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High-throughput technologies produce gene expression time-series data that need fast and specialized algorithms to be processed. While current methods already deal with different aspects, such as the non-stationarity of the process and the temporal correlation, they often fail to take into account the pairing among replicates. We propose PairGP, a non-stationary Gaussian process method to compare gene expression time-series across several conditions that can account for paired longitudinal study designs and can identify groups of conditions that have different gene expression dynamics. We demonstrate the method on both simulated data and previously unpublished RNA sequencing (RNA-seq) time-series with five conditions. The results show the advantage of modeling the pairing effect to better identify groups of conditions with different dynamics. The pairing effect model displays good capabilities of selecting the most probable grouping of conditions even in the presence of a high number of conditions. The developed method is of general application and can be applied to any gene expression time series dataset. The model can identify common replicate effects among the samples coming from the same biological replicates and model those as separate components. Learning the pairing effect as a separate component, not only allows us to exclude it from the model to get better estimates of the condition effects, but also to improve the precision of the model selection process. The pairing effect that was accounted before as noise, is now identified as a separate component, resulting in more accurate and explanatory models of the data., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: The use of cell-free DNA (cfDNA) as a noninvasive biomarker to detect allograft damage is expanding rapidly. However, quantifying the low fraction of donor-derived cfDNA (ddcfDNA) is challenging and requires a highly sensitive technique. ddcfDNA detection through unique donor single nucleotide polymorphisms (SNPs) is a recent new approach, however there are limited data in pediatric solid organ transplant (SOT) recipients., Methods: We developed an assay using a combination of 61 SNPs to quantify the ddcfDNA accurately using a custom R script to model for both the patient and donor genotypes requiring only a single sample from the allograft recipient. Performance of the assay was validated using genomic DNA (gDNA), cfDNA and donor samples where available., Results: The R "genotype-free" method gave results comparable to when using the known donor genotype. applicable to both related and unrelated pairs and can reliably measure ddcfDNA (limit of blank, below 0.12%; limit of detection, above 0.25%; limit of quantification 0.5% resulting in 84% accuracy). 159 pediatric SOT recipients (kidney, heart, and lung) were tested without the need for donor genotyping. Serial sampling was obtained from 82 patients., Conclusion: We have developed and validated a new assay to measure the fraction of ddcfDNA in the plasma of pediatric SOT recipients. Our method can be applicable in any donor-recipient pair without the need for donor genotyping and can provide results in 48 h at a low cost. Additional prospective studies are required to demonstrate its clinical validity in a large cohort of pediatric SOT recipients., (© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Noninvasive prenatal diagnosis (NIPD) for monogenic disorders has a high uptake by families. Since 2013, our accredited public health service laboratory has offered NIPD for monogenic disorders, predominantly for de novo or paternally dominantly inherited mutations. Here we describe the extension of this service to include definitive NIPD for a recessive condition, cystic fibrosis (CF)., Methods: Definitive NIPD for CF was developed using next-generation sequencing. Validation was performed on 13 cases from 10 families before implementation. All cases referred for CF NIPD were reviewed to determine turnaround times, genotyping results, and pregnancy outcomes., Results: Of 38 referrals, 36 received a result with a mean turnaround of 5.75 days (range, 3-11 days). Nine cases were initially inconclusive, with 3 reported unaffected because the low-risk paternal allele was inherited and 4 cases in which the high-risk paternal allele was inherited, receiving conclusive results following repeat testing. One case was inconclusive owing to a paternal recombination around the mutation site, and one case was uninformative because of no heterozygosity. Before 2016, 3 invasive referrals for CF were received annually compared with 38 for NIPD in the 24 months since offering a definitive NIPD service., Conclusions: Timely and accurate NIPD for definitive prenatal diagnosis of CF is possible in a public health service laboratory. The method detects recombinations, and the service is well-received as evidenced by the significant increase in referrals. The bioinformatic approach is gene agnostic and will be used to expand the range of conditions tested for., (© 2019 American Association for Clinical Chemistry.)

We recently described a new neurodevelopmental syndrome (TAF1/MRXS33 intellectual disability syndrome) (MIM# 300966) caused by pathogenic variants involving the X-linked gene TAF1, which participates in RNA polymerase II transcription. The initial study reported eleven families, and the syndrome was defined as presenting early in life with hypotonia, facial dysmorphia, and developmental delay that evolved into intellectual disability (ID) and/or autism spectrum disorder (ASD). We have now identified an additional 27 families through a genotype-first approach. Familial segregation analysis, clinical phenotyping, and bioinformatics were capitalized on to assess potential variant pathogenicity, and molecular modelling was performed for those variants falling within structurally characterized domains of TAF1. A novel phenotypic clustering approach was also applied, in which the phenotypes of affected individuals were classified using 51 standardized Human Phenotype Ontology (HPO) terms. Phenotypes associated with TAF1 variants show considerable pleiotropy and clinical variability, but prominent among previously unreported effects were brain morphological abnormalities, seizures, hearing loss, and heart malformations. Our allelic series broadens the phenotypic spectrum of TAF1/MRXS33 intellectual disability syndrome and the range of TAF1 molecular defects in humans. It also illustrates the challenges for determining the pathogenicity of inherited missense variants, particularly for genes mapping to chromosome X. This article is protected by copyright. All rights reserved., (This article is protected by copyright. All rights reserved.)

