Question: Decreased sensitivity to interleukin-2 in regulatory T cells is linked to increased resilience against autoimmune disorders like Type 1 Diabetes.

Interleukin-2 Genetic Polymorphisms Disrupt Regulatory T Cell Function and Induce Autoimmune Disorders
Imbalances in the immune system's regulatory mechanisms are implicated in the onset of autoimmune diseases. This study demonstrates that susceptibility and resistance to autoimmune conditions, particularly those linked to chromosome 3 (Idd3) in mice, are associated with variations in the expression of interleukin-2 (IL-2), a crucial cytokine for immune regulation. To ascertain that a roughly halved IL-2 production is responsible for the immune instability attributed to Idd3, we engineered a reduction in Il2 gene expression that mirrors the effects of naturally occurring susceptibility variants. This engineered haplodeficiency not only halves IL-2 production by T cells but also replicates the autoimmune dysregulation seen with the susceptibility alleles of Il2. Both genetic alterations result in diminished IL-2 production and are linked to compromised function of CD4+ CD25+ regulatory T cells, which are essential for maintaining immune equilibrium. This reduced responsiveness to IL-2 in regulatory T cells is associated with a heightened resistance to autoimmune diseases, such as Type 1 Diabetes, by maintaining a more stable immune environment.

Objective: To investigate the regulation of CD4+CD25+ Regulatory T cells (Tregs) on pro-inflammatory adhesion molecules, Krüppel-Like Factor-2 (KLF-2) and its downstream transcriptional targets in human umbilical vein endothelial cells (HUVECs) impaired by ox-LDL and the mechanisms of it. Methods and results: HUVECs were cultured in the continuous presence of ox-LDL(0 mg/L,25 mg/L,50 mg/L,100 mg/L) for 4, 6, 12 and 24 hours to allow identification of early-and late-induced genes, respectively, whereas non-stimulated controls were taken at 0 hours. The expression of pro-inflammatory adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), E-selectin, KLF-2 and its target genes eNOS, PAI-1 were determined by real time RT-PCR and/or western-blot analysis. Expression of pro-inflammatory adhesion molecules, KLF-2, eNOS and PAI-1 in HUVEC cultured alone or with anti-CD3 mAbs activated Tregs, followed by addition of ox-LDL (50 mg/L) for 6 hours, are compared to expression levels in control cultures. Ox-LDL treated HUVECs increased pro-inflammatory adhesion molecules expression, as well as increased PAI-1 but decreased eNOS expression accompanied with significant downregulating of KLF-2 at a dose and time dependent manner. Furthermore, ox-LDL increased pro-inflammatory adhesion molecules but inhibited KLF2 expression was reversed by addition of Tregs. Small interfering RNA reduced endogenous KLF-2 expression and partly reversed the suppressive effect of Tregs on HUVECs activation, which strongly implicate KLF-2 as a transcriptional regulator of the Tregs-mediated effects in endothelial cells. Mechanism studies reveal that Treg-mediated KLF2 expression in HUVECs impaired by ox-LDL requires cell contact as well as soluble factors. Conclusions: Tregs could protect endothelial function that is largely dependent on KLF2 and its downstream transcriptional targets regulation involving cell-to-cell contact and soluble factors.

OBJECTIVES Non-celiac wheat sensitivity (NCWS) is defined as a reaction to ingested wheat after exclusion of celiac disease and wheat allergy. As its pathogenesis is incompletely understood, we evaluated the inflammatory response in the rectal mucosa of patients with well-defined NCWS. METHODS The prospective study included 22 patients with irritable bowel syndrome (IBS)-like clinical presentation, diagnosed with NCWS by double-blind placebo-controlled challenge. Eight IBS patients not improving on wheat-free diet were used as controls. Two weeks after oral challenge was performed with 80 grams of wheat daily, cells were isolated from rectal biopsies and thoroughly characterized by fluorescence-activated cell sorting analysis for intracellular cytokines and surface markers. RESULTS Rectal biopsies from wheat-challenged NCWS patients showed that a significant mucosal CD45(+) infiltrate consisted of CD3(+) and CD3(-) lymphocytes, with the latter spontaneously producing more interferon (IFN)-γ than IBS controls. About 30% of IFN-γ-producing CD45(+) cells were T-bet(+), CD56(-), NKP44(-), and CD117(-), defining them as a type-1 innate lymphoid cells (ILC1). IFN-γ-producing ILC1 cells significantly decreased in 10 patients analyzed 2 weeks after they resumed a wheat-free diet. CONCLUSIONS These data indicate that, in patients with active NCWS, IFN-γ-producing ILC1 cells infiltrate rectal mucosa and support a role for this innate lymphoid cell population in the pathogenesis of NCWS.

OBJECTIVE Multiple sclerosis (MS) is a disease of the central nervous system with marked heterogeneity in several aspects including pathological processes. Based on infiltrating immune cells, deposition of humoral factors and loss of oligodendrocytes and/or myelin proteins, four lesion patterns have been described. Pattern II is characterized by antibody and complement deposition in addition to T-cell infiltration. MS is considered a T-cell-mediated disease, but until now the study of pathogenic T cells has encountered major challenges, most importantly the limited access of brain-infiltrating T cells. Our objective was to identify, isolate, and characterize brain-infiltrating clonally expanded T cells in pattern II MS lesions. METHODS We used next-generation sequencing to identify clonally expanded T cells in demyelinating pattern II brain autopsy lesions, subsequently isolated these as T-cell clones from autologous cerebrospinal fluid and functionally characterized them. RESULTS We identified clonally expanded CD8(+) but also CD4(+) T cells in demyelinating pattern II lesions and for the first time were able to isolate these as live T-cell clones. The functional characterization shows that T cells releasing Th2 cytokines and able to provide B cell help dominate the T-cell infiltrate in pattern II brain lesions. INTERPRETATION Our data provide the first functional evidence for a putative role of Th2/Tc2 cells in pattern II MS supporting the existence of this pathogenic phenotype and questioning the protective role that is generally ascribed to Th2 cells. Our observations are important to consider for future treatments of pattern II MS patients.

In type 1 diabetes (T1D), there is an intense inflammatory response that destroys the β cells in the pancreatic islets of Langerhans, the site where insulin is produced and released. A therapy for T1D that targets the specific autoimmune response in this disease while leaving the remainder of the immune system intact, has long been sought. Proinsulin is a major target of the adaptive immune response in T1D. We hypothesized that an engineered DNA plasmid encoding proinsulin (BHT-3021) would preserve β cell function in T1D patients through reduction of insulin-specific CD8⁺ T cells. We studied 80 subjects over 18 years of age who were diagnosed with T1D within the past 5 years. Subjects were randomized 2:1 to receive intramuscular injections of BHT-3021 or BHT-placebo, weekly for 12 weeks, and then monitored for safety and immune responses in a blinded fashion. Four dose levels of BHT-3021 were evaluated: 0.3, 1.0, 3.0, and 6.0 mg. C-peptide was used both as an exploratory efficacy measure and as a safety measure. Islet-specific CD8⁺ T cell frequencies were assessed with multimers of monomeric human leukocyte antigen class I molecules loaded with peptides from pancreatic and unrelated antigens. No serious adverse events related to BHT-3021 were observed. C-peptide levels improved relative to placebo at all doses, at 1 mg at the 15-week time point (+19.5% BHT-3021 versus -8.8% BHT-placebo, P < 0.026). Proinsulin-reactive CD8⁺ T cells, but not T cells against unrelated islet or foreign molecules, declined in the BHT-3021 arm (P < 0.006). No significant changes were noted in interferon-γ, interleukin-4 (IL-4), or IL-10 production in CD4 T cells. Thus, we demonstrate that a plasmid encoding proinsulin reduces the frequency of CD8⁺ T cells reactive to proinsulin while preserving C-peptide over the course of dosing.

BACKGROUND Two inflammatory disorders, type 1 diabetes and celiac disease, cosegregate in populations, suggesting a common genetic origin. Since both diseases are associated with the HLA class II genes on chromosome 6p21, we tested whether non-HLA loci are shared. METHODS We evaluated the association between type 1 diabetes and eight loci related to the risk of celiac disease by genotyping and statistical analyses of DNA samples from 8064 patients with type 1 diabetes, 9339 control subjects, and 2828 families providing 3064 parent-child trios (consisting of an affected child and both biologic parents). We also investigated 18 loci associated with type 1 diabetes in 2560 patients with celiac disease and 9339 control subjects. RESULTS Three celiac disease loci--RGS1 on chromosome 1q31, IL18RAP on chromosome 2q12, and TAGAP on chromosome 6q25--were associated with type 1 diabetes (P<1.00x10(-4)). The 32-bp insertion-deletion variant on chromosome 3p21 was newly identified as a type 1 diabetes locus (P=1.81x10(-8)) and was also associated with celiac disease, along with PTPN2 on chromosome 18p11 and CTLA4 on chromosome 2q33, bringing the total number of loci with evidence of a shared association to seven, including SH2B3 on chromosome 12q24. The effects of the IL18RAP and TAGAP alleles confer protection in type 1 diabetes and susceptibility in celiac disease. Loci with distinct effects in the two diseases included INS on chromosome 11p15, IL2RA on chromosome 10p15, and PTPN22 on chromosome 1p13 in type 1 diabetes and IL12A on 3q25 and LPP on 3q28 in celiac disease. CONCLUSIONS A genetic susceptibility to both type 1 diabetes and celiac disease shares common alleles. These data suggest that common biologic mechanisms, such as autoimmunity-related tissue damage and intolerance to dietary antigens, may be etiologic features of both diseases.

