NB3-Bahia: janeiro 2011

segunda-feira, 31 de janeiro de 2011

excesso de higiene também não ajuda

Moms' Infection Helps Kids' Skin

Can we be too clean? According to what’s called the hygiene hypothesis, yes. Without being challenged as kids, our immune systems don’t flourish. Scientists think it could be part of the rise of allergies and asthma.

Now a new study supports the hygiene hypothesis: infants in Uganda had a lower chance of developing the skin allergy condition eczema if their moms had helminth worm infections while pregnant. The research is in the journal Pediatric Allergy and Immunology. [Harriet Mpairwe et al., "Anthelminthic treatment during pregnancy is associated with increased risk of infantile eczema: randomised-controlled trial results"]

A 2005 study showed that the kids of women treated for worm infections had more eczema. Twenty-five hundred pregnant women took part in this follow-up research. Some got one worm-killing drug. Others took a different drug. And a third group received a placebo. One drug nearly doubled the kids’ risk of eczema. The other more than doubled the odds.

Helminth worm infections can give the mothers symptoms such as mild anemia or stomach pain and vomiting. Although many people have no symptoms at all. The scientists say more research is needed before they would recommend not treating worm infections. But the work lends additional support to the idea that hygiene may be a balancing act rather than a goal.

Um show de imagens em biologia celular - Cell

Image: Omentum was harvested from a C57BL/6 mouse and stained with anti-CD31 (green) and anti-DARC, followed by an Alexa 647 conjugated second stage (red). Whole omentum was placed between coverslips to acquire a Z-stack image using an inverted confocal microscope. Green and red images were processed and then merged.

Image: In the dermis of a mouse ear, the afferent lymphatic vessels (white) are entangled with a vast network of blood vessels (red). Dermal dendritic cells expressing class II MHC (green) are strategically positioned as immune sentinels, continuously probing the microenvironment, including extracellular spaces and into the lumen of the afferent lymph vessel. CD31⁺ endothelial cells (red) and LYVE-1+ lymphatic endothelial cells (white) delineate the blood vessels and lymphatic vessels, respectively. Cellular nuclear are stained blue with DAPI.Image: In the dermis of a mouse ear, the afferent lymphatic vessels (white) are entangled with a vast network of blood vessels (red). Dermal dendritic cells expressing class II MHC (green) are strategically positioned as immune sentinels, continuously probing the microenvironment, including extracellular spaces and into the lumen of the afferent lymph vessel. CD31⁺ endothelial cells (red) and LYVE-1+ lymphatic endothelial cells (white) delineate the blood vessels and lymphatic vessels, respectively. Cellular nuclear are stained blue with DAPI.

Dendritic cells are the "shakers and movers" of the immune system. They detect pathogens and then get the immune response rolling by activating T cells. Dendritic cells in the skin and other epithelial surfaces are called Langerhans cells (purple). Like all dendritic cells, Langerhans cells use receptors on their cell surface (e.g., Toll receptors) to identify and uptake pathogens (red) by phagocytosis or endocytosis. Once triggered, Langerhans cells then emigrate from the epithelia into the blood or lymph to launch an adaptive immune response.une response rolling by activating T cells. Dendritic cells in the skin and other epithelial surfaces are called Langerhans cells (purple). Like all dendritic cells, Langerhans cells use receptors on their cell surface (e.g., Toll receptors) to identify and uptake pathogens (red) by phagocytosis or endocytosis. Once triggered, Langerhans cells then emigrate from the epithelia into the blood or lymph to launch an adaptive immune response.

domingo, 30 de janeiro de 2011

Tuberculose o desafio para descoberta de novas drogas - Revisão Nature

The challenge of new drug discovery for tuberculosis

Anil Koul, Eric Arnoult, Nacer Lounis, Jerome Guillemont & Koen Andries
AffiliationsContributionsCorresponding author
Nature 469, 483–490 (27 January 2011) doi:10.1038/nature09657
Published online 26 January 2011

Tuberculosis (TB) is more prevalent in the world today than at any other time in human history. Mycobacterium tuberculosis, the pathogen responsible for TB, uses diverse strategies to survive in a variety of host lesions and to evade immune surveillance. A key question is how robust are our approaches to discovering new TB drugs, and what measures could be taken to reduce the long and protracted clinical development of new drugs. The emergence of multi-drug-resistant strains of M. tuberculosis makes the discovery of new molecular scaffolds a priority, and the current situation even necessitates the re-engineering and repositioning of some old drug families to achieve effective control. Whatever the strategy used, success will depend largely on our proper understanding of the complex interactions between the pathogen and its human host. In this review, we discuss innovations in TB drug discovery and evolving strategies to bring newer agents more quickly to patients.

Introduction

Introduction Emerging challenges in TB treatment Identifying new chemical scaffolds Targeting host–pathogen signalling pathways In vivo screens and preclinical validation Evolving science of TB clinical development Reassessment and time for acceleration

In 1882, Robert Koch identified Mycobacterium tuberculosis as the causative agent of TB, but since his discovery the global TB epidemic seems unabated; this year it is anticipated that there will be about 9.8 million new cases, more than in any other year in history1. This situation highlights the relative shortcomings of the current treatment strategies for TB and the limited effectiveness of public health systems, particularly in resource-poor countries where the main TB burden lies. The ease with which TB infection spreads (for example, by inhalation of a few droplet nuclei 2–5 μm in diameter containing as few as 1–3 bacilli2), has helped to sustain this scourge at current levels. In spite of half a century of anti-TB chemotherapy, one-third of the world’s population asymptomatically still harbour a dormant or latent form of M. tuberculosis with a lifelong risk of disease reactivation (Fig. 1). Reactivation of latent TB, even after decades of subclinical persistence, is a high risk factor for disease development particularly in immunocompromised individuals such as those co-infected with human immunodeficiency virus (HIV), on an anti-tumour necrosis factor therapy or with diabetes3 (Box 1). In recent years, the TB epidemic has been further fuelled by the emergence of multi- and extensively-drug-resistant (MDR-TB and XDR-TB) strains and dwindling treatment options that are decades old. The last drug with a new mechanism of action approved for TB was rifampicin (discovered in 1963). Further complicating the situation are drug–drug interactions that preclude the co-administration of some available TB drugs with certain anti-HIV treatments or other chronic disease medications, such as those used in diabetics.

M. tuberculosis aerosol transmission and progression to infectious TB or non-infectious (latent) disease. A sizeable pool of latently infected people may relapse into active TB, years after their first exposure to the bacterium. Latent TB is commonly activated by immune suppression, as in the case of HIV. In cases of drug-susceptible (DS)-TB (denoted by an asterisk), 95% of patients recover upon treatment, whereas 5% relapse. If untreated (denoted by two asterisks), high mortality results.

To achieve global control of this epidemic, there is an urgent need for new TB drugs, which can: (1) shorten treatment duration; (2) target MDR or XDR strains; (3) simplify treatment by reducing the daily pill burden; (4) lower dosing frequency (for example, a once-weekly regimen); and (5) be co-administered with HIV medications (Table 1). The challenge of meeting the expectations of this desired target product profile complicates drug discovery efforts. Considering how few drugs from the discovery stage successfully enter the TB clinical pipeline, an increased understanding of the drug discovery hurdles should facilitate development of novel intervention strategies. The situation is further hampered by the unfavourable economics of TB drug development and the lack of proper policy incentives. In this review, we present our perspective on how to refocus discovery and development efforts and identify the underlying knowledge gaps and scientific obstacles in TB drug development. Finally, we highlight some emerging chemical scaffolds, which will hopefully fuel the TB clinical pipeline.

Table 1: Desired target product profile for a new TB drug

Emerging challenges in TB treatment

The world’s two most populous countries, India and China, account for more than 50% of the world’s MDR-TB cases and as such these countries are encountering a high and increasing TB disease burden4. The sheer size of their TB case populations results in the highest estimated numbers of MDR-TB cases (about 100,000 each) emerging annually from these two countries. Moreover, the emergence of XDR strains of M. tuberculosis (5.4% of MDR-TB cases are found to be XDR-TB4) is challenging TB treatment programmes in several other countries and even raises the possibility of a return to a situation akin to the pre-antibiotic TB era. At present, MDR-TB is treated by a combination of eight to ten drugs with therapies lasting up to 18–24 months; only four of these drugs were actually developed to treat TB5. Such suboptimal therapy leads to almost 30% of MDR-TB patients experiencing treatment failure6. The treatment options for XDR-TB are very limited as XDR-TB bacilli are resistant not only to isoniazid and rifampicin, but also to fluoroquinolones and injectables such as aminoglycosides. In addition, there are serious side effects with most MDR-TB and XDR-TB drugs, including nephrotoxicity and ototoxicity with aminoglycosides, hepatotoxicity with ethionamide and dysglycaemia with gatifloxacin7. Thus, the current situation necessitates the immediate identification of new scaffolds that can address emerging resistance and also demands the conduct of appropriate clinical trials as historically very few clinical studies have been performed to evaluate the efficacy of drugs in MDR-TB or XDR-TB patient groups. Improving the diagnostics with wider coverage of drug susceptibility testing will also help to address the high mortality of MDR/XDR-TB and curb the emergence of resistance.

TB accounts for about one in four of the deaths that occur among HIV-positive people8. Of the 9.4 million TB cases in 2009, 11–13% were HIV positive with approximately 80% of these co-infections confined to the African region8. The frequent co-infection of TB in HIV patients further complicates the selection of an appropriate treatment regimen because: (1) increased pill burden diminishes compliance; (2) drug–drug interactions lead to sub-therapeutic concentrations of antiretrovirals; and (3) overlapping toxic side effects increase safety concerns. The main interaction between HIV and TB anti-infectives is rifampicin-induced increased expression of the hepatic cytochrome (CYP) P450 oxidase system9. This CYP induction results in increased metabolism and decreased therapeutic concentrations of many co-medications such as HIV protease inhibitors10. Even in the presence of CYP450 inhibitors such as ritonavir, normal trough levels of various classes of protease inhibitors cannot be rescued and consequently, standard protease inhibitor regimens, whether boosted or not, cannot be given with rifampicin. The only treatments for HIV-infected TB patients with minimal drug–drug interactions are non-nucleoside-reverse-transcriptase-inhibitor (NNRTI) containing regimens. However, there are fewer options for patients with NNRTI-resistant mutations and therefore new chemistry approaches are being used to identify new rifamycins, such as rifabutin, with reduced CYP-induction properties7. However, the presence of ritonavir in the protease cocktail increases the serum concentration of rifabutin, thereby increasing its accompanying toxicity11.

To discover newer rifamycin analogues with minimal interaction with HIV and other co-medications, the upfront screening of newer molecules in a CYP profiling (pregnane-X receptor) assay can be performed12. This receptor drives transcription of CYP genes and can identify chemical analogues with minimal interactions with drug metabolizing enzymes like CYP450. Further, availability of co-crystal structures of rifampicin with bacterial RNA polymerase13 can help to design molecules with better drug-resistance profiles. In HIV patients harbouring MDR- or XDR-TB strains, drug–drug interaction studies are not well established, as most of these second-line TB drugs (for example, ethionamide, cycloserine, kanamycin, amikacin, capreomycin and para-amino salicylate) were discovered several decades ago14. Thus, there is a clear need for new studies to investigate the interaction of antiretrovirals with second-line TB drugs and with those currently in clinical development.

Confounding these issues is the association of TB with other chronic diseases such as diabetes, which is known to increase the risk of developing active TB by threefold1. The biological rationale for the slower response of diabetics to anti-TB drugs and for their increased risk of developing MDR-TB is poorly understood, although it is well known that cell-mediated immunity is suppressed in diabetes, which could explain higher TB rates. Attainment of bacterial culture negativity, relapse rates and mortality are significantly higher in diabetic TB patients15 so we need to identify new TB molecules that are strongly bactericidal and have minimal drug–drug interactions with oral anti-diabetic drugs16. Further, diabetics tend to be heavier and more obese, which may in part lead to lower TB drug exposure17. Where there is a poor response to TB treatment in diabetic patients, therapeutic drug monitoring may be useful in TB management.

Identifying new chemical scaffolds

The poor efficiency of identifying new TB drugs by screening pharmaceutical library collections has been linked to the limited chemical diversity within these collections18. Additionally, most TB drugs and antibacterials in general do not follow Lipinski’s ‘rule of 5’, which defines the optimal drug-like features; whereas pharmaceutical compound collections are biased towards these properties19. In spite of these challenges, the current TB pipeline (Fig. 2) is slowly expanding, although it is inadequate for the development of a completely novel regimen. A key question is: how to search for new TB drugs and where to look for them?

Figure 2: A bull’s-eye representation of the current clinical pipeline for TB.