Importance: Neuroinflammatory disorders are a range of severe neurological disorders causing brain and spinal inflammation and are now increasingly recognized in the pediatric population. They are often characterized by marked genotypic and phenotypic heterogeneity, complicating diagnostic work in clinical practice and molecular diagnosis., Objective: To develop and evaluate a next-generation sequencing panel targeting genes causing neuroinflammation or mimicking neuroinflammation., Design, Setting, and Participants: Cohort study in which a total of 257 genes associated with monogenic neuroinflammation and/or cerebral vasculopathy, including monogenic noninflammatory diseases mimicking these entities, were selected. A customized enrichment capture array, the neuroinflammation gene panel (NIP), was created. Targeted high-coverage sequencing was applied to DNA samples taken from eligible patients referred to Great Ormond Street Hospital in London, United Kingdom, between January 1, 2017, and January 30, 2019, because of onset of disease early in life, family history, and/or complex neuroinflammatory phenotypes., Main Outcomes and Measures: The main outcome was the percentage of individuals with definitive molecular diagnoses, variant classification, and clinical phenotyping of patients with pathogenic variants identified using the NIP panel. The NIP panel was initially validated in 16 patients with known genetic diagnoses., Results: The NIP was both sensitive (95%) and specific (100%) for detection of known mutations, including gene deletions, copy number variants, small insertions and deletions, and somatic mosaicism with allele fraction as low as 3%. Prospective testing of 60 patients (30 [50%] male; median [range] age, 9.8 [0.8-20] years) presenting with heterogeneous neuroinflammatory phenotypes revealed at least 1 class 5 (clearly pathogenic) variant in 9 of 60 patients (15%); 18 of 60 patients (30%) had at least 1 class 4 (likely pathogenic) variant. Overall, a definitive molecular diagnosis was established in 12 of 60 patients (20%)., Conclusions and Relevance: The NIP was associated with molecular diagnosis in this cohort and complemented routine laboratory and radiological workup of patients with neuroinflammation. Unexpected genotype-phenotype associations in patients with pathogenic variants deviating from the classic phenotype were identified. Obtaining an accurate molecular diagnosis in a timely fashion informed patient management, including successful targeted treatment in some instances and early institution of hematopoietic stem cell transplantation in others.

Down syndrome (DS), trisomy of human chromosome 21 (Hsa21), results in a broad range of phenotypes. A recent study reported that DS cells show genome-wide transcriptional changes in which up- or down-regulated genes are clustered in gene expression dysregulation domains (GEDDs). GEDDs were also reported in fibroblasts derived from a DS mouse model duplicated for some Hsa21-orthologous genes, indicating cross-species conservation of this phenomenon. Here we investigate GEDDs using the Dp1Tyb mouse model of DS, which is duplicated for the entire Hsa21-orthologous region of mouse chromosome 16. Our statistical analysis shows that GEDDs are present both in DS cells and in Dp1Tyb mouse fibroblasts and hippocampus. However, we find that GEDDs do not depend on the DS genotype but occur whenever gene expression changes. We conclude that GEDDs are not a specific feature of DS but instead result from the clustering of co-regulated genes, a function of mammalian genome organisation.

