Geert van Loo

Epithelial NEMO links innate immunity to chronic intestinal inflammation

Deregulation of intestinal immune responses seems to have a principal function in the pathogenesis of inflammatory bowel disease. The gut epithelium is critically involved in the maintenance of intestinal immune homeostasis—acting as a physical barrier separating luminal bacteria and immune cells, and also expressing antimicrobial peptides. However, the molecular mechanisms that control this function of gut epithelial cells are poorly understood. Here we show that the transcription factor NF-κB, a master regulator of pro-inflammatory responses, functions in gut epithelial cells to control epithelial integrity and the interaction between the mucosal immune system and gut microflora. Intestinal epithelial-cell-specific inhibition of NF-κB through conditional ablation of NEMO (also called IκB kinase-γ (IKKγ)) or both IKK1 (IKKα) and IKK2 (IKKβ)—IKK subunits essential for NF-κB activation—spontaneously caused severe chronic intestinal inflammation in mice. NF-κB deficiency led to apoptosis of colonic epithelial cells, impaired expression of antimicrobial peptides and translocation of bacteria into the mucosa. Concurrently, this epithelial defect triggered a chronic inflammatory response in the colon, initially dominated by innate immune cells but later also involving T lymphocytes. Deficiency of the gene encoding the adaptor protein MyD88 prevented the development of intestinal inflammation, demonstrating that Toll-like receptor activation by intestinal bacteria is essential for disease pathogenesis in this mouse model. Furthermore, NEMO deficiency sensitized epithelial cells to tumour-necrosis factor (TNF)-induced apoptosis, whereas TNF receptor-1 inactivation inhibited intestinal inflammation, demonstrating that TNF receptor-1 signalling is crucial for disease induction. These findings demonstrate that a primary NF-κB signalling defect in intestinal epithelial cells disrupts immune homeostasis in the gastrointestinal tract, causing an inflammatory-bowel-disease-like phenotype. Our results identify NF-κB signalling in the gut epithelium as a critical regulator of epithelial integrity and intestinal immune homeostasis, and have important implications for understanding the mechanisms controlling the pathogenesis of human inflammatory bowel disease.

Toxic proteins released from mitochondria in cell death

A plethora of apoptotic stimuli converge on the mitochondria and affect their membrane integrity. As a consequence, multiple death-promoting factors residing in the mitochondrial intermembrane space are liberated in the cytosol. Pro- and antiapoptotic Bcl-2 family proteins control the release of these mitochondrial proteins by inducing or preventing permeabilization of the outer mitochondrial membrane. Once released into the cytosol, these mitochondrial proteins activate both caspase-dependent and -independent cell death pathways. Cytochrome c was the first protein shown to be released from the mitochondria into the cytosol, where it induces apoptosome formation. Other released mitochondrial proteins include apoptosis-inducing factor (AIF) and endonuclease G, both of which contribute to apoptotic nuclear DNA damage in a caspase-independent way. Other examples are Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP-binding protein with low PI) and the serine protease HtrA2/OMI (high-temperature requirement protein A2), which both promote caspase activation and instigate caspase-independent cytotoxicity. The precise mode of action and importance of cytochrome c in apoptosis in mammalian cells has become clear through biochemical, structural and genetic studies. More recently identified factors, for example HtrA2/OMI and Smac/DIABLO, are still being studied intensively in order to delineate their functions in apoptosis. A better understanding of these functions may help to develop new strategies to treat cancer.

Mitochondrial intermembrane proteins in cell death.

Apoptosis is a form of programmed cell death important in the development and tissue homeostasis of multicellular organisms. Mitochondria have, next to their function in respiration, an important role in the apoptotic-signaling pathway. Malfunctioning at any level of the cell is eventually translated in the release of apoptogenic factors from the mitochondrial intermembrane space resulting in the organized demise of the cell. Some of these factors, such as AIF and endonuclease G, appear to be highly conserved during evolution. Other factors, like cytochrome c, have gained their apoptogenic function later during evolution. In this review, we focus on the role of cytochrome c, AIF, endonuclease G, Smac/DIABLO, Omi/HtrA2, Acyl-CoA-binding protein, and polypyrimidine tract-binding protein in the initiation and modulation of cell death in different model organisms. These mitochondrial factors may contribute to both caspase-dependent and caspase-independent processes in apoptotic cell death.

