Zfp362 potentiates murine colonic inflammation by constraining Treg cell function rather than promoting Th17 cell differentiation.

Mucosal barrier integrity and pathogen clearance is a complex process influenced by both Th17 and Treg cells. Previously, we had described the DNA methylation profile of Th17 cells and identified Zinc finger protein (Zfp)362 to be uniquely demethylated. Here, we generated Zfp362-/- mice to unravel the role of Zfp362 for Th17 cell biology. Zfp362-/- mice appeared clinically normal, showed no phenotypic alterations in the T-cell compartment, and upon colonization with segmented filamentous bacteria, no effect of Zfp362 deficiency on Th17 cell differentiation was observed. By contrast, Zfp362 deletion resulted in increased frequencies of colonic Foxp3+ Treg cells and IL-10+ and RORγt+ Treg cell subsets in mesenteric lymph nodes. Adoptive transfer of naïve CD4+ T cells from Zfp362-/- mice into Rag2-/- mice resulted in a significantly lower weight loss when compared with controls receiving cells from Zfp362+/+ littermates. However, this attenuated weight loss did not correlate with alterations of Th17 cells but instead was associated with an increase of effector Treg cells in mesenteric lymph nodes. Together, these results suggest that Zfp362 plays an important role in promoting colonic inflammation; however, this function is derived from constraining the effector function of Treg cells rather than directly promoting Th17 cell differentiation.

The thymic microenvironment gradually modulates the phenotype of thymus-homing peripheral conventional dendritic cells.

BACKGROUND & AIMS: Thymic conventional dendritic cells (t-DCs) are crucial for the development of T cells. A substantial fraction of t-DCs originates extrathymically and migrates to the thymus. Here, these cells contribute to key processes of central tolerance like the clonal deletion of self-reactive thymocytes and the generation of regulatory T (Treg) cells. So far, it is only incompletely understood which impact the thymic microenvironment has on thymus-homing conventional DCs (cDCs), which phenotypic changes occur after the entry of peripheral cDCs into the thymus and which functional properties these modulated cells acquire. MATERIALS & METHODS: In the present study, we mimicked the thymus-homing of peripheral cDCs by introducing ex vivo isolated splenic cDCs (sp-DCs) into reaggregated thymic organ cultures (RTOCs). RESULTS: Already after two days of culture, the transcriptomic profile of sp-DCs was modulated and had acquired certain key signatures of t-DCs. The regulated genes included immunomodulatory cytokines and chemokines as well as costimulatory molecules. After four days of culture, sp-DCs appeared to have at least partially acquired the peculiar Treg cell-inducing capacity characteristic of t-DCs. DISCUSSION & CONCLUSION: Taken together, our findings indicate that peripheral cDCs possess a high degree of plasticity enabling them to quickly adapt to the thymus-specific microenvironment. We further provide indirect evidence that thymus-specific properties such as the efficient induction of Treg cells under homeostatic conditions can be partially transferred to thymus-homing peripheral cDC subsets.

Profiling of epigenetic marker regions in murine ILCs under homeostatic and inflammatory conditions.

Epigenetic modifications such as DNA methylation play an essential role in imprinting specific transcriptional patterns in cells. We performed genome-wide DNA methylation profiling of murine lymph node-derived ILCs, which led to the identification of differentially methylated regions (DMRs) and the definition of epigenetic marker regions in ILCs. Marker regions were located in genes with a described function for ILCs, such as Tbx21, Gata3, or Il23r, but also in genes that have not been related to ILC biology. Methylation levels of the marker regions and expression of the associated genes were strongly correlated, indicating their functional relevance. Comparison with T helper cell methylomes revealed clear lineage differences, despite partial similarities in the methylation of specific ILC marker regions. IL-33-mediated challenge affected methylation of ILC2 epigenetic marker regions in the liver, while remaining relatively stable in the lung. In our study, we identified a set of epigenetic markers that can serve as a tool to study phenotypic and functional properties of ILCs.

Recirculating IL-1R2+ Tregs fine-tune intrathymic Treg development under inflammatory conditions.

The vast majority of Foxp3+ regulatory T cells (Tregs) are generated in the thymus, and several factors, such as cytokines and unique thymic antigen-presenting cells, are known to contribute to the development of these thymus-derived Tregs (tTregs). Here, we report the existence of a specific subset of Foxp3+ Tregs within the thymus that is characterized by the expression of IL-1R2, which is a decoy receptor for the inflammatory cytokine IL-1. Detailed flow cytometric analysis of the thymocytes from Foxp3hCD2xRAG1GFP reporter mice revealed that the IL-1R2+ Tregs are mainly RAG1GFP- and CCR6+CCR7-, demonstrating that these Tregs are recirculating cells entering the thymus from the periphery and that they have an activated phenotype. In the spleen, the majority of IL-1R2+ Tregs express neuropilin-1 (Nrp-1) and Helios, suggesting a thymic origin for these Tregs. Interestingly, among all tissues studied, the highest frequency of IL-1R2+ Tregs was observed in the thymus, indicating preferential recruitment of this Treg subset by the thymus. Using fetal thymic organ cultures (FTOCs), we demonstrated that increased concentrations of exogenous IL-1ß blocked intrathymic Treg development, resulting in a decreased frequency of CD25+Foxp3+ tTregs and an accumulation of CD25+Foxp3- Treg precursors. Interestingly, the addition of IL-1R2+ Tregs, but not IL-1R2- Tregs, to reaggregated thymic organ cultures (RTOCs) abrogated the IL-1ß-mediated blockade, demonstrating that these recirculating IL-1R2+ Tregs can quench IL-1 signaling in the thymus and thereby maintain thymic Treg development even under inflammatory conditions.

Dynamic Imprinting of the Treg Cell-Specific Epigenetic Signature in Developing Thymic Regulatory T Cells.

Regulatory T (Treg) cells mainly develop within the thymus and arise from CD25+Foxp3- (CD25+ TregP) or CD25-Foxp3+ (Foxp3+ TregP) Treg cell precursors resulting in Treg cells harboring distinct transcriptomic profiles and complementary T cell receptor repertoires. The stable and long-term expression of Foxp3 in Treg cells and their stable suppressive phenotype are controlled by the demethylation of Treg cell-specific epigenetic signature genes including an evolutionarily conserved CpG-rich element within the Foxp3 locus, the Treg-specific demethylated region (TSDR). Here we analyzed the dynamics of the imprinting of the Treg cell-specific epigenetic signature genes in thymic Treg cells. We could demonstrate that CD25+Foxp3+ Treg cells show a progressive demethylation of most signature genes during maturation within the thymus. Interestingly, a partial demethylation of several Treg cell-specific epigenetic signature genes was already observed in Foxp3+ TregP but not in CD25+ TregP. Furthermore, Foxp3+ TregP were very transient in nature and arose at a more mature developmental stage when compared to CD25+ TregP. When the two Treg cell precursors were cultured in presence of IL-2, a factor known to be critical for thymic Treg cell development, we observed a major impact of IL-2 on the demethylation of the TSDR with a more pronounced effect on Foxp3+ TregP. Together, these results suggest that the establishment of the Treg cell-specific hypomethylation pattern is a continuous process throughout thymic Treg cell development and that the two known Treg cell precursors display distinct dynamics for the imprinting of the Treg cell-specific epigenetic signature genes.