Most epigenome-wide association studies (EWAS) quantify DNA methylation (DNAm) in peripheral tissues such as whole blood to identify positions in the genome where variation is statistically associated with a trait or exposure. As whole blood comprises a mix of cell types, it is unclear whether trait-associated DNAm variation is specific to an individual cellular population. We collected three peripheral tissues (whole blood, buccal epithelial and nasal epithelial cells) from thirty individuals. Whole blood samples were subsequently processed using fluorescence-activated cell sorting (FACS) to purify five constituent cell-types (monocytes, granulocytes, CD4+ T cells, CD8+ T cells, and B cells). DNAm was profiled in all eight sample-types from each individual using the Illumina EPIC array. We identified significant differences in both the level and variability of DNAm between different sample types, and DNAm data-derived estimates of age and smoking were found to differ dramatically across sample types from the same individual. We found that for the majority of loci variation in DNAm in individual blood cell types was only weakly predictive of variance in DNAm measured in whole blood, although the proportion of variance explained was greater than that explained by either buccal or nasal epithelial samples. Covariation across sample types was much higher for DNAm sites influenced by genetic factors. Overall, we observe that DNAm variation in whole blood is additively influenced by a combination of the major blood cell types. For a subset of sites, however, variable DNAm detected in whole blood can be attributed to variation in a single blood cell type providing potential mechanistic insight about EWAS findings. Our results suggest that associations between whole blood DNAm and traits or exposures reflect differences in multiple cell types and our data will facilitate the interpretation of findings in epigenetic epidemiology.

Dendritic cell (DC) derived cytokines play a key role in specifying adaptive immune responses tailored to the type of pathogen encountered and the local tissue environment. However, little is known about how DC perceive the local environment. We investigated whether endogenous Notch signaling could affect DC responses to pathogenic stimuli. We demonstrate that concurrent Notch and TLR stimulation results in a unique cytokine profile in mouse bone-marrow derived DC characterized by enhanced IL-10 and IL-2 and reduced IL-12 expression compared to TLR ligation alone. Unexpectedly, modulation of cytokine production occurred through a non-canonical Notch signaling pathway, independent of γ-secretase activity. Modulation required de novo protein synthesis and PI3K, JNK and ERK activity were necessary for enhanced IL-2 expression while modulation of IL-10 only required PI3K activity. Further, we show that this γ-secretase independent Notch pathway can induce PI3K activity. In contrast, expression of the canonical Notch target gene Hes1 was suppressed in DC stimulated with Notch and TLR ligands simultaneously. Thus, our data suggest that Notch acts as an endogenous signal that modulates cytokine expression of DC through a non-canonical pathway and therefore has the potential to tailor the subsequent adaptive immune response in a tissue and/or stage dependent manner.

Campylobacter jejuni is the leading cause of human bacterial gastroenteritis worldwide, mainly caused by handling and consumption of contaminated poultry. However, the immune response to infection is poorly understood. Here, the impact of the C. jejuni capsule, flagella and the N-linked glycosylation system on cytokine production by dendritic cells was investigated. Bone marrow-derived murine dendritic cells (BMDCs) infected with C. jejuni lacking the N-linked glycosylation system produced similar amounts of cytokines compared to cells infected with C. jejuni 11168H wild-type (WT) cultures. C. jejuni flagellin FlaA mutants elicited reduced IL-6 and IL-10 production in BMDCs compared to C. jejuni WT and this reduction was more pronounced in TLR4(-/-) BMDCs. An acapsular C. jejuni mutant as well as a mutant lacking the O-methyl phosphoramidate modification of the capsule elicited a higher cytokine response in BMDCs. Experiments with TLR4(-/-) BMDCs revealed that this increased cytokine production was not solely dependent on signalling through TLR4. Therefore, the C. jejuni capsule is important to prevent excessive cytokine production by BMDCs and even minor changes in capsule composition such as the lack of the O-methyl phosphoramidate modification can lead to increased cytokine production.

Recently it has been shown that dendritic cells (DC) express both Notch and Notch ligands, allowing for the possibility that Notch signaling may influence their maturation. We show that although both Jagged (Jgd) and Delta-like (DlL) ligands were able to activate the canonical Notch pathway in mouse DC, only Jgd1 could induce the production of certain cytokines. Maturation of DC via Jgd1 resulted in an entirely different maturation program from that induced through TLR (via LPS) signaling, promoting the production of high levels of IL-2 and IL-10. DC matured by Jgd1 (Jgd1-conditioned DC) promoted the survival and proliferation of CD4+CD25+ regulatory T cells that were able to suppress efficiently the proliferation of CD25− cells. Further, CD25+ cells cultured with Jgd1-conditioned DC produced very high levels of IL-17 in an IL-2-dependent fashion. Our data suggest a new and important role for the Notch pathway in the regulation of the DC phenotype.

Chaos and oscillations continue to capture the interest of both the scientific and public domains. Yet despite the importance of these qualitative features, most attempts at constructing mathematical models of such phenomena have taken an indirect, quantitative approach, for example, by fitting models to a finite number of data points. Here we develop a qualitative inference framework that allows us to both reverse-engineer and design systems exhibiting these and other dynamical behaviours by directly specifying the desired characteristics of the underlying dynamical attractor. This change in perspective from quantitative to qualitative dynamics, provides fundamental and new insights into the properties of dynamical systems.