Distribution of segmental duplications in the context of higher order chromatin organisation of human chromosome 7.

BACKGROUND: Segmental duplications (SDs) are not evenly distributed along chromosomes. The reasons for this biased susceptibility to SD insertion are poorly understood. Accumulation of SDs is associated with increased genomic instability, which can lead to structural variants and genomic disorders such as the Williams-Beuren syndrome. Despite these adverse effects, SDs have become fixed in the human genome. Focusing on chromosome 7, which is particularly rich in interstitial SDs, we have investigated the distribution of SDs in the context of evolution and the three dimensional organisation of the chromosome in order to gain insights into the mutual relationship of SDs and chromatin topology. RESULTS: Intrachromosomal SDs preferentially accumulate in those segments of chromosome 7 that are homologous to marmoset chromosome 2. Although this formerly compact segment has been re-distributed to three different sites during primate evolution, we can show by means of public data on long distance chromatin interactions that these three intervals, and consequently the paralogous SDs mapping to them, have retained their spatial proximity in the nucleus. Focusing on SD clusters implicated in the aetiology of the Williams-Beuren syndrome locus we demonstrate by cross-species comparison that these SDs have inserted at the borders of a topological domain and that they flank regions with distinct DNA conformation. CONCLUSIONS: Our study suggests a link of nuclear architecture and the propagation of SDs across chromosome 7, either by promoting regional SD insertion or by contributing to the establishment of higher order chromatin organisation themselves. The latter could compensate for the high risk of structural rearrangements and thus may have contributed to their evolutionary fixation in the human genome.

TLR2-activated human langerhans cells promote Th17 polarization via IL-1beta, TGF-beta and IL-23.

The cytokines IL-6, IL-1beta, TGF-beta, and IL-23 are considered to promote Th17 commitment. Langerhans cells (LC) represent DC in the outer skin layers of the epidermis, an environment extensively exposed to pathogenic attack. The question whether organ-resident DC like LC can evoke Th17 immune response is still open. Our results show that upon stimulation by bacterial agonists, epidermal LC and LC-like cells TLR2-dependently acquire the capacity to polarize Th17 cells. In Th17 cells, expression of retinoid orphan receptor gammabeta was detected. To clarify if IL-17(+)cells could arise per se by stimulated LC we did not repress Th1/Th2 driving pathways by antibodies inhibiting differentiation. In CD1c(+)/langerin(+) monocyte-derived LC-like cells (MoLC), macrophage-activating lipopeptide 2, and peptidoglycan (PGN) induced the release of the cytokines IL-6, IL-1beta, and IL-23. TGF-beta, a cytokine required for LC differentiation and survival, was found to be secreted constitutively. Anti-TLR2 inhibited secretion of IL-6, IL-1beta, and IL-23 by MoLC, while TGF-beta was unaffected. The amount of IL-17 and the ratio of IL-17 to IFN-gamma expression was higher in MoLC- than in monocyte-derived DC-cocultured Th cells. Anti-IL-1beta, -TGF-beta and -IL-23 decreased the induction of Th17 cells. Interestingly, blockage of TLR2 on PGN-stimulated MoLC prevented polarization of Th cells into Th17 cells. Thus, our findings indicate a role of TLR2 in eliciting Th17 immune responses in inflamed skin.

Inhibition of CD1 antigen presentation by human cytomegalovirus.

The betaherpesvirus human cytomegalovirus (HCMV) encodes several molecules that block antigen presentation by the major histocompatibility complex (MHC) proteins. Humans also possess one other family of antigen-presenting molecules, the CD1 family; however, the effect of HCMV on CD1 expression is unknown. The majority of CD1 molecules are classified on the basis of homology as group 1 CD1 and are present almost exclusively on professional antigen-presenting cells such as dendritic cells, which are a major target for HCMV infection and latency. We have determined that HCMV encodes multiple blocking strategies targeting group 1 CD1 molecules. CD1 transcription is strongly inhibited by the HCMV interleukin-10 homologue cmvIL-10. HCMV also blocks CD1 antigen presentation posttranscriptionally by the inhibition of CD1 localization to the cell surface. This function is not performed by a known HCMV MHC class I-blocking molecule and is substantially stronger than the blockage induced by herpes simplex virus type 1. Antigen presentation by CD1 is important for the development of the antiviral immune response and the generation of mature antigen-presenting cells. HCMV present in antigen-presenting cells thus blunts the immune response by the blockage of CD1 molecules.

On the role of co-inhibitory molecules in dendritic cell: T helper cell coculture assays aimed to detect chemical-induced contact allergy.

