Stepwise acquisition of unique epigenetic signatures during differentiation of tissue Treg cells

Regulatory T cells in non-lymphoid tissues are not only critical for maintaining self-tolerance, but are also important for promoting organ homeostasis and tissue repair. It is proposed that the generation of tissue Treg cells is a stepwise, multi-site process, accompanied by extensive epigenome remodeling, finally leading to the acquisition of unique tissue-specific epigenetic signatures. This process is initiated in the thymus, where Treg cells acquire core phenotypic and functional properties, followed by a priming step in secondary lymphoid organs that permits Treg cells to exit the lymphoid organs and seed into non-lymphoid tissues. There, a final specialization process takes place in response to unique microenvironmental cues in the respective tissue. In this review, we will summarize recent findings on this multi-site tissue Treg cell differentiation and highlight the importance of epigenetic remodeling during these stepwise events

Lymph node stromal cell subsets-Emerging specialists for tailored tissue-specific immune responses.

The effective priming of adaptive immune responses depends on the precise dispatching of lymphocytes and antigens into and within lymph nodes (LNs), which are strategically dispersed throughout the body. Over the past decade, a growing body of evidence has advanced our understanding of lymph node stromal cells (LNSCs) from viewing them as mere accessory cells to seeing them as critical cellular players for the modulation of adaptive immune responses. In this review, we summarize current advances on the pivotal roles that LNSCs play in orchestrating adaptive immune responses during homeostasis and infection, and highlight the imprinting of location-specific information by micro-environmental cues into LNSCs, thereby tailoring tissue-specific immune responses

Acute neonatal Listeria monocytogenes infection causes long-term, organ-specific changes in immune cell subset composition.

Listeria monocytogenes (Lm) is a food-borne pathogen with a high chance of infecting neonates, pregnant women, elderly and immunocompromised individuals. Lm infection in neonates can cause neonatal meningitis and sepsis with a high risk of severe neurological and developmental sequelae and high mortality rates. However, whether an acute neonatal Lm infection causes long-term effects on the immune system persisting until adulthood has not been fully elucidated. Here, we established a neonatal Lm infection model and monitored the composition of major immune cell subsets at defined time points post infection (p.i.) in secondary lymphoid organs and the intestine. Twelve weeks p.i., the CD8+ T cell population was decreased in colon and mesenteric lymph nodes (mLNs) with an opposing increase in the spleen. In the colon, we observed an accumulation of CD4+ and CD8+ effector/memory T cells with an increase of T-bet+ T helper 1 (Th1) cells. In addition, 12 weeks p.i. an altered composition of innate lymphoid cell (ILC) and dendritic cell (DC) subsets was still observed in colon and mLNs, respectively. Together, these findings highlight organ-specific long-term consequences of an acute neonatal Lm infection on both the adaptive and innate immune system

Microbiome Dependent Regulation of T and Th17 Cells in Mucosa.

Mammals co-exist with resident microbial ecosystem that is composed of an incredible number and diversity of bacteria, viruses and fungi. Owing to direct contact between resident microbes and mucosal surfaces, both parties are in continuous and complex interactions resulting in important functional consequences. These interactions govern immune homeostasis, host response to infection, vaccination and cancer, as well as predisposition to metabolic, inflammatory and neurological disorders. Here, we discuss recent studies on direct and indirect effects of resident microbiota on regulatory T cells (Tregs) and Th17 cells at the cellular and molecular level. We review mechanisms by which commensal microbes influence mucosa in the context of bioactive molecules derived from resident bacteria, immune senescence, chronic inflammation and cancer. Lastly, we discuss potential therapeutic applications of microbiota alterations and microbial derivatives, for improving resilience of mucosal immunity and combating immunopathology

The microbiota is dispensable for the early stages of peripheral regulatory T cell induction within mesenteric lymph nodes.

Intestinal Foxp3+ regulatory T cell (Treg) subsets are crucial players in tolerance to microbiota-derived and food-borne antigens, and compelling evidence suggests that the intestinal microbiota modulates their generation, functional specialization, and maintenance. Selected bacterial species and microbiota-derived metabolites, such as short-chain fatty acids (SCFAs), have been reported to promote Treg homeostasis in the intestinal lamina propria. Furthermore, gut-draining mesenteric lymph nodes (mLNs) are particularly efficient sites for the generation of peripherally induced Tregs (pTregs). Despite this knowledge, the direct role of the microbiota and their metabolites in the early stages of pTreg induction within mLNs is not fully elucidated. Here, using an adoptive transfer-based pTreg induction system, we demonstrate that neither transfer of a dysbiotic microbiota nor dietary SCFA supplementation modulated the pTreg induction capacity of mLNs. Even mice housed under germ-free (GF) conditions displayed equivalent pTreg induction within mLNs. Further molecular characterization of these de novo induced pTregs from mLNs by dissection of their transcriptomes and accessible chromatin regions revealed that the microbiota indeed has a limited impact and does not contribute to the initialization of the Treg-specific epigenetic landscape. Overall, our data suggest that the microbiota is dispensable for the early stages of pTreg induction within mLNs

MicroRNA-142-3p Negatively Regulates Canonical Wnt Signaling Pathway.

