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CD4+ TH cells develop into subsets that are specialized in the secretion of particular cytokines to mediate restricted types of inflammation and immune responses. Among the subsets that promote development of allergic inflammatory responses, IL-9–producing TH9 cells are regulated by a number of transcription factors. We have previously shown that the E26 transformation-specific (Ets) family members PU.1 and Ets translocation variant 5 (ETV5) function in parallel to regulate IL-9. In this study we identified a third member of the Ets family of transcription factors, Ets-related gene (ERG), that mediates IL-9 production in TH9 cells in the absence of PU.1 and ETV5. Chromatin immunoprecipitation assays revealed that ERG interaction at the Il9 promoter region is restricted to the TH9 lineage and is sustained during murine TH9 polarization. Knockdown or knockout of ERG during murine or human TH9 polarization in vitro led to a decrease in IL-9 production in TH9 cells. Deletion of ERG in vivo had modest effects on IL-9 production in vitro or in vivo. However, in the absence of PU.1 and ETV5, ERG was required for residual IL-9 production in vitro and for IL-9 production by lung-derived CD4 T cells in a mouse model of chronic allergic airway disease. Thus, ERG contributes to IL-9 regulation in TH9 cells.

Differing from the mouse Foxp3 gene that encodes only one protein product, human FOXP3 encodes two major isoforms through alternative splicing—a longer isoform (FOXP3 FL) containing all the coding exons and a shorter isoform lacking the amino acids encoded by exon 2 (FOXP3 ΔE2). The two isoforms are naturally expressed in humans, yet their differences in controlling regulatory T cell phenotype and functionality remain unclear. In this study, we show that patients expressing only the shorter isoform fail to maintain self-tolerance and develop immunodeficiency, polyendocrinopathy, and enteropathy X-linked (IPEX) syndrome. Mice with Foxp3 exon 2 deletion have excessive follicular helper T (T FH ) and germinal center B (GC B) cell responses, and develop systemic autoimmune disease with anti-dsDNA and antinuclear autoantibody production, as well as immune complex glomerulonephritis. Despite having normal suppressive function in in vitro assays, regulatory T cells expressing FOXP3 ΔE2 are unstable and sufficient to induce autoimmunity when transferred into Tcrb -deficient mice. Mechanistically, the FOXP3 ΔE2 isoform allows increased expression of selected cytokines, but decreased expression of a set of positive regulators of Foxp3 without altered binding to these gene loci. These findings uncover indispensable functions of the FOXP3 exon 2 region, highlighting a role in regulating a transcriptional program that maintains T reg stability and immune homeostasis.

Preserving appropriate function and metabolism in regulatory T (Treg) cells is crucial for controlling immune tolerance and inflammatory responses. Yet how Treg cells coordinate cellular metabolic programs to support their functional specification remains elusive. In this study, we report that BATF couples the TH2-suppressive function and triglyceride (TG) metabolism in Treg cells for controlling allergic airway inflammation and IgE responses. Mice with Treg-specific ablation of BATF developed an inflammatory disorder characterized by TH2-type dominant responses and were predisposed to house dust mite–induced airway inflammation. Loss of BATF enabled Treg cells to acquire TH2 cell–like characteristics. Moreover, BATF-deficient Treg cells displayed elevated levels of cellular TGs, and repressing or elevating TGs, respectively, restored or exacerbated their defects. Mechanistically, TCR/CD28 costimulation enhanced expression and function of BATF, which sustained IRF4 activity to preserve Treg cell functionality. Thus, our studies reveal that BATF links Treg cell functional specification and fitness of cellular TGs to control allergic responses, and suggest that therapeutic targeting of TG metabolism could be used for the treatment of allergic disease.

Asthma is a chronic inflammatory lung disease with intermittent flares predominately mediated through memory T cells. Yet, the identity of long-term memory cells that mediate allergic recall responses is not well defined. In this report, using a mouse model of chronic allergen exposure followed by an allergen-free rest period, we characterized a subpopulation of CD4+T cells that secreted IL-9 as an obligate effector cytokine. IL-9–secreting cells had a resident memory T cell phenotype, and blocking IL-9 during a recall challenge or deleting IL-9 from T cells significantly diminished airway inflammation and airway hyperreactivity. T cells secreted IL-9 in an allergen recall–specific manner, and secretion was amplified by IL-33. Using scRNA-seq and scATAC-seq, we defined the cellular identity of a distinct population of T cells with a proallergic cytokine pattern. Thus, in a recall model of allergic airway inflammation, IL-9 secretion from a multicytokine-producing CD4+T cell population was required for an allergen recall response.

Despite IL-9 functioning as a pleiotropic cytokine in mucosal environments, the IL-9–responsive cell repertoire is still not well defined. Here, we found that IL-9 mediates proallergic activities in the lungs by targeting lung macrophages. IL-9 inhibits alveolar macrophage expansion and promotes recruitment of monocytes that develop into CD11c + and CD11c − interstitial macrophage populations. Interstitial macrophages were required for IL-9–dependent allergic responses. Mechanistically, IL-9 affected the function of lung macrophages by inducing Arg1 activity. Compared with Arg1-deficient lung macrophages, Arg1-expressing macrophages expressed greater amounts of CCL5. Adoptive transfer of Arg1 + lung macrophages but not Arg1 − lung macrophages promoted allergic inflammation that Il9r −/− mice were protected against. In parallel, the elevated expression of IL-9, IL-9R, Arg1, and CCL5 was correlated with disease in patients with asthma. Thus, our study uncovers an IL-9/macrophage/Arg1 axis as a potential therapeutic target for allergic airway inflammation.

