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. 2015 Oct;16(10):1051-9.
doi: 10.1038/ni.3244. Epub 2015 Aug 31.

Innate Immunological Function of TH2 Cells in Vivo

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Free PMC article

Innate Immunological Function of TH2 Cells in Vivo

Liying Guo et al. Nat Immunol. .
Free PMC article

Abstract

Type 2 helper T cells (TH2 cells) produce interleukin 13 (IL-13) when stimulated by papain or house dust mite extract (HDM) and induce eosinophilic inflammation. This innate response is dependent on IL-33 but not T cell antigen receptors (TCRs). While type 2 innate lymphoid cells (ILC2 cells) are the dominant innate producers of IL-13 in naive mice, we found here that helminth-infected mice had more TH2 cells compared to uninfected mice, and thes e cells became major mediators of innate type 2 responses. TH2 cells made important contributions to HDM-induced antigen-nonspecific eosinophilic inflammation and protected mice recovering from infection with Ascaris suum against subsequent infection with the phylogenetically distant nematode Nippostrongylus brasiliensis. Our findings reveal a previously unappreciated role for effector TH2 cells during TCR-independent innate-like immune responses.

Conflict of interest statement

Completing financial interests

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Administration of IL-33 and IL-7 induces TH2 cells to produce IL-13
(a) Naïve CD4 T cells were sorted from OT-II–4C13R reporter mice and cultured under TH2 conditions for 3 rounds. Cells were cultured in IL-2-containing medium for 10 days and then injected i.v. into C57BL/6 recipient mice. 24h later, mice were intratracheally challenged with PBS, or indicated cytokines (150 ng IL-33 and/or 100 ng IL-7 each mouse) for 3 consecutive days, or OVA (100 μg endotoxin-free OVA in PBS) once. Lungs were collected and cytokine production was examined 24h after last cytokine administration (72h after OVA challenge). Cells shown were gated on transferred OT-II TH2 cells. (b) Statistical analysis of the cytokine production. Error bars represent standard deviation from the mean. ****, P <0.0001 by two tailed student’s t-test. Data are representative of three independent experiments with 3–6 mice in each group (a, b).
Figure 2
Figure 2. In vivo generated OVA-specific TH2 cells respond to papain to produce IL-13 in an MHC-independent manner
(a, b) 0.5×106 sorted naïve CD4 T cells from OT-II–4C13R reporter mice were injected i.v. into C57BL/6 mice. One day after cell transfer, mice were immunized subcutaneously with a mixture of 500 N. brasiliensis (N.b.) infective larvae L3 and 100 μg endotoxin-free OVA and then boosted intratracheally 5 days later with OVA (100 μg) in PBS. Twenty-five days after N. brasiliensis infection, mice were challenged intratracheally with PBS, OVA (100 μg, endotoxin-free) once, or papain (25 μg) for 3 consecutive days with or without an anti-MHCII antibody. 500 μg anti-MHCII antibody was administered i.v. on day 1 and day 3 of papain challenge. Lungs were collected 24h after last papain challenge; DsRed and AmCyan expression by transferred OT-II TH2 cells were analyzed. Statistical analysis of the cytokine production by OT-II TH2 cells. **, P<0.01; ns, not significant, P>0.05; ****, P<0.0001. Data are representative of three independent experiments with 2–4 mice in each group.
Figure 3
Figure 3. IL-33 acts directly on in vivo generated TH2 cells causing them to produce IL-13 in response to papain