Purpose: To develop a comprehensive next-generation sequencing panel assay that screens genes known to cause developmental eye disorders and inherited eye disease and to evaluate its diagnostic yield in a pediatric cohort with malformations of the globe, anterior segment anomalies, childhood glaucoma, or a combination thereof., Design: Evaluation of diagnostic test., Participants: Two hundred seventy-seven children, 0 to 16 years of age, diagnosed with nonsyndromic or syndromic developmental eye defects without a genetic diagnosis., Methods: We developed a new oculome panel using a custom-designed Agilent SureSelect QXT target capture method (Agilent Technologies, Santa Clara, CA) to capture and perform parallel high-throughput sequencing analysis of 429 genes associated with eye disorders. Bidirectional Sanger sequencing confirmed suspected pathogenic variants., Main Outcome Measures: Collated clinical details and oculome molecular genetic results., Results: The oculome design covers 429 known eye disease genes; these are subdivided into 5 overlapping virtual subpanels for anterior segment developmental anomalies including glaucoma (ASDA; 59 genes), microphthalmia-anophthalmia-coloboma (MAC; 86 genes), congenital cataracts and lens-associated conditions (70 genes), retinal dystrophies (RET; 235 genes), and albinism (15 genes), as well as additional genes implicated in optic atrophy and complex strabismus (10 genes). Panel development and testing included analyzing 277 clinical samples and 3 positive control samples using Illumina sequencing platforms; more than 30× read depth was achieved for 99.5% of the targeted 1.77-Mb region. Bioinformatics analysis performed using a pipeline based on Freebayes and ExomeDepth to identify coding sequence and copy number variants, respectively, resulted in a definitive diagnosis in 68 of 277 samples, with variability in diagnostic yield between phenotypic subgroups: MAC, 8.2% (8 of 98 cases solved); ASDA, 24.8% (28 of 113 cases solved); other or syndromic, 37.5% (3 of 8 cases solved); RET, 42.8% (21 of 49 cases solved); and congenital cataracts and lens-associated conditions, 88.9% (8 of 9 cases solved)., Conclusions: The oculome test diagnoses a comprehensive range of genetic conditions affecting the development of the eye, potentially replacing protracted and costly multidisciplinary assessments and allowing for faster targeted management. The oculome enabled molecular diagnosis of a significant number of cases in our sample cohort of varied ocular birth defects., (Copyright © 2019 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Interleukin (IL)-36α, IL-36β, and IL-36γ are innate mediators of acute epithelial inflammation. We sought to demonstrate that these cytokines are also required for the pathogenesis of plaque psoriasis, a common and chronic skin disorder, caused by abnormal T helper 17 (T H 17) cell activation. To investigate this possibility, we first defined the genes that are induced by IL-36 cytokines in primary human keratinocytes. This enabled us to demonstrate a significant IL-36 signature among the transcripts that are up-regulated in plaque psoriasis and the susceptibility loci associated with the disease in genome-wide studies. Next, we investigated the impact of in vivo and ex vivo IL-36 receptor blockade using a neutralizing antibody or a recombinant antagonist. Both inhibitors had marked anti-inflammatory effects on psoriatic skin, demonstrated by statistically significant reductions in IL-17 expression, keratinocyte activation, and leukocyte infiltration. Finally, we explored the potential safety profile associated with IL-36 blockade by phenotyping 12 individuals carrying knockout mutations of the IL-36 receptor gene. We found that normal immune function was broadly preserved in these individuals, suggesting that IL-36 signaling inhibition would not substantially compromise host defenses. These observations, which integrate the results of transcriptomics and model system analysis, pave the way for early-stage clinical trials of IL-36 antagonists., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background: Monogenic autoinflammatory diseases (AID) are a rapidly expanding group of genetically diverse but phenotypically overlapping systemic inflammatory disorders associated with dysregulated innate immunity. They cause significant morbidity, mortality and economic burden. Here, we aimed to develop and evaluate the clinical impact of a NGS targeted gene panel, the "Vasculitis and Inflammation Panel" (VIP) for AID and vasculitis., Methods: The Agilent SureDesign tool was used to design 2 versions of VIP; VIP1 targeting 113 genes, and a later version, VIP2, targeting 166 genes. Captured and indexed libraries (QXT Target Enrichment System) prepared for 72 patients were sequenced as a multiplex of 16 samples on an Illumina MiSeq sequencer in 150bp paired-end mode. The cohort comprised 22 positive control DNA samples from patients with previously validated mutations in a variety of the genes; and 50 prospective samples from patients with suspected AID in whom previous Sanger based genetic screening had been non-diagnostic., Results: VIP was sensitive and specific at detecting all the different types of known mutations in 22 positive controls, including gene deletion, small INDELS, and somatic mosaicism with allele fraction as low as 3%. Six/50 patients (12%) with unclassified AID had at least one class 5 (clearly pathogenic) variant; and 11/50 (22%) had at least one likely pathogenic variant (class 4). Overall, testing with VIP resulted in a firm or strongly suspected molecular diagnosis in 16/50 patients (32%)., Conclusions: The high diagnostic yield and accuracy of this comprehensive targeted gene panel validate the use of broad NGS-based testing for patients with suspected AID.

RNA-binding proteins of the ZFP36 family are best known for inhibiting the expression of cytokines through binding to AU-rich elements in the 3' untranslated region and promoting mRNA decay. Here we identified an indispensable role for ZFP36L1 as the regulator of a post-transcriptional hub that determined the identity of marginal-zone B cells by promoting their proper localization and survival. ZFP36L1 controlled a gene-expression program related to signaling, cell adhesion and locomotion; it achieved this in part by limiting expression of the transcription factors KLF2 and IRF8, which are known to enforce the follicular B cell phenotype. These mechanisms emphasize the importance of integrating transcriptional and post-transcriptional processes by RNA-binding proteins for maintaining cellular identity among closely related cell types.

The aryl hydrocarbon receptor (AhR), a transcription factor known for mediating xenobiotic toxicity, is expressed in B cells, which are known targets for environmental pollutants. However, it is unclear what the physiological functions of AhR in B cells are. We show here that expression of Ahr in B cells is up-regulated upon B-cell receptor (BCR) engagement and IL-4 treatment. Addition of a natural ligand of AhR, FICZ, induces AhR translocation to the nucleus and transcription of the AhR target gene Cyp1a1, showing that the AhR pathway is functional in B cells. AhR-deficient (Ahr -/- ) B cells proliferate less than AhR-sufficient (Ahr +/+ ) cells following in vitro BCR stimulation and in vivo adoptive transfer models confirmed that Ahr -/- B cells are outcompeted by Ahr +/+ cells. Transcriptome comparison of AhR-deficient and AhR-sufficient B cells identified cyclin O (Ccno), a direct target of AhR, as a top candidate affected by AhR deficiency., (© 2016 The Authors. Published under the terms of the CC BY 4.0 license.)

