Acetyl-CoA-Carboxylase 1-mediated de novo fatty acid synthesis sustains Lgr5+ intestinal stem cell function.

Basic processes of the fatty acid metabolism have an important impact on the function of intestinal epithelial cells (IEC). However, while the role of cellular fatty acid oxidation is well appreciated, it is not clear how de novo fatty acid synthesis (FAS) influences the biology of IECs. We report here that interfering with de novo FAS by deletion of the enzyme Acetyl-CoA-Carboxylase (ACC)1 in IECs results in the loss of epithelial crypt structures and a specific decline in Lgr5+ intestinal epithelial stem cells (ISC). Mechanistically, ACC1-mediated de novo FAS supports the formation of intestinal organoids and the differentiation of complex crypt structures by sustaining the nuclear accumulation of PPARδ/ß-catenin in ISCs. The dependency of ISCs on cellular de novo FAS is tuned by the availability of environmental lipids, as an excess delivery of external fatty acids is sufficient to rescue the defect in crypt formation. Finally, inhibition of ACC1 reduces the formation of tumors in colitis-associated colon cancer, together highlighting the importance of cellular lipogenesis for sustaining ISC function and providing a potential perspective to colon cancer therapy.

K2P18.1 translates T cell receptor signals into thymic regulatory T cell development.

It remains largely unclear how thymocytes translate relative differences in T cell receptor (TCR) signal strength into distinct developmental programs that drive the cell fate decisions towards conventional (Tconv) or regulatory T cells (Treg). Following TCR activation, intracellular calcium (Ca2+) is the most important second messenger, for which the potassium channel K2P18.1 is a relevant regulator. Here, we identify K2P18.1 as a central translator of the TCR signal into the thymus-derived Treg (tTreg) selection process. TCR signal was coupled to NF-κB-mediated K2P18.1 upregulation in tTreg progenitors. K2P18.1 provided the driving force for sustained Ca2+ influx that facilitated NF-κB- and NFAT-dependent expression of FoxP3, the master transcription factor for Treg development and function. Loss of K2P18.1 ion-current function induced a mild lymphoproliferative phenotype in mice, with reduced Treg numbers that led to aggravated experimental autoimmune encephalomyelitis, while a gain-of-function mutation in K2P18.1 resulted in increased Treg numbers in mice. Our findings in human thymus, recent thymic emigrants and multiple sclerosis patients with a dominant-negative missense K2P18.1 variant that is associated with poor clinical outcomes indicate that K2P18.1 also plays a role in human Treg development. Pharmacological modulation of K2P18.1 specifically modulated Treg numbers in vitro and in vivo. Finally, we identified nitroxoline as a K2P18.1 activator that led to rapid and reversible Treg increase in patients with urinary tract infections. Conclusively, our findings reveal how K2P18.1 translates TCR signals into thymic T cell fate decisions and Treg development, and provide a basis for the therapeutic utilization of Treg in several human disorders.

Protection against autoimmunity is driven by thymic epithelial cell-mediated regulation of Treg development.

Medullary thymic epithelial cells (mTECs) are key antigen-presenting cells mediating T cell tolerance to prevent harmful autoimmunity. mTECs both negatively select self-reactive T cells and promote the development of thymic regulatory T cells (tTregs) that mediate peripheral tolerance. The relative importance of these two mechanisms of thymic education to prevent autoimmunity is unclear. We generated a mouse model to specifically target the development and function of mTECs by conditional ablation of the NF-κB­inducing kinase (NIK) in the TEC compartment. In contrast to germline-deficient NIK−/− mice, Foxn1CreNIKfl/fl mice rapidly developed fatal T cell­dependent multiorgan autoimmunity shortly after birth. Thymic transplantation and adoptive transfer experiments demonstrated that autoimmunity arises specifically from the emergence of dysfunctional tTregs. Thus, Treg function, rather than negative selection, enforces the protection of peripheral tissues from autoimmune attack.

The Transcription Factor MAZR/PATZ1 Regulates the Development of FOXP3+ Regulatory T Cells.

