Tissue-specific pathways extrude activated ILC2s to disseminate type 2 immunity

Abstract

Group 2 innate lymphoid cells (ILC2s) are tissue-resident cells prominent at barrier sites. Although precursors are found in blood, mature ILC2s can enter the circulation after small intestinal perturbation by migratory helminths and move to distant tissues to influence the local reparative response. Using fate-mapping and methods to bypass the lung or intestinal phases of Nippostrongylus brasiliensis infection, we show that blood ILC2s comprise heterogeneous populations derived from distinct tissues that are dependent on alarmins matched to the receptor profile of the specific tissue ILC2s. Activation of local ILC2s by tissue-specific alarmins induced their proliferation, lymph node migration, and blood dissemination, thus systemically distributing type 2 cytokines. These studies uncover a possible mechanism by which local innate responses transition to systemic type 2 responses by extrusion of activated sentinel ILC2s from tissue into the circulation.

Figure 1.

Circulating ILC2s phenotypically differ during the course of N. brasiliensis infection. (A–D) Arg1^YargIl5^Red5Il13^Sm13 (YRS) triple-reporter mice were infected with N. brasiliensis and analyzed at the indicated time points. Uninfected mice (Naive) were examined as controls. (A) Expression of KLRG1 and IL-5 by cells gated on CD45⁺Lin⁻ cells isolated from peripheral blood (left) or small intestine lamina propria and lung (right). Expression of ST2 and IL-17RB or Arg1 by ILC2s gated as indicated in the top panels. (B and C) Frequencies of KLRG1⁺IL-5 (Red5)⁺ ILC2s (B) and percentages of IL-13 reporter–positive ILC2s (C) in peripheral blood. DL, detection limit. (D) Flow cytometry plots of blood (left) or lung (right) ILC2s gated on CD45⁺Lin⁻IL-5⁺ cells from d5 or d12, highlighting activation as assessed by IL-13 (Sm13) expression and the expression of ST2, Arg1 (Yarg), and IL-17RB. (E) Serum IL-13 levels from WT C57BL/6J mice infected with N. brasiliensis treated with or without FTY720 (FTY) and analyzed at the indicated time points. The x axis represents days after infection. Data displayed as mean ± SEM. u.i., uninfected. Data are from one experiment representative of at least two independent experiments (A, D, and E) or pooled from multiple independent experiments (B and C). *, P < 0.05.

FigureS1.

ILC2 gating strategy for experiments using reporter and B6 WT mice. (A and B) Gating strategy for experiments using Arg1^YargIl5^Red5Il13^Sm13 (YRS) triple-reporter mice (A) or C57BL/6J mice (B). Data are representative plots for the indicated organs at d5 after N. brasiliensis. Data shown are representative of more than three independent experiments with n ≥ 3 individual mice per group. PE, phycoerythrin.

Figure S2.

Circulating ILC2s phenotypically differ during the course of N. brasiliensis infection. (A) Arg1^YargIl5^Red5Il13^Sm13 (YRS) triple-reporter mice were infected with N. brasiliensis, and blood (left panels) and lungs (right panels) were analyzed on d5 after infection for IL-5 (Red5) and IL-13 (Sm13) expression in ILCs (gated on CD45⁺Lin⁻) and CD4 T cells (gated on CD45⁺CD4⁺). (B) Mice were infected with N. brasiliensis in the presence or absence of FTY720 (1 mg/kg), and blood (top panels) and lung (bottom panels) ILC2s (gated on CD45⁺Lin⁻GATA3⁺) were examined on d5 or d12 for IL-13 (Sm13) expression and Ki-67 labeling. (C and D) Mice were infected with N. brasiliensis and were i.v. injected with 3 µg of APC-Cy7–labeled CD45.2 antibody on d5 and euthanized 3 min later. (C) Top panels indicate the intravascular (IV) or tissue-resident (Tissue) ILC2s gated on CD45⁺Lin⁻IL5 (Red5)⁺ from the blood, lung, or mLNs (mesLN). Middle and bottom panels show the expression of ST2 and IL-17RB (middle panels) or ST2 and CD69 (bottom panels) from the intravascular or tissue-resident fraction from tissues as indicated. (D) Percentage of tissue and blood (IV) ILC2s in the lung on d5. Data are from one experiment representative of at least two independent experiments. *, P < 0.05; **, P < 0.005. ns, no significant difference.

