IL-36α induced the expression of T cell costimulatory molecules in splenic CD11c⁺ cells.

<p>A–C) Transcript expression of the co-stimulatory molecules CD80, CD86 and CD40 in splenic CD11c⁺ cells 2 h following incubation with increasing concentrations of IL-36α. Transcript expression was evaluated by SYBR-Green based quantitative real-time PCR. *Indicates significant differences (<i>P<0.05</i>) compared to 0 µg/mL group. Data represent mean±SD from quadruplicate samples from one of two representative experiments. D) Flow cytometric evaluation of splenic CD11c⁺ cells 24 h following incubation with increasing concentrations of IL-36α for 2 h. E) Cell surface expression of co-stimulatory molecules in splenic CD11c⁺ cells 24 h following incubation with increasing concentrations of IL-36α for 2 h. MFI – mean fluorescence intensity. *Indicates significant differences (<i>P<0.05</i>) compared to 0.1 µg/mL group. Data represent mean±SD from triplicate samples from one of two representative experiments.</p

Intratracheal instillation of IL-36α increased the mRNA expression of proinflammatory mediators in the lungs of wild-type C57BL/6 mice.

<p>A–H) Transcript expression of early response cytokines (TNFα, IL-1α, IL-1β, IL-36γ), the classical IL-1 receptor IL-1R1, the novel IL-1 cytokine cluster receptor IL-36R and the neutrophil specific chemokines CXCL1 and CXCL2 in the lungs of mice 24 h following a single i.t instillation of PBS or 10 µg IL-36α. Transcript expression was evaluated by SYBR-Green based quantitative real-time PCR. I–K) Protein expression of TNFα, IL-1α, IL-1β and CXCL1 in the BAL fluid recovered from mice 24 h following a single i.t instillation of PBS or 10 µg IL-36α. Protein expression was quantified by multiplexed cytometric bead arrays. *Indicates significant differences (<i>P<0.05</i>) compared to PBS treated mice. Data represent mean±SEM from 4–5 mice/group.</p

Enhanced proliferation and cytokine production by Apaf1-deficient naïve T cells.

<p>(A and B) LN cells from unimmunized <i>Apaf1</i>^f/f-OTII or Lck-<i>Cre</i>-<i>Apaf1</i>^f/f -OTII mice were labeled with CFSE and stimulated with anti-CD3ε antibody or OVA peptide at the indicated concentrations. CFSE intensity was analyzed and cells with lower intensity (in cell division) were measured. Representative figures with percentages of cells with lower CFSE intensity are shown in A and results with triplicated samples are shown in B. (C) IFN-γ production by LN cells from unimmunized mice stimulated with anti-CD3ε antibody or OVA peptide. Open columns; <i>Apaf1</i>^f/f-OTII mice and closed columns; Lck-<i>Cre</i>-<i>Apaf1</i>^f/f-OTII mice. Experiments were repeated three times with similar results. Shown are mean+SD. Experiments were repeated three times with similar results. *; p<0.05, **; p<0.01, and ***; p<0.002 compared between <i>Apaf1</i>f/f and Lck-<i>Cre</i>-<i>Apaf1</i>^f/f cells, <i>t</i>-tests.</p

IL-36α directly induced NF-κB activation in mouse macrophage cell lines.

<p>A) Cells from a mouse macrophage NF-κB reporter cell line (RAW-ELAM cells) were stimulated with increasing concentrations of IL-36α. Green fluorescent protein (GFP) expression, indicative of NF-κB activation, was increased in a dose-dependent manner upon incubation with IL-36α. Flow cytometry plot presented is representative of triplicate samples in one out of two independent experiments.</p

Intratracheal instillation of IL-36α does not induce airway hypresponsiveness.

<p>A) Total lung resistance and B) lung compliance were not significantly different in the lungs of IL-36α challenged mice compared to PBS controls. Airway responses in mice were measured using invasive plethysmography 24 h following i.t instillation of IL-36α or PBS. Data presented are percentage changes from baseline (0mg/mL) measurements. Data represent mean±SEM from 5–7 mice/group.</p

IL-36α induced the expression of proinflammatory cytokines and chemokines in splenic CD11c⁺ cells.

<p>A–H) Transcript expression of early response cytokines (TNFα, IL-1α, IL-1β, IL-36γ), the classical IL-1 receptor IL-1R1, the novel IL-1 cytokine cluster receptor IL-36R as well as the neutrophil specific chemokines CXCL1 and CXCL2 in splenic CD11c⁺ cells 2 h following incubation with increasing concentrations of IL-36α. Transcript expression was evaluated by SYBR-Green based quantitative real-time PCR. *Indicates significant differences (<i>P<0.05</i>) compared to 0 µg/mL group. Data represent mean±SD of quadruplicate samples from one of two representative experiments. I) Diff-quik stained cells from cytospun BAL cells from the lungs of naïve mice demonstrates that the majority of lung resident immune cells are alveolar macrophages. J) PCR on cDNA from naïve mouse alveolar macrophages demonstrating the constitutive mRNA expression of an endogenous control (β-actin), IL-1R1, IL-36R and IL-1RAcP. Image of a DNA electrophoresis gel has been color-inverted for clarity. bp – base pairs.</p

Incubation with IL-36α enhances the ability of splenic CD11c⁺ cell mediated CD4⁺ T cell proliferation.

