Fig 2: Schematic summary of local immune environment during progression of endometriosis. Tissue injury results in the release of alarmins such as IL-25, IL-33, and TSLP, which, individually or collectively, promote a type 2 immune response. In particular, platelets and Tregs together promote the Type 2 immunity predominance, which is exemplified by recruitment and aggregation of of Th2 cells, M2 macrophages, and possibly group 2 innate lymphocytes (ILC2s) in the lesional immune microenvironment. These type 2 immune cells subsequently release type 2 cytokines, such as IL-4 and IL-13, which polarizes macrophages into alternatively activated M2 macrophages. M2 macrophages can release copious TGF-β1 and PDGF, inducing EMT, FMT, SMM, and fibrogenesis. Platelets and Tregs, in and by themselves and also by the induction of a type 2 immune response, induce the TGF-β1 and PDGF signaling pathways to promote fibrogenesis in endometriosis. See text for more details. IL, interleukin; PDGF-BB, platelet-derived growth factor-BB; ILC2, group 2 innate lymphoid cells; MΦ, macrophage; Tregs, regulatory T cells; Th, T helper cell; TGF-β1, transforming growth factor β1; IFN-γ, interferon-γ; TNF-α, tumor necrosis factor-α; CCL17, CC chemokine ligand 17; CXCL, CXC chemokine ligand; FGL-2, fibrinogen-like protein 2; TXA2, thromboxane A2; TSLP, thymic stromal lymphopoietin; EMT, epithelial-mesenchymal transition; FMT, fibroblast-to-myofibroblast transdifferentiation; SMM, smooth muscle metaplasia.

Fig 3: R. intestinalis promotes DC-induced differentiation of Tregs to inhibit inflammation through a mechanism requiring TSLP expression in IECs.(a) Caco-2 cells transfected with the TSLP-siRNA or TGFβ siRNA were stimulated with LPS+R.I in the presence or absence of the recombinant TSLP/TGFβ proteins. The supernatant of the Caco-2 cells was used to induce human monocyte-derived DCs. TGFβ and IL-10 levels in DC culture supernatants were measured using ELISAs.(b and c) DCs were treated with the supernatants described above in (a) and then extensively washed and cocultured with human naive CD4+ T cells (Th0). The proportion of Tregs was analysed using flow cytometry (gated on CD4+ cells), and a statistical analysis was performed.(d) Schematic of C57BL/6 WT mice treated with the anti-TSLP (neutralization of TSLP) mAb, TNBS and R. intestinalis.(e and f) Changes in body weight (e) and DAI score (f) of each group (n=6). ***p<0.001, control vs. TNBS; #p<0.05, TNBS vs. TNBS+R.I; &p<0.05, anti-TSLP+TNBS vs. anti-TSLP+TNBS+R.I; $p<0.05, TNBS+R.I vs. anti-TSLP+TNBS+R.I.(g and h) The colon histopathological score and representative images of colon tissues stained with H&E (n=6).(i and j) Representative flow cytometry plots of Treg cells (gated on CD4+ cells) in colon tissues and statistical analysis (n=6).(k) ELISAs of TSLP, TGFβ and IL-10 levels in colon tissue homogenates (n=6).*p<0.05, **p<0.01, ***p<0.001, and ns, non‑significant. Data represent the cumulative results from two or three independent experiments.

Fig 4: A. fumigatus–stimulated DCs activates JAK/STAT through TSLP. (A) DCs were treated with A. fumigatus and then co-cultured with CD4+ T cells for 4 days, a Western blot analysis was performed to determine the protein levels of RORγt, p-JAK1, JAK1, p-JAK2, JAK2, p-STAT3, STAT3, and β-actin in the CD4+ T cells. (B) CD4+ T cells were cultured with rmTSLP (100 ng/mL) for 3 days, protein levels of RORγt, p-JAK1, JAK1, p-JAK2, JAK2, p-STAT3, STAT3, and β-actin were analyzed by western blot. Quantification of relative protein levels in (A) and (B) were shown in (C) and (D), respectively. Cells were divided into four groups and were treated as described in Figure 4C. (E) Western blot analysis of the protein levels of RORγt, p-JAK1, JAK1, p-JAK2, JAK2, p-STAT3, STAT3, and β-actin in CD4+ T cells. (F) CD4+ T cells were incubated with TLSPR siRNA (80 nM) or NC siRNA for 24 hours, then co-cultured with A. fumigatus–stimulated DCs for 4 days. A Western blot was performed to detect the protein levels of RORγt, p-JAK1, JAK1, p-JAK2, JAK2, p-STAT3, STAT3, and β-actin in CD4+ T cells. Quantification of relative protein levels in (E) and (F) were shown in Supplementary Figure S4C and D. (Data are mean ± SEM, *P < 0.05, **P < 0.01, n = 3).

Fig 5: Platelets, macrophage subsets, and T cell subsets in endometriotic lesions of the four groups of mice in Experiment 2. (A) Representative immunohistochemical staining of CD41, CD68, CD163, iNOS, TSLP, FOXP3, T-bet, GATA-3, and RORγT in the four groups. All magnifications were ×400. Scale bar = 50 µm. (B) Boxplots showing the density of CD41+ (platelet), CD68+ (macrophage), CD163+ (M2), and iNOS+ (M1), FOXP3+ (Treg), T-bet+ (Th1), GATA-3+ (Th2), RORγT+(Th17) cells, and the staining level of TSLP. *p < 0.05; **p < 0.01; ***p < 0.001; NS, not statistically significant (p > 0.05) for the difference between the testing group and the Control group (by Wilcoxon’s test). n = 8 for each group. CT, Control group; PD, platelet depletion; TD, Treg depletion; JD, joint (simultaneous) depletion of platelet and Treg; TSLP, thymic stromal lymphopoietin.