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

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

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