Protection by natural IgG: a sweet partnership with soluble lectins does the trick!

EMBO J 32: 2905–2919 10.1038/emboj.2013.199; published online September032013Some B cells of the adaptive immune system secrete polyreactive immunoglobulin G (IgG) in the absence of immunization or infection. Owing to its limited affinity and specificity, this natural IgG is thought to play a modest protective role. In this issue, a report reveals that natural IgG binds to microbes following their opsonization by ficolin and mannan-binding lectin (MBL), two carbohydrate receptors of the innate immune system. The interaction of natural IgG with ficolins and MBL protects against pathogenic bacteria via a complement-independent mechanism that involves IgG receptor FcγRI expressing macrophages. Thus, natural IgG enhances immunity by adopting a defensive strategy that crossovers the conventional boundaries between innate and adaptive microbial recognition systems.The adaptive immune system generates protective somatically recombined antibodies through a T cell-dependent (TD) pathway that involves follicular B cells. After recognizing antigen through the B-cell receptor (BCR), follicular B cells establish a cognate interaction with CD4⁺ T follicular helper (T_FH) cells and thereafter either rapidly differentiate into short-lived IgM-secreting plasmablasts or enter the germinal centre (GC) of lymphoid follicles to complete class switch recombination (CSR) and somatic hypermutation (SHM) (Victora and Nussenzweig, 2012). CSR from IgM to IgG, IgA and IgE generates antibodies with novel effector functions, whereas SHM provides the structural correlate for the induction of affinity maturation (Victora and Nussenzweig, 2012). Eventually, this canonical TD pathway generates long-lived bone marrow plasma cells and circulating memory B cells that produce protective class-switched antibodies capable to recognize specific antigens with high affinity (Victora and Nussenzweig, 2012).In addition to post-immune monoreactive antibodies, B cells produce pre-immune polyreactive antibodies in the absence of conventional antigenic stimulation (Ehrenstein and Notley, 2010). These natural antibodies form a vast and stable repertoire that recognizes both non-protein and protein antigens with low affinity (Ehrenstein and Notley, 2010). Natural antibodies usually emerge from a T cell-independent (TI) pathway that involves innate-like B-1 and marginal zone (MZ) B cells. These are extrafollicular B-cell subsets that rapidly differentiate into short-lived antibody-secreting plasmablasts after detecting highly conserved microbial and autologus antigens through polyreactive BCRs and nonspecific germline-encoded pattern recognition receptors (Pone et al, 2012; Cerutti et al, 2013).The most studied natural antibody is IgM, a pentameric complement-activating molecule with high avidity but low affinity for antigen (Ehrenstein and Notley, 2010). In addition to promoting the initial clearance of intruding microbes, natural IgM regulates tissue homeostasis, immunological tolerance and tumour surveillance (Ochsenbein et al, 1999; Zhou et al, 2007; Ehrenstein and Notley, 2010). Besides secreting IgM, B-1 and MZ B cells produce IgG and IgA after receiving CSR-inducing signals from dendritic cells (DCs), macrophages and neutrophils of the innate immune system (Cohen and Norins, 1966; Cerutti et al, 2013). In humans, certain natural IgG and IgA are moderately mutated and show some specificity, which may reflect the ability of human MZ B cells to undergo SHM (Cerutti et al, 2013). Yet, natural IgG and IgA are generally perceived as functionally quiescent.In this issue, Panda et al show that natural IgG bound to a broad spectrum of bacteria with high affinity by cooperating with ficolin and MBL (Panda et al, 2013), two ancestral soluble lectins of the innate immune system (Holmskov et al, 2003). This binding involved some degree of specificity, because it required the presence of ficolin or MBL on the microbial surface as well as lower pH and decreased calcium concentration in the extracellular environment as a result of infection or inflammation (see Figure 1).Open in a separate windowFigure 1Ficolins and MBL are produced by hepatocytes and various cells of the innate immune system and opsonize bacteria after recognizing conserved carbohydrates. Low pH and calcium concentrations present under infection-inflammation conditions promote the interaction of ficolin or MBL with natural IgG on the surface of bacteria. The resulting immunocomplex is efficiently phagocytosed by macrophages through FcγR1 independently of the complement protein C3, leading to the clearance of bacteria.Ficolins and MBL are soluble pattern recognition receptors that opsonize microbes after binding to glycoconjugates through distinct carbohydrate recognition domain (CRD) structures (Holmskov et al, 2003). While ficolins use a fibrinogen domain, MBL and other members of the collectin family use a C-type lectin domain attached to a collagen-like region (Holmskov et al, 2003). Similar to pentraxins, ficolins and MBL are released by innate effector cells and hepatocytes, and thus may have served as ancestral antibody-like molecules prior to the inception of the adaptive immune system (Holmskov et al, 2003; Bottazzi et al, 2010). Of note, MBL and the MBL-like complement protein C1q are recruited by natural IgM to mediate complement-dependent clearance of autologous apoptotic cells and microbes (Holmskov et al, 2003; Ehrenstein and Notley, 2010). Panda et al found that a similar lectin-dependent co-optation strategy enhances the protective properties of natural IgG (Panda et al, 2013).By using bacteria and the bacterial glycan N-acetylglicosamine, Panda et al show that natural IgG isolated from human serum or T cell-deficient mice interacted with the fibrinogen domain of microbe-associated ficolins (Panda et al, 2013). The resulting immunocomplex was phagocytosed by macrophages via the IgG receptor FcγRI in a complement-independent manner (Panda et al, 2013). The additional involvement of MBL was demonstrated by experiments showing that natural IgG retained some bacteria-binding activity in the absence of ficolins (Panda et al, 2013).Surface plasmon resonance provided some clues regarding the molecular requirements of the ficolin–IgG interaction (Panda et al, 2013), but the conformational changes required by ficolin to interact with natural IgG remain to be addressed. In particular, it is unclear what segment of the effector Fc domain of natural IgG binds to ficolins and whether Fc-associated glycans are involved in this binding. Specific glycans have been recently shown to mitigate the inflammatory properties of IgG emerging from TI responses (Hess et al, 2013) and this process could implicate ficolins and MBL. Moreover, it would be important to elucidate whether and how the antigen-binding Fab portion of natural IgG regulates its interaction with ficolins and MBL.The in vivo protective role of natural IgG was elegantly demonstrated by showing that reconstitution of IgG-deficient mice lacking the CSR-enzyme activation-induced cytidine deaminase with natural IgG from T cell-insufficient animals enhanced resistance to pathogenic Pseudomonas aeruginosa (Panda et al, 2013). This protective effect was associated with reduced production of proinflammatory cytokines, occurred independently of the complement protein C3 and was impaired by peptides capable to inhibit the binding of natural IgG to ficolin (Panda et al, 2013). Additional in vivo studies will be needed to determine whether natural IgG exerts protective activity in mice lacking ficolin, MBL or FcγRI, and to ascertain whether these molecules also enhance the protective properties of canonical or natural IgG and IgA released by bone marrow plasma cells and mucosal plasma cells, respectively.In conclusion, the findings by Panda et al show that natural IgG adopts ‘crossover'' defensive strategies that blur the conventional boundaries between the innate and adaptive immune systems. The sophisticated integration of somatically recombined and germline-encoded antigen recognition systems described in this new study shall stimulate immunologists to further explore the often underestimated protective virtues of our vast natural antibody repertoire. This effort may lead to the development of novel therapies against infections.     