The RNA-binding proteins Zfp36l1 and Zfp36l2 act redundantly to enforce the β-selection checkpoint during thymopoiesis, yet their molecular targets remain largely unknown. In this study, we identify these targets on a genome-wide scale in primary mouse thymocytes and show that Zfp36l1/l2 regulate DNA damage response and cell cycle transcripts to ensure proper β-selection. Double-negative 3 thymocytes lacking Zfp36l1/l2 share a gene expression profile with postselected double-negative 3b cells despite the absence of intracellular TCRβ and reduced IL-7 signaling. Our findings show that in addition to controlling the timing of proliferation at β-selection, posttranscriptional control by Zfp36l1/l2 limits DNA damage responses, which are known to promote thymocyte differentiation. Zfp36l1/l2 therefore act as posttranscriptional safeguards against chromosomal instability and replication stress by integrating pre-TCR and IL-7 signaling with DNA damage and cell cycle control., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2016 by The American Association of Immunologists, Inc.)

RNA-binding proteins (RBPs) facilitate post-transcriptional control of eukaryotic gene expression at multiple levels. The RBP tristetraprolin (TTP/Zfp36) is a signal-induced phosphorylated anti-inflammatory protein guiding unstable mRNAs of pro-inflammatory proteins for degradation and preventing translation. Using iCLIP, we have identified numerous mRNA targets bound by wild-type TTP and by a non-MK2-phosphorylatable TTP mutant (TTP-AA) in 1 h LPS-stimulated macrophages and correlated their interaction with TTP to changes at the level of mRNA abundance and translation in a transcriptome-wide manner. The close similarity of the transcriptomes of TTP-deficient and TTP-expressing macrophages upon short LPS stimulation suggested an effective inactivation of TTP by MK2, whereas retained RNA-binding capacity of TTP-AA to 3'UTRs caused profound changes in the transcriptome and translatome, altered NF-κB-activation and induced cell death. Increased TTP binding to the 3'UTR of feedback inhibitor mRNAs, such as Ier3, Dusp1 or Tnfaip3, in the absence of MK2-dependent TTP neutralization resulted in a strong reduction of their protein synthesis contributing to the deregulation of the NF-κB-signaling pathway. Taken together, our study uncovers a role of TTP as a suppressor of feedback inhibitors of inflammation and highlights the importance of fine-tuned TTP activity-regulation by MK2 in order to control the pro-inflammatory response., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Epithelia function as barriers against environmental insults and express the transcription factor aryl hydrocarbon receptor (AhR). However, AhR function in these tissues is unknown. Here we show that AhR regulates multiciliogenesis in both murine airway epithelia and in Xenopus laevis epidermis. In air-exposed airway epithelia, induction of factors required for multiciliogenesis, including cyclin O (Ccno) and Multicilin (Mcidas), is AhR dependent, and air exposure induces AhR binding to the Ccno promoter. Submersion and hypoxic conditions impede AhR-dependent Ccno induction. This is mediated by the persistence of Notch signalling, as Notch blockade renders multiciliogenesis and Ccno induction by AhR independent from air exposure. In contrast to Ccno induction, air exposure does not induce the canonical AhR target cytochrome P450 1a1 (Cyp1a1). Inversely, exposure to AhR ligands induces Cyp1a1 but not Ccno and impeded ciliogenesis. These data indicate that AhR involvement in detoxification of environmental pollutants may impede its physiological role, resulting in respiratory pathology.

Motivation: Mechanistic models based on ordinary differential equations provide powerful and accurate means to describe the dynamics of molecular machinery which orchestrates gene regulation. When combined with appropriate statistical techniques, mechanistic models can be calibrated using experimental data and, in many cases, also the model structure can be inferred from time-course measurements. However, existing mechanistic models are limited in the sense that they rely on the assumption of static network structure and cannot be applied when transient phenomena affect, or rewire, the network structure. In the context of gene regulatory network inference, network rewiring results from the net impact of possible unobserved transient phenomena such as changes in signaling pathway activities or epigenome, which are generally difficult, but important, to account for., Results: We introduce a novel method that can be used to infer dynamically evolving regulatory networks from time-course data. Our method is based on the notion that all mechanistic ordinary differential equation models can be coupled with a latent process that approximates the network structure rewiring process. We illustrate the performance of the method using simulated data and, further, we apply the method to study the regulatory interactions during T helper 17 (Th17) cell differentiation using time-course RNA sequencing data. The computational experiments with the real data show that our method is capable of capturing the experimentally verified rewiring effects of the core Th17 regulatory network. We predict Th17 lineage specific subnetworks that are activated sequentially and control the differentiation process in an overlapping manner., Availability and Implementation: An implementation of the method is available at http://research.ics.aalto.fi/csb/software/lem/, Contacts: jukka.intosalmi@aalto.fi or harri.lahdesmaki@aalto.fi., (© The Author 2016. Published by Oxford University Press.)

