TSLP promotes interleukin-3-independent basophil haematopoiesis and type 2 inflammation

Abstract

CD4(+) T-helper type 2 (T(H)2) cells, characterized by their expression of interleukin (IL)-4, IL-5, IL-9 and IL-13, are required for immunity to helminth parasites and promote the pathological inflammation associated with asthma and allergic diseases. Polymorphisms in the gene encoding the cytokine thymic stromal lymphopoietin (TSLP) are associated with the development of multiple allergic disorders in humans, indicating that TSLP is a critical regulator of T(H)2 cytokine-associated inflammatory diseases. In support of genetic analyses, exaggerated TSLP production is associated with asthma, atopic dermatitis and food allergies in patients, and studies in murine systems demonstrated that TSLP promotes T(H)2 cytokine-mediated immunity and inflammation. However, the mechanisms through which TSLP induces T(H)2 cytokine responses remain poorly defined. Here we demonstrate that TSLP promotes systemic basophilia, that disruption of TSLP-TSLPR interactions results in defective basophil responses, and that TSLPR-sufficient basophils can restore T(H)2-cell-dependent immunity in vivo. TSLP acted directly on bone-marrow-resident progenitors to promote basophil responses selectively. Critically, TSLP could elicit basophil responses in both IL-3-IL-3R-sufficient and -deficient environments, and genome-wide transcriptional profiling and functional analyses identified heterogeneity between TSLP-elicited versus IL-3-elicited basophils. Furthermore, activated human basophils expressed TSLPR, and basophils isolated from eosinophilic oesophagitis patients were distinct from classical basophils. Collectively, these studies identify previously unrecognized heterogeneity within the basophil cell lineage and indicate that expression of TSLP may influence susceptibility to multiple allergic diseases by regulating basophil haematopoiesis and eliciting a population of functionally distinct basophils that promote T(H)2 cytokine-mediated inflammation.

Figure 1. TSLP promotes peripheral basophilia

IL-4/eGFP reporter mice were treated with PBS or rTSLP and splenic basophils were (a) identified and (b) quantified. WT mice were treated with control-cDNA or TSLP-encoding (TSLP-cDNA) plasmid and splenic basophils were (c) identified and (d) quantified. Serum (e) IL-4, (f) IL-5, (g) IL-13 and (h) total IgE were quantified. (i), Histologic analysis of small intestine. Scale bars, 100 μm. (j), Splenic basophils from control mice (WT), or (SPC-TSLP) mice were identified (j) and (k) quantified. Results are representative of at least three (a-d and j-k) or two (e-i) separate experiments containing 3-5 (a-d), 5 (e-i) or 6-12 (j-k) mice per group. Statistical analyses performed using a two-tailed students t test (*, p<0.05), (**, p<0.01).

Figure 2. TSLP preferentially expands basophil populations from bone marrow- resident cells

Bone marrow-resident cells were taken from IL-4/eGFP reporter mice and cultured in the presence of (a) media, (c) IL-3 or (f) TSLP, and the frequency of IL- 4/eGFP⁺ NBNT cells was determined. (b, d, g), The frequencies of IL-4/eGFP⁺, NBNT, Siglec-F^- basophils and mast cells were determined. Cytology of IL-4/eGFP⁺, CD49b⁺, FcεRI⁺, c-Kit^- (e) IL-3-elicited or (h) TSLP-elicited basophils. Results are representative of at least three separate experiments. Italicized numbers in basophil gates represent the MFI levels of CD49b staining. Scale bar, 10 μm.

Figure 3. TSLP-elicited basophilia is independent of IL-3-IL-3R signaling

Bone marrow cultures from (a) WT or (b) Csf2rb2^-/-/ Csf2rb^-/- mice. WT or Csf2rb2^-/-/ Csf2rb^-/- mice were treated with PBS or rTSLP and (c,e) blood and splenic (g,i) basophils were identified. (d,f,h,j), Total numbers of basophils were quantified. Flow cytometry plots are gated on live NBNT cells. (a,b), Results are representative of at least 3 separate experiments. (c-j), Results are representative of 3 separate experiments. (WT + PBS n=7, Csf2rb2^-/-/ Csf2rb^-/- + PBS n=7, WT + TSLP n=7, Csf2rb2^-/-/Csf2rb^-/- + TSLP n=7). Statistical analysis was performed using a two-tailed students t test (*, p<0.05).

Figure 4. Murine and human basophil populations exhibit heterogeneity

IL-3- elicited or TSLP-elicited murine basophils were stained (shaded) for (a), CD200R, CD69, CD11b, CD62L, (b) CD123, T1/ST2 and IL-18Rα expression and compared to fluorescence minus one (FMO) controls. Numbers represent MFI levels. Results are representative of at least 3 separate experiments. (c), GSEA of microarray data comparing IL-3-elicited and TSLP-elicited basophils. (d), Activated human basophils stained for TSLPR. (e), Representative histograms demonstrating ST2 expression (shaded) compared to isotype controls. (f), Percentage of basophils from control or EoE patients expressing ST2 compared to isotype controls. Statistical analysis was performed using a two-tailed students t test.