Manolis Pasparakis

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.

Identification of oxidative stress and toll-like receptor 4 signaling as a key pathway of acute lung injury

Summary Multiple lung pathogens such as chemical agents, H5N1 avian flu, or SARS cause high lethality due to acute respiratory distress syndrome. Here we report that Toll-like receptor 4 (TLR4) mutant mice display natural resistance to acid-induced acute lung injury (ALI). We show that TLR4-TRIF-TRAF6 signaling is a key disease pathway that controls the severity of ALI. The oxidized phospholipid (OxPL) OxPAPC was identified to induce lung injury and cytokine production by lung macrophages via TLR4-TRIF. We observed OxPL production in the lungs of humans and animals infected with SARS, Anthrax, or H5N1. Pulmonary challenge with an inactivated H5N1 avian influenza virus rapidly induces ALI and OxPL formation in mice. Loss of TLR4 or TRIF expression protects mice from H5N1-induced ALI. Moreover, deletion of ncf1, which controls ROS production, improves the severity of H5N1-mediated ALI. Our data identify oxidative stress and innate immunity as key lung injury pathways that control the severity of ALI.

Necroptosis and its role in inflammation

Regulated cell death has essential functions in development and in adult tissue homeostasis. Necroptosis is a newly discovered pathway of regulated necrosis that requires the proteins RIPK3 and MLKL and is induced by death receptors, interferons, toll-like receptors, intracellular RNA and DNA sensors, and probably other mediators. RIPK1 has important kinase-dependent and scaffolding functions that inhibit or trigger necroptosis and apoptosis. Mouse-model studies have revealed important functions for necroptosis in inflammation and suggested that it could be implicated in the pathogenesis of many human inflammatory diseases. We discuss the mechanisms regulating necroptosis and its potential role in inflammation and disease.

SHARPIN forms a linear ubiquitin ligase complex regulating NF-κB activity and apoptosis

SHARPIN is a ubiquitin-binding and ubiquitin-like-domain-containing protein which, when mutated in mice, results in immune system disorders and multi-organ inflammation. Here we report that SHARPIN functions as a novel component of the linear ubiquitin chain assembly complex (LUBAC) and that the absence of SHARPIN causes dysregulation of NF-κB and apoptotic signalling pathways, explaining the severe phenotypes displayed by chronic proliferative dermatitis (cpdm) in SHARPIN-deficient mice. Upon binding to the LUBAC subunit HOIP (also known as RNF31), SHARPIN stimulates the formation of linear ubiquitin chains in vitro and in vivo. Coexpression of SHARPIN and HOIP promotes linear ubiquitination of NEMO (also known as IKBKG), an adaptor of the IκB kinases (IKKs) and subsequent activation of NF-κB signalling, whereas SHARPIN deficiency in mice causes an impaired activation of the IKK complex and NF-κB in B cells, macrophages and mouse embryonic fibroblasts (MEFs). This effect is further enhanced upon concurrent downregulation of HOIL-1L (also known as RBCK1), another HOIP-binding component of LUBAC. In addition, SHARPIN deficiency leads to rapid cell death upon tumour-necrosis factor α (TNF-α) stimulation via FADD- and caspase-8-dependent pathways. SHARPIN thus activates NF-κB and inhibits apoptosis via distinct pathways in vivo.

Osteoclast differentiation factor RANKL controls development of progestin-driven mammary cancer

Breast cancer is one of the most common cancers in humans and will on average affect up to one in eight women in their lifetime in the United States and Europe. The Women’s Health Initiative and the Million Women Study have shown that hormone replacement therapy is associated with an increased risk of incident and fatal breast cancer. In particular, synthetic progesterone derivatives (progestins) such as medroxyprogesterone acetate (MPA), used in millions of women for hormone replacement therapy and contraceptives, markedly increase the risk of developing breast cancer. Here we show that the in vivo administration of MPA triggers massive induction of the key osteoclast differentiation factor RANKL (receptor activator of NF-κB ligand) in mammary-gland epithelial cells. Genetic inactivation of the RANKL receptor RANK in mammary-gland epithelial cells prevents MPA-induced epithelial proliferation, impairs expansion of the CD49fhi stem-cell-enriched population, and sensitizes these cells to DNA-damage-induced cell death. Deletion of RANK from the mammary epithelium results in a markedly decreased incidence and delayed onset of MPA-driven mammary cancer. These data show that the RANKL/RANK system controls the incidence and onset of progestin-driven breast cancer.