T cells play a pivotal role in sensitization and elicitation of type IV allergic reactions. While T helper cells sustain and maintain the differentiation of further effector cells, regulatory T cells are involved in control of cytokine release and proliferation, and T killer cells execute cellular lysis, thereby leading to certain levels of tissue damage. According to their central role, the widely applied and OECD-supported test method for the assessment of the sensitization potential of a chemical, i.e., the local lymph node assay (LLNA), relies on the detection of the immune-responsive proliferation of lymphocytes. However, most sensitization assays recently developed take advantage of the initiators of sensitization, dendritic cells (DCs) or DC-like cell lines. Here, we focus on inhibitory molecules expressed on the surface of DCs and their corresponding receptors on T cells. We summarize insight into the function of CTLA-4, the ligands of inducible co-stimulators (ICOSs), and on the inhibitory receptor programmed death (PD). The targeting of immune cell surface receptors by inhibitory molecules holds some promise with regard to the development of T cell-based sensitization assays. Firstly, a broader and more sensitive dynamic range of detection could be achieved by blocking inhibitors or by removing inhibiting regulatory T cells from the assays. Secondly, the actual expression levels of inhibitory molecules could be also a valuable indicator for the process of sensitization. Finally, inhibitory molecules in coculture test systems are supposed to have a major influence on DCs by reverse signaling, thereby affecting their differentiation and maturation status in a feedback loop. In conclusion, inhibitory ligands of DC surface receptors and/or their cognate receptors on T cells could serve as useful tools in cell-based assays, directly influencing toxicological endpoints such as sensitization.

Evaluation of selected biomarkers for the detection of chemical sensitization in human skin: a comparative study applying THP-1, MUTZ-3 and primary dendritic cells in culture.

Dendritic cells (DCs) exhibit the unique capacity to induce T cell differentiation and proliferation, two processes that are crucially involved in allergic reactions. By combining the exclusive potential of DCs as the only professional antigen-presenting cells of the human body with the well known handling advantages of cell lines, cell-based alternative methods aimed at detecting chemical sensitization in vitro commonly apply DC-like cells derived from myeloid cell lines. Here, we present the new biomarkers programmed death-ligand 1 (PD-L1), DC immunoreceptor (DCIR), IL-16, and neutrophil-activating protein-2 (NAP-2), all of which have been detectable in primary human DCs upon exposure to chemical contact allergens. To evaluate the applicability of DC-like cells in the prediction of a chemical's sensitization potential, the expression of cell surface PD-L1 and DCIR was analyzed. In contrast to primary DCs, only minor subpopulations of MUTZ-3 and THP-1 cells presented PD-L1 or DCIR at their surface. After exposure to increasing concentrations of nickel and cinnamic aldehyde, the expression level of PD-L1 and DCIR revealed much stronger affected on monocyte-derived DCs (MoDCs) or Langerhans cells (MoLCs) when compared to THP-1 and MUTZ-3 cells. Applying protein profiler arrays we further identified the soluble factors NAP-2, IL-16, IL-8 and MIP-1α as sensitive biomarkers showing the capacity to discriminate sensitizing from non-sensitizing chemicals or irritants. An allergen-specific release of IL-8 and MIP-1α could be detected in the supernatants of MoDCs and MoLCs and also in MUTZ-3 and THP-1 cells, though at much lower levels. On the protein and transcriptional level, NAP-2 and IL-16 indicated sensitizers most sensitively and specifically in MoDCs. Altogether, we have proven the reciprocal regulated surface molecules PD-L1 and DCIR and the soluble factors MIP-1α, NAP-2 and IL-16 as reliable biomarkers for chemical sensitization. We further show that primary DCs are significantly different in their phenotype and function compared to DC-like cell lines. Since they demonstrated higher absolute values and a broader range in biomarker expression, we propose that MoDCs represent an optimal and robust sensor test system well suited to identify and classify chemicals with an allergic potential.

Human Langerhans cells control Th cells via programmed death-ligand 1 in response to bacterial stimuli and nickel-induced contact allergy.

Langerhans cells (LCs) are suspected to initiate inflammatory immune responses to contact allergens and pathogenic bacteria. In chronic infectious diseases, programmed death ligand (PD-L) 1 exhibits both inhibitory and costimulatory functions on T cell-mediated activation and tolerance. Here, we investigated the effects of contact allergens and bacterial stimuli on PD-L1 expression in LCs and the effects of altered PD-L1 expression on cytokine release of subsequently cocultured T cells. Monocyte-derived LCs (MoLCs), LCs, and skin sections of patients suffering from allergic contact dermatitis were challenged with nickel and then analyzed for PD-L1 expression by confocal laser scanning microscopy and flow cytometry. In blocking experiments, we found that the release of Th cell specific cytokines was dependent on both stimulation of LCs and inhibition of PD-L1-PD-1 interactions. Stimulation with peptidoglycan (PGN) or lipopolysaccharide (LPS) and blockage of PD-L1 with a specific antibody triggered the release of high levels of IL-17, IL-22, TNF-α, and IFN-γ in CD4(+)T cells. If nickel was used as a stimulus, blockage of PD-L1 led to high amounts of TNF-α and IL-22. A closer look revealed PD-L1-dependent upregulation of IL-17 secretion in FACS-sorted CCR6(+)/CCR4(+) T memory cells. In the presence of anti-PD-L1, PGN induced secretion of IFN-γ and IL-17 in total CCR6(+) cells, while nickel triggered secretion of IFN-γ and IL-17 exclusively in CCR6(+)/CCR4(+) cells. Our findings suggest that PD-L1 on LCs plays a crucial role in type IV allergic reactions and in response to bacterial stimuli by controlling the nature of inflammatory Th cell responses.