Each drug candidate is shown with its current clinical phase of development along with the target family. TMC207 is in phase IIb trials for MDR-TB and in phase IIa trials for DS-TB. The structure of AZD-5847 has not been disclosed. NDA, new drug application (for regulatory approval).

Advances in the identification of new TB drug targets have been driven largely by the availability of the genome sequence of M. tuberculosis20, but unfortunately this approach has yet to lead to the identification of new drug candidates. Genome-derived, target-based approaches have had little success in the antibacterial therapeutic area in general18. The essentiality of a target for replication may be a prerequisite but it does not ensure its druggability; for many essential targets we are unable to identify specific inhibitors with drug-like properties. For example, several high-throughput screening campaigns for identifying inhibitors of isocitrate lyases, which are key glyoxylate-shunt-pathway enzymes found to be essential for mycobacterial intracellular growth and their long-term persistence in mice, were discontinued owing to lack of druggability of these targets21. Second, we have often failed to understand how to convert good bacterial enzyme inhibitors into a compound that can easily penetrate the highly impermeable bacterial cell wall. Without proper understanding of the entry mechanisms of antibiotics across bacterial cell walls, any medicinal chemistry approach to engineer (via chemical modifications) a ‘permeability property’ into an enzymatic inhibitor has proven to be quite challenging.

Over time, it has emerged that shifting the screening strategy from single-enzyme targets to phenotypic screens at a whole bacterial cell level is a much more successful strategy18. Such a strategy recognizes the potential holistic interactions of a drug target(s) with one or more components in a bacterial cell and defines its essentiality in a more relevant physiological space. One of the drawbacks of the whole-cell-screening approach is that upfront knowledge regarding the mechanism of action remains largely lacking, thereby preventing any input from structural biology into medicinal chemistry efforts around drug design. Another challenge of whole-cell screening is to identify the right in vitro growth conditions that are relevant for in vivo infections, as certain metabolic targets behave differently depending on the composition of the growth medium22. Whole-cell screening can deliver many hits, but several of these may work via non-specific mechanisms (such as detergent effects) and have cytotoxic effects. As such, the key in a whole-cell-screening campaign is to identify the ‘quality hits’ by certain counter-screening assays (for example, cytotoxicity across several cell lines, monitoring non-specific membrane leakage, analysing red-blood-cell haemolysis), so as to account for good selectivity and specificity.

The recent success with the whole-cell-screening approach is particularly exemplified by the identification of new TB drug candidates such as diarylquinolines (TMC207), which target ATP synthesis, and benzothiazines (BTZ043), which target essential cell-wall arabinan synthesis23, 24, 25. An interesting feature of both these molecules is that they target membrane-associated proteins that may be more easily accessible to drugs from the periplasmic space (that is, the target binding sites are exposed to the periplasm) and this to some extent may overcome certain issues of mycobacterial membrane permeability.

Interestingly, more refined multi-target ‘pathway’ screens can be initiated to search for inhibitors blocking validated metabolic or signalling pathways. In this regard, respiratory membrane vesicles of M. tuberculosis, which have been grown in a variety of conditions in order to simulate the host microenvironment, can be used to screen drug classes or analogues inhibiting respiratory chain components (Fig. 3). For example, such a pathway screen could monitor a drug’s influence on diverse mycobacterial respiratory chain functions such as ATP synthesis, redox homeostasis and proton gradients26. Modulating external growth stimuli, such as the carbon source, micronutrients, or oxygen levels in such an assay, results in target respiratory proteomes that can be used to screen against functions essential during those metabolic states. For instance, ATP synthase is highly downregulated during hypoxic conditions, and its inhibition by TMC207 indicates an essential role of ATP synthesis in the generation of energy in the dormant bacteria, which may explain the potent in vivo sterilizing effect of the drug27. Dormant M. tuberculosis seems to be exceptionally susceptible to inhibition of respiratory chain processes such as ATP synthesis or interference with the cellular redox state28, but it still remains to be determined if such inhibition leads to potent sterilization in human lesions with varied microenvironments. Because most TB drugs are less efficient in killing slowly replicating or dormant bacilli in the chronic phase of TB infection, a key challenge for identifying sterilizing drugs is to translate information about the chronic state mycobacterial metabolome and proteome adaptations into drug discovery screening platforms. This strategy will not only facilitate development of proper drug discovery tools that might eventually lead to a faster cure, but may also help us to understand the life cycle of mycobacteria in their host (for example, their switch to anaerobic metabolism29).

Figure 3: Screening for mycobacterial respiratory pathway inhibitors.

Schematic flowchart of a multiple-target screen for the identification of hits targeting the mycobacterial respiratory pathway. Enriched pharmaceutical compound libraries, or compound family analogues, can be used to screen inverted membrane vesicles for inhibition of NADH dehydrogenase, ATP synthase, or other targets impairing electron flow or proton-motive force. The enlargement shows the graphic view of the mycobacterial respiratory chain proteins with menaquinone (MQ) being reduced by NADH dehydrogenase or succinate dehydrogenase (SDH) and oxidized by a supercomplex consisting of complex III and IV (cytochrome bc1 and aa3)29. The transcriptional profiling in infected mice lungs during chronic phase showed downregulation of proton-pumping type-I NADH dehydrogenase (NDH1) and low-affinity aa3-type cytochrome c oxidase, but upregulation of alternative target enzymes such as high-affinity cytochrome bd oxidase (cyd bd) and non-proton pumping NADH dehydrogenase (NDH2), which can serve as effective targets for latent or persistent infections68. Small molecules such as antipsychotic phenothiazine and diarylquinolines (for example, TMC207) have been shown to target NDH2 (ref. 69) and the transmembrane subunit-c of ATP synthase24, respectively, with potent antimycobacterial activity on actively metabolizing and non-growing cells.

Engineering existing scaffolds

Many new antibiotic candidates are chemical molecules reengineered from old drug classes discovered decades ago30. This approach has identified new TB drugs from existing antibacterial drug classes and either involved the redesign of accessible scaffolds to improve their antimycobacterial potencies or, more directly, the repositioning of known antibacterial drugs with good antimycobacterial activity for testing in TB clinical trials (Fig. 4). During re-engineering of known scaffolds, chemical modifications are introduced into the core structure that may lead to improved bactericidal activities, better resistance profiles, safety, tolerability or superior pharmacokinetic/pharmacodynamic properties.

Chemical tailoring of existing drugs or drug classes has led to the identification of new molecules with potent antimycobacterial activities. The oxazolidinones also include the recently discovered AZD-5847, the structure of which has not been disclosed yet and is therefore not listed here. SQ109, an orally active cell-wall-targeting diamine antibiotic, identified via combinatorial chemistry approaches, is currently being tested in humans.

The modified versions of the oxazolidinones (such as linezolid, a marketed product from this class with activity against Gram-positive infections) have led to new structures such as PNU-100480 and AZD-5847 with better activity against M. tuberculosis31. These oxazolidinone TB candidates, currently in phase I studies, must address in their clinical development plan the known toxicity issues of linezolid, namely inhibition of mitochondrial protein synthesis, thrombocytopenia and myelosuppression, which has been observed in patients treated for longer than the recommended 14 days32. Because TB treatment can take months, safety is of paramount importance with any new tailored oxazolidinone and it will be important to monitor for bone marrow toxicity early in clinical trials. The good human pharmacokinetic profile of linezolid (for example, excellent oral bioavailability, low CYP inhibition and good distribution to lung epithelial lining fluid33) raises the hope that this drug class can penetrate the difficult to reach thick-walled lung cavities and lesions where TB bacilli normally hide.

Nitroimidazoles, traditionally used to treat anaerobic bacteria and parasitic infections, represent another established scaffold for which synthetic modifications have been introduced to increase their antimycobacterial potential. An interesting feature of nitroimidazoles relates to their unique mechanism of action, mimicking host defence strategies by producing microbicidal molecules, such as nitric oxide and other reactive nitrogen intermediates, which damage multiple targets including respiratory chain cytochrome oxidases34. The target specificity of such a mechanism of action is achieved through bioactivation of these prodrugs by flavin-dependent nitroreductases, which are absent in mammalian cells but present in M. tuberculosis35. Two candidates from this class, PA-824 and OPC-67683, are currently in clinical studies and may potentially shorten treatment duration as this mechanism of action is operational even in hypoxic induced dormant mycobacteria34 (that are not killed by drugs such as isoniazid). This indicates that in spite of general transcriptional downregulation during mycobacterial dormancy, these nitroreductases are still sufficiently expressed.

For many years, the lack of activity of the natural or semi-synthetic β-lactams against TB was thought to be due to their poor penetration into the organism, with β-lactamase-mediated resistance only a minor confounding factor36. However, a recent genetic knockout of blaC, encoding the extended-spectrum Ambler class A β-lactamase from M. tuberculosis, showed improved sensitivity to β-lactams, particularly carbapenems37. By combining a second-generation carbapenem (meropenem) with a β-lactamase inhibitor (clavulanic acid), good in vitro bactericidal activity on replicating, non-replicating and resistant clinical isolates of M. tuberculosis was obtained37. Availability of structural and mechanistic knowledge around BlaC will help researchers design potent and M.-tuberculosis-specific inhibitors to be used in combination with classical β-lactam antibiotics. At the same time, a newer generation of broad-spectrum β-lactamase inhibitors (for example, current clinical candidates such as NXL104 (ref. 38)) should be explored for mycobacterial BlaC inhibition. Concurrently, medicinal chemistry approaches to improve the antimycobacterial activity of β-lactams, their tissue distribution and oral bioavailability, will be necessary as current drugs such as meropenem require parenteral administration37, thereby limiting their use in more serious MDR/XDR-TB cases.

Although incremental improvements of existing scaffolds is a good strategy to fill a drug development pipeline, the increasing resistance to some of these existing drug classes, such as the fluoroquinolones39, indicates that discovery of new chemical scaffolds is a more attractive approach. To facilitate the identification of new chemical scaffolds, a proper understanding of the physicochemical features of the existing TB drugs and analysis of their chemical space is desirable.

The physicochemical space of TB drugs

Antibacterial drugs in general occupy a unique physicochemical space that is markedly different from the space occupied by drugs in other therapeutic areas40. Specific physicochemical features in antibiotic drug classes are required because of the unique architecture of bacterial cell walls (especially in Gram negatives), which affects the permeability of drug molecules across these membranes. Antibacterial drugs are unique in a number of physicochemical properties, such as lower lipophilicities, higher molecular weights and increased total polar surface areas when compared to drugs for human host targets40. It has been proposed that screening libraries for antibacterial targets should have more polar characteristics to achieve penetration through certain bacterial cell walls40.

We studied 14 different physicochemical features, including molecular weight, lipophilicity and polar surface area of first- and second-line TB drugs, and compared these properties to known marketed non-antibacterial compounds (1,663) identified from the Prous Integrity database (http://integrity.prous.com) (details in Fig. 5). A mathematical tool called principal component analysis (PCA) was used to study the relationships between various physicochemical properties41 and to identify regions of physicochemical space required to achieve antimycobacterial activity. A two-dimensional graph indicates that TB drugs actually occupy a broad chemical space and do not fall into any defined chemical area. As expected, natural-product-based molecules such as rifamycins and aminoglycosides occupy a peripheral region of the plotted area, whereas fluoroquinolones, having more ‘drug-like’ features, are located among the drug bulk (Fig. 5). With no defined optimal physicochemical space for TB drugs, chemistry for the discovery of new scaffolds should be less restricted and more diverse. At the same time, the wide scatter within the PCA plot may reflect to some extent the fact that most TB drugs were discovered several decades ago without much consideration of optimal physicochemical and other drug-like features.

To understand the physicochemical space occupied by TB compounds, we studied 14 different physicochemical features of TB drugs and compared these properties to 1,663 marketed non-antibacterial unique compounds identified from the Prous Integrity database. The physicochemical parameters calculated using the Molecular Operating Environment software (MOE)70 were: log of the octanol/water partition coefficient (lipophilicity evaluated by SlogP), molecular weight, number of hydrogen bond donors and hydrogen bond acceptors, number of rotatable bonds, topological polar surface area, solubility, atomic polarizabilities, absolute atomic polarizabilities, connectivity topological index, density, radius, petitjean (diameter − radius)/diameter) and molecular refractivity. A PCA analysis, a mathematical procedure that transforms a number of (possibly) correlated variables into a number of uncorrelated variables called principal components, was done using a software program (SIMCA-P+12; Umetrics)71. A PCA analysis was carried out using the 14 calculated properties mentioned earlier and variation in the properties between the compounds is mapped onto two axes, principal components PC1 and PC2, which contain most of the variance (in this case 70% for PC1 and PC2 combined). The axes are linear combinations of the original 14 properties and each data point in the two-dimensional graph corresponds to one compound. The complete graph shows the chemical space occupied by the different compounds. This figure indicates that TB drugs are widely distributed within the chemical space.