Inborn errors of the genes encoding two of the four human JAKs (JAK3 and TYK2) and three of the six human STATs (STAT1, STAT3, and STAT5B) have been described. We review the disorders arising from mutations in these five genes, highlighting the way in which the molecular and cellular pathogenesis of these conditions has been clarified by the discovery of inborn errors of cytokines, hormones, and their receptors, including those interacting with JAKs and STATs. The phenotypic similarities between mice and humans lacking individual JAK-STAT components suggest that the functions of JAKs and STATs are largely conserved in mammals. However, a wide array of phenotypic differences has emerged between mice and humans carrying biallelic null alleles of JAK3, TYK2, STAT1, or STAT5B. Moreover, the high degree of allelic heterogeneity at the human JAK3, TYK2, STAT1, and STAT3 loci has revealed highly diverse immunological and clinical phenotypes, which had not been anticipated.

Although the etiology of multiple sclerosis (MS) remains enigmatic, the role of T cells is unquestionably central in this pathology. Immune cells respond to pathogens and danger signals via pattern-recognition receptors (PRR). Several reports implicate Nlrp12, an intracellular PRR, in the development of a mouse MS-like disease, called Experimental Autoimmune Encephalomyelitis (EAE). In this study, we used induced and spontaneous models of EAE, as well as in vitro T cell assays, to test the hypothesis that Nlrp12 inhibits Th1 response and prevents T-cell mediated autoimmunity. We found that Nlrp12 plays a protective role in induced EAE by reducing IFNγ/IL-4 ratio in lymph nodes, whereas it potentiates the development of spontaneous EAE (spEAE) in 2D2 T cell receptor (TCR) transgenic mice. Looking into the mechanism of Nlrp12 activity in T cell response, we found that it inhibits T cell proliferation and suppresses Th1 response by reducing IFNγ and IL-2 production. Following TCR activation, Nlrp12 inhibits Akt and NF-κB phosphorylation, while it has no effect on S6 phosphorylation in the mTOR pathway. In conclusion, we propose a model that can explain the dual immunoregulatory function of Nlrp12 in EAE. We also propose a model explaining the molecular mechanism of Nlrp12-dependent regulation of T cell response.

Allogeneic haematopoietic stem cell transplantation is used to treat a variety of disorders, but its efficacy is limited by the occurrence of graft-versus-host disease (GVHD). The past decade has brought impressive advances in our understanding of the role of stimulatory and suppressive elements of the adaptive and innate immune systems from both the donor and the host in GVHD pathogenesis. New insights from basic immunology, preclinical models and clinical studies have led to novel approaches for prevention and treatment. This Review highlights the recent advances in understanding the pathophysiology of GVHD and its treatment, with a focus on manipulations of the immune system that are amenable to clinical application.

The CC genotype of the interleukin (IL)-28B.rs12979860 gene has been associated with spontaneous hepatitis C virus (HCV) clearance and treatment response. The distribution and correlation of an IL28B.rs12979860 single-nucleotide polymorphism (SNP) with HCV-specific cell-mediated immune (CMI) responses among Egyptian healthcare workers (HCWs) is not known. We determined this relationship in 402 HCWs who serve a patient cohort with ~85 % HCV prevalence. We enrolled 402 HCWs in four groups: group 1 (n = 258), seronegative aviremic subjects; group 2 (n = 25), seronegative viremic subjects; group 3 (n = 41), subjects with spontaneously resolved HCV infection; and group 4 (n = 78), chronic HCV patients. All subjects were tested for an HCV-specific CMI response using an ex-vivo interferon-gamma (IFNγ) ELISpot assay with nine HCV genotype-4a overlapping 15-mer peptide pools corresponding to all of the HCV proteins. All subjects were tested for IL28B.rs12979860 SNP by real-time PCR. An HCV-specific CMI was demonstrated in ~27 % of the seronegative aviremic HCWs (group 1), suggesting clearance of infection after low-level exposure to HCV. The frequency of IL28B.rs12979860 C allele homozygosity in the four groups was 49 %, 48 %, 49 %, and 23 %, while that of the T allele was 14 %, 16 %, 12 and 19 %, respectively, suggesting differential distributions among subjects with different HCV status. As reported, IL28B.rs12979860 predicted the outcome of HCV infection (p < 0.05), but we did not find any relationship between the IL28B genotypes and the outcome of HCV-specific CMI responses in the four groups (p > 0.05). The data show differential IL28B.rs12979860 genotype distribution among Egyptian HCWs with different HCV status and could not predict the outcome of HCV-specific CMI responses.

Missense mutations in the nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing family of gene 12 (Nlrp12) are associated with periodic fever syndromes and atopic dermatitis in humans. Here, we have demonstrated a crucial role for NLRP12 in negatively regulating pathogenic T cell responses. Nlrp12(-/-) mice responded to antigen immunization with hyperinflammatory T cell responses. Furthermore, transfer of CD4(+)CD45RB(hi)Nlrp12(-/-) T cells into immunodeficient mice led to more severe colitis and atopic dermatitis. NLRP12 deficiency did not, however, cause exacerbated ascending paralysis during experimental autoimmune encephalomyelitis (EAE); instead, Nlrp12(-/-) mice developed atypical neuroinflammatory symptoms that were characterized by ataxia and loss of balance. Enhanced T-cell-mediated interleukin-4 (IL-4) production promotes the development of atypical EAE disease in Nlrp12(-/-) mice. These results define an unexpected role for NLRP12 as an intrinsic negative regulator of T-cell-mediated immunity and identify altered NF-κB regulation and IL-4 production as key mediators of NLRP12-associated disease.


Question: Decreased sensitivity to interleukin-2 in regulatory T cells is linked to heightened resilience against autoimmune disorders like Type 1 Diabetes.

Genetic Mapping and Genotype-Phenotype Links Reveal Polymorphisms in the IL2RA Region as a Key Factor in Type 1 Diabetes Susceptibility. Large-scale genetic fine mapping and genotype-phenotype correlation studies have pinpointed specific polymorphisms in the IL2RA gene region as significantly associated with type 1 diabetes (T1D). This extensive research, involving comprehensive resequencing, extensive genotyping, and rigorous statistical analysis on large cohorts, has narrowed down the T1D association to two independent clusters of single nucleotide polymorphisms (SNPs). These SNPs are situated in overlapping regions spanning 14 and 40 kb, including IL2RA intron 1 and the 5′ regions of IL2RA and RBM17, with an odds ratio of 2.04 (95% confidence interval = 1.70–2.45; P = 1.92 × 10−28; control frequency = 0.635). Additionally, this study has established a link between T1D susceptibility genotypes and reduced levels of the biomarker, soluble IL-2RA (P = 6.28 × 10−28), indicating that an inherited reduced immune responsiveness to interleukin-2 is a predisposing factor for T1D.

CD4 memory T cells surviving in the absence of MHC class II contact lose their characteristic memory function. To investigate the mechanisms underlying the impaired function of memory T cells in the absence of MHC class II molecules, we analyzed gene expression profiles of resting memory T cells isolated from MHC class II-competent or -deficient hosts. The analysis focused on five transcripts related to T cell activation, metabolism, and survival that are underexpressed in resting memory T cells from MHC class II-deficient hosts compared with MHC class II-competent hosts. CD4 memory cells isolated from MHC class II-deficient hosts display alterations in their degree of differentiation as well as metabolic activity, and these changes are already manifest in the effector phase despite the presence of Ag-expressing dendritic cells. Our data suggest that the absence of interactions with noncognate MHC class II molecules compromises the progressive accumulation of signals that ensure optimal survival and fitness to sustain the metabolic activity of activated T cells and shape the functional capacity of the future memory compartment. Signals via AKT coordinate survival and metabolic pathways and may be one of the crucial events linking interaction with MHC class II molecules to the successful generation of a long-lived functional memory CD4 T cell population.

Type 1 diabetes mellitus (T1DM) is a chronic metabolic disease that results from cell-mediated autoimmune destruction of insulin-producing cells. In T1DM animal models, it has been shown that the systemic administration of multipotent mesenchymal stromal cells, also referred as to mesenchymal stem cells (MSCs), results in the regeneration of pancreatic islets. Mechanisms underlying this effect are still poorly understood. Our aims were to assess whether donor MSCs (a) differentiate into pancreatic β-cells and (b) modify systemic and pancreatic pathophysiologic markers of T1DM. After the intravenous administration of 5 × 10(5) syngeneic MSCs, we observed that mice with T1DM reverted their hyperglycemia and presented no donor-derived insulin-producing cells. In contrast, 7 and 65 days post-transplantation, MSCs were engrafted into secondary lymphoid organs. This correlated with a systemic and local reduction in the abundance of autoaggressive T cells together with an increase in regulatory T cells. Additionally, in the pancreas of mice with T1DM treated with MSCs, we observed a cytokine profile shift from proinflammatory to antinflammatory. MSC transplantation did not reduce pancreatic cell apoptosis but recovered local expression and increased the circulating levels of epidermal growth factor, a pancreatic trophic factor. Therefore, the antidiabetic effect of MSCs intravenously administered is unrelated to their transdifferentiation potential but to their capability to restore the balance between Th1 and Th2 immunological responses along with the modification of the pancreatic microenvironment. Our data should be taken into account when designing clinical trials aimed to evaluate MSC transplantation in patients with T1DM since the presence of endogenous precursors seems to be critical in order to restore glycemic control.

