S1P-dependent interorgan trafficking of group 2 innate lymphoid cells supports host defense

Abstract

Innate lymphoid cells (ILCs) are innate counterparts of adaptive T lymphocytes, contributing to host defense, tissue repair, metabolic homeostasis, and inflammatory diseases. ILCs have been considered to be tissue-resident cells, but whether ILCs move between tissue sites during infection has been unclear. We show here that interleukin-25- or helminth-induced inflammatory ILC2s are circulating cells that arise from resting ILC2s residing in intestinal lamina propria. They migrate to diverse tissues based on sphingosine 1-phosphate (S1P)-mediated chemotaxis that promotes lymphatic entry, blood circulation, and accumulation in peripheral sites, including the lung, where they contribute to anti-helminth defense and tissue repair. This ILC2 expansion and migration is a behavioral parallel to the antigen-driven proliferation and migration of adaptive lymphocytes to effector sites and indicates that ILCs complement adaptive immunity by providing both local and distant tissue protection during infection.

Fig. 1.. IL-25– or helminthic infection–induced inflammatory ILC2s are circulating cells that differ from tissue-resident ILCs.

(A to C) CD45.1⁺ and CD45.2⁺ mice were surgically connected to generate parabiotic partners. Percentages of host-derived cells were analyzed among ILC2s and ILC3s 2 to 3 weeks, 2 months, and 6 to 8 months after surgery. ILC2s were gated as lineage-negative (Lin⁻) CD127⁺ GATA-3^hi, and ILC3s were gated as Lin⁻ CD127⁺ RORγt⁺. MLNs, mesenteric lymph nodes. (D) B6 mice were treated with IL-25 intranasally or intraperitoneally (i.p.) daily for 3 days. Lung leukocytes were then analyzed by flow cytometry. Cells were gated on CD45.2⁺ Lin⁻ CD127⁺. (E) CD45.1⁺ CD45.2⁺ mice were treated with IL-25 i.p. daily for 3 days. Mice were intravenously injected with phycoerythrin-labeled antibodies against CD45.1 and euthanized 5 minutes later for analysis of CD45.2 on Lin⁻ CD127⁺ ST2⁺ KLRG1^int naturally lung-residing ILC2s (nILC2s), Lin⁻ CD127⁺ ST2⁻ KLRG1^hi inflammatory ILC2s (iILC2s), and CD4⁺ Tcells in the lungs without perfusion. (F and G) Two months after surgery, the CD45.1⁺ partner of each parabiotic pair was treated i.p. with IL-25 (F) or IL-33 (G) daily for 3 days. Cells in the lungs were then analyzed. nILC2s were gated as Lin⁻ CD127⁺ Thy1^hi KLRG1^int, and iILC2s were gated as Lin⁻ CD127⁺ Thy1^lo KLRG1^hi. (H) Two months after surgery, both mice in each parabiotic pair were inoculated with ~200 infective N. brasiliensis larvae (L3). Cells in the lungs were analyzed on day 5 postinoculation. Means ± SEM from six to eight mice at each time point in (A) to (C) and from three mice in (D). ****P ≤ 0.0001; NS, not significant; unpaired two-tailed t test. Results are representative of at least two independent experiments.

Fig. 2.. Intestinal ILC2s are the immediate source of iILC2s.