Although antibacterial agents are generally quite polar, water-soluble molecules, the question is whether TB medicinal chemistry should try to engineer the physicochemical characteristics of newer molecules or screening libraries towards a common parameter such as polarity. In light of this question it is worth considering that TMC207, even with its lipophilic nature (logD at pH 6.0 is 5.14), has potent bactericidal activities. Therefore, biasing our library screens towards compounds with a particular physicochemical parameter could actually be detrimental and decrease the diversity of our screening campaigns and chemistry. Nevertheless, a detailed understanding of the influence of polarity on drug penetration into the highly impermeable mycobacterium cell wall (for example, Mycobacterium smegmatis is about 20 times less permeable than Escherichia coli42) may guide us to improved permeability. An important question is how the unique mycobacterial membrane architecture, with its high lipid content, influences drug uptake and efflux compared to the cell walls of other Gram-positive and Gram-negative bacteria.

Targeting host–pathogen signalling pathways

Subversion of host-cell signalling pathways is one of the strategies used by pathogenic mycobacteria to survive long term in host cells43. As such, targeting the key signalling molecules, either bacterial- or host-derived, may lead to new antimycobacterial therapies.

On the basis of the knowledge that M. tuberculosis has 11 serine/threonine kinases and several other ATP- (or GTP-)using enzymes, researchers have screened enriched kinase libraries for inhibition of mycobacterial growth in vitro or in macrophages, with limited success44. However, a recent kinase library screen in E. coli led to the identification of a pyridopyrimidine scaffold as a competitive inhibitor of the ATP-binding site of acetyl-coenzyme-A carboxylase45. For mycobacteria, the search for kinase inhibitors with potent in vitro bactericidal activity has not been successful, although chemical optimization towards the uniquely conserved ATP-binding pockets of protein kinase G did identify a new chemical scaffold, tetra-hydro-benzothiophene (AX20017), but with activity restricted to infected macrophages46 (Fig. 6). A related concept within bacterial research is to identify inhibitors of bacterial virulence factors or host targets that can modulate pathogen survival inside the infected cells. Recently, it was revealed that the innate immune response within macrophages can be modulated by specifically inhibiting the mycobacterial tyrosine phosphatase (mptpB), which blocks host ERK1/2 and P38 signalling and promotes intramacrophage survival of mycobacteria47 (Fig. 6). At the same time, genome-wide RNA interference screening has identified key host kinase networks and an autophagic/xenophagic machinery that is severely inhibited on mycobacterial infection48. This research showed that pharmacological activation of the xenophagic pathway in infected macrophages by certain drugs led to the killing of intracellular mycobacteria. However, in the absence of any in vivo validation and also any extracellular bactericidal activity, such drugs, if proven to be clinically efficacious, would probably be used in an adjunctive therapy along with a direct antibacterial agent. It is not known if the intracellular dwelling of mycobacteria contributes to its prolonged treatment duration and whether strategies targeting host-cell factors will lead to better bactericidal activity and shorter treatment time in patients.

Figure 6: Representative underexplored and new chemical scaffolds.

Some of the chemical structures shown are mentioned in the text. CPZEN-45, a streptomyces-derived natural product, is a semi-synthetic nucleoside antibiotic from the caprazamycin family with TB activity72. Re-engineering of riminophenazine’s chemical scaffold can lead to interesting energy metabolism inhibitors with the potential to kill non-replicating bacilli. Azamethylquinolones have demonstrated activity on mycobacteria and further chemical optimization may lead to interesting lead candidates hopefully with better resistance profiles73.

In vivo screens and preclinical validation

Animal models that mimic various metabolic stages of human infection have proven to be extremely important for TB drug discovery as some functions deemed to be essential in vitro (such as mycobacterial glycolysis) are not essential in vivo49. However, no animal model is perfect as each model only incompletely reproduces different aspects of human disease. The mouse model is considered imperfect because certain elements of human disease pathogenesis such as organized granulomas, caseous necrosis and hypoxia are not replicated3. In the absence of these features, the challenging question is whether the mycobacterial metabolic repertoire present in mice is less heterogeneous than in humans? Despite not exactly replicating the host-tissue microenvironment, the mouse model has served as a cost-effective tool to assess the bactericidal and sterilizing potencies of individual drugs and drug combinations50.

The mouse model was recently also used to identify bacterial targets that impair or enhance mycobacterial persistence upon treatment with isoniazid51. Such an approach illustrates the potential role of mouse model screens for identifying factors responsible for drug tolerance, which could be easily missed in regular in vitro screens. At the same time, a key feature of mouse models that is not properly understood is to what extent the route of administration of TB inocula determines the relapse rates upon drug withdrawal52. Alternative animals such as guinea pigs, rabbits and even cynomolgus monkeys have been used as preclinical models as they mimic TB disease pathogenesis better than mice with features such as hypoxic lesions and solid necrotic granulomas. Although guinea pigs do not acquire TB naturally, it was the first TB infection model to be used in 1944, when the efficacy of streptomycin was tested in just four animals before treating patients53. We still need rigorous studies comparing the bactericidal and sterilizing efficacy of different drug regimens in different animal models infected by different routes of infection, to enhance our ability to predict treatment outcomes in clinical trials and to validate the models themselves. Only after we have shown for several drug classes that animal model studies are congruent with efficacy seen in clinical trials will we achieve confidence in their predictive power.

Advances in imaging technologies that can map, in real time, the response of individual granulomas to drug treatment will facilitate our understanding of TB pathogenesis and may also help in developing better models to assess relapses. Live imaging tools were recently used to reveal the initial events leading to granuloma formation in a zebrafish model upon infection with Mycobacterium marinum54 and, in another instance, the lungs of patients with pulmonary TB were imaged to study the progression of disease after two months of chemotherapy55.

Evolving science of TB clinical development

A challenge in the clinical development of new TB therapies is the lack of specific biomarkers or surrogate endpoints that are sensitive and specific enough to reliably predict success or failure early in the course of treatment. Historically, clinical development of TB drugs has relied heavily on early bactericidal activity (EBA) trials, which measured reduction in bacterial load in the sputum of patients within 2–5 days of treatment56. The EBA kinetics of newer TB drugs such as TMC207 do not seem to follow the fast bactericidal activity observed with isoniazid and rifampicin and therefore studies with treatment durations of less than one week may be less informative for experimental agents showing a delayed bactericidal response57. Such delayed responses may be due to time-dependent killing, or a killing mechanism that requires depletion of energy reserves, or to physicochemical properties of the drug that delay its distribution to the bacilli in target sites. On the other hand, EBA studies of more than 2 weeks using monotherapy may be considered unethical because of the likelihood of the emergence of resistance.

In the second phase of clinical development, experimental drugs are administered over 8 weeks, on top of a standardized regimen, to estimate the effect of the drug(s) being tested on time to sputum conversion (positive to negative mycobacterial growth in patient sputum samples). In this setting, strong bactericidal activity and safety is a prerequisite for the further extension of therapy to more than 6–12 months for drug-susceptible (DS-)TB and 12–24 months for MDR-TB. A lengthy follow-up period (up to 2 years after the end of treatment) is needed to access the primary clinical endpoint of sterilization as measured by relapse rates. The absence of any other validated surrogate endpoints or biomarkers immensely extends the clinical development timelines and this is preventing any rapid progress in the field and at the same time substantially increasing the costs of running these trials. Sputum conversion to negativity after 2 or 6 months of treatment for DS- or MDR-TB, respectively, may give an early indication of microbiological sterilization58, 59. Although this sputum conversion rate probably represents the best surrogate marker for estimating the sterilizing efficacy of a regimen, it has been recently observed in mice that bactericidal potency of a regimen does not necessarily predict its sterilizing potency50 and, as such, culture status after 2 or 6 months of therapy still needs further validation, particularly in diverse patient populations60.

One of the factors that might explain the discrepancy between bactericidal and sterilizing activities is the heterogeneous nature of the bacterial population in patients with differential growth rates. Recent clinical trial data confirmed the increased sensitivity of liquid cultures (for example, mycobacteria growth indicator tube) to detect sputum TB bacilli, as compared to solid cultures61, 62, clearly indicating that even sputum samples may contain different subpopulations of TB bacilli with different growth kinetics. It remains to be seen to what extent cultivation of sputum samples on liquid cultures will allow a more accurate estimation of the sterilizing activity of a regimen.

To accelerate TB drug development and reduce the long clinical development path, research into non-sputum biomarkers, such as bacterial DNA sequences in urine samples or host-derived markers, such as toll-like receptor activation, should be prioritized. The recently identified interferon-inducible blood transcriptional TB signature, which correlates with the extent of disease in active TB and diminishes upon treatment, has great potential as a diagnostic and prognostic tool63. This TB signature was also observed in a subset of 10–20% of patients with latent TB and may identify those individuals who will develop active disease, and thereby facilitate targeted preventative therapy. Such biomarkers need further validation to determine if they are sufficiently sensitive and specific to allow monitoring of therapy responses in adults with active TB, or in individuals who are at risk of TB reactivation, or in children with active TB but who often do not excrete mycobacteria in their sputum60.

Drug combination trials and standardization of regimens

At present, the global TB development pipeline has nine candidates, but a key issue is how to develop them concomitantly in combination trials to identify the best regimen in the shortest period of time. In this regard, a recent initiative (Critical Path to New TB Regimens (CPTR)), involving several pharmaceutical companies and nongovernmental organizations, aspires to the development of new regimens of investigational drugs with existing TB drugs or drug candidates to avoid developing each drug sequentially and thereby shortening the development timelines that might otherwise spread over decades64. The CPTR approach will undoubtedly lead to improved efficiencies, but only if we can identify drugs that share similar or non-interfering pharmacokinetic features, synergistic or simply additive mechanisms of action, and non-overlapping toxicity profiles. For instance, some TB drugs in clinical development (moxifloxacin) have cardiovascular risks (prolonged QT intervals), and combining them with another drug with a similar liability will raise safety concerns. Even drugs with different mechanisms of action may interact synergistically or antagonistically with each other, and may even induce cross-resistance by common efflux mechanisms.

Reassessment and time for acceleration

Recent research into the pathogenesis of M. tuberculosis has led to the identification of a range of bacterial pathogenic mechanisms that permit it to escape certain host-control measures65. To counteract this we need innovative tools including newer drugs, vaccines, and improved diagnostics and biomarkers. The ultimate goal of the TB drug discovery effort is to eradicate both active and latent disease, possibly within a few weeks, like other more common bacterial infections. However, there are tremendous challenges to achieving this goal considering our lack of understanding of how to target heterogeneous M. tuberculosis populations using a single drug or a drug combination. In this regard, it is helpful to consider that TB in humans is a disease of subpopulations, with each population requiring a different drug or therapeutic approach. At the same time, it still remains to be determined to what extent M. tuberculosis persisters, which are phenotypically and stochastically antibiotic resistant66, determine relapse rates following a drug’s withdrawal. Any future drug discovery efforts should address the questions of how the goal of shortening the treatment durations can be linked to drug activity on latent or persistent bacterial populations. We still do not know if disrupting their energized membranes or targeting key anaerobic respiratory components such as those involved in energy generation can effectively kill these persisters.

At present, drug treatment developments show some promise owing to a renewed interest from pharmaceutical companies in researching new drugs, coupled with effective support from governmental and nongovernmental organizations. The TB vaccine pipeline is also showing progress, with seven vaccine candidates currently in clinical development including candidates being evaluated in paediatric populations67. However, we still need more drugs and vaccines to move from discovery into the development pipeline because of the high rate of drug attrition in clinical development and the potential for post-approval failures. Importantly, we also need more drugs from different classes so as to enable the creation of successful drug regimens, and realize the World Health Organization’s and United Nations millennium development goal of halting the incidence, prevalence and death rates associated with TB by 2015 and eliminating the disease altogether by 20508. However, any new drug or vaccine for TB will fail to make a significant impact if it is not accompanied by proper support from local healthcare systems. And finally, a key societal and economic challenge will be to ensure the proper access of these drugs or vaccines to the patients most in need in resource-poor countries.

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Acknowledgements
Introduction Emerging challenges in TB treatment Identifying new chemical scaffolds Targeting host–pathogen signalling pathways In vivo screens and preclinical validation Evolving science of TB clinical development Reassessment and time for acceleration References Acknowledgements Author i

quinta-feira, 27 de janeiro de 2011

vacina experimental contra Tuberculose latente - nature medicine

RESEARCH HIGHLIGHTS

Immunology: TB vaccine with a long view

Tuberculosis (TB) infection can enter an asymptomatic 'latent' phase and re-emerge later. The only approved TB vaccine, BCG, targets just the disease's early, active stage. A vaccine candidate, H56, that contains an antigen from the latent phase affords mice longer-lasting protection against the disease than does BCG, report Claus Aagaard and Peter Andersen at the Statens Serum Institute in Copenhagen and their co-workers.

