Fig 2: IL-1β treatment increased IL-1R1 and p-IRAK-1 expression in RBECs (n=4). (A) Immunoreactive bands of IL-1R1 (80 kDa) and β-actin (42 kDa). (B) The bar graph shows increased IL-1R1 expression in the IL-1β group compared with that in the control group (**P<0.01). (C) Immunofluorescence images showing the expression of CD31+ RBECs (a and d, green), IL-1R1 (b and e, red), and the co-localization of IL-1R1 and RBECs (c and f). Enhanced IL-1R1 immunofluorescence was evident in the IL‑1β group compared with the control group. Scale bars (a-f): 10 μm. (D) Immunoreactive bands of p-RIAK-1 (77 kDa), t-IRAK-1 (77 kDa) and β-actin (42 kDa). (E) The bar graph shows increased p-IRAK-1 expression in the IL-1β group compared with the control group (**P<0.01). The protein expression of p‑IRAK‑1 was significantly suppressed with IL‑1Ra treatment (*P<0.05). RBECs, rat brain capillary endothelial cells; IL-1β, interleukin-1β; IL‑1R, interleukin‑1 receptor; IL‑1Ra, interleukin‑1 receptor antagonist; HC, high concentration of carbon dioxide; ns, non‑significant.

Fig 3: IL‐1β secreted by Mφ promotes tumor C‐C motif chemokine ligand 2 (CCL2) expression and Mφ infiltration. (A) Representative images of IF staining with serial sections of ovarian cancer mouse tissues. IL‐1β (purple) and DAPI were co‐stained in one section (left two); PD‐L1, CCL2, and DAPI were co‐stained in another section (right three). (B) Scatter plot of IL‐1β and CCL2 secretion levels in ovarian cancer mouse tissues, five fields of each tissue were selected randomly. (C‐D) SKOV3 and OVCAR8 cell lines were co‐cultured with THP1 Mφ in the presence or absence of pretreatment with IL1RA, and then the CCL2 mRNA expression in tumor cells was measured using real‐time PCR. E‐F. The CCL2 concentration in SKOV3 and OVCAR8 culture medium was measured using ELISA after the two cells were treated with THP1 Mφ supernatant activated with lactate (L‐THP1 supernatant) in the presence or absence of IL1RA. G‐H. The CCL2 concentration in the SKOV3 and OVCAR8 culture medium was measured using ELISA after the two cells were treated with IL‐1β in the presence or absence of pretreatment with BAY11‐7085 (NF‐κB inhibitor, 5 μM). (I) Representative images of migration assays of THP1 Mφ and PBMC Mφ in fresh medium or tumor culture medium (TCM) or IL‐1β‐activated TCM from SKOV3. (J‐K) Bar charts of migration assays of THP1 Mφ (J) and PBMC Mφ (K) in fresh medium or TCM or IL‐1β‐activated TCM from SKOV3. Data were assessed by unpaired Student's t‐test or linear regression analysis. ** p < 0.01.

Fig 4: IL‐1β promotes tumor programmed death‐ligand 1 (PD‐L1) expression. (A) Heatmap showing relative mRNA expression of immune checkpoint signature genes in SKOV3 cell line. (B‐C) The CD274 mRNA expression in SKOV3 and OVCAR8 cells after co‐culturing with peripheral blood mononuclear cell‐derived Mφ (PBMC Mφ) with or without IL‐1 receptor antagonist (IL‐1RA) pretreatment was determined using real‐time polymerase chain reaction (PCR; 48 h, n = 4). (D‐E). Representative histograms of MFI and bar charts showing membrane PD‐L1 expression in SKOV3 and OVCAR8 cells after 72 h incubation with PBMC Mφ with or without IL‐1RA pretreatment (n = 4). (F) Representative images of immunofluorescence (IF) staining of IL‐1β and PD‐L1 in ID8 ovarian cancer mouse tissues. (G) Scatter plot of IL‐1β secretion and PD‐L1 expression levels in ID8 ovarian cancer mouse tissues, three to five fields of each tissue were selected randomly. (H‐I) Representative images of IHC staining of IL‐1β and PD‐L1 in tumor tissues from patients with high‐grade serous ovarian cancer (HG‐SOC) (H); scatter plot of PD‐L1 and IL‐1β expression levels in tumor tissues from patients with HG‐SOC (n = 41), five fields of each tissue were selected randomly for calculating the mean value (I). (J‐K) NY‐ESO‐1‐positive SKOV3 cells were incubated with or without IL‐1β for 48 h and then directly planted together with NY‐ESO‐1 CAR‐T cells at a ratio of 1:2 for 5 h in the presence of anti‐PD‐L1 antibodies or not. Apoptosis of tumor cells was determined with annexin V and propidium iodide staining (J), and the ratio of annexin V‐positive tumor cells was quantified (K). All graphs show mean ± SEM. Data were assessed using an unpaired Student's t‐test or linear regression analysis. CSF, colony‐stimulating factor. OC, ovarian cancer. * p < 0.05; ** p < 0.01.

Fig 5: Blocking IL-1β in the RA serum attenuates glycolysis-HIF-1α axis mediated IL-1β. (A)qPCR analysis of HK2, HIF-1α, and IL-1β mRNA expression in HMDMs stimulated for 24 h with IL-1β, TNF-α, GM-CSF, IFN-γ, or IL-6 (n = 7).(B)Representative image of IL-1β and HIF-1α expression in HMDMs pretreated with or without 2-DG (5 mM, 3 h) followed by 24 h stimulation with IL-1β, TNF-α, or GM-CSF (n = 4).(C)Extracellular acidification rate (ECAR) measured by Seahorse assay in HMDMs exposed to RA serum with or without IL-1RN, infliximab, or lenzilumab (n = 6).(D-E) Western blot analysis of HIF-1α and IL-1β expression in macrophages treated with RA serum in the presence or absence of (D) IL-1RN or (E) infliximab for 24 h (n = 6).Data were shown as mean ± SEM and were analyzed using one-way ANOVA or Two-tailed student’s t-test. *, p < 0.05; **, p < 0.01. 2-DG: 2-Deoxy-D-glucose; IL-1RN: Interleukin-1 receptor antagonist; IFX: Infliximab