(A) Total leukocytes from lung, bone marrow, or siLP of CD45.1⁺ Rag1^−/− mice were labeled with carboxyfluorescein succinimidyl ester (CFSE) and transferred into CD45.2⁺ Rag1^−/− mice; this was followed by a 3-day treatment with IL-25 injected i.p. Cells in the lungs were analyzed for CD45.1⁺ donor cells. Blue line, Lin⁻ KLRG1^hi iILC2s; gray shaded area, CD45.1⁺ cells labeled with CFSE before transfer. (B) siLP leukocytes were divided into the three indicated groups by cell sorting and subjected to a similar transfer experiment to that described in (A). Left, cells were gated on CD45.1⁺ Lin⁻; right, CD45.1⁺ iILC2 numbers in the lungs. (C) Equal numbers of lung nILC2, siLP ILC2, and bone marrow (BM) ILC2 progenitors were sorted and subjected to a similar transfer experiment to that described in (A). (D) Comparison of surface markers on lung iILC2s in IL-25–treated mice and lung nILC2s and intestinal ILC2s in naïve mice by flow cytometry. (E) Principal component analysis of the transcriptomes of different ILC2 populations identified by RNA-seq. The analysis is based on log₁₀[transcripts per million (TPM) + 1] of 53 immune-related genes. (F) Scatter plot comparing the gene expression patterns from RNA-seq between activated lung nILC2s and lung iILC2s. The x and y axes show the log₁₀(TPM + 1) values for each sample. Only genes with at least a twofold difference were plotted. Means ± SEM from three mice in (A) to (C). Data are representative of two independent experiments in (A) to (D).

Fig. 3.. Intestinal ILC2s proliferate and enter lymphatics in response to IL-25.

(A) Immunofluorescence staining of ilea from B6 mice 0, 36, and 60 hours (h) after treatment with IL-25. The lower panels are magnified views of the boxed areas in the upper panels. (B) Quantification of Ki-67⁺ ILC2s in the small intestine. (C) Comparison of CD69 expression on siLP ILC2s, lung iILC2s, and MLN iILC2s by flow cytometry. (D) Immunofluorescence staining of ilea from mice 0 and 60 hours after treatment with IL-25. Arrows, KLRG1⁺ ILC2s within a lymphatic vessel. (E) iILC2s in peripheral blood or lungs were analyzed from B6 mice 0, 36, and 60 hours after treatment with IL-25. Images are representative of three gut sections from two mice in each group. Data are representative of two independent experiments in (C) and (E). Means ± SEM; n = 6 to 9 and 3 for each group in (B) and (E), respectively. ***P ≤ 0.001; unpaired two-tailed t test.

Fig. 4.. Intestinally derived iILC2s migrate and provide protection to the lung in a S1P-dependent manner.

(A) S1P receptor expression by the indicated cell populations was assessed by real-time polymerase chain reaction. Intestinal ILC2s and ILC3s and MLN CD4 T cells were obtained from naïve mice, whereas iILC2s in the lung or MLNs were from IL-25–treated mice. (B) Immunofluorescence staining of ilea from mice that were untreated, treated with IL-25, or treated with IL-25 plus FTY720 for 3 days. (C) Quantification of Ki-67⁺ ILC2s in (B). (D) B6 mice were treated with IL-25 or with IL-25 plus FTY720 i.p. for 3 days, and the iILC2s in indicated tissues were analyzed by flow cytometry. (E) Worm burden on day 14 in the small intestine of the mice infected with ~300 N. brasiliensis L3. IL-25 treatment was given every other day in the third group. UD, undetected. (F) Rag1^−/− mice were inoculated with ~500 N. brasiliensis L3 (Nb), and FTY720 treatment started on day 3 and continued daily until day 10, in two groups. In the transfer group, ~3 × 10⁶ iILC2s sorted from IL-25–treated mice were transferred intravenously into each animal on the same day as worm inoculation. Death and survival were monitored until day 13, and the difference between two groups was determined by the log-rank (Mantel-Cox) test. (G) Histology of lung sections from Rag1^−/− mice inoculated with ~300 N. brasiliensis L3. Arrows, worm larvae; asterisk, tissue destruction. (H) Amphiregulin mRNA levels in nILC2s and iILC2s, which were sorted from lungs on day 5 postinoculation with N. brasiliensis. Means ± SEM; n =3 in (A), (D), (E), and (H) and 4 to 6 in (C). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; unpaired two-tailed t test. Data in (A), (D), and (E) are representative of three experiments; data in (F) are the summary of two experiments (n = 10). Images are representative of three sections from two mice of each group in (B) and (G).