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. 2016 Dec;17(12):1388-1396.
doi: 10.1038/ni.3566. Epub 2016 Oct 31.

Fibroblastic reticular cells regulate intestinal inflammation via IL-15-mediated control of group 1 ILCs

Cristina Gil-Cruz  1 , Christian Perez-Shibayama  1 , Lucas Onder  1 , Qian Chai  1 , Jovana Cupovic  1 , Hung-Wei Cheng  1 , Mario Novkovic  1 , Philipp A Lang  2 , Markus B Geuking  3 , Kathy D McCoy  3 , Shinya Abe  4   5 , Guangwei Cui  4 , Koichi Ikuta  4 , Elke Scandella  1 , Burkhard Ludewig  1
Affiliations

Fibroblastic reticular cells regulate intestinal inflammation via IL-15-mediated control of group 1 ILCs

Cristina Gil-Cruz et al. Nat Immunol. 2016 Dec.

Abstract

Fibroblastic reticular cells (FRCs) of secondary lymphoid organs form distinct niches for interaction with hematopoietic cells. We found here that production of the cytokine IL-15 by FRCs was essential for the maintenance of group 1 innate lymphoid cells (ILCs) in Peyer's patches and mesenteric lymph nodes. Moreover, FRC-specific ablation of the innate immunological sensing adaptor MyD88 unleashed IL-15 production by FRCs during infection with an enteropathogenic virus, which led to hyperactivation of group 1 ILCs and substantially altered the differentiation of helper T cells. Accelerated clearance of virus by group 1 ILCs precipitated severe intestinal inflammatory disease with commensal dysbiosis, loss of intestinal barrier function and diminished resistance to colonization. In sum, FRCs act as an 'on-demand' immunological 'rheostat' by restraining activation of group 1 ILCs and thereby preventing immunopathological damage in the intestine.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Effect of FRC-specific MyD88 ablation on PP and mLN organization.
(a,b) Confocal microscopy of PPs from 8- to 10-week -old Ccl19EYFP mice (a) or Ccl19EYFPMyd88-cKO mice (b) showing expression of the EYFP reporter, and the distribution of CD4+ T cells and B220+ B cells in PPs (left) and the PDPN staining of the interfollicular region of FRCs (right). Scale bars, 100 μm (left) or 20 μm (right). (c) Ccl19-Cre transgene activity (assessed as EYFP fluorescence) in CD45−EPCAM−Ter119−EYFP+ stromal cells from Ccl19EYFP and Ccl19EYFPMyd88-cKO mice (above plots, right), analyzed by flow cytometry. Numbers in bottom left quadrants (right plot group) indicate percent PDPN+CD31− FRCs (mean ± s.e.m.). (d) Absolute number of Ccl19-Cre-transgene-expressing (EYFP+) CD45−EPCAM−Ter119− stromal cells in mice as in c, assessed by flow cytometry. (e,f) Flow cytometry of CD45−EPCAM−Ter119−EYFP+ stromal cells from mice as in c. Numbers in plots (e) indicate percent CD35+ follicular dendritic cells among PDPN+EYFP+ cells; numbers in top right quadrants (f) indicate percent CD157+MAdCAM1+ marginal reticular cells among PDPN+EYFP+ cells. (g) Expression of canonical FRC markers (horizontal axes) on CD45−PDPN+EYFP+ cells in PPs and mLNs of mice as in c, assessed by flow cytometry. Isotype, isotype-matched control antibody. NS, not significant (P > 0.05) (Student's t-test). Data are from two experiments with one mouse representative of four mice (a,b), are pooled from two independent experiments with n = 6 mice per genotype (c,d; mean + s.e.m. in d) or are from two experiments (e) or two experiments with one mouse representative of eight mice (f,g). Source data
Figure 2
Figure 2. Population expansion and reactivity of group 1 ILCs in the absence of MyD88 signaling in FRCs.
(a,b) Viral titers in the PPs and mLNs of Myd88-cKO mice, their Cre-negative littermates (Ctrl), Tlr7−/− mice or MyD88−/− mice (key) at day 3 (a) or day 6 (b) after oral infection with 5 × 104 plaque-forming units (PFU) of MHV. (c) Flow cytometry of the ILC1 subset in in PPs of Myd88-sufficient (Ctrl) and Myd88-cKO mice on day 3 after infection as in a, gated from CD45+CD3−CD19−GR1− cells. Numbers adjacent to outlined areas indicate percent NKp46+NK1.1+ cells (mean ± s.e.m.). (d) Quantification of results in c. (e) IFN-γ-producing cells in the CD45+CD3−NK1.1+ population in mice as in c, assessed by flow cytometry. (f–h) Confocal microscopy of EYFP fluorescence and the localization of NK1.1+ cells in the PPs of