<p>A) Flow cytometric evaluation of CD4⁺ T cell proliferation responses induced by IL-36α stimulated splenic CD11c⁺ cells. Splenic CD11c⁺ cells were incubated with increasing doses of IL-36α for 2 h. Following incubation, media containing IL-36α was removed and CFSE-labeled CD4⁺ T cells were co-cultured with IL-36α stimulated CD11c⁺ cells. CFSE dilution was used to evaluate T cell proliferation responses 96 h following co-culture. CD4⁺ T cell proliferation was proportional to the concentration of IL-36α used for stimulating CD11c⁺ cells used in the co-culture. Flow cytometry plot presented is representative of quadruplicate samples in one out of two independent experiments. B) Flow cytometric evaluation of antigen-specific CD4⁺ T cell proliferation responses induced by IL-36α stimulated splenic CD11c⁺ cells. Splenic CD11c⁺ cells were incubated with increasing doses of IL-36α and 100 ng/mL OVA_323-339 for 2 h. Following incubation, media containing IL-36α and the OVA peptide was removed and CFSE-labeled CD4⁺ T cells from OTII TCR transgenic mice were co-cultured with IL-36α stimulated, OVA peptide pulsed CD11c⁺ cells. CFSE dilution in the culture was used to evaluate T cell proliferation responses 96 h following co-culture. CD4⁺ T cell proliferation was proportional to the concentration of IL-36α used for stimulating CD11c⁺ cells used in the co-culture. Flow cytometry plot presented is representative of quadruplicate samples in one out of two independent experiments.</p

Generation of T cell-specific Apaf1-deficient mice.

<p>(A) Southern blot analysis of genomic DNA from <i>Apaf1</i>^f-Neo/+ thymocytes (left), <i>Apaf1</i>^f/+ thymocytes (right), and <i>Apaf1</i>^f/+ ES cells, in which Neo gene was removed by transient expression of FLPe recombinase (middle). DNA was digested with EcoRI and detected with the probe at exon 4. (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195119#pone.0195119.s001" target="_blank">S1 Fig</a> for detail.) (B) Western blot analysis of proteins from LN T cells and thymocytes of <i>Apaf1</i>^f/f mice and Lck-<i>Cre</i>-<i>Apaf1</i>^f/f mice. (C) Thymocytes from <i>Apaf1</i>^f/f mice (open columns) or Lck-<i>Cre</i>-<i>Apaf1</i>^f/f mice (closed columns) were stimulated with indicated doses of anti-Fas antibodies plus cycloheximide (αFas + CHX), dexamethasone (Dex), staurosporine (Stauro), γ-irradiation, or left untreated. Apoptotic cells were detected by flow cytometry. (D) Purified T cells from LN of <i>Apaf1</i>^f/f (open columns) or Lck-<i>Cre</i>-<i>Apaf1</i>^f/f (closed columns) were activated for 48 hours with anti-CD3ε antibody plus anti-CD28 antibody. Activated cells, after removal of dead cells, were cultured in the presence of conditioned medium (CM), in the fresh medium for growth factor deprivation (Media), or re-stimulated with anti-CD3ε antibody in fresh medium for activation-induced cell death, for 20 hours. Apoptotic cells were detected by flow cytometry. Data show means + SD of triplicated samples. Experiments were repeated three times with similar results. *; p<0.05, **; p<0.01, and ***; p<0.002 compared between <i>Apaf1</i>^f/f and Lck-<i>Cre</i>-<i>Apaf1</i>^f/f cells, <i>t</i>-tests. ###; p’ (corrected p value) < 0.002, <i>t</i>-tests with Bonferroni correction.</p

Caspase-independent role of Apaf1 in T cell activation.

<p>LN cells from immunized <i>Apaf1</i>^f/f-OTII or Lck-<i>Cre</i>-<i>Apaf1</i>^f/f -OTII mice were stimulated with indicated doses of OVA or anti-CD3ε antibody in the presence or absence of z-VAD-<i>fmk</i> (z-VAD) for 48 hours. (A) Cell proliferation, cell viability (Annexin V-negative and PI-negative), production of IFN-γ and IL-17, or expression of CD69, CD44, and CD62L were analyzed. Open columns; <i>Apaf1</i>^f/f-OTII mice and closed columns; Lck-<i>Cre</i>-<i>Apaf1</i>^f/f-OTII mice. Data show means + SD of triplicated samples. Experiments were repeated three times with similar results. #; p’<0.05, ##; p’<0.01, and ###; p’<0.002, <i>t</i>-tests with Bonferroni correction compared among indicated samples. (B) Cell lysates were prepared from cells in A, electrophoresed, and blotted. Pro- or cleaved form of caspase 3 and 7, and pro-caspase 9 were detected with respective antibodies. Tubulin was detected as an internal control. Experiments were repeated two times with similar results.</p

Enhanced <i>ex vivo</i> recall responses of Apaf1-deficient T cells.

<p>(A and B) LN cells from OVA-immunized <i>Apaf1</i>^f/f-OTII or Lck-<i>Cre</i>-<i>Apaf1</i>^f/f-OTII mice were assessed for expression of CD69, CD44 and CD62L. Percentages (A) and absolute numbers (B) of CD69⁺ cells and CD44^highCD62L^low cells in TCR Tg-positive population (OTII population) are shown. (C) LN cells from immunized mice were re-stimulated with OVA or anti-CD3ε antibody at the indicated concentration for 48 hours. Percentages of CD69⁺ cells and CD44^highCD62L^low cells are shown. Open columns; <i>Apaf1</i>^f/f-OTII mice and closed columns; Lck-<i>Cre</i>-<i>Apaf1</i>^f/f-OTII mice. Shown are mean+SD of triplicated samples. Experiments were repeated three times with similar results. *; p<0.05, **; p<0.01, and ***; p<0.002 compared between <i>Apaf1</i>^f/f and Lck-<i>Cre</i>-<i>Apaf1</i>^f/f cells, <i>t</i>-tests.</p