Central B-cell tolerance: where selection begins

The development of an adaptive immune system based on the random generation of antigen receptors requires a stringent selection process that sifts through receptor specificities to remove those reacting with self-antigens. In the B-cell lineage, this selection process is first applied to IgM⁺ immature B cells. By using increasingly sophisticated mouse models, investigators have identified the central tolerance mechanisms that negatively select autoreactive immature B cells and prevent inclusion of their antigen receptors into the peripheral B-cell pool. Additional studies have uncovered mechanisms that promote the differentiation of nonautoreactive immature B cells and their positive selection into the peripheral B-cell population. These mechanisms of central selection are fundamental to the generation of a naïve B-cell repertoire that is largely devoid of self-reactivity while capable of reacting with any foreign insult.B-cell generation in the bone marrow of adult mammals occurs through a tightly controlled developmental process (Fig. 1). Productive rearrangement of immunoglobulin heavy (IgH) and light (IgL) chain gene segments in B lymphocyte precursor cells, in addition to the expression of Ig-α (CD79a) and Ig-β (CD79b), result in the generation and expression on the cell surface of a mature B-cell antigen receptor (BCR). Whereas the combination of Ig H and L chains determines the antigenic specificity of the newly formed BCR, their association with Ig-α and Ig-β allows transduction of a signal inside the cell that directs cell fate. Developing B cells first express a mature BCR on the cell surface in the form of IgM and as such are classified as immature B cells (Fig. 1) (Hardy et al. 1991; Pelanda et al. 1996). It is at the immature B-cell stage that the BCR is tested for the first time for reactivity against autoantigens. This test determines whether the immature B cell and the antibody it expresses on the surface will be selected into the peripheral B-cell repertoire. Central B-cell tolerance, in fact, refers to the process that negatively selects newly generated immature B cells that react with a self-antigen in the bone marrow environment. This is considered the first checkpoint of B-cell tolerance, and the results of this checkpoint are fundamental to the generation of a naïve repertoire that contains foreign reactive antibodies and is largely devoid of self-reactive specificities.Open in a separate windowFigure 1.Schematic representation of B-cell development and Ig loci in mice. Large pro-B cells initiate Ig gene rearrangement at the IgH locus. Expression of a H chain following a productive V_HD_HJ_H recombination event promotes the differentiation of large pre-B cells in which the expression of pre-BCR (H chain pairing with surrogate light chains) results in the clonal expansion of H chain-positive pre-B cells and the development of small pre-B cells. Expression of conventional L chains following productive rearrangements at the IgL chain loci in small pre-B cells promotes the development of a diverse population of IgM⁺ immature B cells, which then differentiate into IgM⁺IgD⁺ transitional B cells. The scheme of mouse Ig H, κ, and λ loci (not to scale) indicate the presence of V (white rectangles), D (black vertical lines), J (brown vertical lines; a dashed line indicates a nonfunctional element), and C (black rectangles; a gray rectangle indicates a nonfunctional element) gene segments. The scheme does not represent the number of V_H, D_H, and Vκ gene segments in the actual Ig loci.On passing this central checkpoint, immature B cells continue to differentiate into transitional and mature B cells before and after they travel to the spleen (Loder et al. 1999; Allman et al. 2001; Su and Rawlings 2002; Tarlinton et al. 2003). Analysis of the bone marrow early immature B-cell repertoire indicates that a staggering 50%–75% of these cells express BCRs that are specific for self-antigens, both in mice and humans (Grandien et al. 1994; Wardemann et al. 2003). Similar studies performed on cell populations at the other end of this central checkpoint, namely, transitional and naïve mature B cells in spleen and blood, show a much lower frequency (20%–40%) of cells expressing autoreactive antibodies (Grandien et al. 1994; Wardemann et al. 2003), demonstrating the stringency and limitation of this initial selection step. Moreover, individuals affected by autoimmune disease such as lupus erythematosus or rheumatoid arthritis bear many more autoreactive cells in their new emigrant and naïve B-cell populations (Samuels et al. 2005; Yurasov et al. 2005), indicating a defect in central (and/or peripheral) B-cell selection. Thus, it seems important that the development of autoreactive immature B cells be constrained to prevent the potential occurrence of autoimmunity. However, there are also reasons to believe that the high frequency of autoreactive specificities generated during primary Ig gene rearrangements may be necessary for the generation of the peripheral B-cell repertoire (Pelanda et al. 1997; Kohler et al. 2008). Indeed, a fraction of autoreactive immature B cells, those manifesting a low level of self-reactivity, do bypass the central checkpoint of tolerance and differentiate into mature B cells (Hayakawa et al. 2003; Wardemann et al. 2003; Wen et al. 2005). The inclusion of these weakly self-reactive B cells in the peripheral B-cell repertoire may allow recognition of a broader spectrum of foreign molecules, potentially decreasing the negative impact of infections, especially at early stages (Mouquet et al. 2010).What are the rules that govern the selection of immature B cells? Most studies of central tolerance have been conducted by following the selection of B cells expressing BCRs displaying well-defined reactivity for natural or synthetic self-antigens. This has been accomplished through the use of Ig transgenic mice in which developing B cells have been altered to carry prerearranged Ig H and L chain genes encoding antibodies of defined antigen specificity and reactivity. Here we review some of these studies, what we have learned from them, and open questions that still await answers.     

Amphisomes: out of the autophagosome shadow?

EMBO J (2013) 32: 3130–3144 doi: 10.1038/emboj.2013.233; published online November012013Amphisomes are intermediate organelles, formed during autophagy through the fusion between autophagosomes and endosomes. Complex multivesicular vacuoles that resemble amphisomes have been observed in various cell types, but whether they have cellular roles other than being a precursor structure is still enigmatic. While autophagy-related (ATG) proteins interact with the endocytic pathways in other processes different from autophagy, Patel and colleagues now report that these factors come together to generate amphisome-like compartments that regulate mucin secretion in goblet cells.ATG and endosomal proteins have been linked to secretion, and the specific loss of them impairs the function of different secretory cell types (Jung et al, 2008; DeSelm et al, 2011; Ushio et al, 2011; Sasidharan et al, 2012). ATG proteins have also been shown to interact with the endocytic pathway in few situations that do not involve autophagy. For example in phagocytic cells, the surface of bacteria-containing phagosomes acquires LC3/Atg8 through the concerted action of a subpopulation of ATG proteins. This process, which has been termed LC3-associated phagocytosis (LAP), promotes the fusion of phagosomes with lysosomes (Sanjuan et al, 2007). Something similar occurs during entotic cell death, an engulfment programme leading to the elimination of cells into lysosomes. The entotic vacuole membranes surrounding the internalized cells also recruit LC3 through a mechanism that depends on several ATG proteins, but not on autophagosome formation (Florey et al, 2011).In their work aimed to understand the function of ATG proteins in goblet cells, Patel et al (2013) show that the autophagy and endocytic machinery converge at the amphisomes to promote the secretion of mucins. In the gastrointestinal tract, secretory cells have a crucial role in providing the mucus barrier that protects against intestinal pathogens. Mucins, the main components of the mucus, are produced in goblet cells where large polymers of these highly glycosylated proteins are packed into secretory granules that accumulate at the apical surface. The release of these mucin granules relies on a series of cellular events that are tightly coordinated. Patel et al (2013) show that knockout mice lacking ATG5 in the intestinal epithelium, that is, Atg5VC mice, exhibit both a dramatic accumulation of mucin granules in goblet cells and a diminished mucus secretion. Taking advantage of a newly developed in vitro system to culture and differentiate intestinal epithelial stem cells into secretory goblet cells, the authors also demonstrate that the ablation of other ATG proteins causes the same phenotype showing that the autophagy machinery is required for mucin secretion in these specialized cells (Patel et al, 2013). Interestingly, ATG proteins affect the functionality of another gastrointestinal secretory lineage, the Paneth cells. Paneth cells homozygous for the atg16L1 risk allele, associated with Crohn disease, produce less secretory granules than in controls (Cadwell et al, 2008). This suggests that although ATG proteins regulate secretion in the two most abundant secretory lineages in the intestinal tract, two different mechanisms are probably involved.A microarray analysis of mRNA from Atg5VC mouse colonic epithelial cells revealed a possible alteration in the endocytic pathway. Indeed, blocking endocytosis also provoked an accumulation of mucin granules. While LC3B has been previously found on the surface of secretory granules (Ushio et al, 2011; Ishibashi et al, 2012), immuno-electron microscopy of wild-type mouse intestinal tissue revealed a distribution of LC3B not on mucin granules, but on multivesicular vacuoles positive for several endosomal proteins (Patel et al, 2013). Because of the morphological and molecular characteristics of these compartments, it appears that the ATG proteins together with the endocytic pathway regulate secretion in goblet cells by converging in what could be a new amphisome-like organelle (Figure 1).Open in a separate windowFigure 1Schematic representation for the regulated secretion of mucin granules by amphisome-like structures in goblet cells. ROS generated by NADPH oxidases promote the fusion of LC3-positive vesicles with endosomes marked by Rab5 and containing the NADPH oxidase subunit p22phox. The resulting amphisomes-like organelles are decorated with LC3, endosomal proteins (Rab5, Rab7 and EEA1) and p22phox and localize near the mucin granules. The formation of these copartments probably prolong and/or enhance the production of ROS by the NADPH oxidase, which in turn increases the levels of cytoplasmic calcium through an unknown mechanism leading to the release of the mucin granules.NADPH oxidases are known to be present in endosomes, and NADPH oxidase-generated reactive oxygen species (ROS) are necessary for LC3 recruitment to phagosomes.(Huang et al, 2009). Patel et al (2013) thus explored whether these enzymes played a role in mucin granule secretion in goblet cells. Indeed, expression of a mutant form of p22phox, a transmembrane subunit of several NADPH oxidase complexes, altered the exocytosis of these carriers. Moreover, p22phox was found to localize to Rab5-positive endosomes and also with the observed amphisome-like structures (Figure 1). Because a mutant form of p22phox also caused a misslocalization of both LC3 and the early-endosomal marker protein EEA1, the obvious conclusion was that ROS production by endosomes is necessary to trigger the formation of the amphisome-like organelles via the acquisition of the ATG machinery (Figure 1). Interestingly, addition of H₂O₂ that mimics ROS generation was able to induce mucin granule exocytosis in the p22phox mutant cells, showing that ROS was also required to regulate secretion in goblet cells (Patel et al, 2013). Furthermore, H₂O₂ bypassed as well the mucin granule secretion defect in autophagy and endocytosis-deficient goblet cells through an increase of cytosolic calcium levels (Patel et al, 2013). This, together with the observation that the loss of ATG5 and the block of the endocytic pathway impair the production of ROS has led Patel et al (2013) to propose that amphisome-like organelles are a signalling platform, where NADPH oxidase-driven ROS production promotes the release of the mucin granules.Amphisomes have been characterized and defined as autophagic vacuoles formed upon fusion between autophagosomes and endosomes. Given that ATG and endosomal proteins converge in multivesicular and/or vacuolar compartments resembling amphisomes in cellular processes independent of autophagy, one could consider to use the term amphisomes to describe a more heterogenous and ampler population of unnamed compartments where part of the autophagy and endosomal machineries co-localize. Based on this consideration, the study by Patel et al (2013) has identified an amphisome-like structure where molecular events interconnect to trigger granule secretion. While their work adds to the still limited number of non-degradative roles of the autophagic pathway, which include unconventional secretion (Subramani and Malhotra, 2013), it is one of the first reports highlighting that amphisomes (or any autophagosomal intermediate structure) could be more than just a transport intermediate, and at least in goblet cells, they could act as a platform where signals integrating some aspects of the cell physiology are elicited.Though it remains to be establish whether the organelles described by Patel et al (2013) are indeed amphisomes, especially as they are formed by fusion of endosomes with LC3-positive single-membrane vesicles rather than LC3-positive double-membrane autophagosomes, their study raises some intriguing questions. Are these compartments persistent or will they eventually fuse with lysosomes? Why has the cell opted to signal from amphisomes and not from endosomes, where the NADPH oxidases are normally present? Maybe the answer to these questions is hidden in the transient life of amphisomes. In the most classical signalling pathways, the transduction cascade amplifies the initial cue but it also turn it off subsequently through negative feedback loops. This permits to precisely modulate the signal output temporally (and locally). The amphisome-like structures observed in goblet cells could also act as the molecular switch for the signal-stimulating mucin granule secretion. The ROS generated initially from endosomes would trigger the recruitment of LC3 through vesicle fusion events, and the production of this second messenger will be prolonged and/or enhanced in the resulting amphisomes-like structure, leading to a stimulation of mucin granule exocytosis (Figure 1). The subsequent fusion of the amphisomes with lysosomes could lead to the termination of the signal. Other scenarios, however, cannot be excluded like, for example, the delivery of a protein enhancing the NADPH oxidase activity to the endosomes by the LC3-positive vesicles.While these are just hypotheses, it is clear that Patel et al (2013) have opened a window on a new and unexplored area of the autophagy field. Future investigations will tell us whether what observed in goblet cells is a unique situation or the intermediate organelles characterizing autophagy can carry out cellular functions different from the one delivering unwanted structures into the lysosome interior for degradation, including to serve as signalling platforms.     

