Gene Expression Analysis of Peripheral Cells for Subclassification of Pediatric Inflammatory Bowel Disease in Remission

Objective

In current clinical practice, optimal treatment of inflammatory bowel disease (IBD) aims at the induction and maintenance of clinical remission. Clinical remission is apparent when laboratory markers of inflammation are normal and clinical symptoms are absent. However, sub-clinical inflammation can still be present. A detailed analysis of the immune status during this inactive state of disease may provide a useful tool to categorize patients with clinical remission into subsets with variable states of immune activation.

Design

By using Affymetrix GeneChips, we analysed RNA gene expression profiles of peripheral blood leukocytes from pediatric IBD patients in clinical remission and controls. We performed (un)supervised clustering analysis of IBD-associated genes and applied Ingenuity® pathway software to identify specific molecular profiles between patients.

Results

Pediatric IBD patients with disease in clinical remission display heterogeneously distributed gene expression profiles that are significantly distinct from controls. We identified three clusters of IBD patients, each displaying specific expression profiles of IBD-associated genes.

Conclusion

The expression of immune- and IBD-associated genes in peripheral blood leukocytes from pediatric IBD patients in clinical remission was different from healthy controls, indicating that sub-clinical immune mechanisms are still active during remission. As such, RNA profiling of peripheral blood may allow for non-invasive patient subclassification and new perspectives in treatment regimes of IBD patients in the future.     

Epigenomic association analysis identifies smoking-related DNA methylation sites in African Americans

Cigarette smoking is an environmental risk factor for many chronic diseases, and disease risk can often be managed by smoking control. Smoking can induce cellular and molecular changes, including epigenetic modification, but the short- and long-term epigenetic modifications caused by cigarette smoking at the gene level have not been well understood. Recent studies have identified smoking-related DNA methylation (DNAm) sites in Caucasians. To determine whether the same DNAm sites associate with smoking in African Americans, and to identify novel smoking-related DNAm sites, we conducted a methylome-wide association study of cigarette smoking using a discovery sample of 972 African Americans, and a replication sample of 239 African Americans with two array-based methods. Among 15 DNAm sites significantly associated with smoking after correction for multiple testing in our discovery sample, 5 DNAm sites are replicated in an independent cohort, and 14 sites in the replication sample have effects in the same direction as in the discovery sample. The top two smoking-related DNAm sites in F2RL3 (factor II receptor-like 3) and GPR15 (G-protein-coupled receptor 15) observed in African Americans are consistent with previous findings in Caucasians. The associations between the replicated DNAm sites and smoking remain significant after adjusting for genetic background. Despite the distinct genetic background between African Americans and Caucasians, the DNAm from the two ethnic groups shares common associations with cigarette smoking, which suggests a common molecular mechanism of epigenetic modification influenced by environmental exposure.     

Comparison of the DNA methylation profiles of human peripheral blood cells and transformed B-lymphocytes

Epidemiological studies of DNA methylation (DNAm) profiles may hold substantial promise for identifying mechanisms through which genetic and environmental factors jointly contribute to disease risk. Different cell types are likely to have different DNAm patterns. We investigate the DNAm differences between two types of biospecimens available in many genetic epidemiology studies. We compared DNAm patterns in two different DNA samples from each of 34 participants in the Genetic Epidemiology Network of Arteriopathy study (20 Caucasians and 14 African-Americans). One was extracted from peripheral blood cells (PBC) and the other from transformed B-lymphocytes (TBL). The genome-wide DNAm profiles were compared at over 27,000 genome-wide methylation sites. We found that 26 out of the 34 participants had correlation coefficients higher than 0.9 between methylation profiles of PBC and TBL. Although a high correlation was observed in the DNAm profile between PBC and TBL, we also observed variation across samples from different DNA resources and donors. Using principal component analysis of the DNAm profiles, the two sources of the DNA samples could be accurately predicted. We also identified 3,723 autosomal DNAm sites that had significantly different methylation statuses in PBC compared to TBL (Bonferroni corrected p value <0.05). Both PBC and TBL provide a rich resource for understanding the DNAm profiles in humans participating in epidemiologic studies. While the majority of DNAm findings in PBC and TBL may be consistent, caution must be used when interpreting results because of the possibility of cell type-specific methylation modification.     