Botrytis cinerea induced expression of 15 LrWRKY genes; overexpression of LrWRKY39 and LrWRKY41a increased resistance and susceptibility, respectively, to B. cinerea in a manner related to SA and JA signaling. WRKY transcription factors (TFs), a large family, play important roles in coping with biotic stresses. Lilium regale Wilson is a lily species with strong resistance to fungi and viruses; however, functional characterization of LrWRKY TFs remains very limited. Here, a total of 25 LrWRKY members were identified from the L. regale transcriptome, and 15 LrWRKY genes were significantly induced by Botrytis cinerea. Based on their structural features, B. cinerea-responsive LrWRKY genes could be classified into six subgroups (Groups I, IIa-d, and III), and sequence alignment showed that 12 LrWRKY proteins have a well-conserved WRKYGQK domain, while 3 LrWRKYs have a variant sequence (WRKYGKK or WRMYEQK). Quantitative RT-PCR analysis revealed tissue-specific expression of B. cinerea-responsive LrWRKY genes and their expression profiles in response to defense-related hormones salicylic acid (SA), methyl jasmonate (MeJA) and hydrogen peroxide. LrWRKY39 and LrWRKY41a, which encode two LrWRKY TFs with different three-dimensional (3D) models of the WRKY domain, were cloned, and both proteins were targeted to the nucleus. Overexpression of LrWRKY39 and LrWRKY41a in Arabidopsis thaliana increased the resistance and susceptibility to B. cinerea, respectively, compared to the wild type. Similar results were also observed in tobacco and lily (L. longiflorum 'Snow Queen') by transient transformation analyses. Their distinct roles may be related to changes in the transcript levels of SA-/JA-responsive genes. Our results provide new insights into B. cinerea-responsive LrWRKY members and the biological functions of two different 3D models of LrWRKY TFs in defense responses to B. cinerea infection.

Atopic dermatitis results in profound changes in the function of the skin that include diminished barrier function and altered production of antimicrobial peptides. Our previous work in a model of allergic skin inflammation identified a defect in the wound healing process that was dependent on IL-4. In this report, we show that allergic skin inflammation results in a dramatic decrease in the presence of the Vγ3+ dendritic epidermal T-cell (DETC) population of γδ T cells in the skin. In mice that express an active signal transducer and activator of transcription 6 in T cells, DETCs are lost early in life. The loss of DETCs is entirely dependent on IL-4 and is recovered with a genetic deficiency of IL-4. Moreover, injection of IL-4 into wild-type mice results in acute loss of the DETC population. A similar loss of DETCs was observed in mice treated topically with MC903. Wounding of skin from Stat6VT-transgenic or MC903-treated mice resulted in decreased production of DETC-dependent cytokines in the skin, coincident with diminished wound closure. Importantly, intradermal injection of the DETC-produced cytokine fibroblast GF 7 rescued the rate of wound closure in mice with allergic skin inflammation. Together, these results suggest that the atopic environment diminishes prohealing T-cell populations in the skin, resulting in attenuated wound healing responses.

The basic leucine zipper (bZIP) transcription factor BATF is expressed in multiple Th subsets and cooperates with other factors to regulate gene transcription. BATF activates lineage-specific cytokines in Th subsets, activating IL-9 in Th9 cells and IL-17 in Th17 cells, but not IL-9 or IL-17 in the reciprocal subset. The mechanism for this restricted activity is unclear. In this report, we define BATF binding partners that contribute to Th subset–specific functions. Although BATF and IRF4 are expressed in greater amounts in Th9 than Th17, increased expression of both factors is not sufficient to induce IL-9 in Th17 cells. BATF also requires heterodimer formation with Jun family members to bind DNA and induce gene expression. Using primary mouse T cell culture, we observed that JunB and c-Jun, but not JunD, promote IL-9 production in Th9 cells. Ectopic expression of BATF with either JunB or c-Jun generates modest, but significant, increases in IL-9 production in Th17 cells, suggesting that the low expression of Jun family members is one factor limiting the ability of BATF to induce IL-9 in Th17 cells. We further identified that Bach2 positively regulates IL-9 production by directly binding to the Il9 gene and by increasing transcription factor expression in Th9 cells. Strikingly, cotransduction of Bach2 and BATF significantly induces IL-9 production in both Th9 and Th17 cells. Taken together, our results reveal that JunB, c-Jun, and Bach2 cooperate with BATF to contribute to the specificity of BATF-dependent cytokine induction in Th subsets.

IL-9 is an important mediator of allergic disease that is critical for mast cell–driven diseases. IL-9 is produced by many cell types, including T cells, basophils, and mast cells. Yet, how IL-9 is regulated in mast cells or basophils is not well characterized. In this report, we tested the effects of deficiency of a mouse Il9 gene regulatory element (Il9 CNS-25) in these cells in vivo and in vitro. In mast cells stimulated with IL-3 and IL-33, the Il9 CNS-25 enhancer is a potent regulator of mast cell Il9 gene transcription and epigenetic modification at the Il9 locus. Our data show preferential binding of STAT5 and GATA1 to CNS-25 over the Il9 promoter in mast cells and that T cells and mast cells have differing requirements for the induction of IL-9 production. Il9 CNS-25 is required for IL-9 production from T cells, basophils, and mast cells in a food allergy model, and deficiency in IL-9 expression results in decreased mast cell expansion. In a Nippostrongylus brasiliensis infection model, we observed a similar decrease in mast cell accumulation. Although decreased mast cells correlated with higher parasite egg burden and delayed clearance in vivo, T cell deficiency in IL-9 also likely contributes to the phenotype. Thus, our data demonstrate IL-9 production in mast cells and basophils in vivo requires Il9 CNS-25, and that Il9 CNS-25–dependent IL-9 production is required for mast cell expansion during allergic intestinal inflammation.