The H56 vaccine contains three antigens, including one, Rv2660c, that is expressed during TB's latent stage. When given to mice before infection, H56 generated more diverse T-cell responses than the BCG vaccine, and also yielded a lower bacterial load by 24 weeks post-infection. When administered after the mice had been infected and treated with antibiotics, H56 protected the animals against developing reactivated infections. The team hopes to test the vaccine in humans.

Nature Med. doi: 10.1038/nm.2285 ( 2011 )

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quarta-feira, 26 de janeiro de 2011

Inovação e tecnologia foram enfatizadas no discurso de Obama

Ciência e avanço tecnológico foram enfatizados no Discurso de Obama - State of the Union Address

Estado da União: Obama pede inovação para enfrentar mundo competitivo

WASHINGTON (AFP) - O presidente americano, Barack Obama, clamou nesta terça-feira (hora local) por um maior impulso à inovação nos Estados Unidos, em um mundo cada vez mais competitivo com potências emergentes como Índia e China, em seu discurso sobre o Estado da União perante o Congresso.

Mais do que anunciar políticas específicas, Obama buscou enunciar de maneira franca a direção que os Estados Unidos devem tomar neste novo século, em meio a uma recuperação econômica do país mais lenta que o previsto.

No âmbito regional, Obama anunciou que viajará em março para Brasil, Chile e El Salvador, o que significará sua primeira viagem à América do Sul e Central desde que assumiu o poder, em janeiro de 2009.

"As regras mudaram. Em apenas uma geração, as revoluções em tecnologia transformaram a maneira como vivemos, trabalhamos e fazemos negócios", disse Obama, afirmando que as potências emergentes como Índia e China agora são altamente competitivas.

"Este mundo mudou, e para muitos, esta mudança foi dolorosa".

"Nos Estados Unidos, a inovação não muda apenas nossas vidas. É a maneira como vivemos", disse Obama, que convocou a oposição republicana a apoiar o investimento em educação, inovação e infraestrutura.

O discurso ocorre após a dura derrota eleitoral do mês de novembro, quando os republicanos conquistaram a maioria na Câmara de Representantes.

A oposição, que por enquanto quer falar apenas em como cortar o gasto público, conseguiu horas antes do discurso aprovar uma moção com o apoio de 20 democratas, na qual pede simbolicamente ao governo que gaste o mesmo que em 2008.

Obama deixou claro que entende que a realidade mudou.

"Com seus votos, os americanos determinaram que governar agora será uma responsabilidade compartilhada entre os dois partidos", disse.

"Novas leis serão aprovadas apenas com o apoio de republicanos e democratas", afirmou. E acrescentou que "está em jogo não apenas quem ganha a próxima eleição (...) e sim se os novos empregos e as indústrias fincam raízes em nosso país ou em outro lugar".

Mas os republicanos não se mostraram convencidos e reiteraram suas críticas ao que dizem ser um gasto desenfreado do governo.

"Nenhuma economia pode manter estes altos níveis de dívida e impostos. A próxima geração vai herdar uma economia estancada e uma nação diminuída", disse a legisladora Ileana Ros-Lehtinen, representante da Flórida, na resposta republicana ao discurso sobre o Estado da União.

A imagem do presidente melhorou progressivamente desde a derrota eleitoral de novembro, até ultrapassar os 50% de aprovação, segundo os últimos números da Gallup.

Obama começou seu discurso referindo-se ao recente massacre de Tucson (Arizona), onde um jovem assassinou seis pessoas e feriu outras 14 ao tentar atirar contra uma representante democrata. Em meio a um clima político polarizado, "Tucson nos lembrou que não importa quem somos nem de onde viemos: todos somos parte de algo maior", declarou.

Um primeiro sinal de um possível espírito de cooperação foi a decisão conjunta de alguns democratas e republicanos de se misturar nas bancadas do Congresso durante o discurso, como símbolo de unidade nacional.

Na questão internacional, Obama anunciou que viajará ao Brasil, Chile e El Salvador "para forjar novas alianças para o progresso nas Américas".

Obama fez um gesto à crescente população hispânica nos Estados Unidos: afirmou que os EUA devem resolver "de uma vez por todas" a imigração ilegal, pedindo um esforço bipartidário para uma reforma migratória.

"Estou preparado para trabalhar com republicanos e democratas para proteger nossas fronteiras, fazer cumprir as leis e nos ocuparmos dos milhões de trabalhadores ilegais que atualmente vivem nas sombras", afirmou.

Do New York Times

On Jan. 25, 2011, President Obama was to deliver his second State of the Union address -- his first to a Congress partially in Republican hands.

Advisers say Mr. Obama will lay out his case for investment in education and infrastructure, while tempering his call for new initiatives with an acknowledgment of the country’s long-term fiscal challenges. They said he will propose a five-year freeze on “non-security discretionary spending” though they did not disclose the details of that proposal in advance of the speech.

A White House official called the proposal “a down payment toward reducing the deficit” and said the president will “also will be looking for cuts and efficiencies. For instance, the President is putting forward a five-year plan developed by Secretary Gates to achieve $78 billion in defense savings.”

The move would be a step in the direction of some in Congress who have called for spending controls. But it would fall short of what House Republicans are calling for: a reduction in federal spending to 2008 levels.

But along with his specific proposals, the speech will give Mr. Obama a chance to try to set the tone for the coming political debate, and to partake in one of the most elaborate of Washington's rituals. Every year for decades, presidents have traveled to Capitol Hill to deliver their State of the Union address. They've done it because the Constitution told them to.

More or less.

Actually, all the Constitution says, in Article II, Section 3, is that the president shall "from time to time give to the Congress information of the State of the Union, and recommend to their consideration such measures as he shall judge necessary and expedient."

Not a word about the pageantry of the president’s slow walk down the aisle to front of the House chamber, getting handshakes and hugs from members of his party. Not a word about the major TV networks pre-empting regular prime-time coverage to broadcast the speech. Not a word about tallying up how long the president spoke and how many times he was interrupted by applause.

In fact, if a president wants to literally mail in his remarks, that would be fine.

While the first two presidents, George Washington and John Adams, delivered their messages in person, Thomas Jefferson sent a written message. Other presidents followed Jefferson’s lead until 1913 when Woodrow Wilson decided that he would deliver his speech in person

sábado, 22 de janeiro de 2011

autofagia na imunidade e inflamação

Figure 1: Schematic overview of autophagy and its regulation.

Figure 2: Possible autophagy-protein-dependent pathways of pathogen degradation.

Figure 3: Functions of the autophagy pathway and/or proteins in immunity.

Figure 4: Autophagy/autophagy proteins act to achieve a balance between activation and inactivation of innate immune signalling.

Figure 5: The link between mutations in autophagy regulators and the chronic inflammatory disorder Crohn's disease.

Autophagy in immunity and inflammation

BETH LEVINE, NOBORU MIZUSHIMA & HERBERT W. VIRGIN

Autophagy is an essential, homeostatic process by which cells break down their own components. Perhaps the most primordial function of this lysosomal degradation pathway is adaptation to nutrient deprivation. However, in complex multicellular organisms, the core molecular machinery of autophagy — the 'autophagy proteins' — orchestrates diverse aspects of cellular and organismal responses to other dangerous stimuli such as infection. Recent developments reveal a crucial role for the autophagy pathway and proteins in immunity and inflammation. They balance the beneficial and detrimental effects of immunity and inflammation, and thereby may protect against infectious, autoimmune and inflammatory diseases.

Autofagia é um processo essencial homeostático pelo qual as células quebrar seus próprios componentes. Talvez a função mais primordial desta via de degradação lisossomal é a adaptação à privação de nutrientes. Entretanto, em organismos multicelulares complexos, o mecanismo molecular da autofagia - 'as proteínas autofagia "o - orquestra diversos aspectos da resposta celular e organismal a outros estímulos perigosos, tais como infecção. Os desenvolvimentos recentes revelam um papel crucial para a via autofagia e proteínas na imunidade e inflamação. Eles equilibrar os efeitos benéficos e prejudiciais da imunidade e inflamação, e assim pode proteger contra doenças infecciosas, autoimunes e inflamatórias.

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There is only one known mechanism that eukaryotic cells possess to dispose of intracellular organelles and protein aggregates that are too large to be degraded by the proteasome. It is therefore not surprising that this mechanism — the lysosomal degradation pathway known as autophagy — is also used to degrade microorganisms (such as viruses, bacteria and protozoa) that invade intracellularly1, 2. Indeed, the mutation of autophagy genes increases susceptibility to infection by intracellular pathogens in organisms ranging from plants to flies to worms to mice, and possibly to humans. Perhaps less apparent, but teleologically as intuitive, the autophagy pathway or unique functions of autophagy proteins also have a central role in controlling other diverse aspects of immunity in multicellular organisms.

The autophagy machinery is thought to have evolved as a stress response that allows unicellular eukaryotic organisms to survive during harsh conditions, probably by regulating energy homeostasis and/or by protein and organelle quality control. The same machinery might therefore be expected to diversify functionally in complex metazoan organisms, so as to regulate new layers of defences used by multicellular organisms to confront different forms of stress. A plethora of genetic, biochemistry, cell biology, systems biology and genomic studies have recently converged to support this notion. The autophagy machinery interfaces with most cellular stress-response pathways3, including those involved in controlling immune responses and inflammation. This interface is not only at the level of the autophagy pathway, but also entails direct interactions between autophagy proteins and immune signalling molecules4. There is a complex reciprocal relationship between the autophagy pathway/proteins and immunity and inflammation; the autophagy proteins function in both the induction and suppression of immune and inflammatory responses, and immune and inflammatory signals function in both the induction and suppression of autophagy. Moreover, similar to cancer, neurodegenerative diseases and ageing5, defects in autophagy — through autophagy gene mutation and/or microbial antagonism — may underlie the pathogenesis of many infectious diseases and inflammatory syndromes.

In this Review, we describe recent advances in our evolving comprehension of the interface between autophagy, immunity and inflammation. We discuss how emerging concepts about the functions of the autophagy pathway and the autophagy proteins may reshape our understanding of immunity and disease. This set of proteins not only orchestrates the lysosomal degradation of unwanted cargo, but also exerts intricate effects on the control of immunity and inflammation. Thus, the autophagy pathway and autophagy proteins may function as a central fulcrum that balances the beneficial and harmful effects of the host response to infection and other immunological stimuli.

Mechanisms and membrane dynamics of autophagy

Autophagy is a general term for pathways by which cytoplasmic material, including soluble macromolecules and organelles, is delivered to lysosomes for degradation6. There are at least three different types of autophagy, including macroautophagy, chaperone-mediated autophagy and microautophagy. Macroautophagy, usually referred to simply as autophagy, is the subject of this Review (Fig. 1). In this pathway, a portion of cytoplasm (usually 0.5–1 μm in diameter) is engulfed by an isolation membrane, or 'phagophore', resulting in the formation of a double-membrane structure known as the autophagosome. The outer membrane of the autophagosome fuses with the lysosome to become an autolysosome, leading to the degradation of autophagosomal contents by lysosomal enzymes. Autophagosomes can also fuse with endosomes or multivesicular bodies and major histocompatibility complex (MHC)- class-II-loading compartments7. Autolysosomes become larger as more autophagosomes and lysosomes fuse, but at a termination phase lysosomes are tubulated and fragmented for renewal8.

Figure 1: Schematic overview of autophagy and its regulation.

Overview of the autophagy pathway. The top right box shows a model of our current understanding of the molecular events involved in membrane initiation, elongation and completion of the autophagosome. The major membrane source is thought to be the endoplasmic reticulum (ER), although several other membrane sources, such as mitochondria and the plasma or nuclear membrane, may contribute. After induction of autophagy, the ULK1 complex (ULK1–ATG13–FIP200–ATG101) (downstream of the inhibitory mTOR signalling complex) translocates to the ER and transiently associates with VMP1, resulting in activation of the ER-localized autophagy-specific class III phosphatidylinositol-3-OH kinase (PI(3)K) complex, and the phosphatidylinositol-3-phosphate (PtdIns(3)P) formed on the ER membrane recruits DFCP1 and WIPIs. WIPIs and the ATG12–ATG5–ATG16L1 complex are present on the outer membrane, and LC3–PE is present on both the outer and inner membrane of the isolation membrane, which may emerge from subdomains of the ER termed omegasomes. The cellular events that occur during autophagy are depicted in the bottom diagram, including the major known cellular and microbial proteins that regulate autophagy initiation, cargo recognition and autophagosome maturation. Only those cellular proteins known to be adaptors for targeting microbes are shown; other proteins (not shown) also function in cargo recognition of mitochondria and other organelles. CMV, cytomegalovirus; DAMP, danger-associated molecular pattern; DAP, death-associated protein; EBV, Epstein–Barr virus; HBV, hepatitis B virus; HSV-1, herpes simplex virus 1; KSHV, Kaposi's sarcoma-associated herpesvirus; LIR, LC3-interacting region (motif); LPS, lipopolysaccharide; MDP, muramyl dipeptide; Pam3Cys4, a synthetic TLR2 agonist; PAMP, pathogen-associated molecular pattern; PERK, an eIF2α kinase; PGN, peptidoglycan; PRGP-LE, a peptidoglycan-recognition protein; PRR, pathogen-recognition receptor; ROS, reactive oxygen species; Ub, ubiquitin; UBA, ubiquitin-associated domain; UBZ, ubiquitin-binding zinc finger; v-FLICE, viral FLICE.