C-reactive protein (CRP) gene polymorphisms: implication in CRP plasma levels and susceptibility to acute myocardial infarction

C-reactive protein (CRP) is one of the many molecular factors involved in pathogenesis of coronary artery disease which its plasma levels are associated with increased risk of cardiovascular events. The present study designed to determine whether polymorphisms in the CRP gene are associated with plasma CRP levels and susceptibility to acute myocardial infarction (AMI). Plasma CRP levels were measured in patients with AMI and control subjects and genomic DNA and peripheral blood mononuclear cells (PBMCs) were extracted. The −717A/G and 1059G/C CRP polymorphisms were detected. The mRNA expression of CRP gene and plasma levels of CRP and interleukin-6 (IL-6) were also analyzed. The −717A/G variation was significantly associated with higher CRP levels, but 1059G/C variation was associated with lower CRP levels. The AA genotype frequency of −717A/G variation was significantly more frequent in the patients than control subjects. By contrast, the genotype and allele distribution in 1059G/C of patient were not statistically different between patients and controls. There were significant differences in circulating levels of CRP and IL-6 in the patients than in controls. The mRNA expression levels of CRP were significantly higher in the patient plasma compared with controls. Our results indicate relationship between many polymorphisms in CRP gene and risk of AMI which suggest that genetic variations in CRP might be helpful for determining susceptibility to AMI in Iranian patients. In addition, CRP gene polymorphisms are associated with plasma CRP levels and susceptibility to AMI might be related to CRP gene expression which affects its plasma levels.     

Autosomal sex-associated co-methylated regions predict biological sex from DNA methylation

Sex is a modulator of health that has been historically overlooked in biomedical research. Recognizing this knowledge gap, funding agencies now mandate the inclusion of sex as a biological variable with the goal of stimulating efforts to illuminate the molecular underpinnings of sex biases in health and disease. DNA methylation (DNAm) is a strong molecular candidate for mediating such sex biases; however, a robust and well characterized annotation of sex differences in DNAm is yet to emerge. Beginning with a large (n = 3795) dataset of DNAm profiles from normative adult whole blood samples, we identified, validated and characterized autosomal sex-associated co-methylated genomic regions (sCMRs). Strikingly, sCMRs showed consistent sex differences in DNAm over the life course and a subset were also consistent across cell, tissue and cancer types. sCMRs included sites with known sex differences in DNAm and links to health conditions with sex biased effects. The robustness of sCMRs enabled the generation of an autosomal DNAm-based predictor of sex with 96% accuracy. Testing this tool on blood DNAm profiles from individuals with sex chromosome aneuploidies (Klinefelter [47,XXY], Turner [45,X] and 47,XXX syndrome) revealed an intimate relationship between sex chromosomes and sex-biased autosomal DNAm.     

Non-linear patterns in age-related DNA methylation may reflect CD4+ T cell differentiation

DNA methylation (DNAm) is an important epigenetic process involved in the regulation of gene expression. While many studies have identified thousands of loci associated with age, few have differentiated between linear and non-linear DNAm trends with age. Non-linear trends could indicate early- or late-life gene regulatory processes. Using data from the Illumina 450K array on 336 human peripheral blood samples, we identified 21 CpG sites that associated with age (P<1.03E-7) and exhibited changing rates of DNAm change with age (P<1.94E-6). For 2 of these CpG sites (cg07955995 and cg22285878), DNAm increased with age at an increasing rate, indicating that differential DNAm was greatest among elderly individuals. We observed significant replication for both CpG sites (P<5.0E-8) in a second set of peripheral blood samples. In 8 of 9 additional data sets comprising samples of monocytes, T cell subtypes, and brain tissue, we observed a pattern directionally consistent with DNAm increasing with age at an increasing rate, which was nominally significant in the 3 largest data sets (4.3E-15<P<0.039). cg07955995 and cg22285878 reside in the promoter region of KLF14, which encodes a protein involved in immune cell differentiation via the repression of FOXP3. These findings may suggest a possible role for cg07955995 and cg22285878 in immunosenescence.     