a Ccl19EYFP mouse at day 3 after infection with MHV (f), and enlargement of the interfollicular region (outlined area in f) to identify Ccl19-Cre-transgene-expressing cells in close proximity to NK1.1+ cells in Ccl19EYFP mice (g) and Ccl19EYFPMyd88-cKO mice (h) at day 3 after infection with MHV. Scale bars, 100 μm (f) or 10 μm (g,h). (i) Flow cytometry of CD45+CD3−CD19−GR1− cells from the PPs of Myd88-sufficient (Ctrl) and Myd88-cKO mice at day 3 after infection with MHV. Numbers adjacent to outlined areas indicate percent NK1.1+IL-17Rα− NK cells (left) or NK1.1+IL-17Rα+ cells (ILC1) (right) (left column); GATA3+NK1.1− cells (ILC2) (middle column); or IL-17Rα+RORγt+ cells (ILC3) (right column). (j) Frequency of ILC subsets as in i. (k) Viral titers in the PPs and mLNs of Myd88-sufficient (Ctrl) and Myd88-cKO mice treated with NK1.1-depleting antibody (+ α-NK1.1) or isotype-matched control antibody (+ isotype), assessed at day 6 after oral infection with MHV. *P < 0.05, **P < 0.01 and ***P < 0.001 (Student's t-test (a–e,j) or one-way analysis of variance (ANOVA) with Tukey's post-test (k)). Data are pooled from four independent experiments (a,b; mean + s.e.m.) or three experiments (c–e,k; mean + s.e.m. in d,e,k), are from two experiments with one mouse representative of three mice (f–i) or are representative of two experiments (j; mean + s.e.m.). Source data
Figure 3
Figure 3. MyD88-dependent regulation of IL-15 in PP FRCs.
(a) Flow cytometry of fibroblastic stromal cells isolated from the PPs of C57BL/6 Myd88+/+ or Myd88−/− mice and cultured in vitro for 7 d. Numbers indicate percent CD31−PDPN+ cells (bottom right quadrant). (b) Production of the chemokine CCL2 and the cytokines IL-6 and IL-15 by CD45−CD31− stromal cells from the PPs of Myd88+/+ (Ctrl) or Myd88−/− mice without R848 or at 24 h after exposure to R848 (key). (c) Expression of IL-15Rα on CD45−PDPN+ cells after exposure to medium, R848 (100 ng/ml), single-stranded RNA (ssRNA) (100 ng/ml) or IL-1β (10 ng/ml) (above lines), assessed by flow cytometry. Numbers, mean fluorescence intensity of IL-15Rα (± s.e.m.). Top line, staining with isotype-matched control antibody. (d,e) Quantitative RT-PCR analysis of Il15 mRNA in CD45− PP stromal cells (d) or CD45−EYFP+PDPN+ PP FRCs (e) sorted by flow cytometry from Ccl19EYFP and Ccl19EYFPMyd88-cKO mice before infection (Naive) and on day 3 after infection with MHV. (f) Binding of isotype-matched control antibody (Isotype) on CD44hi cells from a Ccl19EYFP mouse (left), and expression of membrane-bound IL-15 (mIL-15) (middle) and IL-15Rα (right) on CD44hi cells from Ccl19EYFP and Ccl19EYFPMyd88-cKO mice on day 3 after infection with MHV, assessed by flow cytometry. Numbers adjacent to outlined areas indicate percent mIL-15+CD44+ cells (middle) or IL-15Rα+CD44+ cells (right) (mean ± s.e.m.). (g) Expression of membrane-bound IL-15 on PDPN+ FRCs, assessed with back-gating (red). Numbers adjacent to outlined areas indicate percent CD44+PDPN+ cells among mIL15+ cells (± s.e.m.). *P < 0.05, **P < 0.01 and ***P < 0.001 (one- way ANOVA with Tukey's post-test (b,c) or Student's t-test (d–g)). Data are from one experiment representative of four independent experiments (a) or two experiments (b; mean + s.e.m.) or are pooled from three independent experiments (c) or two independent experiments with n ≥ 4 mice per group (d–g; mean + s.e.m. in d,e). Source data
Figure 4
Figure 4. FRC-dependent population expansion and activation of group 1 ILCs and NK cells.
(a,b) Frequency (a) and absolute number (b) of group 1 ILCs among CD45+CD3−CD19−GR1− cells from Myd88-cKO mice and their Cre-negative littermates (Ctrl) treated with neutralizing antibody to IL-15 (α-IL-15) or isotype-matched control antibody (isotype) before and during infection with MHV, assessed on day 3 after infection by staining for NK1.1, NKp46 and IL-7Rα. Numbers adjacent to outlined areas (a) indicate percent NK1.1+NKp46+ cells (mean ± s.e.m.). (c) Quantification of RORγt-expressing group 3 ILCs in mice as in a,b. (d) Expression of CD122 (IL-15Rβ) on IL-7Rα+EOMES− group 1 ILCs and IL-7Rα−EOMES+ NK cells (brackets and arrows above plots) from the PPs of Myd88-cKO mice and their Cre-negative littermates (key), assessed before (Naive) and on day 3 after infection (MHV). Shaded curves, isotype-matched control antibody. (e) Viral titers in PPs and mLNs of mice as in a,b, assessed on day 6 after infection. *P < 0.05, **P < 0.01 and ***P < 0.001 (Student's t-test (a–c) or one-way ANOVA with Tukey's post-test (e)). Data are pooled from two independent experiments with n = 6 mice per group (a–c,e; mean + s.e.m. in b,c,e) or are from one experiment representative of two experiments with similar results (d). Source data