Retinoids (e.g., vitamin A and its derivatives) can regulate immune responses. The aim of this study was to determine whether all-trans retinaldehyde (retinal), a vitamin A derivative, can inhibit inflammatory responses and joint destruction in DBA/1J mice with collagen-induced arthritis (CIA). The arthritis score and incidence of arthritis were lower in mice treated with retinal compared to those treated with cottonseed oil. Histopathologic evidence of joint damage was lower in mice treated with retinal, corresponding with a reduction in the infiltration of immune cells in mice treated with retinal type II collagen (CII)-stimulated spleen cells. In addition, the expression of proinflammatory cytokines, oxidative stress proteins, and osteoclast markers were significantly reduced in mice treated with retinal. In vitro, retinal induced increased Foxp3 expression and inhibited Th17 development. The proportion of Foxp3(+) Treg cells was increased in the spleens of mice treated with retinal, whereas the proportion of Th17 cells was reduced. In both mice and a human culture system, tartrate-resistant acid phosphatase (TRAP) positive mononuclear cells and multinucleated cells were significantly reduced after treatment with retinal. The expression of osteoclast differentiation markers was dramatically decreased upon addition of retinal. This is the first study to demonstrate the therapeutic effect of retinal on an autoimmune arthritis model in mice through reciprocal regulation of Th17 and regulatory T cells and protection of differentiation and activation of osteoclasts. Taken together, our findings indicate that retinal has profound immunoregulatory functions and potential value for the treatment of autoimmune inflammatory disorders.

In type 1 diabetes (T1D), there is an intense inflammatory response that destroys the β cells in the pancreatic islets of Langerhans, the site where insulin is produced and released. A therapy for T1D that targets the specific autoimmune response in this disease while leaving the remainder of the immune system intact, has long been sought. Proinsulin is a major target of the adaptive immune response in T1D. We hypothesized that an engineered DNA plasmid encoding proinsulin (BHT-3021) would preserve β cell function in T1D patients through reduction of insulin-specific CD8⁺ T cells. We studied 80 subjects over 18 years of age who were diagnosed with T1D within the past 5 years. Subjects were randomized 2:1 to receive intramuscular injections of BHT-3021 or BHT-placebo, weekly for 12 weeks, and then monitored for safety and immune responses in a blinded fashion. Four dose levels of BHT-3021 were evaluated: 0.3, 1.0, 3.0, and 6.0 mg. C-peptide was used both as an exploratory efficacy measure and as a safety measure. Islet-specific CD8⁺ T cell frequencies were assessed with multimers of monomeric human leukocyte antigen class I molecules loaded with peptides from pancreatic and unrelated antigens. No serious adverse events related to BHT-3021 were observed. C-peptide levels improved relative to placebo at all doses, at 1 mg at the 15-week time point (+19.5% BHT-3021 versus -8.8% BHT-placebo, P < 0.026). Proinsulin-reactive CD8⁺ T cells, but not T cells against unrelated islet or foreign molecules, declined in the BHT-3021 arm (P < 0.006). No significant changes were noted in interferon-γ, interleukin-4 (IL-4), or IL-10 production in CD4 T cells. Thus, we demonstrate that a plasmid encoding proinsulin reduces the frequency of CD8⁺ T cells reactive to proinsulin while preserving C-peptide over the course of dosing.

Evidence has accumulated that cytokines have a fundamental role in the differentiation of memory T cells. Here, we follow the CD8+ T cell from initial activation to memory-cell generation, indicating the checkpoints at which cytokines determine the fate of the T cell. Members of the common cytokine-receptor γ-chain (γc)-cytokine family — in particular, interleukin-7 (IL-7) and IL-15 — act at each stage of the immune response to promote proliferation and survival. In this manner, a stable and protective, long-lived memory CD8+ T-cell pool can be propagated and maintained.

BACKGROUND Interleukin (IL)-17F, produced in IL-23R-expressing Th17 cells, is a novel member of the IL-17 cytokine family. Given the association of IL23R with inflammatory bowel disease (IBD), we characterized the role of IL-17F in IBD including its intestinal gene expression and the effect of the IL17F p. His161Arg polymorphism on disease susceptibility and phenotype of Crohn's disease (CD) and ulcerative colitis (UC). In addition, we analyzed the IL17F p. His161Arg polymorphism for potential epistasis with IL23R and NOD2/CARD15 variants. METHODS Intestinal IL-17F mRNA expression was measured by quantitative polymerase chain reaction (PCR). Genomic DNA from 1682 individuals (CD: n = 499; UC: n = 216; controls: n = 967) was analyzed for the presence of the IL17F p. His161Arg polymorphism, the 3 NOD2 variants, p. Arg702Trp, p. Gly908Arg, and p. Leu1007fsX1008, and 10 CD-associated IL23R variants. RESULTS Intestinal IL-17F mRNA expression was 4.4-fold increased in inflamed colonic lesions compared to uninflamed biopsies in CD (P = 0.016) but not in UC. However, the mean intestinal IL-17F mRNA expression was higher in UC than in CD (P < 0.0001). The IL17F p. His161Arg substitution was observed with similar frequencies in IBD patients and controls and was not associated with a certain disease phenotype, but weakly associated with a low body mass index (BMI; P = 0.009) and an earlier age of disease onset (P = 0.039) in UC. There was no evidence for epistasis between the IL17F p. His161Arg polymorphism and IBD-associated single nucleotide polymorphisms within the IL23R gene. CONCLUSIONS Intestinal IL17F gene expression is increased in active CD. The IL17F p. His161Arg polymorphism is not associated with IBD susceptibility and has no epistatic interaction with CD-associated IL23R variants.

Little is known about the inter-individual variation of cytokine responses to different pathogens in healthy individuals. To systematically describe cytokine responses elicited by distinct pathogens and to determine the effect of genetic variation on cytokine production, we profiled cytokines produced by peripheral blood mononuclear cells from 197 individuals of European origin from the 200 Functional Genomics (200FG) cohort in the Human Functional Genomics Project (http://www.humanfunctionalgenomics.org), obtained over three different years. We compared bacteria- and fungi-induced cytokine profiles and found that most cytokine responses were organized around a physiological response to specific pathogens, rather than around a particular immune pathway or cytokine. We then correlated genome-wide single-nucleotide polymorphism (SNP) genotypes with cytokine abundance and identified six cytokine quantitative trait loci (QTLs). Among them, a cytokine QTL at the NAA35-GOLM1 locus markedly modulated interleukin (IL)-6 production in response to multiple pathogens and was associated with susceptibility to candidemia. Furthermore, the cytokine QTLs that we identified were enriched among SNPs previously associated with infectious diseases and heart diseases. These data reveal and begin to explain the variability in cytokine production by human immune cells in response to pathogens.

The thymic medulla provides a microenvironment where medullary thymic epithelial cells (mTECs) express autoimmune regulator and diverse tissue-restricted genes, contributing to launching self-tolerance. Positive selection is essential for thymic medulla formation via a previously unknown mechanism. Here we show that the cytokine RANK ligand (RANKL) was produced by positively selected thymocytes and regulated the cellularity of mTEC by interacting with RANK and osteoprotegerin. Forced expression of RANKL restored thymic medulla in mice lacking positive selection, whereas RANKL perturbation impaired medulla formation. These results indicate that RANKL produced by positively selected thymocytes is responsible for fostering thymic medulla formation, thereby establishing central tolerance.

One major role of the thymus is to provide the peripheral immune system with mature T cells, but the mechanisms involving the cellular export are not fully understood. In this study, we examined the ability of a novel immunosuppressive reagent, FTY720, to inhibit T cell export from the thymus. Daily administration of FTY720 at a dose of 1 mg / kg resulted in a marked decrease in the number of peripheral blood T lymphocytes. In the thymus, long-term daily administration of FTY720 caused a three- to fourfold increase in the proportion of mature medullary thymocytes (CD4(+)CD8(-) and CD4(-)CD8(+)) as well as a slight decrease in the double-positive cell (CD4(+)CD8(+)) ratio. Phenotypic analysis (TCRalpha beta, H-2K(d), CD44, CD69 and CD24) revealed that these increased subsets represent possible peripheral recent thymic emigrants. High level expression of L-selectin by these subsets further suggests that they were prevented from leaving the thymus. By intrathymic labeling with fluorescein isothiocyanate, only one fourth of labeled cells could be detected in the lymph nodes and in the spleen of FTY720-treated mice compared to saline-treated control mice. Taken together, these results suggest that the immunosuppressive action of FTY720, at least in part, could be due to its inhibitory effect on T cell emigration from the thymus to the periphery.