The simple, rapid, sensitive, and specific detection of cancer cells plays a pivotal role in the diagnosis and prognosis of cancer. We developed a novel DNA-labeled sandwich electrochemical biosensor based on a glassy carbon electrode modified with 3D graphene and a hybrid of Au nanocages (Au NCs)/amino-functionalized multiwalled carbon nanotubes (MWCNT-NH2) for label-free and selective detection of MCF-7 breast cancer cells via differential pulse voltammetry. The layer-by-layer assembly and cell-detection performance of the Au NCs/MWCNTs-NH2-based biosensor were investigated using scanning electron microscopy and electrochemical methods including cyclic voltammetry and electrochemical impedance spectroscopy. Owing to the advantages of DNA-labeled antibodies and a nanomaterial-based signal amplification strategy, the fabricated cytosensor exhibited high specificity and sensitivity when detecting MCF-7 cells in the range of 1.0 × 102 to 1.0 × 106 cells mL−1 with a low detection limit of 80 cells mL−1 (3σ/slope). Furthermore, the biosensor exhibited high selectivity when detecting MCF-7 cells and showed considerable potential for practical applications. The proposed DNA-labeled sandwich electrochemical biosensor provides a stable, sensitive approach to detecting cancer cells and is promising in terms of potential applications to cancer diagnosis.

T helper cell differentiation requires lineage-defining transcription factors and factors that have shared expression among multiple subsets. BATF is required for development of multiple Th subsets but functions in a lineage-specific manner. BATF is required for IL-9 production in Th9 cells but in contrast to its function as a pioneer factor in Th17 cells, BATF is neither sufficient nor required for accessibility at the Il9 locus. Here we show that STAT5 is the earliest factor binding and remodeling the Il9 locus to allow BATF binding in both mouse and human Th9 cultures. The ability of STAT5 to mediate accessibility for BATF is observed in other Th lineages and allows acquisition of the IL-9-secreting phenotype. STAT5 and BATF convert Th17 cells into cells that mediate IL-9-dependent effects in allergic airway inflammation and anti-tumor immunity. Thus, BATF requires the STAT5 signal to mediate plasticity at the Il9 locus., BATF is a transcription factor that is needed for IL-9 production by T helper 9 cells. Here the authors show that STAT5 is needed at the Il9 locus to enable BATF to function in this manner and that this interaction can reprogram other T helper subsets into IL-9 producing cells, thus regulating the immune response to disease.

In plants, genes involved in the Phenylpropanoid/monolignol pathway play important roles in lignin biosynthesis and plant immunity. However, their biological function in Lilium remains poorly characterized. Comparative RNA sequencing of the expression profiles of the monolignol pathway genes from fungi-resistant species Lilium regale after inoculation with Botrytis cinerea was performed. One upregulated caffeoyl-CoA O-methyltransferase gene, LrCCoAOMT, was cloned for functional characterization by reverse genetic methods. LrCCoAOMT encodes a putative protein of 246 amino acids and is highly expressed in stem tissues and responsive to salicylic acid (SA) signaling and B. cinerea infection. LrCCoAOMT was largely directed to the cytoplasm. LrCCoAOMT overexpression in Arabidopsis resulted in an increased lignin deposition in vascular tissues and conferred resistance to B. cinerea infection in transgenic plants. Transient transformation of LrCCoAOMT in nonresistant Lilium sargentiae leaves also identified the defense function to B. cinerea. In addition, transcript levels of genes involved in the monolignol and SA-dependent signaling pathways were altered in transgenic Arabidopsis, suggesting that LrCCoAOMT might play vital roles in the resistance of L. regale to B. cinerea related to the levels of lignin and the regulation of SA signaling. This is the first report to functionally characterize a CCoAOMT gene in Lilium, a potential molecular target for lily molecular improvement.

Eucalyptus is a worldwide hard-wood species which increasingly focused on. To adapt to various biotic and abiotic stresses, Eucalyptus have evolved complex mechanisms, increasing the cellular concentration of reactive oxygen species (ROS) by numerous ROS controlling enzymes. To better analyse the ROS gene network and discuss the differences between four Eucalyptus species, ROS gene network including 11 proteins families (1CysPrx, 2CysPrx, APx, APx-R, CIII Prx, Diox, GPx, Kat, PrxII, PrxQ and Rboh) were annotated and compared in an expert and exhaustive manner from the genomic data available from E. camaldulensis, E. globulus, E. grandis, and E. gunnii. In addition, a specific sequencing strategy was performed in order to determine if the missed sequences in at least one organism are the results of gain/loss events or only sequencing gaps. We observed that the automatic annotation applied to multigenic families is the source of miss-annotation. Base on the family size, the 11 families can be categorized into duplicated gene families (CIII Prx, Kat, 1CysPrx, and GPx), which contain a lot of gene duplication events and non-duplicated families (APx, APx-R, Rboh, DiOx, 2CysPrx, PrxII, and PrxQ). The gene family sizes are much larger in Eucalyptus than most of other angiosperms due to recent gene duplications, which could give higher adaptability to environmental changes and stresses. The cross-species comparative analysis shows gene gain and loss events during the evolutionary process. The 11 families possess different expression patterns, while in the Eucalyptus genus, the ROS families present similar expression patterns. Overall, the comparative analysis might be a good criterion to evaluate the adaptation of different species with different characters, but only if data mining is as exhaustive as possible. It is also a good indicator to explore the evolutionary process.