Stimulating cROSstalk between commensal bacteria and intestinal stem cells

EMBO J (2013) 32 23, 3017–3028 10.1038/emboj.2013.224; published online October182013Commensal gut bacteria benefit their host in many ways, for instance by aiding digestion and producing vitamins. In a new study in The EMBO Journal, Jones et al (2013) report that commensal bacteria can also promote intestinal epithelial renewal in both flies and mice. Interestingly, among commensals this effect is most specific to Lactobacilli, the friendly bacteria we use to produce cheese and yogurt. Lactobacilli stimulate NADPH oxidase (dNox/Nox1)-dependent ROS production by intestinal enterocytes and thereby activate intestinal stem cells.The human gut contains huge numbers of bacteria (∼10¹⁴/person) that play beneficial roles for our health, including digestion, building our immune system and competing with harmful microbes (Sommer and Backhed, 2013). Both commensal and pathogenic bacteria can elicit antimicrobial responses in the intestinal epithelium and also stimulate epithelial turnover (Buchon et al, 2013; Sommer and Backhed, 2013). In contrast to gut pathogens, relatively little is known about how commensal bacteria influence intestinal turnover. In a simple yet elegant study reported recently in The EMBO Journal, Jones et al (2013) show that among several different commensal bacteria tested, only Lactobacilli promoted much intestinal stem cell (ISC) proliferation, and it did so by stimulating reactive oxygen species (ROS) production. Interestingly, the specific effect of Lactobacilli was similar in both Drosophila and mice. In addition to distinguishing functional differences between species of commensals, this work suggests how the ingestion of Lactobacillus-containing probiotic supplements or food (e.g., yogurt) might support epithelial turnover and health.In both mammals and insects, ISCs give rise to intestinal enterocytes, which not only absorb nutrients from the diet but must also interact with the gut microbiota (Jiang and Edgar, 2012). The metazoan intestinal epithelium has developed conserved responses to enteric bacteria, for instance the expression of antimicrobial peptides (AMPs; Gallo and Hooper, 2012; Buchon et al, 2013), presumably to kill harmful bacteria while allowing symbiotic commensals to flourish. In addition to AMPs, intestinal epithelial cells use NADPH family oxidases to generate ROS that are used as microbicides (Lambeth and Neish, 2013). High ROS levels during enteric infections likely act non-discriminately against both commensals and pathogens, but controlled, low-level ROS can act as signalling molecules that regulate various cellular processes including proliferation (Lambeth and Neish, 2013). In flies, exposure to pathogenic Gram-negative bacteria has been reported to result in ROS (H₂O₂) production by an enzyme called dual oxidase (Duox; Ha et al, 2005). Duox activity in the fly intestine (and likely also the mammalian one) has recently been discovered to be stimulated by uracil secretion by pathogenic bacteria (Lee et al, 2013). In the mammalian intestine another enzyme, NADPH oxidase (Nox), has also been shown to produce ROS in the form of superoxide (O₂⁻), in this case in response to formylated bacterial peptides (Lambeth and Neish, 2013). A conserved role for Nox in the Drosophila intestinal epithelium had not until now been explored.Jones et al (2013) checked seven different commensal bacterial to see which would stimulate ROS production by the fly''s intestinal epithelium, and found that only one species, a Gram-positive Lactobacillus, could stimulate significant production of ROS in intestinal enterocytes. Five bacterial species were checked in mice or cultured intestinal cells, and again it was a Lactobacillus that generated the strongest ROS response. Although not all of the most prevalent enteric bacteria were assayed, those others that were—such as E. coli—induced only mild, barely detectable levels of ROS in enterocytes. Surprisingly, although bacteria pathogenic to Drosophila, like Erwinia caratovora, were expected to stimulate ROS production via Duox, Jones et al (2013) did not observe this using the ROS detecting dye hydrocyanine-Cy3, or a ROS-sensitive transgene reporter, Glutatione S-transferase-GFP, in flies. Further, Jones et al (2013) found that genetically suppressing Nox in either Drosophila or mice decreased ROS production after Lactobacillus ingestion. Consistent with the important role of Nox, Duox appeared not to be required for ROS production after Lactobacillus ingestion. In addition, Jones et al (2013) found that Lactobacilli also promoted DNA replication—a metric of cell proliferation and epithelial renewal—in the fly''s intestine, and that this was also ROS- and Nox-dependent. Again, the same relationship was found in the mouse small intestine. Together, these results suggest a conserved mechanism by which Lactobacilli can stimulate Nox-dependent ROS production in intestinal enterocytes and thereby promote ISC proliferation and enhance gut epithelial renewal.In the fly midgut, uracil produced by pathogenic bacteria can stimulate Duox-dependent ROS production, which is thought to act as a microbicide (Lee et al, 2013), and can also promote ISC proliferation (Buchon et al, 2009). However, Duox-produced ROS may also damage the intestinal epithelium itself and thereby promote epithelial regeneration indirectly through stress responses. In this disease scenario, ROS appears to be sensed by the stress-activated Jun N-terminal Kinase (JNK; Figure 1A), which can induce pro-proliferative cytokines of the Leptin/IL-6 family (Unpaireds, Upd1–3) (Buchon et al, 2009; Jiang et al, 2009). These cytokines activate JAK/STAT signalling in the ISCs, promoting their growth and proliferation, and accelerating regenerative repair of the gut epithelium (Buchon et al, 2009; Jiang et al, 2009). It is also possible, however, that low-level ROS, or specific types of ROS (e.g., H₂O₂) might induce ISC proliferation directly by acting as a signal between enterocytes and ISCs. Since commensal Lactobacillus stimulates ROS production via Nox rather than Duox, this might be a case in which a non-damaging ROS signal promotes intestinal epithelial renewal without stress signalling or a microbicidal effect (Figure 1B). However, Jones et al (2013) stopped short of ruling out a role for oxidative damage, cell death or stress signalling in the intestinal epithelium following colonization by Lactobacilli, and so these parameters must be checked in future studies. Perhaps even the friendliest symbiotes cause a bit of ‘healthy'' damage to the gut lining, stimulating it to refresh and renew. Whether damage-dependent or not, the stimulation of Drosophila ISC proliferation by commensals and pathogens alike appears to involve the same cytokine (Upd3; Buchon et al, 2009), and so some of the differences between truly pathogenic and ‘friendly'' gut microbes might be ascribed more to matters of degree than qualitative distinctions. Future studies exploring exactly how different types of ROS signals stimulate JNK activity, gut cytokine expression and epithelial renewal should be able to sort this out, and perhaps help us learn how to better manage the ecosystems in our own bellies. From the lovely examples reported by Jones et al (2013), an experimental back-and-forth between the Drosophila and mouse intestine seems an informative way to go.Open in a separate windowFigure 1Metazoan intestinal epithelial responses to commensal and pathogenic bacteria. (A) High reactive oxygen species (ROS) levels generated by dual oxidase (Duox) in response to uracil secretion by pathogenic bacteria. (B) Low ROS levels generated by NADPH oxidase (Nox) in response to commensal bacteria. In addition to acting as a microbiocide, ROS in flies may stimulate JNK signaling and cytokine (Upd 1–3) expression in enterocytes, thereby stimulating ISC proliferation and epithelial turnover or regeneration. Whether this stimulation required damage to or loss of enterocytes has yet to be explored.     

