Intratracheal instillation of IL-36α induced neutrophil influx in the lungs of IL-1αβ^−/− mice.

<p>A) Total cell counts from BAL fluid recovered from mice 24 h following a single i.t. instillation of PBS or 10 µg IL-36α. B) Differential cell count percentages and C) Differential cell count numbers in the BAL fluid recovered from mice 24 h following a single i.t. instillation of PBS or 10 µg IL-36α. *Indicates significant differences (<i>P<0.05</i>) compared to PBS treated mice. Data represent mean±SEM from 3–4 mice/group. D) Flow cytometry on cells recovered from BAL fluid from mice 24 h following a single i.t. instillation of PBS or 10 µg IL-36α. A majority of cells from the IL-36α instilled lungs were CD11c⁻CD11b⁺Ly6G⁺ neutrophils. Depicted flow cytometry plots are representative of 3–4 mice/group. H) Hematoxylin & Eosin stained lung sections isolated from mice 24 h following a single i.t instillation of PBS or 10 µg IL-36α. Depicted sections are representative of 3–4 mice/group.</p

Intratracheal instillation of IL-36α induced neutrophil influx in the lungs of wild-type C57BL/6 mice.

<p>A) Coomassie blue stained gel demonstrating the purity of IL-36α preparation. Ten µg IL-36α was loaded on the lane. B) Western immunoblotting of IL-36α protein preparation detects a band around 18 KDa, the predicted molecular weight of mouse IL-36α. C) Cytospin preparations demonstrating neutrophil influx in the BAL fluid recovered from mice 24 h following a single i.t. instillation of PBS or 10 µg IL-36α. D) Total cell counts from BAL fluid recovered from mice 24 h following a single i.t. instillation of PBS or 10 µg IL-36α. E) Differential cell count percentages and F) Differential cell count numbers in the BAL fluid recovered from mice 24 h following a single i.t. instillation of PBS or 10 µg IL-36α. *Indicates significant differences (<i>P<0.05</i>) compared to PBS treated mice. Data represent mean±SEM from 4–5 mice/group. G) Flow cytometry on cells recovered from BAL fluid from mice 24 h following a single i.t. instillation of PBS or 10 µg IL-36α. A majority of cells from the IL-36α instilled lungs were CD11c⁻CD11b⁺Ly6G⁺ neutrophils. Depicted flow cytometry plots are representative of 4–5 mice/group. H) Hematoxylin & Eosin stained lung sections isolated from mice 24 h following a single i.t instillation of PBS or 10 µg IL-36α. Depicted sections are representative of 4–5 mice/group.</p

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

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

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

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

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

A human tissue-based functional assay platform to evaluate the immune function impact of small molecule inhibitors that target the immune system

<div><p>While the immune system is essential for the maintenance of the homeostasis, health and survival of humans, aberrant immune responses can lead to chronic inflammatory and autoimmune disorders. Pharmacological modulation of drug targets in the immune system to ameliorate disease also carry a risk of immunosuppression that could lead to adverse outcomes. Therefore, it is important to understand the ‘immune fingerprint’ of novel therapeutics as they relate to current and, clinically used immunological therapies to better understand their potential therapeutic benefit as well as immunosuppressive ability that might lead to adverse events such as infection risks and cancer. Since the mechanistic investigation of pharmacological modulators in a drug discovery setting is largely compound- and mechanism-centric but not comprehensive in terms of immune system impact, we developed a human tissue based functional assay platform to evaluate the impact of pharmacological modulators on a range of innate and adaptive immune functions. Here, we demonstrate that it is possible to generate a qualitative and quantitative immune system impact of pharmacological modulators, which might help better understand and predict the benefit-risk profiles of these compounds in the treatment of immune disorders.</p></div

Immune function impact profiles of small molecule inhibitors.

<p>Small molecule inhibitors were evaluated in six different functional assays with nine different read-outs. For a given compound on the Y-axis, each symbol on the X-axis represents the protein binding corrected cellular potency of that compound in the corresponding assay. The potencies of the evaluated compounds in these assays are displayed as IC₅₀ values in the X-axis. The reported IC₅₀ values were generated from a composite of 8–10 point dose response curves from n = 6–8 donors for each compound in each assay. Immune function impact of a broader set (A) or a subset (B) of inhibitors are presented.</p