Fig 2: TSLP is differentially implicated for Th2/Tfh responses induced by epicutaneous or dermacutaneous HDM sensitization.a Hematoxylin & eosin (H&E) stained sections of ears collected immediately after laser microporation (LMP). Red arrow points to a micropore. NT, non-treated. b TSLP protein level in ears (n = 5, 12, 6 mice). c RNAscope in situ hybridization for TSLP mRNA. Black arrows point to one of the positive signals. Dashed lines indicate the dermal/epidermal junction. d Experimental protocol. House dust mites (HDM) or PBS were applied on LMP_30 μm ears to realize e.c. sensitization, or on LMP_91 μm ears to realize d.c. sensitization, at day (D) 0, 3, 7 and 10. Mice were challenged intranasally (i.n.) with HDM every day from D9 to D12, and analysed at D13. e H&E staining, immunohistochemistry (IHC) staining with anti-MBP antibody (specific for eosinophils), or anti-MCPT8 antibody (specific for basophils) of ear sections. Black arrows point to one of the positive signals. Scale bar = 50μm (a, c, e). NS, non-sensitized. f Frequency of IL-4+ (AmCyan) or IL-13+ (DsRed) cells among CD45+ TCR-β+ cells in ears of mice (n = 6, 4, 3, 5, 1, 3, 4 mice). g Total cell number in ear-draining lymph nodes (EDLNs). h Frequency and cell number of Tfh cells and IL-4+ Tfh cells in EDLNs. i Numbers of GL7+Fas+ GC B cells, IgE+ B cells and IgG1+ B cells in EDLNs. For g–i, n = 4, 4, 5, 6, 2, 4, 4 mice. j Serum levels of HDM-specific IgE and IgG1 (n = 5, 6, 4, 4 mice). Graphs in b, f–i show mean ± SEM, two-sided Student’s t-test. Graphs in j show median, two-sided Mann–Whitney rank sum test. All data are representative of three independent experiments with similar results. Source data are provided as a Source Data file.

Fig 3: Co-administration of IL-1β enhances e.c. HDM-induced Th2/Tfh responses in a TSLP-independent manner.a RNAscope in situ hybridization for IL-1β mRNA in non-treated (NT), LMP_30μm and LMP_91 μm ears at 48 h after the microporation. Dashed lines indicate the dermal/epidermal junction. b ELISA measurement of IL-1β protein levels in ears at 48 h after LMP_30 μm and 91 μm (n = 5, 12, 6 mice). c Experimental protocol. HDM with or without IL-1β was applied on LMP_30μm ears (e.c. HDM ± IL-1β), at day (D) 0, D3, D7 and D10. Mice were intranasally (i.n.) challenged with HDM every day from D9 to D12 to induce allergic asthma, and analysed at D13. d ELISA measurement of TSLP protein levels in 30 μm-LMP ears co-administrated with recombinant IL-1β or PBS (n = 4 mice). e Hematoxylin & eosin (H&E) staining and immunohistochemistry (IHC) staining for MBP or MCPT8 on ear sections. Black arrows point to one of the positive signals. Scale bar = 50 μm for all pictures. f Comparison of CXCR5+PD1+ Tfh cells, GL7+Fas+ GC B cells, IgE+ B and IgG1+ B cells in EDLNs (n = 4, 5, 3, 3, 3, 4 mice). g Serum levels of HDM-specific IgG1 and IgE in HDM-treated mice (n = 5, 5, 6, 4 mice). Graphs in b, d, f show mean ± SEM. Two-sided Student’s t-test. Graphs in g show median. Two-sided Mann–Whitney rank sum test. All data are representative of two independent experiments with similar results. Source data are provided as a Source Data file.

Fig 4: Co-administration of IL-1β exacerbates e.c. HDM-induced asthmatic inflammation in a TSLP-independent manner.a Total cell number in BAL fluid. b Cell number of eosinophils, neutrophils, lymphocytes and macrophages in BAL fluid. For a, b n = 4, 5, 3, 3, 3, 4 mice. c Quantitative RT-PCR analyses of BAL cells (n = 3, 4, 3, 3, 3, 4 mice). Graphs in a–c show mean ± SEM. Two-sided Student’s t-test. d Lung sections were stained with hematoxylin & eosin (H&E), analysed by immunohistochemistry (IHC) staining for MBP or MCPT8 (stained as dark red, pointed by black arrows), or by Periodic Acid Schiff (PAS) staining (stained as purple). B: bronchiole. V: blood vessel. Scale bar = 50 μm for all pictures. All data are representative of three independent experiments with similar results. Source data are provided as a Source Data file.

Fig 5: A schematic representation of the context-dependent role of TSLP and IL-1β in promoting skin allergic sensitization and the atopic march.When allergen HDM sensitization occurs superficially in the skin (epicutaneous sensitization), TSLP derived from keratinocytes located in the epidermis plays a dominantly crucial role for allergic sensitization through the lymph node (LN; generating Th2, Tfh and B cell responses) and the subsequent allergic inflammation in the lung. When allergen sensitization occurs deeply in the skin (dermacutaneous sensitization), IL-1β derived from the infiltrated neutrophils and monocytes/macrophages contributes together with TSLP, to generate a stronger allergen sensitization and subsequently a more severe lung allergic inflammation.