Continuous IL-23 stimulation drives ILC3 depletion in the upper GI tract and, in combination with TNFα, induces robust activation and a phenotypic switch of ILC3

<div><p>Mutations in the Interleukin (IL)-23/IL-23 receptor loci are associated with increased inflammatory bowel disease (IBD) susceptibility, and IL-23 neutralization has shown efficacy in early clinical trials. To better understand how an excess of IL-23 affects the gastrointestinal tract, we investigated chronic systemic IL-23 exposure in healthy wildtype mice. As expected, IL-23 exposure resulted in early activation of intestinal type 3 innate lymphoid cells (ILC3), followed by infiltration of activated RORγt+ T helper cells. Surprisingly, however, sustained IL-23 stimulus also dramatically reduced classical ILC3 populations within the proximal small intestine, and a phenotypically distinct T-bet expressing ILC3 population emerged. TNFα neutralization, a widely used IBD therapy, reduced several aspects of the IL-23 driven ILC3 response, suggesting a synergy between IL-23 and TNFα in ILC3 activation. <i>In vitro</i> studies supported these findings, revealing previously unappreciated effects of IL-23 and TNFα within the intestine.</p></div

Both CCR6+ and NCR+ ILC3 subsets are reduced following IL-23 stimulation, and remaining ILC3 are phenotypically distinct.

<p>Mice were injected with sham mc (open symbols) or IL-23 mc (filled symbols), and flow cytometry was performed on LP cells from the proximal SI. (A) Representative staining shows the frequency of ILC3 amongst total ILC at 3 days and 2 weeks post mc injection, with compiled geometric MFI of RORγt expression by ILC3 shown. (B) Representative staining shows the frequency of ILC3 subsets amongst total ILC3 at 3 days (top) and 2 weeks (bottom) post mc injection with compiled absolute numbers. (C) Representative staining shows the frequency of IL-23 mc elicited ILC3 (ILC3²³; IL-7R- T-bet+) amongst CCR6- NCR- ILC3 at 3 days post mc injection, with compiled absolute numbers of these cells shown at 3 days and 2 weeks post mc injection. (D) Representative staining shows the frequency of Ki67+ cells amongst ILC3 subsets, with compiled data at 3 days post mc injection. Scatter plots show means ± SEM for all mice from one of at least 3 similar experiments, 4–5 mice per group, with each symbol representative of a single mouse.</p

Chronic IL-23 elevation results in intestinal inflammation within the upper GI tract of wildtype B6 mice.

<p>Mice were injected with sham mc (open symbols) or IL-23 mc (filled symbols). (A) IHC (top) and H+E staining (bottom) of duodenal tissue. For IHC, CD3e+ staining (T cells) is black, and IBA-1 staining (macrophages) is red. (B) SI length 2 weeks post mc injection. (B-C) Flow cytometry was performed on LP cells from the proximal SI and the absolute numbers of (B) hematopoietic and (C) cytokine producing ILC3 and RORγt+ T cells were quantified (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182841#pone.0182841.s001" target="_blank">S1B Fig</a> for typical gating strategy). Scatter plots show means ± SEM for all mice from one of 2–4 similar experiments, 4–5 mice per group, with each symbol representative of a single mouse.</p

ILC3 are rapidly activated and depleted from the proximal small intestine of IL-23 mc injected mice.

<p>Mice were injected with sham mc (open symbols) or IL-23 mc (filled symbols), and flow cytometry was performed on LP cells from the proximal SI. (A) The absolute number of ILC3 (defined as CD45+ CD90+ CD11b- CD11c- F4/80- Gr-1- Ter119- B220- CD3e- RORγt+ cells; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182841#pone.0182841.s001" target="_blank">S1B Fig</a> for typical gating) is shown. (B) Top: representative staining shows protein expression at 24 and 48 hours post mc injection. Bottom: compiled staining intensities are depicted as geometric MFI at 72 hours post mc injection. (C) Representative staining shows cytokine expression three days and 2 weeks post mc injection (left) along with compiled data (right). (D) Expression of cell death associated receptors by ILC3 is shown three days post mc injection. Representative and compiled staining intensities are shown. Scatter plots show means ± SEM for all mice from one of 2–4 similar experiments, 4–5 mice per group, with each symbol representative of a single mouse.</p

TNFα promotes cytokine production by ILC3 and ILC3²³ formation <i>in vivo</i>, and synergizes with IL-23 to directly activate ILC3 <i>in vitro</i>.