(a, b) 0.5×106 naïve CD4 cells from OT-II–4C13R reporter mice were transferred i.v. into wild-type (WT) recipient mice or IL-33-deficient (Il33−/−) recipient mice. Mice were immunized and challenged as described in 2a. 150 ng IL-33 was administered twice i.v. on Day 1 and Day 5 of infection. Lungs were collected 24h after last papain challenge; DsRed and AmCyan expression by transferred OT-II TH2 cells were analyzed. Statistical analysis of the cytokine production by the transferred OT-II TH2 cells. ns, not significant, P>0.05; **, P<0.01. (c–e) Naïve CD4 T cells were sorted from wild-type (WT) CD90.1+CD90.2+ DO11.10 mice or Il1rl1−/−CD90.1CD90.2+ DO11.10 mice. Cells were cultured under TH2 conditions with OVA peptide and APC for three rounds. After being cultured in IL-7-containing medium for 7 days, wild-type TH2 cells and Il1rl1−/− TH2 cells were mixed at 1:1 ratio and i.v. injected into wild-type CD90.1CD90.2+ BALB/c recipients. 24h later, mice were intratracheally challenged with IL-33 (150 ng), IL-7 (100 ng), papain (25 μg), heat-inactivated papain (25 μg) or PBS for 3 consecutive days, or with endotoxin-free OVA (100 μg) once. 24h after IL-33 plus IL-7, papain or PBS challenge or 4h after OVA challenge, lungs were isolated and single cell suspensions were prepared. Transferred WT and Il1rl1−/−DO11.10 TH2 cells were sorted from lung cell suspensions. The amounts of Il13 mRNA and Il4 mRNA were measured by real time PCR. Il13 (d) and Il4 (e) mRNA amount in wild-type cells in PBS-treated recipients were set as 1. Data are representative of two (a, b) or one (c–e) independent experiments with 2–5 mice in each group.
Figure 4
Figure 4. TH2 cells generated by infection with N. brasiliensis respond to papain to produce TCR-independent IL-13
(a) Lung TH2 cells expanded dramatically upon N. brasiliensis infection. Lungs were harvested from mice either unimmunized or 10 days after N. brasiliensis infection. TH2 cells were identified as CD4+CD44+Foxp3GATA3+IL-33R+. Markers for ILC2 cells were LinCD44+GATA3+IL-33R+CD127+Thy1+. Data are representative of at least three independent experiments with 3 mice in each group. (b) Number of lung-resident TH2 and ILC2 cells in individual mice that were unimmunized or 13 days or 21–31 days post-N. brasiliensis infection. ****, P<0.0001; ***, P<0.001 by paired t-test. Data are compiled from multiple independent experiments. (c, d) Twenty-five days after N. brasiliensis infection, 4C13R reporter mice were challenged intratracheally with PBS, or papain (25 μg in PBS) for 3 consecutive days with or without anti-MHCII antibody. 500 μg anti-MHCII antibody was administered i.v. on day 1 and day 3 of papain challenge. DsRed and AmCyan expression by lung-resident TH2 and ILC2 cells were analyzed 24h after last papain challenge. (d) Comparison of percent of DsRed+ TH2 and ILC2 cells among total live lymphocytes. ns, not significant, P>0.05; **, P<0.01; ***, P<0.001. Data are representative of three independent experiments with 4 mice in each group (c–d).
Figure 5
Figure 5. Short-term HDM challenge induces prompt eosinophilic airway inflammation in mice recovering from N. brasiliensis infection
(a, b) C57BL/6 mice were uninfected or infected with N. brasiliensis. Twenty-three days later, mice were challenged intratracheally with PBS or HDM (25 μg in PBS) daily for 3 consecutive days. 24h after the last challenge, BAL fluids and lungs were collected and analyzed. N.b., N. brasiliensis only; HDM, HDM challenge in uninfected mice; N.b.+HDM, HDM challenge 23 days after N. brasiliensis infection; N.b.+HDM+anti-MHCII, 500 μg anti-MHCII antibody was injected i.v. on day 1 and day 3 of HDM challenge. Percentages shown were calculated as a percent of the live cells. ****P<0.0001; **, P<0.01; ***, P<0.001; *, P<0.05; ns, not significant, P>0.05. (c) Lung histology analysis. Top 3 panels, lung sections were stained with H&E; left panel arrow - mucus metaplasia; center panel arrow - perivascular infiltration; right panel arrow - alveolar infiltration. Lower left panel, PAS staining arrow - mucus metaplasia; lower middle, Luna staining arrow - eosinophils; and lower right, Ym1 staining arrow – Ym1+ macrophages. Data are representative of two (a, c) or three (b) independent experiments with 3–4 mice in each group.
Figure 6
Figure 6. HDM-induced eosinophilic airway inflammation is independent of TCR, partially dependent on CD4 T cells, and mediated through IL-33
