Fig 2: Clinical consequences of BC patients with different IFN expression levels. a The GSE21653 and the GSE6532 datasets were obtained and analyzed in the CTGS website (http://ctgs.biohackers.net/). Kaplan–Meier analysis was carried out in the group of patients stratified by ER status. Patients were categorized into a low (lower quartile) and a high (upper quartile) IFN expression groups. To analyze statistical differences, Log-rank (Mantel–Cox) tests were performed. b Gene expression analysis was performed using GSE22219 dataset obtained from the NCBI GEO website. Expression levels of IFNA1 and IFNB1 were analyzed in different grades of BC specimens stratified by ER status. Bars in red indicate the median expression level in each group. Statistical analysis was performed using one-way ANOVA followed by Tukey’s HSD post hoc test. c Gene expression analysis was conducted using E-TABM-158 dataset obtained from ArrayExpress website. A case that lacked age information record was excluded from data analyses. Correlation between the expression of IFN and aromatase was analyzed in ER-positive BC patients by age group. Pearson’s correlation coefficient (r) was calculated

Fig 3: In vivo activation of the TI-IFN pathway in the kidney of mice or humans with kidney tubulointerstitial disease. (A,B) Kidney mRNA expression of ISGs (Ifit1, Usp18, Oasl2) and Ifnb measured by qRT-PCR in murine tubulointerstitial kidney injury induced by folic acid (FAN) (A) or LPS (B). Bar charts represent the Mean ± SEM in control and kidney injured mice. *p ≤ 0.05 vs vehicle-injected control mice. (C) Mice with FAN (left) or LPSN (right) show higher renal IFNAR1 and IFNAR2 mRNA levels than controls (Cont), detected by qRT-PCR at different timepoints (LPSN: 1–24 h; FAN: 24–96 h). *p ≤ 0.05 vs control untreated animals (n = 3-7/temporal group). (D) Kidney IFNAR2 was overexpressed in LPS and FAN mice. Representative optical microscopy pictures show an increased tubular location of IFNAR2 detected by immunohistochemistry, especially in sections of intense tubular damage (boxed areas which are shown at higher magnification below). Yellow arrows point to damaged/dilated tubules expressing IFNAR2. The bar chart shows the signal quantification in the entire set of control and LPS-treated (LPSN, 24 h; n = 5) or folic acid-treated (FAN, 48 h; n = 4) animals. Results are expressed as the Mean ± SEM. *p ≤ 0.05 vs control animals without kidney injury. Original magnification ×200. Scale bar 100 µm. (E) Increased kidney IFNβ expression during LPSN and FAN. Representative optical microscopy pictures of immunohistochemical detection of IFNβ in LPS (n = 3) and FAN (n = 3) kidneys. Boxed sections and the corresponding magnified images below highlight areas of intense staining surrounding tubules (yellow arrows), the Bowman’s capsule (red arrow), and inside glomeruli (black arrow), suggestive of endothelial location. *p ≤ 0.05 vs control untreated group. Original magnification ×200. Increased IFNβ was also detected by confocal microscopy (white arrows). Scale bar 100 µm. (F) Confocal microscopy images of representative LPSN and FAN kidneys stained for IFNβ (green) and CD31 (red) (upper panel) and for IFNβ (red) and F4/80 (green) (middle panel). Endothelial cells expressing CD31, but not mononuclear phagocytes (F4/80), display a marked IFNβ signal. Colocalization was assessed by Mander’s overlap coefficient (MOC) (ImageJ Fiji, JACOP plugging). Bar chart represents the Mean ± SEM, n = 3 mice/group) (lower panel). White arrows indicate cells tracked through the IFNβ/CD31 or IFNβ/F4/80 fluorescence channels. Original magnification ×200. Scale bar 10 µm. (G) Increased IFNAR2 expression in acute human tubulointerstitial nephropathy (AHN). Representative optical microscopy pictures from renal biopsies (AHN1 and AHN2) show damaged tubules with strong tubular IFNAR2 staining. Boxed areas are enlarged below. In situ (yellow arrows) or detached (white arrows) tubular cells show high IFNAR content. The graph shows the Mean ± SEM of IFNAR2 signal in the entire set of patients. *p ≤ 0.05 vs control group, n = 5. Original magnification ×200. Scale bar 100 µm.

Fig 4: TWEAK and LPS activate the TBK1/IKKε and TI-IFN pathways in cultured kidney tubular cells and in vivo in the kidneys. (A,B) TWEAK and LPS activated the TBK1/IKKε/IRF3 pathway as assessed by phosphorylated TBK1 and IKKε detected by western blot (A) and phosphorylated IRF3 detected by western blot (B, upper panel) or immunofluorescence (B, lower panel) as compared with vehicle-treated control cells (Cont). Tubular MCT cells were stimulated with 100 ng/ml TWEAK or 1 μg/ml LPS for 15-60 min (m). Figures are representative of experiments repeated at least thrice. Original magnification ×400 in B (lower panel). Scale bar 10 µM. (C–E) TWEAK and LPS activated the TI-IFN pathway. (C) Quantitative RT-PCR for mRNA expression of Ifna, Ifnb1, and Ifnar1/2 in MCT cells stimulated with TWEAK or LPS for 6 and 24 h, and TWEAK-dependent IFNβ secretion assessed in cell culture supernatants (24 h) by ELISA (right upper panel). Results are expressed as the Mean ± SEM of at least three individual experiments. *p ≤ 0.05 vs control untreated cells. (D) Representative western blots of increased levels of phosphorylated STAT1 (pSTAT1) and TYK2 (pTYK2) in total protein extracts from MCT cells stimulated with 100 ng/ml TWEAK or 1 μg/ml LPS. Stimulation times are expressed in hours (H). (E) Extended gene expression study of samples analyzed in panel C shows that TWEAK and LPS also upregulated ISG genes (Ifit1, Usp18, Oasl2, Cxcl10). Results are the Mean ± SEM. *p ≤ 0.05 vs control untreated cells. (F) The contribution of the autocrine/paracrine recruitment of the TI-IFN pathway to TWEAK- and LPS-elicited responses in MCT cells was studied by blocking IFNAR with 10 μg/ml neutralizing anti-IFNAR antibody before stimulation with 100 ng/ml TWEAK or 1 μg/ml LPS. Gene expression was evaluated by q-RT-PCR after 6 h (Cxcl10, Ccl2) or 24 h (Oasl2). Results are expressed as Mean ± SEM of four or five experiments. *p ≤ 0.05 vs control untreated cells, # p ≤ 0.05 vs TWEAK- or LPS-stimulated cells. (G) Pharmacological inactivation of canonical IKKs limits TBK1/IKK-dependent ISG transcription in TWEAK- or LPS-stimulated MCT cells. Cultured MCT cells were pretreated for 1 h with chemical inhibitors of IKKα/β, namely 10 µM parthenolide (Parth) or 2.5 µM IKK16, before the addition of TWEAK or LPS for 24 h. Gene expression of ISGs (Usp18, Oasl2) was assessed by q-RT-PCR. Bar chart represents the Mean ± SEM. *p ≤ 0.05 vs control vehicle-stimulated cells and # p ≤ 0.05 vs TWEAK- or LPS-stimulated cells (n = 3/4).