<p>(A-C) Mice were injected with sham mc (open symbols) or IL-23 mc (filled symbols), with some groups receiving anti-TNFα (or PBS) as indicated. (A) SI length 2 weeks post mc injection (left), and cytokine secretion from SI explants harvested 3 days post mc injection (right) are shown. (B) Flow cytometry was performed on LP cells from the proximal SI three days post mc injection. Absolute numbers of cytokine producing lymphocytes are shown. (C) Flow cytometry was performed on LP cells from the proximal SI 2 weeks post mc injection. Top: absolute numbers are shown. Bottom: representative staining shows the frequency of ILC3²³ amongst CCR6- NCR- ILC3. (D-E) FACS purified ILCs enriched for ILC3 (~90% RORγt+) were sorted from the proximal SI LP of RAG KO mice and cultured overnight with the indicated cytokines prior to flow cytometry. (D) Protein expression by ILC3 is shown for triplicate wells with staining intensity depicted as geometric MFI (top), and representative IL-7R and T-bet staining is shown for total ILC3 (bottom). (E) Cytokine expression by ILC3 is shown for triplicate wells treated with protein inhibitors for the last 3 hours of culture. Scatter plots show means ± SEM for all mice from one of 2–3 similar experiments, 4–5 mice per group, with each symbol representative of a single mouse. Bar graphs show means ± SEM for triplicate wells from one of two similar experiments.</p

A New Approach for the Study of Lung Smooth Muscle Phenotypes and Its Application in a Murine Model of Allergic Airway Inflammation

<div><p>Phenotypes of lung smooth muscle cells in health and disease are poorly characterized. This is due, in part, to a lack of methodologies that allow for the independent and direct isolation of bronchial smooth muscle cells (BSMCs) and vascular smooth muscle cells (VSMCs) from the lung. In this paper, we describe the development of a bi-fluorescent mouse that permits purification of these two cell populations by cell sorting. By subjecting this mouse to an acute allergen based-model of airway inflammation that exhibits many features of asthma, we utilized this tool to characterize the phenotype of so-called asthmatic BSMCs. First, we examined the biophysical properties of single BSMCs from allergen sensitized mice and found increases in basal tone and cell size that were sustained <i>ex vivo</i>. We then generated for the first time, a comprehensive characterization of the global gene expression changes in BSMCs isolated from the bi-fluorescent mice with allergic airway inflammation. Using statistical methods and pathway analysis, we identified a number of differentially expressed mRNAs in BSMCs from allergen sensitized mice that code for key candidate proteins underlying changes in matrix formation, contractility, and immune responses. Ultimately, this tool will provide direction and guidance for the logical development of new markers and approaches for studying human lung smooth muscle.</p></div

Separation of BSMCs and VSMCs from the lung using the bi-fluorescent <i>αSMA-hrGFP;NG2-DsRed</i> mouse.

<p>(<b>A</b>) Expression pattern of hrGFP and DsRed in BSMCs and VSMCs in the lung of an <i>αSMA-hrGFP;NG2-DsRed</i> mouse. Both BSMCs and VSMCs are hrGFP⁺. BSMCs are DsRed⁻ whereas VSMCs are DsRed⁺. Asterisk (*) indicates the blood vessel in the lung the airway. Arrows (>) indicate the airway. Scale bar: 20 µm. (<b>B</b>) Algorithm for bronchial and vascular smooth muscle cell identification by flow cytometry. CD31⁺ endothelial cells and CD45⁺ immune cells were separated from dissociated lung preparation (left panel) followed by evaluation of hrGFP and DsRed distribution within CD31⁻CD45⁻ population (middle panel). CD31⁻CD45⁻hrGFP⁺DsRed⁻ population corresponds to BSMCs (pointed by an arrow). CD45⁻CD31⁻hrGFP⁺DsRed⁺ population is enriched in vascular smooth muscle cells (VSMCs). (<b>C</b>) Relative <i>Notch3</i> mRNA expression in isolated singly hrGFP⁺ cells and doubly hrGFP⁺DsRed⁺ cells from lung cell preparation as determined by qPCR followed by normalization to 18S. Data show mean ± SD and are representative of three independent experiments. ***p<0.001.</p

Measurement of changes in physical properties of individual BSMCs from OVA sensitized mice.

<p>BSMCs isolated from PBS-sensitized control and OVA sensitized <i>αSMA-hrGFP;NG2-DsRed</i> mice were allowed to attach to collagen I gel in culture for 72 hrs before assays. (<b>A</b>) Images of hrGFP⁺ BSMCs from PBS control and OVA sensitized mice (insert) and their traction maps on collagen I gels. (<b>B</b>) Contractile moment measurement of individual BSMCs from control and sensitized mice based on their traction maps. The line represents the mean of individual measurements of control (n = 20) and acute asthmatic (n = 22) cells. (<b>C</b>) Measurement of cell size of BSMCs from PBS sensitized control and OVA sensitized mice. The line represents the mean of individual measurements. **p<0.008.</p

Gene sets misregulated in BSMCs from OVA sensitized mice as compared to PBS sensitized control.

<p>The five most significantly down- and up-regulated gene sets among the MSigDB canonical pathways, Gene Ontology categories, and motif gene sets are shown, ordered in each subsection by CAMERA <i>p</i>-value. No motif gene set was up-regulated with p<0.05, so these are excluded. The size column gives the number of genes in the gene set after restricting to members with mouse orthologs represented by probe sets on the arrays.</p