Split decisions: oesophageal progenitor cell behaviour

Science advance online publication July192012; doi:10.1126/science.1218835The maintenance and regeneration of continually shedding epithelial tissues that make up the linings and barriers of our bodies requires rapid and continual input of proliferative progenitor cells for tissue homeostasis. The mechanisms by which epithelial progenitors cells maintain tissues remain controversial. In a recent Science paper, Doupé et al (2012) demonstrate that a population of equivalent progenitor cells support tissue homeostasis of the oesophagus without the need for slow cycling cells as described in other rapidly dividing epithelia.In tissues such as blood and skin in which differentiated cells constantly turnover, proliferative progenitor populations are required to continually produce lost differentiated cells. Several models have been proposed to explain mechanisms by which progenitor cells contribute to tissue maintenance (Figure 1). A hierarchical model has been suggested in which longer lived stem cells, which may also cycle slowly, produce highly proliferative cells with less self-renewal potential that differentiate into a restricted number of cells. Following proliferative cells in pulse-chase experiments and genetic lineage tracing has supported a hierarchical model in the blood, epidermis and intestine (Fuchs, 2009). Alternatively, an equivalency model has been proposed in which all proliferative progenitor cells are equally able to produce proliferative and differentiated progeny in a stochastic manner. Analysis of labelled clones has supported an equivalency model for progenitors in the interfollicular epidermis and intestine (Clayton et al, 2007; Doupé et al, 2010; Snippert et al, 2010).Open in a separate windowFigure 1Two types of models have been put forward to describe the pattern of progenitor behaviour within mammalian tissues. In the hierarchical model, a stem cell can produce proliferative progenitors with less self-renewal potential that differentiate into lineage-specific cells. Alternatively, an equivalency model has been proposed that assumes equal behaviour of progenitor cells to maintain tissue homeostasis.An elevated interest in understanding the dynamics of oesophageal epithelium has resulted, in part, from the rapid increase in the incidence of oesophageal adenocarcinoma (Devesa et al, 1998). The oesophagus is a stratified epithelium that lacks any appendages or glands, and thus consists of a basal layer of proliferative keratinocytes and several suprabasal layers of differentiated cells, which are continually shed. Previously, labelling of proliferative cells with DNA analogues has demonstrated that proliferation is restricted to the basal cells, which all proliferate in 5 days seemingly stochastically, supporting an equivalency model (Marques-Periera and Leblond, 1965). In contrast, studies using chimeric mice have suggested that proliferation of labelled progenitor cells occurs in a hierarchical manner (Thomas et al, 1988; Croagh et al, 2008).To address this controversy, a recent study in Science uses several genetic mouse models to define the contribution of proliferative basal cells to oesophageal homeostasis (Doupé et al, 2012). In one mouse model, the authors utilized a genetic pulse-chase system based on the tetracycline-regulated expression of the histone H2B-GFP (Tumbar et al, 2004). They find that the rapidly dividing epithelial cells of the oesophagus lose H2B-GFP expression after 4 weeks. These data suggest that either H2B-GFP is degraded (Waghmare et al, 2008) or oesophageal progenitor cells proliferate faster than their counterparts in skin epithelial appendages or blood lineages, which retain H2B-GFP after 4 weeks (Tumbar et al, 2004; Foudi et al, 2009).To analyse the properties of oesophageal progenitor cells in more detail, the authors label single cells using an inducible cre-lox genetic system and followed clones for a year. Similar to their results with this system in the tail and ear epidermis (Clayton et al, 2007; Doupé et al, 2010), the authors find that the size of the persistent clones is linear with time. Statistical analysis of the clone size data supports the ability of the cells to contribute to proliferative and non-proliferative (i.e., differentiated) progeny with equal probability. Thus, these data support a model in which all of the labelled cells are equivalent.In addition to homeostasis, the authors explore how proliferative progenitors contribute to alterations in tissue homeostasis. After inflicting wounds by biopsy, marked clones span both proliferative and non-proliferative zones of the healing oesophageal epithelium, suggesting that they maintain a progenitor fate with distinct phenotypes. With atRA treatment, the authors show that suprabasal cell formation increases, which is consistent with the known effect of atRA on the oesophagus (Lasnitzki, 1963). Statistical analysis reveals that the probability of forming basal and suprabasal cells was not altered with atRA administration. However, since proliferative cells exist in suprabasal layers during epithelial hyperplasia, additional analyses of cell state are required to determine if atRA maintains stochastic fate decisions of progenitor cells. Furthermore, the progenitor response to atRA treatment might be limited by niche space along the basement membrane like in intestinal crypt progenitor cells (Snippert et al, 2010).In summary, this study together with the authors'' previous work provides additional support for the existence of equivalent progenitor cells within stratified epithelium in several tissues. Additional studies revealing how epithelial progenitor cells behave when proliferation and differentiation are altered in the oesophagus could shed light on mechanisms for the pathogenesis of oesophageal tumours or diseases such as Barrett''s oesophagus.     

Enlightening the impact of immunogenic cell death in photodynamic cancer therapy

EMBO J 31 5, 1062–1079 (2012); published online January172012In this issue of The EMBO Journal, Garg et al (2012) delineate a signalling pathway that leads to calreticulin (CRT) exposure and ATP release by cancer cells that succumb to photodynamic therapy (PTD), thereby providing fresh insights into the molecular regulation of immunogenic cell death (ICD).The textbook notion that apoptosis would always take place unrecognized by the immune system has recently been invalidated (Zitvogel et al, 2010; Galluzzi et al, 2012). Thus, in specific circumstances (in particular in response to anthracyclines, oxaliplatin, and γ irradiation), cancer cells can enter a lethal stress pathway linked to the emission of a spatiotemporally defined combination of signals that is decoded by the immune system to activate tumour-specific immune responses (Zitvogel et al, 2010). These signals include the pre-apoptotic exposure of intracellular proteins such as the endoplasmic reticulum (ER) chaperon CRT and the heat-shock protein HSP90 at the cell surface, the pre-apoptotic secretion of ATP, and the post-apoptotic release of the nuclear protein HMGB1 (Zitvogel et al, 2010). Together, these processes (and perhaps others) constitute the molecular determinants of ICD.In this issue of The EMBO Journal, Garg et al (2012) add hypericin-based PTD (Hyp-PTD) to the list of bona fide ICD inducers and convincingly link Hyp-PTD-elicited ICD to the functional activation of the immune system. Moreover, Garg et al (2012) demonstrate that Hyp-PDT stimulates ICD via signalling pathways that overlap with—but are not identical to—those elicited by anthracyclines, which constitute the first ICD inducers to be characterized (Casares et al, 2005; Zappasodi et al, 2010; Fucikova et al, 2011).Intrigued by the fact that the ER stress response is required for anthracycline-induced ICD (Panaretakis et al, 2009), Garg et al (2012) decided to investigate the immunogenicity of Hyp-PDT (which selectively targets the ER). Hyp-PDT potently stimulated CRT exposure and ATP release in human bladder carcinoma T24 cells. As a result, T24 cells exposed to Hyp-PDT (but not untreated cells) were engulfed by Mf4/4 macrophages and human dendritic cells (DCs), the most important antigen-presenting cells in antitumour immunity. Similarly, murine colon carcinoma CT26 cells succumbing to Hyp-PDT (but not cells dying in response to the unspecific ER stressor tunicamycin) were preferentially phagocytosed by murine JAWSII DCs, and efficiently immunized syngenic BALB/c mice against a subsequent challenge with living cells of the same type. Of note, contrarily to T24 cells treated with lipopolysaccharide (LPS) or dying from accidental necrosis, T24 cells exposed to Hyp-PDT activated DCs while eliciting a peculiar functional profile, featuring high levels of NO production and absent secretion of immunosuppressive interleukin-10 (IL-10) (Garg et al, 2012). Moreover upon co-culture with Hyp-PDT-treated T24 cells, human DCs were found to secrete high levels of IL-1β, a cytokine that is required for the adequate polarization of interferon γ (IFNγ)-producing antineoplastic CD8⁺ T cells (Aymeric et al, 2010). Taken together, these data demonstrate that Hyp-PDT induces bona fide ICD, eliciting an antitumour immune response.By combining pharmacological and genetic approaches, Garg et al (2012) then investigated the molecular cascades that are required for Hyp-PDT-induced CRT exposure and ATP release. They found that CRT exposure triggered by Hyp-PDT requires reactive oxygen species (as demonstrated with the ¹O₂ quencher L-histidine), class I phosphoinositide-3-kinase (PI3K) activity (as shown with the chemical inhibitor wortmannin and the RNAi-mediated depletion of the catalytic PI3K subunit p110), the actin cytoskeleton (as proven with the actin inhibitor latrunculin B), the ER-to-Golgi anterograde transport (as shown using brefeldin A), the ER stress-associated kinase PERK, the pro-apoptotic molecules BAX and BAK as well as the CRT cell surface receptor CD91 (as demonstrated by their knockout or RNAi-mediated depletion). However, there were differences in the signalling pathways leading to CRT exposure in response to anthracyclines (Panaretakis et al, 2009) and Hyp-PDT (Garg et al, 2012). In contrast to the former, the latter was not accompanied by the exposure of the ER chaperon ERp57, and did not require eIF2α phosphorylation (as shown with non-phosphorylatable eIF2α mutants), caspase-8 activity (as shown with the pan-caspase blocker Z-VAD.fmk, upon overexpression of the viral caspase inhibitor CrmA and following the RNAi-mediated depletion of caspase-8), and increased cytosolic Ca²⁺ concentrations (as proven with cytosolic Ca²⁺ chelators and overexpression of the ER Ca²⁺ pump SERCA). Moreover, Hyp-PDT induced the translocation of CRT at the cell surface irrespective of retrograde transport (as demonstrated with the microtubular poison nocodazole) and lipid rafts (as demonstrated with the cholesterol-depleting agent methyl-β-cyclodextrine). Of note, ATP secretion in response to Hyp-PDT depended on the ER-to-Golgi anterograde transport, PI3K and PERK activity (presumably due to their role in the regulation of secretory pathways), but did not require BAX and BAK (Garg et al, 2012). Since PERK can stimulate autophagy in the context of ER stress (Kroemer et al, 2010), it is tempting to speculate that autophagy is involved in Hyp-PDT-elicited ATP secretion, as this appears to be to the case during anthracycline-induced ICD (Michaud et al, 2011).Altogether, the intriguing report by Garg et al (2012) demonstrates that the stress signalling pathways leading to ICD depend—at least in part—on the initiating stimulus (Figure 1). Speculatively, this points to the coexistence of a ‘core'' ICD signalling pathway (which would be common to several, if not all, ICD inducers) with ‘private'' molecular cascades (which would be activated in a stimulus-dependent fashion). Irrespective of these details, the work by Garg et al (2012) further underscores the importance of anticancer immune responses elicited by established and experimental therapies.Open in a separate windowFigure 1Molecular mechanisms of immunogenic cell death (ICD). At least three processes underlie the immunogenicity of cell death: the pre-apoptotic exposure of calreticulin (CRT) at the cell surface, the secretion of ATP, and the post-apoptotic release of HMGB1. ICD can be triggered by multiple stimuli, including photodynamic therapy, anthracycline-based chemotherapy, and some types of radiotherapy. The signalling pathways elicited by distinct ICD inducers overlap, but are not identical. In red are indicated molecules and processes that—according to current knowledge—may be required for CRT exposure and ATP secretion in response to most, if not all, ICD inducers. The molecular determinants of the immunogenic release of HMGB1 remain poorly understood. ER, endoplasmic reticulum; P-eIF2α, phosphorylated eIF2α; PI3K, class I phosphoinositide-3-kinase; ROS, reactive oxygen species.     