Forkhead box protein P3+ (FOXP3+) regulatory T cells (Treg cells) play a key role in maintaining tolerance and immune homeostasis. Here, we report that a T cell-specific deletion of the transcription factor MAZR (also known as PATZ1) leads to an increased frequency of Treg cells, while enforced MAZR expression impairs Treg cell differentiation. Further, MAZR expression levels are progressively downregulated during thymic Treg cell development and during in-vitro-induced human Treg cell differentiation, suggesting that MAZR protein levels are critical for controlling Treg cell development. However, MAZR-deficient Treg cells show only minor transcriptional changes ex vivo, indicating that MAZR is not essential for establishing the transcriptional program of peripheral Treg cells. Finally, the loss of MAZR reduces the clinical score in dextran-sodium sulfate (DSS)-induced colitis, suggesting that MAZR activity in T cells controls the extent of intestinal inflammation. Together, these data indicate that MAZR is part of a Treg cell-intrinsic transcriptional network that modulates Treg cell development.

Blimp1 Prevents Methylation of Foxp3 and Loss of Regulatory T Cell Identity at Sites of Inflammation.

Foxp3+ regulatory T (Treg) cells restrict immune pathology in inflamed tissues; however, an inflammatory environment presents a threat to Treg cell identity and function. Here, we establish a transcriptional signature of central nervous system (CNS) Treg cells that accumulate during experimental autoimmune encephalitis (EAE) and identify a pathway that maintains Treg cell function and identity during severe inflammation. This pathway is dependent on the transcriptional regulator Blimp1, which prevents downregulation of Foxp3 expression and "toxic" gain-of-function of Treg cells in the inflamed CNS. Blimp1 negatively regulates IL-6- and STAT3-dependent Dnmt3a expression and function restraining methylation of Treg cell-specific conserved non-coding sequence 2 (CNS2) in the Foxp3 locus. Consequently, CNS2 is heavily methylated when Blimp1 is ablated, leading to a loss of Foxp3 expression and severe disease. These findings identify a Blimp1-dependent pathway that preserves Treg cell stability in inflamed non-lymphoid tissues.

Transmaternal Helicobacter pylori exposure reduces allergic airway inflammation in offspring through regulatory T cells.

BACKGROUND: Transmaternal exposure to tobacco, microbes, nutrients, and other environmental factors shapes the fetal immune system through epigenetic processes. The gastric microbe Helicobacter pylori represents an ancestral constituent of the human microbiota that causes gastric disorders on the one hand and is inversely associated with allergies and chronic inflammatory conditions on the other. OBJECTIVE: Here we investigate the consequences of transmaternal exposure to H pylori in utero and/or during lactation for susceptibility to viral and bacterial infection, predisposition to allergic airway inflammation, and development of immune cell populations in the lungs and lymphoid organs. METHODS: We use experimental models of house dust mite- or ovalbumin-induced airway inflammation and influenza A virus or Citrobacter rodentium infection along with metagenomics analyses, multicolor flow cytometry, and bisulfite pyrosequencing, to study the effects of H pylori on allergy severity and immunologic and microbiome correlates thereof. RESULTS: Perinatal exposure to H pylori extract or its immunomodulator vacuolating cytotoxin confers robust protective effects against allergic airway inflammation not only in first- but also second-generation offspring but does not increase susceptibility to viral or bacterial infection. Immune correlates of allergy protection include skewing of regulatory over effector T cells, expansion of regulatory T-cell subsets expressing CXCR3 or retinoic acid-related orphan receptor γt, and demethylation of the forkhead box P3 (FOXP3) locus. The composition and diversity of the gastrointestinal microbiota is measurably affected by perinatal H pylori exposure. CONCLUSION: We conclude that exposure to H pylori has consequences not only for the carrier but also for subsequent generations that can be exploited for interventional purposes.

Roquin Suppresses the PI3K-mTOR Signaling Pathway to Inhibit T Helper Cell Differentiation and Conversion of Treg to Tfr Cells.