Cellular therapy with chimeric antigen receptor (CAR)-redirected cytotoxic T cells has shown impressive efficacy in the treatment of hematologic malignancies. We explored a regulatory T cell (Treg)-based therapy in the treatment of allergic airway inflammation, a model for asthma, which is characterized by an airway hyper-reactivity (AHR) and a chronic, T helper-2 (Th2) cell-dominated immune response to allergen. To restore the immune balance in the lung, we redirected Tregs by a CAR toward lung epithelia in mice upon experimentally induced allergic asthma, closely mimicking the clinical situation. Adoptively transferred CAR Tregs accumulated in the lung and in tracheobronchial lymph nodes, reduced AHR and diminished eosinophilic airway inflammation, indicated by lower cell numbers in the bronchoalveolar lavage fluid and decreased cell infiltrates in the lung. CAR Treg cells furthermore prevented excessive pulmonary mucus production as well as increase in allergen-specific IgE and Th2 cytokine levels in exposed animals. CAR Tregs were more efficient in controlling asthma than non-modified Tregs, indicating the pivotal role of specific Treg cell activation in the affected organ. Data demonstrate that lung targeting CAR Treg cells ameliorate key features of experimental airway inflammation, paving the way for cell therapy of severe allergic asthma.


Question: Decreased sensitivity to interleukin-2 in regulatory T cells correlates with increased resilience against autoimmune disorders like Type 1 Diabetes.

Association of Type 1 Diabetes-linked IL2RA Variants with Reduced IL-2 Signaling and Impaired Regulatory T Cell Function. Extensive studies have shown that CD4+CD25+ regulatory T cells (Tregs) from patients with various autoimmune diseases, including Type 1 Diabetes, exhibit a reduced capacity to suppress autologous proinflammatory responses compared to healthy controls. This Treg dysfunction may either be a primary cause or a consequence of immune system alterations during disease progression. Genetic variations in genes crucial for Treg function, such as IL2RA, have been linked to an increased risk of autoimmune disorders. While this suggests a primary role for dysfunctional Tregs in autoimmunity, the connection between IL2RA genetic variations and Treg function remains unexplored. To investigate this, we analyzed the impact of an IL2RA haplotype associated with Type 1 Diabetes on Treg performance and regulatory capabilities. The study was conducted using healthy individuals to eliminate any disease-related confounding factors. Our findings revealed that the presence of the autoimmune disease-linked IL2RA haplotype is associated with decreased IL-2 responsiveness in antigen-experienced CD4+ T cells, as evidenced by reduced STAT5a phosphorylation. This variant also correlates with lower FOXP3 expression levels in Tregs and a diminished capacity to suppress the proliferation of autologous effector T cells. These results provide insights into the molecular and cellular mechanisms by which IL-2RA gene polymorphisms influence immune regulation, thereby impacting susceptibility to autoimmune and inflammatory diseases.

The human cytomegalovirus UL111A gene is expressed during latent and productive infections, and it codes for homologs of interleukin-10 (IL-10). We examined whether viral IL-10 expressed during latency altered differentiation of latently infected myeloid progenitors. In comparison to infection with parental virus or mock infection, latent infection with a virus in which the gene encoding viral IL-10 has been deleted upregulated cytokines associated with dendritic cell (DC) formation and increased the proportion of myeloid DCs. These data demonstrate that viral IL-10 restricts the ability of latently infected myeloid progenitors to differentiate into DCs and identifies an immunomodulatory role for viral IL-10 which may limit the host's ability to clear latent virus.

Metastatic melanoma is a rapidly progressing disease with high mortality rate and limited treatment options. Immunotherapy based on tumor-targeting cytotoxic T cell responses represents a promising strategy. To assist in its development, we examined the possibility and efficacy of using CD4+ cytotoxic T cells. The regulatory mechanisms controlling CD4+ T cell-mediated cytotoxicity were also investigated. We found that naturally occurring granzyme B and perforin-expressing CD4+ cytotoxic T cells can be recovered from metastatic melanoma patients at significantly elevated frequencies compared to those from healthy controls. These CD4+ cytotoxic T cells were also capable of killing autologous tumor cells harvested from metastatic melanoma, independent of CD8+ T cells or any other cell types. However, several restricting factors were observed. First, the cytolytic activity by CD4+ T cells required high MHC class II expression on melanoma cells, which was not satisfied in a subset of melanomas. Second, the granzyme B and perforin release by activated CD4+ cytotoxic T cells was reduced after coculturing with autologous melanoma cells, characterized by low LAMP-1 expression and low granzyme B and perforin secretion in the supernatant. This suggested that inhibitory mechanisms were present to suppress CD4+ cytotoxic T cells. Indeed, blockade of PD-1 and CTLA-4 had increased the cytolytic activity of CD4+ T cells but was only effective in MHC class II high but not MHC class II low melanomas. Together, our study showed that CD4+ T cell-mediated cytotoxicity could eliminate primary melanoma cells but the efficacy depended on MHC class II expression.

OBJECTIVES Non-celiac wheat sensitivity (NCWS) is defined as a reaction to ingested wheat after exclusion of celiac disease and wheat allergy. As its pathogenesis is incompletely understood, we evaluated the inflammatory response in the rectal mucosa of patients with well-defined NCWS. METHODS The prospective study included 22 patients with irritable bowel syndrome (IBS)-like clinical presentation, diagnosed with NCWS by double-blind placebo-controlled challenge. Eight IBS patients not improving on wheat-free diet were used as controls. Two weeks after oral challenge was performed with 80 grams of wheat daily, cells were isolated from rectal biopsies and thoroughly characterized by fluorescence-activated cell sorting analysis for intracellular cytokines and surface markers. RESULTS Rectal biopsies from wheat-challenged NCWS patients showed that a significant mucosal CD45(+) infiltrate consisted of CD3(+) and CD3(-) lymphocytes, with the latter spontaneously producing more interferon (IFN)-γ than IBS controls. About 30% of IFN-γ-producing CD45(+) cells were T-bet(+), CD56(-), NKP44(-), and CD117(-), defining them as a type-1 innate lymphoid cells (ILC1). IFN-γ-producing ILC1 cells significantly decreased in 10 patients analyzed 2 weeks after they resumed a wheat-free diet. CONCLUSIONS These data indicate that, in patients with active NCWS, IFN-γ-producing ILC1 cells infiltrate rectal mucosa and support a role for this innate lymphoid cell population in the pathogenesis of NCWS.

RATIONALE The myeloid differentiation factor (MyD)88/interleukin (IL)-1 axis activates self-antigen-presenting cells and promotes autoreactive CD4(+) T-cell expansion in experimental autoimmune myocarditis, a mouse model of inflammatory heart disease. OBJECTIVE The aim of this study was to determine the role of MyD88 and IL-1 in the progression of acute myocarditis to an end-stage heart failure. METHODS AND RESULTS Using alpha-myosin heavy chain peptide (MyHC-alpha)-loaded, activated dendritic cells, we induced myocarditis in wild-type and MyD88(-/-) mice with similar distributions of heart-infiltrating cell subsets and comparable CD4(+) T-cell responses. Injection of complete Freund's adjuvant (CFA) or MyHC-alpha/CFA into diseased mice promoted cardiac fibrosis, induced ventricular dilation, and impaired heart function in wild-type but not in MyD88(-/-) mice. Experiments with chimeric mice confirmed the bone marrow origin of the fibroblasts replacing inflammatory infiltrates and showed that MyD88 and IL-1 receptor type I signaling on bone marrow-derived cells was critical for development of cardiac fibrosis during progression to heart failure. CONCLUSIONS Our findings indicate a critical role of MyD88/IL-1 signaling in the bone marrow compartment in postinflammatory cardiac fibrosis and heart failure and point to novel therapeutic strategies against inflammatory cardiomyopathy.

IL-12 has been identified as a major cytokine influencing the differentiation of CD4 cells to a Th1 phenotype, whereas a role for IFN-gamma is controversial. We investigated the interrelationship between IL-12 and IFN-gamma in promoting Th1 responses using naive CD4 cells reactive with pigeon cytochrome c from TCR transgenics and memory CD4 cells derived by in vivo priming with KLH. Without exogenous rIL-12 or rIFN-gamma, primary and memory effectors induced by Ag or anti-CD3 and anti-CD28 secreted variable levels of IL-2 and IFN-gamma. The level of IFN-gamma secreted by effectors correlated with endogenous IFN-gamma produced in primary cultures, and anti-IFN-gamma largely inhibited the development of effectors producing IFN-gamma. With optimal TCR stimulation and costimulation, endogenous IFN-gamma, without IL-12, was sufficient to elicit Th1 cells via an autocrine mechanism, whereas with suboptimal stimulation, exogenous rIFN-gamma or rIL-12 was required for Th1 development. However, rIL-12 was more effective than rIFN-gamma, partially because rIL-12 greatly enhanced autocrine production of IFN-gamma, and optimal development of the Th1 phenotype was mediated by the synergistic actions of both cytokines. Thus, both IFN-gamma and IL-12 can independently regulate Th1 development, but because of IFN-gamma-mediated feedback, their relative contributions are determined by the conditions of T cell stimulation. The extent of differentiation to a Th1 phenotype may, therefore, depend on the availability of both APC-derived IL-12 and autocrine IFN-gamma consequent to the overall strength of T cell stimulation.

The persistence of naive and memory T cells has long been of interest to immunologists, but the factors that influence the survival and homeostasis of these subsets have remained obscure. In recent years, it has become evident that the homeostasis of both naive and memory T-cell pools is highly dynamic and tightly regulated by internal stimuli, including cytokines and self-peptide–MHC ligands for the T-cell receptor. These homeostatic mechanisms might have a vital influence on the capacity of the T-cell pool to respond to both foreign and self-antigens.