How two become one: HJURP dimerization drives CENP‐A assembly

EMBO J 32 15, 2113–2124 doi:10.1038/emboj.2013.142; published online June142013Curr Biol 23 9, 764–769 doi:10.1016/j.cub.2013.03.037; published online May062013Curr Biol 23 9, 770–774 doi:10.1016/j.cub.2013.03.042; published online May062013CENP-A containing nucleosomes epigenetically specify centromere position on chromosomes. Deposition of CENP-A into chromatin is mediated by HJURP, a specific CENP-A chaperone. Paradoxically, HJURP binding sterically prevents dimerization of CENP-A, which is critical to form functional centromeric nucleosomes. A recent publication in The EMBO Journal (Zasadzińska et al, 2013) demonstrates that HJURP itself dimerizes through a C-terminal repeat region, which is essential for centromeric assembly of nascent CENP-A.CENP-A containing nucleosomes have a well-established role in the epigenetic specification of centromere position. However, the composition of the CENP-A nucleosome has been the subject of intense investigation and debate (as has been extensively reviewed, e.g., in Black and Cleveland, 2011). X-ray crystallography data, biochemical interaction experiments and in vivo mutational analysis provide strong evidence that CENP-A nucleosomes are octameric (CENP-A/H4/H2A/H2B)₂, analogous to their histone H3-containing counterparts (Tachiwana et al, 2011; Bassett et al, 2012). Alternatively, based primarily on AFM data and nucleosome crosslinking assays, a tetrameric CENP-A/H4/H2A/H2B ‘hemisome'' has been proposed to be present at centromeres, at least during part of the cell cycle (Dalal et al, 2007; Bui et al, 2012). Whether both nucleosome types exist under specific conditions remains an unresolved question. However, recent studies by the Maddox and Black labs have reported single-molecule fluorescence measurements of CENP-A nucleosomes and high-resolution DNA protection assays of centromeric chromatin, respectively, both of which indicate that octamers are the predominant species of CENP-A in vivo (Hasson et al, 2013; Padeganeh et al, 2013).HJURP is the centromeric histone chaperone that is responsible for timely assembly of CENP-A nucleosomes. HJURP binds to soluble CENP-A and is recruited to centromeric chromatin in early G1 phase, concurrently with nascent CENP-A (Stellfox et al, 2013). Importantly, HJURP facilitates CENP-A nucleosome formation in vitro and its transient targeting to non-centromeric chromatin is sufficient to stably deposit CENP-A at these sites in vivo (Barnhart et al, 2011). Together, these observations identify HJURP as a bona fide centromeric CENP-A histone assembly factor.However, there is an apparent discrepancy between the role of HJURP in CENP-A assembly and the octameric nature of CENP-A nucleosomes. The crystal structure of the human prenucleosomal complex clearly shows that HJURP binds to CENP-A/H4 dimers in a manner that precludes CENP-A/H4 hetero-tetramerization (Hu et al, 2011). Interestingly, however, mutational analysis of CENP-A has shown that tetramerization is crucial for centromere assembly (Bassett et al, 2012). Thus, a mechanism must exist to allow for two trimeric HJURP/CENP-A/H4 complexes to coordinately assemble a tetrameric (CENP-A/H4)₂ particle.In this issue, a study by the Foltz lab sheds light on these paradoxical observations (Zasadzińska et al, 2013). Human HJURP contains two C-terminal repeat regions (HJURP C-terminal domains; HCTD). Expression of short fragments of HJURP containing either of these was sufficient to allow for centromere targeting. However, depletion of endogenous HJURP abolished centromere targeting of the C-terminally located HCTD2 fragment, without affecting the localization of the fragment containing HCTD1. These observations suggest that HCTD1 is required for centromere targeting, while HCTD2 allows for HJURP dimerization. Indeed, the authors go on to show that the latter fragment is both necessary and sufficient to form functional dimers of HJURP. RNAi replacement experiments show that HJURP lacking the HCTD2 dimerization domain is incapable of loading nascent CENP-A into centromeres. Importantly, Zasadzińska et al (2013) demonstrate that the defect in CENP-A loading can be directly attributed to a lack of HJURP dimerization. In an elegant experiment where the HCTD2 containing domain is replaced by an unrelated dimerization domain (that of bacterial LacI), CENP-A assembly is rescued to wild-type levels (Figure 1). This indicates that dimerization of HJURP is an essential step in centromeric chromatin assembly and provides a potential mechanism for the assembly of tetrameric (CENP-A/H4)₂ structures into chromatin as precursors to octameric nucleosomes.Open in a separate windowFigure 1Human HJURP contains separate protein domains that are responsible for CENP-A/H4 binding (blue), centromere targeting (brown) and dimerization (red). Full-length HJURP containing all these domains is capable of assembling CENP-A nucleosomes at centromeres (left). Zasadzińska et al (2013) now show that HJURP lacking the dimerization domain is still able to localize to centromeres, but is unable to assemble CENP-A nucleosomes (middle). However, replacement of the HJURP dimerization domain by an exogenous dimerization domain fully rescues the capability to form CENP-A nucleosomes at centromeres (right). These findings show that HJURP dimerization is an essential feature in the process of nucleosome formation, and explain how (CENP-A/H4)₂ tetramers can be formed by a chaperone that exclusively binds to CENP-A/H4 dimers.While the composition of the HJURP complex suggests a likely mechanism for the formation of octameric nucleosomes, this poses a new challenge to the field. Future studies will be needed to dissect how the shielded HJURP-bound state of CENP-A/H4 can transition to a tetramer on DNA. Interestingly, HJURP is not the only histone chaperone that exclusively binds to histone dimers. Crystal structures of trimeric complexes of both Asf1a/H3.1/H4 (English et al, 2006) as well as DAXX/H3.3/H4 (Elsässer et al, 2012) clearly show sterical incompatibility between chaperone binding and histone tetramerization. It follows that efficient chromatin assembly requires a mode for two histone chaperones to deposit histone dimers in a coordinated fashion, e.g., through dimerization as has been shown for Nap1 (McBryant and Peersen, 2004) and now for HJURP. However, dimerization does not appear to be a universal feature for histone chaperones, as a single CAF1 chaperone is able to bind two H3/H4 dimers as well as (H3/H4)₂ tetramers (Winkler et al, 2012). Thus, while deposition of H3.1/H4 at the replication fork may be driven by the high density of pre-assembly complexes, assembly of nucleosomes containing the replacement variant H3.3, H3.1 nucleosomes at DNA damage sites, and CENP-A at the centromere would require a more active form of coordination. Histone chaperone dimerization may therefore be a common feature in the pipeline to chromatin formation.In summary, Zasadzińska et al (2013) propose a solution to a paradox in the assembly pathway of CENP-A. They show that while each HJURP molecule can exclusively bind a single CENP-A/H4 dimer, HJURP itself dimerizes, ultimately allowing for the formation of tetrameric (CENP-A/H4)₂ structures in chromatin. Interestingly, exclusive dimer binding has been observed for a number of histone chaperones, suggesting that chaperone dimerization may be a more general process in the nucleosome assembly pathway.     