Roquin proteins preclude spontaneous T cell activation and aberrant differentiation of T follicular helper (Tfh) or T helper 17 (Th17) cells. Here we showed that deletion of Roquin-encoding alleles specifically in regulatory T (Treg) cells also caused the activation of conventional T cells. Roquin-deficient Treg cells downregulated CD25, acquired a follicular Treg (Tfr) cell phenotype, and suppressed germinal center reactions but could not protect from colitis. Roquin inhibited the PI3K-mTOR signaling pathway by upregulation of Pten through interfering with miR-17∼92 binding to an overlapping cis-element in the Pten 3' UTR, and downregulated the Foxo1-specific E3 ubiquitin ligase Itch. Loss of Roquin enhanced Akt-mTOR signaling and protein synthesis, whereas inhibition of PI3K or mTOR in Roquin-deficient T cells corrected enhanced Tfh and Th17 or reduced iTreg cell differentiation. Thereby, Roquin-mediated control of PI3K-mTOR signaling prevents autoimmunity by restraining activation and differentiation of conventional T cells and specialization of Treg cells.

Unique properties of thymic antigen-presenting cells promote epigenetic imprinting of alloantigen-specific regulatory T cells.

Regulatory T cells (Tregs) are potential immunotherapeutic candidates to induce transplantation tolerance. However, stability of Tregs still remains contentious and may potentially restrict their clinical use. Recent work suggested that epigenetic imprinting of Foxp3 and other Treg-specific signature genes is crucial for stabilization of immunosuppressive properties of Foxp3+ Tregs, and that these events are initiated already during early stages of thymic Treg development. However, the mechanisms governing this process remain largely unknown. Here we demonstrate that thymic antigen-presenting cells (APCs), including thymic dendritic cells (t-DCs) and medullary thymic epithelial cells (mTECs), can induce a more pronounced demethylation of Foxp3 and other Treg-specific epigenetic signature genes in developing Tregs when compared to splenic DCs (sp-DCs). Transcriptomic profiling of APCs revealed differential expression of secreted factors and costimulatory molecules, however neither addition of conditioned media nor interference with costimulatory signals affected Foxp3 induction by thymic APCs in vitro. Importantly, when tested in vivo both mTEC- and t-DC-generated alloantigen-specific Tregs displayed significantly higher efficacy in prolonging skin allograft acceptance when compared to Tregs generated by sp-DCs. Our results draw attention to unique properties of thymic APCs in initiating commitment towards stable and functional Tregs, a finding that could be highly beneficial in clinical immunotherapy.

Imprinting of Skin/Inflammation Homing in CD4+ T Cells Is Controlled by DNA Methylation within the Fucosyltransferase 7 Gene.

E- and P-selectin ligands (E- and P-ligs) guide effector memory T cells into skin and inflamed regions, mediate the inflammatory recruitment of leukocytes, and contribute to the localization of hematopoietic precursor cells. A better understanding of their molecular regulation is therefore of significant interest with regard to therapeutic approaches targeting these pathways. In this study, we examined the transcriptional regulation of fucosyltransferase 7 (FUT7), an enzyme crucial for generation of the glycosylated E- and P-ligs. We found that high expression of the coding gene fut7 in murine CD4+ T cells correlates with DNA demethylation within a minimal promoter in skin/inflammation-seeking effector memory T cells. Retinoic acid, a known inducer of the gut-homing phenotype, abrogated the activation-induced demethylation of this region, which contains a cAMP responsive element. Methylation of the promoter or mutation of the cAMP responsive element abolished promoter activity and the binding of CREB, confirming the importance of this region and of its demethylation for fut7 transcription in T cells. Furthermore, studies on human CD4+ effector memory T cells confirmed demethylation within FUT7 corresponding to high FUT7 expression. Monocytes showed an even more extensive demethylation of the FUT7 gene whereas hepatocytes, which lack selectin ligand expression, exhibited extensive methylation. In conclusion, we show that DNA demethylation within the fut7 gene controls selectin ligand expression in mice and humans, including the inducible topographic commitment of T cells for skin and inflamed sites.

The Treg-specific demethylated region stabilizes Foxp3 expression independently of NF-κB signaling.

Regulatory T cells (Tregs) obtain immunosuppressive capacity by the upregulation of forkhead box protein 3 (Foxp3), and persistent expression of this transcription factor is required to maintain their immune regulatory function and ensure immune homeostasis. Stable Foxp3 expression is achieved through epigenetic modification of the Treg-specific demethylated region (TSDR), an evolutionarily conserved non-coding element within the Foxp3 gene locus. Here, we present molecular data suggesting that TSDR enhancer activity is restricted to T cells and cannot be induced in other immune cells such as macrophages or B cells. Since NF-κB signaling has been reported to be instrumental to induce Foxp3 expression during Treg development, we analyzed how NF-κB factors are involved in the molecular regulation of the TSDR. Unexpectedly, we neither observed transcriptional activity of a previously postulated NF-κB binding site within the TSDR nor did the entire TSDR show any transcriptional responsiveness to NF-κB activation at all. Finally, the NF-κB subunit c-Rel revealed to be dispensable for epigenetic imprinting of sustained Foxp3 expression by TSDR demethylation. In conclusion, we show that NF-κB signaling is not substantially involved in TSDR-mediated stabilization of Foxp3 expression in Tregs.