The membrane dynamics of autophagosome formation involve complex processes, which are not completely understood. Nonetheless, the molecular dissection of autophagy membrane dynamics, stimulated by the discovery of ATG (autophagy-related) genes in yeast9, has shed considerable light on this topic (Table 1). Several recent studies suggest that the endoplasmic reticulum (ER) is crucial for autophagosome formation. The ER cisternae often associate with developing autophagosomes, and electron tomography analysis has demonstrated direct connections between the ER and autophagosomal membranes10, 11. Moreover, the function of several key autophagy proteins seems to be at the level of the ER (Fig. 1).

Table 1: Key proteins involved in mammalian autophagosome formation and their immune functions

Autophagy is induced by nutrient starvation through the inhibition of mammalian target of rapamycin (mTOR), resulting in translocation of the mTOR substrate complex (ULK1/2, ATG13, FIP200 (also known as RB1CC1) and ATG101) from the cytosol to certain domains of the ER or closely attached structures12, 13. This leads to the recruitment of the class III phosphatidylinositol-3-OH kinase (PI(3)K) complex, which includes at least VPS34 (also known as PIK3C3), VPS15 (PIK3R4 and p150), beclin 1 and ATG14, to the ER13, 14. The PI(3)K complex produces phosphatidylinositol-3-phosphate (PtdIns(3)P), which recruits effectors such as double FYVE-containing protein 1 (DFCP1) and WD-repeat domain phosphoinositide-interacting (WIPI) family proteins. DFCP1 is diffusely present on the ER or the Golgi, but translocates to the autophagosome formation site in a PtdIns(3)P-dependent manner to generate ER-associated Ω-like structures termed omegasomes15. Among the four WIPI isoforms, WIPI2 is the major form in most cell types and functions downstream of DFCP1, and it may promote the development of omegasomes into isolation membranes or autophagosomes16.

An ER-associated multispanning membrane protein, VMP1, is also important for autophagosome formation. Although VMP1 interacts with beclin 1 and is present at the autophagosome formation site at an early stage, it seems to function at a late stage in autophagy13, 17, 18. This is perhaps consistent with other evidence that beclin 1–class III PI(3)K complexes may function in autophagosomal maturation (in addition to vesicle nucleation), a process that can be regulated by other beclin-1-interacting partners such as rubicon (Table 1). At the final step of autophagosome formation, elongation of the isolation membrane and/or completion of enclosure require two ubiquitin-like conjugates. The first is the ATG12–ATG5 conjugate, which is produced by the ATG7 (E1-like) and ATG10 (E2-like) enzymes, and functions as a dimeric complex together with ATG16L1 (ref. 19) . The second is the phosphatidylethanolamine (PE)-conjugated ATG8 homologues — LC3, GATE16 and GABARAP — which are produced by the ATG7 and ATG3 (E2-like) enzymes9, 20. Although the proteins involved in autophagosome membrane formation have been characterized as discrete complexes (Table 1), several potential interconnections between components of the different complexes were identified by a recent proteomics study21. Such interconnections may function in autophagosome membrane formation or other distinct cellular functions. For example, the ATG12–ATG3 conjugate is implicated in mitochondrial homeostasis but not in autophagosome membrane formation22.

In addition to the ER, other membranes may be involved in autophagosome formation (Fig. 1). ATG9, another multispanning membrane protein, is essential for autophagy23 and traffics between the trans-Golgi network, endosomes and autophagosome precursors24. Studies suggest that mitochondria, the plasma membrane and the nuclear membrane could also be membrane sources for autophagosome formation25, 26, 27. However, the lack of detection of specific protein markers for these structures on the autophagosomal membrane leaves the decades-old question of the membrane source of the autophagosome unanswered. It is possible that cells may use different membrane sources to form the autophagosome in different contexts, thereby permitting specialization of membrane dynamics in a manner that allows divergent autophagy-inducing signals to stimulate the capture of spatially distinct cargo.

Selective autophagy tackles microbes

Autophagy was originally considered to be a non-selective bulk degradation process, but it is now clear that autophagosomes can degrade substrates in a selective manner28. In addition to endogenous substrates, autophagy degrades intracellular pathogens in a selective form of autophagy, termed xenophagy. Similar to bulk autophagy (such as that induced by nutrient deprivation) and other forms of selective autophagy (such as degradation of damaged mitochondria, peroxisomes, aggregate-prone proteins or damaged ER), the precise membrane dynamics and specificity determinants of xenophagy are not fully understood. Nonetheless, considerable advances have been made, and interesting similarities and differences are beginning to emerge between cellular recognition and degradation of self versus foreign microbial components through autophagy-like pathways (Figs 1 and 2).

Figure 2: Possible autophagy-protein-dependent pathways of pathogen degradation.

Possible pathways involving the autophagy machinery by which viruses, bacteria (and damaged membranes of bacteria-containing vacuoles) and parasites may be targeted to the lysosome. Adaptor refers to the proteins shown in the cargo-recognition box in Fig. 1; however, as yet undiscovered adaptors may be involved in pathogen recognition, and pathogen targeting may involve ubiquitin-dependent or -independent mechanisms.

The vacuoles used for the engulfment of intracytoplasmic bacteria are similar to autophagosomes, and their formation requires the core autophagy machinery. But one apparent difference is the vacuole size; for example, the diameter of group A Streptococcus-containing autophagosome-like vacuoles (GcAV) can be as big as 10 μm. These large GcAVs are generated by the RAB7-dependent fusion of small isolation membranes29. By contrast, the formation of starvation-induced autophagosomes requires RAB7 later in the autophagy process, at the autophagosome–lysosome fusion step.

A more complex question is how autophagosomes (or components of the autophagy pathway) capture pathogens that are inside vacuolar compartments (Fig. 2). There are at least four general pathways that may be used for autophagy-protein-dependent targeting of bacteria to the lysosome. These include autophagy-protein-facilitated fusion of bacteria-containing phagosomes with lysosomes, the envelopment of bacteria-containing phagosomes or endosomes by autophagosomal membranes, the fusion of bacteria-containing phagosomes or endosomes with autophagosomes, or the xenophagic capture of bacteria that have escaped inside the cytoplasm. In some cases, the route of autophagy-dependent targeting to the lysosome has been well defined, such as for group A Streptococcus that escapes from endosomes30. For several bacteria, however, the precise route is unclear. Many studies define bacterial autophagy as the co-localization of bacteria and LC3, but we now know that LC3 can decorate membranous compartments other than autophagosomes (including phagosomes).

Several lines of evidence suggest that autophagy proteins function more broadly, not only in classical macroautophagy, but also in the process of phagolysosomal maturation during antigen presentation and microbial invasion. Autophagy proteins are required for the fusion of phagosomes that contain Toll-like receptor (TLR)-ligand-enveloped particles with lysosomes in macrophages31, and for the fusion of phagosomes that contain TLR-agonist-associated apoptotic cell antigens with lysosomes in dendritic cells during MHC class II antigen presentation32. The self ligand and cell-surface receptor SLAM functions as a microbial sensor that recruits the beclin 1–class III PI(3)K complex to phagosomes containing Gram-negative bacteria, facilitating phagolysosomal fusion and activation of the antibacterial NADPH oxidase (NOX2) complex33. In addition, the engagement of TLR or Fcγ receptors during phagocytosis recruits LC3 (and ATG12) to the phagosome through NOX2-dependent generation of reactive oxygen species (ROS)34. Thus, in bacterial infections, a paradigm is emerging in which the coordinated regulation of microbial sensing, phagolysosomal maturation and antibacterial activity involves the recruitment of autophagy proteins to the phagosome. As a corollary, an interesting speculation is that impaired recruitment of autophagy proteins to the phagosome may contribute to the pathogenesis of chronic granulomatous disease, a genetic disorder caused by mutations in the NOX2 gene (also known as CYBB) and characterized by recurrent bacterial and fungal infections and inflammatory complications, such as inflammatory bowel disease.

Another autophagosome-independent function of autophagy proteins in pathogen destruction has been described in interferon-γ (IFN-γ)-treated macrophages infected with the parasite Toxoplasma gondii. The parasite-derived membrane, termed the parasitophorous vacuole, undergoes destruction through a mechanism that involves ATG5-dependent recruitment of the immunity-related GTPase proteins to the parasitophorous vacuole35, 36, leading to the death of the parasite in the infected cell35, 37. Together, these studies suggest that autophagy proteins have diverse roles in membrane dynamics to benefit the host in the removal of invading pathogens (Fig. 2), through xenophagy, phagolysosomal maturation, the recruitment of molecules that damage pathogen-derived membranes, and presumably, many other as yet undiscovered mechanisms.

The mechanisms that cells use to target intracellular bacteria (and probably viruses) to autophagosomal compartments are notably similar to those used for selective autophagy of endogenous cargo. Cellular cargo is commonly targeted to autophagosomes by interactions between a molecular tag (such as polyubiquitin), adaptor proteins such as p62 (also known as SQSTM1 or sequestome 1) or NBR1 (which recognize these tags and contain an LC3-interacting region (LIR) characterized by a WXXL or WXXI motif), and LC3 (ref. 28). These adaptor molecules enable autophagy to target designated cargo selectively to nascent LC3-positive isolation membranes. As reviewed elsewhere38, a similar mechanism involving ubiquitin and p62 seems to be involved in the targeting of intracellular bacteria, such as Salmonella enterica serotype Typhimurium (S. Typhimurium), Shigella flexneri and Listeria monocytogenes, to autophagosomes.

After escape into the cytoplasm or in vacuolar membrane compartments damaged by type III secretion system (T3SS) effectors, bacteria or bacteria-containing compartments, respectively, may become coated with ubiquitin and associate with p62 and nascent LC3-positive isolation membranes. The autophagosomal targeting of Salmonella also requires another cellular factor, NDP52 (nuclear dot protein 52), an autophagy adaptor protein that, like p62, contains an LIR and ubiquitin-binding domains and restricts intracellular bacterial replication. A ubiquitin-independent pathway (that does not involve p62 or NDP52) could also function in targeting damaged Salmonella-containing vacuoles (SCVs) to the autophagosome. In this pathway, a lipid second messenger, diacylglycerol, acts as a signal for the co-localization of SCVs with LC3-positive autophagosomes by a mechanism that involves protein kinase C and its downstream targets, JNK and NADPH oxidase39. The autophagic targeting of a cytoplasmic positive-strand RNA virus, Sindbis virus, also occurs in a ubiquitin-independent manner, but involves the interaction of p62 with the viral capsid protein40. Thus, diverse molecular strategies, including ubiquitin-dependent and -independent mechanisms, may be used to target microbes inside the cytoplasm or vacuolar compartments to the autophagosome.

Beyond targeting intracellular pathogens for degradation, p62 may have further beneficial effects in infected host cells. For example, Shigella vacuolar membrane remnants generated by bacterial T3SS-dependent membrane damage are targeted by polyubiquitination, p62 and LC3 for autophagosomal degradation41 (Fig. 2). These membrane remnants also accumulate numerous molecules involved in sensing and transduction of pathogen-associated molecular pattern (PAMP) and danger-associated molecular pattern (DAMP) signals, and there is an increase in nuclear factor-κB (NF-κB)-dependent cytokine production, ROS production and necrotic cell death in autophagy-deficient cells. Thus, the ubiquitin–p62-dependent autophagic targeting of pathogen-damaged membranes could help to control detrimental downstream inflammatory signalling during bacterial invasion into host cells. Another emerging concept is that selective autophagy of viral proteins, similar to selective autophagy of aggregate-prone toxic cellular proteins, may protect post-mitotic cells such as neurons against cell death. For example, in Sindbis-virus-infected mice with neuron-specific inactivation of Atg5, there is an accumulation of Sindbis virus antigens (without increased levels of infectious virus), increased neuronal cell death and increased animal mortality40. Moreover, p62 is required for starvation and IFN-γ-induced targeting of Fau (and perhaps other ubiquitylated protein complexes) to mycobacteria-containing phagosomes, resulting in the generation of antimycobacterial Fau-derived peptides42. The role of p62 in innate immunity is probably evolutionarily ancient, as the Drosophila p62 orthologue REF(2)P was originally identified in a screen for modifiers of sigma virus replication43.