Figure 5
Figure 5. Activity of group 1 ILCs and NK cells in the absence of IL-15 expression in FRCs.
(a) Flow cytometry of CD45+CD3−CD19−GR1− cells from the PPs of Il15-cKO mice and their Il15-sufficient littermates (control (Ctrl)) to distinguish ILC subsets (above plots) through the use of various combinations of staining for NK1.1, NKp46, IL-7Rα and GATA3. Numbers adjacent to outlined areas or quadrants indicate percent cells in each subset (mean ± s.e.m.). (b) Absolute number of various ILC populations (horizontal axis) in the PPs of naive Il15-sufficient or Il15-cKO mice. (c,d) Flow cytometry of CD45+CD3−CD19−GR1−NK1.1+ cells in the PPs (c) and mLNs (d) of Il15-sufficient or Il15-cKO mice on day 3 after infection with MHV. Numbers adjacent to outlined areas (left) indicate percent NK1.1+NKp46+ cells; numbers in top right quadrants (right) indicate percent IFN-γ+ cells (mean ± s.e.m. for both). (e) Viral titers in the PPs and mLNs of Il15-sufficient or Il15-cKO mice at day 6 after infection with MHV. *P < 0.001 (Student's t-test). Data are pooled from two or three independent experiments with n = 5 mice per genotype (a–d; mean + s.e.m. in b) or are pooled from two independent experiments with n = 7–8 mice per group (e; mean + s.e.m.). Source data
Figure 6
Figure 6. Innate immunological signaling in FRCs regulates intestinal homeostasis.
(a,b) Frequency of IFN-γ-producing cells among CD4+ T cells obtained from the PPs (a) and mLNs (b) of Myd88-cKO mice and their Cre-negative littermates (Ctrl) (key) at day 10 after oral infection with MHV and then stimulated in vitro with phorbol ester PMA and ionomycin (PI) or the MHV peptide M133 (above plots). (c) Flow cytometry of cells from the PPs of Myd88-cKO mice and their Cre-negative littermates (above plots) at day 10 after oral infection with MHV. Numbers adjacent to outlined areas indicate percent Foxp3+CD4+ T cells (mean ± s.e.m.). (d,e) Weight change in Myd88-cKO mice and their Cre-negative littermates (key) at various times (horizontal axis) after oral infection with MHV, relative to initial weight (set as 100%). (e) Colon length of Myd88-cKO mice and their Cre-negative littermates (key) left uninfected (Naive) or after infection as in c. (f) Histopathological analysis of ileal sections from mice as in e. (g) Microscopy of ileal sections from mice as in e: arrowheads indicate muscular layer; arrows indicate villi. Scale bar, 100 μm. (h) Binding of serum IgG to microbiota (left) or E. coli (right) in mice as in e, as determined by flow cytometry. (i) Ileal microbiota composition (horizontal axis) in mice infected as in c, relative to that in their naive counterparts. (j) Bacterial load in the feces, spleen and mLNs of Myd88-cKO mice and their Cre-negative littermates (key) infected with 2 × 109 colony-forming units of C. rodentium on day 12 after infection with MHV, assessed at day 6 after bacterial infection. Each symbol (e,f,h) represents an individual mouse; small horizontal lines indicate the mean. *P < 0.05, **P < 0.01 and ***P < 0.001 (Student's t-test (a–c,e,j), two-way ANOVA with Bonferroni's post-test (d), Mann-Whitney U-test (f, i) or one-way ANOVA with Tukey's post analysis (h)). Data are pooled from three independent experiments with n = 6–8 mice per group (a,b; mean + s.e.m.) or two independent experiments with n = 6–9 mice per group (c–e,j; mean ± s.e.m. in d), are representative of two experiments with n = 5, 6 or 11 mice per group (f,h) or two experiments (g) or are from one experiment representative of two independent experiments with n = 6 mice per group (i; mean ± interquartile range). Source data
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