Divergent expression patterns of IL-4 and IL-13 define unique functions in allergic immunity

Abstract

Interleukin 4 (IL-4) and IL-13 are critical for responses to parasitic helminthes. We used genetically engineered reporter mice to assess the temporal and spatial production of these cytokines in vivo. In lymph nodes, IL-4, but not IL-13, was made by follicular helper T cells (T(FH) cells). In contrast, tissue type 2 helper T cells (T(H)2 cells) produced both cytokines. There was also divergent production of IL-4 and IL-13 among cells of the innate immune system, whereby basophils produced IL-4, whereas innate helper type 2 cells (Ih2 cells) produced IL-13. IL-13 production by T(H)2 and Ih2 cells was dependent on the transcription factor GATA-3, which was present in large amounts in these cells, and in contrast to the small amount of GATA-3 in T(FH) cells and basophils. The distinct localization and cellular expression of IL-4 and IL-13 explains their unique roles during allergic immunity.

Figure 1. Eosinophil recruitment, worm expulsion and antibody production during N. brasiliensis infection after depletion of IL-13-producing cells

(a) The cell fate, cytokine expression, worm clearance, eosinophil recruitment and IgE production in the indicated reporter mice 9 days post N. brasiliensis infection.[IV1] (b) Total number of worms in the small intestine of N. brasiliensis infected mice, 9 days post infection. (n = 5 mice per group; *P < 0.001, Two-tailed t test). Graph representative of at least 2 independent experiments. (c) Total number of eosinophils (Siglec-F^hi, CD49b^neg) in the lung of N. brasiliensis infected mice, 12 days post-infection. (n = 2–3 mice per group; *P < 0.01, Two-tailed t test). Graph representative of at least 3 independent experiments. (d) The percentage and total number of eosinophils (SiglecF/Bc^hi, DX5^neg), basophils (SiglecF/Bc^int, DX5^high) and CD4⁺ T cells in the lung of indicated mice on day 9 after N. brasiliensis infection. Plots are representative of two independent experiments (n = 2–4 mice). (e) IgE level in the serum of indicated mice 9 days after N. brasiliensis infection. (n = 5 mice). Graph is representative of at least 3 independent experiments. (f) Expression of high-affinity NP-specific IgG1 in serum of indicated mice 28 days after OVA-NP(15) injection in the footpad (n = 4–15 mice; *P < 0.05, **P < 0.005, Two-tailed t test). Graph is representative of at least 2 independent experiments. (g) Affinity maturation of serum anti-NP-specific IgG1 antibodies from the indicated mice following OVA-NP(15) challenge. Ratio depicts binding of serum IgG1 to BSA-NP(3)/BSA-NP(23) (n = 4–15 mice; *P < 0.05, **P < 0.005, Two-tailed t test). Graph is representative of at least 3 independent experiments. Each data point represents an individual mouse (b, e, f, g) .

Figure 2. The localization and enumeration of cytokine-producing CD4⁺ T cells during parasitic infection

(a) The percentage of IL-4- (huCD2) or IL-13 (huCD4)-positive CD4⁺ T cells in the lung and mediastinal lymph nodes from indicated mice infected 8 days previously with N. brasiliensis. Plots are representative of at least 4 independent experiments (n = 3–5 mice per experiment).(b) Percentage and total number of cytokine-positive CD4⁺ T cells in the lung and draining mediastinal LN 9 days after N. brasiliensis infection. Graphs represent data points combined from two independent experiments (n = 9; *P < 0.05; **P < 0.0001, two-tailed t test). Each data point represents an individual mouse. (c) Ratio of IL-4/IL-13 producing-CD4+ T cells within the mediastinal lymph nodes and lung 9 days post infection with N. brasiliensis. Graph represents data points combined from two independent experiments (n = 9; **P < 0.0001, two-tailed t test). Data points represent individual mice. (d) Immunohistochemistry of popliteal lymph nodes (Top panel) 21 days after L. major infection and lung (bottom panel) 8 days after N. brasiliensis infection. Wild-type tissues served as negative controls for huCD2 and fluorescent reporter staining. Image magnification is 100x; insets magnification 200x. Images are representative of tissues from 3–4 animals.