The decline in immunocompetence with age is accompanied by the increase in the incidence of autoimmune diseases. Aging of the immune system, or immunosenescence, is characterized by a decline of both T and B cell function, and paradoxically the presence of low-grade chronic inflammation. There is growing evidence that epigenetics, the study of inherited changes in gene expression that are not encoded by the DNA sequence itself, changes with aging. Interestingly, emerging evidence suggests a key role for epigenetics in human pathologies, including inflammatory and neoplastic disorders. Here, we will review the potential mechanisms that contribute to the increase in autoimmune responses in aging. In particular, we will discuss how epigenetic alterations, especially DNA methylation and histone acetylation, are accumulated during aging and how these events contribute to autoimmunity risk.

Allergic airway inflammation is characterized by marked in situ changes in gene and protein expression, yet the role of microRNAs (miRNAs), a new family of key mRNA regulatory molecules, in this process has not yet been reported. Using a highly sensitive microarray-based approach, we identified 21 miRNAs with differential expression between doxycycline-induced lung-specific IL-13 transgenic mice (with allergic airway inflammation) and control mice. In particular, we observed overexpression of miR-21 and underexpression of miR-1 in the induced IL-13 transgenic mice compared with control mice. These findings were validated in two independent models of allergen-induced allergic airway inflammation and in IL-4 lung transgenic mice. Although IL-13-induced miR-21 expression was IL-13Ralpha1 dependent, allergen-induced miR-21 expression was mediated mainly independent of IL-13Ralpha1 and STAT6. Notably, predictive algorithms identified potential direct miR-21 targets among IL-13-regulated lung transcripts, such as IL-12p35 mRNA, which was decreased in IL-13 transgenic mice. Introduction of pre-miR-21 dose dependently inhibited cellular expression of a reporter vector harboring the 3'-untranslated region of IL-12p35. Moreover, mutating miR-21 binding sites in IL-12p35 3'-untranslated region abrogated miR-21-mediated repression. In summary, we have identified a miRNA signature in allergic airway inflammation, which includes miR-21 that modulates IL-12, a molecule germane to Th cell polarization.

The mammalian gastrointestinal tract harbors a microbial community with metabolic activity critical for host health, including metabolites that can modulate effector functions of immune cells. Mice treated with vancomycin have an altered microbiome and metabolite profile, exhibit exacerbated T helper type 2 cell (Th2) responses, and are more susceptible to allergic lung inflammation. Here we show that dietary supplementation with short-chain fatty acids (SCFAs) ameliorates this enhanced asthma susceptibility by modulating the activity of T cells and dendritic cells (DCs). Dysbiotic mice treated with SCFAs have fewer interleukin-4 (IL4)-producing CD4+ T cells and decreased levels of circulating immunoglobulin E (IgE). In addition, DCs exposed to SCFAs activate T cells less robustly, are less motile in response to CCL19 in vitro, and exhibit a dampened ability to transport inhaled allergens to lung draining nodes. Our data thus demonstrate that gut dysbiosis can exacerbate allergic lung inflammation through both T cell- and DC-dependent mechanisms that are inhibited by SCFAs.

The expression of self-antigen in the thymus is believed to be responsible for the deletion of autoreactive T lymphocytes, a critical process in the maintenance of unresponsiveness to self. The Autoimmune regulator (Aire) gene, which is defective in the disorder autoimmune polyglandular syndrome type 1, has been shown to promote the thymic expression of self-antigens. A clear link, however, between specific thymic self-antigens and a single autoimmune phenotype in this model has been lacking. We show that autoimmune eye disease in aire-deficient mice develops as a result of loss of thymic expression of a single eye antigen, interphotoreceptor retinoid-binding protein (IRBP). In addition, lack of IRBP expression solely in the thymus, even in the presence of aire expression, is sufficient to trigger spontaneous eye-specific autoimmunity. These results suggest that failure of thymic expression of selective single self-antigens can be sufficient to cause organ-specific autoimmune disease, even in otherwise self-tolerant individuals.


Question: Substituting histone H2A with H2A.Z decelerates gene activation in yeasts by stabilizing nucleosomes at the +1 position.

Nucleosome Stability Influenced by Histone Variants H3.3 and H2A.Z in Transcriptional Regulation. Histone variant H3.3, often found clustered around transcriptionally active genes and regulatory elements in vivo, has been implicated in unique transcriptional properties, though the precise role of H3.3-containing nucleosomes has remained unclear. Our findings indicate that H3.3 nucleosomes, isolated from vertebrate sources, exhibit heightened sensitivity to salt-mediated disruption, leading to the loss of H2A/H2B or H2A.Z/H2B dimers. Further, immunoprecipitation analysis of nucleosome core particles (NCPs) reveals that NCPs harboring both H3.3 and H2A.Z are notably less stable than those with H3.3 and H2A. Interestingly, NCPs comprising H3 and H2A.Z demonstrate stability similar to or greater than those with H3 and H2A. These observations establish a stability hierarchy for native nucleosomes based on their variant composition, shedding light on the versatile roles of H2A.Z in the context of its partner within the NCP. Moreover, the findings support the notion that H3.3 actively contributes to the maintenance of accessible chromatin structures in enhancer and transcribed regions. In line with this hypothesis, transcriptionally active genes' promoters and enhancers, as well as coding regions in highly expressed genes, feature nucleosomes that carry both H3.3 and H2A.Z, making them highly susceptible to disruption.

E2F proteins can either activate or repress transcription. Following mitogenic stimulation, repressive E2F4-p130-histone deacetylase complexes dissociate from, while activating species (E2F1, -2, and -3) associate with, target promoters. Histones H3 and H4 simultaneously become hyperacetylated, but it remains unclear whether this is a prerequisite or a consequence of E2F binding. Here, we show that activating E2F species are required for hyperacetylation of target chromatin in human cells. Overexpression of a dominant-negative (DN) E2F1 mutant in serum-stimulated T98G cells blocked all E2F binding, H4 acetylation, and, albeit partially, H3 acetylation. Target gene activation and S-phase entry were also blocked by DN E2F1. Conversely, ectopic activation of E2F1 rapidly induced H3 and H4 acetylation, demonstrating a direct role for E2F in these events. E2F1 was previously shown to bind the histone acetyltransferases (HATs) p300/CBP and PCAF/GCN5. In our hands, ectopically expressed E2F1 also bound the unrelated HAT Tip60 and induced recruitment of five subunits of the Tip60 complex (Tip60, TRRAP, p400, Tip48, and Tip49) to target promoters in vivo. Moreover, E2F-dependent recruitment of Tip60 to chromatin occurred in late G(1) following serum stimulation. We speculate that the activities of multiple HAT complexes account for E2F-dependent acetylation, transcription, and S-phase entry.

Nuclear transcription is not restricted to genes but occurs throughout the intergenic and noncoding space of eukaryotic genomes. The functional significance of this widespread noncoding transcription is mostly unknown. We show that Arabidopsis RNA polymerase IVb/Pol V, a multisubunit nuclear enzyme required for siRNA-mediated gene silencing of transposons and other repeats, transcribes intergenic and noncoding sequences, thereby facilitating heterochromatin formation and silencing of overlapping and adjacent genes. Pol IVb/Pol V transcription requires the chromatin-remodeling protein DRD1 but is independent of siRNA biogenesis. However, Pol IVb/Pol V transcription and siRNA production are both required to silence transposons, suggesting that Pol IVb/Pol V generates RNAs or chromatin structures that serve as scaffolds for siRNA-mediated heterochromatin-forming complexes. Pol IVb/Pol V function provides a solution to a paradox of epigenetic control: the need for transcription in order to transcriptionally silence the same region.

RNA processing is carried out in close proximity to the site of transcription, suggesting a regulatory link between transcription and pre-mRNA splicing. Using an in vitro transcription/splicing assay, we demonstrate that an association of RNA polymerase II (Pol II) transcription and pre-mRNA splicing is required for efficient gene expression. Pol II-synthesized RNAs containing functional splice sites are protected from nuclear degradation, presumably because the local concentration of the splicing machinery is sufficiently high to ensure its association over interactions with nucleases. Furthermore, the process of transcription influences alternative splicing of newly synthesized pre-mRNAs. Because other RNA polymerases do not provide similar protection from nucleases, and their RNA products display altered splicing patterns, the link between transcription and RNA processing is RNA Pol II-specific. We propose that the connection between transcription by Pol II and pre-mRNA splicing guarantees an extended half-life and proper processing of nascent pre-mRNAs.

Telomeres are the physical ends of eukaryotic chromosomes. Genetic studies have established that the baker's yeast Pif1p DNA helicase is a negative regulator of telomerase, the specialized reverse transcriptase that maintains telomeric DNA, but the biochemical basis for this inhibition was unknown. Here we show that in vitro, Pif1p reduces the processivity of telomerase and releases telomerase from telomeric oligonucleotides. The released telomerase is enzymatically active because it is able to lengthen a challenger oligonucleotide. In vivo, overexpression of Pif1p reduces telomerase association with telomeres, whereas depleting cells of Pif1p increases the levels of telomere-bound Est1p, a telomerase subunit that is present on the telomere when telomerase is active. We propose that Pif1p helicase activity limits telomerase action both in vivo and in vitro by displacing active telomerase from DNA ends.