Progression through the stages of lymphocyte development requires coordination of the cell cycle. Such coordination ensures genomic integrity while cells somatically rearrange their antigen receptor genes [in a process called variable-diversity-joining (VDJ) recombination] and, upon successful rearrangement, expands the pools of progenitor lymphocytes. Here we show that in developing B lymphocytes, the RNA-binding proteins (RBPs) ZFP36L1 and ZFP36L2 are critical for maintaining quiescence before precursor B cell receptor (pre-BCR) expression and for reestablishing quiescence after pre-BCR-induced expansion. These RBPs suppress an evolutionarily conserved posttranscriptional regulon consisting of messenger RNAs whose protein products cooperatively promote transition into the S phase of the cell cycle. This mechanism promotes VDJ recombination and effective selection of cells expressing immunoglobulin-μ at the pre-BCR checkpoint., (Copyright © 2016, American Association for the Advancement of Science.)

Uncontrolled Th17 cell activity is associated with cancer and autoimmune and inflammatory diseases. To validate the potential relevance of mouse models of targeting the Th17 pathway in human diseases we used RNA sequencing to compare the expression of coding and non-coding transcripts during the priming of Th17 cell differentiation in both human and mouse. In addition to already known targets, several transcripts not previously linked to Th17 cell polarization were found in both species. Moreover, a considerable number of human-specific long non-coding RNAs were identified that responded to cytokines stimulating Th17 cell differentiation. We integrated our transcriptomics data with known disease-associated polymorphisms and show that conserved regulation pinpoints genes that are relevant to Th17 cell-mediated human diseases and that can be modelled in mouse. Substantial differences observed in non-coding transcriptomes between the two species as well as increased overlap between Th17 cell-specific gene expression and disease-associated polymorphisms underline the need of parallel analysis of human and mouse models. Comprehensive analysis of genes regulated during Th17 cell priming and their classification to conserved and non-conserved between human and mouse facilitates translational research, pointing out which candidate targets identified in human are worth studying by using in vivo mouse models.

Background: The differentiation of naive CD 4(+) helper T (Th) cells into effector Th17 cells is steered by extracellular cytokines that activate and control the lineage specific transcriptional program. While the inducing cytokine signals and core transcription factors driving the differentiation towards Th17 lineage are well known, detailed mechanistic interactions between the key components are poorly understood., Results: We develop an integrative modeling framework which combines RNA sequencing data with mathematical modeling and enables us to construct a mechanistic model for the core Th17 regulatory network in a data-driven manner., Conclusions: Our results show significant evidence, for instance, for inhibitory mechanisms between the transcription factors and reveal a previously unknown dependency between the dosage of the inducing cytokine TGF β and the expression of the master regulator of competing (induced) regulatory T cell lineage. Further, our experimental validation approves this dependency in Th17 polarizing conditions.

IL-22 is a cytokine that regulates tissue homeostasis at barrier surfaces. A variety of IL-22-producing cell types is known, but identification on the single-cell level remains difficult. Therefore, we generated a fate reporter mouse that would allow the identification of IL-22-producing cells and their fate mapping in vivo. To trace IL-22-expressing cells, a sequence encoding Cre recombinase was cloned into the Il22 locus, and IL22(Cre) mice were crossed with reporter mice expressing enhanced yellow fluorescence protein (eYFP) under control of the endogenous Rosa26 promoter. In IL22(Cre)R26R(eYFP) mice, the fluorescent reporter permanently labels cells that have switched on Il22 expression, irrespective of cytokine production. Despite a degree of underreporting, eYFP expression was detectable in nonimmune mice and restricted to group 3 innate lymphoid cells (ILC3) in the gut and γδ T cells in skin or lung. Upon skin challenge with imiquimod, eYFP(+) γδ and CD4 T cells expanded in the skin. Infection with Citrobacter rodentium initially was controlled by ILC3, followed by expansion of eYFP(+) CD4 T cells, which were induced in innate lymphoid follicles in the colon. No eYFP expression was detected in small intestinal Th17 cells, and they did not expand in the immune response. Colonic eYFP(+) CD4 T cells exhibited plasticity during infection with expression of additional cytokines, in contrast to ILC3, which remained largely stable. Single-cell quantitative PCR analysis of eYFP(+) CD4 T cells confirmed their heterogeneity, suggesting that IL-22 expression is not confined to particular subsets or a dedicated Th22 subset., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Enterovirus infections are usually mild but can also cause severe illnesses and play a role in chronic diseases, such as cardiomyopathies and type 1 diabetes. Host response to the invading virus can markedly modulate the course of the infection, and this response varies between individuals due to the polymorphism of immune response genes. However, it is currently not known if virus strains also differ in their ability to stimulate the host immune system. Coxsackievirus B1 (CBV1) causes severe epidemics in young infants and it has recently been connected with type 1 diabetes in seroepidemiological studies. This study evaluated the ability of different field isolates of CBV1 to induce innate immune responses in PBMCs. CBV1 strains differed markedly in their capacity to induce innate immune responses. Out of the 18 tested CBV1 strains two induced exceptionally strong alpha interferon (IFN-α) response in PBMC cultures. The responding cell type was found to be the plasmacytoid dendritic cell. Such a strong innate immune response was accompanied by an up-regulation of several other immune response genes and secretion of cytokines, which modulate inflammation, and adaptive immune responses. These results suggest that enterovirus-induced immune activation depends on the virus strain. It is possible that the immunotype of the virus modulates the course of the infection and plays a role in the pathogenesis of chronic immune-mediated enterovirus diseases., (© 2014 Wiley Periodicals, Inc.)