Figure 3. Differential IL-4 and IL-13 production by T_FH and canonical T_H2 cells

(a) The percentage of IL-4- (huCD2) or IL-13 (huCD4)-producing cells within GFP⁺ CD4⁺ T cells in the lymph nodes and lung of IL-4^KN2/4get and IL-4^4get/+IL-13^Smart/+ mice 8 days post N. brasiliensis infection. Arrow indicates the percentage of T_FH cells among the IL-4- or IL-13-producing cells. Plots are representative of two independent experiments (n = 6 mice). (b) The percentage of (PD-1⁺ CXCR5⁺) T_FH cells among total IL-4- or IL-13-producing CD4⁺ T cells in the lymph node or lung from IL-4^KN2/4get and IL-4^4get/+IL-13^Smart/+ mice 8 days after N. brasiliensis infection (Percentage of cells ± S.E.M. (n = 4; *P < 0.01, two-tailed t test). Data points combined from two independent experiments. (c) Relative expression of mRNA transcripts plotted against housekeeping gene GAPDH for the indicated genes from CD4⁺ T cells isolated from the lymph nodes and lungs of IL-4^KN2/4get mice 9 days post infection with N. brasiliensis. Data are representative of at least 3 independent experiments. (d) Percentage of intracellular GATA-3 and Bcl-6 in CD4⁺ T cells isolated from the mediastinal lymph node and lung from IL-4^KN2/4get mice 8 days post N. brasiliensis infection. Plots are representative of 5 mice from 2 independent experiments.

Figure 4. GATA-3 expression in IL-4- and IL-13-producing CD4⁺ T cells after N. brasiliensis infection

(a) The percentage of intracellular GATA-3^high and IL-4- (huCD2) and IL-13 (huCD4)-producing CD4⁺ T cells from the draining lymph nodes and Lung of WT or IL-4^KN2/+IL-13^Smart/+ mice 9 days after N. brasiliensis. Plots are representative of at least 5 independent experiments (n = 3–4 mice per experiment). (b) percentage of GATA-3^high cells among total IL-4- (huCD2) and IL-13- (huCD4) producing CD4⁺ T cells in the mediastinal lymph nodes and lung 9 days after N. brasiliensis infection.

Figure 5. GATA-3 expression and cytokine production by CD4⁺ T cells in the absence of STAT6

(a) Percentage of GATA-3^high CD4⁺ T cells from wild-type and STAT6-deficient IL-4^KN2/+IL-13^Smart/+ mice 8 days post N. brasiliensis infection. Data points are combined from two independent experiments. (n = 8; *P < 0.0001, two-tailed t test). Each data point represents an individual mouse. (b) Percentage of GATA-3^high IL-4- or IL-13-secreting CD4⁺ T cells from wild-type and Stat6-deficient IL-4^KN2/+IL-13^Smart/+ mice 8 days post N. brasiliensis infection. Data points are combined from two independent experiments. (n = 8; *P < 0.0001; **P < 0.0002, two-tailed t test). Each data point represents an individual mouse. (c) Percentage and total number of IL-4- and IL-13-producing CD4+ T cells in the lymph nodes and lung of wild-type and Stat6-deficient IL-4^KN2/+IL-13^Smart/+ mice 8 days post N. brasiliensis infection. Data points are combined from two independent experiments. (Percentage of cytokine-positive cells or total number ± S.E.M.; n = 8; *P < 0.001; **P < 0.01; ***P < 0.05, two-tailed t test).

Figure 6. IL-4 and IL-13 production by innate type 2 effector cells after N. brasiliensis infection

(a) Top panel shows the percent of ICOS⁺ CD4⁺ or ICOS⁺ CD4⁻ cells among total IL-4- or IL-13-producing lung cells. Bottom panel shows the percentage of CD4⁺ CD49b⁻ or CD4⁻ CD49b⁺ cells among total IL-4- or IL-13-producing lung cells from IL-4^KN2/4get or IL-4^4get/+IL-13^Smart/+ mice 8 days post N. brasiliensis infection. Gates represent CD4⁺ T cells (CD4⁺, CD49b⁻, CD278⁺), Ih2 cells (CD4⁻, CD49b⁻, CD278⁺), or basophils (CD4⁻, CD49b⁺, CD278⁻). Plots are representative of at least three independent experiments. (b) Percentage of CD4⁺ T cells, Ih2 cells, or basophils among total IL-4- and IL-13-producing cells in the lung of IL-4^KN2/4get or IL-4^4get/+IL-13^Smart/+ mice 8 days post N. brasiliensis. Graphs are representative of at least 3 independent experiments, percentage of cells ± S.E.M. (n = 3). (c) Percentage of eosinophils (CD4^neg,SiglecF/βc^hi, DX5^neg), basophils (CD4^neg,SiglecF/βc^int, DX5^high), and Ih2 cells (CD4^neg,SiglecF/βc^neg, DX5^neg, ICOS^high) among CD4⁻ cells in the lung of IL-13^{YetCre/YetCre} 9 days after N. brasiliensis infection. Plots are representative two independent experiments (n = 9 lungs). (d) The percentage and total number of total and YFP-expressing CD4⁺ T cells, Ih2 cells, basophils, eosinophils, and NK/NKT cells among total or YFP-positive (IL-13-producing) lymphocytes in the lung of IL-13^{YetCre/YetCre} mice 9 days post N. brasiliensis infection. Graphs are representative of 3 independent experiments, percentage of cells ± S.E.M. (n = 3). (e) The total number of Ih2 cells (SiglecF/Bc^neg, DX5^neg, CD4^neg, ICOS^high) in the lung of IL-13^{YetCre/YetCre} mice 9 days after N. brasiliensis infection. Plots are representative two independent experiments (n = 2–4 lungs).