Cilia born out of shock and stress

EMBO J (2013) 32 23, 3029–3040 10.1038/emboj.2013.223; published online October112013Primary cilia are cell surface sensory organelles, whose dysfunction underlies various human genetic diseases collectively termed ciliopathies. A new study in The EMBO Journal by Villumsen et al now reveals how stress–response pathways converge to stimulate ciliogenesis by modulating protein composition of centriolar satellites. Better understanding of these mechanisms should bring us closer to identifying the cellular defects that underlie ciliopathies caused by mutations in centriolar satellite proteins.Centrioles are barrel-shaped structures with two distinct identities. In proliferating cells centrioles provide structural support for the centrosome, a key microtubule-organizing centre, whereas in quiescent cells centrioles are converted into basal bodies and promote the assembly of primary cilia. In centrosomes, centrioles are embedded in pericentriolar material (PCM), a dynamic structure responsible for microtubule nucleation. PCM proteins exhibit cell cycle-dependent localisation, achieved at least in part by the regulation of their transport. Centriolar satellites, dense fibrous granules frequently clustered around the interphase centrosome, have been implicated in microtubule-dependent protein transport to centrosomes (Kubo et al, 1999). In particular, PCM-1, the core constituent of centriolar satellites, is required for centrosomal accumulation of several PCM components (Dammermann and Merdes, 2002). Although the proteomic composition of satellites is still elusive, the growing list of satellite proteins includes CEP131/AZI1 (Staples et al, 2012), CEP290 (Stowe et al, 2012), Bardet-Biedl syndrome protein 4 (BBS4) and Oral facial digital syndrome protein (OFD1; Lopes et al, 2011). Mutations in OFD1, CEP290 and BBS4 cause ciliopathies (Kim et al, 2008), underscoring a functional link between satellites and ciliogenesis. So far, two roles have been proposed for satellites in cilia formation: First, in cycling cells they may serve to sequester essential ciliary proteins (Stowe et al, 2012). Second, upon initiation of the ciliogenesis programme, centriolar satellite components seem to promote the recruitment of specific ciliary proteins to basal bodies (Ferrante et al, 2006; Lopes et al, 2011; Stowe et al, 2012).In a new study in The EMBO Journal, Villumsen et al (2013) now describe how stress–response pathways conspire to control ciliogenesis. The authors observed that specific environmental stresses, such as ultraviolet light radiation (UV) or heat shock, but not ionizing radiation (IR), trigger rapid displacement of PCM-1, AZI1 and CEP290 from centriolar satellites. However, OFD1 remained associated with satellites, indicating that centriolar satellites persist despite UV-induced removal of PCM-1. This might come as some surprise, since PCM-1 depletion by RNA interference (RNAi) is thought to disrupt satellite integrity (Kim et al, 2008; Lopes et al, 2011); however, satellite loss upon PCM-1 RNAi may be a consequence of prolonged depletion of PCM-1, while acute PCM-1 displacement by stress might only ‘remodel'' centriolar satellites. It is also possible that not all satellites are created equal, and they do vary in protein composition (Kim et al, 2008; Staples et al, 2012). If so, UV-induced PCM-1 removal may disrupt some, but not all satellites.A good candidate regulator of centriolar satellite remodelling was the stress-activated MAP kinase p38, and indeed, Villumsen et al (2013) found p38 MAPK activity to be stimulated by both UV and heat shock but not IR in U2OS cells, mirroring those very stress pathways that also cause displacement of AZI1 and PCM-1 from satellites. Furthermore, p38 MAPK was essential for UV-induced dispersal of PCM-1 and AZI1. The authors then tested the hypothesis that stress-induced centriolar satellite remodelling could involve changes in the interactome of AZI1, and—consistent with an earlier proteomics study (Akimov et al, 2011)—identified PCM-1, CEP290 and the mindbomb E3 ubiquitin protein ligase 1 (MIB1) as the main AZI1 binding partners. GFP-MIB1 localized to centriolar satellites and mono-ubiquitylated AZI1, PCM-1 and CEP290 in cycling cells. In response to UV, both ubiquitylation of these proteins and MIB1 activity were reduced; notably, UV-induced MIB1 inactivation was independent of p38 MAPK activity, indicating that these two enzymes may act via distinct pathways (Figure 1A).Open in a separate windowFigure 1(A) Regulation of centriolar satellite remodelling. (B) Schematic summary of how centriolar satellite remodelling might facilitate ciliogenesis. See text for details.What could be the purpose of MIB1-dependent ubiquitylation of these satellite proteins? It certainly does not seem to regulate subcellular targeting, as in MIB1-depleted cells, AZI1 and PCM-1 both localised normally to centriolar satellites and could still be displaced by UV. Instead, ubiquitylation seems to suppress the interaction between AZI1 and PCM-1, consistent with the observation that UV, a condition that also reduces their ubiquitylation, enhances the binding of AZI1 to PCM-1.PCM-1, CEP290 and AZI1 all participate in ciliogenesis (Kim et al, 2008; Wilkinson et al, 2009; Stowe et al, 2012), raising the possibility that MIB1 might also affect this process. Indeed, serum starvation, which is known to promote cilia formation, attenuated MIB1 activity. Furthermore, MIB1 overexpression reduced the ciliogenesis observed in serum-starved cells, while MIB1 depletion in proliferating cells triggered a marked increase in the proportion of cells that formed cilia; this seems to reflect a direct effect of MIB1 on ciliogenesis, since neither MIB1 depletion nor overexpression altered cell cycle progression. Taken together, downregulation of MIB1 enzymatic activity appears to be a pre-requisite for efficient ciliogenesis, regardless of whether it is triggered by physiological ciliogenesis-promoting signals or by environmental stresses, making MIB1 a novel negative regulator of cilia formation.The recent discovery of ciliopathy-associated mutations in constituents of the DNA damage response signalling pathway pointed to a connection between DNA damage and ciliogenesis (Chaki et al, 2012). With the new link between UV and centriolar satellites, the authors next asked if UV radiation might affect ciliogenesis. Remarkably, UV and heat shock both triggered cilia assembly in RPE-1 cells in a p38 MAPK-dependent manner. MIB1 depletion further enhanced ciliogenesis after UV radiation, again implying an additive effect of p38 MAPK signalling and MIB1 suppression (Figure 1A).While finer details on the precise role of centriolar satellite components in cilia formation are still lacking, a more coherent picture is finally starting to emerge. In cycling cells, ubiquitination by MIB1 could serve to limit the interaction between AZI1 and PCM-1 on centriolar satellites (Figure 1B). Under these conditions PCM-1 may bind and sequester CEP290, an essential ciliogenic protein, thereby precluding untimely cilia formation (Stowe et al, 2012). Both during normal and stress-induced ciliogenesis programs, remodelling of centriolar satellites creates a permissive environment for cilia formation, and a key step in this process is downregulation of MIB1 activity. While it remains to be established how the latter is achieved, it is clear that MIB1 inactivation causes loss of ubiquitylation and increased binding between AZI1 and PCM-1. Preferential interaction of PCM-1 with AZI1 could in turn facilitate release of CEP290 from centriolar satellites and its subsequent accumulation at the centrosome. Once CEP290 reaches the optimum concentration at the centriole/basal body, it could serve to tether AZI1–PCM-1 complexes. PCM-1 could then concentrate Rab8 GTPase near centrosomes, allowing CEP290 to recruit Rab8 into the cilium, where it acts to extend the ciliary membrane (Kim et al, 2008).Collectively, the findings reported here provide strong experimental support to the notion that centriolar satellites are negative regulators of ciliogenesis in proliferating cells. Their role is central to limit untimely formation of cilia in cells. Environmental strains elicit stress–response pathways that converge to relieve the ciliogenesis block imposed by satellites. It is tempting to speculate that stress-induced cilia might serve as signalling platforms and contribute to checkpoint activation or perhaps initiation of repair mechanisms, but more work is needed to establish the true purpose of ciliogenesis in this context. It is of considerable interest that a recent study reports that autophagy, another stress-induced pathway, selectively removes OFD1 from satellites to promote ciliogenesis (Tang et al, 2013). Therefore stress-mediated centriolar satellite remodelling seems to be an evolving theme in the control of ciliogenesis.     