(a) Mice were infected with N. brasiliensis and challenged with HDM as described in Fig. 4a. Anti-MHCII antibodies were injected i.v. at the time of 1st and 3rd HDM challenge. BAL fluid was collected for cellular constituents analysis. Lungs were harvested and lung sections were analyzed by H&E staining and lunar staining. ns, not significant, P>0.05. (b) Mice were infected and challenged as in Fig. 4a. 500 μg of anti-CD4 neutralizing antibody was injected i.v. on day 1 and day 3 of HDM challenge. *, P<0.05. (c) Wild-type mice or IL-33-deficient (Il33−/−) mice were infected with N. brasiliensis and challenged with HDM as described in Fig. 4a. 150 ng IL-33 was injected i.v. into IL-33-deficient mice on day 1 and day 5 of N. brasiliensis infection. One group of IL-33-deficient mice infected with N. brasiliensis was challenged with HDM without supplemental IL-33 while a second group received IL-33 supplementation (100 ng per mouse on day 1 of HDM challenge). BAL cellular constituents were analyzed. ***, P<0.001; ns, not significant, P>0.05. (d) Left, 5×106 in vitro cultured OT-II TH2 cells were injected i.v. into Rag2−/−Il2rg−/− recipient mice. Some groups of mice were challenged intratracheally with HDM (25 μg in PBS) daily for 4 weeks. 500 μg anti-CD4 antibody was injected into one group on days 2, 8 and 15. BAL cellular constituents were analyzed 24h after the last challenge. Right, 0.5×106 ILC2 derived from IL-25-injected mice and cultured for 3 days in IL-7 plus IL-33 were injected i.v. into Rag2−/−Il2rg−/− recipient mice. Mice were similarly treated and analyzed as those receiving primed OT-II TH2 cells. ns, P>0.05; ***, P<0.001. Data are representative of three (a–c) or two (d) independent experiments with 3–7 mice in each group.
Figure 7
Figure 7. Tissue-resident TH2 cells contribute to innate host defense against helminths
(a) Mice were inoculated with A. summ eggs orally and 11 days later received a subsequent subcutaneous inoculation with 500 N. brasiliensis L3. The mice in control groups were only inoculated subcutaneously with 500 N. brasiliensis L3 or only A. suum eggs. (b) N. brasiliensis adult worms in the small intestine and eggs in the fecal pellets were determined on day 8 post N. brasiliensis inoculation. ***, P<0.001; *, P<0.05. (c) Total number of lung-resident TH2 and ILC2 cells. (d) Lungs were harvested on day 6 post N. brasiliensis inoculation. DsRed and AmCyan expression by lung-resident TH2 and ILC2 cells were analyzed by flow cytometry. (e) Statistics of cell number of DsRed-expressing TH2 and ILC2 cells. *, P<0.05. (f) 5×106 in vitro cultured OT-II TH2 cells were injected i.v. into Rag2−/−Il2rg−/− recipient mice. 24h later, mice were inoculated with N. brasiliensis and adult worms in the small intestine were determined on day 8 after N. brasiliensis inoculation. Data were compiled from two independent experiments. **, P<0.01. (g) BM chimeras were generated by injecting wild-type or RORα-deficient BM cells into Rag2−/−Il2rg−/− recipient mice. 8 weeks after reconstitution, chimeras were inoculated with N. brasiliensis L3 alone or with A. summ eggs followed by N. brasiliensis as described in Fig. 7a. Adult worms in small intestine were determined on day 8 after N. brasiliensis inoculation. Some mice received 500 μg anti-CD4 antibodies i.v. on day 0 and day 4 after inoculation with N. brasiliensis. RORα-deficient BM chimeras that have less than 3% ILC2 cells in lung with and without injection of anti-CD4 antibody are shown. Data were compiled from three independent experiments with 4–5 mice in each group. *, P<0.05; **, P<0.01. (h) Mice were inoculated with N. brasiliensis L3 and 12 days later with A. summ eggs. The mice in the control group were only inoculated with A. suum eggs. The parasitic third-stage larvae in lung were determined 8 days after A. summ inoculation. ***, P<0.001. Data are representative of five (b) or two (c–e, h) independent experiments with 3–5 mice in each group.

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