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each other, in contrast to the promoter of the gene encoding IFN-λ1, which is evolutionarily more divergent from those of the other two cytokines in the type III interferon family.
Collectively, results from the present study by Odendall et al.3 have important implica-tions for virus-host interactions and eluci-date the mechanism of the differences in the induction of interferon from distinct cellular organelles. They have solved an important piece of the IFN-λ puzzle and have shed light on the broader question of why type III inter-ferons exist and how they may complement the canonical type I interferons. Together with the published finding that signaling by IFN-λ is quantitatively and dynamically distinct from that of IFN-α and IFN-β16, there is now greater understanding about this functional dichotomy, which calls for further exploration of the specific role of IFN-λ in the immune system. This important study also provides insight into how cells not of the immune sys-tem are able to spatially coordinate the ini-tiation of interferon signaling to achieve an optimal antiviral response.
COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.
1. Sommereyns, C. et al. PLoS Pathog. 4, e1000017 (2008).2. Ding, S. et al. PLoS Biol. 12, e1001758 (2014).3. Odendall, C.D. et al. Nat. Immunol. 15, 717–726 (2014). 4. Dixit, e. et al. Cell 141, 668–681 (2010).5. Liu, S.Y. et al. Immunity 38, 92–105 (2013).6. Ablasser, A. et al. Nature 503, 530–534 (2013).7. Delorme-Axford, e. et al. Proc. Natl. Acad. Sci. USA
110, 12048–12053 (2013).8. Li, J. et al. Nat. Immunol. 14, 793–803 (2013).9. Ueki, I.F. et al. J. Exp. Med. 210, 1929–1936 (2013).10. Cai, X. et al. Cell 156, 1207–1222 (2014).11. Gao, S.J. et al. Oncogene 15, 1979–1985 (1997).12. Horner, S.M. et al. Proc. Natl. Acad. Sci. USA 108,
14590–14595 (2011).13. Stone, A.e. et al. PLoS Pathog. 9, e1003316 (2013).14. Marukian, S. et al. Hepatology 54, 1913–1923 (2011).15. Thomas, e. et al. Gastroenterology 142, 978–988 (2012).16. Bolen, C.R. et al. Hepatology 59, 1262–1272 (2014).
with biochemical and imaging tools. Finally, the question remains open of whether the induction of IFN-λ in cells of the immune system, such as plasmacytoid dendritic cells, in which Toll-like receptors have a critical role in sensing incoming pathogens, follows a dif-ferent pattern than that observed for the cells that are not part of the immune system, such as those examined in this study3.
On the pathogen side, it is possible to speculate that viruses have evolved strategies to target the RIG-I–MAVS-pex–IRF1–IFN-λ axis to suppress initiation of the type III interferon response. For example, Kaposi’s sarcoma–associated herpesvirus encodes ORF K9, a viral mimetic of host IRF1 that inhibits IFN-β expression11, which may antagonize the induction of type I interferons and type III interferons differently in certain cell types. Another example is the NS3/4A protease from hepatitis C virus, known to efficiently cleave MAVS localized to mitochondria-associated endoplasmic reticulum membranes and to block events downstream of RIG-I signaling12. Whether NS3/4A also cleaves MAVS-pex and thus attenuates the peroxisome-derived IFN-λ response is not known. Interestingly, although the 3′ untranslated region–encoded RNA pathogen-associated molecular pattern of hepatitis C virus can activate the expres-sion of both IFN-α and IFN-λ in plasmacytoid dendritic cells13, infection of hepatocytes with hepatitis C virus seems to readily induce IFN-λ expression but not IFN-α expression14,15, which suggests the possibility of virus- and cell type–specific activation of IFN-λ. Finally, it remains unclear whether the same regulatory mechanism that controls IFN-λ1 also applies to IFN-λ2 and IFN-λ3, especially given that the promoter regions of the genes encoding IFN-λ2 and IFN-λ3 are almost identical to
knocked down by small interfering RNA with MAVS localized to specific cellular compart-ments, they demonstrate that MAVS present in mitochondria acts upstream of both type I interferons and type III interferons, whereas signaling via MAVS-pex activates exclusively IFN-λ and is sufficient to suppress the repli-cation of vesicular stomatitis virus. In addi-tion, they find that during the differentiation of polarized intestinal epithelial cells, there is a functional link between peroxisome bio-genesis and the ability of these cells to express IFN-λ but not IFN-β in response to viral infection. Furthermore, the chemical disrup-tion of mitochondria shifts cells from a type I interferon–dominant expression paradigm to a solely IFN-λ pattern. Finally, in cells from patients with Zellweger syndrome, which lack functional peroxisomes to various degrees, the authors make the interesting finding that a misformed mitochondria-peroxisome ‘hybrid’ organelle leads to enhanced signaling via RIG-I–MAVS, although they do not report whether these cells are defective in IFN-λ production.
