Nematode-Infected Mice Acquire Resistance to Subsequent Infection With Unrelated Nematode by Inducing Highly Responsive Group 2 Innate Lymphoid Cells in the Lung

Abstract

The immune responses against helminths have been investigated individually, and it is well-established that infected hosts develop an immunological memory to resist reinfection by the same pathogen. In contrast, it is poorly understood how the host immune system responds to subsequent infection by unrelated parasites after elimination of the first infection. We previously reported that infection of mice with Strongyloides venezuelensis induces the accumulation of group 2 innate lymphoid cells (ILC2s) in the lung. Here, we demonstrated that S. venezuelensis-experienced (Sv-exp) mice became significantly resistant against infection by Nippostrongylus brasiliensis. N. brasiliensis infection induced enhanced accumulation of ILC2s and eosinophils with increased expressions of mRNA for Th2 cytokines in the lungs of Sv-exp mice. The resistance was dependent on ILC2s, and eosinophils but not on CD4⁺ T cells. Furthermore, pulmonary ILC2s in Sv-exp mice acquired a highly responsive "trained" phenotype; in response to N. brasiliensis infection, they rapidly increased and produced IL-5 and IL-13, which in turn induced the early accumulation of eosinophils in the lungs. IL-33 was required for the accumulation of ILC2s and the resistance of mice against N. brasiliensis infection but insufficient for the induction of trained ILC2s. In conclusion, animals infected with one type of lung-migratory nematodes acquire a specific-antigen-independent resistance to another type of lung-migrating nematodes, providing animals with the capacity to protect against sequential infections with various lung-migratory nematodes.

Keywords: IL-33; eosinophils; innate immune memory; intestinal nematode; trained immunity.

Figure 1

Strongyloides venezuelensis-experienced mice develop an increased pulmonary inflammation and a resistance against Nippostrongylus brasiliensis infection. (A) Experimental workflow for sequential nematode infection. S. venezuelensis (Sv)-infected or uninfected mice were inoculated with 500 N. brasiliensis (Nb) L3 at day 28. B6; C57BL/6, WT; wild type, sac; sacrificed. (B) The number of lung stage N. brasiliensis larva. Two days after N. brasiliensis infection, migrating larva were isolated from the lungs and counted (n = 11). cont, control (uninfected at day 0). Pooled data from two independent experiments are shown (Mean ± SD). (C) The numbers of worms in the intestine were counted at indicated days (n = 5). (D) The numbers of eggs per gram feces (EPG) from each group at day 7 post-N. brasiliensis infection (n = 7). (E–G) The numbers of eosinophils and ILC2s (E), the amounts of IL-5 and IL-13 in the BALF (F), and the Il5, Il13, and Il33 mRNA expression levels in the lungs as analyzed by Q-PCR (G) from day 35 are shown (n = 4–5). uninf.; uninfected at day 28. Statistical analyses were conducted using two-way ANOVAs with Bonferroni post-hoc tests (B,E,G), Wilcoxon matched-pairs signed rank tests, and two-tailed (C) or unpaired t-tests (F). Data are representative of two independent experiments. (H) C57BL/6 WT mice (n = 5–6) were infected with 5000 L3 S. venezuelensis at day 0. The numbers of ILC2s, eosinophils, CD3⁺/B220⁺ cells, and monocytes among the BALF cells at the indicated days were analyzed by flow cytometry (FACScalibur). Cell populations were defined as follows: ILC2s, FSC^loSSC^loLin(CD3, CD4, CD8, CD19, NK1.1, IgE, Gr-1, siglecF)⁻Sca-1⁺ST2⁺; Eosinophils, CD45⁺CD3⁻B220⁻CCR3⁺, CD3/B220: CD45⁺CCR3⁻CD3⁺/B220⁺; and Monocytes, CD45⁺Autofluorescence^high. Data were compared with day 0 data by one-way ANOVA. Data are representative of two independent experiments.

Figure 2

ILC2s are critical for the enhanced inflammation and the protection against Nippostrongylus brasiliensis infection. (A) Experimental workflow for sequential nematode infection in bone marrow chimera mice. BMT; bone marrow transplantation, sac; sacrificed. S. venezuelensis-infected (Sv) or uninfected (cont) mice (Rora^sg/+, n = 6–7; Rora^sg/sg, n = 7) were infected with N. brasiliensis (Nb). Five days after N. brasiliensis infection, (B) ILC2s and eosinophils among the BALF cells were analyzed by flow cytometry (LSRFortessa). Cell populations were defined as follows: ILC2s, FSC^loSSC^loCD45⁺CD4⁻Lin(CD3, CD8, CD19, NK1.1, IgE, siglec F, Gr-1)⁻Sca-1⁺ST2⁺ and Eosinophils, CD45⁺CD11c^loCD3⁻B220⁻CCR3⁺. (C) Il33, Il5, and Il13 mRNA expression levels in the lungs were examined. (D) The numbers of worms in the intestine were counted at day 5 post-N. brasiliensis infection. Data are representative of two independent experiments. (E) S. venezuelensis-infected or uninfected WT mice were infected with N. brasiliensis after anti-CD4 Ab treatment (αCD4) as in Figure S5. The numbers of worms in the intestine were counted at day 5 after N. brasiliensis infection (n = 10–11). Pooled data from two independent experiments are shown (Mean ± SD).