NEMO/IKKγ-Deficient Mice Model Incontinentia Pigmenti

Disruption of the X-linked gene encoding NF-kappa B essential modulator (NEMO) produces male embryonic lethality, completely blocks NF-kappa B activation by proinflammatory cytokines, and interferes with the generation and/or persistence of lymphocytes. Heterozygous female mice develop patchy skin lesions with massive granulocyte infiltration and hyperproliferation and increased apoptosis of keratinocytes. Diseased animals present severe growth retardation and early mortality. Surviving mice recover almost completely, presumably through clearing the skin of NEMO-deficient keratinocytes. Male lethality and strikingly similar skin lesions in heterozygous females are hallmarks of the human genetic disorder incontinentia pigmenti (IP). Together with the recent discovery that mutations in the human NEMO gene cause IP, our results indicate that we have created a mouse model for that disease.

Regulation of tissue homeostasis by NF‑κB signalling: implications for inflammatory diseases

The nuclear factor-κB (NF-κB) signalling pathway regulates immune responses and is implicated in the pathogenesis of many inflammatory diseases. Given the well established pro-inflammatory functions of NF-κB, inhibition of this pathway would be expected to have anti-inflammatory effects. However, recent studies in mouse models have led to surprising and provocative results, as NF-κB inhibition in epithelial cells resulted in the spontaneous development of severe chronic inflammatory conditions. These findings indicate that NF-κB signalling acts in non-immune cells to control the maintenance of tissue immune homeostasis. This Review discusses the mechanisms by which NF-κB activity in non-immune cells regulates tissue immune homeostasis and prevents the pathogenesis of inflammatory diseases.

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.

Inhibition of NF-kappaB activation in macrophages increases atherosclerosis in LDL receptor-deficient mice

Atherosclerosis is now generally accepted as a chronic inflammatory condition. The transcription factor NF-kappaB is a key regulator of inflammation, immune responses, cell survival, and cell proliferation. To investigate the role of NF-kappaB activation in macrophages during atherogenesis, we used LDL receptor-deficient mice with a macrophage-restricted deletion of IkappaB kinase 2 (IKK2), which is essential for NF-kappaB activation by proinflammatory signals. These mice showed increased atherosclerosis as quantified by lesion area measurements. In addition, the lesions were more advanced and showed more necrosis and increased cell number in early lesions. Southern blotting revealed that deletion of IKK2 was approximately 65% in macrophages, coinciding with a reduction of 50% in NF-kappaB activation, as compared with controls. In both groups, the expression of differentiation markers, uptake of bacteria, and endocytosis of modified LDL was similar. Upon stimulation with LPS, production of TNF was reduced by approximately 50% in IKK2-deleted macrophages. Interestingly, we also found a major reduction in the anti-inflammatory cytokine IL-10. Our data show that inhibition of the NF-kappaB pathway in macrophages leads to more severe atherosclerosis in mice, possibly by affecting the pro- and anti-inflammatory balance that controls the development of atherosclerosis.

Mechanisms regulating skin immunity and inflammation

Immune responses in the skin are important for host defence against pathogenic microorganisms. However, dysregulated immune reactions can cause chronic inflammatory skin diseases. Extensive crosstalk between the different cellular and microbial components of the skin regulates local immune responses to ensure efficient host defence, to maintain and restore homeostasis, and to prevent chronic disease. In this Review, we discuss recent findings that highlight the complex regulatory networks that control skin immunity, and we provide new paradigms for the mechanisms that regulate skin immune responses in host defence and in chronic inflammation.