While the novel discovery of selective induc-tion of IFN-λ from peroxisomes is mechanis-tically intriguing, several new questions arise from this work. Despite the evidence that IFN-λ fits the profile of the secreted antivi-ral molecule downstream of MAVS-pex, the possibility cannot be excluded that another undefined cytokine or growth factor may also activate a similar Jak-STAT pathway and thereby confer the protective effect. In addi-tion, it has been reported that the formation of higher order structures resembling those of prions and inflammasomes is necessary for intact MAVS signaling10. Whether the oligomerization of MAVS at peroxisomes is required for the induction of type III interfer-ons is unknown and calls for further analysis
enhancing the understanding of asthmaGolnaz Vahedi, Arianne C Richard & John J O’Shea
The chromatin signature of genomic enhancers in CD4+ T cells distinguishes asthmatic patients from healthy subjects.
Golnaz vahedi, Arianne C. Richard and John J.
O’Shea are with the Molecular immunology and
inflammation Branch, national institute of Arthritis
and Musculoskeletal and Skin Diseases, national
institutes of Health, Bethesda, Maryland, USA.
e-mail: [email protected]
Allergic diseases, particularly asthma, pose a growing medical challenge worldwide.
CD4+ T cells, which are critical for host
defense against infection, can also instigate a detrimental inflammatory response to harm-less environmental substances such as pollens. This misdirected immune response may then lead to allergic disease. A strong genetic basis has been established for asthma, but despite advances in genomics identifying associated loci, a substantial proportion of the causal-ity remains unexplained. In addition to the effect of genetics, the environment also greatly
affects disease, with asthma and allergies serv-ing as profound examples of this influence. In this issue, Seumois et al. establish the fea-sibility of measuring modifications of DNA-packaging proteins in populations of primary human CD4+ T cells and using this informa-tion to better understand the foundations of asthma1. The authors focus on a marker of genomic enhancers, histone 3 dimethylated at Lys4 (H3K4me2). This study therefore
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other exposures, including smoking and pol-lution, as well as hygiene and antibiotic use, seem to be associated with the development and progression of this disease4.
The link between genetic and environ-mental influences on asthma can be captured by epigenetic profiling. Epigenetics refers to the study of the packaging of DNA into a structure called ‘chromatin’ and the effects of variable chromatin organization on activity of genetic regulatory elements. In addition to a set of inherited epigenetic marks, there are probably nonheritable epigenetic marks that are more dynamic and change in response to environmental stimuli4,5.
During the past few years, advances in the epigenomic annotation of functional non-coding elements, commonly referred to as ‘enhancers’, have begun to shed light on gene regulation at the genetic and epigenetic levels. Enhancers are associated with distinct chro-matin structures that display unique histone modifications. Various developments in genomic technology, such as chromatin immu-noprecipitation followed by high-throughput sequencing (ChIP-seq), have enabled the con-struction of genome-wide maps of enhancers through the use of global patterns of such his-tone modifications. Comparisons of enhancer maps in a variety of cell types have revealed
represents an important step in delineating how genetic risk and the effect of the environ-ment on the T cell epigenome come together to affect disease.
More than a decade ago, geneticists envi-sioned principles for genetic mapping using populations rather than families to study com-mon diseases such as asthma. This approach, coined ‘genome-wide association study’, tests a comprehensive catalog of common genetic variants in patients and control subjects in a population to identify variants associated with a particular disease. Although genome-wide association studies have had key roles in dis-covering a few major genes and pathways that drive immune system–mediated disorders2, the outcomes for asthma3 and many other diseases have been largely sobering: much susceptibility cannot be explained by common genetic vari-ants, and of the disease-associated variants identified, the majority are noncoding, which complicates assessment of their function.