The lily, a famous bulbous flower, is seriously affected by high temperatures, which affect their growth and production. To date, the signalling pathways and the molecular mechanisms related to heat response in Lilium have not been elucidated. In this study, a comparative transcriptome analysis was performed in an important thermo-tolerant flower, L. longiflorum, and a thermo-sensitive flower, L. distichum. Lily seedlings were first exposed to heat stress at 42°C for different lengths of time, and the optimal time-points (2 h and 24 h) were selected for RNA sequencing (RNA-seq). Approximately 66.51, 66.21, and 65.36 Mb clean reads were identified from three libraries of L. longiflorum (LL_CK, LL_T2h and LL_T24h, respectively) and 66.18, 66.03, and 65.16 Mb clean reads were obtained from three libraries of L. distichum (LD_CK, LD_T2h and LD_T24h, respectively) after rRNA removing. A total of 34,301 unigenes showed similarity to known proteins in the database NCBI non-redundant protein (NR), Swiss-Prot proteins, InterPro proteins, Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG). In addition, 1,621 genes were differentially expressed in the overlapping libraries between LL_DEGs and LD_DEGs; of these genes, 352 DEGs were obviously upregulated in L. longiflorum and downregulated in L. distichum during heat stress, including 4-coumarate, CoA ligase (4CL), caffeoyl-CoA O-methyltransferase (CCoAOMT), peroxidase, pathogenesis-related protein 10 family genes (PR10s), 14-3-3 protein, leucine-rich repeat receptor-like protein kinase, and glycine-rich cell wall structural protein-like. These genes were mainly involved in metabolic pathways, phenylpropanoid biosynthesis, plant-pathogen interactions, plant hormone signal transduction, and kinase signalling pathways. Quantitative RT-PCR was performed to validate the expression profiling of these DEGs in RNA-seq data. Taken together, the results obtained in the present study provide a comprehensive sequence resource for the discovery of heat-resistance genes and reveal potential key components that are responsive to heat stress in lilies, which may help to elucidate the heat signal transcription networks and facilitate heat-resistance breeding in lily.

Cytokine genes are regulated by multiple regulatory elements that confer tissue-specific and activation-dependent expression. The cis-regulatory elements of the gene encoding IL-9, a cytokine that promotes allergy, autoimmune inflammation and tumor immunity, have not been defined. Here we identify an enhancer (CNS-25) upstream of the Il9 gene that binds most transcription factors (TFs) that promote Il9 gene expression. Deletion of the enhancer in the mouse germline alters transcription factor binding to the remaining Il9 regulatory elements, and results in diminished IL-9 production in multiple cell types including Th9 cells, and attenuates IL-9-dependent immune responses. Moreover, deletion of the homologous enhancer (CNS-18) in primary human Th9 cultures results in significant decrease of IL-9 production. Thus, Il9 CNS-25/IL9 CNS-18 is a critical and conserved regulatory element for IL-9 production., Interleukin-9 (IL-9) is important for allergy, autoimmunity and tumor immunity, but how its expression is regulated is unclear. Here the authors show the essential function of an enhancer, CNS-25 in mouse and CNS-18 in human, for IL-9 expression, with the deletion of this enhancer severely hampering IL-9 production in mice or human cells.

Incompatibility between the stigma and pollen is the key factor limiting distant cross-breeding to produce Lilium Asiatic × Oriental hybrids (AO hybrids). To date, AO hybrids have not been available on the flower market. To produce AO hybrids, three pollination methods were applied to perform distant crosses between the Asiatic (A) hybrid ‘Pollyanna’ and the Oriental (O) hybrid ‘Siberia’ in this study. The seed-setting rate was significantly improved after a salt solution treatment. To clarify the mechanism by which the salt solution treatment allowed cross-incompatibility to be overcome, the effects of NaCl and KCl on the papilla cell morphology, pollen tube growth and seed setting rate were studied in the ‘Pollyanna’ × ‘Siberia’ crosses. After a salt treatment, the papilla cells at each stage swelled significantly, and the viscid exudates covering the papilla cell surface were diluted or dissolved. Furthermore, the pollen germination percentage increased, and the pollen tubes grew rapidly, causing more pollen tubes to enter the treated styles than the control styles. Hybridity of seedlings of ‘Pollyanna’ × ‘Siberia’ were confirmed by simple sequence repeat analysis, and their leaves showed intermediate traits between both the parents. These results suggest that salt solution treatments could be utilized for overcoming cross-incompatibility between A and O lilies to achieve AO hybrids, and that they provide insights into achieving other distant hybridizations in lilies.

Citrus bacterial canker (CBC) caused by Xanthomonas citri subsp. citri (Xcc) is a systemic bacterial disease that affects citrus plantations globally. Biotic stress in plants has been linked to a group of important transcription factors known as Basic Leucine Zippers (BZIPs). In this study, CsBZIP40 was functionally characterized by expression analysis, including induction by Xcc and hormones, subcellular localization, over-expression and RNAi silencing. CsBZIP40 belongs to group D of the CsBZIP family of transcription factors and localizes in the nucleus, potentially serving as a transcriptional regulator. In wild type (WT) plants CsBZIP40 can be induced by plant hormones in addition to infection by Xcc which has given insight into its involvement in CBC. In the present study, over-expression of CsBZIP40 conferred resistance to Xcc while its silencing led to Xcc susceptibility. Both over-expression and RNAi affected salicylic acid (SA) production and expression of the genes involved in the SA synthesis and signaling pathway, in addition to interaction of CsBZIP40 with CsNPR1, as detected by a GST pull-down assay. Taken together, the results of this study confirmed the important role of CsBZIP40 in improving resistance to citrus canker through the SA signaling pathway by the presence of NPR1 to activate PR genes. Our findings are of potential value in the breeding of tolerance to CBC in citrus fruits.