Figure 3

Eosinophils are essential for resistance against Nippostrongylus brasiliensis. (A–C) S. venezuelensis-infected (Sv) or uninfected (cont) mice (n = 5) were treated with anti-IL-5 Ab (TRFK5, αIL-5) or control Ab (cont IgG1) at day 0 and day 2 post-infection with N. brasiliensis (Nb). (A) The numbers of worms in the intestine were counted at day 5 after N. brasiliensis infection. (B) ILC2s, eosinophils, and CD4⁺ cells among the BALF cells were analyzed by flow cytometry (SP6800). Cell populations were defined as follows: ILC2s, FSC^loSSC^loCD45⁺CD4⁻Lin(CD3, CD8, CD19, NK1.1, IgE, siglec F, Gr-1)⁻Sca-1⁺ST2⁺; Eosinophils, CD45⁺CD11c^loCD3⁻B220⁻CCR3⁺; and CD4⁺ cells, FSC^loSSC^loCD45⁺CD4⁺. (C) Il33, Il13, and Epx mRNA expression levels in the lungs were examined. Data are representative of two independent experiments that had similar results. (D–F) S. venezuelensis-infected or uninfected (cont) mice (WT, n = 7; ΔdblGATA, n = 5–6) were infected with N. brasiliensis. (D) The numbers of worms in the intestine were counted at day 5 after N. brasiliensis infection. (E) Eosinophils, ILC2s, and CD4⁺ cells among the BALF cells were analyzed as in (B). (F) Il33, Il5, and Il13 mRNA expression levels in the lungs were examined. Data are representative of two independent experiments that had similar results.

Figure 4

IL-33 is critical for the enhanced inflammation and the protection against Nippostrongylus brasiliensis infection. WT and Il33^−/− mice (n = 3) were infected with S. venezuelensis (Sv) at day 0. (A) BALF cells from uninfected (day 0) or S. venezuelensis-infected mice at the indicated days were analyzed as in Figure 1H. Data are representative of two independent experiments that had similar results. (B) Il5 and Il13 mRNA expression levels in the lungs were examined. (C–E) WT and Il33^−/− mice were infected with N. brasiliensis 4 weeks after S. venezuelensis (Sv) infection. S. venezuelensis-uninfected mice were used as a control (cont). Five days after N. brasiliensis infection, the number of ILC2s and eosinophils (n = 10–12) (C) and the Il5 and Il13 mRNA expression levels (n = 5–7) in the lungs were examined (D). (E) The numbers of worms in the intestine were counted (n = 10–12). Pooled data from (C,E) or representative of (D) two independent experiments are shown (Mean ± SD).

Figure 5

Strongyloides venezuelensis-infection induces highly reactive ILC2s in the lungs. (A) S. venezuelensis-infected (Sv) or uninfected control (cont) mice (n = 3) were inoculated with 500 N. brasiliensis L3 (Nb). Cell populations of lung cells at the indicated days were analyzed by flow cytometry (SP6800) (Figure S6). (B) Eight weeks after S. venezuelensis infection, lung cells were stimulated with PMA and ionomycin for 3 h in the presence of brefeldin A. Intracellular IL-5 and IL-13 in ILC2s were analyzed by flow cytometry (SP6800). ILC2s were gated on Fixable Viability Dye⁻CD45⁺Lin(CD3, CD4, CD8, CD11b, B220, NK1.1, IgE, Gr-1)⁻ST2⁺Sca-1⁺Thy1.2⁺ cells. (C) The proportions of IL-5⁺IL-13⁻, IL-5⁻IL-13⁺, and IL-5⁺IL-13⁺ ILC2s in the population of CD45⁺ cells (n = 4–5). (D) Lung ILC2s were sorted from S. venezuelensis-infected or uninfected mice (n = 4–5) before or 2 days after infection with N. brasiliensis L3. The expression levels of Il5 and Il13 mRNA were analyzed by Q-PCR. Data are representative of two independent experiments that had similar results (Mean ± SD). (E) The expression levels of transcription factors and TSLPR (Crlf2) in lung ILC2s were analyzed by Q-PCR. Statistical analyses were performed using Student's t-tests. Data are representative of two independent experiments (Mean ± SD).

Figure 6

IL-33 is not sufficient for the induction of trained ILC2s. WT C57BL/6 mice were subcutaneously inoculated with 5000 S. venezuelensis L3 at day 0 (Sv) or were intranasally administrated 100 ng of IL-33 at days 0, 1, 2, and 3 (IL-33). cont, untreated control. (A) Cell populations in the lung cells, BALF cells, and peripheral blood leukocytes (PBLs) at indicated days were analyzed by flow cytometry (SP6800) (n = 4). Cell populations were defined as follows: Eosinophils, PI⁻CD45⁺CD11c^intCD3⁻B220⁻CCR3⁺ and ILC2s, PI⁻FSC^loSSC^loCD45⁺Thy1.2⁺Lin⁻Sca-1⁺ST2⁺. (B) Four weeks after treatment, lung cells were stimulated with PMA and ionomycin for 3 h in the presence of brefeldin A. Intracellular levels of IL-5 and IL-13 in ILC2s were analyzed by flow cytometry (SP6800) as in Figure S8. (C) The proportions of IL-5⁺IL-13⁻, IL-5⁺IL-13⁺, and IL-5⁻IL-13⁺ cells within the population of ILC2s (n = 4). (D,E) Mice were infected with N. brasiliensis 4 weeks after S. venezuelensis infection or IL-33 treatment. Control mice were treated with PBS (PBS) instead of IL-33. Five days after N. brasiliensis infection, (D) the numbers of worms in the intestine were counted (n = 7). (E) The numbers of ILC2s, eosinophils, and macrophages (n = 5–7) among the BALF cells were examined. Statistical analyses were performed using a two-way ANOVA (A,C) or one-way ANOVA (D) with Bonferroni post-hoc tests. Data are representative of three independent experiments (Mean ± SD).