Telomere dysfunction puts the brakes on oncogene-induced cancers

EMBO J 31 13, 2839–2851 (2012); published online May082012Senescence represents a major tumour suppressor checkpoint activated by telomere dysfunction or cellular stress factors such as oncogene activation. In this issue of The EMBO Journal, Suram et al (2012) reveal a surprising interconnection between oncogene activation and telomere dysfunction induced senescence. The study supports an alternative model of tumour suppression, indicating that oncogene-induced accumulation of telomeric DNA damage contributes to the induction of senescence in telomerase-negative tumours.Telomere shortening limits the proliferative capacity of primary human cells after 50–70 cell divisions by induction of replicative senescence activated by critically short, dysfunctional telomeres. Different mechanisms were thought to initiate senescence in response to oncogene activation, which occurs abruptly within a few cell doublings (Serrano et al, 1997). Oncogene-induced senescence (OIS) involves an activation of DNA damage signals at stalled replication forks induced by DNA replication stress (Bartkova et al, 2006; Di Micco et al, 2006). Replication fork stalling in response to oncogene activation preferentially affects common fragile sites of the DNA (Tsantoulis et al, 2008). The ends of eukaryotic chromosomes—the telomeres–represent common fragile sites that are sensitive to replication fork stalling (Sfeir et al, 2009). These data made it tempting to speculate whether replication fork stalling at telomeres was causatively involved in OIS. Studies on replicative senescence in human fibroblast also supported this possibility showing that mitogenic signals amplify DNA damage responses in senescent cells (Satyanarayana et al, 2004).Multiple studies revealed experimental evidences that senescence suppresses tumour progression in mouse models and early human tumours (for review see Collado and Serrano, 2010). The relative contribution of OIS and telomere dysfunction induced senescence (TDIS) to tumour suppression and possible interconnections between the two pathways at the level of checkpoint induction were not investigated in previous studies. In this issue of The EMBO Journal, Suram et al (2012) describe the presence of TDIS in human precursor lesions but not in the corresponding malignant tumours. Mechanistically, the study shows that oncogenic signals cause replication fork stalling, resulting in telomeric DNA damage accumulation and activation of DNA damage checkpoints reminiscent to TDIS. Telomerase expression does not rescue replication fork stalling but prevents the accumulation of DNA damage at telomeres allowing a bypass of OIS.The study has several important implications for molecular pathways and therapeutic approaches in cancer that need to be further explored (Figure 1):Open in a separate windowFigure 1Traditional and new models of senescence in tumour suppression. (A) Traditional model of replicative senescence: Telomerase-negative tumour cell clones experience telomere shortening as a consequence of cell division. After a lack period depending on the initial telomere length, tumour cells accumulate telomere dysfunction and activation of senescence impairs tumour growth. Telomerase activation represents a late event allowing tumour progression. (B) New model of oncogene induced, telomere-dependent senescence: Oncogene activation leads to abrupt accumulation of DNA damage at telomeres resulting in senescence and tumour suppression. Telomerase-positive stem cells could be resistant to OIS and may be selected as the cell type of origin of tumour development.(i) Telomere length independent roles of telomeres in tumour suppressionThe classical model of telomere-dependent tumour suppression indicates that proliferation-dependent telomere shortening leads to telomere dysfunction, activation of DNA damage checkpoints, and induction of senescence suppressing the growth of telomerase-negative tumour clones. Studies on mouse models supported this concept showing that telomere shortening impairs the progression of initiated tumours in a telomere length-dependent manner (Feldser and Greider, 2007). The new data from Suram et al (2012) indicate that oncogene-induced replication fork stalling activates a telomere-dependent senescence checkpoint, which is independent of telomere length. The study shows that replication forks stall in response to oncogene activation throughout the genome. However, stalled replication forks are resolved in non-telomeric regions, whereas fork stalling inside telomeres leads to un-repairable DNA damage in telomerase-negative cells. These findings are in line with recent publication showing accumulation of un-repairable DNA damage in telomeric DNA in response to aging and stress-induced DNA damage (Fumagalli et al, 2012).(ii) Telomere length independent roles of telomerase in tumour progressionFollowing the classical model telomeres in tumour suppression (Figure 1A), telomerase re-activation is required for tumour progression by limiting telomere dysfunction and the induction of DNA damage checkpoints in response to telomere shortening. The new data from Suram et al (2012) indicate that telomerase has an additional telomere length independent role in tumour progression. The study shows that catalytically active telomerase prevents the activation of DNA damage signals originating from stalled replication forks inside telomeres in response to oncogene activation (Figure 1B). The exact mechanisms of telomerase-dependent healing of stalled replication forks at telomeres remain to be elucidated. It is also unclear whether telomerase activity can prevent any type of DNA damage at telomeres as an over-expression of TERT could not suppress irradiation-induced cellular senescence or the persistence of telomeric DDR following irradiation, H₂O₂, or chemotherapy induced DNA damage (Hewitt et al, 2012).The data could provide a plausible explanation for the increased tumorigenesis in telomerase transgenic mice—a finding which is difficult to explain by telomere length dependent effects of telomerase given the long telomere reserves in mouse tissues (Gonzalez-Suarez et al, 2001). According to the findings of Suram et al (2012), anti-telomerase therapies could have immediate anti-cancer effects in tumours depending on telomerase-mediated healing of stalled replication forks at telomeres. Specific markers for this dependency could be of clinical value. In addition, the data support the concept that somatic stem cells could represent the cell type of origin of cancers. In contrast to differentiated somatic cells, tissues stem cells are often telomerase-positive, indicating that stem cells might be less sensitive to OIS.     

Senescent cells spread the word: non‐cell autonomous propagation of cellular senescence

Nat Cell Biol advance online publication, June162013; doi:10.1038/ncb2784Senescence has long been considered a cell autonomous arrest programme restricting the propagation of damaged cells in tissues. Now there is accumulating evidence that senescent cells can communicate with their environment. In a recent report by Gil and colleagues (Acosta et al, 2013), it now seems senescence can be transmitted in a paracrine fashion in several in vitro and in vivo contexts. In addition to broadening our understanding of the biology of senescence, these new findings may have interesting implications for tissue homeostasis and future cancer therapies.Senescence is a form of stress-induced cell cycle arrest that restricts the proliferative capacity of damaged and/or potentially harmful cells (Rodier and Campisi, 2011), thereby promoting tissue homeostasis and tumour suppression. While the senescence-associated cell cycle arrest involves the well-studied Rb and p53 pathways, senescent cells also possess the less understood ability to secrete growth factors, cytokines and chemokines into their environment. This process, collectively known as the senescence-associated secretory phenotype (SASP; Rodier and Campisi, 2011), was originally used to mark senescent cells, but is now known to enforce cell cycle arrest, modify the microenvironment and trigger immune surveillance of senescent cells (Xue et al, 2007; Krizhanovsky et al, 2008; Rodier and Campisi, 2011).Adding to our understanding of this process, a recent report by Gil and colleagues showed that the SASP can also mediate paracrine transmission of cellular senescence (Acosta et al, 2013). By co-culturing cells undergoing oncogene-induced senescence (OIS) with normal cells, the authors showed that the senescence phenotype could be transmitted to surrounding cells via the soluble SASP proteins. Coupling quantitative proteomics with small-molecule inhibitor screens, they identified key players mediating the paracrine transmission of senescence, including TGFB, VEGF and CCL2 pathways. A search for upstream regulators of SASP pointed at IL-1 signalling and the inflammasome, molecules that operate cell autonomously to control SASP production and non-cell autonomously to spread the senescent phenotype via the SASP (Figure 1). Complementing the in vitro senescence findings, experiments using mouse and human models of OIS demonstrated evidence for paracrine senescence transmission in vivo.Open in a separate windowFigure 1Cell autonomous and non-cell autonomous effects of cellular senescence. Stress stimuli such as activation of oncogenes and DNA damage can trigger normal mitotic cells to go into senescence. This involves inflammosome-mediated activation of IL-1 signalling, which initiates the SASP response. The SASP acts cell autonomously (autocrine) to reinforce the senescent phenotype via cytokines such as IL-6. The SASP also acts non-cell autonomously (paracrine) to influence the cells in the surrounding environment. For example, SASP components such as VEGF, TGFB and CCL2 can trigger bystander senescence on neighbouring cells. Paradoxically, the SASP can also exert pro-mitogenic stimulation of neighbouring cells via cytokines like IL-6, which appear to play dual roles depending on the context. Furthermore, the SASP can act on the immune system via pro-inflammatory cytokines, leading to immune cell recruitment and subsequent targeting and clearance of senescent cells. Alternatively, the SASP can trigger upregulation of p16 and p21 levels on neighbouring immune cells, the functional consequences of which are not yet so clear.The ability of senescent cells to propagate their phenotype is consistent with previous studies identifying IGFBP7 as a paracrine senescence regulator (Wajapeyee et al, 2008) and provides important insights into senescence biology. It is conceivable to think that the induction of paracrine cell cycle arrest could expand the senescence footprint of the pre-neoplastic lesion to the surrounding epithelium. This could potentially serve to amplify the tissue damage signal, recruit more immune cells and ensure more efficient clearance of damaged cells. In parallel, the induction of paracrine senescence in other cell types within the tissue, for example tumour-associated fibroblasts, could repress their reported paracrine tumour-promoting effects (Krtolica et al, 2001).Despite the biological implications, a number of questions remain. Why, for instance, is paracrine senescence triggered in some cells surrounding pre-neoplastic lesions but not in others? Similarly, what is the functional significance of paracrine senescence induction in the surrounding immune cells? Intriguingly, recent evidence implies that p16 can also be induced in tumour-infiltrating immune cells (Burd et al, 2013). It will be important to determine whether the paracrine p16 induction in immune cells leads to the same consequences as in non-immune cells and whether the induction of a potential arrest programme compromises the ability of the immune cell to clear senescent cells.Beyond the biological implications, the key regulators of paracrine senescence have potential to be manipulated therapeutically. It is commonly believed, for instance, that senescent cells accumulate in aging tissues and disrupt tissue architecture and function (Rodier and Campisi, 2011). In this context, antagonists of paracrine senescence might limit the spread of senescence and prove beneficial for some age-associated disorders. In the context of cancer, both chemotherapeutic drugs and radiation are known to induce senescence in tumour cells (Schmitt, 2007; Prise and O''Sullivan, 2009). The use of agents agonizing paracrine senescence as adjunctive therapy could potentially increase the effectiveness of chemo- and radiotherapy by triggering a bystander response.Nonetheless, it is critical to keep in mind that the SASP may not always relay an arrest-inducing message onto the surrounding cells. Indeed, the SASP component IL-6 has been shown to elicit a pro-mitogenic response in a paracrine fashion (Kuilman et al, 2008). Similarly, the SASP has been shown to be pro- and anti-tumorigenic depending on the microenvironment (Krtolica et al, 2001; Xue et al, 2007; Lujambio et al, 2013; Figure 1). Collectively, these findings suggest that the ultimate outcome of senescence within a tissue is highly dependent on the context. But what then determines this context? One decisive factor could be whether or not the senescence signal engages in sufficient modulation of the immune system to provoke clearance. In cases where the senescent cells in a tissue are not cleared, the pro-mitogenic arm of the SASP signal could persist long enough to have an overall pro-tumorigenic effect. It will thus be important to understand all the flavours of SASP to modulate it safely for therapeutic purposes.     