Beyond genetics, environment is also a key participant in the pathogenesis of asthma. Moreover, the considerable increase in the prevalence of asthma over the past few decades, especially in developing countries, suggests that environmental factors also influ-ence disease development. Although allergens are classically associated with asthma, many
Figure 1 epigenomic profiling of T cells indicates a different enhancer landscape in asthmatic patients than in healthy control subsets. enhancer maps (right) of peripheral blood mononuclear cells (left) collected from healthy control subjects (top) and patients with asthma (bottom) and sorted as CD4+ T cells, then as naive T cells and CCR4− or CCR4+ memory T cells (middle), followed by profiling of active and poised enhancers in all cell types by a micro-scaled H3K4me2 ChIP-Seq assay. The maps of asthmatic patients differ from those of healthy control subjects mostly for CCR4+ T cell populations representing TH2 cells.
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substantial cell-type specificity. Particularly important for understanding genetic asso-ciations with disease was the realization that a DNA variant can disrupt or generate a transcription factor–binding site within an enhancer. This notion suggests that with the enhancer map of a disease-relevant cell type, the functionality of noncoding variants associ-ated with the disease might be assessed.
ChIP-seq experiments begin with immu-noprecipitation of genomic fragments, followed by the preparation of a library for high-throughput sequencing to identify isolated genomic regions6. Due to inefficien-cies at the steps of immunoprecipitation and library preparation, this procedure requires large amounts of starting material, which prevents its application to the study of rare, biologically important cell types.
Several groups have developed small-scale immunoprecipitation protocols to overcome the challenges related to inefficiencies at the ChIP step6, but Seumois et al. are the first to tackle this problem in primary popula-tions of cells of the immune system obtained from patients1. The chromatin landscapes of helper T cells have been extensively studied for cells generated in vitro from both humans and mice5,7,8; however, Seumois et al. present the first enhancer maps of T cells generated in vivo1 (Fig. 1). Their work focuses on the T helper type 2 (TH2) subset of CD4+ T cells, which are key participants in asthma. The authors isolate circulating naive and memory CD4+ T cells from peripheral blood of healthy control subjects and patients with asthma and then further subdivide the CD4+ memory T cells on the basis of their surface expression of the chemokine receptor CCR4 to enrich for TH2 cells. They use the histone modification H3K4me2 for ChIP-seq as a reliable indicator of enhancer elements, following a number of studies highlighting the utility of H3K4me2 in identifying poised and active enhancer domains across the genome.
The message that emerges from this study is that enhancers in cell populations that have undergone enrichment for TH2 cells harbor a large portion of asthma-associated genetic vari-ants. Furthermore, comparison of enhancers identified in healthy control subjects versus patients with asthma high lights the finding that the majority of differences in H3K4me2 distribution appear in populations enriched for TH2 cells rather than in other memory or naive CD4+ T cell populations.
The most intriguing aspect of this study is its proof-of-concept for generating histone modification–based enhancer maps for pri-mary cells of the immune system, particularly for small cell populations in tissues from
Naive
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increasingly routine for doctors to perform whole-exome sequencing. No doubt sooner than expected, it will be reasonable for a patient to ask, “Hey doc, should you check my epig-enome?” Due to compositional heterogeneity of tissue samples from patients, methods that use sorted cell populations will probably prove more effective for epigenomic approaches. The development of an assay for transpos-ase-accessible chromatin using sequencing (ATAC-seq)13, which allows the profiling of ‘open’ chromatin from blood populations in a timeline on the order of days, has brought progress toward this goal. Now a technology for epigenome mapping in small cell popula-tions from clinical samples can be added to the list. As these technologies emerge, it becomes increasingly important to understand how all of these ‘-omic’ measurements relate to each other and to downstream physiological conse-quences if such technology is to be used one day to determine a means of intervention.
COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.
1. Seumois, G. et al. Nat. Immunol. 15, 777–788 (2014).2. Zenewicz, L.A., Abraham, C., Flavell, R.A. & Cho, J.H.
Cell 140, 791–797 (2009).3. Moffatt, M.F. et al. N. Engl. J. Med. 363, 1211–1221
(2010).4. Yang, I.v. & Schwartz, D.A. J. Allergy Clin. Immunol.
130, 1243–1255 (2012).5. vahedi, G. et al. Cell 151, 981–993 (2012).6. Adli, M. & Bernstein, B.e. Nat. Protoc. 6, 1656–1668
(2011).7. Hawkins, R.D. et al. Immunity 38, 1271–1284 (2013).8. wei, G. et al. Immunity 30, 155–167 (2009).9. Gaffney, D.J. et al. Genome Biol. 13, R7 (2012).10. Buecker, C. & wysocka, J. Trends Genet. 28, 276–284
(2012).11. Parker, S.C. et al. Proc. Natl. Acad. Sci. USA 110,
17921–17926 (2013).12. whyte, w.A. et al. Cell 153, 307–319 (2013).13. Buenrostro, J.D., Giresi, P.G., Zaba, L.C., Chang, H.Y. &
Greenleaf, w.J. Nat. Methods 10, 1213–1218 (2013).
patients. Although Seumois et al. restrict their focus to a particular histone mark and cell type1, their approach can easily be extended to other histone modifications and cell popu-lations, which opens the door for epigenomic studies of asthma and other diseases.
