Hedgehog signalling within airway epithelial progenitors and in small-cell lung cancer

Embryonic signalling pathways regulate progenitor cell fates in mammalian epithelial development and cancer. Prompted by the requirement for sonic hedgehog (Shh) signalling in lung development, we investigated a role for this pathway in regeneration and carcinogenesis of airway epithelium. Here we demonstrate extensive activation of the hedgehog (Hh) pathway within the airway epithelium during repair of acute airway injury. This mode of Hh signalling is characterized by the elaboration and reception of the Shh signal within the epithelial compartment, and immediately precedes neuroendocrine differentiation. We reveal a similar pattern of Hh signalling in airway development during normal differentiation of pulmonary neuroendocrine precursor cells, and in a subset of small-cell lung cancer (SCLC), a highly aggressive and frequently lethal human tumour with primitive neuroendocrine features. These tumours maintain their malignant phenotype in vitro and in vivo through ligand-dependent Hh pathway activation. We propose that some types of SCLC might recapitulate a critical, Hh-regulated event in airway epithelial differentiation. This requirement for Hh pathway activation identifies a common lethal malignancy that may respond to pharmacological blockade of the Hh signalling pathway.     

Role of toll-like receptors in regulatory functions of T and B cells

Pathogens can find their ways to most sites in the host. Pathogen sensors, such as Toll-like receptors （TLRs）, must be equally and broadly distributed on immune cells to combat them through innate and adaptive immunity. Most classes of TLRs are found in innate immune cells to obtain an immediate response against pathogens, but recent studies indicate that a number of TLRs are wildly expressed in T and B cells, suggesting TLRs also directly regulate adaptive immune responses. Due to the rapid increase of new information on the multiple roles of TLRs, in this paper we aim to review several main properties of TLRs and their direct role in T and B cells. This review consists of 6 parts： （i） Characteristics of Toll-like receptors （TLRs） and signaling; （ii） signalling pathways of TLRs; （iii） TLR expressions on human leukocytes; （iv） TLR expressions and functions in the Thl, CD4^＋CD45RO^＋ memory T cells and regulatory/suppressor T as well as B cell populations; （v） therapeutic potential of TLR agonists; （Vi） discussion and perspective. The latest findings and potential therapeutic applications are discussed. There is growing evidence supporting the concept that TLR activation contributes not only to innate immunity but also to adaptive immunity, including direct regulation of both T and B lymphocytes by TLRs.     

HVEM signalling at mucosal barriers provides host defence against pathogenic bacteria

The herpes virus entry mediator (HVEM), a member of the tumour-necrosis factor receptor family, has diverse functions, augmenting or inhibiting the immune response. HVEM was recently reported as a colitis risk locus in patients, and in a mouse model of colitis we demonstrated an anti-inflammatory role for HVEM, but its mechanism of action in the mucosal immune system was unknown. Here we report an important role for epithelial HVEM in innate mucosal defence against pathogenic bacteria. HVEM enhances immune responses by NF-κB-inducing kinase-dependent Stat3 activation, which promotes the epithelial expression of genes important for immunity. During intestinal Citrobacter rodentium infection, a mouse model for enteropathogenic Escherichia coli infection, Hvem?/? mice showed decreased Stat3 activation, impaired responses in the colon, higher bacterial burdens and increased mortality. We identified the immunoglobulin superfamily molecule CD160 (refs 7 and 8), expressed predominantly by innate-like intraepithelial lymphocytes, as the ligand engaging epithelial HVEM for host protection. Likewise, in pulmonary Streptococcus pneumoniae infection, HVEM is also required for host defence. Our results pinpoint HVEM as an important orchestrator of mucosal immunity, integrating signals from innate lymphocytes to induce optimal epithelial Stat3 activation, which indicates that targeting HVEM with agonists could improve host defence.     

