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

Relationship between sCD4 sensitivity, CD4-Ig binding, infection of CD4^low cells and sCD4-induced gp120 release of BR24 viruses.

<p>(A) The relationship between sgp120 binding to CD4-Ig, sCD4 sensitivity, infection of RC49 cells and primary macrophages (mΦ) of BR24 dervied viruses is illustrated. Values above the bars indicate fold increase in sCD4 sensitivity of BR24 viruses compared to viruses in the SHIV_SF162P3N inoculum (P3N). (B) Extent of sCD4-induced gp120 from surface of 293T cells transiently expressing BR24-derived envelope glycoproteins. Percentage difference in gp120 release in the presence of sCD4 relative to that in the absence of sCD4 is shown. The data are the means and standard deviations of two independent experiments. The vertical dashed line in (A) and (B) indicates the time of coreceptor switching, and the dotted area highlights the time when the relationship between sCD4 sensitivity, sgp120 binding to CD4-Ig and infection of CD4^low cells dissipates.</p

Entry efficiency, PSC-RANTES and sCD4 sensitivity of R5 viruses evolving over time in BR24.

<p>Entry of luciferase reporter viruses expressing CCR5-using envelopes into TZM-bl cells expressed as relative light unit (RLU)(A), and susceptibility of the reporter viruses to neutralization with PSC-RANTES (B) and sCD4 (C) were determined. The dashed vertical line indicates time of tropism switch in BR24 (20 wpi), and the numbers in the brackets indicate the number of clones analyzed at each time point. Envelope clones from the SHIV_SF162P3N inoculum (P3N) were also included in the characterization for comparison. Absolute CD4+ T-cell count in the animal over the course of infection is shown in (C) for reference, and values above the bars indicate fold increase in sCD4 sensitivity relative to that of the w2 viruses. * P<0.05 (Mann-Whitney <i>U</i> test). Data are representative of 2–3 independent experiments (error bars, s.d.).</p

Entry efficiency, PSC-RANTES and sCD4 sensitivity of R5 viruses evolving over time in CA28.

<p>Entry of luciferase reporter viruses expressing CCR5-using envelopes into TZM-bl cells (A), and susceptibility of the reporter viruses to neutralization with PSC-RANTES (B) and sCD4 (C) were determined. The solid and dashed vertical lines indicate the two switch events in CA28 leading to the emergence of distinct dual-tropic and X4 viruses, respectively. The numbers in the brackets denote the number of envelope clones analyzed at each time point. Absolute CD4+ T-cell count in the animal over the course of infection is shown in (C), and values above the bars indicate fold increase in sCD4 sensitivity of CA28 viruses compared to viruses in the SHIV_SF162P3N inoculum (P3N). *<i>P</i><0.05 (Mann-Whitney <i>U</i> test). Data are representative of at least three independent experiments (error bars, s.d.).</p

Changes in neutralization sensitivity of R5 viruses evolving over time in macaque BR24.

<p>Susceptibility of BR24 R5 pseudoviruses to neutralization with b12, 447-52D and T20 was determined, with sensitivity of variants from the inoculating virus SHIV_SF162P3N (P3N) shown for reference. The vertical dashed line indicates the time of coreceptor switching, and the dotted area designates the period of marked envelope conformational changes. Data are representative of at least two independent experiments (error bars, s.d.). * above the bars indicate IC₅₀ values that are statistically different between the acute (w2) and the evolving R5 viruses.</p

SHIV-infected macrophages identified with double-label SIVnef and Iba-1 immunohistochemistry.

<p>Tissue macrophages are the primary SHIV infected cells at end stage disease in BR24 (A) and CA28 (B). Double-labeled immunohistochemical staining for SIVnef (brown) and the macrophage marker lba-1 (red) was performed. Arrows mark representative double-positive cells.</p