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Chromatin targeting drugs in cancer and immunity

New information about the rules that guide the formation of multimolecular chromatin-bound complexes has helped to delineate gene-regulating pathways and describe how these pathways are altered in various pathological conditions. In this review, Tarakhovsky and Prinjha discuss approaches to therapeutically interfere with chromatin function for the purpose of cancer treatment and immunomodulation.

Recent advances in the enzymology of transcription and chromatin regulation have led to the discovery of proteins that play a prominent role in cell differentiation and the maintenance of specialized cell functions. Knowledge about post-synthetic DNA and histone modifications as well as information about the rules that guide the formation of multimolecular chromatin-bound complexes have helped to delineate gene-regulating pathways and describe how these pathways are altered in various pathological conditions. The present review focuses on the emerging area of therapeutic interference with chromatin function for the purpose of cancer treatment and immunomodulation.
The immune system plays a pivotal role in the control of cancer growth and metastasis (Vesely et al. 2011); the failure of the immune system to detect and eliminate cancer cells that are phenotypically different from the surrounding tissues is a major cause of cancer. Therefore, therapeutic agents that negatively affect immune cell development or activation may have a negative effect on immune surveillance of malignant cells and thereby promote cancer growth . However, most of the currently licensed anti-cancer drugs have a negative impact on hematopoiesis and hence on the immune system. Chemotherapeutic-induced immunosuppression increases susceptibility to infections and may prevent the successful usage of cell-based therapies that rely on cancer cell killing by activated lymphocytes (Mellman et al. 2011). Cancer immunotherapy is poised to become increasingly relevant due to the ascent of potent approaches that rely on rampant T-cell activation caused by suppression of the inhibitory signaling pathways in these cells (Sharma et al. 2011).
Immune cell function is regulated by a large number of specialized transcription factors, which overlap only partially with transcription factors that operate in normal and malignantly transformed cells (Busslinger 2004;Naito et al. 2011;Smale 2012). However, the basic chromatin processes that control DNA accessibility to transcription factors or support the chromatin-coupled processes such as RNA polymerase II (Pol II) pausing, elongation, or splicing as well as many others are likely to be common among every cell type in the human body. Consequently, the systemic administration of drugs that target chromatin regulators in cancer cells is likely to affect the same set of proteins in immune cells and vice versa. However, the degree of dependence of various immune cell subsets on the activity of specific transcription factors and chromatin regulators is likely to be different than that of tumor cells. For example, the dependence of rapidly dividing and actively metabolizing tumor cells on transcriptional regulators of the Myc family distinguishes malignant and nonmalignant cells (Shaughnessy 2008;Dang 2012). Distinct requirements for transcriptional circuits in immune versus tumor cells may present an opportunity for the therapeutic targeting of chromatin processes involved in tumor cell growth or pathologically activated immune cells. Ultimately, the source for the selective drug effect may lie not in the nature of the target chromatin protein, which could be common among cell types, but in the differential dependence of individual genes on a particular chromatin regulator. In this context, knowledge of chromatin-based mechanisms of immune cell regulation may serve as a blueprint for the rational design of therapies to selectively target cancer cells with little impact on immune cell function. Furthermore, information about the negative effects of anti-cancer drugs on immunity may contribute to the development of comprehensive supplementary therapies aimed at alleviating immunological side effects.

A brief overview of chromatin processes associated with immune cell responses
The ultimate aim of the immune system is to achieve a degree of diversity that mirrors the complexity of pathogens in the environment. In cells of the adaptive immune system, represented by B and T lineage cells, this diversity is achieved largely through the generation of highly selective antigen receptors, which collectively cover the complexity of environmental antigens (Rajewsky 1996;Abbas and Janeway 2000). The interaction of antigens with the corresponding receptors on B or T lineage cells results in cell activation followed by eventual elimination of the antigen.
During antigen-driven immune responses, cells of the adaptive immune system engage in multiple intercellular interactions that involve nonimmune cells as well as cells of the innate immune system (Iwasaki and Medzhitov 2010). The latter are represented largely by the migrating or tissue-specific myeloid lineage cells (e.g., macrophages, glia cells, or neutrophils) which possess the ability to recognize pathogens or damaged tissues through the pattern recognition receptor system (Palm and Medzhitov 2009). Initially described by Medzhitov and Janeway (1997), the pattern recognition system senses chemically distinct pathogen components such as nucleic acids, membrane lipids, or, in the case of damaged host tissues, cellular proteins such as high-mobility group protein 1 (Palm and Medzhitov 2009). The specificity of pattern recognition relies on membrane-bound receptors such as Toll-like receptors (TLRs), intracellular nucleic acid sensors such as RIG-I and MDA5, or NOD-like receptors (Medzhitov 2001;Pasare and Medzhitov 2005;Meylan et al. 2006).
The diverse nature of immune cell receptor signaling is further augmented by the existence of a cytokine-driven signaling system that modulates immune and nonimmune cell responses (Bezbradica and Medzhitov 2009). This cytokine signaling is functionally intertwined with antigen and pattern receptor signaling and is essential for optimal immune responses. Cytokine involvement also allows for the generation of whole-body awareness of local inflammatory processes, whereby changes in the cytokine concentration in the blood can affect the function of the nonimmune organs such as the heart, liver, or brain (Medzhitov 2008).
Immune system diversification uses signaling-coupled and RNA transcription-coupled processes to increase cellto-cell variability in individual gene expression within a range imposed by the immune cell lineage constraints. In turn, the efficacy of the immune response requires the existence of mechanisms that can markedly and coordinately activate a large number of genes (Zak and Aderem 2009;Smale 2012). Execution of transcription factor activity at the chromatin template largely depends on posttranslational histone modifications, which define the level of DNA accessibility and contribute to the recruitment of proteins that support RNA transcription (Jenuwein and Allis 2001;Kouzarides 2007;Ruthenburg et al. 2007). Similar to other cell types, active genes in immune cells are associated with nucleosomes enriched for histone H3 trimethylated at Lys 4 (H3K4me3) and acetylated histones H3 and H4 (Wei et al. 2009;Cuddapah et al. 2010). Opposite to these ''positive'' histone marks, histone H3 dimethylation at Lys 9 (H3K9me2) or H3K27me3 has been implicated in gene silencing in immune cells (De Santa et al. 2007;Wei et al. 2009;Kruidenier et al. 2012), which contributes to cytokine production by activated macrophages and other cell types. Early studies by the Natoli group (Saccani and Natoli 2002) show rapid demethylation of H3K9me2 at promoters of genes activated by macrophage treatment with bacteria-derived lipopolysac-charide (LPS). An important role for H3K9me2 in the regulation of cytokine expression was confirmed by findings showing lower levels of H3K9me2 at inflammatory gene promoters in innate immune cells as compared with nonimmune cells (Fang et al. 2012).
Pathogen-derived proteins that target various arms of the immune response frequently antagonize immune cells' drive to achieve maximum diversity and efficiency. Both bacteria and viruses express proteins that can enter the nucleus and interfere with distinct processes, including transcription and chromatin regulation (Bierne et al. 2012). One of the most glaring examples is human adenovirus, which employs viral E1A protein to hijack host transcription by binding to the histone acetyltransferase CBP (Ferrari et al. 2008) or the ubiquitin ligase hBre1 (Fonseca et al. 2012). Bovine and human papilloma viruses express E2 protein that binds to the C-terminal portion of the bromodomain-containing BRD4 protein, coupling histone lysine acetylation to RNA Pol II activation and RNA elongation (You et al. 2004). Similarly, HIV Tat protein employs Brd4 for the activation of virus transcription (Bisgrove et al. 2007;Sobhian et al. 2010;Rice 2013). Pathogen interference with the host epigenome may also involve proteins that carry histone-like sequences or ''histone mimics''-short amino sequences that are similar to the amino acid sequences within the N-terminal histone portions (Sampath et al. 2007). Histone mimics are present in numerous mammalian and pathogen-derived proteins . In some cases, such as the histone methyltransferase G9a, the histone mimic can fully recapitulate the protein-binding capacity of its histone H3 counterpart (Sampath et al. 2007). In other cases, histone mimics serve as recognition modules that enable post-translational modification of nonhistone proteins for purposes not directly linked to chromatin function (Lee et al. 2010Donlin et al. 2012).
Pathogen-derived histone mimics can compete with host histones for common binding partners required for transcriptional activation. For example, the histone mimic within the C-terminal portion of the immunosuppressive NS1 protein of the H3N2 influenza virus binds directly to the transcriptional elongation regulator PAF1 (polymerase-associated factor 1) (Zhou et al. 2012). By binding to the Paf1, which represents an essential part of the PAF1C (PAF1 complex) elongation complex (Krogan et al. 2003;Kim et al. 2010), NS1 inhibits elongation of virus-induced genes and attenuates the antiviral response (Marazzi et al. 2012). Accordingly, siRNA-induced suppression of Paf1 also attenuates the antiviral response and increases viral replication (Marazzi et al. 2012).
The ability of pathogen-derived proteins to control gene expression by interfering with gene transcription is reminiscent of drug-induced suppression of transcription in mammalian cells. Proteins such as BRD4 (see below) have been the focus of studies aimed at suppressing tumor growth and even reversing dedifferentiation associated with malignant transformation. However, the rise of novel anti-cancer drugs targeting transcriptional circuits in tumor cells requires an assessment of the potential effect of these drugs on similar transcriptional processes in immune cells.
Recent reviews have comprehensively described the multitude of chromatin processes that could be targeted for the purpose of cancer treatment. Here, we focus exclusively on transcription circuits that involve the histone methyltransferase Ezh2 and double-bromodomaincontaining transcriptional regulators (BET [bromodomain and extraterminal]). Compounds that target these two classes of proteins display anti-cancer activity in vitro and in vivo and are considered potential candidates for cancer treatment in humans. Additionally, BET protein inhibitors have a marked impact on proinflammatory gene expression and can attenuate systemic inflammatory processes in mice (Nicodeme et al. 2010;Belkina et al. 2013).