FADD prevents RIP3-mediated epithelial cell necrosis and chronic intestinal inflammation

Intestinal immune homeostasis depends on a tightly regulated cross talk between commensal bacteria, mucosal immune cells and intestinal epithelial cells (IECs). Epithelial barrier disruption is considered to be a potential cause of inflammatory bowel disease; however, the mechanisms regulating intestinal epithelial integrity are poorly understood. Here we show that mice with IEC-specific knockout of FADD (FADDIEC-KO), an adaptor protein required for death-receptor-induced apoptosis, spontaneously developed epithelial cell necrosis, loss of Paneth cells, enteritis and severe erosive colitis. Genetic deficiency in RIP3, a critical regulator of programmed necrosis, prevented the development of spontaneous pathology in both the small intestine and colon of FADDIEC-KO mice, demonstrating that intestinal inflammation is triggered by RIP3-dependent death of FADD-deficient IECs. Epithelial-specific inhibition of CYLD, a deubiquitinase that regulates cellular necrosis, prevented colitis development in FADDIEC-KO but not in NEMOIEC-KO mice, showing that different mechanisms mediated death of colonic epithelial cells in these two models. In FADDIEC-KO mice, TNF deficiency ameliorated colon inflammation, whereas MYD88 deficiency and also elimination of the microbiota prevented colon inflammation, indicating that bacteria-mediated Toll-like-receptor signalling drives colitis by inducing the expression of TNF and other cytokines. However, neither CYLD, TNF or MYD88 deficiency nor elimination of the microbiota could prevent Paneth cell loss and enteritis in FADDIEC-KO mice, showing that different mechanisms drive RIP3-dependent necrosis of FADD-deficient IECs in the small and large bowel. Therefore, by inhibiting RIP3-mediated IEC necrosis, FADD preserves epithelial barrier integrity and antibacterial defence, maintains homeostasis and prevents chronic intestinal inflammation. Collectively, these results show that mechanisms preventing RIP3-mediated epithelial cell death are critical for the maintenance of intestinal homeostasis and indicate that programmed necrosis of IECs might be implicated in the pathogenesis of inflammatory bowel disease, in which Paneth cell and barrier defects are thought to contribute to intestinal inflammation.

The ubiquitin-editing enzyme A20 (TNFAIP3) is a central regulator of immunopathology

Nuclear factor (NF)-kappaB has an important role in immunity and inappropriate NF-kappaB activity has been linked with many autoimmune and inflammatory diseases. Multiple mechanisms normally ensure the proper termination of NF-kappaB activation. In this context, the intracellular ubiquitin-editing protein A20 (also known as Tumor Necrosis Factor Alpha-Induced Protein 3 or TNFAIP3) is a key player in the negative feedback regulation of NF-kappaB signaling in response to multiple stimuli. Moreover, A20 also regulates tumor necrosis factor (TNF)-induced apoptosis. Recent genetic studies demonstrate a clear association between several mutations in the human A20 locus and immunopathologies such as Crohns disease, rheumatoid arthritis, systemic lupus erythematosus, psoriasis and type 1 diabetes. These findings further illustrate the importance of A20 in the resolution of inflammation and the prevention of human disease.

Farm dust and endotoxin protect against allergy through A20 induction in lung epithelial cells

How farming protects against allergies People who grow up on dairy farms only rarely develop asthma or allergies. This is probably because as children, they breathe air containing bacterial components, which reduce the overall reactivity of the immune system. Schuijs et al. chronically exposed mice to bacterial endotoxin before they received an allergic stimulus. The protocol indeed protected them from developing an allergic response. Protection relied on a particular enzyme: A20. In humans, a variant of A20 correlates with increased susceptibility to asthma and allergy in children growing up on farms. Science, this issue p. 1106 Chronic exposure to bacterial endotoxin protects against allergy through the enzyme A20. Growing up on a dairy farm protects children from allergy, hay fever, and asthma. A mechanism linking exposure to this endotoxin (bacterial lipopolysaccharide)–rich environment with protection has remained elusive. Here we show that chronic exposure to low-dose endotoxin or farm dust protects mice from developing house dust mite (HDM)–induced asthma. Endotoxin reduced epithelial cell cytokines that activate dendritic cells (DCs), thus suppressing type 2 immunity to HDMs. Loss of the ubiquitin-modifying enzyme A20 in lung epithelium abolished the protective effect. A single-nucleotide polymorphism in the gene encoding A20 was associated with allergy and asthma risk in children growing up on farms. Thus, the farming environment protects from allergy by modifying the communication between barrier epithelial cells and DCs through A20 induction.

