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

Degradation of IκBα is not required for TSDR enhancer activity.

<p>Luciferase plasmids integrating either the NF-κB-RE or TSDR-FoxPro were co-transfected with either an empty vector or with a vector encoding the super-repressor, a non-degradable form of IκBα, into RLM-11 cells. Dual luciferase assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088318#pone-0088318-g001" target="_blank">Figure 1</a> and unstimulated cells served as controls. Luciferase activities are shown as percent of empty vector controls and standard deviations of performed triplicates are shown. One representative experiment out of at least two independent experiments is depicted.</p

B cells and macrophages fail to induce transcriptional enhancer activity of the TSDR.

<p>Dual luciferase assays were performed after transfecting reporter plasmids carrying the indicated inserts or an empty pGL3 vector (EV) into RLM-11 cells (T cell line), A20 cells (B cell line) or RAW 264.7 cells (macrophage cell line). Three hrs (RLM-11, A20) or 20 hrs (RAW 264.7) after transfection, cells were stimulated for 16 hrs with PMA/iono (RLM-11, A20) or for 14 hrs with LPS/IFN-γ (RAW 264.7), followed by measurement of luciferase activities (mean±SD, n = 3). Data are representative of two to four independent experiments.</p

The postulated NF-κB binding site of the TSDR is not transcriptionally responsive to activated NF-κB.

<p>(<b>A</b>) RLM-11 cells were stimulated with PMA/iono for indicated time periods and applied to subcellular fractionation. Nuclear and cytoplasmic extracts were analyzed by Western blotting using the indicated antibodies. p44/42 and lamin B served as loading and purity controls for cytoplasmic and nuclear fractions, respectively. (<b>B</b>) A luciferase reporter plasmid integrating the NF-κB-RE was transfected into RLM-11 cells and dual luciferase assays were performed in triplicates as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088318#pone-0088318-g001" target="_blank">Figure 1</a>. Mean luciferase activity is shown as fold increase to unstimulated control. Results are representative of four independent experiments. (<b>C</b>) A schematic view on the first part of the <i>Foxp3</i> gene locus is depicted. White boxes indicate untranslated exons, the first translated exon is indicated in black. Evolutionary conserved non-coding sequences (CNS) are indicated in grey. The distended region of the TSDR includes the previously described NF-κB binding site (black frame), which is flanked by the seventh CpG motif (underlined) of the TSDR. (<b>D</b>) A tandem of five repetitive sequences of the putative NF-κB binding site was inserted into the pCpGL luciferase reporter plasmid upstream of the <i>EF</i> promoter (tandem-EFPro). Dual luciferase assays were performed as described in (B) using pCpGL-TSDR-EFPro as a positive control. Data represent one out of two independent experiments.</p

Kinase activity of IKKα and IKKβ is largely dispensable for TSDR enhancer activity.

<p>(<b>A</b>) Luciferase plasmids encoding NF-κB-RE or TSDR-FoxPro were co-transfected with plasmids encoding kinase dead (KD) or wild-type (WT) forms of IκB kinase α and β (IKKα and IKKβ) into RLM-11 cells. Cells were cultured for one day allowing efficient protein expression before cells were stimulated overnight with PMA/iono and dual luciferase assays were performed. Luciferase activities are given as percent of luciferase activity of WT samples and standard deviations were calculated from three replicates. (<b>B</b>) Dual luciferase assays as described in (A) were performed co-transfecting the indicated luciferase constructs with a plasmid encoding the constitutively active form of IKKβ (IKK-CA) or empty vector as control (mean±SD, n = 3). One representative out of three independent experiments is shown.</p

c-Rel^−/− Tregs show a stable phenotype.

<p>(<b>A</b>) CD4⁺CD25^hi Tregs and CD4⁺CD25⁻ Tconv were isolated from wild-type (WT) or c-Rel^−/− mice. Genomic DNA was isolated and subjected to bisulfite sequencing in order to determine the methylation status of CpG dinucleotides within the TSDR. (<b>B</b>) CD4⁺CD8⁻CD62L^hiCD25^hi Tregs from spleen and lymph nodes of c-Rel^−/− or WT mice were sorted and an aliquot was analyzed for Foxp3 expression by flow cytometry (top panel). Cells were cultured in the presence of IL-2 and stimulated by plate-bound α-CD3/CD28 for six days followed by flow cytometric analysis of Foxp3 expression. Cells depicted were pregated to viable CD4⁺ T cells. Results represent one out of two independent experiments.</p

Motor coordination in Theiler’s murine encephalomyelitis virus (TMEV)-infected SJL mice.