Targeting Ezh2 for cancer treatment and its effect on immunity
The histone methyltransferase Ezh2, which catalyzes HK27me3 (Cao et al. 2002;Czermin et al. 2002), could be legitimately considered as a poster child of modern cancer ''epigenetics,'' which largely studies the transcriptional response and has little in common with ''epigenetics'' in Waddington's sense of it. At the time of writing this review, a PubMed search for ''Ezh2 and cancer'' yielded 620 references; the initial prominence of Ezh2 as a potential target for cancer therapy has been largely driven by findings of Ezh2 overexpression in metastatic prostate cancer cells as well as in rapidly progressing cancers of other types (van Kemenade et al. 2001;Varambally et al. 2002;Kleer et al. 2003).
Ezh2 operates within the PRC2 protein complex, where the presence of other PRC2 components such as Eed, Suz12, and RbAp48 is essential for the catalysis of H3K27me3 (Cao and Zhang 2004;Margueron and Reinberg 2011). In addition to the core components of PRC2, targeting PRC2 to specific gene loci in mammalian cells involves auxiliary proteins such as Jarid2 (Peng et al. 2009;Landeira et al. 2010;Li et al. 2010;Pasini et al. 2010).
Most of the studies that deal with Ezh2 overexpression in tumors, including the initial description of Ezh2 overexpression in prostate cancer cells (Varambally et al. 2002), omit a detailed analysis of Ezh2-associated PRC2 components. Therefore, it is not clear how Ezh2 overexpression itself could be sufficient to affect H3K27me3, which requires the stochiometrically assembled PRC2.
It is generally assumed that Ezh2 expression controls cell division (van Kemenade et al. 2001;Pasini et al. 2004). The expression levels of PRC2 increase in dividing cells, and suppression of the Ezh2 in vitro by siRNA or specific inhibitors frequently leads to growth arrest (Varambally et al. 2002;Martinez-Garcia and Licht 2010). However, the mechanism of this suppression as well as the nature of genes or signaling processes that mediate it remain largely elusive. Confusion about the role of H2K27me3 and Ezh2 in cancer is compounded by studies showing the potential oncogenic function of both the loss-of-function and gain-of-function Ezh2 mutant pro-teins (Ernst et al. 2010;Martinez-Garcia and Licht 2010;Morin et al. 2010;Guglielmelli et al. 2011;Jankowska et al. 2011;Vainchenker et al. 2011;Yap et al. 2011;Ntziachristos et al. 2012). Recent work by Allis' group (Lewis et al. 2013) raised the question about the overall role of H3K27me3 in the regulation of tumor phenotypes. In highly malignant human gliomas, interactions between Ezh2 and a mutated histone H3 variant (in which the lysine at position 27 is substituted by the methionine) inactivate Ezh2 (Lewis et al. 2013). Tumors that carry H3K2K7-M mutations display negligible levels of H3K27me3, but this does not affect either cell division or the degree of malignancy. Essentially, these studies, along with the independent observation of Ezh2 inactivation by H3K2K7-M (Chan et al. 2013), show that neither Ezh2 activity nor H3K27me3 is required to support tumor growth and maintenance of the malignant phenotype.
The tumor-suppressive effect of Ezh2 inhibitors may also reflect the growth-promoting effect of Ezh2 up-regulation of specific targets. A limited set of data suggests that PRC2 can directly stimulate gene expression. In muscle cells, the Ezh2 homolog Ezh1 positively supports transcriptional elongation (Mousavi et al. 2012). Similarly, PRC2 has been implicated in the positive regulation of actively transcribed cytokine genes (Jacob et al. 2008(Jacob et al. , 2011. Similarly, the oncogenic function of EZH2 in castrationresistant prostate cancer is independent of its role as a transcriptional repressor but involves the ability of EZH2 to act as a coactivator for critical transcription factors, including the androgen receptor (Xu et al. 2012).
Despite the lack of sufficient clarity about the mechanism of Ezh2 involvement in carcinogenesis and tumor progression, a significant effort has been put into Ezh2 inhibition for the purpose of cancer therapy (Melnick 2012). Treatment of diffuse large B-cell lymphoma (DLBCL) that harbors Ezh2-activating mutations with a potent small-molecule inhibitor of EZH2 methyltransferase activity decreased global H3K27me3 levels, reactivated silenced PRC2 target genes, and inhibited the proliferation of these EZH2 mutant DLBCL cells (McCabe et al. 2012).
Before Ezh2 inhibitors gain further support as antitumor drugs, the potential side effect for relatively broad immunosuppression should be considered. Ezh2 deficiency in developing B lineage cells diminishes the antibody repertoire (Su et al. 2003) due to the selective impairment of V H gene rearrangement residing at the 59 end of the 2.3-Mb-long immunoglobulin heavy chain (igH) gene locus (Malin et al. 2010). These so-called ''distal'' V H genes comprise a large portion of the immunoglobulin repertoire in mice and humans (Ebert et al. 2011). Perturbations in distal V H rearrangement cause partial humoral immunodeficiency associated with a paucity of certain antibody classes. In addition to V H gene rearrangement, Ezh2 is also involved in rearrangement of immunoglobulin light chain genes via a mechanism involving IL-7 receptor signaling (Mandal et al. 2011). Ezh2 is expressed at high levels in germinal center (GC) B cells, which are involved in the generation of antibodies with high affinities to their antigen (Velichutina et al. 2010). In GC B cells, EZH2 targets a large number of GC-specific targets, thus suggesting a key role for Ezh2 in GC B-cell function. Consequently, Ezh2 deficiency in GC B cells may reduce the efficacy of the long-lasting humoral immune response that relies on the presence of B cells expressing high-affinity antibodies.
In addition to its impact on B cells, Ezh2 deficiency also has a negative effect on T-cell immunity. Lack of Ezh2 in early hematopoietic progenitors prevents expansion of early T-cell precursors in the thymus (Su et al. 2005). Furthermore, Ezh2 deficiency reduces the T-cell antigen receptor (TCR)-driven proliferation of T cells in vitro and abrogates antigen-driven T-cell responses in vivo . Administration of the histone methylation inhibitor 3-deazaneplanocin A (DZNep) arrests ongoing T-cell-induced graft rejection in mice after allogeneic bone marrow transplantation and selective apoptosis in alloantigen-activated T cells mediating host tissue injury.
The nonnuclear function of Ezh2 in T-cell activation has been linked to the ability of cytosolic Ezh2 to control signal-driven actin polymerization in an H3K27me3-independent but methyltransferase-dependent fashion (Su et al. 2005). A cytosolic presence of PRC2 components has been reported for Eed (Witte et al. 2004) and Ezh1 (Ogawa et al. 2003) in addition to Ezh2 (Su et al. 2005;Bryant et al. 2008;Gonzalez et al. 2011). In T cells, cytosolic Ezh2 binds to the signaling protein Vav1, which plays an essential role in actin polymerization (Hobert et al. 1996a,b;Nolz et al. 2005;Su et al. 2005). Binding to Vav1 may explain the ability of overexpressed Ezh2 to activate the phosphoinositide 3-kinase (PI3K)/Akt pathway in breast cancer cells (Gonzalez et al. 2011) and control actin polymerization in the prostate cancer cells (Bryant et al. 2008). The exact nature of cytosolic Ezh2 substrates is not known, but the likelihood of these substrates' existence is highlighted by the recent identification of several nonhistone targets of Ezh2, such as RORa . It is tempting to speculate that Ezh2 involvement in actin polymerization-a process that plays a prominent role in tumor cell invasion (Kim et al. 2009)-may contribute more to tumor spreading than do putative changes in gene expression caused by altered Ezh2 expression or activity levels.
Ezh2 has been indirectly implicated in the regulation of the inflammatory gene expression in the innate immune cells. Activation of macrophages by LPS is associated with a selective increase in the H3K27me3-specific demethylase JMJD3 (De Santa et al. 2007). Conversely, JMJD3 deficiency or pharmacological inhibition affects macrophage gene expression (Kruidenier et al. 2012). In light of these findings, one would expect to see an increase in proinflammatory gene expression following suppression of Ezh2.
Potential effects of Ezh2 on the immune system have to be viewed differently in the context of solid tumors and blood malignancies. Unless delivered in a targeted fashion, systemically applied Ezh2 inhibitors may have a more immediate impact on the immune cells than on tumor cells. In such a case, immunodeficiency may precede a potential anti-tumor effect. In fact, in the case of blood malignancies, the immunosuppressive function of Ezh2 inhibition could be advantageous for the treatment of B-or T-cell lymphomas that rely on Ezh2-mediated cytosolic and nuclear signaling networks.
Targeting bromodomain-containing transcriptional regulators and effects on immunity Lysine acetylation on various histone molecules as well as on nonhistone nuclear proteins is a hallmark of and prerequisite for transcription in mammalian cells (Cheung et al. 2000;Bannister and Kouzarides 2011). Acetyl-lysines bind to highly conserved bromodomains present in numerous cellular proteins and play an essential role in the assembly of protein networks that control gene expression (Zeng and Zhou 2002;Mujtaba et al. 2007). Recently, the tandem BET domain-containing BET proteins BRD2, BRD3, BRD4, and BRDT became the focus of studies on the pharmacological control of gene expression (Arrowsmith et al. 2012;Prinjha et al. 2012). Before gaining prominence as potential drug targets, BET proteins were known as regulators of gene expression in vitro and in vivo (Belkina and Denis 2012). Some of the BET proteins, such as BRD4, were found in association with the Mediator complex to play a prominent role in transcription (Jiang et al. 1998). In addition to its association with Mediator, BRD4 gained additional importance as the only ubiquitously expressed BET protein that can bind directly to p-TEFb through an extended C-terminal domain (Dey et al. 2003;Peterlin and Price 2006;Zhou et al. 2012). Connection to p-TEF-B provided a direct link between BRD4 binding to the acetyl-lysines and transcriptional elongation (Zhou et al. 2012). BRD2 and BRD3 can also participate in elongation through their association with the RNA Pol II-associated elongation complex PAF1C (Dawson et al. 2011). The PAF1C/BET interaction likely relies on bromodomain-unrelated sequences such as ET or other not yet identified motifs (Rahman et al. 2011). The interaction of the ET domain of BETs with numerous effector proteins such as NSD3 (a SET domain-containing histone methyltransferase), JMJD6 (a histone arginine demethylase), and CHD4 (a catalytic component of the NuRD nucleosome remodeling complex) points to active BET involvement in chromatin modifications that might be required for transcriptional elongation (Rahman et al. 2011). The complexity of BET-mediated regulation is further increased by the ability of BET proteins such as BRD4 or BRD2 to bind directly to acetylated lysines within transcription factors such as NF-kB or GATA1 (Huang et al. 2009;Lamonica et al. 2011). It is important to note that the nucleus may not be the only place where BET functions. Brd2 translocation from the cytoplasm to the nucleus is controlled by serum factors (Guo et al. 2000), which suggests a signal-dependent control of Brd2mediated gene regulation. In the developing mouse neural tube and dorsal root ganglia, Brd2 localized to the nucleus during proliferation but was predominantly cytoplasmic when cells were terminally differentiated (Crowley et al. 2004).
The involvement of BET proteins in transcriptional regulation and the specificity of BET bromodomain binding to acetyl-lysines provided a foundation for the development of synthetic compounds that control gene expression by inhibiting BET binding to acetylated lysines. The first generation of BET inhibitors was developed independently by several groups, including GlaxoSmithKline (I-BET) in collaboration with our group (Nicodeme et al. 2010) and Bradner's group (Filippakopoulos et al. 2010) (JQ1) in collaboration with the Structural Genomics Consotium (SGC). The lion's share of studies that use BET inhibitors describe the effect of the inhibitors on tumor growth (Dawson et al. 2011;Delmore et al. 2011;Zuber et al. 2011;Dawson and Kouzarides 2012;Loven et al. 2013). It appears that MYC-overexpressing tumors are especially sensitive to BET inhibitors, which suppress MYC expression followed by a dampening of the magnitude of the MYC-driven transcriptional response (Delmore et al. 2011;Ott et al. 2012;Loven et al. 2013;Puissant et al. 2013). In addition, BET inhibitors act effectively against tumors that express a rare form of an oncogenic BRD4-NUT fusion protein (Filippakopoulos et al. 2010). The ability of BET inhibitors to suppress tumor growth is not entirely unexpected in light of the earlier studies of Ozato's group (Maruyama et al. 2002;Dey et al. 2003), which demonstrated a crucial role for BRD4 during mitosis.
One of the first studies of BET inhibitors (GSK525762 compound, designated as I-BET) revealed a potent impact on inflammatory gene expression (Nicodeme et al. 2010). This finding was partly expected due to previous studies revealing an important role for BRD4 in the regulation of proinflammatory gene expression. BRD4-supported cotranscriptional mRNA splicing is important for controlling LPS-inducible inflammatory gene expression in macrophages (Hargreaves et al. 2009). In the absence of stimulation, RNA Pol II generates low levels of fulllength but unspliced and untranslatable transcripts at many of the LPS-induced response genes (Hargreaves et al. 2009). Compared with BRD4, which appears to be a generic regulator of elongation, BRD2, which also binds to I-BET, could play a more selective role in the regulation of immune response genes. The positioning of the BRD2 gene within the myosin heavy chain (MHC) class II gene cluster on human chromosome 6 or in syntenic regions of other organisms (Belkina and Denis 2012) could be seen as a sign of specialized BRD2 involvement in immune responses. In support of this model, low levels of BRD2 are associated with the reduced cytotoxic cytokine production by in vitro triggered macrophages (Belkina et al. 2013).
One of the surprising outcomes of studies on the effect of I-BET on LPS-triggered macrophages was the rather selective impact of I-BET on gene expression (Nicodeme et al. 2010). A common theme that has emerged from studies of LPS-inducible genes is a connection between the timing of gene expression and the state of the chromatin associated with the promoter and transcriptional start site of the inducible gene. Different temporal patterns of gene expression in response to LPS appear to be embedded within the CpG content of inducible gene promoters (Natoli et al. 2011;Smale 2012). In macrophages, CpG island-rich promoters are prevalent among primary and weakly induced secondary response genes, while CpG-low promoters are much more prevalent among more highly induced secondary response genes (Bhatt et al. 2012). BET proteins are associated with both primary and secondary response genes at relatively similar levels before LPS induction of macrophages (Nicodeme et al. 2010). However, treatment of macrophages in vitro with I-BET resulted in the strong and selective attenuation of secondary response gene expression while leaving the expression of primary response genes largely unaffected (Nicodeme et al. 2010). Most significantly, the selective effect of I-BET on secondary response genes holds true for macrophage as well as fibroblast responses to not only LPS, but secondary mediators of the inflammatory responses such as TNF or type I interferon (IFN).
The ability of I-BET to profoundly suppress numerous proinflammatory genes in cells of the innate as well as adaptive immune system (Bandukwala et al. 2012) should be considered, as it might affect the host response to pathogens. The negative impact of I-BET on immunity is particularly relevant in cancer patients who may additionally suffer from the effects of tumor-and/or chemotherapy-induced immunosuppression.

