Canonical miRNAs stabilize FoxP3 expression.

<p>Flow cytometric analysis of FoxP3 stability in lymphocytes isolated from FoxP3-GFP-hCre:DGCR8^wt/lox (het) and FoxP3-GFP-hCre:DGCR8^lox/lox (KO) mice. (a) Representative FACS histograms of FoxP3 intracellular staining of CD4⁺GFP⁻ Tconv and CD4⁺GFP⁺ Treg with heterozygous (het) or homozygous deletion of Dgcr8 (KO) isolated from lymph nodes (LN). (b) Quantification of FoxP3 median fluorescence intensity (MFI) in het and KO Treg cells isolated from LN. The MFI was normalized due to inter-experimental variability of the relative FoxP3 MFI. For normalization the MFI of the control Treg was set to 1 and the relative reduction of MFI was calculated for the ko. In experiments with more than one control the mean MFI of the het controls was set to 1. In each individual experiment the het control Treg displayed a higher FoxP3 MFI than ko Treg. Statistical analysis was performed on pooled normalized data from four independent experiments. p = 0.0075 (Two-tailed Mann-Whitney Test). (c) FACS sorted CD4⁺GFP⁺ Treg were cultured with beads coated with anti-CD3 and anti-CD28 antibodies and 2000 U/ml IL-2 and live cells were counted. (d) Treg were purified and cultured under expansion conditions as described for panel c. At 41 h and after 4 days samples were stained for intracellular FoxP3 as in panel a. The bar indicates the gate used to define the cutoff for FoxP3 staining. Numbers indicate the % of FoxP3 expressing cells. Representative growth curve (c) and representative FACS plots (d) from 2 independent experiments.</p

Early lethality in mice with a Treg-specific <i>Dgcr8</i> deficiency.

<p>Survival curve of pooled female and male FoxP3-GFP-hCre:DGCR8^wt/lox (het) or FoxP3-GFP-hCre:DGCR8^lox/lox (KO) mice. Mice found dead or required to be euthanized due to severe body condition as per institutional requirements were collectively flagged as “dead” for the analysis. P<0.0001 (Log-rank (Mantel-Cox) Test.</p

Mice with Treg lacking canonical miRNAs develop scurfy-like disease.

<p>Macroscopic (a), flow cytometric (b,c) and histologic (d) analysis of the disease spontaneously occurring in FoxP3-GFP-hCre:DGCR8^lox/lox (KO) mice. FoxP3-GFP-hCre:DGCR8^wt/lox (het) served as control mice. (a) Splenomegaly (top) and lymphadenopathy (bottom) in het and KO mice. (b) Lymph nodes (LN) were harvested from ≥3 week old het and KO mice. Single cell suspensions stained with anti-CD44 and anti-CD62L to determine activation of CD4⁺ Tconv (gated on CD4⁺GFP⁻) as an indirect readout of Treg function were analyzed by flow cytometry (FACS). The increased frequency of CD44^hiCD62L^lo cells among CD4⁺GFP⁻ lymphocytes represents spontaneous activation of Tconv in lymph node cells. Representative FACS plots from >10 independent experiments. (c) Quantification of frequency of activated CD44^hiCD62L^lo among CD4⁺GFP⁻ Tconv in peripheral blood of 3–4 week old mice. n = 35 (het), n = 27 (KO). p<0.0001 (Two-tailed Mann-Whitney Test). (d) Representative paraffin-embedded sections of liver and lung tissues stained with Hematoxylin & Eosin of het and KO mice. Pictures were taken with 100×optical magnification. 4/4 KO livers and 4/4 KO lungs had infiltrates, 0/4 het livers or lungs were infiltrated.</p

Efficient and Treg-specific deletion of <i>Dgcr8</i> and miR-150.

<p>Conventional CD4⁺GFP⁻ (Tc) and CD4⁺GFP⁺ regulatory (Tr) T cells were FACS sorted from FoxP3-GFP reporter mice (a) and FoxP3-GFP-hCre:DGCR8^wt/lox (het) or FoxP3-GFP-hCre:DGCR8^lox/lox (KO) mice (b-d). qPCR analysis of Dgcr8 mRNA in Tc and Tr (a,b) and miR-150 (c,d). Pooled data from 3 independent experiments with 3 mice total (a,b) and representative data from 2 independent experiments with 2 mice total (c,d).</p

Pancreatic regeneration following acinar injury is impaired in Rag1^−/− mice.