A role for the C3a anaphylatoxin receptor in the effector phase of asthma

Asthma is a chronic inflammatory disease of the airways and lung mucosa with a strong correlation to atopy and acquired (IgE) immunity. However, many features of bronchial asthma, such as smooth muscle contraction, mucus secretion and recruitment of inflammatory cells, are consistent with the actions of complement anaphylatoxins, in particular C3a and C5a. Complement activation forms a central core of innate immune defence against mucosal bacteria, viruses, fungi, helminths and other pathogens. As a system of 'pattern-recognition molecules', foreign surface antigens and immune complexes lead to a proteolytic cascade culminating in a lytic membrane attack. The anaphylatoxins C3a and C5a are liberated as activation byproducts and are potent pro-inflammatory mediators that bind to specific cell surface receptors and cause leukocyte activation, smooth muscle contraction and vascular permeability. Here we show that in a murine model of allergic airway disease, genetic deletion of the C3a receptor protects against the changes in lung physiology seen after allergen challenge. Furthermore, human asthmatics develop significant levels of ligand C3a following intra-pulmonary deposition of allergen, but not saline. We propose that, in addition to acquired immune responses, the innate immune system and complement (C3a in particular) are involved in the pathogenesis of asthma.     

A systems biology analysis of the Drosophila phagosome

Phagocytes have a critical function in remodelling tissues during embryogenesis and thereafter are central effectors of immune defence. During phagocytosis, particles are internalized into 'phagosomes', organelles from which immune processes such as microbial destruction and antigen presentation are initiated. Certain pathogens have evolved mechanisms to evade the immune system and persist undetected within phagocytes, and it is therefore evident that a detailed knowledge of this process is essential to an understanding of many aspects of innate and adaptive immunity. However, despite the crucial role of phagosomes in immunity, their components and organization are not fully defined. Here we present a systems biology analysis of phagosomes isolated from cells derived from the genetically tractable model organism Drosophila melanogaster and address the complex dynamic interactions between proteins within this organelle and their involvement in particle engulfment. Proteomic analysis identified 617 proteins potentially associated with Drosophila phagosomes; these were organized by protein-protein interactions to generate the 'phagosome interactome', a detailed protein-protein interaction network of this subcellular compartment. These networks predicted both the architecture of the phagosome and putative biomodules. The contribution of each protein and complex to bacterial internalization was tested by RNA-mediated interference and identified known components of the phagocytic machinery. In addition, the prediction and validation of regulators of phagocytosis such as the 'exocyst', a macromolecular complex required for exocytosis but not previously implicated in phagocytosis, validates this strategy. In generating this 'systems-based model', we show the power of applying this approach to the study of complex cellular processes and organelles and expect that this detailed model of the phagosome will provide a new framework for studying host-pathogen interactions and innate immunity.     

RICK/Rip2/CARDIAK mediates signalling for receptors of the innate and adaptive immune systems

The immune system consists of two evolutionarily different but closely related responses, innate immunity and adaptive immunity. Each of these responses has characteristic receptors-Toll-like receptors (TLRs) for innate immunity and antigen-specific receptors for adaptive immunity. Here we show that the caspase recruitment domain (CARD)-containing serine/threonine kinase Rip2 (also known as RICK, CARDIAK, CCK and Ripk2) transduces signals from receptors of both immune responses. Rip2 was recruited to TLR2 signalling complexes after ligand stimulation. Moreover, cytokine production in Rip2-deficient cells was reduced on stimulation of TLRs with lipopolysaccharide, peptidoglycan and double-stranded RNA, but not with bacterial DNA, indicating that Rip2 is downstream of TLR2/3/4 but not TLR9. Rip2-deficient cells were also hyporesponsive to signalling through interleukin (IL)-1 and IL-18 receptors, and deficient for signalling through Nod proteins-molecules also implicated in the innate immune response. Furthermore, Rip2-deficient T cells showed severely reduced NF-kappaB activation, IL-2 production and proliferation on T-cell-receptor (TCR) engagement, and impaired differentiation to T-helper subtype 1 (TH1) cells, indicating that Rip2 is required for optimal TCR signalling and T-cell differentiation. Rip2 is therefore a signal transducer and integrator of signals for both the innate and adaptive immune systems.     