Summary
Rapidly emerging information about the transcriptional control of tumor growth and activated immune cells is likely to continue to fuel excitement about the therapeutic promise of drugs that target catalytic activity of the chromatin-modifying enzymes as well as protein-protein interactions within chromatin-bound regulatory complexes. Additional data on the aberrant structure of tumorexpressed chromatin regulators may further the rational design of therapeutics to achieve a therapeutic effect in tumor cells but spare healthy tissues. The effort to affect tumor growth by interfering with overexpressed-but structurally unaltered-chromatin regulators may cause potential side effects, including immunosuppression. However, the likely differences in gene regulation in tumor cells and cells of the immune system may provide an opportunity for the selective targeting of gene circuits involved in a particular type of tumor cell. Ultimately, however, using chromatin targeting drugs to control systemic immune disorders without causing broad immunosuppression or persistent damage to nonimmune cells will require a better understanding of the transcriptional pathways that govern pathological functions of immune cell subsets that drive the disease.


The effects of pyridaben pesticide on the histomorphometric, hormonal alternations and reproductive functions of BALB/c mice.

Objective(s): The adverse effects of pyridaben on reproductive system in male animals are not well established. This study was designed to elucidate how pyridaben can effects the histomorphometric, hormonal alternations and reproductive functions of BALB/c mice. Materials and Methods: For this study, 80 adult and apparently healthy male BALB/c mice were divided into three groups Viz, control, test group 1 and test group 2. Test groups 1 and 2 were received the toxin at doses of 53 mg/kg. BW, and 212 mg/kg. BW, respectively. The experiment period for both groups was 10, 25 and 45 days. Results: The levels of FSH, LH and testosterone were significantly (P<0.05) decreased on the dose and time dependant means. The levels of the ROS and NOS were significantly (P<0.05) increased in all test groups. The percent body weight gains significantly (P<0.05) reduced, whereas weights significantly (P<0.05) increased in test groups in a dose and time dependant manner. The histomorphometric and stereologic findings, including diameters of somniferous tubules, thickness of somniferous tubules epithelium, the leydig's cell distribution, TDI, SI, RI revealed that, all these parameters are also significantly (P<0.05) reduces in test groups in a dose and time dependant manner. Conclusion: Pyridaben causes histomorphometric and stereologic changes in testis, as well as hormonal and reproductive functional alternations in BALB/c mice.