<p>(A) <i>Experiment I</i>: RotaRod^® performance test after early application of the IL-10 receptor blocking antibody (IL-10R Ab) at 0, 7, 14 and 21 dpi (arrows) revealed motor coordination deficits in TMEV-infected animals compared to mock-infected mice at 42 dpi. However, no differences were determined between IL-10R Ab- and isotype-treated animals. white box = TMEV-infected mice without IL-10R Ab treatment (group “isotype_early/TMEV”), grey box = TMEV-infected mice with IL-10R treatment (group “IL10R↓_early/TMEV”), black box = mock-infected mice with IL-10R treatment (group “IL10R↓_early/mock”). (B) <i>Experiment II</i>: application of IL-10R Ab at 35 and 42 dpi (arrows) caused also deterioration of motor coordination starting at 42 dpi. However, no differences were observed between IL-10R Ab- and isotype-treated animals (groups “IL-10R↓_late/TMEV” and “isotype_late/TMEV”). white box = TMEV-infected mice without IL-10R Ab treatment (group “isotype_late/TMEV”), grey box = TMEV-infected mice with IL-10R treatment (group “IL10R↓_late/TMEV”), black box = mock-infected mice with IL-10R treatment (group “IL10R↓_late/mock”). Box and whisker plots display median, minimum and maximum values as well as upper and lower quartiles, 5 animals used at all investigated time points, Wilcoxon rank-sum tests, *  =  p < 0.05.</p

Theiler’s murine encephalomyelitis virus (TMEV)-infection exacerbates enteric disease following interleukin-10 receptor (IL-10R) blockade.

<p>(A) Note changes of colonic mucosa with severe lymphohistiocytic to neutrophilic inflammation (arrow heads), crypt abscess formation (#), and loss of crypt epithelium (arrow). H&E staining, bar = 60 μm (B) Significantly increased severity of colitis at 14 days post infection (dpi) in TMEV-infected mice with IL10R antibody (Ab) treatment (group “IL10R↓_early/TMEV”) compared to Ab treated animals without infection (group “IL10R↓_early/mock”). Animals receiving isotype control instead of IL-10R Ab did not show any intestinal inflammation. IL-10R Ab treated SJL mice with TMEV-infection (group “IL10R↓_early/TMEV”), TMEV-infected mice without IL-10R Ab treatment (group “isotype_early/TMEV”), IL-10R Ab treated SJL mice without TMEV-infection (group “IL10R↓_early/mock”). Box and whisker plots display median, minimum and maximum values as well as upper and lower quartiles, 5 animals used in all groups, Wilcoxon rank-sum tests, *  =  p < 0.05.</p

Effects of acute Theiler’s murine encephalomyelitis virus (TMEV)-infection (<i>experiment I</i>) upon cytokine expression and phenotypical changes in spleens of interleukin-10 receptor (IL-10R) blocked SJL mice.

<p>(A-I) Significantly elevated mRNA levels of IL-1α, IL-2, IL-4, IL-5, IL-6, TNF, IFN-γ, TGF-β and IL-10 in spleens of infected mice (group “IL-10R↓_early/TMEV”) compared to non-infected animals following IL-10R antibody (Ab) treatment (group “IL10R↓_early/mock”). (J) Flow cytometry revealed a relative increase of CD19⁺ B cells and a simultaneous decrease of CD4⁺ T cells (K) in the spleen of infected mice following IL-10R blockade at 14 dpi. (L) Additionally, the relative numbers of CD4⁺ Foxp3⁺ Treg in the spleen were increased following TMEV-infection and IL-10R blockade. Moreover, gMFI of CD69 (M) and CD44 (N) gated on CD4⁺ cells and gMFI of CD69 (O) and CD44 (P) gated on CD8⁺ cells were increased at 14 dpi in infected mice following IL-10R Ab treatment. For gating strategy see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161883#pone.0161883.s001" target="_blank">S1 Fig</a>. IL-10R Ab treated animals without TMEV-infection (group “IL10R↓_early/mock”). IL-10R Ab treated mice with additional TMEV-infection (group “IL-10R↓_early/TMEV”). Box and whisker plots display median, minimum and maximum values as well as upper and lower quartiles, 5 animals used in both groups and at all investigated time points, Wilcoxon rank-sum tests, *  =  p < 0.05.</p

Leukocyte subsets in the colon of interleukin-10 receptor (IL-10R) neutralized SJL mice.

<p>(A) Application of IL-10R antibody (Ab) leads to increased numbers of CD3⁺ T cells in the colon at all investigated time points. (B). An increase of CD45R⁺ B cells can be observed at day 21 after the first Ab application. (C) Foxp3⁺ regulatory T cells and (D) Iba-1⁺ macrophages are elevated at 7, 14, and 21 dpi. grey box with vertical lines = control animals (group “isotype”), white box with vertical lines = IL-10R blocked animals (group “IL-10R↓”). Box and whisker plot display median, minimum and maximum values as well as upper and lower quartiles, 5 animals used at all investigated time points, Wilcoxon rank-sum tests, *  =  p < 0.05.</p