A Human Embryonic Kidney 293T Cell Line Mutated at the Golgi ��-Mannosidase II Locus

Disruption of Golgi α-mannosidase II activity can result in type II congenital dyserythropoietic anemia and induce lupus-like autoimmunity in mice. Here, we isolated a mutant human embryonic kidney (HEK) 293T cell line called Lec36, which displays sensitivity to ricin that lies between the parental HEK 293T cells, in which the secreted and membrane-expressed proteins are dominated by complex-type glycosylation, and 293S Lec1 cells, which produce only oligomannose-type N-linked glycans. Stem cell marker 19A was transiently expressed in the HEK 293T Lec36 cells and in parental HEK 293T cells with and without the potent Golgi α-mannosidase II inhibitor, swainsonine. Negative ion nano-electrospray ionization mass spectra of the 19A N-linked glycans from HEK 293T Lec36 and swainsonine-treated HEK 293T cells were qualitatively indistinguishable and, as shown by collision-induced dissociation spectra, were dominated by hybrid-type glycosylation. Nucleotide sequencing revealed mutations in each allele of MAN2A1, the gene encoding Golgi α-mannosidase II: a point mutation that mapped to the active site was found in one allele, and an in-frame deletion of 12 nucleotides was found in the other allele. Expression of the wild type but not the mutant MAN2A1 alleles in Lec36 cells restored processing of the 19A reporter glycoprotein to complex-type glycosylation. The Lec36 cell line will be useful for expressing therapeutic glycoproteins with hybrid-type glycans and as a sensitive host for detecting mutations in human MAN2A1 causing type II congenital dyserythropoietic anemia.Mammalian N-linked glycosylation is characterized by significant chemical heterogeneity generated by an array of competing glycosidases and glycosyltransferases (1). The structural analysis of recombinant glycoproteins, such as human erythropoietin (2, 3), has illustrated the capacity of mammalian expression systems for generating diverse N-linked glycans.Heterogeneity develops during egress of a glycoprotein through the secretory system (1). N-linked glycosylation is initiated in the rough endoplasmic reticulum (ER)⁴ by the co-translational transfer of Glc₃Man₉GlcNAc₂ to the asparagine residues of the glycosylation sequon. In the absence of protein misfolding, hydrolysis by ER α-mannosidase I plus α-glucosidase I and II results in the transfer of glycoproteins dominated by the D1,D3 isomer of Man₈GlcNAc₂ glycans to the Golgi apparatus (4). Further processing by Golgi α-mannosidases IA–C generates Man₅GlcNAc₂ (5–7), the principle substrate for UDP-N-acetyl-d-glucosamine:α-3-d-mannoside β1,2-N-acetylglucosaminyltransferase I (GnT I). The action of this enzyme yields classic hybrid-type glycans with mannosyl 6-antennae and processed 3-antennae (1). In the absence of the GnT III-mediated addition of bisecting GlcNAc, the two terminal α-mannose residues of the 6-antenna of hybrid-type glycans are cleaved by Golgi α-mannosidase II, forming mono-antennary complex-type glycans. These may then be processed by N-acetylglucosaminyltransferases, generating multiantennary complex-type glycans of enormous potential heterogeneity following the sequential transfer of monosaccharides such as galactose, N-acetylgalactosamine, fucose, and N-acetylneuraminic acid (8).The importance of this carbohydrate diversity in metazoan biology is illustrated by the disease phenotypes that manifest when the biosynthesis of particular glycoforms is disrupted. In humans, about 12 congenital disorders of glycosylation (CDG) have been identified with defects in the biosynthesis of N-linked glycans (9). One disorder characterized by changes in glycosylation is congenital dyserythropoietic anemia type II (hereditary erythroblastic multinuclearity with a positive acidified serum lysis test (HEMPAS)) (10, 11). HEMPAS is a heterogenous autosomal recessive disorder that renders erythrocytes prone to lysis. Although the precise molecular basis of HEMPAS remains to be determined, it is characterized by either a reduction in β1→4-galactosyltransferase, GnT II, or, in some patients, Golgi α-mannosidase II activity (11, 12). Interestingly, the increase in cell surface terminal mannose in mice deficient in Golgi α-mannosidase II leads to autoimmunity through chronic activation of the innate immune system (13, 14).Lectin-resistant (Lec) cell lines harboring loss- or gain-of-function mutations affecting the biosynthesis of N-glycans have emerged as powerful tools for the investigation of these disorders (15). For example, genetic complementation using Lec2, containing a mutation in the cytosine monophosphate sialic acid transporter, was used to identify a novel CDG, type IIf (16). Lectin-resistant cell lines can also be used as hosts to study naturally occurring mutations, as in the case of CHO Lec23 cells used to screen α-glucosidase I mutations in CDG, type IIb (17). Other applications of lectin-resistant cell lines include the expression of specific glycoforms of therapeutic glycoproteins. Manipulating the structure of their carbohydrate moieties modulates the pharmacological properties of glycoproteins by altering their bioactivity, serum half-life, and/or tissue tropism (18). For example, β-glucocerebrosidase expressed in CHO Lec1 cells (deficient in GnT I activity) exhibits mannosylation and improved macrophage uptake for the treatment of Gaucher disease (19). Lectin-resistant CHO cell lines have also been used to improve the crystallizability of glycoproteins for structural determination by x-ray crystallography (20–24).The expression of therapeutic glycoproteins as one or more defined “glycoforms” is essential for their optimization and may even be necessary to obtain regulatory approval (25). To this end, eukaryotic expression systems have been developed that allow glycosylation to be controlled. Recently, Pichia pastoris-based strains with human glycosyltransferases have been established, allowing the expression of glycoforms with oligomannose-, hybrid-, and some complex-type glycans (26, 27) and even sialylated complex-type structures (28). However, mammalian expression remains the dominant technology in industrial settings, presumably because of its reliability for the expression of human secreted glycoproteins.Although the majority of lectin-resistant cell lines have been generated using CHO cells, no Golgi α-mannosidase II-deficient CHO cell line has been generated thus far (15). Furthermore, only one human lectin-resistant cell line, i.e. GnT I-deficient (Lec1) HEK 293S cells (29), has been produced. Hybrid-type glycosylation has been reported to accumulate in ricin-resistant baby hamster kidney cells (30, 31); however, these cells contain a reduced but detectable level of cell-surface complex-type glycans, consistent with an incomplete ablation of Golgi α-mannosidase II activity (31). Moreover, the hybrids from one of these lines contain a trimannosyl rather than pentamannosyl core and appear to be heavily influenced by GnT II deficiency, resulting in the formation of what are now commonly called monoantennary complex-type glycans (32–34). We now describe the isolation of an HEK 293T cell line mutated at the MAN2A1 locus and deficient in Golgi α-mannosidase II activity via selection with ricin.