Pyridaben has been studied for its acute toxicity in different species (3,4). The inhabitation of complex I by pyridaben directs mtNOS to loose it's NO producing activity and to become an O2source (4)(5)(6).
Pyridaben is two times more potent than rotenone in inhibiting mitochondrial respiration, and is a competitive inhibitor of DHR (dihydrorotenone) binding, (5,7,8). Pyridaben causes significantly greater oxidative damage than a similar dose of rotenone as measured by protein carbonyl. Partial inhibition of complex I by pyridaben is sufficient to produce significant increase in reactive oxygen species (ROS) production (9)(10)(11)(12)(13)(14)(15)(16). Pyridaben has not been fully evaluated for its possible immunotoxic effects, but it has been demonstrated that, it is a potent inducer of apoptosis in ST486 and in EW36 cells in combination with heat stress (17). It encompasses anticancer action (18).
The production of reactive oxidative oxygen species in the male reproductive system due to their high toxic effects on the quality and potential of action of sperms is an important concept in andrology (19). Although small amount of ROS is necessary for sperm potentiality in fertilization, acromosomal reaction, motility and capacitation (20), some studies have been revealed that 25-45% of infertile men showing high levels of ROS in their semens (21,22). The spermatozoa are showing special sensitivity to oxidative stresses damages, because their plasma membrane exhibiting profound amounts of polyunsaturated fatty acids (PUFAs), and in their cytoplasm small amount of inhibitory enzymes is present (23,24).
Apart from this, the antioxidant enzymes present inside the cells are not able to protect surrounding plasma membranes of acrosome and tails of spermatozoa, thus, in order to reinforce their limited intracellular antioxidant system, cells get help from protective mechanisms of semen plasma (25)(26)(27). Previous studies have revealed that there is a direct relationship between sperm quality and increased ROS production (28).
Pyridaben inhibits NADH-ubiquinone oxidoreductase activity, leading to the level of induced ornithine decarboxylase activity which in turn causes the antiproliferative effect, and since the ROS causes mitochondrial membrane damage, damaged mitochondrial membrane exaggerates ROS production (29). ROS invade polyunsaturated fatty acids located at cell membrane, which in turn leads to lipid peroxidation (30,31), and finally free radicals liberation starts (32). One of the lipid peroxidation byproducts is malondialdehyde (MDA). This by-product is used in different biochemical assays for detection of peroxidative damages (33). Increased ROS production leads to reduction in sperm motility (34). Increased ROS level leads to sperm DNA damage and this damage causes structural changes in DNA bases such as removal of DNA bases in some loci, removal or DNA bases frame shift, formation of unnatural bonds between two DNA strands, chromosomal recombinant (35). Apart from these, oxidative stress enhances breaks in one DNA strand or both of them (36). Increase in the amounts of ROS also is able to cause different forms of gene mutations such as point mutation and multiforms, which lead to reduction in sperm quality (37). If the amounts of damages to DNA are negligible, sperm or even oocyte will be able to repair DNA (38), but in cases the damages are more pronounced the apoptosis and fragmentation of embryonic cells nuclei which are obtained proceeding such sperms and or oocytes fertilization are more likely to take place. Reductions in fertility and quality of embryo are in direct proportional relation with the size of DNAs damages (39). DNA damage can also take place in Y chromosome and may lead to gene deletions in offsprings and finally lead to infertility (36). The elevated levels of ROS can also cause disturbances in inner and outer mitochondrial membranes, consequently cytochrome c seepage could take place, and activation of caspase enzymes causes apoptosis (40). Previous studies revealed that, reaction of ROS with mitochondria -induces liberation of apoptosis inducing factor (AIF) which directly reacts with DNA and causes DNA fragmentation (41). Another study revealed that there is a direct relationship between ROS-induced damage to the sperms and elevated levels of cytochrome c and caspases 3 and 9 (42). As the deleterious effects of pyridaben on histomorphometric, hormonal alternations and reproductive functions of male mammals have not been studied, this is the first investigation from this aspect.

Materials and Methods
For this study, 80 adult and apparently healthy male BALB/c mice with body weight of 20±5.0 g were used. Before commencing the experiment, animals were left without any treatment for seven days for adaptation. Animals were fed on standard mice pellet and with tap water ad libitum, 12 hr light and 12 hr dark, temperature 20-23°C, conditions were strictly monitored.
Animal groups Animals were divided into viz (3 groups), control, test group 1 and test group 2. The control group comprised of 20 mice, but the other groups comprised of 30 mice. The living conditions were equal for all groups, but experimental groups received pyridaben as described below.

Experiment
Mice in test group 1(treated with low dose of pyridaben) received pyridaben (53 mg/kg. BW), dissolved in distilled water and fed on orally (by gavages) for 45 consecutive days. Mice in test group 2 (treated with high dose of pyridaben) received pyridaben (212mg/kg BW, dissolved in distilled water), orally (by gavages) for 45 consecutive days. Mice in control group received the same volume of distilled water.

Specimens
In each stage, the body weights of mice were recorded, and then the animals sacrificed by advocated human means. Immediately after the sacrifice of animals all the blood present in their circulation was aspirated from the heart by special needle. Collecting the specimens was carried out in all groups on 10 th , 25 th and 45 th day of experiment. In each occasion 10 mice from each group were sacrificed for acquiring specimens. Then, their abdominal region was cut opened and their testes along with epididymides were taken out and fixed in formalin saline solution. The other testis was stored in deep freeze for further biochemical analyses.

Tissue preparation
The formalin fixed testes were processed through routine paraffin embedding, cut at 6 µm and stained with Hematoxilin -Eosine and Weigert's -Iron techniques. All the morphometric parameters including; diameters of semniferous tubules (SFTs), height of SFTs, diameters of tunica albuginea testis, and the thicknesses of interstitial tissues were recorded.

Cytomorphometry
The distribution of leydig's cells as well as lymphocytes in mm 2 of testicular tissue, the different testicular indices of spermatogenesis including RI Iran J Basic Med Sci, Vol. 16, No. 10, Oct 2013 (repopulations index), TDI (tubular differentiation index), and SI (sperm index) were recorded.

Hormonal assays
The hormonal assays including LH, FSH (Pishtaz teb kits -Zaman Diagnostics Tehran, Iran), were carried out on blood serum by ELISA techniques. Testosterone assay was carried out on blood serum by electrochemiluminescence techniques (Roshd kits: 05200067 190).

Assessment of total antioxidant capacity (TAOC)
The testicular tissues were processed for the evaluation of NOS, ROS, FRAP, and Thiol. To determine the effect of pyridaben on oxidative stress, testicular antioxidant capacity (TAOC) was measured. The assay is based on the assessment of ferric reduction antioxidant power (FRAP) assay (43). Briefly, at low pH which was achieved by using acetate buffer (300mM, pH 3.6), reduction of Fe III -TPTZ complex to the ferrous form produces an intensive blue color that could be measured at 593 nm. Aqueous solution of Fe II (FeSO4.7H2O) and appropriate concentration of freshly prepared ascorbic acid were used as blank and standard solutions, respectively. The TAOC was expressed as mM per mg protein of the samples. The protein content of the samples was measured according to Lowry method (44).

Histomorphology
In male rats from control group, the microarchitecture and morphologic appearance of the testicular interstitial tissue, including tunica albuginea, leydig's cells and spermatogenic cell series of somniferous tubules were normal (Figure 1-A).
In male rats treated with low dose of pyridaben, alterations in the microarchitecture and morphologic appearance of the testicular interstitial tissue, including tunica albuginea, leydig's cells and spermatogenic cell series of semniferous tubules were obvious. In this group round spermatids were observed, but spermatozoa not seen in the luminal provinces of somniferous tubules (Figure 1-C).
In rats treated with high dose of pyridaben, profound reduction in populations of all types of the spermatogenic cell series including spermatogonia, spermatocytes, round and elongated spermatids was obsereved, but the spermatozoa were absolutely absent. In the interstitial tissue, the integrity and population of leydig's were reduced; however, infiltration of lymphocytes was profound (Figure 1-B). In rats exposed to high dose of pyridaben for a period of 45 days, the epithelia of semniferous tubules were reduced to below 4 cell layers (TDI negative) ( Figure 1-D).

Histomorphometry and Stereology
Data analyses of weight gain percentages between test and control groups revealed that, after 45 days of exposure to the pyridaben (212 mg/kg.BW/days) significant difference (P<0.05) is attains, but there was no difference(P>0.05) between control with other test groups concerning this parameter (Figure 2).There was no significant difference (P>0.05) between the body weights of control and both of test groups after 10 days, but there was a significant difference between test groups and control after 25 and 45 days of exposure to pyridaben (Figure 2).
There was significant difference (P<0.001) regarding tunica alboginea thicknesses between control and high dose group after 45 days. However, there was no significant difference (P>0.05) between control and other test groups after 25 and 45 days of exposure ( Figure 3).
There was a significant difference (P<0.001) in diameters of seminiferous tubules between control and low dose-treated group after 10 days of exposure, but there was not a significant difference between control and low dose-treated groups after 25 and 45 days and there was significant difference between control and high dose-treated group after 25 and 45 days of exposure, but there was not a significant difference following 10 days of exposure ( Figure 3).
Data analyses regarding diameters of seminiferus tubules epithelium revealed that there is a significant (P<0.001) difference between control and both of the test groups only after 45 days of exposure ( Figure 3).
There was not a significant difference in thicknesses of interstitial tissue between control and all test groups ( Figure 3).
There was significant difference (P<0.001) in leydig's cells distribution in mm 2 of testicular interstitial tissue between control and high dosetreated group after 45 days of exposure, but there was no significant difference between control and other test groups (P >0.05) ( Figure 4).
There was a significant difference in lymphocytes distribution in mm 2 of testicular interstitial tissue between controls with both test groups only after 10 days of exposure ( Figure 4). Data analyses concerning TDI(testicular differential index), revealed that there is no significant difference between control and test groups after 10 days of exposure, but there is a significant(P<0.001) difference between control and other test groups after 25 and 45 days of exposure ( Figure 5).
Data analyses concerning SI(sperm index) revealed that, there is not a significant (P>0.05) difference between control and low dose-treated group after 10 and 25 days ,but there was a significant difference between control and low dosetreated test group after 45 days. In addition, there was a significant difference between control and high dose-treated group in 10, 25 and 45 days ( Figure 5).
Analyses of RI (repopulation index) data revealed that, there is not a significant difference (P>0.05) between control and low dose-treated group after 10 days of exposure, but there was significant difference between control and other test groups ( Figure 5).

Hormonal analyses
Significant decreases were seen in FSH level following 10 days (P<0.05), 25 days (P<0.001) and 45 days (P<0.001) in low dose-treated group, but in high dose-treated groups the reduction in profile of this hormone, 10, 25, and 45 days after exposure was significant (P<0.001). In both low and high dosetreated groups, significant (P<0.001) decrease in profile of LH was evident ( Figure 6). Profile of testosterone in control and different test groups in low dose-treated group revealed significant difference (P<0.01) after 45 days exposure, but in high dose-treated group, it was significant (P<0.01) after 25 days of exposure, whereas it was significant (P<0.001) after 45 days of exposure ( Figure 6).

TTM (Total Thiol Molecules)
Data analyses revealed that there is no significant difference in the amounts of TTM between all groups after 10 days of exposure to pyridaben, whereas, after 25 days of exposure there was significant (Figure 7) difference between control and test groups regarding the amount of TTM, whereas there was not any difference between test groups. On the other hand, after 45 days of exposure there was significant difference between control and test groups from the aspect of the amount of TTM (Figure 7).

