The TAM family receptor tyrosine kinase TYRO3 is a negative regulator of type 2 immunity

Abstract

Host responses against metazoan parasites or an array of environmental substances elicit type 2 immunity. Despite its protective function, type 2 immunity also drives allergic diseases. The mechanisms that regulate the magnitude of the type 2 response remain largely unknown. Here, we show that genetic ablation of a receptor tyrosine kinase encoded byTyro3in mice or the functional neutralization of its ortholog in human dendritic cells resulted in enhanced type 2 immunity. Furthermore, the TYRO3 agonist PROS1 was induced in T cells by the quintessential type 2 cytokine, interleukin-4. T cell-specificPros1knockouts phenocopied the loss ofTyro3 Thus, a PROS1-mediated feedback from adaptive immunity engages a rheostat, TYRO3, on innate immune cells to limit the intensity of type 2 responses.

Fig. 1. Tyro3^−/− mice hyperrespond to HDM-induced asthma

(A) Total numbers of infiltrating leukocytes (CD45⁺), eosinophils (CD45⁺CD3⁻Ly6G⁻CD11c⁻CD11b⁺SiglecF⁺) and lymphocytes (CD45⁺CD3⁺) in the BAL of HDM-challenged WT, Axl^−/−Mertk^−/−, and Tyro3^−/− mice. (B) Percentage of CD4⁺IL-4⁺, CD4⁺IL-5⁺, CD4⁺IL-13⁺, and CD4⁺IFNγ⁺ cells in medLNs and lung as indicated from phosphate-buffered saline (PBS) controls and HDM-treated mice. (C) Concentration of circulating IgE antibodies as determined by enzyme-linked immunosorbent assay (ELISA) in PBS controls and HDM-treated WT and Tyro3^−/− mice. (D) Representative images and independent histological scores of lung sections stained with hematoxylin and eosin from PBS controls and HDM-treated WT and Tyro3^−/− mice. Scale bar, 5 μm. Data are representative of two or three independent experiments. In all panels, each data point is from a different animal, and bars indicate means ± SEM. Data were analyzed with analysis of variance (ANOVA) and Bonferroni multiple comparison test (A) and unpaired Student’s t test [(B) to (D)]. *P 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., nonsignificant.

Fig. 2. Reduced damage and increased clearance in N. brasiliensis–infected Tyro3^−/− mice

WT and Tyro3^−/− mice were infected with 625 N. brasiliensis larvae. N.I., noninfected; N.b., N. brasiliensis. (A) RBC total numbers in BAL at 7 dpi. (B) Representative fluorescence-activated cell sorting (FACS) plots of eosinophil (CD45⁺CD3⁻Ly6G⁻CD11c⁻CD11b⁺SiglecF⁺) percentages and total numbers of eosinophils in BAL at 7 dpi. (C) Percentage of CD4⁺IL-4⁺, CD4⁺IL-5⁺, and CD4⁺IL-13⁺ cells from medLNs at 2 dpi. (D) Circulating L3- and L5-specific IgG1 antibodies and total IgE antibodies as determined by ELISA. O.D., optical density. (E) Kinetics of the total parasite numbers in the lung and small intestine (S.I.) at 1 dpi, 2 dpi, 3 dpi, and 7 dpi. Data are representative of two or three independent experiments. In all graphs, each data point is from a different animal, means ± SEM. Data were analyzed using unpaired Student’s t test [(A) to (E)]. *P < 0.05; **P < 0.01; n.s., nonsignificant.

Fig. 3. TYRO3 is expressed by and functions in mouse and human DCs

(A) TYRO3 expression by FACS in sorted PDL2⁺ and PDL2⁻ DCs from BM DC cultures. (B) Total number of leukocytes from draining LNs of mice receiving WT or Tyro3^−/− PDL2⁺ or PDL2⁻ OVA-loaded DCs. (C) OVA recalls production of IL-4, IL-5, and IL-13 from CD4⁺ T cells in the draining LNs of mice receiving indicated DCs as measured by ELISA. (D) WT or Tyro3^−/− PDL2⁺ or PDL2⁻ DCs as indicated were cocultured with naïve CD4⁺ T cells for 24 hours in the presence of α-CD3; CCL17, CCL22, and CCL5 were measured in the supernatant by ELISA. (E) TYRO3 expression by FACS on human circulating DCs (CD11c^hiCD11b^loCD14^lo) from healthy controls and parasite-infected patients. Data are representative of two or three independent experiments. In all panels, each data point is from a different mouse or human. Means ± SEM. Data were analyzed using unpaired Student’s t test [(B), (C), and (E)] or paired Student’s t test (D).*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., nonsignificant.

Fig. 4. T cell–derived PROS1 mediates TYRO3 regulation of type 2 immunity

(A to D) Cd4-Cre⁻Pros1^flox/flox (Ctrl) and Cd4-Cre⁺Pros1^flox/flox mice were infected with 625 N. brasiliensis larvae, and all parameters are shown at 7 dpi. (A) RBC total numbers in BAL. (B) Representative FACS plots of eosinophil (CD45⁺CD3⁻Ly6G⁻ CD11c⁻CD11b⁺SiglecF⁺) percentages and total numbers of eosinophils in the BAL. (C) mLNs were restimulated in vitro with antibody against CD3 for 48 hours, and cytokines were measured in the supernatant by ELISA. (D) Total parasite numbers in the small intestine. (E) Representative FACS plots and independent data of the percentage of CD2⁺CD4⁺IL-13⁺ and CD2⁺CD4⁺IFNγ⁺ cells after treating an MLR of human monocyte-derived DCs cocultured with human CD4⁺ T cells with either isotype antibody or antibody against TYRO3 (α-TYRO3) for 10 days. Data are representative of two or three independent experiments. In all panels, each data point is from a different mouse or human, means ± SEM. Data were analyzed using unpaired Student’s t test [(A) to (D)] or paired Student’s t test (E).*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s., nonsignificant.