Allergic asthma is a chronic inflammatory lung disease with intermittent flares where patients experience symptoms mediated by memory T cells. Allergen-specific CD4+ tissue resistant memory (Trm) cells in peripheral tissues have been reported to facilitate mucosal barrier immunity. However, the precise role of Trm CD4+ T cells in allergic exacerbations is not clearly defined. In this study we identified interleukin-9 (IL-9)-secreting allergen-specific CD4 T cells with a memory phenotype that regulate recall response to Aspergillus fumigatus. Inhibition of circulating T cells by administering FTY720 in the last month of rest, diminished the total CD4 T cells population in the lung, while the IL-9 secreting CD4 tissue resident cells remained stable. Single-cell RNA-seq and ATAC-seq confirmed the presence of multi-cytokine producing IL-9 secreting CD4 Trms in early recall responses to the allergen. Blockade of IL-9 prior to recall challenge phase reduced overall allergic lung inflammation observed through both flow-cytometry and single cell-RNA-seq analysis. IL-9 neutralization reduced cellularity of several populations in the lung including granulocytes and lymphocytes. Moreover, the expression of genes associated with mucus metaplasia including Muc2, Muc5ac, Muc5b, and Bpifb1 were significantly reduced in type-2 alveolar and epithelial cells with anti-IL-9 treatment. These findings demonstrate that IL-9-producing Trms play an important role in mediating allergic memory responses. IL-9 could be a promising therapeutic target specifically in patients showing symptoms of intermittent allergic response.

Lysin motif receptor-like kinases (LYKs) plays a crucial role in plant-microbe interaction. During microbial infection, LYK recognizes the microbial entry into the host cell and triggers plant defense responses against pathogens. The citrus LYK family and its roles in the citrus bacterial canker (CBC) caused by Xanthomonas citri subsp. citri (Xcc) remains uncertain. In this study, we have performed a systematic annotation of CsLYK family and analyzed the altered expression patterns of genes induced by Xcc and biotic stress-related phytohormones (abscisic acid, methyl-jasmonate, and salicylic acid). We found that the Citrus sinensis genome harbors 9 LYK genes, which were further divided into 3 subgroups, and localized on 5 chromosomes and the unassembled scaffolds. They were characterized by the classic lysM and a Pkinase domain, and were found to be primarily localized on the plasma membrane. Most of the CsLYK family members were involved in a putative protein-protein interaction network. Expression profiles showed that CsLYKs could be induced by Xcc infection and some phytohormones. This highlights the correlation between the LYKs and the CBC resistance. These findings extend our understanding of LYKs in citrus, notably their roles in CBC studies. Besides, this study highlights the potential role of plant LYKs involved in pathogen resistance.

BACKGROUND: Bcl6 is required for the development of T follicular helper cells and T follicular regulatory (Tfr) cells that regulate germinal center responses. Bcl6 also affects the function of regulatory T (Treg) cells. OBJECTIVE: The goal of this study was to define the functions of Bcl6 in Treg cells, including Tfr cells, in the context of allergic airway inflammation. METHODS: We used a model of house dust mite sensitization to challenge wild-type, Bcl6(fl/fl) Foxp3-Cre, and Prdm1 (Blimp1)(fl/fl) Foxp3-Cre mice to study the reciprocal roles of Bcl6 and Blimp1 in allergic airway inflammation. RESULTS: In the house dust mite model, Tfr cells repress the production of IgE and Bcl6(+) Treg cells suppress the generation of type 2 cytokine–producing cells in the lungs. In mice with Bcl6-deficient Treg cells, twice as many ST2(+) (IL-33R(+)) Treg cells develop as are observed in wild-type mice. ST2(+) Treg cells in the context of allergic airway inflammation are Blimp1 dependent, express type 2 cytokines, and share features of visceral adipose tissue Treg cells. Bcl6-deficient Treg cells are more susceptible, and Blimp1-deficient Treg cells are resistant, to acquiring the ST2(+) Treg–cell phenotype in vitro and in vivo in response to IL-33. Bcl6-deficient ST2(+) Treg cells, but not Bcl6-deficient ST2(+) conventional T cells, strongly promote allergic airway inflammation when transferred into recipient mice. Lastly, ST2 is required for the exacerbated allergic airway inflammation in Bcl6(fl/fl) Foxp3-Cre mice. CONCLUSIONS: During allergic airway inflammation, Bcl6 and Blimp1 play dual roles in regulating Tfr-cell activity in the germinal center and in the development of ST2(+) Treg cells that promote type 2 cytokine responses.