TAOC (Total antioxidant capacity)
In both test groups, after 25 days (P<0.05) and 45 days (P<0.01) of exposure, significant reduction was evident in the amounts of TAOC (Figure 7).

MAD (malondealdehyde)
Statistical analysis on the profile of malondialdehyde (MDA) in control and test groups revealed that in the low dose-treated test group significant (P<0.01) increase following 25 days of exposure and highly significant (P<0.001) increase at 45 days exposure are evident, but in high dosetreated group, significant increase after 25 (P<0.001) and 45 days (P<0.001) are evident (Figure 7).

Discussion
Pyridaben, a pyridazinone derivative, is an acaricide and insecticide (45). The other compounds commonly used as pesticide are atrazine, benomyl, carbaryl, carbofuran, chlorpyrifos, endosulfan, dibromochloropropane(DBCP), 2, 4-dichlrophenoxy acetic acid, dimethoate, dioxin, hexachlorocyclohexane(HCH), lindane, malathione, mancozeb, methoxychlor, methyl parathion, organophosphate and pyrethyroid. The reproductive toxic effects of these compounds are widely studied (46). At present, Pyridaben is widely used as miticide and insecticide all over the world, although this compound is toxic for different biological systems (18, 45, and 47) but its noxious effects on male and female reproductive systems are inadequately known. For this reason, we designed this investigation to find out the deleterious effects of this compound on different parameters of male mice reproductive system, including body weight, testis weight, the histomorphologic, histometric and stereological evidences (including diameters of tunica albuginea, semniferous tubules, testicular interstitial tissue, Leydig´s cells and lymphocyte distribution on testicular tissue, TDI, SI, and RI). Besides, the hormonal assays (including FSH, LH and testosterone) and evaluation of ROS and NOS biomarkers were carried out.
According to the results of our study, pyridaben time and dose dependently causes reduction in percentage of body weight gain in male mice. According to previous studies, body weight decreases in methyl parathion treated rats (48). Results of our study revealed that increase in testes weight occurs in a dose and time dependant pattern. The increase in testes weight is highly likely due to the edemas of testes caused by the pyridaben. Our morphological observations confirmed this phenomenon. The edema of testes is reported by Joshi et al (49) and Ngoula et al (50) in male rat by chloropyrifos and pirimiphos -methyl, respectively. It was shown that, a carbamate pesticide (2isopropoxy-phenyl-N-methylcarbamate) which administered to adult male Wistar rats for 90 successive days leads to a concentration-dependent increase in relative weights of testis and epididymis and a decrease in sperm density, serum and intratesticular total cholesterol concentrations, and intratesticular total proteins (50).
According to results of this study, pyridaben results in an increase in thickness of the tunica albugina of the testes in test groups in a concentration and time dependent pattern.
According to Kulshrestha and Neelam (51), carbaryl and endosulfan pesticides cause increases in ovarian tunica albugina thickness in fresh water teleost Chana straitus, following the exposure for a period of 2-30 days during the spawning season.
Our studies on the effects of pyridaben on the thickness of the seminiferous epithelium revealed that, this parameter decreases in a concentration and time dependent pattern. There were sloughing and disorganization of spermatogenic cells with their exfoliation in luminae of seminiferous tubules. It was demonstrated that degenerative changes in the seminiferous tubules occur in rats receiving malathion (27mg/kg/day for 4 weeks) (52). Thus, the reduction in thickness of the somniferous tubules epithelium is due to the degenerative effects of the pesticides.
There were no considerable changes in the testicular interstitial tissues in test groups treated by pyridaben. Also, malation does not lead to changes in the interstitial tissue of the testes (52).
Our evaluation on the distribution of the Leydig´s cells in the interstitial tissue of the testes revealed that significant decrease in the population of these cells occurs only after 45 days administration of the pyridaben at high dose (212 mg/kg). Ngoula et al (50), stated that, pirimiphos-methyl, an organophosphothioate pesticide, causes rarefaction of Leydig's cells.
Our assessments on the distribution of the lymphocytes in the interstitial tissue of the testes revealed that significant increase in the population of these cells occurs after 10 days of daily exposure to the pyridaben at low and high doses but not after 25 and 45 days. It appears more likely that, by first contact with the toxin, a shock like increase in the lymphocytes happens and consequently adjustment and adaptation occurs, that is why in both doses after 25 and 45 days of treatment population of lymphocyte distribution are the same as in control group.
The results of this study revealed that, pyridaben results in an increase in TDI percentage in a dose and time dependant mode. It is to be noticed that, when the TDI percentage increases, it indicates that the epithelial layer is undergoing degeneration and thinning. The previous studies reported that, dimethoate (53) causes dose related testicular damage characterized by moderate to severe seminiferous tubule degeneration as sloughing, atrophy, germ cell degeneration and partial arrest of spermatogenesis. Farag et al (54) reported that, dimethoate causes adverse effects on reproductive performance of male mice, including sperm viability, motility and density. Such changes are reported by Joshi et al (49), which were caused by Chlorpyrifos.
We noticed that the RI decreases in a dose and time dependant manner. This indicates that, the ratio of spermatogonia type B to the spermatogonia type A is reducing by the pyridaben, thus this toxin causes decrease in spermatogonia type B population. High dose of 2-bromopropane decreases spermatogenesis by adversely affecting spermatogonia followed by depletion of spermatocytes, spermatids, and spermatozoa with subsequent testicular atrophy (55).
The SI percentage, i.e., percentage of seminiferous tubules without sperms, was increased by pyridaben as the dose and time increase. Dimethoate was shown to be able to decrease sperm viability, motility and density (56).
Our results indicated that, pyridaben causes decrease in the profiles of FSH, LH and testosterone levels based on concentration and exposure duration of the received toxin. It was reported that carbamate poisoning in male rats causes a significant decrease in the level of testosterone, whereas the levels of the FSH and LH increase (57). Quinalphos, a commonly used organophosphorus insecticide caused a reduction in plasma levels of testosterone, FSH, and LH hormones (58).
According to our results, the malondialdehyde (MDA) and NO levels are increased based on the dose and exposure duration of the toxin. The decrease in antioxidant enzymes by induction of ROS production causes increase in the levels of MDA and NO by pyridaben toxin. The total thiol molecules (TTM), and total antioxidant capacity (TAOC), are decreased in our experiment. The activities of antioxidant enzymes such as superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase decrease in testes proceeding methoxychlor pesticide intoxication male rats (59).

Conclusion
According to the results of this stud we conclude that, pyridaben causes histomorphometric changes of testis, as well as hormonal alternations and increase in the levels of ROS and NOS along with changes in reproductive functional in BALB/c mice.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
TNF superfamily member TL1A elicits type 2 innate lymphoid cells at mucosal barriers

Immune responses at mucosal barriers are regulated by innate type 2 lymphoid cells (ILC2s) that elaborate effector cytokines interleukins 5 and 13 (IL5 and IL13). IL25 and IL33 are key cytokines that support ILC2s; however, mice deficient in these pathways retain some functional ILC2s. Analysis of human and murine cells revealed that ILC2s highly express tumor necrosis factor (TNF)-receptor superfamily member DR3 (TNFRSF25). Engagement of DR3 with cognate ligand TL1A promoted ILC2 expansion, survival, and function. Exogenous protein or genetic overexpression of TL1A activated ILC2s independent of IL25 or IL33. Dr3−/− mice failed to control gut helminthic infections, and failed to mount ILC2 responses in the lung after nasal challenge with papain. Our data demonstrate a key role for TL1A in promoting ILC2s at mucosal barriers.

