tripartite motif-containing (TRIM) protein Pyrin/TRIM20, nucleate the formation of inflammasomes. These cytosolic scaffolds serve to recruit and oligomerize the cysteine protease caspase-1 in filaments that promote its proximity-induced autoactivation. This oligomerization occurs either directly or indirectly through intervention of the bipartite adaptor protein ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD), which is needed for the domain interaction. Caspase-1 cleaves the precursors of the inflammatory cytokines interleukin (IL)-1β and IL-18 and triggers their release into the extracellular space, where they act on effector cells to promote both local and systemic immune responses. Additionally, inflammasome activation gives rise to a lytic mode of cell death, named pyroptosis, which is thought to contribute to initial host defense against infection by eliminating replication niches of intracellular pathogens and exposing them to the immune system. Inflammasome-induced host defense responses are the subject of intense investigation, and understanding their physiological roles during infection and the regulatory circuits that are involved is becoming increasingly detailed. Here, we discuss current understanding of the activation mechanisms and biological outcomes of inflammasome activation.