Melanocortin receptor expression is associated with reduced CRP in response to resistance training

The existing paradigm of exercise-induced decreases in chronic inflammation focuses on the expression of inflammatory receptors on systemic monocytes in response to exercise training, with the role of anti-inflammatory receptors largely ignored. Our recent preliminary studies indicate that the anti-inflammatory melanocortin receptors (MCRs) may play a role in modulating exercise-induced decreases in chronic inflammation. Here, we present a study designed to determine the effect of intense, resistance exercise training on systemic monocyte MCR expression. Because low-grade chronic inflammation is associated with elevated cardiometabolic risk in healthy populations and exercise decreases chronic inflammation, we investigated the associations between systemic monocyte cell surface expression of MCRs and inflammatory markers as a possible mechanism for the beneficial anti-inflammatory effects of resistance training. To this end, the present study includes 40 adults (aged 19-27 yr) and implements a 12-wk periodized, intensive resistance training intervention. Melanocortin 1 and 3 receptor expression on systemic monocytes and inflammatory markers, including C-reactive protein (CRP), interleukin (IL)-6, IL-1β, and IL-10, were measured before and after the intervention. Resistance training significantly altered MCR systemic monocyte cell surface expression, had no chronic effects on IL-6, IL-1β, or IL-10 expression, but significantly decreased CRP levels from a moderate to a low cardiovascular disease risk category. More specifically, decreased melanocortin 3 receptor expression significantly correlated with decreased CRP, independent of changes in adiposity. These data suggest that the observed responses in MCR expression and decreases in cardiovascular disease risk in response to resistance training represent an important anti-inflammatory mechanism in regulating exercise-induced decreases in chronic inflammation that occur independent of chronic changes in systemic cytokines.     

Association of cigarette smoking and CRP levels with DNA methylation in α-1 antitrypsin deficiency

Alpha-1 antitrypsin (AAT) deficiency and tobacco smoking are confirmed risk factors for Chronic Obstructive Pulmonary Disease. We hypothesized that variable DNA methylation would be associated with smoking and inflammation, as reflected by the level of C-Reactive Protein (CRP) in AAT-deficient subjects. Methylation levels of 1,411 autosomal CpG sites from the Illumina GoldenGate Methylation Cancer Panel I were analyzed in 316 subjects. Associations of five smoking behaviors and CRP levels with individual CpG sites and average methylation levels were assessed using non-parametric testing, linear regression and linear mixed effect models, with and without adjustment for age and gender. Univariate linear regression analysis revealed that methylation levels of 16 CpG sites significantly associated with ever-smoking status. A CpG site in the TGFBI gene was the only site associated with ever-smoking after adjustment for age and gender. No highly significant associations existed between age at smoking initiation, pack-years smoked, duration of smoking, and time since quitting smoking as predictors of individual CpG site methylation levels. However, ever-smoking and younger age at smoking initiation associated with lower methylation level averaged across all sites. DNA methylation at CpG sites in the RUNX3, JAK3 and KRT1 genes associated with CRP levels. The most significantly associated CpG sites with gender and age mapped to the CASP6 and FZD9 genes, respectively. In summary, this study identified multiple potential candidate CpG sites associated with ever-smoking and CRP level in AAT-deficient subjects. Phenotypic variability in Mendelian diseases may be due to epigenetic factors.     