INTRODUCTION
Innate lymphoid cells (ILCs) are a recently described group of innate immune cells that regulate immunity, inflammation, and tissue repair. Innate immune responses provide first-line immunity to invading pathogens. Although only a small fraction of the total immune cell population, they are found throughout the immune system, particularly at epithelial barriers, suggesting they have a critical role in maintaining the integrity of the skin, airways, and intestine. ILC subsets have been defined based on distinct pattern of transcription factor expression, cell surface markers, and cytokines, much like CD4 þ T helper (Th) cell subsets. As such, ILC1, ILC2, and ILC3 produce the same signature cytokines as Th1, Th2, and Th17/Th22 cells, respectively. The fact that these ILC subsets elaborate different cytokines yet lack rearranged antigenspecific receptors implicate them as evolutionary precursor of cells of the adaptive immune system that may facilitate crosstalk between the innate and adaptive arms of the immune response. 1,2 ILC2s in particular play a critical role in protection against helminthic infections, in tissue repair after influenza virus infection, and contribute to the pathophysiology of allergic inflammation and asthma. [3][4][5][6][7][8] Interleukin-25 (IL25) and IL33 are two key cytokines that support ILC2 differentiation and function. 6,9 All ILCs lack lineage markers (Lin À ) but express the lymphoid progenitor marker IL7 receptor a chain (IL7Ra; CD127) and the cytokine common gamma (gc) receptor chain, and require IL7 for development. 10 In addition, ILC2s express the receptor for IL33, T1/ST2, and the receptor for IL25, IL17RB. 2 ILC2s were originally identified in Rag-deficient mice after stimulation with IL25 or IL33, as the cellular source of IL5 and IL13. 11 IL25R/IL33R doubly deficient mice have a severely impaired response to helminth infection. Interestingly, although mice deficient for IL25 or IL33 or the receptors for IL25 or IL33 have impaired ILC2 responses, [12][13][14] some functional ILC2s still remain, indicating the existence of additional pathways that support ILC2 biology. As ILC2s are found predominantly at epithelial barriers, which when perturbed elicit a flux of inflammatory factors including IL25 and IL33, suggest that other inflammatory mediators may regulate their generation and function. Indeed, it has been recently reported that ILC2s found in the skin respond synergistically to thymic stromal lymphopoietin (TSLP) and IL33. 15 TL1A belongs to tumor necrosis factor (TNF) superfamily (TNFSF), a family of cytokines that are evolutionally conserved and play important roles in regulating both innate and adaptive immune cells. 16 TL1A is highly expressed by activated myeloid cells and stressed epi-and endothelial cells in the gut and lung barriers. Genome-wide association studies have identified single-nucleotide polymorphisms in TL1A associated with risk for inflammatory bowel disease (IBD). 17,18 TL1A is known to regulate adaptive immune responses in the gut and lung. Mice deficient for TL1A or DR3 (TNFRSF25; the only known receptor for TL1A) have reduced severity of diseases in autoimmune disease models such as asthma model, colitis model, or experimental autoimmune encephalomyelitis model, suggesting that this pathway is important in shaping various immune responses, including type 2 immune responses. 16 TL1A has been ascribed a key role in mucosal immunity. 16 Overexpression of TL1A results in small intestinal inflammation, suggesting their importance at mucosal barriers. [19][20][21] is expressed on CD161 þ gut-homing T cells that respond to TL1A in vitro by expressing proinflammatory cytokines such as interferon-g, TNF, IL6, and IL17. 22 Moreover, recent data suggest that TL1A induces IL13 production also from non-T/ natural killer T-cell compartment in vivo, 19 suggesting innate cells may also respond to TL1A.
Here we elucidate that TL1A is a novel player in ILC2 mucosal biology. We subjected ILC2 cells to microarray and report that DR3 is constitutively expressed on ILC2s. Engagement of DR3 with TL1A promoted ILC2 expansion, survival, and function in vitro and in vivo. We demonstrate that the TL1A/DR3 pathway plays a key role in promoting ILC2s at mucosal barriers and, using relevant receptor-deficient and Rag-deficient mice, we show this was specific to TL1A and independent of IL25 and IL33. Moreover, mice deficient for DR3 not only had impaired responses to control helminthic infections in the gut due to reduced functions of ILC2, but also failed to mount ILC2mediated type 2 inflammation in the lung after nasal challenge with papain, providing a functional relevance for this pathway in physiological inflammatory responses. Our data demonstrate a key role for TL1A in promoting ILC2 proliferation and activation at mucosal barriers.

ILC2s constitutively express DR3
IL25 and IL33 are two key cytokines that support ILC2 differentiation and function. 23 Interestingly, although mice deficient for IL25 or IL33 or their receptors have impaired ILC2 responses, 6,12-14 some functional ILC2s still remain, indicating the existence of additional pathways that support ILC2 biology. Consequently, we determined whether other pathways are necessary for eliciting ILC2 responses. We identified dr3 as a highly expressed gene on human ILC2s (Lin À CRTH2 þ CD161 þ CD127 þ cells) by microarray analysis and corroborated this by real-time quantitative PCR (Supplementary Figure S1a-d online). We confirmed cell-surface DR3 protein expression by fluorescence-activated cell sorting (FACS) on human ILC2s from human blood (Lin À CRTH2 þ CD161 þ CD127 þ cells; (Figure 1a) and murine ILC2s (Lin À Sca1 þ CD127 þ ST2 þ cells; Supplementary Figure S2a online) from mesenteric lymph node (mLN) and lung as well as in bone marrow (KLRG1 À ILC2 precursors 24,25 ) from naive wild-type (WT) mice ( Figure 1b).
We next determined a functional role for DR3 on ILC2s. Murine ILC2s were purified, expanded in vitro with ILC2  cytokine cocktail (IL2, IL7, IL25, and IL33), and their lineage confirmed by IL5 production in response to IL25 and IL33 (Supplementary Figure S2b online). FACS analysis confirmed that these cells retained surface expression of DR3 ( Figure 1c). Importantly, stimulation of the ILC2s with recombinant TL1A (rTL1A) alone, in the absence of exogenous IL2 and IL7, significantly induced the production of IL5 and IL13 in vitro (Figure 1c).

TL1A directly promotes ILC2s in vivo
Given ILC2 responsiveness to TL1A, we reasoned that TL1Amediated immunopathology might be driven, in part, by innate lymphocytes, such as ILC2s. In order to further study the functions of TL1A/DR3 pathway on ILC2s, we generated transgenic mice expressing TL1A, under the CD11c promoter, and crossed the transgenic mice with Rag2 À / À mice (Supplementary Figure S3a To further confirm these results, we directly administered rTL1A into WT, Rag2 À / À , and Rag2 À / À Il2rg À / À mice intraperitoneally (i.p.) to assess IL5 production in the peritoneum. Exogenous TL1A delivered i.p. elicited levels of IL5 in Rag2 À / À mice quantitatively similar to that in WT mice, thereby excluding a contribution from T cells, whereas alymphoid Rag2 À / À Il2rg À / À , which lack ILC2s, failed to elicit IL5 production in response to rTL1A ( Figure 2a). As expected, rTL1A failed to promote IL5 in Dr3 À / À (Figure 2b). In addition to increased IL5 production, rTL1A also increased the number of ILC2s in the mLN or lungs of WT and Rag2 À / À mice after i.p. or intranasal challenge with rTL1A, respectively (Figure 2c,d). Together, these data demonstrate that TL1A is sufficient to induce ILC2s directly and independent of adaptive immune cells.
TL1A/DR3 pathway is required for Nippostrongylus brasiliensis worm clearance Several studies have shown that ILC2s are a prominent source of IL5 and IL13 during a protective immune response to helminth infection. 5,6,12 N. brasiliensis is a helminthic worm infection that elicits a type 2 immune response, characterized by induction of ILC2s in the lung and the small intestine, which is necessary for worm clearance. 26 We confirmed that DR3 is expressed on ILC2s-from mLN, lung, and bone marrow-in mice infected with N. brasiliensis (Figure 3a). Cohorts of WT and Dr3 À / À mice were exposed to stage-3 larvae of N. brasiliensis and by day 10, WT mice had efficiently cleared all worms from their intestine, whereas the Dr3 À / À mice had not ( Figure 3b). Analysis of infected mice on day 7 revealed significantly reduced numbers of ILC2s in the bronchoalveolar lavage fluid (BALF) of Dr3 À / À compared with WT mice, whereas the total CD4 þ Th cells were unaffected (Figure 3c). In concordance with reduced ILC2 numbers, the lungs of infected Dr3 À / À mice had significantly less inflammation (Supplementary Figure S4a,b online). Despite comparable Th cell numbers in the BALF, levels of the type 2 cytokines IL5 and IL13 were all reduced in the BALF in Dr3 À / À mice upon infection, implying that TL1A/DR3 pathway may act to amplify the overall type 2 immune response in the infected lungs ( Figure 3d). These data suggest that Dr3 À / À mice were unable  to effectively clear the helminth worm infection because of the reduced numbers of ILC2s, although, as CD4 þ T cells are still present in the Dr3 À / À mice, we cannot exclude an additional contribution of DR3-driven IL5 and IL13 from these adaptive immune cells to worm clearance. Together, this supports a functional role for DR3 in regulating ILC2s in vivo.
Tl1A/DR3 pathway is required for ILC2 responses in lung to papain challenge Studies have shown that intranasal administration of papain will induce robust lung inflammation that depends on ILC2s. 27 Therefore, we used this model to evaluate whether DR3 is required for the generation of ILC2s in the lung. WT and Dr3 À / À mice were challenged with papain (intranasal) three times, and on day 7 immune cells were quantitated in BALF and lung tissue. ILC2s were easily enumerated in the BALF from WT mice, but were undetectable in Dr3 À / À mice ( Figure 4a). Eosinophils respond to IL5 and are commonly associated with mucosal immune responses. Eosinophils in the BALF after papain challenge were significantly reduced in the Dr3 À / À mice ( Figure 4b). Although some ILC2s were still detected in the lung tissue in Dr3 À / À mice, the numbers were significantly less than WT ( Figure 4c,d). In contrast, the total T-cell number and natural killer T cell number in lung tissues remain similar in WT and Dr3 À / À mice (Supplementary Figure S5a-c). To exclude the possibility that the reduced ILC2 numbers in Dr3 À / À mice, after papain challenge, were simply a reflection of lower CD127 levels used to enumerate these cells, we performed similar assessment in Dr3 À / À mice, lacking adaptive immune cells, using additional ILC2 FACS markers. Dr3 À / À mice were crossed to Rag2 À / À (Rag2 À / À Dr3 À / À ) and subjected to intranasal papain challenge together with Rag2 À / À control mice. ILC2s, identified as Lin À sca1 þ ST2 þ CD278 þ , and eosinophils were enumerated in the lung tissue and BALF, respectively. As seen above, ILC2s in Rag2 À / À Dr3 À / À were also significantly reduced after papain challenge compared with Rag2 À / À mice carrying WT Dr3 alleles (Figure 4e,f), as were eosinophils in the BALF (Figure 4g). These results suggest that DR3 is required for ILC2 survival/expansion in the lung to papain challenge.
TL1A synergizes with IL25 and IL33 to enhance ILC2 effector function As our data support a role for TL1A on ILC2 expansion and function, we asked whether TL1A worked independently or in
To exclude the indirect effects of IL25 and/or TL1A on adaptive immune cells, we tested the synergy of IL25 and TL1A in promoting ILC2s in Rag-deficient mice. Our data showed that, as seen in Rag-sufficient Dr3 À / À mice, IL25 induced less ILC2s in Rag2 À / À deficient for DR3, likely accounting for the significant reduction of IL5 in these mice (Figure 5d,e). Recently, it has been shown that GATA3 is critical for ILC2 differentiation and maintenance. 24,25 GATA3 expression upon IL25 stimulation was significantly decreased in ILC2s that lack DR3 (Figure 5f). Consequently, expression of ILC2-associated genes downstream of GATA3, in ILC2s sorted from IL25-treated Dr3 À / À mice, were also inhibited, including IL13, amphiregulin, and ST2 (Supplementary Figure S6a-c). IL17RB mRNA in ILC2s in response to IL25 was, however, unaltered by DR3 deficiency (Supplementary Figure S6d). Although we observed a predominant synergy of TL1A with IL25 rather than with IL33 in ILC2s in vivo, we did observe that both IL25 and IL33 synergized with TL1A in in vitro expanded murine ILC2s (Supplementary Figure S6e,f). To confirm whether TL1A synergized with IL25 in vivo, we repeated the N. brasiliensis infection in Dr3 À / À as above, but with the addition of anti-IL17RB (an IL25 receptor blocking antibody; Figure 5g). As before, the lack of DR3 resulted in delayed worm clearance, as enumerated from the small intestines. Blocking of IL25 in WT mice also significantly delayed worm clearance as previously reported. 6,7 The addition of anti-IL17RB to DR3-deficient mice resulted in significantly impaired clearance of the parasite and higher worm burden than in mice inhibited for DR3 or IL25 (c) Enumeration of ILC2s from mesenteric lymph nodes (mLNs) of WT littermates (Dr3 þ / þ ) or Dr3 À / À mice treated i.p. with IL33 (gray), IL25 (striped), or PBS (white) daily for 4 consecutive days (n ¼ 5 per group). (d, e) Enumeration of (d) mLN ILC2s and (e) serum IL5 levels in Dr3 À / À mice (black bars) or littermates (Dr3 þ / þ , white bars), and Dr3 À / À Rag2 À / À mice (gray bars) or littermates (Dr3 þ / þ Rag2 À / À , striped bars) treated with IL25 daily for 4 consecutive days (n ¼ 4-5 per group). (f) GATA3 mRNA expression in sorted mLN ILC2s from IL25-treated mice in d (Dr3 À / À , black bars; Dr3 þ / þ littermates, white bars; and Dr3 À / À Rag2 À / À , gray bars; and Dr3 þ / þ Rag2 À / À , striped bars; n ¼ 4-5 per group). Data in a-f are plotted as mean and s.e.m. (g) Total number of worms in small intestine of N. brasiliensis-infected Dr3 À / À mice or Dr3 þ / þ WT littermates treated with anti-IL17RB blocking antibody or isotype control, 10 days post infection. Each dot represents data obtained from an individual animal. Mean is plotted as bar graph (n ¼ 5 per group). *Po0.05 and **Po0.01 by Student's t-test. NS, not significant; SI, small intestine.