Wnt/β-catenin signaling pathway plays essential roles in mammalian development and tissue homeostasis. MicroRNAs (miRNAs) are a class of regulators involved in modulating this pathway. In this study, we screened miRNAs regulating Wnt/β-catenin signaling by using a TopFlash based luciferase reporter. Surprisingly, we found that miR-142 inhibited Wnt/β-catenin signaling, which was inconsistent with a recent study showing that miR-142-3p targeted Adenomatous Polyposis Coli (APC) to upregulate Wnt/β-catenin signaling. Due to the discordance, we elaborated experiments by using extensive mutagenesis, which demonstrated that the stem-loop structure was important for miR-142 to efficiently suppress Wnt/β-catenin signaling. Moreover, the inhibitory effect of miR-142 relies on miR-142-3p rather than miR-142-5p. Further, we found that miR-142-3p directly modulated translation of Ctnnb1 mRNA (encoding β-catenin) through binding to its 3' untranslated region (3' UTR). Finally, miR-142 was able to repress cell cycle progression by inhibiting active Wnt/β-catenin signaling. Thus, our findings highlight the inhibitory role of miR-142-3p in Wnt/β-catenin signaling, which help to understand the complex regulation of Wnt/β-catenin signaling

DataSheet_1_Inflammatory perturbations in early life long-lastingly shape the transcriptome and TCR repertoire of the first wave of regulatory T cells.pdf

The first wave of Foxp3+ regulatory T cells (Tregs) generated in neonates is critical for the life-long prevention of autoimmunity. Although it is widely accepted that neonates are highly susceptible to infections, the impact of neonatal infections on this first wave of Tregs is completely unknown. Here, we challenged newborn Treg fate-mapping mice (Foxp3eGFPCreERT2xROSA26STOP-eYFP) with the Toll-like receptor (TLR) agonists LPS and poly I:C to mimic inflammatory perturbations upon neonatal bacterial or viral infections, respectively, and subsequently administrated tamoxifen during the first 8 days of life to selectively label the first wave of Tregs. Neonatally-tagged Tregs preferentially accumulated in non-lymphoid tissues (NLTs) when compared to secondary lymphoid organs (SLOs) irrespective of the treatment. One week post challenge, no differences in the frequency and phenotypes of neonatally-tagged Tregs were observed between challenged mice and untreated controls. However, upon aging, a decreased frequency of neonatally-tagged Tregs in both NLTs and SLOs was detected in challenged mice when compared to untreated controls. This decrease became significant 12 weeks post challenge, with no signs of altered Foxp3 stability. Remarkably, this late decrease in the frequency of neonatally-tagged Tregs only occurred when newborns were challenged, as treating 8-days-old mice with TLR agonists did not result in long-lasting alterations of the first wave of Tregs. Combined single-cell T cell receptor (TCR)-seq and RNA-seq revealed that neonatal inflammatory perturbations drastically diminished TCR diversity and long-lastingly altered the transcriptome of neonatally-tagged Tregs, exemplified by lower expression of Tigit, Foxp3, and Il2ra. Together, our data demonstrate that a single, transient encounter with a pathogen in early life can have long-lasting consequences for the first wave of Tregs, which might affect immunological tolerance, prevention of autoimmunity, and other non-canonical functions of tissue-resident Tregs in adulthood.</p

Among candidate miRNAs, miR-142 negatively regulates Wnt/β-catenin signaling.

