Maternal retinoids control type 3 innate lymphoid cells and set the offspring immunity.

The impact of nutritional status during fetal life on the overall health of adults has been recognized; however, dietary effects on the developing immune system are largely unknown. Development of secondary lymphoid organs occurs during embryogenesis and is considered to be developmentally programmed. Secondary lymphoid organ formation depends on a subset of type 3 innate lymphoid cells (ILC3) named lymphoid tissue inducer (LTi) cells. Here we show that mouse fetal ILC3s are controlled by cell-autonomous retinoic acid (RA) signalling in utero, which pre-sets the immune fitness in adulthood. We found that embryonic lymphoid organs contain ILC progenitors that differentiate locally into mature LTi cells. Local LTi cell differentiation was controlled by maternal retinoid intake and fetal RA signalling acting in a haematopoietic cell-autonomous manner. RA controlled LTi cell maturation upstream of the transcription factor RORγt. Accordingly, enforced expression of Rorgt restored maturation of LTi cells with impaired RA signalling, whereas RA receptors directly regulated the Rorgt locus. Finally, we established that maternal levels of dietary retinoids control the size of secondary lymphoid organs and the efficiency of immune responses in the adult offspring. Our results reveal a molecular link between maternal nutrients and the formation of immune structures required for resistance to infection in the offspring.

Different properties of skin of different body sites: The root of keloid formation?

The purpose of this study was to examine extracellular matrix composition, vascularization, and immune cell population of skin sites prone to keloid formation. Keloids remain a complex problem, posing esthetical as well as functional difficulties for those affected. These scars tend to develop at anatomic sites of preference. Mechanical properties of skin vary with anatomic location and depend largely on extracellular matrix composition. These differences in extracellular matrix composition, but also vascularization and resident immune cell populations might play a role in the mechanism of keloid formation. To examine this hypothesis, skin samples of several anatomic locations were taken from 24 human donors within zero to 36 hours after they had deceased. Collagen content and cross-links were determined through high-performance liquid chromatography. The expression of several genes, involved in extracellular matrix production and degradation, was measured by means of real-time PCR. (Immuno)histochemistry was performed to detect fibroblasts, collagen, elastin, blood vessels, Langerhans cells, and macrophages. Properties of skin of keloid predilections sites were compared to properties of skin from other locations (nonpredilection sites [NPS]). The results indicated that there are site specific variations in extracellular matrix properties (collagen and cross-links) as well as macrophage numbers. Moreover, predilection sites (PS) for keloid formation contain larger amounts of collagen compared to NPS, but decreased numbers of macrophages, in particular classically activated CD40 positive macrophages. In conclusion, the altered (histological, protein, and genetic) properties of skin of keloid PS may cause a predisposition for and contribute to keloid formation.

Inhibited early immunologic response is associated with hypertrophic scarring.

This study aimed to examine changes in the inflammatory response in early hypertrophic compared to normal wound healing. The immune system is thought to be involved in hypertrophic scar formation. However, the exact mechanism and time of onset of the derailment remain unknown. In a prospective observational study, skin biopsies were taken directly postwounding and 3 hours later from patients who had elective cardiothoracic surgery. The skin biopsies were analysed for mRNA, proteins and cells involved in the early inflammatory phase of wound healing. The endpoint was scar outcome (hypertrophic (HTS) or normal (NTS)) at one year after surgery. There were significant differences between the NTS and HTS groups regarding the fold changes of mRNA expression of P-selectin during surgery. Postoperative skin concentrations of inflammatory proteins IL-6, IL-8 and CCL2 were significantly lower in the HTS compared to the NTS group. Also, a trend of higher pre-operative M2 macrophage numbers was observed in the HTS group. Neutrophil numbers increased equally during surgery in both groups. The increase of P-selectin mRNA in hypertrophic wound healing could affect leucocyte migration. The decreased concentrations of inflammatory proteins in hypertrophic wound healing indicate a reduced inflammatory response, which has consequences for the treatment of hypertrophic scarring during the early inflammatory phase. In a conclusion, alterations of wound healing associated with hypertrophic scarring are visible as early as 3 hours postwounding and include a reduced rather than increased inflammatory protein response.

Involvement of neurons and retinoic acid in lymphatic development: new insights in increased nuchal translucency.

OBJECTIVE: Increased nuchal translucency originates from disturbed lymphatic development. Abnormal neural crest cell (NCC) migration may be involved in lymphatic development. Because both neuronal and lymphatic development share retinoic acid (RA) as a common factor, this study investigated the involvement of NCCs and RA in specific steps in lymphatic endothelial cell (LEC) differentiation and nuchal edema, which is the morphological equivalent of increased nuchal translucency. METHODS: Mouse embryos in which all NCCs were fluorescently labeled (Wnt1-Cre;Rosa26(eYfp) ), reporter embryos for in vivo RA activity (DR5-luciferase) and embryos with absent (Raldh2(-/-) ) or in utero inhibition of RA signaling (BMS493) were investigated. Immunofluorescence using markers for blood vessels, lymphatic endothelium and neurons was applied. Flow cytometry was performed to measure specific LEC populations. RESULTS: Cranial nerves were consistently close to the jugular lymph sac (JLS), in which NCCs were identified. In the absence of RA synthesis, enlarged JLS and nuchal edema were observed. Inhibiting RA signaling in utero resulted in a significantly higher amount of precursor-LECs at the expense of mature LECs and caused nuchal edema. CONCLUSIONS: Neural crest cells are involved in lymphatic development. RA is required for differentiation into mature LECs. Blocking RA signaling in mouse embryos results in abnormal lymphatic development and nuchal edema.

High-throughput analysis of the C4 polymorphism by a combination of MLPA and isotype-specific ELISA's.

Complement factor C4 exists as two main isotypes, C4A and C4B, with different functional properties and encoded by two separate genes. In addition, C4A and C4B genes can occur in multiple copies and may carry a retroviral HERV-K(C4) insertion in intron 9. To study association of C4 polymorphism with disease, accurate genotyping and phenotyping is important. However, current techniques are very laborious and not suitable to study large patient groups. Therefore, we aimed to develop novel assays for C4 geno- and phenotyping, to make high throughput possible. To study C4 gene copy number variation, a novel Multiplex Ligation-dependent Probe Amplification (MLPA) assay was set up with three synthetic probe parts. C4A and C4B protein levels were measured by isotype-specific ELISA's. The relationship between C4 genotype with C4A and C4B serum concentrations was examined in 104 healthy lab workers and 66 children with meningococcal disease. As expected, a strong positive correlation was found between C4A and C4B gene copy number and serum levels of total C4, C4A and C4B. In the healthy controls, 95.3% of C4A genes and 53.7% of C4B genes carried the HERV-K(C4) insertion. Presence of HERV-K(C4) resulted in less C4B protein expression, while there was no effect on total C4 levels. In the meningitis patients, no increased incidence of hetero- or homozygous deficiency of either C4A or C4B was found. In conclusion, the combination of MLPA and ELISA is very suitable to study the geno- and phenotype of complement C4 in large patient groups.