ARTICLES
MucosalImmunology | VOLUME 7 NUMBER 3 | MAY 2014 pathways alone. These data show that TL1A can synergize with IL25, and maybe IL33 to influence ILC2 generation and effector function, and suggests that TL1A may play feedforward role on ILC2s at different locations.

Human ILC2s
To extend the observation that TL1A acts alone and in synergy with IL25 and IL33, we performed similar experiments with in vitro expanded human ILC2s (Supplementary Figure S1a,d  online). FACS analysis confirmed that human ILC2s retained surface expression of DR3 after expansion with ILC2 cytokine cocktail ( Figure 6a). As seen with murine ILC2s, stimulation of human ILC2s with rTL1A alone, in the absence of exogenous IL2 and IL7, also significantly induced the production of IL5 and IL13 in vitro (Figure 6a). Importantly, rTL1A and IL25 or IL33 together elicit significantly higher IL5 production from ILC2s than IL25 or IL33 alone (in the presence of suboptimal IL2 plus IL7; Figure 6b). This is further confirmed by intracellular FACS analysis (Figure 6c,d). These observations indicate that TL1A can regulate human ILC2s and function synergistically with the two key ILC2 regulators IL25 and IL33 to enhance human ILC2 effector functions.

DISCUSSION
Here we elucidate a novel player in ILC2 mucosal biology, the TNFSF member TL1A. The expression of TL1A in lung and gut inflammation and its genetic association with IBD in genomewide association studies place it in a key role in regulating mucosal immune response. Our data provide the first link of the TNFSF with innate lymphoid cells activation and function, and   Figure S7 online). Our data show that TL1A can synergize with IL25, and to a lesser extent IL33. TSLP was recently identified to play a synergistic role with IL33 in ILC2s in the skin of atopic dermatitis patients. 15 These data suggest that tissue-specific cytokines may work together with IL25 and IL33 in a feed forward manner to reinforce type 2 immune response. All ILCs lack lineage markers (Lin À ) but express the lymphoid progenitor marker IL7Ra (CD127) and the cytokine common gamma (gc) receptor chain, and require IL7 for development. 10 ILC2s additionally express the receptor for IL33, T1/ST2, and the receptor for IL25, IL17RB, 2 and these cytokines are important drivers of ILC2 in the gut and lung. 6,9 Here we used these markers to identify mouse ILC2s from BALF, lung, and mLN and found that DR3 is constitutively expressed by ILC2s at these tissues. DR3 was also expressed by immature ILC2s (KLRG1 À ) in mouse bone marrow. However, DR3-deficient mice had similar numbers of ILC2s as WT littermates, suggesting that DR3 is not essential for ILC2 development. Nevertheless, DR3 is important for ILC2 activation and effector function. We show that the ligand for DR3, TL1A, could elicit ILC2-derived effector cytokines in vitro and in vivo in WT or Rag2-deficient mice. Although the overall magnitude of TL1A effects on IL5 and IL13 was less than IL25 or IL33, the TL1A/DR3 pathway was essential for generation of ILC2s in vivo, as DR3-deficient mice not only had impaired responses to control helminthic infections in the gut, but also failed to mount ILC2-mediated type 2 inflammation in the lung after nasal challenge with papain. Moreover, Rag-deficient Dr3 À / À mice that lack adaptive immune cells were also defective in ILC2 induction upon papain challenge, further supporting a direct functional role for DR3 on ILC2 generation and function. Notably in these models, DR3 deficiency did not alter the number of CD4 þ T cells or natural killer T cells, suggesting that DR3 was required specifically for the generation of pathogenic ILC2s only.
Despite the dependence of ILC2 on IL25 and IL33 for activation and competent effector function, mice deficient in either of these signaling pathways retained some functional ILC2s. [12][13][14] TL1A was capable of inducing ILC2s and IL5 in Il17rb À / À or ST2 À / À mice. Furthermore, we found that TL1A can significantly synergize with IL25 in vivo. ILC2s are detectable in Dr3 À / À mice, and IL33 or IL25 were capable of expanding these cells in vivo. Interestingly, ILC2 numbers induced by IL33 were similar in WT and Dr3 À / À mice; however, ILC2 induced by IL25 were significantly reduced in the Dr3 À / À mice. Although we observed a predominant synergy of TL1A with IL25 rather than with IL33 in ILC2s in vivo, we did observe that both IL25 and IL33 synergized with TL1A in in vitro expanded murine and human ILC2s. Notably, we show that mice in which both DR3 and IL25 were inhibited were unable to clear helminth worm infections, and worm burdens were significantly higher on day 10 after infection compared with inhibition of DR3 or IL25 alone. This may be because of the fact that exogenous recombinant IL33 is a potent inducer of other cytokines, including IL25 as well as TSLP, 28 that may be sufficient to elaborate a maximal ILC2 response, in the absence of DR3. Collectively, the data suggest that TL1A can synergize with IL25 and IL33, and possibly other cytokines, to influence ILC2 generation and effector function.
The expression of TL1A at mucosal sites places it in a key position to regulate the magnitude of the innate response in a feedforward manner. Indeed, other TNFSF members not only regulate adaptive immune cells, but also play essential roles in regulating innate immune responses, providing a link and interplay between cells in these different immune responses. LTi cells are innate lymphoid tissue inducer cells and, as the name implies, induce lymphoid tissue and are therefore cells that can regulate early inflammatory responses, especially in mucosal tissue. TNF, LT, OX40, and LIGHT are all known to regulate LTi cells in the gut. 29 Interestingly, TL1A has been shown to be able to regulate OX40 expression on LTi cells in vitro, implicating a broader effect of TL1A on innate immune cells. 30 Unlike these other TNFSF members, TL1A also plays an important role in regulating ILC2s. Thus, TL1A may have a unique role among TNFSF members in mediating the crosstalk of innate and adaptive immune cells during type 2 responses.
Genome-wide association studies have identified singlenucleotide polymorphisms in TL1A associated with the risk for IBD. 17,18 Intriguingly, transgenic overexpression of TL1A results in intestinal inflammation, specifically ileitis, associated with elevated IL13. [19][20][21] Studies with DR3-or TL1A-deficient mice also highlight an essential role of the DR3/TL1A pathway in amplifying adaptive immune responses in various chronic inflammatory diseases such as in asthma and IBD. 19,20,31,32 DR3 is not essential for Th cell activation or polarization of Th cells to effector Th1, Th2, and Th17 cells, but DR3 is expressed on all these Th subsets and synergizes with other signaling pathways to elaborate effector cytokines from these cells. 33 Intriguingly, pathogenic gut-homing T cells, defined by the surface marker CD161 þ , have been recently shown to express DR3, and to respond to TL1A but not TNF. 22 Although a direct role for ILC2s in IBD pathogenesis is undocumented, these cells and type 2 effector cytokines, such as IL5 and IL13, can contribute to goblet cell proliferation and fibrosis. As such, activated ILC2s may underscore pathogenesis in Crohn's disease in which the ileum is affected. Interestingly, transgenic overexpression of TL1A mice result in ileitis. [19][20][21] In our hands, the ileum of transgenic mice overexpressing TL1A expressed elevated IL5 and IL13, even when crossed onto a Rag2-deficient background. Moreover, Dr3 À / À mice failed to fully expulse N. brasiliensis worms, a helminth infection model that provokes a potent type 2 response and in which worm clearance is dependent on ILC2s in the gut. 6 Our findings demonstrate a key role for TL1A in promoting ILC2 proliferation and activation at mucosal barriers and reveal a previous uncharacterized function for this TNFSF member in regulating primordial ILC biology. We show that TL1A alone is sufficient to activate ILC2s, but that it can also synergize with other inflammatory mediators in local mucosal and epithelial tissue environments. A parallel role for TSLP has been identified for ILC2s in the skin of atopic dermatitis patients. 15 The significant role of TL1A in activating both adaptive immune cells and ILC2 implicate the TL1A/DR3 axis in the pathogenesis of IBD and asthma, and blockade of this pathway may afford clinical benefit in these diseases.