Figure 2.

Circulating ILC2s are dependent on tissue extrusion. (A–H) C57BL/6J mice were infected with N. brasiliensis, injected i.p. with saline or FTY720 (1 mg/kg daily from day of infection), and analyzed at the indicated time points. (A) Gating of ILC2s (pregated on live CD45⁺Lin⁻) from blood or lung. (B) Total number of ILC2s in the blood (left) or lung (right) at d5. (C–E) Flow cytometry plots showing the expression of ST2 and IL-17RB on blood and lung ILC2s from d5 (C) and d12 (E) and quantification of the percentage of IL-17RB⁺ST2⁻ ILC2s in the blood and lung on d5 (D). (F) Total number of ILC2s in the blood (left) or lung (right) at d12. Data are representative of two independent experiments with n ≥ 3 individual mice per group. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.0001. N.b., N. brasiliensis;ns, no significant difference.

Figure 3.

Differential cytokine-dependent tissue ILC2 activation correlates with appearance of ILC2s. (A–H) WT, Il25^−/−, or Il1rl1^−/− (ST2^−/−) mice on Arg1^YargIl5^Red5Il13^Sm13 triple-reporter strain were infected with N. brasiliensis and analyzed on d5 and d12. (A) Quantification of KLRG1⁺IL-5 (Red5)⁺ blood ILC2s as a percentage of CD45⁺ cells on d5. DL, detection limit. (B) Percentages of ST2⁺ cells among KLRG1⁺IL-5⁺ blood ILC2s on d5. (C) Quantification of KLRG1⁺IL-5⁺ blood ILC2s as percentage of CD45⁺ cells on d12. (D) Expression of ST2 and IL-17RB by ILC2s gated on Lin⁻CD45⁺IL-5⁺ cells isolated from the lung on d5 and d12. (E) Expression of Arg1 and IL-17RB by ILC2s gated on Lin⁻CD45⁺IL-5⁺ cells isolated from the lung on d5 and d12. (F and G) Quantification of total IL-5⁺Arg1⁺ and Arg1⁻ ILC2s in the lung (F) and their expression of IL-13 (Sm13; G) on d5 and d12. (H) Frequencies of IL-5⁺Arg1⁺ and IL-5⁺Arg1⁻ ILC2s in the mLN. Data are pooled from multiple independent experiments and displayed as mean ± SEM of 11–14 individual mice per group and time point (A–C, F, and H) or from one experiment representative of at least three independent experiments (D, E, and G). **, P < 0.005; ***, P < 0.0005. ns, no significant difference.

Figure 4.

Circulating ILC2s can be traced back to the tissue. (A–H) Arg1^RFP-CreERT2R26R^YFP mice were tamoxifen treated on postnatal day (p)10, p11, and p12 according to the schedule in A. 10 wk later, mice were infected with N. brasiliensis (N.b.), and the ratios of fate-mapped and non–fate-mapped cells were analyzed on d5, d13, and d22 after infection. (B) Frequencies of fate-mapped (black bars) and non–fate-mapped (gray bars) ILC2s in the lung, small intestine lamina propria (SI), or BM from naive mice or d22 after infection with N. brasiliensis. (C) Total numbers (left) and frequencies (right) of fate-mapped ILC2s (CD45⁺Lin⁻KLRG1⁺IL-17RB⁺ and/or ST2⁺) in the lung d5, d13, or d22 after infection. (D) Frequencies of fate-mapped KLRG1⁺IL-17RB⁺ ILC2s from the small intestine. (E) Frequencies of fate-mapped KLRG1⁺ and KLRG1⁻ ILC2s (CD45⁺Lin⁻IL-7Rα⁺ST2⁺Arg1-RFP⁺) in BM from naive mice on d5, d13, or d22 after infection. (F) Percentages of fate-mapped ILC2s (CD45⁺Lin⁻KLRG1⁺IL-17RB⁺ and/or ST2⁺) in the blood at d5 or d13 after infection. Dotted lines represent the mean frequency of fate-mapped percentage of ILC2s for indicated tissues of uninfected mice. (G) Frequencies of fate-mapped KLRG1⁺IL-17RB⁺ ILC2s from mLN. (H) Frequencies of fate-mapped cells among gated ILC2 subsets in the lung on d5 and d13. Data are from one experiment representative of at least two independent experiments displayed as mean ± SEM of four or five individual mice per group and time point. **, P < 0.005; ***, P < 0.0005. ns, no significant difference.