We speculate that p62, as well as the other known LC3-interacting adaptor proteins (NBR1 and NDP52), may represent the tip of the iceberg in terms of cellular adaptor proteins that bind to ubiquitin (or other molecular tags) and target microbial substrates and cytosolic material to autophagosomes to coordinate innate immune responses. A recent proteomics study showed that the mammalian ATG8 family, which includes LC3, GATE16 and GABARAP, has 67 high-confidence interactions with other cellular proteins21. Some of these new ATG8-family-member-interacting partners may have an as yet undiscovered role in innate immunity. Another open question is whether the known proteins involved in selective autophagy of mitochondria (called mitophagy), such as Nix (also known as BNIP3L) and parkin44, also function in microbial autophagy.

Autophagy and resistance to infection

The autophagy pathway and/or autophagy proteins have a crucial role in resistance to bacterial, viral and protozoan infection in metazoan organisms. The genetic deletion or knockdown of autophagy genes protects plants from viral, fungal and bacterial infection by preventing the uncontrolled spread of programmed cell death during the plant innate immune or hypersensitive response45. In other organisms, autophagy proteins function in a cell-autonomous manner to control infection by intracellular pathogens. In Drosophila, autophagy gene mutation increases susceptibility to viral (vesicular stomatitis virus)46 and bacterial (L. monocytogenes)47 infection. In Dictyostelium and Caenorhabditis elegans, autophagy gene mutation increases susceptibility to lethal S. Typhimurium infection48. In mice, knockout of Atg5 in macrophages and neutrophils increases susceptibility to infection with L. monocytogenes and the protozoan T. gondii35, and neuron-specific Atg5 knockout increases susceptibility to central nervous system Sindbis virus infection40. As noted in the next section, the autophagy pathway and proteins may also have 'proviral' or 'probacterial' effects in in vitro studies; however, in vivo evidence for such effects is so far lacking. The mechanisms by which autophagy genes mediate in vivo resistance to infection are not fully understood, but are likely to involve a combination of xenophagy, other autophagy-protein-dependent effects on microbial replication or survival, activation of innate and adaptive immune responses, and/or alterations in pathogen-induced cell death (Fig. 3).

Figure 3: Functions of the autophagy pathway and/or proteins in immunity.

A summary of the known functions of the autophagy pathway and/or proteins in adaptive and innate immunity, and as effectors during infection.

An important question is whether this function of autophagy in broad resistance to infection with intracellular pathogens extends to humans. Recent human genetic studies provide some clues. The immunity-related GTPase human IRGM, which regulates autophagy-dependent clearance of mycobacteria in vitro49, was identified as a genetic risk locus for tuberculosis in a West African population50. Numerous studies have shown a crucial role for autophagy in defence against mycobacterial infection in human cells1, and a genome-wide analysis of host genes that regulate Mycobacterium tuberculosis replication demonstrated that a predominance of factors were autophagy regulators51. Thus, it is possible that autophagy has a central role in resistance to one of the most important global infectious diseases — tuberculosis. Mutations in NOD2, which encodes an intracellular pathogen-recognition receptor (nucleotide-binding oligomerization-domain-containing protein 2) that functions in bacterial autophagy52, 53, are also associated with susceptibility to infection with another mycobacterial agent, Mycobacterium leprae, the aetiological agent of leprosy54. An exciting future venture will be to confirm whether IRGM, NOD2 and other autophagy-related genes are involved in resistance to infection with mycobacteria and other infections in further human populations and, if so, whether this resistance is mediated by autophagy.

Microbes fight back

Microbes undergo strong selective pressure to develop strategies to block host defence mechanisms; the number of such strategies is a surrogate measure of the importance of the host defence mechanism in immunity. As reviewed elsewhere1, 55, viruses and intracellular bacteria have evolved several ways to adapt to host autophagy. They can antagonize autophagy initiation or autophagosomal maturation, evade autophagic recognition, or use components of the autophagy pathway to facilitate their own replication or intracellular survival. An emerging theme is that microbial antagonism of autophagy not only blocks the xenophagic degradation of intracellular pathogens, but also blocks the functions of autophagy in innate and adaptive immunity. A relatively unexplored yet crucially important frontier is how microbial antagonism may contribute more broadly to the role of microbes in diseases characterized by defective autophagy, such as cancer, neurodegenerative diseases, ageing and, potentially, autoimmune and inflammatory diseases.

Viral strategies to shut off autophagy include the blockade of positive upstream regulators of autophagy (such as the IFN-inducible RNA-activated eIF2α protein kinase (PKR) signalling pathway), the activation of negative upstream regulators of autophagy (such as the nutrient-sensing TOR kinase signalling pathway) or direct antagonism of the autophagy machinery55. The overlapping functions of the eIF2α kinase signalling pathway in stress-induced general translational arrest, transcriptional activation of stress-response genes and stress-induced autophagy enable viruses to disarm several facets of the cellular stress response to infection by one mechanism — that is, antagonism of eIF2α kinase signalling. The mTOR signalling pathway has a central role in the control of cell growth and metabolism, and interestingly, many of the viruses that activate mTOR are oncogenic (for example, Epstein–Barr virus, Kaposi's sarcoma-associated herpesvirus, hepatitis B virus and retroviruses). This suggests another type of pluripotent viral weapon — one that can promote oncogenesis by simultaneously inactivating autophagy and promoting cell growth through TOR activation. HIV envelope protein-dependent activation of mTOR signalling is also proposed to be a mechanism for HIV evasion of innate and adaptive immune responses in dendritic cells, including the degradation of incoming virions by lysosomes, blockade of HIV transfer to CD4+ T cells, stimulation of TLR4 and TLR8 ligand responses, and presentation of HIV Gag antigen to CD4+ T cells56. It will be important to determine whether these effects of HIV-mediated mTOR activation and autophagy inhibition contribute to impaired dendritic cell function during HIV infection in vivo.

Several viral proteins target the core autophagy protein beclin 1. Autophagosome initiation is blocked by interactions between beclin 1 and the herpes simplex virus 1 (HSV-1) neurovirulence factor ICP34.5 or the oncogenic γ-herpesvirus-encoded viral BCL2-like proteins, whereas autophagosome maturation is blocked by interactions between beclin 1 and the HIV accessory protein Nef or the influenza virus matrix protein 2 (ref. 55). The interactions between beclin 1, HSV-1 ICP34.5 and viral BCL2 are probably physiologically important in vivo; a mutant HSV-1 virus lacking the beclin-1-binding domain of ICP34.5 is attenuated in mouse models of encephalitis (presumably through its failure to control xenophagy and innate immunity)57 and of corneal disease (through its failure to control adaptive immunity)58. Moreover, a mouse γ-herpesvirus that encodes a mutant viral BCL2 unable to bind to beclin 1 demonstrates impaired ability to maintain chronic infection59. Thus, viral antagonism of host autophagy can manipulate distinct aspects of viral pathogenesis and immunity in vivo.

It is not yet clear whether compared with other autophagy proteins, beclin 1 is preferentially targeted by viral virulence proteins because of its central role in autophagosome formation, or more likely, whether we are just beginning to identify pairs of viral proteins and their autophagy pathway targets. In support of the latter, viral FLICE-like inhibitors encoded by Kaposi's sarcoma-associated herpesvirus and molluscum contagiosum virus suppress autophagy by interacting with the ATG3 E2-like enzyme, thereby preventing it from binding and processing LC3 (ref. 60).

Bacteria possess diverse strategies to avoid degradation by autophagolysosomal pathways. As reviewed elsewhere1, 38, many bacteria that reside in phagosomes or other vacuolar compartments have methods to inhibit lysosomal fusion or maturation, which, in the case of mycobacteria, can be partially overcome by treatments that stimulate autophagy. Another possible mechanism for bacterial evasion of autophagy has emerged from a genome-wide screen to identify host factors that regulate the intracellular survival of M. tuberculosis51. According to bioinformatics analyses, M. tuberculosis infection of human macrophage-like cells activates cellular pathways that inhibit autophagy. Intracellular bacteria that escape into the cytoplasm, such as S. flexneri and L. monocytogenes, use strategies to camouflage themselves to avoid autophagic recognition. The Shigella T3SS effector IcsB competitively binds to VirG, thereby preventing the interaction between ATG5 and VirG, a bacterial surface protein required for actin-based motility and Shigella targeting to autophagosomes38. The Listeria protein ActA interacts with cytosolic actin polymerization machinery (ARP2/3, VASP and actin), which blocks bacterial association with ubiquitin, p62 recruitment and autophagic targeting61. The precise mechanism of this block is unknown, but it has been proposed that the ActA-dependent recruitment of host cell cytoskeletal proteins may enable the bacterium to disguise itself as a host cell organelle61. This concept sheds light on the autophagy pathway in a fundamental aspect of immunology — the basis for discrimination between self and non-self.

Microbes have evolved not only to antagonize autophagy (as a cellular defence mechanism that threatens their survival), but also to exploit its components and functions in membrane trafficking for their own self-serving purposes1, 55. An early concept in the field is that autophagosomes may serve as a protected niche for intracellular bacteria (provided fusion with acidic compartments is blocked) and/or serve as a source of nutrients for intracellular pathogens (which would require intact autophagolysosomal fusion)1. Trafficking of the intracellular bacteria Yersinia pseudotuberculosis to acidic compartments was recently shown to be enhanced by genetic inhibition of autophagy62. This seemingly contradicts other evidence that the autophagy proteins promote phagosomal maturation, but is consistent with the concept that autophagosomes function as a protected intracellular niche for bacteria. The role of the autophagy machinery in promoting and/or inhibiting vacuolar acidification — and the counter effects of microbes that reside in vacuolar compartments — needs to be explored further.

The function of autophagy proteins in membrane formation and/or trafficking is exploited by numerous viruses, including poliovirus, rotavirus, HIV, coronaviruses, Dengue virus, and the hepatitis B and C viruses55. Autophagosomes (defined as LC3-positive membranes, see caveat below) may act as a scaffold for intracellular membrane-associated replication of certain cytoplasmic RNA viruses55. Autophagy may assist in HIV biogenesis, because the processing of the HIV envelope precursor protein Gag and extracellular viral release are enhanced by the autophagy machinery63. Similarly, autophagy proteins are required for maximal levels of poliovirus egress55. Another newly defined proviral function of autophagy is its role in productive hepatitis C virus replication; several different autophagy proteins (such as beclin 1, ATG4B, ATG5, ATG7 and ATG12) assist in the translation of incoming, but not progeny, viral RNA64. ATG7 and class III PI(3)K activity also enhance hepatitis B virus DNA replication65.

The mechanisms by which autophagy proteins facilitate the replication and/or egress of certain viruses are not yet understood. Some observations may relate to the role of autophagy proteins in remodelling the ER (vis-à-vis viral replication) or the role of autophagosomes in fusing with multivesicular bodies (vis-à-vis viral egress). It is possible that autophagy proteins function to provide membrane for viral replication complexes or translation machinery. This may be true for viruses such as hepatitis C virus, for which genetic knockdown of several different autophagy genes decreases productive replication64. However, for other viruses such as coronaviruses, the biogenesis of double-membrane, ER-derived vesicles used for replication proceeds through a pathway that involves the non-lipidated form of LC3 (LC3-I) but not the general autophagy machinery66. Thus, caution must be exercised in interpreting the significance of the co-localization (or biochemical interaction) of viral proteins and LC3, as LC3 may have autophagy-independent roles in membrane dynamics.

Autophagy regulation by immune signalling molecules

The central importance of autophagy in immunity is further underscored by the multitude of immune-related signalling molecules that regulate autophagy. As reviewed in detail elsewhere2, 3, 4, 38, autophagy is induced by different families of pathogen-recognition receptors (such as TLRs, NOD-like receptors and the double-stranded RNA-binding protein PKR), DAMPs (such as ATP, ROS and misfolded proteins), pathogen receptors (such as CD46), IFN-γ and downstream immunity-related GTPases, and DAP kinase, JNK, CD40, tumour necrosis factor-α (TNF-α), inhibitor of NF-κB (IKK) and NF-κB (Fig. 1). High mobility group box (HMGB) proteins have also been shown to function as both universal sensors of nucleic acids in innate immune signalling67 and inducers of autophagy68. Autophagy is inhibited by BCL2, NF-κB, T helper 2 (TH2) cytokines and the canonical nutrient-sensing insulin–AKT–TOR pathway. Inactivation of this nutrient-sensing pathway may contribute to vesicular stomatitis virus stimulation of autophagy in Drosophila46, and autophagy activation in C. elegans with loss-of-function mutations in this pathway may mediate pathogen resistance in long-lived mutant nematodes48. Thus, both 'immune-specific' and more general nutrient-response signals control autophagy in response to infection.