<p>(A) Caerulein injection regime, time of tissue harvesting and schematic depiction of acinar de-differentiation and regeneration following caerulein-induced injury in WT mice. (B and C) Representative H&E staining of pancreas from WT and Rag1^−/− mice treated with PBS control or 2- and 7-days following caerulein-induced injury. (D) Pancreatic immuno-fluorescent staining for CK19, β-catenin and amylase from WT and Rag1^−/− mice at CaeD7. (E and F) Combined and individual scoring of pancreatic histological parameters from WT and Rag1^−/− mice at CaeD2 and CaeD7 (<i>n</i> = 5–20, *P<0.05, **P<0.01, <i>t</i> test).</p

γPMNs are present during caerulein-induced injury in WT mice and are elevated in Rag1^−/− mice.

<p>(A) IFNγ levels in serum isolated from WT and Rag1^−/− mice with and without caerulein treatment (<i>n</i> = 6–8, *P<0.05, **P≤0.01, <i>t</i> test). (B) FACS analysis of the percentage of F4/80⁻Thy1.2⁻ cells expressing Ly6G⁺ and IFNγ⁺ in the pancreas of WT and Rag1^−/− mice with or without caerulein treatment (<i>n</i> = 5–8, *P<0.05, **P<0.01, <i>t</i> test). (C) Percentage of Ly6G⁺IFNγ⁺ (CD45⁺F4/80⁻Thy1.2⁻) amongst live CD45⁺ cells infiltrating the pancreas with or without caerulein treatment as indicated. (D) Sort strategy isolating indicated populations from the pancreas of WT and Rag1^−/− mice and cytospin preparation visualizing May-Grünwald-Giemsa staining.</p

Decreasing inflammatory macrophage activation improves pancreatic injury.

<p>(A) H&E staining of pancreas from Rag1^−/− mice treated with control or the iNOS inhibitor 1400 W 2-days following caerulein-induced injury. Injection regime of 1400 W (W*) is indicated in adjacent cartoon. (B) Combined and individual pancreatic histology scores from Rag1^−/− mice treated with control or iNOS inhibitor 1400 W (<i>n</i> = 6–9). (C) Representative FACS analysis of macrophages expressing iNOS isolated from the pancreas of Rag1^−/− mice treated with α-IFNγR and IgG2 isotype control at CaeD2 (<i>n</i> = 4–8). (D) H&E staining of pancreas from Rag1^−/− mice treated with α-IFNγR and IgG2 isotype control at CaeD2. Injection regime of α-IFNγR (Y) is indicated in adjacent cartoon. (E) Combined and individual pancreatic histology scores from Rag1^−/− mice treated with α-IFNγR and IgG2 isotype control (<i>n</i> = 8–11). (F) Schematic depicting how increased innate inflammation impacts exocrine de-diferentiation.</p

Macrophages are the predominant immune cell infiltrating the pancreas following caerulein-induced injury.

<p>(A) Representative FACS plot showing the percentage of CD45⁺ cells that are F4/80⁺ compared to F4/80⁻CD11c⁺ dendritic cells and F4/80⁻Gr1⁺ myeloid cells in the pancreatic immune infiltrate at CaeD1. (B) Immuno-histochemical staining of F4/80⁺ cells from WT and Rag1^−/− treated with PBS or caerulein. (C and D) Immuno-fluorescent staining of the pancreas from WT and Rag1^−/− mice showing F4/80⁺ cells are associated with areas of the de-differentiated epithelium (visualized by β-catenin and Ecadherin) present only in Rag1^−/− mice 7-days after caerulein treatment and completely surrounding the lobular epithelium within 14-days. (E and F) Representative H&E and corresponding areas stained with F4/80 from the pancreas of Rag1^−/− mice treated with liposomes filled with either PBS or clodronate during caerulein treatment and harvested 2-days after the last caerulein injection.</p

Inflammatory factors that influence effective versus ineffective pancreatic regeneration.

<p>Schematic depicting the impact increased innate inflammation has on exocrine de-differentiation and regeneration in (A) the presence (WT mice) and absence (B) of adaptive immune cells (Rag1^−/− mice).</p

Inflammatory activation of macrophages is increased in Rag1^−/− mice before and after injury.

<p>(A and B) Representative FACS plots characterizing expression of the CD45⁺ immune cells that are F4/80⁺ and CD206⁺ in the pancreatic immune infiltrate at CaeD1 and CaeD2 (<i>n</i> = 8). (C) Representative immuno-histochemical staining of CD301⁺ and CD206⁺ cells in Rag1^−/− mice treated with caerulein at designated time points. (D) Representative FACS plots characterizing F4/80⁺ macrophages isolated from the pancreas of WT and Rag1^−/− mice at CaeD2 (<i>n</i> = 4–8). (E) Representative FACS plots of iNOS expression from macrophages isolated from the pancreas of WT and Rag1^−/− mice in untreated and CaeD2 treated mice (<i>n</i> = 4–8).</p