Association of cigarette smoking and CRP levels with DNA methylation in α-1 antitrypsin deficiency

Alpha-1 antitrypsin (AAT) deficiency and tobacco smoking are confirmed risk factors for Chronic Obstructive Pulmonary Disease. We hypothesized that variable DNA methylation would be associated with smoking and inflammation, as reflected by the level of C-Reactive Protein (CRP) in AAT-deficient subjects. Methylation levels of 1,411 autosomal CpG sites from the Illumina GoldenGate Methylation Cancer Panel I were analyzed in 316 subjects. Associations of five smoking behaviors and CRP levels with individual CpG sites and average methylation levels were assessed using non-parametric testing, linear regression and linear mixed effect models, with and without adjustment for age and gender. Univariate linear regression analysis revealed that methylation levels of 16 CpG sites significantly associated with ever-smoking status. A CpG site in the TGFBI gene was the only site associated with ever-smoking after adjustment for age and gender. No highly significant associations existed between age at smoking initiation, pack-years smoked, duration of smoking, and time since quitting smoking as predictors of individual CpG site methylation levels. However, ever-smoking and younger age at smoking initiation associated with lower methylation level averaged across all sites. DNA methylation at CpG sites in the RUNX3, JAK3 and KRT1 genes associated with CRP levels. The most significantly associated CpG sites with gender and age mapped to the CASP6 and FZD9 genes, respectively. In summary, this study identified multiple potential candidate CpG sites associated with ever-smoking and CRP level in AAT-deficient subjects. Phenotypic variability in Mendelian diseases may be due to epigenetic factors.     

Epigenetic Signatures at AQP3 and SOCS3 Engage in Low-Grade Inflammation across Different Tissues

BackgroundElevated levels of C-reactive protein (CRP, determined by a high-sensitivity assay) indicate low-grade inflammation which is implicated in many age-related disorders. Epigenetic studies on CRP might discover molecular mechanisms underlying CRP regulation. We aimed to identify DNA methylation sites related to CRP concentrations in cells and tissues regulating low-grade inflammation.ResultsGenome-wide DNA methylation was measured in peripheral blood in 1,741 participants of the KORA F4 study using Illumina HumanMethylation450 BeadChip arrays. Four CpG sites (located at BCL3, AQP3, SOCS3, and cg19821297 intergenic at chromosome 19p13.2, P ≤ 1.01E-07) were significantly hypomethylated at high CRP concentrations independent of various confounders including age, sex, BMI, smoking, and white blood cell composition. Findings were not sex-specific. CRP-related top genes were enriched in JAK/STAT pathways (Benjamini-Hochberg corrected P < 0.05). Results were followed-up in three studies using DNA from peripheral blood (EPICOR, n = 503) and adipose tissue (TwinsUK, n = 368) measured as described above and from liver tissue (LMU liver cohort, n = 286) measured by MALDI-TOF mass spectrometry using EpiTYPER. CpG sites at the AQP3 locus (significant p-values in peripheral blood = 1.72E-03 and liver tissue = 1.51E-03) and the SOCS3 locus (p-values in liver < 2.82E-05) were associated with CRP in the validation panels.ConclusionsEpigenetic modifications seem to engage in low-grade inflammation, possibly via JAK/STAT mediated pathways. Results suggest a shared relevance across different tissues at the AQP3 locus and highlight a role of DNA methylation for CRP regulation at the SOCS3 locus.     

DNA methylation and chromatin accessibility predict age in the domestic dog

Across mammals, the epigenome is highly predictive of chronological age. These “epigenetic clocks,” most of which have been built using DNA methylation (DNAm) profiles, have gained traction as biomarkers of aging and organismal health. While the ability of DNAm to predict chronological age has been repeatedly demonstrated, the ability of other epigenetic features to predict age remains unclear. Here, we use two types of epigenetic information—DNAm, and chromatin accessibility as measured by ATAC-seq—to develop age predictors in peripheral blood mononuclear cells sampled from a population of domesticated dogs. We measured DNAm and ATAC-seq profiles for 71 dogs, building separate predictive clocks from each, as well as the combined dataset. We also use fluorescence-assisted cell sorting to quantify major lymphoid populations for each sample. We found that chromatin accessibility can accurately predict chronological age (R²_ATAC = 26%), though less accurately than the DNAm clock (R²_DNAm = 33%), and the clock built from the combined datasets was comparable to both (R²_combined = 29%). We also observed various populations of CD62L+ T cells significantly correlated with dog age. Finally, we found that all three clocks selected features that were in or near at least two protein-coding genes: BAIAP2 and SCARF2, both previously implicated in processes related to cognitive or neurological impairment. Taken together, these results highlight the potential of chromatin accessibility as a complementary epigenetic resource for modeling and investigating biologic age.     