Environmental stimuli are known to contribute to psoriasis pathogenesis and that of other autoimmune diseases, but the mechanisms are largely unknown. Here we show that the aryl hydrocarbon receptor (AhR), a transcription factor that senses environmental stimuli, modulates pathology in psoriasis. AhR-activating ligands reduced inflammation in the lesional skin of psoriasis patients, whereas AhR antagonists increased inflammation. Similarly, AhR signaling via the endogenous ligand FICZ reduced the inflammatory response in the imiquimod-induced model of skin inflammation and AhR-deficient mice exhibited a substantial exacerbation of the disease, compared to AhR-sufficient controls. Nonhematopoietic cells, in particular keratinocytes, were responsible for this hyperinflammatory response, which involved upregulation of AP-1 family members of transcription factors. Thus, our data suggest a critical role for AhR in the regulation of inflammatory responses and open the possibility for novel therapeutic strategies in chronic inflammatory disorders., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

IL-9 fate reporter mice established type 2 innate lymphoid cells (ILC2s) as major producers of this cytokine in vivo. Here we focus on the role of IL-9 and ILC2s during the lung stage of infection with Nippostrongylus brasiliensis, which results in substantial tissue damage. IL-9 receptor (IL-9R)-deficient mice displayed reduced numbers of ILC2s in the lung after infection, resulting in impaired IL-5, IL-13, and amphiregulin levels, despite undiminished numbers of Th2 cells. As a consequence, the restoration of tissue integrity and lung function was strongly impaired in the absence of IL-9 signaling. ILC2s, in contrast to Th2 cells, expressed high levels of the IL-9R, and IL-9 signaling was crucial for the survival of activated ILC2s in vitro. Furthermore, ILC2s in the lungs of infected mice required the IL-9R to up-regulate the antiapoptotic protein BCL-3 in vivo. This highlights a unique role for IL-9 as an autocrine amplifier of ILC2 function, promoting tissue repair in the recovery phase after helminth-induced lung inflammation.

The aryl hydrocarbon receptor (AhR) has been attributed with anti-inflammatory effects in the development of pathological immune responses leading to experimental autoimmune encephalomyelitis (EAE) via the induction of regulatory T cells. In agreement with previously published findings, we find that TCDD administration confers protection from EAE, however, this immuno-modulatory effect was not the consequence of de novo Treg generation, but the inhibition of Th17 cell differentiation. Systemic application of FICZ at the time of immunization also reduced EAE pathology albeit to a lesser degree than TCDD. In vitro Th17 differentiation in the presence of AhR agonists, including TCDD, promoted IL-17 and IL-22 expression, but did not induce Treg differentiation. AhR affinity influenced the amounts of IL-17 and IL-22 protein that was secreted by Th17 cells, but did not seem to affect susceptibility to EAE in vivo. Making use of conditional AhR-deficient mice, we show that the anti-inflammatory effect of TCDD depends on AhR activation in both T cells and dendritic cells, further emphasising the ability of TCDD to interfere with T effector cell differentiation in vivo. The dichotomy between the in vivo and in vitro effects of AhR reveals the complexity of the AhR pathway, which has the capacity of affecting different AhR-expressing cell types involved in mounting immune responses, thus participating in defining their outcome.

The potential impact of nanoparticles on the environment and on human health has attracted considerable interest worldwide. The amount of transcriptomics data, in which tissues and cell lines are exposed to nanoparticles, increases year by year. In addition to the importance of the original findings, this data can have value in broader context when combined with other previously acquired and published results. In order to facilitate the efficient usage of the data, we have developed the NanoMiner web resource (http://nanominer.cs.tut.fi/), which contains 404 human transcriptome samples exposed to various types of nanoparticles. All the samples in NanoMiner have been annotated, preprocessed and normalized using standard methods that ensure the quality of the data analyses and enable the users to utilize the database systematically across the different experimental setups and platforms. With NanoMiner it is possible to 1) search and plot the expression profiles of one or several genes of interest, 2) cluster the samples within the datasets, 3) find differentially expressed genes in various nanoparticle studies, 4) detect the nanoparticles causing differential expression of selected genes, 5) analyze enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) terms for the detected genes and 6) search the expression values and differential expressions of the genes belonging to a specific KEGG pathway or Gene Ontology. In sum, NanoMiner database is a valuable collection of microarray data which can be also used as a data repository for future analyses.

Interleukin-17 (IL-17)-mediated immune responses play a crucial role in the mucosal host defence against microbial and fungal pathogens. However, the chronic activation of IL-17-producing T helper cells can cause autoimmune disease. In addition, recent studies have highlighted key roles of innate cell-mediated IL-17 responses in various inflammatory settings. Besides inflammation, there have also been intriguing findings regarding the involvement of IL-17 responses in the pathogenesis of cardiovascular diseases and tumour formation. Here, we discuss the latest discoveries in regulation and function of innate and adaptive IL-17-producing cells.