Figure S3.

Circulating ILC2s can be traced back to the tissue. (A–C) Mice were tamoxifen treated on p10, p11, and p12 as in Fig. 4. 10 wk later, mice were analyzed naive or after infection with N. brasiliensis at the indicated time points. (A) Expression of CD25 and Arg1-RFP or R26R-YFP by Id2-GFP⁺IL-7Rα⁺ cells gated on CD45⁺Lin⁻ cells isolated from the BM of naive Id2^GFPArg1^RFP-CreERT2R26R^YFP reporter mice. (B) Expression of ST2 and R26R-YFP by Id2-GFP⁺IL-7Rα⁺ cells gated on CD45⁺Lin⁻ cells isolated from the BM of Id2^GFPArg1^RFP-CreERT2R26R^YFP reporter mice on d5, d13, and d22 after infection with N. brasiliensis. (C) Gating strategy that was used to determine the fate-mapping frequency in ILC2s (CD45⁺Lin⁻KLRG1⁺IL-17RB⁺ and/or ST2⁺) from the blood of Arg1^RFP-CreERT2R26R^YFP reporter mice on d5 and d13 after infection with N. brasiliensis. Data are from one experiment are representative of at least two independent experiments.

Figure 5.

Phenotypically distinct ILC2s arise by niche extrusion during local tissue perturbation. (A) Schematic of the infection protocol to isolate lung or small intestine exposure to N. brasiliensis. Groups of Arg1^YargIl5^Red5Il13^Sm13 (YRS) triple-reporter mice with individual treatments are color-coded. Black, standard s.c. infection (s.c.); red, s.c. infection followed by oral pyrantel pamoate treatment d1–d12 (s.c. + pyrantel); blue, oral gavage of adult N. brasiliensis worms (o.g.). Timeline relative to s.c. (B) Frequency of KLRG1⁺IL-5 (Red5)⁺ blood ILC2s. DL, detection limit. (C) Expression of ST2 and IL-17RB or Arg1 by IL-5⁺ cells gated on Lin⁻CD45⁺ cells isolated from peripheral blood on d5 (left panels) and d12 (right panels). (D and E) Quantification of total IL-5⁺ ILC2s in the lung (D) and their expression of IL-13 (Sm13; E) analyzed on d12. (F and G) Frequency (F) or absolute number (G) of lung IL-5⁺ CD4⁺ T cells was analyzed on d12. (H) Expression of ST2 and IL-17RB (top panels) or ST2 and IL-13 (Sm13; bottom panels) by IL-5⁺ cells gated on Lin⁻CD45⁺ cells isolated from d5 lung of naive (uninfected) or o.g. adult N. brasiliensis–infected mice as described in A. (I) Serum IL-13 levels from WT C57BL/6J mice infected with N. brasiliensis as described in A and displayed as mean ± SEM. n.d., not detected. Data are pooled from two independent experiments and 9–12 individual mice per group (B) or from one experiment representative of two independent experiments (C–I). **, P < 0.005; ***, P < 0.0005. ns, no significant difference; N.b., N. brasiliensis.