So far, genomic studies addressing the role of regulatory elements in the human genome have generally taken one of two approaches. Studies such as that of Seumois et al. aim to annotate functional enhancer elements in pathogenic cells using techniques such as ChIP-seq1. These studies focus on identifying genomic loci with differences in the enrichment of histone modifications in patients versus con-trol subjects. In contrast, other studies strive to map various quantitative trait loci, such as those associated with gene expression9, and thereby identify genetic variants that function-ally affect gene regulation. Ultimately, it will be enormously valuable to have a resource of people who have been both comprehensively genotyped and epigenomically characterized across a wide range of tissue types. Without genetic variation data, a critical gap remains in efforts to explain the difference in histone-modification patterns observed in patients with asthma and in healthy controls, and it remains unclear whether such differences in the enhancer landscapes are ‘footprints’ of the external environment.
Even after disease-associated enhanc-ers are identified, it remains a considerable challenge to link these functional noncoding elements to target genes. Distal regulatory elements often physically interact with the promoters of their target genes, and tech-nologies to detect these interactions, such as chromatin-conformation capture and chroma-tin-interaction paired-end tagging, are being
rapidly developed. Nonetheless, technical limitations, such as a requirement for very large quantities of DNA, now hinder their use in primary human cells.
An additional complication of the present study is that while H3K4me2 modification is thought to mark active and poised enhancers, it does not capture a comprehensive map of regulatory elements. Given the combinatorial nature of the regulation of gene expression, it is now evident that no single modifica-tion, but rather integration of multiple layers of genomic information (such as H3K27ac, binding of acetyltransferase p300 and hyper-sensitivity to DNase I), will provide the most complete annotation of enhancer elements10.
The ability to perform epigenomic profiling of cell type–specific populations of samples from patients widens the possibilities open to ‘-omic’ disease research. The potential com-binations of cell types, diseases and histone modifications seem endless, not to mention the transcription factors that could be studied by the same ChIP-seq procedure. Matching those data with cell type–specific gene expres-sion, DNA methylation and DNA-enhancer interactions will illuminate processes specific to particular cell types and diseases. With such comprehensive maps, investigation can begin into how the subset of enhancers known as ‘stretch enhancers’ or ‘super-enhancers’11,12 has a role in asthma and other diseases.
The potential for genomics to contribute to clinical care has long been recognized. With genomic characterization technologies, common diseases can be subcategorized by their altered pathways. Genomic diagnostics could allow more personalized understand-ing of a patient’s condition and thereby inform the choice of treatment. It is now becoming
An antitumor boost to TH9 cellsSergio A Quezada & Karl S Peggs
Cells of the TH9 subset of helper T cells differentiated in the presence of interleukin 1 (IL-1) produce large amounts of IL-9 and IL-21 in a manner dependent on the transcription factors STAT1 and IRF1 and exhibit potent anticancer effects.
Sergio A. Quezada and Karl S. Peggs are with
the Cancer immunology Unit, Research
Department of Haematology, University College
London Cancer institute, London, UK.
e-mail: [email protected]
The immune response to pathogens, tumors and healthy tissues is tightly regulated by
the ability of the CD4+ T cell compartment
to sense specific environmental cues, which ‘instruct’ the differentiation of CD4+ T cells into various subsets of helper and regula-tory cells. The classical helper T cell subsets induced by interleukin 12 (IL-12) (TH1 cells) or IL-4 (TH2 cells) were initially described in 1986 (ref. 1), followed by the later description of additional subsets, including regulatory T cells2, follicular helper T cells3, TH17 cells4 and
TH9 cells5,6. Although the biology and pathophysiological relevance of most of these helper T cell subsets is relatively well character-ized, much less is known about the transcrip-tional control of the genesis, activity and fate of the TH9 subset of helper T cells. In this issue of Nature Immunology, Végran and colleagues clarify the differentiation of TH9 cells and dem-onstrate their potent anticancer functions7.
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