Allergic asthma is a chronic intermittent inflammatory lung disease where patients experience relatively few symptoms between exacerbations precipitated by allergen-challenge of allergic memory responses. Allergen-specific CD4+ T cells are believed to be a major mediator of allergic memory responses through the production of type 2 cytokines including IL-4, IL-5, IL-13 and IL-9. Recently, tissue-resident memory (Trm) cells have been identified in peripheral tissues that mediate mucosal barrier immunity. However, the precise role of Trm CD4+ T cells in allergic exacerbations is not clearly defined. In studies to examine the role of IL-9 in allergen-specific Trm responses to Aspergillus fumigatus we observed that all IL-9-secreting T cells had a resident memory phenotype. Inhibition of circulating T cells by administering FTY720 in the last month of rest, diminished the total CD4 T cells population in the lung, while the Th9 cells remained stable. Blockade of IL-9 prior to recall challenge phase reduced overall allergic lung inflammation as assessed both by flow cytometry and single cell RNA-seq. Within eight hours after challenge, IL-9 is one of the top genes identified by scRNA-seq that distinguishes ST2+ and ST2− CD4+ T cell populations in the lung. Using IL-9-reporter mice we also compared gene expression in isolated IL-9+ T cells and other type 2 T cells. These findings demonstrate that IL-9-producing Trms play an important role in mediating allergic memory responses. IL-9 could be a promising therapeutic target specifically in patients showing symptoms of intermittent allergic response.

IL-9 promotes to the development of allergic lung disease through several responsive populations including mast cells, lymphocytes and eosinophils. However, it is not known if these represent the entire IL-9-responsive cell repertoire. In this study, we detected high IL-9R expression on macrophages and these cells occupied a large proportion of IL-9R+ cells in allergic lung. Allergic inflammation leads to a decrease in the number of alveolar macrophages (AM) in wild type mice. However, in Il9r−/− mice, the AM population is not diminished as greatly, suggesting that IL-9 represses AM self-renewal. The level of the IL-9R expression on blood monocyte is corelated with CCR2 expression, which is required for blood monocyte migration to the inflammatory site. In the chronic allergic airway disease model, the circulating monocyte population decreased coincident with an increase in the number of interstitial macrophages in the control mice compared to the Il9r−/− mice, suggesting IL-9 promotes monocyte migration and differentiation in the lung. Interestingly, IL-9 also induces a CD11c+ interstitial macrophage population in the allergic lung. Adoptive transfer of CD11c+ wild type macrophages to the Il9r−/− mice increased the inflammatory response. Thus, our study demonstrated that macrophages are important IL-9-responsive populations in allergic lung disease, and that IL-9 inhibits AM numbers as it promotes the development of pro-inflammatory CD11c+ and CD11c− interstitial macrophages. This balance is important in determining the level of allergic inflammation in the lung.

Mediated by immune responses in the airways, symptoms of allergic airway diseases include shortness of breath, difficulty breathing, and wheezing. Responses in distant tissues, such as increased lymphocyte proliferation and cytokine secretion, are also exhibited. How airway sensitization mediates these distant effects is unclear. We recently described the development and function of granzyme A-expressing T cells that control intestinal inflammation. Using models of chronic and memory allergic airway inflammation, we now show that a granzyme A expressing CD4 T cell population in the intestine increases during these diseases. This response is amplified in mice that lack IL-9 signaling through a deficiency in IL-9 receptor levels (IL9RKO) or receive an IL-9 antibody blockade. In a chronic intranasal allergen challenge model, we saw elevated levels of granzyme A expressing CD4 T cells in IL9RKO mice when compared to wild-type controls. In a memory internasal allergen model using wild-type mice (chronic allergen exposure, followed by an allergen-free rest period, and an allergen re-exposure), granzyme A expressing cells in the intestine were increased upon IL-9 antibody blockade. Blocking IL-9 or administering IL-9 to naïve mice or cell cultures that generate granzyme A expressing T cells did not affect cell prevalence. Therefore, concurrent allergic airway inflammation seems to be required for the proliferation of these granzyme A expressing T cells. Together, these studies suggest that allergic airway inflammation increases granzyme A expressing CD4 T cells in the intestine and that IL-9 attenuates this effect. Moreover, these results show that allergic airway inflammation impacts immunity at a distant mucosal site.

Alzheimer's disease (AD) is the most common cause of dementia. Mice in the transgenic AβPPswe/PS1dE9 mouse line express a chimeric mouse/human amyloid-β protein precursor (Mo/HuAβPP695swe) and mutant human presenilin 1 (PS1-dE9) associated with early-onset AD. Knowing the protein expression in these mice may offer better understanding of the pathological changes in AD. In this study, we used two-dimensional gel electrophoresis combined with mass spectrometry techniques to compare protein expression in AβPPswe/PS1dE9 mice with age-matched wild-type mice throughout the disease progression. We identified 15 proteins that were significantly different between the AβPPswe/PS1dE9 mice and age-matched controls and also changed with disease development. Among those, the expression levels of the following proteins in AβPPswe/PS1dE9 mice were at least 1.5 times higher than those in normal mice: DCC-interacting protein 13-beta, serum albumin, creatine kinase B-type, heat shock 70 kDa protein 1A, T-complex protein 1 subunit beta, adenylate kinase isoenzyme 1, pyruvate dehydrogenase E1 component subunit beta mitochondrial, and V-type proton ATPase catalytic subunit A. Levels of the following proteins in AβPPswe/PS1dE9 mice were at least 1.5 times lower than those in normal mice: dihydropyrimidinase-related protein 2, actin cytoplasmic 2, isoform 1 of V-type proton ATPase catalytic subunit, tubulin alpha-1C chain, F-actin-capping protein subunit alpha-2, ubiquitin carboxyl-terminal hydrolase isozyme L1, and actin cytoplasmic 1. These proteins are involved in regulating various cellular functions, including cytoskeletal structure, energy metabolism, synaptic components, and protein degradation. These findings indicate altered protein expression in the pathogenesis of AD and illuminate novel therapeutic avenues for treatment in AD.