Helper T cells regulate type-2 innate immunity in vivo

Type-2 immunity requires orchestration of innate and adaptive immune responses to protect mucosal sites from pathogens. Dysregulated type-2 responses result in allergy or asthma. T helper 2 (T(H)2) cells elaborate cytokines, such as interleukin (IL)-4, IL-5, IL-9 and IL-13, which work with toxic mediators of innate immune cells to establish environments that are inhospitable to helminth or arthropod invaders. The importance of T(H)2 cells in coordinating innate immune cells at sites of inflammation is not known. Here we show that polarized type-2 immune responses are initiated independently of adaptive immunity. In the absence of B and T cells, IL-4-expressing eosinophils were recruited to tissues of mice infected with the helminth Nippostrongylus brasiliensis, but eosinophils failed to degranulate. Reconstitution with CD4 T cells promoted accumulation of degranulated IL-4-expressing cells, but only if T cells were stimulated with cognate antigen. Degranulation correlated with tissue destruction, which was attenuated if eosinophils were depleted. Helper T cells confer antigen specificity on eosinophil cytotoxicity, but not cytokine responses, so defining a novel mechanism that focuses tissue injury at sites of immune challenge.     

植物天然免疫系统研究进展

很多植物病原菌严重地损害植物的生长和繁殖。植物与病原体协同进化过程中,也逐渐形成了一系列复杂高效的保护机制来抵御病原物的侵染。植物中抵抗外界微生物刺激所形成的系统被称为植物天然免疫系统,可分为两个层次。第1个层次是植物模式识别受体(PRRs)识别病原相关分子模式(PAMPs),触发病原相关分子模式触发的免疫反应（PTI）,激活植物体中促丝裂原活化蛋白激酶(MAPK)信号通路使植物产生早期应答反应。PTI适应性较广,可识别和响应包括非致病菌的许多类微生物。第2个层次是病原菌产生效应因子抑制基础免疫响应PTI,而植物产生针对性更强的抗性蛋白(R蛋白)识别效应因子,并通过效应因子触发型免疫(ETI)来重建植物的抗性。笔者综述了近年来植物天然免疫系统的研究进展,认为随着对植物天然免疫系统研究的深入,应重视PTI和ETI的结合利用,有效扩大植物抗菌谱,改良植物ETI抗性。     

Innate immune recognition and regulation in liver injury: A brief report from a series of studies

The discovery of innate immune receptors and the emergence of liver immunology （high content of NK and NKT cells in liver） led to the second research summit in innate immunity since the finding of NK cells in the middle 1970s. Liver disease is one of the most dangerous threats to humans, and the progress in innate immunology and liver immunology made it possible to re-explain the cellular and mo- lecular immune mechanisms of liver disease. In the past ten years, we have found that innate recognition of hepatic NK and NKT subsets were involved in murine liver injury. We established a novel NK cell-dependent acute murine hepatitis model by activating Toll-like receptor-3 （TLR-3） with an injection of poly I：C, which may mimic mild viral hepatitis （such as Chronic Hepatitis B）. We observed that a network of innate immune cells including NK, NKT and Kupffer cells is involved in liver immune injury in our established NK cell-dependent murine,model. We noted that TLR-3 on Kupffer cells activated by pretreatment with poly I： C might protect against bacterial toxin （LPS）-induced fulminant hepatitis by down-regulating TLR-4 function, while TLR-3 pre-activation of NK cells might reduce Con A-induced NKT cell-mediated fulminant hepatitis by blocking NKT cell recruitment to the liver. We also found that the oversensitivity to injury by immune stimulation in HBV （hepatitis B virus） transgenic mice （full HBV gene-tg or HBs-tg） correlated to the over-expression of Real, an NKG2D （natural killer cell group 2D） ligand of NK cells or CDld, a ligand of TCR-V14 of NKT cells, on HBV＋ hepatocytes, which leads to an innate immune response against hepatocytes and is critical in liver immune injury and regeneration.     