METHODS
Mice. TL1A transgenic mice were made using mouse TL1A complementary DNA (accession: NM_177371.3) driven by murine CD11c promoter. 34 The 750 bp complementary DNA fragment encoding fulllength mouse TL1A was amplified by PCR to the 3 0 of the 5.5 kb CD11c promoter. The construct was linearized by ApoI digestion and purified by gel electrophoresis for microinjection. Founder mice were identified by PCR using primers specific for the human growth hormone sequence in the construct and bred to C57BL/6 and Rag2 À / À mice. The TL1A mRNA expression was confirmed by RT-PCR with TL1Aspecific primers (Mm00770031_m1, Life Technologies, Carlsbad, CA).
Dr3 À / À mice and Il17rb À / À mice were obtained from Lexicon Pharmaceuticals (The Woodlands, TX) 35 and backcrossed onto C57BL/6 or BALB/c background, respectively. ST2 À / À mice were kindly provided by Andrew McKinsey at the Medical Research Council (Cambridge, UK). Rag2 À / À and Rag2 À / À Il2rg À / À mice were from Taconic (Cambridge City, IN). TNFR-deficient mice lacking both TNFR1 (TNFRSF1a) and TNFR2 (TNFRSF1b) (Tnfrsf1a À / À 1b À / À ), and wild-type B6129SF2/J, and BALB/c and C57BL/6 mice were all purchased from The Jackson Laboratory (Bar Harbor, ME). All animals used in this study were housed and maintained in pathogen-free conditions at Genentech (South San Francisco, CA) in accordance with the American Association of Laboratory Animal Care guidelines. All experimental studies were conducted under the approval of the Institutional Animal Care and Use Committees of Genentech Lab Animal Research.
Reagents. The extracellular domain portion of murine TL1A (amino acids (a.a.) 72-270) was cloned onto pST239 vector. The protein was purified from Escherichia coli on NiNTA column. The formation of trimer was confirmed by size-exclusion chromatography/multiangle light analysis. For recombinant murine IL33, the IL33 cytokine domain (a.a. 109-266) was cloned into a pET28 E. coli expression vector containing the N-terminal 6 Â His-tag. For recombinant human IL33, the cytokine domain (a.a. 112-270) was cloned into the pET28 E. coli expression vector containing the N-terminal 6 Â His-tag. For recombinant murine IL25, the murine IL25 cytokine domain (a.a. 17-169) was subcloned into a pRK expression vector containing the C-terminal 8 Â His-tag. For recombinant human IL25, the cytokine domain (a.a. 1-177) was cloned into pRK vector with the C-terminal FLAG tag and also produced in Chinese hamster ovary (CHO) cells. The E. coli-and CHO cell-produced protein was purified under native condition over a NiNTA column followed by purification over a gel filtration column. Recombinant human IL2 is from Peprotech (Rocky Hill, NJ). Other recombinant cytokines used for cell culture are all from R&D Systems (Minneapolis, MN).
Generation of anti-IL17RB blocking antibody. Monoclonal mouse anti-mouse IL17RB antibody was generated by immunizing IL17RBdeficient mice with fusion protein composed of mouse IL17RB extracellular domain (a.a. 18-282) and mouse IgG2a Fc domain. Clone 1E8 was selected based on its ability to block the interaction between IL17RB-Fc and plate-coated mouse IL25 and its ability to block IL25induced ILC2 expansion and IL5 production in vivo. The selected anti-IL17RB antibody was then sub-cloned into vector of mouse IgG2a backbone with 2 mutations (D265A/N297A) known to abolish FcR binding and expressed in CHO cells for purification.
Flow cytometry. Single-cell suspensions from mLNs), bone marrow, isolated lung lymphocytes, or BALF were washed in phosphatebuffered saline (PBS) containing 5 mM EDTA and 0.5% bovine serum albumin. Cells were incubated for 15 min with Fc receptor block (clone 2.4G2, BD Pharmingen, San Diego, CA) before antibody staining. Propidium iodide was used to discriminate between viable and dead cells. All the antibodies and isotype-matched controls, unless otherwise addressed, were from BD Biosciences (San Diego, CA), eBiosciences (San Diego, CA), or Biolegend (San Diego, CA). Cells were stained with indicated antibodies and sorted on a BD FACS Aria or analyzed on a FACSCalibur or LSRII (BD Biosciences). Flow cytometry data were analyzed using FlowJo software (Treestar, Ashland, OR). For determination of absolute cell numbers, CaliBRITE APC Beads (BD Biosciences) were added before analyzing samples by flow cytometry, and total cell numbers were determined according to the manufacturer's instructions.
Real-time PCR. RNA was isolated from in vitro expanded human ILC2s from five different donors or sorted murine MLN ILC2s with an RNeasy mini kit (Qiagen). An ABI 7500 Real-Time PCR system (Applied Biosystems) was used for real-time RT-PCR. Primers for human DR3, human TNFR1, murine GATA3, murine amphiregulin, murine IL17RB, murine ST2, and murine IL13 are all from ABI (Life Technologies). Data were normalized to results obtained for ribosomal protein L19, 38 and are presented as 2 À DCt .
Ileum organ culture. Ileum was removed, cut open longitudinally, and washed in PBS containing penicillin and streptomycin. The ileum was then placed in 24-well flat-bottom tissue culture plate containing 1 ml of fresh RPMI-1640 with 1% penicillin and streptomycin and incubated at 37 1C for 24 h. Culture supernatants were harvested and assayed for cytokines.
Cytokine injections. For intraperitoneal injections, rmTL1A (5 mg), rmIL25 (500 ng), or rmIL33 (500 ng) were administrated as indicated. PBS was used as control. The mLNs were collected for FACS and serum were collected for cytokine analysis. For intranasal administration, 10 mg of rmTL1A was used. Peritoneal lavage was collected by flushing the abdominal cavity with 1 ml PBS for cytokine analysis, or with 4 ml of PBS for cell enumeration and FACS. BALF was performed by flushing out the lung with 1.5 ml PBS via a tracheal cannula, and lung lymphocytes were isolated after collagenase D digestion. The cells and BALF were then subjected to FACS and/or cytokine analysis.
Cytokine assays. Intracellular detection of IL5 and IL13 was performed using the BD Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Pharmingen) following the manufacturer's protocol with the following antibodies: PE-IL5 (clone TRFK5, eBioscience) and AF647-IL13 (clone eBio13A, eBioscience). Cytokine concentrations were measured in cell culture supernatants with ELISA kit (eBioscience) or with Bio-plex beads (Bio-Rad, Hercules, CA) and subsequent detection in Luminex 100 system according to the manufacturer's recommended protocol (Luminex, Austin, TX).
Intranasal papain challenge. Groups of mice were anesthetized with isoflurane and exposed intranasally to 25 mg papain (Wako, Richmond, VA) in 50 ml PBS on days 0, 3, and 6. On day 7, BAL was performed by flushing out the lung with 1.5 ml PBS via a tracheal cannula. Diced lung fragments were digested in buffer containing collagenase D for 30 min at 37 1C. Lung homogenate was obtained by meshing through a 40-mm cell strainer and the cells were further purified using a 37.5% percoll gradient. Cells were stained for surface antigens.
N. brasiliensis infection. Infective, third-stage larvae of N. brasiliensis (L3) (specimens on file at the US National Parasite Collection, US National Helminthological Collection, Collection 81930, Beltsville, MD) were propagated and stored at room temperature in fecal/ charcoal/peat moss culture plates until use. Then, 8-10-week-old Dr3 À / À mice and Dr3 þ / þ WT littermates were infected subcutaneously with 500 N. brasiliensis L3 larvae in 200 ml of saline on the flank. Animals were placed on polymixin b and neomycin-medicated water for 5 days after infection. When indicated, each mouse was given 200 mg antibodies i.p. every 2 days starting 1 day before infection. Mice were killed and studied at day 7 or day 10 after infection. The mLNs, BALF, lungs, and serum were harvested 7 days after infection. At day 10 after infection, adult worms were detected quantitatively by incubating the opened small intestine in 1 Â Hanks' Balanced Salt Solution (HBSS; Gibco) at 37 1C for 1 h and counting under a microscope. In general, WT mice completely expelled worms by day 10 after inoculation, and therefore, the presence of adult worms in the intestine indicates delayed expulsion.
Histology. Tissue samples were fixed in formalin and processed to paraffin blocks. Sections (4 mm) were stained with hematoxylin and eosin. Pulmonary lesions were scored for inflammation, granuloma formation, and airway goblet cell hyperplasia. Aggregate lesion severity scores from 0 to 10 were determined for each sample and used for groupwise comparison of score means.
Statistics. Statistical analyses were performed using JMP version 8.0.2 software (SAS Institute, San Francisco, CA). We made comparisons for each pair with Student's t-test, with P-values o0.05 considered significant.
SUPPLEMENTARY MATERIAL is linked to the online version of the paper at http://www.nature.com/mi