Special AT-rich binding protein 1 (SATB1) is a global chromatin organizer and a transcription factor regulated by interleukin-4 (IL-4) during the early T helper 2 (Th2) cell differentiation. Here we show that SATB1 controls multiple IL-4 target genes involved in human Th cell polarization or function. Among the genes regulated by SATB1 is that encoding the cytokine IL-5, which is predominantly produced by Th2 cells and plays a key role in the development of eosinophilia in asthma. We demonstrate that, during the early Th2 cell differentiation, IL-5 expression is repressed through direct binding of SATB1 to the IL-5 promoter. Furthermore, SATB1 knockdown-induced up-regulation of IL-5 is partly counteracted by down-regulating GATA3 expression using RNAi in polarizing Th2 cells. Our results suggest that a competitive mechanism involving SATB1 and GATA3 regulates IL-5 transcription, and provide new mechanistic insights into the stringent regulation of IL-5 expression during human Th2 cell differentiation.

Dissecting the molecular mechanisms by which T helper (Th) cells differentiate to effector Th2 cells is important for understanding the pathogenesis of immune-mediated diseases, such as asthma and allergy. Because the STAT6 transcription factor is an upstream mediator required for interleukin-4 (IL-4)-induced Th2 cell differentiation, its targets include genes important for this process. Using primary human CD4(+) T cells, and by blocking STAT6 with RNAi, we identified a number of direct and indirect targets of STAT6 with ChIP sequencing. The integration of these data sets with detailed kinetics of IL-4-driven transcriptional changes showed that STAT6 was predominantly needed for the activation of transcription leading to the Th2 cell phenotype. This integrated genome-wide data on IL-4- and STAT6-mediated transcription provide a unique resource for studies on Th cell differentiation and, in particular, for designing interventions of human Th2 cell responses., (Copyright 2010 Elsevier Inc. All rights reserved.)

IL-12 and IL-18 are essential for Th1 differentiation, whereas the role of IFN-alpha in Th1 development is less understood. In this microarray-based study, we searched for genes that are regulated by IFN-alpha, IL-12, or the combination of IL-12 plus IL-18 during the early differentiation of human umbilical cord blood CD4(+) Th cells. Twenty-six genes were similarly regulated in response to treatment with IL-12, IFN-alpha, or the combination of IL-12 plus IL-18. These genes could therefore play a role in Th1 lineage decision. Transcription factor activating transcription factor (ATF) 3 was upregulated by these cytokines and selected for further study. Ectopic expression of ATF3 in CD4(+) T cells enhanced the production of IFN-gamma, the hallmark cytokine of Th1 cells, whereas small interfering RNA knockdown of ATF3 reduced IFN-gamma production. Furthermore, ATF3 formed an endogenous complex with JUN in CD4(+) T cells induced to Th1. Chromatin immunoprecipitation and luciferase reporter assays showed that both ATF3 and JUN are recruited to and transactivate the IFNG promoter during early Th1 differentiation. Collectively, these data indicate that ATF3 promotes human Th1 differentiation.

T helper (Th) cells differentiate into functionally distinct effector cell subsets of which Th1 and Th2 cells are best characterized. Besides T cell receptor signaling, IL-12-induced STAT4 and T-bet- and IL-4-induced STAT6 and GATA3 signaling pathways are the major players regulating the Th1 and Th2 differentiation process, respectively. However, there are likely to be other yet unknown factors or pathways involved. In this study we used quantitative proteomics exploiting cleavable ICAT labeling and LC-MS/MS to identify IL-4-regulated proteins from the microsomal fractions of CD4(+) cells extracted from umbilical cord blood. We were able to identify 557 proteins of which 304 were also quantified. This study resulted in the identification of the down-regulation of small GTPases GIMAP1 and GIMAP4 by IL-4 during Th2 differentiation. We also showed that both GIMAP1 and GIMAP4 genes are up-regulated by IL-12 and other Th1 differentiation-inducing cytokines in cells induced to differentiate toward Th1 lineage and down-regulated by IL-4 in cells induced to Th2. Our results indicate that the GIMAP (GTPase of the immunity-associated protein) family of proteins is differentially regulated during Th cell differentiation.

Macrophages and dendritic cells (DC) are APC, which regulate innate and adaptive immune responses. Macrophages function locally mainly, maintaining inflammatory responses in tissues, whereas DC take up microbes, mature, and migrate to local lymph nodes to present microbial antigens to naïve T cells to elicit microbe-specific immune responses. Blood monocytes can be differentiated in vitro to macrophages or DC by GM-CSF or GM-CSF + IL-4, respectively. In the present study, we performed global gene expression analyses using Affymetrix HG-U133A Gene Chip oligonucleotide arrays during macrophage and DC differentiation. During the differentiation process, 340 and 350 genes were up-regulated, and 190 and 240 genes were down-regulated in macrophages and DC, respectively. There were also more that 200 genes, which were expressed differentially in fully differentiated macrophages and DC. Macrophage-specific genes include, e.g., CD14, CD163, C5R1, and FcgammaR1A, and several cell surface adhesion molecules, cytokine receptors, WNT5A and its receptor of the Frizzled family FZD2, fibronectin, and FcepsilonR1A were identified as DC-specific. Our results reveal significant differences in gene expression profiles between macrophages and DC, and these differences can partially explain the functional differences between these two important cell types.

