Fig 2: IL-17D promotes infiltration of tumor-associated macrophages (TAMs). (A) Representative flow cytometry profiles and percentages of TAMs in CD45+ TILs in subcutaneous tumors (n = 3). Mean ± SD. **P < 0.01. (B) Representative flow cytometry profiles and percentages of CD206+ cells in CD11b+ F4/80+ cell populations in subcutaneous tumors (n = 2). Mean ± SD. *P < 0.05. (C and D) Flow cytometric analysis and quantification of M-MDSCs (C) and G-MDSCs (D) in CD45+ TILs in subcutaneous tumors (n = 3). Mean ± SD. (E, F) Flow cytometric analysis and quantification of CD8+ T cells (E) and CD4+ T cells (F) in CD45+ TILs in subcutaneous tumors (n = 3). Mean ± SD. *P < 0.05. **P < 0.01. (G) Flow cytometric analysis and quantification of NK cells in CD45+ TILs in subcutaneous tumors (n = 3). Mean ± SD. (H) Images of subcutaneous tumors. (I) Subcutaneous tumor size was measured and compared. **P < 0.01. (J) Representative flow cytometry profiles and percentages of TAMs in CD45+ TILs in subcutaneous tumors (n = 2). Mean ± SD. *P < 0.05.

Fig 3: IL-17D induces lung cancer cells to express a series of TAM recruitment– and polarization–related genes. (A–C) The relative mRNA levels of genes related to macrophages recruitment and polarization was measured via quantitative real-time PCR. RNA was purified from A549 cells expressing IL-17D or empty vector (A), LLC1 cells expressing IL-17D or empty vector (B), and H1155 cells with knockdown of IL-17D or control (C). Mean ± SD. *P < 0.05. **P < 0.01. (D) Representative images and quantification of recruited macrophages. Mean ± SD. ***P < 0.001. Scale bars, 100 µm.

Fig 4: Interleukin (IL)-17D is highly expressed in lung cancer cell lines and tissues and correlates with poor prognosis in human lung cancer. (A) Heatmap of 856 differentially expressed transcripts (710 genes) among normal airway epithelial cell lines (Normal, n = 59), non-small cell lung cancer cell lines (NSCLC, n = 118), and small cell lung cancer cell lines (SCLC, n = 29). (B) RNA-seq data (GSE32036) of IL-17D gene expression of normal airway epithelial cells (n = 59) and NSCLC (n = 118) and SCLC (n = 29) cell lines (value = quantile-normalized and log2-transformed signal). **P < 0.01. ***P < 0.001. (C) Immunoblotting was performed to examine the expression level of IL-17D in normal, NSCLC, and SCLC cells. (D) Representative images of immunostaining with anti-IL-17D antibody in tissue sections of human lung tumor adjacent tissue (a), NSCLC tissue (b) and SCLC tissue (c). Scale bars are 20 µm. (E) The frequency of cases with no (0), low (0.1–3.9), or high (4.0–9.0) IL-17D staining stratified by immunohistochemically-defined lung cancer subtype. (F) The frequency of cases with no (0), low (0.1–3.9), or high (4.0–9.0) IL-17D staining stratified by tumor stage. (G) Kaplan-Meier survival rates for 66 subjects with lung cancer disease with low (staining scores < 2, n = 46, blue line) versus high (staining scores = 2, n = 20, red line) IL-17D expression were compared. Median survivals were undefined months (low IL-17D) versus 34.52 months (high IL-17D; P = 0.0309).

Fig 5: IL-17D promotes TAM infiltration via the p38 MAPK signaling pathway in lung cancer. (A and B) The relative mRNA levels of genes related to macrophages recruitment and polarization were measured via quantitative real-time polymerase chain reaction. RNA was purified from A549 cells expressing IL-17D treated with SB203580, IL-17D treated with DMSO, or empty vector treated with DMSO (A). RNA was purified from LLC1 cells expressing IL-17D treated with SB203580, IL-17D treated with DMSO, or empty vector treated with DMSO (B). Mean ± SD. *P < 0.05. **P < 0.01. (C) Representative images of recruited macrophages. Scale bars, 100 µm. (D) Quantification of recruited macrophages. Mean ± SD. ***P < 0.001. (E) Proposed model for mechanism of IL-17D inducing TAMs infiltration into the TME. High expression of IL-17D in lung cancer cells activates p38 MAPK signaling pathway through binding to receptor complexes of lung cancer cells, which upregulates TAM recruitment– and polarization–related genes expression and promotes infiltration of TAMs in lung cancer.