Prion diseases are neurodegenerative disorders caused by the accumulation of misfolded prion proteins [scrapie form of PrP (PrP(Sc))]. PrP(Sc) accumulation in the brain causes neurotoxicity by inducing mitochondrial-apoptotic pathways. Neurodegeneration can be prevented by imatinib mesylate (Gleevec or STI571) that regulates c-Abl tyrosine kinases, which elicit protective effects in neurodegenerative disease models. However, the protective effect of STI571 against prion disease remains unknown. In the present study, the effect of STI571 on prion peptide-induced neuronal death was investigated. Results showed that STI571 rescued neurons from PrP106-126-induced neurotoxicity by preventing mitochondrial dysfunction. STI571-inhibited c-Abl tyrosine kinases prevented PrP106-126-induced reduction in mitochondrial potential, Bax translocation to the mitochondria and cytochrome c release. The protective effect of STI571 against mitochondrial dysfunction was related to the activation of BIM expression. This study is the first to demonstrate the protective effect of STI571 against prion-mediated neurotoxicity. Our results suggested that imatinib mesylate treatment may be a novel therapeutic strategy to treat prion-mediated neurotoxicity.

Auxin response factors (ARFs) are important regulators modulating the expression of auxin-responsive genes in various biological processes in plants. In the Populus genome, a total of 39 ARF members have been identified, but their detailed functions are still unclear. In this study, six poplar auxin response factor 2 (PtrARF2) members were isolated from P. trichocarpa. Expression pattern analysis showed that PtrARF2.1 is highly expressed in leaf tissues compared with other PtrARF2 genes and significantly repressed by exogenous auxin treatment. PtrARF2.1 is a nuclear-localized protein without transcriptional activation activity. Knockdown of PtrARF2.1 by RNA interference (RNAi) in poplars led to the dwarf plant, altered leaf shape, and reduced size of the leaf blade, while overexpression of PtrARF2.1 resulted in a slight reduction in plant height and the similar leaf phenotype in contrast to the wildtype. Furthermore, histological staining analysis revealed an ectopic deposition of lignin in leaf veins and petioles of PtrARF2.1-RNAi lines. RNA-Seq analysis showed that 74 differential expression genes (DEGs) belonging to 12 transcription factor families, such as NAM, ATAF and CUC (NAC), v-myb avian myeloblastosis viral oncogene homolog (MYB), ethylene response factors (ERF) and basic helix&ndash, loop&ndash, helix (bHLH), were identified in PtrARF2.1-RNAi leaves and other 24 DEGs were associated with the lignin biosynthetic pathway. Altogether, the data indicate that PtrARF2.1 plays an important role in regulating leaf development and influences the lignin biosynthesis in poplars.

T cells are critical regulators of inflammatory diseases and immunity. CD4+ T helper cells develop into various subsets that are specialized in the secretion of particular cytokines and mediate restricted types of inflammation. One of these subsets recently identified to play an important role in development of allergic inflammatory response, is the IL-9 producing TH9 subset. TH9 cells differentiate in the presence of IL-4 and TGFβ, which activate a network of transcription factors downstream of STAT6 and SMAD signaling pathways to regulate IL-9 secretion. Previous studies showed that PU.1, an Ets transcription factor, can bind at the Il9 promoter region to regulate TH9 function during pro-allergic inflammation. Furthermore, members of the Ets family of transcription factors Etv5 and PU.1 appear to share overlapping functions to promote IL-9 production and TH9 development. We screened for additional ETS factors that might play a role in IL-9 regulation. Jabeen et. al. showed the Ets transcription factor termed Erg (Ets-related gene) is expressed preferentially in TH9 subset. Accordingly, we determined that siRNA knock down of Erg in TH9 cells decreased IL-9 production in vitro. Moreover, chromatin immunoprecipitation assay demonstrated that Erg binding at the Il9 promoter and enhancer region is enriched in TH9 cells. Furthermore, ectopic expression of Erg induced IL-9 secretion in TH2 cells. In addition to Erg, we identified Fli-1, another Ets transcription factor, that can enhance IL-9 secretion in TH2 subset and whose expression is altered by Erg-deficiency. Thus, multiple Ets transcription factors regulate Il9 expression in TH9 cells, suggesting that Erg and other Ets transcription factors may cooperate to promote TH9 function.

Atopic dermatitis (AD) is a common Th2-biased chronic inflammatory skin disease. Major clinical manifestations include erythema, severe itchiness, and dry skin that impose a burden on patients and impairs their quality of life. Extensive research has been conducted to identify possible underlying mechanisms and develop therapeutic approaches. γδT cells, an understudied T cell population, play important roles in maintaining skin integrity in both human and mice. They are among the first responders to epithelial wounding and are a source of keratinocyte/fibroblast growth factors that promote wound repair. Hence, we wanted to investigate their behavior during wounding in an AD-like environment. Using a mouse model, we observed that γδT cell numbers are drastically decreased in atopic skin. Multiple factors contribute to this decrease, including insufficient activation, less proliferative capacity and increased cell death. These changes in γδT cell function are IL-4 dependent. Administration of IL-4 to wild type (WT) mice resulted in significant loss of γδT cells, and Il4−/− mice in the AD model showed numbers of γδT cells similar to WT controls. We further observed that the levels of keratinocyte/fibroblast growth factors are lower in AD animals, suggesting that loss of γδT cells could compromise re-epithelialization and repair following wounding. Collectively, our data indicate that in an atopic environment, γδT cells decrease drastically in an IL-4 dependent fashion, which may contribute to the aberrant wound healing process in AD animals.