Th cell subtypes, Th1 and Th2, are involved in the pathogenesis or progression of many immune-mediated diseases, such as type 1 diabetes and asthma, respectively. Defining the molecular networks and factors that direct Th1 and Th2 cell differentiation will help to understand the pathogenic mechanisms causing these diseases. Some of the key factors regulating this differentiation have been identified, however, they alone do not explain the process in detail. To identify novel factors directing the early differentiation, we have studied the transcriptomes of human Th1 and Th2 cells after 2, 6, and 48 h of polarization at the genome scale. Based on our current and previous studies, 288 genes or expressed sequence tags, representing approximately 1-1.5% of the human genome, are regulated in the process during the first 2 days. These transcriptional profiles revealed genes coding for components of certain pathways, such as RAS oncogene family and G protein-coupled receptor signaling, to be differentially regulated during the early Th1 and Th2 cell differentiation. Importantly, numerous novel genes with unknown functions were identified. By using short-hairpin RNA knockdown, we show that a subset of these genes is regulated by IL-4 through STAT6 signaling. Furthermore, we demonstrate that one of the IL-4 regulated genes, NDFIP2, promotes IFN-gamma production by the polarized human Th1 lymphocytes. Among the novel genes identified, there may be many factors that play a crucial role in the regulation of the differentiation process together with the previously known factors and are potential targets for developing therapeutics to modulate Th1 and Th2 responses.

We examined responses of the B-cell antigen receptor-dependent intracellular signaling network to targeted perturbations induced through siRNA-mediated depletion of select signaling intermediates. The constituent nodes displayed graded sensitivities, which resulted from the differential effects of perturbations on the kinetic and quantitative aspects of phosphorylation at each node. By taking the rate of initial phosphorylation, rate of subsequent dephosphorylation, and the total intensity of phosphorylation at each node as separate signaling parameters, we generated data-driven models that accurately predicted the cellular responses of apoptosis, proliferation, and cytokine secretion. Importantly, the effects of perturbation on the primary target alone did not yield successful models. Rather, it also required incorporation of secondary effects on many other nodes. A significant feature of these models was that the three signaling parameters derived from each node functioned largely as independent entities, making distinctive contributions to the cellular response. Thus, the kinetic and quantitative features of phosphorylation at a node appear to play discrete roles during signal processing.

The differentiation of naïve T helper (Th) cells is induced by TCR activation and IL-12/STAT4 or IL-4/STAT6 signaling pathways, forming Th1 and Th2 cells, respectively. In this study, oligonucleotide arrays were used to identify genes regulated during the initiation of human Th1 and Th2 cell differentiation at 2 and 6 h in presence or absence of immunosuppressive TGF-beta. As a result the immediate targets of IL-12, IL-4 and TGF-beta were identified. The effects of IL-12 at this early stage were minimal and consistent with the known kinetics of IL-12Rbeta2 expression. IL-4, however, was observed to rapidly regulate 63 genes, 26 of which were differentially expressed at both the 2- and 6-h time points. Of these IL-4 regulated genes, one-third have previously been observed to display expression changes in the later phases of the polarization process. Similarly to the key regulators, TBX21 and GATA3, the transcription factors SATB1, TCF7 and BCL6 were differentially regulated at the protein level during early Th1 and Th2 cell polarization. Moreover, the developing Th1 and Th2 cells were demonstrated to be responsive to the immunosuppressive TGF-beta and IL-10. In this study, a panel of novel factors that may be important regulators of the differentiation process was identified.

Latent Epstein-Barr virus (EBV) infection is strongly associated with B-cell proliferative diseases such as Burkitt's lymphoma. Here we show that the oncogenic serine/threonine kinases Pim-1 and Pim-2 enhance the activity of the viral transcriptional activator EBNA2. During EBV infection of primary B-lymphocytes, the mRNA expression levels of pim genes, especially of pim-2, are upregulated and remain elevated in latently infected B-cell lines. Thus, EBV-induced upregulation of Pim kinases and Pim-stimulated EBNA2 transcriptional activity may contribute to the ability of EBV to immortalize B-cells and predispose them to malignant growth.

The Erwinia chrysanthemi oligogalacturonate-specific monomeric porin, KdgM, does not present homology with any porins of known structure. A model of this protein, based on sequence similarity and the amphipathy profile, was constructed. The model depicts a beta-barrel composed of 14 antiparallel beta-strands. The accuracy of this model was tested by the chemical labelling of cysteine residues introduced by site-directed mutagenesis. The protein has seven surface-exposed loops. They are rather small with the exception of one, loop L6. Deletion of this loop allowed the entry of maltopentaose into the bacteria, a molecule too large to enter through the wild-type KdgM. Loop L6 could fold back into the lumen of the pore and play the role of the constriction loop L3 of general porins. With 14 transmembrane segments, the KdgM porin family could represent the smallest porin characterized to date.