Studies with vitamin D3 have uncovered a possible link between nutrition, innate immunity and the control of autophagy during mycobacterial infection. Low vitamin D3 levels are associated with increased susceptibility to tuberculosis. Vitamin D3 generates an antimycobacterial peptide, cathelicidin, and induces autophagy and mycobacterial killing in human monocytes through cathelicidin-dependent effects69. Although cathelicidin is required for vitamin-D3-dependent transcriptional upregulation of autophagy genes such as BECN1 and ATG5, and vitamin D3 enhances the recruitment of cathelicidin to autophagosomes, it is not yet clear how cathelicidin promotes autophagy. Nonetheless, these observations may begin to provide some insight into the century-old Nobel prize award (Niels Ryberg Finsen, 1903) for the use of ultraviolet-light therapy (which generates active vitamin D3) in the treatment of diseases such as tuberculosis.

In most instances, the mechanisms of autophagy control by immune-related signalling molecules are not understood. However, there are some examples of specific interactions between immune signals and autophagy proteins that may be relevant to these mechanisms. For example, the interaction between beclin 1 and BCL2 (which inhibits its activity) is thought to be disrupted by the TLR adaptors MyD88 and TRIF, as well as by HMGB1, which bind to beclin 1 and displace BCL2 (refs 3, 68). Two intracellular sensors responsible for inducing autophagy in response to bacterial infection, NOD1 and NOD2, interact with ATG16L1 and recruit it to the plasma membrane, resulting in enhanced association of invasive bacteria (S. flexneri) with LC3 (ref. 53). Interestingly, a NOD2 mutation associated with Crohn's disease impairs ATG16L1 plasma membrane recruitment and bacterial co-localization with LC3 (ref. 53).

The identification of other possible protein–protein interactions between core autophagy proteins and immune signals by a large proteomics screen21 has the potential to foster further advances in understanding how different immune signals regulate the autophagy machinery. For example, tectonin proteins with multivalent β-propeller folds are known to function in pathogen recognition and innate immunity in invertebrates70. Thus, the interactions between previously uncharacterized human proteins of this tectonin family, TECPR1 and TECPR2, with the ATG5–ATG12–ATG16L1 complex and ATG8 family members, respectively21, may contribute to pathogen-induced autophagy stimulation or selective autophagic targeting of pathogens in mammals.

Further links between immune signalling molecules and autophagy regulation were suggested by a genome-wide short interfering RNA screen71. The analysis identified 219 genes that suppressed basal autophagy, largely in a mammalian TOR complex 1 (mTORC1)-independent fashion. These included several cytokines such as CLCF1, LIF, IGF1, FGF2 and the chemokine SDF1 (also known as CXCL12), as well as cellular signalling molecules regulated by cytokines such as STAT3. These findings raise the possibility that cytokines may have a broader role in the control of autophagy than previously thought. Moreover, because these cytokine signalling pathways are important in immune cells, another central question is to what extent cytokine-mediated regulation of autophagy governs immune cell function. Given the general function of autophagy in cellular homeostasis5, and the more specific functions in regulating immune and inflammatory signalling (discussed in 'Regulation of immune signalling by autophagy proteins'), cytokine-mediated changes in autophagy levels in immune cells may have a central role in immunity and inflammation.

Autophagy and adaptive immunity

Autophagy proteins function in adaptive immunity, including in the development and homeostasis of the immune system and in antigen presentation (Table 1 and Fig. 3). The knockout of different autophagy genes in specific lymphocyte populations in mice has shown a crucial role for autophagy proteins in the maintenance of normal numbers of B1a B cells, CD4+ T cells, CD8+ T cells and fetal haematopoietic stem cells2, 44, 72. In T cells, in which mitochondrial numbers are developmentally regulated during the transition from thymocyte to mature circulating T cell, the developmental defect in autophagy-deficient cells may be related to the defective clearance of mitochondria44. Another crucial function of autophagy in the development and homeostasis of the immune system is the elimination of autoreactive T cells in the thymus44. High levels of autophagy are present in thymic epithelial cells, in which autophagy participates in the delivery of self-antigens to MHC class II loading compartments. Genetic disruption of Atg5 in thymic epithelial cells leads to the altered selection of certain MHC class II restricted T-cell specificities and autoimmunity73. Beyond these functions in lymphocyte survival and thymic negative selection, autophagy may exert other functions in lymphocyte differentiation, perhaps, in part, indirectly through effects on cytokine expression (see the next section). It is not yet known whether autophagy is involved in the development and/or homeostasis of immune cell populations other than lymphocytes and haematopoietic stem cells.

Autophagy proteins may participate in different facets of antigen presentation, including the delivery of endogenous antigens for MHC class II presentation to CD4+ T cells74, 75, the enhancement of antigen donor cell cross-presentation to CD8+ T cells75, dendritic cell cross-presentation of phagocytosed antigens to CD4+ T cells32 and, in one report, MHC class I presentation of intracellular antigens to CD8+ T cells27. The discovery that autophagosomes could deliver endogenous antigens to MHC class II loading compartments sheds light on one of the central mysteries of antigen presentation — how the immune system elicits CD4+ T-cell responses to antigens that originate in all parts of the cell. The autophagic delivery of endogenously synthesized antigens for MHC class II presentation has been demonstrated in vitro for certain viral antigens75, and probably explains the essential role of Atg5 in vivo in negative thymic selection73. However, the relative importance of this pathway in antigen presentation during infection in vivo is not yet known. There is nonetheless interest in exploiting this pathway for optimizing vaccine-elicited CD4+ T-cell responses, by either pre-treating dendritic cells with autophagy-inducing agents in cell-based vaccine strategies or fusing antigens with LC3 to enhance their targeting to autophagosomes1.

Of note, autophagy proteins are required for antigen cross-presentation during infection in vivo32. Dendritic-cell-specific deletion of Atg5 in mice results in defects in priming CD4+ T-cell responses after HSV and Listeria infections, and mice succumb more rapidly to lethal disease after intravaginal HSV infection. Atg5-deficient dendritic cells have normal migration, innate responses, endocytic and phagocytic activity and cross-presentation of peptides on MHC class I molecules. However, they exhibit defects in phagosome-to-lysosome fusion and in cross-presentation by MHC class II molecules of phagocytosed antigens containing TLR ligand. Thus, the interior of the phagosome, like that of the autophagosome, is a cellular compartment that autophagy-protein-dependent antigen presentation accesses to generate peptides for presentation to CD4+ T cells. A potential evolutionary advantage of this autophagy-protein-dependent cross-presentation is that, by delegating antigen presentation duties to uninfected dendritic cells, the host can bypass the blockade of antigen presentation that may result from microbial antagonism of autophagy in infected cells.

Regulation of immune signalling by autophagy proteins

In response to infection, the host must activate those arms of the innate and adaptive immune system (including autophagy-dependent functions; Fig. 3) that help to control infection while, in parallel, triggering specific responses that limit detrimental, uncontrolled immune activation and inflammation. An exciting new frontier in autophagy research is the growing recognition of the function of autophagy proteins in achieving this balance (Fig. 4).

Figure 4: Autophagy/autophagy proteins act to achieve a balance between activation and inactivation of innate immune signalling.

A general model in which the levels of autophagy and autophagy proteins control disease in response to stressors. Normal autophagy protein function (green) contributes to balanced inflammatory and metabolic responses, resulting in protection against disease. Altered autophagy protein function (red) results in maladaptive inflammatory and metabolic responses, increased inflammation and more severe disease.

Autophagy proteins function in both the activation and inactivation of innate immune signalling4. The autophagy pathway activates type I IFN production in plasmacytoid dendritic cells by delivering viral nucleic acids to endosomal TLRs76. By contrast, autophagy proteins negatively regulate RIG-I-like receptor (RLR)-mediated induction of type I IFN production through the autophagic elimination of damaged mitochondria (and reduction of ROS)77, and by the binding of ATG5–ATG12 to caspase recruitment domains of RLR signalling molecules78. Moreover, the autophagy protein ATG9A, but not ATG7, negatively regulates the activation of STING, a transmembrane protein that is required for efficient activation of type I IFN and pro-inflammatory cytokine production in response to stimulatory DNA23. Thus, it seems that autophagy proteins can negatively regulate IFN production by both autophagy-dependent and -independent mechanisms. With respect to the latter, different autophagy proteins may be specialized to target different innate immune signalling molecules.

The autophagy pathway and/or proteins also have a crucial role in the control of inflammatory signalling. A major effect is on the regulation of inflammatory transcriptional responses. Increased levels of the adaptor protein p62, which accumulates in autophagy-deficient cells, activate the pro-inflammatory transcription factor NF-κB through a mechanism involving TRAF6 oligomerization79. The accumulation of p62 in Atg7-deficient hepatocytes results in enhanced activity of the stress-responsive transcription factor NRF2 and NRF2-dependent liver injury80. In addition, Paneth cells (intestinal immune epithelial cells) from mice hypomorphic for Atg16l1 (Atg16l1HM) show enhanced transcription of pro-inflammatory cytokines and adipokines81.

A second important effect of autophagy proteins on inflammatory signalling is at the level of the inflammasome. This complex contains NOD-like receptor cryopyrin proteins, the adaptor protein ASC and caspase 1, and is activated by cellular infection or other stress to promote the maturation of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and IL-18 (ref. 4). Atg16l1- or Atg7-deficient mouse macrophages produce increased levels of mature IL-1β and IL-18 after TLR4 stimulation by endotoxin82. In addition, mouse chimaeras engrafted with Atg16l1−/− fetal liver haematopoietic progenitors have increased serum concentrations of IL-1β and IL-18 after treatment with dextran sodium sulphate (DSS), which contributes to increased DSS-induced colitis82.

The mechanism(s) by which autophagy proteins negatively regulate inflammasome activation are not yet understood. Mutually non-exclusive possibilities include direct interactions between autophagy proteins and inflammasome components, indirect inhibition of inflammasome activity through autophagic suppression of ROS accumulation, or autophagic degradation of danger signals that activate the inflammasome. In line with the latter model, the autophagic degradation of amyotrophic-lateral-sclerosis-linked mutant superoxide dismutase has been proposed to limit caspase 1 activation and IL-1β production83.

In addition to regulating inflammatory signalling, the autophagy pathway may prevent tissue inflammation through its role in apoptotic corpse clearance. The efficient clearance of apoptotic corpses during development and tissue homeostasis prevents secondary necrosis, which releases danger signals (DAMPs) that trigger inflammation. Autophagy genes are essential for the heterophagic clearance of dying apoptotic cells during developmental programmed cell death (by the generation of ATP-dependent engulfment signals)84, and the retinas and lungs of embryonic mice lacking Atg5 have a defect in apoptotic corpse engulfment that is associated with infiltration of inflammatory cells84. On the basis of growing evidence that autophagy proteins function in TLR-mediated phagolysosomal pathways, it is possible that autophagy also functions in phagocytes to facilitate apoptotic corpse clearance. Thus, in tissues such as the intestine, in which physiological regeneration involves continuous shedding or apoptosis of epithelial cells, autophagy-dependent functions in dying cells and/or phagocytic cells may promote efficient corpse clearance, thereby limiting inflammation.

Autophagy and inflammatory disease

Perturbations in autophagy-protein-dependent functions in immunity may contribute not only to increased susceptibility to infection, but also to chronic inflammatory diseases and autoimmune diseases. The only well-characterized link thus far is between mutations in autophagy regulators and Crohn's disease, a chronic inflammatory disorder of the small intestine, in which a breakdown in clearance or recognition of commensal bacteria, as well as altered mucosal barrier function and cytokine production, is thought to lead to intestinal inflammation (Fig. 5). Other emerging links include the autoimmune disease systemic lupus erythematosus (SLE), inflammation-associated metabolic diseases such as obesity and diabetes, and inflammation associated with cystic fibrosis lung disease (Fig. 4).

Figure 5: The link between mutations in autophagy regulators and the chronic inflammatory disorder Crohn's disease.

An overview of the many possible mechanisms by which defects in autophagy and autophagy protein function may contribute to the pathogenesis of a type of inflammatory bowel disease, Crohn's disease. A micrograph of a human small intestine from a patient with Crohn's disease is shown (centre), demonstrating the severe transmural inflammation that is characteristic of this disease. The postulated mechanisms by which defects in autophagy protein function might contribute to the development or perpetuation of intestinal inflammation are based on studies in vitro and animal models. There is no direct evidence that autophagy defects contribute to human Crohn's disease, although mutations in three autophagy-related genes, ATG16L1, NOD2 and IRGM, are known to enhance risk of the disease. E, epithelium; IgM, immunoglobulin M; L, lumen; LA, lymphoid aggregates; TM, thickened muscle. Scale bar, 200 μm.