Asthma is a chronic intermittent inflammatory lung disease for which there is no cure. Allergen-specific CD4 T helper (Th) cells that secrete cytokines including IL-4, IL-13, and IL-9 mediate asthma pathogenesis. In a six week chronic model of allergic airway disease, we were able to detect a robust IL-9+CD4+ T cell population in the lungs. Interestingly, the in vivo derived IL-9+CD4+ T cells had a multi-cytokine profile including the production of IL-5, IL-13, and IL-10. IL-9-reporter-positive cells demonstrated high gene expression levels of innate cytokine receptors Il1rl1, Il17rb, and Crlf2 and the transcription factor Gata3. IL-9+CD4+ T cells were found to be tissue-resident through intravenous labeling and had CD4+ tissue-resident memory (Trm) cell markers CD69 and CD11a. To determine the Trm phenotype of lung resident IL-9+ T cells, we developed a memory model using a five week period of rest after six weeks of chronic challenge, before a recall allergen challenge. In the memory model, IL-9+ T cells maintained tissue residency and demonstrated increased cell frequency and IL-9 production, characteristic of a secondary response. Antibody blockade of IL-9 immediately prior to the recall challenge significantly reduced overall allergic lung inflammation, suggesting that IL-9 plays an obligate role in the allergic memory response following pulmonary allergen challenge. These observations further suggest that IL-9 from Trm cell populations plays a novel role in allergen recall responses and is a potential therapeutic target for patients suffering from chronic intermittent asthma.

The basic leucine zipper transcription factor BATF is expressed in multiple Th subsets and cooperates with other factors to regulate gene transcription. BATF activates lineage-specific cytokines in Th subsets, activating IL-9 in Th9 cells and IL-17 in Th17 cells. The mechanism for this restricted activity is unclear. In this report we define BATF binding partners that contribute to Th subset-specific functions. Although BATF and IRF4 are expressed in greater amounts in Th9 than Th17, increased expression of both factors is not sufficient to induce IL-9 in Th17 cells. BATF also requires heterodimer formation with Jun family members to bind DNA and induce gene expression. We observed that JunB and c-Jun, but not JunD, promote IL-9 production in Th9 cells. Ectopic expression of BATF with either JunB or c-Jun generates modest but significant increases in IL-9 production in Th17 cells, suggesting that the low expression of Jun family members is one factor limiting the ability of BATF to induce IL-9 in Th17 cells. We further identified that Bach2 positively regulates IL-9 production by directly binding to the Il9 gene and by increasing transcription factor expression in Th9 cells. Strikingly, co-transduction of Bach2 and BATF significantly induces IL-9 production in both Th9 and Th17 cell. Taken together, our results reveal that JunB, c-Jun and Bach2 cooperate with BATF to contribute to the specificity of BATF-dependent cytokine induction in Th subsets.

The inflammatory responses in Alzheimer's disease and prion diseases are dominated by microglia activation. Three different phenotypes of microglial activation, namely classical activation, alternative activation, and acquired deactivation, have been described. In this study, we investigated the effect of amyloidogenic fragments of amyloid β and prion proteins (Aβ1-42 and PrP106-126) on various forms of microglial activation. We first examined the effect of Aβ1-42 and PrP106-126 stimulation on the mRNA expression levels of several markers of microglial activation, as well as the effect of cytochalasin D, a phagocytosis inhibitor, on microglial activation in Aβ1-42- and PrP106-126-stimulated BV2 microglia. results showed that Aβ1-42 and PrP106-126 induced the classical activation of BV2 microglia, decreased the expression level of alternative expression markers, and had no effect on the expression of acquired deactivation markers. Cytochalasin D treatment significantly reduced Aβ1-42- and PrP106-126-induced up-regulation of proinflammatory factors, but did not change the expression profile of the markers of alternative activation or acquired deactivation in BV2 cells which were exposed to Aβ1-42 and PrP106-126. Our results suggested that microglia interact with amyloidogenic peptides in the extracellular milieu-stimulated microglial classical activation and reduce its alternative activation, and that the uptake of amyloidogenic peptides from the extracellular milieu amplifies the classical microglial activation.

The immune system provides resistance to the myriad of pathogens in the environment, but can also respond inappropriately causing allergic inflammation and autoimmune disease. CD4+ T cells, which play a crucial role in adaptive immune system, can be divided into subsets based on their effector functions. T helper 9 (Th9) cells, derived by the IL-4/STAT6 and TGFβ signaling pathways, produce IL-9 as a hallmark cytokine. Through IL-9 production, Th9 cells protect against parasite infection but are also involved in allergic inflammation and autoimmune diseases. In this study, we demonstrate that a conserved noncoding sequence (CNS) located 25kb upstream of the Il9 transcription start site, termed Il9 CNS-25, is critical for regulating Il9 expression in Th cell subsets and IL-9 producing CD8+ T cells (Tc9). Th9 cells derived from Il9 CNS-25 mutant (Il9ΔCNS-25) mice produce significantly less IL-9. Il9 CNS-25 was bound by factors that promote Il9 gene expression including STATs, IRF4 and BATF, promoted chromatin modifications and transcription factor binding at the promoter, and mediated accessibility of the locus. Il9ΔCNS-25 mice showed attenuated airway inflammation compared to control mice. The Il9 CNS-25 region in mice is conserved with an IL9 CNS-18 region in the human genome. We deleted CNS-18 in primary human Th9 cells and observed diminished IL-9 production. Thus, we have demonstrated that the Il9 CNS-25/IL9 CNS-18 elements are respectively critical for Il9/IL9 gene expression in Th9 cells and other IL-9 producing T cell subsets.