Figure 7. Requirements for STAT6 and GATA-3 on cytokine production by innate type 2 effector cells

(a) Percentage of intracellular GATA-3^high Ih2 (ICOS⁺) cells and basophils (CD49b⁺) among total non-T cells in the lung of IL-4^KN2/4get and IL-4^4get/+IL-13^Smart/+ mice 9 days post N. brasiliensis infection. Plots are representative of at least three independent experiments (n = 3–4 mice per experiment). (b) Percentage GATA-3^high IL-4-(huCD2) and IL-13 (huCD4)-producing CD4⁺ T cells, Ih2 cells, and basophils from the lungs 8 days after N. brasiliensis infection. (c) Total number of Ih2 cells (ICOS⁺, CD4⁻, CD8⁻, CD19⁻) in the lung of wild-type and Stat6-deficient IL-4^KN2/+IL-13^Smart/+ mice. Data points are combined from two independent experiments. (Total Ih2 cells ± S.E.M.; n = 8). (d) The percentage and total number of IL-13 (huCD4)-secreting Ih2 cells in the lungs of wild-type and Stat6-deficient IL-4^KN2/+IL-13^Smart/+ mice 8 days after N. brasiliensis infection. Data points are combined from two independent experiments. (Total number or percentage of cells ± S.E.M.; n = 8; *P < 0.01, two-tailed t test). (e) Percentage of GATA-3^high IL-13-producing Ih2 cells in the mediastinal lymph nodes and lung of wild-type and Stat6-deficient IL-4^KN2/+IL-13^Smart/+ mice 8 days post N. brasiliensis infection. Data points are combined from two independent experiments. (n = 8; *P < 0.01, two-tailed t test). Each data point represents an individual mouse.

Figure 8. GATA-3 is required for IL-13 expression, innate cell recruitment and worm expulsion, but not IgE production

(a) Percentage of IL-13- (YFP) producing CD4⁺ T cells and Ih2 cells in the lung of GATA-3^fl/+, IL-13^YetCre/+GATA-3^fl/+, IL-13^YetCre/+GATA-3^fl/fl mice 9 days post N. brasiliensis infection. Plots are representative of 2 independent experiments with 7–9 mice per group. (b) The number of Total or IL-13-(YFP) producing basophils (CD4^neg, Siglec-F^neg, CD49b^pos, CD90.2^int), eosinophils (CD4^neg, Siglec-F^pos, CD49b^neg, SSC^high), CD4⁺ T cells (CD4^pos, ICOS^high, CD90.2^pos), and Ih2 cells (CD4^neg, Siglec-F^neg, CD49b^neg, ICOS^high, CD90.2^pos) in the lung of IL-13^YetCre/+GATA-3^fl/+ (white bars) or IL-13^YetCre/+GATA-3^fl/fl (black bars) of mice 9 days post N. brasiliensis infection. Graphs are a composite of two independent experiments (+/− SEM, n = 7–9 mice per group, *P < 0.05, **P < 0.005, two-tailed t test). Dashed line represents the limit of detection as scored by the reporter negative wild-type control. (c) Eosinophil recruitment, worm expulsion, and total serum IgE assessed in IL-13^YetCre/+GATA-3^fl/+ (white bars) or IL-13^YetCre/+GATA-3^fl/fl (black bars) mice 9 days post N. brasiliensis infection. (+/− SEM, n = 4–5 mice per group, *P < 0.05, **P < 0.005, two-tailed t test).