IκB(NS) protein mediates regulatory T cell development via induction of the Foxp3 transcription factor.

Forkhead box P3 positive (Foxp3(+)) regulatory T (Treg) cells suppress immune responses and regulate peripheral tolerance. Here we show that the atypical inhibitor of NFκB (IκB) IκB(NS) drives Foxp3 expression via association with the promoter and the conserved noncoding sequence 3 (CNS3) of the Foxp3 locus. Consequently, IκB(NS) deficiency leads to a substantial reduction of Foxp3(+) Treg cells in vivo and impaired Foxp3 induction upon transforming growth factor-ß (TGF-ß) treatment in vitro. Moreover, fewer Foxp3(+) Treg cells developed from IκB(NS)-deficient CD25(-)CD4(+) T cells adoptively transferred into immunodeficient recipients. Importantly, IκB(NS) was required for the transition of immature GITR(+)CD25(+)Foxp3(-) thymic Treg cell precursors into Foxp3(+) cells. In contrast to mice lacking c-Rel or Carma1, IκB(NS)-deficient mice do not show reduced Treg precursor cells. Our results demonstrate that IκB(NS) critically regulates Treg cell development in the thymus and during gut inflammation, indicating that strategies targeting IκB(NS) could modulate the Treg cell compartment.

Neuropilin 1 is expressed on thymus-derived natural regulatory T cells, but not mucosa-generated induced Foxp3+ T reg cells.

Foxp3 activity is essential for the normal function of the immune system. Two types of regulatory T (T reg) cells express Foxp3, thymus-generated natural T reg (nT reg) cells, and peripherally generated adaptive T reg (iT reg) cells. These cell types have complementary functions. Until now, it has not been possible to distinguish iT reg from nT reg cells in vivo based solely on surface markers. We report here that Neuropilin 1 (Nrp1) is expressed at high levels by most nT reg cells; in contrast, mucosa-generated iT reg and other noninflammatory iT reg cells express low levels of Nrp1. We found that Nrp1 expression is under the control of TGF-ß. By tracing nT reg and iT reg cells, we could establish that some tumors have a very large proportion of infiltrating iT reg cells. iT reg cells obtained from highly inflammatory environments, such as the spinal cords of mice with spontaneous autoimmune encephalomyelitis (EAE) and the lungs of mice with chronic asthma, express Nrp1. In the same animals, iT reg cells in secondary lymphoid organs remain Nrp1(low). We also determined that, in spontaneous EAE, iT reg cells help to establish a chronic phase of the disease.

Foxp3⁺ Treg cells in the inflamed CNS are insensitive to IL-6-driven IL-17 production.

Foxp3(+) T regulatory (Treg) cells can be induced to produce interleukin (IL)-17 by in vitro exposure to proinflammatory cytokines, drawing into question their functional stability at sites of inflammation. Unlike their splenic counterparts, Treg cells from the inflamed central nervous system (CNS-Treg cells) during EAE resisted conversion to IL-17 production when exposed to IL-6. We show that the highly activated phenotype of CNS-Treg cells includes elevated expression of the Th1-associated molecules CXCR3 and T-bet, but reduced expression of the IL-6 receptor α chain (CD126) and the signaling chain gp130. We found a lack of IL-6 receptor on all CNS CD4(+) T cells, which was reflected by an absence of both classical and trans-IL-6 signaling in CNS CD4(+) cells, compared with their splenic counterparts. We propose that extinguished responsiveness to IL-6 (via down-regulation of CD126 and gp130) stabilizes the regulatory phenotype of activated Treg cells at sites of autoimmune inflammation.

Methylation matters: binding of Ets-1 to the demethylated Foxp3 gene contributes to the stabilization of Foxp3 expression in regulatory T cells.