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The role of autophagy proteins in Crohn's disease was not suspected until genome-wide association studies identified three Crohn's disease susceptibility genes, IRGM, NOD2 and ATG16L1, that are involved in autophagy85. The IRGM risk allele contains a deletion in the promoter region of the gene that may be associated with changes in IRGM protein expression and may contribute to Crohn's disease, given IRGM's role in autophagy-dependent control of bacterial infection49. However, this hypothesis has not yet been tested. The three major Crohn's-disease-associated NOD2 variants (a frameshift mutant and two missense mutants) may be loss-of-function mutants, with impaired muramyl dipeptide (MDP)-induced inflammatory signalling86. How the loss of function of a pro-inflammatory signal mechanistically contributes to an inflammatory disorder has been unclear, but the recently discovered links between NOD2 and autophagy may solve this conundrum. In primary immature human dendritic cells, NOD2 is required for MDP-induced autophagy, a process that is essential for the MHC class II presentation of bacterial antigens to CD4+ T cells and for bacterial targeting to lysosomes52. Dendritic cells expressing Crohn's disease NOD2 risk variants are defective in both of these functions52. Thus, in patients with Crohn's disease and NOD2 risk variants, aberrant autophagy-dependent bacterial clearance and immune priming could act as a trigger for intestinal inflammation.

A mechanistic link may also exist between ATG16L1 mutation and Crohn's disease pathogenesis. Similar to findings with NOD2 variants, dendritic cells from patients with the Crohn's-disease-associated ATG16L1(T300A) risk variant are defective in presenting bacterial antigen to CD4+ T cells52. However, it is not yet known how the T300A mutation affects the function of the mammalian ATG16L1 protein. This mutation resides in the carboxy-terminal WD-repeat domain that is absent in yeast Atg16 and is dispensable for autophagy. Although some studies have suggested that the ATG16L1(T300A) variant has reduced autophagic clearance of enteric pathogens such as adherent-invasive Escherichia coli87 or S. Typhimurium88, it remains controversial whether the risk versus protective alleles of ATG16L1 have differences in stability or antibacterial autophagic activity89.

Despite the uncertain nature of the effects of the T300A mutation on ATG16L1 function, Atg16l1 mutation (null or hypomorphic alleles) in mice results in abnormalities that are relevant to Crohn's disease pathogenesis. As noted earlier, loss of Atg16l1 function in mice results in enhanced TLR-agonist-induced pro-inflammatory cytokine production by macrophages82, enhanced DSS-induced colitis82, 90 and altered inflammatory gene transcriptional profiles in Paneth cells81, 90. In addition, the Paneth cells of mice expressing low Atg16l1 levels (Atg16l1HM) show defects in the packaging and extrusion of antimicrobial granules into the gut lumen; Paneth cells from patients with Crohn's disease and the ATG16L1(T300A) risk variant show similar defects81. This suggests that, in addition to the overlapping functions of NOD2 and ATG16L1 in a common bacterial-sensing pathway that promotes bacterial antigen presentation, ATG16L1 may have unique protective functions, including Paneth cell antimicrobial peptide release and the negative regulation of pro-inflammatory cytokine production. To connect the striking phenotypes in Atg16l1-mutant mice and the pathogenesis of Crohn's disease in humans with the ATG16L1(T300A) risk allele, the precise effects of the T300A mutation on ATG16L1 protein function need to be uncovered.

A new dimension in understanding the multifactorial basis of chronic inflammatory diseases such as Crohn's disease has emerged from the discovery that a virus trigger is required to observe intestinal abnormalities in Atg16l1HM mice90. In mice raised in a pathogen-free facility, only Atg16l1HM mice (and not wild-type mice) infected with a virus found in routine conventional animal facilities, a murine norovirus, showed abnormal Paneth cell granule secretion, Paneth cell pro-inflammatory gene-expression profiles, and intestinal inflammation in response to DSS treatment90. This mucosal inflammation depended on the presence of the microbiome and pro-inflammatory cytokines, as it was reversed by antibiotic treatment or by TNF-α or IFN-γ inhibition. Thus, variations in a host autophagy gene, exposure to a specific virus and the microbiome can act together to trigger intestinal inflammation in mice that is similar to that in patients with Crohn's disease. Although environmental factors, including the gut microbiome, have long been suspected to contribute to Crohn's disease in genetically susceptible individuals, formal proof of this concept was lacking, and viruses were a previously unsuspected trigger. Another implication of this work is the concept that autophagy proteins, through their diverse roles in immunity and the control of inflammation, may serve as a central rheostat that prevents inflammatory diseases triggered by environmental stress (Fig. 4).

An important unanswered question is whether perturbations in autophagy may also result in inflammatory autoimmune disease. Genome-wide association studies have linked several single nucleotide polymorphisms (SNPs) in ATG5 to SLE susceptibility91, 92, 93. SLE is a multifactorial, heterogeneous disease characterized by autoimmune responses against self-antigens generated from dying cells. Although the effects of these SNPs on ATG5 expression and function are not known, the lack of Atg5-dependent negative thymic selection generates autoimmunity and multi-organ inflammation in mice73. Loss of other ATG5-dependent effects, including regulation of IFN and pro-inflammatory cytokine secretion77, 78, clearance of dying cells84 and dendritic cell antigen presentation32, might also contribute to the autoimmunity and inflammation associated with SLE. Thus, a link between ATG5 mutation (or mutation of other autophagy genes) and SLE pathogenesis is biologically plausible, although not yet proven.

Defects in autophagy may contribute to inflammation-associated metabolic diseases such as diabetes and obesity, which are both linked to insulin resistance. The metabolic inflammasome — a complex composed of signalling molecules such as PKR, eIF2α, JNK, IRS and IKK— may act as a link between ER stress and more global stress responses, including inflammation and metabolic dysfunction (as observed in insulin resistance and obesity)94. Although most components of the metabolic inflammasome promote autophagy, the induction of autophagy by this signalling complex would be expected to serve as a negative-feedback mechanism that limits ER stress and disease progression. Consistent with this postulated protective effect of autophagy, hepatic suppression of the autophagy gene Atg7 in mice results in increased ER stress and insulin resistance95, and mice deficient in the autophagy adaptor protein p62 develop mature-onset obesity and insulin resistance96. Furthermore, obesity is associated with the accumulation and activation of macrophages and subsets of T cells in adipose tissue and the production of cytokines such as TNF-α and IL-6 (ref. 97). Thus, the failure of autophagy-dependent control of ER stress, immune cell homeostasis, immune cell activation and/or pro-inflammatory cytokine secretion may contribute to inflammation-associated responses that underlie the pathogenesis of metabolic diseases.

Another potential link between autophagy deficiency and chronic inflammation is in cystic fibrosis98, a life-threatening genetic disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). Mutations in CFTR lead to autophagy inhibition in lung epithelial cells through a mechanism that may involve ROS-mediated sequestration of the beclin 1–class III PI(3)K complex in perinuclear aggregates (redirecting it from its site of autophagy action at the ER). Restoration of beclin 1 and autophagy in cystic fibrosis epithelial cells rescues the disease phenotype, and antioxidants reverse the airway inflammation in a cystic fibrosis mouse model by a mechanism postulated to involve autophagy.

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Yuk, J. M. et al. Vitamin D3 induces autophagy in human monocytes/macrophages via cathelicidin. Cell Host Microbe 6, 231–243 (2009).

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Low, D. H. et al. A novel human tectonin protein with multivalent β-propeller folds interacts with ficolin and binds bacterial LPS. PLoS ONE 4, e6260 (2009).

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Liu, F. et al. FIP200 is required for the cell-autonomous maintenance of fetal hematopoietic stem cells. Blood 116, 4806–4814 (2010).

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This study provided the first evidence that the autophagy machinery functions in MHC class II antigen presentation in vivo, specifically in shaping the CD4+ T-cell repertoire during negative thymic selection, and thereby preventing autoimmunity and multi-organ inflammation.

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Paludan, C. et al. Endogenous MHC class II processing of a viral nuclear antigen after autophagy. Science 307, 593–596 (2005).

This study provided the first evidence that the autophagy machinery can deliver endogenously synthesized antigens for presentation on MHC class II molecules to CD4+ T cells.

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Munz, C. Antigen processing via autophagy—not only for MHC class II presentation anymore? Curr. Opin. Immunol. 22, 89–93 (2010).

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Lee, H. K., Lund, J. M., Ramanathan, B., Mizushima, N. & Iwasaki, A. Autophagy-dependent viral recognition by plasmacytoid dendritic cells. Science 315, 1398–1401 (2007).

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Tal, M. C. et al. Absence of autophagy results in reactive oxygen species-dependent amplification of RLR signaling. Proc. Natl Acad. Sci. USA 106, 2770–2775 (2009).

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Jounai, N. et al. The Atg5–Atg12 conjugate associates with innate antiviral immune responses. Proc. Natl Acad. Sci. USA 104, 14050–14055 (2007).

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Moscat, J. & Diaz-Meco, M. T. p62 at the crossroads of autophagy, apoptosis, and cancer. Cell 137, 1001–1004 (2009).

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Komatsu, M. et al. The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1. Nature Cell Biol. 12, 213–223 (2010).

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Cadwell, K. et al. A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells. Nature 456, 259–263 (2008).

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Saitoh, T. et al. Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1β production. Nature 456, 264–268 (2008).

This study demonstrates that autophagy proteins negatively control endotoxin-induced inflammasome activation. References 81 and 82 also show that autophagy gene deficiency increases susceptibility to experimentally induced inflammatory bowel disease.

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Meissner, F., Molawi, K. & Zychlinsky, A. Mutant superoxide dismutase 1-induced IL-1β accelerates ALS pathogenesis. Proc. Natl Acad. Sci. USA 107, 13046–13050 (2010).

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This paper shows that autophagy genes are necessary for apoptotic cells to generate engulfment signals required for successful apoptotic corpse clearance and the prevention of tissue inflammation.

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Barrett, J. C. et al. Genome-wide association defines more than 30 distinct susceptibility loci for Crohn's disease. Nature Genet. 40, 955–962 (2008).

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Brain, O., Allan, P. & Simmons, A. NOD2-mediated autophagy and Crohn disease. Autophagy 6, 412–414 (2010).

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Lapaquette, P., Glasser, A. L., Huett, A., Xavier, R. J. & Darfeuille-Michaud, A. Crohn's disease-associated adherent-invasive E. coli are selectively favoured by impaired autophagy to replicate intracellularly. Cell. Microbiol. 12, 99–113 (2010).

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Cadwell, K. et al. Virus-plus-susceptibility gene interaction determines Crohn's disease gene Atg16L1 phenotypes in intestine. Cell 141, 1135–1145 (2010).

This study shows that both hypomorphic expression of an autophagy protein and a viral-infection trigger are necessary for experimentally induced inflammatory bowel disease, suggesting that the interaction between host defects in autophagy and environmental stressors such as infection may be crucial for the pathogenesis of certain inflammatory diseases.

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Harley, J. B. et al. Genome-wide association scan in women with systemic lupus erythematosus identifies susceptibility variants in ITGAM, PXK, KIAA1542 and other loci. Nature Genet. 40, 204–210 (2008).

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Gateva, V. et al. A large-scale replication study identifies TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL10 as risk loci for systemic lupus erythematosus. Nature Genet. 41, 1228–1233 (2009).

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Han, J. W. et al. Genome-wide association study in a Chinese Han population identifies nine new susceptibility loci for systemic lupus erythematosus. Nature Genet. 41, 1234–1237 (2009).

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Nakamura, T. et al. Double-stranded RNA-dependent protein kinase links pathogen sensing with stress and metabolic homeostasis. Cell 140, 338–348 (2010).

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Yang, L., Li, P., Fu, S., Calay, E. S. & Hotamisligil, G. S. Defective hepatic autophagy in obesity promotes ER stress and causes insulin resistance. Cell Metab. 11, 467–478 (2010).

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Hotamisligil, G. S. Inflammation and metabolic disorders. Nature 444, 860–867 (2006).

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Luciani, A. et al. Defective CFTR induces aggresome formation and lung inflammation in cystic fibrosis through ROS-mediated autophagy inhibition. Nature Cell Biol. 12, 863–875 (2010).

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Gutierrez, M. G. et al. Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages. Cell 119, 753–766 (2004).

Reference 99, together with reference 30, provides the first evidence that autophagy has a key role in bacterial infection. Autophagy induction by IFN-γ, rapamycin or starvation results in the conversion of mycobacterial phagosomes into phagolysosomes, thereby enhancing mycobacterial killing.

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Ogawa, M. et al. Escape of intracellular Shigella from autophagy. Science 307, 727–731 (2005).

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