C反应蛋白与动脉粥样硬化

C反应蛋白（C-reactiveprotein,CRP）是人类非特异性急性期蛋白,是判断组织损伤和炎症反应的敏感指标之一。CRP的表达水平与动脉粥样硬化（atherosclerosis,AS）和心血管疾病的发生具有冠著的相关性。但是关于CRP是否是AS的独立危险因素并参与AS的发病机制,目前尚存在很大争议。新近的研究发现,CRP与某些特定的配体结合后,五聚体结构CRP可分离形成单体结构CRP。这一发现为研究CRP蛋白与AS的相互关系提供了新的线索,对CRP及其单体结构的深入研究,将有可能帮助人们找到治疗心血管疾病的有效方法。就炎性反应标志物CRP及其单体（monomeric CRP,mCRP）与动脉粥样硬化的相关研究进展进行综述,以探讨分析CRP在AS中的作用。     

C-reactive Protein

C-reactive protein (CRP) is a phylogenetically highly conserved plasma protein, with homologs in vertebrates and many invertebrates, that participates in the systemic response to inflammation. Its plasma concentration increases during inflammatory states, a characteristic that has long been employed for clinical purposes. CRP is a pattern recognition molecule, binding to specific molecular configurations that are typically exposed during cell death or found on the surfaces of pathogens. Its rapid increase in synthesis within hours after tissue injury or infection suggests that it contributes to host defense and that it is part of the innate immune response. Recently, an association between minor CRP elevation and future major cardiovascular events has been recognized, leading to the recommendation by the Centers for Disease Control and the American Heart Association that patients at intermediate risk of coronary heart disease might benefit from measurement of CRP. This review will largely focus on our current understanding of the structure of CRP, its ligands, the effector molecules with which it interacts, and its apparent functions.     

Galectins in parasite infection and allergic inflammation

Galectins are increasingly recognised as important immunological mediators of homeostasis and disease regulation. This paper gives an overview of current knowledge of galectin involvement in parasite infection and allergic inflammation, two very different but immunologically linked phenomena. Galectins are produced by both the parasite and the host and appear to be intimately involved in parasite establishment, as well as directing the course of infection and the immune response. Host galectins have also been shown to be active participants in the recruitment of cells to sites of inflammation and modulating the effector function of mast cells, neutrophils and eosinophils. Moreover, the ability of galectins to induce differential expression of cytokine genes in leukocytes suggests that they are able to direct the nature of an adaptive immune response, in particular towards a T2-type allergic response.     

DNA methylation provides insight into intergenerational risk for preterm birth in African Americans

African Americans are at increased risk for spontaneous preterm birth (PTB). Though PTB is heritable, genetic studies have not identified variants that account for its intergenerational risk, prompting the hypothesis that epigenetic factors may also contribute. The objective of this study was to evaluate DNA methylation from maternal leukocytes to identify patterns specific to PTB and its intergenerational risk. DNA from peripheral leukocytes from African American women that delivered preterm (24–34 weeks; N = 16) or at term (39–41 weeks; N = 24) was assessed for DNA methylation using the HumanMethylation450 BeadChip. In maternal samples, 17,829 CpG sites associated with PTB, but no CpG site remained associated after correction for multiple comparisons. Examination of paired maternal-fetal samples identified 5,171 CpG sites in which methylation of maternal samples correlated with methylation of her respective fetus (FDR < 0.05). These correlated sites were enriched for association with PTB in maternal leukocytes. The majority of correlated CpG sites could be attributed to one or more genetic variants. They were also significantly more likely to be in genes involved in metabolic, cardiovascular, and immune pathways, suggesting a role for genetic and environmental contributions to PTB risk and chronic disease. The results of this study may provide insight into the factors underlying intergenerational risk for PTB and its consequences.