The transcriptional status of a gene can be maintained through multiple rounds of cell division during development. This epigenetic effect is believed to reflect heritable changes in chromatin folding and histone modifications or variants at target genes, but little is known about how these chromatin features are inherited through cell division. A particular challenge for maintaining transcription states is DNA replication, which disrupts or dilutes chromatin-associated proteins and histone modifications. PRC1-class Polycomb group protein complexes are essential for development and are thought to heritably silence transcription by altering chromatin folding and histone modifications. It is not known whether these complexes and their effects are maintained during DNA replication or subsequently re-established. We find that when PRC1-class Polycomb complex-bound chromatin or DNA is replicated in vitro, Polycomb complexes remain bound to replicated templates. Retention of Polycomb proteins through DNA replication may contribute to maintenance of transcriptional silencing through cell division.

Recent structures of the nucleosome core particle reveal details of histone-histone and histone-DNA interactions. These structures have now set the stage for understanding chromatin assembly and dynamics during replication and transcription. Histone chaperones and chromatin remodeling complexes are important in both of these processes. The nucleosome and its protein core, the histone octamer, have twofold symmetry, which histone chaperones may use to bind core histones. Recent studies suggest that the nucleoplasmin pentamer may mediate histone storage, sperm chromatin decondensation and nucleosome assembly, by dimerizing to form a decamer. In this model, histone binding on the lateral surface of the chaperone involves stereospecific interactions and a shared twofold axis.

Publisher Summary This chapter summarizes the development and characterization of rabbit polyclonal antibodies named histone that are directed against the methylated H3-K9 position. It provides protocols for peptide design, rabbit immunizations, and quality controls of methyl-lysine histone antibodies, followed by their in vivo characterization using indirect IF of inter-and metaphase chromatin in wild-type (wt) and mutant mouse cells that are deficient for the Suv39h histone methyltransferases (HMTases). Histone amino-termini (tails) protrude from the nucleosome core and are subject to a variety of post-translational modifications, including acetylation (on lysine residues), phosphorylation (on serine and threonine residues), methylation (on lysine and arginine residues), ubiquitination (on lysine residues), and ADP-ribosylation (on glutamic acid residues). In addition to their structural roles, histones play important functions in the control of gene expression by regulating access to the underlying nucleosomal template. It is without doubt that the development of high-quality, position-specific methyl-lysine histone antibodies can provide important tools for the further decoding of the epigenetic information, which is in part, indexed by distinct methylation states of selective lysine residues in the histone amino-termini. A comparative analysis indicates significant discrepancies in the specificity and avidity of the available methyl-lysine histone antibodies and highlights the need for extensive quality controls, such that experimental data can be correctly interpreted despite the exquisite complexity of histone lysine methylation.

Nuclear actin-related proteins (Arps) are subunits of several chromatin remodelers, but their molecular functions within these complexes are unclear. We report the crystal structure of the INO80 complex subunit Arp8 in its ATP-bound form. Human Arp8 has several insertions in the conserved actin fold that explain its inability to polymerize. Most remarkably, one insertion wraps over the active site cleft and appears to rigidify the domain architecture, while active site features shared with actin suggest an allosterically controlled ATPase activity. Quantitative binding studies with nucleosomes and histone complexes reveal that Arp8 and the Arp8-Arp4-actin-HSA sub-complex of INO80 strongly prefer nucleosomes and H3-H4 tetramers over H2A-H2B dimers, suggesting that Arp8 functions as a nucleosome recognition module. In contrast, Arp4 prefers free (H3-H4)(2) over nucleosomes and may serve remodelers through binding to (dis)assembly intermediates in the remodeling reaction.

Interphase nuclear repositioning of chromosomes has been implicated in the epigenetic regulation of RNA polymerase (pol) II transcription. However, little is known about the nuclear position–dependent regulation of RNA pol I–transcribed loci. Trypanosoma brucei is an excellent model system to address this question because its two main surface protein genes, procyclin and variant surface glycoprotein (VSG), are transcribed by pol I and undergo distinct transcriptional activation or downregulation events during developmental differentiation. Although the monoallelically expressed VSG locus is exclusively localized to an extranucleolar body in the bloodstream form, in this study, we report that nonmutually exclusive procyclin genes are located at the nucleolar periphery. Interestingly, ribosomal DNA loci and pol I transcription activity are restricted to similar perinucleolar positions. Upon developmental transcriptional downregulation, however, the active VSG promoter selectively undergoes a rapid and dramatic repositioning to the nuclear envelope. Subsequently, the VSG promoter region was subjected to chromatin condensation. We propose a model whereby the VSG expression site pol I promoter is selectively targeted by temporal nuclear repositioning during developmental silencing.

Proper execution of transcriptional programs is a key requirement of gene expression regulation, demanding accurate control of timing and amplitude. How precisely the transcription machinery fulfills this task is not known. Using an in situ hybridization approach that detects single mRNA molecules, we measured mRNA abundance and transcriptional activity within single Saccharomyces cerevisiae cells. We found that expression levels for particular genes are higher than initially reported and can vary substantially among cells. However, variability for most constitutively expressed genes is unexpectedly small. Combining single-transcript measurements with computational modeling indicates that low expression variation is achieved by transcribing genes using single transcription-initiation events that are clearly separated in time, rather than by transcriptional bursts. In contrast, PDR5, a gene regulated by the transcription coactivator complex SAGA, is expressed using transcription bursts, resulting in larger variation. These data directly demonstrate the existence of multiple expression modes used to modulate the transcriptome.


Question: Substituting histone H2A with H2A.Z decelerates gene activation in yeasts by stabilizing the +1 nucleosomes.

Genome-Scale Analysis of H2A.Z Dynamics: A Histone Variant that Primes Repressed Gene Promoters for Activation through Nucleosome Disassembly. The specialized roles of histone variants in chromatin organization and gene regulation remain largely unexplored. In this study, we have mapped the genome-wide distribution and dynamics of H2A.Z, a histone H2A variant in yeast. H2A.Z is predominantly found at hundreds of repressed or constitutively expressed RNA polymerase II promoters, with a preference for TATA-less promoter regions. The deposition of H2A.Z is specifically mediated by the SWR1 complex, which largely overlaps with H2A.Z binding sites. The presence of H2A.Z is associated with distinct histone modifications, and its localization is partly dependent on Gcn5, an acetyltransferase for histones, and Bdf1, a component of the SWR1 complex that recognizes acetylated histones. Upon shifts in growth conditions, H2A.Z undergoes a dramatic relocalization from active to repressed or constitutively expressed promoters. Interestingly, H2A.Z facilitates complete gene activation but does not significantly affect gene repression. Notably, H2A.Z is selectively dislodged from chromatin in vitro under conditions where H2A and H3 remain bound. Our findings suggest that H2A.Z-containing nucleosomes are preferentially deposited at repressed promoters but promote activation by their propensity to be lost, potentially aiding in the exposure of promoter DNA for transcription.

BACKGROUND Open chromatin regions are correlated with active regulatory elements in development and are dysregulated in diseases. The BAF (SWI/SNF) complex is essential for development, and has been demonstrated to remodel reconstituted chromatin in vitro and to control the accessibility of a few individual regions in vivo. However, it remains unclear where and how BAF controls the open chromatin landscape to regulate developmental processes, such as human epidermal differentiation. RESULTS Using a novel "on-plate" ATAC-sequencing approach for profiling open chromatin landscapes with a low number of adherent cells, we demonstrate that the BAF complex is essential for maintaining 11.6 % of open chromatin regions in epidermal differentiation. These BAF-dependent open chromatin regions are highly cell-type-specific and are strongly enriched for binding sites for p63, a master epidermal transcription factor. The DNA sequences of p63 binding sites intrinsically favor nucleosome formation and are inaccessible in other cell types without p63 to prevent ectopic activation. In epidermal cells, BAF and p63 mutually recruit each other to maintain 14,853 open chromatin regions. We further demonstrate that BAF and p63 cooperatively position nucleosomes away from p63 binding sites and recruit transcriptional machinery to control tissue differentiation. CONCLUSIONS BAF displays high specificity in controlling the open chromatin landscape during epidermal differentiation by cooperating with the master transcription factor p63 to maintain lineage-specific open chromatin regions.

Posttranslational modifications mediate important regulatory functions in biology. The acetylation of the p53 transcription factor, for example, promotes transcriptional activation of target genes including p21. Here we show that the acetylation of two lysine residues in p53 promotes recruitment of the TFIID subunit TAF1 to the p21 promoter through its bromodomains. UV irradiation of cells diacetylates p53 at lysines 373 and 382, which in turn recruits TAF1 to a distal p53-binding site on the p21 promoter prior to looping to the core promoter. Disruption of acetyl-p53/bromodomain interaction inhibits TAF1 recruitment to both the distal p53-binding site and the core promoter. Further, the TFIID subunits TAF4, TAF5, and TBP are detected on the core promoter prior to TAF1, suggesting that, upon DNA damage, distinct subunits of TFIID may be recruited separately to the p21 promoter and that the transcriptional activation depends on posttranslational modification of the p53 transcription factor.