A Xanthomonas uridine 5'-monophosphate transferase inhibits plant immune kinases

Plant innate immunity is activated on the detection of pathogen-associated molecular patterns (PAMPs) at the cell surface, or of pathogen effector proteins inside the plant cell. Together, PAMP-triggered immunity and effector-triggered immunity constitute powerful defences against various phytopathogens. Pathogenic bacteria inject a variety of effector proteins into the host cell to assist infection or propagation. A number of effector proteins have been shown to inhibit plant immunity, but the biochemical basis remains unknown for the vast majority of these effectors. Here we show that the Xanthomonas campestris pathovar campestris type III effector AvrAC enhances virulence and inhibits plant immunity by specifically targeting Arabidopsis BIK1 and RIPK, two receptor-like cytoplasmic kinases known to mediate immune signalling. AvrAC is a uridylyl transferase that adds uridine 5'-monophosphate to and conceals conserved phosphorylation sites in the activation loop of BIK1 and RIPK, reducing their kinase activity and consequently inhibiting downstream signalling.     

Lymphoid tissue genesis induced by commensals through NOD1 regulates intestinal homeostasis

Intestinal homeostasis is critical for efficient energy extraction from food and protection from pathogens. Its disruption can lead to an array of severe illnesses with major impacts on public health, such as inflammatory bowel disease characterized by self-destructive intestinal immunity. However, the mechanisms regulating the equilibrium between the large bacterial flora and the immune system remain unclear. Intestinal lymphoid tissues generate flora-reactive IgA-producing B cells, and include Peyer's patches and mesenteric lymph nodes, as well as numerous isolated lymphoid follicles (ILFs). Here we show that peptidoglycan from Gram-negative bacteria is necessary and sufficient to induce the genesis of ILFs in mice through recognition by the NOD1 (nucleotide-binding oligomerization domain containing 1) innate receptor in epithelial cells, and beta-defensin 3- and CCL20-mediated signalling through the chemokine receptor CCR6. Maturation of ILFs into large B-cell clusters requires subsequent detection of bacteria by toll-like receptors. In the absence of ILFs, the composition of the intestinal bacterial community is profoundly altered. Our results demonstrate that intestinal bacterial commensals and the immune system communicate through an innate detection system to generate adaptive lymphoid tissues and maintain intestinal homeostasis.     

A protective role for protease-activated receptors in the airways

The protection of cells in the upper intestine against digestion by pancreatic trypsin depends on the prostanoid prostaglandin E2 (PGE2) and is mediated by protease-activated receptors in the epithelium. As the airway epithelium is morphologically similar and also expresses one of these receptors, PAR2, and is a major source of PGE2, we reasoned that bronchial epithelial PAR2 might also participate in prostanoid-dependent cytoprotection in the airways. Here we show that activation of PAR2, which co-localizes immunohistochemically with trypsin(ogen) in airway epithelium, causes the relaxation of airway preparations from mouse, rat, guinea-pig and humans by the release of a cyclooxygenase product from the epithelium. This physiological protective response in isolated airways also occurred in anaesthetized rats, where activation of PAR2 caused a marked and prolonged inhibition of bronchoconstriction. After desensitization of PAR2, the response to trypsin recovered rapidly by mechanisms dependent on de novo synthesis and trafficking of proteins. Our results indicate that trypsin released from the epithelium can initiate powerful bronchoprotection in the airways by activation of epithelial PAR2.     