The forkhead-box protein P3 (Foxp3) is a key transcription factor for the development and suppressive activity of regulatory T cells (Tregs), a T cell subset critically involved in the maintenance of self-tolerance and prevention of over-shooting immune responses. However, the transcriptional regulation of Foxp3 expression remains incompletely understood. We have previously shown that epigenetic modifications in the CpG-rich Treg-specific demethylated region (TSDR) in the Foxp3 locus are associated with stable Foxp3 expression. We now demonstrate that the methylation state of the CpG motifs within the TSDR controls its transcriptional activity rather than a Treg-specific transcription factor network. By systematically mutating every CpG motif within the TSDR, we could identify four CpG motifs, which are critically determining the transcriptional activity of the TSDR and which serve as binding sites for essential transcription factors, such as CREB/ATF and NF-κB, which have previously been shown to bind to this element. The transcription factor Ets-1 was here identified as an additional molecular player that specifically binds to the TSDR in a demethylation-dependent manner in vitro. Disruption of the Ets-1 binding sites within the TSDR drastically reduced its transcriptional enhancer activity. In addition, we found Ets-1 bound to the demethylated TSDR in ex vivo isolated Tregs, but not to the methylated TSDR in conventional CD4(+) T cells. We therefore propose that Ets-1 is part of a larger protein complex, which binds to the TSDR only in its demethylated state, thereby restricting stable Foxp3 expression to the Treg lineage.

Quantitative DNA methylation analysis of FOXP3 as a new method for counting regulatory T cells in peripheral blood and solid tissue.

Regulatory T-cells (Treg) have been the focus of immunologic research due to their role in establishing tolerance for harmless antigens versus allowing immune responses against foes. Increased Treg frequencies measured by mRNA expression or protein synthesis of the Treg marker FOXP3 were found in various cancers, indicating that dysregulation of Treg levels contributes to tumor establishment. Furthermore, they constitute a key target of immunomodulatory therapies in cancer as well as transplantation settings. One core obstacle for understanding the role of Treg, thus far, is the inability of FOXP3 mRNA or protein detection methods to differentiate between Treg and activated T cells. These difficulties are aggravated by the technical demands of sample logistics and processing. Based on Treg-specific DNA demethylation within the FOXP3 locus, we present a novel method for monitoring Treg in human peripheral blood and solid tissues. We found that Treg numbers are significantly increased in the peripheral blood of patients with interleukin 2-treated melanoma and in formalin-fixed tissue from patients with lung and colon carcinomas. Conversely, we show that immunosuppressive therapy including therapeutic antibodies leads to a significant reduction of Treg from the peripheral blood of transplantation patients. In addition, Treg numbers are predictively elevated in the peripheral blood of patients with various solid tumors. Although our data generally correspond to data obtained with gene expression and protein-based methods, the results are less fluctuating and more specific to Treg. The assay presented here measures Treg robustly in blood and solid tissues regardless of conservation levels, promising fast screening of Treg in various clinical settings.

DNA demethylation in the human FOXP3 locus discriminates regulatory T cells from activated FOXP3(+) conventional T cells.

The transcription factor FOXP3 is critical for development and function of regulatory T cells (Treg). Their number and functioning appears to be crucial in the prevention of autoimmunity and allergy, but also to be a negative prognostic marker for various solid tumors. Although expression of the transcription factor FOXP3 currently constitutes the best-known marker for Treg, in humans, transient expression is also observed in activated non-Treg. Extending our recent findings for the murine foxp3 locus, we observed epigenetic modification of several regions in the human FOXP3 locus exclusively occurring in Treg. Importantly, activated conventional CD4(+) T cells and TGF-beta-treated cells displayed no FOXP3 DNA demethylation despite expression of FOXP3, whereas subsets of Treg stable even upon extended in vitro expansion remained demethylated. To investigate whether a whole set of genes might be epigenetically imprinted in the Treg lineage, we conducted a genome-wide differential methylation hybridization analysis. Several genes were found displaying differential methylation between Treg and conventional T cells, but none beside FOXP3 turned out to be entirely specific to Treg when tested on a broad panel of cells and tissues. We conclude that FOXP3 DNA demethylation constitutes the most reliable criterion for natural Treg available at present.