The centromeric histone H3 variant (CenH3) is essential for chromosome segregation in eukaryotes. We identify posttranslational modifications of Saccharomyces cerevisiae CenH3, Cse4. Functional characterization of cse4 phosphorylation mutants shows growth and chromosome segregation defects when combined with kinetochore mutants okp1 and ame1. Using a phosphoserine-specific antibody, we show that the association of phosphorylated Cse4 with centromeres increases in response to defective microtubule attachment or reduced cohesion. We determine that evolutionarily conserved Ipl1/Aurora B contributes to phosphorylation of Cse4, as levels of phosphorylated Cse4 are reduced at centromeres in ipl1 strains in vivo, and in vitro assays show phosphorylation of Cse4 by Ipl1. Consistent with these results, we observe that a phosphomimetic cse4-4SD mutant suppresses the temperature-sensitive growth of ipl1-2 and Ipl1 substrate mutants dam1 spc34 and ndc80, which are defective for chromosome biorientation. Furthermore, cell biology approaches using a green fluorescent protein-labeled chromosome show that cse4-4SD suppresses chromosome segregation defects in dam1 spc34 strains. On the basis of these results, we propose that phosphorylation of Cse4 destabilizes defective kinetochores to promote biorientation and ensure faithful chromosome segregation. Taken together, our results provide a detailed analysis, in vivo and in vitro, of Cse4 phosphorylation and its role in promoting faithful chromosome segregation.

The behavior of gene modules in complex synthetic circuits is often unpredictable. After joining modules to create a circuit, downstream elements (such as binding sites for a regulatory protein) apply a load to upstream modules that can negatively affect circuit function. Here we devised a genetic device named a load driver that mitigates the impact of load on circuit function, and we demonstrate its behavior in Saccharomyces cerevisiae. The load driver implements the design principle of timescale separation: inclusion of the load driver's fast phosphotransfer processes restores the capability of a slower transcriptional circuit to respond to time-varying input signals even in the presence of substantial load. Without the load driver, we observed circuit behavior that suffered from a 76% delay in response time and a 25% decrease in system bandwidth due to load. With the addition of a load driver, circuit performance was almost completely restored. Load drivers will serve as fundamental building blocks in the creation of complex, higher-level genetic circuits.

A major goal of biology is to provide a quantitative description of cellular behaviour. This task, however, has been hampered by the difficulty in measuring protein abundances and their variation. Here we present a strategy that pairs high-throughput flow cytometry and a library of GFP-tagged yeast strains to monitor rapidly and precisely protein levels at single-cell resolution. Bulk protein abundance measurements of >2,500 proteins in rich and minimal media provide a detailed view of the cellular response to these conditions, and capture many changes not observed by DNA microarray analyses. Our single-cell data argue that noise in protein expression is dominated by the stochastic production/destruction of messenger RNAs. Beyond this global trend, there are dramatic protein-specific differences in noise that are strongly correlated with a protein's mode of transcription and its function. For example, proteins that respond to environmental changes are noisy whereas those involved in protein synthesis are quiet. Thus, these studies reveal a remarkable structure to biological noise and suggest that protein noise levels have been selected to reflect the costs and potential benefits of this variation.

The growing epidemic of obesity and metabolic diseases calls for a better understanding of adipocyte biology. The regulation of transcription in adipocytes is particularly important, as it is a target for several therapeutic approaches. Transcriptional outcomes are influenced by both histone modifications and transcription factor binding. Although the epigenetic states and binding sites of several important transcription factors have been profiled in the mouse 3T3-L1 cell line, such data are lacking in human adipocytes. In this study, we identified H3K56 acetylation sites in human adipocytes derived from mesenchymal stem cells. H3K56 is acetylated by CBP and p300, and deacetylated by SIRT1, all are proteins with important roles in diabetes and insulin signaling. We found that while almost half of the genome shows signs of H3K56 acetylation, the highest level of H3K56 acetylation is associated with transcription factors and proteins in the adipokine signaling and Type II Diabetes pathways. In order to discover the transcription factors that recruit acetyltransferases and deacetylases to sites of H3K56 acetylation, we analyzed DNA sequences near H3K56 acetylated regions and found that the E2F recognition sequence was enriched. Using chromatin immunoprecipitation followed by high-throughput sequencing, we confirmed that genes bound by E2F4, as well as those by HSF-1 and C/EBPα, have higher than expected levels of H3K56 acetylation, and that the transcription factor binding sites and acetylation sites are often adjacent but rarely overlap. We also discovered a significant difference between bound targets of C/EBPα in 3T3-L1 and human adipocytes, highlighting the need to construct species-specific epigenetic and transcription factor binding site maps. This is the first genome-wide profile of H3K56 acetylation, E2F4, C/EBPα and HSF-1 binding in human adipocytes, and will serve as an important resource for better understanding adipocyte transcriptional regulation.

Nuclear lamin B1 (LMNB1) constitutes one of the major structural proteins in the lamina mesh. We silenced the expression of LMNB1 by RNA interference in the colon cancer cell line DLD-1 and showed a dramatic redistribution of H3K27me3 from the periphery to a more homogeneous nuclear dispersion. In addition, we observed telomere attrition and an increased frequency of micronuclei and nuclear blebs. By 3D-FISH analyses, we demonstrated that the volume and surface of chromosome territories were significantly larger in LMNB1-depleted cells, suggesting that LMNB1 is required to maintain chromatin condensation in interphase nuclei. These changes led to a prolonged S phase due to activation of Chk1. Finally, silencing of LMNB1 resulted in extensive changes in alternative splicing of multiple genes and in a higher number of enlarged nuclear speckles. Taken together, our results suggest a mechanistic role of the nuclear lamina in the organization of chromosome territories, maintenance of genome integrity and proper gene splicing.

Synthesis of ribosomal RNA (rRNA) by RNA polymerase (Pol) I is the first step in ribosome biogenesis and a regulatory switch in eukaryotic cell growth. Here we report the 12 A cryo-electron microscopic structure for the complete 14-subunit yeast Pol I, a homology model for the core enzyme, and the crystal structure of the subcomplex A14/43. In the resulting hybrid structure of Pol I, A14/43, the clamp, and the dock domain contribute to a unique surface interacting with promoter-specific initiation factors. The Pol I-specific subunits A49 and A34.5 form a heterodimer near the enzyme funnel that acts as a built-in elongation factor and is related to the Pol II-associated factor TFIIF. In contrast to Pol II, Pol I has a strong intrinsic 3'-RNA cleavage activity, which requires the C-terminal domain of subunit A12.2 and, apparently, enables ribosomal RNA proofreading and 3'-end trimming.

Nonspecific protein-DNA interactions are inherently dynamic and involve both diffusion of the protein along the DNA and hopping of the protein from one DNA molecule or segment to another. Understanding how gene regulatory proteins interact nonspecifically with DNA in terms of both structure and dynamics is challenging because the experimental observables are an ensemble average of many rapidly exchanging states. By using a variety of NMR spectroscopic techniques, including relaxation analysis, paramagnetic relaxation enhancement, and residual dipolar couplings, we have characterized structural and kinetic aspects of the interaction of the HoxD9 homeodomain with a nonspecific, 24-bp DNA duplex in a system in which the protein is not constrained to any particular site. The data reveal that HoxD9 binds to nonspecific DNA with the same binding mode and orientation as that observed in the specific complex. The mobility, however, of Arg side-chains contacting the DNA is increased in the nonspecific complex relative to the specific one. The kinetics of intermolecular translocation between two different nonspecific DNA molecules have also been analyzed and reveal that at high DNA concentrations (such as those present in vivo) direct transfer from one nonspecific complex to another nonspecific DNA molecule occurs without going through the intermediary of free protein. This finding provides a simple mechanism for accelerating the target search in vivo for the specific site in a sea of nonspecific sites by permitting more effective sampling of available DNA sites as the protein jumps from one segment to another.

Kinetochores attach the replicated chromosomes to the mitotic spindle and orchestrate their transmission to the daughter cells. Kinetochore–spindle binding and chromosome segregation are mediated by the multi-copy KNL1Spc105, MIS12Mtw1 and NDC80Ndc80 complexes that form the so-called KMN network. KMN–spindle attachment is regulated by the Aurora BIpl1 and MPS1Mps1 kinases. It is unclear whether other mechanisms exist that support KMN activity during the cell cycle. Using budding yeast, we show that kinetochore protein Cnn1 localizes to the base of the Ndc80 complex and promotes a functionally competent configuration of the KMN network. Cnn1 regulates KMN activity in a spatiotemporal manner by inhibiting the interaction between its complexes. Cnn1 activity peaks in anaphase and is driven by the Cdc28, Mps1 and Ipl1 kinases.


Question: Ribosomopathies display a minimal level of cell and tissue-specific pathologies.

Ribosomal Protein-Mediated Regulation in Hox mRNA Translation and Its Impact on Tissue Differentiation
Traditionally, the ribosome has been considered a non-regulatory ribozyme in mRNA translation, with a uniform function across all mRNAs. However, we have discovered that mutations in the Ribosomal Protein L38 (Rpl38) gene in mice lead to specific and surprising defects in tissue patterning, notably severe homeotic transformations in the axial skeleton. In Rpl38 mutant embryos, overall protein synthesis remains unaffected; yet, the translation of a distinct group of Homeobox mRNAs is significantly disrupted. Our findings indicate that RPL38 acts as a regulatory element of the ribosome, aiding in the formation of the 80S complex on these specific mRNAs to exert selective translational control. Moreover, we observed that Rpl38 expression is notably elevated in the embryonic regions where loss-of-function phenotypes manifest. Interestingly, an extensive analysis of ribosomal protein (RP) expression patterns in the vertebrate embryo reveals a dynamic regulation of individual RPs. These results suggest that the activity of ribosomal proteins could be intricately controlled to introduce a novel level of specificity in gene expression regulation and the development of mammals.