Caspase-8 regulates TNF-α-induced epithelial necroptosis and terminal ileitis

Dysfunction of the intestinal epithelium is believed to result in the excessive translocation of commensal bacteria into the bowel wall that drives chronic mucosal inflammation in Crohn's disease, an incurable inflammatory bowel disease in humans characterized by inflammation of the terminal ileum. In healthy individuals, the intestinal epithelium maintains a physical barrier, established by the tight contact of cells. Moreover, specialized epithelial cells such as Paneth cells and goblet cells provide innate immune defence functions by secreting mucus and antimicrobial peptides, which hamper access and survival of bacteria adjacent to the epithelium. Epithelial cell death is a hallmark of intestinal inflammation and has been discussed as a possible pathogenic mechanism driving Crohn's disease in humans. However, the regulation of epithelial cell death and its role in intestinal homeostasis remain poorly understood. Here we demonstrate a critical role for caspase-8 in regulating necroptosis of intestinal epithelial cells (IECs) and terminal ileitis. Mice with a conditional deletion of caspase-8 in the intestinal epithelium (Casp8(ΔIEC)) spontaneously developed inflammatory lesions in the terminal ileum and were highly susceptible to colitis. Casp8(ΔIEC) mice lacked Paneth cells and showed reduced numbers of goblet cells, indicating dysregulated antimicrobial immune cell functions of the intestinal epithelium. Casp8(ΔIEC) mice showed increased cell death in the Paneth cell area of small intestinal crypts. Epithelial cell death was induced by tumour necrosis factor (TNF)-α, was associated with increased expression of receptor-interacting protein 3 (Rip3; also known as Ripk3) and could be inhibited on blockade of necroptosis. Lastly, we identified high levels of RIP3 in human Paneth cells and increased necroptosis in the terminal ileum of patients with Crohn's disease, suggesting a potential role of necroptosis in the pathogenesis of this disease. Together, our data demonstrate a critical function of caspase-8 in regulating intestinal homeostasis and in protecting IECs from TNF-α-induced necroptotic cell death.     

Adherens junction protein nectin-4 is the epithelial receptor for measles virus

Measles virus is an aerosol-transmitted virus that affects more than 10 million children each year and accounts for approximately 120,000 deaths. Although it was long believed to replicate in the respiratory epithelium before disseminating, it was recently shown to infect initially macrophages and dendritic cells of the airways using signalling lymphocytic activation molecule family member 1 (SLAMF1; also called CD150) as a receptor. These cells then cross the respiratory epithelium and transport the infection to lymphatic organs where measles virus replicates vigorously. How and where the virus crosses back into the airways has remained unknown. On the basis of functional analyses of surface proteins preferentially expressed on virus-permissive human epithelial cell lines, here we identify nectin-4 (ref. 8; also called poliovirus-receptor-like-4 (PVRL4)) as a candidate host exit receptor. This adherens junction protein of the immunoglobulin superfamily interacts with the viral attachment protein with high affinity through its membrane-distal domain. Nectin-4 sustains measles virus entry and non-cytopathic lateral spread in well-differentiated primary human airway epithelial sheets infected basolaterally. It is downregulated in infected epithelial cells, including those of macaque tracheae. Although other viruses use receptors to enter hosts or transit through their epithelial barriers, we suggest that measles virus targets nectin-4 to emerge in the airways. Nectin-4 is a cellular marker of several types of cancer, which has implications for ongoing measles-virus-based clinical trials of oncolysis.     

Chloride and potassium channels in cystic fibrosis airway epithelia

Cystic fibrosis, the most common lethal genetic disease in Caucasians, is characterized by a decreased permeability in sweat gland duct and airway epithelia. In sweat duct epithelium, a decreased Cl- permeability accounts for the abnormally increased salt content of sweat. In airway epithelia a decreased Cl- permeability, and possibly increased sodium absorption, may account for the abnormal respiratory tract fluid. The Cl- impermeability has been localized to the apical membrane of cystic fibrosis airway epithelial cells. The finding that hormonally regulated Cl- channels make the apical membrane Cl- permeable in normal airway epithelial cells suggested abnormal Cl- channel function in cystic fibrosis. Here we report that excised, cell-free patches of membrane from cystic fibrosis epithelial cells contain Cl- channels that have the same conductive properties as Cl- channels from normal cells. However, Cl- channels from cystic fibrosis cells did not open when they were attached to the cell. These findings suggest defective regulation of Cl- channels in cystic fibrosis epithelia; to begin to address this issue, we performed two studies. First, we found that isoprenaline, which stimulates Cl- secretion, increases cellular levels of cyclic AMP in a similar manner in cystic fibrosis and non-cystic fibrosis epithelial cells. Second, we show that adrenergic agonists open calcium-activated potassium channels, indirectly suggesting that calcium-dependent stimulus-response coupling is intact in cystic fibrosis. These data suggest defective regulation of Cl- channels at a site distal to cAMP accumulation.