CCR2 recruits an inflammatory macrophage subpopulation critical for angiogenesis in tissue repair

Monocytes/macrophages are critical in orchestrating the tissue-repair response. However, the mechanisms that govern macrophage regenerative activities during the sequential phases of repair are largely unknown. In the present study, we examined the dynamics and functions of diverse monocyte/macrophage phenotypes during the sequential stages of skin repair. By combining the analysis of a new CCR2-eGFP reporter mouse model with conditional mouse mutants defective in myeloid cell-restricted CCR2 signaling or VEGF-A synthesis, we show herein that among the large number of inflammatory CCR2(+)Ly6C(+) macrophages that dominate the early stage of repair, only a small fraction strongly expresses VEGF-A that has nonredundant functions for the induction of vascular sprouts. The switch of macrophage-derived VEGF-A during the early stage of tissue growth toward epidermal-derived VEGF-A during the late stage of tissue maturation was critical to achieving physiologic tissue vascularization and healing progression. The results of the present study provide new mechanistic insights into CCR2-mediated recruitment of blood monocyte subsets into damaged tissue, the dynamics and functional consequences of macrophage plasticity during the sequential repair phases, and the complementary role of macrophage-derived VEGF-A in coordinating effective tissue growth and vascularization in the context of tissue-resident wound cells. Our findings may be relevant for novel monocyte-based therapies to promote tissue vascularization.

A20 in inflammation and autoimmunity

Although known for many years as a nuclear factor (NF)-κB inhibitory and antiapoptotic signaling protein, A20 has recently attracted much attention because of its ubiquitin-regulatory activities and qualification by genome-wide association studies (GWASs) as a susceptibility gene for inflammatory disease. Here, we review new findings that have shed light on the molecular and biochemical mechanisms by which A20 regulates inflammatory signaling cascades, and discuss recent experimental evidence characterizing A20 as a crucial gatekeeper preserving tissue homeostasis.

A20 (TNFAIP3) deficiency in myeloid cells triggers erosive polyarthritis resembling rheumatoid arthritis

A20 (TNFAIP3) is a protein that is involved in the negative feedback regulation of NF-κB signaling in response to specific proinflammatory stimuli in different cell types and has been suggested as a susceptibility gene for rheumatoid arthritis. To define the contribution of A20 to rheumatoid arthritis pathology, we generated myeloid-specific A20-deficient mice and show that specific ablation of Tnfaip3 in myeloid cells results in spontaneous development of a severe destructive polyarthritis with many features of rheumatoid arthritis. Myeloid-A20–deficient mice have high levels of inflammatory cytokines in their serum, consistent with a sustained NF-κB activation and higher TNF production by macrophages. Destructive polyarthritis in myeloid A20 knockout mice was TLR4-MyD88 and IL-6 dependent but was TNF independent. Myeloid A20 deficiency also promoted osteoclastogenesis in mice. Together, these observations indicate a critical and cell-specific function for A20 in the etiology of rheumatoid arthritis, supporting the idea of developing A20 modulatory drugs as cell-targeted therapies.

Negative regulation of the NLRP3 inflammasome by A20 protects against arthritis

Rheumatoid arthritis is a chronic autoinflammatory disease that affects 1–2% of the world’s population and is characterized by widespread joint inflammation. Interleukin-1 is an important mediator of cartilage destruction in rheumatic diseases, but our understanding of the upstream mechanisms leading to production of interleukin-1β in rheumatoid arthritis is limited by the absence of suitable mouse models of the disease in which inflammasomes contribute to pathology. Myeloid-cell-specific deletion of the rheumatoid arthritis susceptibility gene A20/Tnfaip3 in mice (A20myel-KO mice) triggers a spontaneous erosive polyarthritis that resembles rheumatoid arthritis in patients. Rheumatoid arthritis in A20myel-KO mice is not rescued by deletion of tumour necrosis factor receptor 1 (ref. 2). Here we show, however, that it crucially relies on the Nlrp3 inflammasome and interleukin-1 receptor signalling. Macrophages lacking A20 have increased basal and lipopolysaccharide-induced expression levels of the inflammasome adaptor Nlrp3 and proIL-1β. As a result, A20-deficiency in macrophages significantly enhances Nlrp3 inflammasome-mediated caspase-1 activation, pyroptosis and interleukin-1β secretion by soluble and crystalline Nlrp3 stimuli. In contrast, activation of the Nlrc4 and AIM2 inflammasomes is not altered. Importantly, increased Nlrp3 inflammasome activation contributes to the pathology of rheumatoid arthritis in vivo, because deletion of Nlrp3, caspase-1 and the interleukin-1 receptor markedly protects against rheumatoid-arthritis-associated inflammation and cartilage destruction in A20myel-KO mice. These results reveal A20 as a novel negative regulator of Nlrp3 inflammasome activation, and describe A20myel-KO mice as the first experimental model to study the role of inflammasomes in the pathology of rheumatoid arthritis.