Emerging data suggest that microRNAs (miRNAs) are instrumental in a variety of stress responses in addition to their more recognized role in development. Surprisingly, miRNAs, which normally suppress expression of target transcripts, may become activators of expression during stress. This might be partially explained by new interactions of miRNA/Argonaute complexes with RNA-binding proteins that relocate from different subcellular compartments during stress.

The biogenesis of the many functional compartments contained in the mammalian cell nucleus is poorly understood. More specifically, little is known regarding the initial nucleation step required for nuclear body formation. Here we show that RNA can function as a structural element and a nucleator of nuclear bodies. We find that several types of coding and noncoding RNAs are sufficient to de novo assemble, and are physiologically enriched in, histone locus bodies (with associated Cajal bodies), nuclear speckles, paraspeckles and nuclear stress bodies. Formation of nuclear bodies occurs through recruitment and accumulation of proteins resident in the nuclear bodies by nucleating RNA. These results demonstrate that transcription is a driving force in nuclear body formation and RNA transcripts can function as a scaffold in the formation of major nuclear bodies. Together, these data suggest that RNA-primed biogenesis of nuclear bodies is a general principle of nuclear organization.

Mesenchymal niche cells may drive tissue failure and malignant transformation in the hematopoietic system, but the underlying molecular mechanisms and relevance to human disease remain poorly defined. Here, we show that perturbation of mesenchymal cells in a mouse model of the pre-leukemic disorder Shwachman-Diamond syndrome (SDS) induces mitochondrial dysfunction, oxidative stress, and activation of DNA damage responses in hematopoietic stem and progenitor cells. Massive parallel RNA sequencing of highly purified mesenchymal cells in the SDS mouse model and a range of human pre-leukemic syndromes identified p53-S100A8/9-TLR inflammatory signaling as a common driving mechanism of genotoxic stress. Transcriptional activation of this signaling axis in the mesenchymal niche predicted leukemic evolution and progression-free survival in myelodysplastic syndrome (MDS), the principal leukemia predisposition syndrome. Collectively, our findings identify mesenchymal niche-induced genotoxic stress in heterotypic stem and progenitor cells through inflammatory signaling as a targetable determinant of disease outcome in human pre-leukemia.

Ribonucleoprotein complexes consisting of Argonaute-like proteins and small regulatory RNAs function in a wide range of biological processes. Many of these small regulatory RNAs are predicted to act, at least in part, within the nucleus. We conducted a genetic screen to identify factors essential for RNA interference (RNAi) in nuclei of Caenorhabditis elegans and identified the Argonaute protein NRDE-3. In the absence of small interfering RNAs (siRNAs), NRDE-3 resides in the cytoplasm. NRDE-3 binds siRNAs generated by RNA-dependent RNA polymerases acting on messenger RNA templates in the cytoplasm and redistributes to the nucleus. Nuclear redistribution of NRDE-3 requires a functional nuclear localization signal, is required for nuclear RNAi, and results in NRDE-3 association with nuclear-localized nascent transcripts. Thus, specific Argonaute proteins can transport specific classes of small regulatory RNAs to distinct cellular compartments to regulate gene expression.

The ability to maintain a functional proteome, or proteostasis, declines during the ageing process. Damaged and misfolded proteins accumulate with age, impairing cell function and tissue homeostasis. The accumulation of damaged proteins contributes to multiple age-related diseases such as Alzheimer's, Parkinson's or Huntington's disease. Damaged proteins are degraded by the ubiquitin-proteasome system or through autophagy-lysosome, key components of the proteostasis network. Modulation of either proteasome activity or autophagic-lysosomal potential extends lifespan and protects organisms from symptoms associated with proteostasis disorders, suggesting that protein clearance mechanisms are directly linked to ageing and age-associated diseases.

During the many cell divisions that precede formation of plant gametes, their apical-meristem and floral antecedents are continually exposed to endogenous and environmental mutagenic threats. Although some deleterious recessive mutations may be eliminated during growth of haploid gametophytes and functionally haploid early embryos ("haplosufficiency quality-checking"), the multiplicity of plant genome-maintenance systems suggests aggressive quality control during prior diploid growth. To test in Arabidopsis a hypothesis that prior mismatch repair (MMR) is paramount in defense of plant genetic fidelity, we propagated in parallel 36 MMR-defective (Atmsh2-1) and 36 wild-type lines. The Atmsh2-1 lines rapidly accumulated a wide variety of mutations: fifth-generation (G5) plants showed abnormalities in morphology and development, fertility, germination efficiency, seed/silique development, and seed set. Only two Atmsh2-1, but all 36 wild-type lines, appeared normal at G5. Analyses of insertion/deletion mutation at six repeat-sequence (microsatellite) loci showed each Atmsh2-1 line to have evolved its own "fingerprint," the results of as many as 10 microsatellite mutations in a single line. Thus, MMR during diploid growth is essential for plant genomic integrity.

MicroRNAs (miRNAs) are a family of short, non-coding RNA molecules (∼22nt) involved in post-transcriptional control of gene expression. They act via base-pairing with mRNA transcripts that harbour target sequences, resulting in accelerated mRNA decay and/or translational attenuation. Given miRNAs mediate the expression of molecules involved in many aspects of normal cell development and functioning, it is not surprising that aberrant miRNA expression is closely associated with many human diseases. Their pivotal role in driving a range of normal cellular physiology as well as pathological processes has established miRNAs as potential therapeutics, as well as potential diagnostic and prognostic tools in human health. MicroRNA-7 (miR-7) is a highly conserved miRNA which displays restricted spatiotemporal expression during development and in maturity. In humans and mice, mature miR-7 is generated from three different genes, illustrating unexpected redundancy and also the importance of this miRNA in regulating key cellular processes. In this review we examine the expanding role of miR-7 in the context of health, with emphasis on organ differentiation and development, as well as in various mammalian diseases, particularly of the brain, heart, endocrine pancreas and skin, as well as in cancer. The more we learn about miR-7, the more we realise the complexity of its regulation and potential functional application both from a biomarker and therapeutic perspective.

Cellular circular RNAs (circRNAs) are generated by head-to-tail splicing and are present in all multicellular organisms studied so far. Recently, circRNAs have emerged as a large class of RNA which can function as post-transcriptional regulators. It has also been shown that many circRNAs are tissue- and stage-specifically expressed. Moreover, the unusual stability and expression specificity make circRNAs important candidates for clinical biomarker research. Here, we present a circRNA expression resource of 20 human tissues highly relevant to disease-related research: vascular smooth muscle cells (VSMCs), human umbilical vein cells (HUVECs), artery endothelial cells (HUAECs), atrium, vena cava, neutrophils, platelets, cerebral cortex, placenta, and samples from mesenchymal stem cell differentiation. In eight different samples from a single donor, we found highly tissue-specific circRNA expression. Circular-to-linear RNA ratios revealed that many circRNAs were expressed higher than their linear host transcripts. Among the 71 validated circRNAs, we noticed potential biomarkers. In adenosine deaminase-deficient, severe combined immunodeficiency (ADA-SCID) patients and in Wiskott-Aldrich-Syndrome (WAS) patients' samples, we found evidence for differential circRNA expression of genes that are involved in the molecular pathogenesis of both phenotypes. Our findings underscore the need to assess circRNAs in mechanisms of human disease. KEY MESSAGES: circRNA resource catalog of 20 clinically relevant tissues. circRNA expression is highly tissue-specific. circRNA transcripts are often more abundant than their linear host RNAs. circRNAs can be differentially expressed in disease-associated genes.

Transcript regulation is essential for cell function, and misregulation can lead to disease. Despite technologies to survey the transcriptome, we lack a comprehensive understanding of transcript kinetics, which limits quantitative biology. This is an acute challenge in embryonic development, where rapid changes in gene expression dictate cell fate decisions. By ultra-high-frequency sampling of Xenopus embryos and absolute normalization of sequence reads, we present smooth gene expression trajectories in absolute transcript numbers. During a developmental period approximating the first 8 weeks of human gestation, transcript kinetics vary by eight orders of magnitude. Ordering genes by expression dynamics, we find that "temporal synexpression" predicts common gene function. Remarkably, a single parameter, the characteristic timescale, can classify transcript kinetics globally and distinguish genes regulating development from those involved in cellular metabolism. Overall, our analysis provides unprecedented insight into the reorganization of maternal and embryonic transcripts and redefines our ability to perform quantitative biology.

Nova proteins are neuron-specific antigens targeted in paraneoplastic opsoclonus myoclonus ataxia (POMA), an autoimmune neurologic disease characterized by abnormal motor inhibition. Nova proteins regulate neuronal pre-messenger RNA splicing by directly binding to RNA. To identify Nova RNA targets, we developed a method to purify protein-RNA complexes from mouse brain with the use of ultraviolet cross-linking and immunoprecipitation (CLIP).Thirty-four transcripts were identified multiple times by Nova CLIP.Three-quarters of these encode proteins that function at the neuronal synapse, and one-third are involved in neuronal inhibition. Splicing targets confirmed in Nova-/- mice include c-Jun N-terminal kinase 2, neogenin, and gephyrin; the latter encodes a protein that clusters inhibitory gamma-aminobutyric acid and glycine receptors, two previously identified Nova splicing targets. Thus, CLIP reveals that Nova coordinately regulates a biologically coherent set of RNAs encoding multiple components of the inhibitory synapse, an observation that may relate to the cause of abnormal motor inhibition in POMA.