<p>(A) Schematic representation of the pGL3-TopFlash reporter. Eight tandem repeats of TCF/LEF response elements were introduced upstream of the SV40 promoter and firefly luciferase gene (Luc) in pGL3 promoter vector. (B) Screening for inhibitors of Wnt/β-catenin signaling. Plasmids pGL3-TopFlash, pRL-TK and miRNA-expressing vectors were cotransfected into HEK293T cells. Cells were treated with 25 mM LiCl 6 h posttransfection, and lysed 24 h posttransfection for dual-luciferase analysis (RLUs, Fluc/Rluc). (C, E) miR-142 inhibits the activated Wnt/β-catenin signaling. Wnt/β-catenin signaling was activated by 25 mM LiCl (C) or increasing doses of Wnt3a (E). TopFlash-mediated firefly luciferase activities (B, C, E) were normalized to the activity of Renilla luciferase (pRL-TK); error bars mark the SEM (n = 3; **P < 0.01, *P < 0.05, t test). (D) miR-142 represses expression of <i>Axin2</i>. miR-142 expressing vector or empty vector (EF) was transfected into HEK293T cells with or without 25 mM LiCl treatment. <i>Axin2</i> mRNA expression was analyzed by quantitative RT-PCR, the data shown were normalized by <i>Actb</i> expression; error bars mark the SEM (n = 2; *P < 0.05, t test).</p

Pre-miR-142 is essential for suppressing Wnt/β-catenin signaling.

<p>A and B, schematic of the MDH1-142 (A) and EF-miR-142, EF-del-miR-142, EF-pre-edited-miR-142 (B) constructs. Pre-miR-142 (59 nt) plus flanking sequences were introduced downstream of the H1 or EF-1a promoter, followed by T5 or polyA termination signal. Whole 59 nucleotides of pre-miR-142 were deleted in the del-miR-142 construction. Four Adenosine residues within pre-miR-142 were substituted by guanosine residues (A➔G) constructed the pre-edited-miR-142 plasmid. (C) HEK293T cells were transfected with MDH1-142 expression vector or control MDH1 vector along with the pGL3-TopFlash or pGL3 empty vector and the pRL-TK vector as a normalization control. Cells were treated with 25 mM LiCl and lysed 24 h later for dual-luciferase analysis. Normalized TopFlash values were further divided by normalized pGL3 control values; error bar mark the SEM (n = 3; *P < 0.05, t test). (D) HEK293T cells were transfected with EF-miR142 expression vector or miR-142-mutant vectors along with the pGL3-TopFlash or pGL3-FopFlash vector and the pRL-TK vector as a normalization control. Normalized TopFlash values were further divided by normalized FopFlash values; error bars mark the SEM (n = 3; ***P < 0.001, **P < 0.01, t test). (E) Schematic diagram showing the structure of pre-miR-142. The part highlighted in green indicates miR-142-5p, and in red shows miR-142-3p. Bases with light-gray background represent the seed sequences of these two distinct miRNAs. The frames above M1 to M5 mark the positions of five structure-changing mutants within pre-miR-142, purple bases within the frames indicate the mutant sequences. (F) TopFlash-mediated reporter assay was performed as described in (D) with M1 ~ M5 mutants; error bars mark the SEM (n = 3; ***P < 0.001, **P < 0.01, *P < 0.05, ns P > 0.05, t test).</p

Postnatal expansion of mesenteric lymph node stromal cells towards reticular and CD34(+) stromal cell subsets

Pezoldt J, Wiechers C, Zou M, et al. Postnatal expansion of mesenteric lymph node stromal cells towards reticular and CD34(+) stromal cell subsets. Nature Communications. 2022;13(1): 7227.Gut-draining mesenteric lymph nodes (LN) provide the framework to shape intestinal adaptive immune responses. Based on the transcriptional signatures established by our previous work, the composition and immunomodulatory function of LN stromal cells (SC) vary according to location. Here, we describe the single-cell composition and development of the SC compartment within mesenteric LNs derived from postnatal to aged mice. We identify CD34(+) SC and fibroblastic reticular stromal cell (FRC) progenitors as putative progenitors, both supplying the typical rapid postnatal mesenteric LN expansion. We further establish the location-specific chromatin accessibility and DNA methylation landscape of non-endothelial SCs and identify a microbiota-independent core epigenomic signature, showing characteristic differences between SCs from mesenteric and skin-draining peripheral LNs. The epigenomic landscape of SCs points to dynamic expression of Irf3 along the differentiation trajectories of FRCs. Accordingly, a mesenchymal stem cell line acquires a Cxcl9(+) FRC molecular phenotype upon lentiviral overexpression of Irf3, and the relevance of Irf3 for SC biology is further underscored by the diminished proportion of Ccl19(+) and Cxcl9(+) FRCs in LNs of Irf3(-/-) mice. Together, our data constitute a comprehensive transcriptional and epigenomic map of mesenteric LNSC development in early life and dissect location-specific, microbiota-independent properties of non-endothelial SCs. Lymph nodes in various locations of the body differ in their cell composition and gene expression signatures. Here authors show that the rapid postnatal expansion of lymph nodes is governed by CD34 (+) stromal cells and fibroblastic reticular stromal cell progenitors, distinguished by intrinsic, microbiome-independent core epigenetic blueprints