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